Rhfgf2

The cerebral cortices of mouse embryos at gestational day 14.5 (E14.5) were dissected and mechanically triturated with a P1000 micropipette into single cells as previously described [100 (link),101 (link),103 (link)]. The cells were seeded into 60-mm low-attachment PrimeSurface dishes (Sumilon, Sumitomo Bakelite, Tokyo, Japan) at a density of 1 × 10⁶ cells/dish and cultured in neural stem cell-growing medium containing NeuroBasal medium, 2% B-27 without vitamin A, 2 mM glutamine, 20 ng/mL of rhEGF (Pepro Tech, Cranbury, NJ, USA), and 10 ng/mL of rhFGF2 (Pepro Tech) in a humidified incubator containing 5% CO₂. After 4–5 days, the formed neurospheres were collected and used for further experiments.

We sacrificed pregnant mice at gestational day 14.5 (E14.5) and removed the embryos [28 (link), 29 (link)]. Forebrains (cerebellar cortex or ganglionic eminence) were dissected from the embryonic brains and triturated with a P1000 micropipette for dispersal into single cells. The dispersed cells (1 x10⁶ cells) were seeded into 60-mm low-attachment PrimeSurface (Sumilon) dishes and cultured in neural stem cell growing medium consisting of NeuroBasal medium, 2% B-27 without vitamin A, 2 mM glutamine, 20 ng/mL rhEGF (Pepro Tech), and 10 ng/mL rhFGF2 (Pepro Tech) in a humidified incubator containing 5% CO₂. After 4–6 days, the cells aggregated, proliferated, and formed neurospheres. The neurospheres were collected and used for further experiments.

Bovine PSCs (line #A) [28 , 31 (link)] were cultured in bESC culture medium (bESCM) [N2B27 medium, 1xNEAA, 1xGlutamax (Gibco, #35050061) 0.1 nM β-mercaptoethanol, pen/strep (Gibco, #15140122), 10% AlbumiNZ low fatty acid BSA (MP Biochemicals, #0219989925), 20 ng/μl rhFGF2 (Peprotech, #100-18B), 20 ng/μl rhActivinA (Qkine, Qk001), 2.5 μM IWR-1 (Sigma, #I0161)] on a layer of mitotically-inactivated MEFs (plated on gelatinised tissue culture plastic at a density of 6 × 10⁴/cm²). The MEFs were washed twice with PBS prior to plating PSCs in bESCM. For passaging, PSCs were incubated 1 h with bESCM containing 10 μM Y-27632 prior to dissociating then washed twice with PBS and incubated for 3 min in TrypLE Express (Gibco, #12604013) at 37 °C/5%CO₂. Cells were dispersed to single cell by pipetting and pelleted in 6x volume of bESCM at 300×g for 4 min. Resuspended cells were plated at 1:5 in bESCM + 10 μM Y-27632 overnight then changed to bESCM without Y-27632 and fed daily. Bovine PSCs were passaged every 3–4 days.

Cells were placed in an EPC‐colony‐forming assay (CFA) semisolid culture medium, MethoCult SF H4236 (Stem Cell Tech.), supplemented with rhVEGF (50 ng/mL), rhFGF‐2 (50 ng/mL), rhSCF (100 ng/mL), rhIGF‐1 (50 ng/mL), rhIL‐3 (20 ng/mL), rhEGF (50 ng/mL) (PeproTech), 30% FBS (CCB, Nichirei Biosci., Tokyo, Japan), heparin (2 IU/mL) (Ajinomoto, Tokyo, Japan), and an antibiotic cocktail. EPC‐CFA working medium was diluted with 30% FBS‐IMDM, and 1 mL of suspended PbMNCs was seeded with blunt‐end needles (Nipro, Osaka, Japan) attached to 1‐mL syringes into a 35‐mm Primaria culture dish to promote cell adhesion (2 × 10⁵ cells/dish). On day 16, the number of adherent colonies per dish was measured using a gridded scoring dish (Nunc, Thermo Fisher Scientific) using a phase‐contrast light microscope (Eclipse Ti‐U; Nikon, Tokyo, Japan). Based on the maturation stage of colony‐forming cells, EPCs were classified as primitive EPC colony‐forming units (pEPC‐CFUs) or definitive EPC (dEPC)‐CFUs.

hBM-MNCs were obtained from 4 patients (2M/2F, median age 68) and processed for MPC isolation as described above, applying two different sera batches: LZM, already screened for good performance and BIOW2 that produced cultures with consistent MSC-like cell “contamination.” LZM cultures were also performed in presence of 50 ng/ml rhEGF (ThermoFisher Scientific, Waltham, MA USA), 20 ng/ml rhFGF-2 (ThermoFisher Scientific), 50 ng/ml rhPDGF-AB (PeproTech EC Ltd., London, UK) or 50 ng/ml rhVEGF (ThermoFisher). Combined treatments were also performed adding rhEGF and rhFGF-2, or rhVEGF and rhPDGF-AB. In parallel BIOW2 cultures were also supplemented with 100 ng/ml rhIGF-1 (PeproTech EC Ltd., London, UK), 30 ng/ml rhIGF-2 (PeproTech EC Ltd.) alone or in combination. The culture media were changed every 2 days restoring concentrations of the hGFs. After 7 days cells were detached by TrypLE® Select digestion and processed for flow cytometry to quantify MPC and MSC percentages in the cultures, as described above. Data were collected in duplicate and reported as median values ± standard error (SE). One-way ANOVA test, coupled with Dunnett's post-test, was applied to identify significant differences.