Clinical Application - Novel Indications and Present Ethical Concerns

#### **Chapter 8**

## Ethics of International Stem Cell Treatments and the Risk-Benefit of Helping Patients

*Neil H. Riordan, Luis Gerardo Jiménez Arias and Ramón Coronado*

#### **Abstract**

Numerous and diverse participants are involved in the development of novel therapies: patients, physicians, scientists, sponsors, governing bodies, lawmakers, institutional review boards, and bioethics proponents. While the welfare of the patient must always and unquestionably be at the forefront of any intervention along with informed consent, their wishes, their requests, and their expectations should also be considered at every step. The availability of stem cell research in various countries with dissimilar regulatory agencies has opened the door for thought-provoking questions about their validity from an ethical, legal, and moral perspective, which will be addressed in this chapter, framed within the doctor-patient relationship.

**Keywords:** ethics, stem cell therapy, regenerative medicine, international bioethics, mesenchymal stem cells

#### **1. Introduction**

In 2010, the Ministry of Health of Costa Rica disallowed stem cell therapy, citing concerns about the experimental nature of these procedures. This abrupt decision left several patients unable to continue receiving treatment at the San José hospital – most notably, a young pilot suffering from spinal cord injury after a 2008 airplane accident that rendered him paraplegic with no perspective of ever regaining mobility, according to two independent physicians. Having experienced encouraging progress in muscle recovery and bowel and sexual function following stem cell therapy, [1] he promptly filed a legal remedy for protecting his constitutional rights to the Supreme Court of Costa Rica, along with other patients in a similar predicament. In their appeal, they argued that patients had *"a right to exhaust all technically feasible procedures to recover [their] health and quality of life"*, citing Article XI of the 1948 American Declaration of the Rights and Duties of Man (the right to the preservation of health and well-being) [2]. This right should not, they contended, be limited by a political authority. Additionally, they invoked the American Convention on Human Rights, namely Articles 5 (the right to physical integrity) and 2 (whereby States Parties undertake to adopt (...) legislative or other measures to grant the rights or

freedoms enshrined in The Convention). Lastly, none of the plaintiffs declared having experienced any adverse effects from their therapy at the time of the appeal; they had, in fact, perceived improvements in their health condition.

The Supreme Court ruled that medical treatments had to be permitted by law before their implementation, as had been argued by the defendant (the Ministry of Health). While the judges acknowledged the principle of patient autonomy, the doctor/patient relationship, human dignity, and the right to health, selecting a treatment should rely on the law. However, the Court also recognized that no adverse events had been noted throughout these treatment cycles with stem cells and that they had no factual or legal arguments to halt the treatments that had already begun. The plaintiffs were therefore permitted to continue their therapy.

Six years later, presidential decree n°39,986-S authorized regenerative therapies with adult stem cells in Costa Rica, based on national scientific recommendations, under the human right to health access principles, and citing decades of past research on the safety of hematopoietic stem cells transplants [3]. Therein are outlined the requirements to be submitted by those seeking stem cell therapy administration: safety profile, scientific rationale, cellular characterization, administration, and requirements for the qualifications of health professionals and facilities. While the decree makes a distinction between minimally manipulated and more than minimally manipulated cells, both are permitted with different avenues for authorization.

This Costa Rican case is pivotal because the Supreme Court ruling marks the first legal precedent in a Latin American country that allows patients who are already being treated with stem cells to continue their treatment – a first step, perhaps, to implement international legislation on stem cells. Following the law to the letter in that instance proved to be rather impractical, falling almost into irrationality as it limited the right of access to health for patients who required treatment as a last resort, and going against international law, which stipulated that Costa Rica must have taken the necessary legislative, economic and political measures to facilitate access to the said right to health. The subsequent presidential decree is also a landmark case since it authorizes using a non-minimally manipulated therapeutical product currently not permitted in other jurisdictions, notably in the USA according to the Federal Drugs Administration (FDA) guidance. The compassionate use of or access to drugs and new therapies remains limited on the grounds of minimizing harm to terminally ill patients [4]. How much evidence is necessary to release a given drug or a given experimental therapy? Are Phase I studies sufficient? Can effectiveness be shown at this stage? Answers remain unclear [4].

This chapter will examine legal and moral issues arising from stem cell treatments, the patient/doctor relationship, and the right of access to health and patient welfare in the context of current international regulations and medical tourism – and how conflicting regulations between countries pose conflicting views on ethics regarding patient access to new therapeutics.

#### **2. Stem cells: definitions, applications, and considerations**

Human "stem cells" is a broad term that may refer to various types of cells differing in their origin, applications, and characteristics. Hematopoietic stem cells, multipotent, self-renewing, and capable of generating blood cells, were first described in the early 60s [5]. Soon after, another type of multipotent stem cells differing from those of hematopoietic lineages were observed in bone marrow [6] – these would

#### *Ethics of International Stem Cell Treatments and the Risk-Benefit of Helping Patients DOI: http://dx.doi.org/10.5772/intechopen.108541*

later become known as mesenchymal stem cells [7] or mesenchymal stromal cells (MSCs), with a limited capacity to differentiate into specific types of adult cells. Pluripotent embryonic stem cells (ESCs), capable of giving rise to all cells in the body, were derived in the late 90s from blastocysts [8]. Finally, induced pluripotent stem cells (iPSCs) were introduced in 2006: somatic cells reprogrammed to have the embryonic capacity for differentiation [9]. While much of the research in the 20th century was focused on isolating and identifying techniques to culture stem cells, the early 21st century saw a growing number of case reports and clinical trials seeking to establish their therapeutic potential. This line of research was not without challenges or controversy (**Table 1**).

Most of the earlier bioethical controversy was focused, understandably, on the use of embryonic stem cells due to the loss of viable embryos in the process of isolation and derivation of the stem cells, which led to bans or limitations imposed on research in several nations. Mesenchymal stem cells, in contrast, share little of those ethical concerns related to their tissue origin since they can be derived from adult tissue, notably for autologous use, or from perinatal tissue, such as the umbilical cord that would routinely be discarded after a normal birth, for allogeneic use. Consequently, the ethical controversies surrounding the use of mesenchymal stem cells have shifted to considerations applicable to clinical trials and the development and commercialization of novel experimental therapies.

Recent clinical trials with mesenchymal stem cells cover a broad range of tissue sources and administration routes, complicating comparisons between published results. In brief, mesenchymal stem cells appear safe for administration at least in the short term, [21–23], and some long-term reports are also available [24, 25]. Adverse


#### **Table 1.**

*Biological and ethical concerns arising from the administration or transplantation of various stem cell types.*

events are reportedly transient, usually fever or fatigue, and no tumorigenicity or malignancy has been observed thus far [23]. While the mechanisms of action of mesenchymal stem cells are not entirely understood, current research views stem cell secretions as key for their immuno-modulatory, anti-inflammatory, and therapeutic properties. The conditions treated in various clinical trials or case reports include cardiovascular, neurological, autoimmune, orthopedic, pulmonary, and graftversus-host disease, [26] and more recently, COVID-19 [27]. At least ten products derived from mesenchymal stem cells have received regulatory approval in South Korea, Japan, India, Canada, Australia, and Europe [12]. The wide variety of conditions researched as well as the legitimacy of some products, may give the erroneous impression that mesenchymal stem cells are a "cure-all": more research is necessary to standardize isolation and culture techniques, as well as to establish risk-benefits and true efficacy for certain conditions to avoid these pitfalls. Most importantly, trials should be conducted ethically, ensuring that disappointing results are also published, that all adverse events are carefully documented and reported, and that patients duly consent with a proper understanding of the procedures and the potential benefits and limits.

Throughout this chapter, "stem cells" should be understood to mean mesenchymal stem cells or adult stem cells unless otherwise indicated.

#### **3. International regulation and medical tourism**

The use of stem cells in Costa Rica has been commercially permitted since 2016. The current law does not specify the effectiveness of a treatment or the amounts to be charged, if any. The broad wording was designed to position and cement the place of Costa Rica as a safe biotechnological or medical tourism hub, thereby disregarding ideological debates arising in other countries regarding the restrictions on the use of stem cells. In Turkey, the use of stem cells has been open and regulated as long as they are not embryonic cells, giving way to laws increasingly open to medical tourism [28]. South Korea is a pioneer in the production of stem cell-derived products, [29] notably a composite of allogeneic umbilical cord blood-derived mesenchymal stem cells and hyaluronate to treat cartilage defects in osteoarthritis [24, 30]. In 2014, Japan introduced a "fast-track" regulatory path for regenerative medical products in the Pharmaceutical and Medical Devices Act [31], whereby products can be commercially available following a short trial: patients recruited into that scheme are enrolled in a registry to be followed up for a period during which efficacy must be demonstrated. This law and the subsequent applications of this law were criticized in the international scientific community, [32, 33] using terms such as an *"obsession [with staying at the forefront of regenerative medicine]*" [34]. Nevertheless, Japan stood its ground [35] and continued research with induced pluripotent stem cells, announcing the start of prospective trials for spinal cord injury and heart conditions in Japanese university hospitals [36, 37], among others. Japanese scientists appear to have followed up on these developments and have published some relevant self-reflection [38–40].

Irrespective of stem cells, medical tourism is hardly a new phenomenon, and a wealth of literature has been written about ethical considerations and repercussions both in the country of origin and the destination country [41, 42]. Certain countries such as Thailand, Singapore, and India have developed this medical tourism industry to the extent that it represents a non-negligible percentage of their GDP. Contributors to the debate should examine their own biases when referring to foreign countries,

#### *Ethics of International Stem Cell Treatments and the Risk-Benefit of Helping Patients DOI: http://dx.doi.org/10.5772/intechopen.108541*

avoiding terms such as 'third world' or assuming higher possibilities of substandard care, infections, or fraudulence as this may alienate so-called developing nations from much-needed transparency and open cooperation. The perspectives of physicians or researchers from "destination" nations must be included and perhaps should even be the primary source for scientific output, as they should be considered capable of producing critical literature from their own cultural and scientific perspectives. There is a marked inequality in availability and volume of research produced in more affluent countries compared to underprivileged ones, [43] though a process of "catching up" appears to be in place [44].

Nevertheless, medical tourism is a cause of concern, [45] particularly in the case of stem cell therapy, since prospective patients may not be sufficiently informed about the risk and benefits of the treatments they are receiving, or may be charged disproportionate amounts for dubious or unregulated procedures – a form of preying on their state of mind due to their illnesses. Many patients who travel abroad to be treated with stem cells do so as a last resort and in some cases desperately seek a cure or at least an improvement for their medical condition. However, medical malpractice or dishonesty can occur regardless of whether a country approves a given drug or procedure. While medical tourism may contribute or aggravate the proliferation of questionable practices, it can hardly be considered the only reason. Every country has an obligation to implement and uphold good medical practices promoting principles of medical ethics and the deontological standards of medicine. Moreover, the proliferation of fraudulent clinics in some destination clinics is likely hindering the development of legitimate stem cell research [45]. The case of Stamina in Italy is an example where internal regulations successfully identified a fraudulent clinical practice; this operation needed to be halted because of an individual holding no medical qualifications, poor quality standards for manufacturing and no sound scientific rationale, not because of an inherent evil or penchant for fraudulence in stem cell research.

Should medical tourism then be regulated? There is currently no consensus or homogeneity in criteria among countries on regulatory affairs of therapeutic stem cell treatment. The implantation of international regulations would not be a trivial exercise. The suggestion of a recognized agency such as the FDA or the European Medicines Agency (EMA) granting certificates of approval to international clinics, while desirable in theory, would not be possible in practice as it would interfere with the sovereignty of each territory to regulate and legislate their internal affairs. Similarly, the jurisdiction of national agencies is limited to their borders, and allocating a portion of their budget to oversee practices in other countries would not be economically feasible. Some international networking organizations such as the International Society for Cell and Gene Therapy (ISCT), have recently established working groups to tackle regulatory and ethical issues with committee members primarily based in North America, [46] but unlike charters of international intergovernmental organizations, binding for the undersigning nations, their advisories are ultimately non-binding. The Pan American Health Organization (PAHO) has issued recommendations for the use of "advanced therapy medicinal products", [47] acknowledging that regulatory bodies are still developing in many countries and stressing that ethical implications must be considered, that treatments must take place in authorized, specialized centers, and that a risk-based approach could be considered for establishing regulations. This document notably states that *"continual advances in scientific research generally keep it several steps ahead of regulatory mechanisms, a situation that occurs worldwide,"* and calls for open communication between scientists and regulatory bodies [47].

Another possibility to curb medical tourism would be to introduce restrictions into the national health network for those patients who have traveled and received treatment in clinics unrecognized by regulatory bodies of e.g., the USA – such punitive measures might, however, not be conducive to the best interests of the patients. Patients may feel they have to lie or be reluctant to request their physician's opinion, thereby losing an opportunity for education or more extensive research into the treatments they seek abroad. A more constructive solution would then be to actively educate prospective patients before their decision, ensuring that the cost-benefits of stem cell treatment are carefully considered, including the possibility that effectiveness may be small, limited, or non-existent for their particular pathology. The education of patients is hardly a novel idea [48]. The information should be presented in a clear, easily understandable manner rather than a dry scientific report, as this is likely not the format that prospective patients are used to consuming in an era where multimedia is pervasive. Neither should the tone of the information provided be perceived as scolding or belittling, as this may have the unintended effect of the valuable advice being dismissed altogether to the detriment of the patient; efforts should be focused on empowering the patients to make their own decisions (to "know best" or to "be in the know") – or any other desirable traits that a sociological study into the characteristics of this population may reveal, along with decision-aid studies. For example, they could be more receptive to someone they perceive as a peer than to a disengaged physician or a removed academic seemingly removed from the daily struggles of their conditions. The same narrative and visual techniques employed to attract patients can also be used to educate. Recent efforts, e.g., by the International Society for Stem Cell Research (ISSCR) [49], remain very text-heavy and potentially unengaging for the general public.

However, if after considering the risk/benefits, the patient still decides to seek treatment abroad, be it a clinical trial or a treatment approved by the destination country's regulatory bodies, at a cost or not, it then becomes the right of a patient to health and the right to access health, to improve their quality of life – this is, perhaps, far more complex to regulate or legislate in an international context. There is a delicate balance between governmental control (imposition or restriction) of therapeutics and the reach of these controls inflicting upon the freedom of the patients to choose what they (along with their medical doctors) want to pursue for their health, under the sole assumption that the patients are fully informed about the risk and possible benefits associated with the treatment.

#### **4. Let doctors be doctors: the patient-doctor relationship**

The 2018 Right to Try Act of the USA creates a legal framework for access to unapproved medical products by patients with life-threatening illnesses who have exhausted all their options and may not participate in a clinical trial, provided said product has completed a Phase I study [50, 51]. Opponents of this law have argued that it creates conditions for physicians to prey on desperate patients by creating false hope, that the burden of treatment costs is shifted to patients and manufacturers, and that existing health disparities may be exacerbated, ultimately leading to greater patient suffering [52, 53]. Additionally, valuable information or data collection about product development or adverse events may be lost due to the lack of FDA oversight [52]. The legislation does not compel a manufacturer to provide access to treatment; some manufacturers may outright refuse, [54] and early reports seem to indicate that

#### *Ethics of International Stem Cell Treatments and the Risk-Benefit of Helping Patients DOI: http://dx.doi.org/10.5772/intechopen.108541*

drugs are still being requested in greater volume under Expanded Access rather than with the Right to Try provision [55, 56]. This legislation raised much controversy, often politically charged, sparking ethical debates about what it was trying to achieve, how much it would truly help, or how it would be implemented in practice. The ethical problems of stem cell therapy then seem no different from that of any other experimental therapy (such as those for cancer or rare diseases contemplated by this law) where the patient's autonomy, the cost/benefit of the treatment and any possible abuses or misuses must be weighed in. Advocates of the Right to Try Act emphasized the liberty of patients to choose a treatment and to eliminate bureaucracy; in a similar vein, Texas House Bill 810 (85R) authorized the *"provision of certain investigational stem cell treatments"* under investigation in clinical trials to patients *"with certain severe chronic diseases or terminal illnesses."* [57]. The patients were required to provide written informed consent, and the treatment was to be overseen by an Institutional Review Board (IRB), and administered by a certified physician in a hospital, surgical center, or medical school. The IRBs were to submit annual reports of treatments enacted under this law. *"Why can't someone that is of age have the ability to sign off, so to speak, with regard to a proper medical release on the ability to do something that can make such a dramatic difference in their life, and their lifespan, and their quality of life?"* argued the proponents [58]. This would also encourage *"medical innovation"* [58] in the state of Texas – not unlike the intent of countries who have passed similar laws allowing stem cell research or therapy. These two landmark legislations of the United States exemplify a movement to put the patient and their doctor's relationship at the forefront, which has not been without controversy [59, 60].

If a therapy exists, every effort should be undertaken to implement a way to access said therapy as a last resort for patients. The requirement of having successfully completed Phase I of scientific research may be a reasonable compromise, as long as the patients are sufficiently and objectively educated about the risks and cost/benefits before reaching a decision. And yet, as the example of Japan has shown, a national law, a patient registry, and clinical trials overseen by universities, have still not been considered enough by the academic community. When is it enough? What and whose criteria drive this quantification? Would the debate not be enriched from the participation of physicians and patients who are, so to speak, in the front lines of the battle? One may argue that a "desperate" patient cannot adequately provide informed consent due to their state of mind, but this is a particularly thorny argument that toes close to discrimination, paternalism, [61] or, to use a more modern term, ableism: is having an illness ever sufficient to render a person incapable of making decisions regarding their own welfare? Is a person's dignity and mental ability lessened or invalidated when faced with a significant loss of quality of life or an eventual end of life? This decisionmaking process perhaps belongs more in the sphere of a qualified psychiatrist or therapist for each particular case rather than a broad stroke ruling in an academic setting or legislated by a government body. Broadly qualifying a disadvantaged person (in this case, one with an illness) as intrinsically "vulnerable" or incapable of making informed decisions for themselves may be an insult that reinforces the injustices and stigma that they already face [62]. A decision to use or seek new treatments is not in itself irrational; if prospective patients would be considered competent enough to consent to enter a phase I/II trial in a research setting, why could they not consent in a non-research setting under a physician's supervision? [63]. And if patients have the right to refuse a treatment, surely from this right to refuse follows a reciprocal right to choose or access an intervention [59, 64, 65]. It is an infringement on the right of patient to procure treatment that they understand would be useful for their condition

after being presented with clear and truthful information about said treatment. Instead of paternalistic protection, prospective patients need empowerment: a greater voice in setting research agendas and designing studies [62].

Perhaps the most common concern is the potential harm of stem cells themselves or the problems of their commercialization, as the non-maleficence principle must always be kept in mind. The relative safety of mesenchymal stem cells has been sufficiently covered, as reported by systematic reviews [21–23]. Still, more research is needed for standardization of dosage, culture methods, and source of the stem cells, as well as a need for quantifying effectiveness for clearly defined conditions and thorough documenting of adverse events. Regarding commercialization, as long as there is a demand, there will always be a market to fulfill that demand with various degrees of ethical and legal shades. Stem cell therapy is no stranger to such a conundrum in the face of market greed. A problem of commercialization may derive from health providers being unwilling to fully inform patients about the risk/benefits for fear of losing business; however, not all practices are the same, comparable to medical practices running legally in which their marketing strategies might present skewed information to capture more clients. Within legal boundaries, there could still be unethical behaviors and vice-versa. On the other hand, patients who have gone to great lengths to receive treatment may experience a placebo effect or convince themselves it was worthwhile. Yet incurring in costs to access a treatment is not inherently unethical or fraudulent; the FDA has published a guidance outlining requirements where this practice may be authorized, notably when the costs would be extraordinary to the sponsor because of "*manufacturing complexity, scarcity of a natural resource, the large quantity of the drug needed (e.g., based on the size or duration of the trial), or some combination of these or other extraordinary circumstances (e.g., resources available to a sponsor)*" [66].

The pressure to find new therapies for illnesses with limited, insufficient, or no current treatment options comes precisely from the physicians, the scientific and medical industry, and patients seeking relief for their conditions. Herein lies the more significant risk: a race for supply and greed of demand when faced with pain or eventual death. Suppose a particular country's laws or guidelines are restrictive enough to hinder the physician/patient relationship. In that case, doctors may find it impossible to consider alternative therapies, even under compassionate use, due to the lack of adequate protocols. At the same time, patients feel powerless in the face of government regulations. It is precisely at this point where, in desperation, abuses or misuses arise, not from the new therapies themselves, but from a lack of expectations or incomplete information about the possibility of a cure or relief. And one may ask: what's the rush? Why do some patients insist on seeking a treatment that is not readily available or approved instead of waiting for the due process of clinical research? *"The rush is the daily necessity to help sick people. (...) The 'rush' arises from our human compassion for our fellow man who needs immediate help," as "their illnesses will not wait for a more convenient time"* [67].

If a legally qualified doctor in his professional authority, after having read results of recent advances in the field, well within the boundaries of the regulatory bodies of the country where they operate concludes that such treatment could help a particular patient, a patient who is willing and fully informed to the best of the current understanding – would they be morally justified in refusing said treatment? Can moral objections ever be a sufficient basis for denying the right to healthcare? Patients who seek stem cell treatments may have spiritual distress or therapeutic hope, [68] an aspect some medical doctors may not be equipped to manage. But what of

#### *Ethics of International Stem Cell Treatments and the Risk-Benefit of Helping Patients DOI: http://dx.doi.org/10.5772/intechopen.108541*

compassion? This use of "compassionate" may be close to that employed by ecclesiastical authorities who do not oppose but promote treatments that offer at least a better quality of life: a physician's duty is not limited to knowledge and technical expertise, but also compassion [69]. The compassionate act and the treatment as compassion, coming from the good judgment of a doctor seeking the best for his patient, must therefore be left in that sphere of the doctor's relationship with his patient. Depersonalized rulings do not allow the physician to exercise his art, profession, and oath. The relationship between the physician, or a team of physicians, and their patients is thus an essential aspect of making an informed decision: one the one hand, the patient exercising his autonomy to make medical decisions, and on the other, the physician, upholding his medical oath and not creating false expectations by promising more than what is expected to be achieved with a given treatment or to create hopes beyond what can be offered. Thus, the final decision to access an intervention must lie in the hands of the patient and their physician, based on real world evidence for safety, and within a sound legal and ethical framework.

"*Ethics in both research and clinical settings is most effective when it is preventive"* [70]: indeed, bioethicists do not go ahead with scientific developments but discuss scientific issues that are already on the table. Conversely, neither should physicians regard ethical questions *as "removed from their daily work at bench or bedside,"* as the purpose of new treatments is a societal benefit [70]. *"Market will efficiently allocate the resources, but not always in an ethical manner"* concerning medical tourism; [41] ethical considerations should therefore be contemplated before the application of treatments by creating a legal framework that promotes scientific research and keeps the welfare of the patient at the forefront. The fear of possible misuse or medical misconduct should not deny the patients' right to health, particularly when their lives are at risk or when they are the most vulnerable to their condition. Accumulated clinical experience and evidence-based medicine about safety, dosage, and efficacy would be more appropriate when determining whether to offer or withhold access to treatment.

#### **5. Legal and moral issues**

The international legal system is derived from ethical principles, at the heart of which lies the dignity of the human person, and modern bio-law too draws from ethical and axiological foundations. Human dignity as a concept is fundamentally imprecise, as its definition necessitates defining first what is dignified or worthy, and what is unworthy – an anthropocentric, Judeo-Christian notion [71]. Subsequently, the western concept of human dignity has evolved and dissociated itself from any deity to accept the teleological interpretation of the end of man (Kant): each person is an end in itself and not a means to satisfy the end of another person. In any case, a more pertinent application would be establishing what makes an act respectful of the dignity of others. A philosophical question arises as to whether human dignity is opening up in the last century, whether it is facing threats as never before, or whether both are occurring simultaneously [72]. But if dignity is being threatened, it is first necessary to seek and recognize, that is to say, to pinpoint these threats. One of the possible threats to human dignity comes precisely from biotechnological development and the interference of politicized legal systems that could be restricting fundamental rights, such as the right to health and the right to life. International treaties regarding human dignity appear to have different intensities and interpretations when applied to concrete cases [73], in legal cases ranging from political disappearances to in-vitro

fertilization [74]. Interestingly, in the latter, a court of law and not science has defined a biological fact. Are we then facing an ideological system of human rights? Do human rights serve to protect life and health? Are all lives of equal value or are some worth less than others? Are scientific truths at the service of the law or should the law adjust to the reality of biotechnological progress?

The Catholic Church has been a notable exponent of the ethics of stem cell research and therapy, with contributions from the various Congregations of the Holy See and various speeches and interviews of the Popes after the Second Vatican Council promoting scientific development hand in hand with ethics. *"Progress becomes true progress only if it serves the human person and if the human person grows: not only in terms of his or her technical power, but also in his or her moral awareness*," Pope Benedict XVI declared in 2006. Science shows its usefulness most strongly and richly when its end is to alleviate human suffering through new findings and resources: the efforts of the researchers result in the improvement of the affected and the different conditions or diseases. Stem cell research was deemed deserving of encouragement when it combined scientific knowledge, the most advanced technology in the biological field, and the ethics advocating respect for the human being in all phases of his existence [75]. Man is the actor of scientific research, but he is often the object of this same research; he must consequently be the beneficiary of scientific research, but never a mere instrument. Man cannot be disposed of as an object of research or commercialization, especially when his state is more vulnerable, in accordance with all the principles of personalist ethics; the main interest is the well-being of all and, in particular, of each individual [76]. Research initiatives with adult stem cells were deemed to be free of ethical problems, and clinical use presented no moral objections as long as *"scientific rigor and prudence [reduced] any risks to the patient to the bare minimum and [facilitated] the interchange of information among clinicians and full disclosure to the public at large"* [77]. A repeated call is thus made for dialog between science and ethics, particularly when the fruits of research remain inaccessible to those who lack the means to access them. "*Advances in medical science,"* it was noted, *"go hand in hand with just and equitable provision of health-care services,*" [75] and one may add to this, with public health policies that are willing and open to enable access to those fruits of research. The path of advancement thus leads to the promotion structures and economic means conducive to scientific achievements.

The morality of medical tourism from the perspective of prospective patients has been examined before, considering whether it is moral to "jump the queue" in countries with socialized medicine to seek care in countries where ordinary citizens may not afford the facilities offered to medical tourists. While some patients understood the perspective of the greater good, they were more willing to solve their problems (pain) than to consider fairness or morality at the time of their decision [78]. At the heart of this conflict lie two divergent ethical frameworks: the rights-based and the communitarian frameworks. In rights-based approach, "the rights and dignity of the individual should never (or rarely) be sacrificed to the interests of the larger society", whereas the "common good" will be at the center of communitarian views, where policies will be shaped to promote these "shared values, ideals and goals of a community" [64]. Having a thorough regulatory process protects the needs of the community, or more precisely, of the future members of the community who will become sick – paradoxically to the detriment of those who are currently ill or suffering and consequently have good reasons to prefer a quicker process or an alternative approach [59]. To what extent can the goal of seeking relief be considered immoral? When a patient fully and objectively informed decides to try an experimental or

#### *Ethics of International Stem Cell Treatments and the Risk-Benefit of Helping Patients DOI: http://dx.doi.org/10.5772/intechopen.108541*

unproven therapy in a country where it is regulated, within any means reasonably available to them, should their freedom to choose be curtailed? Is there a price to feeling well, to regain mobility? To life?

The case of Ashya King, [79, 80] a young patient from the UK suffering from a brain tumor in 2015, is a dramatic example of the difficult decisions patients and their families encounter when at odds with current regulations or laws. Unwilling to subject him to radiation and chemotherapy at such a young age, the parents expressed a desire to try proton beam therapy in Prague, which was at that time not approved in the UK. When they were denied this option, the parents signed the child out of the hospital and attempted to travel to Prague – but an arrest warrant was issued against them because of potential child endangerment. After a short judicial deliberation, they were able to reach Prague. The intervention was successful; as of 2018, the child was reportedly in good health with cancer in remission [81]. The UK approved proton beam therapy shortly after positive results from a clinical trial, as it was safe and had fewer side effects than chemotherapy [82]. Randomized trials comparing proton therapy (desired by the parents) and traditional radiation (proposed by the UK health system) were deemed to be "*unethical and not feasible*" [82] and this was "*likely to be the best evidence available*" [83]. Clinical trials are the gold standard of research, but of what good would that have been to the child in 2015? He did not have the time to wait for this approval as he needed treatment as a matter of life or death. Was it morally justified to withhold this treatment option from him? Was it fair to arrest parents for seeking treatment, unapproved in their country, that ultimately would save their child's life and preserve his quality of life? The intervention was successful, but even if it had not been, was it immoral for them to try? Those who are healthy and not living with a significant disability should, perhaps, strive to achieve a greater understanding of the mindset and the goals of someone who is in pain or significantly impaired before they cast moral judgments on their actions, or seek to limit their liberties as self-appointed moral arbitrators. In the late 80s, when genetic therapy was in its infancy, the parent of three sick children wrote in the context of sickle cell anemia research, *"I resent the fact that a few well-meaning individuals have presented arguments strong enough to curtail the scientific technology which promises to give some hope. Aren't they deciding what's best for me without any knowledge of my suffering?"* [84]. Prospective patients may feel similarly bewildered when being deprived of their autonomy and their capacity to make their own decisions, and so the detached intellectual debate should be balanced with compassion [67] and with respect for the self-determination of patients.

#### **6. Future perspectives**

The case of Japan merits more consideration rather than outright dismissal. Following a desire to regulate the proliferation of stem cell clinics with dubious practices, a law was passed after various committee reports, some including medical professionals, lawyers, and lay participants [38]. The development of this legislative action must be understood in the Japanese context, which recognizes the right to access to health care and protects the freedom of discretion and academic freedom (physician's discretion) [38]. Subsequently, pertinent arrests have been made in cases where cells were processed without proper regulatory authorization [38, 85]. The approval of one ophthalmological trial with induced pluripotent stem cells was subject to a committee review process, where risks to patients were examined, along with how information was to be presented to prospective patients (who additionally had significant visual impairments) for informed consent [40]. The process in the latter was not perfect, [40] and neither is the current law [38]. Still, as Japanese researchers note, there is potential for suggestions, amendments, and recommendations to strengthen the practice of regenerative medicine in Japan*: "If various case studies on the review processes of [stem cell trials] or other cutting-edge biotherapeutic trials from around the world were similarly discussed, such case studies would contribute to establishing or improving guidelines for review committees to further improve the quality of these discussions*" [40]. That is, perhaps, the most tragic aspect of the case of Japan: a country exercising its national sovereignty, enacted legislation with reasonable regulatory controls, but it was maligned by academics in other countries since its inception. Yet Japanese researchers have demonstrated they are capable of internal criticism and suggestions for improvement. Rather than jumping to conclusions of fraud, obsessions, or lack of oversight, countries should be encouraged and trusted to develop their regulatory laws with guidance from recognized international health bodies, and scientists of said countries can be held accountable for upholding ethical, medical, and scientific standards.

