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Animal Models to Study Adult Stem Cell-derived, In Vitro-generated Islet Implantation Ammon B. Peck, Li Yin, and Vijayakumar Ramiya Abstract Type 1 diabetes (T1D) is an autoimmune disease characterized by hyperglycemia following the destruction of the insulin-producing beta cells of the pancreatic islets of Langerhans by the body’s own immune system. Although routine insulin injections can provide diabetic patients with their daily insulin requirements, this treatment is not always effective in maintaining normal glucose levels. A true “cure” is considered possible only through replacement of the beta cell mass, by pancreas transplantation, islet implantation, or implantation of nonendocrine cells modified to secrete insulin. With the recent success of islet implantation to reverse T1D, this procedure has become a welcome therapy for T1D patients. Unfortunately, this procedure is hampered by the limited number of transplantation quality pancreata available for the harvesting of islets. This shortage has sparked great interest in finding a replacement for organ donation, primarily the possible use of stem cellderived islets starting with stem cells, or alternatively the harvesting of nonhuman islets. This review focuses on progress with growing islets in the laboratory from stem cells and a comparison between this developing technology and the current use of islets harvested from nonhuman sources. Key Words: in vitro-generated islets; islet implantation; pancreatic stem cells; type 1 diabetes; xenogeneic transplantation Introduction D iabetes mellitus refers to a group of chronic metabolic diseases characterized by hyperglycemia due to the absence of or resistance to insulin. This state may result from defects in insulin secretion, insulin action, or Ammon B. Peck, Ph.D., is a Professor, and Li Yin, M.D., is an Assistant Research Professor, in the Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida. Vijayakumar Ramiya, Ph.D., is an Assistant Professor in the Department of Pediatrics, University of Florida College of Medicine, Gainesville. All authors are affiliated with, and have a financial interest in, Ixion Biotechnology, Inc., Alachua, Florida. Volume 45, Number 3 2004 both (Harris 1999). Type 1 diabetes (T1D1), also known as juvenile-onset, autoimmune, or insulin-dependent diabetes, is a common chronic disease of children and adolescents in both Europe and North America, and an increasing problem elsewhere. The recent implementation of new genetic screening programs for families and newborns to identify “high-risk” individuals has revealed an apparent increase in the incidence of T1D worldwide. T1D is an especially insidious disease because clinical symptoms are usually not recognized until after the patient’s own immune system has destroyed more than 90% of the total insulin-producing beta cells of the endocrine pancreas (Eisenbarth 1986). Although routine insulin injections can provide diabetic patients with their daily insulin requirements, blood glucose excursions are common and result in hyperglycemic episodes. Hyperglycemia currently represents the major health problem for the diabetic patient. When inadequately controlled, chronic hyperglycemia can lead to microvascular complications (i.e., retinopathy and blindness, nephropathy and renal failure, neuropathy, foot ulcers and amputation) and/or macrovascular complications (i.e., atherosclerotic cardiovascular, peripheral vascular, and cerebrovascular disease). Both the Diabetes Control and Complications Trial and the UK Prospective Diabetes Study demonstrated a strong relation between good metabolic control and the rate/progression of complications (DCCT Research Group 1993; UKPDS Group 1998). Unfortunately, it is difficult for most patients to achieve adequate control of hyperglycemic excursions, and attempts to maintain euglycemia through intensive insulin treatment lead to increased incidences of hypoglycemia. The lack of tight glycemic control resulting in acute and chronic metabolic complications costs the healthcare system in the United States more than one hundred billion dollars per year and highlights the urgency for a cure. A true “cure” for T1D relies on replacement of the beta cell mass. Currently, beta cell replacement is accomplished either by ectopancreas transplantation or islet implantation. Pancreas organ transplantation is often successful in normalizing fasting and postprandial blood glucose levels, HbA1C levels, as well as secretion of insulin and C-peptide 1 Abbreviations used in this article: AcXR, acute cellular xenograft rejection; AhXR, acute humoral xenograft rejection; HAR, hyperacute reaction; IPC, islet progenitor cell; IPSC/IPC, islet-producing stem cells/islet progenitor cells; NOD, nonobese diabetic; PERV, procine endogenous retrovirus; T1D, type 1 diabetes. 