The maintenance and passaging of iPSCs were performed using a TECAN liquid handling platform as described41 (link). Briefly, iPSCs were generated by the nucleofection (Lonza) of episomal vectors expressing OCT-4, SOX2, KLF4, L-MYC, LIN28, and shRNA against p53122 (link) in feeder- and serum-free conditions using TeSR™-E7™medium (Stem Cell Technologies) as described41 (link). Pluripotent cells were selected using anti-human TRA-1-60 Microbeads (Miltenyi)43 (link) and subsequently maintained onto vitronectin XF™-coated plates (Stem Cell Technologies) in StemFlex™ (Thermo Fisher Scientific), with media changes every second day and weekly passaging using ReLeSR™ (Stem Cell Technologies). Pluripotency was assessed by expression of the markers OCT3/4 (sc-5279, dilution 1/40, Santa Cruz Biotechnology) and TRA-1-60 (MA1-023-PE, dilution 1/100, Thermo Fisher Scientific) by immunocytochemistry, and virtual karyotyping by CNV array on all lines, as described in ref. 41 (link). Only geographic atrophy lines were generated for this study, as all control lines were already generated, and characterized in ref. 44 (link).
Vitronectin xf coated plates
Manufactured by STEMCELL
Vitronectin XF™-coated plates are specialized cell culture plates designed to support the growth and maintenance of various cell types. The plates are coated with vitronectin, a naturally occurring extracellular matrix protein, which provides a defined and xeno-free substrate for cell attachment and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Tesr e8 medium, by STEMCELL (2 mentions)
Tesr e7 medium, by STEMCELL (1 mentions)
Relesr, by STEMCELL (1 mentions)
Sc 5279, by Santa Cruz Biotechnology (1 mentions)
Ma1 023 pe, by Thermo Fisher Scientific (1 mentions)
Stemflex, by Thermo Fisher Scientific (1 mentions)
Nucleofection, by Lonza (1 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (1 mentions)
Hescs h1 and h9, by WiCell (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
3 protocols using vitronectin xf coated plates
1
Generation and Characterization of iPSCs
2
Characterization of Control hiPSC Lines
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Two independent control hiPSC lines were used in the current study. Cells were generated from skin fibroblasts of a female: LUMC0030iCTRL12 (030) and a male: LUMC0004iCTRL10 (004) by the LUMC iPSC core facility and registered at the Human pluripotent stem cell registry. Cells were characterized according to pluripotent potential and spontaneous differentiation capacity by the iPSC core facility (Dambrot et al. 2013 (link)) and were karyotyped after 15 passages in culture.
hiPSCs were maintained under standard conditions (37 °C, 5% CO2) in TeSR-E8 medium (STEMCELL Technologies) on VitronectinXF-coated plates (STEMCELL Technologies). The medium was refreshed daily, and cells were passaged in aggregates using Gentle Cell Dissociation Reagent (STEMCELL Technologies) upon reaching approximately 80% confluency. Human BMSCs and hPACs were collected from OA patients undergoing joint replacement surgery as part of the RAAK study. Collection and expansion of the primary cells has been previously described (Bomer et al. 2016 (link)).
hiPSCs were maintained under standard conditions (37 °C, 5% CO2) in TeSR-E8 medium (STEMCELL Technologies) on VitronectinXF-coated plates (STEMCELL Technologies). The medium was refreshed daily, and cells were passaged in aggregates using Gentle Cell Dissociation Reagent (STEMCELL Technologies) upon reaching approximately 80% confluency. Human BMSCs and hPACs were collected from OA patients undergoing joint replacement surgery as part of the RAAK study. Collection and expansion of the primary cells has been previously described (Bomer et al. 2016 (link)).
3
Metabolic Profiles of PSCs and Somatic Cells
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To compare the metabolic differences between PSCs and somatic cells, hESCs (H1 and H9; WiCell Research Institute), hiPSCs (CMC-hiPSC-003 and CMC-hiPSC-009; (National Center for Stem Cell and Regenerative Medicine) and fibroblast cell lines (BJ1 and MRC5) were used. This study was approved by the Institutional Review Board (IRB No: P01-202309-03-001). These PSCs were maintained in complete TeSR-E8 Medium (STEMCELL Technologies) as the glucose-free culture. Cells were cultured on Vitronectin XF-coated plates (STEMCELL Technologies) and dissociated by incubation with TrypLE Express (Gibco) at 37 for 3 minutes. The fibroblast cell lines, BJ1 and MRC5, were maintained in Dulbecco's modified Eagle medium (high-glucose DMEM; Corning Life Sciences) supplemented with 10% fetal bovine serum (FBS; Corning Life Sciences) and 100× penicillin-streptomycin (Corning Life Sciences). hESCs and hiPSCs were routinely passaged every 3 days using TrypLE Express, whereas fibroblasts were passaged every 3 5 days using 0.05% trypsin-ethylenediaminetetraacetic acid. All cells were maintained in an incubator at 37 and 5% CO 2 .
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