Method for directionally inducing and differentiating pig pluripotent stem cells into male germ cells and special culture medium

A technology for inducing culture medium and pluripotent stem cells, which can be used in germ cells, cell culture active agents, artificially induced pluripotent cells, etc., and can solve the problems of no function, low differentiation efficiency, and undetected embryonic development.

Active Publication Date: 2016-10-26
CHINA AGRI UNIV
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Problems solved by technology

The article does not examine the ability of differentiated spermatozoa to activate egg cells
In 2004, NielsGeijsen et al. isolated primordial germ cells (PGCs) from EBs cultured with retinoic acid (RA), and continued to culture them to obtain embryonic germ cell lines; PGCs in EBs can develop into haploid sperm, and the resulting haploid Injection of ploidy sperm into the oocyte can activate the oocyte to form a blastocyst-like structure, but the article did not examine the subsequent embryonic development
In 2010, Masanori Imamura et al. co-cultured induced pluripotent stem cells (iPSC) with cells expressing BMP4 protein for differentiation, but the differentiation efficiency was not high; in order to improve the induction efficiency, the researchers modified the cells expressing BMP4, that is, through Three genes GDNF / SCF / EGF are transferred by retrovirus, and then iPSCs are co-cultured with the modified cells, and VASA-positive cells can appear more quickly
In 2012, Yong Zhu et al. combined in vitro induction of differentiation and in vivo seminiferous tubule injection to differentiate mouse iPSCs into spermatogonial stem cells (SSCs) and male germ cells in the later stages of differentiation, but the literature did not conduct functional experiments Research
Although breakthroughs have been made in the directed differentiation of mouse and human pluripotent stem cells to germ cells, the induction of porcine pluripotent stem cells into germ cells is still very challenging

Method used

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  • Method for directionally inducing and differentiating pig pluripotent stem cells into male germ cells and special culture medium
  • Method for directionally inducing and differentiating pig pluripotent stem cells into male germ cells and special culture medium
  • Method for directionally inducing and differentiating pig pluripotent stem cells into male germ cells and special culture medium

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Experimental program
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Embodiment 1

[0104] Example 1. Porcine pluripotent stem cells are directed to differentiate into male germ cells

[0105] 1. Configuration of directional induction differentiation medium

[0106] Table 1 is the formulation of iPSCs cell culture medium (iPSC medium)

[0107]

[0108] Table 2 is the formulation of EpiLC medium

[0109]

[0110] Table 3 is the formula of PGCLC induction medium

[0111]

[0112]

[0113] Table 4 is SSCLC induction medium

[0114]

[0115] 2. Orientation induced differentiation

[0116] In this study, in order to track exogenous cells in vivo, pig iPSCs containing ZsGreen (green) were used. The cells have pluripotency and differentiation potential, and can initially differentiate into neural and lipid cells, see figure 1 .

[0117] On the basis of directional induction and differentiation of mouse and human pluripotent stem cells into germ cells, optimize the culture system, and form a method and process for inducing pig iPSCs into germ cell...

Embodiment 2

[0141] Embodiment 2, detection

[0142] Ⅰ. Molecular level and epigenetic detection of PGCLC and SSCLC

[0143] 1. Detection methods of PGCLC and SSCLC marker genes and marker proteins

[0144] 1. Collection of cell samples

[0145] Collection of iPSC samples: Because iPSCs grow on feeder cells, when collecting samples to extract RNA, the feeder cells need to be removed first. Using the difference in the attachment time of the two kinds of cells, they were separated. First, digest iPSCs into single cells, resuspend the cell pellet with fresh medium after centrifugation, mix the cell suspension evenly, transfer it to a cell culture dish, and put it in a cell culture incubator. After 30 minutes, most of the feeder layer The cells have adhered to the wall, but the iPSCs are still suspended in the culture medium. Transfer the cell suspension in the culture dish to a centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant.

[0146] Collection of EpiLC / PGCLC...

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Abstract

The invention discloses a method for directionally inducing and differentiating pig pluripotent stem cells into male germ cells and a special culture medium. The invention provides the special culture medium. The special culture medium comprises an EpiLC inducing medium, a PGCLC (primordial germ cell-like cell) inducing medium and an SSCLC (spermatogonial stem cell-like cell) inducing medium. The invention provides the method for directionally inducing and differentiating the pig pluripotent stem cells into PGCLC in vitro; experiments prove that the PGCLC can be further induced and differentiated to form SSCLC; in vivo, induced cells are injected into seminiferous tubules of mice of which endogenous sperms are removed, and the PGCLC and the SSCLC can survive and are further developed. Therefore, the invention provides a feasible and useful scheme for directional differentiation from the pig pluripotent stem cells to the germ cells, and lays a foundation for exploring a development mechanism and the like of the germ cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for directional induction and differentiation of porcine pluripotent stem cells into male germ cells and a special culture medium. Background technique [0002] In 2003, Yayoi Toyooka et al. cultured mouse embryonic stem cells (Embronic stem cells, ESCs) to form embryoid bodies (Embryoid bodies, EBs), which can detect germ cells that are equivalent to the accumulation of fetal gonads during in vivo development; In in vivo experiments, the researchers detected sperm differentiated from exogenous cells, which was verified by polymerase chain reaction (PCR). The article does not examine the ability of induced differentiation sperm to activate egg cells. In 2004, NielsGeijsen et al. isolated primordial germ cells (PGCs) from EBs cultured with retinoic acid (RA), and continued to culture them to obtain embryonic germ cell lines; PGCs in EBs can develop into haploid sperm, and th...

Claims

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Application Information

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IPC IPC(8): C12N5/076C12N5/0735C12N5/071
CPCC12N5/061C12N5/0611C12N2500/32C12N2500/44C12N2500/84C12N2501/11C12N2501/115C12N2501/125C12N2501/13C12N2501/155C12N2501/16C12N2501/235C12N2501/30C12N2506/45
Inventor 韩建永王寒凝相金柱
Owner CHINA AGRI UNIV
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