Natalia Pacienza

Objetivo En los últimos años, estudiamos las propiedades regenerativas, anti-inflamatorias e inmuno-moduladoras de las vesículas extracelulares (EVs) secretadas por células madre mesenquimales (MSCs). Debido a que los procesos que conllevan a la opacidad corneal se deben básicamente a una desregulación de mecanismos regenerativos e inflamatorios, en el presente trabajo, nos propusimos evaluar el potencial terapéutico de las EVs derivadas de MSCs en un modelo experimental de injuria corneal. Métodos Previamente, desarrollamos un sistema de aislamiento de vesículas extracelulares y posterior análisis de su potencial anti-inflamatorio (in vitro). Este sistema de producción y aislamiento de EVs comprendido por: cultivo controlado de MSCs (de cordón umbilical humano), recolección de medio condicionado y purificación de EVs mediante cromatografía de intercambio iónico. En este estudio, pusimos a punto un modelo murino de regeneración corneal mediante aplicación tópica de 10ul de NaOH (0,125 M) y posterior tratamiento con igual volumen de EVs (1x109 partículas/ml) por 5 días consecutivos. La acción terapéutica de las EVs se evaluó mediante tinción tisular (hematoxilina-eosina) y determinación de los niveles de expresión de citoquinas inflamatorias por real-time RT-PCR. Resultados Tanto en la observación clínica, como en la tisular, se observa que el NaOH indujo injuria moderada del tejido corneal con un claro restablecimiento del tejido luego del tratamiento con vesículas (vs vehículo). Respecto a las citoquinas evaluadas, estas aumentaron significativamente su expresión luego de la injuria química y descendieron fuertemente tras el tratamiento con EVs (n=5, p<0.05). Conclusión De los resultados obtenidos, se desprende, que logramos establecer un modelo sencillo de injuria del epitelio corneal en el cual testear diversas estrategias terapéuticas y, a su vez, demostramos el enorme potencial regenerativo de las EVs en patologías oculares de orden inflamatorio.

New therapeutic options for liver cirrhosis are needed. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising tool for delivering therapeutic factors in regenerative medicine. Our aim is to establish a new therapeutic tool that employs EVs derived from MSCs to deliver therapeutic factors for liver fibrosis. EVs were isolated from supernatants of adipose tissue MSCs, induced-pluripotent-stem-cell-derived MSCs, and umbilical cord perivascular cells (HUCPVC-EVs) by ion exchange chromatography (IEC). To produce engineered EVs, HUCPVCs were transduced with adenoviruses that code for insulin-like growth factor 1 (AdhIGF-I-HUCPVC-EVs) or green fluorescent protein. EVs were characterized by electron microscopy, flow cytometry, ELISA, and proteomic analysis. We evaluated EVs’ antifibrotic effect in thioacetamide-induced liver fibrosis in mice and on hepatic stellate cells in vitro. We found that IEC-isolated HUCPVC-EVs have an analogous phenotype and antifibrotic activity to those isolated by ultracentrifugation. EVs derived from the three MSCs sources showed a similar phenotype and antifibrotic potential. EVs derived from AdhIGF-I-HUCPVC carried IGF-1 and showed a higher therapeutic effect in vitro and in vivo. Remarkably, proteomic analysis revealed that HUCPVC-EVs carry key proteins involved in their antifibrotic process. This scalable MSC-derived EV manufacturing strategy is a promising therapeutic tool for liver fibrosis.

Mechanisms mediating mesenchymal stromal/stem cells’ (MSCs) multipotency are unclear. Although the expression of the pluripotency factor OCT4 has been detected in MSCs, whether it has a functional role in adult stem cells is still controversial. We hypothesized that a physiological expression level of OCT4 is important to regulate MSCs’ multipotency and trigger differentiation in response to environmental signals. Here, we specifically suppressed OCT4 in MSCs by using siRNA technology before directed differentiation. OCT4 expression levels were reduced by 82% in siOCT4-MSCs, compared with controls. Interestingly, siOCT4-MSCs also presented a hypermethylated OCT4 promoter. OCT4 silencing significantly impaired the ability of MSCs to differentiate into osteoblasts. Histologic and macroscopic analysis showed a lower degree of mineralization in siOCT4-MSCs than in controls. Moreover, OCT4 silencing prevented the up-regulation of osteoblast lineage-associated genes during differentiation. Similarly, OCT4 silencing resulted in decreased MSC differentiation potential towards the adipogenic lineage. The accumulation of lipids was reduced 3.0-fold in siOCT4-MSCs, compared with controls. The up-regulation of genes engaged in the early stages of adipogenesis was also suppressed in siOCT4-MSCs. Our findings provide evidence of a functional role for OCT4 in MSCs and indicate that a basal expression of this transcription factor is essential for their multipotent capacity.

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment.