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EMBRYONIC STEM CELLS |
Department of Genetics, The Institute of Life Sciences, The Hebrew University, Jerusalem, Israel
Key Words. Embryonic stem cells • Endoderm • Pancreas • Development • Genetic manipulation
Correspondence: Nissim Benvenisty, M.D., Ph.D., Department of Genetics, The Hebrew University, Jerusalem 91904, Israel. Telephone: 972-2-6586774; Fax: 972-2-6584972; e-mail: nissimb{at}mail.ls.huji.ac.il
Received August 17, 2005;
accepted for publication April 28, 2006.
First published online in STEM CELLS EXPRESS May 4, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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, ß, 
, and pancreatic polypeptide cells, secreting glucagon, insulin, somatostatin, and pancreatic polypeptide (PPY), respectively. In the mature pancreas, these cells form clusters that comprise the islets of Langerhans. Many transcription factors are involved in the development of the pancreatic cell lineages (reviewed in [2]). In humans at 8 weeks of embryonic development, insulin-containing cells appear, and they coexpress glucagon and somatostatin [3]. The majority of the insulin-containing cells stain negative for the other pancreatic hormones after 9 weeks of embryonic development, which is regarded as a sign of maturation [3]. Human embryonic stem cells (HESCs) are pluripotent cells capable of differentiating into the three embryonic germ layers, that is, ectoderm, mesoderm, and endoderm. The pluripotency of HESCs has been proven both in vivo and in vitro [4–6]. HESCs injected in vivo into immune-deficient mice generate teratomas that are composed of derivatives of all three embryonic germ layers [4, 6]. In vitro, aggregation of HESCs brings about the formation of embryoid bodies (EBs) expressing molecular markers specific to the three embryonic germ layers [5]. Since the differentiation of HESCs is spontaneous, it needs to be directed in order to enrich a specific subset of cells. The ability to induce specific differentiation was demonstrated upon the addition of various growth factors to differentiating HESCs [7]. The isolation of a desired population of differentiated cells was lately demonstrated by genetic labeling of hepatic-like cells derived from HESCs. The hepatic-like cells were labeled by a reporter gene, controlled by hepatic promoter, and sorted from the heterogeneous population of cells using fluorescence-activated cell sorter (FACS) [8].
Differentiation of HESCs into insulin-producing cells was demonstrated by spontaneous differentiation in adherent or suspension culture conditions and by a stepwise protocol of various culture conditions [9, 10]. In mouse embryonic stem cells, there are also several reports of insulin-producing cells by spontaneous and directed differentiation (reviewed in [11]). The conclusions obtained from these experiments are controversial because it was shown that immunostaining for the insulin protein in culture is misleading, since the insulin from the growth media adheres to the cells, and insulin may not be produced by the cells themselves [12, 13].
In this study, by using genetic manipulation methodologies, we have analyzed the potential of HESCs to differentiate into the various cell lineages comprising the pancreas. By introducing a reporter gene under the control of the pancreatic and duodenal homeobox factor-1 (Pdx1) promoter, we could trace putative pancreatic precursor cells during the differentiation of HESCs into EBs. Next, we constitutively expressed either the endodermal transcription factor forkhead box A2 (Foxa2) or Pdx1 in HESCs and examined their pancreatic differentiation. In culture, HESCs overexpressing Pdx1 differentiate into cells expressing pancreatic markers earlier than the control cells. Moreover, we show that further differentiation can be achieved in teratomas derived from the Pdx1- or Foxa2-expressing cells.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was extracted as described [14], and 1 µg of RNA was reverse-transcribed by random hexamer priming using EZ-First Strand cDNA Synthesis Kit (Biological Industries, Kibbutz Beit Haemek, Israel, http://www.bioind.com). cDNA samples were subjected to polymerase chain reaction (PCR) amplification with human-specific primers from different exons. All reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed under nonsaturating conditions. PCR conditions include a first step of 3 minutes at 94°C; a second step of 20–35 cycles of 30 seconds at 94°C, 30 seconds (annealing step) at 55°C–62°C, and 45 seconds at 72°C; and a final step of 5 minutes at 72°C. Primer sequences and the size of the final products are described in Table 1.
