Donor Bone Marrow Stromal Cells Research Articles

The Potential Use of THP-1, a Monocytic Leukemia Cell Line, to Predict Immune-Suppressive Potency of Human Bone-Marrow Stromal Cells (BMSCs) In Vitro: A Pilot Study.

Adoptive transfer of cultured BMSCs was shown to be immune-suppressive in various inflammatory settings. Many factors play a role in the process, but no master regulator of BMSC-driven immunomodulation was identified. Consequently, an assay that might predict BMSC product efficacy is still unavailable. Below, we show that BMSC donor variability can be monitored by IL-10 production of monocytes/macrophages using THP-1 cells (immortalized monocytic leukemia cells) co-cultured with BMSCs. Using a mixed lymphocyte reaction (MLR) assay, we also compared the ability of the different donor BMSCs to suppress T-cell proliferation, another measure of their immune-suppressive ability. We found that the BMSCs from a donor that induced the most IL-10 production were also the most efficient in suppressing T-cell proliferation. Transcriptome studies showed that the most potent BMSC batch also had higher expression of several known key immunomodulatory molecules such as hepatocyte growth factor (HGF), PDL1, and numerous members of the PGE2 pathway, including PTGS1 and TLR4. Multiplex ELISA experiments revealed higher expression of HGF and IL6 by the most potent BMSC donor. Based on these findings, we propose that THP-1 cells may be used to assess BMSC immunosuppressive activity as a product characterization assay.

Engraftment of skeletal progenitor cells by bone-directed transplantation improves osteogenesis imperfecta murine bone phenotype.

Osteogenesis imperfecta (OI) is a genetic disorder most commonly caused by mutations associated with type I collagen, resulting in a defective collagen bone matrix. Current treatments for OI focus on pharmaceutical strategies to increase the amount of defective bone matrix, but do not address the underlying collagen defect. Introducing healthy donor stem cells that differentiate into osteoblasts producing normal collagen in OI patients has the potential to increase bone mass and correct the mutant collagen matrix. In this study, donor bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) expressing both αSMACreERT2/Ai9 progenitor reporter and osteoblast reporter Col2.3GFP were locally transplanted into the femur of OI murine (OIM) mice. One month post-transplantation, 18% of the endosteal surface was lined by donor Col2.3GFP expressing osteoblasts indicating robust engraftment. Long-term engraftment in the marrow was observed 3 and 6 months post-transplantation. The presence of Col1a2-expressing donor cell-derived cortical bone matrix was detected in transplanted OIM femurs. Local transplantation of BMSCs increased cortical thickness (+12%), the polar moment of inertia (+14%), bone strength (+30%), and stiffness (+30%) 3 months post-transplantation. Engrafted cells expressed progenitor markers CD51 and Sca-1 up to 3 months post-transplantation. Most importantly, 3 months post-transplantation donor cells maintained the ability to differentiate into Col2.3GFP+ osteoblasts in vitro, and in vivo following secondary transplantation into OIM animals. Locally transplanted BMSCs can improve cortical structure and strength, and persist as continued source of osteoblast progenitors in the OIM mouse for at least 6 months.

P62-ZZ Domain Signaling Inhibition Rescues MM-Induced Epigenetic Repression at the Runx2 promoter and Allows Osteoblast Differentiation of MM Patient Pre-Osteoblasts In Vitro

