A Whole Mechanical Method To Generate A Human Embryonic Stem Cell Line And Its Differentiation To Neural Cells

Human embryonic stem cells (hESCs) are valuable resource fordevelopmental biology and regenerative medicine research. HESCs linesvaried on their gene expression profile, epigenetic modify profile, anddifferentiation tendency. Generation of more hESCs lines will enhanceour knowledge of hESCs innate character. Besides, it is crucial tooptimize the establish method aiming to obtain clinical grade hESCslines.Objective: Establish a hESCs line with an optimized method, andcheck the differential capability to neurons. Method: Bad quality embryosfrom in vitro fertilization-embryo transfer (IVF-ET) were collected toestablish hESCs lines with informed consent. HESCs line was establishedwith a whole-mechanical method, by only a1ml syringe. The hESCs linewas cultured on mouse embryonic fibroblast (MEF) and human foreskinfibroblast (HFF) and feeder layer, and mechanical and enzymetic methods were used to propagate the newly established hESCs line. The new cellline was checked for pluripotent characteristics with detecting theexpression of “stemness” genes and observing spontaneous differentiationboth in vitro and in vivo. Finally, step-wise protocol was used to checkthe cell line’s ability to directly differentiate into neural cells. Result: Wegenerated a new hESCs line, HN4. The cell line satisfied the criteria ofpluripotent hESCs lines: typically morphological characteristics; theexpression of alkaline phosphatase, hESCs-specific markers includingOct-4,Nanog, Rex-1,SSEA-3, SSEA-4, Tra-1-60and Tra-1-81；maintenance of a stable karyotype after long-term cultivation; and robustthree lineages developmental potentials both in vitro and in vivo. TheHN4cells could be directly differentiated towards neural stem cells andneural cells. Significance: The innovative whole-mechanical method toestablish hESCs lines is time saving, labor saving, economical and exoticcomponents-free. The HN4cell line has high potential to differentiateinto neural cells, thus it should be an ideal cell line for human embryoand neural system developmental research, as well as neural diseasetherapy and research.