Human induced pluripotent stem cells(hiPSCs)are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use.They are particularly useful for st...Human induced pluripotent stem cells(hiPSCs)are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use.They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research.However,significant limitations exist when using conventional flat culturing methods especially concerning cell expansion,differentiation efficiency,stability maintenance and multicellular 3D structure establishment,differentiation prediction.Embryoid bodies(EBs),the multicellular aggregates spontaneously generated from iPSCs in the suspension system,might help to address these issues.Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve,EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing,differentiation efficiency enhancement,ex vivo simulation,and organoid establishment.EBs can potentially also be used in early prediction of iPSC differentiation capability.To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs,critical factors including iPSC pluripotency maintenance,generation of uniform morphology using micro-pattern 3D culture systems,proper cellular density inoculation,and EB size control are discussed on the basis of both published data and our own laboratory experiences.Collectively,the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.展开更多
Embryonic stem （ES） cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy.The main catalyst for ES c...Embryonic stem （ES） cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy.The main catalyst for ES cells differentiation is the growth into embryoid bodies （EBs）,which are utilized widely as the trigger of in vitro differentiation.In this study,a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established.When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds,they grew into aggregates gradually and formed simple EBs with circular structures.After 7 days＇ culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers.Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types;they were also able to form into tissue-like structures.Moreover,with introduction of ascorbic acid,ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19.The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.展开更多
Embryonic stem(ES)cells have the ability to differ-entiate into all germ layers,holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapie...Embryonic stem(ES)cells have the ability to differ-entiate into all germ layers,holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening.Embryoid body (EB)formation from ES cells is a common method for producing different cell lineages for further applications. However,conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation.For standardized mass EB production,a well defined scale-up platform is necessary.Recently,novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems(spinner flasks),rotating cell culture system and rotary orbital culture have allowed large-scale EB formation.Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods.This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems.Furthermore,an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently,new insights in induced pluripotent stem(iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research.These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity.Hence,culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells,although direct evidence of their use for iPS cells is still limited.展开更多
Mouse extended pluripotent stem(EPS)cells have demonstrated signifi cant potential for generating embryo models in vitro.However,their limited capacity for extraembryonic trophoblast development has hindered their use...Mouse extended pluripotent stem(EPS)cells have demonstrated signifi cant potential for generating embryo models in vitro.However,their limited capacity for extraembryonic trophoblast development has hindered their use in constructing whole embryo models,particularly post-implantation embryoids.Here,we establish a stepwise induction protocol to generate trophectoderm-like cells from mouse EPS cells.These cells retain trophectodermspecific transcriptomic features and can differentiate into trophoblast lineages in viuo.Moreover,combining these trophectoderm-like cells with EPS cell-derived primitive endoderm/epiblast bilineage structures enabled the robust generation of post-implantation embryoids in a transgene-free manner.EPS-derived embryoids recapitulate key developmental events of post-implantation mouse embryos,including the formation of the pro-amniotic cavity,anterior-posterior axis,primitive streak,gastrulation,and complex extraembryonic tissues.Notably,single-cell transcriptomic analysis revealed a high degree of transcriptional similarity between EPS-derived embryoids at day 6 and natural E7.5 mouse embryos.Our study presents a novel platform for modeling post-implantation mouse embryogenesis in vitro.展开更多