Some notable lessons may be learned from the history of excimer laser. More commonly known as "LASIK", it was developed in the 1970s and applied to ophthalmology in the late 80s. Compared to other countries, the FDA was notoriously slow to authorize this intervention, or more precisely, the manufacturing process for the laser technology. During the lengthy review process, interesting questions such as "*what complication rates are acceptable?*"; "*what length of follow-up is acceptable [for complications]?*"; "*what is an excellent study design for a clinical trial (...)?*"; "*how good must a surgical procedure (...) be before it will be made widely available for clinical use in this country?*" [86]. These questions have many similarities with the current debates regarding stem cell trials. Excimer laser investigators "*expressed frustration with the deliberate pace of the review process, while outside the United States large numbers of patients [were] having the surgery*" in Canada, Europe, Australia, and Asia [86]. Indeed, clinics from those countries established *"systems of referral in which Americans fly in for surgery and return home for post-operative care*" [86]. The FDA approved the procedure between 1995 and 1999 following the usual clinical trial phase format. Excimer laser surgeries were extremely popular in the 2000s in the USA, then declined notably, [87] with a resurgence during the pandemic presumably due to mask wearing. The reasons for this decline in popularity are not well understood, but some patients cite concerns about complications after reading about the experiences of other patients [88]. There are lessons to be learned here: patients WILL find a way within their means to pursue a novel treatment or procedure they believe will help them, particularly if regulatory bodies are slow on the uptake. The conclusion is not necessarily that regulatory bodies should be less careful or quicker, but given that patients will seek treatment where it is available, once again education and information are of utmost importance. Indeed, once more information about excimer laser became available, even with the low rate of complications, some patients decided on their own that the procedure was not worth it or that they were unwilling to risk it. Thus, patients too should be trusted in their capability to exercise their own educated decisions when it comes to their access to health provided that they are given sufficient guidance.

The development, introduction and "fine-tuning" of stem cell interventions are perhaps not so different from the history of other medical breakthroughs, but this debate has become peculiarly heated, sometimes emotional. All stakeholders, *Ethics of International Stem Cell Treatments and the Risk-Benefit of Helping Patients DOI: http://dx.doi.org/10.5772/intechopen.108541*

patients, patient advocates, scientists, regulators, and clinicians, even those currently offering unproven therapies (as not all are fraudulent or untrustworthy, and may desire fair regulation) must engage in a constructive dialog to define acceptable policies [89]. This dialog would ideally lead to humane, ethical, scientifically sound, and commercially viable regulations, as long as every party listens actively and does not react with frustration when presented with differing points of views. The Colombian Xaverian university recently collaborated with multiple countries to develop an ISO for "biobanking of human mesenchymal stromal cells derived from bone marrow," [90] with debates and meetings in Berlin, Tokyo and Toronto, [91] proving cross-border collaboration is possible to develop international standards for stem cells. We are past the time of pointing fingers or engaging in unproductive academic accusations: all stakeholders must arrive at a consensus with the ultimate goal of developing sound regulatory policies at the international and national level, from which the rest will derive: non-fraudulent, ethically-driven clinics, physicians exercising their medical criteria, and patients empowered to make their own informed decisions.

#### **Acknowledgements**

The authors would like to thank Ms. Dorita Avila for manuscript preparation for publication.

#### **Conflict of interest**

NHR is a shareholder of Medistem Panama. RC and LGJA declare no conflicts of interests.

### **Author details**

Neil H. Riordan1 \*, Luis Gerardo Jiménez Arias2 and Ramón Coronado3,4,5,6

1 Medistem Panama, Panama, Panama

2 International Institute of Human Rights Americas Chapter, San José, Costa Rica

3 University of Texas Health Science Center (Transplant Department) at San Antonio, Texas, USA

4 Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, USA

5 Crown Scientific, San Antonio, Texas, USA

6 Signature Biologics, Irving, Texas, USA

\*Address all correspondence to: neil@aidanresearch.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Ethics of International Stem Cell Treatments and the Risk-Benefit of Helping Patients DOI: http://dx.doi.org/10.5772/intechopen.108541*

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### **Chapter 9**

## Therapeutic Features of Mesenchymal Stem Cells and Human Amniotic Epithelial Cells in Multiple Sclerosis

*Reza ArefNezhad and Hossein Motedayyen*

#### **Abstract**

Imbalance in immune responses plays an indispensable role in pathogenesis and development of multiple sclerosis (MS), as a neurodegenerative disorder. Current treatments are not always successful in preventing MS development and treating the disease. Stem cell-based cell therapy has provided a new window for treating neurodegenerative disorders. Stem cells can regulate the immune system and improve axonal remyelination. They can be isolated from different origins such as bone marrow, embryonic, neural, and adipose tissues. However, there is a challenge in choosing the best cell source for stem cell therapy. Mesenchymal stem cells (MSCs) derived from different origins have significant immunoregulatory impacts on different cells from the immune system. A growing body of evidence indicates that adipose tissue and umbilical cord can be a suitable source for obtaining MSCs. Moreover, human amniotic epithelial cell (hAEC), as a novel stem cell with immunoregulatory effects, regenerative effects, and low antigenicity, can be a candidate for MS treatment. This chapter discusses therapeutic impacts of MSCs and hAECs in MS disease.

**Keywords:** multiple sclerosis, mesenchymal stem cell, human amniotic epithelial cell, regenerative impacts, immunomodulatory effects

#### **1. Introduction**

Neurodegenerative disorders are considered as a chronic and progressive inflammatory condition resulting in the deposition of abnormal forms of specific proteins in the nervous system and destruction of neurons in motor, sensory, or cognitive systems [1, 2]. These disorders mainly involve women and are observed in subjects with age ranging from 20 to 30 years [3]. It is reported that more than 2.5 million individuals suffer from multiple sclerosis (MS) around the world who require effective therapeutic approaches to control disability and recover the central nervous system (CNS) functions [3]. The major problem of the management of the disease is the lack of successful regeneration of neurons [4].

Until now, several therapeutic approaches for MS have been suggested to control abnormal immune responses including natalizumab, interferon-β (IFN-β), glatiramer acetate, and fingolimod (FTY720). These treatments mainly exert their inhibitory effects on immune reactions and thereby reduce the number of relapses and modulate the progression of neurologic disability. However, they have not been consistently successful and are suitable in arresting the disease in approximately 30% of relapsingremitting (RR) MS patients as the most common form of MS [5, 6]. It is reported that these treatments fail to control the degeneration of nerve tissue in an aggressive form of MS [7]. Among these approaches, stem cell-based therapies show a hopeful outlook for decreasing neural damages in the neurological diseases through regenerative roles for remyelination, the secretions of neurotrophic mediators with immunomodulatory impacts, and differentiation into astrocytes and oligodendrocytes effectively *in vivo* and *in vitro* [8]. Previous studies have shown some challenges for using stem cells as a curative treatment in clinical trials such as tumorigenicity and immunogenicity [9, 10]. However, extensive data of the literature have indicated that stem cell therapy exerts positive effects on animal models with neurological disorders [11, 12]. Clinical uses of adult stem cells, particularly mesenchymal stem cells (MSCs) and human amniotic epithelial cells (hAEC), have been recommended for the management of neurological diseases such as MS [13–15]. Several advantages have been reported for their therapeutic applications including the following: (1) their relative safety and low immunogenicity in comparison with other stem cell sources [16, 17]; (2) the ease of their accessibility, isolation, expansion, and manipulation *ex vivo* [18]; (3) their potency in differentiation into mesodermal lineages [16]; and (4) their capability to transport from the blood to damaged sites. Hereby, this chapter aimed to describe and discuss evidence regarding MSC- and hAEC-based therapies and their mechanisms for treating MS.

#### **2. MS and its pathogenesis**

Multiple sclerosis (MS) is the most common non-traumatic disabling disease, resulting in axonal loss and myelin disruption. The frequent features of MS are formations of lesions and sclerotic plaques in the central nervous system (CNS) and the cerebrospinal cord. The immune system plays a critical role in neural evolution through regulating oligodendrogenesis, neurogenesis, and synaptic organization. Therefore, immune cells can participate in the pathogenesis and development of MS [19, 20]. The pathogenesis of MS is largely related to hormone, environmental, and genetic factors. It is reported that alterations in expressions and functions of some immune agents such as major histocompatibility complex (MHC), immunoglobulin (Ig), T-cell receptor (TCR), and cytokines can contribute to the increased risk of MS [6, 21]. Today, studies on MS have indicated that autoreactive T-cell migration to the CNS occurs upon autoimmune cascade initiation and blood-brain barrier (BBB) disruption, which leads to destroy myelin sheath and creates sclerotic lesions and plaques [6, 22]. Destruction of the myelin sheath, which plays a significant role in survival and integration of axon, is a major reason for the development of MS [3]. T helper 1 (Th1) and T helper 17 (Th17) cells are the main effector cells that participate in the demyelination and destruction of the CNS [19, 20]. Th1 and Th17 produce some pro-inflammatory cytokines, including inerleukine-1 (IL-1), IL-17, interferongamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) [23]. Moreover, CD8+ T cells are found in MS lesions, especially around the blood vessels. Previous studies

*Therapeutic Features of Mesenchymal Stem Cells and Human Amniotic Epithelial Cells… DOI: http://dx.doi.org/10.5772/intechopen.110221*

**Figure 1.** *The impacts of immune responses in MS pathogenesis.*

have revealed that the proliferation of CD8+ T cell in patients with MS is more than CD4+ T cell, which is largely associated with axon injury [1]. Besides the roles of T cells in the pathogenesis of MS, other immune cells play important roles in the formations of lesions and plaques. The activation of macrophage by Th1 cytokines leads to the destruction of the myelin and thereby exposes more CNS antigens. Although it is demonstrated that autoreactive T cells are the major effector cells for the pathogenesis of MS, some reports have indicated that autoreactive B cells have critical roles in disappearing the myelin sheaths and axonal loss, through cytokine secretions, antigen presentations, and autoantibody productions [24]. Autoantibodies can be major immune mediators that can be found in MS plaques. There are some reports pointing toward the association of immunoglobulin G (IgG) with MS signs. Furthermore, it is shown that IgG, especially IgG against proteolipid proteins (PLP) and myelin basic proteins (MBP), can be considered as the features of the disease, although their roles in MS pathogenesis are not well identified yet (**Figure 1**) [25].

#### **3. Mesenchymal stem cells**

MSCs can be obtained from different tissues such as bone marrow, adipose tissue, umbilical cord, brain, dental tissue, and fetal lung [26–28]. MSCs can differentiate into monocytes and neurons *in vitro* and *in vivo* [29]. These cells can migrate to injured tissue *via* expressions of the receptors for chemokines such as CXCR4, CXCR5, CXCR6, CCR1, and some growth factors [7]. In line with potential therapeutic effects of MSCs, it is revealed that these cells possess anti-oxidant and anti-apoptotic impacts and are able to secrete trophic factors, which can contribute to support axon and increase neural stability [30]. They improve neural cell differentiation, promote angiogenesis, inhibit neuron apoptosis, and repair the CNS in MS patients [25].

**Figure 2.** *Immunoregulatory and therapeutic effects of MSCs.*

Furthermore, these cells can recruit oligodendrocyte precursors to the CNS and induce their differentiation into neuronal cells [31, 32]. MSCs exert immunomodulatory impacts through suppressing the activity of B, T, and other immune cells [33]. Intravascular MSC therapy improves CNS tissue repair through the induction of T-cell tolerance to myelin glycoproteins [34]. Studies on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, indicated that intravenous injection of syngeneic MSCs induces tolerance in MOG-specific T cells and thereby reduces immune cell infiltrations to the CNS and increases the clinical course [35, 36]. Others have revealed that the immunoinhibitory effects of MSCs are mediated through the secretions of anti-inflammatory cytokines such as TGF-β, prostaglandinE-2 (PGE-2), and indoleamine-pyrrole 2, 3-dioxygenase (IDO) [37]. Previous studies have demonstrated that MSCs can impair B-cell proliferation and antibody production through inhibiting the activation and proliferation of Th1 cells [38]. These cells also improve the activation of suppressor of cytokine signaling 3 (SOCS3) and decrease the differentiation of Th17 via the IFN-γ pathway [39]. IDO, as a mechanism used by MSC for controlling immune responses, depletes tryptophan from the environment of lymphocytes, which plays a key role in lymphocyte activations [40]. MSC participates in the development of regulatory T cells (Treg) through inducing IL-10 secretion of peripheral dendritic cells (DCs) [40]. In line with the improvement of peripheral tolerance, it is reported that MSCs inhibit the differentiation and function of DCs, resulting in the inhibition of clonal expansion of autoreactive T cells via the reduction of antigen presentation [41]. Hepatocyte growth factor (HGF) produced by MSC increases tolerogenic DCs [42]. MSCs with HGF can reduce immune cell infiltrations and CNS inflammation in EAE mice [42]. Thus, HGF derived from MSC may be effective in MS treatment. Several studies on genetically modified MSCs have shown that over-expressed anti-inflammatory cytokines such as IL-10 and IL-4 can participate in suppressing immune responses, reducing BBB injury, and improving remyelination of neurons in EAE mice (**Figure 2**) [43].

#### **4. Human bone marrow-derived MSCs (hBM-MSCs)**

MSCs obtained from bone marrow have multiple properties, which make them an attractive cell source for therapeutic applications (**Table 1**). These cells are the most

*Therapeutic Features of Mesenchymal Stem Cells and Human Amniotic Epithelial Cells… DOI: http://dx.doi.org/10.5772/intechopen.110221*


#### **Table 1.**

*The pros and cons of MSC-based therapies.*

frequent cell sources used in clinical settings [49]. Given therapeutic features of hBM-MSCs in neurological disorders, it is revealed that they can promote disease recovery in relapsing-remitting and chronic types of MS in EAE mice, due perhaps to reduce demyelination regions and inflammatory infiltrates, induce oligodendrogenesis, and

enhance brain-derived neurotrophic factor (BDNF) production [65]. Several studies have reported that BM-MSCs have immunomodulatory effects in EAE through preventing the maturation of antigen-presenting cells (APCs) and proliferation of B and T cells [39]. These immunosuppressive impacts are mainly mediated by releasing various bioactive mediators [66]. Moreover, their neuroprotective effects can induce local progenitor cells and suppress scar creation, gliosis, and neuron apoptosis [67]. Besides having immunomodulatory impacts, they have the ability to differentiate into the neurons and improve the replacement of the cells [67]. Nonetheless, the isolation of BM-MSC is painful, invasive, and low efficiency [45], which may be considered a disadvantage in their clinical applications*.* In the first phase of clinical trial using autologous *ex vivo* expanded BM-MSCs on patients with advanced MS, it was reported that 30% of patients were unable to grow an acceptable number of these cells (< 2 × 106 ) despite several bone marrow aspirations. This observation has reflected an inherent deficiency of MSCs in the bone marrow of participants [67]. Thus, MSCs derived from other tissues can be considered for MS treatment. In EAE, BM-MSCs are notable curative effects if they are used before disease initiation due to a significant suppression on effector T cells and the induction of peripheral tolerance. However, these cells fail to control disease development in the stabilized stage of MS [16].

#### **5. Human umbilical cord (hUC)-MSCs**

HUC-MSCs have significant characteristics, which distinguish them from other sources of MSCs (**Table 1**). Several lines of evidence suggest the administration of hUC-MSCs in autoimmune disorders such as encephalomyelitis, type 1 diabetes, and rheumatoid arthritis due to its immunoregulatory impacts [68–71]. Immunomodulatory effects of these cells have fundamental roles in tissue recovery [72]. They have the decreased expression of HLA-I, increased capacity of proliferation, and more rapid growth *in vitro*, compared with BM-MSCs [73]. *In vitro* and *in vivo* studies have indicated that hUC-MSCs have a positive effect on Treg proliferation [74]. hUC-MSCs can increase behavioral activities and reduce the histopathological impairments of EAE. Furthermore, they exert a positive effect on the productions of IL-4 and IL-10, unlike IL-1 and IL-6 [70]. *In vitro* studies have demonstrated that hUC-MSCs can enhance the frequency of Treg and secretion of anti-inflammatory

**Figure 3.** *Immunomodulatory and therapeutic impacts of hUC-MSCs.*

*Therapeutic Features of Mesenchymal Stem Cells and Human Amniotic Epithelial Cells… DOI: http://dx.doi.org/10.5772/intechopen.110221*

cytokines of peripheral blood mononuclear cells (PBMCs) (**Figure 3**) [74, 75]. In addition to their immunoregulatory effects, this source of stem cells is able to release several nerve growth factors, for example, glial cell-derived neurotrophic factor (GDNF) and BDNF. Moreover, their capacity to differentiate into oligodendrocyte precursor cells can improve axonal growth [50].

#### **6. Human adipose-derived MSCs (AD-MSCs)**

AD-MSC can be obtained from adipose tissue by collagenase digests. Adipose tissue contains high levels of MSCs (approximately 100–1000 MSCs per gram of fat) and is easily accessible for use. Thus, this tissue is an important source of the cell for cellular therapy. AD-MSCs show the adipogenic, cardiogenic, neurogenic, myogenic, chondrogenic, and osteogenic features *in vitro*, which make them a fantastic cell source for stem cell therapy [8, 76]. Unlike BM-MSCs, these cells are able to migrate to different organs due to express α4 integrin, an adhesive molecule [55]. It is suggested that autologous and allogeneic AD-MSCs are effective in the treatment of the diseases with immunopathogenesis such as MS and autoimmune encephalomyelitis [77–80]. Study on EAE mice revealed that intravascular AD-MSC participates in the reduction of immune infiltration in the CNS and decreases demyelination and axonal loss [9]. Various growth factors released from AD-MSCs, such as anti-apoptotic, angiogenic, and neurotrophic mediators, play critical roles in cell differentiation, proliferation, and maturation [56]. It is thought that AD-MSCs have more capabilities for stem cell-based cell therapy, due perhaps to the expression of integrin α4β1; pass the BBB; and exert their anti-inflammatory, immunoregulatory, and neurodegenerative impacts (**Figure 4**) [7]. Until now, several studies have been performed to find a standard method for the treatment of MS by these cells [81, 82]. Nonetheless, there are some concerns regarding the clinical application of MSCs such as tumorigenesis and immune rejection after use that must be addressed in future studies.

#### **7. Human amniotic epithelial cells (hAECs)**

hAECs are easily isolated from the amniotic membrane, the inner layer of the fetal membranes, and possess some stem cell-like properties [83–86]. These cells express

#### **Figure 4.** *Immunoregulatory and therapeutic impacts of AD-MSCs in degenerative disorders.*

some markers of pluripotent stem cells, including FGF-T, Sox-2, Nanog, Rex-1, SSEA-4, and Oct4. Some of these markers have important roles in pluripotency and self-renewal properties in induced pluripotent stem (iPS) cells and embryonic stem cells (ESCs) [87]. hAEC can differentiate into different cells such as the pancreatic cells, neural cells, hepatocytes, cardiomyocytes, adipocytes, and myocytes, which originate from the endoderm, ectoderm, and mesoderm [18]. It is reported that hAECs have immunomodulatory impacts on adaptive and innate immune systems [17, 88, 89]. They exert suppressive effects on the activations of natural killer (NK) and CD4+ T cells, migrations of neutrophil and macrophage, secretions of pro-inflammatory cytokines of CD4+ T cells, and proliferation of B cells [23, 61, 90, 91]. These impacts are primarily mediated through the productions of immunoregulatory mediators, such as IL-4, PG-E2, and transforming growth factor-beta (TGF-β), which may participate in the increase of Tregs and Th2 cells, inhibition of pathogenic T-cell reactions, and protection of the peripheral naive CD4+ T-cell source [61, 63, 92–95]. These effects suggest that hAECs may be considered as an effective cell source for MS treatment [62, 95]. To support this notion, they can contribute to a shift from Th1-type responses to Th2-type responses [95]. EAE mice treated with hAECs experienced significant reductions in demyelination and immune infiltration into the CNS [95]. It is indicated that hAECs have a negative effect on Th17 differentiation through reducing the productions of TGF-β and IL-6, which play indispensable roles in the differentiation of these cells [88]. Studies on animal models of MS have revealed that alpha-fetoprotein (AFP) produced from hAECs participates in the reduction of lymphocyte function and neuroinflammation [96, 97]. Others have indicated that these cells can reduce gray and white matter damages through residing in inflammation locations such as the brain [98]. Furthermore, they can release neurotrophic agents such as neurotrophin-3 (NT-3), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) (**Figure 5**) [59, 60]. These features along

#### **Figure 5.** *Immunomodulatory and stem cell characteristics of hAECs.*

*Therapeutic Features of Mesenchymal Stem Cells and Human Amniotic Epithelial Cells… DOI: http://dx.doi.org/10.5772/intechopen.110221*

with low antigenicity provide additional confirmations to clarify therapeutic properties of hAECs in the treatment and management of inflammatory neurological disorders such as MS [99]. hAECs possess a limited proliferative potential due perhaps to the lack of telomerase [87, 92, 100, 101], which helps to reduce potential tumorigenicity of stem cell-based therapies. Nevertheless, it should be noted that further works and more information are needed to illustrate the possible capability of these cells in treating diseases with immune pathophysiology.

#### **8. Comparison of hAECs with MSCs derived from different sources**

There are some differences and similarities between hAECs and MSCs derived from different sources, for example, morphologic and tumorigenic properties, immunoregulatory characteristics, angiogenesis capacities, and ethical issues associated with their isolations and applications [45, 102]. In line with morphology, hAECs show a cobblestone-like morphology, while the cultured hAMSCs have a spindle fibroblast-like morphology [103]. The morphologic feature of the cultured MSCs derived from BM can range from fibroblast-like spindle-shaped cells to large flat cells [104]. MSCs from other sources, such as AD-MSCs and hUC-MSCs, indicate spindle shapes in the culture [105, 106]. The amniotic membrane can be collected by standard isolation methods following cesarean section, which is not invasive and does not have unfavorable effects on human embryos and ethical issues [102]. The isolation of amniotic cells can simply be performed upon prenatal testing. However, there are some ethical problems in regard to clinical applications of MSCs and the isolation of some sources of MSCs [104, 107]. As mentioned above, the isolation of hBM-MSCs is done by invasive techniques with low efficiency, which is painful [45]. Today, there is no document pointing to the tumorigenicity of amnion membrane or membraneoriginated cells after clinical applications [17]. However, some reports have shown that MSCs may raise tumor growth in some cancer mouse models [108]. Several lines of evidence propose that hAECs, BM-MSCs, and AD-MSCs participate in enhancing angiogenesis through the productions of some cytokines and angiogenic factors, such as VEGF, HGF, and EGF, and mechanisms associated with protease [103, 109]. According to evidence, hAECs possess better immunomodulatory impacts but lesser osteogenic effects than BM-MSCs and MSCs derived from the human amniotic fluid (hAF) [110]. hAECs express some MSC markers such as CD90, CD44, and CD105. However, the levels of SSEA4 and SSEA3 expressions are higher on hAECs than those on hBM-MSCs and hAFMSC, revealing more multipotent potential of these cells [17, 111]. Furthermore, hAECs and hAFMSC possess higher levels of PD-L1 and PD-L2 than hBM-MSCs, which may make them more successful in providing peripheral tolerance in immune cells [110, 112].

#### **9. Mesenchymal stem cell-based cell therapy and clinical trials**

Until now, several clinical trials were carried out using MSCs as a therapeutic approach for MS. In a phase II clinical trial, intravascular MSCs were employed in the treatment of nine relapsing-remitting multiple sclerosis (RRMS) patients. After 6 months, the results revealed a significant reduction in MS lesions in magnetic resonance imaging (MRI) [67]. In a phase IIa clinical trial, autologous BM-MSCs were injected to one RRMS and nine secondary progressive multiple sclerosis (SPMS) patients. After 3 months to 1 year, authors observed that BM-MSCs improved clinical features in the treated patients. In this clinical trial, 10 SPMS patients were treated with intravascular MSCs for 6 months, and the results revealed neuroprotection effect of MSCs and remyelination [67]. Furthermore, in a study conducted by Bonab et al., in 2007, therapeutic impacts of intrathecal injection of MSC were studied on 10 MS patients. This study indicated that the disease progression was gradually reduced in half of the participants [113]. Another study on 22 patients with primary progressive multiple sclerosis (PPMS) demonstrated that intravascular and intrathecal injections of BM-MSCs were effective in MS treatment [35]. In a triple-blind and placebo-controlled study on 30 patients with SPMS, the researchers indicated that AD-MSC injection is a possible and safe method in the treatment of SPMS patients [114]. Staff et al*.* reported the safety of intrathecal administration of AD-MSCs in amyotrophic lateral sclerosis (ALS) patients [115]. In a study conducted by Li et al*.,* it was demonstrated that hUC-MSC transplantation is able to reduce MS symptoms and relapse occurrence in comparison with control individuals. In addition, the researchers observed that hUC-MSC administration results in a shift in Th1 responses toward Th2 immunity [116]. In line with the therapeutic impacts of MSCs, Riordan et al*.* indicated that hUC-MSC transplantation is safe and exerts suitable impacts on life quality and brain lesion in MS patients [117].

#### **10. Conclusion**

There are many documents pointing to stem cell-based cell therapy as a treatment for MS and other neurological disorders. However, an inconsistency in the results of these studies is observed. Among different types of stem cells, MSCs are more possible to consider as a therapeutic approach for MS treatment because they utilize different mechanisms involved in regulating immune responses and repairing CNS damages. Furthermore, MSCs have anti-oxidant and anti-apoptotic properties and trophic factor secretion, which exert positive effects on the axon and neural stability. Numerous studies have recommended that MSCs derived from umbilical cord and adipose tissue can be more effective for stem cell therapy. Moreover, hAECs are mentioned as a novel source of the cells, which have immunoregulatory effects and show a potential for differentiation into the cells originating from three germinal layers. Consequently, hAECs may be considered a therapeutic method to manage and control MS. However, more experimental studies should be done to illustrate their efficiency and mechanisms involved in the treatment of MS.

#### **Finding**

This study was not financially supported and was performed in personal capacity.

#### **Conflicts of interest**

The authors declare that there is no conflict of interest.

*Therapeutic Features of Mesenchymal Stem Cells and Human Amniotic Epithelial Cells… DOI: http://dx.doi.org/10.5772/intechopen.110221*

#### **Author details**

Reza ArefNezhad1 and Hossein Motedayyen<sup>2</sup> \*

1 Department of Anatomy, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

2 Autoimmune Diseases Research Center, Shahid Beheshti Hospital, Kashan University of Medical Sciences, Kashan, Iran

\*Address all correspondence to: hmotedayyen@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 10**

## From the Classification of Stem Cells to the Release of Potential in Cell Therapies: Limits, Considerations and Future Aspects in Regenerative Medicine

*Arnaud Martino Capuzzo, Riccardo Ossanna, Lindsey Alejandra Quintero Sierra, Federica Virla, Alessandro Negri, Anita Conti, Andrea Sbarbati and Sheila Veronese*

#### **Abstract**

Regenerative medicine aims to repair organs or tissues that have congenital abnormalities, or that have been damaged by disease, aging, or trauma, and to restore or at least improve their native function. One of the strategies used in regenerative medicine is stem cell therapy, due to the enormous regenerative potential of stem cells. A staminal cell line is a group of cells that can replicate for an extended period *in vitro*, that is outside the body. These cells are grown in incubators using a culture medium that should have a temperature and an oxygen/carbon dioxide composition that simulates the desired environment. This chapter describes the main characteristics of stem cells, the main fields of application, and outlines what could be the future developments of their use, also considering the ethical and technical problems that currently limit their use. There is still much to be done in the field of stem cell research, and researchers are working tirelessly to remain leaders and innovators in it. A struggle, step by step, will make it possible to have more information on current knowledge by expanding the scientific literature and push current limits ever further.

**Keywords:** stem cells, regenerative medicine, clinical studies, research strategies, therapeutic method

#### **1. Introduction**

In recent years, increasing attention has been paid to the study of various types of cells, with particular attention to their properties, to promote regenerative processes and/or to be used for the cellular treatment of many diseases [1]. Great interest in

research has been aroused by stem cells; their ability to self-renew, and differentiate into mature adult cells has made them, since their discovery, extremely promising for the regeneration of human tissue [2]. It is no coincidence that the first results of their use have contributed to the real definition of "regenerative medicine" [3]. Today, stem cells and their differentiated derivates are increasingly used in an ever-widening field of cellular studies, often with the aim of treating the condition of cell loss related to various diseases [4].

Stem cell division can give birth to an asymmetrical offspring with an additional progenitor cell and a daughter stem cell. For this reason, they exhibit both selfrenewal and regeneration capabilities. The differentiation capacity of stem cells depends on their specification potential.

Generally, the renewal of adult stem cells (ASCs) is limited because they can only differentiate into specific cells of a single tissue. Finding stem cells capable of differentiating into all tissue types is a challenge. In the event that all terminal cell populations can be reached the cells are said to have the property of totipotency, while the ability to pursue a more restricted pattern of phenotypes is the property of multipotency [5] (**Figure 1**).

