The Combined Expression of Pdx1 and MafA with Either Ngn3 or NeuroD Improves the Differentiation Efficiency of Mouse Embryonic Stem Cells into Insulin-Producing Cells

Abstract

The use of pancreatic β-cells differentiated from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells is a promising strategy in cell therapy. Pancreatic β-cell development is regulated by the sequential expression of a molecular network of transcription factors. In this experiment, we adopted a three-step differentiation protocol to differentiate mES (mouse ES) cells into insulin-secreting cells and overexpressed transcription factors by adenoviral vectors at various combinations at different time of differentiation. We found that the coexpression of Pdx1 and MafA with either Ngn3 or NeuroD, especially at the final stage of the three-step differentiation, significantly increased the differentiation efficiency. It also increased the glucose-stimulated insulin and C-peptide secretion in insulin-secreting cells derived from mES cells compared to the control green fluorescent protein (GFP) vector-transduced group. For the first time, we have demonstrated that the coexpression of Pdx1 and MafA during a specific time window of development can act synergistically with either Ngn3 or NeuroD to promote the differentiation of mES cells into insulin-secreting cells.

Keywords

Transcription factors Differentiation efficiency Embryonic stem cells Insulin-secreting cells

Introduction

Islet transplantation has been shown to reduce or abrogate the insulin requirement in type 1 diabetes; however, the scarcity of islet donors limits the propensity of this treatment modality (12, 17, 23, 26). Embryonic stem (ES) cells derived from the inner cell mass of the mammalian blastocyst stage embryo can differentiate into all three germ layer lineages in vitro and in vivo, thus providing a promising source of β-cells (7, 10, 30). In recent years, both mouse and human ES cells have been successfully induced to differentiate into insulin-secreting islet-like or β-like cells in vitro (14, 16, 18, 29, 32). The efficiency of ES cell differentiation models, however, remains low.

During vertebrate embryogenesis, the development of various cell lineages of the pancreas is regulated by the sequential expression of a molecular network of transcription factors (5, 35). Among them, pancreatic and duodenal homeobox 1 (Pdx1) is a key transcription factor involved in the early pancreas development as well as the differentiation and maturation of β-cells. During the development of the mouse pancreas, Pdx1 is initially expressed at around embryonic day E8.5, the expression is then reduced at about E9.5, and a second wave of expression occurs during β-cell differentiation and maturation (25). Neurogenin 3 (Ngn3) is crucial in determining the endocrine lineage fates of these precursor cells and is expressed only in premature endocrine cells (39). Neurogenic differentiation (NeuroD), a transcription factor that regulates insulin expression, is first expressed at about E9.5 and continues to be expressed in β-cells throughout their development (40). v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) is a recently discovered transcription factor that is specifically expressed in pancreatic β-cells and plays an important role in the maintenance of β-cell function (19, 24).

The overexpression of some of the key transcription factors involved in pancreas development, such as paired box gene 4 (Pax4) (4), Pdx1 (28), Nkx2.2 (33), and NeuroD (22), has been shown to promote the differentiation of ES cells into insulin-producing cells. Interestingly, the conditional expression of Pdx1 or Ngn3 is more effective in inducing ES cells into insulin-secreting cells (3, 38). Given the complexity of these molecular networks, however, it seems unlikely that the sequential expression of various transcription factors can be efficiently triggered by a single transcription factor during β-cell development in vitro. Additionally, the timing of expression of these factors may also be crucial to mimic the natural processes that occur during embryogenesis.

In this study, we adopted a three-step induction method to differentiate mES cells into insulin-secreting cells. Through the ectopic expression of the key transcription factors Pdx1, Ngn3, NeuroD, and MafA in multiple combinations during distinct differentiation stages of ES cells, we found that the coexpression of Pdx1 and MafA with either Ngn3 or NeuroD during the early differentiation (step 2) and late differentiation (step 3) steps increased insulin expression, insulin, and C-peptide release in a glucose-dependent manner, especially at the third stage of differentiation, which the increase of insulin-producing cells was apparent.

Materials and Methods

ES Cell Culture and Differentiation

mES cell line E14Tg2a (CRL-1821, ATCC) (37) was cultured in knockout D-MEM (Invitrogen) supplemented with 15% fetal calf serum (FCS, Millipore), 2 mM l-glutamine, nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μM β-mercaptoethanol (all from Invitrogen), and 1,000 U/ml of leukemia inhibitory factor (LIF, Millipore) on feeder cells treated with mytomycin C (Sigma).

