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WORLD STEM CELL REPORT 2009 GENETICS POLICY INSTITUTE73
ROAD TO CURES: SCIENCE, TREATMENTS AND ECONOMICS
ype I diabetes has been acknowledged,from very early on, as one the toppotential targets of stem cell-basedregenerative therapies. While the treatment of other diseases would typically require extensivebioengineering and/or the recapitulation of arather complex cellular niche, the case for type Idiabetes was solidly grounded on the fact that, inprinciple, replacement was needed for only onecell type – the insulin-producing beta cell. Atremendous body of work spanning decades of basic and clinical research had established thatbeta cells can exert glycemic control in a variety of ectopic locations, effectively simplifying theproblem. Finally, successful clinical therapiesresulting in insulin-independence (islet transplan-tation) had already been in use for two decades,demonstrating the feasibility of cell-basedapproaches for type I diabetes. However, adecade after the isolation of the first humanembryonic stem (huES) cells, other
a priori 
less-likely targets seem to have taken an early lead inthe race to clinical applications. Informallyorganized in “top stories”, this article will reviewthe reasons behind this apparent paradox,describing the not-so-simple nature of theproblem, the most promising avenues of research,and the challenges that still lie ahead.
Islet Transplantation: The Proof of Concept
Pancreatic transplantation is effective at eliminating the need forexogenous insulin in type I diabetic patients
1
, but the limitationscommon to all solid organ transplantation procedures (includingscarcity of donors and complications inherent to major surgery 
2-7
)make it hardly a therapy of choice. Islet transplantation, in contrast,is a much less invasive approach that allows for the selectiveheterotopic engraftment of the small percentage of pancreatic cellsthat 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 forthe isolation of islets from a donor pancreas, which led to dramatic
T
yield increases and the first reports on insulin independence inhumans.
14
The second was the development of a steroid-freeimmunosuppresive regime that helped preserve the survival of engrafted islets, extending their function for 5 years or longer in asignificant percentage of transplanted patients.
15,16
In short, theprocedure is based on the enzymatic/mechanical dissociation of isletsfrom the pancreatic parenchyma, using a digestion chamber shakenat a pre-determined speed. Once the digestion is completed, islet cellclusters are separated by gradient centrifugation and then culturedand infused in the liver of the patient via the portal vein. Islets areknown to lodge in the pre-sinusoidal capillaries of the liver, wherethey get revascularized within weeks of the procedure.
9,11,12,17,18
If carried out by skilled teams, the procedure has a very low incidenceof complications, resulting in immediate blood glucose control
15
anda 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 digestionand isolation procedure may destroy one half of the islets present inany given donor pancreas; and by some estimates, up to 80% of thosethat are infused may also die within hours of the procedure.
20
By thisaccount, the observation that 5 years later only 10-50% of thepatients are off-insulin
15,21
is not as surprising as the fact that up to80%
are 
insulin-independent 1 year after the procedure.
15
Insummary, our clinical experience is that even very little can go a long way, which bodes well for the applicability of stem cell therapies totreat this disease.To avoid redundancy with other articles in this publication, we will spare the reader yet another introduction on the basics of stemcells, its many types and prospective uses, proceeding instead todescribe progress in their use for the treatment of diabetes. In noparticular order of importance, here are some of the most significantadvances reported over the last couple of years that are likely to shapethe overall direction of the field:
Functional Beta-like Cells Derived FromhuES Cells.
In a triad of groundbreaking communications, the Novocellteam led by E. Baetge described in quick succession a method for theefficient generation of definitive endoderm from huES cells
22
, the firstreport on “canonical” huES cell differentiation into beta-like cells
23
and, finally, a convincing demonstration of 
in vivo
maturation andfunction of huES cell-derived pancreatic progenitors.
24
The latterapproach was designed to address the fact that the
in vitro
protocol,for all its virtues, was highly inefficient (less than 10% of insulin-producing cells) and did not result in measurable glucose-stimulatedinsulin release. The rationale for transplanting immature progenitors
Stem Cells and Diabetes:New Trends and Clinical Prospects
By Juan Domínguez-Bendala, Ph.D. and Camillo Ricordi, M.D.
 
WORLD STEM CELL REPORT 2009 GENETICS POLICY INSTITUTE74
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 totherapies that would tackle two out of the three major problems weface 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 tothe disease.
Infusion of Bone Marrow Stem Cells forType I Diabetes?
It has been hypothesized that some adult stem cells (chiefly of the ubiquitous mesenchymal lineage, obtained from the bonemarrow and other locations) might contribute to the regeneration of pancreatic beta cells if delivered either systemically or locally. Thepotential benefits of this approach are clear inasmuch as these cells areproliferative enough to yield therapeutic loads, can be potentially obtained from the patient, and pose no ethical concerns. On theother hand, premature claims that mesenchymal stem cells (MSCs)cells are a safer, non-tumorigenic alternative to huES and iPS cellshave proven wrong. Indeed, their adaptation to
in vitro
culture canlead to the development of chromosomal abnormalities resulting inmalignant transformation as early as in a few passages.
