Stemmacs ips brew xf

Human iPSC/ESCs (Table S1) were maintained on dishes coated with Matrigel in mTeSR1 or StemMACS iPS-Brew XF (Miltenyi Biotec) media with 1% penicillin/streptomycin. iPSCs were passaged weekly with ReLeSR (STEMCELL Technologies) with daily media changes except for the day following passaging. Accutase (Innovative Cell Technologies) and media with 10 μM Rho-associated kinase (ROCK) inhibitor (Y-27632, STEMCELL Technologies) was used for any single-cell dissociation. Routine mycoplasma testing was performed.

Approval for the use of hESCs used in this study was granted by the MRC Steering Committee for the UK Stem Cell Bank and for the Use of Stem Cell Lines (ref, SCSC13‐19). RC17 hESCs (~passages 25‐30) and AST18 iPSCs were maintained in self‐renewing conditions on Laminin‐521 (L521, 5 μg/ml, Biolamina) coated 6‐well plates (Corning), and fed daily with StemMACS iPS‐Brew XF (iPS‐B, Miltenyi Biotec). Every 2 or 3 days, once the hESCs reached 70%–90% confluency, the cells were passaged as clumps with EDTA (0.5 mM, Thermo Fisher Scientific) at a split ratio of 1:3 to 1:8. See Supporting Information Table S1 for all catalogue numbers.

The human iPSC cell line WTC11 carrying a doxycycline-inducible Ngn2 transgene (Miyaoka et al. 2014 (link); Wang et al. 2017 (link)) were cultivated as previously described (Bell et al. 2021 (link); Buchholz et al. 2022 ). Briefly, iPSCs were cultured on GelTrex™-coated plates (1X, ThermoFisher Scientific) in StemMACS™ iPS-Brew XF (Miltenyi). When reaching confluence, the cultures were passaged with Versene™ passaging solution (ThermoFisher Scientific) and seeded in thiazovivine-supplemented (Axon Medchem) iPS-Brew for 1 day. Cells were grown at 37 °C and 5% CO₂ in a humidified incubator.

Mesenchymal stromal cells were isolated from bone marrow (caput femoris) of patients undergoing orthopedic surgery and culture-expanded in standard medium consisting of Dulbecco’s Modified Eagle Medium (DMEM; 1 g/l glucose; PAA, Pasching, Austria), 1% L-glutamine (PAA), 1% penicillin/streptomycin (PAA), and 10% pooled human platelet lysate (hPL) [35 (link)]. The medium was supplemented with 0.1% heparin (5000 IU/ml; Ratiopharm, Ulm, Germany) to prevent coagulation [36 (link)].
Induced pluripotent stem cells were generated from three MSC preparations with episomal plasmids [37 (link)] and thoroughly characterized as described before [38 (link), 39 (link)]. iPSCs were cultured on tissue culture plastic (TCP) coated with vitronectin (0.5 mg/cm²) in StemMACS iPS-Brew XF (all Miltenyi Biotec, Bergisch Gladbach, Germany). Pluripotency was validated by in vitro differentiation and Epi-Pluri-Score (Cygenia GmbH, Aachen, Germany) [40 (link)].
Generation of iPSC-derived MSCs (iMSCs) was performed by switching culture conditions to standard hPL-medium and further passaging on 0.1% gelatin-coated plates [20 (link)]. Three-lineage differentiation potential of MSCs and iMSCs was validated as described before [41 (link), 42 (link)]. Cell images were taken on a digital EVOS FL Auto microscope (Thermo Fisher Scientific, Carlsbad, California, USA).

The hESC lines, H1 and H9, and hiPSC lines, 253G4 and 4A (HiPS-RIKEN-4A), were maintained in the human ESC medium as previously described.^{23 (link)} The hESC lines (H1 and H9) were purchased from WiCell Research Institute (WI, United States). The cell lines 253G4 and 4A were kindly provided by Prof. Shinya Yamanaka (Kyoto University, Kyoto, Japan) and Dr. Yukio Nakamura (RIKEN BioResource Center, Tsukuba, Japan), respectively. All hPSC lines were transferred to feeder-free conditions. The hPSC colonies were detached from the feeder cells by CTK solution (0.1% collagenase IV, 0.25% trypsin, 20% KSR, and 1 mM CaCl₂ in phosphate-buffered saline [PBS]). They were also passed through a 40-μm cell strainer to remove feeder cells, and the colonies remaining on the cell strainer were plated on vitronectin-coated (VTN; Life Technologies), Matrigel-coated (BD Matrigel hESC-qualified Matrix; BD Biosciences) or iMatrix-511-coated (nippi, Inc.) dishes in StemMACS™ iPS-Brew XF (Miltenyi Biotec) or Pluripro MATRIX-coated (CELL guidance systems) dishes in Pluripro media (CELL guidance systems). For single-cell-state culture, hPSC colonies were detached using Accutase (Innovative Cell Technologies) and were plated on VTN-, Matrigel-, or iMatrix-511-coated dishes in StemMACS iPS-Brew XF or Pluripro MATRIX-coated dishes in Pluripro media with 10 μM Y-27632 (Nacalai Tesque).