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.

Bioengineered skin constructs based on mesenchymal stromal cells and acellular dermal matrix exposed to inflammatory microenvironment releasing growth factors involved in skin repair.

BACKGROUND: Skin tissue engineering is a rapidly evolving field of research that effectively combines stem cells and biological scaffolds to replace damaged tissues. Human Wharton's jelly mesenchymal stromal cells (hWJ-MSCs) are essential to generate tissue constructs, due to their potent immunomodulatory effects and release of paracrine factors for tissue repair. Here, we investigated whether hWJ-MSC grown on human acellular dermal matrix (hADM) scaffolds and exposed to a proinflammatory environment maintain their ability to produce in vitro growth factors involved in skin injury repair and promote in vivo wound healing. METHODS: We developed a novel method involving physicochemical and enzymatic treatment of cadaveric human skin to obtain hADM scaffold. Subsequently, skin bioengineered constructs were generated by seeding hWJ-MSCs on the hADM scaffold (construct 1) and coating it with human platelet lysate clot (hPL) (construct 2). Either construct 1 or 2 were then incubated with proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α) for 12, 24, 48, 72 and 96 h. Supernatants from treated and untreated constructs and hWJ-MSCs on tissue culture plate (TCP) were collected, and concentration of the following growth factors, bFGF, EGF, HGF, PDGF, VEGF and Angiopoietin-I, was determined by immunoassay. We also asked whether hWJ-MSCs in the construct 1 have potential toward epithelial differentiation after being cultured in an epithelial induction stimulus using an air-liquid system. Immunostaining was used to analyze the synthesis of epithelial markers such as filaggrin, involucrin, plakoglobin and the mesenchymal marker vimentin. Finally, we evaluated the in vivo potential of hADM and construct 1 in a porcine full-thickness excisional wound model. RESULTS: We obtained and characterized the hADM and confirmed the viability of hWJ-MSCs on the scaffold. In both constructs without proinflammatory treatment, we reported high bFGF production. In contrast, the levels of other growth factors were similar to the control (hWJ-MSC/TCP) with or without proinflammatory treatment. Except for PDGF in the stimulated group. These results indicated that the hADM scaffold maintained or enhanced the production of these bioactive molecules by hWJ-MSCs. On the other hand, increased expression of filaggrin, involucrin, and plakoglobin and decreased expression of vimentin were observed in constructs cultured in an air-liquid system. In vivo experiments demonstrated the potential of both hADM and hADM/hWJ-MSCs constructs to repair skin wounds with the formation of stratified epithelium, basement membrane and dermal papillae, improving the appearance of the repaired tissue. CONCLUSIONS: hADM is viable to fabricate a tissue construct with hWJ-MSCs able to promote the in vitro synthesis of growth factors and differentiation of these cells toward epithelial lineage, as well as, promote in a full-thickness skin injury the new tissue formation. These results indicate that hADM 3D architecture and its natural composition improved or maintained the cell function supporting the potential therapeutic use of this matrix or the construct for wound repair and providing an effective tissue engineering strategy for skin repair.

A novel therapeutic management for diabetes patients with chronic limb-threatening ischemia: comparison of autologous bone marrow mononuclear cells versus allogenic Wharton jelly-derived mesenchymal stem cells.

