KR102468360B1 - Neural stem cells derived from 3D brain organoid and use thereof - Google Patents

Abstract

The present invention relates to a method for obtaining a large amount of finally differentiated cells by patterning and dismantling 3D organoids prepared from human pluripotent stem cells, culturing the corresponding stem cells or progenitor cells, and inducing differentiation, wherein the cells obtained in large quantities are Compared to cells differentiated by existing differentiation methods, it is expected to be very useful for screening cell therapy drugs or therapeutic drugs by showing significantly superior effects in terms of reproducibility, stability, and functionality.

Description

Neural stem cells derived from 3D brain organoid and use thereof {Neural stem cells derived from 3D brain organoid and use thereof}

The present invention relates to a differentiation method for securing a large amount of terminally 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 of the midbrain. It is known to be the most suitable disease for stem cell transplantation treatment because the lesion site and lesion cells are clear.

For the treatment of Parkinson's disease cells, technology to differentiate from stem cells into human midbrian-type dopamine neurons (mDA neurons) is required, and human pluripotent stem cells (hPSC, human embryonic stem cells (hESC) or human derived Differentiation methods for differentiating mDA neurons from pluripotent stem cells (hiPSC) have been developed for a long time [Non-Patent Document 1]. However, the following several problems have been raised in using this differentiation method for clinical application.

First, there is a problem with reproducibility. Existing two-dimensional methods are very difficult to reproduce in other laboratories, and in all developed methods, only some pluripotent stem cell lines such as H9 used for development are applied, and in several other pluripotent stem cell lines, differentiation into midbrain dopaminergic neurons didn't 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 the survival, function, and maintenance of midbrain dopaminergic neurons and are closely related to the ability to treat Parkinson's disease after transplantation. Because of this fact, all of the recently developed hPSC-mDA neuron differentiation protocols were developed with a focus on the expression of mesencephalic factors in midbrain dopaminergic neurons. However, since the expression of this midbrain factor is very unstable, midbrain dopaminergic neurons are easily lost after long-term culture or transplantation. Therefore, only some midbrain factors (mainly Foxa2) are expressed in the transplanted midbrain dopaminergic neurons by transplanting differentiated cells by methods developed so far.

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

Finally, there is a problem with the amount of neurons that can be obtained by differentiating neurons. In the dopaminergic neuron differentiation methods developed so far, there is a limit to the amount of finally differentiated neurons that can be obtained through one-time differentiation by inducing differentiation into neurons directly without a neural stem cell stage capable of proliferating 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 have focused on the fact that three-dimensional culture using organoids more closely implements the actual brain environment and enables healthy culture of cells with respect to two-dimensional cell culture. Each organoid that can sufficiently contain progenitor cells or stem cells, which are target cells, is individually patterned to make a cell group of a desired fate into a plurality of cell groups (target cell enriched), dismantle the organoid tissue, and progenitor cells or stem cells Stem cells were cultured. While other technologies obtain cells quantitatively by patterning them in a two-dimensional culture method, or borrow only a three-dimensional sphere shape in the intermediate stage and apply them to the development of a differentiation method, the present invention goes through perfect organoids, Compared to other existing technologies, the cells isolated in the present invention pass through a complete 3-dimensional artificial brain region called organoid, so that each cell is It was confirmed that it is more functionally superior.

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

Another object of the present invention is to provide a cell therapy agent 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 obtaining a large amount of terminally differentiated cells by patterning and disassembling 3D organoids prepared from human pluripotent stem cells, culturing the corresponding stem cells or progenitor cells, and inducing differentiation.

"Pluripotent stem cell (PSC)" as described herein means a stem cell capable of induced differentiation into any type of cell constituting the body, and pluripotent stem cell includes embryonic stem cell (ESC) ) and induced pluripotent stem cells (Ipsc, dedifferentiated stem cells).

The "organoid" described in this specification is a 'mini-like organ' made to perform minimal functions using stem cells, and is characterized by being made in a three-dimensional structure and creating an environment similar to actual body organs in the laboratory. . That is, "organoid" refers to cells having a 3D three-dimensional structure, and refers to a model similar to organs such as nerves and intestines prepared through an artificial culture process that is not collected or obtained from animals. The origin of the cells constituting it is not limited. The organoid may have an environment that allows it to interact with the surrounding environment during the cell growth process. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, the 3D organoids in the present invention almost perfectly mimic organs that actually interact in vivo, and can be an excellent model for observing the development of treatments for diseases and the like.

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 in the form of neuroectodermal spheres or from induced pluripotent stem cells derived from Parkinson's disease to true endoderm and hindgut Differentiation can be induced into intestinal organoids through differentiation.

As used herein, the term "differentiation" refers to a phenomenon in which a cell divides and proliferates, and the structure or function of a cell is specialized during the growth of an entire object. In other words, it refers to the process by which cells, tissues, etc. of living organisms change into a form and function suitable for performing the role given to each. etc.) and the process of changing into endoderm cells, as well as the process of changing hematopoietic stem cells into red blood cells, leukocytes, platelets, etc., that is, progenitor cells expressing specific differentiation traits, can all be included in differentiation.

In the case of directly inducing differentiation from existing human pluripotent stem cells and differentiating them into corresponding cells, there is a problem with reproducibility depending on the cell line or laboratory (experiment environment), and stable maintenance factor expression is not achieved. For example, in the case of midbrain dopaminergic neurons, actual Unlike the in vivo environment, 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 one embodiment of the present invention, organoids capable of sufficiently containing cerebral astrocytes, midbrain astrocytes, midbrain dopaminergic neural stem cells, or hypothalamic neural stem cells are each patterned and produced (target cell enriched), that is, Organoids containing the maximum number of target cells are secured, and the organoid tissue is dismantled and stem cells or progenitor cells are cultured to prepare for a cell group derived from two-dimensional differentiation, which is more similar to cells isolated from the actual brain. It is possible to secure differentiation into a cell group with similar, well-maintained characteristics, and secured viability.

In the present invention, the term "patterning" refers to the characteristics of the origin tissue of cells to be finally extracted from the detailed brain tissues in this way, such as mesocerebral pattern, cerebral cortical pattern, or hypothalamic pattern, when organoids are produced. It means that the organoid is prepared so that the cell group of the fate body having a target cell enriched is contained in a large number of cell groups. In addition, patterning markers include Lmx1a, Foxa2, Nurr1, En1; Pax6, Foxg1 in cortical patterns; There are Rax, Nkx2.2, etc. at the time of the hypothalamic pattern.

Therefore, in the present invention, a large amount of finally differentiated cells can be secured at once by disassembling the prepared 3D organoid, separating and culturing stem cells capable of proliferation from the organoid and proliferating.

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 prepared 3D organoid; and

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

A method of disassembling 3D organoids prepared from human pluripotent stem cells containing a method of securing a large amount of stem cells or progenitor cells and finally differentiating them.

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

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

The cells may be neurons or astrocytes, but are 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 0.3 to 0.7 μg/cm ² of vitronectin. 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 reported in the latest research on mass production of dopaminergic neurons for transplantation to date. 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)), 0.5 μg/cm ² of Lamin521 (LN-521 from BioLamina) was used for the proliferation and cultivation of human embryonic stem cells, and after differentiation into mesencephalic neurons, Lamin511 (from BioLamina) was used. LN-511) was reported to be very important to use 1 μg/cm ² . In contrast, in the examples of the present invention, 0.5 μg/cm ² of vitronectin (Gibco A31804) was used for both the culture of human embryonic stem cells and the culture of mesencephalic neural stem cells. In terms of the coating unit price of a 60 mm 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 mesencephalic neural stem cell culture is 23,832 won/dish, whereas with continued use of vitronectin of the present invention, it is 1,480 won/dish. Looking at the coating cost alone, the method of the present invention has a 5-fold reduction effect when culturing human embryonic stem cells and a whopping 16-fold reduction effect when culturing mesencephalic neural stem cells.

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

"Gila" as described herein is a cell that occupies the largest part of the cells present in the brain, and means "astrocyte". The astrocytes are also referred to as astrocytes, and are involved in the protection and nutrient supply of nerve cells and inflammation, 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 may be obtained from mammalian ventral midbrain, cerebral cortex, or lateral ganglionic eminence (striatal anlage). It can also be obtained separately.

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

A "nerve cell" is a cell of the nervous system, and the terms "neuron" or "neuronal cell" are used interchangeably.

"Dopamine (DA) neurons" refers to neurons expressing tyrosine hydroxylase (TH). In the present invention, "dopaminergic neurons", "dopamine neurons", "DA", etc. are used interchangeably. Dopaminergic neurons are specifically located in the substantia nigra of the midbrain 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 agent comprising cells secured by the above method.

"Cell therapy" refers to cells and tissues manufactured from a subject through isolation, culture, and special chewing, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention. It refers to drugs used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating or selecting allogeneic or heterogeneous cells in vitro, or changing the biological characteristics of cells in other ways. Cell therapy is largely classified into somatic cell therapy and stem cell therapy depending on the degree of cell differentiation.

As used herein, “subject” refers to a vertebrate, preferably a mammal, which is a subject of treatment, observation or experimentation, such as cows, pigs, horses, goats, dogs, cats, mice, mice, rabbits, guinea pigs, may be a human, etc.

In the present invention, "treatment" means any action that suppresses, alleviates, or beneficially changes the clinical situation associated with a disease. Treatment can also mean increased survival compared to expected survival if not treated. Treatment includes both therapeutic and prophylactic means.

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

Degenerative diseases of the nervous system include, 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, cortico-basal ganglia degeneration, diffuse Lew's disease. It may include those selected from the group consisting of body diseases and Pick's diseases, but is not limited thereto.

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

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

The cells obtained by the method of the present invention can be applied as a cell therapy agent, and can 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 living organisms 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 therapy agent is a buffer, preservative, analgesic agent, solubilizer, isotonic agent, stabilizer, base, excipient, lubricant, etc., if known in the art, can be used without limitation. can The cell therapy agent of the present invention can be prepared according to commonly used techniques in the form of various formulations. The cell therapy agent of the present invention can be administered through any route as long as it can induce migration to the disease site. In some cases, loading the stem cells into a vehicle equipped with a means to direct them to the lesion may be considered. Therefore, the cell therapy product of the present invention can be administered locally (including buccal, sublingual, cutaneous and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and intracerebrospinal fluid) or transdermal administration. It can be administered through various routes, including administration, and is preferably administered directly to the affected area. In one embodiment, the cells may be suspended in a suitable diluent and administered to a subject, which is used to protect and maintain the cells and facilitate their use when injected into a tissue of interest. Examples of the diluent may include physiological saline, phosphate buffer, buffer solutions such as HBSS, and cerebrospinal fluid. In addition, the pharmaceutical composition can be administered by any device that allows the movement of the active agent to target cells. A preferred mode of administration and preparation is an injection. Injections are aqueous solvents such as physiological saline solution, IV 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 permeation, a non-penetrating agent known in the art suitable for the barrier to pass through may be used, and as a stabilizer for preventing deterioration, ascorbic acid, sodium hydrogen sulfite, BHA, tocopherol, EDTA Pharmaceutical carriers such as emulsifiers, buffers for pH control, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and preservatives for preventing the growth of microorganisms such as benzyl alcohol may additionally be included.

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

Important characteristics of the cells obtained by the present invention include the possibility of securing a large amount of cell production, the possibility of maintaining the same cell group for a long period of time by maintaining the characteristics even during cryopreservation, and the differentiation more similar to living cells. This characteristic is particularly suitable for simultaneous screening of multiple types of drugs, which requires a large amount of cells in the same state and requires long-term securing of the same cells for repeated analysis. It is very suitable for screening cells because the cell population with the same characteristics in which key markers are maintained can be continuously used from the start of screening to the end.

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

Degenerative diseases of the nervous system include, 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, cortico-basal ganglia degeneration, diffuse Lew's disease. Various neurological diseases may be included, including but not limited to body diseases and Pick's disease.

The inflammatory degenerative disease may include, but is not limited to, those selected from the group consisting of dementia, Lewy body dementia, frontotemporal dementia, leukomatosis, adrenal leukodystrophy, multiple sclerosis, and Lou Gehrig's disease.

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

In addition, the ratio of MAP2-positive cells, which is a neuronal cell marker, among DAPI-positive cells in the total cell population after induction of differentiation of the present invention is 75% or more, and among the MAP2-positive cells, the ratio of TH-positive cells, a marker of dopaminergic neurons, is 25% or more, , The ratio of NURR1-positive cells among the TH-positive cells is 70% or more, or the ratio of cells positive for one or more markers selected from the group consisting of FOXA2, LMX1A and EN1 is 75% or more, a mesencephalic organoid Derived neural stem cells are provided.

The neural stem cells may be characterized in that the ratio of cells positive for one or more markers selected from the group consisting of FOXA2/LMX1A, KI67/NESTIN, OTX2, and SOX2 among the DAPI-positive cells is 75% or more.

