Stem Cells And Diabetes: New Trends And Clinical Prospects

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Stem Cells and Diabetes: New Trends and Clinical Prospects By Juan Domínguez-Bendala, Ph.D. and Camillo Ricordi, M.D.

ype I diabetes has been acknowledged, from very early on, as one the top potential targets of stem cell-based regenerative therapies. While the treatment of other diseases would typically require extensive bioengineering and/or the recapitulation of a rather complex cellular niche, the case for type I diabetes was solidly grounded on the fact that, in principle, replacement was needed for only one cell type – the insulin-producing beta cell. A tremendous body of work spanning decades of basic and clinical research had established that beta cells can exert glycemic control in a variety of ectopic locations, effectively simplifying the problem. Finally, successful clinical therapies resulting in insulin-independence (islet transplantation) had already been in use for two decades, demonstrating the feasibility of cell-based approaches for type I diabetes. However, a decade after the isolation of the first human embryonic stem (huES) cells, other a priori lesslikely targets seem to have taken an early lead in the race to clinical applications. Informally organized in “top stories”, this article will review the reasons behind this apparent paradox, describing the not-so-simple nature of the problem, the most promising avenues of research, and the challenges that still lie ahead.

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yield increases and the first reports on insulin independence in humans.14 The second was the development of a steroid-free immunosuppresive regime that helped preserve the survival of engrafted islets, extending their function for 5 years or longer in a significant percentage of transplanted patients.15,16 In short, the procedure is based on the enzymatic/mechanical dissociation of islets from the pancreatic parenchyma, using a digestion chamber shaken at a pre-determined speed. Once the digestion is completed, islet cell clusters are separated by gradient centrifugation and then cultured and infused in the liver of the patient via the portal vein. Islets are known to lodge in the pre-sinusoidal capillaries of the liver, where they get revascularized within weeks of the procedure.9,11,12,17,18 If carried out by skilled teams, the procedure has a very low incidence of complications, resulting in immediate blood glucose control15 and a significant improvement in the patient’s quality of life.19 Despite the continued refinements of the technique, to this day islet transplantation remains a “brute force” approach. The digestion and isolation procedure may destroy one half of the islets present in any given donor pancreas; and by some estimates, up to 80% of those that are infused may also die within hours of the procedure.20 By this account, the observation that 5 years later only 10-50% of the patients are off-insulin15,21 is not as surprising as the fact that up to 80% are insulin-independent 1 year after the procedure.15 In summary, our clinical experience is that even very little can go a long way, which bodes well for the applicability of stem cell therapies to treat this disease. To avoid redundancy with other articles in this publication, we will spare the reader yet another introduction on the basics of stem cells, its many types and prospective uses, proceeding instead to describe progress in their use for the treatment of diabetes. In no particular order of importance, here are some of the most significant advances reported over the last couple of years that are likely to shape the overall direction of the field:

Islet Transplantation: The Proof of Concept

Functional Beta-like Cells Derived From huES Cells.

Pancreatic transplantation is effective at eliminating the need for exogenous insulin in type I diabetic patients1, but the limitations common to all solid organ transplantation procedures (including scarcity of donors and complications inherent to major surgery2-7) make it hardly a therapy of choice. Islet transplantation, in contrast, is a much less invasive approach that allows for the selective heterotopic engraftment of the small percentage of pancreatic cells that secrete insulin.8-13 Two major breakthroughs shaped the history of this practice after the first tentative attempts in the late 70s and early 80s. The first one was the invention of a semi-automated method for the isolation of islets from a donor pancreas, which led to dramatic

In a triad of groundbreaking communications, the Novocell team led by E. Baetge described in quick succession a method for the efficient generation of definitive endoderm from huES cells22, the first report on “canonical” huES cell differentiation into beta-like cells23 and, finally, a convincing demonstration of in vivo maturation and function of huES cell-derived pancreatic progenitors.24 The latter approach was designed to address the fact that the in vitro protocol, for all its virtues, was highly inefficient (less than 10% of insulinproducing cells) and did not result in measurable glucose-stimulated insulin release. The rationale for transplanting immature progenitors

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ROAD TO CURES: SCIENCE, TREATMENTS AND ECONOMICS destroy its own beta cells, immunosuppresion would still be necessary no matter the origin of the replacement. It can be argued, however, that effective iPS-to-beta cell differentiation methods could lead to therapies that would tackle two out of the three major problems we face right now (supply and allorejection), singling out autoimmunity as the only remaining hurdle. This would simplify the problem, allowing for a much more effective use of resources to find a cure to the disease.