Multiple myeloma (MM) bone disease is characterized by non-healing lytic bone lesions with highly suppressed or absent osteoblast (OB) function. These lesions persist in the absence of active disease, significantly contributing to patient morbidity and mortality after patients achieve hematologic remission. We previously reported that MM cells induce epigenetic changes at the Runx2 promoter in bone marrow stromal cells (BMSC), which are preOB. We also demonstrated that Gfi1, a transcriptional repressor of the key OB differentiation factor, Runx2, is induced in BMSC by MM. Gfi1 is highly expressed in the MM bone marrow microenvironment and directly binds the Runx2 promoter, recruiting chromatin corepressors such as HDAC1 in preOB to induce epigenetic repression of Runx2 and prevent OB differentiation. Importantly, we recently showed that primary MM patient BMSC cultured in the absence of MM cells have decreased levels of the chromatin activation mark H3K9Ac at the Runx2 gene promoter when compared to healthy donor BMSC, suggesting that MM cells induce persistent epigenetic changes in patient BMSC.We reported that p62 (sequestosome-1) in BMSC is critical for the formation of MM-induced signaling complexes that mediate OB suppression and identified XRK3F2, an inhibitor of the p62 ZZ domain. XRK3F2 blunts MM cell-induced Runx2 suppression and Gfi1 induction in murine preOB in vitro, and induced new bone formation and remodeling in the presence of tumor in vivo. In addition, coculture experiments using human MM cells and murine preOB showed that XRK3F2 both prevents and reverses Gfi1 upregulation. Recently, we demonstrated that XRK3F2 prevents the epigenetic suppression of Runx2 in murine preOB cocultured (48h) with MM cells. Using ChIP-qPCR analysis we found that XRK3F2 prevented MM-induced Gfi1 occupancy at the Runx2 promoter, recruitment of the chromatin corepressor HDAC1, and histone de-acetylation.We now report that XRK3F2 restores OB suppression in persistently suppressed BMSC from MM patients. ChIP analysis of primary MM patient-derived BMSC cultured in the presence of XRK3F2 showed that XRK3F2 rescues H3K9ac levels at the Runx2 promoter. XRK3F2 did not alter H3K9ac levels in healthy donor BMSC or enhance OB differentiation. Importantly, XRK3F2 treatment of long-term primary MM patient BMSC cultures allowed osteogenic differentiation and mineralization, as evidenced by alizarin red staining. Further analysis of these cultures demonstrated that XRK3F2 treatment reduced Gfi1 protein expression.We conclude that XRK3F2 blocks MM-induced signaling in BMSC, resulting in decreased Gfi1 levels, thereby reducing recruitment of Gfi1 and HDAC1 to the Runx2 promoter, and reversing MM-induced epigenetic suppression of Runx2. These results suggest that targeting the p62 ZZ domain may reverse Gfi1 upregulation and rescue MM-induced epigenetic suppression of Runx2 in BMSC, allowing restoration of OB function in patients with MM bone disease. DisclosuresRoodman:Amgen: Consultancy.

Local transplantation is an effective method for cell delivery in the osteogenesis imperfecta murine model

Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model. Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after four weeks. We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells. We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.

Modulation of Host Osseointegration during Bone Regeneration by Controlling Exogenous Stem Cells Differentiation Using a Material Approach.

Stem cell-based tissue engineering for large bone defect healing has attracted enormous attention in regenerative medicine. However, sufficient osseointegration of the grafts combined with exogenous stem cells still remains a major challenge. Here we developed a material approach to modulate the integration of the grafts to the host tissue when exogenous bone marrow stromal cells (BMSCs) were used as donor cells. Distinctive osseointegration of bone grafts was observed as we varied the content of hydroxyapatite (HA) in the tissue scaffolds implanted in a mouse femur model. More than 80% of new bone was formed in the first two weeks of implantation in high HA content scaffold but lack of host integration while only less than 5% of the new bone was formed during this time period in the no HA group but with much stronger host integration. Cell origin analysis leveraging GFP reporter indicates new bone in HA containing groups was mainly derived from donor BMSCs. In comparison, both host and donor cells were found on new bone surface in the no HA groups which led to seamless bridging between host tissue and the scaffold. Most importantly, host integration during bone formation is closely dictated to the content of HA present in the scaffolds. Taken together, we demonstrate a material approach to modulate the osseointegration of bone grafts in the context of exogenous stem cell-based bone healing strategy which might lead to fully functional bone tissue regeneration.