End-stage liver disease and acute liver failure are some of the most common causes of death worldwide,affecting over 40,000 patients in the United States,for most of whom liver transplantation is the only treatment.Tr...End-stage liver disease and acute liver failure are some of the most common causes of death worldwide,affecting over 40,000 patients in the United States,for most of whom liver transplantation is the only treatment.Transplantable livers are obtained primarily from deceased donors in the west and living donors in the east,with demand outstripping supply,leading to thousands of deaths each year for those on the transplant waiting lists.As a bridge to liver transplantation,human primary hepatocytes have been transplanted with low success,owing to their inability to grow and expand in vitro,their high sensitivity to cold storage-induced damage,and their dedifferentiation following two-dimensional culture.In the past decade,human induced pluripotent stem cells(hiPSCs)have been studied as a potential alternative to liver and primary hepatocyte transplantation through their differentiation into hepatocyte-like cells(HLCs).Differentiation of hiPSCs into HLCs is limited by the low percentage of differentiated cells that reach a mature hepatic phenotype,poor reproducibility of existing differentiation protocols,and inadequate long-term viability and function in vitro and in vivo.In this review,we will discuss the mechanisms of the various techniques that aim to improve the hepatic differentiation of hiPSCs into mature and genotypically stable HLCs for use in drug studies,as a potential bridge for liver transplantation after liver failure or as therapy for liver regeneration and replacement.展开更多
[Objective] The aim of this study is to reveal the disinfectants and disinfection methods,medium components,and embryoid culture method on dissociative microspore culture.[Method] B5 as basic medium appended with diff...[Objective] The aim of this study is to reveal the disinfectants and disinfection methods,medium components,and embryoid culture method on dissociative microspore culture.[Method] B5 as basic medium appended with different concentrations of sucrose,agar and different hormone combinations was used to optimize the culture technique for DH line in Brassica napus L.[Result] Both the 15 min disinfection of NaClO containing 5% Cl-and 10 min disinfection of 0.1% HgCl2 performed well in disinfection and subsequent embryo production;in the extraction process of dissociative microspores,B5 medium containing 2% sucrose could achieve a good embryo production effect;under dark condition microspores were firstly incubated at 32 ℃ 5-7 d,then at 25 ℃ 12-15 d,and finally transferred to 25 ℃ oscillator(60-65 r/min)for 3-7d,when the embryoid would become full ripeness;1/2MS medium appended with 1.2% agar,0.02% NAA,2.0 mg/L 6-BA,3.4 mg/L AgNO3 and 2% sucrose was helpful for embryoid differentiation and plantlet generation,presenting low degree of browning and slight vitrification.[Conclusion] The results may facilitate DH Line in rape production in large scale and high efficient transformation system.展开更多
[Objective]In order to increase anther culture efficiency of pepper.[Method]MS culture media and Bolajiaohong were used in this experiment to study the influences of carbon sources and concentrations on anther callus ...[Objective]In order to increase anther culture efficiency of pepper.[Method]MS culture media and Bolajiaohong were used in this experiment to study the influences of carbon sources and concentrations on anther callus induction of pepper.Jiayu was taken as a material to study influences of plant growth regulators and concentrations on anther callus induction of pepper according to L16（4^5） orthogonal design.[Result]The average callus and embryoid induction rates of maltose at all concentrations were higher than these of sucrose but the difference was not significant.Taking maltose or sucrose as a carbon source,3% to 6% concentration was good for increasing induction frequencies of calli and embryoids.However,If the concentration was over 6%,the induction rates were declined dramatically with the increase of sugar concentration.The influences of growth regulators on induction rate of calli were listed as 2,4-D﹥ZT﹥NAA﹥KT﹥6-BA;the influences on induction rates of embryoids were listed as 2,4-D﹥NAA﹥ZT﹥KT﹥6-BA.The 2,4-D,ZT,NAA and KT had signficant or extremely significant influences on induction rates of calli and embryoids.2,4-D,ZT at 1.0 mg/L and NNA,KT at 0.5 mg/L had the best effects.The influences of ZT on calli and embryoids were better than those of KT and 6-BA.1.0 mg/L 2,4-D ＋1.0 mg/L ZT ＋0.5 mg/L KT ＋0.5 mg/L 6-BA was the best regulator combination for induction culture of Jiayu anther.[Conclusion]The experiment provided research basis for anther culture of pepper.展开更多
基金Supported by National Natural Science Foundation of China,No.81770621,No.81573053Ministry of Education,Culture,Sports,Science,and Technology of Japan,KAKENHI,No.16K15604,No.18H02866Natural Science Foundation of Jiangsu Province,No.BK20180281
文摘Human induced pluripotent stem cells(hiPSCs)are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use.They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research.However,significant limitations exist when using conventional flat culturing methods especially concerning cell expansion,differentiation efficiency,stability maintenance and multicellular 3D structure establishment,differentiation prediction.Embryoid bodies(EBs),the multicellular aggregates spontaneously generated from iPSCs in the suspension system,might help to address these issues.Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve,EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing,differentiation efficiency enhancement,ex vivo simulation,and organoid establishment.EBs can potentially also be used in early prediction of iPSC differentiation capability.To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs,critical factors including iPSC pluripotency maintenance,generation of uniform morphology using micro-pattern 3D culture systems,proper cellular density inoculation,and EB size control are discussed on the basis of both published data and our own laboratory experiences.Collectively,the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.
