Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine.
ABSTRACT Human bone marrow mesenchymal stromal cells (hMSC) are currently investigated for a variety of therapeutic applications. However, most expansion protocols still use fetal calf serum (FCS) as growth factor supplement which is a potential source of undesired xenogeneic pathogens. We established an expansion protocol for hMSC based on the use of GMP-produced basic medium LP02 supplemented with 5% of platelet lysate (PL) obtained from human thrombocyte concentrates. Compared to FCS-supplemented culture conditions, we found a significant increase in both colony forming unit-fibroblast (CFU-F) as well as cumulative cell numbers after expansion. This accelerated growth is optimized by pooling of at least 10 thrombocyte concentrates. A minimal requirement is the use of 5% of PL with an optimal platelet concentration of 1.5 x 10(9)/ml, and centrifugation of thawed lysate at high speed. Cells expanded by this protocol meet all criteria for mesenchymal stromal cells (MSCs), e.g. plastic adherence, spindle-shaped morphology, surface marker expression, lack of hematopoietic markers, and differentiation capability into three mesenchymal lineages. MSC at passage 6 were cytogenetically normal and retained their immune-privileged potential by suppressing allogeneic reaction of T-cells. Additionally, gene expression profiles show increased mRNA levels of genes involved in cell cycle and DNA replication and downregulation of developmental and differentiation genes, supporting the observation of increased MSC-expansion in PL-supplemented medium. In summary, we have established a GMP-compatible protocol for safe and accelerated expansion of hMSC to be used in cell and tissue therapy.
- SourceAvailable from: Tomoyuki Kawase[Show abstract] [Hide abstract]
ABSTRACT: For successful cell transplantation therapy, the quality of cells must be strictly controlled. Unfortunately, to exclude inappropriate cells that possess structurally abnormal chromosomes, currently only karyotyping functions as an assessment. Unfortunately, this methodology is time-consuming and only effective for metaphasic cells. To develop a more efficient, inclusive and sensitive methodology, we examined the phosphorylation of histone H2AX and the p53 levels in normal human periosteal cells exposed to x-rays or other oxidative stressors.Cytotherapy 10/2014; · 3.06 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Current cell-based cartilage therapies relay on articular cartilage-derived autologous chondrocytes as a cell source, which possesses disadvantages, such as, donor site morbidity and dedifferentiation of chondrocytes during in vitro expansion. Due to these and other limitations, novel cell sources and production strategies are needed. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a fascinating alternative, but they are not spontaneously capable of producing hyaline cartilage-like repair tissue in vivo. In vitro pre-differentiation of BM-MSCs could be used to produce chondrocytes for clinical applications. However, clinically compatible defined and xeno-free differentiation protocol is lacking. Hence, this study aimed to develop such chondrogenic differentiation medium for human BM-MSCs. We assessed the feasibility of the medium using three human BM-MSCs donors and validated the method by comparing BM-MSCs to three other cell types holding potential for articular cartilage repair. The effectiveness of the method was compared to conventional serum-free and commercially available chondrogenic differentiation media. The results show that the defined xeno-free differentiation medium is at least as efficient as conventionally used serum-free chondrogenic medium and performed significantly better on all cell types tested compared to the commercially available chondrogenic medium.Cytotechnology 04/2014; · 1.45 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: We aim to identify current in vitro research exploring platelet-rich plasma (PRP) effects in human Mesenchymal Stem Cells (MSCs) that may encourage or limit the clinical application of MSCs along with PRP. After a systematic search, we identified 57 in vitro studies, focused on optimization of MSC manufacturing, and expanding knowledge about how PRP modifies MSCs behavior for translational purposes. Influences of PRP on proliferation, migration, stemness, preservation of MSC immune-modulatory properties and appearance of senescence phenotype have been explored. Overall PRP stimulates MSC proliferation, preserves MSCs multipotency and does not interfere with any lineage differentiation. PRP (as platelet lysate or releasate) preserves the immune-privileged potential of MSCs and may delay the appearance of the senescent phenotype. Currently there are few data linking precise molecules and biological mechanisms. Various gaps of knowledge need to be addressed in order to obtain enough useful information for translational purposes.Muscles, ligaments and tendons journal. 4(1):52-62.