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

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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.

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Acknowledgements

The authors kindly acknowledge GenXPro GmbH (Frankfurt am Main, Germany) for their assistance in aptamers sequencing. The authors also thank FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for funding.

Funding

This study was funded by FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

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Author notes

  1. Mariane Izabella Abreu de Melo & Pricila da Silva Cunha

    Present address: Centro de Desenvolvimento da Tecnologia Nuclear, Comissão Nacional de Energia Nuclear, Av. Antônio Carlos, 6627, Belo Horizonte, 31270-901, Brazil

Authors and Affiliations

  1. Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901, Brazil

    Mariane Izabella Abreu de Melo, Pricila da Silva Cunha, Marcelo Coutinho de Miranda, Joana Lobato Barbosa, Alfredo Miranda de Goes & Dawidson Assis Gomes

  2. Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus, 69080-900, Brazil

    Jerusa Araújo Quintão Arantes Faria

  3. Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901, Brazil

    Michele Angela Rodrigues

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  1. Mariane Izabella Abreu de Melo
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Contributions

All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by Mariane Izabella Abreu de Melo, Pricila da Silva Cunha, Marcelo Coutinho de Miranda, Joana Lobato Barbosa, Jerusa Araújo Quintão Arantes Faria, and Dawidson Assis Gomes. Funding acquisition, resources, and supervision were provided by Michele Angela Rodrigues, Pricila da Silva Cunha, Alfredo Miranda de Goes, and Dawidson Assis Gomes. Mariane Izabella Abreu de Melo wrote the first draft of the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Mariane Izabella Abreu de Melo.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Research Ethics Committee of the Universidade Federal de Minas Gerais (No. 02508018.1.0000.5149).

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The original version of this article unfortunately contained a mistake in the supplementary files and figure 1. The supplementary files and figure 1 has been corrected.

Supplementary Information

Fig. S1

Determination of the optimal number of PCR cycles in the positive selection. The figure shows electrophoresis images of the amplicons produced by PCR in the positive selection cycle tests. Agarose gel (2%) was stained with ethidium bromide. The order in the gel is: (M): Step-Ladder marker 50 bp, (12) to (20): amplicons produced from 12,14,16, 18 and 20 cycles of the PCR reaction, respectively, (NTC):no-template control. Bp= base pair. (A) 3rd round, (B) 4th round, (C) 5th round, (D) 6th round, (E) 7th round and (F) 8th round (PNG 24663 kb)

High resolution image (TIF 1.41 mb)

Fig. S2

Determination of the optimal number of PCR cycles in the negative selection. The figure shows electrophoresis images of the amplicons produced by PCR in the negative selection cycle tests. Agarose gel (2%) was stained with ethidium bromide. The order in the gel is: (M): Step-Ladder marker 50 bp, (16) to (22): amplicons produced from 16, 18, 20 and 22 cycles of the PCR reaction, respectively, (NTC):no-template control. Bp= base pair. (A) 3rd round, (B) 4th round, (C) 5th round, (D) 6th round, (E) 7th round and (F) 8th round. (PNG 24663 kb)

High resolution image (TIF 1.29 mb)

Fig. S3

Melting profiles of aptamers in all selection rounds. (a) Melt curve of aptamers from the library and round 1 (b) Melt curve of aptamers from rounds 1 and 2. (c) Melt curve of aptamers from rounds 2 and 3. (d) Melt curve of aptamers from rounds 3 and 4. (e) Melt curve of aptamers from rounds 4 and 5. (f) Melt curve of aptamers from rounds 5 and 6. (g) Melt curve of aptamers from rounds 6 and 7. (h) Melt curve of aptamers from rounds 7 and 8. (i) Melt curve from the library and round 8. X-axis: temperature (°C); Y-axis: Derivative reporter (-Rn); NTC (no-template control). The graphs of fluorescence intensity versus temperature were generated on the 7.500 Software v. 2.0.6 (Applied Biosystems) (PNG 11591 kb)

High resolution image (TIF 6.30 mb)

Fig. S4

Apta 99 shows a higher binding capacity to ASC compared to FIB. Total internal reflection microscopy (TIRF) was used to investigate the ability of the aptamers to bind at the regions of the plasma membrane of the cells. ASC and FIB were incubated for 30 min with 250 nm of aptamers at 37°C. Next, cells were washed and observed using a Leica TIRF infinity high power microscope (Leica Microsystems, Germany) with a 100x objective, N.A. 1.47. (a) The representative images show that the fluorescence of Apta 99 was higher in ASC compared to FIB. (b) The average quantification of the fluorescence intensity of the TIRF images confirms that Apta 99 can bind more specifically in ASC (429,7 ± 69,84) than in FIB (118 ± 3,786). (c) The representative images show that the fluorescence of Apta 21 was similar between ASC and FIB. (d) The average quantification of the fluorescence intensity of the TIRF images does not show any difference between ASC (314,7 ± 142,6) and FIB (371 ± 82,87). Unpaired t-test with Welch’s correction; n=3; * p<0.05. Scale bar = 10 μm (PNG 23212 kb)

High resolution image (TIF 2.40 mb)

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de Melo, M.I.A., da Silva Cunha, P., de Miranda, M.C. et al. Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts. Appl Biochem Biotechnol 193, 3704–3718 (2021). https://doi.org/10.1007/s12010-021-03618-5

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