Scientists have traditionally dealt with two types of animal and human stem cells: embryonic stem cells (ESCs) and non-embryonic "somatic" or "adult" stem cells. Almost 30 years ago, in 1981, researchers discovered how to obtain ESCs from early mouse embryos [7]. A method to extract stem cells from human embryos, and

#### **Figure 1.**

*Totipotent stem cells generate all the cell types of the organism (e.g., zygote or fertilized egg). Pluripotent stem cells produce all the embryonic germ layers (endoderm, ectoderm, and mesoderm). Multipotent stem cells generate a limited number of cell types based on their tissue of origin. Mesenchymal stem cells give rise to fat, bone, muscle, and cartilage. Hematopoietic stem cells give rise to different types of blood cells (for example platelets, and red and white cells). Neural stem cells give rise to neurons, oligodendrocytes, and astrocytes. Oligopotent stem cells generate some closely related cell types (for example myeloid stem cells). Unipotent stem cells generate a single cell type (e.g., epidermal stem cells or muscle stem cells) [6].*

#### *From the Classification of Stem Cells to the Release of Potential in Cell Therapies: Limits… DOI: http://dx.doi.org/10.5772/intechopen.110572*

growing the cells in the laboratory was discovered in 1998, as a result of a deepen examination of mouse stem cell biology [8]. Human embryonic stem cells (hESCs) were the name given to these cells. The *in vitro* fertilization techniques utilized in these investigations were used to produce the embryos for use in reproduction. When they were no longer needed for that function, they were given to study with the informed consent of the donor. ESCs possess powerful properties, but their use goes against ethical principles [9], and for this reason it has been limited. Today there is already and will probably continue to be in the future, a debate on their use, on the possibility of keeping them in culture for more than 14 days, and on all the social, moral and ethical problems connected to them [10].

A possible way to overcome this obstacle was already found in 2006 by Takahashi and Yamanaka. Under unknown circumstances, some specialized adult cells were genetically "reprogrammed" to take on a stem cell-like form. The current name of this novel form of stem cell is induced pluripotent stem cell (iPSCs). iPSCs exhibit morphology, growth properties, and cell marker gene expression of ESCs, without ethical concerns. iPSCs can differentiate in various human tissues and exploit regenerate properties [11, 12], given their pluripotency capability. Unlike hESCs, iPSCs do not raise any ethical concerns regarding the timing of human personality initiation [13, 14].

However, both ESCs and IPSCs carry the risk of tumor formation, a risk related to both pluripotency and self-renewal. This is a critical factor of both cells types [15]. Furthermore, iPSCs still present some technical issues related to immune rejection after transplantation. This means that research in this field needs to be expanded because more studies are needed before using iPSCs as a viable tool for *in vivo* tissue regeneration [16, 17].

#### **2. Origin of stem cells**

Stem cells have the ability to self-renew, i.e. to create copies of themselves, and to differentiation into lineage populations, i.e. to develop into more specialized cells, which allow cell turnover in the respective tissues present in multicellular organisms [18]. The production of tissue-specific stem cells, which generally assist the cell renewal of all tissue types for the development of the organism, is a necessary step in the life cycle of a complex organism [19].

The core cells of the 3- to 5-day-old embryo, known as blastocysts, give rise to the complete body of an organism, including the numerous specialized cell types and organs such as the heart, lung, skin, sperm, eggs, and other tissues [20].

Distinct populations of ASCs provide replacements for cells lost due to natural wear and tear, injury, or disease in different adult tissues, such as bone marrow, muscle, and brain. Stem cells allow novel therapeutic possibilities for addressing these conditions thanks to their exceptional ability to regenerate [21].

Scientists can study the basic characteristics of stem cells and what distinguishes them from other specialized cell types through laboratory investigations. In laboratories, stem cells are already being used by researchers to test new drugs, create models/systems to study healthy growth, and pinpoint the origins of birth abnormalities. Understanding of how an organism grows from a single cell and how healthy cells replace damaged ones in adult creatures has been advanced through stem cell research [19, 22].

One of the most promising areas of modern biology is stem cell biological product research. It has recently been ascertained that miRNA-containing vesicles, such as exosomes, could induce a change in some pathologies [23–25] (**Figure 2**).

#### **Figure 2.**

*Adult adipose tissue derived murine stem cells ADAS. Here shown, ADAS cultured with complete DMEM containing 10% FBS and 1% P/S mix 1:1, and incubated at 37°C in a 5% CO2 atmosphere. These cells have the characteristic of producing exosomes, which in recent studies have shown to have unique characteristics for some neuropathologies [23]. The image acquisition was done using a bright field optical microscope, Olympus BX-51 (Olympus, Tokyo, Japan) equipped with a digital camera (DKY-F58 CCD JVC, Yokohama, Japan) and connected with a PC endowed with image-pro plus 7.0 software.*

There is still much work to be done in laboratories and clinics to improve the efficacy of using these cells and their byproducts in what are termed cellular treatments, or even regenerative or reparative medicine treatments, of a diverse pool of diseases [26].

#### **3. Stem cells subpopulations**

All stem cells can self-renew and develop, as described in the previous chapter, but they differ greatly in what they can and cannot become and in the conditions under which they can and cannot perform certain functions. This is one reason why scientists employ different kinds of stem cells in their study.

#### **3.1 Adult stem cells**

ASCs are undifferentiated cells found in some differentiated tissues of the body and have the possible property of self-renewing or producing new cells to replace damaged or dead tissue [27]. Alternatively, ASCs are sometimes referred to as "somatic stem cells", where the term "somatic" refers to the non-reproductive cells of the body (eggs or sperm). Some examples of ASCs are: Epithelial and Skin Stem Cells, Neural Stem Cells, Hematopoietic Stem Cells (Blood Stem Cells), Mesenchymal Stem Cells [28] (**Figure 3**).

ASCs are often insufficient in native tissues, making them difficult to study and harvest for research [29]. Distinct populations of ASCs, which are present in most tissues in the human body, produce new cells to replace those lost as a result of natural repair, disease, or damage.

All tissues in a person, including the umbilical cord, placenta, bone marrow, muscle, brain, adipose tissue and lipoaspirates, skin, stomach, etc. include ASCs. *From the Classification of Stem Cells to the Release of Potential in Cell Therapies: Limits… DOI: http://dx.doi.org/10.5772/intechopen.110572*

#### **Figure 3.**

*Fat harvesting via liposuction. Here shown, human adipose derived mesenchymal stem cells cultured with DMEM complete medium for 2 weeks, fixed with PFA 3%, washed PBS 1x stained with hematoxylin, the protocol involved the seeding of ADSCs on a 12-wells plate with sterile slides on the bottom of each well. The cells were seeded and incubated with 1 ml of complete culture medium for 24 hours at 37° C and 5% CO2. At the end the cells were fixed with paraformaldehyde 4%, stained with Mayer's hematoxylin (bio-Optica, Milan, Italy) for 5 min. Finally, the cells were washed with tap water for 5 min and mounted with mount quick aqueous solution (bio-Optica, Milan, Italy). The image acquisition was done using a bright field optical microscope, Olympus BX-51 (Olympus, Tokyo, Japan) equipped with a digital camera (DKY-F58 CCD JVC, Yokohama, Japan) and connected with a PC endowed with image-pro plus 7.0 software. Slides were gently cleaned with ethanol, acquired using a 20X.*

In 1948, the first ASCs were removed and utilized to create blood [30]. When the first adult bone marrow cells were employed in clinical therapy for blood disorders in 1968, this process was expanded [31].

For more than 40 years, treatments for blood disorders such as leukemia and lymphoma have included transplantation of peripheral blood stem cell and bone marrow [32].

There is an ongoing debate. According to some studies [33], ASCs can only produce the cell types of the tissue in which they reside. However, other studies suggest that ASCs may be able to produce cells of other tissue types [34]. More research is required to clarify this aspect.

Scientists have demonstrated that ASCs are present in most body tissues. Scientific research is looking for ways to locate, isolate, and multiply these cells for therapeutic use.

Most of the biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence surrounding cells. The current development of exosome therapies requires efficient and non-invasive methods to localize, monitor, and trace exosomes [25] (**Figure 4**). The idea behind these therapies is that the exosomes and the chemicals released are the stem cells' way of manifesting their therapeutic function.

#### *3.1.1 What are exosomes?*

Exosomes are vesicles that include peptides, mRNA, and microRNAs [35, 36], range in size from 50 to 150 nm, and are essential for intercellular communication [37].

#### **Figure 4.**

*TEM images of ADAS incubation with nanoparticles (NPs) were morphologically analyzed through a transmission electron microscope (TEM) in order to confirm the intracellular uptake of NPS and visualize their intracellular localization. The scale bar in the left and right pictures is 5000 nm, and the Centre picture is focused on the endocytic invagination containing nanoparticles and the internalized nanoparticles inside the endosome (scale bar 2000 nm). Cell pellets were fixed for 1 h in 2% glutaraldehyde in 0.1 M phosphate buffer (PB) and, after washed, postfixed for 1 h in 1% OsO4 diluted in 0.2 M K3Fe (CN)6. After rinsing in 0.1 M PB, the samples were dehydrated in graded concentrations of acetone and embedded in a mixture of Epon and araldite (electron microscopic sciences, Fort Washington, PA, USA). Ultrathin sections were cut at 70 nm thickness on a Ultracut E ultramicrotome (Reichert-Jung, Heidelberg, Germany), placed on Cu/Rh grids and contrasted with lead citrate. Samples were observed with Pa Philips Morgagni 268 D electron microscope (Fei company, Eindhoven, the Netherlands) equipped with a mega view II camera to acquire digital images [25].*

They mimic the effects of stem cell transplantation by delivering physiologically active chemicals to recipient cells, which change their gene expression and behavior.

According to several studies, stem cell-derived exosomes may have a role in synaptic plasticity, nerve regeneration, neuronal protection, and neurological recovery [38, 39].

By using these vesicles as a treatment, rather than their generated parental cells, restrictions and dangers for cell transplantation are avoided.

#### **3.2 Embryonic stem cells**

The embryo, known as a blastocyst at this stage, contains an inner cell mass capable of growing all the specialized tissues that make up the human body, 3 to 5 days after fertilization and prior to implantation [40]. ESCs are produced from the inner cell mass of an *in vitro* fertilized embryo, donated for scientific research. ESCs are not made from eggs that have been fertilized inside a female's body [41].

Isolable only in the early stages of development, these pluripotent stem cells can develop into virtually any cellular form (**Figure 5**). One of the research objectives is to understand how these cells differentiate during development [42]. The progressive increase in knowledge about these stages of development, could allow researchers to use ESCs generated *in vitro* to rebuild different types of tissue, such as neurons, skin, gut, and liver for transplantation [43].

*From the Classification of Stem Cells to the Release of Potential in Cell Therapies: Limits… DOI: http://dx.doi.org/10.5772/intechopen.110572*

**Figure 5.**

*Schematic representation of ESCs. ESCs are pluripotent, and are derived from the inner cell mass of a blastocyst. Human embryos reach the blastocyst stage 4–5 days after fertilization and at that time consist of approximately 50–150 cells [40].*

In the future, ESCs could be used to treat a broader spectrum of disorders. It is hoped that once this technique is well understood, the information will be applied to vehicular ESCs, i.e., induce them to differentiate into the specific cell type required for patient therapy [44]. Currently, diseases treated with ESCs transplantation include diabetes, spinal cord injury, muscular dystrophy, heart disease, and vision/hearing loss [45].

#### **3.3 Reprogrammed pluripotent stem cells**

Halfway between ASCs and ESCs are iPSCs, which are stem cells produced in a laboratory, by introducing embryonic genes into a somatic cell, such as a skin cell, so that it returns to a "stem cell-like" state [46].

The production of these cells is an innovative technique of genetic reprogramming. First identified in 2006 [47], several years of study will be required before they can be used therapeutically.

The potential to alter recipient somatic cells into a "ESC-like" state undoubtedly makes therapies using iPSCs attractive [48]. The cells required for the therapies could be produced using appropriate differentiation processes of these iPSC cells. What makes this technique attractive is that it circumvents the need for lifelong histocompatibility immunosuppression, as is the case with transplanted cells of donor stem cells [49].

iPSC cells are regarded as pluripotent, making them similar to most ESCs. However, unlike for ESCs, manipulation of iPSCs has not been successful in growing the outer layer of an embryonic cell, which is required for the cell to develop into a full human individual [50]. But, iPSC research is rapidly moving towards translational and clinical applications [51].

#### **4. Advantages of different stem cell lineages**

It is possible to list some advantages and disadvantages associated with the three distinct stem cell types (ASCs, ESCs, and iPSC) previously described. If we start from the analysis of the disadvantages and ongoing debates on the use of different stem cells, we are immediately redirected towards their advantages.

ASCs – Among the positive characteristics of ASCs is their ability to transdifferentiate and reprogram themselves. Also they are less likely to be rejected when used in transplants [52]. Their efficiency in the therapeutic field has already been proven in numerous clinical applications [53].

ESCs – Among their advantageous properties, ESCs have the potential to be maintained in culture, and to grow even for more than a year. Numerous protocols have been established for their maintenance in culture, protocols that consider the ability of these cells to produce most cell types in the body [54–57]. There are numerous studies relating to these cells, which appear to be among the most investigated stem cells. A further increase in these studies may lead to more knowledge about how living things develop and thrive.

iPSCs – iPSCs are mostly derived from donor somatic cells. This means that they can be utilized in large quantities, avoiding histocompatibility issues in transplants. These cells have performed well in preclinical drug testing and research/development studies [58–60]. The definition of the new cellular "reprogramming" procedure, and the new knowledge deriving from it, could be applied to define *in vivo* therapies for the reprogramming of damaged or diseased cells and tissues.

The currently best-known cell therapy for the treatment of blood malignancies and other blood problems is bone marrow transplantation, which transplants blood stem cells [61].

Theoretically, Parkinson's disease, spinal cord damage, stroke, burns, heart disease, Type 1 diabetes, osteoarthritis, rheumatoid arthritis, muscular dystrophy, and liver disease are all possible candidates for stem cell therapy [62]. Additionally, regeneration of the retina using isolated ocular stem cells could one day contribute to the reversal of blindness, providing a potential treatment for distressed or injured eyes [63].

Cell therapy, which replaces unhealthy cells with healthy ones to treat disease, is one potential use of stem cells; it is comparable to organ transplantation, except that the cells rather than the organs are transplanted (**Figure 6**).

#### **Figure 6.**

*Autologous human iPSCs can be derived from individual patients and differentiate into different cell types. To develop new therapies, isolated and cultured cells are used to observe specific disease phenotypes and identify possible new pathological mechanisms. Cell therapy includes therapies based on stem and non-stem cells, unicellular and multicellular, with different immunophenotypic profiles, isolation techniques, mechanisms of action and regulatory levels. The use of human iPSCs, autologous to the patient, offers an innovative approach for regenerative medicine ([35, 36]).*

*From the Classification of Stem Cells to the Release of Potential in Cell Therapies: Limits… DOI: http://dx.doi.org/10.5772/intechopen.110572*

#### **5. Limitations and obstacles to their use**

Many factors still need to be considered and many studies are still needed to fully understand how to use stem cells.

ASCs – The differentiating capacity of ASCs are not yet fully elucidated [64]. This may mean that a different interpretation of their properties is needed, considering them multi- or unipotent.

Furthermore, ASCs cannot be grown for long periods of time in culture without an observable phenotypic change occurring and fail to prevent their immortalization.

Another limitation of the use of these cells is that they are present in small quantities in the tissues. This makes it difficult to identify the niche and the purification/ isolation process is tedious. Currently few technologies are available to generate large quantities of cultured stem cells and keep them incubating. We have previously successfully tested the combination of CELLviewer [65] with Spin∞, a new bioreactor still in the prototype stage [66].

ESCs – The limitations and obstacles that can be found in the use of ESCs concern various aspects, among which the fundamental one is that the cell line generation process is inefficient. Moreover, their use is strictly regulated [67].

It is uncertain whether they would be rejected if used in transplants. Therapies that use ESC pathways are largely new, and much more research and testing is needed to ascertain their effectiveness as alternative pathways to conventional clinical treatments [68].

Finally, when used directly from the undifferentiated culture, ESCs for tissue transplants can generate and cause tumors (teratomas) or the development of cancer.

iPSCs – The reasoning on iPSCS turns out to be more complex. Since the transcription and reprogramming factors of stem cells were identified in 2006 [47], several methods to obtain iPSCs have been certified. These methods have been duly included in protocols that ensure cell reproducibility and maintenance [49]. New discoveries are not uncommon, given that the tissues into which they differentiated are not known *a priori* [69].

An important limit to the use of these cells are the viruses that are currently used to introduce embryonic genes into somatic cells. Studies in mouse models have shown that these viruses can cause tumors.

The use of iPSCs has given rise to several controversies because, however, there could be numerous application possibilities in the research field and in the market.

As far as ethical aspects are concerned, for the use of ASCs has not raised any significant issue. For the use of ESC, to acquire the inner cell mass, the embryo is destroyed. Therefore, donor consent is required. Relevant problem may concern iPSC cells because, when exposed to the right conditions, they have the potential to become embryos.

It should be noted that there can be many possible hitches related to the cellular therapy. For example, in the presence of insufficient synthesis of stem cells in the bone marrow, a poor transfusion is obtained, i.e. a transfusion with few cells. This is typical of elderly individuals who, compared to younger individuals, have a lower capacity of the bone marrow to produce stem cells. Regardless of the number of cells, the quality of these cells is also crucial.

Finally, not all existing health problem can be solved with stem cell treatment. Each person responds differently; the fundamental goal of this method is to provide the body with the means and the optimal environment to repair damaged tissue on its own. The knowledge of the type of patients to be treated in the different modalities to optimize therapeutic outcomes has significantly improved. For some pathologies/

patients, on the other hand, there has been no progress and it is not yet known what the most suitable cell therapy might be. Additionally, the outcome of any procedure relies on the participant's individual body resilience. Each body can respond differently. Therefore, it is currently not possible to generalize the results of a treatment or to determine it as the ideal treatment for a given pathological condition. There is no universal treatment plan.

#### **6. Future perspectives of culturing stem cells**

Faced with the possibility of using different stem cells, it should be remembered that the cells can be used in abundant quantities of donor somatic cells, thus making autologous treatments possible. In other words, the histocompatibility problems typical of transplants from donors other than the recipient, which are normally the main reasons for rejection, could be avoided.

Stem cells can be useful for several areas of interest such as drug development or development studies.

Finding a suitable stem cell source is the first step. Finding, isolating, and cultivating the proper type of stem cell, such as a rare cell in adult tissue, requires painstaking work.

Tissue-specific stem cells are thought to be less adaptable than embryonic and foetal stem cells, in general. The correct environment must be created after the stem cells have been identified and separated, an environment where the cells can differentiate into the specialized cells needed for a specific therapy.

A transport/migration system for cells to the area of the body where they are to act must be created. It is only in this area that the cells must perform their action, integrating with the body's native cells. However, although cells can chemically recognize injured tissue, there may be a physical barrier, such as a blocked artery, preventing these cells from "traveling" to damaged regions. The rate of tissue regeneration will be poor, slow, or non-existent if the root cause of the blockage is not removed.

Like organ transplants, it may be necessary to suppress the immune system of the body to lessen the immune response triggered by the donated cells. It should be considered that the body may react negatively to the addition of progenitor cells causing the formation of tumors or accelerating their growth rate. The oncological field is, without a doubt, a field in which cell therapy research must be strengthened, given its potential, but with the appropriate measures.

Since 2001, hundreds of stem cell lines have been developed; these lines are much more adaptable and easier to deal with than lines established nearly a decade ago [70]. They also have the advantage of not being "contaminated" by being produced from other cells. Therefore, the National Institutes of Health (NIH) or other competitive funding organizations are unlikely to support experiments limited to older cell lines, given the advantages of newer ones [71]. The study of stem cells and their potential uses for the treatment of various human diseases is still in its infancy, despite encouraging results from animal models. To guarantee long-term efficacy and safety, a thorough research process should be followed.

#### **7. Conclusion and final considerations**

Fundamental knowledge of how organisms grow and develop, as well as how tissues are maintained throughout adult life, is aided by stem cell research. *From the Classification of Stem Cells to the Release of Potential in Cell Therapies: Limits… DOI: http://dx.doi.org/10.5772/intechopen.110572*

Understanding what goes wrong during disease and damage and, ultimately, how to treat these diseases, is necessary. In the future, researchers will have the means to simulate diseases, test medications, and create increasingly effective treatments with the help of developing human tissue and stem cell lines, and related biological products.

In conclusion, there are two factors that make the creation of disease- or patientspecific pluripotent stem cells extremely therapeutically promising. First, these cells may offer a powerful new tool for researching the causes of human diseases and for developing new drugs. Secondly, the generated ESCs could be transformed into a specific cell type and, if transplanted into the original donor, would be recognized as "autologous," eliminating the issues with immunosuppression and rejection that arise with transplants from unrelated donors. The research must go on and unlock the potential of stem cells to further advance regenerative medicine.

#### **Acknowledgements**

We gratefully acknowledge the Department of Pathology and Diagnostics at the University of Verona, which allow to perform research.

#### **Conflict of interest**

The author declares no conflict of interest.

#### **Thanks**

To Natasha, who in her modest comprehension of things knows how to pique my curiosity about the areas of my life that have not yet been thoroughly explored.

Thanks to all my dearest supporters who in the dark moment know how to encourage and spur me to work more and more. I would also like to thank all dear colleagues in the Department of Diagnostics and Public Health for providing knowledge and insights into some of the considerations in the chapter.

A special thanks to Sheila who took care to correct and revise the chapter several times, leaving no mistakes.

Special thanks to the IntechOpen Team for giving me the opportunity to write this chapter.

#### **Author details**

Arnaud Martino Capuzzo1 \*, Riccardo Ossanna1 , Lindsey Alejandra Quintero Sierra2 , Federica Virla2 , Alessandro Negri1 , Anita Conti1 , Andrea Sbarbati<sup>2</sup> and Sheila Veronese2

1 Department of Pathology and Diagnostics, Nanoscience and Advanced Technologies, University of Verona, Verona, Italy

2 Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy

\*Address all correspondence to: arnaudmartino.capuzzo@univr.it

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*From the Classification of Stem Cells to the Release of Potential in Cell Therapies: Limits… DOI: http://dx.doi.org/10.5772/intechopen.110572*

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#### **Chapter 11**

## A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial Mesenchymal Stem Cells

*Manuela Cristina Russu*

### **Abstract**

High rate complications and recurrences in reconstructive surgery using *in situ* synthetic/polypropylene (PP) meshes have driven to a new concept based on mesenchymal stem cells (MSCs) for homeostasis repair in pelvic floor disorders (PFD). Prevention and therapy with MSCs are up to date analyzed on small and large animal models, less in women trials. Cell based-vaginal/intraurethral, or systemically introduced, tissue engineering (TE) with new generation meshes/scaffolds MSCs seededbone marrow, adipose tissue and recently proposed the endometrial/menstrual MSCs (eMSCs/MenSCs) for PFDs, management. Easy collected, isolated with specific markers, cultured for number harvesting, without ethic and immune compatibility issues, with unique biologic properties eMSCs/MenSCs differentiate in many cellular types—smooth muscle, and fibroblast-like cells, preserving cell shape, and phenotype, without oncogenic risks, and collagen, elastin fibers; eMSCs/MenSCsare appropriate for PFDs management, respecting good protocols for human safety. The quick appeared regenerative effect-mediated by angiogenesis, apoptosis inhibition, cell proliferation, no chronic inflammation and low/no foreign body reactions, less thick collagen fibers, and fibrosis improve connective/neuromuscular tissues; less pelvic structures stiffness with more elasticity are advantages for new meshes/ scaffolds generation in TE. Human eSMCs/MenSCs deliver bioactive factors by their exosomes/microvesicles/secretome for paracrine effects to injury site, facilitating in vivo tissue repair.

**Keywords:** pelvic floor disorders, endometrial/menstrual mesenchymal stem cells, cell based therapy, tissue engineering, new generation meshes/scaffolds

#### **1. Introduction**

Pelvic Floor Disorders (PFDs) are a continuous gynecologist's challenge, reason for this paper to review their correction attempts, by better knowledge of pathophysiology, pelvic connective tissue structure and biology with the new concepts of regenerative medicine by mesenchymal stem cells (MSCs) and/or their bioactive mollecules, sometimes crucial. All these objectives are after the era of reconstructive surgery with native tissue or *in situ* synthetic/polypropylen (PP), non-absorbable meshes for fascia repair. The surgical procedures have immediate, and subsequent (within 5 years) complications (10–30% rate) [1], disorder's recurrence or a new disorder, and host reactions to mesh—as a foreign body reaction (FBR), with fibrosis, mainly to transvaginal type, explaining abnormal functions after interventions. FDA (USA) had two public health notifications, warning since 2010 against vaginal mesh use in POP, because complications, recurrences, and litigations [2]; PP mesh use is prohibited for transvaginal POP surgery in Australia, New Zealand, USA, and UK [3]. Stem cells, as endometrial/menstrual mesenchymal stem cells (eMSCs/MenSCs) with their remarkable unique biological properties are considered a new potential tool for PFDs prevention and therapy, in a properly response to pathophysiology. MSCs are key regulatory components in the regenerating stem cell local microenvironment termed "stem cell niche" or for MSCs culture conditions, *per se* or by their secretome, facts that positively influence altered pelvic floor connective tissue, and contribute to tissue homeostasis restoration [4]. Allogeneic and autologus MSCS/MenSCs are proposed for these aims, being easy collected, isolated, purified with known perivascular surface markers for pericytes [5], and with the novel single perivascular marker Sushi Domain Containing 2 (SUSD2) for purifying eMSCs [6], and maintaining cells clonogenicity, reduced by flow cytometry used for their sorting [5, 6]. Different to other MSCs culture expandation imposing presence in culture medium of some constituents of their secretome [7, 8], for eMSCs and ERCs one adds transforming growth factor-β receptor inhibitor (TGF-β R inhibitor), that limits cells spontaneous differentiation to fibroblasts, and maintains the undifferentiated cells status in days following administration, to ensure efficacy [9]. eMSCs and ERCs have no ethic issue, or incompatibility risk, no need of immunosuppressive adjuvant drug, they intervene in repair and regeneration by new blood vessels formation, modulating host immune system, reducing chronic inflammation, FBRs, and fibrosis, with no scar as endometrium regenerates after each menstruation from the menarcha to menopause, under a normal estrogen/progesterone balance, and in postmenopause when on menopausal hormone therapy (MHT). One proposes cell based therapy-eMSCs/ MenSCs direct placement in vaginal walls or intraurethral, or systemic administration [10, 11] to prevent postpartum POP or SUI, and tissue engineering (TE)/tissue grafts with eMSCc/MenSCs seeded in a new meshes/scaffold generation preferable biodegradable, obtained by a variety of technologies, as knitting (like old ones) from alternative synthetic and natural polymers, electrospinning, and three-dimensional printing [12], and a composite mesh of polyamide plus a gelatin layer [13], instead of PP meshes, with no ectopic tissue formation, or malignant tumor [14]. There are many small and large animal models, and few human clinical studies on MSCs for tissue restoration and repair when PFDs.

#### **2. Contemporary burden of women's pelvic floor disorders**

PFDs named also Pelvic Floor Dysfunctions represent a women old pathology, with high incidence and prevalence in the last 70 years, associated to worldwide lifespan increase, independently to their different definitions and diagnosis criteria, last updated in 2010 by the International Urogynecological Association and International Continence Society [15], with over 250 definitions for clinical categories and subclassifications, and alphanumeric code for each definition. PFDs is an umbrella term for a

#### *A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial… DOI: http://dx.doi.org/10.5772/intechopen.108010*

group of clinical conditions caused by pelvic floor supportive tissue weakening, sometimes degenerative, occurring independently or simultaneously, connected to genetics, childbirth and aging, and it includes pelvic organs prolapse (POP) into vagina, as a hernia in the endopelvic fascia [16], and alterations in sensory or emptying abnormalities of the lower urinary and gastrointestinal tracts: urinary incontinence (UI) with clinical manifestations of stress urinary incontinence (SUI), overactive bladder syndrome (ORB), detrusor instability, urinary retention, and fecal incontinence, and sexual dysfunctions. PFDs are diagnosed in middle-aged women as stress urinary incontinence (SUI)—incidence peak around 46 years, and in elderly women when a second SUI peak between 70–71 years, being described in young women, also, around 18 years, with annual risk of 3.8 and 3.9 per 1000 women at that bimodal peaks; the risk for pelvic organ prolapse (POP) is increasing progressively from 50 to 60 years in Swedish population [17], to ages of 71 and 73 years, when annual risk is 4.3 per 1000 North American women evaluated for lifetime risks for SUI, or POP, or both surgery between 2007 and 2011 [18], and higher in institutionalized, and when dementia [19]. The recent Swedish national register-based cohort study [20] estimated a 12–19% lifetime risk for surgery for POP, similar to the earliear 19%-for Australian women [21]. PFDs have serious negative influences on women's quality of life at any age [22]. SUI and POP are highly related to childbirth-associated pelvic floor injury [23], less influenced by route of delivery (vaginal, cesarean section), sometimes inherited, as a familial disorder, associated to other hereditary conditions—joint hypermobility and even Ehlers–Danlos syndrome, a mutation in the gene for collagen III [24, 25]. One recommends the extension of familial conditions lower than first degree generation relationship [26] for familial risk reduction and prolapse prevention, many cases being more frequently in postmenopause, without any pregnancy/delivery, when the association of ovarian aging hypoestrogenism, plus vasomotor syndrome, depressive mood, chronic constipation and cough, obesity, characteristics for this life period, are increasing PDFs medical and financial burden [27].

