The nerve-dependency of Merkel cell proliferation in cultured human fetal glabrous skin.

Merkel cells are thought to function as slowly adapting mechanoreceptors and are known as targets for sensory nerves. However, the nerve-dependency of Merkel cells remains controversial. In this respect, some investigators have found interregional differences between hairy and glabrous skin and others have shown intraregional differences within denervated rat touch domes. Differences between species have also been reported. This study was performed to determine whether Merkel cells proliferate in vitro in the absence of the systemic factors, blood vessels and the intact nerves in human skin. Suspension organ culture was performed using fetal digits to investigate their in vitro proliferation. Merkel cells and cutaneous nerves were identified using antibodies to cytokeratin 20 and protein gene product 9.5 (PGP 9.5), respectively. Fetal digits of 56-82 day gestational age were cultured in serum free medium in a high O2 (45%) environment. Tissues were harvested before starting culture (D0) and 1, 4, 7, 14, 28 d after culture. Merkel cells were observed in the volar pads and dorsal nail matrices at D0. After 28 d of suspension organ culture, digits looked healthy structurally and the number of Merkel cells had increased. However, PGP 9.5-immunoreactive nerves were markedly diminished after 1 day of culture and almost disappeared after 4 days. Merkel cell proliferation in vitro suggested that Merkel cell development is probably nerve-independent in human fetal glabrous skin.     

Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration

Introduction of autologous stem cells into the site of a nerve injury presents a promising therapy to promote axonal regeneration and remyelination following peripheral nerve damage. Given their documented ability to differentiate into Schwann cells (SCs) in vitro, we hypothesized that skin-derived precursor cells (SKPs) could represent a clinically-relevant source of transplantable cells that would enhance nerve regeneration following peripheral nerve injury. In this study, we examined the potential for SKP-derived Schwann cells (SKP–SCs) or nerve-derived SCs to improve nerve regeneration across a 12 mm gap created in the sciatic nerve of Lewis rats bridged by a freeze-thawed nerve graft. Immunohistology after 4 weeks showed survival of both cell types and early regeneration in SKP seeded grafts was comparable to those seeded with SCs. Histomorphometrical and electrophysiological measurements of cell-treated nerve segments after 8 weeks survival all showed significant improvement as compared to diluent controls. A possible mechanistic explanation for the observed results of improved regenerative outcomes lies in SKP–SCs' ability to secrete bioactive neurotrophins. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cells for transplantation therapies which act as functional Schwann cells and show great promise in improving regeneration following nerve injury.     

Merkel cell tumor of the skin. Ultrastructural and immunohistochemical studies

A skin tumor of a 66-year-old female was investigated morphologically and immunohistochemically. The tumor was located within the dermis and comprised of rounded cells with scanty cytoplasm, which proliferated forming a small nest or trabecular arrangement. Electron microscopic observation indicated the presence of dense-core granules within the tumor cell cytoplasm suggesting that the tumor was derived from Merkel cells. Occasionally clusters or bundles of the intermediate filaments were found in the perinuclear cytoplasm of the tumor cells. Each tumor cell was connected with desmosomes. Immunohistochemical staining with anti-keratin antiserum showed positive reaction at the perinuclear cytoplasm of the tumor cells indicating that the cluster of the microfilaments presumably contains keratin. Conversely S-100 protein was negative in the tumor cells. The results obtained strongly suggest that the tumor or Merkel cell was considered to be derived from the epidermal immature cells rather than from the neural crest.     

Lymphocyte migration through cultured endothelial cell monolayers derived from the blood-retinal barrier.

Lymphocyte migration across endothelial monolayers, derived from the rat blood-retinal barrier, was recorded in vitro using time-lapse video microscopy. Syngeneic lymphocytes were plated out onto endothelial cell monolayers for 4 hr and their surface motility and transmonolayer migration recorded and quantified. Under resting conditions lymphocytes, obtained from peripheral lymph nodes (PLN), were small, rounded and static with less than 5% migrating across the monolayer. Activation of the lymphocytes with concanavalin A (Con A) increased their size and surface motility on both interferon-gamma (IFN-gamma)-treated and resting endothelia, but did not alter the number migrating across the monolayer. Similar results were also found for phytohaemagglutinin (PHA)-activated lymphocytes. Interleukin-2 (IL-2)-dependent CD4+ T-cell lines specifically recognizing either retinal soluble antigen (S-Ag) or bovine serum albumin (BSA) exhibited significantly greater surface motility over the endothelial monolayers than the mitogen-activated PLN lymphocytes. By 4 hr, in excess of 50% of the T-cell line lymphocytes had migrated across the endothelial monolayer. Treatment of the endothelial cells with IFN-gamma caused a small, but not significant, increase in the level of T-cell line lymphocyte migration. These results suggest that the migration of lymphocytes across central nervous system-derived endothelia is primarily dependent upon the state and mode of lymphocyte activation.     