259 in response to glucose. However, this procedure requires long-term immunosuppression, thereby restricting it generally to patients who have end-stage renal disease and are listed to receive a second organ transplant, or to those with long-standing T1D who have failed insulin therapy. Islet implantation has several notable advantages over pancreatic organ transplantation: 1. The islets can be implanted by percutaneous catheterization of the portal vein under local anesthesia, thus relieving the patient from undergoing general anesthesia and invasive surgery. 2. Theoretically, islets can be genetically manipulated in vitro to resist immune attack, either by reducing potential islet-associated antigens or by expressing immunomodulators. 3. The islets can be encapsulated as a way to protect them from the immune system, but still permit insulin secretion (Drachenberg et al. 1999; Lanza and Chick 1997; Scharp et al. 1994; Sutherland et al. 1996). Currently, however, islet cell implantations also require immunosuppression and have been severely limited by a shortage of implantable islets from donated cadaveric pancreata. This shortage is underscored by the recent estimation that mortality rates for patients waiting for pancreatic grafts or pancreatic plus kidney grafts are 42.2 and 36.2 per 1000 patients, respectively (UNOS 2002). Interestingly, the recent success using new islet implantation protocols has popularized this intervention, a situation that no doubt has the potential to exacerbate the shortage of implantable islets further (Bertuzzi et al. 2003; Bretzel et al. 2001; Ricordi et al. 2003; Ryan et al. 2001). The possibility that stem cell-derived, in vitro-generated islets may soon be an alternative to cadaver-derived islets for treating diabetic patients is gaining acceptance, and has been the topic of several recent reviews (Bonner-Weir and Sharma 2002; Bretzel 2003; Efrat 2002; Lechner and Habener 2003; Peck et al. 2001; Peshavaria and Pang 2000; Soria 2001). Over the past few years, several studies have reported the production of endocrine cells, endocrine tissues and/or islet-like clusters from embryonic stem cells, adult stem cells isolated from the pancreas, as well as adult stem cells from nonpancreas origins (Table 1). Although none of these products are considered perfect yet, major advances have been made in understanding the regulation of growth and differentiation of beta cells and the islets of Langerhans (reviewed in Wilson et al. 2003), thus raising hope for this science to reach the diabetic patient in the near future. In Vitro Generation of Islet-like Clusters from Adult Pancreas—The Human Model As early as the mid-1990s, Peck and coworkers presented evidence indicating that immature, yet functional, islet-like clusters could be grown in vitro from stem/progenitor cells isolated from the pancreatic ducts and islets presumed to contain epithelial stem cells from which islets of Langerhans are derived during embryogenesis (Cornelius et al. 1997; Peck and Cornelius 1995). The protocol that proved most successful consisted of several sequential steps that permitted stem cell/progenitor cell selection with subsequent successive growth, differentiation, and maturation of the cultured cell populations. Today, this process has been divided into four steps: 1. Ductal epithelial cells (in combination with islets), isolated from digested pancreas, are cultured in a growthrestrictive medium to enrich for epithelial cell subpopulations that can form monolayers with a neuroendocrine cell-like phenotype (referred to as isletproducing stem cells, or IPSCs1). 2. Islet progenitor cells (referred to as IPCs1) are induced to bud from the epithelial-like monolayers. Table 1 Stem cells capable of forming endocrine-like tissues and cells Source of stem/ progenitor cells Differentiated tissue References (see text) Mouse embryo Insulin-secreting cells, endocrine hormone-secreting cells and islet-like clusters Human embryo Insulin-secreting cells and islet-like clusters Adult mouse pancreas Endocrine hormone-secreting cells and islet-cell clusters Islet-like clusters Endocrine-hormone-producing cells, ductal cell buds and islet-cell clusters Islet-cell clusters Islet-cell clusters Kahan et al. 2003; Kim et al. 2003; Lumelsky et al. 2001; Soria et al. 2000 Assady et al. 2001; Shiroi et al. 2002 Cornelius et al. 1997; Ramiya et al. 2000 Peck and Ramiya 2003 Bonner-Weir et al. 2000; Peck et al. 2001 Zulewski et al. 2001 Yang et al. 2002 Adult pig pancreas Adult human pancreas Adult rat and human islets Adult rat liver oval cells 260 ILAR Journal 3. Given a favorable growth medium that permits proliferation of IPCs and their daughter cells, islet-like cell clusters will form that contain various endocrine cells in various differentiation states and that are capable of exhibiting regulated insulin responses to glucose challenges. 