Methods for identifying mDA progenitor cells

Abstract

The present disclosure provides a method for identifying, isolating and / or enriching brain dopaminergic neural progenitor cells, the method comprising determining whether a candidate cell is APCDD1 + (Downregulated 1 of Aminogenic Colonopolyposis); and a cell product obtained by using the method. The invention further provides a method for evaluating a cell product and optimizing a cell product preparation process. The products and methods can be used to treat diseases or conditions of the nervous system.

Description

Background Art

[0001] Parkinson's disease (PD) is the second most common degenerative disease of the central nervous system after Alzheimer's disease. There is no effective treatment for Parkinson's disease clinically, and current treatment methods are mainly symptomatic treatment, including drugs such as levodopa and operations such as deep brain stimulation. However, drug treatment is only effective in the early stages, and deep brain stimulation is only suitable for some patients and may cause side effects such as depression. At present, one of the most promising PD treatments is cell replacement therapy (cell therapy). In PD cell therapy, human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC) and people's cumulative induced pluripotent stem cells (hiPSC), are generally used to obtain midbrain dopamine (mDA) neural precursor cells by in vitro differentiation. It is expected that these cells will differentiate and mature into mDA neurons in the graft after transplantation, thereby alleviating the motor dysfunction of patients with Parkinson's disease.

[0002] However, there are still many obstacles that limit the widespread clinical application of PD cell therapy, one of which is the lack of effective cell preparation quality control and evaluation methods. In PD cell therapy, immature neural progenitor cells are usually transplanted, rather than mature terminal neurons. Due to the limitations of currently available differentiation protocols, the cell preparations derived from hPSC are not colonies of a single cell type, but a mixture of target neural progenitor cells and other non-target cell types. Assessing the ratio of target cells (i.e., mDA progenitor cells) is crucial for controlling the purity (an important aspect of quality) of cell products and predicting the efficacy of cell products. The characteristics used to identify mDA progenitor cells can also be used to enrich these cells from mixed populations during the manufacturing process.

[0003] Therefore, there is an urgent need for a method to identify mDA progenitor cells, which will facilitate the quality control, enrichment, or evaluation of cell products for PD cell therapy. Summary of the Invention

[0004] In one aspect, the present disclosure provides a method for identifying mDA (midbrain dopaminergic) neural progenitor cells, the method comprising determining whether a candidate cell is APCDD1+ (adenomatous polyposis coli downregulated protein), and identifying the APCDD1+ cell as an mDA neural progenitor cell.

[0005] In another aspect, the present disclosure provides a method for identifying mDA neural progenitor cells, the method comprising determining whether a candidate cell is APCDD1 + and TPBG + (trophoblast glycoprotein), and identified APCDD1+ and TPBG+ cells as mDA neural progenitors.

[0006] In some embodiments, the candidate cell is a neural progenitor cell.

[0007] In some embodiments, the candidate cells are derived from pluripotent stem cells.

[0008] In some embodiments, the candidate cells are derived from human pluripotent stem cells.

[0009] In some embodiments, the method comprises directly or indirectly detecting the presence and / or activity level of APCDD1 in the candidate cell.

[0010] In some embodiments, the method comprises directly or indirectly detecting the presence and / or activity level of TPBG in the candidate cell.

[0011] In some embodiments, the expression and / or activity level of APCDD1 includes the expression and / or activity level of a nucleic acid encoding APCDD1, and / or the presence and / or activity level of an APCDD1 protein.

[0012] In some embodiments, the expression and / or activity level of TPBG includes the expression and / or activity level of a nucleic acid encoding TPBG, and / or the presence and / or activity level of a TPBG protein.

[0013] In some embodiments, the detecting comprises modifying the candidate cell.

[0014] In some embodiments, the detecting comprises using a marker.

[0015] In some embodiments, the marker comprises a protein, a nucleic acid, and / or a small molecule compound.

[0016] In some embodiments, the marker comprises a fluorescent reporter gene.

[0017] In some embodiments, the method comprises contacting the candidate cell with an agent capable of specifically binding to an APCDD1 protein and / or capable of testing the activity of an APCDD1 protein.

[0018] In some embodiments, the method comprises contacting the candidate cell with primers capable of specifically amplifying a nucleic acid molecule encoding APCDD1 and / or a probe capable of specifically recognizing a nucleic acid molecule encoding APCDD1.

[0019] In some embodiments, the method comprises contacting the candidate cell with an agent capable of specifically binding to a TPBG protein and / or capable of testing the activity of a TPBG protein.

[0020] In some embodiments, the method comprises contacting the candidate cell with primers capable of specifically amplifying a nucleic acid molecule encoding TPBG and / or a probe capable of specifically recognizing a nucleic acid molecule encoding TPBG.

[0021] In another aspect, the present application provides a method for isolating mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) isolating APCDD1+ cells from the candidate cell population.

[0022] In another aspect, the present application provides a method for isolating mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) isolating APCDD1+ and TPBG+ cells from the candidate cell population.

[0023] In another aspect, the present application provides a method for enriching mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) enriching APCDD1+ cells from the candidate cell population.

[0024] In another aspect, the present application provides a method for enriching mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) enriching APCDD1+ and TPBG+ cells from the candidate cell population.

[0025] In some embodiments, the candidate cell is a neural progenitor cell.

[0026] In some embodiments, the candidate cell is an mDA neuron.

[0027] In another aspect, the present application provides a population of mDA neural progenitor cells obtained by the method of the present application.

[0028] In another aspect, the present application provides a cell product comprising mDA neural progenitor cells obtained by the method of the present application.

[0029] In another aspect, the present application provides a cell product comprising APCDD1+ neural progenitor cells.

[0030] In another aspect, the present application provides a cell product comprising APCDD1+ and TPBG+ neural progenitor cells.

[0031] In another aspect, the present application provides a method for preparing a cell product, comprising (a) providing neural progenitor cells, and (b) isolating and / or enriching APCDD1+ neural progenitor cells.

[0032] In another aspect, the present application provides a method for preparing a cell product, comprising (a) providing neural progenitor cells, and (b) isolating and / or enriching APCDD1+ and TPBG+ neural progenitor cells.

[0033] In some embodiments, the method comprises obtaining the neural progenitor cells by differentiating the cell population.

[0034] In some embodiments, the cell population is derived from rodent cells, primate cells, or human cells.

[0035] In some embodiments, the cell population is derived from pluripotent stem cells.

[0036] In some embodiments, the cell population is derived from human pluripotent stem cells.

[0037] In some embodiments, the differentiating comprises in vivo or in vitro differentiation.

[0038] In another aspect, the present application provides a method for evaluating a cell product, comprising testing the proportion of APCDD1+ cells in the cell product.

[0039] In another aspect, the present application provides a method for evaluating a cell product, comprising testing the ratio of APCDD1+ and TPBG+ cells in the cell product.

