Method For Separation Of Dopaminergic Neural Cells And Pharmaceutical Composition Comprising Dopaminergic Neural Cells For Treatment Of Parkinson's Disease

Abstract

The present disclosure addresses a method for separating dopaminergic neural cells and a pharmaceutical composition comprising the dopaminergic neural cells separated by the method for treatment of Parkinson's disease, wherein the method for separating dopaminergic neural cells comprises a step of separating TPBG-positive dopaminergic neural cells, whereby the dopaminergic neural cells separated according to the method of the present invention are enhanced in efficacy for transplantation and have advanced transplantation safety and thus can find useful applications in transplantation for Parkinson's disease.

Claims

1. A method for preparation of dopaminergic neural cells, the method comprising: (a) contacting a cell population with a TPBG (trophoblast glycoprotein) antibodies; and (b) separating TPBG-positive dopaminergic neural cells, which bind to the TPBG antibodies.

2. The method of claim 1, wherein the cell population comprises at least one selected from the group consisting of: human stem cells; progenitors or precursors; and dopaminergic neural progenitors, mature dopaminergic neurons, and neural derivatives, which are derived from the human stem cells or progenitors.

3. The method of claim 2, wherein the human stem cells or progenitors are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells (iPSCs), adult stem cells, or fetal cells.

4. The method of claim 3, wherein the fetal cells are derived from a fetal neural tissue or derivatives thereof.

5. The method of claim 1, wherein the TPBG-positive dopaminergic neural cells alleviate symptoms of Parkinson's disease.

6. The method of claim 1, wherein the TPBG-positive dopaminergic neural cells improve safety for transplantation.

7. The method of claim 1, wherein dopaminergic neural cells are dopaminergic neural progenitors or dopaminergic neural precursor cells, or mature dopaminergic neurons.

8. The method of claim 1, wherein the dopaminergic neural cells are midbrain dopaminergic neural cells.

9. The method of claim 8, wherein the midbrain dopaminergic neural cells are A9 region-specific midbrain dopaminergic neural cells.

10-12. (canceled)

13. A method for enhancing the efficacy and improving transplantation safety of dopaminergic neural cells in transplantation for Parkinson's disease, the method comprising: (a) contacting a cell population with a TPBG (trophoblast glycoprotein) antibodies; and (b) separating TPBG-positive dopaminergic neural cells, which bind to the TPBG antibodies.

14. The method of claim 13, wherein the cell population comprises at least one selected from the group consisting of: human stem cells; progenitors or precursors; and dopaminergic neural progenitors, mature dopaminergic neurons, and neural derivatives, which are derived from the human stem cells or progenitors.

15. The method of claim 14, wherein the human stem cells or progenitors are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells (iPSCs), adult stem cells, or fetal cells.

16. The method of claim 15, wherein the fetal cells are derived from a fetal neural tissue or derivatives thereof.

17. The method of claim 13, wherein dopaminergic neural cells are dopaminergic neural progenitors or dopaminergic neural precursor cells, or mature dopaminergic neurons.

18. The method of claim 13, wherein the dopaminergic neural cells are midbrain dopaminergic neural cells.

19. The method of claim 18, wherein the midbrain dopaminergic neural cells are A9 region-specific midbrain dopaminergic neural cells.

20-24. (canceled)

25. A method for treating Parkinson's disease, the method comprising administering, a pharmaceutical composition comprising TPBG (trophoblast glycoprotein)-positive dopaminergic neural cells.

26. The method of claim 25, wherein the dopaminergic neural cells are midbrain dopaminergic neural cells.

27. The method of claim 26, wherein the midbrain dopaminergic neural cells are A9 region-specific midbrain dopaminergic neural cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG. 1 is a schematic diagram of a method for preparation of dopaminergic neural cells.

[0070] FIG. 2 is a view schematically illustrating a method for construction of LMX1A-eGFP reporter hESC lines according to a Preparation Embodiment of the present disclosure.

[0071] FIG. 3 is a view schematically illustrating a method for construction of PITX3-mCherry reporter hESC lines according to a Preparation Example of the present disclosure

[0072] FIG. 4 shows views identifying a procedure of mDA neural precursor differentiation of the LMX1A-eGFP reporter hESC lines constructed according to a Preparation Example of the present disclosure.

[0073] FIG. 5 shows views identifying a procedure of mDA neuronal cell (neuron) differentiation of the PITX3-mCherry reporter hESC lines constructed according to a Preparation Example of the present disclosure.

[0074] FIGS. 6A and 6B are views characterizing LMX1A-expressing mDA neural precursor cells differentiated according to an Example of the present disclosure.

[0075] FIGS. 7A and 7B are views characterizing LMX1A-expressing mDA neural precursor cells (II) differentiated according to an Example of the present disclosure.

[0076] FIGS. 8A and 8B are views characterizing post-terminal differentiation LMX1A-expressing cells differentiated according to an Example of the present disclosure.

[0077] FIGS. 9A, 9B and 9C are views characterizing PITX3-expressing mDA neuronal cells (neurons) differentiated according to an Example of the present invention.

[0078] FIG. 10 shows views characterizing PITX3-expressing mDA neuronal cells (neurons) differentiated according to an Example of the present disclosure.

