Background: Multipotent adult progenitor cells (MAPC) are an adherent adult stem cell being evaluated as a treatment for ischemic stroke in humans under the name MultiStem®. However, the efficacy of MAPC cells for the treatment of intracerebral hemorrhage (ICH), the most devastating form of stroke for which there is no effective treatment, is not clear Method: The therapeutic efficacy of MAPC administration was evaluated in both autologous blood injection (ABI) and collagenase (COL) rat ICH models. We treated rats intravenously with 1.2x10 6 cells (sub-optimal dose based on MAPC efficacy in ischemic stroke) and 1.2x10 7 cells (optimal dose) at either 2 or 24h after ICH, and used 2 different doses of collagenase to better understand the dose responses. Outcome measurements included 4 sensorimotor tests (up to 28d), ventricular hypertrophy, spleen size, and body weight (N=128 rats tested across 4 separate experiments). Results: MAPC offered a robust benefit in both ICH models in a dose-dependent fashion. (1) ABI model: at the sub-optimal dose MAPCs had no significant effect on behavioral performance, but effectively reduced ventricular hypertrophy. At an optimal dose, MAPCs at 2h or 24h after ICH, robustly reduced deficits in all 4 behavioral tests, and reduced ventricular hypertrophy by 59% and 35% in 2h and 24h post-treatment groups, respectively. No difference in body weight and spleen size was observed. (2) COL model: MAPC administered 2h after high collagenase dose, reduced hematoma volume (hemispheric hemoglobin level), as measured at 48h after collagenase injection. In addition, MAPC administration significantly reduced neurological deficit in the COL model. Conclusions: MAPC provide a uniquely robust therapeutic effect on clinically relevant neurological and morphological outcomes in two different ICH models. MAPC also reduced bleeding in the COL model, suggesting the potential for MAPC as a safe acute therapeutic treatment after ICH. In addition to having beneficial effects on recovery processes, MAPC could be further evaluated as a candidate to limit the hematoma enlargement during the initial postictal period. We are currently investigating the mechanism of MAPC-induced post-ICH recovery as well as hemostasis using tissue microarray analysis.

Involvement of the cerebellum in the pathophysiology of hypoxic-ischemic encephalopathy (HIE) in preterm infants is increasingly recognized. We aimed to assess the neuroprotective potential of intravenously administered multipotent adult progenitor cells (MAPCs) in the preterm cerebellum. Instrumented preterm ovine fetuses were subjected to transient global hypoxia-ischemia (HI) by 25 minutes of umbilical cord occlusion at 0.7 of gestation. After reperfusion, two doses of MAPCs were administered intravenously. MAPCs are a plastic adherent bone-marrow-derived population of adult progenitor cells with neuroprotective potency in experimental and clinical studies. Global HI caused marked cortical injury in the cerebellum, histologically indicated by disruption of cortical strata, impeded Purkinje cell development, and decreased dendritic arborization. Furthermore, global HI induced histopathological microgliosis, hypomyelination, and disruption of white matter organization. MAPC treatment significantly prevented cortical injury and region-specifically attenuated white matter injury in the cerebellum following global HI. Diffusion tensor imaging (DTI) detected HI-induced injury and MAPC neuroprotection in the preterm cerebellum. This study has demonstrated in a preclinical large animal model that early systemic MAPC therapy improved structural injury of the preterm cerebellum following global HI. Microstructural improvement was detectable with DTI. These findings support the potential of MAPC therapy for the treatment of HIE and the added clinical value of DTI for the detection of cerebellar injury and the evaluation of cell-based therapy.

