*2.9. Lipid Extraction*

The cells were grown to confluency on 150 mm cell culture dishes and harvested by scraping into 700 μL PBS. The cell suspension was transferred into 16 mm × 100 mm glass tubes, and 1 mL chloroform and 2.4 mL methanol were added. After water bath sonication, protein was precipitated by centrifuging at 2400× *g* for 30 min. The supernatant was transferred to a new glass tube and 4.5 mL chloroform and 1.2 mL 0.9% NaCl were added. The sample was centrifuged at 900× *g* for 5 min. The upper aqueous phase was discarded, and the lower organic phase was washed twice with 2 mL methanol and 0.8 mL 0.9% NaCl. The lower phase was extracted using a 1 mL glass syringe (Hamilton, Reno, NV, USA) and transferred to a new glass tube. Lipids were dried under a stream of nitrogen.

#### *2.10. Quantification of Gb3 by Thin Layer Chromatography (TLC)*

First, 100 nmole of total phospholipid was applied to a silica high performance TLC plate (Sigma-Aldrich, St. Louis, MO, USA). The plate was first developed in a solvent system consisting of chloroform/methanol (98:2), and air-dried. The plate was then developed in a solvent system consisting of chloroform/methanol/acetic acid/water (61/31/5/3) and air-dried. Plates were submerged in 8% cupric sulfate pentahydrate in water/methanol/H3PO4 (60:32:8), and charred for 10 min at 150 ◦C, or were sprayed with 1% orcinol in 11% H2SO4 and charred at 130 ◦C for 5 min. Plates were scanned and densinometry measured using ImageJ software. Lipids were quantified by running Gb3 standards on a plate (Matreya LLC, State College, PA, USA).

#### *2.11. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS*/*MS) Analysis*

LC-MS/MS analysis was performed using Orbitrap Mass Analyzer (Thermo Fisher Scientific), according to the manufacturer's protocol. Briefly, each sample of digested peptides was reconstituted in 20 μL of 0.1% formic acid. Peptides were first separated by the nanoflow HPLC on Agilent 1100 (Agilent Technologies, Santa Clara, CA, USA) using C18 column (Agilent Technologies) with a flow rate of 0.4 μL/min, and were ionized after passing through the nanospray tip (New Objective, Woburn, MA, USA). LC gradient for the LC-MS/MS system ramped from 2–40% ACN in 120 min, and the system was set up for automated data-dependent acquisition, with a mode of 200–2000 m/z full scan for the maximum three most intense peaks from each Orbitrap MS scan. Peptides with +2 or +3 charge were further subjected to CID. Spectra were obtained in raw data files with Xcalibur (version 2.0 SR2). Protein identification was accomplished by TurboSEQUEST (Thermo Fisher Scientific) using the UniProt database. A protein was confirmed once three peptides with Xcorr >2.5 were matched in sequencing.

#### *2.12. Transmission Electron Microscopy*

The morphology of differentiated cardiomyocytes was characterized using JEM-2000 EX II transmission electron microscope (JEOL, Tokyo, Japan). The cardiomyocytes were covered with 400 mesh carbon-coated copper TEM grid. After 15 min, the grid was tapped with filter paper to remove the excess water followed by staining with 1% phosphotungstic acid (Sigma-Aldrich) for 20 min. The samples were allowed to air-dry for 24 h and then observed under TEM.

#### *2.13. Array-Based Comparative Genomic Hybridization (CGH-Array*

Genomic DNA was isolated and intermittently sonicated using a Digital Sonifier 450 sonicator probe (Branson Ultrasonics, Danbury, CT, USA). DNA samples were amplified using the GenomePlex WGA kit (Thermo Fisher Scientific). Genomic DNA ULS Labeling Kit (Agilent) was used to label the amplified DNA with either Cy3 or Cy5. As recommended by Agilent, 2.0–2.5 μg of amplified DNA was used as the input starting material for each labeling reaction. Scanning and image analysis were conducted according to Agilent Oligonucleotide Array-based CGH for Genomic DNA analysis Protocol (version 4.0). Microarrays were scanned using an Agilent G2565BA DNA Microarray Scanner (Agilent). Agilent Feature Extraction software (v9.1.3) was used to extract data from raw microarray image files. Agilent CGH Analytics software (v3.4) was used to visualize, detect and analyze the aberration patterns from CGH microarray profiles.

#### *2.14. Exosome Isolation and Characterization*

Exosomes were isolated from cell culture supernatants using Total Exosome Isolation Reagent (Thermo Fisher Scientific) following the manufacturer's protocol. Culture media samples were centrifuged at 2000× *g* for 30 min to remove cells and debris. The supernatant was transferred to sterile tubes and an exosome precipitation solution was added at a 2:1 ratio. Samples were mixed and left overnight at 4 ◦C. Samples were then centrifuged at 10,000× *g* for 60 min and supernatant carefully removed. The precipitated exosome pellets were re-suspended with PBS and either used immediately or stored at −80 ◦C until required. Immunoaffinity capture assay was used to characterize the purity of exosomes using CD63 antibody-conjugated dynabeads. Exosomes were incubated with antibody-conjugated dynabeads overnight and washed by PBS containing 0.1% BSA two times. Further, CD63 PE-conjugated antibody was used to stain the exosome-bound dynabeads. All samples were examined by flow cytometry using FACSCanto System (BD Biosciences) and FACSDIVA software was used to analyze the population of exosomes.
