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Taz protects aged hematopoietic stem cells from exhaustion [scRNA-seq]

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. Young hematopoietic stem cells (HSCs) were isolated by flow cytometry, transduced ex vivo with different shRNA constructs (shRen, shPU1 No1, shPU1 No2) and transplanted into lethally irradiated mice. Two months after transplantation, transduced (GFP+)HSPCs were isolated by flow cytometry. The different samples were pooled and the shRNA per cell was identified by a specific dialout PCR (amplifying the mirE shRNA construct together with the 10x cell barcode) to link the cell barcodes with the the different shRNA constructs. Here mirE_R1 contains the UMI and the cell barcode whereas mirE_R2 contains the shRNA sequence.

ORGANISM(S): Mus musculus

PROVIDER: GSE196176 | 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 [scRNA-seq]

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