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

Publication ,  Journal Article
Dicks, A; Wu, C-L; Steward, N; Adkar, SS; Gersbach, CA; Guilak, F
Published in: Stem cell research & therapy
February 2020

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.

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Published In

Stem cell research & therapy

DOI

EISSN

1757-6512

ISSN

1757-6512

Publication Date

February 2020

Volume

11

Issue

1

Start / End Page

66

Related Subject Headings

  • Prospective Studies
  • Induced Pluripotent Stem Cells
  • Humans
  • Chondrogenesis
  • Chondrocytes
  • Cells, Cultured
  • Cell Differentiation
  • CRISPR-Cas Systems
  • 31 Biological sciences
  • 11 Medical and Health Sciences
 

Citation

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Dicks, A., Wu, C.-L., Steward, N., Adkar, S. S., Gersbach, C. A., & Guilak, F. (2020). Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter. Stem Cell Research & Therapy, 11(1), 66. https://doi.org/10.1186/s13287-020-01597-8
Dicks, Amanda, Chia-Lung Wu, Nancy Steward, Shaunak S. Adkar, Charles A. Gersbach, and Farshid Guilak. “Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.Stem Cell Research & Therapy 11, no. 1 (February 2020): 66. https://doi.org/10.1186/s13287-020-01597-8.
Dicks A, Wu C-L, Steward N, Adkar SS, Gersbach CA, Guilak F. Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter. Stem cell research & therapy. 2020 Feb;11(1):66.
Dicks, Amanda, et al. “Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.Stem Cell Research & Therapy, vol. 11, no. 1, Feb. 2020, p. 66. Epmc, doi:10.1186/s13287-020-01597-8.
Dicks A, Wu C-L, Steward N, Adkar SS, Gersbach CA, Guilak F. Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter. Stem cell research & therapy. 2020 Feb;11(1):66.
Journal cover image

Published In

Stem cell research & therapy

DOI

EISSN

1757-6512

ISSN

1757-6512

Publication Date

February 2020

Volume

11

Issue

1

Start / End Page

66

Related Subject Headings

  • Prospective Studies
  • Induced Pluripotent Stem Cells
  • Humans
  • Chondrogenesis
  • Chondrocytes
  • Cells, Cultured
  • Cell Differentiation
  • CRISPR-Cas Systems
  • 31 Biological sciences
  • 11 Medical and Health Sciences