Research On A New Method Of Multipotent Stem Cell Induced From Mouse Embryonic Fibroblasts Which Incubated With The ESC-like Extraction

This study was to establish the incubated methods for reprogramming of mouse embryonic fibroblasts (MEFs) which were finally de-differentiated into pluripotent stem cells and to improve the efficiency of reprogramming, MEFs permeabilized with streptolysin O were exposed to the extract of mouse embryonic stem cells-like (ESC-like). The effective method for preparing ESC-like extract, the suitable concentration of streptolysin O (SLO)-mediated permeabilization of MEFs and the effects of different incubated system on reprogramming of MEFs with ESC-like extract were mainly studied in this paper.1. Study on effective pathways and effect factors of obtaining mouse ESC-like3.5dpc (days post coitum),4dpc or4.5dpc mouse embryos were collected respectively, and those embryos were cultured in DMEM, DMEM+LIF or the co-culture system of MEFs, the hatching time of inner cell mass (ICM) from the zona pellucida, attachment efficiency of hatching blastocyst and the formation efficiency of ICM colonies in different treatment groups were compared in this study, in order to finding out an effective method for obtaining high-quality ICM colony, and therefore laying a solid foundation for further separation and culture of the ESC. Results indicated that61%of3.5dpc mouse embryos were morula which could be transformed into ICM after being cultured in vitro for4-5days. In contrast,4dpc mouse expanded blastocysts and4.5dpc mouse hatching blastocysts could be transformed into ICM colonies after the co-culture for3-4days and1～2days, respectively. Also we found that the hatching rates of ICM colonies are significantly different (p<0.01) among those treatment groups, the formation rates of ICM colonies from4.5dpc mouse embryos were higher than that from4dpc mouse embryos, However, the number of collected hatching blastocysts from4.5dpc mouse embryos reduced dramatically. The co-culture system of MEFs is better than the other two culture media in the isolation and culture of ICM colonies. Therefore, we concluded that4dpc mouse embryos were collected and co-cultured with MEFs for3～4days, high-quality ICM colonies could be obtained for further studies of ESC.2. Study on the methods for preparing mouse ESC-like extract and the cell-free incubated culture systemESC-like colonies were obtained from the high-quality ICM colonies, then ESC-like were repeatedly frozen and thawed in liquid nitrogen for three times, five times, seven times or nine times, the extract of ESC-like were observed under a microscope to find the number of the cells which could maintain the integrity cell morphology. The aim of this study was to find out the suitable number of times of freezing-thawing that all cells and nuclei were lysed and intracellular substances were destroied as less as possible, and to provide a cell-free incubated culture system for reprogramming of MEFs. Results showed that the number of the cells which could maintain the integrity cell morphology after being frozen-thawed in liquid nitrogen for three times was significantly higher than that after being frozen-thawed for five times, seven times or nine times, respectively (p<0.01), there were no significant differences in the number of the cells which could maintain the integrity cell morphology among those treatment groups that the ESC-like were frozen-thawed for five times, seven times or nine times (p>0.05), but the extract still containing the cells which could maintain the integrity cell morphology after ESC-like were frozen-thawed for five times, there were cell-free ESC-like extract after ESC-like were frozen-thawed for seven times or nine times. Therefore, all cells and nuclei were lysed and intracellular substances were destroied as less as possible after ESC-like were frozen-thawed for seven times, it provided a cell-free incubated culture system for reprogramming of MEFs.3. Study on the optimized selection of MEFs and the suitable concentration of streptolysin O (SLO) for the permeabilization of MEFsMEFs were isolated from13.5dpc Kunming mouse embryos, the third passage MEFs were more suitable to be permeabilized with0.1U·μL-1,0.25·μL-1,0.5·μL-1and1·μL-1SLO respectively, then the number of dead cells was counted under a microscope, in order to find out an appropriate concentration of SLO for the permeabilization of MEFs. Results indicated that there were lots of dead cells after MEFs were permeabilized with0.5·μL-1or1·μL-1SLO, the reverse transformation efficiency of MEFs would be affected, there were a few dead cells after MEFs were permeabilized with0.1·μL-1or0.25·μL-1and no significantly difference in the number of dead cells between those two treatment groups. But if the concentration of SLO is too lower and it likely more lesser affected the reverse transformation efficiency of MEFs. Therefore, MEFs were more suitable to be permeabilized with0.25·μL-1SLO considering the efficiency of permeabilization and the negative effects of SLO on MEFs, it laid the foundation for the establishment of co-culture system of the ESC-like extract and MEFs.4. The effects of different incubated system on de-differentiated of MEFs into pluripotent stem cellsThe MEFs permeabilized with SLO were coincubated with the extract of mouse ESC-like (treatment group Ⅰ), were pretreatmented with trichostatin A (TSA) and coincubated with the extract of ESC-like (treatment group Ⅱ), were pretreatmented with5-Aza-2’-deoxycytidine (5-Aza-dC) and coincubated with the extract of ESC-like (treatment group Ⅲ), and were pretreatmented with TSA and5-Aza-dC and coincubated with the extract of ESC-like (treatment group Ⅳ), were exposed to medium without pretreatmented with TSA and5-Aza-dC and coincubated with the extract of ESC-like (control group), cell morphology was observed under a microscope and the pluripotent stem cells from MEFs were identified, in order to explore an effective incubation method for reprogramming of MEFs which were finally de-differentiated into pluripotent stem cells. The results showed that the number of non-circular cell in treatment group Ⅳ was significantly lower than that in other treatment groups (P<0.01), the number of ESC-like colonies in treatment group Ⅳ was significantly higher than that in other treatment groups (P<0.01). Therefore, MEFs were pretreatmented with TSA and5-Aza-dC and coincubated with the extract of ESC-like was the better method to improve the efficiency of reprogramming of MEFs into the pluripotent stem cells.In conclusion,4dpc mouse embryos were collected and co-cultured with MEFs for3～4days, high-quality ICM colonies could be obtained for further studies of ESC, all cells and nuclei were lysed and intracellular substances were destroied as less as possible after ESC-like were frozen-thawed for seven times, MEFs were were pretreatmented with TSA and5-Aza-dC, then were permeabilized with0.25·μL-1SLO, and finally were coincubated with the extract of ESC-like, MEFs could be reprogramming with the some characteristics of pluripotent stem cells. Those studies indicated that the differentiated somatic cells could be reprogrammed to became pluripotent stem cells using the ESC extract, this method will became an new way to produce the autologous stem cells without destroying the embryos.