WO2019212201A1 - Method for isolating dopamine neurons and pharmaceutical composition for treating parkinson's disease, containing dopamine neurons isolated using same - Google Patents

Abstract

The present invention relates to a method for isolating dopamine neurons and a pharmaceutical composition for treating Parkinson's disease, containing dopamine neurons isolated using same. The method for isolating dopamine neurons comprises a step for isolating trophoblast glycoprotein (TPBG)-positive dopamine neurons, such that the dopamine neurons isolated by the method have increased cell efficacy when transplanted and enhanced transplantation safety so as to be effectively used for cell transplantation for the treatment of Parkinson's disease.

Description

Method for isolating dopamine neurons and pharmaceutical composition for treating Parkinson's disease comprising dopamine neurons isolated using the same

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 task number HI18C0829 under the support of the Ministry of Health and Welfare of the Republic of Korea, the research and management institution of the task is the Korea Health Industry Development Institute, the research project name is "advanced medical technology development", the research title is "neural system of pluripotent stem cells In vivo differentiation monitoring and minimum transplant cell number prediction technology for disease application ", The lead organization is Yonsei University Industry-Academic Cooperation Group, Research period 2018.04.30. ~ 2021.12.31.

This patent application is filed with the Korean Patent Application No. 10-2018-0050918 filed with the Korean Patent Office on May 2, 2018 and the Korean Patent Application No. 10-2019-0048784 filed with the Korean Patent Office on April 25, 2019. Priority is claimed, the disclosures of which are incorporated herein by reference.

Parkinson's disease (PD) is one of the neurodegenerative disorders due to focal degeneration of midbrain dopaminergic (mDA) neurons, most suitable for cell-based therapies.

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.

Although these studies have shown that grafts can lead to recovery of motor function, there is consensus that cell transplantation results are inconsistent and in some cases also have side effects, with cell-based therapies further improved. Was formed.

In particular, the development of transplanted cells, which have been pointed out as a problem in previous studies, to develop in close proximity to cells present in vivo, and to compensate for the disadvantages such as limited solubility and batch-to-batch inconsistency of fetal tissue. Alternative studies continued.

The results include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which show endogenous proliferation in vitro and have a wide range of differentiation into various neurons. Human pluripotent stem cells (hPSCs) have attracted attention.

Accordingly, until recently, differentiation techniques of mDA neurons have been developed and evolved to produce high yields of cells. However, other products other than mDA neurons have been developed in the differentiation-induced products from hPSCs through recent development of differentiation techniques. Heterogeneity, in which cells are quite mixed, has been observed, making it difficult to consider clinical applications based on current differentiation techniques.

Thus, identification and isolation of hPSC-derived mDA neurons from various cell populations is critical for successful transplantation research as well as standardization of transplanted cells.

Meanwhile, a strategy for purely separating (concentrating) differentiated mDA neurons derived from early hPSCs is based on fluorescence-activated cell sorting (FACS) targeting multiple surface antigens. It was made. This approach increased the neuronal population expressing tyrosine hydroxylase (TH), but it was unclear whether this cell population retained mDA neuronal properties.

Recently, transcriptomes of mouse fVM tissue and mouse embryonic stem cell (mESC) -derived mDA neuronal progenitor cells have been analyzed to find specific surface markers that can differentiate and enrich mDA neurons. .

These studies have suggested some possible cell surface markers, which have shown that dopamine neurons can be isolated and enriched, but at the same time, cell surface markers expressing exactly what stage of neurons are expressed during neuronal development. It is not yet clear whether it should be excavated. The development of differentiation-specific markers is also an important task, since it is a common opinion in the industry that the survival rate or differentiation of isolated neurons differs according to the developmental stage, and this may affect the recovery of function after transplantation. Must be.

Therefore, the study of surface cerebral dopamine neurons and surface markers of differentiation stages is urgent.

The present inventors have tried to differentiate the process of differentiating cerebral dopamine neurons and develop cell surface markers at each stage. As a result, LMX1A-eGFP and PITX3-mCherry reporter establish hESC cell lines, and to differentiate them LMX1A ⁺ mDA neural progenitor cells and PITX3 ⁺ mDA separate the nerve cells, cell surface markers associated therefrom and midbrain St. dopamine neurons (TPBG ), The present invention was completed.

Accordingly, it is an object of the present invention to provide a method for producing dopaminergic neural cells.

Another object of the present invention is to provide a pharmaceutical composition for treating Parkinson's disease, including Trophoblast glycoprotein (TPBG) -positive dopamine neurons.

It is still another object of the present invention to provide a method of enhancing the efficacy of dopamine neurons and improving transplant safety for cell transplantation therapy of Parkinson's disease.

Another object of the present invention is to provide a composition for transplanting dopamine neurons comprising TPBG (Trophoblast glycoprotein) -positive dopamine neurons.

The present inventors have tried to differentiate the process of differentiating cerebral dopamine (mDA) neurons and develop cell surface markers at each stage. As a result, LMX1A-eGFP and PITX3-mCherry reporter establish hESC cell lines, and to differentiate them LMX1A ⁺ mDA neural progenitor cells and PITX3 ⁺ mDA separate the nerve cells, cell surface markers associated therefrom and midbrain St. dopamine neurons (TPBG ).

More specifically, the inventors have shown that the LMX1A-eGFP reporter hESC cell line engineered to express green fluorescent protein (eGFP) at the same time when the mDA progenitor step-specific gene LMX1A is expressed, and mature mDA neurons ( neuronal step-specific gene PITX3 expression, at the same time the red fluorescent protein (mCherry) engineered to express the PITX3-mCherry reporter hESC cell line, respectively, and established the LMX1A ⁺ mDA neuro precursor cells and PITX ⁺ mDA neurons Through a comparative analysis of carcasses, cell surface marker candidates specifically expressed in the precursors (neuronal precursor cells) of mDA neurons were selected. Among them, TPBG was found as a new cell surface marker, and as a result of cell separation targeting TPBG, it was confirmed that mDA neuroprogenitor cells were concentrated. In addition, TPBG-positive cells isolated by magnetic-activated cell sorting (MACS) at the mDA neuroprogenitor cell stage were transplanted into 6-OHDA-damaged Parkinson's disease (PD) rat model. It was confirmed that motor dysfunction was recovered without tumor formation.

