CHAPTER 41

Generation of PancreaticIslets from Stem Cells

Bernat Soria, Daniela Pezzolla, Javier Lopez, Anabel Rojas andAbdelkrim HmadchaAndalusian Center for Molecular Biology and Regenerative Medicine (CABIMER),Department of Stem Cells and CIBERDEM, Sevilla, Spain

INTRODUCTION

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Diabetes mellitus is one of the most prevalent chronic diseases. Glucose homeostasis

disruption occurs when b-cells fail to secrete the insulin necessary to maintain the homeostasis

of glucose in the blood flow. Over time, diabetes can lead to the rise of different long-termcomplications, such as diabetic foot, retinopathy, neuropathy, nephropathy and arterio-

sclerosis. Nowadays, the only treatments for diabetes consist of exogenous insulin supply or

pancreas/islet transplantation, but the inability to achieve a tight control over glucose regu-lation by exogenous insulin administration and the shortage of pancreatic islets donors have

motivated recent efforts to develop renewable sources and protocols for effective b-cell

replacement.

Embryonic stem cells are non-specialized cells that share two important characteristics: self-

renewal, which allows them to expand indefinitely while maintaining the undifferentiatedstate; and pluripotency, which is the capacity to differentiate into almost all specialized cell

types. Proof-of-concept experiments demonstrate that embryonic stem cells have the ability to

differentiate into insulin-producing cells, even if at a very low frequency.

In this chapter, we review the attempts that have beenmade thus far to convert embryonic stem

cells into pancreatic endocrine cell types of potential use in the treatment of type I diabetes.

FIRST ATTEMPTS TO OBTAIN B-CELL LIKE CELLS BYDIFFERENTIATIONThe first attempt to promote the differentiation of insulin-producing cells was carried out

using a combination of directed differentiation and cell selection methods. Mouse embryonicstem cells (ESCs) expressing antibiotic resistance under control of either the insulin or the

Nkx6.1 promoter [1,2] were driven to differentiate into nutrient-induced insulin-secreting cells

which rescue streptotozotocin-diabetic mice from hyperglycemia when transplanted eitherinto the spleen or under the kidney capsule. Furthermore, the cell type selection protocol

allowed no tumor formation by the presence of non-differentiated ESCs. In contrast, most of

the initial differentiation techniques that relied upon embryoid body (EB) formation appearedto be successful using either mouse ESCs [3e5] or human ESCs [6e7], but the absence of C-

peptide, tumor formation and lack of demonstration of any rescue of diabetic animals revealed

Principles of Tissue Engineering. http://dx.doi.org/10.1016/B978-0-12-398358-9.00041-0

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838

PART 10Endocrinology and Metabolism

that the observed intracellular insulin in these cells did not originate from de novo synthesis,but rather from uptake from the culture media [7e11].

Although pioneering results [1e2] showed that pancreatic b-cell-like cells may be obtainedfrom ESCs, new differentiation strategies that can be used with human stem cells needed to be

developed. Differentiation strategies were based on knowledge of early mouse development,

the sequential expression of the transcription factors [12e14] and the signaling pathways [15]involved in b-cell formation. The application of such developmental principles to stem cell

biology seem to be the key to obtaining a successful differentiation process, thus recent ap-

proaches chosen by the majority of investigators working on human ESC differentiation toproduce insulin-secreting cells are based in a multi-stage protocol attempting to mimic all

phases of in vivo pancreas development. Thus, the aim is to induce human ESCs to transition

sequentially through mesendoderm, definitive endoderm, gut-tube endoderm, pancreaticendoderm and endocrine precursor stages, resulting in a final, functional, insulin-

expressing cell.

STEPS TOWARDS b-CELLS: PROTOCOL COMPARISONObtaining mesendoderm and definitive endoderm

In order to make human ESCs differentiate into insulin-producing cells, the first goal is the

efficient generation of definitive endoderm, which has been readily achieved by D’Amour et al.[16] using a combination of a TGFb family member, Activin A, to activate Nodal signaling, and

low serum concentration of media to avoid the activation of PI3K. Furthermore, to improve

the yield of definitive endoderm cells, the activity of PI3K could be inhibited using twodifferent inhibitors; LY 294002 [4,17] or wortmannin [18]. Wnt3a-mediated Brachyury

expression is also important for the migration of precursor cells through the anterior region of

the primitive streak (PS) and the formation of a mesendoderm population from which bothendoderm and mesoderm will be generated depending on the magnitude and duration of

