Cell-free Therapy Research Articles

Adipose-Derived Stem Cells Secretome and Its Potential Application in “Stem Cell-Free Therapy”

Adipose-derived stem cells (ASCs) secrete many cytokines, proteins, growth factors, and extracellular vesicles with beneficial outcomes that can be used in regenerative medicine. It has great potential, and the development of new treatment strategies using the ASCs secretome is of global interest. Besides cytokines, proteins, and growth factors, the therapeutic effect of secretome is hidden in non-coding RNAs such as miR-21, miR-24, and miR-26 carried via exosomes secreted by adequate cells. The whole secretome, including ASC-derived exosomes (ASC-exos) has been proven in many studies to have immunomodulatory, proangiogenic, neurotrophic, and epithelization activity and can potentially be used for neurodegenerative, cardiovascular, respiratory, inflammatory, and autoimmune diseases as well as wound healing treatment. Due to limitations in the use of stem cells in cell-based therapy, its secretome with emphasis on exosomes seems to be a reasonable and safer alternative with increased effectiveness and fewer side effects. Moreover, the great advantage of cell-free therapy is the possibility of biobanking the ASCs secretome. In this review, we focus on the current state of knowledge on the use of the ASCs secretome in stem cell-free therapy.

AC electrokinetic isolation and detection of extracellular vesicles from dental pulp stem cells: Theoretical simulation incorporating fluid mechanics.

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles involved in intracellular communication and the transportation of biomarkers. EVs released by mesenchymal stem cells have been recently reported to play a role in cell-free therapy of many diseases. However, the demand for better research tools to replace the tedious conventional methods used to study EVs is getting stronger. EVs' manipulation using alternating current (AC) electrokinetic forces in a microfluidic device has appeared to be a reliable and sensitive diagnosis and trapping technique. Given that different AC electrokinetic forces may contribute to the overall motion of particles and fluids in a microfluidic device, EVs' electrokinetic trapping must be examined considering all dominant forces involved depending on the experimental conditions. In this paper, AC electrokinetic trapping of EVs using an interdigitated electrode arrays is investigated. A 2D numerical simulation incorporating the two significant AC electrokinetic phenomena (Dielectrophoresis and AC electroosmosis) has been performed. Theoretical predictions are then compared with experimental results and allow for a plausible explanation of observations inconsistent with DEP theory. It is demonstrated that the inconsistencies can be attributed to a significant extent to the contribution of the AC electroosmotic effect.

Current status of myocardial restoration via the paracrine function of mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have been studied for nearly two decades as a therapy for myocardial restoration. An emerging direction to repair myocardium is through their paracrine function, which includes the utilization of MSC-derived conditioned medium or extracellular vesicles. In this review, we go over the unique characteristics of MSCs that make it suitable for "off the shelf," cell-free regenerative therapy, current MSC-derived cell-free approaches including their advantages and disadvantages, and the known mechanisms of action of the paracrine effect of MSCs. With a summary of the clinical trials and preclinical studies of MSC-derived cell-free therapy, we classify the aforementioned mechanisms into angiogenesis, immunomodulation, extracellular matrix remodeling, antiapoptosis, and antioxidation. Particularly, we discuss on ways researchers have worked toward enhancing these desired properties to improve the therapeutic outcomes and the investigation of mechanobiology involved in MSC paracrine function. Lastly, we bring up the remaining challenges in this arising field and suggestions for future directions to improve our understanding and control over the potential of MSC paracrine function for myocardial restoration.

Mesenchymal Stem Cells Lysate as a Cell-Free Therapy: A Review

Abstract: Mesenchymal Stem Cells (MSCs), a self-renewing and multipotent population of cells have proved to be a potential regenerative medical therapy option. Initially, it was assumed that the primary mechanism behind MSCs action was cell substitution by differentiation and engraftment at the injury site. However, recent trials have demonstrated that implanted cells don’t last long and that the benefits of MSCs therapy may be triggered by a wide number of bioactive factors, essential for regulating main biological processes. Thus, supporting the paracrine mechanism of action of MSCs. Application of secretome could eliminate the potential risk factors associated with MSCs transplantation, like the unknown fate of cells after transplantation, malignant transformation, and undesired differentiation of MSCs. However, the use of a secretome might not give long-lasting and desired results. Thus, it may intuitively suggest the need for other treatment options. The cellular lysate is considered as an alternate cell-free treatment strategy for the cure of multiple disorders. The present review describes the MSCs secretome/extracellular vesicles and MSCs lysate, derived from bone marrow and adipose, as a cell-free therapy with the application of MSCs lysate for various diseases from 2010 to 2020. Moreover, it also highlights the advantages and limitations of each cell-free therapy.

