Different types of feeder cells for maintenance of human embryonic stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To compare the effects of different types of feeder cells on supporting undifferentiation and high proliferation of human embryonic stem cells (hESC).</p><p><b>METHODS</b>hESC were seeded on mouse embryonic fibroblasts (MEF), human marrow stromal cells (hMSC), and human foreskin fibroblasts (hFF), respectively. Colony number, cell quantity after digestion, and survival rate were observed by alkaline phosphatase (AP) staining and Trypan blue, and the biological properties of hESC after 5 passages were observed by immunofluorescence staining.</p><p><b>RESULTS</b>Although all the three feeder layers could support the formation of hESC colonies and maintain pluripotency, the morphology of colonies on different feeder layers remarkably varied. The stage-specific embryonic antigen-3 and AP staining were positive on three types of feeders. The number of colonies, number of cells produced, and cell survival rates were significantly higher on MEF than on human feeder cells (P < 0.01). Furthermore, the number of AP-positive colonies and cell quantity were also significantly higher on hMSC than on hFF (P < 0.01).</p><p><b>CONCLUSIONS</b>All three types of feeder cells are able to support the growth of hMSC, although MEF are more favourable for the proliferation. Two types of human feeder cells lay the foundation for the removal of animal-derived hESC culture system. hMSC is superior to hFF in supporting the proliferation of hESC.</p>

Removing Murine Embryonic Stem Cells From the Differentiating Cell Culture By Using Magnetic Activated Cell Sorting / 中国生物工程杂志

Objective:To remove murine embryonic stem cells(mESC)from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting(MACS).Methods:Neural differentiation of mESC was induced by a 5-stage method.The specific cell surface marker,SSEA-1,was used to identify ES cells in the differentiating cells.The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test.The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate.After the optimization,stage 4 cells were dissociated into single cell suspension,incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM,and then sorted through the magnetic field.The rate of SSEA-1 positive cells in pre-and post-separation groups was assessed by flow cytometry,and the viability of cells was evaluated by trypan blue staining counting under light microscopy.Results:The proportion of SSEA-1 positive cells in the separated cells can be reduced from(7.19?1.36)% to(1.34?0.80)%.The survival rate of sorted cells was more than 92%,similar to that of pre-separation cells.Conclusions:The MACS system we used can effectively remove mESC from the differentiated cells.The sorted cells will be well provided for the subsequent studies about transplantation therapy.