Rie Onodera

Intravenous administration of auto serum-expanded autologous mesenchymal stem cells in stroke

Transplantation of human mesenchymal stem cells has been shown to reduce infarct size and improve functional outcome in animal models of stroke. Here, we report a study designed to assess feasibility and safety of transplantation of autologous human mesenchymal stem cells expanded in autologous human serum in stroke patients. We report an unblinded study on 12 patients with ischaemic grey matter, white matter and mixed lesions, in contrast to a prior study on autologous mesenchymal stem cells expanded in foetal calf serum that focused on grey matter lesions. Cells cultured in human serum expanded more rapidly than in foetal calf serum, reducing cell preparation time and risk of transmissible disorders such as bovine spongiform encephalomyelitis. Autologous mesenchymal stem cells were delivered intravenously 36-133 days post-stroke. All patients had magnetic resonance angiography to identify vascular lesions, and magnetic resonance imaging prior to cell infusion and at intervals up to 1 year after. Magnetic resonance perfusion-imaging and 3D-tractography were carried out in some patients. Neurological status was scored using the National Institutes of Health Stroke Scale and modified Rankin scores. We did not observe any central nervous system tumours, abnormal cell growths or neurological deterioration, and there was no evidence for venous thromboembolism, systemic malignancy or systemic infection in any of the patients following stem cell infusion. The median daily rate of National Institutes of Health Stroke Scale change was 0.36 during the first week post-infusion, compared with a median daily rate of change of 0.04 from the first day of testing to immediately before infusion. Daily rates of change in National Institutes of Health Stroke Scale scores during longer post-infusion intervals that more closely matched the interval between initial scoring and cell infusion also showed an increase following cell infusion. Mean lesion volume as assessed by magnetic resonance imaging was reduced by >20% at 1 week post-cell infusion. While we would emphasize that the current study was unblinded, did not assess overall function or relative functional importance of different types of deficits, and does not exclude placebo effects or a contribution of recovery as a result of the natural history of stroke, our observations provide evidence supporting the feasibility and safety of delivery of a relatively large dose of autologous mesenchymal human stem cells, cultured in autologous human serum, into human subjects with stroke and support the need for additional blinded, placebo-controlled studies on autologous mesenchymal human stem cell infusion in stroke.

Long-term clinical outcome after intramuscular implantation of bone marrow mononuclear cells (Therapeutic Angiogenesis by Cell Transplantation [TACT] trial) in patients with chronic limb ischemia.

BACKGROUND Angiogenic cell therapy by intramuscular injection of autologous bone marrow mononuclear cells was first attempted in patients with peripheral artery disease (PAD) with critical limb ischemia, and the feasibility was shown by a randomized controlled Therapeutic Angiogenesis by Cell Transplantation (TACT) study. METHODS AND RESULTS The present study was designed to assess the 3-year safety and clinical outcomes of this angiogenic cell therapy by investigating the mortality and leg amputation-free interval as primary end points. The median follow-up time for surviving patients was 25.3 months (range, 0.8-69.0 months), and 3-year overall survival rates were 80% (95% CI 68-91) in patients with atherosclerotic peripheral arterial disease (11 died in 74 patients) and 100% (no death) in 41 patients with thromboangiitis obliterans (TAO; Buergers disease). Three-year amputation-free rate was 60% (95% CI 46-74) in PAD and 91% (95% CI 82-100) in patients with TAO. The multivariate analysis revealed that the severity of rest pain and repeated experience of bypass surgery were the prognostic factors negatively affecting amputation-free interval. The significant improvement in the leg pain scale, ulcer size, and pain-free walking distance was maintained during at least 2 years after the therapy, although the ankle brachial index and transcutaneous oxygen pressure value did not significantly change. CONCLUSIONS The angiogenic cell therapy using bone marrow mononuclear cells can induce a long-term improvement in limb ischemia, leading to extension of amputation-free interval. The safety and efficacy are not inferior to the conventional revascularization therapies.

