Effects Of Puramatrix Hydrogel Three-dementional Culture On The Differentiation Of Human Umbilical Cord-derived MSCs Towards Islet-like Clusters

Objective：Screening out the maturity-promoting protocol to direct the differentiation of humanumbilical cord-derived mesenchymal stem cells (hUC-MSCs）towards islet-like clusters (ILCs)expressing prohormone convertases（PC）PC1and PC2through biological reagents in thetwo-dimensional condition. Culturing hUC-MSCs in PuraMatrix (PM) and induing thehUC-MSCs with the selected protocol toward the ILCs to explore the effects of thethree-dimensional culture on the differentiation from hUC-MSCs into ILCs.Methods：Primary hUC-MSCs were isolated from the whole human umbilical cord and thenwere identified by the methods of flow cytometry,RT-PCR and immunocyto-fluorescence.Protocol A, B and C were adopted to induce the differentiation of hUC-MSCstowards ILCs in the two-dimensional condition.Before and after induction, PC1and PC2weremeasured by qPCR and Western blot and the maturity-promoting protocol was screened out.Culturing hUC-MSCs in pretreated PM with different dilutions, and determined the optimalconcentration and time point through the mophorlogical observation,cck8/live-dead kit assayand RT-PCR/qPCR examinations. The maturity-promoting protocol was used to induce thedifferentiation of hUC-MSCs in PM towards ILCs，the changes of pc1, pc2and nestin genewere tested with qPCR.Results：hUC-MSCs expressed Nestin broadly and Isl1to a less degree. qPCR and Western blotshowed only ILCs induced by Protocol C express PC1gene, ILCs induced by Protocol B andProtocol C both express PC2gene but the expression was significantly higher in Protocol Cthan that in Protocol B (P <0.01).The results of cck8showed that the proliferation rates ofhUC-MSCs at the low concentrations of PM(0.125%and0.25%) were higher than at the highconcentrations of PM (0.5%and0.75%). hUC-MSCs at the high concentrations of PM wouldgrow into spheroids and expressed a small amount of pc1, pc2and insulin in0.5%PM. Whenusing protocol C to induce hUC-MSCs in0.5%PM to differentiate towards ILCs, theexpressions of pc1, pc2and insulin were higher compared to those that cultured and induced intraditional two-dimensional enviroment(P <0.01).Conclusion：hUC-MSCs possess the potential to differentiate into ILCs under certain inducingconditions. Compared to Protocol A and B, Protocol C proved to be the optimalmaturity-promoting protocol in the current study. The hUC-MSCs grow in0.5%PM tend to aggregate into spheroids and express a small amount of islet development related genes.ILCsexpress more genes after induction with protocol C,which indicates that0.5%PMthree-dimensional culture could promote hUC-MSCs to differentiate into ILCs.