KR102296446B1 - Differentiation method of securing large amount of cells by chopping target cell enriched 3D organoids prepared from human pluripotent stem cells - Google Patents

Abstract

The present invention relates to a method for securing a large amount of finally differentiated cells by patterning and disassembling a 3D organoid prepared from human pluripotent stem cells, culturing the stem cells or progenitor cells, and inducing differentiation. It is expected to be very useful for screening cell therapies or therapeutic drugs as it exhibits significantly superior effects in terms of reproducibility, stability, and functionality compared to cells differentiated by existing differentiation methods.

Description

{Differentiation method of securing large amount of cells by chopping target cell enriched 3D organoids prepared from human pluripotent stem cells}

The present invention relates to a differentiation method for securing a large amount of finally differentiated cells by separating and disassembling 3D organoids prepared from human pluripotent stem cells.

Parkinson's disease is a neurodegenerative disease caused by loss of dopaminergic neurons in the substantia nigra in the midbrain. It is known as the most suitable disease for stem cell transplantation because the lesion site and lesion cells are clear.

For the treatment of Parkinson's disease cells, a technology to differentiate from stem cells into human midbrain-type dopamine neurons (mDA neurons) is required, and human pluripotent stem cells (hPSCs, human embryonic stem cells (hESCs) or human induction A differentiation method for differentiating pluripotent stem cells (hiPSCs) into mDA neurons has been developed for a long time [Non-Patent Document 1]. However, in using this differentiation method for clinical application, several problems have been raised as follows.

First, there is a problem with reproducibility. The existing two-dimensional method is very difficult and cannot be reproduced well in other laboratories. In all of the developed methods, it is applied only to some pluripotent stem cell lines such as H9 used for development. did not happen well.

Second, there is a problem of stable expression of midbrain-specific factors. Midbrain factors such as Nurr1 Foxa2 and Lmx1a are known to be critically important for survival, function, and maintenance of midbrain dopaminergic neurons, and thus are closely related to their ability to treat Parkinson's disease after transplantation. Because of this fact, all of the recently developed hPSC-mDA neuronal differentiation protocols were developed focusing on the expression of midbrain factors in midbrain dopaminergic neurons. However, the expression of this midbrain factor is very unstable and is easily lost after long-term culture or transplantation of midbrain dopaminergic neurons. Therefore, by transplanting differentiated cells by the method developed so far, only some midbrain factor expression (mainly Foxa2) is shown in the transplanted midbrain dopaminergic neurons.

Third, there is a problem in that astrocytes do not exist in the differentiated mDA neuronal cell culture. Astrocytes are not included in the culture of mDA neurons differentiated by the methods developed so far. Astrocytes are cells that are responsible for the survival and function enhancement of neurons such as mDA neurons, and are essential for normal survival and function maintenance of neurons in actual brain tissue. In addition, astrocytes are required to maintain the expression of midbrain factors in mDA neurons.

Finally, there is a problem in the amount of nerve cells that can be obtained through nerve cell differentiation. In the dopaminergic neuron differentiation method developed so far, there is a limit to the amount of finally differentiated neurons that can be obtained through one differentiation by inducing differentiation into neurons without a neural stem cell stage capable of proliferation in the middle.

3D brain Organoids derived from pluripotent stem cells: promising experimental models for brain development and neurodegenerative disorders, Journal of Biomedical Science (2017) 24:59

Accordingly, the present inventors made research efforts focusing on the fact that the three-dimensional culture using organoids more closely emulates the actual brain environment and the healthy culture of cells is possible for the two-dimensional cell culture. By patterning each organoid capable of sufficiently containing target cells, progenitor cells or stem cells, a cell group of the desired fate body is prepared as a plurality of cell groups (target cell enriched), and this organoid tissue is disassembled to form progenitor cells or Stem cells were cultured. While other technologies are applied to the development of differentiation methods by patterning and quantitatively securing cells by a two-dimensional culture method, or by borrowing only the three-dimensional sphere form in the intermediate stage to secure cells, the present invention provides an organoid in a perfect form, In order to sufficiently enrich the cells in the desired state with appropriate culture, the cells isolated in the present invention, compared to other conventional techniques, undergo a complete three-dimensional artificial brain called an organoid, so that each cell is It was confirmed that it was more functionally superior.

Accordingly, it is an object of the present invention to provide a method for securing a large amount of finally differentiated cells by patterning and disassembling 3D organoids prepared from human pluripotent stem cells, culturing the stem cells or progenitor cells, and inducing differentiation.

Another object of the present invention is to provide a cell therapy product comprising the differentiated cells obtained by the above method as an active ingredient.

Another object of the present invention is to provide a drug screening method using the differentiated cells obtained by the above method.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for patterning and disassembling 3D organoids prepared from human pluripotent stem cells, culturing the stem cells or progenitor cells, and inducing differentiation to secure a large amount of finally differentiated cells.

As used herein, "pluripotent stem cell (PSC)" refers to a stem cell capable of induced differentiation into any type of cell constituting the body, and the pluripotent stem cell includes an embryonic stem cell (ESC). ) and induced pluripotent stem cells (Ipsc).

The "organoid" described herein is a 'mini-like organ' made to have minimal functions using stem cells. . That is, "organoid" refers to a cell having a 3D three-dimensional structure, and refers to a model similar to organs such as nerves and intestines manufactured through an artificial culture process that is not collected or acquired from animals. The origin of the cells constituting it is not limited. The organoid may have an environment that is allowed to interact with the surrounding environment in the process of cell growth. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, the 3D organoid in the present invention can be an excellent model for observing the development of therapeutic agents for diseases and the like by almost completely mimicking the organs that actually interact in vivo.

Organoids can generally be prepared by culturing human pluripotent stem cells. Specifically, differentiation from induced pluripotent stem cells derived from Parkinson's disease into neuroectodermal spheres or from induced pluripotent stem cells derived from Parkinson's disease to definitive endoderm and hindgut Differentiation can be induced into intestinal organoids through differentiation.

As used herein, the term "differentiation" refers to a phenomenon in which cells divide and proliferate, and the structure or function of cells is specialized while the entire individual grows. In other words, it refers to the process by which cells, tissues, etc. of living organisms change into a suitable form and function to perform their respective roles. For example, pluripotent stem cells transform into ectoderm (cortex, midbrain, hypothalamus, etc.) etc.) and endoderm cells, as well as the process in which hematopoietic stem cells change into red blood cells, white blood cells, platelets, etc., that is, the expression of specific differentiation traits by progenitor cells, can all be included in differentiation.

In the case of direct differentiation from existing human pluripotent stem cells and differentiation into corresponding cells, there is a problem with reproducibility depending on the cell line or laboratory (experimental environment), and stable expression of maintenance factors is not achieved. For example, in the case of midbrain dopaminergic neurons, Unlike the in vivo environment of , astrocytes do not exist together, and there is a limit to the amount of final cells that can be obtained through one differentiation. As in an embodiment of the present invention, each of the organoids that can sufficiently contain cerebral astrocytes, midbrain astrocytes, midbrain dopaminergic neural stem cells, or hypothalamic neural stem cells is patterned and produced (target cell enriched), that is, Securing an organoid containing the maximum amount of each target cell, dismantling the organoid tissue and culturing the stem or progenitor cells to prepare for a cell group induced two-dimensionally, compared to cells isolated from the actual brain. It is similar, its characteristics are well maintained, and it is possible to secure differentiation into a cell group with secured viability.

In the present invention, the term "patterning" refers to the characteristics of the origin of cells to be finally extracted from among the brain sub-tissues in this way, the midbrain pattern, the cortical pattern, or the hypothalamus pattern when the organoid is manufactured. It means that the organoid is prepared so that the cell group of the fate body having In addition, patterning markers include Lmx1a, Foxa2, Nurr1, En1; In cortical patterns, Pax6, Foxg1; In the case of hypothalamic patterns, there are Rax and Nkx2.2.
In the present invention, the term "chopping" means to physically cut and disperse the 3D organoids fabricated before subculture into several pieces (eg, using a needle, etc.).

Therefore, in the present invention, it is possible to secure a large amount of finally differentiated cells at once by disassembling the produced 3D organoid, separating and culturing stem cells capable of proliferating from the organoid, and proliferating them.

According to one embodiment of the present invention,

1) Proliferating and culturing human pluripotent stem cells to produce 3D organoids;

2) patterning and disassembling the fabricated 3D organoid; and

3) culturing, proliferating, and inducing differentiation of stem cells or progenitor cells in the cells extracted from the released organoids to secure a large amount of finally differentiated cells

It includes a method of disassembling 3D organoids produced from human pluripotent stem cells, including a method for securing a large amount of stem cells or progenitor cells, and finally differentiate them.

The stem cells may be ectodermal stem cells (skin, nerve cells), mesodermal stem cells (muscle, bone cells), or endoderm stem cells (digestive and respiratory cells). Specifically, it may be a midbrain dopaminergic neural stem cell, or a hypothalamic neural stem cell.

The progenitor cells may be cerebral astrocytes, or midbrain astrocytes, but are not limited thereto.

The cell may be a neuron or an astrocyte, but is not limited thereto.

More preferably, the human pluripotent stem cells and stem cells are cultured using vitronectin. At this time, it is appropriate to use vitronectin 0.3 to 0.7 ug/cm ^{2 .} Parmar group's CellStemCell (A. Kirkeby et al. , Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease. Cell Stem Cell 20 , 135-148 (2017).), Nature Protocol (S. Nolbrant, A. Heuer, M. Parmar, A. Kirkeby, Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nature protocols 12 ^{, 1962-1979 (2017)) reported that 0.5ug/cm 2 of} Lamin521 (BioLamina LN-521) was used for proliferation and culture of human embryonic stem cells, and after differentiation into midbrain neurons, Lamin511 ( BioLamina's LN-511) was reported to be very important to use ^{1ug/cm 2 . In contrast, in the examples of the present invention, 0.5 ug/cm 2 of} vitronectin (Gibco's A31804) was used for both culturing of human embryonic stem cells and culturing of mesencephalic neural stem cells. In terms of the coating unit price of a 60mm dish generally used for culture, Lamin521 for human embryonic stem cell culture is 7,282 won/dish Vitronectin in the present invention is 1,480 won/dish, and Lamin511 for culturing midbrain neural stem cells is While it is 23,832 won/dish, it is 1,480 won/dish with continued use of vitronectin of the present invention. Looking at the coating cost alone, the method of the present invention has an effect of reducing 5 times when culturing human embryonic stem cells and 16 times when culturing midbrain neural stem cells.

The term "large amount" used in the present invention differs depending on each patterned cell group when introduced from the start of one culture dish of pluripotent stem cells used for the first time, but it is difficult to secure about 1000-7200 vials of frozen cells It is possible, and this generally means an increased amount of about 1,000 to 14,000 times when one or two culture dish cells are frozen in one vial. In particular, it includes not only simple quantitative proliferation, but also maintenance of characteristics.

As used herein, the term "glia (gila)" refers to a cell that occupies the largest portion of cells present in the brain, and refers to an "astrocytic cell (astrocyte)". The astrocytes are also referred to as astrocytes, and are involved in the protection and supply of nutrients and inflammation of nerve cells, and the microglia are cells responsible for inflammation in the brain.

The astrocytes may be obtained by differentiation from embryonic or adult stem cells, and in the mammalian midbrain (ventral midbrain), cerebral cortex (cortex) or lateral ganglionic eminence (striae anlage) It can also be obtained by isolation.

"Neural stem/precursor cells (NSCs/NPCs)" are isolated and cultured from brain tissue in developing or adults, and the cultured NPCs produce large quantities of dopaminergic neurons for research and drug screening. can be used to form.

A “neuron” is a cell of the nervous system, and the terms “neuron” or “neuronal cell” can be used interchangeably.

"Dopamine (DA) nerve cell" refers to a nerve cell expressing Tyrosine Hydroxylase (TH). In the present invention, "dopaminergic neuron", "dopaminergic neuron", "DA" and the like are used interchangeably. Dopaminergic neurons are specifically located in the midbrain substantia nigra and stimulate the striatum, limbic system, and neocortex in vivo to regulate postural reflexes, movement, and reward-related behaviors.

The present invention also includes a cell therapy product comprising the cells obtained by the above method.

"Cell therapy product" refers to cells and tissues manufactured through isolation, culture, and special masturbation from a subject, and is a drug used for treatment, diagnosis, and prevention purposes (US FDA regulations). , refers to a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting allogeneic or xenogeneic cells in vitro, or changing the biological properties of cells in other ways. Cell therapy products are largely classified into somatic cell therapy and stem cell therapy according to the degree of cell differentiation.

As used herein, a "subject" is a vertebrate, preferably a mammal, which is the subject of treatment, observation or experiment, for example, cattle, pigs, horses, goats, dogs, cats, rats, mice, rabbits, guinea pigs, It may be a human or the like.

In the present invention, "treatment" refers to any action that suppresses, alleviates, or advantageously changes the clinical situation related to a disease. Treatment can also mean increased survival compared to the expected survival rate if not receiving treatment. Treatment includes simultaneously prophylactic means in addition to therapeutic means.

The cell therapy agent of the present invention exhibits a therapeutic effect on a disease selected from the group consisting of neurodegenerative diseases, inflammatory degenerative diseases and metabolic diseases.

The neurodegenerative disease is, for example, Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, memory loss, myasthenia gravis, progressive supranuclear palsy, multiple system atrophy, essential tremor, cortical-basal nucleus degeneration, diffuse Lewy. It may include one selected from the group consisting of bodily disease and Pick's disease, but is not limited thereto.

The inflammatory degenerative disease may include one selected from the group consisting of dementia, Lewy body dementia, frontotemporal dementia, white matter degeneration, adrenal leukodystrophy, multiple sclerosis, and Lou Gehrig's disease, but is not limited thereto.

The metabolic disease may include, but is not limited to, one selected from the group consisting of diabetes, obesity and metabolic disorders.

The cells obtained by the method of the present invention may be applied as a cell therapeutic agent, and may be formulated by further including a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not significantly stimulate the organism and does not inhibit the biological activity and properties of the administered component. In the present invention, the pharmaceutically acceptable carrier that can be included as a cell therapeutic agent is used without limitation as long as it is known in the art, such as a buffer, preservative, analgesic agent, solubilizer, isotonic agent, stabilizer, base, excipient, lubricant, etc. can The cell therapeutic agent of the present invention may be prepared in the form of various formulations according to commonly used techniques. The cell therapy agent of the present invention can be administered through any route as long as it can induce movement to the diseased site. In some cases, it may be possible to consider loading the stem cells into a vehicle equipped with a means for directing the lesion. Therefore, the cell therapy agent of the present invention is topical (including buccal, sublingual, dermal and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and intracerebrospinal fluid) or transdermal It can be administered through several routes including administration, and is preferably administered directly to the site of disease. In one embodiment, the cells may be administered to an individual by suspending the drug in a suitable diluent, and the diluent is used to protect and maintain the cells, and to facilitate use when injected into a target tissue. The diluent may include physiological saline, a phosphate buffer solution, a buffer solution such as HBSS, and cerebrospinal fluid. In addition, the pharmaceutical composition may be administered by any device to allow the active agent to migrate to the target cell. A preferred mode of administration and formulation are injections. Injections include aqueous solutions such as physiological saline, Ringel's solution, Hank's solution or sterilized aqueous solution, vegetable oils such as olive oil, higher fatty acid esters such as ethyl oleic acid, and non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerin For mucosal penetration, a non-penetrating agent known in the art suitable for a barrier to pass through may be used, and ascorbic acid, sodium hydrogen sulfite, BHA, tocopherol, EDTA as a stabilizer for preventing deterioration It may further include a pharmaceutical carrier such as an emulsifier, a buffer for pH control, a preservative for inhibiting the growth of microorganisms such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzyl alcohol.

The present invention also provides a drug screening method using the cells or progenitor cells obtained by the above method.

Important features of the cells secured by the present invention include the possibility of securing the production of a large amount of cells, the maintenance of the characteristics even during cryopreservation, the possibility of maintaining the same cell group for a long period of time, and differentiation more similar to the cells derived from the living body. This property is particularly suitable for simultaneous screening of multiple drugs, which requires a large amount of cells in the same state and the key to obtaining the same cells for a long period of time for repeated analysis thereof. It is very suitable for screening cells as a cell population with the same characteristics in which key markers are maintained can be used continuously from the beginning to the end of the screening operation.

The drug is a drug for treating a disease selected from the group consisting of a neurodegenerative disease, a neurodegenerative disease, an inflammatory degenerative disease, and a metabolic disease, and a disease selected from the group consisting of the neurodegenerative disease, a neurodegenerative disease, an inflammatory degenerative disease and a metabolic disease shows the therapeutic effect on

The neurodegenerative disease is, for example, Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, memory loss, myasthenia gravis, progressive supranuclear palsy, multiple system atrophy, essential tremor, cortical-basal nucleus degeneration, diffuse Lewy. Various neurological diseases may include, but are not limited to, bodily disease and Pick's disease.

The inflammatory degenerative disease may include one selected from the group consisting of dementia, Lewy body dementia, frontotemporal dementia, white matter degeneration, adrenal leukodystrophy, multiple sclerosis, and Lou Gehrig's disease, but is not limited thereto.

The metabolic disease may include, but is not limited to, one selected from the group consisting of diabetes, obesity and metabolic disorders.

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. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

In the present invention, by disassembling the produced 3D organoid, separating and culturing stem cells or progenitor cells from the organoids and proliferating them, the terminally differentiated cells can be differentiated in a large amount, so that a large amount of cells can be secured at once. For example, when the method of the present invention is applied to mDA neuronal differentiation, differentiation can be easily induced without cell line-dependent or batch-to-batch variation, and thus can be easily reproduced in other laboratories. In addition, a series of Nurr1 Foxa2, Lmx1a, etc., which are known to be critical for survival, function, and maintenance of mDA neurons, are faithfully expressed in the transplanted mDA neurons when the mesencephalic neural stem cells differentiated according to the present invention are transplanted into Parkinson's disease. It was confirmed that the cell transplantation treatment effect was significantly improved. In addition, astrocytes, which are cells responsible for the survival and function enhancement of neurons along with the mDA neurons, also exist, which protect and enhance the function of mDA neurons, which seems to have contributed to the enhancement of cell transplantation therapy during transplantation.

