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Optimized Approaches for Generation of Integration-free iPSCs from Human Urine-derived Cells with Small Molecules and Autologous Feeder

ABSTRACT: We generated iPSCs from human urine cells (hUCs) with the aid of small molecules and autologous hUC feeders. A compound cocktail including Cyclic Pifithrin-a, a p53 inhibitor and other compounds known for benefiting reprogramming like A-83-01, CHIR99021, Thiazovivin, NaB and PD0325901 was used to aid hUC reprogramming (Plan B). Aided by this cocktail, we achieved significantly improved efficiency (170 folds more) for hUC reprogramming and iPSC generation. In addition, to enable iPSC generation in some cases that massive cell death occurred during delivering reprogramming factors, we replaced Matrigel with autologous hUCs as feeder for reprogramming and iPSC generation (Plan C). Replacing Matrigel with autologous feeder not only enhanced reprograming, but also avoided concern using animal components for human iPSC generation. These were efficient approaches to enable iPSC generation from hUCs that were otherwise difficult for reprogramming, which would be valuable for banking patient’s specific iPSCs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE79134 | GEO | 2017/02/10

SECONDARY ACCESSION(S): PRJNA314996

REPOSITORIES: GEO

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Project description:Emergence of induced pluripotent stem cells (iPSC) technology has paved novel routes for regenerative medicine. iPSCs offer the possibilities of disease modeling, drug toxicity studies as well as cell replacement therapies by autologous transplantation. Classical protocols of iPSC generation harness infection by retro- or lenti-viruses. Although such integrating viruses represent very robust tools for reprogramming, the presence of viral transgenes in iPSCs is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Moreover, remaining reprogramming transgenes have been shown to affect the differentiation potential of iPSCs. More recently, alternative protocols have been explored to derive transgene-free iPSC, including use of transposons, mRNA transfection, episomal plasmid transfection, and infection with non-integrating viruses such as Sendai virus. However, the utility of such protocols remains limited due to low efficiency and narrow range of cell specificity. In this study we aim at combining the robustness of lentiviral reprogramming with the high efficacy of Cre recombinase protein transduction to readily delete reprogramming transgenes from iPSCs. We demonstrate rapid generation of transgene-free human iPSCs by excising the loxP-flanked reprogramming cassette employing direct delivery of biologically active Cre protein. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs do resemble more to human ESCs and has better differentiation potential than iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of superior transgene-free iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies. mRNA extracted from human Fibroblasts (AR1034ZIMA), human Embryonic Stem Cell line I3 (hES I3), three human induced Pluripotent Stem Cell clones 1, 1.2 and 1.4 (fl-ARiPS cl1, del-ARiPS cl 1.2, del-ARiPS cl1.4) has been hybridized on Illumina Human HT-12 (version 4 revision 2) arrays for genome wide expression analysis. Samples were run at least as duplicate technical replicates. Differential gene expression analysis has been performed on the grouped expression data with the human embryonic stem cells (hES I3) group as the reference.

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Optimized Approaches for Generation of Integration-free iPSCs from Human Urine-derived Cells with Small Molecules and Autologous Feeder

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