Commentary : mesenchymal stem cells : a new piece in the puzzle of COVID-19 treatment

A Commentary on Mesenchymal Stem Cells: A New Piece in the Puzzle of COVID-19 Treatment By Saldanha-Araujo F, Melgaço Garcez E, Silva-Carvalho AE and Carvalho JL (2020). Front. Immunol. 11:1563. doi: 10.3389/fimmu.2020.01563

Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987. Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/ Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP

Segurança clínica da aplicação intra-ovariana bovina de células-tronco mesenquimais alogênicas

A basic premise of reproductive physiology is that females have a finite and non-renewable stock of germinative cells, which results in a decrease in reproductive capacity over time. For female bovines, a further factor associated with this decrease is follicular puncture (ovum pickup - OPU), a technique widely used for production of embryos in vitro. As such, it is necessary to seek therapeutic or preventive options for fertility problems, and one potential option is treatment with mesenchymal stem cells (MSC), which exercise a paracrine effect in combating inflammatory and degenerative processes. However, as important as evaluating the efficacy of such treatments is an evaluation of safety. In this context, the current study was carried out with the application of 2.5 x 106 allogenic MSC derived from adipose tissue, to the ovarian cortex of healthy nelore (n = 5) and girolando (n = 5) cows. The animals were subsequently evaluated by ultrasonography, clinical examination, number of viable oocytes collected, and rate of embryo production. None of the animals presented any clinical alteration or any alteration on ultrasonography after receiving the MSC. Furthermore, comparison between the number of viable oocytes, embryos produced, and rate of embryo production before and after MSC application did not show a difference. Based on these data, it can be concluded that intraovarian application of 2.5 x 106 adipose-derived MSC is safe, and this technique represents a potential for study as a therapy in cases of ovarian degeneration or lesions.Una premisa básica de la fisiología reproductiva es que las hembras tienen un stock finito y no renovable de células germinativas, lo que resulta en una disminución de la capacidad reproductiva con el tiempo. Para las hembras bovinas, otro factor asociado a esta disminución es la punción folicular (ovum pickup - OPU), técnica ampliamente utilizada para la producción de embriones in vitro. Por tanto, es necesario buscar opciones terapéuticas o preventivas para los problemas de fertilidad, y una opción potencial es el tratamiento con células madre mesenquimales (MSC), que ejercen un efecto paracrino en la lucha contra los procesos inflamatorios y degenerativos. Sin embargo, tan importante como evaluar la eficacia de tales tratamientos es una evaluación de la seguridad. En este contexto, el presente estudio se llevó a cabo con la aplicación de 2,5 x 106 MSC alogénicas derivadas de tejido adiposo, a la corteza ovárica de vacas nelore (n = 5) y girolando (n = 5) sanas. Posteriormente, los animales se evaluaron mediante ultrasonografía, examen clínico, número de ovocitos viables recogidos y tasa de producción de embriones. Ninguno de los animales presentó alteración clínica ni alteración ecográfica tras recibir la MSC. Además, la comparación entre el número de ovocitos viables, los embriones producidos y la tasa de producción de embriones antes y después de la aplicación de MSC no mostró ninguna diferencia. En base a estos datos, se puede concluir que la aplicación intraovárica de 2,5 x 106 MSC derivadas de tejido adiposo es segura, y esta técnica representa un potencial de estudio como terapia en casos de degeneración o lesiones ováricas.Uma premissa básica da fisiologia reprodutiva é que as fêmeas possuem um estoque finito e não renovável de células germinativas, o que resulta em uma diminuição da capacidade reprodutiva ao longo do tempo. Para as fêmeas bovinas, outro fator associado a essa diminuição é a punção folicular (ovo pickup - OPU), técnica amplamente utilizada para produção de embriões in vitro. Assim, é necessário buscar opções terapêuticas ou preventivas para problemas de fertilidade, sendo uma opção potencial o tratamento com células-tronco mesenquimais (CTM), que exercem efeito parácrino no combate a processos inflamatórios e degenerativos. No entanto, tão importante quanto avaliar a eficácia de tais tratamentos é a avaliação da segurança. Nesse contexto, o presente estudo foi realizado com a aplicação de 2,5 x 106 MSC alogênicas derivadas do tecido adiposo, no córtex ovariano de vacas nelore (n = 5) e girolando (n = 5) hígidas. Os animais foram posteriormente avaliados por ultrassonografia, exame clínico, número de oócitos viáveis coletados e taxa de produção de embriões. Nenhum dos animais apresentou qualquer alteração clínica ou ultrassonográfica após receber o MSC. Além disso, a comparação entre o número de oócitos viáveis, embriões produzidos e taxa de produção de embriões antes e depois da aplicação de MSC não mostrou diferença. Com base nesses dados, pode-se concluir que a aplicação intraovariana de MSC de origem adiposa 2,5 x 106 é segura, e essa técnica representa um potencial para estudo como terapia em casos de degeneração ou lesões ovarianas