BACKGROUND: Chronic limb-threatening ischemia (CLTI) represents the final stage of peripheral arterial disease. Approximately one-third of patients with CLTI are not eligible for conventional surgical treatments. Furthermore, patients with advanced stage of CLTI are prone to amputation and death. Thus, an effective therapeutic strategy is urgently needed. In this context, autologous bone marrow mononuclear cell (auto-BM-MNC) and allogeneic mesenchymal stem cells represent a promising therapeutic approach for treating CLTI. In this study, we compared the safety and beneficial therapeutic effect of auto-BM-MNC versus allogeneic Wharton jelly-derived mesenchymal stem cells (allo-WJ-MSCs) in diabetic patients with CLTI. METHODS: We performed a randomized, prospective, double-blind and controlled pilot study. Twenty-four diabetic patients in the advanced stage of CLTI (4 or 5 in Rutherford's classification) and a transcutaneous oxygen pressure (TcPO2) below 30 mmHg were randomized to receive 15 injections of (i) auto-BM-MNC (7.197 × 106 ± 2.984 × 106 cells/mL) (n = 7), (ii) allo-WJ-MSCs (1.333 × 106 cells/mL) (n = 7) or (iii) placebo solution (1 mL) (n = 10), which were administered into the periadventitial layer of the arterial walls under eco-Doppler guidance. The follow-up visits were at months 1, 3, 6, and 12 to evaluate the following parameters: (i) Rutherford's classification, (ii) TcPO2, (iii) percentage of wound closure, (iv) pain, (v) pain-free walking distance, (vi) revascularization and limb-survival proportion, and (vii) life quality (EQ-5D questionnaire). RESULTS: No adverse events were reported. Patients with CLTI who received auto-BM-MNC and allo-WJ-MSCs presented an improvement in Rutherford's classification, a significant increase in TcPO2 values‬, a reduction in the lesion size in a shorter time, a decrease in the pain score and an increase in the pain-free walking distance, in comparison with the placebo group. In addition, the participants treated with auto-BM-MNC and allo-WJ-MSCs kept their limbs during the follow-up period, unlike the placebo group, which had a marked increase in amputation. CONCLUSIONS: Our results showed that patients with CLTI treated with auto-BM-MNC and allo-WJ-MSCs conserved 100% of their limb during 12 months of the follow-up compared to the placebo group, where 60% of participants underwent limb amputation in different times. Furthermore, we observed a faster improvement in the allo-WJ-MSC group, unlike the auto-BM-MNC group. Trial registration This study was retrospectively registered at ClinicalTrials.gov (NCT05631444).

Three-Dimensional Bioprinting of an In Vitro Lung Model.

In December 2019, COVID-19 emerged in China, and in January 2020, the World Health Organization declared a state of international emergency. Within this context, there is a significant search for new drugs to fight the disease and a need for in vitro models for preclinical drug tests. This study aims to develop a 3D lung model. For the execution, Wharton's jelly mesenchymal stem cells (WJ-MSC) were isolated and characterized through flow cytometry and trilineage differentiation. For pulmonary differentiation, the cells were seeded in plates coated with natural functional biopolymer matrix as membrane until spheroid formation, and then the spheroids were cultured with differentiation inductors. The differentiated cells were characterized using immunocytochemistry and RT-PCR, confirming the presence of alveolar type I and II, ciliated, and goblet cells. Then, 3D bioprinting was performed with a sodium alginate and gelatin bioink in an extrusion-based 3D printer. The 3D structure was analyzed, confirming cell viability with a live/dead assay and the expression of lung markers with immunocytochemistry. The results showed that the differentiation of WJ-MSC into lung cells was successful, as well as the bioprinting of these cells in a 3D structure, a promising alternative for in vitro drug testing.

Hyperglycemia affects neuronal differentiation and Nestin, FOXO1, and LMO3 mRNA expression of human Wharton's jelly mesenchymal stem cells of children from diabetic mothers.

Pregestational Diabetes Mellitus (PDM) during pregnancy constitutes an unfavorable embryonic and fetal development environment, with a high incidence of congenital malformations (CM). Neural tube defects are the second most common type of CM in children of diabetic mothers (CDM), who also have an elevated risk of developing neurodevelopmental disorders. The mechanisms that lead to these neuronal disorders in CDM are not yet fully understood. The present study aimed to know the effect of hyperglycemia on proliferation, neuronal differentiation percentage, and expression of neuronal differentiation mRNA markers in human umbilical cord Wharton's jelly mesenchymal stem cells (hUCWJMSC) of children from normoglycemic pregnancies (NGP) and PDM. We isolated and characterized hUCWJMSC by flow cytometry, immunofluorescence, RT-PCR and were induced to differentiate into adipocytes, osteocytes, and neurons. Proliferation assays were performed to determine the doubling time, and Nestin, TUBB3, FOXO1, KCNK2, LMO3, and MAP2 mRNA gene expression was assessed by semiquantitative RT-PCR. Hyperglycemia significantly decreased proliferation and neuronal differentiation percentage in NGP and PDM cells treated with 40 mM d-glucose. Nestin mRNA expression decreased under control glycemic conditions, while FOXO1, KCNK2, LMO3, and MAP2 mRNA expression increased during neuronal differentiation in both NGP and PDM cells. On the other hand, under hyperglycemic conditions, Nestin was significantly decreased in cells from NGP but not in cells from PDM, while mRNA expression of FOXO1 and LMO3 was significantly increased in cells from NGP, but not in cells from PDM. We found evidence that maternal PDM, with hyperglycemia in culture, affects the biological properties of fetal cells. All these results could be part of fetal programming.

