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KR20210078704A - Method of production for midbrain ogarnoid - Google Patents

️Tue Jun 29 2021

KR20210078704A - Method of production for midbrain ogarnoid - Google Patents

Method of production for midbrain ogarnoid Download PDF

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Publication number

KR20210078704A

KR20210078704A KR1020190170415A KR20190170415A KR20210078704A KR 20210078704 A KR20210078704 A KR 20210078704A KR 1020190170415 A KR1020190170415 A KR 1020190170415A KR 20190170415 A KR20190170415 A KR 20190170415A KR 20210078704 A KR20210078704 A KR 20210078704A Authority

South Korea

Prior art keywords

midbrain

stem cells

organoids

medium composition

differentiation

Prior art date

2019-12-19

Application number

KR1020190170415A

Other languages

Korean (ko)

Inventor

한동욱

권정우

Original Assignee

주식회사 오간텍

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2019-12-19

Filing date

2019-12-19

Publication date

2021-06-29

2019-12-19 Application filed by 주식회사 오간텍 filed Critical 주식회사 오간텍

2019-12-19 Priority to KR1020190170415A priority Critical patent/KR20210078704A/en

2021-06-29 Publication of KR20210078704A publication Critical patent/KR20210078704A/en

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108010073969 valyllysine Proteins 0.000 description 1
229940088594 vitamin Drugs 0.000 description 1
229930003231 vitamin Natural products 0.000 description 1
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DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1

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Abstract

The present invention relates to a medium composition for inducing differentiation of stem cells into midbrain organoids, comprising CHIR99021, an agonist of an SMAD inhibitor and WNT signaling system, as an active ingredient, and to a method for differentiating the stem cells into the midbrain organoids using the medium composition. When culturing the stem cells using the medium composition of the present invention, it is possible to differentiate the stem cells into homogeneous midbrain cells with high efficiency, and the midbrain organoids manufactured by the method of the present invention can be used as a platform that can be used for various drug development and disease simulation research, and can also be used in the field of tissue transplantation.

Description

중뇌 오가노이드 제조방법{METHOD OF PRODUCTION FOR MIDBRAIN OGARNOID}METHOD OF PRODUCTION FOR MIDBRAIN OGARNOID

본 발명은 SMAD 억제제와 WNT 신호전달체계의 아고니스트(agonist)인 CHIR99021을 유효성분으로 포함하는 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물 및 상기 배지 조성물을 이용하여 줄기세포를 중뇌 오가노이드로 분화시키는 방법에 관한 것이다.The present invention relates to a medium composition for inducing differentiation of stem cells into midbrain organoids comprising SMAD inhibitor and CHIR99021, which is an agonist of the WNT signaling system, as active ingredients, and stem cells using the medium composition to transform the stem cells into midbrain organoids. How to differentiate into

인간의 장기는 한 가지의 세포가 아닌 수십종의 세포가 유기적으로 결합되어 특이적인 기능을 수행하는 고도로 발달된 구조로 이루어져 있다. 그러므로, 줄기세포기반 질환모델링 및 신약스크리닝 연구의 정확성과 신뢰성을 극대화하기 위해서는 실제 장기의 구조생리학적, 환경적 특성을 모사할 수 있는 3차원 분화기술 기반 생체모방형 연구시스템 개발이 필수적으로 요구되고, 이는 향후 질환모델링 및 신약스크리닝의 차세대 플랫폼을 개발하는데 요긴하게 사용될 것으로 판단된다.Human organs are not a single cell, but a highly developed structure in which dozens of types of cells are organically combined to perform specific functions. Therefore, in order to maximize the accuracy and reliability of stem cell-based disease modeling and drug screening research, it is essential to develop a biomimetic research system based on 3D differentiation technology that can simulate the structural, physiological and environmental characteristics of an actual organ. , which is expected to be used in the future to develop a next-generation platform for disease modeling and drug screening.

3D 오가노이드 기술은 생체조직의 복잡한 구조를 높은 수준으로 모사할 수 있는 분화기술로써, 기존의 2차원적 분화기법에 비해서 실제 조직의 구조적, 생리학적 및 기능적 특성을 보다 완벽하게 재현할 수 있는 기술로 평가받고 있다. 이러한 이유로 3D 오가노이드 기술은 최근 전세계적으로 연구가 활발히 이루어지고 있고, 지속적인 연구 및 개발가능성이 큰 줄기세포 분화기술로 주목 받고 있다. 3D organoid technology is a differentiation technology that can simulate the complex structure of living tissue at a high level, and it is a technology that can more perfectly reproduce the structural, physiological and functional characteristics of the actual tissue compared to the existing two-dimensional differentiation method. is evaluated as For this reason, 3D organoid technology has recently been actively studied worldwide and is attracting attention as a stem cell differentiation technology with great potential for continuous research and development.

최근 인간 배아줄기세포(ESC) 및 역분화줄기세포(iPSC)를 이용한 다양한 중추신경계 조직(대뇌피질, 안배, 해마, 소뇌, 뇌하수체)을 모사하고 있는 3D 오가노이드의 생산기술이 개발되고 있고, 그 유도된 3D 오가노이드는 실제 중추신경계의 발생학적, 구조적 및 생리학적 특성을 매우 높은 수준으로 모사하고 있다. 특히, 최근 개발된 중뇌 오가노이드는 도파민성 신경세포와 그 전구세포로 이루어진 신경세포층 및 중뇌 특이적 뉴로멜라닌 축적을 체내 조직과 매우 유사한 수준으로 모사함으로써, 인간 중뇌의 구조생리학적 특성연구 및 퇴행성 뇌질환 기전연구에 적합하다는 것이 증명되었다.Recently, a 3D organoid production technology that mimics various central nervous system tissues (cerebral cortex, oculum, hippocampus, cerebellum, pituitary gland) using human embryonic stem cells (ESC) and iPSCs has been developed, and the The derived 3D organoids mimic the developmental, structural and physiological properties of the actual central nervous system to a very high level. In particular, the recently developed midbrain organoid simulates the neuronal cell layer composed of dopaminergic neurons and their precursor cells and the midbrain-specific neuromelanin accumulation at a level very similar to that of the body tissue, thereby studying the structural and physiological characteristics of the human midbrain and degenerative brain. It has been proven to be suitable for disease mechanism studies.

다만, 현재 대부분의 중추신경계 3D 오가노이드 생산기술의 경우, 분화과정을 정밀하게 제어할 수 있는 기술의 부재로 인한 비균질한 구조의 한계점을 가지고 있는 실정이다. 현재 ESC로부터 중뇌 오가노이드로의 분화시 중뇌 특이적 바닥판(floor plate) 구조 뿐 아니라, 대뇌피질의 신경세포상피층, 전두전엽(prefrontal lobe), 맥락총(choroid plexus), 망막(retina) 등과 같은 다양한 중추신경조직들로의 비균질한 분화가 빈번히 관찰되며, 이러한 비균질 분화과정은 중뇌 등 특정 뇌구조 기반의 질환모델링 등을 위해서 반드시 개선되어야 하는 문제점으로 지적되고 있으나, 현재 단일 오가노이드 내에서 특정 신경계 구조(예: 중뇌)로만 유도 가능한 고순도 분화기법의 개발은 전무한 실정이다. 최근 배아줄기세포에서 일차적으로 분화시킨 신경줄기세포에서 균질한 중뇌 오가노이드로를 생산한 사례가 있지만, 이 경우 성체줄기세포인 신경줄기세포를 사용하였기 때문에 배아줄기세포 및 역분화줄기세포에서 직접 중뇌 오가노이드를 만든 경우와는 다르게 중뇌의 구조가 재현되지 못하는 문제를 보였고 뉴로멜라닌의 분비 측면에서는 여전히 비균질한 양상을 보였다.However, most of the current central nervous system 3D organoid production technologies have limitations in their heterogeneous structure due to the absence of a technology capable of precisely controlling the differentiation process. Currently, during differentiation from ESCs into midbrain organoids, not only the midbrain-specific floor plate structure, but also the neuronal epithelial layer of the cerebral cortex, the prefrontal lobe, the choroid plexus, the retina, etc. Heterogeneous differentiation into central nervous systems is frequently observed, and this heterogeneous differentiation process is pointed out as a problem that must be improved for disease modeling based on specific brain structures such as the midbrain. There is no development of a high-purity differentiation technique that can only be induced (eg, midbrain). Recently, there has been a case of producing homogeneous midbrain organoids from neural stem cells differentiated primarily from embryonic stem cells, but in this case, adult stem cells, neural stem cells, were used, so midbrain organoids were directly derived from embryonic stem cells and dedifferentiated stem cells. In contrast to the case of making , the midbrain structure could not be reproduced, and neuromelanin secretion was still non-homogeneous.

이에, 본 발명자들은 특정 화합물을 이용하여 줄기세포로부터 높은 효율로 균질한 중뇌 오가노이드로 분화시키는 기술을 개발하여 본 발명을 완성하였다.Accordingly, the present inventors have completed the present invention by developing a technique for differentiating from stem cells into homogeneous midbrain organoids with high efficiency using specific compounds.

본 발명의 목적은 SMAD 억제제 및 WNT 신호전달체계의 아고니스트(agonist)인 CHIR99021을 유효성분으로 포함하는 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물을 제공하는 것이다. It is an object of the present invention to provide a medium composition for inducing differentiation of stem cells into midbrain organoids comprising CHIR99021, which is an agonist of a SMAD inhibitor and a WNT signaling system, as an active ingredient.

본 발명의 또 다른 목적은 줄기세포를 SMAD 억제제를 포함하는 배지 조성물에서 배양하여 중뇌로 분화키는 단계를 포함하는 것을 특징으로 하는 중뇌 오가노이드 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a midbrain organoid comprising the step of culturing stem cells in a medium composition containing a SMAD inhibitor to differentiate into the midbrain.

본 발명의 또 다른 목적은 상기 중뇌 오가노이드 제조방법에 의해서 제조된 중뇌 오가노이드를 제공하는 것이다.Another object of the present invention is to provide a midbrain organoid prepared by the method for preparing the midbrain organoid.

상기 목적을 달성하기 위하여, 본 발명은 SMAD 억제제를 유효성분으로 포함하는 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물을 제공한다.In order to achieve the above object, the present invention provides a medium composition for inducing differentiation of stem cells comprising a SMAD inhibitor as an active ingredient into a midbrain organoid.

또한, 본 발명은 줄기세포를 SMAD 억제제를 포함하는 배지 조성물에서 배양하여 중뇌로 분화키는 단계를 포함하는 것을 특징으로 하는 중뇌 오가노이드 제조방법을 제공한다.In addition, the present invention provides a method for producing a midbrain organoid comprising the step of culturing stem cells in a medium composition containing a SMAD inhibitor to differentiate into the midbrain.

또한, 본 발명은 상기 중뇌 오가노이드 제조방법에 의해서 제조된 중뇌 오가노이드를 제공한다.In addition, the present invention provides a midbrain organoid prepared by the method for preparing the midbrain organoid.

이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다.All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention.

본 발명의 하나의 양태는 SMAD 억제제를 유효성분으로 포함하는 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물에 관한 것이다.One aspect of the present invention relates to a medium composition for inducing differentiation of stem cells into midbrain organoids comprising a SMAD inhibitor as an active ingredient.