We used a three-step approach adopted from published protocols (15, 32) to induce mES cells toward insulin-producing cell fates. The protocol was as follows:

Step 1: To induce the formation of embryoid bodies (EBs), mES cells were dissociated with trypsin and suspended in Petri dishes (NUNC) with mES cell medium without LIF. After 48 h, EBs were collected without being dissociated and directly replated with serum free X-VIVO™ 10 medium (Lonza) onto 1% Matrigel-coated dishes (BD Biosciences). Two hours later, EBs began to spread onto the dishes and were cultured in the same medium with 100 ng/ml activin A (Sigma) for 2 days. The differentiated EBs were then cultured in 2% fetal bovine serum (FBS)/DMEM with 2 mM retinoid acid (RA, Sigma) and 1% insulin–transferrin–selenium (ITS, Invitrogen) for 1 day.

Step 2: To expand insulin-producing precursor cells, the differentiated cells from step 1 were cultured in 10% FBS/ DMEM (low glucose, Invitrogen) with 10 ng/ml basic fibroblast growth factor (bFGF, Sigma), 20 ng/ml epidermal growth factor (EGF, Sigma), and 1% ITS for 5 days.

Step 3: To induce the maturation of insulin-producing cells, the expanded cells in step 2 were switched to DMEM/F12 with N2 supplement, B27 supplement (all from Gibco-BRL), 10 ng/ml bFGF, 1% ITS, and 10 mM nicotinamide (Sigma) and cultured for 5 days.

Differentiated cells were infected with adenoviral particles carrying the appropriate transcription factors during step 2 or step 3.

Adenovirus Construction

The adenoviral vectors pAd nGFP, pAd Pdx1-I-nGFP, pAd NeuroD-I-nGFP, pAd MafA-I-nGFP, and pAd Ngn3-I-nGFP, which were constructed in Prof. Douglas A. Melton's lab and obtained from Addgene (41). The adenoviral vectors were packaged and amplified in 293A cells. The virus titration was assessed by TCID₅₀, and the number of p.f.u. ml⁻¹ was derived from 0.69 × TCID₅₀ as described (11). The virus titration was about 10⁹–10¹⁰ p.f.u. ml⁻¹. The packaged virus particles were stored at −80°C as previously described (13). The viral particles were used to transduce differentiated mES cells grown on Matrigel-coated dishes. The infection efficiency of cells was about 20–30% as determined by the numbers of GFP-positive cells.

RT-PCR and Real-Time PCR Analysis

Total RNA from ES cells and differentiated cells were extracted in TRIzol Reagent (Invitrogen), and cDNA were amplified by RT-PCR. The sequences of the gene primer sets are available upon request.

Immunofluorescence Assay

The differentiated cells growing on glass slips were fixed with 4% paraformaldehyde (PFA, Sigma) and washed with PBS. They were then treated with 0.1% TritonX-100 (Sigma) in PBS for 15 min, washed with PBS, and then blocked with 3% bovine serum albumin (BSA, Sigma) at room temperature for 30 min. Cells were incubated with the primary antibodies or corresponding IgG (Jackson) overnight at 4°C. The information of the primary antibodies was as follows: goat anti-sex-determining region Y-box 17 (Sox17; 1:200, R&D), rabbit anti-forkhead box A2 (FoxA2; 1:500, Millipore), rabbit anti-Pdx1 (1:100, Cell Signaling), mouse anti-Nkx6.1 (1:200, DSHB, Developmental Studies Hybridoma Bank, Iowa, USA), mouse anti-insulin (1:200, Invitrogen), rabbit anti-C-peptide (1:200, Cell Signaling).

Upon completion of thrice washings, signals were detected by the corresponding secondary antibodies, Alexa Fluor^® 546 goat anti-rabbit IgG (Invitrogen), Alexa Fluor^® 546 goat anti-mouse IgG (Invitrogen), and rhodamine (TRITC)-conjugated mouse anti-goat IgG (Jackson). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI, Sigma) and visualized using an inverted fluorescence microscope.

