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Cell Regeneration

Table 1 Human stem cell-based embryo models

From: Human embryos in a dish – modeling early embryonic development with pluripotent stem cells

Reference

Starting cells

Culture media

Culture condition

Blastoids formation

Fan et al. (2021)

hEPS in LCDM + hEPS-derived TE-like cells

IVC1, IVC2

AggreWell

6 days

Liu et al. (2021)

hiPSC in t2iLGoY

iBlastoid

AggreWell

6 days

Yu et al. (2021)

Naïve hPSC in 5i/L/A or PXGL

HDM, TDM

AggreWell

9 days

Yanagida et al. (2021)

Naïve hPSC in PXGL

PD + A83 + Y, A83, N2B27

U-bottom 96-well

3 days

Sozen et al. (2021)

hEPS in hEP

IVF + hEP + hTS (5% O2)

AggreWell

6 days

Kagawa et al. (2021)

Naïve hPSC in PXGL

N2B27 + Y, PALLY, LY

Non-adherent hydrogel microwell

4 days

  1. LCDM: N2B27 with 10 ng/ml human LIF, 3 μM CHIR99021, 2 μM (S)-( +)-Dimethindene maleate and 2 μM minocycline hydrochloride, 1 μM IWR endo-1, and 2 μM Y-27632
  2. IVC1: Advanced DMEM/F12, 20% Heat-inactivated FBS, 2 mM L-glutaMAX, 0.5% penicillin/streptomycin, 1% ITS-X, 1% sodium pyruvate, 8 nM β-estradiol, 200 ng/mL progesterone and 25 μM N-acetyl-L-cysteine
  3. IVC2: Advanced DMEM/F12, 30% Knockout serum, 2 mM L-glutaMAX, 0.5% penicillin/streptomycin, 1% ITS-X, 1% sodium pyruvate, 8 nM β-estradiol, 200 ng/mL progesterone, 2 μM Y27632 and 25 μM N-Acetyl-L-cysteine
  4. t2iLGoY: 50:50 mixture of DMEM/F-12 (ThermoFisher) and neurobasal medium (ThermoFisher), supplemented with 2 mM L-glutamine (ThermoFisher), 0.1 mM 2-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), 1% penicillin–streptomycin (ThermoFisher), 10 ng/ml human leukaemia inhibitory factor (LIF, made in house), 250 μM L-ascorbic acid (Sigma), 10 μg/ml recombinant human insulin (Sigma), 1 μM PD0325901 (Miltenyi Biotec), 1 μM CHIR99021 (Miltenyi Biotec), 2.5 μM Gö6983 (Tocris), and 10 μM Y-27632 (ROCK inhibitor; Selleckchem)
  5. iBlastoid: 50:50 mixture of DMEM/F-12 (ThermoFisher) and neurobasal medium (ThermoFisher), supplemented with 2 mM l-glutamine (ThermoFisher), 0.1 mM 2-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), and 1% penicillin–streptomycin (ThermoFisher))
  6. 5i/L/A: N2B27 basal medium with 1 × GlutaMAX (Gibco), 1 × nonessential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 0.5% penicillin–streptomycin (Gibco), 50 mg/ml bovine serum albumin (BSA, Sigma) and the following small molecules and cytokines: 1 μM PD0325901 (Stemgent), 0.5 or 1 μM IM-12 (Enzo), 0.5 μM SB590885 (R&D systems), 1 μM WH-4–023 (A Chemtek), 20 ng/ml recombinant human LIF (Peprotech) and 10 ng/ml activin A (Peprotech). PXGL medium: N2B27 basal medium with 1 × GlutaMAX, 1 × nonessential amino acids, 0.1 mM β-mercaptoethanol, 0.5% penicillin–streptomycin and the following small molecules and cytokines: 1 μM PD0325901, 2 μM XAV939 (Sigma), 2 μM Go6983 (Sigma) and 20 ng/ml recombinant human LIF
  7. HDM: 1:1 (v/v) mixture of DMEM/F12 and neurobasal medium, 1 × N2 supplement, 1 × B27 supplement, 1 × GlutaMAX, 1 × nonessential amino acids, 0.1 mM β-mercaptoethanol, 0.5% penicillin–streptomycin, 20 ng/ml bFGF (Peprotech), 20 ng/ml activin A and 3 μM CHIR99021
  8. TDM: 1:1 (v/v) mixture of DMEM/F12 and neurobasal medium, 0.5 × N2 supplement, 0.5 × B27 supplement, 0.5% ITS-X, 0.5 × GlutaMAX, 0.5 × nonessential amino acids, 0.1 mM β-mercaptoethanol, 0.5% knockout serum replacement (KSR, Gibco), 0.1% FBS, 50 mg ml − 1 BSA, 0.5% penicillin–streptomycin, 1 μM PD0325901, 0.5 μM A83-01, 0.25 μM SB590885, 0.5 μM WH-4–023, 0.25 μM IM-12, 1 μM CHIR99021, 0.5 μM SB431542, 10 ng ml − 1 recombinant human LIF, 25 ng ml − 1 EGF, 0.75 μg ml − 1 l-ascorbic acid and 0.4 mM VPA. For certain experiments the following chemicals were added to the TDM: 100 nM Go6976 (Selleckchem), 1 μM Go6983 (Selleckchem), 10 μM PKCα (C2-4) inhibitor peptide (Cayman), 10 μM PKCζ pseudosubstrate inhibitor (Cayman), 10 μM PKCη pseudosubstrate inhibitor (Sigma) or 10 μM PKCδ inhibitor KAI-9803 (delcasertib hydrochloride, MedChemExpress)
  9. PD + A83 + Y: N2B27 supplemented with 1.5 mM PD0325901, 1 mM A83-01 and 10 mM Y-27632
  10. A83: N2B27 supplemented with 0.5 mM A83-01
  11. N2B27 + PALLY: N2B27 supplemented with PD0325901 (1 μM), A 83–01 (1 μM, MedChemExpress, HY-10432), 1-Oleoyl lysophosphatidic acid sodium salt (LPA)32 (500 nM, Tocris, 3854), hLIF (10 ng/ml), and Y-27632 (10 μM)
  12. hEP: N2B27 supplemented with 10 ng/ml recombinant human LIF (L, 10 ng/ml; Peprotech, 300–05), CHIR99021 (C, 1 mM; Stem Cell Technologies), (S)-( +)-Dimethindenemaleate (D, 1 mM; Tocris, 1425) and Minocycline hydrochloride (M, 2 mM; Santa Cruz Biotechnology, sc-203339), Y-27632 (5 µM)
  13. IVF + hEP + hTS: 50% IVF media (Continuous Single Culture-NX Complete (CSCM-NXC)) (90,168, FUJIFILM), 25% hEP media, and 25% hTS media, supplemented with CHIR99021 (2 μM), Y27632 (5 μM), BMP4 (20 ng/mL), FGF2 (40 ng/mL), and A83-01 (2 μM). After 48 h, FGF2 is lowered to 20 ng/mL and A83-01 is omitted
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