Defining the Human Articular Chondrocyte Lineage

进行性关节变性，如骨关节炎中发生的，是常见的和当前用于治疗关节衰竭的疗法 是有限的。原位诱导修复受损关节软骨的尝试效果有限， 这种组织在出生后似乎缺乏内在的再生能力。我们假设了解了 多能干细胞（PSC）在体外成为关节软骨细胞（AC），并在体内维持这种表型。 体内，将导致修复和再生软骨的改进策略。因此我们将识别基因 以及负责使人（h）PSC成为AC的生物学机制。我们还将确定 使AC能够在体外挑战时更好地抵抗变成肥大软骨细胞的因子， 植入体内。使用我们先前发表的方法， hPSC，我们将确定这些细胞何时完全定型为AC谱系。我们会完成的 通过使用TGFβ3诱导关节软骨细胞分化不同时间长度，然后评估 当细胞在体外暴露于BMP 4并植入到 免疫缺陷小鼠体内。为了确定维持AC身份和增强AC弹性的因素， 在这些挑战的存在下，我们将确定hPSC衍生的软骨细胞的分子谱， 在它们成为AC后使用RNA和ATAC测序。我们还将比较 来源于小鼠ESC的成软骨祖细胞、关节软骨细胞和生长板软骨细胞 我们对CRISPR/Cas9进行了编辑，使其在谱系特异性调控下表达荧光蛋白。 发起人。从这些数据中，我们将识别并优先考虑下游研究的候选转录 参与AC谱系的因子、染色质修饰酶和信号通路组分 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），以及在可能的情况下，药理学方法 (e.g.,途径激动剂和拮抗剂）。通过了解如何 AC是从hPSC诱导的，以及AC表型如何可以在存在外部刺激的情况下维持。 挑战，我们将发现新的方法来修复，替换或再生患者受损的软骨。

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.