ES and iPS cells could potentially overcome the chronic shortage of cadaveric organs if we are able to establish efficient differentiation protocols. Derivation of specific cell types and tissues from ES and iPS cells could end the need for donated cadaveric tissue. However, the current protocols for the in vitro differentiation of ES and iPS cells into IPCs are still very inefficient, time consuming and very expensive. Currently, there are two different approaches that allow the generation of IPCs using ES cells: (a) embryoid body (EB) formation  and (b) definitive endoderm (DE) formation . Here, we investigated the generation of IPCs using the EB formation approach. The EB represents an early differentiation stage that is characterized by the formation of a three-dimensional cluster of cells, which give rise to tissues representing all the three primary germ layers.
A number of differentiation protocols for the generation of IPCs using ES cells have been reported [14, 27, 36, 38, 41–49]. However, a major caveat is the lack of pancreatic β-cell-specific surface markers that allow purification of the IPCs. A second obstacle is that the ES cell differentiation procedures used are extremely inefficient. Consequently, in some of these studies, ES cells could be differentiated into IPCs but their transplantation in diabetic mice seldom led to a correction of the hyperglycemic state [27, 37, 38, 46]. Moreover, the insulin production by the ES cell-derived IPCs has been recently challenged and suggested to be rather an artifact because the cells could uptake insulin from the culture media and release it when they apoptose [50, 51], leading to false positive results.
The embryonic development of pancreatic β cells requires regulated expression of multiple transcription factors. However, Pdx1 has been shown to be the master regulator of the pancreas development and plays a crucial role during the development and function of pancreatic β cells [16, 17, 25, 28]. Recently, ectopic Pdx1 expression in bone marrow derived mesenchymal stromal cells, human ES cells and adipose tissue derived stem cells has been demonstrated to promote their differentiation into IPCs [26, 52–55]. One of the critical steps in the differentiation of ES cells into IPCs is the derivation of the Pdx1 expressing pancreatic progenitors. We, therefore, hypothesized that ectopic over-expression of Pdx1 in ES cells in vitro enforces lineage commitment and progressive differentiation of ES cells into IPCs. Here, we report an alternative approach for the efficient differentiation of murine ES cells into IPCs. The unique features about our differentiation strategy are the use of the pancreatic β-cell-specific transcription factor Pdx1 and the development of a new protocol that eliminates the nestin selection stage, thus reducing the overall differentiation time.
Here, we employed a multistep differentiation strategy to study and optimize the differentiation of mouse ES cells into IPCs. The R1Pdx1AcGFP/RIP-Luc ES cell line was subjected to in vitro differentiation using the EB formation protocol. We compared four different protocols with modifications post-EB formation for the directed differentiation of the R1Pdx1AcGFP/RIP-Luc ES cells into IPCs. The gene expression analysis of the ES cells undergoing differentiation into IPCs indicates a selective up-regulation of pancreatic β-cell-specific genes, including Pax4, Isl1, insulin 1, insulin 2 and PC2/3, thereby implying a lineage commitment towards β-cells. These findings support the view that ES cells can be coaxed to differentiate into physiologically responsive IPCs. Our immunostaining results indicate that the ES cells undergoing differentiation express Foxa2, Sox17, Ngn3 and NeuroD, as well as C-peptide. These results confirm that our differentiation protocol triggers a temporally regulated signaling cascade mediated by multiple pancreatic β-cell-specific transcription factors, which ultimately leads to the robust generation of IPCs. In some cases, the IPCs are typically arranged in the form of small pancreatic islet-like clusters.
Interestingly, despite ectopic Pdx1 expression in our R1Pdx1AcGFP/RIP-Luc ES cells, we found a significant number of glucagon-expressing cells in addition to the IPCs. Similar results were observed in transplanted diabetic mice following spontaneous in vivo differentiation of R1Pdx1AcGFP/RIP-Luc ES cell-derived PELCs . However, at present we do not fully understand the molecular mechanism underlying the generation of glucagon expressing cells in our differentiation studies. Our results are not entirely surprising because in an earlier study, inducible biphasic expression of Pdx1 led to the differentiation of mouse ES cells into both insulin and glucagon expressing cells . It is possible that sustained Pdx1 expression in our ES cells leads to the development of a bihormonal progenitor cell population during differentiation, which in turn gives rise to both insulin- and glucagon-expressing cells. These findings suggest that ectopic Pdx1 expression alone may not be sufficient to allow for the complete maturation of the IPCs in vitro. We, however, anticipate that these cells eventually mature in vivo post-transplantation and become mono-hormonal. In a recent study, adenoviral-mediated coexpression of Pdx1 and MafA with either Ngn3 or NeuroD has been shown to improve the generation of IPCs . However, the critical transplantation experiments to demonstrate the therapeutic efficacy of the ES cell-derived IPCs were not performed.
The insulin secretion, as well as glucose responsiveness, of the R1Pdx1AcGFP/RIP-Luc ES cell-derived IPCs was confirmed by an ultrasensitive ELISA. Our ELISA data suggest that constitutive Pdx1 expression in our R1Pdx1AcGFP/RIP-Luc ES cell-derived IPCs leads to a robust glucose responsive insulin secretion as compared to the biphasic Pdx1 expression or combined inducible Pdx1 and Ngn3 expression as reported earlier [26, 57]. It is possible that constitutive Pdx1 expression maintains the insulin processing and secretion mechanism in ES cell-derived IPCs in a dynamic state, thereby facilitating glucose responsive insulin secretion. Moreover, in these earlier studies, the much needed transplantation experiments to evaluate the therapeutic efficacy of ES cell-derived IPCs to correct hyperglycemia in diabetic mice were not performed.
The therapeutic efficacy of the IPCs derived using our new differentiation protocol was tested in streptozotocin-treated 129/SvJ syngeneic diabetic mice by transplanting the cells under the kidney capsule. The 129/SvJ mice administered STZ for five consecutive days become progressively diabetic, do not recover from severe hyperglycemia and die between Days 10 and 15 due to failure of endogenous β-cell regeneration as reported earlier . In the present studies, R1Pdx1AcGFP/RIP-Luc ES cell-derived IPCs were transplanted under the kidney capsule of the syngeneic 129/SvJ diabetic mice. The blood glucose levels in mice transplanted with the IPCs demonstrated a steady decline until Day 20 when they finally stabilized throughout the duration of the study. However, complete normoglycemia was not observed in the IPCs transplanted mice. We speculate that the base-line hyperglycemia could easily be overcome by transplanting a greater number of IPCs. To further validate our results, we performed immunofluorescence analysis of the kidney that was transplanted with the IPCs. Our immunofluorescence data demonstrate the presence of positive insulin staining of the R1Pdx1AcGFP/RIP-Luc ES cell-derived IPCs in the kidney. Our results confirm that the transplanted IPCs were functional and able to correct hyperglycemia.
Thus, our studies highlight the need to develop novel strategies to selectively enrich IPCs by eliminating the tumor-causing cells prior to transplantation. Possible candidate cell surface molecules that could be used to remove non-differentiated and partially differentiated ES cells are CXCR4, CD326 and surface specific early antigen.