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Plasmid Construction
INS-eGFP expression vector was constructed by deleting the cytomegalovirus promoter sequence from peGFP-NI (basic-eGFP) (Clontech, Franklin Lakes, NJ, http://www.clontech.com) [15] and inserting a 410-base pair rat insulin 1 (NM_019129
[GenBank]
) minimal promoter sequence [16] into the BamHI and HindIII restriction sites. The construct contained an SV40-driven neomycin selectable marker, which confers resistance to G418 antibiotics. PDX1-eGFP expression vector was established by inserting 4.5 kilobases (kb) of the mouse Pdx1 promoter (NM_008814
[GenBank]
) [17] into the Sal1 restriction site of basic-eGFP. Actin-PDX1 was constructed by insertion of mouse Pdx1 coding region, 0.86 kb long (NM_008814
[GenBank]
) [18], into pCAGGS and adding the SV40-driven neomycin selectable marker. PGK-FOXA2 plasmid was based on the plasmid pCA1037 containing the complete rat cDNA sequence of Foxa2 (NM_012743
[GenBank]
), 2.2 kb long, under the control of phosphoglycerate kinase 1 (Pgk1) [19] and adding the SV40-driven neomycin selectable marker. Transfection and establishment of cell lines were performed as previously described [15].
Immunostaining
HESCs were washed three times with saline and fixed onto the plate with 4% paraformaldehyde. Either mouse anti-chick Foxa2, 1:50 (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/
dshbwww), or rabbit anti-human Pdx1, 1:200 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) were used as primary antibodies. As secondary antibodies, we used Cy-3-conjugated goat anti-mouse IgG (H+L), 1:200 (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com), or Cy-3-conjugated donkey anti-rabbit IgG (H+L), 1:200 (Jackson Immunoresearch Laboratories). The first antibody was incubated with the cells overnight at 4°C. The next morning, the cells were washed twice with saline for 5 minutes. Then, the cells were incubated with the second antibody for 1 hour at room temperature. The cells were washed as described above, and nucleus staining was performed by incubation with 1 µg/ml bis-benzimide, Hoechst 33258 (Sigma-Aldrich) for 10 minutes.
FACS Analysis
To analyze the cells by FACS, the HESCs and their differentiated derivatives were trypsinized, transferred to phosphate-buffered saline, and passed through 70-µm nylon mesh (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd. com). The negative control was nontransfected HESCs that were grown in the same conditions as the PDX/INS/ALB-eGFP cells. Each sample was analyzed twice to verify the results. Analysis was performed on FACSCalibur system (Becton, Dickinson and Company) according to green fluorescent emission, for detection of enhanced green fluorescent protein (eGFP)-positive cells. Analysis was performed by CellQuest software (Becton, Dickinson and Company). Forward and side scatter plots were used to exclude dead cells and debris from the histogram analysis plots.
Statistical Analysis
The significance of the differences in the expression of pancreatic markers between the wild-type (WT) cells and the cells overexpressing Pdx1 was tested using t test analysis. Differences with p < .05 are labeled with an asterisk (*).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Pdx1 is expressed early in the endocrine differentiation in pancreas progenitor cells and not only in differentiated ß cells. To discriminate between the pancreas progenitors and the ß cells, we labeled HESCs by eGFP under the control of the insulin promoter (INS-eGFP). Several clones of HESCs carrying the INS-eGFP plasmid were subjected to differentiation conditions through EBs and were examined for eGFP expression at different time points during the differentiation. We searched for eGFP-positive cells within the EBs using both fluorescent inverted and confocal microscopes. eGFP-positive cells could not be detected during the spontaneous differentiation of HESCs, even in late, 30-day-old EBs (Fig. 1A, VI). Insulinoma cells transformed with the INS-eGFP plasmid expressed the eGFP and served as a positive control (Fig. 1A, V). RNA extracted from the HESCs tested by RT-PCR showed low expression of PDX1 and no expression of insulin (data not shown).
Overexpression of Endoderm Transcription Factors in HESCs
The expression of insulin could not be detected at significant levels following spontaneous differentiation of HESCs through EBs, even by using the reporter gene. Hence, we hypothesized that we should look for specific conditions inducing the differentiation toward ß cells. We thus tested whether the overexpression of major transcription factors involved in pancreatic development might enhance the differentiation and enable us to obtain, in culture, insulin-expressing cells. We constitutively expressed two different transcription factors, Foxa2 and Pdx1, that are involved in different phases of early endoderm and pancreatic differentiation (Fig. 2A).
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Pdx1 is a major transcription factor involved in pancreatic development [21]. It is expressed downstream of Foxa2, and its expression in the endoderm lineage is enriched in pancreatic cells [1]. Pdx1 binds the insulin promoter together with other transcription factors, enabling the expression of insulin in the ß cells [21, 22]. The precise combination of many transcription factors (e.g., Pdx1, Pax4, and Nkx2.2) is unique to the ß cell, and the interaction among them activates transcription in a cooperative manner [22]. Pdx1 regulated by the ß actin promoter was introduced into HESCs, and several clones were isolated. These HESCs expressed the exogenous Pdx1, as we show by RT-PCR analysis and by immunostaining (Fig. 2C).
