Tra 1 81

Cells were dissociated into single cells with Accutase (Invitrogen) and stained with primary antibodies for Tra-1–81 (Santa Cruz) and SSEA4 (Santa Cruz) for 1h on ice. Secondary antibody staining was performed for 30 min with R-PE-goat anti-mouse IgG and Alexa Fluor 488-donkey anti-mouse IgG&M. Data were collected with BD Accuri C6 flow cytometry (BD biosciences).

To confirm pluripotency, iPSC colonies were fixed (3% paraformaldehyde × 30 min), washed with phosphate-buffered saline (PBS) and permeabilized with 0.5% Triton. After blocking with 5% BSA, the colonies were incubated overnight with primary antibodies (OCT3/4, 1:50; NANOG, 1:50; TRA-1-60, 1:200; and TRA-1-81,1:200; all from Santa Cruz, Dallas, TX) followed by secondary antibody (anti-mouse Cy3, anti-rabbit FITC, 1:1,000). Isotype control antibodies were: anti-mouse IgG2b for OCT3/4, anti-goat IgG for Sox2 and Nanog, and anti-mouse IgM for TRA-1-60 and TRA-1-81 (all Abcam, Cambridge, UK). Images were taken using a confocal laser scanning microscope (LSM 710, Carl Zeiss, Germany).
For immunocytochemistry, 4% paraformaldehyde-fixed iPSCs were incubated with 3% donkey serum in 0.3% Triton X-100 in PBS (blocking) and exposed to rat anti-human αKlotho monoclonal antibody (1:1000, KM2076, overnight at 4°C, Trans Genic, Fukuoka, Japan), followed by incubation with secondary donkey anti-rat antibody conjugated to Alexa Fluor 555 (A31570, Invitrogen, Carlsbad, CA) at room temperature × 60min. Filamentous actin was labeled with rhodamine phalloidin (R415, Invitrogen), and nuclei with 4′,6-diamidino-2-phenylindole (DAPI, P36931, Thermo Fisher, Waltham, MA), and imaged with a laser scanning microscope (Zeiss LSM 880, Advanced Imaging Microscopy, Germany).

IPSCs-derived neurons from SCA-3 patients were fixated with 4% paraformaldehyde for 20 min. Cells were blocked in 5% normal goat serum and 2% fetal calf serum; subsequently, samples were probed with primary antibodies: TRA-1–81 (Santa Cruz; sc-21706), TRA-2–54 (made by group Peter Andrews lab, The University of Sheffield), OCT-4 (Santa Cruz; sc-5279), SOX-2 (Cell Signaling; #4900S) and βIII-tubulin (Abcam; ab7751). Alexa 488 and Cy3-conjugated secondary antibodies were used in combination with Hoechst nuclear staining (1:1000). Images were obtained using a Leica TCS SP8 confocal microscope (Leica Microsystems).

We used primary antibodies for Oct4, Nanog, Sox2, tropomyosin 1 (Cell Signaling Technology), β-actin, Tra1–60, Tra1–81, SSEA4, cardiac troponin T, and alpha sarcomeric actin (Santa Cruz Biotechnology, Inc.) to perform in vitro analysis. Secondary antibodies such as HRP-conjugated donkey anti–mouse, anti–rabbit, anti–goat (Santa Cruz Biotechnology, Inc.); TRITC-, FITC-, and Cy-5-conjugated donkey anti–mouse, anti–goat, and anti–rabbit (Jackson Immunoresearch Laboratories, Inc.) were used. DAPI (Life-Tech); Matrigel (BD Biosciences) NutriStem medium, alkaline phosphatase assay kit (Stemgent, Cambridge, MA); RPMI medium, Mytomycin C (Sigma-Aldrich, USA). StemRNA Reprogramming kit (Reprocell USA Inc).