Tryple express

Natural and ETX embryos were transferred to Falcon tubes, washed with PBS and incubated in TrypLE Express (12604013; Gibco) for 15 min at 37 °C to dissociate them into single cells. If clumps remained, the incubation was extended for an additional 5 min at 37 °C and the sample pipetted further. Samples were filtered to remove large clumps, centrifuged at 200g for 5 min and resuspended in PBST (containing 0.02% Tween 20) and then processed for encapsulation, as previously reported^{29 (link),53 (link)}. For E5.5 embryos, one litter of 12 embryos was dissociated together. A total of 15 ETX embryos were dissociated for sequencing. Cells in culture were dissociated into a single-cell suspension using TrypLE Express (12604013; Gibco) and multiplexed using MULTI-seq lipid-modified oligos before running on two 10X Genomics lanes using single-cell 3′ version 3 reagents as reported^{54 (link)}.

For single-cell sequencing of the tissue, mouse thyroid was dissociated with collagenase I (Sigma-Aldrich) as described above and subsequently resuspended in TrypLE Express (GIBCO) preheated to 37 °C and dissociated under repeated pipetting. For single-cell sequencing of TFCOs, organoid droplets were incubated in Cell Recovery solution (354253, Corning) for 30 min in order to dissolve the BME. Then, organoids were pelleted and resuspended in TrypLE Express (GIBCO) preheated to 37 °C and dissociated under repeated pipetting. When the gland and the organoids were fully dissociated into single cells, samples were pelleted, washed, resuspended in fluorescence-activated cell sorting (FACS) buffer (advanced DMEM/F12, 10 μM Y-27632, and DAPI) and strained (35 μm).
DAPI-negative cells were immediately sorted into 384-well plates containing External RNA Controls Consortium (ERCC) spike-ins (Agilent), reverse transcription (RT) primers, and deoxynucleotide triphosphates (dNTPs) (Promega) using a FACSFusion (BD Biosciences). Plates were prepared using Mosquito HTS (TTP Labtech). Single-cell RNA-sequencing libraries were prepared following the SORT-seq protocol (26 (link)), which is based on the CEL-seq2 method (53 (link)). Extensive explanation of the protocol can be found in SI Appendix.

Initially, the H1 ES cell line was differentiated to PP cells using the STEMdiff Pancreatic Progenitor Kit (STEMCELL Technologies, 05120) according to the manufacturer’s instructions. In short, the hES cell colonies were dissociated into single cells using TrypLE Express (Gibco, 12604-013) and seeded on Matrigel-coated plates as described above at a concentration of 95,000 cells/cm² in mTeSR supplemented with 20 µM ROCKi. The medium was replaced the next day by mTeSR and differentiation was initiated by replacing it with the S1d1 differentiation medium when cells were 60–70% confluent, typically 2 d after the initial seeding. Daily washes with DPBS (Gibco, 14190250) and media changes were done until S4d5 when the cells reached the end of PP stage. The monolayer of PP cells was then dissociated using Accumax (STEMCELL Technologies, 07921), and cells were used for expansion under INI conditions.
Later, H1, H9, and CRTD1 iPS cells were differentiated into PP cells using an adaptation of published procedures (Mahaddalkar et al., 2020 (link); Rezania et al., 2014 (link); Shi et al., 2017 (link); Supplementary file 1d). The monolayer of PP cells was then dissociated using TrypLE Express (Gibco, 12604-013) for 2 min, and cells were used for expansion under C0 to C8 conditions.

Brains and spinal cords were dissected from canine fetuses between E32–37. Briefly, the tissue was incubated in pre-warmed TrypLE Express (Gibco) with 10 mg/ml DNase-1 (A&A Biotechnology, Gdansk, Poland) for 10–12 mins, gently triturated and incubated in 37 °C for 10 min. Next, 5 ml of GRP medium (Gibco) was added and the suspension was centrifuged at 1000 rpm for 5 mins. Obtained pellet of cells was resuspended in 10 ml of GRP medium with 10 mg/ml of DNase, incubated at 37 °C in humified atmosphere with 5% CO₂ for 10 mins. Then, the pellet was triturated again and centrifuged at 1000 rpm for 5 mins, resuspended in 10 ml of GRP medium and plated on coated PLL/Laminin 25 ml flasks in 37 °C in humified incubator with 5% CO₂. Cells were cultured for 5–10 days (1–2 passages) in GRP medium with bFGF, harvested with TrypLE Express (Gibco), cryopreserved in ATCC medium (LGC Standards, Teddington, UK), and stored in vapor phase liquid nitrogen until transplantation.

The hESC cell line H9 (WiCell Research Institute, Madison, WI, USA) and hiPSC cell line CMC-hiPSC-003 (Korea Centers for Disease Control and Prevention, Osong, Korea) were maintained in complete TeSR-E8 medium (StemCell Technologies, Vancouver, Canada) for feeder-free culture. These cells were cultured on vitronectin XFTM- (hPSC-qualified; StemCell Technologies) coated plates and dissociated by 3 min incubation at 37 °C with TrypLE Express (Gibco, Grand Island, NY, USA). The fibroblast MRC5 cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM high glucose; Corning, Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum (PSCs quality; Corning) and 100× penicillin-streptomycin (Corning). Thapsigargin (TG, Sigma, St. Louis, MO, USA) was diluted at 10 nM to complete growth media. hESCs and hiPSCs were routinely passaged every 3 days with TrypLE Express (Gibco); fibroblasts were passaged every 4–5 days with 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA). All cells were maintained in an incubator at 37 °C with 5% CO₂.

Human hPSCs, at a confluence of 60–70%, were washed with PBS three times, followed by the application of 1 mL of TrypLE Express (GIBCO) at 37 °C for 3 m for dissociation. Once dissociated into single cells, 9 mL of DMEM/F12 medium supplemented with 20% FBS (GIBCO) was added for neutralization and TrypLE Express washing. The cells were then centrifuged at 300 RCF for 3 m After discarding the supernatant, a second wash was applied by adding 10 mL of DMEM/F12 medium containing 20% FBS followed by centrifugation at 300 RCF for 3 m. The supernatant was discarded and the cells were resuspended in 1 mL E8-Flex medium supplemented with 10 μM of ROCKi. A cell count was performed and the volume of medium was adjusted to obtain cell density of 500,000 cells/mL. A volume of 1 mL was added to a Matrigel-coated well of a 6-well-plate. Immediately after, 1 mL of the 2× MagC solution was added and gently mixed with the cells using a 1000 μL pipette. The same protocol was followed for the control cells, except the final 1 mL medium addition contained no MagCs. The cells were incubated for 24 h (37 °C, 5% CO₂) to obtain mhPSCs and control hPSCs.