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BACKGROUND:Human multipotent adult progenitor cells (MAPC®) are an emerging therapy for traumatic brain injury (TBI); however, clinically translating a therapy involves overcoming many factors in vivo which are not present in pre-clinical testing. In this study we examined clinical parameters in vitro that may impact cell therapy efficacy. METHODS:MAPC were infused through varying gauged needles and catheters with and without chlorhexidine, and their viability tested with trypan blue exclusion. MAPC were co-cultured with phenytoin and celecoxib at relevant clinical concentrations for 1 h and 24 h. Anti-inflammatory potency was tested using a stimulated rat splenocyte co-culture and ELISA for TNF-α production. MAPC were cultured under different osmolar concentrations and stained with propidium iodide for viability. Anti-inflammatory potency was tested by co-culture of MAPC with naïve lymphocytes activated by CD3/CD28 beads, and Click-iT® Plus EdU was used to quantify proliferation by flow cytometry. RESULTS:The mean viability of the MAPC infused via needles was 95 ± 1%; no difference was seen with varying flow rate, but viability was notably reduced by chlorhexidine. MAPC function was not impaired by co-culture with phenytoin, celecoxib, or combination with both. Co-culture with phenytoin showed a decrease in TNF-α production as compared to the MAPC control. MAPC cultured at varying osmolar concentrations all had viabilities greater than 90% with no statistical difference between them. Co-culture of MAPC with CD3/CD28 activated PBMCs showed a significant reduction in proliferation as measured by EdU uptake. DISCUSSION:Needle diameter, phenytoin, celecoxib, and a relevant range of osmolarities do not impair MAPC viability or anti-inflammatory potency in vitro.

There is an urgent need for therapies that could reduce the disease burden of preterm hypoxic-ischemic encephalopathy. Here, we evaluate the long-term effects of multipotent adult progenitor cells (MAPC) on long-term behavioral outcomes in a preterm rat model of perinatal asphyxia. Rats of both sexes were treated with two doses of MAPCs within 24 h after the insult. Locomotor, cognitive and psychiatric impairments were evaluated starting at 1.5 (juvenile) and 6 months (adult). Hypoxia-ischemia affected locomotion, cognition, and anxiety in a sex-dependent manner, with higher vulnerability observed in males. The MAPC therapy partially attenuated deficits in object recognition memory in females of all tested ages, and in the adult males. The hypoxic insult caused delayed hyperactivity in adult males, which was corrected by MAPC therapy. These results suggest that MAPCs may have long-term benefits for neurodevelopmental outcome after preterm birth and global hypoxia-ischemia, which warrants further preclinical exploration.

Following spinal cord injury (SCI), inflammation amplifies damage beyond the initial insult, providing an opportunity for targeted treatments. An ideal protective therapy would reduce both edema within the lesion area and the activation/infiltration of detrimental immune cells. Previous investigations demonstrated the efficacy of intravenous injection of multipotent adult progenitor cells (MAPC®) to modulate immune response following SCI, leading to significant improvements in tissue sparing, locomotor and urological functions. Separate studies have demonstrated that tissue inhibitor of matrix metalloproteinase-3 (TIMP3) reduces blood-brain barrier permeability following traumatic brain injury in a mouse model, leading to improved functional recovery. This study examined whether TIMP3, delivered alone or in concert with MAPC cells, improves functional recovery from a contusion SCI in a rat model. The results suggest that intravenous delivery of MAPC cell therapy 1 day following acute SCI significantly improves tissue sparing and impacts functional recovery. TIMP3 treatment provided no significant benefit, and further, when co-administered with MAPC cells, it abrogated the therapeutic effects of MAPC cell therapy. Importantly, this study demonstrated for the first time that acute treatment of SCI with MAPC cells can significantly reduce the incidence of urinary tract infection (UTI) and the use of antibiotics for UTI treatment.

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.

