Sirolimus-loaded exosomes as a promising vascular delivery system for the prevention of post-angioplasty restenosis.

Restenosis remains the main reason for treatment failure of arterial disease. Sirolimus (SIR) as a potent anti-proliferative agent is believed to prevent the phenomenon. The application of exosomes provides an extended-release delivery platform for SIR intramural administration. Herein, SIR was loaded into fibroblast-derived exosomes isolated by ultracentrifugation. Different parameters affecting drug loading were optimized, and exosome samples were characterized regarding physicochemical, pharmaceutical, and biological properties. Cytotoxicity, scratch wound assays, and quantitative real-time PCR for inflammation- and migration-associated genes were performed. Restenosis was induced by carotid injury in a rat carotid model and then exosomes were locally administered. After 14 days, animals were investigated by computed tomography (CT) angiography, morphometric, and immunohistochemical analyses. Western blotting confirmed the presence of specific protein markers in exosomes. Characterization of empty and SIR-loaded exosomes verified round and nanoscale structure of vesicles. Among prepared formulations, desired entrapment efficiency (EE) of 76% was achieved by protein:drug proportion of 2:1 and simple incubation for 30 min at 37 °C. Also, the optimal formulation released about 30% of the drug content during the first 24 h, followed by a prolonged release for several days. In vitro studies revealed the uptake and functional efficacy of the optimized formulation. In vivo studies revealed that %restenosis was in the following order: saline > empty exosomes > SIR-loaded exosomes. Furthermore, Ki67, alpha smooth muscle actin (α-SMA), and matrix metalloproteinase (MMP) markers were less expressed in the SIR-exosomes-treated arteries. These findings confirmed that exosomal SIR could be a hopeful strategy for the prevention of restenosis.

Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.

Immunomodulatory potential of human clonal mesenchymal stem cells and their extracellular vesicle subpopulations in an inflammatory-mediated diabetic Rhesus monkey model.

AIMS: This study aimed to investigate the therapeutic potential of a homogenous clonal population of mesenchymal stem cells (cMSC) and their extracellular vesicles (cMSC-EV) subpopulations on isolated rat islets in vitro and in inflammatory-mediated type 1 diabetes (T1D) non-human primate models. MAIN METHODS: EV subpopulations were isolated from human bone marrow-derived cMSC supernatant by low- and high-speed ultracentrifuge (EV-20K and EV-U110K) and sucrose density gradient (EV-S110K). The EVs were characterized generally and for the level of albumin, acetylcholinesterase (AChE) activity, co-isolate apoptotic markers, and expression of CD63+/annexin V+. Rat islet-derived single cells (iSCs) proliferation was measured using a Ki-67 proliferation assay. Diabetes was induced by multiple low-dose administrations of streptozotocin in rhesus monkeys. The diabetic monkeys were divided into three groups: the cMSC group, received two injections of 1.5 × 106 cMSC/kg body weight; the EV group received two injections of EVs isolated from 1.5 × 106 cMSC/kg, and the vehicle group received phosphate-buffered saline. KEY FINDINGS: EV-S110K showed higher AChE activity, lower expression of CD63+/annexin V+, and lower apoptotic co-isolates. EV-S110K induced ß-cell proliferation in vitro in a dose-dependent manner. The administration of EV-S110K and/or cMSC in diabetic monkeys demonstrated no significant changes in general diabetic indices and ß-cell mass in the pancreas of the monkeys. Both treatments demonstrated a lowering trend in blood glucose levels and reduced pro-inflammatory cytokines. In contrast, regulatory T cells and anti-inflammatory cytokines were increased. SIGNIFICANCE: cMSC and cMSC-EV provided initial evidence to attenuate clinical symptoms in inflammatory-mediated T1D non-human primates through immunomodulation.

Clonal mesenchymal stem cell-derived extracellular vesicles improve mouse model of weight drop-induced traumatic brain injury through reducing cistauosis and apoptosis.

