A Serum- and Feeder-Free System to Generate CD4 and Regulatory T Cells from Human iPSCs
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ABSTRACT:
PROVIDER: PRJNA1018432 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26087341_1.fastq.gz | Fastqsanger.gz | |||
| SRR26087341_2.fastq.gz | Fastqsanger.gz | |||
| SRR26087342_1.fastq.gz | Fastqsanger.gz | |||
| SRR26087342_2.fastq.gz | Fastqsanger.gz | |||
| SRR26087343_1.fastq.gz | Fastqsanger.gz |
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Project description:While feeder-free generation of clinical grade iPSC-derived CD8 T cells has been achieved, differentiation of iPSC-derived CD4sp and regulatory T cells requires mouse stromal cells in an artificial thymic organoid. We developed a serum- and feeder-free differentiation process suitable for large-scale production and used scRNAseq to study the cell identity of the iPSC-derived cells and compared their transcriptional profile to primary human cells.
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Project description:Induced pluripotent stem cells (iPSCs) have been generated from various somatic cells under feeder-layer conditions. These feeder-derived iPSCs generated in different labs exhibit greater variability than between different traditional embryo derived hESC lines. For that reason, it is important to develop a standard and defined system for deriving autologous patient stem cells. We have generated iPSCs under feeder-free conditions using Matrigel coated vessels in chemically defined medium, mTeSR1. These feeder-free derived iPSCs are in many ways similar to feeder-derived iPSCs and also to hESCs, with respect to their pluripotent gene expression (OCT4, NANOG, SOX2), protein expression (OCT4, NANOG, SSEA4, TRA160) and differentiation capabilities. We conducted a whole genomic transcript analysis using Affymetrix Human Gene 1.0 ST arrays to elucidate the important differences between traditional feeder-derived iPSCs and feeder-free derived iPSCs. We reveal that feeder-free iPSCs have over-represented terms belonging to DNA replication and cell cycle genes which are lacking in feeder-derived iPSCs. Feeder-free iPSCs are in many aspects more similar to hESCs including; apoptosis, chromatin modification enzymes and mitochondrial energy metabolism. We have also identified potential biomarkers for fully reprogrammed iPSCs (FRZB) and partially reprogrammed iPSCs (POTEG, MX2) based on their expression trends across all cell types. In conclusion, feeder-free derived iPSCs is transcriptomically more similar to hESCs than feeder derived iPSCs, in many biological functions.
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Project description:Induced pluripotent stem cells (iPSCs) have been generated from various somatic cells under feeder-layer conditions. These feeder-derived iPSCs generated in different labs exhibit greater variability than between different traditional embryo derived hESC lines. For that reason, it is important to develop a standard and defined system for deriving autologous patient stem cells. We have generated iPSCs under feeder-free conditions using Matrigel coated vessels in chemically defined medium, mTeSR1. These feeder-free derived iPSCs are in many ways similar to feeder-derived iPSCs and also to hESCs, with respect to their pluripotent gene expression (OCT4, NANOG, SOX2), protein expression (OCT4, NANOG, SSEA4, TRA160) and differentiation capabilities. We conducted a whole genomic transcript analysis using Affymetrix Human Gene 1.0 ST arrays to elucidate the important differences between traditional feeder-derived iPSCs and feeder-free derived iPSCs. We reveal that feeder-free iPSCs have over-represented terms belonging to DNA replication and cell cycle genes which are lacking in feeder-derived iPSCs. Feeder-free iPSCs are in many aspects more similar to hESCs including; apoptosis, chromatin modification enzymes and mitochondrial energy metabolism. We have also identified potential biomarkers for fully reprogrammed iPSCs (FRZB) and partially reprogrammed iPSCs (POTEG, MX2) based on their expression trends across all cell types. In conclusion, feeder-free derived iPSCs is transcriptomically more similar to hESCs than feeder derived iPSCs, in many biological functions. For each cell sample, 2 or 3 biological replicates were obtained.
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Project description:Graphene has been selected as a candidate for synthetic feeder-free culture substrate guiding human/mouse multipotent stem cell lineage specification, and culturing pluripotent stem cells in a number of studies. However, conventional graphene is not an ideal biomaterial to maintain the pluripotency of human pluripotent stem cells (hPSC) including hESCs/hiPSCs due to its intrinsic hydrophobicity and relatively flat surface topography. Here, we applied morphology-controlled nanocrystalline graphene (NG) coating onto the culture substrates via diffusion-associated synthesis (DAS) process and cultivated hPSCs. It is found that enhanced hydrophilicity and controlled surface roughness of DAS-NG enabled tight focal adhesion of hPSCs onto the DAS-NG coated culture substrate and retained pluripotency for over 2 weeks. It is also found hPSCs grown on DAS-NG shared comparable global gene expression profile with hPSCs grown on mouse embryonic fibroblast (MEF). Importantly, the similarities in cell adhesion gene expression between hPSCs grown on DAS-NG and hPSCs on MEF suggest DAS-NG may provide comparable physical cues with MEF for sustaining pluripotency. Taken together, our findings show a new reliable method for culturing hPSCs in feeder-free condition using DAS-graphene. Human iPSCs and human ESC H9 were seeded onto DAS-NG coated glass, ITO or QU, CVD-grown graphene coated glass, uncoated glass and mitomycin-C treated CF1. Human pluripotent stem cells seeded on each culture substrate were cultured in human embryonic stem cell medium composed of knockout DMEM (GIBCO-10829) supplemented with 20% knockout serum replacement (GIBCO-10828), 1mM L-glutamine, 1% penicillin/streptomycin (PAA-P11-010), 1% MEM-non essential amino acid (PAA-M11-003), 0.1mM beta-mercaptoethanol, and 5ng/ml human basic fibroblast growth factor.
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