The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses （ICMs） and fetal neural stem （FNS） cells. In ...The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses （ICMs） and fetal neural stem （FNS） cells. In result, three rat chimeras were produced by day 5 （D5） Sprague-Dawley （SD） blastocysts injected with ICMs derived from day 6 （D6） and D5 Dark Agouti （DA） blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs arc able to contribute to chimeras, but their potential decreases significantly after culture in vitro （P〈0.05）, and rFNS cells only have the potential to contribute to early fetal development.展开更多
Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture ...Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.展开更多
Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at ...Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at present limited by xeno-contamination during the in vitro derivation and propagation of these cells. In this review, we summarize the current methodologies for the derivation and the propagation of hESC in conditions that will eventually enable the generation of clinical-grade cells for future therapeutic applications.展开更多
Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identic...Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identical and distributed symmetrically in the mammalian embryo. Functionally, in some species, they are all totipotent. However, due to the compaction of blastomeres and the asymmetrical cell division at the late phase of the eight-cell embryo, the blastomeres of the morula are no longer identical. During the transition from morula to blastocyst, blastomeres differentiate, resulting in the first cell fate decision in embryogenesis, namely, the segregation of the inner cell mass and the tropheetoderm. In this review, we will discuss the regulatory mechanisms essential for the cell fate choice during blastocyst development, including transcriptional regulation, epigenetic regulation, mieroRNAs, and signal transduction.展开更多
The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embry...The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure. Bovine embryos formed four kinds of ICM colonies with different morphology including the mass-like, the net-like, the stream-like and the展开更多
Background： Despite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization （IVF） therapy, it is not yet clear which blastocyst morphological paralneters best...Background： Despite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization （IVF） therapy, it is not yet clear which blastocyst morphological paralneters best predict the outcomes of single blastocyst transfer. In addition. most of the previous studies did not exclude the effect of embryo aneuploidy on blastocysts transfer. Thus, the present study investigated the predictive value of various parameters on the pregnancy outcomes achieved via the transfer of frozcn euploid blastocysts. Methods： The study retrospectively analyzed 914 single euploid blastocyst transfer cycles that were performed at the Peking U laivcrsity Third Hospital Reproductive Medical Center between June 2011 and May 2016. The expansion, trophectoderm （TE）. and inner cell mass （ICM） quality of the blastocysts were assessed based on blastocyst parameters, and used to differentiate between ＂excellent＂, ＂good＂, ＂average＂, and ＂poor＂-quality embryos. The relationship between these embryo grades and the achieved pregnancy outcomes was then analyzed via the Chi-square and logistic regression tests. Results： For embryo grades of excellent, good, average and poor, the clinical pregnancy rates were 65.0%. 50.3%, 50.3% and 33.3%. respectively; and the live-birth rates were 50.0%, 49.7%, 42.3% and 25.0%, respectively. Both the clinical pregnancy ratc （x2= 21.28. P = 0.001） and live-birth rate （x2 = 13.50, P 〈 0.001） increased with the overall blastocyst grade. Both rates were significanlly higher after the transfer era blastocyst that exhibited either an A-grade or B-grade TE, and similarly, an A-grade ICM. than after the transfer el a blastocyst that exhibited a C-grade TE and/or ICM. The degree of blastocysl expansion had no apparent effect on the clinical pregnancy or live-birth rate. All odds ratio were adjusted for patient age, body mass index, length （years） of infertility history, and infertility type. Conclusions： A higher overall euploid blastocyst quality is shown to correlate most strongly with optimal pregnancy outcomes. The study thus supports the use of the described TE and ICM morphological grades to augment current embryo selection criteria.展开更多
In the field of developmental biology and regenerative medicine,mammalian interspecific chimeras have been proved very useful for investigating early embryonic development and the immune system establishment,and exten...In the field of developmental biology and regenerative medicine,mammalian interspecific chimeras have been proved very useful for investigating early embryonic development and the immune system establishment,and extended to a promising potential for human organ generation（Rossant et al.,1982）.展开更多
The intra- or inter-strain reconstitutedblastocysts were produced by replacing the inner cell mass of Kunming mouse blastocysts with that of Kunming orC57BL/6 mouse strain blastocysts. A total of 192 intra-strain reco...The intra- or inter-strain reconstitutedblastocysts were produced by replacing the inner cell mass of Kunming mouse blastocysts with that of Kunming orC57BL/6 mouse strain blastocysts. A total of 192 intra-strain reconstituted blastocysts were transferred into 17pseudopregnant Kunming mice, and 2 reconstituted embryos were developed into term; while 115 inter-strain reconstituted blastocysts were produced, analysis of thereconstituted blastocysts showed that the morphology andcytoskeleton srtucture of the blastocysts were not differentfrom those of normal blastocysts, however, no viableoffspring was obtained after embryo transfer for these inter-strain reconstituted blastocysts. The resultsdemonstrated that the intra-strain reconstituted blastocystscould normally develop into term, whereas the inter-strain reconstituted blastocysts possessed less developmental potential as the intra-strain reconstituted blastocysts. Thisstudy may give light to solve the problem of low implantation rate and placenta abnormality in mammal cloning.展开更多
文摘The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses （ICMs） and fetal neural stem （FNS） cells. In result, three rat chimeras were produced by day 5 （D5） Sprague-Dawley （SD） blastocysts injected with ICMs derived from day 6 （D6） and D5 Dark Agouti （DA） blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs arc able to contribute to chimeras, but their potential decreases significantly after culture in vitro （P〈0.05）, and rFNS cells only have the potential to contribute to early fetal development.
