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.

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.

Total Gastrectomy for CDH-1 Mutation Carriers: An Institutional Experience.

BACKGROUND: Gastric cancer is a leading cause of cancer-related death across the world. A subset of gastric cancers demonstrates an inherited genetic predisposition. Individuals with germline mutations in the CDH1 gene incur a lifetime risk for diffuse gastric cancer and benefit from prophylactic gastrectomy. The results for this operative intervention remain relatively undescribed in the literature, despite guidelines supporting its use. METHODS: We present a single-institution series of patients with confirmed CDH1 mutations who underwent gastrectomy. We describe their presenting symptoms, preoperative screening, clinicopathologic features, and outcomes. Focal outcomes of interest are weight loss and postoperative morbidity. RESULTS: Between 2010 and 2018, ten patients with a confirmed CDH1 mutation underwent total gastrectomy with intestinal pouch reconstruction at our institution. Two patients had clinical gastric cancer at the time of their operation at 21 and 60 y of age. Eight patients had prophylactic gastrectomy. All prophylactic patients had undergone prior endoscopic screening without detection of cancer; however, three had occult gastric cancer on pathological examination. Median weight loss after gastrectomy was 10 kg at 6 mo and 11 kg at 1 y. Postoperative morbidity was limited to one anastomotic leak, one hematoma, and one case of pneumonia. All patients remain disease-free with median follow-up of 19 mo. CONCLUSIONS: Total gastrectomy for patients with a CDH1 mutation is a cancer-preventing operation for a high-risk population. For this series, jejunal pouch reconstruction was performed with encouragingly low postoperative morbidity, weight loss, and good subjective function.

Metastasis-promoting role of extravasated platelet activation in tumor.

BACKGROUND: The last decade has focused attention on the central role of platelets interacting with the tumor cells and the immune system in promoting tumor progression and distant spread through release of growth factors, such as transforming growth factor beta, vascular endothelial growth factor A, and plasminogen activator inhibitor 1, into the tumor microenvironment. We focused on the potential metastasis-promoting role of extravasated platelet aggregation in pancreatic cancer and stroma. MATERIALS AND METHODS: Resected pancreatic cancer specimens from 40 patients were used in this study. To examine the expression and localization of platelet aggregation in the epithelial-mesenchymal transition (EMT) region in cancer and stroma, CD42b, Snail1, and E-cadherin were assessed using immunohistochemistry. We determined the correlation of these expressed proteins with clinical features. RESULTS: CD42b expression was detected at the invasive front of the tumor, which was in 73% of the EMT portion, but not in the region of tubular formation. Increased Snail1 and reduction and/or loss of E-cadherin expressions were noted in 85% and 75% of the EMT portion, respectively. There was a significant correlation between CD42b and Snail1 expressions (P = 0.02) and CD42b and reduction and/or loss of E-cadherin expressions (P = 0.008). CONCLUSIONS: We demonstrate that extravasated platelet aggregation is associated with the first step in the formation of the EMT. These data suggest a potential role for antiplatelet agents to suppress EMT and metastasis by changing the tumor microenvironment.

Strengthening the skin with topical delivery of keratinocyte growth factor-1 using a novel DNA plasmid.

Fragile skin, susceptible to decubitus ulcers and incidental trauma, is a problem particularly for the elderly and for those with spinal cord injury. Here, we present a simple approach to strengthen the skin by the topical delivery of keratinocyte growth factor-1 (KGF-1) DNA. In initial feasibility studies with the novel minimalized, antibiotic-free DNA expression vector, NTC8385-VA1, the reporter genes luciferase and enhanced green fluorescent protein were delivered. Transfection was documented when luciferase expression significantly increased after transfection. Microscopic imaging of enhanced green fluorescent protein-transfected skin showed green fluorescence in hair follicles, hair shafts, and dermal and superficial epithelial cells. With KGF-1 transfection, KGF-1 mRNA level and protein production were documented with quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Epithelial thickness of the transfected skin in the KGF group was significantly increased compared with the control vector group (26 ± 2 versus 16 ± 4 µm) at 48 hours (P = 0.045). Dermal thickness tended to be increased in the KGF group (255 ± 36 versus 162 ± 16 µm) at 120 hours (P = 0.057). Biomechanical assessment showed that the KGF-1-treated skin was significantly stronger than control vector-transfected skin. These findings indicate that topically delivered KGF-1 DNA plasmid can increase epithelial thickness and strength, demonstrating the potential of this approach to restore compromised skin.

Endothelial expression of hypoxia-inducible factor 1 protects the murine heart and aorta from pressure overload by suppression of TGF-ß signaling.

