TrendsStem cell therapies are finally coming ofage in the context of pancreatic endo-crine regeneration for diabetes. Clinicaltrials aimed at testing the safety andefficacy of human embryonic stem cell-derived islet surrogates are alreadyongoing.

If successful, these approaches areexpected to lead to the phasing outof the use of cadaveric islets for trans-plantation, exponentially extending ourability to treat millions of type 1 dia-betes – and potentially also type 2 dia-betes – patients.

Different cell populations within thepancreas can regenerate the endo-crine compartment through repro-gramming, replication, or stimulationof resident progenitors.

The field has recently advanced to thepoint where these phenomena can beinduced without the need for geneticmanipulation, getting us closer to thedesign of viable clinical trials for b cellreplenishment or endogenousregeneration.

1Diabetes Research Institute,University of Miami Miller School ofMedicine, Miami, FL, USA2Department of Surgery, University ofMiami Miller School of Medicine,Miami, FL, USA3Department of Cell Biology andAnatomy, University of Miami MillerSchool of Medicine, Miami, FL, USA

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ReviewThe Human EndocrinePancreas: New Insights onReplacement andRegenerationJuan Domínguez-Bendala,1,2,3,* Giacomo Lanzoni,1

Dagmar Klein,1 Silvia Álvarez-Cubela,1 andRicardo L. Pastori1,4,5,*

Islet transplantation is an effective cell therapy for type 1 diabetes (T1D) but itsclinical application is limited due to shortage of donors. After a decade-longperiod of exploration of potential alternative cell sources, the field has onlyrecently zeroed in on two of them as the most likely to replace islets. These arepluripotent stem cells (PSCs) (through directed differentiation) and pancreaticnon-endocrine cells (through directed differentiation or reprogramming). Herewe review progress in both areas, including the initiation of Phase I/II clinicaltrials using human embryonic stem cell (hESc)-derived progenitors, advances inhESc differentiation in vitro, novel insights on the developmental plasticity of thepancreas, and groundbreaking new approaches to induce b cell conversionfrom the non-endocrine compartment without genetic manipulation.

Diabetes Therapies Require Alternative Sources of IsletsThe insulin-producing b cells of the pancreas (see Glossary), alongside other endocrine cell typeswithin the islets of Langerhans, are responsible for the maintenance of glucose homeostasis. Inthe autoimmunedisorder knownasT1D,bcells are targetedanddestroyedbyautoreactiveTcells.Chronic insulin administration is a life-saving intervention but one that fails to prevent long-termcomplications that include blindness, vascular disease, and kidney failure. Islet transplantationfrom cadaveric donors is a successful cell therapy for T1D, especially since the development ofsteroid-free immunosuppression protocols [1] and, more recently, T cell-depleting interventionsthat ensure insulin independence rates up to 5 years post-transplantation with minimal compli-cations [2]. However, its general clinical application is limited by the need for lifelong immunesuppression and the shortage of donors [1,3,4]. The latter – andarguablymost pressing –problemcould be addressed by using self-renewable cells with the potential to become b cells. Thedevelopment of any such therapy may also have applications beyond T1D for type 2 diabetes(T2D), the most prevalent form of the disease. Notwithstanding its different etiology, many formsand/or stages of T2Dare also characterized by insufficient insulin production and/or b cell loss andthus could benefit from the transplantation/regeneration of b cells.

Several cell sources (e.g., mesenchymal, hematopoietic, fetal) scrutinized throughout the pastdecade and a half are no longer a primary focus for the development of b cell replacementstrategies. Some of the key approaches presently explored include the use of PSCs (throughdirected differentiation), pancreatic non-endocrine cells (through directed differentiation orreprogramming), and expansion of pancreatic islet cells.

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4Department of Microbiology andImmunology, University of Miami MillerSchool of Medicine, Miami, FL, USA5Department of Medicine, University ofMiami Miller School of Medicine,Miami, FL, USA

*Correspondence:jdominguez2@med.miami.edu(J. Domínguez-Bendala) andrpastori@med.miami.edu (R.L. Pastori).

The differentiation of PSC lines (chiefly hEScs) along the b cell lineage has taken an early lead.The initiation of nationwide Phase I/II clinical trials for T1D using hESc-derived pancreaticprogenitors (Viacyte, Inc., based on the results reported in [5]) as well as the recent publicationof key advances in the maturation of functional b cells in vitro [6–8] have understandablygalvanized the field. However, none of these differentiation methods has yet yielded a cellproduct that is equivalent to native b cells and capable of reversing diabetes right aftertransplantation. Here we present a careful analysis of the potential and shortcomings of thecurrent state of the art.

Another area of research that has gained traction in recent years, both as a source of surrogateislets and for endogenous repair/regenerative medicine applications, is the non-endocrinecompartment of the pancreas. Therapeutic strategies based on this concept might potentiallytap into the non-endocrine pancreatic tissue (>95% of the pancreas) that is currently discardedafter islet isolation. These tissues could be converted into endocrine cell types and thentransplanted [9,10]. Alternatively, if conversion could be induced in situ (and in the presenceof sufficient functional progenitors), the need for transplantation would be circumventedaltogether. Conversion encompasses two fundamentally different approaches: (i) the reprog-ramming of non-endocrine tissues; and (ii) the stimulation of progenitor cells within the non-endocrine compartment of the pancreas [11]. We review here progress made in both areas,which includes novel insights on the developmental plasticity of the pancreas and groundbreak-ing new approaches to induce b cell conversion from the exocrine pancreas using nongeneticmethods (Figure 1, Key Figure). Some of these avenues of research could find their way to theclinic earlier than expected. Recent advances have unveiled unexpected potential for cross-pollination between this area of research and the differentiation of PSCs.

