The present invention relates to a differentiation method, which separates and chops 3D organoids prepared from human pluripotent stem cells to secure a large amount of finally differentiated cells.
Parkinson's disease is a neurodegenerative disease caused by loss of dopamine neurons in the substantia nigra in the midbrain. It is known to be the most suitable disease for stem cell transplantation because the lesion site and lesion cells are clear.
For the cell therapy of Parkinson's disease, a technology to differentiate from stem cells into human midbrain-type dopamine neurons (mDA neurons) is required, and differentiation methods have been developed for a long time to differentiate from human pluripotent stem cells (hPSCs, human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs)) into mDA neurons [3D brain Organoids derived from pluripotent stem cells: promising experimental models for brain development and neurodegenerative disorders, Journal of Biomedical Science (2017) 24:59]. However, in using these differentiation methods, for clinical application, several problems have been presented as follows.
First, there is a problem of reproducibility. Because the existing two-dimensional method is very complicated, it is not well reproduced in other laboratories. In addition, the existing two-dimensional method was applied only to some pluripotent stem cell lines such as H9, which was used for development in all the developed methods, and differentiation into midbrain dopamine neurons did not occur well in several other pluripotent stem cell lines.
Second, there is a problem of stable expression of midbrain-specific factors. Midbrain-specific factors such as Nurr1, Foxa2, and Lmx1a are known to be critically important for the survival, function, and maintenance of midbrain dopamine neurons, and thus they are closely related to the ability to treat Parkinson's disease after transplantation. Because of this fact, all of the recently developed protocols for differentiating hPSC-mDA neurons were developed focusing on the expression of midbrain-specific factors in midbrain dopamine neurons. However, the expression of these midbrain-specific factors is very unstable and is easily reduced after long-term culture or transplantation of midbrain dopamine neurons. Therefore, in transplanting cells differentiated by the methods developed up to now, only the expression of some midbrain-specific factors (mainly Foxa2) is shown in the transplanted midbrain dopamine neurons.
Third, there is a problem in that astrocytes do not exist in the culture of differentiated mDA neurons. Astrocytes are not included in the culture of mDA neurons differentiated by the methods developed up to now. Astrocytes are cells that are responsible for the enhancement of survival and function of neurons such as mDA neurons, and are essential for the maintenance of normal survival and function of neurons in actual brain tissue. In addition, astrocytes are required to maintain the expression of midbrain-specific factors in mDA neurons.
Finally, there is a problem in the amount of neurons that can be obtained through differentiation of neurons. Since the dopaminergic neuron differentiation methods developed up to now induce differentiation into neurons directly without a neural stem cell stage capable of proliferating in the middle, there is a limit to the amount of finally differentiated neurons that can be obtained through one differentiation.
Accordingly, the present inventors made research efforts focusing on the fact that three-dimensional culture using organoids more closely implements the actual brain environment than two-dimensional cell culture, and enables healthy cell culture. Each organoid capable of sufficiently containing target cells, i.e., progenitor cells or stem cells, was patterned to prepare a cell population of a desired fate into a plurality of cell populations (target cell enriched). This organoid tissue was chopped, and progenitor cells or stem cells were cultured. Other technologies are applied to the development of a differentiation method by either patterning and quantitatively securing cells by a two-dimensional culture method, or borrowing only a three-dimensional sphere form in the intermediate stage in order to obtain cells, whereas in the present invention, cells in a desired state are sufficiently enriched (target cells) by appropriate culture through the organoid in a perfect form. Therefore, it was confirmed that each cell isolated in the present invention was more functionally superior by going through a complete three-dimensional artificial brain region called an organoid compared to other existing technologies.
Therefore, an object of the present invention is to provide a method of patterning and chopping 3D organoids prepared from human pluripotent stem cells, culturing the stem cells or progenitor cells, and inducing the differentiation thereof to obtain a large amount of finally differentiated cells.
In addition, another object of the present invention is to provide a cell therapeutic agent comprising the differentiated cells obtained by the above method as an active ingredient.
In addition, another object of the present invention is to provide a drug screening method using the differentiated cells obtained by the above method.
In the present invention, the finally differentiated cells may be differentiated in a large amount by chopping the prepared 3D organoids, separating stem cells or progenitor cells from the organoids and culturing the same to proliferate them, and thus a large amount of cells may be obtained at once. For example, when the method of the present invention is applied to mDA neuron differentiation, differentiation may be easily induced without cell line-dependent or batch-to-batch variation, and thus the method of the present invention may be easily reproduced in other laboratories. In addition, it was confirmed that, when the midbrain-type neural stem cells differentiated according to the present invention are transplanted into Parkinson's disease, a series of Nurr1, Foxa2, Lmx1a, and the like, which are known to be critically important for the survival, function, and maintenance of mDA neurons, are faithfully expressed in the transplanted mDA neurons, and thus the therapeutic effect of cell transplantation is remarkably improved. In addition, astrocytes, which are cells responsible for enhancing the survival and function of neurons, also exist along with mDA neurons, and thus astrocytes protect mDA neurons and enhance the function of mDA neurons, which seems to have contributed to the enhancement of cell transplantation treatment during transplantation.
Therefore, the differentiated cells obtained in such a large amount (including cerebral astrocyte progenitor cells, midbrain astrocyte progenitor cells, midbrain dopaminergic neural stem cells, hypothalamus neural stem cells, and the like, in addition to mDA neurons) may be used as a cell therapeutic agent or for the screening of drugs capable of being treated with cells.
Hereinafter, the present invention will be described in more detail.
The present invention relates to a method of patterning and chopping 3D organoids prepared from human pluripotent stem cells, culturing the stem cells or progenitor cells, and inducing the differentiation thereof to obtain a large amount of finally differentiated cells.
As used herein, the term “pluripotent stem cell (PSC)” refers to a stem cell capable of inducing differentiation into any type of cell constituting the body, and the pluripotent stem cell includes an embryonic stem cell (ESC) and an induced pluripotent stem cell (Ipsc, dedifferentiated stem cell).
As used herein, the term “organoid” refers to a ‘mini-organ like’ made to have a minimum function using stem cells, and is characterized in that it is made in a three-dimensional structure and may create an environment similar to an actual body organ even in a laboratory. That is, “organoid” refers to a cell having a 3D stereostructure, and refers to a model similar to organs such as nerves and intestines prepared through an artificial culture process that is not collected or acquired from animals and the like. The origin of the cells constituting it is not limited. The organoid may have an environment that is allowed to interact with the surrounding environment in the process of cell growth. Unlike 2D culture, 3D cell culture allows cells to grow in all directions ex vivo. Accordingly, in the present invention, the 3D organoid may be an excellent model for observing the development of therapeutic agents for diseases and the like by almost completely mimicking the organs that actually interact in vivo.
An organoid may generally be prepared by culturing human pluripotent stem cells. Specifically, it may differentiate from induced pluripotent stem cells derived from Parkinson's disease into neuroectodermal spheres, or differentiate into intestinal organoids through step-by-step differentiation from induced pluripotent stem cells derived from Parkinson's disease into definitive endoderm and hindgut.
As used herein, the term “differentiation” refers to a phenomenon in which the structure or function of cells is specialized during division and proliferation of cells and growth of the entire individual. In other words, it refers to a process of a change into a suitable form and function of cells, tissues, and the like of organisms in order to perform their respective given roles. For example, differentiation may include a process in which pluripotent stem cells are transformed into ectoderm (cerebral cortex, midbrain, hypothalamus, etc.), mesoderm (yolk sac, etc.) and endoderm cells, as well as a process in which hematopoietic stem cells are transformed into red blood cells, white blood cells, platelets, and the like, that is, a process in which progenitor cells express specific differentiation traits.
The existing method of inducing differentiation directly from human pluripotent stem cells to differentiate them into the corresponding cells has a problem in reproducibility depending on the cell line or laboratory (experiment environment), and does not achieve stable expression of maintenance factors. For example, in the case of midbrain dopamine neurons, unlike the actual in vivo environment, astrocytes do not exist together, and there is a limit to the amount of final cells that may be obtained through one differentiation. As in one embodiment of the present invention, by patterning and preparing organoids capable of sufficiently containing cerebral astrocyte progenitor cells, midbrain astrocyte progenitor cells, midbrain dopaminergic neural stem cells, or hypothalamus neural stem cells, respectively (target cell enriched), that is, securing organoids containing the maximum amount of each target cell, and chopping the organoid tissues to culture the corresponding stem cells or progenitor cells, as compared to the two-dimensionally differentiation-induced cell population, they may be more similar to the cells isolated from the actual brain, their properties may be well maintained, and it is possible to obtain differentiation into a cell population in which viability is obtained.
