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
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 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
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 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 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.
