**2.4 iPSC-based therapies**

*Innovations in Cell Research and Therapy*

carried out using iPSCs.

**2.3 iPSC disease modeling**

isogenic iPSCs and NT-ESCs have demonstrated that NT-ESCs more closely resemble bona fide ESCs derived from fertilized embryos [12, 13]. Moreover, a most important difference between iPSCs and NT-ESCs is the source of mitochondrial DNA (mtDNA). The mtDNA in NT-ESCs is of an oocyte germline origin, while in iPSCs is of a parental somatic origin. Due to the random nature of somatic mtDNA mutations, the frequency of mtDNA defects in iPSCs has been demonstrated to increase with the age of somatic cells [14]. Thus, NT-ESCs, carrying mutation-free mtDNA and closely resembling ESCs, represent an invaluable stem cell source for regenerative medicine. However, as the derivation of NT-ESCs requires donor oocytes, which are more technically challenging than iPSCs and are also subject to ethical and/or legal restrictions, the majority of current PSC research has been

These groundbreaking discoveries have revolutionized our understanding of stem cell development and created novel opportunities for human disease modeling and drug screening in "disease-in-a-dish" models (**Figure 1**) [15]. To date, significant progress has been made utilizing human iPSCs to model various neurological disorders, inherited heart diseases, and other genetic diseases such as Duchene muscular dystrophy and recessive dystrophic epidermolysis bullosa (RDEB) [16, 17]. Utilizing amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease as an example, ALS is a neurodegenerative disease that primarily affects corticospinal "upper" motor neurons (UMNs) and spinal cord "lower" motor neurons (LMNs), resulting in progressive muscle weakness [18]. In about 10% of patients with ALS, the disease runs in the family (familiar ALS) with mutations in around 20 genes including SOD1, TARDBP, FIS, and C9orf72 identified as common causes [18]. The remaining 90% of the patients are classified as sporadic ALS, with the causative mutations largely unidentified. As the iPSCs generated from ALS patients and differentiated into motor neurons carry the same genetic background as the patients, it represents a novel tool for studying disease pathology of ALS, particularly the sporadic form, which is not possible in the other model systems. A proof-of-principle study on derivation of iPSCs from an ALS patient and differentiation into LMNs was reported in 2008 [19]. Subsequently, Kiskinis et al. and Chen et al., respectively, established in vitro models of ALS by generating iPSC-derived LMNs from patients carrying different SOD1 mutations. Both studies recapitulated the spontaneous and progressive decrease in cell viability and ALS-related morphological changes including reduction in soma size and altered dendrites, which was linked to neurofilament aggregation [20, 21]. Chen et al. further demonstrated the pathological features of mutated SOD1 in patient-derived MNs, but not in non-MNs. Only in MNs, mutated SOD1 bound to the 3′UTR region of neurofilament (NF)-L mRNA resulted in neurofilament aggregation, restoring the expression of NF-L mitigated neurite degeneration of the ALS-iPSC-derived MNs. Meanwhile, Wainger et al. generated iPSC-derived LMNs from patients carrying SOD1, C9orf72, or FUS mutations [22]. All these ALS-iPSC-derived LMNs with distinct genetic mutations have recapitulated essential disease features and discovered common molecular pathways driving ALS pathogenesis, opening the possibility of new and effective drug screening [23]. However, challenges still remain for in vitro modeling for ALS using iPSCs [18]. Different protocols have been reported in deriving LMNs from iPSCs; thus, criteria need to be established to compare the MNs generated using different methods. Moreover, generation of UMNs from PSCs involves a series of steps and

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The most significant advantage of iPSCs lies in its application in cell-based therapies. iPSCs can be developed without destroying human embryos, therefore circumventing the ethical obstacles of utilizing and generating human ES cells. Being able to differentiate into all cell types in the body similar to ES cells, iPSCs theoretically provide an unlimited source of cells for autologous transplantation, eliminating the need for immunosuppression. Moreover, scientists have established robust directed differentiation protocols with sequential activation and inhibition of molecular differentiation pathways to generate a wide range of somatic cells from iPSCs, such as β cells and cardiomyocytes (**Figure 1**).

