Background: Pre-clinical studies in rodents and pigs indicate that the self-assembling microtissues known as cardiospheres (CSp), when administered intramyocardially, may be more effective than dispersed CSp-derived cells (CDCs). However, the more desirable intracoronary (IC) route has been assumed to be unsafe for CSp delivery: CSp are large (>35 μm), raising concerns about likely microembolization. Objective: We sought to evaluate the safety and efficacy of IC delivery of allogeneic CSp in a porcine model of convalescent MI. Methods: Dosage was optimized by infusing CSp (3.25x10 5 particles [n=2], 6.5 x10 5 [n=3] and 1.3x10 6 [n=2], size=44±23, 29%>50μm) in the LAD of naïve pigs, looking for acute adverse effects (troponin I [TnI] leak, low TIMI flow, stunning). We next tested the efficacy of IC allogeneic Csp (1.3x10 6 ; n=7) or vehicle (n=8) in a minipig model of chronic MI. Animals underwent MRI before infusion and 1 month later. Left ventricular (LV) ejection fraction (EF), scar mass and viable mass were evaluated at both time points. Results: In the dosing study, we observed no impairment of TIMI flow or LVEF after CSp infusion. TnI at 24 hours was 0.7±0.5ng/mL and did not differ among groups (P=0.11). In the post-MI study, EF was identical in the two groups at baseline. One month post-infusion, LV function was preserved in the CSp group but not in controls (ΔEF=+0.5±1.6% vs. -4.5±1.8%, p<0.001). CSp reduced scar mass (P<0.001) and increased viable mass (+17±8% vs. +6±6% from baseline, P=0.04) compared to controls. IC CSp also decreased LV end diastolic pressure (-7±4mmHg vs. -1±4 mmHg in control, P<0.01)) and increased cardiac output (+0.5±0.4 mL/min vs. -0.1±0.3mL/min, P<0.01. Conclusions: IC delivery of allogeneic CSp is safe and preserves LV function after MI. In addition, global hemodynamic improvement is observed, which may have significant clinical implications. The decision to use CDCs or CSp is not forced, therefore, by an inability to infuse CSp safely via the IC route.
Bone marrow stromal cells (BMSCs) are a promising component for engineered bone tissues, but in vitro formation of a bonelike tissue requires culture conditions that direct these multipotent cells toward osteoblastic maturation. Fluid flow has been postulated to stimulate bone tissue development in vivo, but the effect of shear stress on proliferation and differentiation of osteoprogenitor cell cultures in vitro has not been examined closely. In this study BMSCs were cultured on fibronectin-coated substrates and exposed intermittently (for 30 min 3, 5, 7, 9, 11, and 13 days after seeding) to a spatially dependent range of shear stresses (0.36 to 2.7 dyn/cm2) using a radial-flow chamber. After 7 days cell density did not vary between sheared and control cell layers. In contrast, after 21 days the accumulation of osteocalcin protein (OC) in cell layers was increased significantly relative to static controls, while the quantity of multilayer cell aggregates (i.e., bone nodules) was diminished. Neither of these effects varied systematically with shear magnitude. Finally, pretreatment of cultures with the cyclooxygenase (COX)-2-specific inhibitor NS-398 blocked prostaglandin secretion in response to shearing flow and significantly reduced OC accumulation in cell layers. These results provide evidence that flow stimulates osteoblastic maturation but not proliferation of bone marrow stromal cells and that prostaglandin signaling is involved in this effect.
Background: Since the first intracoronary delivery of cells to the heart in 2001, the stop-flow technique has been used by default. A balloon angioplasty catheter is inflated in the target vessel and cells infused via the luminal port. However, the need for balloon occlusion has never been demonstrated. Objective: We sought to compare the safety and efficacy of intracoronary delivery of allogeneic cardiosphere derived cells (CDCs) under stop-flow versus nonocclusive intracoronary delivery in a porcine model of myocardial infarction (MI). Methods: MI was created by 2.5 hr occlusion of the mid-LAD in adult female Yucatan minipigs. Three weeks later, 12.5M CDCs were intracoronarily infused in the LAD under stop-flow (in 3 3-min occlusions with 3 min rest intervals) or nonocclusive conditions (continuous-flow infusion over 10 min; n=5 in each group). During the course of the study, animals underwent contrast enhanced magnetic resonance imaging at baseline (just before infusion) and 4 weeks post infusion. Left ventricular ejection fraction, scar mass and viable mass were evaluated at both time points. Results: No adverse events (arrhythmias, death) were observed during or soon after cell infusion in any of the animals infused. Coronary blood flow evaluated by TIMI grade was TIMI 3 in all animals following completion of infusion. TnI and CK-MB values were within normal range 1 day post-infusion in all animals. One month post-infusion, allogeneic CDCs reduced scar mass in both groups (continuous flow p=0.015 vs. baseline; stop-flow p=0.044). The effects on ejection fraction (p=0.08) and viable mass (p=0.88) were equivalent in the two groups. Conclusions: Nonocclusive continuous-flow delivery is equally efficient to stop-flow method as a means of cell delivery to the infarcted myocardium, at least for this particular cell type. The need for stop-flow delivery, while traditional, is therefore questionable.
