Culture medium for inducing directional differentiation of human embryonic stem cells into liver-like tissue, inducing method and application
A technology of human embryonic stem cells and differentiation medium, which is applied in the field of medium for inducing human embryonic stem cells to differentiate into liver-like tissues, and can solve the problems of single liver cells, inability to further differentiate into tissue organoids, and complicated culture
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Embodiment 1
[0048] Example 1 Medium for Inducing Directed Differentiation of Human Embryonic Stem Cells into Liver-like Tissues in Vitro
[0049] The culture medium for inducing the directed differentiation of human embryonic stem cells into liver-like tissue, including the following five mediums used in sequence:
[0050] (1) Differentiation medium I, directional endoderm differentiation medium A: based on RPIM1640 medium, 25% MTeSR™1, 100ng / mL Activin A (ActivinA), and 10ng / mL bone morphogenetic protein were added 4 (BMP4), 2% B27 (-insulin);
[0051] (2) Differentiation Medium II, i.e. Directed Endoderm Differentiation Medium B: Based on RPIM1640 medium, 25% MTeSR™1, 100ng / mL Activin A (ActivinA), 2% B27 (-insulin) were added ;
[0052] (3) Differentiation medium III, that is, liver-directed differentiation medium: on the basis of RPIM1640 medium, 25% MTeSR™1, 20ng / mL bone morphogenetic protein-2 (BMP2), 30ng / mL human fibroblast Cell growth factor-4 (FGF4), 2% insulin-free B27 (-ins...
Embodiment 2
[0055] Example 2 Induction of Directed Differentiation of Human Embryonic Stem Cells into Liver-like Tissues in Vitro
[0056] Using the method of the present invention to induce the directed differentiation of human embryonic stem cells (humanembryonicstemcells, hEScells) into liver-like tissues in vitro, the specific process includes the following steps:
[0057] 1. Obtainment of Committed Endoderm Cells
[0058] Day 1~2:
[0059] (1) Human embryonic stem cells were induced 2 to 3 days after H1 passage, and cells with good growth status were selected for differentiation experiments;
[0060] (2) Discard the human embryonic stem cell medium (MTeSR™1), and wash twice with DMEM / F12;
[0061] (3) Replace with differentiation medium I (endoderm induction medium A), at 37°C, 5% CO 2 conditions for two days.
[0062] Day 3-4:
[0063] (4) Discard yesterday's medium, replace with differentiation medium II (endoderm induction medium B), and culture for two days to obtain endoder...
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