The capability of human pluripotent stem cell(hPSC)lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research an...The capability of human pluripotent stem cell(hPSC)lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine.However,variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost.To screen the qualified cell lines and exclude problematic cell lines,their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by highthroughput gene expression profiling assay.Additionally,their differentiation potential,particularly the lineage-specific differentiation propensities of hPSC lines,should be predicted in an early stage.As a complement to the teratoma assay,RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo.Moreover,multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation.For clinical application of hPSCs,the malignant potential of the cells must also be evaluated.A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas,malignancy marker expression,and other parameters.Although various prediction methods are available,distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost,restricting their wide applications in routine studies.Therefore,simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.展开更多
Whereas conservative therapies aim to stall the advance of disease,regenerative medicine strives to reverse it.The capacity of most tissues to regenerate derives from stem cells,but there are a number of barriers whic...Whereas conservative therapies aim to stall the advance of disease,regenerative medicine strives to reverse it.The capacity of most tissues to regenerate derives from stem cells,but there are a number of barriers which have to be circumvented before it will be possible to use stem-cell-based therapies.Such therapies,however,are expected to improve human health enormously, and knowledge gained from studying stem cells in culture and in model organisms is now laying the groundwork for a new era of regenerative medicine.One of the most prominent methods to study stem cell differentiation is to let them to form embryoid bodies.Under favourable conditions any stem cell line will form embryoid bodies.However,the mechanism of the formation of embryoid bodies is not very well understood,and to produce them in the laboratory is in no way trivial-an important technical barrier in stem cell research.Recently,the embryoid body cultivation step has been successfully circumvented for the derivation of osteogenic cultures of embryonic stem cells.Here we report on a simple and reusable system to cultivate embryoid bodies in extremely short times.The method is inspired by the principles that lead to the establishment of the biomimetic triangle.展开更多
The differentiation and screening methodology proposed here is an efficient in vitro system to screen and study effects of small molecules and bioagents and is an alternative to studies that use live animals and embry...The differentiation and screening methodology proposed here is an efficient in vitro system to screen and study effects of small molecules and bioagents and is an alternative to studies that use live animals and embryos. The method is based on engineering a stable murine embryonic stem (ES) cell line expressing lineage-specific promoters that drive selection and reporter genes. Additionally, uniform embryoid bodies (EBs) are used for differentiation studies that allow synchronous differentiation. The reporter and selection marker genes are expressed only in lineages where the promoter is functional. The differentiated cell type can be identified by reporter gene expression and the selection marker can be used for selective enrichment of that particular cell population. The method described here is useful in screening small molecules or bioagents that can differentiate stem cells into particular lineages or cell types. Identified compounds are useful in areas such as stem cell-based regenerative medicine and therapeutics. The method described here has been applied to neuronal cell differentiation.展开更多
Human pluripotent stem cells(hPSCs)are important resources for cell-based therapies and pharmaceutical applications.In order to realize the potential of hPSCs,it is critical to develop suitable technologies required f...Human pluripotent stem cells(hPSCs)are important resources for cell-based therapies and pharmaceutical applications.In order to realize the potential of hPSCs,it is critical to develop suitable technologies required for specific applications.Most hPSC technologies depend on cell culture,and are critically influenced by culture medium composition,extracellular matrices,handling methods,and culture platforms.This review summarizes the major technological advances in hPSC culture,and highlights the opportunities and challenges in future therapeutic applications.展开更多
Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies ...Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of 展开更多
基金Supported by National Natural Science Foundation of China,No.81770621Ministry of Education,Culture,Sports,Science,and Technology of Japan,KAKENHI,No.16K15604 and No.18H02866Natural Science Foundation of Jiangsu Province,No.BK20180281
文摘The capability of human pluripotent stem cell(hPSC)lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine.However,variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost.To screen the qualified cell lines and exclude problematic cell lines,their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by highthroughput gene expression profiling assay.Additionally,their differentiation potential,particularly the lineage-specific differentiation propensities of hPSC lines,should be predicted in an early stage.As a complement to the teratoma assay,RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo.Moreover,multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation.For clinical application of hPSCs,the malignant potential of the cells must also be evaluated.A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas,malignancy marker expression,and other parameters.Although various prediction methods are available,distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost,restricting their wide applications in routine studies.Therefore,simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.
文摘Whereas conservative therapies aim to stall the advance of disease,regenerative medicine strives to reverse it.The capacity of most tissues to regenerate derives from stem cells,but there are a number of barriers which have to be circumvented before it will be possible to use stem-cell-based therapies.Such therapies,however,are expected to improve human health enormously, and knowledge gained from studying stem cells in culture and in model organisms is now laying the groundwork for a new era of regenerative medicine.One of the most prominent methods to study stem cell differentiation is to let them to form embryoid bodies.Under favourable conditions any stem cell line will form embryoid bodies.However,the mechanism of the formation of embryoid bodies is not very well understood,and to produce them in the laboratory is in no way trivial-an important technical barrier in stem cell research.Recently,the embryoid body cultivation step has been successfully circumvented for the derivation of osteogenic cultures of embryonic stem cells.Here we report on a simple and reusable system to cultivate embryoid bodies in extremely short times.The method is inspired by the principles that lead to the establishment of the biomimetic triangle.
文摘The differentiation and screening methodology proposed here is an efficient in vitro system to screen and study effects of small molecules and bioagents and is an alternative to studies that use live animals and embryos. The method is based on engineering a stable murine embryonic stem (ES) cell line expressing lineage-specific promoters that drive selection and reporter genes. Additionally, uniform embryoid bodies (EBs) are used for differentiation studies that allow synchronous differentiation. The reporter and selection marker genes are expressed only in lineages where the promoter is functional. The differentiated cell type can be identified by reporter gene expression and the selection marker can be used for selective enrichment of that particular cell population. The method described here is useful in screening small molecules or bioagents that can differentiate stem cells into particular lineages or cell types. Identified compounds are useful in areas such as stem cell-based regenerative medicine and therapeutics. The method described here has been applied to neuronal cell differentiation.
基金Supported by University of Macao Multi-Year Research Grants,No.MYRG2015-00228-FHS and MYRG2018-00135-FHSMacao Science and Technology Development Fund,No.FDCT/131/2014/A3 and FDCT/056/2015/A2
文摘Human pluripotent stem cells(hPSCs)are important resources for cell-based therapies and pharmaceutical applications.In order to realize the potential of hPSCs,it is critical to develop suitable technologies required for specific applications.Most hPSC technologies depend on cell culture,and are critically influenced by culture medium composition,extracellular matrices,handling methods,and culture platforms.This review summarizes the major technological advances in hPSC culture,and highlights the opportunities and challenges in future therapeutic applications.
文摘Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of
