Vascular smooth muscle cell-derived exosomes promote osteoblast-to-osteocyte transition via ß-catenin signaling.

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via ß-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/ß-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function.

Amorphous TiO2nano-coating on stainless steel to improve its biological response.

This study delves into the potential of amorphous titanium oxide (aTiO2) nano-coating to enhance various critical aspects of non-Ti-based metallic orthopedic implants. These implants, such as medical-grade stainless steel (SS), are widely used for orthopedic devices that demand high strength and durability. The aTiO2nano-coating, deposited via magnetron sputtering, is a unique attempt to improve the osteogenesis, the inflammatory response, and to reduce bacterial colonization on SS substrates. The study characterized the nanocoated surfaces (SS-a TiO2) in topography, roughness, wettability, and chemical composition. Comparative samples included uncoated SS and sandblasted/acid-etched Ti substrates (Ti). The biological effects were assessed using human mesenchymal stem cells (MSCs) and primary murine macrophages. Bacterial tests were carried out with two aerobic pathogens (S. aureusandS. epidermidis) and an anaerobic bacterial consortium representing an oral dental biofilm. Results from this study provide strong evidence of the positive effects of the aTiO2nano-coating on SS surfaces. The coating enhanced MSC osteoblastic differentiation and exhibited a response similar to that observed on Ti surfaces. Macrophages cultured on aTiO2nano-coating and Ti surfaces showed comparable anti-inflammatory phenotypes. Most significantly, a reduction in bacterial colonization across tested species was observed compared to uncoated SS substrates, further supporting the potential of aTiO2nano-coating in biomedical applications. The findings underscore the potential of magnetron-sputtering deposition of aTiO2nano-coating on non-Ti metallic surfaces such as medical-grade SS as a viable strategy to enhance osteoinductive factors and decrease pathogenic bacterial adhesion. This could significantly improve the performance of metallic-based biomedical devices beyond titanium.

Exploring the Therapeutic Potential of Extracellular Vesicles Derived from Human Immature Dental Pulp Cells on Papillary Thyroid Cancer.

Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been increasingly investigated for cancer therapy and drug delivery, and they offer an advanced cell-free therapeutic option. However, their overall effects and efficacy depend on various factors, including the MSC source and cargo content. In this study, we isolated EVs from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) and investigated their effects on two papillary thyroid cancer (PTC) cell lines (BCPAP and TPC1). We observed efficient uptake of hIDPSC-EVs by both PTC cell lines, with a notable impact on gene regulation, particularly in the Wnt signaling pathway in BCPAP cells. However, no significant effects on cell proliferation were observed. Conversely, hIDPSC-EVs significantly reduced the invasive capacity of both PTC cell lines after 120 h of treatment. These in vitro findings suggest the therapeutic potential of hIDPSC-EVs in cancer management and emphasize the need for further research to develop novel and effective treatment strategies. Furthermore, the successful internalization of hIDPSC-EVs by PTC cell lines underscores their potential use as nanocarriers for anti-cancer agents.

Cellular In Vitro Responses Induced by Human Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Obtained from Suspension Culture.

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-ß and IL-1ß was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.

Applications of Modified Mesenchymal Stem Cells as Targeted Systems against Tumor Cells.

Combined gene and cell therapy are promising strategies for cancer treatment. Given the complexity of cancer, several approaches are actively studied to fight this disease. Using mesenchymal stem cells (MSCs) has demonstrated dual antitumor and protumor effects as they exert massive immune/regulatory effects on the tissue microenvironment. MSCs have been widely investigated to exploit their antitumor target delivery system. They can be genetically modified to overexpress genes and selectively or more efficiently eliminate tumor cells. Current approaches tend to produce more effective and safer therapies using MSCs or derivatives; however, the effect achieved by engineered MSCs in solid tumors is still limited and depends on several factors such as the cell source, transgene, and tumor target. This review describes the progress of gene and cell therapy focused on MSCs as a cornerstone against solid tumors, addressing the different MSC-engineering methods that have been approached over decades of research. Furthermore, we summarize the main objectives of engineered MSCs against the most common cancers and discuss the challenges, limitations, risks, and advantages of targeted treatments combined with conventional ones.

Additive manufacturing of bioactive and biodegradable poly (lactic acid)-tricalcium phosphate scaffolds modified with zinc oxide for guided bone tissue repair.

Bioactive and biodegradable scaffolds that mimic the natural extracellular matrix of bone serve as temporary structures to guide new bone tissue growth. In this study, 3D-printed scaffolds composed of poly (lactic acid) (PLA)-tricalcium phosphate (TCP) (90-10 wt.%) were modified with 1%, 5%, and 10 wt.% of ZnO to enhance bone tissue regeneration. A commercial chain extender named Joncryl was incorporated alongside ZnO to ensure the printability of the composites. Filaments were manufactured using a twin-screw extruder and subsequently used to print 3D scaffolds via fused filament fabrication (FFF). The scaffolds exhibited a homogeneous distribution of ZnO and TCP particles, a reproducible structure with 300 µm pores, and mechanical properties suitable for bone tissue engineering, with an elastic modulus around 100 MPa. The addition of ZnO resulted in enhanced surface roughness on the scaffolds, particularly for ZnO microparticles, achieving values up to 241 nm. This rougher topography was responsible for enhancing protein adsorption on the scaffolds, with an increase of up to 85% compared to the PLA-TCP matrix. Biological analyses demonstrated that the presence of ZnO promotes mesenchymal stem cell (MSC) proliferation and differentiation into osteoblasts. Alkaline phosphatase (ALP) activity, an important indicator of early osteogenic differentiation, increased up to 29%. The PLA-TCP composite containing 5% ZnO microparticles exhibited an optimized degradation rate and enhanced bioactivity, indicating its promising potential for bone repair applications.

Guarana (Paullinia cupana) as a potential tool for mesenchymal stromal cells priming in regenerative medicine.

Mesenchymal stromal cells (MSCs) have therapeutic potential due to their abilities of differentiation, immunomodulation, and migration to injured tissues, potentiating such effects when cells are activated. Guarana (Paullinia cupana) is a tropical plant species found in South America that is known for its antioxidant, stimulant, and cicatricial effects. The guarana extract is composed of many substances and caffeine is the main component. The objective was to evaluate the effects of guarana and caffeine on MSCs. After the initial characterization, MSCs were treated with Paullinia cupana (10, 100, and 1000 µg/mL) or caffeine (0.4, 4, and 40 µg/mL) for 24 h. MSCs treatment with 1000 µg/mL guarana increased cell polarity, viability, cell migration to chemoattractant, antioxidant potential, and liberation of extracellular vesicles (EVs), while it reduced the levels of autophagy. MSCs treated with 100 and 1000 µg/mL guarana or 40 µg/mL caffeine showed a decrease of cell proliferation. No treatment affected the cellular area and cell cycle of MSCs. The study shows in vitro evidence that guarana could be a promising alternative for activating MSCs to promote better cellular products for future clinical therapies.

Cellular Therapy in Experimental Autoimmune Encephalomyelitis as an Adjuvant Treatment to Translate for Multiple Sclerosis.

This study aims to evaluate and compare cellular therapy with human Wharton's jelly (WJ) mesenchymal stem cells (MSCs) and neural precursors (NPs) in experimental autoimmune encephalomyelitis (EAE), a preclinical model of Multiple Sclerosis. MSCs were isolated from WJ by an explant technique, differentiated to NPs, and characterized by cytometry and immunocytochemistry analysis after ethical approval. Forty-eight rats were EAE-induced by myelin basic protein and Freund's complete adjuvant. Forty-eight hours later, the animals received intraperitoneal injections of 250 ng/dose of Bordetella pertussis toxin. Fourteen days later, the animals were divided into the following groups: a. non-induced, induced: b. Sham, c. WJ-MSCs, d. NPs, and e. WJ-MSCs plus NPs. 1 × 105. Moreover, the cells were placed in a 10 µL solution and injected via a stereotaxic intracerebral ventricular injection. After ten days, the histopathological analysis for H&E, Luxol, interleukins, and CD4/CD8 was carried out. Statistical analyses demonstrated a higher frequency of clinical manifestation in the Sham group (15.66%) than in the other groups; less demyelination was seen in the treated groups than the Sham group (WJ-MSCs, p = 0.016; NPs, p = 0.010; WJ-MSCs + NPs, p = 0.000), and a lower cellular death rate was seen in the treated groups compared with the Sham group. A CD4/CD8 ratio of <1 showed no association with microglial activation (p = 0.366), astrocytes (p = 0.247), and cell death (p = 0.577) in WJ-MSCs. WJ-MSCs and NPs were immunomodulatory and neuroprotective in cellular therapy, which would be translated as an adjunct in demyelinating diseases.

