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Purification and functional characterization of novel human skeletal stem cell lineages

Nature Protocols volume 18, pages 2256–2282 (2023)Cite this article

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Abstract

Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1–2 days. The downstream functional assays can be performed within a range of 1–2 months.

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Fig. 1: Overview of the protocol for human skeletal stem cell isolation and functional assessment.
Fig. 2: hSSC sources, hierarchy and FACS gating strategy.
Fig. 3: Subcutaneous human xenograft model and FACS isolation of hSSC lineages.
Fig. 4: Renal capsule xenograft model and in vitro skeletal differentiation assays.
Fig. 5: ScRNAseq platform for analyzing hSSCs.

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Data availability

All raw and processed scRNAseq data presented in this study have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus online repository and are available under accession number GSE212609. We will share all other raw data upon request.

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Acknowledgements

We thank B. Barres, J. Janas and R. Mann for their support and mentorship; A. McCarty and C. Wang for mouse colony management; H. Gentner, P. Pereira, T. Storm, T. Naik, L. Quinn, L. Jerabek, S. Kantoff and C. McQuarrie for lab management; P. Lovelace, J. Ho and S. Weber for FACS support; and J.C. Wu for the kind gift of iPSC line. This study was supported by NIH (R01DE027323, R56 DE025597, R01 DE026730, R01 DE021683, R21 DE024230, U01HL099776, U24DE026914 and R21DE019274), CIRMTR1-01249, Oak Foundation, Stanford Wu Tsai Human Performance Alliance Funds, Hagey Laboratory, Pitch Johnson Fund and Gunn/Olivier Research Fund to M.T.L., NIH (U01 HL099999, R01 CA86065, and R01 HL058770), Siebel Fellowship, the Heritage Medical Foundation, Prostate Cancer Foundation, the American Federation for Aging Research (AFAR)–Arthritis National Research Foundation (ANRF), a seed grant from the WHSDM Stanford Women’s Health and Ses Differences in Medicine Center, an endowment from the DiGenova Family to C.K.F.C., PCF YI Award, Stinehart/Reed and NIH NIAK99AG049958-01A1 to C.K.F.C., NIH (I01)VA I01BX003754 and NIH K08GM069677 to G.Y., HHMI Fellowship to G.S.G., PSRF to M.P.M., the German Research Foundation (DFG-Fellowship) 399915929 and NIH/NIA 1K99AG066963 to T.H.A., NIH P50-HG007735 to H.Y.C., NIH (R01 AR055650 and R01 AR063717) and the Ellenburg Chair to S.B.G., NIH S10 RR02933801 to Stanford Stem Cell FACS core, European Union’s Horizon 2020 research innovation program (grant agreement no. 733006 to DS and no. 731377 to KS) and Land Salzburg WISS 2025 F 2000237-FIP “STEBS” to DS. Additional support came from NIH S10 1S10OD028493-01A1 for acquisition of a microfluidic chip-based system for cluster sorting and dispensing (Principal Investigator: Charles. K.F. Chan) and NIH S10 1S10OD02349701.

Author information

Author notes

  1. These authors contributed equally: Malachia Y. Hoover, Thomas H. Ambrosi, Holly M. Steininger, Lauren S. Koepke, Yuting Wang.

Authors and Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Malachia Y. Hoover, Thomas H. Ambrosi, Holly M. Steininger, Lauren S. Koepke, Yuting Wang, Liming Zhao, Matthew P. Murphy, Alina A. Alam, Elizabeth J. Arouge, M. Gohazrua K. Butler, Eri Takematsu, Suzan P. Stavitsky, Rahul Sinha, Michael Longaker & Charles K. F. Chan

  2. Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA

    Thomas H. Ambrosi, Michael Longaker & Charles K. F. Chan

  3. Department of Orthopaedic Surgery, UC Davis Health, Sacramento, CA, USA

    Thomas H. Ambrosi

  4. Department of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Yuting Wang & Liming Zhao

  5. Blond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UK

    Matthew P. Murphy

  6. Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, USA

    Serena Hu, Julius Bishop, Michael Gardner & Stuart Goodman

  7. Department of Pathology, University of California San Diego, La Jolla, CA, USA

    Debashis Sahoo

  8. Chan Zuckerberg BioHub, San Francisco, CA, USA

    Maurizio Morri & Norma Neff

  9. Altos Labs, Redwood City, CA, USA

    Maurizio Morri

  10. Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford University, Stanford, CA, USA

    Michael Longaker & Charles K. F. Chan

Authors
  1. Malachia Y. Hoover
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Contributions

C.K.F.C., T.H.A., M.T.L. and R.S. conceived the isolation strategy and functional assays. C.K.F.C. and M.T.L. supervised the project. T.H.A., M.Y.H, H.M.S., L.S.K., M.P.M. and Y.W. developed the protocol, performed the experiments and analyzed the data. M.Y.H. wrote the manuscript. A.A.A., E.J.A., L.Z., M.G.K.B., E.T. and S.P.S. assisted with flow cytometry, in vitro assays and manuscript preparation. J.B., M.G., S.H. and S.G. provided clinical skeletal specimens and edited the manuscript. D.S., R.S., M.M. and N.N. assisted in the bioinformatics and single-cell sequencing platforms.

Corresponding author

Correspondence to Charles K. F. Chan.

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Nature Protocols thanks Matthew Greenblatt, Noriaki Ono, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Chan, C. K. F. et al. Cell 175, 43–56.e21 (2018): https://doi.org/10.1016/j.cell.2018.07.029

Murphy, M. P. et al. Nat. Med. 26, 1583–1592 (2020): https://doi.org/10.1038/s41591-020-1013-2

Ambrosi, T. H. et al. Aging Cell 19, e13164 (2020): https://doi.org/10.1111/acel.13164

Extended data

Extended Data Fig. 1 FACS gating strategy for other hSSC sources.

FACS gating plots for other three hSSC sources, including: fracture callus (28-year-old clavicle), femoral head (87-year-old) and iPSCs (human-monocyte-derived line, SCVI 113).

Extended Data Fig. 2 FACS gating strategy for SSCs of bone marrow reamings.

FACS gating plots for SSCs collected from human bone marrow reamings of 22-year-old femur (top) and 87-year-old hip (bottom).

Extended Data Fig. 3 Fluorescence Minus One (FMO) gating strategy.

FMO controls help to define the negative signal for the given antibody and allow for a more informed gating strategy. Cells were isolated from a 76-year-old male femoral head. Representative plots were generated on a FACS Aria II for each FMO (eg. anti-Tie2/CD31 in APC-Cy7, anti-CD235/CD45/DAPI in Pacific Blue, anti-PDPN in APC, anti-CD146 in PE-Cy7, anti-CD73 in FitC, and anti-CD164 in PE).

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Hoover, M.Y., Ambrosi, T.H., Steininger, H.M. et al. Purification and functional characterization of novel human skeletal stem cell lineages. Nat Protoc 18, 2256–2282 (2023). https://doi.org/10.1038/s41596-023-00836-5

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