KR20150104688A - Preparation method of intracellular neural stem cell from induced pluripotent stem cell using chimera - Google Patents

Preparation method of intracellular neural stem cell from induced pluripotent stem cell using chimera Download PDF

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KR20150104688A
KR20150104688A KR1020140026317A KR20140026317A KR20150104688A KR 20150104688 A KR20150104688 A KR 20150104688A KR 1020140026317 A KR1020140026317 A KR 1020140026317A KR 20140026317 A KR20140026317 A KR 20140026317A KR 20150104688 A KR20150104688 A KR 20150104688A
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도정태
최현우
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건국대학교 산학협력단
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Abstract

The present invention relates to a producing method of neural stem cells in the body from induced pluripotent stem cells (iPSCs) and the method is to prepare chimeric mice using a method which injects iPSCs into blastocyst (or an aggregation method) and to produce neural stem cells by extracting neural stem cells included brain tissues of the chimeric mice from iPSCs. The present invention can be used as a new differentiation technology of iPSCs which cultures neural stem cells in the body by separating neural stem cells from brain tissues of the chimeric mice using iPSCs.

Description

키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법{Preparation method of intracellular neural stem cell from induced pluripotent stem cell using chimera}[0001] The present invention relates to a method for producing an inducible pluripotent stem cell using chimera,

본 발명은 키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법에 관한 것이다.The present invention relates to a method for producing an in vivo pluripotent stem cell-derived neural stem cell using chimera.

유도만능줄기세포(induced pluripotent stem cells; iPSCs)는 분화된 체세포를 역분화 과정에 의해 모든 세포로 분화가 가능한 초기 배아단계로 유도된 만능세포이다. 유도만능줄기세포가 모든 세포로 분화가 가능한 특징 때문에 환자 유래 세포를 이용한 면역거부반응이 없이 난치성 질환 세포치료제로써의 연구가 활발히 진행되고 있다. 유도만능줄기세포를 이용한 연구 중, 신경질환 치료가 상당 부분을 차지하고 있다. 따라서, 유도만능줄기세포를 신경세포 또는 신경줄기세포로 분화시키는 기술이 많이 개발되어 있다. 즉 체외에서 신경구(neurosphere)를 형성하여 단일층(monolayer) 배양 방법으로 bFGF, EGF를 첨가한 배지에서 배양하여 신경줄기세포로 분화하는 방법이 이용되고 있다. 신경줄기세포로 분화하면, 지속적으로 신경세포로 분화할 수 있는 전구세포를 공급할 수 있기 때문에 완전히 분화된 신경세포로 분화시키는 것보다 여러 면에서 장점을 가지고 있다. 현재까지 개발된 유도만능줄기세포 분화방법은 체외에서 배양하는 방법으로 효율이 낮다.
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.

이에, 본 발명자들은 두 단계를 거치는 유도만능줄기세포 유래 신경줄기세포 생산 기술을 개발하였다. 유도만능줄기세포를 배반포에 주입하는 방법(또는 aggregation 방법)을 이용한 키메라 생쥐를 생산하고, 이 키메라 생쥐의 뇌조직에 포함되어 있는 유도만능줄기세포 유래의 신경줄기세포를 추출하여 신경줄기세포를 생산하는 방법이다. 이렇게 수립된 유도만능줄기세포 키메라 유래 신경줄기세포는 신경줄기세포 마커 (Nestin, Musashi)를 발현하며, 정상적으로 뉴런(neuron), 성상세포(astrocyte), 희소돌기아교세포(oligodendrocyte)로의 분화가 가능하다. 또한 유전자 발현 패턴이 유도만능줄기세포의 체외 분화된 신경줄기세포보다 뇌조직에서 유도된 일반 신경줄기세포와 더 유사한 것으로 나타났다. 따라서, 본 발명은 유도만능줄기세포를 이용한 키메라 생쥐의 뇌조직에서 신경줄기세포를 분리하여 체내 신경줄기세포를 배양하는 새로운 유도만능줄기세포 분화 기술이다.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.

한국공개특허 10-2012-0126463(2012.11.21 공개)Korean Patent Laid-Open No. 10-2012-0126463 (published Nov. 21, 2012)

본 발명의 목적은 키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법을 제공하는데 있다. 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.

본 발명의 다른 목적은 상기 방법에 따라 생산된 키메라를 이용한 유도만능줄기세포 유래 신경줄기세포를 제공하는데 있다. 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.

