Biological methods

Abstract

The present invention relates to a method for identifying one or more caudal ventral midbrain (cVM) progenitor cells by measuring the presence and / or amount of adenomatosis polyposis coli down-regulated 1 (APCDD1) expressed by one or more cells. The present invention also provides a method for enriching cVM cells, the use of said cells in the treatment of disease, the use of APCDD1 as a biomarker, and a kit.

Description

[Technical Field]

[0001] The present invention relates to a method for identifying one or more caudal ventral midbrain (cVM) progenitor cells by measuring the presence and / or amount of adenomatosis polyposis coli down-regulated 1 (APCDD1) expressed by one or more cells. The present invention also provides a method for enriching cVM cells, the use of said cells in the treatment of disease, the use of APCDD1 as a biomarker, and a kit. [Background technology]

[0002] Parkinson's disease (PD) is a common neurodegenerative motor disorder. Levodopa is the most common treatment strategy for improving the motor symptoms of this disease, but it is associated with serious complications, and its effectiveness is variable and progressively decreases (Connolly and Lang 2014). Since the motor symptoms of PD may be due to the relatively selective and localized loss of dopaminergic (DA) neurons in the ventral midbrain (VM), cell replacement therapy is a promising alternative treatment strategy to levodopa.

[0003] The feasibility of cell replacement therapy has been demonstrated by using fetal VM-derived cells (Barker et al. 2019; Bjorklund et al. 1980; Bolam et al. 1987; Dunnet et al. 1983; Freund et al. 1985; Strecker et al. 1987). However, due to tissue-related issues, the focus has recently shifted to using human pluripotent stem cells (hPSCs) as a nearly inexhaustible source of VM DA cells (Kefalopoulou et al. 2014; Kurowska et al. 2011; JYLi et al. 2008; W.Li et al. 2016). The cells are transplanted as precursors that mature in the host brain and replace the functions of cells that have been lost endogenously.

[0004] hPSCs are a promising source of caudal ventral midbrain (cVM) progenitor cells used in cell replacement therapy for Parkinson's disease. However, some heterogeneity in cell composition is inevitable during the cell differentiation process. Cell surface markers that predict the functional maturation of cVM precursors into ventral midbrain (VM) dopaminergic (DA) neurons can be used as a convenient quality control to enrich target cell types. However, to date, the use of such markers has not been successful in providing genuine cVM precursor markers.

[0005] To obtain safe, effective, and reproducible results, a pure population of cells must be transplanted. Genes indicating VM DA precursors, such as the transcription factors LMX1A, FOXA2, and OTX2, are commonly used as surrogate markers for functional maturation (Arenas, Denham, and Netherlandsescusa 2015). These markers are incorporated into intracellular-based flow cytometry quality control (QC) panels to assess the purity of differentiated cells. However, only LMX1A / FOXA2 / OTX2 triple-positive precursors derived from caudal VM (cVM) give rise to VM DA neurons, while triple-positive cells from the rostral adjacent subthalamic nucleus (STN), then called rostral VM (rVM), produce glutamatergic neurons (Nolbrant et al. 2017). Current flow cytometry-based QC assays using LMX1A / FOXA2 / OTX2 cannot distinguish between the fate of cVM and rVM. In fact, the expression levels of FOXA2, OTX2, and LMX1A cannot predict graft outcomes at the time of transplantation (Kirkeby et al. 2017).

[0006] There are several publicly available putative surface markers that predict VMDA neurons (Bye et al. 2015; Doi et al. 2014; Fathi et al. 2018; Gennet et al. 2016; Kikuchi et al. 2017; Lehnen et al. 2017; Paik et al. 2018; Samata et al. 2016; Sundberg et al. 2013; Yoo et al. 2021), some of which improve behavioral impairment in animal models of Parkinson's disease (Doi et al. 2014; Kikuchi et al. 2017; Lehnen et al. 2017; Sundberg et al. 2013; Yoo et al. 2021). Apart from the surface marker CORIN, most of these are not broadly characterized, and their region specificity has not been sufficiently studied.

[0007] While some of these studies have focused on enriching LMX1A+ and FOXA2+ cells from mixed initiation progenitor populations, few have focused on evaluating the specificity of the markers against various other localized neural progenitor cell populations to investigate the specificity of the markers against potential contaminating populations of other neural fates. In addition, there has been little focus on evaluating the specificity of published markers against true DA progenitor cells of cVM that are LMX1A+FOXA2+EN1+ against adjacent non-DA rVM progenitor cells that are LMX1A+FOXA2+EN1- (Kee et al. 2017; Kirkeby et al. 2017).

[0008] Therefore, the inventors identified the need to identify truly rich markers of true VMDA fate in order to help develop accurate and efficient QC assays for cell products used in Phase I clinical trials exploring cell replacement therapy as a treatment for PD.

[0009] In this specification, the inventors identified a novel cell surface marker for cVM precursors, adenomatosis polyposis coli down-regulated 1 (APCDD1). The inventors compared the performance of this marker with seven previously published cVM precursor surface markers. The markers were evaluated for cVM specificity, sensitivity to different dissociation enzymes, and reproducibility.

[0010] The inventors showed that among the surface markers tested, APCDD1 and trophoblast glycoprotein (TPBG) were most enriched in cVM progenitor cells compared to neural progenitor cells derived from other adjacent regions of the brain. APCDD1 has been shown to have higher reproducibility than TPBG and is therefore more useful as a quality control marker. APCDD1 was highly correlated with markers indicating the fate of cVM. Selection of APCDD1+ cells enhances the cVM gene while depleting the contaminated cell population. Transplanted APCDD1+ cells, selected from mixed cells rather than APCDD1- cells, resulted in complete behavioral improvement in a Parkinson's disease rat model. Animals with APCDD1+-derived grafts had an increase in true VM DA neurons compared to APCDD1- grafts.

[0011] In summary, the inventors demonstrated that APCDD1 surpasses previously published surface markers and would be useful in identifying cVM progenitor cells for quality control of cell products to be used in future clinical trials.

[0012] A first aspect of the invention is a method for identifying one or more caudal ventral midbrain (cVM) progenitor cells, (i) A step of providing one or more cells, (ii) The step of measuring the presence and / or amount of adenomatosis polyposis coli down-regulated 1 (APCDD1) expressed by one or more cells, and the step of including or comprising the steps thereof The presence and / or amount of APCDD1 expressed by one or more cells provides a method for indicating that one or more cells are cVM progenitor cells.

[0013] During the development of the human central nervous system, the neural plate curls to form the neural tube as part of neural tube formation. From anterior to posterior, the neural tube consists of the forebrain (telencephalon and diencephalon), midbrain (midbrain), and hindbrain (rhomboencephalon). The midbrain-hindbrain boundary is located between the midbrain and the hindbrain. The base of the midbrain is the ventral midbrain (VM), and posterior to the VM is the caudal VM (cVM), located immediately rostral (anterior) to the midbrain-hindbrain boundary. The caudal VM (cVM) can be distinguished from the non-dopaminergic rostral VM (rVM) through EN1 expression. Only cVM progenitor cells give rise to VM dopaminergic neurons belonging to the substantia nigra of the human brain. A characteristic feature of Parkinson's disease is the relatively selective loss of VM dopaminergic neurons in the substantia nigra, which causes the motor symptoms of the disease.

[0014] "cVM progenitor cells" include any cells on the differentiation pathway from stem cells to mature cVM cells or ventral midbrain dopaminergic neurons. Identifying cVM progenitor cells is challenging in this field, and to date, no cell surface markers have been identified that allow for reliable distinction of cVM progenitor cells from the fate of other midbrain progenitor cells. This invention solves this problem by identifying the marker APCDD1, which has been shown herein to be specific to cVM progenitor cells.

[0015] "Providing one or more cells" includes any cells or cell populations from which cVM precursor cells are identified. This can take the form of, for example, a population of cells that have differentiated from an initial population of cells derived from stem cells or fetal tissue to the fate of cVM precursors. One or more cells may also be a single cell suspected to be a cVM precursor cell.

[0016] "Measuring the presence and / or amount of APCDD1 expressed by one or more cells" includes performing any method for detecting or measuring APCDD1 expression by one or more cells. In some embodiments, APCDD1 expression is on the surface of one or more cells. In some embodiments, APCDD1 expression is intracellular. This term also includes measuring the presence and / or amount of either the APCDD1 protein or the corresponding mRNA precursor or mature mRNA. In some preferred embodiments, the method involves measuring the presence of APCDD1 expressed by one or more cells.

[0017] "APCDD1" refers to the adenomatosis polyposis coli down-regulated 1 protein (also known as DRAPC1, B7323, or FP7019). APCDD1 is a single-pass type I membrane protein that has been previously shown to be expressed in both the epidermal and dermal compartments of the gastrointestinal tract, placental trophoblast cells, and human hair follicles.

[0018] In some embodiments, APCDD1 is mammalian APCDD1. In some preferred embodiments, APCDD1 is human APCDD1. In some embodiments, APCDD1 has the amino acid sequence shown in UniProt entry Q8J025 (human APCDD1), where SEQ ID NO: 1: MSWPRRLLLRYLFPALLLHGLGEGSALLHPDSRSHPRSLEKSAWRAFKESQCHHMLKHLH NGARITVQMPPTIEGHWVSTGCEVRSGPEFITRSYRFYHNNTFKAYQFYYGSNRCTNPTY TLIIRGKIRLRQASWIIRGGTEADYQLHNVQVICHTEAVAEKLGQQVNRTCPGFLADGGP WVQDVAYDLWREENGCECTKAVNFAMHELQLIRVEKQYLHHNLDHLVEELFLGDIHTDAT QRMFYRPSSYQPPLQNAKNHDHACIACRIIYRSDEHHPPILPPKADLTIGLHGEWVSQRC EVRPEVLFLTRHFIFHDNNNTWEGHYYHYSDPVCKHPTFSIYARGRYSRGVLSSRVMGGT EFVFKVNHMKVTPMDAATASLLNVFNGNECGAEGSWQVGIQQDVTHTNGCVALGIKLPHT EYEIFKMEQDARGRYLLFNGQRPSDGSSPDRPEKRATSYQMPLVQCASSSPRAEDLAEDS This is represented as GSSLYGRAPGRHTWSLLLAALACLVPLLNIRR (sequence number 1).

[0019] "APCDD1" refers to both the APCDD1 protein and the corresponding mRNA expressed in one or more cells. In some preferred embodiments, APCDD1 refers to the APCDD1 protein. In some preferred embodiments, APCDD1 refers to the APCDD1 protein expressed on the surface of one or more cells. Therefore, in some preferred embodiments, the method involves measuring the presence and / or amount of APCDD1 expressed on the surface of one or more cells.

[0020] In the claimed method, one or more cells are identified as expressing APCDD1 and can then be classified as cVM progenitor cells based on this expression. This classification is useful for enriching cVM progenitor cell populations for applications such as cell replacement therapy where there is a need for a high-purity population of cVM progenitor cells.

[0021] Therefore, in some embodiments, the method is for enriching a population of cVM progenitor cells, and the method is (iii) Further comprising isolating one or more cells identified as cVM progenitor cells to obtain cVM progenitor cells or a population enriched with cVM progenitor cells.

[0022] "Enriching a population of cVM progenitor cells" includes methods for obtaining a population of cells containing a higher proportion of cVM progenitor cells than an initial population of one or more cells. "Enrichment" may be used interchangeably with "purification" in the art.

[0023] In some embodiments, isolating one or more cells involves selecting cVM progenitor cells in the starting population, thereby excluding other contaminating cell types. In some other embodiments, this may involve actively removing contaminating cell types from a population of one or more cells, leaving an enriched population of cVM progenitor cells.

[0024] In some embodiments, the present invention provides a method for enriching a population of cVM progenitor cells, the method being (i) providing one or more cells, (ii) Measuring the presence and / or amount of adenomatosis polyposis coli down-regulated 1 (APCDD1) expressed by one or more cells, (iii) comprising isolating one or more cells identified as cVM progenitor cells to obtain cVM progenitor cells or a population enriched with cVM progenitor cells.

[0025] In some embodiments, the one or more cells provided in step (i) are neural progenitor cells. In some embodiments, the one or more cells provided in step (i) are a population comprising neural progenitor cells.

[0026] Neural progenitor cells are precursor cells of the central nervous system (CNS) that give rise to mature CNS cells. In some specific embodiments, neural progenitor cells may be midbrain progenitor cells. In some specific embodiments, neural progenitor cells may be selected from a group including dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral-ventral midbrain (rVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB) progenitor cells.

[0027] In some embodiments, the population comprising neural progenitor cells includes a mixture of different types of neural progenitor cells, such as those described in the preceding paragraph.

[0028] In some embodiments, one or more cells provided in step (i) are derived from fetal tissue or are neural progenitor cells differentiated from stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ES).

[0029] In some embodiments, the fetal tissue includes one or more fetal stem cells from which one or more cells in step (i) may be derived or differentiated. In some embodiments, the fetal tissue includes fetal neural stem cells. In this embodiment, one or more cells in step (i) may differentiate from fetal neural stem cells to form one or more neural progenitor cells from which, for example, cVM progenitor cells are derived.

[0030] In some other embodiments, one or more neural progenitor cells can be obtained directly from fetal tissue.

[0031] In some preferred embodiments, one or more cells provided in step (i) are cells differentiated from iPSCs, for example, neural progenitor cells differentiated from iPSCs. In some preferred embodiments, the iPSCs are human iPSCs (hiPSCs).