Nodal signaling [19e20]. Hence, the efficiency of definitive endoderm generation can be

further improved by exposure of human ESCs to a combination of Activin A andWint3a in theabsence of serum on the first day, followed by one day of culture in a medium supplemented

with Activin A and 0.2% serum, and then three days in a medium supplemented with Activin A

and 2% serum [21]. In contrast to Wnts, bone morphogenic proteins (BMPs) inhibit endo-derm induction. Therefore, inhibition of BMP signaling using the BMP antagonist, Noggin,

resulted in increased expression of PS/endoderm markers and in a rapidly reduced expression

of PS/mesoderm markers, thus demonstrating the cooperative intertalk of canonical Wnt/b-catenin, Activin/Nodal and BMP signaling pathways during ESCs specification of PS,

mesoderm and endoderm [22]. A different approach to inducing definitive endoderm hasbeen recently published [23], and uses two small molecules identified as endoderm inducers

(IDE1 and IDE2) with an efficiency similar to that obtained with the Activin A treatment

described above.

Obtaining foregut patterning

Once definitive endoderm has been obtained, the next step is to trigger DE to foregut

patterning, which results from the complex crosstalk between mesoderm and endoderm,

involving gradients of fibroblast growth factors (FGFs), BMPs, retinoic acid (RA) and sonichedgehog (SHH) [24]. During foregut patterning, high concentrations of FGF4 promote

a posterior/intestinal endoderm cell fate, whereas lower FGF4 levels induce a more anterior/

pancreas-duodenal cell fate [24]. In the same way, it has been shown that FGF2 specifieshuman ESCs-derived definitive endoderm into different foregut lineages in a dose-dependent

manner. Specifically low doses of FGF2 promote a hepatic cell fate, intermediate FGF2 levels

induce a pancreatic cell fate and high concentrations of FGF2 induce midgut endoderm smallintestinal progenitors [25]. Retinoids are known as morphogens and differentiation inducers

CHAPTER 41Generation of Pancreatic Islets from Stem Cells

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during embryonic development and ESC differentiation, as reported by many investigators.These studies often used retinoic acid to induce pancreatic endoderm commitment in the

definitive endoderm obtained by Activin A treatment [18,21,26e29]. In early development,

SHH is highly expressed in the stomach and duodenal endoderm, but not in the pancreaticendoderm; hence specific inhibition of sonic hedgohog signaling has been shown to promote

pancreatic differentiation by expanding the endoderm population of Pdx1-expressing cells[30]. In the same way, inhibition of SHH prevents stomach and duodenal endoderm speci-

fication, and the inhibition of BMP signaling pathway by Noggin has been shown to block

hepatic commitment. Hence, to promote pancreatic endoderm specification at the expense ofother foregut endoderm lineages, Cyclopamine, firstly introduced by Leon-Quinto et al. [2] for

in vitro differentiation, and Noggin are often used in conjunction with RA treatment in human

ESC differentiation protocols [18e29]. Curiously, anti-sonic hedgehog, which displayed betterresults than the non-specific small molecule cyclopamine, is not used.

Obtaining endocrine precursors

Notch signaling regulates the consecutive cell fate decisions required for the formation ofspecialized tissues, including b-cell generation. Once pancreatic endoderm is obtained, Notch

inhibition seems to be critical for further differentiation towards an endocrine fate. This is

consistent with the fact that Notch signaling represses transcription of Ngn3, a critical tran-scription factor for the formation of pancreatic endocrine cells. Notch activity serves to expand

the pool of pancreatic progenitors preventing premature endocrine differentiation. During in

vitro differentiation, it has been recently proved that EGF treatment can be used to expand thePdx1-positive population of progenitor cells, which is necessary to obtain a large number of

endocrine cells [18]. After EGF or FGF10 treatment, inhibition of Notch signaling using N-[N-

(3,5-difluorophenacetyl)-L-alanyl-S-phenylglycine t-butyl ester (DAPT) a g-secretase inhibitorhad only a slight impact on promoting endocrine differentiation [21,31,32] probably due to

a non-specific inhibition of Notch.

