Understanding skeletal diseases using human induced pluripotent stem cells

多发性骨骺发育不良（MED）和假性软骨发育不全（PACH）是临床上相似的骨骼疾病，涉及身材矮小和骨关节炎在年轻的时候。MED和PSACH可以由两种细胞外基质蛋白matrilin-3和软骨寡聚基质蛋白（COMP）的突变引起。我们通过对MED和PSACH患者的血细胞进行重新编程来制造干细胞，并开发了一种在培养皿中从这些干细胞中生产软骨的方法。因此，我们产生了一个易于处理的模型，用于分析这些疾病的病理机制，可以与其他骨骼疾病或骨关节炎患者的软骨进行比较。我们发现，骨骼疾病患者产生的软骨细胞（软骨内的细胞）与健康人产生的软骨细胞反应不同。我们的目标是现在建立在这些数据的基础上，以了解这些骨骼疾病的分子机制，并确定潜在的治疗药物。在这个项目中，我们将从其他MED和PSACH患者中产生额外的干细胞[称为诱导多能干细胞（iPSC）]，并作为其健康亲属的对照。我们需要看看几个不同的家庭，以确保症状不是由诊断的突变以外的其他遗传因素引起的。我们还将使用一种称为基因编辑的技术，在健康的iPSC中产生相同的致病突变，这也使我们能够确定我们看到的差异是由突变引起的。此外，我们将纠正疾病iPSC中的突变，之后这些细胞形成的软骨应该与健康干细胞形成的软骨相同。然后，我们将对突变和健康干细胞产生的软骨细胞和软骨中的分子进行深入分析。这将包括观察编码蛋白质的所有RNA分子（通过一种称为RNA-Seq的技术），并评估来自疾病iPSC软骨细胞的软骨颗粒的不同区域，并将这些与我们培养系统中产生的健康软骨和成人软骨进行比较。先进的超微结构分析将用于揭示软骨蛋白的结构变化，这些变化有助于在患者中观察到的差异。我们还将确定和量化蛋白质相互作用，特别是促进软骨形成的小蛋白（BMP-2），在健康和突变的Matrilin-3之间的差异，我们有初步的数据，并找出这是否也是COMP突变的情况。基于我们的发现，我们将选择药物来纠正突变细胞的疾病表型，并在健康干细胞衍生的软骨中实验诱导它，以确认所鉴定的病理机制。这种结构和机制分析将增加对软骨发育和疾病的一般理解，并有助于确定MED和PSACH的新药物靶点，并可能用于骨关节炎患者的子集。

Multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH) are clinically similar skeletal diseases, involving short stature and osteoarthritis at a young age. MED and PSACH can be caused by mutations in two extracellular matrix proteins matrilin-3 and cartilage oligomeric matrix protein (COMP). We made stem cells by reprogramming blood cells from patients with MED and PSACH, and developed a method to produce cartilage from these stem cells in a dish. Thus we produced a tractable model for analysis of the mechanisms underlying the pathology in these diseases which can be compared with cartilage from people with other skeletal diseases or osteoarthritis. We have found that the chondrocytes (cells within cartilage) produced from patients with skeletal disease respond differently to those produced from healthy people. Our aim is to now build on these data to understand the molecular mechanisms underlying these skeletal diseases and to identify potential therapeutic drugs. In this project, we will generate additional stem cells [known as induced pluripotent stem cells (iPSCs)] from other patients with MED and PSACH and as controls their healthy relatives. We need to look at several different families to make sure the symptoms are not caused by other genetic factors than the diagnosed mutation. We will also use a technique called gene editing to generate the same disease causing mutations in healthy iPSCs also allowing us to determine that the differences we see are caused by the mutation. Further, we will correct mutations in disease iPSCs, after which the formation of cartilage by those cells should be the same as cartilage from healthy stem cells. We will then perform a deep analysis of the molecules in chondrocytes and cartilage produced by both mutant and healthy stem cells. This will include looking at all the RNA molecules that code for protein (by a technique called RNA-Seq) and evaluating different regions of the cartilage pellets from the disease iPSC-chondrocytes, and comparing these to healthy cartilage produced in our culture system and to adult cartilage. Advanced ultrastructural analysis will be used to reveal structural changes in cartilage proteins contributing to the differences seen in the patients. We will also identify and quantify how protein interactions, particularly that of a small protein which promotes cartilage formation (BMP-2), differ between healthy and mutant Matrilin-3, for which we have preliminary data and find out if this is also the case for COMP mutation. Based on our findings, we will select drugs to correct the disease phenotype of the mutant cells and experimentally induce it in healthy stem cell derived cartilage to confirm the identified mechanism(s) of pathology. This structural and mechanistic analysis will increase general understanding of cartilage development and disease, and help identify new drug targets for MED and PSACH and potentially for a subset of patients with osteoarthritis.