[0032] "iPSC" refers to any pluripotent stem cell obtained by somatic cell reprogramming. "ES cell" refers to any pluripotent stem cell derived from embryonic tissue. For the purposes of this application, it will be understood that the ES cells used in the claimed method may be derived from parthenogenetically activated human oocytes without causing destruction of a human embryo.

[0033] In some preferred embodiments, the fetal tissues, iPSCs, and ES cells described herein are mammalian tissues / cells. In some preferred embodiments, the fetal tissues, iPSCs, and ES cells described herein are human tissues / cells.

[0034] "Differentiated" means that the stem cells described herein are directed to form a specific type of cell, such as cVM progenitor cells, using external factors. In some embodiments, this is called directed differentiation. Those skilled in the art will recognize that the direction of differentiation is usually achieved by treating stem cells with specific growth factors and / or cytokines and / or tailing factors and / or ventralizing factors. Those skilled in the art will recognize methods for directing the differentiation of cVM progenitor cells.

[0035] Therefore, in some embodiments, one or more cells in step (i) may include any neural progenitor cells derived from fetal tissue or differentiated from stem cells. These may be any of the neural progenitor cell types considered herein, mature neurons (e.g., mature rVM cells), or undifferentiated cells derived from fetal tissue or stem cells.

[0036] In some embodiments, the method is for quality control during the preparation of cells for cell replacement therapy.

[0037] In some embodiments, the method is for providing cVM progenitor cells for cell replacement therapy.

[0038] In some embodiments, the method is for determining the suitability of cells for use in treatment, and optionally, the treatment is cell replacement therapy.

[0039] Cell replacement therapy involves the administration or transplantation of cells (or appropriate progenitor cells) to enable the replacement of dead, damaged, or non-functional cells with healthy, functional cells. For example, in the context of this application, cVM progenitor cells identified or enriched using the method of the present invention may be transplanted or administered to a patient's brain to replace lost or non-functional dopamine cells.

[0040] Cell replacement therapy has been proposed as an important treatment for Parkinson's disease because it has the ability to replace dead or dying dopaminergic neurons, thereby restoring lost function and minimizing symptoms. cVM progenitor cells can be administered to patients and then differentiate into mature dopaminergic neurons in situ.

[0041] To maximize the effectiveness of the procedure and ensure the absence of undesirable side effects, it is crucial to ensure that transplanted cVM progenitor cells are highly pure and homogeneous. However, when differentiating stem cells into cVM progenitor cells, other neural progenitor cell types are often formed, and some stem cells remain undifferentiated. These contaminated cell populations can impair the cells available for transplantation. Therefore, it is important to ensure that a very pure population of cVM cells is obtained, which was previously difficult to achieve when enriching cVM precursor populations based on cell surface markers. This process of ensuring a very pure population of cVM precursors is called quality control.

[0042] However, the present invention solves this problem by presenting a novel marker, APCDD1, which can successfully distinguish between cVM precursors and contaminated cell types. Importantly, APCDD1 can distinguish between cVM precursor cells and similar rVM precursor cells with high efficiency.

[0043] Therefore, APCDD1 is particularly suitable as a marker for quality control of cVM precursor cell production and enrichment.

[0044] In some embodiments, step (ii) includes measuring the presence or amount of APCDD1 expression on the surface of one or more cells.

[0045] APCDD1 is a cell surface protein, and therefore, in some preferred embodiments, the method of the present invention involves detecting the expression of the APCDD1 protein on the surface of cells. The advantage of this method is that it allows for APCDD1 measurement in intact cells without requiring lysis.

[0046] In some embodiments, step (ii) includes measuring the presence of APCDD1. In some preferred embodiments, step (ii) includes measuring the presence of APCDD1 using an antibody-based detection method, such as flow cytometry. In some embodiments, step (ii) includes any method that can determine whether a cell is APCDD1 positive. "APCDD1 positive" means that a cell has measurable expression of APCDD1.

[0047] In some embodiments, the presence and / or quantity of APCDD1 is measured using a first binding portion specific to APCDD1.

[0048] In some embodiments, the measurement is performed using a second binding site specific to the first binding site. For example, if the first binding site is an antibody, the second binding site may be a further antibody or its antigen-binding fragment specific to the Fc portion of the first binding site.

[0049] In some embodiments, the first and / or second binding sites are an antibody or its antigen-binding fragment, or a variant thereof.

[0050] Methods for the production and use of antibodies are well known in the art; see, for example, Antibodies: A Laboratory Manual, 1988, Harlow & Lane, Cold Spring Harbor Press, ISBN-13: 978-0879693145, Using Antibodies: A Laboratory Manual, 1998, Harlow & Lane, Cold Spring Harbor Press, ISBN-13: 978-0879695446 and Making and Using Antibodies: A Practical Handbook, 2006, Howard & Kaser, CRC Press, ISBN-13: 978-0849335280 (these disclosures are incorporated herein by reference).

[0051] Therefore, the fragment has a variable weight (V H ) Domain or variable light (V L It may contain one or more of the domains. For example, the term antibody fragment is used to describe Fab-like molecules (Better et al (1988) Science 240, 1041), Fv molecules (Skerra et al (1988) Science 240, 1038), V H and V L This includes single-chain Fv(scFv) molecules in which the partner domain is linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423, Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879), and single-domain antibodies (dAb) containing isolated V domains (Ward et al (1989) Nature 341, 544).

[0052] For example, the binding site may be an scFv molecule. In some preferred embodiments, the binding site is a whole antibody (i.e., containing both Fc and Fv).

[0053] The term "antibody variant" includes, but is not limited to, any synthetic antibody, recombinant antibody, or antibody hybrid, single-chain antibody molecules produced by phage presentation of immunoglobulin light chain and / or heavy chain variable and / or constant regions, or other immune interaction molecules that can bind to an antigen in an immunoassay format known to those skilled in the art.

[0054] A general review of the techniques involved in the synthesis of antibody fragments that retain these specific binding sites is described in Winter & Milstein (1991) Nature 349, 293-299.

[0055] Molecular libraries such as antibody libraries (Clackson et al, 1991, Nature 352, 624-628; Marks et al, 1991, J Mol Biol 222(3): 581-97), peptide libraries (Smith, 1985, Science 228(4705): 1315-7), expression cDNA libraries (Santi et al (2000) J Mol Biol 296(2): 497-508), libraries on scaffolds other than antibody frameworks such as affibodies (Gunneriusson et al, 1999, Appl Environ Microbiol 65(9): 4134-40), or aptamer-based libraries (Kenan et al, 1999, Methods Mol Biol 118, 217-31) can be used as sources from which binding sites specific to a given motif can be selected for use in the method of the present invention.

[0056] In one embodiment of the method of the present invention, step (ii) is carried out using an assay comprising a first binding portion capable of binding to APCDD1. The first binding portion may also comprise a detectable portion. In some alternative embodiments, the first binding portion does not comprise a detectable portion, and a second binding portion is used which comprises a detectable portion specific to the first binding portion.

[0057] The second binding portion can be as described above in relation to the (first) binding portion, such as an antibody or an antigen-binding fragment thereof.

[0058] Alternatively or additionally, the first binding portion and / or the second binding portion may be labeled with a detectable moiety.

[0059] A "detectable moiety" is a moiety by which the moiety can be detected and includes the meaning that the relative amount and / or position of the moiety (e.g., a position on an array) can be determined.

[0060] Suitable detectable moieties are well known in the art. For example, the detectable moiety may be selected from the group consisting of a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety, an enzyme moiety.

[0061] The detectable moiety may be a fluorescent moiety and / or a luminescent moiety and / or a chemiluminescent moiety that can be detected when exposed to specific conditions. For example, the fluorescent moiety is exposed to radiation (i.e., light) at a specific wavelength and intensity to cause excitation of the fluorescent moiety, thereby enabling it to emit detectable fluorescence at a specific wavelength.

[0062] Alternatively, the detectable moiety may be an enzyme that can convert a (preferably undetectable) substrate into a detectable product that can be visualized and / or detected.

[0063] In a further alternative, the detectable moiety may be a radioactive atom useful for imaging. Suitable radioactive atoms include 99m Tc and 123 I. Other readily detectable moieties include, for example, again 123 I, 131 I, 111 In, 19 F, 13 C, 15 N, 17Examples of spin labels for magnetic resonance imaging (MRI) include 0, gadolinium, manganese, or iron. Clearly, the drug to be detected (e.g., one or more biomarkers in the test and / or control samples described herein, and / or antibody molecules for use in detecting selected proteins) must have sufficient suitable atomic isotopes so that the detectable portion is readily detectable.

[0064] In some preferred embodiments, the first and / or second binding portions are fluorescently labeled. Suitable fluorescent probes are well known in the art and include, but are not limited to, CF568, Alexa Fluor 488, 568, 405, 647 and 700, R-PE, PerCP-Cy5.5, R-PE-Cy7, and APC.

[0065] In some other embodiments, the first and / or second bonding portions are magnetically labeled. Suitable magnetic probes are well known in the art and include paramagnetic nanoparticles.

[0066] In some embodiments, the presence and / or amount of APCDD1 is measured by flow cytometry, qRT-PCR, RNA sequencing or other RNA detection methods, immunostaining, or chromatography.

[0067] In some preferred embodiments, the presence and / or quantity of APCDD1 is measured by APCDD1-specific flow cytometry.

[0068] Flow cytometry is a method that uses laser light to sort a sample of stained or labeled cells based on the cell labeling / staining profile. For example, in the case of APCDD1-expressing cells, flow cytometry can be used to detect and isolate cells labeled with an APCDD1-specific antibody.

[0069] In some embodiments, the presence and / or quantity of APCDD1, and / or the isolation of cells expressing APCDD1, are performed by flow cytometry, which is optionally fluorescence-activated cell sorting (FACS) or magnetoaffinity cell sorting (MACS).

[0070] In some embodiments, when step (ii) is carried out by FACS, the bonded portion is fluorescently labeled as described herein. In some other embodiments, when step (ii) is carried out by MACS, the bonded portion is magnetically labeled as described herein.

[0071] In some other embodiments, APCDD1 is detected using qRT-PCR, RNA sequencing, or another RNA detection method. Therefore, in these embodiments, what is detected is APCDD1-specific mRNA, not the protein.

[0072] In some other embodiments, APCDD1 is detected using immunostaining. “Immunostaining” means the process of identifying cells expressing APCDD1 by treating a sample with a binding site specific to APCDD1 and then visualizing the identified cells, for example, by using a labeled primary or secondary labeled antibody. The antibody may be labeled with a fluorescent label as described herein. Immunostaining can be a particularly useful technique when cVM progenitor cells are isolated from a tissue sample, for example, a fetal tissue sample.

[0073] In some other embodiments, APCDD1 is detected using chromatography. "Chromatography" means any technique that relies on the separation of materials in a mixture based on the properties of the materials, such as whether cells bind to the anti-APCDD1 binding site. Thus, step (ii) of the method may involve the use of an affinity chromatography-based method.

[0074] Naturally, those skilled in the art can combine any of the techniques described herein to carry out step (ii) of the method. For example, a crude first step may involve immunostaining or chromatography, followed by a more elaborate second step of flow cytometry, yielding a very pure sample of cVM progenitor cells.

[0075] In some embodiments, the method further comprises administering identified cVM progenitor cells to the brain of a subject, preferably a human subject.

[0076] The “brain” includes the cerebrum, cerebellum, and brainstem. In some preferred embodiments, cVM progenitor cells are directly administered to the putamen, which is a target site of human midbrain dopamine neurons.

[0077] In some embodiments, cVM progenitor cells are administered to a portion of the target brain where target processes derived from cVM progenitor cells are typically found. In some embodiments, cVM progenitor cells are administered directly to the forebrain. In some embodiments, cVM progenitor cells are administered directly to the basal ganglia region of the forebrain. In some embodiments, cVM progenitor cells are administered directly to the putamen region of the forebrain. In some embodiments, cVM progenitor cells are administered directly to the substantia nigra pars compacta.

[0078] In some embodiments, the method is for the treatment of a neurological disorder or condition. In some preferred embodiments, the neurological disorder or condition is Parkinson's disease. In some embodiments, the neurological disorder or condition is another Parkinsonian condition. "Parkinsonian condition" means any disorder or syndrome that shares clinical features with Parkinson's disease but has a different pathology.

[0079] Parkinson's disease (PD) is a neurodegenerative condition characterized by cell death in the basal ganglia of the brain, particularly the death of dopaminergic neurons in the midbrain (e.g., caudal-ventral midbrain). Cell death is typically caused by overexpression and / or misfolding of the protein alpha-synuclein, which aggregates and leads to cell death. PD is characterized by the following symptoms: tremor, bradykinesia, rigidity, limping / stumbling, autonomic dysfunction (autonomic dysphagia), neuropsychiatric problems (changes in mood, cognition, behavior, or thought), as well as sensory disturbances (e.g., changes in smell) and difficulty sleeping.

[0080] In some embodiments, the subject has diagnosed Parkinson's disease. In some other embodiments, the subject may be suspected of having Parkinson's disease. In some other embodiments, the subject may have early Parkinson's disease. In some other embodiments, the subject may have late Parkinson's disease. In some other embodiments, the subject may have another Parkinsonian condition.

[0081] In some embodiments, the method generates a substantially enriched population of cVM progenitor cells. In some embodiments, the method generates a substantially pure population of cVM progenitor cells.

[0082] "Substantially enriched" or "substantially pure" means that the population of cVM progenitor cells is highly pure and contains little to no contaminating cell types. In some embodiments, this means that the population of cVM progenitor cells contains a substantially higher proportion of cVM progenitor cells after performing the claimed method.