4. The islet-like structures are implanted into an in vivo environment to promote final maturation of the islet cells. The three in vitro stages of growth of islet-like clusters are presented in Figure 1A. The establishment of islet-forming cultures from human pancreata is highly reproducible despite variations in the health status and quality of donated pancreata. However, one drawback to working with human cultures is the difficulty in carrying out required efficacy and toxicity studies to determine the behavior of stem cell-derived, in vitrogenerated islets when implanted into an appropriate in vivo environment. Thus, many aspects of using cells and tissues grown from human stem cells must be extrapolated from animal studies and/or studies involving implantations across species. As a result, we turned originally to the nonobese diabetic (NOD1) mouse model of T1D (Atkinson and Leiter 1999) to investigate a number of questions that might apply to the use of in vitro-generated islets in treating diabetic patients. In Vitro Generation of Islets from Adult Pancreatic Stem Cells—The NOD Mouse Model We have used the NOD mouse as a model for human T1D. In our studies, we have shown the following: the feasibility of obtaining IPSCs from prediabetic, postdiabetic, or normal adult mice; the ability of duplicating the human IPSC/ IPC cultures by establishing stem cell cultures for the growth of immature functional islet-like structures; and the potential of in vitro-generated mouse islets to reverse insulin-dependent diabetes when implanted into diabetic NOD mice (Ramiya et al. 2000). In Figure 1B, the temporal growth and differentiation of epithelial monolayers derived from isolated mouse pancreatic ducts plus islets are shown, revealing the growth characteristics observed with human IPSC/IPC cultures. These monolayers contain IPSCs capable of producing immature islet cell-like clusters that are similar (but not identical) to the human-derived IPSC/IPC cultures. We have noted that the immature islets maintain their structural integrity ranging from a few days to several weeks, but they can easily be dissociated into single cell suspensions that, if recultured, start the process again. Thus, the clusters contain cells retaining the IPSC/IPC phenotype, including the capacity for self-renewal, a phenomenon shown to be true for adult islets freshly isolated from the pancreas (Zulewski et al. 2001). Interestingly, the IPCs appear to bud from the epithelial Volume 45, Number 3 2004 cells (IPSCs), and then proliferate into the threedimensional cell clusters. However, mouse islet cell-like clusters appear to be more uniform and stable than human islet cell-like clusters. The mouse IPSC/IPC cultures have been maintained up to 3 yr through constant expansion via repeated serial transfers. Each subculture exhibited the ability to produce increasing numbers of islet-like structures. Based on the number of these immature islets produced within the secondary cultures, we calculated that between 10,000 and 15,000 pancreas equivalents were produced within the 3 yr of growth of a single cell line from five pancreata used for initiating the primary culture. It is critical to note that stem cell-derived, in vitro-grown islet-like clusters exhibit temporal gene expressions and cellular organizations that mimic natural islets of Langerhans during their organogenesis. During differentiation of cultured ductal epithelial cells to islet-like clusters, the IPSC/ IPC-derived cells temporally express various endocrine hormones and islet cell-associated factors. A comparison of these gene expressions for mouse and human cultures, as detected by measuring transcripts by reverse transcriptasepolymerase chain reaction, is provided in Table 2. Included are the genes encoding insulin I and II, insulin receptor, hepatocyte growth factor and its receptor c-MET, glucagon, somatostatin, glucose transporter-2, GAD-67, and insulinlike growth factors I and II. In addition, genes related to development and differentiation are also detected, including those encoding Reg-1, Pdx-1, Ngn-3, Isl-1, and beta2/ neuroD. Despite detectable expression of these mRNA transcripts for islet-associated genes, the cells within the islet structures remain mostly immature. The fact that the mouse islets generated in vitro fail to achieve full maturation before implantation into an in vivo environment is supported by the observations that (1) many of the cells coexpressed more than a single endocrine hormone while in culture, and (2) insulin secretion in response to glucose challenge remained minimal. However, when