Canalis Ernesto, Yu Jungeun, Schilling Lauren, Jafar-Nejad Paymaan, Carrer Michele
Departments of Orthopaedic Surgery, UConn Musculoskeletal Institute, UConn Health, Farmington, CT, United States of America.
Departments of Medicine, UConn Musculoskeletal Institute, UConn Health, Farmington, CT, United States of America.
PLoS One. 2025 Jan 3;20(1):e0316644. doi: 10.1371/journal.pone.0316644. eCollection 2025.
Lateral Meningocele Syndrome (LMS), a disorder associated with NOTCH3 pathogenic variants, presents with neurological, craniofacial and skeletal abnormalities. Mouse models of the disease exhibit osteopenia that is ameliorated by the administration of Notch3 antisense oligonucleotides (ASO) targeting either Notch3 or the Notch3 mutation. To determine the consequences of LMS pathogenic variants in human cells and whether they can be targeted by ASOs, induced pluripotent NCRM1 and NCRM5 stem (iPS) cells harboring a NOTCH36692-93insC insertion were created. Parental iPSCs, NOTCH36692-93insC and isogenic controls, free of chromosomal aberrations as determined by human CytoSNP850 array, were cultured under conditions of neural crest, mesenchymal and osteogenic cell differentiation. The expected cell phenotype was confirmed by surface markers and a decline in OCT3/4 and NANOG mRNA. NOTCH36692-93insC cells displayed enhanced expression of Notch target genes HES1, HEY1, 2 and L demonstrating a NOTCH3 gain-of-function. There was enhanced osteogenesis in NOTCH36692-93insC cells as evidenced by increased mineralized nodule formation and ALPL, BGLAP and BSP expression. ASOs targeting NOTCH3 decreased both NOTCH3 wild type and NOTCH36692-93insC mutant mRNA by 40% in mesenchymal and 90% in osteogenic cells. ASOs targeting the NOTCH3 insertion decreased NOTCH36692-93insC by 70-80% in mesenchymal cells and by 45-55% in osteogenic cells and NOTCH3 mRNA by 15-30% and 20-40%, respectively. In conclusion, a NOTCH3 pathogenic variant causes a modest increase in osteoblastogenesis in human iPS cells in vitro and NOTCH3 and NOTCH3 mutant specific ASOs downregulate NOTCH3 transcripts associated with LMS.
外侧脑脊膜膨出综合征（LMS）是一种与NOTCH3致病变体相关的疾病，表现为神经、颅面和骨骼异常。该疾病的小鼠模型表现出骨质减少，通过给予靶向Notch3或Notch3突变的Notch3反义寡核苷酸（ASO）可改善这种情况。为了确定LMS致病变体在人类细胞中的后果以及它们是否可以被ASO靶向，创建了携带NOTCH36692 - 93insC插入的诱导多能NCRM1和NCRM5干细胞（iPS细胞）。通过人类CytoSNP850阵列确定无染色体畸变的亲代iPSC、NOTCH36692 - 93insC和同基因对照，在神经嵴、间充质和成骨细胞分化条件下培养。通过表面标志物以及OCT3/4和NANOG mRNA的下降证实了预期的细胞表型。NOTCH36692 - 93insC细胞显示出Notch靶基因HES1、HEY1、2和L的表达增强，表明NOTCH3功能获得。NOTCH36692 - 93insC细胞中矿化结节形成增加以及ALPL、BGLAP和BSP表达增加，证明成骨作用增强。靶向NOTCH3的ASO在间充质细胞中使NOTCH3野生型和NOTCH36692 - 93insC突变体mRNA均降低40%，在成骨细胞中降低90%。靶向NOTCH3插入的ASO在间充质细胞中使NOTCH36692 - 93insC降低70 - 80%，在成骨细胞中降低45 - 55%，使NOTCH3 mRNA分别降低15 - 30%和20 - 40%。总之，NOTCH3致病变体在体外导致人类iPS细胞中成骨细胞生成适度增加，并且NOTCH3和NOTCH3突变体特异性ASO下调与LMS相关的NOTCH3转录本。
