KR20150104689A - Preparation method of intracellular neural stem cell from teratoma induced from pluripotent stem cell - Google Patents

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

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KR20150104689A
KR20150104689A KR1020140026318A KR20140026318A KR20150104689A KR 20150104689 A KR20150104689 A KR 20150104689A KR 1020140026318 A KR1020140026318 A KR 1020140026318A KR 20140026318 A KR20140026318 A KR 20140026318A KR 20150104689 A KR20150104689 A KR 20150104689A
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도정태
홍연주
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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 derived from teratoma formed from pluripotent stem cells and the method is to induce teratoma formation by injecting pluripotent stem cells into subcutaneous or a testis capsule of mice and to produce neural stem cells in the body by extracting neural stem cells from among various tissue cells derived from pluripotent stem cells included in the teratoma. The present invention can be used as a new differentiation technology of pluripotent stem cells which cultures neural stem cells formed in the body by separating neural stem cells from the teratoma after inducing the teratoma formation from embryonic stem cells or induced pluripotent stem cells.

Description

만능줄기세포로부터 형성된 테라토마 유래 체내 신경줄기세포 생산 방법{Preparation method of intracellular neural stem cell from teratoma induced from pluripotent stem cell}[0001] The present invention relates to a method for producing a teratoma-derived neural stem cell produced from pluripotent stem cells,

본 발명은 만능줄기세포로부터 형성된 테라토마 유래 체내 신경줄기세포 생산 방법에 관한 것이다.The present invention relates to a method for producing a teratoma derived body neural stem cell formed from pluripotent stem cells.

만능줄기세포 즉, 배아줄기세포 또는 유도만능줄기세포(induced pluripotent stem cells; iPSCs)는 태반 조직을 제외한 모든 세포로 분화가 가능한 세포로 다분화능을 가지고 있다. 만능줄기세포가 성체의 모든 세포로 분화가 가능한 특징 때문에 만능줄기세포를 이용한 세포치료제 연구가 활발하다. 즉, 환자 유래 세포에서 만능줄기세포를 만들어 이용하면 면역거부반응이 없이 난치성 질환의 세포치료제가 가능하다. 유도만능줄기세포를 이용한 연구 중, 신경질환 치료가 상당 부분을 차지하고 있다. 따라서, 만능줄기세포를 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.
Allogeneic stem cells, or embryonic stem cells or induced pluripotent stem cells (iPSCs), are capable of differentiating into all but the placental tissue. Because of the ability of pluripotent stem cells to differentiate into all adult cells, research on cell therapy using pluripotent stem cells is active. In other words, when pluripotent stem cells are prepared from patient-derived cells, a cell therapy agent for intractable diseases is possible without an immune rejection reaction. Among the studies using inducible pluripotent stem cells, treatment of neurological diseases accounts for a large part. Therefore, many techniques for differentiating pluripotent stem cells into neural 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 만능줄기세포 유래 신경줄기세포 생산 기술을 개발하였다. 만능줄기세포를 생쥐의 피하(subcutaneous) 또는 고환 캡슐(testis capsule)에 주입하여 테라토마(teratoma) 형성을 유도한다. 테라토마는 만능세포로부터 증식 및 분화된 세포로 형성된 종양의 일종으로 내배엽, 중배엽, 외배엽 조직을 모두 포함하고 있다. 따라서, 이 테라토마에 존재하고 있는 만능줄기세포 유래의 여러 조직세포 중에서 신경줄기세포를 추출하여 체내 신경줄기세포를 생산하는 방법이다. 이렇게 수립된 만능줄기세포로부터 만들어진 테라토마 유래 신경줄기세포는 신경줄기세포 마커 (Nestin, Musashi)를 발현하며, 정상적으로 뉴런(neuron), 성상세포(astrocyte), 희소돌기아교세포(oligodendrocyte)로의 분화가 가능하다. 또한 유전자 발현 패턴 또한 뇌조직에서 유도된 일반 신경줄기세포와 유사한 것으로 나타났다. 따라서, 본 발명은 배아줄기세포 또는 유도만능줄기세포로부터 테라토마 형성을 유도한 후, 테라토마에 존재하는 신경줄기세포를 분리하여 체내에서 형성된 신경줄기세포를 배양하는 새로운 만능줄기세포 분화 기술이다.Accordingly, the present inventors have developed a technique for producing pluripotent stem cell-derived neural stem cells through two steps. Allogeneic stem cells are injected into the subcutaneous or testis capsule of mice to induce teratoma formation. Teratoma is a type of tumor formed by pluripotent cells that proliferate and differentiate into cells. It contains endoderm, mesoderm, and ectodermal tissue. Therefore, it is a method of producing neural stem cells in the body by extracting neural stem cells from various pluripotent stem cell-derived tissue cells present in the teratoma. Teratoma-derived neural stem cells from these established pluripotent stem cells express neural stem cells (Nestin, Musashi) and are normally capable of differentiating into neurons, astrocytes, and oligodendrocytes. In addition, gene expression pattern was similar to that of normal neural stem cells derived from brain tissue. Accordingly, the present invention is a novel pluripotent stem cell differentiation technique that induces the formation of teratoma from embryonic stem cells or induced pluripotent stem cells, and then separates the neural stem cells existing in the teratoma and cultivates neural stem cells formed in the body.

