Microencapsulated VEGF gene–modified umbilical cord mesenchymal stromal cells promote the vascularization of tissue-engineered dermis: an experimental study

Background aimsTissue-engineered dermis (TED) is thought to be the best treatment for skin defect wounds; however, lack of vascular structures in these products can cause slow vascularization or even transplant failure. We assessed the therapeutic potential of microencapsulated human umbilical cord mesenchymal stromal cells (hUCMSCs) expressing vascular endothelial growth factor (VEGF) in vascularization of TED.MethodshUCMSCs were isolated by means of enzymatic digestion and identified by means of testing biological characteristics. hUCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. Collagen-chitosan laser drilling acellular dermal matrix (ADM) composite scaffold was prepared by means of the freeze dehydration and dehydrothermal cross-linking method. hUCMSC-derived fibroblasts were implanted on composite scaffolds to construct TED. TED with microencapsulated VEGF gene–modified hUCMSCs was then transplanted into skin defect wounds in pigs. The angiogenesis of TED at 1 week and status of wound healing at 3 weeks were observed.ResultsThe collagen-chitosan laser ADM composite has a uniform microporous structure. This composite has been used to grow hUCMSC-derived fibroblasts in vitro and to successfully construct stem cell–derived TED. Microencapsulated VEGF gene–modified hUCMSCs were prepared with the use of a sodium alginate–barium chloride one-step encapsulation technology. Seven days after the transplantation of the stem cell–derived TED and microencapsulated VEGF gene–modified hUCMSCs into the skin defect wounds on the backs of miniature pigs, the VEGF expression increased and the TED had a higher degree of vascularization. Re-epithelialization of the wound was completed after 3 weeks.ConclusionsMicroencapsulated VEGF gene–modified hUCMSCs can effectively improve the vascularization of TED and consequently the quality of wound healing.     

Telocytes in human skin – are they involved in skin regeneration?

Telocytes (TCs), a particular interstitial cell type, have been recently described in a wide variety of mammalian organs ( www.telocytes.com ). The TCs are identified morphologically by a small cell body and extremely long (tens to hundreds of μm), thin prolongations (less than 100 nm in diameter, below the resolving power of light microscopy) called telopodes. Here, we demonstrated with electron microscopy and immunofluorescence that TCs were present in human dermis. In particular, TCs were found in the reticular dermis, around blood vessels, in the perifollicular sheath, outside the glassy membrane and surrounding sebaceous glands, arrector pili muscles and both the secretory and excretory portions of eccrine sweat glands. Immunofluorescence screening and laser scanning confocal microscopy showed two subpopulations of dermal TCs; one expressed c‐kit/CD117 and the other was positive for CD34. Both subpopulations were also positive for vimentin. The TCs were connected to each other by homocellular junctions, and they formed an interstitial 3D network. We also found TCs adjoined to stem cells in the bulge region of hair follicles. Moreover, TCs established atypical heterocellular junctions with stem cells (clusters of undifferentiated cells). Given the frequency of allergic skin pathologies, we would like to emphasize the finding that close, planar junctions were frequently observed between TCs and mast cells. In conclusion, based on TC distribution and intercellular connections, our results suggested that TCs might be involved in skin homeostasis, skin remodelling, skin regeneration and skin repair.     

Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

1. Download high-res image (188KB)
2. Download full-size image

     

Three specific antigens to isolate endothelial progenitor cells from human liposuction material

Background aimsHuman endothelial progenitor cells (EPC) play an important role in regenerative medicine and contribute to neovascularization on vessel injury. They are usually enriched from peripheral blood, cord blood and bone marrow. In human fat tissue, EPC are rare and their isolation remains a challenge.MethodsFat tissue was prepared by collagenase digestion, and the expression of specific marker proteins was evaluated by flow cytometry in the stromal vascular fraction (SVF). For enrichment, magnetic cell sorting was performed with the use of CD133 microbeads and EPC were cultured until colonies appeared. A second purification was performed with CD34; additional isolation steps were performed with the use of a combination of CD34 and CD31 microbeads. Enriched cells were investigated by flow cytometry for the expression of endothelial specific markers, by Matrigel assay and by the uptake of acetylated low-density lipoprotein.ResultsThe expression pattern confirmed the heterogeneous nature of the SVF, with rare numbers of CD133+ detectable. EPC gained from the SVF by magnetic enrichment showed cobblestone morphology of outgrowth endothelial cells and expressed the specific markers CD31, CD144, vascular endothelial growth factor (VEGF)R2, CD146, CD73 and CD105. Functional integrity was confirmed by uptake of acetylated low-density lipoprotein and the formation of tube-like structures on Matrigel.ConclusionsRare EPC can be enriched from human fat tissue by magnetic cell sorting with the use of a combination of microbeads directed against CD133, an early EPC marker, CD34, a stem cell marker, and CD31, a typical marker for endothelial cells. In culture, they differentiate into EC and hence could have the potential to contribute to neovascularization in regenerative medicine.     