Therefore, differentiated cells obtained in such a large amount (including cerebral astrocytes, midbrain astrocytes, midbrain dopaminergic neural stem cells, hypothalamic neural stem cells, etc.) can

O₂ *p=0.013-0.165 (E), p=0.000032-0.11 (F) n=3, Student's t-test. G-P: 도파민성 신경세포 성숙도와 기능성 분석. G, Og-NSCs 발현 특이적인 유전자의 Heatmap 분석 (vs 2D-NSCs). H, Neurolucida 분석 프로그램으로 대표적으로 분석된 Og-NSCs 유래 성숙도 높은 도파민성 신경세포. J, 12일 동안 분화 유도된 도파민성 신경세포 길이 분석. 23-58 세포/배양물로부터의 TH+ 섬유 길이를 측정하였다. *p=0.013, n= 7 (Og), 3(2D) independent cultures, t-test. I, K, 도파민성 신경세포의 시냅스 분석. 실제 시냅스를 형성하는 수를 synaptophysin 염색으로 분석함. (27-43 TH+ fibers/culture) *p=0.03, n= 5 cultures for each group. L-O, 신경세포 기능을 기반으로 한 Ca²⁺ influx 분석. L, Og-NSCs와 2D NSCs로 부터의 1AP을 내는 신경세포를 관찰. Og-NSCs (red) 및 2D-NSCs (black). 화살표 1AP point. M, 1 AP response amplitudes 평균값: [Ca²⁺ influx ]Og = 0.60 ± 0.09 (n=10), [Ca²⁺ influx]2D = 0.16 ± 0.02 (n=12). N, 자극에 의한 Ca²⁺ influx traces. O, 100AP response 동안 Ca2+ influx kinetics 평균값: [τ of Ca²⁺ influx]3D=0.85s ± 0.11 (n=10), [τ of Ca²⁺ influx]2D=2.35s ± 0.43 (n=12). *p=0.0009(M), p=0.005(O), Student's t-test. P, Pre-synaptic DA 분비, DA levels은 분화 13~15일 배지 내의 도파민 분비를 그 24시간 동안의 조건화 배지로 측정함. 30분 동안 KCl-depolarization 유도함. *p=0.0006-0.0009, n=4 (Og), 5-6 (2D) independent cultures. Q, R, Og-NSCs의 성상교세포 분화능. Single RNA-seq 의 cluster 1 에서 성상전구세포군이 관찰됨 (Q). CD44+ 성상 전구세포군은 신경줄기세포군에 포함됨 (R), 분화 이후 도파민성 신경세포(TH)와 함께 성상교세포(GFAP)가 같이 분화됨 (S). Og-NSC 군에서 관찰되고 2D-NSC 군에서는 거의 없음. *p=0.004(R), p=0.0003(S), n=3-4 independent cultures. scale bars, 10μm(I), 25μm(B, R), 50μm(C, D, S), 100μm(H)].
도 4는 파킨슨병의 생체적 특징인 α-synucleiopathy 모델에서 관찰하는 경우, 본 발명의 방법으로 유도된 신경줄기세포는 심화된 파킨슨 병인인α-synuclein 전파가 잘 되지 않는 상태의 세포임을 볼 때, 파킨슨 세포치료제로 사용 시 유리하다고 할 수 있다 [오가노이드 유래 중뇌형 신경줄기세포(Og-NSCs) 유래 신경세포는 상대적으로 α-syn이 덜 퍼지는 것으로 관찰됨. α-syn 질환 환경에서 보다 저항성 가질 수 있음. A-D, α-syn이 신경세포 내로 적게 침투되는 특성 관찰. Og-NSCs (vs 2D-NSC). A, 세포간에 α-syn 이동을 관찰할 수 있는 듀얼 쳄버 시스템. B: α-syn (GFP) 강도를 768 개 (Og)와 740 개 (2D)의 처리된 세포에서 비교함. *p=0.000004. Og-NSCs에서 상대적으로 덜 침투됨을 보임. C-F: 파킨슨 병인이 될 수 있는 α-syn 올리고머화를 바이러스를 이용한 과발현 시스템으로 유발시켰을 때, 특히 병인이 될 수 있는 p129-α-syn levels은 상대적으로 Og-NSCs에서 적게 관찰됨 (C), α-syn 올리고머를 바이러스 과발현 유발 후 (왼쪽) 혹은 임의로 PFF(preformed α-syn fibril) 처리 후 (오른쪽)에 Western blot 분석. 파킨슨병 주요 병인이 될 수 있는 α-syn 올리고머가 Og-NSCs에서 덜 발생함을 보임 (D), Bi-FC 시스템 (E, F). Bi-FC-녹색 발현되는 inclusions이 상대적으로 Og-NSCs에서 덜 관찰됨. 이로서 같은 양의 α-syn 이라도 뭉쳐서 병인으로 될 확률이 낮음을 보여줌 (F). *significance at p=0.001 (C) and 0.02 (F), n= 3 independent experiments. G, PFF-a syn 모델 동물에다가 Og-NSCs(left hemisphere)와 2D-NSCs(right-hemisphere)를 각각 이식 후 조직학적으로 분석함. 동물은 2주간 PFF와 a-syn AAV 바이러스로 파킨슨병 모델 유도하고, 각 세포 이식 후 1달 후 희생시킴. Og-NSCs 이식한 부위에서 상대적으로 적은 악성 α-syn (p129-α-syn)이 관찰되고, 건강한 도파민성 신경세포 관찰됨. scale bars, 50μm(B, C, F), 100μm(G)].
도 5는 Og-NSC 이식 후 나타날 수 있는 결과에 대해 다각도의 in vivo 예측 연구를 나타낸 것이다 [A: 도파민 신경세포의 이식 시 성공의 중요한 인자로 보고되어 있는 인자들의 발현을 Single cell RNA-seq data 에서 분석 결과, Og-NSCs 에서 상대적으로 높은 발현을 보임. (vs 2D-NSCs). 결과값은 색깔의 농도로 표시. log2 (expression of unique molecular identifier (UMI) values) (좌), log2 Og/2D 발현값의 비율 평균으로 표시 (우). B: pSyn-GCaMP6s-발현하는 신경세포를 dorsal striatum 에 이식하여, Two-photon 형광 endomicroscopy 관찰한 이미지 결과 (상). GCaMP6s 형광의 각 다른색의 ROI(regions of interest)는 도면 위쪽 이미지의 동그라미로 표시됨 (B, 하). C-F: Og-NSC-유래된 이식 부위의 Action potential firing 와 synaptic transmission. pDll1-GFP-hESCs로부터 분화되어, 신경줄기세포에서 형광을 발현하는 Og-NSCs 을 hemi parkinsonian 모델 동물의 선조체에 이식. 이식 1.5개월 후의 동물 뇌 슬라이스에서 Whole-cell patch clamp recording. 이식된 GFP+ 세포들의depolarizing current injection 으로 intrinsic excitability 관찰 (C), -70 mV holding potential로 sEPSC 기록 (D). Og-NSC 유래 이식부위의 sEPSCs의 평균 amplitude(E)와 frequency(F)를 이식하지 않은 부위의 정상 세포와 비교(좌). n=5 (graft) 및 4 (control, unlesioned) from 2 rats. G, H: 6-OHDA 파킨슨병 유발 동물에 이식된 Og-NSCs를 5개월 후에 ¹⁸F FP-CIT DAT binding 을 MRI와 PET 스캔으로 분석함. *p=0.021, n=8 (graft) 및 5 (control, lesioned). T: 이식 부위, scale bars, 200μm.
도 6은 Og-NSCs 를 실제 파킨슨병 모델 동물에 이식 후 결과를 관찰한 것이다 [Og-NSCs 은 6-OHDA-유발 hemi parkinsonian 모델 랫트에 이식 (A-O), 또한 필리핀 원숭이에도 이식함 (P). A-C: 파킨슨병 동물의 이식 후, 행동학적 분석. A: 이식 6 개월 후, 암페타민-유발 rotation 스코어 관찰함. 시작 시점에 14마리 파킨슨병 모델 동물이 Og-NSCs 이식받았고, 암페타민-유래 행동학적 회복 스코어는 이식 후 1-6 달 동안 매달 분석함. 1-4개월 동안, 6 마리의 행동학적 회복 스코어가 >60% 이면, 분석을 위해 희생시켰음. 이식받기 전의 행동학적 스코어 분석 자료를 근거로 회복을 그것의 %로 표시함. 대조군으로는 생리식염수 이식 그룹으로 7마리를 사용하였다. (sham-(PBS-injected) controls (n=7) 분석은 *p=7.8e-10-0.037, one-way ANOVA.) Og-NSC-이식된 동물 모델의 다른 행동학적 실험으로 이식 6개월 후에 step adjustment tests 실시함, *p=5.58e-07-0.0006, one-way ANOVA, Tukey's post hoc analysis (B) 또한 cylinder tests도 실시함, *p=1.63e-08-0.000424, one-way ANOVA, Tukey's post hoc analysis (C) (n=8). D: Og-NSC-이식된 동물 모델 이식 후 6개월에 도파민 신경세포의 형태를 Neurite tracing (Neurolucida) 이미지로 관찰한 결과 성숙한 형태로 분화되었음을 확인함. E-O: 조직 형태학적 분석을 형광염색으로 확인함. E-G: 이식 부위의 TH+/FOXA2+, TH+/LMX1A+, TH+/NURR1+ 도파민성 신경세포 확인. 아래 확대된 이미지를 통해 이식된 도파민성 신경세포가 진정한 중뇌형의 신경세포임을 확인함. I: 중뇌성 인자들과의 형광 염색 결과 발현%를 8마리의 동물 이식 부위에서 확인함 (13,697 TH+ 세포 개수 분석함). L-P: 이식 6개월 후에 이식 부위를 부피로 계산 (L), TH+ 신경세포의 개수 (M), density (N), body size (O) (n=8 rats). P, Og-NSC-이식된 성체 필리핀 원숭이(cynomolgus)에 이식 1개월 후에, TH+ 도파민 신경세포 중에 인간세포 유래 이식된 세포임을 나타내는, STEM121을 발현하고, 중뇌성 마커로 FOXA2 와 NURR1을 발현하는 이식된 도파민성 신경세포 확인. scale bars, 50μm(E-G bottom, H, J, K, P bottom), 200μm(P top), 500μm(D-G)].
도 7은 hPSC로부터의 중뇌 유사 오가노이드의 생성 및 특성을 나타낸 것이다[A: 중뇌 유사 오가노이드를 생성하기 위한 실험 절차의 개략도. 시험관내 (DIV) 배양 일에 걸친 오가노이드의 B-D, 위상차 이미지. 스케일 바, 200 μm. E-O: 오가노이드에 형성된 신경 구조에 대한 대표적인 면역 형광 이미지. 오가노이드는 시험관내 표시된 날에 동결 절편화되고 지시된 항체로 염색되었다. V, 심실; VZ, 심실 구역; IZ, 중간 구역; MZ, 맨틀 존, 스케일 바, 100μm (E-P), 200μm (B-D)].
도 8은 본 발명의 방법에 의해 중뇌 신경 발생에 중요한 FGF8의 처리 시간 조절로 중뇌 신경줄기세포 유지가 효율적으로 개선됨을 나타낸 것이다 [A: 프로토콜 A 및 B의 FGF8b 처리 계획. B: 2개의 상이한 FGF8b 처리 계획으로 산출된 오가노이드(Og)-NSC에서 복부 중뇌 (VM)-특이적 및 시상 하부-특이적 마커의 발현에 대한 RNA-seq 데이터. C: 프로토콜 A 및 B로 유도된 EN1+/TH+ mDA 뉴런에 대한 대표 이미지. 스케일 바 (50μm)].
도 9는 복부 중뇌(VM) 신경 줄기/전구 세포(NSC) 또는 2D 배양 환경에서 hPSC로부터 2D-NSC 생성을 나타낸 것이다 [A: 2D에서 분화 hPSC의 세포 계대 동안 성상 세포 조절 배지(ACM) 처리의 요구. NSC를 제조하기 위해, ACM의 부재(-) 또는 존재(+)에서 VM-패터닝 화학 처리 (상단 전, 상부 패널)로 분화된 hPSC를 분리하고 도금(통과 후)하였다. 표시된 중뇌-특이적 인자를 발현하는 세포의 위상차 및 면역 형광 염색의 이미지가 제시된다. B: 2D 배양 기반 hPSC-NSC/mDA 뉴런 분화 프로토콜의 도식적 개요. C에 의해 추정된 2D-NSC에서의 VM-특이적 NSC 마커 발현 NSC 확장 2주 후 면역 형광 염색. D: 집단 배가 수준 (PDL, 막대) 및 누적 PDL (선)에 의해 표현된 2D-NSC의 세포 확장. E-F: mDA 뉴런은 분화 후 12-15일 후에 Og-NSC로부터 산출되었다. DA 뉴런 (TH+ 세포의 %)의 중뇌-특이적 마커 발현을 3개의 독립적인 실험 (F)으로부터 정량화하였다. 스케일 바, 50μm].
도 10은 신경 줄기/전구 세포(NSC) 및 오가노이드-(상) 및 2D 기반 배양 (하)에서 중뇌 관련 마커를 표현하는 mDA 신경세포에 대한 대표 이미지를 나타낸 것이다 [스케일 바, 50μm].
도 11은　개발된 본 프로토콜이 인간 배아줄기세포/유도만능 줄기세포에 다양한 줄기세포주에 모두 범용 가능한 프로토콜임을 보여주는 결과이다 [복부 중뇌(VM) 신경 줄기/전구 세포(NSC) 및 중뇌-특이적 마커를 발현하는 mDA 신경세포는 시험된 모든 hPSC 라인에 걸쳐 생성된다. 3개의 hESC 및 4 hiPSC 라인을 도 1의 a에 요약된 오가노이드 프로토콜을 사용하여 VM-타입 NSC로 분화하도록 유도하였다. NSC는 2-3 세포 통과로 확장되었고 mDA 신경세포로 NSC의 최종 분화가 유도되었다. 말단 분화 후 7-12일 후 중뇌 마커 FOXA2, LMX1A, NURR1 및 EN1을 발현하는 NSC 단계 (확장) 및 TH+ mDA 신경세포에서 FOXA2 +, LMX1A + NSC에 대한 대표적인 면역 염색 이미지가 도시되어 있다. 스케일 바, 50μm].
도 12는 오가노이드(Og)-신경줄기/전구 세포(NSC)는 인간 mDA 신경세포에 대한 확장 가능하고, 양적인 면에서나 그 세포생물학적 특성 유지에 있어서, 범용의 세포 치료제로의 사용 가능할 정도의 공급이 가능함을 보여주는 결과이다. [A: 5 세포 계대를 갖는 NSC 확장을 사용하여 미분화된 hPSC 1 다쉬로부터 mDA 뉴런 7,260 바이알 (9,075 디쉬)을 생성하는 오가이드-기반 프로토콜의 도식적 개요. B: 다수의 NSC 계대 동안 Og-NSC의 mDA 신경원 용량의 유지. 　좌측에는 FOXA2+, NSC 단계에서의 LMX1A+ NSC (확장) 및 중뇌 마커 FOXA2, LMX1A, NURR1 및 EN1을 발현하는 TH+ mDA 뉴런에 대한 대표적인 면역 염색 이미지가 말단 분화 후 10-18일에 도시되어 있다. NSC 및 mDA 뉴런에서의 중뇌 마커 발현은 우측의 그래프에서 정량화되었다. n = 3 개의 독립적인 실험. C: mDA 신경원성 전위를 변경시키지 않으면서 액체 N2에 Og-NSC의 저장. 액체 N2 저장 있는(+) 그리고 없는 (-) H9 Og-NSC로부터 분화된 mDA 뉴런의 대표적인 면역 염색 이미지가 도시되어 있다. 스케일 바, 50μm].
도 13은 PD-iPSC로부터 유래된 mDA 신경세포를 이용한 PD 약물의 후보 화합물 스크리닝을 나타낸 것이다 [A: 실험 절차의 개략도. PD 환자 (PD-ips1)로부터 유래된 hiPSC를 오가노이드-기반 프로토콜을 사용하여 mDA 신경세포로 분화시켰다. PD-iPSC-mDA 신경세포의 세포 생존을 MTT 분석 및 TH- 양성 DA 신경세포의 면역 형광 강도-기반 카운팅을 사용하여 화합물의 존재 하에 평가하였다. Image Express Microconfocal (Molecular Devices, San Jose, CA)을 사용한 고함량 스크리닝(HCS) 분석을 TH 강도-기반 mDA 신경세포 생존 분석에 사용하였다. B: MTT 분석 (상부) 및 TH+ 강도 측정 (하부)에 의해 얻어진 스크리닝 데이터의 예. 노란색 선, 대조군의 평균값 (비히클). C: 비히클 (상부) 및 화합물 #9를 사용하여 10일에 PD-iPSC 유래 mDA 신경세포의 대표적인 TH/MAP2 이미지].
도 14는 계대 배양에 따라, 오가노이드(Og) 신경 줄기/전구 세포(NSC) 분화 발달 중뇌(VM)에서 mDA 신경세포의 개발 그 세포의 특성을 요약한 것이다. Og-NSC의 말단 분화는 bFGF의 철회 및 신경 영양 인자 BDNF, GDNF 및 cAMP의 존재에 의해 유도되었다. 면역 세포 화학적 분석은 증식 세포 (Ki67), 성숙 신경세포 (MAP2), DA 신경세포 (TH) 및 중뇌 (FOXA2, LMX1A, NURR1)에 특이적인 항체를 사용하여 지시된 분화 일에 수행되었다. 스케일 바, 50μm.
도 15는 오가노이드(Og) 및 2D-NSC에서 신경 줄기/전구 세포(NSC)-(A), 만능 세포-(B) 및 세포 노화-특이적 유전자 (D)의 단일 세포 RNA 발현 패턴을 나타낸 것이다 [다능성 유전자 SSEA4 및 TRA1-60의 낮은 발현 수준은 FACS 분석 (C)에 의해 추가로 평가되었다].
도 16은 3D 대뇌 피질 오가노이드를 이용하여 대뇌 피질 신경줄기세포 분리를 위한 세부 프로토콜을 나타낸 것이다.
도 17은 3D 대뇌 피질 오가노이드를 이용하여 신경줄기세포를 확보함을 나타낸 것이다 [좌 그래프: 분화 프로토콜 적합성을 대뇌 마커인 pax6 발현을 통해 확인하였다. 우: 위쪽 사진은 오가노이드 해체(chop) 전에 사진으로 제대로 된 형태의 오가노이드 형성을 확인하였고, 아래 사진은 해체한 이후에 정상적인 신경줄기세포 형태로 성장함을 확인하였다].
도 18은 2D 및 3D 유래 배양으로부터 중뇌-신경줄기세포로의 최종 확립된 직접 분화 프로토콜을 나타낸 것이다[A: 인간 배아줄기세포를 이용해 단계적으로 배아줄기세포로부터 중뇌-신경줄기세포로의 직접 분화(Direct differentiation), 성상전구세포 확보, 그리고 그 이후에 성상 세포로의 말단 분화(Terminal differentiation)기술을 확립, B: 대뇌피질유래 성상교세포 확보의 프로토콜. (A)는 (B)와 그 패턴화 법에 있어서 상이].
도 19는 2D 및 3D 유래 배양으로부터 중뇌-신경줄기세포로의 직접 분화 프로토콜을 이용한 성상교세포의 제조를 나타낸 것으로, 기존의 이차원적 배양법에 대비하여 본 발명의 방법으로 유래된 세포들이 보다 건강하고 마커 유지가 잘되는 행상된 형태의 세포임을 보여준다.
도 20은 in vitro 배양된 대뇌 피질형 및 중뇌형 성상교세포의 특성을 분석한 것으로, 둘 간에는 실제적인 신경세포의 지지 기능면에서는 중뇌 유래 성상세포가 유전자 분석 시 우수함을 보여준다.
도 21은 in vivo 이식 후 대뇌 피질형 및 중뇌형 성상교세포의 특성을 분석한 것으로, 둘 간에는 실제적인 신경세포의 지지 기능면에서는 중뇌 유래 성상세포가 유전자 분석 시 우수함을 보여준다.
도 22는 in vivo 성상전구세포의 이식으로부터 미세아교세포의 M2 타입의 양질의 세포로의 변화를 나타낸 것으로, 이는 향후 성상세포의 이식 시 그 환경의 개선이 가능하므로, 활용도가 클 것임을 예측할 수 있다.
도 23은 Xeno-free 시스템을 이용한 다른 종류의 유도만능 줄기세포의 적응 배양 결과를 나타낸 것이다. 실제 세포치료제 개발 시에는 Xeno-free 시스템을 이용해서 다양한 라인에 적용이 중요하므로, 기본적인 결과이다.
도 24는 다른 종류의 유도만능 줄기세포(CMC-iPSC#11)의 중뇌성 성상세포군으로의 프로토콜 적용 확인(좌) 및 정상적인 신경세포 분화 확인(우)을 나타낸 것이다.
도 25는 다른 종류의 유도만능 줄기세포(CMC-iPSC#11)의 프로토콜 적용을 확인한 것이다.
도 26은 TGF-β와 LIF를 이용한 효율적인 분화 유도를 통해 프로토콜 적용을 확인한 것이다.
도 27은 생성한 중뇌 성상교세포의 생리학적 특성을 분석한 것이다.
도 28은 중뇌형 성상교세포의 조건화 배지를 활용한 미세아교세포의 변화를 관찰한 것이다. 이를 통해 성상세포에 의한 항염증 효과를 in vitro로 검증하였다.
도 29는 염증 유도 후 성상교세포 조건화 배지로 사멸 인자 억제 가능성을 확인한 것이다. 이를 통해 성상세포에 의한 항염증 효과를 in vitro로 검증하였다.
도 30은 염증 유도 후 성상교세포 조건화 배지로 항염증 효과를 RT-PCR 을 이용하여 확인한 것이다. 이를 통해 성상세포에 의한 항염증 효과를 in vitro로 검증하였다.
도 31은 선조체에 성상교세포 이식 후 비이식 부위와 염증반응 관련 RNA 발현을 분석한 것이다. 이를 통해 성상세포에 의한 항염증 효과를 in vivo로 검증하였다.
도 32는 In vivo 성상교세포 이식 후, M1 마커 iNOS 발현을 나타낸 것이다. 신경줄기세포 이식 부위에 대비하여, 염증이 덜 발생되는 것을 확인하였다.
도 33은 In vivo 성상교세포 이식 후, M1 마커 CD11b 발현을 나타낸 것이다. 신경줄기세포 이식 부위에 대비하여, 염증이 덜 발생되는 것을 확인하였다.
도 34는 In vivo 성상교세포 이식 후, M1 마커 CD16.32 발현을 나타낸 것이다. 신경줄기세포 이식 부위에 대비하여, 염증이 덜 발생되는 것을 확인하였다.
도 35는 In vivo 성상교세포 이식 후, M2 마커 CD206 발현을 나타낸 것이다. 신경줄기세포 이식 부위에 대비하여, 염증이 덜 발생되는 것을 확인하였다.
도 36은 3D 오가노이드를 이용하여 시상하부 신경줄기세포 분리를 위한 세부 프로토콜을 나타낸 것이다.
도 37은 3D 시상하부 오가노이드를 확보하고, 이에 맞는 마커의 발현을 확인한 것이다. 특이 마커로서 nestin, Rax, Sox2 의 발현이 잘 일어난다.
도 38은 증식 가능한 시상하부 신경줄기세포 확보됨을 확인한 것이다 [a: 시상하부 신경줄기세포 마커인 nestin/Rax를 97% 발현하는 4계대까지 증식 가능한 신경줄기세포 확보; b: 시상하부 신경줄기세포의 시상하부 관련 유전자 발현 확인].
도 39는 시상하부 신경줄기세포의 분화 유도 후 정상적인 신경세포와 성상교세포 분화를 확인한 것이다.
도 40은 시상하부 신경줄기세포로부터 분화 유도 후 렙틴(leptin)에 반응성 있는 세포로의 분화를 확인한 것이다. 이러한 기능 분석으로 시상하부세포의 기능적 특징을 잘 보이고, 분화후에 시상하부 신경세포 마커인 NPY, AGRP의 발현이 증진되어 있음으로, 기능적으로도 마커 발현 측면에서도 원하는 시상하부 신경줄기세포로의 패턴화가 잘 되었음을 확인하였다.
도 41은 3D 오가노이드 이용으로 개발된 시상하부 신경줄기세포가 이차원적 2D 환경에서 개발된 것 보다 생체 내의 마커 유전자군을 많이 발현함을 나타낸 것이다.
도 42는 3D 오가노이드 이용으로 개발된 시상하부 신경줄기세포의 생체 이식 가능성을 확인한 것이다.1 shows a method of preparing a midbrain organoid and extracting mesencephalic neural stem cells therefrom. B and C: Expression of mesencephalic (OTX2, FOXA2, LMX1A, PAX2, PAX5) markers and neural stem cell (SOX2, NESTIN, KI67) markers was confirmed by fluorescent staining of mesencephalic neural stem cells proliferated from organoids for 2 weeks. D; Calculation of the proliferation rate of mesencephalic neural stem cells proliferated from organoids. PDL (population doubling level, bars) and cumulative PDL (line). EG: Confirmation of accurate differentiation of midbrain dopaminergic neurons after 12-15 days of differentiation induction of mesencephalic neural stem cells proliferated from organoids. Identification of midbrain markers (FOXA2, LMX1A, NURR1, EN1) and fully mature neuronal differentiation markers (AADC, SYPT, DAT) along with dopaminergic neuronal markers (TH). F: Verification of expression % compared to neurons differentiated from 2D neural stem cells. G: It was confirmed that the protocol was stably implemented when various human embryonic stem cells and induced pluripotent stem cells were used. H: Confirmation of stable differentiation success rate of organoid-derived neural stem cell differentiation technology compared to 2D differentiation method. (When performing each method as a criterion of success, >70% (>30%) TH+ of MAP2+ neurons and >70% (>20%) NURR1+ of TH+ mDA neurons). IL: Organoid-derived neural stem cell differentiation protocol can more stably inhibit apoptosis and differentiate. EthD-1+ dead cells (I), cleaved caspase 3+ apoptotic cells (J), FACS-using Annexin+/PI+ cell counting (K), and β-galactosidase-stained cells for cell senescence (L) ). * P = 0.0045 (I), 0.02 (J), 0.008-0.044 (L), 0.003 (K), n= 3 independent experiments, one-tailed Student's t-test. scale bars, 25 μm (J), 50 μm (B, E, I, K)].
Figure 2 shows the analysis of the transcriptional regulation (Transcriptome characteristics) of organoid-derived neural stem cells [A and B: midbrain-like organoid (MLO)), organoid-derived neural stem cells (Og-NSCs), and 2D Produced neural stem cells (2D-NSCs), transcription factors from the RNA, and previously reported transcriptome datasets of prenatal midbrain, adult dopaminergic neurons (adult substantia nigra) , Analysis of differences in transcription factor regulation in adult cortex and hindbrain by bulk RNA sequencing. A: Spearman's correlation plot. Analysis of the differentially expressed genes (DEGs) of 2D neural stem cells and organoid-derived neural stem cells. B: Principle component analysis (PCA) of bulk RNA sequencing data. The PCA score is centered on the 1,000 genes with the greatest difference in expression. CG: Single cell RNA sequencing analysis of transcriptional regulation of organoid-derived neural stem cells (Og-NSCs) and 2D-derived neural stem cells (2D-NSCs). (13,793 and 12,166 cells analyzed, respectively) C: t-SNE (t-distributed stochastic neighbor embedding) map is expressed in data-based color. D: Three separate k-means clustered by t-SNE projection. (Loupe Cell Browser ver. 3.0.1, 10X Genomics, Inc.). The analysis of Og- and 2D-neuronal stem cells is analyzed as a Van diagram, respectively (below). E: Hierarchical clustering analysis of 40 genes overexpressed compared to other groups in each cluster. F: Top-ranked GOs based on overexpressed genes in each group (UMI>1, log ₂ FC>0.5, P<0.05, DAVID). G, Representative gene expression specifically regulated compared to other groups. Data are expressed in dark colors (log2 expression (UMI). FH, Og-neuronal stem cells showed more pronounced expression compared to 2D-neuronal stem cell analysis (DEGs) GOs (vs 2D-NSCs, log ₂ FC >1, padj<0.01) analyzed bulk (H) and single-cell RNA-seq datasets (I), 'cell-cell adhesion,' increased expression in both bulk- and single-cell RNA-seq data, and analyzed heatmap ( J).
3 shows the results of analyzing the maturity and functionality of Og-NSCs and dopaminergic neurons in terms of mitochondrial function, and the results showing the difference in neuronal maturity between the two groups. In conclusion, it shows that Og-NSCs can differentiate into a healthier and more mature form compared to the conventionally produced NSCs. [AF: Organoid neural stem cells (Og-NSCs)-derived neurons maintain healthy mitochondria even under mitochondrial toxin experiments. A: STRING analysis of mitochondrial factors overexpressed in Og-NSCs (vs 2D-NSCs). B: Synthesis analysis of mitochondria (Mitochondrial biogenesis (MitoTimer)). *p=0.0000005 (48hr), 0.024 (24hr), n=4 cultures. C: Mitochondrial ROS (MitoSox), D, Mitochondrial membrane potential (JC-1). *p=1.9e-46, independent cultures, 310-314 cells (C), p=3.5e-21, 283-311 cell analysis results from three or more independent experiments (D), Student's t-test. E, F, Survival analysis of dopaminergic neurons differentiated for 4 days under mitochondrial toxin treatment. Cell survival/death (E), dopaminergic neuron length analysis (F). ROT, rotenone; CC, cccp; HO, H

