Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of ...Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stern cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, ~lll-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neuregenesis related genes （Sox-1, Nestin, GABA, Pax6, Zic5 and Pitxl） in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.展开更多
Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells （ESCs）, which may be an important source ofhistocompatible cells and tissues for cell therapy. Here we describe the derivat...Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells （ESCs）, which may be an important source ofhistocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines （hPES-1 and hPES-2） from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA- 1-60, and TRA- 1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies （EBs） of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers （hPES-2 did not）. Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat （STR） results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.展开更多
A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was d...A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was demonstrated to confer a HIV resistance, resulting in discontinuation of antiretroviral therapy. In search for an unlimited source of CCR5-del32 cells for transplantation purposes, we tested 137 human embryonic stem cell (hESC) lines from the Reproductive Genetics Institute’s hESC lines collection, and report here the finding of 12 hESC lines with the CCR5-del32 allele, one of which represents a unique partenogenetic ESC line containing two copies of this deletion and may be studied for utility in stem cell transplantation treatment of HIV.展开更多
Parthenogenetic embryonic stem cells（pE SCs）, as ＂seed cells＂ for regenerative medicine, are an effective way to build patient-specific pluripotent stem cells, due to the fact that characteristics of self-renewal ...Parthenogenetic embryonic stem cells（pE SCs）, as ＂seed cells＂ for regenerative medicine, are an effective way to build patient-specific pluripotent stem cells, due to the fact that characteristics of self-renewal and pluripotent are similar to embryonic stem cells（ESCs）. Parthenogenetic activation can be performed at meiosis I or meiosis II describing the embryos with distinct patterns of homozygosity and heterozygosity.Heterozygous pE SCs are expected to be used for autologous transplantation, while homozygous pE SCs enable to be used for allogeneic gene therapy in theory but is hampered by immunological barriers defined by the recognition of natural killer（NK）cells. In this review, we describe the mechanism of deriving heterozygous and homozygous pE SCs, and summarize the advantages and limitations of pE SCs in the area of cell therapy.展开更多
Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem(iPS) cells present an important model for biomedical research and provide potential resources fo...Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem(iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies.However,the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells.Embryonic stem cells(ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy.Recent studies on human parthenogenetic embryonic stem cells(hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics,but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions.To generate various pluripotent stem cells,diverse reprogramming techniques and approaches will be developed and integrated.This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology,and ultimately benefit cell therapy and regenerative medicine.展开更多
Parthenogenetic embryos,created by activation and diploidization of oocytes,arrest at mid-gestation for defective paternal imprints,which impair placental development.Also,viable offspring has not been obtained withou...Parthenogenetic embryos,created by activation and diploidization of oocytes,arrest at mid-gestation for defective paternal imprints,which impair placental development.Also,viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells(pESCs)derived from parthenogenetic embryos,presumably attributable to their aberrant imprinting.We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring.Moreover,normal expression of imprinted genes is found in the germ cells and the mice.pESCs exhibited imprinting consistent with exclusively maternal lineage,and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background.pESCs differentiated into primordial germ cell-like cells(PGCLCs)and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function.The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs,consistent with efficient reprogramming of methylation and genomic imprinting.These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting,offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.展开更多
MicroRNAs（miRNAs） are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions.In this study,miRNA and messenger RNA（mRNA） profiles were examined by Illumin...MicroRNAs（miRNAs） are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions.In this study,miRNA and messenger RNA（mRNA） profiles were examined by Illumina microarray in mouse embryonic stem cells（ESCs） derived from parthenogenetic,androgenetic,and fertilized blastocysts.The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression.Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs（aESCs） and fertilized ESCs（fESCs）,a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs（pESCs） and fESCs,and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs.In addition,a total of 575,5 and 376 miRNA-mRNA target pairs were observed in aESCs vs.fESCs,pESCs vs.fESCs,and aESCs vs.pESCs,respectively.Furthermore,15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR.Finally,transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3,Dyrklb,and Prrg2 mRNA,inhibition of miR-363 increased the expression of Nfat5 and Soatl mRNA,and inhibition of miR-883b-5p increased Nfat5,Tacstd2,and Ppapdc1 mRNA.These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.展开更多
Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology.However,the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate th...Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology.However,the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches.Recently,mouse haploid embryonic stem(haES)cells have been successfully isolated from parthenogenetic and androgenetic embryos,providing an ideal tool for genetic analyses.In these studies,mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination.In particular,haES cells from androgenetic embryos can be employed as novel,renewable form of fertilization agent for yielding live-born mice via injection into oocytes,thus showing the possibility that genetic analysis can be extended from cellular level to organism level.展开更多
Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro a...Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential.Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated(IVO)oocytes or from the in vitro-matured(IVM)oocytes with that of fertilized embryonic stem(fES)cells derived from fertilized embryos.A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells,whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value.No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes.Notably,no differences were found in the protein expression of imprinted genes between the pES and fES cells,suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages.The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.展开更多
基金supported by the National Natural Science Foundation of China,No. 30900155 and 81070496the Research Foundation of Education Bureau of Shaanxi Province,China,No. 09JK785+1 种基金Foundation of Interdisciplinary for Postgraduates from Northwest University,No. 08YJC22the Key Laboratory Funding of Northwestern University,Shaanxi Province in China
文摘Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stern cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, ~lll-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neuregenesis related genes （Sox-1, Nestin, GABA, Pax6, Zic5 and Pitxl） in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.