[0040] In another aspect, the present application provides a method for optimizing a process for manufacturing a cell product, the method comprising testing the cell product for a proportion of APCDD1+ cells.

[0041] In another aspect, the present application provides a method for optimizing a process for preparing a cell product, the method comprising testing the cell product for a ratio of APCDD1+ and TPBG+ cells.

[0042] In some embodiments, the process for preparing a cell product comprises optimizing the production, differentiation, isolation, and / or purification process of the cell product.

[0043] In another aspect, the present application provides a cell preparation obtained by further expansion and proliferation of the cell product of the present application.

[0044] In another aspect, the present application provides a kit comprising an APCDD1+ indicator, wherein the kit is used to examine the proportion of mDA neural progenitor cells in a cell product.

[0045] In another aspect, the present application provides use of an APCDD1+ indicator for preparing a cell product, wherein the cell product comprises mDA neural progenitor cells.

[0046] In another aspect, the present application provides use of the cell product of the present application in screening drugs, wherein the drugs are used to prevent and / or treat nervous system diseases or disorders.

[0047] In another aspect, the present application provides a pharmaceutical composition comprising APCDD1+ neural progenitor cells.

[0048] In some embodiments, the neural progenitor cells are capable of differentiating into neural cells, wherein the neural cells contain at least 30% mDA neural cells.

[0049] In some embodiments, the differentiating comprises in vivo or in vitro differentiation.

[0050] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0051] In another aspect, the present application provides a method for preventing and / or treating a nervous system disease or disorder, comprising administering the cell product of the present application and / or the pharmaceutical composition of the present application to a subject in need thereof.

[0052] In another aspect, the present application provides the cell product of the present application and / or the pharmaceutical composition of the present application, which is used for preventing and / or treating nervous system diseases or disorders.

[0053] In some embodiments, the neurological disease or disorder comprises Parkinson's disease.

[0054] In another aspect, the present application provides use of the cell product of the present application and / or the pharmaceutical composition of the present application in the preparation of a medicament, wherein the medicament is used to prevent and / or treat a neurological disease or disorder.

[0055] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0056] Incorporated by reference

[0057] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and accompanying drawings (also referred to herein as "figures"), which set forth illustrative embodiments embodying the principles of the present invention, wherein:

[0059] Figure 1A-1J Identification of surface markers representing fate-committed ventral midbrain neural progenitors to improve transplant outcomes.

[0060] (1A) Volcano plot identifying II-NPs in stage I vMB Fate-committed progenitors (left), stage II III-NP vMB Differentially expressed genes in fate-committed progenitors (center) and stage III IV-mDA fate-committed progenitors (right). FDR-corrected P values ​​are shown. Selected genes are labeled.

[0061] (1B) Schematic diagram of the generation of the APCDD1-tdtomato hESC line and an overview of the experiments used to mature APCDD1+ cells in vitro and in vivo.

[0062] (1C) Representative immunofluorescence images of APCDD1-sorted and unsorted cells after in vitro maturation. Ho, Hoechst. Scale bar, 50 μm.

[0063] (1D) The percentage of TH+ cells in the total cell population after in vitro maturation. Data are expressed as mean ± SEM; ****P < 0.0001, unpaired t-test.

[0064] (1E) Representative immunofluorescence images of TH in transplants derived from APCDD1-sorted and unsorted cells. hN, human nucleus. (i) and (ii) indicate the periphery and central regions of the transplant, respectively. Scale bars, 100 μm in the full micrographs and 20 μm in the insets.

[0065] (1F) Percentage of TH+ cells in the hN+ population in transplants derived from APCDD1-sorted and unsorted cells. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ****P < 0.0001, unpaired t-test.

[0066] (1G and 1H) Representative immunofluorescence images of COL1A1 (1G) and vGlut2 (1H) in transplants derived from APCDD1-sorted and unsorted cells. hN, human nucleus. Scale bar, 20 μm.

[0067] (I and J) Percentage of COL1A1+ (1I) and TH– / vGlut2+ (1J) cells in the hN+ population in transplants derived from APCDD1-sorted and unsorted cells. Data are presented as mean ± SEM.

[0068] Figures 2A-2D The ventral midbrain lineage trajectory and associated biological processes are shown.

[0069] (2A) Top: UMAP plots showing clonal trajectories of the ventral midbrain lineage from stage I to II (clone number = 112) and from stage II to III (clone number = 212). Bottom: Expression of marker genes for the ventral midbrain lineage (LMX1A, EN1, and OTX2). Clones with 100% progenitor cells in a given progenitor cluster were selected as progenitor cluster-associated clones, and all cluster-associated clones were plotted on the UMAP plots for both stages.

[0070] (2B to 2D) II-NP is enriched in stage I relative to other fate-committed progenitors. vMB Fate-committed progenitors (2B) are enriched in III-NP relative to other fate-committed progenitors in stage II vMB Fate-committed progenitors (2C) and top biological processes enriched for IV-mDA fate-committed progenitors (2D) relative to other fate-committed progenitors in stage III.

[0071] Figure 3A-Figure 3E Shown is the generation of the APCDD1-tdtomato hESC line.

[0072] (3A) Right: Stage I progenitor clusters in UMAP space and gene expression of EN1, OTX2, and FOXA2 (markers of ventral midbrain progenitors) and APCDD1 (identified surface marker).

[0073] (3B) Strategy used to generate APCDD1-tdtomato knock-in hESC line. Exons are shown as gray boxes.

[0074] (3C) PCR genotyping of APCDD1-tdtomato hESC clones. Clones with horizontal arrows indicate the expected size of the PCR products used to assess targeted locus insertion and homozygosity, respectively. Heterozygous clones were selected for downstream experiments.

[0075] (3D) Image of neural rosettes in APCDD1-tdtomato cells at stage I. Scale bar, 500 μm.

[0076] (3E) Representative FACS plots of the sorting protocol used to isolate APCDD1-positive cells at stage I during hPSC-based ventral midbrain-hindbrain differentiation. Negative controls were cells derived from the wild-type H9 hESC line. DETAILED DESCRIPTION

[0077] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions may occur to those skilled in the art without departing from the present invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0078] As used herein, the term "APCDD1 (APC Down-Regulated 1)" generally refers to adenomatous polyposis coli down-regulated protein, which is a negative regulator of the Wnt signaling pathway. The term "APCDD1" can include full-length APCDD1, as well as truncations, functional fragments, different transcripts, splice variants and isoforms of APCDD1, naturally occurring APCDD1, artificially modified or mutated APCDD1 proteins.

[0079] As used herein, the term "TPBG" generally refers to trophoblast glycoprotein, which is an inhibitor of Wnt / β-catenin signaling and is reported as a cell surface marker for ventral midbrain progenitor cells. The term "TPBG" may include full-length TPBG, as well as truncations, functional fragments, different transcripts, splice variants and isoforms of TPBG, naturally occurring TPBG, artificially modified or mutated TPBG proteins.

