ScienceDirect - Seminars in Cell & Developmen...

Review

Kevin Docherty^{a, ,} , Andreia S. Bernardo^a and Ludovic Vallier^b

^aSchool of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK

^bDepartment of Surgery and Cambridge Institute for Medical Research, Addenbrooke's Hospital, University of Cambridge, Cambridge CB2 0XY, UK

Abstract

There is a compelling need to develop novel therapies for diabetes mellitus. Recent successes in the transplantation of islets of Langerhans are seen as a major breakthrough. However, there is huge disparity between potential recipients and the availability of donor tissue. Human embryonic stem cells induced to form pancreatic β cells could provide a replenishable supply of tissue. Early studies on the spontaneous differentiation of mouse embryonic stem cells have laid the foundation for a more directed approach based on recapitulating the events that occur during the development of the pancreas in the mouse. A high yield of definitive endoderm has been achieved, and although β-like cells can be generated in a step-wise manner, the efficiency is still low and the final product is not fully differentiated. Future challenges include generating fully functional islet cells under Xeno-free and chemically defined conditions and circumventing the need for immunosuppression.

Keywords: Differentiation; Pdx1; Insulin gene; Regenerative medicine; Cell therapy; Pancreas development

Article Outline

1.

Introduction

2.

Islets of Langerhans

3.

Initial studies on ES-derived insulin-secreting cells

4.

Developmental biology of the pancreas

5.

Recapitulating pancreatic development in ES cells

6.

Conclusions and future challenges

6.1. Variation between human ES cell lines

6.2. Xeno-free and chemically defined culture conditions

6.3. Functional characterisation of DE

6.4. Requirement for human ES cell reporter lines

6.5. The criteria for a functional β cell

6.6. Generation of mature β cells

6.7. Importance of the niche

6.8. ES cell-derived β cells required for a single transplant

6.9. Avoidance of immunosuppression

Acknowledgements

References

1. Introduction

2. Islets of Langerhans

Before describing how one might go about making a new insulin-secreting cell it would be appropriate to describe what the end-product should look like. Islets of Langerhans are discrete clusters of endocrine cells scattered throughout the pancreas. Islet numbers vary between species but in general range from 100,000 to 2.5 million per pancreas and vary in size from about 50–300 μm in diameter. Each islet contains several thousand hormone-secreting cells, comprising insulin-secreting β cells, glucagon-secreting α cells, somatostatin-secreting δ cells and pancreatic polypeptide-secreting PP cells. The β cell which comprises about 70% of the endocrine cells in the islet is unique in its ability to express the preproinsulin gene by mechanisms that are fairly well, if not completely, understood [10]. It contains the proteases PC2, PC1/PC3 and carboxypeptidase H, which are necessary for the efficient processing of proinsulin to insulin, and it can store insulin as a hexameric zinc crystal in specialised granules. Secretion of insulin occurs in two phases in a strictly regulated pulsatile manner in response to continuously varying levels of circulating nutrients, particularly glucose. Insulin has been detected at low levels in other tissues but it is unlikely that it would be secreted in a regulated manner or function in fuel metabolism and we know very little about how the insulin gene is regulated in these cells [11]. Glucose-stimulated insulin secretion (GSIS) in the β cell is driven predominantly by glucose-metabolism derived changes in the ratio of ATP:ADP. This, along with signals from the sympathetic nervous system and incretins secreted from the gut, affects the electrical properties (K_ATP channel activity) of the plasma membrane leading to changes in the cytoplasmic Ca²⁺ concentration that trigger exocytosis. Between meals the pool of insulin is replenished predominantly through translational mechanisms. The insulin gene is also sensitive to nutrients with changes in insulin mRNA levels that occur over longer time periods.

There are important differences between rodent and human islets that should be emphasised. In rodent islets the β cells are clustered in the core of the islet surrounded by a mantle of α, δ and PP cells. The islet is highly vascularised, and while the pancreatic artery supplies both islets and the surrounding exocrine tissue, the islets receive up to 20 times more blood flow than the acinar tissue [12]. Within the islet the blood flows in the direction β cell to α cell to δ cell [13]. This presumably ensures that the β cells of the central core are protected from the powerful inhibitory effects of glucagon and somatostatin. Recent data, however, have prompted a shift in ideas concerning intraislet cellular interactions in humans. In humans and non-human primates the various cell types are scattered throughout the islet [14]. The majority (71%) of β cells are found in direct contact with other endocrine cell types, suggesting a more important role for paracrine interactions in the human [15]. These differences between rodent and human islets are raised because of their implications for the derivation of insulin-secreting cell clusters from mouse and human ES cells. Other differences include the glucose transporter GLUT2, which is highly abundant in rat islets, but in human islets is present at very low levels [16] and [17]. The relevance of this is unclear but may reflect differences in glucose sensing mechanisms. There are also differences in the way islet cell mass is maintained in rodents and humans. Rodents appear to have a substantial capacity for β cell replication [18] and [19], whereas in humans, where the ability to measure these parameters is much more limited, β cell replication does not appear to be common [20]. This may be relevant to approaches that might be used to scale up the production of ES cell-derived β cells.

One important question concerns whether transplanted ES cell-derived β cells would function as well, in the absence of α and δ cells, as would human islets. It is difficult to answer this question. The only available data are from flow cytometry-sorted rat β cells, which appeared to function in rats rendered diabetic following treatment with streptozotocin almost as well as intact islets [21]. Equivalent data are not available for purified human β cells; because of their high endogenous fluorescence it is almost impossible to purify human β cells by flow cytometry.

How high must the bar be set? This question has already been answered in part [22]; nothing less than a fully functional β cell will do. In excessive amounts insulin can be fatal. It is absolutely essential therefore that the sophisticated mechanisms that regulate insulin production and secretion are recreated in all their aspects in any ES-derived β cell. Myocardiocyte-like or neuronal-like cells may have some therapeutic roles in diseases of the cardiovascular and nervous systems, but there is no therapeutic role for a functionally imperfect β-like cell. For these reasons differentiation protocols should best be adapted to mimic the normal process of islet ontogeny. Before reviewing recent progress in this area the following section will review the first phase of publications on the derivation of ES-derived insulin-secreting cells. With the benefit of hindsight it will be clear that there were conceptual flaws in some of these experiments, but as in every other area of research they served to influence the design of future studies.

3. Initial studies on ES-derived insulin-secreting cells

ES cells are lines derived from the inner cell mass of preimplantation embryos that have been allowed to reach the blastocyst stage [23] and [24]. They can be expanded in culture indefinitely while retaining the functional attributes of pluripotent cells of the embryo, i.e. the ability to differentiate into any cell type in the body. The first publication to describe the differentiation of ES cells into insulin-secreting cells made use of the natural propensity of ES cells to spontaneously differentiate into a large variety of different cell types [25]. A cell trapping strategy, in which the gene conferring resistance to neomycin was placed under the control of the insulin promoter, was used to select for insulin-expressing clones. The selected cells were able to normalise blood glucose levels when placed under the kidney capsule of mice rendered hyperglycaemic following treatment with streptozotocin. This was a seminal study in so far as it was the first to describe the differentiation of ES cells into insulin-expressing cells. However, the efficiency of generation of insulin-expressing clones was extremely low, they did not survive well, and the animal studies have been criticised on several counts including the failure to demonstrate that removal of the kidney containing the grafted cells would reverse the beneficial effects of the graft on hyperglycaemia [4].

A number of other studies have described the derivation of insulin-secreting cells from mouse ES cells [26], [27], [28], [29], [30], [31], [32], [33] and [34]. Several of these were based on protocols that had been developed to differentiate mouse ES cells towards neurons [35]. The approach involved generating a highly enriched population of nestin-positive cells from embryoid bodies (EBs). The EBs were then plated into serum-free medium supplemented with insulin, transferrin, selenium, and fibronectin (ITSFn) for 6–7 days to enrich for nestin⁺ cells and then sequentially in N₂ medium containing B27 and FGF2 (6 days) and N₂ medium containing B27 without FGF2 (6 days) [26]. About 10–30% of the cells stained positive for insulin and formed islet-like clusters. Modifications to the protocol, which led to increased levels of insulin, involved introducing an exogenous pax4 gene into the cells prior to differentiation [29] or addition of a phosphatidyl inositol 3-kinase inhibitor (LY 294002) to the final stage of differentiation [27]. Nestin is a filament protein that was originally identified as a marker for neuroepithelial progenitors [36] but subsequently found to be up-regulated in progenitor cells of other lineages. The rationale for deriving insulin-secreting cells from a nestin-enriched population was based on the reported presence of nestin in adult islets [37] and in the developing pancreas [38]. However, although nestin appears to be present in many cell types in the developing pancreas there is no convincing evidence that it directly affects the differentiation of islet cells. The experiments were also influenced by the similarities that exist between neural and islet cell types, including shared expression of transcription factors such as Isl1, Ngn3, Pax6, Pax4 and β2, glucose transporters [39] and other genes related to the storage and processing of neurotransmitters [40]. These similarities, however, do not reflect a common embryonic origin of islets and neurons, and as such differentiation via neurons would not be an obvious route to generate β cells from ES cells. It has also been argued that the results of the studies using what has become known as the Lumelsky protocol, have been misinterpreted due to potential artefacts resulting from the uptake of insulin from the medium [41], [42] and [43]. The take home message from these studies was that C-peptide biosynthesis and secretion should be demonstrated to substantiate claims that insulin-expressing cells can be derived from ES cells [42], and that pre-selection via a nestin-enriched population, involving the use of ITSFn and FGF2, should be avoided, since even if bona fide insulin expression was detected it could likely come from insulin-expressing neurons [44], [45] and [46].