The Challenges of Functional b Cell Differentiation In VitroThe 2006 method reported by Novocell (currently ViaCyte, Inc.) for the differentiation of hEScsinto insulin-producing cells [12] wasmet with high expectations as it was the first report of its kindin which pluripotent cells were first specified along definitive endoderm (the developmental pathfor pancreatic endocrine cells), and the resulting product had an insulin content almost as high asthat of native b cells. However, the insulin-producing cells did not respond to glucose stimulation[12]. Box 1 depicts the basic differentiation sequence of PSCs along the b cell lineage, on whichall subsequent protocols are based. The widespread belief was that minor refinements of thisprotocol would inevitably result in the necessary gains in efficiency for b-like function, both in vitroand after transplantation into diabetic mice. A decade later, this goal has not yet been reached.Multiple variations on the above method have been published, including those calling for the useof protein kinase C (PKC) activators to improve the yield of hESc-derived pancreatic progenitorcells (PPcs) [13,14]. Today, the procurement of a high percentage of PDX1+[4_TD$DIFF]/NKX6.1+ PPc-like(stage 4) cells is no longer perceived as a bottleneck. However, the conditions required for thefunctional maturation of PPcs into glucose-responsive insulin-producing cells remain elusive.Insulin and glucagon coexpression (a sign of dysfunction or immaturity) and lack of glucoseresponsiveness (possibly owing to defects in KATP channels and lack of the glucose transporterGLUT1) were posited as potential causes [15].

Several groups have recently [2_TD$DIFF] focused on these concerns. In early 2014, Cechin et al. showedextensive gains in differentiation efficiency, //b cell separation and b cell function in vitro [16]versus the state-of-the-art method at the time [14] by simply adjusting the environmentaloxygenation of stage 4 PPcs to that of human islets in their in vivo niche. This report wasthe first to capitalize on our recent understanding of oxygen not just as an electron acceptor inrespiration but also as a key developmental modifier [17,18]. Two almost back-to-back reportsat the end of 2014 also reported significant improvements on the prevailing methods [6,7],leading to measurable in vitro b-like function and improved terminal differentiation efficiency.

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Glossaryb cells: islet cells that secrete insulininto the bloodstream in a glucose-responsive manner. They are primarytargets in autoimmune T1D. b celldemise results in pathologicalhyperglycemia and lifelong need forexogenous insulin administration. InT2D, peripheral insulin resistanceoften leads to b cell exhaustion anddeath. As exogenous insulin is alsorequired in severe T2D cases,strategies for b cell replenishment/regeneration are also potentiallyapplicable.Exocrine pancreas: also known asthe non-endocrine pancreaticcompartment; comprises >95% ofthe organ, with the secretion ofdigestive enzymes as its primaryfunction. This tissue comprises acinarand ductal cells. Acini are connectedto the ductal system by centroacinarcells. The ductal tree drains digestivesecretions to the duodenum throughducts of increasing caliber.Human embryonic stem cells(hEScs): the archetypical PSCs,obtained from the inner cell mass(ICM) of a human preimplantationembryo (d5–6 blastocyst). These cellsare widely considered the goldstandard of pluripotency. Embryonicgerm (EG) cells are another exampleof native PSCs.Induced pluripotent stem cells(iPScs): new PSC types that do notexist in nature, derived byreprogramming differentiated cells.iPScs can theoretically be obtainedfrom any individual. To most practicaleffects, iPScs and hEScs arefunctionally equivalent.Islets of Langerhans: also knownas the endocrine pancreas, the isletsof Langerhans are micro-organ-likecell clusters interspersed within theexocrine pancreas. They maintainglucose homeostasis by secretingspecific hormones into thebloodstream. The main islet cell typesare / (glucagon secreting), b (insulinsecreting), d (somatostatin secreting),PP (pancreatic polypeptide secreting),and e (ghrelin secreting).Islet transplantation: a medicalprocedure by which islets from acadaveric pancreas are separatedfrom the non-endocrine fraction andthen transplanted. Enzymaticdigestion of the organ is followed bygradient separation of the variouspancreatic fractions. Isolated isletsare implanted in the liver through

Key Figure

Graphic Representation of Current and Potential Future Cell Therapiesfor b Cell Replacement

Islets

Pancreas

β-cell

Ductal cell

Key:

Acinar cell

Progenitor-like cell

hESc-derived pancrea�c progenitor

hESc-derived β-like cell

hESc

6

Non-islet frac�on

6

5 1

4

3

2 1 5

Figure 1. Islet transplantation (1) is the current standard for b cell replacement in type 1 diabetes (T1D). Islets (red) arepurified and isolated from a cadaveric pancreas and infused into the liver of the patient through the portal vein. Strategies forislet dedifferentiation into progenitor-like cells followed by proliferation and redifferentiation, as well as b cell replication (2),are currently being investigated to expand b cell mass before transplantation. The non-islet fraction (exocrine pancreas)contains multiple subpopulations that are amenable to conversion into b cells. Such is the case for acinar (dark green) orductal (blue) cells by means of reprogramming (3). Putative progenitor-like cells present in the exocrine compartment(yellow) could also be subjected to in vitro expansion for transplantation (4). The most developed alternative source of new bcells outside the pancreas is human embryonic stem cells (hEScs). Ongoing Phase I/II clinical trials are exploring the safetyand functionality of hESc-derived pancreatic progenitor cells (lime green) implanted in the subcutaneous space of patientswithin an immune isolation device (5). Strategies are under development to explore the potential use of hESc-derived b-likecells (orange) matured in vitro (6). Only (1) and (5) are presently in clinical trials.