[0080] As used herein, when referring to a cell, a molecular marker followed by a superscript "+" (such as "APCDD1+" and / or "TPBG+") generally means that the cell is positive for the molecular marker, i.e., the cell transcribes the mRNA of the molecular marker and / or translates the protein of the molecular marker. For example, APCDD1+ with respect to a cell means that the cell is positive for APCDD1, e.g., APCDD1 protein can be detected in the cell, or the cell can transcribe APCDD1 RNA. When a cell is positive for more than one marker, for example, when APCDD1+ and TPBG+ expression is used, the cell is positive for both APCDD1 and TPBG. The term "positive" refers to an assay that measures the expression and / or activity of a molecular marker, wherein the result is above the threshold or cutoff value of the assay for samples that are considered to reproducibly contain detectable levels of the molecular marker.

[0081] By detecting the expression of a certain molecular marker (including protein and / or nucleic acid) in a cell, it can be determined whether the cell is positive for the molecular marker. In some cases, for molecular markers (e.g., "APCDD1" and / or "TPBG"), a molecule that can specifically recognize or bind to the molecular marker is used, which may include proteins, nucleic acids, macromolecules and / or small molecules. For example, the molecule can be a detectably labeled antibody that can specifically bind to a molecular marker protein. The antibody is mixed with the cell to be examined, and if the antibody binds to the cell and is therefore detected, it means that the cell is positive for the molecular marker. As another example, the molecule can be a probe (such as a fluorescent probe) with a detectable label that can specifically hybridize with the nucleic acid encoding the molecular marker, and if the detectable label is detected, it means that the cell is sensitive to the molecule and the marker is positive. The expression and / or activity of the molecular marker in the cell can be qualitatively or quantitatively compared with positive or negative control cells. In some cases, the expression of the target molecular marker can also be assessed by the presence of a reporter protein (such as a fluorescent protein) engineered into the genomic locus of the target molecular marker.

[0082] As used herein, the term "marker" generally refers to a gene / protein that can be used to indicate an expressed molecule (e.g., "APCDD1" and / or "TPBG") and / or an active substance, including those that can directly recognize or bind to a molecular marker protein or nucleic acid and react with the molecular marker protein or nucleic acid through its own expression and / or activity. For example, the marker molecule can be a radioisotope, a fluorophore, a chemiluminescent substance, a chromophore, an antibody, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a chromophore, a dye, a metal ion, a metal sol, a ligand (such as biotin, avidin, streptavidin, or a hapten), etc.

[0083] As used herein, the term "midbrain dopaminergic neural progenitor cells" generally refers to cells that are capable of proliferating and / or differentiating into dopaminergic neurons in vitro or in vivo. Midbrain dopaminergic neural progenitor cells can be differentiated from pluripotent stem cells. Dopaminergic neural progenitor cells can also be differentiated or reprogrammed from other cell types.

[0084] As used herein, the term "midbrain dopaminergic (mDA) neurons" generally refers to dopaminergic neurons residing in the midbrain region, such as, but not limited to, dopaminergic neurons observed in the ventral midbrain region. In addition, mDA neurons may be specific to the A9 group. A9 is the most densely packed group of dopaminergic cells, located in the substantia nigra pars compacta in the ventral midbrain and involved in the control of motor function. It is noteworthy that in PD patients, dopaminergic neurons in this region preferentially degenerate.

[0085] As used herein, the term "cell population" may include human stem cells; progenitor cells or their precursors; dopaminergic neural progenitor cells and / or dopaminergic neural precursor cells or mature dopaminergic neurons derived from human stem cells or precursors, as well as neural derivatives derived therefrom, but is not limited thereto. Specifically, examples of human stem cells or precursor cells may include, but are not limited to, embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells (iPSCs), adult stem cells, and fetal cells.

[0086] As used herein, the term "pluripotent stem cell" generally refers to a class of cells that have the potential to differentiate into any cell type in the human body. Pluripotent stem cells can be derived from fertilized eggs or somatic cells, which may include blood cells, urine cells, skin cells, and / or umbilical cord blood cells. Depending on their source, pluripotent stem cells may include human embryonic stem cells (derived from fertilized eggs) and human induced pluripotent stem cells (somatic cells). Pluripotent stem cells have the ability to sustainably proliferate and differentiate into a variety of cells.

[0087] As used herein, the term "candidate cell" generally refers to a cell identified as a midbrain dopaminergic neural progenitor cell. A "candidate cell" can be a neural progenitor cell.

[0088] As used herein, the term "neural progenitor cell" generally refers to an undifferentiated progenitor cell that has not yet exhibited terminal differentiation characteristics and is capable of proliferating and / or differentiating into a mature neuronal cell. As used herein, "progenitor cell," "precursor," and "precursor cell" are used interchangeably. Depending on the neurotransmitters of the differentiated neurons, neural progenitor cells may include cholinergic progenitor cells, adrenergic progenitor cells, GABAergic progenitor cells, glutamatergic progenitor cells, and dopaminergic progenitor cells, serotonergic progenitor cells, and / or purinergic progenitor cells. Depending on the synchronization of the differentiation zones, neural precursor cells may include midbrain ventral floor plate neural progenitor cells, hindbrain floor plate neural progenitor cells, and / or midbrain floor plate neural progenitor cells.

[0089] As used herein, the term "modification" generally refers to labeling candidate cells, for example, using a marker molecule. The modification can be at the gene level, RNA level, or protein level of the candidate cell. For example, the modification can refer to inserting a reporter gene into an expression cassette for a molecular marker gene.

[0090] As used herein, the term "Parkinson's disease (PD)" generally refers to a disease associated with a dopamine deficiency in the basal ganglia (the part of the brain that controls movement). Symptoms include tremors, bradykinesia (extremely slow movements), flexion, postural instability, and rigidity. A diagnosis of Parkinson's disease requires the presence of at least two of these symptoms, one of which must be tremors or bradykinesia. Parkinson's disease includes idiopathic or typical Parkinson's disease and Parkinson's plus syndrome (atypical Parkinson's disease). In general, Parkinson's disease involves dysfunction and death of important nerve cells, primarily in an area of ​​the brain called the substantia nigra. Many of these important nerve cells produce dopamine, and when these neurons die, the amount of dopamine in the brain decreases, making it impossible to control movement normally. The symptom clusters experienced by individuals vary from person to person. The main motor symptoms of Parkinson's disease include the following: tremors in the hands, arms, legs, jaw, and face, bradykinesia or slow movements, stiffness or rigidity of the limbs and trunk, and postural instability or impaired balance and coordination. The rate of progression of Parkinson's disease can be quantified by the President-Parkinson's Disease Rating Scale (Total UPDRS) score.

