Transplantation of autologous mesenchymal stromal cells in complete cervical spinal cord injury: a pilot study.

Objective: Spinal cord injury (SCI) is a serious condition that can lead to partial or complete paraplegia or tetraplegia. Currently, there are few therapeutic options for these conditions, which are mainly directed toward the acute phase, such as surgical intervention and high-dose steroid administration. Mesenchymal stromal cells (MSC) have been shown to improve neurological function following spinal cord injury. The aim of the study was to evaluate the safety, feasibility, and potential efficacy of MSC transplantation in patients with cervical traumatic SCI. Methods: We included seven subjects with chronic traumatic SCI (> 1 year) at the cervical level, classified as American Spinal Cord Injury Association impairment scale (AIS) grade A. Subjects received two doses of autologous bone marrow derived MSC, the first by direct injection into the lesion site after hemilaminectomy and the second three months later by intrathecal injection. Neurologic evaluation, spinal magnetic resonance imaging (MRI), urodynamics, and life quality questionnaires were assessed before and after treatment. Results: Cell transplantation was safe without severe or moderate adverse effects, and the procedures were well tolerated. Neurological evaluation revealed discrete improvements in sensitivity below the lesion level, following treatment. Five subjects showed some degree of bilateral sensory improvement for both superficial and deep mechanical stimuli compared to the pretreatment profile. No significant alterations in bladder function were observed during this study. Conclusion: Transplantation of autologous MSC in patients with chronic cervical SCI is a safe and feasible procedure. Further studies are required to confirm the efficacy of this therapeutic approach. Clinical trial registration: https://clinicaltrials.gov/study/NCT02574572, identifier NCT02574572.

Comprehensive analysis of secretome and transcriptome stability of Wharton jelly mesenchymal stromal cells during good manufacturing practice-compliant production.

BACKGROUND: Mesenchymal stromal cells (MSCs) hold promise for cell-based therapies due to their ability to stimulate tissue repair and modulate immune responses. Umbilical cord-derived MSCs from Wharton jelly (WJ) offer advantages such as low immunogenicity and potent immune modulatory effects. However, ensuring consistent quality and safety throughout their manufacturing process remains critical. RNA sequencing (RNA-seq) emerges as a crucial tool for assessing genetic stability and expression dynamics in cell-based therapeutic products. METHODS: We examined the secretome and transcriptome of WJ-MSC signatures throughout Good Manufacturing Practice (GMP) production, focusing on the performance of total RNA or Massive Analysis of cDNA Ends (MACE) sequencing. RESULTS: Through extensive transcriptomic analysis, we demonstrated consistent stability of WJ-MSC expression signatures across different manufacturing stages. Notably, MACE-seq showed improved identification of key expression patterns related to senescence and immunomodulation. CONCLUSIONS: These findings highlight the potential of MACE-seq as a quality assessment tool for WJ-MSC-based therapies, ensuring their efficacy and safety in clinical applications. Importantly, MACE-seq demonstrated its value in characterizing WJ-MSC-derived products, offering insights that traditional assays cannot provide.

Mesenchymal Stem/Stromal Cells Derived from Dental Tissues Mediate the Immunoregulation of T Cells through the Purinergic Pathway.

Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to bone marrow-derived mesenchymal stem cells (BM-MSCs) for potential clinical applications because of their accessibility and anti-inflammatory capacity. We previously demonstrated that DT-MSCs from dental pulp (DP-MSCs), periodontal ligaments (PDL-MSCs), and gingival tissue (G-MSCs) show immunosuppressive effects similar to those of BM, but to date, the DT-MSC-mediated immunoregulation of T lymphocytes through the purinergic pathway remains unknown. In the present study, we compared DP-MSCs, PDL-MSCs, and G-MSCs in terms of CD26, CD39, and CD73 expression; their ability to generate adenosine (ADO) from ATP and AMP; and whether the concentrations of ADO that they generate induce an immunomodulatory effect on T lymphocytes. BM-MSCs were included as the gold standard. Our results show that DT-MSCs present similar characteristics among the different sources analyzed in terms of the properties evaluated; however, interestingly, they express more CD39 than BM-MSCs; therefore, they generate more ADO from ATP. In contrast to those produced by BM-MSCs, the concentrations of ADO produced by DT-MSCs from ATP inhibited the proliferation of CD3+ T cells and promoted the generation of CD4+CD25+FoxP3+CD39+CD73+ Tregs and Th17+CD39+ lymphocytes. Our data suggest that DT-MSCs utilize the adenosinergic pathway as an immunomodulatory mechanism and that this mechanism is more efficient than that of BM-MSCs.

Characterization of ibrutinib's effects on the morphology, proliferation, phenotype, viability, and anti-inflammatory potential of adipose-derived mesenchymal stromal cells.

Ibrutinib (IB) is a tyrosine kinase inhibitor (TKI) that has immunomodulatory action and can be used as second-line therapy for steroid-refractory or steroid-resistant chronic Graft versus Host Disease (cGVHD). Mesenchymal stromal cells (MSCs) are distributed throughout the body and their infusion has also been explored as a second-line therapeutic alternative for the treatment of cGVHD. Considering the currently unknown effects of IB on endogenous MSCs, as well as the possible combined use of IB and MSCs for cGVHD, we investigated whether adipose tissue-derived MSCs present IB-targets, as well as the consequences of treating MSCs with this drug, regarding cell viability, proliferation, phenotype, and anti-inflammatory potential. Interestingly, we show for the first time that MSCs express several IB target genes. Also of note, the treatment of such cells with this TKI elevated the levels of CD90 and CD105 surface proteins, as well as VCAM-1. Furthermore, IB-treated MSCs presented increased mRNA expression of the anti-inflammatory genes PD-L1, TSG-6, and IL-10. However, continued exposure to IB, even at low doses, compromised the viability of MSCs. These data indicate that the use of IB can stimulate an anti-inflammatory profile in MSCs, but also that a continued exposure to IB can compromise MSC viability over time.

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.

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.

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.

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.

Mesenchymal Stromal Cells Nanovesicles Carry microRNA with Nephroprotective Proprieties Regardless of Aging.

Containing information molecules from their parent cells and inclining to fuse with targeted cells, bone marrow mesenchymal stromal cells-derived extracellular vesicles (MSCs- EV) are valuable in nanomedicine. BACKGROUND: The effects of aging on the paracrine mechanism and in the production and action of MSCs-EV and their cargos of miR-26a and siRNA-26a for the treatment of tubular renal cells under nephrotoxicity injury remain unelucidated. OBJECTIVE: The purpose of this study was to evaluate MSCs-EV of different ages and their ability to deliver the cargos of miR-26a and siRNA-26a to target renal tubular cells affected by nephrotoxicity injury. METHODS: In a model of gentamicin-induced nephrotoxicity, renal tubular cells treated with MSCs-EV expressing or not expressing microRNA-26a were analyzed. Western blotting was utilized to evaluate cell cycle markers, and MTT assay was utilized to evaluate auto-renovation capacity. RESULTS: Tubular cells under nephrotoxicity injury showed decreased proliferative capacity, but the treatment in the tubular renal cells under nephrotoxicity injury with MSCs-EV expressing microRNA-26a showed nephroprotective effects, regardless of EV age. While the treatment with EV-mediated siRNA-26a failed to preserve the nephroprotective effects equally, regardless of age. CONCLUSION: Mesenchymal stromal cell nanovesicles carry microRNA with nephroprotective proprieties regardless of aging.

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.

Immunomodulatory-associated gene transcripts to multipotency of bovine amniotic fluid mesenchymal stem cells.

The adnexa fetal tissues are sources of mesenchymal stromal cells (MSCs) due to their noninvasive harvest, with all biological material discarded most of the time. MSCs are a promise regarding to their plasticity, self-renewal, differentiation potentials, immunomodulatory and anti-inflammatory properties, which have made clinical stem cell therapy a reality. The present study aimed to characterize and evaluate the immunomodulation ability of bovine mesenchymal cells collected from bovine amniotic fluid (bAFMSCs) isolated and subjected to sixth consecutive culture passages in vitro. The multilineage properties of the bAFMSCs collections confirmed the ability to undergo adipogenic, chondrogenic and osteogenic differentiation. The mesenchymal gene transcription CD106, CD73, CD29, CD90 and CD166 were detected in bAFMSCs, whereas CD34 and CD45 were not detected. Regarding cytokine mRNA expression, IL2, IL6, INFα, INFß, INFγ, TNFα and TNFß were downregulated, while IL10 was highly regulated in all studied passages. The present study demonstrated the immunological properties and multipotency of in vitro bAFMSCs collections, and thus, they can be tested in cattle pathological treatments or multiplication by nuclear transfer cloning.

