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. 2022 Mar;2(3):e400.
doi: 10.1002/cpz1.400.

A New Method of Bone Stromal Cell Characterization by Flow Cytometry

Chirayu Patel  1 , Lihong Shi  1 , John F Whitesides  2   3 , Brittni M Foster  1 , Roberto J Fajardo  4 , Ellen E Quillen  5 , Bethany A Kerr  1   3   6
Affiliations

A New Method of Bone Stromal Cell Characterization by Flow Cytometry

Chirayu Patel et al. Curr Protoc. 2022 Mar.

Abstract

The bone microenvironment cellular composition plays an essential role in bone health and is disrupted in bone pathologies, such as osteoporosis, osteoarthritis, and cancer. Flow cytometry protocols for hematopoietic stem cell lineages are well defined and well established. Additionally, a consensus for mesenchymal stem cell flow markers has been developed. However, flow cytometry markers for bone-residing cells-osteoblasts, osteoclasts, and osteocytes-have not been proposed. Here, we describe a novel partial digestion method to separate these cells from the bone matrix and present new markers for enumerating these cells by flow cytometry. We optimized bone digestion and analyzed markers across murine, nonhuman primate, and human bone. The isolation and staining protocols can be used with either cell sorting or flow cytometry. Our method allows for the enumeration and collection of hematopoietic and mesenchymal lineage cells in the bone microenvironment combined with bone-residing stromal cells. Thus, we have established a multi-fluorochrome bone marrow cell-typing methodology. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Partial digestion for murine long bone stromal cell isolation Alternate Protocol 1: Partial digestion for primate vertebrae stromal cell isolation Alternate Protocol 2: Murine vertebrae crushing for bone stromal cell isolation Basic Protocol 2: Staining of bone stromal cells Support Protocol 1: Fluorescence minus one control, isotype control, and antibody titration Basic Protocol 3: Cell sorting of bone stromal cells Alternate Protocol 3: Flow cytometry analysis of bone stromal cells Support Protocol 2: Preparing compensation beads.

Keywords: bone marrow; cell sorting; flow cytometry; osteoblast; osteoclast; osteocyte.

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Conflict of interest statement

CONFLICT OF INTEREST STATEMENT:

The authors declare there are no conflicts of interest.

Figures

Figure 1.
Figure 1.. Staining Scheme for Bone Marrow and Residing Stromal Cells.
Based upon existing literature, a panel of markers was developed to identify and differentiate bone marrow stromal cells of the hematopoietic and mesenchymal lineages. Created with Biorender.com.
Figure 2.
Figure 2.. Murine Digestion Yields across Age, Sex, and Genotype.
For this study, male and female Balb/c mice aged 8 weeks and C57Bl/6 mice aged 18 or 28 weeks were purchased to optimize the protocol across strains, sex, and age under an approved Wake Forest School of Medicine IACUC protocol. Long bones were isolated from mice and processed as described in Basic Protocol 1. The numbers of cells recovered from 3–5 individual animals are represented as mean±SEM.
Figure 3.
Figure 3.. Representative Gating for Murine Bone-Residing Stromal Cells.
To isolate murine stromal cells, gating was performed to differentiate A) Singlets, B) Live/Dead Staining, C) hematopoietic stem cells (HSC), D) mesenchymal stem cells (MSC), E) osteoblasts, F) osteocytes, G) macrophages, and H) osteoclast.
Figure 4.
Figure 4.. Representative Gating for Primate Bone-Residing Stromal Cells.
To isolate primate stromal cells, gating was performed to differentiate A) Singlets, B) Live/Dead Staining, C) hematopoietic stem cells (HSC), D) mesenchymal stem cells (MSC), E) osteoblasts, F) osteocytes, G) macrophages, and H) osteoclast.
Figure 5.
Figure 5.. Murine Long Bone Stromal Cell Percentages across Age, Sex, and Genotype.
Isolated bone-residing stromal cells were stained and analyzed by cell sorting as described in Basic Protocols 2 and 3. The percentages of bone stromal cells from 3–5 individual animals are represented as mean±SEM.
Figure 6.
Figure 6.. Primate Vertebrae Bone Stromal Cell Percentages.
Archival baboon lumbar vertebrae (collected during routine necropsy at the Southwest National Primate Research Center (SNPRC) housed at Texas Biomedical Research Institute) and human lumbar vertebrae (from donated, deidentified cadaveric tissue to the Willed Body Program) were processed and stained prior to cell sorting. The percentages of bone stromal cells in three pooled samples (N=3 in each pool) are represented as mean±SEM.
See this image and copyright information in PMC

References

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INTERNET RESOURCES:

    1. https://www.stemcell.com/human-hematopoietic-stem-and-progenitor-cell-ph... A regularly updated list of phenotypic markers for progenitor cells of the hematopoietic lineage.
    1. https://www.rndsystems.com/research-area/osteoblast-and-osteoclast-markers A regularly updated list of surface and secreted markers for osteoblasts and osteoclasts.
    1. https://www.biolegend.com/en-us/blog/titrating-totalseq-antibodies A recommended antibody titration protocol.
    1. https://www.biolegend.com/Files/Images/BioLegend/literature/images/02-00... A resource describing how best to optimize multi-fluorophore flow cytometry staining.
    1. https://www.bio-rad-antibodies.com/introduction-to-flow-cytometry.html A good overview of the principles of flow cytometry with detailed explanations on preparing controls and optimizing flow data.

LinkOut - more resources