Adult stem cells, on the contrary, could be easily derived from the patient and expanded in vitro prior to re-differentiation and implantation. As would be the case with patient-derived huES/iPS cells, this approach would theoretically circumvent the immune rejection of the tissue. In both cases, however, the problem of autoimmunity would still require additional interventions. The use of mesenchymal stem cells (MSCs) in an allotransplantation setting has also been proposed based on the observation that these cells have immunomodulatory properties. The hypothesis was that these cells might be able to engraft even in allogeneic recipients, down-regulating the immune response. However, this theory has been proven incorrect in a number of in vivo experiments and now the prevailing view is that the effects of MSCs in mismatched settings might be due to transient trophic/secretory effects. Some argue that a solution to the autoimmunity component of type I diabetes might be just enough to cure the disease. After all, it has been observed that insulin-positive cells persist even decades after the onset of the disease, suggesting that regeneration mechanisms are at play throughout the course of the disease, but perhaps kept at bay by the autoimmune response. It is likely, however, that regeneration will not be possible unless a critical mass of beta cells remains in the pancreas.This would explain why the “reeducation” of the immune system proposed by Voltarelli et al. was not hypothesized to work in patients with long-standing diabetes. Therefore, a boost of exogenous cells will be required even after the autoimmunity problem has been solved. The jury is still out regarding whether this second component of the cure (replacement) will come from embryonic stem cells, adult stem cells, transdifferentiation, or regeneration. It might also be the case that success is the result of a combination of several approaches. A theoretical clinical intervention, for instance, may involve the differentiation of huES cells into both pancreatic endocrine tissue and hematopoietic cells with the ability to induce chimerism – and tolerance – in any given patient. Provided that concerns about teratomas are conveniently addressed, the stem cells could thus be used both as a replacement and a tolerance inducing tool . The same approach would be applicable to a situation where hematopoietic cells (bone marrow or cord blood) are differentiated into beta cells and co-transplanted with undifferentiated aliquots for tolerance induction. In a different setting, we cannot entirely rule out the possibility that some forms of transdifferentiation may result in the generation of insulin-producing cells that, not being true beta cells, may actually be in a better position to evade the immune response. Finally, the use of MSCs is likely to help in virtually any therapeutic intervention by virtue of their immunomodulatory and “feeder” effects. The bottom line is that the several strategies presented in previous chapters, far from being exclusive of each other, are expected to work in a synergistic fashion; and that the knowledge gathered from each field will certainly cross-fertilize the others. The themes herein discussed are so new and rapidly evolving that this book could not have been written a decade ago. We can only hope that, 10 years from now, all of these novel concepts will have finally settled and the field will have advanced much closer to a definitive cure for type I diabetes.

ES cells homozygous for the most widely represented HLA haplotypes can be generated by means of parthenogenesis. This process is the result of the fusion of a haploid egg with an haploid polar body generated during the maturation of the former.

Theoretical co-transplantation of hematopoietic and pancreatic beta cells derived from a single universal huES cell donor.

A typical adult pancreas contains approximately one million islets, which represent around 1–2% of the total mass of the organ. It has been estimated that only 50% will survive the harsh process of isolation, with up to 60–80% of the remaining mass perishing in the immediate posttransplantation period due to inflammatory processes not yet fully understood. For instance, it has been shown that islets express tissue factor which may contribute to early islet loss by stimulating coagulation upon their contact with the blood. Considering the many insults that may invariably result in islet cell death from the time of the pancreas procurement to the actual infusion, the fact that only 10% of the transplanted patients are insulin-free 5 years after the procedure is much less surprising than the observation that up to 80% are insulinfree after 1 year. While we define alternative sources of islets that are either plentiful (xenotransplantation) or self-renewable (stem cells), there is an imperative need to “make every islet count” and to minimize their destruction upon implantation. The field of islet cytoprotection is a fertile one, with a large number of chemical gene-based, and protein transduction strategies proven successful in many experimental models. However, it is still necessary to gather much more information about the basic mechanisms that drive beta cell destruction upon implantation. In this context, another limitation of islet transplantation has been the difficulty to explore in real time the causes of their demise once implanted. An important breakthrough was reported just recently by Speier et al., who transplanted islets in the anterior chamber of the eye of recipient mice. This location was supportive of islet engraftment and function, as evidenced by the reversal of diabetes. More importantly, this system allows investigators to follow in real time events such as vascularization, inflammation, and rejection, which can be studied in living animals by microscopic examination of the eyes where the islets are transplanted. Interventions designed to interfere with physiological responses leading to islet destruction can now be implemented by means of eye drops and monitored as they exert their effects.

Limitations of Islet Transplantation: Immunosuppression and Tolerance

General Considerations About Islet Rejection

As much as islet transplantation improves upon exogenous insulin administration as a way to regulate glucose homeostasis, unless both the allo-rejection and the autoimmune processes are arrested, it will not be considered a cure. The paradox about immunosuppressants is that, while they have made islet allotransplantation possible, they are also diabetogenic, impairing beta cell survival, function, and replication. Thus, the ultimate goal of any cell therapy for type I diabetes is that the replaced cells are accepted as “self” by the recipient, and that the immune system of the patient is reeducated to prevent recurrence of autoimmunity. When T cells contact either a self-antigen or a harmless exogenous antigen, the absence of co-stimulatory signals or the presence of co-inhibitory ones will prevent the unleashing of the immune response. At this point, the T cell may get inactivated or even apoptose. As a growing body of evidence suggests that tolerance or rejection are determined by the balance between positive and negative co-signaling, the receptors involved are potentially useful targets to minimize allo-rejection. As for the mechanisms responsible for the recurrence of autoimmunity, it is known that in the nonobese diabetic mice, a model of type I diabetes, beta cell destruction is mediated by autoreactive T cells. The two major costimulatory pathways involved in this rogue T-cell response are CD28–B7 and CD40 ligand–CD40. In fact, the use of antibodies that interfere with these pathways has been proven effective at preventing the onset of diabetes in this animal model. There is a balance between normal immune regulation and autoimmunity that appears to be mediated by regulatory T cells (Tregs), a subset of T cells characterized by the expression of the master transcriptional regulator Foxp3 and identifiable by the surface expression of CD4 and CD25. These cells confer protection against autoimmune diabetes. Unfortunately, the experimental blockade of the co-stimulatory response that triggers allo-rejection has also a negative effect on Treg function. In this context, the identification of co-inhibitory molecules might offer a safer and more effective therapeutic alternative .

