Isolation and functional assessment of mouse skeletal stem cell lineage

Nature Protocols
Gunsagar S GulatiCharles K F Chan

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 ...Continue Reading

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Citations

Mar 5, 2019·The Journal of Clinical Investigation·Yujiao HanWeiguo Zou
Sep 24, 2019·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·Regis J O'KeefeRosa Serra
Jan 30, 2020·Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society·Regis J O'KeefeRosa Serra
Jul 10, 2019·Stem Cells International·Jun-Qi LiuZhen Tan
Aug 19, 2020·Nature Medicine·Matthew P MurphyCharles K F Chan
Oct 2, 2019·Frontiers in Cell and Developmental Biology·Thomas H AmbrosiCharles K F Chan
Jul 17, 2020·Frontiers in Bioengineering and Biotechnology·Stuart B Goodman, Tzuhua Lin
Nov 23, 2020·Bone·Laura DohertyArchana Sanjay
Oct 15, 2020·Biological Reviews of the Cambridge Philosophical Society·Shuo HuangLin Chen
Apr 19, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Wenbin WuSong Guo Zheng
Aug 1, 2021·Nature Communications·Siddharth MenonNatalina Quarto
Aug 13, 2021·Nature·Thomas H AmbrosiCharles K F Chan

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