[0083] In some embodiments, substantially enriched / pure populations contain cell types other than cVM precursor cells at undetectable levels. For example, these cell types other than cVM precursor cells may not be detectable by flow cytometry.

[0084] In some embodiments, the cVM progenitor cells identified by this method can differentiate into ventral midbrain dopaminergic neurons.

[0085] In some embodiments, ventral midbrain dopaminergic neurons express tyrosine hydroxylase (TH).

[0086] Ventral midbrain dopaminergic neurons are crucial for controlling movement, processing, and memory. These neurons are involved in dopamine production, and disruption of this process can lead to functional loss, as seen in Parkinson's disease, for example. These neurons are often associated with TH expression because this enzyme is essential for the conversion of tyrosine to dopamine. Therefore, ventral midbrain dopaminergic neurons are any neuronal cells in the ventral midbrain that produce or are capable of producing dopamine.

[0087] In some embodiments of the method, cVM progenitor cells may be further characterized according to the expression of one or more known markers previously known to be associated with cVM progenitor cells. However, none of these previously identified markers were true markers of cVM progenitor cells because they are also expressed on other neural progenitor cells, such as rVM progenitor cells. For example, previous QC assays of cVM progenitor cells relied on the detection of LMX1A, FOXA2, and OTX2, but these three markers are also markers of rVM cells that give rise to glutamatergic neurons instead of dopaminergic neurons.

[0088] In some embodiments, the cVM progenitor cells identified by this method also express one or more of the following markers: FOXA2, OTX2, LMX1A, and EN1.

[0089] The inventors also found herein that a combination of APCDD1-positive cells and CORIN-negative cells can distinguish between cVM precursor cells and rVM precursor cells with the highest efficiency.

[0090] Therefore, in some preferred embodiments, the cVM progenitor cells identified by this method do not express CORIN. In some preferred embodiments, the method further includes measuring the presence / absence and / or amount of CORIN.

[0091] In some embodiments, the cVM progenitor cells identified by this method do not express CORIN.

[0092] In some embodiments, cVM progenitor cells also express FOXA2 and / or OTX2, and the method further comprises measuring the presence and / or amount of FOXA2 and / or OTX2 expression in one or more cells, and the presence and / or amount of APCDD1 and FOXA2 and / or OTX2 on or in one or more cells indicates that one or more cells are cVM progenitor cells.

[0093] In some embodiments, cVM progenitor cells also express FOXA2, and the method further comprises measuring the presence and / or amount of FOXA2 expression in one or more cells, and the presence and / or amount of APCDD1 and FOXA2 on or in one or more cells indicates that one or more cells are cVM progenitor cells.

[0094] In some embodiments, cVM progenitor cells also express OTX2, and the method further comprises measuring the presence and / or amount of OTX2 expression in one or more cells, and the presence and / or amount of APCDD1 and OTX2 on or in one or more cells indicates that one or more cells are cVM progenitor cells.

[0095] In some embodiments, cVM progenitor cells do not express CORIN, and the method further comprises measuring the presence and / or amount of CORIN on the surface of one or more cells, and the presence and / or amount of APCDD1 expression on one or more cells, as well as the absence and / or amount of CORIN expression on one or more cells, indicates that one or more cells are cVM progenitor cells.

[0096] In some preferred embodiments, cVM progenitor cells do not express CORIN, and the method further comprises measuring the presence and / or amount of CORIN on the surface of one or more cells, and the presence and / or amount of APCDD1 expression on one or more cells, as well as the absence and / or amount of CORIN expression on one or more cells, indicates that one or more cells are cVM progenitor cells.

[0097] In some embodiments, cVM progenitor cells have one of the following marker profiles: (a) APCDD1 positive and CORIN negative; (b) Positive for APCDD1, FOXA2, and OTX2; (c) APCDD1 positive, CORIN negative, FOXA2 positive, and OTX2 positive.

[0098] In some preferred embodiments, cVM progenitor cells are APCDD1-positive and CORIN-negative. In these embodiments, the methods described herein can distinguish between cVM and rVM progenitor cells with high efficiency.

[0099] In some embodiments, the method can distinguish between cVM progenitor cells and neural progenitor cells, or between cVM progenitor cells and one or more neurons from the following lineages: dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral-ventral midbrain (rVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB).

[0100] In some preferred embodiments, the method can distinguish between cVM precursor cells and rVM precursor cells.

[0101] A second aspect of the present invention provides cells or a population of cells produced according to the method of the present invention.

[0102] In some embodiments, this population of cells produced according to the method of the present invention is a highly enriched or pure population of cVM progenitor cells.

[0103] A third aspect of the present invention provides a population of substantially enriched populations of cVM progenitor cells produced according to the method of the present invention.

[0104] A fourth aspect of the present invention provides isolated cells or a population of isolated cells that are cVM precursor cells, the cells expressing APCDD1 on their surface.

[0105] In some embodiments, cells or populations of cells are intended for use in a method of treating a neurological condition or disease. In some preferred embodiments, the neurological condition or disease is Parkinson's disease.

[0106] A fifth aspect of the present invention provides cVM progenitor cells or populations thereof for use in a method of treating a neurological condition or disease of a subject requiring treatment, the method comprising carrying out a method of the present invention as discussed herein and administering a therapeutically effective amount of identified cVM cells to the subject.

[0107] A sixth aspect of the present invention provides the use of cVM progenitor cells or populations thereof for the manufacture of pharmaceuticals for the treatment of neurological conditions or diseases in subjects requiring treatment.

[0108] A seventh aspect of the present invention provides a method for treating a subject having a neurological condition or disease, comprising administering a therapeutically effective amount of cells or a population of cells described herein to the subject.

[0109] The therapeutic uses and methods described herein preferably involve the administration of cVM progenitor cells as part of a pharmaceutical composition.

[0110] In some preferred embodiments, the neurological condition or disease is Parkinson's disease.

[0111] In some embodiments, the use or method includes administering cells or a population of cells into the brain of a target.

[0112] The present invention also provides pharmaceutical compositions comprising cells or populations of cVM progenitor cells as described herein, and pharmaceutically acceptable carriers or excipients.

[0113] In some embodiments, cVM progenitor cells are administered to a portion of the brain in which cells derived from cVM progenitor cells are typically found. In some embodiments, cVM progenitor cells are administered directly to the forebrain. In some embodiments, cVM progenitor cells are administered directly to the basal ganglia region of the forebrain. In some embodiments, cVM progenitor cells are administered directly to the putamen region of the forebrain. In some embodiments, cVM progenitor cells are administered directly to the substantia nigra pars compacta.

[0114] Pharmaceutical compositions can be prepared in ways known in the art, which are sufficiently storage stable and suitable for administration to humans and animals. For example, pharmaceutical compositions may be freeze-dried, for example, by lyophilization, spray drying, spray cooling, or by the use of particle formation from supercritical particle formation.

[0115] "Pharmacologically acceptable" means a non-toxic substance that does not impair the efficacy of cVM progenitor cells. Such pharmaceutically acceptable buffers, carriers, or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., Mack Publishing Company (1990) and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated by reference).

[0116] The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture for the purpose of stabilizing the pH. Examples of buffers include Trizma, Bicin, Tricin, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphates, carbonates, acetates, citrates, glycolates, lactates, borates, ACES, ADA, tartrates, AMP, AMPD, AMPSO, BES, CABS, cacodylates, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazoleacetic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0117] The term “diluent” is intended to mean an aqueous or non-aqueous solution used for diluting a drug in a pharmaceutical preparation. The diluent may be one or more of the following: physiological saline, water, polyethylene glycol, propylene glycol, ethanol, or oil (e.g., safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0118] The term "adjuvant" is intended to mean any compound added to a formulation to increase the biological effect of the drug of the present invention. Adjuvants include, but are not limited to, one or more zinc salts, copper salts, or silver salts having different anions, such as fluorides, chlorides, bromides, iodides, thiocyanates, sulfites, hydroxides, phosphates, carbonates, lactates, glycolates, citrates, borates, tartrates, and acetates of different acyl compositions. Adjuvants may also be cationic synthetic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, and poly(vinylimidazole), as well as cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0119] Excipients may be one or more of carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrin, which are added to the composition, for example, to promote freeze-drying. Examples of polymers include starch, cellulose ether, cellulose carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, ethyl hydroxyethylcellulose, alginates, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinylpyrrolidone, all of which may have different molecular weights, and are added to the composition for viscosity control, achieving bioadhesion, or protecting lipids from chemical and protease degradation. Examples of lipids include fatty acids, phospholipids, mono, di, and triglycerides, ceramides, sphingolipids, and glycolipids (all of which may have different acyl chain lengths and saturation), egg lecithin, soy lecithin, hydrogenated egg, and soy lecithin, which are added to compositions for similar reasons as polymers. Examples of minerals include talc, magnesium oxide, zinc oxide, and titanium dioxide, which are added to compositions to obtain benefits such as reduced liquid accumulation or favorable pigment properties.

[0120] The drug of the present invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for its delivery.

[0121] In one embodiment, the pharmaceutical composition of the present invention may be in the form of liposomes, in which the drug is combined with an amphiphilic agent such as a lipid existing in aggregate form as a micelle, an insoluble monolayer, or a liquid crystal, in addition to other pharmaceutically acceptable carriers. Suitable lipids for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, and bile acids. Suitable lipids also include the above lipids modified with poly(ethylene glycol) at the polar head group to extend blood flow circulation time. The preparation of such liposomal formulations can be found, for example, in U.S. Patent No. 4,235,871, the disclosure of which is incorporated herein by reference.

[0122] The pharmaceutical compositions of the present invention may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(caprolactone) (PCL), and polyanhydrides are widely used as biodegradable polymers in the production of microspheres. The preparation of such microspheres can be found in U.S. Patent No. 5,851,451 and European Patent No. 0213303, the disclosures thereof are incorporated herein by reference.

[0123] In further embodiments, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, and starch, cellulose ether, cellulose carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, ethyl hydroxyethylcellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polyvinylimidazole, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate of different degrees of hydrolysis, and polyvinylpyrrolidone are used to thicken the solution containing the drug. The polymer may also contain gelatin or collagen.

[0124] Alternatively, the drug may simply be dissolved in physiological saline, water, polyethylene glycol, propylene glycol, ethanol, or oil (e.g., safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil), tragacanth gum, and / or various buffers.

[0125] It will be understood that the pharmaceutical composition of the present invention may include ions and a defined pH for enhancing the action of the activator. In addition, the composition may undergo conventional pharmaceutical procedures such as sterilization and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, and fillers.

[0126] The pharmaceutical composition according to the present invention may be administered via any suitable route known to those skilled in the art.

[0127] In some preferred embodiments, the composition is administered by injection into a desired area of ​​the brain (for example, during brain surgery). In some embodiments, the composition is administered by injection into the putamen region of the forebrain.

[0128] Suitable formulations for administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the recipient's CSF, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. The formulations may be presented in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a frozen state requiring the addition of a sterile liquid carrier, such as sterile water for injection, immediately before use. Immediate injection solutions and suspensions may be prepared from the sterile powders, granules, and tablets of the types described above.

[0129] Pharmaceutical compositions are administered to patients in pharmaceutically effective doses. As used herein, “therapeutic effective dose,” “effective dose,” or “therapeutably effective” refers to an amount that provides a therapeutic effect for a given condition and administration regimen. This is a predetermined amount of active material calculated to produce the desired therapeutic effect in relation to the necessary additives and diluents, i.e., carriers or administration vehicles. Furthermore, it is intended to mean an amount sufficient to reduce, and most preferably prevent, clinically significant deficiencies in the host’s activity, function, and response. Alternatively, a therapeutic effective dose is sufficient to cause a clinically significant improvement in the host’s condition. As understood by those skilled in the art, the amount of a compound may vary depending on its specific activity. An appropriate dosage may include a predetermined amount of the active composition calculated to produce the desired therapeutic effect in relation to the necessary diluents. In the methods for preparing and using the compositions of the present invention, a therapeutic effective dose of the active ingredient is provided. The therapeutic effective dose may be determined by those skilled in the art based on patient characteristics such as age, weight, sex, condition, complications, and other diseases, as is well known in the art. The administration of a pharmaceutically effective dose may be carried out by single doses in the form of individual dose units or otherwise several smaller dose units, and by multiple doses in divided doses at specific intervals. Alternatively, the dose may be provided as a continuous infusion over a long period of time.

[0130] The compositions of the present invention can be formulated at various concentrations depending on the efficacy / toxicity of the compounds used. Those skilled in the art will recognize appropriate techniques for determining the formulation and dosage actually used in patient administration.

[0131] Those skilled in the art will understand that the pharmaceutical compositions of the present invention may be administered alone or in combination with other therapeutic agents used to treat neurological disorders or conditions. In particular, the therapeutic agent(s) may be one known to be effective for the target indication, for example, Parkinson's disease (e.g., levodopa).

[0132] In some embodiments, the subject has diagnosed Parkinson's disease. In some other embodiments, the subject may be suspected of having Parkinson's disease. In some other embodiments, the subject may have early Parkinson's disease. In some other embodiments, the subject may have late Parkinson's disease.

[0133] cVM progenitor cells may be administered in a single dose or in multiple doses to achieve the desired therapeutic effect.

[0134] The success of the treatments for Parkinson's disease described herein may involve improvement (to any degree) or complete relief of Parkinson's disease symptoms over any period of time. The improvement may be short-term or, more preferably, long-term. In some embodiments, the improvement may be permanent.