The weaknesses in these early studies served to emphasise the need to recapitulate the normal series of events that occur during embryonic development. There is no doubt that insulin-secreting cells can arise spontaneously from mouse EBs by a process that fails to mimic pancreatic development [32] and extra-embryonic tissue may be the source of some of these cells [34]. It is unlikely, however, that these cells will ever become β cells. Since the pancreas is derived from endoderm it would make more sense to focus efforts on inducing the in vitro formation of this germ layer. The next section will describe the developmental biology of the pancreas, anticipating that this will underpin more recent studies on the differentiation of ES cells.

4. Developmental biology of the pancreas

Shortly after fertilisation of the egg the blastocyst, a spherical structure of about 50–60 cells, forms. This consists of an outer cell layer, the trophectoderm surrounding a cluster of cells called the inner cell mass (ICM) and a hollow cavity known as the blastocoel (Fig. 1). The trophectoderm gives rise to the yolk sac and the placenta while the ICM generates the embryo [47]. The ICM forms a bilaminar structure in which the cells closer to the blastocoel are know as the hipoblast or visceral endoderm (VE) and contribute only to the formation of extra-embryonic tissue, whereas the cells closer to the trophectoderm are known as the epiblast or primitive ectoderm and give rise to the entire embryo. Around embryonic day 6.5 (e6.5) in the mouse the epiblast undergoes gastrulation, whereby a region of proliferating and migrating cells (the primitive streak), gives rise to the three germ cell layers, ectoderm, mesoderm and definitive endoderm (DE). The pancreas is formed from the DE. Mesoderm and DE originate from an intermediate population of bipotential cells called the mesendoderm [48]. One of the challenges of stem cell research has been to identify robust markers that can distinguish ectoderm, mesendoderm, mesoderm, VE and DE and to identify the factors involved in their formation. Several families of growth factors, including fibroblast growth factors (FGFs) and the transforming growth factor β (TGFβ) super family regulate gastrulation in the mouse. TGFβs are particularly important in the generation of DE through an indirect effect on the production of mesendoderm [49], while the Sry-related HMG box gene Sox17 plays a determinant role in the formation of DE [50]. At the end of gastrulation the DE is an undetermined sheet of cells. It then forms a primitive gut tube, which becomes regionalised along its anterior–posterior axis in response to retinoic acid (RA) and FGFs released from the lateral plate mesoderm [51].

Fig. 1. Gastrulation in the mouse. The morula (16 cell stage embryo) forms a blastocyst which contains the inner cell mass (ICM) and the trophoectoderm. The cells of the ICM then start to delaminate into hypoblast and the epiblast (early gastrula). The epiblast gives rise to the ectoderm, mesoderm and endoderm (late gastrula). An intermediate stage involves delamination of the epiblast cells and formation of a bipotential intermediate cell called the mesendoderm.

At around e8.5 signals from the adjacent notochord and mesenchyme induce patterning of the forward region of the gut tube resulting in the formation of the dorsal and ventral pancreatic buds [52] and [53]. During the next 10 days of foetal development the pancreatic buds expand, the two lobes fuse, and individual cells of the branching epithelial network differentiate into acinar and ductal tissue of the exocrine pancreas as well as the islets of Langerhans [54]. These events are controlled by the sequential activation of transcription factors, most of which function as positive activators but some, such as Nkx2.2 can act as inhibitory factors [55]. Strategies towards recapitulating these events in vitro have been based on our understanding of the role played by specific transcription factors in establishing cell lineages, and the on the identification of the growth factors and signalling molecules emanating from the surrounding mesoderm and mesenchyme that regulate their activity.

FoxA2 (Hnf3β), Hnf1α, Hnf1β and Hnf4α delineate regions of the foregut endoderm from which the pancreas forms. The pancreatic domain is further refined by the expression of the homeodomain protein Pdx1 [56] and [57] in sub-regions of the foregut in which Hedgehog (Hh) signalling is repressed [58]. The basic helix-loop-helix (bHLH) transcription factor Ptf1a and the homeodomain protein Hlxb9 are also involved in specification of the pancreas [59], [60] and [61]. In the Ptf1a knockout mouse, the Pdx1⁺ domain of the foregut adopts a duodenal fate, suggesting that Ptf1a may act along with Pdx1 to drive endoderm towards a pancreatic fate [62]. Pdx1 under some conditions can interact physically with the TALE homeodomain protein Pbx1 [63] and Pdx:Pbx complexes may be important for normal proliferation of cells during pancreatic development [64]. Pbx1 null mice display pancreatic hypoplasia and marked defects in exocrine and endocrine cell differentiation prior to death at e15 or e16 suggesting that Pbx1 may also play a role at this early stage in the development of the pancreas [65].

The Pdx1⁺ and Ptf1a⁺ cells within the pancreatic buds represent a population of progenitor cells that undergo a period of expansion under the influence of FGF10 produced by the surrounding mesenchyme [66] and [67]. At around e14.5 the commitment to expand further or differentiate is governed by Delta-Notch signalling on adjacent cells. Notch-mediated signalling ensures maintenance of the progenitor cell population through activation of the bHLH factor Hes1 [68]. In a population of Pdx1⁺ cells that evade Notch signalling, repression of Hes1 leads to activation of the bHLH factor neurogenin 3 (ngn3) that specifies the endocrine lineage [69] and [70]. Ptf1a expression on the other hand is restricted to cells that will form the exocrine pancreas. The HMG box transcription factor Sox9 may play an important role in maintaining the pancreatic progenitor cell population [71]. Inactivation of SOX9 results in depletion of the progenitor pool similar to that seen in the Hes-1 null embryos, suggesting that Sox9 may regulate the progenitor pool by governing its release into a programme of differentiation [71] and [72]. This may be the stage in development at which islet progenitors generated in vitro from ES cells might best be expanded.

The expression of neurogenin 3 (ngn3), insulinoma 1 (Insm1, which is also known as IA1) [73] and [74] and β2 (a bHLH protein which forms a heterodimeric complex with bHLH partners such as E47) [75] and [76] at around e14 specifies the endocrine lineage. Within this lineage NKx2.2, Nkx6.1, Pax4, Isl1, MafA and Pdx1 (in a second wave of expression) drive formation of β cells, while Pax6, Arx and Brn4 drive formation of α cells [55]. Very little is known about the transcription factors that drive formation of δ and PP cells. In the adult mouse expression of Pdx1 is restricted to β cells, some (20%) δ cells and rare endocrine cells in the gut, while Nkx6.1 is restricted to β cells and some neurones. MafA is also enriched in β cells [77]. Interestingly, MafB is expressed earlier in pancreatogenesis but there is a switch to MafA at a late stage of differentiation concomitant with the acquisition of glucose sensing and other characteristics of the mature β cell [77], [78] and [79].

5. Recapitulating pancreatic development in ES cells

During embryogenesis cells pass through a series of checkpoints in their progress towards a specific lineage (Fig. 2). For islet cells these checkpoints, starting from undifferentiated ES cells, include definitive endoderm (DE), posterior foregut (PF), pancreatic endoderm (PE), islet precursors, and differentiated islet cells. Each checkpoint would be expected to express a specific set of genes that would serve as specific markers. One of the bottlenecks in driving ES cells towards a pancreatic lineage has been the lack of robust markers for DE. The reason for this is that several of the genes used as markers of DE are also expressed by visceral endoderm (VE) [80]. The fact that DE but not VE originates from a Mixl1⁺/Bry⁺ mesendoderm population [81] has been important in devising and monitoring differentiation protocols. In the absence of single unique markers the following combinations have proved useful: DE, Sox17⁺, Foxa2⁺, SOX7⁻ and Bry⁻; VE, Sox17(lo), Foxa2⁺, Sox7⁺, Bry⁻; and mesoderm, Sox17⁻, Foxa2⁻, Sox7⁻, Bry⁺. Markers for primitive foregut include Foxa2 (Hnf1β) and HNF4α, while Pdx1 and Nkx6.1 serve as markers for pancreatic endoderm [82], and Ngn3 as a marker for islet precursor cells. A fully differentiated β cell would express insulin, Pdx-1, islet amyloid polypeptide (IAPP) and MafA, while the other hormone-secreting islet cells could be identified on the basis of specific hormone expression.

