Methods for Providing Cell Populations Enriched for Neurons and Their Precursors - Patent application

Abstract

The present invention relates to a method for providing a cell population enriched for neurons and / or their precursors, comprising obtaining a cell population comprising neuronal precursors and selecting positive cells, which indicate whether the cells have a high propensity to develop into neurons.

Description

[Technical field]

[0001] The present invention relates generally to the field of stem cells, such as human pluripotent stem cells (hPSCs). Specifically, methods are provided for enriching cell populations in stem cell-derived neurons and their precursors. [Background technology]

[0002] Human pluripotent stem cells have the potential to revolutionize the treatment of a variety of intractable diseases and disorders of the human body. Treatments include cell replacement therapy for neurological conditions such as Parkinson's disease. However, for such treatments to become viable, the development of in vitro methods to artificially produce stem cell-derived products for their delivery to the central nervous system (CNS) is required.

[0003] Differentiation of hPSCs into defined cell types is a difficult process to control, and often the progeny generated from a particular protocol are heterogeneous and contain a mixture of cell types. Currently, several methods have been described for differentiating hPSCs into neural stem cells and subsequently into neurons. However, all protocols generate heterogeneous populations, i.e., not all cells are necessarily neuronal progenitors and may specialize into different cell types, or the asynchronous nature of in vitro differentiation and in vivo development results in different cell stages existing simultaneously. Heterogeneous cultures are not optimal when transplantation of a single / specific cell type is required, such as to investigate disease and normal biology in a single / specific cell type of interest, or in the context of cell replacement therapy, and especially when neurons are the intended therapeutic cell type. Transplantation of mixed populations of cells reduces the safety and efficacy profile of cell therapy and increases the risk of undesired side effects from undesired cell types. Methods for separating cell populations to obtain higher purity neurons and their precursors are described in US Pat. No. 5,333,635. However, there remains a need for additional methods for obtaining enriched cell populations that contain neural cells with an increased propensity to develop into neurons.

[0004] It is therefore an object of the present invention to improve the homogeneity of cell populations comprising neural cells, towards cell types committed to developing into neurons, such as ventral midbrain neurons, and their precursors. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2021 / 042027 Summary of the Invention

[0006] The above-outlined objectives are achieved by the aspects of the present invention. In addition, the present invention can also solve further problems that will become apparent from the disclosure of the exemplary embodiments.

[0007] A first aspect of the present invention relates to a method for providing a cell population enriched for neurons and / or their precursors, such as intermediate progenitor cells, comprising obtaining a cell population comprising neuronal precursors and detecting the activity of PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR8. selecting cells positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

[0008] The inventors have found that it is possible to increase the proportion of cells in a cell population that have a tendency to develop into neurons by isolating certain cells, which are isolated by selecting positively and / or negatively on certain surface markers. Furthermore, the inventors have found that the timing of selecting and / or excluding cells can improve the outcome, especially when applied to cell populations that differentiate into neural cells such as neurons. Specifically, cells that develop into neurons, such as in an in vitro differentiation process, progress through different stages, first the cells become neural stem cells, and then turn into neurons after further differentiation into intermediate progenitor cells. The inventors have found that certain surface markers that indicate whether a neural stem cell has a neuronal fate are only reliably expressed after the neural stem cell has further developed. In a cell population undergoing differentiation into a neuronal identity, individual cells are at different developmental stages, and the cell population may include subpopulations of cells such as pluripotent stem cells, neural stem cells, intermediate progenitor cells, and neurons. Furthermore, individual cells that transition from one stage to another may express markers indicative of both stages. The present inventors have found that cell selection and / or exclusion is preferably applied to the cell population, and at least a portion of the cells are further developed from being neural stem cells. The expression marker SOX2 is characteristic of neural stem cell identity. As neural stem cells further develop, the cells at a certain point no longer express SOX2. This is consistent with certain surface markers being expressed or silenced on cells with neuronal fate. Thus, the decrease in the expression of SOX2 in the cell population indicates that neural stem cells are further developed, and can be used to identify the best opportunity to isolate cells that tend to develop into neurons.