SMAD 단백질은 TGF-β(transforming growth factor-β) 신호 전달에서 중요한 역할을 하며, 신호전달을 위해 원형질 막(plasma membrane)에서 핵으로 이동한다. SMAD 단백질은 TGF-β와 상호작용하여 분화 및 세포자살 등을 조절한다. TGF-β수용체 키나제는 리간드가 결합하면 SMAD1과 SMAD5 또는 SMAD2와 SMAD3의 C-말단에 위치한 세린 잔기를 인산화시킨다[Liu et al., Proc. Natl. Acad. Sci. USA., 1997, 64:10669-74; Souchelnytskyi et al., J. Biol. Chem., 1997, 272:2810 7-15; Abdollah et al., J. Biol. Chem., 1997, 272:27678-85]. 상기 인산화된 SMAD들은 SMAD4와 헤테로다이머(heterodimers)를 형성하여 핵으로 이동하게 되고[Lagna et al., Nature, 1996, 383:832-6], 여기서 SMAD는 목적 유전자의 전사(transcription)를 촉진한다[Attisano and Wrana, Curr. Opin. Cell Biol., 2000, 12:235-43; Kijke et al., Trends Biochem Sci., 2000, 25:64-70]. SMAD에 대한 목적 모티프(motif)가 인간 PAI-1(plasminogen activator inhibitor-1)과 같은 TGF-β 반응 유전자의 프로모터에 존재하기 때문에 수용체-특이 SMAD3 및 SMAD4는 DNA에 직접 결합한다[Dennler et al., EMBO J., 1998, 17:3091-100]. TGF-β는 SMAD3 및 SMAD4 전사 인자가 인접한 프로모터에 결합하는 것을 통해 유도 억제자인 SMAD7 유전자의 전사를 유도한다[von Gersdorff et al., J. Biol. Chem., 2000, 275:11320-6].The SMAD protein plays an important role in transforming growth factor-β (TGF-β) signaling and translocates from the plasma membrane to the nucleus for signaling. SMAD protein interacts with TGF-β to regulate differentiation and apoptosis. TGF-β receptor kinase phosphorylates serine residues located at the C-terminus of SMAD1 and SMAD5 or SMAD2 and SMAD3 upon ligand binding [Liu et al., Proc. Natl. Acad. Sci. USA., 1997, 64:10669-74; Souchelnytskyi et al., J. Biol. Chem., 1997, 272:2810 7-15; Abdollah et al., J. Biol. Chem., 1997, 272:27678-85]. The phosphorylated SMADs form heterodimers with SMAD4 and migrate to the nucleus [Lagna et al., Nature, 1996, 383:832-6], where SMAD promotes the transcription of the target gene. [Attisano and Wrana, Curr. Opin. Cell Biol., 2000, 12:235-43; Kijke et al., Trends Biochem Sci., 2000, 25:64-70]. Receptor-specific SMAD3 and SMAD4 bind directly to DNA because the target motif for SMAD is present in the promoter of a TGF-β-responsive gene such as human plasminogen activator inhibitor-1 (PAI-1) [Dennler et al. , EMBO J., 1998, 17:3091-100]. TGF-β induces transcription of the SMAD7 gene, an induction repressor, through binding of the SMAD3 and SMAD4 transcription factors to adjacent promoters [von Gersdorff et al., J. Biol. Chem., 2000, 275:11320-6].

상기 SMAD 억제제는 SMAD 단백질의 활성을 억제하는 화합물, 항체, 펩티드,또는 압타머를 의미할 수 있다.The SMAD inhibitor may refer to a compound, antibody, peptide, or aptamer that inhibits the activity of a SMAD protein.

본 발명의 바람직한 구체예로서, 상기 SMAD 억제제는 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride(도르소모핀, Dorsomorphin), 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline(LDN193189), 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide(SB431542), 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide(A83-01) 및 Noggin 단백질(서열번호 1)로 이루어진 군에서 선택되는 어느 하나 이상을 포함 일 수 있고, 더욱 바람직하게는 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride(Dorsomorphin) 및 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide(A83-01)일 수 있다. In a preferred embodiment of the present invention, the SMAD inhibitor is 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (dor Somophine, Dorsomorphin), 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (LDN193189), 4-[4-(1,3-benzodioxol) -5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole It may contain any one or more selected from the group consisting of -1-carbothioamide (A83-01) and Noggin protein (SEQ ID NO: 1), more preferably 6-[4-[2-(1-Piperidinyl)ethoxy ]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin) and 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole- 1-carbothioamide (A83-01).

상기 도르소모핀(Dorsomorphin)은 1 uM 내지 10 uM의 농도로 포함될 수 있고, 보다 바람직하게는 1 내지 4 uM의 농도이고, 보다 더욱 바람직하게는 1 내지 2 uM의 농도일 수 있으나, 이에 제한되는 것은 아니다.The dorsomorphin may be included in a concentration of 1 uM to 10 uM, more preferably in a concentration of 1 to 4 uM, and even more preferably in a concentration of 1 to 2 uM, but is limited thereto. it is not

상기 LDN193189은 5 nM 내지 10 uM의 농도로 포함될 수 있고, 보다 바람직하게는 5 nM 내지 500 nM의 농도이고, 보다 더욱 바람직하게는 50 nM 내지 100 nM의 농도일 수 있으나, 이에 제한되는 것은 아니다.The LDN193189 may be included in a concentration of 5 nM to 10 uM, more preferably in a concentration of 5 nM to 500 nM, and even more preferably in a concentration of 50 nM to 100 nM, but is not limited thereto.

상기 SB431542은 50 nM 내지 50 uM의 농도로 포함될 수 있고, 보다 바람직하게는 1 uM내지 25 uM의 농도이고, 보다 더욱 바람직하게는 5 uM 내지 10 uM의 농도일 수 있으나, 이에 제한되는 것은 아니다.The SB431542 may be included in a concentration of 50 nM to 50 uM, more preferably in a concentration of 1 uM to 25 uM, and even more preferably in a concentration of 5 uM to 10 uM, but is not limited thereto.

상기 A83-01은 1 내지 10 uM의 농도로 포함될 수 있고, 보다 바람직하게는 1 내지 4 uM의 농도이고, 보다 더욱 바람직하게는 1 내지 2 uM의 농도일 수 있으나, 이에 제한되는 것은 아니다.The A83-01 may be included in a concentration of 1 to 10 uM, more preferably in a concentration of 1 to 4 uM, and even more preferably in a concentration of 1 to 2 uM, but is not limited thereto.

상기 Noggin 단백질은 10 ng/mL 내지 1 ug/mL의 농도로 포함될 수 있고, 보다 바람직하게는 50 ng/mL 내지 500 ng/mL의 농도이고, 보다 더욱 바람직하게는 100 내지 200 ng/mL의 농도일 수 있으나, 이에 제한되는 것은 아니다.The Noggin protein may be included at a concentration of 10 ng/mL to 1 ug/mL, more preferably a concentration of 50 ng/mL to 500 ng/mL, and even more preferably a concentration of 100 to 200 ng/mL. may be, but is not limited thereto.

본 발명의 상기 배지 조성물은 SMAD 억제제 이외에 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile(CHIR99021)를 추가적으로 더 포함할 수 있고, 상기 CHIR99021은 다양한 염 형태, 약학적으로 허용가능한 염 형태로 존재할 수 있으며, 예를 들어, 소듐, 칼륨 등의 알칼리 금속염 형태로 존재할 수 있다.In addition to the SMAD inhibitor, the medium composition of the present invention is 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino] It may further include ethyl]amino]-3-pyridinecarbonitrile (CHIR99021), and the CHIR99021 may exist in various salt forms, pharmaceutically acceptable salt forms, for example, in the form of alkali metal salts such as sodium and potassium. can exist as

상기 CHIR99021는 0.1 uM 내지 20 uM의 농도로 포함될 수 있고, 보다 바람직하게는 0.1 uM 내지 10 uM의 농도이고, 보다 더욱 바람직하게는 2 uM 내지 3 uM의 농도일 수 있으나, 이에 제한되는 것은 아니다.The CHIR99021 may be included in a concentration of 0.1 uM to 20 uM, more preferably in a concentration of 0.1 uM to 10 uM, and even more preferably in a concentration of 2 uM to 3 uM, but is not limited thereto.

또한, 상기 도르소모핀, LDN193189, SB431542, A83-01, Noggin 단백질, 또는 CHIR99021, 이들의 약학적으로 허용 가능한 염, 또는 그 용매화물은 본원의 목적에 부합하는 한, 목적하는 구체적 세포의 종류에 따라 적절한 농도로 포함할 수 있다.In addition, the dorsomorphin, LDN193189, SB431542, A83-01, Noggin protein, or CHIR99021, a pharmaceutically acceptable salt thereof, or a solvate thereof is suitable for the specific cell type of interest as long as it meets the purpose of the present application. Depending on the concentration, it may be included in an appropriate concentration.

본원에서 도르소모핀, LDN193189, SB431542, A83-01 및 Noggin 단백질로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 배지 조성물에서 줄기세포를 배양하는 것에 비해서 도르소모핀, LDN193189, SB431542, A83-01 및 Noggin 단백질로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 배지 조성물에 CHIR99021을 추가하여 줄기세포를 배양하면, Wnt 신호의 활성화를 유도하여 중뇌 오가노이드로의 분화가 더욱 현저하게 촉진될 수 있다.Compared to culturing stem cells in a medium composition comprising any one or more selected from the group consisting of dorsomorphin, LDN193189, SB431542, A83-01 and Noggin protein herein, dorsomorphin, LDN193189, SB431542, A83-01 and When the stem cells are cultured by adding CHIR99021 to a medium composition containing at least one selected from the group consisting of Noggin proteins, the differentiation into midbrain organoids can be more remarkably promoted by inducing activation of Wnt signals.

본원의 용어 "줄기세포"는 한 개의 세포가 여러 종류의 다른 세포를 생산해 낼 수 있는 다중 분화능을 가진 세포로서, 우리 몸의 손상받은 부위의 세포들을 새로 재생할 수 있는 세포들을 통칭한다. 줄기세포는 자신과 동일한 세포를 지속적으로 만들어 낼 수 있는 자가 재생산 능력, 특정 환경에서 기능성 특정 세포로 분화할 수 있는 분화능, 면역세포와 반응하여 면역반응을 조절하는 면역 조절능을 갖는다. 줄기세포의 종류는 분화되는 세포의 영역에 따라 인체를 구성하는 200여 가지의 세포로 모두 분화할 능력을 가진 전분화능 줄기세포(pluripotent stem cell)와 특정 종류의 세포로 분화할 수 있도록 특화된 조직-특이적 줄기세포(tissue-specific stem cell)로 나눌 수 있다. 또한 줄기세포를 획득하기 위한 공급원에 따라 수정란에서 출발한 배아 또는 배반포(blastocyte)에서 얻어지는 배아줄기세포(embryonic stem cell)와 발생 과정이 끝난 신생아 또는 성인의 신체 각 조직에서 얻어지는 성체줄기세포(adult stem cell)로 분류될 수 있다. As used herein, the term “stem cell” refers to a cell that has multiple differentiation capacity in which one cell can produce different types of cells, and collectively refers to cells that can regenerate cells in damaged areas of our body. Stem cells have the self-renewal ability to continuously create cells identical to themselves, the differentiation ability to differentiate into functional specific cells in a specific environment, and the immune modulatory ability to regulate immune responses by reacting with immune cells. The types of stem cells are pluripotent stem cells, which have the ability to differentiate all of the 200 types of cells that make up the human body, and specialized tissues that can differentiate into specific types of cells, depending on the area of the cell to be differentiated. It can be divided into tissue-specific stem cells. In addition, depending on the source for obtaining stem cells, embryonic stem cells obtained from embryos or blastocytes from a fertilized egg and adult stem cells obtained from each tissue of a newborn or adult body after the development process has been completed cells) can be classified as

본원에서 용어 "배아줄기세포(embryonic stem cell)"는 수정란이 모체의 자궁에 착상하기 직전인 포배기 배아에서 내세포괴(inner cell mass)를 추출하여 체외에서 배양한 것으로서, 개체의 모든 조직의 세포로 분화할 수 있는 다능성(pluripotent)이거나 전능성(totipotent)일 수 있는 세포를 의미하며, 넓은 의미로는 배아줄기세포로부터 유래한 배아체(embryoid bodies)도 포함한다. 배아체는 배아줄기세포의 다양한 조직 형태로의 자발적 분화 과정에서 줄기세포에 의해 형성된 중간구조이며, 배아 줄기 세포의 배양 중에 형성된 응집물(aggregate) 형태이다. 한편, 본 발명의 배아줄기세포는 인간을 포함한 포유동물로부터 유래할 수 있고, 바람직하게는 인간 배아줄기세포이다.As used herein, the term "embryonic stem cell" refers to extracting an inner cell mass from a blastocyst embryo just before implantation of a fertilized egg in the mother's uterus and culturing it in vitro, and is a cell of all tissues of an individual. It refers to a cell that can be pluripotent or totipotent capable of differentiating, and in a broad sense includes embryoid bodies derived from embryonic stem cells. The embryonic body is an intermediate structure formed by stem cells in the process of spontaneous differentiation of embryonic stem cells into various tissue types, and is in the form of an aggregate formed during culturing of embryonic stem cells. On the other hand, the embryonic stem cells of the present invention may be derived from mammals including humans, preferably human embryonic stem cells.

