The Basic Research In The Separation, Differentiation Of Human Muse Cells And Differentiation Into Muse-NPCs

Objective Using the latest reports of stem cells-multilineage differentiating stress enduring cells (Muse cells) have the properties of pluripotent, and can be able to differentiate into the endodermal, mesodermal, and ectodermal lineages, isolating Muse cells from human bone marrow cells, inducing into neural precursor cells (Muse-NPCs) in vitro, and establishing a simple and reliable system of separation, induced differentiation and identification in vitro, to provide new seed cells for cells therapy of repairing nerve injury in the future.Methods The density gradient centrifugation method and differential adhesion method were used to isolate and culture the human bone marrow stromal cells (hBMSCs) from healthy adult bone marrow. The cells phenotypes were detected by immunocy-tochemistry and flow cytometry; After expansion in vitro, Muse cells were separated from hBMSCs by fluorescence activated cell sorting. The long-term trypsin incubation method was used to identified their biological characteristics. Cell counting Kit-8 (CCK-8) method and flow cytometry technology were used to test the cells biological characteristics, immunocytochemistry and qPCR technologies used to detect the pluripotent properties of Muse cells, and their ability to differentiate into the endodermal, mesodermal, and ectodermal lineages; Muse cells were induced by neural induction medium into Muse-NPCs in vitro, using immunecytoch-emistry and qPCR technologies to detect the expression of neural stem cell markers, CCK-8 and flow cytometry technology were used to test the cells biological characteristics; Transfecting the red marker protein cherry into Muse-NPCs by lentivirus in vitro, for the transplant experiment in vivo.Results The result showed that CD90 was positive expression, while CD45 and CDllb were negative in hBMSCs. Muse cells were successfully separated by fluorescence activated cell sorting, the immunocytochemistry result showed that Muse cells espressed SSEA-3 and CD 105, The cells growing in suspension culture spontaneously formed characteristic cell clusters, in good status and stabilization, also in a continuous appreciation state in a certain time. Typsin-EDTA incubation after 8h, the vast majority of Muse cells grew well, the cell body maintained a big and bright morphology. Immunocytochemistry and qPCR results showed that Muse cells were positive for some pluripotent markers such as Nanog, Oct4 and Sox2, and they were much higher than hBMSCs in mRNA levels; Muse cells differentiated into irregular shapes when grew in adherent culture, immunocytochemistry and qPCR results showed that the cells were positive for the markers of the three germ layers, α-fetoprotein, α-SMA and NF-M respectively. Induced Muse-NPCs by neural induction medium, growing in suspension culture spontaneously formed characteristic stem cell clusters, immunocytochemistry and qPCR results showed that they expressed the neural sterm cell markers Nestin, NCAM and DCX, and they were much higher than hBMSCs and Muse cells in mRNA levels; Muse-NPCs were in good status and proliferated rapidly within 3 days, and then they were gradually into the plateau state. We could see that the cells spontaneously showed red fluorescence when transfected the red marker protein cherry into Muse-NPCs by lentivirus in vitro, the fluorescent tags rate was above 95%, as well as the cells were in a good status.Conclusions Muse cells were successfully separated from the adult bone marrow, which had the properties of pluripotent stem cells, the cell clusters were in good status, We successfully induced Muse cells into Muse-NPCs, had the properties of neural stem cell, and abled to label cherry protein in order to the later experimence.