RESUMEN
Hematopoietic stem cell (HSC) mutations can result in clonal hematopoiesis (CH) with heterogeneous clinical outcomes. Here, we investigate how the cell state preceding Tet2 mutation impacts the pre-malignant phenotype. Using an inducible system for clonal analysis of myeloid progenitors, we find that the epigenetic features of clones at similar differentiation status are highly heterogeneous and functionally respond differently to Tet2 mutation. Cell differentiation stage also influences Tet2 mutation response indicating that the cell of origin's epigenome modulates clone-specific behaviors in CH. Molecular features associated with higher risk outcomes include Sox4 that sensitizes cells to Tet2 inactivation, inducing dedifferentiation, altered metabolism and increasing the in vivo clonal output of mutant cells, as confirmed in primary GMP and HSC models. Our findings validate the hypothesis that epigenetic features can predispose specific clones for dominance, explaining why identical genetic mutations can result in different phenotypes.
Asunto(s)
Proteínas de Unión al ADN , Dioxigenasas , Epigénesis Genética , Células Madre Hematopoyéticas , Mutación , Proteínas Proto-Oncogénicas , Dioxigenasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Animales , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Humanos , Hematopoyesis/genética , Ratones , Diferenciación Celular/genéticaRESUMEN
Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.