Effects of adipose-derived stromal cells and endothelial progenitor cells on adipose transplant survival and angiogenesis

Background A paracrine mechanism is thought to mediate the proangiogenic capacity of adipose-derived stromal/stem cells (ASCs). However, the precise mechanism by which ASCs promote the formation of blood vessels by endothelial progenitor cells (EPCs) is unclear. Methods The EPCs-ASCs cocultures prepared in different ratios were subjected to tube formations assay to verify whether ASCs could directly participate in the tube genesis. The supernatant from cultured ASCs was used to stimulate EPCs to evaluate the effects on the angiogenic property of EPCs, as well as capacity for migration and invasion. A coculture model with transwell chamber were used to explore the regulation of angiogenesis markers expression in EPCs by ASCs. We then mixed ASCs with EPCs and transplanted them with adipose tissue into nude mice to evaluate the effects on angiogenesis in adipose tissue grafts. Results In the EPCs-ASCs cocultures, the tube formation was significantly decreased as the relative abundance of ASCs increased, while the ASCs was found to migrate and integrated into the agglomerates formed by EPCs. The supernatant from ASCs cultures promoted the migration and invasion of EPCs and the ability to form capillary-like structures. The expression of multiple angiogenesis markers in EPCs were significantly increased when cocultured with ASCs. In vivo, ASCs combined with EPC promoted vascularization in the fat transplant. Immunofluorescence straining of Edu and CD31 indicated that the Edu labeled EPC did not directly participate in the vascularization inside the fat tissue. Conclusions ADSC can participate in the tube formation of EPC although it cannot form canonical capillary structures. Meanwhile, Soluble factors secreted by ASCs promotes the angiogenic potential of EPCs. ASCs paracrine signaling appears to promote angiogenesis by increasing the migration and invasion of EPCs and simultaneously upregulating the expression of angiogenesis markers in EPCs. The results of in vivo experiments showed that ASCs combined with EPCs significantly promote the formation of blood vessels in the fat implant. Remarkably, EPCs may promote angiogenesis by paracrine regulation of endogenous endothelial cells (ECs) rather than direct participation in the formation of blood vessels.

Human adipose-derived stromal cells transplantation prolongs reproductive lifespan on mouse models of mild and severe premature ovarian insufficiency

Background Although recent studies have investigated the ability of Mesenchymal Stromal Cells (MSCs) to alleviate short-term ovarian damage in animal models of chemotherapy-induced Premature Ovarian Insufficiency (POI), no data are available on reproductive lifespan recovery, especially in a severe POI condition. For this reason, we investigated the potential of MSCs isolated from human adipose tissue (hASCs), since they are easy to harvest and abundant, in ameliorating the length and performance of reproductive life in both mild and severe chemotherapy-induced murine POI models. Methods Mild and severe POI models were established by intraperitoneally administering a light (12 mg/kg busulfan + 120 mg/kg cyclophosphamide) or heavy (30 mg/kg busulfan + 120 mg/kg cyclophosphamide) dose of chemotherapy, respectively, in CD1 mice. In both cases, a week later, 1 × 106 hASCs were transplanted systemically through the tail vein. After four additional weeks, some females were sacrificed to collect ovaries for morphological evaluation. H&E staining was performed to assess stroma alteration and to count follicle numbers; immunofluorescence staining for αSMA was used to analyse vascularization. Of the remaining females, some were mated after superovulation to collect 2-cell embryos in order to evaluate their pre-implantation developmental capacity in vitro, while others were naturally mated to monitor litters and reproductive lifespan length. F1 litters’ weight, ovaries and reproductive lifespan were also analysed. Results hASC transplantation alleviated ovarian weight loss and size decrease and reduced alterations on ovarian stroma and vasculature, concurrently preventing the progressive follicle stockpile depletion caused by chemotherapy. These effects were associated with the preservation of the oocyte competence to develop into blastocyst in vitro and, more interestingly, with a significant decrease of chemotherapy-induced POI features, like shortness of reproductive lifespan, reduced number of litters and longer time to plug (the latter only presented in the severe POI model). Conclusion Human ASC transplantation was able to significantly reduce all the alterations induced by the chemotherapeutic treatment, while improving oocyte quality and prolonging reproductive functions, thus counteracting infertility. These results, strengthened by the use of an outbred model, support the potential applications of hASCs in women with POI, nowadays mainly induced by anticancer therapies. Graphic abstract

3D Poly(Lactic Acid) Scaffolds Promote Different Behaviors on Endothelial Progenitors and Adipose-Derived Stromal Cells in Comparison With Standard 2D Cultures

Tissue engineering is a branch of regenerative medicine, which comprises the combination of biomaterials, cells and other bioactive molecules to regenerate tissues. Biomaterial scaffolds act as substrate and as physical support for cells and they can also reproduce the extracellular matrix cues. Although tissue engineering applications in cellular therapy tend to focus on the use of specialized cells from particular tissues or stem cells, little attention has been paid to endothelial progenitors, an important cell type in tissue regeneration. We combined 3D printed poly(lactic acid) scaffolds comprising two different pore sizes with human adipose-derived stromal cells (hASCs) and expanded CD133+ cells to evaluate how these two cell types respond to the different architectures. hASCs represent an ideal source of cells for tissue engineering applications due to their low immunogenicity, paracrine activity and ability to differentiate. Expanded CD133+ cells were isolated from umbilical cord blood and represent a source of endothelial-like cells with angiogenic potential. Fluorescence microscopy and scanning electron microscopy showed that both cell types were able to adhere to the scaffolds and maintain their characteristic morphologies. The porous PLA scaffolds stimulated cell cycle progression of hASCs but led to an arrest in the G1 phase and reduced proliferation of expanded CD133+ cells. Also, while hASCs maintained their undifferentiated profile after 7 days of culture on the scaffolds, expanded CD133+ cells presented a reduction of the von Willebrand factor (vWF), which affected the cells’ angiogenic potential. We did not observe changes in cell behavior for any of the parameters analyzed between the scaffolds with different pore sizes, but the 3D environment created by the scaffolds had different effects on the cell types tested. Unlike the extensively used mesenchymal stem cell types, the 3D PLA scaffolds led to opposite behaviors of the expanded CD133+ cells in terms of cytotoxicity, proliferation and immunophenotype. The results obtained reinforce the importance of studying how different cell types respond to 3D culture systems when considering the scaffold approach for tissue engineering.

Repair of Osteoporotic Bone Defects Using Adipose-Derived Stromal Cells and Umbilical Vein Endothelial Cells Seeded in Chitosan/Nanohydroxyapatite-P24 Nanocomposite Scaffolds

Background. The cell regeneration and blood supply of bone defect lesions are restricted under osteoporotic pathological conditions, which make the healing of bone defect of osteoporosis still a great challenge. The current therapeutic strategies that mainly inhibit bone resorption are not always satisfactory for osteoporotic bone defects, which make the development of new therapies an urgent need. Methods. Previously, we prepared chitosan/nanohydroxyapatite (CS/nHA) biomimetic nanocomposite scaffolds for controlled delivery of bone morphogenetic protein 2-derived peptide (P24). In this study, we determined the effect of coculturing adipose-derived stromal cells (ADSCs) and human umbilical vein endothelial cells (HUVECs) with the CS-P24/nHA nanocomposite scaffolds on osteoporotic bone defect healing. In vitro mixed coculture models were employed to assess the direct effects of coculture. Results. ADSCs cocultured with HUVECs showed significantly greater osteogenic differentiation and mineralization compared with ADSCs or HUVECs alone. The CS-P24/nHA scaffold cocultured with ADSCs and HUVECs was more effective in inducing osteoporotic bone repair, as demonstrated by micro-computed tomography and histology of critical-sized calvariae defects in ovariectomized rats. Calvariae defects treated with the CS-P24/nHA nanocomposite scaffold plus ADSC/HUVEC coculture had a greater area of repair and better reconstitution of osseous structures compared with defects treated with the scaffold plus ADSCs or the scaffold plus HUVECs after 4 and 8 weeks. Conclusion. Taken together, coculture of ADSCs and HUVECs with the CS-P24/nHA nanocomposite scaffold is an effective combination to repair osteoporotic bone defects.

Adipose-Derived Stromal Cells and Mineralized Extracellular Matrix Delivery by a Human Decellularized Amniotic Membrane in Periodontal Tissue Engineering

Periodontitis is a prevalent disease characterized by the loss of periodontal supporting tissues, bone, periodontal ligament, and cementum. The application of a bone tissue engineering strategy with Decellularized Human Amniotic Membrane (DAM) with adipose-derived stromal cells (ASCs) has shown to be convenient and valuable. This study aims to investigate the treatments of a rat periodontal furcation defect model with DAM, ASCs, and a mineralized extracellular matrix (ECM). Rat ASCs were expanded, cultivated on DAM, and with a bone differentiation medium for four weeks, deposited ECM on DAM. Periodontal healing for four weeks was evaluated by micro-computed tomography and histological analysis after treatments with DAM, ASCs, and ECM and compared to untreated defects on five consecutive horizontal levels, from gingival to apical. The results demonstrate that DAM preserves its structure during cultivation and healing periods, supporting cell attachment, permeation, bone deposition on DAM, and periodontal regeneration. DAM and DAM+ASCs enhance bone healing compared to the control on the gingival level. In conclusion, DAM with ASC or without cells and the ECM ensures bone tissue healing. The membrane supported neovascularization and promoted osteoconduction.