Maturation of pancreatic endocrine cells

The last step of human ESC differentiation into insulin-producing cells consists of directing

the maturation of endocrine precursors into specialized and functional hormone-secretingcells. However, despite of the great number of biologically active compounds that have been

used in published endocrine pancreas differentiation protocols, as yet an in vitro differentia-

tion of ESCs into functional b-cells has not been achieved. D’Amour et al. [21] used a mix ofdifferent ’maturation factors’ such as IGF1, Exendin-4, HGF and B27 supplement during

terminal differentiation stages, but observed only minor effects on differentiation when these

factors were omitted. On the other hand, Cho et al. [32] demonstrated that the application ofbetacellulin and nicotinamide to D’Amour’s protocol resulted in sustained Pdx1 expression

and led to subsequent insulin production. Nevertheless, insulin-producing cells obtained by in

vitro differentiation protocols are commonly immature and non-functionally glucose-responsive. As a consequence, in vitro terminal differentiation steps were omitted from the

protocol published by Kroon et al. [29], where pancreatic progenitors were allowed to mature

into functional b-cell by in vivo maturation after transplantation in streptozotocin-inducedhyperglycemic mice.

ALTERNATIVE STRATEGIES FOR PROTOCOL OPTIMIZATIONAll the signaling pathways and factors described above are the result of more than ten years ofresearch into ESCs differentiation with the aim of obtaining functional insulin-secreting cells.

The fact that this aim has still not been achieved demonstrates the complexity of the differ-

entiation process (Fig. 41.2). New factors and different culture conditions will be probablyrequired to induce the complete differentiation and maturation of ESC-derived b-cells.

FIGURE 41.1Schematic representationshowing the transcriptionfactors and signalingpathways identified in thedevelopment of mousepancreatic b-cells.

FIGURE 41.2Overview of the signaling pathways and factors that have been shown to efficiently differentiate ESCs to a b-cell fate. Resveratrol, miR-7 and Reg-1are proposed factors to improve the maturation stage. ESC: embryonic stem cells; ME: mesendoderm; DE: definitive endoderm; PG: primitive gut; PF: posterior

foregut; PE: pancreatic endoderm; EP: endocrine precursors; BC: beta cells; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl-S-phenylglycine t-butyl ester; BTC:

betacelulin. Adapted from Champeris Tsaniras S et al. (2010). (*) Differentiation factors contributed by our group.

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CHAPTER 41Generation of Pancreatic Islets from Stem Cells

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Here we mention some novel approaches that could be useful in improving definitiveendoderm generation, and the final maturation of the endocrine precursors, resulting in

a more efficient insulin-secreting cell differentiation strategy. In addition, stem and somatic

cells other than ESCs have been used to obtain a b-cell phenotype. Some of these strategies areoverviewed below.

Increase of the glucose-stimulated insulin secretion pathway

Recent studies have shown the impact of Resveratrol (RSV) on insulin secretion and the way

that this compound potentiates glucose-stimulated insulin secretion. RSV (3,5,4’-trihydroxy-trans-stilbene) is a polyphenol that has been shown to activate Sirt-1, a NADþ-dependent

deacetylase [33]. Sirt-1 plays a role not only in the maturation process, but also in the initial

differentiation process [34]. The effect of RSVon insulin secretion was studied for the first timeby Zhang Y et al. [35] and recently by Vetteli et al. using the INS-1E cell line and human islets

[36]. Bordone et al. demonstrated that Sirt-1 represses transcription of the mitochondrial

uncoupling protein 2 (UCP2) by binding directly to its promoter [37]. Lower levels of UCP2,induced by Sirt-1 overexpression, result in increased ATP production and enhanced insulin

secretion in INS-1E [38]. In humans, the transcriptional pathway regulating b-cell UCP2 gene

expression is activated by the transcriptional cofactor peroxisome proliferator-activated re-ceptor-g coactivator-1 a (PGC-1 a) [39] which is another target of both Sirt-1 and RSV

(Fig. 41.3). Hence RSV could be considered as a good candidate for improving the maturation

process of human, ESC-derived, insulin-secreting cells.

Effects of new soluble factors on the maturation process

Of all the differentiation steps described above, the most difficult one to promote seems to bethe maturation stage. Despite the large number of factors and their combinations that have

been used in current protocols, functional b-like cells have not been produced. Screening for

new active molecules to be used as ’maturation factors’ could be helpful. In this context,a previous study described a fetal soluble factor, released by pancreatic buds, that has been

used to induce in vitro endocrine pancreatic differentiation in mouse ESCs [40]. Subsequent

proteomic studies (data not published) have demonstrated that one of the most abundantproteins present in the soluble factors released by pancreatic buds was Regenerating 1 (Reg-1).

Reg-1 is normally induced in pancreatic b-cells and acts as an autocrine/paracrine growth

factor for b-cell regeneration [41,42]. Based on this information, Reg-1 could be used in dif-ferentiation protocols to induce human ESC-derived b-cell maturation.

