Technical field

The present invention relates to a method for isolating dopamine neurons and a pharmaceutical composition for treating Parkinson's disease comprising dopamine neurons isolated using the same.

The present invention was made by the project number HI18C0829 under the support of the Ministry of Health and Welfare of the Republic of Korea. The research management institution of the project is the Korea Health Industry Development Institute, the name of the research project is "Development of advanced medical technology", and the name of the research project is "The nervous system of pluripotent stem cells. Establishment of in vivo differentiation monitoring and prediction technology for the minimum number of transplanted cells for disease application” ~ 2021.12.31.
[3]
This patent application is based on Korean Patent Application No. 10-2018-0050918 filed with the Korean Intellectual Property Office on May 2, 2018 and Korean Patent Application No. 10-2019-0048784 filed with the Korean Intellectual Property Office on April 25, 2019. Priority is claimed, and the disclosure of the patent application is incorporated herein by reference.
Background
[4]
Parkinson's disease (PD) is one of the neurodegenerative disorders caused by focal degeneration of midbrain dopaminergic (mDA) neurons, most suitable for cell-based therapies.
[5]
Since the early 1980s, attempts have been made to restore Parkinson's disease-related motor function by implanting fetal ventral mesencephalon (VM) tissue into the patient's striatal.
[6]
According to these studies, it was confirmed that recovery of motor function can be induced by grafts, but the results of cell transplantation are inconsistent in each research institute, and in some cases, side effects also appear, and there is consensus that the cell-based therapy should be further improved afterwards. Was formed.
[7]
In particular, in order to develop the cells, which are transplant materials, which have been pointed out as problems in the previous studies to a level close to the cells existing in the living body, and to compensate for the disadvantages such as limited availability and inconsistency of fetal tissues. Alternative research continued.
[8]
As a result, it includes embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which exhibit infinite proliferation capacity in vitro and have a wide differentiation capacity into various neurons. Human pluripotent stem cells (hPSCs) have attracted attention.
[9]
Accordingly, until recently, various differentiation technologies for mDA neurons have been developed and evolved to a level capable of producing high-yield cells, but other types other than mDA neurons in the result of differentiation induced from hPSCs through the recently developed differentiation technology It is difficult to consider clinical application based on the current differentiation technology because heterogeneity in which cells are quite mixed is observed.
[10]
Therefore, the identification and isolation of hPSC-derived mDA neurons from various cell populations is very important for successful transplant therapy studies as well as standardization of transplanted cells.
[11]
On the other hand, the strategy to purely separate (concentrate) mDA neurons induced differentiation from hPSCs is based on fluorescence-activated cell sorting (FACS) targeting multiple surface antigens. I did it. This approach increased the neuronal population expressing tyrosine hydroxylase (TH), but it was unclear whether these cell populations possessed mDA neuronal properties.
[12]
Recently, a study was conducted to discover a specific surface marker capable of discriminating and enriching mDA neurons by analyzing the transcriptome of mouse fVM tissue and mouse embryonic stem cells (mESC)-derived mDA neuronal progenitor cells. .
[13]
These studies have suggested several possible cell surface markers, and have shown that dopaminergic neurons can be isolated and enriched using them, but at the same time, the cell surface markers expressed by neurons at exactly certain stages of the neuronal development process were identified. Whether it should be excavated has not yet been clearly stated. Since it is a common opinion in the industry that the survival rate and degree of differentiation after transplantation of isolated neurons depending on the stage of development are different, and this may affect the recovery of function after transplantation, the discovery of differentiation stage-specific markers is also an important task. Must be.
[14]
Accordingly, there is an urgent need to study the surface markers of mesocerebral dopamine neurons and their differentiation stages.
Detailed description of the invention
Technical challenge
[15]
The present inventors differentiated the differentiation process of mesocerebral dopamine neurons and tried to develop cell surface markers for each step. As a result, LMX1A-eGFP and PITX3-mCherry reporter hESC cell lines were established and differentiated to isolate LMX1A + mDA neuroprogenitor cells and PITX3 + mDA neurons, from which cell surface markers related to mesocerebral dopamine neurons (TPBG ), thereby completing the present invention.
[16]
Accordingly, an object of the present invention is to provide a method for producing dopaminergic neural cells.
[17]
Another object of the present invention is to provide a pharmaceutical composition for treating Parkinson's disease, including TPBG (Trophoblast glycoprotein)-positive dopamine neurons.
[18]
Another object of the present invention is to provide a method of enhancing the efficacy of dopaminergic neurons for cell transplant therapy of Parkinson's disease and improving transplantation safety.
[19]
Another object of the present invention is to provide a composition for transplanting dopamine neurons comprising TPBG (Trophoblast glycoprotein)-positive dopamine neurons.
Means of solving the task
[20]
The present inventors differentiated the differentiation process of mesocerebral dopamine (mDA) neurons, and tried to develop cell surface markers for each step. As a result, LMX1A-eGFP and PITX3-mCherry reporter hESC cell lines were established and differentiated to isolate LMX1A + mDA neuroprogenitor cells and PITX3 + mDA neurons, from which cell surface markers related to mesocerebral dopamine neurons (TPBG ) Was excavated.
[21]
More specifically, the present inventors described the LMX1A-eGFP reporter hESC cell line engineered to simultaneously express green fluorescent protein (eGFP) when LMX1A, the stage-specific gene of mDA neurons (progenitor), is expressed, and mature mDA neurons ( neuronal) PITX3-mCherry reporter hESC cell lines engineered to express red fluorescent protein (mCherry) at the same time when PITX3, a stage-specific gene, is expressed, respectively, and transfer of LMX1A + mDA neuron progenitor cells and PITX + mDA neurons. Through cadaver comparative analysis, candidates for cell surface markers specifically expressed on precursors (neural progenitor cells) of mDA neurons were selected. Among them, TPBG was discovered as a new cell surface marker, and as a result of cell separation targeting TPBG, it was confirmed that mDA neural progenitor cells were concentrated. In addition, TPBG-positive cells isolated by magnetic-activated cell sorting (MACS) at the stage of mDA neuronal progenitor cells were transplanted into a 6-OHDA-damaged Parkinson's disease (PD) rat model. It was confirmed that the symptoms of motor function abnormalities were recovered without tumor formation.
[22]
Therefore, TPBG is a new surface marker protein for isolating transplantable mDA neuronal progenitor cells, and mDA cells isolated using TPBG are expected to provide a safe and effective cell replacement therapy for Parkinson's disease treatment.
[23]
The present invention is a method for isolating dopamine neurons, a pharmaceutical composition for the treatment of Parkinson's disease comprising dopamine neurons isolated using the same, improving the efficacy of dopamine neurons for cell transplantation therapy of Parkinson's disease and improving the safety of transplantation It relates to a method, and a composition for transplanting dopamine neurons prepared using the same.
[24]
Hereinafter, the present invention will be described in more detail.
[25]
[26]
One aspect of the present invention relates to a method for producing dopaminergic neural cells comprising the following steps.
[27]
(a) contacting the cell population with a Trophoblast glycoprotein (TPBG) antibody; And
[28]
(b) separating TPBG-positive dopamine neurons that bind to the TPBG antibody.
[29]
In the present invention, "neural cells" are cells constituting the nervous system and may be used in the same meaning as neurons, and "dopaminergic neural cells" refers to dopamine, a neurotransmitter. It refers to the secreting nerve cells.
[30]
The dopaminergic neurons may be dopaminergic neural progenitors or dopaminergic neural precursor cells or mature dopaminergic neurons, but are not limited thereto.
[31]
In the present invention, "neural progenitor cells" refer to undifferentiated progenitor cells that have not yet expressed differentiation traits, and "progenitors", "precursors" and "precursor cells" may all be used with the same meaning.
[32]
The dopamine neuron may be a midbrain dopamine neuron.
