| The process of de-differentiating somatic cells to a pluripotent state whereby they adopt characteristics of embryonic stem cells is referred to as’cellular reprogramming’ The advantages of cell extracts-mediated reprogramming of somatic cells are:(1) Chromosome reprogramming of imported pluripotent cells does not exist,(2) Manipulating cellular composition of the extract to identify cytokines which are relevant to reprogramming,(3) Since the extract is derived from cells, its role is biological, rather than chemical, so security is higher. More and more researchers pay attention to this method, which is very worthwhile to study and develop in depth, and there are bright prospects of application.Embryonic stem cells were isolated from the high-quality inner cell mass (ICM) from blastocysts derived from in vivo fertilization, parthenogenetic activation, and reconstruction of embryo using somatic cell nuclear transfer, and were identified with morphological observation, alkaline phosphatase assay, karyotype analysis, immunocytochemistry staining for Oct-4and SSEA-1, RT-PCR analysis for Oct-4and Sox-2, and examination the in vivo differentiation of embryoid body. MEFs were pretreated with trochostatin A (TSA) and5-Aza-deoxycytidine (5-Aza-dc) and permeabilized with streptolysin O, then those cells were incubated with reprogramming cell-free extracts which were prepared by repeatedly freezing and thawing ESCs in liquid nitrogen. Reprogrammed MEFs were continually cultured in ESCs medium and were identified whether those cells were reprogrammed to became a pluripotential stem cells or not. To rule out the possibility of ESCs contamination during the preparation of ESCs extracts, further pluripotential stem cells generation were performed using male MEFs which were identified by dual PCR for sex identification system of a different gender from extract-donor ESCs. The donors of ESCs-derived extract were the parthenogenetic embryonic stem cells (pESCs) from the oocytes which been parthenogenetic activated and female ESCs with the identified by dual PCR for sex identification system from the blastocystosts derived from in vivo fertilization. Therefore, we can decided the origin of the pluripotential stem cells according to their gender using dual PCR for sex identification system. Finally, chicken embryo fibroblasts which being act as the target cells of reprogramming and were incubated with mouse ESCs extracts, to explore the feasibility of reprogramming between the different species, the origin of cells resulted from reprogramming mediated by mouse ESCs extracts was identified by karyotype analysis.The aim of this study was to screening the appropriate incubation method to improve the efficiencies of reprogramming which being mediated by ESCs extracts, to assess the reliability of the reprogramming system, and to explore the reprogramming mechanism about the effects of paternal gene on the efficiencies of reprogramming and The feasibility of reprogramming which be induced with the effective components in the ESCs extracts between the different species etc. The main results in this paper were as follows:1. Effective generation pathways of blastocysts which were used to isolate the ESCs1.1Optimization of generation pathways of ICM from mouse blastocysts which produced from in vivo fertilizationThe effects of different gestational age and continuous passage on isolation, culture and growth status of MEFs were explored in this study in order to establish a effective system for the isolation and culture of MEFs, and to obtain the high-quality MEFs for preparing the feeder layer and acting as the target cells of reprogramming.3.5dpc (days post coitum),4dpc and4.5dpc mouse embryos were collected and cultured in different culture system including DMEM, DMEM+LIF and co-culture with MEFs respectively, the hatching time of ICM, attachment efficiencies of hatching blastocysts and formation efficiencies of ICM outgrowth were compared, in order to find out an effective method for obtaining high-quality ICM outgrowth for the isolation of the ESCs. Results indicated that12.5dpc to14.5dpc mouse embryos were appropriate for isolating the primary MEFs, the third generation MEFs were suitable for preparing the feeder layer.4dpc embryos were collected and co-cultured with MEFs for3days to4days, high-quality ICM outgrowth could be obtained for the isolation of ESCs.1.2Optimization of generation pathways of mouse heterozygous diploid blastocystsOocytes were activated with five methods of parthenogenetic activation, including ethanol, ethanol+6-DMAP, ethanol+6-DMAP+CB, SrCl2, SrCl2+CB. Then, activated oocytes were observed under the inverted microscope and karyotype analysis of activated oocytes were done to identify the parthenogenetic activation types of activated oocytes, including uniform haploid, heterozygous diploid with one pronucleus, mosaic haploid, and heterozygous diploid with two pronuclei. The development rate of morula and blastocyst from activated oocytes with different parthenogenetic activation types were compared to screen the appropriate activated method to obtain heterozygous diploid blastocysts. Results indicated that when oocytes were treated with10%alcohol for5min united with6-DMAP (6-dimethylaminopyridine) or with lOmM SrCl2and5μg/mL CB for4h, the extrusion of second polar body would be inhibitted, higher rate of diploid parthenogenetic embryos, overcome the two-cell block and generation of morula and blastocyst were produced. But, the morula and blastocyst development rate of oocytes were treated with lOmM SrCl2and5u g/ml CB for4h was higher than that treated with10%alcohol for5min united with6-DMAP. Therefore, those two parthenogenetic activation methods are the appropriate method to obtain the higher rate of mouse heterozygous diploid blastocysts.1.3Reconstruction of mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyteThe selection of suitable method for enucleating the germinal vesicle (GV) of MⅠ oocyte, GV oocyte being treated with cytochalasin B (CB) at appropriate concentrations before being enucleated, adhesion system between primary oocyte of new-born mouse and GV oocyte without GV, electrofusion parameteres for fusion of reconstructed oocyte and chemical activation of reconstructed embryo were explored in this study, in order to investigate the method for reconstructing mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyte. Results indicated that GV oocyte was pretreated with10μg/mL CB, GV oocyte was treated with0.5%pronase for removing the zona pellucida, GV oocyte without germinal vesicle was adhesived to primary oocyte of new-born mouse in the hollowness culture system, the adhesived oocyte was treated with the appropriate parameters (electric field strength was between1.5KV/cm and2.0KV/cm and the pulse width was40μs). The nuclear region of reconstructed oocyte which had been expelled the first polar body was transferred into the perivitelline space of MⅡ oocyte which the first polar body had been enucleated. The nuclear transfer embryo was fused by electrofusion, reconstructed embryo was activated with7%ethanol for5min united with2.5mmol/L6-DMAP for4h, then we can obtain the mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyte.2. Isolation, culture and identification of ESCs from blastocysts derived from in vivo fertilization, parthenogenetic activation, and reconstruction of embryo2.1Isolation, culture and identification of ESCs from blastocysts derived from in vivo fertilization13.5dpc mouse embryos were collected for the isolation of MEFs. Mitomycin C-treated MEFs at the third passages for preparing the feeder layer. ICMs were isolated from4dpc mouse embryos with intact embryo method and immunosurgury method, then they were transferred on the feeder layer and cultured in ESCs medium, finally cell colonies on the feeder layer were identified. Results indicated that there were no significantly differences in colony formation rate between intact embryo method and immunosurgury method (30.26±2.77%vs32.92±4.37%, P>0.05). The pluripotent nature of ESCs isolated with intact embryo method and immunosurgury method was identified by typical cellular and colonial morphology of ESCs, positive AKP staining, normal karyotype, positive Oct-4and SSEA-1immunocytochemistry staining, expression of Oct-4and Sox-2. These ESCs could also spontaneously differentiate into embryoid bodies which could express Fgf-5、Bra T and Afp.2.2Isolation, culture and identification of ESCs from blastocysts derived from parthenogenetic activationConsidering it was not easy for parthenogenetic blastocyst to hatch out from zona pellucida because of long time operation in vitro, immunosurgury method was used to isolated pESCs from uniform haploid blastocysts which developed from oocytes activated with SrCl2for4h, and pESCs from heterozygous diploid blastocysts which developed from oocytes activated with lOmM SrCl2and5μg/mL CB for4h, then cell colonies on the feeder layer were identified. Isolation efficiencies of pESCs from uniform haploid blastocysts and heterozygous diploid blastocysts were compared in order to select parthenogenetic blastocysts with suitable chromosome ploidy for the isolation of pESCs. Results indicated that the pluripotent nature of pESCs obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization. The colonies formation rate of pESCs from heterozygous diploid blastocysts was significantly higher than that from uniform haploid blastocysts (13.54±4.43%vs7.17±4.45%, P<0.05)。 