More comprehensive strategies go one step beyond the mere tipping of the balance between autoimmunity and regular immune function. Bone marrow transplantation, for instance, has been shown to cure diabetes in animals, but for the new cells to home, the resident stem cells must be wiped out by lethal or sublethal irradiation. This is obviously not a therapeutic option for diabetes as it is for other fulminant illnesses, as diabetes is a chronic and (to some extent) manageable disease. Hence the search for gentler ways of making room for “healthy” bone marrow cells without completely ablating the recipient’s hematopoietic compartment; and inducing graft-versus-host disease. The latest approach (already discussed in the chapter “Adult stem cells & pancreatic differentiation”) is that of using hematopoietic stem cells extracted from the same patient, who is subsequently treated with a strong immunosuppressive regime prior to stem cell reinfusion. The safety and long-term effectiveness of these treatments are still in question, and more studies will be necessary before they are translated into widely used clinical practice.

Finally, another area of research aims at encapsulating the transplanted islets with a physical barrier that will protect them from both allo- and auto-rejection (including immune-effector cells, complement system, and immunoglobulins), while allowing the free transit of insulin, sugar, nutrients, and oxygen. Considering the sophistication of the cellular and molecular strategies described above to interfere with the immune response, this could be considered a “brute force” approach. If successful, however, it could even open the door to xenotransplantation of nonhuman islets, which could potentially be a valid alternative to that of using stem cells for directed differentiation into pancreatic endocrine cells. The different strategies presented thus far (intravascular implant, macroencapsulation, and microencapsulation) are posted here. Several materials have already been tested with variable degrees of success. The workhorse of these studies is alginate, derived from kelp, a member of the brown algae (Phaeophyceae) group.

Antigens are detected by T cells by means of their interaction with antigen-presenting cells (APCs). At the molecular level, such interaction occurs between a T-cell receptor (TCR) and a major histocompatibility complex (MHC). Without any other signal, optimal activation does not occur, and may even induce tolerance or anergy. However, secondary interactions (costimulatory signals) cannot activate the T cell by themselves. A combination between the primary TCR-MHC and a co-stimulatory signal are necessary for optimal response. Below is represented the balance between co-stimulation and co-inhibition. The net result of these opposing forces will ultimately determine the response of the T cell .

General encapsulation strategies. The intravascular implant (also known as biohybrid artificial pancreas) is a perfusion chamber directly connected to the blood vessels through an arteriovenous shunt. Islets are within the diffusion area of influence of the blood vessel, while protected by a membrane. However, implantation is not a straightforward procedure, and there is a significant risk of clotting. More conventional strategies involve the protection of islets by means of immunobarriers (macroencapsulation and microencapsulation) not in direct contact with existing vasculature .

This compound is already in clinical trials, and offers the advantage that it can be conjugated to materials such as polyethylene glycol and poly-lysine to reduce plasma adsorption and decrease the formation of fibrotic tissue around the capsule (recently reviewed by Beck et al ).

Polysulphone is another promising material for islet encapsulation, although it needs to be chemically modified in order to minimize insulin adsorption. All of these mechanisms, however, suffer from two important limitations, namely: their inability to prevent the circulation of cytokines across the barrier (which would be especially problematic in xenotransplantation settings) and nutrient deprivation and hypoxia. The latter has already been acknowledged as one of the main determinants of islet cell death upon transplantation, and islet cell encapsulation can only exacerbate this problem. Conventional approaches induce large void volumes where the islet ( ~ 150 m m) is engulfed in a much larger structure (400–800 m m). Considering the ensuing delay in nutrient and oxygen delivery, as well as the unusually high metabolic demands of islets, it is hardly surprising that encapsulated islets tend to exhibit starvation-induced apoptosis and/or impaired function. In an effort to retain the critical advantages of encapsulation while maximizing transport of oxygen and nutrients across the barrier, a promising area of research is that of designing thin, conformal polymeric layers that can be “coated” directly onto the islet surface. Doing so may reduce the diffusion distance between the islet and the capsule by up to 1,000- fold thus improving the chances for islet survival in the immediate posttransplantation period.

In a paradoxical reversal of the “miniaturization” trend, the recent development of a subcutaneously implantable biocompatible device may also help address many of the problems of immune rejection. In preliminary studies in rats, a cylindrical stainless-steel mesh was implanted and allowed to be vascularized for 40 days. Islets were then placed in the device, where they supported reversal of diabetes in a syngeneic setting. Although a device of this nature is not designed to prevent the access of the immune effectors (cells, complement, and immunoglobulins), a major advantage over noncontained transplantation sites is that immunosuppression could be potentially delivered in a local fashion. Systemic medication to prevent rejection is based on the administration of very large doses of immunosuppressants, due to the need of reaching biologically active concentrations at the site of the graft. The well-known side effects of this treatment include higher incidence of infection and cancer. With this system, much smaller doses could be delivered locally in the site of the graft, allowing for immunoprotection with no or little systemic effects. In fact, this approach might allow for the delivery of potentially powerful drugs not approved for systemic administration.

Islet transplantation • Autoimmunity • Rejection • Nanoencapsulation • Diabetes

While the exploration of the mechanisms behind the development of the pancreas would have been fully warranted from a purely scientific point of view, it cannot be disputed that the prevalence of diabetes has very significantly stoked our progress in the field. Two more almost simultaneous circumstances have aligned to make pancreatic development one of the best studied examples of organogenesis: the advent of human embryonic stem cells and the development of protocols for the long-term survival and function of transplanted islets. Unlike many other conditions for which potential stem cell therapies have been conceived – but not put into practice yet – type I diabetes is arguably the perfect target of regenerative therapies: only one cell type needs to be replaced and there is an already existing cell therapy. The success of islet transplantation as a viable treatment of type I diabetes has led to the valid assumption that, if stem cells can be coaxed to produce insulin in a glucose-regulated manner, ensuing therapies are likely to work as well as native islets do. Such an approach would provide an immediate solution to the most pressing problem that stands in the way of the widespread implementation islet transplantation, namely the shortage of organs for islet processing and transplantation. Here we review the challenges and clinical perspectives of stem cell research in the context of the current status of islet transplantation.

Diabetes and Islet Transplantation

Type I diabetes is an autoimmune disorder whereby the immune system of the affected individual attacks and destroys the pancreatic beta cells that secrete insulin in response to elevated blood sugar levels. Because it is usually (but not always) diagnosed during childhood or early teenage years, it is also referred to as juvenile diabetes. Type II diabetes differs from it in that it does not usually start as an autoimmune response, but rather as a consequence of the inability of the cells of the body to respond adequately to otherwise normally synthesized and secreted insulin. In some cases, the beta cells will also produce less insulin than required to maintain glucose homeostasis. Type II diabetes tends to affect individuals at older ages, and it is commonly associated with obesity.

The only “conventional” treatment for type I diabetes is insulin administration. This is a life-saving procedure, but one that unfortunately fails to replicate the exquisite native regulation that islets exert over blood sugar levels. Years of exogenous insulin use cannot prevent the occurrence of complications that are generally based on a compromised integrity of the vasculature, including renal failure, amputations, and blindness.