#### **2.1 Synthetic mesh unfavorable results in surgery for pelvic floor disorders**

In front of PFDs burden, one tried to correct the supportive tissue of cardinal, utero-sacral ligaments, levatorani muscle, and urethral sphincter damaged by gestation, parturition, and aging initially by non-surgical interventions: physical exercises (after delivery), pessaries—first choice for POP symptomatic women [28], or as a tool for decision of mid-urethral slim mesh, when one decides POP surgery, to avoid over and under-treatment if vaginal mesh is used for an nonexistent SUI or a urinary leakage when surgery for POP [29], pharmacological therapies, laser, nonablative monopolar radiofrequency [30], and surgery, when all these failure; either vaginal or abdominal route, or both with native tissue repair, through open surgery or laparoscopy, or reconstructive surgery with synthetic, monofilament PP mesh respecting the principles for different PFDs manifestations, classified in anterior and posterior compartment, and vaginal vault disorders. It was evident that the addition of mesh as reinforcement to vaginal walls provides better prolapse correction, compared with colporrhaphy using native tissue alone, by both objective and subjective criteria [31], as it was demonstrated for abdominal wall defects/hernia repair [32]. Unfortunately medical staff and patients wishes for pelvic floor normal functions restoration with PP meshes were not accomplished, or the results were not better than after native tissue repair, being reported high risks—immediate, subsequent (within first 5 years) readmissions for later postoperative complications, such as intractable pain, or mesh

erosion or extrusion into the bladder, bowel or vagina [33], requiring surgical excision in ≥10% [28], or deterioration of vaginal biomechanical properties by high stiffness mesh implanted for prolapse [34], and further recurrences of previous disorder, or a *de novo* one, as incontinence surgery-incidence of 9.9% after surgery for POP without occult SUI (apical and non-apical) [35] or further prolapse surgery, sacro-spinous fixation with synthetic mesh being a risk factor for POP recurrence [36], andtransvaginal PP mesh having a higher risk than vaginal vault sacro–colpopexy [37, 38]. The population-based cohort study from Scotland [39] compared the primary outcome—immediate postoperative complications and subsequent (within 5 years) readmissions for later postoperative complications, further incontinence surgery, or further prolapse surgery in women older than 20 years, after first, single PP mesh (retropubic mesh, or single prolapse mesh procedure) to non-mesh procedures during 20 years. Immediate complications were lower after mesh procedures [adjusted relative risk (aRR) 0.44 (95%CI 0.36–055) and subsequent prolapse surgery [adjusted incidence rate ratio (air) 0.30 (0.24–0.39)], and a similar risk for further incontinence surgery [0.90 (0.73–1.11)], and later complications [1.12 (0.98–1.27)]. Anterior compartment prolapse PP mesh repair was associated to a similar risk of immediate complications as non-mesh surgery [aRR 0.93 (95%CI 0.49–1.79)], but with an increased risk of further incontinence [air 3.20 (2.06–4.96)], and prolapse surgery [1.69 (1.29–2.20)], and a substantially higher risk of later complications [3.15 (2.46–4.04)]. Posterior compartment mesh repair was associated to a similarly increased risk of repeat prolapse surgery and later complications as non-mesh surgery. Vaginal vault prolapse had similar outcome when vaginal and, separately, abdominal mesh repair were compared with vaginal non-mesh repair. Both vaginal and abdominal mesh procedures for vaginal vault prolapse repair are associated with similar effectiveness and complication rates to non-mesh repair.

The synthetic meshes used for pelvic floor reinforcement or reconstruction may provide the necessary mechanical support for damaged tissue, but implants' biological actions interfere with host biology, inducing the growth of a fibrous tissue layer, as an additional physical support, but the scar may contract the mesh, and surrounding tissue up to 60% [40], Mesh parameters influence host tissues: microstructureporosity (permeability for host's cells, mainly immune cells, fibroblasts, macrophages, metabolites, oxygen at repair place, and also bacteria effects, by chronic inflammation), fiber filament type (monofilament: polyamide and polyetheretherketone monofilaments or multifilaments; synthetic/natural/composite), and diameter, mesh weight (light/ultralight/heavy), mechanical properties as stiffness, and elasticity, chemical properties, materials biodegradability, and integration in host organ by new blood vessels formation, facts that contributed to the new concept of stem cells in cells based therapy, and tissue engineering (TE) for PFDs prevention or repair [13, 41].

#### **3. A new concept of stem cell use in prevention and therapy of women's pelvic floor disorders**

Tissue engineering is a new option in the field of pelvic floor repair when soft tissue reinforcement or reconstruction, or normal function are necessary. MSCs and/or their secretome are proposed after many studies regarding pelvic tissue biology, materials properties associated to stem cells designed to restore the anatomical functions, to provide a real pelvic floor mechanical support after damage, usually to be like a hammock, and to offer both the lost stiffness when under tension and the flexibility under bending [42].

*A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial… DOI: http://dx.doi.org/10.5772/intechopen.108010*

#### **3.1 Tissue homeostasis/remodeling behind repair in pelvic floor disorders**

Pelvic floor—a complex resistance piece, keeps pelvic organs within the body, still allowing passage through urethra, vagina and anal canal, around which are designated striated muscles—levatorani, with its three fascicles, and superficialperinealmuscles or urogenital diaphragm [43], forming a functional neuromuscular unit, and fibrous connective tissue, generating endopelvic fascia, cardinal and utero-sacral suspensory ligaments, and vaginal dense fibromuscular-connective tissue. Pelvic connective tissue maintains the position of organs adjacent to vagina, and the close anatomical relationship among vagina, bladder, and rectum may contribute to the emergence of anatomical-functional failure of adjacent organs/systems, in PFDs [44], according to their normal different stiffness/elasticity. Animal models indicate that molecular changes in tissue composition, mainly protein content, coincide to altered biomechanical properties, in PFDs, mainly in POP, as one cites in Australian [45], European [46], North American analyses [47, 48]. Human and mouse pelvic floor provided similarities of pathological changes, centered on deep and superficial muscles, ligaments, and connective tissue, mainly on vaginal walls. Pelvic floor connective tissue contains stromal cells and a very complex extracellular matrix (ECM). The balance between ECM synthesis and degradation during women/females life is a key in pelvic floor properties, and the vaginal structures are strongly influenced during women, small and large animals life, ovine models demonstrating architectural and functionaldifferences according the reproductive status. The Australian ovine model [45] revealed the lowest total collagen content in virgin vaginal tissue, in contrast to parous tissue with highest total collagen and lowest elastin content with concomitant high maximum stress, in contrast to pregnant sheep with lowest collagen and highest elastin contents, and thickest smooth muscle layer and low maximum stress, and poor dimensional recovery following repetitive gestational loading. The vaginal tissue is anisotropic with some biomechanical properties—loading pressure, deformation rates, resistance to rupture, which were tested in ewes [49], and in vaginal specimens collected during surgery for POP [50] compared to specimens from cadavers without noticed PFDs (non-pelvic organ prolapse)—the first experimental study providing vaginal tissue mechanical behavior. The results highlight the non-linear relationship between stress (force per unit of surface) and strain, the vagina being hyperelastic and supporting very large deformation before rupture appearance, as in labor, and fetal expulsion. The vaginal walls tissue is stiffer in patients with POP than non-POP [51]. Comparison of biomechanical properties of the crucial organs of pelvic support [52], showed significant differences at large strain levels: vagina is more rigid, and less extendible than rectum, which, is more rigid than the bladder. The anterior and posterior vaginal walls have different stiffness, and the bladder tissue was anisotropic at large strain levels, facts very important for tissue repair: region with dysfunction/ disorder, or procedure type.

ECM molecules are arranged in a matrix/scaffold, surrounding stromal cells (fibroblasts, myofibroblasts, smooth muscle) that synthesize collagen, as tropocollagen and elastin, as tropoelastin to form the fibers complex network, plus proteoglycans, and matricellular proteins, enzymes, according to their genes. All forming the complex network of pelvic floor support, recently updated [53]. EMC contains:

• tropocollagen, self-assembled into fibrils, aggregating to form a collagen type I (for tensile strength) and type III collagen fiber (for elastic properties, and increased collagen III reduces mechanical strength [54]; collagen form a cross-linked

network intertwined with elastin—the elastic fibers core component, secreted by elastogenic cells as the monomer tropoelastin, and undergoes self aggregation, cross-linking and deposition on to microfibrils assemble into insoluble elastin polymers. A microfibril scaffold-primarily formed by the protein fibrilin-1, is required for elastic fibers formation [53]. Collagen and elastin fibers are surrounded by a viscous substance of proteoglycans-consisting of a core protein to which one or more glycosaminoglycan (GAG) chains—ashyaluronan or hyaluronic acid, heparan, dermatan sulfate and the small leucine-rich repeat proteoglycans (SLRPs)-decorin, lumican and fibromodulin [17] are covalently attached; SLRPs cover the surface of collagen fibers, contributing to fiber optimal formation [55]. Proteoglycans have key roles in controlling gradients and availability of potent growth factors, chemokines, cytokines, and morphogens, very important in tissue's homeostasis, mechanical strength, development, and repair. One or more proteoglycans are cell surface or transmembrane receptors for adhesion molecules in all mammalian extracellular matrices [56], contributing to progenitor stem cells microenvironment/niche [57, 58].

	- matrix proteases as matrix metalloproteinase (MMP)-2, MMP-9 involved indisruption of collagen and elastin fibers, and particularly increased in POP, and in postmenopause comparative to premenopauseal asymptomatic cases; estrogens withdrawal or antiestrogenic therapy upregulates MMP-9; and TIMP-1, TIMP-2
	- lysyl oxidase-like–1 (LOXl-1)—predominantly catalyzes elastin cross-linking, its inhibition associated to increased MMP-9 led to subclinical POP [27, 64], because tropoelastin accumulation, according to the theory of antielastaseelastase inbalance in mice lacking LOXl-1, and the lack of deposit with normal elastic fibers in the uterine tract, and an abnormal postpartum heal of elastin, In LOXL1 knockout mice, smooth muscle cells stiffness and cells adhesion are altered, being proved the interplay between smooth muscle mechanics and ECM remodeling, mainly in postpartum [65, 66].

The content, aspect and cross-link of collagen and elastic fibers, matricellular proteins, proteoglycans, and enzymes specially MMPs, LOXl-1 are negatively changed *A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial… DOI: http://dx.doi.org/10.5772/intechopen.108010*

when POP; elastic and mechanical strength are decreased during gestation, and with age, beingconceivable that a loss of elastic fiber–associated proteins in pelvic floor connective tissues with aging, may disrupt the optimal balance between synthesis and degradation of vaginal elastic fibers, and lead to POP, fact associated to the critical negative proteases role in POP progression [48]. The fibers amount is less interested initially after vaginal delivery; their histomorphology is first changed, regarding length (shorter), and cross-linking in net-work [61], fibers density decreases later, by aging [67]. The quantification of collagen and elastic fibers shows a more important decrease of elastic fibers in superficial epithelial layers near vaginal epithelium, and less in the deepness of pelvic cavity, around muscles, and thin, irregular and disrupted collagen bundles, higher levels of collagen type III in the vaginal wall, and fragmentation of collagen fibers [68], being appreciated that epithelial-stromal interactions, and fibulin-5-integrin interactions—that suppress ROS generation, are critical in regulation of MMP-9 in mice vaginal wall [48], with an increased level of MMP-2, -9 in advanced prolapse [69, 70]. It is sure that such molecular changes are not corrected by surgical techniques with native tissue or with PP mesh, and the procedures of tissue engineering by different MSCs types, and/or their secretome may change pelvic floor future histomorphology and functionality with normal/near normal connective tissue appearance, that will be discussed in Section 2.5.

#### **3.2 Genetics, gestational and postmenopausal influences on pelvic floor connective tissue disorders**

Genes, sexual steroid hormones with their receptors, ligands and co-activators modulate pelvic floor structures, and volumes entire women's life. One analyses Homeobox genes (*HOXA-11*involved in utero sacral ligaments fibroblasts proliferation and p53 regulation [71]), gene encoding *LOXl1-*generating a primarily failure of elastin postpartum healing in knockout mice, the decrease gene signal for production of three SLRPs-decorin, lumican and fibromodulin, which are collagen fiber assembly regulators with affected collagen fibrilogenesis and collagen fibrils shape, and impairment in elastic fiber assembly by down regulation of fibulin-5 in POP [17]. Genes encoding *fibulin-5*, *fibulin-3*, *Upii-sv40t*—involved in elastin fiber structure, are analyzed in PFDs associated to knockout mice aging [27].

Pregnancy induces adaptations in pelvic floor structures for vaginal delivery to withstand deformations with minimum damages, but vaginal delivery leads to floor disorders, damaging nerves, connective tissue, pelvic smooth and striated muscles. Pelvic connective tissue reduced stiffness and elasticity is essential, being demonstrated that the load carrying response (other than the functional response to the pelvic organs) of each fascia component, pelvic organ, smooth muscle, and ligament are assumed to be isotropic, hyperelastic, and incompressible [72]. There are parallel gestational changes in levatorani muscle: sarcomerogenesis, fiber elongation, and an increased ECM collagen content, with muscular stiffness [73], to avoid sarcomere hyperelongation resulted from mechanical strains imposed by vaginal delivery. Sometimes delivery related strains lead to acute sarcomere hyperelongation, and pregnancy pelvic floor muscles (PFMs) adaptation is exceeded [74]; with pelvic floor muscles (PFMs) avulsions discovered postpartum [73]. Human parturition needs PFMs elongation of 300% in resting muscle length to achieve fetal vaginal delivery, as computational models revealed [75]. An instrumental vaginal delivery with forceps may induce important damages of levator ani muscle visible at 3D/4D postpartum ultrasound [76], and one considers that the majority of vaginal deliveries are followed by subclinical damages. The postpartum

pelvic floor repair is different from *cervix uteri* repair, with loss of pregestational EMC composition restoration. Each vaginal delivery, in special genes and familial heredity conditions, and also without these risk factors, may contribute to EMC damages, with changes of pelvic floor shape and in biochemical structure in pregnancy, and post delivery versus nullipara [77], gestation has the greatest impact on vaginal tissue composition and biomechanical properties proved in animal models (mice, sheep), and women. Important differences of pelvic floor EMC structures are described [45] between virgin, pregnant and parous females regarding total collagen, collagen III/I + III ratios, GAGs, and elastin, and in passive biomechanical properties-compliance and elasticity, and maximum stress and strain, with permanent strain following cyclic loading after each gestation. Vaginal tissue of virgin sheep had the lowest total collagen content and permanent strain, and parous tissue had the highest total collagen, and lowest elastin content with concomitant high maximum stress in contrast to pregnant sheep, that had the highest elastin and lowest collagen contents, and thickest smooth muscle layer, situation associated with low maximum stress, and poor dimensional recovery following repetitive loading. Vaginal biomechanical properties do not recover after pregnancy to those of virgins [44], and tensile strength appears to be linked to vaginal content: total collagen, elastin, and smooth muscles show a direct influence on tissue compliance—reduced after ovine consecutive pregnancies, different to rectum and bladder compliance which are stiffer than vaginal walls after many deliveries [78]. Vaginal distensibility pregnancyinduced and along vaginal delivery by tissue vaginal pressure is not recovered in late postpartum rats [79]. It was demonstrated in mice vaginal culture, the POP appearance and progression after each pregnancy and vaginal delivery, caused by a combination of inhibited elastin linking with tropoelastin accumulation [47], because inability to initiate damaged tissue necessary clear and replacement, with poor elastin properly self repair after each delivery, through abnormal enzymatic actions of MMP-2 (decrease)— TMP-4 (rise) [48], after an initial total elastin amount preservation.

Ageing associated hypoestrogenism is worsening pelvic floor condition. Sexual steroid hormones have receptors in all pelvic organs, not only in genitalia (**Table 1**) [80]. Uterine prolapse, but not SUI is diagnosed in nuliparous postmenopausal women.


*Adapted from Rechberger and Skorupski [80] (creative common: License CC BY4.0 for adapt). ERs: estrogen receptors; P4Rs: progesterone receptors; ARs: androgens receptors.*

#### **Table 1.**

*Sexual steroid hormones receptors distribution in pelvic floor structures.*

#### *A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial… DOI: http://dx.doi.org/10.5772/intechopen.108010*

Aging is associated to intrinsic aging stem cells-meaning self renewal reducing, through their genes aging [81] in the general dysfunctional frailty syndrome, where pro-inflammatory cytokines-TNF-α, IL-6 and C-reactive, are increased [82], and one may delay aging effects by menopausal hormone therapy (MHT), started from perimenopause for urogenital aging, with local estriol to reduce vaginal atrophy, and some symptoms of bladder aging [83, 84], and it is adjuvant in pre and long term postoperative care, associated to systemic MHT, around age of 50′ as new protocols advice, and different MSCs, types treatments, for frailty delay to safe health and function of organs/tissues, and one speaks about safe proper frailty treatment with MSCs, to increase health and function of organs/tissues [85, 86].

#### **3.3 Mesenchymal stem/stromal cells for pelvic tissue repair and regeneration**

High number surgical gynecological procedures proposed along 100 years showed limitations, low adequacy to PFDs pathophysiology, no restoring organs' normal positions and functions, and better understanding by three dimensional digital models combining DeLancey JO's theory [16] to Petros P integral theory of continence [interdependence between pelvic organ support systems, linking ligament fascia lesions, and clinical expression, with less critics after TVT (tension free vaginal tape) technique] [87], and tissue structure continuing to deteriorate by aging after correction, being far to be restored by surgery either by native tissue or by PP meshes. MSCs, and their secretome, are discussed since more than 10 years, specially after techniques for their potency enhancement by specific culture systems, including three-dimensional culture conditions, or their priming preconditioning with some molecules of their secretome [7, 65]. MSCs represent a pathophysiologic correction, and limitation of PDFs progression [88]. Tissue engineering is a new option to restore and maintain micturition normality via direct effects on damaged or dysfunctional tissues, or pelvic floor repair when soft tissue reinforcement is necessary [89, 90], to improve outcomes in POP management [91]. MSCs also referred as mesenchymal stromal cells belong to the pool of progenitor and adult stem cells (ASCs) family, from all postnatal organs and tissues, with specific properties for each one, ensuring the capacity of renewal after damage, and in aging [7]. Collected and isolated from various anatomical sites, more or less easy accessible, few ethics-related issues, MSCs are actually easily separated/ purified [92], with specific markers, and cultured. Some consider multipotent MSCs to have a limited self-renewal capacity [93], in a specific microenvironment termed "stem cells niche", first described by Schofield R [94] as "adult stem cell niche hypothesis", which is reconsidered to be more dynamically than originally appreciated, with a bilateral influence from healthy or damaged tissue to MSCs, mainly by immunological and inflammatory signals in conjunction to MSCs' paracrine effects. Others [95] consider that MSCs unique properties-high proliferative ability, self-renewal, differentiation to mesodermal lineages, appropriate to their location, are supporting tissue regeneration in physiologic and pathologic conditions, and are contributing to tissue homeostasis. MSCs are key regulatory components in the regenerating stem cell niche, by the increase of their own compound, or increasing physiologic cells turn-over [96] to support tissue regeneration after injury, or are activated in injured tissue, where they are inactive [97], or are attracting supporting cells to niche [4], or are activating tissue's own cells to facilitate repair [98], capabilities that are different according to tissue type. These biological properties determined the change of "stem" cell nomination to "stromal" for a more appropriate connotation [7], and earlier Caplan AI [99] proposed the name of "Medicinal Signaling Cells" for a more accurate presentation:

when systemically administered MSCs home in on sites of injury/disease, and exhibit a paracrine action, by secreting bioactive molecules as regulatory and growth factors, chemokines, cytokines, nucleic acids, packaged into extracellular vesicles or MSCsderived exosomes, with trophic and immunomodulatory actions, reflecting that MSCs make therapeutic drugs *in situ* that are medicinal, important for tissue repair. MSCs fate in a tissue is influenced by local microenvironment or niche fixed compartment, where ASCs are in a dormant state (G0) through signaling pathways inhibitory for growth and differentiation, often involving transforming growth factor-ß (TGF-ß), and bone morphogenetic protein (BMP) family members [57], being anchored to niche cells by adhesion molecules—cadherins, integrins [100] during stem cells' periods of inactivity, and niche cells differentiation signals to resident stem cells [58]. The bioactive molecules produced through MSCs homing when systemic administrated, or when are added in cultures to potentiate MSCs action, as MSCs primers [65], are considered more important than cell engraftment and replacement. MSCs and their bioactive molecules have proangiogenic [101], antifibrotic, anti-inflammatory, and pro-inflammatory actions, which sustain proliferation [102], and stimulate effect of resident progenitor cells, in relationship to disease/organ/tissue type [103].

#### **Figure 1.**

*MSCs role in damaged connective tissue repair. MSCs activation after tissue injury. The damaged tissue activates MSCs after injury through different inflammatory signals (hypoxia, cytokines asIL-1β, IFN-γ, TNF-α, LPS). MSCs activation leads microenvironment to coordinate the production of immunomodulatory factors, that sustain inflammation progression, and of growth factors to stimulate endothelial cells, fibroblasts, and tissue progenitor cells to differentiate, and all contribute to tissue repair, in an orderly action by angiogenesis, EMC remodeling, and functional tissue restoration. Adapted from Miceli et al. [7]. It is an open access article, distributed under the terms and conditions of the creative commons attribution (CCBY), licensee MDPI, Basel, Switzerland.*

*A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial… DOI: http://dx.doi.org/10.5772/intechopen.108010*

The damaged, ischemic tissue activates MSCs after injury, through different inflammatory signals (hypoxia, proinflammatory cytokines as IL-1β, IFN-γ, TNF-α, lipopolysaccharide) from host innate immune system and leads microenvironment to coordinate the production of immunomodulatory factors to sustain inflammation progression and rapid remove of allogenic MSCs, and production of growth factors to stimulate endothelial cells, fibroblasts, and tissue progenitor cells' differentiation from MSCs niche, all contributing to tissue repair, in an orderly action by angiogenesis, EMC remodeling, and functional tissue restoration [7], MSCs have the ability to home to injured tissues to exert their paracrine actions when systemically administered, a very attractive feature for this chapter discussions (**Figure 1**) [104].

MSCs do not impose immunosuppresion, being immune-privileged due to their low expression of *CD40*, *CD80*, *CD86*, and major histocompatibility complex I (*MHC I*), and the lack of *MHC II* [92], or because they are immune evasive [105].

#### **4. Endometrial and menstrual mesenchymal stem cells for prevention and therapy of pelvic floor disorders**

MSCs from different tissues exhibit many common characteristics, their biological activity and some markers are different and depend on tissue origin: bone marrow (obtained by aspirate), adipose tissue (obtained by liposuction), placenta (for maternal MSCs), and umbilical cord, amniotic membranes and liquid (for fetal MSCs) collected at birth. All these MSCs have some limitations, as in vitro expansion [106] for their rarity in original tissue, invasive methods to harvest bone marrow aspirates, donor aging affects MSCs proliferative capacity [86], and the necessity to add their secretome's active molecules in culture medium [7, 8, 107].

#### **4.1 Unique characteristics of endometrial mesenchymal stromal/stem cells and menstrual mesenchymal stromal cells**

In the last 15–20 years, a MSCs subpopulation of stem/progenitor/regenerative cells has been identified and characterized in human endometrium (eMSCs) and in menstrual blood (MenMSCs or ERCs), comparable to bone marrow and adipose tissue MSCs [95], but with unique biologic characteristics [1, 108], and knowledge plus new technique capabilities made them a very promising MSCs category in autologous and allogeneic cellular therapy, and TE. Uterine fragments of shedding endometrial tissue with their remarkable cells turn-over—like hematopoietic bone marrow, intestinal epithelium, epidermis, contribute to endometrial repair and renewal without scar, and gene profiling has demonstrated that the lysed stroma is enriched in genes involved in EMC dynamics, biosynthesis and degradation [109, 110], very promising in pelvic floor connective tissue repair/restoration, and other endometrial fragments from menstrual blood contain MenSCs. The concept of endometrial renewing after each menstruation (~400 cycles in woman's life) by endometrial stem/progenitor cells located perivascular [6], in the basalis of endometrial glands near myometrium was first hypothetized by Prianishnikov VA, 1978 [111], reloaded by Padykula HA, 1989 [112], and it is continued in Australia at the Department of Obstetrics—Gynecology, Monash University and Centre for Women's Health Research (Melbourrne, Victoria) by a team led by Gargett CE [113], who presented the first direct evidence that human endometrium contains rare populations of epithelial (0.22%), mesenchymal/stromaleMSCs (1.25%), and endothelial progenitor cells, which exhibit the adult stem cells

behavior in vitro, as clonogenicity, and later their differentiation potential (reviewed in 2016) (**Figure 2**) [115].

One may add a small side population (SP) cells which enriches endometrial stem cells fractions, according to their identity and differentiation potential [116]. eMSCs are responsible for cyclic regeneration of human, mice, and ovine endometrium [117–120]. Other research group [121] demonstrated that the low number of human endometrial stromal/stem cells seems to belong to the family of MSCs, by possessing the minimal criteria of MSCs assessment [122] clonogenity, self-renewal, plastic adherence in culture, high proliferative potential and capacity and ability to differentiate into at least one type of mature functional progeny, but eMSCs have multilineage differentiation capacity [123]. eMSCs have proliferative capability to undergo 30 populations doubling before reaching senescence, generating 6.5 × 1011 cells (**Table 2**) [124].

Taylor HS [125] at Yale University (USA) presented bone marrow (BM) as an exogenous source of eMSCs, which appear histologically as epithelial and stromal endometrial cells, expressing appropriate markers of endometrial cell differentiation, and cyclic mobilization of BM-derived stem cells is considered a normal physiologic process [126]. Menstrual blood, an usual waste tissue, but a "bio-waste" as recently reconsidered [127] (endometrial functionalis layer shed during menstruation) is an easy obtained source of MSCs, with no ethic issue, and isolated, cultured similarly to bone marrow aspirated.

#### **Figure 2.**

*Schematic perivascular localisation of human eMSCs. Co-expressing CD146 and PDGFRβ/CD140b and SUSD2+ eMSC in the endometrial basalis and functionalis layer, indicating eMSC will be shed into menstrual blood. Reprinted from Gargett and Masuda [114] with license permission: 535199193277 for Copyright Oxford University Press, 2022, July.*


*International Society for Cellular Therapy position statement [122].*

#### **Table 2.**

*Minimal criteria for defining multipotent MSCs.*

#### *A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial… DOI: http://dx.doi.org/10.5772/intechopen.108010*

Menstrual stem cells (MenSCs), named also endometrial regenerative cells (ERCs) by the team from Bio-Communications Research Institute (Wichita, USA), who first isolated and cultured them [128], have the classic properties, and pattern of MSCs surface markers, are multipotent [129], retain a stable karyotype in culture [130], proved at more than 68 doublings without any karyotype or functional abnormalities [128, 131]. ECRs have similar capabilities for tissue repair and restoration as eMSCs, according to their secretome that ensure cells paracrine actions on endometrium (after menstruation, and Asherman syndrome) [132], ovaries [133], and on different organ [134, 135]. There are many controversies on eMSCs and ERCs makers [108], being demonstrated that endometrial/menstrual MSCs clones express MCSs markers [ITGB1 (CD29), CD44, NT5E (CD73), THY1 (CD90), ENG (CD105), PDGFRB (CD140B), MCAM (CD146)], but not endothelial or hemopoietic markers PECAM1 (CD31), CD34, PTPRC (CD45), and the pan-leukocyte marker CD45 [128]. The Australian team used these markers for eMSCs isolation and culture [124], plus two perivascular cell surface markers—CD146, and platelet-derived growth factor-receptor β (PDGF-Rβ) or CD140B, and have determined eMSCs location near blood vessels in human endometrium [114, 136, 137], blood vessel wall is considered the eMSCs niche, as it is for all MSCs [138, 139]. These markers were used in association to Sushi Domain Containing 2 (SUSD2) or W5C5, a special marker for eMSCs and MenSCs isolation, proposed as a novel single marker for purifying eMSCs, and to reconstitute endometrial stromal tissue in vivo from endometrial biopsies [6]. MenSCs need a selective marker enrichment to be consistent and efficacious as eMSCs obtained by endometrial biopsy. It is known that perivascular MSCs or pericytes are rare cells, difficult to harvest from adult tissues, and necessitate substantial ex-vivo culture expansion to achieve a sufficient number of potent cells, and prolonged culture of MSCs determine a spontaneous differentiation to fibroblasts, which limits culture expansion, and these significant limits challenged the special add in culture medium of a novel small molecule-transforming growth factor-β receptor inhibitor, namely A83-01, that limits the inconvenient and maintains eMSCs and other MSCs undifferentiated in the days following administration and ensure the therapeutic efficacy of a small proportion (2%) of cells which are estimated to remain in vivo in the days following administration [9, 91, 114, 140]. MenSCs secretome needs a special attention for its exceptional therapeutic effects, due to extracellular vesicles (EV) [135], including microvesicles, exosomes and apoptotic bodies transporting bioactive molecules. A total of 895 molecules are identified in exosomes [141], as micro RNA, lipids, growth factors (vascular endothelial growth factor, insulin-like growth factor-1, hepatocyte growth factor), chemokines, cytokines as regulators of immune response in different tissues [142], which can be isolated from menstrual blood as are MenSCs isolated [143]. The human MenSCs transcriptome and methylome profiles showed their most distinctive expression and epigenetic signature compared to human bone marrow and adipose MSCs [144]. MenSCs trandifferentiation capacity is extensively discussed, associated to their possibility to differentiate into mesodermal lineage (including chondrogenic, osteogenic, adipogenic, and cardiomyogenic fate), endodermal lineage (hepatocyte), and ectodermal lineage (neural and glial) [145], processes that varies considerably between each type, and it is different when one compares them to bone marrow and adipocyte MSCs [128]. Recently there were proved the beneficial effects of MenSCs and their secretome in pulmonary healing in severe acute adults lung cells injury (ARDS) from COVID, by increasing number of CD4 lymphocytes, reducing expression of inflammatory markers (C Reactive Proteine, ferritine, LDH), absorption of bilateral pleural exudates, better than other MSCs types (BM, adipose tissue) when systemic transplanted [146], having also the advantage of easy collection in emergency.

#### **4.2 Potential application of endometrial and menstrual mesenchymal stem cells in pelvic floor disorders prevention and therapy**

eMSCs with autologous and allogeneic origins are easily procured from endometrial biopsies—during reproductive years, under contraceptives, in postmenopause [115], without anesthesia or from menstrual blood (source that can be repeatedly used at every menstrual cycle, much easier obtained vs. other sources of ASC). with remarkable differentiation capabilities, plus paracrine actions from their secretome, proved by preclinical and some phase III trials—much discussed and criticized in USA [147], because industry commercial enthusiasm [148]. FDA (USA) approved in 2011 clinical trials with ERCs, which can be used for PFDs prevention and therapy. Actually eMSCs are not accessible for human trials all over the world, or there are no legally approved banks for eMSCs/MenMSCs, these cells being used in some countries only for academic/scientific health centers or not-for profit public institutions. European Medicine Agency has a "hospital exemption" clause, with existence of many unknowns/controversies on eMSCs/MenMSCsbased therapies in human (allogeneic or autologous), as they are used in animal models. Actually one must consider that amalgamation of highly specialized disciplines such as tissue engineering, stem cell therapy and personalized medicine provide important approaches and tools to respond to these challenges in PFDs prevention and therapy, as there were found regulatory approval and deployment for disorders with unmet medical needs [149].

