Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.

Journal Article (Journal Article)


Articular cartilage shows little or no capacity for intrinsic repair, generating a critical need of regenerative therapies for joint injuries and diseases such as osteoarthritis. Human-induced pluripotent stem cells (hiPSCs) offer a promising cell source for cartilage tissue engineering and in vitro human disease modeling; however, off-target differentiation remains a challenge during hiPSC chondrogenesis. Therefore, the objective of this study was to identify cell surface markers that define the true chondroprogenitor population and use these markers to purify iPSCs as a means of improving the homogeneity and efficiency of hiPSC chondrogenic differentiation.


We used a CRISPR-Cas9-edited COL2A1-GFP knock-in reporter hiPSC line, coupled with a surface marker screen, to identify a novel chondroprogenitor population. Single-cell RNA sequencing was then used to analyze the distinct clusters within the population. An unpaired t test with Welch's correction or an unpaired Kolmogorov-Smirnov test was performed with significance reported at a 95% confidence interval.


Chondroprogenitors expressing CD146, CD166, and PDGFRβ, but not CD45, made up an average of 16.8% of the total population. Under chondrogenic culture conditions, these triple-positive chondroprogenitor cells demonstrated decreased heterogeneity as measured by single-cell RNA sequencing with fewer clusters (9 clusters in unsorted vs. 6 in sorted populations) closer together. Additionally, there was more robust and homogenous matrix production (unsorted: 1.5 ng/ng vs. sorted: 19.9 ng/ng sGAG/DNA; p < 0.001) with significantly higher chondrogenic gene expression (i.e., SOX9, COL2A1, ACAN; p < 0.05).


Overall, this study has identified a unique hiPSC-derived subpopulation of chondroprogenitors that are CD146+ /CD166+ /PDGFRβ+ /CD45- and exhibit high chondrogenic potential, providing a purified cell source for cartilage tissue engineering or disease modeling studies.

Full Text

Duke Authors

Cited Authors

  • Dicks, A; Wu, C-L; Steward, N; Adkar, SS; Gersbach, CA; Guilak, F

Published Date

  • February 18, 2020

Published In

Volume / Issue

  • 11 / 1

Start / End Page

  • 66 -

PubMed ID

  • 32070421

Pubmed Central ID

  • PMC7026983

Electronic International Standard Serial Number (EISSN)

  • 1757-6512

International Standard Serial Number (ISSN)

  • 1757-6512

Digital Object Identifier (DOI)

  • 10.1186/s13287-020-01597-8


  • eng