45-50
 Also, themesodermal origin of both MSCs and hematopoietic places a bigquestion mark on their purported ability to become endodermaltissues such as pancreatic beta cells. Early observations that donorbone marrow cells could migrate to the damaged pancreas andcontribute to the regeneration of islets
51
 were subsequently disputedby studies that confirmed the migration but not the differentiation.
52-54
The latter of such studies, in fact, provided evidence that the cellsrecruited to the pancreas were rather of endothelial origin. As is thecase with MSCs -which have well-documented trophic, angiogenic,immunomodulatory and anti-inflammatory effects
55-61
, thesemobilized bone marrow-derived cells could indeed aid in theendogenous regeneration of beta cells, but not by directdifferentiation. Whichever the mechanism may be, autologous intra-pancreaticbone marrow transplantation is currently enjoying the limelight as apotential new treatment for type I and II diabetes. Much of thisattention is due to the controversial for-profit clinical practice of thetechnique in some countries at a time when there is no evidence of safety and efficacy in humans. Fortunately, there are already a numberof Phase I/II trials looking at the potential benefit of local delivery of MSCs and bone marrow cells (www.clinicaltrials.gov), including onein the USA for type II diabetes in which autologous intra-pancreaticbone marrow cell transplantation is done in conjunction withhyperbaric treatment. The role of oxygen in promoting beta celldifferentiation and survival has been recently established
62-64
, andpreliminary results of this combination therapy in human subjects arehighly encouraging.
65
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 abovehow bone marrow stem cells may actually help in the regeneration of 
ROAD TO CURES: SCIENCE, TREATMENTS AND ECONOMICS
 was that perhaps the
in vivo
microenvironment would provide them with the necessary cues to complete their differentiation intofunctional beta cells. Success was achieved at the expense of a ratherlong lag period till function (up to three months) and a very highincidence of teratomas, which would obviously hamper the clinicaltranslation of this method.
25
It is unclear at this point the directionthe field will take. On the one hand, the full differentiation protocolhas been reportedly difficult to reproduce with cell lines other thanthose indicated in the original articles, which would argue in favor of additional research into developing more robust methods that couldbe used with many cell lines. Few would dispute that a fully functional, ready-to-transplant cell product would be preferable in aclinical setting -not only because of the expected reduction in the rateof tumorigenic events, but also because of the elimination of the lagbetween transplantation and function. In this context, othercompeting methods
26
may still prove more viable than Novocell’s. Onthe other hand, progress at sorting out the fully mature cells from thecarry-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 therapeuticimplementation of the
in vivo
maturation approach.
Directed Differentiation of iPS Cells.
Nothing epitomizes better the frantic pace of stem cell researchthan the advent and overnight success of induced pluripotent stem(iPS) cells. Barely two years after the first breakthrough reports on thereprogramming of adult fibroblasts by viral transduction of a few master genes
27,28
, this technique -which allows for the generation of custom-made, patient-matched embryonic stem (ES)-like cells- hasalmost relegated to oblivion the much more cumbersome theoreticalalternative of somatic cell nuclear transfer (SCNT) for “therapeuticcloning”.
29,30
The biotechnological feat of reprogramming adultsomatic cells in such simple fashion is likely to have enormousmedical implications, provided that (a) the newly reprogrammed cellsare comparable to blastocyst-derived ES cells; and (b) the procedurecan be done safely. Despite some reports indicating that themolecular signature of iPS cells might be, after all, somewhatdifferent to that of ES cells
31
, there is very little question now thatboth pluripotent stem cell types are, to a large extent,interchangeable. As for the safety concerns, ongoing efforts ataddressing them include the use of non-viral delivery methods
32
,episomal vectors
33
, protein transduction
34,35
, the progressivereplacement of reprogramming genes by chemical factors
36-39
and theuse of adult stem cells as substrates for reprogramming.
40
 As these techniques quickly became mainstream, laboratoriesaround the world endeavored to replicate with iPS cells resultspreviously attained with huES cells.
41-43
In this context, the eventualuse of these cells for direct differentiation into beta-like cells wasnothing short of inevitable. The first report was published last year by Tateishi and colleagues
44
, who drove skin-derived iPS cells along thebeta cell lineage using a protocol similar to that described by Jiang etal 26 for huES cells.The fact that type I diabetes is an autoimmune disorder certainly limits enthusiasm for the prospective uses of patient-matched iPScells. After all, since the patient’s immune system is already poised to
 
WORLD STEM CELL REPORT 2009 GENETICS POLICY INSTITUTE75
ROAD TO CURES: SCIENCE, TREATMENTS AND ECONOMICS
islets72 and then by the unexpected finding that the Ngn3 endocrinedifferentiation program could be reactivated in the adult pancreasfollowing partial duct ligation.
