Understanding skeletal diseases using human induced pluripotent stem cells

使用人类诱导多能干细胞了解骨骼疾病

• 批准号: MR/S002553/1
• 负责人: Susan Kimber
• 金额: $ 79.46万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FS002553%2F1
• 关键词: Understanding; skeletal; diseases; using; human

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.

多发性骨骺发育不良（MED）和假性软骨发育不全（PSACH）是临床上类似的骨骼疾病，在年轻时涉及身材矮小和骨关节炎。MED和PSACH可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产生软骨的方法。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，可以与患有其他骨骼疾病或骨关节炎的人的软骨进行比较。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。我们的目标是建立在这些数据的基础上，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，我们将从其他患有MED和PSACH的患者及其健康亲属身上产生额外的干细胞（称为诱导多能干细胞（iPSCs））。我们需要研究几个不同的家庭，以确保症状不是由诊断出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iPSCs中产生导致突变的相同疾病，这也使我们能够确定我们所看到的差异是由突变引起的。此外，我们将纠正疾病iPSCs的突变，之后由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。这将包括观察所有编码蛋白质的RNA分子（通过一种称为RNA- seq的技术），评估来自疾病ipsc软骨细胞的软骨颗粒的不同区域，并将其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，导致患者的差异。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质（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COMP突变是否也是如此。基于我们的研究结果，我们将选择药物来纠正突变细胞的疾病表型，并在健康的干细胞衍生软骨中实验诱导其，以确认已确定的病理机制。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一部分。

项目成果

Optogenetic control of the Bone Morphogenetic Protein signalling pathway through engineered blue light-sensitive receptors

• DOI: 10.1101/2020.04.27.063073
• 发表时间: 2020-04
• 期刊: bioRxiv
• 作者: Paul A Humphreys;Steven Woods;Christopher A Smith;S. Cain;Robert J. Lucas;S. Kimber

Characterization of the mechanism by which a nonsense variant in RYR2 leads to disordered calcium handling.

• DOI: 10.14814/phy2.15265
• 发表时间: 2022-04
• 期刊: PHYSIOLOGICAL REPORTS
• 影响因子: 2.5
• 作者: Hopton, Claire;Tijsen, Anke J.;Maizels, Leonid;Arbel, Gil;Gepstein, Amira;Bates, Nicola;Brown, Benjamin;Huber, Irit;Kimber, Susan J.;Newman, William G.;Venetucci, Luigi;Gepstein, Lior
• 通讯作者: Gepstein, Lior

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs

• DOI: 10.1016/j.celrep.2023.113502
• 发表时间: 2023-11-28
• 期刊: CELL REPORTS
• 影响因子: 8.8
• 作者: Humphreys，Paul E. A.;Woods，Steven;Kimber，Susan J.
• 通讯作者: Kimber，Susan J.

Emulating Human Tissues and Organs: A Bioprinting Perspective Toward Personalized Medicine.

• DOI: 10.1021/acs.chemrev.0c00342
• 发表时间: 2020-10-14
• 期刊: Chemical reviews
• 影响因子: 62.1
• 作者: Fonseca AC;Melchels FPW;Ferreira MJS;Moxon SR;Potjewyd G;Dargaville TR;Kimber SJ;Domingos M
• 通讯作者: Domingos M

Optogenetic Control of the BMP Signaling Pathway.

• DOI: 10.1021/acssynbio.0c00315
• 发表时间: 2020-11-20
• 期刊: ACS synthetic biology
• 影响因子: 4.7
• 作者: Humphreys PA;Woods S;Smith CA;Bates N;Cain SA;Lucas R;Kimber SJ
• 通讯作者: Kimber SJ