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Animals : an open access journal from MDPI2022; 12(16); 2049; doi: 10.3390/ani12162049

Comparison of Sources and Methods for the Isolation of Equine Adipose Tissue-Derived Stromal/Stem Cells and Preliminary Results on Their Reaction to Incubation with 5-Azacytidine.

Abstract: Physiological particularities of the equine heart justify the development of an in vitro model suitable for investigations of the species-specific equine cardiac electrophysiology. Adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point from which to develop such a cardiomyocyte (CM)-like cell model. Therefore, we compared abdominal, retrobulbar, and subcutaneous adipose tissue as sources for the isolation of ASCs applying two isolation methods: the collagenase digestion and direct explant culture. Abdominal adipose tissue was most suitable for the isolation of ASCs and both isolation methods resulted in comparable yields of CD45-/CD34-negative cells expressing the mesenchymal stem cell markers CD29, CD44, and CD90, as well as pluripotency markers, as determined by flow cytometry and real-time quantitative PCR. However, exposure of equine ASCs to 5-azacytidine (5-AZA), reportedly inducing CM differentiation from rats, rabbits, and human ASCs, was not successful in our study. More precisely, neither the early differentiation markers and , nor the late CM differentiation markers , and were upregulated in equine ASCs exposed to 10 µM 5-AZA for 48 h. Hence, further work focusing on the optimal conditions for CM differentiation of equine stem cells derived from adipose tissue, as well as possibly from other origins, are needed.
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Publication Date: 2022-08-11 PubMed ID: 36009640PubMed Central: PMC9404420DOI: 10.3390/ani12162049Google Scholar: Lookup
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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 paper outlines a study investigating the most effective sources and methods for obtaining stem cells from horse adipose tissue and tests their potential transformation into cardiomyocyte-like cells using 5-azacytidine as an accelerator. This is with the aim to create an in vitro model to study equine heart electrophysiology.

Objective and Methods

The main objectives of the research were to compare different adipose tissues (abdominal, retrobulbar, and subcutaneous) as sources for isolating stem cells, and to compare two isolation methods: collagenase digestion and direct explant culture. Further, the researchers aimed to test the effect of 5-Azacytidine (5-AZA) on inducing stem cells’ differentiation into cardiomyocyte-like (CM) cells, a process already established in rats, rabbits, and humans.

  • The researchers took adipose tissue samples from the three specified areas and applied two different methods of isolation.
  • The isolated stem cells were then subjected to flow cytometry and real-time quantitative PCR to identify the expression of stem cell markers such as CD29, CD44, and CD90, and pluripotency markers.
  • Isolated stem cells were then exposed to a compound called 5-AZA, a molecule known to induce CM differentiation in other species.

Findings and Conclusion

The research concluded that out of the three sources of adipose tissue, the abdominal adipose tissue provided the most promising samples for isolating stem cells. The two isolation methods being compared yielded comparable results in terms of the number of “CD45-/CD34-negative cells” that expressed the stem cell and pluripotency markers.

  • The success of abdominal adipose tissue in yielding ASCs implies that it might be the preferable site for obtaining horse-derived stem cells in future research.
  • Results indicated no significant difference between the collagenase digestion method and the direct explant culture method, making either applicable for future research.”
  • However, treating equine ASCs with 5-AZA did not successfully induce differentiation into CM cells. Notably, both the early and late differentiation markers were not upregulated after exposure to 5-AZA.

Overall, although the study found successes in ASCs isolation, it showed negative results for 5-AZA’s differentiation-inducing properties in equine ASCs. Therefore, the researchers suggest that future work should focus on identifying optimal conditions for facilitating CM differentiation in horse-derived stem cells, possibly exploring sources beyond adipose tissue.

Cite This Article

APA
Trachsel DS, Stage HJ, Rausch S, Trappe S, Söllig K, Sponder G, Merle R, Aschenbach JR, Gehlen H. (2022). Comparison of Sources and Methods for the Isolation of Equine Adipose Tissue-Derived Stromal/Stem Cells and Preliminary Results on Their Reaction to Incubation with 5-Azacytidine. Animals (Basel), 12(16), 2049. https://doi.org/10.3390/ani12162049

Publication

Animals : an open access journal from MDPI
ISSN: 2076-2615
NlmUniqueID: 101635614
Country: Switzerland
Language: English
Volume: 12
Issue: 16
PII: 2049

Researcher Affiliations

Trachsel, Dagmar S
  • Equine Clinic: Surgery and Radiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
  • Clinical Unit of Equine Internal Medicine, Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria.
Stage, Hannah J
  • Equine Clinic: Surgery and Radiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Rausch, Sebastian
  • Institute of Immunology, Department of Veterinary Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7, 14163 Berlin, Germany.
Trappe, Susanne
  • Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Söllig, Katharina
  • Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Sponder, Gerhard
  • Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Merle, Roswitha
  • Institute for Veterinary Epidemiology and Biostatistics, Department of Veterinary Medicine, Freie Universität Berlin, Königsweg 67, 14163 Berlin, Germany.
Aschenbach, Jörg R
  • Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Gehlen, Heidrun
  • Equine Clinic: Surgery and Radiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.

Grant Funding

  • grant Number 071045 / scholarship of the Elsa-Neumann foundation
  • n/a / Internal Research Funding, Research Committee of the Freie Universitu00e4t Berlin

Conflict of Interest Statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Citations

This article has been cited 1 times.
  1. Stage HJ, Trappe S, Söllig K, Trachsel DS, Kirsch K, Zieger C, Merle R, Aschenbach JR, Gehlen H. Multilineage Differentiation Potential of Equine Adipose-Derived Stromal/Stem Cells from Different Sources.. Animals (Basel) 2023 Apr 15;13(8).
    doi: 10.3390/ani13081352pubmed: 37106915google scholar: lookup
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