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Highly efficient reprogramming to pluripotency and directed differentiation using synthetic mRNA

ABSTRACT: Clinical application of induced pluripotent stem (iPS) cells is limited by low efficiency of iPS derivation, and protocols that permanently modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-mutagenic strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem (RiPS) cells into terminally differentiated myogenic cells. Our method represents a safe, efficient strategy for somatic cell reprogramming and directing cell fates that has broad applicability for basic research, disease modeling and regenerative medicine. We isolated RNA from human RNA derived iPS cells, viral derived iPS cells, different human fibroblasts and human embryonic stem cells for hybridization to the Affymetrix gene expression microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Hu Li

PROVIDER: E-GEOD-23583 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Highly efficient reprogramming to pluripotency and directed differentiation using synthetic mRNA

Project description:Clinical application of induced pluripotent stem (iPS) cells is limited by low efficiency of iPS derivation, and protocols that permanently modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-mutagenic strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem (RiPS) cells into terminally differentiated myogenic cells. Our method represents a safe, efficient strategy for somatic cell reprogramming and directing cell fates that has broad applicability for basic research, disease modeling and regenerative medicine.

2010-09-30 | GSE23583 | GEO

Transcription profiling of IPS cells derived from peripheral blood cells: Reprogramming of T Cells from Human Peripheral Blood

Project description:Human induced pluripotent stem (iPS) cells derived from somatic cells of patients hold great promise for modelling human diseases. Dermal fibroblasts are frequently used for reprogramming, but require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Here, we report the derivation of iPS cells from multiple human blood sources including peripheral blood mononuclear cells (PBMCs) harvested by routine venipuncture. Peripheral blood-derived human iPS lines are comparable to human embryonic stem (ES) cells with respect to morphology, expression of surface antigens, activation of endogenous pluripotency genes, DNA methylation and differentiation potential. Analysis of Immunoglobulin and T-cell receptor gene rearrangement revealed that some of the PBMC iPS cells were derived from T-cells, documenting derivation of iPS cells from terminally differentiated cell types. Importantly, peripheral blood cells can be isolated with minimal risk to the donor and can be obtained in sufficient numbers to enable reprogramming without the need for prolonged expansion in culture. Reprogramming from blood cells thus represents a fast, safe and efficient way of generating patient-specific iPS cells. We isolated RNA from multiple human blood sources including peripheral blood mononuclear iPS cells, multiple somatic cells and iPS cells and human embryonic cells and hybridized them to Affymetrix gene expression microarrays.

2010-06-23 | E-GEOD-22167 | biostudies-arrayexpress

Transcription profiling of human fibroblast cell line CCD27SK before and after pluripotent reprogramming by lentiviral-mediated insertion of SOX2 C-MYC and TCL-1A

Project description:Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used four transcription factors, OCT4, SOX2, KLF4 and C-MYC, can induce pluripotency in mouse and human fibroblasts. Here we report that a forced expression of a new combination of transcription factors (TCL-1A, C-MYC and SOX2) is sufficient to promote the reprogramming of human fibroblast into pluripotent cells. These three-factor pluripotent cells are similar to human embryonic stem cells (hESC) in morphology, in the ability to differentiate into cells of the three embryonic layers, and at the level of global gene expression. Induced pluripotent human cells generated by combination of other factors will be of great help for the understanding of reprogramming pathways. This in turn will allow us to better control cell-fate and apply this knowledge to cell therapy.

2010-10-26 | E-MEXP-2422 | biostudies-arrayexpress

Reprogramming of T Cells from Human Peripheral Blood

2010-06-10 | GSE22167 | GEO

Therapeutic globin expression in thalassemia patient induced pluripotent stem cells from genomic safe harbors

Project description:The advent of human induced pluripotent stem (iPS) cells enables for the first time the derivation of unlimited numbers of patient-specific stem cells and holds great promise for regenerative medicine. However, realizing the full potential of iPS cells requires robust, precise and safe strategies for their genetic modification. Safe human iPS cell engineering is especially needed for therapeutic applications, as stem cell-based therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify iPS cells from patients with beta-thalassemia in a potentially clinically relevant manner. Our approach is based on the identification and selection of “safe harbor” sites for transgene expression in the human genome. We show that thalassemia patient iPS cell clones harboring a transgene can be isolated and screened according to chromosomal position. We next demonstrate that iPS cell clones that meet our “safe harbor” criteria resist silencing and allow for therapeutic levels of beta-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. Combined bioinformatics and functional analyses thus provide a robust and dependable approach for achieving desirable levels of transgene expression from selected chromosomal loci. This approach may be broadly applicable to introducing therapeutic or suicide genes into patient specific iPS cells for use in cell therapy.