Cell-Free Blood Cell Secretome (BCS) Counteracts Skin Aging and Restores Firmness and Elasticity.

Blood Cell Secretome, BCS (also Autologous Conditioned Serum, ACS) is efficacious in treatment of musculoskeletal disorders. It contains inflammation resolving cytokines, growth factors, exosomes, and lipid mediators. Skin aging is associated with reduced TGF-ß signaling and collagen synthesis and chronic (sub-acute) inflammation, among other factors. Pre-clinically, BCS counteracts these mechanisms, suggesting it as a treatment against cutaneous aging. This 24-week study evaluated the effects of deep dermal to immediate sub-dermal micropuncture injections of cell-free BCS in patients with age-related reduced facial skin elasticity. In this prospective, single-armed, mono-center study, 21 women underwent 4 BCS treatment sessions over 12 weeks with follow-up at 24 weeks. The primary endpoint was skin elasticity measured by cutometry. Secondary endpoints were safety, skin hydration, and aesthetic assessments using global aesthetic improvement scale. Skin firmness increased significantly between baseline and 12 weeks (P<0.001) and further increased by 24 weeks (P<0.001). Skin tiring was congruently reduced (P<0.001). Skin hydration and aesthetic ratings improved significantly. No BCS-related adverse reactions occurred. BCS treatment resulted in increased firmness and hydration, usually attributed to younger skin. BCS is potentially the first cell-free autologous therapy for skin rejuvenation derived from patients’ own blood. J Drugs Dermatol. 2021;20(6):682-688. doi:10.36849/JDD.5018.

369-P: Wound Repair by P53 Silenced Human Endothelial Cell-Derived Conditioned Media, In Vitro

Introduction: Diabetes is associated with impaired wound healing. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature human endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment and p53 gene silencing prevents cellular senescence and helps vascular regeneration. Our goal was to test whether cell conditioned media obtained from p53 silenced human cells can also improve cellular regeneration. Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control. To collect condition media, different human cells such as HEK, human CD14+ve cells and HUVEC (human umbilical vein endothelial cells) were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% BSA (with no FBS and growth factors) for only 24 h and the collected supernatant was concentrated 10-fold to obtain the conditioned media (CM). Endothelial regeneration or wound healing was tested using endothelial wound healing assay kit (from Cell Biolabs # CBA-120-T). Results: We compared CM obtained from null and p53sh in HEK, CD14+ve and HUVEC cells on human endothelial cells. Proliferation capacity of the endothelial cells was greater with p53sh CM compared to null and non-transduced CM. The CM obtained from the untreated and null samples did not show increased proliferation in any of the 3 kinds of cells tested. The CM from p53sh HUVEC was more effective than CM from other cells. We are analyzing the CM (exosome and non-exosome fraction) for identification of specific secretory proteins. Conclusion: P53 silencing appears to improve HUVEC paracrine factors and this particular modality of cell free therapy may have therapeutic role in treating diabetes complications and even aging. Disclosure D. K. Rivera: None. S. Nandula: None. S. Sen: None. Funding American Heart Association

Exosomes derived from pioglitazone-pretreated MSCs accelerate diabetic wound healing through enhancing angiogenesis