The promise of cellular therapies as a treatment for stroke and other injuries and diseases of the central nervous system (CNS) has compelled researchers, clinicians, patients, and the public for the last 2 decades. Various types of cells isolated from numerous tissue sources have demonstrated benefit in improving outcomes in animal stroke models when administered intravenously in an acute time frame (minutes to days) after stroke onset. Data supporting the homing of cells to the brain and proximal to the infarct after intravenous administration is lacking, begging the question “How are cells from an array of tissue sources functioning to improve neurological recovery without the presence of the cells at the primary site of injury?” The last 10 years have seen an increased focus on understanding the importance the peripheral immune system plays in exacerbating and complicating treatment and recovery of patients with stroke. Based on seminal observations from the Willing, Pennypacker, and Offner laboratories, we now know that the spleen participates in the immune response in the acute time frame after stroke onset in animal studies, and the involvement of spleen has now been confirmed in observational human stroke studies.1–3 The accumulation of laboratory and clinical data supports the contention that acute intravenous administration of cell therapies provides long-term benefit after stroke by modulating the initial peripheral immune response, potentiating tissue repair and recovery, and these data may collectively signal the emergence of a new class of therapies for the treatment of ischemic stroke. After the initial injury in the brain after ischemic stroke, a complex set of inflammatory cascades are initiated resulting in the engagement of both innate and adaptive immune systems. Glial cells—the endogenous phagocytic immune cells of the brain—become activated and in concert with the injured parenchymal tissue, signal the peripheral immune system …

Background: Traumatic brain injury (TBI) is a major cause of death and disability. TBI results in a prolonged secondary central neuro-inflammatory response. Previously, we have demonstrated that multiple doses (2 and 24 h after TBI) of multipotent adult progenitor cells (MAPC) delivered intravenously preserve the blood-brain barrier (BBB), improve spatial learning, and decrease activated microglia/macrophages in the dentate gyrus of the hippocampus. In order to determine if there is an optimum treatment window to preserve the BBB, improve cognitive behavior, and attenuate the activated microglia/macrophages, we administered MAPC at various clinically relevant intervals. Methods: We administered two injections intravenously of MAPC treatment at hours 2 and 24 (2/24), 6 and 24 (6/24), 12 and 36 (12/36), or 36 and 72 (36/72) post cortical contusion injury (CCI) at a concentration of 10 million/kg. For BBB experiments, animals that received MAPC at 2/24, 6/24, and 12/36 were euthanized 72 h post injury. The 36/72 treated group was harvested at 96 h post injury. Results: Administration of MAPC resulted in a significant decrease in BBB permeability when administered at 2/24 h after TBI only. For behavior experiments, animals were harvested post behavior paradigm. There was a significant improvement in spatial learning (120 days post injury) when compared to cortical contusion injury (CCI) in groups when MAPC was administered at or before 24 h. In addition, there was a significant decrease in activated microglia/macrophages in the dentate gyrus of hippocampus of the treated group (2/24) only when compared to CCI. Conclusions: Intravenous injections of MAPC at or before 24 h after CCI resulted in improvement of the BBB, improved cognitive behavior, and attenuated activated microglia/macrophages in the dentate gyrus.

BACKGROUND:Multipotent adult progenitor cells are a bone marrow-derived, allogeneic, cell therapy product that modulates the immune system, and represents a promising therapy for acute stroke. We aimed to identify the highest, well-tolerated, and safest single dose of multipotent adult progenitor cells, and if they were efficacious as a treatment for stroke recovery. METHODS:We did a phase 2, randomised, double-blind, placebo-controlled, dose-escalation trial of intravenous multipotent adult progenitor cells in 33 centres in the UK and the USA. We used a computer-generated randomisation sequence and interactive voice and web response system to assign patients aged 18-83 years with moderately severe acute ischaemic stroke and a National Institutes of Health Stroke Scale (NIHSS) score of 8-20 to treatment with intravenous multipotent adult progenitor cells (400 million or 1200 million cells) or placebo between 24 h and 48 h after symptom onset. Patients were ineligible if there was a change in NIHSS of four or more points during at least a 6 h period between screening and randomisation, had brainstem or lacunar infarct, a substantial comorbid disease, an inability to undergo an MRI scan, or had a history of splenectomy. In group 1, patients were enrolled and randomly assigned in a 3:1 ratio to receive 400 million cells or placebo and assessed for safety through 7 days. In group 2, patients were randomly assigned in a 3:1 ratio to receive 1200 million cells or placebo and assessed for safety through the first 7 days. In group 3, patients were enrolled, randomly assigned, and stratified by baseline NIHSS score to receive 1200 million cells or placebo in a 1:1 ratio within 24-48 h. Patients, investigators, and clinicians were masked to treatment assignment. The primary safety outcome was dose-limiting toxicity effects. The primary efficacy endpoint was global stroke recovery, which combines dichotomised results from the modified Rankin scale, change in NIHSS score from baseline, and Barthel index at day 90. Analysis was by intention to treat (ITT) including all patients in groups 2 and 3 who received the investigational agent or placebo. This study is registered with ClinicalTrials.gov, number NCT01436487. FINDINGS:The study was done between Oct 24, 2011, and Dec 7, 2015. After safety assessments in eight patients in group 1, 129 patients were randomly assigned (67 to receive multipotent adult progenitor cells and 62 to receive placebo) in groups 2 and 3 (1200 million cells). The ITT populations consisted of 65 patients who received multipotent adult progenitor cells and 61 patients who received placebo. There were no dose-limiting toxicity events in either group. There were no infusional or allergic reactions and no difference in treatment-emergent adverse events between the groups (64 [99%] of 65 patients in the multipotent adult progenitor cell group vs 59 [97%] of 61 in the placebo group). There was no difference between the multipotent adult progenitor cell group and placebo groups in global stroke recovery at day 90 (odds ratio 1·08 [95% CI 0·55-2·09], p=0·83). INTERPRETATION:Administration of multipotent adult progenitor cells was safe and well tolerated in patients with acute ischaemic stroke. Although no significant improvement was observed at 90 days in neurological outcomes with multipotent adult progenitor cells treatment, further clinical trials evaluating the efficacy of the intervention in an earlier time window after stroke (<36 h) are planned. FUNDING:Athersys Inc.

