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Immortalization of Mesenchymal Stromal Cells by TERT Affects Adenosine Metabolism and Impairs their Immunosuppressive Capacity

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Abstract

Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.

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Acknowledgements

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001; All students are recipients of fellowships from CAPES. MRW, GL and RPC are recipients of research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brasil (CNPq). This study was supported by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - Brasil (FAPERGS/CAPES 06/2018 - Programa de Internacionalização da pós-graduação no RS (19/2551-0000679-9) and FAPERGS/MS/CNPq/SESRS n.03/2017 – PPSUS (17/2551-0001)); and CNPqMS-SCTIE-Decit/CNPqn°12/2018(441575/2018-8). JS received support from the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2016-05867).

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Authors and Affiliations

  1. Laboratory of Cell Biology, Universidade Federal de Ciências da Saúde de Porto Alegre- UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS, CEP 90050-170, Brazil

    L. R. Beckenkamp, D. M. S. da Fontoura, V. G. Korb, I. C. Iser, A. P. S. Bertoni & Márcia Rosângela Wink

  2. Department of Biophysics and Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil

    R. P. de Campos, G. R. Onzi & G. Lenz

  3. Département de Microbiologie-Infectiologie et d’Immunologie, Faculté de Médecine, Université Laval, Québec city, QC, G1V 0A6, Canada

    J. Sévigny

  4. Centre de recherche du CHU de Québec, Université Laval, Québec city, QC, G1V 4G2, Canada

    J. Sévigny

Authors
  1. L. R. Beckenkamp
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  2. D. M. S. da Fontoura
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  3. V. G. Korb
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  4. R. P. de Campos
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  6. I. C. Iser
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  7. A. P. S. Bertoni
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  8. J. Sévigny
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  9. G. Lenz
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  10. Márcia Rosângela Wink
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Contributions

LRB performed cell culture experiments, HPLC assays, flow cytometry, immunosuppression assay and wrote the manuscript. GRO assisted the stable transfection cell assays and wrote the manuscript. DMF and VGK performed cell culture experiments, enzymatic activity and cell differentiation assay, flow cytometry and doubling population experiments. RPC performed the stable transfection of cells. ICI performed the ADA enzymatic assay. APSB performed RT-qPCR and HPLC assays. JS contributed to the interpretation of the results and provided the NTPDases antibodies. MRW and GL supervised the experiments, assisted in drafting and critical reading. All the authors discussed the results and contributed to the writing of the manuscript.

Corresponding author

Correspondence to Márcia Rosângela Wink.

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Supplementary Figure 1

– CD73 activity and expression in a new cell transduction with TERT. To confirm that cell immortalization process alters the activity and expression of CD73 enzyme, a new stable transfection with the TERT gene was performed. (A) The TERT gene insertion was confirmed by RT-qPCR, as demonstrated by melt curve peak chart collected using the StepOnePlusTM (Applied Biosystems). (B) Flow cytometry data analysis also confirmed a decrease of CD73 MFI in MSCs-TERT (n = 3). (C) Specific enzymatic activity from MSCs and MSCs-TERT measured by release of inorganic phosphate after incubation with AMP (n = 3). Data are expressed as nmol Pi/min/mL, using mean ± SD. T-test was used to determine the statistical difference. (PNG 285 kb)

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Beckenkamp, L.R., da Fontoura, D.M.S., Korb, V.G. et al. Immortalization of Mesenchymal Stromal Cells by TERT Affects Adenosine Metabolism and Impairs their Immunosuppressive Capacity. Stem Cell Rev and Rep 16, 776–791 (2020). https://doi.org/10.1007/s12015-020-09986-5

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