基金supported by the National High Technology Research and Development Program of China (No 2006AA02A105 to CW)the National Nature Science Foundation of China (No 30530220)Beijing Nature Science Foundation of China (No 7062053)
文摘Embryonic stem （ES） cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy.The main catalyst for ES cells differentiation is the growth into embryoid bodies （EBs）,which are utilized widely as the trigger of in vitro differentiation.In this study,a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established.When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds,they grew into aggregates gradually and formed simple EBs with circular structures.After 7 days＇ culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers.Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types;they were also able to form into tissue-like structures.Moreover,with introduction of ascorbic acid,ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19.The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.
基金Supported by Grants from EU FP6("MEDRAT"-LSHG-CT-2005-518240"CLONET",MRTN-CT-2006-035468),EU FP7("Partn ErS",PIAP-GA-2008-218205+6 种基金"InduHeart",EU FP7-PEOPLE-IRG-2008-234390"InduStem",PIAP-GA-2008-230675"Plurisys",HEALTH-F4-2009-223485)NKFP_07_1-ES2HEART-HU,No.OM-00202-2007 CHE-TRF senior scholarship,No.RTA 5080010supported by grant under the program Strategic Scholarships for Frontier Research Network for the Joint Ph.D.Program Thai Doctoral degree from the Office of the Higher Education Commission,Thailand,No.CHE-PhD-SW-2005-100
文摘Embryonic stem(ES)cells have the ability to differ-entiate into all germ layers,holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening.Embryoid body (EB)formation from ES cells is a common method for producing different cell lineages for further applications. However,conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation.For standardized mass EB production,a well defined scale-up platform is necessary.Recently,novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems(spinner flasks),rotating cell culture system and rotary orbital culture have allowed large-scale EB formation.Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods.This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems.Furthermore,an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently,new insights in induced pluripotent stem(iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research.These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity.Hence,culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells,although direct evidence of their use for iPS cells is still limited.
基金supported by the National Key R&D Program of China(2021YFA1100300)the National Natural Science Foundation of China(32370843,32270603,32288102,32025006)+2 种基金the Beijing Natural Science Foundation(JQ24042)the Beijing Natural Science Foundation(BJNSF,5242010)the Fundamental Research Funds for the Central Universities(BMU2021YJ057).
文摘Mouse extended pluripotent stem(EPS)cells have demonstrated signifi cant potential for generating embryo models in vitro.However,their limited capacity for extraembryonic trophoblast development has hindered their use in constructing whole embryo models,particularly post-implantation embryoids.Here,we establish a stepwise induction protocol to generate trophectoderm-like cells from mouse EPS cells.These cells retain trophectodermspecific transcriptomic features and can differentiate into trophoblast lineages in viuo.Moreover,combining these trophectoderm-like cells with EPS cell-derived primitive endoderm/epiblast bilineage structures enabled the robust generation of post-implantation embryoids in a transgene-free manner.EPS-derived embryoids recapitulate key developmental events of post-implantation mouse embryos,including the formation of the pro-amniotic cavity,anterior-posterior axis,primitive streak,gastrulation,and complex extraembryonic tissues.Notably,single-cell transcriptomic analysis revealed a high degree of transcriptional similarity between EPS-derived embryoids at day 6 and natural E7.5 mouse embryos.Our study presents a novel platform for modeling post-implantation mouse embryogenesis in vitro.
