Oct3 4

Human iPSCs used in this study have been previously described [20 ]. iPSC lines were generated using non-integrating Sendai virus carrying the Yamanaka factors: OCT3/4, SOX2, KLF4, and cMYC (Life Technologies) [21 , 22 (link)]. The following parameters were used for the characterization of each of the iPSC lines using standard methods [21 ]: pluripotency markers by immunocytochemistry (ICC) and quantitative PCR (qPCR); spontaneous or TriDiff differentiation into the three germ layers by ICC and qPCR; assessment of chromosomal abnormalities by karyotyping; and MAPT mutation status confirmation by Sanger sequencing (characterization data previously reported [15 (link)]).
To determine the impact of the MAPT mutant allele on molecular phenotypes, we used CRISPR/Cas9-edited isogenic controls in which the mutant allele was reverted to the wild-type (WT) allele in each of the donor iPSC lines as previously described [15 (link), 20 ]. The resulting edited iPSC lines were characterized as described above in addition to on- and off-target sequencing (characterization data previously reported [15 (link)]). All iPSC lines used in this study carry the MAPT H1/H1 common haplotype. All cell lines were confirmed to be free of mycoplasma.

We carried out immunoblotting using the Wes™ Simple Western System (Protein Simple, San Jose, CA) and specific antibodies against HDAC1-8 (Bethyl Laboratories Inc, Montgomery, TX), LSD1 (Cell Signaling Technology, Beverly, MA), Oct3/4 (Invitrogen), ßIII-tubulin, SOX17, ɑ-smooth muscle actin (Gene Tex Inc, Irvine, CA), di-methylated H3K4 (Active Motif, Carlsbad, CA), di-methylated H3K9 (Millipore), and GAPDH (Cell Signaling Technology).

The iPSC clones were confirmed as IPSCs with a battery of rabbit anti-mouse IPSC markers including Oct3/4, Sox2, c-Myc, mKlf4, Nestin and SSEA-1 (Thermo Fisher Scientific, Waltham, MA, USA). The secondary antibody was an Alexa Fluor 594-conjugated goat anti-rabbit (Thermo Fisher Scientific), all used in accordance with the manufacturer’s conditions. To immobilize the iPSC clones, glass-bottom dishes were coated with Cell-TEK adhesive. The adherent iPSC was then fixed with 4% paraformaldehyde, after permeabilizing with TX-100 and blocking with normal goat serum. The iPSC clones were then incubated with the primary antibodies, washed, and incubated with the secondary antibodies. The dishes were finally mounted with Vectorshield mounting medium with DAPI (#H-1200) (Vector Laboratories, Burlingame, CA, USA) and viewed with an Olympus Fluoview-1000 confocal scanning system under different wavelengths.

The iPSC clones were confirmed as IPSCs with a battery of rabbit anti-mouse IPSC markers including Oct3/4, Sox2, c-Myc, mKlf4, nestin, and SSEA-1 (Thermo Fisher Scientific, Waltham, MA) The secondary antibody was an Alexa Fluor 594-conjugated goat anti-rabbit (Thermo Fisher Scientific), all used with the manufacturer’s conditions. In order to immobilize the iPSC clones, glass-bottom dishes were coated with Cell-TEK adhesive. The adherent iPSC cells were then fixed with 4% paraformaldehyde, after permeabilizing with TX-100 and blocking with normal goat serum. The iPSC clones were then incubated with the primary antibodies, washed, and followed by the secondary antibodies. The dishes were finally mounted with Vectorshield mounting medium with DAPI (#H-1200) (Vector Laboratories, Burlingame, CA) and viewed with an Olympus Fluoview-1000 confocal scanning system under different wave lengths.