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Culture medium and method for inducing human pluripotent stem cells to be differentiated into alveolar cells or alveolar organs

A technology of human pluripotent stem cells and alveolar cells, applied in the field of medium for inducing human pluripotent stem cells to differentiate into alveolar cells or alveolar organoids, can solve the problems of cell separation, immature purification, and difficulty in obtaining aborted fetuses. Gentle, easily accessible effect

Active Publication Date: 2021-08-06
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, human alveolar cells have always been a very scarce resource, and the technologies for the isolation, purification and laboratory expansion of related cells are immature
Although the current experimental procedures for isolating lung epithelial precursor cells from human fetuses are relatively mature, since alveolar epithelial cells generally appear at 26 weeks of gestation, it is extremely difficult to obtain aborted fetuses that can be used for sampling, and a set that can efficiently obtain human cells is urgently needed. Methods for alveolar cells or alveolar organoids

Method used

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  • Culture medium and method for inducing human pluripotent stem cells to be differentiated into alveolar cells or alveolar organs
  • Culture medium and method for inducing human pluripotent stem cells to be differentiated into alveolar cells or alveolar organs
  • Culture medium and method for inducing human pluripotent stem cells to be differentiated into alveolar cells or alveolar organs

Examples

Experimental program
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Effect test

Embodiment 1

[0084] Example 1 Inducing Human Pluripotent Stem Cells to Differentiate into Definitive Endoderm Cells

[0085] All the cells in the present invention are cultured at 37° C. in a cell incubator containing 5% carbon dioxide.

[0086] The initial cells used in Example 1 were induced human pluripotent stem cells DYR0100 (Cell Bank / Stem Cell Technology Platform, Chinese Academy of Sciences, SCSP-1301).

[0087] 10 mL of mTesR1 (STEMCELL Technologies, 85850) medium was used in a Matrigel (Corning, 356231)-coated 24-well plate to maintain culture of DYR0100. When the confluence of DYR0100 clones reached 90%, differentiation was induced.

[0088] The basal medium used to induce differentiation was LD basal, and the formula of LD basal was: 100 mL DMEM / F12 (Gibco, C1410500BT330500BT) medium was added with 1 mL GlutaMAX (Gibco, 35050061) (100×), 1 mL N2 supplement (Gibco, 17502001) ( 100×), 2 mL of B27 supplement (Gibco, 17504044) (50×), 0.1 mL of ascorbic acid-2-phosphate (Yuan Ye, ...

Embodiment 2

[0091] Example 2 Induction of defined endoderm cells to differentiate into lung precursor cells

[0092] For the defined endoderm cells finally obtained in Example 1, LDM-3 was used in the first stage to induce differentiation of the defined endoderm cells obtained in Example 1 for 3 days to become front foregut cells. The formula of LDM-3 is: SB431542 (Selleck, S2924) (10 μM) and dorsomorphin (Selleck, S7306) (2 μM) are added to LD basal medium.

[0093] In the second stage, LDM-4 was used to induce the differentiation of the frontal foregut cells obtained above for 9 days to become lung precursor cells ( figure 2 ). The formula of LDM-4 is: add CHIR99021 (3μM), retinoic acid (Sigma, R2625) (50nM), BMP4 (Gibco, PHC9531) (10ng / mL), FGF7 (R&D, 251-KG- 050) (10 ng / mL), FGF10 (R&D, 345-FG-025) (10 ng / mL).

Embodiment 3

[0094] Example 3 Induces differentiation of lung precursor cells into monolayer alveolar cells

[0095] For the lung precursor cells finally obtained in Example 2, use LDM-5 to induce the differentiation of lung precursor cells for about 16 days, and finally become alveolar cells ( image 3 ). The formula of LDM-5 is: add CHIR99021 (3μM), FGF7 (10ng / mL), dexamethasone (Sigma-Aldrich, D4092) (50nM), cAMP (Sigma-Aldrich, B5386) (0.1mM) to LD basal medium ), 3-isobutyl-1-methylxanthine (Sigma-Aldrich, I5879-250MG) (0.1 mM).

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Abstract

The invention provides a culture medium and method for inducing human pluripotent stem cells to be differentiated into alveolar cells or alveolar organs, and particularly relates to a culture medium and method for inducing the human pluripotent stem cells to be differentiated into lung precursor cells or alveolar cells by using a small molecule activator / inhibitor. The cells obtained by induced differentiation express typical lung precursor cells and alveolar cell biomarkers, and a three-dimensional human alveolar organ has an alveolar-like blister structure and can be used as a material for toxicological research, drug screening and lung disease research.

Description

technical field [0001] The invention belongs to the cross field of cell biology and developmental biology, and relates to a culture medium and a method for inducing human pluripotent stem cells to differentiate into alveolar cells or alveolar organoids. Background technique [0002] The lung is the main respiratory organ of humans, and the alveolar composed of alveolar epithelial cells is the basic structural and functional unit of the lung to realize the gas exchange function. The cultivation of lung cells in vitro for drug screening, toxicity testing and disease research is a common demand in the fields of clinical medicine, pharmacy, developmental biology and cell biology. However, human alveolar cells have always been a very scarce resource, and the technologies for the isolation, purification and laboratory expansion of related cells are immature. Although the current experimental procedures for isolating lung epithelial precursor cells from human fetuses are relativel...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0688C12N2506/45C12N2501/16C12N2501/405C12N2501/15C12N2501/155C12N2500/38C12N2501/117C12N2501/119C12N2500/40C12N2501/39C12N2501/415C12N2501/115C12N2501/01
Inventor 杨仁君刘抒羽殷诺雅费凡
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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