[0091] As used herein, the term "midbrain" refers to the region between the forebrain (front) and hindbrain (back) in the vertebrate brain. The midbrain region produces many brain regions, including but not limited to the reticular formation (which is part of the tegmentum), the area of ​​the brainstem that affects motor function, the cerebral peduncle (cms cerebri) (which consists of nerve fibers connecting the cerebral hemispheres to the cerebellum) and the so-called cerebrum and the large pigmented nucleus of the substantia nigra. A unique feature of the developing ventral midbrain is the co-expression of floor plate markers FOXA2 and LMX1A.

[0092] As used herein, the term "dopamine neuron" or "dopaminergic neuron" generally refers to a cell that contains and is capable of releasing dopamine. "Midbrain dopamine neuron" or "mDA" refers to a neuronal cell in the midbrain structure that contains and releases dopamine.

[0093] As used herein, the terms "separation," "sorting," and "screening" are used interchangeably when applied to cells, and generally refer to separating a subpopulation of cells from a mixture of cells based on the properties of the specific subpopulation of cells rather than the rest of the cells in the mixture. For example, in the present application, separation refers to separating a group of cells having one or more of the following characteristics: "APCDD1" and / or "TPBG" from a cell population. The cell population to be purified typically includes other cells that do not have the above characteristics in addition to cells having the above characteristics. For example, the cell population can be heterogeneous neural precursor cells, or undifferentiated neural precursor cells (e.g., pluripotent stem cells), or fully or partially differentiated neural precursor cells. Common cell separation methods may include methods based on antibody-antigen recognition properties and / or methods based on physical properties of cells. For example, separation methods may include flow cytometry sorting, immunomagnetic cell sorting, and / or density gradient centrifugation.

[0094] As used herein, the term "enrichment" generally refers to increasing the proportion of cells in a cell population that share certain characteristics. Enrichment can be achieved by separating cells that share the common characteristics from cells that do not share the common characteristics.

[0095] method

[0096] In one aspect, the present application provides a method for identifying mDA (midbrain dopaminergic) neural progenitor cells, the method comprising determining whether a candidate cell is APCDD1+ (adenomatous polyposis coli downregulated 1), and identifying the APCDD1+ cell as an mDA neural progenitor cell.

[0097] In another aspect, the present application provides a method for identifying mDA neural progenitor cells, the method comprising determining whether a candidate cell is APCDD1+ and TPBG+ (trophoblast glycoprotein), and identifying the APCDD1+ and TPBG+ cells as mDA neural progenitor cells.

[0098] In the present application, the candidate cells may be derived from pluripotent stem cells, such as human pluripotent stem cells, or embryonic stem cells. For example, induced pluripotent stem cells may be derived from autologous or allogeneic cells.

[0099] In the present application, candidate cells can be a neural progenitor cell colony of appropriate differentiation. For example, the neural progenitor cell colony can be heterogeneous. Heterogeneous neural progenitor cell colony refers to a cell colony comprising two or more neural progenitor cell types, or even neuroblasts and / or neurons. In the present application, candidate cells can be differentiated into neural progenitor cells from pluripotent stem cells (for example, human pluripotent stem cells) in vitro. In the present application, pluripotent stem cells can be human embryonic stem cells and / or human induced pluripotent stem cells. In the present application, cells to be differentiated (for example, pluripotent stem cells) can be derived from rodent cells, primate cells, human cells. For example, cells to be differentiated (for example, pluripotent stem cells) can be derived from cells of healthy people or patients with Parkinson's disease symptoms or patients suffering from Parkinson's disease.

[0100] Methods for differentiating neural progenitor cells from stem cells or other types of cells are known, for example, by using small molecules, growth factor proteins and other growth conditions to promote the transition of cells from a pluripotent state to a more mature or specialized state. Depending on the different differentiation conditions and differentiation methods, the differentiation time of cells in a pluripotent state can be at least about 10 days, about 12 days, about 13 days, about 14 days, 15 days, 18 days, 21 days, 25 days, 28 days, 30 days, 35 days or longer, such as 14 days or 21 days. Those skilled in the art can determine whether neural progenitor cells differentiated from pluripotent stem cells can be used to evaluate whether they have one or both of the following characteristics under different differentiation conditions and methods: APCDD1+ and / or TPBG+.

[0101] In another aspect, the present application provides a method for isolating mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) isolating APCDD1+ cells from the candidate cell population.

[0102] In another aspect, the present application provides a method for isolating mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) isolating APCDD1+ and TPBG+ cells from the candidate cell population.

[0103] In another aspect, the present application provides a method for enriching mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) enriching APCDD1+ cells in the candidate cell population.

[0104] In another aspect, the present application provides a method for enriching mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) enriching the candidate cell population for APCDD1+ and TPBG+ cells.

[0105] In the present application, "identification", "separation" or "enrichment" may include the use of molecular markers that can specifically recognize and / or bind to reagents (e.g., APCDD1 and / or TPBG). The reagents can be proteins, such as antibodies or antigen-binding fragments thereof, affinity ligands, which can be labeled with fluorescein (for fluorescence-activated cell sorting) or used with magnetic beads (for magnetic-activated cell sorting). When the cell population to be separated is mixed with the reagent, cells with one or more of the above characteristics are specifically bound by the reagent and change in some characteristics (e.g., molecular weight, polarity, charge, fluorescence wavelength, etc.), thereby distinguishing them from cells that do not have the above characteristics.

[0106] The neural progenitor cells identified, isolated or enriched in the present application can be further differentiated into neurons, and the neural cells can contain at least 10% or more (e.g., 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more) of midbrain dopaminergic neurons.

[0107] In another aspect, the present application provides a method for preparing a cell product, comprising (a) providing neural progenitor cells, and (b) isolating and / or enriching APCDD1+ neural progenitor cells.

[0108] In another aspect, the present application provides a method for preparing a cell product, comprising (a) providing neural progenitor cells, and (b) isolating and / or enriching APCDD1+ and TPBG+ neural progenitor cells.

[0109] In the present application, the cell products prepared by the methods described in the present application include neural precursor cells having one or more of the following characteristics: APCDD1+ and / or TPBG+. When the cell products are further differentiated into neurons in vivo or in vitro, the neurons may contain at least 10% or more (e.g., 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more) of midbrain dopaminergic neurons. After differentiation in vivo (transplantation) or in vitro, the cell products of the present application have a clear and stable cell composition.

[0110] In another aspect, the present application provides a method for evaluating a cell product, comprising testing the proportion of APCDD1+ cells in the cell product.

[0111] In another aspect, the present application provides a method for evaluating a cell product, comprising testing the ratio of APCDD1+ and TPBG+ cells in the cell product.

