| Cattle is important economic animals. The pluripotent stem cells of bovine has broad application value, and is the biological materials of research significance, such as used in genetic engineering, embryo engineering and cloning research, etc. So far, after many attempts, bovine pluripotent cell lines has not effectively been established. Recently, several groups had reported that a new type of stem cells, named Muse cells, which was isolated from human skin fibroblasts and mesenchymal stem cells. It has the self-renewal and multi-directional differentiation potential. Muse cells have shown a certain degree of pluripotency. In this study, we tried to use magnetic activated cell sorting(MACS) technology and trypsin digestion method to isolate Muse cells from bovine fetal fibroblasts for the further research of bovine stem cells differentiation, gene regulation and reprogrammed. The main contents of this research are as follows:1. Isolationof SSEA4-positive cells from bovine fetal fibroblasts by MACSThere is a few cells expression SSEA4 in bovine fibroblast cells, which was confirmed by immunofluorescence staining. The SSEA4-positive cells had been separated and sorted by magnetic activated cell sorting from bovine fibroblasts. About 2~3% SSEA4-positive cells was obtained. Positive cells in the triangle or polygon, small, has the high nuclear mass ratio. The results showed that they expressed of alkaline phosphatase(AP), Oct4, Nanog, Sox2, and SSEA4 pluripotent- related marker and could differentiated into three germ layer cell. But the SSEA4-positive cells could not form teratomas in nude mice experiment. In addition, SSEA4-positive cells expressed CD105 and other mesenchymal marker and could differentiated to fat, bone and cartilage cell.2. Isolation and characterization of Muse cells by trypsin digestionWe explored that bovine fetal fibroblasts were treated with 0.25% trypsin/EDTA to separate Muse cells. The results showed that the pluripotency of cells increased with the increase of processing time. The pluripotency of cells was best when dealing with 12 h. The cells were rarely after trypsin/EDTA treated. Muse cells was small. They exhibited triangle or polygon. The results showed that they expressed of alkaline phosphatase(AP), Oct4, Nanog, Sox2, and SSEA4 pluripotent- related marker and could differentiated into three germ layer cell. But the SSEA4-positive cells could not form teratomas in nude mice experiment. But their telomerase activity was low. By transmission electron microscopy(SEM) and WB detection, trypsin stress during the processing of the existence of autophagy occurs, and with the increase of processing time, the expression of LC3 quantity increase gradually.3. Optimizing culture conditions of Muse cellBy comparing the effect of bFGF of 4 ng/mL, 10 ng/mL, 20 ng/mL, 40 ng/mL, and 100 ng/mL on Muse cells proliferation and pluripotency, we found that high concentrations of bFGF(>10 ng/mL) has obvious promoting effect to Muse cell proliferation. BrdU incorporation experiments also proved that 20 ng/mL and 40 ng/mL concentration was best. AP staining and QRT-PCR results also showed that 40 ng/mL of bFGF is more advantageous. 40 ng/mL of bFGF improved the expression level of plurupotrnt related markers of Muse cells. On the Muse cell suspension culture, cell formed by the low adsorption dish of ball size is uneven, prone to fusion, and the high cost. Muse cells ball were more uniform under sphere culture by using methyl cellulose suspension culture in PolyHEMA package. In addition, after testing, methyl cellulose suspension culture was conducive to the further purification of Muse cells and maintaining their undifferentiated state. It can be used as a Muse cell cultivation method for a long time.4. Developmental potential of cloned bovine embryos from muse cells in vitroCloned embryos derived from SSEA-4 cells(85.62%) showed significant differences in cleavage rate and blastocyst development when compared with those from BEF(70.36%) and SSEA4- cells(66.32%). Moreover, blastocysts derived from SSEA4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA4-derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle... |
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