Isolation And Identification Of Embryonic-like Stem Cells (ELSCs) From Rat Marrow And Their Effects On Cardiac Repair After Transplantation For Myocardial Infarction

Objective:Myocardial infarction (MI) is a major health problem and the leading cause of death and disability in industrialized and developing nations. Stem cell therapy is a promising therapeutic strategy for treating MI. However, it is important to select optimal stem cells and explore effectiveness and mechanisms after transplantation. Methods:Bone marrow mononuclear cells were labeled with SSEA-1, the positive cells were sorted by flow cytometry. The biological characteristics of ELSCs were studied in many aspects, the ultrastructural characteristics were observed under transmission electron microscope (TEM) and scanning electron microscope (SEM), the expreesion of transcriptional factors of embryonic stem cells (ESCs) in ELSCs were detected by immunocytochemistry and RT-PCR, prliferative ability was studied on feeder of mouse embryonic fibroblasts (MEF), formation of embryoid-like bodies were observed in agarose and the expression of trilineage markers was dectected in the cells of embryoid-like bodies, the possible formation of tumor and teratoma of ELSCs was investigated by injection into subcutaneous of severe combined immunodeficiency mice. Differentiation of ELSCs toward cardiomyocytes and endothelial cells was induced by cytokines. The expression of specific protein and mRNA was detected in induced cells. On the basis of in vitro experiments, differentiation of ELSCs in vivo and therapeutic effects was explored. MI female rat models were transplantated by PBS, MMSCs, ELSCs and induced-ELSCs. The cardiac function was examed by echocardiography after transplantation. Scar area was evaluated by Masson’s trichrome staining. The survival of the transplanted cells was detected by Y chromosome in situ hybridization. The differentiation of cardiomyocytes and endothelial cells in vivo was evaluated by the colocalization of the Y chromosome and cTnT, Cx43 and CD31. Results:ELSCs had typical features for ESCs, the nucleus was large, the ratio of nucleus to cytoplasm was very high under TEM, immunocytochemistry and RT-PCR analysis demonstrated that ELSCs expressed transcription factors Oct4, Nanog and Sox2, the cells proliferated significantly of feeder of MEF, the cells formed embryoid-like bodies after incubation in agarose with LIF and bFGF and expressed Nestin, Brachyury and E-cadherin. However, anatomical observation showed there was neither tumor nor teratoma in the body for 4 weeks after transplantation of ELSCs for SCID mice. After induction with cytokines for 4 weeks, ELSCs expressed cTnT and Cx43 for differentiation toward cardiomyocytes and expressed CD31 and vWF for differentiation toward endothelial cells. After transplantation for MI female rat models, cardiac function was improved and scar area was reduced significantly in the induced-ELSC group as compared with control, non-induced ELSC and MMSC groups. In induced-ELSC group the differentiated cells were parallel alignment and connected with host cardiomyocytes. Y chromosome fluorescence in situ hybridization demonstrated that the positive cells were colocalizated with cTnT and Cx43. Cx43 was located between Y chromosome positive cells and recipient cardiomyocytes. This structural basis was the premise for synchronized contraction of transplanted cells with host cells. Colocalization of the Y chromosome and CD31 revealed that transplanted stem cells also underwent endothelial cells differentiation in the infarcted and peri-infarcted regions. And ELSCs were directly participated in angiogenesis in infarcted and peri-infarcted regions. Conclusions:This is the first time to isolate ELSCs from adult rats. ELSCs have similar biological characteristics with ESCs, ELSCs express transcriptional factors Oct4, Nanog and Sox2, proliferate significantly on feed of MEF, form embryoid-like bodies in agarose and the cells in the embryoid-like bodies can differentiate into cells of trilineage. ELSCs can differentiate into cardiomyocytes and endothelial cells under induction with cytokines. Cardiac function is significantly improved, scar area is reduced and ventricular remodeling is effective controlled by transplantation of induced ELSCs. The transplanted cells differentiate into cardiomyocytes and endothelial cells, and the cells are directly participated in angiogenesis. These biological characteristics enable ELSCs to be an optimal source of seed cells for stem cell research and regenerative medicine, and this study provide a theoretical basis for MI by ELSC transplantation.