However, neither reported islet-like reversal of diabetes on transplantation into diabetic mice. Inthe first study [7], it took approximately 40 days for the cells to achieve competence afterimplantation, suggesting the need for a significant degree of maturation in the host. The authorsof the other study [6] did not attempt the reversal of diabetes in chemically induced diabetic mice,reporting instead on its prevention in a rather unusual model for this type of study (the Akitamouse, where amutation in the insulin gene results in progressively severe hyperglycemia). Morerecently, Russ et al. [8] reported that the suppression of bone morphogenetic protein (BMP)inhibitors commonly used in previous protocols resulted in a drastic reduction in the number ofdysfunctional and/or polyhormonal cells. The removal of these inhibitors solved this problem,although the resulting cells also failed to correct diabetes in a mouse model. As shown in Table 1(left) and described in Box 1, there remain significant limitations that stand in the way of obtaininga clinical substitute for islets, as in vitro-differentiated hEScs still differ from human maturepancreatic islets [6–8]. However, steadfast progress in the refinement of differentiation methods,

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portal vein catheterization, althoughcurrent clinical trials are exploringalternative placements such as theomentum (a visceral, highlyvascularized fold of the peritoneum).Islet transplantation remains thestandard cell therapy for T1D but islimited by the shortage of organs andthe necessity for lifelongimmunosuppression.Pluripotent stem cells (PSCs):cells with the ability to proliferateindefinitely in vitro under the rightconditions while maintainingpluripotency; that is, the capacity todifferentiate along all lineages of thethree embryonic layers (endoderm,ectoderm, and mesoderm).Progenitor cells: non-terminallydifferentiated cells characterized by avariable degree of potency. Putativeprogenitor cells within the adultpancreas (PPcs) may be oligopotent(e.g., they would give rise toendocrine cells only) or multipotent(e.g., with multilineage differentiationpotential spanning endocrine/exocrinefates).Reprogramming: also calledtransdifferentiation; refers to a switchin cellular fate that is not broughtabout by differentiation. It typicallyrefers to experimental methods(chiefly genetic/epigeneticmanipulation) inducing the conversionof one cell type into another.

Box 1. Stepwise Differentiation of PSCs Along the b Cell Lineage

Experimental approaches for the differentiation of PSCs have been devised primarily using hEScs. Please note that thedivision into four stages presented in Figure I is generic and publications on this topic do not always adopt exactly thesame terms. More recent advances include a variable number of additional steps following stage 4 (represented by anasterisk) leading to the generation of b-like cells that exhibit functionality in vitro, albeit with limitations (see Table 1 fordetails). Although these protocols yield b cells that resemble primary human b cells in terms of in vitro function and keymarker expression (such as colocalization of the transcription factors PDX-1 and NKX6.1 and insulin), global genetranscript expression still differs from that of primary cells. For example, UCN3 (an important differentiation marker thatdistinguishes immature frommature b cells [54]) was downregulated in one study [7] and could not be studied in the otherbecause the signal in the gene array study was too low [6]. More comprehensive analyses were suggested for a morerigorous comparison between primary and differentiated cells, including detailed studies on proteins/enzymes regulatingglucose sensing and insulin secretion, electrophysiology of insulin secretion and the composition of the exocytoticmachinery, and themetabolomics profile of mitochondrial function [55]. Of note, islets isolated from human pancreata arecurrently used as the gold standard against which the maturity of differentiated cells is compared. However, there is alarge variability in the quality and potency of primary islets for many reasons, including the time of ischemia, previousclinical history and demographics of the donor, and processing factors.

hESc DE PGT PF PPc β-like cell

OCT4NANOG

SOX2E-CAD

SOX17FOXA2CXCR4

HNF1βHNF4α

PDX1HNF6

PROX1SOX9

PDX1 NKX6.1NKX2.2

NEUROD1

PDX1 INS

MAFAGK

∗Stage 1 Stage 2 Stage 3 Stage 4

Figure I. General Strategies Used for the Differentiation of Pluripotent Stem Cells. While state-of-the-artprotocols exhibit multiple variations, they all have in common the coaxing of stem cells into sequential developmentalstages, namely: definitive endoderm (DE), primitive gut tube (PGT), posterior foregut (PF), and pancreatic progenitor cells(PPcs). The temporal pattern of expression of key genes is indicated under each developmental stage.

fueled by substantial public and private funding for these initiatives, makes defining a clinicallyrelevant cell product a distinct possibility within the next 5 years.

In vivo Maturation of hEScsShortly after their seminal report [12], and in parallel to the efforts described above, the scientistsat ViaCyte, Inc. initiated an alternative path to the clinic that has finally borne fruit in the shape ofongoing Phase I/II trials [6_TD$DIFF] (clinicaltrials.gov identifier: NCT02239354). In short, cells at the pan-creatic progenitor stage (stage 4), represented by an asterisk in Figure I in Box 1, weretransplanted into T1D patients. The idea behind these studies was that the environmentalmilieu provided by a living host might be conducive to functional b cell maturation in a way that invitro conditions may be unable to do. In a 2008 report, the same group reported that, around 3months after the transplantation of hESc-derived pancreatic progenitors, diabetes was pre-vented in mice treated with streptozotocin (stz), a toxin that at certain dosages selectivelydestroys murine, but not human, b cells [5]. Graft retrieval and analysis conclusively demon-strated that progenitor cells had undergone functional maturation throughout the post-trans-plantation period andwere able tomaintain normoglycemia after the native b cells of the host hadbeen ablated. The downside, however, was a relatively high incidence of teratoma-like lesionsaround the graft. In a subsequent embodiment of this approach, designed with a view towardfuture therapeutic applications, the cells were inserted within an immunoisolated drug deliverysystem adapted from the TheraCyte device [19,20] termed Encaptra® [5_TD$DIFF]. This is a thin, retrievable,

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Table 1. Main Features of In Vitro versus In Vivo hESc-to-b [1_TD$DIFF]Cell Differentiation

Terminal Differentiation In Vitro Refs Terminal Differentiation In Vivo Refs

4–6-week protocol [6–8,16] 2-week protocol [5,14,22,23]

Cell product exhibits glucoseresponsiveness before transplantation

[6–8] Cell product is not functional beforetransplantation

[5,14,22,23]

In the best scenario, it takes �40 days toreverse already established diabetes in mice

[7] Several months to mature aftertransplantation

[5,14,22,23]

Overall endocrine cell proportion larger thanin islets in one study [7]; substantialproportion of b-like cells (30–40%) [6,7] butlower proportion of other endocrine celltypes in relation to islets and higherproportion of PDX1+ undifferentiated cells inanother study [6]

[6,7] Grafts contain predominantly endocrinecells; 50% of the grafts after maturationare b-like cells but there are alsosignificant percentages of other isletendocrine cell types

[21]

Non-endocrine cell types yet to be fullycharacterized

[6] Low incidence of non-endocrine cells [21]