[0009] Accordingly, one aspect of the present invention is a method for isolating neurons and / or their precursors from a cell population, comprising obtaining a cell population comprising neural stem cells expressing the marker SOX2, further culturing the cell population until expression of SOX2 in the cell population is reduced, and detecting the presence of PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, selecting cells positive for one or more of the markers selected from CLDN5, CHRNA5, and GPR85, and / or excluding cells positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

[0010] Alternatively, the timing of cell selection and / or exclusion may be established based on markers indicating that a portion of neural stem cells have further developed into intermediate progenitor cells and / or neurons. In one embodiment, the cell population is first obtained by differentiating pluripotent stem cells (PSCs) into neural stem cells, and cell selection may be applied once the culture has progressed to a certain stage in differentiation consisting of unipotent and / or multipotent cells, such as intermediate progenitor cells, and / or terminally differentiated cells, such as neurons. Thus, in one embodiment, when cells are selected and / or excluded, at least 5% of the cell population expresses a marker of intermediate progenitor identity. In one embodiment, when selecting and / or eliminating cells, at least 5% of the cell population is positive for one or more of the following markers: ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2, and NHLH1.The inventors have identified the expression of these markers as a strong indication that the cell population has matured to a stage where positive and / or negative selection of cells with a strong tendency for neuronal development can be performed based on the identified surface markers.Cells before developing into intermediate progenitor cells may not fully express or silence the identified surface markers for cell isolation to be performed.

[0011] The present inventors have found that cell selection and / or exclusion can be applied to all regions of the brain to increase neurons in cell populations based on identified surface markers. Specifically, we found that exclusion of cells expressing the surface marker CD47 increases the percentage of neurons in cell populations that are committed to becoming ventral midbrain neural system cells. Furthermore, we found that exclusion of cells expressing the surface marker CD99 increases the percentage of neurons in cell populations that are committed to the forebrain, hindbrain, or spinal cord.

[0012] In another aspect of the invention, a cell population of neurons and / or their precursors is provided, wherein at least 50% of the cells are positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85, and / or less than 50% of the cells are positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1. The cell population according to the above is characterized by an increased propensity to develop into neurons.