Intramuscular Transplantation of G‐CSF‐Mobilized CD34+ Cells in Patients With Critical Limb Ischemia: A Phase I/IIa, Multicenter, Single‐Blinded, Dose‐Escalation Clinical Trial

A number of preclinical studies have indicated the therapeutic potential of endothelial progenitor cells for vascular regeneration in ischemic diseases. A phase I/IIa clinical trial of transplantation of autologous CD34+ cells, the endothelial and hematopoietic progenitor‐enriched fraction, was performed in no‐option patients with atherosclerotic peripheral artery disease or Buergers disease with critical limb ischemia (CLI). CD34+ cells were isolated from the G‐CSF‐mobilized apheresis product using a magnetic cell sorting system. CD34+ cells (105/kg, n = 6; 5 × 105/kg, n = 8; or 106/kg, n = 3) were injected i.m. into the leg with more severe ischemia. The Efficacy Score, representing changes in the toe brachial pressure index (TBPI), Wong‐Baker FACES pain rating scale, and total walking distance 12 weeks after cell transplantation, the primary endpoint, was positive, indicating improvement in limb ischemia in all patients, although no significant dose‐response relationship was observed. During the 12‐week observation after cell therapy, the Wong‐Baker FACES pain rating scale, TBPI, transcutaneous partial oxygen pressure, total or pain‐free walking distance, and ulcer size serially improved in all patients. No death or major amputation occurred, and severe adverse events were rare, although mild to moderate events relating to G‐CSF and leukapheresis were frequent during the 12‐week follow‐up. In conclusion, the outcomes of this prospective clinical study indicate the safety and feasibility of CD34+ cell therapy in patients with CLI. Favorable trends in efficacy parameters encourage a randomized and controlled trial in the future. STEM CELLS 2009;27:2857–2864

Spinal cord injury treatment with intrathecal autologous bone marrow stromal cell transplantation: the first clinical trial case report.

Spinal cord injury often results in devastating dysfunction and disability. When a spinal cord is injured, various symptoms are presented depending on the segments of the damage and the degree. If cervical spinal damage is severe, tetraplegia results. If damage occurs at levels higher than C4, diaphragmatic movement will be impaired, and the patient has to live being connected with the ventilator on the bed. Patients will suffer from acute hyperesthesia or severe chronic pain, urinary and rectal dysfunction, and autonomic dystonia as well as motor and sensory deficits. In Japan, there are more than 100,000 victims suffering from spinal injury, and a new 5,000 to 6,000 patients are added every year. In the Unites States, about 250,000 to 400,000 people are living with spinal cord injury, and there are about 11,000 to 13,000 new injuries every year. The number of incidence is increasing. The majority of them result from motor vehicle or sports injuries, violence, or falls. An injured central nervous system never regenerates. This has long been thought as a medical common sense terms. Therefore, the principal object for the treatment of spinal injury was mainly purposed how to minimize the progression of secondary injuries and maintain the remnant function of the spine. For the purpose of preventing secondary spinal cord injury, spine stabilization for the fracture or dislocation and rehabilitation were the main strategy in the treatment. There has been no successful treatment for the severe spinal cord injury to recover the function satisfactorily. However, if spinal cord damage is functionally improved even at the minimum, it will affect not only the physical, mental, and economic status of patients and their families, but also the medical resources of society. Recently, regenerative treatments with stem cells are in the limelight. However, there are some serious problems such as ethical ones to be solved for the study with stem cells. We reported significant recovery of motor function in rats with experimental spinal cord injury treated by transplanting bone marrow stromal cells (BMSCs) in the cerebrospinal fluid (CSF). Based on that study, we aimed at the clinical application of this treatment, and actually planned a clinical trial of spinal cord injury treatment by transplanting patient’s autologous BMSCs into CSF in the acute phase after spinal cord injury, at Kansai Medical University Hospital. We have developed a detailed protocol for the clinical trial. The medical ethics committees of the institutions have approved the protocol officially. This clinical trial aims to treat a damaged spinal cord by a novel method of injecting BMSCs into CSF through the lumbar puncture, and assess the safety and efficacy of the procedure. Although we have experienced only a single case, a committee that monitors the data to assess the efficacy and safety of the trial with members independent of this study team has evaluated the safety of the trial in this case, approved to continue the study, and agreed to submit a report of the first case. In addition, Japan Spinal Cord Foundation strongly requested to disclose the course of the first case. Therefore, we would like to publish the report of the first case to enhance research work on the new strategy for the difficult treatment of spinal cord injury. Submitted for publication July 6, 2007. Accepted for publication September 18, 2007. Copyright

Mesenchymal stem cells: therapeutic outlook for stroke.