Chronic systemic hypertension causes cardiac pressure overload leading to increased myocardial O(2) consumption. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of O(2) homeostasis. Mouse embryos lacking expression of the O(2)-regulated HIF-1α subunit die at midgestation with severe cardiac malformations and vascular regression. Here we report that Hif1a(f/f);Tie2-Cre conditional knockout mice, which lack HIF-1α expression only in Tie2(+) lineage cells, develop normally, but when subjected to pressure overload induced by transaortic constriction (TAC), they manifest rapid cardiac decompensation, which is accompanied by excess cardiac fibrosis and myocardial hypertrophy, decreased myocardial capillary density, increased myocardial hypoxia and apoptosis, and increased TGF-ß signaling through both canonical and noncanonical pathways that activate SMAD2/3 and ERK1/2, respectively, within endothelial cells of cardiac blood vessels. TAC also induces dilatation of the proximal aorta through enhanced TGF-ß signaling in Hif1a(f/f);Tie2-Cre mice. Inhibition of TGF-ß signaling by treatment with neutralizing antibody or pharmacologic inhibition of MEK-ERK signaling prevented TAC-induced contractile dysfunction and pathological remodeling. Thus, HIF-1 plays a critical protective role in the adaptation of the heart and aorta to pressure overload by negatively regulating TGF-ß signaling in endothelial cells. Treatment of wild-type mice with digoxin, which inhibits HIF-1α synthesis, resulted in rapid cardiac failure after TAC. Although digoxin has been used for decades as an inotropic agent to treat heart failure, it does not improve survival, suggesting that the countertherapeutic effects of digoxin observed in the TAC mouse model may have clinical relevance.

Impact of inflammation-metaplasia-adenocarcinoma sequence and inflammatory microenvironment in esophageal carcinogenesis using surgical rat models.

BACKGROUND AND AIMS: Chronic inflammation has been demonstrated to correlate with tumor onset and progression. Tumor-associated macrophages (TAMs) play an important role in inflammatory tumor microenvironment. We hypothesized that an inflammatory microenvironment around TAMs may promote the development of esophageal carcinomas when induced by duodenal content reflux without carcinogens. ANIMALS AND METHODS: A total gastrectomy followed by esophagojejunostomy was performed on rats in order to induce chronic duodenal content reflux esophagitis. The animals were sacrificed sequentially, at the 20th, 30th, 40th and 50th week after surgery, and their esophagi were examined. The primary antibodies against CD68, CD163, pStat3 and Foxp3 were used. Expression and localization of infiltrated cells was assessed by immunohistochemical analysis. RESULTS: At 20-weeks' post-surgery, squamous proliferative hyperplasia (PHP) and Barrett's metaplasia (BM) were observed. Adenocarcinoma (ADC) associated with BM, and squamous cell carcinoma (SCC) were observed 30-50 weeks' post-surgery. Numerous CD68 and pStat3-positive cells were identified surrounding PHP and BM after 20 weeks, and around ADC and SCC after 30 weeks. Moderate infiltration of CD163-positive macrophages was seen with BM, ADC, and SCC after 30 weeks. However, very few Foxp3-positive cells were observed around ADC and SCC. CONCLUSION: Macrophages infiltrate the esophagus and activate the pStat3 pathway in stromal cells and epithelium. M2 phenotype macrophages infiltrate following infiltration of M1 macrophage and contribute to tumor development through regulatory T cells (Tregs). The involvement of immune cells such as TAMs and Tregs in the inflammatory microenvironment promotes esophageal carcinogenesis.

Dextran hydrogel scaffolds enhance angiogenic responses and promote complete skin regeneration during burn wound healing.

Neovascularization is a critical determinant of wound-healing outcomes for deep burn injuries. We hypothesize that dextran-based hydrogels can serve as instructive scaffolds to promote neovascularization and skin regeneration in third-degree burn wounds. Dextran hydrogels are soft and pliable, offering opportunities to improve the management of burn wound treatment. We first developed a procedure to treat burn wounds on mice with dextran hydrogels. In this procedure, we followed clinical practice of wound excision to remove full-thickness burned skin, and then covered the wound with the dextran hydrogel and a dressing layer. Our procedure allows the hydrogel to remain intact and securely in place during the entire healing period, thus offering opportunities to simplify the management of burn wound treatment. A 3-week comparative study indicated that dextran hydrogel promoted dermal regeneration with complete skin appendages. The hydrogel scaffold facilitated early inflammatory cell infiltration that led to its rapid degradation, promoting the infiltration of angiogenic cells into the healing wounds. Endothelial cells homed into the hydrogel scaffolds to enable neovascularization by day 7, resulting in an increased blood flow significantly greater than treated and untreated controls. By day 21, burn wounds treated with hydrogel developed a mature epithelial structure with hair follicles and sebaceous glands. After 5 weeks of treatment, the hydrogel scaffolds promoted new hair growth and epidermal morphology and thickness similar to normal mouse skin. Collectively, our evidence shows that customized dextran-based hydrogel alone, with no additional growth factors, cytokines, or cells, promoted remarkable neovascularization and skin regeneration and may lead to novel treatments for dermal wounds.

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.

The severity of duodeno-esophageal reflux influences the development of different histological types of esophageal cancer in a rat model.

