We have generated an embryonic stem (ES) cell line in which sequences encoding green fluorescent protein (GFP) were targeted to the locus of the pancreatic-duodenal homeobox gene (Pdx1). Analysis of chimeric embryos derived from blastocyst injection of Pdx1GFP/w ES cells demonstrated that the pattern of GFP expression was consistent with that reported for the endogenous Pdx1 gene. By monitoring GFP expression during the course of ES cell differentiation, we have shown that retinoic acid (RA) can regulate the commitment of ES cells to form Pdx1+ pancreatic endoderm. RA was most effective at inducing Pdx1 expression when added to cultures at day 4 of ES differentiation, a period corresponding to the end of gastrulation in the embryo. RT-PCR analysis showed that Pdx1-positive cells from day 8 cultures expressed the early endoderm markers Ptf1a, Foxa2, Hnf4α, Hnf1β, and Hnf6, consistent with the notion that they corresponded to the early pancreatic endoderm present in the embryonic day 9.5 mouse embryo. These results demonstrate the utility of Pdx1GFP/w ES cells as a tool for monitoring the effects of factors that influence pancreatic differentiation from ES cells.

Insulin-producing cells generated from in vitro–differentiated embryonic stem (ES) cells have been advanced as a potential alternative to cadaveric-derived pancreatic islets in transplantation therapies for treatment of type 1 diabetes. The development of protocols that facilitate the reliable and efficient derivation of such cells has been the focus of several studies over the last 5 years. Soria et al. (1) used a “cell-trapping” protocol to select for insulin-producing cells expressing the NeoR gene under the control of the human insulin gene promoter. This strategy was refined by placing the NeoR gene under the control of the promoter of Nkx6.1 (2), a gene found to be important in the development of cells from endocrine precursors. Taking into account the close evolutionary and developmental relationship between endocrine and neural cell lineages, Lumelsky et al. (3) developed a five-step protocol based on methods known to promote the generation of neural cell types from ES cells. Although the nature of insulin-staining cells derived by this method remains controversial (4,5), other groups have successfully used variations on this procedure to isolate similar cells from differentiating ES cells (6,7). Enforced expression of transcription factors with a role in pancreatic development has also been used to increase the frequency with which insulin-producing cells were isolated from differentiating ES cells (8,9).

We have taken an alternative approach to optimize the efficiency of the intermediate stages traversed by ES cells differentiating toward the pancreatic lineages. Pancreatic endocrine cells originate from definitive endoderm that expresses the pancreatic-duodenal homeobox gene (Pdx1) (10,11). In the absence of Pdx1, the pancreas fails to develop beyond the formation of ventral and dorsal buds (12,13). Thus, Pdx1 expression marks a critical step in pancreatic organogenesis, and Pdx1+ cells are likely to represent an obligate intermediate population in the generation of β-cells from ES cells. Therefore, we generated a reporter cell line by inserting the gene encoding green fluorescent protein (GFP) into exon 1 of the Pdx1 gene to facilitate the optimization of ES cell differentiation toward the pancreatic lineage. Using this cell line, we now show that retinoic acid (RA) promotes the generation of Pdx1+ cells that express a repertoire of genes indicative of early foregut endoderm.

Construction of targeted ES cells.

The Pdx1-GFP targeting vector comprised a 2.8-kb DNA fragment encompassing sequences upstream of the Pdx1 initiation codon, positioned 5′ of a cassette encoding GFP and a hygromycin resistance gene (HygroR) flanked by flp recombinase target sites. The 3.3-kb 3′ arm of the targeting vector encompassed sequences from an MluI site in exon 1 to an XbaI site immediately 5′ of exon 2. The targeting vector was electroporated into W9.5 ES cells, and targeted clones were identified by a PCR-based approach. Correctly targeted ES cells were transiently transfected with a vector encoding flp recombinase, and a clone of normal karyotype in which the HygroR cassette had been excised was characterized by Southern blot analysis.

