Aggrewell plate

To measure cell–cell contact angles, 1200 dissociated ES, TS or XEN cells were mixed in pairs and seeded onto AggreWell plates (34411; STEMCELL Technologies) pretreated with rinsing solution (07010; STEMCELL Technologies). Cells were centrifuged at 100g for 3 min. After 1 h incubation at 37 °C, cells were collected and fixed for immunostaining.

Single cell suspensions of TRA-1-60 positive cells were deposited into AggreWell plates (Stem Cell Technologies) and allowed to aggregate into spheroids. Briefly, 1x10⁶ cells positive for TRA-1-60 were suspended in 5 ml of EB Aggrewell medium (Stem Cell technologies #05893) and added into a well of an AggreWell400Ex plate (Stem Cell Technologies, # 27840) containing approximately 4,700 microwells with a diameter of 400 μm and pretreated with AggreWell rinsing solution (Stem Cell Technologies, # 07010). AggreWell plates were centrifuged at 100 x g for 3 minutes to deposit the cells in the microwells and incubated at 37°C for 24 hours, during which time spherical aggregates of deposited pluripotent cells (spheroids) were formed.

NtES cells were trypsinized into single-cell suspensions and plated on gelatin-coated plates for 30 min to remove feeder cells. To create uniform-sized EBs, 3–5×10⁵ unattached ES cells were added to AggreWell plates (Stemcell Technologies, Vancouver, BC, Canada) for 24 h. EBs and cystic EBs formed after 5 days of culture and were cultured for an additional 5 days. Total RNA was extracted and two representative markers from each of the three germ layers were selected for RT-PCR analysis: Nes and Msi1 for ectoderm, Acta2 (smooth muscle) and Bra for mesoderm, and Afp and Alb for endoderm.

This was carried out in Aggrewell 400 plates (STEMCELL Technologies, France). The Aggrewell plates were first incubated in anti‐adherence rinsing solution (500 µL/well—STEMCELL Technologies, France) for 30 minutes—2 hours, during which the ucMSC were harvested from 2D culture. After incubation, the Aggrewell plate was washed with PBS, followed by either hPL‐ or KO‐medium (500 µL/well). Fresh hPL‐ or KO‐medium containing ucMSC at a density of 1.2 × 10⁵ cells/mL/well were added to each well of the Aggrewell plate and mixed thoroughly by pipetting. The plate was centrifuged to collect the cells at the bottom of the microwells and kept in the incubator undisturbed for at least 3 days. The medium was changed after 3 days, during which the cells were imaged. The medium was then changed every 2‐3 days and the culture was maintained for 24 days. Conditioned media (CM) were collected and stored at 4°C for subsequent exosome isolation. ucMSC spheroids were harvested by thorough pipetting using 1 mL pipette tips (ends cut to provide larger orifice) to resuspend the spheroids.

iPSC line generation and characterization were described previously (Ku et al., 2010 (link)). Neuronal differentiation was performed as described (Lai et al., 2019 (link)). Briefly, iPSCs grown on Matrigel (Corning, Coming, NY) and mTeSR (Stem Cell Technologies, Vancouver, Canada) were treated for 10 days in E6 medium (Stem Cell Technologies, Vancouver, Canada) with 0.5 μM LDN-193189, 10 μM SB431542, and 20 μg/ml FGF2. iPSC colonies were dissociated with Accutase (Innovative Cell Technologies, San Diego, CA) and plated in AggreWell plates (Stem Cell Technologies, Vancouver, Canada) at a density of 1,000 cells per microwell. Neurospheres were grown in suspension for 1 week in Neurobasal-A medium (Thermo Fisher Scientific, Waltham, MA), supplemented with N2 and B27 supplements (Thermo Fisher Scientific, Waltham, MA) and FGF2 and EGF, both at 20 μg/ml. Neurospheres were then plated on Matrigel and grown in the same medium as mentioned earlier until rosettes appeared. Rosettes were manually isolated, grown for 4–7 days in suspension, then dissociated with Accutase, and plated on Matrigel at 200,000 cells/cm2. To induce neuronal differentiation, cells were grown in the same medium as mentioned earlier without FGF2 and EGF for 14 days.

Immediately following shipment, human islets were dissociated with Versene (Thermo); then live β cells were FACS-sorted based on negativity for propidium iodide and HIC3-2D12 and positivity for HIC1-2B4, as described (65 (link)). Collected β cells were seeded into V-bottom 96-well plates and exposed overnight to pLKO.1-encoded lentiviruses producing either nontargeting shRNA or a pool of lentiviruses producing LSD1 shRNA (Supplemental Table 7). Four days later, islet aggregates were starved for 1 hour in KRBH supplemented with 2.8 mM glucose, then were incubated with KRBH containing either 2.8 mM glucose or 16.8 mM glucose for 1 hour, after which insulin content of media and lysates was determined as described above. Knockdown was verified in whole dissociated islets infected as above and reaggregated in AggreWell plates (STEMCELL Technologies), using GFP-expressing pLKO.1-encoded lentivirus for visualization of transduction efficiency in control islets 72 hours following transduction.