Upregulating CXCR4 in human fetal mesenchymal stem cells enhances engraftment and bone mechanics in a mouse model of osteogenesis imperfecta.

Stem cells have considerable potential to repair damaged organs and tissues. We previously showed that prenatal transplantation of human first trimester fetal blood mesenchymal stem cells (hfMSCs) in a mouse model of osteogenesis imperfecta (oim mice) led to a phenotypic improvement, with a marked decrease in fracture rate. Donor cells differentiated into mature osteoblasts, producing bone proteins and minerals, including collagen type Iα2, which is absent in nontransplanted mice. This led to modifications of the bone matrix and subsequent decrease of bone brittleness, indicating that grafted cells directly contribute to improvement of bone mechanical properties. Nevertheless, the therapeutic effect was incomplete, attributing to the limited level of engraftment in bone. In this study, we show that although migration of hfMSCs to bone and bone marrow is CXCR4-SDF1 (SDF1 is stromal-derived factor) dependent, only a small number of cells present CXCR4 on the cell surface despite high levels of internal CXCR4. Priming with SDF1, however, upregulates CXCR4 to increase the CXCR4(+) cell fraction, improving chemotaxis in vitro and enhancing engraftment in vivo at least threefold in both oim and wild-type bone and bone marrow. Higher engraftment in oim bones was associated with decreased bone brittleness. This strategy represents a step to improve the therapeutic benefits of fetal cell therapy toward being curative.

IFN-γ directly inhibits TNF-α-induced osteoclastogenesis in vitro and in vivo and induces apoptosis mediated by Fas/Fas ligand interactions.

Cytokines secreted by T cells play a pivotal role in inflammatory bone destruction. Tumor necrosis factor-α (TNF-α) is a major proinflammatory cytokine produced by macrophages following T cell activation, and directly promotes osteoclast differentiation resulting in accelerated bone resorption. Interferon-γ (IFN-γ) attenuates RANKL-initiated cellular signals through osteoclast formation and counterbalances aberrant bone resorption. With respect to this crosstalk during osteoclastogenesis, the direct interruption of IFN-γ in TNF-α-induced osteoclast formation still requires elucidation. We have demonstrated that IFN-γ directly inhibits osteoclastogenesis induced by TNF-α stimulation and accelerates apoptosis mediated by Fas/Fas ligand signals. There were a decreased number of osteoclasts and reduced mRNA levels encoding Nfatc1 in cultured bone marrow macrophages. Apoptotic responses of cultured cells were observed, with accelerated nuclear fragmentation in osteoclast precursor cells and increased FasL mRNA levels in bone marrow cells stimulated with TNF-α evident. IFN-γ reduced the level of osteoclastogenesis in response to TNF-α treatment in vivo. IFN-γ inhibited TNF-α-induced osteoclastogenesis in mice with T cells that had been exposed to anti-CD4 and -CD8 antibodies. These results provide evidence that IFN-γ directly inhibits osteoclastogenesis and induces cells apoptosis by Fas/FasL signals, leading to the indirect regulation of bone resorption, which is required for protective roles in bone destruction at an inflammation site.

Amelioration of a mouse model of osteogenesis imperfecta with hematopoietic stem cell transplantation: microcomputed tomography studies.

OBJECTIVE: To test the hypothesis that hematopoietic stem cells (HSCs) generate bone cells using bone marrow (BM) cell transplantation in a mouse model of osteogenesis imperfecta (OI). OI is a genetic disorder resulting from abnormal amount and/or structure of type I collagen and is characterized by osteopenia, fragile bones, and skeletal deformities. Homozygous OI murine mice (oim; B6C3Fe a/a-Col1a2(oim)/J) offer excellent recipients for transplantation of normal HSCs, because fast turnover of osteoprogenitors has been shown. MATERIALS AND METHODS: We transplanted BM mononuclear cells or 50 BM cells highly enriched for HSCs from transgenic enhanced green fluorescent protein mice into irradiated oim mice and analyzed changes in bone parameters using longitudinal microcomputed tomography. RESULTS: Dramatic improvements were observed in three-dimensional microcomputed tomography images of these bones 3 to 6 months post-transplantation when the mice showed high levels of hematopoietic engraftment. Histomorphometric assessment of the bone parameters, such as trabecular structure and cortical width, supported observations from three-dimensional images. There was an increase in bone volume, trabecular number, and trabecular thickness with a concomitant decrease in trabecular spacing. Analysis of a nonengrafted mouse or a mouse that was transplanted with BM cells from oim mice showed continued deterioration in the bone parameters. The engrafted mice gained weight and became less prone to spontaneous fractures while the control mice worsened clinically and eventually developed kyphosis. CONCLUSIONS: These findings strongly support the concept that HSCs generate bone cells. Furthermore, they are consistent with observations from clinical transplantation studies and suggest therapeutic potentials of HSCs in OI.

Potential role of mesenchymal stromal cells in pediatric hematopoietic SCT.

Mesenchymal stromal cells (MSCs) can be isolated from several human tissues and expanded for clinical use. MSCs are identified by phenotypic and functional characteristics, and are poor Ag-presenting cells not expressing MHC class II or co-stimulatory molecules. MSCs have potent immune-modulatory effects and in vitro induce a more anti-inflammatory or tolerant phenotype. Clinical studies have exploited both the immune-modulatory properties of MSCs as well as their hematopoietic supportive role. MSCs have been safely administered for the treatment of severe steroid refractory GVHD. A phase I/II multicenter study included 25 children in whom 80% responded to either one or two infusions of MSCs derived mainly from third party donors. Twenty children have undergone co-transplantation of haploidentical MSCs with PBSC in a phase I/II study, which has overcome the problems of graft failure in HLA-disparate grafts. Similarly, co-transplantation of MSCs and cord blood stem cells is under investigation. MSCs may have important future potential for the treatment of pediatric autoimmune disease as well as inborn errors such as osteogenesis imperfecta. Currently, much needed randomized studies under the auspices of the EBMT are ongoing to determine the optimal use of these exciting new modalities of treatment.

Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone.

Treatment with isolated allogeneic mesenchymal cells has the potential to enhance the therapeutic effects of conventional bone marrow transplantation in patients with genetic disorders affecting mesenchymal tissues, including bone, cartilage, and muscle. To demonstrate the feasibility of mesenchymal cell therapy and to gain insight into the transplant biology of these cells, we used gene-marked, donor marrow-derived mesenchymal cells to treat six children who had undergone standard bone marrow transplantation for severe osteogenesis imperfecta. Each child received two infusions of the allogeneic cells. Five of six patients showed engraftment in one or more sites, including bone, skin, and marrow stroma, and had an acceleration of growth velocity during the first 6 mo postinfusion. This improvement ranged from 60% to 94% (median, 70%) of the predicted median values for age- and sex-matched unaffected children, compared with 0% to 40% (median, 20%) over the 6 mo immediately preceding the infusions. There was no clinically significant toxicity except for an urticarial rash in one patient just after the second infusion. Failure to detect engraftment of cells expressing the neomycin phosphotransferase marker gene suggested the potential for immune attack against therapeutic cells expressing a foreign protein. Thus, allogeneic mesenchymal cells offer feasible posttransplantation therapy for osteogenesis imperfecta and likely other disorders originating in mesenchymal precursors.