Chimera analysis.

Chimeric embryos generated by injecting Pdx1GFP/w ES cells into C57BL/6 blastocysts were harvested at 7 and 10 days’ postimplantation and fixed in 4% paraformaldehyde on ice for 5 min. Images of embryos expressing GFP were captured with a Leica fluorescence microscope. This work involving animals was conducted in accordance with Monash University guidelines.

Cell culture.

ES cells were maintained on primary mouse embryonic fibroblasts in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum and 103 units/ml leukemia inhibitory factor (LIF). For differentiations, feeder-depleted ES cells were seeded at 10,000 cells/ml in 5 ml differentiation medium (14) in 6-cm Petri dishes (Phoenix Biomedical). For RA treatments, embryoid bodies (EBs) were harvested, washed once in PBS, and returned to Petri dishes in chemically defined medium (CDM) (15) supplemented with all-trans RA (2625; Sigma). The following day EBs were washed in PBS and a single EB picked into each well of a gelatin-coated 96-well tissue culture plate in CDM. Each EB was subsequently scored for GFP expression using a Zeiss Axiovert fluorescence microscope.

Gene expression analysis.

ES cells differentiated for 8 days were stained with an anti–E-cadherin (E-cad) antibody (13-1900; Chemicon), and GFP+E-cad+, GFPE-cad+, and GFPE-cad cells were isolated by flow cytometry using a FACSAria (BD Biosciences). cDNA was generated using a Cells-to-cDNA II (Ambion) kit and samples standardized essentially as described (16). For PCR analysis, the primer sequences and product sizes are listed in Table 1. Following an initial denaturation step of 95°C (2 min), PCRs were performed for 33 cycles with conditions of 95°C (30 s), 55° (30 s), and 72° (60 s) using High Fidelity Platinum Taq polymerase in the presence of 25 mmol/l MgSO4 and 200 μmol/l dNTPs in the buffer supplied (Invitrogen). PCR products were separated by electrophoresis on a 2% agarose gel.

Sequences encoding GFP were inserted into exon 1 of the Pdx1 locus in mouse ES cells using homologous recombination (Fig. 1A), and correct targeting was verified by Southern blotting (Fig. 1B). We examined the pattern of GFP expression in chimeric embryos derived by blastocyst injection of Pdx1GFP/w ES cells. Embryos that recovered at day 7 postimplantation (developmentally equivalent to embryonic day [E] 9.5) showed two areas of GFP expression (Fig. 1C) associated with the forming gut tube. This pattern of expression in prospective dorsal and ventral pancreatic buds was identical to that reported for the endogenous Pdx1 gene (17,18). Robust GFP fluorescence was observed in the dorsal and ventral pancreatic anlage, and lower levels were present in the duodenum of day 10 (developmentally E12.5) postimplantation embryos (Fig. 1D). GFP expression was not detected in other embryonic tissues.

Studies in zebrafish and Xenopus showed that the proportion of cells allocated to pancreatic endoderm could be increased by treating embryos with RA toward the end of gastrulation (19,20). Preliminary studies in our laboratory indicated that a 24-h pulse with RA was also able to induce GFP expression in differentiating Pdx1GFP/w ES cells and that continuous presence of RA was not required (data not shown). To determine the optimal concentration of RA in our system, ES cells differentiated for 4 days were treated with various concentrations of RA for 24 h and observed for subsequent GFP expression (Fig. 2A). The highest proportion of GFP+ EBs (>90%) formed when cultures were treated with 10−5 mol/l RA. This number peaked 3–4 days following RA treatment and decreased gradually over the next 7 days. To determine whether the time of treatment influenced the frequency of GFP+ EBs, cultures were pulsed with RA for 24 h between days 2 and 10 of differentiation. These experiments indicated that exposure of EBs to RA at day 4 yielded the highest percentage of GFP+ EBs (Fig. 2B). Examination of day 8 EBs from these cultures revealed that GFP expression was localized to epithelial structures (Fig. 2C), often in close proximity to cardiac mesoderm (data not shown).

