Purification and functional characterization of novel human skeletal stem cell lineages
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 functiona...
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| Published in: | Nature protocols Vol. 18; no. 7; pp. 2256 - 2282 |
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| Main Authors: | , , , , , , , , , , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
London
Nature Publishing Group UK
01-07-2023
Nature Publishing Group |
| Subjects: | |
| Online Access: | Get full text |
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| Summary: | 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.
The authors present a FACS-based protocol for the purification of human skeletal stem cell lineages from a variety of tissue sources, alongside in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-3 content type line 23 ObjectType-Review-2 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 Author contributions |
| ISSN: | 1754-2189 1750-2799 |
| DOI: | 10.1038/s41596-023-00836-5 |