We describe an approach to cancer therapy based on exploitation of common losses of genetic material in tumor cells (loss of heterozygosity) (Basilion et al., 1999; Beroukhim et al., 2010). This therapeutic concept addresses the fundamental problem of discrimination between tumor and normal cells and can be applied in principle to the large majority of tumors. It utilizes modular activator/blocker elements that integrate signals related to the presence and absence of ligands displayed on the cell surface (Fedorov et al., 2013). We show that the targeting system works robustly in vitro and in a mouse cancer model where absence of the HLA-A*02 allele releases a brake on engineered T cells activated by the CD19 surface antigen. This therapeutic approach potentially opens a route toward a large, new source of cancer targets.
Magnetic resonance imaging (MRI) in the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial revealed that cardiosphere-derived cells (CDCs) decrease scar size and increase viable myocardium after myocardial infarction (MI), but MRI has not been validated as an index of regeneration after cell therapy. We tested the validity of contrast-enhanced MRI in quantifying scarred and viable myocardium after cell therapy in a porcine model of convalescent MI.
Intracoronary delivery of cardiosphere-derived cells (CDCs) has been demonstrated to be safe and effective in porcine and human chronic myocardial infarction. However, intracoronary delivery of CDCs after reperfusion in acute myocardial infarction has never been assessed in a clinically-relevant large animal model. We tested CDCs as adjunctive therapy to reperfusion in a porcine model of myocardial infarction.First, escalating doses (5, 7.5, and 10 million cells) of allogeneic CDCs were administered intracoronary 30 minutes after reperfusion. Forty-eight hours later, left ventriculography was performed and animals euthanized to measure area at risk, infarct size (IS), and microvascular obstruction. Second, identical end points were measured in a pivotal study of minipigs (n=14) that received 8.5 to 9 million allogeneic CDCs, placebo solution, or sham. Multiple indicators of cardioprotection were observed with 7.5 and 10 million allogeneic CDCs, but not 5 million CDCs, relative to control. In the pivotal study, IS, microvascular obstruction, cardiomyocyte apoptosis, and adverse left ventricular remodeling were all smaller in the CDC group than in sham or placebo groups. In addition, serum troponin I level at 24 hours was lower after CDC infusion than that in the placebo or sham groups, consistent with the histologically-demonstrated reduction in IS.Intracoronary delivery of allogeneic CDCs is safe, feasible, and effective in cardioprotection, reducing IS, preventing microvascular obstruction, and attenuating adverse acute remodeling. This novel cardioprotective effect, which we call cellular postconditioning, differs from previous strategies to reduce IS in that it works even when initiated with significant delay after reflow.
Introduction: Osteonecrosis of the femoral head, which involves the death of cells in trabecular bone and marrow, leads to fracture of subchondral bone and loss of the femur articulating surface in the hip and ultimately leads to total hip replacement (THR). Retrospective clinical studies show that osteonecrosis in 80–90% of affected patients inevitably progresses to destroy the femur head, usually within 2–3 years of diagnosis. None of the current treatment options are effective at terminating or reversing the disease process. Two reports (Hernigou and Beaujean, 2002 and Gangji, et al 2004) using fresh autologous bone marrow tissue injected directly into the necrotic femoral head, reported a high rate of success, especially in early stage osteonecrosis, in patients at most risk for disease progression. As a more standardized alternative to fresh bone marrow, Aastrom Biosciences has developed a proprietary automated process to expand autologous bone marrow cells. The ex vivo expanded cells referred to as Bone Repair Cells (BRC) are based on Aastrom Tissue Repair Cell (TRC) technology. BRC are a mixture of stem and early progenitor cells including cells of hematopoietic, mesenchymal, and endothelial lineages derived from a small sample of the patient’s own bone marrow. Materials and Methods: Fresh bone marrow mononuclear cells from normal donors were purchased from Poietics Inc. (Gaithersburg, Maryland) for BRC culture. After ex vivo expansion, BRC viability and cell phenotype characterization was performed by flow cytometry. The frequency of mesenchymal and hematopoietic stem cells within BRC was determined using CFU-F and CFU-GM assays. The osteogenic and vascular in vitro potential of BRC was measured using standard osteogenic differentiation assays and tube formation assays. The bone formation potential of BRC was determined using an ectopic bone formation model involving subcutaneous implantation. Based on the in vitro and in vivo potential of BRC, a mixing procedure was developed to implant BRC and bone matrix into osteonecrotic sites during standard core decompression surgery. The viability of BRC within the bone matrix was measured using standard cell metabolic assays. Results: BRC possess a diverse range of cell phenotypes with the potential to differentiate down the osteogenic and angiogenic lineage under the right conditions. BRC also has the potential for in vivo bone formation. In addition, examination of several cell-surface markers revealed a strong correlation between the frequency of cell surface markers CD105+, CD166+, CD90+ and in vivo bone formation scores when implanted with a ceramic matrix material. This BRC product can be mixed with a bone matrix for the implantation into long-bone defects or osteonecrotic sites without loss in cell viability. Discussion: Aastrom BRCs have both in vivo and in vitro bone and vascular potential; thus, it is our intent to demonstrate clinical safety and efficacy in treating osteonecrosis patients with BRC. Aastrom’s ON-CORE trial is a 120 patient Phase III clinical trial for the treatment of University of Pennsylvania radiographic classification stage IIb and IIc osteonecrosis patients. The primary efficacy endpoint of this trial is to delay disease progression of osteonecrosis to fracture for at least 24 months post-treatment, and potentially prevent collapse of the femur head, which will be measured by a blinded third-party reviewer through magnetic resonance imaging. Patients will be followed for a total of 5 years, post-treatment.
