**1.1. Allogeneic islet transplantation**

treat the disease [2,3,4]. It is, however, apparent that islet transplantation is not currently a viable option for the treatment of all potential recipients, due to the limited source of islet cells, i.e. limited number of available donors. Another feature of islet transplantation, as currently performed, is the requirement for life-long immunosuppression that limits the patients' eligibility to individuals with the most severe cases of IDDM. These issues have driven the investigation of alternative cell sources, which include xenografts from other species, embry‐ onic and adult stem cells, and gene therapy products. Such therapies will also likely require immunological protection provided by means such as conventional immunosuppression, administration of immunomodulatory cell subsets or a combination and manipulation of the islets by shielding and/or encapsulation, which can protect transplanted cells from recognition

minor part of the organ, i.e. 2-3 % of the pancreatic tissue. Islets designated for transplantation must be isolated from the whole pancreas using the method that combines enzymatic digestion with mechanical disruption. Despite considerable improvements made in the islet isolation process (the process itself, the reagents used during the procedure), that led to improved quantity and quality of islet preparations, it still remains a largely inefficient process. Clinical symptoms of Type 1 diabetes do not develop until 60-80% of the β-cell mass is lost to the autoimmune attack [5]. This means that adequate glycemic control can be maintained with as little as 20-40% of the normal β-cell mass. Intrahepatic islet transplantation is the accepted gold standard at the present time. Ample scientific evidence suggests [4] that a significant number of islets are lost during the immediate post-transplant period, mostly due to the inflammation and thrombosis following initial islet-blood contact and activation of hepatic microenviron‐ ment. Thus, if the goal of islet transplantation is to replace 1 x 106 islets to achieve long-term normoglycemia, several donors may be requires for each recipient. In fact, it has been previ‐ ously demonstrated [2,3] that insulin independence is achieved with ≥13,000 islet equivalents (IEQ)/kg of recipient body weight, using more than one islet preparation per recipient, at the same time or in succession. This means that a single islet transplant may require 3-4 donor pancreata. At the present time, the only source of islet cells are pancreata obtained from a deceased, heart-beating, brain-dead donor. This type of donor, especially of suitable age, is rare, making current protocols for human islet transplantation an unlikely candidate for widespread treatment for patients with IDDM. In the US alone, there are approximately 2 million people diagnosed with Type 1 diabetes. This demand is driving the current research trends into alternative functionally competent, i.e. insulin secreting and sensing, β -cell sources

A number of different cell types have been proposed as a starting material to generate sufficient cell mass for transplantation; these include insulin-secreting cell lines, non-β-cell sources engineered through gene therapy, β-cells from non-human species, and β-cells generated from adult (bone marrow, pancreas, liver and neural tissue) and embryonic stem cells [5]. Regardless of the cell source, i.e. β- or non-β cells, many agree that the optimal treatment for Type 1 diabetes should ideally consist of an autologous cell source, which can synthesize, store and release insulin in a highly regulated fashion to maintain glucose homeostasis. Too much or too

) islet cells, which represent a

by the immune system and, in particular, from recurrence of autoimmunity.

An adult pancreas contains approximately one million (1 x 106

584 Type 1 Diabetes

as potential replacement therapies for IDDM.

Diabetes Mellitus (DM) poses a significant challenge in the United States and around the world. It's increasing in prevalence and, at the present time, affects almost 20 million peo‐ ple in the United States alone [1]. DM is considered to be the sixth leading cause of death in the USA and is a major morbidity hazard [6,7] because of its associated complications that may negatively impact a patient's quality of life. Presently, the disease lowers aver‐ age life expectancy by about 15 years, increases cardiovascular disease (CVD) risk by about two- to four-fold, and is the main cause of kidney failure, lower limb amputations, and adult-onset blindness. DM is a costly disease: its estimated attributable costs in 2010 were approximately 135 billion dollars [6].

IDDM has an early childhood or young adulthood onset, although it can be diagnosed at any age. It is characterized by profound deficiency in insulin secretion caused by the autoimmune destruction of insulin-producing cells in the pancreas, the pancreatic β-cells. IDDM accounts for approximately 5-10% of all disease cases. Factors that have been associated with the development of Type 1 DM are both genetic and environmental [8-10]. In animal models such as the NOD mouse and BB rat, and in human Type 1 diabetes, there is strong evidence of a role of the class II gene, I-A in NOD mouse (equivalent to human DQ beta gene), most probably in combination with lack of I-E expression (equivalent to human DR) [11]. Although it is entirely possible that the genetic response can be triggered by environmental factors such as infections or drastic change in diet, the clear definition of such factors has been elusive to date [11]. Ultimately, though, it is the autoimmune component of Type 1 diabetes that is responsible for the progressive and selective autoimmune destruction of insulin-producing β-cells in the pancreas. Due to the fact that the disease is the result of the loss of a single cell type, i.e. β-cell, it is considered to be amenable to treatment by cell replacement therapy.

The discovery of insulin in 1922 by the Canadian physician Frederic Banting brought about the realization that it was the pancreas that produced the "sugar-reducing sub‐ stance" [1], i.e. insulin. Since then scientists have been interested in how this hormone is synthesized and secreted, and the main therapeutic approach to IDDM has been focused on insulin replacement. Until recently, the only available treatment for Type 1 diabetes was the administration of exogenous insulin. The Diabetes Control and Complications Tri‐ al (DCCT) [12] demonstrated that, in patients with Type 1 diabetes, intensive insulin re‐ placement therapy can control blood glucose levels to a certain extent [12]. Unfortunately, even intensive care it is not able to mimic normal hormone release that regulates glucose homeostasis [8] and results in the fine-tuned physiological balance [13]. Even in patients with good glycemic control achieved through intensive insulin therapy blood glucose lev‐ els can vary greatly outside the normal range [13]. In addition, tight control of blood glu‐ cose levels often results in frequent episodes of hypoglycemia. The DCCT trial [12] clearly demonstrated that although intensive insulin therapy is able to delay the onset of diabe‐ tes-associated complications, it doesn't result in complete prevention of their development [12,13]. It is also not clear as to how early in the progression of the disease glucose ho‐ meostasis must be restored to affect a near-positive outcome.

In the 1990's, the International Islet Registry [25] reported that 10% of the patients receiving allogeneic islet grafts could maintain insulin independence at ≥1 year following transplant. Although the majority of transplant recipients continued to require some exogenous insulin, their daily insulin intake was reduced, HbA1c decreased, and they reported fewer episodes of hypoglycemia unawareness. At this point, transplantation of allogeneic islet cells became a reality. However, questions related to partial graft function and eventual graft failure due to recurrence of auto-immunity or rejection - both difficult to predict - remained. Animal studies of glucose metabolism in rat [26], dog [27-29] and cynomolgus monkey [30,31] models demonstrated that long-term normoglycemia could be achieved provided that a sufficient islet mass was transplanted. These studies also showed that, in dog and simian models, the site of implantation did not play a significant role in graft failure. These findings demonstrated that islet transplantation could be successful, and represented a sustainable cell-based treatment

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Of significant, positive impact was the introduction of the Ricordi automated method for islet isolation [32] which allowed for continuous release of large numbers of islet cells during the digestion phase, protecting them from any further enzymatic action, thereby preventing overdigestion of the islet tissue, and significantly reducing islet cell loss as a result of the isolation process. The digestion process was allowed to proceed until only a fibrous network of ducts and vessels of the pancreas remained. In contrast with previous methods utilized to isolate human islets, the Ricordi method allowed for the digestion of the whole pancreas and a significant improvement in the quantity and quality of the isolated cells [32]. Introduction of more efficient enzyme blends [33,34], development of more effective organ preservation methods [35-37], effective use of semi-automated large-scale purification techniques [38-41], and the introduction of additional reagents during various phases of islet cell processing, all contributed to the improved islet recovery and the utilization of islet preparations for trans‐ plantation. Islet preparations can be transplanted fresh, i.e. immediately following isolation, or following culture [2,3], which is of substantial benefit. This window offers sufficient time for both the detailed characterization and quality assessment of the islet preparation, and

Islet preparations of various degree of purity are normally implanted into the recipient's liver portal vein by transhepatic cannulation using minimally invasive interventional radiological techniques [42-47]. This approach has been demonstrated to be safe, is associated with low morbidity and is well tolerated by the transplant recipients. In fact, when additional islet mass is required to improve recipient's metabolic control, additional preparations of islet cells are

New immunosuppressive protocols designed for the recipients of solitary islet allografts, i.e. islet transplant alone (ITA), and the publication of the results of the Edmonton Protocol in the year 2000 [2,3] lead to further improvement in the clinical outcomes reported by a number of centers [42,48-50]. These new protocols moved away from the use of glucocorticoids and calcineurin inhibitors (CNI, cyclosporin A (CyA)) that have diabetogenic effects, and potential islet toxicity [2,3,42,48-50]; and utilized alternative strategies as immunosuppressive therapy. On-going clinical studies clearly demonstrated that allogeneic islet transplantation has the

shipment of the cells to satellite transplant centers, when necessary.

delivered using the same route of administration.

for patients with Type 1 diabetes.

Thus, the need for alternative or additional therapies has been apparent for some time. Endocrine replacement has been but one approach in the quest for tight glycemic control. Achieved either through transplant of a whole pancreas [14] or allogeneic islet cells [13-15], it has been investigated for quite some time now. There is little doubt that pan‐ creas transplants, especially when performed simultaneously with a kidney, favorably im‐ pact metabolic control [14]. Eighty percent of the patients receiving simultaneous kidneypancreas transplants demonstrate good graft function and insulin independence at one year following surgery, with 50% of the recipients maintaining euglycemia at 5 years [16]. Pancreas transplantation results in independence from exogenous insulin, normalization of glucose levels (both fasting and post-prandial), normal Hemoglobin A1c (HbA1c) lev‐ els, and freedom from hypoglycemia [16]. However, pancreas transplantation is still asso‐ ciated with significant morbidity and mortality rates [16-18]. Thus, most patients with Type 1 diabetes are not candidates for pancreas transplantation.

In contrast, islet transplantation requires only a safe interventional radiology technique to implant the graft, and doesn't require general anesthesia, does not call for post-transplant management of pancreatic secretions, and is not associated with post-transplant morbidity and mortality. In addition, in patients with Type 1 diabetes, pancreatic exocrine tissue, which represents the vast majority of the organ, is not affected. These are all factors that contribute favorably toward a wider application of islet cell transplantation.

Of the 159 islet cell allografts reported to the International Pancreas Transplant Registry [18] in 1983, none resulted in insulin independence that could be clearly linked to the implanted graft. These unsatisfactory results could be attributed to the suboptimal islet isolation methods and variable immunosuppressive regiments utilized at the time. It is now apparent that islet isolation methods used at that time - originally developed for the isolation of rat islets by Moskalewski19 and further improved upon by Lacy [20] - were not entirely adequate for the isolation of human islet cells. The use of unpurified islet preparations was not particularly safe, resulting in reported cases of portal hypertension and even death [21].

Introduction of collagenase through the pancreatic duct during the distension of the organ, and purification of the islet cells from the exocrine tissue using discontinuous Ficoll gradients [22, 23] resulted in the optimization of the islet isolation method, i.e. improved isolation yield and islet purity of up to 90% [9]. These continued improvements in the islet isolation meth‐ odology provided a new impetus to continued attempts at islet transplantation during the 1980's. Although none of the islet allografts resulted in insulin independence, clinical trials conducted during this period proved islet cell transplantation to be safe, and for the first time demonstrated a sustained C peptide production [24].

In the 1990's, the International Islet Registry [25] reported that 10% of the patients receiving allogeneic islet grafts could maintain insulin independence at ≥1 year following transplant. Although the majority of transplant recipients continued to require some exogenous insulin, their daily insulin intake was reduced, HbA1c decreased, and they reported fewer episodes of hypoglycemia unawareness. At this point, transplantation of allogeneic islet cells became a reality. However, questions related to partial graft function and eventual graft failure due to recurrence of auto-immunity or rejection - both difficult to predict - remained. Animal studies of glucose metabolism in rat [26], dog [27-29] and cynomolgus monkey [30,31] models demonstrated that long-term normoglycemia could be achieved provided that a sufficient islet mass was transplanted. These studies also showed that, in dog and simian models, the site of implantation did not play a significant role in graft failure. These findings demonstrated that islet transplantation could be successful, and represented a sustainable cell-based treatment for patients with Type 1 diabetes.

els can vary greatly outside the normal range [13]. In addition, tight control of blood glu‐ cose levels often results in frequent episodes of hypoglycemia. The DCCT trial [12] clearly demonstrated that although intensive insulin therapy is able to delay the onset of diabe‐ tes-associated complications, it doesn't result in complete prevention of their development [12,13]. It is also not clear as to how early in the progression of the disease glucose ho‐

Thus, the need for alternative or additional therapies has been apparent for some time. Endocrine replacement has been but one approach in the quest for tight glycemic control. Achieved either through transplant of a whole pancreas [14] or allogeneic islet cells [13-15], it has been investigated for quite some time now. There is little doubt that pan‐ creas transplants, especially when performed simultaneously with a kidney, favorably im‐ pact metabolic control [14]. Eighty percent of the patients receiving simultaneous kidneypancreas transplants demonstrate good graft function and insulin independence at one year following surgery, with 50% of the recipients maintaining euglycemia at 5 years [16]. Pancreas transplantation results in independence from exogenous insulin, normalization of glucose levels (both fasting and post-prandial), normal Hemoglobin A1c (HbA1c) lev‐ els, and freedom from hypoglycemia [16]. However, pancreas transplantation is still asso‐ ciated with significant morbidity and mortality rates [16-18]. Thus, most patients with

In contrast, islet transplantation requires only a safe interventional radiology technique to implant the graft, and doesn't require general anesthesia, does not call for post-transplant management of pancreatic secretions, and is not associated with post-transplant morbidity and mortality. In addition, in patients with Type 1 diabetes, pancreatic exocrine tissue, which represents the vast majority of the organ, is not affected. These are all factors that contribute

Of the 159 islet cell allografts reported to the International Pancreas Transplant Registry [18] in 1983, none resulted in insulin independence that could be clearly linked to the implanted graft. These unsatisfactory results could be attributed to the suboptimal islet isolation methods and variable immunosuppressive regiments utilized at the time. It is now apparent that islet isolation methods used at that time - originally developed for the isolation of rat islets by Moskalewski19 and further improved upon by Lacy [20] - were not entirely adequate for the isolation of human islet cells. The use of unpurified islet preparations was not particularly safe,

Introduction of collagenase through the pancreatic duct during the distension of the organ, and purification of the islet cells from the exocrine tissue using discontinuous Ficoll gradients [22, 23] resulted in the optimization of the islet isolation method, i.e. improved isolation yield and islet purity of up to 90% [9]. These continued improvements in the islet isolation meth‐ odology provided a new impetus to continued attempts at islet transplantation during the 1980's. Although none of the islet allografts resulted in insulin independence, clinical trials conducted during this period proved islet cell transplantation to be safe, and for the first time

meostasis must be restored to affect a near-positive outcome.

