Endogenous pluripotent factor expression after reprogramming cat fetal fibroblasts using inducible transcription factors

Abstract

Incomplete transgene‐silencing remains a challenge in the generation of induced pluripotent stem cells (iPSC) in felids—a critical family in biomedical and biodiversity conservation science. In this study doxycycline‐inducible transgenes (NANOG, POU5F1, SOX2, KLF4, and cMYC) were used to reprogram cat fetal fibroblasts with the objective of obtaining iPSC with fully silenced transgenes. Colony formation was slower (14 vs. 8 days) and at lower efficiency than mouse embryonic fibroblasts (0.002% vs. 0.02% of seeded cells). Alkaline‐phosphatase positive colonies were grown on feeder cells plus LIF and GSK3, MEK, and ROCK inhibitors. Cells could be passaged singly and transgene expression was silenced at passage 3 (P3) after doxycycline removal at P2. NANOG, POU5F1, and SOX2 were expressed at P3, P6, and P10, although at lower immunostaining intensities than in cat inner cell masses (ICM). Transcripts related to pluripotency (NANOG, POU5F1, SOX2, KLF4, cMYC, and REX1) and differentiation (FGF5, TBXT, GATA6, SOX17, FOXF1, PAX6, and SOX1) were assessed by a reverse transcription‐quantitative polymerase chain reaction in iPSC and embryoid bodies. The immunostaining patterns, relatively low levels of NANOG and REX1 in comparison to ICM along with the expression of TBXT (mesoderm) suggested that cells were a mix of reprogrammed pluripotent and differentiating cells.

Viability, Yield and Expansion Capability of Feline MSCs Obtained from Subcutaneous and Reproductive Organ Adipose Depots

“…In cats, Gata6 is assumed to be involved in differentiation similar to humans, but that has yet to be examined. Studies have described the presence of Gata6 in feline induced pluripotent stem cells [41] and in cat embryos [36], but ours is the first study to report the presence of Gata6 in feline adipose MSCs. SOX17, identified in human adipose derived MSCs [43], is involved in the capacity of the cells to differentiate into endoderm and eventually to hepatocytes [44] or pancreatic tissue [33].…”

“…In comparison with canine cells, feline amniotic MSCs showed a much lower expression of Sox2 and less immunostaining of the protein compared to canine amniotic stem cells [39]. In addition, a number of labs have shown that induced pluripotent stem cells derived from feline fibroblasts express Sox2 [40,41]. However, the presence of Sox2 in feline adipose-derived MSCs is controversial.…”

“…Like Gata6, Sox17 has been detected in feline induced pluripotent stem cells [41], but has not been investigated in feline adipose MSCs. Thus, this is the first study to identify Sox17 in feline adipose-derived MSCs.…”

The use of induced pluripotent stem cells in domestic animals: a narrative review

“…iPSCs have been developed from porcine [16], equine [17], canine [18], bovine [19], galline [20], caprine [21], ovine [22], and feline [23] tissue. Successful iPSC production from domestic species was first reported in 2009 by Wu and colleagues in porcine, and the field has since expanded to other species (Fig.…”

“…1). iPSC derivation protocols have been developed in species including porcine [16], equine [17], canine [18], bovine [19], galline [20], caprine [21], ovine [22], and feline [23]. Aside from their importance in treating veterinary pathologies, porcine, canine, and equine models have been shown to be valuable for the study and treatment of human disease [24][25][26].…”