This work has identified differentially expressed proteins specific to KS tissue extracts which can potentially be used as specific targets for therapeutic intervention.

In this study we describe the derivation and immunological characterization of a primary epithelial cell type from the human umbilical cord membrane. These cord lining epithelial cells (CLECs) expressed and/or secreted isoforms of the nonclassical human leukocyte antigen class I (HLA-1b) glycoproteins, HLA-G and E. Conditioned media from CLECs inhibited mitogen-stimulated T-lymphocyte responses, and in a mixed leukocyte reaction (MLR) assay, cocultured CLECs inhibited allogeneic responses with a concomitant reduction in proinflammatory cytokines. Using a transwell coculture system, it was demonstrated that these immunoregulatory effects were mediated by soluble factors secreted by CLECs, in a dose-dependent manner. Functional studies using HLA-G blocking antibody showed that the effects of CLEC-secreted products could be inhibited, thus demonstrating a significant and important role for soluble HLA-G. In vivo, we show that transplanted CLECs could be maintained for extended periods in immunocompetent mice where xenorejection rapidly destroyed primary keratinocytes, a control human epithelial cell type. Additionally, CLECs delayed the rejection of keratinocytes and extended their survival when cotransplanted, indicating an ability to protect adjacent human cell types that would otherwise be rejected if transplanted alone. We also show that CLECs transduced with a modified human proinsulin gene were transplanted intraperitoneally into streptozotocin (STZ)-induced diabetic mice, resulting in significantly lower levels of serum glucose compared to control mice. This study has characterized the immunological properties of CLECs and tested a potential therapeutic application in the treatment of a type 1 diabetes mouse model.

These data indicate that the SCF/c-KIT system plays an important role in scar pathogenesis, and underscore the role of imatinib as a key therapeutic agent in keloid scars.

Hepatoma-derived growth factor (HDGF) is a novel mitogenic growth factor that has been implicated in many different carcinomas. Its role in keloid biology has not yet been investigated. The present study is aimed at examining the role of HDGF in keloid pathogenesis. Immunohistochemical staining and Western blot analyses were used to examine in vivo localization and expression of HDGF in keloid and normal skin tissue. This was followed by the detection of HDGF expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial–mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were co-cultured with the fibroblasts. HDGF expression levels in all cell extracts and conditioned media were assayed through Western blot analysis. In another set of experiments, the effect of exogenous recombinant HDGF on keloid fibroblasts (KF) and normal fibroblasts (NF) was examined. Cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by quantifying proliferating cell nuclear antigen (PCNA) expression. Downstream targets of HDGF were identified by detecting their expression through Western blot analysis. Our results indicate that there was an increase in HDGF expression in the dermis of keloid compared with normal skin tissue. The application of serum and epithelial–mesenchymal interactions did not seem to have any effect on intracellular HDGF expression levels. However, co-culturing keloid keratinocytes with KFs resulted in increased HDGF secretion when compared with monoculture or normal controls. Furthermore, treatment with exogenous recombinant HDGF was found to increase the proliferation of KFs, activate the extracellular signal-regulated kinase (ERK) pathway and up-regulate the secretion of vascular endothelial growth factor (VEGF).

Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious.

Nanofibers for tissue scaffolding and wound dressings hold great potential in realizing enhanced healing of wounds in comparison with conventional counterparts. Previously, we demonstrated good fibroblast adherence and growth on a newly developed scaffold, Tegaderm™-Nanofiber (TG-NF), made from poly ɛ-caprolactone (PCL)/gelatin nanofibers electrospun onto Tegaderm (TG). The purpose of this study is to evaluate the performance and safety of TG-NF dressings in partial-thickness wound in a pig healing model. To evaluate the rate of reepithelialization, control TG, human dermal fibroblast-seeded TG-NF(+) and -unseeded TG-NF(-) were randomly dressed onto 80 partial-thickness burns created on four female and four male pigs. Wound inspections and dressings were done after burns on day 7, 14, 21, and 28. On day 28, full-thickness biopsies were taken for histopathological evaluation by Masson-Trichrome staining for collagen and hematoxylin-eosin staining for cell counting. No infection and severe inflammation were recorded. Wounds treated with TG-NF(+) reepithelialized significantly faster than TG-NF(-) and control. Wound site inflammatory responses to study groups were similar as total cell counts on granulation tissues show no significant differences. Most of the wounds completely reepithelialized by day 28, except for two wounds in control and TG-NF(-). A higher collagen coverage was also recorded in the granulation tissues treated with TG-NF(+). With better reepithelialization achieved by TG-NF(+) and similar rates of wound closure by TG-NF(-) and control, and the absence of elevated inflammatory responses to TG-NF constructs, TG-NF constructs are safe and demonstrated good healing potentials that are comparable to Tegaderm.

We investigated the safety of using umbilical cord-lining stem cells for liver regeneration and tested a novel method for stem cell delivery. Stem cells are known by their ability to repair damaged tissues and have the potential to be used as regenerative therapies. The umbilical cord’s outer lining membrane is known to be a promising source of multipotent stem cells and can be cultivated in an epithelial cell growth medium to produce cell populations which possess the properties of both epithelial cells and embryonic stem cells—termed cord-lining epithelial cells (CLEC). Hepatocytes are epithelial cells of the liver and their proliferation upon injury is the main mechanism in restoring the liver. Earlier studies conducted showed CLEC can be differentiated into functioning hepatocyte-like cells (HLC) and can survive in immunologically competent specimens. In this study, we chose a porcine model to investigate CLEC as a treatment modality for liver failure. We selected 16 immune competent Yorkshire-Dutch Landrace pigs, with a mean weight of 40.5 kg, for this study. We performed a 50% hepatectomy to simulate the liver insufficient disease model. After the surgery, four pigs were transplanted with a saline scaffold while seven pigs were transplanted with a HLC scaffold. Five pigs died on the surgical table and were omitted from the study analysis. This study addressed the safety of transplanting human CLEC in a large animal model. The transplant interfaces were evaluated and no signs of cellular rejection were observed in both groups.

Objective: To investigate the effect of glucose and insulin concentrations on differentiation of umbilical cord lining progenitor cells to adipocyte-like cells (ALCs). Methods: Cord lining mesenchymal cells (CLMCs) were isolated from the explant of human umbilical cord amniotic membrane. CLMCs were subjected to differentiation under various culture conditions for 20 days. Lipid droplets were confirmed with Oil Red O staining. Gene expressions of adipsin and peroxisome proliferator-activated receptor gamma (PPARg) were analyzed using reverse transcription-PCR. Leptin and adiponectin secretions were detected using enzyme-linked immunosorbent assay kit. Results: CLMCs became irregular, cuboidal-shaped cells that resemble adipocytes, and Oil Red O staining showed the presence of lipid droplets. The gene expressions of PPARg and adipsin were upregulated. Leptin and adiponectin secretions by naive CLMCs were below the limits of detection. Matured ALCs cultured in low-glucose medium significantly secreted leptin and adiponectin, whereas those in high-glucose medium significantly secreted only leptin. Insulin concentration affects leptin but not adiponectin secretion. Conclusions: Under different culture conditions, CLMCs can differentiate into ALCs that resemble adipocytes in either normalweight or obese individuals. Hence, these ALCs have the potential to be used as an in vitro model to study adipogenesis and obesity, and possibly as a drug discovery model for metabolic disorders.