Differentiation Of Human Umbilical Cord-derived Mesenchymal Stem Cells Into Corneal Endothelium Like Cells In Vitro

Tissue engineering of the cornea has been presented as a promising opportunityfor overcoming the limitations of conventional corneal replacement, such as shortageof donor corneas and possible allograft rejection. One of the important elements aboutartificial cornea construction is the source of seed cells. However, the acquisition ofabundant of human corneal endothelial cells (CECs) by in vitro cultivation isconfronted with major challenges. Thus, finding other cell types analogous to humanCECs become an urgent issue.Umbilical cord-derived mesenchymal stem cells (UCMSCs) are a unique,accessible, and non-controversial source of embryonic stem cells that can bewidespread availability. UCMSCs can differentiate into several mesenchymal cellsand used in regenerative medicine for tissue and cell replacement therapies. It isknown that both human and rodent CECs originate from neural crest derivedperiocular mesenchymal tissue. Thus, multiple differentiation ability in vitro andextended sources make UCMSCs a suitable cell source for CECs induction.In this study, we present co-culture techniques for the generation of cornealendothelium like cells with morphologic, genetic, and functional characteristics ofCECs from UCMSCs.Part1Isolation, Culture and Identification of Human UmbilicalCord-derived Mesenchymal Stem Cells in VitroObjective: To establish the methods of isolation, culture and identification ofmesenchymal stem cells from human umbilical cord. Methods: Human umbilical cords were collected to isolate human umbilicalcord-derived mesenchymal stem cells (hUCMSCs) using digestion by collagenase at37℃temperature. During the whole culturing and passaging period, morphologiccharacteristics of hUCMSCs were observed under inverted microscope. Theimmunephnotype of hUCMSCs was detected by flow cytometry. The hUCMSCswere differentiated into osteoblasts and adipocytes in the specific-induction medium.Alkaline phosphatase, alizarin red and oil red O staining were used to observe calciumdeposition and lipid droplet formation to identify the hUCMSCs.Results: We successfully isolated and cultured mesenchymal stem cells from humanumbilical cord by using collagenase digestion method. hUCMSCs expressed themarker of mesenchymal, such as CD29, CD44, CD90and CD105; but not expresshematopoietic and graft rejection phenotypes, like CD14, CD31, CD34, CD40, CD45and HLA-DR. The hUCMSCs had positive results of alkaline phosphatase, Alizarinred and Oil Red O staining after induction in the specific-induction medium.Conclusions: HUCMSCs can be isolated from umbilical cord by using collagenasedigestion method. They had the immunephnotype of MSCs and multilineagedifferentiation potentials.Part2Isolation, Culture and Identification of Rabbit Corneal EndothelialCells in VitroObjective: To establish the methods of isolation, culture and Identification of rabbitcorneal endothelial cells (RCECs).Methods: Descemet’s membrane of cornea was striped to isolate RCECs usingdigestion by0.25%Tyrisin/EDTA at37℃temperature for5min. During the wholeculturing and passaging period, morphologic characteristics of RCECs were observedby using the inverted microscope and HE staining. The ultramicrostructure of RCECswere observed by using scanning electron microscope. Marker of endothelial cell: Zonula Occludens-1(ZO-1) and Neuron Specific Enolase (NSE) was detected byimmunofluorescence and immunohistochemistry.Results: We successfully isolated and cultured RCECs by striping Descemet’smembrane and digestion by0.25%Tyrisin/EDTA. Primary culture of RCECsexhibited polygonal morphology, and reached near confluency within10days. Thesurface of RCECs had abundance of microvillus. The RCECs can express markerscharacteristics of corneal endothelium: ZO-1and NSE.Conclusions: RCECs can be isolated by using the method of striping Descemet’smembrane and enzyme digestion. They had the characteristics of native RCECs.Part3Induced Differentiation of hUCMSCs into Corneal Endotheliumlike Cells by Co-culture with RCECsObjective: To investigate the potential differentiation of human umbilical cordmesenchymal stem cells into corneal endothelium like cells by co-culture with rabbitcorneal endothelial cells.Methods: Using the transwell co-culture system of hUCMSCs with RCECs for7days, we induced hUCMSCs to differentiate into CEC-like cells. The morphologicalcharacterizes of differentiated cells was observed under inverted microscope andatomic force microscopy. Differentiated cell proliferation was monitored by CellCounting Kit-8(CCK-8) assay. Then, differentiated cells expressed the cornealendothelium differentiation marker Zonula Occludens-1, N-cadherin, Neuron SpecificEnolase and transcription factors FoxC1and Pitx2were detected by RT-PCR andWestern blot assay.Results: Cell proliferation, cell cycle and apoptosis of differentiated cells weresimilar to hUCMSCs. Upon co-culture of hUCMSCs with RCECs using transwellco-culture system for7days, these spindle-like hUCMSCs turned to oval and polygonal corneal endothelium–like shape and expressed ZO-1, N-cadherin, NSE,FoxC1and Pitx2.Conclusions: HUCMSCs can differentiate into CEC-like cells after havingbeen co-cultured with RCECs.