Dyslipidemia induced by lipid diet in late gestation donor impact on growth kinetics and in vitro potential differentiation of umbilical cord Wharton's Jelly mesenchymal stem cells in goats.

Mesenchymal stem cells (MSC) from the umbilical cord (UC) have several attractive properties for clinical use. This study aimed to verify the impact of a lipid-rich diet during late gestation of donor goats on the growth and differentiation of MSCs from UC. From the 100th day of pregnancy to delivery, 22 goats were grouped based on their diet into the donor-lipid (DLD; n = 11) and donor-baseline (DBD; n = 11) diet groups. Diets were isonitrogenous and isoenergetic, differing in fat content (2.8% vs. 6.3% on a dry matter basis). Wharton's jelly (WJ) fragments were cultured. After primary culture, samples of WJ-MSCs were characterized by the expression of CD90, CD73, CD34, CD45, CD105, and Fas genes, mitochondrial activity using MitoTracker (MT) fluorescence probe, and growth kinetics. Population doubling time (PDT) was also determined. WJ-MSCs were differentiated into chondrocytes, adipocytes and osteocytes, and the mineralized area and adipocytes were determined. The lipid diet significantly increased triglyceride and cholesterol levels during pregnancy. The DLD group showed sub-expression of the CD90 gene, a high MT intensity, and a low proliferation rate at the end of the subculture. The mean PDT was 83.9 ± 1.3 h. Mineralized area and lipid droplet stain intensity from osteogenic and adipogenic differentiations, respectively, were greater in DLD. We conclude that in donor goats, dietary dyslipidemia during late pregnancy affects the ability of UC-derived MSCs to express their developmental potential in vitro, thus limiting their possible use for therapeutic purposes.

Secretome from Human Mesenchymal Stem Cells-Derived Endothelial Cells Promotes Wound Healing in a Type-2 Diabetes Mouse Model.

Tissue regeneration is often impaired in patients with metabolic disorders such as diabetes mellitus and obesity, exhibiting reduced wound repair and limited regeneration capacity. We and others have demonstrated that wound healing under normal metabolic conditions is potentiated by the secretome of human endothelial cell-differentiated mesenchymal stem cells (hMSC-EC). However, it is unknown whether this effect is sustained under hyperglycemic conditions. In this study, the wound healing effect of secretomes from undifferentiated human mesenchymal stem cells (hMSC) and hMSC-EC in a type-2 diabetes mouse model was analyzed. hMSC were isolated from human Wharton's jelly and differentiated into hMSC-EC. hMSC and hMSC-EC secretomes were analyzed and their wound healing capacity in C57Bl/6J mice fed with control (CD) or high fat diet (HFD) was evaluated. Our results showed that hMSC-EC secretome enhanced endothelial cell proliferation and wound healing in vivo when compared with hMSC secretome. Five soluble proteins (angiopoietin-1, angiopoietin-2, Factor de crecimiento fibroblástico, Matrix metallopeptidase 9, and Vascular Endothelial Growth Factor) were enriched in hMSC-EC secretome in comparison to hMSC secretome. Thus, the five recombinant proteins were mixed, and their pro-healing property was evaluated in vitro and in vivo. Functional analysis demonstrated that a cocktail of these proteins enhanced the wound healing process similar to hMSC-EC secretome in HFD mice. Overall, our results show that hMSC-EC secretome or a combination of specific proteins enriched in the hMSC-EC secretome enhanced wound healing process under hyperglycemic conditions.

Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector.