Compared to 2D culture-derived neural stem cells, the neural stem cells showed marked expression of one or more genes selected from the group consisting of DBN1, HSP90AB, CCT8, SERBP1, KIF5B, RDX, MACFA, EIF4G2, HNRNPK, HSPAB, BZW1, ZC3H15, and RSL1D1. It can be characterized as an increase in

The present invention also provides dopaminergic neurons terminally differentiated from the mesencephalic organoid-derived neural stem cells.

The present invention also provides a cell population comprising the dopaminergic neurons.

In the cell population, the dopaminergic neurons may be differentiated together with astrocytes (GFAP ⁺ ).

The present invention also provides a pharmaceutical composition for treating neurodegenerative diseases and/or inflammatory degenerative diseases, comprising the mesencephalic organoid-derived neural stem cells, the dopaminergic neurons, or the cell population as an active ingredient.

In the pharmaceutical composition, the neurodegenerative disease is 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, cortico-basal ganglia degeneration It may be one selected from the group consisting of schizophrenia, diffuse Lewy body disease, and Pick's disease.

In the pharmaceutical composition, the inflammatory degenerative disease may be 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.

The present invention also provides hypothalamic organoid-derived neural stem cells capable of proliferating up to 4 passages with a ratio of Rax-positive cells, a hypothalamic neural stem cell marker, of 80% or more among DAPI-positive cells in the entire cell population after induction of differentiation.

The hypothalamic organoid-derived neural stem cells were terminally differentiated from hypothalamic organoid-derived neural stem cells, and compared to 2D culture-derived neural cells, expression of one or more markers selected from the group consisting of NKX2.1, RAX1, ISL, SF1, and NGN3 was remarkable. It can be characterized as an increase in

The present invention also provides hypothalamic organoid-derived neural cells that are differentiated from the hypothalamic organoid-derived neural stem cells and are responsive to leptin.

The present invention also provides a cell population comprising the hypothalamic organoid-derived neurons.

In the cell population, the hypothalamic organoid-derived neurons may be differentiated together with astrocytes (GFAP ⁺ ).

The present invention also provides a pharmaceutical composition for treating metabolic diseases comprising the hypothalamic organoid-derived neural stem cells or the hypothalamic organoid-derived neural cells or the cell population as an active ingredient.

In the pharmaceutical composition, the metabolic disease may be one selected from the group consisting of diabetes, obesity and metabolic disorders.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

In the present invention, the prepared 3D organoid is dismantled, and stem cells or progenitor cells are separated from the organoid, cultured, and proliferated to differentiate terminally differentiated cells 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 neuron differentiation, differentiation can be easily induced without cell line or batch-to-batch variation, so it can be easily reproduced in other laboratories. In addition, when mesencephalic neural stem cells differentiated according to the present invention are transplanted to Parkinson's disease, a series of Nurr1 Foxa2, Lmx1a, etc. known to be critically important for the survival, function, and maintenance of mDA neurons are faithfully expressed in the transplanted mDA neurons. It was confirmed that the cell transplantation treatment effect was remarkably improved. In addition, along with mDA neurons, astrocytes, which are cells responsible for the survival and function enhancement of neurons, also exist, protecting mDA neurons and enhancing their functions, which seems to have contributed to the enhancement of cell transplantation treatment during transplantation.

Therefore, the differentiated cells (including cerebral astrocytes, midbrain astrocytes, midbrain dopaminergic neural stem cells, hypothalamic neural stem cells, etc., other than mDA neurons) can be used for cell therapy or screening for drugs that can be treated with cells. 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(O g), 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, 50 μm(B, C, F), 100 μm(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, 200 μm.
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, 50 μm(E-G bottom, H, J, K, P bottom), 200 μm(P top), 500 μm(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, 100 μm(E-P), 200 μm(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, 50 μm].
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, 50 μm].
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, 50 μm].
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, 50 μm].
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, 50 μm.
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 for preparing mesencephalic organoids and extracting mesencephalic neural stem cells from them [A: A diagram showing a method for proliferating and securing neural stem cells from organoids with bFGF in a 2D environment. B and C: Expression of mesencephalic (OTX2, FOXA2, LMX1A, PAX2, PAX5) and neural stem cell (SOX2, NESTIN, KI67) markers confirmed by fluorescent staining of mesencephalic neural stem cells proliferated from organoids for 2 weeks. D; Calculation of proliferation rate of mesencephalic neural stem cells grown from organoids. Population doubling level (bars) and cumulative PDL (line). EG: Accurate differentiation of mesencephalic dopaminergic neurons after induction of differentiation of mesencephalic neural stem cells proliferated from organoids for 12 to 15 days. Identification of mesencephalic markers (FOXA2, LMX1A, NURR1, EN1) and fully mature neuronal differentiation markers (AADC, SYPT, DAT) along with dopaminergic neuron 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 using various human embryonic stem cells and induced pluripotent stem cells. H: Confirmation of stable differentiation success rate of organoid ^- derived neural stem cell differentiation technology compared to 2D differentiation method ^. >20%) NURR1 ⁺ of TH ⁺ mDA neurons). IL: Organoid-derived neural stem cell differentiation protocol more stably suppresses apoptosis and enables differentiation. EthD-1 ⁺ dead cells (I), cleaved caspase 3 ⁺ apoptotic cells (J), FACS-used Annexin+/PI+ cell counting (K), and cell senescence as β-galactosidase-stained cells (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 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 Derived neural stem cells (2D-NSCs) were prepared, transcription factors from the RNA, previously reported transcriptome datasets of prenatal midbrain, and adult substantia nigra with dopaminergic neurons in adults , Analyzing differences in transcription factor regulation in the adult cerebral cortex and hindbrain by bulk RNA sequencing. A: Spearman's correlation plot. Analysis of DEGs (differentially expressed genes) 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: Transcriptional regulation of organoid-derived neural stem cells (Og-NSCs) and 2D-derived neural stem cells (2D-NSCs) was analyzed by single cell RNA sequencing (analyzed 13,793 and 12,166 cells, respectively). C: t-SNE (t-distributed stochastic neighbor embedding) map with data-based color representation. D: 3 separate k-means clustering with t-SNE projection. (Loupe Cell Browser ver. 3.0.1, 10X Genomics, Inc.). The analysis of Og- and 2D-neural stem cells was analyzed by Venn diagram respectively (below). E: Hierarchical clustering analysis of 40 genes overexpressed compared to other groups in each cluster. F: Top-ranked GOs (UMI > 1, log ₂ FC > 0.5, P < 0.05, DAVID) centering on overexpressed genes in each group. G, Representative gene expression specifically regulated compared to other groups. Data are shown in dark color (log ₂ expression (UMI). GOs (vs 2D-NSCs, log ₂ FC > 1) showing more pronounced expression (DEGs) in FH, Og-neural stem cells compared to the analysis of 2D-neural stem cells , 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).
Figure 3 is a result of analyzing the maturity and functionality of Og-NSCs and dopaminergic neurons in terms of mitochondrial function and showing the difference in the maturity of neurons between the two groups. In conclusion, compared to NSCs produced in the past, Og-NSCs show that a more healthy and mature type of differentiation is possible. [AF: Even under the mitochondrial toxin experiment, organoid neural stem cells (Og-NSCs)-derived neurons maintain their mitochondria in a healthy state. A: STRING analysis of mitochondrial factors overexpressed in Og-NSCs (vs 2D-NSCs). B: Analysis of mitochondrial synthesis (Mitochondrial biogenesis (MitoTimer)). *p=0.0000005 (48 hr), 0.024 (24 hr), 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: Analysis of dopaminergic neuron maturity and functionality. G, Heatmap analysis of Og-NSCs expression-specific genes (vs 2D-NSCs). H, Highly mature dopaminergic neurons derived from Og-NSCs, representatively analyzed with the Neurolucida analysis program. J, Analysis of the length of dopaminergic neurons induced to differentiate for 12 days. TH+ fiber lengths from 23-58 cells/culture were measured. *p=0.013, n=7 (O g), 3 (2D) independent cultures, t-test. Synapse analysis of I, K, and dopaminergic neurons. The number of actual synapses formed was analyzed by synaptophysin staining. (27-43 TH ⁺ fibers/culture) *p=0.03, n= 5 cultures for each group. LO, analysis of Ca ²⁺ influx 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, stimulus-induced Ca ²⁺ influx traces. Mean values of Ca2+ influx kinetics during O, 100AP response: [τ of Ca ²⁺ 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, and DA levels were measured by dopamine secretion in the culture medium on day 13-15 of differentiation in the conditioned medium for 24 hours. Induces KCl-depolarization for 30 minutes. *p=0.0006-0.0009, n=4 (Og), 5-6 (2D) independent cultures. Astrocytic differentiation capacity of Q, R, and Og-NSCs. An astrocyte progenitor cell group was observed in cluster 1 of Single RNA-seq (Q). The CD44 ⁺ astrocyte progenitor cell group is included in the neural stem cell group (R), and dopaminergic neurons (TH) and astrocytes (GFAP) differentiate together after differentiation (S). Observed in the Og-NSC group and almost absent 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 observed in the α-synucleiopathy model, which is a biological feature of Parkinson's disease, when the neural stem cells induced by the method of the present invention are cells in a state in which α-synuclein propagation, which is an advanced Parkinson's disease, is not well carried out, Parkinson's disease It can be said to be advantageous when used as a cell therapy [Observed that α-syn spreads relatively less in neurons derived from organoid-derived mesencephalic neural stem cells (Og-NSCs). May be more resistant in the setting of α-syn disease. AD, α-syn infiltrates into nerve cells. Og-NSCs (vs 2D-NSCs). A, Dual-chamber system capable of observing α-syn movement between cells. B: Comparison of α-syn(GFP) intensity in 768 (Og) and 740 (2D) treated cells. *p=0.000004. Relatively less infiltration was shown in Og-NSCs. CF: When α-syn oligomerization, which can be the cause of Parkinson's disease, was induced with a viral overexpression system, relatively few p129-α-syn levels, which can be the cause, were observed in Og-NSCs (C), Western blot analysis after viral overexpression of α-syn oligomers (left) or after treatment with preformed α-syn fibril (PFF) (right). α-syn oligomers, which can be a major cause of Parkinson's disease, are less likely to occur in Og-NSCs (D) and in the 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 becoming a cause of aggregation (F). *significance at p=0.001(C) and 0.02(F), n= 3 independent experiments. G, PFF-a syn model animals were transplanted with Og-NSCs (left hemisphere) and 2D-NSCs (right-hemisphere), respectively, and histologically analyzed. Animals were induced in a Parkinson's disease model with PFF and a-syn AAV virus for 2 weeks, and sacrificed 1 month after each cell transplant. Relatively few malignant α-syn (p129-α-syn) were observed at the site where Og-NSCs were transplanted, and healthy dopaminergic neurons were observed. scale bars, 50 μm (B, C, F), 100 μm (G)].
Figure 5 shows multi-faceted in vivo prediction studies on the results that may appear after Og-NSC transplantation [A: single cell RNA-seq data of 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 the average ratio of log2 Og/2D expression values (right). B: Two-photon fluorescence endomicroscopy (upper) image obtained by transplanting pSyn-GCaMP6s-expressing neurons into the dorsal striatum. Regions of interest (ROIs) 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 of 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 implantation. Intrinsic excitability observed by depolarizing current injection of transplanted GFP+ cells (C), sEPSC recording with -70 mV holding potential (D). Comparison of average amplitude (E) and frequency (F) of sEPSCs in Og-NSC-derived transplanted areas with normal cells in non-transplanted areas (left). n=5 (graft) and 4 (control, unlesioned) from 2 rats. G, H: Og-NSCs transplanted into 6-OHDA Parkinson's disease-induced animals were analyzed for ¹⁸ F FP-CIT DAT binding by MRI and PET scans after 5 months. *p=0.021, n=8 (graft) and 5 (control, lesioned). T: implantation site, scale bars, 200 μm.
Figure 6 is The results were observed after transplanting Og-NSCs into actual Parkinson's disease model animals [Og-NSCs were transplanted into 6-OHDA-induced hemi parkinsonian model rats (AO) and also transplanted into Filipino monkeys (P). AC: post-implantation behavioral analysis of Parkinson's disease animals. A: 6 months after transplantation, amphetamine-induced rotation scores were observed. At baseline, 14 Parkinson's disease model animals were transplanted with Og-NSCs, and amphetamine-induced behavioral recovery scores were analyzed monthly for 1-6 months after transplantation. During months 1-4, 6 animals with a behavioral recovery score >60% were sacrificed for analysis. Based on pre-implantation behavioral score analysis data, recovery expressed as % of it. As a control group, 7 animals were used as a saline transplant group. (Sham-(PBS-injected) controls (n = 7) Analysis *p = 7.8e-10-0.037, one-way ANOVA.) Og-NSC- As another behavioral experiment of the transplanted animal model, step adjustment tests were conducted 6 months after transplantation, *p=5.58e-07-0.0006, one-way ANOVA, Tukey's post hoc analysis (B) and cylinder tests were also conducted, * p=1.63e-08-0.000424, one-way ANOVA followed by Tukey's post hoc analysis (C) (n=8). D: As a result of observing the morphology of dopaminergic neurons with Neurite tracing (Nurolucida) images 6 months after Og-NSC-implanted animal model transplantation, it was confirmed that they were differentiated into mature forms. EO: Histomorphometric analysis confirmed by fluorescence staining. EG: Confirmation 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 mesencephalic neurons. I: As a result of fluorescence staining with mesencephalic factors, expression % was confirmed in the transplantation sites of 8 animals (13,697 TH ⁺ cell count analysis). LP: 6 months after transplantation, transplanted area calculated by volume (L), number of TH+ neurons (M), density (N), and body size (O) (n=8 rats). P, Og-NSC-transplanted adult Philippine monkeys (cynomolgus) 1 month after transplantation, among TH+ dopaminergic neurons, transplantation expressing STEM121, expressing FOXA2 and NURR1 as mesencephalic markers, indicating human cell-derived transplanted cells identified dopaminergic neurons. scale bars, 50 μm (EG bottom, H, J, K, P bottom), 200 μm (P top), 500 μm (DG)].
Figure 7 shows the generation and characterization of midbrain-like organoids from hPSCs [A: Schematic diagram 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 on organoids. Organoids were cryosectioned on the indicated days in vitro and stained with the indicated antibodies. V, ventricles; VZ, ventricular zone; IZ, middle zone; MZ, mantle zone, scale bar, 100 μm (EP), 200 μm (BD)].
Figure 8 shows that maintenance of mesencephalic neural stem cells is efficiently improved by controlling the treatment time of FGF8, which is important for mesencephalic neurogenesis, by the method of the present invention [A: FGF8b treatment plan of protocols A and B. B: RNA-seq data for the expression of ventral midbrain (VM)-specific and hypothalamic-specific markers in organoid (Og)-NSCs generated with two different FGF8b treatment regimens. C: Representative images for EN1 ⁺ /TH ⁺ mDA neurons induced with protocols A and B. Scale bar (50 μm)].
Figure 9 shows the generation of 2D-NSCs from ventral midbrain (VM) neural stem/progenitor cells (NSCs) 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, differentiated hPSCs were isolated and plated (after passage) with VM-patterning chemical treatment (before, top panel) in the absence (−) or presence (+) of ACM. 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. Expression of VM-specific NSC markers in 2D-NSCs as estimated by C immunofluorescence staining after 2 weeks of NSC expansion. D: Cell expansion of 2D-NSCs expressed by population doubling level (PDL, bars) and cumulative PDL (line). EF: mDA neurons were generated from Og-NSCs 12-15 days after differentiation. Midbrain-specific marker expression of DA neurons (% of TH ⁺ cells) was quantified from three independent experiments (F). scale bar, 50 μm].
Figure 10 shows representative images for mDA neurons expressing midbrain-related markers in neural stem/progenitor cells (NSCs) and organoid- (top) and 2D-based culture (bottom) [scale bar, 50 μm].
11 is a result showing that the developed protocol is universally usable for human embryonic stem cells/pluripotent stem cells as well as various stem cell lines [ventral midbrain (VM) neural stem/progenitor cells (NSC) and midbrain-specific markers mDA neurons expressing β are generated across all hPSC lines tested. Three hESC and four hiPSC lines were induced to differentiate into VM-type NSCs using the organoid protocol outlined in Figure 1A. NSCs were expanded in 2-3 cell passages and terminal differentiation of NSCs into mDA neurons was induced. Representative immunostaining images for FOXA2 ⁺ , LMX1A ⁺ NSCs in NSC stages (expanded) and TH ⁺ mDA neurons expressing the midbrain markers FOXA2, LMX1A, NURR1 and EN1 7-12 days after terminal differentiation are shown. scale bar, 50 μm].
12 shows that organoid (Og)-neural stem/progenitor cells (NSC) are scalable to human mDA neurons, and can be supplied as a general-purpose cell therapy in terms of quantity or maintenance of cell biological properties. This result shows that this is possible. [A: Schematic outline of an Auger-based protocol to generate 7,260 vials (9,075 dishes) of mDA neurons from 1 dash of undifferentiated hPSCs using NSC expansion with 5 cell passages. B: Maintenance of mDA neuronal capacity of Og-NSCs over multiple NSC passages. On the left, representative immunostaining images for LMX1A ⁺ NSC at the FOXA2+, 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 neurogenic 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 for PD drugs using mDA neurons derived from PD-iPSCs [A: Schematic diagram of the experimental procedure. hiPSCs derived from a PD patient (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 the MTT assay and immunofluorescence intensity-based counting of TH-positive DA neurons. High content screening (HCS) analysis using Image Express Microconfocal (Molecular Devices, San Jose, Calif.) was used for TH intensity-based mDA neuron survival assay. B: Examples of screening data obtained by MTT assay (top) and TH ⁺ intensity measurements (bottom). Yellow line, mean value of control group (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 organoid (Og) neural stem/progenitor cell (NSC) differentiation and development of mDA neurons in the developing midbrain (VM) of the cells following subculture. Terminal differentiation of Og-NSCs was induced by the withdrawal of bFGF and the presence of the neurotrophic factors BDNF, GDNF and cAMP. Immunocytochemical analysis was 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 cellular senescence-specific genes (D) in organoids (Og) and 2D-NSCs. [The low expression levels of the pluripotency genes SSEA4 and TRA1-60 were further evaluated by FACS analysis (C)].
16 shows a detailed protocol for isolating cortical neural stem cells using 3D cortical organoids.
17 shows that neural stem cells were obtained using 3D cortical organoids [Left graph: suitability of the differentiation protocol was confirmed through the expression of pax6, a cerebral marker. Right: The upper photo confirms the formation of organoids in proper form before organoid disassembly (chop), and the lower photo confirms that they grow into normal neural stem cell types after dismantling].
18 shows the final established direct differentiation protocol from 2D and 3D-derived cultures to midbrain-nerve stem cells [A: direct differentiation from embryonic stem cells into midbrain-nerve stem cells step by step using human embryonic stem cells (Direct differentiation ), securing astrocytes, and thereafter establishing terminal differentiation technology into astrocytes, B: protocol for securing cortical-derived astrocytes. (A) is in (B) and its patterning method disparity].
Figure 19 shows the production of astrocytes using a direct differentiation protocol from 2D and 3D-derived culture to mesencephalon-neural stem cells. Compared to the existing two-dimensional culture method, the cells derived from the method of the present invention are healthier and markers are maintained shows that it is a peddled type of cell that works well.
20 is an analysis of the characteristics of in vitro cultured cerebral cortical and mesencephalic astrocytes, and between them, it is shown that mesencephalic astrocytes are superior in gene analysis in terms of actual nerve cell support function.
21 shows the analysis of the characteristics of cerebral cortical and mesencephalic astrocytes after in vivo transplantation, and between the two shows that the mesencephalic-derived astrocytes are superior in gene analysis in terms of actual nerve cell support function.
22 shows the change of microglia from transplantation of astrocytes in vivo to M2 type of high-quality cells, which can be used to improve the environment when transplanting astrocytes in the future, so it can be predicted that the utilization will be great .
23 shows the results of adaptive culture of different types of induced pluripotent stem cells using a Xeno-free system. This is a basic result because it is important to apply to various lines using a Xeno-free system when developing actual cell therapy products.
FIG. 24 shows confirmation of protocol application (left) and normal neuronal differentiation (right) of different types of induced pluripotent stem cells (CMC-iPSC#11) to mesencephalic astrocytes.
25 confirms the application of the protocol to other types of induced pluripotent stem cells (CMC-iPSC#11).
26 confirms the application of the protocol through efficient induction of differentiation using TGF-β and LIF.
27 is an analysis of the physiological characteristics of the generated midbrain astrocytes.
28 is the observation of changes in microglial cells using a conditioned medium for mesencephalic astrocytes. Through this, the anti-inflammatory effect by astrocytes was verified in vitro.
29 confirms the possibility of suppressing apoptosis factors with an astrocyte conditioned medium after inflammation induction. 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 non-transplanted sites and inflammatory response-related RNA expression after transplantation of astrocytes into the striatum. Through this, the anti-inflammatory effect by astrocytes was verified in vivo.
32 shows the expression of the M1 marker iNOS after in vivo transplantation of astrocytes. In preparation for the neural stem cell transplant site, it was confirmed that less inflammation occurred.
33 shows the expression of the M1 marker CD11b after transplantation of astrocytes in vivo. In preparation for the neural stem cell transplant site, it was confirmed that less inflammation occurred.
34 shows the expression of the M1 marker CD16.32 after transplantation of astrocytes in vivo. In preparation for the neural stem cell transplant site, it was confirmed that less inflammation occurred.
35 shows the expression of the M2 marker CD206 after transplantation of astrocytes in vivo. In preparation for the neural stem cell transplant site, it was confirmed that less inflammation occurred.
36 shows a detailed protocol for isolating hypothalamic neural stem cells using 3D organoids.
FIG. 37 shows the 3D hypothalamic organoids obtained and the expression of markers suitable for them was confirmed. Expression of nestin, Rax, and Sox2 as specific markers occurs well.
38 confirms that hypothalamic neural stem cells capable of proliferating were obtained [a: securing neural stem cells capable of proliferating up to 4 generations expressing 97% of hypothalamic neural stem cell marker nestin/Rax; b: Verification of hypothalamic-related gene expression in hypothalamic neural stem cells].
Figure 39 confirms the differentiation of normal neurons and astrocytes after differentiation induction of hypothalamic neural stem cells.
40 confirms differentiation from hypothalamic neural stem cells into cells responsive to leptin after induction of differentiation. This functional analysis showed the functional characteristics of hypothalamic cells, and after differentiation, the expression of hypothalamic neuronal markers, NPY and AGRP, was enhanced. confirmed that it was.
41 shows that hypothalamic neural stem cells developed using 3D organoids express more in vivo marker gene groups than those developed in a two-dimensional 2D environment.
42 confirms the biotransplantation potential of hypothalamic neural stem cells developed using 3D organoids.