文摘Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells （ESCs）, which may be an important source ofhistocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines （hPES-1 and hPES-2） from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA- 1-60, and TRA- 1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies （EBs） of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers （hPES-2 did not）. Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat （STR） results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.
文摘A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was demonstrated to confer a HIV resistance, resulting in discontinuation of antiretroviral therapy. In search for an unlimited source of CCR5-del32 cells for transplantation purposes, we tested 137 human embryonic stem cell (hESC) lines from the Reproductive Genetics Institute’s hESC lines collection, and report here the finding of 12 hESC lines with the CCR5-del32 allele, one of which represents a unique partenogenetic ESC line containing two copies of this deletion and may be studied for utility in stem cell transplantation treatment of HIV.
文摘Parthenogenetic embryonic stem cells（pE SCs）, as ＂seed cells＂ for regenerative medicine, are an effective way to build patient-specific pluripotent stem cells, due to the fact that characteristics of self-renewal and pluripotent are similar to embryonic stem cells（ESCs）. Parthenogenetic activation can be performed at meiosis I or meiosis II describing the embryos with distinct patterns of homozygosity and heterozygosity.Heterozygous pE SCs are expected to be used for autologous transplantation, while homozygous pE SCs enable to be used for allogeneic gene therapy in theory but is hampered by immunological barriers defined by the recognition of natural killer（NK）cells. In this review, we describe the mechanism of deriving heterozygous and homozygous pE SCs, and summarize the advantages and limitations of pE SCs in the area of cell therapy.
基金Supported by the National High Technology Research and Development Program of China (Grant No. 2006AA02A101)
文摘Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem(iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies.However,the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells.Embryonic stem cells(ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy.Recent studies on human parthenogenetic embryonic stem cells(hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics,but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions.To generate various pluripotent stem cells,diverse reprogramming techniques and approaches will be developed and integrated.This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology,and ultimately benefit cell therapy and regenerative medicine.
基金This work was supported by China National Key R&D Program(2018YFC1003004,2018YFA0107002)the National Natural Science Foundation of China(31430052,91749129)as well as the Stanley H.Kaplan Research Fund at NYU School of Medicine.
文摘Parthenogenetic embryos,created by activation and diploidization of oocytes,arrest at mid-gestation for defective paternal imprints,which impair placental development.Also,viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells(pESCs)derived from parthenogenetic embryos,presumably attributable to their aberrant imprinting.We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring.Moreover,normal expression of imprinted genes is found in the germ cells and the mice.pESCs exhibited imprinting consistent with exclusively maternal lineage,and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background.pESCs differentiated into primordial germ cell-like cells(PGCLCs)and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function.The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs,consistent with efficient reprogramming of methylation and genomic imprinting.These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting,offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.
基金supported by the grants from the NextGeneration BioGreen 21 Program(Nos.PJ009080 and PJ00909801)Rural Development Administration,Republic of Korea
文摘MicroRNAs（miRNAs） are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions.In this study,miRNA and messenger RNA（mRNA） profiles were examined by Illumina microarray in mouse embryonic stem cells（ESCs） derived from parthenogenetic,androgenetic,and fertilized blastocysts.The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression.Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs（aESCs） and fertilized ESCs（fESCs）,a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs（pESCs） and fESCs,and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs.In addition,a total of 575,5 and 376 miRNA-mRNA target pairs were observed in aESCs vs.fESCs,pESCs vs.fESCs,and aESCs vs.pESCs,respectively.Furthermore,15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR.Finally,transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3,Dyrklb,and Prrg2 mRNA,inhibition of miR-363 increased the expression of Nfat5 and Soatl mRNA,and inhibition of miR-883b-5p increased Nfat5,Tacstd2,and Ppapdc1 mRNA.These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.
基金supported by grants from the Ministry of Science and Technology of China(No.2009CB941101 to J.L.)the "Strategic Priority Research Program" of the Chinese Academy of Sciences(No.XDA01010403 to J.L.).
文摘Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology.However,the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches.Recently,mouse haploid embryonic stem(haES)cells have been successfully isolated from parthenogenetic and androgenetic embryos,providing an ideal tool for genetic analyses.In these studies,mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination.In particular,haES cells from androgenetic embryos can be employed as novel,renewable form of fertilization agent for yielding live-born mice via injection into oocytes,thus showing the possibility that genetic analysis can be extended from cellular level to organism level.
基金supported by MOST National Major Basic Research Program(Grant Nos.2010CB94500,2009CB941000(to LL),and 2010CB833703(to FY)).
文摘Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential.Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated(IVO)oocytes or from the in vitro-matured(IVM)oocytes with that of fertilized embryonic stem(fES)cells derived from fertilized embryos.A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells,whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value.No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes.Notably,no differences were found in the protein expression of imprinted genes between the pES and fES cells,suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages.The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.
