203 IDENTIFICATION OF A POTENTIAL MULTILINEAGE-DIFFERENTIATING STRESS ENDURING CELL POPULATION IN PORCINE FETAL FIBROBLASTS

Abstract

Physiologically, humans and pigs are very similar, which makes cell model systems such as porcine-induced pluripotent stem (iPS) cells an attractive model for studying human diseases. Recently, it has been shown that a finite population of cells, termed multilineage-differentiating stress enduring (MUSE) cells, which are particularly prone for reprogramming, exists in human fibroblasts. Human MUSE cells co-express the cell surface markers, SSEA-3 and CD105; however, it is unknown if a similar subpopulation of cells exists in porcine fibroblasts. Therefore, we examined the expression level of varying cell surface markers, including SSEA-1, SSEA-3, CD105, and Vimentin (positive control), in 9 porcine fetal fibroblast lines from 3 different breed backgrounds (Danish Landrace lines 2, 4, 5; Gottingen minipig lines A1, B2, B3; Yucatan minipig lines 1, 3, 4) by performing chromogen immunocytochemistry. The results revealed that all the 9 cell lines were 100% positive for Vimentin and negative for SSEA-3. However, a small positive population of SSEA-1 cells could be detected in Danish Landrace lines 2, 4, 5 (0.267%, 0.441%, 0.292%, respectively) and in Gottingen minipig lines A1, B2, B3 (0.207%, 0.214%, 0.325%, respectively). Furthermore, CD105 could only be detected in the Yucatan minipig lines (0.170, 0.177, and 0.233%, in lines 1, 3, and 4, respectively). To identify if any of these lines were more receptive to reprogramming, we performed non-integrating vector-based reprogramming. A total of 1 × 10⁵ cells from each cell line was electroporated with 3 episomal plasmids (pCXLE-hOCT3/4-shp53; pCXLE-hSK; pCXLE-hUL), which were diluted to 1 µg µL^–1, and then plated in DMEM-AQ + 10% FBS without antibiotics. The medium was replaced with the addition of penicillin/streptomycin on Day 2, and on Day 7 cells were trypsinized and passaged onto mitomycin C-treated mouse embryonic fibroblasts in iPS culture medium (DMEM-F12 + 20%KSR + NEAA + Pen/strep + 2 µL mL^–1 β-Me + 10 ng mL^–1 bFGF). Only a select number of Danish Landrace lines 2 and 4 formed ES-like colonies after 14 days, and few colonies with neural-like morphology emerged in Yucatan minipig line 3, but all these colonies lost their proliferative potential after a few passages. The reprogramming efficiency observed on Day 21 was 0.0787, 0.0170, and 0.0033% for Danish Landrace 2, Danish Landrace 4, and Yucatan 3 lines, respectively. The NANOG immunostaining was also performed at passage 1, and a minor proportion of the colonies in Danish Landrace line 2 was found to contain small populations of cells with nuclear staining for NANOG. Interestingly, Danish Landrace lines 2 and 4, which formed ES-like colonies, also expressed SSEA1, which may suggest that SSEA1 could potentially be a marker of a MUSE cell population. Further studies are ongoing to investigate this hypothesis. This research may further support the elite hypothesis for how reprogramming may occur.