Patent Application
20210093673
The present disclosure relates to a method for separation of dopaminergic neural cells and a pharmaceutical composition comprising dopaminergic neural cells separated thereby for treatment of Parkinson's disease.
The present disclosure was made with the support of the Ministry of Health and Welfare of the Republic of Korea, under Project No. HI18C0829, which was conducted under the research subject named “In vivo Differentiation Monitoring and Prediction of Minimal Number of Transplant Cell for Application of Pluripotent Stem Cell to Neurological Disorder”, within the research project entitled “Development of Advanced Medical Technology” by the Yonsei University-Industry Foundation under the management of the Korea Health Industry Development Institute, from Apr. 30, 2018 to Dec. 31, 2021
This application claims priority to and the benefit of Korean Patent Application No. 10-2016-0032283 filed in the Korean Intellectual Property Office on 17 Mar. 2016, the disclosure of which is incorporated herein by reference.
Parkinson's disease (PD) is one of the most felicitous neurodegenerative disorders for cell-based therapies due to the focal degeneration of midbrain dopaminergic (mDA) neurons.
Since the early 1980s, efforts have been made to restore Parkinson's disease-related motor functions by engrafting fetal ventral mesencephalon (VM) tissue to patient's striatum.
Although the graft-induced recovery of motor functions was achieved, inconsistent outcomes were reported among research institutes or organizations, even with the production of side effects for some outcomes. Thereafter, a consensus regarding the need of improvement for cell-based treatments has been established.
Particularly, research has been continued to develop solutions to the problems indicated in previous studies, that is, to advance the grafting material cells to a level similar to that of the cells existing in the living body and avoid disadvantages of fetal tissue which includes limited availability and batch-to-batch inconsistency.
As a result, human pluripotent stem cells (hPSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have attracted attention for their abundance and ability to limitlessly proliferate in vitro and differentiate into a variety of neural cells.
Accordingly, techniques for differentiation into mDA neurons have been developed and advanced in recent years to a level of producing cells at high yield. However, the hPSC-derived mDA cell populations induced by recently developed differentiation techniques were found yet to be subject to the risk of heterogeneity, that is, to have mixed different kinds of cells in addition to mDA neurons. Thus, clinical application based on the current differentiation techniques is premature to consider.
Thus, identification and isolation of hPSC-derived mDA cells from diverse cell populations are critical for standardization of the cell source and successful transplantation.
The initial strategies for purely isolating (enriching) differentiated mDA cells from hPSCs were based on fluorescence-activated cell sorting (FACS) targeting multiple surface antigens. Although these early approaches enriched neuronal population expressing tyrosine hydroxylase (TH), it was unclear whether these neurons retained mDA neuronal properties.
Recent studies took much more sophisticated approaches by analyzing transcriptome of mouse NM tissue and mouse embryonic stem cell (mESC)-derived mDA neuronal progenitors to identify specific surface markers that could distinguish and enrich mDA neurons.
While these studies suggest some feasible cell surface markers and support the feasibility of dopaminergic neuron isolation and enrichment for standardization by using the markers, it has not yet been made clear which stage of the neural cell development expresses pertinent cell surface markers that should be excavated. Because it is a common view in this field that the isolated neural cells vary in post-grafting survival rate and differentiation extent, depending on developmental stages thereof, and have an influence on post-grafting function restoration, identification of differentiation stage-specific markers is also a critical task.
Therefore, there is an urgent need for research into the differentiation stage of mDA neurons and the surface marker of the differentiation stage.
The present inventors endeavored to develop a differentiation protocol with stepwise specifications of mDA neurons and develop cell surface markers in each stage. As a result, an LMX1A-eGFP and a PITX3-mCherry reporter hESC lines were established and differentiated to isolate LMX1A+ mDA progenitors and PITX3+ mDA neurons from which an mDA neuron-related cell surface marker (TPBG) was identified, leading to the present invention.
Therefore, the purpose of the present disclosure is to provide a method for preparation of dopaminergic neural cells.
Another purpose of the present disclosure is to provide a pharmaceutical composition comprising TPBG (trophoblast glycoprotein)-positive dopaminergic neurons for treatment of Parkinson's disease.
Another purpose of the present disclosure is to provide a method for advancing the efficacy of dopaminergic neurons in cell replacement therapy for Parkinson's disease and enhancing transplantation safety.
Another purpose of the present disclosure is to provide a composition comprising TPBG (trophoblast glycoprotein)-positive dopaminergic neurons for dopaminergic neuron replacement.
The present inventors endeavored to develop a differentiation protocol with stepwise specifications of mDA neurons and develop cell surface markers in each stage. As a result, an LMX1A-eGFP and a PITX3-mCherry reporter hESC lines were established and differentiated to isolate LMX1A+ mDA progenitors and PITX3+ mDA neurons from which an mDA neuron-related cell surface marker (TPBG) was identified.
