Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts.

Mariane Izabella Abreu De Melo,Michele Angela Rodrigues,Dawidson Assis Gomes,Marcelo Coutinho De Miranda,Joana Lobato Barbosa,Pricila Da Silva Cunha,Jerusa Araújo Quintão Arantes Faria,Alfredo Miranda De Goes

doi:10.1007/s12010-021-03618-5

Abstract

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.

Highlights

Nucleic acid aptamers can be defined as single-stranded DNA or RNA oligonucleotides, generally smaller than 100-mer, capable of binding to a wide variety of target molecules with high affinity and specificity [1]
We developed aptamers capable of recognize mesenchymal stem cells derived from adipose tissue (ASC) using the Cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique
Once the target molecules are anchored to the cell surface, the bound aptamers can be separated from unbound oligonucleotides by washing and centrifugation during the Cell-SELEX process

Summary

Introduction

Nucleic acid aptamers can be defined as single-stranded DNA or RNA oligonucleotides, generally smaller than 100-mer, capable of binding to a wide variety of target molecules with high affinity and specificity [1]. Once the target molecules are anchored to the cell surface, the bound aptamers can be separated from unbound oligonucleotides by washing and centrifugation during the Cell-SELEX process. This method supports the development of in vitro probes and the discovery of cell biomarkers [1, 4]. Adipose tissue is an accessible source that provides higher yields of stem cells than bone-marrow [5] These human adipose-derived stem cells (ASC) are promising self-renewing and multipotent mesenchymal stem cells. In the therapeutic application of stem cells, there is a high risk of contamination with fibroblasts (FIB), which can lead to a series of losses for the therapy, such as the decrease in the stem cell differentiation capacity and the induction of senescence [7,8,9]

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