Kenichi Yamahara

Direct Intramyocardial But Not Intracoronary Injection of Bone Marrow Cells Induces Ventricular Arrhythmias in a Rat Chronic Ischemic Heart Failure Model

Background— Therapeutic efficacy of bone marrow (BM) cell injection for treating ischemic chronic heart failure has not been established. In addition, experimental data are lacking on arrhythmia occurrence after BM cell injection. We hypothesized that therapeutic efficacy and arrhythmia occurrence induced by BM cell injection may be affected by the cell delivery route. Methods and Results— Three weeks after left coronary artery ligation, wild-type female rats were injected with 1×107 mononuclear BM cells derived from green fluorescent protein–transgenic male rats through either a direct intramyocardial or a retrograde intracoronary route. Both intramyocardial and intracoronary injection of BM cells demonstrated similar improvement in left ventricular ejection fraction measured by echocardiography and a similar graft size analyzed by real-time polymerase chain reaction for the Y chromosome–specific Sry gene. Noticeably, intramyocardial injection of BM cells induced frequent ventricular premature contractions (108±73 per hour at 7 days after BM cell injection), including multiform, consecutive ventricular premature contractions and ventricular tachycardia for the initial 14 days; intracoronary injection of BM cells and intramyocardial injection of phosphate-buffered saline rarely induced arrhythmias. Immunohistochemistry demonstrated that intramyocardial BM cell injection formed distinct cell clusters containing donor-derived cells and accumulated host-derived inflammatory cells in the infarct border zone, whereas intracoronary BM cell injection provided more homogeneous donor cell dissemination with less inflammation and without disrupting the native myocardial structure. Conclusions— BM cell injection is able to improve cardiac function in ischemic chronic heart failure but has a risk of arrhythmia occurrence when the intramyocardial route is used. Such arrhythmias may be prevented by using the intracoronary route.

Choice of cell-delivery route for skeletal myoblast transplantation for treating post-infarction chronic heart failure in rat

Background Intramyocardial injection of skeletal myoblasts (SMB) has been shown to be a promising strategy for treating post-infarction chronic heart failure. However, insufficient therapeutic benefit and occurrence of ventricular arrhythmias are concerns. We hypothesised that the use of a retrograde intracoronary route for SMB-delivery might favourably alter the behaviour of the grafted SMB, consequently modulating the therapeutic effects and arrhythmogenicity. Methods and Results Three weeks after coronary artery ligation in female wild-type rats, 5×106 GFP-expressing SMB or PBS only (control) were injected via either the intramyocardial or retrograde intracoronary routes. Injection of SMB via either route similarly improved cardiac performance and physical activity, associated with reduced cardiomyocyte-hypertrophy and fibrosis. Grafted SMB via either route were only present in low numbers in the myocardium, analysed by real-time PCR for the Y-chromosome specific gene, Sry. Cardiomyogenic differentiation of grafted SMB was extremely rare. Continuous ECG monitoring by telemetry revealed that only intramyocardial injection of SMB produced spontaneous ventricular tachycardia up to 14 days, associated with local myocardial heterogeneity generated by clusters of injected SMB and accumulated inflammatory cells. A small number of ventricular premature contractions with latent ventricular tachycardia were detected in the late-phase of SMB injection regardless of the injection-route. Conclusion Retrograde intracoronary injection of SMB provided significant therapeutic benefits with attenuated early-phase arrhythmogenicity in treating ischaemic cardiomyopathy, indicating the promising utility of this route for SMB-delivery. Late-phase arrhythmogenicity remains a concern, regardless of the delivery route.

Heterogeneic nature of adult cardiac side population cells

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, alpha-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2(+) cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin(-) cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin(-) cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.