In greater detail, the present inventors established an LMX1A-eGFP reporter hESC line which is manipulated to express green fluorescent protein (eGFP) concurrently with LMXIA, which is an mDA neural progenitor stage-specific gene, and a PITX3-mCherry reporter hESC line which is manipulated to express a red fluorescent protein (mCherry) concurrently with PITX3, which is a mature mDA neuronal stage-specific gene. Transcriptome analysis of LMX1A+ mDA neural precursor cells and PITX+ mDA neural cells revealed cell surface marker candidates specifically expressed on progenitors of mDA neural cells (neural precursor cells). Among them, TPBG was discovered as a novel cell surface marker. Cell separation targeting TPBG revealed the enrichment of mDA neural precursor cells. In addition, magnetic-activated cell sorting (MACS)-purified TPBG-positive cells at the mDA neural precursor cell stage were transplanted into 6-OHDA-lesioned Parkinson's disease (PD) rat models, with the significant recovery of motor deficits without tumor formation in the PD rats.
Therefore, the identification and use of TPBG as a new surface marker protein to isolate transplantable mDA neural precursor cells is expected to provide a safe and effective cell replacement therapy for PD.
The present disclosure relates to a method for separation of dopaminergic neural cells, a pharmaceutical composition comprising dopaminergic neural cells separated using the same method, a method for advancing the efficacy of dopaminergic neurons in cell replacement therapy for Parkinson's disease and enhancing transplantation safety, and a composition comprising TPBG (trophoblast glycoprotein)-positive dopaminergic neurons for dopaminergic neuron replacement.
Below, a detailed description will be given of the present disclosure.
An embodiment of the present disclosure pertains to a method for preparing dopaminergic neural cells, the method comprising the following steps:
(a) contacting a cell population with an TPBG (trophoblast glycoprotein) antibodies; and
(b) separating TPBG-positive dopaminergic neural cells, which bind to the TPBG antibodies.
As used herein, the term “neural cells” refers to cells constituting the nervous system and is used in the same meaning as neurons, and “dopaminergic neural cells” means neural cells secreting the neurotransmitter dopamine.
The dopaminergic neural cells may be dopaminergic neural progenitors or dopaminergic neural precursor cells, or mature dopaminergic neurons, but are not limited thereto.
As used herein, the term “neural progenitors or neural precursor cells” means undifferentiated precursor cells that have not yet expressed a differentiated characteristic, and “progenitors”, “precursors”, and “precursor cell” may be used interchangeably.
The dopaminergic neural cells may be midbrain dopaminergic neural cells.
As used herein, the term “mDA neural cells” refers to dopaminergic neural cells observed in the midbrain region, for example, dopaminergic neural cells observed in the midbrain ventral region, but are not limited thereto.
Further, the mDA neural cells may be A9 region specific.
The “A9 region” is a midbrain ventrolateral region, which corresponds to the pars compacta part of the substantia nigra. Hence, the cells prepared by the preparation method of the present disclosure are midbrain cells.
The A9 region is an area in which dopaminergic neural cells are abundantly found and is related to the control of motor function. Particularly for PD patients, dopaminergic neural cells are specifically degenerated in this region. The cells prepared by the preparation method of the present disclosure may be used for preventing and/or treating PD.
The method for preparing dopaminergic neural cells of the present disclosure is described in detail, below.
Step (a)
The “cell population” includes human stem cells; progenitors or precursors thereof; and/or dopaminergic neural progenitors or mature dopaminergic neurons derived from human stem cells or precursors, and neural derivatives derived therefrom, but are not limited thereto.
In detail, examples of the human stem cells or precursors may include embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells (iPSCs), adult stem cells, and fetal cells, but are not limited thereto.
The fetal cells may be derived from a fetal neural tissue and/or derivatives thereof and may be, for example, fetal ventral mesencephalic cells (NM cells), but not limited thereto.
Step (b)
The TPBG″ may be used in the same meaning as in Wnt-Activated Inhibitory Factor 1 or WAIF1 and is known as an antagonist of the Wnt/β-catenin signaling pathway. However, there have been no reports on the separation of dopaminergic neural cells through the expression of TPBG.
The gene has the nucleotide sequence represented by SEQ ID NO: 53. In addition, the gene may be easily available to a person skilled in the art because the nucleotide sequence is registered in the GenBank.
As used herein, the term “TPBG-positive dopaminergic neural cells” means dopaminergic neural cells bound by a TPBG antibody.
The term “TPBG antibody”, as used herein, refers to an antibody that binds specifically to TPBG.
