They expressed all tested endogenous pluripotency markers, as detected by RT-PCR (Fig
They expressed all tested endogenous pluripotency markers, as detected by RT-PCR (Fig. processes are covered by a specialized epithelium that is of neuroepithelial origin. Sphere formation assays in a serum-free medium were first described as a method to select and expand stem cells [5,6]. Previous studies exhibited that main ciliary body epithelial (CE) cells have the ability to Trichostatin-A (TSA) form clonal neurospheres, suggesting stem cells-like cells residing in the ciliary body epithelium [7]. CE sphere-derived cells expressed some retinal stem/progenitor cell markers in the culture [7,8]. However, the expression seems to be induced by growth factors in the medium. It remains unclear if these cells are bona fide quiescent retinal stem cells [8,9]. Generation of induced pluripotent stem (iPS) cells through ectopic expression of defined transcription factors holds great potential for regenerative medicine and disease modeling [10]. We previously found that mouse CE-derived cells, produced in monolayer cultures, express Sox2, Klf4, and c-Myc, and that these monolayer CE cells can be reprogrammed into iPS cells with ectopic Oct4 and Klf4 (2F), but not with Oct4 alone [1]. Given that adult neural stem cells, capable of sphere formation, have been previously reprogrammed into iPS cells with Oct4 alone [11,12], and that CE cells have the amazing plasticity to form spheres displaying certain characteristics of neuroepithelial progenitors [7,8], we attempted to reprogram CE sphere-derived cells with Oct4 alone. == Materials and Methods == == Animals == All procedures on mice were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Care and Use Committee at the University or college of North Carolina at Chapel Hill. Wild-type (WT) CD-1 strain mice, aged 56 weeks, were utilized Trichostatin-A (TSA) for the study. == CE spheres and monolayer cultures == Dissection of adult mouse Trichostatin-A (TSA) ciliary body was performed as previously explained [7]. Dissociated cells were resuspended in a serum-free growth medium (SFM) made up of Neurobasal medium, 1 B27 product, 1% penicillin/streptomycin, 2 mMl-glutamine, 20 ng/mL basic fibroblast growth factor (Peprotech), 20 ng/mL epidermal growth factor (Peprotech), and 2 g/mL heparin (Sigma). To generate floating spheres, CE cells were plated at a density of 20 cells per microliter in ultra-low attachment 24-well plates (Corning) [8]. To establish monolayer cultures, cells were produced in SFM supplemented with 1% fetal bovine serum (FBS) as previously explained [1]. The medium was changed every 3 days. All cell culture reagents were from Invitrogen, unless otherwise noted. == Induction of iPS cells == The procedure of iPS cells generation was essentially as previously explained with minor modifications [1]. In brief, all spheres at day 7 were dissociated into single cells with Accutase (Sigma) and plated at 1105cells per well of gelatin-coated six-well plates in 1% FBS-containing SFM. CE monolayer cultures at passage 2 (7 days after isolation) were plated the same as above. The next day, for the two-factor transduction, concentrated lentiviruses made up of CMV promoter-driven human Oct4 and Klf4 or Sall4 (Cellomics Technology) were added to the cells at a multiplicity of contamination of 10 with 4 g/mL polybrene (Sigma) in 1% FBS-containing SFM, whereas for the one-factor transduction, only lentivirus coding Trichostatin-A (TSA) human Oct4 was added. Twenty-four hours post contamination, the viral contamination mix was exchanged for new 1% FBS-containing SFM. The following day, transduced cells were subcultured onto mitomycin C-treated SNL feeder cells (Cell Biolabs) in six-well plates at a split TRUNDD ratio of 1 1:4 in embryonic stem (ES) cell medium minus leukemia inhibitory factor (LIF) made up of Dulbecco’s altered Eagle’s medium (DMEM) supplemented with 15% FBS, 2 mMl-glutamine, 100 M non-essential amino acids, 100 M 2-mercaptoethanol, 50 U/mL penicillin, and 50 g/mL streptomycin [13]. At day 21, the ES cell medium was replaced with serum-free N2B27 medium supplemented with LIF and 2i inhibitors [14], CHIR99021 (3 M; Stemgent), and PD0325901 (1 M; Stemgent). After another 7 days, ES cell-like colonies were picked and dissociated with trypsin and expanded on SNL feeders in KnockOut DMEM supplemented with 15% knockout serum replacement (KSR), 100 M minimum essential medium (MEM) nonessential amino acids, 100 M 2-mercaptoethanol, 2 mMl-glutamine, 50 U penicillin, and 50 g/mL streptomycin [15]. The efficiency of generating iPS cells was calculated by dividing the number of alkaline phosphatase (or Nanog)-positive ES cell-like colonies by the seeded cell number. == Viral vector integration analysis == Genomic DNA was isolated with the Quick-gDNA MiniPrep (Zymo.