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.

Fig 1. Pluripotent characterization of two horse iPSC lines, L2 and L3.

a) A representative colony of equine iPSCs 2 weeks after reprogramming. b) Alkaline phosphatase activity analysis of the L2 iPSC line, I) A 60 mm petri dish full of iPSC colonies observed in pink; II) Two iPSC colonies with high alkaline phosphatase activity; III) Horse fibroblasts negative for alkaline phosphatase activity. c) Inmunostaining of both iPSC lines and horse fibroblasts with the pluripotent markers OCT-4, SOX-2 and c-MYC. In blue nucleous are stained with DAPI d) RT-qPCR analysis comparing the expression of OCT-4, NANOG and REX-1 among the original fibroblasts and the L2 and L3 iPSC lines. Results are presented as means ± SEM (n = 3). Data were relativized to fibroblasts. Different letters indicate significant differences (p<0.05).

Fig 2. End point PCR analysis of iPSCs.

a) the pluripotent genes OCT-4, NANOG and REX1 in fibroblasts (Fib) and both iPSCs lines L2 and L3; and b) representative genes from the three germ layers in L2 and L3 iPSCs, and embryo bodies (EB), derived from these both iPSCs lines (EB-L2 and EB-L3, respectively). RPL7 was used as the housekeeping gene.

Fig 3. In vitro differentiation of L2 and L3 iPSCs to embryo bodies (EB).

a) Generation of EBs, I) EBs in suspension for 1 week in DMEM medium, II) EBs after 3 days in adherent dishes, III) EBs after 2 weeks in adherent dishes; b) Inmmunofluorescence of endodermal, ectodermal and mesodermal markers in EBs derived from L2 (EB-L2), L3 (EB-L3) iPSCs lines and fibroblasts as controls. In blue nucleous are stained with DAPI c) RT-qPCR analysis of pluripotent markers in L2 vs. EB-L2 and L3 vs. EB-L3. Results are presented as means ± SEM (n = 3). Data were relativized to L2 or L3 for EB-L2 and EB-L3, respectively. *Statistically different (p<0.05).

Fig 4. RT-qPCR analysis of 7 microRNAs in the L2 and L3 iPSCs lines and the original fibroblasts.

RT-qPCR analysis of 7 microRNAs in the L2 and L3 iPSCs lines and the original fibroblasts. Results are presented as means ± SEM (n = 3). Data were relativized to fibroblasts. Different letters indicate significant differences (p<0.05).

Fig 5. RT-qPCR analysis of 7 microRNAs in the iPSCs lines and the embryo bodies (EBs) derived from them, iPSC-L2 vs. EB-L2 and iPSC-L3 vs. EB-L3.

Results are presented as means ± SEM (n = 3). EB-L2 data were relativized to iPSC-L2 data and EB-L3 data were relativized to iPSC-L3 data. *Statistically different (p<0.05).

Fig 6. In-silico analysis of equine microRNAs targets.

The green bars and the black zones represent the homology between sequences. a) Comparison between equine miR-302/367 cluster (eca-miR-302/367) and human miR-302/367 cluster (hsa-miR-302/367) in the genome. In orange are the different microRNAs positioned in the cluster. Below this alignment, the seed sequence of the miR-302 family is aligned to the 3‘UTR of 4 mRNAs in the equine (CDK2, E2F1, RHOC and CYCLIN D). b) Seed alignment of the differentiation-related microRNAs miR-9, miR-96 and miR-145, to the 3‘UTR of the equine mRNAs HES1, PAX6 and KLF4 and OCT-4, respectively.