Estratégias moleculares para a caracterização e diferenciação entre células-tronco derivadas de tecido adiposo e fibroblastos dérmicos humanos

Abstract

Human adipose-derived stem cells (hASC) are undifferentiated and self-renewing cells that have the potential to differentiate into several cell lines. Due to these particularities, these cells are considered promising in regenerative medicine application. One concern about the therapeutic application of stem cells is the risk of contamination with dermal fibroblasts, which can lead to some impairment to cell therapy. Thus, it is imperative to develop new strategies that allow the differentiation between these two cell types. In the present study, which consists of three chapters, we used molecular strategies to characterize and distinguish between hASC and human dermal fibroblasts in vitro. Cells were isolated by mechanical and enzymatic dissociation from the liposuction and abdominoplasty samples, respectively. In the first chapter, immunophenotyping analyzes performed by flow cytometry showed similar markers between the two cells. Differently, growth-curve assays, induction of adipogenic and osteogenic differentiation for 21 days with specific inducing media and alkaline phosphatase activity using BCIP-NBT were shown to be promising in the phenotypic differentiation between these two cells. In the second chapter, Cell-SELEX (System-Based Evolution of Ligands by Exponential Cell-Based Enrichment), DNA melting and bioinformatics analyzes were used to select two DNA aptamers with higher efficiency and affinity for hASC binding compared to human dermal fibroblasts. Also, real-time quantitative PCR technique proved to be robust and efficient for the validation of aptamers with high affinity for target cells. In the third chapter, three unpaired samples of hASC and human fibroblasts were sequenced by RNA-Seq using the Illumina platform. Bioinformatics analyzes contributed to the identification of more than 2,000 differentially expressed genes (GDEs) between the two cells, in addition to enhancing knowledge about functions and enriched pathways (GO and KEGG) related to distinct biological and molecular processes in hASC and fibroblasts. In this chapter, 16 genes present in the list of GDEs were also validated by RT-qPCR with paired samples. Therefore, this study contributed to the expansion of scientific knowledge and the development of important tools not only for the distinction, but also for better understanding of both cells’ biology.