Thus, TPBG is a novel surface marker protein for isolating implantable mDA neuronal progenitor cells, and mDA cells isolated using TPBG are expected to provide safe and effective cell replacement therapy for Parkinson's disease.

The present invention provides a method for isolating dopamine neurons, a pharmaceutical composition for treating Parkinson's disease comprising isolated dopamine neurons, enhancing the efficacy of dopamine neurons for improving the cell transplantation therapy of Parkinson's disease and improving graft safety. It relates to a method, and a composition for transplanting dopamine neurons prepared using the same.

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

One aspect of the invention relates to a method for producing dopaminergic neural cells comprising the following steps.

(a) contacting a cell population with a Trophoblast glycoprotein (TPBG) antibody; And

(b) isolating TPBG-positive dopamine neurons that bind to TPBG antibodies.

In the present invention, "neural cells" are cells constituting the nervous system and may be used in the same sense as neurons, and "dopaminergic neural cells" refers to a neurotransmitter dopamine (dopamine). Means a secreted neuron.

The dopamine neurons may be dopaminergic neural progenitors or dopaminergic neural precursor cells or mature dopaminergic neurons, but are not limited thereto.

In the present invention, "neural progenitor cells" refers to undifferentiated progenitor cells that have not yet expressed differentiation traits, and "progenitors", "precursors" and "precursor cells" may be used in the same sense.

The dopamine neuron may be a midbrain dopamine neuron.

In the present invention, "middle cerebral dopamine neurons" refers to dopamine neurons observed in the midbrain region, and for example, may refer to dopamine neurons observed in the midbrain ventral region. It is not limited.

In addition, the mesenchymal dopamine neurons can be expressed A9 region-specific (A9 region-specific).

The "A9 region" is a ventrolateral region of the midbrain, and means a part corresponding to the pars compacta of the substantia nigra (substantia nigra), and the cells produced by the manufacturing method of the present invention are mesothelial. It can be seen that the cell.

In addition, the A9 region is a region where dopamine neurons are concentrated, and is related to the regulation of motor function, and particularly, in the case of Parkinson's disease patients, the dopamine neurons at this region are specifically killed. The cells produced by the method for preparing may be used for the purpose of preventing and / or treating Parkinson's disease.

Hereinafter, the dopamine neuron manufacturing method of the present invention will be described in detail.

(a) step

The "cell population" includes human stem cells; Progenitors or precursors; And / or dopaminergic neural progenitors differentiated from human stem cells or progenitor cells, dopaminergic neurons and neural derivatives derived therefrom, but are not limited thereto. It is not.

Specifically, the human stem cells or progenitor cells are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells (iPSCs). In addition, the stem cells may be adult stem cells or fetal cells, but are not limited thereto.

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. .

(b) step

The "TPBG" can 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 has not been reported for the isolation of dopamine neurons through the expression of TPBG. none.

The nucleotide sequence of the gene is shown in SEQ ID NO: 53. In addition, the gene is readily available to those skilled in the art because the nucleotide sequence is registered in the gene bank.

In the present invention, "TPBG-positive dopamine neurons" means dopamine neurons that bind to TPBG antibodies.

The "TPBG antibody" refers to an antibody that specifically binds to TPBG.

In this step, the method for separating TPBG-positive dopamine neurons may be used as long as it is a method for 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.

The TPBG-positive dopamine neurons can alleviate the symptoms of Parkinson's disease.

The TPBG-positive dopamine neurons can enhance the safety of cell transplantation therapy.

Another aspect of the present invention relates to a pharmaceutical composition for treating Parkinson's disease, including Trophoblast glycoprotein (TPBG) -positive dopamine neurons.

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 preparation, 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, and the like. In addition to the above components, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives and the like may be further included.

The pharmaceutical compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “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 may be applied to the type, severity, drug activity, or drug of the patient. Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.

The pharmaceutical composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in combination.

Another aspect of the invention relates to a method for treating Parkinson's disease comprising administering to a subject said TPBG-positive dopamine neurons.

The term "individual" means a subject in need of treatment of a disease, and more specifically, a mammal, such as a primate, mouse, dog, cat, horse and cow, which is human or non-human.

Another aspect of the invention relates to the use of said TPBG-positive dopamine neurons for the treatment of Parkinson's disease.

In the pharmaceutical composition for treating Parkinson's disease comprising the TPBG-positive dopamine neurons, the overlapping with the method for producing the dopamine neurons is omitted in consideration of the complexity of the present specification.

Another aspect of the invention is directed to a method of enhancing the efficacy and improving transplant safety of dopamine neurons for cell transplantation therapy of Parkinson's disease, comprising the following steps.

(a) contacting a cell population with a Trophoblast glycoprotein (TPBG) -antibody; And

(b) isolating TPBG-positive dopamine neurons that bind to TPBG antibodies.

In the method of improving the efficacy of the dopamine neurons and improving the safety of transplantation, the content overlapping with the production method of the dopamine neurons is omitted in consideration of the complexity of the present specification.

Another aspect of the present invention relates to a composition for transplanting dopamine neurons comprising TPBG (Trophoblast glycoprotein) -positive dopamine neurons.

The TPBG-positive dopamine neurons may be cultured by a method for producing dopamine neurons, and the TPBG-positive dopamine neurons cultured by the above method may be enhanced cell efficacy and improved transplant safety.

On the other hand, the transplantation of the dopamine neurons is selected in the appropriate graft site known in the art (for example, the putamen or the caudate nucleus of the brain, or a striatum including all of them, etc.), It may be carried out through a method known at the site of implantation (eg, a stereotactic system, etc.).

The composition of the present invention can be used for the treatment of Parkinson's disease.