Given the fact that only the islet can provide the glucose regulation required for long-term avoidance of complications, replacement beta cell therapies would be indicated not only for type I diabetes, but also for insulin-dependent type II diabetes and other conditions such as cystic fibrosis, hemochromatosis, liver cirrhosis, or iatrogenic diabetes after pancreatectomy. While whole pancreas transplantation is usually effective at reversing the symptoms of diabetes, it is rarely indicated as a treatment for the disease unless the patient is simultaneously receiving another organ (typically a kidney) or is already in an immunosuppressive regime. It is also considered major surgery and has a relatively high risk of complications. Islet transplantation, in contrast, is a much safer and easier procedure that offers the possibility of preconditioning the “organ” prior to transplantation. In short, this approach is based on the enzymatic digestion of a pancreas (from a deceased organ donor or from a living related one ) using a semiautomated method that makes use of mechanical agitation to separate the islets from the exocrine and ductal components of the pancreas .

A subsequent gradient centrifugation enriches for fractions with a high proportion of islets, which are subsequently cultured and infused through the portal vein of the patient using minimally invasive interventional radiology methods. The islets lodge in the vessels of the liver, where they get revascularized within 2–3 weeks.

In the case of allogenic islet transplantation (i.e., islets obtained from deceased donors), immunosuppression is necessary to prevent rejection. Before 2000, islet transplantation was successful only for a limited window of time, due to the deleterious effects of immunosuppressive steroids on islet cells. The development of a novel, glucocorticoid-free regime for islet transplantation enhanced very substantially the long-term viability and function of transplanted islets, with a great majority of patients reporting a significant improvement in their quality of life. Total insulin independence and full metabolic control is typically achieved after a critical mass of islets has been transplanted, which may require more than one donor.

Islet transplantation. Islets are isolated from a donor pancreas. Isolated preparations are typically cultured to allow islets to recover from the procedure, and then they are infused through the portal vein of recipients. This is an outpatient procedure, and requires the expertise of an interventional radiologist. Islets lodge in the microvasculature of the liver, and get revascularized within weeks

The molecular basis of this phenomenon remained unclear until very recently, when Meivar-Levy et al. presented compelling evidence that Pdx1-mediated transdifferentiation requires an intermediate “de-differentiation” step. Pdx1, but not other pancreatic genes delivered using the same system, induced a substantial down-regulation of C/EBPbeta and LAP, two redundant proteins of a family of transcription factors known to play important roles during liver embryonic development as well as in adult hepatocytes. Overexpression of LAP in primary cultures of human hepatic cells prevented the Pdx1-mediated de-differentiation and activation of the pancreatic program. However, down-regulation of C/ EBPbeta was insufficient by itself to trigger the activation of pancreatic genes, with the exception of Ngn3. As expected, the simultaneous down-regulation of C/ EBPbeta and the administration of Pdx1 had a synergistic effect in inducing transdifferentiation.

Liver transdifferentiation approach as reported by Ferber et al. (2000) . A Pdx1 cassette is delivered systemically through an adenoviral vector. Expression of this cassette in the liver yields insulin-producing cells. When the procedure is done in streptozotocin (STZ)-treated diabetic mice, it results in a very significant reduction of blood glucose levels.

The question remains of whether this approach is targeting bona fide hepatocytes or perhaps more undifferentiated progenitors that might be more amenable to transdifferentiate. In vitro experiments are not conclusive because hepatocyte cultures become fibroblastic in appearance very rapidly, perhaps because of the adaptation of the cells to attachment in plastic. In addition, it is very likely that the insulinproducing cells generated in this manner are not true beta cells, but rather hybrids between hepatic and pancreatic cells.

Parallel experiments conducted by Horb and colleagues confirmed that transdifferentiation is indeed feasible in other contexts. In short, the authors created transgenic frogs where a Pdx1 (Xlhbox8)-VP16 fusion cassette is expressed under the control of the liver-specific promoter transthyretin (TTR) . The rationale for the use of VP16 – a potent transcriptional transactivator from the herpes simplex virus – is that nonpancreatic cells may lack the appropriate molecular partners for Pdx1 to exert its biological function. An additional marker was added to screen for successful transdifferentiation, namely the green fluorescent protein (GFP) under the control of the elastase promoter, a pancreas-specific regulatory element. Up to 60% of transgenic tadpoles showed partial or total conversion of liver to pancreas, as evidenced not only by the expression of GFP but also by that of pancreatic endocrine (insulin and glucagon) and exocrine (amylase) markers. It is important to note that no transdifferentiation was observed when Pdx1, without VP16, was used. This observation suggests that the assertion that Pdx1 is necessary, but not sufficient to promote pancreatic differentiation, remains true for the liver.

Experimental conversion of liver to pancreas in a frog transgenic model, as described by Horb et al. The Xlhbox8 gene, the amphibian homolog of Pdx1, was fused to the VP16 transactivation domain, and placed under the control of the liver-specific promoter TTR. An additional marker was added to follow up successful conversion events (GFP driven by the promoter of elastase, a gene expressed in the pancreas but not the liver). Transgenic tadpoles exhibited various degrees of liver transdifferentiation into pancreas.

Given that the timing was such that the onset of the ectopic Pdx1 expression was coincident in time with the initiation of liver development, this event could in theory be considered an induced redirecting of early organogenesis, rather than a proper transdifferentiation event. The authors, however, used the same construct to transfect immortalized human hepatocytes (HepG2), which led to elastase activation in approximately 65% of the cells that received it. Of these, approximately 15% were insulin positive. These results were subsequently expanded by characterizing the transdifferentiated cells. Confirming the observations of Ferber and colleagues, it was found that the hepatic phenotype was lost upon ectopic expression of Pdx1; that the requirement for the transgene was not permanent, as an initial trigger was sufficient to activate the pancreatic lineage; and that the insulin-positive cells obtained through this approach had PC 1/3, C-peptide, and glucagon-like receptor 1 (GLP-1), among other functional markers of true beta cells. These cells were glucose responsive and increased insulin expression upon treatment with GLP-1 and beta-cellulin.

