257 A novel polysaccharide-based porous scaffold for cell delivery into the infarcted heart

BackgroundCellular cardiomyoplasty has been proposed as an attractive strategy to repair myocardial damage. One of the crucial point is the optimal delivery strategy. In the present study, we examined the use of an implantable porous scaffold for promoting bone marrow-derived mesenchymal stem cells (MSCs) survival and functions in a rat model of acute myocardial infarction.Methods and ResultsCardiac patch was based on biodegradable polysaccharides porous scaffold. After ligation of the left anterior coronary artery, the fate of 1x106 GFP+ MSC administered either using cellularized scaffold implantation or direct injection was examined at 1 and 2 months. The number of residual GFP+ cells in the sample studied was estimated on the basis of the fluorescence emitted by a defined number of GFP+ cells used for calibration. Cellularized scaffold allowed a more efficient delivery and the difference with direct injection was significant at 2 months, with respectively 2100±1300×103 and 215±85x103 residual GFP+ cells (p<0.03). Cardiac tissue levels of matrix metalloproteinase-2 and -9 mRNA were similar whatever the administration conditions but a slight increase in the local production of vascular endothelial growth factor was observed at 2 months after patch implantation in comparison to direct injection (p<0.05). In animals having received MSC implemented on scaffold, clusters of GFP+ cells, mainly phenotypically consistent with immature MSC cells, were detected in the peri-infarct area. The increased survival using scaffold was not translated in an improved myocardial remodelling and functions with no significant difference in the LVEDD, LVESD, and FS between the 2 groups as in comparison with animals implanted with non cellularized scaffold.ConclusionsThese findings demonstrate that the implantation of cellularized grafts is safe and effective for delivering mesenchymal stem cells into damaged myocardium, and results in a better cellular engraftment compared to direct injection

Lack of evidence of sustained hematopoietic reconstitution after transplantation of unmanipulated adult liver stem cells in monkeys.

International audienceThe aim of this study was to search for hematopoietic potential in the liver of non-human primates. Lethally irradiated (2 x 5 Gy gamma) macaque monkeys were given autologous hepatic mononuclear cells (HMNC) isolated from a liver lobe by perfusion and digestion with 0.1% collagenase. Two monkeys were given intramedullary injections of HMNC (18.6 x 10(6)/kg, 20.4 x 10(6)/kg) and two others were co-transplanted with HMNC (14.35 x 10(6)/kg, 96.5 x 10(6)/kg) and bone marrow mesenchymal stem cells (0.42 x 10(6)/kg, 1.16 x 10(6)/kg). All monkeys exhibited a transient neutrophil recovery from day 22 for 10 days, but failed to produce platelets and remained transfusion-dependent. In conclusion, adult liver stem cells from a monkey model show a low level of in vivo hematopoietic potential, suggesting ex vivo manipulation will be required before clinical use of such cells

Calcium-phosphate ceramics and polysaccharide-based hydrogel scaffolds combined with mesenchymal stem cell differently support bone repair in rats

International audienceResearch in bone tissue engineering is focused on the development of alternatives to autologous bone grafts for bone reconstruction. Although multiple stem cell-based products and biomaterials are currently being investigated, comparative studies are rarely achieved to evaluate the most appropriate approach in this context. Here, we aimed to compare different clinically relevant bone tissue engineering methods and evaluated the kinetic repair and the bone healing efficiency supported by mesenchymal stem cells and two different biomaterials, a new hydrogel scaffold and a commercial hydroxyapatite/tricalcium phosphate ceramic, alone or in combination. Syngeneic mesenchymal stem cells (5 × 10 5) and mac-roporous biphasic calcium phosphate ceramic granules (Calciresorb C35 ® , Ceraver) or porous pullulan/dextran-based hydrogel scaffold were implanted alone or combined in a drilled-hole bone defect in rats. Using quantitative microtomography measurements and qualitative histologi-cal examinations, their osteogenic properties were evaluated 7, 30, and 90 days after implantation. Three months after surgery, only minimal repair was evidenced in control rats while newly mineralized bone was massively observed in animals treated with either hydrogels (bone volume/tissue volume = 20%) or ceramics (bone volume/tissue volume = 26%). Repair mechanism and resorption kinetics were strikingly different: rapidly-resorbed hydrogels induced a dense bone mineralization from the edges of the defect while ceramics triggered newly woven bone formation in close contact with the ceramic surface that remained unre-sorbed. Delivery of mesenchymal stem cells in combination with these biomaterials enhanced both bone healing (>20%) and neovascularization after 1 month, mainly in hydrogel. Osteogenic and angiogenic properties combined with rapid resorption make hydrogels a promising alternative to ceramics for bone repair by cell therapy

Heterozygous and homozygous JAK2(V617F) states modeled by induced pluripotent stem cells from myeloproliferative neoplasm patients.

JAK2(V617F) is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2(V617F), respectively. In the patient with homozygous JAK2(V617F), additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA(+)CD41(+) cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy

JAK2^V617F induces an increased sensitivity to EPO and TPO

<p>(A) GPA⁺CD41⁺ cells from P1(H), P2(h) or control were plated in methylcellulose in the presence of SCF (25 ng/mL) and increasing concentrations of EPO. EryP colonies were counted 12 days later. (B) The percentage of large EryP (>50 cells per colony) in total EryP was also calculated. Results are the mean ± SEM of 3 independent experiments. (C) Cloning efficiency of GPA⁺CD41⁺ cells for each genotype was calculated (D) GPA⁺ cells were cytokine-deprived overnight in serum-free medium and then seeded in IMDM alone for 4 hours. Cells were then stimulated with 10 U/mL EPO or not. (E) CD41⁺ cells from P1(H), P2(h) or control were plated in plasma clots without or with increasing concentrations of TPO. CFU-MK colonies were counted at day 10 after CD41a indirect staining. Results are the mean ± SEM of 3 independent experiments (*P<0.05). (F) Primary cells from one control and 4 patients (P1(H), P2(h), P3(H) and P4(h)) were grown either with SCF (25 ng/mL) ±EPO (1 U/mL) or with SCF (25 ng/mL) ± TPO (20 ng/mL), and cloned at one progenitor cell/well. The percentage of endogenous erythroid colonies (EEC) or endogenous CFU-MK was calculated for each condition.</p

Impact of different inhibitors on erythroid growth

<p>GPA⁺CD41⁺ cells were plated in methylcellulose with various inhibitors, in the presence of SCF (25 ng/mL), without EPO for P1(H), and with EPO (1 U/mL) for P1(H), P2(h) and control. EryP colonies were counted 12 days later. (A) Ruxolitinib ; (B) TG101348 ; (C) LY294002 ; (D) RAD001 ; (E) AUY922. Results are the mean ± SEM of 3 independent experiments (*P<0.05).</p