Stromal Cell Isolation From Hematopoietic Organs

Published: January 26, 2024

doi:

Trine Kristiansen*^1,2,3, Christina Mayerhofer*^1,2,3, Karin Gustafsson^2,3, David T. Scadden^2,3

¹Center for Regenerative Medicine,Massachusetts General Hospital, ²Harvard Stem Cell Institute, ³Department of Stem Cell and Regenerative Biology,Harvard University

Summary

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

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.

Introduction

In the healthy adult, de novo production of blood cells occurs in the bone marrow and the thymus. Stromal cells at these sites are essential for maintenance of hematopoiesis, but stroma constitutes less than 1% of the tissue¹^,²^,³^,⁴. Obtaining pure isolates of hematopoiesis supporting stroma therefore constitutes a significant challenge, particularly for single-cell multiomics that requires expedient processing to obtain samples of high quality. Components of different digestion cocktails may interfere with certain steps in multiomics analysis⁵^,⁶. The protocols presented here detail the isolation of a wide variety of stromal cells from bone marrow and thymic tissues.

Perturbations of stromal constituents in both bone marrow and thymus result in profound disruption in blood cell development and can result in malignancies⁷^,⁸^,⁹. Hematopoiesis supporting stroma is damaged following cytotoxic conditioning and bone marrow transplantation, resulting in reduced secretion of cytokines and growth factors that sustain hematopoietic stem and progenitor cells (HSPCs)²^,¹⁰^,¹¹. Furthermore, aging affects bone marrow and thymus stromal cells likely contributing to aged hematopoietic phenotypes. The thymus is the first organ to undergo extensive age-associated involution. Fat and fibrotic tissue start replacing T cell supportive stroma as early as the onset of puberty¹²^,¹³. In the bone marrow, adipocyte content increases with age and the vascular and endosteal niches are significantly remodeled¹⁴^,¹⁵^,¹⁶.

To enable study of hematopoiesis supportive stroma across multiple stress states and in the case of the thymus of both human and murine tissue, we have optimized previously published digestion protocols¹^,²^,⁸^,¹⁷^,¹⁸. These protocols ensure efficient and reproducible isolation of cells, and they are compatible with single-cell RNAsequencing (scRNAseq) and other types of multiomics.

Protocol

All work with human tissue was conducted after approval by the Massachusetts General Hospital Internal Review Board (IRB). All animal procedures were conducted in accordance with the Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) guidelines. C57Bl/6 mice, 8-10 weeks old, and both males and females, were used for the present study. The animals were obtained from a commercial source (see Table of Materials). 1. Preparation of murine thym…

Representative Results

These protocols yield reproducible stromal cell varieties from the thymus and bone marrow suitable for flow cytometric analysis, as well as single-cell multiomics, such as scRNA sequencing. Murine thymic tissue undergoes significant remodeling in response to stressors, such as the cytotoxic conditioning that precedes bone marrow transplantation or the natural aging process. As a consequence, thymic cellularity is drastically reduced in both of these settings (Figure 1A). While a thymus from …

Discussion

Stromal cells in hematopoietic organs are critical for normal blood production and hematopoietic stroma perturbations can result in severe impairments in hematopoietic maintenance and response to stress⁹^,²³^,²⁴. Insight into hematopoietic stromal cells is essential for understanding hematological diseases⁷^,⁹^,¹⁰^,<sup class="xre…

Divulgations

The authors have nothing to disclose.

Acknowledgements

We were supported with expert technical assistance by the HSCI-CRM Flow Cytometry facility at Massachusetts General Hospital and the Bauer Core Facility at Harvard University. T.K and K. G were supported by the Swedish Research Council and C.M. by the German Research Foundation. We thank Sergey Isaev and I-Hsiu Lee for assistance in analysis of single-cell RNA sequencing data.

Materials

0.25% Trypsin-EDTA	Thermo Fisher Scientific	25200-072
7AAD (7-aminoactinomycin D)	BD Biosciences	559925
Anti-Human Lineage Cocktail 3-FITC	BD Biosciences	643510
Bovine Serum Albumin	Millipore Sigma	A9647
C57Bl/6 mice	Jackson	664	Males or females, 8-12 weeks old
Calcein	Fisher Scientific	65-0853-78
Collagenase IV	Millipore Sigma	C5138
Corning Sterile Cell Strainers, White, Mesh Size: 70 µm	Fisher Scientific	08-771-2
DAPI (4',6-Diamidino-2-Phenylindole, Dilactate)	Biolegend	422801
Dispase II	Thermo Fisher Scientific	17105041
Dnase I Solution	Thermo Fisher Scientific	90083	2500 U/mL
Easysep mouse streptavidin RapidSpheres Isolation kit	StemCell Technologies	19860
Fetal Bovine Serum	Gibco	A31605-01	Qualified One Shot
Human Fc Block	BD Biosciences	564220
Liberase TM	Millipore Sigma	5401127001	Research Grade
Medium 199	Gibco	12350
Mouse anti-human CD235a-BV77	BD Biosciences	740785
Mouse anti-human CD31-PE/Dazzle594	Biolegend	303130
Mouse anti-human CD45-BV77	Biolegend	304050
Mouse anti-human CD4-BV605	BD Biosciences	562658
Mouse anti-human CD66b-FITC	BD Biosciences	555724
Mouse anti-human CD8-APC/Cy7	BD Biosciences	557760
Mouse anti-human EpCam-BV421	Biolegend	324220
Protector RNase Inhibitor	Millipore Sigma	3335402001
Rat anti-mouse CD105-PE /dazzle594	Biolegend	120424
Rat anti-mouse CD11b-Biotin	Biolegend	101204
Rat anti-mouse CD140a-APC	Fisher Scientific	17-1401-81
Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)	BD Biosciences	553142
Rat anti-mouse CD31-BUV737	BD Biosciences	612802
Rat anti-mouse CD31-BV421	Biolegend	102424
Rat anti-mouse CD3-Biotin	Biolegend	100244
Rat anti-mouse CD45.2-Biotin	Biolegend	109804
Rat anti-mouse CD45-PE/Cy7	Biolegend	103114
Rat anti-mouse CD45-PE/Cy7	Biolegend	103114
Rat anti-mouse CD45R/B220-Biotin	Biolegend	103204
Rat anti-mouse CD51-PE	Biolegend	104106
Rat anti-mouse EpCam-BV711	BD Biosciences	563134
Rat anti-mouse Ly-6A/E(Sca-1)-AF700	Biolegend	108142
Rat anti-mouse Ly-6G/Ly-6C(Gr1)-Biotin	Biolegend	108404
Rat anti-mouse Ter119-Biotin	Biolegend	116204
Rat anti-mouse Ter119-PE	Biolegend	116208
Rat anti-mouse Ter119-PE/Cy7	Biolegend	116222
Stemxyme	Worthington Biochemical	LS004107

