PMID- 37316563
OWN - NLM
STAT- MEDLINE
DCOM- 20230712
LR  - 20231108
IS  - 1750-2799 (Electronic)
IS  - 1754-2189 (Print)
IS  - 1750-2799 (Linking)
VI  - 18
IP  - 7
DP  - 2023 Jul
TI  - Purification and functional characterization of novel human skeletal stem cell 
      lineages.
PG  - 2256-2282
LID - 10.1038/s41596-023-00836-5 [doi]
AB  - 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.
CI  - (c) 2023. Springer Nature Limited.
FAU - Hoover, Malachia Y
AU  - Hoover MY
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Ambrosi, Thomas H
AU  - Ambrosi TH
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
AD  - Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford 
      University School of Medicine, Stanford, CA, USA.
AD  - Department of Orthopaedic Surgery, UC Davis Health, Sacramento, CA, USA.
FAU - Steininger, Holly M
AU  - Steininger HM
AUID- ORCID: 0000-0001-8098-3753
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Koepke, Lauren S
AU  - Koepke LS
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Wang, Yuting
AU  - Wang Y
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
AD  - Department of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, 
      Huazhong University of Science and Technology, Wuhan, China.
FAU - Zhao, Liming
AU  - Zhao L
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
AD  - Department of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, 
      Huazhong University of Science and Technology, Wuhan, China.
FAU - Murphy, Matthew P
AU  - Murphy MP
AUID- ORCID: 0000-0003-0885-4089
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
AD  - 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.
FAU - Alam, Alina A
AU  - Alam AA
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Arouge, Elizabeth J
AU  - Arouge EJ
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Butler, M Gohazrua K
AU  - Butler MGK
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Takematsu, Eri
AU  - Takematsu E
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Stavitsky, Suzan P
AU  - Stavitsky SP
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Hu, Serena
AU  - Hu S
AD  - Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, 
      USA.
FAU - Sahoo, Debashis
AU  - Sahoo D
AUID- ORCID: 0000-0003-2329-8228
AD  - Department of Pathology, University of California San Diego, La Jolla, CA, USA.
FAU - Sinha, Rahul
AU  - Sinha R
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
FAU - Morri, Maurizio
AU  - Morri M
AD  - Chan Zuckerberg BioHub, San Francisco, CA, USA.
AD  - Altos Labs, Redwood City, CA, USA.
FAU - Neff, Norma
AU  - Neff N
AD  - Chan Zuckerberg BioHub, San Francisco, CA, USA.
FAU - Bishop, Julius
AU  - Bishop J
AD  - Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, 
      USA.
FAU - Gardner, Michael
AU  - Gardner M
AD  - Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, 
      USA.
FAU - Goodman, Stuart
AU  - Goodman S
AD  - Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, 
      USA.
FAU - Longaker, Michael
AU  - Longaker M
AUID- ORCID: 0000-0003-1430-8914
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA.
AD  - Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford 
      University School of Medicine, Stanford, CA, USA.
AD  - Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School 
      of Medicine, Stanford University, Stanford, CA, USA.
FAU - Chan, Charles K F
AU  - Chan CKF
AUID- ORCID: 0000-0001-6570-7574
AD  - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University 
      School of Medicine, Stanford, CA, USA. chazchan@stanford.edu.
AD  - Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford 
      University School of Medicine, Stanford, CA, USA. chazchan@stanford.edu.
AD  - Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School 
      of Medicine, Stanford University, Stanford, CA, USA. chazchan@stanford.edu.
LA  - eng
GR  - S10 OD028493/OD/NIH HHS/United States
GR  - K99 AG049958/AG/NIA NIH HHS/United States
GR  - R01 DE026730/DE/NIDCR NIH HHS/United States
GR  - U01 HL099999/HL/NHLBI NIH HHS/United States
GR  - U01 HL099776/HL/NHLBI NIH HHS/United States
GR  - R00 AG049958/AG/NIA NIH HHS/United States
GR  - I01 BX003754/BX/BLRD VA/United States
GR  - R01 DK115600/DK/NIDDK NIH HHS/United States
GR  - R01 CA086065/CA/NCI NIH HHS/United States
GR  - R01 HL058770/HL/NHLBI NIH HHS/United States
GR  - R21 DE019274/DE/NIDCR NIH HHS/United States
GR  - R01 AR063717/AR/NIAMS NIH HHS/United States
GR  - R56 DE025597/DE/NIDCR NIH HHS/United States
GR  - R01 DE021683/DE/NIDCR NIH HHS/United States
GR  - K99 AG066963/AG/NIA NIH HHS/United States
GR  - R21 DE024230/DE/NIDCR NIH HHS/United States
GR  - R01 DE027323/DE/NIDCR NIH HHS/United States
GR  - K08 GM069677/GM/NIGMS NIH HHS/United States
GR  - P50 HG007735/HG/NHGRI NIH HHS/United States
GR  - R00 AG066963/AG/NIA NIH HHS/United States
GR  - R01 AR055650/AR/NIAMS NIH HHS/United States
GR  - U24 DE026914/DE/NIDCR NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Review
DEP - 20230614
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
SB  - IM
MH  - Humans
MH  - Mice
MH  - Animals
MH  - Cell Lineage
MH  - Reproducibility of Results
MH  - *Mesenchymal Stem Cells
MH  - Cell Differentiation/physiology
MH  - Bone and Bones
MH  - Bone Marrow Cells
MH  - Cells, Cultured
PMC - PMC10495180
MID - NIHMS1925629
EDAT- 2023/06/15 01:08
MHDA- 2023/07/12 06:42
PMCR- 2023/09/11
CRDT- 2023/06/14 23:21
PHST- 2022/04/13 00:00 [received]
PHST- 2023/03/21 00:00 [accepted]
PHST- 2023/07/12 06:42 [medline]
PHST- 2023/06/15 01:08 [pubmed]
PHST- 2023/06/14 23:21 [entrez]
PHST- 2023/09/11 00:00 [pmc-release]
AID - 10.1038/s41596-023-00836-5 [pii]
AID - 10.1038/s41596-023-00836-5 [doi]
PST - ppublish
SO  - Nat Protoc. 2023 Jul;18(7):2256-2282. doi: 10.1038/s41596-023-00836-5. Epub 2023 
      Jun 14.