[0135] An eighth aspect of the present invention provides the use of APCDD1 as a biomarker for identifying and / or enriching cVM progenitor cells from a population comprising one or more cells.

[0136] A ninth aspect of the present invention provides the use of an APCDD1-specific binding site for identifying and / or enriching cVM progenitor cells from a population of one or more cells.

[0137] A tenth aspect of the present invention is: (i) A binding site specific to APCDD1, which optionally has a label attached to it, (ii) Washing and blocking buffer, (iii) Positive controls and negative controls (multiple controls are allowed), (iv) Optionally, provide a kit that includes instructions for use.

[0138] In some embodiments, the binding portion is an antibody or its antigen-binding fragment as described herein.

[0139] Embodiments of the present invention are described in the following numbered paragraphs. 1. A method for identifying one or more caudal ventral midbrain (cVM) progenitor cells, (i) A step of providing one or more cells, (ii) The step of measuring the presence and / or amount of adenomatosis polyposis coli down-regulated 1 (APCDD1) expressed by one or more cells, and the step of including or comprising the steps thereof The presence and / or amount of APCDD1 expressed by one or more cells indicates that one or more cells are cVM progenitor cells, a method.

[0140] 2. The method is for enriching a population of cVM precursor cells, and the method is (iii) The method according to paragraph 1, further comprising isolating one or more cells identified as cVM progenitor cells to obtain cVM progenitor cells or a population enriched with cVM progenitor cells.

[0141] 3. The method according to any one of paragraphs 1-3, wherein one or more cells provided in step (i) are neural progenitor cells.

[0142] 4. The method according to any one of paragraphs 1-3, wherein one or more cells provided in step (i) are neural progenitor cells obtained from fetal tissue or differentiated from stem cells, and optionally the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ES).

[0143] 5. The method according to any one of paragraphs 1 to 4, wherein the method is for quality control during the preparation of cells for cell replacement therapy.

[0144] 6. The method according to any one of paragraphs 1 to 5, wherein the method is for providing cVM progenitor cells for cell replacement therapy.

[0145] 7. The method according to any one of paragraphs 1-6, wherein the method is for determining the suitability of cells for use in treatment, and optionally the treatment is cell replacement therapy.

[0146] 8. The method according to any one of paragraphs 1 to 7, wherein step (ii) comprises measuring the presence or amount of APCDD1 expression on the surface of one or more cells.

[0147] 9. The method according to any one of paragraphs 1 to 8, wherein step (ii) includes measuring the presence of APCDD1.

[0148] 10. The method according to any one of paragraphs 1 to 9, wherein the presence and / or amount of APCDD1 is measured using a first binding portion specific to APCDD1.

[0149] 11. The method according to paragraph 10, wherein the measurement is performed using a second ligament specific to the first ligament.

[0150] 12. The method according to paragraph 10 or 11, wherein the first and / or second binding site is an antibody or its antigen-binding fragment.

[0151] 13. The method according to any one of paragraphs 10-12, wherein the first and / or second junction is fluorescently labeled.

[0152] 14. The method according to any one of paragraphs 1-13, wherein the presence and / or amount of APCDD1 is measured by flow cytometry, qRT-PCR, RNA sequencing, or other RNA detection methods, immunostaining, or chromatography.

[0153] 15. The method according to paragraph 14, wherein the presence and / or amount of APCDD1 is measured, and / or cells expressing APCDD1 are isolated by flow cytometry, and optionally, flow cytometry is fluorescence-activated cell sorting (FACS) or magnetoaffinity cell sorting (MACS).

[0154] 16. The method according to any one of paragraphs 1 to 15, further comprising administering identified cVM progenitor cells to the brain of a subject, preferably the subject being human.

[0155] 17. The method according to paragraph 16, wherein the method is for the treatment of a neurological disorder or condition, and optionally the neurological disorder or condition is Parkinson's disease.

[0156] 18. The method according to any one of paragraphs 1 to 17, wherein the method generates a substantially enriched population of cVM progenitor cells.

[0157] 19. The method according to any one of paragraphs 1-18, wherein cVM progenitor cells identified by the method can differentiate into ventral midbrain dopaminergic neurons.

[0158] 20. The method according to paragraph 19, wherein ventral midbrain dopaminergic neurons express tyrosine hydroxylase (TH).

[0159] 21. The method according to any one of paragraphs 1 to 20, wherein the cVM progenitor cells identified by the method express one or more of the markers FOXA2, OTX2, LMX1A, and EN1.

[0160] 22. The method according to any one of paragraphs 1 to 21, wherein the cVM progenitor cells identified by the method do not express CORIN.

[0161] 23. The method according to any one of paragraphs 1 to 22, wherein cVM progenitor cells also express FOXA2 and / or OTX2, and the method further comprises measuring the presence and / or amount of FOXA2 and / or OTX2 expression in one or more cells, and the presence and / or amount of APCDD1 and FOXA2 and / or OTX2 on or in one or more cells indicates that one or more cells are cVM progenitor cells.

[0162] 24. The method according to any one of paragraphs 1 to 23, wherein cVM progenitor cells do not express CORIN, and the method further comprises measuring the presence and / or amount of CORIN on the surface of one or more cells, the presence and / or amount of APCDD1 expression on one or more cells, and the absence and / or amount of CORIN expression on one or more cells, indicating that one or more cells are cVM progenitor cells.

[0163] 25. The method according to any one of paragraphs 1 to 24, wherein the cVM progenitor cells have one of the following marker profiles: (a) APCDD1 positive and CORIN negative; (b) Positive for APCDD1, FOXA2, and OTX2; (c) APCDD1 positive, CORIN negative, FOXA2 positive, and OTX2 positive.

[0164] 26. The method according to any one of paragraphs 1 to 25, wherein the method can distinguish between cVM progenitor cells and neural progenitor cells, or between cVM progenitor cells and one or more neurons from the lineages of the dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral ventral midbrain (rVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB), and preferably the method can distinguish between cVM progenitor cells and rVM progenitor cells or rVM cells.

[0165] 27. Cells or populations of cells produced according to the methods of paragraphs 1-26.

[0166] 28. A population of substantially enriched populations of cVM progenitor cells produced according to the methods of paragraphs 1-26.

[0167] 29. Isolated cells or isolated cell populations that are cVM precursor cells, wherein the cells express APCDD1 on their surface.

[0168] 30. Cells or populations of cells as described in paragraphs 27-29, for use in methods of treating neurological conditions or diseases.

[0169] 31. cVM progenitor cells or populations for use in a method of treating a neurological condition or disease of a subject requiring treatment, wherein the method comprises performing the method described in any one of paragraphs 1 to 26 and administering a therapeutically effective dose of identified cVM cells to the subject.

[0170] 32. Use of cVM progenitor cells or populations thereof as described in paragraphs 27-29 for the manufacture of pharmaceuticals for the treatment of neurological conditions or diseases in subjects requiring Chiro-to.

[0171] 33. A method for treating a subject having a neurological condition or disease, comprising administering a therapeutically effective dose to the cells or population of cells described in paragraphs 27-29.

[0172] 34. The use of cells or populations of cells for use as described in paragraphs 30-31, the use as described in paragraph 32, or the method as described in paragraph 33, wherein the neurological condition or disease is Parkinson's disease.

[0173] 35. A cell or cell population for use as described in paragraphs 30-31, a use as described in paragraph 32, or a method as described in paragraph 33, wherein the use or method involves administering cells or a population thereof into the brain of a subject.

[0174] 36. Use of APCDD1 as a biomarker to identify and / or enrich cVM progenitor cells from a population containing one or more cells.

[0175] 37. Use of APCDD1-specific binding sites to identify and / or enrich cVM progenitor cells from a population of one or more cells.

[0176] 38. (i) A binding site specific to APCDD1, which optionally has a label attached to it, (ii) Washing and blocking buffer, (iii) Positive controls and negative controls (multiple controls are allowed), (iv) Optionally, a kit including instructions for use.

[0177] 39. The use described in paragraph 37 or the kit described in paragraph 38, wherein the binding site is an antibody or its antigen-binding fragment.

[0178] Herein, preferred, non-limiting examples embodying a particular aspect of the present invention will be described with reference to the accompanying drawings. [Brief explanation of the drawing]