Hereinafter, the present application will be described in detail through examples. The following examples are merely illustrative of 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 (mesencephalic dopaminergic) neurons using midbrain organoids

[Test method]

Cultivation 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, Republic of Korea). The hESCs and hiPSCs used in this experiment are shown in Table 1 below.

hESC lines
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 lines Name used in this paper original name Source (Human) Reprogramming factors Methods Establishing Institute Retro-1 Rv-hiPS 01-1 Newborn fibroblasts 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 Univ. (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.
(Ore own)

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

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

Briefly, a system was used in which mesencephalic 3D organoids were first prepared, cut into small pieces, and then massively proliferated in a mesencephalic neural stem cell state in a culture dish.

When subcultured hESC and hiPSC, which were normally cultured in an undifferentiated state, were removed using Accutase ^TM and undifferentiated human embryonic stem cells were dispensed at 10,000 cells/well on a 96 well plate with a round bottom that does not stick to the bottom. Organoids were formed. The composition of the medium on the first day was the seeding base medium in Table 4 below and the additive conditions corresponding to Day 0 in Table 2 (SB431542, 10 μM; Noggin, 100 ng/ml; ascorbic acid, 200 μM; Doxycyclin, 1 μg/ml; Y27632, 20 μM) to initiate organoid development. The next day, the medium was set as neural inducing base media (Table 3), and among the components added on the first day, Doxycyclin, 1 μg/ml, Y27632, 20 μM were added, and purmophamine 2 μM and sonic hedgehog 100 ng/ml were added to induce occurrence (See Table 2) The culture medium was neural inducing base media until day 11, and the additives were induced by adjusting the additives according to the concentration with reference to Table 2. The medium was changed every day according to the date. The correct formation of organoids was confirmed by the formation of cell aggregations on the 2nd and 3rd day, and the budding to the cell periphery before the 11th day. On the 11th day of organoid differentiation, organoids formed one by one in a 96-well plate were grouped into groups of 8 and cultured separately in one well of a 6-well low binding plate. Generation was induced under midbrain patterning base media (Table 3) containing the components of Table 2 while rotating with an orbital shaker (80-100 rpm). On day 18, cut one organoid into 4 to 10 pieces with a 30G needle, treat with Accutase ^TM for 10 minutes, pipet 5 times with a 1 ml pipette, remove Accutase ^TM by centrifugation, and then collect 3 wells of organoid cells. Poly-L ornithine (PLO; 15 μg/cm ² )/Fibronectin (FN; 1 μg/cm ² ) or vitronectin (VN; 0.5 μg/cm ² ) coated 60 mm culture dishes were center plated.