Cells from different origins behave differently regarding the incorporation of exogenous DNA and formation of transgenic cells. Milk production of recombinant antibody may benefit from efficient transfection protocols to produce transgenic animals. In this context, the objective of this study was to verify the transfection potential of bovine mesenchymal stem cells from Wharton's jelly (MSC-WJ) and adipose tissue (MSC-AT), comparing co-transfection protocols with vectors pBC1-anti-CD3 and pEF-NEO-GFP, using transfection reagents Lipofectamine LTX with Plus Reagent or Xfect. Skin fibroblasts (FIB) were used as the control group. Forty-eight hours after transfection, neomycin was added and cells cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than fibroblasts, for both the Xfect (20.057 ± 1.620,7 and 10.601 ± 702,86, respectively, p < 0.05) and LTX (19.590 ± 113,84 and 10.518 ± 442,65, respectively, p < 0.05). These results, associated with evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than other cells, independent of the kit used. Performing PCR on co-transfected cells demonstrated the presence of anti-CD3, making this approach feasible for future experiments.

Impact of donor nutritional balance on the growth and development of mesenchymal stem cells from caprine umbilical cord Wharton´s jelly.

Mesenchymal stem cells (MSCs) from the umbilical cord (UC) have aroused considerable interest. However, little is known about the maternal effect on these cells. The aim of this study was to verify the impact of the nutritional status of donor goats on the growth and differentiation of MSCs from the UC. At parturition, 19 goats were grouped based on their low or high body mass index (low BMI, LBMI, n = 9; and high BMI, HBMI, n = 10). UCs were collected during delivery and Wharton's jelly (WJ) fragments cultured. WJ-MSCs were differentiated into osteocytes, adipocytes, chondrocytes, and the population doubling time (PDT) was determined. Samples of WJ-MSCs were also used to verify the expression of the CD90, CD73, CD34, CD45, and CD105 genes. Media used for WJ-MSC primary cultures were analyzed using near-infrared spectroscopy. The lag phase was 7.5 ± 0.6 days and the entire culture took 26.7 ± 1.3 days, with a cell proliferation rate of 8.500 cells/day. The mean PDT from subculture was 30.0 ± 0.7 h. The CD105 gene was sub-expressed in LBMI, and the spectra of the spent media from the second to fourth day of WJ-MSC primary culture were segregated into negative scores by multivariate analysis. We conclude that, in goats, the nutritional balance of the donor did not affect the in vitro growth of MSCs derived from the UC. However, the molecular profile observed in the low BMI group suggests that the use of MSCs for therapeutic purposes should be considered more carefully.

Efecto del factor de crecimiento fibroblástico dos (FGF-2), en la reducción de la senescencia en células madre mesenquimales aisladas de gelatina de Wharton / Fibroblastic growth factor two (FGF-2), in reducing the senescent effect on mesenchymal stem cell isolated from Wharton ́s jelly

Resumen Introducción. Las células madre mesenquimales han generado interés en la ingeniería de tejidos, debido a sus propiedades proliferativas y capacidad de reparación de tejidos, sin embargo, para un trasplante exitoso, es necesario aumentar el número de células mediante un cultivo in-vitro. Durante este proceso la capacidad proliferativa disminuye, provocando cambios en la morfología y funcionalidad celular y afectando la viabilidad del cultivo, este estado se conoce como senescencia celular y como posibles causales, se ha considerado el estrés oxidativo y la falta de factores de crecimiento. Objetivos: Evaluar el efecto de FGF-2 sobre la senescencia de un cultivo de células madre mesenquimales aisladas de gelatina de Wharton y su papel en la regulación del estrés oxidativo. Metodología. Se añadieron dosis de 3,5 y 7,5 ng de FGF-2 al cultivo. Durante los pasajes 5 y 7, se estimó tanto la senescencia celular como la presencia de ROS (especies reactivas de oxígeno). Resultados.Se obtuvo en el pasaje 5, una diferencia significativa del 99,5% entre el control (+) con respecto a los tratamientos con FGF-2, sin embargo, en el pasaje 7 se observó un aumento en la producción de la enzima ß-galactosidasa y cambios morfológicos, confirmando un estado senescente en el cultivo en todos los tratamientos evaluados. Conclusión. Las dosis utilizadas en este estudio contribuyeron positivamente a disminuir el proceso senescente en el cultivo celular, además se determinó, que el FGF-2 puede prolongar el tiempo de cultivo, retardando parcialmente la concentración de especies reactivas de oxígeno

