Melanocyte transplantation to skin prepared by controlled PUVA-induced sunburn-like blistering for vitiligo treatment - A pilot clinical trial.

Vitiligo is a common acquired disease of pigment loss. In lesions recalcitrant to non-invasive treatment, transplantation of cultured autologous melanocytes is an emerging choice. Conventionally, the recipient site is often prepared by laser-mediated or mechanical dermabrasion. Such preparation procedures have disadvantages including prolonged transplantation duration, long period for reepithelialization and potential scarring. We propose a method of preparing recipient sites by psoralen and controlled ultraviolet A (PUVA)-induced blistering followed by transplanting suspended melanocytes. We introduced this method in 10 patients with segmental vitiligo on their recipient site 3 to 5 days before transplantation and blistering developed in 2 to 3 days afterwards. On the day of transplantation, the blister roof could be peeled off easily without bleeding and the recipient site preparation could be completed in 20 min. The recipient site became reepithelialized within 1 week. Progressive repigmentation was observed for up to 6 months, with an average of 65.06% repigmentation in the recipient site without scarring at the end of follow-up. Hence, preparation of the recipient site by controlled PUVA-induced sunburn-like blistering can potentially facilitate melanocyte transplantation and prevent scarring.

Bringing Safe and Standardized Cell Therapies to Industrialized Processing for Burns and Wounds.

Cultured primary progenitor cell types are worthy therapeutic candidates for regenerative medicine. Clinical translation, industrial transposition, and commercial implementation of products based on such cell sources are mainly hindered by economic or technical barriers and stringent regulatory requirements. Applied research in allogenic cellular therapies in the Lausanne University Hospital focuses on cell source selection technique optimization. Use of fetal progenitor cell sources in Switzerland is regulated through Federal Transplantation Programs and associated Fetal Biobanks. Clinical applications of cultured primary progenitor dermal fibroblasts have been optimized since the 1990s as "Progenitor Biological Bandages" for pediatric burn patients and adults presenting chronic wounds. A single organ donation procured in 2009 enabled the establishment of a standardized cell source for clinical and industrial developments to date. Non-enzymatically isolated primary dermal progenitor fibroblasts (FE002-SK2 cell type) served for the establishment of a clinical-grade Parental Cell Bank, based on a patented method. Optimized bioprocessing methodology for the FE002-SK2 cell type has demonstrated that extensive and consistent progenitor cell banks can be established. In vitro mechanistic characterization and in vivo preclinical studies have confirmed potency, preliminary safety and efficacy of therapeutic progenitor cells. Most importantly, highly successful industrial transposition and up-scaling of biobanking enabled the establishment of tiered Master and Working Cell Banks using Good Manufacturing Practices. Successive and successful transfers of technology, know-how and materials to different countries around the world have been performed. Extensive developments based on the FE002-SK2 cell source have led to clinical trials for burns and wound dressing. Said trials were approved in Japan, Taiwan, USA and are continuing in Switzerland. The Swiss Fetal Transplantation Program and pioneer clinical experience in the Lausanne Burn Center over three decades constitute concrete indicators that primary progenitor dermal fibroblasts should be considered as therapeutic flagships in the domain of wound healing and for regenerative medicine in general. Indeed, one single organ donation potentially enables millions of patients to benefit from high-quality, safe and effective regenerative therapies. This work presents a technical and translational overview of the described progenitor cell technology harnessed in Switzerland as cellular therapies for treatment of burns and wounds around the globe.

Repair of porcine articular cartilage defect with autologous chondrocyte transplantation.

