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Thrombospondin-1 protein silencing in adipose stem cell spheroids
Osteoarthritis (OA) is caused by mechanical and biological events that destabilize the joint tissues homeostasis. This pathology is commonly associated with loss of joint cartilage, synovial inflammation, and the formation of osteophytes. Furthermore, angiogenesis has been described as main event that leads to these effects. Aiming to bridge the gap in joint cartilage repair, strategies that target events of angiogenesis has emerged as OA therapeutics approach. Our research group has performed the secretome of adipose stem cells spheroids induced to the chondrogenesis and identified several molecules related to skeletal development, osteogenesis, chondrogenesis and anti-angiogenesis, such as the protein Thrombospondin-1. This protein has been described as having anti-angiogenic and anti-hypertrophic properties.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
Therefore, our objective is to support the importance of TSP-1 in chondrogenesis from ASC spheroids.
Human ASCs were obtained from subcutaneous lipoaspirate samples, from healthy donors (18 to 55 years old), according to the Research Ethics Committee of the Clementino Fraga Filho University Hospital (UFRJ-protocol 146/09). After ASCs isolation, the monolayer culture was maintained on 24 well plates, at 37ºC with 5% CO2 and 21% of O2. Immunohistochemistry (IHC) for anti-TSP-1 and Western blot analysis in the culture supernatant was performed in ASC spheroids (i.e. 3D culture system). The silencing standardization was made in the kinetics of 18,48,36 and 72 h, using the kit HiPerfect Transfection reagent (Qiagen). The quantification of gene expression was performed with Real-Time PCR System (7500-Applied Biosystems). The ASCs viability and morphology - after mRNA silencing- was examined using phase contrast microscopy and the kit LIVE/DEAD (Thermo Fisher).
Western Blot analysis from the culture supernatant of induced ASC spheroids revealed a high intensity of bands correlated with TSP-1, corroborating the secretome analysis. The IHC attested the presence of TSP-1 in induced ASC spheroids. The folding change for the kinetics of 48 h showed a significant (p=0,00394704) decrease for ASC monolayer that underwent silencing, compared with not silenced ASC . ASC morphology and viability was not altered by the TSP-1 silencing.
Considerações Finais/Final considerations/Consideraciones finales
Preliminary results showed an efficient silencing of the TSP-1 protein in ASC monolayer. TSP-1 silencing is being performed in ASC spheroids.
Palavras-chave/Key words/Palabras clave
Protein silencing; Thrombospondin-1 (TSP-1); Adipose derived stem cells (ASC); Chondrogenesis; Spheroids.
LETICIA EMILIANO CHARELLI, Renata Akemi Matsui, Leandra Santos Baptista