Osteosarcoma cells exhibit functional interactions with stromal cells, fostering a lung microenvironment conducive to the establishment of metastatic tumor cells.

BACKGROUND: Osteosarcoma (OS) stands out as the most common bone tumor, with approximately 20% of the patients receiving a diagnosis of metastatic OS at their initial assessment. A significant challenge lies in the frequent existence of undetected metastases during the initial diagnosis. Mesenchymal stem cells (MSCs) possess unique abilities that facilitate tumor growth, and their interaction with OS cells is crucial for metastatic spread. METHODS AND RESULTS: We demonstrated that, in vitro, MSCs exhibited a heightened migration response toward the secretome of non-metastatic OS cells. When challenged to a secretome derived from lungs preloaded with OS cells, MSCs exhibited greater migration toward lungs colonized with metastatic OS cells. Moreover, in vivo, MSCs displayed preferential migratory and homing behavior toward lungs colonized by metastatic OS cells. Metastatic OS cells, in turn, demonstrated an increased migratory response to the MSCs' secretome. This behavior was associated with heightened cathepsin D (CTSD) expression and the release of active metalloproteinase 2 (MMP2) by metastatic OS cells. CONCLUSIONS: Our assessment focused on two complementary tumor capabilities crucial to metastatic spread, emphasizing the significance of inherent cell features. The findings underscore the pivotal role of signaling integration within the niche, with a complex interplay of migratory responses among established OS cells in the lungs, prometastatic OS cells in the primary tumor, and circulating MSCs. Pulmonary metastases continue to be a significant factor contributing to OS mortality. Understanding these mechanisms and identifying differentially expressed genes is essential for pinpointing markers and targets to manage metastatic spread and improve outcomes for patients with OS.

A Phase I Dose-Escalation Clinical Trial to Assess the Safety and Efficacy of Umbilical Cord-Derived Mesenchymal Stromal Cells in Knee Osteoarthritis.

Osteoarthritis (OA) is the most common degenerative joint disease. Mesenchymal stromal cells (MSC) are promising cell-based therapy for OA. However, there is still a need for additional randomized, dose-dependent studies to determine the optimal dose and tissue source of MSC for improved clinical outcomes. Here, we performed a dose-dependant evaluation of umbilical cord (UC)-derived MSC (Celllistem) in a murine model and in knee OA patients. For the preclinical study, a classical dose (200.000 cells) and a lower dose (50.000 cells) of Cellistem were intra-articularly injected into the mice knee joints. The results showed a dose efficacy response effect of Cellistem associated with a decreased inflammatory and degenerative response according to the Pritzker OARSI score. Following the same approach, the dose-escalation phase I clinical trial design included 3 sequential cohorts: low-dose group (2 × 106 cells), medium-dose group (20 × 106), and high-dose group (80 × 106). All the doses were safe, and no serious adverse events were reported. Nonetheless, 100% of the patients injected with the high-dose experienced injection-related swelling in the knee joint. According to WOMAC total outcomes, patients treated with all doses reported significant improvements in pain and function compared with baseline after 3 and 6 months. However, the improvements were higher in patients treated with both medium and low dose as compared to high dose. Therefore, our data demonstrate that the intra-articular injection of different doses of Cellistem is both safe and efficient, making it an interesting therapeutic alternative to treat mild and symptomatic knee OA patients. Trial registration ClinicalTrials.gov NCT03810521.

Mesenchymal Stromal Cells Derived from Dental Tissues: Immunomodulatory Properties and Clinical Potential.

Mesenchymal stem/stromal cells (MSCs) are multipotent cells located in different areas of the human body. The oral cavity is considered a potential source of MSCs because they have been identified in several dental tissues (D-MSCs). Clinical trials in which cells from these sources were used have shown that they are effective and safe as treatments for tissue regeneration. Importantly, immunoregulatory capacity has been observed in all of these populations; however, this function may vary among the different types of MSCs. Since this property is of clinical interest for cell therapy protocols, it is relevant to analyze the differences in immunoregulatory capacity, as well as the mechanisms used by each type of MSC. Interestingly, D-MSCs are the most suitable source for regenerating mineralized tissues in the oral region. Furthermore, the clinical potential of D-MSCs is supported due to their adequate capacity for proliferation, migration, and differentiation. There is also evidence for their potential application in protocols against autoimmune diseases and other inflammatory conditions due to their immunosuppressive capacity. Therefore, in this review, the immunoregulatory mechanisms identified at the preclinical level in combination with the different types of MSCs found in dental tissues are described, in addition to a description of the clinical trials in which MSCs from these sources have been applied.

