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.

Extracellular vesicles: a comprehensive review of their roles as biomarkers and potential therapeutics in psoriasis and psoriatic arthritis.

Psoriasis is a chronic immune-mediated condition that affects the skin and joints, with current treatments still unable to offer a cure and long-term use of treatments posing health risks. Understanding the pathogenesis of the disease has helped identify new targets that have allowed for the expansion of the therapeutic arsenal. Extracellular vesicles (EVs) have recently emerged as pathophysiological mediators of psoriasis, and there have been increasing reports of EVs as potential biomarkers and therapeutics. Given their innate role as natural vehicles for cell-to-cell communication, EVs have vast potential in their ability to determine disease status based on EV-specific cargo as well as act as therapeutics because of their anti-inflammatory properties and potential for enhancement. In this review we summarize the role of EVs in the pathogenesis of psoriasis and discuss EVs as both diagnostic and therapeutic agents.

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.

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.

Preconditioning of surgical pedicle flaps with DNA plasmid expressing hypoxia-inducible factor-1α (HIF-1α) promotes tissue viability.

Ischemic necrosis of surgical flaps after reconstruction is a major clinical problem. Hypoxia-inducible factor-1α (HIF-1α) is considered the master regulator of the adaptive response to hypoxia. Among its many properties, it regulates the expression of genes encoding angiogenic growth factors, which have a short half-life in vivo. To achieve a continuous application of the therapeutic, we utilized DNA plasmid delivery. Transcription of the DNA plasmid confirmed by qRT-PCR showed significantly increased mRNA for HIF-1α in the transfected tissue compared to saline control tissue. Rats were preconditioned by injecting with either HIF-1α DNA plasmid or saline intradermally in the designated flap region on each flank. Seven days after preconditioning, each rat had two isolated pedicle flaps raised with a sterile silicone sheet implanted between the skin flap and muscle layer. The flaps preconditioned with HIF-1α DNA plasmid had significantly less necrotic area. Angiogenesis measured by CD31 staining showed a significant increase in the number of vessels per high powered field in the HIF-1α group (p < 0.05). Our findings offer a potential therapeutic strategy for significantly promoting the viability of surgical pedicle flaps by ischemic preconditioning with HIF-1α DNA plasmid.

Targeting the Hedgehog Pathway Using Itraconazole to Prevent Progression of Barrett's Esophagus to Invasive Esophageal Adenocarcinoma.

OBJECTIVE: The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma. BACKGROUND: The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC. METHODS: The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry. RESULTS: BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively. CONCLUSION: Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.

Basal cell carcinosarcoma: a systematic review and reappraisal of its challenges and the role of Mohs surgery.

Basal cell carcinosarcoma (BCCS) is a rare malignant biphasic tumor of the skin, composed of epithelial and mesenchymal components, and may be underdiagnosed. We sought to summarize the current understanding of BCCS including its reported history, clinical presentation, diagnosis, and treatment. We also reappraise and present our recommendations of histological interpretation for its diagnosis and treatment. A systematic review of PubMed and EMBASE, from inception of databases to December 1, 2022, identified all reported cases of basal cell carcinosarcoma. A total of 34 reports containing 54 patients with basal cell carcinosarcoma were included. The neoplasm was most commonly associated in areas of sun-exposed skin and primarily affected the elderly. Diagnosis was made on histology specimens using H&E. To address underdiagnosis, additional immunohistochemical markers have been proposed due to unreliable phenotypic appearance in this poorly differentiated neoplasm. Treatment consists of excision of the tumor, typically with Mohs surgery, and is curative in most cases. There are limited treatment options for metastatic disease. There were limitations to this study as various immunohistochemical stains used on suspected BCCS without providing an explanation as to why certain markers were included and others were excluded. Continued efforts in characterizing this complex neoplasm are critical in establishing reliable and accurate diagnostic tests and accompanying treatment options, especially in cases of metastatic disease.

A case of cutaneous Scedosporium apiospermum infection in a patient on a janus kinase inhibitor.

Scedosporium apiospermum is a mold that is usually found in soil and polluted water, but has also been linked to contaminated ambient air in hospitals. This fungus typically behaves as a rare opportunistic pathogen affecting immunocompromised patients in whom disseminated disease can readily occur, causing shock and multiorgan failure. We report the first case of cutaneous Scedosporium apiospermum infection in a patient with rheumatoid arthritis treated with a Janus kinase inhibitor. We also reviewed other cutaneous manifestations of Scedosporium apiospermum reported between 2003 and 2022.

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.

Clinical outcomes in 'diabese' burn patients: A systematic review and meta-analysis.

