Dnase 1

As described above, animals were perfused transcardially and brains were fixed overnight in 4% PFA, then gradually dehydrated and stored in 70% ethanol (EtOH). Paraffin-embedded brain tissue was sectioned on a microtome (Leica, RM45) for 7-µm-thick sections. For further experiments following sections from the region of the frontal cortex, anterior and posterior hippocampus were collected. Next, chosen microscope slides with tissue sections were deparaffinized in xylene and declining concentrations of EtOH and remained in 0.01 M PBS. The apoptotic signal was assessed using Click-iT™ Plus TUNEL Assay for in situ apoptosis detection with Alexa Fluor™ 488 picolyl azide dye. For positive control, brain slices were incubated with 1 unit of DNAse I (A&A Biotechnology, Gdansk, Poland) as recommended by manufacturer. Vectashield Mounting Medium (Vector Laboratories) with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) was used for slipping and counterstaining the slides. Stained slides were examined under a fluorescence microscope (Nikon Eclipse50i, Japan) equipped with a camera and NIS Elements software.

Forty-eight hours post-transfection, total RNA was isolated from cells using GeneMATRIX Universal RNA Purification Kit (Cat No. E3598, EURx, Gdansk, Poland) with the step of the on-column DNase I (Cat No. 1009-100, A&A Biotechnology, Gdynia, Poland) digestion according to the producer’s instructions. Five hundred nanograms of total RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Cat No. 4374966, ThermoFisher Scientific, Inc.), following the recommended protocol. The expression of the genes was quantified by RT-qPCR using a 5x HOT FIREPol EvaGreen qPCR Mix Plus (Cat No. 08-25-00020, Solis BioDyne, Tartu, Estonia), 6-times diluted cDNA, and 0.5 µM of specific oligonucleotide primers (listed in Table 2). RT-qPCR was performed in the CFX96 Detection System (Bio-Rad, Hercules, CA, USA) with one cycle of 15 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 20 s at 58 °C, and 20 s at 72 °C. The expression of target genes was calculated using the 2^−ΔΔCt method and normalized to the 18S rRNA reference gene.

Murine GRP cells were received as a gift from the Department of Radiology and Radiological Science, Johns Hopkins School of Medicine (Baltimore, MD, USA). Cells were isolated as previously described by Srivastava, Bulte et al. [17 (link)] from spinal cords dissected from ^{Luc+/PLP/GFP+} mice between E12.5 and E14 stage. Tissue samples were plated on a Petri dish in DMEM/F12 medium (Thermo Fisher, Waltham, MA, USA), then incubated in pre-warmed TrypLE Express (Thermo Fisher, Waltham, MA, USA) with 10 mg/mL DNase-1 (A&A Biotechnology, Gdansk, Poland) for 10–12 min and incubated for a further 10 min. Next, 5 mL of GRP medium was added and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in 10 mL of GRP medium with 10 mg/mL of DNase, and incubated at 37 °C in a humidified incubator with 5% CO₂ for 10 min. The pellet was then mechanically agitated and centrifuged at 1000 rpm for 5 min; resuspended in 10 mL of GRP medium (DMEM/F12 medium (Thermo Fisher, Waltham, MA, USA); supplemented with N2 and B27 (Life Technologies, Carlsbad, CA, USA), 1% BSA (Abcam Cambridge, Great Britain), Penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA), and 20 ng per mL bFGF (Peprotech, Rocky Hill, NJ, USA)); and plated on coated with poly-L-lysine and laminin (Sigma, Saint Louis, MI, USA) 25 mL flasks in a humidified incubator at 37 °C with 5% CO₂.

Brains and spinal cords were dissected from canine fetuses between E32–37. Briefly, the tissue was incubated in pre-warmed TrypLE Express (Gibco) with 10 mg/ml DNase-1 (A&A Biotechnology, Gdansk, Poland) for 10–12 mins, gently triturated and incubated in 37 °C for 10 min. Next, 5 ml of GRP medium (Gibco) was added and the suspension was centrifuged at 1000 rpm for 5 mins. Obtained pellet of cells was resuspended in 10 ml of GRP medium with 10 mg/ml of DNase, incubated at 37 °C in humified atmosphere with 5% CO₂ for 10 mins. Then, the pellet was triturated again and centrifuged at 1000 rpm for 5 mins, resuspended in 10 ml of GRP medium and plated on coated PLL/Laminin 25 ml flasks in 37 °C in humified incubator with 5% CO₂. Cells were cultured for 5–10 days (1–2 passages) in GRP medium with bFGF, harvested with TrypLE Express (Gibco), cryopreserved in ATCC medium (LGC Standards, Teddington, UK), and stored in vapor phase liquid nitrogen until transplantation.