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Method for controlling stem cell differentiation
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A technology of stem cell differentiation and cell differentiation, applied in non-embryonic pluripotent stem cells, biochemical equipment and methods, embryonic cells, etc.
Active Publication Date: 2017-02-22
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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However, only very limited studies have shown inhibition of differentiation into specific cell types
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Embodiment 1
[0117] Differentiation of hESCs to definitive endoderm was inhibited by TGFβ inhibitors.
[0118] The cell culture conditions during the differentiation process are as follows: Undifferentiated human H1ES cells with a confluence of nearly 50% were subcultured with Accutase for 24 hours after inoculation, and the confluence reached nearly 90% at 37°C and 5% CO2. Then, H1 cells were cultured for 3 days in RPMI / B27 medium (without insulin) supplemented with Activin A in the presence or absence of 2 μM Repsox.
[0119] On the 0th day when hESCs started to differentiate, RepSox, a TGFβ signaling pathway inhibitor, was added at a final concentration of 2 μM.
[0120] In the process of cell differentiation, the expression of various marker genes of different layer types of cells, namely SOX1 / PAX6 (ectoderm marker gene), GATA2 / BMP4 (mesoderm marker gene), SOX17 / FOXA2 (endoderm marker gene) Assays were used to assess differentiated cells.
[0121] Techniques like single-cell qPCR and...
Embodiment 2
[0127] Inhibition of hESCs differentiation to definitive endoderm by SNAI1 siRNAs.
[0128] The culture conditions of the cells during differentiation are shown below. H1 cells were cultured in RPMI / B27 medium (without insulin) supplemented with 20 ng / ml activin A for 3 days after the third transfection.
[0129] During cell differentiation, the expression of various marker genes of definitive endoderm, namely SOX17, FOXA2, GSC, GATA4, GATA6, etc., was detected to evaluate differentiated cells.
[0130] Methods include siRNA treatment and real-time RT-PCR. A detailed description of the experimental method is given in the section on the experimental method.
[0131] See the results of the study picture 4G -H and picture 7 . Among the candidate genes, SNAI1 is the most critical gene for activating CDH2 and DE marker genes such as SOX17 and FOXA2 ( picture 4H ). Furthermore, expression of all detected mesendoderm / endoderm marker genes was similarly suppressed when SNAI1...
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Abstract
The invention belongs to the field of biological medicine, and relates to a method for controlling multi-potential stem cell differentiation. The method is used for controlling multi-potential stem cells to be differentiated to entoderm source cells, and particularly, the multi-potential stem cells are prevented from being differentiated to the entoderm source cells through a specific inhibitor.
Description
[0001] field of invention [0002] The invention belongs to the field of biomedicine and provides a method for controlling the differentiation of pluripotent stem cells. In particular, the present invention provides methods for controlling the differentiation of pluripotent stem cells into cells of endoderm origin. [0003] Background of the invention [0004] Stem cells have the ability to differentiate into many different types of cells that make up an organism, and they can differentiate into cells derived from the three germ layers (endoderm, mesoderm and ectoderm). [0005] There has been a great deal of interest in directed differentiation of stem cells. The first intermediate stage of differentiation is the formation of definitive layer type cells. In order to obtain purified cells of interest, it is advantageous to inhibit differentiation of unwanted cell types. However, only very limited studies have shown inhibition of differentiation into specific cell types. Co...
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