Fig. 2. Step-wise differentiation of ES cells towards an islet phenotype. ES cells are cultured as a monolayer and treated with various growth factors and inhibitors over a period of time that can vary from 15 to 35 days to generate the intermediate cell populations. Progress through the pathway can be controlled by the addition to the culture medium of: (1) activin A and wnt3a (to drive ES cells towards mesendoderm and from there to DE); (2) FGFs (that are known to affect patterning of the primitive foregut); (3) the Hh inhibitor cyclopamine (to induce formation of Pdx1⁺ cells) and RA (to affect further patterning of the foregut cells); (4) The Notch (γ secretase) inhibitor DAPI (to induce formation of Ngn3⁺ islet progenitor cells); and (5) factors known to affect differentiation of islet cells, including nicotinamide, exendin 4, IGFs and HGF.

With a view to optimising protocols aimed at directing ES cells through the various checkpoints, a number of groups used homologous recombination to introduce fluorescent reporter probes into the genomic locus of particular marker genes. These included the generation of mouse ES cells expressing green fluorescent protein (GFP) or β galactosidase at the Bry [83], Gsc [84], pdx1 [85] and [86] and Mixl1 [81] loci. The anticipation was that these reporter lines would provide assays that would lead to the identification of factors that would drive cells towards a specific checkpoint. The subsequent study using the Bry-GFP line represented a major breakthrough in attempts to generate endoderm from ES cells. When EBs were allowed to differentiate in the presence of serum there was a substantial enrichment in the proportion of GFP-expressing cells. The serum was then replaced with various members of the TGFβ family, leading to the identification of activin A as an important inducer of mesendoderm formation. The amount of activin A required to replace the actions of serum was relatively high (30 ng/ml) and its effects very much concentration-dependent. Grafting the mesendoderm-enriched cells under the kidney capsule of mice led to the development of endoderm-derived structures, leading to the conclusion that Bry⁺ cells could give rise to both DE and mesoderm [87]. The Gsc-GFP cell line was used to show that activin A in the absence of serum could induce formation of mesendodermal Gsc⁺ cells that co-expressed E-cadherin and platelet derived growth factor receptor 1α (PDGFRα). This population could be further differentiated to endoderm and mesoderm [84].

The pdx1-GFP cells were used to identify a population of Pdx1⁺ cells that are present in the foregut as the pancreas begins to form. Treatment of the developing EBs with retinoic acid (RA), which is known to pattern the primitive gut, generated a small population of GFP⁺ cells that expressed early pancreatic endodermal markers but not markers associated with further differentiation of the pancreas [85]. Similarly, the ES cells harbouring the LacZ gene integrated at the pdx1 locus differentiated towards a gut endodermal fate when co-cultured with mouse pancreatic rudiments. TGFβ2 was able to mimic the effects of the pancreatic rudiments further confirming the role of this family of growth factors in patterning the primitive foregut towards a pancreatic fate [86]. In keeping with the localisation of Mixl1 in the posterior region of the primitive streak, the Mixl1-GFP mouse lines differentiated into mesoderm that contributed to the haematopoietic lineage [81].

The studies using the Bry-GFP and Gsc-GFP cells were particularly important in confirming that mouse ES cells could differentiate through a mesendoderm-enriched population to generate either mesoderm or DE and that activin A could play an important role. This was taken a step further by using mouse ES cells that contained fluorescent (GFP) or cell surface markers (e.g. CD25) inserted at the Gsc or Sox17 loci of the same cell. This permitted the purification and characterisation of Gsc⁺Sox17⁺ DE and Gsc⁻Sox17⁺ VE and the further optimisation of culture conditions that differentially induced formation of DE and VE. Comparing the gene expression profiles of these two cell populations led to the identification of markers, including CXCR4, that distinguished the two populations [88]. Further support that DE could be efficiently generated came from studies on human ES cells [89]. Differentiation of the human ES cells in the presence of high doses of activin A and low serum produced cultures that were up to 80% DE. This population could be further enriched using the cell surface marker CXCR4.

It had been previously been shown that human ES cells could spontaneously differentiate into insulin-secreting cells [90] and [91], and that this could be improved by grafting the differentiated cells along with dorsal pancreatic rudiments under the kidney capsule of immunocompromised SCID mice [92]. With the success in generating a highly enriched population of DE cells, the way was open to extend the differentiation of human ES cells towards a β cell fate using a more systematic step-wise approach (Fig. 2). This involved sequential exposure of the cells in monolayer culture to: (i) activin A and wnt3a to form mesendoderm followed by activin A in low serum (0.2%) to form DE; (ii) FGF10 and the Hh inhibitor cyclopamine (CYC) to form primitive gut; (iii) RA, CYC and FGF10 to form posterior foregut; (iv) the Notch (γ-secretase) inhibitor DAPT and exendin 4 (Ex4) to form pancreatic endoderm; (v) Ex4, IGF1 and HGF to form islet hormone expressing cells [93]. The protocol was performed over 18 or so days and generated a population of cells, of which around 12% stained positive for insulin as assessed by flow cytometry. The insulin content of the purified insulin-expressing cells was around 14–208 pmol/μg DNA which is roughly similar to the insulin content of adult human islets. There were many positive outcomes to this study in that during the differentiation process the cultures tended to recapitulate expression in the correct sequence of key endodermal and pancreatic markers [94]. However, the final cell population failed to exhibit GSIS and many of the insulin⁺ cells co-expressed other islet hormones. It could be argued that the protocol generated immature β cells, and that a further period of differentiation or “tweaking” the protocol might be required. It is worth noting that although the protocol worked well in one particular human ES cell line (Cyt203), the efficiency was markedly reduced in five other lines. This emphasises the major differences that exist between various human ES cells in their capacity to differentiate towards a particular lineage or even germ layer, and may reflect the time after fertilisation at which the ES cells were generated (see below).

Using the H1 and H9 human ES cells, a protocol was designed whereby cells grown in monolayer were differentiated over 20 days in the absence of serum in medium supplemented with activin A (to make DE), and then sequentially with RA, FG2, and nicotinamide. A final period in suspension culture to encourage maturation of hormone expressing cells was employed [95]. The percentage of C-peptide positive cells was around 15%, they contained about 0.1 pmol C-peptide per mg protein and exhibit an acute (15 min), albeit weak, secretory response to glucose. When transplanted under the kidney capsule of streptozotocin-treated nude mice, normalisation of blood glucose was seen in 30% of the animals for up to 6 weeks after engraftment and this effect was reversed when the kidney bearing the graft was removed. A further study, in which human ES cells (H1, H7 and H9) were differentiated in suspension culture in the absence of serum, produced islet-like clusters (ILCs) over a 36 day period [96]. A DE-enriched population, which was obtained using activin A and the deacetylase inhibitor Na-butyrate, was directed down a pancreatic lineage using epidermal growth factor (EGF), FGF2, Noggin, nicotinamide and IGF2. The ILCs, of which roughly 2–8% were C-peptide⁺, contained 70 ng (10 pmol) insulin per μg DNA and secreted insulin (C-peptide) in response to glucose over a 3.5 h period. Electron microscopy was used to show the presence of secretory granules. Finally, a protocol has been developed whereby human ES cells (HES3) were cultured as EBs suspended in matrigel supplemented with activin A and BMP-4 to produce a DE-enriched population (day 4), which was further differentiated towards a PDX-1 enriched population (day 20) [141]. A subset (between 5 and 20%) of the Pdx-1⁺ cells persisted throughout a subsequent culture period (day 34) and a small percentage of these cells differentiated further to express Nkx6.1, C-peptide and Glut-2 and exhibited a very modest secretory response to glucose. When grafted into streptozotocin-treated SCID mice the C-peptide expressing EBs failed to lower blood glucose levels, but human C-peptide was detected in the blood up to 2 weeks after transplantation.

6. Conclusions and future challenges

Tremendous progress has been made but there are many challenges before the goal of using human ES cell-derived islet cells for transplantation in the treatment of diabetes can be achieved. These include, obtaining functional fully differentiated β cells in sufficient quantities under Xeno-free and GMP conditions, and circumventing the requirement for immunosuppression. The following section describes some of the arising issues with comments on how they might be addressed.

6.1. Variation between human ES cell lines

The efficiency of methods to generate specific cell type varies between human ES cell lines [93] and often between laboratories growing the same line. In addition, some lines appear to prefer to differentiate into derivatives of a specific germ layer. These observations are often based on anecdotal reports and are rarely discussed in published reports. This issue has been systematically addressed in a comparative study performed on 59 independently derived human ES cell lines from 17 laboratories worldwide [97]. The results showed little variation in gene expression, including those involved in pluripotency such as Nanog, Oct4 and Sox2. However, gene expression profiling of differentiated cells showed marked differences between the various lines. In addition, some epigenetic variations could be observed, especially for specific imprinted genes and for X inactivation in the female lines. The differences observed between the various human ES cell lines could have several origins, including the genetic background and variations in the techniques for their derivation and subsequent cell culture [98] and [99]. Thus, a major challenge is to develop a universal protocol for the derivation of human ES cells and chemically defined culture media that can be applied to all human ES cell lines [100], [101], [102] and [103].