BackgroundEnhanced angiogenesis can promote diabetic wound healing. Mesenchymal stem cells (MSCs)-derived exosomes, which are cell-free therapeutics, are promising candidates for the treatment of diabetic wound healing. The present study aimed to investigate the effect of exosomes derived from MSCs pretreated with pioglitazone (PGZ-Exos) on diabetic wound healing.ResultsWe isolated PGZ-Exos from the supernatants of pioglitazone-treated BMSCs and found that PGZ-Exos significantly promote the cell viability and proliferation of Human Umbilical Vein Vascular Endothelial Cells (HUVECs) injured by high glucose (HG). PGZ-Exos enhanced the biological functions of HUVECs, including migration, tube formation, wound repair and VEGF expression in vitro. In addition, PGZ-Exos promoted the protein expression of p-AKT, p-PI3K and p-eNOS and suppressed that of PTEN. LY294002 inhibited the biological function of HUVECs through inhibition of the PI3K/AKT/eNOS pathway. In vivo modeling in diabetic rat wounds showed that pioglitazone pretreatment enhanced the therapeutic efficacy of MSCs-derived exosomes and accelerated diabetic wound healing via enhanced angiogenesis. In addition, PGZ-Exos promoted collagen deposition, ECM remodeling and VEGF and CD31 expression, indicating adequate angiogenesis in diabetic wound healing.ConclusionsPGZ-Exos accelerated diabetic wound healing by promoting the angiogenic function of HUVECs through activation of the PI3K/AKT/eNOS pathway. This offers a promising novel cell-free therapy for treating diabetic wound healing.Graphic abstract

Evaluation of the performance, operability, and safety of Plasautoμ, a new type of machine for cell-free and concentrated ascites reinfusion therapy, in a postmarketing clinical study.

Cell‐free and concentrated ascites reinfusion therapy (CART) is performed by collecting the ascites from the patient, followed by filtration and concentration. Thereafter, concentrated cell‐free ascites is reinfused into the patient intravenously. The new type of machine, Plasauto μ, for managing the process of CART was launched onto the market. We have evaluated the machine through postmarketing clinical study in 17 patients with malignant ascites. The amounts of original and concentrated ascites were 3673 ± 1920 g and 439 ± 228 g, respectively. Recovery rates were acceptable regarding values of total protein, albumin, and IgG that were 55.6% ± 17.3%, 60.2% ± 20.8%, and 58.2% ± 20.5%, respectively. Recovery rates were positively associated with amounts of original ascites and negatively associated with total protein concentration. No adverse events related to the machine were observed. The new type of machine showed preferable performance in processing malignant ascites.

TRAIL-Armed ER Nanosomes Induce Drastically Enhanced Apoptosis in Resistant Tumor in Combination with the Antagonist of IAPs (AZD5582).

Although mesenchymal stem cells (MSCs) can be engineered to deliver the TNF-related apoptosis-inducing ligand (TRAIL) as an effective anticancer therapy, the clinical application is hampered by the costly manufacturing of therapeutic MSCs. Therefore, it is needed to find an alternative cell-free therapy. In this study, TRAIL-armed endoplasmic reticulum (ER)-derived nanosomes (ERN-T) are successfully prepared with an average size of 70.6nm in diameter from TRAIL transduced MSCs. It is demonstrated that the ERN-T is significantly more efficient for cancer cell killing than the soluble recombinant TRAIL (rTRAIL). AZD5582 is an antagonist of the inhibitors of apoptosis proteins (IAPs), and its combination with ERN-T induces strikingly enhanced apoptosis in cancerous but not normal cells. AZD5582 sensitizes resistant cancer cells to TRAIL through concomitant downregulation of IAP members like XIAP and the Bcl2 family member Mcl-1. Intravenously infused ERN-Ts accumulate in tumors for over 48 h indicating good tumor tropism and retention. The combination of ERN-T and AZD5582 drastically promotes therapeutic efficacy comparing with the cotreatment by rTRAIL and AZD5582 in a subcutaneous MDA-MB-231 xenograft tumor model. The data thus demonstrate that ERN-T can be a novel cell-free alternative to TRAIL-expressing MSC-based anticancer therapy and its efficacy can be drastically enhanced through combination with AZD5582.

