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RNA-seq of uncultured CD112high and CD112low and cultured long-term (LT) and short-term (ST) human hematopoietic stem cells (HSC) overexpressing INKA-1

ABSTRACT: RNA-seq of uncultured CD112high and CD112low long-term(LT) human hematopoietic stem cells (HSC) and cultured and lentiviral transduced (CTRL, INKA1-OE) LT- and short-term HSC from umbilical cord blood We performed RNA-sequencing to elucidate the biological pathways (1) altered by INKA1 overexpression in LT-HSC and ST-HSC that may contribute to the induced transient restraint in generating proliferative hematopoietic output in vivo and in vitro; and (2) that underlie the differential in vitro priming and regeneration phenotypes of CD112high and CD112low LT-HSC. Therefore, we prospectively isolated indicated subpopulations from 3 independent human umbilical cord blood pools and subjected them either to RNA-sequencing directly or following culture, lentiviral transduction and purification of the transduced cell fractions. We show that distinct subsets of human LT-HSC respond differently to regeneration-mediated stress and that INKA1 governs these distinct stemness states where CD112low LT-HSC are marked by an alternative state of quiescence with high INKA1 preserving self-renewal and regenerative capacity upon successive rounds of regeneration-mediated stress.

ORGANISM(S): Homo sapiens

PROVIDER: GSE148883 | GEO | 2021/03/25

REPOSITORIES: GEO

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RNA-seq of uncultured CD112high and CD112low long-term(LT) human hematopoietic stem cells (HSC) and cultured and lentiviral transduced (CTRL, INKA1-OE) LT- and short-term HSC from umbilical cord blood

Project description:We performed RNA-sequencing to elucidate the biological pathways (1) altered by INKA1 overexpression in LT-HSC and ST-HSC that may contribute to the induced transient restraint in generating proliferative hematopoietic output in vivo and in vitro; and (2) that underlie the differential in vitro priming and regeneration phenotypes of CD112high and CD112low LT-HSC. Therefore, we prospectively isolated indicated subpopulations from 3 independent human umbilical cord blood pools and subjected them either to RNA-sequencing directly or following culture, lentiviral transduction and purification of the transduced cell fractions.

| EGAS00001004768 | EGA

ATAC-seq of uncultured CD112high and CD112low long-term(LT) human hematopoietic stem cells (HSC) from umbilical cord blood

Project description:We performed ATAC-sequencing to elucidate the chromatin accessibility that underlie the differential in vitro priming and regeneration phenotypes of CD112high and CD112low LT-HSC. Therefore, we prospectively isolated these subpopulations from 3 independent human umbilical cord blood pools and subjected to ATAC-sequencing directly. We show that distinct subsets of human LT-HSC respond differently to regeneration-mediated stress and that INKA1 governs these distinct stemness states where CD112low LT-HSC are marked by an alternative state of quiescence with high INKA1 and low H4K16ac preserving self-renewal and regenerative capacity upon successive rounds of regeneration-mediated stress.

2021-04-13 | GSE172005 | GEO

ATAC-seq of uncultured CD112high and CD112low long-term(LT) human hematopoietic stem cells (HSC) from umbilical cord blood

Project description:ATAC-sequencing was performed to establish chromatin accessibility signatures that underlie the differential in vitro priming and regeneration phenotypes of CD112high and CD112low LT-HSC. CD112high and CD112low LT-HSC from 2-3 independent human umbilical cord blood pools were prospectively isolated for DNA extraction, library preparation and sequencing.

| EGAS00001005121 | EGA

Transcriptomic analysis of MYC overexpression in LT-HSC.

Project description:Long-term hematopoietic stem cells (LT-HSC) were sorted from human cord blood and cultured overnight before transduction with lentiviral vectors (empty E CTRL or MYC). Six days after transduction, BFP+ transduced cells were sorted for RNA extraction and sequencing

| EGAS00001004970 | EGA

Transcriptomic analysis of TFEB knockdown in LT-HSC.

