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Isolation and functional assessment of mouse skeletal stem cell lineage

Nature Protocols volume 13, pages 1294–1309 (2018)Cite this article

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Abstract

There are limited methods available to study skeletal stem, progenitor, and progeny cell activity in normal and diseased contexts. Most protocols for skeletal stem cell isolation are based on the extent to which cells adhere to plastic or whether they express a limited repertoire of surface markers. Here, we describe a flow cytometry–based approach that does not require in vitro selection and that uses eight surface markers to distinguish and isolate mouse skeletal stem cells (mSSCs); bone, cartilage, and stromal progenitors (mBCSPs); and five downstream differentiated subtypes, including chondroprogenitors, two types of osteoprogenitors, and two types of hematopoiesis-supportive stroma. We provide instructions for the optimal mechanical and chemical digestion of bone and bone marrow, as well as the subsequent flow-cytometry-activated cell sorting (FACS) gating schemes required to maximally yield viable skeletal-lineage cells. We also describe a methodology for renal subcapsular transplantation and in vitro colony-formation assays on the isolated mSSCs. The isolation of mSSCs can be completed in 9 h, with at least 1 h more required for transplantation. Experience with flow cytometry and mouse surgical procedures is recommended before attempting the protocol. Our system has wide applications and has already been used to study skeletal response to fracture, diabetes, and osteoarthritis, as well as hematopoietic stem cell–niche interactions in the bone marrow.

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Figure 1: Overview of the protocol for skeletal stem cell lineage isolation and functional assessment.
Figure 2: Schema for mechanical digestion of skeletal tissue.
Figure 3: Skeletal stem cell lineage hierarchy and flow cytometry gating scheme.
Figure 4: Fluorescence-minus-one (FMO) controls for mouse skeletal lineage gating strategy.
Figure 5: Schema for renal subcapsular transplantation of skeletal-lineage cells.
Figure 6: Movat pentachrome staining of renal subcapsular grafts.
Figure 7: In vitro colony-formation assays with p-mSSCs.

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Acknowledgements

We thank A. McCarty and C. Wang for mouse-colony management; P. Pereira, T. Storm, E. Seo, and T. Naik for laboratory management; and P. Lovelace, J. Ho, and S. Weber for FACS management. This study was supported by the National Institutes of Health (NIH; grants R56 DE025597, R01 DE021683, R21 DE024230, R01 DE019434, RC2 DE020771, U01 HL099776, and R21 DE019274 to M.T.L.; grants U01HL099999, 5 R01 CA86065, and 5 R01 L058770 to I.L.W.); a Siebel Fellowship from the Thomas and Stacey Siebel Foundation, a Prostate Cancer Foundation Young Investigator Award, and a National Institute on Aging Research Career Development Award (grant 1K99AG049958-01A1) to C.K.F.C.; the California Institute for Regenerative Medicine (CIRM; grant TR1-01249), the Oak Foundation, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Gunn/Olivier Research Fund to M.T.L.; a Howard Hughes Medical Institute Medical Student Research Fellowship to G.S.G.; and The Plastic Surgery Research Foundation National Endowment for Plastic Surgery to M.P.M.

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Author notes

  1. Gunsagar S Gulati, Matthew P Murphy, Owen Marecic and Michael Lopez: These authors contributed equally to this work.

Authors and Affiliations

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

    Gunsagar S Gulati, Matthew P Murphy, Owen Marecic, Michael Lopez, Rachel E Brewer, Lauren S Koepke, Anoop Manjunath, Ryan C Ransom, Ankit Salhotra, Irving L Weissman, Michael T Longaker & Charles K F Chan

  2. Plastic and Reconstructive Surgery Division, Department of Surgery, Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University, Stanford, CA, USA

    Gunsagar S Gulati, Matthew P Murphy, Owen Marecic, Michael Lopez, Rachel E Brewer, Lauren S Koepke, Anoop Manjunath, Ryan C Ransom, Ankit Salhotra, Michael T Longaker & Charles K F Chan

  3. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Irving L Weissman

  4. Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA

    Irving L Weissman

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Contributions

C.K.F.C., G.S.G., M.T.L., and I.L.W. conceived the isolation strategy and functional assays. M.T.L. and I.L.W. supervised the project. G.S.G., M.P.M., O.M., and M.L. developed the protocol, performed the experiments, and analyzed the data. G.S.G. wrote the manuscript. R.E.B., L.S.K., R.C.R., A.S., and A.M. assisted with flow cytometry, in vitro assays, and manuscript preparation.

Corresponding author

Correspondence to Charles K F Chan.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Effect of successive chemical digests on total cell yield.

Total cell yield from crushed bones increases with each successive digest but saturates after three digests [Steps 11-15]. Briefly, crushed 8-week mouse skeletal tissue was separated from whole bone marrow and digested in digest buffer containing 3000 U ml−1 type II collagenase. Total cell number before and after each successive digest was counted by hemocytometer and mean and standard deviation for n = 4 was calculated. All animal experiments in this figure are in accordance with the Stanford's Administrative Panel on Laboratory Animal Care (APLAC) and received approval from the Institutional Review Board (IRB).

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1. (PDF 199 kb)

Supplementary Data

Estimated total number of cells obtained from individual 8-week-old C57BL/6 mice. The data in this file were used to obtain the averages ± standard deviation stated in Table 1. (XLSX 9 kb)

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Gulati, G., Murphy, M., Marecic, O. et al. Isolation and functional assessment of mouse skeletal stem cell lineage. Nat Protoc 13, 1294–1309 (2018). https://doi.org/10.1038/nprot.2018.041

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