Li, Mark

Introduction: The process of engaging stem cells towards a cardiopoietic (CP) state was established using a protein-based cocktail consisting of multiple growth factors. Although this regenerative enhancement protocol has been successfully tested in advanced clinical trials, it remains resource-intensive. Recent efforts have focused on streamlining cardiopoiesis through a protein-independent alternative that leverages a transfection-based mRNA approach. Microencapsulated modified messenger RNA (M3RNA) was utilized to deliver brachyury (T), an early mesodermal transcription factor to provoke early lineage specification in pluripotent cells. Hypothesis: Transfection of a human pluripotent stem cell source with T induces early expression of CP transcription factors to accelerate cardiac differentiation. Methods: Human pluripotent stem cells were transfected with T and maintained for 14 days. Expression of the cardiopoietic markers MEF2C, GATA4, and Nkx2.5 was analyzed using RT-qPCR, in tandem with MYH6 at days 10 and 20. Differentiation proficiency was independently corroborated by assessing Mef2c and Nkx2.5 expression by IF. Results: Following transfection with T, Nkx2.5, MEF2C and GATA4 were significantly induced at 72h (60-, 20- and 40-fold, respectively, p<0.01 for all 3 markers vs baseline), reflecting timing of differentiation initiation. At 5 days post differentiation, expression of the cardiac-specific Nkx2.5 increased by 5 fold vs control and the cell cycle regulator CHIR (p<0.01), suggesting successful persistent induction of cardiopoeisis. At 10 and 20 days of differentiation, RNA expression of MYH6 was 10-15 times induced in human pluripotent cells transduced with T (p < 0.0001, and p < 0.001 vs control, respectively), compared to 2 times (p<0.05, p<0.01 vs control, respectively) in CHIR treated cohorts. Accordingly, at 42 days post-induction of differentiation, a greater number of cells expressed MLC2V and cTnT (p<1e-05, <1e-07, respectively vs control), driving a robust beating pattern. Conclusion: This study implements a single gene T M3RNA-based approach to readily induce cardiac differentiation from pluripotent sources, exceeding available commercial platforms.

Introduction: Platelets are heavily implicated in angiogenesis; however, the underlying mechanism remains relatively uncharacterized. The goal of this study was to evaluate the angiogenic potency of a platelet-derived regenerative exosome product (PEP) in an effort to develop a novel treatment for peripheral arterial disease. Here, we evaluated the mechanistic basis of PEP in angiogenesis and it’s use In Vivo in a rodent hind limb ischemia model. Methods/Results: Under Current Good Manufacturing Practices, conditioned medium from apheresis platelet cultures was purified to acquire the exosomes. PEP, enriched in exosome markers CD63, CD9 and Flotillin, induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and vascular tube formation In Vitro. Proteomic evaluation of PEP highlighted receptor tyrosine kinase (RTK) pathways and western blot validation demonstrated induction of phospho-MAPK and phospho-AKT following PEP treatment of HUVECs. Systematic evaluation of RTK receptor involvement was performed using small molecule inhibitors and receptor knock-down in HUVECs, ruling out RTK activation as a mode of action. However, small molecule inhibition of PI3 kinase demonstrated elimination of PEP-induced AKT phosphorylation and reduction in PEP-induced angiogenesis In Vitro measured by HUVEC proliferation and migration. In Vivo, evaluation of angiogenesis was performed in a rodent model of hindlimb ischemia treated with PEP embedded in fibrin glue (PEP/TISSEEL). Blood flow was evaluated by SPY angiography on day 0 pre- and post-operation, day 21, and day 28. Animals treated with PEP/TISSEEL showed a significant increase in blood flow compared to sham and TISSEEL treated animals (percent perfusion at 28 days: sham 40.9%, TISSEEL 44.3%, PEP/TISSEEL 100.2%), with significant increase in vascular area on histology (at 28 days vascular area (μm 2 )/muscle area(μm 2 ): healthy control 12.71, sham 4.66, TISSEEL 4.76, PEP/TISSEEL 15.54). Conclusions: Exosome driven activation of PI3 kinase can induce angiogenesis both In Vitro and In Vivo. PEP provides a novel, clinical-grade, off-the-shelf therapy to promote angiogenesis for the treatment of peripheral arterial disease.

