Suppression of IL-7-dependent Effector T-cell Expansion by Multipotent Adult Progenitor Cells and PGE2.

T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1ß-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients.

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.

Human Wharton's Jelly-Derived Stem Cells Display a Distinct Immunomodulatory and Proregenerative Transcriptional Signature Compared to Bone Marrow-Derived Stem Cells.

Mesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive and trophic support functions. While MSCs from different sources frequently display a similar appearance in culture, they often show differences in their surface marker and gene expression profiles. Although bone marrow is considered the "gold standard" tissue to isolate classical MSCs (BM-MSC), MSC-like cells are currently also derived from more easily accessible extra-embryonic tissues such as the umbilical cord. In this study, we defined the best way to isolate MSCs from the Wharton's jelly of the human umbilical cord (WJ-MSC) and assessed the mesenchymal and immunological phenotype of BM-MSC and WJ-MSC. Moreover, the gene expression profile of established WJ-MSC cultures was compared to two different bone marrow-derived stem cell populations (BM-MSC and multipotent adult progenitor cells or MAPC®). We observed that explant culturing of Wharton's jelly matrix is superior to collagenase tissue digestion for obtaining mesenchymal-like cells, with explant isolated cells displaying increased expansion potential. While being phenotypically similar to adult MSCs, WJ-MSC show a different gene expression profile. Gene ontology analysis revealed that genes associated with cell adhesion, proliferation, and immune system functioning are enriched in WJ-MSC. In vivo transplantation confirms their immune modulatory effect on T cells, similar to BM-MSC and MAPC. Furthermore, WJ-MSC intrinsically overexpress genes involved in neurotrophic support and their secretome induces neuronal maturation of SH-SY5Y neuroblastoma cells to a greater extent than BM-MSC. This signature makes WJ-MSC an attractive candidate for cell-based therapy in neurodegenerative and immune-mediated central nervous system disorders such as multiple sclerosis, Parkinson's disease, or amyotrophic lateral sclerosis.

Genetic ablation of phosphatidylinositol transfer protein function in murine embryonic stem cells.

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITP alpha and PITP beta, two highly homologous proteins that are encoded by distinct genes. Insights into PITP alpha and PITP beta function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITP alpha and PITP beta in murine cells. Contrary to expectations, we find that ablation of PITP alpha function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITP alpha does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITP alpha-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITP beta alleles from murine cells and show that PITP beta deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITP beta is an essential housekeeping PITP in murine cells, whereas PITP alpha plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.

Crosstalk with Inflammatory Macrophages Shapes the Regulatory Properties of Multipotent Adult Progenitor Cells.

Macrophages and microglia are key effector cells in immune-mediated neuroinflammatory disorders. Driving myeloid cells towards an anti-inflammatory, tissue repair-promoting phenotype is considered a promising strategy to halt neuroinflammation and promote central nervous system (CNS) repair. In this study, we defined the impact of multipotent adult progenitor cells (MAPC), a stem cell population sharing common mesodermal origin with mesenchymal stem cells (MSCs), on the phenotype of macrophages and the reciprocal interactions between these two cell types. We show that MAPC suppress the secretion of tumor necrosis factor alpha (TNF-α) by inflammatory macrophages partially through a cyclooxygenase 2- (COX-2-) dependent mechanism. In turn, we demonstrate that inflammatory macrophages trigger the immunomodulatory properties of MAPC, including an increased expression of immunomodulatory mediators (e.g., inducible nitric oxide synthase (iNOS) and COX-2), chemokines, and chemokine receptors. Macrophage-primed MAPC secrete soluble factors that suppress TNF-α release by macrophages. Moreover, the MAPC secretome suppresses the antigen-specific proliferation of autoreactive T cells and the T cell stimulatory capacity of macrophages. Finally, MAPC increase their motility towards secreted factors of activated macrophages. Collectively, these in vitro findings reveal intimate reciprocal interactions between MAPC and inflammatory macrophages, which are of importance in the design of MAPC-based therapeutic strategies for neuroinflammatory disorders in which myeloid cells play a crucial role.