#### *4.2.1 Endometrial/menstrual mesenchymal stem cells based for pelvic floor disorders prevention and therapy*

Cell therapy is an emerging field in clinical practice, bone marrow, adipose tissue being subjects for trials in chronic and degenerative disease. Basic evidences provide the preventive role of MSCs autologous cell based therapy in rats SUI by local-urethral administration [150], which is a minimally invasive procedure, and intravenous (i.v.) route [10]. MSCs are homing in damaged pelvic organs, where they are attracted by cytokines [151] and chemokines [152]. After i.v. administration, post vaginal distension (VD)—a model for childbirth injury, GFP-labeled cells were depicted at 4 to 10 days in urethra, vagina, rectum, and levator ani muscle of sacrificed animals, with significantly more MSCs homing at 4 days *versus* after sham VD, and reduction of GFP intensity at 10 days after VD [153]. It is discussed PFDs prevention with MSCs in high risk women by genetic predisposition, with postpartum SUI and/or POP, and the possibility to induce homing a long time after injury or to increase homing after an acute injury *via* stem cells genetic modification to express a greater number of homing ligands [154] or an electrical stimulation to the paravaginal region, which induces neural stem cells migration [155], or by MSCs paracrine effects in damaged tissues [growth factors as Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), angiopoietin-2, and Platelet-Derived Growth Factor (PDGF-BB)], which were proved to be in a rate of about 10–100,000 times more for MenMSCs than the control mesenchymal cell lines derived from umbilical cord blood, and by stimulating metalloproteinases involved in elastin postpartum [128]. Post infusion febrile reaction is the sole adverse event associated to bone marrow MSCs i.v. [156]. One discusses the i.v. dose differentiation between small (rodents), large (ovine) animals, and human: 50 million MSCs/kg for rodents, and 1–2 million cells/kg and never more than 12 million cells/kg for human. MSCs pulmonary entrapment, with their quick

*A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial… DOI: http://dx.doi.org/10.5772/intechopen.108010*

phagocytisation, by lung tissue macrophages [157], and the genetic instability and tumorigenicity [156], not valuable for eMSCs/MenMSCs [14]. ERCs presenting more than 68 doublings without any karyotype or functional abnormalities [128, 131].

Australia has programs based on techniques to purify eMSCs by magnetic beads and special markers, for production of large number of cells under Good Manufacturing Practice (GMP) conditions to offer women's own eMSCs when they need for PFDs, for TE. Actually there are only animal studies, no human trial on eMSCs, /MenSCs for PFDs, being discussed other MSCs types. The Cochrane Database Syst Rev. (2017) [158] found in Cochrane Incontinence Group Specialized Trials Register only one small RCT for SUI, on injection of autologous MSCs with fat origin *vs placebo*, terminated early because of safety concerns, and afterwards there are mentioned some clinical trials for SUI treated with autologous muscle derived MSCs compared to *placebo* [159], or transurethral and periurethral intrasphincteric injections of cellular suspension for SUI, with limited accuracy of results [160], or a preliminary randomized study of stem cells from adipose tissue implanted for fecal incontinence [161].

#### **4.3 Tissue engineering with endometrial and menstrual mesenchymal stromal cells for pelvic floor disorders**

Tissue engineering (TE) for pelvic floor disorders treatment combines principles of eMSCs/MenSCs biology with materials science (scaffolds, meshes for pelvic implantation), and biomedical engineering [162]. The most discussed PFDs beneficiary of eMSCs therapy are POP and SUI—*de novo*, persistent or recurrent after failure of surgery with native tissue or reconstruction. There are necessary multidisciplinary trained teams. and special protocols, as are presenting Ichim T (2008) CEO at Medistem Laboratory (San Diego, USA), and Gargett CE (2013)—head of the Australian Stem Cell Centre.

The key to safe and efficacious TE in PFDs was the generation of some tissue substitute by materials with nano-architecture/nanofiber technology [163] or 3D printing, mimicking EMC pelvic floor topography, mainly vaginal EMC, or to induce favorable tissue mechanical responses, or to add some EMC constituents (as tropoelastin-the elastin core in EMC network, besides collagen, lost in POP) [164], for production of mesh/scaffold new generation, which allows entrapment, and persistence of seeded eMSCs/MenSCs up to 14 days after implant [41]. eMSCs are in vitro optimized in serum free conditions—fibronectine is the optimal substrate for human eMSCs attachment [91], and eMSCs transcriptome reveals improved potential for cell based therapy after adding TGF-β receptor inhibitor in culture medium (to prolong their undifferentiated status after implantation, by eMSCs apoptosis, and senescence prevention, and maintaining the percentage of SUSD2+ cells to more than 90% for all samples) [165]. Another proposal for eMSCs tissue repair efficacy augmentation is the add of a protective delivery system, as a compatible bio-hydrogel carrier that encapsulates eMSCs in mesh/scaffold and improves cells retention at site from host immune system actions to rapid their remove, due to loss of vascular niches [107], and by their encapsulation in hydrogel MSCs can promote endogenous cellular repair [140]. There are many composite meshes produced from different materials: nondegradable polyamide meshes, as those of polyamide/gelatin seeded with 100,000 human or ovine MenSCs/cm2 , which stimulate angiogenesis, host synthesis type 1 and type III collagen, lower leukocytes infiltrate at 90 days postimplantation (when tested on rats) [166], andthe new biomimetic tissue generation of degradable nano/microstructured meshes [167], meshes obtained through new technologies of

electrospinning and 3 D bioprinted endometrial stem cells on an aloe vera–alginate (AV-ALG) injectable hydrogel or on melt electrospun poly epsilon-caprolactone mesh, with the largest open pore diameter and the lowest thickness that promotes eMSCs encapsulated in the hydrogel attachment, which reduces FBRs associated to eMSCs same action [168, 169]. The meshes designed with nanoscale fibers using electrospinning techniques promote cell–cell and cell-biomaterial interactions, being appreciated in Australia [91, 167, 170], and Nederland studies [171] that biometric properties of this nanostructured mesh can improve the integration, overcome erosion, and offer good outcomes in POP reconstructive surgery. The mesh type added to eMSCs have different persistence time after implantation, the natural ones have the shortest "life" duration after implant; the non-degradable polyamide/gelatin mesh plus autologuse MSC persisted 3 days in immunocompetent mice, 1–2 weeks in immunocompromised rodent model when xenogenic human eMSCs, and 90 days in ovine; the longest duration is at 3 D printed mesh [168]. The Chinese study [12] shows that a composite mesh based on synthetic and natural polymers seems to provide the best combination for an ideal pelvic floor mesh material, because natural polymers can provide ligands for cell adhesion and growth factors that promote tissue remodeling, while synthetic polymers provide mechanical strength. New scaffolds proposed for POP provide a threedimensional environment, and are mimicking EMC network, specially by their micro/ nanoscale architecture [172], offering a larger area for EMC constituent proteins, and more binding sites for cell membrane receptors, and adhesion molecules, growth factors, genes, immunomodulatory agents, and external stimuli (electrical, or magnetic pulses), which are delivered simultaneously to target sites after scaffold implantation for promotion of new healthy tissue [173]. No ideal mesh/scaffold for PFDs exists to day, and one may choose the mesh/scaffold according to patient history, and implants' properties, such as material type: natural (as purified human/animal collagen, chitosan, gelatin, elastin, fibrin, silk, and fibronectin for human eSMCs [174, 175], or synthetic, or by the old criteria used for PP meshes, as pore sizes, mesh's weight, and the host tissue response to mesh/scaffold material or to eMSCS/ERCs, understanding that reaction to implant materials are crucial for balance between their own elasticity and stiffness, vaginal tissue capacity of strain, which are absent when PP meshes are used. Novel blends of electrospun synthetic and natural polymers combined with eMSC in new generation of implants show that this approach promotes host cell infiltration and slows biomaterial degradation that has potential to strengthen the vaginal wall during healing [164, 165], actioning like intrinsic ECM, but with some limitation regarding small pore size of electrospun nanofiber meshes and toxic organic solvents used for their production [12]. /Human eMSCs/MenSCs modulate host tissue response to implanted materials, by stimulating tissue proper stem cells proliferation, their own high proliferation rate [128], and scaffolds' eMSCs infiltration, and constituents of their secretome as growth factors, enzymes as MMPs—important for elastin postpartum recovery [128], influence mesh mechanical behavior after implantation [166], by fibrosis and FBRs reducing, when nondegradable polyamide mesh implant, through influencing macrophage polarization switching from an M1 to M2 phenotype, as in rodent and ovine models [166, 176, 177]. eMSCs seeded in degradable nano/microstructured meshes improve mesh tissue integration, eMSCs are entraped over 6 weeks in vivo, by cells with immunomodulatory effects, and by increasing local angiogenesis reduce FBRs to mesh implanted in mice with POP [166, 167, 169], and induce an up-regulation of M2 markers—as CD206 and *Arg1*, *Mrc1*, and *Il10* genes in host tissue macrophages, parallel to reduction of cellular infiltration and secretion of inflammatory cytokines Il-1β and Tnf-α [175].

#### *A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial… DOI: http://dx.doi.org/10.5772/intechopen.108010*

The Australian researchers [140] appreciate that tissue engineered mesh inserted transvaginally in large animal models will aid the validation of these constructs prior to clinical translation by assessing their integration with host tissue, and FBRs through histological analysis, immunoassays and gene profiling. Research has commenced with the completion of multiple xenogenic small and large animals studies assessing eMSC/PAG constructs, as mentioned above [166]. These animal models will be crucial in further assessing the efficacy of locally delivered eMSC and further determination of their action mechanisms. Based on these findings, multiple heterologous small and large animal studies are underway to assess the efficacy of other biomimetic degradable materials such as PLCL and 3D-PCL meshes seeded with eMSC/MenSCs.

#### **5. Future proposals for stem cell prevention and therapy in pelvic floor disorders**

The Australian research teams recognize some unknown data about eMSCs/ MenSCs, mediation on cellular migration and recruitment by their paracrine effects, how eMSCs mediate M2 immunomodulatory responses during the FBR after implantation of bioengineered constructs. One discusses eMSCs secretome constituents as future associated to new generation implants for tissue engineering in PFDs. The Canadian and North American and Chinese researchers recognize the high financial burden for the studies and introduction in human clinical practice according to legislation issues which are incomplete resolved in Europe and North America, in comparison to Australia and Japan.

#### **Conflict of interest**

None.

#### **Author details**

Manuela Cristina Russu "Dr I. Cantacuzino" Discipline of Obstetrics and Gynecology, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania

\*Address all correspondence to: manuela\_russu@yahoo.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 12**

## Therapeutic Approaches Targeting Cancer Stem Cells

*Shin Mukai*

#### **Abstract**

Cancer stem cells (CSCs) have been identified in many types of cancer since their discovery in leukemia in the 1990s. CSCs have self-renewal and differentiation capacity, and are thought to be a key driver for the establishment and growth of tumours. Several intracellular signalling pathways are reported to play a significant role in the regulation of the biological activities of CSCs. Thus, many researchers have considered CSCs to be a compelling therapeutic target for cancer, and blockade of CSCrelated signalling pathways can be efficacious for the treatment of multiple cancer types. This chapter succinctly summarises the recent progress in the development of treatments targeting signalling pathways related to the functions of CSCs.

**Keywords:** therapeutic modalities, drug development, signalling pathways, self-renewal, cancer stem cells

#### **1. Introduction**

Cancer is a life-threatening disease in which abnormal cells grow and divide, resulting in the destruction of normal body tissues. The last several decades have seen advancements in cancer treatments [1]. However, the conventional therapeutic methods often fail due to cancer recurrence and metastasis [2]. This can be explained by the existence of cancer stem cells (CSCs), which are a minor population in tumours and can survive most traditional cancer therapies killing cancer cells with proliferative properties [3]. Cancer relapse, metastasis, multidrug resistance, and radiation resistance can be induced by the transient arrest of CSCs at the G0 phase, leading to the production of new malignant tumours [4]. The ability of CSCs to self-renew and differentiate into multiple cellular subtypes allows them to generate tumours [4]. Thus, researchers have regarded CSCs as a promising target for the treatment of cancer since they were discovered in leukemia in the 1990s [5, 6]. CSCs have also been found to be a subpopulation of many types of tumours, and tissue-specific expression of CSC markers has been reported [7]. It is known that the biological functions of CSCs are controlled by several signalling pathways [8]. This chapter focuses on the research status in cancer therapies targeting the signalling pathways that are believed to control the properties of CSCs.

### **2. General biology of CSCs**

Since CSCs were found in leukemia in the 1990s, they have been studied intensively [5, 6]. However, the origin of CSCs remains to be elucidated [9]. It has been reported that (a) CSCs possess self-renewal capacity and high proliferation rate, (b) CSCs are able to generate and maintain tumours, and (c) cancer recurrence may be induced by the unlimited self-renewal capacity of CSCs [10]. CSCs are shown to be a distinct subpopulation in haematologic malignancies and solid tumours, and cell surface markers of CSCs in various types of cancer have been reported and can be used for the identification and isolation of CSCs (**Figure 1**) [11–15]. Signalling pathways such as Wnt/β-catenin, Notch and Hedgehog signalling pathways are thought to regulate the properties of CSCs [8]. Emerging evidence supports the clinical relevance of CSCs [16]. In particular, CSCs are shown to be resistant to conventional chemotherapy and radiation therapy, and they are believed to be a key player for cancer recurrence and metastasis [16]. Thus, understanding of signalling pathways that control the functions of CSCs can be useful for the creation of novel therapeutic interventions for cancer.

**Figure 1.** *Cell surface markers for CSCs in various organs. This figure was created with BioRender.*

### **3. Development of therapeutic modalities targeting CSCs**

#### **3.1 Notch signalling**

The Notch signalling pathway is a highly conserved pathway in mammals and controls a variety of cellular functions [17]. In mammals, the Notch pathway comprises four Notch receptors (Notch 1–4) and five Notch ligands (Jagged 1, Jagged 2, Deltalike ligand (DLL)-1, DLL-3, and DLL-4), and its primary effector is the transcription factor CSL (CBF1/RBP, Su(H)/Lag-1), which is crucial for the activation of the genes downstream of the Notch signalling pathway [18]. The Notch signalling pathway is divided into canonical and noncanonical pathways: the CSL-dependent pathway

*Therapeutic Approaches Targeting Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.108963*

(canonical Notch signalling pathway) and non-CSL-dependent pathway (non-canonical Notch signalling pathway) [19]. Evidence suggests that these pathways play an instrumental role in preserving the existence of stem cells and initiating embryonic or foetal cell differentiation [20].

In the canonical Notch signalling pathway (**Figure 2**), the binding of receptor and ligand is induced through metalloproteinase- and γ-secretase-mediated proteolytic activation of the Notch receptor, leading to the release of the intracellular NOTCH domain (NICD) [21]. Subsequently, NICD migrates to the nucleus and forms a complex with CSL. As a result, the transcription and expression of the downstream target genes are triggered, leading to self-renewal, differentiation and proliferation [21].

Recent studies suggest that there is non-canonical Notch signalling, which can be activated either with or without ligand interaction [22]. In addition, the activation of non-canonical Notch signalling can occur in a γ-secretase-dependent or -independent manner [22]. In the case where non-canonical Notch signalling occurs in a γ-secretase-independent way, Notch remains bound to membrane [22]. Noncanonical Notch signalling does not require CSL [23, 24]. Instead, NICD or membrane bound Notch interacts with (a) Wnt, PI3K, mTORC2 and/or AKT pathways in the cytoplasm, and (b) NF-κB, YY1 and/or HIF-1α pathways in the nucleus [23, 24]. It is suggested that non-canonical Notch signalling plays a role in cell survival, metabolism and differentiation [23, 24]. Compared with the canonical Notch signalling pathway, there is less information on the non-canonical Notch signalling pathway [25]. Thus, more work will be needed for the identification of potential therapeutic targets in the non-canonical Notch signalling pathway [25].

Notch inhibition is believed to be a promising therapeutic approach to target CSCs, which are resistant to conventional methods such as chemotherapy and radiation [26]. As γ-secretase is a key player in Notch signalling, a great deal of effort has been invested in the development of γ-secretase inhibitors (GSIs) (**Figure 3**) [27]. It should be noted that GSIs show anti-CSC effects and that they were the first Notch inhibitors to reach clinical development [27]. However, one of the major concerns is the toxicity of GSIs [28]. In particular, serious toxicity in the gastrointestinal tract

#### **Figure 2.** *Brief diagram of the canonical Notch signalling pathway. This figure was created with BioRender.*

**Figure 3.** *Structures of Notch signalling inhibitors in clinical development.*

can be caused by GSIs [28]. Several GSIs have entered clinical trials thus far. Data from a Phase II clinical trial suggest that RO4929097 did not show sufficient efficacy for the treatment of metastatic melanoma and platinum-resistant ovarian cancer as monotherapy [29, 30]. Nirogacestat (PF-3084014) is another GSI undergoing clinical trials for desmoid tumours [31]. The result of Phase II clinical trials indicates that (a) treatment of desmoid tumour fibromatosis patients with Nirogacestat could be a promising approach, (b) the objective response rate was 71.4% and (c) relatively low doses and high tolerability were achieved, resulting in prolonged disease control [31, 32]. Another study shows the antitumour and antimetastatic effects of Nirogacestat in hepatocellular carcinoma [33]. The potent pan-Notch Inhibitor BMS-906024 has advanced into clinical trials, and the data suggest that it could be effective for the treatment of leukemia and solid tumours [34]. The clinical development status of other small molecule inhibitors is as follows: (a) The GSI MK-0752 is in a Phase I clinical trial for the treatment of pancreatic ductal adenocarcinoma [27], (b) the GSI LY900009 is in a Phase I clinical trial for ovarian cancer [35], (c) CB-103, which inhibits the interaction between NICD and CSL, is in a Phase I clinical trial for adenoid cystic carcinoma (ACC), colorectal cancer, breast cancer and prostate cancer [36], and (d) Crenigacestat (LY3039478), which is an oral Notch and GSI inhibitor, is in a Phase I clinical trial for solid tumours [37]. Recently, cryoelectron microscopy (cryo-EM) structures of γ-secretase in complex with each of the two GSI clinical candidates Semagacestat and Avagacestat have been reported, and these pieces of information might be useful for the design of novel GSIs [38].

A study suggests that the expression of the Notch ligand DLL4 is increased in gastric cancer, enhancing self-renewal ability of CSCs [39]. Therefore, inhibition of DLL4 can be an effective approach to treating cancer [39]. Currently, Enoticumab (REGN421), a fully human IgG1 monoclonal antibody against DLL4, is in a phase I clinical trail for the treatment of solid tumours [40].

#### **3.2 Wnt signalling**

The Wnt signalling pathway is known to play a pivotal role in embryogenesis and tissue repair by controlling proliferation, differentiation, apoptosis and cell-to-cell interactions [41, 42]. The Wnt signalling pathway is classified into the canonical

#### *Therapeutic Approaches Targeting Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.108963*

Wnt pathway (β-catenin dependent) and the noncanonical Wnt pathway (β-catenin independent) [43]. Wnt ligands are required for the activation of Wnt signalling, and the acyltransferase Porcupine is known to be essential for the production of Wnt ligands [44]. In canonical Wnt signalling (**Figure 4**), the absence of Wnt ligands leads to the degradation of β-catenin due to phosphorylation by glycogen synthase kinase 3β (GSK3β), and thereby translocation of β-catenin from the cytoplasm to the nucleus does not occur [45]. In the presence of Wnt ligands, their ligation to Frizzled proteins and LRP5/6 receptors induces the activation of the cytoplasmic protein DVL and the subsequent suppression of GSK3β [46]. It enables β-catenin to migrate to the nucleus and trigger target gene transcription by binding to TCF/LEF transcription factors [46]. Noncanonical Wnt signalling does not require the cytoplasmic stabilisation of β-catenin or its translocation into the nucleus [47]. The noncanonical Wnt signalling is subdivided into the following two well-characterised pathways: the planar cell polarity (Wnt/PCP) pathway and the Wnt-Calcium (Wnt/Ca2+) pathway [48]. In the Wnt/PCP pathway, Wnt ligands bind to the Frizzled receptor, leading to the activation of Dishevelled (DVL) protein [49]. Activated DVL forms the DVL-Rac complex and DVL-Rho complex [49]. The former stimulates the Rho kinase (ROCK), and the latter stimulates the c-Jun N-terminal Kinase (JNK) [50]. JNK is known to translocate into the nucleus and trigger the transcription of target genes [51]. The Wnt/Ca2+ pathway involves the activation of PLC and PKC, and an increase in intracellular Ca2+ [52]. The phosphatase calcineurin is stimulated by Ca2+ and dephosphorylates the transcriptions factor NF-AT, resulting in the migration of NF-AT to the nucleus [52].

Dysregulation of Wnt signalling is observed in many types of cancer [53]. The Wnt signalling cascade is reported to play an important role in controlling the properties of CSCs [53]. Thus, many scholars have been striving to create therapeutic modalities to target Wnt signalling for safe and effective elimination of CSCs, and several small molecule inhibitors and monoclonal antibodies have entered clinical trials (**Figure 5**) [53]. Evidence suggests that pharmacological inhibition of Porcupine can selectively block Wnt signalling and suppress tumour growth [54]. Thus, Porcupine is considered to be a promising therapeutic target for cancer [54]. LGK-974, a small molecule disrupting the enzymatic activity of Porcupine, is currently in Phase I clinical trials for pancreatic cancer, melanoma and triple-negative breast cancer [55]. Cryo-electron microscopy (cryo-EM) structures of Porcupine in complex with LGK-974 are available in the PDB database (PDB ID: 7URD) [55], and this information could be useful for creation of novel Porcupine inhibitors. Another Porcupine inhibitor ETC-159 is

**Figure 5.**

*Structures of Wnt signalling inhibitors in clinical development.*

**Figure 6.** *Brief diagram of the canonical Hedgehog signalling pathway. This figure was created with BioRender.*

in a Phase I clinical trial for the treatment of solid tumours [56]. The small molecule inhibitor PRI-724 is reported to block canonical Wnt signalling by preventing the interaction between β-catenin and its coactivator CREB binding protein (CBP) [57]. PRI-724 is in a Phase II clinical trial for advanced myeloid malignancies [58]. The therapeutic monoclonal antibody OMP-18R5 (vantictumab), which can target the Frizzled receptors, is now in a Phase I clinical trial for the treatment of non-small-cell lung cancer (NSCLC), pancreatic cancer and breast cancer [59–62]. The recombinant fusion protein ipafricept (OMP-54F28) can inhibit Wnt signalling by binding to Wnt ligands, and its safety and effectiveness are being evaluated in clinical trials [63–65].

#### **3.3 Hedgehog signalling**

The Hedgehog (Hh) signalling pathway contributes to the control of cell proliferation, cell survival, cell differentiation, and stem cell maintenance and development [66]. In Hh signalling (**Figure 6**), the autoproteolytic cleavage of Hh ligand precursor proteins leads to the production of an N-terminal protein, followed by dual lipid modification [67]. Subsequently, active Hh ligands are released through mediation of Dispatched and Scube2 [67]. In the absence of Hh ligand, Patched (PTCH) prevents the activation and ciliary localisation of Smoothened (SMO) [68, 69]. As a result, the Glioma-Associated Oncogene Homolog (GLI) forms a complex with Suppressor of Fused (SUFU), which precludes GLI from translocating into the nucleus [68, 69]. In the presence of Hh ligand, the binding of Hh to PTCH allows SMO to interact with

#### *Therapeutic Approaches Targeting Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.108963*

β-arrestin (Arrb2) and kinesin family member 3A (KIF3A), leading to the ciliary localisation of SMO [68, 69]. As a result, GLI is released from SUFU and subsequently migrates to the nucleus, triggering the transcription of Hh target genes [68, 69].

Evidence suggests that the dysregulation of Hh signalling is associated with detrimental events such as the self-renewal and metastasis of cancer stem cells [67]. Thus, therapeutic targeting of Hh signalling has accorded a great deal of attention from many researchers, and SMO has been regarded as the most promising pharmacological target [68]. Indeed, several SMO inhibitors have been approved by the Food and Drug Administration (FDA) or are undergoing clinical trials (**Figure 7**) [70]. Vismodegib (GDC-0449) was the first SMO inhibitor that was granted FDA approval to treat basal cell carcinoma in 2011 [71]. Mounting evidence suggests the inhibitory activity of Vismodegib against self-renewal and mammosphere formation of breast CSCs [72]. Data from Phase II clinical trials demonstrate that (a) Vismodegib could be used as a neoadjuvant chemotherapy agent for patients with triple-negative breast cancer (NCT02694224) [73], (b) Vismodegib could be efficacious for the treatment of pancreatic cancer by suppressing self-renewal, proliferation and survival of pancreatic CSCs [74] and (c) Vismodegib could be effective for untreated metastatic colorectal cancer by reducing the stem cell markers of colon CSCs [75, 76]. These results support the notion that Vismodegib can inhibit the activities of CSCs by blocking the Hh signalling pathway. The SMO inhibitor Sonidegib (LDE225) was approved by FDA for the treatment of advanced basal cell carcinoma in 2015 [77]. A recent study indicates that Sonidegib can make triple-negative breast cancer more sensitive to Paclitaxel and improve clinical outcomes by reducing the expression of CSC markers [78]. Glasdegib (PF-04449913) was an FDA-approved SMO antagonist for the treatment of acute myeloid leukemia and launched in the USA in 2018 [79]. Glasdegib is also undergoing a Phase II clinical trial for the treatment of myelodysplastic syndrome and chronic myelomonocytic leukemia [80]. Although the development of SMO inhibitors is beneficial for cancer patients, monotherapy with each of the FDA-approved antagonists can cause SMO mutations in tumour tissues, leading to drug resistance [80]. Hence, novel therapeutic methods inhibiting SMO will need

**Figure 7.**

*Structures of Hedgehog signalling inhibitors in clinical development.*

to be created in order to overcome this setback. The clinical development status of other SMO inhibitors is as follows [81]: (a) BMS-833923 is in a Phase I clinical trial for extensive-stage small cell lung cancer [82], (b) Itraconazole is in a Phase II clinical trial for prostate cancer [83], (c) Saridegib (IPI-926) is in a Phase I clinical trial for advanced and/or metastatic solid tumours [84], (d) LEQ-506 is in a Phase I clinical trial for advanced solid tumours [85], (e) Taladegib (LY2940680) is in a Phase I clinical trial for advanced solid tumours [86] and (f) TAK-441 is in a Phase I clinical trial for advanced nonhematologic malignancies [87]. In addition to the SMO inhibitors, arsenic trioxide is reported to inhibit the Hh signalling pathway and tumour growth by binding to GLI [81]. Arsenic trioxide is in a Phase II clinical trial for the treatment of advanced neuroblastoma or other childhood solid tumours [88]. Although there are no other GLI inhibitors undergoing clinical trials, *in vitro* and *in vivo* preclinical investigation into the GLI inhibitor GANT61 suggests its inhibitory activity against pancreatic cancer stem cells [89].

#### **3.4 NF-κB signalling**

The transcription factor nuclear factor kappa B (NF-κB) is a rapidly inducible transcription factor and a family of heterodimers or homodimers [90]. The heterodimers or homodimers are produced from different combinations of the five related proteins: p65/RelA, RelB, c-Rel, p105/p50 (NF-κB1) and p100/p52 (NF-κB2) [90]. The p50/p65 complex is thought to the most abundant form of NF-κB and serve main physiological functions [91]. The activation of NF-κB signalling induces the translocation of the transcription factor complexes from the cytoplasm to the nucleus [92]. The NF-κB signalling pathway diversifies into the canonical NF-κB signalling pathway and the noncanonical NF-κB signalling pathway [92]. The canonical NF-κB pathway is activated by the binding of ligands to their receptors such as the binding of (a) bacterial cell components to toll-like receptors (TLRs), (b) TNF-α to the TNF receptor (TNFR), (c) lipopolysaccharides to their respective receptors such as TLRs and (d) IL-1β to the IL-1 receptor (IL-1R) [93]. In response to the stimulation of these receptors, the kinase TGFβ-activated kinase 1 (TAK1) is activated, leading to the subsequent phosphorylation and activation of the IκB kinase (IKK) proteins [94]. The activated IKK proteins then phosphorylate IκB proteins. It induces the degradation of IκB, leading to the translocation of the activated p50/p60 complex into the nucleus [95]. The noncanonical NF-κB pathway is initiated by stimulation of receptors such as CD40, receptor activator for NF-κB (RANK), B cell activation factor (BAFF), TNFR2 and lymphotoxin β-receptor (LTBR) [94]. Subsequently, the kinase NF-κB-inducing kinase (NIK) is activated, resulting in the phosphorylation and activation of IKKα [94], which induces the phosphorylation of carboxy-terminal serine residues of p100 [94]. As a result, the C-terminal IκB-like structure of p100 is selectively degraded, which generates p52 and causes the p52-RelB complex to migrate to the nucleus [94].

A previous study shows that the production of cytokines, growth and angiogenic factors and proteases is promoted in the tumour development and progression, activating NF-κB signalling [96]. The NF-κB pathway is reported to contribute to self-renewal, maintenance and metastasis of CSCs [11]. Ovarian CSCs can display the enhanced capability of self-renewal, metastasis and maintenance due to the increased expression of RelA, RelB and IKKα [97]. In breast cancer, NIK expression is augmented, and the noncanonical NF-κB pathway is activated, leading to the self-renewal and metastasis of breast CSCs [98]. Regarding the development of

*Therapeutic Approaches Targeting Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.108963*

therapeutic modalities to inhibit self-renewal, proliferation and metastasis of CSCs by targeting the NF-κB pathway (**Figure 8A**), Disulfiram is known to inhibit the activity of NF-κB in breast CSCs and is in a Phase II clinical trial for the treatment of metastatic breast cancer [99, 100]. Sulforaphane is suggested to prevent the translocation of p50/p65 and reduce the expression and transcriptional activity of p52/ RelB, resulting in the inhibition of self-renewal of triple-negative breast CSCs [101]. Sulforaphane is in a Phase II clinical trial for the treatment of breast cancer [102]. The NF-κB pathway inhibitor curcumin is reported to impede self-renewal and metastasis of CSCs and is undergoing clinical trials for the treatment of breast cancer [103–105].

