| Embryonic stem (ES) cell lines isolated from primitive embryonic cells are highly undifferentiated and totipotent cells. In vitro, ES cells can spontaneously differentiate into embryoid bodies (EBs) containing derivatives of three germ layers. The researchers in blood area are trying promoting hematopoietic differentiation of embryonic stem cells. The main methods of promoting hematopoietic differentiation are to coculture ES cells with the mouse fibroblast lines such as OP9, RP0.10, MS-5, ST2, NIH-3T3, PA6. There are few studies on bone marrow endomelial cell line of inducing ES cells differentiation into hematopoietic cells. In our study, we have applied the mouse bone marrow endothelial cell-conditioned medium (mBMEC-CM) to promote hematopoietic differentiation of mouse ES cells, in order to eliminate contamination of exogenous cells and provide experimental basis on inducing human ES cells differentiation into hematopoietic cells. Day 4 embryoid body (4dEB) cells were derived from ES cells and then induced with mBMEC-CM into hematopoietic precursor cells. Immunocytochemistry staining and flow cytometry were adopted to observe the antigen expressions. RT-PCR method was used to detect the expressions of hematopoietic genes. And hematopoietic progenitor assay was used to examine hematopoietic differentiation. The effect of mBMEC-CM when combined with cytokines or mouse fetal liver stromal cell-conditionalmedium (mFLSC-CM) on inducing ES cells differentiation into hematopoietic cells was detected. The hematopoietic reconstitution of ES cell-derived HSCs was detected in vivo in irradiated recipient femalemice.Part 1: The culture and identification of ES-D3 cells and the study of the efficiency of EB formation from ES cellsWhen grown on MEF feeder layer in ES culture medium or cultured in ES culture medium supplemented with LIF 1000U/ml, ES-D3 cells being used in our experiments formed normal clones, expressed AKP and kept their normal karyotype over many passages. The in vitro and in vivo differentiation experiments showed that ES-D3 cells could differentiate into variety of cell types derived from three primary germ layers. ES-D3 cells could form chimeras. We referred that the ES-D3 cells being used in our experiments maintained undifferentiated and totipotent.The enhancing factors of EB formation were studied. Fifty thousand ES-D3 cells per milliliter were incubated in DMEM-high glucose supplemented with 20% FBS and 2mM glutamine. The influencing factors which included Hyclone FBS and homemade Green Season FBS, feeder cell layer, culture containers and 3 -mercaptoethanol ( -ME) were investigated. ES-D3 cells differentiated into EBs only when depleted of feeder cell layer by being cultured three generations in the presence of leukemia-inhibitory factor (LIF). The numhers of day-4EBs per milliliter culture media are 44.83 1.22 (n=6) and 43.17 1.05(n=6) respectively when cultured by Hyclone FBS and homemadeGreen Season FBS. There is no significant difference between them(P>0.05). The numbers of day-4 EBs per milliliter culture media are49.33 1.76 (n=6 ) and 273.50 4.06 (n=6) when cultured in absence andpresent of -ME respectively. There is significant difference betweenthem (P<0.001) . The numbers of day-4 EBs per milliliter culture mediaare 47.33 l.48(n=6)when cultured in glass container and 310.83 2.92( n=6 ) in Petriperm bacterial-culture dish and there is no EB formationin cell-culture dish. There is significant difference when cultured inPetriperm bacterial-culture dish compared with the glass container ortissue-culture dish (P<0.001) . After being cultured three generations anddepleted feeder cell layers, the ES-D3 cells differentiated into EBsefficiently in the culture media supplemented with homemade GreenSeason FBS and -ME in Petriperm bacterial-culture dish.Part 2: Induction of hematopoietic differentiation of mouse ES cellswith mBMEC-CMTo promote hematopoiesis, the 4dEB cells were cocultured with mBMEC-CM.
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