Extracellular Vesicles in chondrogenesis and Cartilage regeneration

Extracellular vesicles (EVs), mainly exosomes and microvesicles, are bilayer lipids containing biologically active information, including nucleic acids and proteins. They are involved in cell communication and signalling, mediating many biological functions including cell growth, migration and proliferation. Recently, EVs have received great attention in the field of tissue engineering and regenerative medicine. Many in vivo and in vitro studies have attempted to evaluate the chondrogenesis potential of these microstructures and their roles in cartilage regeneration. EVs derived from mesenchymal stem cells (MSCs) or chondrocytes have been found to induce chondrocyte proliferation and chondrogenic differentiation of stem cells in vitro. Preclinical studies have shown that exosomes derived from MSCs have promising results in cartilage repair and in cell‐free therapy of osteoarthritis. This review will focus on the in vitro and in vivo chondrogenesis and cartilage regeneration of EVs as well as their potential in the treatment of osteoarthritis.

Combined topical and systemic administration with human adipose-derived mesenchymal stem cells (hADSC) and hADSC-derived exosomes markedly promoted cutaneous wound healing and regeneration

BackgroundCutaneous wound healing and regeneration have become a recognized health challenge in the world, which causes severe damage to the mental and physical health of patients. Human adipose-derived mesenchymal stem cells (hADSC) play an essential role in wound healing via their paracrine function. Exosomes secreted by hADSC may contribute to this progress. In this study, we investigated the potential clinical application roles of hADSC and hADSC-derived exosomes (hADSC-Exo) in cutaneous wound healing.MethodshADSC-Exo was isolated from human hADSC by ultracentrifugation. Mice were subjected to a full-thickness skin biopsy experiment and treated with either control vehicle or hADSC or hADSC-Exo by smearing administration (sm) or subcutaneous administration (sc) or intravenous administration (iv). The efficacy of hADSC and hADSC-Exo on wound healing was evaluated by measuring wound closure rates, histological analysis.ResultsCombined application of local hADSC-Exo smearing and hADSC/hADSC-Exo intravenous administration offered the additional benefit of promoting wound healing, accelerating re-epithelialization, reducing scar widths, and enhancing angiogenesis and collagen synthesis. Either topical application of hADSC-Exo or systemic administration with hADSC/hADSC-Exo appeared more effective in stimulating cell proliferation, inhibiting cell apoptosis and inflammation, and promoting skin elasticity and barrier integrity, with increased genes expression of PCNA, VEGF, collagen III, Filaggrin, Loricrin, and AQP3, with decreased genes expression of TNF-alpha.ConclusionOur findings suggest that the combined administration of hADSC/hADSC-Exo can facilitate cutaneous wound healing and reduce scar formation. These data provide the first evidence for the feasibility of smearing of hADSC-Exo as a cell-free therapy in treating cutaneous wounds, and the potential clinical value of combined administration of hADSC/hADSC-Exo.

Extracellular Vesicles as Promising Carriers in Drug Delivery: Considerations from a Cell Biologist's Perspective.

Simple SummaryExtracellular vesicles are promising nanocarriers of active pharmaceutical ingredients in precision medicine. However, to develop safe and effective extracellular vesicles-based therapies, it is crucial to gain fundamental knowledge on key cell biology mechanisms governing the extracellular vesicles-mediated cell-cell exchange of macromolecules. In this review, we present five aspects we believe are worth taking into account before considering extracellular vesicles as candidate vectors of pharmaceutical ingredients in cell-free therapies.The use of extracellular vesicles as cell-free therapy is a promising approach currently investigated in several disease models. The intrinsic capacity of extracellular vesicles to encapsulate macromolecules within their lipid bilayer membrane-bound lumen is a characteristic exploited in drug delivery to transport active pharmaceutical ingredients. Besides their role as biological nanocarriers, extracellular vesicles have a specific tropism towards target cells, which is a key aspect in precision medicine. However, the little knowledge of the mechanisms governing the release of a cargo macromolecule in recipient cells and the Good Manufacturing Practice (GMP) grade scale-up manufacturing of extracellular vesicles are currently slowing their application as drug delivery nanocarriers. In this review, we summarize, from a cell biologist’s perspective, the main evidence supporting the role of extracellular vesicles as promising carriers in drug delivery, and we report five key considerations that merit further investigation before translating Extracellular Vesicles (EVs) to clinical applications.

Is Extracellular Vesicle-Based Therapy the Next Answer for Cartilage Regeneration?

