Method for obtaining hepatic stellate cells from embryonic stem cells through differentiation
A technology of hepatic stellate cells and embryonic stem cells, applied in embryonic cells, biochemical equipment and methods, animal cells, etc., can solve the problems of unfavorable research on the characteristics and mechanism of HSC initial activation, and achieve stable quality and high safety Effect
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Embodiment 1
[0054] Example 1, the acquisition and detection of hepatic stellate cells
[0055] 1. Embryonic stem cells differentiated to obtain hepatic stellate cells
[0056] The inventors of the present invention established a method for obtaining hepatic stellate cells by differentiating embryonic stem cells through a large number of experiments. Specific steps are as follows:
[0057] 1. Take embryonic stem cells (W09, Wicell, USA) and culture them with STEMdiff Mesoderm Induction Medium (Stemcell, 05221) and STEMdiff Definitive Endoderm Kit (StemCell, 05110) respectively (refer to the following literature for the culture method: Wu X, Dao Thi VL, Huang Y , et al.Intrinsic Immunity Shapes Viral Resistance of Stem Cells.Cell.2018; 172(3):423-438.e25.doi:10.1016 / j.cell.2017.11.018), and sequentially obtained mesoderm cells and endoderm cells. This time was recorded as day 0 of differentiation (D0).
[0058] 2. After completing step 1, inoculate endoderm cells and / or mesoderm cells in...
Embodiment 2
[0105] Example 2. Activation of hepatic stellate cells after FBS and TGFβ stimulation
[0106] 1. Activation of hepatic stellate cells obtained in Step 1 differentiation in Example 1
[0107] (1) Take the hepatic stellate cells differentiated in Step 1 in Example 1, and add FBS to obtain System 1; the concentration of FBS in System 1 is 10%. The hepatic stellate cells obtained in Step 1 differentiation in Example 1 were taken, and TGFβ was added to obtain system 2; the concentration of TGFβ in system 2 was 50 ng / ml.
[0108] (2) Take the hepatic stellate cells (as a control) obtained from System 1, System 2 and step 1 differentiation in Example 1, respectively, at 37°C, 5% CO 2 Cultured for 2 days, 4 days or 6 days.
[0109] 2. Take the hepatic stellate cells that completed step 1, and detect collagen I, Desmin, α-SMA and Nestin by immunofluorescence staining. The detection steps are the same as those in Step 2 of Example 1.
[0110] 3. Take the hepatic stellate cells that...
Embodiment 3
[0117] Example 3, the activation of hepatic stellate cells under the action of thioacetamide
[0118] 1. Take the hepatic stellate cells differentiated in step 1 in Example 1, and add culture medium to obtain treatment system 1. The hepatic stellate cells obtained in the step 1 differentiation in Example 1 were taken, and the medium containing TAA solution (solvent was DMSO) was added to obtain the treatment system 2; in the treatment system 2, the concentration of TAA was 25mM or 75mM. The hepatic stellate cells obtained in the step 1 differentiation in Example 1 were taken, and the medium containing DMSO was added to obtain the treatment system 3. The amount of DMSO added in treatment system 3 is exactly the same as that in treatment system 2.
[0119] TAA is a product of Sigma Company, the catalog number is Cat#163678.
[0120] 2. After completing step 1, treat the treatment system (treatment system 1, treatment system 2 or treatment system 3) for 5 days.
[0121] 3. Aft...
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