As used herein, the term “patterning” refers to the preparation of an organoid such that, when preparing an organoid, the cell population with the fate having the property of the origin tissue of the cell to be finally extracted from among the brain detailed tissues in this way, such as midbrain-type pattern, cerebral cortex-type pattern, or hypothalamus-type pattern, is contained as a plurality of cell populations (target cell enriched). In addition, patterning markers include Lmx1a, Foxa2, Nurr1, and En1 for midbrain-type patterning; Pax6 and Foxg1 for cerebral cortex-type patterning; and Rax and Nkx2.2 for hypothalamus-type patterning, and the like.
Therefore, in the present invention, the finally differentiated cells may be obtained in a large amount at once by chopping the prepared 3D organoids, and separating stem cells capable of being proliferated from the organoids and culturing the same to proliferate them.
According to one embodiment of the present invention, it includes a method, which chops the 3D organoids prepared from human pluripotent stem cells to obtain a large amount of stem cells or progenitor cells and finally differentiates them, comprising:
1) proliferating and culturing human pluripotent stem cells to prepare 3D organoids;
2) patterning and chopping the prepared 3D organoids; and
3) culturing and proliferating stem cells or progenitor cells in the cells extracted from the chopped organoids, and inducing the differentiation thereof to obtain a large amount of the finally differentiated cells.
The stem cell may be an ectodermal stem cell (skin and nerve cells), a mesodermal stem cell (muscle and bone cells), or an endodermal stem cell (digestive and respiratory cells). Specifically, it may be a midbrain dopaminergic neural stem cell or a hypothalamus neural stem cell.
The progenitor cell may be a cerebral astrocyte progenitor cell or a midbrain astrocyte progenitor cell, but is not limited thereto.
The cell may be a neuron or an astrocyte, but is not limited thereto.
More preferably, the human pluripotent stem cells and stem cells are cultured using vitronectin. In this regard, it is appropriate to use vitronectin at 0.3 to 0.7 μg/cm2. Up to now, according to the report of CellStemCell of Parmar group (A. Kirkeby et al., Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease. Cell Stem Cell 20, 135-148 (2017).), Nature Protocol (S. Nolbrant, A. Heuer, M. Parmar, A. Kirkeby, Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nature protocols 12, 1962-1979 (2017)), which has reported the latest research on mass-producing dopaminergic neurons for transplantation, it was reported that it is very important to use Lamin521 (BioLamina LN-521) at 0.5 μg/cm2 for proliferation and culture of human embryonic stem cells and to use Lamin511 (BioLamina LN-511) at 1 μg/cm2 after differentiation into midbrain neurons. In contrast, in the Examples of the present invention, vitronectin (Gibco A31804) at 0.5 μg/cm2 was used for both culturing of human embryonic stem cells and culturing of midbrain neural stem cells. In terms of the coating unit price of a 60 mm dish generally used for culture, the price of Lamin521 for culturing human embryonic stem cells is 7,282 won/dish, the price of vitronectin in the present invention is 1,480 won/dish, and the price of Lamin511 for culturing midbrain neural stem cells is 23,832 won/dish, whereas the price of vitronectin continuously used in the present invention is 1,480 won/dish. Even if only the coating cost is taken into consideration, the method of the present invention has an effect of saving 5 times when culturing human embryonic stem cells and 16 times when culturing midbrain neural stem cells.
As used herein, the term “large amount” means that it is possible to obtain about 1,000 to 7,200 vials of frozen cells when introduced from the start of one culture dish of pluripotent stem cells used for the first time although there is a difference depending on each patterned cell population, and refers to an 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 proliferation, but also maintenance of properties.
As used herein, the term “glia” refers to a cell that occupies the largest portion of cells present in the brain, and refers to an “astrocyte.” The astrocyte is also referred to as an microglia and is involved in the protection of neurons and supply of nutrients 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 also obtained by isolation from midbrain (ventral midbrain), cerebral cortex (cortex) or lateral ganglionic eminence (striatum anlage) in mammals.
“Neural stem/precursor cells (NSCs/NPCs)” are isolated and cultured from brain tissue in developing or adult brain tissue, and the cultured NPCs may be used for mass formation of dopamine neurons for research and drug screening.
A “nerve cell” is a cell of the nervous system, and the term “neuron” or “neuron cell” may be used interchangeably.
“Dopamine (DA) nerve cell” refers to a nerve cell expressing tyrosine hydroxylase (TH). In the present invention, “dopaminergic nerve cell,” “dopamine neuron,” “DA,” and the like are used interchangeably. Dopamine nerve cells are specifically located in the midbrain substantia nigra, and stimulate the striatum, limbic system, and neocortex in vivo to control postural reflexes, migration, and reward-related behaviors.
The present invention also includes a cell therapeutic agent comprising cells obtained by the above method.
“Cell therapeutic agent” is a drug used for treatment, diagnosis, and prevention with cells and tissues prepared through isolation, culture, and special modification from a subject (U.S. FDA regulations). It refers to a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating or sorting living autologous, allogeneic, or xenogeneic cells ex vivo, or changing the biological properties of cells in other ways to restore the function of cells or tissues. Cell therapeutic agents are largely classified into somatic cell therapeutic agents and stem cell therapeutic agent according to the degree of cell differentiation.
As used herein, the term “subject” may be a vertebrate to be treated, observed or experimented, preferably a mammal, for example, cattle, pigs, horses, goats, dogs, cats, rats, mice, rabbits, guinea pigs, human, and the like.
As used herein, the term “treatment” refers to any action that inhibits, alleviates, or advantageously alters a clinical situation related to a disease. In addition, treatment may mean increased survival compared to the expected survival rate if not receiving treatment. Treatment simultaneously includes prophylactic means in addition to therapeutic means.
The cell therapeutic agent of the present invention exhibits a therapeutic effect on a disease selected from the group consisting of a neurodegenerative disease, an inflammatory degenerative disease, and a metabolic disease.
The neurodegenerative disease may include, for example, one selected from the group consisting of 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, corticobasal degeneration, diffuse Lewy body disease, and Pick disease, but is not limited thereto.
The inflammatory degenerative disease may include one selected from the group consisting of dementia, Lewy body dementia, frontotemporal dementia, white matter degeneration, adrenoleukodystrophy, multiple sclerosis, and Lou Gehrig's disease, but is not limited thereto.
The metabolic disease may include one selected from the group consisting of diabetes mellitus, obesity, and a metabolic disorder, but is not limited thereto.
The cells obtained by the method of the present invention may be applied as a cell therapeutic agent, and may be formulated by further including a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not considerably stimulate the organism and does not inhibit the biological activity and properties of the administered component. In the present invention, the pharmaceutically acceptable carrier that may be included in a cell therapeutic agent may be used without limitation as long as it is known in the art, such as a buffering agent, a preservative, an analgesic agent, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and the like. The cell therapeutic agent of the present invention may be prepared in the form of various formulations according to commonly used techniques. The cell therapeutic agent of the present invention may be administered through any route as long as it may induce migration to the diseased site. In some cases, a method of loading the stem cells into a vehicle equipped with a means for directing the lesion may be also considered. Therefore, the cell therapeutic agent of the present invention may be administered through several routes including topical (including buccal, sublingual, dermal and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and intracerobrospinal fluid) or transdermal administration, and is preferably administered directly to the site of disease. In one embodiment, the cells may be administered to an individual by suspending the drug in a suitable diluent, and the diluent is used to protect and maintain the cells and to facilitate use when injected into a target tissue. The diluent may include physiological saline, a phosphate buffer solution, a buffer solution such as HBSS, cerebrospinal fluid, and the like. In addition, the pharmaceutical composition may be administered by any device to allow the active substance to migrate to the target cell. A preferred mode of administration and preparation are injections. Injections may be prepared using aqueous solutions such as physiological saline, Ringer's solution, Hank's solution or sterilized aqueous solution, vegetable oils such as olive oil, higher fatty acid ester such as ethyl oleic acid, and non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerin, and the like. For mucosal penetration, a non-penetrating agent known in the art suitable for a barrier to pass through may be used, and may further include a pharmaceutical carrier such as ascorbic acid, sodium hydrogen sulfite, BHA, tocopherol, EDTA, and the like as a stabilizer for preventing deterioration, and an emulsifier, a buffering agent for pH adjustment, and a preservative for inhibiting the growth of microorganisms such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzyl alcohol.
The present invention also provides a drug screening method using the cells or progenitor cells obtained by the above method.
Important features of the cells obtained by the present invention include the possibility of securing the production of a large amount of cells, the maintenance of their properties even during cryopreservation, the possibility of maintaining the same cell population for a long period of time, and differentiation more similar to those of cells derived from a living body. This property is particularly suitable for simultaneous screening of multiple drugs, which requires a large amount of cells in the same state and is the key to securing the same cells for a long period of time for repeated analysis thereof. It is very suitable for screening cells because a cell population with the same features in which key markers are maintained may be used continuously from the beginning to the end of the screening operation.
The drug is a drug for treating a disease selected from the group consisting of a neurodegenerative disease, an inflammatory degenerative disease, and a metabolic disease, and exhibits a therapeutic effect on the disease selected from the group consisting of a neurodegenerative disease, an inflammatory degenerative disease, and a metabolic disease.
