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Preclinical evaluation of mesenchymal stem cells overexpressing VEGF to treat critical limb ischemia.

ABSTRACT: Numerous clinical trials are utilizing mesenchymal stem cells (MSC) to treat critical limb ischemia, primarily for their ability to secrete signals that promote revascularization. These cells have demonstrated clinical safety, but their efficacy has been limited, possibly because these paracrine signals are secreted at subtherapeutic levels. In these studies the combination of cell and gene therapy was evaluated by engineering MSC with a lentivirus to overexpress vascular endothelial growth factor (VEGF). To achieve clinical compliance, the number of viral insertions was limited to 1-2 copies/cell and a constitutive promoter with demonstrated clinical safety was used. MSC/VEGF showed statistically significant increases in blood flow restoration as compared with sham controls, and more consistent improvements as compared with nontransduced MSC. Safety of MSC/VEGF was assessed in terms of genomic stability, rule-out tumorigenicity, and absence of edema or hemangiomas in vivo. In terms of retention, injected MSC/VEGF showed a steady decline over time, with a very small fraction of MSC/VEGF remaining for up to 4.5 months. Additional safety studies completed include absence of replication competent lentivirus, sterility tests, and absence of VSV-G viral envelope coding plasmid. These preclinical studies are directed toward a planned phase 1 clinical trial to treat critical limb ischemia.

SUBMITTER: Beegle JR

PROVIDER: S-EPMC5003097 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Preclinical evaluation of mesenchymal stem cells overexpressing VEGF to treat critical limb ischemia.

Beegle Julie R JR Magner Nataly Lessa NL Kalomoiris Stefanos S Harding Aja A Zhou Ping P Nacey Catherine C White Jeannine Logan JL Pepper Karen K Gruenloh William W Annett Geralyn G Nolta Jan A JA Fierro Fernando A FA

Molecular therapy. Methods & clinical development 20160824

Numerous clinical trials are utilizing mesenchymal stem cells (MSC) to treat critical limb ischemia, primarily for their ability to secrete signals that promote revascularization. These cells have demonstrated clinical safety, but their efficacy has been limited, possibly because these paracrine signals are secreted at subtherapeutic levels. In these studies the combination of cell and gene therapy was evaluated by engineering MSC with a lentivirus to overexpress vascular endothelial growth fact ...[more]

PMID: 27610394

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Project description:BackgroundCritical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1% to 10% of the population with peripheral arterial disease. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However, no randomized, controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. This may be due to poor cell quality (ie, inability to generate a sufficient number of angiogenic MSCs) or to the inadequate retention and viability of MSCs after delivery, or both. Because concerns remain about the expansion and angiogenic potential of autologous MSCs in the CLI population, the objective of this study was to examine the effect of our novel culture media supplement, pooled human platelet lysate (PL), in lieu of the standard fetal bovine serum (FBS), to improve the expansion potential of MSCs from CLI patients. We also characterized the in vitro angiogenic activity of MSCs from the tibia of amputated CLI limbs compared with MSCs from healthy donors.MethodsMSCs were obtained from the tibia of four CLI patients (ISC) and four ISC patients with diabetes mellitus (ISC+DM) undergoing major amputation. Healthy MSCs were aspirated from the iliac crest of four young and healthy donors. MSCs were isolated and expanded in culture with PL or FBS. MSCs from passage 3 to 6 were used for phenotypic marker expression and for adipogenic and osteogenic differentiation and were tested for their in vitro angiogenic activity on human microdermal endothelial cells. In parallel MSCs were cultured to passage 11 for population-doubling calculations.ResultsMSCs from ISC and ISC+DM patients and from healthy patients exhibited appropriate expression of cell surface markers and differentiation capacity. Population doublings were significantly greater for PL-stimulated compared with FBS-stimulated MSCs in all groups. Biologically active amounts of angiogens were identified in the secretome of all MSCs without consistent trends among groups. PL expansion did not adversely affect the angiogenic activity of MSCs compared with FBS. The ISC and ISC+DM MSCs demonstrated angiogenic effects on endothelial cells similar to those of healthy and ISC MSCs.ConclusionsPL promotes the rapid expansion of MSCs from CLI and healthy persons. Importantly, MSCs expanded from CLI patients demonstrate the desired angiogenic activity compared with their healthy counterparts. We conclude that autologous MSCs from CLI patients can be sufficiently expanded with PL and be expected to deliver requisite angiogenic effects in vivo. We expect the improved expansion of ISC and ISC+DM with PL to be helpful in improving the successful delivery of autologous MSCs to patients with CLI.

Project description:ObjectiveRegenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb.Methods and resultsIn this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism.ConclusionIn conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.

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Preclinical evaluation of mesenchymal stem cells overexpressing VEGF to treat critical limb ischemia.

Publications

Preclinical evaluation of mesenchymal stem cells overexpressing VEGF to treat critical limb ischemia.

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