Development of Advanced Dressings for the Delivery of Progenitor Cells.

Culture surfaces that substantially reduce the degree of cell manipulation in the delivery of cell sheets to patients are described. These surfaces support the attachment, culture, and delivery of multipotent adult progenitor cells (MAPC). It was essential that the processes of attachment/detachment to the surface did not affect cell phenotype nor the function of the cultured cells. Both acid-based and amine-based surface coatings were generated from acrylic acid, propanoic acid, diaminopropane, and heptylamine precursors, respectively. While both functional groups supported cell attachment/detachment, amine coated surfaces gave optimal performance. X-ray photoelectron spectroscopy (XPS) showed that at a primary amine to carbon surface ratio of between 0.01 and 0.02, greater than 90% of attached cells were effectively transferred to a model wound bed. A dependence on primary amine concentration has not previously been reported. After 48 h of culture on the optimized amine surface, PCR, functional, and viability assays showed that MAPC retained their stem cell phenotype, full metabolic activity, and biological function. Consequently, in a proof of concept experiment, it was shown that this amine surface when coated onto a surgical dressing provides an effective and simple technology for the delivery of MAPC to murine dorsal excisional wounds, with MAPC delivery verified histologically. By optimizing for cell delivery using a combination of in vitro and in vivo techniques, we developed an effective surface for the delivery of MAPC in a clinically relevant format.

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

: MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8-CD69+ T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. SIGNIFICANCE: Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8+ T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy.

Using miRNA-mRNA Interaction Analysis to Link Biologically Relevant miRNAs to Stem Cell Identity Testing for Next-Generation Culturing Development.

UNLABELLED: Therapeutic benefit of stem cells has been demonstrated in multiple disease models and clinical trials. Robust quality assurance is imperative to make advancements in culturing procedures to enable large-scale cell manufacturing without hampering therapeutic potency. MicroRNAs (miRNAs or miRs) are shown to be master regulators of biological processes and are potentially ideal quality markers. We determined miRNA markers differentially expressed under nonclinical multipotent adult progenitor cell (MAPC) and mesenchymal stem cell (MSC) culturing conditions that regulate important stem cell features, such as proliferation and differentiation. These bone marrow-derived stem cell types were selected because they both exert therapeutic functions, but have different proliferative and regenerative capacities. To determine cell-specific marker miRNAs and assess their effects on stem cell qualities, a miRNA and mRNA profiling was performed on MAPCs and MSCs isolated from three shared donors. We applied an Ingenuity Pathway Analysis-based strategy that combined an integrated RNA profile analysis and a biological function analysis to determine the effects of miRNA-mRNA interactions on phenotype. This resulted in the identification of important miRNA markers linked to cell-cycle regulation and development, the most distinctive being MAPC marker miR-204-5p and MSC marker miR-335-5p, for which we provide in vitro validation of its function in differentiation and cell cycle regulation, respectively. Importantly, marker expression is maintained under xeno-free conditions and during bioreactor isolation and expansion of MAPC cultures. In conclusion, the identified biologically relevant miRNA markers can be used to monitor stem cell stability when implementing variations in culturing procedures. SIGNIFICANCE: Human adult marrow stromal stem cells have shown great potential in addressing unmet health care needs. Quality assurance is imperative to make advancements in large-scale manufacturing procedures. MicroRNAs are master regulators of biological processes and potentially ideal quality markers. MicroRNA and mRNA profiling data of two human adult stem cell types were correlated to biological functions in silico. Doing this provided evidence that differentially expressed microRNAs are involved in regulating specific stem cell features. Furthermore, expression of a selected microRNA panel was maintained in next-generation culturing platforms, demonstrating the robustness of microRNA profiling in stem cell comparability testing.