The neurodegenerative disease may include, for example, various nervous system diseases including 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, corticobasal degeneration, diffuse Lewy body disease, and Pick disease, but is not limited thereto.
The inflammatory degenerative disease may include one selected from the group consisting of dementia, Lewy body dementia, frontotemporal dementia, white matter degeneration, adrenoleukodystrophy, multiple sclerosis, and Lou Gehrig's disease, but is not limited thereto.
The metabolic disease may include one selected from the group consisting of diabetes mellitus, obesity, and a metabolic disorder, but is not limited thereto.
All technical terms used in the present invention, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. In addition, although preferred methods and samples are described herein, ones similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications described herein as a reference are incorporated herein by reference.
Hereinafter, the present application will be described in detail through examples. The following examples are only for illustrating the present application, and the scope of the present application is not limited to the following examples.
[Experimental Method]
Culturing of human embryonic stem cells or human induced pluripotent stem cells hESCs and hiPSCs were cultured based on the hESC research guidelines approved by the institutional review board (IRB) of Hanyang University (Seoul, Republic of Korea). The hESCs and hiPSCs used in this experiment are shown in Table 1 below.
For proliferation and maintenance of undifferentiation hESC/iPSC, the cells were cultured on Matrigel™ or on a vitronectin (Human; Gibco Fisher Scientific, Waltham, Mass.) (Gibco A31804; 0.5 μg/cm2)-coated 6 cm dish (Thermo Fisher Scientific, Waltham, Mass.) using mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) in a CO2 incubator set at 37° C. without a feeder layer, and medium replacement was performed daily. The undifferentiated stem cells maintained their differentiation ability by replacing the medium daily, and were subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.
Preparation of Midbrain-Type Neural Stem Cells Using 3D Organoid Preparation Method
Briefly, a system was used in which a midbrain-type 3D organoid was first prepared, cut into small pieces, and proliferated in a large amount in a culture dish in a state of midbrain-type neural stem cells.
hESCs and hiPSCs, which were normally cultured in an undifferentiated state, were detached using Accutase™ during subculture, and human embryonic stem cells in an undifferentiated state were aliquoted into a 96 well plate with a round bottom that does not stick to the bottom at 10,000 cells/well to form organoids once. In the medium composition of the first day, the generation of organoids was started with the additive conditions (SB431542, 10 μM; Noggin, 100 ng/ml; ascorbic acid, 200 μM; doxycyclin, 1 μg/ml; Y27632, 20 μM) corresponding to Day 0 of Table 2 in the seeding base medium of Table 4 below. The next day, the neural inducing base media (Table 3) was used as a medium, and development was induced by using a medium in which purmorphamine 2 μM, sonic hedgehog 100 ng/ml were additionally added to the components except for doxycyclin, 1 μg/ml, Y27632, 20 μM among the components added on the first day (see Table 2). The culture medium was the neural inducing base media until day 11, and development was induced by adding or subtracting the additives according to the concentration with reference to Table 2 for the addition components. The medium was replaced daily according to the date. Correct formation of organoids was confirmed by the formation of a cell mass on days 2 to 3, and was checked by budding into the outside of the cell before day 11. On day 11 of organoid differentiation, eight organoids formed one by one in a 96 well plate were grouped into each group and aliquoted and cultured into respective wells of a 6 well low binding plate. Development was induced under the midbrain patterning base media (Table 3) containing the components of Table 2 while rotating with an orbitary shaker (80 to 100 rpm). On day 18, one organoid was cut into about 4 to 10 pieces with a 30 G needle, treated with Accutase™ for 10 minutes, and then pipetted 5 times with a 1 ml pipette, and then Accutase™ was removed by centrifugation, and then 3 wells of organoid cells were center-plated in one 60 mm culture dish coated with poly-L ornithine (PLO; 15 μg/cm2)/fibronectin (FN; 1 μg/cm2) or vitronectin (VN; 0.5 μg/cm2).
Mass Proliferation of Midbrain 3D Organoid-Derived Midbrain-Type Neural Stem Cells
The organoid-derived midbrain-type neural stem cells were physically cut with a 30 G needle. After the first plating for subculture, 3×106 cells were cultured in plane in a 60 mm culture dish coated with PLO/FN or PLO/VN using Accutase™ once a week for subculture when the cells were about 80 to 90% full. For the culture medium of midbrain-type neural stem cells, a medium in which mitogen (bFGF 20 ng/ml, FGF-8 50 ng/ml), BDNF (20 ng/ml), GDNF (20 ng/ml), ascorbic acid 200 μM, doxycyclin, 1 μg/ml were added to the midbrain patterning base medium of Table 4 was used as a proliferation medium. However, on the day of subculture, Y27632, 20 μM was added to this medium. At every subculture, 8×104 cells per well were plated on a PLO/VN 24 well coated plate and proliferated for 2-3 days. When 80-90% cell confluency was reached, differentiation of NSCs was induced by fluorescence staining of midbrain-type markers or removal of mitogens (bFGF, FGF-8). For the differentiation medium, BDNF (20 ng/ml), GDNF (20 ng/ml), Ascorbic acid 200 μM, and cAMP 500 μM were added to the midbrain patterning base medium shown in Table 4 above to induce the differentiation of neurons for 12 days, and then it was checked whether the dopaminergic neuron and midbrain-type markers were co-expressed.
Frozen Storage of 3D Organoid-Derived Midbrain-Type Neural Stem Cells
Since organoid-derived midbrain-type neural stem cells generally do not change their properties at least until passage 5, the cells can be thawed and used without change in properties after frozen storage of the cells at every passage after the first passage. The frozen storage of the cells was made during subculture, the cells were detached using Accutase™, the number of cells was counted, and 6×106 cells were dissolved and frozen in a freezing medium mixed with a proliferation medium and DMSO at a concentration of 10% in one freezing vial. The cells were stored frozen at −70° C. for the first 24 hours and then stored in a cryogenic nitrogen tank the next day until use. If necessary, one vial was quickly thawed in a 37° C. water bath, and the cells were cultured in plane in a 60 mm culture dish coated with PLO/FN or PLO/VN. For thawing, a neural stem cell proliferation medium was used, and on the day of thawing, Y27632, 20 μM was added to this medium.
Immunocytochemistry
The cells were fixed with 4% paraformaldehyde in PBS (phosphate-buffered saline), cultured in a blocking solution (1% BSA/0.03% Triton X-100 in PBS), and then cultured together with the following primary antibodies. Tuj 1 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 fluorescence molecules were used for visualization. Other antibodies are listed in Table 4. Alexa 488 (1:200, Invitrogen) and Cy3 (1:200, Jackson ImmunoResearch Laboratories). The stained sample was mounted in a Vectashield (Vector Laboratories) with DAPI mounting medium and photographed using an epifluorescence microscope (Leica).
1Santa Cruz
2DSHB
3Covance
4Immunostar
5Novocastra
6BD Biosciences
7Chemicon
8Sigma
9Dr Martha Marvin,
10Pel-freez
11DAKO
12Cell signaling
Cell Transplantation and Histological Procedure in Parkinson's Disease Model Animal
Experiments were performed according to NIH (National Institutes of Health) guidelines. Hemi-parkinsonian was induced in mature female Sprague-Dawley rats (220-250 g) by stereotaxic injection of 3 μl of 6-OHDA (6-hydroxydopamine, 8 μg/μl; Sigma) alone into the right substantia nigra (AP-4.8 mm, ML-1.8 mm, V-8.2 mm) and MFB (median forebrain bundle) (AP −4.4 mm, ML −1.2 mm, V −7.8 mm). The incisor bar was set to −3.5 mm, and the AP and ML coordinates were given relative to the bregma. The rats with 300 rotations/hour on the same side for this disorder were selected in the amphetamine-induced rotation test. For transplantation, rat E12 VM-NPCs were proliferated and mixed with Ctx-, VM-astrocytes, N+F-VM-astrocytes or E14 Ctx-NPCs (control group) in a 2:1 ratio. 3 μl of the mixed cells (1.5×105 cells/μl) was injected into two sites of 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), respectively, over 10 minutes under anesthesia induced by 100 μl/100 g (50 mg/ml) of Zoletil mixed with 100 μl/100 g (23.32 mg/ml) of Rompun. The needle (22 gauge) was left in place for 5 minutes after completion of each injection. The rats were administered daily with cyclosporine A (10 mg/kg, i.p.) from one day before transplantation for 1 month and maintained without immunosuppressants for the remainder of the post-transplantation period. Six months after transplantation, the animals were anesthetized and transdermally perfused with 4% paraformaldehyde. The brains were removed, soaked in 30% sucrose in PBS overnight, frozen in Tissue-Tek® (Sakura Finetek, Torrance, Calif., USA), and then sliced in a frozen microtome (Leica). Free-floating brain sections (thickness of 30 μm) were subjected to immunohistochemistry as described above and imaged by a confocal microscope (Leica). In experiments to test the host environment of transplanted brains, the animals were sacrificed one month after transplantation and sliced to a thickness of 1 mm on a rat brain slice matrix (ZIVIC Instruments, Pittsburgh, Pa.). In order to observe the changes after transplantation, the tissue at transplantation site was extracted. 8-12 regions of the graft-host interface (ca 2×2 mm)/graft were dissected, and qPCR analysis was performed. In addition, cells immunoreactive to neurotrophic and pro-inflammatory glia markers were counted along the graft-host interface of the freeze cleaved brain slices 7-10 days after transplantation.
