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.
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ABSTRACT: Mesenchymal stem cells (MSCs) ameliorate injury and accelerate repair in many organs, including the kidney, although the reparative mechanisms and interaction with macrophages have not been elucidated. This study investigated the reparative potential of human bone marrow-derived MSCs and traced their homing patterns following administration to mice with ischemia/reperfusion (IR) injury using whole body bioluminescence imaging. The effect of MSCs on macrophage phenotype following direct and indirect co-culture was assessed using qPCR. Human cytokine production was measured using multiplex arrays. After IR, MSCs homed to injured kidneys where they afforded protection indicated by decreased proximal tubule kidney injury molecule-1 expression, blood urea nitrogen and serum creatinine levels. SDS-PAGE and immunofluorescence labeling revealed MSCs reduced collagen α1(I) and IV by day 7 post-IR. Gelatin zymography confirmed that MSC treatment significantly increased matrix metalloproteinase-9 activity in IR kidneys, which contributed to a reduction in total collagen. Following direct and indirect co-culture, macrophages expressed genes indicative of an anti-inflammatory 'M2' phenotype. MSC-derived human GM-CSF, EGF, CXCL1, IL-6, IL-8, MCP-1, PDGF-AA and CCL5 were identified in culture supernatants. In conclusion, MSCs home to injured kidneys and promote repair, which may be mediated by their ability to promote M2 macrophage polarization.AJP Renal Physiology 03/2014; · 4.42 Impact Factor
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ABSTRACT: Introduction: Bidirectional interactions between cells and fluidic surroundings regulate cellular functions and maintain tissue or organ architecture. Accordingly, the synovial fluid is the primary source of environmental signals and determines to a great extent the molecular interactions within the joint capsule, both in homeostasis and pathology. Areas covered: We provided an update on hyaluronic acid (HA) and platelet-rich plasma (PRP) concepts necessary to build the rationale for creating a combined treatment. The information is based on a PubMed search using the terms 'platelet-rich plasma', 'hyaluronic acid', 'knee pathology', 'knee osteoarthritis' (OA). Expert opinion: In OA, a deleterious fluidic microenvironment is established, with presence of HA fragments, catabolic enzymes and inflammatory molecules. The central concept underlying intra-articular injection is to modify deleterious fluidic microenvironments. PRP administration has shown pain remission and function improvement, but less than half of the patients showed clinically significant improvement. PRP exceeds HA, the comparator used in PRP clinical trials, albeit both HA and PRP alleviate symptoms in mild-to-moderate OA patients. Combining PRP and HA may benefit from their dissimilar biological mechanisms and help in controlling delivery and presentation of signaling molecules. Three armed randomized studies, using both HA and PRP as comparators, will provide information about the impact of this approach.Expert opinion on biological therapy 02/2014; · 3.22 Impact Factor
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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; · 1.32 Impact Factor