[0079] FIG. 11 shows views comparing in vitro cell viability between LMX1A-expressing mDA neural precursor cells and PITX3-expressing mDA neuronal cells (neurons), both differentiated according to an Example of the present disclosure.

[0080] FIG. 12 shows views accounting for results of the transcriptome analysis performed with regard to LMX1A-expressing mDA neural precursor cells and PITX3-expressing mDA neuronal cells (neurons), both differentiated according to an Example of the present disclosure.

[0081] FIG. 13 is a schematic diagram illustrating a procedure of identifying candidates of mDA markers.

[0082] FIGS. 14 and 15 show evaluation results of target validation for identifying candidates of mDA markers.

[0083] FIGS. 16 and 17 are views accounting for results of MACS targeting candidates (CORIN, TPBG, CD47, ALCAM) of mDA markers.

[0084] FIG. 18 is a view accounting for behavioral recovery in PD animal models after transplantation of TPBG-positive cells derived from hESC according to an Example of the present disclosure.

[0085] FIG. 19 shows views characterizing grafts in PD-animal models after transplantation of TPBG-positive cells derived from hESC according to an Example of the present disclosure.

[0086] FIG. 20 shows views identifying cell proliferative potentials of unsorted cell grafts compared with TPBG-positive cell grafts in PD-animal models after transplantation of TPBG-positive cells derived from hESC according to an Example of the present disclosure.

[0087] FIG. 21 is a view characterizing TPBG-positive cells separated from human NM cells according to an Example of the present disclosure.

[0088] FIG. 22 shows views characterizing TPBG-positive cells derived from human iPSC according to an Example of the present disclosure.

DETAILED DESCRIPTION

[0089] Human Embryonic Stem Cell (hESC) Culture

[0090] Undifferentiated hESCs (H9; WiCell Inc., USA) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco-Thermo Fisher Scientific) supplemented with 20% knockout-serum replacement (KSR) (Invitrogen, USA), 1× nonessential amino acid (Gibco-Thermo Fisher Scientific, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 4 ng/ml of basic fibroblast growth factor (bFGF) (R&D System, USA) on the layer of mitomycin-C(Sigma-Aldrich, USA) treated mouse STO fibroblasts (ATCC, USA).

[0091] Genotyping of Clonal Cell

[0092] Genomic DNA was extracted using DNeasy Blood & Tissue kit (QIAGEN, Germany) according to the manufacturer's instruction. Genomic DNA PCR was performed with EmeraldAmp® GT PCR Master Mix (TAKARA Bio Inc., Japan) in the GeneAmp PCR System 2720 (Applied Biosystems-Thermo Fisher Scientific).

[0093] FACS

[0094] FACS was performed using BD FACSAria III cell sorter and FACSDiva software (BD Bioscience). Using a 488-nm laser, an eGFP-positive fraction was determined depending on fluorescence intensity. Using a 561-nm laser, an mCherrypositive fraction was determined depending on fluorescence intensity.

[0095] MACS

[0096] In order to block antibodies from binding nonspecifically thereto, cells were incubated in 1% FBS-PBS solution (4° C., 30 min) and then with primary antibodies (see Table 1, below) for 30 min at 4° C.

TABLE-US-00001 TABLE 1 Protein Species Company Cat. no. Dilution OCT4 Rabbit Santa Cruz sc-9081 1:200 SOX2 Rabbit Millipore AB5603 1:200 NANOG Goat R&D Systems AF1997 1:50  (human) SSEA4 Mouse Millipore MAB4304 1:200 TRA-1-81 Mouse Millipore MAB4381 1:100 TRA-1-60 Mouse Millipore MAB4360 1:100 NESTIN Rabbit Millipore ABD69 .sup. 1:1,000 (human) 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 4G11 1:50  Bank FOXA2 (HNF3P) 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 .sup. 1:1,000 eGFP Mouse Rockland 600-301-215 .sup. 1:1,000 PITX3 Rabbit NOVUS NBP1-92274 1:500 mCherry Rabbit Rockland 600-401-P16S .sup. 1:1,000 mCherry Rat Thermo M11217 .sup. 1:1,000 KI67 Rabbit Vision Biosystem NCL-K67P .sup. 1:1,000 TUBB3 Mouse Covance (BioLegend) MMS-435P .sup. 1:1,000 (801201) TH Rabbit Pel-freez P40101-0 .sup. 1:1,000 TH Mouse Sigma T1299   1:10,000 NURR1 Rabbit Santa Cruz sc-990 .sup. 1:1,000 MAP2 Rabbit Millipore AB5622 .sup. 1:1,000 AADC Rabbit Chemicon AB1569 1:500 VMAT2 Rabbit Abcam AB81855 .sup. 1:1,500 DAT Rabbit Pel-freez P40501-0 1:500 KCNJ6 Rabbit Almone Labs APC-006 1:500 CALB Rabbit Millipore AB1778 .sup. 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 2.5 μg/10.sup.6 cells TPBG Mouse R&D Systems MAB49751 2.5 μg/10.sup.6 cells CORIN Mouse R&D Systems MAB2209 2.5 μg/10.sup.6 cells CD47 Mouse Santa Cruz sc-12730 1.0 μg/10.sup.6 cells

[0097] After washing, the primary antibody-labeled cells were incubated with 20 μL of microbeads (Miltenyi Biotec) per 1×10.sup.7 cells. After washing, the cell suspension was loaded on the separation column (LS column) (Miltenyi Biotec) that was attached to a magnetic stand. Negatively labeled cells which were passed-through during column washing were collected in a separate tube, and positively labeled cells that remained in the column were eluted to another tube, together with the culture medium after removing the column from the magnetic stand.