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, 100 ng/ml) + Ascorbic Acid (Sigma, #A4544, 200 μM) 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, 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 7~10 Neural Induction Base Media (recipe below) + Noggin (Peprotech, #120-10, 100 ng/ml) + Purmorphamine (Calbiochem, #540223, 2 µM) + SonicHedgeHog (Peprotech, #100-45, 100 ng/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, 100 ng/ml) + Purmorphamine (Calbiochem, #540223, 2 µM) + SonicHedgeHog (Peprotech, #100-45, 100 ng/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, 5 0ng/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, 200 μM) Day 18 Chop&Plating

Seeding Base Media Neural Induction Base Media Midbrain Patterning Base Media Neurobasal/N2 2X (1:1)
(Neurobasal, Gibco, #21103049)
(N2 2X recipe as follows(1 L)
DW 1L
DMEM-F12 12g
Sodium Bicarbonate 1.69 g
D-glucose 1.55 g
L-Glutamine 0.073 g
Apo-transferrin 200 mg
Putrescine 200 μl
Selenite 120 μl
Progesterone 400 μl)

MEM-NEAA
(Sigma, #M7145, 1:100)
β-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(1 L)
DW 1L
DMEM-F12 12g
Sodium Bicarbonate 1.69 g
D-glucose 1.55 g
L-Glutamine 0.073 g
Apo-transferrin 200mg
Putrescine 200 μl
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(1 L)
DW 1L
DMEM-F12 12g
Sodium Bicarbonate 1.69 g
D-glucose 1.55 g
L-Glutamine 0.073 g
Apo-transferrin 200 mg
Insulin 312 µL
Putrescine 100 μL
Selenite 60 μl
Progesterone 200 μl)

Mass proliferation of mesencephalic neural stem cells derived from midbrain 3D organoids

Organoid-derived mesencephalic neural stem cells are physically cut with a 30G needle and after the first plating for subculture, PLO/FN or PLO using Accutase ^TM once a week for subculture when the cells are 80-90% full. 3 X 10 ⁶ cells were cultured flat on a 60 mm culture dish coated with /VN. The culture medium of mesencephalic neural stem cells was mitogen (bFGF 20 g/ml, FGF-8 50 ng/ml), BDNF (20 ng/ml), GDNF (20 ng/ml) in the midbrain patterning base medium shown in Table 4. , Ascorbic acid 200 μM, Doxycyclin, 1 μg/ml was added as a growth medium. However, on the day of subculture, Y27632, 20 μM was added to this medium. For each subculture, 8 X 10 ⁴ cells per well were plated on PLO/VN 24 well coated plates, and after 2-3 days of proliferation, when 80-90% cell confluency was reached, mesencephalic markers Differentiation of NSCs was induced by fluorescent staining or removal of mitogens (bFGF, FGF-8). Differentiation medium was added to the midbrain patterning base medium shown in Table 4 with BDNF (20ng/ml), GDNF (20ng/ml), Ascorbic acid 200uM, and cAMP 500uM for 12 days to induce neuronal differentiation, followed by dopaminergic neurons and midbrain It was checked whether the phenotypic markers were co-expressed.

Cryopreservation of 3D organoid-derived mesencephalic neural stem cells

Organoid-derived mesencephalic neural stem cells generally have no change in their characteristics until the 5th passage, so they can be thawed and used after cryopreservation of the cells in each passage after the first passage without any change in their characteristics. Cell cryopreservation is performed at the time of subculture, using Accutase ^TM to detach cells, count the number of cells, and freeze 6 X 10 ⁶ cells in one vial mixed with growth medium and DMSO at 10% concentration. It was poured onto a medium and frozen. After the first 24 hours of freezing storage at -70 ° C, it was stored in a cryogenic nitrogen tank until use the next day. If necessary, one vial was quickly thawed in a 37 degree water bath and cultured flat on a PLO/FN or PLO/VN coated 60mm petri dish. Neural stem cell proliferation medium was used as the medium used during thawing, and Y27632, 20 μM was added to this medium on the day of thawing.

immunocytochemistry

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), incubated in blocking solution (1% BSA/0.03% Triton X-100 in PBS), and then with the following primary antibodies cultured 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 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 Abs 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 ^11DAKO 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, 28 kDa (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 in accordance with National Institutes of Health (NIH) guidelines. Hemi-parkinsonian was administered with 6-OHDA to the right side of the substantia nigra (AP-4.8 mm, ML-1.8 mm, V-8.2 mm) and the 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 adult 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 AP and ML coordinates are given relative to bregma. Rats with ipsilateral 300 rotations/time for this impairment 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 ^- astrocytes or E14 Ctx ^- NPCs (control) at a 2:1 ratio. 3 μl of the mixed cells (1.5 x 10 ⁵ cells/μl) was mixed with Rompun 100 μl/100 g (23.32 mg/ml) of 100 μl/100 g (50 mg/ml) of Zoletil under induced anesthesia in the striatum. (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. A needle (22 gauge) was left in place for 5 minutes after completion of each injection. Rats received daily doses of cyclosporine A (10 mg/kg, ip) starting 1 day before transplantation and continuing for 1 month, and were maintained without immunosuppressive agents for the remainder of the post-transplantation period. Six months after implantation, 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 then sliced on a cryomicrotome (Leica). Free-floating brain sections (30 μm thick) were subjected to immunohistochemistry as described above and imaged by confocal microscopy (Leica). In an experiment to test the host environment of the transplanted brain, animals were sacrificed at 1 month after transplantation and sliced at 1 mm thickness on a rat brain slice matrix (ZIVIC Instruments, Pittsburgh, PA), and transplanted to observe changes after transplantation. The tissue of the site was excised. 8-12 regions of the graft-host interface (ca 2x2 mm)/graft were dissected and qPCR analysis was performed. In addition, cells immunoreactive for neurotrophic and pro-inflammatory glial markers were counted along the graft-host interface of cryosectioned brain slices 7-10 days post transplant.

behavioral test

Animal behavior was reviewed by YH Rhee et al. , evaluated using an apomorphine/amphetamine-induced rotation test as described in the 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. It became. Apomorphine (Sigma) was injected subcutaneously at a dose of 0.5 mg/kg and observed while rotating for 60 minutes. Results are expressed in units of final revolutions/60 minutes.

Animal PET/MRI imaging and analysis

PET-MRI fusion imaging was performed using the nanoScanPET/MRI system (1T, Mediso, Hungary). To keep the mice warm, 0.2 mL of FP-CIT was administered intravenously via the tail vein 6.5±1.0 MBq and the 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) acquired during the FP-CIT absorption period, weighted T1 and fast spin echoes ( FSE) 3D sequence (TR = 2400 ms, TE _eff = 110, FOV = 50 mm, matrix = 256x256) weighted T2 images were obtained. Twenty minutes of static PET images were acquired with 1–3 congruence in a single field of view in the MRI range. Body temperature was maintained with heated air from an animal bed (Multicell Mediso, Hungary) and a pressure-sensing pad was used to trigger respiration. PET images were reconstructed by Tera-Tomo 3D with all corrections and high normalization and 8 iterations in full detector mode. Three-dimensional volume of interest (VOI) analysis of the reconstructed images 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 striatum and VOI for the striatum were derived. The SUV of each VOI site was calculated using the formula: SUVmean = (tumor radioactivity in tumor volume of interest in Bq/cc × body weight)/radioactivity injected.

High-throughput whole-transcriptome RNA sequencing

In the case of RNA, total RNA was isolated using Trizol reagent (Invitrogen), and rRNA removal was performed using 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 with 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. To determine the expression level of the gene region, FPKM (fragments per kb of exon per million fragments) was used. Global normalization was used for comparison between samples. Gene 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. GO and KEGG pathway analyzes of enriched categories were presented up to a p-value 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 performed after the experiment. Cells separated into 15,000 single cells were placed on a Single Cell A chip with Master mix solution. In each single cell, transcription factors inside the cell were reverse transcribed, and oligo dT-encoded UMIs were emulsioned and RNA barcoded. Barcoded Libraries were sequenced on an Illumina HiSeq 4000 platform (Macrogen Inc., Seoul, Korea). The results were analyzed with Cell Ranger (v2.1.1, 10 × Genomics). When analyzing 2D-NSCs, with 2D_cellRanger, 12,166 cells averaged about 57,343 reads and 2,284 genes per cell (out of 24,055 total genes) and when Og-NSCs were analyzed (with 3D_cellRanger), 13,739 cells averaged approximately 39,191 reads and 2,353 genes per cell ( of 23,860 total genes) were analyzed. Transcription factor expression in cells was expressed as UMI count. Cell diversity analysis was performed by t-distributed stochastic neighbor embedding (t-SNE) analysis and k-means clustering (performed by Loupe Cell Browser ver. 3.0.1, 10× Genomics, Inc.). The top 40 genes with increased expression showing significant differences 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, log ₂ FC > 0.5, P < 0.05) were analyzed. Gene 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).

Mitochondrial function assay

Mitochondrial regeneration dynamics are 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 It was analyzed using the tool for monitoring mitochondrial turnover (Autophagy 9, 1852-1861). pMitoTimer vector (Addgene52659; Addgene, Watertown, MA) was injected into NSCs cells by electroporation (NEPA21, Nepagene, Japan) to express them, and 2 days later, mitochondria produced by red versus green fluorescence were examined. Mitochondrial ROS and membrane potential were measured using MitoSox (Thermo Scientific Inc., Waltham, MA) and NucleoCounter3000 (NC3000; Chemometec, Allerod, Denmark), respectively. ROS production was analyzed after treatment with H ₂ O ₂ (500 μM) 2 hours before analysis. Toxic resistance studies of mitochondria were analyzed by TH fluorescence staining by treatment with 10 μM Rotenone, 4 μM CCCP or 250 μM H ₂ O ₂ for 1 hour.

DA release assay

Presynaptic activity of DA neurons was determined by measuring the levels of DA neurotransmitters released in differentiated VM-NPC cultures. The 24-hour culture medium (12-13 days of differentiation) 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 evaluated by incubating the cells in fresh N2 medium for 30 min in the presence or absence of 56 mM KCl (day 12 of culture). Evoked DA release was calculated as the DA level with KCl minus the DA release without KCl.

Intercellular α-syn transfer assay

Confirmation of intracellular movement of α-synuclein aggregates by factors secreted from astrocytes

SH-SY5Y (Professor Sang-Myun Park, Ajou University) cells derived from neuroblastoma were analyzed by co-culture with cells overexpressing A53Tα-syn-EGFP.

SH-SY5Y cells, which were induced to differentiate using RA for 5 days to express A53Tα-syn-EGFP, were co-cultured with neural stem cells induced for 10 days in a dual chamber system for 24 hours to obtain A53Tα-syn-EGFP expression. Migration was analyzed comparatively to the degree of GFP expression. The 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 PFF (pre-formed fibrils) were treated (8 μg/ml of medium) to induce a-synuclein aggregation, and the degree of a-synuclein aggregation was analyzed after 20 days of differentiation. Alternatively, overexpression was induced using lentiviruses expressing α-syn (pEF1α-α-syn) and analyzed. Thioflavin S (staining method for measuring protein aggregation; sigma Aldrich) and a-synuclein antibody were used for immunostaining (Immunoncytochemistry) and protein electrophoresis (Western blot) to confirm the presence and aggregation was observed as a change in protein size.

Bimolecular fluorescence complementation (Bi-FC)

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

In vivo a-syn propagation into transplanted grafts

AAV expressing human hα-syn (AAV2-CMV-hα-syn, 2×10 ¹³ genome copies) and PFF (10 μg) were injected into the striatum of female SD (Sprague Dawley) rats (225-250 g) to evaluate expression and Aggregation was induced. anteroposterior (AP), -0; mediolateral (ML), -3.0; dorsoventral (DV), -5.0. After 2 weeks, Og-NSCs and 2D-NSCs were grafted on both sides of the striatum. One month after transplantation, the transfer of α-syn to 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 a synapsin promoter (pSyn-GCaMP6s) and used to transduce cultures 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. The cells were then transplanted into four wild-type SD rats, and the rats were analyzed one 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 tunable wavelengths 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, the plate was translated and the brain was inserted from above. A 10× dry objective (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 to 900 nm for two-photon excitation of GCaMP6. An excitation laser was injected into the brain to perform two-photon imaging. The imaging field of view (FOV) and imaging speed were 387.50 μm x 387.50 μm with 512 × 512 pixels and 9.03 frames/s, respectively. Emission light from the brain was collected by the GRIN lens and coupled to the microscope system through an objective lens. The microscope system had four detection channels (Ch1: 430-480 nm, Ch2: 500-550 nm, Ch3: 565-605 nm and Ch4: 625-675 nm) and GCaMP6 fluorescence was collected in the second channel (Ch2). . The excitation laser power is 58.08 mw.

Calcium imaging assay

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 the fluorescence with a confocal microscope, the fluorescence intensity was taken at intervals of 1 to 3 seconds, and the ROI was measured for each cell in the photo at each time point to confirm that the fluorescence intensity was changed. Gated by FACS as needed.

