Αn optimized, simplified and clinically approved culture system to produce, in large scale, dendritic cells capable of priming specific T cells.

Cancer immunotherapy using dendritic cells (DCs) able to induce specific immune responses to naïve T lymphocytes raises great research interest. However, the extremely complex and expensive methods used to produce DCs, combined with the limited number of autologous DCs in the circulation make any application almost impossible. Aim of the study is the development of an optimized and simplified system to easily produce in large scale cord blood-derived DCs, loaded with common tumor antigens, capable of promoting controlled Th1 immunoresponses following clinically approved maturation with vaccines. CD34+cells cultured in the presence of a cytokine cocktail in miniPERM® bioreactors and the generated DCs were matured using anti-flu vaccines. Autologous T cells plated with DCs pulsed with overlapping peptides CEA and WT1 for multiple stimulations. 200 billion of myeloid DCs were produced and matured in just 8 h in bioreactors, presenting an increased expression of the co-stimulatory molecules and also high levels of Th1 related cytokines. Upon just the 2nd stimulation, the T cells exhibited specificity following stimulation with the CEA/WT1 peptides and strong cytotoxic capacity in co-culture with a colorectal cancer (CRC)-cell line. The high produced doses of DCs, easily maturated with clinically approved agents, and capable of priming specific T cells, could potentially strengthen the further progress in DCs-mediated cancer immunotherapy field.

Intravitreal Administration Effect of Adipose-Derived Mesenchymal Stromal Cells Combined with Anti-VEGF Nanocarriers, in a Pharmaceutically Induced Animal Model of Retinal Vein Occlusion.

Antiangiogenic therapeutic agents (anti-VEGF) have contributed to the treatment of retinal vein occlusion (RVO) while mesenchymal stromal cell- (MSCs-) mediated therapies limit eye degeneration. The aim of the present study is to determine the effect of adipose-derived MSCs (ASCs) combination with nanocarriers of anti-VEGF in a pharmaceutically induced animal model of RVO. Nanoparticles (NPs) of thiolated chitosan (ThioCHI) with encapsulated anti-VEGF antibody were prepared. ASCs were isolated and genetically modified to secrete the green fluorescence GFP. Twenty-four New Zealand rabbits were divided into the I-IV equal following groups: ASCs, ASCs + nanoThioCHI-anti-VEGF, RVO, and control. For the RVO induction, groups I-III received intravitreal (iv) injections of MEK kinase inhibitor, PD0325901. Twelve days later, therapeutic regiments were administered at groups I-II while groups III-IV received BSS. Two weeks later, the retinal damage evaluated via detailed ophthalmic examinations, histological analysis of fixed retinal sections, ELISA for secreted cytokines in peripheral blood or vitreous fluid, and Q-PCR for the expression of related to the occlusion and inflammatory genes. Mild retinal edema and hemorrhages, limited retinal detachment, and vasculature attenuation were observed in groups I and II compared with the pathological symptoms of group III which presented a totally disorganized retinal structure, following of positive immunostaining for neovascularization and related to RVO markers. Important reduction of the high secreted levels of inflammatory cytokines was quantified in groups I and II vitreous fluid, while the expression of the RVO-related and inflammatory genes has been significantly decreased especially in group II. GFP+ ASCs, capable of being differentiated towards neural progenitors, detected in dissociated retina tissues of group II presenting their attachment to damaged area. Conclusively, a stem cell-based therapy for RVO is proposed, accompanied by sustained release of anti-VEGF, in order to combine the paracrine action of ASCs and the progressive reduction of neovascularization.

Isolation of a novel embryonic stem cell cord blood-derived population with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Recent studies highlight the existence of a population of cord blood (CB)-derived stem cells that bare embryonic features (very small embryonic-like stem cells [VSELs]) as the most primitive CB-stem cell population. In the present study, we present for the first time a novel and high purity isolation method of VSELs with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). METHODS: The experimental procedure includes isolation upon gradually increased centrifugation spins and chemotaxis to Stromal cell-derived factor 1a (SDF-1a). Τhis cell population is characterized with flow cytometry, alkaline phosphatase (ALP) staining and qRT-PCR. The functional role of the isolated VSELs is assayed following co-culture with WJ-MSCs or bone marrow-derived mesenchymal stromal cells (BM-MSCs), whereas the stimulation of the quiescent VSEL population is verified via cell cycle analysis. The in vitro hematopoietic capacity is evaluated in methylcellulose cultures and also through induction of erythroid differentiation. RESULTS: The final isolated subpopulation is characterized as a small-sized CD45/Lineage-/CXCR4+/CD133+/SSEA-4+cell population, positive in ALP staining and overexpressing the Oct3/4, Nanog and Sox-2 transcription factors. Upon the co-culture with MSCs, a stimulation of the quiescent VSEL population is observed. An impressive increase in the co-expression of the CD34+/CD45+ markers is observed following the co-culture with the WJ-MSCs, which is confirmed by the intense clonogenic ability suggesting in vitro differentiation toward all of the hematopoietic cell lineages and successful differentiation toward erythrocytes. DISCUSSION: Conclusively, we propose a novel, rapid and rather simplified isolation method of CB-VSELs, capable of in vitro hematopoiesis.

Accelerated healing of a diabetic foot ulcer using autologous stromal vascular fraction suspended in platelet-rich plasma.

We describe the case of a Type I diabetic patient with a refractory foot ulcer that remained unhealed for 2 years despite conventional therapy. Autologous adipose-derived stromal vascular fraction suspended in autologous platelet-rich plasma was applied to the wound, which completely healed within 1 month. The wound remained closed with no complications for a 2-year follow-up. Reporting of this and similar cases may lead to larger clinical trials that will prove the efficacy of this therapy that may offer accelerated healing and lessen the financial burden of more expensive therapeutic modalities.