#### **3.5 mTOR signalling**

The mammalian target of rapamycin (mTOR) signalling pathway plays a pivotal role in cellular growth and metabolism in mammalian cells within various environments [106]. The activation of mTOR signalling can promote cell survival by (a) increasing cellular metabolism and the synthesis of proteins and lipids and (b) blocking apoptotic pathways [106]. The mTOR pathway is initiated by the binding of growth factors to tyrosine kinase receptors in the cell membrane, leading to the phosphorylation and activation of phosphatidylinositol 3-kinase (PI3K) [107]. Subsequently, protein kinase B (AKT) is phosphorylated and activated, leading to the activation of mTOR [107]. It activates a variety of transcription factors and enables them to migrate to the nucleus in order to promote the transcription of the target genes [108]. mTOR is divided into two protein complexes. mTORC1 is a complex of RAPTOR, mLST8, PRAS40 and DEPTOR, and mTORC2 is a complex of RICTOR, mLST8, DEPTOR, mSin1 and PROCTOR [109, 110]. mTORC1 is reported to (a) activate the ribosomal protein S6K, which promotes protein synthesis, (b) facilitate lipid synthesis and mitochondrial biogenesis and (c) reduce autophagy [109, 110].

#### **Figure 8.**

*Structures of CSC-related signalling inhibitors in clinical development. (A) NF-κB, (B) mTOR, (C) JAK/STAT, (D) ROCK.*

mTORC2 is known to promote actin cytoskeleton and cell migration by phosphorylating various proteins [109, 110].

Evidence suggests that the mTOR signalling pathway is correlated with the functions of CSCs. According to previous studies, activation of mTOR signalling contributes to (a) prostate cancer radioresistance due to the enhancement of CSC properties and (b) the tumourigenicity of breast CSCs [111, 112]. Dysregulation of the PI3K/ Akt/mTOR signalling pathway is reported to enhance the expression of chemokine (C-X-C motif) receptor 4 (CXCR4), and CXCR4-mediated STAT3 signalling is then activated, promoting the self-renewal of CSCs in non-small-cell lung cancer [113]. Furthermore, the activity of mTOR through the PI3K feedback loop is associated with the survival of prostate CSCs [114].

The mTOR signalling pathway has been regarded as a promising candidate for therapeutic modalities targeting CSCs, and several mTOR inhibitors have been approved for the treatment of cancer or undergoing clinical development (**Figure 8B**) [115]. As demonstrated by a series of *in vitro* and *in vivo* experiments, the mTOR inhibitor Everolimus can suppress the expression and phosphorylation of AKT-1, and thereby inhibit the activity of HER2-overexpressing breast CSCs [116]. Everolimus has been approved by FDA for the treatment of advanced breast cancer [117]. The mTOR inhibitor Rapamycin is shown to suppress the properties of colon CSCs [118] and inhibit the stemness of haemangioma stem cells [119]. Rapamycin is undergoing clinical trials for advanced or metastatic colorectal cancer and infantile hepatic haemangioendothelioma [120, 121]. Previous reports show the inhibitory effects of the mTOR antagonist Metformin on breast and pancreatic CSCs [122, 123]. Clinical trials of Metformin are in progress or completed for the treatment of breast and pancreatic cancer [124, 125]. Considering previous studies show that mTOR signalling plays an important role in controlling the functions of CSCs, further investigation into a link between mTOR signalling and CSCs could lead to the development of better therapeutic strategies for cancer.

#### **3.6 JAK/STAT signalling**

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway contributes to a variety of biological processes such as embryonic development, stem cell maintenance, haematopoiesis and inflammatory response [126]. It is known that the JAK family in mammals comprises four members (JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2)) and that the STAT family in mammals consists of seven members (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6) [127]. The JAK/STAT signalling pathway is initiated by the binding of cytokines to their corresponding receptors [128]. The receptors then undergo dimerisation, and the JAKs bound to the receptors come close to each other, leading to the activation of the JAKs through the interaction of tyrosine phosphorylation [128]. The activated JAKs induce the phosphorylation of the tyrosine residues of the catalytic receptor, resulting in the formation of a docking site with the SH2 domain of the STAT protein [128]. Subsequently, the STAT protein bound to the receptor is phosphorylated and dimerised, culminating in the translocation of the activated STAT to the nucleus and the transcription of the target genes [128].

Literature precedent indicates that JAK/STAT signalling plays a role in controlling the properties of CSCs [129]. It has been suggested that (a) the self-renewal and survival of breast CSCs are facilitated by the persistent activation of JAK/ STAT signalling [112], (b) IL-10-mediated JAK1/STAT1 signalling promotes the

*Therapeutic Approaches Targeting Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.108963*

self-renewal and migration of non-small-cell lung cancer and colorectal CSCs [130, 131], (c) the OCT4-activated JAK1/STAT6 pathway is associated with the functions of ovarian CSCs [132], (d) OCT4, which is a gene downstream of IL-6-mediated JAK1/ STAT6 signalling, is involved in the transformation of bulk cancer cells to CSCs in breast cancer [133] and (e) JAK2/STAT3 signalling plays a role in the regulation of the properties of breast and colorectal CSCs [134–136].

With respect to the development of therapeutic strategies to target JAK/STAT signalling for the treatment of cancer, the JAK1/2 inhibitor Ruxolitinib is shown to inhibit the functions of CSCs, leading to a decrease in the number of CSCs (**Figure 8C**) [137]. Ruxolitinib is undergoing clinical development for the treatment of solid tumours [138]. Many studies have shown that the JAK/STAT signalling pathway is correlated with the survival, self-renewal and metastasis of CSCs. Thus, more endeavours will be needed to develop novel therapeutic modalities targeting CSCs through the inhibition of JAK/STAT signalling.

#### **3.7 ROCK signalling**

The Rho-associated coiled-coil-containing protein kinase (ROCK) signalling pathway plays a pivotal role in various cellular activities such as cell survival and apoptosis [139]. It is known that there are two types of ROCK in mammals: ROCK1 and ROCK2 [139]. Diverse extracellular stimuli activate guanine nucleotide exchange factors (GEFs), leading to the conversion of Rho-GDP to Rho-GTP [140]. Rho-GTO subsequently activates ROCK1 and ROCK2, resulting in the phosphorylation of their substrates and the induction of a range of cellular responses [140].

Dysregulation of ROCK signalling is reported to be involved in the pathogenesis of a variety of diseases such as cancer [139]. According to an *in vitro* study, the properties of CSCs can be reduced by pharmacological inhibition of ROCK with the ROCK inhibitors ML7 or Y-27632, supporting the involvement of ROCK signalling in controlling the functions of CSCs [141]. With regard to the clinical development of ROCK inhibitors, the dual ROCK1/2 antagonist AT13148 is undergoing clinical trials for patients with advanced cancer [142, 143]. There is still sparce information on the roles of the ROCK signalling pathway in the regulation of the functions of CSCs. Thus, further investigation into a correlation between ROCK signalling and CSCs could be beneficial for the development of novel cancer treatments.

#### **3.8 TGF-β signalling**

The transforming growth factor β (TGF-β) signalling pathway contributes to diverse biological processes including cell proliferation and differentiation [11]. When dimeric TGF-β binds to the TGF-β receptor type-2 (TβRII), TβRII is dimerised with the TGF-β receptor type-1 (TβRI), leading to the phosphorylation and activation of the receptorregulated SMADs (R-SMADs) SMAD2 and SMAD3 [11]. Subsequently, SMAD2 and SMAD3 undergo trimerisation with the common-partner SMAD, SMAD4 [11]. The trimer migrates to the nucleus and promotes the transcription of target genes [11].

It has been suggested that TGF-β may have contradictory functions in the properties of CSCs [144]. A study using a breast cancer xenograft model indicates that the activation of TGF-β signalling can reduce the number and the self-renewal potential of breast CSCs [145]. In addition, another *in vivo* study suggests that the number of CSCs in diffuse-type gastric carcinoma can be reduced by the activation of TGF-β signalling, leading to the suppression of tumour formation [146]. In contrast, it is

reported that the activation of TGF-β signalling leads to an increase in CSC counts and the enhancement of CSC properties in various types of cancer such as breast cancer liver cancer, gastric cancer, skin cancer, glioblastoma and leukaemia [147–156]. Considering these pieces of evidence, the therapeutic targeting of the TGF-β signalling pathway might be a promising strategy to eliminate CSCs. However, more work will be required to gain a better understanding of a link between TGF-β signalling and CSCs for the development of novel therapeutic modalities.

### **4. Conclusion**

CSCs are a minor population in tumours, and their self-renewal capacity and differentiation potential contribute to tumour relapse, metastasis, chemoresistance and radioresistance. This chapter has provided a succinct summary of (a) major signalling pathways that are reported to be associated with the functions of CSCs and (b) clinical development of inhibitors targeting CSC-related signalling pathways for the purpose of encouraging research scientists (medicinal chemists, biologists, immunologists and others) to create new treatments (**Table 1**). Therapeutic targeting of CSCs *via* these signalling pathways has been considered to be a compelling strategy, and several small molecule inhibitors such as Vismodegib (GDC-0449), Sonidegib, Glasdegib (PF-04449913) and Everolimus have been approved by FDA for the treatment of cancer in the clinic. However, the development of such therapeutic interventions is challenging, and there is still vast scope for improvement. It is in part because signalling pathways interact with each other and because the CSC properties are thought to be controlled by the signalling network. AL/ML has been applied to the drug discovery, and it is reported that AL/ML is highly beneficial for target discovery, drug design and so on. These pieces of evidence can provide a scientific rational for applying AL/ML to the development of new therapeutic interventions targeting CSCs. The signal regulatory mechanisms of CSCs remain to be elucidated, and continuing studies of CSC-related pathways will lead to the creation of novel therapeutic modalities for various types of cancer.

#### **Table 1.** *Landscape of clinical development of inhibitors targeting CSC-related pathways.*

*Therapeutic Approaches Targeting Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.108963*

#### **Author details**

Shin Mukai New Wind Therapeutics L3C, Boston, United States of America

\*Address all correspondence to: shin.mukai@new-wind-ther.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine

*Igor Kizub, Andrii Rozhok and Ganna Bilousova*

#### **Abstract**

Reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) through the ectopic expression of reprogramming factors offers truly personalized cellbased therapy options for numerous human diseases. The iPSC technology also provides a platform for disease modeling and new drug discoveries. Similar to embryonic stem cells, iPSCs can give rise to any cell type in the body and are amenable to genetic correction. These properties of iPSCs allow for the development of permanent corrective therapies for many currently incurable disorders. In this chapter, we summarize recent progress in the iPSC field with a focus on potential clinical applications of these cells.

**Keywords:** cell differentiation, cell-based therapy, genetic correction, induced pluripotent stem cells, iPSCs, regenerative medicine, stem cell reprogramming

#### **1. Introduction**

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) through the ectopic expression of reprogramming factors [1–5] has had a dramatic impact on the field of regenerative medicine and has opened a new era in research and therapy. Human iPSCs represent an unprecedented source of patient-specific pluripotent stem cells suitable for disease modeling and tissue replacement therapy.

Stem cells (SCs) have the ability to self-renew through cell division and can differentiate into various cell types. Based on their origin, SCs are divided into embryonic SCs (ESCs), induced pluripotent SCs (iPSCs), and adult SCs. ESCs are pluripotent cells derived from the inner cell mass of the blastocyst. They can give rise to tissues of the three germ layers and are regarded as a renewable potent cell source for the regeneration of all bodily tissues [6]. Adult SCs are multipotent cells of adult tissues that are also essential for regenerative medicine [7]. iPSCs share many similarities with ECSs, including pluripotency, differentiation potential, and the capability to form teratomas and viable chimeras [8].

Human ESCs are isolated by the use of surplus *in vitro* fertilization embryos [9]. Therefore, unlike the iPSC technology, ESC-based techniques do not allow for the generation of genetically diverse patient-specific cells. Additionally, the use of ESCs is obstructed by the need to destroy human embryos in the process of cell isolation, which raises ethical considerations. Primary human ESCs, therefore, are a suboptimal SC source for therapy and tissue engineering. ESC-based cell therapies may also result in immune rejection, which theoretically can be avoided if autologous iPSC-derived cells are used instead.

Similar to ESCs, iPSCs can proliferate indefinitely and differentiate into all three germ layers. Thus, the iPSC technology solves many problems associated with the use of ESCs and provides an unlimited source of autologous pluripotent SCs, which can be genetically corrected, differentiated into adult lineages, and returned to the same patient as an autograft [10]. Significant advances have been achieved in recent years in improving the safety of the iPSC technology; thus, expanding the opportunities for its clinical application. However, despite the tremendous potential of iPSCs, extensive analyses of their safety and reliability are still required. This chapter discusses the current progress and prospects of using the iPSC technology in tissue replacement therapy and as a tool for studying human pathologies.

#### **2. Reprograming of somatic cells into iPSC**

The first attempts to derive pluripotent SCs from adult cells were stimulated by early experiments that demonstrated the feasibility of reprogramming adult frog somatic cell nuclei by the cytoplasm of an enucleated unfertilized frog oocyte [11]. Later findings showed that reprogramming of somatic cells back to the pluripotent state is possible by transferring somatic cell nuclei into oocytes or by fusing somatic cells with pluripotent SCs [12–14]. Finally, the successful cloning of Dolly the sheep showed the feasibility of complete reprogramming of a mammalian somatic nucleus back to a pluripotent state from which it can develop a new animal [15].

#### **2.1 Main reprograming factors**

The success of animal cloning has demonstrated that unfertilized eggs and ESCs contain a set of factors that can confer pluripotency to somatic cells. Oct4, Sox2, c-Myc, and Klf4 were later identified by Takahashi and Yamanaka as sufficient to induce pluripotency in mouse somatic cells, resulting in iPSCs that were functionally equivalent to mouse ESCs [1, 2].

OCT4 is a key transcriptional factor, which maintains pluripotency in both early embryos and ESCs [16]. The level of OCT4 expression is vital for regulating pluripotency and it can both activate or repress the promoter of the *REX1* gene, also a critical regulator of pluripotency [17]. The transcription factor SOX2 (sex determining region Y (SRY)-box2) is also essential for maintaining cell pluripotency. It comprises a regulatory complex with OCT4 and REX1 that cooperatively binds to DNA to activate transcription of other pluripotency factors [18]. The proto-oncogene c-MYC has multiple downstream targets that enhance cell proliferation, resulting in SC self-renewal [19]. The c-MYC protein can also induce global histone acetylation allowing OCT4 and SOX2 to bind to otherwise inaccessible sites [20]. Krüppel-like factor 4 (KLF4) is an oncogene that contributes to the long-term maintenance of the ESC phenotype. The role of KLF4 in cell reprogramming is probably to downregulate the expression of the tumor suppressor protein p53 [21]. KLF4 can also activate transcription by interacting with histone acetyltransferases, suppressing cell proliferation, and reciprocally acting with c-MYC [22].

The combination of OCT4, SOX2, KLF4, and c-MYC (abbreviated as OSKM), is widely used to reprogram various types of somatic cells into a pluripotent state. Under specific conditions, reprogramming can also be achieved without c-MYC or with

*Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.109274*

only one or two factors from the OSKM set [23–25]. An alternative combination of OCT4, SOX2, NANOG, and LIN28 has also been shown to be sufficient to reprogram human cells into iPSCs [5]. Additional factors can be used in combination with OSKM to enhance reprogramming efficiencies, such as LIN28, human telomerase reverse transcriptase (hTERT), and SV40 large T antigen [26–28]. Regardless of their combination, reprogramming factors remodel the epigenetic configuration of somatic cells in a way that allows for the conversion of these mature somatic cells into immature pluripotent SCs. Mechanisms of reprogramming into iPSCs are reviewed in detail by Meir and Li [29].

#### **2.2 Reprograming approaches**

Early approaches to obtain iPSCs relied on the use of integrating retro- and lentiviral vectors to deliver reprogramming factors into somatic cells [1]. However, the expression of these exogenous factors is only essential in the initial step of reprogramming, and their silencing must occur to ensure the stability of iPSCs [2]. The use of retroviral vectors can not only result in the reactivation of exogenous reprogramming factors and in turn destabilization of iPSCs [2] but also increase the risk of insertional mutagenesis and cancer transformation in iPSC-derived tissues. The development of integration-free reprogramming approaches made the production of iPSCs safer for potential clinical applications. Somatic cells have been successfully reprogrammed into iPSCs using episomal plasmids encoding the reprogramming factors and adenoviral vectors [30, 31]. A number of other non-integrating vectors of viral origins have been utilized for reprogramming, such as those based on Sendai virus [32], Epstein– Barr virus [26], and various circular plasmid constructs [33, 34]. While these methods produce integration-free iPSCs, many of these approaches suffer from extremely low efficiency, which hampers their potential clinical application [35].

The generation of iPSCs has been recently accomplished with defined chemicals that can functionally replace reprogramming factors [36]. iPSCs can also be generated by fusing reprogramming factors with cell-penetrating proteins that allow for the efficient transport of reprogramming proteins through cell membrane [37]. Despite their safety, these DNA-free approaches also suffer from low reprogramming efficiency.

A more promising approach for the transgene-free generation of iPSCs can be the use of synthetic modified mRNAs encoding the reprogramming factors. This approach has been shown to reprogram a variety of cell lines with an efficiency superior to that of other integration-free approaches [38]. The disadvantage of this method is that RNAs have to be delivered into the cells daily during the reprogramming process. MicroRNA (miRNAs), such as miR-200c, miR-291-3p, miR-294, miR-295, and miR-302a-d, have also been shown to significantly enhance the efficiency of pluripotency induction [39]. When combined with modified mRNAs encoding reprogramming factors, miR-367 and miR-302a-d have been shown to increase the reprogramming efficiency of human primary fibroblasts to an unprecedented level, and reprogramming up to 90.7% of individually plated cells [40].

#### **2.3 Reprogramming process**

Different cell types have been used for the generation of iPSCs, albeit with different reprogramming efficiencies. Fibroblasts are the most commonly used cells due to their availability and easy culture conditions. Keratinocytes, melanocytes, blood cells, hepatocytes, and gastric epithelial cells are also suitable for reprogramming [41].

The reprogramming process has been extensively studied in fibroblasts and has been shown to follow an organized sequence of events, which begins with the downregulation of somatic gene expression [42]. The first step requires a phenotype transition initiated by the activation of the early pluripotency stage-specific embryonic antigen (SSEA1) and alkaline phosphatase, and the inactivation of the differentiation-related antigen Thy-1 (CD90), followed by the activation of *NANOG* and *OCT4* [31, 42]. OCT4, SOX2, and NANOG further induce the expression of stemness genes, such as *STAT3* and *ZIC3*, and repress differentiation-associated genes [17, 43]. The expression level and balance of reprogramming factors are also important for iPSC generation. For example, the increased relative expression of OCT4 enhances reprogramming efficiency [44].

Reprogramming somatic cells often results in the generation of heterogeneous iPSCs with different molecular phenotypes and differentiation potentials [45]. The duration of the reprogramming process also affects the characteristics of the resulting iPSC. Prolonged cultivation of iPSCs yields phenotypes closer to those of ESCs as compared to cells in the early phase of reprogramming. This suggests that reprogramming continues even after iPSCs have been established [46]. Alterations in epigenetic modifications, such as DNA methylation, are also important for iPSC induction [47]. Interestingly, epigenetic profiling of iPSCs has revealed that reprogrammed cells retain epigenetic marks of the cell type of origin [48] although these marks disappear upon continued passaging [42].

#### **3. Clinical applications of iPSCs**

The rapid progress of the iPSC technology increases efforts to translate autologous iPSC-based therapies into the clinic. While only a few clinical trials have directly tested the delivery of iPSC-derived cells into patients, the technology continues to develop and researchers from virtually every field develop iPSC-based cell therapies for relevant diseases. These therapies are still at different stages of development and rely on the feasibility to derive functional somatic cell types from iPSCs. Examples of the efforts toward the development of iPSC-based therapies for a variety of tissues and organs are summarized for selected fields below.

#### **3.1 Dermatology**

The skin may represent an ideal tissue for testing novel iPSC-based therapies. It is readily accessible, highly proliferative, and can be easily monitored. Multiple skin cell lineages have been generated from iPSCs, such as keratinocytes [49], melanocytes [50], fibroblasts [51], and ectodermal precursor cells [52]. Mouse iPSC-derived keratinocytes display characteristics similar to those of primary keratinocytes and can regenerate differentiated epidermis and skin appendages when grafted together with mouse fibroblasts into athymic mice [49]. Human iPSC-derived keratinocytes have also been shown to establish functional organotypic skin in culture and 3D skin models [53]. These and other studies have demonstrated the potential of iPSCs to generate autologous donor cells for cell-based therapies for skin diseases.

As an essential step toward future clinical use, *in vitro* 3D skin equivalents have been generated using iPSC-derived components. These equivalents exhibit normal skin morphology, stratification, and terminal differentiation [54], and can potentially be used for drug screening. Skin organoids with stratified epidermal and dermal skin

#### *Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.109274*

layers that are able to spontaneously produce *de novo* hair and sebaceous glands have also been generated from murine [55] and human [56] iPSCs.

Many of the most devastating forms of inherited skin diseases that are caused by known mutations, such as Epidermolysis Bullosa (EB), can be treated with genetically corrected iPSC-derived cells [54]. EB is a group of inherited skin blistering diseases that results in severe blistering and scarring [57]. Precise gene editing techniques, such as those based on CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associate (Cas) systems, can be used for the generation of these patient-specific genetically corrected iPSCs. These corrected iPSCs can then be differentiated into skin cells and transplanted back to the same patient in need of treatment. Similar strategies can be implemented for a variety of other diseases affecting many other organs (**Figure 1**). In fact, the generation of iPSCs, coupled with gene targeting, can solve many obstacles that are associated with gene correction in somatic cells. Unlike somatic cells, iPSCs can be expanded indefinitely, allowing for easier selection and expansion of corrected clones. In addition, iPSCs derived from very old patients can differentiate into "rejuvenated" cells [58].

While currently there are no approved clinical trials to test iPSC-based therapies for the treatment of skin diseases, EB is likely to be the first skin disease to benefit from the iPSC-based therapy due to the severity of this disorder. For

#### **Figure 1.**

*Cell therapy strategies using iPSC-derived cells. Patient-specific primary cells of different origin can be isolated, cultured in vitro, reprogrammed to iPSCs, and if needed, genetically corrected. Genetically corrected or unmodified iPSC clones can be differentiated into desired cell lineages. These iPSC-derived cells can then be used for either autologous transplantation or the development of new therapeutic strategies, for example, by testing new drug modalities.*

example, Anthony Oro's team at Stanford University has received an award from the California Institute for Regenerative Medicine (CIRM) to translate an iPSC-based gene correction therapy for the severe recessive dystrophic form of EB into the clinic by producing transplantable epidermal sheets from genetically corrected EB iPSCs [59]. Dr. Oro's team is currently generating data for an investigational new drug application (IND) with the Food and Drug Administration (FDA) to initiate a clinical trial.

#### **3.2 Vascular therapy**

Endothelial cells, pericytes, and vascular smooth muscle cells have been derived from animal and human iPSCs [60, 61]. iPSCs can also be directed into cord-blood endothelial colony-forming cells that can be used to derive highly proliferative blood vessel-forming cells applicable for the restoration of endothelial function in patients with vascular diseases [62].

Coronary artery disease continues to be the leading cause of death and morbidity around the world, with the existing therapy being not always efficient. Studies have shown that transplanted iPSCs can promote angiogenesis and effective tissue revascularization [63].

In diabetes, prolonged hyperglycemia causes aberrant angiogenesis in both microand macro-vessels, resulting in deficient functionality of endothelial progenitor cells and leading to decreased neovascularization. iPSC-derived endothelial cells have been widely explored as a model to study the mechanisms and novel treatments for endothelial dysfunction in type 1 and 2 diabetes and maturity-onset diabetes of the young (MODY) [64, 65].

iPSC-derived endothelial cells have also been used to study the mechanisms of macular degeneration and ischemic retinopathies [66, 67]. iPSC-derived spinal motor neurons and cerebral microvascular endothelial cells seeded into the spinal cord lead to vascular-neural interaction with specific maturation effects of endothelial cells on the neural tissue [68].

Vascular grafts have been successfully developed from iPSC-derived cells, recapitulating the cellular composition and orientation, as well as the anti-inflammatory properties, of functional blood vessels [69, 70].

Endothelial derived from iPSCs are yet to be evaluated in clinical trials. However, these cells are now successfully used for drug testing [71].

#### **3.3 Cardiology**

The recent advances in iPSC reprogramming into cardiomyocytes and other types of cardiac cells have provided potential avenues for cardiac repair, and functional cardiomyocytes have been successfully generated from iPSCs [72]. iPSC-derived cardiomyocyte-like cells demonstrate spontaneous contractility and exhibit molecular and structural similarities to cardiomyocytes.

Many studies have focused on testing iPSC-derived cells for post-myocardial infarction repair [73], which is one of the leading causes of morbidity and death throughout the world. In patients with extensive myocardial infarction, more than a billion cardiomyocytes can be lost, overwhelming the heart's repair capacity. Such massive cell death in the myocardium initiates the replacement of cardiomyocytes with fibrous tissue, resulting in heart failure. Beating iPSC-derived cardiomyocytes have been generated from patients with hypertrophic cardiomyopathy associated

*Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.109274*

with diastolic dysfunction to study the cellular mechanisms and potential therapeutic targets of diastolic dysfunction [74].

Patient-specific iPSC-derived cardiomyocytes have also been generated from patients with different types of diabetes and used for modeling and studying the molecular mechanisms underlying diabetic cardiomyopathies [75]. Human iPSC-derived cardiomyocytes, endothelial cells, and cardiac fibroblasts have been generated and integrated in beating 3D cardiac microtissues as a platform for cardiovascular disease modeling [76].

Human iPSC lines have also been generated from patient-derived cells to study ventricular and atrial arrhythmias, which often lead to sudden cardiac death [77].

A few clinical trials to test the efficacy of iPSC-derived cardiomyocytes have been initiated. For example, an Osaka University spin-off company, Cuorips, Inc. has recently initiated a clinical trial to determine the efficacy and safety of a human allogeneic iPSC-derived cardiomyocyte sheet for ischemic cardiomyopathy patients (NCT04696328). Heartseed, Inc. is currently testing iPSC-derived cardiomyocyte spheroids in patients with severe heart failure in Phase I/II study (NCT04945018). Since both studies are still ongoing, no results have been reported.

#### **3.4 Skeletal muscle regeneration**

Converting iPSCs into skeletal muscle cells can offer a tool for *in vitro* modeling of muscular diseases and potential hope for patients afflicted with skeletal muscle dystrophic diseases. Myogenic progenitor cells have been derived from iPSCs in many studies [78, 79].

Skeletal muscle satellite cells, which drive skeletal muscle regeneration, have been shown to play an important role in the early regeneration of damaged skeletal muscles in muscular dystrophies and have been generated from human iPSCs for the identification of new therapeutic targets for the treatment of these disorders [80]. 3D functional skeletal muscle tissues have also been successfully generated from human iPSC-derived skeletal myotubes with sarcomeric structures as an *in vitro* model of contractile myofibrils for disease modeling and drug screening to study muscular and neuromuscular diseases [78].

Developing treatments for muscular dystrophy is a priority topic for researchers. Vita Therapeutics, Inc. has recently received an orphan drug designation from the FDA to initiate a clinical study that will test the efficacy of iPSC-derived myogenic stem cells to treat Duchenne muscular dystrophy [59]. More iPSC-based therapies for muscular dystrophies are currently in development [81].

#### **3.5 Neurology**

Several types of well-differentiated and functional populations of neural cells and neuronal multipotent progenitors have been generated from human and murine iPSCs. These progenitors have also been tested in cell replacement studies in rodent models with promising results [82]. Spinal neural progenitors have been differentiated from human iPSCs and together with human iPSC-derived brain microvascular endothelial cells were included in the dual-channel spinal cord chip system as an *in vitro* model of human vascularized motor neuron tissue [68]. Similarly, the blood– brain barrier chip system has been created for modeling neurological disorders and drug screening [83]. Cells in such systems can be generated from the same iPSC donor source, producing an isogenic *in vitro* model [84].

iPSCs are becoming an important source for the development of personalized therapeutic and preclinical strategies for research focusing on neurodegeneration. Parkinson's disease is one of the most common neurodegenerative disorders, resulting from the loss of dopamine neurons in substantia nigra. Cell replacement therapy, such as the transplantation of iPSC-derived neural progenitors, provides an alternative treatment strategy for Parkinson's disease (PD) [85]. An ongoing clinical trial in Japan is testing iPSC-derived dopaminergic neurons for PD [86]. In 2018, midbrain dopaminergic progenitor cells derived from autologous iPSCs were successfully transplanted into the brain of a patient with PD, and clinical symptoms improved in this patient at 18 to 24 months after implantation [85]. Aspen Neuroscience is currently developing two iPSC-based therapies for PD: an autologous iPSC-derived dopaminergic neuron therapy for idiopathic PD and an autologous gene-corrected iPSC-derived dopaminergic neuron therapy for genetic PD [87]. These and other therapies currently in development provide hope in the treatment of many neurological conditions.

#### **3.6 Hearing loss**

Hearing loss is a common impairment in humans that mainly results from the irreversible loss of sensory hair cells and auditory neurons. Patient-specific iPSCs are a promising tool for the regeneration of sensory hair cells and spiral ganglion neurons of the affected cochlea. iPSCs can be successfully reprogrammed into otic epithelial progenitors and otic neuroprogenitors that can subsequently be differentiated into inner ear hair cells [88, 89]. Functional cochlear supporting cells that can be important therapeutic targets for the treatment of hereditary deafness have also been successfully generated from mouse iPSCs [90]. Human iPSC-derived neural progenitors have been shown to innervate early postnatal cochlear hair cells *in vitro*, forming functional synapses [91].

Sensorineural hearing loss is a prevalent form of deafness, commonly arising from damage to the cochlear sensory hair cells and degeneration of the spiral ganglion neurons. iPSCs can serve as an autologous source of replacement neurons in an injured cochlea for the treatment of sensorineural hearing loss and as a model system to develop therapies to treat hereditary hearing loss [89]. While many iPSC-based treatment options are being developed in research settings, there are no approved clinical trials for hearing loss using iPSCs.

#### **3.7 Ophthalmology**

Transplantation of ocular cells derived from both autologous and allogeneic iPSCs in animal models and clinical trials showed great promise for cell-based therapies and disease modeling in ophthalmology. Using patient's own cells and the ability to correct disease-related gene mutations in patient-derived iPSCs provided a powerful approach for the treatment of ophthalmologic diseases. Various ocular cells have been generated from iPSCs, including corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells [92], conjunctival epithelial cells, and conjunctival goblet-like cells [93], retinal pigment epithelial cells [92], photoreceptors, and retinal ganglion cells among others [94]. Human iPSCs can, in an autonomous manner, recapitulate the main steps of retinal development and form 3D retinal cups containing all major retinal cell types arranged in layers via retinal progenitors [95].

iPSCs hold a promise for the treatment of various degenerative eye disorders by filling clinical gaps in the use of adult limbal SCs or ESCs [96]. iPSC-derived

*Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.109274*

photoreceptor cells and retinal pigment epithelium cells provide a cell replacement therapy for visual impairment associated with inherited retinal degeneration and age-related degeneration of photoreceptors [97].