“Extracellular vesicles” (EVs) is a term gathering biological particles released from cells that act as messengers for cell-to-cell communication. Like cells, EVs have a membrane with a lipid bilayer, but unlike these latter, they have no nucleus and consequently cannot replicate. Several EV subtypes (e.g., exosomes, microvesicles) are described in the literature. However, the remaining lack of consensus on their specific markers prevents sometimes the full knowledge of their biogenesis pathway, causing the authors to focus on their biological effects and not their origins. EV signals depend on their cargo, which can be naturally sourced or altered (e.g., cell engineering). The ability for regeneration of adult articular cartilage is limited because this avascular tissue is partly made of chondrocytes with a poor proliferation rate and migration capacity. Mesenchymal stem cells (MSCs) had been extensively used in numerous in vitro and preclinical animal models for cartilage regeneration, and it has been demonstrated that their therapeutic effects are due to paracrine mechanisms involving EVs. Hence, using MSC-derived EVs as cell-free therapy tools has become a new therapeutic approach to improve regenerative medicine. EV-based therapy seems to show similar cartilage regenerative potential compared with stem cell transplantation without the associated hindrances (e.g., chromosomal aberrations, immunogenicity). The aim of this short review is to take stock of occurring EV-based treatments for cartilage regeneration according to their healing effects. The article focuses on cartilage regeneration through various sources used to isolate EVs (mature or stem cells among others) and beneficial effects depending on cargos produced from natural or tuned EVs.

Exosome-shuttled miR-7162-3p from human umbilical cord derived mesenchymal stem cells repair endometrial stromal cell injury by restricting APOL6

BackgroundRecent studies have shown that exosomes (Exos) derived from stem cells can be used as paracrine factors to regenerate cells and tissues via shuttling miRNAs. Exos derived from human umbilical cord derived mesenchymal stem cells (UCMSCs) have been found to alleviate mifepristone-induced endometrial stromal cell (ESC) injury in vitro. Information on the functions and mechanisms of Exos from UCMSC-induced endometrial repair is limited and requires more study. MethodsUCMSC-Exos were isolated and identified by Transmission Electron Microscopy, Nanoparticle Tracking Analysis software, and western blot assays. The damaged-ESC model and the UCMSC co-culture system were established, while GW4869, a noncompetitive neutral sphingomyelinase (N-SMase) inhibitor, was used to investigate the effects of UCMSC-Exos on mifepristone-induced ESC injury. Cell apoptosis of damaged ESCs treated with UCMSCs was detected using the TUNEL assay and flow cytometry analysis. Then, miRNA microarrays were performed to detect differentially expressed miRNA profiles in both UCMSCs and ESCs after co-culturing. A subset of upregulated miRNAs was validated by qRT-PCR, and miRNA mimics/inhibitor were used to investigate the functions of miR-7162-3p. The miRNA-mRNA interactions were predicted by Targetscan software, while the miRNA binding sites were predicted by miRcode software. Moreover, dual-luciferase reporter, western blot assays and qPCR were conducted to identify the regulatory mechanisms between miR-7162-3p and APOL6. ResultsUCMSCs attenuated mifepristone-induced endometrial stromal cell apoptosis by Exos, while three miRNAs (miR-6831-5p, miR-4669, and miR-7162-3p) were both upregulated in UCMSCs and ESCs after co-culture, and were candidate effectors of UCMSC-Exos-mediated endometrial repair. We showed that miR-7162-3p was shuttled by Exos from UCMSCs and regulated the expression of APOL6 by targeting its 3ʹ-UTR in ESCs. ConclusionsThese results showed UCMSC-Exos protected ESCs from mifepristone-induced apoptosis and played an active role in repairing the damaged ESCs by in vitro shuttling of miR-7162-3p. The miR-7162-3p-overexpressed UCMSC-Exos may therefore be used in cell-free therapy of endometrial injury.

Large-volume cell-free and concentrated ascites reinfusion therapy improves venous flow in patients with liver cirrhosis.