nicotinamide, a reagent known to enhance proliferation and maturation of mouse beta cells, was added to cultures of differentiating mouse IPSCs, the number of islets increased, the islet cells exhibited increased insulin synthesis, and the islet clusters responded with increased insulin secretion to glucose challenges (Ramiya et al. 2000). Functional Activity of Stem Cell-derived, in Vitro-generated Islet Implants—A Syngeneic and Allogeneic Mouse Model One potential drawback of stem cell-derived, in vitro-grown islets has been the inability to identify a culture condition that permits full differentiation of the immature beta cells to mature cells within the islet clusters. This last step has required exposure to an in vivo environment, suggesting that in vitro environments do not simulate the in vivo environment and are missing important factors. The functional capacity of in vitro-grown, stem cell-derived mouse islets has 261 Figure 1 In vitro generation of islet-like clusters from (A) an adult human pancreas and (B) an adult mouse pancreas. Growth of islet-like clusters in primary islet-producing stem cells/islet progenitor cell cultures begin with establishment and growth of epithelial-like cells from partially digested pancreatic tissue (panels a-b). A number of islands of cells begin to produce cells that proliferate on top of the underlying monolayers (panels c-d), which tend to aggregate into clusters of cells (panels e-f). The aggregated clusters express markers associated with immature/mature endocrine cells, whereas the adherent monolayers express markers associated primarily with epithelial cells. When cells within the primary cultures are dispersed with trypsin and seeded to new cultures, the entire process is repeated, thus permitting expansion of the cultures. 262 ILAR Journal Table 2 Gene expression profiles of long-term IPSC/IPC culturesa Human Gene/factor Isl-1 Reg-1 pdx-1 Hlxb9 (HB9) ngn-3 beta2/neuroD pax-4 pax-6 Nkx2.2 Nkx6.1 glut-2 Tyrosine hydroxylase -galactosidase Insulin growth factor-1 Insulin growth factor-2 Npy (Neuropeptide-Y) Carbonic anhydrase-11 Cytokeratin-19 Insulin receptor HGF c-MET c-Kit CD34 INGAP Insulin Glucagon Somatostatin Pancreatic polypeptide Amylase Mouse Whole pancreas IPSC/IPC cultures Whole pancreas IPSC/IPC cultures ++ ++ ++ + (+)b ++ ++ − (+) − Variable Variable Variable − + + + + +/− + ++ Variable ++ + + + + ++ ++ ++ ++ + + + + + ++ ++ ++ + ++ + + ++ ++ ++ (+) (+) + ++ ++ ++ ++ ++ ++ ++ (+) (+) − ++ ++ ++ + ++ Variable (+) − + ++ ++ Variable + ++ ++ ++ ++ + ++ ++ + a IPSC/IPC, islet-producing stem cells/islet progenitor cells. Gene expressions were determined by reverse transcriptase-polymerase chain reaction. b (+) = weakly positive; variable = not all lines are positive for this gene/factor. been investigated in a set of implantation experiments (Ramiya et al. 2000). In the first set of experiments, 300 islet-like clusters from IPSC/IPC cultures originating from NOD mice were implanted into the spleen, the leg muscle, or the subcapsular region of the left kidneys of individual female diabetic NOD mice that had been maintained on daily insulin injections for >3 wk. Within 1 wk of being weaned from their insulin injections, mice implanted with islets to the kidney or the spleen exhibited stable blood glucose levels of 180 to 220 mg/dL and remained insulin independent until euthanized for analysis of the implants. Diabetic mice that had islets implanted into the muscle or had not received any islet implants exhibited severe wasting syndrome when weaned from their insulin and had to be euthanized early. In a second experiment, female diabetic NOD mice maintained on insulin for 4 wk were implanted subcutaneVolume 45, Number 3 2004 ously with in vitro-grown, stem cell-derived mouse islets with similar results, except that the blood glucose stabilized at more normal levels (100-150 mg/dL). Islets placed subcutaneously required approximately 2 to 4 wk to achieve a homeostatic state with the recipient. We have speculated (Ramiya et al. 2000) that this is the time required for the islets to become fully vascularized and establish the necessary glucose-sensing machinery for rapid and regulated insulin responses. An interesting observation from the implant studies is the fact that none of the syngeneic implants of in vitrogrown islets reactivated the autoimmune response in the diabetic recipient mice, as evidenced by a lack of immune cells in histological examinations of implant sites at time of euthanization. In contrast, allogeneic implants of in vitrogrown islets originating from either BALB/c or C57BL/6 mice were destroyed in what appeared to be an allogeneic 263 immune response. Thus, there is clear indication that these two immune responses are mutually distinct and that successful