[0098] Immunocytochemistry

[0099] First, cells were fixed in 4% paraformaldehyde-PBS solution.

[0100] For smooth permeabilization of antibody into cytosols, then, cells were treated with 0.1% Triton X-100-PBS solution, blocked with 2% bovine serum albumin (BSA) (Bovogen, Australia)-PBS solution for 1 hr at room temperature, and incubated overnight at 4° C. with primary antibodies (see Table 1 above). Appropriate fluorescence-tagged secondary antibodies (Molecular Probes-Thermo Fisher Scientific and Vector Laboratories, USA) were used for visualization of the primary antibody-labeled protein.

[0101] Finally, in order to identify cell nuclei, cells were mounted in 4′,6-diamidino-2-phenylindole mounting medium (Vector Laboratories), and images were obtained using an Olympus IX71 microscope equipped with a DP71 digital camera, Olympus FSX100 system (Olympus Corp., Japan) or LSM710 confocal microscope (Carl Zeiss, Germany).

[0102] Flow Cytometry

[0103] Cells were dissociated into single cells using Accutase (Merck Millipore, Germany) and fixed with 4% paraformaldehyde-PBS solution. For detecting intracellular markers, cells were permeabilized with 1×Perm/Wash buffer (BD Biosciences) and incubated with the appropriate antibodies for 1 hr in 2% BSA-PBS solution. Appropriate fluorescence-tagged secondary antibodies were used. Flow cytometry was performed using LSRII (BD Biosciences) and analyzed using FlowJo software.

[0104] Gene Expression Analysis

[0105] Total RNA was isolated using the Easy-Spin® Total RNA Extraction kit (iNtRON Biotechnology, South Korea). cDNA was synthesized from 1 μg of total RNA using the PrimeScript™ RT Master Mix (TAKARA Bio, Inc.). mRNA levels were quantified by real-time RT-PCR assays using the SYBR® Premix Ex Taq™ (TAKARA Bio, Inc.) and CFX96 Real-Time System (Bio-Rad, USA). Ct values for each targeted gene were normalized according to those of GAPDH, and the normalized expression levels of the targeted genes were compared between the sorted/unsorted samples and control samples based on the comparative Ct method. Data are expressed as the mean relative expression ±standard error of the mean (SEM) from three independent experiments. The sequences of the primers used for gene expression analysis are listed in Table 2, below.

TABLE-US-00002 TABLE 2 Symbol Gene name Sequence(5′ to 3′) SEQ ID No. GAPDH Glyceraldehyde-3- F: CAA TGA CCC CTT CAT TGA CC SEQ ID No. 1 Phosphate R: TTG ATT TTG GAG GGA TCT CG SEQ ID No. 2 Dehydrogenase 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 SEQ ID No. 6 GC 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 F: CTG CAG CTG TCT TGA TCG AGT TAT SEQ ID No. 9 dioxygenase 1 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- F: CCG TTC TCC ATC AAC AAC CT SEQ ID No. 15 3β) R: GGG GTA GTG CAT CAC CTG TT SEQ ID No. 16 LMX1A LIM homeobox F: CGC ATC GTT TCT TCT CCT CT SEQ ID No. 17 transcription factor 1a R: CAG ACA GAC TTG GGG CTC AC SEQ ID No. 18 eGFP Enhanced green F: CAT CAA GGT GAA CTT CAA GAT CCG SEQ ID No. 19 fluorescent protein CCA CAA C R: CTT GTA CAG CTC GTC CAT GCC GAG SEQ ID No. 20 AGT GAT C PITX3 Paired like homeodomain F: GCC AAC CTT AGT CCG TG SEQ ID No. 21 3 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 F: GGA AGC ACT GTT TGC CAA GAC C SEQ ID No. 25 2 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 F: CTT AAC CAG CCT CAG CGA CT SEQ ID No. 33 transcription factor 1b 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 F: AAA CTG CCC AGT GGA CAA GCG T SEQ ID No. 39 subfamily 4 group A R: GCT CTT CGG TTT CGA GGG CAA A SEQ ID No. 40 member 2 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 F: GCT ATG CCT TCC TGC TGA TTG C SEQ ID No. 45 member A2(vesicular R: CCA AGG CGA TTC CCA TGA CGT T SEQ ID No. 46 monoamine transporter 2) HTR2B 5-hydroxytryptamine F: GCT GGT TGG ATT GTT TGT GAT GC SEQ ID No. 47 receptor 2B R: CCA CTG AAA TGG CAC AGA GAT GC SEQ ID No. 48 NeuN RNA binding fox-1 F: TAC GCA GCC TAC AGA TAC GCT C SEQ ID No. 49 homolog 3 R: TGG TTC CAA TGC TGT AGG TCG C SEQ ID No. 50 MAP2 Microtubule associated F: AGG CTG TAG CAG TCC TGA AAG G SEQ ID No. 51 protein 2 R: CTT CCT CCA CTG TGA CAG TCT G SEQ ID No. 52

[0106] Microarray: Transcriptome Profiling

[0107] Ten μg of total RNA from each sample was processed and analyzed by Macrogen, Inc. (Korea), and the samples were hybridized to the Affymetrix Human U133 Plus 2.0 array.

