Citation

Kristiansen T, Mayerhofer C, Gustafsson K, Scadden DT. 2024. Stromal Cell Isolation from Hematopoietic Organs. Journal of visualized experiments : JoVE. Pubmed: 38345255 DOI:10.3791/66231

Abstract

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.

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David Scadden’s laboratory is dedicated to discovering the principles governing blood cell production, with the ultimate goal of guiding the development of therapies for blood disorders and cancer.

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