The composition of the present invention may include only dopamine neurons alone as transplanted cells, or may include bioderived and / or biodegradable stabilizers in addition to the active ingredient (graft cells). The stabilizer is to stably disperse the dopamine neurons, and is a bio-derived material so that there are no side effects when transplanted in the body, or it should be biodegradable. Biodegradability in the present invention means a property that is slowly decomposed and absorbed in the body, and does not have a meaning in particular the rate of degradation.

The stabilizer may include hyaluronic acid, collagen, thrombin, elastin, chondroitin sulfate, albumin and mixtures thereof. In particular, the hyaluronic acid, collagen, thrombin, elastin, chondroitin sulfate, albumin, and the like are bio-derived substances, and have biodegradable properties that can be naturally degraded in vivo. However, as long as it satisfies the properties of imparting biodegradability and viscosity in the medium, the synthesized compound is also a biodegradable material and may be used for the purposes of the present invention.

When formulated with the stabilizer dopamine neurons, dopamine neurons may be present evenly dispersed without floating or sedimentation in the medium.

In the composition for implantation of dopamine neurons comprising the TPBG-positive dopamine neurons, the description overlapping with the preparation method of the dopamine neurons is omitted in consideration of the complexity of the present specification.

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 separating dopamine neurons is a step of separating TPBG-positive dopamine neurons. By including, dopamine neurons isolated according to the present method is characterized in that the efficiency of the cells at the time of transplantation and improved transplant safety, it can be usefully used for cell transplantation for the treatment of Parkinson's disease.

1 is a diagram schematically illustrating a method for producing dopamine neurons of the present invention.

2 is a diagram schematically illustrating a method for preparing an LMX1A-eGFP hES reporter cell line according to one embodiment of the present invention.

3 is a diagram schematically illustrating a method for preparing a PITX3-mCherry hES reporter cell line according to one embodiment of the present invention.

Figure 4 is a diagram confirming the mDA neuroprogenitor differentiation process of the LMX1A-eGFP hES reporter cell line prepared according to one embodiment of the present invention.

5 is a diagram confirming the mDA neuronal (neuron) differentiation process of the PITX3-mCherry hES reporter cell line prepared according to an embodiment of the present invention.

6A and 6B are diagrams confirming the characteristics of differentiated LMX1A-expressing mDA neuroprogenitor cells according to one embodiment of the present invention.

7A and 7B are diagrams confirming the characteristics of differentiated LMX1A-expressing mDA neuroprogenitor cells according to one embodiment of the present invention.

8A and 8B confirm the characteristics after terminal differentiation of differentiated LMX1A-expressing cells according to one embodiment of the present invention.

9A to 9C are diagrams illustrating the characteristics of differentiated PITX3-expressing mDA neurons (neurons) according to one embodiment of the present invention.

10 is a diagram confirming the characteristics of differentiated PITX3-expressing mDA neurons (neurons) according to an embodiment of the present invention.

FIG. 11 is a diagram comparing in vitro apoptosis with respect to differentiated LMX1A-expressing mDA neuroprogenitor cells and PITX3-expressing mDA neurons (neurons) according to an embodiment of the present invention.

12 is a diagram showing the results of transcriptome analysis on differentiated LMX1A-expressing mDA neuroprogenitor cells and PITX3-expressing mDA neurons (neurons) according to an embodiment of the present invention.

13 is a schematic diagram illustrating a process of identifying a candidate group of mDA markers.

14 and 15 show the results of evaluating the target effectiveness to identify candidate groups of mDA markers.

16 and 17 are diagrams showing MACS results targeting a candidate group of mDA markers (CORIN, TPBG, CD47, and ALCAM).

FIG. 18 is a diagram illustrating behavioral recovery after TPBG-positive cell transplantation for a PD-animal model transplanted with TPBG-positive cells isolated from hESCs according to an embodiment of the present invention. FIG.

19 is a diagram showing the characteristics of the graft after TPBG-positive cell transplantation for the PD-animal model transplanted with TPBG-positive cells isolated from hESC according to one embodiment of the present invention.

FIG. 20 is not sorted in comparison to TPBG-positive cell grafts after TPBG-positive cell transplants for PD-animal models implanted with TPBG-positive cells isolated from hESCs according to one embodiment of the invention (FIG. Unsorted diagram showing the possibility of cell proliferation in cell grafts.

Figure 21 is a diagram confirming the characteristics of TPBG-positive cells isolated from human fVM cells in accordance with an embodiment of the present invention.

Figure 22 is a diagram confirming the characteristics of TPBG-positive cells isolated from human iPSC in accordance with an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Human Embryonic Stem Cells (hESC) Culture

Undifferentiated hESCs (H9, WiCell Inc., USA) were treated with mitomycin-C (Sigma-Aldrich, USA) treated mouse STO fibroblasts (ATCC, USA) 20% Knockout-Serum Replacement; Invitrogen , USA), 1x non-essential amino acids (Gibco-Thermo Fisher Scientific, USA), 0.1 mM β-mercapto ethanol (Sigma-Aldrich) and 4 ng / mL bFGF (basic fibroblast growth factor; R & D System, USA) Cultured in DMEM (Dulbecco's modified Eagle's medium) / F12 medium (Gibco-Thermo Fisher Scientific).

Genotyping of Clonal Cells

Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to the manufacturer's instructions. Genomic DNA PCR was performed using the EmeraldAmp® GT PCR Master Mix (TAKARA Bio Inc., Japan) on GeneAmp PCR System 2720 (Applied Biosystems-Thermo Fisher Scientific).

FACS

Cell separation was performed using a BD FACSAria III flow cytometer and FACSDiva software (BD Bioscience). eGFP-positive fractions were determined according to fluorescence intensity using a 488 nm laser, and mCherry-positive fractions were determined according to fluorescence intensity using a 561 nm laser.

MACS

To inhibit nonspecific binding of the antibodies, cells were incubated in 1% FBS-PBS solution (4 ° C., 30 minutes) and then bound with primary antibody (see Table 1 below) at 4 ° C. for 30 minutes.