Adult bone marrow-derived mesenchymal stem cells (MSCs) display a spectrum of functional properties. Transplantation of these cells improves clinical outcome in models of cerebral ischemia and spinal cord injury via mechanisms that may include replacement of damaged cells, neuroprotective effects, induction of axonal sprouting, and neovascularization. Therapeutic effects have been reported in animal models of stroke after intravenous delivery of MSCs, including those derived from adult human bone marrow. Initial clinical studies on intravenously delivered MSCs have now been completed in human subjects with stroke. Here, we review the reparative and protective properties of transplanted MSCs in stroke models, describe initial human studies on intravenous MSC delivery in stroke, and provide a perspective on prospects for future progress with MSCs.

Topical insulin-like growth factor 1 treatment using gelatin hydrogels for glucocorticoid- resistant sudden sensorineural hearing loss: a prospective clinical trial

BackgroundSudden sensorineural hearing loss (SSHL) is a common condition in which patients lose the hearing in one ear within 3 days. Systemic glucocorticoid treatments have been used as standard therapy for SSHL; however, about 20% of patients do not respond. We tested the safety and efficacy of topical insulin-like growth factor 1 (IGF1) application using gelatin hydrogels as a treatment for SSHL.MethodsPatients with SSHL that showed no recovery to systemic glucocorticoid administration were recruited. We applied gelatin hydrogels, impregnated with recombinant human IGF1, into the middle ear. The primary outcome measure was the proportion of patients showing hearing improvement 12 weeks after the test treatment. The secondary outcome measures were the proportion of patients showing improvement at 24 weeks and the incidence of adverse events. The null hypothesis was that 33% of patients would show hearing improvement, as was reported for a historical control after hyperbaric oxygen therapy.ResultsIn total, 25 patients received the test treatment at a median of 23 days (range 15-32) after the onset of SSHL, between 2007 and 2009. At 12 weeks after the test treatment, 48% (95% CI 28% to 69%; P = 0.086) of patients showed hearing improvement, and the proportion increased to 56% (95% CI 35% to 76%; P = 0.015) at 24 weeks. No serious adverse events were observed.ConclusionsTopical IGF1 application using gelatin hydrogels is well tolerated and may be efficacious for hearing recovery in patients with SSHL that is resistant to systemic glucocorticoids.

Long-term clinical outcomes for patients with lower limb ischemia implanted with G-CSF-mobilized autologous peripheral blood mononuclear cells

BACKGROUND Many studies have described the clinical effects of treating critical limb ischemia with granulocyte colony-stimulating factor-mobilized autologous peripheral blood mononuclear cells (M-PBMNC); however, there are no long-term data available on survival, limb salvage, or prognostic factors. METHODS To investigate the long-term clinical outcomes of M-PBMNC implantation, we reviewed data for 162 consecutive patients with limb ischemia who were treated with M-PBMNC implantation at 6 hospitals between 2001 and 2006. A subset of 123 patients with homogenous clinical profiles was selected for prognostic factor analysis. RESULTS Of the 162 patients, 50 died during the follow-up period. The median follow-up time for surviving patients was 26.4 months. The 2-year survival rate was 65% for the 140 patients with arteriosclerosis obliterans (ASO), and 100% for the 11, 4 and 7 patients with thromboangiitis obliterans (TAO), diabetic gangrene (DG) and connective tissue disease (CTD), respectively. The 1-year amputation-free rates for ASO, TAO, DG and CTD were 70%, 79%, 75% and 83%, respectively. Common serious adverse events included heart failure (15 cases), myocardial infarction (15 cases), serious infection (13 cases), stroke (10 cases), and malignant tumor (9 cases). Significant negative prognostic factors associated with overall survival were ischemic heart disease and collection of a small number of CD34-positive cells. Factors associated with time-to-amputation and amputation-free survival were a combination of Fontaine classification and lower limb gangrene, and history of dialysis. CONCLUSIONS Collection of a small number of CD34-positive cells and ischemic heart disease were associated with a reduction in overall survival.

Bone marrow mononuclear cells versus G-CSF-mobilized peripheral blood mononuclear cells for treatment of lower limb ASO: pooled analysis for long-term prognosis.

