Method for establishing kidney organoids through stem cell induced differentiation

A technology for inducing differentiation and organoids, applied in the field of cell engineering, can solve the problems of low differentiation efficiency and unstable differentiation, and achieve the effect of high differentiation efficiency, stable differentiation, and stable establishment

Pending Publication Date: 2022-01-18
SHANXI MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has the problems of unstable

Method used

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  • Method for establishing kidney organoids through stem cell induced differentiation
  • Method for establishing kidney organoids through stem cell induced differentiation
  • Method for establishing kidney organoids through stem cell induced differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Induced differentiation of stem cells to establish kidney organoids

[0043] 1.1 Establishment of 2D kidney organoids

[0044] 1. Human induced pluripotent stem cells were cultured in mTesR medium at 37°C with 5% CO 2 In the incubator, change the medium every day to keep the cell growth in good condition (such as figure 1 As shown in the light microscope), immunofluorescence staining showed that the markers of the pluripotent state were well expressed (such as figure 1 Fluorescent staining is shown).

[0045]2. When the cell density reaches 70%-80%, digest with Accutase to prepare a single cell suspension, add 10μM Rock inhibitor according to 1.5×10 4 / cm 2 Seed the cells at the cell density. at 5% CO 2 After culturing in an incubator at 37°C for 24 hours, change the medium and continue culturing for 48 hours.

[0046] 3. When the cells grow to 50% density (differentiation day 0), add wnt signaling pathway activator CHIR-99021 (10 μM) and TGF-signaling ...

Embodiment 2

[0057] Example 2 compares the effects of different reagents on stem cell induced differentiation

[0058] The present invention compares the influence of different reagents on the differentiation of human induced pluripotent stem cells, and the specific experimental conditions are shown in the following table:

[0059]

[0060]

[0061] The differentiation results of the 24 differentiation methods in the above table are as follows: Figure 4 As shown, only the conditions 16, 17, and 18 were used, and the differentiation of human induced pluripotent stem cells was successful. Among them, compared with differentiation condition 16 and 17 and 18, the structure obtained by differentiation under condition 16 was clearer and more mature.

Embodiment 3

[0062] Example 3 Comparison of Induced and Differentiated Results of Stem Cells

[0063] Experimental group: using the method of Example 1 of the present invention to induce differentiated stem cells;

[0064] Control group: for differentiation day0-day4, add CHIR-99021 10μM to the differentiation medium; day4-day7, add ActivinA 10ng / ml to the differentiation medium; day7-day9, add FGF9 10ng / m to the differentiation medium; From day9 to day11, FGF9 10ng / ml and CHIR-99021 3μM were added to the differentiation medium; from day9 to day20, FGF9 10ng / ml was added to the differentiation medium.

[0065] Using the differentiation method of the control group, the differentiation results are unstable, and differentiation failure (without any structure) often occurs in the late stage of differentiation or occasionally some incomplete results appear (such as Figure 5 shown on the left), Figure 5 The figure on the right shows the results obtained by the method of the experimental grou...

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Abstract

The invention belongs to the field of cell engineering, and particularly relates to a method for establishing kidney organoids through stem cell induced differentiation. By adjusting Wnt, TGF-beta and FGF signal channels, human pluripotent stem cells are directionally induced and differentiated into kidney progenitor cells, then the progenitor cells are made into a single-cell suspension, and the kidney organoids are formed through self-assembly. The method through stem cell induced differentiation, provided by the invention can efficiently and stably establish the kidney organoids, and can provide a powerful platform for researching kidney diseases and evaluating kidney toxicity.

Description

technical field [0001] The invention belongs to the field of cell engineering, and in particular relates to a method for establishing kidney organoids by inducing differentiation of stem cells. Background technique [0002] The kidney is an important human organ, which has physiological functions such as removing metabolic waste in the body, regulating electrolyte balance, and secreting renin and erythropoietin. Studies have shown that the kidneys of adults can filter about 200L of fluid a day. However, kidney tissue gradually loses its regenerative function after birth. Aging, disease, environment and other factors cause irreversible damage to the kidney, and the kidney function is gradually impaired, leading to kidney disease. As the disease progresses, it eventually leads to end-stage renal disease, requiring dialysis or a kidney transplant. Studies have shown that about 10% of the world's population suffers from kidney disease. Kidney disease seriously endangers publ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2513/00C12N2506/45C12N2501/415C12N2501/15C12N2501/115C12N2501/727
Inventor 解军周冰蕊何生刘志贞赵虹侯淑琳赵陶然
Owner SHANXI MEDICAL UNIV
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