586 Type 1 Diabetes

Type 1 diabetes are not candidates for pancreas transplantation.

favorably toward a wider application of islet cell transplantation.

resulting in reported cases of portal hypertension and even death [21].

demonstrated a sustained C peptide production [24].

Of significant, positive impact was the introduction of the Ricordi automated method for islet isolation [32] which allowed for continuous release of large numbers of islet cells during the digestion phase, protecting them from any further enzymatic action, thereby preventing overdigestion of the islet tissue, and significantly reducing islet cell loss as a result of the isolation process. The digestion process was allowed to proceed until only a fibrous network of ducts and vessels of the pancreas remained. In contrast with previous methods utilized to isolate human islets, the Ricordi method allowed for the digestion of the whole pancreas and a significant improvement in the quantity and quality of the isolated cells [32]. Introduction of more efficient enzyme blends [33,34], development of more effective organ preservation methods [35-37], effective use of semi-automated large-scale purification techniques [38-41], and the introduction of additional reagents during various phases of islet cell processing, all contributed to the improved islet recovery and the utilization of islet preparations for trans‐ plantation. Islet preparations can be transplanted fresh, i.e. immediately following isolation, or following culture [2,3], which is of substantial benefit. This window offers sufficient time for both the detailed characterization and quality assessment of the islet preparation, and shipment of the cells to satellite transplant centers, when necessary.

Islet preparations of various degree of purity are normally implanted into the recipient's liver portal vein by transhepatic cannulation using minimally invasive interventional radiological techniques [42-47]. This approach has been demonstrated to be safe, is associated with low morbidity and is well tolerated by the transplant recipients. In fact, when additional islet mass is required to improve recipient's metabolic control, additional preparations of islet cells are delivered using the same route of administration.

New immunosuppressive protocols designed for the recipients of solitary islet allografts, i.e. islet transplant alone (ITA), and the publication of the results of the Edmonton Protocol in the year 2000 [2,3] lead to further improvement in the clinical outcomes reported by a number of centers [42,48-50]. These new protocols moved away from the use of glucocorticoids and calcineurin inhibitors (CNI, cyclosporin A (CyA)) that have diabetogenic effects, and potential islet toxicity [2,3,42,48-50]; and utilized alternative strategies as immunosuppressive therapy. On-going clinical studies clearly demonstrated that allogeneic islet transplantation has the potential to become a viable therapy for patients with severe forms of Type 1 diabetes. However significant challenges need to be overcome before islet transplantation can be considered as the treatment of choice.

the PERV scare may have been overestimated: long-term immunosuppressive regimens and exposure to porcine islet grafts did not result in any detectable PERV transmission. These data clearly showed (a) no expression of PERV in porcine islets in either *in vivo* or *in vitro* studies, and (b) no integration of PERV sequences into recipient cell or organs [55, 57-58]. Additionally, Koulmanda et al successfully demonstrated that, following anti-CD4 treatment, pig islet grafts became resistant to autoimmune destruction in non-obese diabetes (NOD) recipients, sug‐ gesting that CD4-mediated autoimmunity, rather than hyper-acute immunological response,

Cell Replacement Therapy in Type 1 Diabetes

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589

It is also difficult to overlook the fact that large numbers of porcine islets can be isolated with considerable ease, using protocols similar to those developed for the bulk isolation of human islet cells [32,60]. Since the introduction of highly efficient semi-automated methods for bulk islet isolation of pig pancreatic islets by Ricordi et al [32], the quantity and quality of islet preparations from a pig donor has been consistently higher. Pig donors are healthy and avoid cell senescence due to various co-morbidities, brain death, and cold and warm ischemia injury, as these factors can be controlled and, under normal circumstances, kept to a minimum [60,61]. Using standard purification methods [61] a purity of 70-90% of islet can be achieved. Islet cells isolated from adult pigs are functionally competent, and graft function can be recorded shortly following transplantation [61]. In addition, adult pig islets have appropriate glucose-sensing and insulin release mechanisms, as demonstrated by prolonged diabetes reversal when porcine islets were transplanted into nonhuman primates [534,55]. However, the fragile nature of adult porcine islets leads to significant loss as a result of ischemia and inflammation, during cell culture and early engraftment process. It also makes it challenging to maintain them in culture, and may result in the loss of a significant proportion of cells following isolation. Although a reduction in islet mass and cell viability has been reported when adult porcine islets were maintained in culture, short-term culture is desirable to reduce cell immunogenicity

In comparison to adult pig islets, fetal islets have been isolated with even greater ease. Once isolated these require several weeks of culture to facilitate re-aggregation of the endocrine tissue and elimination of exocrine tissue, and to mature to glucose-sensing and insulin production [60]. Additionally, immature cells are much more resistant to the ischemia and inflammation-related injury. Fetal islet isolation is very simple and highly reproducible, and can be accomplished using an exogenous solution of digestive enzymes with minimal loss of immature islet cells [61]. This is due to the fact that fetal islet tissue is not prone to ischemic damage, most probably because of its inherent relative lack of exocrine tissue, capable of inducing damage as a result of the release of proteolytic enzymes from damaged exocrine cells [62,63]. At the same time, there is a relative abundance of endocrine tissue which makes the isolation of fetal islets an easier and more efficient process. Additionally, the copiousness of immature precursor cells in the ductal tissue and their possible presence in the islet-like cell clusters (ICCs) that form during culture, contributes to high capacity of ICC tissue for posttransplant proliferation, a key feature lost in adult pig islets [63]. Thus, small numbers of ICCs can eventually produce large-size grafts, provided that rejection, recurrence of autoimmunity and hyperglycemia can be overcome and controlled [63]. Considering the small size of the fetal

might be the cause of the destruction of xenogeneic islet grafts [59].

or combine preparations from several donors, prior to transplant [61].

Some of the critical questions that remain to be addressed include: (i) definition of an adequate supply of donor organs which can meet the existing need; (ii) isolation of a sufficient number of high quality islet cells from the exocrine tissue, which comprises 98-99% of the pancreas; (iii) improvements in the immunosuppressive strategies that are currently used, by either the development of less toxic drugs or the induction of tolerance; (iv) preventing the recurrence of autoimmunity, demonstrated to have successful outcomes in murine models [5]; (v) identifying the early occurrence of immune rejection, which is quite challenging to monitor given the very small volume of the transplanted tissue and our limited ability to characterize the process.

#### **1.2. Islet cells from xenogeneic sources**

At the present time xenogeneic islet cells isolated from pig pancreata offer the most promising alternative to human islets as a treatment for Type 1 diabetes. This is based on a number of observations: (i) there is a large number of facilities in the US with capabilities for highthroughput breeding, rearing and slaughter of pigs; (ii) pig insulin differs from human insulin by just one amino acid and has been successfully utilized as a source of exogenous insulin for many years before the advent of recombinant insulin; (iii) large numbers of islet cells can be isolated from a single pig pancreas using techniques similar to those developed for human islet isolation; (iv) pig donors can be genetically manipulated to increase insulin production, and to protect the islet cells from immune and cytokine assault [5,51].

Several limitations have restricted the use of pig islets in human recipients. The first one is the hyperimmune response, possibly mediated by the galactoseα -1,3-galactose (Gal) epitope. Elimination of this epitope was shown to prevent hyperacute rejection of pig-tononhuman primate solid organ xenografts. Immune protection of xenografts utilizing en‐ capsulation techniques resulted in progressive loss of graft viability and insulin secretion over prolonged period of time, during which transplanted islets were expected to func‐ tion [52]. The second one is the possibility of transmission of porcine endogenous retrovi‐ ruses (PERV), several copies of which are present in the genome of all pigs and able to infect human cell *in vitro*, with unknown consequences [53]. The possibility of novel viral infections in recipients of porcine islet grafts raised serious safety and ethical concerns, as C-type retroviruses related to PERV have been demonstrated to associate with hemato‐ poietic cell malignancies in the natural hosts [53].