Influence of the Surface Topography of Titanium Dental Implants on the Behavior of Human Amniotic Stem Cells.

The dental implant surface plays a crucial role in osseointegration. The topography and physicochemical properties will affect the cellular functions. In this research, four distinct titanium surfaces have been studied: machined acting (MACH), acid etched (AE), grit blasting (GBLAST), and a combination of grit blasting and subsequent acid etching (GBLAST + AE). Human amniotic mesenchymal (hAMSCs) and epithelial stem cells (hAECs) isolated from the amniotic membrane have attractive stem-cell properties. They were cultured on titanium surfaces to analyze their impact on biological behavior. The surface roughness, microhardness, wettability, and surface energy were analyzed using interferometric microscopy, Vickers indentation, and drop-sessile techniques. The GBLAST and GBLAST + AE surfaces showed higher roughness, reduced hydrophilicity, and lower surface energy with significant differences. Increased microhardness values for GBLAST and GBLAST + AE implants were attributed to surface compression. Cell viability was higher for hAMSCs, particularly on GBLAST and GBLAST + AE surfaces. Alkaline phosphatase activity enhanced in hAMSCs cultured on GBLAST and GBLAST + AE surfaces, while hAECs showed no mineralization signals. Osteogenic gene expression was upregulated in hAMSCs on GBLAST surfaces. Moreover, α2 and ß1 integrin expression enhanced in hAMSCs, suggesting a surface-integrin interaction. Consequently, hAMSCs would tend toward osteoblastic differentiation on grit-blasted surfaces conducive to osseointegration, a phenomenon not observed in hAECs.

Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries.

BACKGROUND AND AIMS: Ovum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions. METHODS: MSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4). RESULTS: Characterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions. CONCLUSIONS: MSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo.

Stem cell-conditioned medium improves methylation patterns and quality of caprine preantral follicles.

In brief: Conditioned medium from Wharton's jelly mesenchymal stem cells improved tissue and preantral follicle outcomes, preventing adverse effects of oxidative stress, apoptosis, and epigenetic changes. Abstract: This study investigated the methylation patterns of H3K4me3 and H3K9me3, as well as the mRNA expression of genes encoding the epigenetic regulators KDM1AX1, KDM1AX2, and KDM3A in goat preantral follicles developed in vivo (Uncultured control) or after in vitro culture for 7 days in either the absence (α-MEM+) or presence of conditioned medium (α-MEM+ + CM) from Wharton's jelly mesenchymal stem cells (WJ-MSCs). In the invivo setting, all follicular categories exhibited similar H3K4me3 and H3K9me3 patterns, and transcripts of KDM1AX1, KDM1AX2, and KDM3A were detected in all samples. During in vitro culture, α-MEM+ + CM enhanced several important aspects. It increased the percentage of normal growing follicles, oocyte diameters across all categories, stromal cell density, and the H3K4me3 methylation pattern in preantral follicles. Simultaneously, it decreased the levels of reduced thiols and reactive oxygen species in the spent media, diminished the presence of lipofuscin aggresomes, lowered granulosa cell apoptotic rates, and reduced the H3K9me3 methylation pattern in preantral follicles. In conclusion, the findings from this study provide compelling evidence that supplementing the in vitro culture medium (α-MEM+) with CM from WJ-MSCs has a protective effect on goat preantral follicles. Notably, CM supplementation preserved follicular survival, as evidenced by enhanced follicular and oocyte growth and increased stromal cell density when compared to the standard culture conditions in the α-MEM+ medium. Furthermore, CM reduced oxidative stress and apoptosis and promoted alterations in H3K4me3 and H3K9me3 patterns.