Insulin content similar to that of humancultured islets but higher proinsulin:C-peptide ratio, suggesting deficiency in insulinprocessing

[7] Insulin content at 20–30 weeks stillaround 40% lower than that of humancultured islets; lower degree ofgranulation

[21]

Differentiated cells exhibit glucose sensitivity,although in a manner that is quantitativelyand qualitatively different from that of humanb cells

[6–8] Functionality impaired relative to humanislets; similar basal insulin secretion butlower glucose-induced insulinbiosynthesis [21]; however, in anotherstudy similar function in terms of glucose-stimulated human C-peptide release wasobserved [14]

[14,21]

No teratomas detected after transplantation [6–8] The most recent studies do not showsignificant incidence of teratogeniclesions

[5,14,22,23]

biocompatible bilaminar container designed to protect enclosed cells against the immunesystem of the host while allowing the free exchange of oxygen and nutrients. Later studiesrevealed additional insights on the process of in vivo maturation, such as the fact that hormoneproduction increases by an entire order of magnitude within the 20–30-week post-transplanta-tion period [21]. At this point, grafts largely comprise monohormonal pancreatic endocrine cellsthat exhibit islet-like basal insulin production, although insulin content on a per-cell basis is stillless than half that of human islets and glucose-induced insulin biosynthesis remains suboptimal(Table 1, right). Despite these limitations, ViaCyte, Inc. developed controlled and scalablemethods for hESc expansion, banking, and suspension-based differentiation into pancreaticprogenitors for clinical applications [22]. Another group developed a similar strategy based onthe in vivomaturation of cells that were differentiated up to the pancreatic progenitor stage. PPcswere transplanted into immunodeficient mice with stz-induced diabetes. Glycemia was initiallycontrolled with exogenous insulin and eventually the production of human C-peptide by the graftwas sufficient to control blood glucose levels [14]. However, amajor limitation of this methodwasthe formation of cells resembling bone and cartilage. Further modifications of this protocolallowed in vivo maturation of PPcs within a TheraCyte device in diabetic mice without theformation of mesoderm-derived tissue [23].

In August 2014, ViaCyte, Inc. secured FDA approval for a combination product (VC-01TM)comprising hESc-derived pancreatic endoderm cells (PEC-01TM) delivered within the Encaptra®

immunoisolation device. The first transplantation was performed 2 months later in the context of

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a multicenter Phase I/II trial (clinicaltrials.gov identifier: NCT02239354) designed to evaluateVC-01TM [6_TD$DIFF] directly in T1D patients. There is understandable excitement about these clinical trials,as taking a basic concept to the clinic in less than a decade – and using hEScs, no less – is a tourde force by any measurable standard. However, many aspects that may be ‘lost in translation’from mice to humans call for caution. Although the risk of iatrogenic tumorigenesis seemsnegligible in later versions of the protocol (with the device and the immune competence ofthe host being secondary and tertiary safeguards), only long-term follow up will give a definitiveanswer. Other areas of uncertainty include cell dosage, the durability/effectiveness of the devicein an autoimmune setting, and the fact that, for all we know, this approach may just not work inhumans as it does in mice (see Outstanding Questions). Of note, induced PSCs (iPScs) alsorepresent a distinct therapeutic opportunity, as they have been shown to be capable of PPc [24]and b-like cell differentiation [25]. Since the procurement of iPScs from the very patients who weintend to treat is now feasible [26], we could envision ‘personalized’ therapies in whichalloimmunity (although not autoimmunity) will be taken out of the equation.

The Pancreatic Non-endocrine Compartment as a Potential Source of b CellsThe use of PSCs that can be induced to adopt a b-like phenotype has clearly taken an early leadamong the approaches with therapeutic potential for T1D. However, as discussed above,the path to translation is not without roadblocks. The pancreas itself contains at leastthree categories of cells that may provide an alternative to hEScs as a source of new b cells.These are residual b cells (through expansion; Box 2), progenitor-like cells (through endogenousregeneration; Box 3), and non-endocrine cells (acinar/ductal; through reprogramming). At thecell level, reprogramming (also termed transdifferentiation) isdefinedasaphenotypicchange that isnot brought about by differentiation and usually entails conversion of cells of one tissue into cells ofanother through ectopic expression of specific transcription factors [27]. However, the distinctionbetween reprogramming and differentiation is not clear cut in the case of the pancreas. As wereview progress in the reprogramming of cells that reside within the exocrine compartment intoinsulin-producing cells, it is often unclear whether the conversion occurs across the gamut of celltypes within the exocrine tissue or in specific subpopulations of respondent cells –which could beconsidered ‘progenitor-like’ for all practical purposes (Box 3).

Acinar-to-Endocrine ConversionPancreatic acinar cells are known to be highly malleable [28,29], as shown by their reportedability to give rise to liver [30], ductal [31], and b-like cells [32,33]. In a breakthrough 2008 study,Zhou and colleagues came up with a combination of three key pancreatic transcription factors(PDX1, NGN3, and MAFA, a variation of which had been used in earlier liver-to-pancreasreprogramming attempts [34]) that, when shuttled aboard adenoviral vectors and injected into

Box 2. Expansion of Insulin-Producing Cells from Human Islets

Once considered post-mitotic, human pancreatic b cells have been reported to proliferate in vitro in response to themanipulation of cell cycle activator genes such as cyclin D3 and CDK6 [56] or, more recently, small molecules such asharmine analogs probably acting through dual-specificity tyrosine-regulated kinase-1a (DYRK1A) on the nuclear factorsof activated T cells (NFAT) family of transcription factors [57]. Enhancing the potency and b cell specificity of thesecompounds are important future challenges. Questions remain regarding the specificity and potency of this approach, aswell as the possibility of applying it for b cell replication in vivo versus on isolated b cells for subsequent transplantation.Nonetheless, this approach represents a step forward in the identification of elusive replication methods for human islets.The most-characterized strategy developed to expand human islet cells (specifically b cells) includes a sequence of stepsthat starts with the dissociation of islets into single cells followed by culture in which the b cells enter into a process ofdedifferentiation. This process resembles EMT and is associated with extensive cell proliferation (up to 16-fold). Finally,the expanded dedifferentiated cells are redifferentiated in culture with factors that have been shown to promote b celldifferentiation such as activin A, exendin-4, nicotinamide, and high glucose concentrations, in a process involvingmesenchymal–epithelial transition (MET) [58]. Redifferentiated b cells express the transcription factors PDX-1 and MAFAat levels comparable with those of human islets, are glucose responsive in vitro, and exhibit insulin storage in exocytoticvesicles with >90% of it being processed. However, the insulin content is around 10% of that of human islets.

