Human TSC2-null fibroblast-like cells induce hair follicle neogenesis and hamartoma morphogenesis

Abstract

Hamartomas are composed of cells native to an organ but abnormal in number, arrangement or maturity. In the tuberous sclerosis complex (TSC), hamartomas develop in multiple organs because of mutations in TSC1 or TSC2. Here we show that TSC2-null fibroblast-like cells grown from human TSC skin hamartomas induced normal human keratinocytes to form hair follicles and stimulated hamartomatous changes. Follicles were complete with sebaceous glands, hair shafts and inner and outer root sheaths. TSC2-null cells surrounding the hair bulb expressed markers of the dermal sheath and dermal papilla. Tumour xenografts recapitulated characteristics of TSC skin hamartomas with increased mammalian target of the rapamycin complex 1 (mTORC1) activity, angiogenesis, mononuclear phagocytes and epidermal proliferation. Treatment with an mTORC1 inhibitor normalized these parameters and reduced the number of tumour cells. These studies indicate that TSC2-null cells are the inciting cells for TSC skin hamartomas, and suggest that studies on hamartomas will provide insights into tissue morphogenesis and regeneration.

Figure 1. Hair follicle formation in grafts containing TSC2-null cells

(a) Haematoxylin and eosin (H&E) stained sections of TSC patient normal-appearing skin (NL), fibrous forehead plaque (FP), angiofibroma (AF), and periungual fibroma (PF). Scale bar, 320 μm. (b) Levels of TSC2, phosphorylated ribosomal protein S6 (pS6), S6, and ß-actin assessed by western blotting in serum-starved fibroblast-like cells isolated from NL, AF, FP and PF from different patients (P1-4). (c) Absence of hair follicles in dermal-epidermal composites grafted onto mice when made with TSC normal fibroblasts and human neonatal keratinocytes. Scale bar, 320 μm. (d) Formation of hair follicles in dermal-epidermal composites grafted onto mice when composed of TSC2-null cells from a TSC fibrous forehead plaque and human neonatal keratinocytes. Scale bar, 320 μm.

Figure 2. Characterization of hair follicles in grafts containing TSC2-null cells

Representative stained sections of grafts obtained 17 weeks after grafting dermal-epidermal composites. (a) Hair follicles with sebaceous gland. (b) Longitudinal section of hair follicle with hair shaft. (c) Oblique section of hair follicle sebaceous glands and outer root sheath, inner root sheath, and hair shaft. (d) Hair bulb with dermal papilla, lower dermal sheath, matrix, and inner and outer root sheaths. (e) Epithelial cells and scattered dermal cells reactive with human-specific COX IV antibody. (f) Hair follicle and dermal sheath cells reactive with COX IV antibody. (g) Signals for a human Y chromosome probe (red) mark foreskin-derived keratinocytes observed in the epidermis and hair follicles but not in dermal cells; DAPI (blue) nuclear stain. (h) Signals for Y chromosome probe in follicular epithelium but not surrounding dermal sheath. (i) Human-specific nestin antibody reactive with cells in the dermal papilla and lower dermal sheath region. (j) Nestin antibody reactive with cells in the dermal papilla and hair bulb dermal sheath. (k) Human-specific versican antibodies reactive with mesenchymal cells of the dermal papilla and lower dermal sheath region of hair follicles. (l) Alkaline phosphatase activity localized in dermal papilla and lower sheath region of hair follicles. (m) Reactivity with antibodies against Ki-67, scattered in basal layer of the epidermis and dense in the hair follicle matrix. (n) Keratin 15 immunoreactivity below follicular infundibulum. (o) Keratin 15 immunoreactivity in the basal layer of the outer root sheath. (p) Keratin 75 immunoreactivity in hair follicle companion layer. Results shown are from patient 4; similar patterns of staining were observed in at least four tumour xenografts from patient 4 and two tumour xenografts from patient 5. Scale bars in a, b, e, and g are 130 μm; for i, k, l, m, n, o, and p are 65μm; for c, d, f, and j are 35 μm and h is 20 μm.

