Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation

Bone Marrow Transplantation (2004) 33, 597–604 & 2004 Nature Publishing Group All rights reserved 0268-3369/04 $25.00 www.nature.com/bmt Mesenchymal stem cells Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation B Maitra1, E Szekely1, K Gjini1, MJ Laughlin1,2, J Dennis3, SE Haynesworth3 and ON Koc¸1,2 1 Comprehensive Cancer Center, Case Western Reserve University, University Hospitals of Cleveland, Cleveland, OH, USA; 2Division of Hematology/Oncology, Department of Medicine, Cleveland, OH, USA; and 3Department of Biology, Skeletal Research Center, Case Western Reserve University, Cleveland, OH, USA Summary: Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and represent potential cellular therapy to enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease (GVHD). We investigated the role of human MSCs in NOD-SCID mice repopulation by unrelated human hematopoietic cells and studied the immune interactions between human MSCs and unrelated donor blood cells in vitro. When hematopoietic stem cell numbers were limited, human engraftment of NOD-SCID mice was observed only after coinfusion of unrelated human MSCs, but not with coinfusion of mouse mesenchymal cell line. Unrelated human MSCs did not elicit T-cell activation in vitro and suppressed T-cell activation by Tuberculin and unrelated allogeneic lymphocytes in a dose-dependent manner. Cellfree MSC culture supernatant, mouse stromal cells and human dermal fibroblasts did not elicit this effect. These preclinical data suggest that unrelated, human bone marrow-derived, culture-expanded MSCs may improve the outcome of allogeneic transplantation by promoting hematopoietic engraftment and limiting GVHD and their therapeutic potential should be tested in clinic. Bone Marrow Transplantation (2004) 33, 597–604. doi:10.1038/sj.bmt.1704400 Published online 12 January 2004 Keywords: stem cell; MSC; stromal cell; GVHD; immunosuppression Graft failure and graft-versus-host disease (GVHD) remain significant obstacles to a successful outcome in allogeneic hematopoietic stem cell transplantation. There is a growing interest in cotransplantation of allogeneic stromal and hematopoietic progenitors to improve the patient outcome after allogeneic transplantation.1 Here we investigated the Correspondence: Dr ON Koc¸, Case Western Reserve University, BRB-3 Hematology/Oncology, 10900 Euclid Ave., Cleveland, OH 44106, USA; E-mail: Received 25 April 2003; accepted 07 August 2003 Published online 12 January 2004 interaction between bone marrow-derived mesenchymal stem cells (MSC) and the hematopoietic stem cells to determine if MSCs could support allogeneic hematopoietic stem cell transplantation by facilitating engraftment without increasing the risk of GVHD. Several groups have described nonhematopoietic, plastic-adherent progenitor cells derived from adult human bone marrow aspirates, which are capable of differentiating into mature mesenchymal cells.2–6 It is thought that these progenitors give rise to adventitial and other mesenchymal cells in the marrow that fabricate the connective tissue scaffolding, and produce cytokines, chemokines and extracellular matrix proteins that regulate hematopoietic homing and proliferation.7–10 Therefore, we hypothesized that MSCs could support the survival of hematopoietic stem cells and tested this using an in vivo model of human hematopoietic stem cell engraftment and survival using NOD-SCID mice. Furthermore, several groups have recently shown that MSCs suppress T-lymphocyte proliferation in response to allogeneic stimuli.11–14 This suppressive effect of MSCs appears to be partially mediated by soluble factors, although there are conflicting data. Therefore, we investigated the immune interactions between MSCs and unrelated allogeneic lymphocytes in vitro using Elispot assay. In experiments described here, we used a human bone marrow-derived population of adherent cells described by Haynesworth et al3 that have extensive proliferative capacity and ability to differentiate along the osteogenic, chondrogenic and adipogenic lineages both in vitro and in vivo.15 In unstimulated cultures, MSCs appear as fusiform fibroblasts with the expression of unique surface proteins (recognized by monoclonal antibodies SH2 and SH3)16 not found on hematopoietic precursors. We have previously demonstrated the feasibility of culturing large numbers of these cells ex vivo and safety of their intravenous infusion into patients.17,18 Therefore, our results are directly relevant to acceptable cellular therapy that can be tested in clinic. In this manuscript, we show that coinfusion of human MSCs and unrelated umbilical cord blood (UCB) cells into NODSCID mice results in improved frequency and degree of human engraftment when UCB dose is limiting. We also found that allogeneic-unrelated MSCs do not elicit T-cell activation in vitro. On the contrary, unrelated donor MSCs inhibited T-cell activation normally seen in mixed lympho- MSC support HSC B Maitra et al 598 cyte reactions. This effect could not be elucidated with MSC culture supernatant alone. centrifugation. All cells were used fresh, without cryopreservation. Materials and methods NOD-SCID mice infusion and detection of human engraftment Mesenchymal stem cell procurement Human hematopoietic engraftment was evaluated in irradiated NOD-SCID mice after infusing human UCB cells with or without coinfusion of allogeneic human MSCs. Recipient 6–8-week-old NOD-SCID mice were given sublethal total body irradiation (250 cGy) 24 h prior to infusion using a cesium source. Cohorts of 4–8 mice were injected intravenously with UCB cells at doses of 2 to 8
106 per mouse, alone or mixed with 1
106 human MSCs (unrelated donor to UCB cells) or 1
106 conditionally immortalized mouse mesenchymal progenitor cells (clone BMC9).20 Mice were kept in microisolator cages and fed irradiated food and water containing penicillin. Mice were killed 6–7 weeks after cell infusion and long bones of hind legs were extracted. Bone marrow cells were flushed into medium and used for flow cytometry or plated for growth of human MSCs as described above. Human cell engraftment was detected by flow cytometry, using anti-human CD45 antibody conjugated to PE (Pharmingen, San Diego, CA, USA) and isotype control antibody, Mouse IgG1-PE (Becton Dickinson, Parsippany, NJ, USA). Percent human engraftment was calculated after subtraction of the background detected in control mice and 0.5% was designated as the lower threshold for unequivocal human engraftment. Human MSC cultures were established as described previously.17 Briefly, bone marrow was aspirated (10– 30 ml) under local anesthesia from healthy volunteer donors after consent approved by the IRB of the University Hospitals of Cleveland. Mononuclear cells (MNCs) were isolated by Percoll (d 1.07 (...truncated)