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 GATA4 and NKX2-5, nor the late CM differentiation markers TNNI3, MYH6, and MYH7 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.

Multilineage Differentiation Potential of Equine Adipose-Derived Stromal/Stem Cells from Different Sources

“…In the present study, however, not only induced cells were affected by this downregulation, but also NC, so that cardiomyogenic differentiation activity was excluded. That this downregulation can be associated with the occurrence of senescence in cells has already been discussed in detail in Trachsel et al [59]. While some studies demonstrated that BMPs possess a cardiomyogenesis-promoting effect [106,111,112], other research groups showed that transient inhibition of BMPs is necessary for in vitro cardiomyogenesis [25,113].…”

“…Sample collection and isolation of equine ASCs have already been published in our previous study [ 59 ]. In brief, equine abd, rb and sc adipose tissue from n = 16 horses (mares, geldings, stallions; median age [interquartile range]: 13.5 [18.0] years) was harvested after euthanasia at the Equine Clinic: Surgery and Radiology, Department of Veterinary Medicine, Freie Universität (FU) Berlin or slaughter in commercial slaughterhouses up to a maximum of 8 h post mortem.…”

“…ASCs were harvested from abd, rb and sc adipose tissue using EXP and SVF techniques as described in Trachsel et al [ 59 ]. Briefly, cell harvest with the SVF method relied on classical collagenase digestion.…”

“…The medium was changed every two to three days. In the case of adipogenic and osteogenic differentiation, cell culture vessels were coated with 2 µg/cm 2 murine laminin (Sigma Aldrich, Taufkirchen, Germany) in order to avoid cell detachment as far as possible, which was observed in Trachsel et al due to the absence of any coating [ 59 ]. To ensure reproducibility, experiments were carried out in duplicate.…”

“…It was hypothesized that equine MSCs behave equally, regardless of the isolation method, and differ from each other depending on the tissue source used. Furthermore, based on the unsuccessful induction of cardiomyogenic differentiation using 5-azacytidine (5-AZA) in our previous study [ 59 ], a different protocol for cardiomyogenic differentiation relying on BMP, activin (Act) A and Dickkopf (DKK)-1 was tested in the present study on equine ASCs.…”