Generation and characterization of horse iPSCs using a lentiviral vector for their application in reproductive biotechnologies

LUCÍA MORO; CARLOS LUZZANI; RAMIRO OLIVERA; GABRIEL VICHERA; GUSTAVO SEVLEVER; SANTIAGO G. MIRIUKA

Tours

Congreso; 18th International Congress of Animal Reproduction; 2016

ICAR

Equine pluripotent stem cells offer very valuable potential for their application in regenerative medicine, in human disease modeling and could also be a more effective source of nuclear donor for horse cloning. Until now, no embryonic stem cell lines (ESC) have been obtained in the horse, but the generation of equine induced pluripotent stem cells iPSCs is a promising alternative as they are functionally similar to ESC and they can be generated from an adult animal in a patient-specific manner. The aim of this work is to generate and characterize horse iPSC lines by using a single lentiviral vector expressing the human Yamanaka´s factors (OCT4-SOX2-cMYC-KLF4). Firstly, 1x105 horse fetal fibroblasts were cultured in DMEM 10% FBS in one gelatin-coated well of a 6 well plate. Lentiviral infection was performed the next day using the STEMCCA vector (Sommer et al. 2009) with a MOI=2 and 10 µg/ml polybrene for 24 h; after that time the virus was removed and the cells were cultured with DMEM 10% FBS for 3 more days. On day 4, the medium was changed to DMEM-F12 supplemented with 20% knockout serum replacement, 55 µM 2-Mercaptoethanol, 8 ng/ml bFGF and 10 ng/ml hLIF. On day 15, 2x105 irradiated murine embryonic fibroblasts (MEF) were added to the well and on day 25, 12 colonies with stem-cell like morphology were picked and transferred individually over MEF for their clonal growth. We validated the bona fide legitimacy of 2 of the cell lines generated, by evaluating the expression of stemness marker genes like OCT4, NANOG and REX1. Furthermore, under the appropriate conditions, the equine iPSCs readily formed embryoid bodies and differentiated in vitro into cells expressing markers of endoderm, ectoderm and mesoderm including NESTIN, TUBB3, AFP and VIMENTIN, both by RT-PCR and inmunofluorescence staining. We are now evaluating their efficiency in generating embryos by nuclear transfer and also their capacity of colonizing the inner cell mass of in vitro embryos by injecting the equine iPSC in morulae and early blastocysts. In conclusion, we could obtain equine iPSCs by using a lentiviral vector not reported before in this species, with high efficiency and quality of pluripotency, ready to be used in veterinary medicine, as preclinical models or to demonstrate their potential in nuclear transfer.