KR101588109B1 - Preparation method of intracellular neural stem cell from induced pluripotent stem cell using chimera - Google Patents

Abstract

The present invention relates to a method for producing an inducible pluripotent stem cell-derived neural stem cell using a chimera, which comprises producing a chimeric mouse using a method (or an aggregation method) of injecting pluripotent stem cells into a blastocyst, Derived pluripotent stem cell-derived neural stem cells are extracted to produce neural stem cells. The present invention can be utilized as a new inducible pluripotent stem cell differentiation technique in which neural stem cells are isolated from brain tissue of chimeric mice using induced pluripotent stem cells and cultured in the body of neural stem cells.

Description

[0001] The present invention relates to a method for producing an inducible pluripotent stem cell using chimera,

The present invention relates to a method for producing an in vivo pluripotent stem cell-derived neural stem cell using chimera.

Induced pluripotent stem cells (iPSCs) are pluripotent stem cells (IPSCs) derived from early embryonic stages that can differentiate into differentiated somatic cells by the differentiation process. Because inducible pluripotent stem cells can differentiate into all cells, studies as therapeutic agents for intractable disease cells have been actively conducted without immunological rejection using patient-derived cells. Among the studies using inducible pluripotent stem cells, treatment of neurological diseases accounts for a large part. Therefore, many techniques for differentiating induced pluripotent stem cells into nerve cells or neural stem cells have been developed. In other words, a method of forming a neurosphere in vitro and culturing it in a monolayer culture medium supplemented with bFGF and EGF to differentiate into neural stem cells has been used. Differentiation into neural stem cells has several advantages over differentiating into fully differentiated neurons because they can supply precursor cells that can differentiate into neurons continuously. The induction pluripotent stem cell differentiation method developed so far is ineffective because it is cultured in vitro.

Thus, the present inventors have developed a technique for producing an inducible pluripotent stem cell-derived neural stem cell through two steps. A method for producing chimeric mice using induction pluripotent stem cells (or an aggregation method) by injecting pluripotent stem cells into a blastocyst and extracting the pluripotent stem cells derived from the pluripotent stem cells of the chimeric mouse to produce neural stem cells to be. The established pluripotent stem cell chimeric neural stem cells express neural stem cells (Nestin, Musashi) and are capable of differentiating into neurons, astrocyte, and oligodendrocyte. In addition, the gene expression pattern was more similar to that of normal neural stem cells derived from brain tissue than in vitro differentiated neural stem cells of induced pluripotent stem cells. Accordingly, the present invention is a new inducible pluripotent stem cell differentiation technique in which neural stem cells are isolated from brain tissue of chimeric mice using inducible pluripotent stem cells to cultivate the body neural stem cells.

Korean Patent Laid-Open No. 10-2012-0126463 (published Nov. 21, 2012)

It is an object of the present invention to provide a method for producing an in vivo pluripotent stem cell-derived neural stem cell using chimera.

Another object of the present invention is to provide an inducible pluripotent stem cell-derived neural stem cell using the chimera produced according to the above method.

In order to achieve the above object, the present invention provides a method for producing a chimeric embryo, comprising the steps of: (1) injecting induced pluripotent stem cells into a blastocyst or inducing aggregation to obtain a chimeric embryo; (2) transplanting the chimeric embryo into the uterus to produce chimeric mice; (3) separating single cells from the brain tissue of the chimeric mice and forming neurospheres; And (4) separating the induced pluripotent stem cell-derived neural stem cells from the formed neural tube. The present invention also provides a method for producing an in vivo stem cell-derived neural stem cell using the chimera.

Specifically, the inducible pluripotent stem cells express heterozygous fibroblast reprogramming factors Oct4, Sox2, Klf4, and c-Myc produced through mating with OG2 (Oct4-GFP) and ROSA26 (neo / lacZ) But not limited to, a stem cell, a stem cell, and a stem cell.

Specifically, the step of isolating the pluripotent stem cell-derived neural stem cells of step (4) may include, but is not limited to, adding G418 to the culture medium.