Experiment result

Preparation of mesencephalic organoids from human embryonic stem cells

Conventional differentiation techniques based on two-dimensional 2D culture cannot represent the three-dimensional complex network in the actual brain body. Only the development of a protocol based on 3D generation can represent the tissue characteristics of the actual brain. Therefore, first of all, the research team produced the existing 3D mesencephalic organoids first. Successfully prepared mesencephalic organoids expressed ZO-1 and N-cadherin along with neural stem cell markers PLZF ⁺ and Sox2 ⁺ in the early stages of development (E&F in Fig. 7). Anterior embryonic brain marker (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 (FIG. 7 GI). When the development is further induced, the proliferative marker Ki67 ⁺ , SOX2 ⁺ and the neuronal marker MAP2 are expressed in the outer layer, and more specifically, PCNA ⁺ (the proliferative ventricular zone; VZ), MASH1 ⁺ (VZ, Intermediate zone (IZ) and mantle zone (MZ)), and structures that were specifically expressed in NURR1 ⁺ cells (IZ and MZ) were confirmed (L and M in FIG. 7). Characteristically, in IZ and MZ, FOXA2 ⁺ , LMX1A ⁺ and NURR1 ⁺ cells, which are major midbrain-specific factors, are expressed together with the dopaminergic neuron marker TH (tyrosine hydroxylase), confirming that they are true midbrain-type dopaminergic neurons (N, O, P in FIG. 7).

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

As a more evolved type of neural stem cells when used as a cell therapy for Parkinson's disease, a separate culture technology for organoid-derived mesencephalic neural stem cells (Og-NSCs) was developed. As a previous study, we tried to extract neural stem cells from organoids between 10 and 35 days of development, but the efficiency was not high. It was believed that an appropriate amount of cells could be secured by increasing the ratio of neural stem cells in organoids, and the protocol was modified in an effort to increase the amount of cells.

(1) When differentiation is continuously induced, the amount of stem cell population automatically decreases, so the final developmental differentiation protocol proceeding from the 10th day was stopped and improved in the direction of proliferation. (2) From the 17th day, it was modified by adding bFGF, which can specifically increase neural stem cells. (3) By increasing the treatment period of midbrain-specific factors (SHH/Purmorphamine/CHIR999021) (days 1 to 18) up to), that is, by increasing the process of midbrain patterning, proliferation of midbrain-specific neural stem cells was induced. (4) Unlike the conventional method, FGF8b treatment started from the 7th day (Fig. 8A).

Through such fine control of timing, occurrence in the hypothalamus, which is prone to contamination during midbrain induction, can be reduced, and expression of EN1, one of the specific markers, can be remarkably increased (FIG. 8). By applying these various modifications, on the 18th day of induction of mesencephalic organoids, they were proliferated and cultured in a 2D environment. Plates coated with human vitronectin were cultured in 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, NESTIN, and OTX2 mesencephalic markers, FOXA2, which is very important for early acquisition of dopaminergic neuron characteristics, 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). Organoid-derived mesencephalic neural stem cells (Og-NSCs) were observed to proliferate while faithfully maintaining their characteristics during subculture (Fig. 1D) through the expression of midbrain-specific markers such as FOXA2 ⁺ and LMX1A ⁺ (Fig. 12). VM type NSCs were prepared. Treatment of medium conditioned by VM-like astrocytes (Astrocyte conditioned medium (ACM) was important for cell survival and expression of midbrain markers during cell passage (Fig. 9A). ACM treatment and Along with the modifications to enrich the NSC population described above, we were also able to generate scalable FOXA2+/LMX1A+VM-NSCs from differentiation of hESCs in conventional 2D culture (FIG. 9).

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

Through the final differentiation process, mesencephalic Og-NSCs can efficiently differentiate into complete dopaminergic neurons (Fig. 1E, F). Og-NSC-derived dopaminergic neurons expressed FOXA2 (95%), LMX1A (96%), NURR1 (91%), and EN1 (86%) along with TH, a dopaminergic neuron marker, as mesencephalic markers. . On the other hand, in the conventional 2D culture environment, the expression of mesencephalic factor was as low as 44-61% (E, F of FIG. 9 and FIG. 10). Moreover, this organoid-derived mesencephalic neural stem cell isolation protocol can be applied to various human embryonic stem cells and dedifferentiated stem cells (Fig. 1G and Fig. 11). 111 out of 117 experiments conducted 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 28 out of 63 success rates were recorded (Fig. 1H).

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

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

Og-NSCs are patterned into a mesencephalic type and can differentiate into complete dopaminergic neurons after differentiation, and can maintain their characteristics for more than 5 generations (Fig. 1D and Fig. 12A, B), arithmetic It is possible to secure 7,260 vials (9,075 culture dishes) of Og-NSCs from one culture dish (A in FIG. 12). In addition, it can be stored in a nitrogen tank without losing its function (FIG. 12C). As a result, it is actually used by leading pharmaceutical companies (Chong Kun Dang Pharm., Seoul, KOREA) and candidate Parkinson's disease drug development (FIG. 13). The scalable and storable properties of the Og-NSC culture system were confirmed by collaborating with a pharmaceutical company (Chong Kun Dang Pharm., Seoul, KOREA) to develop mDA neurons from hESC (H9) and PD-hiPSC (PD-ips1) lines. underwent mass screening of candidate chemicals to develop PD drugs using (FIG. 13).

Undifferentiated Og-NSCs fully express the appropriate mesencephalic neural stem cell markers (Fig. 1B, C and Fig. 14A, E, F). Prior to differentiation, no differentiation neuronal/DA neuronal markers were expressed (MAP2, TH) (FIG. 14B, C). After induction of final differentiation, the expression of Ki67+, a proliferation marker, decreases, and morphological changes such as neurite elongation occur as characteristics of neurons (AC in FIG. 14). As in the developmental stage of actual mesencephalic neural stem cells, the specific marker NURR1 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 differentiation stage) (FIG. 14D). In fact, FOXA2 and LMX1A were continuously expressed in accordance with the biogenic rhythm in the midbrain, and after differentiation, the expression of LMX1A+ tended to decrease slightly (FIG. 14E, F). In the case of NURR1, FOXA2, and LMX1A, which are important markers of mesencephalic cells, they were well expressed overlapping in the terminally differentiated dopaminergic neurons (E-G in FIG. 1). Taken together, these findings indicate that in vitro NSC differentiation recapitulates physiologic mDA neuron development in VM, and may be useful as an in vitro model to study previously unexisted human mDA neuron development.

Analysis of transcriptional regulatory properties of Og-NSCs using bulk and single cell RNA sequencing

For molecular biological transcriptional analysis of Og-NSCs, RNA sequencing (RNA-seq) analysis was performed. Organoid-derived mesencephalic neural stem cells (Og-NSCs) and neural stem cells (2D-NSCs) generated by conventional differentiation methods were compared and analyzed for differences in transcriptional regulators from conventional human fetal and adult brain tissues (Carithers, L.J., Ardlie, K., Barcus, M., Branton, P.A., Britton, A., Buia, S.A., Compton, C.C., DeLuca, D.S., Peter-Demchok, J., Gelfand, E.T., et al. (2015). Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project.Biopreserv Biobank 13, 311-319.;Jo, J., Xiao, Y., Sun, A.X., Cukuroglu, E., Tran, H.D., Goke, J. , Tan, Z.Y., Saw, T.Y., Tan, C.P., 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, A.X., Cukuroglu, E., Tran, H.D., Goke, J., Tan, Z.Y., Saw, T.Y., Tan, C.P., 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.), as a result of PCA (principal component analysis) and Spearman's correlation analysis Organoids were remarkably similar to human fetal brains (Fig. 2A, B). Og-NSCs are similar to mesencephalic organoids and retain their characteristics (Spearman's correlation co-efficiencies: 0.79-0.97), and are also similar to the fetal midbrain (0.70-0.85), so they are actually extracted as neural stem cells. It can be seen that the characteristics of the Og-NSCs produced are maintained even during the isolation and subculture process.

To confirm the characteristics of individual cells 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, 3.8×10 ⁵ reads after normalization per cell and 2,227 genes detected in each individual 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). POU5F1, NANOG, and KLF4, which are markers of pluripotent cells, were expressed only in 0.09%, 0.45%, and 0.91% of Og-NSC cells, and even in the expressed cells, the expression levels were insignificant (average unique molecular identifiers (UMI) were <0.005 ) (B in FIG. 15). Although most of them were neural stem cells, conventional 2D-NSCs showed 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 express the three markers at the same time, this shows that only neural stem cells can be isolated and cultured. FACS analysis confirmed that there was almost no expression of TRA-1-60 and SSEA-4 (FIG. 15C). Previously, senescent cells (pre-apoptosis) were also significantly less in Og-NSC culture (Fig. 1L), and it was analyzed that the number of cells expressing the apoptosis gene was very small (Fig. 15D). In particular, compared to the cells of 2D-NSC, a very low percentage of the cells of Og-NSC expressed p16/INK4a, which is related to 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). Senescence and apoptosis block hematopoietic activation of quiescent hematopoietic stem cells with short telomeres. Blood 124, 3237-3240) decrease in stem cell repair ability is an important feature of

The Og-NSCs cell population analyzed through t-distributed stochastic neighbor embedding (t-SNE) analysis can be classified into three clusters (CE in FIG. 2). To confirm the characteristics of each cell cluster, GO (gene ontology) analysis of gene groups with increased expression was performed (UMI > 1, log ₂ FC > 0.5, P < 0.05) (FIG. 2 F), cluster 3 25% (Og-NSC) and 12% (2D-NSC) (Fig. 2D), and as a result of genetic analysis, it is a cell group already destined to become a neuron, and contains a significant number of differentiation factors. In the case of the gene group overexpressed in cluster 1, many gene groups important for the development of the 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), so patterning to the cluster 1 midbrain can be said to be the most complete cell group. In particular, 71% of Og-NSCs cells and 13% of existing 2D-NSCs cells belonged to cluster 1. These genetic analysis results also showed that the Og-NSC cultures were much more mesencephalic patterned and could stably secure cell populations 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, the gene group that is relatively highly expressed in Og-NSCs (compared to 2D-NSCs) is a gene group that emphasizes its importance in the development and maintenance of stem cell function through cell adhesion and ECM. The expression of this gene group, which is highly expressed by both methods of seq data, was studied by unsupervised hierarchal clustering (Fig. 2J). Its special function is also described (Table 5).