Nitric oxide and definitive endoderm induction

The relevant role of nitric oxide (NO) in developmental processes in the embryo has been

previously described [43,44]. NO has also been reported to play a role in the induction of ESCs

FIGURE 41.3Proposed links beteen Resveratrol, Sirt1, UCP2 and insulin secretion. The indirect activation of Sirt1 by Resveratrol

regulates insulin signaling pathways via repression of UCP2 transcription and through phosphorylation and subsequent

deacetylation of PPARg coactivator 1a (PGC1a), leading to mitochondrial function modulation, ATP increase and insulin

secretion.

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842

to differentiate into cardiomyocytes [45,46]. Recently the mechanism by which NO inducesESC differentiation has been described, and it has been observed that the exposure of ESCs to

exogenous donors of NO like 1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-

ium-1,2-diolate (DETA-NO) for a short time induces early differentiation towards a definitiveendoderm phenotype. Treatment with DETA-NO induced the expression of endoderm

markers such as Pdx1 and GATA4 after a very short period of exposure (19 hrs): this is aninteresting approach which could offer a suitable alternative for the generation of endoderm to

the 3e5 days of Activin/Wnt3a treatment [47].

Endothelial cell co-culture

A less studied area in the field of pancreatic differentiation is the effect of endothelial cell

signaling in b-cell maturation; this is increasingly being appreciated as an important contri-buting factor in in vivo pancreatic islet maturation. Recent studies have shown the effect of

endothelial cell signaling in the maturation of human ESC-derived pancreatic progenitor cells

into insulin-producing islet-like cells [48e50].

Microenvironment considerations

Despite the great influence of oxygen tension and extracellular matrix (ECM) on islet survival,

the physiological environment has been largely ignored in b-cell differentiation protocols. Islet

cells are extremely sensitive to both hypoxia and hyperoxia, and at the same time they needa three-dimensional (3D) cell-to-cell interaction structure to achieve a functional phenotype

[51]. Unfortunately the in vitro condition used until now could not reproduce an optimal in

vivo microenvironment, thus the 3D islet-like structure is not compatible with physiologicaloxygen distribution because of the lack of capillary vessels in the cluster structure. Novel

differentiation approaches that take into account the role of both the ECM and oxygen dis-

tribution could be important for improving the maturation process of the endocrine pre-cursors derived from human ESC differentiation. For these reasons, studies of the use of ECM

components, such as laminins, and silicone rubber membranes to control oxygen tension are

being carried out [52,53].

MicroRNAs

MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression by post-

transcriptional interference with specific messenger RNAs (mRNA). Study of miRNAs as reg-

ulators of complex gene expression networks is an emerging field that could be of great impactin both the differentiation and maintenance of cell phenotype. Studies in b-cell development

demonstrate that miR-7 is highly expressed in both mouse and human developing pancreas

[54], in the same way it contributes to in vivo b-cell development, consequentially miR-7 couldbe considered as an important player for the achievement of a complete differentiated human

ESCs-derived b-cell.

ALTERNATIVE CELL SOURCESInduced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are a new source of embryonic-like stem cells obtained

by reprogramming somatic cells. Similar to ESCs, iPSCs can differentiate into many differentcell types, including insulin-secreting cells [55], suggesting that patient-specific functional b-

cells might be generated [56]. Furthermore, b-cells generated from iPSCs were able to reverse

hyperglycemia after transplantation into diabetic mice [57]. One of the most important ad-vantages of using patient-specific b-cells is that they avoid the risk of immunological rejection

[58]. However, unexpectedly, rejection of autologous iPSCs transplanted in genetically iden-

tical mice has been observed [59] hence, further studies are required to ensure consistency andsafety of iPSCs before they can be used in future cell regenerative therapy.

CHAPTER 41Generation of Pancreatic Islets from Stem Cells

Mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that are

being explored as a promising new treatment for tissue regeneration. However, the ability ofMSCs to differentiate into insulin-producing cells when treated with different soluble factors is

still under question [60]. Nevertheless the efficacy of MSCs in the treatment of diabetes could

derive from different abilities of these cells, such as their immunomodulatory properties ortheir capability to differentiate into endothelial cells, thus providing environmental support

for pancreatic regeneration [61]. Actually several lines of evidence have demonstrated that

cotransplantation of islets and MSCs exhibits a better outcome then islet transplantationalone, by promoting vascularization of the graft and hence preventing rejection [62,63].

