Graft-Host Interaction and Its Effect on Wound Repair Using Mouse Models.

Autologous skin grafting has been commonly used in clinics for decades to close large wounds, yet the cellular and molecular interactions between the wound bed and the graft that mediates the wound repair are not fully understood. The aim of this study was to better understand the molecular changes in the wound triggered by autologous and synthetic grafting. Defining the wound changes at the molecular level during grafting sets the basis to test other engineered skin grafts by design. In this study, a full-thickness skin graft (SKH-1 hairless) mouse model was established. An autologous full-thickness skin graft (FTSG) or an acellular fully synthetic Biodegradable Temporising Matrix (BTM) was grafted. The wound bed/grafts were analysed at histological, RNA, and protein levels during the inflammation (day 1), proliferation (day 5), and remodelling (day 21) phases of wound repair. The results showed that in this mouse model, similar to others, inflammatory marker levels, including Il-6, Cxcl-1, and Cxcl-5/6, were raised within a day post-wounding. Autologous grafting reduced the expression of these inflammatory markers. This was different from the wounds grafted with synthetic dermal grafts, in which Cxcl-1 and Cxcl-5/6 remained significantly high up to 21 days post-grafting. Autologous skin grafting reduced wound contraction compared to wounds that were left to spontaneously repair. Synthetic grafts contracted significantly more than FTSG by day 21. The observed wound contraction in synthetic grafts was most likely mediated at least partly by myofibroblasts. It is possible that high TGF-ß1 levels in days 1-21 were the driving force behind myofibroblast abundance in synthetic grafts, although no evidence of TGF-ß1-mediated Connective Tissue Growth Factor (CTGF) upregulation was observed.

A Comparative Study of Engineered Dermal Templates for Skin Wound Repair in a Mouse Model.

Engineered dermal templates have revolutionised the repair and reconstruction of skin defects. Their interaction with the wound microenvironment and linked molecular mediators of wound repair is still not clear. This study investigated the wound bed and acellular "off the shelf" dermal template interaction in a mouse model. Full-thickness wounds in nude mice were grafted with allogenic skin, and either collagen-based or fully synthetic dermal templates. Changes in the wound bed showed significantly higher vascularisation and fibroblast infiltration in synthetic grafts when compared to collagen-based grafts (P ≤ 0.05). Greater tissue growth was associated with higher prostaglandin-endoperoxide synthase 2 (Ptgs2) RNA and cyclooxygenase-2 (COX-2) protein levels in fully synthetic grafts. Collagen-based grafts had higher levels of collagen III and matrix metallopeptidase 2. To compare the capacity to form a double layer skin substitute, both templates were seeded with human fibroblasts and keratinocytes (so-called human skin equivalent or HSE). Mice were grafted with HSEs to test permanent wound closure with no further treatment required. We found the synthetic dermal template to have a significantly greater capacity to support human epidermal cells. In conclusion, the synthetic template showed advantages over the collagen-based template in a short-term mouse model of wound repair.

Xeno-free expansion of adult keratinocytes for clinical application: the use of human-derived feeder cells and serum.

Cultured epithelial autograft (CEA) was the birth of skin tissue engineering and encompassed methodologies for the isolation and expansion of autologous basal keratinocytes for burn treatment that are still practiced at some specialised units around the world. One of the limitations of CEA, however, is the reliance on animal-derived material during the manufacturing process and despite all efforts to date, no xeno-free alternative with proven efficacy has been reported. Here, we investigate whether human-derived fibroblast feeder cells and human serum can sufficiently and effectively provide a suitable microenvironment for adult keratinocyte isolation and expansion. Human dermal fibroblasts and epidermal keratinocytes were isolated from discarded skin during abdominoplasty and breast reduction procedures and cultured in xeno-free conditions. We report that these xeno-free adult keratinocytes form similar numbers of colony-forming units as those cultured using the Green's methods; however, xeno-free keratinocytes express lower levels of α6 integrin (CD49f; a progenitor and stem cell marker). We identified IL-8 as a potential growth factor secreted by adult human fibroblasts that may enhance keratinocyte colony formation in human serum. Finally, we propose a step-by-step xeno-free isolation and cultivation methodology for adult keratinocytes that can be tested further in serial cultivation for clinical application.

A phase IIa study of HA-irinotecan, formulation of hyaluronic acid and irinotecan targeting CD44 in extensive-stage small cell lung cancer.

