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Home / Equine Research Bank / PLoS One / MicroRNA characterization in equine induced pluripotent stem...
PloS one2018; 13(12); e0207074; doi: 10.1371/journal.pone.0207074

MicroRNA characterization in equine induced pluripotent stem cells.

Abstract: Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. We aim to generate horse iPSCs, characterize them and evaluate the expression of different microRNAs (miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b and miR-296). Two equine iPSC lines (L2 and L3) were characterized after the reprogramming of equine fibroblasts with the four human Yamanaka's factors (OCT-4/SOX-2/c-MYC/KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2 and c-MYC. In vitro differentiation to embryo bodies (EBs) showed the capacity of the iPSCs to differentiate into ectodermal, endodermal and mesodermal phenotypes. MicroRNA analyses resulted in higher expression of the miR-302 family, miR-9 and miR-96 in L2 and L3 vs. fibroblasts (p<0.05), as previously shown in human pluripotent cells. Moreover, downregulation of miR-145 and miR-205 was observed. After differentiation to EBs, higher expression of miR-96 was observed in the EBs respect to the iPSCs, and also the expression of miR-205 was induced but only in the EB-L2. In addition, in silico alignments of the equine microRNAs with mRNA targets suggested the ability of miR-302 family to regulate cell cycle and epithelial mesenchymal transition genes, miR-9 and miR-96 to regulate neural determinant genes and miR-145 to regulate pluripotent genes, similarly as in humans. In conclusion, we could obtain equine iPSCs, characterize them and determine for the first time the expression level of microRNAs in equine pluripotent cells.
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Publication Date: 2018-12-03 PubMed ID: 30507934PubMed Central: PMC6277106DOI: 10.1371/journal.pone.0207074Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article is centered around the characterization of microRNAs in equine (horse) induced pluripotent stem cells, aimed at understanding their role in pluripotency maintenance and cell differentiation, generating and characterizing horse iPSCs and gauging the expression of specific microRNAs.

Objective of the Study

  • The main goal of this study was to investigate, generate, and characterize induced pluripotent stem cells (iPSCs) in horses, and analyze specific microRNA expression in these cells. The microRNAs studied were miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b, and miR-296.

Methodology

  • The scientists reprogrammed equine fibroblasts using four human Yamanaka’s factors (OCT-4/SOX-2/c-MYC/KLF4).
  • Two different equine iPSC lines (L2 and L3) were created and characterised. This was done by evaluating the phosphatase alkaline activity and the expression levels of OCT-4, NANOG and REX1 via RT-PCR as well as by immunofluorescence of OCT-4, SOX-2 and c-MYC.
  • In vitro differentiation was conducted to form embryo bodies (EBs), which evaluated the iPSCs’ capacity to differentiate into ectodermal, endodermal and mesodermal phenotypes.

Findings

  • MicroRNA analysis showed higher expression levels of the miR-302 family, miR-9 and miR-96 in the L2 and L3 lines in comparison to fibroblasts. This corresponds with findings in human pluripotent cells.
  • A decrease in the expression level of miR-145 and miR-205 was observed.
  • The EBs exhibited higher miR-96 expression compared to iPSCs. MiR-205 expression was induced but only in the EB-L2 line.
  • In silico (computer simulated) alignments of the equine microRNAs with mRNA targets suggested the miR-302 family’s ability to regulate cell cycle and epithelial mesenchymal transition genes; miR-9 and miR-96’s ability to regulate neural determinant genes; and miR-145’s role in regulating pluripotent genes, a behaviour similar to humans.

Conclusion

  • This study accomplished the generation and characterization of equine iPSCs, and elucidated for the first time, the expression level of microRNAs in equine pluripotent cells.

Cite This Article

APA
Moro LN, Amin G, Furmento V, Waisman A, Garate X, Neiman G, La Greca A, Santín Velazque NL, Luzzani C, Sevlever GE, Vichera G, Miriuka SG. (2018). MicroRNA characterization in equine induced pluripotent stem cells. PLoS One, 13(12), e0207074. https://doi.org/10.1371/journal.pone.0207074

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 13
Issue: 12
Pages: e0207074
PII: e0207074

Researcher Affiliations

Moro, Lucia Natalia
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Amin, Guadalupe
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Furmento, Veronica
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Waisman, Ariel
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Garate, Ximena
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Neiman, Gabriel
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
La Greca, Alejandro
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Santín Velazque, Natalia Lucia
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Luzzani, Carlos
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Sevlever, Gustavo E
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
Vichera, Gabriel
  • Kheiron Biotech, Pilar, Buenos Aires, Argentina.
Miriuka, Santiago Gabriel
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.

MeSH Terms

  • Animals
  • Cell Differentiation / genetics
  • Fibroblasts / cytology
  • Gene Expression Profiling
  • Horses
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism
  • Kruppel-Like Factor 4
  • MicroRNAs / genetics
  • Nuclear Transfer Techniques

Conflict of Interest Statement

We have the following interests: Author Gabriel Vichera is employed by Kheiron Biotech. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

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