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Box 3. Endogenous PPcs

A large body of data supports the hypothesis that embryonic-like PPcs residing in the pancreas persist in adult life, buttheir identity is actively debated [59]. The unambiguous identification of an adult cell population endowed with isletmaturation potential would have major therapeutic implications for diabetes.

Sustained islet cell proliferation occurs after birth but turnover becomes slow in adulthood. However, compensatorygrowth can occur in response to increased demand in both the endocrine and exocrine compartments [60,61].Proliferation of preexisting cells of the same lineage seems to be the main route for cell replacement during tissuehomeostasis and in response tominor injuries.Lineage tracing in rodents showed thatpreexistingacinar cellsgive rise tonewacinar cells but not islets [62] and that preexisting islet b cells replicate to generate new ones [63,64]. Nevertheless, thesefindingsdonot preclude an upstreamcontribution frommultipotent populations of stem/progenitor cells [46,65–68] or otheradult cell types [52], especially after major regenerative demands. b cell impairment/loss, peripheral insulin resistance, andhyperglycemia may stimulate regeneration from endogenous progenitors [69].

Multipotent cells have been observed within the pancreas [65,66] but are rare and elusive in adult life [59]. Cells withpancreatic stem cell traits have been observed in the biliary tree, primarily in the hepatopancreatic ampulla (ampulla ofVater), which is formed by the union of the common bile duct and the major pancreatic duct [67]. In the pancreas proper,PPcs have been observed integrated in ducts [46,67] and in pancreatic duct glands (PDGs) [67]. PDGs are associatedwith the main pancreatic duct and its immediate ramifications and are abundant in proximity to the hepatopancreaticampulla [67,70]. Remarkably, PDGs undergo major hyperplasia in murine models of chronic pancreatitis [70]. Some cellsin adult human PDGs along the larger pancreatic ducts present a phenotype consistent with that of PPcs and PDGs arefound to harbor NGN3+ cells, putative islet progenitors [67]. Taken together, these observations suggest that the adultpancreas contains niches of progenitors associated with ducts and PDGs, which may mediate regenerative responses.

the pancreatic parenchyma, gave rise to new b-like cells in vivo [35]. These were detected asearly as 3 days after the injection and their number kept expanding for up to 3 months, long afterthe clearance of the vectors. They were indistinguishable from native b cells in terms of size,morphology, insulin granule content, and molecular markers. Diabetic mice treated with thistranscription factor cocktail showed a significant and permanent improvement in blood glucoselevels even if diabetes was not completely reversed. The latter observation could be explained bythe fact that the newly created b cells remained isolated and did not cluster to form islets, as b cellcommunication is essential to stimulate glucose-mediated insulin secretion [36]. More recently,the above results were confirmed in vitro using the AR42J acinar cell line [37–39]. Although signsof rudimentary endocrine differentiation were observed in these studies, the resulting cells werenot glucose responsive. Subsequently, acinar-to-endocrine conversion was reported usingprimary human pancreatic exocrine cells cultured in conditions (TGF-b and ROCK inhibitors)that preventedepithelial-to-mesenchymal transition (EMT) [40]. The latter results are remarkable astheyestablishedproof of principle thatpancreatic reprogrammingcanbeeffectively achievedusinghuman material. However, successful reprogramming required the ectopic expression of fourtranscription factors (PDX1,NGN3,MAFA,andPAX4)plus twochromatin-wide remodelingagents(5-aza-20-deoxycytidine and sodiumbutyrate) in addition to the aforementioned TGF-b andROCKinhibitors [40]. Approximately 18% of the cells generated through this method expressed insulinand were glucose responsive in vitro and in vivo, preventing diabetes in stz-treated immunode-ficient mice. A small number of glucagon-expressing cells were also found within the islet-likestructures. However, as the authors did not study their gene expression profile these cannotbe compared to human islets. More recent attempts at inducing human exocrine-to-endocrineconversionalso entailed geneticmanipulationby lentiviral transductionofMAPKandSTAT3 [33]. Inthis case, cells were able to start endocrine differentiation only when grown in 3D culture.Differentiated cells exhibited insulin expression at the protein level but did not express thetranscription factor MAFA, an indicator of b cell maturity. However, 3D-cultured cells maturedin vivo into glucose-responsive islet-like clusters and could prevent chemically induced diabetes inimmunodeficient mice, suggesting that MAPK and STAT3 lentiviral transduction of human acinarcells led to the formation of PPc-like cells similar to those derived from hESc at stage 4.

In only two recent instances has substantial acinar-to-endocrine reprogramming been shown inthe absence of genetic manipulation. The first study was conducted in mice following chemically

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Outstanding QuestionsWill in vitro differentiated hEScs provesuperior to those that undergo terminalmaturation in vivo? How critical is it forthe cells tomaturewithin a living host? Isa human diabetic/autoimmune micro-environment conducive to or harmfulfor functional b cell differentiation?

What are the functional implications ofdifferent endocrine cell compositionsafter PSC differentiation? What is theexact cell composition of the non-endocrine fraction after terminal hEScdifferentiation in vitro?

Is potential teratogenesis a problem,especially when transplanting imma-ture progenitor cells? Do undesiredor dysfunctional cells pose a risk?

Will immune isolationdevices sustain thelong-term viability and function ofengrafted cells, particularly in the pres-ence of autoimmunity? If not, how oftenwould they have to be replaced? Thesubcutaneous placement of these devi-ces is ideal for retrievability but is it goodenough for long-term survival and accu-rate maintenance of normoglycemia?