A challenge for the PSC or iPSC differentiation, as also mentioned above in the iPSC-derived MNs, is that the PSC-derived cells tend to be immature. This is indeed the major limitation for translating iPSC-derived red blood cells into the clinic [25]. In 2008, Lu et al. reported differentiation of human ES cells into functional oxygencarrying erythrocytes on a large scale with up to 60% enucleation rate [26]. In comparison, differentiation of iPSCs along the erythroid lineage generated orthochromatic (nucleated) erythroblasts and reticulocytes. In most reports, the differentiated red blood cells express mainly fetal and embryonic globins, but very little adult-type (β-) globin [27]. This is likely due to the low level of erythroid Kruppellike factor 1 (EKLF1) and absence of BCL11A in these iPSC-derived red blood cells. These two factors have been demonstrated to be essential for the developmental switch from fetal to adult globin expression [28]. Inducible expression of KLF1 during later stages of the differentiation process has been recently demonstrated to enhance differentiation and maturation of red blood cells from both human ES cells and iPSCs [29].

Recent advances in the development of programmable site-specific nucleases, including zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas)9 system, have enabled target-specific introduction of transgene or correction of disease-specific mutations by homologous recombination, creating novel opportunities not only for disease modeling and drug testing but also generation of genetically corrected cells for autologous transplantation (**Figure 2**).

The development of iPSC technology has also revolutionized the future treatment for end-stage organ failure. Takebe et al. recently reported vascularized and functional mini-livers or liver buds created in vitro based on human iPSCs [30]. In this proof-of-concept demonstration, the authors first prepared hepatic endoderm cells from human iPSCs by directed differentiation. About 80% of the differentiated cells express liver-specific marker HNF4A. To recapitulate early organogenesis, the investigators next cultured the iPSC-derived hepatic endoderm cells with two stromal cell populations, i.e., human umbilical vein endothelial cells and human mesenchymal stem cells (MSCs), in a traditional two-dimensional culture condition. Intriguingly, the iPSC-derived hepatic cells

### **Figure 2.**

*Development of iPSC gene correction and autologous transplantation therapy. Biopsies such as the skin or blood can be obtained from a patient with genetic mutations and reprogrammed into patient-specific iPSCs. Through targeted nuclease technologies, such as zinc-finger nuclease which contains sequence-specific DNAbinding domain fused to a non-specific* Fok1 *endonuclease enzyme, transcription activator-like effector proteins consisted of tandem DNA-binding repeats linked with the* Fok1 *enzyme, and the most recent method of CRISPR/Cas system that utilizes a single-guide RNA (sgRNA) and a protospacer adjacent motif for efficient genome targeting and binding followed by activity of the Cas enzyme, a double-stranded break (DSB) occurs at the target site of genome, inducing activation of internal DNA repair mechanism. Homologous recombination (HR) can then be achieved to incorporate exogenously transduced donor DNA to repair the mutations in the genome. After validation of the target-specific gene correction, the corrected iPSCs can be further differentiated into a cell type of preference for autologous transplantation.*

self-organized into three-dimensional cell clusters that resemble in vivo liver buds during embryonic development. Moreover, within 48 hours of transplantation into the nonobese diabetic/severe combined immunodeficiency disease (NOD/ SCID) mice, the vasculatures in the iPSC-derived liver buds became functional by connecting to the host vessels, which further stimulated the maturation of iPSCderived liver buds into the tissue resembling the adult liver. Considering critical shortage of donor organs, development of iPSC-derived organoids suggests an alternative and innovative regenerative approach for patients with end-stage organ failure.

