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Method for obtaining haploid germ cells through in-vitro differentiation of human skin stem cells

A technology of skin stem cells and germ cells, applied in the field of haploid germ cells

Inactive Publication Date: 2016-03-02
QINGDAO AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, both ESCs and iPSCs have their own disadvantages in clinical application, while human skin-derived stem cells (hSDSCs) are derived from patients themselves, and there are no histocompatibility, tumorigenicity and ethics in clinical application. question

Method used

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  • Method for obtaining haploid germ cells through in-vitro differentiation of human skin stem cells
  • Method for obtaining haploid germ cells through in-vitro differentiation of human skin stem cells
  • Method for obtaining haploid germ cells through in-vitro differentiation of human skin stem cells

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Embodiment 1

[0018] 1. Tissue localization of human skin-derived stem cells

[0019] Fix the human skin tissue with 4% paraformaldehyde overnight at 4°C. After fixing, wrap the tissue piece with gauze, put it in a jar and put it under the tap, let the tap water slowly pour into the bottle, and carry out Rinse, rinse for 2-3h. After washing, dehydrate according to the following steps: 50% alcohol (15min) - 70% alcohol (15min) - 80% alcohol (20min) - 90% alcohol (20min) - 95% alcohol I (10min) - 95% alcohol II ( 20min)—absolute alcohol I (30min)—absolute alcohol II (30min)—alcohol:xylene (volume ratio 1:1, 20min). Then go through two steps of xylene transparency (15min for each step). Afterwards, the tissue block was put into a wax cup, and after two immersions in wax for 1.5 hours each time, the tissue block was embedded in an embedding box. Embedded tissues were sliced ​​into 5 μm white slices with a paraffin microtome. For HE staining, first place the cut white slices on a 60-degree h...

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Abstract

The invention relates to a method for obtaining haploid germ cells through differentiation of human skin stem cells. The method comprises the following operation steps: after in-vitro separation, in-vitro culture is carried out on human skin derived stem cells: wherein separated human skin derived stem cells form a spherical cloning sample structure during in-vitro suspension culture, subculturing is carried out every four days and human skin derived stem cell cloning spheres are obtained after subculturing; human skin derived stem cells are induced to germ cell sample cells; human skin derived stem cells are induced and differentiated towards haploid germ cells, and after differentiation is carried out for about 18 days, DNA ploidy analysis can detect that 1.79% of haploid sample cells are formed. With the adoption of the method provided by the invention, primordial germ cells, of which the morphology and specific molecular marker expression are similar to those of normal primordial germ cells in the human body are obtained, the human haploid germ cells are obtained in a differentiation process of a proper condition culture medium and a good in-vitro model is provided for research on a generating mechanism of human germ cells.

Description

Technical field: [0001] The invention relates to a method for in vitro differentiation of human skin stem cells into haploid germ cells, in particular to a method comprising in vitro isolation of human skin-derived stem cells, suspension culture in vitro, induction of germ cell-like cells with the obtained clone structure, and haploid A method for obtaining human haploid germ cells by means of somatic induction. It belongs to the field of biomedical technology of reproductive medicine. Background technique: [0002] The research on stem cells has a long history. One of the research hotspots of stem cells in the field of reproduction is to use stem cells to differentiate reproductive gametes in order to solve the problem of human infertility; on the other hand, so far, the process of reproductive gametes There are still many mechanisms that have not been discovered. The establishment of a platform for using stem cells to differentiate into reproductive gametes in vitro will ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076C12N5/075
CPCC12N5/0609C12N5/061C12N5/0611C12N2506/1392
Inventor 葛伟孙源超沈伟
Owner QINGDAO AGRI UNIV
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