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Perturb-seq delineates gene regulatory programs governing the biology of hematopoietic stem and progenitor cells

ABSTRACT: To sustain blood formation, hematopoietic stem and progenitor cells (HSPCs) coordinate a multitude of cell biological processes, from cell-cycle control and stress responses to lineage priming. How genetic perturbations shape these elemental programs in HSPCs, and how such programs relate to stem-cell-associated phenotypes, remains incompletely understood. Here we use Perturb-seq to profile the transcriptional consequences of targeting 520 genes by CRISPRi in primary mouse HSPC cultures. To resolve perturbation effects in this heterogeneous primary-cell system, we develop an analysis strategy that separates changes in cell-state abundance from local transcriptional effects within cell states, and use these local perturbation signatures to identify 19 gene regulatory programs (GRPs) comprising genes respond coherently to genetic perturbations. Most GRPs map to defined cell biological programs, including protein biosynthesis, cell cycle, inflammatory signaling, and HSC identity. By decomposing gene expression data from functional and clinical studies into program activity, we show that GRP activities are associated with and predict phenotypes such as clonal output after transplantation, as well as survival and drug response in AML cohorts. In sum, our study defines perturbation-derived programs of genetic co-regulation in HSPCs and shows that these programs inform stem cell function across physiological and malignant contexts.

ORGANISM(S): Mus musculus Escherichia coli

PROVIDER: GSE320250 | GEO | 2026/08/27

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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Perturb-seq delineates gene regulatory programs governing the biology of hematopoietic stem and progenitor cells

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