[0013] Another embodiment relates to a composition comprising the cell population according to the invention. In a further embodiment, a cell population or composition according to the invention is disclosed for use as a medicament, such as in the treatment of neurological conditions. Transplantation of a homogeneous cell population enhances the safety and efficacy profile of cell therapy and reduces the risk of undesirable side effects from undesirable cell types. Treatment with a cell population or composition according to the invention increases the proportion of cells bearing neurons and / or their precursors, thereby making them highly suitable for the prevention or treatment of conditions requiring the administration of such cells. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 shows a simplified schematic illustrating the differentiation steps from hPSCs (A), to early NSCs (B), to heterogeneous intermediate stage cultures (C), and finally, to terminally differentiated cell types (D). The time frames during which scRNA-seq analysis was performed are marked with boxes (E). [Figure 2A]Figure 2A shows a Venn diagram obtained by scRNA-seq analysis of hESCs, showing the percentage of cells expressing one or more of POU5F1, SOX2, ASCL1, and INA. [Figure 2B] Figure 2B shows a t-SNE plot obtained by scRNA-seq analysis of hESCs. The plot shows the expression profile of genes POU5F1, SOX2, ASCL1, and INA. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 3A] Figure 3A shows a Venn diagram obtained by scRNA-seq analysis of DIV16 ventral midbrain NSCs, showing the percentage of cells expressing one or more of POU5F1, SOX2, ASCL1, and INA. [Figure 3B] Figure 3B shows a t-SNE plot obtained by scRNA-seq analysis of DIV16 ventral midbrain NSCs. The plot shows the expression profile of genes POU5F1, SOX2, ASCL1, and INA. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 4A] Figure 4A shows a Venn diagram obtained by scRNA-seq analysis of DIV24 ventral midbrain neural system cells, showing the percentage of cells expressing one or more of POU5F1, SOX2, ASCL1, and INA. [Figure 4B] Figure 4B shows a t-SNE plot obtained by scRNA-seq analysis of DIV24 ventral midbrain neural cells. The plot shows the expression profile of genes POU5F1, SOX2, ASCL1, and INA. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Diagram 5]Figure 5 shows the integrated UMAP plot obtained by scRNA-seq analysis of DIV22, 24, and 26 ventral midbrain nervous system cells. The plot shows the expression profile of genes LMX1A, SOX2, ASCL1, and INA. LMX1A-negative cells are marked with circles. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 6] Figure 6 shows the integrated UMAP plot obtained by scRNA-seq analysis of DIV22, 24, and 26 ventral midbrain nervous system cells. The plot shows the expression profile of genes ADGRG1, EPHB2, GAP43, ADCYAP1, C1QL1, and NRXN1. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 7] Figure 7 shows the integrated UMAP plot obtained by scRNA-seq analysis of DIV22, 24, and 26 ventral midbrain nervous system cells. The plot shows the expression profile of genes FZD1, CACNA2D1, DDC, GPR85, and LRRC4C. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 8] Figure 8 shows the integrated UMAP plot obtained by scRNA-seq analysis of DIV22, 24, and 26 ventral midbrain neural system cells. The plot shows the expression profile of genes TNFRSF21, TRPM8, FREM2, DLL3, DLL1, CHRNA5, and CLDN5. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 9]Figure 9 shows an integrated UMAP plot obtained by scRNA-seq analysis of DIV22, 24, and 26 ventral midbrain nervous system cells. The plot shows the expression profile of genes SLIT2, NTN1, CMTM7, CMTM8, CD36, and CD47. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 10] Figure 10 shows the integrated UMAP plot obtained by scRNA-seq analysis of DIV22, 24, and 26 ventral midbrain nervous system cells. The plot shows the expression profile of genes CD99, TNFRSF1A, TMEM123, LRP10, HLA-A, and RAMP1. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 11] Figure 11 shows the integrated UMAP plot obtained by scRNA-seq analysis of DIV22, 24, and 26 ventral midbrain neural cells. The plot shows the expression profile of genes CMTM6, TNFRSF12A, CRLF1, ITGB8, and CD83. Expression levels are expressed as UMI per transcript (UTP) and displayed using a color gradient ranging from no expression (very light gray) to high expression (dark gray). [Figure 12] Figure 12 outlines a fluorescence-activated cell sorting (FACS) strategy for obtaining populations enriched for CD47-positive or CD99-negative cells. The figure shows hierarchical gates that first (A) select events representing cells and exclude debris, second (B) select single cells