O₂ *p=0.013-0.165 (E), p=0.000032-0.11 (F) n=3, Student's t-test. GP: Dopaminergic neuronal maturity and functional analysis. G, Heatmap analysis of Og-NSCs expression-specific genes (vs 2D-NSCs). H, High-maturity dopaminergic neurons derived from Og-NSCs typically analyzed by the Neurolucida analysis program. J, Length analysis of differentiation-induced dopaminergic neurons for 12 days. TH+ fiber lengths from 23-58 cells/culture were measured. *p = 0.013, n = 7 (Og), 3 (2D) independent cultures, t-test. I, K, Synaptic analysis of dopaminergic neurons. The number of actual synapses was analyzed by synaptophysin staining. (27-43 TH+ fibers/culture) *p=0.03, n=5 cultures for each group. ^{LO, Ca 2+} influx analysis based on neuronal function. Observation of 1AP-producing neurons from L, Og-NSCs and 2D NSCs. Og-NSCs (red) and 2D-NSCs (black). Arrow 1AP point. Mean values of M, 1 AP response amplitudes: [Ca ²⁺ influx ]Og = 0.60 ± 0.09 (n=10), [Ca ²⁺ influx]2D = 0.16 ± 0.02 (n=12). N, Ca ²⁺ influx traces by stimulation. O, mean values of Ca2+ influx kinetics during 100AP response: [τ of Ca2 ⁺ influx]3D=0.85s ± 0.11 (n=10), [τ of Ca2 ⁺ influx]2D=2.35s ±0.43 (n=12). *p=0.0009(M), p=0.005(O), Student's t-test. P, Pre-synaptic DA secretion, DA levels were measured as dopamine secretion in the medium for 13-15 days of differentiation with the conditioned medium for 24 hours. Induce KCl-depolarization for 30 min. *p=0.0006-0.0009, n=4 (Og), 5-6 (2D) independent cultures. Q, R, Astrocyte differentiation capacity of Og-NSCs. Astrocytes were observed in cluster 1 of Single RNA-seq (Q). The CD44+ astrocyte group is included in the neural stem cell group (R), and after differentiation, astrocytes (GFAP) are differentiated together with dopaminergic neurons (TH) (S). Observed in the Og-NSC group and rarely in the 2D-NSC group. *p=0.004(R), p=0.0003(S), n=3-4 independent cultures. scale bars, 10 μm (I), 25 μm (B, R), 50 μm (C, D, S), 100 μm (H)].
Figure 4 is when observed in the α-synucleiopathy model, which is a biological characteristic of Parkinson's disease, the neural stem cells induced by the method of the present invention are cells in a state in which α-synuclein propagation, which is the intensified Parkinson's disease, is not well propagated. It can be said to be advantageous when used as a cell therapy. May be more resistant to α-syn disease environments. AD, Observation of the characteristic that α-syn penetrates less into neurons. Og-NSCs (vs 2D-NSCs). A, A dual-chamber system capable of observing the movement of α-syn between cells. B: Comparison of α-syn (GFP) intensity in 768 (Og) and 740 (2D) treated cells. *p=0.000004. Og-NSCs showed relatively less penetration. CF: When α-syn oligomerization, which may be a Parkinson’s etiology, was induced with a viral overexpression system, particularly etiological p129-α-syn levels were observed relatively low in Og-NSCs (C), Western blot analysis of α-syn oligomers after viral overexpression (left) or optionally after PFF (preformed α-syn fibril) treatment (right). Showing that α-syn oligomers, which may be the main etiology of Parkinson's disease, occur less in Og-NSCs (D), Bi-FC system (E, F). Bi-FC-green expressed inclusions were relatively less observed in Og-NSCs. This shows that even the same amount of α-syn has a low probability of aggregation and pathogenesis (F). *significance at p=0.001 (C) and 0.02 (F), n= 3 independent experiments. G, Histologically analyzed after transplantation of Og-NSCs (left hemisphere) and 2D-NSCs (right-hemisphere) in PFF-a syn model animals, respectively. Animals were induced in the Parkinson's disease model with PFF and a-syn AAV virus for 2 weeks, and sacrificed 1 month after transplantation of each cell. Relatively little malignant α-syn (p129-α-syn) was observed at the site of transplantation of Og-NSCs, and healthy dopaminergic neurons were observed. scale bars, 50 μm (B, C, F), 100 μm (G)].
Figure 5 shows a multi-angle in vivo prediction study for the results that may appear after Og-NSC transplantation [A: Single cell RNA-seq data for the expression of factors reported as important factors for success in transplantation of dopaminergic neurons. As a result of the analysis, Og-NSCs showed relatively high expression. (vs 2D-NSCs). The result is displayed as a color intensity. log2 (expression of unique molecular identifier (UMI) values) (left), expressed as a ratio average of log2 Og/2D expression values (right). B: Images obtained by two-photon fluorescence endomicroscopy of pSyn-GCaMP6s-expressing neurons transplanted into dorsal striatum (top). ROIs (regions of interest) of different colors of GCaMP6s fluorescence are indicated by circles in the upper image of the figure (B, lower). CF: Action potential firing and synaptic transmission at Og-NSC-derived graft sites. Og-NSCs differentiated from pDll1-GFP-hESCs and expressing fluorescence in neural stem cells were transplanted into the striatum of a hemi parkinsonian model animal. Whole-cell patch clamp recordings from animal brain slices 1.5 months after transplantation. Observation of intrinsic excitability by depolarizing current injection of transplanted GFP+ cells (C), sEPSC recording with -70 mV holding potential (D). Mean amplitude (E) and frequency (F) of sEPSCs from Og-NSC-derived graft sites compared to normal cells from non-transplanted sites (left). n=5 (graft) and 4 (control, unlesioned) from 2 rats. G, H: After 5 months of Og-NSCs transplanted into 6-OHDA Parkinson's disease-induced animals, ¹⁸ F FP-CIT DAT binding was analyzed by MRI and PET scans. *p=0.021, n=8 (graft) and 5 (control, lesioned). T: implantation site, scale bars, 200 μm.
6 is The results were observed after transplantation of Og-NSCs into actual Parkinson's disease model animals [Og-NSCs were transplanted into 6-OHDA-induced hemi parkinsonian model rats (AO), and also into Philippine monkeys (P). AC: Behavioral analysis after transplantation of Parkinson's disease animals. A: Six months after transplantation, amphetamine-induced rotation scores were observed. At the start, 14 Parkinson's disease model animals were transplanted with Og-NSCs, and amphetamine-derived behavioral recovery scores were analyzed monthly for 1-6 months post-transplantation. For months 1-4, 6 animals were sacrificed for analysis if their behavioral recovery score was >60%. Recovery is expressed as a percentage of it based on pre-transplant behavioral score analysis. As a control group, 7 mice were used in the physiological saline transplantation group. (sham-(PBS-injected) controls (n=7) analysis was *p=7.8e-10-0.037, one-way ANOVA.) Another behavioral experiment in an Og-NSC-transplanted animal model 6 months after transplantation. Step adjustment tests were performed, *p=5.58e-07-0.0006, one-way ANOVA, Tukey's post hoc analysis (B) Also cylinder tests were performed, *p=1.63e-08-0.000424, one-way ANOVA, Tukey's post hoc analysis (C) (n=8). D: Og-NSC-transplanted animal model 6 months after transplantation, the morphology of dopaminergic neurons was observed with a Neurite tracing (Neurolucida) image, confirming that they were differentiated into a mature form. EO: Confirmed by fluorescence staining for tissue morphological analysis. EG: Identification of TH+/FOXA2+, TH+/LMX1A+, TH+/NURR1+ dopaminergic neurons at the transplant site. The enlarged image below confirms that the transplanted dopaminergic neurons are true midbrain type neurons. I: As a result of fluorescence staining with mesencephalic factors, expression % was confirmed at the transplantation site of 8 animals (13,697 TH+ cell number analysis). LP: 6 months after transplantation, the transplant site was calculated by volume (L), number of TH+ neurons (M), density (N), body size (O) (n=8 rats). One month after transplantation into P, Og-NSC-transplanted adult cynomolgus, transplantation expressing STEM121 and expressing FOXA2 and NURR1 as mesencephalic markers, indicating human cell-derived transplanted cells among TH+ dopaminergic neurons. identified dopaminergic neurons. scale bars, 50 μm (EG bottom, H, J, K, P bottom), 200 μm (P top), 500 μm (DG)].
7 shows the generation and characterization of midbrain-like organoids from hPSCs [A: Schematic of the experimental procedure for generating midbrain-like organoids. BD, phase contrast images of organoids over days of in vitro (DIV) culture. Scale bar, 200 μm. EO: Representative immunofluorescence images of neural structures formed in organoids. Organoids were frozen sectioned in vitro on the indicated days and stained with the indicated antibodies. V, ventricle; VZ, ventricular zone; IZ, middle zone; MZ, mantle zone, scale bar, 100 μm (EP), 200 μm (BD)].
8 shows that the maintenance of midbrain neural stem cells is efficiently improved by controlling the processing time of FGF8, which is important for midbrain neurogenesis, by the method of the present invention [A: FGF8b treatment scheme of protocols A and B. B: RNA-seq data for expression of ventral midbrain (VM)-specific and hypothalamic-specific markers in organoid (Og)-NSCs generated with two different FGF8b treatment regimens. C: Representative images of EN1+/TH+ mDA neurons induced with protocols A and B. Scale bar (50 μm)].
9 shows 2D-NSC generation from ventral midbrain (VM) neural stem/progenitor cells (NSC) or hPSCs in a 2D culture environment [A: Astrocyte conditioned medium (ACM) treatment during cell passage of differentiated hPSCs in 2D. request. To prepare NSCs, hPSCs differentiated with VM-patterning chemical treatment (top before, top panel) in the absence (-) or presence (+) of ACM were isolated and plated (after passage). Images of phase contrast and immunofluorescence staining of cells expressing the indicated midbrain-specific factors are shown. B: Schematic overview of the 2D culture-based hPSC-NSC/mDA neuron differentiation protocol. VM-specific NSC marker expression in 2D-NSCs estimated by C. Immunofluorescence staining 2 weeks after NSC expansion. D: Cell expansion of 2D-NSCs expressed by population doubling level (PDL, bars) and cumulative PDL (lines). EF: mDA neurons were generated from Og-NSCs 12-15 days after differentiation. Midbrain-specific marker expression in DA neurons (% of TH+ cells) was quantified from three independent experiments (F). Scale bar, 50 μm].
10 shows representative images of neural stem/progenitor cells (NSCs) and mDA neurons expressing midbrain-related markers in organoid- (top) and 2D-based cultures (bottom) [scale bar, 50 μm].
11 is a result showing that the developed protocol is a protocol that is universally applicable to human embryonic stem cells/induced pluripotent stem cells and various stem cell lines [ventral midbrain (VM) neural stem/progenitor cells (NSC) and midbrain-specific markers. mDA neurons expressing mDA are generated across all hPSC lines tested. Three hESC and 4 hiPSC lines were induced to differentiate into VM-type NSCs using the organoid protocol outlined in Figure 1a. NSCs expanded with 2-3 cell passage and induced terminal differentiation of NSCs into mDA neurons. Representative immunostaining images for FOXA2 +, LMX1A + NSCs in NSC stage (expanded) and TH+ mDA neurons expressing midbrain markers FOXA2, LMX1A, NURR1 and EN1 are shown 7-12 days after terminal differentiation. Scale bar, 50 μm].
12 is an organoid (Og)-neuronal stem/progenitor cell (NSC) that is expandable to human mDA neurons, and in terms of quantity or maintaining its cell biological properties, supply enough to be used as a general-purpose cell therapy The results show that this is possible. [A: Schematic overview of an oguide-based protocol to generate 7,260 vials (9,075 dishes) of mDA neurons from undifferentiated hPSC 1 dash using NSC expansion with a 5-cell passage. B: Maintenance of mDA neuronal capacity of Og-NSCs during multiple NSC passages. On the left, representative immunostaining images for FOXA2+, LMX1A+ NSCs at the NSC stage (expanded) and TH+ mDA neurons expressing the midbrain markers FOXA2, LMX1A, NURR1 and EN1 are shown 10-18 days after terminal differentiation. Midbrain marker expression in NSC and mDA neurons was quantified in the graph on the right. n = 3 independent experiments. C: Storage of Og-NSCs in liquid N2 without altering the mDA neuronal potential. Representative immunostaining images of mDA neurons differentiated from H9 Og-NSCs with (+) and without (-) liquid N2 storage are shown. Scale bar, 50 μm].
Figure 13 shows the screening of candidate compounds of PD drugs using mDA neurons derived from PD-iPSCs [A: Schematic of the experimental procedure. hiPSCs derived from PD patients (PD-ips1) were differentiated into mDA neurons using an organoid-based protocol. Cell survival of PD-iPSC-mDA neurons was assessed in the presence of compounds using MTT assay and immunofluorescence intensity-based counting of TH-positive DA neurons. A high content screening (HCS) assay using Image Express Microconfocal (Molecular Devices, San Jose, CA) was used for TH intensity-based mDA neuronal survival assays. B: Examples of screening data obtained by MTT analysis (top) and TH+ intensity measurements (bottom). Yellow line, mean value of control (vehicle). C: Representative TH/MAP2 images of PD-iPSC-derived mDA neurons at day 10 using vehicle (top) and compound #9].
Figure 14 summarizes the characteristics of mDA neurons in the developmental midbrain (VM) of organoid (Og) neural stem/progenitor cells (NSC) differentiation developmental midbrain (VM) following subcultures. Terminal differentiation of Og-NSCs was induced by the withdrawal of bFGF and the presence of the neurotrophic factors BDNF, GDNF and cAMP. Immunocytochemical analyzes were performed on the indicated days of differentiation using antibodies specific for proliferating cells (Ki67), mature neurons (MAP2), DA neurons (TH) and midbrain (FOXA2, LMX1A, NURR1). Scale bar, 50 μm.
15 shows single cell RNA expression patterns of neural stem/progenitor cells (NSC)-(A), pluripotent cells-(B) and senescence-specific genes (D) in organoids (Og) and 2D-NSCs. [low expression levels of the pluripotency genes SSEA4 and TRA1-60 were further assessed by FACS analysis (C)].
16 shows a detailed protocol for isolating cortical neural stem cells using 3D cortical organoids.
Figure 17 shows that neural stem cells are obtained using 3D cortical organoids [left graph: suitability of the differentiation protocol was confirmed through expression of pax6, a cerebral marker. Right: In the upper photo, before organoid disassembly (chop), proper organoid formation was confirmed, and in the lower photo, normal neural stem cell growth was confirmed after disassembly].
Figure 18 shows the final established direct differentiation protocol from 2D and 3D derived culture to mesencephalic-neuronal stem cells [A: Direct differentiation from embryonic stem cells to mesencephalic-neuronal stem cells in stages using human embryonic stem cells. ), securing astrocytes, and then establishing terminal differentiation technology into astrocytes, B: Protocol for securing cerebral cortex-derived astrocytes. (A) differs from (B) in its patterning method].
19 shows the preparation of astrocytes using a direct differentiation protocol from 2D and 3D-derived culture to mesencephalic-neuronal stem cells. Compared to the conventional two-dimensional culture method, the cells derived by the method of the present invention are healthier and maintain markers. shows that the cells are well-paced.
20 is an analysis of the characteristics of cortical-type and midbrain astrocytes cultured in vitro, and shows that midbrain-derived astrocytes are superior in gene analysis in terms of the actual support function of neurons between the two.
21 shows the characteristics of cortical-type and midbrain astrocytes after transplantation in vivo, showing that midbrain-derived astrocytes are superior in gene analysis in terms of the actual support function of neurons between the two.
Figure 22 shows the change of microglia into high-quality cells of M2 type from transplantation of astrocytes in vivo, which can be expected to be of great utility because the environment can be improved during transplantation of astrocytes in the future. .
23 shows the results of adaptive culture of different types of induced pluripotent stem cells using the Xeno-free system. This is a basic result because it is important to apply the Xeno-free system to various lines when developing an actual cell therapy product.
24 shows confirmation (left) and confirmation of normal neuronal differentiation (right) of protocol application of different types of induced pluripotent stem cells (CMC-iPSC#11) to the mesencephalic astrocyte group.
Figure 25 confirms the application of the protocol of different types of induced pluripotent stem cells (CMC-iPSC#11).
Figure 26 confirms the application of the protocol through efficient differentiation induction using TGF-β and LIF.
27 is an analysis of the physiological characteristics of the generated midbrain astrocytes.
28 is an observation of changes in microglia using a conditioned medium for midbrain type astrocytes. Through this, the anti-inflammatory effect by astrocytes was verified in vitro.
Figure 29 confirms the possibility of inhibiting apoptosis factors with an astrocyte conditioned medium after induction of inflammation. Through this, the anti-inflammatory effect by astrocytes was verified in vitro.
30 shows the anti-inflammatory effect of an astrocyte conditioned medium after induction of inflammation using RT-PCR. Through this, the anti-inflammatory effect by astrocytes was verified in vitro.
31 is an analysis of RNA expression related to non-transplantation site and inflammatory response after transplantation of astrocytes into the striatum. Through this, the anti-inflammatory effect by astrocytes was verified in vivo.
Figure 32 shows the expression of M1 marker iNOS after transplantation of astrocytes in vivo. Compared to the neural stem cell transplantation site, it was confirmed that less inflammation occurred.
33 shows the expression of the M1 marker CD11b after transplantation of astrocytes in vivo. Compared to the neural stem cell transplantation site, it was confirmed that less inflammation occurred.
34 shows the expression of the M1 marker CD16.32 after transplantation of astrocytes in vivo. Compared to the neural stem cell transplantation site, it was confirmed that less inflammation occurred.
35 shows the expression of the M2 marker CD206 after transplantation of astrocytes in vivo. Compared to the neural stem cell transplantation site, it was confirmed that less inflammation occurred.
36 shows a detailed protocol for isolating hypothalamic neural stem cells using 3D organoids.
Figure 37 is to secure the 3D hypothalamic organoids, confirming the expression of the appropriate marker. As specific markers, the expression of nestin, Rax, and Sox2 occurs well.
Figure 38 confirms that proliferative hypothalamic neural stem cells are secured [a: securing proliferative neural stem cells up to passage 4 expressing 97% nestin/Rax, a hypothalamic neural stem cell marker; b: Confirmation of hypothalamus-related gene expression in hypothalamic neural stem cells].
Figure 39 confirms the differentiation of normal neurons and astrocytes after induction of differentiation of hypothalamic neural stem cells.
Figure 40 confirms the differentiation into cells responsive to leptin (leptin) after induction of differentiation from hypothalamic neural stem cells. This functional analysis shows the functional characteristics of hypothalamic cells well, and the expression of hypothalamic neuronal markers, NPY and AGRP, is enhanced after differentiation. confirmed that it was.
41 shows that hypothalamic neural stem cells developed using 3D organoids express more marker gene groups in vivo than those developed in a two-dimensional 2D environment.
Figure 42 confirms the in vivo transplantation potential of hypothalamic neural stem cells developed by using 3D organoids.