Therefore, we should select the parthenogenetic activation method which could result in higher formation rate of heterozygous diploid blastocysts for the isolation of pESCs. 2.3Isolation, culture and identification of pESCs from blastocysts derived from reconstructed embryosIntact embryo method was used to isolated pESCs from mouse diploid parthenogenetic blastocysts contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MII oocyte. Results indicated that180morula and blastocyst derived from reconstructed embryos, after being cultured for48h morula and blastocyst developed into hatched blastocyst, after being cultured for96h ICM outgrowth were generated on the MEFs feeder layer,26pESCs colonies were observed after the ICM were dissociated into clusters of cells and transferred on the MEFs feeder layer, the pESCs colonies formation rate was14.43±4.36%. The pluripotent nature of pESCs obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization.3. Cell-free ESCs extracts or-mediated derivation of multipotent stem cells from MEFs3.1Effects of SLO at different concentration on growth state of MEFsThe aim of this study was to explore the comparatively suitable SLO concentration for reversible permeabilization of MEFs before those cells were used to reprogram into pluripotent stem cells using cell-free extracts, MEFs were incubated in PBS without Ca2+and Mg2+supplemented with SLO which was added to a final concentration of10U,25U,50U and100U SLO, cells density, survival rate and adherence rate of MEFs were measured. Results were as follows:No significant difference was found in cells density of MEFs between25U SLO treated group and10U SLO treated group (p>0.05), but the cells density of MEFs permeabilized with10U SLO or25U SLO was significantly higher than those permeabilized with50U SLO or100U SLO respectively (p<0.01). There was no significantly difference in the survival rate of MEFs permeabilized with25U SLO compared with the0U or10U SLO group respectively (p>0.05), but was significantly higher than those permeabilized with50U SLO (p<0.05). The adherence rate of MEFs permeabilized with25U SLO and resealed in2mM CaCl2was significantly higher than that with50U or100U SLO (p＜0.01). Proliferation of MEFs permeabilized with25U SLO was normal. Therefore,25U SLO was better suited to permeabilize MEFs considering both improving the efficiency of permeabilization and reducing the damage to MEFs.3.2Preparation of ESCs extract from Kunming mouse blastocysts derived from in vivo fertilization Effects of ultrasonic waves treatment and repeatedly freezing and thawing in liquid nitrogen on preparation of ESCs extract were explored in this study to obtain the high-efficiency lysis method to prepare the ESCs extract for programming of somatic cells. In order to detect and reduce the possibility of ESCs contamination during the preparation of ESCs extracts, ESCs extracts were transferred on the feeder layer and cultured in vitro, and MEFs without being permeablizated with streptolysin O were added to the ESCs extracts and were used to perform immunocytochemistry staining for Oct-4. Results were as follows:the vast majority of ESCs could be lysed after being repeatedly frozen and thawed in liquid nitrogen for seven times, the protein concentration were higher than that in ultrasonic waves treatment group (35.6mg/ml vs14.3mg/ml). There were no ESCs colonies observed on the feeder layer, MEFs without being permeablizated with streptolysin O revealed negative reaction to Oct-4immunocytochemstry staining. Therefore, ESCs were lysed and intracellular substances were destroyed as less as possible after ESCs were repeatedly freezing and thawing in liquid nitrogen for seven times, it provided a incubated culture system for reprogramming of somatic cells.3.3Studies on incubated system for mouse ESCs extract and MEFsEfficiencies of male MEFs which being pretreated with TSA and5-aza-dC or not being reprogrammed into multipotent stem cells by three kinds of female ESCs extracts respectively were compared in this study, male MEFs treated with male MEFs extracts and male MEFs without being treated with extracts were acted as controls, the pluripotent nature of multipotent stem cells obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization, the aim of the study was to optimize the reprogramming system which mediated by ESCs extracts. Results were as follows:male MEFs could be reprogrammed into multipotent stem cells by the female ESCs which be identified by dual PCR for sex