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.

Keywords: adipose tissue differentiation; cardiomyocyte-like cells; horse; mesenchymal stem cells; preadipocytes.

Figure 1

Timeline showing the protocol for the induction experiment with 5-azacytidine (5-AZA). E-M, expansion medium; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle Medium.

Figure 2

Isolation success of adipose tissue-derived stem/stromal cells (ASCs) harvested by explant culture (ASCs-_EXP, red) and collagenase digestion (ASCs-_SVF, black) from abdominal (abd), retrobulbar (rb), and subcutaneous (sc) adipose tissue based on the isolation success scoring.

Figure 3

Characterization of equine abdominal adipose tissue-derived stem/stromal cells (ASCs) by flow cytometry. (A) Surface marker expression by ASCs-_EXP and ASCs-_SVF. Representative overlay plots including fully stained cells (target, blue) and isotype controls (black) are shown. (B) Marker expression by ASCs generated with the two protocols. Data pooled from two (ASCs-_SVF) and four (ASCs-_EXP) independent experiments, each performed with cells from one to three individual donors. (C) Equine peripheral blood mononuclear cells (PBMC, n = 5) were used as positive controls to survey the binding of the antibodies applied for the characterization of equine stem cells. Representative overlay plots report fluorescence signals detected with fully stained cells (target, red) and isotype controls (black). The CD90 was stained in all samples. Numbers indicate the percentage of cells expressing the target molecules. (D) Marker expression by PBMC isolated from five donors. Gating strategies are reported in Supplementary Figure S1. FSC, forward scatter; SSC, side scatter.

Figure 4

Morphology of abdominal adipose tissue-derived stem/stromal cells (ASCs) obtained by collagenase digestion before (day 0) and after (day 7, 14 and 21) exposure to 5-azacytidine (5-AZA) (exemplary for n = 2 donors). The cells of horse 1 exhibited the typical fibroblast-like morphology and moderate “dome” formation. Cell detachment was seen in horse 2 after two to three weeks as well as atypical small cells indicative of this. No differences between the induced and negative control cells were visible in either horse. Scale bar: 0.1 mm.

Figure 5

Results of the SYBR Green real-time quantitative PCR for the pluripotency, cardiac, and myogenic markers expressed in equine adipose-derived stem/stromal cells (ASCs) after exposure to 5-azacytidine (5-AZA) for 48 h. Gene expression at T0 (nonexposed) and T3 (exposed T3_ind, induced and T3_nc, negative control) as well as positive (p, red color symbols) and negative control (nc as NRT and H₂O controls, blue color symbols) are shown. Dot plots represent Ct mean values with standard deviation from two experimental runs (n = 5 donors). One-way ANOVA (post hoc tests: Hochberg, Tukey’s HSD) was used for the statistical analysis. Statistical significance was set at p < 0.05.