Enhanced anti-inflammatory effects of mesenchymal stromal cells mediated by the transient ectopic expression of CXCR4 and IL10.

BACKGROUND: Mesenchymal stromal cells (MSCs) constitute one of the cell types most frequently used in cell therapy. Although several studies have shown the efficacy of these cells to modulate inflammation in different animal models, the results obtained in human clinical trials have been more modest. Here, we aimed at improving the therapeutic properties of MSCs by inducing a transient expression of two molecules that could enhance two different properties of these cells. With the purpose of improving MSC migration towards inflamed sites, we induced a transient expression of the C-X-C chemokine receptor type 4 (CXCR4). Additionally, to augment the anti-inflammatory properties of MSCs, a transient expression of the anti-inflammatory cytokine, interleukin 10 (IL10), was also induced. METHODS: Human adipose tissue-derived MSCs were transfected with messenger RNAs carrying the codon-optimized versions of CXCR4 and/or IL10. mRNA-transfected MSCs were then studied, first to evaluate whether the characteristic phenotype of MSCs was modified. Additionally, in vitro and also in vivo studies in an LPS-induced inflamed pad model were conducted to evaluate the impact associated to the transient expression of CXCR4 and/or IL10 in MSCs. RESULTS: Transfection of MSCs with CXCR4 and/or IL10 mRNAs induced a transient expression of these molecules without modifying the characteristic phenotype of MSCs. In vitro studies then revealed that the ectopic expression of CXCR4 significantly enhanced the migration of MSCs towards SDF-1, while an increased immunosuppression was associated with the ectopic expression of IL10. Finally, in vivo experiments showed that the co-expression of CXCR4 and IL10 increased the homing of MSCs into inflamed pads and induced an enhanced anti-inflammatory effect, compared to wild-type MSCs. CONCLUSIONS: Our results demonstrate that the transient co-expression of CXCR4 and IL10 enhances the therapeutic potential of MSCs in a local inflammation mouse model, suggesting that these mRNA-modified cells may constitute a new step in the development of more efficient cell therapies for the treatment of inflammatory diseases.

Engraftment and in vivo proliferation advantage of gene-corrected mobilized CD34+ cells from Fanconi anemia patients.

Previous Fanconi anemia (FA) gene therapy studies have failed to demonstrate engraftment of gene-corrected hematopoietic stem and progenitor cells (HSPCs) from FA patients, either after autologous transplantation or infusion into immunodeficient mice. In this study, we demonstrate that a validated short transduction protocol of G-CSF plus plerixafor-mobilized CD34+ cells from FA-A patients with a therapeutic FANCA-lentiviral vector corrects the phenotype of in vitro cultured hematopoietic progenitor cells. Transplantation of transduced FA CD34+ cells into immunodeficient mice resulted in reproducible engraftment of myeloid, lymphoid, and CD34+ cells. Importantly, a marked increase in the proportion of phenotypically corrected, patient-derived hematopoietic cells was observed after transplantation with respect to the infused CD34+ graft, indicating the proliferative advantage of corrected FA-A hematopoietic repopulating cells. Our data demonstrate for the first time that optimized protocols of hematopoietic stem cell collection from FA patients, followed by the short and clinically validated transduction of these cells with a therapeutic lentiviral vector, results in the generation of phenotypically corrected HSPCs capable of repopulating and developing proliferation advantage in immunodeficient mice. Our results suggest that clinical approaches for FA gene therapy similar to those used in this study will facilitate hematopoietic repopulation in FA patients with gene corrected HSPCs, opening new prospects for gene therapy of FA patients.

Comparative analysis of the immunomodulatory capacities of human bone marrow- and adipose tissue-derived mesenchymal stromal cells from the same donor.

BACKGROUND AIMS: The immunomodulatory properties of mesenchymal stromal cells (MSCs), together with their tissue regenerative potential, make them interesting candidates for clinical application. METHODS: In the current study, we analyzed the in vitro immunomodulatory effects of MSCs derived from bone marrow (BM-MSCs) and from adipose tissue (AT-MSCs) obtained from the same donor on both innate and acquired immunity cells. BM-MSCs and AT-MSCs were expanded to fourth or fifth passage and co-cultured with T cells, monocytes or natural killer (NK) cells isolated from human peripheral blood and stimulated in vitro. The possible differing impact of MSCs obtained from distinct sources on phenotype, cell proliferation and differentiation, cytokine production and function of these immune cells was comparatively analyzed. RESULTS: BM-MSCs and AT-MSCs induced a similar decrease in NK-cell proliferation, cytokine secretion and expression of both activating receptors and cytotoxic molecules. However, only BM-MSCs significantly reduced NK-cell cytotoxic activity, although both MSC populations showed the same susceptibility to NK-cell-mediated lysis. AT-MSCs were more potent in inhibiting dendritic-cell (DC) differentiation than BM-MSC, but both MSC populations similarly reduced the ability of DCs to induce CD4(+) T-cell proliferation and cytokine production. BM-MSCs and AT-MSCs induced a similar decrease in T-cell proliferation and production of inflammatory cytokines after activation. CONCLUSIONS: AT-MSCs and BM-MSCs from the same donor had similar immunomodulatory capacity on both innate and acquired immunity cells. Thus, other variables, such as accessibility of samples or the frequency of MSCs in the tissue should be considered to select the source of MSC for cell therapy.