In this study, we report the comparative result of long-term clinical prognoses for patients with no-option critical limb ischemia (CLI) caused by arteriosclerosis obliterans, who are implanted with autologous bone marrow mononuclear cells (BMMNC; n=74) or G-CSF-mobilized (M)-PBMNC (n=111), as no information is available on how the two treatments compare in terms of long-term prognosis, such as survival or amputation. We performed pooled analysis using data from two previous cohort studies. All patients had disease of Fontaine classification III or IV. The endpoints were OS and amputation-free survival (AFS). After adjustment for history of dialysis and Fontaine classification, there was no significant difference between the two treatments with respect to OS (hazard ratio (HR)=1.49; 95% confidence interval (CI)=0.74–3.03, P=0.26) or AFS (HR=0.96; 95% CI=0.61–1.51, P=0.87). The negative prognostic factors affecting OS or AFS were the small number of CD34-positive cells collected, history of dialysis, Fontaine classification, male sex and older age. These results suggest that there was no significant difference in long-term prognosis between patients treated with BMMNC and those treated with M-PBMNC. The number of CD34-positive cells collected was an important prognostic factor for amputation and death.

Bilateral cortical hyperactivity detected by fMRI associates with improved motor function following intravenous infusion of mesenchymal stem cells in a rat stroke model.

Intravenous transplantation of mesenchymal stem cells (MSCs) derived from bone marrow ameliorates functional deficits in rat cerebral infarction models. In this study, MSCs were intravenously administered 6h after right middle cerebral artery occlusion (MCAO) induction in rat. Functional MRI (fMRI) during electrical stimulation of the left forepaw and behavioral testing (treadmill stress test) were carried out at day 1, 4, 7, 14, 21, 28 and 42 following MCAO. In medium infused group (n=20) electrical stimulation of the left forepaw elicited a unilateral (right cortex) activated signal detected by fMRI in the infarcted somatosensory cortex. In the MSC infused animals two fMRI patterns were observed: unilateral (n=17) and bilateral (n=19) activation of sensorimotor cortex. In the MSC group both unilateral and bilateral cortical activated animals displayed significantly improved motor function compared to the medium infused group. However, the bilateral activated pattern in the MSC group showed the greatest functional recovery. Lesion volume as calculated from high intensity signals using T2WI was less in the MSC groups as compared to the medium group, but the lesion volume for the unilateral and bilateral signals in the MSC group was the same. These results suggest that the presence of a bilateral signal in sensorimotor cortex as detected by fMRI was more predictive of improved functional outcome than lesion volume alone.

Intravenous infusion of mesenchymal stem cells inhibits intracranial hemorrhage after recombinant tissue plasminogen activator therapy for transient middle cerebral artery occlusion in rats

OBJECTIVE Reperfusion therapy with intravenous recombinant tissue plasminogen activator (rtPA) is the standard of care for acute ischemic stroke. However, hemorrhagic complications can result. Intravenous infusion of mesenchymal stem cells (MSCs) reduces stroke volume and improves behavioral function in experimental stroke models. One suggested therapeutic mechanism is inhibition of vascular endothelial dysfunction. The objective of this study was to determine whether MSCs suppress hemorrhagic events after rtPA therapy in the acute phase of transient middle cerebral artery occlusion (tMCAO) in rats. METHODS After induction of tMCAO, 4 groups were studied: 1) normal saline [NS]+vehicle, 2) rtPA+vehicle, 3) NS+MSCs, and 4) rtPA+MSCs. The incidence rate of intracerebral hemorrhage, both hemorrhagic and ischemic volume, and behavioral performance were examined. Matrix metalloproteinase-9 (MMP-9) levels in the brain were assessed with zymography. Quantitative analysis of regional cerebral blood flow (rCBF) was performed to assess hemodynamic change in the ischemic lesion. RESULTS The MSC-treated groups (Groups 3 and 4) experienced a greater reduction in the incidence rate of intracerebral hemorrhage and hemorrhagic volume 1 day after tMCAO even if rtPA was received. The application of rtPA enhanced activation of MMP-9, but MSCs inhibited MMP-9 activation. Behavioral testing indicated that both MSC-infused groups had greater improvement than non-MSC groups had, but rtPA+MSCs provided greater improvement than MSCs alone. The rCBF ratio of rtPA groups (Groups 2 and 4) was similar at 2 hours after reperfusion of tMCAO, but both were greater than that in non-rtPA groups. CONCLUSIONS Infused MSCs may inhibit endothelial dysfunction to suppress hemorrhagic events and facilitate functional outcome. Combined therapy of infused MSCs after rtPA therapy facilitated early behavioral recovery.