and exclude doublets, third (C) select live cells and exclude dead cells, and finally (D) select CD47-positive or CD47-negative populations. [Figure 13] Figure 13 shows the post-sorting purity immediately after FACS. The purity of CD47 positive (A) and CD47 negative (B) cells was assessed by re-analysing the samples on a flow cytometer. [Figure 14] FIG. 14 shows the percentage of cells expressing CD47 in sorted CD47-positive (A) and CD47-negative (B) samples analyzed by intracellular flow cytometry immediately after FACS. [Figure 15] FIG. 15 shows the percentage of cells expressing the neural stem cell marker SOX2 in sorted CD47 positive (A) and negative (B) samples analyzed by intracellular flow cytometry immediately after FACS. [Figure 16] FIG. 16 shows the percentage of cells expressing the proliferation marker Ki67 in sorted CD47 positive (A) and negative (B) samples analyzed by intracellular flow cytometry immediately after FACS. [Figure 17] FIG. 17 shows the percentage of cells expressing the midbrain floor plate marker FOXA2 in sorted CD47 positive (A) and negative (B) samples analyzed by intracellular flow cytometry immediately after FACS. [Figure 18] FIG. 18 shows a bar graph summarizing the flow cytometry results obtained by intracellular flow cytometry immediately after FACS of CD47 positive and negative cells. [Figure 19] Figure 19 shows cell morphology in low (A, B, C) and high magnification (Ai, Bi, Ci) phase contrast images obtained 24 hours after FACS of sorted CD47 positive (A, Ai), CD47 negative (B, Bi) and "pass-through" control cells (C, Ci). Scale bar: 50 μm. [Figure 20] Figure 20 shows cell morphology in low (A, B, C) and high magnification (Ai, Bi, Ci) phase contrast images of sorted CD47 positive (A, Ai), CD47 negative (B, Bi) and "pass-through" control cells (C, Ci) obtained 5 days after FACS. Scale bar: 50 μm. [Figure 21]Figure 21 shows representative immunofluorescence images of sorted CD47 positive cells (A), CD47 negative cells (B), and "pass-through" control cells (C) stained for Ki67 and DAPI 5 days after FACS. Scale bar: 50 μm. [Figure 22] Figure 22 shows representative immunofluorescence images of sorted CD47 positive cells (A), CD47 negative cells (B), and "pass-through" control cells (C) stained for FOXA2 and DAPI 5 days after FACS. Scale bar: 50 μm. [Figure 23] Figure 23 shows representative immunofluorescence images of sorted CD47 positive cells (A), CD47 negative cells (B), and "pass-through" control cells (C) stained for TH and DAPI 5 days after FACS. Scale bar: 50 μm. [Figure 24] Figure 24 outlines a fluorescence-activated cell sorting (FACS) strategy for obtaining populations enriched for CD99-positive or CD99-negative cells. The figure shows hierarchical gates that first (A) select events representing cells and exclude debris, second (B) select single cells and exclude doublets, third (C) select live cells and exclude dead cells, and finally (D) select CD99-positive or CD99-negative populations. [Diagram 25] Figure 25 shows the post-sorting purity immediately after FACS. The purity of CD99 positive (A) and CD47 negative (B) cells was assessed by reanalysing the samples on a flow cytometer. [Figure 26] FIG. 26 shows the post-FACS viability of cells sorted on the basis of CD99 expression. [Figure 27] Figure 27 shows cell morphology in low (A, B, C) and high magnification (Ai, Bi, Ci) phase contrast images of sorted CD99 positive (A, Ai), CD99 negative (B, Bi) and "pass-through" control cells (C, Ci) obtained 24 hours after FACS. Scale bar: 50 μm. [Figure 28]Figure 28 shows cell morphology in low (A, B, C) and high magnification (Ai, Bi, Ci) phase contrast images obtained 48 hours after FACS of sorted CD99 positive (A, Ai), CD99 negative (B, Bi) and "pass-through" control cells (C, Ci). Scale bar: 50 μm. [Figure 29] Figure 29 shows cell morphology in low (A, B) and high magnification (Ai, Bi) phase contrast images obtained 5 days after FACS of sorted CD99 positive (A, Ai) and CD99 negative (B, Bi) cells. Scale bar: 50 μm. [Diagram 30] Figure 30 shows representative immunofluorescence images of sorted CD99 positive cells (A), CD99 negative cells (B), and "pass-through" control cells (C) stained for Ki67 and DAPI 48 hours after FACS. Scale bar: 50 μm. [Diagram 31] Figure 31 shows representative immunofluorescence images of sorted CD99 positive cells (A) and CD99 negative cells (B) stained for Ki67 and DAPI 12 days after FACS. Scale bar: 50 μm. [Diagram 32] Figure 32 shows representative immunofluorescence images of sorted CD99 positive cells (A), CD99 negative cells (B), and "pass-through" control cells (C) stained for FOXA2 and DAPI 48 hours after FACS. Scale bar: 50 μm. [Diagram 33] Figure 33 shows representative immunofluorescence