文摘End-stage liver disease and acute liver failure are some of the most common causes of death worldwide,affecting over 40,000 patients in the United States,for most of whom liver transplantation is the only treatment.Transplantable livers are obtained primarily from deceased donors in the west and living donors in the east,with demand outstripping supply,leading to thousands of deaths each year for those on the transplant waiting lists.As a bridge to liver transplantation,human primary hepatocytes have been transplanted with low success,owing to their inability to grow and expand in vitro,their high sensitivity to cold storage-induced damage,and their dedifferentiation following two-dimensional culture.In the past decade,human induced pluripotent stem cells(hiPSCs)have been studied as a potential alternative to liver and primary hepatocyte transplantation through their differentiation into hepatocyte-like cells(HLCs).Differentiation of hiPSCs into HLCs is limited by the low percentage of differentiated cells that reach a mature hepatic phenotype,poor reproducibility of existing differentiation protocols,and inadequate long-term viability and function in vitro and in vivo.In this review,we will discuss the mechanisms of the various techniques that aim to improve the hepatic differentiation of hiPSCs into mature and genotypically stable HLCs for use in drug studies,as a potential bridge for liver transplantation after liver failure or as therapy for liver regeneration and replacement.
基金Supported by the Fund of Science and Technology in Guizhou Province[(2007)0281]the Fund of Guizhou Academy of Agricultural Sciences[(2007)013]+2 种基金the Nomarch Special Foundation for the Excellent Science and Technology Talents of Guizhou Province[(2005)40]Key Science and Technology Project in Guizhou Province[(2006)3008]Program from Science and Technology Department of Guizhou Province[(2008)-008]~~
文摘[Objective] The aim of this study is to reveal the disinfectants and disinfection methods,medium components,and embryoid culture method on dissociative microspore culture.[Method] B5 as basic medium appended with different concentrations of sucrose,agar and different hormone combinations was used to optimize the culture technique for DH line in Brassica napus L.[Result] Both the 15 min disinfection of NaClO containing 5% Cl-and 10 min disinfection of 0.1% HgCl2 performed well in disinfection and subsequent embryo production;in the extraction process of dissociative microspores,B5 medium containing 2% sucrose could achieve a good embryo production effect;under dark condition microspores were firstly incubated at 32 ℃ 5-7 d,then at 25 ℃ 12-15 d,and finally transferred to 25 ℃ oscillator(60-65 r/min)for 3-7d,when the embryoid would become full ripeness;1/2MS medium appended with 1.2% agar,0.02% NAA,2.0 mg/L 6-BA,3.4 mg/L AgNO3 and 2% sucrose was helpful for embryoid differentiation and plantlet generation,presenting low degree of browning and slight vitrification.[Conclusion] The results may facilitate DH Line in rape production in large scale and high efficient transformation system.
文摘[Objective]In order to increase anther culture efficiency of pepper.[Method]MS culture media and Bolajiaohong were used in this experiment to study the influences of carbon sources and concentrations on anther callus induction of pepper.Jiayu was taken as a material to study influences of plant growth regulators and concentrations on anther callus induction of pepper according to L16（4^5） orthogonal design.[Result]The average callus and embryoid induction rates of maltose at all concentrations were higher than these of sucrose but the difference was not significant.Taking maltose or sucrose as a carbon source,3% to 6% concentration was good for increasing induction frequencies of calli and embryoids.However,If the concentration was over 6%,the induction rates were declined dramatically with the increase of sugar concentration.The influences of growth regulators on induction rate of calli were listed as 2,4-D﹥ZT﹥NAA﹥KT﹥6-BA;the influences on induction rates of embryoids were listed as 2,4-D﹥NAA﹥ZT﹥KT﹥6-BA.The 2,4-D,ZT,NAA and KT had signficant or extremely significant influences on induction rates of calli and embryoids.2,4-D,ZT at 1.0 mg/L and NNA,KT at 0.5 mg/L had the best effects.The influences of ZT on calli and embryoids were better than those of KT and 6-BA.1.0 mg/L 2,4-D ＋1.0 mg/L ZT ＋0.5 mg/L KT ＋0.5 mg/L 6-BA was the best regulator combination for induction culture of Jiayu anther.[Conclusion]The experiment provided research basis for anther culture of pepper.