OBJECTIVE: Traumatic brain injury (TBI) is a major risk factor for disabilities globally with no effective treatment thus far. Recently, homogenous population of clonal mesenchymal stem cells (cMSC) and their derived extracellular vesicles (cMSC-EVs) have been proposed as a promising TBI treatment strategy. We herein investigated possible therapeutic effect of cMSC-EVs in TBI treatment and the underlying mechanisms considering cis p-tau as an early hallmark of TBI. METHODS: We examined the EVs morphology, size distribution, marker expression, and uptake. Moreover, the EVs neuroprotective effects were studied in both in-vitro and in-vivo model. We also examined the anti-cis p-tau antibody-loading characteristics of the EVs. We treated TBI mouse model with EVs; prepared from cMSC-conditioned media. TBI mice were given cMSC-EVs intravenously and their cognitive functions were analyzed two months of the treatment. We employed immunoblot analysis to study the underlying molecular mechanisms. RESULTS: We observed a profound cMSC-EVs uptake by primary cultured neurons. We found a remarkable neuroprotective effect of cMSC-EVs upon nutritional deprivation stress. Furthermore, cMSC-EVs were effectively loaded with an anti-cis p-tau antibody. There was a significant improvement in cognitive function in TBI animal models treated with cMSC-EVs compared to the saline-treated group. There was a decreased cis p-tau and cleaved caspase3 as well as increased p-PI3K in all treated animals. CONCLUSIONS: The results revealed that cMSC-EVs efficiently improved animal behaviors after TBI by reducing cistauosis and apoptosis. Moreover, the EVs can be employed as an effective strategy for antibody delivery during passive immunotherapy.

Allogenic mesenchymal stromal cells and their extracellular vesicles in COVID-19 induced ARDS: a randomized controlled trial.

BACKGROUND AND AIMS: The main causes of death in patients with severe Coronavirus disease-2019 (COVID-19) are acute respiratory distress syndrome (ARDS) and multiorgan failure caused by a severe inflammatory cascade. Novel treatment strategies, such as stem-cell-based therapy and their derivatives can be used to relieve inflammation in these cases. In this study, we aimed to evaluate the safety and efficacy of therapy using mesenchymal stromal cells (MSCs) and their derived extracellular vesicles in COVID-19 patients. MATERIALS AND METHODS: COVID-19 patients with ARDS were included in this study and allocated into two study and control groups using block randomization. While all patients received recommended treatment based on guidelines from the national advisory committee for COVID-19 pandemic, the two intervention groups received two consecutive injections of MSCs (100 × 106 cells) or one dose of MSCs (100 × 106 cells) followed by one dose of MSC-derived extracellular vesicles (EVs). Patients were assessed for safety and efficacy by evaluating clinical symptoms, laboratory parameters, and inflammatory markers at baseline and 48 h after the second intervention. RESULTS: A total number of 43 patients (the MSC alone group = 11, MSC plus EV group = 8, and control group = 24) were included in the final analysis. Mortality was reported in three patients in the MSC alone group (RR: 0.49; 95% CI 0.14-1.11; P = 0.08); zero patient in the MSC plus EV group (RR: 0.08; 95% CI 0.005-1.26; P = 0.07) and eight patients in the control group. MSC infusion was associated with a decrease in inflammatory cytokines such as IL-6 (P = 0.015), TNF-α (P = 0.034), IFN-γ (P = 0.024), and CRP (P = 0.041). CONCLUSION: MSCs and their extracellular vesicles can significantly reduce the serum levels of inflammatory markers in COVID-19 patients, with no serious adverse events. Trial registration IRCT, IRCT registration number: IRCT20200217046526N2. Registered 13th April 2020, http://www.irct.ir/trial/47073 .

Homogenous subpopulation of human mesenchymal stem cells and their extracellular vesicles restore function of endometrium in an experimental rat model of Asherman syndrome.

