Differentiation Of RhSCNT-ESCs Into Hematopoietic Lineages

Part I Differentiation of rhSCNT-ESCs into hematopoietic lineages Objective:rhSCNT-ESCs which has the same genotype with the donor can be induced to hematopoietic lineages either by stromal cell coculture or by formation of EBs. Here, we detected the expression level of CD34 with flow cytometry, immunocytochemical and immunofluorescent stain to discover a more effective differentiation method. Methods:1. rhSCNT-ESCs were cultured with pre-treated MEF, FLSC and BMSC from aborted embryos as feeder layers respectively. Flow cytometry analysis was used to detect the expression of CD34 at 3d. 7d,11d and 14d after coculture.2. rhSCNT-ESCs were planted into low adherent 6-well plates with or without BMP₄. 4d later, we transferred EBs into low adherent tissue culture plates containing 0.9% methylcellulose in 1MDM with various cytokines. Immunocytochemical and immunofluorescent stain were performed to detect the expression of CD34 and KDR.Results:1. rhSCNT-ESCs cocultured with FLSC and BMSC demonstrated similar hematopoietic development. We observed hematopotic clusters after 3-4 days and CFU-GEMM at the 14th day. The percentage of CD34+ hematopoietic stem cells increased little by little, peaked at the 7th day, decreased more and more and reached another lower peak at the 14th day. While rhSCNT-ESCs with MEF as feeder layer displayed spontaneous differentiation.2. In the differentiation media, simple EBs can be observed 2d later and cylist EBs with 100-150 μm in diameter at the 4th day. Single cell suspension was made and we could...