LL-37 boosts immunosuppressive function of placenta-derived mesenchymal stromal cells

Background: Although promising for graft-versus-host disease (GvHD) treatment, MSC therapy still faces important challenges. For instance, increasing MSC migratory capacity as well as potentializing immune response suppression are of interest. For GvHD management, preventing opportunistic infections is also a valuable strategy, since immunocompromised patients are easy targets for infections. LL-37 is a host defense peptide (HDP) that has been deeply investigated due to its immunomodulatory function. In this scenario, the combination of MSC and LL-37 may result in a robust combination to be clinically used. Methods: In the present study, the effects of LL-37 upon the proliferation and migratory capacity of human placenta-derived MSCs (pMSCs) were assessed by MTT and wound scratch assays. The influence of LL-37 over the immunosuppressive function of pMSCs was then investigated using CFSE cell division kit. Flow cytometry and real-time PCR were used to investigate the molecular mechanisms involved in the effects observed. Results: LL-37 had no detrimental effects over MSC proliferation and viability, as assessed by MTT assay. Moreover, the peptide promoted increased migratory behavior of pMSCs and enhanced their immunomodulatory function over activated human PBMCs. Strikingly, our data shows that LL-37 treatment leads to increased TLR3 levels, as shown by flow cytometry, and to an increased expression of factors classically related to immunosuppression, namely IDO, IL-10, TGF-β, IL-6, and IL-1β. Conclusions: Taken together, our observations may serve as groundwork for the development of new therapeutic strategies based on the combined use of LL-37 and MSCs, which may provide patients not only with an enhanced immunosuppression regime, but also with an agent to prevent opportunistic infections

GVHD-derived plasma as a priming strategy of mesenchymal stem cells

Background: Mesenchymal stem cell (MSC) therapy is an important alternative for GVHD treatment, but a third of patients fail to respond to such therapy. Therefore, strategies to enhance the immunosuppressive potential of MSCs constitute an active area of investigation. Here, we proposed an innovative priming strategy based on the plasma obtained from GVHD patients and tested whether this approach could enhance the immunosuppressive capacity of MSCs. Methods: We obtained the plasma from healthy as well as acute (aGVHD) and chronic (cGVHD) GVHD donors. Plasma samples were characterized according to the TNF-α, IFN-γ, IL-10, IL-1β, IL-12p40, and IL-15 cytokine levels. The MSCs primed with such plasmas were investigated according to surface markers, morphology, proliferation, mRNA expression, and the capacity to control T cell proliferation and Treg generation. Results: Interestingly, 57% of aGVHD and 33% of cGVHD plasmas significantly enhanced the immunosuppressive potential of MSCs. The most suppressive MSCs presented altered morphology, and those primed with cGHVD displayed a pronounced overexpression of ICAM-1 on their surface. Furthermore, we observed that the ratio of IFN-γ to IL-10 cytokine levels in the plasma used for MSC priming was significantly correlated with higher suppressive potential and Treg generation induction by primed MSCs, regardless of the clinical status of the donor. Conclusions: This work constitutes an important proof of concept which demonstrates that it is possible to prime MSCs with biological material and also that the cytokine levels in the plasma may affect the MSC immunosuppressive potential, serving as the basis for the development of new therapeutic approaches for the treatment of immune diseases

Pouteria macrophylla fruit extract microemulsion for cutaneous depigmentation : evaluation using a 3D pigmented skin model

Here, we verify the depigmenting action of Pouteria macrophylla fruit extract (EXT), incorporate it into a safe topical microemulsion and assess its effectiveness in a 3D pigmented skin model. Melanocytes-B16F10- were used to assess the EXT effects on cell viability, melanin synthesis, and melanin synthesis-related gene transcription factor expression, which demonstrated a 32% and 50% reduction of intra and extracellular melanin content, respectively. The developed microemulsion was composed of Cremophor EL®/Span 80 4:1 (w/w), ethyl oleate, and pH 4.5 HEPES buffer and had an average droplet size of 40 nm (PdI 0.40 ± 0.07). Skin irritation test with reconstituted epidermis (Skin Ethic RHETM) showed that the formulation is non-irritating. Tyrosinase inhibition was maintained after skin permeation in vitro, in which microemulsion showed twice the inhibition of the conventional emulsion (20.7 ± 2.2% and 10.7 ± 2.4%, respectively). The depigmenting effect of the microemulsion was finally confirmed in a 3D culture model of pigmented skin, in which histological analysis showed a more pronounced effect than a commercial depigmenting formulation. In conclusion, the developed microemulsion is a promising safe formulation for the administration of cutite fruit extract, which showed remarkable depigmenting potential compared to a commercial formulation