BACKGROUND: Asherman syndrome (AS), or intrauterine adhesions, is a main cause of infertility in reproductive age women after endometrial injury. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) are promising candidates for therapies that repair damaged endometria. However, concerns about their efficacy are attributed to heterogeneity of the cell populations and EVs. A homogenous population of MSCs and effective EV subpopulation are needed to develop potentially promising therapeutic options in regenerative medicine. METHODS: AS model was induced by mechanical injury in adult rat uteri. Then, the animals were treated immediately with homogeneous population of human bone marrow-derived clonal MSCs (cMSCs), heterogenous parental MSCs (hMSCs), or cMSCs-derived EV subpopulations (EV20K and EV110K). The animals were sacrificed two weeks post-treatment and uterine horns were collected. The sections were taken, and hematoxylin-eosin was used to examine the repair of endometrial structure. Fibrosis was measured by Masson's trichrome staining and α-SMA and cell proliferation by Ki67 immunostaining. The function of the uteri was explored by the result of mating trial test. Expression changes of TNFα, IL-10, VEGF, and LIF were assayed by ELISA. RESULTS: Histological analysis indicated fewer glands, thinner endometria, increased fibrotic areas, and decreased proliferation of epithelial and stroma of the uteri in the treated compared with intact and sham-operated animals. However, these parameters improved after transplantation of both types of cMSCs and hMSCs and/or both cryopreserved EVs subpopulations. The cMSCs demonstrated more successful implantation of the embryos in comparison with hMSCs. The tracing of the transplanted cMSCs and EVs showed that they migrated and localized in the uteri. Protein expression analysis results demonstrated downregulation of proinflammatory factor TNFα and upregulation of anti-inflammatory cytokine IL-10, and endometrial receptivity cytokines VEGF and LIF in cMSC- and EV20K-treated animals. CONCLUSION: Transplantation of MSCs and EVs contributed to endometrial repair and restoration of reproductive function, likely by inhibition of excessive fibrosis and inflammation, enhancement of endometrial cell proliferation, and regulation of molecular markers related to endometrial receptivity. Compared to classical hMSCs, cMSCs were more efficient than hMSCs in restoration of reproductive function. Moreover, EV20K is more cost-effective and feasible for prevention of AS in comparison with conventional EVs (EV110K).

Regenerative potential of different extracellular vesicle subpopulations derived from clonal mesenchymal stem cells in a mouse model of chemotherapy-induced premature ovarian failure.

AIMS: Some studies have shown that mesenchymal stem cells (MSCs) and their derived extracellular vesicles (MSC-EVs) can restore ovarian function in premature ovarian failure (POF), however, concerns about their efficacy are attributed to the heterogeneity of the cell populations and EVs. Here, we assessed the therapeutic potential of a homogeneous population of clonal MSCs (cMSCs) and their EVs subpopulations in a mouse model of POF. MAIN METHODS: Granulosa cells were treated with cyclophosphamide (Cy) in the absence or presence of cMSCs, or cMSCs-derived EV subpopulations (EV20K and EV110K, isolated by high-speed centrifugation and differential ultracentrifugation, respectively). In addition, POF mice were treated with cMSCs, EV20K and/or EV110K. KEY FINDINGS: cMSC and both EV types protected granulosa cells from Cy-induced damage. Calcein-EVs were detected in the ovaries. Moreover, cMSC and both EV subpopulations significantly increased body weight, ovary weight, and the number of follicles, restored FSH, E2, and AMH levels, increased the granulosa cell numbers and restored the fertility of POF mice. cMSC, EV20K, and EV110K alleviated inflammatory-related genes expression (Tnf-α and IL8), and improved angiogenesis via upregulation expression of Vegf and Igf1 at the mRNA level and VEGF and αSMA at the protein level. They also inhibited apoptosis through the PI3K/AKT signaling pathway. SIGNIFICANCE: The administration of cMSCs and two cMSC-EVs subpopulations improved ovarian function and restored fertility in a POF model. EV20K is more cost-effective and feasible in terms of isolation, particularly in good manufacturing practice (GMP) facilities for treatment of POF patients in comparison with conventional EVs (EV110K).