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

본 발명의 목적은 만능줄기세포로부터 형성된 테라토마 유래 체내 신경줄기세포 생산 방법을 제공하는데 있다. It is an object of the present invention to provide a method for producing a teratoma derived body neural stem cell formed from pluripotent stem cells.

본 발명의 다른 목적은 상기 방법에 따라 생산된 테라토마 유래 신경줄기세포를 제공하는데 있다. Another object of the present invention is to provide a teratoma derived neural stem cell produced according to the above method.

상기 목적을 달성하기 위하여, 본 발명은 (1) 만능줄기세포를 생쥐에 주입하여 테라토마(teratoma) 형성을 유도하는 단계; (2) 상기 테라토마의 조직으로부터 단일세포를 분리하고, 신경구(neurosphere)를 형성시키는 단계; 및 (3) 상기 신경구로부터 테라토마 유래 신경줄기세포를 분리하는 단계를 포함하는 테라토마 유래 체내 신경줄기세포 생산 방법을 제공한다.In order to accomplish the above object, the present invention provides a method for producing a stem cell comprising the steps of: (1) inducing teratoma formation by injecting pluripotent stem cells into a mouse; (2) separating single cells from the tissue of the teratoma and forming neurospheres; And (3) separating the teratoma-derived neural stem cells from the nerve root.

상세하게는, 상기 만능줄기세포는 유도만능줄기세포 또는 배아줄기세포일 수 있다. 보다 상세하게는, 상기 만능줄기세포는 Olig2-GFP 형질전환 만능줄기세포일 수 있다.Specifically, the pluripotent stem cells may be induced pluripotent stem cells or embryonic stem cells. More particularly, the pluripotent stem cells may be Olig2-GFP transgenic pluripotent stem cells.

상세하게는, 상기 테라토마 유래 신경줄기세포를 분리하는 단계는 Olig2-GFP 형광 발현 세포를 분리하는 것을 특징으로 한다.
Specifically, the step of isolating the teratoma-derived neural stem cells comprises separating Olig2-GFP fluorescence-expressing cells.

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

본 발명에서 사용된 "Olig2-GFP 형질전환 만능줄기세포"는 신경줄기세포에서 발현하는 Olig2 유전자 프로모터에 의해 초록형광단백질(GFP)의 발현이 조절되는 형질전환 세포를 말한다. 따라서, Olig2-GFP 형질전환 만능줄기세포가 신경줄기세포로 분화하면, GFP를 발현한다. 상기 만능줄기세포의 제조방법은 Mech Dev. Dec; 2000 99(1-2):143-8 등에 공지되어 있다.
As used herein, "Olig2-GFP transformed pluripotent stem cell" refers to a transformed cell in which the expression of green fluorescent protein (GFP) is regulated by the Olig2 gene promoter expressed in neural stem cells. Thus, when Olig2-GFP-transformed pluripotent stem cells differentiate into neural stem cells, they express GFP. The method for producing pluripotent stem cells is described in Mech Dev. Dec; 2000 99 (1-2): 143-8.