Reduced efficacy of mesenchymal stromal cells in preventing graft-versus-host disease in an in vivo model of haploidentical bone marrow transplant with leukemia.

Mesenchymal stromal cell (MSC) immunosuppressive properties have been applied to treat graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplants (HSCTs). We have previously demonstrated that MSC infusions early after haplo-HSCT prevent GVHD in a haploidentical-HSCT mouse model. Now, we investigated the impact that MSCs' immunosuppressive properties have on the graft-versus-leukemia (GVL) effect. First, to mimic a chronic myeloid leukemia (CML) relapse after a haploidentical HSCT, lethally irradiated mice were coinfused with haploidentical donor bone marrow cells plus syngenic hematopoietic progenitors transduced with a retroviral vector encoding both the BCR/ABL oncogene and the ΔNGFR marker gene. As expected, a CML-like myeloproliferative syndrome developed in all the recipient animals. The addition of haploidentical splenocytes to the transplanted graft prevented CML development by a GVL effect, and all transplanted recipients died of GVHD. This GVL mouse model allowed us to investigate the impact of MSCs infused to prevent GVHD on days 0, 7, and 14 after HSCT, on the GVL effect, expecting an increase in leukemic relapse. Strikingly, a high mortality of the recipients was observed, caused by GVHD, and only few leukemic cells were detected in the recipient animals. In contrast, GVHD prevention by MSCs in the absence of BCR/ABL leukemic cells resulted in a significant survival of the recipients. In vitro data pointed to an inability of MSCs to control strong CTLs responses against BCR/ABL. Our results show that, although an evident increase in leukemic relapses induced by MSCs could not be detected, they showed a reduced efficacy in preventing GVHD that precluded us to draw clear conclusions on MSCs' impact over GVL effect.

Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients.

Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).

Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease.

Previous studies have shown the relevance of bone marrow-derived MSCs (BM-MSCs) in controlling graft-versus-host disease (GVHD) after allogeneic transplantation. Since adipose tissue-derived MSCs (Ad-MSCs) may constitute a good alternative to BM-MSCs, we have expanded MSCs derived from human adipose tissue (hAd-MSCs) and mouse adipose tissue (mAd-MSCs), investigated the immunoregulatory properties of these cells, and evaluated their capacity to control GVHD in mice. The phenotype and immunoregulatory properties of expanded hAd-MSCs were similar to those of human BM-MSCs. Moreover, hAd-MSCs inhibited the proliferation and cytokine secretion of human primary T cells in response to mitogens and allogeneic T cells. Similarly, ex vivo expanded mAd-MSCs had an equivalent immunophenotype and exerted immunoregulatory properties similar to those of hAd-MSCs. Moreover, the infusion of mAd-MSCs in mice transplanted with haploidentical hematopoietic grafts controlled the lethal GVHD that occurred in control recipient mice. These findings constitute the first experimental proof that Ad-MSCs can efficiently control the GVHD associated with allogeneic hematopoietic transplantation, opening new perspectives for the clinical use of Ad-MSCs.

Genetic modification of hematopoietic stem cells: recent advances in the gene therapy of inherited diseases.

Hematopoietic stem cells constitute a rare population of precursor cells with remarkable properties for being used as targets in gene therapy protocols. The last years have been particularly productive both in the fields of gene therapy and stem cell biology. Results from ongoing clinical trials have shown the first unquestionable clinical benefits of immunodeficient patients transplanted with genetically modified autologous stem cells. On the other hand, severe side effects in a few patients treated with gene therapy have also been reported, indicating the usefulness of further improving the vectors currently used in gene therapy clinical trials. In the field of stem cell biology, evidence showing the plastic potential of adult hematopoietic stem cells and data indicating the multipotency of adult mesenchymal precursor cells have been presented. Also, the generation of embryonic stem cells by means of nuclear transfer techniques has appeared as a new methodology with direct implications in gene therapy.