Hereinafter, the present application will be described in detail through examples. The following examples only illustrate the present application, but the scope of the present application is not limited to the following examples.

[Example]

Example 1: Differentiation of human induced pluripotent stem cells into mDA (midbrain dopamine) neurons using midbrain organoids

[Test method]

Culturing of human embryonic stem cells or human induced pluripotent stem cells

hESCs and hiPSCs were cultured based on the hESC research guidelines approved by the institutional review board (IRB) of Hanyang University (Seoul, Korea). The hESCs and hiPSCs used in this experiment are shown in Table 1 below.

For proliferation and maintenance of undifferentiated hESCs/iPSCs, in a CO ₂ incubator set at 37° C. without a feeder layer, using mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) ^{on Matrigel TM} or vitronectin (Human; Gibco) Fisher Scientific, Waltham, MA) (Gibco A31804; 0.5 ug/cm ² )-coated 6 cm dishes (Thermo Fisher Scientific, Waltham, MA), medium change was performed daily. The undifferentiated stem cells were maintained in differentiation capacity by daily medium replacement and subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.

Fabrication of midbrain neural stem cells using 3D organoid manufacturing method

Briefly, a system was used in which a midbrain-type 3D organoid was first produced, cut into small pieces, and mass-proliferated in a medium-brain type neural stem cell state in a culture dish.

When subculturing hESCs and hiPSCs, which were normally cultured in an undifferentiated state, they were ^{removed using Accutase TM} , and undifferentiated human embryonic stem cells were dispensed at 10,000 cells/well in a 96-well plate with a round bottom that does not stick to the bottom. to form organoids. The medium composition of the first day was added to the seeding base medium of Table 4 below with the additive conditions corresponding to Day 0 of Table 2 (SB431542, 10uM; Noggin, 100ng/ml; ascorbic acid, 200uM; Doxycyclin, 1ug/ml; Y27632, 20uM). Noid generation started. The next day, the medium was used as neural inducing base media (Table 3), and purmophamine 2uM and sonic hedgehog 100ng/ml were added in addition to components other than Doxycyclin, 1ug/ml, Y27632, and 20uM among the components added on the first day to induce development. (See Table 2) The culture medium was used as neural inducing base media until the 11th day, and the additive components were added or subtracted according to the concentration with reference to Table 2 to induce generation. The medium was changed daily according to the date. Correct formation of organoids was confirmed by the formation of a cell mass on the 2nd to 3rd day, and by buding out of the cell before the 11th day. On the 11th day of organoid differentiation, eight organoids each formed in a 96-well plate were grouped and cultured in one well of a 6-well low binding plate. Generation was induced under the midbrain patterning base media (Table 3) containing the components of Table 2 while rotating with an orbitary shaker (80-100 rpm). On the 18th day, one organoid was cut into 4-10 pieces with a 30G needle, ^{treated with Accutase TM} for 10 minutes, pipetted 5 times with a 1ml pipette, ^{removed Accutase TM} by centrifugation, and then 3 wells of organoid cells were poly -L ornithine (PLO; 15 ug/cm ² )/Fibronectin (FN; 1 ug/cm ² ) or vitronectin (VN; 0.5 ug/cm ² ) was center-plated in a single 60 mm culture dish.

Day 0 Seeding Base Media (recipe below) + SB431542 (Tocris, #1614 10 μM) + Noggin (Peprotech, #120-10, 100 ng/ml) + Ascorbic Acid (Sigma, #A4544, 200 μM) + Doxycycline (Sigma, #D9891, 1) μg/ml) + Y27632 (Sigma, #Y0503, 20 μM) Day 1 Neural Induction Base Media (recipe below) + SB431542 (Tocris, #1614 10 μM) + Noggin (Peprotech, #120-10, 100 ng/ml) + Purmorphamine (Calbiochem, #540223, 2 μM) + SonicHedgeHog (Peprotech, #100-45) , 100ng/ml) + Ascorbic Acid (Sigma, #A4544, 200μM) Day 2~4 Neural Induction Base Media (recipe below) + SB431542 (Tocris, #1614 10 μM) + Noggin (Peprotech, #120-10, 100 ng/ml) + Purmorphamine (Calbiochem, #540223, 2 μM) + SonicHedgeHog (Peprotech, #100-45) , 100 ng/ml) + CHIR99021 (Stemgent, #04-0004, 0.8 μM) + Ascorbic Acid (Sigma, #A4544, 200 μM) Day 5~6 Neural Induction Base Media (recipe below) + Noggin (Peprotech, #120-10, 100ng/ml) + Purmorphamine (Calbiochem, #540223, 2μM) + SonicHedgeHog (Peprotech, #100-45, 100ng/ml) + CHIR99021 (Stemgent) , #04-0004, 0.8 μM) + Ascorbic Acid (Sigma, #A4544, 200 μM) Day 7-10 Neural Induction Base Media (recipe below) + Noggin (Peprotech, #120-10, 100ng/ml) + Purmorphamine (Calbiochem, #540223, 2μM) + SonicHedgeHog (Peprotech, #100-45, 100ng/ml) + Fgf8b (Peprotech) , #AF-100-25, 100 ng/ml) + CHIR99021 (Stemgent, #04-0004, 0.8 μM) + Ascorbic Acid (Sigma, #A4544, 200 μM) Day 11-14 Midbrain Patterning Base Media (recipe below) + Noggin (Peprotech, #120-10, 100ng/ml) + Purmorphamine (Calbiochem, #540223, 2μM) + SonicHedgeHog (Peprotech, #100-45, 100ng/ml) + Fgf8b (Peprotech) , #AF-100-25, 100 ng/ml) + CHIR99021 (Stemgent, #04-0004, 1.5 μM) + Ascorbic Acid (Sigma, #A4544, 200 μM) Day 15~16 Midbrain Patterning Base Media (recipe below) + Purmorphamine (Calbiochem, #540223, 2 μM) + Fgf8b (Peprotech, #AF-100-25, 50 ng/ml) + CHIR99021 (Stemgent, #04-0004, 1.5 μM) + Ascorbic Acid (Sigma, #A4544, 200 μM) Day 17 Midbrain Patterning Base Media (recipe below) + Purmorphamine (Calbiochem, #540223, 2 μM) + Fgf8b (Peprotech, #AF-100-25, 50 ng/ml) + CHIR99021 (Stemgent, #04-0004, 1.5 μM) + bFGF ( R&D systems, #233-FB, 20ng/ml) + Ascorbic Acid (Sigma, #A4544, 200μM) Day 18 Chop&Plating

Seeding Base Media Neural Induction Base Media Midbrain Patterning Base Media

-mercaptoethanol
(Sigma, #M6250, 0.1%)
B27 w/o VitA
(Gibco, #12587010, 1:50)
GlutaMAX
(Gibco, #35050061, 1:100)
Insulin
(Gibco, #12585014, 0.31㎕/ml)
Neurobasal/N2 2X (1:1)
(Neurobasal, Gibco, #21103049)
(N2 2X recipe as follows(1L)
DW 1L
DMEM-F12 12g
Sodium Bicarbonate 1.69g
D-glucose 1.55g
L-Glutamine 0.073g
Apo-transferrin 200mg
200 μl of Putrescine
Selenite 120 μl
Progesterone 400 μl)

MEM-NEAA
(Sigma, #M7145, 1:100)

-mercaptoethanol
(Sigma, #M6250, 0.1%)
B27 w/o VitA
(Gibco, #12587010, 1:50)
GlutaMAX
(Gibco, #35050061, 1:100)
Insulin
(Gibco, #12585014, 0.31 μl/ml)
Neurobasal/N2 2X (1:1)
(Neurobasal, Gibco, #21103049)
(N2 2X recipe as follows(1L)
DW 1L
DMEM-F12 12g
Sodium Bicarbonate 1.69g
D-glucose 1.55g
L-Glutamine 0.073g
Apo-transferrin 200mg
200 μl of Putrescine
Selenite 120 μl
Progesterone 400 μl)

B27 w/o VitA
(Gibco, #12587010, 1:50)
Insulin
(Gibco, #12585014, 0.31 μl/ml)



Human N2
(human N2 recipe as follows(1L)
DW 1L
DMEM-F12 12g
Sodium Bicarbonate 1.69g
D-glucose 1.55g
L-Glutamine 0.073g
Apo-transferrin 200mg
Insulin 312 μl
Putrescine 100 μl
Selenite 60 μl
200 μl of Progesterone)

Mass proliferation of midbrain 3D organoid-derived midbrain neural stem cells

Organoid-derived midbrain neural stem cells are physically cut with a 30G needle and subcultured. After the first plating, once a week for subculture, when 80-90% cells are filled, PLO/FN or PLO using ^{Accutase TM.} /VN-coated 60mm culture dish 3 X 10 ⁶ cells were flat-cultured. The medium brain-type neural stem cell culture medium is mitogen (bFGF 20ng/ml, FGF-8 50ng/ml), BDNF (20ng/ml), GDNF (20ng/ml), Ascorbic acid 200uM in the midbrain patterning base medium shown in Table 4. , Doxycyclin, 1ug/ml added was used as a growth medium. However, on the day of subculture, Y27632, 20 uM was added to this medium. ^{At every subculture, 8 X 10 4} cells per well were plated on a PLO/VN 24-well coated plate and proliferated for 2-3 days. When 80-90% cell confluency was reached, The differentiation of NSCs was induced by fluorescent staining or removal of mitogens (bFGF, FGF-8). For the differentiation medium, BDNF (20ng/ml), GDNF (20ng/ml), Ascorbic acid 200uM, and cAMP 500uM were added to the midbrain patterning base medium shown in Table 4 above to induce neuronal differentiation for 12 days, and then dopaminergic neurons and midbrain It was checked whether the phenotype marker was co-expressed.

Freeze storage of 3D organoid-derived mesencephalic neural stem cells

In general, organoid-derived mesencephalic neural stem cells do not change their properties at least until passage 5. Cryopreservation of cells are passaged made during the culture, Accutase ^TM using frozen, remove the cell mixture of DMSO to be the coefficient of frozen single vial cells of ⁶ X 10 6 to by the cell in growth medium with 10% concentration Frozen in the medium. After being frozen at -70°C for the first 24 hours, it was stored in a cryogenic nitrogen tank the next day until use. If necessary, one vial was quickly thawed in a 37 degree water bath, and flat cultured in a 60 mm culture dish coated with PLO/FN or PLO/VN. For thawing, a neural stem cell proliferation medium was used, and on the day of thawing, Y27632 and 20 uM were added to this medium.

immunocytochemistry

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and cultured in a blocking solution (1% BSA/0.03% Triton X-100 in PBS), and then with the following primary antibodies incubated together. Tuj1 anti-rabbit (1:2000, Babco), TH anti-mouse (1:1000, Immunostar), GFAP anti-mouse (1:100, ICN Biochemicals), microtubule-associated protein 2 (MAP2, 1:200, Sigma) Secondary antibodies labeled with the following fluorescent molecules were used for visualization. Other antibodies are listed in Table 4. Alexa 488 (1:200, Invitrogen) and Cy3 (1:200, Jackson ImmunoResearch Laboratories). Stained samples were mounted in a Vectashield (Vector Laboratories) with DAPI mounting medium and photographed using an epifluorescence microscope (Leica).