배아줄기세포는 외배엽, 중배엽 및 내배엽성 줄기세포로 분화할 수 있다.Embryonic stem cells can differentiate into ectoderm, mesoderm and endoderm stem cells.

본원에서 상기 줄기세포는 배아줄기세포(Embryonic stem cell) 또는 유도만능줄기세포(induced Pluripotent stem cell)를 포함하는 전분화능 줄기세포에서 유래한 것일 수 있고, 상기 줄기세포는 통상적으로 당업계에 공지된 어떠한 방법을 이용하여 획득할 수 있다.The stem cells herein may be derived from pluripotent stem cells including embryonic stem cells or induced pluripotent stem cells, and the stem cells are commonly known in the art. It can be obtained using any method.

본원에서 용어 “분화(differentiation)”란 세포가 분열 증식하여 성장하는 동안에 세포의 구조나 기능이 특수화되는 현상을 의미한다. 다능성 베아줄기세포는 계통이 한정된 전구세포(예컨대, 외배엽성 세포, 중배엽성 세포 또는 내배엽성 세포 등)로 분화한 후, 다른 형태의 전구세포로 더 분화될 수 있고, 그 뒤 특정 조직(예컨대, 중뇌 등)에서 특징적인 역할을 수행하는 말기 분화세포(예컨대, 중뇌세포 등)로 분화될 수 있다. 한편, 본 발명에서는 특정 조합의 SMAD 억제제 및 WNT 신호전달체계의 아고니스트(agonist)인 CHIR99021을 유효성분으로 포함하는 배지 조성물에서 줄기세포를 배양하는 경우 중뇌 오가노이드 또는 중뇌 세포로 분화가 촉진됨을 확인하였다.As used herein, the term “differentiation” refers to a phenomenon in which the structure or function of a cell is specialized while the cell divides and proliferates and grows. After differentiation into lineage-defined progenitor cells (eg, ectoderm cells, mesoderm cells, or endoderm cells, etc.), pluripotent embryonic stem cells can further differentiate into other types of progenitor cells, and then into specific tissues (eg, , midbrain, etc.) can be differentiated into terminally differentiated cells (eg, midbrain cells, etc.) that play a characteristic role. On the other hand, in the present invention, when culturing stem cells in a medium composition containing a specific combination of a SMAD inhibitor and CHIR99021, an agonist of the WNT signaling system, as an active ingredient, it is confirmed that differentiation into midbrain organoids or midbrain cells is promoted. did.

본원에서 용어 "유도만능줄기세포" 또는 "iPSC"는 체세포 또는 이미 분화된 세포를 처리하여 만능 분화성을 갖게 된 세포를 의미한다. 여기에서 처리하는 방법은 화합물, 유전적 변환 또는 특정 조건으로 배양하는 방법 등을 포함하나 이에 한정되는 것은 아니다. "인간 유도만능줄기세포" 또는 "hiPSC"는 인간의 체세포 또는 인간의 분화된 세포를 처리하여 만능분화성을 갖게 된 세포를 의미한다. 상기의 인간 유도만능줄기세포는 섬유아세포 유래일 수 있지만, 이에 한정되는 것은 아니며, 혈액 등 다양한 기원으로부터 유래할 수 있다. 또한, 상기 인간 유도만능줄기세포는 인간 섬유아세포에 Oct4, Sox2, Klf4 및 c-Myc 등 리프로그래밍 관련 유전자를 발현시켜 제작될 수 있다. 이때, Oct4, Sox2, Klf4 및 c-Myc 유전자 등의 발현은 레트로바이러스 감염 또는 에피조말 시스템(episomal system)을 통해 유래될 수 있다.As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a cell having pluripotent differentiation by treating somatic cells or already differentiated cells. Here, the treatment method includes, but is not limited to, a compound, a genetic transformation, or a method of culturing under specific conditions. “Human induced pluripotent stem cells” or “hiPSCs” refer to cells obtained by pluripotent differentiation by treating human somatic cells or human differentiated cells. The human induced pluripotent stem cells may be derived from fibroblasts, but are not limited thereto, and may be derived from various sources such as blood. In addition, the human induced pluripotent stem cells can be produced by expressing reprogramming-related genes such as Oct4, Sox2, Klf4 and c-Myc in human fibroblasts. In this case, the expression of Oct4, Sox2, Klf4 and c-Myc genes may be derived through retroviral infection or an episomal system.

본원에서 특정 조합의 SMAD 억제제는 줄기세포를 중뇌 세포 또는 중뇌 오가노이드로 분화시키는 효과를 나타내기 위해서, 줄기세포배양 또는 줄기세포를 중뇌세포로 분화시키는데 사용되는 통상의 배지에 첨가되어 사용될 수 있다.The SMAD inhibitor of a specific combination herein may be used by adding to a conventional medium used for culturing stem cells or differentiating stem cells into midbrain cells in order to exhibit the effect of differentiating stem cells into midbrain cells or midbrain organoids.

구체적으로, 본원의 SMAD 억제제를 유효성분으로 포함하는 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물에서 줄기세포를 배양하는 경우 줄기세포에서 중뇌 세포 또는 중뇌 오가노이드로 분화되는 효율이 현저하게 증가하고, 균질하고 우수한 기능의 중뇌 세포로 분화되는 비율이 현저하게 증가한다.Specifically, when culturing stem cells in a medium composition for inducing differentiation of stem cells into midbrain organoids containing the SMAD inhibitor of the present application as an active ingredient, the efficiency of differentiation from stem cells into midbrain cells or midbrain organoids is significantly increased. and the rate of differentiation into homogeneous and well-functioning midbrain cells is significantly increased.

또한, 본원의 SMAD 억제제를 유효성분으로 포함하는 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물에서 줄기세포를 배양하여 중뇌 오가노이드로 분화시키는 경우 생체내에서 분화되어 존재하는 중뇌와 유사한 특성을 가지면서, 균질한 특성의 중뇌 세포들로 구성된 중뇌 오가노이드를 수득할 수 있다.In addition, when stem cells are differentiated into midbrain organoids by culturing stem cells in a medium composition for inducing differentiation of stem cells containing the SMAD inhibitor of the present application into midbrain organoids as an active ingredient, properties similar to those of midbrain differentiated in vivo while having, it is possible to obtain a midbrain organoid composed of midbrain cells of homogeneous properties.

본원에서 줄기세포 배양에 사용되는 배지는 이 기술분야의 통상의 기술자들에게 널리 알려진 배지라면 제한없이 사용될 수 있다. 상기 배지는 인위적으로 합성하여 제조할 수 있으며, 상업적으로 제조된 배지를 사용할 수도 있다. 상업적으로 제조되는 배지의 예를 들면, DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal Essential Medium), BME(Basal Medium Eagle), RPMI 1640, F-10, F-12, α―MEM(α―Minimal essential Medium), G-MEM(Glasgow's Minimal Essential Medium), IMDM(Isocove's Modified Dulbecco's Medium), 또는 mTESR(Stabilized feeder-free maintenance medium for human ES and iPS cells), E8(TESR-E8 medium for hESC/hiPSC maintenance), AlphaSTEM™ Naive hPSC Medium(Biocompare), PLUSTEM(HumanES/iPSC medium, Life science resaerch)을 예로 들 수 있으나 이에 한정되는 것은 아니다.The medium used for culturing the stem cells herein may be used without limitation as long as it is a medium well known to those skilled in the art. The medium may be artificially synthesized and prepared, or a commercially prepared medium may be used. Examples of commercially prepared medium include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal) essential Medium), Glasgow's Minimal Essential Medium (G-MEM), Isocove's Modified Dulbecco's Medium (IMDM), or Stabilized feeder-free maintenance medium for human ES and iPS cells (mTESR), E8 (TESR-E8 medium for hESC/hiPSC maintenance) ), AlphaSTEM™ Naive hPSC Medium (Biocompare), and PLUSTEM (HumanES/iPSC medium, Life science resaerch), but are not limited thereto.

본원에서 용어 " 분화(differentiation)"는, 세포가 분열 증식하여 성장하는 동안에 서로 구조나 기능이 특수화하는 현상, 즉 생물의 세포, 조직 등이 각각에게 주어진 일을 수행하기 위하여 형태나 기능이 변해가는 것을 말한다. 본 발명에서 분화는 줄기세포에서 특정세포로 완전히 분화가 유도된 경우뿐만 아니라 줄기세포에서 특정세포로의 완전 분화되기 전 중간 단계에서 형성되는 배아유사구조체(embryonic body)의 형성도 포함하는 것이다.As used herein, the term "differentiation" refers to a phenomenon in which the structure or function of cells is specialized to each other during division and proliferation, that is, the form or function of cells, tissues, etc. of living organisms change in order to perform a given task. say that In the present invention, differentiation includes the formation of an embryonic body formed in an intermediate stage before complete differentiation from stem cells into specific cells as well as when the differentiation is completely induced from stem cells to specific cells.

또 하나의 양태로 본 발명은 줄기세포를 SMAD 억제제를 포함하는 배지 조성물에서 배양하여 중뇌로 분화키는 단계를 포함하는 것을 특징으로 하는 중뇌 오가노이드 제조방법에 관한 것이다.In another aspect, the present invention relates to a method for producing a midbrain organoid comprising the step of culturing stem cells in a medium composition containing a SMAD inhibitor to differentiate into the midbrain.

상기 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물에 관한 사항은 중뇌 오가노이드 제조방법의 본질에 벗어나지 않는 한도에서 중뇌 오가노이드 제조방법에 대해서도 동일하게 적용될 수 있다.The medium composition for inducing differentiation of the stem cells into midbrain organoids can be equally applied to the midbrain organoid manufacturing method as long as it does not deviate from the essence of the midbrain organoid manufacturing method.

본원의 줄기세포를 SMAD 억제제를 포함하는 배지 조성물에서 배양하여 중뇌로 분화키는 단계에서 도르소모핀, LDN193189, SB431542, A83-01 및 Noggin 단백질로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 배지 조성물에서 줄기세포를 배양하는 것에 비해서 도르소모핀, LDN193189, SB431542, A83-01 및 Noggin 단백질로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 배지 조성물에 CHIR99021을 추가하여 줄기세포를 배양하면, Wnt 신호의 활성화를 유도하여 중뇌 오가노이드로의 분화가 더욱 현저하게 촉진될 수 있다.In the step of culturing the stem cells of the present application in a medium composition containing a SMAD inhibitor to differentiate into midbrain, dorsomorphin, LDN193189, SB431542, A83-01, and a medium composition comprising any one or more selected from the group consisting of Noggin proteins Compared to culturing stem cells in dorsomorphin, LDN193189, SB431542, A83-01, and when culturing stem cells by adding CHIR99021 to a medium composition containing any one or more selected from the group consisting of Noggin proteins, By inducing activation, differentiation into midbrain organoids can be more markedly promoted.

본원에서 상기 줄기세포는 배아줄기세포(Embryonic stem cell) 또는 유도만능줄기세포(induced Pluripotent stem cell)일 수 있고, 상기 줄기세포는 통상적으로 당업계에 공지된 어떠한 방법을 이용하여 획득할 수 있다. Herein, the stem cells may be embryonic stem cells or induced pluripotent stem cells, and the stem cells may be obtained using any method commonly known in the art.

구체적으로, 상기 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물을 줄기세포에 처리하여 배양하는 경우 중뇌 세포로 분화되는 효율이 현저하게 증가하고, 우수한 기능을 가진 균질한 중뇌 세포로 분화되는 비율이 현저하게 증가한다.Specifically, when the culture medium composition for inducing differentiation of the stem cells into midbrain organoids is treated and cultured, the efficiency of differentiation into midbrain cells is remarkably increased, and the rate of differentiation into homogeneous midbrain cells with excellent functions This increases significantly.

또 하나의 양태로 본 발명은 상기 중뇌 오가노이드 제조방법에 의해서 제조된 중뇌 오가노이드에 관한 것이다.In another aspect, the present invention relates to a midbrain organoid prepared by the method for preparing the midbrain organoid.