Using a lentiviral vector, Tang et al. were able to transdifferentiate rat hepatic stem-like WB cells into pancreatic beta cells both with Pdx1 and Pdx1–VP16. This study was the first to systematically compare the transdifferentiation potential of the two versions of the gene. While they found that cell lines expressing either Pdx1 or Pdx1–VP16 long-term had comparable gene expression profiles as well as a similar capacity to correct hyperglycemia in recipient diabetic mice, short-term expression gave a marked edge to the VP16-fused version. Additional studies were significantly consistent with the first set of data first published by Ferber and colleagues, and showed the cumulative effect of adding other pancreatic endocrine factors to the mix, such as Ngn3, NeuroD, or MafA. Interestingly, Wang et al. reported their inability to induce liver-to-pancreas transdifferentiation in vivo when using adeno-associated viruses as vectors to co-deliver Pdx1 and Ngn3. However, when they delivered these cassettes using plasmid vectors with an irrelevant adenoviral vector, they reported correction of hyperglycemia in diabetic rodents. The authors postulated that the antigen-dependent immune response elicited by the adenoviral capsid (but not other viruses) was instrumental in the induction of transdifferentiation.

The concept of liver transdifferentiation has attracted significant attention for several reasons. First, liver cells are easier to obtain and expand than those derived from the pancreas. Therefore, they provide an easily accessible source (a biopsy might provide enough cells to manipulate ex vivo) that could be extracted from the very same patients we want to treat, thus eliminating the risk of allogeneic rejection . Second, it is becoming clear that, despite some degree of functionality, transdifferentiated liver cells are not true beta cells. Some evidence indicates that the ability of these cells to appropriately regulate insulin secretion in a glucoseregulated manner might not stand comparison with that of true beta cells. However, clinical therapies could be devised even if these cells worked just as a “pump” (i.e., continuously secreting a basal amount of insulin in a nonregulated fashion). Most importantly, in type I diabetic patients, the immune system is poised to attack and destroy any cell that resembles a beta cell. From this perspective, these “hybrids” might have a selective advantage over native beta cells, because they could be able to elude the autoimmune response. Of course, this hypothesis hinges on the assumption that the autoimmune response will spare non-beta cells that express insulin, which might not be the case in view of the fact that insulin has been shown to be an auto-antibody in type I diabetes.

A more recent attempt at transdifferentiating non-endocrine tissue into beta cells used a different starting material, one that – at least in theory – should be more closely related to the desired end product. Based on the screening of at least 20 transcription factors (of which nine gave rise to gross phenotypic changes in beta cells when knocked out) expressed either in terminally differentiated beta cells or their progenitors Zhou et al. were able to reprogram pancreatic exocrine tissue into islet cell types using a combination of three genes (Pdx1, Ngn3, and MafA) delivered by means of adenoviral vehicles. New insulin-producing cells were detected as early as 3 days after the injection of the adenoviral mix into the pancreata of Rag1 −/− mice, a strain typically used to minimize the occurrence of viral-induced immune responses such as those described earlier by Wang et al. The number of these cells kept expanding for up to 3 months, long after the adenoviruses had been cleared from the recipients. They were indistinguishable from native beta cells in terms of size, morphology, presence, and distribution of insulin granules and molecular markers. Unlike in other transdifferentiation settings, the original exocrine phenotype appeared to be completely abrogated (i.e., they were not “hybrids”). Diabetic mice subjected to the treatment showed a significant and permanent improvement in blood glucose levels, even if diabetes was not completely reversed. The latter observation could be explained by the fact that the newly created beta cells remained isolated and did not cluster to form islets. Indeed, beta cell communication is essential to stimulate glucose-mediated insulin secretion.

As promising as this rapidly evolving field is, safety concerns may still preclude its immediate clinical translation. The observation that the ectopic genes need only be expressed transiently in order to activate transdifferentiation is encouraging. However, the use of adenoviruses may have serious side effects by eliciting immune responses in the host. Also, the ability of Pdx1 to induce exocrine tissue as well as endocrine derivatives proved harmful by inducing fulminant hepatitis in animal models. In a transgenic setting, ectopic expression of the gene resulted in widespread liver dysmorphogenesis, with abnormal lobe structures and polycystic lesions.Certainly, the systemic infusion of viral vectors containing master pancreatic regulators into human patients does not seem a clinical possibility in the near future. However, the extraction of liver tissue for ex vivo transdifferentiation and subsequent reimplantation in the patient appears to be a more reasonable course of action. It is likely that in this setting the adenoviruses would have already been cleared from the cells at the time of transplantation, increasing the overall safety of the procedure.

Liver transdifferentiation • Pdx1 • VP16 • Reprogramming • Hepatocytes

Expression of Pdx1 in the foregut (e8) is one of the earlier molecular events that mark the specification of the pancreas as a separate organ (see the chapter “Pancreatic Development”). The role of Pdx1 as a “master regulator” of pancreatic development has led many investigators to test whether its ectopic expression would induce pancreatic differentiation by itself. This strategy has yielded somewhat modest results in most cellular substrates examined which suggests that Pdx1 expression is necessary, but not sufficient, to initiate pancreatic development. The conclusion from the observation that the initial evagination of the pancreatic epithelium occurs even in the absence of Pdx1.A possible exception to this rule, however, is observed when the target tissue is liver.In fact, there is a wealth of studies indicating that the liver and pancreas are especially susceptible to interconversion. Many invertebrates have a single organ that comprises both hepatic and pancreatic functions, which suggests that the separation of these two organs is a relatively late evolutionary event. In vertebrates, fibroblast growth factor (FGF) signals from the cardiac mesoderm have been shown to play an essential role for the ventral endoderm to differentiate into early hepatic cells and it has been demonstrated that both organs originate from common endodermal progenitors in the early foregut.According to the model described by Deutsch et al., 662 cardiac FGF will have inductive and blocking effects on liver and pancreas specification, respectively.

In general, hepatocytes and beta cells share not only many developmental features but also similar molecular machinery for glucose sensing and secretion. Many studies confirm that interconversion of liver and pancreas occurs under a variety of experimental conditions, including copper depletion in rats treatment with dexamethasone or diethylnitrosamine and certain tumoral processes.

FGF signalling from the cardiac mesoderm will induce liver specification proximally ( dotted region ), but will have a blocking effect on the distal portion of the ventral foregut, which will become pancreas ( stripes ) (Adapted from Deutsch et al.

As for the non-MSC compartment, HSCs are presently being used for the treatment of diabetes in clinical trials for diabetes, but from a different angle: the reeducation of the immune system. In clinical trials conducted on 15 newly diagnosed type I diabetic patients, HSCs were mobilized, collected from peripheral blood, and cryopreserved. Patients were then subjected to a very aggressive immunosuppressive regime prior to the reinfusion of their own stem cells, which were assumed to be at a stage prior to the onset of the disease. As a result of this treatment, 14 out of 15 patients became insulin-free for extended periods of time. Others are already transplanting hematopoietic cells directly into the pancreas of diabetic patients, but the claimed beneficial effects of this treatment on the overall glycemic control still need to be independently validated– and a mechanistic explanation investigated. As was the case with MSCs, since the hematopoietic compartment is derived from the mesoderm, there are doubts that HSCs may become true endodermal or ectodermal derivatives. To this date, there is no conclusive evidence in one way or another that transplanted HSCs that migrate to target tissues help in their regeneration by differentiation/ transdifferentiation, cell fusion, or simply by supporting endogenous regeneration through revascularization and/or as feeders.