Antibodies working dilution Company Notes Mouse monoclonal Antibody (Ab) Oct4 1:200 ¹ Santa Cruz Biotechnology Use only Cy3 2nd antibody Pax6 1:100 ² DSHB Cdh2 (N-cadherin) 1:500 Santa Cruz Biotechnology Neuron-specific class III beta-tubulin (TuJ1) 1:500 ³ Covance Tyrosine Hydroxylase (TH) 1:1000 ⁴ Immunostar Incubation for 2 days at 4℃ Ki67 1:100 ⁵ Novocastra Pax5 1:50 ⁶ BD Biosciences Human Neural Cell Adhesion Molecule (hNCAM) 1:100 Santa Cruz Biotechnology HuC/D 1:100 ⁷ Chemicon Microtubule-Associated Protein 2 (MAP2) 1:200 ⁸ Sigma Dopamine Transporter (DAT) 1:100 BD Biosciences Blocking with 0.1% Saponin Human Nuclei antigen (HN) 1:1000 Chemicon Rabbit polyclonal Ab Nanog 1:200 Santa Cruz Biotechnology Nestin #130 1:50 ⁹ Dr Martha Marvin, Dr Ron McKay Sox2 1:100 Chemicon TuJ1 1:2000 Covance Pax2 1:100 Covance TH 1:500 ¹⁰ Pel-freez GFAP 1:400 ¹¹ DAKO Serotonin 1:4000 Sigma γ-aminobutyric acid (GABA) 1:700 Sigma GAD67 1:500 Chemicon Vesicular monoamine transporter 2 (VMAT2) 1:500 Pel-freez Use only cy3 2nd antibody Nurr1 1:500 Chemicon 0.1% SDS treatment for 5min before blocking EN1 1:200 Chemicon Incubation for 2 days at 4℃ Calbindin1, 28kDa (calbindin D28K) 1:250 Chemicon potassium inwardly-rectifying channel, subfamily J, member 6 (GIRK2) 1:200 Sigma Cleaved Caspase-3 1:500 ¹² Cell signaling

Cell Transplantation and Histological Procedures in Parkinson's Disease Model Animals

Experiments were performed according to National Institutes of Health (NIH) guidelines. Hemi-parkinsonian 6-OHDA in the right substantia nigra (AP-4.8 mm, ML-1.8 mm, V-8.2 mm) and median forebrain bundle (MFB) (AP-4.4 mm, ML -1.2 mm, V -7.8 mm). (6-hydroxydopamine, 8 μg/μl; Sigma) was induced in mature female Sprague-Dawley rats (220-250 g) by a single stereotaxic injection of 3 μl. The incisor bar was set to -3.5 mm, and the AP and ML coordinates are given relative to the bregma. Rats with 300 reps/hour on the same side for this disorder in the amphetamine-induced rotation test were selected. For transplantation, rat E12 VM-NPCs were propagated and mixed with Ctx-, VM-astrocytes, N + F-VM-astroglia or E14 Ctx-NPCs (control) in a 2:1 ratio. 3 μl of mixed cells (1.5×10 ⁵ cells/ul) were mixed with 100 μl/100 g (23.32 mg/ml) of Rompun striatum under anesthesia induced by Zoletil at 100 μl/100 g (50 mg/ml). (coordinates of AP, ML, and V relative to bregma and dura: [1] 0.07, -0.30, -0.55; [2] -0.10, -0.40, -0.50; incisor bar set to 3.5 mm below zero) Each site was injected over 10 minutes. The needle (22 gauge) was left in place for 5 minutes after each injection was completed. Rats received daily cyclosporine A (10 mg/kg, ip) starting 1 day before transplantation and continuing for 1 month, and were maintained without immunosuppressants for the remainder of the post-transplantation period. Six months after transplantation, animals were anesthetized and perfused percutaneously with 4% paraformaldehyde. Brains were removed, soaked in 30% sucrose in PBS overnight, frozen in Tissue-Tek® (Sakura Finetek, Torrance, CA, USA) and sliced in a frozen microtome (Leica). Free-floating brain Free-floating brain sections (30 μm thick) were subjected to immunohistochemistry as described above and imaged by confocal microscopy (Leica). In experiments to test the host environment of transplanted brains, animals were sacrificed one month after transplantation and sliced 1 mm thick on a rat brain slice matrix (ZIVIC Instruments, Pittsburgh, PA), and transplanted to observe changes after transplantation. Tissues from the site were excised. 8-12 regions of the graft-host interface (ca 2x2 mm)/graft were dissected and qPCR analysis was performed. In addition, cells immunoreactive to neurotrophic and pro-inflammatory glial markers were counted along the graft-host interface of cryosectioned brain slices 7-10 days after transplantation.

behavioral test

Animal behavior was described by YH Rhee et al. , using the apomorphine/amphetamine-induced rotation test as described in a paper by LIN28A enhances the therapeutic potential of cultured neural stem cells in a Parkinson's disease model. Brain: a journal of neurology 139, 2722-2739 (2016). was evaluated. Apomorphine (Sigma) was injected subcutaneously at a dose of 0.5 mg/kg and observed while rotating for 60 minutes. The results are expressed in units of the number of final revolutions/60 minutes.

Animal PET/MRI Imaging and Analysis

PET-MRI fusion imaging was performed using a nanoScanPET/MRI system (1T, Mediso, Hungary). To keep mice warm, 0.2 mL of FP-CIT was administered intravenously via tail vein 6.5±1.0 MBq and mice were maintained under anesthesia (1.5% isoflurane in _{100% O 2 gas).} MR brain imaging was performed using gradient echo (GRE) 3D sequences (TR = 25 ms, TE _eff = 3.4, FOV = 50 mm, matrix = 256x256) obtained during FP-CIT absorption, weighted T1 and fast spin echo ( FSE) 3D sequence (TR = 2400 ms, TE _eff = 110, FOV = 50 mm, matrix = 256x256) weighted T2 images were obtained. A 20-minute static PET image was acquired with a 1-3 concordance in a single field of view within the MRI range. Body temperature was maintained with heated air from the animal bed (Multicell Mediso, Hungary) and pressure-sensitive pads were used to induce respiration. PET images were reconstructed by Tera-Tomo 3D in full detector mode with all corrections and high normalization and 8 iterations. Three-dimensional VOI (volume of interest) analysis of the reconstructed image was performed using the InterView Fusion software package (Mediso, Hungary) and applying standard uptake value (SUV) analysis. The VOI was fixed with a 2 mm sphere in the coronal image, and the VOI for the striatum and for it was derived. The SUV of each VOI site was calculated using the following equation. SUVmean = (Bq/cc × tumor volume of interest in body weight)/injected radioactivity.

High-throughput whole transcriptome RNA sequencing

For RNA, total RNA was isolated using Trizol reagent (Invitrogen), and then rRNA removal was performed using the Ribo-Zero Magnetic kit (Epicentre Inc., Madison, WI, USA). Library construction was performed using the SENSE mRNA-Seq Library Prep Kit (Lexogen Inc., Vienna, Austria). High-throughput sequencing was performed as paired-end 100 sequencing using a HiSeq 2000 (Illumina, San Diego, CA, USA). RNA-Seq reads were mapped using TopHat software to obtain bam alignment files. Read counts mapped to transcriptional regions were extracted from alignment files using BEDTools (v2.25.0) and R/Bioconductor (version 3.2.2; R Development Core Team, 2011). Alignment files were used to assemble transcripts, estimate their abundance, and detect differential expression of genes, linc RNAs or isoforms. FPKM (fragments per kb of exon per million fragments) was used to determine the expression level of the gene region. Global normalization was used for comparison between samples. Genetic classification is based on searches submitted to DAVID (http://david.abcc.ncifcrf.gov/).

GO and KEGG pathway analyzes were performed using DAVID Bioinformatics Resources version 6.8. Analyzes of rich categories of GO and KEGG pathways were presented up to p-values of 0.05.

10× Genomics single-cell RNA sequencing (scRNA-seq) and data analysis

Single cell transcription factor regulation analysis (scRNA-seq) was performed using Chromium Controller (10 × Genomics, San Francisco, CA) program, Chromium Single Cell Gene Expression Solution and Chromium Single Cell 3' GEM, Library and Gel Bead Kit v2 ( 10 × Genomics) was carried out after the experiment. Cells separated into 15,000 single cells were placed on Single Cell A chip with Master mix solution. In each single cell, transcription factors inside the cell were reverse transcribed, and RNA barcodes were encoded by emulsion of oligo dT-encoded UMIs. The barcoded Libraries were sequenced with the Illumina HiSeq 4000 platform (Macrogen Inc., Seoul, Korea). The results were analyzed with Cell Ranger (v2.1.1, 10 × Genomics). For 2D-NSCs analysis, 12,166 cells with 2D_cellRanger averaged 57,343 reads and 2,284 genes per cell (out of 24,055 total genes), and for Og-NSCs (with 3D_cellRanger) 13,739 cells averaged 39,191 reads and 2,353 genes per cell (with 3D_cellRanger) 23,860 of the total genes) were analyzed. Cellular transcription factor expression was expressed as UMI count. Cell diversity analysis was performed by k-means clustering for t-distributed stochastic neighbor embedding (t-SNE) analysis (performed by Loupe Cell Browser ver. 3.0.1, 10 × Genomics, Inc.). The top 40 genes with increased expression showing a significant difference in expression were analyzed by hierarchical clustering analysis, and each cluster was divided based on this. For the GO and KEGG pathway analysis of Og-NSCs, 580 differentially upregulated genes (UMI >1, log2FC>0.5, P<0.05) were analyzed. Genetic analysis was mainly performed using DAVID (http://david.abcc.ncifcrf.gov/). Network analysis was performed using Cytoscape (version 3.7.1+JAVA v8, provided by NIGMS).

Analysis of mitochondrial function

The regenerative dynamics of mitochondria are described in integrated portions of young (green) and old (red) MitoTimer proteins (Ferree, AW, Trudeau, K., Zik, E., Benador, IY, Twig, G., Gottlieb, RA, and Shirihai, OS (2013).MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age.Autophagy 9, 1887-1896; Hernandez, G., Thornton, C., Stotland, A., Lui, D., Sin, J., Ramil, J., Magee, N., Andres, A., Quarato, G., Carreira, RS , et al. (2013). MitoTimer: a novel Tool for monitoring mitochondrial turnover was analyzed using Autophagy 9, 1852-1861). pMitoTimer vector (Addgene52659; Addgene, Watertown, MA) was injected into NSCs cells by electroporation (NEPA21, Nepagene, Japan) to express them, and then 2 days later, mitochondria generated by red versus green fluorescence were investigated. Mitochondrial ROS and membrane potential were measured with MitoSox (Thermo Scientific Inc., Waltham, Mass.) and NucleoCounter3000 (NC3000; Chemometec, Allerod, Denmark), respectively. ROS production was analyzed for induction after treatment with _{H 2} O _{2 (500 μM) 2 hours before analysis.} For the study of mitochondrial toxicity resistance, 10 μM Rotenone, 4 μM CCCP, or 250 μM H ₂ O ₂ was treated for 1 hour and analyzed by TH fluorescence staining.

DA release assay

The presynaptic activity of DA neurons was determined by measuring the level of DA neurotransmitter released in differentiated VM-NPC cultures. The 24-hour culture medium (differentiation day 12-13) was collected and the DA level was measured using an ELISA kit (BA E-5300, LDN). In addition, DA release induced by membrane depolarization was assessed by incubation (day 12 of culture) in fresh N2 medium for 30 min in the presence or absence of 56 mM KCl. The induced DA release was calculated as the DA level with KCl minus the DA release without KCl.

Intercellular α-syn Transduction Assay

Confirmation of intercellular α-synuclein aggregate migration by factors secreted from astrocytes

An analysis experiment was conducted using a co-culture with cells overexpressing A53Tα-syn-EGFP in SH-SY5Y (Professor Park Sang-myeon, Ajou University) derived from neuroblastoma.

SH-SY5Y cells, which were differentiated using RA for 5 days and induced to express A53Tα-syn-EGFP, were co-cultured with neural stem cells induced to differentiate for 10 days in a dual chamber system for 24 hours to produce A53Tα-syn-EGFP. Migration was compared to the extent of GFP expression. Transmission of αsynuclein was confirmed by immunostaining (Immunoncytochemistry) through the brightness of α-syn-GFP using an antibody.

Detection of pathological α-syn aggregates

α-synuclein aggregates during differentiation A-synuclein aggregation was induced by treatment with PFF (pre-formed fibrils) (8 μg/ml of medium), and the degree of a-synuclein aggregation was analyzed after differentiation for 20 days. Alternatively, overexpression was induced using α-syn (pEF1α-α-syn) expressing lentiviruses and analyzed. Thioflavin S (staining method for measuring protein aggregation; sigma Aldrich) and immunostaining (Immunoncytochemistry) and protein electrophoresis (Western blot) using a-synuclein antibody were used to confirm the presence and the aggregation was observed as a change in protein size.

Bi-FC (Bimolecular fluorescence complementation)

Venus1-α-syn (V1S; N-terminal of α-syn) and α-syn-Venus2 (SV2; C-terminal of α-syn) expressing lentiviruses were induced in Og-NSCs or 2D-NSCs, and on the 25th day Aggregated α-syn in differentiated dopaminergic neurons was analyzed by GFP.

In vivo a-syn propagation to transplanted grafts

In female SD (Sprague Dawley) rats (225-250 g), AAV expressing human hα-syn (AAV2-CMV-hα-syn, 2×10 ¹³ genome copies) and PFF (10 μg) were injected into the striatum. Aggregation was induced. anteroposterior (AP), -0; medialateral (ML), -3.0; dorsoventral (DV), -5.0. Two weeks later, Og-NSCs and 2D-NSCs were grafted onto both sides of the striatum. One month after transplantation, the transfer of α-syn to the transplanted cells was analyzed by immunohistochemistry (α-syn and p129-α-syn).

Two-photon imaging of GCaMP6 expressing neurons in grafts

GCaMP6 (Addgene, #40753) was generated under the control of the synapsin promoter (pSyn-GCaMP6s) and used to transduce the culture in vitro. 2 ml/6-cm dishes or 200 μl/well (24-well plate) and 10 ⁶ transduction units (TU/ml (60-70 ng/ml) were used for each transduction reaction. AAV2-CMV- Packaging and production of hα-syn was performed at the Korea Institute of Science and Technology (Seoul).

Og-NSCs at passage 3 were transduced with lentivirus (pSyn-GCaMP6) 3 days before transplantation. Cells were then transplanted into 4 wild-type SD rats, and the rats were analyzed 1 month after transplantation. Two-photon imaging was performed using a commercial microscope system (SP-5) equipped with a Ti:Sapphire femtosecond laser (Chameleon Vision, Coherent Inc., USA) with a tunable wavelength from 680 to 1080 nm, 80 MHz repetition rate, and 140 fs pulse width. , Leica, Germany).

For in vivo brain imaging, a single GRIN lens (NEM-100-25-10-860-S-1.0p-ST, GRINTECH GmbH) with a diameter of 1.0 mm and a length of 9.20 mm was used. Under gas anesthesia, rats were mounted in a rat head holder with the surgically exposed brain facing up. The GRIN lens was fixed to the aluminum plate through the hole in the plate and the plate was translated and the brain was inserted from above. A 10× dry objective lens (Leica HC PL FLUOTAR 10.0 × 0.30) was placed close to one end of the GRIN lens to couple the excitation laser from the microscope system. The excitation wavelength was set at 900 nm for the two-photon excitation of GCaMP6. Two-photon imaging was performed by injecting an excitation laser into the brain. The imaging field of view (FOV) and imaging speed were 387.50 um x 387.50 um with 512 x 512 pixels and 9.03 frames/s, respectively. Emission light from the brain was collected by a GRIN lens and coupled to a microscope system through an objective. The microscopy system had four detection channels (Ch1:430-480nm, Ch2:500-550nm, Ch3:565-605nm and Ch4:625-675nm) and GCaMP6 fluorescence was collected in the second channel (Ch2). . The excitation laser power is 58.08mw.

Calcium Imaging Analysis

Fluo3-AM was treated for 1 hour to be absorbed into the cells and washed three times with PBS so that no fluorescence remained in the medium. While observing fluorescence with a confocal microscope, fluorescence intensity was taken at intervals of 1 to 3 seconds, and ROI was measured for each cell in each time-framed picture, and it was confirmed that the intensity of fluorescence was changed. Gated by FACS as needed.

Experiment result

Production of midbrain organoids from human embryonic stem cells

The existing differentiation technology based on two-dimensional 2D culture does not represent the three-dimensional complex network in the brain in vivo. Only the development of a protocol based on 3D generation can represent the tissue characteristics of the real brain. Therefore, first of all, this research team produced the existing 3D midbrain organoid first. In the early development of the successfully produced mesencephalic organoids, ZO-1 and N-cadherin were expressed along with the neural stem cell markers PLZF+ and Sox2+ (FIG. 7E&F). Anterior embryonic brain markers (OTX2) and ventral VM floor plate markers (FOXA2, LMX1A) are expressed along with Nestin and Sox2, which can be called general neural stem cell markers (G-I in FIG. 7). When development is further induced, MAP2, a neuronal marker, is expressed as a layer outside the proliferation marker Ki67+ and SOX2+, and more specifically, PCNA+ (the proliferative ventricular zone; VZ), MASH1+ (VZ, intermediate zone (IZ) ) and mantle zone (MZ)), and structures specifically expressed in NURR1+ cells (IZ and MZ) were confirmed (L, M in FIG. 7). Characteristically, in IZ and MZ, FOXA2+, LMX1A+, and NURR1+ cells, which are canonical midbrain-specific factors, were expressed together with TH (tyrosine hydroxylase), a dopaminergic neuron marker, confirming that they were true midbrain-type dopaminergic neurons (Fig. 7 N, O, P).

Midbrain type neural stem cells (Og-NSCs) from midbrain organoids separation

When used as a cell treatment for Parkinson's disease, a technology for separating and culturing organoid-derived midbrain type neural stem cells (Og-NSCs) as a more evolved form of neural stem cells was developed. As a previous study, an attempt was made to extract neural stem cells from organoids during 10 to 35 days of development, but the efficiency was not high. By increasing the ratio of neural stem cells in the organoid, it is believed that it is possible to secure an appropriate amount of cells, and the protocol was modified in an effort to increase the amount of cells.

(1) Since the amount of stem cell population is automatically reduced when differentiation is continuously induced, the final developmental differentiation protocol from day 10 was stopped and improved in the direction of proliferation, (2) neural stem cell-specific from day 17 It was modified by adding bFGF, which can be extended to , (3) by increasing the processing time of midbrain-specific factors (SHH/Purmorphamine/CHIR999021) (from day 1 to day 18), that is, the process of patterning the midbrain was increased to induce proliferation of midbrain-specific neural stem cells. (4) Unlike the conventional method, the FGF8b treatment period started from day 7 (FIG. 8A).

Through the control of such a detailed time period, the occurrence of the hypothalamus, which is prone to contamination during midbrain induction, can be reduced, and the expression of EN1, one of the specific markers, can be significantly increased ( FIG. 8 ). By applying these various modifications, growth and culture were carried out in a 2D environment on the 18th day of induction of mesencephalic organoid generation. Human vitronectin-coated plates were cultured with bFGF-supplemented hN2 medium. After isolation, the cells contained 84-97% of organoid-derived neural stem cells (Og-NSCs) (expressing neural stem cell markers SOX2 and NESTIN, and FOXA2, which is very important for initial acquisition of dopaminergic neuronal characteristics as an OTX2 mesencephalic marker, It was confirmed by the expression of LMX1A (FIG. 1 B, C). By confirming the expression of PAX2 and PAX5 factors around the neural stem cells, it was confirmed that they developed into true mesencephalic cells (FIG. 1 B). It was observed that (Og-NSCs) can proliferate while faithfully maintaining their properties during subculture through the expression of midbrain-specific markers such as FOXA2+ and LMX1A+ (FIG. 12) (FIG. 1D) VM type in 2D hESC culture To prepare NSC.Treatment of medium conditioned by VM-like astrocyte conditioned medium (ACM) was important for cell survival and midbrain marker expression during cell passage (FIG. 9A).ACM treatment and described above With modifications to enrich the NSC population, we were also able to prepare scalable FOXA2+/LMX1A+VM-NSCs from the differentiation of hESCs in conventional 2D culture (Figure 9).

Organoid-derived mesencephalic neural stem cells (Og-NSCs) can be differentiated into complete dopaminergic neurons that fully express mesencephalic markers through the final differentiation process.

Through the final differentiation process, mesencephalic Og-NSCs can be efficiently differentiated into complete dopaminergic neurons (FIG. 1E, F). Og-NSC-derived dopaminergic neurons simultaneously expressed FOXA2 (95%), LMX1A (96%), NURR1 (91%), and EN1 (86%) along with TH, a dopaminergic neuron marker, as a mesencephalic marker. . On the other hand, in the conventional 2D culture environment, the expression of midbrain factor was as low as 44-61%. (FIG. 9E, F, and FIG. 10). Moreover, this organoid-derived mesencephalic neural stem cell isolation protocol is applicable to a variety of human embryonic stem cells and dedifferentiated stem cells (FIG. 1G and FIG. 11). The success rate of 111 out of 117 experiments performed with 8 human pluripotent stem cells (3 hESCs and 5 hiPSCs) showed a fairly high success rate. On the other hand, the 2D method was much more difficult to apply, and the success rate of 28 out of 63 was recorded (H in FIG. 1 ).

The H9 hESC line showing the highest midbrain factor expression was mainly used throughout this study. This suggests that the protocol developed from organoids is very stable, and only a small amount undergoes the process of apoptosis during general processes such as cell isolation, proliferation, and differentiation. This, in contrast to the 2D method, showed a significant percentage of cell death during the procedure. In fact, in culture, ethidium heterodimer-stained dead cells (I in Fig. 1), cleaved caspase 3+ dead cells (J in Fig. 1) and Annexin+/PI+ dead cells analysis through FACS analysis after cell separation were also analyzed. A relatively small number of stable results were shown (FIG. 1K). Senescent cells (pre-apoptotic stage) represented by β-galactosidase+ cells are also significantly less in Og-NSC culture (FIG. 1L). Compared to the existing protocol, the newly secured protocol can more stably secure midbrain type neurons.