The interest in porcine islets peaked when it was demonstrated that T-cell immunomodulatory therapies which target indirect co-stimulatory pathway, i.e. CD28-CD154, supported pro‐ longed engraftment of unmodified pig islet cells in non-human primates [54, 55] Furthermore, published data drew attention to the fact that, in contrast to human islets that produce copious amounts of islet amyloid polypeptide (IAPP) capable of inducing β -cell apoptosis, pig neonatal and adult islets do not form amyloid deposits. This could be due to the fact that pig IAPP is considerably less amyloidogenic [56]. Recently published data, however, have suggested that the PERV scare may have been overestimated: long-term immunosuppressive regimens and exposure to porcine islet grafts did not result in any detectable PERV transmission. These data clearly showed (a) no expression of PERV in porcine islets in either *in vivo* or *in vitro* studies, and (b) no integration of PERV sequences into recipient cell or organs [55, 57-58]. Additionally, Koulmanda et al successfully demonstrated that, following anti-CD4 treatment, pig islet grafts became resistant to autoimmune destruction in non-obese diabetes (NOD) recipients, sug‐ gesting that CD4-mediated autoimmunity, rather than hyper-acute immunological response, might be the cause of the destruction of xenogeneic islet grafts [59].

potential to become a viable therapy for patients with severe forms of Type 1 diabetes. However significant challenges need to be overcome before islet transplantation can be

Some of the critical questions that remain to be addressed include: (i) definition of an adequate supply of donor organs which can meet the existing need; (ii) isolation of a sufficient number of high quality islet cells from the exocrine tissue, which comprises 98-99% of the pancreas; (iii) improvements in the immunosuppressive strategies that are currently used, by either the development of less toxic drugs or the induction of tolerance; (iv) preventing the recurrence of autoimmunity, demonstrated to have successful outcomes in murine models [5]; (v) identifying the early occurrence of immune rejection, which is quite challenging to monitor given the very small volume of the transplanted tissue and our limited ability to characterize

At the present time xenogeneic islet cells isolated from pig pancreata offer the most promising alternative to human islets as a treatment for Type 1 diabetes. This is based on a number of observations: (i) there is a large number of facilities in the US with capabilities for highthroughput breeding, rearing and slaughter of pigs; (ii) pig insulin differs from human insulin by just one amino acid and has been successfully utilized as a source of exogenous insulin for many years before the advent of recombinant insulin; (iii) large numbers of islet cells can be isolated from a single pig pancreas using techniques similar to those developed for human islet isolation; (iv) pig donors can be genetically manipulated to increase insulin production,

Several limitations have restricted the use of pig islets in human recipients. The first one is the hyperimmune response, possibly mediated by the galactoseα -1,3-galactose (Gal) epitope. Elimination of this epitope was shown to prevent hyperacute rejection of pig-tononhuman primate solid organ xenografts. Immune protection of xenografts utilizing en‐ capsulation techniques resulted in progressive loss of graft viability and insulin secretion over prolonged period of time, during which transplanted islets were expected to func‐ tion [52]. The second one is the possibility of transmission of porcine endogenous retrovi‐ ruses (PERV), several copies of which are present in the genome of all pigs and able to infect human cell *in vitro*, with unknown consequences [53]. The possibility of novel viral infections in recipients of porcine islet grafts raised serious safety and ethical concerns, as C-type retroviruses related to PERV have been demonstrated to associate with hemato‐

The interest in porcine islets peaked when it was demonstrated that T-cell immunomodulatory therapies which target indirect co-stimulatory pathway, i.e. CD28-CD154, supported pro‐ longed engraftment of unmodified pig islet cells in non-human primates [54, 55] Furthermore, published data drew attention to the fact that, in contrast to human islets that produce copious amounts of islet amyloid polypeptide (IAPP) capable of inducing β -cell apoptosis, pig neonatal and adult islets do not form amyloid deposits. This could be due to the fact that pig IAPP is considerably less amyloidogenic [56]. Recently published data, however, have suggested that

and to protect the islet cells from immune and cytokine assault [5,51].

poietic cell malignancies in the natural hosts [53].

considered as the treatment of choice.

**1.2. Islet cells from xenogeneic sources**

the process.

588 Type 1 Diabetes

It is also difficult to overlook the fact that large numbers of porcine islets can be isolated with considerable ease, using protocols similar to those developed for the bulk isolation of human islet cells [32,60]. Since the introduction of highly efficient semi-automated methods for bulk islet isolation of pig pancreatic islets by Ricordi et al [32], the quantity and quality of islet preparations from a pig donor has been consistently higher. Pig donors are healthy and avoid cell senescence due to various co-morbidities, brain death, and cold and warm ischemia injury, as these factors can be controlled and, under normal circumstances, kept to a minimum [60,61]. Using standard purification methods [61] a purity of 70-90% of islet can be achieved. Islet cells isolated from adult pigs are functionally competent, and graft function can be recorded shortly following transplantation [61]. In addition, adult pig islets have appropriate glucose-sensing and insulin release mechanisms, as demonstrated by prolonged diabetes reversal when porcine islets were transplanted into nonhuman primates [534,55]. However, the fragile nature of adult porcine islets leads to significant loss as a result of ischemia and inflammation, during cell culture and early engraftment process. It also makes it challenging to maintain them in culture, and may result in the loss of a significant proportion of cells following isolation. Although a reduction in islet mass and cell viability has been reported when adult porcine islets were maintained in culture, short-term culture is desirable to reduce cell immunogenicity or combine preparations from several donors, prior to transplant [61].

In comparison to adult pig islets, fetal islets have been isolated with even greater ease. Once isolated these require several weeks of culture to facilitate re-aggregation of the endocrine tissue and elimination of exocrine tissue, and to mature to glucose-sensing and insulin production [60]. Additionally, immature cells are much more resistant to the ischemia and inflammation-related injury. Fetal islet isolation is very simple and highly reproducible, and can be accomplished using an exogenous solution of digestive enzymes with minimal loss of immature islet cells [61]. This is due to the fact that fetal islet tissue is not prone to ischemic damage, most probably because of its inherent relative lack of exocrine tissue, capable of inducing damage as a result of the release of proteolytic enzymes from damaged exocrine cells [62,63]. At the same time, there is a relative abundance of endocrine tissue which makes the isolation of fetal islets an easier and more efficient process. Additionally, the copiousness of immature precursor cells in the ductal tissue and their possible presence in the islet-like cell clusters (ICCs) that form during culture, contributes to high capacity of ICC tissue for posttransplant proliferation, a key feature lost in adult pig islets [63]. Thus, small numbers of ICCs can eventually produce large-size grafts, provided that rejection, recurrence of autoimmunity and hyperglycemia can be overcome and controlled [63]. Considering the small size of the fetal pancreas, the capability of a small number of ICCs to mature into a functionally competent graft speaks to the use of this tissue. As mentioned above, a major drawback with using functionally immature cells is their delayed function. ICCs require several weeks, and even months, of development before normal glucose levels in the recipient can be achieved, during which time a poor response to physiological glucose has been observed [64]. This on-going hyperglycemic state during the period of functional maturation can lead to possible damage of the transplanted fetal tissue. Thus, while transplanting immature ICCs in diabetic recipients who are early in the course of their disease might not represent a problem, it is potentially a serious drawback for patients with brittle diabetes and declining kidney function [51,62]. A second disadvantage to using ICCs is the high expression of α-1,3-Gal epitope on the surface of fetal islets, making these cells more susceptible to rejection than adult pig islets, which in contrast express little Gal [62].