843

Transdifferentiation

In addition to studies using stem cells and iPSCs, recent reports suggest that pancreatic duct

cells, liver cells, acinar cells, and other mature cell types (Fig. 41.4) have the ability to trans-

differentiate into insulin-producing cells [64e66]. Even adult monocytes retain this capability[67]. However is difficult to demonstrate that these ’insulin-producing cells’ possess the criteria

to be considered pancreatic b-cells. The difficulties of differentiating adult cells into insulin-producing pancreatic cells have been bypassed by using in vivo gene transfer of the pdx-1 gene

into liver cells to induce hepatocyte transdifferentiation [68,69]. Experiments are not

conclusive because cells generated in this manner are not true b-cells, but rather hybrids ofhepatic and pancreatic cells. To avoid the problem of viral infection, a very recent study shows

that is possible to induce liver transdifferentiation by using a hydrodynamic approach to

deliver genes such as pdx1, ngn3 andmafA [70]. On the other hand, a more efficient attempt attransdifferention of non-endocrine tissue into b-cells has been achieved using exocrine cells as

the starting material. In this context, it was feasible to reprogram pancreatic exocrine tissue into

islet cell types using a combination of three genes (Pdx1, Ngn3, and MafA) [71,72]. Unlikein other transdifferentiation settings, the original exocrine phenotype appeared to be

completely abrogated, and diabetic mice subjected to transplantation of these cells showed

a significant and permanent improvement in blood glucose levels, even if their diabetes wasnot completely reversed.

De novo organ formation

While it has been shown that insulin-producing cells may benefit glucose homeostasis, in

human physiology the actual micro-organs controlling blood glucose are the pancreatic islets.

FIGURE 41.4Cell sources that have been proved able totransdifferentiate into insulin-producing cellsunder in vitro and/or in vivo conditions.

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Pancreatic islets are well-vascularized and innervated structures in which endocrine and non-endocrine cells are responsible for an integral response to blood glucose oscillations. Insulin-

secreting b-cells respond in synchrony to stimulatory nutrient increases within the islet

building a well-tuned response [73e75], simultaneously glucagon- and somatostatin-secretingcells modulate their activity to other nutrient ranges [76,77]. It is generally accepted that

pancreatic islets better represent the overall physiology than isolated b-cells, but so far no-onehas been able to produce a pancreatic islet in vitro. A complex and exciting strategy is appearing

in the field of regenerative medicine which consists of the generation of entire transplantable

organs. The complex cellular interactions among and within tissues that are required fororganogenesis are extremely difficult to recapitulate in vitro so, as an alternative, promising new

studies on blastocyst complementation are being undertaken [78]. The aim is to generate

pluripotent stem cell (PSC)-derived donor organs in vivo by injection of PSCs into blastocystsobtained from mutant mice in which the development of a certain organ was precluded by

genetic manipulation. The PSC-derived cells would developmentally compensate for the defect

and form the missing organ [78]. Nakauchi’s group has shown proof-of-principle findings forpancreas generation through the injection of PSCs into pancreas-deficient Pdx1(�/�) mouse

blastocysts [79]. This innovative approach poses not only technical but ethical questions; for

example, could the production of human organs in human-pig chimeras be an alternativeapproach? Obviously, we are still far from such an attempt.

CONCLUSIONDevelopment of human ESC-based therapy for diabetes represents one of the most chal-

lenging areas of stem cell research. Mimicking the complex developmental processes of b-cell

formation has been demonstrated to be the most promising and effective approach toobtaining insulin-secreting cells. However, our understanding of the signals which are

important in the final phase of pancreatic endoderm specification is still incomplete. Study of

endothelial cell-released factors and cell matrix interactions during pancreatic differentiationwill be required to generate functionally competent insulin-secreting pancreatic cells. On the

other hand, direct or indirect reprogramming of somatic cells through transdifferentiation or

iPSCs derivation has been shown to be an effective strategy to produce b-cell surrogates.However, the problem of genetic manipulation that characterizes both these two techniques

represents a safety concern that is still unlikely to be acceptable for clinical applications. In

conclusion, despite all the investigations efforts and promising progress reported in thisreview, further studies are required to generate new transplantable insulin-producing cells that

are safe and able to mimic extremely closely the complex functions of an endogenous b-cell.

AcknowledgmentsAuthors are supported by the Fundacion Progreso y Salud, Consejerıa de Salud, Junta de Andalucıa (Grant PI-0022/

2008); Consejerıa de Innovacion Ciencia y Empresa, Junta de Andalucıa (Grant CTS-6505; INP-2011e1615e900000);

Ministry of Science and Innovation (Red TerCel-FEDER Grant RD06/0010/0025; Instituto de Salud Carlos III GrantPI10/00964) and the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120).

CIBERDEM is an initiative of the Instituto de Salud Carlos III.

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