Introduction: Stem cell therapies have been investigated as potential treatment modalities for chronic wounds however there has been limited success to date. Multipotent Adult Progenitor Cells (MAPCs©) have been identified as having potential as an allogenic stem cell product due to their high population doubling number and their characteristic dampening of T-cell proliferation. This helps to prevent autoimmunity and graft/cell rejection. Methods: We have developed a dressing, consisting of medical grade silicone coated with a heptylamine plasma polymer, which supports the growth and transfer of MAPCs to skin. To determine if the dressing can deliver functional stem cells into diabetic wounds, they were loaded with MAPCs and then placed over excisional wounds in both normal and diabetic mice. Results and discussion: Accelerated healing was observed in both the normal and diabetic wounds with wound gape being significantly smaller at day 3 when compared to controls. Wound analysis showed that treatment with the MAPC dressings dampened the inflammatory response with reduced numbers of neutrophils and macrophages observed. Additionally, an increase in pro-angiogenic VEGF and CD31 positive endothelial cells was observed indicating improved new blood vessel formation. The MAPC dressings had no effect on fibrosis with collagen I and III being equally affected in both control and treated wounds. Overall, the functionalized MAPC dressings improve healing responses particularly in diabetic mice with impaired healing responses and therefore, show potential for development as an advanced therapeutic approach for the treatment of chronic diabetic wounds.

Stem cells have been shown to have potential as a new therapy for burns and promote wound healing through decreasing inflammation and increasing angiogenesis. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived stem cells with outstanding self-renewal and differentiation capacity. MAPC cells also secrete a wide range of cytokines which can affect cellular activities. This article aimed to examine the effects of MAPC cells treatment on burn injury repair using a mouse model of partial thickness burn injury. The immunomodulatory effect of MAPC cells was investigated in vitro using a simultaneous T-cell proliferation assay. Partial thickness burns were created on the dorsal surface of mice and MAPC cells were administered via intradermal injection to the wound margins 24 h post-burn injury. The burn tissues were analysed macroscopically to determine wound area and histologically assessed to determine wound width and rate of re-epithelialisation. Immunohistochemistry and ELISA were employed to assess cell proliferation, inflammation and angiogenesis and collagen deposition in the burn area. MAPC cells inhibit the proliferation of stimulated T cells in culture. Burns intradermally injected with MAPC cells showed a significant reduction in the macroscopic wound area, histologic wound width and had an increased rate of re-epithelialisation. Immunohistochemistry and ELISA analysis of burn tissues showed dampened inflammation evidenced by a reduction in neutrophilic infiltration and modulation of inflammatory cytokines. Angiogenesis within the burn area was also improved in MAPC cell treated mice. However, no significant effect of MAPC cell treatment was observed on extracellular matrix production. Treatment of burns with MAPC cells improved burn injury repair with reduced time to healing, decreased inflammation and increased angiogenesis. These findings demonstrate the promising effects of MAPC cells on burn injury repair and suggest MAPC cells as a candidate source for clinical cell therapies.

Dysregulation of the immune system can initiate chronic inflammatory responses that exacerbate disease pathology. Multipotent adult progenitor cells (MAPC cells), an adult adherent bone-marrow derived stromal cell, have been observed to promote the resolution of uncontrolled inflammatory responses in a variety of clinical conditions including acute ischemic stroke, acute myocardial infarction (AMI), graft vs host disease (GvHD), and acute respiratory distress syndrome (ARDS). One of the proposed mechanisms by which MAPC cells modulate immune responses is via the induction of regulatory T cells (Tregs), however, the mechanism(s) involved remains to be fully elucidated. Herein, we demonstrate that, in an in vitro setting, MAPC cells increase Treg frequencies by promoting Treg proliferation and CD4 + T cell differentiation into Tregs. Moreover, MAPC cell-induced Tregs (miTregs) have a more suppressive phenotype characterized by increased expression of CTLA-4, HLA-DR, and PD-L1 and T cell suppression capacity. MAPC cells also promoted Treg activation by inducing CD45RA + CD45RO + transitional Tregs. Additionally, we identify transforming growth factor beta (TGFβ) as an essential factor for Treg induction secreted by MAPC cells. Furthermore, inhibition of indoleamine 2, 3-dioxygenase (IDO) resulted in decreased Treg induction by MAPC cells demonstrating IDO involvement. Our studies also show that CD14 + monocytes play a critical role in Treg induction by MAPC cells. Our study describes MAPC cell dependent Treg phenotypic changes and provides evidence of potential mechanisms by which MAPC cells promote Treg differentiation.