Considering that hESc-based clinicaltrials have gone directly from mouseto human, has the right cell dosagebeen achieved?

What is the true nature of adult pancre-atic progenitors?Can theybe expandedin a therapeutically meaningful way? Arethey available and can they be stimu-lated in situ in diabetic patients?

To what extent can cell therapies forT1D be applied to T2D?

induced diabetes. The simultaneous intraperitoneal administration of epidermal growth factor(EGF) and ciliary neurotrophic factor (CNTF) – which collectively stimulate the STAT3 pathway –resulted in diabetes reversal in �64% of the animals (�33% when diabetes was induced by stzinstead of alloxan) [32]. Lineage-tracing experiments led to the conclusion that newly createdb-like cells arose from the acinar fraction. The effect was dependent on the CNTF-mediatedphosphorylation of STAT3 in acinar cells and the degree of success of the treatment was directlyrelated to the amount of cells within the acinar parenchyma in which NGN3 expression wasreactivated. The second study was conducted using cultured human non-endocrine cellsobtained as a byproduct of islet isolation [41]. Exposure to BMP-7 (a clinical product withthe dual ability to inhibit TGF-b and activate BMP signaling) led to the generation of glucose-responsive cell clusters that expressed insulin within the published range for research islets[42] – 50-fold higher than in previously reported genetic methods for the endocrine conversion ofthe non-endocrine fraction of the pancreas [40]. Preliminary lineage-tracing results suggestedthat the cells that responded to BMP-7 were not adult acinar or ductal but rather PPc-like innature (Box 3). Further anatomical and molecular characterization of these cells may shedadditional light on their potential ability to regenerate the endocrine pancreas on stimulation insitu. Linking this field of research to that of hESc differentiation is the recent finding by Russ et al.[8] that BMP inhibition, commonly used in earlier hESc-to-b cell differentiation methods topromote efficient PPc generation, does in fact result in dysfunctional/polyhormonal b-like cells.The removal of these inhibitors corrected this problem, further suggesting that BMP signaling isimportant for functional b cell differentiation.

Ductal-to-Endocrine ConversionApproximately 30% of the human pancreas comprises ductal cells [43]. Originally, it wasproposed that these could be a repository of b cell progenitors as well as other mature celltypes in the adult pancreas [44]. Indeed, the ‘budding’ of islet cells from pancreatic ducts is not arare finding [45,46]. However, the contribution of ductal cells to islet endocrine cell turnoverremains a matter of debate [45–47]. Intriguingly, in response to pathological conditions,structures with heterogeneous ductal phenotypes expand in the pancreas [48,49]. Recentobservations indicate that the aging pancreas loses the potential for ductal-to-b cell differentia-tion [50].

In general, the limited capability of these cells to replicate in culture stands in the way of theirdevelopment as an alternative to islets. The latest meaningful advance on this front is afluorescence-activated cell sorting (FACS)-based method for the isolation of human adultpancreatic ductal cells followed by clonal expansion and genetic conversion into endocrinecells via ectopic expression of NGN3, MAFA, and PDX-1 [51]. These cells shared features offunctional neonatal b cells, including high-level pre-proinsulin expression, proinsulin processing,and dense-core granule formation.

Concluding Remarks and Future PerspectivesGlancing back at a decade and a half of work on pancreatic regeneration, we have witnessedfirsthand the crystallization of the most promising avenues of research, some of which arealready undergoing clinical trials with others to follow suit in the not-so-distant future. hEScs,against all odds, have taken an early lead (see Outstanding Questions). So far, the functionality ofin vivo-matured cells seems more backed by evidence than that of in vitro-differentiated ones.However, once the latter are shown to be equivalent to the former, it will be difficult to arguethe case against their preferential use, even if the current trials with hESc-derived PPcs prove tobe successful. Meanwhile, approaches to convert the plentiful non-endocrine fraction of thepancreas into functional endocrine cell types are also starting to bear fruit. While geneticmanipulation strategies will not make it to the clinic anytime soon, alternative strategies thatmake use of cytokines, synthetic RNAs, or growth factors may get there much sooner. The

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appeal of exocrine-to-endocrine conversion is not somuch the prospect of transplanting severalpatients with a single organ as it is to be able to stimulate such conversion within the patient.Needless to say, the latter approach would not work unless simultaneous steps were taken toprevent the recurrence of autoimmunity in T1D – which is a daunting task in itself. The sameholds true for the interconversion of endocrine cell types (such as / to b), which has beenreported in animal models [52,53] but is beyond the scope of this review.

The transplantation of exogenously generated cells, a road paved by 30 years of islet trans-plantation, is likely to be the treatment of choice in the short term. In this context, the immediategoal is to define a mode of delivery and protection that is conducive to the long-term survival andfunction of the transplanted cells. Recent refinements in immune suppression have significantlyimproved on old standards but their inherent risks still keep islet transplantation in children offlimits. Retrievable immune isolation devices might bridge the gap to the ultimate goal of immunetolerance but their worth in autoimmune settings remains to be tested. In this context, the broadT2D spectrum also presents us with an opportunity to test these approaches in the absence ofautoimmunity. The next few years will be key in shaping the next generation of b cell replacementtherapies.

AcknowledgmentsThe authors acknowledge funding from the Diabetes Research Institute Foundation (DRIF), the University of Miami's Miller

School of Medicine, the Fred and Mabel R. Parks Foundation, the Foundation for Diabetes Research (FDR), the Michael J.

and Katherine E. Franco Foundation, the Frank Strick Foundation, and Mildred Graff, as well as NIH grant

1R43DK105655-01.