Og-NSC cultured cells can be used as platform cells for drug development

Og-NSCs are patterned in the midbrain type and can be differentiated into complete dopaminergic neurons after differentiation and can maintain their characteristics for more than 5 passages. (FIG. 1D and FIG. 12A, B), arithmetic, it is possible to secure Og-NSCs of 7,260 vials (9,075 culture dish) from one culture dish (FIG. 12A). In addition, it can be stored in a nitrogen tank without loss of its function. (Fig. 12C). As a result of this, it is actually being used with a leading pharmaceutical company (Chong Kun Dang Pharm., Seoul, KOREA) to develop a candidate Parkinson's disease drug (FIG. 13). The scalable and storable properties of the Og-NSC culture system, in cooperation with a pharmaceutical company (Chong Kun Dang Pharm., Seoul, KOREA), were made to produce mDA neurons from hESC (H9) and PD-hiPSC (PD-ips1) lines. was used to undergo a large-scale screening of candidate chemicals to develop PD drugs (Figure 13).

Og-NSCs in an undifferentiated state perfectly express an appropriate mesencephalic neural stem cell marker (FIG. 1B, C and FIG. 14A, E, F). Prior to differentiation, no differentiation neuronal/DA neuronal markers were expressed (MAP2, TH) ( FIGS. 14B and 14C ). After induction of the final differentiation, the expression of the proliferation marker Ki67+ is reduced, and morphological changes such as neurites increase due to the characteristics of neurons occur ( FIGS. 14A-C ). As in the developmental stage of actual mesencephalic neural stem cells, NURR1, a specific marker, starts to be expressed from the progenitor cell stage and continues to be expressed until after differentiation. (from day 2 of differentiation to the final stage of differentiation) (Fig. 14D). Actually, in accordance with the biological rhythm of development in the midbrain, FOXA2 and LMX1A were continuously expressed, and after differentiation, LMX1A+ expression showed a tendency to slightly decrease (FIG. 14E, F). In the case of NURR1, FOXA2, and LMX1A, which are important markers of midbrain cells, they were well expressed overlaid on the terminally differentiated dopaminergic neurons (FIG. 1E-G). These findings collectively indicate that in vitro NSC differentiation recapitulates physiological mDA neuron development in VM, and may be useful as an in vitro model to study human mDA neuron development that did not exist.

Analysis of transcriptional regulation characteristics of Og-NSCs using bulk and single cell RNA sequencing

To analyze the molecular biological transcriptional regulation of Og-NSCs, RNA sequencing (RNA-seq) analysis was performed. Organoid-derived midbrain neural stem cells (Og-NSCs) and neural stem cells (2D-NSCs) generated by the conventional differentiation method were compared and analyzed for differences in transcriptional regulatory factors with those of human fetal and adult brain tissues (Carithers, LJ, Ardlie, K., Barcus, M., Branton, PA, Britton, A., Buia, SA, Compton, CC, DeLuca, DS, Peter-Demchok, J., Gelfand, ET, et al. (2015). Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project. Biopreserv Biobank 13, 311-319.; Jo, J., Xiao, Y., Sun, AX, Cukurolu, E., Tran, HD, Goke, J. , Tan, ZY, Saw, TY, Tan, CP, Lokman, H., et al. (2016). Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons. Cell Stem Cell 19, 248- 257.). Previous reports (Jo, J., Xiao, Y., Sun, AX, Cukuroglu, E., Tran, HD, Goke, J., Tan, ZY, Saw, TY, Tan, CP, Lokman, H., et al. Organoids were remarkably similar to human fetal brains (FIG. 2A, B). Og-NSCs are similar to midbrain type organoids and retain their characteristics (Spearman's correlation co-efficiencies: 0.79-0.97) and are also similar to fetal midbrain (0.70-0.85), so they are actually extracted as neural stem cells. It can be seen that the obtained Og-NSCs retain their properties even in the process of isolation and subculture.

In order to confirm the individual characteristics of the neural stem cell community, single-cell RNA-seq was performed (Og-NSCs: total 13,793 cells analyzed; 2D-NSCs: 12,166 cells ;. On average, there were 3.8 × 10 ⁵ post -normalization reads per cell and 2,227 genes detected in each individual cell) On average, 2,227 genes were detected in each individual cell and 3.8 × 10 ⁵ reads after normalization per cell. As expected, ~99.7% of Og-NSC and conventional 2D-NSC cells expressed neural stem cell markers NESTIN, VIMENTIN, NCAM1, SOX2, NOTCH1, and MUSASHI1 (FIG. 15A). The markers of pluripotent cells, POU5F1, NANOG, and KLF4, were expressed in only 0.09%, 0.45%, and 0.91% of Og-NSC cells, and even the expressed cells had insignificant expression levels (average unique molecular identifiers (UMI) were <0.005). ) (FIG. 15B). Although most of them are neural stem cells, the conventional 2D-NSCs showed a relatively high expression of pluripotent cells (0.19%, 2.69%, and 1.24%, respectively, with an average UMI of <0.031). Since neither Og-NSCs nor 2D-NSCs have cells expressing the three markers at the same time, this shows that only neural stem cells can be isolated and cultured. Through FACS analysis, it was confirmed that the expression of TRA-1-60 and SSEA-4 was almost absent (FIG. 15C). Previously, senescent cells (stage before apoptosis) were also analyzed to be significantly less in Og-NSC culture (FIG. 1L), and cells expressing apoptosis genes were also very small (FIG. 15D). In particular, compared with cells of 2D-NSC, a very low proportion of cells of Og-NSC expressed p16/INK4a, which resulted in cell aging/senescence and brain (Molofsky, AV, Slutsky, SG, Joseph, NM, He, S ., Pardal, R., Krishnamurthy, J., Sharpless, NE, and Morrison, SJ (2006). Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during aging. Nature 443, 448-452.; Nishino, J., Kim, I., Chada, K., and Morrison, SJ (2008).Hmga2 promotes neural stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf Expression. Cell 135, 227-239.) and other tissues (Sousa) -Victor, P., Gutarra, S., Garcia-Prat, L., Rodriguez-Ubreva, J., Ortet, L., Ruiz-Bonilla, V., Jardi, M., Ballestar, E., Gonzalez, S. ., Serrano, AL, et al. (2014).Geriatric muscle stem cells switch reversible quiescence into senescence.Nature 506, 316-321.;Wang, J., Lu, X., Sakk, V., Klein, CA, and Rudolph, KL (2014).Stem cell recovery in Senescence and apoptosis block hematopoietic activation of quiescent hematopoietic stem cells with short telomeres. Blood 124, 3237-3240.) It is an important characteristic for the deterioration of ability.

The Og-NSCs cell population analyzed through t-SNE (t-distributed stochastic neighbor embedding) analysis can be classified into three clusters ( FIG. 2 CE ). To confirm the characteristics of each cell cluster, GO (gene ontology) analysis of the gene group with increased expression was performed. (UMI>1, log ₂ FC>0.5, P<0.05) (FIG. 2F), in the case of cluster 3, 25% (Og-NSC ) and 12% (2D-NSC) (FIG. 2D) accounted for However, as a result of genetic analysis, it is a group of cells that have already been destined to be nerve cells, and contains a significant number of differentiation factors. In the case of the overexpressed gene group in cluster 1, many gene groups important for the development of substantia nigra (SN), where dopaminergic neurons are generated, are expressed (ENO3, RSPO2, FGF9, LDHA, NR4A2, INA, LMX1A, OTX2, HSPA5, SYNGR3, RAD1, UCHL1, EN1, NDUFS3, and SEC16A), and thus the cluster 1 midbrain is the most complete cell group. In particular, 71% of Og-NSCs cells and 13% of existing 2D-NSCs cells belong to cluster 1. The results of this genetic analysis also showed that the Og-NSC culture had a much more mesencephalic pattern, and it was possible to stably secure a cell population without apoptosis and without contamination of pluripotent cells.

Through GO and KEGG analysis, bulk and single cell RNA-seq data (Og-NSCs vs 2D-NSCs) were analyzed in more detail (Fig. 2H, I). According to the seq data of two methods (compared to 2D-NSCs), the gene group that is expressed relatively in Og-NSCs is a gene group that emphasizes the importance of stem cell development and maintenance of function due to cell adhesion and ECM. The expression of this gene group, which is highly expressed by the seq data of both methods, was studied by unsupervised hierarchal clustering (FIG. 2J) and its special function was also described (Table 5).

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Also, as a group of genes that are specifically expressed a lot in the Og-NSC cell group, 'SN development' and 'negative regulation of cell death' (I in FIG. 2) related to midbrain patterning. In the analysis of single cell RNA-seq analysis, it was found that the gene group related to mitochondrial biological function was largely regulated in the Og-NSCs group (mitochondria function, ER and protein folding, and autophagy), which resulted in cell aging and death. The importance of mitochondria can be emphasized because mitochondria are closely related to the organ that is typically affected in the normalization of neurodegenerative diseases closely related to . This data can be inferred by KEGG analysis as it is ranked as the highest change in 'Parkinson's disease', 'Huntington's disease', and 'Alzheimer's disease', which are representative neurodegenerative diseases (I in FIG. 2).

The function of mitochondria is very important in the differentiation of human embryonic stem cells and induced pluripotent stem cells. In particular, the mitochondria of neural stem cells are known to be very important in the progression and pathogenesis of neurodegenerative diseases. Mitochondrial genes were all regulated compared to cells induced by the conventional method in two different bulk and single-RNA-seq analyzes of Og-NSCs, including those related to mitochondrial generation and dynamics (FIG. 3A). Similar to genetic analysis, it can also be analyzed with Mitotimer, and it was found that the Og-NSCs cell group was much healthier and functionally superior to the conventional 2D-NSCs ( FIG. 3B ). The healthy and functional superiority of Og-NSC can also be seen by mitochondrial ROS analysis (MitoSox, FIG. 3C). In addition, the mitochondrial membrane potential was also high in the Og-NSC group, showing superiority in all aspects (JC-1, FIG. 3D). With improved mitochondrial turnover and antioxidant capacity, mDA neurons differentiated from Og-NSCs exhibited apoptosis (Fig. 3E) and neurite degeneration (Fig. 3E) induced by a variety of mitochondrial toxins than those derived from 2D-NSCs. 3 showed greater resistance to F).

Differentiation-induced dopaminergic neurons from Og-NSCs mDA neurons coexist with astrocytes, resulting in improved neuronal function and maturation

The transcriptional regulation of genes actually shows early changes before the shape of the cell population changes. For example, in the case of actual gene analysis (in differentially expressed genes (DEGs)), changes that can be seen from neural stem cells can be seen from the embryonic stem cell stage. In bulk RNA-seq analysis, genes related to neuronal maturation were highly expressed in Og-NSCs (vs 2D-NSCs) cells (FIG. 2H and FIG. 3G). As in the gene expression profile, compared with their 2D counterparts, differentiation-induced mesencephalic dopaminergic neurons in Og-NSCs showed a more mature morphology after neuronal differentiation (Fig. 3H, J), and synapse formation ( 3I, K), action potential-induced Ca ²⁺ dynamics (LO of FIG. 3), and pro-synaptic dopaminergic neuron function (FIG. 3P) evaluated by depolarization-induced dopamine secretion. looks in shape

In all existing hPSC-mDA differentiation protocols, mDA neurons are generated without astrocyte differentiation. Similarly, no astrocyte differentiation was found in cultured 2D-NSCs. (R, S in Fig. 3). Interestingly, 17% of the subpopulation of Og-NSCs cluster 1 (the SN-generating cell population of the midbrain, a cluster with higher SN developmental gene expression) contained genes related to glia development (EIF2B5, MT3, PLP1, CDK6, SCL1A3, HES1). , HES5, and SOX8 ) showed a characteristic of high expression in common ( FIG. 3 Q ). It was shown that Og-NSC cultures contained a VM (SN)-type glia precursor population. Consistently, some of the cells in Og-NSC culture expressed CD44 ( FIG. 3S ), which is specifically found in neurogenic precursors ( FIG. 3R ) that differentiate into astrocytes upon peripheral differentiation. These cells express CD44, a marker of astrocytes (FIG. 3R), and can be expected to differentiate into astrocytes during final differentiation in the future (FIG. 3S). It can be seen that, like the actual pattern of midbrain development, mDA neurons are generated by mixing with GFAP + astrocytes ( FIG. 3S ).

Therefore, mDA neurons were mixed with GFAP + astrocytes in differentiated cultures as in the midbrain in vivo ( FIG. 3S ). In addition to the general neurotrophic action of astrocytes, VM-type astrocytes are known to exert specific dopaminotrophic functions superior to astrocytes from other brain regions.

Considering the cytokine secretion ability of astrocytes based on previous reports, astrocytes can be expected to have a positive effect when dopaminergic neurons are generated. Based on this information, adjacent astrocytes can exert neurotrophic support for mDA neuron maturation, survival, presynaptic function and midbrain-specific factor expression, as shown in E-P of FIG. 3 and E-L of FIG. 1 .

Dopaminergic neurons differentiated from Og-NSCs exhibit less oligomerization and pathological propagation of α-syn.

In order to prevent the spread of α-synucleinopathy existing in the original patient’s brain to transplanted cells, and for the long-term success of Parkinson’s disease cell therapy, the It is important to secure Pathogenic propagation of α-syn is associated with intercellular migration of α-syn, and α-syn aggregates are also associated with migration into transplanted cells. To measure the intercellular migration ability of α-syn, a dual chamber system (Choi, YR, Cha, SH, Kang, SJ, Kim, JB, Jou, I., and Park, SM (2018b). Prion-like propagation) of alpha-Synuclein Is Regulated by the FcgammaRIIB-SHP-1/2 Signaling Pathway in Neurons. Cell reports 22, 136-148) were analyzed in Og-NSCs and 2D-NSCs, respectively. GFP-labeled α-syn (protein overexpressed in SH-SY5Y neuronal cells) was placed on the chamber, and cultured Og-NSC (2D-NSC)-derived neurons were placed underneath to observe expansion of uptake after protein migration. As a result (FIG. 4A), the fluorescence value of GFP-labeled α-syn was observed to be significantly lower in Og-NSC-derived neurons (FIG. 4B).

The pathological spread of α-syn into transplanted cells as well as α-syn migration as well as other important issues were analyzed as toxic α-syn aggregates. The degree of aggregation was compared and analyzed by dividing the cell into an environment in which the amount of α-syn inside the cell increases or when α-syn is introduced from the outside (exogenous α-syn fibril seed).

Og-NSCs and 2D-NSCs cell groups were infected with a lentivirus overexpressing α-syn, _{and oxidative stress was induced with H 2} O ₂ after differentiation of dopaminergic neurons, and toxic α-syn aggregation and diffusion were analyzed. Phosphorylated α-syn at serine 129 (p129-α-syn), which may be toxic α-syn, but GFP+ α-syn shift was low in Og-NSC ( FIG. 4C ). Therefore, toxic α-syn aggregation was also observed less in the differentiated Og-NSC cell group (Fig. 4D, left). By injecting α-syn fibril (PFF), conditions were prepared for external α-syn inflow (exogenous α-syn fibril seed), and the observed results showed that toxic α-syn aggregation was higher than that of conventional 2D-NSCs. As it can be seen (Fig. 4D, right), toxic α-syn aggregation under various conditions was observed to be low in the Og-NSC cell group. Next, toxic α-syn aggregation was observed using a Bi-FC (bimolecular fluorescence complementation) system. This system is a system for observing α-syn by showing intense fluorescence when the amino (N) terminus (V1S) or carboxy (C) terminus (SV2) Venus fragment is expressed and aggregated, respectively (FIG. 4E, F). Oxidative stress and mitochondrial destruction are known to cause protein misfolding and aggregation of α-syn. The relative resistance of Og-NSC-derived dopaminergic neurons to α-synucleinopathy can be attributed to low oxidative and mitochondrial stress (FIG. 3 BF). In addition, the inclusion of astrocytes in the differentiated Og-NSC cell population can also reduce toxic α-syn aggregation as an original function of astrocytes, thus contributing to the reduction of α-synucleinopathy. Overall, Og-NSCs-derived neurons exhibit relatively suppressed toxic α-syn diffusion, which was confirmed by the results of intracellular and intracellular α-syn studies.

Finally, Og-NSCs or 2D-NSCs were implanted in living brain tissue and α-syn diffusion and aggregation were observed in the real brain environment. Parkinson's disease caused by α-syn disease was induced by injecting human-specific α-syn overexpressing AAV virus and PFF into the rat striatum, and 2 weeks later, Og-NSCs and 2D-NSCs were transplanted into the model animal striatum, respectively, and the living brain Differences within the environment were observed. As a result of analysis of the transplanted tissue after 1 month, under severe α-synucleinopathy, the existing 2D-NSC-derived transplant site had almost no surviving dopaminergic neurons (Fig. 4G, lower panel), α-syn+ and As Lewy bodies co-expressed with p129-α-syn+ were observed and neurites were hardly differentiated, it was shown that the existing cell population is not suitable as a cell therapy. On the other hand, in the case of the newly developed Og-NSC cell group-derived transplantation site, the shape of the nerve cells was much healthier and there was no severe neurite degeneration. (G in Fig. 4, upper panel). Moreover, less toxic α-syn+ (p129-α-syn+) aggregation was observed at various sites in neurons. In patients with Parkinson's disease, even after transplantation of cell therapy, the spread of patient-derived α-syn into the transplanted cells (host-to-graft α-syn propagation) is severe during the patient's survival (11-16 years after transplantation). raised as an issue In the case of animal models, α-syn diffusion aggregation is observed at a much faster period of about one month. Although it does not fully reflect the patient's brain environment, the results of in vitro and in vivo assays show that Og-NSCs-derived cells are toxic. It shows that α-syn is more resistant to diffusion and aggregation.

Og-NSC transplantation provides healthy dopaminergic neurons in an animal model of Parkinson's disease

Finally, the possibility of Og-NSCs-derived cell population as a cell therapy was observed after transplantation in a model animal system induced by Parkinson's disease with 6-OHDA (6-hydroxydopamine). Recently, there has been a study on genes that can measure the success of human embryonic stem cell-derived cells after transplantation. As expected, as a result of single cell RNA-seq analysis, it was found that this gene group was expressed relatively highly in the newly developed Og-NSCs cell group. (Compared to the conventional 2D cell population) (FIG. 5A). Synaptic plasticity of transplanted dopaminergic neurons is very important for future cell survival and success of transplantation. To analyze their function as neurons, GCaMP6s (calcium indicator protein, which emanates green fluorescence by Ca++ binding) with GFP fluorescence was expressed in the Og-NSCs cell group, and then transplanted into the brain striatum. The function of the nerve cells was observed by two-photon endomicroscopy one month after transplantation without special stimulation to the nerve cells. (B, top of Fig. 5), it was possible to see the signal transduction through the spontaneous neurons in the transplanted Og-NSCs cells. Ca ²⁺ transients (ΔF/F) show an action potential frequency of 2-5Hz of neurons (Fig. 5B, bottom), which is a dopaminergic neuron that originally occurred in SNpc (substantia nigra pars compacta) of brain tissue. It is similar to that. To further confirm synaptic plasticity by transplanted Og-NSC cells, we constructed Og-NSCs from a group of human embryonic stem cells expressing the (Dll1; pDll1-GFP-hESC) gene with GFP attached to delta-like canonical Notch ligand 1. And, since it is fluorescently expressed only in differentiated neurons, only cells expressing fluorescence were analyzed after cell transplantation into a Parkinson's disease model animal. After one and a half months of patch clamp analysis, action potentials (FIG. 5C) and spontaneous excitatory postsynaptic currents (sEPSCs) (FIG. 5D) were observed in transplanted GFP-expressing neurons. The synaptic plasticity of transplanted neurons was not significantly different from that of normal cells without contralateral lesions (FIG. 5E, F), which showed that even after transplantation of Og-NSCs, neurons had a functionally perfect morphology. ^[18 F]FP-CIT PET scan of dopamine uptake by dopamine transporters to see whether transplanted dopaminergic neurons are fully matured and have perfect synaptic functions such as dopamine secretion. Although the absorption capacity (2% of the intact hemisphere) was relatively restored to 40% of the normal Og-NSCs cell population after 5 months of transplantation (FIG. 5G, H).