Cell-based therapy for the treatment of inflammatory disorders has focused on the application of mesenchymal stromal cells (MSCs) and multipotent adult progenitor cells (MAPCs). Despite the recent positive findings in industry-sponsored clinical trials of MSCs and MAPCs for graft vs host disease (GvHD), cell therapy is efficacious in some but not all patients, highlighting the need to identify strategies to enhance cell-based therapeutic efficacy. Here, we demonstrate the capacity for interferon (IFN)-γ licensing to enhance human MAPC efficacy and retention following early administration in a humanized mouse model of acute GvHD (aGvHD). Activation of the nuclear receptor peroxisome proliferator-activated receptor delta (PPARδ) negatively influenced the retention and efficacy of human MAPCs as well as IFN-γ-licensed MAPCs in the aGvHD model. PPARδ antagonism significantly enhanced the efficacy of human MAPCs when administered early in the humanized aGvHD model. COX-2 expression in human MAPC was significantly decreased in IFN-γ licensed MAPCs exposed to a PPARδ agonist. Importantly, MAPC exposure to the PPARδ antagonist in the presence of a COX-2 inhibitor indomethacin before administration significantly reduced the efficacy of PPARδ antagonized MAPCs in the aGvHD humanized mouse model. This is the first study to demonstrate the importance of PPARδ in human MAPC efficacy in vivo and highlights the importance of understanding the disease microenvironment in which cell-based therapies are to be administered. In particular, the presence of PPARδ ligands may negatively influence MAPC or MSC therapeutic efficacy.

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo β7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

Background Stem cell therapies have been widely investigated for their healing effects. However, the translation of these therapies has been hampered by the requirement to deliver live allogeneic or autologous cells directly to the wound in a clinical setting. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived adherent stem cells that secrete a wide range of factors known to accelerate the wound healing process. The aim of this study was to determine the impact of MAPC cells secretome on healing outcomes without the presence of MAPC cells. Methods The effect of MAPC-conditioned medium (MAPC-CM) on the capacity of keratinocytes, fibroblasts and endothelial cells to migrate and proliferate was determined in vitro using scratch wound closure and WST1 assay, respectively. The effect of MAPC-CM on collagen deposition and angiogenesis was also assessed using in vitro methods. Additionally, two excisional wounds were created on the dorsal surface of mice ( n = 8/group) and 100 μL of 20× MAPC-CM were intradermally injected to the wound margins. Wound tissues were collected at 3, 7 and 14 days post-wounding and stained with H&E for microscopic analysis. Immunohistochemistry was performed to investigate inflammation, angiogenesis and collagen deposition in the wounds. Results Skin fibroblasts, keratinocytes and endothelial cells treated with MAPC-CM all showed improved rates of scratch closure and increased cellular proliferation. Moreover, fibroblasts treated with MAPC-CM deposited more collagens I and III and endothelial cells treated with MAPC-CM showed increased capillary tube formation. Murine excisional wounds intradermally injected with MAPC-CM showed a significant reduction in the wound area and an increase in the rate of reepithelialisation. The results also showed that inflammatory cell infiltration was decreased while an increase in angiogenesis, as well as collagens I and III expressions, was observed. Conclusion These findings suggest that factors produced by MAPC cells can have an important effect on cutaneous wound healing by affecting skin cell proliferation and migration, balancing inflammation and improving the formation of extracellular matrix and angiogenesis. Development of stem cell-free therapy for the treatment of wounds may be a more clinically translatable approach for improving healing outcomes.