Specifically, the induced pluripotent stem cell-derived neural stem cells are characterized by expressing neural stem cell markers Nestin and Sox2.

The "OG2 (Oct4-GFP) transgenic mouse" used in the present invention refers to a mouse transformed with GFP (Oct4-GFP) expressed by the regulation of the Oct4 promoter. Methods for preparing such mice are known from, for example, Methods in Molecular Biology, vol. 254, Germ Cell Protocols, Volume 2: Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning.

In the present invention, "induced pluripotent stem cells (iPSCs)" refers to cells that have been reprogrammed into early embryonic stages capable of differentiating into all cells in differentiated somatic cells.

In the present invention, the term "chimera " refers to a compound animal in which an individual has genetic characteristics such as a tissue, cell, nucleus, chromosome or gene derived from a genetically different entity.

The present invention also provides an inducible pluripotent stem cell-derived neural stem cell using the chimera produced according to the above method.

The present invention relates to a method for producing an inducible pluripotent stem cell-derived neural stem cell using a chimera, which comprises producing a chimeric mouse using a method (or an aggregation method) of injecting pluripotent stem cells into a blastocyst, Derived pluripotent stem cell-derived neural stem cells are extracted to produce neural stem cells. The present invention can be utilized as a new inducible pluripotent stem cell differentiation technique in which neural stem cells are isolated from brain tissue of chimeric mice using induced pluripotent stem cells and cultured in the body of neural stem cells.

FIG. 1 shows an experimental procedure of chimera formation to establish a neural stem cell line differentiated in the body from degenerated stem cell retained in vitro.
FIG. 2 shows the result of forming a chimera using degenerated stem cells. (A) A chimeric embryo obtained by inducing aggregation of a general embryo and a degenerated stem cell colony in a culture dish. (B) Chimera embryos were transplanted into the uterus of a pregnant mouse, and the embryo was confirmed in the uterus at 13.5 dpc. (C) X-gal staining of the body except for the head to see if the degenerated stem cells contributed to the embryo formation.
FIG. 3 shows expanded neural stem cells from neural nerves formed in neural tube cultures to obtain degenerated stem cell-derived neural stem cells differentiated in the body from chimeric embryos.
FIG. 4 shows the results of confirming the expression of Nestin and Sox2 genes, which are neural stem cell markers, in neural stem cells differentiated from the degenerated stem cells.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> chimera ( chimera ) formation

Chimeric formation experiments were used to establish neural stem cell lines differentiating in the body from degenerative stem cells maintained in vitro (Fig. 1). The dedifferentiated stem cells used in the present invention are heterozygous fibrin bacterium reprogramming factors Oct4, Sox2, Klf4, c-Myc, which are produced by mating with OG2 (Oct4-GFP) and ROSA26 (neo / lacZ) Was overexpressed using retrovirus. Thus, when pluripotent stem cells remain pluripotent, Oct4-GFP is expressed and X-gal staining experiments can be used to confirm the positive response, even if the G418 drug is treated in the medium. By using the characteristics of these degenerative stem cells, it is possible to establish a neural stem cell line derived from a degenerative stem cell derived from the body. In order to form a chimera using dedifferentiated stem cells, chimeric embryos were obtained by inducing aggregation of general embryos and dedifferentiated stem cell colonies in a culture dish (FIG. 2A). Since the degenerative stem cells were universal, they were found to enter the inner cell mass (ICM), and these chimeric embryos were transplanted into the uterus of the gestational mouse. The embryo could be identified in the uterus at 13.5 dpc (Fig. 2B). In order to confirm whether or not the degenerated stem cells contributed to the embryo formation, the X-gal staining experiment was carried out on the body except for the head. As a result, it was confirmed that the embryo was mixed with the X- It was confirmed that the chimera was formed into stem cells (Fig. 2C).