As a result of transplanting the Og-NSCs cell group into an animal model of Parkinson's disease, and performing a behavioral study, the amphetamine-induced rotational behavioral analysis showed a consistent recovery effect. At ∼6 months of transplantation, rotation scores of 11 of 14 animals were >50% before transplantation. In the case of 8 animals observed 6 months after transplantation, all animals showed >79% results ( FIG. 6A ). In the transplanted animals, the recovery effect was also shown in the stepping test (FIG. 6B) and the cylinder test (FIG. 6C).

As a result of analyzing Og-NSCs 6 months after transplantation into a model animal, healthy dopaminergic nerve cell tissue was confirmed at the transplant site (graft volume: 4.50±1.56 mm ³ , TH+ cells: 9031±3773 cells, n=8 from 6) rounds of independent transplantation experiments, FIG. 6 DO). No specific proliferation was observed at the site of transplantation. This is the same as the differentiation into Og-NSCs-derived dopaminergic neurons in vitro (H in Fig. 2 and GI in Fig. 3). In particular, the transplanted dopaminergic neurons synapse with cells in the original brain tissue. to mature and extend normal neurites (FIG. 6D). Although midbrain-specific factors are very important for the survival and maturation of dopaminergic neurons, it is known that their expression is not maintained well after transplantation. For this reason, most of the existing papers did not show the expression of the entire mesencephalic factor in the tissue after transplantation, but showed the image of the maximum FOXA2/TH+ expressing cells. Expresses all midbrain factors. Even 6 months after transplantation, 96.7% (3,106/3,196, FOXA2), 93.8% (3,338/3,549, LMX1A), 95.0% (3,496/3,678, NURR1), and 91.5% (2,989/3,274, EN1) of major midbrain factors were present. It is expressed in common with TH+, a marker of dopaminergic neurons. (from 8 animals; EI in FIG. 6). In particular, they express GIRK2 (Fig. 6J), a marker of A9 nigral dopaminergic neurons, and transplanted dopaminergic neurons have a mature form extending neurites in multiple directions, as are the main characteristics of A9 nigral dopaminergic neurons ( 6 EG, H, and FIG. 6 P), it is shown that mature neurites are stretched even with the original brain tissue cells. (Fig. 6D). Human GFAP (hGFAP)-expressing astrocytes are clustered around the transplanted dopaminergic neurons (Fig. 6K), which means that the transplanted Og-NSC-derived astrocytes, dopaminergic neurons, and neurites extend It is shown that it forms synapses and helps the maturation of cells. It is the same as the previous co-transplantation of astrocytes showed similar effects. To see the effect in primates, the Og-NSCs-derived cell population was transplanted into the striatum of two adult Filipino monkeys, and one month later, mature dopaminergic neurons co-expressing FOXA2 and NURR1, important midbrain markers, were identified. (Fig. 6P).

discussion

Cultured organoids may be a source of donor transplantation in regenerative medicine, in addition to their applications in tissue development and human disease modeling studies. However, transplantation of brain organoids will not be applicable for the treatment of brain disorders, as transplantation of brain organoids into deep brain regions is not feasible without damaging the host brain tissue. Moreover, the therapeutic outcomes of transplanted brain organoids should be able to establish new neural networks in which self-organized structures in transplanted organoids interact with the host brain in a highly precise manner. In contrast, transplanted organoids from other tissues (liver, intestine, kidney, pancreas, etc.) may exert therapeutic functions as isolated functional units (liver buds, pancreatic islets, renal nephrons, etc.) after transplantation. Therefore, although organoid transplantation may have the potential to treat other extra-CNS disorders, direct transplantation of brain organoids to treat brain disorders is unreasonable.

All tissue development is achieved through sequential generative processes, proliferation of tissue-specific stem/progenitor cells and their differentiation into tissue-specific cells. Cultured tissue-specific stem/progenitor cells can provide donor cells for regenerative medicine as well as bioassay platforms for developmental studies and drug screening. However, the induction of NSC cultures with appropriate VM-patterning from hPSCs was not achieved in previous studies. We realized that the difficulty in preparing VM-specific NSC cultures was due to the instability of VM region specificity in the culture. Since maintenance of VM-specific marker expression is very sensitive to cell density, it is easily lost during the cell separation and replating process required for NSC preparation ( FIG. 9A ).

Therefore, hPSC-mDA neuron protocols were developed with very high cell densities or specific plate coating materials such as Laminin-511 or 111 (Biolamina, LN-511, LN-111). Various cell dissociation conditions (Acutase, collagenase, Ca++, Mg++-free HBSS), cell survival cytokines (TGFβ, doxycycline, cAMP, ascorbic acid, Y27632), chemicals that induce open epigenetic conditions (5-azacytidine, VPA) ), TSA), β-estradiol or DAPT (Notch inhibitor) did not solve the problem.

Recent studies (Song, JJ, Oh, SM, Kwon, OC, Wulansari, N., Lee, HS, Chang, MY, Lee, E., Sun, W., Lee, SE, Chang, S., et al (2018).Cografting astrocytes improves cell therapeutic outcomes in a Parkinson's disease model.The Journal of clinical investigation 128, 463-482.), the present inventors found that factors secreted from cultured astrocytes, especially factors derived from VM tissue It was observed to exert a strong trophic effect to promote cell survival and midbrain-specific factor expression in primary NSC and mDA neuron cultures.

Therefore, it was possible to develop the 2D-NSC culture protocol used in this study through the treatment of conditioned medium prepared from cultured astrocytes (ACM) (Fig. 9). However, the induction of 2D-NSC culture still had the aforementioned stability and reproducibility issues. In contrast, NSCs with stable VM marker expression were readily isolated and cultured from rodent embryonic VM tissues, stimulated to isolate human VM-specific NSCs from hPSC-derived midbrain organoids, and placed in an environment similar to the physiological structure of the embryonic brain. It has an established structure and region specificity.

Organoid-based formulations have had great success with respect to stability of VM-patterns in NSC cultures with faithful midbrain-specific marker expression as well as general culture stability with very low levels of apoptosis, senescence, oxidative and mitochondrial stress. all. Another notable observation was that Og-NSCs contain an astrocyte progenitor cell population with a VM-specific gene expression profile that differentiates into astrocytes intermixed with mDA neurons. In contrast, mDA neuronal differentiation was induced without astrocyte differentiation in all existing methods for hPSC-mDA differentiation, and consequently there was no astroglia in differentiated mDA neuronal cultures. Therefore, in the absence of astroglial support, neural maturity and function could not be expected to be identical to that of differentiated mDA neurons in their in vivo counterparts. Indeed, insufficient maturation and function have been proposed as critical drawbacks in the utility of hPSC-differentiated mDA neurons in in vitro disease modeling and therapy. In contrast, under nutritional support from mixed astrocytes, Og-NSC-derived mDA neurons exhibited improved synaptic maturation, functionality, resistance to toxic insult, and long midbrain specific factor expression in vivo and in vivo after transplantation. showed an improved set. All these properties of Og-NSC ultimately contribute to superior long-term therapeutic efficacy after transplantation in a reproducible manner. Furthermore, it has been shown that organoid-derived NSCs are a scalable source of cells needed for the preparation of donor cells for therapy as well as bioassay platforms for drug screening, developmental studies, and disease modeling.

Based on the present invention, organoid-based methods may become a next-generation common strategy to produce tissue-specific stem/progenitor cells with strong therapeutic potential for CNS as well as non-CNS disorders.

In other words, there is no doubt that the cells of organoids are healthier compared to the two-dimensional culture and that excellent results can be obtained during cell therapy. Since it is not preferred, it can be said that the present invention is the most appropriate model for a cell therapy product for the nervous system.

Example 2: Differentiation from human pluripotent stem cells into cortical neural stem cells using cortical organoids

[Experimental process]

Human pluripotent stem cell (hESCs/hiPSCs) culture

hESCs and hiPSCs were cultured based on the hESC research guidelines approved by the institutional review board (IRB) of Hanyang University (Seoul, Korea).

The hESCs and hiPSCs used in this experiment are shown in Table 6 below.

hESC line Name used in this paper original name Karyotype Establishing institute HSF6 UC06 Female (46, XX) UCSF H9 WA09 Female (46, XX) Wi cell HUES6 HUES6 Female (46, XX) Harvard university (HSCI iPS Core) hiPSC line Name used in this paper original name Source
(Human) Reprogramming factors Methods Establishing institute Retro-1 Rv-hiPS 01-1 Newborn fibroblast OCT4, SOX2, KLF4, MYC retrovirus Harvard Epi-ips episomal-ips1 Skin OCT4, SOX2, KLF4, shp53 Episomal vector Hanyang Univ. (Our own) CMC-ips#11 CMC-hipsc-011 bone marrow blood OCT4, SOX2, KLF4, MYC Sendai virus Catholic University. (Korea NIH SCRM) PD-ips1 PD patient derived ips-1 Skin OCT4, SOX2, KLF4, shp53 Episomal vector Hanyang Univ. (Our own) PD-#22 Corr. PD-ips#22 Skin OCT4, SOX2, KLF4, shp53 Episomal vector and genome edited Hanyang Univ. (Our own)

For proliferation and maintenance of undifferentiated hESCs/iPSCs, in a CO ₂ incubator set at 37° C. without a feeder layer, using mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) ^{on Matrigel TM} or vitronectin (Human; Gibco) Fisher Scientific, Waltham, MA) (Gibco A31804; 0.5 ug/cm ² )-coated 6 cm dishes (Thermo Fisher Scientific, Waltham, MA), medium change was performed daily. The undifferentiated stem cells were maintained in differentiation capacity by daily medium replacement, and subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.

Fabrication of 3D cortical organoids

Human embryonic/induced pluripotent stem cells were removed from the dish using Acutase, placed in a neuronal differentiation induction medium, and injected into a low attachment 96-well round bottom plate (Corning, Corning, NY) with 150 μL of 10,000 cells per well (differentiation 0). day car). The composition of the nerve cell differentiation induction medium is shown in the table below. For the first seeding, KSR-hES media (DMEM 40ml, KSR 10ml, 2-mercapto 20ul, MEM-NEAA 500ul, GlutaMAX 500ul), bFGF (4 ng/ml), ROCK inhibitor Y27632 (20 μM), Doxycyclin 1ug/ml put it in Y27632 was treated only for 24 hours on the first day. On day 2, retinoic acid-free B27, Insulin (15.6ul/50ml), SB431542 (10 μM), LDN (10 μM), and A-83 (10 μM) are added to induce full-fledged nervous system differentiation. Between 2 and 3 days, only 1/2 of the basal medium was used as N2: Neurobasal (1:1, Gibco), and then the entire basal medium was used as N2: Neurobasal (N2: Neurobasal ( 1:1, Gibco). The entire medium was replaced whenever the medium composition was changed. From day 8, bFGF (20 ng/ml) and EGF (20 ng/ml) were added.

Organoids were formed by culturing according to the medium shown in the table below until day 18.

Separation of cortical neural stem cells and astrocytes from cortical organoids and differentiation into neurons and astrocytes

On the 18th day, the organoids were digested with Acutase at 37°C for 7 minutes, then chopped with a 30-gauge needle and spread on vitronectin-coated 6 cm plates (Corning, cells from 24 organoids/plate). At this point, the cells were at the Neural Stem Cell (NSC) stage, proliferative and passaged every 5-7 days. The medium used for NSC culture is an N2 expansion medium, and the composition is as follows (1X N2 supplement containing, 200 μM ascorbic acid, 20 ng/ml EGF, 20 ng/ml bFGF, and 1 μg/ml doxycycline). The medium was changed daily and Y27632 was treated at a concentration of 5 μM for one day at the first conversion to 2D culture and at each subculture. Final differentiation into neurons was induced with N2 differentiation medium (10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid).

Isolation and culture of cortical astrocytes from cortical organoids

If the passage of the created cortical-type Og-NSCs is further extended to match the actual brain development stage, it is possible to culture Og-strocytes mainly containing astrocytes after passage 9 without changing the medium composition. Generations 9 to 11 can be used in a healthy state. As with cortical Og-NSCs, the growth medium is N2 expansion medium, and the composition is 1X N2 supplement containing, 200 μM ascorbic acid, 20 ng/ml EGF, 20 ng/ml bFGF, and 1 μg/ml doxycycline. The medium was changed daily and Y27632 was treated at a concentration of 5 μM for one day at the first conversion to 2D culture and at each subculture. Like organoid-derived astrocytes of the midbrain (see Example 3), cells in the proliferative stage with an older passage are used for transplantation studies. For analysis, the high passage cell population as the final differentiation medium above assumes the characteristics of astrocytes.

Confirmation of differentiation into cortical neurons and astrocytes

Cortical-type neurons were prepared, and through this, cortical-type patterning was preferentially performed to secure astrocytes through several passages. After induction of differentiation of the nervous system from human embryonic stem cells, the confirmation thereof was preferentially confirmed by patterning, and the expression of cortical markers such as PAX6 and FOXG1 was confirmed. In addition, when the astrocytes were developed through several passages, expression of astrocytic factors such as GFAP, AQP4, ALDH1L1, and GLAST was confirmed. Identification of the marker is possible through a process such as simultaneous expression confirmation using -real time PCR or immunostaining after RNA extraction.

[result]

3D Organoid Creation

The point of the result is to confirm that the differentiation into a normal organoid form into a group of cerebral cortex cells is preferentially formed, and the differentiation into astrocytes is completed in the future. As shown in FIG. 17 , it was confirmed that the normal form of the organoid was completed, and the expression of the patterning markers PAX6 and FOXG1 was maintained even after the organoid was dismantled.

Securing cortical neural stem cells and astrocytes from the fabricated cortical 3D organoids

As in the protocol mentioned, SB431542 (10 μM), LDN (10 μM), and A-83 (10 μM) are added to induce full-scale nervous system differentiation (Fig. 16 below, cortical induction scheme in Fig. 18) and normal morphology It was confirmed as shown in FIG. 17 that organoids of To confirm this, the expression of PAX6 and FOXG1, which are cortical region patterning factors, was confirmed by RNA-real time PCR ( FIGS. 17 and 25 ). As mentioned above, through several passages, the generation of astrocytes can be induced and secured. Using immunostaining, sufficient expression of GFAP, AQP4, ALDH1L1, GLAST, etc. can be confirmed. and at the same time, it was possible to confirm the expression of the patterning factor in this cell group (FIG. 25).

Confirmation of cortical neuron and astrocyte differentiation

Nervous system patterning factors SB431542 (10 μM), LDN (10 μM), and A-83 (10 μM) were added to induce full-scale nervous system differentiation and patterned into cortical brain organoids, but The process after differentiation into astrocytes through division of neurons and multiple passages differs only in initial patterning and is not different from the process of differentiation through midbrain organoids. When compared with the differentiation of midbrain astrocytes, using immunostaining, sufficient expression of GFAP, AQP4, ALDH1L1, GLAST, etc. could be confirmed, and at the same time, expression of patterning factors in each cell group could be confirmed ( Fig. 25). It can be seen that differentiation into activated astrocytes can be completed in terms of functional and electrophysiological functions.

In this example, a method using a 3D organoid capable of producing cerebral cortex-specific neural stem cells was established. By utilizing 3D organoids, neural stem cells are extracted from tissues with the same structure as the actual living cerebrum, so it can be expected to secure more healthy and realistic cells.

Example 3: Confirmation of differentiation from human induced pluripotent stem cells into midbrain astrocytes using midbrain organoids

[Experimental process]

Human pluripotent stem cell (hESCs/hiPSCs) culture

hESCs and hiPSCs were cultured based on the hESC research guidelines approved by the institutional review board (IRB) of Hanyang University (Seoul, Korea).

The hESCs and hiPSCs used in this experiment are shown in Table 7 below.

hESC line Name used in this paper original name Karyotype Establishing institute HSF6 UC06 Female (46, XX) UCSF H9 WA09 Female (46, XX) Wi cell HUES6 HUES6 Female (46, XX) Harvard university (HSCI iPS Core) hiPSC line Name used in this paper original name Source
(Human) Reprogramming factors Methods Establishing institute Retro-1 Rv-hiPS 01-1 Newborn fibroblast OCT4, SOX2, KLF4, MYC retrovirus Harvard Epi-ips episomal-ips1 Skin OCT4, SOX2, KLF4, shp53 Episomal vector Hanyang Univ. (Our own) CMC-ips#11 CMC-hipsc-011 bone marrow blood OCT4, SOX2, KLF4, MYC Sendai virus Catholic University. (Korea NIH SCRM) PD-ips1 PD patient derived ips-1 Skin OCT4, SOX2, KLF4, shp53 Episomal vector Hanyang Univ. (Our own) PD-#22 Corr. PD-ips#22 Skin OCT4, SOX2, KLF4, shp53 Episomal vector and genome edited Hanyang Univ. (Our own)

For proliferation and maintenance of undifferentiated hESCs/iPSCs, in a CO ₂ incubator set at 37° C. without a feeder layer, using mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) ^{on Matrigel TM} or vitronectin (Human; Gibco) Fisher Scientific, Waltham, MA) (Gibco A31804; 0.5 ug/cm ² )-coated 6 cm dishes (Thermo Fisher Scientific, Waltham, MA), medium change was performed daily. The undifferentiated stem cells were maintained in differentiation capacity by daily medium replacement and subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.

3D midbrain organoid fabrication

It was prepared in the same manner as in Example 1 until the midbrain type organoid was produced. In the early development of the successfully produced mesencephalic organoids, ZO-1 and N-cadherin were expressed along with the neural stem cell markers PLZF+ and Sox2+ (FIG. 7E&F). Anterior embryonic brain markers (OTX2) and ventral VM floor plate markers (FOXA2, LMX1A) are expressed along with Nestin and Sox2, which can be called general neural stem cell markers (G-I in FIG. 7). When development is further induced, MAP2, a neuronal marker, is expressed as a layer outside the proliferation marker Ki67+ and SOX2+, and more specifically, PCNA+ (the proliferative ventricular zone; VZ), MASH1+ (VZ, intermediate zone (IZ) ) and mantle zone (MZ)), and structures specifically expressed in NURR1+ cells (IZ and MZ) were confirmed (L, M in FIG. 7). Characteristically, FOXA2+, LMX1A+ and NURR1+ cells, which are midbrain-specific factors, are also expressed in the organoid, confirming that the organoid is directly produced as a true midbrain type organoid.

