Real-Time PCR analysis of Embryoid Body differentiation of Human pluripotent stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE97963 | GEO | 2017/06/20
SECONDARY ACCESSION(S): PRJNA383535
REPOSITORIES: GEO
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Project description:The International Stem Cell Initiative compared three common approaches for assessing pluripotent stem cells (PSC). The formation of teratomas in vivo, or embryoid bodies (EB) in vitro, provide direct tests of differentiation, whereas PluriTest predicts pluripotency through bioinformatic analysis of transcriptomes of undifferentiated cells. We studied the teratomas by histology and TeratoScore, which analyzes gene expression in each tumor. For the EB assay, we assessed differentiation under neutral conditions and under conditions promoting differentiation to ectoderm, mesoderm, or endoderm lineages. Comparison of each method showed that all assays predict pluripotency, but they have different endpoints that provide different insights. For example, tumors from a subset of lines also contained undifferentiated cells and yolk sac elements that indicate possible malignant potential. This characteristic was not detected by the other two methods. Our results highlight the need for multiple assays to assess both pluripotency and potential malignancy in clinical applications of PSCs.
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Project description:Geminin is a small nucleoprotein that neuralizes ectoderm in the Xenopus embryo. Geminin promotes neural fate acquisition of mouse embryonic stem cells: Geminin knockdown during neural fate acquisition decreased expression of neural precursor cell markers (Pax6, Sox1), while increasing expression of Pitx2, Lefty1 and Cited2, genes involved in formation of the mouse node. Here we differentiated mouse embryonic stem cells into embryoid bodies to study Geminin's ability to repress primitive streak mesendoderm fate acquisition. We used microarrays to define the sets of genes that are regulated by Geminin during cell fate acquisition in embryoid bodies, using Dox-inducible Geminin knockdown or overexpression mouse embryonic stem cell lines. ES cell lines for Geminin over-expression (GemOE) were treated without or with Dox from day 3 to day 5 of EB differentiation and were collected on days 4 or 5 for microarray analysis. Gem knockdown (KD) ES cell lines were treated without or with Dox from day 0 to day 4 of EB differentiation and were collected on day 4 for microarray analysis.
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Project description:Stem cell differentiation strategies and optimization for generating lineage-specific cells and tissues most frequently rely on a three-dimensional embryoid body (EB) intermediate. We previously applied nanotechnology tools of photolithography to generate custom microarrays that allow high throughput uniform formation of EBs of custom size for precise downstream analysis. Formation of EBs of 200 or 500 micron size revealed distinct morphological differences that are single or multicystic cores, respectively, independent of method of formation from single cells or two-dimensional (2D) clusters. Here we utilize photolithographic array generated EBs to obtain 3D cultures under a standardized platform for transcriptome analysis to compare EB size and the method of EB formation from single cells or mechanically passaged 2D clusters. Our analysis evaluates RNA expression in EBs formed from the human embryonic stem cell (hESC) line WA09 and from ethnically diverse human induced pluripotent stem cell lines (ED-iPSC) of African American and Hispanic Latino ethnicity recently derived in our laboratory. This is the first comprehensive study on EB transcriptomes including multiple size parameters, EB formation methodologies, and ethnicities. Our analysis indicates upregulation of genes involved in wound healing for mechanically passaged cells and of genes for embryonic tube formation in 500 micron multicystic EBs. We propose that EB maturation may be a longer process then previously realized. In addition, the type or extent of maturation possible may be influenced by EB size, with larger EBs capable of more extensive remodeling as revealed by multicystic morphology and initiation of early tube formation pathways while retaining pluripotency status. We anticipate that this information will be broadly useful to the stem cell and bioengineering communities in optimization of tissue engineering with pluripotent stem cells and understanding sources of variation. mRNA profiles by RNA-seq from embryoid bodies generated by different methods
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Project description:Stem cell differentiation strategies and optimization for generating lineage-specific cells and tissues most frequently rely on a three-dimensional embryoid body (EB) intermediate. We previously applied nanotechnology tools of photolithography to generate custom microarrays that allow high throughput uniform formation of EBs of custom size for precise downstream analysis. Formation of EBs of 200 or 500 micron size revealed distinct morphological differences that are single or multicystic cores, respectively, independent of method of formation from single cells or two-dimensional (2D) clusters. Here we utilize photolithographic array generated EBs to obtain 3D cultures under a standardized platform for transcriptome analysis to compare EB size and the method of EB formation from single cells or mechanically passaged 2D clusters. Our analysis evaluates RNA expression in EBs formed from the human embryonic stem cell (hESC) line WA09 and from ethnically diverse human induced pluripotent stem cell lines (ED-iPSC) of African American and Hispanic Latino ethnicity recently derived in our laboratory. This is the first comprehensive study on EB transcriptomes including multiple size parameters, EB formation methodologies, and ethnicities. Our analysis indicates upregulation of genes involved in wound healing for mechanically passaged cells and of genes for embryonic tube formation in 500 micron multicystic EBs. We propose that EB maturation may be a longer process then previously realized. In addition, the type or extent of maturation possible may be influenced by EB size, with larger EBs capable of more extensive remodeling as revealed by multicystic morphology and initiation of early tube formation pathways while retaining pluripotency status. We anticipate that this information will be broadly useful to the stem cell and bioengineering communities in optimization of tissue engineering with pluripotent stem cells and understanding sources of variation.
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Project description:Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin mono-allelic expression of a small subset of mammalian genes. Imprinted genes have been categorized into two groups: multi lineage (ML) imprinted genes that can show imprinted expression in both embryonic and extra-embryonic lineages, and genes that show extra-embryonic lineage (EXEL) specific imprinted expression restricted to tissues like the placenta and visceral yolk sac (VYS) endoderm. Many genes showing EXEL imprinted expression are silenced by lncRNAs that act over long distances. Thus the analysis of this form of gene silencing is likely to be pivotal for understanding new mechanisms of long range gene silencing by lncRNAs. It has been reported that ES cells differentiated into cystic embryoid bodies (cystic EBs) contain VYS endoderm and here we investigate if cystic EBs could serve as an in vitro model for the analysis of EXEL imprinted expression. Unexpectedly we found that cystic EBs lack EXEL imprinted expression, while retaining normal imprinted expression of ML genes. This shows that cystic EBs do not model VYS imprinted expression and also argues against previous claims that cystic EBs contain VYS endoderm. We further characterized cystic EBs by performing RNA-seq and whole genome bisulphite sequencing. By comparison to various embryonic tissues we found that cystic EBs more resemble embryonic liver, which contains definitive endoderm, than the extra-embryonic endoderm of the VYS.
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