< Example  2> chimera  Differentiated from the embryo in the body De-differentiation  Isolation and culture of stem cell-derived neural stem cells

To obtain neural stem cells derived from stem cells derived from the chimeric embryos, the head was separated into single cells using trypsin / EDTA and cultured in neural tube culture for neural tube formation. Neuroglial cultures were maintained in DMEM / F12 (Gibco) supplemented with 100x N2 (Gibco), 50x B27 (Gibco), 1x penicillin / streptomycin / glutamine (Gibco), 0.6% glucose, 20ng / ml EGF, bFGF . When cultured in the neural tube culture for 10 to 15 days, it can be confirmed that the nerve nerve is formed. When the nerve root is cultured in the culture medium of neural stem cell expansion in a culture dish coated with 0.1% gelatin, Neural stem cells can be identified (Fig. 3A). The neural stem cell expansion medium was supplemented with DMEM / F12 (Gibco) supplemented with 50 × N2 (Gibco), 1 × penicillin / streptomycin / glutamine (Gibco), 50 μg / ml bovine serum albumin (BSA), 10 ng / ml EGF, bFGF Gibco) was added and used. X-gal staining was performed to confirm that the established neural stem cell line contained neural stem cells derived from the de-differentiated stem cells. The neural stem cell line established from the chimeric head was confirmed to be a mixture of positive cells and negative cells in X-gal staining similar to the body part (Fig. 3B, left). To obtain only the neural stem cells derived from degenerated stem cells from chimeric neural stem cells, G418 drug was added to the neural stem cell expansion medium and cultured. As a result, the neural stem cells derived from the normal embryo were found to die in the culture medium containing G418, and it was confirmed that only cells positive for X-gal staining remained (middle of FIG. 3B). After selection through drug treatment, subculture of neural stem cell expansion media confirmed establishment of degenerated stem cell-derived neural stem cell line (Fig. 3B, right). Nestin, Sox2 gene, which is a neural stem cell marker, is expressed in neural stem cells differentiated from degenerated stem cells (Fig. 4). Established stem cells were washed with PBS, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and washed again with PBS. 0.1% Triton-X 100 was treated for 5 minutes and then allowed to stand at room temperature for 30-60 minutes through the stop solution. After washing with washing solution for 5 minutes, the cells were allowed to stand overnight at 4 ° C after the primary antibody treatment. The next day, it was washed with 3 times of washing solution for 10 minutes and left at room temperature for 2 hours and 30 minutes through the secondary antibody. The stem cells were stained with DAPI for 3-5 minutes and washed three times for 10 minutes with washing solution. The stained cells were confirmed by fluorescence microscopy. Anti-Nestin (Chemicon) and anti-Sox2 (Chemicon) were used as the primary antibodies used for neural stem cells derived from stem cells. Referring to the results, it was confirmed that both the cervical stem cells prepared by the method according to the present invention and the neural stem cells derived from the cervical stem cell showed immunocytochemistry for each antibody, And the stem cells were found to have different characteristics.

Claims (5)

(1) an animal transformed with GFP (Oct4-GFP) expressed by the regulation of the Oct4 promoter and a heterozygous fibrinogen produced through mating of neomycin resistance gene with the lacZ gene (neo / lacZ) Introducing the induced pluripotent stem cells, which have been established by overexpression of the bcl-E reprogramming factors Oct4, Sox2, Klf4 and c-Myc, into an animal blastocyst or inducing aggregation to obtain a chimeric animal embryo;
(2) transplanting the chimeric animal embryo into the uterus to produce chimeric animals;
(3) isolating single cells from brain tissue of the chimeric animal and forming neurospheres; And
(4) separating the induced pluripotent stem cell-derived neural stem cells by culturing the neural nerve formed in the culture medium to which G418 is added to induce pluripotent stem cell-derived neural stem cells derived from chimeric animals To produce neural stem cells (note that the animal is not human). 2. The method according to claim 1, wherein the animal is a mouse. 2. A method for producing neural stem cells by separating induced pluripotent stem cell-derived neural stem cells from chimeric animals using the in-vivo chimeric animal differentiation. delete 2. The method according to claim 1, wherein the inducible pluripotent stem cell-derived neural stem cells are continuously viable cell lines and express neural stem cell markers Nestin and Sox2. A method for producing neural stem cells by isolating stem cell-derived neural stem cells. delete

Images