Preclinical studies in small cell lung cancer (SCLC) have shown that hyaluronic acid (HA) can be effectively used to deliver chemotherapy and selectively decrease CD44 expressing (stem cell-like) tumour cells. The current study aimed to replicate these findings and obtain data on safety and activity of HA-irinotecan (HA-IR). Eligible patients with extensive stage SCLC were consented. A safety cohort (n = 5) was treated with HA-IR and Carboplatin (C). Subsequently, the patients were randomised 1:1 to receive experimental (HA-IR + C) or standard (IR + C) treatment, to a maximum of 6 cycles. The second line patients were added to the study and treated with open label HA-IR + C. Tumour response was measured after every 2 cycles. Baseline tumour specimens were stained for CD44s and CD44v6 expression. Circulating tumour cells (CTCs) were enumerated before each treatment cycle. Out of 39 patients screened, 34 were evaluable for the study. The median age was 66 (range 39-83). The overall response rates were 69% and 75% for experimental and standard arms respectively. Median progression free survival was 42 and 28 weeks, respectively (p = 0.892). The treatments were well tolerated. The incidence of grade III/IV diarrhea was more common in the standard arm, while anaemia was more common in the experimental arm. IHC analysis suggested that the patients with CD44s positive tumours may gain survival benefit from HA-IR. HA-IR is well tolerated and active in ES-SCLC. The effect of HA-IR on CD44s + cancer stem-like cells provide an early hint towards a potential novel target.

Characterization of the Human Pancreas Side Population as a Potential Reservoir of Adult Stem Cells.

OBJECTIVES: The side population (SP) contains cells with stem cell/progenitor properties. Previously, we observed that the mouse pancreas SP expanded after pancreatic injury. We aimed to characterize the SP in human pancreas as a potential source of stem cells. METHODS: Human organ donor pancreata were fractionated into islets and exocrine tissue, enriched by tissue culture and dispersed into single cells. Cells were phenotyped by flow cytometry, and the SP was defined by efflux of fluorescent dye Hoechst 33342 visualized by ultraviolet excitation. Cells were flow sorted, and their colony-forming potential measured on feeder cells in culture. RESULTS: An SP was identified in islet and exocrine cells from human organ donors: 2 with type 1 diabetes, 3 with type 2 diabetes, and 28 without diabetes. Phenotyping revealed that exocrine SP cells had an epithelial origin, were enriched for carbohydrate antigen 19-9 ductal cells expressing stem cell markers CD133 and CD26, and had greater colony-forming potential than non-SP cells. The exocrine SP was increased in a young adult with type 1 diabetes and ongoing islet autoimmunity. CONCLUSIONS: The pancreatic exocrine SP is a potential reservoir of adult stem/progenitor cells, consistent with previous evidence that such cells are duct-derived and express CD133.

Adult pancreas side population cells expand after ß cell injury and are a source of insulin-secreting cells.

Pancreas stem cells are a potential source of insulin-producing ß cells for the therapy of diabetes. In adult tissues the 'side population' (SP) of cells that effluxes the DNA binding dye Hoechst 33342 through ATP-binding cassette transporters has stem cell properties. We hypothesised therefore that the SP would expand in response to ß cell injury and give rise to functional ß cells. SP cells were flow sorted from dissociated pancreas cells of adult mice, analysed for phenotype and cultured with growth promoting and differentiation factors before analysis for hormone expression and glucose-stimulated insulin secretion. SP cell number and colony forming potential (CFP) increased significantly in models of type diabetes, and after partial pancreatectomy, in the absence of hyperglycaemia. SP cells, ∼1% of total pancreas cells at 1 week of age, were enriched >10-fold for CFP compared to non-SP cells. Freshly isolated SP cells contained no insulin protein or RNA but expressed the homeobox transcription factor Pdx1 required for pancreas development and ß cell function. Pdx1, along with surface expression of CD326 (Ep-Cam), was a marker of the colony forming and proliferation potential of SP cells. In serum-free medium with defined factors, SP cells proliferated and differentiated into islet hormone-expressing cells that secreted insulin in response to glucose. Insulin expression was maintained when tissue was transplanted within vascularised chambers into diabetic mice. SP cells in the adult pancreas expand in response to ß cell injury and are a source of ß cell progenitors with potential for the treatment of diabetes.

Pancreatic expression and mitochondrial localization of the progestin-adipoQ receptor PAQR10.

Steroid hormones induce changes in gene expression by binding to intracellular receptors that then translocate to the nucleus. Steroids have also been shown to rapidly modify cell function by binding to surface membrane receptors. We identified a candidate steroid membrane receptor, the progestin and adipoQ receptor (PAQR) 10, a member of the PAQR family, in a screen for genes differentially expressed in mouse pancreatic beta-cells. PAQR10 gene expression was tissue restricted compared with other PAQRs. In the mouse embryonic pancreas, PAQR10 expression mirrored development of the endocrine lineage, with PAQR10 protein expression confined to endocrine islet-duct structures in the late embryo and neonate. In the adult mouse pancreas, PAQR10 was expressed exclusively in islet cells except for its reappearance in ducts of maternal islets during pregnancy. PAQR10 has a predicted molecular mass of 29 kDa, comprises seven transmembrane domains, and, like other PAQRs, is predicted to have an intracellular N-terminus and an extracellular C-terminus. In silico analysis indicated that three members of the PAQR family, PAQRs 9, 10, and 11, have a candidate mitochondrial localization signal (MLS) at the N-terminus. We showed that PAQR10 has a functional N-terminal MLS and that the native protein localizes to mitochondria. PAQR10 is structurally related to some bacterial hemolysins, pore-forming virulence factors that target mitochondria and regulate apoptosis. We propose that PAQR10 may act at the level of the mitochondrion to regulate pancreatic endocrine cell development/survival.