[33]
In the present invention, the term "midbrain dopamine neuron" refers to a dopamine neuron observed in the midbrain region, and may mean, for example, a dopamine neuron observed in the ventral region of the midbrain. It is not limited.
[34]
In addition, the mesocerebral dopamine neurons can be expressed in an A9 region-specific manner.
[35]
The "A9 region" is a ventrolateral region of the midbrain, and refers to a part corresponding to the pars compacta of the midbrain black matter (substantia nigra), and the cells produced by the method of the present invention are It can be seen that it is a cell.
[36]
In addition, the A9 region is a region in which dopamine neurons are concentrated, and is related to the regulation of motor function. In particular, in the case of Parkinson's disease patients, dopamine neurons in this region are specifically killed. The cells produced by the manufacturing method of can be used for the purpose of preventing and/or treating Parkinson's disease.
[37]
Hereinafter, a method for preparing dopamine neurons according to the present invention will be described in detail.
[38]
[39]
Step (a)
[40]
The "cell population" refers to human stem cells; Progenitors or precursors; And/or dopaminergic neural progenitors differentiated from human stem cells or progenitor cells, mature dopaminergic neurons, and neural derivatives derived therefrom, but are limited thereto. It is not.
[41]
Specifically, the human stem cells or progenitor cells are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells (iPSCs). , Adult stem cells or fetal cells, but is not limited thereto.
[42]
The fetal cells may be derived from fetal neural tissue or derivatives thereof, and may be, for example, fetal ventral mesencephalic cells (fVM cells), but are not limited thereto. .
[43]
step (b)
[44]
The "TPBG" may be used in the same sense as Wnt-Activated Inhibitory Factor 1 or WAIF1, and is known as an antagonist of the Wnt/β-catenin signaling pathway, but the dopamine neuron isolation through the expression of TPBG has yet to be reported. none.
[45]
The nucleotide sequence of the gene is shown in SEQ ID NO: 53. In addition, since the nucleotide sequence of the gene is registered in the gene bank, it will be readily available to those skilled in the art.
[46]
In the present invention, "TPBG-positive dopamine neurons" means dopamine neurons that bind to TPBG antibodies.
[47]
The "TPBG antibody" refers to an antibody that specifically binds to TPBG.
[48]
In this step, the method of isolating TPBG-positive dopamine neurons may be any method as long as it is a method of separating cells by specifying a target, for example, fluorescence-activated flow cytometry (FACS) and/or magnetic-activated cells. The separation method (MACS) may be used, but is not limited thereto.
[49]
The TPBG-positive dopamine neurons can alleviate symptoms of Parkinson's disease.
[50]
The TPBG-positive dopamine neurons can improve the safety of cell transplant therapy.
[51]
[52]
Another aspect of the present invention relates to a pharmaceutical composition for treating Parkinson's disease, comprising TPBG (Trophoblast glycoprotein)-positive dopamine neurons.
[53]
The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto. In addition, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be additionally included in addition to the above components.
[54]
The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenous, subcutaneous, intraperitoneal or topical application) according to a desired method, and the dosage is It depends on the degree, drug form, administration route and time, but may be appropriately selected by those skilled in the art.
[55]
The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective amount is the type, severity, activity of the drug, and Sensitivity, administration time, route of administration and rate of excretion, duration of treatment, factors including concurrent drugs and other factors well known in the medical field.
[56]
The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.
[57]
Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate and excretion rate, the type of disease, and the drug used in combination.
[58]
Another aspect of the present invention relates to a method for treating Parkinson's disease comprising administering the TPBG-positive dopamine neurons to a subject.
[59]
The "individual" refers to a subject in need of treatment of a disease, and more specifically, refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse, and cow.
[60]
Another aspect of the present invention relates to the use of the TPBG-positive dopamine neurons to treat Parkinson's disease.
[61]
In the pharmaceutical composition for treating Parkinson's disease including the TPBG-positive dopamine neurons, the content overlapping with the method of preparing the dopamine neurons will be omitted in consideration of the complexity of the present specification.
[62]
[63]
Another aspect of the present invention relates to a method of enhancing the efficacy of dopaminergic neurons for cell transplantation therapy of Parkinson's disease and improving transplantation safety, comprising the following steps.
[64]
(a) contacting a cell population with a Trophoblast glycoprotein (TPBG)-antibody; And
[65]
(b) separating TPBG-positive dopamine neurons that bind to the TPBG antibody.
[66]
In the method for enhancing the efficacy of the dopamine neuron and improving the safety of transplantation, the content overlapping with the method for producing the dopamine neuron will be omitted in consideration of the complexity of the present specification.
[67]
[68]
Another aspect of the present invention relates to a composition for transplanting dopamine neurons comprising TPBG (Trophoblast glycoprotein)-positive dopamine neurons.
[69]
The TPBG-positive dopamine neurons may be cultured by a method for producing dopamine neurons, and the TPBG-positive dopamine neurons cultured by the method may have improved cell efficacy and improved transplantation safety.
[70]
On the other hand, for the transplantation of the dopamine neurons, an appropriate transplant site known in the art (eg, putamen or caudate nucleus of the brain, or striatum encompassing all of them) is selected, and It can be performed through a method known at the implantation site (eg, stereotactic system, etc.).
[71]
The composition of the present invention can be used for treatment of Parkinson's disease.
[72]
The composition of the present invention may contain only dopamine neurons alone as transplanted cells, or may contain a bio-derived and/or biodegradable stabilizer in addition to the active ingredient (grafted cells). The stabilizing agent is for stably dispersing the dopamine neurons, and as it is a bio-derived material, there is no side effect upon implantation in the body, otherwise it must have biodegradability. In the present invention, the term "biodegradability" refers to a property that is gradually decomposed and absorbed in the body, and does not particularly mean the rate of decomposition.
[73]
The stabilizer may include hyaluronic acid, collagen, thrombin, elastin, chondroitin sulfate, albumin, and mixtures thereof. In particular, hyaluronic acid, collagen, thrombin, elastin, chondroitin sulfate, albumin, and the like are bio-derived materials and have biodegradable properties that can be naturally degraded in vivo. However, the synthesized compound is also a biodegradable material and can be used for purposes of the present invention as long as the properties of biodegradability and viscosity in the medium are satisfied, so it is not necessarily limited to a material derived from a living body.
[74]
When the stabilizing agent is formulated together with dopamine neurons, dopamine neurons may not float or settle in a medium, but may be evenly dispersed and present.
[75]
In the composition for transplanting dopaminergic neurons including the TPBG-positive dopamine neurons, the content overlapping with the method for preparing the dopaminergic neurons will be omitted in consideration of the complexity of the present specification.
Effects of the Invention
[76]
The present invention relates to a method for isolating dopamine neurons and a pharmaceutical composition for treating Parkinson's disease comprising dopamine neurons isolated using the same, wherein the method for isolating dopamine neurons comprises the steps of isolating TPBG-positive dopamine neurons By including, the dopamine neuron isolated according to the present method is characterized in that the efficacy of the cells during transplantation is improved and the transplantation safety is improved, and thus can be usefully used for cell transplantation for the treatment of Parkinson's disease.
Brief description of the drawing
[77]
1 is a diagram schematically illustrating a method for producing a dopamine neuron according to the present invention.
[78]
2 is a diagram schematically illustrating a method of preparing an LMX1A-eGFP hES reporter cell line according to a preparation example of the present invention.
[79]
3 is a diagram schematically illustrating a method of preparing a PITX3-mCherry hES reporter cell line according to a preparation example of the present invention.
[80]
4 is a diagram illustrating a process of differentiation of mDA neural progenitor cells in an LMX1A-eGFP hES reporter cell line prepared according to a preparation example of the present invention.