was that perhaps the in vivo microenvironment would provide them with the necessary cues to complete their differentiation into functional beta cells. Success was achieved at the expense of a rather long lag period till function (up to three months) and a very high incidence of teratomas, which would obviously hamper the clinical translation of this method.25 It is unclear at this point the direction the field will take. On the one hand, the full differentiation protocol has been reportedly difficult to reproduce with cell lines other than those indicated in the original articles, which would argue in favor of additional research into developing more robust methods that could be used with many cell lines. Few would dispute that a fully functional, ready-to-transplant cell product would be preferable in a clinical setting -not only because of the expected reduction in the rate of tumorigenic events, but also because of the elimination of the lag between transplantation and function. In this context, other competing methods26 may still prove more viable than Novocell’s. On the other hand, progress at sorting out the fully mature cells from the carry-over undifferentiated ones, together with the development of appropriate encapsulation techniques and better controls to identify a teratogenic threshold, may still allow for an early therapeutic implementation of the in vivo maturation approach.

Infusion of Bone Marrow Stem Cells for Type I Diabetes? It has been hypothesized that some adult stem cells (chiefly of the ubiquitous mesenchymal lineage, obtained from the bone marrow and other locations) might contribute to the regeneration of pancreatic beta cells if delivered either systemically or locally. The potential benefits of this approach are clear inasmuch as these cells are proliferative enough to yield therapeutic loads, can be potentially obtained from the patient, and pose no ethical concerns. On the other hand, premature claims that mesenchymal stem cells (MSCs) cells are a safer, non-tumorigenic alternative to huES and iPS cells have proven wrong. Indeed, their adaptation to in vitro culture can lead to the development of chromosomal abnormalities resulting in malignant transformation as early as in a few passages.45-50 Also, the mesodermal origin of both MSCs and hematopoietic places a big question mark on their purported ability to become endodermal tissues such as pancreatic beta cells. Early observations that donor bone marrow cells could migrate to the damaged pancreas and contribute to the regeneration of islets51 were subsequently disputed by studies that confirmed the migration but not the differentiation.52-54 The latter of such studies, in fact, provided evidence that the cells recruited to the pancreas were rather of endothelial origin. As is the case with MSCs -which have well-documented trophic, angiogenic, immunomodulatory and anti-inflammatory effects55-61, these mobilized bone marrow-derived cells could indeed aid in the endogenous regeneration of beta cells, but not by direct differentiation. Whichever the mechanism may be, autologous intra-pancreatic bone marrow transplantation is currently enjoying the limelight as a potential new treatment for type I and II diabetes. Much of this attention is due to the controversial for-profit clinical practice of the technique in some countries at a time when there is no evidence of safety and efficacy in humans. Fortunately, there are already a number of Phase I/II trials looking at the potential benefit of local delivery of MSCs and bone marrow cells (www.clinicaltrials.gov), including one in the USA for type II diabetes in which autologous intra-pancreatic bone marrow cell transplantation is done in conjunction with hyperbaric treatment. The role of oxygen in promoting beta cell differentiation and survival has been recently established62-64, and preliminary results of this combination therapy in human subjects are highly encouraging.65

Directed Differentiation of iPS Cells. Nothing epitomizes better the frantic pace of stem cell research than the advent and overnight success of induced pluripotent stem (iPS) cells. Barely two years after the first breakthrough reports on the reprogramming of adult fibroblasts by viral transduction of a few master genes27,28, this technique -which allows for the generation of custom-made, patient-matched embryonic stem (ES)-like cells- has almost relegated to oblivion the much more cumbersome theoretical alternative of somatic cell nuclear transfer (SCNT) for “therapeutic cloning”.29,30 The biotechnological feat of reprogramming adult somatic cells in such simple fashion is likely to have enormous medical implications, provided that (a) the newly reprogrammed cells are comparable to blastocyst-derived ES cells; and (b) the procedure can be done safely. Despite some reports indicating that the molecular signature of iPS cells might be, after all, somewhat different to that of ES cells31, there is very little question now that both pluripotent stem cell types are, to a large extent, interchangeable. As for the safety concerns, ongoing efforts at addressing them include the use of non-viral delivery methods32, episomal vectors33, protein transduction34,35, the progressive replacement of reprogramming genes by chemical factors36-39 and the use of adult stem cells as substrates for reprogramming.40 As these techniques quickly became mainstream, laboratories around the world endeavored to replicate with iPS cells results previously attained with huES cells.41-43 In this context, the eventual use of these cells for direct differentiation into beta-like cells was nothing short of inevitable. The first report was published last year by Tateishi and colleagues44, who drove skin-derived iPS cells along the beta cell lineage using a protocol similar to that described by Jiang et al 26 for huES cells. The fact that type I diabetes is an autoimmune disorder certainly limits enthusiasm for the prospective uses of patient-matched iPS cells. After all, since the patient’s immune system is already poised to

