Directed differentiation of human embryonic stem cells towards a pancreatic cell fate
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The relative lack of successful pancreatic differentiation of human embryonic stem cells (hESCs) may suggest that directed differentiation of hESCs into definitive endoderm and subsequent commitment towards a pancreatic fate are not readily achieved. The aim of this study was to investigate whether sequential exposure of hESCs to epigenetic signals that mimic in vivo pancreatic development can efficiently generate pancreatic endodermal cells, and whether these cells can be further matured and reverse hyperglycaemia upon transplantation.
Materials and methods
The hESCs were sequentially treated with serum, activin and retinoic acid (RA) during embryoid body formation. The patterns of gene expression and protein production associated with embryonic germ layers and pancreatic endoderm were analysed by RT-PCR and immunostaining. The developmental competence and function of hESC-derived PDX1-positive cells were evaluated after in vivo transplantation.
Sequential treatment with serum, activin and RA highly upregulated the expression of the genes encoding forkhead box protein A2 (FOXA2), SRY-box containing gene 17 (SOX17), pancreatic and duodenal homeobox 1 (PDX1) and homeobox HB9 (HLXB9). The population of pancreatic endodermal cells that produced PDX1 was significantly increased at the expense of ectodermal differentiation, and a subset of the PDX1-positive cells also produced FOXA2, caudal-type homeobox transcription factor 2 (CDX2), and nestin (NES). After transplantation, the PDX1-positive cells further differentiated into mature cell types producing insulin and glucagon, resulting in amelioration of hyperglycaemia and weight loss in streptozotocin-treated diabetic mice.
Our strategy allows the progressive differentiation of hESCs into pancreatic endoderm capable of generating mature pancreatic cell types that function in vivo. These findings may establish the basis of further investigations for the purification of transplantable islet progenitors derived from hESCs.
KeywordsActivin Differentiation Embryoid bodies Endoderm Human embryonic stem cells Hyperglycaemia PDX1 Retinoic acid Serum
caudal type homeobox transcription factor 2
fibroblast growth factor
forkhead box protein A2
human embryonic stem cell
paired box protein 6
pancreatic and duodenal homeobox 1
Human embryonic stem cells (hESCs) have been proposed to be a limitless source of specific types of adult cells to be used in therapies for degenerative diseases. To date, while many studies have reported that hESCs could be efficiently induced to differentiate into mesodermal and ectodermal lineages, such as cardiomyocytes , endothelial cells , blood cells  and nerve cells , few reports have focused on the conversion of hESCs into endodermal lineages, particularly into pancreatic cell types [5, 6, 7, 8], which may provide a limitless stock of insulin-producing cells to treat type 1 diabetes. Moreover, despite increasing knowledge on pancreatic development, there is still debate regarding the origin of beta cells and the markers that can be used to identify their progenitors . Because hESCs are a useful in vitro system for lineage tracing, the development of strategies for the differentiation of hESCs into pancreatic cell types may hold the key to these questions. The relative lack of success in terms of pancreatic differentiation of hESCs, however, indicates that directed differentiation of hESCs into definitive endoderm and subsequent commitment towards a pancreatic fate are not readily achieved.
It has been shown that pancreatic cell types can be generated from mouse and non-human primate ESCs [10, 11, 12, 13, 14, 15, 16, 17, 18]. Based on these findings, recent studies have also suggested the potential of hESCs to differentiate into insulin-producing cells through spontaneous in vitro differentiation  or the use of a multi-stage protocol . However, several studies have claimed that insulin staining of ESC-derived cells is likely due to insulin uptake by apoptotic cells from the culture medium [19, 20, 21], suggesting that more reliable analyses should be considered in terms of beta cell physiology and from an embryological standpoint. To date, many studies using mouse ESCs or hESCs have focused mainly on a relatively late phase of differentiation, rather than addressing the dynamics of differentiation from ESCs into pancreatic endoderm. Unlike stem cells found in the adult body, hESCs are pluripotent and capable of differentiating into various cell types. Thus, early complex events leading to sequential decisions on cell fate are an essential prerequisite for pancreatic specification. In this context, it is therefore relevant to develop an efficient strategy for obtaining an enriched cell population of pancreatic endoderm as a first step for generating functional islet cell types.
More recently, the generation of pancreatic endodermal cells from hESCs was investigated based on fundamental concepts of early pancreatic specification under spontaneous differentiation  and by using chemically defined medium conditions plus activin A . In the present study we investigated whether efficient generation of the pancreatic endoderm can be achieved from hESCs by sequential treatment of developmentally relevant factors using an in vitro system to generate embryoid bodies (EBs).