Separation of midbrain neural stem cells from midbrain organoids and differentiation into neurons

hESCs/hiPSCs were removed from the dish using Acutase, placed in neuronal differentiation induction medium, and injected into a low attachment 96-well round bottom plate (Corning, Corning, NY) with 150 μL of 10,000 cells per well (Day 0 of differentiation). . The composition of the neuronal differentiation induction medium is as follows (N2: Neurobasal (1:1, Gibco) containing B27 without vitamin A (2%, Invitrogen Fisher Scientific, Waltham, MA), GlutaMAX (1%, Invitrogen Fisher Scientific), minimum essential media-nonessential amino acid (1%, MEM-NEAA, Invitrogen Fisher Scientific), β-mercaptoethanol (0.1%, Invitrogen Fisher Scientific), SB431542 (10 μM, Tocris, Bristol, UK), Noggin (200 ng/ml) , Peprotech, Rocky Hill, NJ), ascorbic acid (200 μM, Sigma-Aldrich), insulin (25 mg/L), ROCK inhibitor Y27632 (20 μM, Sigma-Aldrich, St. Louis, MO), Y27632 on day 24 time only). Sonic hedgehog (100 ng/ml, SHH, Peprotech) and purmorphamine (2 μM, Calbiochem, MilliporeSigma, Burlington, MA) were added to the neuronal differentiation induction medium from the first day of differentiation for organoid Ventral Midbrain type neural stem cell expansion. became On day 2, CHIR99021 (0.8 μM, Stemgent, Cambridge, Mass.) was added. The entire medium was replaced whenever the medium composition was changed. From day 5, SB431542 was removed and from day 7, FGF8b (100 ng/ml, Peprotech) was added. On day 11, the basal medium was completely changed to N2, and B27, GlutaMAX, MEM-NEAA, and β-mercaptoethanol were not added. The concentration of CHIR99021 added from the 11th day increased about 2 times (1.5 μM). From this point on, the organoids were transferred from a 96-well plate to a 6-well plate (low attachment, Corning) and cultured on an orbital shaker at a speed of 80 rpm. became Noggin and sonic hedgehog were removed from the 15th day, and bFGF (20 ng/ml) was added from the 17th day. On day 18, the organoids were digested with Acutase at 37°C for 7 min, torn into pieces with a 30-gauge needle, and spread on vitroxectin-coated 6 cm plates (Corning, cells from 24 organoids/plate). At this point, the cells were at the Neural Stem Cell (NSC) stage, proliferative and passaged every 5-7 days. The medium used for NSC culture was N2 growth medium, and the composition was as follows (N2 containing 10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid, 100 ng/ml FGF8b, 20 ng/ml bFGF, and 1 μg/ml doxycycline). The medium was changed daily and Y27632 was treated at a concentration of 5 μM for one day at the first conversion to 2D culture and at each subculture. Final differentiation into neurons was induced with N2 differentiation medium and the composition was as follows (10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid, and 500 μM db-cAMP).

Separation of midbrain astrocytes from midbrain organoids and differentiation into astrocytes

If the passage of the produced midbrain type Og-NSCs is further increased to match the actual brain development stage, it is possible to culture Og-astrocytic progenitor cells mainly containing astrocytes after passage 9 without changing the medium composition. Generations 9 to 11 can be used in a healthy state. As with Og-NSCs, the growth medium is N2 growth medium, and the composition is N2 containing 10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid, 100 ng/ml FGF8b, 20 ng/ml bFGF, and 1 μg/ ml doxycycline. The medium was changed daily and Y27632 was treated at a concentration of 5 μM for one day at the first conversion to 2D culture and at each subculture. Proliferating astrocytes were used for transplantation, and differentiation for research was induced with N2 differentiation medium. The composition was as follows (10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid, and 500 μM). db-cAMP).

[result]

Confirmation of 3D midbrain organoid fabrication

If the mesencephalic organoids shown as a result of FIG. 7 are prepared and passages are further increased according to the actual brain development stage, it is possible to produce mesencephalic organoids capable of differentiation into mesencephalic neurons as well as astrocytes. Therefore, the results regarding the production of mesencephalic organoids and their properties are the same as the results shown during the differentiation of neurons through the mesencephalic organoids.

Confirmation of securing midbrain astrocytes from the manufactured midbrain 3D organoid

① Direct differentiation of midbrain neural stem cells and securing of astrocytes

To induce differentiation from hESC/iPSCs into neural stem cells, midbrain-neuronal stem cells were obtained using Neuroectoderm inducers and midbrain type neural stem cell inducers for about 20 days at the embryonic stem cell stage. The secured neural stem cells are easy to proliferate and stock, and a greater number of neural stem cells can be obtained through subculture. Confirmation of proper differentiation can be confirmed through the expression rate of neural stem cell markers such as Nestin and Sox2 and midbrain-neuronal stem cell markers such as Foxa2 and Lmx1a. When this protocol is applied, differentiation into midbrain neural stem cells can be induced with a high efficiency of 95% or more as follows. In order to obtain astrocytes, several additional passages are performed. During the initial subculture, terminally differentiated cells have a higher differentiation rate into neurons than astrocytes, but the proportion of astrocytes increases as the subculture increases. The existing protocol was able to identify astrocytes upon differentiation after passage 5, but for more efficient establishment of astrocytes, the neural stem cell group of passages 9 to 11 is used as the astrocyte group. At this time, the astrocyte marker CD44 was 90% or more, and when differentiation was induced, the differentiated astrocyte marker group (GFAP, AQP4, GLAST, S100b) was expressed more than 90% as shown in FIG. 19 .

② Observation of higher quality effects of midbrain type astrocytes

As a result of in vitro characterization, it was observed that midbrain type astrocytes overexpress factors related to secretion of more useful cytokines and maintenance of neuronal function ( FIG. 20 ). In vivo results also observed that the secretion of more useful factors promotes expression in midbrain type astrocytes (FIG. 21). In addition, the influence of these astrocytes affects microglia and participates in the change to a better type of microglia, and a synergistic effect of anti-inflammatory effect can be expected (FIG. 22).

Confirmation of differentiation of midbrain astrocytes

Confirmation of protocol application using hiPSC

An experiment was conducted to see if the secured protocol was applied not only to H9 hESC but also to other types of embryonic stem cells. For this purpose, iPSCs were obtained from the Central Center for Disease Control (CMC-hiPSC-003 and 011). In the culture stage, we succeeded in adaptive culture using a Xeno-free system that does not use animal-derived components such as mouse feeder or matrigel, which were previously used for culturing human embryonic stem cells (Fig. 23), and secured a large number of cell stocks did.

As a result of applying the hiPSCs thus obtained to this protocol, it was confirmed that they were well differentiated into neural stem cells, and further, it was confirmed that astrocyte markers such as GFAP increased during subculture, such as H9 hESCs (FIG. 24).

Normal differentiation into midbrain astrocytes was confirmed ( FIG. 25 ). In other words, it was confirmed that this protocol can be applied not only to H9 hESC but also to other types of embryonic stem cells. Among the used iPSC lines, #11 is easier to differentiate and will be a very important step in securing available induced pluripotent stem cells when practically used in the future.

Efficient differentiation induction using TGF-β and LIF.

Through efficient differentiation induction using TGF-β and LIF, a technology for securing a large amount of astrocytes was secured. By treating TGF-β in the proliferation stage and LIF in the differentiation stage, it was shown that a greater amount of astrocytes were differentiated, and a higher amount of neuromodulatory factors were expressed in these astrocytes (by expression of the GFAP marker). cell formation was observed (FIG. 26). In order to confirm the amount of astrocytes, EdU, which can be marked only on dividing cells in the proliferation stage, was treated for only 30 minutes, and after complete differentiation, the increase or decrease of the progenitor cells of astrocytes was confirmed by looking at the amount expressed simultaneously with the astrocytes. As a result, it was confirmed that the amount of astrocytes increased by 1.5 times.

To see the utility of the astrocyte-derived material obtained through the final protocol, preferentially characterize the conditioned medium

- Cell characteristics analysis: To analyze the characteristics of the obtained astrocytes, the physiological activity of the mesencephalic astrocytes was measured by analyzing the physiological characteristics, and through this, it was confirmed that they are immature astrocytes suitable for securing differentiation derivatives and transplantation [ 27]. Considering that fully mature astrocytes can be easily assimilated into a bad environment in a bad environment, these immature types of cells are more suitable for transplantation.

- Functional analysis: After transplantation, it was carried out to check the function of astrocytes, and it was observed in vitro and in vivo whether it was possible to perform functions as high-quality cells in vivo after transplantation. In order to find out whether the in vitro results are suitable for improving the surrounding environment, an experiment using a conditioned medium was conducted.

The results are shown in FIGS. 28, 29 and 30 . 28 is an observation of changes in microglia using a conditioned medium for midbrain astrocytes. Through this, the anti-inflammatory effect by astrocytes was verified in vitro. In cell culture containing microglia, when inflammation is induced by LPS treatment, treatment with conditioned medium for midbrain astrocytes reduces iNOS, Cd11b, and CD16.32, which are microglia markers in a bad direction, The expression of Arg and CD206, which are markers showing transformation into microglia, was increased.

Figure 29 confirms the possibility of inhibiting apoptosis factors with an astrocyte conditioned medium after induction of inflammation. Through this, the anti-inflammatory effect by astrocytes was verified in vitro. Also, in cell culture containing microglia, when inflammation was induced by LPS treatment, the expression of b-gal and p16, apoptosis markers, was decreased by treatment with the conditioned medium of mesencephalic astrocytes.

30 shows the anti-inflammatory effect of an astrocyte conditioned medium after induction of inflammation by RT-PCR. Through this, the anti-inflammatory effect by astrocytes was verified in vitro. Also, in cell culture containing microglia, when inflammation was induced by LPS treatment, it was confirmed that the expression of inflammosome formation-related factors decreased and the expression of neurogenic factors was increased by treatment of the conditioned medium of midbrain astrocytes.

Post-transplant characterization to see the in vivo utility of astrocytes obtained through the final protocol

First, we observed a study on the positive role of astrocytes on the surrounding environment after transplantation into the brain in vivo. After transplanting astrocytes to the striatum region, the results of anti-inflammatory factors, inflammatory factors, and cytokine secretion were confirmed. The expression of inflammatory factors such as TNF-a, IL1b, iNOS, and CD11b was reduced in the astrocyte transplantation site, and the expression of inflammatory factors such as IL1b, iNOS, CD11b was also reduced in the surrounding area, and BDNF, GDNF, Arg1 astrocytes were Cain expression was slightly enhanced ( FIG. 31 ).

The effect of improving the environment after transplantation of astrocytes in vivo was observed in vivo through changes in microglia.

After transplanting the obtained astrocytes into the rat brain striatum, neural stem cells were transplanted to the opposite side as a control, and it was confirmed whether the microglia were converted to M1 to M2 by observing representative environmental changes.

Inflammation was induced by LPS, and as a result of observation after astrocyte transplantation, the expression of iNOS, CD11b, and CD16, which are bad types of M1 markers at the astrocyte transplantation site, was suppressed ( FIGS. 32-34 ), and the expression of CD206 as a marker of M2 By observing that this is increased, a positive effect of astrocytes was seen (FIG. 35).

Example 4: Confirmation of differentiation into hypothalamic neurons and astrocytes from human induced pluripotent stem cells using hypothalamic organoids

[Experimental process]

Human pluripotent stem cell (hESCs/hiPSCs) culture

hESCs and hiPSCs were cultured based on the hESC research guidelines approved by the institutional review board (IRB) of Hanyang University (Seoul, Korea).

The hESCs and hiPSCs used in this experiment are shown in Table 8 below.

hESC line Name used in this paper original name Karyotype Establishing institute HSF6 UC06 Female (46, XX) UCSF H9 WA09 Female (46, XX) Wi cell HUES6 HUES6 Female (46, XX) Harvard university (HSCI iPS Core) hiPSC line Name used in this paper original name Source
(Human) Reprogramming factors Methods Establishing institute Retro-1 Rv-hiPS 01-1 Newborn fibroblast OCT4, SOX2, KLF4, MYC retrovirus Harvard Epi-ips episomal-ips1 Skin OCT4, SOX2, KLF4, shp53 Episomal vector Hanyang Univ. (Our own) CMC-ips#11 CMC-hipsc-011 bone marrow blood OCT4, SOX2, KLF4, MYC Sendai virus Catholic University. (Korea NIH SCRM) PD-ips1 PD patient derived ips-1 Skin OCT4, SOX2, KLF4, shp53 Episomal vector Hanyang Univ. (Our own) PD-#22 Corr. PD-ips#22 Skin OCT4, SOX2, KLF4, shp53 Episomal vector and genome edited Hanyang Univ. (Our own)

For proliferation and maintenance of undifferentiated hESCs/iPSCs, in a CO ₂ incubator set at 37° C. without a feeder layer, using mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) ^{on Matrigel TM} or vitronectin (Human; Gibco) Fisher Scientific, Waltham, MA) (Gibco A31804; 0.5 ug/cm ² )-coated 6 cm dishes (Thermo Fisher Scientific, Waltham, MA), medium change was performed daily. The undifferentiated stem cells were maintained in differentiation capacity by daily medium replacement and subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.

3D hypothalamic organoid fabrication

An organoid was prepared by the same protocol as in FIG. 36 .

Basically, because organoids of the nervous system are produced from embryonic/induced pluripotent stem cells, human embryonic/induced pluripotent stem cells are removed from the dish using Acutase and placed in a neuron differentiation induction medium with a low attachment 96-well round bottom plate (Corning, Corning). , NY) at 150 μL of 10,000 cells per well (day 0 of differentiation). SB -431542 for the production of organoids into the nervous system. Treated with 10 μM, Noggin, 100 ng/mL, induced into the nervous system and patterned by treatment with ventral inducers SHH and Purmophamin. Specific medium composition is shown in Tables 9 and 10 below.

Isolation of hypothalamic neural stem cells from hypothalamic organoids and confirmation of differentiation into neurons

The composition of the differentiation induction medium according to the date is shown in Table 9 below.

Day Media Composition 96-well (seeding: 1 × 10 ⁴ cells/well) D0 Neurobasal: 2Х N2 (1:1) 1×B27 (-RA) 1×MEM NEAA 1×Glutamax β-mercaptoethanol, 55 μM Penicillin/Streptomycin Ascorbic Acid, 200 μM Doxycycline, 1 μg/mL Y-27632, 10 μM SB -431542. 10 μM Noggin, 100 ng/mL D1-6 Neurobasal : 2×N2 (1:1) 1×B27 (-RA) 1×MEM NEAA 1×Glutamax β-mercaptoethanol, 55 μM Penicillin/Streptomycin Ascorbic Acid, 200 μM SB -431542. 10 μM SHH, 100 ng/mL Purmorphamine, 2 μM D7-12 Neurobasal : 2×N2 (1:1) 1×B27 (-RA) 1×MEM NEAA 1×Glutamax β-mercaptoethanol, 55 μM Penicillin/Streptomycin Ascorbic Acid, 200 μM Noggin, 100 ng/mL SHH, 100 ng/mL Purmorphamine, 2 μM Move to 6-well culture plate D13-20 N2 medium Penicillin/Streptomycin Ascorbic Acid, 200 μM FGF2, 20 ng/mL chopping D18-21 N2 medium Penicillin/Streptomycin Ascorbic Acid, 200 μM Doxycycline, 1 μg/mL FGF2, 20 ng/mL

For initial seeding, N2: Neurobasal (1:1, Gibco), B27 (without RA), 2-mercapto 55uM, MEM-NEAA, GlutaMAX, Ascorbic Acid, 200 μM, SB431542 (10 μM), Noggin 100ng/ml, ROCK Inhibitor Y27632 (20 μM) and Doxycyclin 1ug/ml were added. Y27632 and Doxycyclin were treated only for 24 hours on the first day. Between days 1 and 6, SHH, 100 ng/mL, Purmorphamine, and 2 μM were added while using the basal medium. Between 7 and 12 days, a medium without SB431542 was used while using the existing basal medium. It was transferred to a 6-well plate (low binding) on day 13 and stirred with an orbitary shaker. On days 13-20, only Ascorbic Acid, 200 μM, FGF2, and 20 ng/mL were added to N2 medium and cultured, and then chopping in 2D state on the 21st day (possible after the 18th), and the entire basal medium until spread on a plate. Used as N2, and Doxycyclin was added only on the first day of the plate. The entire medium was replaced whenever the medium composition was changed, and the medium used on the 13th day was used as the growth medium. Each subculture was treated with Doxycyclin 1ug/ml and Y27632 at a concentration of 5 μM for one day. Final differentiation into neurons was induced with N2 differentiation medium (10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid).

The basic badges are shown in Table 10 below.

2× N2 medium N2 medium 12 g DMEM/F12 (- HEPES buffer, - sodium bicarbonate) 12 g DMEM/F12 (- HEPES buffer, - sodium bicarbonate) 1.69 g Sodium bicarbonate 1.69 g Sodium bicarbonate 1.55 g D(+) Glucose 1.55 g D(+) Glucose 0.073 g L-Glutamine 0.073 g L-Glutamine 200 mg Apo-transferrin 100 mg Apo-transferrin Progesterone, 40 nM Progesterone, 20 nM Putrescine, 200 μM Putrescine, 100 μM Selenite, 60 nM Selenite, 30 nM Insulin, 200 nM Insulin, 200 nM 1 L water 1 L water

Isolation of hypothalamic astrocytes from hypothalamic organoids to confirm differentiation into astrocytes

If the passage of the hypothalamic-type Og-NSCs produced is further increased to match the actual brain development stage, it is possible to culture Og-progenitor cells, mainly containing astrocytes, after passage 9 without changing the medium composition. Generations 9 to 11 can be used in a healthy state. Like hypothalamic Og-NSCs, the growth medium is N2 growth medium, and the composition is 1X N2 supplement containing, 200 μM ascorbic acid, 20 ng/ml bFGF, and 1 μg/ml doxycycline. Medium change was made daily and Y27632 was treated at a concentration of 5 μM for one day at the first conversion to 2D culture and at each subculture. Cells in the proliferative stage with an older passage, such as organoid-derived astrocytes in the midbrain, are used for transplantation studies.

[result]

Confirmation of 3D hypothalamic organoid production

The prepared hypothalamic organoid has a normal shape as shown in FIG. 37 , and has an initial shape expressing Nestin and Sox2, which are markers of neural stem cells, together with RAX as a hypothalamic marker.

Confirmation of securing hypothalamic neural stem cells and astrocytes from the produced hypothalamic 3D organoid

It was confirmed that the neural stem cells dissected and extracted from the hypothalamic organoids became properly patterned neural stem cells in which 96.8% of the hypothalamic markers were expressed as shown in the result of FIG. 38 . Quantitatively, neural stem cells up to passage 4 can be used for neural cell differentiation, and it is possible to secure about 1,000 times the neural stem cells from the first 10,000 cells (FIG. 38). Neural stem cells express RAX and NKX2.2 as patterning markers.

Confirmation of differentiation of hypothalamic neurons and astrocytes

As shown in the results of FIG. 39, the produced hypothalamic stem cells can be differentiated into MAP2-expressing neurons and GFAP-expressing astrocytes during differentiation. Neurons are differentiated into cells expressing hypothalamus-specific markers NPY and a-MSH.

In order to confirm the formation of functionally complete cells after differentiation, when specifically looking at the leptin reactivity (FIG. 40), the sensitivity to the p-stat3 mechanism was measured, and when looking at the gene response, anorexic-related, POMC, The expression of CARTPT, PCSK1,2, LEPR, and MC4R is increased, and the expression of orexic-related genes NPY, AGRP, and NPY1R is increased, suggesting that it can fully function. In particular, it can be seen through the expression of NKX2.1, RAX, ISL, SF1, and NGN3 that the three-dimensional organoid-derived cells are better patterned compared to the two-dimensional culture (FIG. 41).

Securing hypothalamic neural stem cells as leptin-responsive stem cells shows that they can actually perform their functions similar to those in the living body, and it is likely to be mainly used for research and development of therapeutic agents for metabolic diseases such as obesity and diabetes in the future suggest that there is (FIG. 40).

Confirmation of biotransplantation potential of hypothalamic neural stem cells developed by using 3D organoids

The potential for transplantation of hypothalamic neural stem cells developed by using 3D organoids was confirmed. 1X10 transplant ^five hypothalamic neural stem cells in the 3 ^rd ventricle, after 7 days it was confirmed that the possibility of engraftment. It was confirmed that the hNCAM/Rax-expressing hypothalamic neural stem cells were stably engrafted, and at the same time, it was confirmed that the hGFAP/POMC-expressing hypothalamic neural stem cells had a form ( FIG. 42 ).

Claims (16)

In the method of producing an organoid derived from the midbrain, cerebral cortex or hypothalamus by culturing human pluripotent stem cells, culturing neural stem cells or astrocytes in the produced organoid, and inducing differentiation into neurons or astrocytes,
patterning and disassembling the prepared organoid to secure a large amount of neural stem cells or astrocytes, and culturing and differentiating them;
A differentiation method in which a large amount of nerve cells or astrocytes is secured, comprising a.
The method of claim 1,
The differentiation method, characterized in that the human pluripotent stem cells and stem cells are cultured using vitronectin (Vitronectin).
The method of claim 1,
The differentiation method, characterized in that the nerve cells are present mixed with astrocytes.
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