Abstract
β-cell replacement may efficiently cure type 1 diabetic (T1D) patients whose insulin-secreting β-cells have been selectively destroyed by autoantigen-reactive T cells. To generate insulin-secreting cells we used two cell sources: rat multipotent adult progenitor cells (rMAPC) and the highly similar rat extra-embryonic endoderm precursor (rXEN-P) cells isolated under rMAPC conditions from blastocysts (rHypoSC). rMAPC/rHypoSC were sequentially committed to definitive endoderm, pancreatic endoderm, and β-cell like cells. On day 21, 20% of rMAPC/rHypoSC progeny expressed Pdx1 and C-peptide. rMAPCr/HypoSC progeny secreted C-peptide under the stimulus of insulin agonist carbachol, and was inhibited by the L-type voltage-dependent calcium channel blocker nifedipine. When rMAPC or rHypoSC differentiated d21 progeny were grafted under the kidney capsule of streptozotocin-induced diabetic nude mice, hyperglycemia reversed after 4 weeks in 6/10 rMAPC- and 5/10 rHypoSC-transplanted mice. Hyperglycemia recurred within 24 hours of graft removal and the histological analysis of the retrieved grafts revealed presence of Pdx1-, Nkx6.1- and C-peptide-positive cells. The ability of both rMAPC and HypoSC to differentiate to functional β-cell like cells may serve to gain insight into signals that govern β-cell differentiation and aid in developing culture systems to commit other (pluripotent) stem cells to clinically useful β-cells for cell therapy of T1D.
Highlights
Type 1 diabetes (T1D) is caused by the selective loss of pancreatic b-cells by autoantigen-reactive T cells
We have previously shown that rat multipotent adult progenitor cells (rMAPC) [22] and rHypoSC [25] can be fated towards hepatic endodermal cell types, apparently by refating the cells from a primitive endoderm (PrE) to ME/definitive endoderm (DE) fate
We demonstrate here that culture of rMAPC and rHypoSC with 100 ng/mL activin-A combined with 50 ng/mL BMP4 induces a complement of marker genes (Gsc, Eomes, Mxl1, CXC chemokine receptor type 4 (Cxcr4), Sox17, and FoxA2) which, when combined, represents ME/DE
Summary
Type 1 diabetes (T1D) is caused by the selective loss of pancreatic b-cells by autoantigen-reactive T cells. The pancreas is derived from definitive endoderm (DE), that specifies from pluripotent cells in the blastocyst stage of the embryo by a two-step process, wherein mesendoderm (ME) is generated to the exclusion of ectoderm, followed by specification to CXC chemokine receptor type 4 (Cxcr4) and SRY-related. Specification to pancreatic endoderm is associated with expression of Pancreatic and duodenal homeobox 1 (Pdx). Other TFs important for b-cell differentiation include Paired box gene (Pax), that specifies endocrine pancreatic cells to a b-cell [5], NK6 homeobox (Nkx6). that regulates b-cell development [6]. Musculo aponeurotic fibrosarcoma oncogene homolog A (MafA) is expressed initially at e13.5 and is found only in insulin-positive cells during development or in mature islets. MafA is thought to act in conjunction with other known insulin enhancer regulatory factors (Neurogenic differentiation 1 (NeuroD1) and Pdx1) to promote transcription of the insulin gene [7]
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