6.2. Xeno-free and chemically defined culture conditions

The culture conditions used to generate different germ layer cell types, including DE, represent another issue that needs to be addressed if derivatives of these precursors are to be used for cell based therapy. All the currently available protocols use animal products (foetal bovine serum, mouse feeder cells and bovine serum albumin) or unknown components that are present in Matrigel™, Serum Replacements and conditioned media. Importantly, the serum and the feeders used in these protocols cannot be substituted by serum albumin or by artificial matrices, suggesting that uncharacterised factors are required for the differentiation of human ES cells to DE. These factors obscure analysis of developmental mechanisms and potentially render the resulting tissues incompatible with future clinical applications. The development and validation of Xeno-free and chemically defined culture conditions for achieving specification of hESCs into DE and beyond therefore remains a major challenge.

6.3. Functional characterisation of DE

Recent publications (as described above) have clearly shown that ES cells can be differentiated into a near homogenous population of DE cells and thus, the preliminary step towards the generation of pancreatic progenitors can be achieved in vitro [84], [87], [88] and [89]. The human DE cells generated in these studies expressed the markers (Sox17, CXCR4, Gsc, Mixl1) and exhibited the capacity to differentiate into liver-like and intestine-like cells in vivo and into pancreatic cells in vitro. Consequently, the endodermal nature of these cells cannot be questioned. However, some uncertainties remain concerning their embryonic origin. Thus, although the human DE cells were shown to arise from a Bry⁺ mesendoderm population, further data confirming that the Bry⁺ cells could form mesoderm and differentiate further into cardiac, muscle, endothelial or haematopoietic cells was not presented [89]. In addition, the data concerning the ability of the DE-enriched cells to differentiate further was limited. Transplantation of the DE cells under the kidney capsule of immuno-deficient mice resulted in an organised structure containing cells expressing CDX2, villin and hepatocyte specific antigen (HSA), which mark gut and liver cells. However, these markers are also expressed in other cell types including extra-embryonic cells [104] and histological analyses of the tissues generated by the graft indicated that they were poorly differentiated. Further experiments are therefore required to define more clearly the nature of the differentiated cells obtained in vivo, including the analyses of additional endoderm markers, the ability to rescue animal models of liver and pancreatic failure, and the capacity of these cells to form tumours. Additionally, the ability of DE cells generated from human ES cells to colonise the endoderm germ layer after injection in the primitive streak of gastrulating mouse or chick embryos should be investigated.

6.4. Requirement for human ES cell reporter lines

The rapid progress in the past few years in deriving protocols for inducing differentiation towards DE has emphasised the importance of reporter cell lines. To date no human ES cell knock-in reporter lines have been reported. It is anticipated that progress will be made in this area. Knock-in lines whereby GFP or β-galactosidase are expressed under the control of the sox17, pdx1, sox9, ngn3, mafA, and insulin gene promoters are among those which would prove interesting to study, and hopefully should become generally available. These lines may also provide novel insights into the differentiation of the human pancreas. Human ES cells are possibly the only realistic model to examine cell lineages and organogenesis in human embryogenesis.

6.5. The criteria for a functional β cell

A fully differentiated functional β cell should: express insulin (C-peptide) at levels equivalent to those seen in an average human β cell; contain storage granules as detected by EM microscopy; efficiently process proinsulin to insulin; and exhibit GSIS, i.e. an acute 3-fold stimulatory response to glucose. In vivo studies should include: the ability to detect human C-peptide in the blood of SCID mice for periods up to 4–6 weeks following engraftment; an increase in blood C-peptide upon administration of glucose following engraftment; normalisation of blood glucose levels in a diabetic animal model; and no formation of teratomas [105].

6.6. Generation of mature β cells

Maturation of pancreatic progenitors in vitro represents a key step towards the generation of fully functional β cells from human ES cells. The protocols currently available allow for the production of β cells with the characteristics of immature cells, i.e. low insulin secretion and co-expression of several islet hormones [93] and [95]. The factors and mechanisms that control the final maturation stage are not well understood. Genetic studies in the mouse have demonstrated that a switch of expression between the transcription factors MafA and MafB is required for the maturation of pancreatic progenitors into functional insulin-secreting cells in vivo [78] and [79], while NKx2.2 appears to be necessary for the maintenance of mature pancreatic cells [106]. One possibility might therefore be to overexpress transcription factors, such as MafA or Nkx2.2, during the differentiation process. A similar approach has successfully been used in the generation of haematopoietic stem cells (HSCs) [107] and [108]. HSCs generated from mouse ES cells are not functional insofar as it is impossible to achieve stable long-term blood engraftment of ES cell-derived HSCs in irradiated mice. However, overexpression of the transcription factor HoxB4 during ES cell differentiation was sufficient to induce a switch to a definitive HSC phenotype with engraftment and long-term, multilineage hematopoiesis. This provides proof of principle that the expression of a key transcription factor could be sufficient to drive the maturation of embryonic progenitors into fully functional adult cells. But, the requirement for genetic modification with the potential for stable expression of oncogenes represents a major barrier to the therapeutic application of such an approach. This problem could potentially be overcome, however, by the use of protein transduction domains, similar to that of the HIV transactivating protein (TAT) [109] and [110]. Some preliminary studies on TAT mediated overexpression of ngn3 have also been reported [111].

6.7. Importance of the niche

An alternative approach to generating fully differentiated functional β or islet cells would be to recreate the pancreatic niche. Endothelial cells represent a major component of this niche, playing an essential role in the differentiation of pancreatic cells [112]. Thus, co-culture of human ES cell-derived pancreatic progenitors with endothelial cells might be a solution to the problem of obtaining mature β cells, and possibly also to facilitate the formation of a vascularised islet structure prior to engraftment. Islet neurons play a different role in the niche, modulating insulin secretion. Among the neurotransmitters that reach the islet nerve endings are the vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) which in combination with glucose and incretins regulate post-prandial insulin secretion [113]. Therefore, the presence of such factors might be important for the differentiated cells to exhibit GSIS. Interestingly, current protocols for generating β cells are based on the production of a highly enriched population of endoderm cells, avoiding the generation of mesoderm and ectoderm. In this context a less directed differentiation protocol might generate a more complex environment, including the presence of endothelial cells and islet neurons that would better facilitate pancreatic cell maturation and possibly islet formation as well as better GSIS.

6.8. ES cell-derived β cells required for a single transplant

Based on the Edmonton Protocol, each transplant would require about 600,000 islet equivalents (IE). Since each IE contains about 1000 β cells, this would mean that roughly 1 billion (assuming some loss following engraftment) ES-derived β cells would be required. It is likely that the differentiation protocol will contain a stage at which the cells are expanded. More research is required on the proliferative potential of the various sub-populations (Fig. 2); however based on data from the developing mouse, the Sox9 enriched population might be best suited for this purpose. In order to avoid teratomas formation it would be important to remove any proliferative precursors from the final differentiated population prior to transplantation.

6.9. Avoidance of immunosuppression

Human ES cells and their differentiated derivatives express human leukocyte antigens (HLA) and major histocompatibility complex (MHC) molecules and are thus likely to be rejected by the immune system after transplantation [114]. One method to avoid immunosuppression of islet transplant recipients has been to encase the islet grafts in selectively permeable microcapsules made of sodium alginate or poly-l-ornithine [115]. Preliminary studies in humans have been promising [116], and it may be the case that microencapsulation represents the future for stem cells in the treatment of diabetes. Other possible ways to avoid the need for immunosuppression include reducing expression of MHC molecules by genetic modification of human ES cells [117], mixed chimerism [118], or the use of dendritic cells [119].

Despite some recent setbacks [120], therapeutic cloning remains another potential approach to the generation of immunocompatibe cells. This involves the production of ES cells from blastocysts obtained by introducing nuclei of adult somatic cells into meiotic enucleated oocytes [121]. The resultant ES cell lines and their differentiated derivatives would display the same HLA and MHC haplotype as the donor cell. The feasibility of such an approach has been demonstrated in the mouse in an experiment whereby transplantation of haematopoietic cells generated from cloned ES cells could correct the immunodeficiency caused by a mutation in the Rag2 gene [122]. However, the low efficiency of nuclear transfer [123], [124] and [125], combined with the low efficiency of ES cell derivation, and the relatively small number of available oocytes, could impose limitations on the use of therapeutic cloning in humans. Production of oocytes in vitro from ES cells [126] and the use of zygotes [127] could overcome the shortage of donated gametes, although more studies are required to define the potential of such approaches in human. Collectively, these observations show that the use of cloning for therapeutic purposes will require much more innovation than initially predicted at the time of the birth of Dolly [128].

The ability to convert somatic cells to patient-specific ES cells would have huge implications for cell based therapies. With this in mind three independent research groups have recently demonstrated that ES cells can be generated from somatic cells by overexpression of four transcription factors, namely Oct-4, Sox2, c-Myc and Klf4 [129], [130] and [131]. These induced pluripotent stem cells (iPSCs) show all the hallmarks of ES cells: they express pluripotent markers; they form teratomas; and they are germ line competent. It is anticipated that problems related to the formation of tumors following reactivation of the c-myc in adult mice obtained from iPSCs can be overcome. The generation of human iPSCs, however, may be more challenging. The protocols used to make iPSCs were based on reporter lines established from transgenic mice and the equivalent lines would be technically difficult to develop in human. Over and beyond these technical aspects, the differences between human and mouse ES cells could represent the most important challenge to establish human iPSCs. The divergence of the core transcriptional network controlling pluripotency in mouse and human ES cells [132] and [133], which could indicate a different embryonic origin [134] and [135], suggests that the transcription factors sufficient to re-program mouse somatic cells might not function to reprogram human cells.