AbstractIntroduction. Mesenchymal stem cells have been generated interest in tissue engineering, due to their proliferative properties and tissue repair capacity, however, for a successful transplant process, it is necessary to increase the number of cells in a culture expansion process. During this process the proliferative capacity is limited, causing changes in cell morphology and functionality affecting the viability of the culture, this state is known as cell senescence. Oxidative stress and deregulation of growth factors are considered as reasons. Aims. To evaluate the effect of FGF-2 on the senescence of a mesenchymal stem cells culture isolated from Wharton ́s jelly and its role in the regulation of oxidative stress. Methodology: 3,5 and 7,5 ng doses of FGF-2 were added to the culture medium from passage 2, then the senescence of the culture was evaluated and the presence of reactive oxygen species was determined during passages 5 and 7. Results. We observed that in passage 5, there is a significant difference 99.5% between the control (+) concerning the FGF-2 treatments, however, in passage 7, an increase in the production of the enzyme ß-galactosidase was observed and changes in morphology such as: increase in size and elongated shape of the cell, confirming a senescent state on the culture in all the treatments evaluated. Conclusion. The doses used in this study contributed positively to decrease this process in a cell culture, also, the FGF- 2 can prolong the cultivation time, partially decreasing the concentration of reactive oxygen species

Advances in translational orthopaedic research with species-specific multipotent mesenchymal stromal cells derived from the umbilical cord.

Compliance with current regulations for the development of innovative medicines require the testing of candidate therapies in relevant translational animal models prior to human use. This poses a great challenge when the drug is composed of cells, not only because of the living nature of the active ingredient but also due to its human origin, which can subsequently lead to a xenogeneic response in the animals. Although immunosuppression is a plausible solution, this is not suitable for large animals and may also influence the results of the study by altering mechanisms of action that are, in fact, poorly understood. For this reason, a number of procedures have been developed to isolate homologous species-specific cell types to address preclinical pharmacodynamics, pharmacokinetics and toxicology. In this work, we present and discuss advances in the methodologies for derivation of multipotent Mesenchymal Stromal Cells derived from the umbilical cord, in general, and Wharton's jelly, in particular, from medium to large animals of interest in orthopaedics research, as well as current and potential applications in studies addressing proof of concept and preclinical regulatory aspects.

Human Platelet Lysate Supports Efficient Expansion and Stability of Wharton's Jelly Mesenchymal Stromal Cells via Active Uptake and Release of Soluble Regenerative Factors.

Manufacturing of mesenchymal stromal cell (MSC)-based therapies for regenerative medicine requires the use of suitable supply of growth factors that enhance proliferation, cell stability and potency during cell expansion. Human blood derivatives such as human platelet lysate (hPL) have emerged as a feasible alternative for cell growth supplement. Nevertheless, composition and functional characterization of hPL in the context of cell manufacturing is still under investigation, particularly regarding the content and function of pro-survival and pro-regenerative factors. We performed comparative analyses of hPL, human serum (hS) and fetal bovine serum (FBS) stability and potency to support Wharton's jelly (WJ) MSC production. We demonstrated that hPL displayed low inter-batch variation and unique secretome profile that was not present in hS and FBS. Importantly, hPL-derived factors including PDGF family, EGF, TGF-alpha, angiogenin and RANTES were actively taken up by WJ-MSC to support efficient expansion. Moreover, hPL but not hS or FBS induced secretion of osteoprotegerin, HGF, IL-6 and GRO-alpha by WJ-MSC during the expansion phase. Thus, hPL is a suitable source of factors supporting viability, stability and potency of WJ-MSC and therefore constitutes an essential raw material that in combination with WJ-MSC introduces a great opportunity for the generation of potent regenerative medicine products.

