337 PROMOTER-DEPENDENT SILENCING OF REPROGRAMMING TRANSCRIPTION FACTORS IN MOUSE INDUCED PLURIPOTENT STEM CELLS PRODUCED WITH SLEEPING BEAUTY TRANSPOSON VECTORS

Abstract

Epigenetic silencing of the transgenes has been considered a prerequisite for complete reprogramming of mouse somatic cells to induced pluripotent stem cells (miPSC). Here, we examined the activity status of the reprogramming transcription factors in miPSC produced with Sleeping Beauty (SB) transposon vectors carrying expression cassettes with the porcine OCT4, SOX2, c-MYC, and KLF4 (pOSMK) under the control of doxycycline (DOX)-inducible (TetO) or constitutive (CAG) promoters. Mouse embryo fibroblasts (MEF) were electroporated with SB-TetO-rTA-SV40pA-TetO-pOSMK-IRES-tdTomato-bGHpA (TetO group) or with SB-loxP-CAG-pOSMK-IRES-tdTomato-SV40pA-loxP (CAG group) together with SB100x (SB transposase). The cells were cultured on mitotically inactivated MEF feeders with DMEM supplemented with 20% knockout serum replacement, 2 mM l-glutamine, penicillin-streptomycin, nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1000 U mL^–1 of ESGRO, and 5 µg mL^–1 of DOX. The miPSC colonies were individually picked, disaggregated to single cells, and propagated further under the same culture conditions. Three cell lines from each experimental group were examined for pluripotency characteristics, and the activity of the transgenes was monitored by the presence of tdTomato fluorescence and by RT-PCR. The miPSC produced with TetO vector silenced the transgene expression within 11 days post-transfection (in the presence of DOX) and upregulated the endogenous pluripotency genes Oct4, Sox2, Nanog, Rex1, and Utf1. These cells showed typical miPSC morphology and ability to differentiate into cells from the 3 primary germ layers in vitro and in vivo (teratomas). At the same time, the miPSC from the CAG group did not silence the transgenes even after 20 passages of continuous propagation, although they upregulated the endogenous pluripotency genes similarly to the TetO group. Moreover, these cells also showed ability to differentiate in vitro into cells from the 3 germ layers (contracting cardiac myocytes, neurons, epithelia) expressing differentiation markers Afp, Sox17, Gata4, Gata6, cardiac troponin, nestin, and PGP 9.5. Following Cre-mediated excision of the reprogramming cassette, the miPSC from the CAG group continued to self-renew and the expression of pluripotency markers Oct4, Sox2, Nanog, and Rex1 did not change significantly, as evidenced by real-time RT PCR (all P > 0.1), showing that these cells were not dependent on the transgenes for maintaining their pluripotency characteristics. Currently, we are investigating the ability of the miPSC from the CAG group to differentiate in vivo by producing teratomas and chimeras. The results from our preliminary investigations suggest that porcine transcription factors can be used for production of miPSC and that the silencing of the reprogramming transcription factors in miPSC is promoter-dependent, but may not be absolutely necessary for complete reprogramming to pluripotency.