[81]
5 is a diagram illustrating a process of differentiation of mDA neurons (neurons) of the PITX3-mCherry hES reporter cell line prepared according to a preparation example of the present invention.
[82]
6A and 6B are diagrams illustrating characteristics of differentiated LMX1A-expressing mDA neural precursor cells according to an embodiment of the present invention.
[83]
7A and 7B are diagrams illustrating characteristics of differentiated LMX1A-expressing mDA neural precursor cells according to an embodiment of the present invention.
[84]
8A and 8B are diagrams illustrating characteristics of LMX1A-expressing cells differentiated according to an embodiment of the present invention after terminal differentiation.
[85]
9A to 9C are diagrams illustrating characteristics of differentiated PITX3-expressing mDA neurons (neurons) according to an embodiment of the present invention.
[86]
10 is a diagram illustrating the characteristics of PITX3-expressing mDA neurons (neurons) differentiated according to an embodiment of the present invention.
[87]
11 is a diagram comparing the degree of apoptosis in vitro for differentiated LMX1A-expressing mDA neuron progenitor cells and PITX3-expressing mDA neurons (neurons) according to an embodiment of the present invention .
[88]
12 is a diagram showing the results of transcriptome analysis on LMX1A-expressing mDA neuron progenitor cells and PITX3-expressing mDA neurons (neurons) differentiated according to an embodiment of the present invention.
[89]
13 is a schematic diagram showing a process of identifying candidate groups for mDA markers.
[90]
14 and 15 are results of evaluating target effectiveness in order to identify candidate groups for mDA markers.
[91]
16 and 17 are diagrams showing results of MACS targeting mDA marker candidate groups (CORIN, TPBG, CD47, ALCAM).
[92]
18 is a diagram illustrating behavioral recovery after transplantation of TPBG-positive cells in a PD-animal model transplanted with TPBG-positive cells isolated from hESC according to an embodiment of the present invention.
[93]
19 is a diagram illustrating characteristics of a graft after TPBG-positive cell transplantation with respect to a PD-animal model transplanted with TPBG-positive cells isolated from hESC according to an embodiment of the present invention.
[94]
FIG. 20 shows a PD-animal model transplanted with TPBG-positive cells isolated from hESC according to an embodiment of the present invention, which is not classified as compared with a TPBG-positive cell graft after TPBG-positive cell transplantation ( This is a diagram confirming the possibility of cell proliferation in unsorted) cell grafts.
[95]
21 is a diagram illustrating the characteristics of TPBG-positive cells isolated from human fVM cells according to an embodiment of the present invention.
[96]
22 is a diagram illustrating the characteristics of TPBG-positive cells isolated from human iPSCs according to an embodiment of the present invention.
Mode for carrying out the invention
[97]
Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
[98]
[99]
Human Embryonic stem cells (hESC) culture
[100]
Undifferentiated hESCs (H9, WiCell Inc., USA) were treated with mitomycin-C (Sigma-Aldrich, USA) in a layer of mice STO fibroblasts (ATCC, USA) treated with 20% Knockout-Serum Replacement; Invitrogen. , U.S.), 1x non-essential amino acids (Gibco-Thermo Fisher Scientific, U.S.), 0.1 mM β-mercapto ethanol (Sigma-Aldrich) and 4 ng/mL bFGF (basic fibroblast growth factor; R&D System, U.S.) DMEM (Dulbecco's modified Eagle's medium) / F12 medium (Gibco-Thermo Fisher Scientific).
[101]
[102]
Genotyping of clonal cells
[103]
Genomic DNA was extracted using the DNeasy Blood&Tissue kit (QIAGEN, Germany) according to the manufacturer's instructions. Genomic DNA PCR was performed in GeneAmp PCR System 2720 (Applied Biosystems-Thermo Fisher Scientific) using EmeraldAmp® GT PCR Master Mix (TAKARA Bio Inc., Japan).
[104]
[105]
FACS
[106]
Cell separation was performed using a BD FACSAria III flow cytometer and FACSDiva software (BD Bioscience). The eGFP-positive fraction was determined according to the fluorescence intensity using a 488 nm laser, and the mCherry-positive fraction was determined according to the fluorescence intensity using a 561 nm laser.
[107]
[108]
MACS
[109]
In order to inhibit non-specific binding of the antibody, the cells were incubated in 1% FBS-PBS solution (4° C., 30 minutes) and then bound with the primary antibody (see Table 1 below) at 4° C. for 30 minutes.
[110]
[Table 1]
Protein Species Company Cat. no. Dilution
OCT4 Rabbit Santa Cruz sc-9081 1:200
SOX2 Rabbit Millipore AB5603 1:200
NANOG (human) Goat R&D Systems AF1997 1:50
SSEA4 Mouse Millipore MAB4304 1:200
TRA-1-81 Mouse Millipore MAB4381 1:100
TRA-1-60 Mouse Millipore MAB4360 1:100
NESTIN (human) Rabbit Millipore ABD69 1:1,000
SOX1 Goat R&D Systems AF3369 1:100
SMAα Mouse SIGMA A5228 1:100
BRACHYURY Goat R&D Systems AF2085 1:100
EN1 Mouse Dev. Stud. Hybridoma Bank 4G11 1:50
FOXA2 (HNF3β) Rabbit Abcam AB108422 1:300
FOXA2 (HNF3β) Goat Santa Cruz sc-6554 1:100
LMX1A Goat Santa Cruz sc-54273 1:100
eGFP Goat Rockland 600-101-215 1:1,000
eGFP Mouse Rockland 600-301-215 1:1,000
PITX3 Rabbit NOVUS NBP1-92274 1:500
mCherry Rabbit Rockland 600-401-P16S 1:1,000
mCherry Rat Thermo M11217 1:1,000
KI67 Rabbit Vision Biosystem NCL-K67P 1:1,000
TUBB3 Mouse Covance (BioLegend) MMS-435P (801201) 1:1,000
TH Rabbit Pel-freez P40101-0 1:1,000
TH Mouse Sigma T1299 1:10,000
NURR1 Rabbit Santa Cruz sc-990 1:1,000
MAP2 Rabbit Millipore AB5622 1:1,000
AADC Rabbit Chemicon AB1569 1:500
VMAT2 Rabbit Abcam AB81855 1:1,500
DAT Rabbit Pel-freez P40501-0 1:500
KCNJ6 Rabbit Almone Labs APC-006 1:500
CALB Rabbit Millipore AB1778 1:1,000
NCAM (human) Mouse Santa Cruz sc-106 1:100
PCNA Rabbit Abcam ab18197 1:700
PH3 Rabbit Millipore 06-570 1:500
NeuN Mouse Chemicon MAB377 1:100
NeuroD Mouse Abcam AB60704 1:500
ALCAM Mouse R&D Systems MAB561 2.5μg/10 6 cells
TPBG Mouse R&D Systems MAB49751 2.5 μg /10 6 cells
CORIN Mouse R&D Systems MAB2209 2.5 μg /10 6 cells
CD47 Mouse Santa Cruz sc-12730 1.0 μg /10 6 cells
[111]
[112]
After washing, the primary antibody-labeled cells were incubated with 20 μL of microbeads (Miltenyi Biotec) per 1Х10 7 cells. After washing, the cell suspension was loaded on a separation column (LS column) (Miltenyi Biotec) attached to a magnetic stand. The negative-labeled cells passed during column washing were collected in a separate tube, and after removing the column from the magnetic stand, the positive-labeled cells remaining in the column were eluted with the culture medium into another tube.
[113]
[114]
Immunocytochemistry analysis
[115]
First, cells were fixed in a 4% paraformaldehyde-PBS solution.