Quantitative assessment of initial retention of bone marrow mononuclear cells injected into the coronary arteries

BACKGROUNDnIntracoronary injection of bone marrow mononuclear cells (BMMNC) is a common clinical protocol of cell transplantation for heart disease, but poor engraftment of donor cells in the heart, which will limit its therapeutic efficacy, is a major issue. Initial retention (endothelial adherence and/or extravasation) of BMMNC immediately after intracoronary injection is a key step toward successful engraftment; however, this event has not been fully characterized. The aim of this study is to quantitatively clarify the frequency of retention of BMMNC after intracoronary injection, determine the impact of prior induction of ischemia-reperfusion injury on retention efficiency, and elucidate the underlying mechanisms focusing on adhesion molecule-mediated cell-cell interactions.nnnMETHODSnOne million BMMNC collected from green fluorescent protein (GFP)-transgenic mice were injected into the coronary arteries of syngeneic wild-type mouse hearts under Langendorff perfusion. Retention efficiency was quantitatively estimated from the GFP-positive cell number flushed out into the coronary effluent.nnnRESULTSnWhereas only 13.3 ± 1.2% of injected BMMNC were retained into normal hearts, prior induction of 30-minute ischemia and 30-minute reperfusion increased the retention efficiency to 36.5 ± 1.6% (p < 0.05, n = 8). Immunoconfocal observation further confirmed this enhanced retention after ischemia-reperfusion. Noticeably, the enhanced retention efficiency after ischemia-reperfusion treatment was diminished by administration of anti-P-selectin antibody (8.3 ± 0.8%, p < 0.05), but was not affected by inhibiting intercellular adhesion molecule-1 (39.6 ± 3.3%) or vascular cell adhesion molecule-1 (43.9 ± 2.9%).nnnCONCLUSIONSnRetention efficiency of intracoronary-injected BMMNC was poor in a model of isolated, crystalloid-perfused murine hearts. An antecedent period of global ischemia-reperfusion increased the retention via P-selectin-dependent BMMNC-endothelial interaction.

Oral administration of conditioned medium obtained from mesenchymal stem cell culture prevents subsequent stricture formation after esophageal submucosal dissection in pigs

BACKGROUND AND AIMSnEndoscopic submucosal dissection (ESD) for esophageal cancer often causes postoperative stricture when more than three fourths of the circumference of the esophagus is dissected. Mesenchymal stem cells are a valuable cell source in regenerative medicine, and conditioned medium (CM) obtained from mesenchymal stem cells reportedly inhibits inflammation. In this study we evaluated whether CM could prevent esophageal stricture after ESD.nnnMETHODSnWe resected a semi-circumference of pig esophagus by ESD. We prepared CM gel by mixing with 5% carboxymethyl cellulose and endoscopically applied it onto the wound bed immediately after ESD and on days 8 and 15 (weekly CM group) or administered it orally from days 1 to 4 (daily CM group). We also injected triamcinolone acetonide into the remaining submucosa immediately after ESD (steroid group). We killed the pigs on day 8 or day 22 to measure the stricture rate and to perform histologic analysis.nnnRESULTSnStricture rate in weekly and daily CM groups and steroid groups were significantly lower than in the control group on day 22. Moreover, CM significantly attenuated the number of activated myofibroblasts and fiber thickness on day 22. CM also significantly decreased the infiltration of neutrophils and macrophages compared with the control group on dayxa08.nnnCONCLUSIONSnCM gel prevents esophageal stricture formation by suppressing myofibroblast activation and fibrosis after the infiltration of neutrophils and macrophages. Oral administration of CM gel is a promising treatment for the prevention of post-ESD stricture.

Self-assembling peptide hydrogel enables instant epicardial coating of the heart with mesenchymal stromal cells for the treatment of heart failure

Transplantation of mesenchymal stromal cells (MSCs) is an emerging therapy for the treatment of heart failure. However, the delivery method of MSC is currently suboptimal. The use of self-assembling peptide hydrogels, including PuraMatrix® (PM; 3-D Matrix, Ltd), has been reported for clinical hemostasis and in research models. This study demonstrates the feasibility and efficacy of an advanced approach for MSC-therapy, that is coating of the epicardium with the instantly-produced PM hydrogel incorporating MSCs (epicardial PM-MSC therapy). We optimized the conditions/procedure to produce “instant” 2PM-MSC complexes. After spreading on the epicardium by easy pipetting, the PM-MSC complex promptly and stably adhere to the beating heart. Of note, this treatment achieved more extensive improvement of cardiac function, with greater initial retention and survival of donor MSCs, compared to intramyocardial MSC injection in rat heart failure models. This enhanced efficacy was underpinned by amplified myocardial upregulation of a group of tissue repair-related genes, which led to enhanced repair of the damaged myocardium, i.e. augmented microvascular formation and reduced interstitial fibrosis. These data suggest a potential for epicardial PM-MSC therapy to be a widely-adopted treatment of heart failure. This approach may also be useful for treating diseases in other organs than the heart.

