Critical elements in the development of cell therapy potency assays for ischemic conditions.

A successful potency assay for a cell therapy product (CTP) used in the treatment of ischemic conditions should quantitatively measure relevant biological properties that predict therapeutic activity. This is especially challenging because of numerous degrees of complexity stemming from factors that include a multifactorial complex mechanism of action, cell source, inherent cell characteristics, culture method, administration mode and the in vivo conditions to which the cells are exposed. The expected biological function of a CTP encompasses complex interactions that range from a biochemical, metabolic or immunological activity to structural replacement of damaged tissue or organ. Therefore, the requirements for full characterization of the active substance with respect to biological function could be taxing. Moreover, the specific mechanism of action is often difficult to pinpoint to a specific molecular entity; rather, it is more dependent on the functionality of the cellular components acting in a in a multifactorial fashion. In the case of ischemic conditions, the cell therapy mechanism of action can vary from angiogenesis, vasculogenesis and arteriogenesis that may activate different pathways and clinical outcomes. The CTP cellular attributes with relation to the suggested mechanism of action can be used for the development of quantitative and reproducible analytical potency assays. CTPs selected and released on the basis of such potency assays should have the highest probability of providing meaningful clinical benefit for patients. This White Paper will discuss and give examples for key elements in the development of a potency assay for treatment of ischemic disorders treated by the use of CTPs.

A novel potential therapy for vascular diseases: blood-derived stem/progenitor cells specifically activated by dendritic cells.

BACKGROUND: Vascular diseases are a major cause of morbidity and mortality, particularly in diabetic patients. Stem/progenitor cell treatments with bone marrow-derived cells show safety and promising outcomes, albeit not without some preprocedural adverse events related to cell collection and mobilization. We describe a novel technology for generating a therapeutic population (BGC101) of enriched endothelial progenitor cells (EPCs) from non-mobilized blood, using dendritic cells to specifically direct stem/progenitor cell activity in vitro. METHODS AND RESULTS: Selected immature plasmacytoid and myeloid dendritic cells from 24 healthy and two diabetic donors were activated with anti-inflammatory and pro-angiogenic molecules to induce specific activation signals. Co-culturing of activated dendritic cells with stem/progenitor cells for 12-66 h generated 83.7 ± 7.4 × 10(6) BGC101 cells with 97% viability from 250 mL of blood. BGC101, comprising 52.4 ± 2.5% EPCs (expressing Ulex-lectin, AcLDL uptake, Tie2, vascular endothelial growth factor receptor 1 and 2, and CD31), 16.1 ± 1.9% stem/progenitor cells (expressing CD34 and CD184) and residual B and T helper cells, demonstrated angiogenic and stemness potential and secretion of interleukin-8, interleukin-10, vascular endothelial growth factor and osteopontin. When administered to immunodeficient mice with limb ischemia (n = 40), BGC101 yielded a high safety profile and significantly increased blood perfusion, capillary density and leg function after 21 days. Cell tracking and biodistribution showed that engraftment was restricted to the ischemic leg. CONCLUSIONS: These observations provide preliminary evidence that alternatively activated dendritic cells can promote the generation of EPC-enriched stem/progenitor cells within a 1-day culture. The resulting product BGC101 has the potential for treatment of various vascular conditions such as coronary heart disease, stroke and peripheral ischemia.

Transient extremity ischemia augments CD34+ progenitor cell availability.

Peripheral blood is an easily accessed source for stem cell production; however, the number of cells produced is relatively low. We hypothesized that ischemic preconditioning may serve as a safe method to increase the number of CD34+ cells that can be harvested and cultured in a short period. This study was conducted to test this hypothesis by examining the safety and efficacy of brief, transient ischemia of the lower limbs to augment the number of cells that can be produced from blood of healthy volunteers. Following induction of ischemia, blood samples were withdrawn at baseline, 30 min, 12 h and 24 h. The number of progenitor cells was determined by flow cytometry after the harvested cells were cultured for 5 days. We also analyzed the blood samples to determine IL-8 and VEGF concentrations. No serious adverse events were observed. The total number of cells increased from 0.46 ± 0.1 × 10(6) cells/ml in the pretreatment blood samples to 0.7 ± 0.1 × 10(6) cells/ml in blood taken 12 h after the conclusion of transient ischemia, p = 0.0029. The number of CD34+ cells increased from 4.23 ± 0.8 × 10(4) cells/ml in the pretreatment samples to 7.17 ± 1.34 × 10(4) cells/ml in blood taken 12 h after ischemia, p = 0.0001. The harvested stem cells maintained their ability to construct tubular structures. The augmentation in the number of CD34+ cells was positively correlated with the increase of IL-8, but not with VEGF concentrations. Ischemic preconditioning is a safe and effective technique to increase the availability of stem cells for therapeutic purposes.

Autologous transplantation of arterial cells improves cardiac function in a rabbit model of infarcted myocardium.