[0112] The quality of the cell product can be assessed based on the proportion of cells having one or more of the above characteristics (e.g., 1%-10%, 10%-20% or more than 20%), thereby predicting the content of the cell product after transplantation, the therapeutic effect, or the dosage and administration frequency of the cell product. Generally, when the proportion of cells having one or more of the above characteristics in the cell product is high (e.g., greater than 10%), the administration dose or frequency of the cell product can be reduced. Generally, when the proportion of cells having one or more of the above characteristics in the cell product is low (e.g., less than 10%), the administration dose or frequency of the cell product can be increased.

[0113] In another aspect, the present application provides a method for optimizing a process for preparing a cell product, the method comprising assessing the proportion of APCDD1+ cells in the cell product.

[0114] In another aspect, the present application provides a method for optimizing a process for preparing a cell product, the method comprising assessing the ratio of APCDD1+ and TPBG+ cells in the cell product.

[0115] For example, when the proportion of cells in the cell product having one or more of the above characteristics is low (e.g., less than 10%), it can be considered that the manufacturing process of the cell product needs to be optimized. The manufacturing process may include the production, differentiation, separation and / or purification of the cell product.

[0116] Molecular markers

[0117] For each molecular marker, different detection methods can be used to determine whether the molecular marker is positive, depending on its expression characteristics and activity characteristics, such as whether it is expressed on the cell surface or intracellularly, and whether the protein product is membrane-bound or free. Detection methods include, but are not limited to, immunohistochemistry, PCR, RT-PCR, in situ hybridization, Southern blotting, protein blotting, Northern blotting, spectrophotometry, gene chip, flow cytometry (FACS), protein chip, DNA / RNA sequencing, and ELISA.

[0118] For the provided cells or cell populations, the methods described herein include determining whether the cells (e.g., candidate cells) have the following characteristics: APCDD1+. For example, the method includes detecting the level of APCDD1 protein, the activity of APCDD1 protein and / or the expression level of APCDD1 nucleic acid in the candidate cells. In some cases, the method may include using primers that can amplify nucleic acid molecules encoding APCDD1. The primers can be a pair of primers. In addition, the method may include using probes that can specifically identify nucleic acid molecules encoding APCDD1. The probe may be able to bind to APCDD1 nucleotide sequences or fragments thereof, but not to nucleotide sequences of other genes. The probe may have a detectable signal. In other cases, the method may include using reagents that can specifically recognize APCDD1 protein and / or reagents that can assess the activity of APCDD1 protein, such as antibodies and / or ligands directed to APCDD1 protein and / or fragments thereof.

[0119] For provided cells or cell populations, the methods described herein include determining whether a cell (e.g., a candidate cell) has the following characteristics: APCDD1+ and TPBG+. For example, the method includes detecting the level of APCDD1 and TPBG proteins, the activity level of APCDD1 and TPBG proteins, the expression level of APCDD1 and TPBG nucleic acids, and / or the activity level of APCDD1 and TPBG nucleic acids in the candidate cells. In some cases, the method may include using primers that can specifically amplify nucleic acid molecules encoding APCDD1 and TPBG. The primers can be a pair of primers. In addition, the method may include using probes that can specifically recognize nucleic acid molecules encoding APCDD1 and TPBG. The probe may be able to bind to APCDD1 and TPBG nucleotide sequences or fragments thereof, but not to another nucleotide sequence. The probe may have a detectable signal. In other cases, the method may include using reagents that can specifically recognize APCDD1 and TPBG proteins and / or reagents that can measure the activity of APCDD1 and TPBG proteins, such as antibodies and / or ligands against APCDD1 and TPBG proteins and / or fragments thereof.

[0120] In the present application, the expression of the molecular marker (APCDD1 and / or TPBG) may include the expression amount of the molecular marker in the cell (e.g., candidate cell) and / or the ratio of the number of cells that are positive for the molecular marker to the number of cells in the total cell population. In certain embodiments, under different detection methods, when the content of the molecular marker in the cell is detected to be higher than the detection limit or threshold, the cell can be considered to be positive for the molecular marker. Alternatively, in a cell population, when the ratio of the number of cells that are positive for the molecular marker to the number of cells in the total cell population is detected, it can be used to infer the proportion of dopaminergic neural precursor cells in the cell population, and it can also be used to infer the proportion of terminal dopaminergic neurons produced by differentiation (in vivo and in vitro).

[0121] In another aspect, the present application provides the use of an APCDD1+ indicator and / or a TPBG+ indicator for preparing a cell product, wherein the cell product comprises midbrain dopaminergic neural progenitor cells. The indicator can be used to indicate or detect the level and / or activity of a molecular marker. In the present application, the indicator may include a protein, a nucleic acid and / or a small molecule. For example, the indicator may include a reagent that can specifically bind to a molecular marker protein and / or a reagent that can measure the activity of a molecular marker protein. As another example, the indicator may include a primer that can specifically initiate a polymerization reaction to amplify a nucleic acid molecule encoding a molecular marker and / or a probe that can specifically identify a nucleic acid molecule encoding a molecular marker. For example, the indicator may be an antibody or its antigen-binding protein / compound that can specifically bind to APCDD1 or a combination of APCDD1 and TPBG.

[0122] In another aspect, the present application provides a kit comprising an APCDD1+ indicator, wherein the kit is used to examine the proportion of mDA neural progenitor cells in a cell product.

[0123] In some cases, the kit may further include a reagent capable of culturing and / or preserving the candidate cells. The reagent may be a cell culture medium, such as a neural progenitor cell culture medium and / or a neural progenitor cell differentiation medium. In the kit, the reagent capable of culturing and / or preserving the candidate cells may be packaged separately from the quality control reagent.

[0124] In some cases, the kit may further include a candidate cell as described herein, which may be, for example, a neural progenitor cell (eg, a neural progenitor cell derived from a human pluripotent stem cell).

[0125] Cell products

[0126] In another aspect, the present application provides a population of mDA neural progenitor cells obtained by the methods of the present application. In some cases, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the population of mDA neural progenitor cells express one or more of the markers: APCDD1 and / or TPBG.

[0127] In some cases, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in a population of midbrain dopaminergic neural precursor cells express APCDD1.

[0128] In some cases, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in a population of midbrain dopaminergic neural precursor cells express APCDD1 and TPBG.

[0129] In another aspect, the present application provides a cell product comprising mDA neural progenitor cells obtained by the method of the present application.

[0130] In another aspect, the present application provides a cell product comprising APCDD1+ neural progenitor cells.

[0131] In another aspect, the present application provides a cell product comprising APCDD1+ and TPBG+ neural progenitor cells.

[0132] In another aspect, the present application provides a cell preparation obtained by further expansion and proliferation of the cell product of the present application.

[0133] In some cases, the cell preparation may also include a pharmaceutically acceptable adjuvant.