Hemodynamic change after total paracentesis was investigated because it might lead to various complications. Although cell-free and concentrated ascites reinfusion therapy (CART) is safer and more effective than total paracentesis in theory, hemodynamic change after CART has been never reported. And previous studies did not mention hemodynamics of the venous system. We investigated the hemodynamic change, including that of the venous system, before and after CART using color Doppler ultrasonography and fast Fourier transform analysis. Twenty-eight patients with tensive cirrhotic ascites underwent ultrasonography the day before and after total volume CART. The diameter and velocity of the main, right, and left portal vein; inferior vena cava (IVC); and right renal vein were measured using ultrasonography. A total of 11.8 ± 4.4L of ascites (range 3.6-20.9 L) was filtered and concentrated to 0.85 ± 0.40L (range 0.36-1.50 L). The diameter of the IVC increased from median 13.5 ± 5.4mm (range 4-25mm) to 18.5 ± 4.1mm (range 7-29mm) (p = 0.007). The diameter of the right segmental renal vein significantly increased after KM-CART [from 5.0 ± 1.0 (4-8)mm to 7.0 ± 2.0 (3-10) mm] (p = 0.011). Hemodynamic change of the portal venous system was not significant. The time to the next CART in patients with an IVC diameter ≥ 20mm and < 20mm was 86days and 20.5days (p = 0.035), respectively. Tensive ascites results in venous congestion in patients with cirrhotic ascites. CART improved venous flow, but it did not change the hemodynamics of the portal venous system.

Mesenchymal stem cell derived extracellular vesicles as treatment for osteoarthritis in a rat high fat diet groove model

Purpose: Mesenchymal stromal/stem cells (MSC) hold a great promise to control the imbalance between anabolic and catabolic processes in an OA joint due to their immunomodulatory and regenerative capacities. Recently, intra-articular injections with human MSC entered the clinical trials as a novel, low invasive and relatively cheap therapy for OA. Although, the results of this novel treatment are promising, it remains unclear how MSC exert their beneficial effects in inflammation and tissue (cartilage) repair. Increasing evidence suggests that the therapeutic efficacy of MSC depends on paracrine signaling. Recently we have shown that MSC-derived extracellular vesicles (MSC-EVs) are an important component of this signaling and have a major contribution to cartilage regeneration and inhibition of TNF-alpha stimulated inflammatory responses in chondrocytes in vitro. Extracellular vesicles (EVs) are secreted by all cell types and range in size from 40 - 100 nm (exosomes) and from 100 -1000 nm (microvesicles). They increasingly appear to play an important role in intercellular communication by transportation of important molecules (peptides, mRNA, miRNA, enzymes). MSC-derived EVs have a great potential to serve as a safer cell free therapy for OA. The purpose of this study was to investigate if MSC-derived EVs can inhibit inflammation and promote cartilage repair to a similar extent as MSCs in a rat high fat diet groove model of OA. Methods: 36 male Wistar-han (Crl:WI(Han) rats were fed a high fat diet for 24 weeks, starting at an age of 12 weeks old. In week 12 of the study the rats received unilateral surgery where local cartilage damage was induced at the right knee joint by making five longitudinal grooves on the femoral condyles. In week 13 the rats were randomized into 3 groups and given an intraarticular injection with either PBS, 2 million human bone marrow derived MSCs or EVs derived from 2 million MSCs. The extracellular vesicles group received 5 of these doses with 5 days interval. Before, during and after the injections pain behavior was monitored by dynamic weight bearing and via measuring the left and right hind paw withdrawal responses to the application of von Frey hairs. uCT scans were made at week 0 and week 24, and used to determine the number of osteophytes and cartilage mineralization in the tibia. Additionally, tibial subchondral plate thickness, trabecular bone thickness, and trabecular bone volume fraction (total volume/trabecular bone volume) were measured using ImageJ 1.47v. At endpoint animals were sacrificed and the knee joints were harvested. Joint tissue is currently under preparation for histological staining to determine the OARSI histopathological score for rats. Data is represented as mean +/- standard deviation (SD) and the difference between the means in the three groups was statistically tested using one way ANOVA. Results: Results from the von Frey tests showed a clear decrease in 50% withdrawal threshold in the right hind paw, which was expected as the right side was the grooved limb. However, no difference between the three treatment groups in either left or right paw was seen. The mean osteophyte number in the PBS group was 4.7 (+/- 0.65) versus 3.6 (+/-2.15) in the MSC group and 4.3 (+/-1.8) in the MSC-EV group (figure 1A). In the MSC and MSC-EV treatment groups the variation in osteophyte number and total osteophyte diameter between the animals was large compared to the PBS group. Cartilage mineralization were observed in 9 of 12 animals in the PBS and MSC group and in 5 of 12 animals in the MSC-EV group. Both the number and total diameter was lowest in the MSC-EV group, but there was no significant difference as compared with control groups (Figure 1B). Other parameters measured on uCT like bone volume fraction, subchondral plate and trabecular bone thickness were similar between the 3 groups. Results from the OARSI histopathological score for rats is currently under preparation. Conclusions: Preliminary results from this in vivo study show no significant differences between PBS, MSC or MSC-EV treatment on uCT data or von Frey test. However, we do observe a large spread in osteophyte score and total diameter in the MSC and MSC-EV treated groups, which could be a sign that individual animals are responding differently to the treatments. Data from the OARSI scoring will help us determine if the response really is variable for MSC and MSC-EV treatment groups. This would be in accordance with observations made in humans, as many clinical trials that test MSC-based therapies report a substantial nonresponder group. In summary, although osteophytes are one of the hallmarks of osteoarthritis, we feel a complete picture of the joint damage including cartilage and synovium is needed to draw conclusions on the effect of the treatments in this study.

