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A technical method for skin stem cells to differentiate into sperm through ectopic development

A technology of skin stem cells and sperm, which is applied in the field of biomedical stem cells and tissue engineering, can solve the problems that have not been widely used, and achieve the effects of reducing the difficulty of cultivation, complete nutrition, and less damage to the body

Inactive Publication Date: 2020-04-07
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since it has not been replicated by other laboratories, it has not been widely used so far

Method used

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  • A technical method for skin stem cells to differentiate into sperm through ectopic development
  • A technical method for skin stem cells to differentiate into sperm through ectopic development
  • A technical method for skin stem cells to differentiate into sperm through ectopic development

Examples

Experimental program
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Effect test

Embodiment 1

[0025] A method for differentiation of skin stem cells into sperm through in vivo development, comprising two steps of inducing skin stem cells to differentiate into primordial germ cells, and implanting the primordial germ cells into the testes of recipient mice to obtain sperm cells, wherein:

[0026] 1) Inducing the differentiation of skin stem cells into primordial germ cells:

[0027] ①Skin stem cell preparation: Extract and culture the skin stem cells and wild-type mouse fibroblasts of the first generation of green fluorescent transgenic mice respectively, and treat the wild-type mouse fibroblasts with mitomycin C, and then the skin stem cells Mix with the treated fibroblasts according to the mass ratio of 1:1 before use;

[0028] ② Select DMEM high-glucose medium as the primordial germ cell induction medium, and add 8% fetal bovine serum to the medium based on the total mass of DMEM high-glucose in the medium, 40 mIU / ml follicle-stimulating hormone, 80 IU / ml leukemia i...

Embodiment 2

[0034] A method for skin stem cells to differentiate into sperm through in vivo development, comprising three steps of inducing differentiation of skin stem cells into primordial germ cells, cultivating the primordial germ cells, and implanting the primordial germ cells into the testes of recipient mice to obtain sperm cells, wherein:

[0035] 1) Inducing the differentiation of skin stem cells into primordial germ cells:

[0036] ①Skin stem cell preparation: Extract and culture the skin stem cells and wild-type mouse fibroblasts of the 2nd generation green fluorescence transfer gene mice respectively, and treat the wild-type mouse fibroblasts with mitomycin C, and then the skin stem cells Mix with the treated fibroblasts according to the mass ratio of 1:1 before use;

[0037] ②Select DMEM high-glucose medium as the primordial germ cell induction medium, and add 12% fetal bovine serum to the medium based on the total mass of DMEM high-glucose in the medium, 60 mIU / ml follicle-st...

Embodiment 3

[0045] Identical substantially with embodiment 2, its difference is:

[0046] 2) Primordial germ cells are specially cultivated, specifically:

[0047] The primordial germ cells obtained in step ③ in 1) were cultured in a special-purpose medium, specifically: DMEM high-glucose medium was used as the basis, and DMEM high-sugar total in the medium was added to it. Fetal bovine serum with a mass fraction of 12% by mass, 25 ng / ml bone morphogenetic factor based on the total volume of DMEM high glucose in the medium, 0.25 mmol / L sodium pyruvate, 0.12 mmol / L non-essential amino acids, 2.5mmol / L L-glutamine, 0.12 mmol / L β-mercaptoethanol, 25ng / ml epidermal growth factor, 50 ng / ml basic fibroblast growth factor, 50ng / ml stem cell growth factor; culture time 5 days, temperature 38°C, Humidity 85%; CO in the environment 2 The concentration is 6%; Primordial germ cells that are more purified, more in number and better in shape can be obtained after a certain period of dedicated culture...

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Abstract

The invention relates to a method for inducing skin stem cells to differentiate into sperms. The method comprises the following steps of firstly, co-culturing the skin stem cells of a mouse and fibroblast treated by mitomycin C; inducing to differentiate into PGCs (primordial germ cells); then, reversely injecting PGC cell mixing liquid into a testis seminiferous tubule through an efferent ductule of a recipient mouse; after developing for one month in the testis of the recipient mouse, fetching out the testis of the recipient mouse; after HE staining analysis, finding the recovery of spermatogenesis in the seminiferous tubule, emerging of cells with typical sperm morphology, and expression of GFP and MVH of spermatogenic cells in the seminiferous tubule with recovery of spermatogenesis. The method has the advantages that the injury to a body is little, the limitation of ethical issue is avoided, the skin stem cells are effectively induced to be differentiated into sperm cells, the differentiation efficiency is high, the sperm cells obtained by differentiation have typical sperm morphology, and the specific gene of the sperm cell is expressed.

Description

technical field [0001] The invention relates to a method for skin stem cells to differentiate into sperm through in vivo development, and belongs to the technical field of stem cells and tissue engineering in biomedicine. Background technique [0002] In recent years, male infertility patients account for about 10% of the population of childbearing age in my country, and more than 40% of male infertility is caused by abnormal spermatogenesis. Although assisted reproductive technology has brought hope to many patients, assisted reproduction and drug treatment alone are not enough. Using the directional differentiation of stem cells to germ cells can establish a model of germ cell generation in vitro and finally obtain functional gametes, which is of great significance for the study of the regulatory mechanism of germ cell generation and the treatment of male infertility. [0003] Stem cells (SC) are a type of cells with self-replication ability and multi-lineage differentiat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/076C12N5/074
CPCC12N5/061C12N2500/30C12N2500/32C12N2500/44C12N2501/11C12N2501/115C12N2501/155C12N2501/235C12N2501/31C12N2506/09
Inventor 沈伟孙源超程顺峰孙晓凤李兰
Owner QINGDAO AGRI UNIV
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