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Serum-free induction method of sensory neuronal cells

A neuron cell, sensory nerve technology, applied in nervous system cells, animal cells, nervous system diseases, etc., can solve problems affecting cell purity, drug screening interference, etc.

Pending Publication Date: 2020-12-08
IREGENE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through this method, induced sensory neurons can be obtained in the presence of serum-like components (Knockout Serum Replacement) (Nat Biotechnol., 2013, 30(7): 715-720), although this method does not use trophoblasts, but still Need to use serum components, which have a certain interference effect on drug screening
Moreover, some non-target cells are mixed in the induced cells, which affects the purity of the cells.

Method used

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  • Serum-free induction method of sensory neuronal cells
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  • Serum-free induction method of sensory neuronal cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Preparation of human induced pluripotent stem cells

[0056] Matrigel (STEMCELL Technologies) was used to coat the 6-well culture plate, and after plating, it was placed in a 37°C incubator and incubated for more than one hour. When somatic cell reprogramming was performed using the Epi5 Reprogramming Kit (Invitrogen), the following human adult cells were reprogrammed into induced pluripotent stem cells using the Neon Electroporation Kit (Thermo Fisher): Human mesenchymal cells (Lonza, PT-2501) , human CD34+ cells (PromoCELL, c-12921), and human skin fibroblasts (PromoCELL, c-12302).

[0057] The operation steps are as follows: after centrifugation and washing, the human adult cells were resuspended in the resuspension buffer provided in the electroporation kit, and the electroporation was performed according to the following procedure: 1650V pulse voltage, 10ms pulse width, 3 pulses. After the electroporation, the cells were poured into a tube of 6ml reprog...

Embodiment 2

[0059] Example 2. Counting of induced pluripotent stem cell clones and identification of pluripotency

[0060] (2.1) Alkaline phosphatase staining (Cat#SCR004, Millipore) was used to count induced pluripotent stem cell clones and identify pluripotency. First, remove the cell culture medium in the culture dish, wash it with PBS, add 4% paraformaldehyde to fix it for 1-2 minutes, wash it with TBST three times, and add the staining working solution provided by the kit (taking a 24-well plate as an example) , add 0.5m1 to each well), and place in the dark at room temperature for 15-20min; after staining, the staining solution was sucked away, washed 2-3 times with phosphate buffer, and the staining results were observed under a microscope (DMi8, Leica).

[0061] (2.2) Immunofluorescence identification of Oct3 / 4 induced pluripotent stem cells was performed using fluorescence immunofluorescence.

[0062] The induced pluripotent stem cells in Example 1 were taken and identified by i...

Embodiment 3

[0066] Example 3. Sensory neuron differentiation of induced pluripotent stem cells (3.1) Induction of sensory neural precursor cells

[0067] Matrigel (STEMCELL Technologies) was used to coat T25 cell culture flasks, and placed in a 37°C incubator for more than one hour after plating. A variety of human induced pluripotent stem cells from different sources obtained in Example 1 according to 1x10 6 Cells were seeded in T25 culture flasks.

[0068] When the induced pluripotent stem cells reached 70% coverage, they were digested with 0.05% trypsin / EDTA at 37°C for 5 minutes, and the cell digestion was terminated with DMEM. Cells were washed and centrifuged according to 2×10 5 The proportion of each bottle was re-seeded in T25 culture plate, and cultured with sensory neuron induction medium, the culture condition was 37°C, 5% CO 2 (Panasonic, MCO-18AC) were cultured for 10 days until a neural plate was formed, and the formation of sensory nerve precursor cells was confirmed.

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Abstract

According to the novel human sensory neuron induction culture system provided by the invention, the combination of the small-molecule inhibitor LY2157299 and the growth factor is added into the serum-free basal culture medium, so that compared with a serum-containing induction method, the efficiency of converting pluripotent stem cells into sensory neurons is greatly improved, and the induction efficiency of the pluripotent stem cells is improved. In addition, the expression of various ion channel proteins is obviously improved, so that various induced pluripotent stem cells with different sources are successfully induced into sensory neurons.

Description

technical field [0001] The present invention belongs to the biological field. It specifically relates to a method for inducing pluripotent stem cells into sensory neuron cells, an induction medium and applications thereof. Background technique [0002] In 2006, Shinya Yamanaka's team invented a "cocktail" method composed of four transcription factors, OCT4, SOX2, KLF4 and c-Myc, which can successfully reprogram terminally differentiated skin fibroblasts into differentiated pluripotent cells These stem cells are called induced pluripotent stem cells (induced pluripotent cells) (Cell, 2006, 124(4) pp.663-676; Cell, 2007, 131(5) pp.861-872). These stem cells have similar differentiation potential to embryonic stem cells, and can form the three most basic germ layers of human development: ectoderm, mesoderm and endoderm, and eventually form a variety of adult cells. This invention breaks through the ethical restrictions on the use of human embryonic stem cells in medicine, can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/0793C12Q1/02
CPCC12N5/0619C12N2506/45C12N2500/90C12N5/0623G01N33/5058A61K35/30A61P25/00C12N2500/25C12N2500/32C12N2500/12C12N2506/08C12N2533/52C12N2503/02G01N2500/10C12N2501/405C12N2501/15G01N33/5073G01N33/6896G01N2333/495Y02A50/30C12N5/062C12N2500/98G01N33/5088A61P25/28C12N2506/02C12N2533/54C12N5/0037C12N2500/24C12N2501/13C12N2506/03A61K31/4709C12N5/0696
Inventor 魏君蔡萌
Owner IREGENE THERAPEUTICS LTD
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