implantation of stem cell-derived islets will require special attention to the allogeneic response but possibly not to the autoimmune response. Whether this same observation is eventually observed in human implantation will be of great interest. Regardless, understanding why the autoimmune response is not reactivated in this system could prove valuable for islet transplantation, in general. Furthermore, although our ability to control the growth and differentiation of islet stem cells potentially provides an abundant source for beta cell reconstitution in T1D patients and insulinrequiring patients with type 2 diabetes, obtaining pancreatic tissue for autologous implants poses a number of challenges. Although the use of animal models, like the NOD mouse, provides important information and invaluable insight into how stem cell-derived cells and tissues behave in vitro and in vivo, the all-important question is whether this knowledge can be extrapolated to human stem cell-derived materials. The process will first require examination of human stem cell-derived, in vitro-generated islet implantations into animal models, possibly with confirmation by appropriate cross-species implantations of stem cell-derived, in vitro-generated islets of animal models. Xenogeneic Implants—A Brief Overview The transplantation of organs, tissues, or cells between individuals of different species, referred to as xenotransplantation, has been a topic of great interest and controversy due to the conflict of a worsening shortage of available human organs for transplantation versus perceived risks. In 2002, nearly 6200 patients died in the United States because of the acute organ shortage (UNOS 2002). Major issues that need to be addressed in xenotransplantation procedures include (1) the physiological compatibility between donor and recipient species, (2) immune attack and rejection, (3) the potential microbiological hazards that may “jump” across the species (zoonosis), and (4) the general attitude of society at large toward the ethical and emotional aspects surrounding xenotransplantation. Pancreatic islet xenotransplantation is considered to be technically less demanding than vascularized grafts (MacKenzie et al. 2003). The absence of major physiological incompatibilities between pig and human islets has been established, and porcine insulin has been used clinically in treating diabetic patients for many years. Although, from an immunological perspective, nonhuman primates would be a preferable source of organs for humans, almost all of these species are either endangered or too small to provide organs suitable for transplantation into large adult humans (Cooper et al. 2002). Additional reasons that disfavor primates include zoonosis, their small litter size, the enormous expense involved in breeding them in captivity, and the emotional factor. Due to similar reasons of sentimental attachment, 264 dogs will not become a popular source of islet donors in the clinical setting. There have been limited observations with piscine islets (Brockmann bodies) demonstrating the feasibility of their principal islets in reversing hyperglycemia in mice (Laue et al. 2001). In this issue, Wright and colleagues (2004) report notable progress in piscine research. Nevertheless, fish appear to be less likely to comprise a prime target species for islet xenotransplantation due to their phylogenetic distance from humans. Many investigators now expect the pig to be the species most likely to be the source of islets for xenotransplantation, and numerous reports have already been published using porcine islets in rodents and nonhuman primates (briefly reviewed by Larsen and Rolin 2004, also in this issue). In terms of physiology and size, pig pancreas and heart are both close to that of humans with subtle differences. For instance, pig islets activate the human blood clotting system, inducing severe damage to the islets. This process is associated with activation of platelets, leukocytes, and the complement system (Bennet et al. 2000b). Entrapment of islets in large thrombi will impair neovascularization and engraftment. In addition, there are also molecular incompatibilities, such as incompatibilities at the level of several receptors and ligands, including activated human coagulation factors and the natural anticoagulants expressed by porcine endothelial cells (Robson et al. 2000). One of the major hurdles of xenotransplantation with pig organs is the immune rejection phenomenon. Human immune responses to pig organs is a complex process that involves hyperacute rejection (HAR1), acute humoral xenograft rejection (AhXR1), and acute cellular xenograft rejection (AcXR1). Briefly, the main components in HAR are natural xenoreactive antibodies in combination with complement. In the