Patients with classical melorheostosis exhibit exuberant bone overgrowth in the appendicular skeleton, resulting in pain and deformity with no known treatment. Most patients have somatic, mosaic mutations in MAP2K1 (encoding the MEK1 protein) in osteoblasts and overlying skin. As with most rare bone diseases, lack of affected tissue has limited the opportunity to understand how the mutation results in excess bone formation. The aim of this study was to create a cellular model to study melorheostosis. We obtained patient skin cells bearing the MAP2K1 mutation (affected cells), and along with isogenic control normal fibroblasts reprogrammed them using the Sendai virus method into induced pluripotent stem cells (iPSCs). Pluripotency was validated by marker staining and embryoid body formation. iPSCs were then differentiated to mesenchymal stem cells (iMSCs) and validated by flow cytometry. We confirmed retention of the MAP2K1 mutation in iMSCs with polymerase chain reaction (PCR) and confirmed elevated MEK1 activity by immunofluorescence staining. Mutation-bearing iMSCs showed significantly elevated vascular endothelial growth factor (VEGF) secretion, proliferation and collagen I and IV secretion. iMSCs were then differentiated into osteoblasts, which showed increased mineralization at 21 days and increased VEGF secretion at 14 and 21 days of differentiation. Administration of VEGF to unaffected iMSCs during osteogenic differentiation was sufficient to increase mineralization. Blockade of VEGF by bevacizumab reduced mineralization in iMSC-derived affected osteoblasts and in affected primary patient-derived osteoblasts. These data indicate that patient-derived induced pluripotent stem cells recreate the elevated MEK1 activity, increased mineralization, and increased proliferation seen in melorheostosis patients. The increased bone formation is driven, in part, by abundant VEGF secretion. Modifying the activity of VEGF (a known stimulator of osteoblastogenesis) represents a promising treatment pathway to explore. iPSCs may have wide applications to other rare bone diseases. © 2023 American Society for Bone and Mineral Research (ASBMR).

中文翻译：

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VEGF 分泌促进经典 MAP2K1+ 骨质增生中的骨形成

患有典型的骨质增生症的患者表现出附肢骨骼的过度生长，导致疼痛和畸形，且没有已知的治疗方法。大多数患者的成骨细胞和皮肤上的MAP2K1（编码 MEK1 蛋白）存在体细胞嵌合突变。与大多数罕见的骨病一样，缺乏受影响的组织限制了了解突变如何导致过量骨形成的机会。本研究的目的是创建一个细胞模型来研究melorheostosis。我们获得了携带MAP2K1突变的患者皮肤细胞（受影响的细胞），并使用仙台病毒方法将其与同基因对照正常成纤维细胞一起重新编程为诱导多能干细胞 (iPSC)。通过标记染色和拟胚体形成验证多能性。然后 iPSC 分化为间充质干细胞 (iMSC)，并通过流式细胞术进行验证。我们通过聚合酶链式反应 (PCR) 证实了 iMSC 中保留了MAP2K1突变，并通过免疫荧光染色证实了 MEK1 活性升高。携带突变的 iMSC 表现出血管内皮生长因子 (VEGF) 分泌、增殖以及 I 型和 IV 型胶原蛋白显着升高。然后 iMSC 分化为成骨细胞，在分化 21 天时矿化增加，并在分化 14 天和 21 天时 VEGF 分泌增加。在成骨分化期间向未受影响的 iMSC 施用 VEGF 足以增加矿化。贝伐珠单抗阻断 VEGF 会减少 iMSC 来源的受影响的成骨细胞和受影响的原发性患者来源的成骨细胞中的矿化。这些数据表明，源自患者的诱导多能干细胞重现了在软组织增生患者中观察到的 MEK1 活性升高、矿化增加和增殖增加。骨形成的增加部分是由丰富的 VEGF 分泌驱动的。改变 VEGF（一种已知的成骨细胞生成刺激剂）的活性是一条有希望探索的治疗途径。iPSC 可能对其他罕见骨病有广泛的应用。© 2023 美国骨与矿物质研究学会 (ASBMR)。