[0108] The present disclosure will become more fully understood from the following Examples. The Examples are given by way of illustration only, and thus are not limitative of the present invention.

Example: mDA Neural Cell Differentiation Protocol

[0109] A concrete protocol is depicted in FIG. 1.

[0110] hESCs cultured in the form of colonies were detached with 2 mg/ml of type IV collagenase (Worthington Biochemical Corp., USA) for 30 min and embryoid bodies were induced to form in bFGF-free hES culture medium (EB medium) including 1.5% dimethyl sulfoxide (DMSO; Calbiochem-Merck Millipore) for the first 24 hrs, followed by treating the embryoid bodies with 5 μM dorsomorphin (DM) (Calbiochem-Merck Millipore) and 5 μM SB431542 (SB) (Sigma-Aldrich) for four days.

[0111] On day five (d5), EBs were attached to Matrigel-coated culture dishes in DMEM/F12 1×N2 supplemented media containing 20 ng/mL bFGF and 20 μg/ml human insulin solution (Sigma-Aldrich) (bmN2 medium) and treated with patterning factors (1 μM CHIR99021 (Miltenyi Biotec) and 0.5 μM SAG (Calbiochem-Merck Millipore)) for another 6 days.

[0112] On day 11 (d11), the emerged rosette structures formed within the EB colonies were mechanically isolated using pulled glass pipettes and the isolated neural rosette clumps were crushed by pipetting and replated in Matrigel-coated dishes. The replated cells were then expanded and specified for an additional 2 days in DMEM/F12 1×N2 and 1×B27 media with 20 ng/mL bFGF (bN2B27 medium) supplemented with 1 μM CHIR99021 and 0.5 μM SAG to induce differentiation into mDA neural cells.

[0113] On day 13 (d13) of differentiation, the mDA neural precursor clusters were dissociated to single cells by using Accutase and replated onto Matrigel-coated plates at a density of 3.12×10.sup.5 cells/cm.sup.2 in a bFGF-free N2B27 medium. mDA precursors were expanded in N2B27 medium for additional 7 days at 90% confluence.

[0114] On day 20 (d20), midbrain/ventral mDA neural precursors were cultured in 1×N2, 0.5×B27 and 0.5×G21 supplement (Gemini Bio-Products, USA) (NBG medium) with 1 μM of DAPT (Sigma-Aldrich) for the first 7 days. After that, the cells were cultured in NBG medium with 10 ng/ml of brain-derived neurotrophic factor (BDNF) (ProSpec-Tany TechnoGene, Israel), 10 ng/ml of glial cell line-derived neurotrophic factor (GDNF) (ProSpec-Tany TechnoGene), 200 μM of ascorbic acid (AA), and 1 μM of dibutyryl cyclic-AMP (db-cAMP) (Sigma-Aldrich) for terminal differentiation.

Preparation Example 1: Construction of LMX1A-eGFP Reporter Cell Line

[0115] A concrete protocol is given in FIG. 2.

[0116] 1-1. Design of Programmable Nucleases and Donor DNA Plasmids

[0117] TALEN-encoding plasmids were purchased from ToolGen, Inc. (Korea).

[0118] TALEN sites were designed to cause double-strand breaks (DSBs) near the stop codon, TGA, in exon 9 of LMX1A gene (5′-TCC ATG CAG AAT TCT TAC TT-3′ (left), 5′-TCA CAG AAC TCT AGG GGA AG-3′ (right)). Potential off-target sites were searched using Cas-OFFinder (www.rgenome.net/).

[0119] The donor DNA plasmids were constructed using pUC19 as the plasmid backbone in DH5a as follows: 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).

[0120] 1-2. Generation of LMX1A-eGFP Reporter Line

[0121] hESC colonies on inactivated STO were transferred on plates coated with hESC-qualified Matrigel (BD Biosciences, Bedford, Mass., USA) in StemMACS™ iPS-Brew XF complete medium (Miltenyi Biotec, Germany). Thereafter, cells were passaged when they were 80-90% confluent (split ratio, 1:5). Cells were dissociated into single cells using Accutase and transferred on Matrigel-coated plates with ROCK inhibitor (10 μM, Y-27632) (Calbiochem-Merck Millipore) included in the medium for the first 24 hrs after plating and continued with daily medium changes. Only hESCs that had undergone less than 10 enzymatic passages were used in the experiments.

[0122] hESCs were harvested using Accutase to create single cell suspensions. These cells were then resuspended gently with R buffer from the Neon transfection kit (100 μl kit; Invitrogen) at a final density of 1.0×10.sup.7 cells/ml. 120 μl of resuspended cells were mixed with a pair of TALEN-encoding plasmids of Preparation Example 1-1 (6 μg of each plasmid) and donor LMX1A DNA plasmid and pulsed with a voltage of 850 mV for 30 ms for electroporation (Neon transfection system; Invitrogen).

[0123] Cells were subsequently plated into two or three 35-mm dishes preseeded with STO feeders in hESC medium supplemented with ROCK inhibitor for the first 48 hours. The medium was changed with a fresh one after 2 days, and then the medium was changed every day.

