Anti sca 1

TRI reagent was from Sigma-Aldrich (Madrid, Spain). Antibodies for the detection of murine CD135 (Flt3), CD34, CD16/32, and CD127 (IL-7Rα) were from eBioscience (Barcelona, Spain). The Lineage Cell Depletion Kit for mice, FcR Blocking reagent and murine flow cytometry antibodies (CD3e, CD4, CD8a, CD11b, CD19, CD43, CD45R (B220), CD117 (c-Kit), anti-Gr1, anti-Sca-1, anti-Ter-119, anti-IgM) were from Miltenyi Biotec (Madrid, Spain). Super Script II reverse transcriptase and RNase OUT ribonuclease inhibitor were from Invitrogen and Thermo Fisher Scientific (Madrid, Spain). GoTaq^R qPCR master mix was from Promega (Madrid, Spain).

Cultivated hCSCs were harvested by centrifugation after treatment with trypsin and subsequently stained with PE-coupled anti-CD105, anti-CD117, anti-Sca1 or anti-CD31 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s guidelines. For isotype controls, hCSCs were stained with PE-coupled IgG1 control antibody or APC-coupled IgG1 control antibody. Analysis was done using Gallios Flow Cytometer (Beckmann Coulter Inc., Brea, CA, USA), while Kaluza Acquisition Software (Beckmann Coulter Inc.) was used for subsequent data acquisition and statistical analysis.

Induced pluripotent stem cell-derived-CMs were analyzed using a FACScan instrument (BD, Franklin Lakes, NJ, USA). The dead cells were identified with propidium iodide (PI, BD). Cell debris and dead cells were gated out, and the remaining cells were quantified for expression of eGFP using CellQuest v2.0 software (BD). The MSC surface markers were analyzed using the following fluorescently labeled antibodies: anti-CD29 (phycoerythrin, PE), anti-CD44 (biotin, anti-biotin-PE-Vio770), anti-CD90.2 (VioBlue), anti CD105 (allophycocyanin, APC), anti-Sca-1 (fluorescein isothiocyanate), and anti-CD11b (APC-Vio770) and fluorophore-matched isotype controls (all Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were analyzed using a MACSquant flow cytometer and MACSquantify software (version: 2.4, both Miltenyi Biotec) with a 3% threshold (isotype control vs. specific antibody).