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In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors
Published on: July 19, 2014
Sonic hedgehog and other soluble factors from differentiating embryoid bodies inhibit pancreas development
Josué K Mfopou1, Véronique De Groote, Xiabo Xu
1Cell Differentiation Unit, Diabetes Research Centre, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
Abstract:
Success of cell-replacement therapy for diabetes will largely depend on the establishment of alternative sources of pancreatic islet grafts. Embryonic stem (ES) cell differentiation toward pancreatic insulin-producing cells offers such perspectives, but there are still many challenges to overcome. Our previous studies suggested that the limited amount of insulin-positive cells derived from ES cells is related to the activation of pancreas inhibitory signals. To confirm this hypothesis, we report here that exposure of mouse embryonic pancreas explants to soluble factors from embryoid bodies (EBs) inhibits growth, morphogenesis, and endocrine and exocrine differentiation as evaluated by explant size and mRNA and protein expression. Sonic Hedgehog (Shh), an established pancreas repressor both at early and late developmental stages, was produced and secreted by EBs, and participated in the inhibitory effect by inducing its target Gli1 in the explants. Inhibition of Hedgehog pathway rescued the differentiation of Insulin-positive cells in the explants. In contrast to pancreatic cells, hepatic progenitors exposed to EB-conditioned medium showed improved differentiation of albumin-positive cells. In a model system of ES cell differentiation in vitro, we found that definitive endoderm induction by serum removal or activin A treatment further increased Hedgehog production and activity in EBs. Concomitantly, downregulation of the pancreas marker Pdx1 was recorded in activin-treated EBs, a phenomenon that was prevented by antagonizing Hedgehog signaling with Hedgehog interacting protein. These data strongly suggest that Hedgehog production in EBs limits pancreatic fate acquisition and forms a major obstacle in the specification of pancreatic cells from ES-derived definitive endoderm. Disclosure of potential conflicts of interest is found at the end of this article.
Insights
Embryonic stem cell differentiation for diabetes treatment is hindered by Hedgehog signaling. Inhibiting this pathway promotes insulin-producing cell development from stem cells.
Area of Science:
- Developmental biology
- Stem cell biology
- Endocrinology
Background:
- Cell-replacement therapy for diabetes requires alternative pancreatic islet graft sources.
- Embryonic stem (ES) cell differentiation offers a promising source but faces challenges.
- Previous studies indicated pancreas inhibitory signals limit insulin-positive cell yield from ES cells.
Purpose of the Study:
- To investigate the role of embryoid body (EB)-derived soluble factors in inhibiting pancreatic differentiation.
- To confirm the hypothesis that pancreas inhibitory signals limit insulin-positive cell generation from ES cells.
- To elucidate the mechanism by which Hedgehog signaling impacts pancreatic fate acquisition.
Main Methods:
- Exposure of mouse embryonic pancreas explants to EB-conditioned medium.
- Assessment of explant growth, morphogenesis, and endocrine/exocrine differentiation markers (mRNA and protein).
- Analysis of Hedgehog pathway components (Shh, Gli1) and pancreatic markers (Pdx1, insulin).
- In vitro ES cell differentiation with serum removal or activin A treatment, with and without Hedgehog pathway inhibition.
Main Results:
- EB-derived soluble factors inhibited pancreatic explant growth, morphogenesis, and differentiation.
- Sonic Hedgehog (Shh) secreted by EBs mediated this inhibition by inducing Gli1.
- Inhibiting the Hedgehog pathway rescued insulin-positive cell differentiation in explants.
- Hedgehog pathway activation during definitive endoderm induction in ES cells reduced Pdx1 expression, which was preventable by antagonism.
- Hepatic progenitors showed enhanced albumin-positive cell differentiation upon exposure to EB-conditioned medium.
Conclusions:
- Hedgehog production by embryoid bodies significantly limits pancreatic fate acquisition.
- Hedgehog signaling acts as a major obstacle in specifying pancreatic cells from ES-derived definitive endoderm.
- Targeting Hedgehog signaling is crucial for advancing stem cell-based therapies for diabetes.
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