상기 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물에 관한 사항은 중뇌 오가노이드 제조방법에 의해서 제조된 중뇌 오가노이드의 본질에 벗어나지 않는 한도에서 중뇌 오가노이드 제조방법에 의해서 제조된 중뇌 오가노이드에 대해서도 동일하게 적용될 수 있다.The medium composition for inducing the differentiation of the stem cells into midbrain organoids relates to the midbrain organoids prepared by the midbrain organoid manufacturing method as long as they do not deviate from the essence of the midbrain organoids prepared by the midbrain organoid manufacturing method. The same can be applied to

본 발명의 중뇌 오가노이드 제조방법에 의해서 제조된 중뇌 오가노이드는 균질한 중뇌 세포로 구성되어 있고, 본 발명의 제조방법으로 제조한 중뇌 오가노이드를 다양한 신약개발 및 질환모사 연구에 활용할 수 있는 플랫폼으로 활용할 수 있고 조직이식 분야에도 활용할 수 있다.The midbrain organoid produced by the method for manufacturing a midbrain organoid of the present invention is composed of homogeneous midbrain cells, and the midbrain organoid prepared by the method of the present invention is a platform that can be used for the development of various new drugs and disease simulation research. It can be used in the field of tissue transplantation.

본 발명의 배지 조성물을 이용하여 줄기세포를 배양하는 경우 높은 효율로 균질한 중뇌 오가노이드로 분화시킬 수 있고, 본 발명의 제조방법으로 제조한 중뇌 오가노이드를 다양한 신약개발 및 질환모사 연구에 활용할 수 있는 플랫폼으로 활용할 수 있고 조직이식 분야에도 활용할 수 있다.When stem cells are cultured using the medium composition of the present invention, they can be differentiated into homogeneous midbrain organoids with high efficiency, and the midbrain organoids prepared by the manufacturing method of the present invention can be utilized for various drug development and disease simulation studies. It can be used as a platform that exists and can also be used in the field of tissue transplantation.

도 1은 본 발명의 중뇌 오가노이드 제조방법에 대한 모식도이다.
도 2는 다양한 조합의 SMAD 억제제 처리군 배상체(EB, embryoid bodies)의 형태학적 분석 및 생존율를 나타낸 결과이다.
도 3은 다양한 조합의 SMAD 억제제 처리군의 유전자 발현양상을 나타낸 결과이다.
도 4는 다양한 조합의 SMAD 억제제 처리군에서 도파민성신경세포의 존재를 공초점현미경으로 확인한 결과이다. ACSL1은 도파민전구세포 표시인자이고, TH는 도파민성신경세포의 표시인자를 의미한다.
도 5는 다양한 조합의 SMAD 억제제 처리군의 유전자 발현양상을 나타낸 결과이다.
도 6은 다양한 조합의 SMAD 억제제 처리군의 도파민성신경세포의 분포를 공초점현미경으로 확인한 결과이다. MO는 중뇌 오가노이드이고, MAP2는 신경세포 표시인자이고, TH는 도파민성신경세포의 표시인자를 의미한다.
도 7은 다양한 조합의 SMAD 억제제 처리군의 도파민성신경세포의 분포를 확인한 결과이다.
도 8은 0, 0.8, 2 및 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 전체 유전자의 발현을 분석한 결과이고, 3 uM의 CHIR99021를 처리한 경우 신경세포관련 유전자들이 가장 잘 활성화되었었다.
도 9는 0, 0.8, 1.5, 2, 2.5 및 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 중뇌 특이적 유전자의 발현을 분석한 결과이다.
도 10은 0, 0.85, 2 및 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 중뇌와 대뇌 유전자의 발현을 분석한 결과이다.
도 11은 0, 0.8, 1.5, 2, 2.5 및 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 도파민성신경세포의 분포를 공초점현미경으로 확인한 결과이다.
도 12는 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 중뇌 오가노이드의 구조를 공초점현미경으로 분석한 결과이다.
도 13은 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 중뇌 오가노이드의 구조를 전자현미경으로 분석한 결과이다.
도 14는 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 도파민 생성능력을 분석한 결과이다.
도 15는 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 중뇌 오가노이드의 전기생리학적 분석 결과이다.
도 16은 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 경우 중뇌 오가노이드내 뉴로멜라빈 생성을 분석한 결과이다.
도 17은 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 후 생성된 중뇌 오가노이드에 MPTP를 처리한 후 도파민성신경세포의 선택적 사멸을 공초점 현미경으로 분석한 결과이다.
도 18은 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 후 생성된 중뇌 오가노이드에 MPTP를 처리한 후 도파민성신경세포의 선택적 사멸을 공초점 현미경으로 분석하기 위해서 Caspase 3 양성 뉴런세포의 비율 또는 TUNEL 양성 뉴런세포의 비율을 분석한 결과이다.
도 19는 3 uM의 CHIR99021를 처리하여 줄기세포를 분화시킨 후 생성된 중뇌 오가노이드에 MPTP를 처리한 후 도파민성신경세포 이외의 세포 사멸을 공초점 현미경으로 분석한 결과이다.1 is a schematic diagram of a method for preparing midbrain organoids of the present invention.
Figure 2 is a result showing the morphological analysis and survival rate of various combinations of SMAD inhibitor-treated group embryoid bodies (EB, embryoid bodies).
3 is a result showing the gene expression pattern of the SMAD inhibitor treatment group of various combinations.
4 is a result of confirming the presence of dopaminergic neurons in the SMAD inhibitor-treated group of various combinations with a confocal microscope. ACSL1 is a marker for dopaminergic progenitor cells, and TH is a marker for dopaminergic neurons.
5 is a result showing the gene expression pattern of the SMAD inhibitor treatment group of various combinations.
6 is a result of confirming the distribution of dopaminergic neurons in the SMAD inhibitor-treated group of various combinations with a confocal microscope. MO is a midbrain organoid, MAP2 is a neuronal marker, and TH is a marker of dopaminergic neurons.
7 is a result confirming the distribution of dopaminergic neurons in the SMAD inhibitor-treated group of various combinations.
8 is a result of analyzing the expression of all genes when the stem cells were differentiated by treatment with 0, 0.8, 2, and 3 uM of CHIR99021, and when the CHIR99021 was treated with 3 uM, neuron-related genes were most activated.
9 is a result of analyzing the expression of midbrain-specific genes when stem cells are differentiated by treatment with 0, 0.8, 1.5, 2, 2.5 and 3 uM CHIR99021.
10 is a result of analyzing the expression of midbrain and cerebral genes when the stem cells are differentiated by treatment with 0, 0.85, 2 and 3 uM of CHIR99021.
11 is a result of confirming the distribution of dopaminergic neurons by confocal microscopy when the stem cells were differentiated by treatment with 0, 0.8, 1.5, 2, 2.5 and 3 uM CHIR99021.
12 is a result of analyzing the structure of midbrain organoids by confocal microscopy when stem cells are differentiated by treatment with 3 uM CHIR99021.
13 is a result of analyzing the structure of midbrain organoids with an electron microscope when stem cells are differentiated by treatment with 3 uM CHIR99021.
14 is a result of analyzing dopamine-producing ability when stem cells are differentiated by treatment with 3 uM CHIR99021.
15 is an electrophysiological analysis result of midbrain organoids when stem cells are differentiated by treatment with 3 uM CHIR99021.
FIG. 16 is a result of analyzing neuromelavin production in midbrain organoids when stem cells were differentiated by treatment with 3 uM CHIR99021.
17 is a result of analyzing the selective apoptosis of dopaminergic neurons by confocal microscopy after MPTP treatment in midbrain organoids generated after stem cells were differentiated by treatment with CHIR99021 at 3 uM.
18 shows the ratio of Caspase 3-positive neuronal cells or TUNEL in order to analyze the selective apoptosis of dopaminergic neurons by confocal microscopy after MPTP treatment on midbrain organoids generated after differentiating stem cells by treatment with 3 uM CHIR99021 This is the result of analyzing the percentage of positive neuronal cells.
19 shows the results of analysis of cell death other than dopaminergic neurons by confocal microscopy after MPTP treatment in midbrain organoids generated after stem cells were differentiated by treatment with 3 uM CHIR99021.

이하, 본 발명을 참고예, 실시예 및 실험예에 의해 상세히 설명한다. 단, 하기 참고예, 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명을 제한하는 것으로 해석되어서는 아니 된다.Hereinafter, the present invention will be described in detail by reference examples, examples and experimental examples. However, the following Reference Examples, Examples and Experimental Examples are merely illustrative of the present invention, and should not be construed as limiting the present invention.

실시예 1. SMAD 저해제를 이용하여 줄기세포를 중뇌 오가노이드로 분화유도Example 1. Induction of differentiation of stem cells into midbrain organoids using SMAD inhibitors

단일세포로 해리된 1 x 10⁴개의 인간 배아줄기세포(Human embryonic stem cell line(H9, WiCELl WA09)) 부착저해코팅이 된 U-bottom 96 well plate(Coasta, CLS3474-24EA)에 배양하여 구형의 배양체를 형성시키고, 24시간 후 0.8 uM CHIR99021(Tocris, 4423)를 포함한 신경외배엽분화배지(표 1)에 DA(2uM Dorsomorphin(Biogems, 8666430)+ 2 uM A83-01(Biogems, 9094360)를 첨가하여 제조한 중뇌분화배지를 상기 배양체에 처리하여 초기단계의 중뇌조직으로 분화를 유도하였다(도 1). ^{1 x 10 4} human embryonic stem cell lines (H9, WiCELl WA09) dissociated into single cells were cultured in a U-bottom 96-well plate (Coasta, CLS3474-24EA) coated with an adhesion inhibition coating to form a spherical shape. After forming a culture, DA (2uM Dorsomorphin (Biogems, 8666430) + 2 uM A83-01 (Biogems, 9094360) was added to the neuroectoderm differentiation medium (Table 1) containing 0.8 uM CHIR99021 (Tocris, 4423) after 24 hours. The prepared midbrain differentiation medium was treated with the culture medium to induce differentiation into midbrain tissue in the initial stage (FIG. 1).

성분ingredient DMEM/F12:Neurobasal (1:1) (Gibco)DMEM/F12: Neurobasal (1:1) (Gibco) 1X N2 supplement (Gibco) 1X N2 supplement (Gibco) 1X B27 without vitamin (Gibco)1X B27 without vitamin (Gibco) 1%(v/v) penicillin/streptomycin (Invitrogen)1% (v/v) penicillin/streptomycin (Invitrogen) 1%(v/v) GlutaMAX (Invitrogen),1% (v/v) GlutaMAX (Invitrogen), 1%(v/v) minimum essential media-nonessential amino acid (Invitrogen)1% (v/v) minimum essential media-nonessential amino acid (Invitrogen) 0.1%(v/v) β-mercaptoethanol (Invitrogen)0.1% (v/v) β-mercaptoethanol (Invitrogen) 1μg/ml heparin (Sigma-Aldrich)1μg/ml heparin (Sigma-Aldrich)

분화 4일차에 상기 중뇌분화배지에 100 ng/ul의 FGF8(Peprotech, 100-25)및 100 ng/ul의 SAG(Carbosynth, FS27779)를 첨가하여 중뇌바닥판(midbrain floor plate)으로의 분화를 유도하고, 분화 7일차에 각각의 배양체를 마트리젤(Matrigel, Corning, 354277) 방울로 포매(메트리젤이란 일종의 젤 성분으로 말랑말랑한 방울을 만들고 그 안에 오가노이드를 집어 넣음)하여 뇌조직성숙화 배지(표 2)로 옮겨 배양하였다. 그리고 나서, 분화 9일차에 안정화된 배양체를 16개/배양접시(6 cm petri-dish, SPL 10035)의 비율로 옮긴 후 85 rpm의 속도로 흔들며 장기배양(orbital shaker, Thermo, 25870)하여 중뇌 오가노이드로 성숙화하였다.On the 4th day of differentiation, 100 ng/ul of FGF8 (Peprotech, 100-25) and 100 ng/ul of SAG (Carbosynth, FS27779) were added to the midbrain differentiation medium to induce differentiation into a midbrain floor plate. On the 7th day of differentiation, each culture was embedded with drops of Matrigel (Matrigel, Corning, 354277) (Matrigel is a kind of gel component, soft drops are made and organoids are put in them), and brain tissue maturation medium (Table 2) and cultured. Then, on the 9th day of differentiation, the stabilized culture was transferred at a ratio of 16 / culture dish (6 cm petri-dish, SPL 10035), and then shaken at a speed of 85 rpm and cultured for an organ (orbital shaker, Thermo, 25870). matured into a neuron.

