peri-Adventitial delivery of smooth muscle cells in porous collagen scaffolds for treatment of experimental abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is associated with the loss of vascular smooth muscle cells (SMCs) within the vessel wall. Direct delivery of therapeutic cells is challenging due to impaired mechanical integrity of the vessel wall. We hypothesized that porous collagen scaffolds can be an effective vehicle for the delivery of human-derived SMCs to the site of AAA. The purpose was to evaluate if the delivery of cell-seeded scaffolds can abrogate progressive expansion in a mouse model of AAA. Collagen scaffolds seeded with either primary human aortic SMCs or induced pluripotent stem cell derived-smooth muscle progenitor cells (iPSC-SMPs) had >80% in vitro cell viability and >75% cell penetrance through the scaffold's depth, while preserving smooth muscle phenotype. The cell-seeded scaffolds were successfully transplanted onto the murine aneurysm peri-adventitia on day 7 following AAA induction using pancreatic porcine elastase infusion. Ultrasound imaging revealed that SMC-seeded scaffolds significantly reduced the aortic diameter by 28 days, compared to scaffolds seeded with iPSC-SMPs or without cells (acellular scaffold), respectively. Bioluminescence imaging demonstrated that both cell-seeded scaffold groups had cellular localization to the aneurysm but a decline in survival with time. Histological analysis revealed that both cell-seeded scaffold groups had more SMC retention and less macrophage invasion into the medial layer of AAA lesions, when compared to the acellular scaffold treatment group. Our data suggest that scaffold-based SMC delivery is feasible and may constitute a platform for cell-based AAA therapy.

Human bone marrow-derived, pooled, allogeneic mesenchymal stromal cells manufactured from multiple donors at different times show comparable biological functions in vitro, and in vivo to repair limb ischemia.

BACKGROUND: We have previously demonstrated that a pooled population of bone marrow-derived, allogeneic mesenchymal stromal cells (BMMSC), Stempeucel®-1, produced under good manufacturing practices (GMP) conditions, showed clinical efficacy and safety in patients suffering from critical limb ischemia (CLI) due to Buerger's disease. While Stempeucel®-1 is currently used for CLI and other clinical indications, we wanted to ensure that the product's continuity is addressed by developing and characterizing a second generation of pooled product (Stempeucel®-1A), manufactured identically from second BM aspirates of the same three donors after a 2-year interval. METHODS: The two versions of Stempeucel® were manufactured and subjected to gene and protein expression analysis. The nature of various growth factors/cytokines secreted and immunomodulatory activity of these two cell populations were compared directly by various in vitro assays. The preclinical efficacy of these two cell types was compared in an experimental model of hind limb ischemia (HLI) in BALB/c nude mice. The reversal of ischemia, blood flow, and muscle regeneration were determined by functional scoring, laser Doppler imaging, and immunohistochemical analyses. RESULTS: Qualitative and quantitative analyses of genes and proteins involved in promoting angiogenic activity and immune regulatory functions revealed high levels of correlation between Stempeucel®-1 and Stempeucel®-1A cell populations. Moreover, intramuscular (i.m) administration of these two cell products in the ischemic limbs of BALB/c nude mice showed significant repair (≥ 70%) of toe and foot necrosis, leading to improved ambulatory function and limb salvage. Furthermore, a biodistribution kinetics study showed that Stempeucel®-1 was mostly localized in the ischemic muscles of mice for a significantly longer time compared to normal muscles, thus playing an essential role in modulating and reversing HLI damage. CONCLUSIONS: This study shows that with a reproducible manufacturing procedure, it is possible to generate large numbers of pooled mesenchymal stromal cells from human bone marrow samples to establish product equivalence. We conclude from these results that, for the first time, two pooled, allogeneic BMMSC products can be repeatedly manufactured at different time intervals using a two-tier cell banking process with robust and comparable angiogenic properties to treat ischemic diseases.

Development of a surrogate potency assay to determine the angiogenic activity of Stempeucel®, a pooled, ex-vivo expanded, allogeneic human bone marrow mesenchymal stromal cell product.

BACKGROUND: Mesenchymal stromal cells (MSCs) have emerged as a more beneficial alternative to conventional therapy and may offer a potential cure for unmet medical needs. MSCs are known to possess strong immunomodulatory and anti-inflammatory properties. Moreover, they promote angiogenesis and tissue regeneration through the secretion of trophic factors. For these reasons, the past decade witnessed a sharp increase in the number of clinical trials conducted with stem cells for various vascular diseases requiring angiogenesis. In this study, we evaluated the in vitro angiogenic potency of Stempeucel®, which is an allogeneic pooled human bone marrow-derived mesenchymal stromal cell (phBMMSC) product. We previously established the safety of Stempeucel® in our pre-clinical studies, and clinical trials conducted for critical limb ischaemia and acute myocardial infarction. METHODS: Because the proposed mechanism of action of phBMMSCs is mainly through the secretion of pro-angiogenic cytokines, we developed a surrogate potency assay by screening various batches of large-scale expanded phBMMSCs for the expression of angiogenic factors and cytokines through gene expression and growth factor analyses, followed by in vitro functional assays. RESULTS: The well characterized angiogenic vascular endothelial growth factor (VEGF) was selected and quantified in twenty six manufactured batches of phBMMSCs to establish consistency following the United States Food and Drug Administration recommendations. According to recommendations 21 CFR 211.165(e) and 211.194(a)(2), we also established and documented the specificity and reproducibility of the test methods employed through validation. Moreover, we also attempted to elucidate the mechanism of action of the cell population to ensure appropriate biological activity. The functional role of VEGF has been established through in vitro angiogenic assays and a dose-dependent correlation was observed with in vitro functional results. CONCLUSIONS: The data generated from this study suggest the selection of VEGF as a single surrogate marker to test the angiogenic potency of phBMMSCs. Our study reports the quantification of VEGF in twenty six batches of large-scale manufactured phBMMSCs, and a concentration-dependent correlation of secreted VEGF to endothelial cell functions of migration, proliferation and tube formation, in the conditioned medium obtained from nine phBMMSC batches. To our cognizance, this is the first study in which a single angiogenic factor (VEGF) has been qualified as a surrogate potency marker through all three in vitro functional assays to determine the angiogenic potency of the phBMMSC population.

Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties.

Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3ß and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation.