Nonviral Transfection Strategies for Keratinocytes, Fibroblasts, and Endothelial Progenitor Cells for Ex Vivo Gene Transfer to Skin Wounds

In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas nucleofection was optimal (32%) for porcine cells. For FBs, however, nucleofection resulted in the highest transfection rates in human (46%) and porcine (60%) FBs. For specific epidermal cell studies, Fugene HD was the preferred gene transfer method, whereas Effectene appeared to be the optimal reagent for pro-angiogenic studies. Nucleofection in combination with FBs is the best combination to achieve the highest overall transfection rate and is thus the optimal combination for use in ex vivo gene transfer strategies of wound healing or skin tissue engineering.status: publishe

Integration of Blood Outgrowth Endothelial Cells in Dermal Fibroblast Sheets Promotes Full Thickness Wound Healing

Vascularization is the cornerstone of wound healing. We introduced human blood outgrowth endothelial cells (hBOEC) in a self-assembled human dermal fibroblast sheet (hDFS), intended as a tissue-engineered dermal substitute with inherent vascular potential. hBOEC were functionally and molecularly different from early endothelial progenitor cells and human umbilical vein endothelial cells (HUVEC). hBOEC alone, unlike HUVEC, efficiently revascularized and re-oxygenated the wound bed, both by active incorporation into new vessels and by trophic stimulation of host angiogenesis in a dose-dependent manner. Furthermore, hBOEC alone, but not HUVEC, accelerated epithelial coverage and matrix organization of the wound bed. In addition, integration of hBOEC in hDFS not only further improved vascularization, epithelial coverage and matrix organization but also prevented excessive wound contraction. In vitro analyses with hBOEC, fibroblasts and keratinocytes revealed that these effects were both due to growth factor crosstalk and to short cutting hypoxia. Among multiple growth factors secreted by hBOEC, placental growth factor mediated at least in part the beneficial effects on keratinocyte migration and proliferation. Overall, this combined tissue engineering approach paves the way for clinical development of a fully autologous vascularized dermal substitute for patients with large skin defects that do not heal properly. STEM CELLS 2010;28:1165-1177status: publishe

Reversal of hyperglycemia in diabetic mice by a marginal islet mass together with human blood outgrowth endothelial cells is independent of the delivery technique and blood clot-induced processes

We recently reported that human blood outgrowth endothelial cells (BOEC) are supportive to reverse hyperglycemia in marginal islet mass-transplanted diabetic mice. In this report, we investigated whether the observed effect was evoked by islet packing in a blood clot prior to transplantation or could be mimicked by another method of islet/cell delivery. A marginal islet mass with or without BOEC was grafted underneath the kidney capsule of diabetic recipient mice via a (blood clot-independent) tubing system and compared with previous islet packing in a blood clot. The effect on metabolic outcome of both delivery techniques as well as the additive effect of BOEC was subsequently evaluated. Marginal islet mass transplantation via a tubing system required more islets per recipient than via a blood clot. Using the tubing method, transplantation of a marginal islet mass combined with 5x10 (5) BOEC resulted in reversal of hyperglycemia, improved glucose tolerance and increased kidney insulin content. The present study provides evidence that (1) previous packing in a blood clot results in more effective islet delivery compared with tubing; (2) BOEC exert a beneficial effect on marginal islet transplantation, independent of grafting technique and potential blood clot-induced processes. These data further support the use of BOEC in (pre-) clinical studies that aim to improve current islet transplantation protocols.status: publishe

Neovascularisation Potential of Blood Outgrowth Endothelial Cells From Patients With Severe Ischemic Cardiomyopathy

Dauwe D., Verdonck K., Gilllijns H., Caluwe E., Pokreisz P., van Gastel N., Carmeliet G., Depypere M., Maes F., Droogne W., Vanhaecke J., Luttun A., Janssens S., ''Neovascularisation potential of blood outgrowth endothelial cells from patients with severe ischemic cardiomyopathy'', American Heart Association scientific sessions 2013, November 16-20, 2013, Dallas, Texas, USA.status: publishe

Histological findings compared with magnetic resonance and ultrasonographic imaging in irreversible postmastectomy lymphedema: A case study

Postmastectomy edema is a current complication after axillary lymph node dissection in cases of breast cancer treatment. Staging is important in order to select those patients who can benefit from complex physical therapy (CPT). Different imaging techniques can be used to evaluate the edema. Ultrasonography (US) is a harmless, cheap, and easily applicable technique to visualize the dermal and subcutaneous tissue, but interpretation of the obtained images is not always evident. The aim of this study was to compare ultrasound images of irreversible edema with tissue histology, magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). Ultrasonographic images of the edematous dermis show an homogeneous hypoechogenic dermal layer that appears on tissue histology to be less compact, due to the excess of fluid in the interstitium separating the collagen fibres and making it more transparent on light microscopy. MRI of the dermis gives a hyperintense signal, indicating the presence of fluid. In the subcutis, increase of the adipose tissue could be observed on US, MRI, and tissue histology. In the case of lymphedema, the area and perimeter of fat cells is significantly (p < 0.05) increased. Hypoechogenic areas near the muscle fascia are registrated on US corresponding with epifascial fluid on MRI, and hyperechogenic branches are embedded within the adipose tissue, on tissue histology seen as large fibrotic septa enclosing adipose cells. MRI has a honeycomb picture corresponding with fluid bound to fibrosis. © Mary Ann Liebert, Inc.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Neovascularization Potential of Blood Outgrowth Endothelial Cells From Patients With Stable Ischemic Heart Failure Is Preserved

Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models, but outgrowth characteristics and functionality of BOECs from patients with ischemic cardiomyopathy (ICMP) are unknown. We compared outgrowth efficiency and in vitro and in vivo functionality of BOECs derived from ICMP with BOECs from age-matched (ACON) and healthy young (CON) controls.Dauwe D., Pelacho B., Wibowo A., Walravens A., Verdonck K., Gillijns H., Caluwe E., Pokreisz P., van Gastel N., Carmeliet G., Depypere M., Maes F., Vanden Driessche N., Droogne W., Van Cleemput J., Vanhaecke J., Prosper F., Verfaillie C., Luttun A., Janssens S., ''Neovascularization potential of blood outgrowth endothelial cells from Patients with stable ischemic heart failure is preserved'', Journal of the American Heart Association, vol. 5, no. 4, pp. e002288, April 2016.status: publishe