[0179] [Figure 1] Identification of candidate surface marker APCDD1 for caudal-ventral midbrain cells. A. Single-cell RNA sequencing of ventral MiSTR tissue at day 14 plotted on UMAP dimensions. Expression of previously published caudal-ventral midbrain cell surface marker genes ALCAM, LRTM1, CORIN, FOLR1, CD47 / IAP, CNTN2, candidate marker genes TPBG and APCDD1, ventral midbrain genes FOXA2, LMX1A, OTX2 and EN1, and forebrain and hindbrain markers NKX2-1 and HOXB1, respectively. B. Immunofluorescence labeling of EN1, APCDD1 and OTX2 in human fetal midbrain at gestational week (GW) 5. [Figure 2]Validation of a flow cytometry region specificity panel. A-I. Titration of antibodies against caudal ventral midbrain (cVM) precursor markers ALCAM (A), APCDD1 (b), CD47 / IAP (C), CNTN2 (D), CORIN (e), FOLR1 (F), LRTM1 (G-H), and TPBG (I), as well as representative flow cytometry plots of dorsal forebrain (dFB) and cVM precursor cells. The y-axis shows the modal normalized frequency distribution. Suboptimal 16-day cVM precursors were used for titration. Selected concentrations are marked with arrows. Two anti-LRTM1 antibodies were tested. First, anti-LRTM1 (G) from R&D Systems, with two secondary antibodies tested: anti-rat PE from Thermo Fisher (TF) and anti-rat PE from R&D Systems. Second, anti-LRTM1 (H) from KAN Research Institute (KAN). A common fluorescent dye-specific gating strategy used by employing a fluorescent minus-one (FMO) control. [Figure 3]RNA expression levels of APCDD1 and previously published caudal-ventral midbrain cell surface markers. A. Schematic differentiation protocols for generating 16-day neural progenitor cells in dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral-ventral midbrain (rVM), caudal-ventral midbrain (cVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB). B. Scale-normalized mRNA expression levels (n≧4) for ventral midbrain dopaminergic progenitor cells of dFB, vFB, dMB, rVM, cVM, dHB, and vHB relative to undifferentiated RC17 hESCs, scaled to 100. C~F. Linear regression of log10-converted scale-normalized mRNA expression levels relative to undifferentiated RC17 hESCs in dFB, vFB, dMB, rVM, cVM, dHB, and vHB. C-E. Simple linear regression models between APCDD1 and EN1 (C), APCDD1 and FOXA2 (D), and APCDD1 and LMXA1 (E). F. Multiparametric linear regression between APCDD1 and EN1, LMX1A, and FOXA2 expression levels. Points are sized with respect to FOXA2 and color-scaled by EN1 expression levels, log10 transformed using the Batlow palette. [Figure 4]Benchmark evaluation of caudal-ventral midbrain cell surface markers in flow cytometry regional panels. A-C. Benchmark evaluation of cell surface markers indicating ventral midbrain dopaminergic progenitor cells by flow cytometry analysis of neural progenitor cells at day 16 in the dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral-ventral midbrain (rVM), caudal-ventral midbrain (cVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB). A-C. Percentage of viable single cells positive for putative cell surface markers after dissociation with Accutase (A), papain (B), and TrypLE (C) following 16 days of differentiation in vitro. Bars are color-coded by region. D. Percentage of viable single cells positive for putative surface markers after dissociation with Accutase from paired batches of cVM cells after 11 and 16 days of differentiation in vitro. E. Percentage of APCDD1+ and TPBG+ cells relative to viable single cells of cVM progenitor cells dissociated with Accutase, papain, and TrypLE at day 16, colored by surface markers. F. Standard error of the percentage of positive viable single cells relative to cVM progenitor cells dissociated with Accutase and TrypLE at day 16, grouped by dissociation method and colored by region. Error bars represent the standard error of the mean. [Figure 5] mRNA levels in neural progenitor cells. A-B. Multiplicative changes (n≧4) in mRNA expression relative to undifferentiated RC17 hESCs for selected regional markers in neural progenitor cells at day 16 of the dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral ventral midbrain (rVM), caudal ventral midbrain (cVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB). A. Heatmap of maximum normalized RNA expression levels. B. Pearson correlation between ventral midbrain transcription factors and surface markers. Non-significant regression models are indicated by the absence of r-correlation coefficients. [Figure 6]Effect of dissociation method of caudal-ventral midbrain cell surface markers in flow cytometry region panels and local enrichment of H9 human embryonic stem cells. A-B. Factor analysis of mixed data (FAMD) on flow cytometry region panels. A. Scree plot showing the explained variance in each dimension for the proportion of viable single cells positive for putative surface markers. B. Contribution rate (%) of each variable in the first dimension. C. Proportion of APCDD1+ viable single cells in dorsal forebrain (dFB), rostral-ventral midbrain (rVM), and caudal-ventral midbrain (cVM) cells dissociated with Accutase from H9 cells on day 16. D. Proportion of positive rVM and cVM progenitor cells to viable single cells on day 16, grouped by dissociation method and faceted by cell surface markers and regions. Points are shaped according to region (rVM, dot, cVM, triangle) and colored by batch. An asterisk indicates a statistically significant difference determined by Dunn's multiple comparison test (APCDD1) or pairwise t-tests using the Benjamini-Hochberg method for false detection (CNTN2 and CORIN). *, p≦0.05; **, p≦0.01; ***, p≦0.005. [Figure 7]Benchmark evaluation of APCDD1 as a surface marker for caudal ventral midbrain cells in a regional panel using flow cytometry for comparison with previously reported markers. A-F. Benchmark evaluation of cell surface markers indicating ventral midbrain dopaminergic progenitor cells by flow cytometry analysis of neural progenitor cells at days 11 and 16 in the dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral ventral midbrain (rVM), caudal ventral midbrain (cVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB). A. Percentage of positive rVM and cVM precursors at day 16 relative to viable single cells for surface markers affected by dissociation method. Asterisks indicate a statistically significant difference in the percentage of positive cells between dissociation methods for either or both regions, determined using nested pairwise analysis of variance (ANOVA) by region. B. Percentage of APCDD1+ cells relative to viable single cells at day 11 and day 16 in cVM progenitor cells dissociated with Accutase, obtained from paired batches (n = 3). C. Percentage of positive viable single cells in Accutase-dissociated cells at day 16, colored by region (n≧3). D. Enrichment of cVM progenitor cells after dissociation, determined by specificity index. Points are colored by dissociation method (n=≧5). E. Standard deviation (SD) of the percentage of positive single cells in rVM and cVM regions. Points are colored by dissociation method. APCDD1 had a statistically significantly lower SD compared to TPBG and ALCAM when determined using paired pairwise t-tests with Benjamini-Hochberg false-find rate correction. F~H. Intracellular and extracellular flow cytometry analysis of cVM precursors at day 16, using fully stained dFB precursors as a biological negative control to set up a FOXA2+ / OTX2+ gate. Experimental schematic diagram (F) FOXA2+ / OTX2+ (Q2) (G), and linear regression model (H) between the proportion of FOXA2+ / OTX2+ and APCDD1+ in a single viable cell (n=9). *, p ≤ 0.05. Error bars represent the standard error of the mean. [Figure 8]Verification of the association between APCDD1 and pure cell differentiation in caudal-ventral midbrain (VM) fate sorting, and analysis of enrichment from caudal VM to rostral VM. A-D. Sorting of pure caudal-ventral midbrain (VM) batches for APCDD1. A. Example of experimental design. Briefly, high-purity cryopreserved 16-day-old caudal VM progenitor cells derived from RC17 were sorted for APCDD1. RNA was isolated immediately after sorting, and expression levels were analyzed using quantitative real-time PCR (qRT-PCR). Cells were reseeded and fixed at 42 days for immunocytochemistry (ICC) analysis. B. Flow cytometry plots showing approximate APCDD1+ / - cell percentages in VM batches 1-3 and clinical batches STEM-PD GMP numbers 3 and 5. C. mRNA ratio changes of VM and non-VM genes in undifferentiated human embryonic stem cells for APCDD1 sorted cells. Statistical significance was assessed using a paired t-test (n=5). ICC of terminally differentiated cells at 42 days post-sorting using D.TH, FOXA2, and MAP2. Scale bar, 100 μm. Flow cytometry analysis of regional specificity of E. rVM and cVM. The panel APCDD1+CORIN- best distinguishes between rostral-ventral midbrain (rVM) and caudal-ventral midbrain (cVM). [Figure 9] Enrichment panels of caudal-ventral midbrain and rostral-central midbrain. A. Common fluorescent dye-specific gating strategies used by using a fluorescence minus-one (FMO) control. B. Gating used for the APCDD1+CORIN- panel for caudal-ventral midbrain (cVM, left) and rostral-central midbrain (rVM, right) cells. See the lower right quadrant (circled). C. Percentage of positive cells to viable single cells in caudal-ventral midbrain (cVM) and rostral-central midbrain (rVM) progenitor cells dissociated with Accutase, papain, and TrypLE at day 16. D. Percentage of cells in the gate to viable single cells in dissociated cVM and rVM progenitor cells at day 16. Dots are colored according to whether the batch passed cVM qRT-PCR quality control. [Figure 10]Salvage of contaminated caudal-ventral midbrain batches by sorting APCDD1+ cells. A-C. Sorting of APCDD1 in mixed cell batches. A. Example of experimental design. A 4:3:3 mixture of 16-day-old caudal-ventral midbrain (cVM), rostral-ventral midbrain (rVM), and ventral hindbrain (vHB) progenitor cells from RC17 hESCs was sorted for APCDD1. RNA was isolated immediately after sorting and expression levels were analyzed using quantitative real-time PCR. Cells were transplanted into Parkinson's disease nude rats. B. Flow cytometry plot of approximate APCDD1+ / - cell percentage in mixed batches. C. mRNA ratio changes of VM and non-VM genes in APCDD1-sorted cells compared to undifferentiated human embryonic stem cells. Statistical significance was assessed using paired t-tests (n=4). [Figure 11] Functional maturation of APCDD1+ cells into ventral midbrain dopaminergic neurons by cell sorting of a mixed batch of cells. A-H. Sorting of APCDD1 in a mixed batch of cells and transplantation into Parkinson's disease nude rats. A. Example of experimental design. A 4:3:3 mixture of 16-day-old progenitor cells from the caudal ventral midbrain (cVM), rostral ventral midbrain (rVM), and ventral hindbrain (vHB) derived from RC17 hESCs was sorted for APCDD1. Cells were transplanted into the striatum of Parkinson's disease nude rats. Rats were sacrificed 28 weeks after transplantation. B-C. hNCAM diaminobenzidine (DAB) immunohistochemistry (IHC) in APCDD1+ (B) and APCDD1-sorted cells (C). D-E. TH DAB IHC in APCDD1+ (D) and APCDD1-sorted cells (E). F. Estimated total yield of TH+ cells per 100,000 transplanted cells. G. Total number of TH+ / HuNu+ cells per animal. H. Amphetamine-induced turnover test at 16, 20, 24, and 28 weeks post-transplantation. [Figure 12]Characterization of graft composition of APCDD1-derived grafts sorted from mixed cells. A-C. Characterization of composition of APCDD1-sorted grafts derived from mixed cell batches. A-B. Immunofluorescence of TH, LMX1A, and FOXA2 in APCDD1+ (A) and APCDD1- (B) sorted cells. C. Quantification of single, double, and triple-positive cells for TH, LMX1A, and FOXA2 in APCDD1-derived grafts. Statistical significance was assessed using t-tests (n=5). [Examples]

[0180] The inventors identified adenomatosis polyposis coli down-regulated 1 (APCDD1) as a novel cell surface candidate biomarker to predict the functional maturation of cVM precursors into ventral midbrain (VM) dopaminergic (DA) neurons.

[0181] Background and Summary Parkinson's disease (PD) is a common neurodegenerative motor disorder. Levodopa is the most common treatment strategy for improving the motor symptoms of this disease, but it is associated with serious complications, and its effectiveness is variable and progressively decreases (Connolly and Lang 2014). Since the motor symptoms of PD may be due to the relatively selective and localized loss of dopaminergic (DA) neurons in the ventral midbrain (VM), cell replacement is a promising alternative treatment strategy to levodopa.

[0182] The feasibility of this approach has been demonstrated by using fetal VM-derived cells (Barker et al. 2019; Bjorklund et al. 1980; Bolam et al. 1987; Dunnet et al. 1983; Freund et al. 1985; Strecker et al. 1987). However, due to tissue-related issues, the focus has recently shifted to using human pluripotent stem cells (hPSCs) as a nearly inexhaustible source of VM DA cells (Kefalopoulou et al. 2014; Kurowska et al. 2011; JYLi et al. 2008; W.Li et al. 2016). The cells are transplanted as precursors that mature in the host brain and replace the functions of cells that have been lost endogenously.

[0183] Human pluripotent stem cells are a promising source of caudal-ventral midbrain (cVM) progenitor cells used in cell replacement therapy for Parkinson's disease. However, some heterogeneity in cell composition is inevitable during the cell differentiation process. Cell surface markers that predict the functional maturation of cVM precursors into ventral midbrain (VM) dopaminergic (DA) neurons can be used as a convenient quality control to enrich target cell types.

[0184] To obtain safe, effective, and reproducible results, a pure population of cells must be transplanted. Genes indicating VM DA precursors, such as the transcription factors LMX1A, FOXA2, and OTX2, are commonly used as surrogate markers for functional maturation (Arenas, Denham, and Netherlandsescusa 2015). These markers are incorporated into intracellular-based flow cytometry quality control (QC) panels to assess the purity of differentiated cells. However, only LMX1A / FOXA2 / OTX2 triple-positive precursors derived from caudal VM (cVM) give rise to VM DA neurons, while triple-positive cells from the rostral adjacent subthalamic nucleus (STN), then called rostral VM (rVM), produce glutamatergic neurons (Nolbrant et al. 2017). Current flow cytometry-based QC assays using LMX1A / FOXA2 / OTX2 cannot distinguish between the fate of cVM and rVM. In fact, the expression levels of FOXA2, OTX2, and LMX1A cannot predict graft outcomes at transplantation (Kirkeby et al. 2017). While EN1 specifically indicates VM DA fate, there are no sufficiently specific EN1 antibodies available for flow cytometry. Furthermore, intracellular flow cytometry, compared to the use of cell surface markers, is associated with increased sample processing time and considerable nonspecific background staining, complicating gating in a Good Manufacturing Practice (GMP) environment for pharmaceuticals and quasi-drugs. In fact, under GMP conditions, only gating to a fluorescence minus one (FMO) control is permitted, which does not address the de facto background observed in intracellular flow cytometry. Extracellular-based flow cytometry can be a more convenient QC for cell differentiation than intracellular flow cytometry because it avoids the drawbacks listed above. In addition, while insufficient fixation and permeabilization can be used to select cells for VM DA fate, it can also exclude undesirable cells such as serotonergic cells and stem cells. This would allow for a direct correlation between cell products and transplantation outcomes, but this is currently not possible.Finally, surface markers can be used to enrich the target population of induced PSC (iPSC)-derived differentiation immediately before transplantation.

[0185] Several publicly available putative surface markers predict VMDA neurons (Bye et al. 2015; Doi et al. 2014; Fathi et al. 2018; Gennet et al. 2016; Kikuchi et al. 2017; Lehnen et al. 2017; Paik et al. 2018; Samata et al. 2016; Sundberg et al. 2013; Yoo et al. 2021), some of which improve behavioral impairment in animal models of Parkinson's disease (Doi et al. 2014; Kikuchi et al. 2017; Lehnen et al. 2017; Sundberg et al. 2013; Yoo et al. 2021). Apart from the surface marker CORIN, most of these are not broadly characterized, and their region specificity has not been sufficiently studied. See Table 3 for a summary of the surface markers.

[0186] Some of these studies have focused on LMX1A from mixed initiation progenitor populations. + Cells and FOXA2 + While the focus has been on cell enrichment, there have been few studies that have focused on evaluating the specificity of markers against various other localized neural progenitor cells and investigating the specificity of markers against other potential contaminating populations of neural fate. In addition, LMX1A + FOXA2 + EN1 + The specificity of the published marker for true DA progenitor cells of cVM is LMX1A + FOXA2 + EN1 -There has been little focus on evaluating this against adjacent non-DA rVM progenitor cells (Kee et al. 2017; Kirkeby et al. 2017). Therefore, the inventors identified the need for cell surface marker-to-cell surface marker comparison to identify truly VM-DA fate-rich markers, in order to help develop accurate and efficient QC assays for cell products used in Phase I clinical trials exploring cell replacement therapy as a treatment for PD.

[0187] The inventors herein provide a novel candidate marker for predicting VMDA neurons, adenomatosis polyposis coli down-regulated 1 (APCDD1), which functions as a Wnt / β-catenin antagonist (He and Tang 2020; Kagermeier-Schenk et al. 2011; Shimomura et al. 2010; Nolli et al. 2020). By comparing the regional specificity of APCDD1 and TPBG with previously published putative surface markers for VM DA progenitor cells using quantitative real-time PCR (qRT-PCR), flow cytometry, and region enrichment modeling, the inventors demonstrated that the newly identified surface marker was most enriched for VM DA fate. APCDD1 correlates with EN1 and FOXA2 at the transcriptome level. + / OTX2 + APCDD1 in cells + It also correlates with the proportion of cells. APCDD1 selected from a mixed population of cells. + Animals transplanted with cells showed complete behavioral recovery in a rat model of PD, while APCDD1 - The animals that received the cell transplant did not show improvement. APCDD1 + Cell-derived grafts are APCDD1 - Compared to cell-derived grafts, the yield of true VMDA neurons was higher.

[0188] APCDD1 is the most predictive surface marker for VMDA neuronal fate, making it an attractive candidate for flow cytometry-based QC assays for cell replacement therapy in PD.

[0189] Example 1 material and method hESC differentiation in MiSTR Roslin Cells 17 (RC17, hPSC registration number RCe021-A) hESCs were cultured and differentiated in a custom-designed microfluidic device as described in Rifes et al. 2020. Briefly, this was done using a continuous flow of neuronal patterning medium (NPM) consisting of a 1:1 mixture of DMEM / F12 (Invitrogen) and NeuroMedium (Miltenyi Biotec), an N2 supplement (1:200, Invitrogen), and vitamin A-free NeuroBrew-21 (1:100) supplemented with 10 μM SB431542 (Miltenyi Biotec) and 100 ng / mL rh-Noggin (Miltenyi Biotec). One syringe contained culture medium supplemented with GSK3i (100%, CHIR99021, Miltenyi Biotec), while the other did not contain GSK3i (0%). For aeration, 200 ng / mL of SHH-C24II (Miltenyi Biotec) and 0.5 μM of purmorphamine (Miltenyi Biotec) were added to the culture medium at both inlets.

