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

Abstract

Background: 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. Methods: 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. Results: 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). Conclusions: 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.

Transient Receptor Potential Vanilloid 4 as a Regulator of Induced Pluripotent Stem Cell Chondrogenesis

“…10 A similar pattern of TRPV4 expression has also been observed in human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and adipose stem cells (ASCs) undergoing chondrogenesis. [25][26][27][28] Furthermore, ASCs isolated from Trpv4 À/À mice exhibit reduced chondrogenic differentiation potential and enhanced osteogenic potential compared to ASCs from Trpv4 +/+ mice. 29 Interestingly, the chemical activation of TRPV4 induced Sox9-dependent reporter activity, 10 and iPSCs derived from patients with a lethal skeletal dysplasia caused by TRPV4 mutation show significantly decreased chondrogenesis and expression of COL2A1, SOX9, ACAN, type X collagen (COL10A1), and runt-related protein 2 (RUNX2).…”

Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration

“…For example, using the approach of single-cell analysis, Dicks et al . ( 35 ) have found that at least nine different cell clusters—including chondrogenic, neurogenic, and other nonbone/noncartilage mesenchymal cells—are identified in hiPSC-derived mesodermal chondrocyte culture. Similarly, Umeda et al .…”

Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells

“…To investigate the conditions that differentiate PSCs toward cells that bear more chondrocytic properties, PSCs in which chondrocyte‐specific reporter constructs were integrated have been investigated. The promoter/enhancer sequences of type XI collagen α2 chain gene ( COL11a2 ) (Yamashita et al., 2015) or COL2A1 (Adkar et al., 2019; Dicks et al., 2020) have been used to direct the reporter expression in a chondrocyte‐specific manner. To increase the efficiency of the chondrocytic differentiation, the transfection of non‐viral vectors containing TGFβ3 and BMP2 to promote the chondrogenic differentiation of PSCs (Rim et al., 2020) and lithium‐containing biomaterial to reduce hypertrophy (Hu et al., 2020) have also been applied.…”