Background: Pre-clinical research with multi-potent adult progenitor cells (MAPC® cells, Multistem, Athersys Inc., Cleveland, Ohio) suggests their potential as an anti-inflammatory and immunomodulatory therapy in organ transplantation. Normothermic machine perfusion of the liver (NMP-L) has been proposed as a way of introducing therapeutic agents into the donor organ. Delivery of cellular therapy to human donor livers using this technique has not yet been described in the literature. The primary objectives of this study were to develop a technique for delivering cellular therapy to human donor livers using NMP-L and demonstrate engraftment.Methods: Six discarded human livers were perfused for 6 h at 37°C using the Liver Assist (Organ Assist, Groningen). 50 × 106 CMPTX-labeled MAPC cells were infused directly into the right lobe via the hepatic artery (HA, n = 3) or portal vein (PV, n = 3) over 20 min at different time points during the perfusion. Perfusion parameters were recorded and central and peripheral biopsies were taken at multiple time-points from both lobes and subjected to standard histological stains and confocal microscopy. Perfusate was analyzed using a 35-plex multiplex assay and proteomic analysis.Results: There was no detrimental effect on perfusion flow parameters on infusion of MAPC cells by either route. Three out of six livers met established criteria for organ viability. Confocal microscopy demonstrated engraftment of MAPC cells across vascular endothelium when perfused via the artery. 35-plex multiplex analysis of perfusate yielded 13 positive targets, 9 of which appeared to be related to the infusion of MAPC cells (including Interleukin's 1b, 4, 5, 6, 8, 10, MCP-1, GM-CSF, SDF-1a). Proteomic analysis revealed 295 unique proteins in the perfusate from time-points following the infusion of cellular therapy, many of which have strong links to MAPC cells and mesenchymal stem cells in the literature. Functional enrichment analysis demonstrated their immunomodulatory potential.Conclusion: We have demonstrated that cells can be delivered directly to the target organ, prior to host immune cell population exposure and without compromising the perfusion. Transendothelial migration occurs following arterial infusion. MAPC cells appear to secrete a host of soluble factors that would have anti-inflammatory and immunomodulatory benefits in a human model of liver transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P = .009), decreased expression of injury biomarker NGAL (P = .012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P = .019, medulla P = .001), downregulation of interleukin (IL)-1β (P = .050), and upregulation of IL-10 (P < .047) and Indolamine-2, 3-dioxygenase (P = .050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P < .001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

Cryopreservation is an essential part of tissue banking and effective cryopreservation methods are critical for the development of cost-effective cell therapy products. Cell sheets are an attractive subset of cell therapy types, and cryopreservation has the potential to further drive down costs of allogeneic cell sheet therapy. This is currently a challenge as adhered cell monolayers are more susceptible to membrane damage during the freezing process. In this article, we investigate the performance of a surface-modified dressing for the cryopreservation of cells and strategies to improve cell recovery. Cryopreservation of multipotent adult progenitor cells (MAPC®) was performed on cells following their attachment to a surface for different periods of time. MAPC cells, given just 1 h to attach, washed off and were not recovered on the surface following thawing. Cells attached for longer periods, elongated further, and were more susceptible to damage from cryopreservation. A temporal window was identified that could allow cryopreservation on adherent surfaces where cells had attached to a surface without full elongation. By functionalizing the surface with coupled hyaluronic acid, cell spreading was initially retarded, thereby widening this temporal window. This approach demonstrates a novel method for enhancing the recovery of cryopreserved cell sheets on surfaces.

This article summarizes the immunological properties of adult bone marrow-derived stem cells (BMSCs) and their use in the treatment of immune and neurological diseases and injuries. These cells have a broad range of effects on immune cells from both the innate and adaptive immune systems that allows them to modulate the repair response of the body. BMSCs have demonstrated therapeutic benefit in many animal models of disease, and as a result are currently being evaluated in over 100 clinical trials all over the world for a variety of indications.

Lymphodepletion strategies are used in the setting of transplantation (including bone marrow, hematopoietic cell, and solid organ) to create space or to prevent allograft rejection and graft versus host disease. Following lymphodepletion, there is an excess of IL-7 available, and T cells that escape depletion respond to this cytokine undergoing accelerated proliferation. Moreover, this environment promotes the skew of T cells to a Th1 pro-inflammatory phenotype. Existing immunosuppressive regimens fail to control this homeostatic proliferative (HP) response, and thus the development of strategies to successfully control HP while sparing T cell reconstitution (providing a functioning immune system) represents a significant unmet need in patients requiring lymphodepletion. Multipotent adult progenitor cells (MAPC®) have the capacity to control T cell proliferation and Th1 cytokine production. Herein, this study shows that MAPC cells suppressed anti-thymocyte globulin-induced cytokine production but spared T cell reconstitution in a pre-clinical model of lymphodepletion. Importantly, MAPC cells administered intraperitoneally were efficacious in suppressing interferon-γ production and in promoting the expansion of regulatory T cells in the lymph nodes. MAPC cells administered intraperitoneally accumulated in the omentum but were not present in the spleen suggesting a role for soluble factors. MAPC cells suppressed lymphopenia-induced cytokine production in a prostaglandin E2-dependent manner. This study suggests that MAPC cell therapy may be useful as a novel strategy to target lymphopenia-induced pathogenic T cell responses in lymphodepleted patients.