ELISA Assay

The release of insulin and C-peptide by differentiated cells under glucose stress were tested. Having washed five times with PBS, cells were preincubated in the freshly prepared KRBH buffer [118 mM NaCl, 4.7 mM KCl, 1.1 mM KH₂PO₄, 25 mM NaHCO₃, 3.4 mM CaCl₂, 2.5 mM MgSO₄, 10 mM HEPES, and 1 mg/ml bovine serum albumin (BSA), pH 7.4] with 2.5 mM glucose at 37°C for 90 min. Cells were further cultured for an hour in the KRBH buffer containing glucose in either low or high concentration.

In the assay of insulin release, the cells were cultured in Krebs–Ringer phosphate HEPES (KRPH) buffer containing glucose of either 5.5 or 27.7 mM. The insulin concentrations in the spent media were measured by using the mouse Insulin ELISA kit (Millipore). In the C-peptide release assay, the spent media from cultures supplemented with 3 or 16.8 mM glucose were analyzed by using mouse C-peptide ELISA kit (Yanaihara Institute, Inc., Japan), The total protein content was assayed by using the BCA protein assay kit (Pierce).

Statistical Analysis

All values are shown as means ± SD. To determine the significance between groups, comparison was made using ANOVA test or Student's t test adjusted by Bonferroni correction. For all statistical tests, the 0.05 confidence level was considered statistically significant.

Results

Differentiation of mES Cells Into Insulin-Secreting Cells Using a Three-Step Approach

We adopted a three-step approach to induce the differentiation of mES cells toward an insulin-secreting cell fate (Fig. 1A). First, mES cells in suspension cultures were spontaneously differentiated into EBs, which then were replated onto dishes precoated with 1% Matrigel. EBs were initially induced by 100 ng/ml activin A in X-VIVO 10 medium and then by 2 mM RA, 1% ITS and 2% FBS in DMEM. In the second stage, the medium was changed to low glucose DMEM supplemented with 10% FBS, 10 ng/ ml bFGF, 20 ng/ml EGF, and 1% ITS, which were noted to enhance the production of insulin-secreting precursors. Figure 1B showed the appearance of the cell clusters. In the third stage, the cell clusters were cultured in DMEM/ F12 medium containing 10 ng/ml bFGF, 1% ITS, 1% N2B27 supplement, and 10 mM nicotinamide, which promoted the maturation of pancreatic β-cells as evident of the small islet-like cluster structures (Fig. 1C).

Figure 1.

Protocol of mouse embryonic stem (mES) cell differentiation, phase-contrast images of the cell morphology during induction and RT-PCR for genes expressed by differentiated cells at the three stages of differentiation. (A) The three-stage protocol of mES cell differentiation into insulin-secreting cells is shown. (B) mES cell-derived cell clusters in the second stage of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) induction. Scale bar: 100 μm. (C) mES cell-derived islet-like cell clusters formed in the third stage of induction. Scale bar: 100 μm. (D) RT-PCR analysis of endoderm marker gene expression in the differentiated cells at the end of stage 1. Lane 1, cells were treated with activin A. Lane 2, cells were treated without activin A. BLK, reagent blank. (E) RT-PCR for genes expressed by differentiated cells with (+A) and without activin A (-A) induction at the stage 2 of differentiation. ES, mES cells; BLK, reagent blank. (F) RT-PCR for genes expressed by differentiated cells with (+A) and without activin A (-A) induction at the third stage of differentiation. Ctrl, positive control, Min 6 cells.

RT-PCR revealed the gene expression of Sox17 and FoxA2 derived from cell products harvested at the end of the first stage of cultures suggesting activin A enhanced the endoderm lineage commitment (Fig. 1D). In the second stage, the differentiated cells were noted to express pancreatic progenitor genes, Ngn3, Pdx1, NeuroD, Pax6, Pax4, Nkx2.2, and Isl1 (insulin gene enhancer protein) (Fig. 1E). In the final stage, the expressions of pancreatic β-cell genes, NeuroD, MafA, Nkx2.2, Nkx6.1, Isl1, Insulin1, Insulin2, Glut2 (glucose transporter type 2), and Iapp (islet amyloid polypeptide), were observed in the differentiated cell clusters (Fig. 1F). Besides, the gene expressions of pancreatic endocrine cells including glucagon of β-cells, PP of pancreatic polypeptide (PP) cells and somatostatin (SST) of β-cells, amylase and PTF1a of pancreatic exocrine cells were observed (Fig. 1F). Activin A-induced cells were noted to strongly express pancreatic β-cell genes.