Human uterine fluid lavage-derived extracellular vesicle isolation: a comparative study for minimally invasive endometrial receptivity assessment.

RESEARCH QUESTION: Does pre-implantation uterine fluid lavage (UFL) of patients undergoing IVF and frozen embryo transfer (FET) affect implantation and clinical pregnancy rates? Which methods among ultracentrifugation, sucrose cushion and qEV column are suitable for isolating UFL extracellular vesicles? DESIGN: First, UFL was collected from 20 patients undergoing IVF and FET 2 days before embryo transfer as the case group. The control group consisted of 20 patients undergoing IVF and FET patients without lavage. All patients were monitored for 6 weeks. In the next step, the UFLs (n = 30) were collected and pooled. The UFL-derived extracellular vesicles were extracted by ultracentrifugation, sucrose cushion and qEV column methods and characterized. RESULTS: Preimplantation uterine lavage sampling did not affect implantation and clinical pregnancy rates. Extracellular vesicles were successfully isolated from UFL by all three methods. Scanning electron microscopy and dynamic light scattering analysis showed that the isolated vesicles were morphologically spherical. The qEV technique showed that they were smaller and homogenized in size. SDS-PAGE of extracellular vesicles showed a weaker albumin band in the qEV column. Western blot analysis indicated that the isolated extracellular vesicles by the qEV column were more immunoreactive for all the common extracellular vesicle markers (CD81, CD9, CD63, and TSG101). Six reference genes were compared by real-time polymerase chain reaction in the isolated extracellular vesicle subpopulations, and lowest cycle threshold value was observed for the 18SrRNA gene. CONCLUSIONS: The isolation of endometrial secretome extracellular vesicles is a minimally invasive procedure for individual assessment of endometrial receptivity and can be carried out during conception cycles along with transvaginal ultrasonography. Molecular analysis of UFL-derived extracellular vesicle components could suggest biomarkers to determine precise extracellular vesicle timing.

Pre-clinical investigation of mesenchymal stromal cell-derived extracellular vesicles: a systematic review.

The therapeutic potential of naturally secreted micro- and nanoscale extracellular vesicles (EVs) makes them attractive candidates for regenerative medicine and pharmaceutical science applications. To date, the results of numerous publications have shown the practicality of using EVs to replace mesenchymal stromal cells (MSCs) or liposomes. This article presents a systematic review of pre-clinical studies conducted over the past decade of MSC-derived EVs (MSC-EVs) used in animal models of disease. The authors searched the relevant literature in the PubMed and Scopus databases (9358 articles), and 690 articles met the inclusion criteria. The eligible articles were placed in the following disease categories: autoimmune, brain, cancer, eye, gastrointestinal, heart, inflammation/transplantation, liver, musculoskeletal, pancreas, spinal cord and peripheral nervous system, respiratory system, reproductive system, skin, urinary system and vascular-related diseases. Next, the eligible articles were assessed for the rate of publication and global distribution, methodology of EV isolation and characterization, route of MSC-EV administration, length of follow-up, source of MSCs and animal species. The current review classifies and critically discusses the technical aspects of these MSC-EV animal studies and discusses potential relationships between methodological details and the effectiveness of MSC-EVs as reported by these pre-clinical studies.

Extracellular Vesicles Derived from Human Umbilical Cord Perivascular Cells Improve Functional Recovery in Brain Ischemic Rat via the Inhibition of Apoptosis.

