The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,...The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,a system enabling long-term maintenance of imprints in ESCs has proven challenging.Here,we report that the use of a two-step a2i(alternative two inhibitors of Src and Gsk3β,TSa2i)derivation/culture protocol results in the establishment of androgenetic haploid ESCs(AG-haESCs)with stable DNA methylation at paternal DMRs(differentially DNA methylated regions)up to passage 60 that can efficiently support generating mice upon oocyte injection.We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations.Furthermore,we demonstrate that TSa2itreated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation.Strikingly,AGhaESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells,in part through the enhanced proliferation of H19-DMR hypermethylated cells.Together,we establish AG-haESCs that can longterm maintain paternal imprints.展开更多
While Mek1/2 and Gsk3βinhibition("2i")supports the maintenance of murine embryonic stem cells(EsCs)in a homogenous naive state,prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impa...While Mek1/2 and Gsk3βinhibition("2i")supports the maintenance of murine embryonic stem cells(EsCs)in a homogenous naive state,prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential.Additionally,2i fails to support derivation and culture of fully potent female ESCs.Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin(AlbuMAx)undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture.Mechanisticaily,lipids in AlbuMAx impact intracellular metabolism including nucleotide biosynthesis,lipid biogenesis,and TCA cycle intermediates,with enhanced expression of DNMT3s that prevent DNA hypomethylation.Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation,which also alleviates X chromosome loss in female ESCs.Importantly,both male and female"all-ESc"mice can be generated from de novo derived ESCs using AlbuMAXbased media.Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.展开更多
Mouse embryonic stem cells(mESCs)derived from inner cell mass(ICM)of pre-implantation embryos,can maintain undifferentiated state when cultured in N2B27 medium supplemented with GSK3inhibitor CHIR99021 and MEK inhibit...Mouse embryonic stem cells(mESCs)derived from inner cell mass(ICM)of pre-implantation embryos,can maintain undifferentiated state when cultured in N2B27 medium supplemented with GSK3inhibitor CHIR99021 and MEK inhibitor PD0325901(‘‘2i’’)and leukemia inhibitor factor(LIF).Compare to conventional culture medium,all components of this medium are defined.With the N2B27 medium,‘‘2i’’and LIF,mESCs can contribute to the germline of the chimeric embryos,however,whether the‘‘all-ES cells’’mice can been generated by tetraploid complementation is unclear yet,while the tetraploid complementation serve as a golden standard to assess the pluripotency of ES cells.Here,our study showed that mESCs derived and cultured with the N2B27 complete medium could generate fertile mice by tetraploid complementation.In addition,the survival rate of tetraploid complementation mice produced by inbred mES cell lines is higher than the conventional culture condition,and increased the percentage of Oct4 positive cells contrast to conventional medium either.Therefore,the N2B27 medium supplemented with‘‘2i’’and LIF is an alternative choice forthe derivation and long-term culture of mouse embryonic stem cells.展开更多
用液相沉积法对纳米ZnO/TiO2进行了表面改性。用TEM、XRD和FTIR对产物进行了结构表征,用静态沉淀法分析了改性前后纳米ZnO/TiO2的分散稳定性,用紫外-可见分光光度计对其紫外屏蔽性能进行了检测。结果表明,改性纳米ZnO/TiO2表面存在致密...用液相沉积法对纳米ZnO/TiO2进行了表面改性。用TEM、XRD和FTIR对产物进行了结构表征,用静态沉淀法分析了改性前后纳米ZnO/TiO2的分散稳定性,用紫外-可见分光光度计对其紫外屏蔽性能进行了检测。结果表明,改性纳米ZnO/TiO2表面存在致密的氧化铝膜,产物经充分分散后在有机介质中或水中的稳定时间分别由改性前的2 m in和5 m in提高到2 h和1 d,紫外线透过率由改性前的大于8.5%降低到小于7%。展开更多
基金This study was supported by Genome Tagging Project and grants from the Chinese Academy of Sciences,the National Key Research and Development Program of Chinathe National Natural Science Foundation of China(2019YFA0109900,2020YFA0509000,XDB19010204,QYZDJ-SSW-SMC023,Facility-based Open Research Program,31821004,32030029,and 31730062).
文摘The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,a system enabling long-term maintenance of imprints in ESCs has proven challenging.Here,we report that the use of a two-step a2i(alternative two inhibitors of Src and Gsk3β,TSa2i)derivation/culture protocol results in the establishment of androgenetic haploid ESCs(AG-haESCs)with stable DNA methylation at paternal DMRs(differentially DNA methylated regions)up to passage 60 that can efficiently support generating mice upon oocyte injection.We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations.Furthermore,we demonstrate that TSa2itreated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation.Strikingly,AGhaESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells,in part through the enhanced proliferation of H19-DMR hypermethylated cells.Together,we establish AG-haESCs that can longterm maintain paternal imprints.
基金supported by the New York State Stem Cell Science Program under contract C32581GGthe National Institutes of Health under award numbers:1 R01 GM129380-01 and 1R210OD031973-01 (to D.W.).
文摘While Mek1/2 and Gsk3βinhibition("2i")supports the maintenance of murine embryonic stem cells(EsCs)in a homogenous naive state,prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential.Additionally,2i fails to support derivation and culture of fully potent female ESCs.Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin(AlbuMAx)undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture.Mechanisticaily,lipids in AlbuMAx impact intracellular metabolism including nucleotide biosynthesis,lipid biogenesis,and TCA cycle intermediates,with enhanced expression of DNMT3s that prevent DNA hypomethylation.Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation,which also alleviates X chromosome loss in female ESCs.Importantly,both male and female"all-ESc"mice can be generated from de novo derived ESCs using AlbuMAXbased media.Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.
基金supported by the National Basic Research Program of China(2012CBA01300 and2012CB966500)
文摘Mouse embryonic stem cells(mESCs)derived from inner cell mass(ICM)of pre-implantation embryos,can maintain undifferentiated state when cultured in N2B27 medium supplemented with GSK3inhibitor CHIR99021 and MEK inhibitor PD0325901(‘‘2i’’)and leukemia inhibitor factor(LIF).Compare to conventional culture medium,all components of this medium are defined.With the N2B27 medium,‘‘2i’’and LIF,mESCs can contribute to the germline of the chimeric embryos,however,whether the‘‘all-ES cells’’mice can been generated by tetraploid complementation is unclear yet,while the tetraploid complementation serve as a golden standard to assess the pluripotency of ES cells.Here,our study showed that mESCs derived and cultured with the N2B27 complete medium could generate fertile mice by tetraploid complementation.In addition,the survival rate of tetraploid complementation mice produced by inbred mES cell lines is higher than the conventional culture condition,and increased the percentage of Oct4 positive cells contrast to conventional medium either.Therefore,the N2B27 medium supplemented with‘‘2i’’and LIF is an alternative choice forthe derivation and long-term culture of mouse embryonic stem cells.
文摘用液相沉积法对纳米ZnO/TiO2进行了表面改性。用TEM、XRD和FTIR对产物进行了结构表征,用静态沉淀法分析了改性前后纳米ZnO/TiO2的分散稳定性,用紫外-可见分光光度计对其紫外屏蔽性能进行了检测。结果表明,改性纳米ZnO/TiO2表面存在致密的氧化铝膜,产物经充分分散后在有机介质中或水中的稳定时间分别由改性前的2 m in和5 m in提高到2 h和1 d,紫外线透过率由改性前的大于8.5%降低到小于7%。