Materials and methods
Cell culture and differentiation conditions
Maintenance of hESC lines (Miz-hES4 and Miz-hES6) and generation of human EBs (hEBs) were carried out as previously described . Pancreatic differentiation of Miz-hES6 cells was initiated by treating hEBs sequentially with serum, activin and all-trans retinoic acid (RA; Sigma, St Louis, MO, USA) during EB formation. The hEBs were cultured in the presence of 20% fetal bovine serum (Gibco, Grand Island, NY, USA) for the first 4 days. The serum-treated hEBs were then treated with 10–100 ng/ml activin A (R&D systems, Minneapolis, MN, USA) under serum-free conditions for the following 6 days to determine the optimal concentration of activin A for expression of the gene encoding pancreatic and duodenal homeobox 1 (PDX1). After optimisation of the activin concentration, the effects of RA (10 μmol/l) on pancreatic endodermal differentiation were examined by sequential treatment of hEBs with serum (20%) for 4 days, activin (30 ng/ml) for 4 days and RA for 2 days. The hEBs grown in suspension for 10 days were dissociated by mechanical trituration, and plated on to 35 mm tissue culture dishes (non-coated; Corning, Corning, NY, USA) at a density of 100–150 clusters per dish (500–800 cells/cluster) in insulin–transferrin–selenite (ITS) medium  containing fibronectin (5 μg/ml; Sigma). Over 10–13 days, cells that grew out of the clusters covered large areas of the culture dishes and formed spherical clusters of various sizes. The clusters were then harvested under a dissecting microscope (Stemi 2000; Zeiss, Gottingen, Germany) using a mouth pipette connected to a pulled glass capillary tube (Sigma), and were used for immunostaining and transplantation as described below. Both cell lines showed similar patterns of differentiation, and the data obtained from Miz-hES4 cells are presented in Electronic supplementary material (ESM) Fig. 1.
Semiquantitative RT-PCR analysis
Total RNA was isolated using Trizol (Invitrogen, Grand Island, NY, USA), and cDNA was synthesised from 2 μg of total RNA using the SuperScript III First-Strand Synthesis Kit (Invitrogen). PCR was subsequently carried out using AccuPower PCR-Premix (Bioneer, Daejeon, Korea). The primer sequences and reaction conditions used in this study are listed in ESM Table 1. Relative band intensities were determined using an image analyser (AutoChemi Bioimaging System; UVP, CA, USA). The levels of target mRNA were normalised to the signal obtained for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression.
Cells were fixed in 4% paraformaldehyde in PBS. The hEBs and human adult pancreatic tissues were fixed in 4% paraformaldehyde, transferred to 20% sucrose, frozen in O.C.T compound (Tissue Tek; Sakura, Tokyo, Japan) and cut into 10 μm-thick sections. Immunostaining was carried out using standard protocols, and alkaline phosphatase activity staining was performed with an Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA, USA). The primary antibodies used in the present study are listed in ESM Table 2, and additional methodological details are described in the ESM. Human pancreatic tissues were provided by the Research Institute for Medical Imaging, School of Medicine, Korea University, in accordance with the guidelines set by the Medical Research Ethical Committee (IRB No. MD0604).
Seven-week-old male nude BALB/c mice (Charles River, Wilmington, MA, USA) were treated with a single intraperitoneal injection of streptozotocin (Sigma; 200 mg/kg of body weight). After 6 days, animals were engrafted with 1 × 106 cells differentiated under different culture conditions or received a sham transplantation (10 μl of culture medium) in the left subcapsular renal space. Blood glucose levels were measured every 2–4 days between 09.00 and 11.00 h, under non-fasting conditions, with a portable glucometer (Accu-chek Active; Roche, Basel, Switzerland). Body weight was monitored every 2–4 days. Grafts were removed 4 weeks after transplantation and were analysed by immunohistochemistry or haematoxylin/eosin staining. All experimental procedures involving animals were approved by the Korean Stem Cell Research Centre.
Group means were analysed using the Student’s t test if only one comparison was made between two groups. Statistical differences between several groups were determined using one-way ANOVA followed by Tukey’s multiple comparison tests.
Differentiation of hESCs into endoderm
Differentiation of endoderm into PDX1+ pancreatic endoderm
We next attempted to examine the effects of activin A on the formation of pancreatic endoderm. Based on the finding that different concentrations of activin can induce the formation of different cell types, hEBs were stimulated for the first 4 days with serum and were subsequently further differentiated in the presence of different concentrations of activin for 6 days in serum-free conditions. Although the expression of PDX1, a marker of pancreatic endoderm, was significantly increased in the presence of 30, 50 and 100 ng/ml activin, the highest level of PDX1 expression was observed in hEBs grown with 30 ng/ml activin (Fig. 1c). The biphasic dose–response seen could be explained by the hypothesis that the expression of activin receptor subtypes in the cells are differentially regulated in the presence of high doses of activin, as previously reported in testicular tumour cells .