Culturing protocols for human multipotent adult stem cells.

Culture procedures are presented that support the initiation and controlled expansion of the multipotent adult progenitor cell (MAPC) population within the human bone marrow derived multipotent mesenchymal stromal cell compartment. Culture procedures or conditions and characterization assays that maintain and survey the distinctive primitive MAPC properties are discussed in the context of cell culturing platforms that facilitate controlled expansion of clinical grade human MAPC product to levels required for mid to late stage clinical testing.

Neuroinflammatory signals enhance the immunomodulatory and neuroprotective properties of multipotent adult progenitor cells.

INTRODUCTION: Stem cell-based therapies are currently widely explored as a tool to treat neuroimmune diseases. Multipotent adult progenitor cells (MAPC) have been suggested to have strong immunomodulatory and neuroprotective properties in several experimental models. In this study, we investigate whether MAPC are of therapeutic interest for neuroinflammatory disorders such as multiple sclerosis by evaluating their capacities to modulate crucial pathological features and gain insights into the molecular pathways involved. METHODS: Rat MAPC were treated with combinations of pro-inflammatory cytokines that are closely associated with neuroinflammatory conditions, a process called licensing. mRNA expression of immunomodulatory molecules, chemokines and chemokine receptors was investigated. The migratory potential of licensed rat MAPC towards a broad spectrum of chemokines was tested in a Transwell assay. Furthermore, the effect of licensing on the ability of rat MAPC to attract and suppress the proliferation of encephalitogenic T cells was assessed. Finally, neuroprotective properties of rat MAPC were determined in the context of protection from oxidative stress of oligodendrocytes. Therefore, rat MAPC were incubated with conditioned medium of OLN93 cells subjected to sublethal doses of hydrogen peroxide and the gene expression of neurotrophic factors was assessed. RESULTS: After licensing, a wide variety of immunomodulatory molecules and chemokines, including inducible nitric oxide synthase and fractalkine, were upregulated by rat MAPC. The migratory properties of rat MAPC towards various chemokines were also altered. In addition, rat MAPC were found to inhibit antigen-specific T-cell proliferation and this suppressive effect was further enhanced after pro-inflammatory treatment. This phenomenon was partially mediated through inducible nitric oxide synthase or cyclooxygenase-2. Activated rat MAPC secreted factors that led to attraction of myelin-specific T cells. Finally, exposure of rat MAPC to an in vitro simulated neurodegenerative environment induced the upregulation of mRNA levels of vascular endothelial growth factor and ciliary neurotrophic factor. Factors secreted by rat MAPC in response to this environment partially protected OLN93 cells from hydrogen peroxide-induced cell death. CONCLUSIONS: Rat MAPC possess immune modulatory and neuroprotective properties which are enhanced in response to neuroinflammatory signals. These findings thereby warrant further research to evaluate MAPC transplantation as a therapeutic approach in diseases with an immunological and neurodegenerative component such as multiple sclerosis.

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.

Mutual interaction between human multipotent adult progenitor cells and NK cells.

Human multipotent adult progenitor cells (hMAPCs) are isolated from bone marrow with a more extensive expansion capacity compared to human mesenchymal stem cells (hMSCs) and with the ability to differentiate into endothelium. Like hMSCs, hMAPCs inhibit T-cell proliferation induced by alloantigens. In this study, we tested the interaction between hMAPCs and natural killer (NK) cells. We assessed the susceptibility of hMAPCs to NK cell-mediated lysis and the immunomodulation of hMAPCs on NK cell function during IL-2-driven stimulation and the cytolytic effector phase. Human MAPCs express the ligands PVR and ULBP-2/5/6, which are recognized by activating NK cell receptors. However, they also express MHC class I molecules, which induce inhibitory signals in NK cells. Freshly isolated NK cells at different effector:target ratios did not kill hMAPCs as assessed by an MTT and (51)Cr-release assay, while hMAPCs impaired the cytotoxic activity of resting NK cells against the NK-sensitive K562 leukemia cell line. By contrast, IL-2-stimulated NK cells were capable of killing hMAPCs, and preactivated NK cells were not influenced during their cytotoxic effector function against K562 cells by hMAPCs. When added during the 6-day preactivation phase with IL-2, hMAPCs dose-dependently reduced NK cell proliferation in an IDO-dependent manner, but they did not influence the induction of cytotoxic capacity by IL-2. This study indicates that human MAPCs mutually interact with NK cells.