[116]
Next, in order to facilitate the penetration of the antibody into the cytoplasm, after treatment with 0.1% Triton X-100-PBS solution for 15 minutes, 2% bovine serum albumin (BSA) (Bovogen, Australia) -PBS solution at room temperature 1 After reacting for a period of time, the primary antibody (refer to Table 1 above) and was combined overnight at 4°C. In order to visualize the protein to which the primary antibody is bound, an appropriate fluorescent-labeled secondary antibody (Molecular Probes-Thermo Fisher Scientific and Vector Laboratories, USA) was used.
[117]
Finally, in order to confirm the cell nucleus, it was mounted on a mounting medium (Vector Laboratories) containing 4',6-diamino-2-phenylindole, and an Olympus IX71 microscope equipped with a DP71 digital camera (Olympus Corp., Japan). , Olympus FSX100 system or LSM710 confocal microscope (Carl Zeiss, Germany) to acquire images.
[118]
[119]
Flow cytometry
[120]
Cells were dissociated into single cells using Accutase (Merck Millipore, Germany), and then fixed using 4% paraformaldehyde-PBS solution. To detect intracellular markers, the cell membrane was permeabilized with 1X Perm/Wash buffer (BD Biosciences), and incubated in 2% BSA-PBS solution with an appropriate antibody for 1 hour. A fluorescent-labeled secondary antibody suitable for the antibody was used. Flow cells were counted with LSRII (BD Biosciences) and analyzed using FlowJo software.
[121]
[122]
Gene expression analysis
[123]
Total RNA (total RNA) present in cells was isolated using Easy-Spin ® Total RNA Extraction Kit (iNtRON Biotechnology, Korea). cDNA was synthesized from 1 μg of total RNA using PrimeScript TM RT Master Mix (TAKARA Bio Inc.). mRNA levels SYBR ® Premix for Ex Taq TM (TAKARA a Bio Inc.) and using CFX96 Real-Time System (Bio- Rad, USA) was quantified by real-time RT-PCR analysis. The Ct value for each target gene was normalized according to the value of GAPDH, and the normalized expression level of the target gene was compared with the control sample in the sorted/unsorted group according to the comparative Ct method. Data were expressed as mean relative deviation ± standard deviation of the mean (SEM) obtained from three independent experiments. The sequences of the primers used for gene expression analysis are shown in Table 2 below.
[124]
[Table 2]
Symbol Gene name Sequence(5' to 3') SEQ ID No.
GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase F: CAA TGA CCC CTT CAT TGA CC SEQ ID No.1
R: TTG ATT TTG GAG GGA TCT CG SEQ ID No.2
OCT4 POU class 5 homeobox 1 F: CCT CAC TTC ACT GCA CTG TA SEQ ID No.3
R :CAG GTT TTC TTT CCC TAG CT SEQ ID No.4
SOX2 SRY-box2 F: TTC ACA TGT CCC AGC ACT ACC AGA SEQ ID No.5
R: TCA CAT GTG TGA GAG GGG CAG TGT GC SEQ ID No.6
NANOG Nanog homeobox F: TGA ACC TCA GCT ACA AAC AG SEQ ID No.7
R: TGG TGG TAG GAA GAG TAA AG SEQ ID No.8
TET1 TET methylcytosine dioxygenase 1 F: CTG CAG CTG TCT TGA TCG AGT TAT SEQ ID No.9
R: CCT TCT TTA CCG GTG TAC ACT ACT SEQ ID No.10
REX1 ZFP42 zinc finger protein F: TCA CAG TCC AGC AGG TGT TTG SEQ ID No.11
R: TCT TGT CTT TGC CCG TTT CT SEQ ID No.12
EN1 Engrailed 1 F: CGT GGC TTA CTC CCC ATT TA SEQ ID No.13
R: TCT CGC TGT CTC TCC CTC TC SEQ ID No.14
FOXA2 Forkhead box A2 (HNF-3β) F: CCG TTC TCC ATC AAC AAC CT SEQ ID No.15
R: GGG GTA GTG CAT CAC CTG TT SEQ ID No.16
LMX1A LIM homeobox transcription factor 1a F: CGC ATC GTT TCT TCT CCT CT SEQ ID No.17
R: CAG ACA GAC TTG GGG CTC AC SEQ ID No.18
eGFP Enhanced green fluorescent protein F: CAT CAA GGT GAA CTT CAA GAT CCG CCA CAA C SEQ ID No.19
R: CTT GTA CAG CTC GTC CAT GCC GAG AGT GAT C SEQ ID No.20
PITX3 Paired like homeodomain 3 F: GCC AAC CTT AGT CCG TG SEQ ID No.21
R: GCA AGC CAG TCA AAA TG SEQ ID No.22
mCherry F: ACT ACG ACG CTG AGG TCA AG SEQ ID No.23
R: GTG TAG TCC TCG TTG TGG GA SEQ ID No.24
OTX2 Orthodenticle homeobox 2 F: GGA AGC ACT GTT TGC CAA GAC C SEQ ID No.25
R: CTG TTG TTG GCG GCA CTT AGC T SEQ ID No.26
FOXA1 Forkhead box A1 F: GGG CAG GGT GGC TCC AGG AT SEQ ID No.27
R: TGC TGA CCG GGA CGG AGG AG SEQ ID No.28
SIM1 Single-minded homolog 1 F: AAA GGG GGC CAA ATC CCG GC SEQ ID No.29
R: TCC GCC CCA CTG GCT GTC AT SEQ ID No.30
LHX1 LIM homeobox 1 F: AGG TGA AAC ACT TTG CTC CG SEQ ID No.31
R: CTC CAG GGA AGG CAA ACT CT SEQ ID No.32
LMX1B LIM homeobox transcription factor 1b F: CTT AAC CAG CCT CAG CGA CT SEQ ID No.33
R: TCA GGA GGC GAA GTA GGA AC SEQ ID No.34
NKX2.2 NK2 homeobox 2 F: CCT TCT ACG ACA GCA GCG ACA A SEQ ID No.35
R: ACT TGG AGC TTG AGT CCT GAG G SEQ ID No.36
NKX6.1 NK6 homeobox 1 F: CGA GTC CTG CTT CTT CTT GG SEQ ID No.37
R: GGG GAT GAC AGA GAG TCA GG SEQ ID No.38
NURR1 Nuclear receptor subfamily 4 group A member 2 F: AAA CTG CCC AGT GGA CAA GCG T SEQ ID No.39
R: GCT CTT CGG TTT CGA GGG CAA A SEQ ID No.40
TH Tyrosine hydroxylase F: GCT GGA CAA GTG TCA TCA CCT G SEQ ID No.41
R: CCT GTA CTG GAA GGC GAT CTC A SEQ ID No.42
DAT Dopamine transporter F: CCT CAA CGA CAC TTT TGG GAC C SEQ ID No.43
R: AGT AGA GCA GCA CGA TGA CCA G SEQ ID No.44
VMAT2 Solute carrier family 18 member A2 (vesicular monoamine transporter 2) F: GCT ATG CCT TCC TGC TGA TTG C SEQ ID No.45
R: CCA AGG CGA TTC CCA TGA CGT T SEQ ID No.46
HTR2B 5-hydroxytryptamine receptor 2B F: GCT GGT TGG ATT GTT TGT GAT GC SEQ ID No.47
R: CCA CTG AAA TGG CAC AGA GAT GC SEQ ID No.48
NeuN RNA binding fox-1 homolog 3 F: TAC GCA GCC TAC AGA TAC GCT C SEQ ID No.49
R: TGG TTC CAA TGC TGT AGG TCG C SEQ ID No.50
MAP2 Microtubule associated protein 2 F: AGG CTG TAG CAG TCC TGA AAG G SEQ ID No.51
R: CTT CCT CCA CTG TGA CAG TCT G SEQ ID No.52
[125]
[126]
Microarray Analysis: Transcript Profiling
[127]
10 μg of total RNA of each sample was processed/analyzed by Macrogen (Macrogen Inc., Korea), and the samples were hybridized to Affymetrix Human U133 Plus 2.0 array.
