Abstract
BackgroundArticular 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.MethodsWe 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.ResultsChondroprogenitors 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).ConclusionsOverall, 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.
Highlights
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
Collagen type two alpha chain one (COL2A1)-positive chondroprogenitor cells express PDGFRβ, CD146, and CD166 COL2A1-green fluorescent protein (GFP) reporter Human-induced pluripotent stem cells (hiPSC) were differentiated into chondroprogenitor cells along the mesodermal lineage for 12 days as previously described [26]
The cells were labeled for surface markers commonly associated with Mesenchymal stem cell (MSC) and/or chondroprogenitors in the developing limb bud: BMPR1β, CD73, CD105, CD146, CD166, CD271, and PDGFRβ [22,23,24]
Summary
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. Despite reports of several chondrogenic differentiation protocols for pluripotent stem cells [10,11,12,13,14,15], incomplete differentiation and cell heterogeneity remain as the major obstacles for iPSC chondrogenesis [16, 17] This challenge has been addressed in other stem and progenitor cell types by prospectively isolating cells that exhibit chondrogenic lineage commitment using surface marker expression. Mesenchymal progenitor cells, which express CD105, CD166 (ALCAM), and CD146 (MCAM), have been reported to have a high chondrogenic potential [19,20,21] Adult multipotent cells, such as the bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (ASCs), exhibit chondrogenic potential and have been used extensively for cartilage tissue engineering. Surface marker characteristics of hiPSC-derived chondroprogenitors or chondrocytes remain to be identified
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