The general aim of the approaches described above is that of barrier protection or the generation of isogenic cell lines that would be fully compatible with the transplant recipient. In the case of allogenic cells the graft could be better protected from rejection if the donor and recipient had the same HLA-haplotypes. This could be achieved by creating a bank of HLA-typed human ES cell lines from which a best match could be selected for transplantation. To be efficient such a bank would have to contain at least 150 different lines with specific HLA-haplotypes [136]. The creation of a large international consortium would be a prerequisite for the creation of this bank, and the use of defined culture conditions throughout the process of derivation would be absolutely necessary (see above). The generation of human ES cell lines that are homozygous for common HLA-haplotypes could reduce the number of lines required to 10 [136]. The chances of obtaining such lines from in vitro fertilization clinics would be very low, while the large scale screening of individuals and subsequent derivation of embryos for therapeutic purposes would be ethically unacceptable. Ironically, the failure to achieve nuclear transfer in human ES cells (see above) could bring a solution to this problem. Thus, it has recently been demonstrated that the human ES cell line SCNT-hES-1, previously claimed to have been derived by nuclear transfer, is a human parthenogenetic line [137]. Parthenogenetic embryos are derived from oocytes activated in the absence of sperm and consequently ES cell lines derived from such embryos can theoretically be genetically homozygous since their genetic material is derived exclusively from the donor oocytes [138]. However, failure of independent segregation of sister chromatids during meiosis can results in recombination leading to heterozygosity. Nevertheless, homozygosity should be obtainable for specific loci including HLA. Parthenogenetic ES cell lines have been derived in mouse [137] and non-human primates [139], but efforts to produce human parthenogenetic ES cell lines had been unsuccessful until the publication of the SCNT-hES-1 line. The recent derivation of 6 human ES cell lines from a human blastocyst of parthenogenetic origin confirms the feasibility of such a method [140]. Collectively, these results suggest that a bank of human ES cell lines homozygous for HLA-type could be developed in a short period of time by taking advantage of parthenogenetic human ES cells.

Acknowledgements

This work was supported by grants from the Wellcome Trust and JDRF. A.S.B. was supported by an FCT studentship.

References

[1] A.M. Shapiro, J.R. Lakey, E.A. Ryan, G.S. Korbutt, E. Toth and G.L. Warnock et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med 343 (2000), pp. 230–238. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (2190)

[2] E.A. Ryan, B.W. Paty, P.A. Senior, D. Bigam, E. Alfadhli and N.M. Kneteman et al., Five-year follow-up after clinical islet transplantation, Diabetes 54 (2005), pp. 2060–2069. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (494)

[3] A.M. Shapiro, C. Ricordi, B.J. Hering, H. Auchincloss, R. Lindblad and R.P. Robertson et al., International trial of the Edmonton protocol for islet transplantation, N Engl J Med 355 (2006), pp. 1318–1330. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (412)

[4] A. Colman, Making new beta cells from stem cells, Semin Cell Dev Biol 15 (2004), pp. 337–345. Abstract | View Record in Scopus | Cited By in Scopus (17)

[5] M. Stoffel, L. Vallier and R.A. Pedersen, Navigating the pathway from embryonic stem cells to beta cells, Semin Cell Dev Biol 15 (2004), pp. 327–336. Abstract | View Record in Scopus | Cited By in Scopus (22)

[6] C.J. Burns, S.J. Persaud and P.M. Jones, Stem cell therapy for diabetes: do we need to make beta cells?, J Endocrinol 183 (2004), pp. 437–443. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (21)

[7] E. Roche, R. Ensenat-Waser, J.A. Reig, J. Jones, T. Leon-Quinto and B. Soria, Therapeutic potential of stem cells in diabetes, Handb Exp Pharmacol 174 (2006), pp. 147–167. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (5)

[8] A. Santana, R. Ensenat-Waser, M.I. Arribas, J.A. Reig and E. Roche, Insulin-producing cells derived from stem cells: recent progress and future directions, J Cell Mol Med 10 (2006), pp. 866–883. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (20)

[9] P. Lu, F. Liu, L. Yan, T. Peng, T. Liu and Z. Yao et al., Stem cells therapy for type 1 diabetes, Diabetes Res Clin Pract 78 (2007), pp. 1–7. Article | PDF (139 K) | View Record in Scopus | Cited By in Scopus (8)

[10] C.W. Hay and K. Docherty, Comparative analysis of insulin gene promoters: implications for diabetes research, Diabetes 55 (2006), pp. 3201–3213. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (39)

[11] H. Kojima, M. Fujimiya, T. Terashima, H. Kimura and L. Chan, Extrapancreatic proinsulin/insulin-expressing cells in diabetes mellitus: is history repeating itself?, Endocr J 53 (2006), pp. 715–722. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (6)

[12] N. Lifson, C.V. Lassa and P.K. Dixit, Relation between blood flow and morphology in islet organ of rat pancreas, Am J Physiol 249 (1985), pp. E43–E48. View Record in Scopus | Cited By in Scopus (26)

[13] E. Samols, J.I. Stagner, R.B. Ewart and V. Marks, The order of islet microvascular cellular perfusion is B–A–D in the perfused rat pancreas, J Clin Invest 82 (1988), pp. 350–353. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (86)

[14] M. Brissova, M.J. Fowler, W.E. Nicholson, A. Chu, B. Hirshberg and D.M. Harlan et al., Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy, J Histochem Cytochem 53 (2005), pp. 1087–1097. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (74)

[15] O. Cabrera, D.M. Berman, N.S. Kenyon, C. Ricordi, P.O. Berggren and A. Caicedo, The unique cytoarchitecture of human pancreatic islets has implications for islet cell function, Proc Natl Acad Sci U S A 103 (2006), pp. 2334–2339. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (135)

[16] J. Ferrer, C. Benito and R. Gomis, Pancreatic islet GLUT2 glucose transporter mRNA and protein expression in humans with and without NIDDM, Diabetes 44 (1995), pp. 1369–1374. View Record in Scopus | Cited By in Scopus (31)

[17] A. De Vos, H. Heimberg, E. Quartier, P. Huypens, L. Bouwens and D. Pipeleers et al., Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression, J Clin Invest 96 (1995), pp. 2489–2495. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (121)

[18] A.E. Butler, J. Janson, W.C. Soeller and P.C. Butler, Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formation rather than direct action of amyloid, Diabetes 52 (2003), pp. 2304–2314. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (128)

[19] Y. Dor, J. Brown, O.I. Martinez and D.A. Melton, Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation, Nature 429 (2004), pp. 41–46. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (798)

[20] A.E. Butler, J. Janson, S. Bonner-Weir, R. Ritzel, R.A. Rizza and P.C. Butler, Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes, Diabetes 52 (2003), pp. 102–110. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1004)

[21] D.G. Pipeleers, M. Pipeleers-Marichal, J.C. Hannaert, M. Berghmans, P.A. In’t Veld and J. Rozing et al., Transplantation of purified islet cells in diabetic rats. I. Standardization of islet cell grafts, Diabetes 40 (1991), pp. 908–919. View Record in Scopus | Cited By in Scopus (34)

[22] P.A. Halban, S.E. Kahn, A. Lernmark and C.J. Rhodes, Gene and cell-replacement therapy in the treatment of type 1 diabetes: how high must the standards be set?, Diabetes 10 (2001), pp. 2181–2191. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (59)

[23] M.J. Evans and M.H. Kaufman, Establishment in culture of pluripotential cells from mouse embryos, Nature 292 (1981), pp. 154–156. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (2210)

[24] G.R. Martin, Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells, Proc Natl Acad Sci U S A 78 (1981), pp. 7634–7638. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1581)

[25] B. Soria, E. Roche, G. Berna, T. Leon-Quinto, J.A. Reig and F. Martin, Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice 39, Diabetes 49 (2000), pp. 157–162. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (486)

[26] N. Lumelsky, O. Blondel, P. Laeng, I. Velasco, R. Ravin and R. McKay, Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets 1, Science 292 (2001), pp. 1389–1394. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (809)

[27] Y. Hori, I.C. Rulifson, B.C. Tsai, J.J. Heit, J.D. Cahoy and S.K. Kim, Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells, Proc Natl Acad Sci U S A 99 (2002), pp. 16105–16110. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (254)

[28] A. Shiroi, M. Yoshikawa, H. Yokota, H. Fukui, S. Ishizaka and K. Tatsumi et al., Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone, Stem Cells 20 (2002), pp. 284–292. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (107)

[29] P. Blyszczuk, J. Czyz, G. Kania, M. Wagner, U. Roll and L. St Onge et al., Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells, Proc Natl Acad Sci U S A 100 (2003), pp. 998–1003. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (254)

[30] B.W. Kahan, L.M. Jacobson, D.A. Hullett, J.M. Ochoada, T.D. Oberley and K.M. Lang et al., Pancreatic precursors and differentiated islet cell types from murine embryonic stem cells: an in vitro model to study islet differentiation, Diabetes 52 (2003), pp. 2016–2024. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (75)

[31] Y. Moritoh, E. Yamato, Y. Yasui, S. Miyazaki and J. Miyazaki, Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells, Diabetes 52 (2003), pp. 1163–1168. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (107)

[32] N. Houard, G.G. Rousseau and F.P. Lemaigre, HNF-6-independent differentiation of mouse embryonic stem cells into insulin-producing cells, Diabetologia 46 (2003), pp. 378–385. View Record in Scopus | Cited By in Scopus (20)

[33] D. Kim, Y. Gu, M. Ishii, M. Fujimiya, M. Qi and N. Nakamura et al., In vivo functioning and transplantable mature pancreatic islet-like cell clusters differentiated from embryonic stem cell, Pancreas 27 (2003), pp. E34–E41.