Behavior Test
Animal behavior was evaluated using the apomorphine/amphetamine-induced rotation test as described in the paper by Y. H. Rhee et al. (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)). Apomorphine (Sigma) was injected subcutaneously at a dose of 0.5 mg/kg and observed while rotating for 60 minutes. The results are expressed in a unit of final number of revolutions/60 minutes.
Animal PET/MRI Imaging and Analysis
PET-MRI fusion imaging was performed using a nanoScanPET/MRI system (1T, Mediso, Hungary). In order to keep mice warm, 0.2 mL of FP-CIT was administered intravenously via tail vein 6.5±1.0 MBq, and the rats were maintained under anesthesia (1.5% isoflurane in 100% O2 gas). MR brain imaging obtained T1 images weighted with gradient echo (GRE) 3D sequences (TR=25 ms, Tarr=3.4, FOV=50 mm, matrix=256×256), and T2 images weighted with fast spin echo (FSE) 3D sequences (TR=2400 ms, Tarr=110, FOV=50 mm, matrix=256×256) acquired during the FP-CIT uptake period. A 20 minute static PET image was acquired with a 1-3 concordance in a single field of view within the MRI range. Body temperature was maintained with heated air of the animal bed (Multicell Mediso, Hungary), and pressure-sensitive pads were used to induce respiration. PET images were reconstructed by Tera-Tomo 3D in full detector mode with all corrections and high normalization and 8 repetitions. Three-dimensional VOI (volume of interest) analysis of the reconstructed images was performed by using the InterView Fusion software package (Mediso, Hungary) and applying standard uptake value (SUV) analysis. The VOIs were fixed with 2 mm spheres in the coronal images, and VOIs for the striatum were derived. The SUV of each VOI site was calculated using the following equation. SUVmean=(Bq/cc×tumor radioactivity in the tumor volume of interest in body weight)/radioactivity injected.
High-Throughput Whole Transcriptome RNA Sequencing
For RNA, total RNA was isolated using a Trizol reagent (Invitrogen), and then the removal of rRNA was performed using the Ribo-Zero Magnetic kit (Epicentre Inc., Madison, Wis., USA). Library construction was performed using the SENSE mRNA-Seq Library Prep Kit (Lexogen Inc., Vienna, Austria). High-throughput sequencing was performed as paired-end 100 sequencing using the HiSeq 2000 (Illumina, San Diego, Calif., USA). RNA-Seq read values were mapped using TopHat software to obtain bam alignment files. Read counts mapped to transcription regions were extracted from alignment files using BEDTools (v2.25.0) and R/Bioconductor (version 3.2.2; R Development Core Team, 2011). The alignment files were used to assemble transcriptomes, estimate their abundance, and detect differential expression of genes, linc RNAs or isoforms. FPKM (fragments per kb of exon per million fragments) was used to determine the expression level of the gene region. Global normalization was used for comparison between samples. Genetic classification is based on searches submitted to DAVID (http://david.abcc.ncifcrf.gov/).
GO and KEGG pathway analysis was performed using DAVID Bioinformatics Resources version 6.8. GO and KEGG pathway analysis of the rich categories gave p-value up to 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, Calif.) program after experiments of Chromium Single cell Gene Expression Solution and Chromium Single cell 3′ GEM, Library and Gel Bead Kit v2 (10× Genomics). Cells separated into 15,000 single cells were placed on Single cell A chip with Master mix solution. In each single cell, transcription factors inside the cell were reverse transcribed and RNA barcoded by emulsion of oligo dT encoded UMIs. The barcoded libraries were sequenced with the Illumina HiSeq 4000 platform (Macrogen Inc., Seoul, Korea). The results were analyzed with Cell Ranger (v2.1.1, 10× Genomics). For 2D-NSCs analysis, 12,166 cells with 2D_cellRanger averaged 57,343 reads and 2,284 genes per cell (among 24,055 total genes) were analyzed, and for Og-NSCs analysis (with 3D_cellRanger), 13,739 cells averaged 39,191 reads and 2,353 genes per cell (among 23,860 total genes) were analyzed. Cellular transcription factor expression was expressed as UMI count. Cell diversity analysis was performed by k-means clustering for t-SNE (t-distributed stochastic neighbor embedding) analysis (performed by Loupe Cell Browser ver. 3.0.1, 10× Genomics, Inc.). The top 40 genes with increased expression showing a significant difference in expression were analyzed by hierarchical clustering analysis method, and each cluster was divided based on this. For GO and KEGG pathway analysis of Og-NSCs, 580 differentially upregulated genes (UMI >1, log 2FC>0.5, P<0.05) were analyzed. Genetic analysis was mainly performed using DAVID (http://david.abcc.ncifcrf.gov/). Network analysis was performed using Cytoscape (version 3.7.1+JAVA v8, provided by NIGMS).
Analysis of Mitochondrial Function
The regenerative dynamics of mitochondria was analyzed using integrated portions of young (green) and old (red) MitoTimer proteins (Ferree, A. W., Trudeau, K., Zik, E., Benador, I. Y., Twig, G., Gottlieb, R. A., and Shirihai, O. S. (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, R. S., et al. (2013). MitoTimer: a novel tool for monitoring mitochondrial turnover. Autophagy 9, 1852-1861). The pMitoTimer vector (Addgene52659; Addgene, Watertown, Mass.) was injected into NSCs cells by electroporation (NEPA21, Nepagene, Japan) and expressed, and then 2 days later, mitochondria generated by red versus green fluorescence were investigated. Mitochondria ROS and membrane potential were measured with MitoSox (Thermo Scientific Inc., Waltham, Mass.) and NucleoCounter3000 (NC3000; Chemometec, Allerod, Denmark), respectively. ROS generation was analyzed for induction after treatment with H2O2 (500 μM) 2 hours before analysis. In order to study the toxicity resistance of mitochondria, mitochondria were treated with 10 μM Rotenone, 4 μM CCCP or 250 μM H2O2 for 1 hour and analyzed by TH fluorescence staining.
Analysis of DA Release
The presynaptic activity of DA neurons was determined by measuring the level of DA neurotransmitter released from the differentiated VM-NPC culture. The medium cultured for 24 hours (days 12-13 of differentiation) was collected, and the DA level was measured using an ELISA kit (BA E-5300, LDN). In addition, the DA release induced by membrane depolarization was evaluated by culturing (day 12 of culture) in a fresh N2 medium culture for 30 minutes in the presence or absence of 56 mM KCl. The induced DA release was calculated as the DA level with KCl minus the DA release without KCl.
Analysis of Intercellular α-Syn Transduction
Confirmation of Intercellular α-Synuclein Aggregate Transfer by Factors Secreted from Astrocytes
An analysis experiment was conducted using a co-culture with cells overexpressing A53Tα-syn-EGFP in SH-SYSY (Professor Sang Myun Park, Ajou University) cells derived from neuroblastoma.
SH-SYSY cells, which were induced to differentiate using RA for 5 days and induced to express A53Tα-syn-EGFP, were co-cultured for 24 hours with neural stem cells induced to differentiate for 10 days in a dual chamber system, and the transfer of A53Tα-syn-EGFP was compared by the level of GFP expression. The transfer (transmission) of α-synuclein was confirmed by immunostaining (immunocytochemistry) through the brightness of α-syn-GFP using an antibody.
Detection of Pathological α-Syn Aggregates
During differentiation, the medium was treated (8 μg/ml of medium) with PFF (pre-formed fibrils), α-synuclein aggregates, to induce α-synuclein aggregation, and the level of α-synucelin aggregation was analyzed after 20 days of differentiation. Alternatively, overexpression was induced using lentiviruses expressing α-syn (pEF1α-α-syn) and analyzed. Immunostaining (immunocytochemistry) using thioflavin S (staining method to measure protein aggregation; Sigma Aldrich) and α-synuclein antibody and protein electrophoresis (Western blot) were used to confirm the presence, and the aggregation was observed through a change in protein size.
Bi-FC (Bimolecular Fluorescence Complementation)
Og-NSCs or 2D-NSCs were induced to express lentiviruses expressing Venus1-α-syn (V1S; N-terminal of α-syn), α-syn-Venus2 (SV2; C-terminal of α-syn), and the aggregated α-syn in dopaminergic neurons that were differentiated for 25 days was analyzed by GFP.