Background: Ischemic stroke, as a health problem caused by the reduced blood supply to the brain, can lead to the neuronal death. The number of reliable therapies for stroke is limited. Mesenchymal stem cells (MSCs) exhibit therapeutic achievement. A major limitation of MSC application in cell therapy is the short survival span. MSCs affect target tissues through the secretion of many paracrine agents including extracellular vesicles (EVs). This study aimed to investigate the effect of human umbilical cord perivascular cells (HUCPVCs)-derived EVs on apoptosis, functional recovery, and neuroprotection. Methods: Ischemia was induced by middle cerebral artery occlusion (MCAO) in male Wistar rats. Animals were classified into sham, MCAO, MCAO + HUCPVC, and MCAO + EV groups. Treatments began at two hours after ischemia. Expressions of apoptotic-related proteins (BAX/BCl-2 [B-cell lymphoma-2] and caspase-3 and -9), the amount of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, neuronal density (microtubule-associated protein 2 [MAP2]), and dead neurons (Nissl staining) were assessed on day seven post MCAO. Results: Administration of EVs improved the sensorimotor function (p < 0.001) and reduced the apoptotic rate of Bax/Bcl-2 ratio (p < 0.001), as well as caspases and TUNEL-positive cells (p < 0.001) in comparison to the MCAO group. EV treatment also reduced the number of dead neurons and increased the number of MAP2+ cells in the ischemic boundary zone (p < 0.001), as compared to the MCAO group. Conclusion: Our findings showed that HUCPVCs-derived EVs are more effective than their mother's cells in improving neural function, possibly via the regulation of apoptosis in the ischemic rats. The strategy of cell-free extracts is, thus, helpful in removing the predicaments surrounding cell therapy in targeting brain diseases.

Extracellular vesicles derived from human ES-MSCs protect retinal ganglion cells and preserve retinal function in a rodent model of optic nerve injury.

BACKGROUND: Retinal and/or optic nerve injury is one of the leading causes of blindness due to retinal ganglion cell (RGC) degeneration. There have been extensive efforts to suppress this neurodegeneration. Various somatic tissue-derived mesenchymal stem cells (MSCs) demonstrated significant neuroprotective and axogenic effects on RGCs. An alternative source of MSCs could be human embryonic stem cells (ES-MSCs), which proliferate faster, express lower levels of inflammatory cytokines, and are capable of immune modulation. It has been demonstrated that MSCs secrete factors or extracellular vesicles that may heal the injury. However, possible therapeutic effects and underlying mechanism of human ES-MSC extracellular vesicles (EVs) on optic nerve injury have not been assessed. METHODS: EVs were isolated from human ES-MSCs. Then, ES-MSC EV was applied to an optic nerve crush (ONC) mouse model. Immunohistofluorescence, retro- and anterograde tracing of RGCs, Western blot, tauopathy in RGCs, and function assessments were performed during 2-month post-treatment to evaluate ONC improvement and underlying mechanism of human ES-MSC EV in in vivo. RESULTS: We found that the ES-MSC EV significantly improved Brn3a+ RGCs survival and retro- and anterograde tracing of RGCs, while preventing retinal nerve fiber layer (RNFL) degenerative thinning compared to the vehicle group. The EVs also significantly promoted GAP43+ axon counts in the optic nerve and improved cognitive visual behavior. Furthermore, cis p-tau, a central mediator of neurodegeneration in the injured RGCs, is detectable after the ONC at the early stages demonstrated tauopathy in RGCs. Notably, after EV treatment cis p-tau was downregulated. CONCLUSIONS: Our findings propose that human ES-MSC EVs, as an off-the-shelf and cell-free product, may have profound clinical implications in treating injured RGCs and degenerative ocular disease. Moreover, the possible mechanisms of human ES-MSC EV are related to the rescue of tauopathy process of RGC degeneration.

Mesenchymal stem cell-derived extracellular vesicles alone or in conjunction with a SDKP-conjugated self-assembling peptide improve a rat model of myocardial infarction.

