Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter
Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter
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DOI：
10.1186/s13287-020-01597-8
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发表时间：
2020-02-18
影响因子：
7.5
通讯作者：
Guilak, Farshid
中科院分区：
文献类型：
--
作者：
Dicks, Amanda;Wu, Chia-Lung;Guilak, Farshid
关节软骨几乎或完全没有内在修复能力，因此迫切需要再生疗法来治疗关节损伤和疾病，如骨关节炎。人类诱导的多能干细胞（hiPSC）为软骨组织工程和体外人类疾病建模提供了有前途的细胞来源;然而，在hiPSC软骨形成期间，脱靶分化仍然是一个挑战。因此，本研究的目的是鉴定定义真正软骨祖细胞群体的细胞表面标志物，并使用这些标志物纯化iPSC作为改善hiPSC软骨形成分化的均一性和效率的手段。方法我们使用CRISPR-Cas9编辑的COL 2A 1-GFP敲入报告基因hiPSC系，结合表面标记筛选，以鉴定新的软骨祖细胞群体。然后使用单细胞RNA测序来分析群体中的不同簇。进行Welch校正的非配对t检验或非配对Kolmogorov-Smirnov检验，在95%置信区间报告显著性。结果表达CD 146、CD 166和PDGFR β但不表达CD 45的软骨祖细胞平均占总人群的16.8%。在软骨形成培养条件下，这些三重阳性软骨祖细胞表现出降低的异质性，如通过单细胞RNA测序所测量的，具有更少的簇（未分选群体中的9个簇与分选群体中的6个簇）更靠近在一起。此外，存在更稳健和均匀的基质产生（未分选：1.5ng/ng对比分选：19.9ng/ng sGAG/DNA; p < 0.001），具有显著更高的软骨形成基因表达（即，S0X9、C0L2A1、ACAN; p < 0.05）。结论：总体而言，本研究已经鉴定了独特的hiPSC衍生的软骨祖细胞亚群，其为CD 146（+）/CD 166（+）/PDGFR β（+）/CD 45（-），并表现出高的软骨形成潜力，为软骨组织工程或疾病建模研究提供了纯化的细胞来源。
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 PDGFR beta, 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(+)/PDGFR beta(+)/CD45(-) and exhibit high chondrogenic potential, providing a purified cell source for cartilage tissue engineering or disease modeling studies.