Similar to the skin, the eye may represent an ideal tissue for testing iPSC therapies: it is relatively easy to monitor and access. Unsurprisingly, retinal pigment epithelium (RPE) cells derived from iPSCs were the first autologous iPSC-derived cell type to be transplanted into a human patient [85]. These RPE cells were used to treat age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. A 4-year follow-up demonstrated that the iPSC-derived RPE sheets transplanted into the right eye of a 77-year-old patient had survived post-engraftment. While no improvement in vision was noted, the patient's vision remained stable, emphasizing the safety of the iPSC-based therapy to treat eye diseases [98]. Additional iPSC-based therapies are being assessed for the treatment of AMD in clinical trials in Japan [99] and in the United States by the team at the National Institute of Health (NCT04339764). The use of iPSCderived photoreceptors to cure blindness is also being tested in preclinical research [59].

#### **3.8 Bone and cartilage regeneration**

While autologous bone grafting remains the main approach to reconstruct bone defects, the risk of bone resorption, infection, and insufficient amount of tissue available for transplantation is high. Therefore, iPSC technologies may provide a suitable alternative to grafting autologous bone/cartilage-forming cells. Functional osteoblasts, osteocytes, and chondrocytes have been generated from iPSCs [100, 101].

Human and animal iPSCs, as well as iPSC-derived mesenchymal stem/stromal cells (MSCs), have been differentiated into osteoblast- and osteocyte-like cells, which could be transplanted to achieve bone formation or regeneration of calvarial bone defects in *in vivo* animal models [102]. For bone tissue regeneration, engineered bioactive scaffolds provide mechanical support and components that mimic the extracellular matrix for iPSC-derived osteogenic cell grafting, increasing their adhesion, growth, and survival [103].

iPSCs also represent a potential source of viable chondroprogenitors for articular cartilage repair and engineering [104]. The regenerative potential of iPSCs is attractive for the therapy of intervertebral disc degeneration—a common cause of musculoskeletal diseases, such as low back pain, which is often attributed to a reduced number of nucleus pulposus cells that form the intervertebral disc. Current treatment strategies fail to replenish nucleus pulposus cells. The latter, however, have been successfully differentiated from iPSCs [105].

The development of iPSC-based therapies for bone and cartilage diseases is still ongoing. However, Australian Cynata Therapeutics has already tested allogeneic MSCs derived from iPSCs in a clinical trial for graft vs. host disease (GvHD) (NCT02923375). The infusion of these iPSC-derived MSCs was well tolerated by patients and promoted encouraging improvement in symptoms of GvHD [106]. Cynata therapeutics is also initiating a Phase III trial that will use iPSC-derived MSCs for the treatment of osteoarthritis [107].

#### **3.9 Dentistry**

Human iPSCs can potentially be a source to derive human odontoblasts for tissue engineering and regenerative therapy for the treatment of dental pulp damage [108]. The tooth is formed by sequential reciprocal interactions between epithelial cells

originating from mesenchymal cells and surface ectoderm cells from cranial neural crest. iPSCs can be differentiated into neural crest-like cells, which in turn can be differentiated into odontogenic mesenchymal cells, odontoblast progenitors, and odontoblasts suitable for transplantation [109].

Periodontal disease is an important health problem that ultimately leads to tooth loss. An alternative to the existing artificially manufactured tooth replacements is the generation of complete or partial biological replacements, consisting of living periodontal tissues. Animal and human iPSCs and iPSC-derived MSCs transplanted to animals with a model of molar defects demonstrate periodontal tissue regeneration. Transplanted neural crest-like cells derived from iPSCs have also been shown to form a well-organized vascularized dentin-pulp complex and calcified tooth-like structures, demonstrating the feasibility for iPSCs use in dental tissue regeneration [108]. However, extensive research is still needed to advance the iPSC technology into dental applications.

#### **3.10 Nephrology and urology**

Renal failure is one of the most common causes of mortality and morbidity in the world. An iPSC-based cell therapy may offer an alternative therapeutic approach to kidney transplantation.

iPSCs have been successfully differentiated into nephrogenic intermediate mesoderm and renal progenitor cells [110, 111]. These cells pose the ability to differentiate into multiple cell types that constitute the adult kidney, such as metanephric mesenchyme cells, metanephric stromal cells, nephric duct, ureteric bud cells, proximal tubular cells, mature glomerular podocytes, and other types of cells capable of forming renal tubule-like structures [110]. iPSC-derived kidney organoids have been created containing multiple cell types and mimicking nephrogenesis that have a potential for regenerative medicine and personalized therapy [112]. iPSC-derived cells are also used to recellularize decellularized kidney scaffolds as an approach to bioengineering human replacement kidneys [113]. iPSCs generated from patients with specific kidney disorders have also been used in disease modeling [114]. The iPSC technology is now widely used to model kidney diseases and perform drug screening. However, no clinical trials have been approved to date.

#### **3.11 Pulmonology**

Lung transplantation remains the only treatment for many severe lung diseases. The use of iPSCs may be an effective strategy for developing patient-specific cells for lung cell therapy and lung tissue engineering as an alternative to whole-organ transplantation. iPSC-based models of lung diseases can also help to better understand lung pathologies and identify new therapeutic approaches.

Human alveolar and airway epithelial and basal cells have been successfully generated from iPSCs [115, 116]. Airway basal cells, in particular, can give rise to other airway lineages, such as secretory and ciliated cells, and can restore airway functionality [117].

Human iPSC-derived type II alveolar epithelial cells are capable of repopulating acellular lung matrices prepared from rat and human adult lungs, adhering and proliferating to form alveolar structures as a 3D lung tissue model of the distal lung regions [118]. Recently, scaffold-free structures for airway regeneration were also created using 3D bioprinting and a combination of human native chondrocytes, MSCs, and iPSC-derived endothelial cells [119].

Thus, significant progress has been achieved in deriving alveolar epithelial cells from iPSCs. However the complexity of lung tissue prevents rapid development and clinical translation of iPSC-based therapies for lung diseases.

#### **3.12 Hepatology**

Currently, liver transplants represent the only way to treat patients suffering from terminal liver failure. However, liver transplantation is associated with numerous problems, such as graft failure. As an alternative to the donor liver, human iPSCs may provide a promising source of hepatocytes for autologous cell therapy. iPSCs have been differentiated into hepatocytes [120]. Since the function of hepatocytes depends on their position in the liver globule, methods have been developed to generate iPSCderived hepatocytes with zone-specific hepatic properties [121].

In addition to hepatocytes, the liver parenchyma also consists of many types of nonparenchymal cells, which are essential for maintaining hepatic metabolic activity and other functions. Human iPSC-derived hepatic progenitors have been differentiated into multiple liver cell types and produce functional liver models [122]. *In vitro* liver models are crucial for the study of liver diseases and development of effective therapies. Since liver transplantation is contingent on organ availability and other constraints, transplantable iPSC-derived cells and vascularized 3D organoids capable of repopulating and restoring liver functions have been developed as an alternative [123].

Hepatocyte transplantation is one of the most attractive approaches for the treatment of liver failure [124], and patient-specific iPSC-derived hepatic cells are expected to be used for patient-specific transplantations. The transplantation of hepatocyte-like cells differentiated from genetically corrected iPSCs has also been shown to ameliorate inherited liver diseases in a mouse model [125]. While iPSCderived hepatocytes have not yet been translated into clinical trials, these cells are currently being used for screening drug hepatoxicity [126].

#### **3.13 Gastroenterology**

The gastrointestinal tract is one of the largest and most active systems, which not only breaks down and absorbs macromolecules from the lumen but also functions as an endocrine organ that regulates digestion and metabolism. The gastrointestinal system requires integrated neuronal, lymphatic, immune, and vascular tissues to function properly. The iPSC technology provides unique opportunities for modeling human diseases and novel therapeutic approaches in regenerative gastroenterology. Human iPSCderived intestinal and gastric cells, as well as generated *in vitro* human organoids, may facilitate drug screening and modeling of gastrointestinal diseases. On the other hand, intestinal cell models can be widely used to study drug absorption and metabolism.

iPSCs can be differentiated into various types of intestinal cells and can even form multicell type intestinal tissue [127]. Among other cell types, human iPSCs have been differentiated into gastric epithelial cells and acid-secreting parietal cells [128], mature exocrine pancreatic cells [129], as well as directed along the gastric endocrine cell fate path [127]. Human iPSCs can also be efficiently differentiated into neural crest SCs and various subtypes of mature enteric neurons [130]. Enterocytes derived from human iPSCs have been used as a model system for predicting the pharmacokinetics of the human intestine and drug absorption and metabolism [131, 132].

The developed cell culture protocols allow for the derivation of self-organizing multicellular intestinal organoids from iPSCs, which resemble *in vivo* intestinal crypts [133]. Such organoids, which are composed of various intestinal cells, represent a physiologically relevant *in vitro* model for basic studies of intestinal development and pathophysiology, as well as a tool in personalized regenerative medicine and drug development. Bioengineering the intestine on a vascularized native scaffold can also be a promising approach for intestinal regeneration in patients with intestinal failure [134]. Intestinal 3D organoids derived from human primary digestive samples are currently being tested in a clinical trial to treat ulcerative colitis at Rennes University Hospitals (NCT05294107). The successful completion of this study will pave way for approval to use iPSC-derived intestinal organoids for the treatment of intestinal diseases in clinical trials.

#### **3.14 Metabolic disorders**

Several major types of diabetes are caused by the destruction and decrease in the number of functional insulin-producing β-cells. Therefore, the generation of functional insulin-secreting pancreatic β-cells represents an important goal for the treatment of various types of diabetes. Functional insulin-secreting pancreatic β-cells have been successfully generated from healthy human iPSCs [135], providing an important cell source for personalized drug screening and cell transplantation therapy in diabetes. Human iPSC-derived β-cells exhibit many of the properties of functional pancreatic β-cells, such as expression of specific transcription factors and the presence of mature endocrine secretory granules [136].

Brown adipocytes are promising cell targets for the treatment of obesity and type 2 diabetes due to their ability to actively drain and oxidize circulating glucose and triglycerides, which can prevent hyperglycemia and hypertriglyceridemia. Because of the scarcity of brown adipocytes in adults, iPSCs may be an important potential source of these cells and their progenitors for transplantation. Human iPSCs have been successfully differentiated into adipocytes [137].

MODY is a monogenic autosomal dominant disease caused by a mutation in one of the specific genes. Various mutations in each of these genes affect pancreatic β-cells, resulting in their dysfunction and diabetes development. However, despite extensive research, the mechanism by which the mutant MODY gene results in monogenic diabetes is not yet clear [138]. iPSCs have been generated from patients with different types of MODY to establish a human-based model for studying the molecular manifestations and mechanisms of these diseases [139].

iPSCs harboring disease-specific gene mutations have also been generated from somatic cells of patients with a few types of lipodystrophy and other inherited metabolic disorders for studying the pathogenesis of these diseases [140, 141].

While iPSC-based therapies for metabolic diseases are still being developed, the progress achieved by Melton's group in using human ESCs to derive functional allogeneic insulin-producing β-cells [142] has created a strong foundation for the use of iPSCs in treating type 1 diabetes. Vertex Pharmaceuticals, Inc. is currently testing the glucoseresponsive allogeneic β-cells generated from human ESCs in combination with immunosuppressive therapy in Phase I clinical trial (NCT04786262). The preliminary results of this clinical trial show engraftment and functionality of implanted hESC-derived β-cells [143].

#### **3.15 Gland regeneration**

Hypofunction of salivary glands causes various life-disrupting effects. With no satisfactory therapy available, the therapeutic and regenerative potential of iPSCs has been explored for the treatment of salivary gland dysfunction [144].

*Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.109274*

Inflammatory and degenerative changes in the lacrimal gland often lead to the development of severe dry eye syndrome, a complex disease resulting in visual acuity disruption. Currently, only palliative treatments for this disease exist. iPSCs have been used for developing a therapy for lacrimal gland tissue injuries [145].

The thymus plays a significant role in the establishment of immunological selftolerance and is required for the generation of T cell-mediated immunity. Thyroid progenitors, thymic epithelial cells, and thymic organoids derived from iPSCs can completely regenerate the thymus *in vivo* and demonstrate the potential for regenerative therapy in patients with immunodeficiency and hypothyroidism [146]. Despite the progress in generating iPSC-derived thymic cells, a poor understanding of thymus biology impedes the clinical translation of these cells.

The mammary gland is a primary target for carcinogenesis, and regenerative therapy for damaged mammary gland tissues is the best way to restore breast functions. iPSCs have been successfully reprogrammed into mammary SCs [147]. In addition, human mammary-like organoids have been produced from iPSCs. Such organoids have been shown to regenerate mammary glands upon transplantation [148]. To date, no clinical trials have been approved for the use of iPSCs in gland regeneration.

#### **4. Conclusion**

Despite tremendous progress achieved in the iPSC field, broad applications of iPSC-based therapies will take time to establish. Nevertheless, considerable advances have been made in deriving iPSCs from patients, differentiating them into tissues of interest, and using them as a platform for studying the mechanisms of diseases. The development of iPSC-based therapies is just emerging and only a limited number of clinical studies using the transplantation of iPSC-derived cells have been initiated to date. However, clinical studies related to the iPSC technology are not limited to the studies described above. Other studies worth mentioning include the successful use of autologous iPSC-derived platelets for the treatment of aplastic anemia [149] and the derivation of an off-the-shelf allogeneic chimeric antigen receptor (CAR) T-cell therapy targeting B-cell malignancies developed by Fate Therapeutics (NCT04629729). An iPSC-derived, off-the-shelf, CAR natural killer (NK) cell therapy is currently being tested in a Phase I clinic trial for refractory B-cell lymphoma (NCT 04245722) and is showing promising therapeutic efficacy [150]. Other studies have been reviewed by Kim et al. [99].

Even though iPSCs hold great potential in the field of regenerative medicine and personalized medicine, a number of challenges hinder the widespread clinical applications of these cells. These challenges include the safety of methodologies for the generation, gene correction, and differentiation of iPSCs and the high cost associated with the technology.

The first major challenge is reprogramming and gene correction methods that are known to introduce undesired genetic modifications into the patient genome. Other limitations include the heterogeneity of iPSC lines that impairs the consistency of differentiation during manufacturing of iPSC-based cell products. Establishing selection criteria for iPSC-derived cells, such as cell-specific markers, proliferation rate, lifespan, and genomic analyses, help minimize the variability of iPSC-derived cells. Just like ESCs, iPSCs are also predisposed to forming teratomas when undifferentiated. The available cell purification technologies often do not guarantee the complete eradication of undifferentiated iPSCs during manufacturing. Although many problems concerning the clinical applications of iPSCs still remain, iPSC-based therapies have a tremendous therapeutic potential for many diseases that are difficult to treat otherwise.

### **Acknowledgements**

We are grateful for funding support from the National Institutes of Health (R21 AR074642 and U01AR075932). We also thank Epidermolysis Bullosa (EB) Research Partnership, the EB Medical Research Foundation, the Cure EB Charity, Dystrophic Epidermolysis Bullosa Research Association (DEBRA) International, and the Gates Frontiers Fund.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Igor Kizub, Andrii Rozhok and Ganna Bilousova\* Department of Dermatology, Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA

\*Address all correspondence to: ganna.bilousova@cuanschutz.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.109274*

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#### **Chapter 14**

## Stem Cell Therapy and Its Products Such as Exosomes: Modern Regenerative Medicine Approach

*Leila Dehghani, Amir Hossein Kheirkhah, Arsalan Jalili, Arman Saadati Partan, Habib Nikukar and Fatemeh Sadeghian-Nodoushan*

#### **Abstract**

Regenerative Medicine is a developing and multidisciplinary field of science that uses tissue engineering, biology, and cell or cell-free therapy to regenerate cells, tissues, and organs to restore their impaired or lost function. Regenerative medicine uses a new element linked to stem cells, which call exosomes, introduces it to the healthcare market. Exosomes are present in almost all body fluids, such as synovial fluid and blood. Exosomes and microvesicles are very efficient mediators of cell-to-cell communication by transferring their specific cargo to recipient cells. Furthermore, the modification of extracellular vesicles is possible that can become an excellent choice for drug delivery systems and vaccines. Isolation of exosomes for their use as therapeutic, research, or diagnostic agents for a specific type of disease is of particular importance. Five techniques have been used to isolate exosomes from different sources, including ultracentrifugationbased, size-based, immunoassay, exosome sedimentation, and microfluidic techniques. The use of exosomes in medicine has many applications, including in Bone and cartilage, dental, immune system, liver, kidney, skeletal muscle, nervous, heart systems, skin and wound, microbial and infectious, and also in cancers. This chapter focuses on stem cells, especially exosomes, as novel approaches in disease treatment and regenerative medicine.

**Keywords:** stem cell therapy, exosome, regenerative medicine, disease treatment, organ

#### **1. Introduction**

Regenerative medicine is a developing and multidisciplinary field of science that uses tissue engineering, biology, surgery, and cell or cell-free therapy to repair and regenerate cells, tissues, and organs to restore their impaired or lost function [18]. Cellbased treatment methods, especially stem cell therapy have been recognized for many years as the main methods in regenerative medicine for their characteristics, such as easy isolation, self-renewal, multidirectional differentiation, immunomodulatory function, and stimulation of tissue regeneration. During the last decade, it was discovered that most of the therapeutic effect of Stem cells is due to different paracrine factors such as exosomes. So consequently, cell-free treatment was introduced as a novel approach in regenerative medicine. Although exosomes do not have cell therapy-associated complications such as tumor formation, transplant rejection in the host, and the formation of ectopic tissue, stem cells can differentiate into different types of tissues, which is their main advantage over paracrine factors. This section focuses on stem cells, especially exosomes, as novel approaches in disease treatment and regenerative medicine.

#### **2. Stem cells in clinic, advantages, limitations**

Stem cells (SCs) have been extensively known for their reparative actions. There is enormous global anticipation for stem cell-based therapies that are safe and effective. Numerous pre-clinical studies represent encouraging results on the therapeutic potential of different stem cell types, such as tissue-derived stem cells.

SCs are classified into two broad categories according to their differentiation capacity and tissue of origin. Based on stem cell hierarchy, SCs are classified into totipotent, pluripotent, multipotent, or unipotent cells, depending on their cluster of differentiation [1].

If we want to compare adult stem cells, embryonic stem cells, and induced pluripotent stem cells, the positive points of adult stem cells are the ability of transdifferentiation and reprogramming of these cells which is possible but is not well studied, being less likely to be rejected if used in transplants, and successful results have already been demonstrated in various clinical applications. On the other hand, there are some concerns about them such as limitations on the differentiation ability of ASCs which is still uncertain; they are currently thought to be multi or unipotent, being not able to grow for long periods of time in culture, usually, a very small number in each tissue making them difficult to find and purify. There is no technology available to generate large quantities of stem cells in culture, and no major ethical concerns have been raised [2].

Embryonic stem cells possess remarkable properties, including their ability to maintain and grow in culture for extended periods of one year or more. Wellestablished protocols for their culture maintenance are available, making them a promising research subject. With their pluripotency, they can generate a wide range of cell types, making them an important tool for understanding the process of development. By further studying embryonic stem cells, we can gain a deeper understanding of developmental processes. Also, there are some limitations, such as the inefficient process to generate ESC lines, being unsure whether they would be rejected if used in transplants, therapies using ESC avenues are mainly new, and much more research and testing are needed. Also if they are used directly from the ESC undifferentiated culture prep for tissue transplants, they can cause tumors (teratomas) or cancer development, and finally, the ethical concerns as the embryo is destroyed to acquire the inner cell mass, and the risks for female donors [3].

Also, positive points about induced pluripotent stem cells are that abundant somatic cells of the donor can be used for therapeutic approaches, concerns about histocompatibility mismatch are avoided, they are beneficial for drug development and developmental studies, information learned from the "reprogramming" process may be transferable for *in vivo* therapies to reprogram damaged or diseased cells/tissues.

Furthermore, there are some limitations such as methods for reproducibility and maintenance, differentiated tissues are not specific, viruses are currently used to introduce embryonic genes and have been shown to cause cancers in mouse studies.

#### *Stem Cell Therapy and Its Products Such as Exosomes: Modern Regenerative Medicine Approach DOI: http://dx.doi.org/10.5772/intechopen.111574*

Moreover, as an ethical concern, it should be noted that iPS cells can become embryos if exposed to the right conditions [4].

Although stem cell transplantation has good efficacy, weak immunogenic potential, and high multi-potential differentiation, there are some concerns about that, such as safety considerations in terms of tumorigenicity and transmission of infection, tight regulations, short shelf life, and high cost associated with strict production, transport, and storage conditions. To overcome these challenges, CM's induction of SCs in their native niche to stimulate the regeneration process is a promising cell-free approach [5]. Despite the advantages of this method, such as Immuno-compatibility, improved safety compared with stem cell transplantation, and feasibility of mass production, some limitations should be considered, such as limited therapeutic efficacy due to low concentration of paracrine factors, difficulty in obtaining the CM with a consistent composition, short half-lives of paracrine factors, and requiring frequent administrations with large doses [6]. Stromal Vascular Fraction (SVF) treatment efficacy likely depends upon several patient and treatment-specific characteristics, including the severity and cause of hair loss, treatment frequency, preparation methods, and adjunctive therapies [7]. Literature reviews propose that all kinds of cell types will have the therapeutic application with the potency of regenerative therapy. Different types of cells include many diagnostic and therapy factors. Differential potency of SC such a neurogenesis, synaptogenesis, vasculogenesis, myogenesis, oligodendrogenesis, axonal connectivity, myelin formation, etc.

Although the exact mechanisms by which SCs perform are still unidentified, recent documents have proposed they might be associated with their contents, such as exosomes [8]. Extracellular vesicles (EV) are lipid bilayer-enclosed and small (40–1000 nm) vesicles secreted into biological fluids. EVs are highly heterogeneous in the context of contents, size, and membrane composition, depending on the source of origin. So far, three main categories of EVs have been identified, including exosomes, apoptotic bodies, and cellular microparticles/microvesicles/ ectosomes [8]. EVs have been identified as vital components in intercellular communication and information transfer to other cells, affecting both the recipient and parent cells' physiological and pathological functions. Also, the roles of EVs in cancer and autoimmune disease have been suggested in some research [9].

#### **3. Exosomes: properties and applications**

Extracellular vesicles (EVs) are small lipid particles secreted from all human cell types, both healthy and malignant. They can be released either directly from the plasma membrane or upon fusion among multivesicular bodies (MVBs) and the plasma membrane. Based on their size, origin, and cargo heterogeneity (i.e., DNA, proteins, various types of RNAs), EVs have been classified into several groups, such as exosomes, microvesicles, apoptotic bodies, and other vesicle types [10].

Two scientists named Pan and Johnston first defined intercellular communication by exosomes in 1983. They discovered that during the maturation of sheep reticulocytes into erythrocytes, transferrin receptors were enclosed in nanovesicles of endosomal origin. During these years, scientists considered the term exosome for these nanovesicles, which are between 30 and 150 nm [11].

Exosomes are present in almost all body fluids, such as synovial fluid and blood. Exosomes and microvesicles are very efficient mediators of cell-to-cell communication by transferring their specific cargo to recipient cells. For example, exosomes are involved in the delivery of genetic materials causing epigenetic modifications in the target cells, antigen transfer to dendritic cells (DCs) for cross-presentation to T cells, extracellular matrix remodeling, and several signaling pathways [12, 13].

The most crucial feature of EVS, including exosomes, is to be loaded as delivery systems and vaccines because it can be easily loaded with different molecules, such as drugs, antibodies, miRNAs, and siRNAs, especially in anti-tumor treatments, resulting in more specific and efficient systems compared to the carried molecules alone. Furthermore, the modification of EVs is possible that can become an excellent choice for drug delivery systems and vaccines [14].

Exosomes enter the cell through pinocytosis, endocytosis, or direct fusion with the plasma membrane. Today, stem cells are used significantly in regenerative medicine treatment protocols due to their ability and capacity to differentiate into various cell lines. It has also been proven that the ability to heal and regenerate stem cells is due to exosomes secreted from them, which act in paracrine [15, 16]. Similar to cancer, exosomes act as a double-edged sword due to their ability to carry and deliver molecules to target cells in infectious diseases. Exosomes play a crucial role in the pathogenesis of infection but also trigger immune responses to confer protection against pathogens [17]. In general, the advantages of using exosomes in regenerative medicine include the following:


#### **4. Structure and biogenesis**

The biogenesis of exosomes starts with endocytosis, and the cargo enters the primary endosome membrane by budding. Endocytosis can be dependent or independent of clathrin protein. The early endosome enters the late endosome phase, which has a spherical shape and is located close to the nucleus [18]. The budding of the cargo into the lumen of the endosome causes the formation of intraluminal vesicles (ILVs) with sizes of 30–150 nm, called Multivesicle bodies (MVBs). These multivesicular bodies (MVBs) may fuse with the cell membrane and release their intraluminal vesicles (ILVs), which are exosomes, outside the cell, or they may fuse with the lysosomal membrane to degrade their contents [19]. The membrane components of exosomes that have been identified so far include: lipid rafts containing sphingomyelin, cholesterol, ceramide, phosphatidylserine, and more than 4000 exosomal proteins [20].

Common proteins in all exosomes include transfer proteins such as annexin, Rab GTPase, proteins related to the biogenesis of exosomes such as Alix, TSG101, actin, myosin, and cofilin, as well as tetraspanins such as CD9, CD63, CD81, CD82, CD151, and MHC classes one and two [21, 22]. Sometimes, on the membrane of exosomes, there

are glycoproteins related to targeting lysosomes called Lamp1 or Lamp2, as well as integrins and heat shock proteins such as HSP90 and HSP70. Usually, integrins and tetraspanins play the roles of adhesion and targeting [23, 24].

#### **5. Separation methods**

Isolation of exosomes for their therapeutic use, research, or diagnostic agents for a specific type of disease is of particular importance. With the rapid progress of science and technology, techniques for isolating exosomes in a high-quality and highpurity form have been expanded in large quantities. Each technique uses a particular feature of exosomes, such as size, shape, or surface proteins, for their separation. Five techniques have been used to isolate exosomes from different sources, including ultracentrifugation-based, size-based, immunoassay, exosome sedimentation, and microfluidic techniques. For the investigation of the quality of isolated exosomes, several optical and non-optical techniques have been developed to check the size, shape, and quantity of chemical components [25, 26].

*Ultracentrifuge*: There are usually two types of ultracentrifuge: analytical and preliminary. Analytical ultracentrifuge is used to research the physicochemical properties of particles and molecular interactions of polymeric materials [27]. The preliminary ultracentrifuge is used as the gold standard for the isolation of exosomes because its use is simple and does not require particular expertise. Of course, it is fast and cheap. Separation of exosomes by the type of differential usually includes a series of centrifugation cycles with different centrifugal forces, the duration of which is different from other components based on density and size [28]. A purification step is performed at the beginning of the separation of exosomes from human plasma and serum to get rid of large biological particles. Of course, protease inhibitors are added to the sample to prevent the destruction of exosome membrane proteins [29, 30]. There are two types of density gradient ultracentrifuge: isopycnic and moving zone. The use of density gradient ultracentrifuge to separate extracellular vesicles such as exosomes has received much attention. In the density gradient ultracentrifuge, different densities of the substance are created in the tube, which usually decreases from the bottom to the top, so the exosomes with different densities are placed in a different part of the tubes based on the force exerted on them during the centrifugation [31, 32]. In moving zone ultracentrifuge, samples containing exosomes are placed in a narrow area above the density gradient of the environment, which has a lower density than any of the substances dissolved in the sample, unlike isopycnic ultracentrifuge, which separates only based on density [33, 34].

*Based on size*: Ultrafiltration is one of the most popular methods of separating exosomes based on size [35]. Methods such as western blotting or electron microscopic methods are used to confirm the successful isolation of exosomes. For cell-free samples such as urine, serum, spinal fluid, and cell fluid culture media, kits based on filtration separation have been developed [27, 36]. Sequential filtration is performed to separate exosomes from the solution on the cell culture medium. Initially, filtration with 100 nm filters is used to separate floating cells, and large cell debris, but components with a size larger than 100 nm but flexible are possible [37]. In this method, in the chromatography column, the stationary phase is a porous substance and is used to separate particles and macromolecules that are smaller in size than exosomes. These substances enter the pores, and when washing the column, the exosomes are separated earlier, which are finally separated by western blotting of the isolated exosomes [38].

*Immunoaffinity capture*: For example, the Enzyme-linked immunosorbent assay or ELISA method is usually used to isolate exosomes in body fluids such as serum or urine. The results of absorption values indicate surface biomarkers produced on the membrane of exosomes. In another method, the surface characteristics of exosomes are evaluated by immunoprecipitation methods based on microplates by ultracentrifugation. This method's more accurate results are obtained with a smaller sample, which shows its superiority over the ultracentrifugation method [28]. It also depends on the quality of the exosomes and the environment in which they are located [35, 39]. Examples of these diagnostic kits are used to isolate exosomes from the plasma of acute myeloid leukemia (AML) patients that contain abundant amounts of CD34 on their membrane, and separation is done by magnetic beads coated with the target antibody of this marker [28, 40].

To increase the capacity of immunoaffinity capture, the mass spectrometry method is used along with the immunoassay method. For exosome isolation, antibodies on highly porous silica micropipettes that are integrally immobilized are used to isolate CD9-containing exosomes. Another example demonstrating the combination of immunoaffinity trapping with other exosome isolation methods is a magnetically activated cell sorter that uses epithelial cell adhesion molecules to purify isolated tumor exosomes. It uses plasma samples of lung cancer using the method (SEC) [41, 42].

