Cultural method of crest-derived stem cell of cranial nerve and identification method

A culture method and stem cell technology, which is applied in the field of purification and identification, and the cultivation of cranial neural crest-derived stem cells, can solve the practical application difficulties of neural crest-derived cell isolation and purification, complicated cell acquisition operations, and inability to obtain neural crest-derived cells. To achieve good growth activity and proliferation potential, stable cell phenotype, and no cell proliferation activity

Inactive Publication Date: 2013-09-18
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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  • Application Information

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Problems solved by technology

At present, the specific surface molecules of neural crest-derived stem cells are still being explored, which also brings certain difficulties to the practical application of the isolation and purification of neural crest-derived cells
The patent publication numbers are CN101717750A and CN101717751A. Using mesenchymal stem cell-specific markers stro-1 and CD146 as markers of periodontal ligament stem cells and dental pulp stem cells can only confirm their mesenchymal origin, but cannot prove their neural crest origin
The patent publication number is CN1590537. The cells obtained by using the specific antibody HNK-1 / CD57 need bovine pituitary extract and leukocyte inhibitory factor to maintain the proliferation and biological stability of ectodermal mesenchymal stem cells. Easy to differentiate, and HNK-1 / CD57 is mainly expressed in odontogenic ectodermal mesenchymal cells, and other maxillary processes are not specific

Method used

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  • Cultural method of crest-derived stem cell of cranial nerve and identification method
  • Cultural method of crest-derived stem cell of cranial nerve and identification method
  • Cultural method of crest-derived stem cell of cranial nerve and identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Isolation and purification of sox-2+ cranial neural crest-derived stem cells

[0040] The 11.5-day pregnant SD mice were taken out, killed by dislocation of the neck, soaked in alcohol for 15 minutes, and the embryos were dissected under the ultra-clean table and the mandibular process tissues were excised (see figure 1 -A), rinse with 0.01MPBS, cut the tissue pieces as much as possible, add 0.25% trypsin / 0.1mM EDTA to digest for 5-10 minutes, neutralize with an equal volume of DMEM / F12 medium, gently pipette the discrete cell factor pieces, and put A single cell suspension formed. Filter through a cell sieve with a pore size of 70 μm, centrifuge at 800 r / min for 5 minutes, remove the supernatant, then add 0.01 MPBS solution to resuspend the cells and centrifuge again, repeat this process twice. Collect cells at 1 x 10 5 Inoculate at a density of 50ml into a 50ml culture bottle, and the culture medium is DMEM / F12 (100μg / ml streptomycin and 100U / ml penicillin) medium c...

Embodiment 2

[0043] Phenotypic characterization of sox-2+ cranial neural crest-derived stem cells

[0044] The 3rd generation cells after sorting and the 9th generation cells cultured in vitro sox-2+ cranial neural crest derived stem cells were taken at a rate of 1×10 4 Each well was seeded in a 24-well plate, and each group had three replicate wells. One group was taken every day, digested with 0.25% trypsin, counted, for a total of 9 days, and the growth curve was drawn. The cell population doubling time was calculated as TD=txlog2 / (logNt-logNO), where t was the number of days of cell growth, Nt was the number of cells on day t, and NO was the number of cells inoculated. The culture has the following common characteristics: the cells have good proliferative activity, and the cells are fully stretched on the second day, maintaining a spindle-shaped cell shape; on the 7th to 8th day, they are basically confluent and reach the peak of the growth curve. The population doubling times of sox...

Embodiment 3

[0049] Multilineage differentiation potential of sox-2+ cranial neural crest-derived stem cells

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Abstract

The invention provides a cultural method of a crest-derived stem cell of a cranial nerve. The method is characterized by sorting the crest-derived stem cell of the cranial nerve from a gnathism tissue of a mouse embryo which is 11-12 days old by using a sox-2 gene. The crest-derived stem cell of the cranial nerve obtained by the method has better growth activity and proliferation potential. In the continuous passage process in vitro, characteristics of the stem cell and cell phenotype are stably expressed, and the proliferation efficiency of cells has no remarkable difference. The crest-derived stem cell of the cranial nerve obtained has the double characteristics of the stem cell from the outer embryo and that from the middle embryo, and can be induced and differentiated directionally to fat cell, osteoblast, chondrocyte and neuron-like cell, so that the crest-derived stem cell of the cranial nerve displays multipotency of the stem cell as the stem cell in early stage of the embryo.

Description

technical field [0001] The invention belongs to the field of stem cell biotechnology, and relates to the cultivation, purification and identification of cranial neural crest-derived stem cells. Background technique [0002] Neural crest-derived stem cells are an important cell type during embryonic development. They originate from the cranial neural crest in early embryonic development. Before the neural tube closes in the early embryonic stage, cranial neural crest stem cells migrate extensively and locate in the maxillary and mandibular processes. The neural crest-derived stem cells are also called ectodermal mesenchymal stem cells. Esodermal mesenchymal stem cells belong to the transitional cells during embryonic development, and further differentiate into periodontal ligament stem cells and dental pulp stem cells during tooth development. These stem cells are also called stem cells derived from neural crest. [0003] Migration of neural crest cells can be observed on th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12Q1/34C12Q1/04
Inventor 聂鑫温秀杰行勇军刘鲁川邓蔓菁刘锐
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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