High load of Merkel cell polyomavirus DNA detected in the normal skin of Japanese patients with Merkel cell carcinoma

BackgroundAlthough Merkel cell polyomavirus (MCPyV) has the potential to cause Merkel cell carcinoma (MCC), it is also found in the normal skin of healthy individuals. However, the mechanism for transformation of MCPyV to an oncogenic form is unknown.ObjectivesTo investigate the levels of MCPyV infection in the normal skin patients with MCC compared with those in a control cohort.Study designWe studied a total of six Japanese patients with cutaneous MCC. Sun-exposed and sun-unexposed skin swabs were obtained and analyzed for MCPyV loads using quantitative real-time polymerase chain reaction.ResultsAt first, we found a patient with MCC carrying an extremely high load of MCPyV DNA in normal skin. This unique case prompted us to further explore the levels of MCPyV as skin microbiota in patients with MCC. We showed that MCPyV DNA levels were significantly higher in swabs obtained from normal skin samples of six patients with MCC compared with those from 30 age-matched healthy individuals and 19 patients with other cutaneous cancers. Whereas MCPyV strains obtained from the normal skin of patients with MCC had gene sequences without structural alterations, sequences of the tumor-derived strains showed truncating mutations or deletions.ConclusionsAlthough the number of patients with MCC studied was small, our findings suggest that MCC may occur with a background of high MCPyV load in the skin, and are expected to stimulate further studies on whether such skin virome levels could be one of predictive markers for the development of MCC.     

Merkel cell carcinoma of the skin: the structure and origin of normal Merkel cells

A series of 15 Merkel cell tumours of skin is reported. They occur dominantly on the head and neck and on the extremities of elderly women, frequently presenting as a reddish nodule. Three cases were associated with squamous carcinoma at the same site, an association deserving further study. There are two main patterns: the commoner one takes the form of a trabecular carcinoma in the dermis mimicking metastatic carcinoma, including oat-cell carcinoma and neuroblastoma: a dissociated-cell form mimicks malignant lymphymphoma. The triad of vesicular nuclei with very small nucleoli, abundant mitotic activity and apoptosis is so characteristic as to be virtually pathognomonic in conjunction with structural features. Argyrophilia is common, but Bourn fixation is necessary to demonstrate it regularly. Small round secretory granules (89 ± 18 nm) with narrow haloes, and an abundance of intermediate size filaments are among the ultrastructural hallmarks. There is a close similarity between better differentiated tumour cells and normal Merkel cells. The neural crest origin of MC is in doubt both on the basis of studies of the development and regeneration of MC and from the study of Merkel cell tumours.     

Hair follicle regeneration in skin grafts: current concepts and future perspectives

The repair and management of full-thickness skin defects resulting from burns and chronic wounds remain a significant unmet clinical challenge. For those skin defects exceeding 50%-60% of total body surface area, it is impractical to treat with autologous skin transplants because of the shortage of donor sites. The possibility of using tissue-engineered skin grafts for full-thickness wound repair is a promising approach. The primary goal of tissue-engineered skin grafts is to restore lost barrier function, but regeneration of appendages, such as hair follicles, has to be yet achieved. The successful regeneration of hair follicles in immunodeficient mice suggests that creating human hair follicles in tissue-engineered skin grafts is feasible. However, many limitations still need to be explored, particularly enriching isolated cells with trichogenic capacity, maintaining this ability during processing, and providing the cells with proper environmental cues. Current advances in hair follicle regeneration, in vitro and in vivo, are concisely summarized in this report, and key requirements to bioengineer a hair follicle are proposed, with emphasis on a three-dimensional approach.     

Uncovered skin grafts in mice.

A technique for suturing ear skin on the lateral thoracic area of recipient mice is described. Using this technique the graft was visible from the day of grafting until rejection was complete and mice were not burdened by circumferential body dressings. Consistent mean survival times were observed in a donor-recipient combination over 1 year's time and technical failures could be recognized. This technique was useful to accurately assess accelerated rejection responses in normal mice that occur 4-8 days after grafting as a result of passively transferred effector cells.     

Sarcoma derived from cultured mesenchymal stem cells.

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon-related genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.     

Merkel cell tumor of the skin: an immunohistochemical study

Skin biopsy specimens from 12 elderly patients with Merkel cell tumors were investigated. Conventional light microscopy and immunohistochemical techniques were used. All of the tumors had similar morphologic features. Immunoreactivity for neuronspecific enolase, gastrin, calcitonin, and epithelial membrane-like antigen was demonstrated, and both neurofilaments and keratin filaments were observed. The immunohistochemical findings supported a Merkel cell origin for these Merkel cell tumors. The co-expression of neuroendocrine and epithelial markers in Merkel cell carcinomas is suggestive of neuroendocrine differentiation in a neoplasm of epithelial origin. Merkel cell carcinomas share many characteristics with neuroendocrine tumors of the bronchopulmonary and gastrointestinal tracts. All of these neoplasms may originate from cells of similar types that are present in several organs.     