In Vivo α-Syn Propagation into Transplanted Grafts
Female SD (Sprague Dawley) rats (225-250 g) were injected into the striatum with AAV expressing human ha-syn (AAV2-CMV-ha-syn, 2×1013 genome copies) and PFF (10 μg) to induce the expression and aggregation. anteroposterior (AP), −0; mediolateral (ML), −3.0; dorsoventral (DV), −5.0. Two weeks later, Og-NSCs and 2D-NSCs were transplanted onto both sides of the striatum, respectively. 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 the synapsin promoter (pSyn-GCaMP6s) and used to transduce the culture in vitro. 2 ml/6-cm dish or 200 μl/well (24-well plate) and 106 transduction units (TU/ml (60-70 ng/ml) were used for each transduction reaction. Packaging and production of AAV2-CMV-ha-syn were 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. Thereafter, the cells were transplanted into 4 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, Leica, Germany) equipped with a Ti:Sapphire femtosecond laser (Chameleon Vision, Coherent Inc., USA) having a tunable wavelength from 680 to 1080 nm, 80 MHz repetition rate and 140 fs pulse width.
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 an aluminum plate through a hole in the plate, and the plate was translated and the brain was inserted from above. A 10× dry objective lens (Leica HC PL FLUOTAR 10.0×0.30) was placed close to one end of the GRIN lens in order to couple the excitation laser from the microscope system. The excitation wavelength was set to 900 nm for the two-photon excitation of GCaMP6. Two-photon imaging was performed by scanning the excitation laser into the brain. The imaging field of view (FOV) and imaging speed were 387.50×387.50 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 the 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 Analysis
The medium was treated with Fluo3-AM for 1 hour so that Fluo3-AM was absorbed into the cells, and washed 3 times with PBS so that no fluorescence remained in the medium. While observing fluorescence with a confocal microscope, the intensity of fluorescence was taken at an interval of 1 to 3 seconds, and ROI was measured for each cell in each time-framed photograph, and it was confirmed that the intensity of fluorescence was changed. It was gated by FACS as needed.
Experiment Result
Preparation of Midbrain-Type Organoids from Human Embryonic Stem Cells
The conventional differentiation technology based on two-dimensional 2D culture does not represent a three-dimensional complex network in the actual brain in vivo. Only the development of a protocol based on three-dimensional generation can represent the tissue properties of the actual brain. Therefore, first of all, this research team first prepared an existing 3D midbrain-type organoid. In the early stage of development of the successfully prepared midbrain-type organoids, ZO-1 and N-cadherin were expressed along with PLZF+ and Sox2+, which are neural stem cell markers (
Isolation of Midbrain-Type Neural Stem Cells (Og-NSCs) from Midbrain-Like Organoids
When used as a cell therapeutic agent for Parkinson's disease, a technology for isolating and culturing organoid-derived midbrain-type neural stem cells (Og-NSCs) as a more evolved form of neural stem cells was developed. As a previous study, an attempt was made to extract neural stem cells from organoids during days 10 to 35 of development, but the efficiency was not high. It is believed that it is possible to secure an appropriate amount of cells by increasing the ratio of neural stem cells in the organoid, and the protocol was modified in an effort to increase the amount of cells.
(1) Since the amount of stem cell population is automatically reduced when differentiation is continuously induced, the final developmental differentiation protocol to be performed from day 10 was stopped and improved in the direction of proliferation. (2) From day 17, it was modified by adding bFGF, which can specifically increase neural stem cells. (3) The proliferation of midbrain specific neural stem cells was induced by extending the treatment period of midbrain specific factors (SHH/Purmorphamine/CHIR999021) (from day 1 to day 18), that is, by extending the process of midbrain patterning. (4) Unlike the conventional method, the FGF8b treatment period started from day 7 (A in
By controlling such a detailed time period, the development into the hypothalamus (sunthalamic), which is prone to contamination during induction of the midbrain, can be reduced, and the expression of EN1, one of the specific markers, can be significantly increased (
Organoid-Derived Midbrain-Type Neural Stem Cells (Og-NSCs) can be Differentiated into Complete Dopaminergic Neurons that Fully Express Midbrain-Type Markers Through the Final Differentiation Process
Through the final differentiation process, midbrain-type Og-NSCs can be efficiently differentiated into complete dopaminergic neurons (
The H9 hESC line showing the highest midbrain-specific factor expression was mainly used throughout this study. This is because the protocol developed from organoids is very stable, and it seems that only a small amount undergoes the process of cell death during general processes such as cell isolation, proliferation, and differentiation. This, in contrast to the 2D method, showed a significant percentage of cell death during the procedure. In actual culture, analysis of ethidium heterodimer-stained dead cells (
Og-NSC Cultured Cells can be Used as Platform Cells for New Drug Development
Og-NSCs can be patterned in the midbrain-type and differentiated into complete dopaminergic neurons after differentiation, and can maintain their properties for more than 5 passages (
Og-NSCs in an undifferentiated state perfectly express an appropriate midbrain-type neural stem cell marker (
Analysis of Transcriptional Regulation Properties of Og-NSCs Using Bulk and Single Cell RNA Sequencing
In order to analyze molecular biological transcriptional regulation of Og-NSCs, RNA sequencing (RNA-seq) analysis was performed. Organoid-derived midbrain-type neural stem cells (Og-NSCs) and neural stem cells (2D-NSCs) generated by the conventional differentiation method were compared and analyzed for differences in transcription regulation factors in the existing 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). A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project. Biopresery 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.). As in 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.), PCA (principal component analysis) and Spearman's correlation analysis showed that midbrain-type organoids were remarkably similar to human fetal brains (
In order to confirm the properties of each individual cell in the neural stem cell colony, single-cell RNA-seq was performed (Og-NSCs: a total of 13,793 cells analyzed; 2D-NSCs: 12,166 cells. On average, there were 3.8×105 post-normalization reads per cell and 2,227 genes detected in each individual cell). On average, there were 3.8×105 post-normalization reads per cell and 2,227 genes detected in each individual cell. As expected, ˜99.7% of Og-NSCs and the conventional 2D-NSC cells expressed the neural stem cell markers NESTIN, VIMENTIN, NCAM1, SOX2, NOTCH1, and MUSASHI1 (
The Og-NSCs cell populations analyzed by t-SNE (t-distributed stochastic neighbor embedding) analysis can be classified into three clusters (
Through GO and KEGG analysis, bulk and single cell RNA-seq data (Og-NSCs vs 2D-NSCs) was analyzed in more detail (
In addition, the gene group that is specifically highly expressed in the Og-NSC cell population is ‘SN development’ and ‘negative regulation of cell death’ (
The function of mitochondria is very important in the differentiation of human embryonic stem cells and induced pluripotent stem cells. In particular, the mitochondria of neural stem cells are known to be very important in the progression and pathogenesis of neurodegenerative diseases. Mitochondrial genes were all regulated in Og-NSCs, including those related to mitochondrial generation and dynamics, in two different bulk and single-RNA-seq analyses compared to cells induced by conventional methods (
Dopaminergic Neuron mDA Neurons Differentiation-Induced from Og-NSCs Coexist with Astrocytes, Resulting in Improved Neuronal Function and Maturation
The transcriptional regulation of genes actually shows early changes before the shape of the cell population changes. For example, in actual genetic analysis (in differentially expressed genes (DEGs)), the change that can be seen from neural stem cells is one that can be seen from the embryonic stem cell stage. In bulk RNA-seq analysis, genes related to neuronal maturation were highly expressed in Og-NSCs (vs 2D-NSCs) cells (
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 (
Therefore, mDA neurons were generated along with GFAP+ astrocytes in the differentiated culture as in the midbrain in vivo (
Considering the cytokine secretion ability of astrocytes based on previous reports, astrocytes can be expected to have a positive effect when dopaminergic neurons are generated. Based on this information, as shown in
Dopaminergic Neurons Differentiated from Og-NSCs are Less Prone to α-Syn Oligomerization and Pathogenic Propagation.
In order to prevent the propagation of α-synucleinopathy existing in the original patient's brain into transplanted cells, and for the long-term success of cell therapy for Parkinson's disease, it is important to secure the α-syn propagation resistance of cells to be used (transplanted) as a cell therapeutic agent. Toxic α-syn pathogenic propagation is associated with intercellular transfer of α-syn, and α-syn aggregation is also associated with transfer into transplanted cells. In order to measure the intercellular transfer ability of α-syn, Og-NSCs and 2D-NSCs were analyzed using 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), respectively. GFP-labeled α-syn (a protein overexpressed in SH-SY5Y neuronal cells) was placed on the chamber, and the cultured Og-NSC (2D-NSC)-derived neurons were placed under the chamber, and uptake and expansion was observed after protein transport (
In addition to α-syn transfer, the pathogenic propagation of α-syn into transplanted cells, which is another important issue, was analyzed by toxic α-syn aggregates. The degree of aggregation was compared and analyzed in an environment in which the amount of α-syn inside the cell is increased or in a condition in which α-syn is introduced from the outside (exogenous α-syn fibril seed).