[34] H.M. Milne, C.J. Burns, I. Kitsou-Mylona, M.J. Luther, S.L. Minger and S.J. Persaud et al., Generation of insulin-expressing cells from mouse embryonic stem cells, Biochem Biophys Res Commun 328 (2005), pp. 399–403. Article | PDF (355 K) | View Record in Scopus | Cited By in Scopus (15)

[35] S.H. Lee, N. Lumelsky, L. Studer, J.M. Auerbach and R.D. McKay, Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells 2, Nat Biotechnol 18 (2000), pp. 675–679. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (682)

[36] U. Lendahl, L.B. Zimmerman and R.D. McKay, CNS stem cells express a new class of intermediate filament protein, Cell 60 (1990), pp. 585–595. Abstract | View Record in Scopus | Cited By in Scopus (1583)

[37] H. Zulewski, E.J. Abraham, M.J. Gerlach, P.B. Daniel, W. Moritz and B. Muller et al., Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes 1, Diabetes 50 (2001), pp. 521–533. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (489)

[38] E. Hunziker and M. Stein, Nestin-expressing cells in the pancreatic islets of Langerhans, Biochem Biophys Res Commun 271 (2000), pp. 116–119. Abstract | View Record in Scopus | Cited By in Scopus (121)

[39] H. Edlund, Developmental biology of the pancreas 1, Diabetes 50 (Suppl. 1) (2001), pp. S5–S9. View Record in Scopus | Cited By in Scopus (66)

[40] A.G. Pearse, The APUD concept and hormone production, Clin Endocrinol Metab 9 (1980), pp. 211–222. Abstract | View Record in Scopus | Cited By in Scopus (44)

[41] J. Rajagopal, W.J. Anderson, S. Kume, O.I. Martinez and D.A. Melton, Insulin staining of ES cell progeny from insulin uptake, Science 299 (2003), p. 363. View Record in Scopus | Cited By in Scopus (289)

[42] M. Hansson, A. Tonning, U. Frandsen, A. Petri, J. Rajagopal and M.C. Englund et al., Artifactual insulin release from differentiated embryonic stem cells, Diabetes 53 (2004), pp. 2603–2609. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (120)

[43] H.J. Paek, L.J. Moise, J.R. Morgan and M.J. Lysaght, Origin of insulin secreted from islet-like cell clusters derived from murine embryonic stem cells, Cloning Stem Cells 7 (2005), pp. 226–231. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (6)

[44] S. Sipione, A. Eshpeter, J.G. Lyon, G.S. Korbutt and R.C. Bleackley, Insulin expressing cells from differentiated embryonic stem cells are not beta cells, Diabetologia 47 (2004), pp. 499–508. Full Text via CrossRef

[45] G. Kania, P. Blyszczuk and A.M. Wobus, The generation of insulin-producing cells from embryonic stem cells—a discussion of controversial findings, Int J Dev Biol 48 (2004), pp. 1061–1064. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (27)

[46] E. Roche, P. Sepulcre, J.A. Reig, A. Santana and B. Soria, Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells, FASEB J 19 (2005), pp. 1341–1343. View Record in Scopus | Cited By in Scopus (18)

[47] P. Gadue, T.L. Huber, M.C. Nostro, S. Kattman and G.M. Keller, Germ layer induction from embryonic stem cells, Exp Hematol 33 (2005), pp. 955–964. Article | PDF (250 K) | View Record in Scopus | Cited By in Scopus (31)

[48] A. Rodaway and R. Patient, Mesendoderm. An ancient germ layer?, Cell 105 (2001), pp. 169–172. Article | PDF (172 K) | View Record in Scopus | Cited By in Scopus (60)

[49] D.Y. Stainier, A glimpse into the molecular entrails of endoderm formation, Genes Dev 16 (2002), pp. 893–907. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (85)

[50] M. Kanai-Azuma, Y. Kanai, J.M. Gad, Y. Tajima, C. Taya and M. Kurohmaru et al., Depletion of definitive gut endoderm in Sox17-null mutant mice, Development 129 (2002), pp. 2367–2379. View Record in Scopus | Cited By in Scopus (206)

[51] A. Grapin-Botton and D.A. Melton, Endoderm development: from patterning to organogenesis, Trends Genet 16 (2000), pp. 124–130. Abstract | View Record in Scopus | Cited By in Scopus (150)

[52] M.E. Wilson, D. Scheel and M.S. German, Gene expression cascades in pancreatic development, Mech Dev 120 (2003), pp. 65–80. Article | PDF (329 K) | View Record in Scopus | Cited By in Scopus (190)

[53] P. Collombat, J. Hecksher-Sorensen, P. Serup and A. Mansouri, Specifying pancreatic endocrine cell fates, Mech Dev 123 (2006), pp. 501–512. Article | PDF (511 K) | View Record in Scopus | Cited By in Scopus (47)

[54] R.L. Pictet, W.R. Clark, R.H. Williams and W.J. Rutter, An ultrastructural analysis of the developing embryonic pancreas, Dev Biol 29 (1972), pp. 436–467. Abstract | View Record in Scopus | Cited By in Scopus (164)

[55] J.M. Servitja and J. Ferrer, Transcriptional networks controlling pancreatic development and beta cell function, Diabetologia 47 (2004), pp. 597–613. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (96)

[56] J. Jonsson, U. Ahlgren, T. Edlund and H. Edlund, IPF1, a homeodomain protein with a dual function in pancreas development 4, Int J Dev Biol 39 (1995), pp. 789–798. View Record in Scopus | Cited By in Scopus (49)

[57] M.F. Offield, T.L. Jetton, P.A. Labosky, M. Ray, R.W. Stein and M.A. Magnuson et al., PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum 2, Development 122 (1996), pp. 983–995. View Record in Scopus | Cited By in Scopus (763)

[58] J. Lau, H. Kawahira and M. Hebrok, Hedgehog signaling in pancreas development and disease, Cell Mol Life Sci 63 (2006), pp. 642–652. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (39)

[59] A. Krapp, M. Knofler, B. Ledermann, K. Burki, C. Berney and N. Zoerkler et al., The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas 4, Genes Dev 12 (1998), pp. 3752–3763. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (241)

[60] K.A. Harrison, J. Thaler, S.L. Pfaff, H. Gu and J.H. Kehrl, Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice 2, Nat Genet 23 (1999), pp. 71–75. View Record in Scopus | Cited By in Scopus (174)

[61] H. Li, S. Arber, T.M. Jessell and H. Edlund, Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9 2, Nat Genet 23 (1999), pp. 67–70. View Record in Scopus | Cited By in Scopus (188)

[62] Y. Kawaguchi, B. Cooper, M. Gannon, M. Ray, R.J. MacDonald and C.V. Wright, The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors, Nat Genet 32 (2002), pp. 128–134. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (265)

[63] B. Peers, S. Sharma, T. Johnson, M. Kamps and M. Montminy, The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain, Mol Cell Biol 15 (1995), pp. 7091–7097. View Record in Scopus | Cited By in Scopus (130)

[64] S. Dutta, M. Gannon, B. Peers, C. Wright, S. Bonner-Weir and M. Montminy, PDX:PBX complexes are required for normal proliferation of pancreatic cells during development, Proc Natl Acad Sci U S A 98 (2001), pp. 1065–1070. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (86)

[65] S.K. Kim, L. Selleri, J.S. Lee, A.Y. Zhang, X. Gu and Y. Jacobs et al., Pbx1 inactivation disrupts pancreas development and in Ipf1-deficient mice promotes diabetes mellitus, Nat Genet 30 (2002), pp. 430–435. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (84)

[66] A. Bhushan, N. Itoh, S. Kato, J.P. Thiery, P. Czernichow and S. Bellusci et al., Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis, Development 128 (2001), pp. 5109–5117. View Record in Scopus | Cited By in Scopus (182)

[67] A. Hart, S. Papadopoulou and H. Edlund, Fgf10 maintains notch activation, stimulates proliferation, and blocks differentiation of pancreatic epithelial cells, Dev Dyn 228 (2003), pp. 185–193. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (80)

