Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors.

Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs-one predominant, three variant, and two novel mutant isoforms-into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student's T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.

Chitosan Particles Complexed with CA5-HIF-1α Plasmids Increase Angiogenesis and Improve Wound Healing.

Wound therapies involving gene delivery to the skin have significant potential due to the advantage and ease of local treatment. However, choosing the appropriate vector to enable successful gene expression while also ensuring that the treatment's immediate material components are conducive to healing itself is critical. In this study, we utilized a particulate formulation of the polymer chitosan (chitosan particles, CPs) as a non-viral vector for the delivery of a plasmid encoding human CA5-HIF-1α, a degradation resistant form of HIF-1α, to enhance wound healing. We also compared the angiogenic potential of our treatment (HIF/CPs) to that of chitosan particles containing only the plasmid backbone (bb/CPs) and the chitosan particle vector alone (CPs). Our results indicate that chitosan particles exert angiogenic effects that are enhanced with the human CA5-HIF-1α-encoded plasmid. Moreover, HIF/CPs enhanced wound healing in diabetic db/db mice (p < 0.01), and healed tissue was found to contain a significantly increased number of blood vessels compared to bb/CPs (p < 0.01), CPs (p < 0.05) and no-treatment groups (p < 0.01). Thus, this study represents a method of gene delivery to the skin that utilizes an inherently pro-wound-healing polymer as a vector for plasmid DNA that has broad application for the expression of other therapeutic genes.

Induced Pluripotent Stem Cell-Derived Extracellular Vesicles Promote Wound Repair in a Diabetic Mouse Model via an Anti-Inflammatory Immunomodulatory Mechanism.

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been explored in clinical trials for treatment of diseases with complex pathophysiologies. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, it is initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, it is found that their vascularization bioactivity is similar and their anti-inflammatory bioactivity is superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial is employed. In this in vivo model, iPSC EVs more effectively mediate inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

Induced pluripotent stem cell-derived extracellular vesicles promote wound repair in a diabetic mouse model via an anti-inflammatory immunomodulatory mechanism.

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been widely explored in clinical trials for treatment of diseases with complex pathophysiology. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, we initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, we found that their vascularization bioactivity was similar and their anti-inflammatory bioactivity was superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, we employed a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial. In this in vivo model, iPSC EVs more effectively mediated inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.

Impact of storage conditions and duration on function of native and cargo-loaded mesenchymal stromal cell extracellular vesicles.

BACKGROUND AIMS: As evidenced by ongoing clinical trials and increased activity in the commercial sector, extracellular vesicle (EV)-based therapies have begun the transition from bench to bedside. As this progression continues, one critical aspect of EV clinical translation is understanding the effects of storage and transport conditions. Several studies have assessed the impact of storage on EV characteristics such as morphology, uptake and component content, but effects of storage duration and temperature on EV functional bioactivity and, especially, loaded cargo are rarely reported. METHODS: The authors assessed EV outcomes following storage at different temperatures (room temperature, 4°C, -20°C, -80°C) for various durations as well as after lyophilization. RESULTS: Mesenchymal stromal cell (MSC) EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4-6 weeks at -20°C and -80°C and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA and long non-coding RNA cargo in MSC EVs. CONCLUSIONS: These findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storing MSC EVs for both basic research and translational purposes.

Potential Role of Silencing Ribonucleic Acid for Esophageal Cancer Treatment.

INTRODUCTION: Esophageal cancer is an aggressive malignancy with high mortality. Optimal treatment of esophageal cancer remains an elusive goal. Ribonucleic acid (RNA) interference is a novel potential targeted approach to treat esophageal cancer. Targeting oncogenes that can alter critical cellular functions with silencing RNA molecules is a promising approach. The silencing of specific oncogenes in esophageal cancer cells in the experimental setting has been shown to decrease the expression of oncogenic proteins. This has resulted in cell apoptosis, reduction in cell proliferation, reduced invasion, migration, epithelial-mesenchymal transition, decrease in tumor angiogenesis and metastasis, and overcoming drug resistance. The Hedgehog (Hh) signaling pathway has been shown to be involved in esophageal adenocarcinoma formation in a reflux animal model. In addition to Hh, we will focus on other targets with clinical potential in the treatment of esophageal cancer. MATERIALS AND METHODS: We searched for articles published from 2005 to August 2020 that studied the siRNA effects on inhibiting esophageal cancer formation in experimental settings. We used combinations of the following terms for searching: "esophageal cancer," "RNA interference," "small interfering RNA," "siRNA," "silencing RNA," "Smoothened (Smo)," "Gli," "Bcl-2," "Bcl-XL," "Bcl-W,″ "Mcl-1," "Bfl-1," "STAT3,"and "Hypoxia inducible factor (HIF)". A total of 21 relevant articles were found. RESULTS AND CONCLUSIONS: Several proto-oncogenes/oncogenes including Hh pathway mediators, glioma-associated oncogene homolog 1 (Gli-1), Smoothened (Smo), and antiapoptotic Bcl-2 have potential as targets for silencing RNA in the treatment of esophageal cancer.

HOTAIR-Loaded Mesenchymal Stem/Stromal Cell Extracellular Vesicles Enhance Angiogenesis and Wound Healing.

Chronic wounds remain a substantial source of morbidity worldwide. An emergent approach that may be well-suited to induce the complex, multicellular processes such as angiogenesis that are required for wound repair is the use of extracellular vesicles (EVs). EVs contain a wide variety of proteins and nucleic acids that enable multifactorial signaling. Here, the capability of EVs is leveraged to be engineered via producer cell modification to investigate the therapeutic potential of EVs from mesenchymal stem/stromal cells (MSCs) transfected to overexpress long non-coding RNA HOX transcript antisense RNA (HOTAIR). HOTAIR is previously shown by the authors' group to be critical in mediating angiogenic effects of endothelial cell EVs, and MSCs are chosen as EV producer cells for this study due to their widely reported intrinsic angiogenic properties. The results indicate that MSCs overexpressing HOTAIR (HOTAIR-MSCs) produce EVs with increased HOTAIR content that promote angiogenesis and wound healing in diabetic (db/db) mice. Further, endothelial cells exposed to HOTAIR-MSC EVs exhibit increased HOTAIR content correlated with upregulation of the angiogenic protein vascular endothelial growth factor. Thus, this study supports EV-mediated HOTAIR delivery as a strategy for further exploration toward healing of chronic wounds.