Spheroids from equine amnion mesenchymal stem cells: an in vitro study

Background: Equine amnion mesenchymal stem cells (EAMSCs) from amnion isolated after the foal birth represented an alternative source of easy collection of mesenchymal cells used in equine regenerative medicine. Methods: These cells grown as 2D culture in α-MEM medium supplemented with EGF were differentiated in adipogenic, chondrogenic and osteogenic cells. Half a million cells as pellet were left in 15ml tubes with the same differentiation media for 20 days. After the pellets were collected, embedded in paraffin for morphological study. Results: 2D culture showed EAMSCs with an embryonic phenotype (C-kit+, CD105+, Oct-4+) and a differentiation potential in adipogenic, chondrogenic and osteogenic multipotent cells. By a reproducible method of 3D culture, at day 20 the Authors evidenced a formation of small aggregated spheroids gradually gathering. In cross sections the surface of spheroid evidenced flattened cells embedded in a red matrix by Alizarin staining and occasionally a core of calcium precipitation. A network of apoptotic or necrotic cells in a not mineralized matrix was present into the center of nodules. The 3D spheroids appeared larger (mean diameter of 605±53 µm for gathering spheroids and 1486±79 µm for spheroids already gathered) than those from standard monolayer cultures (mean diameter of 200 ± 73 µm). Conclusions: EAMSCs cultured in 3D method preserve their in vitro multipotent differentiation than adherent 2D culture method. These EAMSCs included in extracellular matrix not mineralized at day 20 seem to be a good source of MSCs for tissue repair and regeneration in equine medicine

Different media and supplements modulate the clonogenic and expansion properties of rabbit bone marrow mesenchymal stem cells

<p>Abstract</p> <p>Background -</p> <p>Rabbits provide an excellent model for many animal and human diseases, such as cardiovascular diseases, for the development of new vaccines in wound healing management and in the field of tissue engineering of tendon, cartilage, bone and skin.</p> <p>The study presented herein aims to investigate the biological properties of bone marrow rabbit MSCs cultured in different conditions, in order to provide a basis for their clinical applications in veterinary medicine.</p> <p>Findings -</p> <p>MSCs were isolated from 5 New Zealand rabbits. Fold increase, CFU number, doubling time, differentiation ability and immunophenotype were analyzed.</p> <p>With the plating density of 10 cells/cm²the fold increase was significantly lower with DMEM-20%FCS and MSCs growth was significantly higher with αMEM-hEGF. The highest clonogenic ability was found at 100 cell/cm²with MSCBM and at 10 cell/cm²with M199. Both at 10 and 100 cells/cm², in αMEM medium, the highest CFU increase was obtained by adding bFGF. Supplementing culture media with 10%FCS-10%HS determined a significant increase of CFU.</p> <p>Conclusion -</p> <p>Our data suggest that different progenitor cells with differential sensitivity to media, sera and growth factors exist and the choice of culture conditions has to be carefully considered for MSC management.</p

Both carotid and peripheral percutaneous transluminal angioplasty mobilize progenitor cells

It is well known that circulating endothelial progenitor cells (EPC) are involved in injured endothelium repair, contributing to angiogenesis in in vitro and in vivo models. The main triggers of EPC mobilization from bone marrow to peripheral blood are ischemia and vascular trauma. Endothelial injury induced by coronary angioplasty has been recently shown as sufficient to trigger EPC mobilization in coronary artery disease (CAD) patients. Aim of our work was to evaluate the effect of internal carotid angioplasty and peripheral transluminal angioplasty on circulating progenitor cells (PC, CD34+ cells) and EPC (CD34+KDR+ cells) mobilization. Methods: 10 patients (7 males and 3 females, mean age 64.2 ± 10.3 years) undergoing elective percutaneous transluminal angioplasty (PTA) were consecutively enrolled. 5 patients underwent internal carotid angioplasty (IC-PTA) and the others peripheral transluminal angioplasty (P-PTA). Inclusion criteria were: carotid stenosis greater than 70 for IC-PTA; peripheral stenosis greater than 70 and documented peripheral arterial disease (PAD), defined by an intermittent claudication (Leriche-Fontaine stage II) for P-PTA. Patients with unstable angina, myocardial infarction or stroke within the preceding 3 months were excluded to avoid any potentially confounding effect. Peripheral blood samples were collected at the time of admission (t0), on day 1 (t1), on day 7 (t7) and on day 30 (t30). C-reactive protein (CRP), white blood cells (WBC) and VEGF were evaluated. Circulating PC and EPC were determined by flow cytometry analysis. Data are expressed as cells number per ml of blood according to the International Society of Hematotherapy and Graft Engineering protocol. Patients were treated according to the international guidelines and the angioplasty outcome was evaluated by Echo colour Doppler ultrasonography at t1, t7 and t30. Results: No significant differences were found in the prevalence of risk factors. The efficacy of PTA was confirmed in all patients, showing stent patency by Echo colour Doppler evaluation. In all patients the PC count at t7 was significantly increased compared with basal level (t0: 1694±141/mL vs. t7: 2748±375/mL, p0.024) and t1 (t1: 1413±215/mL vs. t7: 2748±375/mL, p0.002), in parallel to VEGF concentration (t0: 530±95 vs t7: 759±153 pg/mL, p0.024; t1: 555±92 vs t7: 759±153 pg/mL, p0.037). Regarding EPC levels, at t7 a trend to an increase compared to both t0 (t0: 595±72/mL vs. t7: 737±98/mL, p0.085) and t1 (t1: 529±87/mL vs. t7: 737±98/mL, p0.052) was observed, maintained up to day 30. CRP was significantly increased after angioplasty, with a peak at t1 (t0: 4.3±0.5 vs t1: 42.3±13.8 mg/L, p0.028), remaining elevated at t7 (t0: 4.3±0.5 vs t7: 26.2±4.3 mg/L, p0.001) and decreasing to baseline levels at t30. Considering IC-PTA and P-PTA separately, in the first group we observed both for PC and EPC levels a trend to increase lasting up to 30 days, while in P-PTA patients the mobilization lasted only up to 7 days. Conclusions: Our data show a transient PC and EPC mobilization together with an increase of VEGF and CRP levels following vascular injury, suggesting that it can be part of an inflammatory response PTA-induced. No relevant differences seem to exist between IC-PTA and P-PTA except for 1-month progenitor quantification. Longitudinal studies and a greater number of patients are required to determinate the role that PC mobilization PTA-induced may play as prognostic factor to identify high-risk patients for restenosis and other cardiovascular events after PTA

Are standard cell culture conditions adequate for human umbilical cord blood mesenchymal stem cells?

Dear Sir, Due to the detrimental action of free radicals and reactive oxygen species (ROS), oxidative stress is involved in the pathogenesis of ageing and diseases such as inflammation, cancer, cardiovascular disorders and infections. Although this view remains still valid, during the last decade the understanding of oxidative stress has progressively changed and it has become clear that free radicals and reactive oxygen species also play a key role in cell biology and function. In fact, oxidation/reduction reactions are a primary mechanism for regulation of cell proliferation, death, and most notably, cell differentiation, which involves the function of several redox-sensitive molecular elements1. (...