The latest cell type to join our arsenal of adult stem cells is the amniotic fluid stem (AFS) cell. These cells are naturally shed by the embryo to the surrounding amniotic fluid, and can be easily cultured in vitro. In a recent breakthrough report, De Coppi et al. described a novel subpopulation of amniotic fluid cells with a multilineage differentiation potential spanning the three embryonal layers. These cells can be expanded almost indefinitely without loss of pluripotency, and they coexpress markers of both human embryonic stem cells (Oct3/4, SSEA-4, telomerase, and others) and MSCs (CD105, CD73, and CD90). Unlike huES cells, however, AFSs did not form teratomas in immunocompromised recipients. Their proven ability to generate endodermal derivatives bodes well for the field of pancreatic regeneration, although current efforts at differentiating these cells into pancreatic endocrine cell lineages have not been reported yet.

Transiently Immortalized Beta Cells

A potential alternative to the use of adult stem cells for directed differentiation into beta cells would be to define the conditions to expand fully differentiated beta cells.  we have already reviewed the literature on beta cell regeneration in vivo. However, in vitro “re-differentiation” of beta cells after de-differentiation and expansion phases was a less than convincing strategy, due to the fact that the end product could not stand comparison with the real beta cell. A different approach to the same end was presented in 2005 by Narushima et al., who transfected primary human beta cells with loxP-flanked sequences for the simian virus 40 large T antigen (SV40T) and the human telomerase reverse transcriptase (hTERT). These cells could be expanded without senescence for more than 50 passages in vitro. During the expansion phase, expression of insulin and other critical beta cell genes was downregulated. However, upon Cre-mediated “reversion,” the two immortalizing genes were removed and the cell adopted a quiescent, functional beta cell phenotype again. Reverted cells were able to permanently correct hyperglycemia in streptozotocin-treated SCID mice without any further replication in vivo . A complementary technology that could help the clinical translation of these findings is that of transducible TAT-Cre whose use in this context would preclude the use of viral vehicles to deliver the recombinase.

Transient immortalization of beta cells, as described by Narushima et al. A double round of retroviral transfection allows for the selection of cells that express both the hTERT and the SV40 large T antigen genes, which confer immortality. An EGFP marker can be used to sort these cells. This can be reverted by introducing the Cre recombinase, which will excise out the immortalizing genes in a process that will bring together a constitutive promoter and a neomycinselectable marker. These “reverted” cells can be selected using G418 and by sorting for EGFPnegative signal.

In Vivo Transplantation of Undifferentiated MSCs

As previously discussed in the chapter “Pancreatic Regeneration,” the evidence for bone marrow-mediated regeneration of pancreatic endocrine function is more likely related to a “trophic” activity than it is to direct transdifferentiation. However, several groups have recently carried out a number of intriguing experiments where undifferentiated MSCs are implanted in diabetic animals with the aim of ameliorating the symptoms of the disease. Thus, Ezquer et al. reported that streptozotocin-treated diabetic mice that received an intravenous injection of undifferentiated MSCs showed reduced blood glucose levels as early as 1 week after the intervention. Compared with the animals in the control group, the MSC-injected animals exhibited reduced albuminuria and better renal function. Histological studies suggested that the number of islets is increased. Similar observations were reported by Dong and collaborators in an allogenic rat transplantation model, in which streptozotocin-treated recipients were injected through the tail vein with a single dose of undifferentiated, BrdU-labeled MSCs. A significant reduction of hyperglycemia was observed at day 45, with treated animals showing higher body weight and an increased number of small islets with BrdU + /Insulin + cells. In yet another animal model, Chang et al. injected male porcine MSCs directly in the pancreas of female diabetic pigs. Two weeks after transplantation, blood glucose levels decreased compared with those of sham-treated controls. Confirming the observations of the above groups, the authors reported a higher number of small islets containing insulin-producing cells of male origin, suggesting direct transdifferentiation. Although none of these studies addressed convincingly the possibility of cell fusion (which could explain many of these findings), the observed effects are certainly encouraging and warrant additional investigation.

Mesenchymal stem cells • Cord blood stem cells • Amniotic fluid stem cells • Hematopoietic stem cells • Transient immortalization

The general notion of “adult stem cells” describes populations of cells with poorly defined characteristics that can be found in specific niches of most adult tissues. They are thought to be involved in the native turnover of their tissue, and occasionally show a great mobilization potential in response to insults, leading to regeneration. Unfortunately, our ability to study these cells in their native environment is very limited: we are biased by their apparent behavior after we isolate and expand them in vitro. It can be argued that the majority of the expandable “adult” stem cells described thus far are just the result of an adaptation to in vitro culture, which is probably a misleading indication of their actual nature and potential in vivo. Indeed, stem cell niches tend to be complex and maintain a delicate equilibrium between many different compartments. However, when we extract these cells from their native environment, we disrupt this complexity and favor the expansion of those that will better adapt to whichever culture conditions are set. Since researchers tend to favor attachment to plastic as a desirable characteristic (due to ease of cultivation and passage), perhaps it is not surprising that the end result, regardless of the tissue of origin, is usually quite similar: fibroblastic-like mesenchymal stem cells (MSCs) with a relatively limited differentiation potential and the ability to be propagated extensively without senescence. Only recently have we started to look into the possible in vitro replication of the natural complexity seen in stem cell niches. This would include not only the various participating cell types, but also the right combination of extracellular components. If successful, we might be able to accomplish the ultimate goal of expanding true, “ready-to-differentiate” tissue-specific stem cells – and not their surrogate, artifactual derivatives – for therapeutic purposes. This chapter is not intended as an exhaustive review of adult stem cells and their niches, particularly in view of the fact that very little, if anything, is known about the elusive pancreatic stem cell. Instead, we will describe the attempts at differentiating pancreatic cells from stem cells of “adult” (as opposed to embryonic) origin. In general, these cells will be characterized by some degree of in vitro expansion and differentiation potential, even if typically narrower than that of embryonic stem cells.

