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Taz protects aged hematopoietic stem cells from exhaustion [bulk RNA-seq II]

ABSTRACT: We report here the analysis of young and aged hematopoietic stem and progenitor cells (HSPCs) by transcriptional profiling (bulk RNA-Seq, CITE-Seq) and chromatin profiling (ATAC-Seq, scATAC-Seq). We use here a Clca3a1 as a surface marker to further fractionate HSPCs. Old (Clca3a1_high) hematopoietic stem cells (HSCs) were isolated by flow cytometry, transduced ex vivo with different shRNA constructs (shRen, shTaz_Seq1, shTaz_Seq2) and transplanted into lethally irradiated mice. Four months after transplantation, transduced (GFP+)HSPCs were isolated by flow cytometry and subsequently analyzed by low input RNA-Seq. Per sample two mice were pooled in order to obtain sufficient RNA for library prep.

ORGANISM(S): Mus musculus

PROVIDER: GSE196173 | GEO | 2022/07/14

REPOSITORIES: GEO

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Project description:The occurrence of clonal perturbations and leukemia in patients transplanted with retrovirally-transduced autologous hematopoietic stem and progenitor cells (HSPCs) has stimulated extensive investigation, demonstrating that proviral insertions perturb adjacent proto-oncogene expression. Although enhancer-deleted lentiviruses are less likely to result in insertional oncogenesis, there is evidence that they may perturb transcript splicing, and one patient with a benign clonal expansion of lentivirally-transduced HPSC has been reported. The rhesus macaque model provides an opportunity for informative long-term analysis to ask whether transduction impacts on long-term HSPC properties. We utilized two techniques to examine whether lentivirally-transduced HSPCs from eight rhesus macaques transplanted 1-13.5 years previously are perturbed at a population level, comparing telomere length as a measure of replicative history and gene expression profile of vector positive versus vector negative cells. There were no differences in telomere lengths between sorted GFP+ and GFP- blood cells, suggesting that lentiviral transduction did not globally disrupt replicative patterns. Bone marrow GFP+ and GFP- CD34+ cells showed no differences in gene expression using unsupervised and principal component analysis. These studies did not uncover any global long-term perturbation of proliferation, differentiation, or other important functional parameters of transduced HSPCs in the rhesus macaque model. CD34+ hematopoietic stem and progenitor cells were purified from bone marrow aspirates obtained from 4 rhesus macaques 3-9 years following transplantation with lentivirally-transduced autologous hematopoietic stem and progenitor cells. All lentiviral vectors contained the GFP marker gene. The CD34+ cells from each animal were sorted via flow cytometry into GFP+ and GFP- fractions, and RNA from these cells was used for Affymetrix gene expression analysis as detailed below. There were two samples, GFP+ and GFP-, from each of 4 animals.

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Taz protects aged hematopoietic stem cells from exhaustion [bulk RNA-seq II]

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