Figure 3. Genetic analysis of TSC2-null fibroblast-like cells

Upper panels a-d show results using samples from patient 4, and lower panels e-h show results using samples from patient 2. (a) Sequencing of DNA from TSC2-null cells from a forehead plaque revealed a single base substitution (1074G>A) in exon 10 of the TSC2 gene. (b) Analysis of microsatellite nucleotide repeat polymorphisms flanking TSC2 at 16p13.3. Alleles are marked with arrowheads for patient normal fibroblasts and TSC2-null cells. (c) Restriction enzyme analysis of part of exon 10 of TSC2, using PCR amplified DNA from TSC normal fibroblasts (Normal), TSC2-null cells from a forehead plaque (Tumour), laser microdissected follicular epithelium (FE) or dermal sheath (DS) regions of tumour xenografts, and fibroblast-like cells derived from 2 angiofibromas (AF1 and AF2) and one oral fibroma (OF). The mutation 1074G>A introduces a restriction site for BsmA1, so that mutant but not normal DNA is cleaved. (d) Tumour xenografts were microdissected using laser microdissection (laser track in red), including the dermal sheath (DS) and follicular epithelium (FE). (e) Sequence analysis shows a single base substitution (4830G>A) in exon 36 of TSC2 in fibroblasts from patient normal-appearing skin and TSC2-null cells from a periungual fibroma. (f) Restriction enzyme analysis of part of exon 36 of TSC2, using PCR products amplified from normal human dermal fibroblasts (NHDF), TSC patient fibroblasts (Normal), and TSC2-null periungual fibroma cells (Tumour). The mutation 4830G>A eliminates a restriction site for BslI, so that wild-type but not mutant DNA is cleaved. (g) Sequence analysis of the TSC2 gene in TSC2-null cells reveals a deletion of two base pairs, 1058-1059delTC, in exon 10 indicated by the arrow. (h) Restriction enzyme analysis of part of exon 10 of TSC2, using PCR products from TSC normal fibroblasts (Normal), TSC2-null cells (Tumour), and laser microdissected dermis of normal and tumour xenografts. The mutation 1058_1059delTC eliminates a restriction site for SacI, so that wild-type but not mutant DNA is cleaved.

Figure 4. Effects of rapamycin on TSC2-null cells in vitro and in a xenograft mouse model

(a) Western blot of serum-starved cells treated with or without rapamycin (2 nM) for 24 hours. Levels of phosphorylated S6K1 (pS6K1), total S6K1, pS6, total S6, and ß-actin were assessed in TSC fibroblasts (Normal) and cells from a forehead plaque (Tumour). Similar results were obtained using cells from a periungual fibroma. (b) Inhibition of cell viability measured using MTT assay in TSC normal fibroblasts (white bars) and TSC2-null cells from a fibrous forehead plaque (black bars) treated with or without rapamycin for three days. Results are means ± SD of values from four experiments. *p<0.01. Similar results were obtained using TSC2-null cells from a periungual fibroma. Panels c-k display the in vivo effects of rapamycin in mice with composite grafts containing TSC2-null cells or TSC normal fibroblasts from patient 4. (c) Immunohistochemistry for human COX-IV, pS6, Ki-67 (proliferation), F4/80 (phagocytes), and CD31 (vessels) in four experimental groups: NV, TSC normal fibroblasts with vehicle treatment; NR, TSC normal fibroblasts with rapamycin treatment; TV, TSC2-null cells with vehicle treatment; TR, TSC2-null cells with rapamycin treatment. Scale bar, 35 μm. Staining was quantified as shown in panels d-k. The white and black bars indicate means ± SE for vehicle- and rapamycin-treated animals, respectively. (d) Density of dermal cells reactive with human-specific COX-IV. (e) Density of dermal cells reactive with pS6. (f) Intensity of staining for pS6 in the epidermis. (g) Numbers of non-follicular epidermal cells reactive with Ki-67 relative to epidermal length. (h) Density of dermal cells reactive with F4/80. (i) Density of CD-31 positive vessels. (j) Average cross-sectional area CD-31 positive vessels. (k) The ratio of vessel area to dermal area within grafts. Numbers of mice were 13 for NV, 14 for NR, 12 for TV, and 15 for TR.*, p<0.05; **, p<0.01; ***, p<0.001.