Cellular cardiomyoplasty is a promising approach for the treatment of severe heart failure. However, the question which cell line is the best to use is still a matter of debate. In this study, we aimed to evaluate the efficacy of arterial media-intima cell suspension (AMICS) transplantation in rabbit myocardial infarct model. The study was divided into 2 groups: group A (the cell-treated group, n = 9) and group B (the medium injection group, n = 8). Group A was further divided into 2 subgroups as branch-1 (treated with unlabeled cells) and branch-2 (treated with iron-labeled cells). The experimental myocardial infarction (MI) was induced by ligation of left anterior descending coronary artery with a combination of cryoinjury. Ten days after the MI, cells obtained from autologous femoral arteries were injected into the injured myocardium of group A, while group B received an injection of only DMEM medium. Clinical, echocardiographic, and histopathologic evaluations were done. As compared to the ninth day values, echocardiography showed a significant improvement in systolic functions and left ventricular (LV) dimensions of the cell-treated group on the 30th day. In the heart biopsy sections of branch-1, the immunostained injected cells were observed to exist closely, suggesting an organization. Cells existing separately and lumen-like structure organizations stained positive with both smooth muscle cell (SMC) alpha-actin and Prussian Blue were also showed in the histological observation of branch-2. Autologous AMICS transplantation seems to be a feasible and efficacious method for cellular cardiomyoplasty in our rabbit model.

Enhancing limb salvage by non-mobilized peripheral blood angiogenic cell precursors therapy in patients with critical limb ischemia.

BACKGROUND: Stem cell therapy has been proposed to enhance the salvage of critically ischemic limbs. OBJECTIVE: Assess the efficacy and safety of the implantation of non-mobilized peripheral blood angiogenic cell precursors (NMPB-ACPs) in patients with critical limb ischemia (CLI) who were poor candidates for standard revascularization treatment options. MATERIAL AND METHOD: Six patients with CLI due to the infrapopliteal artery occlusive disease were included in the present study. Intramuscular injections of NMPB-ACPs were administered in the ischemic limbs. The efficacy was evaluated by clinical outcomes, ankle brachial index, toe brachial index, and computerized tomographic angiography. RESULTS: There was no evidence of local or systemic complication related to the procedure. Five patients (83.3%) had clinically significant improvement of adequate circulation at the distal limb for the complete healing. Four of them had complete healing of ischemic ulcers and stumps of toe amputation. However one patient with adequate granulation tissue at the stump of the left first toe amputation subsequently suffered from severe foot infection originating from the other toes and eventually underwent below knee amputation. There was no improvement of circulation at the distal limb after the administration of NMPB-ACPs in one patient (16.7%) who eventually underwent major amputation. CONCLUSION: The preliminary result of NMPB-ACPs therapy may be safe and provide benefits in the improvement of circulation in patients with CLI. A larger controlled trial is required to ascertain these preliminary results.

Human angiogenic cell precursors restore function in the infarcted rat heart: a comparison of cell delivery routes.

BACKGROUND: We recently isolated angiogenic cell precursors (ACPs) from human blood, which can induce angiogenesis in vitro. AIMS: In the present study, we used a nude rat model of ischaemic cardiomyopathy to compare the efficacy of intramyocardial and intracoronary ACP implantation, and to evaluate effects on cardiac function, scar size and angiogenesis. METHODS AND RESULTS: Adult nude rats underwent coronary artery ligation. Six days later, ACPs (characterized in vitro prior to implantation) or culture media were injected directly into the ischaemic myocardial region or into the coronary artery via the aorta. Cardiac function was measured by echocardiography prior to and at 2 and 4 weeks after implantation. Scar morphology, cell engraftment, and myocardial angiogenesis were evaluated at 4 weeks. Two and four weeks after implantation, cardiac function declined in both of the control groups but improved in both the intramyocardial and intracoronary ACP groups. Significant reductions in myocardial scar area were only observed in the intramyocardial ACP group, while increases in blood vessel density, which were observed in all ACP recipients, were greatest in the intracoronary ACP group. CONCLUSIONS: Human ACPs, delivered via intramyocardial or intracoronary injection, engrafted into damaged cardiac tissue and improved cardiac function within 4 weeks through effects on scar morphology and blood vessel formation.

Isolation of an adult blood-derived progenitor cell population capable of differentiation into angiogenic, myocardial and neural lineages.

Blood-derived adult stem cells were previously considered impractical for therapeutic use because of their small numbers. This report describes the isolation of a novel human cell population derived from the peripheral blood, termed synergetic cell population (SCP), and defined by the expression of CD31Bright, CD34+, CD45-/Dim and CD34Bright, but not lineage-specific features. The SCP was capable of differentiating into a variety of cell lineages upon exposure to defined culture conditions. The resulting cells exhibited morphological, immunocytochemical and functional characteristics of angiogenic, neural or myocardial lineages. Angiogenic cell precursors (ACPs) expressed CD34, CD133, KDR, Tie-2, CD144, von Willebrand factor, CD31Bright, concomitant binding of Ulex-Lectin and uptake of acetylated low density lipoprotein (Ac-LDL), secreted interleukin-8, vascular endothelial growth factor and angiogenin and formed tube-like structures in vitro. The majority of CD31Bright ACP cells demonstrated Ac-LDL uptake. Neural cell precursors (NCPs) expressed the neuronal markers Nestin, betaIII-Tubulin, and Neu-N, the glial markers GFAP and O4, and responded to neurotransmitter stimulation. Myocardial cell precursors (MCPs) expressed Desmin, cardiac Troponin and Connexin 43. In conclusion, the simple and rapid method of SCP generation and the resulting considerable quantities of lineage-specific precursor cells makes it a potential source of autologous treatment for a variety of diseases.