Mesenchymal stem cell-derived cell-free technologies: a patent landscape.

Mesenchymal stem/stromal cells (MSC) play a pivotal role in regenerative therapies. Recent studies show that factors secreted by MSC can replicate their biological activity, driving the emergence of cell-free therapy, likely to surpass stem cell therapy. Patents are an objective measure of R&D and innovation activities, and patent mapping allows us to verify the state of the art and technology, anticipate trends, and identify emerging lines of research. This review performed a search on Derwent World Patents Index™ and retrieved 269 patent families related to the MSC-derived cell-free products. Analysis reveals an exponential increase in patents from the mid-2010s, primarily focusing on exosomes. The patent's contents offer a great diversity of applications and associated technologies by using the products as medicinal agents or drug delivery systems. Nevertheless, numerous application branches remain unexplored, suggesting vast potential for cell-free technologies alone or combined with other approaches.

Vascular smooth muscle cells exhibit elevated hypoxia-inducible Factor-1α expression in human blood vessel organoids, influencing osteogenic performance.

Considering the importance of alternative methodologies to animal experimentation, we propose an organoid-based biological model for in vitro blood vessel generation, achieved through co-culturing endothelial and vascular smooth muscle cells (VSMCs). Initially, the organoids underwent comprehensive characterization, revealing VSMCs (α-SMA + cells) at the periphery and endothelial cells (CD31+ cells) at the core. Additionally, ephrin B2 and ephrin B4, genes implicated in arterial and venous formation respectively, were used to validate the obtained organoid. Moreover, the data indicates exclusive HIF-1α expression in VSMCs, identified through various methodologies. Subsequently, we tested the hypothesis that the generated blood vessels have the capacity to modulate the osteogenic phenotype, demonstrating the ability of HIF-1α to promote osteogenic signals, primarily by influencing Runx2 expression. Overall, this study underscores that the methodology employed to create blood vessel organoids establishes an experimental framework capable of producing a 3D culture model of both venous and arterial endothelial tissues. This model effectively guides morphogenesis from mesenchymal stem cells through paracrine signaling, ultimately leading to an osteogenic acquisition phenotype, with the dynamic involvement of HIF-1α.

Mesenchymal Stromal Cell-Based Products: Challenges and Clinical Therapeutic Options.

Mesenchymal stromal cell (MSC)-based advanced therapy medicinal products (ATMPs) are being tried in a vast range of clinical applications. These cells can be isolated from different donor tissues by using several methods, or they can even be derived from induced pluripotent stem cells or embryonic stem cells. However, ATMP heterogeneity may impact product identity and potency, and, consequently, clinical trial outcomes. In this review, we discuss these topics and the need to establish minimal criteria regarding the manufacturing of MSCs so that these innovative therapeutics may be better positioned to contribute to the advancement of regenerative medicine.

IFN-γ-Preconditioned Human Gingival-Derived Mesenchymal Stromal Cells Inhibit Plasmacytoid Dendritic Cells via Adenosine.

Plasmacytoid dendritic cells (pDCs) are vital players in antiviral immune responses because of their high levels of IFN-α secretion. However, this attribute has also implicated them as critical factors behind the immunopathogenesis of inflammatory diseases, and no currently available therapy can efficiently inhibit pDCs' aberrant activation. Mesenchymal stromal cells (MSCs) possess stromal immunomodulatory functionality, regulating immune cell activation through several mechanisms, including the adenosinergic (CD39/CD73/adenosine) pathway. The IFN-γ preconditioning of bone marrow MSCs improves their inhibitory properties for therapy applications; however, isolating human gingival tissue-derived MSCs (hGMSCs) is more accessible. These cells have shown better immunomodulatory effects, yet the outcome of IFN-γ preconditioning and its impact on the adenosinergic pathway has not been evaluated. This study first validated the immunoregulatory properties of primary-cultured hGMSCs, and the results showed that IFN-γ preconditioning strengthens CD39/CD73 coexpression, adenosine production, and the regulatory properties of hGMSC, which were confirmed by describing for the first time their ability to reduce pDC activation and their IFN-α secretion and to increase the frequency of CD73+ pDC. In addition, when CD73's enzymatic activity was neutralized in hGMSCs, adenosine production and the IFN-γ preconditioning effect were restrained. This evidence might be applied to design hGMSCs- and adenosine-based immunotherapeutic strategies for treating inflammatory disorders that are associated with pDC overactivation.