Cardiac Injury Protection from Mouse Bone Marrow Stromal Cells with In Utero Transplantation Followed by Secondary Postnatal Boost

Limited donor-cell engraftment to the injured tissue restricts therapeutic efficacy of stem cell transplantation. Herein, we proposed an alternative strategy by using in utero transplantation (IUT) to create mixed-chimerism environment in recipients and to facilitate donor-cell engraftment followed by postnatal secondary boost with the same cells. Mouse bone marrow stromal cells (BMSCs) were used as the xenogenic donor cells and given into rat fetus as an early exposure of IUT treatment. The engraftment potential was analyzed for the presence of BMSCs by flow cytometry or PCR in recipient tissues. The function of a second boost of mouse BMSCs, in terms of cardioprotection, was tested by given 1×10⁶ cells to rat IUT hearts with ischemia/reperfusion (IR) injury that was induced by a 45 min of left coronary ligation and released for 72 h. Mouse BMSCs demonstrated an immunosuppressive effect when mixed with mouse or rat lymphocytes. IUT treatment only caused few BMSCs engrafted to fetal (embryonic day 20) and adult (4 weeks after birth) rat organs including heart, but engraftment was increased in hearts of the IUT rats after second boost. This was coincided with attenuation of cardiac injury caused by IR. Interestingly, an up-regulation of CXC chemokine receptor type 4 (CXCR4) was seen when BMSCs were exposed to hypoxia. This indicates that enhanced engraftment of mouse BMSCs to post-ischemic rat hearts possibly is dependent on CXCR4. Moreover, results of flow cytometry demonstrated that the presence of CD34⁺ cells in rat IUT hearts with IR injury was increased. These observations suggest that enhanced engraftment of donor BMSCs to rat IR hearts by CXCR4 may recruit endogenous CD34⁺ cells of recipients which in turn protects heart against IR. This also supports the notion of fetal preconditioning with BMSC enhances the efficiency of progenitor cell-mediated organ protection after a postnatal second boost in xeno-transplantation.

Experimental study on osteogenic differentiation of allogeneic bone marrow stromal cells in muscles of rabbits

Objective To study and evaluate the ability of allogeneic bone marrow stromal cells(BMSCs) to survive and regenerate bone in muscles without using immunosuppressive agents. Methods A complete mismatch between donor BMSCs and recipient rabbit was confirmed by one way mixed lymphocyte reaction assaysprior to implantation. And then bone marrow aspirates were obtained from donor rabbits.BMSCs were isolated from the bone marrow, cultured in conditional medium to be ubdyced to become osteogenic, and then to examine these characteristics. After that the donor BMSCs were transplanted into the recipient rabbits. Immunological tests such as lymphocyte transformation rate and cell mediated cytotoxity, histological observation, seeding cells survival and bone formation were performed following transplantation. Results Allogeneic BMSCs transplantation did not actually elicit an adverse immune response, and bone regenerated at the transplantation area, and then the transplanted BMSCs marked with BRDU preoperatively were founded to be living 8 weeks later after transplantation using immunohistochemistric technique. Conclusion Allogeneic BMSCs would not elicit an adverse immune response in vivo without the immunosuppressive therapy, which could survive and form new bone tissue with the help of BMP-2 in the muscles. Key words: Bone marrow stromal cells; Allogeneic; Bone; Tissue engineering; Transplantation

Bone Marrow Stromal Cells Promote Neurite Extension in Organotypic Spinal Cord Slice: Significance for Cell Transplantation Therapy