Immunofluorescence staining demonstrated Sox17 and FoxA2 in activin A-induced cells at the first stage of differentiation cultures (Fig. 2A). In the second stage of differentiation cultures, the differentiated cells displayed the positivity of pancreatic progenitor marker Pdx1, but the less intense pancreatic β-cell marker Nkx6.1 (Fig. 2B). In the final stage of differentiation cultures, dual positivity of Pdx1 and Nkx6.1 were noted among cells (Fig. 2C). Besides, the derived cell clusters were positively stained for C-peptide and insulin (Fig. 2D and E). Taken together, the data suggest that the in vitro three-stage differentiation protocol might mimic the development of pancreatic β-cells.

Figure 2.

Immunofluorescence staining. Nuclei are counterstained with DAPI (blue). Scale bar: 50 μm. (A) Sox17 (red) and FoxA2 (green) on mES cell-derived cells at stage 1 of differentiation. Goat and Rabbit IgG were used as negative control. (B) Pdx1 (green) and Nkx6.1 (red) on derived cells at stage 2. Rabbit and mouse IgG were used as negative control. (C) Pdx1 (green) and NKx6.1 (red) on derived cells at stage 3. Min6 was used as positive control. (D) C-peptide (red). INS-1E and rabbit IgG were used positive control and negative control, respectively. (E) Insulin (red). INS-1E and mouse IgG were used as positive control and negative control, respectively.

Coexpressing Pdx1 and MafA with Either Ngn3 or NeuroD Increased the Transcription of the Insulin Gene

Differentiated cells at stage 3 were overexpressed with Pdx1, Ngn3, NeuroD, and MafA, either alone or in combination, by transduction using adenoviral vectors (Fig. 3A). Ectopic expressions of these transcription factors were confirmed by RT-PCR (Fig. 3B). Their relative levels of expression were quantified by real-time PCR.

Figure 3.

Expression of pancreatic marker genes in the differentiated cells derived from mES cells overexpressing transcription factors in step 3. (A) Schematic representation transcription factors alone or in combination. (B) RT-PCR analysis of the expression of pancreatic transcription factors in differentiated cells. (C) Real-time PCR analysis of Insulin1, Insulin2, Isl1, Iapp, Glut2, Nkx2.2, Nkx6.1, Glucagon, PP, SST, Amylase, and PTF1a mRNA levels. The level of mRNA in cells transduced with GFP was defined as 1. Data are shown as mean ± SD (n ≥ 3 experiments). Statistical significances were tested by ANOVA test (*p < 0.01, **p < 0.001, ***p < 0.0001). P, PDX1; Ng, Ngn3; ND, NeuroD; M, MafA.

There was an approximately threefold increase in the expression of Insulin1 in the two-factor combination groups (Pdx1 + Ngn3, Pdx1 + NeuroD, and Pdx1 + MafA) compared to that of the GFP control group (Fig. 3C). The expression of Insulin1 of differentiated cells transduced with three factors (Pdx1 + MafA + Ngn3 or Pdx1 + MafA + NeuroD) increased dramatically by more than 15-fold. The expression of Insulin2 of differentiated cells transduced with three factors increased significantly by more than eightfold (Fig. 3C). However, the levels of expressions of Insulin1 and Insulin2 in differentiated cells transduced with four factors were inferior to those derived from three-factor combination groups, indicating that Pdx1 and MafA could enhance the expression of Insulin genes synergistically with either Ngn3 or NeuroD.

Significantly elevated levels of expressions of Isl1, Iapp, Glut2, Nkx2.2, and Nkx6.2 in the three-factor combination groups were also evident (Fig. 3C). Readouts indicated that Pdx1 and MafA could also increase significantly the expression of pancreatic β genes synergistically with either Ngn3 or NeuroD. Interestingly, it was noted that the transduction of NeuroD was able to upregulate the expression of Glucagon to the level comparable to those derived from three-factor combination groups (Fig. 3C). Besides, either Pdx1 alone or in combination with MafA could increase significantly the expression of PP in differentiated cells (Fig. 3C). Apparently, there was no significantly difference in the expression of the pancreas exocrine genes, Amylase and PTF1a, derived from differentiated cells transduced with any of factors either alone or in combinations (Fig. 3C). Data suggested that the forced expression of Pdx1, Ngn3, and NeuroD in cells at the third stage of differentiation could promote pancreatic endocrine cell fate but not exocrine cell commitment.

