Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations

Abstract

Neurofibromas are one of the most characteristic features of neurofibromatosis type 1 (NF1), an inherited autosomal-dominant neurogenetic disorder affecting 1 in 3500 individuals worldwide. These benign tumors mainly consist of Schwann cells (SCs) and fibroblasts. Recent evidence demonstrates that somatic mutations at the NF1 gene are found in neurofibromas, but it has not been demonstrated whether SCs, fibroblasts and/or both cell types bear a somatic loss of NF1. We recently established a cell culture system that allows selective expansion of human SCs from neurofibromas. We cultured pure populations of SCs and fibroblasts derived from 10 neurofibromas with characterized NF1 mutations and found that SCs but not fibroblasts harbored a somatic mutation at the NF1 locus in all studied tumors. Furthermore, by culturing neurofibroma-derived SCs under different in vitro conditions we were able to obtain two genetically distinct SC subpopulations: NF1(-/-) and NF1(+/-). These data strongly support the idea that NF1 mutations in SCs, but not in fibroblasts, correlate to neurofibroma formation and demonstrate that only a portion of SCs in neurofibromas have mutations in both NF1 alleles.

The Development of Cutaneous Neurofibromas

“…7,8,18 The skin covering the neurofibroma was removed and the tumor tissue was cut into small pieces and put into preincubation medium containing Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) with 10% fetal calf serum (Invitrogen, Eugene, OR), 2 mol/L forskolin (Sigma-Aldrich, St. Louis, MO), and antibiotics for 5 to 10 days. The tumors were then enzymatically dissociated and the cells suspended.…”

“…18 The cell suspension was cultured overnight in 25-cm 2 culture flasks in proliferation medium with forskolin. 8 The medium was then collected and the unattached cells spun down. The cells were resuspended in medium favoring stem cell growth.…”

“…8 For adipogenesis, cells were incubated in stem cell growth medium supplemented with 1.7 mol/L insulin (Sigma-Aldrich), 0.5 mmol/L 1-Methyl-3-isobutylxanthine (Sigma-Aldrich), 100 mol/L indomethacin (Sigma-Aldrich), and 1 mol/L dexamethasone (SigmaAldrich). After 8 days, the cells were fixed with 10% formalin and stained with Oil Red O stain (Cambrex Bio Science, Walkersville, MD).…”

“…Despite the absence of known Schwann cell growth factors or serum, 8,18 a number of the cells produced by the NFPs cultured in stem cell medium 19 exhibited a Schwann cell-like phenotype in that they were bipolar and S100 positive (Figure 3, A-B …”

“…7,8 This inactivation has been detected in cultured cells displaying characteristics typical of Schwann cells, including a bipolar morphology and the expression of S100 protein.…”