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

After washing, primary antibody-labeled cells were incubated with 20 μL microbeads (Miltenyi Biotec) per 1Х10 ⁷ cells. After washing, the cell suspension was loaded onto a separation column (LS column) (Miltenyi Biotec) attached to a magnetic stand. The negative-labeled cells that passed during the column wash were collected in separate tubes, and the positive-labeled cells remaining in the column were eluted with the culture medium into another tube after removing the column from the magnetic stand.

Immunocytochemistry Analysis

First, cells were fixed in 4% paraformaldehyde-PBS solution.

Next, to facilitate permeation of the antibody into the cytoplasm, treatment with 0.1% Triton X-100-PBS solution for 15 minutes, followed by 2% bovine serum albumin (BSA) (Bovogen, Australia) -PBS solution at room temperature. After reacting for a time, the primary antibody (see Table 1 above) was bound at 4 ° C. overnight. To visualize the protein to which the primary antibody is bound, appropriate fluorescently-labeled secondary antibodies (Molecular Probes-Thermo Fisher Scientific and Vector Laboratories, USA) were used.

Finally, to identify the nucleus, Olympus IX71 microscope (Olympus Corp., Japan) mounted on a mounting medium (Vector Laboratories) containing 4 ', 6-diamino-2-phenylindole and equipped with a DP71 digital camera Images were acquired using an Olympus FSX100 system or LSM710 confocal microscope (Carl Zeiss, Germany).

Flow cytometry

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 membranes were permeated with IX Perm / Wash buffer (BD Biosciences) and incubated in 2% BSA-PBS solution with appropriate antibody for 1 hour. Fluorescently-labeled secondary antibodies appropriate for the antibody were used. Flow cells were counted by LSRII (BD Biosciences) and analyzed using FlowJo software.

Gene expression analysis

Total RNA present in cells was isolated using Easy-Spin ^® Total RNA Extraction Kit (iNtRON Biotechnology, Korea). cDNA was synthesized from 1 μg total RNA using PrimeScript ^™ RT Master Mix (TAKARA Bio Inc.). mRNA levels ^{SYBR ® Premix Ex Taq TM (TAKARA} Bio Inc.) and using CFX96 Real-Time System (Bio- Rad, USA) was quantified by real-time RT-PCR analysis. Ct values for each target gene were normalized according to the value of GAPDH, and standardized expression levels of the target genes were compared to control samples compared to sorted / unsorted groups according to the comparative Ct method. Data are 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.

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

Microarray Analysis: Transcript Profiling

10 μg of total RNA of each sample was processed / analyzed in Macrogen (Macrogen Inc., Korea), and the samples were hybridized to an Affymetrix Human U133 Plus 2.0 array.

Example. Differentiation Protocol to Mesenchymal Dopamine (mDA) Neurons

The specific protocol is shown in FIG.

HESCs incubated in the colony form were treated with 2 mg / mL of type IV collagenase (Worthington Biochemical Corp., USA) for 30 minutes to induce embryonic formation, and bFGF-free hES culture medium (EB medium) Incubated at. At this time, 1.5% dimethyl sulfoxide (DMSO; Calbiochem-Merck Millipore) was treated for the first 24 hours, followed by 5 μm of dosomorphin (DM) (Calbiochem-Merck Millipore) and 5 μm for 4 days. SB431542 (SB) (Sigma-Aldrich) was treated.

From day 5 (d5), attach EB to matrigel-coated culture dish in DMEM / F12 1X N2 supplement medium (bmN2 medium) with 20 ng / mL bFGF and 20 μg / mL human insulin solution (Sigma-Aldrich) added Patterning factors (1 μM CHIR99021 (Miltenyi Biotec) and 0.5 μM SAG (Calbiochem-Merck Millipore)) were treated for 6 days.

On day 11 (d11) mechanically isolated neural rosettes formed in EB colonies using a pulled glass pipette, the isolated neural rosette masses were crushed by pipetting and reattached to the Matrigel-coated petri dish. It was. Reattached cells were expanded and supplemented with 1 μM CHEM99021 and 0.5 μM SAG for 2 days in DMEM / F12 1X N2 and 1X B27 medium (bN2B27 medium) supplemented with 20 ng / mL bFGF, Sex dopamine neuronal specific differentiation was induced.

On day 13 (d13), the mesenchymal dopamine neuroprogenitor cell cluster was separated into single cells using accutase, and Matrigel-coated plates at a density of 3.12x10 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 the cells occupy nearly 90% of the plate total area.

Cerebral dopaminergic precursor cells with midbrain / dorsal characteristics from day 20 (d20) were cultured in a medium (NBG medium) containing 1X N2, 0.5X B27 and 0.5X G21 supplements (Gemini Bio-Products, USA). It was. During the first 7 days, 1 μM of DAPT (Sigma-Aldrich) was added, after which 10 ng / mL of BDNF (brain-derived neurotrophic factor; ProSpec-Tany TechnoGene, Israel), 10 ng / mL of GDNF ( Final differentiation was achieved by addition of a 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).

Preparation Example 1 Preparation of LMX1A-eGFP Reporter Line

The specific protocol is shown in FIG.

1-1. Design of Nuclease and Donor DNA Plasmids

TALEN-encoded plasmids were purchased from Toolen Inc., Korea.

The TALEN site is near TGA, the 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)) was designed to cause double-strand brsaks (DSB), and potential off-target sites were searched using Cas-Offinder (www.rgenome.net/).

The donor DNA plasmid was constructed 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).

1-2. Preparation of LMX1A-eGFP Reporter Cell Line

HESC colonies on inactivated STO were transferred onto plates coated with hESC-compatible matrigel (BD Biosciences, USA) in StemMACS ^™ iPS-Brew XF complete medium (Miltenyi Biotec, Germany). The cells were then subcultured to account for nearly 80-90% of the plate total area (Split ratio, 1: 5). After dissociation into single cells using accutase, transfer to a Matrigel-coated plate in medium supplemented with ROCK inhibitor (10 μM, Y-27632) (Calbiochem-Merck Millipore) for the first 24 hours and daily The medium was freshly replaced. Only hESCs with less than 10 enzymatic passages were used in the experiment.

