Trypsin edta

Each cell was cultured according to the SFEBq protocol [6 (link)]. In brief, mESCs were enzymatically dissociated to single cells in 0.25% Trypsin-EDTA (Wako; Cat# 201–16945) and quickly reaggregated in differentiation medium (3,000 cells per well) using 96-well U-bottom low cell-adhesion plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan; Cat# MS-9096U). The differentiation medium was growth factor-free CDM (gfCDM), which contains Iscove’s modified Dulbecco’s medium (Gibco; Cat# 31980030) /Ham’s F-12 (Gibco; Cat# 31765035) 1:1, 1×chemically defined lipid concentrate (Gibco; Cat# 11905031), monothioglycerol (450 μM; Sigma-Aldrich; Cat# M6145, CAS# 96-27-5) and 5 mg/ml purified bovine serum albumin (BSA, Sigma-Aldrich; Cat# A9418, CAS# 9048-46-8). The differentiation medium was used from days 0–7. To regulate Sonic hedgehog (Shh) signal transduction, 10 nM SAG (Cayman Chemicals, Ann Arbor, MI, USA; Cat# 11914, CAS# 912545-86-9) was added to the culture from day 4 for induction of ventral hypothalamic neurons, such as POMC, AgRP and NPY.

For the positive control of osteocalcin protein and gene expression, the human osteosarcoma-derived osteoblast cell line Saos-2, which exhibits most features of a mature osteoblastic profile^{31 (link)}, was obtained from The Cell Resource Center for Biomedical Research (IDAC, Tohoku University, Miyagi, Japan). Cells were cultured in α-modified minimal essential medium (α-MEM; Wako) supplemented with 10% (v/v) foetal bovine serum (BioWest, Nuaillé, France), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were incubated at 37 °C in a 5% CO₂ environment. Confluent cells were passaged with trypsin-EDTA (Wako) and seeded at 6 × 10⁶ cells per 35-mm plastic culture dish (Falcon; Thermo Fisher Scientific, Waltham, MA, USA). Cells cultured for 1 day were used for confocal microscopy observation, and cells cultured for 3 days were used for total RNA extraction and subsequent real-time PCR analysis.

In each inhibitor treatment/release cycle, cells were treated with 500 μM thymidine (Wako), 1 μM PD-0332991 (MedChemExpress), or 1 μM LY-2835219 (Sigma-Aldrich) for 16 h, then rinsed with and incubated in supplemented IMDM without the inhibitors for 8 h. In the case of roscovitine co-treatment with thymidine, 5 μM roscovitine (Sigma-Aldrich) was introduced 5 h after the introduction of thymidine, treated for 11 h, and then washed out along with thymidine. roscovitine was introduced from the second cycle of the thymidine treatment/release cycles. Cell passage was conducted using 0.05% trypsin-EDTA (Wako) typically once two days while cells were cultured in inhibitors-free medium. For immunofluorescence imaging, cells were fixed and stained 8 h after the release from the third inhibitor treatment. For live imaging, the cell culture medium was exchanged to supplemented phenol red-free IMDM (Thermo Fisher Scientific), and cells were imaged from 2 h after the release from the third inhibitor treatment. For monitoring the dynamics of DNA content in the long-term culture experiments, cells were subjected to flow cytometry 8 h after release from inhibitor treatment at the cycles of inhibitor treatment/release indicated in the main text and the corresponding figure.