images of sorted CD99 positive cells (A), CD99 negative cells (B), and "pass-through" control cells (C) stained for TH and DAPI 48 hours after FACS. Scale bar: 50 μm. [Diagram 34] FIG. 34 shows the percentage of DIV25 hPSC-derived midbrain cells expressing only CD47 (Q3) or CD99 (Q1), or both CD47 and CD99 (Q2). [Diagram 35]Figure 35 shows quantification of Ki67+ cells by immunofluorescence after FACS. The graph shows the percentage of Ki67+ cells among total cells as identified by DAPI in unsorted samples and in CD47-negative and CD47-positive sorted samples. [Diagram 36] Figure 36 shows the quantification of SOX2+ cells by immunofluorescence after FACS. The graph shows the percentage of SOX2+ cells in total cells identified by DAPI in unsorted samples and in CD47-negative and CD47-positive sorted samples. [Figure 37] Figure 37 shows a bar graph of the results from intracellular flow cytometry analysis performed immediately after FACS. The graph shows the percentage of CD47+ cells out of total live cells in unsorted samples, as well as samples sorted based on CD47 (CD47- and CD47+), and samples sorted based on CD47 in combination with CD99 (CD47+CD99+ and CD47-CD99-). [Figure 38] Figure 38 shows a bar graph of the results from intracellular flow cytometry analysis performed immediately after FACS. The graph shows the percentage of CD99+ cells out of total live cells in unsorted samples, as well as samples sorted based on CD47 (CD47- and CD47+), and samples sorted based on CD47 in combination with CD99 (CD47+CD99+ and CD47-CD99-). [Figure 39] Figure 39 shows a bar graph of the results from intracellular flow cytometry analysis performed immediately after FACS. The graph shows the percentage of SOX2+ cells among total live cells in unsorted samples, as well as samples sorted based on CD47 (CD47- and CD47+), and samples sorted based on CD47 combined with CD99 (CD47+CD99+ and CD47-CD99-). [Diagram 40]Figure 40 shows a bar graph of the results from intracellular flow cytometry analysis performed immediately after FACS. The graph shows the percentage of Ki67+ cells out of total live cells in unsorted samples, as well as samples sorted based on CD47 (CD47- and CD47+), and samples sorted based on CD47 combined with CD99 (CD47+CD99+ and CD47-CD99-). [Diagram 41] Figure 41 shows a bar graph of the results from intracellular flow cytometry analysis performed immediately after FACS. The graph shows the percentage of INA+ cells out of total live cells in unsorted samples, as well as samples sorted based on CD47 (CD47- and CD47+), and samples sorted based on CD47 in combination with CD99 (CD47+CD99+ and CD47-CD99-). [Diagram 42] FIG. 42 shows cell morphology in phase contrast images obtained 48 hours after MACS of sorted CD47 negative cells (A) and unsorted control cells (B). [Diagram 43] Figure 43 shows the post-sorting purity immediately after MACS. The purity of CD47 positive cells (A) and CD47 negative cells (B) was assessed by flow cytometry analysis. [Diagram 44] FIG. 44 shows a bar graph summarizing the flow cytometry results obtained by intracellular flow cytometry immediately after MACS of unsorted cells (black bars) and CD47 negative cells (white bars). [Diagram 45] FIG. 45 shows histograms depicting the percentage of DIV35 hPSC-derived forebrain cells expressing CD99 (A) and CD47 (B) as determined by flow cytometry. [Diagram 46] FIG. 46 shows the percentage of co-expression of SOX2 with Ki67 (A) and INA (B) in DIV35 forebrain cells as measured by flow cytometry. [Figure 47]FIG. 47 shows the percentage of co-expression of SOX2 with CD47 (A) and CD99 (B) in DIV35 forebrain cells as measured by flow cytometry. [Figure 48] FIG. 48 shows the percentage of Ki67 co-expression with CD47 (A) and CD99 (B) in DIV35 forebrain cells as measured by flow cytometry. [Figure 49] FIG. 49 shows histograms depicting the percentage of DIV22 hPSC-derived hindbrain / spinal cord cells expressing CD99 (A) and CD47 (B) as determined by flow cytometry. [Figure 50] FIG. 50 shows the percentage of co-expression of SOX2 with Ki67 (A) and INA (B) in DIV22 hindbrain / spinal cord cells as measured by flow cytometry. [Figure 51] FIG. 51 shows the percentage of co-expression of SOX2 with CD47 (A) and CD99 (B) in DIV22 hindbrain / spinal cord cells as measured by flow cytometry. [Figure 52] FIG. 52 shows the percentage of Ki67 co-expression with CD47 (A) and CD99 (B) in DIV22 hindbrain / spinal cord cells as measured by flow cytometry. [Diagram 53] FIG. 53 shows representative immunofluorescence images of unsorted control cells (A) and sorted CD47-negative cells (B) stained for TH and DAPI 10 days after MACS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology known to those skilled in the art.