Preventive effect of mesenchymal stem cell culture supernatant on luminal stricture after endoscopic submucosal dissection in the rectum of pigs

BACKGROUNDnMesenchymal stem cells (MSCs) are valuable in regenerative medicine, and MSC culture supernatant (MSC-CS) reportedly inhibits inflammation and fibrosis. We investigated whether colorectal luminal stricture develops after circumferential endoscopic submucosal dissection (ESD) in the colorectum, and whether the development of luminal stricture could be prevented by using MSC-CS enema.nnnMETHODSnIn the first experiment, we performed circumferential ESD in the rectums or distal colons of pigs (nu200a=u200a4 in each group). We sacrificed the pigs on Dayxa022 and measured the degree of luminal stricture. In the second experiment, we performed circumferential ESD in the rectums of pigs and administered an MSC-CS gel or a control gel enema after ESD for 4xa0days. We sacrificed the pigs on Dayxa08 (nu200a=u200a3 in each group) or 22 (nu200a=u200a3 in each group) to measure the degree of luminal stricture, and performed histological analysis.nnnRESULTSnSevere luminal stricture was observed in the rectum but not in the distal colon. Moreover, fiber accumulation in the submucosa and hypertrophy of the muscularis propria were observed in the rectum but not in the distal colon. The degree of luminal stricture in the rectum was significantly lower in the MSC-CS group than in the control group.u200aFurthermore, MSC-CS attenuated myofibroblast activation and hypertrophy of the muscularis propria on Dayxa022, and reduced inflammatory cell infiltration on Dayxa08.nnnCONCLUSIONSnLuminal stricture after ESD developed only in the rectum because of the difference in myofibroblast activation and fiber accumulation. In addition, MSC-CS enema prevented luminal stricture after ESD, possibly by inhibiting the inflammatory reaction and fibrosis.

Evaluation of amnion-derived mesenchymal stem cells for treatment-resistant moderate Crohn’s disease: study protocol for a phase I/II, dual-centre, open-label, uncontrolled, dose–response trial

Introduction The medical treatment options for patients with Crohn’s disease (CD) are limited and patients resistant to those therapies are left requiring surgical operations that usually only achieve some symptomatic relief. Mesenchymal stem cells (MSC) have been shown to be effective for the treatment of CD, and we have demonstrated in animal experiments that human amnion-derived MSCs (AMSC) are a potential new therapeutic strategy. Therefore, we designed this study to investigate the safety and efficacy of AMSCs in patients with treatment-resistant CD. Methods and analysis This is the protocol for an ongoing phase I/II, dual-centre, open-label, uncontrolled, dose–response study. The estimated enrolment is 6–12 patients with treatment-resistant, moderate CD. A dose of 1.0×106 cells/kg will be administered intravenously in the low-dose group at days 0 and 7. After confirming the safety of low-dose administration, a dose of 4.0×106 cells/kg will be administered intravenously in the high-dose group on days 0 and 7. The primary endpoint will measure the occurrence of adverse events related to acute infusion toxicity, and secondary endpoints will include long-term adverse events and efficacy of AMSC administration. Ethics and dissemination The Institutional Review Board of Hokkaido University Hospital approved this study protocol (approval number H29-6). A report releasing study results will be submitted to an appropriate journal. Discussion This study is the first to investigate the safety and efficacy of AMSC use for CD treatment. Our results will advance studies on more efficient and convenient methods to overcome the limits of available CD treatments. Trial registration number UMIN000029841.