62]. Reversal of diabetes with prolonged restoration of insulin independence has been achieved in several porcine-to-nonhuman primate xenogeneic transplant models [54,55] in recipients that developed diabetes as a result of chemical treatment, surgical intervention, i.e. pancreo‐ tectomy, or spontaneously. Long-term insulin independence has also been achieved when neonatal and fetal pig pancreatic precursors were implanted intraportaly, subcutaneously, and into the peritoneal cavity [66,67]. The choice of the anatomical implantation site for not only porcine, but human islets is crucial. At the present time, the accepted clinical practice is to deliver islets to the liver, through the portal vein. However, it has been demonstrated that using this route of administration, low oxygen tension, and an active innate immune response that results in complement activation and immediate blood-mediated inflammatory response (IBMIR) contribute to significant islet mass loss in the immediate post-transplant period [68,69]. Different challenges arise when the graft is placed under the kidney capsule, i.e. islets in this case may be damaged by stress as a result of ischemic injury. However, implantation of encapsulated porcine islets under the kidney capsule of non-diabetic Cynomolgus Maca‐ ques resulted in low levels of porcine C-peptide, with islet grafts surviving for up to 6 months [70]. Reports of other implantation sites, such as subcutaneous and peritoneal space, have been published, but both have been reported as relatively immunoreactive [51], unless the islets

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Although most of the data regarding the possible use of porcine islets as an alternative treatment modality for Type 1 diabetes became available as a consequence to the extensive effort undertaken in a number of small and large animal models, a number of reports of controversial clinical trials have been published in the last several years. An Australian biotechnology company, Living Cell Technologies Ltd., reported a clinical trial in Moscow where 10,000 encapsulated porcine islet equivalents (IEQ)/kg recipient body weight isolated from adult virus-free pigs (DiabeCell®) were implanted into several adult recipients with brittle form of Type 1 diabetes, leading to reduced insulin requirements and detectable porcine C-peptide 11 months following transplant [71]. Follow-up dose-finding clinical trial conducted in New Zealand, however, produced less optimistic results. Although a statistically significant reduction in hypoglycemic unawareness was demonstrated, insulin requirements and Cpeptide were reported to be largely unchanged. Additional dose-finding trials using Diabe‐ Cell® are currently in progress. Earlier clinical trials conducted in Mexico utilized neonatal islets co-cultured with Sertoli cells in a collagen-coated device which was implanted subcuta‐ neously into 12 adolescent Type 1 diabetic patients [72]. Although pioneering in nature, this work drew a certain amount of criticism with regard to the ethical implications of conducting clinical trials in countries without strict regulatory oversight, the dearth of relevant pre-clinical data to warrant Phase I clinical trials to assess the safety of the investigational therapy, i.e.

It's hard to dispute potential clinical and commercial implications of porcine xenotransplan‐ tation as a potential therapy for patients with severe forms of Type 1 diabetes. Although not a new idea, recent developments in this field are likely to drive larger, more tightly controlled pre-clinical and clinical studies to explore its enormous potential as a substitute for human islets. However, if xenotransplantation is going to be the way to solve inherent supply

were protected by an immune barrier in the form of a capsule.

porcine islet transplants, as well as the efficacy of the treatment [73].

Neonatal pancreatic cell clusters (NPCCs) obtained from 1-5 day-old piglets can be also easily procured and successfully isolated in a relatively quick and efficient manner [61,65], using culture media supplemented with collagenase. Due to their availability and inherent capacity to differentiate *in vitro* and *in vivo,* NPCCs represent an attractive source of xenogeneic tissue for clinical transplantation. Although freshly isolated cell clusters contain only 7% endocrine cells, 11% epithelial cells, and ~74% exocrine tissue, this content undergoes dramatic transfor‐ mation following a 9-day culture [51,61]. Published data indicates that during *in vitro* culture the acinar tissue undergoes apoptosis resulting in the enrichment of the endocrine component to 35% [61] of the total cellular content, with 25% of the cells capable of insulin production. The rest of the tissue is characterized as non-granulated epithelial cells [61]. *In vitro* culture results in the formation of NPCCs [61,65], as well as the proliferation of β-cell as assessed by studies using bromodeoxyuridine (BrdU) [65]. NPCCs have been demonstrated to be more responsive to glucose challenge compared to the fetal ICCs, but not as fully functional as adult islets [61]. Although NPCCs were showed not to correct diabetes immediately following transplantation, the insulin content of the grafts was reported to increase by ~20 fold [61], confirming either NPCCs' capacity for β-cell proliferation, or differentiation of epithelial precursor cells into β-cells, or both. During the period of hyperglycemia, none of the trans‐ planted immunodeficient mice were lost, suggesting that even in the immediate post-trans‐ plant period NPCCs are capable of producing small, but sufficient, amount of insulin to keep the recipients alive, stopping short of achieving normoglycemia [61]. This speaks to the fact that compared to the adult porcine islets, NPCCs have an extensive *in vivo* and *in vitro* proliferative capacity [61,65], as well as the ability to acquire endocrine function in a timedependent manner. In addition, data showing that NPCC can be successfully and reproducibly transfected with a non-immunogenic, non-pathogenic recombinant AAV demonstrated a possible strategy for gene delivery to improve transplantation outcome [65].

On the other hand, NPCCs require long periods of *in vivo* maturation before developing functional competence [65], which represents a potential draw-back with respect to the clinical utilization of this xenogeneic islet cell source.

Small and large animal models to study the potential clinical use of porcine islet transplanta‐ tion to treat Type 1 diabetes have been developed and success has been reported [54,55,59,61, 62]. Reversal of diabetes with prolonged restoration of insulin independence has been achieved in several porcine-to-nonhuman primate xenogeneic transplant models [54,55] in recipients that developed diabetes as a result of chemical treatment, surgical intervention, i.e. pancreo‐ tectomy, or spontaneously. Long-term insulin independence has also been achieved when neonatal and fetal pig pancreatic precursors were implanted intraportaly, subcutaneously, and into the peritoneal cavity [66,67]. The choice of the anatomical implantation site for not only porcine, but human islets is crucial. At the present time, the accepted clinical practice is to deliver islets to the liver, through the portal vein. However, it has been demonstrated that using this route of administration, low oxygen tension, and an active innate immune response that results in complement activation and immediate blood-mediated inflammatory response (IBMIR) contribute to significant islet mass loss in the immediate post-transplant period [68,69]. Different challenges arise when the graft is placed under the kidney capsule, i.e. islets in this case may be damaged by stress as a result of ischemic injury. However, implantation of encapsulated porcine islets under the kidney capsule of non-diabetic Cynomolgus Maca‐ ques resulted in low levels of porcine C-peptide, with islet grafts surviving for up to 6 months [70]. Reports of other implantation sites, such as subcutaneous and peritoneal space, have been published, but both have been reported as relatively immunoreactive [51], unless the islets were protected by an immune barrier in the form of a capsule.

pancreas, the capability of a small number of ICCs to mature into a functionally competent graft speaks to the use of this tissue. As mentioned above, a major drawback with using functionally immature cells is their delayed function. ICCs require several weeks, and even months, of development before normal glucose levels in the recipient can be achieved, during which time a poor response to physiological glucose has been observed [64]. This on-going hyperglycemic state during the period of functional maturation can lead to possible damage of the transplanted fetal tissue. Thus, while transplanting immature ICCs in diabetic recipients who are early in the course of their disease might not represent a problem, it is potentially a serious drawback for patients with brittle diabetes and declining kidney function [51,62]. A second disadvantage to using ICCs is the high expression of α-1,3-Gal epitope on the surface of fetal islets, making these cells more susceptible to rejection than adult pig islets, which in