So long as it targets cells, any separation method for TPBG-positive dopaminergic neural cells can be used in this step. For example, the separation may utilize fluorescence-activated cell sorting (FACS) and/or magnetic-activated cell sorting (MACS), but is not limited thereto.
The TPBG-positive dopaminergic neural cells can alleviate symptoms of Parkinson's disease.
The TPBG-positive dopaminergic neural cells can advance the safety for cell replacement therapy.
Another embodiment of the present disclosure pertains to a pharmaceutical composition comprising TPBG (trophoblast glycoprotein)-positive dopaminergic neural cells for treatment of Parkinson's disease.
The pharmaceutical composition according to the present disclosure may include a pharmaceutically acceptable carrier in addition to the effective ingredient. In this regard, the pharmaceutically acceptable carrier is typically used in preparations and may include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto. In addition, ingredients such as, a lubricant, a humectant, a sweetener, a flavorant, an emulsifier, a suspending agent, a preservative, etc. may be included.
The composition of the present disclosure may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and time, but may be appropriately selected by those skilled in the art.
The pharmaceutical composition of the present disclosure is administrated at a pharmaceutically effective dose. As used herein, the term “pharmaceutically effective dose” means an amount that is sufficient to treat the diseases at a reasonable benefit/risk ratio applicable to medical treatment or improvement, and an effective dose level may be determined according to elements including a kind of disease of the patient, the severity, age, and sex of the patient, activity of a drug, sensitivity to a drug, a time of administration, a route of administration, and an emission rate, duration of treatment, and simultaneously used drugs and other elements well-known in the medical field.
The composition of the present disclosure may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or concurrently with conventional therapeutic agents, and may be administered singly or multiply. It is important to administer an amount at which the maximum effect is able to be obtained with a minimum amount without causing side effects in consideration of all of the above-described factors, and may be easily determined by those skilled in the art.
Specifically, the effective amount of the pharmaceutical composition of the present disclosure may be dependent on a patient's age, sex, condition, and body weight, an absorption rate of the active ingredient in the body, an inactivation rate, an excretion rate, a type of disease, or a drug used in combination.
Another embodiment of the present disclosure pertains to a method for treatment of Parkinson's disease, the method comprising administering the TPBG-positive dopaminergic neural cells to a subject.
The term “subject” used herein refers to a subject in need of treatment, and more specifically, a mammal such as a human, or a non-human primate, a mouse, a rat, a dog, a cat, a horse, and a cow.
Another embodiment of the present disclosure pertains to a use of the TPBG-positive dopaminergic neural cells in treating Parkinson's disease.
In order to avoid undue redundancy leading to the complexity of this specification by the pharmaceutical composition comprising TPBG-positive dopaminergic neural cells for treatment of Parkinson's disease because it uses dopaminergic neural cells as described in the preparation method, the common description between them are omitted.
Another embodiment of the present disclosure pertains to a method for enhancing the efficacy and improving transplantation safety of dopaminergic neural cells in transplantation for Parkinson's disease, the method comprising the following steps:
(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.
In order to avoid undue redundancy leading to the complexity of this specification by the method for advancing the efficacy of dopaminergic neurons and enhancing transplantation safety because it uses the same elements as described in the preparation method for dopaminergic neural cells, the common description between them are omitted.
Another embodiment of the present disclosure pertains to a composition comprising TPBG (trophoblast glycoprotein)-positive dopaminergic neural cells for transplantation.
The TPBG-positive dopaminergic neural cells can be cultured by the preparation method for dopaminergic neural cells, and the TPBG-positive dopaminergic neural cells cultured by the method may be enhanced in cell efficacy and improved in transplantation safety.
For transplantation of the dopaminergic neural cells, an appropriate injection site (e.g., the putamen or caudate nucleus, or the striatum including both of them in the brain) is selected and then, a well-known modality (for example, stereotactic system, etc.) may be taken.
The composition of the present disclosure may be used in treating Parkinson's disease.
The composition of the present disclosure may comprise dopaminergic neural cells as transplanted cells, alone or in combination with a biocompatible and/or biodegradable stabilizer. The stabilizer functions to stably disperse the dopaminergic neural cells and is a bio-derived material that does not cause any side effect after transplantation. Alternatively, the stabilizer should be biodegradable. As used herein, the term “biodegradable” refers to having the property of being slowly degraded in and absorbed into the body, but does not impose special meaning on a degradation speed.
Examples of the stabilizer include hyaluronic acid, collagen, thrombin, elastin, chondroitin sulfate, albumin, and a mixture thereof. Particularly, hyaluronic acid, collagen, thrombin, elastin, chondroitin sulfate, and albumin are bio-derived materials that have biodegradability, e.g., are naturally degraded in vivo. So long as it meets the requirement for being biodegradable and providing viscosity in a medium, even a synthetic compound may be used in the present disclosure. Hence, the stabilizer is not limited only to bio-derived materials.
