Neurociencias.pósters

Tratamiento de heridas complejas en equinos mediante aplicación alogénica de una línea celular derivada de células madre mesenquimales

2024-11-15T00:00:00Z

Tratamiento de heridas complejas en equinos mediante aplicación alogénica de una línea celular derivada de células madre mesenquimales Iribarne, Ailén; Palma, María Belén; Andrini, Laura B.; Martínez, K.; Hernández, H.O.; Muriel, M.G.; López, R.A.; Riccillo, Fernando; Gatti, L.; Rodríguez, Delfina; Muro, Adriana; Mazza, Leandro; Buero, Guillermo; Miriuka, Santiago Gabriel; Carosella, Edgardo D.; García, Marcela Nilda La cicatrización de las heridas tras una lesión cutánea es un proceso complejo, especialmente en los equinos, donde las heridas en los miembros son frecuentes y su reparación es complicada debido a sus especiales características anatómicas. Los tratamientos convencionales, utilizados actualmente, no son suficientemente eficaces. Las células madre mesenquimales (MSC) han surgido como una de las terapias celulares más prometedoras investigadas durante la última década, debido a sus propiedades regenerativas e inmuno-moduladoras. El cordón umbilical es una importante fuente de células madre mesenquimales adultas (ucMSCs). El presente estudio pretende demostrar la seguridad y potencial terapéutico del uso alogénico de una línea celular mesenquimal equina específica, obtenida a partir de ucMSCs, denominada eqMC, en la cicatrización de heridas graves en los miembros. Los métodos empleados fueron el aislamiento, cultivo y expansión de ucMSCs. Para la caracterización y selección de la línea celular se utilizó citometría de flujo, RT-PCR, inmunocitoquímica y evaluación de su capacidad inmunomoduladora. Se realizó un ensayo preclínico murino y, por último, se comenzó con el ensayo clínico en equinos. Nuestros resultados muestran que las eqMCs expresan el EQMHCB2, un homólogo funcional del HLA-G (proteína perteneciente al Complejo mayor de Histocompatibilidad tipo I no clásica, uno de los puntos de control inmunitario más relevantes). En el ensayo de inmunomodulación, las eqMCs inhibieron la proliferación de células mononucleares de sangre periférica equina activadas (e-PBMCs). En el ensayo preclínico murino, las eqMC redujeron el tiempo de cicatrización en un 50%. En el ensayo clínico equino, la inyección de eqMCs en lesiones graves de los miembros, disminuyó el tiempo de cierre de las heridas, observándose una mejora en la calidad de la regeneración de los tejidos implicados, especialmente en la relación dermo-epidérmica y con una disminución de tamaño de las molestas cicatrices de tejido fibroso. En conclusión, los resultados de este estudio sugieren que las eqMC pueden utilizarse de manera segura para su aplicación alogénica en la cicatrización de heridas graves, mejorando no solo el tiempo de resolución de la misma, sino también la calidad del tejido neoformado.

A critical pathway in human development: C19MC micrornas regulates FGF2 response in a model of human pluripotent stem cells cardiac differentiation

2021-11-01T00:00:00Z

A critical pathway in human development: C19MC micrornas regulates FGF2 response in a model of human pluripotent stem cells cardiac differentiation Möbbs, Alan Miqueas; Garate, Ximena; Scarafía, María Agustina; Colli, Carolina; Moro, Lucía Natalia; Luzzani, Carlos Daniel; Waisman, Ariel; La Greca, Alejandro; Miriuka, Santiago Gabriel

Embryo aggregation and adipose-derived mesenchymal donor cells in bovine somatic cell nuclear transfer