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.

또한, 본 발명은 상기의 방법에 따라 생산된 테라토마 유래 신경줄기세포를 제공한다. The present invention also provides a teratoma derived neural stem cell produced by the above method.

본 발명은 만능줄기세포로부터 형성된 테라토마 유래 체내 신경줄기세포 생산 방법에 관한 것으로서, 만능줄기세포를 생쥐의 피하(subcutaneous) 또는 고환 캡슐(testis capsule)에 주입하여 테라토마(teratoma) 형성을 유도하고, 상기 테라토마에 존재하고 있는 만능줄기세포 유래의 여러 조직세포 중에서 신경줄기세포를 추출하여 체내 신경줄기세포를 생산하는 방법이다. 본 발명은 배아줄기세포 또는 유도만능줄기세포로부터 테라토마 형성을 유도한 후, 테라토마에 존재하는 신경줄기세포를 분리하여 체내에서 형성된 신경줄기세포를 배양하는 새로운 만능줄기세포 분화 기술로서 활용될 수 있다.The present invention relates to a method for producing a teratoma-derived neural stem cell derived from pluripotent stem cells, wherein pluripotent stem cells are injected into a subcutaneous or testis capsule of a mouse to induce teratoma formation, Is a method for producing neural stem cells in the body by extracting neural stem cells from various pluripotent stem cell-derived pluripotent stem cells. The present invention can be utilized as a novel pluripotent stem cell differentiation technique in which teratoma formation is induced from embryonic stem cells or induced pluripotent stem cells and then neural stem cells existing in teratoma are isolated to cultivate neural stem cells formed in the body.

도 1A는 정상 정소와 테라토마 형성된 정소를 나타낸다. 도 1B는 테라토마 조직을 분쇄하여 형광 현미경에서 관찰하면, 신경줄기세포 마커인 Olig2-GFP가 발현하는 세포가 관찰됨을 나타낸다.
도 2A는 테라토마 조직에서 유래한 세포 중 약 11.2%의 세포가 Olig2-GFP를 발현하는 신경줄기세포임을 나타낸다. 도 2B는 단일(single) 세포로 분리된 GFP를 발현하는 신경줄기세포를 나타낸다.
도 3은 Olig2-GFP를 발현하는 세포를 이용한 신경구(neurosphere) 형성 결과를 나타낸다. 이렇게 형성된 신경구(neurosphere)를 신경줄기세포 배양액에 배양하면 GFP를 발현하는 신경줄기세포로 자라난다.
도 4는 테라토마에서 Olig2-GFP를 발현하는 세포를 추출한 후에 수립된 신경줄기세포는 신경줄기세포 마커인 Nestin, Sox2를 발현하며, 항체염색법에 의해 염색한 결과이다.Figure 1A shows normal testes and teratoma formed testes. FIG. 1B shows that cells that express Olig2-GFP, a neural stem cell marker, are observed when the teratoma tissue is pulverized and observed under a fluorescence microscope.
FIG. 2A shows that about 11.2% of the cells derived from the teratoma tissue are neural stem cells expressing Olig2-GFP. Figure 2B shows neural stem cells expressing GFP isolated in a single cell.
Figure 3 shows the results of neurosphere formation using cells expressing Olig2-GFP. When the neurospheres are cultured in neural stem cells, they grow into neural stem cells expressing GFP.
FIG. 4 shows the results of neuron stem cells established after extracting cells expressing Olig2-GFP in a teratoma expressing neural stem cell markers Nestin and Sox2, and stained by antibody staining.

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> 만능줄기세포  1> pluripotent stem cells 테라토마Teratoma 유래 신경줄기 세포주 확립 Establishment of the derived neural stem cell line

