Articular Cartilage Tissue Engineering with Human Pluripotent Stem Cells

摘要 损伤后关节软骨功能受损和骨关节炎(OA)等疾病仍然是一项主要健康问题 有问题。基于组织工程的关节软骨损伤治疗的主要缺点之一 是由植入的间充质基质细胞或内源性祖细胞形成的软骨修复组织 不像关节软骨，可能是由于纤维软骨生成和软骨内骨化 进程。关节软骨是在胚胎发育过程中由特殊的GDF5+细胞产生的，这种细胞被称为间带细胞或 “联合祖先”。它们不同于造就生长板软骨细胞的祖细胞。在这份提案中， 我们的目标是确定控制关节样永久软骨细胞形成和生长的分子靶点。 新型人GDF5+间充质细胞形成板状暂时性软骨细胞 多能干细胞(HPSC)。在体外软骨形成过程中，这些细胞表现出原始(或 胚胎)关节软骨细胞，但不是软骨细胞肥大。值得注意的是，在移植了 软骨形成，8周内未见矿化(即永久性软骨)。因此， HPSC来源的GDF5+细胞可能具有联合祖细胞的活性，尽管全基因组RNA测序 (SEQ)分析表明与腱细胞或韧带细胞的发育有关。相比之下，替代方案 HPSC来源的软骨前体细胞，SOX9+细胞，产生的软骨很容易完全 矿化，模仿生长板软骨祖细胞。有趣的是，当与GDF5+细胞混合时， SOX9+细胞来源的软骨以GDF5+细胞剂量依赖的方式表现为永久软骨。 暗示了非细胞自主机制的参与。因此，我们首先提出测试是否 GDF5+细胞具有关节祖细胞样活性，是从它们发育的(永久)软骨的特征， 并阐明了细胞如何生成永久软骨(目标1)。第二，我们计划识别基因 通过比较RNA-SEQ分析可能参与GDF5+细胞的永久软骨形成 从GDF5+和SOX9+细胞发育的软骨颗粒(目标2)。然后我们将从功能上验证 候选基因(以及它们的编码蛋白、抑制物和激活剂，如果可以商业化的话) 通过基因敲除使SOX9+细胞生成关节样永久软骨细胞 过度表达技术(目标3)。然后我们将检测GDF5+细胞和这种基因修饰的SOX9+ 与SOX9+细胞相比，细胞能更持久地促进受损关节软骨的修复(目标4)。最后，任何基因 在这些研究中定义的将在治疗相关的成人间充质基质细胞中进行类似的操作 为了确认靶向相同的机制将成人干细胞转化为关节软骨形成细胞 (目标4)。因此，拟议的研究的成功将提供对关节状 永久软骨可以由各种软骨细胞选择性地形成，有可能导致新的 有效、持续修复受损软骨的治疗策略。

ABSTRACT Impairment of articular cartilage function after injury and disease like osteoarthritis (OA), remains a major health problem. One of the major drawbacks of tissue engineering-based therapies for damaged joint articular cartilage is that the cartilaginous repair tissue formed by implanted mesenchymal stromal cells or endogenous progenitors does not resemble articular cartilage, likely due to fibrochondrogenesis and the endochondral ossification process. Joint cartilage is generated during embryogenesis by specialized GDF5+ cells called ‘interzone’ cells or ‘joint progenitors’. They are distinct from progenitors that give rise to growth plate chondrocytes. In this proposal, we aim to define the molecular targets that control articular-like permanent chondrocyte formation versus growth plate-like transient chondrocyte formation, by using novel GDF5+ mesenchymal cells developed from human pluripotent stem cells (hPSCs). During in vitro chondrogenesis, such cells express signs of primitive (or embryonic) articular chondrocytes but not of chondrocyte hypertrophy. Significantly, after transplantation of the cartilage they develop, no mineralization was observed for 8 weeks (i.e., permanent cartilage). Therefore, the hPSC-derived GDF5+ cells may share the activity of joint progenitors, although genome-wide RNA-sequencing (seq) analyses suggested association with developing tenocytes or ligamentocytes. In contrast, alternative hPSC-derived chondroprogenitors, SOX9+ cells, generated cartilage that readily underwent complete mineralization, mimicking growth-plate chondroprogenitors. Interestingly, when mixed with GDF5+ cells, the SOX9+ cell-derived cartilage behaved as permanent cartilage in a GDF5+ cell-dose-dependent manner, suggesting the involvement of a non-cell autonomous mechanism. Therefore, we first propose to test if the GDF5+ cells have a joint progenitor-like activity, characterize the (permanent) cartilage developed from them, and shed lights on how the cells generate permanent cartilage (Aim 1). Second, we plan to identify genes potentially involved in permanent cartilage formation from GDF5+ cells through comparative RNA-seq analyses of cartilage pellets developed from the GDF5+ and SOX9+ cells (Aim 2). We will then functionally validate the candidate genes (and their encoded proteins, inhibitors and activators if commercially available) for their ability to enable SOX9+ cells to generate articular-like permanent chondrocytes using gene knockout and overexpression techniques (Aim 3). We will then examine whether GDF5+ cells and such gene-modified SOX9+ cells induce more sustained repair of damaged joint cartilage than SOX9+ cells (Aim 4). Lastly, any of the genes defined in these studies will be manipulated similarly in therapeutically relevant adult mesenchymal stromal cells to confirm that targeting the same mechanisms will convert the adult stem cells to articular cartilage-forming cells (Aim 4). Thus, success of the proposed research will provide mechanistic insights into how articular-like permanent cartilage can be selectively formed from various chondrogenic cells, potentially leading to novel therapeutic strategies for effective, sustained repair of damaged cartilage.