Flow cytometric analysis indicated that GFP+ cells present in day 8 EBs treated with RA at day 4 coexpressed the epithelial marker E-cad (Fig. 3A). To determine the developmental stage represented by the GFP+ cells, RT-PCR analysis was performed on RNA from cells isolated on the basis of GFP and E-cad expression. The purity of the sorted populations was verified by RT-PCR analysis, showing that cells expressing Pdx1 and E-cad RNA were confined to GFP+ and E-cad+ fractions, respectively (Fig. 3B). This analysis indicated that the GFP+ population expressed endoderm markers including Foxa2, Hnf4α, Hnf6, and Hnf1β. Although these cells did not express genes associated with later pancreatic differentiation, the expression of Ptf1a suggests that a proportion of the GFP+ population was committed to pancreatic endoderm (21) (Fig. 3B).

The efficient differentiation of ES cells into β-cells will require the optimization of a series of steps corresponding to the sequential stages of pancreatic development (11). To facilitate the isolation of pancreatic endoderm from differentiating ES cells, we used gene targeting to insert the gene encoding GFP into the Pdx1 locus. Analysis of chimeric embryos generated with Pdx1GFP/w ES cells showed that the pattern of GFP expression mirrored that previously reported for Pdx1, and GFP+ cells isolated by flow cytometry expressed Pdx1 RNA. These data led us to conclude that GFP expression faithfully reported expression of the endogenous Pdx1 gene and therefore provided a reliable marker of cells within the pancreatic endoderm differentiation pathway. Consistent with findings of studies in zebrafish and Xenopus (19,20), our experiments show that Pdx1 expression was induced in differentiating ES cells treated with RA. Analysis of these Pdx1+ cells shows that they expressed a suite of transcription factor genes diagnostic of early foregut endoderm present in the mouse embryo between E8.5 and E9.5 (11). Time course analysis showed that GFP expression diminished by day 14 of differentiation, suggesting that additional factors were required to guide further pancreatic differentiation.

However, it is also possible that endogenous factors produced in our cultures actively repressed continuing pancreatic development. Experiments by Deutsch et al. (22) showed that ventral foregut endoderm adopted a default pathway of pancreatic commitment that could be diverted to Pdx1 hepatic endoderm by the proximity of cardiac mesoderm. Their results suggested that mesoderm-derived fibroblast growth factor (FGF) induced the local production of sonic hedgehog (Shh), a factor previously reported to repress Pdx1 expression in the dorsal foregut endoderm (23). However, although cardiac mesoderm was a prominent feature in our cultures, addition of either FGF2 or inhibitors of Shh signaling to day 8 GFP+ EBs did not modulate subsequent GFP expression (data not shown).

Our experiments show that RA can promote the formation of Pdx1+ foregut endoderm that coexpresses Ptf1a, a transcription factor indicative of pancreatic commitment (21). However, the absence of markers of further pancreatic differentiation, such as Ngn3, NeuroD, Nkx2.2, and Insulin, emphasize that these experiments describe only the first step in the development of a protocol for the differentiation of ES cells into pancreatic β-cells. Indeed, studies by Mandel et al. (24) demonstrated that pancreatic tissue from E12 fetal mice required 2 weeks of maturation in vitro before it could contribute to the regulation of blood glucose levels when transplanted into animals. In this context, the Pdx1+ cells characterized in this study are also likely to require further culture before they reach a developmental stage capable of regulating glucose levels in vivo. The definition of culture conditions that will facilitate such further development will form the basis of future work.

Posted on the World Wide Web at http://diabetes.diabetesjournals.org on 7 December 2004.

This work was supported by the Australian Stem Cell Centre, the Juvenile Diabetes Research Foundation, and the National Health and Medical Research Council (NHMRC) of Australia. A.G.E. is an NHMRC Senior Research Fellow.

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