pig-to-nonhuman primate model, HAR is generally due to the existence of naturally occurring antibodies to Gal␣1,3Gal terminal oligosaccharides (Gal). Binding of these natural antibodies to porcine tissues and subsequent activation of complement result in the destruction of the parenchyma, vasculature, wide-spread interstitial hemorrhage, and thorombosis in less than 24 hr after transplantation. Several antibody depletion procedures, use of complement activation inhibitors that include soluble complement receptor-1, complement activation blocker-2 (a chimeric protein from human decay-accelerating factor CD55 and membrane cofactor protein CD46, and treatment with cobra venom factor have been shown to delay graft rejection (Schuurman et al. 2003). AhXR, also known as “acute vascular rejection,” is characterized by antibodies, complement, and polymorphonuclear granulocytes. The antibody involved in AhXR may be anti-Gal or induced antibodies. AcXR is similar to processes seen in allograft rejection. Cellular rejection is a relatively rare phenomenon in pig-to-human primate solid organ transplants. More frequently, AcXR accompanies AhXR but is less severe than AhXR (Pino-Chavez 2001). However, because porcine islets are nonvasuclarized cellular grafts, HAR is not a great problem. Instead, rejection of ILAR Journal islet cells appears several days after implantation and is assumed to be mediated mainly by cellular mechanisms. Essentially, the whole spectrum of mononuclear cells, including T cells (CD4+ and CD8+), B cells, natural killer cells, and macrophages, may be present in the infiltrates. Several published studies describe the potential of islet xenotransplantation with varying degrees of success or failure. In all of these studies, wild-type pigs were used as the organ donors. In future studies, pigs transgenic for human complement inhibitors (Cozzi and White 1995) or cloned pigs lacking the Gal epitope (Phelps et al. 2003) will be favored as donor animals and are expected to enhance engraftment success. Several studies in nonhuman primates have already shown that vascularized organs from transgenic pigs avoid hyperacute rejection, and the same will presumably be true for organs from Gal-knockout pigs. Expression of the Gal epitope in the islets appears to vary depending on age, with reduced levels of expression in the islets of adult animals compared with fetal porcine islets (Bennet et al. 2000a). Ever since porcine endogenous retrovirus (PERV1) was shown to be able to infect human cells in vitro (Patience et al. 1997), a major concern has focused on the possibility that PERV would infect the recipients of porcine xenografts, and that infected patients would therefore pose a public health hazard. Interestingly, earlier studies conducted in the 1990s in which fetal porcine islets were implanted into diabetic renal transplant patients provided an opportunity to examine porcine tissue recipients for zoonosis or crossspecies infections. Because PERV is carried in the pig germline, potentially all recipients of porcine tissues or organs will be exposed to the virus. Using serology, polymerase chain reaction, and reverse transcriptase assays in each islet recipient, it was established that there was no infection with PERV (Heneine et al. 1998). In addition, Paradis and colleagues (1999) analyzed samples collected from 160 patients who had been treated with various living pig tissues up to 12 yr earlier. No viremia was detected in any patient despite a persistent microchimerism (i.e., presence of donor cells in the recipient) in 23 patients for up to 8.5 yr. Finally, a highly controversial Mexican trial wherein pig islets were implanted intraperitoneally inT1D patients was reported at the XIXth International Congress of Transplantation Society (Valdes Gonzalez 2002). In this trial, 17 adolescents were implanted with pig islets derived from specific pathogen-free pigs and isolated by Diatranz Company (New Zealand). Of the recipients, 12 remained insulin free for more than 12 mo, and five required reduced insulin doses. Even when all scientific issues have been satisfactorily addressed in the future, xenotransplantation will not become a commonplace clinical procedure unless public at large, and patients in particular, accept this procedure as ethical and meaningful. Given that some 100 million pigs are slaughtered for food every year in the United States, it will not be a serious issue for the public to accept pigs as donors of vital organs to save the life of patients. The fact that the US Food and Drug Administration did not agree with a Volume 45, Number 3 2004 moratorium demanded by Bach and colleagues (1998) clearly indicates the keen interest and the hope the public has for the potential of xenotransplantation. A better understanding