[0190] hESC culture and differentiation RC17 hESCs were differentiated toward the precursors of the dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rVM, cVM, dorsal hindbrain (dHB), and ventral hindbrain (vHB) fate. For all conditions, the medium composition, coating, seeding density, and reseeding steps were followed up to day 16 as described above in Nolbrant et al., 2017 (Kirkeby et al. 2017; Nolbrant et al. 2017). All conditions received dual SMAD inhibition (SB431542 10 μM and Noggin 100 ng / mL) from days 0–9 of differentiation. Patterning for each of the different regions was achieved by adding the patterning factors CHIR99021 (referred to as CHIR), SHH-C24II (referred to as SHH), and FGF8b (all obtained from Miltenyi Biotec) in the following differential amounts: dFB (no additional factors added), vFB (SHH 300 ng / mL on days 0-9), dMB (CHIR 0.7 μM on days 0-9 + FGF8b 100 ng / mL on days 4-16), rVM (CHIR 0.7 μM on days 0-9 + SHH 300 ng / mL on days 0-9), cVM (CHIR 0.7 μM on days 0-9 + SHH 300 ng / mL on days 0-9 + FGF8b 100 ng / mL on days 9-16), dHB (CHIR 2 μM on days 0-9), and vHB (CHIR 2 μM H9 hESCs (WA09, WiCell Research Institute, Inc.) were differentiated into dFB, rVM, and cVM precursors using a method similar to that of RC17, but with some modifications. The seeding density on day 0 was 15,000 cells / cm³. 2The following patterning factors were added: dFB (N2 + 10 μM SB + 100 ng / mL Noggin on days 0-9, N2 on days 9-11, B27 + 0.2 mM AA + 20 ng / mL BDNF on days 11-16), rVM (N2 + 10 μM SB + 100 ng / mL Noggin + 500 ng / mL SHH + 0.7 μM CHIR on days 0-9, N2 on days 9-11, B27 + 0.2 mM AA + 20 ng / mL BDNF on days 11-16), cVM (N2 + 10 μM SB + 100 ng / mL Noggin + 500 ng / mL SHH + 0.7 μM CHIR on days 0-9, N2 + 100 ng / mL FGF8b on days 9-11, B27 + 0.2 mM AA + 20 ng / mL Cells were treated with BDNF + 100 ng / mL FGF8b (days 11-16). Cells were harvested on day 11 using Accutase (ThermoFisher), or on day 16 using either Accutase, TrypLE (ThermoFisher), or Miltenyi Neural Tissue Dissociation Kit (P) (called papain), all using a 10-minute enzymatic incubation. After dissociation on day 16, the cells were cryopreserved in CryoStor CS10 (Sigma) for later use in flow cytometry. For terminal maturation, cells were retained on laminin-521 coated plates from day 16 and cultured in terminal differentiation medium as described in Nolbrant et al. 2017.

[0191] Creation, sequencing, processing, alignment, and analysis of scRNA-seq data. scRNA-seq of MiSTR tissues was performed as detailed in Rifes et al. 2020. Briefly, cells were loaded lane by lane onto 10X Chromium (10× Genomics) and cDNA libraries were generated according to the manufacturer's instructions. Cells from five regions were mixed in equal proportions and then loaded onto 10X lanes using a 10X V3 chemistry kit. cDNA sequencing was performed on a NextSeq 500 (Illumina) according to the manufacturer's instructions. Post-processing, alignment, and analysis of scRNA-seq data from MiSTR tissues were performed. Uniform manifold approximation and projection (UMAP) was visualized using the RunUMAP() function in Seurat, based on pre-calculated principal components (PCA).

[0192] Flow cytometry For the region-specific panels, we used the same concentrations and manufacturers as publicly available antibodies whenever possible, and employed the highest possible fluorescent dyes. The LRTM1 antibody (KAN Research Institute) was obtained through a material transfer agreement with the supplier. The antibodies were reconstituted according to the manufacturer's instructions. For the region-specific panels, cryopreserved cells patterned to nerve fate were thawed and reconstituted with Buffer A (0.5% (vol / vol) KnockOut® serum replacement (KOSR), Dulbecco's phosphate-buffered saline (DPBS) (-Ca 2+ / -Mg 2+ The cells were resuspended in )) and then centrifuged, followed by buffer B (2.5% (vol / vol) KOSR, DPBS (-Ca 2+ / -Mg 2+The cells were resuspended in )) to a final concentration of 1,000,000 cells / mL. The cells were incubated indirectly with primary antibodies (CORIN, 1:200; LRTM1, 1:20 / 1:200; CNTN2, 1:20; IAP-PE / Vio770, 1:50; ALCAM-PE, 1:100; FOLR1, 1:200 (conjugated with Mix-n-Stain® CF® 568 Dye antibody labeling kit according to manufacturer's instructions); TPBG-PE, 1:50; APCDD1-PE, 1:100) on ice, covered, on a slowly rotating platform for 30 minutes. The cells were then centrifuged at 500xg and 4°C for 5 minutes, and washed three times with buffer A. Secondary antibodies against goat (1:200), mouse (1:200), or donkey (1:500) rats were added to cells labeled with CNTN2, CORIN, and LRTM1, respectively. The cells were then washed as described above. During final resuspension, the cells were resuspended in buffer A + DRAQ7 (1:1,000) and analyzed using BD FACSAria III®.

[0193] Using rVM and cVM progenitor cells from the same batch, rVM vs. cVM panels were performed as described for the region-specific panels above. Cells were incubated with APCDD1-APC (1:100), TPBG (1:200), CORIN (1:200), ZIP8 (1:500), and CD83-PE / Cy7 (1:100) antibodies. Cells stained with TPBG, CORIN, and ZIP8 were then incubated with anti-mouse AF594, anti-rat AF488, and anti-rabbit PE, respectively, as described above. Correction controls were prepared using Ultracomp eBeads™ Plus (BD Biosciences) according to the manufacturer's instructions. Cells were resuspended and incubated with buffer C (0.5% (vol / vol) human serum albumin (HSA), Hanks equilibrium salt solution (HBSS) (-Ca 2+ / -Mg 2+ Wash with buffer D (2.5% (vol / vol) HSA, HBSS (-Ca 2+ / -Mg 2+The cells were stained in )). During final resuspension, the cells were resuspended in buffer C + DRAQ7 (1:1,000) and analyzed with BD FACSAria III (trademark).

[0194] For the intracellular flow cytometry protocol, cells were first prepared, stained with APCDD1-PE antibody, and washed as instructed for the rVM vs cVM panel above. The cells were then diluted to 1,000,000 cells / mL in Buffer E (1% (vol / vol) N-2 (trademark) supplement, CTS (trademark) Neurobasal (trademark) medium). LIVE / DEAD (trademark) Fixable Violet dy (Thermo Fisher) or dimethyl sulfoxide (DMSO) was added, and the sample was incubated at room temperature for 15 minutes. Buffer F (1% (vol / vol) bovine serum albumin (BSA), DPBS (-Ca 2+ / -Mg 2+ After washing with )), the cells were fixed and permeabilized by adding 1X Perm / Wash buffer (BD Biosciences) prepared according to the manufacturer's instructions. After washing twice consecutively with 1× Perm / wash buffer (BD Biosciences) prepared according to instructions, the cells were stored overnight at 4°C in the dark. The cells were then incubated at 4°C for 30 minutes in FOXA2-APC (1:80) and / or OTX2-Vio515 (1:320) antibodies. The cells were washed once with 1x Perm / wash buffer and twice with buffer F. Anti-REA beads (Miltenyi Biotec) were used for FOXA2-APC and OTX2-vio515, ArC-reactive beads (Miltenyi Biotec) for Live / Dead Fixable Violet Dye, and Ultracomp eBeads for APCDD1-PE single stain control. TM Each sample was prepared using Plus (BD Biosciences) as recommended. Cells were analyzed using BD LSRFortessa®, and fully stained dorsal forebrain or ventral hindbrain progenitor cells were used as a biological negative control for FOXA2 analysis. + / OTX2 + The gate was set.

[0195] During FACS, cells were prepared in the same manner as for region-specific panels. However, cells were resuspended in buffer C and stained in buffer D. During final resuspension, when sorting cells on BD FACSAria3® or MACSQuant® Tyto®, cells were resuspended in buffer C + DRAQ7 (1:1,000), and when using BD FACSMelody®, cells were resuspended in buffer C + 100nM DAPI. Sorting for transplantation into SD rats was performed on BD FACSAria3® for 16-day cVM progenitor cells. Sorting for transplantation into nude rats was performed on BD FACSMelody® for a mixture of 16-day cVM, rVM, and vHB progenitor cells combined in a 4:3:3 cell number ratio. Cells were stained with APCDD1-PE antibody, and approximately 50% APCDD1 was detected per viable single cell based on previous flow cytometry analysis of the cells. + Cells were obtained. The top 40% of the viable cells were APCDD1. + Fraction and lower 40% APCDD1 - The fractions were selected. The cells to be transplanted were prepared as described above in Nolbrant et al. 2017 and transplanted immediately after selection.

[0196] Gating was set based on FMO controls collected for each cell population during each analysis, and corrections were determined using single-stain controls. For each sample, ≥10,000 single cells were analyzed. The following common gating strategies were used: (i) FSC-A vs. SSC-A, (ii) FSC-A vs. FSC-W, and (iii) FSC-A vs. live / dead-A. Gating of the region specificity panel was performed using FACSDiva® software, and statistics were subsequently exported. Post-processing, QC, and analysis of the exported .fcs files were performed using the software program FlowJo (version 10.7.2). Results show the percentage of marker-positive cells relative to the population (normal viable single cells). A list of reagents and dilutions used for flow cytometry is shown in Table 1.

[0197] [Table 1-1] [Table 1-2] [Table 1-3]

[0198] qRT-PCR RNA was isolated from neuronal progenitor cells at days 11 and 16 using the RNeasy® Plus Micro Kit (Qiagen). cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher) for RT-qPCR. qRT-PCR was performed using Sybr Green Mastermix (Roche Life Sciences) and primers, with an automated pipetting instrument Bravo (Agilent) or a liquid handler I-DOT (Dispendix). Samples were analyzed on a LightCycler 480 instrument (Roche Life Sciences) using a two-step protocol including a 60°C annealing / extension step. Samples were performed using technical replication, and averaged Ct values ​​were used for calculations. Data are expressed using the DDCt method. Fold change was based on the mean fold change for two housekeeping genes (ACTB and GAPDH) in undifferentiated RC17 hESCs. Heatmap and region panel mRNA expression graph data were normalized with a scaling factor of 100 for the maximum variation metric of each gene. The primers used are summarized in Table 2. Based on qRT-PCR data, only batches of cells that passed rigorous QC criteria were included in the study.

[0199] [Table 2-1] [Table 2-2] [Table 2-3]

[0200] Image acquisition and analysis Images were acquired using the Leica DMI6000 B and PerkinElmer Operetta CLS High-Content Analysis system. (TH in immunohistochemistry) + Cells and HuNu + For cell quantification, Z-stack images were captured at 20x magnification and automatically concatenated. For IHC, graft-derived TH was used. + The cells were counted manually, and HuNu + Cell counts were determined semi-automatically. For immunofluorescence, the number of TH, FOXA2, and LMX1 cells was manually quantified.

[0201] Statistical analysis and diagrams Statistical tests were performed using RStudio. A significance level of 0.05 was used for all statistical tests. Normality was assessed using the Shapiro-Wilk test and frequency distribution histogram testing. Gaussian distributions were analyzed using paired and unpaired t-tests. To determine whether two or more samples of non-normal distributions were statistically significantly different, pairwise Wilcoxon-Mann-Whitney U tests or Dunn's test were used as post-hoc analyses of the Kruskal-Wallis test. Nonlinearity was assessed, and correlations were evaluated using linear regression models.

[0202] result Identifying novel candidate markers for cVM progenitor cells from scRNA-seq data Previous publications identified cell surface markers that label dopaminergic progenitor cells (Bye et al. 2015; Doi et al. 2014; Fathi et al. 2018; Gennet et al. 2016; Kikuchi et al. 2017; Lehnen et al. 2017; Paik et al. 2018; Samata et al. 2016; Sundberg et al. 2013; Yoo et al. 2021) (Table 3).

[0203] To identify candidate cell surface markers that showed the highest specificity for cVM progenitor cells compared to other neural progenitor cells from other brain regions, and in particular to identify rVM cells that do not give rise to DA neurons but are commonly present and very similar, the inventors utilized a previously published microfluidic-based in vitro model of neural tube development that replicates the gradual rostral-caudal patterning of neural progenitor cells from the forebrain to the midbrain and hindbrain (Rifes et al. 2020).

[0204] To identify candidate cell surface markers for cVM fate, we screened scRNA-seq datasets from ventral microfluidic neural tube tissue to find genes specifically expressed in VMs. By screening VM-specific expression patterns, we identified novel cell surface markers APCDD1 and TPBG as candidate markers for common VM progenitor cells. In contrast, vMB / MHB-specific expression was not observed for previously published cell surface markers indicating cVM progenitor cells (Figure 1A). The expression pattern of APCDD1 was validated by immunolabeling in 5-week-old human fetuses, confirming cVM-specific surface marker-like expression (Figure 1B). As expected, APCDD1 was not detected in 11-week-old human fetal VMs, suggesting a transient expression pattern.

[0205] Therefore, scRNA-seq showed that APCDD1 and TPBG are more enriched for VM fate compared to previously published markers, and VM-specific expression of APCDD1 was confirmed in human fetal tissue.

