Cucurbitacin

Differentiation was initiated 72 h after plating when the culture was approximately 80% confluent.
Step 1: Cells were differentiated with 1.5 μM CHIR99021 (CHIR, Stem-RD), 20 ng/mL BMP4 and 20 ng/mL Activin A in RPMI (Cellgro) supplemented with B27 minus insulin, 2 mM GlutaMAX, 1x NEAA and 1x Pen/Strep for 3 days (RB27-INS).
Step 2: For hPSC-V cardiomyocyte differentiation, cells were treated for an additional 3 days with 5 μM XAV939 (Tocris). From day 6 onward, hPSC-V differentiation were carried out in RPMI supplemented with B27, 2 mM GlutaMAX, 1x NEAA and 1x Pen/Strep (RB27+INS). The differentiation efficiency was approximately 85–90% (Tsai et al., 2020 (link)).
For hPSC-SAN differentiation, Step 1 was followed by addition of 0.1 μM cucurbitacin (Sigma Aldrich), 1 μM retinoic acid, 5 μM SU5402 (SU, Tocris) in RB27-INS from day 3–6. 5 μM XAV939 was added from day 5–6. From day 6–9, cells were cultured with 5 μM Tyrphostin AG 490 (Sigma Aldrich) in RB27+INS. At day 7–9, 5 μM chidamide was added to the differentiation cocktail. After day 9, hPSC-SAN differentiation was carried out in RPMI supplemented with B27, 2 mM GlutaMAX, 1x NEAA and 1x Pen/Strep (RB27+INS).

To assess the involvement of JAK/STAT3‐mediated signaling, Cucurbitacin I (Sigma‐Aldrich), a selective inhibitor of the JAK/STAT3 signaling pathway that suppresses the levels of tyrosine phosphorylated STAT3 9, was used at a concentration of 60 nM. Cucurbitacin was supplemented to 48‐hours BCM from NI‐MiMPCs and control MiMPCs, and NI‐MSCs and control MSCs, which were subsequently used to culture chick DRGs for 5 days. Cultures were paraformaldehyde‐fixed and immunostained for heavy neurofilament, and branching complexity of neurite outgrowth was assessed (see below).