The Effect of Endodermal Transcription Factors on the Pancreatic Differentiation
HESCs expressing Foxa2 or Pdx1 were induced to differentiate via EBs and analyzed for the expression pattern of the pancreatic genes. RNA samples were taken from nondifferentiated HESCs, early EBs, mid Ebs, and late EBs. The expression profile of transcription factors involved in pancreatic development and the proteins produced by the various pancreatic cell types were compared between WT cells and the Foxa2 (Fig. 3B) or the Pdx1-overexpressing cells (Fig. 3A, 3B). This analysis revealed that although we had a high expression of Foxa2 in the cells, it did not significantly change the expression pattern of its downstream gene targets during the differentiation of HESCs. However, overall we saw a small induction in few genes; for example, PAX6 is induced in late EBs, and SST is induced in early EBs (Fig. 3B). Foxa2-expressing EBs express low levels of Pdx1. This is in contrast to the cells transfected with the Pdx1 expression vector that transcribe high levels of the Pdx1 gene. Comparison of the expression pattern of pancreatic genes between WT and Pdx1-overexpressing HESCs revealed induction toward pancreatic differentiation (Fig. 3B). The onset of expression of the transcription factor ISL1 in Pdx1 clones was prematurely induced compared to the WT cells. In Pdx1 clones, the ISL1 showed significantly higher levels of expression during the differentiation of HESCs into EBs. The transcription factors PAX4 and NGN3 and the marker for the endocrine polypeptide cells PPY showed higher expression in Pdx1 clones and were mainly induced in the early EBs. The transcription factor PAX6 showed a more prominent expression in the Pdx1 clones in mid- and late EBs. SST and AMY2B of endocrine cells and exocrine cells, respectively, showed a higher level of expression in early, mid-, and late EBs of Pdx1 clones. This induction was significant in several time points. Finally, the transcription factor NKX2.2 was expressed at low or viable levels but only in the Pdx1 clones and not in the WT cells. The overall trend is that the constitutive expression of Pdx1 in the HESCs promoted the expression of most transcription factors involved in the pancreas development and their downstream targets (Fig. 3A, 3B). However, Pdx1-overexpressing cells did not express insulin, and the expression of glucagon was turned off (Fig. 3B).
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, and exocrine cells. However, it was not sufficient to obtain insulin-expressing cells. Further enhancement of the differentiation is problematic since there are various transcription factors involved in the ß cell development, and they can all be candidates for simultaneous over expression. Hence, we decided to investigate whether the in vivo differentiation within teratomas can further differentiate the Pdx1-overexpressing HESCs to become insulin-expressing cells. We injected HESCs from two different Pdx1-overexpressing clones and two Foxa2-overexpressing clones into the kidney capsule of immunocompromised mice and tested the expression of several pancreatic markers in the teratomas. Most of the teratomas, WT and overexpressing Pdx1 or Foxa2 teratomas, expressed the exocrinic marker AMY2B, the pancreatic polypeptide cells marker PPY, and the cell marker somatostatin (Fig. 4). Three Pdx1 teratomas and one Foxa2 teratoma expressed the insulin gene. The transcription factor NKX2.2 was not detected in WT cells, but in three insulin-positive teratomas, we detected the expression of NKX2.2. PDX1 was detected in the teratomas overexpressing Pdx1 and not in WT (Fig. 4). We statistically analyzed the data and found that although the genes NKX2.2 and Insulin had higher expression level in Foxa2 teratomas than in WT teratomas, the results were not statistically significant (.05 < p < .1). Likewise, although the genes PDX1, NKX2.2, Insulin, and PPY had higher expression level in Pdx1 teratomas compared to WT teratomas, the results were not statistically significant (.05 < p < .15).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Thus, we tried to constitutively express transcription factors that play a major role in directing differentiation toward pancreatic cells. For this purpose, we introduced into HESCs either Foxa2 or Pdx1 and assessed their effect by examining the expression level of a panel of pancreatic markers during EB formation. In the cells constitutively expressing Foxa2, we almost could not detect better induction of the pancreatic genes compared to the WT cells. Pdx1, on the other hand, enhanced the differentiation of the HESCs toward the pancreatic cell types. The transcription factors promoting the pancreatic differentiation, which are expressed downstream of Pdx1, showed either premature or more prominent expression. The differentiation was mainly toward exocrine cells, although induced expression of markers for pancreatic polypeptide cells and endocrine cells was also noted. Still, expression of insulin, a marker for ß cells was absent. To test whether HESCs are able to differentiate into ß cells in vivo, we examined the expression of pancreatic markers during differentiation of HESCs into teratomas. We tested teratomas derived from WT cells and cells overexpressing Foxa2 or Pdx1. Only in teratomas were we able to detect mRNA transcript encoding for insulin. The differentiation of pancreatic cells relies on growth factors and also on interaction with adjacent cells [23]. Both of these regulations may be more pronounced in the teratomas than in the EBs. The supply of growth factors by the blood vessels may be critical for the ß cell differentiation in the teratoma. Candidates for such factors are members of the fibroblast growth factor and epidermal growth factor families. Moreover, the mere presence of blood vessels in the teratoma may be important for the pancreatic differentiation, as it was demonstrated that the vascular endothelium is critical for ß cell formation [24]. Future experiments should aim to characterize and isolate the insulin-producing cells either after their differentiation in vivo or in vitro. We conclude that the current culture conditions for differentiating HESCs in vitro into pancreatic cells are still lacking a factor or a combination of factors that exists, at least partially, in vivo. Further differentiation toward a fully matured phenotype might be possible by overexpressing a combination of various transcription factors, together with the identification of factor(s) supplied by the intact animal.