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.

Stem cell therapy modulates not only the local microenvironment of the brain but also the systemic immune responses. We explored the impact of human multipotent adult progenitor cells (MAPC) modulating splenic activation and peripheral immune responses after ischemic stroke. Hundred twenty-six Long-Evans adult male rats underwent middle cerebral artery occlusion. Twenty-four hours later, they received IV MAPC or saline treatment. At 3 days after infusion, RNA was isolated from the injured cortex and spleen for microarray analysis. Spleen mass, splenocyte phenotype, and releasing cytokines were measured. Serum cytokines, MAPC biodistribution, brain lesion sizes and neurofunctional deficits were compared in rats treated with MAPC or saline with and without spleens. Stroked animals treated with MAPC exhibited genes that more closely resembled animals with sham surgery. Gene categories downregulated by MAPC included leukocyte activation, antigen presentation, and immune effector processing, associated with the signaling pathways regulated by TNF-α, IL-1β, IL-6, and IFN-γ within the brain. MAPC treatment restored spleen mass reduction caused by stroke, elevated Treg cells within the spleen, increased IL-10 and decreased IL-1β released by splenocytes. MAPC reduced IL-6 and IL-1β and upregulated IL-10 serum levels. Compared with saline, MAPC enhance stroke recovery in rats with intact spleens but had no effects in rats without spleens. MAPC restores expression of multiple genes and pathways involved in immune and inflammatory responses after stroke. Immunomodulation of the splenic response by the intravenous administration of MAPC may create a more favorable environment for brain repair after stroke. Stem Cells 2017;35:1290-1302.

Introduction: MultiStem is an adult, adherent, stem cell product, having shown safety and efficacy in other clinical indications and pre-clinical models of stroke. We assessed whether it was safe and improved outcomes in patients with ischemic stroke. Methods: B01-02 was a double-blind, placebo-controlled study of ischemic stroke patients (NIHSS 8-20, inclusive) treated within 24-48 hours of symptoms at 33 sites in the U.S. and U.K. Patients were randomized 1:1 and received infusion of 1.2 billion cells or placebo. Efficacy endpoints included Global Recovery (mRS ≤2, NIHSS Δ ≥75% and BI ≥95) and Excellent Outcome between groups (mRS ≤1, NIHSS ≤1, BI ≥95) at Day 90, among others. Safety end points included neurologic worsening, secondary infections, adverse events and mortality. Results: 126 patients formed the Intention-To-Treat (ITT) population, 65 receiving MultiStem, 61 placebo. The cell therapy did not show a significant benefit relative to placebo for the primary and secondary endpoints. However, MultiStem treatment was associated with lower rates of infections and pulmonary events, a reduction in hospitalization, and a reduction in life threatening adverse events and death. A higher proportion of patients receiving MultiStem treatment achieved an Excellent Outcome (p=0.10) compared to placebo. Post-hoc analyses indicate that, compared to placebo subjects (n=52), patients who received cell treatment earlier in the treatment window (≤36 hrs, n=27) had better Global Recovery (41.9% vs. 17.3%, p<0.01) and Excellent Outcome (18.5% v. 3.8%, p=0.03), had significantly better recovery by mRS shift analysis (p=0.03) and significantly reduced hospitalization (6.7d vs. 10.3 d, p<0.01). Conclusions: Administration of MultiStem is safe and well tolerated in ischemic stroke patients within 48 hours of onset and reduces adverse events and death. Post-hoc analyses suggest that earlier administration of MultiStem may provide therapeutic benefit. Additional clinical studies exploring the optimal time frame for MultiStem administration are warranted.