상기 목적을 달성하기 위하여, 본 발명은 (1) 유도만능줄기세포를 배반포에 주입하거나, 응집(aggregation)을 유도하여 키메라 배아를 얻는 단계; (2) 상기 키메라 배아를 자궁에 이식하여 키메라 생쥐를 생산하는 단계; (3) 상기 키메라 생쥐의 뇌조직에서 단일세포를 분리하고, 신경구(neurosphere)를 형성시키는 단계; 및 (4) 상기 형성시킨 신경구로부터 유도만능줄기세포 유래 신경줄기세포를 분리하는 단계를 포함하는 키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법을 제공한다.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.

상세하게는, 상기 유도만능줄기세포는 OG2(Oct4-GFP)와 ROSA26 (neo/lacZ) 형질전환 생쥐와의 교배를 통해 생성된 이형 접합체 섬유아세포에 리프로그래밍 인자 Oct4, Sox2, Klf4 및 c-Myc를 과발현시켜 확립한 역분화 줄기세포일 수 있지만, 이에 제한되는 것은 아니다.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.

상세하게는, 상기 (4) 단계의 유도만능줄기세포 유래 신경줄기세포를 분리하는 단계는 G418을 배양액에 첨가하여 선별할 수 있지만, 이에 제한되는 것은 아니다.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.

상세하게는, 상기 유도만능줄기세포 유래 신경줄기세포는 신경줄기세포 마커인 Nestin 및 Sox2를 발현하는 것을 특징으로 한다.
Specifically, the induced pluripotent stem cell-derived neural stem cells are characterized by expressing neural stem cell markers Nestin and Sox2.

본 발명에서 사용된 "OG2(Oct4-GFP) 형질전환 생쥐"는 Oct4 프로모터의 조절에 의해 발현되는 GFP(Oct4-GFP)로 형질전환된 생쥐를 말한다. 상기 생쥐의 제조방법은 Methods in Molecular Biology, vol 254, Germ Cell Protocols, Volume 2: Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning 등에 공지되어 있다.
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.

본 발명에 있어서, "유도만능줄기세포(induced pluripotent stem cells; iPSCs)"는 분화된 체세포에서 모든 세포로 분화가 가능한 초기 배아단계로 역분화된 세포를 의미한다.
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.

본 발명에 있어서, "키메라(chimera)"는 한 개체가 유전적으로 서로 다른 개체에서 유래하는 조직, 세포, 핵, 염색체 또는 유전자 등의 유전적 특징을 내포하고 있는 복합동물을 말한다.
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.

또한, 본 발명은 상기의 방법에 따라 생산된 키메라를 이용한 유도만능줄기세포 유래 신경줄기세포를 제공한다. The present invention also provides an inducible pluripotent stem cell-derived neural stem cell using the chimera produced according to the above method.

본 발명은 키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법에 관한 것으로서, 유도만능줄기세포를 배반포에 주입하는 방법(또는 aggregation 방법)을 이용한 키메라 생쥐를 생산하고, 이 키메라 생쥐의 뇌조직에 포함되어 있는 유도만능줄기세포 유래의 신경줄기세포를 추출하여 신경줄기세포를 생산하는 방법이다. 본 발명은 유도만능줄기세포를 이용한 키메라 생쥐의 뇌조직에서 신경줄기세포를 분리하여 체내 신경줄기세포를 배양하는 새로운 유도만능줄기세포 분화 기술로서 활용될 수 있다.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.

도 1은 체외에서 유지되는 역분화 줄기세포로부터 체내에서 분화되는 신경줄기세포주를 확립하기 위해 키메라 형성 실험 과정을 나타낸다.
도 2는 역분화 줄기세포를 이용하여 키메라를 형성한 결과이다. (A) 일반 배아와 역분화 줄기세포 콜로니를 배양접시에서 응집(aggregation)을 유도하여 얻은 키메라 배아를 나타낸다. (B) 키메라 배아를 가임신 생쥐 자궁에 이식하여, 13.5 dpc에 자궁에서 배아를 확인한 결과이다. (C) 역분화 줄기세포가 배아를 형성하는 데에 기여하였는지 확인하기 위해 머리를 제외한 몸통 부분을 X-gal 염색 실험한 결과이다.
도 3은 키메라 배아로부터 체내에서 분화된 역분화 줄기세포 유래 신경 줄기세포를 얻기 위해, 신경구 배양액에서 형성시킨 신경구로부터 확장된 신경줄기세포를 나타낸다.
도 4는 역분화 줄기세포로부터 체내 분화한 신경 줄기세포에서 신경 줄기세포 마커인 Nestin, Sox2 유전자가 발현하는 것을 확인한 결과이다.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.