73
The physiological significance of thismodel is unclear, and it is a safe assumption that self-replication is stillthe main engine of adult beta cell regeneration. However, now weknow that the pancreas may still have an “ace in the hole”, analternative pathway that could be activated under specificcircumstances and take over regeneration. Further research will beneeded to decipher its molecular determinants, the reasons that makethis regeneration model different from all the others (which do notinduce the Ngn3 program) and the potential ways to apply suchknowledge to our advantage for therapeutic purposes.
Transdifferentiation
 Also in line with the above strategy, a subtler but equally effectivemethod to induce beta cell neogenesis in the adult pancreas wasrecently reported by Zhou and colleagues
74
, 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 afterthe adenoviral delivery of the aforementioned cassettes, and keptexpanding long after the viruses had been cleared from the host.Clearly inspired in the pioneering work of Ferber and colleaguesin the liver
75-77
, the new approach had the advantage that -perhaps dueto the closeness of the starting material to the desired end product-the reprogramming process completely abrogated the originalphenotypes; 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 notcompletely reversed. This could be due to the fact that, while directcommunication between beta cells appears to be critical for thesynchronicity and effectiveness of glucose-mediated insulinsecretion
78,79
, the newly induced cells failed to aggregate in clusters.The clinical applicability of this strategy is still unclear. Althoughadenoviruses are present in the recipient only transiently, they areknown to elicit serious immune responses. However, theseexperiments are proof of principle that exocrine cell reprogrammingis feasible and perhaps doable ex vivo on cultured acinar cells that would be subsequently transplanted. Non-viral alternatives (such asthe use of chemicals and/or protein transduction) should also beexplored with the goal of improving the safety of this promisingapproach, thus accelerating its clinical translation.
New trends in transplantation
 Although the liver has served as the surrogate for the pancreas forall islet transplantation procedures conducted in humans thus far,there is widespread consensus that this ectopic location is far fromoptimal. As mentioned earlier in this article, islets sustain a massivecell loss upon intra-hepatic infusion. Stem cell-derived beta cells(whichever their origin) are likely to share the same fate, unlessalternative sites are identified. There has been an active search of suchsites in recent years, including an intriguing approach pioneered by Speier and colleagues in Miami
80
by which islets transplanted in theanterior chamber of the eye can restore normoglycemia in diabeticislets by providing endogenous progenitor cells with the chemicalcues required to complete their maturation, or by otherwisesupporting their action (e.g., by creating anti-inflammatory orangiogenic microenvironments). One of the major developments inthe field over the past few years has to do with another well-knownfacet of bone marrow stem cells, namely their ability to modulate thebody’s immune response. Thus, in clinical trials conducted on 15newly diagnosed type I diabetic patients, hemaotopoietic stem cells were mobilized, harvested from peripheral blood and subsequently frozen for later use. Following a harsh immunosuppressive treatmentintended to destroy autoreactive T cell clones, the patients were thenreinfused 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 14out of the 15 patients became insulin-free for extended periods of time.
66,67
 A recent update of the original study with a longer follow-up (2
1
/
2
years) confirmed that C-peptide levels increased significantly in the treated patients, the majority of whom achieved andmaintained 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. Forone, it has been hypothesized to work only in recently diagnosedpatients, who may still have a significant mass of beta cells susceptibleof regeneration. If such mass is below a still undetermined threshold(as would be expected in long-standing diabetes), it may still benecessary 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 inrelation to this therapy, the sample sizes are still very small. A 2005study evaluating the clinical outcome of autologous hematopoieticstem cell transplantation for a variety of autoimmune conditions(including neurological, rheumatologic, hematological andgastrointestinal disorders) concluded that the treatment was effective,but associated with a high rate of morbidity and mortality (7% at 3years).
69
However, better patient selection, refinement of methods,and progressive experience of teams are likely to decrease transplant-related mortality to more acceptable rates.
Regeneration: From The Duct to Replication(And Back?)
Proof of the rapid evolution of an ever-changing field is thecontinuous shift of the paradigm on adult beta cell regeneration. It was the conventional wisdom for decades that beta cells arose fromprogenitor cells residing in the pancreatic ducts, although the supportfor this hypothesis had always been anecdotal and largely based ontwo-dimensional pictures of beta cells seemingly budding out fromthe periductal area.
70
 A series of elegant lineage-tracing experimentsconducted in a transgenic mouse model challenged this notion in2004, indicating that self-replication was the main mechanismbehind adult beta cell turnover and regeneration.
71
 While theproponents of the “adult stem cell” theory still contended that humanbeta cell regeneration might obey to different rules, these clear-cutobservations clearly put the burden of proof on their shoulders. Thenthe new idea was challenged again in 2008, first by new lineagetracing experiments showing ductal offspring in newly created
of 00

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