[0206] Benchmarking the regional specificity of published and novel cell surface markers. It was important to benchmark the regional enrichment of newly discovered candidate markers compared to previously published surface markers. For this purpose, hESCs were patterned to 16-day (d16) progenitor cells of the dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral and caudal VM (rVM and cVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB) neural progenitor fate (Figure 3A), and qRT-PCR was performed using panels of rostral-caudal and dorsal-ventral markers. Consistent with scRNA-seq from ventral MiSTR on day 14, APCDD1 and TPBG were shown to be the most enriched markers in the caudal VM DA fate at the transcriptional level compared to previously published surface markers (Figure 3B, Figures 5A-5B). Notably, there was a statistically significant correlation between TPBG and CORIN, but no correlation between APCDD1 and CORIN. Previously published markers ALCAM, CORIN, CD47 / IAP, CNTN2, and LRTM1 showed enriched expression in rVM progenitor cell cultures, while their expression was relatively low in cVM DA progenitor cells. FOLR1 expression was not enriched in VM cultures compared to other region cultures, and FOLR1 did not appear to be a useful marker for VM cells (Figure 3B). Consistent with this finding, mRNA expression of APCDD1 and TPBG showed good correlation with VM markers such as FOXA2, LMX1A, and EN1 (Figures 3C-3E, Figure 5B). Using a linear regression model, APCDD1 expression levels were highly correlated with LMX1A, FOXA2, and EN1 (adjusted R 2 (=0.95, p=1.79e-19), APCDD1 was further justified as a marker of VM DA fate (Figure 3F).

[0207] To further validate these results, we developed and performed a flow cytometry-based region-specific assay by comparing APCDD1 with previously published antibodies against putative cell surface markers indicating VM progenitor cells (Bye et al. 2015; Doi et al. 2014; Fathi et al. 2018; Gennet et al. 2016; Kikuchi et al. 2017; Lehnen et al. 2017; Paik et al. 2018; Samata et al. 2016; Sundberg et al. 2013). For this purpose, regionized neural progenitor cells at day 16 (the same batch used for qRT-PCR) were analyzed for cell surface protein marker expression by flow cytometry using fluorescent dye conjugated antibodies against each of the candidate cell surface markers (Figures 2A-2J). For previously published markers, the same antibodies and similar concentrations used in the publications were employed. Two anti-LRTM1 antibodies were tested at various concentrations and with several secondary antibodies, and LRTM1 + We confirmed that the proportion of cells was low (Figures 2G to 2H).

[0208] Determining the potential impact of dissociation methods To assess the potential impact of dissociation method on the outcomes, cells from all region fates were dissociated with either Accutase, TrypLE, or papain and analyzed by flow cytometry. Factor analysis of mixed data (FAMD) showed that the dissociation method was a relatively small contributor to the overall observed variance (Figure 6B). However, using appropriate parametric or non-parametric multiple comparison tests, the proportion of positive cells for APCDD1, CORIN, and CNTN2 was statistically significantly lower with papain compared to actase and TrypLE (Figures 7A, 6D).

[0209] Based on the proportion of positive cells, APCDD1 and TPBG appeared to be the most specific in labeling the fate of cVM progenitor cells (Figure 7C, Figures 4A-4C). To quantify regional enrichment, we devised a model to determine the specificity index of the VM population by grouping by dissociation method and batch and summarizing the proportion of positive cells for each cVM and non-cVM marker. Using the Benjamini-Hochberg non-parameterized multiple comparison Dunn test with false-positive rate adjustment, it was shown that APCDD1 and TPBG had statistically significantly higher specificity indices compared to most previously published markers, i.e., were more enriched for cVM progenitor cells (Figure 7D). However, for Accutase-dissociated cells and Triple-LE-dissociated cells, TPBG in cVM + The average percentage of cells is APCDD1 + The cell percentage was lower than the average. Using a level-matched paired t-test, TPBG + Due to the large variability of the cells, the difference was determined not to be statistically significant (Figure 4E). In fact, compared to APCDD1, TPBG was used for cVM progenitor cells. + There was statistically significant batch-to-batch variability in the cell proportions (Figure 7E). In addition, the standard error of the positive proportion of cVM progenitor cells in TPBG was increased across all dissociation methods compared to APCDD1 (Figure 4F). Next, the findings were extended to other cell lines to verify that APCDD1 also enriched cVM progenitor cells in the hESC line H9 (Figure 6C).

[0210] Temporal expression of APCDD1 To investigate the temporal expression of these cell surface markers, cVM precursors on day 11 were analyzed using a surface marker flow cytometry panel, and their expression levels were compared to those of the same batch on day 16. For Accutase-dissociated cVM cells, APCDD1 was used. + The percentage of cells was lower on day 11 compared to day 16, and a greater spread of APCDD1 expression was observed on day 11 (94.3 ± 1.6 and 43.5% APCDD1). +(±21.1 SEM, Figure 7B, Figure 4D). This suggests that day 11 is too early to use APCDD1 and TPBG for QC, as markers of the true cVM precursor are not significantly and firmly upregulated until day 16 (this corresponds to the temporal expression in EN1) (Kirkeby et al. 2017; Nolbrant et al. 2017; Rifes et al. 2020).

[0211] Use of APCDD1 to distinguish between rVM progenitor cells and cVM progenitor cells VM progenitor cells are characterized by the co-expression of two regional transcription factors, FOXA2 and OTX2 (Arenas, Denham, and Whiteescusa 2015). Therefore, we investigated the co-expression of APCDD1 and FOXA2 / OTX2 double-positive cells to validate the feasibility of APCDD1 as a QC purity marker for the VM population. To achieve this, we developed a panel in which APCDD1 was first extracellularly labeled with an APCDD1 antibody, followed by intracellular labeling with antibodies against FOXA2 and OTX2. + The proportion of cells is FOXA2 + / OTX2 + APCDD1 correlated highly with the cell proportion (adjusted r squared = 0.95, p = 6.77e11, Figures 7F-7H), confirming that it can be used as an alternative marker to identify VM populations. Next, to identify a potential dual cell surface marker panel that can rapidly and potently distinguish successful batches of dopaminergic cVM precursors for PD cell therapy from unsuccessful batches of rVM precursor cells, different combinations of APCDD1 and TPBG (enriched in cVM populations, Figure 3B, Figures 7C-7D, Figures 4A-4C) were tested with CORIN (enriched in rVM populations, Figure 3B, Figures 4A-4C). Based on this, dual APCDD1 + CORIN -The panel was able to most clearly separate the two VM cell populations, showing that rVM cells were enriched an average of 57 times more than cVM cells. APCDD1+ showed 2-fold cVM enrichment compared to rVM (Figure 8E, Figure 9D). These samples were intentionally included to confirm the possibility that APCDD1 could identify batches that did not meet the QC criteria. + and APCDD1 + Both CORINs appeared to detect batches that did not meet the cVM QC criteria, as indicated by an abnormal percentage of cells within the gate. TPBG was not useful in this regard (Figure 9D, Figure 8E). In summary, APCDD1 is a useful inclusion and / or exclusion marker for QC of VM DA progenitor cell differentiation.

[0212] Consideration APCDD1 and TPBG were the two markers that showed the highest specificity for cVM progenitor cells compared to all previously published cell surface markers. A higher proportion of cVM progenitor cells were positive for APCDD1 compared to TPBG, and the variance of positive cells was lower for APCDD1. In summary, we can conclude that APCDD1 is a more appropriate QC marker for VM DA fate compared to all previously published markers.

[0213] APCDD1 + The proportion of cells is FOXA2 + OTX2 + Highly correlated with cells, APCDD1 + Cells, especially CORIN - When used with cells, it demonstrated high-precision differentiation between rVM progenitor cells and cVM progenitor cells, and detected failed batches of cVM progenitor cells. In summary, these results justify the use of APCDD1 as a marker for VM DA cell differentiation.

[0214] [Table 3-1] [Table 3-2]

Table 3-3

[0215] Example 2 Background As described in Example 1, caudal ventral midbrain (cVM) progenitor cells can be identified by measuring the presence and / or amount of APCDD1 expressed intracellularly. This method can further be used to purify a population of cVM progenitor cells by isolating cells that express APCDD1 to an amount that identifies them as cVM cells from a cell population.

[0216] The inventors evaluated the possibility of isolating cVM cells based on the expression of APCDD1 from a mixed population of cells and evaluated the level of cVM progenitor cell enrichment and the level of non-cVM progenitor cell contamination.

[0217] Materials and Methods Fluorescence-activated cell sorting (FACS) For FACS, cryopreserved progenitor cells patterned toward neuronal fate were thawed and resuspended in wash buffer (0.5% serum, equilibrium salt solution). The cells were centrifuged and then resuspended in staining buffer (2.5% serum, equilibrium salt solution) to a final density of 1,000,000 cells / mL. The cells were incubated with primary antibody on ice for 30 minutes. The cells were washed three times by adding wash buffer, followed by centrifugation. Secondary antibody was added to cells labeled with indirectly conjugated antibody, and the cells were incubated as described above. The washing process was then repeated. Finally, the cells were resuspended in wash buffer + viability dye (1:1,000), analyzed, and sorted on a FACS sorter. The gate was set based on a fluorescence minus one (FMO) control, and corrections were determined using single-stained and unstained controls. Post-processing, QC, and analysis of the collected data were performed using the software program FlowJo (version 10.7.2). Table 1 lists the reagents and dilutions used for flow cytometry and FACS.

[0218] result To evaluate the value of APCDD1 for predicting dopaminergic cell fate, APCDD1 + Cells and APCDD1 - Cells were selected from high-purity batches of cVM progenitor cells, as well as from STEM-PD GMP3 (a batch of cells intended for use in the STEM-PD Phase I clinical trial) and STEM-PD GMP5 (Figures 8A-8B). qRT-PCR analysis was performed to identify APCDD1 + Cell sorting enriches genes that indicate cVM fate, such as ENEN1, FOXA1, LMX1A, and SHH, while genes that mark contaminated cell populations (i.e., NKX2-1, ISL1, and PITX2) are enriched by APCDD1 - Enrichment was demonstrated in the population (Figure 8C). To evaluate the maturation capacity of cells sorted on day 16, APCDD1 + Cells and APCDD1 -When the cells were further seeded in vitro for terminal maturation, after 42 days in vitro, only the APCDD1 + cultures had TH neurons with typical neuromorphology + and were rich in neurons (Figure 8D).

[0219] APCDD1 + To test whether sorting of cells can rescue mispatterned cVM batches, based on previous flow cytometry analysis of the cells, approximately 50% of APCDD1 + cells were combined with progenitor cells patterned in cVM, rVM, and vHB. Then the cells were sorted for APCDD1 + cells and APCDD1 - cells (Figure 10A - B). On day 18 in vitro, many SOX1 - cells and ISL1 / 2 + cells were observed by ICC. Conversely, at the same time point, few SOX1 + cells were present in APCDD1 + -derived cells, and ISL1 / 2 + cells were completely removed. Similar to the sorting results of a pure batch of cVM progenitor cells, a statistically significant enrichment was observed in the expression levels of genes indicating the VM DA fate in the APCDD1 + fraction, while contaminating non - VM DA genes (when expressed) were depleted (Figure 10C). +

[0220] Example 3 As described in Examples 1 and 2, caudal ventral midbrain (cVM) progenitor cells can be identified by measuring the presence and / or amount of APCDD1 expressed intracellularly, and cells expressing APCDD1 to an amount that identifies them as cVM progenitor cells can be further used to purify a population of cVM progenitor cells by isolating them from a cell population. The isolated cVM progenitor cells can be further used in cell replacement therapy.

[0221] ​material and method Animal experiments All procedures were carried out in accordance with European Union Directive (2010 / 63 / EU) and approved by the local ethics committee of Lund University and the Swedish Ministry of Agriculture (Jordbruksverket). Adult, female, athymogenic nude rat (Hsd:RH-Foxn1) rnu Rats were purchased from Envigo and housed in a 12:12 light-dark cycle with free access to food and water. For all surgical procedures, rats (>225g) were anesthetized by intraperitoneal injection of a 20:1 or 3:2 mixture of fentanyl-domitol or ketaminol-domitol (Apoteksbolaget), according to body weight. Unilateral lesions were induced in the rats by intracranial injection of 10.5 μg of 6-hydroxydopamine into the medial forebrain bundle. The extent of the lesions was assessed by an amphetamine-induced turnover test. For this purpose, 3.5 mg / kg of amphetamine was administered by intraperitoneal injection, and the median net turnover per minute was determined over 90 minutes. Frozen RC17 hESCs were prepared as described above (Kirkeby et al. 2012; Nolbrant et al. 2017). As previously mentioned, 200,000–240,000 cells were injected depending on the yield from FACS (Tiklova et al. 2020). Nude rats were injected at striatal coordinates AP, +0.9 / +1.4, ML, -3.0 / -2.6, DV, -5.0 / -4.0. SD rats were administered 10 mg / kg of cyclosporine by daily intraperitoneal injection two days before transplantation and until euthanasia 18 weeks after transplantation.

[0222] Organizational handling Rat brains were fixed in 4% (wt / vol) paraformaldehyde by perfusion according to a standard protocol. After removal and fixation overnight, the brains were dehydrated in 25% (wt / vol) sucrose. The brains were serially sectioned into 1 / 8 series with a thickness of 35 μm using a cryomicrotome (Leica) and stored at -20°C in buffer G (13 mM NaH2PO4, 38 mM Na2HPO4 30% (vol / vol), glycerol 30% (vol / vol)). Fetal tissue was sectioned to a thickness of 12 μm and stored on glass slides at -20°C.