Neonatal pancreatic cell clusters (NPCCs) obtained from 1-5 day-old piglets can be also easily procured and successfully isolated in a relatively quick and efficient manner [61,65], using culture media supplemented with collagenase. Due to their availability and inherent capacity to differentiate *in vitro* and *in vivo,* NPCCs represent an attractive source of xenogeneic tissue for clinical transplantation. Although freshly isolated cell clusters contain only 7% endocrine cells, 11% epithelial cells, and ~74% exocrine tissue, this content undergoes dramatic transfor‐ mation following a 9-day culture [51,61]. Published data indicates that during *in vitro* culture the acinar tissue undergoes apoptosis resulting in the enrichment of the endocrine component to 35% [61] of the total cellular content, with 25% of the cells capable of insulin production. The rest of the tissue is characterized as non-granulated epithelial cells [61]. *In vitro* culture results in the formation of NPCCs [61,65], as well as the proliferation of β-cell as assessed by studies using bromodeoxyuridine (BrdU) [65]. NPCCs have been demonstrated to be more responsive to glucose challenge compared to the fetal ICCs, but not as fully functional as adult islets [61]. Although NPCCs were showed not to correct diabetes immediately following transplantation, the insulin content of the grafts was reported to increase by ~20 fold [61], confirming either NPCCs' capacity for β-cell proliferation, or differentiation of epithelial precursor cells into β-cells, or both. During the period of hyperglycemia, none of the trans‐ planted immunodeficient mice were lost, suggesting that even in the immediate post-trans‐ plant period NPCCs are capable of producing small, but sufficient, amount of insulin to keep the recipients alive, stopping short of achieving normoglycemia [61]. This speaks to the fact that compared to the adult porcine islets, NPCCs have an extensive *in vivo* and *in vitro* proliferative capacity [61,65], as well as the ability to acquire endocrine function in a timedependent manner. In addition, data showing that NPCC can be successfully and reproducibly transfected with a non-immunogenic, non-pathogenic recombinant AAV demonstrated a

possible strategy for gene delivery to improve transplantation outcome [65].

utilization of this xenogeneic islet cell source.

On the other hand, NPCCs require long periods of *in vivo* maturation before developing functional competence [65], which represents a potential draw-back with respect to the clinical

Small and large animal models to study the potential clinical use of porcine islet transplanta‐ tion to treat Type 1 diabetes have been developed and success has been reported [54,55,59,61,

contrast express little Gal [62].

590 Type 1 Diabetes

Although most of the data regarding the possible use of porcine islets as an alternative treatment modality for Type 1 diabetes became available as a consequence to the extensive effort undertaken in a number of small and large animal models, a number of reports of controversial clinical trials have been published in the last several years. An Australian biotechnology company, Living Cell Technologies Ltd., reported a clinical trial in Moscow where 10,000 encapsulated porcine islet equivalents (IEQ)/kg recipient body weight isolated from adult virus-free pigs (DiabeCell®) were implanted into several adult recipients with brittle form of Type 1 diabetes, leading to reduced insulin requirements and detectable porcine C-peptide 11 months following transplant [71]. Follow-up dose-finding clinical trial conducted in New Zealand, however, produced less optimistic results. Although a statistically significant reduction in hypoglycemic unawareness was demonstrated, insulin requirements and Cpeptide were reported to be largely unchanged. Additional dose-finding trials using Diabe‐ Cell® are currently in progress. Earlier clinical trials conducted in Mexico utilized neonatal islets co-cultured with Sertoli cells in a collagen-coated device which was implanted subcuta‐ neously into 12 adolescent Type 1 diabetic patients [72]. Although pioneering in nature, this work drew a certain amount of criticism with regard to the ethical implications of conducting clinical trials in countries without strict regulatory oversight, the dearth of relevant pre-clinical data to warrant Phase I clinical trials to assess the safety of the investigational therapy, i.e. porcine islet transplants, as well as the efficacy of the treatment [73].

It's hard to dispute potential clinical and commercial implications of porcine xenotransplan‐ tation as a potential therapy for patients with severe forms of Type 1 diabetes. Although not a new idea, recent developments in this field are likely to drive larger, more tightly controlled pre-clinical and clinical studies to explore its enormous potential as a substitute for human islets. However, if xenotransplantation is going to be the way to solve inherent supply problems with allogeneic organs, a much better understanding of the immunological processes involved in the destruction of xenogeneic tissue is necessary.

Glut-2 and Pdx1, did not exactly resemble that observed in the embryonic pancreas; nor was the transcription of other genes normally expressed in β-cells, such as Nkx2-2 and Nkx6-1, detected in the later stage islet-like clusters. Some of these insulin-producing cell clusters [77], while staining positive for insulin, were - in all likelihood - the result of insulin uptake from the culture medium, rather than activation of robust insulin transcription, as demonstrated by other studies [84]. These data pointed to the fact that evidence demonstrating the equivalence of these islet-like cells clusters to mature β-cells was lacking; and that a better understanding of the signaling pathways and transcriptional factors regulating the development of pancreatic

Cell Replacement Therapy in Type 1 Diabetes

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593

In 2005, D'Amour clearly demonstrated that cells closely resembling fully mature native βcells could be generated by replicating the culture conditions that closely mimicked embryonic development [85,86]. Utilizing a step-wise approach, ESCs were first directed into definitive endoderm stage, a pre-requisite for all pancreatic cell types, followed by a pancreatic endo‐ derm, and subsequently into β-cells with an insulin content similar to that observed in native islets [86]. However, similar to fetal β-cells, the resulting cells were able to release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. These studies were followed by others [87-89] in which these cells were implanted into immunocompromised mice half way through the differentiation process. When the cells were allowed to mature *in vivo*, the efficiency of the differentiation process was improved, glucose-responsive insulin secretion observed, and chemically induced diabetes reversed [89]. Progress in this area has been rapid and recently, California-based ViaCyte (previously Novocell) has reported positive preclinical results with Pro-Islet™, a material based on the technology discussed above, in conjunction with a retrievable encapsulation device. On-going efforts to translate this strategy into pre-clinical and clinical applications were supported by a recent \$20 million award from the California Institute of Regenerative Medicine. This works favorably towards ESC-based

Upon demonstration that the mature state of somatic stem cells can be redirected toward the a progenitor state similar to that of ESCs [90-92], the field of stem cell-based strategies has been further expanded and now includes an attractive alternative to ESCs, i.e. induced pluripotent stem cells (iPSCs). Potentially, iPCSs offer a practical solution to the ethical dilemma posed by the destruction of human embryos necessary for the production of ESCs-based cell therapies. These cells, they are virtually undistinguishable from ESCs in terms of their molecular and biological characteristics. They offer a possibility of generating autologous patient-specific cell therapies directed to treat a variety of medical conditions, including diabetes. This means that depending on the specific illness, desired cells can be differentiated from the patient's own cells. This is certainly attractive as the cells designated for re-transplantation would have the same genetic makeup as those of the patient, and would alleviate the challenges posed by the activation of the recipient's immune system that would occur when allogeneic cells are

In 2006, Takahashi et al [90] demonstrated that pluripotent stem (iPS) cells could be generated from mouse fibroblasts by the retrovirus-mediated transfection of four transcription factors, namely Oct3/4, Sox2, c-Myc, and Klf4. Since then, following the main steps of the original β-

β-cell identity during embryogenesis was necessary.

clinical approaches becoming available in the very near future.

transplanted.

