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Method for inducing human embryonic stem cells to differentiate to neuroderm

A technology of human embryonic stem cells and neuroectoderm, applied in embryonic cells, animal cells, vertebrate cells, etc., can solve the problems of no patent issuance and lack of similar research technologies.

Active Publication Date: 2014-12-10
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is even a lack of similar research technologies in China, and no similar patents have been released

Method used

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  • Method for inducing human embryonic stem cells to differentiate to neuroderm
  • Method for inducing human embryonic stem cells to differentiate to neuroderm
  • Method for inducing human embryonic stem cells to differentiate to neuroderm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Subculture of human embryonic stem cells H9 high-density cell mass.

[0044] 1. Method

[0045] 1. Recovery of 1H9 cells

[0046] One hour before cell recovery, each well of the 6-well plate was plated with 1 mL Matrigel solution (160 μL Matrigel dissolved in 12 mL DMEM / F12 medium pre-cooled on ice), and placed at room temperature. At the same time, DMEM / F12 medium, mTeSR TM 1 Take the culture medium out of the refrigerator and place it at room temperature.

[0047] After 1 hour, the H9 cells frozen in liquid nitrogen were taken out and immediately placed in a 37°C water bath. After complete thawing, the cells were mixed into 5 mL of DMEM / F12 medium at room temperature, centrifuged at 300 g×5 minutes with a centrifuge, and then the supernatant was removed. With 2mLmTesR TM 1 Gently resuspend the cells in medium, and transfer the suspended cells to Matrigel-treated culture plates. 37 ° C, 5% CO2 incubator culture.

[0048] 1. Passage of 2H9 cells

[004...

Embodiment 2

[0060] Example 2 The effect of KSR medium and N2 medium combined with SB431542 and NOGGIN in inducing differentiation of high-density human embryonic stem cell clusters was detected by immunofluorescence method.

[0061] 1. Method

[0062] For the 95% fused H9 cells in Example 1, the original medium was discarded, and different ratios (4:0, 3:1, 2:2, 1:3 and 0:4) of KSR medium and N2 medium were given respectively , the time scale is marked as days 1, 2, 3, 4, and 5 of differentiation (d1, d2, d3, d4, and d5) ( figure 2 ).

[0063] The KSR medium of the present invention contains: DMEM / F12 (Hyclone), 20% knockout serum replacer (KSR), 10 ng / ml FGF-2, 0.1 mM beta-mercaptoethanol, 1% Non-essentiala acid (NEAA).

[0064] The N2 medium described in the present invention contains: DMEM / F12 (Hyclone), 1% N2supplement, 10ng / ml FGF2, 0.1mM beta-mercaptoethanol, 1% Non-essential acid (NEAA).

[0065] Add both SB431542 and NOGGIN for the first 5 days. SB431542 was formulated into a...

Embodiment 3

[0069] Example 3 PCR method to detect the effect of SB431542 and NOGGIN on promoting neuroectoderm differentiation of human embryonic stem cells

[0070] 1. Method

[0071] Carry out subculturing of human embryonic stem cells H9 high-density cell clusters according to Example 1, except according to 2X10 4 / cm 2 The method was the same except that the subculture was in Matrigel-coated 6-well cell culture plates. Culture H9 cells until 95% confluence, discard the original medium, and give different ratios (4:0, 3:1, 2:2, 1:3 and 0:4) of KSR medium and N2 medium respectively, time Labeled as days 1, 2, 3, 4, and 5 of differentiation (d1, d2, d3, d4, and d5), respectively. One group of cells was cultured with KSR and N2 medium, and the other group of cells was cultured with KSR and N2 medium combined with SB431542 and NOGGIN. The cells were collected on the 6th day, RNA was extracted with an RNA extraction kit (purchased from Axygen), and cDNA was synthesized with a cDNA synth...

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Abstract

The invention discloses a method for inducing human embryonic stem cells to differentiate to neuroderm. The method comprises the following steps that H9 cells are kept to passage in a cell cluster state, wherein the cell clusters are uniform in size, and high-density culture of the cell clusters can be kept; based on this, two inducing factors of SB431542 and NOGGIN are introduced, a knock out serumreplacer (KSR) culture medium and an N2 culture medium which are determined according to chemical components are used in matched manner according to different proportions at different culture times; and ascorbic acid (vitamin C) is added into the N2 culture medium at induced differentiation later period, so that HESC can be induced within shorter time to differentiate directionally to form an NE cell for expressing a PAX6 protein.

Description

technical field [0001] The invention relates to a method for inducing differentiation of human embryonic stem cells to neuroectoderm, which is mainly applied in the technical field of stem cell directed differentiation. Background technique [0002] The formation of neuroectoderm (Neuroectoderm, NE) is the origin of the nervous system and an important stage in the development of early mammalian embryos. Human embryonic stem cells (HESC) can form NE after induced differentiation in vitro, so they become an important platform for studying the formation of NE in human early embryonic development. HESC has the characteristics of self-renewal (Self-renewal) and pluripotency (Pluripotent), and can differentiate into three germ layers (endoderm, mesoderm and ectoderm) in vitro and then differentiate into any cell in the human body. Directed neural differentiation of HESC has become an important method for the study of human neurodevelopment, and it is also a hot spot in the field ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
Inventor 刘超刘丽华
Owner ANHUI MEDICAL UNIV
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