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Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.
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PMID:Quality evaluation analysis of bioengineered human skin. 1546 8

Transplantation of cultivated limbal epithelium on substrates such as amniotic membrane is an established treatment for severe ocular surface disease with limbal stem cell deficiency. In this study, we adapted an established method to generate sheets of limbal epithelium on amniotic membrane and characterized the cells contained in these sheets and tested them for safety with regard to microbial contamination. Human limbal biopsies were cultivated on denuded amniotic membranes. After three weeks of culture, the phenotypes of cultivated cells were analyzed by immunohistochemistry and real-time RT-PCR for the expression of a panel of specific markers. Cultivated limbal epithelial cell sheets were also analyzed by scanning (SEM) and transmission (TEM) electron microscopy. Sterility tests and mycoplasma assays were conducted for the safety of product. A confluent layer of polygonal cells was formed in 2 weeks and 1-3 stratified layer of cells were observed after three weeks of culture. Cultivated cells were positive for p63, K3, K19, and involucrin but negative for K14, integrin alpha9 and ABCG2 when analyzed by immunohistochemistry. Expression of molecular markers was detectable with real-time RT-PCR. SEM showed multilayer of flat squamous polygonal epithelial cells. Desmosomal and hemidesmosomal attachments were evident. Our study showed that cultivated limbal epithelium consists of limbal progenitors as well as differentiated corneal epithelial cells. SEM and TEM analysis showed cultivated cells demonstrated typical features of corneal epithelium. The risk of contamination is low and can be prevented by culturing the cells in a clean room facility complying to Good Manufacturing Practice standard.
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PMID:Characterization and safety assessment of bioengineered limbal epithelium. 2229 42

In the adult mammalian skin, cells are constantly renewing, differentiating and moving upward, to finally die in a yet not fully understood manner. Here, we provide evidence that macroautophagy/autophagy has a dual role in the skin. In addition to its known catabolic protective role as an evolutionary conserved upstream regulator of lysosomal degradation, we show that autophagy induced cell death (CDA) occurs in epithelial lineage-derived organs, such as the inter-follicular epidermis, the sebaceous- and the Harderian gland. By utilizing GFP-LC3 transgenic and ATG7-deficient mice, we show that CDA is initiated during terminal differentiation at a stage when the cells have become highly resistant to apoptosis. In these transitional cells, the Golgi compartment expands, which accounts for the formation of primary lysosomes, and the nucleus starts to condense. During CDA a burst of autophagosome formation is observed, first the endoplasmic reticulum (ER) is phagocytosed followed by autophagy of the nucleus. By this selective form of cell death, most of the cytoplasmic organelles are degraded, but structural proteins remain intact. In the absence of autophagy, consequently, parts of the ER, ribosomes, and chromatin remain. A burst of autophagy was stochastically observed in single cells of the epidermis and collectively in larger areas of ductal cells, arguing for a coordinated induction. We conclude that autophagy is an integral part of cell death in keratinocyte lineage cells and participates in their terminal cell fate.Abbreviations: Atg7: autophagy related 7; BECN1: beclin 1; CDA: cell death-induced autophagy; Cre: Cre-recombinase; DAPI: 4',6-diamidino-2-phenylindole; ER: endoplasmatic reticulum; GFP: green fluorescent protein; HaGl: haderian gland; IVL: involucrin; KRT14: keratin 14; LD: lipid droplet; LSM: laser scanning microscope; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PN: perinuclear space; RB: residual body; rER: rough endoplasmatic reticulum; SB: sebum; SG-SC: stratum granulosum - stratum corneum; SGl: sebaceous gland; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.
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PMID:Cell death induced autophagy contributes to terminal differentiation of skin and skin appendages. 3137 49