Accutase was used to obtain hESCs and single cell suspensions were prepared. Next, the cells were gently resuspended in a final density of 1.0Х10 ⁷ cells / mL in the R buffer of Neon transfection kit (100 μL, Invitrogen). 120 μL of the resuspended cells were mixed with one pair of TALEN-coding plasmids (6 μg each) and LMX1A donor DNA plasmid (6 μg) of Preparation Example 1-1, and electrophoresis was performed by applying pulses at a voltage of 850 mV for 30 ms. Electroporation was performed (Neon transfection system).

Cells were then transferred to two or three 35 mm plates pre-inoculated with STO feeder in hESC medium and added a ROCK inhibitor for the first 48 hours, after two days of medium replacement, followed by fresh medium replacement.

After 5 days of electroporation, the hESC medium was treated with 0.5 μg / mL puromycin (Sigma-Aldrich). After 10-14 days, colonies showing puromycin resistance were classified as reporter cell line candidates and passaged to expand cell numbers.

Finally, the genotyping of clonal cells confirmed the LMX1A-eGFP reporter cell line.

Preparation Example 2 Preparation of PITX3-mCherry Reporter Cell Line

The specific protocol is shown in FIG. 3.

2-1. Design of Nuclease and Donor DNA Plasmids

Cas9- and sgRNA (CRISPR / Cas9) -coding plasmids were purchased from Tulgen.

The sequence for preparing sgRNAs that mediate PITX3 targeting was characterized by a stop codon TGA (5'-TAC GGG CGG GGC CGC TCA TA C GG -3 ') to cause double-strand cleavage (DSB) near the stop codon TGA. : Designed to be positioned across the PAM)). Potential off-target sites were searched using Cas-Offinder (www.rgenome.net/).

The donor DNA plasmid was constructed at 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).

2-2. Preparation of the PITX3-mCherry Reporter Cell Line

Cas9- and sgRNA-encoding plasmids in place of TALEN-encoding plasmids, PITX3 donor DNA plasmids in place of LMX1A donor DNA plasmids, except 100 μg / mL G418 (Calbiochem-Merck Millipore) instead of 0.5 μg / mL puromycin To prepare a PITX3 reporter cell line in the same manner as in Preparation Example 1-2.

Experimental Example 1. Identification of progenitor stage: LMX1A-eGFP reporter cell line identification

After the LMX1A-eGFP reporter cell line prepared in Preparation Example 1 was differentiated using the differentiation protocol of the above example, the differentiation process was confirmed (Immunocytochemistry and Cytometry).

As can be seen in Figure 4, expression of the midbrain regional marker EN1, the midbrain floor plate marker FOXA2, the dopamine lineage marker LMX1A, and eGFP throughout the differentiation process was observed. In particular, at day 20 of differentiation (d20), ˜41.1% of the cell population appeared as eGFP-positive (eGFP ⁺ ) cells, and eGFP ⁺ cells expressed both EN1 and FOXA2 simultaneously. Progenitor cells (˜46.6% EN1 ⁺ eGFP ⁺ , ˜49.2% FOXA2 ⁺ eGFP ⁺ ) with all three markers (EN1 and FOXA2, and LMX1A) were also detected (see FIG. 6).

These results indicate that cells expressing eGFP are cells expressing LMX1A (LMX1A reporter cell line established), and that hESCs have been directly differentiated into mDA neuroprogenitor cells exhibiting bottom plate (FOXA2) and midbrain (EN1) properties.

Experimental Example 2 Identification of Neuronal Stage: Identification of PITX3-mCherry Reporter Cell Line

The PITX3-mCherry reporter cell line prepared in Preparation Example 2 was differentiated using the differentiation protocol of the above example, and the differentiation process was confirmed (Immunocytochemistry and Cytometry).

As can be seen in Figure 5, the expression of mCherry was not observed until 30 days of differentiation (d30), mCherry-positive (mCherry ⁺ ) neuronal (neuron) clusters were observed at about 40 days of differentiation (d40). On the other hand, the expression pattern of the PITX3 gene was the same as that of the reporter mCherry throughout the differentiation (maturation) process. In particular, ~ 16% of the final differentiated mDA neuron (neuron) populations developed the location and lineage markers (EN1 and FOXA2). , And LMX1A), together with mCherry ⁺ cells (see FIG. 9).

These results indicate that mCherry expressing cells are PITX3 expressing cells (establishment of PITX3 reporter cell line), mES neurons whose hESCs show bottom plate (FOXA2) and midbrain (EN1), and midbrain dopamine (LMX1A) characteristics. Means differentiation directly into (neurons).

Experimental Example 3. Characterization of LMX1A-positive cells

The cells of d20 of Experimental Example 1 were exposed to 10 μm of Y27632 for 1 hour and then dissociated using accutase, and then cells of 40 μm or less were collected using a cell sieve (Cell strainer, BD Science). Dissociated progenitor cells were LMX1A supplemented with 1 × penicillin-streptomycin (P / S) (Gibco-Thermo Fisher Scientific) in 3% fetal bovine serum (FBS) (Gemini Bio-Products) and HBSS (WELGENE Inc., Korea). -Resuspend in Sorting buffer (LMX1A-SB) at a final density of 2Х10 ⁶ cells / mL and perform cell separation (FACS). MRNA expression levels of the Unsorted group, LMX1A ⁻ group and LMX1A ⁺ group were compared.

As can be seen in Figure 6a, ~ 41.1% of the viable cells after cell separation was shown as LMX1A-eGFP ⁺ (LMX1A ⁺ ) fraction. Also, LMX1A ⁺ and LMX1A-eGFP ^- (LMX1A ^-) progenitor cells was shown to maintain the shape (morphology) is similar to the non-sorted cells. In particular, ˜99.4% of isolated LMX1A ⁺ were positive for both EN1 and FOXA2. These results indicate that mDA neuroprogenitor cells were separated by FACS cell separation.