[0124] Five days after electroporation, cells were treated 0.5 μg/ml puromycin (Sigma-Aldrich) in hESC medium. At 10-14 days after electroporation, individual colonies resistant to puromycin were picked as reporter cell line candidates and expanded by passages.

[0125] Finally, genotyping of clonal cells allowed for the selection of LMX1A-eGFP reporter lines.

Preparation Example 2: Generation of PITX3-mCherry Reporter Line

[0126] A concrete protocol is depicted in FIG. 3.

[0127] 2-1. Design of Nuclease and Donor DNA Plasmid

[0128] Cas9- and sgRNA (CRISPR/Cas9)-encoding plasmids were purchased from ToolGen. Inc.

[0129] The sequence for making sgRNA for mediating PITX3 targeting was located so that it spanned across the stop codon TGA(5′-TAC GGG CGG GGC CGC TCA TAC GG-3′ (PAM is underlined)) to cause double strand breaks (DSBs) near the stop codon TGA. Potential off-target sites were searched using Cas-OFFinder (www.rgenome.net).

[0130] The donor DNA plasmids were constructed using pUC19 as the plasmid backbone in DH5a as follows: 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).

[0131] 2-2. Generation of PITX3-mCherry Reporter Line

[0132] A PITX3 reporter line was generated in the same manner as in Preparation Example 1-2, with the exception that a Cas9- and sgRNA-encoding plasmid, a PITX3 donor DNA plasmid, and 100 μg/mL G418 (Calbiochem-Merck Millipore) were used instead of the TALEN-encoding plasmid, the LMX1A donor DNA plasmid, and 0.5 μg/mL puromycin, respectively.

Experimental Example 1: Identification at Progenitor Stage: Identification of LMX1A-eGFP Reporter Line

[0133] The LMX1A-eGFP reporter line generated in Preparation Example 1 was differentiated according to the differentiation protocol of the Example, followed by analyzing the differentiation (Immunocytochemistry and Cytometry).

[0134] As can be seen in FIG. 4, eGFP expression was observed, together with the midbrain regional marker EN1, the midbrain floor plate regional marker FOXA2, and the dopamine lineage marker LMX1A, throughout the progenitor differentiation. Particularly, by 20 days of differentiation (d20), ˜41.1% of the cell population appeared to be eGFP-positive eGFP.sup.+) and the eGFP.sup.+ cells co-expressed EN1 and FOXA2 (FIG. 4 and FIG. 6). Progenitors positive for all three makers (EN1, FOXA2, and LMX1A) were also detected (˜46.6% EN1.sup.+ eGFP.sup.+, ˜49.2% FOXA2.sup.+eGFP.sup.+ (see FIG. 3).

[0135] These data indicated that eGFP-expressing cells expressed LMX1A (construction of LMX1A reporter lines) and hESCs were directed to differentiate into mDA neural progenitors with floor plate (FOXA2) and midbrain (EN1) characteristics.

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

[0136] The PITX3-mCherry reporter line generated in Preparation Example 2 was differentiated according to the differentiation protocol of the Example, followed by analyzing the differentiation (Immunocytochemistry and Cytometry).

[0137] As can be seen in FIG. 5, mCherry expression was absent up until 30 days of differentiation (d30). mCherry-positive (mCherry.sup.+) neuron clusters were visualized on around d40. Meanwhile, the expression pattern of PITX3 gene was identical to that of its reporter mCherry throughout the differentiation (maturation) process. Terminally differentiated mDA neuron cultures were found to consist of ˜16% mCherry.sup.+ cells coexpressing regional and lineage markers (EN1 and FOXA2, and LMX1A) (see FIG. 9).

[0138] Taken together, these data indicated that eGFP and mCherry expressions dependably recapitulated LMX1A and PITX3 expressions, respectively, at different time points during the mDA neuron differentiation.

[0139] Taken together, these data indicated that mCherry-expressing cells expressed PITX3 (construction of PITX3 reporter lines) and hESCs were directed to differentiate into mDA neuron with floor plate (FOXA2), midbrain (EN1), and mDA (LMX1A) characteristics.

Experimental Example 3: Characterization of LMX1A.SUP.+ Cell

[0140] After exposure to 10 μM of Y27632 for 1 hr, the cells on d20 in Experimental Example 1 were dissociated with Accutase and strained through a 40 μm cell strainers (BD Science). The dissociated precursors were resuspended in LMX1A-Sorting Buffer (LMX1A-SB) supplemented with 3% fetal bovine serum (FBS) (Gemini Bio-Products), and 1× Penicillin-Streptomycin (P/S) (Gibco-Thermo Fisher Scientific) in HBSS (WELGENE, Inc., Gyeongsan, South Korea) at a final density of 2×10.sup.6 cells/ml. FACS was performed. Comparison of mRNA expression levels was made among the unsorted group, the LMX1A group, and the LMX1A.sup.+ group.

[0141] As can be seen in FIG. 6a, the LMX1A-eGFP.sup.+(LMX1A.sup.+) fraction yielded ˜41.1% of all viable cells after sorting. In addition, LMX1A.sup.+ and LMX1A-eGFP.sup.−(LMX1A.sup.−) progenitors appeared to retain similar morphologies to those of unsorted cells. Notably, ˜99.4% of isolated LMX1A.sup.+ progenitor populations were positive for both EN1 and FOXA2. These results imply the separation of mDA neural precursor cells through FACS.