*Based on sedimentation*: Exosomes can be precipitated in biological fluids by changing the solutions containing exosomes or by creating a particular type of dispersion in these solutions. For this purpose, special polymers like polyethylene glycol (PEG) can be used [27]. Typically, the incubation of samples with precipitating substances such as PEG, which have a molecular weight of up to 8000 D, for one night at 4°C causes exosomes to precipitate at a low speed by centrifugation or filtration. This method is an easy separation method that does not require special tools [43]. Several exosome precipitation kits have been commercially developed and available that isolate exosomes from body fluids such as plasma, serum, urine, and spinal fluid. The urinary exosomes isolated with these kits have been proven to be quantitatively higher than in ultracentrifugation [44]. The main weakness of the polymers' exosome deposition method is co-precipitation with other non-exosome components, such as proteins and other substances [28].

*Separation based on microfluidics*: Rapid advances in microfluidic technology have led to the development and manufacture of devices for the fast and highyield separation of exosomes; applying such devices savings the use of materials, reagents, and time. An example of such a device is an acoustic nano-filter that uses ultrasound waves to separate the constituents of a sample based on size and density. Larger particles experience more wave pressure and move faster toward the pressure nodes embedded in the device [45]. To take advantage of the size difference between exosomes and other extracellular vesicles, Wang and colleagues have tailored porous nanowire structures on the micropillars of a microfluidic device, which preferentially transport exosomes between 40 nm and 400 nm in diameter. Proteins and other cellular components are filtered out, and the entrapped exosomes are isolated by dissolving the porous silicon nanowires in PBS buffer [46]. To increase the specificity and introduction of exosomes, Chen and his colleagues tried for the first time to integrate the immunoaffinity trapping technique with the microfluidic chip, which is similar to the immunoassay methods of exosome isolation based on the specific interaction between exosome membrane proteins and their proven antibodies on a chip. Based on this method, commercial Exochips have been designed, as shown in **Table 1**, to isolate exosomes. These exochips are immunochips with anti-CD63 function present on the membrane of most exosomes, and a special fluorescent dye called carbocyanine (Dio) stain exosomes are also used in these special chips [47].

*Stem Cell Therapy and Its Products Such as Exosomes: Modern Regenerative Medicine Approach DOI: http://dx.doi.org/10.5772/intechopen.111574*

#### **6. Application of exosomes in regenerative medicine**

The use of exosomes in Medicine has many applications, including in drug delivery to transfer a specific drug or therapeutic molecule, as well as in regenerative medicine and cell therapy, because nanovesicles are made of cell membranes, which are effective due to their genuineness. They have high and typical safety, and also their presence in all body fluids makes them reliable candidates for diagnosing methods (**Figure 1**).

The role of exosomes in diagnosing and treating various diseases lies in their function as carriers of intracellular communication signals. Recent exosome investigations


#### **Table 1.**

*Summery of exosomes separation methods.*

**Figure 1.** *Exosomes and its application in regenerative medicine.*

focused on exosomes derived from humans and plants. Based on registered clinical trials (https://clinicaltrials.gov/), exosomes are used in different issues as biomarkers for cancer diagnosis like, lung, breast, colorectal, thyroid, exosome-therapy, drug delivery, and vaccines. In the case of exosome therapy, 35 studies have been registered which 22 clinical trials used MSCs-derived exosomes and most of them have been used in infectious diseases. Although some stem cell therapies have been approved for blood and immune diseases, but there is no approval for exosome therapy and more clinical trials are needed, yet.

#### **7. Bone and cartilage tissue**

Clinical studies on laboratory animals have shown the therapeutic effect of mesenchymal stem cells (MSCs) in healing cartilage injuries. The reason for these therapeutic effects is the secretion of various exosomes by these cells. It has been found that intra-articular injection of exosomes derived from mesenchymal stem cells improved osteoporotic defects in model rats. Most articular cartilages have a limited capacity to heal after injury. As challenging surgeries should be done to replace the joint, there is always a fear of rejection of the graft in this treatment [48, 49]. Recently, mesenchymal stem cells have been used to treat these defects. However, using mature mesenchymal stem cells as multipotent stem cells may not be able to optimally repair these damages because, usually, with the increasing age of the donor cells, the ability of these cells to self-regenerate and multiply and differentiate decreases dramatically [50].

Another theory suggests using pluripotent stem cells as permanent sources in cell therapy. Although these cells can differentiate into various cell lines clinically, they may cause tumors and teratoma, and they cannot be considered a reliable treatment source. So an alternative method is to use exosomes derived from these types of cells [51].

In a study by Zang et al., osteoporotic defects were created in two groups of model mice, one as a sample and the other as a control of the same size. For 8 weeks, 100 μL

#### *Stem Cell Therapy and Its Products Such as Exosomes: Modern Regenerative Medicine Approach DOI: http://dx.doi.org/10.5772/intechopen.111574*

of exosomes derived from mesenchymal stem cells were injected intra-articularly into the PBS control group and the sample group (the environmental and nutritional conditions of both groups were the same). To observe the progress and effectiveness of the treatment, tissue evaluation was performed. It was observed that four cartilage defects were completely healed within 6 weeks, and hyaline and collagen type 2 were created entirely. In one case, tissue repair was done by fibrosis [48]. In the field of bone regeneration, most of the attention is on exosomes derived from mesenchymal stem cells due to their ability to influence and interact with the bone microenvironment at different levels of regeneration.

At different levels of regeneration, cells such as osteoblasts, osteoclasts, osteocytes, chondrocytes, and endothelial cells are associated with different repair mechanisms [52]. Exosomes derived from MSCs can generally distinguish osteoblasts by transmitting various miRNA cargoes. Exosomes produced by stimulated cells can connect to the extracellular matrix and induce the distinction of osteoblasts through regional interaction with them. In addition, considering the importance of the interaction between osteoclasts and osteoblasts in bone homeostasis, specific targeting molecules to inhibit or induce osteogenesis, such as miR-214-3p, which is secreted from osteoclasts, are of great interest [48, 53]. Another study identified the pathways by which miRNAs in exosomes regulate osteoblastic differentiation.

WNT and PI3K/AKT pathways directly affect the induction of bone formation. One of the essential miRNAs in this field is miR-27A-3P, which affects different signaling pathways in bone, such as TGFβ, BMP, and WNT, which influence proliferation genes involved in bone distinction, such as STAB2, DLX2, OSX, and Runx2 [52, 54]. According to various research, the bone regeneration capacity of exosomes derived from MSCs depends on the type of tissue from which they are isolated. For example, exosomes derived from MSCs isolated from human adipose tissue can increase the speed of healing and regeneration of bone defects [55].

#### **8. Dental tissue**

Recently, studies have been conducted on miR133b derived from certain exosomes of dental dentin cells, which regulate apoptosis in tooth development. This miRNA induces apoptosis in the primary mesenchyme of the dental tissue's upper part and causes the dental tissue's proper formation in the laboratory environment. Such studies highlight exosomes' critical role in signaling growth, differentiation, and regeneration of oral, facial, and cranial tissue [56]. Exosomes derived from adipose tissue stem cells have tremendous healing effects in treating oral and dental diseases. They have opened a clear horizon for dental treatments without the need for surgery. Exosomes may be isolated from mesenchymal stem cells of dental tissues, including dental pulp stem cells (DPSCs). These are multipotent cells that can directly contribute to the regeneration of dental pulp, bone, muscle, nerve, blood vessels, and even the liver [57, 58]. DPCS-derived exosomes play an important role in regenerative medicine. Research has confirmed that these exosomes in primary cell culture and animal models have a modulatory role and support the immune system and anti-apoptotic activity, similar to MSC-derived. They have the unique ability to regenerate dental pulps. Periodontal ligament cells (PDLCs), known as old sources of multipotent stem cells, are other cells in tooth regeneration. One of these cells' unique features is maintaining the ability of self-regeneration when transplanted [59]. Although extensive research has not been done on the effects of exosomes derived from dental ligament cells, there are shreds of evidence about the modulating properties of these cells. In addition, it has been found that exposure of PDLCs to lipopolysaccharide (LPS) produces exosomes that can induce polarity in proinflammatory macrophages [60, 61].

#### **9. Modulation of the immune system**

The ability of exosomes to modulate the immune system, and increase or inhibit inflammation, has introduced them as an attractive choice as therapeutic agents. Exosomes can transport different antigens, load on MHC class I and II complexes, and stimulate immune response through epitope presentation by cell-presented antigens [62, 63]. Exosomes derived from dendritic cells (DC) loaded with viral antigens can activate TCD8+ cells. Exosomes secreted from cells infected by bacterial and viral antigens can stimulate the release of macrophages and determine the activity of T cells. Similarly, after being made inside dendritic cells, cancer-specific epitopes can stimulate the activity of cytotoxic T cells against cancer cell antigens [64]. Exosomes derived from regulatory T cells (Treg) play the role of modulating and sometimes suppressing the immune system. TCD4+, CD25+, and Foxp3+ cells can activate TCRs or receptors on T cells. Exosomes produced by this type of T cells are quantitatively more than other T cells. Treg-derived exosomes can reduce the release of inflammatory cytokines such as IL2 and IFNγ [65]. The suppressive nature of Treg exosomes has been attributed to CD73 ectoenzymes, and the loss of CD73 in Treg exosomes reverses its natural suppressive property. It has been found that the contents of exosomes that move between Treg cells and T effector cells (Teffs) contain miRNAs such as Let-7d, Let-7b, and miR155, which indicate the modulating and inhibitory function of these exosomes [65, 66]. It has been reported that using exosomes derived from cancer cells as a vaccine for chronic myeloid leukemia (CML) patients increases the power of cytotoxic T cells against CML cells. In addition, exosomes derived from mesenchymal stem cells present cancer epitopes on their membrane, which can stimulate the activity of antibody secretion by B cells and Th1 memory cells [67]. Exosomes can be used to transfer molecules that modulate the immune system, such as vitamin D derivatives, which play a role in regulating the immune system in osteoporosis, or as vectors for gene transfer of anti-inflammatory molecules to reduce the damage caused by osteoporosis. Immunotherapy based on exosomes has numerous advantages over cellular immunotherapy because its production is of higher quality, a safer method, and they are more stable and less toxic [68, 69].

#### **10. Liver tissue**

The special features of mesenchymal stem cells, such as multipotency and selfregeneration, have been used as promising tools for treating liver diseases. According to the figure, exosomes derived from these cells can regenerate various tissues, including the liver, in damaged models [70]. Several studies have been conducted on the therapeutic effects of MSC-derived exosomes in mice models of liver fibrosis. Carbon tetrachloride (CCL4) was used to cause this damage. It was determined that exosomes derived from MSCs isolated from umbilical cord blood have improved liver fibrosis by inhibiting the epithelial-mesenchymal transition of hepatocytes and increasing collagen production [71, 72]. It has been found that exosomes significantly restore the activity of the liver aspartate aminotransferase enzyme and inhibit the smad/TGFβ

*Stem Cell Therapy and Its Products Such as Exosomes: Modern Regenerative Medicine Approach DOI: http://dx.doi.org/10.5772/intechopen.111574*

signaling pathway by inactivating phosphorylation and increasing the production of type 1 and 3 collagen [73]. Another study showed that hepatic mesenchymal stem cells could release exosomes containing miR-125b, which are transported between these cells and target cells, such as stellate cells that respond to the Hedgehog (Hh) signaling pathway and heal the fibrosis caused by CCL4 damage in mouse models by inhibiting Hh pathway signaling which preventing SMO protein expression [74]. It has been reported that exosomes derived from adipose-derived MSCs (AD-MSC) contain miR-122, which affects hepatocytes and regulates the expression of specific genes such as P4HA1 and IGF1R, which are effective in collagen production and increase the speed of liver fibrosis treatment [75]. Exosomes derived from AD-MSC can significantly reduce the level of alanine and aspartate aminotransferase and concanavalin A, as well as the serum level of pro-inflammatory cytokines such as TNFα, INFγ, IL6, IL8, and also reduces IL1β, which causes severe liver inflammation. Exosomes derived from MSCs can also improve the acute liver damage caused by acetaminophen or H2O2 by affecting the genes of anti-apoptotic proteins such as BCL-XL and transcription activators such as STAT3 and increasing their expression [70, 76].

#### **11. Renal tissue**

Exosomes secreted by bone marrow mesenchymal stem cells can enhance the growth of cisplatin-damaged proximal tubule epithelial cells by horizontal transfer of IGF-1 receptor mRNA. It has also been shown that exosomes derived from human umbilical cord blood mesenchymal stem cells can improve acute kidney injury by inhibiting kidney oxidative stress and apoptosis, increasing kidney epithelial cells' growth [77, 78]. A scientist named Borges discovered that by placing renal tubule epithelial cells in hypoxia condition, these cells release TGF-β1 mRNA-rich exosomes into the culture medium, which can activate fibroblasts to initiate a fibrotic remodeling response [79]. Burger investigated the therapeutic potential of colony-forming cells derived from umbilical cord blood in acute kidney ischemic injury models, which showed the therapeutic abilities of these cells in treating this type of injury due to miR-486-containing exosomes. 5p is derived from these cells, which can repair this damage by targeting the PTEN gene [80]. Research has also been done on the therapeutic abilities of exosomes derived from stem cells isolated from the urine; which has shown that the damage caused by streptozotocin-induced renal damage models by weekly injection of urine-derived stem cell (USCs) exosomes can inhibit apoptosis and increase survival and vessel regeneration [81].

#### **12. Skeletal muscle tissue**

Recently, the use of mesenchymal stem cell secretomes, especially exosomes derived from mesenchymal stem cells (MSCs), for skeletal muscle regeneration has been researched. *In vivo* studies have shown that exosomes derived from MSCs can increase the speed of muscle regeneration by increasing angiogenesis and reducing muscle fibrosis. Concerning skeletal muscle injuries, researchers have discovered miRNAs with anti-apoptotic activity, such as miR-21, and myogenic activities, such as miR-1, miR-133, miR-206, and miR-494, which were able to reduce these types of injuries in mouse models [82, 83]. Choi and colleagues found that exosomes derived from human skeletal myoblasts (hSKMs) during myotube differentiation could

induce myogenesis response in hASCs. Experiments on skeletal muscle injury model mice confirm that using hSKMs-derived exosomes can accelerate skeletal muscle regeneration by reducing the collagen deposition rate and increasing myofibrils' regeneration in injured muscles. According to studies, exosomes derived from MSCs regenerate skeletal muscles by strengthening myogenesis and angiogenesis; at least part of these effects are caused by miRNAs such as miR-494 [82, 84].

#### **13. Nervous system**

Exosomes have also been investigated to improve regenerative medicine's central and peripheral nerve systems. They can cross the blood-brain barrier as moderators of inflammatory responses and regeneration of nerve damage. Nervous system injuries are very debilitating for patients and often cause severe skeletal muscle disorders, and the management and recovery of these injuries are complicated and unresolved. Peripheral nervous system damage causes inflammation, loss of neuron function, and destruction, resulting in cell death. Today, we know that exosomes derived from MSCs support nerve growth by stimulating the secretion of growth factors needed to support and stimulate Schwann cells, which play an essential role in myelin production [85, 86]. Exosomes derived from MSCs can significantly induce repair of the nervous system by miR-133b, which is modified with lentiviral expression vectors and determine the overexpression or silencing of miR-133b and thus cause the regeneration of neurons [87]. Recently, it has been found that in debilitating diseases such as Parkinson's and Alzheimer's, neurons release exosomes containing α synuclein and β amyloid, respectively. These exosomes can play a role in the nucleation and physical release of these aggregated proteins that cause these diseases [88]. In the research, it has been proven that exosomes can be used as biomarkers of brain damage, for example, exosomes containing miR-9 and miR-124 isolated from blood as biomarkers are used to diagnose acute ischemic stroke (AIS) and also evaluate the amount or degree of damage caused by this ischemia [89]. The effects of exosomes in the regeneration of neurons and the nervous system have also been proven, for example, exosomes derived from oligodendrocytes stimulated with glutamate can increase neurons' survival in hypoxia conditions without glucose. Exosomes derived from bone marrow tissue stem cells can significantly increase the survival of retinal ganglion cells (RGCs) and the regeneration of their axons [90–92]. Usually, after nerve damage, Schwann cells differentiate and grow and direct axons to their target tissue. It has been found that the exosomes derived from these cells inhibit the activity of RhoA, a GTPase that can cause axons to lengthen and repair them [93, 94].

#### **14. Heart muscle tissue**

The protective effects of exosomes in myocardial ischemia re-injury models are being investigated. Scientists have shown that exosomes isolated from cardiospherederived cells (CDCs), when injected into mice model of ischemia, can inhibit apoptosis and induce the growth of heart cells. It has been found that these beneficial effects are due to the richness of these exosomes in miR-146a [95]. During another study, it was determined that exosomes secreted from bone marrow mesenchymal stem cells stimulate the formation of umbilical cord vein endothelial cell tubes and inhibit the production of T cells in vitro. In addition, the severity reduces infarction [96].

*Stem Cell Therapy and Its Products Such as Exosomes: Modern Regenerative Medicine Approach DOI: http://dx.doi.org/10.5772/intechopen.111574*

According to other research on exosomes derived from mouse embryonic stem cells, these exosomes can restore the internal function of the heart after myocardial infarction. With further research, the researchers found that this is due to miR-294 in these exosomes, which are transferred to cardiac progenitor cells [97, 98]. Another research group in China investigated the protective effects of mesenchymal stem cells derived from human umbilical cord blood on acute myocardial infarction (AMI) animal models and noticed the effects of these exosomes in protecting myocardial cells. Apoptosis and increased angiogenesis in the damaged area. They found that these effects are related to the modulation of BCL-2 pro-apoptotic protein family gene expression [99]. Certain exosomes in the heart's pericardial fluid improve the survival, growth, and communication of endothelial cells in the culture medium. It restores the angiogenic capacity of endothelial cells, and even these exosomes improve blood flow and angiogenesis after ischemic injuries in model mice. Further research has shown that these exosomes contain miR let-7b to carry out this process [100].

#### **15. Skin**

Angiogenesis is essential in various physiological processes, including wound healing and skin tissue regeneration. Scientists found that exosomes secreted by mesenchymal stem cells derived from adipose tissue can significantly stimulate the angiogenesis of endothelial cells *in vitro* and *in vivo*. Further research showed that these exosomes transfer miR-125a to endothelial cells, which decreases the expression of inhibitory proteins Delta-like 4 (DLL4) that inhibit angiogenesis [101, 102]. Burns is one of the most common skin injuries that significantly intensify inflammatory reactions by increasing the level of factors such as TNFα, Il-1β, and decreasing the level of IL-10. The scientists found that using exosomes derived from umbilical cord blood stem cells successfully reduced the inflammatory reactions caused by burns, and further research revealed that this effect is due to the presence of miR-181c in these exosomes, which reduces pain and severe inflammation caused by burns by reducing TLR4 signaling [103]. It has been determined that exosomes derived from umbilical cord blood endothelial progenitor cells can heal diabetic wounds in rat models. Microarray analysis has shown that exosomes significantly increase the expression of some genes. They change the ERK1/2 signaling pathway, which is very important in the healing and regeneration of these wounds. Studies by Guo and colleagues have shown that platelet-rich plasma-derived exosomes can effectively induce the proliferation and migration of endothelial cells and fibroblasts to increase angiogenesis and regeneration and repair severe skin wounds. Exosomes can also control skin regeneration bipolarly. They can also prevent scarring caused by burn healing and collagen deposition through the induction of phosphorylation that inhibits the WNT/catenin YAP pathway [84, 104].

#### **16. Cancer**

In the mid-2000s, the first results from clinical trials on exosomes as a treatment for cancer were published. Exosomes derived from dendritic cells (DEXs) are potential targets for cancer therapeutic strategy. DEXs can directly catalyze the transfer of peptide-MHC complexes from their membrane surface to the T cell membrane surface (cross-dressing). Moreover, DEXs can indirectly stimulate T cell responses via cross-dressing with dendritic cells or via exosome uptake and processing, following the peptide-MHC complex presentation to T cells. DEXs can also induce activation and proliferation of NK cells by establishing interaction of the NKG2D ligand on DEXs with NKG2D receptors on the NK cell membrane [105]. In 2005, two phase I clinical trials using DEX vaccines were performed. The first trial reported the use of DEXs loaded with HLA-restricted melanoma-associated antigen (MAGE) peptides, which were infused into patients with HLA A2+ non-small cell lung cancer (NSCLC); the second trial reported the use of DEXs derived from DCs pulsed with MAGE and inoculated them to conduct immunization of melanoma patients [106]. Exosomes exhibit features for application as adjuvant carriers, such as optimal size, biocompatibility, stability in the systemic circulation, and target-specific delivery. Recently, an exosome-based adjuvant delivery system was developed using genetically modified murine melanoma B16BL6 cells. The exosomes derived from these cells containing CpG DNA were injected three times at 3-day intervals and successfully induced immunostimulatory signals in mice 7 days after the last immunization [107, 108]. In a recent study by Shi et al., a vaccine with exosomes derived from IFNɣ-modified RM-1 prostate cancer cells under a vaccination regimen of four injections (on days 0, 4, 8, and 12) decreased the number of Tregs. It reduced the metastatic tumor rate in C57BL male mice with lung metastasis [109]. Recently, a non-randomized phase I/II clinical trial showed promising results with a vaccine designed using exosomes derived from DCs pulsed with SART1, a biomarker of esophagus squamous cell carcinoma. Pulsed DCs obtained from patients could generate exosomes that were well tolerated and induced antigen-specific CTLs in seven patients. One patient in this study remained stable for 20 months after DEXs therapy, although he developed lung metastasis after the stable period. The other six patients had progressive disease and died up to 10 months post-vaccination. These findings indicate that developing personalized exosome-based immunotherapy is feasible, although challenging [110]. A phase I clinical trial reported using exosomes derived from ascites (AEXs) in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) as immunotherapy for colorectal cancer. Injection of AEXs for colorectal cancer was safe and well tolerated by all patients during the four weekly doses administered [111].

#### **17. Applied exosomes on scaffolds**

The short tissue retention of exosomes after *in vivo* implantation is still a significant challenge in clinical applications. Scaffold encapsulation of exosomes can enable continuous delivery in the injured environment, thereby improving the therapeutic effect. Researchers have developed several methods to deliver exosomes to the post-infarct environment sustainably. For example, exosomes isolated from cardiomyocyte-derived induced pluripotent stem cells encapsulated in hydrogel patches were directly delivered to the hearts of infarcted rats [112]. The exosome patches demonstrated prolonged exosome release and promoted recovery of the ejection fraction, prevented cardiomyocyte hypertrophy, alleviated the ischemic injury, and promoted heart recovery. Another study loaded endothelial progenitor cell-derived exosomes into a shear-thinning gel to achieve precise administration and sustained delivery [113]. In a rat model of myocardial infarction, the exosome hydrogels enhanced angiogenesis and myocardial hemodynamics around the infarct. The cell-free scaffold material improved the effects of exosome-mediated myocardial therapy. In another study, exosomes isolated from human umbilical cord-derived

#### *Stem Cell Therapy and Its Products Such as Exosomes: Modern Regenerative Medicine Approach DOI: http://dx.doi.org/10.5772/intechopen.111574*

MSCs were encapsulated in functional peptide hydrogels to increase their stability and provide sustained release. The exosome hydrogels protected cardiomyocytes from oxidative stress induced by H2O2, which improved cardiac function in a rat myocardial infarction model. These studies provide practical and effective methods for exosomeladen scaffolds in myocardial regeneration [114]. Exosome-laden scaffolds are most widely used for skin repair.

Several findings indicate that combining bioactive scaffold materials with the controlled release of exosomes heals skin wounds. For example, exosomes isolated from human umbilical cord-derived MSCs encapsulated in polyvinylalcohol (PVA)/ Alg nanohydrogels were used to heal diabetic wounds. The PVA/Alg nanohydrogel promoted cell proliferation, migration, angiogenesis, enhanced the efficacy of exosomes, and accelerated the healing of diabetic wounds. In another study, exosomes were loaded in a novel injectable bioactive hydrogel called FHE. Exosomes isolated from adipose-derived MSCs were loaded into FHE hydrogel through electrostatic interactions with poly-ε-L-lysine. The exosome hydrogel promoted angiogenesis, cell proliferation, and granulation tissue formation at the wound site and accelerated the healing of diabetic wounds and skin regeneration [115, 116]. In another study, methylcellulose-chitosan hydrogels loaded with exosomes isolated from placentaderived MSCs were shown to heal diabetic wounds and form new tissues similar to natural skin. Similarly, chitosan/silk hydrogels with swelling and moisturizing capabilities loaded with exosomes isolated from gingival-derived MSCs promoted collagen epithelial regeneration and angiogenesis and accelerated the healing of diabetic skin defects [117, 118]. Chitosan scaffolds have also been shown to provide controlled release of exosomes isolated from synovium-derived MSCs, which accelerated wound healing by increasing the formation of granulation tissue and angiogenesis [118, 119]. Modified exosomes also have the potential to stimulate bone regeneration. For example, miR-375 was enriched in exosomes by overexpression in parental cells. The exosomes were loaded into a hydrogel and injected into a rat skull defect model. The exosomes were continuously released into the wound, which enhanced bone regeneration [120]. Liu et al. developed a photoinduced imine functional group cross-linking hydrogel glue to generate a decellularized tissue patch for cartilage regeneration. The patch retained stem cell-derived exosomes in the cartilage for a long time. In addition, the exosome-laden scaffold integrated with the natural cartilage matrix induced cell migration in the cartilage defect and promoted the repair and regeneration of articular cartilage [121]. Another study constructed a cell-free bone tissue engineering system by combining poly (lactic-co-glycolic acid) (PLGA)/polydopamine (pDA) scaffolds and exosomes isolated from human adipose-derived stem cells. The exosomes were slowly and continuously released from the scaffold, which promoted the migration of MSCs and significantly enhanced bone regeneration [112].

#### **18. Exosome for microbial diseases**

MSCs express various types of anti-microbial peptides and proteins (AMPs). Some of them are known for anti-bacterial properties, such as cathelicidin LL-37), β-defensin-2 (BD-2), hepcidin, and Lipocalin-2 (Lcn2) [122]. It is suggested in recent studies that MSCs can improve bacterial infection in preclinical models by AMPs. Therefore, MSCs can enhance the innate immune response against bacteria. It is suggested in recent studies that MSCs can improve bacterial infection in preclinical models by AMPs. Therefore, MSCs can enhance the innate immune response against bacteria [123].

In a study by Yagi et al. in 2020, the anti-microbial activity of human Adiposederived MSCs (AD-MSCs) on *Staphylococcus aureus* was assessed. The findings showed that human AD-MSCs conditioned medium significantly prevented the growth of *S. aureus*. The results also showed the critical anti-microbial activity of cathelicidin LL-37 in AD-MSCs [124]. A previous study also showed that the antimicrobial activity of BM-MSCs against the growth of Gram-negative (*Escherichia coli* and *Pseudomonas aeruginosa*) and Gram-positive (*S. aureus*) bacteria was mediated by LL-37 [125]. On the other hand, human umbilical cord blood-derived MSCs attenuated acute lung injury through *E. coli* infection in mice. The results demonstrated that MSCs secreted BD-2 through the TLR-4 signaling pathway and mediated the antimicrobial effects [126].

Moreover, menstrual-derived MSCs secreting hepcidin in synergy with antibiotics in sepsis were responsible for bacterial clearance. MSCs also secret different growth factors, such as the Keratinocyte growth factor (KGF), to exert anti-bacterial activity. In the research performed by Lee et al. on an *E. coli* infection model in an *ex vivo* perfused human lung, BM-MSCs improved alveolar fluid bacterial clearance and mitigated inflammation [127, 128]. The metabolomics and immunomodulatory effects of MSCs-MVs are performed by enhancing the intracellular ATP levels in injured alveolar epithelial cells and reducing the secretion of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) in human monocytes. It should be considered that MSC-MVs expressed Cyclooxygenase2 (COX2) mRNA. COX2 is the crucial enzyme in prostaglandin E2 (PGE2) synthesis that is a critical factor for transforming the polarization of M1 into M2 macrophages. As articles suggest, the enhancement in PGE2 secretion by monocytes following the transfer of COX2 mRNA from MSC-MVs to these cells caused the phenotype switch from M1 to M2. MSC-MVs, by direct transfer of KGF or indirectly by activating monocytes, mitigated lung inflammation, cytokine permeability, bacterial growth, and improved survival. This therapeutic effect of MVs was abrogated by KFG neutralizing antibody, proposing a possible mechanism for the anti-bacterial effect of MSC-MVs [129, 130]. As the anti-bacterial effect of KGF in MSCs was previously reported [128], these studies supported the hypothesis that MVs can partly conserve the anti-microbial effects of parent cells, by using growth factors, including KGF. Cell-Exo can overcome the shortage of stem cells to treat microbial and other infectious diseases and provide a new generation in medical science from cellular to acellular therapy.

Both intact and engineered exosomes have been applied, and their efficacy on various infectious diseases has been assessed in preclinical studies and limited clinical trials. Although exosomes perform part of their antimicrobial activity by directly transferring mRNA, miRNA, and protein cargos, their beneficial effects are mainly indirectly applied through reprogramming immune cells and activating innate and adaptive immune responses.

### **Author details**

Leila Dehghani1,2, Amir Hossein Kheirkhah3 , Arsalan Jalili4,5, Arman Saadati Partan5 , Habib Nikukar6 \* and Fatemeh Sadeghian-Nodoushan7 \*

1 Department of Applied Cell Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

3 Department of Tissue Engineering and Applied Cell Science, School of Medicine, Qom University of Medical Sciences, Qom, Iran

4 Department of Applied Cell Sciences, Faculty of Basic Sciences and Advanced Medical Technologies, Royan Institute, ACECR, Tehran, Iran

5 Parvaz Research Ideas Supporter Institute, Tehran, Iran

6 Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

7 Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

\*Address all correspondence to: habibnik@gmail.com and fsadeghian@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Diana Kitala*

Although the concept of using advanced therapy products such as stem cells seems to be a key strategy in the treatment of various diseases, much information in this area remains unknown. Stem cell products are highly complex, much more complex than chemical-based drugs. More and more often there are data indicating the risk of using stem cells. These risks are determined by various factors that are related to quality, biological activity, and the use itself, and thus administration. Therefore, it is very important to constantly systematize knowledge in this area. This book was created to present both the perspective of basic research, including the manipulation and changes in the properties of cells, and the changes and novelties in therapies themselves.

### *Miroslav Blumenberg, Biochemistry Series Editor*

Published in London, UK

© 2023 IntechOpen © monsitj / iStock

Possibilities and Limitations in Current Translational Stem Cell Research

IntechOpen Series

Biochemistry, Volume 44

Possibilities and Limitations

in Current Translational Stem

Cell Research

*Edited by Diana Kitala*