[0223] Immunohistochemistry (IHC) IHC was performed on free suspension sections, placed in mesh wells in 12-well plates for most steps. Between each step, sections were washed three times in PBS for 5 minutes each. For antigen recovery, sections were incubated in Buffer H (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0, H2O) followed by incubation at 80°C. Quenching was performed by incubation in Buffer I (10% (V / V) methanol, 3% (V / V) H2O2, PBS). Sections were then blocked in Buffer J (0.244% V / V Triton-X, 5% (V / V) species-specific serum, PBS). For specific markers, sections were then blocked by incubation in avidin / biotin solution for 2 × 15 minutes, according to the manufacturer's instructions (Vector laboratories). Next, the sections were incubated overnight with serum-diluted primary antibody. Following this, the sections were incubated in serum for 15 minutes, followed by incubation in biotinylated secondary antibody for 1 hour. Then, the sections were incubated for 1 hour in ABC horseradish peroxidase complex (Vector Laboratories) prepared as instructed (Vector Laboratories). Subsequently, the sections were incubated for an appropriate time (1–3 minutes) in 0.5 mg / mL DAB solution + 0.125% (vol / vol) H2O2 or in DAB substrate kit solution (Vector Laboratories). Due to the high signal-to-noise ratio, to visualize tissue morphology, DAB / nickel-labeled sections were counterstained with Gill II hematoxylin for 1 minute, followed by two washes of 1 minute each. The sections were mounted, dehydrated, and coverslip-attached using appropriate mounting media according to the standard protocol. A list of reagents and dilutions used for IHC and ICC is shown in Table 1.

[0224] Immunofluorescence Immunofluorescent labeling on rat tissues was performed in the same manner as IHC, but quenching and avidin / biotin blocking were excluded. Slides with fetal tissues were thawed to room temperature and then immersed in PBS for a short time. Sections were blocked in Buffer J for 30 minutes and then incubated overnight at 4 °C in primary antibody diluted with serum. Next, the sections were washed in PBS for 30 minutes. Then, the sections were incubated in secondary antibody diluted with serum (1:500) for 30 minutes. Then, the sections were washed twice in PBS for 30 minutes each. The sections were coverslipped using FluorSave™ (Sigma Aldrich).

[0225] Image acquisition TH in DAB-developed IHC sections + Cells and HuNu + For cell quantification, five Z-stack images (at approximately 3.0 μm intervals) were captured at bright field, 20x magnification, and automatically stitched using Leica DMI6000B with the LAS-X software program. ICC images were acquired with the same microscope, and immunofluorescent images were acquired with an Operetta CLS High-Content Analysis System (PerkinElmer) using a 20x confocal objective lens with six Z-stacks (at approximately 3.4 μm intervals).

[0226] Manual and semi-automatic image analysis DAB-labeled TH + Cells and immunofluorescently labeled TH + , LMX1A + , and FOXA2 + The numbers of single-positive, double-positive, and triple-positive cells for, were manually counted using the software program Fiji (version 2.1.0). HuNu +Cells were semi-automatically counted using the same program. For this purpose, the background was subtracted, the image was binarized by Otsu's method (Smith et al. 1979), and then the particles were segmented using the built-in watershed algorithm. The number of cells was quantified using the "Analyze Particles" option with a particle size range of 0.05 to infinity. TH per animal + The cell yield was TH + The total number of cells was divided by HuNu + the total number of cells, or TH per 100,000 transplanted cells + shown as the estimated total number of cells.

[0227] Convolutional Neural Network-based Automated Image Analysis Pipeline - SIMPLIcity A multiplexed image analysis pipeline was developed based on the SIMPLI pipeline (Bortolomeazzi et al. 2022) to perform preprocessing, single-cell level data extraction (segmentation) using StarDist (Schmidt et al. 2018), and downstream analysis. The pipeline was named SIMPLIcity.

[0228] Images exported from the Operetta Harmony software were processed using Fiji's PerkinElmer Operetta Stitcher (4.1) to reconstruct them for downstream analysis. The stacks were projected using standard deviation projection and exported as single-channel images. The channels were manually cut into 512×512 or 1024×1024 patches and then processed and analyzed independently. The original CellProfiler4 (Sirling et al. 2021) preprocessing was replaced. Instead, for bright-field images, SIMPLI's built-in pixel normalization was used, followed by median filtering to remove salt-and-pepper noise, and then Gaussian noise was removed using a non-local denoising algorithm. Finally, image contrast was enhanced with contrast-limited adaptive histogram equalization. Images acquired with Operetta CLS were preprocessed by normalizing pixel values ​​while maintaining image quality. In addition, the preprocessing script performed thresholding of images using a user-specified thresholding algorithm. To improve segmentation accuracy, the model was trained with several markers. Images were manually annotated using the Fiji Labkit (v.0.3.5) (Arzt et al. 2022) plugin to create corresponding binary masks. To increase the training set size, additional datasets from the StarDist GitHub page were included for training the HuNu and LMX1A models. The entire dataset consisted of 497 single-channel images. Training speed, batch size, number of epochs, and dropout were adjusted to achieve a stable learning process and optimal model performance. Mean absolute error, StarDist's default loss function, was used to ensure a precise learning process. Scripts provided by StarDist were used for training. The HuNu and LMX1A models were trained on the Kebnekaise supercomputer at HPC2N using Intel Xeon E7-8860v4 GPUs.After obtaining a model with good performance, the remaining markers were trained using forward learning from the ZeroCostDL4Mic platform (von Chamier et al. 2021), which is compatible with the StarDist network. A comprehensive LMX1A-TH model was trained to segment both the LMX1A and TH channels.

[0229] To improve the accuracy of image segmentation, each channel was segmented separately instead of merging images before segmentation. A slightly modified version of the original Nextflow pipeline was used. To ensure an interoperability-enabled pipeline, the Singularity container was replaced with a Docker container hosted on Docker Hub to manage dependencies. Singularity can be used as a command-line and web-based tool. Web application dependencies are managed in a Conda environment.

[0230] result Sorting with APCDD1 generates DA-rich grafts after transplantation and removes contaminating cell populations. To validate the predictive value of APCDD1 for in vivo results, we combined cVM, rVM, and vHB patterning precursors as described above. Subsequently, APCDD1 + Cells and APCDD1 - Cells were selected and then transplanted into Parkinson's disease nude rats (Figure 11A). 28 weeks after transplantation, the amphetamine-induced turnover test was used to determine if APCDD1 + Animals injected with cells showed complete behavioral recovery, but APCDD1 - No improvement was observed in rats transplanted with the cells (Figure 11H). APCDD1 + Cell-derived grafts are APCDD1 - Compared to the graft, there were statistically significantly more TH cells per transplanted cell. + The cells contained (2233±532 SEM vs. 292±43 total TH cells per rat).+ Cells, p=0.018), this is 664% TH + This corresponds to the average increase in cells (Figure 11F). Consistent with this finding, human nuclear antigen (HuNu) + More TH per cell + Neurons were present (Figure 11G). However, the APCDD1-derived graft showed many highly expressed TH cells. + The cells had a morphology similar to that of non-A9 DA neurons (Figure 11D', Figure 11-E''). Based on this, the TH group selected for APCDD1 was determined. + The cell belongs to a biologically distinct subtype of TH neuron, APCDD1 - TH in the graft + It can be assumed that at least a subset of the cells were not true VMDA neurons. To confirm this, since the pre-trained model failed to accurately segment the objects, an image analysis pipeline based on SIMPLI (Bortolomeazzi et al. 2022) was developed. By training the model and then using import learning, TH + We were able to accurately identify the cells. After segmentation, we compared the frequency distribution of pixel intensity values. APCDD1 - TH in the graft + A subset of cells is APCDD1 + It was shown that the TH pixel intensity was statistically significantly increased compared to the origin graft. In fact, APCDD1 + The derived graft was highly TH-expressing. + Lacking this subset of cells, TH + This suggests that the cell subsets are essentially different among the APCDD1 sorting groups. Further characterization of graft composition was performed by determining the proportion of cells co-expressing TH / FOXA2 / LMX1A. + The originating graft showed a statistically significant increase in the ratio of TH / LMX1A / FOXA2 triple-positive cells (p=0.033, Figure 12C).

[0231] In summary, both APCDD1 selection groups showed TH after transplantation into Parkinson's disease rats. + Neurons were produced, but APCDD1 + While only cells predict functional VM DA neurons, APCDD1 - The cells produced non-A9-like DA neurons that did not result in improvement of behavioral disorders.

[0232] In summary, Examples 1-3 of this specification demonstrate that APCDD1 is a novel marker for cVM progenitor cells and is useful for quality control and purification of cVM progenitor cells for use in clinical trials and therapeutics. APCDD1 is highly specific to cVM precursors and can therefore be used to isolate these cells with higher reproducibility and purity than previously identified markers.

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Claims

1. A method for identifying one or more caudal ventral midbrain (cVM) progenitor cells, (i) A step of providing one or more cells, (ii) The step of measuring the presence and / or amount of adenomatosis polyposis coli down-regulated 1 (APCDD1) expressed by one or more cells, comprising or consisting of the steps: A method in which the presence and / or amount of APCDD1 expressed by the one or more cells indicates that the one or more cells are cVM progenitor cells.

2. The method described above is for enriching a population of cVM progenitor cells, and the method described above is (iii) The method according to claim 1, further comprising the step of isolating one or more cells identified as cVM progenitor cells to obtain cVM progenitor cells or a population enriched with cVM progenitor cells.

3. The method according to claim 1 or 2, wherein the one or more cells provided in step (i) are neural progenitor cells, optionally, the one or more cells provided in step (i) are neural progenitor cells obtained from fetal tissue or differentiated from stem cells, optionally, the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ES).

4. The method according to any one of claims 1 to 3, wherein the method is for quality control during the preparation of cells for cell replacement therapy, and / or the method is for providing cVM progenitor cells for cell replacement therapy, and / or the method is for determining the suitability of cells for use in therapy, wherein the therapy is optionally cell replacement therapy.

5. The method according to any one of claims 1 to 4, wherein the presence and / or amount of APCDD1 is measured using a first binding site specific to APCDD1, and optionally, the measurement is performed using a second binding site specific to the first binding site, and / or the first and / or second binding sites are an antibody or an antigen-binding fragment thereof, and / or the first and / or second binding sites are fluorescently labeled.

6. The method according to any one of claims 1 to 5, wherein the measurement of the presence and / or amount of APCDD1 is performed by flow cytometry, qRT-PCR, RNA sequencing, or other RNA detection methods, immunostaining, or chromatography, and optionally, the measurement of the presence and / or amount of APCDD1 and / or the isolation of cells expressing APCDD1 is performed by flow cytometry, and optionally, the flow cytometry is fluorescence-activated cell sorting (FACS) or magnetoaffinity cell sorting (MACS).

7. The method according to any one of claims 1 to 6, wherein the method is for the treatment of a neurological disease or condition, and optionally the neurological disease or condition is Parkinson's disease.

8. The method according to any one of claims 1 to 7, wherein the cVM progenitor cells identified by the method can differentiate into ventral midbrain dopaminergic neurons.

9. The method according to any one of claims 1 to 8, wherein the cVM progenitor cells identified by the method also express one or more of the following markers: FOXA2, OTX2, LMX1A, and EN1, and / or the cVM progenitor cells identified by the method do not express one or more of the following markers: CORIN and CNTN2.

10. The method according to any one of claims 1 to 9, wherein the cVM progenitor cells also express FOXA2 and / or OTX2, and the method further comprises measuring the presence and / or amount of FOXA2 and / or OTX2 expression in one or more cells, wherein the presence and / or amount of APCDD1 and FOXA2 and / or OTX2 on or inside the one or more cells indicates that the one or more cells are cVM progenitor cells, and / or the cVM progenitor cells do not express CORIN, and the method further comprises measuring the presence and / or amount of CORIN on the surface of one or more cells, wherein the presence and / or amount of APCDD1 expression on one or more cells, and the absence and / or amount of CORIN expression on one or more cells, indicates that the one or more cells are cVM progenitor cells.

11. The aforementioned cVM progenitor cells, (a) APCDD1 positive and CORIN negative; (b) APCDD1 positive, FOXA2 positive, and OTX2 positive; (c) The method according to any one of claims 1 to 10, having one of the marker profiles of PCDD1-positive, CORIN-negative, FOXA2-positive, and OTX2-positive.

12. The method according to any one of claims 1 to 11, wherein the method can distinguish between cVM progenitor cells and neural progenitor cells, or between cVM progenitor cells and one or more nerve cells from the lineages of the dorsal forebrain (dFB), ventral forebrain (vFB), dorsal midbrain (dMB), rostral ventral midbrain (rVM), dorsal hindbrain (dHB), and ventral hindbrain (vHB), and preferably the method can distinguish between cVM progenitor cells and rVM progenitor cells or rVM cells.

13. cVM progenitor cells or populations for use in a method of treating a neurological condition or disease of a subject requiring treatment, wherein the method comprises performing the method according to any one of claims 1 to 12 and administering a therapeutically effective amount of identified cVM cells to the subject, optionally, the neurological condition or disease is Parkinson's disease, and optionally, the use comprises administering the cells or populations to the brain of the subject.

14. Use of APCDD1 as a biomarker for identifying and / or enriching cVM progenitor cells from a population containing one or more cells.

15. Use of an APCDD1-specific binding site for identifying and / or enriching cVM progenitor cells from a population of one or more cells.

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