#### **1.3. Stem cell as β-cell replacement therapy**

The most promising cell source for β-cell progenitors is embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts during the early stages of embryogenesis. ECSs offer several notable advantages. First, ESCs differ from adult stem cells in that under the right growth and differentiation conditions they have the potential to differentiate into any cell type *in vitro* and *in vivo*, a potential termed pluripotency. Given the capacity for pluripotency, there is an interest to explore guided *in vitro* differentiation into a desired cell type for the purpose of cell replacement therapy, in this case for the treatment of Type 1 diabetes. Second, ESCs' potential to self-renew while maintaining their stem cell properties is of immeasurable advantage, as it allows for unlimited cell expansion, while the cell differentiation capacity is preserved. Given the need for large number of cells for therapeutic applications, this favors ESCs over the cells at more advanced stages of maturation which, in general, are reported to have a much more limited proliferative capacity [74]. Here, of course, certain precautions are necessary. Directed cell differentiation and proliferation also results in the differentiation of associated cell types, which are not necessarily desired and need to be inhibited. This repre‐ sents a challenge. It's been previously postulated that to successfully differentiate a cell type such as insulin-producing β-cells, an ideal protocol would involve culture steps that mimic a differentiation process taking place during normal embryonic development. That involves certain signaling pathways and transcription factors necessary to guide the development of undifferentiated progenitor cells into fully mature, metabolically functional insulin-producing β-cells [74-76].

First attempts to generate insulin producing islet-like cells (IPCs) were centered on the selection of cells positive for nestin, an intermediate filament protein which serves as a neural stem cell/progenitor marker [77-79]. The reason behind the focus on nestin-positive cells is that in some species neural cells, namely brain neurons in Drosophila, are the source of circulating insulin. In addition, insulin gene transcription is found in the vertebrate brain, although it's not clear if vertebrate neurons produce or secrete the actual protein [74,78]. Recent reports, however, have demonstrated that selection of nestin-positive cells from ESCs leads to gener‐ ation of neural cell types [80-82], although differentiation into insulin-producing cells was also achieved. This is consistent with the fact that nestin is a marker of neural and pancreatic exocrine progenitors, but does not indicate endocrine progenitor cells. Attempts to differen‐ tiate brain-derived neural ESCs into insulin-producing cells resulted in the formation of glucose-sensing insulin producing cell clusters following the exposure to multiple signals that regulate *in vivo* islet pancreatic development [83]. Following transplantation into immuno‐ compromised mice islet-like clusters were demonstrated to release insulin and C-peptide. However, the C-peptide content of these islet progenitor clusters was estimated to be 0.3% of the normal level found in isolated human pancreatic β-cells [83], suggesting that the resulting islet-like cell clusters were not bona-fide β -cells. In addition, temporal sequence of expression of gene products active during the development of pancreatic islet cells, such as glucokinase, Glut-2 and Pdx1, did not exactly resemble that observed in the embryonic pancreas; nor was the transcription of other genes normally expressed in β-cells, such as Nkx2-2 and Nkx6-1, detected in the later stage islet-like clusters. Some of these insulin-producing cell clusters [77], while staining positive for insulin, were - in all likelihood - the result of insulin uptake from the culture medium, rather than activation of robust insulin transcription, as demonstrated by other studies [84]. These data pointed to the fact that evidence demonstrating the equivalence of these islet-like cells clusters to mature β-cells was lacking; and that a better understanding of the signaling pathways and transcriptional factors regulating the development of pancreatic β-cell identity during embryogenesis was necessary.

problems with allogeneic organs, a much better understanding of the immunological processes

The most promising cell source for β-cell progenitors is embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts during the early stages of embryogenesis. ECSs offer several notable advantages. First, ESCs differ from adult stem cells in that under the right growth and differentiation conditions they have the potential to differentiate into any cell type *in vitro* and *in vivo*, a potential termed pluripotency. Given the capacity for pluripotency, there is an interest to explore guided *in vitro* differentiation into a desired cell type for the purpose of cell replacement therapy, in this case for the treatment of Type 1 diabetes. Second, ESCs' potential to self-renew while maintaining their stem cell properties is of immeasurable advantage, as it allows for unlimited cell expansion, while the cell differentiation capacity is preserved. Given the need for large number of cells for therapeutic applications, this favors ESCs over the cells at more advanced stages of maturation which, in general, are reported to have a much more limited proliferative capacity [74]. Here, of course, certain precautions are necessary. Directed cell differentiation and proliferation also results in the differentiation of associated cell types, which are not necessarily desired and need to be inhibited. This repre‐ sents a challenge. It's been previously postulated that to successfully differentiate a cell type such as insulin-producing β-cells, an ideal protocol would involve culture steps that mimic a differentiation process taking place during normal embryonic development. That involves certain signaling pathways and transcription factors necessary to guide the development of undifferentiated progenitor cells into fully mature, metabolically functional insulin-producing

First attempts to generate insulin producing islet-like cells (IPCs) were centered on the selection of cells positive for nestin, an intermediate filament protein which serves as a neural stem cell/progenitor marker [77-79]. The reason behind the focus on nestin-positive cells is that in some species neural cells, namely brain neurons in Drosophila, are the source of circulating insulin. In addition, insulin gene transcription is found in the vertebrate brain, although it's not clear if vertebrate neurons produce or secrete the actual protein [74,78]. Recent reports, however, have demonstrated that selection of nestin-positive cells from ESCs leads to gener‐ ation of neural cell types [80-82], although differentiation into insulin-producing cells was also achieved. This is consistent with the fact that nestin is a marker of neural and pancreatic exocrine progenitors, but does not indicate endocrine progenitor cells. Attempts to differen‐ tiate brain-derived neural ESCs into insulin-producing cells resulted in the formation of glucose-sensing insulin producing cell clusters following the exposure to multiple signals that regulate *in vivo* islet pancreatic development [83]. Following transplantation into immuno‐ compromised mice islet-like clusters were demonstrated to release insulin and C-peptide. However, the C-peptide content of these islet progenitor clusters was estimated to be 0.3% of the normal level found in isolated human pancreatic β-cells [83], suggesting that the resulting islet-like cell clusters were not bona-fide β -cells. In addition, temporal sequence of expression of gene products active during the development of pancreatic islet cells, such as glucokinase,

involved in the destruction of xenogeneic tissue is necessary.

**1.3. Stem cell as β-cell replacement therapy**

β-cells [74-76].

592 Type 1 Diabetes

In 2005, D'Amour clearly demonstrated that cells closely resembling fully mature native βcells could be generated by replicating the culture conditions that closely mimicked embryonic development [85,86]. Utilizing a step-wise approach, ESCs were first directed into definitive endoderm stage, a pre-requisite for all pancreatic cell types, followed by a pancreatic endo‐ derm, and subsequently into β-cells with an insulin content similar to that observed in native islets [86]. However, similar to fetal β-cells, the resulting cells were able to release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. These studies were followed by others [87-89] in which these cells were implanted into immunocompromised mice half way through the differentiation process. When the cells were allowed to mature *in vivo*, the efficiency of the differentiation process was improved, glucose-responsive insulin secretion observed, and chemically induced diabetes reversed [89]. Progress in this area has been rapid and recently, California-based ViaCyte (previously Novocell) has reported positive preclinical results with Pro-Islet™, a material based on the technology discussed above, in conjunction with a retrievable encapsulation device. On-going efforts to translate this strategy into pre-clinical and clinical applications were supported by a recent \$20 million award from the California Institute of Regenerative Medicine. This works favorably towards ESC-based clinical approaches becoming available in the very near future.