2021-01-08T00:00:00Z

Embryo aggregation and adipose-derived mesenchymal donor cells in bovine somatic cell nuclear transfer Alberio, Virgilia; Savy, Virginia; Vans Landschoot, Geraldina; Moro, Lucía Natalia; Olea, Fernanda Daniela; Rodríguez Álvarez, L.; Salamone, Daniel Felipe Somatic cell nuclear transfer (SCNT) is a powerful tool, but its efficiency remains low. The use of less differentiated donor cells or the embryo aggregation (EA) strategy improves the SCNT rates in several species. It remains unexplored whether the combined use of both strategies results in a synergistic effect that improves SCNT efficiency in bovine. To evaluate that, we assessed the optimal time of EA using IVF embryos (aim 1) and we evaluated whether the use of adipose-derived mesenchymal stem cells (ASC) as donor for SCNT together with EA improves the blastocyst rates and quality (aim 2). For aim 1, cumulus–oocyte complexes (COCs) were collected from slaughterhouse ovaries, in vitro matured (TCM-199), fertilized (16 × 106 spermatozoa mL−1 for 5 h) and cultured (synthetic oviductal fluid media in a humidified gas mixture at 39°C). After IVF, the zona pellucida was enzymatically removed and zona-free (ZF) embryos were cultured individually (1X) or 2 embryos placed together within a microwell (2X) (Day 0, n = 70). This procedure was performed at Days 3, 4, 5, 6, or 7 (n = 76, 78, 94, 96, 90, respectively) and blastocyst rate was assessed at Day 8. Contribution of both embryos to the 2X blastocyst was confirmed by staining Day 0 IVF embryos either with green or red Mitotracker (ThermoFisher Scientific) before EA. For aim 2, fibroblast (FB) and ASC cells were isolated from the skin and subcutaneous adipose tissue of the same adult animal, respectively. Cloned embryos were produced by ZF enucleation, fusion of one ASC or FB cell, and activation with 5 μM ionomycin/6-(dimethylamino)purine (6DMAP). After activation, cloned embryos were aggregated (FB2X or ASC2X) or individually cultured (FB1X or ASC1X). Blastocyst rates were recorded at Day 7 of in vitro culture. Three biological replicates were evaluated for each aim. Embryo developmental differences were determined using Fisher’s exact test. Relative expression of OCT4, SOX2, and KRT18 was measured by RTqPCR at the blastocyst stage and analysed by Kruskal–Wallis statistical test. Regarding aim 1, no differences for developmental rates were found for Day 0, 3, 4, and 5 groups (57%, 60%, 61.5%, 61%), but the blastocyst rate was only improved in Day 0 and Day 3 relative to their respective 1X controls (Day 0 2X 54.2% vs. Day 0 1X 25.5% and Day 3 2X 52.6% vs. Day 3 1X 25.3%). No aggregation occurred in Day 6 and Day 7 groups. All blastomeres were homogeneously distributed in the 2X blastocyst. Regarding aim 2, no effect of the donor cell was observed on the blastocyst rate (FB1X 26.8%, n = 82; ASC1X 21.7%, n = 198; FB2X 39.7%, n = 126; ASC2X 33%, n = 204), whereas EA improved the blastocyst rate of ASC-derived embryos (ASC1X 21.7% vs. ASC2X 33%). Overall, no synergistic effect of the use of both strategies was observed. Relative expression of KRT18 was significantly different between ASC1X and ASC2X embryos. Although OCT4 and SOX2 expression did not differ between groups, EA tended to bring the values closer to that of an IVF embryo. No effect of the donor cell was observed on the embryo relative expression. Our results suggest that EA at Day 0 improves the blastocyst rate in bovine SCNT and IVF embryos. EA of 2 ASC-derived embryos seemed to normalise the embryo quality and may improve post-implantation development.

Myostatin gene editing by CRISPR/Cas9 technology of Brangus fetal fibroblasts to produce edited embryos by cloning

2021-01-08T00:00:00Z

Myostatin gene editing by CRISPR/Cas9 technology of Brangus fetal fibroblasts to produce edited embryos by cloning Bastón, Juan Ignacio; Viale, Diego Luis; Olguín, Martín; Wiedenmann, Elisabet; Arnold, Victoria; Suva, Mariana; Luzzani, Carlos Daniel; Miriuka, Santiago Gabriel; Moro, Lucía Natalia; Vichera, Gabriel Some European cattle breeds, such as Charolais and Maine Anjou, have natural mutations in the myostatin gene (MSTN) that inhibit its expression and result in an increase in muscle mass and protein content. An innovative hallmark would be the in vitro introduction of this genotype in South America cattle breeds to improve their commercial value. To achieve this, we aimed to disrupt MSTN gene expression in bovine fetal fibroblasts (BFFs) using CRISPR/Cas9 technology and to generate MSTN-edited embryos by somatic cell nuclear transfer (SCNT). BFFs were isolated from a cloned fetus of a Brangus bull with a prized genetic background and nucleofected with the Cas9 ribonucleoprotein-gRNA complex previously assessed to target exon 2 of the bovine MSTN gene. To evaluate MSTN editing, genomic DNA from the wild-type (WT) and nucleofected BFFs were isolated, exon 2 of MSTN was amplified by PCR, and the PCR product was Sanger sequenced. In all cases, the sequencing results were analysed using the indel Synthego software tool. According to indel analysis, MSTN gene editing efficiency of the BFFs was 58.83% ± 3.2 (n = 6). The resulting edit consisted of insertion of thymine in exon 2 of the MSTN gene that shifted the gene reading frame, introducing a premature stop codon and generating a truncated MSTN protein. The nucleofected BFFs were then used to generate embryos by SCNT, and WT BFFs were included as controls. Embryo cleavage and blastocyst development rates were evaluated at Day 2 and 7, respectively (Chi-squared test, P < 0.05). Although lower cleavage rates were obtained in the MSTN-edited BFFs group [65.3% (n = 273/418) vs. 87.6% (n = 169/193)], no differences were observed at the blastocyst stage [19.1% (n = 80/418) vs. 25.4% (n = 49/193)]. To confirm the efficiency of MSTN editing in the cloned embryos, 10 blastocysts generated with the MSTN-edited BFFs were individually analysed for MSTN exon 2 sequence as described before. The results showed that 30% of the blastocysts (3/10) presented a homozygous biallelic edition, which consisted of a thymine base insertion, as expected. In summary, the strategy we used allowed production of MSTN null cloned Brangus embryos avoiding putative undesired integration of exogenous DNA into the bovine genome, such as plasmid sequence, regardless of off-target occurrences. We conclude that CRISPR/Cas9 is an efficient technique to disrupts MSTN gene expression in bovine embryos; this work represents a step toward improving the production efficiency of South American cattle breeds.