References

Baryawno, N., et al. A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia. Cell. 177 (7), 1915-1932 (2019).
Severe, N., et al. Stress-induced changes in bone marrow stromal cell populations revealed through single-cell protein expression mapping. Cell Stem Cell. 25 (4), 570-583 (2019).
Han, J., Zuniga-Pflucker, J. C. A 2020 view of thymus stromal cells in t cell development. J Immunol. 206 (2), 249-256 (2021).
Park, J. E., et al. A cell atlas of human thymic development defines t cell repertoire formation. Science. 367 (6480), (2020).
Denisenko, E., et al. Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus rna-seq workflows. Genome Biol. 21 (1), 130 (2020).
Lischetti, U., et al. Dynamic thresholding and tissue dissociation optimization for cite-seq identifies differential surface protein abundance in metastatic melanoma. Commun Biol. 6 (1), 830 (2023).
Ding, L., Saunders, T. L., Enikolopov, G., Morrison, S. J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature. 481 (7382), 457-462 (2012).
Mendez-Ferrer, S., et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature. 466 (7308), 829-834 (2010).
Raaijmakers, M. H., et al. progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature. 464 (7290), 852-857 (2010).
Himburg, H. A., et al. Distinct bone marrow sources of pleiotrophin control hematopoietic stem cell maintenance and regeneration. Cell Stem Cell. 23 (3), 370-381 (2018).
Zhou, B. O., et al. marrow adipocytes promote the regeneration of stem cells and haematopoiesis by secreting scf. Nat Cell Biol. 19 (8), 891-903 (2017).
Steinmann, G. G. Changes in the human thymus during aging. Curr Top Pathol. 75, 43-88 (1986).
Steinmann, G. G., Klaus, B., Muller-Hermelink, H. K. The involution of the ageing human thymic epithelium is independent of puberty. A morphometric study. Scand J Immunol. 22 (5), 563-575 (1985).
Ambrosi, T. H., et al. Adipocyte accumulation in the bone marrow during obesity and aging impairs stem cell-based hematopoietic and bone regeneration. Cell Stem Cell. 20 (6), 771-784 (2017).
Ho, Y. H., et al. Remodeling of bone marrow hematopoietic stem cell niches promotes myeloid cell expansion during premature or physiological aging. Cell Stem Cell. 25 (3), 407-418 (2019).
Kusumbe, A. P., et al. Age-dependent modulation of vascular niches for haematopoietic stem cells. Nature. 532 (7599), 380-384 (2016).
Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. Purified enzymes improve isolation and characterization of the adult thymic epithelium. J Immunol Methods. 385 (1-2), 23-34 (2012).
Stoeckle, C., et al. Isolation of myeloid dendritic cells and epithelial cells from human thymus. J Vis Exp. (79), e50951 (2013).
Gustafsson, K., Scadden, D. T. Isolation of thymus stromal cells from human and murine tissue. Methods Mol Biol. 2567, 191-201 (2023).
Amend, S. R., Valkenburg, K. C., Pienta, K. J. Murine hind limb long bone dissection and bone marrow isolation. J Vis Exp. (110), e53936 (2016).
Zhu, H., et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc. 5 (3), 550-560 (2010).
Calvi, L. M., et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature. 425 (6960), 841-846 (2003).
Kode, A., et al. Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts. Nature. 506 (7487), 240-244 (2014).
Agarwal, P., et al. Mesenchymal niche-specific expression of cxcl12 controls quiescence of treatment-resistant leukemia stem cells. Cell Stem Cell. 24 (5), 769-784 (2019).
Duarte, D., et al. Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in aml. Cell Stem Cell. 22 (1), 64-77 (2018).
O’flanagan, C. H., et al. Dissociation of solid tumor tissues with cold active protease for single-cell rna-seq minimizes conserved collagenase-associated stress responses. Genome Biol. 20 (1), 210 (2019).
Stoeckius, M., et al. Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 19 (1), 224 (2018).