Cell-cell adhesion Gene ID Alias Functions HSP90AB1 Heat Shock Protein HSP 90 Beta involved in signal transduction, protein folding, and degradation DBN1 Developmentally-Regulated Brain Protein a cytoplasmic actin-binding protein which plays a role in the process of neuronal growth SERBP1 SERPINE1 mRNA Binding Protein 1 play a role in the regulation of mRNA stability CCT8 Chaperoin Containing TCP1 Subunit 8 involved in the transport and assembly of newly synthesized proteins RDX Radixin a cytoskeletal protein important in linking actin to the plasma membrane KIF5B Kinensin Family Member 5B microtubule-dependent motor required for normal distribution of mitochondria and lysosomes MACF1 Microtubule-Actin Crosslinking Factor 1 acts as a positive regulator of Wnt receptor signaling pathway EIF4G2 Eukaryotic Translation Initiation Factor 4 Gamma 2 plays a role in the switch from cap-dependent to IRES-mediated translation during mitosis HNRNPK Heterogeneous Nuclear Ribonucleoprotein K plays an important role in p53/TP53 response to DNA damage HSPA8 Heat Shock 70-kDa Protein 8 plays a pivotal role in the protein quality control system ZC3H15 Zinc Finger CCCH-Type Containing 15 protects DRG1 from proteolytic degradation BZW1 Basic Leucine Zipper and W2 Domains 1 enhances histone H4 gene transcription RSL1D1 Ribosomal L1 Domain Containing 1 regulates cellular senescence through inhibition of PTEN translation HSP90AB1 Heat Shock Protein HSP 90 Beta involved in signal transduction, protein folding, and degradation DBN1 Developmentally-Regulated Brain Protein a cytoplasmic actin-binding protein which plays a role in the process of neuronal growth Neuron Maturity Gene ID Alias Functions CADPS Calcium-dependent secretion activator 1 calcium-binding protein involved in exocytosis of vesicles P2RX7 P2X purinoceptor 7 receptor for ATP that acts as a ligand-gated ion channel CADPS2 Calcium-dependent secretion activator 2 regulates the exocytosis of synaptic and dense-core vesicles in neurons and neuroendocrine cells SYT10 Synaptotagmin 10 calcium sensors in the regulation of neurotransmitter release and hormone secretion UNC13C Unc-13 Homolog C plays a role in vesicle maturation during exocytosis RIMS1 Regulating synaptic membrane exocytosis protein 1 synaptic vesicle protein that regulates synaptic vesicle exocytosis PCLO Protein piccolo regulates the cycling of synaptic vesicles. Similar role to Bassoon, RIMS1/2 KCNMB3 Calcium-activated potassium channel subunit beta-3 modulates the calcium sensitivity and gating kinetics of KCNMA1 SCN1A sodium channel, voltage-gated, type I, alpha subunit mediates the voltage-dependent sodium ion permeability of excitable membrane KCND2 Potassium voltage-gated channel subfamily D member 2 mediates transmembrane potassium transport in excitable membranes ANK3 Ankyrin-3 plays key role in cell motility, activation, and proliferation SCN2A sodium channel, voltage-gated, type II, alpha subunit responsible for the generation and propagation of action potentials in neurons and muscle SCN9A sodium channel, voltage-gated, type 9, alpha subunit mediates the voltage-dependent sodium ion permeability of excitable membranes GPER1 G protein-coupled estrogen receptor 1 stimulates cAMP production, calcium mobilization and tyrosine kinase Src MYO5A Myosin-Va a class of actin-based motor proteins involved in cytoplasmic vesicle transport GRIK1 Glutamate receptor, ionotropic, kainate 1 meidates excitatory neurotransmission and is critical for normal synaptic function AKAP9 A-kinase anchor protein 9 required to maintain the integrity of the Golgi apparatus GABBR2 Gamma-aminobutyric acid (GABA) B receptor, 2 regulates the release of neurotransmitters and the activity of ion channels PMCHL1 Pro-Melanin Concentrating Hormone-Like 1 pseudogene found in fetal newborn and adult brain KCNQ5 Potassium voltage-gated channel subfamily KQT member 5 associates with KCNQ3 to form a potassium channel PCDHB16 Protocadherin beta-16 plays a critical role in the establishment and function of specific cell-cell neural connections DLG2 Disks large homolog 2 part of the postsynaptic protein scaffold of excitatory synapses PCDHB9 Protocadherin beta-9 plays a critical role in the establishment and function of specific cell-cell neural connections PTPRD Receptor-type tyrosine-protein phosphatase delta promotes neurite growth, and regulating neurons axon guidance CEP89 Centrosomal protein 89 plays a role in mitochondrial metabolism PCDHB3 Protocadherin beta-3 plays a critical role in the establishment and function of specific cell-cell neural connections GRIN2A Glutamate [NMDA] receptor subunit epsilon-1 ionotropic glutamate receptor PDYN Prodynorphin preproproteins of ligands for the kappa-type of opioid receptor HOMER1 Homer protein homolog 1 plays a role in synaptic plasticity GRM5 Metabotropic glutamate receptor 5 involved in the regulation of neural network activity and synaptic plasticity ATXN3 Ataxin-3 deubiquitinating enzyme involved in protein homeostasis maintenance GRIA2 Glutamate ionotropic receptor AMPA type subunit 2 function as ligand-activated cation channels LRP6 Low-density lipoprotein receptor-related protein 6 a co-receptor with LRP5 for transducing signals by Wnt protein RIT2 GTP-binding protein Rit2 binds and modulates the activation of POU4F1 HTR2A 5-Hydroxytryptamine Receptor 2A 5-HT _2A receptor OPRD1 δ-opioid receptor inhibits neurotaransmitter release by reducing calcium ion currents DCC Deleted in Colorectal Carcinoma mediates axon guidance of neuronal growth cones toward sources of netrin 1 ligand KIF5B Kinensin Family Member 5B microtubule-dependent motor required for normal distribution of mitochondria and lysosomes LMX1A LIM Homeobox Transcription Factor 1 Alpha plays a role in the development of dopamine-producing neurons during embryogenesis PTPRO Receptor-type tyrosine-protein phosphatase O plays a role in the inhibition of cell proliferation and facilitation of apoptosis SHH Sonic the hedgehog key inductive signal in patterning of the ventral neural tube EPHA5 EPH Receptor A5 functions as an axon guidance molecule during development WNT3 Wnt Family Member 3 functions in the canonical Wnt signaling pathway ISPD Isoprenoid Synthase Domain Containing catalyzes the formaion of CDP-ribotol nucleotide sugar form D-ribotol 5-phosphate SOS1 SOS Ras/Rac Guanine Nucleotide Exchange Factor 1 guanine nucleotide exchange factor for RAS proteins, membrane proteins SPTBN1 Spectrin Beta, Non-Erythrocytic 1 actin crosslinking and molecular scaffold protein OPHN1 Oligophrenin 1 Rho-GTPase-activating protein CNTN1 Contactin 4 axon-associated cell adhesion molecule UNC5D Unc-5 Netrin Receptor D plays a role in cell-cell adhesion and cell guidance SPTA1 Spectrin Alpha, Erythrocytic 1 links the plasma membrane to the actin cytoskeleton CHL1 Cell Adhesion Molecule L1 Like plays a role in nervou system development and in synaptic palsticity FEZ2 Fasciculation and Elongation Protein Zeta 2 involved in axonal outgrowth and fasciculation MYH10 Myosin Heavy Chain 10 plays a role in cytokinesis, cell shape SPTB Spectrin Beta, Erythrocytic plays a role in cell membrane organization and stability

In addition, gene groups specifically highly expressed in the Og-NSC cell group include 'SN development' and 'negative regulation of cell death' related to midbrain patterning (I in FIG. 2). In the single cell RNA-seq analysis, it was found that a large number of genes related to the biological functions of mitochondria were regulated, especially in the Og-NSCs group (mitochondria function, ER and protein folding, and autophagy), which could lead to cell aging and death. In the context of neurodegenerative diseases that are closely related to mitochondria, its importance can be emphasized because it is closely related to mitochondria as an organ where the effects are typically shown. Even in the KEGG analysis, the correlation between these data can be inferred by being ranked as the highest change in representative neurodegenerative diseases such as 'Parkinson's disease', 'Huntington's disease', and 'Alzheimer's disease' (FIG. 2 I).

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

Dopaminergic neurons, mDA neurons, differentiated from Og-NSCs coexist with astrocytes, resulting in enhanced neuronal function and maturation.

Transcriptional regulation of genes actually precedes changes in the shape of the cell population. For example, changes that can be seen from neural stem cells can be seen from the embryonic stem cell stage during actual gene analysis (in differentially expressed genes (DEGs)). 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 to their 2D counterparts, mesencephalic dopaminergic neurons induced to differentiate from Og-NSCs show a more mature morphology after neuronal differentiation (Fig. 3H, J), synapse formation ( I, K in FIG. 3), action potential-evoked Ca ²⁺ dynamics (LO in FIG. 3), and pre-synaptic dopaminergic neuron function assessed by depolarization-induced dopamine secretion (FIG. 3 P). see the shape

In all currently existing hPSC-mDA differentiation protocols, mDA neurons are generated without astrocyte differentiation. Similarly, no astrocyte differentiation was found in cultured 2D-NSCs (Fig. 3R, S). Interestingly, 17% of cells in the subpopulation of cluster 1 of Og-NSCs (mesencephalic SN-producing cell population, cluster with higher SN developmental gene expression) expressed genes associated with glia development (EIF2B5, MT3, PLP1, CDK6, SCL1A3, HES1). , HES5, and SOX8) were commonly expressed at high levels (Q in FIG. 3). It was shown that the Og-NSC cultures contained a VM(SN)-type glia progenitor population. Consistently, some of the cells in the Og-NSC cultures expressed CD44, which is specifically found in neurogenic progenitors (Fig. 3R) that differentiate into astrocytes upon peripheral differentiation (Fig. 3S). By expressing CD44, a marker for astrocytes, these cells 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 in a mixture of GFAP ⁺ astrocytes (Fig. 3 S).

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

Considering the ability of astrocytes to secrete cytokines based on previous reports, astrocytes can be expected to positively influence dopaminergic neurons when they occur. 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 FIG. 3 E-P and FIG. 1 E-L.

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

In order to prevent the spread of α-synucleinopathy existing in the original patient's brain to the transplanted cells, and also for the long-term success of Parkinson's disease cell therapy, the α-syn diffusion resistance of the cells to be used (transplanted) as a cell therapy is required. Acquisition is important Pathogenic propagation of toxic α-syn is associated with cell-to-cell movement of α-syn and movement of α-syn aggregates into transplanted cells. To measure the cell-to-cell movement ability of α-syn, a dual chamber system (Choi, Y.R., Cha, S.H., Kang, S.J., Kim, J.B., Jou, I., and Park, S.M. (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 (a protein overexpressed in SH-SY5Y neuronal cells) was placed on top of the chamber, and cultured Og-NSC (2D-NSC)-derived neurons were placed underneath to observe absorption and expansion after protein transfer. 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).

In addition to α-syn migration, pathological spread of α-syn into transplanted cells as another important issue was analyzed as toxic α-syn aggregates. The degree of aggregation was compared and analyzed by dividing into the environment in which the amount of α-syn inside the cell increases and the condition of exogenous α-syn fibril seed.

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

Finally, Og-NSCs or 2D-NSCs were transplanted into living brain tissue, and α-syn proliferation and aggregation were observed in a 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 striatum of rats, and 2 weeks later, Og-NSCs and 2D-NSCs were transplanted into the model animal striatum, respectively, to develop living brains. Differences within the environment were observed. As a result of the analysis of the transplanted tissue after 1 month, in the severe α-synucleinopathy environment, the existing 2D-NSC-derived graft site had almost no surviving dopaminergic neurons (Fig. 4G, lower panel), and α-syn ⁺ and p129-α-syn ⁺ co-expressed Lewy bodies were observed, and neurites were hardly differentiated, indicating that the existing cell population was not suitable as a cell therapy. On the other hand, in the case of the transplantation site derived from the newly developed Og-NSC cell group, the shape of the nerve cells was much healthier and there was no serious neurite degeneration (Fig. 4G, upper panel). Moreover, a small amount of toxic α-syn ⁺ (p129-α-syn ⁺ ) aggregation was observed in several regions of neurons. In the case of patients with Parkinson's disease, host-to-graft α-syn propagation, even after transplantation of cell therapy products, is severe during the patient's survival (11 to 16 years after transplantation). raised as an issue In the case of animal models, α-syn diffuse aggregation is observed in a much faster period of about a month. Although it does not fully reflect the patient's brain environment, in vitro and in vivo assays have shown that Og-NSCs-derived cells are toxic. It shows that it is more resistant to α-syn diffusion and aggregation.

Og-NSC Transplantation Provides Healthy Dopaminergic Neurons in Animal Models of Parkinson's Disease

Finally, the potential of Og-NSCs-derived cell population as a cell therapy was observed after transplantation in a model animal system induced with Parkinson's disease by 6-OHDA (6-hydroxydopamine). Recently, there has been a study on genes that can measure success after transplantation of human embryonic stem cell-derived cells. As expected, this gene group was found to be relatively highly expressed in the newly developed Og-NSCs cell group as a result of single cell RNA-seq analysis (compared to the existing 2D cell group) (Fig. 5A). Synaptic plasticity of transplanted dopaminergic neurons is very important for future cell survival and success of transplantation. To analyze the function as a neuron, Og-NSCs cells were transplanted into the brain striatum after expressing GCaMP6s (calcium indicator protein, which emanates green fluorescence by Ca ⁺⁺ binding) with GFP fluorescence. Neuronal function was observed by two-photon endomicroscopy one month after transplantation (Fig. 5B, top) without any specific stimulation of neurons, and signal transmission through spontaneous neurons was observed in the transplanted Og-NSCs cells. Ca ²⁺ transients (ΔF/F) show an action potential frequency of 2-5 Hz of neurons (Fig. 5B, bottom), which is the origin of dopaminergic neurons originating from SNpc (substantia nigra pars compacta) of brain tissue. Similar to that. To further confirm synaptic plasticity by the transplanted Og-NSC cells, Og-NSCs were prepared from human embryonic stem cell groups expressing a delta-like canonical Notch ligand 1 with GFP ( Dll1 ; pDll1-GFP-hESC) gene. Since fluorescence is expressed only in differentiated neurons, only cells expressing fluorescence were analyzed after cell transplantation into Parkinson's disease model animals. At the time of patch clamp analysis after one and a half months, 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). This showed that the neurons had a functionally perfect morphology even after Og-NSCs were transplanted. In order to see whether the transplanted dopaminergic neurons are fully mature and have perfect synaptic functions such as dopamine secretion, dopamine uptake by the dopamine transporter was examined by [ ¹⁸ F]FP-CIT PET scan, and the striatum of Parkinson's disease animals showed low dopamine Although absorptive capacity was shown (2% of the intact hemisphere), the Og-NSCs cell population was relatively recovered to 40% of normal 5 months after transplantation (Fig. 5G, H).

As a result of transplanting Og-NSCs cell population into an animal model of Parkinson's disease and conducting behavioral studies, a consistent recovery effect was observed as a result of amphetamine-induced rotational behavioral analysis. By ∼6 months of transplantation, 11 of 14 rats had behavioral rotation scores >50% of pre-transplantation. In the case of 8 animals observed at 6 months after transplantation, all animals showed >79% results (FIG. 6A). In the transplanted animals, the stepping test (FIG. 6B) and the cylinder test (FIG. 6C) also showed a recovery effect.