Optimized BMSC-derived osteoinductive exosomes immobilized in hierarchical scaffold via lyophilization for bone repair through Bmpr2/Acvr2b competitive receptor-activated Smad pathway

Mesenchymal stem cell-derived exosomes (MSC-exos), with its inherent capacity to modulate cellular behavior, are emerging as a novel cell-free therapy for bone regeneration. Herein, focusing on practical applying problems, the osteoinductivity of MSC-exos produced by different stem cell sources (rBMSCs/rASCs) and culture conditions (osteoinductive/common) were systematically compared to screen out an optimized osteogenic exosome (BMSC-OI-exo). Via bioinformatic analyses by miRNA microarray and in vitro pathway verification by gene silencing and miRNA transfection, we first revealed that the osteoinductivity of BMSC-OI-exo was attributed to multi-component exosomal miRNAs (let-7a-5p, let-7c-5p, miR-328a-5p and miR-31a-5p). These miRNAs targeted Acvr2b/Acvr1 and regulated the competitive balance of Bmpr2/Acvr2b toward Bmpr-elicited Smad1/5/9 phosphorylation. On these bases, lyophilized delivery of BMSC-OI-exo on hierarchical mesoporous bioactive glass (MBG) scaffold was developed to realize bioactivity maintenance and sustained release by entrapment in the surface microporosity of the scaffold. In a rat cranial defect model, the loading of BMSC-OI-exo efficiently enhanced the bone forming capacity of the scaffold and induced rapid initiation of bone regeneration. This paper could provide empirical bases of MSC-exo-based therapy for bone regeneration and theoretical bases of MSC-exo-induced osteogenesis mechanism. The BMSC-OI-exo-loaded MBG scaffold developed here represented a promising bone repairing strategy for future clinical application.

Clinical usefulness of cell-free and concentrated ascites reinfusion therapy (CART) in combination with chemotherapy for malignant ascites: a post-marketing surveillance study.

Cell-free and concentrated ascites reinfusion therapy (CART) has been suggested to be able to treat malignant ascites more safely and effectively with chemotherapy because of its ability to retain serum protein and albumin. Although the characteristics of cancer types and CART and the clinical implications of combination therapy with antitumor agents are becoming widespread, there are limited reports on its efficacy and complications. In this prospective observational national post-marketing study, 128 patients with malignancies received 300 CART sessions at 22 centers. After excluding other malignancies, the patients were divided into four groups: gynecological malignancies with chemotherapy (GYC+; 18 cases and 36 times) and without chemotherapy (GYC-; 35 cases and 52 times), and gastrointestinal malignancies with chemotherapy (GIC+; 8 cases and 16 times) and without chemotherapy (20 cases and 58 times). There were significant reductions in the body weight in all groups and significant reductions in abdominal circumference and significant improvements in the diet and Eastern Cooperative Oncology Group performance status only in the GYC+ group. The total serum protein and albumin increased significantly in all groups, except for the GIC+ group, before and after CART. There was no significant difference in the presence or absence of antitumor medication. With CART, there were differences in the improvement of the clinical symptoms between malignancy groups. The combination of CART and antineoplastic agents may be as safe as CART alone in cases of exudative malignant ascites.