Upon demonstration that the mature state of somatic stem cells can be redirected toward the a progenitor state similar to that of ESCs [90-92], the field of stem cell-based strategies has been further expanded and now includes an attractive alternative to ESCs, i.e. induced pluripotent stem cells (iPSCs). Potentially, iPCSs offer a practical solution to the ethical dilemma posed by the destruction of human embryos necessary for the production of ESCs-based cell therapies. These cells, they are virtually undistinguishable from ESCs in terms of their molecular and biological characteristics. They offer a possibility of generating autologous patient-specific cell therapies directed to treat a variety of medical conditions, including diabetes. This means that depending on the specific illness, desired cells can be differentiated from the patient's own cells. This is certainly attractive as the cells designated for re-transplantation would have the same genetic makeup as those of the patient, and would alleviate the challenges posed by the activation of the recipient's immune system that would occur when allogeneic cells are transplanted.

In 2006, Takahashi et al [90] demonstrated that pluripotent stem (iPS) cells could be generated from mouse fibroblasts by the retrovirus-mediated transfection of four transcription factors, namely Oct3/4, Sox2, c-Myc, and Klf4. Since then, following the main steps of the original βcell differentiation protocol and retroviral expression of the same four transcription factors, it was reported that differentiation into insulin-producing islet-like clusters was possible. Isletlike clusters were obtained from iPSCs using a serum-free, feeder-free protocol [93]. Following initial reports, a number of modifications to the original protocol have been introduced. These included substituting the originally-described transcription factors with oncogenic potential with stable recombinant proteins [94], episomal constructs [95], DNA minicircles [96], modified mRNAs [97], and small molecule compounds with re-programming properties [98]. Despite these efforts, generation of patient-specific cell lines form iPSCs remains inefficient and expensive, hindering progress in this area. Additionally, there seem to be an inconsistency in the methods utilized for the successful differentiation of insulin-producing islet-like cells, leaving the field open for a much wanted universal protocol utilized to generate a wide variety of patient-specific cell lines, much like that developed by ViaCyte for the Pro-Islet™ technol‐ ogy. Then, of course, the risks inherent to the use of iPSC-and ESC-based approaches must be carefully considered, as these seem to be almost identical.

native pancreatic islets. Extensive efforts should be undertaken to understand whether the same regulatory mechanisms are in place in stem cell-derived insulin producing cells to control prolonged and uncontrolled insulin release which would result in sever hypoglycemia. Only when it is clearly and unequivocally ascertained that stem cell-derived insulin producing cells are true equivalents of endogenous pancreatic β-cells, can clinical application of such therapies

Cell Replacement Therapy in Type 1 Diabetes

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As Type 1 diabetes is an autoimmune disease characterized by the selective and progressive destruction of insulin-producing β-cells via the cumulative attack by autoantigen-specific CD4+ and CD8+ T-cells, autoantibodies, and functionally defective bone marrow derived antigen-specific cells, development of various immunotherapeutic options has been the major focus for prevention and treatment of IDDM. Multiple studies in the NOD mouse model demonstrate that islets are attacked in step-wise manner, with benign insulitis being the starting point of this assault. With time and not fully defined qualitative changes, overt diabetes characterized by the efficient destruction of β-cells ensues. It is generally acknowl‐ edged that diabetes in animal models and men is strongly associated with the changes in more than 20 genetic loci - with genes encoding MHC class II molecules playing the major role, most probably influenced by a number of environmental factors, although it's been quite challeng‐

Animal studies indicate that a number of pathogenic events contribute to the progressive loss of T-cell tolerance to β-cell proteins, and, therefore, expansion of β-cell specific pathogenic CD4+ and CD8+ T-cells. These events seem to take place during the early stages of the preclinical IDDM. These include defective negative selection in the thymus, inefficient peripheral tolerance characterized by low frequencies of IL-4, IL-10 and TGF-β secreting CD4+ T helper 2 (Th2) cells, as well as diminished numbers of "natural" immunoregulatory FoxP3 expressing CD4+CD25+ Regulatory T (Treg) cells and invariant natural killer T (iNKT) cells. These events, coupled with reduced frequency / function of immunoregulatory effector cells within the islets, reduced sensitivity of T cells to immunoregulation, and increased levels of pro-inflammatory cytokines produced by macrophages and dendritic cells (DCs), result in the severe loss in the balance between pathogenic effector and immunoregulatory T cells, especially during the later stages of the disease [99-101]. Effective prevention / treatment strategies for Type 1 diabetes

The progression course of diabetes offers obvious time points for interventional immunother‐ apeutic strategies. The first opportunity is presented during the pre-clinical stages of diabetes, when the goal is to suppress the on-going β-cell autoimmune process and prevent the development of overt diabetes. Undiagnosed at risk individuals - family members of patients with the previously diagnosed diabetes - can be monitored by screening for autoantibodies specific for several autoantigens found in the serum. These include insulin, glutamic acid decarboxylase 65 (GAD65), and insulinoma–associated tyrosine phosphate (IA-2) [102]. The second time point for intervention is at clinical onset, in an attempt to preserve 10-15% of the

**1.4. Immunotherapy for the prevention and treatment of Type 1 diabetes**

become a reality.

ing to identify either in detail [11].

must focus on the restoration of this balance..

β-cell mass that is usually still present at the time of diagnosis.

First, there are the reports of teratoma formation when undifferentiated (cultured *in vitro* for ~12 days) ESCs are utilized in pre-clinical models [89]. Interestingly, when cells cultured under similar conditions for extended period of time were utilized [87], no teratoma formation was observed in recipient animals suggesting that more extensively differentiated ESCs lose their ability for neoplastic transformation. Hence, teratoma formation can probably be controlled through elimination of less differentiated cells via advanced purification techniques, as well as more efficient machinery for cell differentiation.

Another critical aspect that deserves serious consideration is related to the full cell complement present at the final ESC differentiation stages. Transplantation of pancreatic progenitor cells results in the development of not only the endocrine cell types, the full complement of which are probably required for fully functional islet structures, but also the exocrine pancreas, i.e. acinar and ductal cells, albeit at much lower frequency [89]. The presence of these cells that have the ability to produce and release various enzymatically active proteins is worrisome. In addition, under conditions of stress caused by inflammation and injury, acinar cells can develop into cells with progenitor-like activity, able to result in neoplastic lesions as a result of oncogenic mutations. While the possibility of such events is small, detailed investigation into these issues needs to continue to assure that the function of ESC-derived endocrine cells are not compromised by the cancer-related risks associated with exocrine cell populations.

Another issue that needs to be explored is the immune response of the host following trans‐ plantation of an allogeneic ESC-derived cellular graft. In the last decade or so sophisticated immunosuppressive regiments have been developed to protect allogeneic islet grafts obtained from deceased donors [46-50] long term, following transplantation. In case of ESC-related therapies, not only the graft must be protected from the immune insult by the recipient's immune system, but the cells with tumorigenic capacity need to be isolated and sequestered. This can be, most probably, achieved with the use of sophisticated immunoisolation / encap‐ sulation devices that have become available in the last few years [4].

Finally, what needs to be ascertained is the fact that stem-cell derived β-cells have the same ability to synthesize, store and release insulin in a highly regulated fashion similar to that of native pancreatic islets. Extensive efforts should be undertaken to understand whether the same regulatory mechanisms are in place in stem cell-derived insulin producing cells to control prolonged and uncontrolled insulin release which would result in sever hypoglycemia. Only when it is clearly and unequivocally ascertained that stem cell-derived insulin producing cells are true equivalents of endogenous pancreatic β-cells, can clinical application of such therapies become a reality.