[128]
[129]
Example. Differentiation protocol into mesocerebral dopamine (mDA) neurons
[130]
The specific protocol is shown in FIG. 1.
[131]
[132]
The hESC being cultured in the colony form was treated with 2 mg/mL of type IV collagenase (Worthington Biochemical Corp., USA) for 30 minutes to induce embryoid formation, and bFGF-free hES culture medium (EB medium) Cultured in. At this time, 1.5% dimethyl sulfoxide (DMSO; Calbiochem-Merck Millipore) was treated for the first 24 hours, and for 4 days thereafter, 5 μm of dosomorphin (DM) (Calbiochem-Merck Millipore) and 5 μm of SB431542 (SB) (Sigma-Aldrich) was treated.
[133]
From day 5 (d5), EB was attached to a matrigel-coated culture dish in DMEM/F12 1X N2 supplemented medium (bmN2 medium) to which 20 ng/mL bFGF and 20 μg/mL human insulin solution (Sigma-Aldrich) were added, and , Patterning factors (1 μM CHIR99021 (Miltenyi Biotec) and 0.5 μM SAG (Calbiochem-Merck Millipore)) were treated for 6 days.
[134]
On day 11 (d11), the neural rosette formed in the EB colony was mechanically isolated using a pulled glass pipette, and the isolated neural rosette mass was crushed through pipetting and reattached to a matrigel-coated culture dish. I did. Reattached cells were expanded and cultured in DMEM/F12 1X N2 and 1X B27 medium (bN2B27 medium) supplemented with 20 ng/mL bFGF, supplemented with 1 μM CHEM99021 and 0.5 μM SAG for 2 days, and midbrain Sex dopamine neuronal specific differentiation was induced.
[135]
On the 13th day (d13), the mesocerebral dopamine neural progenitor cell cluster was separated into single cells using Accutase, and a matrigel-coated plate at a density of 3.12×10 5 cells/cm 2 in N2B27 medium (N2B27 medium) without bFGF. Reattached to the top. The cells were amplified and cultured for 7 days so that they occupied nearly 90% of the total area of ​​the plate.
[136]
From the 20th day (d20), the mesocerebral dopamine neural progenitor cells having the characteristics of the midbrain/dorsal side were cultured in a medium (NBG medium) containing 1X N2, 0.5X B27 and 0.5X G21 supplements (Gemini Bio-Products, USA). I did. At this time, 1 μM of DAPT (Sigma-Aldrich) was added for the first 7 days, and after that, 10 ng/mL of BDNF (brain-derived neurotrophic factor; ProSpec-Tany TechnoGene, Israel), 10 ng/mL of GDNF ( glial cell line-derived neurotrophic factor; ProSpec-Tany TechnoGene), 200 μM of ascorbic acid (AA) and 1 μM of dibutylyl cyclic-AMP (db-cAMP) (Sigma-Aldrich) were added for final differentiation.
[137]
[138]
Preparation Example 1. Preparation of LMX1A-eGFP reporter cell line (reporter line)
[139]
The specific protocol is shown in FIG. 2.
[140]
[141]
1-1. Design of nuclease and donor DNA plasmid
[142]
The TALEN-encoding plasmid was purchased from Toolen Inc., Korea.
[143]
The TALEN site is near TGA, a stop codon of exon 9 of the LMX1A gene (5'-TCC ATG CAG AAT TCT TAC TT-3' (left), 5'-TCA CAG AAC TCT AGG GGA AG-3' ( Right)), it was designed to cause double-strand brsaks (DSB), and potential off-target sites were searched using Cas-Offinder (www.rgenome.net/).
[144]
The donor DNA plasmid was constructed as follows in DH5α using pUC19 as the plasmid backbone: 5'homology arm-endogenous LMX1A genomic fragment (left arm)-T2A-eGFP-bGH poly(A)-PGK promoter driven puromycin resistance cassette- bGH poly(A)-3' homology arm (right arm).
[145]
[146]
1-2. Preparation of LMX1A-eGFP reporter cell line
[147]
HESC colonies on inactivated STO were transferred onto plates coated with hESC-compatible matrigels (BD Biosciences, USA) in StemMACS ™ iPS-Brew XF complete medium (Miltenyi Biotec, Germany). Then, the cells were subcultured to occupy close to 80-90% of the total area of ​​the plate (Split ratio, 1:5). After dissociation into single cells using Accutase, transfer to a matrigel-coated plate in medium to which ROCK inhibitor (10 μM, Y-27632) (Calbiochem-Merck Millipore) was added for the first 24 hours, and daily The medium was refreshed. Only hESC with less than 10 enzymatic passages were used in the experiment.
[148]
HESCs were obtained using Accutase, and a single cell suspension was prepared. Then, it was gently resuspended in R buffer of Neon transfection kit (100 μL, Invitrogen) to a final density of 1.0 Х10 7 cells/mL. 120 μL of the resuspended cells were mixed with one pair of TALEN-encoding plasmids (6 μg each) and LMX1A donor DNA plasmid (6 μg) of Preparation Example 1-1, and pulsed at a voltage of 850 mV for 30 ms. Electroporation was performed (Neon transfection system).
[149]
Then, the cells were transferred to 2-3 35 mm plates pre-inoculated with STO feeder in hESC medium, and a ROCK inhibitor was added for the first 48 hours, and the medium was changed after 2 days, and the medium was changed daily.
[150]
After 5 days of electroporation, 0.5 μg/mL puromycin (Sigma-Aldrich) was treated in the hESC medium. After 10-14 days, colonies showing puromycin resistance were classified as reporter cell line candidates, and the number of cells was expanded by subculturing them.
[151]
Finally, the LMX1A-eGFP reporter cell line was confirmed through genotyping of clonal cells.
[152]
[153]
Preparation Example 2. Preparation of PITX3-mCherry reporter cell line
[154]
The specific protocol is shown in FIG. 3.
[155]
[156]
2-1. Design of nuclease and donor DNA plasmid
[157]
Cas9- and sgRNA (CRISPR/Cas9)-encoding plasmids were purchased from Tulgen.
[158]
The sequence for producing sgRNA that mediates PITX3 targeting is the stop codon TGA (5'-TAC GGG CGG GGC CGC TCA TA C GG -3' (underlined ) to cause double-stranded cleavage (DSB) near the stop codon TGA. : PAM)). Potential off-target sites were searched using Cas-Offinder (www.rgenome.net/).
[159]
The donor DNA plasmid was constructed as follows in DH5α using pUC19 as the plasmid backbone: 5'homology arm-endogenous PITX3 genomic fragment (left arm)-T2A-mCherry-bGH poly(A)-PGK promoter driven neomycin resistance cassette- bGH poly(A)-3' homology arm (right arm).
[160]
[161]
2-2. Preparation of PITX3-mCherry reporter cell line
[162]
Except for Cas9- and sgRNA-encoding plasmids instead of TALEN-encoding plasmids, PITX3 donor DNA plasmids instead of LMX1A donor DNA plasmids, and 100 μg/mL G418 (Calbiochem-Merck Millipore) instead of 0.5 μg/mL puromycin. And, a PITX3 reporter cell line was prepared in the same manner as in Preparation Example 1-2.
[163]
[164]
Experimental Example 1. Identification of progenitor stage: LMX1A-eGFP reporter cell line identification
[165]
The LMX1A-eGFP reporter cell line prepared in Preparation Example 1 was differentiated using the differentiation protocol of the above example, and then the differentiation process was confirmed (Immunocytochemistry and Cytometry).
