Comparing The Hematopoietic Potentials Of The Four HESCs Lines

Human embryonic stem cells(hESCs) derived from the inner cell mass of pre-implantation blastocysts have the ability to self-renew as undifferentiated cells for prolonged periods in culture,and retain the potiential to differentiate into any cell type within the adult body.Human embryonic stem cells(hESCs) could potentially represent an alternative source for blood transfusion therapies,and a tool for studying ontogeny and development of hematopoiesis.It has been shown recently that each of the hESCs lines has its own unique transcriptional profile;they may also differ significantly in their doubling time,transfection efficiency, long term culture stability,spontaneous differentiation patterns and X-inactivation status.Here,we compared the hematopoietic differentiation potentials of four hESCs lines((chHES-22,XY; chHES-8,XY;chHES-137,XX;chHES-32,parthenogenetic homozygous) established in our laboratory under spontaneous differentiation and induction conditions by flow cytometry,hematopoietic colony-forming assays and RT-PCR.Part 1 Comparing the hematopoietie potentials of the four hESCs linesObjective:To investigate the hematopoietic potentials of four hESCs lines((chHES-22,XY;chHES-8,XY;chHES-137,XX;chHES-32, parthenogenetic homozygous) under spontaneous differertiation and induction conditions.Methods:1.After spontaneous differertiation for 12 days,EBs were digested into single cells,the immonophenotype,hematopoietic-related transcription factors and colony-forming ability of the trypsinized EB cells was analyzed by flow cytometry,RT-PCR and semisolid culture.2. After induced differertiation for 12 days,EBs were digested into single cells,the immonophenotype,hematopoietic-related transcription factors of the trypsinized EB cells was analyzed by flow cytometry,RT-PCR.3. After induced differertiation for 12 days,EBs were transferred to 12-well plate coated with Matrigel and cultured for 7 days.The adherent and non-adherent cells were harversted by trypsin/EDTA digestion,and plated in MethoCult SF H4436 simisolid medium for hematopoietic colony assay.Result:chHES-22 had the best hematopoietic differentiation potential among the four hESCs linesPart 2 hESCs differentiation into hemangioblastsObjective:To analyze the differentiation potential of chHES-22 into hemangiblasts Methods:Undifferentiated hESCs colonies were harvested off the Modified TeSR^TM1 medium and plated in STEMPRO-34 SFM supplemented with 50ng/ml BMP-4,and 50ng/ml VEGF At 3.5days of culture,EBs were transferred to BGM semisolid medium containing a combination of growth factors.Result:chHES-22 could give rise to blast-conoly consisting of grape-like cells,26 colonies could be generated by per 4×10⁴ EB-derived cells.Conclusion:1.we compared the hematopoietic differentiation potentials of four hESCs lines(chHES-22,chHES-8,chHES-137,chHES-32)under spontaneous differertiation and induction conditions,chHES-22 had the best potential for the hematopoietic induction.2.chHES-22 could differentiate into blast-colonies consisting of grape-like hemangioblasts.