Both primed and na?ve-like PSCs could also be equally differentiated into the neuronal lineage and the hematopoietic lineage (Supplementary Fig. CA). The protein concentration of the cell lysate was determined by BCA assay (Bio-Rad), and the result in RLU (relative luminescent units) was normalized to the protein concentration. 2.8. Three-germ-layer differentiation The na?ve-like and primed iPSCs were plated on Geltrex-coated plates after undergoing single-cell dissociation. Three-germ-layer differentiation was performed by using a STEMdiff? Trilineage Differentiation Kit (STEMCELL Technologies) according to the manufacturer’s protocol. To validate the expression of each germ-layer differentiation, Q-PCR and immunofluorescence assays were performed with the following antibodies: anti-OTX2 (for ectoderm), anti-BRACHYURY (for mesoderm), and anti-SOX17 (for endoderm). All antibodies were purchased from R&D Systems. 2.9. RNA-seq Total RNA was extracted using an RNeasy Plus Micro Kit (Qiagen). cDNA libraries were constructed using an Illumina TruSeq Stranded mRNA Kit with poly-A selection. Libraries were paired-end 100-bp sequenced using an Illumina HiSeq 2500 System. The sequencing Palosuran reads were aligned to human cDNA from ensembl.org by using Kallisto [19] (version 0.43.0) with the default settings. Differentially expressed genes were called using Palosuran the Sleuth R package [20]. 2.10. Transmission electron microscopy Samples were fixed overnight in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1?M sodium cacodylate buffer, pH 7, then post fixed for 1.5?h in 2% osmium tetroxide in 0.1?M cacodylate buffer with 0.3% potassium ferrocyanide. After the tissue was rinsed in the same buffer, it was stained with 4% aqueous uranyl acetate and dehydrated through a graded ethanol series to propylene oxide. It was then infiltrated through a propylene oxide:epon series, ending with 100% epon overnight. This routine processing was performed on a Leica EM TP Tissue Processor. Next day, the tissue was embedded in fresh epon and polymerized at 70?C overnight. Semithin sections (0.5?m) were stained with toluidine blue for light microscope examination. Ultrathin sections (80?nm) were cut and imaged using an FEI Tecnai 200Kv FEG Electron Microscope with an ATM XR41 2K Digital Camera. 3.?Results and discussion 3.1. Generation of human iPSCs and conversion to na?ve-like PSCs Human iPSC lines were generated by treating human female dermal fibroblast cells with a Sendai virus vector, which is an established non-integration method for reprogramming. Once the iPSC lines were established, the cells were cultivated under feeder-free conditions to prevent contamination by mouse feeder cells in downstream functional assays. Palosuran Immunofluorescence assays with an antibody to the canonical pluripotency marker OCT4 and flow cytometry analysis with antibodies to SSEA3/SSEA4 confirmed the pluripotency of the established iPSCs (Fig. 1A). From among these established iPSC lines, single clonal cells that showed non-viral gene integration were used for the subsequent experiments. In an earlier study, primed human iPSCs were converted to a na?ve state by growing them in culture in serum/bFGF-free medium containing a primitive growth factor, NME7AB [12]. We also used NME7AB to generate na?ve-like stem cells, congruent with this previously published method. To verify the conversion, we used the H3K27 trimethylation (H3K27me3) marker. Primed iPSCs have one active and one inactive X chromosome, whereas na?ve stem cells have two active X Rabbit Polyclonal to CKMT2 chromosomes. In primed iPSCs, staining with an anti-H3K27me3 antibody resulted in condensed puncta, signifying X-chromosome inactivation (Fig. 1A). In contrast, X-chromosome reactivation resulted in cloud-like staining with the anti-H3K27me3 antibody (Fig. 1B), and this can be seen after the conversion Palosuran of primed PSCs to na?ve-like (XaXa) PSCs. However, the resulting na?ve-like stem cells still expressed the pluripotent markers OCT4 and SSEA3/SSEA4 at high levels (Fig. 1B). Both primed PSCs and na?ve-like PSCs showed normal karyotyping (Fig. 1C and D). Open in a separate window Fig. 1 Na?ve-like stem cell conversion by adding NME7AB. (A) Human female primed iPSC lines were generated by treating human female dermal fibroblast cells with four reprogramming factors (c-Myc, OCT4, SOX2, and KLF4) encoded by a Sendai virus vector. To confirm the stemness of the iPSCs, immunofluorescence assays were performed with an anti-OCT4 antibody (green). DAPI staining (blue) was used to show nuclei. The scale bar in this image represents 400?m. Antibodies to SSEA-3 and SSEA-4 were used for flow analysis. Primed stem cells had one active X chromosome; the other X chromosome had been inactivated through methylation (indicated by condensed dot/punctate staining with the anti-H3K27me3 antibody; the scale bar in.