References

1. Shapiro, A.M. et al. (2000) Islet transplantation in seven patients

with type 1 diabetes mellitus using a glucocorticoid-free immuno-suppressive regimen. N. Engl. J. Med. 343, 230–238

2. Bruni, A. et al. (2014) Islet cell transplantation for the treatment oftype 1 diabetes: recent advances and future challenges. Diab.Metab. Syndr. Obes. 7, 211–223

3. Ryan, E.A. et al. (2005) Five-year follow-up after clinical islettransplantation. Diabetes 54, 2060–2069

4. Ricordi, C. and Strom, T.B. (2004) Clinical islet transplantation:advances and immunological challenges. Nat. Rev. Immunol. 4,259–268

5. Kroon, E. et al. (2008) Pancreatic endoderm derived from humanembryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat. Biotechnol. 26, 443–452

6. Pagliuca, F.W. et al. (2014) Generation of functional human pan-creatic beta cells in vitro. Cell 159, 428–439

7. Rezania, A. et al. (2014) Reversal of diabetes with insulin-produc-ing cells derived in vitro from human pluripotent stem cells. Nat.Biotechnol. 32, 1121–1133

8. Russ, H.A. et al. (2015) Controlled induction of human pancreaticprogenitors produces functional beta-like cells in vitro. EMBO J.34, 1759–1772

9. Baeyens, L. and Bouwens, L. (2009) Cellular plasticity of thepancreas. Biol. Chem. 390, 995–1001

10. Efrat, S. and Russ, H.A. (2012) Making beta cells from adulttissues. Trends Endocrinol. Metab. 23, 278–285

11. Bonner-Weir, S. et al. (2012) Islet neogenesis: a possible pathwayfor beta-cell replenishment. Rev. Diab. Stud. 9, 407–416

12. D’Amour, K.A. et al. (2006) Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.Nat.Biotechnol. 24, 1392–1401

13. Chen, S. et al. (2009) A small molecule that directs differentiationof human ESCs into the pancreatic lineage. Nat. Chem. Biol. 5,258–265

14. Rezania, A. et al. (2012) Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable oftreating pre-existing diabetes in mice. Diabetes 61, 2016–2029

15. Bruin, J.E. et al. (2014) Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells.Stem Cell Res. 12, 194–208

16. Cechin, S. et al. (2014) Influence of in vitro and in vivo oxygenmodulation on beta cell differentiation from human embryonicstem cells. Stem Cells Transl. Med. 3, 277–289

17. Fraker, C. et al. (2009) Oxygen: a master regulator of pancreaticdevelopment? Biol. Cell 101, 431–440

18. Bigarella, C.L. et al. (2014) Stem cells and the impact of ROSsignaling. Development 141, 4206–4218

19. Boettler, T. et al. (2015) Pancreatic tissue transplanted in Ther-aCyteTM encapsulation devices are protected and prevent hyper-glycemia in a mouse model of immune-mediated diabetes. CellTransplant. Published online August 21, 2015. http://dx.doi.org/10.3727/096368915X688920

20. Kirk, K. et al. (2014) Human embryonic stem cell derived isletprogenitorsmature inside an encapsulationdevicewithout evidenceof increased biomass or cell escape. Stem Cell Res. 12, 807–814

21. Motte, E. et al. (2014) Composition and function of macroencap-sulated human embryonic stem cell-derived implants: comparisonwith clinical human islet cell grafts. Am. J. Physiol. Endocrinol.Metab. 307, E838–E846

22. Schulz, T.C. et al. (2012) A scalable system for production offunctional pancreatic progenitors from human embryonic stemcells. PLoS ONE 7, e37004

23. Bruin, J.E. et al. (2013) Maturation and function of human embry-onic stem cell-derived pancreatic progenitors in macroencapsu-lation devices following transplant into mice. Diabetologia 56,1987–1998

24. Kunisada, Y. et al. (2012) Small molecules induce efficient differ-entiation into insulin-producing cells from human induced pluripo-tent stem cells. Stem Cell Res. 8, 274–284

25. Alipio, Z. et al. (2010) Reversal of hyperglycemia in diabetic mousemodels using induced-pluripotent stem (iPS)-derived pancreaticb-like cells. Proc. Natl. Acad. Sci. U.S.A. 107, 13426–13431

26. Maehr, R. et al. (2009) Generation of pluripotent stem cells frompatients with type 1 diabetes. Proc. Natl. Acad. Sci. U.S.A. 106,15768–15773

Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy 9

TEM 1100 No. of Pages 10

27. Sisakhtnezhad, S. and Matin, M.M. (2012) Transdifferentiation: acell and molecular reprogramming process. Cell Tissue Res. 348,379–396

28. Pin, C.L. et al. (2015) Acinar cell reprogramming: a clinicallyimportant target in pancreatic disease. Epigenomics 7, 267–281

29. Ziv, O. et al. (2013) The plastic pancreas. Dev. Cell 26, 3–7

30. Al-Adsani, A. et al. (2010) Dexamethasone treatment induces thereprogramming of pancreatic acinar cells to hepatocytes andductal cells. PLoS ONE 5, e13650

31. Liou, G.Y. et al. (2015) Protein kinase D1 drives pancreatic acinarcell reprogramming and progression to intraepithelial neoplasia.Nat. Commun. 6, 6200

32. Baeyens, L. et al. (2013) Transient cytokine treatment inducesacinar cell reprogramming and regenerates functional beta cellmass in diabetic mice. Nat. Biotechnol. 32, 76–83

33. Lemper, M. et al. (2015) Reprogramming of human pancreaticexocrine cells to beta-like cells. Cell Death Differ. 22, 1117–1130

34. Kaneto, H. et al. (2005) A crucial role of MafA as a novel therapeutictarget for diabetes. J. Biol. Chem. 280, 15047–15052

35. Zhou, Q. et al. (2008) In vivo reprogramming of adult pancreaticexocrine cells to beta-cells. Nature 455, 627–632

36. Geron, E. et al. (2015) The edges of pancreatic islet beta cellsconstitute adhesive and signaling microdomains. Cell Rep. Pub-lished online January 13, 2015. http://dx.doi.org/10.1016/j.celrep.2014.12.031

37. Zhang, T. et al. (2012) Functional role of an islet transcriptionfactor, INSM1/IA-1, on pancreatic acinar cell trans-differentiation.J. Cell. Physiol. 227, 2470–2479

