Versene solution

Human iPSCs that were maintained under feeder-free culture conditions in growth factor reduced Matrigel and defined lab-made Essential 8 (E8) media. The iPSCs were passaged every 5–7 days using the StemPro EZ Passaging Tool (Life Technologies) or Versene solution (Life Technologies). Every cell line in the iPSC core cultured is tested monthly for mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza; LT07-710). Our annual rate of mycoplasma contamination is <0.2% and only mycoplasma-free cell lines were used in this study for analysis. The iPSC lines once reprogrammed were authenticated using cell line identity assay by short tandem repeat (STR) profiling, and compared with parental tissue source (PBMCs). For STR profiling, the iPSC lines were authenticated by the Cedars-Sinai iPSC Core by contracting with IDEXX Laboratories, Inc. and use of their CellCheck 9 service. This test allows provides a human 9 STR marker profile and for interspecies contamination check for human, mouse, rat, African green monkey, and Chinese hamster cells. The identity of all cell lines validated is matched to tissue source annually and at the time of generation of the distribution cell bank. Certificate of Analysis of all iPSC lines used in this manuscript are available upon request from the Cedars-Sinai iPSC Core.

Flow cytometry was performed essentially as described previously (42 (link)). CHO cells were detached by using a Versene solution (Life Technologies, Inc.), incubated with the antibodies/proteins described in Text S1 in the supplemental material, and analyzed on a FACSCalibur instrument (BD Biosciences). For detection of HS, cells were incubated sequentially with the vesicular stomatitis virus (VSV)-tagged single-chain variable-fragment (scFv) antibody HS4C3 (1:100 [41 (link)]), mouse anti-VSV IgG (1:10; P5D4), and Alexa Fluor 488 fluorochrome-conjugated anti-mouse IgG (20 μg/ml; Life Technologies, Inc.). For detection of FGF2 binding, cells were incubated sequentially with biotinylated FGF2 (1:250 [43 (link)]) and DyLight 488 fluorochrome-conjugated streptavidin (10 μg/ml; Thermo Scientific). Endocytic/phagocytic assays are described in Text S1.
To determine the laminin-binding capacities of different bacteria, laminin-211/221 was concentrated using a 100-kDa-cutoff Amicon centrifugal filter unit (EMD Millipore) and FITC labeled using the FluoReporter FITC protein labeling kit (Life Technologies, Inc.) according to the manufacturer’s instructions. Bacteria (3.75 × 10⁷ CFU) were incubated with 50 μg/ml of the labeled laminin for 30 min at 37°C, washed twice with PBS, and analyzed by flow cytometry on a FACSCalibur instrument.

HiPSC-CMs were maintained in cardio culture medium (RPMI 1640 with Glucose, GlutaMax, HEPES, and B27 supplement) and incubated at 37 °C in a humidified incubator with 5% CO₂. For transient transfection experiments hiPSC-CMs were washed briefly with Versene solution (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), detached using 0.25% Trypsin-EDTA solution, washed once in splitting medium (RPMI 1640 with HEPES and GlutaMax, B27 supplement, 20% FCS (Thermo Fisher Scientific, Waltham, MA, USA), 2 µM Thiazovivin (Merck Millipore, Burlington, MA, USA)) and seeded at the desired cell density. Twenty-four hours after seeding, the cells were switched back to cardio culture medium, allowed to recover for 48 h, and then used for experiments. To modify the expression levels of specific miRNAs, the cells were transfected with miRNA mimics or inhibitors (Qiagen GmbH, Hilden, Germany) at 20 and 50 nM concentrations, respectively. Lipofectamine RNAiMax (Thermo Fisher Scientific, Waltham, MA, USA) was used as the transfection reagent according to the manufacturer’s instructions. All miRNA mimics and inhibitors as well as the corresponding negative controls were purchased from Qiagen (Qiagen GmbH, Hilden, Germany). An overview of miRNA mimics and inhibitors used in this study is listed in Supplementary Table S6.

All human pluripotent stem cells (hPSCs) were maintained in mTeSR1 medium on Matrigel (WiCell) coated tissue culture polystyrene plates (BD Falcon). Cells were passaged every 4–5 days at a ratio of 1:8 using Versene solution (Life Technologies). Patient-derived iPSC line, Pompe GM04192, was a gift from the T. Kamp and M. Suzuki (UW-Madison). Cardiomyocytes derived from hPSC and iPSC cultures were maintained in RPMI/B27 on Matrigel (WiCell) coated polystyrene plates (BD Falcon). Patient-derived fibroblast lines were obtained from Coriell Institute with different GAA mutations (W746X mutation was from Coriell ID: GM04912; D645N mutation was from Coriell ID: GM20090; R660H was from Coriell ID: GM13522) and cultured in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin. All cells were maintained at 37 °C in 5% CO₂, and tested monthly for possible mycoplasma contamination. Samples obtained from the Coriell Institute have been approved by the Working Group of the Human Genetic Cell Repository.