Ldn193189

The neuronal differentiation protocol was modified from a previously published paper (Giulitti et al., 2019 (link)). GV-extract reprogrammed cells were seeded on Matrigel-coated plates at a density of 530 cells/mm² and cultured in mTeSR1 Plus medium (STEMCELL Technology, 100-0276) for 6 days. From day 0 to 2, cells were cultured in neural medium (N2B27, 1% NEAA, 200 ng/ml L-ascorbic acid) added with 20 ng/ml bFGF (STEMCELL Technology, 78003.1) and 0.1 μM LDN 193189 (STEMCELL Technology, 72147). The cells were then cultured in neural medium supplemented with 0.1 μM LDN 193189 and 10 μM SB431542 (STEMCELL Technology, 72234) on day 3. The following 6 days, the same medium used on day 3 was supplemented with 1 μM all-trans retinoic acid (Merck, R2625) and 1 μM SAG (Merck, 566661). On day 10 to 15, the cells were cultured in neural medium supplemented with 5 μM DAPT (Merck, D5942), 4 μM SU-5402 (Merck, SML0443), 1 μM all-trans retinoic acid and 1 μM SAG. On day 16, cells were seeded on glass-bottom chamber slide and cultured in maturation medium, which contained neural medium supplemented with 20 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml CNTF (PeproTech, 450-02, 450-10, 450-13) and 10 μM ROCK inhibitor (STEMCELL Technology, 72302). The cells were cultured in maturation medium up to day 22 and fixed for immunostaining. All the culture media were changed daily.

The neuroectoderm differentiation was based on Douvaras and Fossati (2015 (link)). In brief, 90% confluent hESCs were subjected to neural induction for 8 days using 100 nM RA (Sigma-Aldrich), 10 μM SB431542 (Tocris), and 250 nM LDN193189 (STEMCELL Technologies). The induction of cardiac progenitor differentiation was based on Lin and Zou (2020 ). hESCs at 80%–90% confluence were treated with 5 mM CHIR99021 (Tocris) for 24 h, after which the cells were cultured in medium with 0.6 U/mL heparin (Sigma-Aldrich) for 24 h. Subsequently, the medium was supplemented with 0.6 U/mL heparin and 3 mM IWP2 (Tocris) for another 3 days. The 8-day depatoblast differentiation was based on Boon et al. (2020 (link)). Differentiation was initiated at 40%–50% confluency, and the hESCs were treated with with 50 ng/mL activin A (STEMCELL Technologies), 50 ng/mL WNT3A (PeproTech), and 6 μL/mL DMSO (Sigma-Aldrich) for 48 h. The cells were incubated for an additional 48 h in the same medium without WNT3A. Then, the medium was changed to contain 50 ng/mL BMP4 (STEMCELL Technologies) and 6 μL/mL DMSO for the following 4 days. More details on the differentiation and the media composition can be found in the supplemental information.