[0142] As can be seen in FIG. 6b, significant up-regulations of mDA progenitor-specific genes (EN1, FOXA1, FOXA2, LMX1A, and LMX1B) were observed in LMX1A.sup.+ group while serotonergic progenitor-specific gene (NKX2.2) and red nucleus (an anatomical area in the midbrain) progenitor-specific genes (SIM1, LHX1, and NKX6.1) were up-regulated in unsorted and LMX1A.sup.− groups. These data suggest that LMX1A.sup.+ cells separated by FACS exhibit characteristics of mDA neural precursor cells.

[0143] Additionally, the unsorted group, the LMX1A.sup.− group, and the LMX1A.sup.+ group were in vitro cultured for an additional one day after FACS. The cultured cells were observed for neuron-specific and proliferative cell-specific markers and subjected to BrdU assay to reveal various phases of the cell cycle.

[0144] As can be seen in FIG. 7a, NESTIN-, SOX1-, SOX2- and KI67-positive cells were similar in all the three groups. These data suggest that the sorted LMX1A.sup.+ cells retain characteristics of neural progenitors and a proliferative potential, like the unsorted group and the LMX1A.sup.− group.

[0145] In LMX1A.sup.+ progenitor cultures, as shown in FIG. 7b, 38.5±3.9% and 49.5±6.2% of the viable cells at the mDA precursor stage were in the G0/G1 and S phases, respectively, while 6±2.7% were in the G2/M, indicating that the sorted LMX1A.sup.+ cells actually underwent the cell cycle for proliferation.

[0146] Finally, the unsorted group, the LMX1A.sup.− group, and the LMX1A.sup.+ group after FACS were subjected to terminal differentiation (4 weeks, d52) and then compared with each other with respect to the expression of mDA neuron-specific markers.

[0147] After the final differentiation, as can be seen in FIG. 8a, mDA neuron-specific genes (TH, NURR1, and PITX3) were significantly enriched in the LMX1A.sup.+ group, compared to the unsorted group and the LMX1A.sup.− group, implying that the LMX1A.sup.+ cells are mDA precursors capable of differentiating to mDA neurons.

[0148] Further, the fully matured cells (8 weeks, d75) by the terminal differentiation were analyzed for dopamine release.

[0149] Briefly, cells were washed with a low KCl solution (2.5 mM CaCl.sub.2), 11 mM glucose, 20 mM HEPES-NaOH, 4.7 mM KCl, 1.2 mM KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, and 140 mM NaCl) and incubated in the low KCl solution for 2 min. The solution was subsequently replaced with a high KCl solution (2.5 mM CaCl.sub.2), 11 mM glucose, 20 mM HEPES-NaOH, 60 mM KCl, 1.2 mM KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, and 85 mM NaCl), followed by incubation for an additional 15 min. The solution was collected in 15 ml tubes and centrifuged for 1 min at 2,000 rpm to remove the debris. The supernatant were collected in 1.5 ml tubes and stored at −80° C. before the assay. The concentration of dopamine was detected by Dopamine ELISA kit (Cat. No. KA3838; Abnova, Taiwan) according to the manufacturer's instructions.

[0150] As can be seen in FIG. 8b, an increased release of dopamine was observed in the LMX1A.sup.+ group, compared to the unsorted group and the LMX1A.sup.− group, after terminal differentiation. These data imply the mDA neurons to which the LMX1A.sup.+ cell have finally differentiated are functional neurons that release dopamine.

Experimental Example 4: Characterization of PITX3.SUP.+ Cell

[0151] After being dissociated into single cells using Papain (Worthington Biochemical Corp.) supplemented with 5% trehalose, the cells on d40 of Experimental Example 2 were strained through 70 μm and 40 μm cell strainers, sequentially. The cells thus collected through the 40 μm cell strainer were resuspended in PITX3-Sorting Buffer (PITX3-SB) containing 5% FBS, 1×Glutamax (Gibco-Thermo Fisher Scientific), 5% trehalose, and 1×P/S in HBSS at a concentration of 1×10.sup.7 cells/ml, followed by performing FACS. In addition, observation was made of morphologies of the cells that survived in vitro incubation for an additional 36 hrs after FACS.

[0152] As can be seen in FIG. 9a, the PITX3-mCherry.sup.+(PITX3.sup.+) fraction accounted for ˜16% of all the viable cells after FACS. Although a significant cell loss was encountered during the cell sorting procedures, surviving PITX3.sup.+ and PITX3-mCherry.sup.−(PITX3.sup.−) cells appeared to have similar neuronal morphologies to those of the unsorted cells after in vitro incubation. These results imply the separation of PITX3.sup.+ cells through FACS and the appearance of neuron-specific morphologies in all the three groups (unsorted, PITX3.sup.+, and PITX3.sup.− groups).

[0153] Next, expression levels of mDA neuron-specific markers were compared among the unsorted group, the PITX3.sup.− group, and the PITX3.sup.+ group of the cells on d40.

