Human umbilical cord epithelial cells express Notch1: implications for its epidermal-like differentiation.

BACKGROUND: Notch1 receptor, a member of the Notch signaling pathway, is involved in the terminal differentiation process of epidermal keratinocytes. Human umbilical cord epithelia are continuous with embryonic and fetal epidermis during development, display cellular stratification and express differentiation markers related to the epidermis. As such, we hypothesized that Notch1 may be present in human umbilical cord epithelial cells (HUCEC). OBJECTIVE: To investigate the expression of Notch1 in HUCEC in vivo and in vitro. METHODS: Umbilical cord sections were probed with antibodies specific for Notch1, -2, -3, and -4. Expression of Notch transcripts in HUCEC in vitro was examined by RT-PCR analysis and protein expression was identified using immunocytochemistry and western blotting analysis. Using a three-dimensional organotypic culture system that promotes epidermal terminal differentiation, Notch1 expression was examined and changes in expression level were analysed using real-time quantitative PCR. RESULTS: Immunostaining of cord epithelia revealed expression of all four Notch receptors, with differential spatial distribution. Notch mRNA and protein were expressed in HUCEC in vitro. Specifically, western blotting revealed the presence of the transmembrane unit of the mature Notch1 heterodimeric receptor in HUCEC and epidermal keratinocytes. In organotypic culture, Notch1 mRNA was expressed in HUCEC with protein localised to the upper epithelial layers. Interestingly, Notch1 gene expression was down-regulated in HUCEC in organotypic culture compared to cells in submerged monolayer culture. CONCLUSION: HUCEC express Notch1 as in epidermal keratinocytes. The presence of Notch1 has implications for its involvement in the differentiation program of cord epithelia as a stratified epithelial tissue.

Intractable venous leg ulcer treated successfully with allogeneic cultured dermal substitute.

Venous leg ulcers are resistant to various treatments, including autologous skin grafting. We applied an allogeneic cultured dermal substitute in a patient with such a wound, and the wound improved, healthy granulation tissue formed, and the size of the wound was reduced.

Amnia for intractable skin ulcers with recessive dystrophic epidermolysis bullosa: report of three cases.

Recessive dystrophic epidermolysis bullosa (RDEB) is a disease characterized by recurrent blistering and chronic ulceration of the skin. In these patients, recurrent blisters frequently result in intractable skin ulcers due to impaired wound healing caused by mutations in the type VII collagen gene and malnutrition as well as by increased collagenase activity. To evaluate the efficacy of amnia for intractable ulcers in RDEB, we treated RDEB patients with amnia. The amniotic membrane was simply placed on the cleansed wound surface. The procedure was repeated once a week for up to 10 weeks. As a result, wound conditions improved remarkably after treatment with amnia for 2-10 weeks in all the patients, resulting in total re-epithelization of the ulcers. Amnia could be an effective therapy for intractable skin ulcers in RDEB patients, and should be considered as a re-emerging therapeutic option for the disease.

An allogeneic cultured dermal substitute suitable for treating intractable skin ulcers and large skin defects prior to autologous skin grafting: three case reports.

Intractable skin ulcers that arise as secondary lesions from disease and full-thickness skin defects that result from skin tumor excision often need autologous skin grafting to close the wound. We developed an allogeneic cultured dermal substitute (CDS) to shorten the time needed to prepare a wound bed suitable for autologous skin grafting. The CDS was prepared by plating normal human fibroblasts on a spongy matrix consisting of hyaluronic acid and atelo-collagen. The allogeneic CDS was then placed on the rinsed wound surface. This procedure was repeated twice a week for up to five weeks, until the wounds were closed by autologous skin grafting. In all three cases, after CDS treatment for two to five weeks, the wound conditions became suitable for skin grafting; these conditions had not been improved by conventional topical treatments, including topical basic fibroblast growth factor (bFGF). Healthy granulation tissue developed rapidly, concomitant with wound size reduction. The present results indicate that CDS is an excellent biological wound dressing for improving wound conditions so that they are suitable for subsequent autologous skin grafting as well as for shortening the treatment duration for skin ulcers and full-thickness skin defects.

Engraftment of umbilical cord epithelial cells in athymic mice: in an attempt to improve reconstructed skin equivalents used as epithelial composite.

BACKGROUND: The umbilical cord (UC) is composed of connective tissue called Wharton Jelly, covered by a simple epithelium believed to derive from amniotic membrane epithelium. In previous studies, we observed that the umbilical cord epithelium (UCE) in situ displayed stratified epithelial structures, in some areas that expressed cytokeratins and differentiation markers as characteristic of keratinocytes under airlifted condition in vitro, UCE cells grown on collagen gel displayed more keratinocytes characteristics. OBJECTIVE: To study the ability of UCE cells to undergo terminal differentiation when grown in the most proper environment. METHODS: UCE cells were seeded onto the surface of a fibroblast-populated collagen gel then grafted onto the back of nude mice and examined using immunohistochemical techniques and by transmission electron microscope (TEM). RESULTS: Post-grafted UCE cells formed a stratified epithelial structure similar to that formed by keratinocytes. Although immunohistochemical staining of UCE cells in skin grafts showed a similar pattern to that seen with the keratinocyte controls, UCE cells maintained many of their own intrinsic characteristics, such as stronger expression of mucous membrane cytokeratins and expression of simple epithelial cytokeratin. Notably, with longer transplant periods, expression of keratinocyte characteristics in UCE cells increased while expression of simple epithelial properties decreased. We observed formation of a complete basement membrane, which had not been achieved using an in vitro model. CONCLUSIONS: Grafted UCE cells in an animal model maintain their own intrinsic characteristics, but display the stratified morphogenesis, terminal differentiation and ultrastructures similar to those seen in keratinocytes.