The mechanism through which each histological type of carcinoma arises from the esophageal mucosa remains unknown. This study was designed to investigate whether there is an association between the severity of duodeno-esophageal reflux and the histological type of esophageal cancer. A series of 120 male Fischer rats, weighing ∼180 g, were randomized to receive one of the following procedures: duodeno-forestomach reflux (DFR) with reduced exposure to duodenal contents, duodeno-esophageal reflux (DER) with increased exposure to duodenal contents and three control operations (DFR, DER control and sham). The reflux of bile was estimated with (99m)Tc-PMT scintigraphy. All animals were fed a standard diet without carcinogen. The esophageal mucosa was assessed 50 weeks after surgery for carcinoma. The median scanned fraction rate of duodeno-esophageal reflux was significantly lower for the rodents in the DFR group than those in the DER group. Five of 28 rodents in the DFR group and 17 of the 22 rodents in the DER group developed esophageal carcinoma. None of the controls developed carcinoma. The five rodents in the DFR group developed SCC. Of 22 esophageal carcinomas for the DER group, nine were SCC, 12 ADC and one was adenosquamous carcinoma. The fraction of esophageal SCC for the DFR group was significantly higher than that for the DER group, while the fraction of esophageal ADC for the DFR group was significantly lower than that for the DER group. These observations suggest that the severity of duodeno-esophageal reflux in rodents is related to the development of different histological types of esophageal carcinoma.

Impact of inflammation-metaplasia-adenocarcinoma sequence and prevention in surgical rat models.

The incidence of esophageal cancer continues to rise in the Western world. Prior studies have suggested that gastroduodenal content reflux from gastroesophageal reflux disease induces the inflammation-mediated progression from hyperplasia to metaplasia, and to adenocarcinoma. We further investigated the sequential development of esophageal adenocarcinoma (EADC) with the use of an established surgical rat model. The present paper will describe the impact of the inflammation-metaplasia-adenocarcinoma sequence and chemoprevention in surgical rat models. A clinically relevant rat reflux model was used to investigate the cause of carcinogenesis, the sequential development of adenocarcinoma and chemoprevention with the use of a proton pump inhibitor. We found that duodenal reflux plays an important role in the inflammation-induced transformation of esophageal mucosa to adenocarcinoma. We were able to inhibit this transformation with rabeprazole, a proton pump inhibitor. Duodenal reflux promotes inflammation in the esophagus. The inflammation-metaplasia-adenocarcinoma sequence is important in the progression and development of EADC. Carcinogenesis can be prevented with chemoprevention agents such as rabeprazole. These results will need to be validated in clinical trials.

Do proton pump inhibitors protect against cancer progression in GERD?

Gastro-duodenal content reflux from gastro-esophageal reflux disease (GERD) induces the inflammation-metaplasia-dysplasia-adenocarcinoma sequence. Proton pump inhibitors (PPIs) are potent blockers of gastric acid secretion, which are widely used for treating GERD and peptic ulcer-associated acid-secreting diseases. The effect of PPI therapy on esophageal carcinogenesis remains unclear. While some studies suggest PPIs result in a significant reduction in the risk of developing dysplasia and adenocarcinoma in patients with Barrett's esophagus, others suggest that PPIs have no effect. Recent studies have revealed that PPIs can exert anti-inflammatory effects such as anti-oxidant properties and immunomodulatory effects through their interactions with neutrophils, monocytes, endothelial and epithelial cells. In addition, PPIs have the ability to prevent adhesion molecule binding in malignant cells and suppress metastasis. This article reviews the role of PPIs in esophageal carcinogenesis and their use as antitumor agents.

Pioneering use of genetic analysis for CDH1 to identify candidates for prophylactic total gastrectomy to prevent hereditary diffuse gastric cancer.

Worldwide, gastric cancer results in significant morbidity and mortality. Ten per cent of patients with gastric cancer have a strong family history of the disease. CDH1 (E-cadherin) has been identified as a key gene whose mutation leads to hereditary diffuse gastric cancer. We overviewed 33 articles with prophylactic total gastrectomy and assessed the outcomes and benefits. Families with mutations in CDH1 may benefit from early prophylactic total gastrectomy. Dr Mark Duncan has applied his experience as a high-volume gastric cancer surgeon to treat not only individual patients, but several generations of patients within a family. This use of prophylactic total gastrectomy is well tolerated by patients and prevents the future development of gastric cancer.

Revolutionary transformation lowering the mortality of pancreaticoduodenectomy: a historical review.

The History Maker paper focuses on the extraordinary revolution that dramatically improved the surgical results for the Whipple procedure (pancreaticoduodenectomy) in the 1980s and identifies Dr. Cameron as the leader of this revolution, who reported a mortality rate of approximately 1%. The revolutionary reduction of postoperative mortality for the Whipple procedure was achieved by adherence to gentle and precise Halstedian surgical techniques with adequate drainage of pancreatico-jejunal anastomosis with closed-suction silastic drains, along with the development of high-volume surgeons and hospitals. Excellent teamwork in patient care, including but not limited to preoperative evaluation by multidisciplinary teams, intraoperative communication between surgeons and anaesthesiologists, and postoperative management, contributed to a successful Whipple procedure.

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.

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.

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.