Mesenchymal Stem Cells

Most studies on beta cell differentiation from adult stem cells are based on MSCs. Originally identified in the bone marrow, these cells are multipotent, adhere to plastic, self-replicate for many passages, and can be easily derived from virtually any organ or tissue in the body, perhaps with the exception of peripheral blood. They have an elongated morphology resembling that of fibroblasts and have the potential to differentiate both in vitro and in vivo into a variety of connective fates, including adipose tissue, bone, and cartilage. At one point, it was thought that mesenchymal cells could be as pluripotent as their embryonic counterparts. The general view at this point is that, being mesodermal in origin, these cells might require more than simple soluble factor-driven protocols in order to force their differentiation into endodermal and ectodermal lineages. Earlier this decade, Verfaillie and collaborators isolated and characterized a subset of bone marrow-derived MSCs (multipotent adult progenitor cells [MAPCs]) that could have been an exception to the above rule. When injected into mouse blastocysts, these cells contributed extensively to all three germ layers in a manner consistent with that of a truly totipotent stem cell type. Other groups, however, have had difficulties replicating the methods originally described to establish these cell lines, and the technology has failed to become mainstream.

A colony of human MSCs derived from the umbilical cord blood . After passaging, the cells grow in a monolayer and adopt a mesenchymal-like morphology . Kindly provided by Prabakar Kamalaveni and Luca Inverardi

Due to an explosion on reports on MSC-like cells isolated from almost every possible tissue source, the International Society for Cellular Therapy established recently a set of minimal criteria for MSC standardization. These criteria are the following :

1. MSCs must adhere to plastic when maintained in standard culture conditions.
2. They must express the surface markers CD73, CD90, and CD105.
3. They must not express the surface markers CD34, CD45, CD14, CD11b, CD79 a , and CD19.
4. They must be negative for HLA-DR (major histocompatibility complex class II).
5. They must readily differentiate into osteoblasts, adipocytes, and chondroblasts following standard in vitro methods.

Interestingly, expression of the pluripotent Oct3/4 transcription factor has been occasionally observed in MSCs . However, the prevailing view is that Oct3/4 expression in adult tissues is not representative of pluripotency beyond the inner cell mass (ICM) stage, but rather an indication that this factor is not as embryonic-specific as originally thought.

Given their ease of differentiation toward connective phenotypes, MSCs are already in the pipeline for potential regenerative therapies involving the replacement of lost/damaged tissues such as the skin, the heart, and the gastrointestinal (GI) tract, among others. While we will now review the efforts made thus far at using these cells for beta cell differentiation, there is an increasingly widespread credence that the best use of MSCs in this context is not as building blocks, but rather as immunomodulatory and pro-angiogenic agents. Indeed, MSCs are well known for their ability to secrete many cytokines and growth factors, including vascular endothelial growth factor, brain-derived neurotrophic factor, nerve growth factor, basic fibroblast growth factor, insulin-like growth factor-1, hepatocyte growth factor (HGF), and others. In different contexts, these factors have been found to interact with local microenvironments and have anti-apoptotic, morphogenetic, mitogenic, and angiogenic effects that may favor engraftment and/or endogenous regeneration. A growing area of interest in the field of islet transplantation is that of cocultivation and/or cotransplantation of adult islet tissues with MSCs.

Signal-Driven Approaches

Many groups have attempted the directed differentiation of MSCs of various origins into beta cells by means of adding specific combinations of soluble factors to the culture medium. The general assumption has been that, since these cells are already committed along one of three major differentiation pathways (mesoderm), standard protocols for the differentiation of ES cells may not work as effectively. This, however, remains to be tested.

In 2004, Chen et al. treated rat MSCs with nicotinamide and beta-mercaptoethanol, obtaining “islet-like cluster cells” (ICCs), a term used by many people in the field to describe cellular aggregates (perhaps with the bias that this is how islets look in culture after isolation). When transplanted into syngeneic diabetic recipients, the authors observed a reduction in overall glycemic levels. One year later, Choi et al. reported robust expression of all pancreatic endocrine hormones, as well as glucoseregulated insulin secretion in vitro, after the exposure of bone marrow-derived rat MSCs to pancreatic extracts from rats subjected to partial (60%) pancreatectomy. The rationale behind these experiments, as described in the chapter “Stem Cell Differentiation: General Approaches,” is that pancreatic insults induce a regenerative response that might be accompanied by the release of islet-specific growth factors. Other protocols for the in vitro differentiation of MSCs include cultivation with HGF and activin A (which resulted in the expression of small levels of insulin from human MSCs, as well as glucagon, glucokinase, and a number of markers of early pancreatic development); the use of fibronectin and pellet suspension techniques; the coculture with pancreatic tissues in an artificial extracellular matrix; and a combination of conophylline and betacellulin (BTC)-delta4, which substantially accelerated the pancreatic differentiation of murine bone marrow-derived MSCs when compared with basal differentiation methods making use of standard activin A plus BTC. Cells differentiated in this way were able to reduce hyperglycemia in transplanted animals for up to 4 weeks, an outcome comparable to that resulting from transplanting rat MSCs treated with exendin-4 and nicotinamide.

Genetic Manipulation

As is the case for embryonic stem cells (see the chapter “Embryonic Stem Cells and Pancreatic Differentiation”) and transdifferentiation (see the chapter “Transdifferentiation”), the master pancreatic regulator Pdx1 remains a key player in all approaches involving genetic manipulation of MSCs. Thus, upon transfection of bone marrow-derived rat MSCs with a constitutively active Pdx1 cassette, Sun et al. observed expression of insulin, glucagon, and somatostatin. Transplantation of these cells into diabetic animals resulted in longer survival and maintenance of body weight, even if normoglycemia was not achieved. A similar approach was also used by Li et al. with human MSCs, where the Pdx1 gene was introduced via an adenoviral vector. Several relevant beta cell genes, including Ngn3, insulin, glucagon, glucokinase, and GLUT-2 were significantly up-regulated, and transduced cells exhibited a marginal ability to respond to glucose stimulation in vitro. Upon transplantation, these cells were able to reverse hyperglycemia in diabetic mice for up to 6 weeks. A triple adenovirus-mediated transfection with Pdx1, Hlxb9, and Foxa2 resulted in the detection of insulin in vitro, but with just a small proportion of cells expressing the fundamental component of the pro-insulinprocessing gene PC 1/3. Another group used retroviral vectors to ensure longterm expression of the Pdx1 gene in human bone marrow-derived MSCs, which led to the expression of all four pancreatic hormones but not NeuroD 619 (see the chapter “Pancreatic Development”). Despite the absence of this critical marker, these cells were able to regulate insulin secretion in vitro. In addition, when transplanted into diabetic animals, NeuroD was activated and a significant reduction in hyperglycemic levels was observed. More recently, Masaka et al. developed a chemical protocol based on the administration of HGF and FGF-4, which resulted in the generation of a bipotential, self-renewable “hepato-pancreatic” progenitor cell population. When Pdx1 was overexpressed in these cells, insulin was produced and an in vitro glucose-regulated response observed. In line with the above observations, the intrahepatic transplantation of mouse MSCs expressing a human ectopic insulin cassette reduced glucose levels for up to 6 weeks in diabetic recipients .

