Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

in Cytotherapy (in press)

Background aims. Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous ... [more ▼]

Background aims. Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects. Methods. The ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rg(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed. Results. Injection of 200 106 human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM1 antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 106 MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 106 human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 106 MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Conclusions. Injection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models. [less ▲]

Imatinib and Nilotinib Inhibit Hematopoietic Progenitor Cell Growth, but Do Not Prevent Adhesion, Migration and Engraftment of Human Cord Blood CD34+ Cells

in PLoS ONE (2012), 7(12), 52564

Background: The availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to ... [more ▼]

Background: The availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34+ cells. Design and Methods: After a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rc (null) mice. <br />Results: TKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34+ cell cycle entry. Adhesion of CD34+ cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1a gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT. <br />Conclusions: These data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe. [less ▲]

Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID -repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages

in Haematologica (2010), 95(1), 47-56

Background Bone marrow (BM) mesenchymal stem cells (MSC) support proliferation and differentiation of hematopoietic progenitor cells (HPC) in vitro. Since they represent a rare subset of BM cells, MSC ... [more ▼]

Background Bone marrow (BM) mesenchymal stem cells (MSC) support proliferation and differentiation of hematopoietic progenitor cells (HPC) in vitro. Since they represent a rare subset of BM cells, MSC preparations for clinical purposes involves a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on MSC supportive activity. DESIGN AND METHODS: MSC were expanded for up to 10 passages. MSC and CD34(+) cells were seeded in cytokine-free co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed. RESULTS: Early passage MSC supported HPC expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage MSC did not support HPC and myeloid cell outgrowth but maintained B cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for one week in contact with MSC was effective until the fourth MSC passage and declined afterwards. CD34(+) cells achieved higher levels of engraftment in NOD/SCID mice when co-injected with early passage MSC; however MSC expanded beyond 9 passages were ineffective in promoting CD34(+) cell engraftment. Non-contact cultures indicated that MSC supportive activity involved diffusible factors. Among these, interleukin (IL)-6 and IL-8 contributed to the supportive activity of early passage MSC but not of late passage MSC. MSC phenotype as well as fat, bone and cartilage differentiation capacity did not change during MSC culture. Conclusions Extended MSC culture alters their supportive ability toward HPC without concomitant changes in phenotype and differentiation capacity. [less ▲]

Etude des propriétés hémato-supportives in vitro des cellules souches mésenchymateuses

Doctoral thesis (2009)

Bone marrow (BM) mesenchymal stem cells (MSC) support proliferation and differentiation of hematopoietic progenitor cells (HPC) in vitro. Since they represent a rare subset of BM cells, MSC preparations ... [more ▼]

Bone marrow (BM) mesenchymal stem cells (MSC) support proliferation and differentiation of hematopoietic progenitor cells (HPC) in vitro. Since they represent a rare subset of BM cells, MSC preparations for clinical purposes involves a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on MSC supportive activity. MSC were expanded for up to 10 passages. MSC and CD34+ cells were seeded in cytokinefree co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed. Early passage MSC supported HPC expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage MSC did not support HPC and myeloid cell outgrowth but maintained B cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for one week in contact with MSC was effective until the fourth MSC passage and declined afterwards. CD34+ cells achieved higher levels of engraftment in NOD/SCID mice when co-injected with early passage MSC; however MSC expanded beyond 9 passages were ineffective in promoting CD34+ cell engraftment. Non-contact cultures indicated that MSC supportive activity involved diffusible factors. Among these, interleukin (IL)-6 and IL-8 contributed to the supportive activity of early passage MSC but not of late passage MSC. MSC phenotype as well as fat, bone and cartilage differentiation capacity did not change during MSC culture. Extended MSC culture alters their supportive ability toward HPC without concomitant changes in phenotype and differentiation capacity. [less ▲]

Human bone marrow adipocytes block granulopoiesis through neuropilin-1-induced granulocyte colony-stimulating factor inhibition.

in Stem Cells (2008), 26(6), 1556-64

Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral ... [more ▼]

Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34(+) differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34(+) cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 beta or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. Disclosure of potential conflicts of interest is found at the end of this article. [less ▲]

The hematopoiesis-supporting activity of mesenchymal stem cells increases with the number of passages

in Experimental Hematology (2007, September), 35(9, Suppl. 2), 125-125

Definition of quality standards applicable to the purification and the expansion of mesenchymal stem cells for their use in haematopoietic, immunosuppressive and regenerative cellular therapy

Master of advanced studies dissertation (2005)

Mesenchymal stem cells (MSCs) reside within the stromal compartment of bone marrow and represent a plastic cell population for which pro-haematopoietic, immunosuppressive and regenerative properties are ... [more ▼]

Mesenchymal stem cells (MSCs) reside within the stromal compartment of bone marrow and represent a plastic cell population for which pro-haematopoietic, immunosuppressive and regenerative properties are poorly characterized. A characterization of MSC preparations was carried out with each passage in MSCGM or α-MEM + 20% FBS expansion media. We obtained 3 times more cells after 5 passages in MSCGM than in α-MEM + 20% FBS. We analysed the phenotype of MSCs after each passage by flow cytometry. At all passages and in both media cells were tested negative for CD45, strongly positive for CD73 and CD90 antigens and weakly positive for CD105 and CD106 antigens. We wanted to assess the MSC ability to differentiate into adipocytes, osteoblasts and chondroblasts. At all tested passages and in both media, MSCs differentiated in these three tissues when they were placed in the adequate induction medium. We used RayBio® Human Cytokine Antibody Array to analyse the presence of several cytokines in MSC-conditioned medium. At all passages and in both media, IL-6, IL-8 and VCAM-1 were more strongly expressed than the other cytokines. We used the CFU-F assay to evaluate the frequency and purity of MSCs in culture. We observed that CFU-F number reached a maximum at passages 3 and 4 after expansion in both MSCGM and in α-MEM + 20% FBS. We also observed a greater proportion of CFU-F for the cells expanded in MSCGM compared to the cells expanded in α-MEM + 20% FBS. To assess the haematopoietic supporting ability of MSCs, long term cultures were performed. We observed more colony-forming cells when long-term cultures were done with 1st and 2nd passages MSCs than when cultures were done with 3rd, 4th and 5th passages MSCs. In NOD/SCID mice we transplanted CD34+ uncultured cells or the expansion product of CD34+ cells co-cultured for one week with MSCs. Human chimerism was present in all conditions. We observed a greater human chimerism when CD34+ cells were co-cultured with MSCs from passage 4 than in the other conditions. In all conditions simultaneous expression of CD19 or CD33 antigens on human CD45+ cells demonstrated the presence of repopulating cells with lympho-myeloid potential. Despite the fact that MSCs had the same phenotype, differentiation potential and cytokine secretion profile at each passage and in both media, MSCs did have neither the same capacity to form CFU-F nor the same capacity to support haematopoiesis in vitro and in vivo. [less ▲]