38. Lima, M.J. et al. (2012) Efficient differentiation of AR42J cellstowards insulin-producing cells using pancreatic transcription fac-tors in combination with growth factors.Mol. Cell. Endocrinol. 358,69–80

39. Akinci, E. et al. (2012) Reprogramming of pancreatic exocrine cellstowards a beta (b) cell character using Pdx1, Ngn3 and MafA.Biochem. J. 442, 539–550

40. Lima, M.J. et al. (2013) Suppression of epithelial-to-mesenchymaltransitioning enhances ex vivo reprogramming of human exocrinepancreatic tissue toward functional insulin-producing beta-likecells. Diabetes 62, 2821–2833

41. Klein, D. et al. (2015) BMP-7 induces adult human pancreaticexocrine-to-endocrine conversion. Diabetes 64, 4123–4134

42. Kayton, N.S. et al. (2015) Human islet preparations distributed forresearch exhibit a variety of insulin-secretory profiles. Am. J.Physiol. Endocrinol. Metab. 308, E592–E602

43. Bouwens, L. and Pipeleers, D.G. (1998) Extra-insular beta cellsassociated with ductules are frequent in adult human pancreas.Diabetologia 41, 629–633

44. Bonner-Weir, S. et al. (2004) The pancreatic ductal epitheliumserves as a potential pool of progenitor cells. Pediatr. Diabetes5 (Suppl. 2), 16–22

45. Hosokawa, S. et al. (2015) Impact of Sox9 dosage and Hes1-mediated Notch signaling in controlling the plasticity of adultpancreatic duct cells in mice. Sci. Rep. 5, 8518

46. Inada, A. et al. (2008) Carbonic anhydrase II-positive pancreaticcells are progenitors for both endocrine and exocrine pancreasafter birth. Proc. Natl. Acad. Sci. U.S.A. 105, 19915–19919

47. Kawaguchi, Y. (2013) Sox9 and programming of liver and pancre-atic progenitors. J. Clin. Invest. 123, 1881–1886

48. Li, W.C. et al. (2010) Activation of pancreatic-duct-derived pro-genitor cells during pancreas regeneration in adult rats. J. Cell Sci.123, 2792–2802

10 Trends in Endocrinology & Metabolism, Month Year, Vol. xx, N

49. Wang, G.S. et al. (2005) Tubular complexes as a source for isletneogenesis in the pancreas of diabetes-prone BB rats. Lab.Invest. 85, 675–688

50. El-Gohary, Y. et al. (2015) Intra-islet pancreatic ducts can give riseto insulin-positive cells. Endocrinology Published online October27, 2015. http://dx.doi.org/10.1210/en.2015-1175

51. Lee, J. et al. (2013) Expansion and conversion of human pancreaticductal cells into insulin-secreting endocrine cells. Elife 2, e00940

52. Thorel, F. et al. (2010) Conversion of adult pancreatic alpha-cells tobeta-cells after extreme beta-cell loss. Nature 464, 1149–1154

53. Piran, R. et al. (2014) Pharmacological induction of pancreatic isletcell transdifferentiation: relevance to type I diabetes. Cell DeathDis. 5, e1357

54. Blum, B. et al. (2014) Reversal of beta cell de-differentiation by asmall molecule inhibitor of the TGFb pathway. Elife 3, e02809

55. Kushner, J.A. et al. (2014) Stem cells to insulin secreting cells:two steps forward and now a time to pause? Cell Stem Cell 15,535–536

56. Fiaschi-Taesch, N.M. et al. (2013) Cytoplasmic-nuclear traffickingof G1/S cell cycle molecules and adult human beta-cell replication:a revised model of human beta-cell G1/S control. Diabetes 62,2460–2470

57. Wang, P. et al. (2015) A high-throughput chemical screen revealsthat harmine-mediated inhibition of DYRK1A increases humanpancreatic beta cell replication. Nat. Med. 21, 383–388

58. Russ, H.A. et al. (2009) Epithelial–mesenchymal transition in cellsexpanded in vitro from lineage-traced adult human pancreatic betacells. PLoS ONE 4, e6417

59. Lysy, P.A. et al. (2013) Making beta cells from adult cells within thepancreas. Curr. Diab. Rep. 13, 695–703

60. Fernandes, A. et al. (1997) Differentiation of new insulin-producingcells is induced by injury in adult pancreatic islets. Endocrinology138, 1750–1762

61. Bonner-Weir, S. et al. (1993) A second pathway for regeneration ofadult exocrine and endocrine pancreas. A possible recapitulationof embryonic development. Diabetes 42, 1715–1720

62. Desai, B.M. et al. (2007) Preexisting pancreatic acinar cells con-tribute to acinar cell, but not islet beta cell, regeneration. J. Clin.Invest. 117, 971–977

63. Dor, Y. et al. (2004) Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.Nature 429, 41–46

64. Meier, J.J. et al. (2008) Beta-cell replication is the primary mecha-nism subserving the postnatal expansion of beta-cell mass inhumans. Diabetes 57, 1584–1594

65. Seaberg, R.M. et al. (2004) Clonal identification of multipotentprecursors from adult mouse pancreas that generate neuraland pancreatic lineages. Nat. Biotechnol. 22, 1115–1124

66. Smukler, S.R. et al. (2011) The adult mouse and human pancreascontain rare multipotent stem cells that express insulin. Cell StemCell 8, 281–293

67. Wang, Y. et al. (2013) Biliary tree stem cells, precursors to pan-creatic committed progenitors: evidence for possible life-longpancreatic organogenesis. Stem Cells 31, 1966–1979

68. Xu, X. et al. (2008) Beta cells can be generated from endogenousprogenitors in injured adult mouse pancreas. Cell 132, 197–207

69. Razavi, R. et al. (2015) Diabetes enhances the proliferation of adultpancreatic multipotent progenitor cells and biases their differenti-ation to more beta-cell production. Diabetes 64, 1311–1323

70. Strobel, O. et al. (2010) Pancreatic duct glands are distinct ductalcompartments that react to chronic injury and mediate Shh-induced metaplasia. Gastroenterology 138, 1166–1177

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