New advances have also been made in cardiac tissue engineering for cardiovascular diseases. The protocols for differentiating ES cells or iPSCs into cardiomyocytes, smooth muscle cells, and endothelial cells that are the main functional cell types in the heart have been reported [31, 32]. However, although readily obtainable, the morphology, calcium handling, electric coupling, contraction stress, and electrophysiology of the PSC-derived cardiomyocytes have been demonstrated to be immature compared to adult cardiomyocytes (reviewed in [33]). With that, significant bioengineering efforts have been made to recapitulate environmental cues to enable maturation of newly differentiated cardiomyocytes and to promote vascular network formation (reviewed in [34]). To address the need for tissue and/ or organ transplantation, there are also exciting advances in incorporating biocompatible materials, cells, and supporting components into complex 3D functional living tissues [35]. Excitingly, Noor et al. recently reported a 3D printing of thick, vascularized, and perfusable cardiac patches that fully match the immunological, biochemical, and anatomical properties of the patient [36]. This was the first report on the use of fully personalized, non-supplemented materials as bioink for 3D printing. In this study, fatty tissue was extracted from a patient. The cells from the tissue were reprogrammed to iPSCs, followed by directed differentiation into cardiomyocytes and endothelial cells. The remaining fatty tissue was decellularized and processed to generate a thermo-responsive hydrogel. The iPSC-derived cells

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*Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine*

**2.5 Xenogeneic generation of human organs using PSCs**

and to investigate experimental drugs in different diseases.

allowed to develop past the fetal stage.

**2.6 iPSC banking and allogeneic cell therapies**

Despite all the promises of using human/nonhuman chimeras for regenerative medicine, they also have raised serious ethical dilemmas about the morality of these chimeras. One of the biggest concerns is whether the human cells migrate to the brain and the chimeras end up with a humanlike mind. Such issues could potentially be prevented through genetic editing to avoid the human cell differentiating into the human brain or human gonads. So far, investigations in this field are moving forward with caution, and the reported human/nonhuman chimeras have not been

Although the iPSC cell therapy theoretically enables autologous transplantation, which would eliminate the need for immunosuppression, the inefficiency of iPSC derivation, the time and cost for developing each personalized cell product, and the safety of the products have made such autologous therapies unpractical,

were then encapsulated with the hydrogel and served as the bioink for 3D printing of vascularized patches and complex cellularized structures. The investigators of this study also pointed out the obstacles that need to be overcome for a more applicable 3D printing, including efficient generation of a sufficient number of cells for the organ printing, identifying biochemical and physical cues for cell maturation and conditions for long-term cultivation and a higher-resolution imaging of the entire blood vessels for the blueprint of 3D printing, etc. Nevertheless, the results from these studies have shed light on developing autologous engineered tissue or

With the growing knowledge of organogenesis and the aid of gene editing technologies, scientists are also pushing the boundary and creating interspecies chimeras to grow human organs in animals, which ideally could subsequently be transplanted into people. In 2017, Yamaguchi et al. demonstrated that injection of mouse PSCs in the blastocysts of apancreatic *Pdx1mu/mu* rats (TALEN-mediated disruption in *Pdx1* gene, a master regulator for pancreas development) which resulted in generation of a mouse pancreas in the rat [37]. Moreover, when the mouse pancreas grown in the rat was transplanted into diabetic mice, they were able to cure diabetes in the recipients without the administration of immunosuppression [37]. Wu et al. subsequently reported the creation of the first human-pig chimeras by injecting human PSCs into pig blastocysts [38]. The success rate of generating human-pig chimeras was indeed very low, and the chimeras only carried very few human cells, less than one human cell per 100,000 pig cells. To date, no one has reported using gene editing techniques performed in rat embryos to disable the pigs forming a particular organ and enable the human cells to develop more humanlike organs. Ross et al. demonstrated the generation of sheep-human hybrids, and as a step further than the reported human-pig chimeras, the contribution of human cells in sheep embryos was increased to one in 10,000 sheep cells [39]. Although these studies represent only a preliminary step toward the long-term goal, the results from these studies suggest that such creation may be eventually used to grow human organs. As the pigs and sheep are similar in size to humans, the human organs grown in these animals, the heart, liver, kidney, pancreas, lungs, and brain, could be harvested and transplanted into people, meeting the high demand for organ transplantation in the end-stage diseases. In addition, these human-animal chimeras could also be used to investigate the mechanisms of prenatal development

*DOI: http://dx.doi.org/10.5772/intechopen.88790*

organs for transplantation.

*Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.88790*

were then encapsulated with the hydrogel and served as the bioink for 3D printing of vascularized patches and complex cellularized structures. The investigators of this study also pointed out the obstacles that need to be overcome for a more applicable 3D printing, including efficient generation of a sufficient number of cells for the organ printing, identifying biochemical and physical cues for cell maturation and conditions for long-term cultivation and a higher-resolution imaging of the entire blood vessels for the blueprint of 3D printing, etc. Nevertheless, the results from these studies have shed light on developing autologous engineered tissue or organs for transplantation.