[68] J. Jensen, E.E. Pedersen, P. Galante, J. Hald, R.S. Heller and M. Ishibashi et al., Control of endodermal endocrine development by Hes-1, Nat Genet 24 (2000), pp. 36–44. View Record in Scopus | Cited By in Scopus (486)

[69] G. Gradwohl, A. Dierich, M. LeMeur and F. Guillemot, Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas, Proc Natl Acad Sci U S A 97 (2000), pp. 1607–1611. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (526)

[70] V.M. Schwitzgebel, D.W. Scheel, J.R. Conners, J. Kalamaras, J.E. Lee and D.J. Anderson et al., Expression of neurogenin3 reveals an islet cell precursor population in the pancreas, Development 127 (2000), pp. 3533–3542. View Record in Scopus | Cited By in Scopus (314)

[71] K. Piper, S.G. Ball, J.W. Keeling, S. Mansoor, D.I. Wilson and N.A. Hanley, Novel SOX9 expression during human pancreas development correlates to abnormalities in Campomelic dysplasia, Mech Dev 116 (2002), pp. 223–226. Article | PDF (461 K) | View Record in Scopus | Cited By in Scopus (25)

[72] P.A. Seymour, K.K. Freude, M.N. Tran, E.E. Mayes, J. Jensen and R. Kist et al., SOX9 is required for maintenance of the pancreatic progenitor cell pool, Proc Natl Acad Sci U S A 104 (2007), pp. 1865–1870. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (91)

[73] M.S. Lan, Q. Li, J. Lu, W.S. Modi and A.L. Notkins, Genomic organization, 5′-upstream sequence, and chromosomal localization of an insulinoma-associated intronless gene, IA-1, J Biol Chem 269 (1994), pp. 14170–14174. View Record in Scopus | Cited By in Scopus (15)

[74] G. Mellitzer, S. Bonne, R.F. Luco, M. Van De Casteele, N. Lenne-Samuel and P. Collombat et al., IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas, EMBO J 25 (2006), pp. 1344–1352. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (59)

[75] J.E. Lee, S.M. Hollenberg, L. Snider, D.L. Turner, N. Lipnick and H. Weintraub, Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix–loop–helix protein, Science 268 (1995), pp. 836–844. View Record in Scopus | Cited By in Scopus (706)

[76] F.J. Naya, C.M. Stellrecht and M.J. Tsai, Tissue-specific regulation of the insulin gene by a novel basic helix–loop–helix transcription factor, Genes Dev 9 (1995), pp. 1009–1019. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (394)

[77] L. Zhao, M. Guo, T.A. Matsuoka, D.K. Hagman, S.D. Parazzoli and V. Poitout et al., The islet beta cell-enriched MafA activator is a key regulator of insulin gene transcription, J Biol Chem 280 (2005), pp. 11887–11894. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (61)

[78] W. Nishimura, T. Kondo, T. Salameh, I. El Khattabi, R. Dodge and S. Bonner-Weir et al., A switch from MafB to MafA expression accompanies differentiation to pancreatic beta-cells, Dev Biol 293 (2006), pp. 526–539. Article | PDF (1347 K) | View Record in Scopus | Cited By in Scopus (70)

[79] I. Artner, B. Blanchi, J.C. Raum, M. Guo, T. Kaneko and S. Cordes et al., MafB is required for islet beta cell maturation, Proc Natl Acad Sci U S A 104 (2007), pp. 3853–3858. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (43)

[80] G. Keller, Embryonic stem cell differentiation: emergence of a new era in biology and medicine, Genes Dev 19 (2005), pp. 1129–1155. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (374)

[81] E.S. Ng, L. Azzola, K. Sourris, L. Robb, E.G. Stanley and A.G. Elefanty, The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4-induced ventral mesoderm patterning in differentiating ES cells, Development 132 (2005), pp. 873–884. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (52)

[82] J.K. Pedersen, S.B. Nelson, M.C. Jorgensen, K.D. Henseleit, Y. Fujitani and C.V. Wright et al., Endodermal expression of Nkx6 genes depends differentially on Pdx1, Dev Biol 288 (2005), pp. 487–501. Article | PDF (1757 K) | View Record in Scopus | Cited By in Scopus (32)

[83] H.J. Fehling, G. Lacaud, A. Kubo, M. Kennedy, S. Robertson and G. Keller et al., Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation, Development 130 (2003), pp. 4217–4227. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (174)

[84] S. Tada, T. Era, C. Furusawa, H. Sakurai, S. Nishikawa and M. Kinoshita et al., Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture, Development 132 (2005), pp. 4363–4374. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (140)

[85] S.J. Micallef, M.E. Janes, K. Knezevic, R.P. Davis, A.G. Elefanty and E.G. Stanley, Retinoic acid induces Pdx1-positive endoderm in differentiating mouse embryonic stem cells, Diabetes 54 (2005), pp. 301–305. View Record in Scopus | Cited By in Scopus (54)

[86] N. Shiraki, C.J. Lai, Y. Hishikari and S. Kume, TGF-beta signaling potentiates differentiation of embryonic stem cells to Pdx-1 expressing endodermal cells, Genes Cells 10 (2005), pp. 503–516. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (15)

[87] A. Kubo, K. Shinozaki, J.M. Shannon, V. Kouskoff, M. Kennedy and S. Woo et al., Development of definitive endoderm from embryonic stem cells in culture, Development 131 (2004), pp. 1651–1662. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (264)

[88] M. Yasunaga, S. Tada, S. Torikai-Nishikawa, Y. Nakano, M. Okada and L.M. Jakt et al., Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells, Nat Biotechnol 23 (2005), pp. 1542–1550. View Record in Scopus | Cited By in Scopus (152)

[89] K.A. D’Amour, A.D. Agulnick, S. Eliazer, O.G. Kelly, E. Kroon and E.E. Baetge, Efficient differentiation of human embryonic stem cells to definitive endoderm, Nat Biotechnol 23 (2005), pp. 1534–1541.

[90] S. Assady, G. Maor, M. Amit, J. Itskovitz-Eldor, K.L. Skorecki and M. Tzukerman, Insulin production by human embryonic stem cells, Diabetes 50 (2001), pp. 1691–1697. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (468)

[91] H. Segev, B. Fishman, A. Ziskind, M. Shulman and J. Itskovitz-Eldor, Differentiation of human embryonic stem cells into insulin-producing clusters, Stem Cells 22 (2004), pp. 265–274. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (168)

[92] G.K. Brolen, N. Heins, J. Edsbagge and H. Semb, Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing beta-cell-like cells, Diabetes 54 (2005), pp. 2867–2874. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (69)

[93] K.A. D’Amour, A.G. Bang, S. Eliazer, O.G. Kelly, A.D. Agulnick and N.G. Smart et al., Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells, Nat Biotechnol 24 (2006), pp. 1392–1401.

[94] O.D. Madsen and P. Serup, Towards cell therapy for diabetes, Nat Biotechnol 24 (2006), pp. 1481–1483. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (20)

[95] W. Jiang, Y. Shi, D. Zhao, S. Chen, J. Yong and J. Zhang et al., In vitro derivation of functional insulin-producing cells from human embryonic stem cells, Cell Res 17 (2007), pp. 333–344. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (62)

[96] J. Jiang, M. Au, K. Lu, A. Eshpeter, G. Korbutt and G. Fisk et al., Generation of insulin-producing islet-like clusters from human embryonic stem cells, Stem Cells 25 (2007), pp. 1940–1953. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (103)

[97] O. Adewumi et al., Characterization of human embryonic stem cell lines by the International Stem Cell Initiative, Nat Biotechnol 25 (2007), pp. 803–816. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (218)

[98] D.E. Baker, N.J. Harrison, E. Maltby, K. Smith, H.D. Moore and P.J. Shaw et al., Adaptation to culture of human embryonic stem cells and oncogenesis in vivo, Nat Biotechnol 25 (2007), pp. 207–215. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (109)

[99] C. Allegrucci, Y.Z. Wu, A. Thurston, C.N. Denning, H. Priddle and C.L. Mummery et al., Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome, Hum Mol Genet 16 (2007), pp. 1253–1268. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (53)

[100] L. Vallier, M. Alexander and R.A. Pedersen, Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells, J Cell Sci 118 (2005), pp. 4495–4509. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (200)

[101] S. Yao, S. Chen, J. Clark, E. Hao, G.M. Beattie and A. Hayek et al., Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions, Proc Natl Acad Sci U S A 103 (2006), pp. 6907–6912. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (136)

[102] T.E. Ludwig, M.E. Levenstein, J.M. Jones, W.T. Berggren, E.R. Mitchen and J.L. Frane et al., Derivation of human embryonic stem cells in defined conditions, Nat Biotechnol 24 (2006), pp. 185–187. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (299)

[103] J. Lu, R. Hou, C.J. Booth, S.H. Yang and M. Snyder, Defined culture conditions of human embryonic stem cells, Proc Natl Acad Sci U S A 103 (2006), pp. 5688–5693. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (73)

