Isolation of SSEA-3-positive mesenchymal stem cells from equine bone marrow and evaluation of their pluripotency.

Generally, most mesenchymal stem cells (MSCs) have lower pluripotency and limited differentiation potential than embryonic stem cells (ESCs). However, a small subpopulation of MSCs, called multilineage differentiating stress-enduring (MUSE) cells, exhibit pluripotency. MUSE cells express stage-specific embryonic antigen 3 (SSEA-3), a sphingoglycolipid. Here, we isolated and investigated the pluripotency of SSEA-3-positive MSCs (MUSE cells). Six thoroughbred horses were used as test subjects. MSCs were harvested from the bone marrow of the thoracic vertebrae under ultrasound guidance. Harvested MSCs were sorted into SSEA-3-positive and -negative cells using a cell sorter, and their pluripotency was evaluated by immunostaining for the markers NANOG, OCT4, and SOX2. The results showed that 2.1 % of equine MSCs were SSEA-3-positive, and these cells were successfully isolated using a cell sorter. This study confirms that SSEA-3-positive cells are present in equine bone marrow-derived MSCs, similar to that in other species. Immunostaining revealed SOX2 (88.3 ± 11.6 %) and NANOG expressions (9.0 ± 3.9 %) in the SSEA-3-negative cell group, whereas OCT4 expression was not detected (0.0 ± 0.0 %). Contrastingly, the SSEA-3-positive cell group showed significant NANOG (19.8 ± 3.8 %), OCT4 (9.7 ± 2.6 %), and SOX2 expressions (97.3 ± 4.1 %), with significantly higher values for NANOG and OCT4 compared to the SSEA-3-negative group (p < 0.05).