E8 media

HEK293T cells were purchased from ATCC (CRL-3216) and cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep at 37 °C, 5% CO₂. Human iPS cell line HMGUi001-A2 (sex: female), a sub-clone of HMGUi001-A^{57 (link)}, was kindly provided by Dr Kühn (MDC) and authenticated by karyotyping. Human iPSCs were maintained on Geltrex-coated (Invitrogen) plates in E8 media (STEMCELL Technologies). The medium was changed daily, and cells were passaged every ∼3 days as cell clumps or single cells using 0.5 mM EDTA (Invitrogen) or Accutase (Invitrogen), respectively. Medium was supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Sigma) when iPSCs were thawed or passaged as single cells. All cell lines tested negative for mycoplasma contamination which was carried out routinely.
For transient transfection experiments in HEK293T cells were transfected using Polyethylenimine “Max” (PEI) (Polysciences) in OptiMEM medium (Invitrogen).

The PSC were differentiated into macrophages according to protocol as described previously by our group (Han et al., 2019 (link)). hESC H9 (WiCell) was maintained on a Matrigel-coated 35-mm dish using mTeSR 1 (STEMCELL), and the medium was replenished every day. ThehiPSC line CMC-hiPSC-003 was obtained from Korea National Stem Cell Bank (Kim et al., 2021 (link)) and maintained on a vitronectin-coated 35 mm dish using E8 media (STEMCELL). When colonies of both H9 or CMC-hiPSC-003 grew to approximately 500 μm in diameter, cellular differentiation was induced using mesoderm differentiation medium (APEL 2 supplemented with 1X insulin-transferrin-selenium-X [Invitrogen] and 100 ng/ml BMP4). After 2 days, 100 ng/ml BMP4 was replaced with 20 ng/ml BMP4 and incubated for 2 days. On day 4, BMP4 was replaced with 40 ng/ml VEGF and 50 ng/ml SCF. Two days later, the medium was replaced with hematopoietic differentiation medium (APEL 2 supplemented with 1X insulin-transferrin-selenium-X [Invitrogen], 50 ng/ml SCF, 10 ng/ml TPO, 50 ng/ml IL-3, 50 ng/ml IL-6, and 50 ng/ml Flt-3L). On day 15, floating cells were harvested and incubated in macrophage differentiation medium (RPMI 1640 supplemented with 100 ng/ml M-SCF).

hPSC lines, H1, Hues8, and FUCCI-H9, were cultured under feeder-free on hESC-qualified Matrigel (BD Biosciences) in E8 media (Stemcell Technologies) with 30% of irradiated mouse embryonic fibroblasts (iMEFs) conditional media. FUCCI-H9 hPSCs were kindly provided by S. Dalton29 (link) and were maintained under G418 sulfate (Sigma, 200 ug/ml) and Puromycin (0.1 ug/ml) selections. Cells were passaged every 3–5 days at 80% confluent with TrypLE Express (Invitrogen). After dissociation, cells were plated in E8 media with 10 uM Y-27632, (StemGent) for 24 hrs. After 24 hrs media, without Y-27632, was replenished daily. iMEF conditional media was prepared by incubating iMEFs with hPSC media without bFGF for 24 hrs for 7 days. Collected media was filtered, flash frozen and stored at −80 ^oC.
To maintain neural progenitors, cells were cultured in NPM media: DMEM/F12: Neurobasal media at a 1:1 ratio +0.5xB27 +0.5xN2 + 2 mM Glutamax +20 ng/mL bFGF (R&D Systems) +20 ng/mL EGF (R&D Systems) and media was replenished every four days. To split cells, cells were washed with PBS and incubated for 5 min at 37 ^oC in Accutase (Innovative Cell Technologies). Cells were washed, spun down at 1000 g for 5 min, and replated in NPM media on Geltrex-coated plates.

iPSCs were seeded in ultra-low adherence 96-well plates and cultured in E8 media (Stemcell Technologies) with 0.5% polyvinyl alcohol (Sigma) for 24 h. Subsequently, the cells were cultured for 2 weeks in E8 media, refreshed every 2–3 days, then plated onto vitronectin-coated 8 well chamber slides and cultured in E8 medium for 3 weeks. EBs were fixed with 4% Paraformaldehyde for 10 min, permeabilised in 0.2% Triton X-100 (Sigma) for 10 min and blocked in 2% Bovine Serum Albumin (Life Technologies) for 60 min at room temperature. Cells were then incubated with primary antibodies at 4 °C overnight, followed by an incubation with secondary antibodies for 60 min at room temperature. The coverslips were mounted on slides with DAPI to stain the nuclei (VectorLabs). Images were captured with an LSM 780 confocal microscope running Zen Black software (Zeiss version 2.3 SP1). Antibodies used are detailed in Supplementary Data 4. Images for SMA and SOX17 are a single slice. MAP2 are maximum projections of a stack of 10 slices at 0.8 μm intervals to allow visualisation of axonal projections.