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.

Transitioning from static to suspension culture system for large-scale production of xeno-free extracellular vesicles derived from mesenchymal stromal cells.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC-EV in suspension cultures using spinner flasks and human collagen-coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T-flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold-increase was 5.7-fold for the 3D culture system and 4.6-fold for the 2D culture system, signifying a fold-change of 1.2 (calculated as the ratio of fold-increase 3D to fold-increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T-flask cultures. The total cell production in the spinner flask cultures was 5.2-fold higher than that in T-flask cultures. While the EV specific production (particles/cell) in T-flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC-EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC-EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno-free conditions.

cAMP regulates the progesterone receptor gene expression through the protein kinase A pathway during decidualization in human immortalized endometrial stromal cells.

Decidualization, a crucial process for successful pregnancy establishment and maintenance, involves endometrial stromal cell differentiation. This process is orchestrated by estradiol (E2), progesterone, and other stimuli that increase intracellular cyclic adenosine monophosphate (cAMP) levels. The intracellular progesterone receptor (PR), encoded by the PGR gene, has a key role in decidualization. This study aimed to understand the role of sex steroids and cAMP in regulating PGR expression during the in vitro decidualization of the human immortalized endometrial stromal cell line, T-HESC. We subjected the cells to individual and combined treatments of E2, medroxyprogesterone (MPA), and cAMP. Additionally, we treated cells with PR and estrogen receptor antagonists and a protein kinase A (PKA) inhibitor. We evaluated the expression of PGR isoforms and decidualization-associated genes by RT-qPCR. Our findings revealed that cAMP induced PGR-B and PGR-AB expression by activating the PKA signaling pathway, while MPA downregulated their expression through the PR. Furthermore, downstream genes involved in decidualization, such as those coding for prolactin (PRL), insulin-like growth factor-binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1), exhibited positive regulation via the cAMP-PKA pathway. Remarkably, MPA-activated PR signaling induced the expression of IGFBP1 and DKK1 but inhibited that of PRL. In conclusion, we have demonstrated that the PKA signaling pathway induces PGR gene expression during in vitro decidualization of the T-HESC human endometrial stromal cell line. This study has unraveled some of the intricate regulatory mechanisms governing PGR expression during this fundamental process for implantation and pregnancy maintenance.

Secretome of stem cells from human exfoliated deciduous teeth (SHED) and its extracellular vesicles improves keratinocytes migration, viability, and attenuation of H2 O2 -induced cytotoxicity.

Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H2 O2 in vitro was performed with H2 O2 followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 µg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H2 O2 -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.

Human Bone Marrow Mesenchymal Stem Cells Promote the M2 Phenotype in Macrophages Derived from STEMI Patients.

Acute ST-elevation myocardial infarction (STEMI) leads to myocardial injury or necrosis, and M1 macrophages play an important role in the inflammatory response. Bone marrow mesenchymal stem/stromal cells (BM-MSCs) are capable of modulating macrophage plasticity, principally due to their immunoregulatory capacity. In the present study, we analyzed the capacity of MSCs to modulate macrophages derived from monocytes from patients with STEMI. We analyzed the circulating levels of cytokines associated with M1 and M2 macrophages in patients with STEMI, and the levels of cytokines associated with M1 macrophages were significantly higher in patients with STEMI than in controls. BM-MSCs facilitate the generation of M1 and M2 macrophages. M1 macrophages cocultured with MSCs did not have decreased M1 marker expression, but these macrophages had an increased expression of markers of the M2 macrophage phenotype (CD14, CD163 and CD206) and IL-10 and IL-1Ra signaling-induced regulatory T cells (Tregs). M2 macrophages from patients with STEMI had an increased expression of M2 phenotypic markers in coculture with BM-MSCs, as well as an increased secretion of anti-inflammatory cytokines and an increased generation of Tregs. The findings in this study indicate that BM-MSCs have the ability to modulate the M1 macrophage response, which could improve cardiac tissue damage in patients with STEMI.