[166]
As can be seen in Figure 4, throughout the differentiation process, the expression of EN1 as a regional marker, FOXA2 as a position marker for the midbrain floor plate, LMX1A as a dopamine lineage marker, and eGFP were observed throughout the differentiation process. In particular, on the 20th day of differentiation (d20), ~41.1% of the cell population appeared as eGFP-positive (eGFP + ) cells, and eGFP + cells simultaneously expressed EN1 and FOXA2. Progenitor cells (~46.6% EN1 + eGFP + , ~49.2% FOXA2 + eGFP + ) that showed positive reactions for all of the three markers (EN1 and FOXA2, and LMX1A) were also detected (see FIG. 6).
[167]
These results indicate that the cells expressing eGFP are cells expressing LMX1A (establishment of the LMX1A reporter cell line), and hESC directly differentiated into mDA neural progenitor cells exhibiting floor plate (FOXA2) and midbrain (EN1) characteristics.
[168]
[169]
Experimental Example 2. Identification of neuronal stage: PITX3-mCherry reporter cell line identification
[170]
The PITX3-mCherry reporter cell line prepared in Preparation Example 2 was differentiated using the differentiation protocol of the above example, and then the differentiation process was confirmed (Immunocytochemistry and Cytometry).
[171]
As can be seen in FIG. 5, mCherry expression was not observed until about 30 days (d30) of differentiation, and mCherry-positive (mCherry + ) neurons (neurons) clusters were observed on about 40 days (d40) of differentiation . On the other hand, the expression pattern of the PITX3 gene appeared the same as that of the reporter mCherry throughout the differentiation (maturation) process, and in particular, ~16% of the final differentiated mDA neuron (neuron) population was the occurrence location and lineage markers (EN1 and FOXA2). , And LMX1A) together expressing mCherry + cells (see Fig. 9).
[172]
These results indicate that the cells expressing mCherry are cells expressing PITX3 (establishment of a PITX3 reporter cell line), and hESCs are mDA neurons exhibiting the characteristics of the floor plate (FOXA2) and midbrain (EN1), and mesocerebral dopamine (LMX1A). It means that it is directly differentiated into (neurons).
[173]
[174]
Experimental Example 3. Characterization of LMX1A-positive cells
[175]
The cells of d20 of Experimental Example 1 were exposed to Y27632 of 10 μm for 1 hour, and then dissociated using accutase, and then cells of 40 μm or less were collected using a cell strainer (BD Science). Dissociated progenitor cells were treated with 3% fetal bovine serum (FBS) (Gemini Bio-Products) and 1x penicillin-streptomycin (P/S) (Gibco-Thermo Fisher Scientific) in HBSS (WELGENE Inc., Korea) LMX1A -Sorting buffer (LMX1A-SB) was resuspended at a final density of 2 Х10 6 cells/mL, and cell separation (FACS) was performed. The mRNA expression levels of the Unsorted group, LMX1A - group and LMX1A + group were compared.
[176]
As can be seen in Figure 6a, after cell separation, ~41.1% of the viable cells appeared as LMX1A-eGFP + (LMX1A + ) fraction. Also, LMX1A + and LMX1A-eGFP - (LMX1A - ) progenitor cells have been shown to maintain the shape (morphology) is similar to the non-sorted cells. In particular, ~99.4% of the isolated LMX1A + was found to be positive for both EN1 and FOXA2. These results indicate that mDA neuronal progenitor cells were isolated by FACS cell separation.
[177]
In addition, as can be seen in Figure 6b, in the LMX1A + group, expression of mDA progenitor cell-specific genes (EN1, FOXA1, FOXA2, LMX1A, LMX1B) was significantly upregulated, but serotonin progenitor cell-specific genes (NKX2 .2) and red nucleus (Red nucleus, an anatomical site of the midbrain) progenitor cell-specific genes (SIM1, LHX1, NKX6.1) were upregulated in the unclassified group and in the LMX1A - group. These results indicate that the LMX1A + cells isolated by FACS cell separation exhibit the characteristics of mDA neuronal progenitor cells.
[178]
[179]
In addition, after the cell separation (FACS), the unsorted group, the LMX1A - group and the LMX1A + group were further cultured in vitro for 1 day .
[180]
For the cultured cells, neural-specific and proliferative cell-specific markers were observed, and BrdU analysis was performed to confirm the cell cycle.
[181]
As can be seen in Figure 7a, NESTIN-, SOX1-, SOX2-, and KI67-positive cells were similar in all three groups. These results imply that the sorted LMX1A + cells maintain the characteristics of neuronal progenitor cells and cell proliferation ability, as in the unsorted group and the LMX1A - group.
[182]
In addition, as can be seen in Figure 7b, in the LMX1A + group, 38.5±3.9% and 49.5±6.2% of the surviving cells were in the G0/G1 and S stage in the mDA neuroprogenitor cell stage, and 6±2.7% were in G2/M. there was. These results mean that the sorted LMX1A + cells are actually proliferating cells that go through the cell cycle.
[183]
[184]
Finally, after the cell separation (FACS), the unsorted group, the LMX1A - group and the LMX1A + group were additionally differentiated (4 weeks, d52), and then the expression of mDA neuron-related markers was compared. .
[185]
As can be seen in Figure 8a, after the final differentiation, the ratio of cells expressing mDA neuron-related markers (TH, NURR1, PITX3) in the LMX1A + group increased compared to the unsorted group and the LMX1A - group . These results indicate that LMX1A + cells are mDA neuronal progenitor cells capable of differentiating into mDA neurons.
[186]
[187]
Furthermore, the degree of dopamine secretion (release) was confirmed for cells that were fully matured by the final differentiation (8 weeks, d75).
[188]
Specifically, the cells were washed with a low KCl solution (2.5 mM CaCl 2 , 11 mM glucose, 20 mM HEPES-NaOH, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 and 140 mM NaCl) and a low KCl solution Incubated for 2 minutes at. Then, it was replaced with a high KCl solution (2.5 mM CaCl 2 , 11 mM glucose, 20 mM HEPES-NaOH, 60 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 and 85 mM NaCl) and incubated for 15 minutes. I did. The solution was collected in a 15 mL tube, centrifuged at 2,000 rpm for 1 minute to remove debris, and the supernatant was collected in a 1.5 mL tube and stored at -80 °C. The concentration of dopamine was detected by the dopamine ELISA kit (Cat. No. KA3838; Abnova, Taiwan) according to the manufacturer's instructions.
[189]
As can be seen in Figure 8b, after the final differentiation, the secretion of dopamine was increased in the LMX1A + group compared to the unclassified group and the LMX1A - group . These results indicate that the mDA neurons formed by the final differentiation of LMX1A + cells are functional mDA neurons that secrete dopamine.
[190]
[191]
Experimental Example 4. Characterization of PITX3-positive cells
[192]
The cells of d40 of Experimental Example 2 were dissociated into single cells using Papain (Worthington Biochemical Corp.) to which 5% trehalose was added, and then 40 μm using 70 μm and 40 μm cell bodies sequentially. The following cells were collected. Dissociated cells were added to PITX3-Sorting buffer (PITX3-SB) to which 1x P/S in 5% FBS, 1x Glutamax (Gibco-Thermo Fisher Scientific), 5% trehalose, and HBSS was added to 1 × 10 7 cells/mL. Resuspended at the concentration and cell separation (FACS) was performed. In addition, after cell separation, the cells were cultured in vitro for an additional 36 hours and then the morphology of the surviving cells was observed.
[193]
As can be seen in Figure 9a, after cell separation, ~16% of the viable cells appeared as PITX3-mCherry + (PITX3 + ) fraction. In addition, the cell in spite of the disappearance of the cells there was in the selection process and, further in vitro culture after surviving PITX3 + and PITX3-mCherry - (PITX3 - shown to keep form cells are neurons (neuronal) similar to the non-sorted cells) . These results indicate that PITX3 + cells were isolated by FACS cell separation, and all three groups (unclassified group, PITX3 + group, and PITX3 - group) showed neuronal cell-specific morphology.
[194]
[195]
Next, the expression of mDA neuron-related markers was compared in the unsorted group, PITX3 - group and PITX3 + group of the d40 cells.