We explored the optimal time frame to efficiently differentiate mES cells by repeating the gene transduction to cells at the stage 2 of the expansion of precursor cells (Fig. 4). Ectopic expressions of these transcription factors were confirmed by RT-PCR (Fig. 4A). The expression levels of Insulin1 in cells derived from the three-factor combination groups were more than five times higher than that of the GFP control group (Fig. 4B), but much lower than those observed in cells harvested at the third stage of cultures (Fig. 3C). Moreover, the expression level of Insulin1 was increased by two- to threefold in either single-factor or two-factor group. Readouts indicated that Pdx1 and MafA could not act effectively and synergistically with either Ngn3 or NeuroD to promote the expression of Insulin1 during the pancreatic precursor cell development. Insulin2 was slightly increased by approximately 0.5-fold. Alternately, the expressions of pancreatic endocrine genes including β-cell genes, Glucagon, PP, and SST were modestly higher in cells transduced with Pdx1, Ngn3, and NeuroD alone or in combination than that of GFP control (Fig. 4B). There were no significant differences in the levels of expressions of Amylase and PTF1a in cells transduced with either factors alone or in combinations, compared to the GFP control group (Fig. 4B).

Figure 4.

Expression of pancreatic genes in mES cell-derived cells overexpressing transcription factors in the second stage of differentiation. (A) RT-PCR for pancreatic transcription factors. (B) Real-time PCR for Insulin1, Insulin2, Isl1, Iapp, Glut2, Nkx2.2, Nkx6.1, Glucagon, PP, SST, Amylase, and PTF1a mRNA. The level of mRNA in cells transduced with GFP was defined as 1. Data are shown as mean ± SD (n ≥ 3 experiments). Statistical significance were tested by ANOVA test (*p < 0.01, **p < 0.001, ***p < 0.0001).

Readouts indicated that Pdx1, Ngn3, and NeuroD could promote the definitive endoderm differentiation towards the pancreatic endocrine cell fate. Indeed they are important in the development of pancreatic endocrine cells but less necessary for the development of exocrine cells, the coexpressing Pdx1 and MafA with either Ngn3 or NeuroD at the stage 2 of the expansion of progenitor cells appeared to be less effective compared to the gene transduction conducted in the third stage of differentiation.

Increased Insulin and C-Peptide Release by Differentiated Cells Transduced with Pdx1 and MafA with Either Ngn3 or NeuroD in Response to Glucose Stimulation

mES cell-derived cells harvested at the third stage of differentiation and transduced Pdx1 + MafA + Ngn3 or Pdx1 + MafA + NeuroD were functionally assayed for the release of C-peptide and insulin by using ELISA (Fig. 5). Irrespective of the gene transduction, differentiated cells were noted to release C-peptide and insulin in response to glucose stimulation. The three-factor transducted cells displayed significantly elevated levels of C-peptide and insulin compared to those of GFP control groups, especially when challenged at high concentrations of glucose (Fig. 5). Data indicated that the coexpression of Pdx1 and MafA with either Ngn3 or NeuroD could significantly enhance the differentiation of mES cells to C-peptide-and insulin-secreting cells.

Figure 5.

ELISA of Insulin and C-peptide release by mES cell-derived cells having undergone forced expression of either Pdx1 + MafA + Ngn3 or Pdx1 + MafA + NeuroD subjected to low and high stimulation of glucose. Data are the means ± SD of three independent experiments. Statistical significance was determined by Student's t tests (*p < 0.05). INS-1E was used as the positive control.

Discussion

ES cells are a potential alternative source for β-cell-based therapy for diabetes. The overexpression of key transcription factors has been shown to promote the differentiation of mES cells into insulin-secreting cells (4, 22); however, the efficiency of this process remained low. Based on the understanding of the transcriptional regulation on pancreatic β-cell development, the overexpression of a single transcription factor is not efficient in triggering the sequential expression of the complex signaling network that guides pancreatic β-cell development.

