Changes in the expression of adhesion proteins involved in myoblast differentiation were investigated in monolayer (two-dimensional) and 3D (three-dimensional) cell cultures. The expression of integrin alpha3 subunit, integrin beta1 subunit, ADAM12 (a disintegrin and metalloproteinase 12), tetraspanins CD9 and CD81 and M-cadherin were examined in the murine myoblast cell line C2C12 and in a primary culture of rat satellite cells. Myoblasts in monolayer and 3D cultures showed significant differences in their morphology and cytoskeletal organization. All of the studied proteins participated in myoblast fusion in each culture examined, but differences in their levels of expression were observed. Satellite cell-derived myoblasts exhibited higher expression of adhesion protein mRNAs than C2C12 cells. Also, C2C12 cells from a 3D culture showed slightly higher expression of adhesion protein transcripts than the same cells cultured as a monolayer. Significantly, the levels of adhesion protein mRNAs were found to change in parallel in all cell culture types. Despite this finding, it is important that differences between satellite cell-derived myoblasts and cell line C2C12 grown in monolayer and 3D cultures are taken into account when studying processes of myoblast differentiation in vitro.

Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro.

No abstract

Introduction Mesenchymal stem cells (MSC) are now in the limelight of stem cell researchers. The growing number of preclinical studies gives feedback for using MSC in the various fields of medicine. Their immunomodulatory function gives them a scientific rationale to be used in Graft-versus-Host Disease (GvHD) treatment. The MSC can be isolated from bone marrow (BM), adipose tissue, cord blood (CB) or umbilical cord (UC). However, BM harvest puts a donor at risk of procedure complication. In the contrary, Wharton Jelly (WJ) - derived MSC can be collected safely, easily and they are reach in MSC, which makes them more preferable source than CB. We would like to present the first in Europe international cooperation that led to the clinical application of WJ-derived MSC in patients with GvHD. In the present publication we describe the results of collection, transport, culture, investigation, cryopreservation and the first examples of clinical usage of MSC derived from more than 500 UC collected by our group of stem cells banks (www.famicord.eu). Methods WJ-derived MSC were obtained from third party unrelated donors after natural deliveries as well as caesarian sections. They were collected to the sterile vessel containing 0,9% natrium chloratum and 1% antibiotic and transported in the temperature of 18-24°C. After an isolation by mechanical dissection of cord’s blood vessels, they underwent culture in the 37°C in the atmosphere of 5% CO2 in the air with human MSC growth medium as well as supplement containing fetal bovine serum and antibiotic. They were enumerated and their viability was evaluated. Then the cells were cryopreserved in the presence of DMSO and placed in the vapour phase of liquid nitrogen in <150°C. The repeated cell counting, viability test, flow cytometric immunophenotyping, and functional in vitro differentiation assays were performed from the thawed reference samples. Results Low contamination level (less than 2%) of the UC tissue collected after both natural deliveries and caesarian section was reported. We have not noticed any differences in growth, cell number and morphology in the primary cultures of tissue fragments from placental, central and baby side of the cord. The first adherent cells with fibroblast-like morphology were well-distinguishable within a week after the initiation of the cell culture. The immunophenotype remained stable (CD45-/CD34-/CD19-/CD14-/HLA-DR-/CD73+/CD90+/CD105+) during the whole period of culture (with extreme limit of 15 passages). MSC were capable of differentiation into adipogenic, chondrogenic and osteogenic cells. The WJ-derived MSC have been applied to the ten patients with steroid-refractory GvHD always after approval of bioethical committee. Three patients were diagnosed with chronic form and 7 with acute one. Five children had multiple infusions, up to 4 doses with 1-2 week intervals. No adverse effects were described during infusions apart from low grade fever in 1 adult patient. Conclusion The results described above demonstrate a repeatable method to obtain an adequate number of cells for the clinical use. The international cooperation between Polish, Hungarian, Romanian and Spanish stem cell banks, enabled us to use WJ-derived MSC in the setting of GvHD. No serious adverse effects were described. Third party donor WJ-derived MSCs are safe and effective treatment of GvHD, however further studies are needed. Disclosures: No relevant conflicts of interest to declare.