Solution-Phase Crosstalk and Regulatory Interactions Between Multipotent Adult Progenitor Cells and Peripheral Blood Mononuclear Cells.

UNLABELLED: Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in clinical trials for acute graft versus host disease with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation-related processes. Anti-CD3/anti-CD28 (3/28) activation of T cells within the peripheral blood mononuclear cell (PBMC) compartment was performed in the presence or absence of MAPCs. Liquid chromatography-coupled tandem mass spectrometry was used to characterize the differential secretion of proteins, and transcriptional profiling was used to monitor mRNA expression changes in both cell populations. Overall, 239 secreted and/or ectodomain-shed proteins were detected in the secretomes of PBMCs and MAPCs. In addition, 3/28 activation of PBMCs induced differential expression of 2,925 genes, and 22% of these transcripts were differentially expressed on exposure to MAPCs in Transwell. MAPCs exposed to 3/28-activated PBMCs showed differential expression of 1,247 MAPC genes. Crosstalk was demonstrated by reciprocal transcriptional regulation. Secretome proteins and transcriptional signatures were used to predict molecular activities by which MAPCs could dampen local and systemic inflammatory responses. These data support the hypothesis that MAPCs block PBMC proliferation via cell cycle arrest coupled to metabolic stress in the form of tryptophan depletion, resulting in GCN2 kinase activation, downstream signaling, and inhibition of cyclin D1 translation. These data also provide a plausible explanation for the immune privilege reported with administration of donor MAPCs. Although most components of the major histocompatibility complex class II antigen presentation pathway were markedly transcriptionally upregulated, cell surface expression of human leukocyte antigen-DR is minimal on MAPCs exposed to 3/28-activated PBMCs. SIGNIFICANCE: This study documents experiments quantifying solution-phase crosstalk between multipotent adult progenitor cells (MAPCs) and peripheral blood mononuclear cells. The secretome and transcriptional changes quantified suggest mechanisms by which MAPCs are hypothesized to provide both local and systemic immunoregulation of inflammation. The potential impact of these studies includes development of a robust experimental framework to be used for preclinical evaluation of the specific mechanisms by which beneficial effects are obtained after treatment of patients with MAPCs.

Human multipotent adult progenitor cells transcriptionally regulate fucosyltransferase VII.

BACKGROUND AIMS: Targeted recruitment of leukocytes to sites of inflammation is a crucial event in normal host defense against pathogens, and attachment to and rolling on activated endothelial cells is a prerequisite first step for eventual leukocyte extravasation into sites of inflammation. These key events are mediated by interactions between glycosylated ligands expressed on leukocytes and selectins expressed on activated endothelium. Cell surface expression of selectin ligands on leukocytes is regulated by the rate-limiting enzyme fucosyltransferase VII (Fut7), and in its absence extravasation of leukocytes is severely inhibited. Multipotent adult progenitor cells (MAPCs) are an adherent cell population isolated from adult bone marrow. Intravenous administration of MAPCs provided functional improvement in multiple pre-clinical models of injury or disease, but the mechanisms by which these outcomes were achieved remain poorly understood. METHODS: In vitro cell analysis studies including fluorescence-activated cell sorting, messenger RNA analysis, T-cell proliferation assays and endothelial cell binding assays were performed. RESULTS: The in vitro cell analysis studies characterized the ability of MAPCs to secrete factors that transcriptionally attenuate expression of Fut7 in T cells, blocking the terminal fucosylation event in the biosynthesis of selectin ligands and reducing T-cell binding to endothelial cells. CONCLUSIONS: This study presents the first example of a distinct regulatory mechanism involving transcriptional down-regulation of Fut7 by MAPCs that could modulate the trafficking behavior of T cells in vivo.

Dissection of the human multipotent adult progenitor cell secretome by proteomic analysis.

Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in acute graft versus host disease clinical trials with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation-related processes. Our previous studies documented that MAPCs secrete factors that play a role in regulating T-cell activity. Here we expand our studies using a proteomics approach to characterize and quantify MAPC secretome components secreted over 72 hours in vitro under steady-state conditions and in the presence of the inflammatory triggers interferon-γ and lipopolysaccharide, or a tolerogenic CD74 ligand, RTL1000. MAPCs differentially responded to each of the tested stimuli, secreting molecules that regulate the biological activity of the extracellular matrix (ECM), including proteins that make up the ECM itself, proteins that regulate its construction/deconstruction, and proteins that serve to attach and detach growth factors from ECM components for redistribution upon appropriate stimulation. MAPCs secreted a wide array of proteases, some detectable in their zymogen forms. MAPCs also secreted protease inhibitors that would regulate protease activity. MAPCs secreted chemokines and cytokines that could provide molecular guidance cues to various cell types, including neutrophils, macrophages, and T cells. In addition, MAPCs secreted factors involved in maintenance of a homeostatic environment, regulating such diverse programs as innate immunity, angiogenesis/angiostasis, targeted delivery of growth factors, and the matrix-metalloprotease cascade.

Clinical-grade multipotent adult progenitor cells durably control pathogenic T cell responses in human models of transplantation and autoimmunity.

A major goal of immunotherapy remains the control of pathogenic T cell responses that drive autoimmunity and allograft rejection. Adherent progenitor cells, including mesenchymal stromal cells (MSCs) and multipotent adult progenitor cells (MAPCs), represent attractive immunomodulatory cell therapy candidates currently active in clinical trials. MAPCs can be distinguished from MSCs on the basis of cellular phenotype, size, transcriptional profile, and expansion capacity. However, despite their ongoing evaluation in autoimmune and allogeneic solid organ transplantation settings, data supporting the immune regulatory potential of clinical-grade MAPCs are limited. In this study, we used allogeneic islet transplantation as a model indication to assess the ability of clinical-grade MAPCs to control T cell responses that drive immunopathology in human autoimmune disease and allograft rejection. MAPCs suppressed T cell proliferation and Th1 and Th17 cytokine production while increasing secretion of IL-10 and were able to suppress effector functions of bona fide autoreactive T cells from individuals with type 1 diabetes mellitus, including killing of human islets. Furthermore, MAPCs favored the proliferation of regulatory T cells during homeostatic expansion driven by γ-chain cytokines and exerted a durable, yet reversible, control of T cell function. MAPC suppression required licensing and proceeded via IDO-mediated tryptophan catabolism. Therefore, the common immune modulatory characteristics of clinical-grade MAPCs shown in this study suggest that they can be regarded as an alternative source of adult progenitor cells with similar clinical usefulness to MSCs. Taken collectively, these findings may guide the successful deployment of both MSCs and MAPCs for the amelioration of human autoimmunity and allograft rejection.

Development of a surrogate angiogenic potency assay for clinical-grade stem cell production.

BACKGROUND AIMS: Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. METHODS AND RESULTS: Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors produced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increasing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. CONCLUSIONS: A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria.

Percutaneous adventitial delivery of allogeneic bone marrow-derived stem cells via infarct-related artery improves long-term ventricular function in acute myocardial infarction.

Acute myocardial infarction (AMI) results in ischemic damage and death of cardiomyocytes and loss of vasculature. Stem cell therapy has emerged as a potentially promising strategy for maximizing cardiac function following ischemic injury. Issues of cell source, delivery, and quantification of response have challenged development of clinically viable strategies. In this study we investigate the effects of a well-defined bone marrow-derived allogeneic cell product delivered by catheter directly to the myocardium via the infarct-related vessel on global and regional measures of left ventricular (LV) function in a porcine model of anterior wall myocardial infarction. Multipotent adult progenitor cells (MAPCs) were derived and expanded from the bone marrow of a donor Yorkshire pig. Anterior wall myocardial infarction (AMI) was induced by 90 min of mid-LAD occlusion using a balloon catheter. Two days after AMI was induced, either vehicle (Plasma Lyte-A, n = 7), low-dose (20 million, n = 6), or high-dose (200 million, n = 6) MAPCs were delivered directly to the myocardium via the infarct-related vessel using a transarterial microsyringe catheter-based delivery system. Echocardiography was used to measure LV function as a function of time after AMI. Animals that received low-dose cell treatment showed significant improvement in regional and global LV function and remodeling compared to the high-dose or control animals. Direct myocardial delivery of allogeneic MAPCs 2 days following AMI through the vessel wall of the infarct-related vessel is safe and results in delivery of cells throughout the infarct zone and improved cardiac function despite lack of long-term cell survival. These data further support the hypothesis of cell-based myocardial tissue repair by a paracrine mechanism and suggest a clinically translatable strategy for delivering cells at any time after AMI to modulate cardiac remodeling and function.

