Defining the Human Articular Chondrocyte Lineage

进行性关节退行性变，就像发生在骨性关节炎中一样，是目前治疗关节衰竭的常见方法。 是有限的。诱导损伤关节软骨原位修复的尝试效果有限，因为 这种组织在出生后似乎缺乏内在的再生能力。我们假设理解如何 多能干细胞(PSCs)在体外可分化为关节软骨细胞(ACS)，并在 体内，将导致修复和再生软骨的改进策略。因此，我们将确定基因 以及使人类(H)PSCs成为急性冠脉综合征的生物学机制。我们还将确定 使急性冠脉综合征在体外受到刺激时更好地抵抗成为肥大软骨细胞的因素 植入体内。使用我们之前发表的方法生成关节软骨组织 HPSCs，我们将确定这些细胞何时完全致力于AC谱系。我们会做到这一点的 通过用转化生长因子β3诱导不同时间的关节软骨细胞分化，然后评估 当BMP4体外暴露并植入细胞后，细胞不再肥大 体内免疫缺陷小鼠。为了找出维持交流特性并增强交流弹性的因素 面对这些挑战，我们将定义hPSC来源的软骨细胞的分子图谱，然后 使用RNA和ATAC测序将其转化为急性冠脉综合征。我们还将比较两种基因的表达模式 小鼠胚胎干细胞来源的软骨祖细胞、关节软骨细胞和生长板软骨细胞 我们编辑的CRISPR/Cas9在谱系特异性控制下表达荧光蛋白的株系 推动者。从这些数据中，我们将确定下游研究的候选转录并确定优先顺序 与AC谱系有关的因子、染色质修饰酶和信号通路组件 承诺。然后，我们将使用遗传方法评估单个候选人的重要性(例如， 使用修饰的Cas9蛋白调节基因表达)，并在可能的情况下，药理学方法 (例如，通路激动剂和拮抗剂)在hPSC/mESC分化实验中。通过了解如何 从hPSCs诱导的AC以及在外源细胞存在时AC表型是如何保持的 挑战，我们将发现修复、替换或再生患者受损软骨的新方法。

Progressive joint degeneration, as occurs in osteoarthritis, is common and current therapies for failing joints are limited. Attempts to induce repair of damaged articular cartilage in situ have had limited efficacy because this tissue seems to lack intrinsic regenerative capacity after birth. We hypothesize that understanding how pluripotent stem cells (PSCs) become articular chondrocytes (ACs) in vitro, and maintain this phenotype in vivo, will lead to improved strategies for repairing and regenerating cartilage. We will therefore identify genes and biological mechanisms responsible for enabling human (h)PSCs to become ACs. We will also identify factors that enable ACs to better resist becoming hypertrophic chondrocytes when challenged in vitro and implanted in vivo. Using our previously published methods for generating articular cartilage tissues from hPSCs, we will determine when these cells become fully committed to the AC lineage. We will accomplish this by inducing articular chondrocyte differentiation for different lengths of time using TGFβ3, and then evaluating when the cells no longer become hypertrophic after BMP4 exposure in vitro and implantation into immunodeficient mice in vivo. In order to identify factors that maintain AC identity and enhance AC resilience in the presence these challenges, we will define the molecular profiles of hPSC-derived chondrocytes before and after they become ACs using RNA and ATAC sequencing. We will also compare the expression profiles of chondrogenic progenitors, articular chondrocytes, and growth plate chondrocytes derived from mouse ESC lines that we have CRISPR/Cas9 edited to express fluorescent proteins under the control of lineage-specific promoters. From these data, we will identify and prioritize for downstream studies candidate transcription factors, chromatin modifying enzymes, and signaling pathway components that are involved in AC lineage commitment. We will then assess the importance of individual candidates using genetic approaches (e.g., regulating gene expression using modified Cas9 proteins) and, when possible, pharmacologic approaches (e.g., pathway agonists and antagonists) in the hPSC/mESC differentiation assays. By understanding how ACs are induced from hPSCs and how the AC phenotype can be maintained in the presence of external challenges, we will discover new ways to repair, replace, or regenerate damaged cartilage in patients.