The endodermal progenitors of the various cell types of the pancreas express the transcription factor Pdx1. These cells are the source for the exocrinic and endocrinic cells. Later on, more transcription factors are temporally activated. Of the Pdx1-expressing cells, Ngn3 is expressed only in the cells that are oriented to the endocrinic pathway. In the endocrinic pathway, the expression of Pax6 is detected only in cells that finally become cells, whereas Pax4 is expressed in the progenitors of ß, , and pancreatic polypeptide cells. Nkx2.2 is then activated only in pancreatic ß cells and is followed by insulin expression [2]. During the spontaneous differentiation of HESCs, we detected the expression of the exocrine cells by AMY2B expression. We could also show the expression of glucagon, somatostatin, and PPY, which are expressed by , , and pancreatic polypeptide cells, respectively. Yet we could not detect the expression of NKX2.2 and its downstream target insulin, representing the ß pancreatic cells (Fig. 3). The constitutive expression of Pdx1 caused the differentiating HESCs to express NKX2.2, but it was not enough to cause for expression of insulin. On the other hand, the expression of glucagon was turned off during the differentiation of Pdx1-overexpressing cells in vitro. Since Pdx1 is required for maintaining the hormone-producing phenotype of ß cells by positively regulating insulin and by repressing glucagon in ß cells [21], we speculate that the elimination of glucagon expression is due to the high expression of Pdx1.
During embryonic development, Foxa2 is expressed early during the differentiation of the endodermal lineage [2]. We speculated that the expression of Foxa2 would promote the cascade of expression of transcription factors and effectively induce pancreatic differentiation. Our results show that in vitro rat Foxa2 had little effect on pancreatic differentiation of HESCs. The rat Foxa2 that we constitutively expressed in the HESCs is a close homologue to the human FOXA2 protein (showing amino acid conservation of 96.4% and almost full identify in the winged helix DNA-binding domain) [25]. We thus speculate that although Foxa2 is actively expressed in our cells, it is not sufficient to induce pancreatic differentiation. Hepatic differentiation, which occurs earlier than that of pancreatic differentiation during embryonic development, was previously shown to be induced by overexpression of Foxa2 in the murine ES cells [19]. Our conclusion is that pancreatic differentiation requires pancreatic-specific transcription factors, and induction of early endodermal factors is not sufficient.
We should also bear in mind that most of the markers used to define pancreatic differentiation are also expressed in other cell types. Since the differentiation is heterogeneous, we cannot rule out the possibility of expression due to other cell types. The transcription factors Isl1, Pax6, Ngn3, and Nkx2.2 are also involved in the differentiation of other cell types. Pax4 is the only pancreas-specific transcription factor. Hence, its expression during the spontaneous differentiation of HESCs reflects differentiation toward pancreatic cells.
The differentiation of HESCs into pancreatic ß cells is of major importance to understand the differentiation of the human pancreas and as a source of cells for therapy in diabetes. However, although the differentiation of HESCs was documented for multiple cell types, such as nerve cells and cardiomyocytes [5], the differentiation into ß cells was not trivial, and different groups reported different results or interpretations of the results (reviewed in [11]). We aimed at directing the differentiation using two transcription factors. Although differentiation into pancreatic cells was induced, it was not enough to achieve profound differentiation into ß cells in vitro. Differentiation into endoderm-derived tissues seems more difficult from differentiation into ectoderm-derived cells, either in mouse or human embryonic stem cells. But even differentiation into endodermal hepatic-like cells may be easier than that of pancreatic ß cells. During normal human embryonic development, pancreatic ß cells appear later than hepatic cells, and in our body we have more than 100 times more hepatocytes than ß cells. If differentiation of EBs in a way mimics normal development, it is conceivable that it will not be trivial to obtain these cell types in culture.
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DISCLOSURES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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