Mtesr1

For non-integrating reprogramming, oriP/EBNA1-based pCEP4 episomal vectors (Invitrogen) expressing Oct4, Sox2, Klf4, L-Myc and Lin28 were cotransfected into 10⁶ cells using Amaxa Nucleofector (Lonza, Basel, Switzerland). The cells were then plated onto a Matrigel-coated 10-mm dish and cultured in fibroblast medium. After 24 h, the medium was replaced by N2B27 medium supplemented with 100 ng ml⁻¹ basic fibroblast growth factor. The medium was changed every other day, up to 15 days post-transfection. Then the medium was replaced by mTeSR1 (STEMCELL Technologies, Vancouver, Canada) and changed every day. The hESC-like colonies emerged were picked onto new Matrigel-coated dishes for expansion and characterization. Established human iPSC lines were cultured in mTeSR1 medium on dishes coated with Matrigel (BD, Franklin Lakes, NJ, USA).

The hiPSC lines WD39 and 201B7 were used in this study^{20 (link),38 (link)}. Human iPSCs were cultured in Matrigel-coated 6-well plates with mTeSR-1 (BD Bioscience, CA, USA). Medium was changed daily, and hiPSCs were passaged with collagenase IV (Thermo Fisher Scientific, MA, USA). LT-NCLC induction was slightly modified from that previously described^{37 (link)}. hiPSCs were detached using collagenase IV and were then cultured in neural crest induction medium on 6-well adhesive dishes (Greiner Bio One, Kremsmünster Austria). Induction medium was composed of neurobasal medium (Thermo Fisher Scientific) and Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (Thermo Fisher Scientific) with 1% Gem 21 neuroplex (Gemini Bio-Products, CA, USA), 0.5% of x100 GlutaMax (Thermo Fisher Scientific), 0.5% N2 supplement (Thermo Fisher Scientific), 20 ng/ml of human epidermal growth factor (ReproTech, MO, USA), 20 ng/ml of human basic fibroblast growth factor (ReproTech), 20 ng/ml of insulin (Nacalai Tesque, Kyoto, Japan), and 0.5% penicillin and streptomycin. Induced cells formed spheres until day 4, and then formed spindle-shaped cells 9–10 days after induction.

All cells used in this study was listed in Table S1. These cells were generated and cryopreserved before this study. Specifically, 12 hCiPSC cell lines were used, with 7 for biomarker discovery and 5 for biomarker verification. Additionally, 13 hCiPSC-islets were used, with 8 for biomarkers discovery and 5 for biomarkers verification. When used, these cells were thawed in a 37 °C water bath, centrifuged at 350g for 3 min, and resuspended in different media. hCiPSC-islets were resuspended in DMEM-basic with 1% B27 (Gibco, 12587–010), while hCiPSCs were in mTeSR1 (STEMCELL Technologies, 85850). hCiPSCs were further dissociated into single cells with Accutase (EMD Millipore, SCR005) and both type of cells was counted with Countess II Automated Cell Counter (Invitrogen, AMQAX1000).
hCiPSCs were further differentiated into islets using a modified six-stage protocol^{8 (link)}. Cryopreserved hCiPSCs were recovered and cultured on Matrigel-coated dishes (BD BioSciences, 356231, 1:40 diluted) in mTeSR1 medium with 5% CO₂ at 37 °C. hCiPSCs were then differentiated in vitro. Cells at stage1, 3, and 4 were partially harvested and stage 6 was fully harvested for analysis their RNA expression of target markers.