Heregulin β1

Both canine MSCs and CDCs were cultured in the following differentiation media for 12 days: (i) cardiomyocyte differentiation media: IMDM with 1% N2 (Cat no. 12440; Gibco, Carlsbad, CA, USA), and 100 ng/ml Heregulin-β1 (Cat no. 100-03; Peprotech, Rocky Hill, NJ, USA); (ii) smooth muscle differentiation media: IMDM with 10 ng/ml platelet-derived growth factor-beta (Cat no. 100-14B; Peprotech) and (iii) endothelial differentiation media: IMDM with 50 ng/mL VEGF (Cat no. 100-20A; Peprotech) 18 . After the differentiation process, the cells were fixed with 4% PFA, blocked/permeabilized with Protein Block Solution (DAKO) containing 1% saponin (Sigma-Aldrich), and then stained with mouse anti- α-sarcomeric actin (α-SA) (Sigma-Aldrich), mouse anti-smooth muscle actin (Sigma-Aldrich) and rabbit anti-von Willebrand factor (Abcam) antibodies. FITC or Texas-Red secondary antibodies were obtained from Abcam as well. Cell nuclei were counter-stained with 4’,6-diamidino-2-phenylindole (DAPI).

For Schwann precursors, NBC-NCCs were seeded at 5 × 10⁴ cells/cm² on Matrigel-coated dishes and cultured for 40 days in NC medium supplemented with 20 ng/mL heregulin-β1 (PeproTech) (Liu et al., 2012 (link)); medium was changed every 3 days. To generate peripheral neurons, NBC-NCCs were seeded at 10⁵ cells/cm² on Matrigel-coated dishes, and were differentiated for 10 days in DMEM/F12 supplemented with 1% penicillin-streptomycin solution, 1× N2 supplement (Thermo Fisher Scientific), 10 ng/mL brain-derived neurotrophic factor, 10 ng/mL glial cell line-derived neurotrophic factor, 10 ng/mL NT-3 (PeproTech), 10 ng/mL NGF (BioLegend), 0.5 mM db-cAMP, and 200 μM ascorbic acid (Menendez et al., 2013 (link)). Melanocyte precursors were induced by seeding 5 × 10⁴ cells on Matrigel-coated dishes in E6 medium supplemented with Normocin, 1 μM CHIR99021, 100 nM endothelin-3 (Tocris), and 1 μM SJ000291942 (Sigma). Medium was changed every 3 days.

Primary SCs were prepared from the sciatic nerves of C57BL/6 mice aged 3–5 days as previously described [27 (link)]. Briefly, after removal of the epineurium, sciatic nerves were cut into small slices and then digested at 37 °C in 0.05% collagenase/dispase (Roche, 10,269,638,001, Mannheim, Germany) for 1 h, followed by digestion using 0.25% trypsin (Gibco, 25,200,056, NY, USA) for 15 min. Dissociated cells were resuspended in complete culture medium consisting of DMEM/F12 medium, 10% fetal bovine serum, 10 ng/mL heregulin-β-1 (Peprotech, 100–03, NJ, USA), 2 μM forskolin (Sigma, F6886, MA, USA) and 1% penicillin/streptomycin, and then planted in cell plates pre-coated with poly L-lysine and laminin. After 48 h of culture, half of the culture medium was replaced with fresh culture medium containing 10 μM Ara-C (MCE, HY-13605, NJ, USA) for further culture of 2 days. The media was then switched to a complete culture medium for 2 days for further cell growth. The cell passage was digested with 0.125% trypsin to remove fibroblasts. A purity of SCs greater than 95% identified by immunostaining of S100β and p75 neurotrophin receptor permits the subsequent experiments. Our study was approved by the Ethics Committee of Medical Science Research of the Affiliated Hospital of Jining Medical University (Reference number, 2022B005).

KS-EMPD-1 cells (originally established by Ito et al. ^{60 (link)}) were cultured in Endothelial Cell Growth Medium 2 Kit (C-22111; Takara Bio Inc., Tokyo, Japan) supplemented with 10 ng/mL Heregulin β1 (100-03; PeproTech, Cranbury, NJ). NHEKs (00192907; Lonza, Basel, Switzerland) were cultured in KGM-Gold Basal Medium with SingleQuots supplements (00192060; Lonza). Medium was refreshed every 2–3 days and cells were passaged at 80% confluence using 0.05 w/v% trypsin/EDTA (202-16931; Fujifilm Wako Pure Chemicals). Contamination of mycoplasma was tested using CycleavePCR Mycoplasma Detection Kit (CY232; Takara Bio Inc.). Cells were confirmed to be mycoplasma free.

Undifferentiated CyT49 hESCs (ViaCyte, Inc. San Diego CA) were maintained on EmbryoMAX Primary Mouse Embryo Fibroblasts (MEF) feeder layers (Millipore) in 10/10 media [DMEM/F12 (Cellgro), 10% XenoFree KnockOut Serum Replacement (Life Technologies), 1x MEM non-essential amino acids (Life Technologies), 1x GlutaMAX (Life Technologies), 1x penicillin/streptomycin (10,000 U/mL) (Life Technologies), 10 nM β-mercaptoethanol (Sigma), supplemented with 10 ng/mL Activin A (R&D) and 10 ng/mL Heregulin-β1 (Peprotech)] [31] (link), [32] (link). Cells were split twice weekly and plated at a density of 5×10⁵ or 1×10⁶ on 35 and 60 mm plates, respectively. Cells to be differentiated were plated on Growth Factor Reduced BD Matrigel Matrix (BD Biosciences; 1∶75 in DMEM/F12) coated plates. The cells derived in this study may be obtained upon written consent from ViaCyte Inc.

Human airway organoids were generated from human lung tissue as previously described [37 (link)]. Briefly, the lung tissues were obtained from the Matthay lab (UCSF) and underwent enzymatic digestion to obtain single cells, which were then resuspended in Basement Membrane Extract (BME, R&D Systems). This single-cell mixture was plated into droplets, which were submerged in a specialized human airway organoid (HAO) medium consisting of 1 mM HEPES (Corning), 1x GlutaMAX (Gibco), 1x Penicillin-Streptomycin (Corning), 10% R-spondin1 conditioned medium, 1% B27 (Gibco), 25 ng/mL noggin (Peprotech), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 5 nM heregulin-β1 (Peprotech), and 100 μg/mL Primocin (InvivoGen) in DMEM. This HAO medium was also supplemented with 5 μM Y-27632, 500 nM A83-01, 500 nM SB202190, 25 ng/mL FGF7, and 100 ng/mL FGF10 (all obtained from Stem Cell Technologies). The HAO medium was refreshed bi-weekly, and the droplets were transferred into a single-cell suspension every two weeks to facilitate growth. To induce differentiation, the HAO medium was substituted with a mixture of HAO medium and PneumaCult-ALI Medium (Stem Cell Technologies) at a 1:1 ratio.