Geltrex matrix

Transwell chambers with 24 wells and an 8 μm polycarbonate membrane (Corning, NY, USA) were used, following the manufacturer’s protocol. The upper chambers were coated with 100 µL of DMEM-diluted Geltrex matrix (Gibco) and incubated at 37 °C for 6 h to allow the gel to solidify. The cultured cells were detached using 0.25% trypsin-EDTA solution and counted in a Neubauer chamber. Then, 1 × 10⁵ cells were seeded into the upper chambers in 200 µL of serum-free media. A total of 500 µL of complete medium was added to the lower chamber as a chemo attractant. After 12 h, the cells remaining in the upper side of the polycarbonate membrane were removed with cotton swabs. Bottom chambers containing invasive cells were washed (twice with PBS), fixed in 100% methanol, and stained with DAPI (5 μm) for 5 min. Ten visual fields of each insert were randomly chosen, and photographed at 4x magnification. The number of cells/fields was quantified using ImageJ software (NIH, Bethesda, MD, USA).

NPCs were cultured, as mentioned earlier [76 (link)]. In brief, embryos (E14) were isolated from a Sprague–Dawley rat. Cortices were aseptically dissected from brains and dissociated to produce single-cell suspensions by gentle mechanical pipetting using fire-polished Pasteur pipettes. In the presence of epidermal growth factor (10 ng/mL) and fibroblast growth factor-2 (10 ng/mL), cells were then grown overnight in 5 mL of medium supplemented with DMEM-F12/Glutamax (Gibco, ThemoFisher, Waltham, MA, USA), penicillin–streptomycin (1%, Gibco) and B27 supplement (1%, Gibco) in 25 cm² flasks [77 (link)]. For ICC and PLA, coated coverslips (poly-D-lysine/laminin) were used to plate floating neurospheres. The transfection of neurospheres with specified plasmids was done by Lipofectamine^® 2000 reagent (Invitrogen). Geltrex^® matrix (Gibco) was used for transfection and live-cell imaging. The plated neurospheres were further grown for 48 h to attain differentiated cortical neurons in the medium containing 2% B27 supplement, 500 mM L-glutamine, and 1% penicillin–streptomycin with no growth factors. After culturing for 48–60 h, differentiated cortical neurons cultures were used for live-cell imaging and ICC.

4′-(p-Tolyl)-2,2′:6′,2″-terpyridine,
4-(trifluoromethyl)benzyl bromide, iodomethane, 2-phenylbenzimidazole,
4-chloro-3-nitrobenzoic acid, butylamine, zinc in powder, ammonium
formate, 3,4-(methylenedioxy)benzaldehyde, 2-naphthaldehyde, 1,2-phenylenediamine,
2-thiophenecarboxaldehyde, triethylamine, trifluoroacetic acid, magnesium
sulfate, potassium hexafluorophosphate, dimethyl sulfoxide (DMSO),
and ethylene glycol were obtained from Sigma-Aldrich (Madrid, Spain).
IrCl₃ was obtained from Johnson Matthey. Deuterated solvents
were obtained from Euriso-top.
MDA-MB-231, MDA-MB-231 VIM RFP,
MCF-7, and MCF10A were from ATCC. Cells were maintained in Dulbecco’s
minimum essential medium (Biosera) supplemented with 10% FBS (Biosera)
and gentamicin (Merck). Cells were cultured in a humidified CO₂ (5%) incubator and subcultured 2–3 times per week
according to the proliferation of each cell line, but still at the
sub-confluent state, to maintain exponential growth characteristics.
Sulforhodamine B, doxorubicin, and propidium iodide were from Merck
(Germany). The Geltrex matrix was from Gibco. All the chemicals were
in a purity suitable for cell culture conditions. The purity ≥95%
of the synthesized complexes used for biological evaluation was determined
by NMR and RP-HPLC.

To confirm the iPSC status of reprogrammed donor fibroblasts, we performed a TaqMan iPSC Scorecard Assay^{44 (link)}, which also confirmed the cells’ trilineage potential (Fig. 2b). We followed the protocol described by the manufacturer of the TaqMan hPSC Scorecard Assay (Thermo Fisher Scientific).
Stem cells were cultured on Geltrex matrix (Gibco) in mTeSR^™1 media (StemCell Technologies) under standard incubator conditions of 5% CO2 and humidity. On the day of analysis, the cells were dissociated using Accutase and pelleted by centrifugation. RNA was extracted using a Qiagen extraction kit and cDNA was synthesized as per Scorecard kit instructions. Embryonic bodies were generated as per Scorecard kit instructions, RNA was extracted and cDNA synthesized in the same way as for iPSC pellets. The TaqMan hPSC Scorecard Kit 384w plate was amplified using Lightcycler 480 (Roche Diagnostics) and the data were uploaded to the hPSC Scorecard analysis software available online from Thermo Fisher Scientific. The resulting graphs were downloaded and included in Fig. 2.

Transwell chambers with 24 wells and 8 μm polycarbonate membrane (Corning, New York, USA) were used following the manufacturer’s protocol. Upper chambers were coated with 100 µL of DMEM-diluted Geltrex matrix (Gibco) and incubated at 37 °C for 6 h to allow the gel to solidify. Cultured cells were detached using 0.25% trypsin-EDTA solution. Cells were counted in a Neubauer chamber. Then, 1 × 10⁵ cells were seeded into the upper chambers in 200 µL of serum-free media. A total of 500 µL of complete medium were added to the lower chamber as a chemo attractant. After 12 h, cells remaining in the upper side of the polycarbonate membrane were removed with cotton swabs. Bottom chambers containing invasive cells were washed (twice with PBS), fixed in 100% methanol,and stained with DAPI (5 μm) for 5 min. Ten visual fields of each insert were randomly chosen, and photographed at 40Xmagnification. The number of cells/field was quantified using ImageJ software (NIH, Bethesda, MD, USA).

SH-SY5Y cells were seeded on glass coverslips treated with 0.2℅ gelatin, and NSCs were plated on glass coverslips treated with Geltrex matrix (GIBCO) following the manufacturer's protocol. Cells were fixed with 3.7% formaldehyde, and LDs were stained with 0.3% Oil Red O (diluted in 60% isopropanol) for 2 min at room temperature. The coverslips were mounted on slides using antifade mounting medium (VECTASHIELD®). Nuclear recognition was based on DAPI staining (1 μg/mL) for 5 min. Fluorescence was analyzed by fluorescence microscopy with a 100 × objective lens (Olympus, Tokyo, Japan). The numbers of LDs were automatically quantified by ImageJ software analysis from 15 random fields.