Human multipotent adult progenitor cells are nonimmunogenic and exert potent immunomodulatory effects on alloreactive T-cell responses.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohn's disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

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.

Heart grafts tolerized through third-party multipotent adult progenitor cells can be retransplanted to secondary hosts with no immunosuppression.

Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal-type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor- and organ recipient-independent, third-party cell product. We report that stable allograft survival can be achieved following third-party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long-term accepted heart grafts recovered from MAPC-treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC-mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC-mediated allograft acceptance differs mechanistically from drug-induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC-based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third-party MAPC therapy after liver transplantation.

Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Fourth Meeting: lessons learned from first clinical trials.

The Fourth Expert Meeting of the Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Consortium took place in Barcelona on October 19 and 20, 2012. This meeting focused on the translation of preclinical data into early clinical settings. This position paper highlights the main topics explored on the safety and efficacy of mesenchymal stem cells as a therapeutic agent in solid organ transplantation and emphasizes the issues (proper timing, concomitant immunossupression, source and immunogenicity of mesenchymal stem cells, and oncogenicity) that have been addressed and will be followed up by the MiSOT Consortium in future studies.

Application of MultiStem(®) Allogeneic Cells for Immunomodulatory Therapy: Clinical Progress and Pre-Clinical Challenges in Prophylaxis for Graft Versus Host Disease.

The last decade has seen much progress in adjunctive cell therapy for immune disorders. Both corporate and institutional Phase III studies have been run using mesenchymal stromal cells (MSC) for treatment of Graft versus Host Disease (GvHD), and product approval has been achieved for treatment of pediatric GvHD in Canada and New Zealand (Prochymal(®); Osiris Therapeutics). This effectiveness has prompted the prophylactic use of adherent stem cells at the time of allogeneic hematopoietic stem cell transplantation (HSCT) to prevent occurrence of GvHD and possibly provide stromal support for hematopoietic recovery. The MultiStem(®) product is an adult adherent stem cell product derived from bone marrow which has significant clinical exposure. MultiStem cells are currently in phase II clinical studies for treatment of ischemic stroke and ulcerative colitis, with Phase I studies completed in acute myocardial infarction and for GvHD prophylaxis in allogeneic HSCT, demonstrating that MultiStem administration was well tolerated while the incidence and severity of GvHD was reduced. In advancing this clinical approach, it is important to recognize that alternate models exist based on clinical manufacturing strategies. Corporate sponsors exploit the universal donor properties of adherent stem cells and manufacture at large scale, with many products obtained from one or limited donors and used across many patients. In Europe, institutional sponsors often produce allogeneic product in a patient designated context. For this approach, disposable bioreactors producing <10 products/donor in a closed system manner are very well suited. In this review, the use of adherent stem cells for GvHD prophylaxis is summarized and the suitability of disposable bioreactors for MultiStem production is presented, with an emphasis on quality control parameters, which are critical with a multiple donor approach for manufacturing.

Differentiation assays of bone marrow-derived Multipotent Adult Progenitor Cell (MAPC)-like cells towards neural cells cannot depend on morphology and a limited set of neural markers.