Functional properties of ion channels and transporters in tumour vascularization

Vascularization is crucial for solid tumour growth and invasion, providing metabolic support and sustaining metastatic dissemination. It is now accepted that ion channels and transporters play a significant role in driving the cancer growth at all stages. They may represent novel therapeutic, diagnostic and prognostic targets for anti-cancer therapies. On the other hand, although the expression and role of ion channels and transporters in the vascular endothelium is well recognized and subject of recent reviews, only recently has their involvement in tumour vascularization been recognized. Here, we review the current literature on ion channels and transporters directly involved in the angiogenic process. Particular interest will be focused on tumour angiogenesis in vivo as well as in the different steps that drive this process in vitro, such as endothelial cell proliferation, migration, adhesion and tubulogenesis. Moreover, we compare the ‘transportome’ system of tumour vascular network with the physiological one.     

Effects of human adipose-derived stem cells on the viability of rabbit random pattern flaps

Background aimsFlap necrosis is the most commonly encountered outcome influencing the effect of operations in clinical practice. The advent of cytotherapy and regenerative medicine with stem cells, especially adipose-derived stem cell therapy, appears to be a promising approach in providing multi-lineage differentiating cells. However, autologous stem cells are limited in both quantity and quality in aging individuals. Hence, xenogenic stem cell therapy was used in this study.MethodsRandom pattern flaps (6 cm × 2 cm) were prepared in a rabbit model transplanted either with 4 × 10⁵ human adipose-derived stem cells at five sites or equal volumes of Dulbecco's modified Eagle's medium. At 7 days after operation, the viability of the flaps from both groups was evaluated. We determined the numbers of locally infiltrating T cells, whereas the CD4/CD8 ratio, interferon, interleukin (IL)-2, IL-4 and IL-10 in the serum were determined to evaluate the immunological response of the rabbit. Moreover, Dil labeling was administrated to trace the homing of the transplanted stem cells.ResultsBoth the survival areas and the capillary number of the flaps that were injected with human adipose-derived stem cells significantly increased as compared with the control group (P < 0.05). Additionally, no significant difference in the immune response was detected between the groups. Dil-labeled stem cells were found to participate in the formation of tubular structures, which were further shown to be CD31+, although not predominantly.ConclusionsHuman adipose-derived stem cells could be used therapeutically to improve the viability of random pattern flaps without detection of serious immune rejection of stem cells.     

Cell-based approaches to the engineering of vascularized bone tissue

This review summarizes recent efforts to create vascularized bone tissue in vitro and in vivo through the use of cell-based therapy approaches. The treatment of large and recalcitrant bone wounds is a serious clinical problem, and in the United States approximately 10% of all fractures are complicated by delayed union or non-union. Treatment approaches with the use of growth factor and gene delivery have shown some promise, but results are variable and clinical complications have arisen. Cell-based therapies offer the potential to recapitulate key components of the bone-healing cascade, which involves concomitant regeneration of vasculature and new bone tissue. For this reason, osteogenic and vasculogenic cell types have been combined in co-cultures to capitalize on the function of each cell type and to promote heterotypic interactions. Experiments in both two-dimensional and three-dimensional systems have provided insight into the mechanisms by which osteogenic and vasculogenic cells interact to form vascularized bone, and these approaches have been translated to ectopic and orthotopic models in small-animal studies. The knowledge generated by these studies will inform and facilitate the next generation of pre-clinical studies, which are needed to move cell-based orthopaedic repair strategies into the clinic. The science and application of cytotherapy for repair of large and ischemic bone defects is developing rapidly and promises to provide new treatment methods for these challenging clinical problems.