Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of human embryonic stem cells (hESC) line Amicqui-1 derived and maintained on human amniotic epithelial cells

ABSTRACT: Conventional conditions to maintain human embryonic stem cells (hESC) imply the use of inactive mouse embryonic fibroblast (iMEF) as a feeder layer. However, it has suggested that the culture of hESC on iMEF could be an artifact that does not correspond to the in vitro counterpart of the human epiblast. We previously derived and maintained human embryonic stem cells (Amicqui-1 hESC line) on a feeder layer of human amniotic epithelial cells (hAEC). However, the mechanisms involved in the interaction between both cell types to promote the pluripotency still remain unknown. To elucidate if the transcription profile of hESC on hAEC differs from conventional conditions on iMEF, we carried out RNA-seq of Amicqui-1 hESC on both feeder layer conditions (AMIQ hESC-iMEF and AMIQ hESC-hAEC) and on their respective conditioned media (AMIQ hESC-hAEC-CM and AMIQ hESC-iMEF-CM). In this experiment we included H1-hESC-iMEF and hAEC alone.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Daniela Ávila-González

PROVIDER: E-MTAB-10347 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder-free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation towards hematopoietic lineage than hESCs grown in standard human foreskin fibroblast (HFF)-conditioned media (HFF-CM). We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. Human ESCs grown in MSC-CM showed a decrease of 20% in global DNA methylation and a promoter DNA methylation signature consisting in 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition towards hematopoietic specification. Total DNA isolated by standard procedures from human embryonic stem cells (hESC) cultured in different conditioned media

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RNA-seq of human embryonic stem cells (hESC) line Amicqui-1 derived and maintained on human amniotic epithelial cells

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