In mouse ESCs, day 4 of EB formation is thought to correspond to the end of gastrulation in mouse embryos . Based on the findings that RA signalling is necessary for pancreatic specification during this period, and that hESCs divide more slowly than mouse ESCs, we exposed hEBs pretreated with serum and activin (30 ng/ml) to RA (10 μmol/l) during the last 2 days (day 8–10) of hEB formation. Compared with control cultures (SF10) and cultures treated with serum and activin (S/A), the RA treatment (S/A/RA) significantly downregulated the expression of the neuroectodermal genes paired box gene 6 (PAX6) and SOX1 (Fig. 1d). In contrast, the expression of FOXA2 and SOX17 was significantly upregulated by RA treatment (Fig. 1d). Interestingly, we observed undetectable or very low levels of MIXL1 and T in the S/A/RA-treated hEBs, even though FOXA2 and SOX17 were expressed at high levels (Fig. 1d). Furthermore, together with a marked increase of PDX1 expression, RA treatment upregulated the expression of the gene encoding homeobox HB9 (HLXB9) (Fig. 1d), which is expressed in the early pancreatic bud prior to PDX1 expression.
Formation of cell clusters from PDX1+ endoderm
Differentiation of PDX1+ endoderm into pancreatic cell types in vivo
Twenty-eight days after transplantation, we removed the kidney bearing the grafts and found that removal of grafts from the mice transplanted with S/A/RA-derived cells resulted in the rapid return of hyperglycaemia, suggesting that the graft was responsible for the restoration of glucose levels (Fig. 5b). The removed grafts contained gut tube- and islet-like structures (Fig. 5d,e), suggesting endodermal differentiation in vivo. We also found numerous proinsulin-positive (ProINS+; Fig. 5f) and INS+ cells (Fig. 5g) in the grafts. Additionally, the INS+ cells were immunoreactive to an antibody against human nuclear antigen (Fig. 5h), indicating that these cells were derived from hESCs. INS+ cells in the grafts co-produced ProINS and C-peptide (Fig. 5i,j) and some of the INS+ cells were co-stained for glucagon (Fig. 5k–m), suggesting that these cells synthesise insulin but are still immature, as has been described previously . INS+PDX1+ cells were also often detected, suggesting the presence of more mature islet cells in the grafts (Fig. 5n). Very few INS+ cells, if any, were detected in the grafts of mice that were sham-operated or engrafted with cells derived under SF10 or S/A conditions (data not shown). No teratomas were observed in animals transplanted with cells, although additional long-term data are needed.
Our study demonstrates that sequential exposure to serum, activin and RA stimulates the progressive differentiation of hESCs into pancreatic endoderm capable of generating mature pancreatic cell types that function in vivo. Our results show that the expression of FOXA2, SOX17, MIXL1 and T is markedly increased by limited exposure to serum and activin. In mouse embryogenesis, the loss of FOXA2 or SOX17 activity results in the loss of definitive gut endoderm [29, 30], and FOXA2 is known to be a major upstream regulator of Pdx1 in mice [31, 32]. In Xenopus laevis, Mixer, a paired-like homeobox gene, has been identified as a downstream transcriptional target of Xbra, as well as an upstream factor of Sox17b . Additionally, a recent study has shown that ESCs lacking Mixl1, a murine Mix-like gene, were found to contribute poorly to gut endoderm . In these embryological contexts, our data showed that differentiation of endoderm or mesendoderm can be actively directed by exposing hEBs to serum and activin. Consistent with our study, a recent study using mouse ESCs  reported the positive effect of serum and activin on the differentiation of definitive endoderm. A more recent study using hESCs  also demonstrated that, under two-dimensional differentiation conditions, a population enriched with definitive endoderm can be generated by treatment with activin A at a low serum concentration (0.5%). Because we used a relatively high concentration (20%) of serum during EB formation (three-dimensional differentiation), the optimal range of serum concentrations for endodermal differentiation seems to vary depending on the culture conditions.
RA generated from lateral plate mesoderm has recently been reported to control Pdx1 expression in early pancreatic development in mice and zebrafish [36, 37, 38]. Our study demonstrated that RA treatment at the end of EB formation efficiently upregulated PDX1 expression, together with FOXA2 and SOX17 expression, while strongly downregulating the expression of MIXL1 and T, both of which were previously used as markers of the mesendodermal differentiation of ESCs . Although the exact mechanism underlying this phenomenon is not clear, we speculate that RA may facilitate the conversion of mesendoderm or endoderm into pancreatic endoderm in our system. This is further supported by our observation that RA increased the expression of HLXB9, which is expressed in the dorsal bud of the developing mouse pancreas prior to the onset of Pdx1 expression [39, 40]. However, we cannot rule out the possibility that RA simply increased the endodermal population at the expense of mesodermal differentiation, and the increased expression of PDX1 resulted from differentiation of the enriched endodermal population.