[0016] Please note that all headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0017] The use of any and all examples or exemplary phrases (e.g., "such as") presented herein is intended merely to make the invention more clear and does not limit the scope of the invention unless otherwise claimed.

[0018] In the following, the method according to the invention is described in more detail by means of non-limiting embodiments and examples.

[0019] definition General definition As used herein, "a" or "an" or "the" may mean one or more than one. Unless otherwise indicated herein, terms provided in the singular also include plural contexts.

[0020] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). Furthermore, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted.

[0021] stem cells "Stem cells" should be understood as undifferentiated cells that have differentiation and proliferation capabilities (especially self-renewal capabilities) but maintain differentiation capabilities. Stem cells include classifications such as pluripotent stem cells, multipotent stem cells, unipotent stem cells, and the like, according to their differentiation capabilities.

[0022] As used herein, "pluripotent stem cells" (PSCs) refer to stem cells that can be cultured in vitro and have the potential to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm).

[0023] Pluripotent stem cells can be derived or isolated from fertilized eggs, somatic cell nuclear transfer embryos, germline stem cells, stem cells in tissues, somatic cells, and the like. Examples of pluripotent stem cells (PSCs) include embryonic stem cells (ESCs), embryonic germ cells (EG cells), induced pluripotent stem cells (iPSCs), and the like.

[0024] As used herein, the term induced pluripotent stem cell (also known as iPS cell or iPSC) refers to a type of pluripotent stem cell that is not a PSC and can be generated directly from a cell that has a nucleus. By introducing the products of a specific set of pluripotency-associated genes, non-pluripotent cells can be converted into pluripotent stem cells.

[0025] As used herein, the term "embryonic stem cell" refers to pluripotent stem cells derived from the inner cell mass of a blastocyst. Pluripotent embryonic stem cells may also be derived from parthenogenetic organisms, such as those described in WO2003 / 046141. Embryonic stem cells may be produced by culturing the inner cell mass obtained from a single blastomere or without destroying the embryo. Embryonic stem cells are available from a given tissue and are commercially available. Preferably, the method and product of the present invention are based on human PSCs, i.e., stem cells derived from either human induced pluripotent stem cells or human embryonic stem cells, including parthenogenetic organisms.

[0026] As used herein, the term "pluripotent stem cells" refers to stem cells that have the potential to differentiate into multiple, but not all, types of tissues or cells, and are typically not restricted to one germ layer. Neural stem cells are an example of a multipotent stem cell that is restricted to the neural lineage.

[0027] As used herein, the term "unipotent stem cell" refers to a stem cell that has the potential to differentiate into only one specific cell type.

[0028] As used herein, the term "in vitro" means that the cells are provided and maintained outside the human or animal body, such as in a vessel such as a flask, multi-well, or Petri dish. Thus, the cells are said to be cultured in a cell culture medium.