[196]
As can be seen in Figure 9b, in the PITX3 + group , expression of neurons (neurons)-specific genes (NeuN and MAP2) and mDA neurons (neurons)-specific genes (PITX3, NURR1, TH, DAT and VMAT2) Significantly upregulated, but expression of serotonin neuron (neuron)-specific gene (HTR2B) was downregulated. In addition, as can be seen in Fig. 9c, the proportion of cells expressing TH, which is an mDA neuron marker, and cells expressing TUBB3 (TUJ1) and MAP2, which are neuronal-specific markers, increased in the PITX3 + group. These results indicate that PITX3 + cells are mDA neurons.
[197]
[198]
Finally, after the cell separation (FACS), the unsorted group was additionally cultured, and then double-labeled immunostaining was performed on the 44th day of differentiation (d44).
[199]
As can be seen in Figure 10, it was confirmed that PITX3 + cells express mature mDA neuronal markers NURR1, AADC, VMAT2, and DAT. In addition, KCNJ6 (GIRK2), an A9 regional marker, was expressed, but no cells expressing CALB, an A10 regional marker, were observed. These results indicate that the PITX3 + cells differentiated through the differentiation protocol of the present invention are mature mDA neurons.
[200]
[201]
Experimental Example 5. Transplantation suitability check
[202]
After differentiating the LMX1A-eGFP reporter cell line of Preparation Example 1 and the PITX3-mCherry reporter cell line of Preparation Example 2 using the differentiation protocol of the above example, mDA neural progenitor cells and differentiation 50 days (d50) on day 20 (d20) of differentiation ) Of mature mDA neurons were separated into single cells using the same method as in Experimental Examples 3 and 4, respectively. The degree of apoptosis of the isolated single cells in vitro was compared. At this time, apoptosis was confirmed according to the manufacturer's instructions using a LIVE/DEAD Fixable Violet Dead Cell Stain kit (Thermo Fisher).
[203]
As can be found at 11, the cells showing apoptosis was separated single cells LMX1A + cells in about 8%, PITX3 + from the cells was about 30%. That is, when isolated into single cells, LMX1A + mDA neuronal progenitor cells maintained higher viability than PITX + mDA neurons, and through this, LMX1A + cells and PITX3 + cells had a difference in sensitivity to the single cell separation process for transplantation. I could see that it appeared. These results indicate that transplanting the mDA neural precursor cells LMX1A + cells is more advantageous in terms of apoptosis than the transplantation of mature neurons PITX3 + cells.
[204]
[205]
Experimental Example 6. Identification of mesocerebral dopamine (mDA) marker
[206]
6-1. Transcriptome analysis
[207]
After differentiating the LMX1A-eGFP reporter cell line of Preparation Example 1 and the PITX3-mCherry reporter cell line of Preparation Example 2 using the differentiation protocol of the above example, LMX1A + and LMX1A on day 20 of differentiation (d20, mDA neuron progenitor cell stage) - cells, and the differentiation of the 40 days PITX3 (d40, mDA neuron stage) + and PITX3 - were isolated cell, performing transcript analysis (Microarray) for this (see Fig. 12).
[208]
Meanwhile, cells expressing eGFP and cell cycle markers (Ki67, PCNA, and PH3) were observed in the mDA neuronal progenitor cell stage. In the mDA neuronal stage, cells expressing mCherry and mDA neuronal marker (TH), mature Cells expressing a neuronal cell marker (NeuN) were observed, but no cells expressing an immature neuronal cell marker (NeuroD) and a proliferative cell marker (KI67) were observed.
[209]
[210]
6-2. Identification of mDA marker candidates
[211]
Based on the above results, a candidate group of mDA markers was obtained. The specific process is shown in FIG. 13.
[212]
[213]
As a result of comparative microarray analysis of the four isolated cells, LMX1A - genes that are upregulated in LMX1A + cells (> 2-FC) compared to cells and PITX3 - are upregulated in PITX3 + cells compared to cells (> 2 -FC), and among them, 53 genes encoding surface markers were identified through gene mining. The 53 genes identified included a number of genes ( Corin, Clstn2, Kitlg, Plxdc2, Pcdh7, Ferd3l, Frem1, Alcam and Notch2 ) known to be mouse mDA neuronal precursor cell-specific .
[214]
[215]
Next, target validation was evaluated by confirming the expression of the gene in mDA cells that are actually differentiated. As a result, the LMX1A - cells compared to LMX1A + cells, among the genes upregulated in LMX1A + cells, surface marker genes (Fig. 14), and LMX1A + cells and PITX3 + cells both up-regulated or down-regulated surface marker genes (Fig. 15) were identified. I did. Screening was performed for 18 genes with commercially available antibodies among the 21 genes of FIG. 14.
[216]
As a result, four genes ( CORIN, TPBG, CD47, ALCAM ) of the 18 genes were selected as final surface marker candidates.
[217]
[218]
6-3. Identification of mDA neural progenitor cell-specific markers
[219]
Based on the results, MACS targeting the four genes ( CORIN, TPBG, CD47, ALCAM ) was performed.
[220]
As can be seen in FIGS. 16 and 17, CORIN- and trophoblast glycoprotein (TPBG)-targeted MACS showed statistically significant enrichment of LMX1A + FOXA2 + mDA neuronal progenitor cells. In particular, TPBG was widely expressed in mDA neuronal progenitor cells.
[221]
Meanwhile, the CORIN gene is already known for use for enriching mDA neuronal progenitor cells, but TPBG has never been reported during mDA development, so TPBG was selected as the final mDA neuronal progenitor cell-specific marker.
[222]
[223]
Experimental Example 7. In vivo ( in vivo ) transplantation effect confirmation of TPBG-positive cells isolated from human embryonic stem cells (hESC)
[224]
7-1. 6-OHDA damaged Parkinson's disease (PD)-model manufacturing
[225]
200-250 g of female Sprague-Dawley rats (Orient Bio Inc., Korea) were used as a transplant target. 30 mg/kg Zoletil ® (Virbac, France) and 10 mg/kg Rompun ® (Bayer, Germany) were mixed and used as an anesthetic.
[226]
According to the coordinates (TB -0.45, AP -0.40, ML -0.13, DV -0.70), 3 μL of 30 mM 6-OHDA was injected into the medial forebrain bundle of rats and hemi-parkinsonian disease model (hemi-parkinsonian). model).
[227]
[228]
7-2. Confirmation of behavioral recovery of PD-model after TPBG-positive cell transplantation
[229]
After the hESCs being cultured in the form of colonies were differentiated using the differentiation protocol of the above example, MACS targeting TPBG was performed on day 20 of differentiation (d20).
[230]
The isolated TPBG-positive cells were suspended in 1X HBSS so that the final concentration was 8.75 Х10 4 cells/μL to prepare a cell suspension. At this time, as a control group, a group transplanted with only HBSS was used.
[231]
4 weeks after the 6-OHDA injury of Experimental Example 7-1, the prepared cell suspension (350,000 cells in total) was measured per rat according to coordinates (TB -0.24, AP +0.08, ML -0.30, DV -0.40 and -0.50). Each 4 μL was transplanted by stereotactic method.
[232]
From 2 days before transplantation until the mice were sacrificed, 10 mg/kg of cyclosporine A (cyclosporine A; Chong Kun Dang, Korea) was intraperitoneally injected daily for the duration of the experiment for immunosuppressive treatment.
[233]
Before transplantation, at 4, 8, 12 or 16 weeks after transplantation, amphetamine (2.5 mg/kg, Sigmal-Aldrich) was injected intraperitoneally, and the rat rotation was recorded for 30 minutes.
[234]
As can be seen in FIG. 18, TPBG-positive cells showed significant improvement in motor function for 16 weeks after transplantation compared to the control group. These results indicate that hESC-derived TPBG-positive mDA neural progenitor cells can survive in vivo and improve motor function.