identification system extracts from blastocysts derived from in vitro fertilization, parthenogenetic activation, and reconstruction of embryo respectively. The pluripotent nature of multipotent stem cells obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization. The number of colonies of multipotent stem cells from male MEFs which being treated with female ESCs extracts from blastocysts derived from in vivo fertilizationwere largest (64.33±3.7、80.67±2.08), following that of blastocysts derived from reconstruction of embryo (45.00±4.36、53.67±3.06), and that of blastocysts derived from parthenogenetic activation (35.67±2.52、47.67±4.16). Reprogramming of male MEFs could be improved by the pretreatment with TSA and5-Aza-dC, but it could not obtain the colonies of multipotent stem cells from male MEFs only pretreated with TSA and5-Aza-dC. Colony of multipotent stem cells was not be observed in control groups. Therefore, components which could reprogram the male MEFs into multipotent stem cells were from the ESCs extracts.4. Identification of the origin of multipotent stem cells from MEFs which been reprogrammed by ESCs extractsTo rule out the possibility of ESCs contamination during the preparation of ESCs extracts, and assess the reliability of the reprogramming system, further pluripotential stem cells generation were performed using male MEFs which were identified by dual PCR for sex identification system(which can amplify250bp Sry gene and399bp ZFX gene) of a different gender from extract-donor ESCs. The donors of ESCs-derived extract were a parthenogenetic embryonic stem cells (pESCs) from the oocytes (which can only amplify399bp ZFX gene) which been parthenogenetic activated and the female ESCs identified with dual PCR for sex identification system from the blastocyst produced from in vivo fertilization. Therefore, we can decided the origin of the pluripotential stem cells according to their gender using dual PCR for sex identification system. Results indicated that40colonies of multipotent stem cells from8batch reprogramming (5colonies of multipotent stem cells were randomly selected in every batch) which mediated by pESCs extracts, pESCs from the reconstructed embryos extract and female extract from in vivo fertilization were used to perform dual PCR for sex identification. Multipotent stem cells which had been amplified250bp Sry gene and399bp ZFX gene, that was there were male colonies. Therefore, the origin of multipotent stem cells obtained in our study were male MEFs, ESCs extracts were not the contaminated by donor ESCs during the preparation of pESCs extracts.5. Feasibility of interspecies somatic cells reprogramming-mediated by ESCs extractsChicken embryonic fibroblasts which being act as the target cells of reprogramming and were incubated with mouse ESCs extracts from Kunming mouse blastocysts derived from in vivo fertilization, morphology changes were observed, and the pluripotent nature of cells obtained from chicken embryonic fibroblasts after being reprogrammed by mouse ESCs extracts were identified, in order to explore the feasibility of reprogramming between the different species. Results indicated that after be incubated with mouse ESCs extracts, reprogrammed chicken embryonic fibroblasts turned to be round, fusiform adherent cells were not observed, and42.33±4.5colonies were generated. The pluripotent nature of the colonies was identified by typical cellular and colonial morphology of ESCs, positive AKP staining, and positive Oct-4and SSEA-1immunocytochemistry staining, and results from karyotype analysis indicated that those cells were derived from chicken embryonic fibroblasts. Chicken embryonic fibroblasts incubated with chicken embryonic fibroblasts extracts or without incubated with any extracts were not appeared the pluripotent nature, Therefore, components which could reprogram the chicken embryonic fibroblasts into multipotent stem cells were from the mouse ESCs extracts. Interspecies somatic cells reprogramming between mouse ESCs and chicken embryonic fibroblasts could be mediated by ESCs extracts.Conclusions:MEFs pretreated with SLO, TSA and5-Aza-dC could be reprogram ed into multipotent stem cells with cell-free ESCs extract in a shorter period. One day after treated with ESCs extract, MEFs formed the round cells, colonies with defined edges developed and resembled ESCs colonied within4days, these colonies showed the AKP activity on day3and they express Oct-4, Sox2and Nanog on day7. Chicken embryonic fibroblasts could be reprogrammed into multipotent stem cells using the reprogramming system mediated by mouse ESCs extract. |
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