Project description:Long-term hematopoietic stem cells (LT-HSC) were sorted from human cord blood and cultured overnight before transduction with lentiviral vectors overexpressing an shRNA against Renilla (shCTRL) or TFEB (shTFEB). Six days later, mCherry+ transduced cells were sorted for RNA extraction and sequencing.

| EGAS00001004967 | EGA

RNA-seq of human long-term and short-term hematopoietic stem cells from umblical cord blood with lentiviral overexpression of S1PR3

2021-01-19 | GSE149238 | GEO

RNA-seq study of human long-term and short-term hematopoietic stem cells from umblical cord blood with lentiviral overexpression of S1PR3

Project description:To investigate the molecular and biological pathways altered by S1PR3OE in human hematopoietic stem cells (HSC), we performed RNA-sequencing (RNA-seq) of LT- and ST-HSC 3 days after transduction with control or S1PR3 overexpression (OE) lentiviral vectors. LT-HSC and ST-HSC from 3 pool of CB lin- were FACS-purified, cells were prestimulated for 4 hours and transduced with lentiviral vectors. At day 3, 2000-5300 BFP+ cells were FACS-purified for RNA isolation with a PicoPure kit. We were able to isolate only 1600-1800 BFP+ cells from LT-HSC control samples as opposed to 4000-5400 BFP+ cells from S1PR3OE samples. Thus, we pooled all control BFP+ LT-HSC cells into one sample for RNA-seq analysis. BFP- LT-HSC from control vector transduction were purified from CB1 as an additional LT-HSC control. Nextera libraries generated from 10 ng RNA from 5 LT-HSC samples (2 controls, 3 S1PR3OE) and 6 ST-HSC samples (3 controls, 3 S1PR3OE) were subjected to 125 bp, paired-end RNA-sequencing on the Illumina HiSeq 2500 with an average of 50 million reads/sample at the Center for Applied Genomics, Sick Kids Hospital.

| EGAS00001004798 | EGA

ea9af326-25e0-42af-a639-732a3bafd94b - samples

Project description:To elucidate the epigenetic changes which occur when human long-term hematopoietic stem cells (LT-HSC) become activated we performed Bulk ATAC-Seq on 13 sorted bulk hematopoietic populations from cord bloodas well as single-cell ATAC-Seq upon CD34+CD38-CD45RA- cells enriched for HSC as well as CD34+/CD38+ progenitor cells both from cord blood. These studies revealed gains of chromatin accessibility around CTCF binding sites during HSPC activation, as such we additionally performed Low-C to directly profile the 3D conformation of human cord-blood derived LT-HSC and Short-term hematopoietic stem cells (ST-HSC), as well as Hi-C , ATAC-Seq and CTCF ChIP-Seq upon the OCIAML-2 cell line in which CTCF sites gained during LT-HSC activation are enriched. Finally we transduced human cord-blood LT-HSC with an shCTCF vector; in-vitro cultured LT-HSC cells harbouring shCTCF were used to perform RNA-Seq, and scATAC-Seq was performed on CD34+/CD38- human CB cells transduced with shCTCF, four weeks post xeno-transplantation into mice. Collectively these studies have helped us demonstrate the role of 3D chromatin conformation changes during human LT-HSC activation.

| EGAD00001006447 | EGA

Transcriptomic analysis of TFEB overexpression in LT-HSC, ST-HSC and MEP

Project description:Long-term hematopoietic stem cells (LT-HSC), Short-term hematopietic stem cells (ST-HSC) and Megakaryocyte-erythrocyte progenitors (MEP) were sorted from human cord blood and cultured overnight before transduction with lentivirus to overexpress GP91 (CTRL) or TFEB. Three (LT-HSC ans ST-HSC) or six (MEP) days later, BFP+ transduced cells were sorted for RNA extraction and sequencing.

| EGAS00001004969 | EGA

Time-course analysis of transcriptomes of human long-term (LT) and short-term (ST) hematopoietic stem cells (HSC) after xenotransplantation

Project description:Here we tracked transcriptional changes in LT, ST-HSC and MLP after transplantation into immunocompromised mice. We show that LT- and ST-HSC activate similar pathways upon transplantation but they modulate distinct sets of genes. Total RNA was obtained from flow-sorted populations of human LT-, ST-HSC and MLP isolated from cord blood or immunocompromised mice transplanted with human at hematopoeitic progenitors at different time points after transplantation.

2015-02-20 | E-GEOD-58299 | biostudies-arrayexpress

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