[0154] As can be seen in FIG. 9b, neuron-specific genes (NeuN and MAP2) and mDA neuron-specific genes (PITX3, NURR1, TH, DAT, and VMAT2) were significantly upregulated in the PITX3.sup.+ group, but with the expression of serotonergic neuron-specific gene (HTR2B) downregulated therein. In addition, as can be seen in FIG. 9c, neurons expressing the mDA neuron marker TH and neurons coexpressing the neuron-specific markers TUBB3 (TUJ1) and MAP2 were enriched in the PITX3.sup.+ group, suggesting that the PITX3.sup.+ cells are mDA neurons.

[0155] Finally, the post-FACS, unsorted group was additionally incubated and then subjected to double-labeled immunostaining on d44.

[0156] As can be seen in FIG. 10, PITX3.sup.+ cells were revealed to express NURR1, AADC, VMAT2, and DAT, which are all markers for mature mDA neurons. In addition, the A9 regional marker KCNJ6 (GIRK2) was expressed, but cells expressing the A10 regional marker CALB were not observed. These data suggest that the PITX3.sup.+ cells differentiated through the differentiation protocol of the present invention are mature mDA neural cells.

Experimental Example 5: Identification of Transplantation Suitability

[0157] After the LMX1A-eGFP reporter line of Preparation Example 1 and the PITX3-mCherry reporter line of Preparation Example 2 were differentiated using the differentiation protocol of the Example, mDA neural precursors on d20 and mature mDA neural cells on d50 were dissociated into single cells in the same manners as in Experimental Examples 3 and 4, respectively. Isolated single cells were compared for in vitro cell death. In this regard, cell death was measured using LIVE/DEAD™ Fixable Violet Dead Cell Stain kit (Thermo Fisher) according to the manufacturer's instruction.

[0158] As can be seen in FIG. 11, the cells undergoing cell death after single cell dissociation accounted for about 8% of the LMX1A.sup.+ cells and about 30% of the PITX3.sup.+ cells. That is, when dissociated into single cells, LMX1A.sup.+ mDA neural precursors retained higher viability than PITX.sup.+ mDA neurons, demonstrating that there is a difference in susceptibility to single cell dissociation for transplantation between LMX1A.sup.+ cells and PITX3.sup.+ cells. These results imply that LMX1A.sup.+ cells, which are mDA neural precursors, are more advantageously transplanted than PITX3.sup.+ cells, which are mature neurons, in terms of cell death.

Experimental Example 6: Identification of mDA Marker

[0159] 6-1. Transcriptome Analysis

[0160] After the LMX1A-eGFP reporter line of Preparation Example 1 and the PITX3-mCherry reporter line of Preparation Example 2 were differentiated using the differentiation protocol of the Example, LMX1A.sup.+ and LMX1A.sup.− cells on d20 (mDA neural precursor stage) and PITX3.sup.+ and PITX3.sup.− cells on d40 (mDA neuron state) were separated and subjected to transcriptome analysis (Microarray) (see FIG. 12).

[0161] Meanwhile, coexpression of eGFP and cell cycle markers (Ki67, PCNA, and PH3) were detected in mDA progenitors and cells at the mDA neuron stage were observed to express mCherry and mDA neuronal marker (TH) and mature neuron marker (NeuN), but not to express immature neuron maker (NeuroD) and proliferative cell marker (Ki67).

[0162] 6-2. Identification of mDA Marker Candidates

[0163] Based on the above result, mDA marker candidates were obtained. A concrete procedure is depicted in FIG. 13.

[0164] Comparative microarray analysis of the four isolated cells identified upregulated genes in LMX1A.sup.+ cells and PITX3.sup.+ cells relative to their reference cells LMX1A.sup.− cells and PITX3.sup.− cells (>2-FC). Among the upregulated genes, 53 candidate genes coding for cell membrane proteins having extracellular domains were identified by gene mining. The 53 identified genes included a number of genes known to be specific for mouse mDA progenitors (Corin, Clstn2, KitIg, Plxdc2, Pcdh7, Ferd3l, Frem1, Alcam, and Notch2).

[0165] Subsequently, target validation was assessed by examining whether the genes were expressed in mDA cells that were practically differentiating. As a result, among the targets, surface marker genes were identified to be upregulated in LMX1A.sup.+ cells relative to LMX1A.sup.− cells (FIG. 14) and upregulated or downregulated in both LMX1A.sup.+ cells and PITX3.sup.+ cells (FIG. 15). Commercially available antibodies were screened against 18 genes among 21 genes in FIG. 14.

[0166] Of the 18 genes, 4 (CORIN, TPBG, CD47, and ALCAM) were selected as final surface marker candidates.

[0167] 6-3. Identification of mDA Neural Precursor-Specific Marker

[0168] Based on the above result, MACS targeting the four genes (CORIN, TPBG, CD47, and ALCAM) was performed.

[0169] As can be seen in FIGS. 16 and 17, CORIN- and TPBG (trophoblast glycoprotein)-targeted MACS resulted in statistically significant enrichments of LMX1A.sup.+ FOXA2.sup.+ mDA progenitors. Particularly, TPBG was expressed extensively in mDA progenitor culture population.

[0170] The CORIN gene was already known for use in enriching mDA progenitors whereas TPBG had never been previously reported in the context of mDA development. Finally, TPBG was thus selected as an mDA progenitor-specific marker.