In this study, we systematically screened multiple combinations of transcription factors to optimize an induction method to derive, expand, and differentiate mES cells into insulin-secreting cells (Fig. 6). Previous experiments have shown that the preconditioning of ES cells using either Pdx1 or Ngn3 can effectively induce the formation of pancreatic β-cells. Based on these premises, we modified the methodology by coexpressing various transcription factors in differentiated cells at distinct stages of insulin-secreting cell induction (3, 38). To facilitate the sequential expression of the genes, we transiently expressed these transcription factors with adenoviral vectors that are not integrated into the chromosomes. Based on a three-stage approach, it was noted that coexpressing Pdx1 and MafA with either NeuroD or Ngn3 in derived cells at the third stage of maturation elicited the formation of cell clusters, which possessed a significant increase in efficiency in differentiation and release of insulin and C-peptide (Fig. 6). Aramata et al. have reported that the coexpression of Pdx1, MafA, and NeuroD in a hamster insulinoma cell line had a strong mutual effect on the activation of the human insulin gene promoter (2). A recent study also showed that the ectopic coexpression of Pdx1, MafA, and Ngn3 could reprogram acinar exocrine cells into insulin-secreting cells in vivo (41). Consistent with the studies, we found that coexpressing Pdx1 and MafA with either NeuroD or Ngn3 at the third stage of differentiation of mES cells has a synergistic effect on the expression of insulin. Besides, the coexpression of Pdx1 and MafA, especially in conjunction with either NeuroD or Ngn3, increased significantly the expression of pancreatic β-cell genes. It is suggestive that Pdx1 and MafA may act synergistically with either Ngn3 or NeuroD to promote the maturation of ES cell-derived insulin-secreting cells. Unexpectedly, the coexpression of the four factors did not further augment the levels of gene expressions of pancreatic β-cells. Further studies are needed to elucidate the underlying mechanism.

Figure 6.

Schema depicting stem cell technology in the three-stage induction of mES cells into insulin-producing cells by forced expression of transcription factors of Pdx1, MafA, Ngn3, and NeuroD. M1, one factors; M2, two factors; M3, three factors; M4, four factors (listed in Fig. 3).

Even though the coexpression of Pdx1 and MafA, with either NeuroD or Ngn3, in derived cells at both second and third stages of induction was able to increase the gene expression of insulin, the most dramatic results were observed when these factors were transduced to cells at the third stage of induction. It may be attributed to the signaling of the transcription factors in a time-dependent manner. The forced expression of Pdx1, Ngn3, or NeuroD, either alone or in combination, in derived cells at the second stage of induction, when pancreatic endocrine precursor cells were being formed, enhanced the gene expression level of pancreatic endocrine cells. These findings attest the important role of the three transcription factors in the development of pancreatic endocrine cells (3, 38).

It was noteworthy in this study that insulin and C-peptide released upon glucose stimulation by derived cells having undergone the transduction of three factors, Pdx1 + MafA + Ngn3 or Pdx1 + MafA + NeuroD, were not dramatically elevated as insulin mRNA. It was likely that these derived cells were not fully mature in terms of their secretory capacity. The observation is in line with other studies which demonstrated the immaturity of differentiated cells (1, 32). More efforts are needed to promote the terminal differentiation of β-cells with secretory functions (6, 8).

Recent studies indicated that the ectopic expression of Pdx1, MafA especially when it is coexpressed with either Ngn3 or NeuroD, initiated the trans-differentiation of non-β-cells into insulin-producing cells (9, 21, 27, 31, 34, 41). The studies suggested that Pdx1, MafA act synergistically with either Ngn3 or NeuroD to reprogram non-β-cells to insulin-producing cells. Although the transdifferentiation study provides an alternative means for β-cell replacement therapy, insulin-secreting cells derived from ES cells are still a superior approach in terms of the unlimited proliferation capability and the high differentiation potential of ES cells (20, 36, 42).

In conclusion, we have developed an effective strategy to induce mES cells to insulin-secreting cells by forced expression of transcription factors, Pdx1 and MafA with either Ngn3 or NeuroD. This strategy only partially mimics the time frame of natural development in which the regulatory network of the development of pancreatic β-cells are activated. Data of the study suggested that coexpressing Pdx1 and MafA with either Ngn3 or NeuroD in partially differentiated β-cell clusters can greatly augment the efficiency of differentiation of mES cells into functional β-cells. The strategy emphasizes the importance of the sequential expression of transcription factors at time of the development for the induction and differentiation of ES cells into insulin-secreting cells. Further investigations are required to improve the efficacy of the ES cell induction into insulin-secreting cells with fully secretory capability.

Footnotes

Acknowledgments

This study was supported by the Research Grant Committee (GRF 477008), the Hong Kong Foundation for Research and Development in Diabetes, and the Liao Wun Yuk Diabetes Memorial Fund of the Chinese University of Hong Kong. The authors declare no conflict of interest.

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