Og-NSCs and 2D-NSCs cell populations were infected with a lentivirus overexpressing α-syn, and oxidative stress was induced with H2O2 after differentiation of dopaminergic neurons, and toxic α-syn aggregation and propagation were analyzed. Phosphorylated α-syn at serine 129 (p129-α-syn), which may be a toxic α-syn, or GFP+α-syn transfer was observed to be less in Og-NSCs (
Finally, Og-NSCs or 2D-NSCs were transplanted into the brain tissue in vivo, and α-syn propagation and aggregation in the actual brain environment were observed. Human specific α-syn overexpressing AAV virus and PFF were injected into the rat striatum to induce Parkinson's disease caused by α-syn disease. After two weeks, Og-NSCs and 2D-NSCs were transplanted into the model animal striatum, respectively, and differences within the in vivo brain environment were observed. As a result of analysis of the transplanted tissue after one month, under a serious α-synucleinopathy environment, the conventional 2D-NSC-derived transplantation site had almost no surviving dopaminergic neurons (
Og-NSC Transplantation Provides Healthy Dopaminergic Neurons in Parkinson's Disease Animal Model
Finally, the possibility of Og-NSCs derived cell population as a cell therapeutic agent was observed after transplantation in a model animal system in which Parkinson's disease was induced by 6-OHDA (6-hydroxydopamine). Recently, there has been a study on genes that can expect the success after transplantation of human embryonic stem cell derived cells. As expected, as a result of single cell RNA-seq analysis, it was found that this gene group was expressed relatively highly in the newly developed Og-NSCs cell population (compared to the conventional 2D cell population) (
Og-NSCs cell populations were transplanted into a Parkinson's disease animal model, and behavioral studies were performed. As a result, the amphetamine-induced rotational behavior analysis showed a consistent recovery effect. At ˜6 months of transplantation, behavioral recovery (rotation scores) in 11 of 14 animals was >50% of that before transplantation. In the case of 8 animals observed at 6 months after transplantation, all animals showed >79% results (
6 months after transplantation of Og-NSCs into a model animal, the results were analyzed. Healthy dopaminergic neuron tissue was confirmed at the transplantation site (graft volume: 4.50±1.56 mm3, TH+ cells: 9031±3773 cells, n=8 from 6 rounds of independent transplantation experiments,
Discussion
Cultured organoids may be applied to tissue development and human disease modeling studies and may be also a source of donor transplantation in regenerative medicine. However, because transplantation of brain organoids into deep brain regions is not feasible without damaging host brain tissue, transplantation of brain organoids will not be applicable for the treatment of brain disorders. Moreover, the therapeutic outcome of transplanted brain organoids should be able to establish new neural networks in which self-organized structures in transplanted organoids interact with the host brain in a very precise manner. In contrast, transplanted organoids from other tissues (liver, intestine, kidney, pancreas, etc.) may exert therapeutic functions as isolated functional units (liver buds, pancreatic islets, renal nephrons, etc.) after transplantation. Therefore, although organoid transplantation may have the potential to treat other extra-CNS disorders, direct transplantation of brain organoids to treat brain disorders is unreasonable.
All tissue developments are achieved through sequential generation processes, proliferation of tissue-specific stem/progenitor cells and their differentiation into tissue-specific cells. Cultured tissue-specific stem/progenitor cells can provide donor cells for regenerative medicine as well as bioassay platforms for developmental studies and drug screening. However, the induction of NSC cultures with appropriate VM-patterning from hPSCs was not achieved in previous studies. The present inventors realized that the difficulty in preparing VM-specific NSC cultures was due to the instability of VM region specificity in the culture. Since maintenance of VM-specific marker expression is very sensitive to cell density, it is easily lost during the cell isolation and replating process required for NSC preparation (
Therefore, the hPSC-mDA neuron protocol was developed with very high cell densities or specific plate coating materials such as Laminin-511 or 111 (Biolamina, LN-511, LN-111). Various efforts using various cell dissociation conditions (Acutase, collagenase, Ca++, Mg++-free HBSS), cell survival cytokines (TGFβ, doxycycline, cAMP, ascorbic acid, Y27632), chemicals that induce open epigenetic conditions (5-azacytidine, VPA, TSA), (3-estradiol or DAPT (Notch inhibitor) failed to solve the problem.
In a recent study (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.), the present inventors observed that factors secreted from cultured astrocytes, particularly factors derived from VM tissues, exert strong trophic effects to promote cell survival and midbrain-specific factor expression in primary NSC and mDA neuron cultures.
Therefore, the 2D-NSC culture protocol used this study could be developed through the treatment of conditioned medium prepared from cultured astrocytes (ACM) (
Organoid-based formulations have had great success with respect to stability of VM-patterns in NSC cultures with faithful midbrain-specific marker expression, as well as general culture stability with very low levels of apoptosis, senescence, oxidation and mitochondrial stress. Another notable observation was that Og-NSCs contain an astrocyte progenitor cell population with a VM-specific gene expression profile in which Og-NSCs are 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, there were no astroglia in differentiated mDA neuron cultures. Therefore, in the absence of astroglial support, nerve maturity and function could not be expected to be identical to that of differentiated mDA neurons in their in vivo counterparts. Indeed, insufficient maturation and function have been proposed as critical drawbacks in the utility of mDA neurons differentiated from hPSC in in vitro disease modeling and therapy. In contrast, under nutritional support from mixed astrocytes, Og-NSC-derived mDA neurons exhibited an improved set of improved synapse maturation, functionality, resistance to toxic insult, and long midbrain specific factor expression in vivo and in vivo after transplantation. All these properties of Og-NSCs ultimately contribute to superior and long-term therapeutic efficacy after transplantation in a reproducible manner. In addition, we have shown that organoid-derived NSCs are an expandable cell source needed for the preparation of donor cells for therapy, as well as bioassay platforms for drug screening, developmental studies and disease modeling.
Based on the present invention, organoid-based methods may become a common next-generation strategy to prepare tissue specific stem/progenitor cells with strong therapeutic potential for CNS as well as non-CNS disorders.
In other words, there is no doubt that the cells of organoids will be healthier compared to the two-dimensional culture and that excellent results will be obtained during cell therapy. Since it is not desirable to directly transplant organoids in the form of ‘mass’ due to the characteristics of brain tissue, the present invention can be said to be the most appropriate model of a cell therapeutic agent for the nervous system.
[Experimental Procedure]
Culturing of Human Pluripotent Stem Cells (hESCs/hiPSCs)
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.
For proliferation and maintenance of undifferentiation hESC/iPSC, the cells were cultured on Matrigel™ or on a vitronectin (Human; Gibco Fisher Scientific, Waltham, Mass.) (Gibco A31804; 0.5 μg/cm2)-coated 6 cm dish (Thermo Fisher Scientific, Waltham, Mass.) using mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) in a CO2 incubator set at 37° C. without a feeder layer, and medium replacement was performed daily. The undifferentiated stem cells maintained their differentiation ability by replacing the medium daily, and were subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.
Preparation of Three-Dimensional Cerebral Cortex Organoids
Human embryonic/induced pluripotent stem cells were detached from the dish using Acutase, placed in a neuron differentiation induction medium, and injected into a low attachment 96-well round bottom plate (Corning, Corning, N.Y.) at 150 μL of 10,000 cells per well (day 0 of differentiation). The composition of the neuron 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) and bFGF (4 ng/ml), ROCK inhibitor Y27632 (20 μM), doxycyclin 1 μg/ml were added. The cells were treated with Y27632 only 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) were added to induce a full-scale nervous system differentiation. Between day 2 and day 3, only ½ of the basal medium was used as N2: Neurobasal (1:1, Gibco), and then the entire basal medium was used as N2: Neurobasal (1:1, Gibco) until spread on a plate after chopping in 2D state on day 18. The entire medium was replaced whenever the medium composition was changed. From day 8, bFGF (20 ng/ml) and EGF (20 ng/ml) were added.
Organoids were formed by culturing according to the medium shown in the table below until day 18.
indicates data missing or illegible when filed
Isolation of Cerebral Cortex Neural Stem Cells and Astrocyte Progenitor Cells from Cerebral Cortex Organoids and Differentiation into Neurons and Astrocytes
On day 18, the organoids were digested with Acutase at 37° C. for 7 minutes, and then chopped into pieces with a 30 gauge needle, and spread on vitronectin-coated 6 cm plates (Corning, cells from 24 organoids/plate). At this point, the cells were at the neural stem cell (NSC) stage, capable of being proliferated, and subcultured every 5-7 days. The medium used for NSC culture was an 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). The medium was replaced daily and treated with Y27632 at a concentration of 5 M for one day at the first conversion to two-dimensional culture and at each subculture. The final differentiation into neurons was induced with an N2 differentiation medium (10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid).
