Exosome communication in the transdifferentiation of stem cells in co-culture with neuronal cells

Author(s)

Kelly Cristine Santos Roballo

• Document type: Doctoral Thesis
• Press: Pirassununga.
• Institution: Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT)
• Defense date: 2017-09-22

Examining board members

Carlos Eduardo Ambrosio; Fabiana Fernandes Bressan; Deise Carla Almeida Leite Dellova; Simone Kashima Haddad; Moyses dos Santos Miranda; Paulo Sérgio Monzani

• Advisor: Carlos Eduardo Ambrosio; Juliano Coelho da Silveira

Abstract

Neuronal cells co-cultured with stem cells derived from adipose tissue (ADSC) can induce the latter to neuronal transdifferentiation. However, the cellular communication processes involved in this induction, and the functionality of the transdifferentiated ADSC in vivo, are unknown. Recently, a new type of cellular communication measured by extracellular vesicles was indicated in the modulation of different cellular events like differentiation. Therefore, the hypothesis in this proposal was to identify if the process of cellular differentiation is mediated by extracellular vesicles and if the differentiated cells are able to act in the regeneration of tissues in the lesions of the peripheral nervous system. For this, the research was divided in two phases: the first one consisted of the in vitro process, with the objective of observing the transition process of the ADSC to the neuronal lineage and analyzing the cellular communication function in the differentiation process; the second consisted in the in vivo evaluation of the possible functionality of the differentiated ADSCs. Murine was the animal model used (C57BL/6 and FVB). ADSCs and neuronal cells were isolated, cultured in primary culture and co-cultured for three, seven and 14 days. To confirm the phenotypic changes of ADSC, immunolocalization with beta tubulin III and SNAP25 and real-time PCR (RT-qPCR) of the Map2 and Snap25 genes was performed, followed by analysis of genes related to neurogenesis. In addition, extracellular vesicles were isolated and used for in vitro differentiation and gene and functional analysis. As a result, it has been found that ADSCs in co-culture with neurons can differentiate into neuronal-like. The communication by extracellular vesicles between neurons and ADSCs was verified, and the extracellular vesicles were correlated in the differentiation process by the transport of the protein SNAP25. After these results, the second phase of this work was continued, the in vivo step, which focused on the use of the co-cultivated ADSCs for seven days and functional local and systemic evaluation in the process of sciatic nerve regeneration after neurotmese. As a result of this step, the co-cultured stem cells modulated the lesion and provided an improvement in functionality after injury. It is concluded that ADSCs can transdifferentiate neuronal lines in co-culture with neurons, the extracellular vesicles play a certain role in this process and the transdifferentiated ADSC may be an alternative to aid in the regeneration of peripheral nerves. (AU)

• FAPESP's process: 15/17897-9 - Exosomal communication in the stem cells transdifferentiation in co-culture with neuronal cells
• Grantee: Kelly Cristine Santos Roballo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)