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A kind of ectodermal stem cell line and its establishment method and use

A stem cell line, stem cell technology, applied in embryonic cells, animal cells, germ cells, etc., can solve problems such as transient state and limited application value

Active Publication Date: 2020-03-31
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ectodermal precursor cells cannot self-renew and proliferate in vitro
Moreover, the induced ectoderm-like state is very short-lived and can only last for a few hours, so its application value is limited to a certain extent.

Method used

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  • A kind of ectodermal stem cell line and its establishment method and use
  • A kind of ectodermal stem cell line and its establishment method and use
  • A kind of ectodermal stem cell line and its establishment method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Establishment of Epiblast Stem Cells from Mouse Epiblast Stem Cells

[0032] Based on the research basis of early embryonic development in vivo, we added Nodal inhibitor SB431542 and bFGF signaling pathway factors to the basal medium CDM (Chemically defined medium) of epiblast stem cells to induce mouse epiblast stem cells (EpiSCs) into Ectoderm stem cells (Ectoderm stem cells, EctSCs).

[0033] The preparation method of epiblast stem cells is a prior art, please refer to:

[0034] Brons, I.G., et al., Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature, 2007.448(7150):p.191-5.

[0035] The specific method is as follows: the mouse epiblast stem cells are first digested into small pieces with type IV collagenase, and then the cell agglomerates are divided into about 1.5×10 5 Density seeds were coated overnight with serum (FBS) and rinsed twice with PBS in a petri dish. The culture solution was CDM containing SB431542 (2μM)) and bFGF (...

Embodiment 2

[0037] Example 2 Epiblast stem cell line is similar to mouse ectoderm in gene expression profile

[0038] In order to study which stage and which position of the mouse embryo the ectodermal stem cells obtained in the examples correspond to, we performed Pearson correlation coefficients on the gene expression profiles of the obtained ectoderm stem cells and the gene expression profiles of the tissues in various regions of the embryo. analyze. Correlation coefficient heatmaps reflect embryo locations corresponding to different cell states. The results showed that ectodermal stem cells corresponded to the AP fraction of E7.0 embryos, and the AP and AD fractions of E7.5 embryos (as image 3 B-C shown).

[0039] E is the abbreviation of Embryonic day, and E5.5 means 5.5 days of embryonic development.

[0040] The specific method is as follows:

[0041] Embryo Dissection and Partitioning

[0042] We dissected and cut embryos at E5.5, E6.0, E6.5, E7.0, and E7.5 days, and divided...

Embodiment 3

[0108] Example 3 Ectodermal stem cell lines can specifically differentiate into nerve and epidermis in an in vitro differentiation system

[0109] In order to investigate whether ectoderm stem cells have the differentiation potential of ectoderm, we separately added epiblast stem cells and ectoderm stem cells to culture medium without BMP4 (8% KSR, 2mM glutamine, 1mM pyruvate, 0.1mM Nonessential amino acids, and 0.1mM β-mercaptoethanol) were suspended into cell clusters (EB) for neural differentiation, or suspended in culture medium supplemented with BMP4 into cell clusters for epidermal differentiation. We found that epiblast stem cells are pluripotent and can rapidly differentiate to a neural fate in the absence of BMP4. But under the condition of adding BMP4, it can differentiate into epidermis and mesendoderm. Ectodermal stem cells can also quickly differentiate into neural fate without adding BMP4, and the expression of neural marker genes is slightly higher than that of...

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Abstract

The invention relates to the field of biotechnology, specifically to an ectoderm stem cell line and an establishment method and application thereof. According to the invention, SB431542 and bFGF are added during the culture process of epiblast stem cells, and epiblast stem cells are induced to be differentiated into ectoderm stem cells. The ectoderm stem cells established by the method can be subcultured serially for 40 generations and above, can maintain the morphology similar to the epiblast stem cells, and have differentiation potential of ectoderm. The ectoderm stem cells are similar to ectoderm in the aspect of differentiative capacity, lack the capability of differentiation into mesendoderm but have stronger capability of epidermal differentiation than epiblast stem cells. In comparison with pluripotent epiblast stem cells, the ectoderm pluripotent stem cells of the invention have more specialized differentiation potential and have clinic and market value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an ectoderm stem cell line and its establishment method and application. Background technique [0002] Early mammalian embryonic development is a rapid and orderly process. During the process from the formation of the inner cell mass (ICM) to the implantation of the embryo, the cells of the inner cell mass develop and differentiate into the epiblast. The epiblast differentiates into ectoderm, mesoderm and endoderm by gastrulation. Among them, the ectoderm can be further differentiated into terminally differentiated tissues such as nerve and epidermis. Due to the lack of marker genes in the ectoderm and the short time window during its development, relatively little research has been done on the development of the ectoderm lineage. [0003] Due to the extremely small individual mouse embryos during the gastrulation period, it is difficult to obtain a sufficient amount of embryonic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735
Inventor 景乃禾刘畅
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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