Signal-Driven Approaches

A natural first attempt at directing the differentiation of huES cells toward beta cells was to expose them to the natural milieu of signals that drive the process in vivo. Given the practical difficulties of working with human fetal tissue, Brolen et al. transplanted spontaneously differentiated huES cells containing subpopulations of pancreatic (Pdx1 + /Foxa2 + ) and endocrine (Pdx1 + /Isl1 + ) together with murine fetal pancreatic explants under the kidney capsule of recipient immunocompromised mice. Beta cell-like clusters expressing not only insulin, but also many transcription factors known to participate in the maintenance of the beta cell phenotype, could be consistently observed in the experimental group, but not in control animals that were transplanted with the huES and nonpancreatic tissues, such as the liver or the telencephalon of mouse embryos. The results later reported by Vaca et al. in an in vitro setting are consistent with these observations. As for purely in vitro approaches, the adaptation of the protocol of Lumelsky et al., based on the generation and expansion of intermediate-stage nestinpositive cells, was another logical step. It was first reported by Baharvand et al., who were able to observe glucose-mediated insulin release in vitro, but failed to detect insulin secretory granules. As is the case with mouse ES cells, even the cells that produce bona fide insulin as a result of nestin-based differentiation protocols from huES cells are more related to neuroectoderm than to endodermderived beta cells.

The first reports on “canonical” ES cell differentiation into beta cells came shortly after the description of the conditions for definitive endoderm differentiation in murine ES cells.Using a method based on the administration of Activin A (an analog of Nodal) in the presence of a very low percentage of serum, D’Amour and colleagues obtained cultures consisting of up to 80% of cells with definitive endoderm characteristics (Sox17, Goosecoid, Foxa2, and Mixl1 expression). These cells could be further enriched by sorting for CXCR4, a chemokine receptor expressed in mesoderm and definitive endoderm, but not in primitive/visceral endoderm. Transplantation of these cells into immunodeficient mice resulted in further progression of the endoderm differentiation program, as evidenced by expression of intestinal and liver markers in histological sections of grafts. Arguably, the most critical contribution of this study to the field was the finding that Activin A was most effective at low serum concentrations. Serum-borne factors are known to interfere with specific differentiation pathways, including that leading to the generation of definitive endoderm. The same team of investigators contributed a breakthrough follow-up study shortly after the publication of the first one. Capitalizing on their ability to efficiently differentiate huES cells toward definitive endoderm, they applied FGF7 (a powerful inducer of Notch signaling during pancreatic development ) and cyclopamine (an inhibitor of Shh signaling ) to obtain cells that exhibited features similar to those of the primitive gut tube (chiefly expression of HNF-1beta and HNF-4A). Subsequent addition of retinoic acid (which had been previously found by others to help both the patterning of endoderm and the progression of pancreatic development ) in defined medium resulted in the appearance of Pdx1- positive cells with a expression profile consistent with that of posterior foregut, first, and pancreatic endoderm/endocrine progenitors later. A final maturation step led to the formation of pancreatic endocrine cells with a messenger RNA pattern comparable to that of human adult islets . All of the endocrine cell types found in adult islets were represented in these cultures. Given their importance for prospective cell therapies for diabetes, the authors focused on the characterization of putative beta cells, which constituted approximately 7% of the total population. These cells had very high insulin content, almost equivalent to that of adult islets and much more elevated than in earlier protocols.They had well-defined secretory granules and were shown to secrete insulin/C-peptide in response to a variety of secretagogues, but not glucose. An explanation for the latter observation was that these beta-like cells might not be completely mature.

Another multistep protocol described around the same time showed some degree of glucose-responsiveness, but comparable differentiation efficiency. Shortly thereafter, the authors of the original study completed an unparalleled triad of breakthrough reports. Seemingly accepting defeat at obtaining therapeutic yields in vitro, they decided to transplant huES cell derivatives into immunodeficient mice midway throughout the protocol, in the hope that they would complete their maturation in vivo. The procedure was successful at preventing the development of diabetes in streptozotocin-treated recipients, but the implanted cells took several months to mature upon implantation, and teratogenic lesions were observed in at least 15% of the animals.

The entire field is presently at a crossroads: is there anything else we can do to significantly increase the efficiency of full differentiation in vitro, or should we abandon such efforts and focus instead on making the transplantation of immature progenitors safer and faster acting? Only time will tell, but judging from the almost frenetic pace of research on the subject, a definitive answer might be just around the corner.

Genetic Manipulation

Following the demonstration that constitutive Pax4 expression improves the outcome of pancreatic differentiation in mouse ES cells, Liew et al. selected huES cell clones stably transfected with Pax4. Late-stage EBs generated from these cells were plated on Matrigel ™ and treated with low glucose and nicotinamide, a set of conditions previously found to enhance the differentiation of beta cell progenitors in vitro. They yielded a higher number of cells that stained positive for Newport Green (NG), a zinc-fluorescent probe commonly used to label and sort beta cells.

FACS-enriched NG-positive cells had higher levels of Insulin, C-peptide, and Pdx1 expression than those derived from their nontransfected counterparts. However, they were irresponsive to glucose stimulation. In another recent report, Lavon et al. found that constitutive expression of Foxa2 in huES cells did not significantly alter the pattern of pancreatic specification in spontaneously differentiating EBs. Pdx1 overexpression, in contrast, resulted in an overall acceleration in the onset of the downstream gene Isl1 and the up-regulation of downstream genes such as Ngn3 and Pax4. However, insulin-expressing cells could not be detected unless the cells were allowed to further differentiate in vivo teratomas, and the differences between the genetically modified cells and the wild-type controls were statistically insignificant.

Protein Transduction

The only example thus far of the use of this novel approach to aid in the pancreatic differentiation of huES cells was reported only recently. The authors subjected huES cell-derived EBs to TAT-Pdx1 treatment for 7 days, using Pdx1 protein as a control. Despite the well-known ability of Pdx1 to penetrate cells by virtue of an antennapedia-like protein transduction domain, samples treated with TATPdx1 showed a very significant up-regulation of Pdx1 targets compared with the controls. These included its own endogenous Pdx1 counterpart ( ~ 20-fold), Insulin ( ~ 30-fold), or islet amyloid polypeptide ( ~ 12-fold). C-peptide could be detected by immunofluorescence, but GLUT-2 levels remained unchanged when compared with those of samples treated with Pdx1 protein.

