Eriko Okamoto

Expression of basement membrane components in skin equivalents--influence of dermal fibroblasts.

We have made a skin equivalent constructed of fibroblasts embedded in a type I collagen, with an overlying stratified keratinocyte epithelium to examine formation of the basement membrane. We assessed the influence of the existence and species of fibroblasts in the collagen gel. Cultured human keratinocytes were well attached to the dermal equivalent. Plating efficiency was not clearly different among several types of gel. On the control and mouse fibroblast gel, sheet formation was delayed and epithelial stratification on the human fibroblast gel was more remarkable than on the control gel. On the human fibroblast gel, we observed the expression of basement membrane components (bulbous phemphigoid antigen, laminin, type IV collagen and fibronectin) between the sheet of cultured keratinocytes and the human fibroblast gel earlier than those on the control gel and mouse fibroblast gel. Type VIII collagen was not observed in any of the models at 4 weeks.

Effects of several growth factors on cultured neurofibroma cells

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by abnormalities affecting multiple tissues derived from the neural crest. The peripheral neurofibromas are numerous and sometimes reach several hundred in number. In this study, the possible involvement of several growth factors in neurofibroma growth was investigated in vitro. When explants of neurofibroma tissue were cultured, macrophage-like cells with pseudopodia migrated out first, and later took on a slender fusiform shape. These cells contained S-100 protein and were identified as Schwann cells. They did not proliferate under standard culture conditions. Nerve growth factor (NGF) was helpful in maintaining the differentiated phenotype of Schwann cells, but did not stimulate their proliferation. Immunohistochemical staining for type IV collagen revealed that some large flattened polygonal cells had a mesh of type IV collagen on the surface. These cells were perineurial cells. The proliferation of cells derived from neurofibroma was stimulated by basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and transforming growth factor alpha (TGF-alpha). In comparison with skin fibroblasts, the cells derived from neurofibroma responded to these growth factors at considerably lower concentrations. Stimulation by EGF at physiological concentrations indicated the possible involvement of EGF in the development of neurofibromas.

In vitro Keratin Expression of Hair Cells

Human hair follicles were isolated from the scalp by dispase and collagenase treatment and dispersed into a cell suspension by trypsin. These cells proliferated well and could be subcultured 7 to 8 times. The medium used was MCDB 153 HAA medium further supplemented with some amino acids, hydrocortisone, insulin, EGF, and bovine brain extract. The concentration of Ca++ was adjusted to 0.1 mM. Immunohistochemically, these cells were proved to possess keratins specific to hair forming cells.

Fibroblasts of neurofibromatosis type 1 showed no abnormality in p21ras-mediated response to serum in culture

In order to disclose the possible disturbance in regulation of p21ras function in neurofibromatosis type 1, we investigated the recovery from the quiescent to cycling states of the cell cycle in the skin fibroblasts of the patients. When cells cultured in serum-dependent medium are deprived of the serum, these go into the quiescent state (G0). On being returned to the serum-containing medium, the cells re-enter S phase. The skin fibroblasts from neurofibromatosis type 1 patients, and normal subjects were brought into the quiescent state by serum-starvation, and after the re-addition of the serum, the recovery to cycling states were analysed by flow cytometry. There was no apparent difference between these fibroblasts in the progression from G0 to S phase.