### **2.5 Xenogeneic generation of human organs using PSCs**

With the growing knowledge of organogenesis and the aid of gene editing technologies, scientists are also pushing the boundary and creating interspecies chimeras to grow human organs in animals, which ideally could subsequently be transplanted into people. In 2017, Yamaguchi et al. demonstrated that injection of mouse PSCs in the blastocysts of apancreatic *Pdx1mu/mu* rats (TALEN-mediated disruption in *Pdx1* gene, a master regulator for pancreas development) which resulted in generation of a mouse pancreas in the rat [37]. Moreover, when the mouse pancreas grown in the rat was transplanted into diabetic mice, they were able to cure diabetes in the recipients without the administration of immunosuppression [37]. Wu et al. subsequently reported the creation of the first human-pig chimeras by injecting human PSCs into pig blastocysts [38]. The success rate of generating human-pig chimeras was indeed very low, and the chimeras only carried very few human cells, less than one human cell per 100,000 pig cells. To date, no one has reported using gene editing techniques performed in rat embryos to disable the pigs forming a particular organ and enable the human cells to develop more humanlike organs. Ross et al. demonstrated the generation of sheep-human hybrids, and as a step further than the reported human-pig chimeras, the contribution of human cells in sheep embryos was increased to one in 10,000 sheep cells [39]. Although these studies represent only a preliminary step toward the long-term goal, the results from these studies suggest that such creation may be eventually used to grow human organs. As the pigs and sheep are similar in size to humans, the human organs grown in these animals, the heart, liver, kidney, pancreas, lungs, and brain, could be harvested and transplanted into people, meeting the high demand for organ transplantation in the end-stage diseases. In addition, these human-animal chimeras could also be used to investigate the mechanisms of prenatal development and to investigate experimental drugs in different diseases.

Despite all the promises of using human/nonhuman chimeras for regenerative medicine, they also have raised serious ethical dilemmas about the morality of these chimeras. One of the biggest concerns is whether the human cells migrate to the brain and the chimeras end up with a humanlike mind. Such issues could potentially be prevented through genetic editing to avoid the human cell differentiating into the human brain or human gonads. So far, investigations in this field are moving forward with caution, and the reported human/nonhuman chimeras have not been allowed to develop past the fetal stage.

### **2.6 iPSC banking and allogeneic cell therapies**

Although the iPSC cell therapy theoretically enables autologous transplantation, which would eliminate the need for immunosuppression, the inefficiency of iPSC derivation, the time and cost for developing each personalized cell product, and the safety of the products have made such autologous therapies unpractical,

*Innovations in Cell Research and Therapy*

*into a cell type of preference for autologous transplantation.*

self-organized into three-dimensional cell clusters that resemble in vivo liver buds during embryonic development. Moreover, within 48 hours of transplantation into the nonobese diabetic/severe combined immunodeficiency disease (NOD/ SCID) mice, the vasculatures in the iPSC-derived liver buds became functional by connecting to the host vessels, which further stimulated the maturation of iPSCderived liver buds into the tissue resembling the adult liver. Considering critical shortage of donor organs, development of iPSC-derived organoids suggests an alternative and innovative regenerative approach for patients with end-stage

*Development of iPSC gene correction and autologous transplantation therapy. Biopsies such as the skin or blood can be obtained from a patient with genetic mutations and reprogrammed into patient-specific iPSCs. Through targeted nuclease technologies, such as zinc-finger nuclease which contains sequence-specific DNAbinding domain fused to a non-specific* Fok1 *endonuclease enzyme, transcription activator-like effector proteins consisted of tandem DNA-binding repeats linked with the* Fok1 *enzyme, and the most recent method of CRISPR/Cas system that utilizes a single-guide RNA (sgRNA) and a protospacer adjacent motif for efficient genome targeting and binding followed by activity of the Cas enzyme, a double-stranded break (DSB) occurs at the target site of genome, inducing activation of internal DNA repair mechanism. Homologous recombination (HR) can then be achieved to incorporate exogenously transduced donor DNA to repair the mutations in the genome. After validation of the target-specific gene correction, the corrected iPSCs can be further differentiated* 