When formulated together with the stabilizer, dopaminergic neural cells do not float or settle, but can exist in an evenly dispersed form.
In order to avoid undue redundancy leading to the complexity of this specification by the composition comprising TPBG (trophoblast glycoprotein)-positive dopaminergic neurons for dopaminergic neuron replacement because it uses the same elements as described in the preparation method, the common description between them are omitted.
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.
Human Embryonic Stem Cell (hESC) Culture
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).
Genotyping of Clonal Cell
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).
FACS
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.
MACS
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.
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,500
1:1,000
After washing, the primary antibody-labeled cells were incubated with 20 μL of microbeads (Miltenyi Biotec) per 1×107 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.
Immunocytochemistry
First, cells were fixed in 4% paraformaldehyde-PBS solution.
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.
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).
Flow Cytometry
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.
Gene Expression Analysis
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.
Microarray: Transcriptome Profiling
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.
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.
A concrete protocol is depicted in
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.
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.
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.
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 3.12×105 cells/cm2 in a bFGF-free N2B27 medium. mDA precursors were expanded in N2B27 medium for additional 7 days at 90% confluence.
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.
A concrete protocol is given in
1-1. Design of Programmable Nucleases and Donor DNA Plasmids
TALEN-encoding plasmids were purchased from ToolGen, Inc. (Korea).
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/).
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).
1-2. Generation of LMX1A-eGFP Reporter Line
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.
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 1.0×107 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).
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.
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.
Finally, genotyping of clonal cells allowed for the selection of LMX1A-eGFP reporter lines.
A concrete protocol is depicted in
2-1. Design of Nuclease and Donor DNA Plasmid
Cas9- and sgRNA (CRISPR/Cas9)-encoding plasmids were purchased from ToolGen. Inc.
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 TACGG-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).
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).
2-2. Generation of PITX3-mCherry Reporter Line
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.
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).
As can be seen in
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.
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).
As can be seen in
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.
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.
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 2×106 cells/ml. FACS was performed. Comparison of mRNA expression levels was made among the unsorted group, the LMX1A group, and the LMX1A+ group.
As can be seen in
As can be seen in
Additionally, the unsorted group, the LMX1A− group, and the LMX1A+ 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.
As can be seen in
In LMX1A+ progenitor cultures, as shown in
Finally, the unsorted group, the LMX1A− group, and the LMX1A+ 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.
After the final differentiation, as can be seen in
Further, the fully matured cells (8 weeks, d75) by the terminal differentiation were analyzed for dopamine release.
Briefly, cells were washed with a low KCl solution (2.5 mM CaCl2), 11 mM glucose, 20 mM HEPES-NaOH, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 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 CaCl2), 11 mM glucose, 20 mM HEPES-NaOH, 60 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 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.
As can be seen in
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 1×107 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.
As can be seen in
Next, expression levels of mDA neuron-specific markers were compared among the unsorted group, the PITX3− group, and the PITX3+ group of the cells on d40.
As can be seen in
Finally, the post-FACS, unsorted group was additionally incubated and then subjected to double-labeled immunostaining on d44.
As can be seen in
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.
As can be seen in
6-1. Transcriptome Analysis
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+ and LMX1A− cells on d20 (mDA neural precursor stage) and PITX3+ and PITX3− cells on d40 (mDA neuron state) were separated and subjected to transcriptome analysis (Microarray) (see
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).
6-2. Identification of mDA Marker Candidates
Based on the above result, mDA marker candidates were obtained. A concrete procedure is depicted in
Comparative microarray analysis of the four isolated cells identified upregulated genes in LMX1A+ cells and PITX3+ cells relative to their reference cells LMX1A− cells and PITX3− 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).
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+ cells relative to LMX1A− cells (
Of the 18 genes, 4 (CORIN, TPBG, CD47, and ALCAM) were selected as final surface marker candidates.
6-3. Identification of mDA Neural Precursor-Specific Marker
Based on the above result, MACS targeting the four genes (CORIN, TPBG, CD47, and ALCAM) was performed.
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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.
7-1. Construction of 6-OHDA-Lesioned Parkinson's Disease (PD) Model
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.
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.
7-2. Behavioral Recovery of PD-Model after TPBG-Positive Cell Transplantation
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).
The dissociated TPBG-positive cells were suspended at a final concentration of 8.75×104 cells/μL in 1×HBSS to give a cell suspension. For a control, HBSS alone was used.
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).
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.
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.
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7-3. Characterization of Graft after TPBG-Positive Cell Transplantation
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.
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.
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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).
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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).
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| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0050918 | May 2018 | KR | national |
| 10-2019-0048784 | Apr 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/005058 | 4/26/2019 | WO | 00 |
