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

Abstract

Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Causative mutations in several genes associated with the sonic hedgehog (SHH) signaling pathway, including PTCH1, have been identified in Gorlin syndrome patients. However, no definitive genotype—phenotype correlations are evident in these patients, and their clinical presentation varies greatly, often leading to delayed diagnosis and treatment. 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. GLI1 expression levels were greater in fibroblasts and patient-derived iPSCs than in the corresponding control cells. Patient-derived iPSCs expressed lower basal levels than control iPSCs of the genes encoding the Hh ligands Indian Hedgehog (IHH) and SHH, the Hh acetyltransferase HHAT, Wnt proteins, BMP4, and BMP6. Most of these genes were upregulated in patient-derived iPSCs grown in osteoblast differentiation medium (OBM) and downregulated in control iPSCs cultured in OBM. The expression of GLI1 and GLI2 substantially decreased in both control and patient-derived iPSCs cultured in OBM, whereas GLI3, SHH, and IHH were upregulated in patient-derived iPSCs and downregulated in control iPSCs grown in OBM. Activation of Smoothened by SAG in cells grown in OBM significantly enhanced alkaline phosphatase activity in patient-derived iPSCs compared with control iPSC lines. In summary, patient-derived iPSCs expressed lower basal levels than the control iPSCs of the genes encoding Hh, Wnt, and bone morphogenetic proteins, but their expression of these genes strongly increased under osteogenic conditions. These findings indicate that patient-derived iPSCs are hypersensitive to osteogenic induction. We propose that Hh signaling is constituently active in iPSCs from Gorlin syndrome patients, enhancing their response to osteogenic induction and contributing to disease-associated abnormalities.

Gorlin Syndrome: Recent Advances in Genetic Testing and Molecular and Cellular Biological Research

“…Hasegawa et al [ 119 ] from our group showed that iPS cells specific to patients with GS responded more sensitively to osteostimulation. We showed that basic FGF, IGF-1, and TGF-β stimulation or Smo agonist SAG treatment significantly upregulated osteoblast markers and ALP activity.…”

“…Patient-derived hiPSCs are also used for in vitro disease modeling and drug screening. Recently, iPSCs derived from patients with GS have been reported by several groups [ 31 , 48 , 117 , 118 , 119 , 120 ]. By using iPSCs derived from patients with GS, a model of abnormal tissue development and tumor formation can be constructed.…”

“…BMP2/7 stimulation, a potent bone-induced growth factor, increased RUNX2 expression compared to normal iPS cells. GLI1 and GLI2 transcription factors decreased, while the repressor type GLI3 levels increased [ 119 ]. These findings indicate that Gorlin iPS cells are more prone to osteoblast differentiation than normal iPS cells.…”

Hedgehog Activation Regulates Human Osteoblastogenesis

“…We previously generated induced pluripotent stem cells (iPSCs) derived from patients with Gorlin syndrome (Gorlin iPSCs), who therefore carried heterozygous loss-of-function mutations in PTCH1 ; Gorlin iPSCs showed hypersensitivity to Hh signaling, and Hh signaling may be associated with accelerated osteoblast differentiation ( Hasegawa et al., 2017 ). Gorlin iPSCs also demonstrated enhanced Hh signaling, which in turn suppressed Wnt and BMP signaling ( Hasegawa et al., 2017 ). However, once cultured in a conventional osteogenic differentiation medium, they suddenly acquired steep upregulation of Wnts and BMPs to promote osteoblastogenesis.…”

The Role of Dermal Fibroblasts in Nevoid Basal Cell Carcinoma Syndrome Patients: An Overview

“…It is characterized by jaw keratocysts, palmar and/or plantar pits and calcification of celebral falx, malformations of the ribs, macrocephaly, generalized overgrowth and a predisposition to tumors, specifically multiple basocellular carcinomas, medulloblastoma, meningioma and benign ovarian cysts and cardiac fibromas [4][5][6][7][8][9][10][11][12]. Recently, the model of patient-derived induced pluripotent stem cells (iPSC) clarified some of the molecular mechanisms underlying skeletal abnormalities [13]. Clinical diagnosis of Gorlin syndrome requires either two major criteria (multiple basal cell carcinomas (BCCs) (≥5 in a lifetime) or a BCC before 30 years, lamellar calcification of the falx cerebri, jaw keratocysts, palmar or plantar pits, first-degree relative with NBCCS) or one major and two minor ones (lympho-mesenteric or pleural cysts, macrocephaly, cleft lip/palate, vertebral/rib anomalies, preaxial or postaxial polydactyly, ovarian/cardiac fibromas, medulloblastoma, ocular anomalies).…”