Introduction: B01-02 was a double-blind, placebo-controlled study of ischemic stroke patients (NIHSS 8-20, inclusive) treated within 24-48 hours of symptom onset with MultiStem, an adult, adherent, stem cell product. In pre-clinical models of stroke, MultiStem appears to confer improved neurological benefit through multiple mechanisms, including down-regulation of the peripheral immune response. We sought to determine whether this observation translated to acute ischemic stroke patients treated with MultiStem. Methods: Patients were randomized 1:1 and received IV infusion of 1.2 billion cells or placebo. After randomization, patients had blood drawn to determine baseline levels of circulating levels of CD3+ cells or specific inflammatory cytokines. Blood was drawn again at days 2, 7 and 30 post-treatment to determine the average fold change from baseline in each patient at each time point. Results: 126 patients formed the Intention-To-Treat (ITT) population, 65 receiving MultiStem, 61 placebo. 38 MultiStem patients and 40 placebo patient samples were analyzed for CD3+ cells, while 60 MultiStem patients and 55 placebo patients samples were analyzed for serum cytokine levels. CD3+ cell levels were significantly decreased in the MultiStem treatment group compared to placebo (p=0.001) at Day 2, but not later. IL-6 (p=.031) and IL-12 (p=.035) were both significantly downregulated in the MultiStem treatment group at Day 7. IL-1β (p=.065), TNF-α (p=.068) and IFN-γ (p=.100) also trended towards being significantly reduced at Day 7. Conclusions: IV administration of MultiStem within 24-48 hours of an ischemic stroke results in decreased circulating CD3+ cells, while reducing inflammatory cytokines in the blood during the first 7 days after onset of symptoms. These results support the hypothesis that MultiStem treatment limits the activation of the innate immune system’s response to stroke, which may provide improved neurological and recovery outcomes. Additional studies are required to confirm this observation and optimize the time of administration for MultiStem mediated benefit