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.

19. Chiaki Komatsu, Yolandi van der Merwe, Lin He, Maxine R. Miller, Katie A. Lucy, Huamin Tang, Ian Rosner, Wendy Chen, Jila Noori, Valeria Fu, Michael Steketee, Gadi Wollstein, Mario Solari, Joel S. Schuman, Kevin C. Chan, Kia M. Washington. Retinal and optic nerve viability evaluated with optical coherence tomography, manganese-enhanced MRI and electroretinography after whole eye transplantation

Cell-based therapy has potential therapeutic value in autoimmune diseases such as rheumatoid arthritis (RA). In RA, reduction of disease activity has been associated with improvement in the function of regulatory T cells (Treg) and attenuated responses of proinflammatory effector T cells (Teff). Mesenchymal stem cells (MSCs) and related multipotent adult progenitor cells (MAPC) have strong anti-inflammatory and immunomodulatory properties and may be able to "reset" the immune system to a pre-RA state. MAPC are MSC-like cells that are slightly earlier in lineage, have greater expansion capacity, and can be used as "off-the-shelf" therapy. Assessment of cell-based therapy to treat arthritis and related diseases is limited by the lack of available biological correlates that can be measured early on and indicate treatment response. We set out to develop a functional measure that could be used ex vivo as a biomarker of response. We were able to demonstrate that MAPC products could inhibit Teff responses from patients with active RA and that Treg from RA patients suppressed Teff. This assay used ex vivo can be used with MAPC or Treg alone or in combination and reflects the overall level of Teff suppression. Use of a novel functional biomarker as an exploratory endpoint in trials of cell-based therapy should be of value to detect biological outcomes at a point prior to the time that clinical response might be observed.

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.-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-1 beta-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.

Primary graft dysfunction (PGD) is a significant cause of early morbidity and mortality following lung transplantation. Improved organ preservation techniques will decrease ischemia-reperfusion injury (IRI) contributing to PGD. Adult bone marrow-derived adherent stem cells, including mesenchymal stromal (stem) cells (MSCs) and multipotent adult progenitor cells (MAPCs), have potent anti-inflammatory actions, and we thus postulated that intratracheal MAPC administration during donor lung processing would decrease IRI. The goal of the study was therefore to determine if intratracheal MAPC instillation would decrease lung injury and inflammation in an ex vivo human lung explant model of prolonged cold storage and subsequent reperfusion.

Introduction: Acute respiratory distress syndrome (ARDS) is the most common cause of respiratory failure among critically ill subjects, sepsis and severe bacterial pneumonia being its most common causes. The only interventions that have proven beneficial are protective ventilation strategies and fluid conservation approaches. New therapies are needed to address this common clinical problem. Others and we have previously shown the beneficial effect of infusion of exogenous adult stem cells in different pre-clinical models of ARDS.Methods: In the present study endotoxin was infused intravenously into 14 sheep from which 6 received different doses of adult stem cells by intrabronchial delivery to evaluate the effect of stem cell therapy.Results: After administration of endotoxin, there was a rapid decline in oxygenation to hypoxemic values, indicative of severe-to-moderate ARDS. None of the animals treated with saline solution recovered to normal baseline values during the 6 hours that the animals were followed. In contrast, sheep treated with a dose of 40 million adult stem cells returned their levels of oxygen in their blood to baseline two hours after the cells were infused. Similarly, improvements in carbon dioxide (CO2) clearance, pulmonary vascular pressures and inflammation were observed and confirmed by histology and by the decrease in lung edema.Conclusions: We concluded that instillation of adult non-hematopoietic stem cells can diminish the impact of endotoxin and accelerate recovery of oxygenation, CO2 removal and inflammation in the ovine model, making the use of adult stem cells a real alternative for future therapies for ARDS.