Organotypic culture and surface plantation using umbilical cord epithelial cells: morphogenesis and expression of differentiation markers mimicking cutaneous epidermis.

BACKGROUND: The umbilical cord epithelium (UCE) is composed of a single epithelial layer covering mucous connective tissue and it is thought to derive from the amniotic epithelium. Interestingly, UCE cells express not only simple and mucous epithelial keratins (CK8 and CK4/CK13), but also stratified epithelial keratins (CK1/10) and cornified cell envelope (CCE)-associated proteins. OBJECTIVE: To understand the nature of UCE, UCE cells were cultured under the same conditions of organotypic culture of epidermal keratinocytes and grafted onto the back of nude mice. METHODS: UCE cells isolated from fresh umbilical cord specimens were cultured using serum-free keratinocyte growth medium, and plated on a fibroblast-populated collagen matrix using air-liquid interface methods. UCE cells were transplanted onto the back of Balb C nu/nu mice as a thin epithelial sheet grown on a collagen matrix. RESULTS: UCE cells formed a multi-layered stratified epithelium both in organotypic culture and surface transplantation. Regarding the expression profile of differentiation-specific proteins, such as keratins, the CCE-precursor proteins and junctional proteins, the reconstructed epithelium showed a close similarity to natural epidermis in organotypic culture. CONCLUSION: These results suggest the possibility that UCE cells can differentiate and organize into an epidermis-like structure, when exposed to the appropriate conditions which is similar to those of cutaneous epidermis.

Actin reorganization is abnormal and cellular ATP is decreased in Hailey-Hailey keratinocytes.

Actin reorganization and the formation of adherens junctions are necessary for normal cell-to-cell adhesion in keratinocytes. Hailey-Hailey disease (HHD) is blistering skin disease, resulting from mutations in the Ca2+ ATPase ATP2C1, which controls Ca2+ concentrations in the cytoplasm and Golgi of human keratinocytes. Because actin reorganization is among the first responses to raised cytoplasmic Ca2+, we examined Ca2+-induced actin reorganization in normal and HHD keratinocytes. Even though HHD keratinocytes display raised baseline cytoplasmic Ca2+, we found that actin reorganization in response to Ca2+ was impaired in HHD keratinocytes. Defects in actin reorganization were linked to a marked decrease in cellular ATP in HHD keratinocytes, which persists, in vivo, in HHD epidermis. Defective actin reorganization was reproduced in normal keratinocytes in which the intracellular ATP concentration had been lowered pharmacologically. ATP concentrations in undifferentiated keratinocytes markedly declined after extracellular Ca2+ was increased, but then recovered to a new baseline that was approximately 150% of the previous baseline. In contrast, ATP concentrations in HHD keratinocytes did not change in response to increased extracellular Ca2+. This report provides new insights into how the ATP2C1-controlled ATP metabolism mediates Ca2+-induced cell-to-cell adhesion in normal keratinocytes. In addition, these findings implicate inadequate ATP stores as an additional cause in the pathogenesis of HHD and suggest novel therapeutic options.

Two Japanese cases of localized involutional lipoatrophy.

We present two Japanese cases of involutional lipoatrophy. The first case is that of a 30-year-old woman, who first appeared at our hospital complaining of a localized, well-demarcated depression, approximately 3 x 4 cm in size, normal to slightly erythematous in coloration, on the lateral side of the left upper arm (Fig. 1a). The condition was asymptomatic, and she had noticed this anomaly a month prior to consultation. She received intramuscular injections of corticosteroids of unknown dosage at the affected site for the treatment of allergic rhinitis 4 months prior to her present consultation. The second patient, a 23-year-old woman, appeared at our hospital complaining of a similar macule 4 x 4 cm in size, which she noticed several weeks prior to her most recent consultation. She had no history of injury or injection at the site before the development of the condition (Fig. 1b). She had been under treatment for atopic dermatitis since early childhood and was treated only with topical applications of white petrolatum containing 2% salicylic acid for the past several years. In order to rule out the possibility of acquired partial lipodystrophy associated with localized scleroderma, lupus profundus and the other connective tissue diseases, a histological examination was performed for both patients. Histopathological analysis of the region exhibited a well-defined fat lobule composed of numerous small adipocytes (Fig. 1c) embedded in hyaline connective tissue. Edema and dilated capillaries were noticeable in the subcutaneous tissue surrounding the area. Inflammatory cells were not prominent, although mononuclear cells were observed in both patients. No epidermal change was seen in either patient. Direct and indirect immunofluorescence studies revealed no deposits of immunoreactants in the skin of either patient. Immunohistochemical studies with the antibody against macrophage (anti-CD68 antigen; DAKO.) showed that positive cells were scattered around blood vessels and shrunken lipocytes in the subcutaneous tissues (Fig. 1d). Most of these cells in the fat lobules were also positive for mucin stains such as Alcian blue. No abnormal findings came to light in the ordinary hematological and blood chemistry examinations of both patients. The autoantibody screening tests using antinuclear, anti-DNA, anticentromere, and anti-Scl-70 antibodies were negative in both patients.