Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

Abstract

Introduction: Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species.

Objectives: We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes.

Methods: Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed.

Results: Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells.

Conclusions: We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

Figure 1

(a) Adipose tissue mesenchymal cells, (b) fibroblasts, and (c) umbilical cord tissue mesenchymal cells, 200x. After multilineage differentiation, it is possible to observe (a i, b i, and c i) adipogenic differentiated cells characterized by Sudan black-stained lipid vacuole accumulation, 200x; (a ii, b ii, and c ii) osteogenic differentiated cells characterized by calcium deposition, which were stained with alizarin red, 100x; and (a iii, b iii, and c iii) chondrogenic differentiated cells characterized by chondrogenic pellet development, which were stained with Alcian blue, 200x. Negative control cells maintained the typical spindle-like shape and differed from the treated cells.

Figure 2

Equine iPSCs on day 18 after transduction from (a) adipose tissue mesenchymal cells, (b) fibroblasts, and (c) umbilical cord tissue mesenchymal cells, 200x. Alkaline phosphatase-positive equine iPSC colonies were induced from each cell type: (a) adipose tissue mesenchymal cells, 100x; (b) fibroblasts, 200x; (c) umbilical cord tissue mesenchymal cells, 100x. In addition, images present the immunocytochemistry expression of merged OCT4, OCT4/FITC, and Hoechst staining: (a) adipose tissue mesenchymal cells, 100x; (b) fibroblasts, 200x; (c) umbilical cord tissue mesenchymal cells, 100x. (d) Transcript expression of GAPDH, NANOG, and OCT4 in equine adipose tissue mesenchymal cells, umbilical cord tissue mesenchymal cells, and fibroblasts before and after pluripotency induction. NANOG and OCT4 expression levels are enhanced after cell reprogramming. Also in (d), confirmation of GAPDH and STEMCCA expression in equine iPSCs by conventional PCR. (e) Graph showing the total production of eiPSC colonies using hOSKM. No colonies were formed from the bone marrow mesenchymal cells. Different letters indicate significantly different results (P < 0.05).

Figure 3

Four-day-old EBs produced from equine iPSCs derived from (a) adipose tissue mesenchymal cells, (b) fibroblasts, and (c) umbilical cord tissue mesenchymal cells. After the spontaneous differentiation of embryoid bodies into multiple lineages, the cells presented a more elongated morphology and were immunocytochemically positive for neurofilament. Merged images of neurofilament/FITC and Hoechst staining: (a) eiPSCs-eADmsc-derived cells, (b) eiPSCs-eFibros-derived cells, and (c) eiPSCs-UCmsc-derived cells, 200x.

Figure 4

(a) PCA analysis of all 110 miRNAs commonly detected in eiPSCs derived from adipose tissue mesenchymal cells, umbilical cord tissue mesenchymal cells, fibroblasts, and control cells from each of these groups, suggesting slight clustering and difference between the nonreprogrammed and reprogrammed cells. The more clustered and segregated the groups and points, the greater the similarities among the groups. (b) Venn diagram showing distribution of the miRNAs analyzed on the three cell groups and the presence of the 110 miRNAs commonly detected in all of them. (c) Expression levels of miRNA differently expressed between eiPSCs-eADmsc and eADmsc cells (P < 0.05) and Venn diagram of miRNAs exclusively expressed in eADmsc and eiPSCs-eADmsc. (d) Expression levels of miRNAs differently expressed between eiPSCs-eFibros and eFibros cells (P < 0.05) and Venn diagram of miRNAs exclusively expressed in eFibros and eiPSCs-eFibros. (e) Expression levels of miRNAs differently expressed between eiPSCs-eUCmsc and eUCmsc cells (P < 0.05) and Venn diagram of miRNAs exclusively expressed in eUCmsc and eiPSCs-eUCmsc.