Mass Spectrum Analysis Of Human Embryonic Germ Cells Induced To Differentiate Into Cardiomyocytes

Objectives: 1. To establish a cultivation method for more efficient growth and amplification of human embryonic germ cells (hEGCs) in vitro using human embryonic fibroblasts as feeder layer. 2. To induce hEGCs to differentiate into cardiomyocytes by ascorbic acid. 3. To analyze the difference of protein profiles between hEGCs and cardiomyocytes differentiated from hEGCs.Methods: Through tissue culture of the gonadal ridge taken from human embryos aged 5-10 weeks, hEGCs were obtained. Human embryo fibroblasts were also isolated and cultured from gonadal ridge of the same embryonic origin, hEGCs were subcultured on the human embryo fibroblast feeder layer. hEGCs which grew well and steadily were seeded in the petri dish for suspension culture. After 7 days, embryoid bodies(EBs) formed from hEGCs were placed in gelatin-treated 24-well plate, and incubated with differentiation medium containing 0.1mg/ml retinoic acid. Immunofluorescence was used to detect the expression of cardiac troponin T(cTnT) on hEGCs induced to differentiate for 2 weeks. Cell total proteins of the hEGCs and differentiated cells were extracted respectively and labeled differently by isobaric tags for relative and absolute quantitation (iTRAQ), the proteins of hEGCs were labeled by 115,the proteins of differentiated cells were labeled by 114.The labeled proteins were isolated by liquid chromatography(LC) and then the different expressing of proteins were identified by Tandem mass spectrometry(MS/MS)in hEGCs and differentiated cells.Results: Human embryonic fibroblasts cultured in vitro were fusiform and grew well. hEGCs of subculture proliferated strongly on human embryo fibroblast feeder cells, and the size of the clones was nest-like and no significant different from the ones of primary culture, hEGCs had reached the 6th generation. cTnT was positive expression in some polygonal differentiated cells induced by ascorbic acid for 2 weeks. Mass spectrometry method identified a total of 349 proteins (proteins confidence >95%), of which 27 proteins (21 upregulated proteins, 6 downregulated proteins) in hEGCs and differentiated cells were different expression significantly (difference multiples > 3).Conclusions: A modified culture method of proliferating hEGCs on human embryonic fibroblast feeder layer in vivo is established. Ascorbic acid can induce hEGCs to differentiate into cardiomyocytes. Mass spectrometry is an useful method to screen the important proteins in a high throughput which are related to differentiation of hEGCs induced to cardiomyocytes.