성분ingredient DMEM/F12:Neurobasal (1:1) (Gibco)DMEM/F12: Neurobasal (1:1) (Gibco) 1X N2 supplement (Gibco)1X N2 supplement (Gibco) 1X B27 (Gibco)1X B27 (Gibco) 1%(v/v) penicillin/streptomycin (Invitrogen)1% (v/v) penicillin/streptomycin (Invitrogen) 1%(v/v) GlutaMAX (Invitrogen)1% (v/v) GlutaMAX (Invitrogen) 1%(v/v) minimum essential media-nonessential amino acid (Invitrogen)1% (v/v) minimum essential media-nonessential amino acid (Invitrogen) 0.1%(v/v)

-mercaptoethanol (Invitrogen)0.1% (v/v)

-mercaptoethanol (Invitrogen) 1 μg/ml heparin (Sigma-Aldrich)1 μg/ml heparin (Sigma-Aldrich) 10 ng/ml BDNF (Peprotech)10 ng/ml BDNF (Peprotech) 10 ng/ml GDNF (Peprotech)10 ng/ml GDNF (Peprotech) 100 μM ascorbic acid (Sigma-Aldrich),100 μM ascorbic acid (Sigma-Aldrich), 125 μM db-cAMP (Sigma-Aldrich).125 μM db-cAMP (Sigma-Aldrich).

실시예 2. SMAD 저해제(NS)Example 2. SMAD inhibitor (NS)

실시예 1과 같은 방법으로 줄기세포를 중뇌 오가노이드로 분화유도하되, DA(2uM Dorsomorphin + 2 uM A83-01) 대신에 NS(200ng/ml Noggin(Peprotech, 120-10c) + 10uM SB431542(Peprotech, 3014193)를 신경외배엽분화배지에 처리하였다.Inducing differentiation of stem cells into midbrain organoids in the same manner as in Example 1, NS (200ng/ml Noggin (Peprotech, 120-10c) + 10uM SB431542 (Peprotech, 3014193) was treated in a neuroectoderm differentiation medium.

실시예 3. SMAD 저해제(LS)Example 3. SMAD inhibitor (LS)

실시예 1과 같은 방법으로 줄기세포를 중뇌 오가노이드로 분화유도하되, DA(2 uM Dorsomorphin + 2 uM A83-01) 대신에 LS(100 nM LDN(Biogems, 1066208) + 10 uM SB431542(Peprotech, 3014193)를 신경외배엽분화배지에 처리하였다.Stem cells were differentiated into midbrain organoids in the same manner as in Example 1, but instead of DA (2 uM Dorsomorphin + 2 uM A83-01), LS (100 nM LDN (Biogems, 1066208) + 10 uM SB431542 (Peprotech, 3014193) ) was treated in neuroectoderm differentiation medium.

비교예 1. SMAD 저해제(DS)Comparative Example 1. SMAD inhibitor (DS)

실시예 1과 같은 방법으로 줄기세포를 중뇌 오가노이드로 분화유도하되, DA(2uM Dorsomorphin + 2 uM A83-01) 대신에 DS(10 uM Dorsomorphin(Biogems, 8666430) + 10 uM SB431542(Peprotech, 3014193)를 신경외배엽분화배지에 처리하였다.Stem cells were differentiated into midbrain organoids in the same manner as in Example 1, but instead of DA (2 uM Dorsomorphin + 2 uM A83-01) DS (10 uM Dorsomorphin (Biogems, 8666430) + 10 uM SB431542 (Peprotech, 3014193) were treated in a neuroectoderm differentiation medium.

실시예 4. WNT 신호증가를 이용하여 줄기세포를 중뇌 오가노이드로 분화유도Example 4. Induction of differentiation of stem cells into midbrain organoids using WNT signal increase

실시예 1과 같은 방법으로 줄기세포를 중뇌 오가노이드로 분화유도하되, 0.8 uM CHIR99021 대신에 1.5 uM CHIR99021를 신경외배엽분화배지에 처리하였다.Differentiation of stem cells into midbrain organoids was induced in the same manner as in Example 1, but 1.5 uM CHIR99021 was treated in a neuroectoderm differentiation medium instead of 0.8 uM CHIR99021.

실시예 5.Example 5.

실시예 1과 같은 방법으로 줄기세포를 중뇌 오가노이드로 분화유도하되, 0.8 uM CHIR99021(Tocris, 4423) 대신에 2 uM CHIR99021(Tocris, 4423)를 신경외배엽분화배지에 처리하였다.Stem cells were differentiated into midbrain organoids in the same manner as in Example 1, but 2 uM CHIR99021 (Tocris, 4423) was treated in a neuroectodermal differentiation medium instead of 0.8 uM CHIR99021 (Tocris, 4423).

실시예 6.Example 6.

실시예 1과 같은 방법으로 줄기세포를 중뇌 오가노이드로 분화유도하되, 0.8 uM CHIR99021(Tocris, 4423) 대신에 2.5 uM CHIR99021(Tocris, 4423)를 신경외배엽분화배지에 처리하였다.Stem cells were differentiated into midbrain organoids in the same manner as in Example 1, but 2.5 uM CHIR99021 (Tocris, 4423) was treated in a neuroectodermal differentiation medium instead of 0.8 uM CHIR99021 (Tocris, 4423).

실시예 7.Example 7.

실시예 1과 같은 방법으로 줄기세포를 중뇌 오가노이드로 분화유도하되, 0.8 uM CHIR99021(Tocris, 4423) 대신에 3 uM CHIR99021(Tocris, 4423)를 신경외배엽분화배지에 처리하였다.Stem cells were differentiated into midbrain organoids in the same manner as in Example 1, but 3 uM CHIR99021 (Tocris, 4423) was treated in a neuroectodermal differentiation medium instead of 0.8 uM CHIR99021 (Tocris, 4423).

비교예 2.Comparative Example 2.

실시예 1과 같은 방법으로 줄기세포를 중뇌 오가노이드로 분화유도하되, DA(2uM Dorsomorphin + 2 uM A83-01) 대신에 0.1%(v/v) DMSO(Sigma-Aldrich, D 2650)를 신경외배엽분화배지에 처리하였다.Inducing the differentiation of stem cells into midbrain organoids in the same manner as in Example 1, but using 0.1% (v/v) DMSO (Sigma-Aldrich, D 2650) instead of DA (2uM Dorsomorphin + 2 uM A83-01) neuroectoderm It was treated in differentiation medium.

실험예 1. SMAD 저해제를 이용하여 분화시킨 중뇌 오가노이드의 특성 분석Experimental Example 1. Characterization of midbrain organoids differentiated using SMAD inhibitors

SMAD 저해제를 이용하여 분화시킨 중뇌 오가노이드의 특성을 분석하기 위해서 하기와 같은 실험을 수행하였다.In order to analyze the characteristics of the midbrain organoids differentiated using the SMAD inhibitor, the following experiment was performed.

실험예 1-1. 배상체 형성Experimental Example 1-1. embryoid body formation

실시예 1 내지 3 및 비교예 1과 같은 방법으로 SMAD 저해제를 이용하여 분화시킨 중뇌 오가노이드의 특성을 분석하였다.In the same manner as in Examples 1 to 3 and Comparative Example 1, the characteristics of the midbrain organoids differentiated using the SMAD inhibitor were analyzed.

구체적으로, 실시예 1 내지 3 및 비교예 1과 같은 방법을 이용하여 인간 배아줄기세포를 중뇌 오가노이드로 분화유도하기 위한 첫 단계로 서로다른 SMAD 저해제가 처리된 인간 배아줄기세포의 배상체 형성을 현미경을 이용하여 확인하였다.Specifically, as a first step for inducing differentiation of human embryonic stem cells into midbrain organoids using the same method as in Examples 1 to 3 and Comparative Example 1, the formation of embryoid bodies of human embryonic stem cells treated with different SMAD inhibitors was performed. It was confirmed using a microscope.

그 결과 도 2에 나타난 것과 같이, SMAD 저해제를 NS(실시예 2), DA(실시예 1) 및 LS(실시예 3)로 사용한 경우 배상체 형성이 효율적으로 이루어졌으나, DS(비교예 1)로 사용한 경우 배상체가 잘 형성되지 않고 세포들이 죽는 현상을 확인할 수 있었다.As a result, as shown in FIG. 2, when SMAD inhibitors were used as NS (Example 2), DA (Example 1) and LS (Example 3), embryoid body formation was efficiently achieved, but DS (Comparative Example 1) When used as an embryoid body, it was confirmed that the embryoid body was not well formed and the cells died.

실험예 1-2. 신경분화 확인을 위한 유전자 발현 분석Experimental Example 1-2. Gene Expression Analysis for Confirmation of Neurodifferentiation

신경분화 확인을 위해서 실시예 1 내지 3 및 비교예 1과 같은 방법을 이용하여 분화된 조직을 qPCR을 이용하여 분석하였다.To confirm neuronal differentiation, tissues differentiated using the same method as Examples 1 to 3 and Comparative Example 1 were analyzed using qPCR.

qPCR에 사용한 프라이머 서열은 하기 표 3에 기재되어 있다.The primer sequences used for qPCR are shown in Table 3 below.

이름name 서열(5'*sequence (5'* 서열번호SEQ ID NO: Nanog_FNanog_F TGCAACCTGAAGACGTGTGATGCAACCTGAAGACGTGTGA 22 Nanog _RNanog_R CTATGAGGGATGGGAGGACTATGAGGGATGGGAGGA 33 Oct4_FOct4_F GACAGGGGGAGGGGAGGAGCTAGGGACAGGGGGAGGGGAGGAGCTAGG 44 Oct4_ROct4_R CTTCCCTCCAACCAGTTGCCCCAAACCTTCCCTCCAACCAGTTGCCCCAAAC 55 Sox2_FSox2_F AGACTGCACATGAGCCAGCAAGACTGCACATGAGCCAGCA 66 Sox2_RSox2_R CGTCTCCAGCCAGCTTCAACCGTCTCCAGCCAGCTTCAAC 77 Otx2_FOtx2_F CCCTCACTCGCCACATCTACCCCTCACTCGCCACATCTAC 88 Otx2_ROtx2_R GTTCAGAGTCCTTGGTGGGTGTTCAGAGTCCTTGGTGGGT 99 Plzf_FPlzf_F TCCCGCCCGACTGGAGGATATCCCGCCCGACTGGAGGATA 1010 Plzf_RPlz_R TTCTTTCCTGGCTCCCCGCTCTTCTTTCCTGGCTCCCCGCTC 1111 Eomes_FEomes_F CTCAAAAGGCATGGGAGGGTACTCAAAAGGCATGGGAGGGTA 1212 Eomes_REomes_R CACCACCAAGTCCATCTGCAACACCACCAAGTCCATCTGCAA 1313 Sox17_FSox17_F CCTTAGCTCCAGGAAGTGTGCCTTAGCTCCAGGAAGTGTG 1414 Sox17_RSox17_R GCCAGCTCCGCGGTATATTACTGCCAGCTCCGCGGTATATTACT 1515 T_FT_F CGCCTCATAGCCTCATGGACCGCCTCATAGCCTCATGGAC 1616 T_RT_R CACTGGCTGCCACGACAAACACTGGCTGCCACGACAAA 1717 BOK_FBOK_F GCTGGCCACATCTTCTCTGCGCTGGCCACATCTTCTCTGC 1818 BOK_RBOK_R TGCTGACCACACACTTGAGGACTGCTGACCACACACTTGAGGAC 1919 BAX_FBAX_F TGCTGACGTGGACACGGACTTGCTGACGTGGACACGGACT 2020 BOK_RBOK_R CCAGCCACCCTGGTCTTGGACCAGCCACCCTGGTCTTGGA 2121 BAD_FBAD_F TCGGAGTCGCCACAGTTCGTTCGGAGTCGCCACAGTTCGT 2222 BAD_RBAD_R GCGCTCTTTGGGCGAGGAAGGCGCTCTTTGGGCGAGGAAG 2323

그 결과 도 3에 나타난 것과 같이, SMAD 저해제를 NS(실시예 2), DA(실시예 1) 및 LS(실시예 3)로 사용한 경우 신경 외배엽 관련 마커인 SOX2, N-CAD, OTX2, PLZF 등의 유전자 발현이 증가하여 신경분화가 특이적으로 일어났으나, DS(비교예 1)로 사용한 경우 세포사멸인자인 BOK, BAX, BAD 등의 유전자 발현양의 증가와 EMOES, SOX17, T 유전자 발현양의 증가를 통해 세포 사멸이 유도되면서 내배엽, 중배엽으로의 분화가 일어남을 확인할 수 있었다.As a result, as shown in FIG. 3, when SMAD inhibitors were used as NS (Example 2), DA (Example 1) and LS (Example 3), neuroectoderm-related markers SOX2, N-CAD, OTX2, PLZF, etc. Neuronal differentiation occurred specifically due to the increase in gene expression of It was confirmed that differentiation into endoderm and mesoderm occurred as apoptosis was induced through the increase of .