Isolation and Culture of Cerebral Cortex Astrocyte Progenitor Cells from Cerebral Cortex Organoids
If the passage of the prepared cerebral cortex-type Og-NSCs is further increased according to the actual brain development stage, it is possible to culture Og-astrocyte progenitor cells mainly containing astrocyte progenitor cells after passage 9 without any change in medium composition, etc. Passage 9 to passage 11 can be used in a healthy state. As in cerebral cortex-type Og-NSCs, the proliferation medium was an 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 pg/ml doxycycline. The medium was replaced daily and treated with Y27632 at a concentration of 5 μM for one day at the first conversion to two-dimensional culture and at each subculture. Like organoid-derived astrocyte progenitor cells in the midbrain (see Example 3), cells in the proliferative stage with an older passage are used for transplantation studies. For analysis, the cell population with a high passage with the above final differentiation medium takes on the characteristics of astrocytes.
Confirmation of Differentiation into Cerebral Cortex Neurons and Astrocytes
Cerebral cortex-type neurons were prepared, and through this, cerebral cortex-type patterning was preferentially carried out to secure astrocyte progenitor cells through several subcultures. After induction of differentiation from human embryonic stem cells into the nervous system, the confirmation thereof was preferentially performed by the confirmation of patterning, and the expression of cerebral cortex-type markers such as PAX6 and FOXG1 was confirmed. In addition, when the astrocyte progenitor cells were developed through several subcultures, the expression of astrocyte-type factors such as GFAP, AQP4, ALDH1L1, and GLAST was confirmed. The markers can be confirmed through a process such as an expression confirmation simultaneously using real time PCR or immunostaining method after RNA extraction.
[Result]
Preparation of 3D Organoids
The point of the result is to confirm that the differentiation into a cerebral cortex cell population in a normal organoid form is preferentially performed, patterning is performed, and the differentiation into astrocyte progenitor cells is completed in the future. As shown in
Securing Cerebral Cortex Neural Stem Cells and Astrocyte Progenitor Cells from the Prepared Cerebral Cortex 3D Organoids
As in the mentioned protocol, SB431542 (10 μM), LDN (10 μM), and A-83 (10 μM) were added to induce a full-scale nervous system differentiation (lower drawing in
Confirmation of Differentiation of Cerebral Cortex Neurons and Astrocytes
The nervous system patterning factors SB431542 (10 μM), LDN (10 μM), and A-83 (10 μM) were added to induce a full-scale nervous system differentiation, and patterning into cerebral cortex-type brain organoids was performed. However, the process after differentiation into astrocyte progenitor cells through division of neurons and multiple passages from neural stem cells is not different from the process of differentiation through midbrain-like organoids except that it differs only in initial patterning. When compared with the differentiation of midbrain astrocyte progenitor cells, the sufficient expression of GFAP, AQP4, ALDH1L1, GLAST, and the like could be confirmed using immunostaining method, and at the same time, the expression of the patterning factors could be confirmed in each cell population (
In this example, a method using 3D organoids capable of producing cerebral cortex specific neural stem cells was established. By utilizing 3D organoids, neural stem cells are extracted from tissues with the same structure as the actual in vivo cerebrum, so it can be expected that it is possible to secure healthier and actual cells.
[Experimental Procedure]
Culturing of Human Pluripotent Stem Cells (hESCs/hiPSCs)
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.
For proliferation and maintenance of undifferentiation hESC/iPSC, the cells were cultured on Matrigel™ or on a vitronectin (Human; Gibco Fisher Scientific, Waltham, Mass.) (Gibco A31804; 0.5 μg/cm2)-coated 6 cm dish (Thermo Fisher Scientific, Waltham, Mass.) using mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) in a CO2 incubator set at 37° C. without a feeder layer, and medium replacement was performed daily. The undifferentiated stem cells maintained their differentiation ability by replacing the medium daily, and were subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.
Preparation of Three-Dimensional Midbrain-Like Organoids
It was prepared in the same manner as in Example 1 until the midbrain-type organoids were prepared. In the early stage of development of the successfully prepared midbrain-type organoids, ZO-1 and N-cadherin were expressed along with PLZF+ and Sox2+, which are neural stem cell markers (
Isolation of Midbrain Neural Stem Cells from Midbrain-Like Organoids and Differentiation into Neurons
hESCs/hiPSCs were detached from the dish using Acutase, placed in a neuron differentiation induction medium, and injected into a low attachment 96-well round bottom plate (Corning, Corning, N.Y.) at 150 μL of 10,000 cells per well (day 0 of differentiation). The composition of the neuron differentiation induction medium was as follows (N2: Neurobasal (1:1, Gibco) containing B27 without vitamin A (2%, Invitrogen Fisher Scientific, Waltham, Mass.), GlutaMAX (1%, Invitrogen Fisher Scientific), minimum essential media-nonessential amino acid (1%, MEM-NEAA, Invitrogen Fisher Scientific), P-mercaptoethanol (0.1%, Invitrogen Fisher Scientific), SB431542 (10 μM, Tocris, Bristol, UK), Noggin (200 ng/ml, Peprotech, Rocky Hill, N.J.), ascorbic acid (200 μM, Sigma-Aldrich), insulin (25 mg/L), ROCK inhibitor Y27632 (20 μM, Sigma-Aldrich, St. Louis, Mo.). The cells were treated with Y27632 only for 24 hours on the first day. For the expansion of ventral midbrain type neural stem cells of the organoids, sonic hedgehog (100 ng/ml, SHH, Peprotech) and purmorphamine (2 μM, Calbiochem, MilliporeSigma, Burlington, Mass.) were added to the neuron differentiation induction medium from day 1 of differentiation 1. On the second day, CHIR99021 (0.8 μM, Stemgent, Cambridge, Mass.) was added. The entire medium was replaced whenever the medium composition was changed. SB431542 was removed from day 5, and FGF8b (100 ng/ml, Peprotech) was added from day 7. On day 11, the basal medium was completely replaced with N2; and B27, GlutaMAX, MEM-NEAA, and P-mercaptoethanol were not added. The concentration of CHIR99021 added from day 11 was increased about two-fold (1.5 μM). From this point, the organoids were transferred from a 96 well plate to a 6 well plate (low attachment, Corning) and cultured on an orbital shaker at a speed of 80 rpm. Noggin and sonic hedgehog were removed from day 15, and bFGF (20 ng/ml) was added from day 17. On day 18, the organoids were digested with Acutase at 37° C. for 7 minutes, and then chopped into pieces with a 30 gauge needle, and spread on vitronectin-coated 6 cm plates (Corning, cells from 24 organoids/plate). At this point, the cells were at the neural stem cell (NSC) stage, capable of being proliferated, and subcultured every 5-7 days. The medium used for NSC culture was an N2 proliferation medium, and the composition was as follows (N2 containing 10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid, 100 ng/ml FGF8b, 20 ng/ml bFGF, and 1 μg/ml doxycycline). The medium was replaced daily and treated with Y27632 at a concentration of 5 M for one day at the first conversion to two-dimensional culture and at each subculture. The final differentiation into neurons was induced with an 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 astrocyte progenitor cells from midbrain-like organoids, and differentiation into astrocytes
If the passage of the prepared midbrain-type Og-NSCs is further increased according to the actual brain development stage, it is possible to culture Og-astrocyte progenitor cells mainly containing astrocyte progenitor cells after passage 9 without any change in medium composition, etc. Passage 9 to passage 11 can be used in a healthy state. As in Og-NSCs, the proliferation medium was an N2 proliferation medium, and the composition was N2 containing 10 ng/ml BDNF, 10 ng/ml GDNF, 200 μM ascorbic acid, 100 ng/ml FGF8b, 20 ng/ml bFGF, and 1 μg/ml doxycycline. The medium was replaced daily and treated with Y27632 at a concentration of 5 μM for one day at the first conversion to two-dimensional culture and at each subculture. Astrocyte progenitor cells in the proliferative stage was used for transplantation, and differentiation for research was induced with an 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 Preparation of 3D Midbrain-Like Organoids
If the midbrain-type organoids shown as a result of
Confirmation of Securing Midbrain Astrocyte Progenitor Cells from Prepared Midbrain 3D Organoids
{circle around (1)} Direct Differentiation of Midbrain Neural Stem Cells, and Securing of Astrocyte Progenitor Cells
In order to induce differentiation from hESCs/iPSCs into neural stem cells and into midbrain-neural stem cells, midbrain-neural stem cells were secured using neuroectoderm inducers and midbrain-type neural stem cell inducers for about 20 days at the embryonic stem cell stage. The secured neural stem cells are easy for proliferation and stock, and a greater number of neural stem cells can be obtained through subculture. Confirmation of proper differentiation can be performed through the expression rate of neural stem cell markers such as Nestin and Sox2 and midbrain-neural stem cell markers such as Foxa2 and Lmx1a. When the present protocol is applied, differentiation into midbrain neural stem cells can be induced with a high efficiency of 95% or more as follows. In order to obtain astrocyte progenitor cells, additional subcultures are performed several times. During the initial subculture, terminally differentiated cells have a higher differentiation rate into neurons than astrocytes, but the proportion of astrocyte progenitor cells are increased as the subculture is increased. The conventional protocol was able to identify astrocytes upon differentiation after 5 subcultures, but for more efficient establishment of astrocyte progenitor cells, the neural stem cell population from passage 9 to passage 11 is used as the astrocyte progenitor cell population. At this time, the astrocyte progenitor cell marker CD44 was expressed 90% or more, and when differentiation was induced, the differentiated astrocyte marker group (GFAP, AQP4, GLAST, and S100b) was expressed 90% or more as shown in
