Background Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity. Results After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G 1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation. Conclusion Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

Osteonecrosis is a frequent complication after treatment for childhood leukemia and other steroid-based therapies. The success rate of core decompression surgery is limited. Therefore, we evaluated relevant biological characteristics of human multipotent mesenchymal stromal cells (MSCs) in vitro. MSCs cultured under low-oxygen tensions showed decreased proliferation and differentiation into bone. However, these MSCs secreted significant amounts of vascular endothelial-derived factor in the presence of interferon-γ. These in vitro results with potential effects on neovascularization and bone regeneration as well as findings in animal models prompted us to treat five patients with steroid-induced osteonecrosis of the femur by core decompression surgery and instillation of expanded autologous MSCs. Within 3 weeks of culture, sufficient numbers of MSCs were generated using animal protein-free culture conditions. No chromosomal aberrations were detected by matrix-based comparative genomic hybridization. Application of MSCs during core decompression was feasible and safe. Median follow-up is 16 months and the patients in this pilot study reported clinical improvement. Formation of mineralized bone in the osteonecrotic cavity was proven by computed tomography. Taken together, MSCs display biological properties that may add to the efficiency of surgical treatment in osteonecrosis and should be evaluated in larger patient cohorts.

Multipotent mesenchymal stromal cells (MSC) have immunomodulatory effects. The aim of this study was to demonstrate safety and feasibility of MSC transfusion in pediatric patients who had undergone allogencic stem cell transplantation from MMFD, MUD, MMUD and MSD. Patients with posttransplant complications based on deregulated immune effector cells who may benefit from an immunomodulatory effect of MSC had been selected. MSC were isolated from the hematopoietic stem cell donors in five cases and from a third party parental donor in two cases. We transfused ex vivo-expanded MSC in 11 doses into seven pediatric patients. Cell doses were escalated based on availability from 0.4 x 10(6) to 3.0 x 10(6) per kg bodyweight No adverse effects were detected with a maximum follow-up of 29 months. One out of three patients showed slight improvement of chronic GVHD. Two patients with severe acute GvHD did not progress to cGvHD. One patient received MSC to stabilize graft function after secondary haploidentical transplantation. One patient recovered from trilineage failure due to severe hemophagocytosis. This is the first case of a pediatric patient treated with MSC for trilineage failure after haploidentical stem cell transplantation from her father. We report the first series of 11 transfusions of expanded MSC in pediatric patients with immunological complications after allogeneic transplantation. Transfusion of MSC was safe and encouraging improvements in some patients were observed. (C) 2007 Elsevier Inc. All rights reserved.

Osteonecrosis is a frequent complication after treatment for childhood leukemia.17% of adolescents above the age of 15 years treated in the ALL-BFM 95 trial protocol suffered from avascular necrosis of the bone (AVN). In addition, children receiving steroid-based treatment, e. g. autoimmune diseases, are frequently affected as well. If conservative measures fail, core decompression surgery is required. The success rate of surgery varies depending on the stage from 29% to 84% (Castro et al., 2000). Evidently, improved intervention strategies are warranted. Therefore, we evaluated the safety of implanting autologous MSC during surgical intervention in order to improve bone regeneration. AVN represent an area of absent blood perfusion and consequently low supplies of nutrients and oxygen. Therefore, we cultivated MSC under oxygen tensions of 3% or lower. These cells showed slowed proliferation and differentiation into bone. However, MSC grown under these low-oxygen conditions secreted significant amounts of vascular endothelial derived factor and selected insulin-like growth factors and their binding proteins. These factors might facilitate reperfusion and regeneration of bone at the site of implantation during core decompression surgery. 10 ml of heparinized bone marrow was drawn from the iliac crest of the patient. MSC were expanded under GMP conditions without the use of animal proteins employing platelet lysate and human fresh frozen plasma as source for growth factors (Müller et al., 2006). Recombinant trypsin was used for detachment and harvest of the cells. MSC could be expanded from 5 out of 5 juvenile bone marrow aspiates. Within three weeks of culture, sufficient numbers of cells, i. e. 112·106 ± 67·106, were generated in a certified GMP lab. At each of the three passages diagnostic cultures for aerobic bacteria were established and remained sterile throughout the culture period. MSC grown under these conditions were able to differentiate into osteoblasts. No chromosomal aberrations were detected by matrix-based comparative genomic hybridization using DNA microarrays containing >6,400 genomic DNA fragments. At the day of core decompression surgery, cells were harvested and resuspended to a final volume of 3 ml in saline. As core decompression implies drilling a hole into the necrotic area, the MSC were implanted through the same channel, which was sealed thereafter. Hence, instillation of the MSC did not require additional surgical procedures. Application of MSC was feasible and successful during core decompression surgery in all of the 5 patients. Follow up of the patients is between 14 and 8 months. None of the patients experienced adverse side effects, most importantly, there were no infectious complications. Clinically, no patient deteriorated and all patients reported less or no pain two months after surgery. Although the number of patients in this pilot trial is small, initial data for safety and feasibility of employing MSC in juvenile steroid-induced osteonecrosis is very promising.

BACKGROUNDMultipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements.METHODSAs PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation.RESULTSWe found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP).DISCUSSIONTaken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.