실험예 1-3. 중뇌 도파민성신경세포의 활성 분석Experimental Example 1-3. Analysis of the activity of midbrain dopaminergic neurons

실시예 1 내지 3과 같은 방법을 이용하여 분화된 중뇌 오가노이드를 공초현미경으로 확인하여 중뇌 도파민성신경세포의 활성을 분석하였다.The activity of midbrain dopaminergic neurons was analyzed by confirming the differentiated midbrain organoids using the same method as in Examples 1 to 3 with a confocal microscope.

공초점현미경을 이용하여 관찰하기 위해서 ASCL1(BD Pharmingen, 556604) 및 TH(Abcam, ab152) 항체를 사용하였다.For observation using a confocal microscope, ASCL1 (BD Pharmingen, 556604) and TH (Abcam, ab152) antibodies were used.

한편, 비교예 1의 경우 생존하는 중뇌 오가노이드가 없었기 때문에 분석에 사용하지 않았다.On the other hand, in the case of Comparative Example 1, since there was no surviving midbrain organoid, it was not used for analysis.

그 결과 도 4에 나타난 것과 같이, SMAD 저해제를 NS(실시예 2), DA(실시예 1) 및 LS(실시예 3)로 사용한 경우 중뇌 도파민성신경세포의 표시유전자(TH) 발현이 확인되어 중뇌 오가노이드내에 중뇌 도파민성신경세포(Midbrain dopaminergic neurons, mDA)가 존재함을 확인할 수 있었다.As a result, as shown in FIG. 4 , when SMAD inhibitors were used as NS (Example 2), DA (Example 1) and LS (Example 3), expression of the marker gene (TH) in midbrain dopaminergic neurons was confirmed, and the midbrain It was confirmed that midbrain dopaminergic neurons (mDA) were present in the organoid.

실험예 1-4. 중뇌 활성 마커 유전자의 발현 분석Experimental Example 1-4. Expression analysis of midbrain activity marker genes

실시예 1 내지 3과 같은 방법을 이용하여 분화된 조직을 중뇌 활성 마커 유전자의 발현을 확인하기 위해서 qPCR 분석을 수행하였다.In order to confirm the expression of the midbrain activity marker gene in the differentiated tissue using the same method as in Examples 1 to 3, qPCR analysis was performed.

qPCR에 사용한 프라이머 서열은 하기 표 4에 기재되어 있다.The primer sequences used for qPCR are shown in Table 4 below.

이름name 서열(5'*3')Sequence (5'*3') 서열번호SEQ ID NO: LRTM_FLRTM_F CTGCCATGCCCTTCGTGTGTCTGCCATGCCCTTCGTGTGT 2424 LRTM_RLRTM_R AACACTTGTCCGGGCAGCTGAACACTTGTCCGGGCAGCTG 2525 DAT_FDAT_F CGCCACTGGCTCAAGGTGTACGCCACTGGCTCAAGGTGTA 2626 DAT _RDAT_R CCGGCACGGAAAGGTGTAACCGGCACGGAAAGGTGTAA 2727 TH_FTH_F CTGAGATTCGGGCCTTCGACCTGAGATTCGGGCCTTCGAC 2828 TH _RTH_R TGCACCTAGCCAATGGCACTTGCACCTAGCCAATGGCACT 2929 LMX1B_FLMX1B_F GGCATCAAGATGGAGGAGCAGGCATCAAGATGGAGGAGCA 3030 LMX1B_RLMX1B_R TGGTGAGGGCTTGCTGACACTGGTGAGGGCTTGCTGACAC 3131 LMX1A_FLMX1A_F AAAGCGCGATCGACACCTCAAAGCGCGATCGACACCTC 3232 LMX1A_RLMX1A_R TCCCGGTAGAAGCAGGTGGTTCCCGGTAGAAGCAGGTGGT 3333 ASCL1_FASCL1_F GGTGATCGCACAACCTGCATGGTGATCGCACAACCTGCAT 3434 ASCL1_RASCL1_R GTTCTGAGCGCTTCCCGTTTGTTCTGAGCGCTTCCCGTTT 3535 PAX3_FPAX3_F TTTCCGTTTCGCCTTCACCTTTTCCGTTTCGCCTTCACCT 3636 PAX3_RPAX3_R ACGATCTTGTGGCGGATGTGACGATCTTGTGGCGGATGTG 3737

그 결과 도 5에 나타난 것과 같이, SMAD 저해제를 DA(실시예 1) 와 LS(실시예 3)로 사용한 경우 중뇌 활성 마커 유전자(LRTM1, DAT, TH, LMX1B, LMX1A, ASC1, PAX3)의 발현이 확인되었지만 NS(실시예 2)로 사용한 경우 대뇌 표시유전자(FOXG1, LHX2, SIX3)의 발현이 동시에 이루어져 중뇌 오가노이드 생산에 DA(실시예 1) 와 LS(실시예 3)가 보다 효율적임을 확인할 수 있었다.As a result, as shown in FIG. 5, when SMAD inhibitors were used as DA (Example 1) and LS (Example 3), the expression of midbrain activity marker genes (LRTM1, DAT, TH, LMX1B, LMX1A, ASC1, PAX3) was Although it was confirmed, when used as NS (Example 2), expression of cerebral marker genes (FOXG1, LHX2, SIX3) was simultaneously made, so that DA (Example 1) and LS (Example 3) were more efficient in midbrain organoid production. there was.

실험예 1-5. TH 양성세포의 분석Experimental Example 1-5. Analysis of TH-positive cells

실시예 1 내지 3과 같은 방법을 이용하여 분화된 중뇌 오가노이드를 공초점현미경으로 조사하여 TH 양성세포 숫자 및 분포을 분석하였다.The number and distribution of TH-positive cells were analyzed by irradiating the differentiated midbrain organoids with a confocal microscope using the same method as in Examples 1 to 3.

공초점현미경을 이용하여 관찰하기 위해서 MAP2(Millipore Merck, 05-346) 및 TH(Abcam, ab152) 항체를 사용하였다.For observation using a confocal microscope, MAP2 (Millipore Merck, 05-346) and TH (Abcam, ab152) antibodies were used.

그 결과 도 6 및 7에 나타난 것과 같이, SMAD 저해제 DA(실시예 1)로 사용한 경우, 높은 숫자의 중뇌 도파민성신경세포가 균질하게 존재하였으나, NS(실시예 2) 및 LS(실시예 3)로 사용한 경우 매우 적은 수의 중뇌 도파민성신경세포가 비균질하게 존재함을 확인할 수 있었다.As a result, as shown in FIGS. 6 and 7, when the SMAD inhibitor DA (Example 1) was used, a high number of midbrain dopaminergic neurons were homogeneously present, but NS (Example 2) and LS (Example 3) When used, it was confirmed that a very small number of midbrain dopaminergic neurons existed non-homogeneously.

실험예 2. WNT 신호증가를 이용하여 분화시킨 중뇌 오가노이드의 특성 분석Experimental Example 2. Characterization of differentiated midbrain organoids using WNT signal increase

CHIR99021의 처리 농도를 증가하여 WNT 신호를 증킨 경우 분화된 중뇌 오가노이드의 특성을 분석하기 위해서 하기와 같은 실험을 수행하였다.In order to analyze the characteristics of differentiated midbrain organoids when the WNT signal was increased by increasing the treatment concentration of CHIR99021, the following experiment was performed.

실험예 2-1. 신경관련 유전자의 발현 분석Experimental Example 2-1. Expression analysis of nerve-related genes

실시예 1, 실시예 4 내지 7 및 비교예 2와 같은 방법을 이용하여 분화된 중뇌 오가노이드의 글로벌 유전자 발현양상을 RNA sequencing 기법으로 분석하였다.The global gene expression patterns of the differentiated midbrain organoids were analyzed by RNA sequencing using the same method as in Example 1, Examples 4 to 7, and Comparative Example 2.

그 결과 도 8에 나타난 것과 같이, 2 내지 3 uM 농도의 CHIR99021를 처리했을 때 신경관련 유전자들의 발현이 유의하게 증가됨을 확인할 수 있었다.As a result, as shown in FIG. 8 , it was confirmed that the expression of nerve-related genes was significantly increased when CHIR99021 was treated at a concentration of 2 to 3 uM.

실험예 2-2. 중뇌 유전자의 발현 분석Experimental Example 2-2. Expression analysis of midbrain genes

실시예 1, 실시예 4 내지 7 및 비교예 2와 같은 방법을 이용하여 분화된 중뇌 오가노이드에서 대뇌와 중뇌 관련 유전자의 발현을 qPCR 기법으로 조사하였다.The expression of cerebral and midbrain-related genes in the differentiated midbrain organoids using the same method as in Example 1, Examples 4 to 7 and Comparative Example 2 was investigated by qPCR technique.

qPCR에 사용한 프라이머 서열은 상기 표 4에 기재되어 있다.The primer sequences used for qPCR are shown in Table 4 above.

그 결과 도 9에 나타난 것과 같이, 2, 2.5, 또는 3 uM 농도의 CHIR99021이 처리된 경우 대뇌 관련 유전자는 억제되고 중뇌 특이적 유전자의 발현이 증가함을 확인하였다.As a result, as shown in FIG. 9 , it was confirmed that when CHIR99021 at a concentration of 2, 2.5, or 3 uM was treated, cerebral-related genes were suppressed and the expression of midbrain-specific genes was increased.

실험예 2-3. 대뇌 및 중뇌 관련 유전자의 발현 분석Experimental Example 2-3. Expression analysis of cerebral and midbrain related genes

실시예 1, 실시예 4 내지 7 및 비교예 2와 같은 방법을 이용하여 분화된 중뇌 오가노이드를 RNA sequencing기법을 이용하여 유전자의 발현을 분석하였다.The gene expression of the differentiated midbrain organoids using the same method as in Example 1, Examples 4 to 7 and Comparative Example 2 was analyzed using RNA sequencing technique.

그 결과 도 10에 나타난 것과 같이, 2 내지 3 uM 농도의 CHIR99021를 처리했을 때 대뇌 관련 유전자들의 발현은 완전히 차단되고 중뇌 관련 유전자의 발현이 높은 수준으로 활성화 됨을 확인할 수 있었다.As a result, as shown in FIG. 10 , it was confirmed that the expression of cerebral-related genes was completely blocked and the expression of midbrain-related genes was activated at a high level when CHIR99021 was treated at a concentration of 2 to 3 uM.

실험예 2-4. 면역조직학 분석Experimental Example 2-4. Immunohistological analysis

실시예 1, 실시예 4 내지 7 및 비교예 2와 같은 방법을 이용하여 분화된 중뇌 오가노이드내 중뇌 도파민성신경세포의 분포를 조사하기 위하여 면역조직학 분석을 실시하였다. In order to examine the distribution of midbrain dopaminergic neurons in the differentiated midbrain organoids using the same method as in Examples 1, 4 to 7 and Comparative Example 2, immunohistological analysis was performed.

면역조직한 분석을 위해서 SOX2(R&D system, AF2018), TUJ1(BIOLEGEND, 801201) 및 TH(Abcam, ab152) 항체를 사용하였다.For immunohistochemical analysis, SOX2 (R&D system, AF2018), TUJ1 (BIOLEGEND, 801201) and TH (Abcam, ab152) antibodies were used.

그 결과 도 11에 나타난 것과 같이, 2, 2.5, 또는 3 uM 농도의 CHIR99021를 처리했을 때 많은 수의 중뇌 도파민성신경세포가 균질하게 존재함을 확인할 수 있었다. As a result, as shown in FIG. 11 , it was confirmed that a large number of midbrain dopaminergic neurons were homogeneously present when CHIR99021 at a concentration of 2, 2.5, or 3 uM was treated.

실험예 2-5. 중뇌 오가노이드의 구조적 성숙도 및 균질성 분석Experimental Example 2-5. Analysis of Structural Maturity and Homogeneity of Midbrain Organoids

실시예 7과 같은 방법을 이용하여 분화된 조직의 중뇌 오가노이드의 구조적 성숙도 및 균질성을 확인하기 위해서 공초점현미경을 이용하여 분석하였다.In order to confirm the structural maturity and homogeneity of the midbrain organoids of the differentiated tissue using the same method as in Example 7, analysis was performed using a confocal microscope.