Experimental Example 7: Effect of In Vivo Transplantation of TPBG-Positive Cell Separated from Human Embryonic Stem Cell (hESC)

[0171] 7-1. Construction of 6-OHDA-Lesioned Parkinson's Disease (PD) Model

[0172] Female Sprague-Dawley rats weighing 200-250 g (Orient Bio Inc., Korea) were used as subjects to be transplanted. A combination of 30 mg/kg Zoletil® (Virbac, France) and 10 mg/kg Rompun® (Bayer, Germany) was used as an anesthesia.

[0173] According to 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 the rats to induce a hem i-parkinsonian model.

[0174] 7-2. Behavioral Recovery of PD-Model after TPBG-Positive Cell Transplantation

[0175] The hESC cultured in colonies were differentiated using the differentiation protocol of the Example and MACS targeting TPBG was performed after 20 days of differentiation (d20).

[0176] The dissociated TPBG-positive cells were suspended at a final concentration of 8.75×10.sup.4 cells/μL in 1×HBSS to give a cell suspension. For a control, HBSS alone was used.

[0177] Four weeks postlesion with 6-OHDA of Experimental Example 7-1, 4 μL of the cell suspension (total of 350,000 cells) was stereotaxically transplanted per rat at the coordinates (TB −0.24, AP +0.08, ML −0.30, DV −0.40, and −0.50).

[0178] Immunosuppressive treatment was made for the duration of the experiment by intraperitoneally injecting cyclosporine A (Chong Kun Dang, Korea) every day at a dose of 10 mg/kg from 2 days prior to transplantation to the sacrifice of rats.

[0179] Amphetamine (2.5 mg/kg; Sigma-Aldrich) was intraperitoneally injected before transplantation, 4, 8, 12, or 16 weeks after transplantation and the amphetamine-induced rotation test was recorded for 30 minutes after injection.

[0180] As can be seen in FIG. 18, the TPBG-positive cells exhibited a significantly improved motor function for 16 weeks post-transplantation, compared to the control. These data demonstrate that hESC-derived TPBG-positive mDA neural precursor cells are viable in vivo and improve a motor function.

[0181] 7-3. Characterization of Graft after TPBG-Positive Cell Transplantation

[0182] TPBG-positive cells and unsorted cells were transplanted in the same manner as Example 7-2, with the exception that unsorted cells were used for a control.

[0183] Sixteen weeks post-transplantation, the rats were anesthetized with 25% urethane solution and transcardially perfused with 0.9% saline solution followed by 4% paraformaldehyde. Removed brains were fixed overnight and cryoprotected in 30% sucrose-PBS solution. Cryoprotected brains were embedded in FSC 22® compound (Leica, Nuβloch, Germany), and coronal sections, each 18 μm thick, were made using a cryostat (Thermo Fisher Scientific). Then, immunohistochemistry against hNCAM (human-specific neural cell adhesion molecule) was carried out.

[0184] As can be seen in FIG. 19, the TPBG-positive cell group was composed of a greater number of TH.sup.+hNCAM.sup.+ and PITX3.sup.+ hNCAM.sup.+ mDA neural cells, compared to the unsorted group. The result implies that TPBG-positive cells are more suitable for in vivo differentiation into mDA neurons, compared to the unsorted group.

[0185] In addition, as can be seen in FIG. 20, a graft comprising about 20% or more of KI67.sup.+hNCAM.sup.+ cells was observed in one certain rat in the unsorted group while no KI67.sup.+hNCAM.sup.+ cells were found in the TPBG-positive group. These data indicate that the unsorted group has the feasibility of retaining proliferative potential even after 16 weeks post-transplantation, but proliferative cells can be excluded upon cell sorting by TPBG. The result is very significant in terms of safety for cell therapy.

Experimental Example 8: Characterization of TPBG-Positive Cell Separated from Human Fetal Ventral Mesencephalic Cells (NM Cells)

[0186] When cultured on a laminin-coated plate in NM cells in a nerve stem cell maintenance medium (ReNcell NSC maintenance Medium, Merck) at confluence 80-90%, NM cells were subjected to TPBG-targeting MACS. Then, the expression of EN1 in TPBG-positive cells and TPBG-negative cells was analyzed by qRT-PCR relative to the control hESC (H9) (expression of H9=1).

[0187] As can be seen in FIG. 21, the expression of the mDA neuron-specific regional marker EN1 in TPBG-positive cells was increased compared to TPBG-negative cells, indicating that TPBG can be used for enrichment of NM cells exhibiting midbrain characteristics.

Experimental Example 9: Characterization of Human Induced Pluripotent Stem Cells (iPSC) Separated from TPBG-Positive Cell

[0188] Human iPSC (HDF-epi3) that was being cultured in the same manner as for the human embryonic stem cells was allowed to differentiate using the differentiation protocol of the Example and then subjected to MACS targeting TPBG on d20. The separated TPBG-positive cells were assayed for expression of EN1, FOXA2, and LMX1A (Immunocytochemistry).

[0189] As can be seen in FIG. 22, the expression of the midbrain-specific regional markers EN1 and FOXA2 did not differ before and after MACS, but TPBG-positive cells expressing the mDA lineage marker LMX1A were enriched. In addition, TPBG-positive cells positive for all the three markers (EN1, FOXA2, and LMX1A) were also significantly enriched.