2010-10-26 | GSE24901 | GEO

Therapeutic globin expression in thalassemia patient induced pluripotent stem cells from genomic safe harbors

Project description:The advent of human induced pluripotent stem (iPS) cells enables for the first time the derivation of unlimited numbers of patient-specific stem cells and holds great promise for regenerative medicine. However, realizing the full potential of iPS cells requires robust, precise and safe strategies for their genetic modification. Safe human iPS cell engineering is especially needed for therapeutic applications, as stem cell-based therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify iPS cells from patients with beta-thalassemia in a potentially clinically relevant manner. Our approach is based on the identification and selection of âsafe harborâ sites for transgene expression in the human genome. We show that thalassemia patient iPS cell clones harboring a transgene can be isolated and screened according to chromosomal position. We next demonstrate that iPS cell clones that meet our âsafe harborâ criteria resist silencing and allow for therapeutic levels of beta-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. Combined bioinformatics and functional analyses thus provide a robust and dependable approach for achieving desirable levels of transgene expression from selected chromosomal loci. This approach may be broadly applicable to introducing therapeutic or suicide genes into patient specific iPS cells for use in cell therapy. iPS cell clones were derived from beta-thalassemia patients. A single copy of beta-globin transgene cis-linked to locus control region (LCR) elements and an excisable Neo-eGFP transcription unit were inserted into these cell clones. beta-globin expression was induced by erythroid differentiation.

2010-10-26 | E-GEOD-24901 | biostudies-arrayexpress

Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins

Project description:To date, all methods to generate induced pluripotent stem (iPS) cells require the use of genetic materials and/or potentially mutagenic chemicals. Here we report the generation of stable human iPS cells from human fibroblasts by directly delivering defined reprogramming proteins. This system eliminates the potential risks associated with genetic and/or chemical manipulation, and could provide a safe and physiologically intact source of cells for regenerative medicine.

2009-05-29 | GSE16093 | GEO

Tox4 Modulates Cell Fate Reprogramming to iPS Cells and Direct Reprogramming

Project description:Tox4 Modulates Cell Fate Reprogramming to iPS Cells and Direct Reprogramming

| PRJNA525829 | ENA

Extracellular matrix-dependent generation of integration- and xeno-free iPS cells using a modified mRNA transfection method

Project description:In this study, we sought to examine whether an extracellular matrix (ECM)-based xeno-free culture system that we recently established could be used together with a microRNA-enhanced mRNA reprogramming method for the generation of clinically safe iPS cells. The notable features of this method are (1) the use of a xeno-free/feeder-free culture system for the generation and expansion of iPS cells rather than the conventional labor-intensive culture systems using human feeder cells or human feeder-conditioned medium and (2) the enhancement of mRNA-mediated reprogramming via the delivery of microRNAs. Strikingly, we observed the early appearance of iPS cell colonies (~11 days), substantial reprogramming efficiency (~0.2-0.3%), and a high percentage of ESC-like colonies among the total colonies (~87.5%), indicating enhanced kinetics and reprogramming efficiency. Therefore, the combined method established in this study provides a valuable platform for the generation and expansion of clinically safe (i.e., integration- and xeno-free) iPS cells, facilitating immune-matched cell therapy in the near future.

2016-04-21 | GSE68035 | GEO

Molecular insights into induced pluripotency mediated by the OCT4, SOX2, KLF and c-MYC gene regulatory network

Project description:Human somatic fibroblasts can be reprogrammed to induced pluripotent stem (iPS) cells by exogenic expression of the Yamanaka factors (OCT4, SOX2, KLF4 and MYC) after about 1 month. To gain some insight into the early processes operative in fibroblast reprogramming, we profiled genome-wide transcription levels using Illumina microarrays in the starting donor cells-human foreskin fibroblast (HFF1) cells and at three time points after OSKM transduction (24 h, 48 h, 72 h), as well as two iPS cell lines (iPS2, iPS4) and hES cell lines (H1, H9). We show that within the context of the viral transduction reprogramming protocol, the donor cell response to viral transfection perturbs redox homeostasis, which induces oxidative damage on the donor cells' protein and DNA. This leads to activation of p53, senescence, and apoptosis, greatly reducing the efficiency of reprogramming. Total RNA obtained from HFF1 (human foreskin fibroblast) cells, OSKM-transduced HFF1 cells after 24h, 48h, 72h, undifferentiated hESCs, iPSCs.

2011-10-24 | E-GEOD-28688 | biostudies-arrayexpress

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