공초점현미경을 이용하여 관찰하기 위해서 FOXA2(Abcam, ab108422), ASCL1(BD Pharmingen, 556604), LRTM1(Cusabio, CSB-PA884513LA01HU), LMX1A(SANTA CRUZ, sc-54273), DAT(Millipore Merck, MER-MAB369), MAP2(Millipore Merck, 05-346) 및 TH(Abcam, ab152) 항체를 사용하였다.For observation using a confocal microscope, FOXA2 (Abcam, ab108422), ASCL1 (BD Pharmingen, 556604), LRTM1 (Cusabio, CSB-PA884513LA01HU), LMX1A (SANTA CRUZ, sc-54273), DAT (Millipore Merck, MER- MAB369), MAP2 (Millipore Merck, 05-346) and TH (Abcam, ab152) antibodies were used.

그 결과 도 12에 나타난 것과 같이, 3 uM의 CHIR99021를 처리했을 때 사람의 중뇌와 유사하게 뇌실대(ventricular zone), 중간대(intermediate zone), 변연대(marginal zone)로 구성된 성숙한 층상구조 (laminated structure)를 가지고 있음을 확인하여 도파민성신경세포의 균질한 분포를 통해 3 uM의 CHIR99021를 처리한 중뇌 오가노이드가 매우 균질한 구조를 가지고 있음을 확인할 수 있었다.As a result, as shown in FIG. 12, when 3 uM of CHIR99021 was treated, similar to the human midbrain, a mature layered structure composed of a ventricular zone, an intermediate zone, and a marginal zone. ), and through the homogeneous distribution of dopaminergic neurons, it was confirmed that the midbrain organoids treated with 3 uM CHIR99021 had a very homogeneous structure.

실험예 2-6. 중뇌 오가노이드의 구조적 균일성 및 성숙도 분석Experimental Example 2-6. Analysis of Structural Uniformity and Maturity of Midbrain Organoids

실시예 7과 같은 방법을 이용하여 분화된 조직의 중뇌 오가노이드의 구조적 균일성 및 성숙도을 확인하기 위해서 전자현미경 분석을 실시하였다.In order to confirm the structural uniformity and maturity of the midbrain organoids of the differentiated tissue using the same method as in Example 7, electron microscopic analysis was performed.

그 결과 도 13에 나타난 것과 같이, 3 uM의 CHIR99021를 처리했을 때 뇌실대 (ventricular zone)에는 신경줄기세포들이 존재하고 변연대(marginal zone)에는 신경세포들이 존재하여 사람의 중뇌와 유사한 구조성을 가짐을 확인할 수 있었다.As a result, as shown in FIG. 13 , when 3 uM of CHIR99021 was treated, neural stem cells were present in the ventricular zone and neurons were present in the marginal zone, so it had a structure similar to that of the human midbrain. was able to confirm

실험예 2-7. 중뇌 오가노이드의 기능성 분석Experimental Example 2-7. Functional analysis of midbrain organoids

실시예 7과 같은 방법을 이용하여 분화된 조직의 중뇌 오가노이드의 기능성을 확인하기 위해서 HPLC 분석을 통해 도파만의 생성을 분석하였다.In order to confirm the functionality of the midbrain organoids of the differentiated tissue using the same method as in Example 7, the generation of dopaman was analyzed through HPLC analysis.

그 결과 도 14에 나타난 것과 같이, 3 uM의 CHIR99021를 처리했을 때 도파민 생성이 매우 높은 수준으로 이루어져 3 uM의 CHIR99021의 처리를 통해 생산된 중뇌 오가노이드가 높은 기능성을 가짐을 확인할 수 있었다. As a result, as shown in FIG. 14 , when 3 uM of CHIR99021 was treated, dopamine production was made at a very high level, and it was confirmed that the midbrain organoids produced through the treatment of 3 uM CHIR99021 had high functionality.

실험예 2-8. 중뇌 오가노이드의 기능성 분석Experimental Example 2-8. Functional analysis of midbrain organoids

실시예 7과 같은 방법을 이용하여 분화된 조직의 중뇌 오가노이드의 기능성을 확인하기 위해서 패치 클램프 기록(patch-clamp recording)을 통해 전기생리학적 분석을 실시하였다.Electrophysiological analysis was performed through patch-clamp recording to confirm the functionality of midbrain organoids in differentiated tissues using the same method as in Example 7.

그 결과 도 15에 나타난 것과 같이, 3 uM의 CHIR99021를 처리했을 때 신경세포의 활동전위(action potential)이 관찰되어 전기생리학적으로 활성을 가짐을 확인할 수 있었다.As a result, as shown in FIG. 15, when 3 uM of CHIR99021 was treated, the action potential of the nerve cells was observed and it was confirmed that it had electrophysiological activity.

실험예 2-9. 중뇌 오가노이드의 기능성 분석Experimental Example 2-9. Functional analysis of midbrain organoids

실시예 7과 같은 방법을 이용하여 분화된 조직의 중뇌 오가노이드의 기능성을 확인하기 위해서 뉴로멜라닌의 생성을 분석하여 3 uM의 CHIR99021를 처리했을 때 중뇌 오가노이드 전반에 걸쳐 매우 균질하게 뉴로멜라닌이 생성됨을 확인할 수 있었고(도 16 왼쪽), 뉴로멜라닌의 경우 중뇌 특이적으로 생성되기 때문에 대뇌 중뇌와의 비교분석을 통해서, 대뇌 오가노이드에서는 생성되지 않는 뉴로멜라닌이 만들어진 중뇌 오가노이드에서만 특이적으로 생성됨을 확인고(도 16 가운데), 또한, 전자한미경 사진을 통해서 중뇌 오가노이드에서 전반적인 뉴로멜라닌 과립을(도 16 오른쪽)확인하였다.In order to confirm the functionality of the midbrain organoids of the differentiated tissue using the same method as in Example 7, the generation of neuromelanin was analyzed and when 3 uM of CHIR99021 was treated, neuromelanin was produced very homogeneously throughout the midbrain organoids was confirmed (Fig. 16 left), and in the case of neuromelanin, because it is produced specifically in the midbrain, through comparative analysis with the cerebral midbrain, it was found that neuromelanin, which is not produced in cerebral organoids, is specifically produced only in midbrain organoids. Confirmation (middle of FIG. 16), and overall neuromelanin granules in midbrain organoids (right side of FIG. 16) were confirmed through electron micrographs.

실험예 3. 분화시킨 중뇌 오가노이드의 파킨슨 질환 모델링Experimental Example 3. Parkinson's disease modeling of differentiated midbrain organoids

파킨슨 질병은 주로 중뇌 도파민 뉴런의 세포 사멸로 인해서 발병함이 알려져 있으나, 환자의 뇌에서 선택적인 세포 사멸이 발생하는 자세한 기전에 대해서는 명확히 밝혀져 있지 않다. 파킨슨 질병의 발병기전을 밝히기 위한 연구를 위해서 본 기술을 이용하여 제조된 중뇌 오가노이드가 파킨슨 질병 모델로 유용하게 사용될 수 있는지 여부를 확인하기 위해서 하기와 같은 실험을 수행하였다.Although it is known that Parkinson's disease is mainly caused by apoptosis of dopaminergic neurons in the midbrain, the detailed mechanism by which selective apoptosis occurs in the patient's brain has not been clearly elucidated. For a study to elucidate the pathogenesis of Parkinson's disease, the following experiment was performed to determine whether the midbrain organoid prepared using this technology can be usefully used as a Parkinson's disease model.

구체적으로, 실시예 7과 같은 방법을 이용하여 분화된 조직의 중뇌 오가노이드가 파킨슨 질병의 모델로 기능할 수 있는지 여부를 확인하기 위해서 실시예 7과 같은 방법을 이용하여 분화된 조직의 중뇌 오가노이드를 8주간 배양한 후 약 2일간 0, 10, 50, 또는 100 uM MPTP를 처리하여 중뇌 도파민성신경세포의 선택적 사멸 여부를 면역염색기법으로 분석(도 17, 18, 19) 하였다.Specifically, in order to confirm whether the midbrain organoids of the tissue differentiated using the same method as in Example 7 can function as a model of Parkinson's disease, the midbrain organoids of the tissue differentiated using the same method as in Example 7 was cultured for 8 weeks and then treated with 0, 10, 50, or 100 uM MPTP for about 2 days to determine whether or not the mesencephalic dopaminergic neurons were selectively killed by immunostaining ( FIGS. 17, 18, 19 ).

면역염색기법으로 분석하기 위해서 TUNEL assay(Abcam, ab66108), TUJ1(BIOLEGEND, 801201), Caspase-3(CELL SIGNALING, 9661s), GABA(SIGMA, A2052), AQP4(Merck millipore, AB2118), PLP(Cusabio, CSB-PA018202LA01HU) 및 TH(Abcam, ab152) 항체를 사용하였다.For analysis by immunostaining technique, TUNEL assay (Abcam, ab66108), TUJ1 (BIOLEGEND, 801201), Caspase-3 (CELL SIGNALING, 9661s), GABA (SIGMA, A2052), AQP4 (Merck millipore, AB2118), PLP (Cusabio) , CSB-PA018202LA01HU) and TH (Abcam, ab152) antibodies were used.

그 결과 도 17과 18에 나타난 것과 같이 실시예 7과 같은 방법을 이용하여 분화시킨 중뇌 오가노이드는 MPTP 처리시 MPTP의 농도가 증가됨에 따라 중뇌 도파민성신경세포의 사멸이 증가하였지만(도 17 및 18), 도 19에 나타난 것과 같이 실시예 7과 같은 방법을 이용하여 분화시킨 중뇌 오가노이드는 MPTP 처리시 중뇌 도파민성신경세포 이외의 다른 세포의 사멸은 관찰되지 않아 MPTP의 효과가 중뇌 도파민성신경세포에 특이적임을 확인할 수 있었다. 상기 결과는 중뇌 오가노이드의 파킨슨병 질환모델로써 활용 가능성을 제시한다.As a result, as shown in FIGS. 17 and 18 , in the midbrain organoids differentiated using the same method as in Example 7, the death of midbrain dopaminergic neurons increased as the concentration of MPTP increased during MPTP treatment ( FIGS. 17 and 18 ). As shown in FIG. 19 , in the midbrain organoids differentiated using the same method as in Example 7, the death of cells other than midbrain dopaminergic neurons was not observed during MPTP treatment, so the effect of MPTP is specific to midbrain dopaminergic neurons. was able to confirm The above results suggest the possibility of utilizing midbrain organoids as a Parkinson's disease disease model.

