Articular Cartilage Tissue Engineering with Human Pluripotent Stem Cells

摘要 损伤和疾病（如骨关节炎（OA））后关节软骨功能受损仍然是一个主要的健康问题， 问题.基于组织工程的关节软骨损伤治疗的主要缺点之一 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形成和软骨内骨化 过程关节软骨在胚胎发生过程中由称为“间区”细胞的专门的GDF 5+细胞或 “共同祖先”。它们不同于产生生长板软骨细胞的祖细胞。在这一提议中， 我们的目标是确定控制关节样永久性软骨细胞形成与生长的分子靶点 通过使用从人类发育的新型GDF 5+间充质细胞形成板状瞬时软骨细胞 多能干细胞（hPSC）。在体外软骨形成过程中，这些细胞表达原始（或 胚胎）关节软骨细胞，但不软骨细胞肥大。值得注意的是，移植后， 它们发育的软骨，8周内没有观察到矿化（即，永久软骨）。因此 hPSC衍生的GDF 5+细胞可以共享联合祖细胞的活性，尽管全基因组RNA测序结果显示， (seq)分析表明与发育中的腱细胞或韧带细胞有关。相反，替代 hPSC衍生的软骨祖细胞，SOX 9+细胞，产生的软骨容易经历完全的 矿化，模仿生长板软骨祖细胞。有趣的是，当与GDF 5+细胞混合时， SOX 9+细胞衍生的软骨以GDF 5+细胞剂量依赖性方式表现为永久性软骨， 暗示了非细胞自主机制的参与。因此，我们首先建议测试 GDF 5+细胞具有关节祖细胞样活性，表征了从它们发育的（永久）软骨， 并阐明了细胞如何产生永久性软骨（目标1）。其次，我们计划识别基因 通过比较RNA-seq分析，可能参与GDF 5+细胞的永久性软骨形成 从GDF 5+和SOX 9+细胞发育的软骨颗粒（Aim 2）。然后，我们将在功能上验证 候选基因（及其编码的蛋白质、抑制剂和激活剂，如果可商购获得）， 使用基因敲除使SOX 9+细胞产生关节样永久性软骨细胞， 过表达技术（Aim 3）。然后，我们将研究GDF 5+细胞和这种基因修饰的SOX 9+细胞是否能够被诱导。 细胞比SOX 9+细胞诱导更持久的受损关节软骨修复（目的4）。最后， 将在治疗相关的成人间充质基质细胞中类似地操作 以证实靶向相同的机制将使成体干细胞转化为关节软骨形成细胞 (Aim 4）。因此，所提出的研究的成功将提供关于关节样 永久性软骨可以选择性地从各种软骨细胞形成，潜在地导致新的软骨细胞。 有效的治疗策略，持续修复受损软骨。

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.