In addition, as can be seen in Figure 6b, the expression of mDA progenitor-specific genes (EN1, FOXA1, FOXA2, LMX1A, LMX1B) in the LMX1A ⁺ group was significantly upregulated, but serotonergic progenitor-specific genes (NKX2) .2) and the expression of the red nucleus (anatomical site of the midbrain) progenitor-specific genes (SIM1, LHX1, NKX6.1) were upregulated in the unclassified group and in the LMX1A ^- group. These results indicate that LMX1A ⁺ cells isolated by FACS cell separation show the characteristics of mDA neuroprogenitor cells.

Additionally, after the cell separation (FACS), the Unsorted group, the LMX1A ⁻ group and the LMX1A ⁺ group were incubated for an additional day in vitro .

For the cultured cells, neural-specific and proliferative cell-specific markers were observed and BrdU analysis was performed to confirm the cell cycle.

As can be seen in FIG. 7A, NESTIN-, SOX1-, SOX2- and KI67-positive cells were similar in all three groups. These results indicate that the sorted LMX1A ⁺ cells maintain the properties and cell proliferative capacity of neural progenitor cells as well as the unclassified and LMX1A ⁻ groups.

In addition, as can be seen in Figure 7b, the LMX1A ⁺ group was 38.5 ± 3.9% and 49.5 ± 6.2% of the surviving cells in the G0 / G1 and S phase, 6 ± 2.7% to G2 / M at the mDA neuroprogenitor cell stage there was. These results indicate that the sorted LMX1A ⁺ cells are actually proliferating cells that cycle the cell cycle.

Finally, after the FACS, the unsorted group, the LMX1A ⁻ group and the LMX1A ⁺ group were further finally differentiated (4 weeks, d52) and then the expression of mDA neuron-related markers was compared. .

As can be seen in FIG. 8A, after final differentiation, the proportion of cells expressing mDA neuron-related markers (TH, NURR1, PITX3) in the LMX1A ⁺ group increased compared to the unclassified and LMX1A ⁻ groups. These results indicate that LMX1A ⁺ cells are mDA neuronal progenitor cells capable of differentiating into mDA neurons.

Furthermore, the degree of release of dopamine was confirmed for fully mature cells (8 weeks, d75) by the final differentiation.

Specifically, cells were washed with 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 low KCl solution. Incubated for 2 minutes at. Then replace with 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 incubate for 15 minutes. It was. The solution was collected in a 15 mL tube and 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.

As can be seen in FIG. 8B, after final differentiation, dopamine secretion increased in the LMX1A ⁺ group compared to the unclassified and LMX1A ⁻ groups. These results indicate that mDA neurons formed by the final differentiation of LMX1A ⁺ cells are functional mDA neurons that secrete dopamine.

Experimental Example 4. Characterization of PITX3-positive cells

The cells of d40 of Experimental Example 2 were dissociated into single cells using papain (Papain; Worthington Biochemical Corp.) to which 5% trehalose was added, and then 40 μm using 70 μm and 40 μm cell sieves sequentially. The following cells were collected. Dissociated cells were treated with 1Х10 ⁷ cells / mL in PITX3-Sorting buffer (PITX3-SB) with 1x P / S in 5% FBS, 1x Glutamax (Gibco-Thermo Fisher Scientific), 5% trehalose and HBSS. Resuspend in concentration and perform cell separation (FACS). In addition, after culturing for an additional 36 hours in vitro ( in vitro ) was observed the morphology of the cells survived.

As can be seen in Figure 9a, ~ 16% of the viable cells after cell separation was shown 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 ^- shown to keep form cells are neurons (neuronal) similar to the non-sorted cells ^- (PITX3) . 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-specific morphology.

Next, expression of mDA neuron-related markers in the Unsorted, PITX3 ⁻ and PITX3 ⁺ groups of the d40 cells was compared.

As can be seen in Figure 9b, the expression of neuronal (neuron) -specific genes (NeuN and MAP2) and mDA neuronal (neuron) -specific genes (PITX3, NURR1, TH, DAT and VMAT2) in the PITX3 ⁺ group Significantly upregulated, but expression of serotonergic neurons (neurons) -specific gene (HTR2B) was downregulated. In addition, as can be seen in Figure 9c, the proportion of cells expressing the mDA neuronal marker TH in the PITX3 ⁺ group, and cells expressing the neuronal-specific markers TUBB3 (TUJ1) and MAP2 at the same time increased. These results indicate that PITX3 ⁺ cells are mDA neurons.

Finally, after FACS, unsorted groups were further cultured and then double-labeled immunostaining was performed on day 44 (d44) of differentiation.

As can be seen in Figure 10, PITX3 ⁺ cells were confirmed to express the mature mDA neuronal markers NURR1, AADC, VMAT2 and DAT. In addition, although the A9 region marker KCNJ6 (GIRK2) was expressed, cells expressing the A10 region marker CALB were not observed. These results indicate that PITX3 ⁺ cells differentiated through the differentiation protocol of the present invention are mature mDA neurons.

Experimental Example 5. Confirmation of graft suitability

The LMX1A-eGFP reporter cell line of Preparation Example 1 and the PITX3-mCherry reporter cell line of Preparation Example 2 were differentiated using the differentiation protocol of the above example, and then 20 days of differentiation (d20) of mDA neuroprogenitor cells and 50 days of differentiation (d50). ) Mature mDA neurons were isolated into single cells using the same method as Experimental Example 3 and 4, respectively. The degree of in vitro apoptosis of the isolated single cells 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).

As can be found at 11, the cells showing apoptosis was separated single cells LMX1A ⁺ cells in the from of about 8%, PITX3 ⁺ cells was about 30%. That is, when isolated into single cells, LMX1A ⁺ mDA neuroprogenitors maintained higher viability than PITX ⁺ mDA neurons, and through this, LMX1A ⁺ and PITX3 ⁺ cells showed a difference in susceptibility to single cell separation for transplantation. It appeared that it appeared. These results indicate that the transplantation of LMX1A ⁺ cells, which are mDA neuroprogenitor cells, is advantageous in terms of apoptosis than the transplantation of PITX3 ⁺ cells, which are mature neurons.