As a result of analysis after 6 months of transplantation of Og-NSCs into model animals, healthy dopaminergic neuronal 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, DO in FIG. 6). The transplantation site was constant and no specific proliferation was observed. This is the same aspect as the in vitro differentiation into Og-NSCs-derived dopaminergic neurons (Figure 2H and Figure 3GI). is formed maturely and normal neurites are extended (Fig. 6D). It is known that midbrain-specific factors are very important for the survival and maturation of dopaminergic neurons, but their expression is not well maintained, especially 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 important 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 key midbrain factors were It is expressed in common with TH+, a marker of dopaminergic neurons (from 8 animals; EI in FIG. 6). In particular, transplanted dopaminergic neurons express GIRK2 (Fig. 6J), a marker of A9 nigral dopaminergic neurons, and, like the main characteristics of A9 nigral dopaminergic neurons, are mature forms that extend neurites in multiple directions ( Figure 6 EG, H, lower figure and Figure 6 P), it is shown that the original brain tissue cells are also extending mature neurite. (Fig. 6 D). Human GFAP (hGFAP)-expressing astrocytes were spread around the transplanted dopaminergic neurons (Fig. 6K), indicating that the transplanted Og-NSC-derived astrocytes were dopaminergic neurons extending neurites. It shows that it forms synapse and helps cell maturation. It is the same as the previous cotransplantation of astrocytes showed a similar effect. To see the effect in primates, Og-NSCs-derived cell populations were transplanted into the striatum of two adult Philippine monkeys, and a month later, mature dopaminergic neurons co-expressing key mesencephalic markers FOXA2 and NURR1 were identified. (P in FIG. 6).

discussion

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

All tissue development is achieved through a sequential process of generation, proliferation of tissue-specific stem/progenitor cells and their differentiation into tissue-specific cells. Cultured tissue-specific stem/progenitor cells can provide a bioassay platform for developmental studies and drug screening, as well as donor cells for regenerative medicine. However, derivation of NSC cultures with proper VM-patterning from hPSCs has not been achieved in previous studies. The inventors have realized that difficulties in preparing VM-specific NSC cultures are due to the instability of VM region specificity in culture. Since maintenance of VM-specific marker expression is very sensitive to cell density, it is easily lost during the cell isolation and replating process required for NSC preparation (Fig. 9A).

Therefore, hPSC-mDA neuron protocols have been 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 trigger open epigenetic states (5 Various efforts with -azacytidine (VPA), TSA), β-estradiol or DAPT (Notch inhibitor) did not solve the problem.

Recent studies (Song, J.J., Oh, S.M., Kwon, O.C., Wulansari, N., Lee, H.S., Chang, M.Y., Lee, E., Sun, W., Lee, S.E., 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. It was observed that primary NSC and mDA neuron cultures exert strong trophic effects to promote cell survival and expression of midbrain-specific factors.

Therefore, the 2D-NSC culture protocol used in this study could be developed through the treatment of conditioned medium prepared from cultured astrocytes (ACM) (Fig. 9). However, derivation of 2D-NSC cultures still had the aforementioned stability and reproducibility issues. In contrast, NSCs with stable VM marker expression can be readily isolated and cultured from rodent embryonic VM tissue, stimulating the isolation of human VM-specific NSCs from hPSC-derived midbrain organoids and placed in an environment similar to the structure of the physiological embryonic brain. It has an established structure and region specificity.

Organoid-based preparations have achieved 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 noteworthy observation was that Og-NSCs contained an astrocyte progenitor cell population with a VM-specific gene expression profile that differentiated into astrocytes mixed with mDA neurons. In contrast, mDA neuron differentiation was induced without astrocyte differentiation in all existing methods for hPSC-mDA differentiation, and consequently, differentiated mDA neuron cultures were free of astroglia. Thus, without astroglial support, neuronal maturity and function could not be expected to be equivalent to that of differentiated mDA neurons in their in vivo counterparts. Indeed, insufficient maturation and function have been proposed as critical drawbacks in the usefulness of hPSCs and differentiated mDA neurons in in vitro disease modeling and therapy. In contrast, under trophic support from mixed astrocytes, Og-NSC-derived mDA neurons exhibit improved synaptic maturation, functionality, resistance to toxic insults, and long expression of midbrain-specific factors in vitro and in vivo after transplantation. showed an improved set. All these properties of Og-NSCs ultimately contribute to superior and long-term therapeutic efficacy after transplantation in a reproducible manner. Furthermore, organoid-derived NSCs have been shown to be a scalable cell source necessary for the preparation of donor cells for therapy, as well as a bioassay platform for drug screening, developmental studies, and disease modeling.

Based on the present invention, organoid-based methods may become a common next-generation strategy to generate 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 organoid cells are healthier and can show excellent results in cell therapy compared to two-dimensional culture. Since this is not preferred, the present invention can be said to be the most appropriate model for cell therapy agents for the nervous system.

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

[Experiment 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, Republic of Korea).

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

hESC lines 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 lines Name used in this paper original name Source
(Human) Reprogramming factors Methods Establishing institute Retro-1 Rv-hiPS 01-1 Newborn fibroblasts 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 Univ. (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 hESC/iPSC, mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) was used in a CO ₂ incubator set at 37 °C without a feeder layer on Matrigel ^TM or vitronectin (Human; Gibco Fisher Scientific, Waltham, MA) (Gibco A31804; 0.5 μg/cm ² )-coated 6 cm dishes (Thermo Fisher Scientific, Waltham, MA) were cultured and medium change was performed daily. The differentiation capacity of the undifferentiated stem cells was maintained by changing the medium every day and subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.

Fabrication of 3D cortical organoids

Human embryonic/induced pluripotent stem cells were detached from the dish using Acutase, placed in a neural cell differentiation induction medium, and injected into a low attachment 96-well round bottom plate (Corning, Corning, NY) at 150 μL, 10,000 cells per well ( day 0 of eruption). The composition of the neural cell differentiation induction medium is shown in the table below. For the first seeding, KSR-hES media (DMEM 40 ml, KSR 10 ml, 2-mercapto 20 μl, MEM-NEAA 500 μl, GlutaMAX 500 μl), bFGF (4 ng/ml), ROCK inhibitor Y27632 (20 μM), Add 1 μg/ml of Doxycyclin. Y27632 was only treated for 24 hours on the first day. On the second day, B27 without retinoic acid, Insulin (15.6 μl/50 ml), SB431542 (10 μM), LDN (10 μM), and A-83 (10 μM) are added to induce full-scale nervous system differentiation. Between the 2nd and 3rd days, only 1/2 of the basal medium was used as N2: Neurobasal (1:1, Gibco), and then the entire basal medium was N2: Neurobasal ( 1:1, Gibco). The entire medium was replaced whenever the composition of the medium was changed. From day 8, bFGF (20 ng/ml) and EGF (20 ng/ml) were added.

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

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

On day 18, organoids were digested with Acutase for 7 min at 37°C, then minced with a 30 gauge needle and plated 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 and capable of proliferating and were passaged every 5-7 days. The medium used for NSC culture was N2 expansion medium, and the composition was as follows (1X N2 supplement containing, 200 μM ascorbic acid, 20 ng/ml EGF, 20 ng/ml bFGF, and 1 μg/ml doxycycline). Medium exchange was performed daily, and Y27632 was treated at a concentration of 5 μM for one day at the time of the first transition to the second-dimensional 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 cultivation of cortical astrocytes from cortical organoids

If the passages of the cortical-type Og-NSCs are further increased according to the actual brain development stage, it is possible to culture Og-astrocytic progenitor cells mainly containing astrocytes after 9 passages without changing the medium composition. The 9th to 11th passages can be used in a healthy condition. As with cortical Og-NSCs, the growth medium was N2 expansion medium, and the composition was 1X N2 supplement containing, 200 μM ascorbic acid, 20 ng/ml EGF, 20 ng/ml bFGF, and 1 μg/ml doxycycline. Medium exchange was performed daily, and Y27632 was treated at a concentration of 5 μM for one day at the time of the first transition to the second-dimensional culture and at each subculture. Like organoid-derived astrocytes of the midbrain (see Example 3), cells in the proliferative stage with a long passage are used for transplantation studies. For analysis, the cell population of high passage into the above final differentiation medium will assume the character of astrocytes.

Confirmation of differentiation into cerebral cortical neurons and astrocytes

Cortical-type neurons were produced, and cortical-type patterning was preferentially performed to secure astrocytes through several subcultures. After the induction of differentiation of the nervous system from human embryonic stem cells, confirmation of this was primarily confirmed by confirmation of patterning, and expression of cortical markers such as PAX6 and FOXG1 was confirmed. In addition, when astrocytes were generated through several subcultures, the expression of astrocytic factors such as GFAP, AQP4, ALDH1L1, and GLAST was confirmed. Confirmation 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 fabrication

First of all, the point of the result is to confirm that the differentiation into a normal organoid form into a group of cerebral cortical cells is patterned, 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 cerebral cortical neural stem cells and astrocytes from the cerebral cortical 3D organoids produced

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

Confirmation of differentiation of cerebral cortical neurons and astrocytes

Nervous system patterning factors SB431542 (10 μM), LDN (10 μM), and A-83 (10 μM) were added, and full-scale neural system differentiation was induced and patterned into cortical brain organoids. The process after differentiation into astrocytes through division of neurons and several passages differs only in initial patterning and is not different from the process of differentiation through midbrain organoids. When compared with differentiation of mesencephalic astrocytes, immunostaining was used to confirm sufficient expression of GFAP, AQP4, ALDH1L1, GLAST, etc., and at the same time, expression of patterning factors in each cell group was confirmed ( Figure 25). From the functional aspect, it can be seen that differentiation into activated astrocytes can be completed in terms of electrophysiological function as well.

In this example, a method using 3D organoids capable of producing cerebral cortex-specific neural stem cells was established. By utilizing 3D organoids, since neural stem cells are extracted from tissue with the same structure as the actual living body cerebrum, it can be expected that cells in a healthier and more realistic form can be obtained.

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

[Experiment 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, Republic of Korea).

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

hESC lines 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 lines Name used in this paper original name Source
(Human) Reprogramming factors Methods Establishing institute Retro-1 Rv-hiPS 01-1 Newborn fibroblasts 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 Univ. (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 hESC/iPSC, mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) was used in a CO ₂ incubator set at 37 °C without a feeder layer 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) were cultured and medium change was performed daily. The differentiation capacity of the undifferentiated stem cells was maintained by changing the medium every day and subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.

Fabrication of 3D midbrain organoids

The preparation of mesencephalic organoids was carried out in the same manner as in Example 1. Successfully prepared mesencephalic organoids expressed ZO-1 and N-cadherin along with neural stem cell markers PLZF ⁺ and Sox2 ⁺ in the early stages of development (E&F in Fig. 7). Anterior embryonic brain marker (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 (FIG. 7 GI). When the development is further induced, the proliferative marker Ki67 ⁺ , SOX2 ⁺ and the neuronal marker MAP2 are expressed in the outer layer, and more specifically, PCNA ⁺ (the proliferative ventricular zone; VZ) and MASH1 ⁺ (VZ), like the brain layer in vivo. , intermediate zone (IZ) and mantle zone (MZ)), and structures specifically expressed in NURR1 ⁺ cells (IZ and MZ) were confirmed (L and M in FIG. 7). Characteristically, midbrain-specific factors such as FOXA2 ⁺ , LMX1A ⁺ and NURR1 ⁺ cells were also expressed in organoids, confirming that organoids of true midbrain type were directly prepared.

Isolation of mesencephalic neural stem cells from midbrain organoids and differentiation into neurons

hESCs/hiPSCs were detached from the dish using Acutase, placed in a neural cell differentiation induction medium, and injected into a low attachment 96-well round bottom plate (Corning, Corning, NY) at 150 μL at 10,000 cells per well (differentiation day 0). . 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 the first day 24 treated only for an hour). Sonic hedgehog (100 ng/ml, SHH, Peprotech) and purmorphamine (2 μM, Calbiochem, MilliporeSigma, Burlington, MA) were added to the neural cell differentiation induction medium from the first day of differentiation for the expansion of ventral midbrain type neural stem cells of organoids It became. On day 2, CHIR99021 (0.8 μM, Stemgent, Cambridge, MA) was added. The entire medium was replaced whenever the composition of the medium was changed. SB431542 was dropped from day 5 and FGF8b (100 ng/ml, Peprotech) was added from day 7. On day 11, the basal medium was completely changed to N2, and B27, GlutaMAX, MEM-NEAA, and β-mercaptoethanol were not added. From the 11th day, the concentration of CHIR99021 added increased by about 2 times (1.5 μM), and from this time, the organoids were transferred from a 96-well plate to a 6-well plate (low attachment, Corning) and cultured on an orbital shaker at 80 rpm. It 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, organoids were digested with Acutase for 7 min at 37 °C, then minced with a 30 gauge needle and plated onto vitrosectin-coated 6 cm plates (Corning, cells from 24 organoids/plate). At this point the cells were at the Neural Stem Cell (NSC) stage and were proliferative and were 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). Medium exchange was performed daily, and Y27632 was treated at a concentration of 5 μM for one day at the time of the first transition to the second-dimensional 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).

Isolation of midbrain astrocytes from midbrain organoids and differentiation into astrocytes

If the number of passages of the generated mesencephalic Og-NSCs is increased according to the actual brain development stage, it is possible to culture Og-astrocytic progenitor cells mainly containing astrocytes after 9 passages without changing the medium composition. The 9th to 11th passages can be used in a healthy condition. As with Og-NSCs, the growth medium is N2 growth medium, 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 is doxycycline. Medium exchange was performed daily, and Y27632 was treated at a concentration of 5 μM for one day at the time of the first transition to the second-dimensional culture and at each subculture. At the time of transplantation, proliferating astrocytes were used, and at the time of differentiation for research, they were 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).