이하, 하기 실시예를 통해 본 발명을 보다 상세하게 설명한다. 다만, 이러한 실시예에 의해 본 발명이 한정되는 것은 아니다.
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.

<< 실시예Example 1>  1> 키메라chimera (( chimerachimera ) 형성) formation

체외에서 유지되는 역분화 줄기세포로부터 체내에서 분화되는 신경줄기세포주를 확립하기 위해 키메라 형성 실험을 이용하였다(도 1). 본 발명에서 사용된 역분화 줄기세포는 OG2 (Oct4-GFP)와 ROSA26 (neo/lacZ) 형질전환 생쥐와의 교배를 통해 생성된 이형 접합체 섬유아세포에 리프로그래밍 인자 Oct4, Sox2, Klf4, c-Myc를 레트로바이러스를 이용해 과발현시켜 확립한 세포주이다. 그리하여 역분화 줄기세포는 만능성이 유지되면 Oct4-GFP가 발현하며 X-gal 염색(staining) 실험을 이용하면 양성 반응을 확인할 수 있으며 G418 약물을 배지에 처리하더라도 유지가 된다. 이러한 역분화 줄기세포의 특징을 이용하여 체내에서 분화된 역분화 줄기세포 유래 신경 줄기세포주를 확립할 수 있다. 먼저 역분화 줄기세포를 이용하여 키메라를 형성하기 위해 일반 배아와 역분화 줄기세포 콜로니를 배양접시에서 응집(aggregation)을 유도하여 키메라 배아를 얻을 수 있었다(도 2A). 역분화 줄기세포는 만능성을 가지고 있기 때문에 내부세포덩어리(ICM)로 들어가 있는 것을 확인 하였고, 이러한 키메라 배아를 가임신 생쥐 자궁에 이식하였다. 13.5 dpc에 자궁에서 배아를 확인할 수 있었다(도 2B). 역분화 줄기세포가 배아를 형성하는 데에 기여하였는지 확인하기 위해 머리를 제외한 몸통 부분을 X-gal 염색 실험을 진행한 결과 배아가 X-gal 양성인 부분과 음성인 부분이 섞여 있는 것을 확인하여 역분화 줄기세포로 형성된 키메라인 것을 확인하였다(도 2C).
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).

<< 실시예Example 2>  2> 키메라chimera 배아로부터 체내에서 분화된  Differentiated from the embryo in the body 역분화De-differentiation 줄기세포 유래 신경 줄기세포 분리 및 배양 Isolation and culture of stem cell-derived neural stem cells