Informe diário de evidências COVID-19: busca realizada em 29 de maio de 2020 / COVID-19 daily evidence report: search conducted on May 29, 2020

O Informe Diário de Evidências é uma produção do Ministério da Saúde que tem como objetivo acompanhar diariamente as publicações científicas sobre tratamento farmacológico e vacinas para a COVID-19. Dessa forma, são realizadas buscas estruturadas em bases de dados biomédicas, referente ao dia anterior desse informe. Não são incluídos estudos pré-clínicos (in vitro, in vivo, in silico). A frequência dos estudos é demonstrada de acordo com a sua classificação metodológica (revisões sistemáticas, ensaios clínicos randomizados, coortes, entre outros). Para cada estudo é apresentado um resumo com avaliação da qualidade metodológica. Essa avaliação tem por finalidade identificar o grau de certeza/confiança ou o risco de viés de cada estudo. Para tal, são utilizadas ferramentas já validadas e consagradas na literatura científica, na área de saúde baseada em evidências. Cabe ressaltar que o documento tem caráter informativo e não representa uma recomendação oficial do Ministério da Saúde sobre a temática. Foram encontrados 13 artigos e 13 protocolos.

Intravenous Human Umbilical Cord-Derived Mesenchymal Stromal Cell Administration in Models of Moderate and Severe Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is as a life-threatening condition that can occur in young adults, often causing long-term disability. Recent preclinical data suggest mesenchymal stromal cell (MSC)-based therapies as promising options to minimize brain damage after ICH. However, therapeutic evidence and mechanistic insights are still limited, particularly when compared with other disorders such as ischemic stroke. Herein, we employed a model of collagenase-induced ICH in young adult rats to investigate the potential therapeutic effects of an intravenous injection of human umbilical cord Wharton's jelly-derived MSCs (hUC-MSCs). Two doses of collagenase were used to cause moderate or severe hemorrhages. Magnetic resonance imaging showed that animals treated with hUC-MSCs after moderate ICH had smaller residual hematoma volumes than vehicle-treated rats, whereas the cell therapy failed to decrease the hematoma volume in animals with a severe ICH. Functional assessments (rotarod and elevated body swing tests) were performed for up to 21 days after ICH. Enduring neurological impairments were seen only in animals subjected to severe ICH, but the cell therapy did not induce statistically significant improvements in the functional recovery. The biodistribution of Technetium-99m-labeled hUC-MSCs was also evaluated, showing that most cells were found in organs such as the spleen and lungs 24 h after transplantation. Nevertheless, it was possible to detect a weak signal in the brain, which was higher in the ipsilateral hemisphere of rats subjected to a severe ICH. These data indicate that hUC-MSCs have moderately beneficial effects in cases of less severe brain hemorrhages in rats by decreasing the residual hematoma volume, and that optimization of the therapy is still necessary.

Wharton's jelly-derived mesenchymal stem cells attenuate sepsis-induced organ injury partially via cholinergic anti-inflammatory pathway activation.

Sepsis induces organ dysfunction due to overexpression of the inflammatory host response, resulting in cardiopulmonary and autonomic dysfunction, thus increasing the associated morbidity and mortality. Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) express genes and secrete factors with anti-inflammatory properties, neurological and immunological protection, as well as improve survival in experimental sepsis. The cholinergic anti-inflammatory pathway (CAP) is mediated by α7-nicotinic acetylcholine receptors (α7nAChRs), which play an important role in the control of systemic inflammation. We hypothesized that WJ-MSCs attenuate sepsis-induced organ injury in the presence of an activated CAP pathway. To confirm our hypothesis, we evaluated the effects of WJ-MSCs as a treatment for cardiopulmonary injury and on neuroimmunomodulation. Male Wistar rats were randomly divided into four groups: control (sham-operated); cecal ligation and puncture (CLP) alone; CLP+WJ-MSCs (1 × 106 cells, at 6 h post-CLP); and CLP+methyllycaconitine (MLA)+WJ-MSCs (5 mg/kg body wt, at 5.5 h post-CLP, and 1 × 106 cells, at 6 h post-CLP, respectively). All experiments, including the assessment of echocardiographic parameters and heart rate variability, were performed 24 h after CLP. WJ-MSC treatment attenuated diastolic dysfunction and restored baroreflex sensitivity. WJ-MSCs also increased cardiac sympathetic and cardiovagal activity. WJ-MSCs reduced leukocyte infiltration and proinflammatory cytokines, effects that were abolished by administration of a selective α7nAChR antagonist (MLA). In addition, WJ-MSC treatment also diminished apoptosis in the lungs and spleen. In cardiac and splenic tissue, WJ-MSCs downregulated α7nAChR expression, as well as reduced the phospho-STAT3-to-total STAT3 ratio in the spleen. WJ-MSCs appear to protect against sepsis-induced organ injury by reducing systemic inflammation, at least in part, via a mechanism that is dependent on an activated CAP.