[104] R. Maunoury, S. Robine, E. Pringault, C. Huet, J.L. Guenet and J.A. Gaillard et al., Villin expression in the visceral endoderm and in the gut anlage during early mouse embryogenesis, EMBO J 7 (1988), pp. 3321–3329. View Record in Scopus | Cited By in Scopus (28)

[105] T. Fujikawa, S.H. Oh, L. Pi, H.M. Hatch, T. Shupe and B.E. Petersen, Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells, Am J Pathol 166 (2005), pp. 1781–1791. View Record in Scopus | Cited By in Scopus (127)

[106] M.J. Doyle and L. Sussel, Nkx2.2 regulates beta-cell function in the mature islet, Diabetes 56 (2007), pp. 1999–2007. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (11)

[107] M. Kyba, R.C. Perlingeiro and G.Q. Daley, HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors, Cell 109 (2002), pp. 29–37. Article | PDF (1418 K) | View Record in Scopus | Cited By in Scopus (310)

[108] J. Antonchuk, G. Sauvageau and R.K. Humphries, HOXB4-induced expansion of adult hematopoietic stem cells ex vivo, Cell 109 (2002), pp. 39–45. Article | PDF (316 K) | View Record in Scopus | Cited By in Scopus (319)

[109] S. Amsellem, F. Pflumio, D. Bardinet, B. Izac, P. Charneau and P.H. Romeo et al., Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein, Nat Med 9 (2003), pp. 1423–1427. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (109)

[110] J. Krosl, P. Austin, N. Beslu, E. Kroon, R.K. Humphries and G. Sauvageau, In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein, Nat Med 9 (2003), pp. 1428–1432. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (138)

[111] J. Dominguez-Bendala, D. Klein, M. Ribeiro, C. Ricordi, L. Inverardi and R. Pastori et al., TAT-mediated neurogenin 3 protein transduction stimulates pancreatic endocrine differentiation in vitro, Diabetes 54 (2005), pp. 720–726. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (40)

[112] G. Nikolova, N. Jabs, I. Konstantinova, A. Domogatskaya, K. Tryggvason and L. Sorokin et al., The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation, Dev Cell 10 (2006), pp. 397–405. Article | PDF (732 K) | View Record in Scopus | Cited By in Scopus (98)

[113] K. Filipsson, M. Kvist-Reimer and B. Ahren, The neuropeptide pituitary adenylate cyclase-activating polypeptide and islet function, Diabetes 50 (2001), pp. 1959–1969. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (46)

[114] M. Drukker and N. Benvenisty, The immunogenicity of human embryonic stem-derived cells, Trends Biotechnol 22 (2004), pp. 136–141. Article | PDF (154 K) | View Record in Scopus | Cited By in Scopus (86)

[115] G.S. Korbutt, A.G. Mallett, Z. Ao, M. Flashner and R.V. Rajotte, Improved survival of microencapsulated islets during in vitro culture and enhanced metabolic function following transplantation, Diabetologia 47 (2004), pp. 1810–1818. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (23)

[116] R. Calafiore, G. Basta, G. Luca, A. Lemmi, L. Racanicchi and F. Mancuso et al., Standard technical procedures for microencapsulation of human islets for graft into nonimmunosuppressed patients with type 1 diabetes mellitus, Transplant Proc 38 (2006), pp. 1156–1157. Article | PDF (61 K) | View Record in Scopus | Cited By in Scopus (24)

[117] M. Drukker, Immunogenicity of human embryonic stem cells: can we achieve tolerance?, Springer Semin Immunopathol 26 (2004), pp. 201–213. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (20)

[118] I. Pree, N. Pilat and T. Wekerle, Recent progress in tolerance induction through mixed chimerism, Int Arch Allergy Immunol 144 (2007), pp. 254–266. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (9)

[119] A.E. Morelli and A.W. Thomson, Tolerogenic dendritic cells and the quest for transplant tolerance, Nat Rev Immunol 7 (2007), pp. 610–621. View Record in Scopus | Cited By in Scopus (165)

[120] D. Cyranoski, Hwang takes the stand at fraud trial, Nature 444 (2006), pp. 12–13. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1)

[121] Z. Han, C.A. Vandevoort and K.E. Latham, Therapeutic cloning: status and prospects, Curr Opin Mol Ther 9 (2007), pp. 392–397. View Record in Scopus | Cited By in Scopus (4)

[122] W.M. Rideout III, K. Hochedlinger, M. Kyba, G.Q. Daley and R. Jaenisch, Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy, Cell 109 (2002), pp. 17–27. Article | PDF (422 K) | View Record in Scopus | Cited By in Scopus (328)

[123] C. Simerly, T. Dominko, C. Navara, C. Payne, S. Capuano and G. Gosman et al., Molecular correlates of primate nuclear transfer failures, Science 300 (2003), p. 297. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (136)

[124] M.C. Lavoir, J. Weier, J. Conaghan and R.A. Pedersen, Poor development of human nuclear transfer embryos using failed fertilized oocytes, Reprod Biomed Online 11 (2005), pp. 740–744. Abstract | PDF (196 K) | View Record in Scopus | Cited By in Scopus (31)

[125] S.M. Mitalipov, Q. Zhou, J.A. Byrne, W.Z. Ji, R.B. Norgren and D.P. Wolf, Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling, Hum Reprod 22 (2007), pp. 2232–2242. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (24)

[126] N. Geijsen, M. Horoschak, K. Kim, J. Gribnau, K. Eggan and G.Q. Daley, Derivation of embryonic germ cells and male gametes from embryonic stem cells, Nature 427 (2004), pp. 148–154. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (305)

[127] D. Egli, J. Rosains, G. Birkhoff and K. Eggan, Developmental reprogramming after chromosome transfer into mitotic mouse zygotes, Nature 447 (2007), pp. 679–685. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (88)

[128] I. Wilmut, A.E. Schnieke, J. McWhir, A.J. Kind and K.H. Campbell, Viable offspring derived from fetal and adult mammalian cells, Nature 385 (1997), pp. 810–813. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (2126)

[129] K. Takahashi and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell 126 (2006), pp. 663–676. Article | PDF (1266 K) | View Record in Scopus | Cited By in Scopus (2069)

[130] K. Okita, T. Ichisaka and S. Yamanaka, Generation of germline-competent induced pluripotent stem cells, Nature 448 (2007), pp. 313–317. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (904)

[131] M. Wernig, A. Meissner, R. Foreman, T. Brambrink, M. Ku and K. Hochedlinger et al., In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state, Nature 448 (2007), pp. 318–324. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (802)

[132] L.A. Boyer, T.I. Lee, M.F. Cole, S.E. Johnstone, S.S. Levine and J.P. Zucker et al., Core transcriptional regulatory circuitry in human embryonic stem cells, Cell 122 (2005), pp. 947–956. Article | PDF (477 K) | View Record in Scopus | Cited By in Scopus (954)

[133] Y.H. Loh, Q. Wu, J.L. Chew, V.B. Vega, W. Zhang and X. Chen et al., The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells, Nat Genet 38 (2006), pp. 431–440. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (581)

[134] I.G. Brons, L.E. Smithers, M.W. Trotter, P. Rugg-Gunn, B. Sun and S.M. Chuva de Sousa Lopes et al., Derivation of pluripotent epiblast stem cells from mammalian embryos, Nature 448 (2007), pp. 191–195. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (214)

[135] P.J. Tesar, J.G. Chenoweth, F.A. Brook, T.J. Davies, E.P. Evans and D.L. Mack et al., New cell lines from mouse epiblast share defining features with human embryonic stem cells, Nature 448 (2007), pp. 196–199. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (241)

[136] C.J. Taylor, E.M. Bolton, S. Pocock, L.D. Sharples, R.A. Pedersen and J.A. Bradley, Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching, Lancet 366 (2005), pp. 2019–2025. Article | PDF (136 K) | View Record in Scopus | Cited By in Scopus (122)

[137] K. Kim, P. Lerou, A. Yabuuchi, C. Lengerke, K. Ng and J. West et al., Histocompatible embryonic stem cells by parthenogenesis, Science 315 (2007), pp. 482–486. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (71)

[138] J.B. Cibelli, K. Cunniff and K.E. Vrana, Embryonic stem cells from parthenotes, Methods Enzymol 418 (2006), pp. 117–135. Abstract | View Record in Scopus | Cited By in Scopus (18)

[139] K.E. Vrana, J.D. Hipp, A.M. Goss, B.A. McCool, D.R. Riddle and S.J. Walker et al., Nonhuman primate parthenogenetic stem cells, Proc Natl Acad Sci U S A 100 (Suppl. 1) (2003), pp. 11911–11916. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (94)

[140] E.S. Revazova, N.A. Turovets, O.D. Kochetkova, L.B. Kindarova, L.N. Kuzmichev and J.D. Janus et al., Patient-specific stem cell lines derived from human parthenogenetic blastocysts, Cloning Stem Cells (June) (2007).

[141] B.W. Phillips, H. Hannes, W.L. Rust, C. Qi-Ping, H. Chipperfield and T. Ee-Kim et al., Directed differentiation of human embryonic stem cells into the pancreatic endocrine lineage, Stem Cells and Development 16 (2007), pp. 561–578. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (40)