[result]

Confirmation of fabrication of 3D midbrain organoids

If the mesencephalic organoids shown in FIG. 7 are produced and passages are further increased according to the actual brain development stage, mesencephalic organoids capable of differentiating into not only mesencephalic neurons but also astrocytes can be prepared. Therefore, the results on the preparation and characteristics of mesencephalic organoids are the same as those shown when differentiating neurons through mesencephalic organoids.

Confirmation of securing midbrain astrocytes from prepared midbrain 3D organoids

① Direct differentiation of midbrain neural stem cells and securing astrocytes

To induce the differentiation of hESC/iPSC into neural stem cells into mesencephalon-neuronal stem cells, midbrain-neuronal stem cells were obtained using Neuroectoderm inducer and mesenbrain-type neural stem cell inducer for about 20 days at the embryonic stem cell stage. The secured neural stem cells are easy to proliferate and stock, and a larger number of neural stem cells can be obtained through subculture. Proper differentiation can be confirmed through the expression rates 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 mesencephalic neural stem cells can be induced with high efficiency of 95% or more as follows. In order to obtain astrocytes, several subcultures are additionally performed. Terminally differentiated cells at the time of initial subculture have a higher differentiation rate into neurons than astrocytes, but the proportion of astrocytes increases as subculture progresses. Although the existing protocol was able to identify astrocytes upon differentiation after subculture 5 times, in order to establish more efficient astrocytes, neural stem cells of passages 9 to 11 are used as astrocyte progenitor cells. At this time, CD44, an astrocyte progenitor cell marker, was more than 90%, and when differentiation was induced, as shown in FIG. 19, the differentiated astrocyte marker group (GFAP, AQP4, GLAST, S100b) was expressed more than 90%.

② Observation of higher quality effect of mesencephalic astrocytes

As a result of in vitro characterization, it was observed that mesencephalic astrocytes secreted more useful cytokines and overexpressed factors related to neuronal function maintenance (FIG. 20). As a result of in vivo , it was also observed that the secretion of more useful factors was promoted in mesencephalic astrocytes (FIG. 21). In addition, the effect of these astrocytes affects microglia and is involved in the change to a better type of microglia, so a synergistic effect of anti-inflammatory effect can be expected (FIG. 22).

Confirmation of midbrain astrocyte differentiation

Confirmation of protocol application using hiPSC

An experiment was conducted to determine whether the secured protocol was applied to other types of embryonic stem cells as well as H9 hESC. To this end, iPSCs were obtained from the Centers for Disease Control and Prevention (CMC-hiPSC-003 and 011). In the culturing step, adaptive culture was successfully performed using a Xeno-free system that does not use animal-derived components such as mouse feeder or matrigel previously used for culturing human embryonic stem cells (FIG. 23), and a large number of cell stocks were secured. did

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

Normal differentiation into midbrain astrocytes was confirmed (FIG. 25). That is, 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 iPSC lines used, #11 is more easily differentiated, and will be a very important step in securing usable induced pluripotent stem cells for practical use in the future.

Efficient differentiation induction using TGF-β and LIF

Through efficient induction of differentiation using TGF-β and LIF, a technology to secure a larger amount of astrocytes was secured. By treating TGF-β in the proliferation phase and LIF in the differentiation phase, a greater amount of astrocytes are differentiated (by GFAP marker expression), and a higher amount of neuromodulators are expressed in these astrocytes, resulting in a high quality quality. Cellularity was observed (FIG. 26). In order to confirm the amount of astrocyte progenitor cells, EdU, which can be marked only on dividing cells during the proliferation phase, was treated for only 30 minutes, and after complete differentiation, the amount of astrocytes and simultaneous expression was observed to confirm the increase in the progenitor cells of astrocytes. As a result, it was confirmed that the amount of astrocytes was increased 1.5 times.

In order to see the utilization of the astrocyte-derived material obtained through the final protocol, the characterization of the conditioned medium was firstly analyzed.

- Cell characteristics analysis: To analyze the characteristics of the secured astrocytes, physiological characteristics were analyzed to measure the physiological activity of mesencephalic astrocytes, and through this, it was confirmed that they were immature astrocytes suitable for securing differentiation derivatives and transplantation [ Figure 27]. Given that fully mature astrocytes can easily assimilate badly in adverse environments, these immature forms of cells are more suitable for transplantation.

- Function analysis: After transplantation, it was performed to see the function of astrocytes, and whether they functioned as high-quality cells in vivo after transplantation was observed in vitro and in vivo . Experiments using conditioned media were conducted to determine whether the in vitro results were suitable for improving the surrounding environment.

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

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

30 is RT-PCR to confirm the anti-inflammatory effect of astrocyte conditioned media after inflammation induction. Through this, the anti-inflammatory effect by astrocytes was verified in vitro . Also, in the cell culture containing microglia, when inflammation was induced by LPS treatment, treatment with the conditioned medium of mesencephalic astrocytes reduced the expression of factors related to inflammasome formation and increased the expression of neurogenic factors.

Characterization after transplantation to see the in vivo utilization of astrocytes obtained through the final protocol

First of all, a study was observed to see the effect of the positive role of astrocytes on the surrounding environment after transplantation into the living brain in vivo . After transplanting astrocytes to the striatum area, 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 at the astrocyte transplant site, and the expression of inflammatory factors such as IL1b, iNOS, and CD11b was decreased at the transplant site and periphery. Caine expression was slightly enhanced (FIG. 31).

Observed in vivo the effect of improving the environment after ex vivo transplantation of astrocytes through changes in microglia

After transplanting the secured astrocytes into the brain striatum of the rat, neural stem cells were transplanted to the opposite side as a control group, and the M1 to M2 conversion of microglia was confirmed by observing a representative environmental change.

As a result of inducing inflammation with LPS and observing after astrocyte transplantation, the expression of iNOS, CD11b, CD16, etc., which are bad type M1 markers, was suppressed at the astrocyte transplantation site (FIGS. 32-34), and the expression of CD206 as an M2 marker By observing that this increased, a positive effect of astrocytes was observed (FIG. 35).

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

[Experiment 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, Republic of Korea).

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

hESC lines 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 lines Name used in this paper original name Source
(Human) Reprogramming factors Methods Establishing institute Retro-1 Rv-hiPS 01-1 Newborn fibroblasts 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 Univ. (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 hESC/iPSC, mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) was used in a CO ₂ incubator set at 37 °C without a feeder layer on Matrigel ^TM or vitronectin (Human; Gibco Fisher Scientific, Waltham, MA) (Gibco A31804; 0.5 μg/cm ² )-coated 6 cm dishes (Thermo Fisher Scientific, Waltham, MA) were cultured and medium change was performed daily. The differentiation capacity of the undifferentiated stem cells was maintained by changing the medium every day and subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.

Fabrication of 3D hypothalamic organoids

Organoids were prepared according to the protocol shown in FIG. 36 .

Basically, since 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 neural cell differentiation induction medium in a low attachment 96-well round bottom plate (Corning, Corning , NY) at 150 μL of 10,000 cells per well (differentiation day 0). To prepare organoids for the nervous system, SB-431542 10 μM, Noggin, 100 ng/mL were treated, and the cells were induced to the nervous system and patterned with ventral inducer SHH and Purmophamin treatment. Specific medium compositions are shown in Tables 9 and 10 below.

Isolation of hypothalamic neural stem cells from hypothalamic organoids to confirm 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 55 μM, MEM-NEAA, GlutaMAX, Ascorbic Acid, 200 μM, SB431542 (10 μM), Noggin 100 ng/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, and Purmorphamine, 2 μM were added while using the basic medium. Between days 7 and 12, a medium without SB431542 was used while using the existing basic medium. It was transferred to a 6 well plate (low binding) on day 13 and stirred with an orbitary shaker. On days 13 to 20, only Ascorbic Acid, 200 μM, FGF2, and 20 ng/mL were added to the N2 medium and cultured. After chopping in 2D state on day 21 (possible after day 18), the entire basal medium was used until plated. N2 was used, and Doxycyclin was added only on the first day of the plate. The entire medium was replaced whenever the composition of the medium was changed, and the medium used on the 13th day was used as the growth medium. Each subculture was treated with Doxycyclin 1 μg/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 medium is 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 generated hypothalamic Og-NSCs are passaged further according to the actual brain development stage, it is possible to culture Og-astrocytic progenitor cells mainly containing astrocytes after 9 passages without changing the medium composition. The 9th to 11th passages can be used in a healthy condition. As with the hypothalamic Og-NSCs, the growth medium was N2 growth medium, and the composition was 1X N2 supplement containing, 200 µM ascorbic acid, 20 ng/ml bFGF, and 1 µg/ml doxycycline. Medium replacement was performed daily, and Y27632 was treated at a concentration of 5 μM for one day at the time of the first transition to two-dimensional culture and at each subculture. Cells in the proliferative stage with a long passage, such as organoid-derived astrocytes of the midbrain, are used for transplantation studies.

[result]

Confirmation of fabrication of 3D hypothalamic organoids

As shown in FIG. 37, the fabricated hypothalamic organoid has a normal form and takes on an initial form expressing RAX as a hypothalamic marker and Nestin and Sox2, which are neural stem cell markers.

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

It was confirmed that the neural stem cells dismantled and extracted from the hypothalamic organoids became properly patterned neural stem cells expressing 96.8% of the hypothalamic markers as shown in 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 as many 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 hypothalamic stem cells produced can differentiate into cells expressing MAP2, which are nerve cells, and astrocytes expressing GFAP 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, the sensitivity to which the p-stat3 mechanism reacts was measured when specifically looking at leptin reactivity (FIG. 40), and when looking at gene responses, anorexic-related, POMC, The expression of CARTPT, PCSK1,2, LEPR, and MC4R was increased, and the expression of orexic-related genes NPY, AGRP, and NPY1R was increased, suggesting that it is fully functional. In particular, it can be seen through the expression of NKX2.1, RAX, ISL, SF1, and NGN3 that the 3-dimensional organoid-derived cells are better patterned than in the 2-dimensional culture (FIG. 41).

Securing hypothalamic neural stem cells as stem cells responsive to leptin shows that they can actually perform their functions similar to those in vivo, and are likely to be used primarily for research on metabolic diseases such as obesity and diabetes and development of therapeutic agents in the future. It suggests that there is (FIG. 40).

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

The possibility of in vivo transplantation of hypothalamic neural stem cells developed using 3D organoids was confirmed. After transplanting 1X10 ⁵ hypothalamic neural stem cells into the 3 ^rd ventricle, the engraftment possibility was confirmed 7 days later. It was confirmed that the hypothalamic neural stem cells expressing hNCAM/Rax were stably engrafted, and at the same time, it was confirmed that the hypothalamic neural stem cells expressing hGFAP/POMC were in the form of hypothalamic neural stem cells (FIG. 42).

Claims (20)

After induction of differentiation into dopaminergic neurons, the ratio of MAP2-positive cells, a marker of neuronal cells, among DAPI-positive cells in the entire cell population is 75% or more, and the ratio of TH-positive cells, a marker of dopaminergic neurons, among the MAP2-positive cells is 25% or more And, among the TH-positive cells, the ratio of NURR1-positive cells is 70% or more, or the ratio of cells positive for one or more markers selected from the group consisting of FOXA2, LMX1A, and EN1 is 75% or more, and DAPI before differentiation Derived from mesencephalic organoids with a cumulative population doubling level (PDL) of 9 or more until the 5th passage and the ratio of FOXA2 and LMX1A-positive cells, KI67 and NESTIN-positive cells, OTX2-positive cells and SOX2-positive cells among positive cells is 75% or more neural stem cells. delete According to claim 1,
Compared to 2D culture-derived neural stem cells, the expression of one or more genes selected from the group consisting of DBN1, HSP90AB, CCT8, SERBP1, KIF5B, RDX, MACFA, EIF4G2, HNRNPK, HSPAB, BZW1, ZC3H15, and RSL1D1 is significantly increased. To, mesencephalic organoid-derived neural stem cells. delete delete delete A pharmaceutical composition for the treatment of neurodegenerative diseases or inflammatory degenerative diseases, comprising the mesencephalic organoid-derived neural stem cells of claim 1 or 3 as an active ingredient. According to claim 7,
The neurodegenerative diseases include 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, cortico-basal ganglia degeneration, diffuse Lewy body disease, and A pharmaceutical composition for the treatment of neurodegenerative diseases or inflammatory degenerative diseases, characterized in that one selected from the group consisting of Pick's disease. According to claim 7,
The inflammatory degenerative disease is a pharmacology for the treatment of neurodegenerative diseases or inflammatory degenerative diseases, characterized in that one selected from the group consisting of dementia, Lewy body dementia, frontotemporal dementia, leukodystrophy, adrenoleukodystrophy, multiple sclerosis and Lou Gehrig's disease enemy composition. delete delete delete delete delete delete delete delete delete delete delete

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