基金supported by the National Natural Science Foundation(C36970359).
文摘Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.
文摘Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at present limited by xeno-contamination during the in vitro derivation and propagation of these cells. In this review, we summarize the current methodologies for the derivation and the propagation of hESC in conditions that will eventually enable the generation of clinical-grade cells for future therapeutic applications.
文摘Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identical and distributed symmetrically in the mammalian embryo. Functionally, in some species, they are all totipotent. However, due to the compaction of blastomeres and the asymmetrical cell division at the late phase of the eight-cell embryo, the blastomeres of the morula are no longer identical. During the transition from morula to blastocyst, blastomeres differentiate, resulting in the first cell fate decision in embryogenesis, namely, the segregation of the inner cell mass and the tropheetoderm. In this review, we will discuss the regulatory mechanisms essential for the cell fate choice during blastocyst development, including transcriptional regulation, epigenetic regulation, mieroRNAs, and signal transduction.
基金supported by the National Natural Science Fundation(39670359)the Nation Tackle Key Problems Fund(96C0103)Zhanjiang Ocean University Young Expert Fund.
文摘The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure. Bovine embryos formed four kinds of ICM colonies with different morphology including the mass-like, the net-like, the stream-like and the
文摘Background： Despite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization （IVF） therapy, it is not yet clear which blastocyst morphological paralneters best predict the outcomes of single blastocyst transfer. In addition. most of the previous studies did not exclude the effect of embryo aneuploidy on blastocysts transfer. Thus, the present study investigated the predictive value of various parameters on the pregnancy outcomes achieved via the transfer of frozcn euploid blastocysts. Methods： The study retrospectively analyzed 914 single euploid blastocyst transfer cycles that were performed at the Peking U laivcrsity Third Hospital Reproductive Medical Center between June 2011 and May 2016. The expansion, trophectoderm （TE）. and inner cell mass （ICM） quality of the blastocysts were assessed based on blastocyst parameters, and used to differentiate between ＂excellent＂, ＂good＂, ＂average＂, and ＂poor＂-quality embryos. The relationship between these embryo grades and the achieved pregnancy outcomes was then analyzed via the Chi-square and logistic regression tests. Results： For embryo grades of excellent, good, average and poor, the clinical pregnancy rates were 65.0%. 50.3%, 50.3% and 33.3%. respectively; and the live-birth rates were 50.0%, 49.7%, 42.3% and 25.0%, respectively. Both the clinical pregnancy ratc （x2= 21.28. P = 0.001） and live-birth rate （x2 = 13.50, P 〈 0.001） increased with the overall blastocyst grade. Both rates were significanlly higher after the transfer era blastocyst that exhibited either an A-grade or B-grade TE, and similarly, an A-grade ICM. than after the transfer el a blastocyst that exhibited a C-grade TE and/or ICM. The degree of blastocysl expansion had no apparent effect on the clinical pregnancy or live-birth rate. All odds ratio were adjusted for patient age, body mass index, length （years） of infertility history, and infertility type. Conclusions： A higher overall euploid blastocyst quality is shown to correlate most strongly with optimal pregnancy outcomes. The study thus supports the use of the described TE and ICM morphological grades to augment current embryo selection criteria.
基金supported by the grants from the National Natural Science Foundation of China (Nos.31621004,31501188 and 31422038)the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences (QYZDY-SSW-SMC002)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16000000)
文摘In the field of developmental biology and regenerative medicine,mammalian interspecific chimeras have been proved very useful for investigating early embryonic development and the immune system establishment,and extended to a promising potential for human organ generation（Rossant et al.,1982）.
基金supported by the Climbing Project of the Ministry of Science and Technology of China(Climbing-special-08)the Knowledge Innovation Project of the Chinese Academy of Sciences(Grant No.KSCX1-05-01).
文摘The intra- or inter-strain reconstitutedblastocysts were produced by replacing the inner cell mass of Kunming mouse blastocysts with that of Kunming orC57BL/6 mouse strain blastocysts. A total of 192 intra-strain reconstituted blastocysts were transferred into 17pseudopregnant Kunming mice, and 2 reconstituted embryos were developed into term; while 115 inter-strain reconstituted blastocysts were produced, analysis of thereconstituted blastocysts showed that the morphology andcytoskeleton srtucture of the blastocysts were not differentfrom those of normal blastocysts, however, no viableoffspring was obtained after embryo transfer for these inter-strain reconstituted blastocysts. The resultsdemonstrated that the intra-strain reconstituted blastocystscould normally develop into term, whereas the inter-strain reconstituted blastocysts possessed less developmental potential as the intra-strain reconstituted blastocysts. Thisstudy may give light to solve the problem of low implantation rate and placenta abnormality in mammal cloning.