Objective. Recent reports have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurological function when transplanted into models of central nervous system (CNS) disorders, including traumatic spinal cord injury. In this study, the authors aimed to clarify the underlying mechanism through which BMSCs supported CNS regeneration in the spinal cord. Methods. The authors topically applied mouse BMSCs expressing green fluorescence protein (0.4-4 × 104 cells) on the organotypic spinal cord slice culture prepared from 6-day-old rat pups (n = 17). They were co-cultured for 3 weeks after the slice culture started, and the behavior of the applied BMSCs was serially observed using a fluorescence bioimaging technique. The authors completed a histological analysis at the end of the co-cultures and evaluated the profiles of the cultured BMSCs using microarray and immunocytochemistry techniques. Results. The fluorescence bioimaging showed that the BMSCs survived and made a cluster on the slice during the experiments. They also induced a morphological change in the slice within 48 hours of co-culture. Immunohistochemistry analysis showed that the BMSCs promoted a marked neurite extension toward their cluster and some of the BMSCs expressed Tuj-1, an early neuronal marker. Analysis by microarray and immunocytochemistry revealed that BMSCs highly expressed the matrix metalloproteinases (MMPs), stromal cell—derived factor-1, and its specific receptor CXCR4. Conclusions . These findings suggest that the donor BMSCs can support CNS regeneration due to their acquisition of a suitable environment for differentiation and promotion of neurite extension via MMPs and chemokines.

Facilitation of hematopoietic recovery by bone grafts with intra-bone marrow–bone marrow transplantation

We have previously shown that T cells can acquire donor-type major histocompatibility complex (MHC) restriction and can interact with both donor-type antigen-presenting cells (APCs) and B cells, when adult donor bones are co-grafted with intravenous (IV) injection of bone marrow cells (BMCs) in order to supply donor bone marrow (BM) stromal cells. We have also found that the direct injection of donor BMCs into recipient BM (intra-bone marrow–bone marrow transplantation: IBM–BMT) produces more rapid reconstitution (including T-cell functions) and higher survival rates than IV injection (IV-BMT) even in chimerism-resistant combinations. In the present study, we show that the co-administration of bones from suckling (2–3 days old) donor mice is also effective in the IBM–BMT system. Even when a relatively low number of BMCs were injected into adult (more than 15 weeks old) mice, complete reconstitution was achieved in the mice that had received IBM–BMT+bone grafts, but not in the mice that had received IBM–BMT alone. Most BM and splenic adherent cells obtained from the recipients that had received IBM–BMT+bone grafts were reconstituted by donor-type cells. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice showed donor-type MHC restriction. Moreover, the analyses of thymic sections using confocal microscopy revealed that donor BM stromal cells had migrated into the thymus. Thus, the co-administration of donor bones has great advantages for allogeneic BMT in adult mice.

Canine Cranial Reconstruction Using Autologous Bone Marrow Stromal Cells

Limited-sized transplants of culture-expanded autologous or allogeneic bone marrow stromal cells (BMSCs) form cortico-cancellous bone in rodent models. Initiation of clinical studies using autologous BMSC transplantation requires effective bone formation among sizable transplants in a large animal model as well as noninvasive techniques for evaluating transplant success. Here, we obtained bone marrow from the femurs of six dogs and expanded BMSCs in tissue culture. Autologous BMSC-hydroxyapatite/tricalcium phosphate (HA/TCP) transplants were introduced into critical-sized calvarial defects and contralateral control skull defects received HA/TCP vehicle alone. At intervals ranging from 2 to 20 months, transplants were biopsied or harvested for histological and mechanical analysis. Noninvasive studies, including quantitative computed tomography scans and ultrasound, were simultaneously obtained. In all animals, BMSC-containing transplants formed significantly more bone than their control counterparts. BMSC-associated bone possessed mechanical properties similar to the adjacent normal bone, confirmed by both ultrasound and ex vivo analysis. Evaluation by quantitative computed tomography confirmed that the extent of bone formation demonstrated by histology could be discerned through noninvasive means. These results show that autologous cultured BMSC transplantation is a feasible therapy in clinical-sized bone defects and that such transplants can be assessed noninvasively, suggesting that this technique has potential for use in patients with certain bone defects.

Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein: stromal cells as BMT-facilitating cells.

