Efficacy and safety of human mesenchymal stromal cells in healing of critical-size bone defects in immunodeficient rats.

To evaluate the preclinical efficacy and safety of human mesenchymal stem cells (hMSC) rapidly expanded in growth medium for clinical use with human serum and recombinant growth factors, we conducted a controlled, randomized trial of plasma clots with hMSC vs. plasma clots only in critical segmental femoral defects in rnu/rnu immunodeficient rats. X-ray, microCT and histomorphometrical evaluation were performed at 8 and 16 weeks. MSC were obtained from healthy volunteers and patients with lymphoid malignancy. Human MSC survived in the defect for the entire duration of the trial. MSC from healthy volunteers, in contrast to hMSC from cancer patients, significantly improved bone healing at 8, but not 16 weeks. However, at 16 weeks, hMSC significantly improved vasculogenesis in residual defect. We conclude that hMSC from healthy donors significantly contributed to the healing of bone defects at 8 weeks and to the vascularisation of residual connective tissue for up to 16 weeks. We found the administration of hMSC to be safe, as no adverse reaction to human cells at the site of implantation and no evidence of migration of hMSC to distant organs was detected.

Xenogeneic protein-free cultivation of mesenchymal stromal cells - towards clinical applications.

We have studied a rapid cultivation method for human mesenchymal stromal cells based on CellGroTM medium and human serum, supplemented with insulin, ascorbic acid, dexamethasone, epidermal growth factor, platelet-derived growth factor BB, macrophage colony-stimulating factor and fibroblast growth factor 2. This study has shown that rapid expansion of human multipotent mesenchymal stromal cells using human serum could not be achieved without addition of growth factors. Furthermore, we have found that insulin and, quite probably, epidermal growth factor may be omitted from our formula without loss of colony-forming capacity or total cell yield. On the other hand, dexamethasone, ascorbic acid and fibroblast growth factor 2 were necessary for the growth and colony-forming capacity of multipotent mesenchymal stromal cells, while platelet-derived growth factor BB prevented their differentiation into adipogenic lineage. Moreover, multipotent mesenchymal stromal cells cultivated in our system expressed higher levels of bone morphogenetic protein 2, but not bone morphogenetic protein 7, than cells cultivated in α-MEM with foetal bovine serum. This shows that our system promotes differentiation of mesenchymal cells towards osteogenic and chondrogenic lineages, making them more suitable for bone and cartilage engineering than cells grown in conventional media. Furthermore, we have proved that these cells may be conveniently cultivated in a closed system, in vessels certified for clinical use (RoboFlaskTM), making the transfer of our cultivation technology to good clinical practice easier and more convenient.

[Xenogeneic protein free cultivation of mesenchymal multipotent stromal cells]. / Kultivace mezenchymových kmenových bunek bez xenogenních bílkovin.

PURPOSE OF THE STUDY: The aim of this study was to compare the standard laboratory method of cultivation of mesenchymal multipotent stromal cells (MSC) and a novel technique of rapid MSC expansion focused on simple clinical use. MATERIAL AND METHODS: Bone marrow mononuclear cells of donors were cultured for 14 days by the standard and the new cultivation method. The standard method (STD) was based on an alpha MEM medium supplemented with foetal calf serum (FCS). The new animal protein-free method (CLI) was based on the clinical grade medium CellgroTM, pooled human serum and human recombinant growth factors (EGF, PDGF-BB, M-CSF, FGF-2) supplemented with dexamethasone, insulin and ascorbic acid. The cell product was analyzed by flow cytometry. Furthermore, the cell products of STD and CLI methods were differentiated in vitro, and histochemical and immunohistochemical analyses, electron microscopy and elemental analysis were performed. Some cells were seeded on biodegradable scaffolds, in vivo implanted into immunodeficient mice for 6 weeks and evaluated by histological methods. RESULTS: Yields of the CLI method after 14 days of cultivation were 40-fold higher than those obtained by the STD technique (p<0.05). Cell products of both STD and CLI methods fulfilled the criteria of MSC in terms of antigen expression assessed by flow cytometry, as well as osteogenic, chondrogenic and adipogenic in vitro differentiation assays. Moreover, these cells seeded on three-dimensional scaffolds cultured in osteogenic medium produced mineral deposits and a fibrillar extracellular matrix seen with the electron microscope. Deposits examined by element analysis contained calcium and phosphorus at a ratio of 5 to 3, which corresponded to hydroxyapatite. The cell product seeded on biodegradable scaffolds and implanted into immunodeficient mice was able to form a bone-like calcified tissue with blood supply of mouse origin. DISCUSSION: The currently used methods of cultivation have certain disadvantages compared to the CLI technique, such as a longer cultivation period, need of primary expansion and reseeding and use of FCS with all its potential risks. High yields of cells obtained by the CLI method in a very short time make the use of cultured cells potentially suitable for an acute trauma management. Other therapeutic non-orthotopic applications of CLI-cultured cells have to be further investigated. CONCLUSIONS: The CLI method is unique, rapid, simple and lacking the addition of animal proteins. CLI-cultured cells fulfil the criteria of MSC. The CLI method potentially allows for closed system cultivation in good manufacturing practice (GMP) conditions. It seems to be easily transferable to good clinical practice compared to other protocols and should extend the possibilities of cell therapy and tissue engineering of cartilage and bone. The new method is protected by Czech patent 301 148 and by Europian patent EP 1999250 according to Czech and international laws.

Circulating endothelial precursor cells (EPC) in patients undergoing allogeneic haematopoietic progenitor cell transplantation.

We have studied the number of endothelial precursor cells in eighteen patients undergoing allogeneic haematopoietic stem cell transplantation. Endothelial precursor cells were evaluated by colony-forming assay and compared to healthy controls. Patients undergoing allogeneic haematopoietic stem cell transplantation had significantly lower numbers of endothelial precursor cells before the procedure than healthy controls. The numbers of endothelial precursor cells were even lower in the first year after the treatment and seemed to recover partially after twelve months, but even then, they were lower than in healthy volunteers. On the other hand, the number of circulating CD146+CD31+ mature endothelial cells were higher than in healthy controls after more than a one-year follow-up. We hypothesize that lower numbers of endothelial precursor cells and higher numbers of endothelial cells in patients undergoing allogeneic haematopoietic stem cell transplantation reflect ongoing endothelial damage, probably caused by immunological mechanisms, and that this longterm damage may explain the higher risk of cardiovascular events in allogeneic haematopoietic stem cell transplant survivors.