Resetting the Immune System? Most people tend to think about stem cells as “building blocks” to be used for the repair of damaged tissues. We have described above how bone marrow stem cells may actually help in the regeneration of

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ROAD TO CURES: SCIENCE, TREATMENTS AND ECONOMICS islets by providing endogenous progenitor cells with the chemical cues required to complete their maturation, or by otherwise supporting their action (e.g., by creating anti-inflammatory or angiogenic microenvironments). One of the major developments in the field over the past few years has to do with another well-known facet of bone marrow stem cells, namely their ability to modulate the body’s immune response. Thus, in clinical trials conducted on 15 newly diagnosed type I diabetic patients, hemaotopoietic stem cells were mobilized, harvested from peripheral blood and subsequently frozen for later use. Following a harsh immunosuppressive treatment intended to destroy autoreactive T cell clones, the patients were then reinfused with their own cryopreserved hematopoietic stem cells, which were assumed to be “naïve” (i.e., at a stage prior to the onset of the disease). In the first communication, the authors reported that 14 out of the 15 patients became insulin-free for extended periods of time.66,67 A recent update of the original study with a longer followup (21/2 years) confirmed that C-peptide levels increased significantly in the treated patients, the majority of whom achieved and maintained insulin independence.68 Excitement was and remains justified, as such reports are the first ever suggestive of a definitive “cure”. However, the approach also has substantive limitations. For one, it has been hypothesized to work only in recently diagnosed patients, who may still have a significant mass of beta cells susceptible of regeneration. If such mass is below a still undetermined threshold (as would be expected in long-standing diabetes), it may still be necessary to supplement the patients with an exogenous “boost” of beta cells. Secondly, the immunosuppressive regime is highly toxic, and potentially fatal. Although no deaths have been reported yet in relation to this therapy, the sample sizes are still very small. A 2005 study evaluating the clinical outcome of autologous hematopoietic stem cell transplantation for a variety of autoimmune conditions (including neurological, rheumatologic, hematological and gastrointestinal disorders) concluded that the treatment was effective, but associated with a high rate of morbidity and mortality (7% at 3 years).69 However, better patient selection, refinement of methods, and progressive experience of teams are likely to decrease transplantrelated mortality to more acceptable rates.

islets72 and then by the unexpected finding that the Ngn3 endocrine differentiation program could be reactivated in the adult pancreas following partial duct ligation.73 The physiological significance of this model is unclear, and it is a safe assumption that self-replication is still the main engine of adult beta cell regeneration. However, now we know that the pancreas may still have an “ace in the hole”, an alternative pathway that could be activated under specific circumstances and take over regeneration. Further research will be needed to decipher its molecular determinants, the reasons that make this regeneration model different from all the others (which do not induce the Ngn3 program) and the potential ways to apply such knowledge to our advantage for therapeutic purposes.

Transdifferentiation Also in line with the above strategy, a subtler but equally effective method to induce beta cell neogenesis in the adult pancreas was recently reported by Zhou and colleagues74, who described the “reprogramming” of the acinar tissue of the murine pancreas by ectopically expressing a combination of three genes, namely Pdx1, Ngn3 and MafA. New insulin-producing cells – virtually indistinguishable from the native ones- were detected just days after the adenoviral delivery of the aforementioned cassettes, and kept expanding long after the viruses had been cleared from the host. Clearly inspired in the pioneering work of Ferber and colleagues in the liver75-77, the new approach had the advantage that -perhaps due to the closeness of the starting material to the desired end productthe reprogramming process completely abrogated the original phenotypes; in other words, these were true beta cells, not merely exocrine cells that secreted insulin. Hyperglycemia was significantly ameliorated in the treated animals, even if diabetes was not completely reversed. This could be due to the fact that, while direct communication between beta cells appears to be critical for the synchronicity and effectiveness of glucose-mediated insulin secretion78,79, the newly induced cells failed to aggregate in clusters. The clinical applicability of this strategy is still unclear. Although adenoviruses are present in the recipient only transiently, they are known to elicit serious immune responses. However, these experiments are proof of principle that exocrine cell reprogramming is feasible and perhaps doable ex vivo on cultured acinar cells that would be subsequently transplanted. Non-viral alternatives (such as the use of chemicals and/or protein transduction) should also be explored with the goal of improving the safety of this promising approach, thus accelerating its clinical translation.