Chitosan hydrogel-loaded MSC-derived extracellular vesicles promote skin rejuvenation by ameliorating the senescence of dermal fibroblasts

BackgroundThe senescence of dermal fibroblasts (DFLs) leads to an imbalance in the synthesis and degradation of extracellular matrix (ECM) proteins, presenting so-called senescence-associated secretory phenotype (SASP), which ultimately leads to skin aging. Recently, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been recognized as a promising cell-free therapy for degenerative diseases, which opens a new avenue for skin aging treatment.MethodsIn this study, we utilized chitosan (CS) hydrogel for effective loading and sustained release of EVs. In vitro, we explored the rejuvenation effects of CS hydrogel-incorporated EVs (CS-EVs) on replicative senescence DFLs through a series of experiments such as senescence-associated β-galactosidase (SA-β-gal) staining, RT-PCR, and Western blot analysis. Besides, we employed local multi-site subcutaneous injection to treat skin aging of naturally aged mice with CS-EVs and DiI fluorescent dye was used to label EVs to achieve in vivo real-time tracking.ResultsCS-EVs can significantly improve the biological functions of senescent fibroblasts, including promoting their proliferation, enhancing the synthesis of ECM proteins, and inhibiting the overexpression of matrix metalloproteinases (MMPs). Moreover, CS hydrogel could prolong the release of EVs and significantly increase the retention of EVs in vivo. After CS-EVs subcutaneous injection treatment, the aging skin tissues showed a rejuvenation state, manifested explicitly as the enhanced expression of collagen, the decreased expression of SASP-related factors, and the restoration of tissue structures.ConclusionsCS hydrogel-encapsulated EVs could delay the skin aging processes by ameliorating the function of aging DFLs. Our results also highlight the potential of CS hydrogel-encapsulated EVs as a novel therapeutic strategy for improving aging skin to rejuvenation.

Sequential Release of Small Extracellular Vesicles from Bilayered Thiolated Alginate/Polyethylene Glycol Diacrylate Hydrogels for Scarless Wound Healing.

Excessive scar formation has adverse physiological and psychological effects on patients; therefore, a therapeutic strategy for rapid wound healing and reduced scar formation is urgently needed. Herein, bilayered thiolated alginate/PEG diacrylate (BSSPD) hydrogels were fabricated for sequential release of small extracellular vesicles (sEVs), which acted in different wound healing phases, to achieve rapid and scarless wound healing. The sEVs secreted by bone marrow derived mesenchymal stem cells (B-sEVs) were released from the lower layer of the hydrogels to promote angiogenesis and collagen deposition by accelerating fibroblast and endothelial cell proliferation and migration during the early inflammation and proliferation phases, while sEVs secreted by miR-29b-3p-enriched bone marrow derived mesenchymal stem cells were released from the upper layer of the hydrogels and suppressed excessive capillary proliferation and collagen deposition during the late proliferation and maturation phases. In a full-thickness skin defect model of rats and rabbit ears, the wound repair rate, angiogenesis, and collagen deposition were evaluated at different time points after treatment with BSSPD loaded with B-sEVs. Interestingly, during the end of the maturation phase in the in vivo model, tissues in the groups treated with BSSPD loaded with sEVs for sequential release (SR-sEVs@BSSPD) exhibited a more uniform vascular structure distribution, more regular collagen arrangement, and lower volume of hyperplastic scar tissue than tissues in the other groups. Hence, SR-sEVs@BSSPD based on skin repair phases was successfully designed and has considerable potential as a cell-free therapy for scarless wound healing.