Clinical scale expanded adult pluripotent stem cells prevent graft-versus-host disease.

Adherent bone marrow adult stem cells have been used in the treatment of GVHD. In this study, we investigate the capacity of a newly characterized population of stem cells, the Multipotent Adult Progenitor Cells (MAPC), to modulate acute GVHD. These cells were derived from bone marrow cells and grown extensively without evidence for replicative senescence or loss of differentiating capacity. MAPC significantly decreased mortality of acute GVHD. Moreover, they were non immunogenic and they were not sensitive to NK-lysis. When these cells were added to a mixed lymphocyte reaction (MLR), a dose-dependent suppression of T cell proliferation was observed that was non-MHC restricted, was reversible upon removal of MAPC from culture and was mediated by soluble factors. These data show that in vitro expanded adult stem cells can efficiently control an allo-reactive response associated with acute GVHD, that they are immuno-privileged and present strong immunosuppressive properties.

Global Characterization and Genomic Stability of Human MultiStem, A Multipotent Adult Progenitor Cell.

The therapeutic benefits of adult adherent stem cells are currently being investigated in clinical trials for a variety of diseases. Data from initial clinical studies are promising and as a consequence of moving to later stage clinical studies, have resulted in larger scale clinical-grade cell production strategies. Therefore it becomes imperative to examine the epigenetic flux and genomic stability of stem cells in long-term culture to determine that minimal risk is associated with these therapies. Multipotent adult progenitor cells (MAPC) are an adherent adult stem cell population that can be derived from bone marrow and was the first of a class of adult stem cells that have broad developmental potential both in vitro and in vivo. Here, we report a panel of tests to characterize MultiStem, a multipotent adult stem cell type based on MAPC, and establish its genomic stability during culture expansion. A variety of techniques were employed that consisted of miRNA expression to characterize and define the cell population; chromosomal SNP analysis and G-banding to determine karyotypic stability; and methylation pattern and telomerase expression to examine potential changes in epigenetic and chromosomal stability with prolonged in vitro culture of cells. This panel of test was applied to cultures at early isolation stages and compared to cultures harvested at population doublings greater than those reached in current MultiStem clinical trials. These tests also provide a baseline for quality control of cells prepared from various biological donor sources for subsequent large scale propagation and preparation of cell banks for downstream applications.

Identification of novel and improved antimitotic agents derived from noscapine.

Analogues of the natural product noscapine were synthesized and their potential as antitumor agents evaluated. The discovery of a novel regioselective O-demethylation facilitated the synthesis of the potent aniline 6, which arrests mammalian cells in the G2/M phase of the cell cycle at 0.1 microM and also affects tubulin polymerization. Aniline 6 is orally bioavailable and is 250-fold more potent than noscapine in reducing cell proliferation in rapidly dividing cells.

Discovery of S-phase arresting agents derived from noscapine.

Analogues of the natural product noscapine were synthesized, and their potential as antitumor agents were examined. The discovery of a novel regio- and stereoselective O-demethylation led to the synthesis of several O-alkylated analogues that induced an unexpected S-phase arrest of mammalian cells. Compound 4a was the most potent analogue inhibiting cell proliferation at an EC(50) of 1.9 microM.