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Purification and functional characterization of novel human skeletal stem cell lineages

祖细胞 间充质干细胞 间质细胞 生物 干细胞 计算生物学 细胞生物学 流式细胞术 细胞 细胞分化 生物信息学 免疫学 癌症研究 遗传学 基因
作者
Malachia Hoover,Thomas H. Ambrosi,Holly Steininger,Lauren S. Koepke,Yuting Wang,Liming Zhao,Matthew P. Murphy,Alina Alam,Elizabeth J. Arouge,Morgan Butler,Eri Takematsu,Suzan P. Stavitsky,Serena S. Hu,Debashis Sahoo,Rahul Sinha,Maurizio Morri,Norma Neff,Julius A. Bishop,Michael J. Gardner,Stuart B. Goodman,Michael T. Longaker,Charles K. F. Chan
出处
期刊:Nature Protocols [Nature Portfolio]
卷期号:18 (7): 2256-2282 被引量:10
标识
DOI:10.1038/s41596-023-00836-5
摘要

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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