PURPOSE: The aim of this study was to investigate the cardiac repair effect of human bone marrow mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) after intramyocardial injection in free form or encapsulated within a self-assembling peptide hydrogel modified with SDKP motif, in a rat model of myocardial infarction (MI). METHODS: MSC-EVs were isolated by ultracentrifuge and characterized for physical parameters and surface proteins. Furthermore, cellular uptake and cardioprotective effects of MSC-EVs were evaluated in vitro using neonatal mouse cardiomyocytes (NMCMs). In vivo effects of MSC-EVs on cardiac repair were studied in rat MI model by comparing the vehicle group (injected with PBS), EV group (injected with MSC-EVs) and Gel + EV group (injected with MSC-EVs encapsulated in (RADA)4-SDKP hydrogel) with respect to cardiac function and fibrotic area using echocardiography and Masson's trichrome staining, respectively. Histological sections were assessed by α-SMA and CD68 immunostaining to investigate the angiogenic and anti-inflammatory effects of the MSC-EVs. RESULTS: We observed the uptake of MSC-EVs into NMCMs which led to NMCMs protection against H2O2-induced oxidative stress by substantial reduction of apoptosis. In myocardial infarcted rats, cardiac function was improved after myocardial injection of MSC-EVs alone or in conjunction with (RADA)4-SDKP hydrogel. This functional restoration coincided with promotion of angiogenesis and decrement of fibrosis and inflammation. CONCLUSION: These data demonstrated that MSC-EVs can be used alone as a potent therapeutic agent for improvement of myocardial infarction.

Long-Term Follow-up of Autologous Fibroblast Transplantation for Facial Contour Deformities, A Non-Randomized Phase IIa Clinical Trial.

OBJECTIVE: Recently, the promising potential of fibroblast transplantation has become a novel modality for skin rejuvenation. We investigated the long-term safety and efficacy of autologous fibroblast transplantation for participants with mild to severe facial contour deformities. MATERIALS AND METHODS: In this open-label, single-arm phase IIa clinical trial, a total of 57 participants with wrinkles (n=37, 132 treatment sites) or acne scars (n=20, 36 treatment sites) who had an evaluator's assessment score of at least 2 out 7 (based on a standard photo-guide scoring) received 3 injections of autologous cultured fibroblasts administered at 4-6 week intervals. Efficacy evaluations were performed at 2, 6, 12, and 24 months after the final injection based on evaluator and patient's assessment scores. RESULTS: Our study showed a mean improvement of 2 scores in the wrinkle and acne scar treatment sites. At sixth months after transplantation, 90.1% of the wrinkle sites and 86.1% of the acne scar sites showed at least a one grade improvement on evaluator assessments. We also observed at least a 2-grade improvement in 56.1% of the wrinkle sites and 63.9% of the acne scar sites. A total of 70.5% of wrinkle sites and 72.2% of acne scar sites were scored as good or excellent on patient assessments. The efficacy outcomes remained stable up to 24-month. We did not observe any serious adverse events during the study. CONCLUSION: These results have shown that autologous fibroblast transplantation could be a promising remodeling modality with long-term corrective ability and minimal adverse events (Registration Number: NCT01115634).

Effects of neural stem cell-derived extracellular vesicles on neuronal protection and functional recovery in the rat model of middle cerebral artery occlusion.

Stroke imposes a long-term neurological disability with limited effective treatments available for neuronal recovery. Transplantation of neural stem cells (NSCs) is reported to improve functional outcomes in the animal models of brain ischemia. However, the use of cell therapy is accompanied by adverse effects, so research is growing to use cell-free extracts such as extracellular vesicles (EVs) for targeting brain diseases. In the current study, male Wistar albino rats (20 months old) were subjected to middle cerebral artery occlusion (MCAO). Then, EVs (30 µg) were injected at 2 hours after stroke onset via an intracerebroventricular (ICV) route. Measurements were done at day 7 post-MCAO. EVs administration reduced lesion volume and steadily improved spontaneous locomotor activity. EVs administration also reduced microgliosis (ionized calcium-binding adaptor molecule 1 (Iba1)+ cells) and apoptotic (terminal-deoxynucleotidyl transferase mediated nick end labelling [TUNEL]) positive cells and increased neuronal survival (neuronal nuclear (NeuN)+ cells) in the ischemic boundary zone (IBZ). However, it had no effect on neurogenesis within the sub-ventricular zone (SVZ) but decreased cellular migration toward the IBZ (doublecortin (DCX)+ cells). The results of this study showed neuroprotective and restorative mechanisms of NSC-EVs administration, which may offer new avenues for therapeutic intervention of brain ischemia. SIGNIFICANCE OF THE STUDY: Based on our results, EVs administration can effectively reduce microglial density and neuronal apoptosis, thereby steadily improves functional recovery after MCAO. These findings provide the beneficial effect of NSC-EVs as a new biological treatment for stroke.

