Generation of equine induced pluripotent stem cells from cells of embryonic, perinatal and adult tissues

Background: Regenerative therapies are quickly expanding to application in equine patients because of their importance as sporting and companion Animals. Furthermore, aligning with a One Health concept, veterinary medicine offers a unique platform for preclinical studies. While mesenchymal stem/stromal cells (MSCs) therapies are already used in treating horses, strategies involving induced pluripotent stem cells (iPSCs) are poorly developed. iPSCs present great potential for therapy and disease modelling, but their consistent generation in horses requires further investigation into the source of somatic cells and the reprogramming method and conditions.

Methods: The reprogramming potential of equine cells from tissues of three developmental origins was compared: prenatal (embryo-derived MSCs, eMSCs), perinatal (cord blood-derived MSCs, CB-MSCs) and adult (articular chondrocytes, ACs). Two reprogramming methods (retroviral, lentiviral) and different culture conditions (serum/serum-free, feeder cells/feeder-free, with/without small molecules) were tested. Pluripotent gene expression was analyzed at different time-points to reveal transcriptomic changes associated with reprogramming. The generated equine iPSCs (eqiPSCs) were characterized by Alkaline Phosphatase (AP) staining, expression of pluripotent genes and proteins, three-germ layer differentiation (embryoid body) and karyotype.

Results: Using a lentiviral vector with serum-free media and feeder cells resulted in the most favorable conditions for eqiPSCs reprogramming, but adding small molecules had a negative effect. Equine CB-MSCs and ACs were only partially reprogrammed and could not be efficiently expanded in culture. Only eMSCs generated putative eqiPSCs that met the cellular, molecular and functional criteria of pluripotent cells. Equine eMSCs showed higher proliferation and basal expression of pluripotent genes compared to CB-MSCs and ACs, and showed the highest upregulation of pluripotent genes along reprogramming.

Conclusions: The developmental stage of the starting cell strongly influences their reprogramming potential in equine species. This has been suggested for human and Other animal species, but direct comparison of equine cells from prenatal, perinatal and adult sources has not been reported before. Novel preliminary insight into the transcriptomic changes of different equine cell types during reprogramming, and on the effect of different culture conditions, can contribute improving the generation of eqiPSCs. While transgene-free methods are the goal, putative eqiPSCs are critical to enlarge our knowledge on animal iPSC biology.

Equine; Gene expression; Horse; Pluripotency; Reprogramming; iPSC.