[0134] Medical uses

[0135] In another aspect, the present application provides the use of the cell products described in the present application in screening for the prevention, treatment or alleviation of a drug for a nervous system disease or condition. For example, a nervous system disease or condition may include a disease or condition associated with neuronal degeneration (e.g., Parkinson's disease). In the present application, the cell product can be differentiated into midbrain dopaminergic neurons, which can then be contacted with a drug to be screened. If the drug to be screened has one or more of the following properties: (1) it can prevent the death of midbrain dopaminergic neurons, (2) it can promote the survival of midbrain dopaminergic neurons, and (3) it can improve the metabolism of elements in midbrain dopaminergic neurons, the drug to be screened is selected as a drug that can prevent, treat or alleviate a nervous system disease or condition.

[0136] In another aspect, the present application provides use of the cell product of the present application in screening drugs, wherein the drugs are used to prevent and / or treat nervous system diseases or disorders.

[0137] In another aspect, the present application provides a method for preventing, treating, or alleviating a neurological disease or condition. The method comprises the following steps: identifying whether candidate cells have one or more of the following characteristics: APCDD1+ and TPBG+; selecting cells having the characteristics; and administering an effective dose of the cells having the characteristics to a subject in need.

[0138] In another aspect, the present application provides pharmaceutical compositions comprising neural precursor cells having one or more of the following characteristics: APCDD1+ and TPBG+. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant. In certain embodiments, the cells are capable of differentiating into neural cells, wherein the neural cells comprise at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) mDA neurons. In certain embodiments, differentiation comprises both in vitro and in vivo differentiation.

[0139] Depending on the intended method, the dosage of the pharmaceutical composition, the formulation of the pharmaceutical preparation, the administration route, and the administration time and interval may vary according to the subject's condition, weight, and degree of disease, and can be appropriately selected by those skilled in the art.

[0140] The pharmaceutical composition or cell product of the present application is administered in a pharmaceutically effective dose. A "pharmaceutically effective dose" refers to an amount sufficient to treat a disease and having a reasonable benefit / risk ratio suitable for medical treatment or improvement, and the effective dose level can be determined based on factors including the following categories: the subject's disease, disease severity, age and sex, drug activity, subject's sensitivity to the drug, administration time, administration route, discharge rate, duration of treatment, and concomitant drug use, as well as other factors well-known in the medical field. The term "subject" generally refers to a subject in need of treatment, and more specifically refers to a mammal, such as a human or non-human primate, mouse, rat, dog, cat, horse and / or cattle.

[0141] In the present application, the methods or products of the present application can be used to treat diseases or conditions of the nervous system. Nervous system diseases or conditions may include degenerative diseases. Degenerative diseases are diseases in which a specific cell type (e.g., neurons) degenerates (e.g., functionally, structurally, biochemically), leading to adverse clinical conditions. For example, Parkinson's disease is a degenerative disease of the basal ganglia in the central nervous system.

[0142] The cell product of the present application and / or pharmaceutical composition can be for example transplanted or placed in the central nervous system (for example, brain or spinal cord) or peripheral nervous system.The implantation position of cell product and / or pharmaceutical composition in nervous system is determined based on specific nervous system disease, for example, is directly injected into striatum, spinal cord substance or dorsal ganglion.For example, the cell product of the present application and / or pharmaceutical composition can be transplanted in or near the striatum of the experimenter suffering from Parkinson's disease.Depending on the position of nervous system disease and the medical condition of the patient, those skilled in the art will be able to determine the most appropriate mode of transplanted cells.The cell product of the present application and / or pharmaceutical composition can be used together with other therapies to treat nervous system disease or disease.

[0143] Example

[0144] The following examples are set forth so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (such as amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acid; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; etc.

[0145] Materials and methods

[0146] Human ventral midbrain-hindbrain differentiation based on hESCs

[0147] The hESC neural differentiation protocol to mimic human ventral midbrain-hindbrain embryonic development was based on previously described methods with some modifications (Chen, Y., Xiong, M., Dong, Y., Haberman, A., Cao, J., Liu, H., Zhou, W., and Zhang, SC (2016). Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson's Disease. Cell Stem Cell 18, 817–826.; Xiong, M., Tao, Y., Gao, Q., Feng, B., Yan, W., Zhou, Y., Kotsonis, T. A., Yuan, T., You, Z., Wu, Z. et al. (2021). Human Stem Cell-Derived NeuronsRepair Circuits and Restore Neural Function. Cell Stem Cell 28, 112-126.e6; Xu, P., Xiong, M., and Chen, Y. (2022a). Human midbrain dopaminergic neuronal differentiation markers predict cell therapy outcome in a Parkinson's disease model.). One day after passaging, the culture medium of hESCs was changed to neural induction medium (NIM) containing DMEM / F12, 1× N2 supplement (Gibco), and 1× NEAA, and supplemented with SHH (C25II, R&D Systems, 500 ng / ml), CHIR99021 (Tocris, 0.4 μM), DMH-1 (Tocris, 2 μM), and SB431542 (Stemgent, 2 μM) for 8 days (days 1 to 9). On day 9, single colonies were gently blown off and transferred to 6-well dishes coated with fresh MEFs in NIM and supplemented with SHH (100 ng / ml), SAG (Millipore, 1 μM), and CHIR99021 (0.4 μM) for 4 days until stage I (day 13).In stage I, single colonies were gently blown off and transferred to non-adherent 25 ml culture dishes cultured in suspension medium containing NIM supplemented with SHH (20 ng / ml), SAG (Millipore, 0.5 μM), and FGF8b (PeproTech, 100 ng / ml) for 8 days until stage II (day 21). From stage II to stage III (day 30), neurospheres were allowed to continue differentiation and proliferation in suspension medium containing NIM supplemented with SHH (20 ng / ml) and FGF8b (20 ng / ml). In stage III, neurospheres were dissociated with Accutase (Innovative Cell Technologies) at 37°C for 6 minutes and then re-plated onto 24-well culture dishes coated with Matrigel (BD Biosciences). From stage III to stage IV (day 45), differentiated cells were fed with neural differentiation medium (NDM), which contained neural basal medium, 1× N2 supplement (Gibco), and 1× B27 (Life Technologies) supplemented with brain-derived neurotrophic factor (BDNF, Peprotech, 10 ng / ml), glial-derived neurotrophic factor (GDNF, Peprotech, 10 ng / ml), transforming growth factor 3 (TGFβ3, R&D Systems, 1 ng / ml), ascorbic acid (AA, Sigma-Aldrich, 200 μM), cAMP (Sigma-Aldrich, 1 μM), and compound E (Calbiochem, 1 μM). In addition, to improve cell survival during passage, vitamin A-free B-27 supplement (Life Technologies) and Rho-kinase (ROCK) inhibitor (Tocris, 0.5 mM) were added to the culture medium.