Extracellular vesicles derived from human Wharton's jelly mesenchymal stem cells protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-ß oligomers.

BACKGROUND: Mesenchymal stem cells (MSCs) have been explored as promising tools for treatment of several neurological and neurodegenerative diseases. MSCs release abundant extracellular vesicles (EVs) containing a variety of biomolecules, including mRNAs, miRNAs, and proteins. We hypothesized that EVs derived from human Wharton's jelly would act as mediators of the communication between hMSCs and neurons and could protect hippocampal neurons from damage induced by Alzheimer's disease-linked amyloid beta oligomers (AßOs). METHODS: We isolated and characterized EVs released by human Wharton's jelly mesenchymal stem cells (hMSC-EVs). The neuroprotective action of hMSC-EVs was investigated in primary hippocampal cultures exposed to AßOs. RESULTS: hMSC-EVs were internalized by hippocampal cells in culture, and this was enhanced in the presence of AßOs in the medium. hMSC-EVs protected hippocampal neurons from oxidative stress and synapse damage induced by AßOs. Neuroprotection by hMSC-EVs was mediated by catalase and was abolished in the presence of the catalase inhibitor, aminotriazole. CONCLUSIONS: hMSC-EVs protected hippocampal neurons from damage induced by AßOs, and this was related to the transfer of enzymatically active catalase contained in EVs. Results suggest that hMSC-EVs should be further explored as a cell-free therapeutic approach to prevent neuronal damage in Alzheimer's disease.

Regenerative treatment with umbilical cord mesenchymal stem cells from Wharton's jelly in chronic ulcer caused by dermolipectomy.

BACKGROUND: Procedures to remove adiposities and skin, such as dermolipectomy, can develop wounds that are difficult to heal by conventional therapies. Mesenchymal stem cells are indicated as potential candidates for regenerative therapy in wounds, due to their multipotentiality, low immunogenicity, modulating capacity of inflammation and tissue modeling processes. CASE REPORT: Patient with dehiscent chronic ulcer secondary to dermolipectomy, who received cutaneous treatment with mesenchymal stem cells. The therapy induced scar formation and neovascularization, as well as the decrease of infiltrated leukocytes and proinflammatory cytokines. Mesenchymal cells are proposed as an interesting alternative for the treatment of postoperative lesions.

INTRODUCCIÓN: Los procedimientos para retirar adiposidades y piel, como la dermolipectomía, pueden desarrollar heridas difíciles de sanar mediante tratamientos convencionales. Se ha señalado que es posible utilizar las células madre mesenquimales en el tratamiento regenerativo en heridas, en virtud de su multipotencialidad, baja inmunogenicidad, capacidad moduladora de inflamación y procesos modeladores de tejidos. CASO CLÍNICO: Paciente con dehiscencia en úlcera crónica secundaria a dermolipectomía, sometida a tratamiento cutáneo con células madre mesenquimales. Se indujo formación de cicatriz y neovascularización, así como la disminución de leucocitos infiltrados y citocinas proinflamatorias. Se propone a las células mesenquimales como una alternativa interesante para el tratamiento de lesiones postoperatorias.

Conditioned medium of ovine Wharton's jelly-derived mesenchymal stem cells improves growth and reduces ROS generation of isolated secondary follicles after short-term in vitro culture.