The safety profile of mesenchymal stem cell therapy administered through intrathecal injections for treating neurological disorders: a systematic review and meta-analysis of randomised controlled trials.

BACKGROUND: Based on previous in vivo studies and human trials, intrathecal cell delivery is a safe and relevant therapeutic tool for improving patient's quality of life with neurological conditions. We aimed to characterise the safety profile of intrathecally delivered Mesenchymal stem cells (MSCs). METHODS: Ovid MEDLINE, Embase, Scopus, Cochrane Library, KCI-Korean Journal Database, and Web of Science. Databases were searched from their inception until April 13, 2023. Randomised Controlled Trials (RCTs) that compared intrathecal delivery of MSCs to controls in adult populations were included. Adverse events (AEs) were pooled and meta-analysed using DerSimonian-Laird random effects models with a correction factor 0.5 added to studies with zero count cells. Pooled AEs were described using Risk ratio (RR) and 95% confidence intervals (95% CI). Then, a random-effects meta-regress model on study-level summary data was performed to explore the relationship between the occurrence of AEs and covariates thought to modify the overall effect estimate. Finally, publication bias was assessed. RESULTS: 303 records were reviewed, and nine RCTs met the inclusion criteria and were included in the quantitative synthesis (n = 540 patients). MSCs delivered intrathecally, as compared to controls, were associated with an increased probability of AEs of musculoskeletal and connective tissue disorders (categorised by Common Terminology Criteria for Adverse Events-CTCAE version 5.0) (RR: 1.61, 95% CI 1.19-2.19, I2 = 0%). The random-effects meta-regress model suggested that fresh MSCs increased the probability of occurrence of AEs compared to cryopreserved MSCs (RR: 1.554; p-value = 0.048; 95% CI 1.004-2.404), and the multiple-dose, decreased the probability of AEs by 36% compared to single doses (RR: 0.644; p-value = 0.048; 95% CI 0.416-0.996); however, univariate random effects meta-regression models revealed a not significant association between the occurrence of AEs from MSCs intrathecal delivery and each covariate. CONCLUSIONS: Intrathecal delivery of MSCs was associated with a slight increase in AEs associated with musculoskeletal and connective tissue disorders, albeit without serious AEs. We conclude that intrathecal MSCs delivery is safe for patients with neurological conditions. However, further high-quality, large-scale RCTs are needed to confirm these findings.

Comparative analysis of uninduced and neuronally-induced human dental pulp stromal cells in a 6-OHDA model of Parkinson's disease.

BACKGROUND: In recent years, dental pulp stromal cells (DPSCs) have emerged as a promising therapeutic approach for Parkinson's disease (PD), owing to their inherent neurogenic potential and the lack of neuroprotective treatments for this condition. However, uncertainties persist regarding the efficacy of these cells in an undifferentiated state versus a neuronally-induced state. This study aims to delineate the distinct therapeutic potential of uninduced and neuronally-induced DPSCs in a rodent model of PD induced by 6-Hydroxydopamine (6-OHDA). METHODS: DPSCs were isolated from human teeth, characterized as mesenchymal stromal cells, and induced to neuronal differentiation. Neuronal markers were assessed before and after induction. DPSCs were transplanted into the substantia nigra pars compacta (SNpc) of rats 7 days following the 6-OHDA lesion. In vivo tracking of the cells, evaluation of locomotor behavior, dopaminergic neuron survival, and the expression of essential proteins within the dopaminergic system were conducted 7 days postgrafting. RESULTS: Isolated DPSCs exhibited typical characteristics of mesenchymal stromal cells and maintained a normal karyotype. DPSCs consistently expressed neuronal markers, exhibiting elevated expression of ßIII-tubulin following neuronal induction. Results from the animal model showed that both DPSC types promoted substantial recovery in dopaminergic neurons, correlating with enhanced locomotion. Additionally, neuronally-induced DPSCs prevented GFAP elevation, while altering DARPP-32 phosphorylation states. Conversely, uninduced DPSCs reduced JUN levels. Both DPSC types mitigated the elevation of glycosylated DAT. CONCLUSIONS: Our results suggested that uninduced DPSCs and neuronally-induced DPSCs exhibit potential in reducing dopaminergic neuron loss and improving locomotor behavior, but their underlying mechanisms differ.