Ischemic stroke is caused by a blockage of blood flow to the brain. A leading cause of death and disability globally, each year more than 15 million people are estimated to suffer a stroke, including more than two million people in the United States, Japan, and European Union combined. According to the American Heart Association, ischemic strokes comprise more than 85% of all strokes. Current standard of care for ischemic stroke involves the administration of a thrombolytic agent such as tissue plasminogen activator (tPA) within 3 to 4 hours after a stroke has occurred, a narrow window that results in only a small percentage of patients receiving such treatment. In light of this significant unmet clinical need, Athersys has developed a cell therapeutic, MultiStem, as a platform for treating acute central nervous system injury including stroke, traumatic brain injury, and spinal cord injury.1-6 The therapeutic premise reflects the strong immunomodulatory and anti-inflammatory influence shown by adherent (mesenchymal stromal cell-like) stem cells and posits that administration of MultiStem early after injury can diminish the systemic inflammatory response contributing to significant pathology and comorbidity in stroke. This hypothesis is supported by preclinical data demonstrating the role of splenic lymphocytes in onset of stroke pathology. When this population is absent or modulated, a striking prevention of injury and recovery benefit is seen.7-9 Accordingly, an exploratory Phase II clinical study was designed to test the core hypotheses and to determine patient stratification and dose regimens for subsequent development. MultiStem is a cell therapeutic isolated from adult marrow and expanded ex vivo for clinical use. Cells in this class have shown utility when used allogeneically and without patient matching, based on expression of metabolic and biologic factors which modulate the adaptive immune system away from immune sensitization.10 MultiStem has shown the ability to promote tissue repair and healing in a variety of ways, such as through the production of therapeutic factors produced in response to signals of inflammation and tissue damage. MultiStem therapy's potential for multidimensional therapeutic impact distinguishes it from traditional biopharmaceutical therapies focused on a single mechanism of benefit. The product represents a unique "off-the-shelf" stem cell product that can be manufactured in a scalable manner, may be stored for years in frozen form, and is administered without tissue matching or the need for immune suppression. The randomized, double-blind, placebo-controlled Phase II clinical trial was conducted at 33 sites in the United States and the United Kingdom.11 The study was conducted in two parts—a small dose selection phase involving 16 patients in two cohorts, followed by a larger efficacy phase of 118 patients. The evaluable patient population included eight patients from Cohort 2 and the Cohort 3 patients, who all received a high dose of MultiStem cells or placebo. The study enrolled subjects who received either MultiStem treatment or placebo 1 to 2 days after the stroke. The primary endpoints for the study include safety over the first 7 days after treatment and global stroke recovery at Day 90, which assesses disability (modified Rankin score ≤ 2), neurologic deficit (NIH Stroke Scale [NIHSS], Δ ≥ 75%), and activities of daily living (Barthel index [BI] ≥ 95%). Additionally, there are secondary and exploratory endpoints evaluating elements of recovery and dysfunction, including biomarkers associated with subject condition and recovery and safety variables over the study period. Of the patients evaluated in the study, 65 patients were in the MultiStem treatment group and 61 patients were in the placebo group. Among the enrolled patients, the groups were generally evenly balanced in terms of baseline stroke characteristics (Table 1). As described in Table 2, patients who received MultiStem treatment earlier in the treatment window (24-36 hr poststroke) exhibited more favorable recovery on the primary and key secondary endpoints than patients who received placebo or patients who received MultiStem treatment later (e.g., excellent outcome; p = 0.03), and this treatment effect was even more pronounced the earlier the MultiStem administration within the 24- to 36-hr time frame. As noted above, post hoc analyses show that earlier MultiStem administration appears to provide substantial benefit, as evident in Table 2. This exploratory trial represents the first cell based Phase II clinical study for stroke and was designed to evaluate the safety and efficacy of a single dose of MultiStem 24 to 48 hours after the occurrence of the stroke. This treatment window extends well beyond the limits of current standard of care, treatment with tPA, which may only be administered within the first several hours after a stroke. The core hypothesis for mode of action was validated, with the finding that induction of circulating T cells by stroke was dramatically and statistically reduced after MultiStem administration. This correlated with the significant reduction in death and infectious complications and validates this core understanding. Interim analysis of core inflammatory biomarkers, notably IL-6, show their down regulation correlating with MultiStem treatment. While the trial did not achieve the primary or component secondary endpoints, the evidence indicating that patients who received MultiStem treatment early appeared to exhibit meaningfully better clinical outcomes and recovery is promising. The results appear to confirm that the window of intervention with MultiStem therapy may extend beyond the limits of current care using tPA. RM is an employee of Athersys, Inc. RD is a consultant for Athersys, Inc.