Articular cartilage is known to have poor healing capacity after injury. Autologous chondral grafting remains the mainstay to treat well-defined, full-thickness, symptomatic cartilage defects. We demonstrated the utilization of gelatin microbeads to deliver autologous chondrocytes for in vivo cartilage generation. Chondrocytes were harvested from the left forelimbs of 12 Lee-Sung pigs. The cells were expanded in monolayer culture and then seeded onto gelatin microbeads or left in monolayer. Shortly before implantation, the cell-laden beads were mixed with collagen type I gel, while the cells in monolayer culture were collected and re-suspended in culture medium. Full-thickness cartilage defects were surgically created in the weight-bearing surface of the femoral condyles of both knees, covered by periosteal patches taken from proximal tibia, and sealed with a porcine fibrin glue. In total, 48 condyles were equally allotted to experimental, control, and null groups that were filled beneath the patch with chondrocyte-laden beads in gel, chondrocytes in plain medium solution, or nothing, respectively. The repair was examined 6 months post-surgery on the basis of macroscopic appearance, histological scores based on the International Cartilage Repair Society Scale, and the proportion of characteristic chondrocytes. Tensile stress-relaxation behavior was determined from uniaxial indentation tests. The experimental group scored higher than the control group in the categories of matrix nature, cell distribution pattern, and absence of mineralization, with similar surface smoothness. Both the experimental and control groups were superior to the null group in the above-mentioned categories. Viable cell populations were equal in all groups, but the proportion of characteristic chondrocytes was highest in the experimental group. Matrix stiffness was ranked as null > native cartilage > control > experimental group. Transplanted autologous chondrocytes survive and could yield hyaline-like cartilage. The application of beads and gel for transplantation helped to retain the transferred cells in situ and maintain a better chondrocyte phenotype.

Association of the growth-arrest-specific protein Gas7 with F-actin induces reorganization of microfilaments and promotes membrane outgrowth.

The growth-arrest-specific gene, Gas7, is required for neurite outgrowth in cerebellar neurons. Here we report that Gas7 can induce the formation of extended cellular processes in NIH3T3 cells by interacting with actin and mediating reorganization of microfilaments. The Gas 7 protein, which increased markedly during growth arrest of NIH3T3 cells and persisted transiently at high levels upon reentry of cells into the cell cycle, localized near the plasma membrane and selectively colocalized with microfilaments in membrane ruffles. Process extensions induced by ectopic overexpression of Gas7 were blocked by the actin-depolymerizing agent cytochalasin D, suggesting that membrane extensions produced by Gas7 require actin polymerization. Association of endogenous Gas7 protein with microfilaments was verified by F-actin affinity chromatography; direct binding of purified His-Gas7 to actin also was demonstrated and shown to be mediated by the Gas7 C-terminal domain. Similarly, localization of Gas7 in membrane ruffles was mediated by the C-terminal domain, although neither this region nor the N-terminal domain was individually sufficient to induce process formation. Biochemical studies and electron microscopy showed that both full-length Gas7 protein and its C-terminal region can promote actin assembly as well as the crosslinking of actin filaments. We propose that Gas7 localized near the plasma membrane induces the assembly of actin and the membrane outgrowth.

Isolation and properties of Gas8, a growth arrest-specific gene regulated during male gametogenesis to produce a protein associated with the sperm motility apparatus.

Growth arrest-specific (Gas) genes are expressed during serum starvation or contact inhibition of cells grown in culture. Here we report the isolation and characterization of Gas8, a novel gene identified on the basis of its growth arrest-specific expression in murine fibroblasts. We show that production of Gas8 mRNA and protein occurs in adult mice predominantly in the testes, where expression is regulated during postmeiotic development of male gametocytes. Whereas a low level of Gas8 mRNA was detected by Northern blotting in testes of murine male neonates and young adolescents, Gas8 mRNA increased rapidly postmeiotically. In adult males, both Gas8 mRNA and protein reached steady state levels in testes that were 10-fold higher than in other tissues. Immunohistochemical analyses showed that Gas8 protein accumulates in gametocytes as they approach the lumen of seminiferous tubules and is localized to the cytoplasm of round spermatids, the tails of elongating spermatids, and mature spermatid tail bundles protruding into the lumen; in epididymal spermatozoa Gas8 protein was present in the flagella. However, premeiotic murine gametocytes lacked detectable Gas8 protein, as did seminiferous tubules in biopsy specimens from seven human males having cytological evidence of non-obstructive azoospermia secondary to Sertoli cell-only syndrome. Our findings, which associate Gas8 production developmentally with the later stages of spermatogenesis and spatially with the sperm motility apparatus, collectively suggest that this growth arrest-specific gene product may have a role in sperm motility. This postulated role for Gas8 is supported by our observation that highly localized production of Gas8 protein occurs also in the cilia of epithelial cells lining pulmonary bronchi and fallopian tubes and by the flagellar association of a Trypanosoma brucei ortholog of Gas8.