[0148] Donor plasmid construction

[0149] To generate the APCDD1-tdtomato donor plasmid, a DNA fragment with left or right homology arms was amplified by PCR from H9 hESC genomic DNA immediately upstream or downstream of the stop codon of the APCDD1 gene (with certain mutations on the right homology arm to avoid self-targeting of the sgRNA on the donor plasmid). A DNA fragment of the P2A-tdTomato gene was amplified by PCR from pAAV-FLEX-ArchT-tdTomato (plasmid #28305) (Han et al., 2011). The DNA fragments with left and right homology arms of APCDD1 and P2A-tdtomato were then cloned into the multiple cloning site of plasmid pL552 to obtain the APCDD1-tdtomato donor plasmid.

[0150] Generation of hESC reporter lines

[0151] After pretreatment with ROCK inhibitor (0.5 mM) for 24 hours, H9 hESCs were digested with TrypLETM Express enzyme at 37°C for 6 minutes and dissociated into single cells. The single cells were then resuspended in 500 ul of electroporation buffer (5 mM MgCl2, 5 mM KCl, 102.94 mM Na2HPO4, 47.06 mM NaH2PO4, and 15 mM HEPES, pH = 7.2) and supplemented with Cas9 plasmid, donor plasmid, and sgRNA. Electroporation was performed using the GenePulser Xcell system (Bio-Rad) in a 4 mm cuvette (Phenix Research Products) at 250 V and 500 mF. The cells were then immediately plated in a 6-well culture dish coated with fresh MEFs and cultured in MEF-conditioned ESC medium containing ROCK inhibitor (0.5 mM, 24 hours). After 3 days, cells were treated with G418 (50-100 μg / ml) or puromycin (0.5 μg / ml) for two weeks for selection. Subsequently, cells were treated with ROCK inhibitor for 24 hours and then individually picked for further proliferation. Clones were then genotyped to check for transgene integration.

[0152] To generate APCDD1-tdtomato, a cassette including the tdtomato sequence linked to a P2A peptide followed by loxP-flanked PGK-Pur was inserted immediately upstream of the stop codon of the endogenous APCDD1 gene in H9 hESCs by wild-type Cas9.

[0153] Example 1 Discovery of novel molecular markers for mDA progenitor cells

[0154] The molecular basis of mDA lineage fate determinants was investigated. DEGs were tested and genes enriched in mDA lineage fate-committed progenitors, including stage I II-NPs, were identified. vMB Fate-committed progenitor cells, mediating II-III-NP vMB Fate-committed progenitor cells and IV-mDA-mediated fate-committed progenitor cells ( Figure 1A These genes include some known markers such as EN1, OTX2, and LMX1A, as well as other genes not yet reported in ventral midbrain development, suggesting a potential role for these genes in mDA lineage specification ( Figure 1A ).

[0155] Metascape analysis of these DEGs revealed biological processes enriched in these mDA lineage-fate-committed progenitors ( Figure 2B-2D As expected, biological processes involved in dopaminergic neuronal differentiation were enriched in all of these fate-committed progenitors. Tyrosine kinase signaling, WNT signaling, or synapse assembly-related processes were enriched in early or late mDA lineage fate-committed progenitors, respectively ( Figure 2B-2D Notably, two surface markers, APCDD1 (APC downregulated 1) and TPBG (trophoblast glycoprotein), were identified as being expressed in stage I II-NPs. vMB Fate-committed progenitor cells and II-NP vMB Cluster-specific expression (Figure 6A and Figure 3AAPCDD1, also known as adenomatous polyposis coli downregulated protein, is a negative regulator of the Wnt signaling pathway. TPBG is an inhibitor of Wnt / β-catenin signaling and has been reported as a cell surface marker for ventral midbrain progenitor cells. Transplantation of progenitor cells from late differentiation stages (post-stage III) has been used to treat PD mouse models (Chen, Y., Xiong, M., Dong, Y., Haberman, A., Cao, J., Liu, H., Zhou, W., and Zhang, SC (2016). Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson's Disease. Cell Stem Cell 18, 817–826.; Xiong, M., Tao, Y., Gao, Q., Feng, B., Yan, W., Zhou, Y., Kotsonis, TA, Yuan, T., You, Z., Wu, Z. et al. (2021). Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural Function. Cell Stem Cell 28, 112-126.e6.). The finding that the mDA lineage may be fate-committed as early as stage I suggests that early progenitor cells sorted by APCDD1 could give rise to mDA neuron-enriched engraftments following transplantation. Importantly, the tripotentiality of ventral midbrain progenitors suggests that even when we transplant highly purified ventral midbrain progenitors, glutamatergic neurons and VLMCs will be present in the grafts alongside mDA neurons. To test this hypothesis, an APCDD1-tdTomato reporter hESC line was generated, allowing expression of a fluorescent protein to report endogenous APCDD1 ( Figure 3B-3E ). Stage I Tdtomato+ cells were sorted, reaggregated into neurospheres, and then matured in vitro ( Figure 1B ).

[0156] TH+ mDA neurons were highly enriched in neurospheres from the in vitro APCDD1 sorted group (APCDD1, sorted: 60% ± 2%, unsorted: 24% ± 1%, P < 0.001, unpaired t-test) ( Figure 1C and Figure 1D ). Marker-sorted or unsorted progenitor cells were transplanted into the striatum of PD mouse models ( Figure 1BImmunohistochemistry revealed that four months after transplantation, the proportion of TH+ mDA neurons in marker-sorted grafts was significantly higher than that in unsorted grafts (APCDD1, sorted: 49% ± 4%; unsorted: 15% ± 2%; P < 0.001, unpaired t-test) ( Figure 1E and Figure 1F As expected, in addition to mDA neurons, VLMCs (COL1A1+) and glutamatergic neurons (TH- / VGLUT2+) were detected in APCDD1-sorted transplants ( Figure 1G-1J ), which is consistent with the tripotency of ventral midbrain progenitors inferred by clonal lineage analysis.

[0157] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not limited by the specific embodiments provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not intended to be interpreted in a limiting sense. Those skilled in the art will now appreciate that many variations, changes, and replacements do not depart from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration, or relative proportions described herein that depend on various conditions and variables. It should be understood that, in practicing the present invention, various alternatives to the embodiments of the present invention described herein may be adopted. Therefore, it is contemplated that the present invention will also encompass any such alternatives, modifications, variations, or equivalents. This means that the appended claims define the scope of the present invention and that methods and structures within the scope of these claims and their equivalents are therefore encompassed.

Claims

1. A method for identifying mDA (midbrain dopaminergic) neural progenitor cells, the method comprising determining whether a candidate cell is APCDD1+ (adenomatous polyposis coli downregulated protein), and identifying the APCDD1+ cell as an mDA neural progenitor cell.

