Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

CRISPR activation enables high fidelity reprogramming into human pluripotent stem cells (bulk cells)

ABSTRACT: Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSC). Forced expression of transcription factors might lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSC. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can potentially reduce this heterogeneity. Here we describe a high efficiency reprogramming of human somatic cells into iPSC using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells with more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high quality reprogramming of human iPSC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE162529 | GEO | 2021/12/23

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

CRISPR activation enables high fidelity reprogramming into human pluripotent stem cells (single cells)

Project description:Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSC). Forced expression of transcription factors might lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSC. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can potentially reduce this heterogeneity. Here we describe a high efficiency reprogramming of human somatic cells into iPSC using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells with more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high quality reprogramming of human iPSC.

2021-12-23 | GSE162528 | GEO

CRISPR activation enables high fidelity reprogramming into human pluripotent stem cells

2021-12-23 | GSE162530 | GEO

Nanog is Dispensable for the Generation of Induced Pluripotent Stem Cells

Project description:Cellular reprogramming from somatic cells to induced pluripotent stem cells (iPSCs) can be achieved through forced expression of the transcription factors Oct4, Klf4, Sox2 and c-Myc (OKSM). These factors, in combination with environmental cues, induce a stable intrinsic pluripotency network that confers indefinite self-renewal capacity on iPSCs. In addition to Oct4 and Sox2, the homeodomain-containing transcription factor Nanog is an integral part of the pluripotency network. Although Nanog expression is not required for the maintenance of pluripotent stem cells, it has been reported to be essential for the establishment of both embryonic stem cells (ESCs) from blastocysts and iPSCs from somatic cells. Here we revisit the role of Nanog in direct reprogramming. Surprisingly, we find that Nanog is dispensable for iPSC formation under optimized culture conditions. We further document that Nanog-deficient iPSCs are transcriptionally highly similar to wild-type iPSCs and support the generation of teratomas and chimeric mice. Lastly, we provide evidence that the presence of ascorbic acid in the culture media is critical for overcoming the previously observed reprogramming block of Nanog knockout cells. Comparison of Nanog KO iPSCs to WT pluripotent cells.

2014-01-28 | E-GEOD-53832 | biostudies-arrayexpress

Expression data from human keratinocyte and PBMC-derived iPS cells

Project description:Induced pluripotent stem cell (iPSC) technology allows for the generation of patient-specific pluripotent stem cells, from somatic cell sources, thereby providing a novel cell therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming and subsequent feasiblity of reprogramming into a pluripotent state. We used microarrays to detail the global gene expression profiles from blood cells. The use of blood cells allows for minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming, mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation. We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and mononuclear myeloid cells and tested the pluripotency of derived iPS clones.

2011-11-09 | E-GEOD-33536 | biostudies-arrayexpress

STRT RNAseq of pluripotent stem cells reprogrammed with CRISPRa

Project description:Transcriptome comparison of CRISPRa induced human pluripotent stem cell lines, control iPSC line induced with Sendai viral vectors, H9 embryonic stem cell line and human foreskin fibroblasts.

2018-06-07 | E-MTAB-6185 | biostudies-arrayexpress

DNA methylome analysis of human pluripotent stem cells reprogrammed with CRISPRa

Project description:Comparison of DNA methylation in CRISPRa induced human pluripotent stem cell lines, control iPSC line induced with Sendai viral vectors, H9 embryonic stem cell line and human foreskin fibroblasts.

2018-06-07 | E-MTAB-6194 | biostudies-arrayexpress

Epigenetic and transcriptional aberrations in human pluripotent stem cells reflect differences in reprogramming mechanisms [methylation array]

Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but existing cell types have imitations. Human embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the “gold standard”, but are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) can be produced from somatic cells by forced expression of pluripotency-associated factors, but are prone to genetic and epigenetic aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming, or secondary to the reprogramming method, we employed an alternative approach by somatic cell nuclear transfer (SCNT). SCNT-based reprogramming to NT-ESCs is mediated by factors present in oocyte’s cytoplasm, thus mimicking early embryogenesis. We generated genetically matched pluripotent stem cells and conducted genome-wide genetic, epigenetic and transcriptional analyses. We discovered that unlike iPSCs, NT-ESCs have a low burden of de novo copy number variations (CNVs), reflecting superior maintenance of genetic stability. Moreover, DNA methylation and transcriptome profiles of NT-ESCs corresponded closely to those of IVF-ESCs. In contrast, iPSCs harbored methylation abnormalities including residual CpG methylation typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT with the potential to satisfy the clinical requirements for cell replacement therapies. Bisulphite converted DNAs of two IVF-ESCs, two sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, and the parental fibroblast were hybridized to the Illumina Infinium HumanMethylation 450K Beadchip

2014-07-08 | E-GEOD-53060 | biostudies-arrayexpress

Deterministic direct reprogramming of somatic cells to pluripotency [Microarray/Expression]

Project description:Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Samples include Mbd3+/+, Mbd3flox/- and Mbd3-/- cells from mouse ES cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors).

2013-09-12 | E-GEOD-45352 | biostudies-arrayexpress

Coordination of engineered factors with TET1/2 promotes early stage epigenetic modification during somatic cell reprogramming

Project description:Somatic cell reprogramming towards induced pluripotent stem cells (iPSCs) holds great promise in future regenerative medicine, however, the reprogramming process mediated by the traditional defined factors (OSNK) is slow and extremely inefficient. Here we show that a combination of modified reprogramming factors (OySyNyK), in which the transactivation domain of the Yes-associated protein is fused to OCT4, SOX2 and NANOG respectively, could be used to establish a highly efficient and rapid reprogramming system for iPSC generation. We show that the efficiency of OySyNyK-induced iPSCs was up to 100-fold higher than that of induction by traditional OSNK. Moreover we show that the reprogramming by OySyNyK is very rapid (initiated at 24 h versus 5 d by OSNK). Compared with OSNK, we found that OySyNyK factors significantly increased the expression of Tet1/2 at the early stage, which will interact with OSNK factors, and co-occupy pluripotency loci in the genome for somatic cell reprogramming. Our studies not only establish a rapid and highly efficient iPSC reprogramming system, but also uncover a novel mechanism by which OSNK factors coordinate with TET proteins to regulate 5hmC-mediated epigenetic control, thereby promoting somatic cell reprogramming. We examined DNA hydroxymethylation progression of reprogramming intermediates. To this end, we profiled the genome-wide 5hmC distribution in MEFs, the reprogramming intermediates at different stages induced by either the OSNK or OySyNyK methods, and iPSCs using the chemical capture approach

2013-08-08 | E-GEOD-46202 | biostudies-arrayexpress

A serial screen for roadblocks to reprogramming identifies the sumoylation effector protein Sumo2

Project description:The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of Oct4, Klf4, Sox2 and c-Myc (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an ubiased serial shRNA enrichment screen to identify novel repressors of somatic cell reprogramming into iPSCs. This effort uncovered the sumoylation effector protein Sumo2 as one of the strongest roadblocks to iPSC formation. Depletion of Sumo2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 36 hours of OKSM expression. We further show that the Sumo2 pathway acts independently of exogenous c-Myc expression and in parallel with small molecule enhancers of reprogramming. Critically, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors. Microarray analysis was performed during reprogramming or of iPSC lines derived upon Sumo2 knockdown Total RNA was isolated from day 6 reprogramming fibroblasts with or without Sumo2 knockdown; as well as stable iPSC clones derived from Sumo2 knockdown fibroblasts.

2016-03-03 | E-GEOD-76699 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data