Method for in-vitro differentiation from induced pluripotent stem cells to oocyte stagnating in meiosis II stage

A technology of human pluripotent stem cells and meiosis, applied in the field of cell engineering, can solve the problems of time-consuming and low efficiency, and achieve the effect of high efficiency and short time.

Active Publication Date: 2019-07-12
NINGXIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the process of EB is inefficient and time-consuming

Method used

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  • Method for in-vitro differentiation from induced pluripotent stem cells to oocyte stagnating in meiosis II stage
  • Method for in-vitro differentiation from induced pluripotent stem cells to oocyte stagnating in meiosis II stage
  • Method for in-vitro differentiation from induced pluripotent stem cells to oocyte stagnating in meiosis II stage

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Embodiment 1

[0042] A method for inducing human pluripotent stem cells to differentiate in vitro into oocytes arrested in meiosis II, comprising the following steps:

[0043] (1) Inducing human pluripotent stem cells to form primordial germ cell-like cells: inoculate pluripotent stem cells into PGC-m medium, 2 Cultured under conditions for 10 days to obtain primordial germ cell-like cells (iPGC), the medium was replaced every day during the culture; the PGC-m medium was α-MEM medium supplemented with 5% KSR and 5% bFF, Moreover, the following components are also added to the PGC-m medium:

[0044] 1% L-glutamine, 1% non-essential amino acids, 0.1 mM b-mercaptoethanol, 1% penicillin, 50 ng / mL bone morphogenetic protein, 200 ng / mL leukocyte inhibitory factor, 100 ng / mL stem cells factor, 50ng / mL epidermal growth factor and 10μmol / L ROCK inhibitor;

[0045] (2) Inducing primordial germ cells to form a follicle-like structure: transfer the primordial germ cells induced in step (1) to PF-m me...

Embodiment 2

[0050] A method for inducing human pluripotent stem cells to differentiate in vitro into oocytes arrested in meiosis II, comprising the following steps:

[0051] (1) Inducing human pluripotent stem cells to form primordial germ cell-like cells: inoculate pluripotent stem cells into PGC-m medium, 2 Cultured under conditions for 10 days to obtain primordial germ cell-like cells (iPGC), the medium was replaced every day during the culture; the PGC-m medium was α-MEM medium supplemented with 3% KSR and 6% bFF, Moreover, the following components are also added to the PGC-m medium:

[0052] 0.5% L-glutamine, 2% non-essential amino acids, 0.15mM b-mercaptoethanol, 1.5% penicillin, 40ng / mL bone morphogenetic protein, 250ng / mL leukocyte inhibitory factor, 80ng / mL stem cells Factor, 60ng / mL epidermal growth factor and 5μmol / L ROCK inhibitor;

[0053] (2) Inducing primordial germ cells to form a follicle-like structure: transfer the primordial germ cells induced in step (1) to PF-m med...

Embodiment 3

[0058] A method for inducing human pluripotent stem cells to differentiate in vitro into oocytes arrested in meiosis II, comprising the following steps:

[0059] (1) Inducing human pluripotent stem cells to form primordial germ cell-like cells: inoculate pluripotent stem cells into PGC-m medium, 2 Cultured under conditions for 10 days to obtain primordial germ cell-like cells (iPGC), the medium was replaced every day during the culture; the PGC-m medium was α-MEM medium supplemented with 6% KSR and 3% bFF, Moreover, the following components are also added to the PGC-m medium:

[0060] 2% L-glutamine, 0.5% non-essential amino acids, 0.05mM b-mercaptoethanol, 0.5% penicillin, 60ng / mL bone morphogenetic protein, 150ng / mL leukocyte inhibitory factor, 120ng / mL stem cells factor, 40ng / mL epidermal growth factor and 15μmol / L ROCK inhibitor;

[0061] (2) Inducing primordial germ cells to form a follicle-like structure: transfer the primordial germ cells induced in step (1) to PF-m m...

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Abstract

The invention discloses a method for in-vitro differentiation from induced pluripotent stem cells to oocyte stagnating in the meiosis II stage. The method includes the three steps of firstly, puttingpluripotent stem cells with the cell density of 80% in a PGC-m culture medium to be cultured for 10 days at 35-40 DEG C in CO2 with the concentration of 3-6% to obtain original germ cell-like cells; secondly, transferring the formed original germ cell-like cells into a PF-m culture medium to be cultured for 5 days at 35-40 DEG C in CO2 with the concentration of 8-12% to obtain a follicle-like structure; finally, transferring the formed follicle-like structure into an OLC-m culture medium to be incubated for 10-15 days to obtain oocyte-like cells stagnating in the meiosis II stage. The method is high in efficiency and short in time, and the secondary oocyte can be formed by removing first polar bodies from the induced oocyte-like cells. The method provides new means for studying the formation of human female germ cells and the generation of ova.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and specifically relates to a method for inducing human pluripotent stem cells to differentiate in vitro into oocytes stagnated in meiosis II. Background technique [0002] Generation of germ cells in vitro in the absence of morphogenetic events associated with gastrulation is one of the effective ways to understand the entire process of in vitro gametogenesis. Numerous studies have demonstrated that human and mouse pluripotent stem cells can generate functional male gametes and oocyte-like cells (OLCs) by reconstituting the in vitro gametogenesis process. Usually, pluripotent stem cells are differentiated into embryoid bodies (EBs) by suspension culture, and the steroid hormones produced by them can promote the formation of primordial germ cell-like cells (PGCLCs), and then PGCLCs are treated with cytokines (such as BMP4, SCF, EGF and LIF) culture medium to develop and form aggregates ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2500/32C12N2500/44C12N2501/155C12N2501/125C12N2501/11C12N2501/31C12N2500/25
Inventor 俞晓丽王宁王翔任亚辉王华岩张樱馨邱意开蒲静刘心蕊裴秀英王燕蓉
Owner NINGXIA MEDICAL UNIV
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