{circle around (2)} OBSERVATION of higher quality effects of midbrain-type astrocyte progenitor cells
As a result of analysis of in vitro properties, it was observed that midbrain-type astrocytes overexpress factors related to secretion of more useful cytokines and maintenance of neuron function (
Confirmation of Differentiation of Midbrain Astrocytes
Confirmation of Protocol Application Using hiPSCs
An experiment was conducted to confirm whether the secured protocol was applied not only to H9 hESCs but also to other types of embryonic stem cells. To this end, iPSCs were secured from the Korea Centers for Disease Control and Prevention (CMC-hiPSC-003 and 011). In the culture stage, we succeeded in adaptive culture using a xeno-free system that does not use animal-derived components such as mouse feeder or matrigel, which were previously used for culturing human embryonic stem cells (
As a result of applying the secured hiPSCs to the present protocol, it was confirmed that they were well differentiated into neural stem cells, and furthermore, it was confirmed that astrocyte markers such as GFAP were increased during the subculture process, as in H9 hESCs (
Normal differentiation into midbrain astrocytes was confirmed (
Efficient Differentiation Induction Using TGF-β and LIF
Through efficient differentiation induction using TGF-β and LIF, a technology for securing a large amount of astrocytes was secured. By treating with TGF-β in the proliferation stage and with LIF in the differentiation stage, it was observed that a greater amount of astrocytes were differentiated (by expression of the GFAP marker), and it was observed that these astrocytes became high-quality cells in which a greater amount of neuromodulatory factors were expressed (
In Order to Confirm the Utility of the Original Material of Astrocytes Secured Through the Final Protocol, the Properties of the Conditioned Medium are Preferentially Analyzed
The results are shown in
Analysis of Property after Transplantation to Confirm In Vivo Utility of Astrocytes Secured Through Final Protocol
First, we studied the positive effect of astrocytes on the surrounding environment after transplantation into the in vivo brain. After transplantation of astrocytes into the striatum site, the results of anti-inflammatory factors, inflammatory factors, and cytokine secretion, and the like were confirmed. The expression of inflammatory factors such as TNF-a, IL1b, iNOS, and CD11b was reduced at the astrocyte transplantation site, and the expression of inflammatory factors such as IL1b, iNOS, and CD11b was reduced at the surrounding site, and the expression of BDNF, GDNF, and Arg1 cytokines was slightly increased at the transplantation site and the surrounding site (
The Effect of Improving the Environment after Transplantation of Astrocytes In Vivo was Observed In Vivo Through Changes in Microglia.
The secured astrocytes were transplanted into the rat brain striatum, and then neural stem cells were transplanted into the opposite side as a control group. It was confirmed whether the microglia were converted to M1 and M2 by observing representative environmental changes.
Inflammation was induced by LPS, and astrocytes were transplanted. As a result, it was observed that the expression of the poor type M1 markers iNOS, CD11b, CD16, and the like, was inhibited at the astrocyte transplantation site (
[Experimental Procedure]
Culturing of Human Pluripotent Stem Cells (hESCs/hiPSCs)
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 9 below.
For proliferation and maintenance of undifferentiation hESC/iPSC, the cells were cultured on Matrigel™ or on a vitronectin (Human; Gibco Fisher Scientific, Waltham, Mass.) (Gibco A31804; 0.5 μg/cm2)-coated 6 cm dish (Thermo Fisher Scientific, Waltham, Mass.) using mTESR-1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) in a CO2 incubator set at 37° C. without a feeder layer, and medium replacement was performed daily. The undifferentiated stem cells maintained their differentiation ability by replacing the medium daily, and were subcultured using Acutase (Stemcell Technologies Inc.) every 4 to 5 days.
Preparation of Three-Dimensional Hypothalamus Organoids
Organoids were prepared by the same protocol as in
Basically, because organoids of the nervous system were prepared from embryonic/induced pluripotent stem cells, human embryonic/induced pluripotent stem cells were detached from the dish using Acutase, placed in a neuron differentiation induction medium, and injected into a low attachment 96-well round bottom plate (Corning, Corning, N.Y.) at 150 L of 10,000 cells per well (day 0 of differentiation). For the preparation of organoids into the nervous system, they were treated with SB-431542, 10 μM, and Noggin, 100 ng/mL, induced into the nervous system, and patterned by treatment with the ventral inducers SHH and purmorphamine. The specific composition of the medium is shown in Tables 10 and 11 below.
Isolation of Hypothalamus Neural Stem Cells from Hypothalamus Organoids, and Confirmation of Differentiation into Neurons
The composition of the differentiation induction medium according to the date is shown in Table 10 below.
For the first seeding, N2: Neurobasal (1:1, Gibco), B27 (without RA), 2-mercapto 55 M, MEM-NEAA, GlutaMAX, ascorbic acid, 200 μM, SB431542 (10 μM), Noggin 100 ng/ml, ROCK inhibitor Y27632 (20 μM), doxycyclin 1 μg/ml were added. The cells were treated with Y27632 and doxycyclin only for 24 hours on the first day. Between day 1 and day 6, SHH, 100 ng/mL, and Purmorphamine, 2 μM were added while using the basal medium. Between day 7 and day 12, a medium only without SB431542 was used while using the conventional basal 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, and FGF2, 20 ng/mL were added to the N2 medium and the cells were cultured, and the entire basal medium was used as N2 until spread on a plate after chopping in 2D state on day 21 (possible after day 18), and doxycyclin was added only on the first day of plating. The entire medium was replaced whenever the medium composition was changed, and the medium used on day 13 was used as the proliferation medium. It was treated with doxycyclin at a concentration of 1 μg/ml and Y27632 at a concentration of 5 μM for one day at each subculture. The final differentiation into neurons was induced with an N2 differentiation medium (10 ng/ml BDNF, 10 ng/ml GDNF, and 200 μM ascorbic acid). The basal medium is shown in Table 11 below.
Isolation of Hypothalamus Astrocyte Progenitor Cells from Hypothalamus Organoids, and Confirmation of Differentiation into Astrocytes
If the passage of the prepared hypothalamus-type Og-NSCs is further increased according to the actual brain development stage, it is possible to culture Og-astrocyte progenitor cells mainly containing astrocyte progenitor cells after passage 9 without any change in medium composition, etc. Passage 9 to passage 11 can be used in a healthy state. As in hypothalamus-type Og-NSCs, the proliferation medium was an N2 proliferation medium, and the composition was 1X N2 supplement containing 200 μM ascorbic acid, 20 ng/ml bFGF, and 1 μg/ml doxycycline. The medium was replaced daily and treated with Y27632 at a concentration of 5 μM for one day at the first conversion to two-dimensional culture and at each subculture. Like organoid-derived astrocyte progenitor cells in the midbrain, cells in the proliferative stage with an older passage are used for transplantation studies.
[Result]
Confirmation of Preparation of 3D Hypothalamus Organoids
As shown in
Confirmation of Securing Hypothalamus Neural Stem Cells and Astrocyte Progenitor Cells from Prepared Hypothalamus 3D Organoids
It was confirmed that the neural stem cells chopped and extracted from the hypothalamus organoids became properly patterned neural stem cells in which 96.8% of the hypothalamus markers were expressed as shown in the results of
Confirmation of Differentiation of Hypothalamus Neurons and Astrocytes
As shown in the results of
In order to confirm the formation of functionally complete cells after differentiation, when specifically observing leptin reactivity (
Securing hypothalamus neural stem cells as leptin-responsive stem cells shows that they can actually perform their functions similar to those in the living body, and suggests that they have the possibility to be mainly used for research and development of therapeutic agents for metabolic diseases such as obesity and diabetes in the future (
Confirmation of In Vivo Transplantation Possibility of Hypothalamus Neural Stem Cells Developed by Using 3D Organoids
The possibility of in vivo transplantation of hypothalamus neural stem cells developed by using 3D organoids was confirmed. After transplanting 1×105 hypothalamus neural stem cells into the 3rd ventricle, the engraftment possibility was confirmed 7 days later. It was confirmed that the hypothalamus neural stem cells expressing hNCAM/Rax were stably engrafted, and at the same time, it was confirmed that they took the form of hypothalamus neural stem cells expressing hGFAP/POMC (
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/KR2020/000581 | 1/13/2020 | WO |