Experimental Example 6 Identification of Cerebral Dopamine (mDA) Markers

6-1. Transcript Analysis

The LMX1A-eGFP reporter cell line of Preparation Example 1 and the PITX3-mCherry reporter cell line of Preparation Example 2 were differentiated using the differentiation protocol of the above example, and then LMX1A ⁺ and LMX1A of 20 days of differentiation (d20, mDA neuroprogenitor cell stage) ^- to separate the cells, performing transcript analysis (Microarray) for this (see Fig. ¹²⁾ cells, and differentiation 40 days PITX3 ⁺ and PITX3 of (d40, mDA neuron stage).

Meanwhile, cells expressing eGFP and cell cycle markers (Ki67, PCNA, and PH3) were observed in the mDA neuronal progenitor cell stage, and cells expressing mCherry and mDA neuronal marker (TH) in the mDA neuronal cell stage, mature. Cells expressing neuronal markers (NeuN) were observed, but no cells expressing immature neuronal markers (NeuroD) and proliferating cell markers (KI67).

6-2. Identify candidates for mDA markers

Based on the above results, a candidate group of mDA markers was secured. A detailed process is shown in FIG. 13.

The cost for the four types of cells isolated comparing microarray analysis, ^{LMX1A-upregulated} in LMX1A ⁺ cells compared to the cells (> 2-FC) is genetically and PITX3 ^{in-upregulated} in PITX3 ⁺ cells compared to the cells (> 2 -FC) genes were identified and 53 genes encoding surface markers were identified through gene mining. The 53 genes identified included a number of genes known to be mouse mDA neuronal progenitor cells ( Corin, Clstn2, Kitlg, Plxdc2, Pcdh7, Ferd3l, Frem1, Alcam and Notch2 ).

Next, for the gene, target validation was evaluated by confirming whether expression in mDA cells that are actually differentiated. As a result, we identified surface marker genes (FIG. 14) among the genes that are upregulated in LMX1A ⁺ cells as compared to LMX1A ⁻ cells and surface marker genes that are up or down regulated in both LMX1A ⁺ cells and PITX3 ⁺ cells (FIG. 15). It was. Screening was performed for 18 genes with commercially available antibodies among the 21 genes of FIG. 14.

As a result, four of the 18 genes ( CORIN, TPBG, CD47, ALCAM ) were selected as the final surface marker candidate group.

6-3. Identification of mDA Neuroprogenitor Cell-specific Markers

Based on the results, MACS targeting the four genes ( CORIN, TPBG, CD47, ALCAM ) was performed.

As can be seen in Figures 16 and 17, CORIN- and trophoblast glycoprotein (TPBG) -target MACS showed a statistically significant enrichment of LMX1A ⁺ FOXA2 ⁺ mDA neuronal progenitor cells. In particular, TPBG was widely expressed in mDA neuroprogenitor cells.

On the other hand, the CORIN gene is already known for mDA neuroprogenitor cell enrichment, but TPBG has never been reported in the mDA development process, TPBG was selected as the final mDA neuroprogenitor cell-specific marker.

Experimental Example 7 In vivo of TPBG-positive cells isolated from human embryonic stem cells (hESCs) in vivo ) Check the transplant effect

7-1. 6-OHDA Impaired Parkinson's Disease (PD) -Model Preparation

200-250 g of female Sprague-Dawley rats (Orient Bio Inc., Korea) were used for transplantation. 30 mg / kg Zoletil ^® (Virbac, France) and 10 mg / kg Rompun ^® (Bayer, Germany) were mixed and used as anesthetics.

Injecting 3 μL of 30 mM 6-OHDA into the medial forebrain bundle of rats according to the coordinates (TB -0.45, AP -0.40, ML -0.13, DV -0.70) to the hemi-parkinsonian model model).

7-2. Confirmation of Behavioral Recovery of PD-Model After TPBG-positive Cell Transplantation

HESCs in culture in the colony form were differentiated using the differentiation protocol of the above example, and then MACS targeting TPBG was performed on day 20 (d20) of differentiation.

Cell suspensions were prepared by suspending the isolated TPBG-positive cells in 1 × HBSS to a final concentration of 8.75Х10 ⁴ cells / μL. At this time, a control group was used as a group transplanted with only HBSS.

After 4 weeks of 6-OHDA injury of Experimental Example 7-1, the prepared cell suspension (total 350,000 cells) was prepared per rat according to the coordinates (TB -0.24, AP +0.08, ML -0.30, DV -0.40 and -0.50). 4 μL was implanted by stereotactic method.

Immunosuppressive treatment was performed by intraperitoneal injection of 10 mg / kg of cyclosporine A (Ceun Kun Dang, Korea) daily during the experimental period from 2 days before transplantation to the sacrifice of mice.

Amphetamine (2.5 mg / kg, Sigmal-Aldrich) was injected intraperitoneally before 4, 8, 12 or 16 weeks after transplantation and rat rotation was recorded for 30 minutes.

As can be seen in Figure 18, TPBG-positive cells showed a significant improvement in motor function compared to the control for 16 weeks after transplantation. These results indicate that TPBG-positive mDA neuroprogenitor cells derived from hESC are viable in vivo and improve motor function.

7-3. Characterization of Grafts After TPBG-positive Cell Transplantation

TPBG-positive cells and unclassified cells were transplanted in the same manner as in Example 7-2, except that the group transplanted with Unsorted cells was used as a control.

After 16 weeks of implantation, rats were anesthetized with 25% urethane solution and 0.9% saline and 4% paraformaldehyde were perfused with perfusion. The removed brains were fixed overnight and cryoprotected with 30% sucrose-PBS solution. Cryoprotected brains were fixed in FSC 22 ^® compounds (Leica, Nußloch, Germamy) and coronal sections were made to 18 μm thickness using Thermo Fisher Scientific. Next, immunohistochemical staining was performed to target human-specific neural cell adhesion molecules (hNCAMs).

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 unclassified groups.