The aim of this study was to evaluate the effect of the conditioned medium of ovine Wharton's jelly-derived mesenchymal stem cells (oWJ-MSCs) on the morphology, growth, reactive oxygen species (ROS) and glutathione (GSH) intracellular levels, active mitochondria, and meiotic resumption of isolated ovine secondary follicles in vitro. The oWJ-MSCs were isolated and the medium where they were cultured was recovered (conditioned medium). Isolated ovine secondary follicles were cultured for 6 days in 1) supplemented α-MEM+ (control); 2) 50% α-MEM+ + 50% conditioned medium (α-MEM + CM group) or 3) conditioned medium only (CM group). The parameters analyzed were morphology, antrum formation, follicle and oocyte growth, ROS and GSH levels, mitochondrial activity and meiotic resumption. The percentage of normal follicles, antrum formation, and fully grown oocytes did not differ (P > 0.05) among treatments. Follicles cultured in α-MEM + CM group had greater (P < 0.05) diameter than other treatments after culture. Moreover, the diameter of the follicles cultured in CM alone was higher (P < 0.05) than in the α-MEM+. In addition, α-MEM + CM and CM treatments increased the growth rate compared to the α-MEM+. Treatments containing conditioned medium (α-MEM + CM or CM) significantly reduced ROS levels compared to the control medium. Moreover, mitochondrial activity was higher in α-MEM+ and α-MEM + CM than in CM alone. All treatments showed oocytes in GV, GVBD and MI. In conclusion, oWJ-MSCs conditioned medium, especially when associated with α-MEM, improves the growth of secondary follicles and reduces ROS generation after short-term culture.

Extracellular vesicles from pluripotent stem cell-derived mesenchymal stem cells acquire a stromal modulatory proteomic pattern during differentiation.

Mesenchymal stem/stromal cells (MSCs) obtained from pluripotent stem cells (PSCs) constitute an interesting alternative to classical MSCs in regenerative medicine. Among their many mechanisms of action, MSC extracellular vesicles (EVs) are a potential suitable substitute for MSCs in future cell-free-based therapeutic approaches. Unlike cells, EVs do not elicit acute immune rejection, and they can be produced in large quantities and stored until ready to use. Although the therapeutic potential of MSC EVs has already been proven, a thorough characterization of MSC EVs is lacking. In this work, we used a label-free liquid chromatography tandem mass spectrometry proteomic approach to identify the most abundant proteins in EVs that are secreted from MSCs derived from PSCs (PD-MSCs) and from their parental induced PSCs (iPSCs). Next, we compared both datasets and found that while iPSC EVs enclose proteins that modulate RNA and microRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion, and influence cell-substrate adhesion. Moreover, compared to their respective cells, iPSCs and iPSC EVs share a greater proportion of proteins, while the PD-MSC proteome appears to be more specific. Correlation and principal component analysis consistently aggregate iPSCs and iPSC EVs but segregate PD-MSC and their EVs. Altogether, these findings suggest that during differentiation, compared with their parental iPSC EVs, PD-MSC EVs acquire a more specific set of proteins; arguably, this difference might confer their therapeutic properties.

Comparison of Advanced Platelet Rich Fibrin (A-PRF) and Culture Media Conditioned Warton's Jelly (CMCWJ) on Fibroblast Cells Proliferation

Objective: To compare the potency of fibroblast cells proliferation in 12.5% and 25% Culture Media Conditioned Warton's Jelly (CMCWJ) and Advanced Platelet Rich Fibrin (A-PRF) cultured medium. Material and Methods: Fibroblast cells were divided into five groups: Group I (Control Group): serum-starved fibroblast without any treatment as a negative control; Group II: fibrolast that supplemented in 12.5% CMCWJ medium; Group III: fibrolast that supplemented 12.5% A-PRF medium; Group IV: fibrolast that supplemented 25% CMCWJ medium, and Group V: fibrolast that supplemented 25% A-PRF medium. The fibroblasts proliferation was counted by an automated cell counter. Statistical analysis was performed using One-way ANOVA and Post hoc Tamhane test was conducted to analyze the potential fibroblast proliferation differences in different concentration of CMCWJ and A-PRF group. Results: There were no significant differences in the fibroblast cell proliferation between GI and GIV, GII and GIV, GII and GIII, GII and GV, also GIV and GV. There were significant differences between GI and GII, GI and GIII, GI and GV, also GIII and GIV. Conclusion: The 12.5% CMCWJ group, 12.5% A-PRF group and 25% A-PRF group has excellent potential ability of fibroblast cells proliferation, meanwhile 25% CMCWJ group has the lowest mean potency of fibroblast cells proliferation compared to other groups. The 12.5% A-PRF Group has the highest mean of fibroblast cell proliferation amongst other groups.