[235]
[236]
7-3. Confirmation of graft characteristics after TPBG-positive cell transplantation
[237]
As a control group, TPBG-positive cells and unsorted cells were transplanted in the same manner as in Example 7-2, except that a group transplanted with unsorted cells was used.
[238]
After 16 weeks of implantation, rats were anesthetized with 25% urethane solution and perfused with 0.9% saline and 4% paraformaldehyde by transcardial perfusion. The removed brain was fixed overnight and cryoprotected with 30% sucrose-PBS solution. The cryoprotected brain was immobilized on an FSC 22 ® compound (Leica, Nußloch, Germamy), and coronal sections were prepared at 18 μm thickness using a cryostat (Thermo Fisher Scientific). Then, immunohistochemical staining was performed targeting a human-specific neural cell adhesion molecule (hNCAM).
[239]
As can be seen in FIG. 19, the TPBG-positive cell group consisted of a greater number of TH + hNCAM + and PITX3 + hNCAM + mDA neurons compared to the unclassified group . These results indicate that TPBG-positive cells are more suitable for differentiation into mDA neurons in vivo compared to the unclassified group.
[240]
In addition, as can be seen in FIG. 20, a graft with about 20% or more of KI67 + hNCAM + cells was observed in one specific rat of the unclassified group, but KI67 + hNCAM + cells were observed in the TPBG-positive group. Didn't. These results indicate that, if not classified, it is possible to maintain the proliferative ability even after 16 weeks of transplantation, but proliferating cells can be excluded when cells are sorted with TPBG. This would be a very important result in terms of the safety of cell therapy.
[241]
[242]
Experimental Example 8. Characterization of TPBG-positive cells isolated from human fetal ventral mesencephalic cells (fVM cells)
[243]
For fVM cells being cultured on a laminin-coated plate in a neural stem cell maintenance medium (ReNcell NSC maintenance Medium, Merck), when the cells are cultured to occupy close to 80-90% of the total plate area, MACS targeting TPBG is performed. I did. Then, the relative expression of EN1 in the isolated TPBG-positive cells and TPBG-negative cells was confirmed by qRT-PCR using hESC (H9) as a control (expression of H9 = 1).
[244]
As can be seen in FIG. 21, the expression of EN1, which is a marker for the development of mDA neurons, was increased in TPBG-positive cells compared to TPBG-negative cells. These results imply that TPBG can be used to enrich cells exhibiting midbrain characteristics among fVM cells.
[245]
[246]
Experimental Example 9. Characterization of TPBG-positive cells isolated from human induced pluripotent stem cells (iPSC)
[247]
Human iPSCs (HDF-epi3) being cultured in the same manner as the human embryonic stem cells were differentiated using the differentiation protocol of the above example, and then MACS targeting TPBG was performed on day 20 of differentiation (d20). Expression of EN1, FOXA2, and LMX1A in the isolated TPBG-positive cells was confirmed (Immunocytochemistry).
[248]
As can be seen in Figure 22, the expression of the midbrain developmental location markers EN1 and FOXA2 did not show a difference before and after MACS, but the cells expressing the mDA development lineage marker LMX1A was found to be enriched in TPBG-positive cells. In addition, the proportion of cells in which all of the three markers (EN1 and FOXA2, and LMX1A) responded positively were also significantly enriched in TPBG-positive cells.
Industrial availability
[249]
The present invention relates to a method for isolating dopamine neurons and a pharmaceutical composition for treating Parkinson's disease comprising dopamine neurons isolated using the same, wherein the method for isolating dopamine neurons is TPBG (Trophoblast glycoprotein)-positive dopamine neurons By including the step of isolating, dopamine neurons isolated according to the present method are characterized in that the efficacy of the cells during transplantation is improved and the transplantation safety is improved, and thus can be usefully used for cell transplantation for the treatment of Parkinson's disease.
Claims
[Claim 1]
A method for producing dopaminergic neural cells comprising the following steps: (a) contacting a cell population with a Trophoblast glycoprotein (TPBG) antibody; And (b) separating TPBG-positive dopamine neurons that bind to the TPBG antibody.
[Claim 2]
According to claim 1, wherein the cell population is human stem cells (human stem cells); Progenitors or precursors; And dopaminergic neural progenitors differentiated from the human stem cells or progenitor cells, mature dopaminergic neurons, and neural derivatives derived therefrom; The method comprising at least one selected from the group consisting of.
[Claim 3]
The method of claim 2, wherein the human stem cells or progenitor cells are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells. , iPSCs), adult stem cells or fetal cells.
[Claim 4]
4. The method of claim 3, wherein the fetal cells are derived from fetal neural tissue or derivatives thereof.
[Claim 5]
The method of claim 1, wherein the TPBG-positive dopamine neurons alleviate symptoms of Parkinson's disease.
[Claim 6]
The method of claim 1, wherein the TPBG-positive dopamine neurons enhance the safety of cell transplant therapy.
[Claim 7]
The method of claim 1, wherein the dopamine neurons are dopaminergic neural progenitors or dopaminergic neural precursor cells or mature dopaminergic neurons.
[Claim 8]
The method of claim 1, wherein the dopamine neuron is a midbrain dopamine neuron.
[Claim 9]
The method of claim 8, wherein the mesocerebral dopamine neuron is an A9 region-specific mesocerebral dopamine neuron.
[Claim 10]
A pharmaceutical composition for the treatment of Parkinson's disease, including TPBG (Trophoblast glycoprotein)-positive dopaminergic neural cells.
[Claim 11]
11. The pharmaceutical composition of claim 10, wherein the dopamine neurons are midbrain dopamine neurons.
[Claim 12]
The pharmaceutical composition according to claim 11, wherein the mesocerebral dopamine neurons are A9 region-specific mesocerebral dopamine neurons.
[Claim 13]
A method of enhancing the efficacy of dopaminergic neural cells and improving transplantation safety for cell transplant therapy for Parkinson's disease, comprising the following steps: (a) The cell population is changed to TPBG Trophoblast glycoprotein)-contacting with an antibody; And (b) separating TPBG-positive dopamine neurons that bind to the TPBG antibody.
[Claim 14]
14. The method of claim 13, wherein the cell population comprises human stem cells; Progenitors or precursors; And dopaminergic neural progenitors differentiated from the human stem cells or progenitor cells, mature dopaminergic neurons, and neural derivatives derived therefrom; The method comprising at least one selected from the group consisting of.
[Claim 15]
The method of claim 14, wherein the human stem cells or progenitor cells are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells. , iPSCs), adult stem cells or fetal cells.
[Claim 16]
16. The method of claim 15, wherein the fetal cells are derived from fetal neural tissue or derivatives thereof.
[Claim 17]
The method of claim 13, wherein the dopamine neurons are dopaminergic neural progenitors or dopaminergic neural precursor cells or mature dopaminergic neurons.
[Claim 18]
14. The method of claim 13, wherein the dopamine neuron is a midbrain dopamine neuron.
[Claim 19]
19. The method of claim 18, wherein the mesocerebral dopamine neuron is an A9 region-specific mesocerebral dopamine neuron.
[Claim 20]
A composition for transplanting dopamine neurons, including TPBG (Trophoblast glycoprotein)-positive dopaminergic neural cells.
[Claim 21]
21. The composition of claim 20, wherein the dopamine neurons are midbrain dopamine neurons.
[Claim 22]
22. The composition of claim 21, wherein the mesocerebral dopamine neuron is an A9 region-specific mesocerebral dopamine neuron.
[Claim 23]
The composition of claim 20, wherein the composition is for treatment of Parkinson's disease.
[Claim 24]
The composition of claim 20, wherein the composition enhances the efficacy of dopamine neurons and improves transplant safety.