만능성을 가진 배아줄기세포로부터 체내에서 분화한 신경줄기세포를 얻기 위해 테라토마 형성 실험을 수행하였다. 본 발명에서는, Olig2-GFP 만능 줄기 세포 1x10*6 개를 면역 억제 생쥐의 고환(testis) 혹은 내피 사이에 주입하여 테라토마 형성을 유도하였다. 신경줄기세포는 Olig2 유전자를 발현하기 때문에 Olig2-GFP 형광 발현에 따라 신경줄기세포를 분리할 수 있다. 만능줄기세포를 주입한 지 4-6주 후, 발생한 테라토마를 적출하였다(도 1A). 테라토마는 내배엽, 중배엽, 외배엽 조직을 모두 포함하고 있다. 따라서 테라토마 조직 내에 신경 줄기 세포가 존재함이 Olig2-GFP를 통해 확인된다(도 1B). 이 중 신경 줄기 세포를 분리하기 위해, 조직을 작게 조각내어 0.25% Trypsin/EDTA와 PBS를 1:1로 혼합한 분해 용액에 넣어 37℃에 5분간 배양, Pipetting을 통해 단일 세포로 분리하는 작업을 2회 반복하였다. 분리된 세포는 mash로 걸러진 후 FACs sorting을 통해 Olig2-GFP 형광을 발현하는 세포와 발현하지 않는 세포로 나누어졌다. 이 중 11.2%의 세포가 Olig2-GFP 형광을 발현하는 것으로 확인된다(도 2). 이 세포를 신경줄기세포 배양액에서 약 10-15일간 부유 배양하여 Olig2-GFP를 발현하는 신경구가 형성됨을 확인하였다(도 3A). 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. 형성된 신경구는 신경 줄기세포 확장 배양액과 함께 0.15% 젤라틴(gelatin)으로 코팅된 배양접시에 5-10일 동안 붙여졌다. 이후, 0.25% Trypsin/EDTA를 이용해 확장된 신경구를 단일 세포로 분리하여 계대 배양, Olig2-GFP를 발현하는 테라토마 유래 신경줄기 세포주를 확립할 수 있었다(도 3B). 신경줄기세포 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. 이렇게 수립된 만능줄기세포 테라토마 유래 신경줄기세포는 체외에서 분화된 신경줄기 세포주와 유사한 형태와 특징을 보였다.
Teratoma formation experiments were carried out to obtain differentiated neural stem cells from pluripotent embryonic stem cells. In the present invention, 1x10 * 6 Olig2-GFP pluripotent stem cells were injected into the testis or endothelium of immunosuppressed mice to induce teratoma formation. Since neural stem cells express the Olig2 gene, neural stem cells can be isolated according to the expression of Olig2-GFP fluorescence. After 4-6 weeks of injection of the pluripotent stem cells, the resulting teratoma was harvested (Fig. 1A). Teratoma contains endodermal, mesodermal, and ectodermal tissues. Thus, the presence of neural stem cells in the teratoma tissue is confirmed by Olig2-GFP (Fig. 1B). In order to isolate neural stem cells, the tissue was cut into small pieces and incubated at 37 ° C for 5 minutes in a 1: 1 mixture of 0.25% Trypsin / EDTA and PBS, and then separated into single cells by pipetting And repeated twice. The isolated cells were divided into mash-expressing cells and Olig2-GFP fluorescence-expressing cells and FACs sorting-free cells. 11.2% of the cells express Olig2-GFP fluorescence (Fig. 2). These cells were cultured in neural stem cells for about 10-15 days to induce the formation of neurons expressing Olig2-GFP (FIG. 3A). 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 . The formed nerve fibers were stuck to a culture dish coated with 0.15% gelatin together with neural stem cell expansion media for 5-10 days. Then, the expanded neural nerve was separated into single cells using 0.25% trypsin / EDTA, and subculture and establishment of a teratoma-derived neural stem cell line expressing Olig2-GFP could be established (Fig. 3B). 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 ) Was added and used. The established pluripotent stem cell teratoma - derived neural stem cells showed similar morphology and characteristics to the neural stem cell line differentiated in vitro.