GMP-compliant extracellular vesicles derived from umbilical cord mesenchymal stromal cells: manufacturing and pre-clinical evaluation in ARDS treatment.

BACKGROUND AIMS: Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model. METHODS: hUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model. RESULTS: hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury. CONCLUSIONS: hUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.

3D Bioprinting of Biomimetic Alginate/Gelatin/Chondroitin Sulfate Hydrogel Nanocomposites for Intrinsically Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

3D-printed hydrogel scaffolds biomimicking the extracellular matrix (ECM) are key in cartilage tissue engineering as they can enhance the chondrogenic differentiation of mesenchymal stem cells (MSCs) through the presence of active nanoparticles such as graphene oxide (GO). Here, biomimetic hydrogels were developed by cross-linking alginate, gelatin, and chondroitin sulfate biopolymers in the presence of GO as a bioactive filler, with excellent processability for developing bioactive 3D printed scaffolds and for the bioprinting process. A novel bioink based on our hydrogel with embedded human MSCs presented a cell survival rate near 100% after the 3D bioprinting process. The effects of processing and filler concentration on cell differentiation were further quantitatively evaluated. The nanocomposited hydrogels render high MSC proliferation and viability, exhibiting intrinsic chondroinductive capacity without any exogenous factor when used to print scaffolds or bioprint constructs. The bioactivity depended on the GO concentration, with the best performance at 0.1 mg mL-1. These results were explained by the rational combination of the three biopolymers, with GO nanoparticles having carboxylate and sulfate groups in their structures, therefore, biomimicking the highly negatively charged ECM of cartilage. The bioactivity of this biomaterial and its good processability for 3D printing scaffolds and 3D bioprinting techniques open up a new approach to developing novel biomimetic materials for cartilage repair.

Evaluation of the therapeutic effect of very small stem cells from peripheral blood on the treatment of Premature Ovarian Failure: A pilot study.

OBJECTIVE: Premature ovary failure (POF) is a severe health condition with multiple negative outcomes, which deteriorate a patient's life. The current study aimed to evaluate the therapeutic effect of mesenchymal stem cells (MSCs) derived from peripheral blood in the treatment of women with the POF background. METHODS: The current study was a pilot study carried-out on women younger than 40 with premature ovarian failure. Study participants underwent 4-months cell therapy using Mesenchymal stem cells extracted from peripheral bloods. Serum level of Follicle-stimulating hormone (FSH), Estradiol (E2), Anti-mullerian hormone (AMH), and Antral follicle count (AFC) were the main investigated outcomes that were assessed at baseline, month two and month four of the very small stem cell intervention. RESULTS: Average serum level of FSH was 45.0 (12.1) mIU/mL at baseline and continually decreased during the study and reached 33.2 (12.4) mIU/mL in the fourth month. The average AMH level was 0.10 ng/mL prior to the intervention and increased to 0.13 ng/mL in the 2nd month and 0.15 ng/mL in the fourth month. The level E2 was 85.7 (23.6) pg/ml on average at baseline, while the average E2 reduced to 77.2 (25.6) pg/ml in the fourth month. Average number of AFC was 2.0 (0.8) at baseline. We observed a gradual increase in the second month (Mean AFC=2.2) and after four months it increased to 3.1 (1.8) as the highest menstrual restoration and pregnancy was observed in 10% of our study participants. CONCLUSIONS: MSCs could significantly improve hormone secretion in women with POF. Implantation of MSCs in women with POF background was associated with an increase in AMH and AFC, while it downed the serum level of E2 and FSH. MSCs could also lead to menstrual restoration and pregnancy in women with POF.