Importantly, RA treatment significantly increased the number of PDX1+ cells in hEBs, but decreased the number of T+ cells. Moreover, our data also demonstrate that, at the end of hEB formation, PDX1+ cells co-produced FOXA2 or CDX2. Although Cdx2 is known to be expressed in the mid- and hindgut, and also in the placenta, a recent study has shown that Cdx2 and Pdx1 exhibit spatio-temporal overlap of expression during mouse gut development . Taken together, our results suggest that RA may be a critical factor for the conversion of gut endoderm into pancreatic endoderm. Consistent with our findings, a recent report using mouse ESCs demonstrated the important role of RA in pancreatic differentiation, and also emphasised that the timing of RA treatment is crucial for the formation of Pdx1+ endoderm [17, 18].
Recently, several studies have demonstrated that NES is produced in human pancreatic progenitors and that insulin-producing cells can be derived from the NES+ cells in vitro [42, 43]. However, other reports have claimed that, in rodent pancreas, NES is not produced in the developing pancreatic epithelial cells , but is produced only in the adjacent mesenchymal cells  and pancreatic exocrine cell lineages . Our results have shown that almost all PDX1+ cells present at the end of hEB formation produce NES, but these cells later become heterogeneous (PDX1+NES+, PDX1−NES+, and PDX1+NES−). Similarly, a recent study using clonal analysis showed that NES+ populations from the postnatal rat pancreatic islet were heterogeneous, but a small proportion of the NES+ cells produced PDX1, and suggested that the NES+PDX1+ population may play a role in islet formation . However, it remains unclear whether the PDX1+ cells producing NES in our system gave rise to pancreatic endocrine cells, exocrine cells or other cell types after transplantation. Further investigations including lineage-tracing studies using additional markers or genes associated with human pancreatic endocrine cells will be required to address this question.
To date, only a few studies have suggested the potential of hESCs to differentiate into insulin-producing cells [5, 6, 7, 8]. It was previously reported that insulin-producing cells were found in EBs after spontaneous in vitro differentiation of hESCs . A more recent study using the spontaneous differentiation strategy without EB formation showed that differentiated hESCs containing PDX1+ progenitors were able to differentiate into cells expressing islet cell markers when co-transplanted with mouse fetal dorsal pancreas . Another study demonstrated that hESCs can be coaxed to differentiate into islet-like clusters by modifying the multi-stage method that was previously used to generate insulin-secreting clusters from mouse ESCs . However, it is not clear whether the ESCs followed normal developmental sequences in these studies due to a lack of initial specification using developmentally relevant factors known to regulate pancreatic endoderm development. It has recently been shown that treatment of feeder-free hESC colonies with activin directs the differentiation of hESCs into PDX1+ pancreatic endoderm, although the developmental competence of the PDX1+ endoderm was not characterised in this study . In our study, hESC-derived PDX1+ clusters were further differentiated into cells that produce insulin, proinsulin, C-peptide and glucagon upon transplantation. We also showed that the grafted cells ameliorated hyperglycaemia and weight loss, suggesting the competence of hESC-derived PDX1+ endoderm to differentiate into more mature islet cell types that function in vivo.
Although the results presented here suggest the potential use of hESCs in the treatment of diabetes, further studies are needed both in rodent and primate systems to address the long-term safety and efficacy of these cells. Inappropriate cells may be included along with PDX1+ cells upon transplantation. In particular, tumour formation is a problem associated with the grafting of differentiated ESCs. Under the conditions we used, no teratomas were seen in the graft, but long-term data are needed to show that ESC-derived cells do not form tumours.
In summary, our results show that the differentiation of pancreatic endoderm can be efficiently directed from hESCs in defined culture conditions corresponding to fundamental concepts of early pancreatic development. Our data also suggest that these cells appear to have the developmental competence to differentiate into mature pancreatic cell types that function in vivo. These findings are expected to facilitate further investigations for the purification of transplantable islet progenitors and the generation of large numbers of mature insulin-producing cells in vitro.
This work was supported by a Korea Research Foundation Grant funded by the Korean government (MOEHRD) (KRF-2004-205-E00030) and a grant (SC2050) from the Stem Cell Research Centre of the 21C Frontier R&D Programme funded by the Ministry of Science and Technology, Republic of Korea.
Conflict of interest statement
The authors declare that there is no duality of interest with regard to this study.