2. A method for identifying mDA neural progenitor cells, the method comprising determining whether a candidate cell is APCDD1+ and TPBG+ (trophoblast glycoprotein), and identifying the APCDD1+ and TPBG+ cell as an mDA neural progenitor cell. The method according to claim 1 , wherein the candidate cells are neural progenitor cells. The method according to claim 1 , wherein the candidate cells are derived from pluripotent stem cells. The method according to claim 1 , wherein the candidate cells are derived from human pluripotent stem cells. 6 . The method according to claim 1 , comprising directly or indirectly detecting the expression and / or activity of APCDD1 in the candidate cells.

7. The method according to any one of claims 1 to 6, comprising directly or indirectly detecting the expression and / or activity of TPBG in the candidate cells.

8. The method according to any one of claims 1 to 7, wherein the expression and / or activity of APCDD1 comprises the expression and / or activity of a nucleic acid encoding APCDD1, and / or the presence and / or activity of an APCDD1 protein.

9. The method according to any one of claims 1 to 8, wherein the expression and / or activity of TPBG comprises the expression and / or activity of a nucleic acid encoding TPBG, and / or the presence and / or activity of a TPBG protein.

10. The method of any one of claims 1-9, wherein the detecting comprises modifying the candidate cell.

11. The method of any one of claims 1 to 10, wherein the detecting comprises using a marker.

12. The method according to claim 11, wherein the marker comprises a protein, a nucleic acid and / or a small molecule compound.

13. The method of any one of claims 11-12, wherein the marker comprises a fluorescent reporter gene. 14 . The method according to claim 1 , comprising contacting the candidate cell with an agent capable of specifically binding to APCDD1 protein and / or capable of testing the activity of the APCDD1 protein. 15 . The method according to claim 1 , comprising contacting the candidate cell with a primer capable of specifically amplifying a nucleic acid molecule encoding APCDD1 and / or a probe capable of specifically recognizing a nucleic acid molecule encoding APCDD1. 16 . The method according to claim 1 , comprising contacting the candidate cell with an agent capable of specifically binding to a TPBG protein and / or capable of testing the activity of the TPBG protein.

17. The method according to any one of claims 1 to 16, comprising contacting the candidate cell with primers capable of specifically amplifying a nucleic acid molecule encoding TPBG and / or a probe capable of specifically recognizing a nucleic acid molecule encoding TPBG.

18. A method for isolating mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) isolating APCDD1+ cells of the candidate cell population.

19. A method for isolating mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) isolating APCDD1+ and TPBG+ cells of the candidate cell population.

20. The method of any one of claims 18-19, wherein the candidate cell is a neural progenitor cell.

21. The method of any one of claims 18-20, wherein the mDA neural progenitor cells are mDA neurons.

22. A method for enriching mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) enriching the candidate cell population for APCDD1+ cells.

23. A method for enriching mDA neural progenitor cells, the method comprising (a) providing a candidate cell population, and (b) enriching the candidate cell population for APCDD1+ and TPBG+ cells.

24. The method of any one of claims 22-23, wherein the candidate cell is a neural progenitor cell.

25. The method of any one of claims 22-24, wherein the candidate cell is an mDA neuron. 26 . An mDA neural progenitor cell population, obtained by the method of any one of claims 1 to 25 .

27. A cell product comprising mDA neural progenitor cells obtained by the method of any one of claims 1-25.

28. A cell product comprising APCDD1+ neural progenitor cells.

29. A cell product comprising APCDD1+ and TPBG+ neural progenitor cells.

30. A method for preparing a cell product, the method comprising (a) providing neural progenitor cells, and (b) isolating and / or enriching APCDD1+ neural progenitor cells.

31. A method for preparing a cell product, the method comprising (a) providing neural progenitor cells, and (b) isolating and / or enriching APCDD1+ and TPBG+ neural progenitor cells.

32. The method according to any one of claims 30-31, comprising obtaining the neural progenitor cells by differentiation of a cell population.

33. The method of any one of claims 30-32, wherein the cell population is derived from rodent cells, non-human primate cells, or human cells.

34. The method of any one of claims 30-33, wherein the cell population is derived from pluripotent stem cells.

35. The method of any one of claims 30-34, wherein the cell population is derived from human pluripotent stem cells.

36. The method of any one of claims 30-35, wherein the differentiation comprises in vivo or in vitro differentiation.

37. A method for evaluating a cell product, the method comprising testing the cell product for the proportion of APCDD1+ cells.

38. A method for evaluating a cell product, the method comprising testing the cell product for the ratio of APCDD1+ and TPBG+ cells.

39. A method for optimizing a process for preparing a cell product, the method comprising testing the cell product for the proportion of APCDD1+ cells.

40. A method for optimizing a process for preparing a cell product, the method comprising testing the cell product for the ratio of APCDD1+ and TPBG+ cells.

41. The method of any one of claims 30-36, wherein the process for preparing a cell product comprises optimizing a process for producing, differentiating, isolating, and / or purifying the cell product.

42. A cell preparation, obtained by further expansion and proliferation of the cell product according to any one of claims 27 to 29.

43. A kit comprising an APCDD1+ indicator, wherein the kit is used to examine the proportion of mDA neural progenitor cells in a cell product.

44. Use of an APCDD1+ indicator for preparing a cell product, wherein the cell product comprises mDA neural progenitor cells.

45. Use of the cell product according to any one of claims 27 to 29 in screening drugs, wherein the drugs are used to prevent and / or treat diseases or disorders of the nervous system.

46. ​​A pharmaceutical composition comprising APCDD1+ neural progenitor cells.

47. The pharmaceutical composition of claim 46, wherein the neural progenitor cells are capable of differentiating into neural cells, wherein the neural cells contain at least 30% mDA neural cells.

48. The pharmaceutical composition of claim 47, wherein the differentiation comprises in vivo or in vitro differentiation.

49. The pharmaceutical composition of any one of claims 46-48, comprising a pharmaceutically acceptable carrier.

50. A method for preventing and / or treating a neurological disease or disorder, the method comprising administering the cell product of any one of claims 27-29 and / or the pharmaceutical composition of any one of claims 46-49 to a subject in need thereof.

51. The method of claim 50, wherein the neurological disease or disorder comprises Parkinson's disease.

52. The cell product according to any one of claims 27 to 29, and / or the pharmaceutical composition according to any one of claims 46 to 49, for use in preventing and / or treating a disease or disorder of the nervous system.

53. The cell product of any one of claims 27-29, and / or the pharmaceutical composition of any one of claims 46-49, wherein the neurological disease or disorder comprises Parkinson's disease.

54. Use of the cell product and / or pharmaceutical composition according to any one of claims 27 to 29 in the preparation of a medicament, wherein the medicament is used to prevent and / or treat a disease or disorder of the nervous system.

55. The use according to claim 54, wherein the neurological disease or disorder comprises Parkinson's disease.

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