We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day-14 colony-forming unit-spleen (CFU-S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU-S formation nor the hemopoietic colonies in the recipient liver were observed. These findings suggest that the interaction of HSCs with stromal cells in the liver is the first crucial step for successful engraftment of allogeneic HSCs. It is likely that donor stromal cells and HSCs trapped in the liver migrate into the recipient BM and spleen, where they form CFU-BM and CFU-S, respectively.

Evidence for migration of donor bone marrow stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: A role of stromal cells in positive selection.

Intrathymic T-cell differentiation is characterized by two selection events: positive and negative selection. It has been shown that thymic epithelial cells in the cortex are involved in the positive selection, while macrophages and dendritic cells, derived from hemopoietic stem cells, are involved in the negative selection. Here we investigate whether donor-derived bone marrow stromal cells can migrate into the thymus and participate there in positive selection after bone marrow transplantation plus bone grafts (to recruit bone marrow stromal cells). Allogeneic bone marrow transplantation with or without bone grafts was carried out in the [C57BL/6-->C3H] combination. Fluorescence-activated cell sorter analyses of recipient thymic adherent cells showed that donor-type bone marrow stromal cells exist in the thymus of mice that received bone marrow plus bone grafts but not in the mice that received bone marrow cells alone. Histological examination using confocal microscopy also confirmed the existence of donor-type stromal cells in the thymus of mice that received bone marrow cells plus bones. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice show donor-type major histocompatibility complex-restriction. These findings strongly suggest that stromal cells can migrate from the bone marrow to the thymus, where they participate in the positive selection of thymocytes.

Characterization of bone marrow stromal cells from multiple myeloma

We have cultured multiple myeloma (MM) bone marrow (BM) stromal cells that are able to sustain the in vitro growth of monoclonal B-cells. Our aim was to evaluate which adhesion molecules are expressed and which extracellular matrix proteins are produced by these cells and whether they differ from the stromal cells that can be grown under the same experimental conditions from the BM of monoclonal gammopathies of undetermined significance (MGUS) and of normal donors. MM BM stromal cells that support malignant B-cell development have a striking proliferative ability that is absent in MGUS and normal donors of the same age group and are formed by four major different cell populations. Two kinds of HLA-DR+, CD10+ fibroblast-like cells can be recognized through the expression (or the lack) of α-smooth muscle actin isoform; further, macrophages and osteoclasts can be identified. Fibroblast-like cells that express α-smooth muscle actin isoform, often organized along stress fibers in a periodic fashion, may be considered as myofibroblasts. Fibroblast-like cells react strongly with antibodies to CD54 (ICAM-1), integrin β1, β3, β5 and some of associated α chains. Integrin β1 is diffusely exposed on the surface while β3 is clustered in focal contacts in association with vinculin. A still undetermined subpopulation of fibroblasts is highly positive for ανβ5 that is clustered at focal contacts as shown by association with stress fiber termini and by interference reflection microscopy. A major difference between MM and normal donor BM stromal cells involves lower deposition and simpler organization of the extracellular matrix proteins (fibronectin, laminin, collagen type IV) deposited by MM fibroblast-like cells. CD14+ macrophages from MM, MGUS and normal donor BM are CD11a+ (αL), CD11b+ (αM), CD11c+ (αX), CD54+ (ICAM-1), CD56+ (N-CAM), β1 and β2 (CD18) integrin positive. The integrin β1 is diffusely expressed on the surface, while β2 is concentrated in podosomes. MM osteoclasts show a weak diffuse staining with CD54 and CD56 MoAbs; β1 integrin has a diffuse surface expression, while β3 integrin is concentrated in the podosomes. Normal donor osteoclasts are CD54- and the staining with CD56 is barely visible. These findings lead us to suggest that the microenvironment provided by MM BM may be significantly different from that of normal BM indicating its potential role in controlling the local proliferation and differentiation of malignant B-lineage cells.