New advances have also been made in cardiac tissue engineering for cardiovascular diseases. The protocols for differentiating ES cells or iPSCs into cardiomyocytes, smooth muscle cells, and endothelial cells that are the main functional cell types in the heart have been reported [31, 32]. However, although readily obtainable, the morphology, calcium handling, electric coupling, contraction stress, and electrophysiology of the PSC-derived cardiomyocytes have been demonstrated to be immature compared to adult cardiomyocytes (reviewed in [33]). With that, significant bioengineering efforts have been made to recapitulate environmental cues to enable maturation of newly differentiated cardiomyocytes and to promote vascular network formation (reviewed in [34]). To address the need for tissue and/ or organ transplantation, there are also exciting advances in incorporating biocompatible materials, cells, and supporting components into complex 3D functional living tissues [35]. Excitingly, Noor et al. recently reported a 3D printing of thick, vascularized, and perfusable cardiac patches that fully match the immunological, biochemical, and anatomical properties of the patient [36]. This was the first report on the use of fully personalized, non-supplemented materials as bioink for 3D printing. In this study, fatty tissue was extracted from a patient. The cells from the tissue were reprogrammed to iPSCs, followed by directed differentiation into cardiomyocytes and endothelial cells. The remaining fatty tissue was decellularized and processed to generate a thermo-responsive hydrogel. The iPSC-derived cells

**102**

organ failure.

**Figure 2.**

particularly for diseases that require an immediate treatment. For the clinical use of iPSC-based cell therapies, it is essential to produce high-quality and safe (no induced mutations in the genome) iPSCs. As will be mentioned below, the pioneering iPSC clinical study in Japan using patients' own iPSC-derived retinal epithelial cells for the treatment of macular degeneration was put on hold due to genomic mutations in the iPSCs. Therefore, the most feasible application of iPSC-based cell therapy would rely on the banked and human leukocyte antigen (HLA)-typed iPSCs, in which the quality and safety have been validated in advance, in the setting of an allogeneic transplantation. This use of allogenic iPSCs however means that immunosuppression would have to be applied to prevent immune rejection. Kawamura et al. recently demonstrated that even though the immunogenicity of allogenic iPSC-derived cardiomyocytes was reduced by major histocompatibility complex (MHC) class I- and class II-matched transplantation in the macaque (monkey), the recipients still required substantial and highly toxic immunosuppression for sustained allogeneic cell engraftment [40]. It has been suggested that the MHC-matched iPSC-derived cardiomyocytes were still susceptible to natural killer (NK) cell destruction, leading to their rejection in the recipients in the absence of immunosuppression [40]. Forced expression of HLA alpha chain E (HLA-E) in PSCs and their differentiated derivatives has been demonstrated to prevent allogeneic response and lysis by NK cells [41]. Recently, Deuse et al. looked into the expression of genes in syncytiotrophoblast, an interface between fetus and mother, and identified low MHC class I and II expression and a high CD47 expression as the features that are responsible for the immune tolerance of syncytiotrophoblast toward allogenic fetal antigens [42]. CD47 is a membrane protein that interacts with several cell surface receptors to inhibit phagocytosis [43]. Indeed, CD47 is a "don't eat me" signal highly expressed on the surface of cancer cells to escape the innate immune responses [44]. The authors then inactivated MHC class I and II genes through CRISPR-Cas9 targeting and overexpressed CD47 via lentiviral transduction in both human and mouse iPSCs [43]. Importantly, the engineered iPSCs and derivatives (endothelial cells, smooth muscle cells, and cardiomyocytes) lost their immunogenicity and persisted long term in fully MHC-mismatched recipients without the use of immunosuppression [43]. This suggests that hypoimmunogenic cell grafts can be engineered from iPSCs for universal transplantation without immunosuppression. These approaches are associated with potential risks of uncontrollable malignant transformation or impaired immune reactions using hypoimmunogenic cell grafts, and consideration of designing an inducible killing switch in the engineered cells to ensure overall safety should be taken into account.