There are accumulating studies that report a neurogenic potential of bone marrow-derived cells both in vitro as well as in vivo. Most claims of neural "transdifferentiation" have based their conclusions on morphology and neural gene expression. Recently, doubts have been raised about the validity of both outcome parameters since non-neural cells can extend neurites and show aberrant neural gene expression as a response to stress inducing factors. In this study, we compared bone marrow-derived Multipotent Adult Progenitor Cell (MAPC)-like cells and neural stem cells (NSC) in their morphology and neural gene expression profile after neural differentiation using three differentiation protocols. We evaluated the expression of five neuroglial antigens [neurofilament 200 (NF200); beta III tubulin (beta3 tub); tau; Glial Fibrillary Acidic Protein (GFAP); Myelin Basic Protein (MBP) and RIP antigen] using real-time PCR (RT-PCR) and immunocytochemistry (ICC). MAPC-like cells adopted a neural-like morphology in one protocol but a fibroblast-like morphology in the two other protocols. RT-PCR and ICC show that MAPC-like cells already express the neural antigens beta III tubulin and NF200 at baseline, but no upregulation of these genes after exposure to three distinct differentiation protocols was seen. In contrast, NSC adopt neural and glial morphologies with a clear increase in expression of all neuroglial genes in all differentiation protocols used. In conclusion, our data demonstrate that neural-like morphology and expression of a limited set of neural marker genes by MAPC-like cells after differentiation are not absolute proof of neural transdifferentiation because MAPC-like cells only partially meet the criteria which are fulfilled by NSC after neural differentiation.

Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells.

The inositol lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] is involved in a myriad of cellular processes, including the regulation of exocytosis and endocytosis. In this paper, we address the role of PtdIns(4,5)P2 in compound exocytosis from rat peritoneal mast cells. This process involves granule-plasma membrane fusion as well as homotypic granule membrane fusion and occurs without any immediate compensatory endocytosis. Using a novel quantitative immunofluorescence technique, we report that plasma membrane PtdIns(4,5)P2 becomes transiently depleted upon activation of exocytosis, and is not detected on the membranes of fusing granules. Depletion is caused by phospholipase C activity, and is mandatory for exocytosis. Although phospholipase C is required for Ca2+ release from internal stores, the majority of the requirement for PtdIns(4,5)P2 hydrolysis occurs downstream of Ca2+ signalling - as shown in permeabilised cells, where the inositol (1,4,5)-trisphosphate-Ca2+ pathway is bypassed. Neither generation of the PtdIns(4,5)P2 metabolite, diacylglycerol (DAG) or simple removal and/or sequestration of PtdIns(4,5)P2 are sufficient for exocytosis to occur. However, treatment of permeabilised cells with DAG induces a small potentiation of exocytosis, indicating that it may be required. We propose that a cycle of PtdIns(4,5)P2 synthesis and breakdown is crucial for exocytosis to occur in mast cells, and may have a more general role in all professional secretory cells.

Four stage liquid chromatographic selection of methionyl peptides for peptide-centric proteome analysis: the proteome of human multipotent adult progenitor cells.

Serial application of strong cation-exchange and diagonal reversed-phase chromatography selecting methionyl peptides by stepwise shifting them from their reduced to their sulfoxide and sulfone forms generates a four-stage fractionation system, allowing high coverage analysis of complex proteome digests by LC-MALDI-MS/MS. Application to the proteome of a human multipotent adult progenitor cell line (MAPC) identified 2151 proteins with high confidence as on average four MS/MS-spectra were linked to each protein. Our dataset contains several novel, potential marker proteins that may be evaluated as affinity-anchors for isolating different adult stem cells in further studies. Furthermore, at least 2 tyrosine kinases that were previously linked to the self-renewal potential of stem cells were identified, validating the stemness of the analyzed cells. We also present data hinting at possible involvement of the ubiquitin/proteasome machinery in steering proliferation and/or differentiation of MAPC. Finally, following comparison of the MAPC proteome with proteomes of four human differentiated cell lines reveals differential usage of chromosomal information: compared to differentiated cells, MAPC do not appear to hold any preference for expressing genes located on specific chromosomes.