OBJECTIVE: The objective of this study was to update the current status of clinical outcomes in diabetic (type II) and obese (BMI: 30-39.9 kg/m2) burn patients. METHODS: We adhered to Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. We searched MEDLINE (PubMed), Google Scholar, Scopus, and Embase for studies related to a number of comorbidities and burn outcomes. Search terms for each of these databases are listed in the Appendix. From this search, we screened 6923 articles. Through our selection criteria, 12 articles focusing on either diabetes or obesity were selected for systematic review and meta-analysis. Data was analyzed using the "meta" package in R software to produce pooled odds ratios from the random effect model. RESULTS: Diabetic patients had 2.38 times higher odds of mortality [OR: 2.38, 95% CI:1.66, 3.41], however no statistically significant difference was found in mortality in obese patients [OR: 2.49, 95% CI: 0.36, 17.19]. Obese patients had 2.18 times higher odds of inhalation injury [95%CI: 1.23, 3.88], whereas diabetic patients did not show a difference in odds of inhalation injury [OR:1.02, 95% CI: 0.57, 1.81]. Diabetic patients had higher odds of complications resulting from infection: 5.47 times higher odds of wound, skin, or soft tissue infections [95% CI:1.97, 15.18]; 2.28 times higher odds of UTI or CAUTI [95% CI:1.50, 3.46]; and 1.78 times higher odds of pneumonia or respiratory tract infections [95% CI:1.15, 2.77]. Obese patients also had similar complications related to infection: 2.15 times higher odds of wound infection [95% CI: 1.04, 4.42] and 1.96 times higher odds of pneumonia [95% CI: 1.08, 3.56]. Other notable complications in diabetic patients were higher odds of amputation [OR: 37, 95% CI: 1.76, 779.34], respiratory failure [OR: 4.39, 95% CI: 1.85, 10.42], heart failure [OR: 6.22, 95% CI: 1.93, 20.06], and renal failure [OR: 2.95, 95% CI: 1.1, 7.86]. CONCLUSIONS: Diabetic patients have higher odds of mortality, whereas no statistically significant difference of mortality was found in obese patients. Obese patients had higher odds of inhalation injury, whereas odds of inhalation injury was unchanged in diabetic patients. Diabetic patients had higher odds of failure in multiple organs, whereas such failure in obese patients was not reported. Both diabetic and obese patients had multiple complications related to infection.

Sustained released of bioactive mesenchymal stromal cell-derived extracellular vesicles from 3D-printed gelatin methacrylate hydrogels.

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate the therapeutic potential of MSC EV-laden GelMA bioinks.

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.

Therapeutic potential of extracellular vesicle-associated long noncoding RNA.

Both extracellular vesicles (EVs) and long noncoding RNAs (lncRNAs) have been increasingly investigated as biomarkers, pathophysiological mediators, and potential therapeutics. While these two entities have often been studied separately, there are increasing reports of EV-associated lncRNA activity in processes such as oncogenesis as well as tissue repair and regeneration. Given the powerful nature and emerging translational impact of other noncoding RNAs such as microRNA (miRNA) and small interfering RNA, lncRNA therapeutics may represent a new frontier. While EVs are natural vehicles that transport and protect lncRNAs physiologically, they can also be engineered for enhanced cargo loading and therapeutic properties. In this review, we will summarize the activity of lncRNAs relevant to both tissue repair and cancer treatment and discuss the role of EVs in enabling the potential of lncRNA therapeutics.

Towards rationally designed biomanufacturing of therapeutic extracellular vesicles: impact of the bioproduction microenvironment.

Extracellular vesicles (EVs), including exosomes, microvesicles, and others, have emerged as potential therapeutics for a variety of applications. Pre-clinical reports of EV efficacy in treatment of non-healing wounds, myocardial infarction, osteoarthritis, traumatic brain injury, spinal cord injury, and many other injuries and diseases demonstrate the versatility of this nascent therapeutic modality. EVs have also been demonstrated to be effective in humans, and clinical trials are underway to further explore their potential. However, for EVs to become a new class of clinical therapeutics, issues related to translation must be addressed. For example, approaches originally developed for cell biomanufacturing, such as hollow fiber bioreactor culture, have been adapted for EV production, but limited knowledge of how the cell culture microenvironment specifically impacts EVs restricts the possibility for rational design and optimization of EV production and potency. In this review, we discuss current knowledge of this issue and delineate potential focus areas for future research towards enabling translation and widespread application of EV-based therapeutics.

Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments.

Green Fluorescent protein (GFP), used as a cellular tag, provides researchers with a valuable method of measuring gene expression and cell tracking. However, there is evidence to suggest that the immunogenicity and cytotoxicity of GFP potentially confounds the interpretation of in vivo experimental data. Studies have shown that GFP expression can deteriorate over time as GFP tagged cells are prone to death. Therefore, the cells that were originally marked with GFP do not survive and cannot be accurately traced over time. This review will present current evidence for the immunogenicity and cytotoxicity of GFP in in vivo studies by characterizing these responses.