The Immunogenicity Changes Of Insulin-producing Cells Differentiated From Human Umbilical Cord Mesenchymal Stem Cells

Objective：In order to provide a theoretical basis for the cells treatment of diabetes，we establisha protocol for the efficient and directional differentiation from human umbilical cordmesenchymal stem cells into Insulin-producing cells，and observe immunogenicity changes ofIPCs differentiated from hUCMSCs.Methods: HUCMSCs were analysed phenotypic and molecular characterization by FACS andthe ability to differentiate into a variety of cell types by osteogenic and adipogenicdifferentiation,which were separated from human umbilical cord Wharton’s Jelly. ThenhUCMSCs were induced to differentiate to IPCs by a improved protocol based on an improvedprotocol with addition of islet neogenessis-associated protein-pp. The IPCs identification wasincluding morphology changes,differentiation efficiency by FACS，important transcriptionfactors mRNA and protein expression in pancreatic development detected by Real-time PCR andimmunofluorescence, and the Insulin and C peptide concentration by ELISA. CTL killing assaywas performed to observe apoptosis rate of IPCs in the co-culture with the mice pre-sensitizedspleen lymphocyte cells, while the leves of pro-inflammatory cytokines (IFN-γ and IL-2) andanti-inflammatory cytokines (IL-4) in culture media were detected by ELISA. Afterintraperitoneal injection of IPCs, the number of white blood cells and T cells of Peritoneal CellPopulation were detected by FACS,and the biopsy of tissues was stained by hematoxylin andeosin (HE) after transplanting hUCMSCs or IPCs to mouse left renal subcapsular, to observeinflammatory changes in the graft site.Results: Our study successfully separated and purified hUCMSCs from Wharton’s Jelly.Phenotypic characterization, osteogenic and adipogenic differentiation confirmed the hUCMSCsas MSCS. INGAP-PP improved induction protocol was effective in the differentiation to IPCsfrom hUCMSCs. During the differentiation IPCs expressed transcription factors mRNA of thepancreas development in turn, and C peptide，PDX-1，Nkx6.1protein. IPCs could secrete Cpeptide and insulin after glucose stimulation. HUCMSCs differentiation efficiency was up to12%. Both hUCMSCs and IPCs expressed HLA-ABC, and didn’t express HLA-DR, CD40andCD80; IPCs had low expression of HLA-DR. In one-way MLR under the condition of that hPBMCs: hUCMSCs or IPCs were10:1and50:1, hUCMSCs and IPCs inhibited theproliferation of hPBMCs, while the inhibition of hUCMSCs was stronger than IPCs. Fortunately,when the ratio reached100:1,both hUCMSCs and IPCs didn’t cause proliferation of hPBMCs.Pre-sensitized spleen lymphocytes could lead to apoptosis of IPCs by dose-dependent manner inCTL kill assay, pro-inflammatory cytokines IFN-gamma and IL-2concentration in culturesupernatant were increased with the spleen lymphocytes.On the contrary, anti-inflammatorycytokines IL-4reduced. Four hours after peritoneal transplantation of IPCs, leukocyte infiltrationcould be detected, but there was not mainly an increase of lymphocytes. On days15aftermouse left renal subcapsular transplantation of IPCs, the transplanted cells in the injected sitesbegan to decrease,and on days30, almost no transplanted cells were observed. IPCs hadstronger ability in immune rejection than hUCMSCs.Conclusions: INGAP-pp promoted the differentiation into IPCs from hUCMSCs. Compared tohUCMSCs，the IPCs almost had a low immunogenic profile in vitro, while after transplantationIPCs could react an immune rejection in vivo.