In addition, as can be seen in Figure 20, grafts with at least about 20% of KI67 ⁺ hNCAM ⁺ cells were observed in certain rats of one of the unclassified groups, whereas KI67 ⁺ hNCAM ⁺ cells were observed in the TPBG-positive group. It wasn't. These results indicate that, if not classified, proliferative capacity may be maintained even after 16 weeks of transplantation, but proliferating cells may be excluded when sorting cells by TPBG. This is a very important result in terms of safety of cell therapy.

Experimental Example 8. Characterization of TPBG-positive cells isolated from human fetal ventral mesencephalic cells (fVM cells)

For fVM cells incubated on laminin-coated plates in ReNcell NSC maintenance Medium (Merck), perform TPBG-targeted MACS when cells are cultured to approximately 80-90% of the plate's total area. It was. Next, relative EN1 expression in isolated TPBG-positive and TPBG-negative cells was confirmed by qRT-PCR method with hESC (H9) as a control (expression of H9 = 1).

As can be seen in Figure 21, compared with TPBG-negative cells, the expression of EN1, the developmental location marker of mDA neurons increased in TPBG-positive cells. These results indicate that TPBG can be used to enrich cells exhibiting midbrain characteristics among fVM cells.

Experimental Example 9. Characterization of TPBG-positive cells isolated from human induced pluripotent stem cells (iPSCs)

Human iPSC (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 (d20) of differentiation. Expression of EN1, FOXA2, LMX1A in isolated TPBG-positive cells was confirmed (Immunocytochemistry).

As can be seen in Figure 22, expression of the midbrain development markers EN1 and FOXA2 did not show a difference before and after MACS, cells expressing LMX1A, mDA-generating line markers were found to be concentrated in TPBG-positive cells. In addition, the percentage of cells that tested positive for all three markers (EN1 and FOXA2, and LMX1A) was also significantly enriched in TPBG-positive cells.

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 separating dopamine neurons is TPBG (Trophoblast glycoprotein) -positive dopamine neurons. By including the step of separating, dopamine neurons isolated according to the present method is characterized in that the efficacy of the cells at the time of transplantation and improved transplant safety, it can be useful for cell transplantation for the treatment of Parkinson's disease.

Claims (24)

1. Method for preparing dopaminergic neural cells, comprising the following steps:

   (a) contacting a cell population with a Trophoblast glycoprotein (TPBG) antibody; And

   (b) isolating TPBG-positive dopamine neurons that bind to TPBG antibodies.
2. The method of claim 1, wherein the cell population is

   Human stem cells;

   Progenitors or precursors; And

   Dopaminergic neural progenitors (dopaminergic neural progenitors), mature dopaminergic neurons and neural derivatives derived therefrom from the human stem cells or progenitor cells;

   It comprises one or more selected from the group consisting of.
3. According to claim 2, wherein the human stem cells or progenitor cells are embryonic stem cells (Embryonic stem cells), embryonic germ cells (Embryonic germ cells), embryonic carcinoma cells (Embryonic carcinoma cells), induced pluripotent stem cells (Induced pluripotent stem cells) , iPSCs), adult stem cells or fetal cells.
4. The method of claim 3, wherein the fetal cell is derived from fetal neural tissue or derivatives thereof.
5. The method of claim 1, wherein the TPBG-positive dopamine neurons alleviate the symptoms of Parkinson's disease.
6. The method of claim 1, wherein the TPBG-positive dopamine neurons enhance the safety of cell transplantation therapy.
7. The method of claim 1, wherein the dopamine neurons are dopaminergic neural progenitors or dopaminergic neural precursor cells or mature dopaminergic neurons.
8. The method of claim 1, wherein the dopamine neurons are midbrain dopamine neurons.
9. The method of claim 8, wherein the mesothelial dopamine neurons are A9 region-specific mesothelial dopamine neurons.
10. Pharmaceutical composition for treating Parkinson's disease, including Trophoblast glycoprotein (TPBG) -positive dopaminergic neural cells.
11. The pharmaceutical composition of claim 10, wherein the dopamine neurons are midbrain dopamine neurons.
12. The pharmaceutical composition of claim 11, wherein the mesothelial dopamine neurons are A9 region-specific mesothelial dopamine neurons.
13. How to improve the efficacy of dopaminergic neural cells and improve transplant safety for cell transplantation therapy of Parkinson's disease, comprising the following steps:

    (a) contacting a cell population with a Trophoblast glycoprotein (TPBG) -antibody; And

    (b) isolating TPBG-positive dopamine neurons that bind to TPBG antibodies.
14. The method of claim 13, wherein the cell population is

    Human stem cells;

    Progenitors or precursors; And

    Dopaminergic neural progenitors (dopaminergic neural progenitors), mature dopaminergic neurons and neural derivatives derived therefrom from the human stem cells or progenitor cells;

    It comprises one or more selected from the group consisting of.
15. The method of claim 14, wherein the human stem cells or progenitor cells are embryonic stem cells (Embryonic stem cells), embryonic germ cells (Embryonic germ cells), embryonic carcinoma cells, induced pluripotent stem cells (Induced pluripotent stem cells) , iPSCs), adult stem cells or fetal cells.
16. The method of claim 15, wherein the fetal cell is derived from fetal neural tissue or derivatives thereof.
17. The method of claim 13, wherein the dopamine neurons are dopaminergic neural progenitors or dopaminergic neural precursor cells or dopaminergic neurons.
18. The method of claim 13, wherein the dopamine neurons are midbrain dopamine neurons.
19. The method of claim 18, wherein the mesothelial dopamine neurons are A9 region-specific mesothelial dopamine neurons.
20. Composition for dopamine neuron transplantation comprising TPBG (Trophoblast glycoprotein) -positive dopaminergic neural cells.
21. The composition of claim 20, wherein the dopamine neurons are midbrain dopamine neurons.
22. The composition of claim 21, wherein the mesothelial dopamine neurons are A9 region-specific mesothelial dopamine neurons.
23. The composition of claim 20, wherein the composition is for the treatment of Parkinson's disease.
24. The composition of claim 20, wherein the composition enhances the efficacy of dopamine neurons and enhances transplant safety.

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