Experimental Study Of Differentiation Of EBD Cells From Human PGCs Into Human Cardiomyocytes

Objective To investigate whether human primordial germ cells( hPGCs) can be obtained from human embryonic genital ridge and cultured in vitro with multiple-potency, whether human embryoid bodies- derived cells( hEBDs) from hPGCs has multipotency and can be induced to differentiate into human cardiomyocyte in vivo, and whether hEBD cells can be used in cellular cardiomyoplasty in future.Methods (1) Cells were isolated from the genital ridge of human developed embryos of 5-7 weeks and cultured on mouse STO feeder in vitro; (2) For identifying hPGCs, cells were assayed in morphology , alkaline phosphatase(AKP) activity and expression of cellular marker of stage-specific embryonic antigen-4 (SSEA-4) and tumor related antigen -1-60(TRA-1-60); (3) Cells derived from embroid bodies formed when hPGCs were cultured in suspension were cultured in vitro and analyzed for myogenic regulator factor 5 (MEF-5), a-fetoprotein (a-FP) and glial fibrillary acidic protein (GFAP) with a method of reverse transcription polymerase chain reaction (RT-PCR ); (4) Human EBD cells labeled by DAPI were transplanted into the myocardium of developed chick embryos of day 4; (5) With Alu probes, DNA in suit hybridization (ISH ) was employed in myocardium in human being , mice, rat, chick, rabbit, dog, sheep and pig for human specificity; (6) DNA ISH with Alu probes was used for detecting human cells in hEBDs-grafted myocardium in survived chick embryos of post-operation ; (7) Human myocardial specific marker atrial natriuretic peptide (ANP), cardiac troponin t (cTnT) and NKx-2.5 were analyzed in adjacent ones in ISH positive sections with a method of fluorescent immunohistochemistry ; (8)Using RT-PCR , human myocardial specific gene myosin light chain 2v (MLC-2V)> myosin light chain 2A(MLC-2A), a-myosin heavy chain (a-MHC), cTnT , ANP and NKx-2.5 were measured in myocardium of survived chick embryos of post-operation .Results (1) Cells isolated from genital ridge of human developed embryos of 5-7 weeks could be cultured, formed clones and EB in vitro, had positive AKP activity and expressed SSEA-4 and TRA-1-60; (2) RT-PCR showed that cells derived from EB expressed MEF-5, a-FP and GFAP; (3) In DNA ISHwith Alu probes, it was positive in myocardium in human being , but not in mice, rat, chick, rabbit, dog, sheep and pig; (4) DNA ISH with Alu probes demonstrated that positive cells lied in hEBDs -grafted myocardium in survived chick embryos of post-operation ; (6) Human specific ANP, cTnT and NKx-2.5 were found positive in adjacent ones of ISH positive sections with a method of fluorescent immunohistochemistry; (7) It was clear in RT-PCR that human myocardial specific gene MLC-2V. MLC-2A. a-MHC. CTnT. ANP and NKx-2.5 lied in hEBDs -grafted myocardium in survived chick embryos of post-operation.Conclusions HPGCs isolated from human embryonic genital ridge is multipotent and can be cultured in vitro. hEBDs from hPGCs has multipotency , can be induced to differentiate into human cardiomyocyte when transplanted in vivo and may be used for cellular therapy for coronary heart disease in future.