키메라 배아로부터 체내에서 분화된 역분화 줄기세포 유래 신경 줄기세포를 얻기 위해 머리부분을 Trypsin/EDTA를 이용해 단일세포로 분리하고, 신경구 형성을 위해 신경구 배양액에서 배양을 시도하였다. 신경구 배양액은 DMEM/F12 (Gibco) 에 100x N2 (Gibco), 50x B27 (Gibco), 1x 페니실린/스트렙토마이신/글루타민 (Gibco), 0.6% 글루코즈 (sigma), 20ng/ml EGF, bFGF (Gibco)를 첨가하여 사용하였다. 신경구 배양액에서 10~15일 정도 배양을 하게 되면 신경구가 형성된 것을 확인 할 수 있으며, 형성된 신경구를 0.1% 젤라틴(gelatin)이 코팅되어 있는 배양 접시에서 신경 줄기세포 확장 배양액에서 배양하게 되면 신경구로부터 확장되는 신경줄기세포를 확인할 수 있다(도 3A). 신경 줄기세포 확장 배양액은 DMEM/F12 (Gibco)에 50x N2 (Gibco), 1x 페니실린/스트렙토마이신/글루타민 (Gibco), 50㎍/ml 소혈청알부민 (BSA; Gibco), 10ng/ml EGF, bFGF (Gibco)를 첨가하여 사용하였다. 확립된 신경 줄기세포주에 역분화 줄기세포로부터 유래된 신경줄기세포가 포함되어 있는지 확인하기 위해 X-gal 염색을 실시하였다. 키메라 머리로부터 확립된 신경 줄기세포주는 몸통 부분과 유사하게 X-gal 염색에 양성인 세포와 음성인 세포가 섞여 있는 것을 확인하였다(도 3B 왼쪽). 키메라 신경 줄기세포로부터 역분화 줄기세포로부터 유래된 신경 줄기세포만을 획득하기 위해 G418 약물을 신경 줄기세포 확장 배양액에 첨가하여 배양을 시도하였다. 그 결과 일반 배아로부터 유래된 신경 줄기세포는 G418이 함유된 배양액에서 죽는 것을 확인하였고, X-gal 염색에 양성인 세포만 남는 것을 확인하였다(도 3B 중간). 약물처리를 통해 선발한 이후 신경 줄기세포 확장 배양액에 계대배양 하게 되면 역분화 줄기세포 유래 신경 줄기세포주를 확립한 것을 확인할 수 있었다(도 3B 오른쪽). 역분화 줄기세포로부터 체내 분화한 신경 줄기세포에서 신경 줄기세포 마커인 Nestin, Sox2 유전자가 발현하는 것으로 확인된다(도 4). 확립된 줄기세포를 PBS로 세척 후, 4% 파라포름알데히드를 이용하여 상온에서 20분간 고정하고 다시 PBS로 세척하였다. 0.1% Triton-X 100을 5분 동안 처리한 후 정지 용액을 통해 30-60분 동안 상온에 방치하였다. 5분 동안 세척용액으로 세척하고 1차 항체 처리 후 4℃에서 하룻밤 동안 방치하였다. 다음날 10분씩 3번 세척용액으로 세척하고 2차 항체를 통해 2시간 30분 동안 상온에 방치하였다. 3-5분 동안 DAPI를 처리하고 다시 10분씩 3번 세척용액으로 세척한 후 염색시킨 줄기세포들은 형광 현미경을 통해 확인하였다. 역분화 줄기세포에서 유래한 신경 줄기세포에 사용된 1차 항체는 항-Nestin (Chemicon), 항-Sox2 (Chemicon)를 사용하였다. 각 결과를 참조하면, 본 발명에 따른 방법으로 제조된 낭배외피 줄기세포와 낭배외피 줄기세포에서 유래한 신경줄기세포는 모두 각각의 항체에 대한 면역세포화학(Immunocytochemistry) 반응을 보이는 것으로 확인되는바, 제조된 줄기세포들이 각각의 특성을 나타내는 것으로 판단된다.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) 유도만능줄기세포를 배반포에 주입하거나, 응집(aggregation)을 유도하여 키메라 배아를 얻는 단계;
(2) 상기 키메라 배아를 자궁에 이식하여 키메라 생쥐를 생산하는 단계;
(3) 상기 키메라 생쥐의 뇌조직에서 단일세포를 분리하고, 신경구(neurosphere)를 형성시키는 단계; 및
(4) 상기 형성시킨 신경구로부터 유도만능줄기세포 유래 신경줄기세포를 분리하는 단계를 포함하는 키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법.(1) inducing pluripotent stem cells into blastocysts or inducing aggregation to obtain chimeric embryos;
(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 nerve. 제 1 항에 있어서, 상기 유도만능줄기세포는 OG2(Oct4-GFP)와 ROSA26 (neo/lacZ) 형질전환 생쥐와의 교배를 통해 생성된 이형 접합체 섬유아세포에 리프로그래밍 인자 Oct4, Sox2, Klf4 및 c-Myc를 과발현시켜 확립한 역분화 줄기세포인 것을 특징으로 하는 키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법.2. The method of claim 1, wherein said induced pluripotent stem cells are selected from the group consisting of heterozygous fibrin baculovirus reprogramming factors Oct4, Sox2, Klf4 and cfc produced by mating with OG2 (Oct4-GFP) and ROSA26 (neo / lacZ) -Myc overexpressing stem cells derived from the stem cells of the present invention. 제 1 항에 있어서, 상기 (4) 단계의 유도만능줄기세포 유래 신경줄기세포를 분리하는 단계는 G418을 배양액에 첨가하여 선별하는 것을 특징으로 하는 키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법.The method according to claim 1, wherein the step of isolating the induced pluripotent stem cell-derived neural stem cells in step (4) comprises the step of selecting G418 by adding to the culture medium to select the inducible pluripotent stem cell-derived neural stem cells. 제 1 항에 있어서, 상기 유도만능줄기세포 유래 신경줄기세포는 신경줄기세포 마커인 Nestin 및 Sox2를 발현하는 것을 특징으로 하는 키메라를 이용한 유도만능줄기세포 유래 체내 신경줄기세포 생산 방법.The method according to claim 1, wherein the induced pluripotent stem cell-derived neural stem cells express neural stem cell markers Nestin and Sox2. 제 1 항 내지 제 4 항 중 어느 한 항의 방법에 따라 생산된 키메라를 이용한 유도만능줄기세포 유래 신경줄기세포. An inducible pluripotent stem cell-derived neural stem cell using a chimera produced according to the method of any one of claims 1 to 4.
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