<< 실시예Example 2> 면역세포화학( 2> Immunocytochemistry ImmunocytochemistryImmunocytochemistry ) 염색을 통한 신경줄기세포의 특성 확인) Characterization of neural stem cells by staining

만능줄기세포 테라토마 유래 신경줄기세포는 신경줄기세포 마커인 Nestin, Sox2 유전자가 발현하는 것으로 확인된다(도 4). 확립된 신경줄기 세포주를 PBS 세척 후 4% 파라포름알데히드로 4℃에서 20분간 고정하였다. 0.3% Triton-X 100을 5분 동안 처리한 후 정지 용액을 넣고 상온에서 30-60분간 방치하였다. 세척 용액으로 5분 세척 후 1차 항체를 넣고 상온에서 2-4 시간 방치 후 10분 간격으로 3회 세척용액으로 세척하였다. 이후, 2차 항체를 넣고 상온에서 2-3시간 방치시킨 후 5분동안 DAPI를 처리하고 다시 10분 간격으로 3회 세척용액으로 세척하였다. 염색시킨 신경줄기세포는 형광 현미경을 통해 확인하였다. 본 발명에서 사용된 1차 항체는 항-Nestin (Chemicon), 항-sox2 (Chemicon)를 사용하였다. 본 발명을 통하여 만능줄기세포 테라토마 유래 신경줄기세포가 신경줄기세포의 특징을 가진다는 것이 확인된다.
The pluripotent stem cell-derived neural stem cells are found to express the neural stem cell markers Nestin, Sox2 gene (Fig. 4). Established neural stem cell lines were washed with PBS and fixed with 4% paraformaldehyde at 4 ° C for 20 min. After 0.3% Triton-X 100 was treated for 5 minutes, the stop solution was added and left at room temperature for 30-60 minutes. After washing with washing solution for 5 minutes, the primary antibody was added and incubated at room temperature for 2-4 hours and then washed with washing solution three times at intervals of 10 minutes. Then, the secondary antibody was added and allowed to stand at room temperature for 2-3 hours, followed by treatment with DAPI for 5 minutes and washing with washing solution three times at intervals of 10 minutes. The stained neural stem cells were identified by fluorescence microscopy. The primary antibodies used in the present invention were anti-Nestin (Chemicon) and anti-sox2 (Chemicon). It is confirmed by the present invention that pluripotent stem cell-derived neural stem cells are characterized by neural stem cells.

Claims (6)

(1) 만능줄기세포를 생쥐에 주입하여 테라토마(teratoma) 형성을 유도하는 단계;
(2) 상기 테라토마의 조직으로부터 단일세포를 분리하고, 신경구(neurosphere)를 형성시키는 단계; 및
(3) 상기 신경구로부터 테라토마 유래 신경줄기세포를 분리하는 단계를 포함하는 테라토마 유래 체내 신경줄기세포 생산 방법.(1) inducing teratoma formation by injecting pluripotent stem cells into mice;
(2) separating single cells from the tissue of the teratoma and forming neurospheres; And
(3) separating the teratoma-derived neural stem cells from the nerve root. 제 1 항에 있어서, 상기 만능줄기세포는 유도만능줄기세포 또는 배아줄기세포인 것을 특징으로 하는 테라토마 유래 체내 신경줄기세포 생산 방법.The method according to claim 1, wherein the pluripotent stem cell is an inducible pluripotent stem cell or an embryonic stem cell. 제 2 항에 있어서, 상기 만능줄기세포는 Olig2-GFP 형질전환 만능줄기세포인 것을 특징으로 하는 테라토마 유래 체내 신경줄기세포 생산 방법.3. The method according to claim 2, wherein the pluripotent stem cells are Olig2-GFP transformed pluripotent stem cells. 제 1 항에 있어서, 상기 테라토마 유래 신경줄기세포를 분리하는 단계는 Olig2-GFP 형광 발현 세포를 분리하는 것을 특징으로 하는 테라토마 유래 체내 신경줄기세포 생산 방법. The method according to claim 1, wherein the step of isolating the teratoma-derived neural stem cells comprises separating Olig2-GFP fluorescence-expressing cells. 제 1 항에 있어서, 상기 테라토마 유래 신경줄기세포는 신경줄기세포 마커인 Nestin 및 Sox2를 발현하는 것을 특징으로 하는 테라토마 유래 체내 신경줄기세포 생산 방법.The method according to claim 1, wherein the teratoma-derived neural stem cells express neural stem cell markers Nestin and Sox2. 제 1 항 내지 제 5 항 중 어느 한 항의 방법에 따라 생산된 테라토마 유래 신경줄기세포.
A teratoma derived neural stem cell produced according to the method of any one of claims 1 to 5.
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