Hydrogel-Mediated Sustained Systemic Delivery of Mesenchymal Stem Cell-Derived Extracellular Vesicles Improves Hepatic Regeneration in Chronic Liver Failure.

Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have been widely reported as promising cell-free products that show therapeutic effects of the parental cells but not their limitations. Due to the intrinsic liver tropism of MSC-EVs, they have been widely used as therapeutics or drug carriers for treatment of liver diseases. However, rapid clearance from the target site may attenuate the efficiency of systemically administered MSC-EVs. Herein, sustained release into the peritoneum has been proposed as a new strategy to prolong the bioavailability of the MSC-EVs in the target liver. During intraperitoneal injection, clickable polyethylene glycol (PEG) macromeres were mixed with MSC-EVs to form EV-encapsulated PEG hydrogels via a fast, biocompatible click reaction. Upon biodegradation, the EV-laden hydrogels were swollen gradually to release EVs in a sustained manner over 1 month. In vivo tracking of the labeled EVs revealed that the accumulation of EVs in the liver was extended by hydrogel-mediated delivery for 1 month. Four weeks after injection in a rat model of chronic liver fibrosis, the physical and histopathological investigations of the harvested liver showed superior antifibrosis, anti-apoptosis, and regenerative effects of the EVs when delivered by the sustained systemic release (Gel-EV) to the conventional bolus injection (Free-EV). Specifically, the Gel-EV system improved the antifibrosis, anti-inflammation, anti-apoptosis, and regenerative effects of the EVs to nearly 40, 50, 40, and 50% compared to Free-EV, respectively, as was specified by quantification of the fibrotic area, α-SMA density, and caspase-3 density in the harvested tissues and ALT enzyme in serum. This study may potentiate the use of MSC-EVs as cell-free therapeutics for chronic liver failure. The sustained systemic delivery strategy may open a new paradigm to extend the effects of disease-targeting EVs over time.

Treatment of Hypertrophic Scar in Human with Autologous Transplantation of Cultured Keratinocytes and Fibroblasts along with Fibrin Glue.

OBJECTIVE: Hypertrophic scar involves excessive amounts of collagen in dermal layer and may be painful. Nowadays, we can't be sure about effectiveness of procedure for hypertrophic scar management. The application of stem cells with natural scaffold has been the best option for treatment of burn wounds and skin defect, in recent decades. Fibrin glue (FG) was among the first of the natural biomaterials applied to enhance skin deformity in burn patients. This study aimed to identify an efficient, minimally invasive and economical transplantation procedure using novel FG from human cord blood for treatment of hypertrophic scar and regulation collagen synthesis. MATERIALS AND METHODS: In this case series study, eight patients were selected with hypertrophic scar due to full-thickness burns. Human keratinocytes and fibroblasts derived from adult skin donors were isolated and cultured. They were tested for the expression of cytokeratin 14 and vimentin using immunocytochemistry. FG was prepared from pooled cord blood. Hypertrophic scars were extensively excised then grafted by simply placing the sheet of FG containing autologous fibroblast and keratinocytes. Histological analyses were performed using Hematoxylin and eosin (H&E) and Masson's Trichrome (MT) staining of the biopsies after 8 weeks. RESULTS: Cultured keratinocytes showed a high level of cytokeratin 14 expression and also fibroblasts showed a high level of vimentin. Histological analyses of skin biopsies after 8 weeks of transplantation revealed re-epithelialization with reduction of hypertrophic scars in 2 patients. CONCLUSION: These results suggest may be the use of FG from cord blood, which is not more efficient than previous biological transporters and increasing hypertrophic scar relapse, but could lead to decrease pain rate.