Consistent chromosomal anomalies in keratinocyte cell lines derived from untreated malignant lesions of the oral cavity

Cytogenetic analysis has been carried out on 8 early passage cell lines derived from 8 untreated human oral squamous cell carcinomas. Clonal aberrations were detected in the karyotypes of each cell line. A high frequency of breakpoints were noted on chromosomes 1, 7, 8, 9, 11, and X. An isochromosome 8 was present in 6 out of 8 cell lines; isochromosome 9 (3 cell lines) and isochromosome 11 (1 cell line) were also found. In 4 out of 8 cell lines the X chromosome harboured breakpoints, a novel finding in oral squamous cell carcinomas. Breakpoints were common on chromosome 1, with 1p12–p13 most frequently involved. Tandem duplication of 11q13–q23, which contains a number of growth regulatory genes, was also noted in 2 cases. We correlate the sites of proto-oncogenes and other growth control genes with chromosomal breakpoints and suggest that several of these may play a role in the pathogenesis of oral cancer. © 1993 Wiley-Liss, Inc.     

Human embryonic stem cell lines derived from discarded embryos

Human pluripotent embryonic stem (ES) cells have important potential in regenerative medicine and as models for human preimplantation development; however, debate continues over whether embryos should be destroyed to produce human ES cells. We have derived four ES cell lines on mouse embryonic fibroblast cells in medium supplemented with basic fibroblast growth factor, human recombinant leukemia inhibitory factor, and fetal bovine serum. The source of these cell lines was poor-quality embryos that in the course of routine clinical practice would have been discarded. After continuous proliferation in vitro for more than 12 months, these ES cell lines maintained their developmental potential to form trophoblast and somatic cells, including cardiac muscle and neuronal tissue.     

T cell clones from psoriasis skin lesions can promote keratinocyte proliferation in vitro via secreted products

Psoriasis vulgaris has been recognized lately as an immunologically mediated inflammatory skin disease. To analyze the pathogenetic role of T lymphocytes in the generation of psoriatic skin lesions, 105 T cell clones (TCC) and 10 T cell lines (TCL) were differentially isolated from dermis and epidermis of psoriatic skin specimens. Supernatants prepared from these T cells were studied for their effects on keratinocyte proliferation in vitro. Conditioned media from 14 of 77 epidermal TCC, 7 of which were CD8⁺, and from 8 of 28 dermal TCC, 5 of which were CD8⁺, reproducibly enhanced keratinocyte proliferation, with more pronounced mitogenic activities found in dermal TCC. Another 9 epidermal and 3 dermal TCC did not affect keratinocyte growth and supernatants from the remaining clones, as well as from the 5 epidermal and 5 dermal TCL, inhibited keratinocyte replication to varying degrees. Both mitogenic and suppressive activities were largely abolished by addition of an antiserum to interferon-gamma (IFN-γ), while addition of epidermal growth factor or irradiated psoriatic TCL had little effect on the activities of the supernatants. These studies reveal that a subpopulation of lesional psoriatic T lymphocytes is capable of enhancing keratinocyte proliferation in vitro via secreted products. Their mitogenic capacity most likely requires IFN-γ, but the ultimate effect is apparently determined by the presence of additional cytokines. Activation of T cells secreting such combinations of factors in vivo may contribute to the keratinocyte alterations characteristic of psoriatic skin lesions.     

Intrastriatal grafts derived from fetal striatal primordia

Cocaine, a catecholamine agonist, has been shown to produce a transient induction of the immediate-early gene c-fos and its protein product Fos in the striatum of normal rats. In the present study we report that the expression of Fos can be induced by cocaine challenge in intrastriatal grafts derived from cell suspensions of embryonic striatal primordia. Fos-like immunoreactivity in the nuclei of grafted neurons was detected 2 hr after the injection of 50 mg/kg cocaine into the host rats. Neurons with Fos-immunoreactive nuclei tended to form clusters in the striatal grafts. The Fos-rich clusters were aligned with acetylcholinesterase (AChE)-rich and tyrosine hydroxylase (TH)-rich patches demonstrated in adjoining sections. Previous studies have shown that presynaptic and postsynaptic cellular markers of the dopaminergic system in the striatum, including immunostaining for TH and dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein (DARPP-32), and binding for high affinity dopamine uptake sites and for dopamine D1 and D2 receptor sites, are all concentrated in the AChE-rich patch regions (P regions) of such embryonic striatal grafts. The preferential expression of Fos in neurons of the P regions of the grafts thus implies that the induction of Fos was cell-type specific in being concentrated in the parts of the grafts that express striatal phenotype and that are innervated by catecholamine-containing fibers.(ABSTRACT TRUNCATED AT 250 WORDS)