Objectives: Peripheral arterial disease is a significant cause of morbidity and mortality. The goal of this study was to develop a novel treatment to promote angiogenesis. Platelets have been previously implicated in angiogenic events; however, platelet activation rather than presence of growth factors has been associated with new blood vessel formation. This study evaluated exosomes purified from activated platelets to determine their potential in promoting angiogenesis both in vitro and in vivo. Methods & Results: Under Current Good Manufacturing Practices, conditioned medium processed from apheresis platelet cultures yielded a purified exosome product (PEP) enriched for CD63, CD9 and Alix. In vitro, PEP drove HUVEC proliferation, migration, and vascular tube formation. Proteomic evaluation showed Flk1 (VEGFR2) was one of the key angiogenic proteins in PEP. HUVECs treated with Flk1 antibody neutralized PEP (anti-Flk1-PEP) showed reduced vascular tube formation compared to PEP. To evaluate in vivo angiogenesis, a rodent model of hindlimb ischemia was treated with sustained release PEP embedded in fibrin glue (PEP/TISSEEL) with blood flow evaluated by SPY angiography at 28 days post-treatment. Animals treated with PEP/TISSEEL showed improved blood flow with significant difference (p<0.001) as compared to sham, TISSEEL, and anti-Flk1-PEP/TISSEEL. Muscle tissue stained with α smooth muscle actin showed a significant increase (p<0.001) in vascular area in PEP/TISSEEL treated animals compared to TISSEEL alone. To further evaluate clinical translatability, a rabbit ear ischemia wound model was then treated with PEP/TISSEEL. Rabbit ears showed accelerated wound healing and restored blood flow on SPY angiography. Histologic evaluation of vascular area showed a significant increase in vascular area (p<0.05) compared to sham and TISSEEL alone animals. Conclusions: Exosome driven donation of Flk1 here promoted angiogenic events and was successfully translated into healing outcomes in the setting of two animal models of peripheral ischemia. This novel insight into biotherapy achievable pro-angiogenesis establishes a clinical-grade off-the-shelf exosome technology for the treatment of peripheral vascular disease.

Abstract To optimize the regenerative proficiency of stem cells, a cardiopoietic protein-based cocktail consisting of multiple growth factors has been developed and advanced into clinical trials for treatment of ischemic heart failure. Streamlining the inductors of cardiopoiesis would address the resource intensive nature of the current stem cell enhancement protocol. To this end, the microencapsulated-modified-mRNA (M3RNA) technique was here applied to introduce early cardiogenic genes into human adipose-derived mesenchymal stem cells (AMSCs). A single mesodermal transcription factor, Brachyury, was sufficient to trigger high expression of cardiopoietic markers, Nkx2.5 and Mef2c. Engineered cardiopoietic stem cells (eCP) featured a transcriptome profile distinct from pre-engineered AMSCs. In vitro, eCP demonstrated protective antioxidant capacity with enhanced superoxide dismutase expression and activity; a vasculogenic secretome driving angiogenic tube formation; and macrophage polarizing immunomodulatory properties. In vivo, in a murine model of myocardial infarction, intramyocardial delivery of eCP (600 000 cells per heart) improved cardiac performance and protected against decompensated heart failure. Thus, heart repair competent stem cells, armed with antioxidant, vasculogenic, and immunomodulatory traits, are here engineered through a protein-independent single gene manipulation, expanding the available regenerative toolkit.

The management of patients with heart failure focuses on halting the progression of disease. To date, no therapeutic approach has been able to restore cardiac function to its original baseline state. Cell therapies, such as cardiopoietic (CP) stem cells, have shown to be promising platforms to restore cardiac function. The CP platform enhances the cardioregenerative potency of mesenchymal stem cells (MSCs). CP cells are characterized by the robust expression and nuclear translocation of cardiac transcription factors such Mesoderm Posterior Basic Helix‐Loop‐Helix Transcription Factor 1 (MESP1), early cardiac transcription factor Nkx2.5, myocyte enhancer factor 2C (Mef2C), and zinc finger‐containing transcription factor GATA4. Clinical trials using CP cells showed signs of safety and efficacy in improving cardiac function in patients with heart failure. However, the costs, time, and complexities associated with the manufacturing process have precluded the adoption of the therapy as a large‐scale clinical practice. In response, we adopted a microencapsulated‐modified‐messenger RNA (M3RNA) platform as a cost‐effective and time‐saving alternative to induce high expression of cardiopoietic markers. We hypothesized that the transfection of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) with M3RNA encoding mesodermal and/or cardiac transcription factor(s) will induce the expression of cardiopoietic markers (MESP1, Nkx2.5, Mef2C, and GATA4). hUC‐MSCs were transfected with M3RNA encoding Brachyury (this protein plays a key role in cardiomyocyte differentiation) and MESP1, Nkx2.5, Mef2C, and GATA4 using the transfection reagent Lipofectamine STEM. Protein expression was measured and quantified using fluorescence microscopy and flow cytometry. hUC‐MSCs transfected with M3RNA encoding mesodermal and/or cardiac transcription factor(s) demonstrated rapid protein expression at 4 hours post‐transfection that reached peak levels between 24 hours and 48 hours post‐transfection. Immunocytochemistry demonstrated similar morphology between transfected and non‐transfected cells. Thus, hUC‐MSCs can be transfected with M3RNA with high efficiency to produce rapid and predictable protein expression in vitro. We here demonstrate a simple, cost‐effective, and time‐saving approach to induce the expression of cardiopoietic markers in UC‐MSCs using M3RNA, which can serve as a highly translatable platform to circumvent many burdens associated with current strategies and development of cell‐based therapies for future clinical trials and practice. Support or Funding Information Mayo Clinic Center for Regenerative Medicine, Van Cleve Cardiac Regenerative Medicine Program, Marriott Foundation, Michael S. and Mary Sue Shannon Family, NIH NHLBI HL134664, Mayo Clinic Regenerative Sciences Post‐Baccalaureate Training Program