A caveat of these studies – which could explain the partial effects observed – is that Pdx1 was applied at a very early differentiation stage, prior to the initial specification of primitive gut/posterior foregut cells. At this point, in the absence of the molecular partners that would normally act in concert with Pdx1 during pancreatic specification the protein alone might be insufficient to fully trigger by itself the onset of such program. It is plausible, however, that the use of TAT-Pdx1 at the appropriate huES cell differentiation stage might improve the yield and function of beta cells in the context of signal-driven approaches. In this context, it would be interesting to test whether TAT-Pdx1-VP16, a recently described version of transducible Pdx1 that includes the VP16 transactivation domain would improve ES cell differentiation efficiency.

iPS Cells

With the cloning of Dolly the sheep and the advent of SCNT the possibility of tailoring ES cells to each patient was immediately acknowledged. The nuclei of easily harvested somatic cells from any given donor would undergo a reprogramming process by microinjection into enucleated oocytes. The activation of the reconstituted embryo would lead to the in vitro development of blastocysts from which ES cells – genetically identical to the donor – would be harvested. The principle of “therapeutic cloning” was readily tested in several species, including nonhuman primates but, despite initial reports suggesting otherwise huES cells could never be derived from SCNT-derived embryos. Alternatives to SCNT included the use of cellular extracts or fusion with ES cells but none of these approaches was completely successful. Very recently, however, the group of Takahashi and Yamanaka were able to reprogram murine somatic cells by introducing four critical components of the core ES cell circuitry, namely Oct3/4, Sox2, c-Myc, and Klf4. Similar results with human cells were presented shortly thereafter by two independent groups – the second using Sox2, Oct3/4, Lin28, and Nanog in the reprogramming mixture . The biotechnological feat of reprogramming adult somatic cells in such simple fashion (the top scientific breakthrough of the year 2008 of the journal Science ) might have enormous medical implications, provided that the newly reprogrammed cells are comparable to blastocyst-derived ES cells; and the procedure can be done safely. While all preliminary evidence seems to confirm that these induced pluripotent stem (iPS) cells are functionally indistinguishable from their embryo-derived counterparts, the use of retroviral vectors to deliver the critical genes is still unsafe in the context of human therapies . Ongoing efforts at addressing this concern include the use of nonviral delivery methods and protein transduction. The use of iPS cells for directed differentiation is now a fertile field, as dozens of laboratories around the world attempt to replicate results previously obtained with huES cells.Of special interest is the recently reported proof of principle that iPS cells can undergo pancreatic differentiation using a protocol similar to that used by Jiang et al.

Safety of ES Cell Therapies

The translation of basic ES cell research into clinical therapies remains hindered by a number a safety concerns that arise from their very nature. Indeed, these cells are characterized by an unlimited proliferation potential under the appropriate conditions. If unchecked, even minute percentages of undifferentiated cells could keep dividing upon transplantation in immunosuppressed recipients, yielding teratomas. These tumors develop either from carry-over undifferentiated cells or from cells that de-differentiate upon transplantation. Some groups have approached this problem by screening the number of undifferentiated cells in each transplantable preparation. If such number is below the threshold known to produce teratomas in nude mice, they argue, these preparations should be considered safe for clinical use. However, this method does not take into account the risk of dedifferentiation after transplantation.Other teams have addressed the teratogenic potential of ES cells by integrating suicide genes into them. Such elements sensitize ES cells to specific pro-drugs, which can be used to induce their selective ablation both in vitro and in vivo. However, if teratomas were to form in vivo due to de-differentiation of implanted cells, administration of the pro-drug would kill the entire graft. The risk of accumulation of genomic instabilities as a result of long-term cell culture might be subtler, but not less dangerous. In view of later observations, the first reports on the karyotypic stability of huES cells turned out to be inexact. As it happens with other cells, their adaptation to proliferative conditions in vitro invariably results in the selection of traits – perhaps not surprisingly – similar to those responsible for malignant transformation.These include chromosomal and subchromosomal alterations not always detectable in standard G-banding tests.Rushing ES cell therapies to the clinic would not be advisable until steps are taken to minimize these risks.

Line-to-Line Variability

Despite the overall similarities between different huES cell lines when kept under conditions that help maintain their pluripotency, several groups have reported differences in a number of parameters, ranging from population doubling time to susceptibility to spontaneous differentiation. These differences become even more striking when the cells start to specify. In a comprehensive study of huES cell lines allowed to spontaneously differentiate, Osafune et al. found >100-fold line-to-line variations in the expression of markers specific for the three germ layers. Three cell lines, for example, had a higher propensity to develop mesodermal lineages; two exhibited a marked preference for ectodermal or neural genes; another two showed a tendency to give rise to endodermal cell types; and so on. Out of the latter, line huES turned out to be the best in terms of differentiation potential into pancreatic cells. One potential conclusion from these results is that we ought to look for the right cell line for each differentiation protocol. This would argue in favor of banking an extensive number of ES cell lines and use only the ones that are better suited for each purpose. The alternative view, however, is that the results manifest the need for developing more robust protocols that will work in a cell line-independent fashion. After all, prospective personalized therapies where ES-like (iPS) cells are derived from the patient might not afford the clinician the freedom to choose the most appropriate cell line for the desired developmental outcome.

Spontaneously differentiated ES cells containing endocrine and exocrine progenitors are able to terminally mature in vivo when engrafted together with fetal pancreatic tissue. Controls in which other tissues unrelated to the pancreas were transplanted together with the ES cells did not yield insulin-producing cells.

huES cell differentiation protocol described by D’Amour and colleagues (2006). FCS fetal calf serum; FGF fibroblast growth factor; RA retinoic acid; Cyc cyclopamine; Ex-4 exendin-4 IGF-1 insulin-like growth factor 1; HGF hepatocyte growth factor.

huES cell differentiation protocol reported by Jiang et al. . EGF epithelial growth factor; bFGF basic fibroblast growth factor; IGFII insulin-like growth factor II.

Original protocols for the generation of iPS from adult fibroblasts. Retroviruses were used for the delivery of two different sets of reprogramming genes. Both combinations included the master regulators Oct3/4 and Sox2.

Undifferentiated ES cells can proliferate indefinitely under the appropriate conditions. Upon differentiation and transplantation, undifferentiated escapees may keep proliferating, especially if the recipient is immunosuppressed. These cells will give rise to a tumor termed teratoma . There is evidence that such tumors may also originate by de-differentiation of differentiated cells . Strategies to prevent this undesired outcome may involve the engineering of suicide genes in the genome of the donor cell line ( black nuclei ), which will destroy the host cell in the presence of a pro-drug . Current research focuses on the design of strategies to activate these suicide genes only in the cells that remain undifferentiated, as opposed to the entire graft.

Gene-trap approach to selectively ablate non-insulin producing cells, as described by Soria et al. ES cell clones expressing an Ins–Neo cassette are allowed to spontaneously differentiate. Cells that differentiated along the insulin-producing lineage were resistant to the drug G418, and could be selected for transplantation into streptozotocin-treated mice.