Background: In a phase II randomized, placebo controlled trial, MultiStem, an adult stem cell, was administered IV within 24 to 48 hrs after stroke onset. Earlier administration of MultiStem (<36 hrs) may have provided a benefit. In preclinical studies, we explored immune targets during this time frame that may underlie potential treatment effects. Methods: Rats underwent tandem CCA/MCA occlusion and then at 24 hrs were randomized to receive IV MultiStem or saline (N=3-6 per group). At 3 days after infusion, rats were sacrificed and RNA was isolated from the injured cortex and spleen. To assess differential expression, noninformative probes were removed with a variance cut-off and genes having p-value < 0.01 were retained for gene set enrichment analysis. Principal component analysis and hierarchical clustering were performed. Results: Among 364 selected genes within the brain, the gene expression profile of MultiStem treated stroke animals aligned with sham animals while there was a clear divergent profile of expression in vehicle treated stroke rats (fig). At 28d, samples from MultiStem treated animals clustered even more closely with shams and remained distinct from vehicle treated animals. Highly enriched gene expression categories downregulated by MultiStem included leukocyte activation, antigen presentation, and immune effector processing. Gene expression profiles in spleens derived from MultiStem treated rats closely resembled shams at 3 and 28 days. Using Ingenuity Pathway Analysis, MultiStem was shown to suppress the activation of various inflammatory signaling pathways regulated by TNF-α, IL-1β, IL-6, and IFN-γ, within peri-infarct tissue at 3 days after infusion. Conclusion: MultiStem restores expression of multiple genes and pathways involved in immune and inflammatory responses after stroke. Optimal therapeutic windows for acute stroke may depend upon targeting the activation of immune responses from peripheral tissues such as the spleen.

Following spinal cord injury (SCI), immune-mediated secondary processes exacerbate the extent of permanent neurological deficits. We investigated the capacity of adult bone marrow-derived stem cells, which exhibit immunomodulatory properties, to alter inflammation and promote recovery following SCI. In vitro, we show that human multipotent adult progenitor cells (MAPCs) have the ability to modulate macrophage activation and prior exposure to MAPC secreted factors can reduce macrophage-mediated axonal dieback of dystrophic axons. Using a contusion model of SCI, we found that intravenous delivery of MAPCs one day, but not immediately, after SCI significantly improves urinary and locomotor recovery, which was associated with marked spinal cord tissue sparing. Intravenous MAPCs altered the immune response in the spinal cord and periphery, however biodistribution studies revealed that no MAPCs were found in the cord and instead preferentially homed to the spleen. Our results demonstrate that MAPCs exert their primary effects in the periphery and provide strong support for the use of these cells in acute human contusive SCI.

BACKGROUND:Preterm infants are at risk for hypoxic-ischemic encephalopathy. No therapy exists to treat this brain injury and subsequent long-term sequelae. We have previously shown in a well-established pre-clinical model of global hypoxia-ischemia (HI) that mesenchymal stem cells are a promising candidate for the treatment of hypoxic-ischemic brain injury. In the current study, we investigated the neuroprotective capacity of multipotent adult progenitor cells (MAPC®), which are adherent bone marrow-derived cells of an earlier developmental stage than mesenchymal stem cells and exhibiting more potent anti-inflammatory and regenerative properties.METHODS:Instrumented preterm sheep fetuses were subjected to global hypoxia-ischemia by 25 min of umbilical cord occlusion at a gestational age of 106 (term ~147) days. During a 7-day reperfusion period, vital parameters (e.g., blood pressure and heart rate; baroreceptor reflex) and (amplitude-integrated) electroencephalogram were recorded. At the end of the experiment, the preterm brain was studied by histology.RESULTS:Systemic administration of MAPC therapy reduced the number and duration of seizures and prevented decrease in baroreflex sensitivity after global HI. In addition, MAPC cells prevented HI-induced microglial proliferation in the preterm brain. These anti-inflammatory effects were associated with MAPC-induced prevention of hypomyelination after global HI. Besides attenuation of the cerebral inflammatory response, our findings showed that MAPC cells modulated the peripheral splenic inflammatory response, which has been implicated in the etiology of hypoxic-ischemic injury in the preterm brain.CONCLUSIONS:In a pre-clinical animal model MAPC cell therapy improved the functional and structural outcome of the preterm brain after global HI. Future studies should establish the mechanism and long-term therapeutic effects of neuroprotection established by MAPC cells in the developing preterm brain exposed to HI. Our study may form the basis for future clinical trials, which will evaluate whether MAPC therapy is capable of reducing neurological sequelae in preterm infants with hypoxic-ischemic encephalopathy.

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.