Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to Hedgehog-mediated osteogenic induction

Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Patched-1 (PTCH1) is a Hedgehog (Hh) receptor that acts as a negative regulator of constitutive Hh signaling by preventing the G protein-coupled receptor Smoothened from entering the cilium in the absence of Hh protein binding. We generated iPSCs from four unrelated Gorlin syndrome patients with loss-of-function mutations in PTCH1 using the Sendai virus vector (SeVdp(KOSM)302). The patient-derived iPSCs exhibited basic iPSC features, including stem cell marker expression, totipotency, and the ability to form teratomas. To check the Patient-derived iPSCs mimic patinet phenotype, We performted osteoblast differentiation and checked mRNA expression by hedgehog PCR array systems. qPCR gene expression profiling. 2 control hiPSC (KD iPS and 201B7) and 4 disease specific hiPSC (G1-1,G1-6,G2-5,G2-8) were used and treated separately as indicated in the summary. Two disease specific iPSc (G1-1,G1-6) were established by same patient fibroblasts. Two disease specific iPSc (G2-5,G2-8) were established by other patient fibroblasts. There were two experimental group named iPS and OBM. iPS group cultured EB and then collect samples. OBM group cultured EB, performed osteoblast differentiation until day 10 and then collect samples. Equal amount total RNA from each donor was pooled prior to gene expression analysis.

戈林综合征（Gorlin Syndrome）是一种常染色体显性遗传综合征，可使患者易患基底细胞癌（basal cell carcinoma）、牙源性角化囊肿（odontogenic keratocyst）及骨骼发育异常。Patched-1（PTCH1）是刺猬信号（Hedgehog, Hh）受体，在未结合Hh蛋白的情况下，可通过阻止G蛋白偶联受体平滑蛋白（Smoothened）进入纤毛，作为组成型Hh信号通路的负调控因子发挥作用。本研究使用仙台病毒载体（Sendai virus vector，SeVdp(KOSM)302），从4例携带PTCH1功能丧失性突变的无关戈林综合征患者体内构建了诱导多能干细胞（induced pluripotent stem cells, iPSCs）。该患者源性iPSCs具备诱导多能干细胞的基本特征，包括干细胞标志物表达、全能性以及畸胎瘤形成能力。为验证患者源性iPSCs能够模拟患者表型，我们开展了成骨细胞分化实验，并通过刺猬信号通路PCR芯片系统检测mRNA表达水平，同时进行了实时定量PCR（qPCR）基因表达谱分析。本研究使用2株对照人类诱导多能干细胞（hiPSC，KD iPS和201B7）及4株疾病特异性人类诱导多能干细胞（hiPSCs，G1-1、G1-6、G2-5、G2-8），并按研究摘要所述分别进行处理。其中，G1-1与G1-6两株疾病特异性iPSCs均由同一患者的成纤维细胞构建获得，G2-5与G2-8则由另外两名患者的成纤维细胞构建得到。本研究设置两个实验组：iPS组与OBM组。iPS组先培养胚胎体（embryoid body, EB）后收集样本；OBM组先培养胚胎体，再进行成骨细胞分化至第10天后收集样本。在开展基因表达分析前，将每位供体的等量总RNA进行混合。