of PERV infection and the development of protocols to control immune responses against xenografts should convince even skeptics to appreciate the power of xenotransplantation in the treatment of insulin-dependent diabetes. Thus, available data from xenotransplantation provide a basis for expectations in xenogeneic stem cell implantation. In the meantime, stem cell-based therapies remain reasonable and practical alternatives. Future Direction For many years, it has been speculated that a small population of islet stem/progenitor cells persisted after organogenesis within the pancreatic ducts of both healthy and diabetic individuals (Bonner-Weir et al. 1993). Nevertheless, the ability to culture and stimulate these stem cells into functional islets in vitro represents a major breakthrough that has great potential for the therapeutic intervention of T1D. Yet, results from a number of laboratories indicate that although we are able to initiate expansion and differentiation of such stem cells, we lack some of the basic understanding to control the process fully. Identification of temporal gene expressions during the developmental stages of islets may provide the information necessary for sequentially stimulating stem cells to mature to end-stage islets. Alternatively, the immature islets may represent the more ideal reagent for implantation because they appear to have the potential to respond to their in vivo environment, thereby establishing homeostasis with the recipient through vascularization, expansion of the beta cell mass, and differentiation to insulin-secreting cells. In this regard, stem cellderived IPSC/IPC cultures and their daughter islets starting with animal tissues clearly mimic the human cultures. Fully understanding each stage of differentiation and identifying the best impant material will require further investigations. At this time, this area appears to be best studied using animal models in which syngeneic, allogeneic, and xenogeneic implantation can be examined to answer questions pertinent to bringing this technology to the clinics and the patients. A second aspect of this technology is the use of autologous/allogeneic islets versus xenogeneic islets. Although our mouse studies using syngeneic islets generated in vitro indicate that they may be masked from the autoimmune response, it is definitely possible for allogeneic islets to be rejected. This potential suggests an active T cell-mediated allogeneic response, even if there is no reactivation of the autoimmune response. It means that potentially, the recipient of allogeneic stem cell-derived implants would still require immunosuppression or, alternatively, the implant would have to be isolated via a form of encapsulation, no differently than recipients of xenogeneic implants. Although the ideal situation would be to isolate the recipient’s 265 own stem cells to provide autologous implants, “one patient at a time,” this situation is not feasible if the goal is to treat a large number of diabetic patients. So, although most patients would probably prefer to receive autologous islet implants, the use of allogeneic islets, or possibly xenogeneic islets, is most expedient. In the near future, as stem cells and stem cell-derived tissues become a legitimate source of “islets” for preclinical trials, two different animal models are expected to precede human trials for safety and efficacy studies. The small animal model will no doubt be the diabetic mouse model, most likely the NOD mouse; and it appears likely that the larger animal model will be the miniature swine model of diabetes (Larsen and Rolin 2004). This situation could change rapidly, however, should regulatory agencies insist on studies in nonhuman primates prior to initiating human trials. The test article in these trials will be human stem cells or their derivatives. Our current understanding about stem cells and xenotransplantation will be helpful in predicting outcomes. Because stem cells are cultured in vitro for longer periods of time, the antigenicity may be diminished enough to permit less threatening immunosuppression therapy, making the procedure ethically acceptable to a larger patient population. However, the longer in vitro culturing may lead to substantial deviations of the stem cell-derived tissue from normal cellular phenotypes, deviations from regulated insulin secretion, abnormal migratory patterns after implantation in vivo, and/or formation of transformed cells, just to mention a few scenarios. Finally, one of the advantages of being able to grow endocrine pancreas in culture from stem cells is the ease with which the IPSC/IPC cells can be genetically modified, possibly to create daughter cells resistant to immunological attack. 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