<110> Organtech <120> METHOD OF PRODUCTION FOR MIDBRAIN OGARNOID <130> SP19-0111KR <160> 37 <170> KoPatentIn 3.0 <210> 1 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> Noggin protein <400> 1 Met Gln His Tyr Leu His Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro 1 5 10 15 Leu Val Asp Leu Ile Glu His Pro Asp Pro Ile Phe Asp Pro Lys Glu 20 25 30 Lys Asp Leu Asn Glu Thr Leu Leu Arg Ser Leu Leu Gly Gly His Tyr 35 40 45 Asp Pro Gly Phe Met Ala Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly 50 55 60 Gly Gly Gly Ala Ala Gly Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln 65 70 75 80 Leu Leu Arg Gln Arg Pro Ser Gly Ala Met Pro Ser Glu Ile Lys Gly 85 90 95 Leu Glu Phe Ser Glu Gly Leu Ala Gln Gly Lys Lys Gln Arg Leu Ser 100 105 110 Lys Lys Leu Arg Arg Lys Leu Gln Met Trp Leu Trp Ser Gln Thr Phe 115 120 125 Cys Pro Val Leu Tyr Ala Trp Asn Asp Leu Gly Ser Arg Phe Trp Pro 130 135 140 Arg Tyr Val Lys Val Gly Ser Cys Phe Ser Lys Arg Ser Cys Ser Val 145 150 155 160 Pro Glu Gly Met Val Cys Lys Pro Ser Lys Ser Val His Leu Thr Val 165 170 175 Leu Arg Trp Arg Cys Gln Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile 180 185 190 Pro Ile Gln Tyr Pro Ile Ile Ser Glu Cys Lys Cys Ser Cys 195 200 205 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog_F <400> 2 tgcaacctga agacgtgtga 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Nanog_R <400> 3 ctatgaggga tgggagga 18 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oct4_F <400> 4 gacaggggga ggggaggagc tagg 24 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Oct4_R <400> 5 cttccctcca accagttgcc ccaaac 26 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2_F <400> 6 agactgcaca tgagccagca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2_R <400> 7 cgtctccagc cagcttcaac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Otx2_F <400> 8 ccctcactcg ccacatctac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Otx2_R <400> 9 gttcagagtc cttggtgggt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Plzf_F <400> 10 tcccgcccga ctggaggata 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Plzf_R <400> 11 ttctttcctg gctccccgct c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Eomes_F <400> 12 ctcaaaaggc atgggagggt a 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Eomes_R <400> 13 caccaccaag tccatctgca a 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox17_F <400> 14 ccttagctcc aggaagtgtg 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Sox17_R <400> 15 gccagctccg cggtatatta ct 22 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T_F <400> 16 cgcctcatag cctcatggac 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> T_R <400> 17 cactggctgc cacgacaaa 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BOK_F <400> 18 gctggccaca tcttctctgc 20 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BOK_R <400> 19 tgctgaccac acacttgagg ac 22 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAX_F <400> 20 tgctgacgtg gacacggact 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAX_R <400> 21 ccagccaccc tggtcttgga 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAD_F <400> 22 tcggagtcgc cacagttcgt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAD_R <400> 23 gcgctctttg ggcgaggaag 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRTM_F <400> 24 ctgccatgcc cttcgtgtgt 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRTM_R <400> 25 aacacttgtc cgggcagctg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DAT_F <400> 26 cgccactggc tcaaggtgta 20 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DAT_R <400> 27 ccggcacgga aaggtgtaa 19 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TH_F <400> 28 ctgagattcg ggccttcgac 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TH_R <400> 29 tgcacctagc caatggcact 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMX1B_F <400> 30 ggcatcaaga tggaggagca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMX1B_R <400> 31 tggtgagggc ttgctgacac 20 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LMX1A_F <400> 32 aaagcgcgat cgacacctc 19 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMX1A_R <400> 33 tcccggtaga agcaggtggt 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ASCL1_F <400> 34 ggtgatcgca caacctgcat 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ASCL1_R <400> 35 gttctgagcg cttcccgttt 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX3_F <400> 36 tttccgtttc gccttcacct 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX3_R <400> 37 acgatcttgt ggcggatgtg 20 <110> Organtech <120> METHOD OF PRODUCTION FOR MIDBRAIN OGARNOID <130> SP19-0111KR <160> 37 <170> KoPatentIn 3.0 <210> 1 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> Noggin protein <400> 1 Met Gln His Tyr Leu His Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro 1 5 10 15 Leu Val Asp Leu Ile Glu His Pro Asp Pro Ile Phe Asp Pro Lys Glu 20 25 30 Lys Asp Leu Asn Glu Thr Leu Leu Arg Ser Leu Leu Gly Gly His Tyr 35 40 45 Asp Pro Gly Phe Met Ala Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly 50 55 60 Gly Gly Gly Ala Ala Gly Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln 65 70 75 80 Leu Leu Arg Gln Arg Pro Ser Gly Ala Met Pro Ser Glu Ile Lys Gly 85 90 95 Leu Glu Phe Ser Glu Gly Leu Ala Gln Gly Lys Lys Gln Arg Leu Ser 100 105 110 Lys Lys Leu Arg Arg Lys Leu Gln Met Trp Leu Trp Ser Gln Thr Phe 115 120 125 Cys Pro Val Leu Tyr Ala Trp Asn Asp Leu Gly Ser Arg Phe Trp Pro 130 135 140 Arg Tyr Val Lys Val Gly Ser Cys Phe Ser Lys Arg Ser Cys Ser Val 145 150 155 160 Pro Glu Gly Met Val Cys Lys Pro Ser Lys Ser Val His Leu Thr Val 165 170 175 Leu Arg Trp Arg Cys Gln Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile 180 185 190 Pro Ile Gln Tyr Pro Ile Ile Ser Glu Cys Lys Cys Ser Cys 195 200 205 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog_F <400> 2 tgcaacctga agacgtgtga 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Nanog_R <400> 3 ctatgaggga tgggagga 18 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oct4_F <400> 4 gacaggggga ggggaggagc tagg 24 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Oct4_R <400> 5 cttccctcca accagttgcc ccaaac 26 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2_F <400> 6 agactgcaca tgagccagca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2_R <400> 7 cgtctccagc cagcttcaac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Otx2_F <400> 8 ccctcactcg ccacatctac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Otx2_R <400> 9 gttcagagtc cttggtgggt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Plzf_F <400> 10 tcccgcccga ctggaggata 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Plzf_R <400> 11 ttctttcctg gctccccgct c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Eomes_F <400> 12 ctcaaaaggc atgggagggt a 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Eomes_R <400> 13 caccaccaag tccatctgca a 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox17_F <400> 14 ccttagctcc aggaagtgtg 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Sox17_R <400> 15 gccagctccg cggtatatta ct 22 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T_F <400> 16 cgcctcatag cctcatggac 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> T_R <400> 17 cactggctgc cacgacaaa 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BOK_F <400> 18 gctggccaca tcttctctgc 20 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BOK_R <400> 19 tgctgaccac acacttgagg ac 22 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAX_F <400> 20 tgctgacgtg gacacggact 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAX_R <400> 21 ccagccaccc tggtcttgga 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAD_F <400> 22 tcggagtcgc cacagttcgt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAD_R <400> 23 gcgctctttg ggcgaggaag 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRTM_F <400> 24 ctgccatgcc cttcgtgtgt 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRTM_R <400> 25 aacacttgtc cgggcagctg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DAT_F <400> 26 cgccactggc tcaaggtgta 20 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DAT_R <400> 27 ccggcacgga aaggtgtaa 19 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TH_F <400> 28 ctgagattcg ggccttcgac 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TH_R <400> 29 tgcacctagc caatggcact 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMX1B_F <400> 30 ggcatcaaga tggagggagca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMX1B_R <400> 31 tggtgagggc ttgctgacac 20 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LMX1A_F <400> 32 aaagcgcgat cgacacctc 19 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMX1A_R <400> 33 tcccggtaga agcaggtggt 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ASCL1_F <400> 34 ggtgatcgca caacctgcat 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ASCL1_R <400> 35 gttctgagcg cttcccgttt 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX3_F <400> 36 tttccgtttc gccttcacct 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX3_R <400> 37 acgatcttgt ggcggatgtg 20

Claims (15)

SMAD 억제제를 유효성분으로 포함하는 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
A medium composition for inducing differentiation of stem cells into midbrain organoids comprising a SMAD inhibitor as an active ingredient.
제1항에 있어서, 상기 SMAD 억제제는 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride(Dorsomorphin), 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline(LDN193189), 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide(SB431542), 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide(A83-01) 및 Noggin 단백질로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
The method of claim 1, wherein the SMAD inhibitor is 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline(LDN193189), 4-[4-(1,3-benzodioxol-5-yl)- 5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide (A83 -01) and Noggin protein, characterized in that it contains any one or more selected from the group consisting of, a medium composition for inducing differentiation of stem cells into midbrain organoids.
제1항에 있어서, 상기 SMAD 억제제는 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride(Dorsomorphin) 및 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide(A83-01)인 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
The method of claim 1, wherein the SMAD inhibitor is 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin) and Medium for inducing differentiation of stem cells into midbrain organoids, characterized in that 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide (A83-01) composition.
제1항에 있어서, 상기 도르소모핀(Dorsomorphin)은 1 uM 내지 2 uM의 농도인 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
The medium composition for inducing differentiation of stem cells into midbrain organoids according to claim 1, wherein the dorsomorphin is at a concentration of 1 uM to 2 uM.
제1항에 있어서, 상기 LDN193189은 50 nM 내지 100 nM의 농도인 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
The medium composition for inducing differentiation of stem cells into midbrain organoids according to claim 1, wherein the LDN193189 is at a concentration of 50 nM to 100 nM.
제1항에 있어서, 상기 SB431542은 5 uM 내지 10 uM의 농도인 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
According to claim 1, wherein the SB431542 is characterized in that the concentration of 5 uM to 10 uM, the medium composition for inducing differentiation of stem cells into midbrain organoids.
제1항에 있어서, 상기 A83-01은 1 내지 2 uM의 농도인 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
The medium composition for inducing differentiation of stem cells into midbrain organoids according to claim 1, wherein the A83-01 has a concentration of 1 to 2 uM.
제1항에 있어서, 상기 Noggin 단백질은 100 ng/ml 내지 200 ng/mL의 농도인 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
According to claim 1, wherein the Noggin protein is characterized in that the concentration of 100 ng/ml to 200 ng/mL, the medium composition for inducing differentiation of stem cells into midbrain organoids.
제1항에 있어서, 상기 줄기세포는 배아줄기세포(Embryonic stem cell), 또는 유도만능줄기세포(induced Pluripotent stem cell)인 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
According to claim 1, wherein the stem cells are embryonic stem cells (Embryonic stem cells), or induced pluripotent stem cells (induced Pluripotent stem cells), characterized in that, the medium composition for inducing differentiation of stem cells into midbrain organoids.
제1항에 있어서, 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile(CHIR99021)를 추가적으로 포함하는 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물.
The method of claim 1 , wherein 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino] A medium composition for inducing differentiation of stem cells into midbrain organoids, characterized in that it further comprises -3-pyridinecarbonitrile (CHIR99021).
제10항에 있어서, 상기 CHIR99021는 0.8 uM 내지 3 uM의 농도인 것을 특징으로 하는, 줄기세포를 중뇌 오가노이드로 분화 유도하기 위한 배지 조성물
The medium composition for inducing differentiation of stem cells into midbrain organoids according to claim 10, wherein the CHIR99021 has a concentration of 0.8 uM to 3 uM.
줄기세포를 SMAD 억제제를 포함하는 배지 조성물에서 배양하여 중뇌로 분화키는 단계를 포함하는 것을 특징으로 하는 중뇌 오가노이드 제조방법.
A method for producing a midbrain organoid comprising the step of culturing stem cells in a medium composition containing a SMAD inhibitor to differentiate into the midbrain.
제12항에 있어서, 상기 SMAD 억제제는 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride(Dorsomorphin), 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline(LDN193189), 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide(SB431542), 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide(A83-01) 및 Noggin 단백질로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 것을 특징으로 하는, 중뇌 오가노이드 제조방법.
13. The method of claim 12, wherein the SMAD inhibitor is 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline(LDN193189), 4-[4-(1,3-benzodioxol-5-yl)- 5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide (A83 -01) and Noggin protein, characterized in that it comprises any one or more selected from the group consisting of, midbrain organoid manufacturing method.
제12항에 있어서, 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile(CHIR99021)를 추가적으로 포함하는 것을 특징으로 하는, 중뇌 오가노이드 제조방법.
13. The method of claim 12, 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino] -3-pyridinecarbonitrile (CHIR99021), characterized in that it further comprises, midbrain organoid manufacturing method.
제12항 내지 제14항중 어느 한 항의 제조방법에 의해서 제조된 중뇌 오가노이드.A midbrain organoid prepared by the method of any one of claims 12 to 14.

KR1020190170415A 2019-12-19 2019-12-19 Method of production for midbrain ogarnoid KR20210078704A (en)

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KR20240175165A (en)	2023-06-12	2024-12-19	고려대학교 산학협력단	Method for preparing sheeted taste bud organoids
KR20240176372A (en)	2023-06-15	2024-12-24	고려대학교 산학협력단	Method for preparing sheeted oral mucosa organoids

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KR20240175165A (en)	2023-06-12	2024-12-19	고려대학교 산학협력단	Method for preparing sheeted taste bud organoids
KR20240176372A (en)	2023-06-15	2024-12-24	고려대학교 산학협력단	Method for preparing sheeted oral mucosa organoids
CN117778313A (en) *	2024-02-23	2024-03-29	成都云测医学生物技术有限公司	Differentiation method and application of mesenchymal stem cells obtained from brain organoids
CN117778313B (en) *	2024-02-23	2024-05-24	成都云测医学生物技术有限公司	Differentiation method and application of mesenchymal stem cells obtained from brain organoids

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KR20210078704A - Method of production for midbrain ogarnoid - Google Patents