Regeneration: From The Duct to Replication (And Back?) Proof of the rapid evolution of an ever-changing field is the continuous shift of the paradigm on adult beta cell regeneration. It was the conventional wisdom for decades that beta cells arose from progenitor cells residing in the pancreatic ducts, although the support for this hypothesis had always been anecdotal and largely based on two-dimensional pictures of beta cells seemingly budding out from the periductal area.70 A series of elegant lineage-tracing experiments conducted in a transgenic mouse model challenged this notion in 2004, indicating that self-replication was the main mechanism behind adult beta cell turnover and regeneration.71 While the proponents of the “adult stem cell” theory still contended that human beta cell regeneration might obey to different rules, these clear-cut observations clearly put the burden of proof on their shoulders. Then the new idea was challenged again in 2008, first by new lineage tracing experiments showing ductal offspring in newly created

New trends in transplantation Although the liver has served as the surrogate for the pancreas for all islet transplantation procedures conducted in humans thus far, there is widespread consensus that this ectopic location is far from optimal. As mentioned earlier in this article, islets sustain a massive cell loss upon intra-hepatic infusion. Stem cell-derived beta cells (whichever their origin) are likely to share the same fate, unless alternative sites are identified. There has been an active search of such sites in recent years, including an intriguing approach pioneered by Speier and colleagues in Miami80 by which islets transplanted in the anterior chamber of the eye can restore normoglycemia in diabetic

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ROAD TO CURES: SCIENCE, TREATMENTS AND ECONOMICS currently do when infused through the portal vein. This would enable the clinician to better monitor the graft while minimizing the inflammatory responses. Secondly, the use of these devices may eventually allow for the administration of immunosuppresants locally at much smaller effective doses than those required if taken systemically. This would largely prevent the occurrence of the side effects associated with oral immunosuppresion83. Finally, if using huES/iPS cell-derived tissues as a source of insulin-producing cells, any teratogenic lesion potentially arising from carryover undifferentiated cells would be contained within the device.

mice while allowing for a real-time, in vivo follow-up of their engraftment, vascularization and response to the immune attack. Unfortunately, the generalized view of the eye as an immunoprivileged site81 has not proven true for islet transplantation. This, together with the rather limited load of islets that could be transplanted in a clinical setting, makes the eye an unlikely candidate to replace the liver as a preferred location. An alternative to finding a suitable site is to create one. Thus, Pileggi et al82 demonstrated recently that islets infused into a biocompatible, subcutaneously implanted device – previously allowed to vascularize through its stainless-steel mesh walls – were able to reverse hyperglycemia in diabetic rats. The advantages of such an approach are many-fold: first, the islets (or the stem cell-derived beta cells) would not spread throughout an entire organ, as they

Acknowledgements CR and JDB are funded by the NIH, JDRF and the Diabetes Research Institute Foundation (DRIF).

Juan Domínguez-Bendala, Ph.D. Juan Domínguez-Bendala is Director of the Pancreatic Development & Stem Cells laboratory at the Diabetes Research Institute, University of Miami. He obtained his Ph.D. at the Roslin Institute (UK) under the supervision of Dr. McWhir, co-author of the 1997 report on

the cloning of Dolly the sheep. During his training, he acquired considerable experience in ES cell research and state-of-the-art genetic engineering techniques. His current projects focus on the use of stem cells to obtain pancreatic islets. He co-invented the “oxygen sandwich”, a culture device designed to promote differentiation. He is also working on long-term culture/regeneration of pancreatic stem cells and reprogramming.

Camillo Ricordi, M.D. Camillo Ricordi, M.D., is Professor of Surgery, Medicine, Biomedical Engineering, Microbiology and Immunology, Director of the Cell Transplant Center and head of the Diabetes Research Institute at the University of Miami. Dr. Ricordi is

known for developing the method for large scale isolation of human pancreatic islets, and for leading the team that performed the first series of successful clinical islet allografts in 1990. Dr. Ricordi’s research interests include induction of immune tolerance, cellular therapies, stem cells and regenerative medicine strategies. Dr. Ricordi received numerous honors and awards, authored over 600 scientific publications and, as inventor, holds nine patents.

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