[0029] As used herein, the term "non-natural" means that the cells derived from pluripotent stem cells, but may be of human origin, are artificial constructs that do not exist in nature. In general, in the field of stem cell therapy, the aim is to provide cells that are as similar as possible to the cells of the human body. However, it may not be possible to mimic the development that pluripotent stem cells undergo in embryonic and fetal stages to the extent that mature cells are indistinguishable from natural cells of the human body. Essentially, in one embodiment of the present invention, the cells are artificial.

[0030] As used herein, the term "artificial" with respect to cells can include materials that naturally occur in nature, but that have been modified into non-naturally occurring constructs, including human stem cells that differentiate into non-naturally occurring cells that mimic cells in the human body.

[0031] When referring to a particular percentage of a cell population, e.g., X% of the cell population, expressing a particular marker, what is meant is the percentage of cells in that cell population, i.e., X% of the cells in that cell population.

[0032] protocol Throughout this application, the terms "method" and "protocol" when referring to a process for culturing or differentiating cells may be used interchangeably.

[0033] As used herein, the term "day" with respect to a protocol, and similarly days in vitro (DIV), refers to a specific time for performing a particular step during a differentiation procedure.

[0034] Generally, and unless otherwise specified, "day 0" refers to the initiation of the protocol, for example, but not limited to, by plating the stem cells, or transferring the stem cells to an incubator, or contacting the stem cells in the current cell culture medium with a compound prior to transplantation of the stem cells. Typically, the initiation of the protocol is performed by transferring the undifferentiated stem cells to another cell culture medium and / or container, for example, but not limited to, by plating or incubating, and / or by first contacting the undifferentiated stem cells with a compound(s) that affect the undifferentiated stem cells in such a way that the differentiation process is initiated.

[0035] When referring to "cells" in the methods, it means all cells of a cell population, regardless of cell type.

[0036] When referring to "day X", such as day 1, day 2, etc., this is relative to the start of the protocol on day 0. One of skill in the art will recognize that the exact date and time for performing a step may vary unless otherwise specified. Thus, "day X" is meant to encompass a time range such as ±10 hours, ±8 hours, ±6 hours, ±4 hours, ±2 hours, or ±1 hour.

[0037] Stem cell culture As used herein, the term "culture" refers to a continuous procedure, which is used throughout the method, to maintain the viability of cells at various stages. After the cells of interest are isolated, for example, but not limited to, from living tissue or embryos, they are subsequently maintained under carefully controlled conditions. These conditions vary for each cell type, but generally include the provision of essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO ). 2 , O 2 ) and regulate the physicochemical environment (pH buffer, osmolarity, temperature).

[0038] As used herein, the term "cell culture medium" refers to a liquid or gel designed to support the growth of cells. Cell culture media generally contain an appropriate energy source and compounds that regulate the cell cycle.

[0039] As used herein, the term "incubator" refers to any suitable incubator that can support cell culture.Non-limiting examples include culture dishes, Petri dishes, and plates (such as 6-well, 24-well, 48-well, 96-well, 384-well, 9600-well and the like microtiter plates, microplates, deep-well plates, etc.), flasks, chamber slides, tubes, Cell Factory systems, roller bottles, spinner flasks, hollow fibers, microcarriers, or beads.

[0040] As used herein, the term "providing stem cells" when referred to in the protocol means obtaining a batch of cells by the method as described above, and optionally transferring the cells to a different environment, such as by seeding them on a new substrate. Those skilled in the art will readily recognize that stem cells are vulnerable to such transfer, the procedure requires care, and that maintaining the stem cells in the original cell culture medium may promote a more sustainable transfer of the cells before replacing the cell culture medium with another cell culture medium that is more suitable for further differentiation processes.

[0041] Stem Cell Differentiation As used herein, the term "expressed" in relation to a gene or protein refers to the presence of an RNA molecule that can be detected using assays such as reverse transcription quantitative polymerase chain reaction (RT-qPCR), RNA sequencing, and the like, and / or a protein that can be detected using antibody-based assays such as flow cytometry, immunocytochemistry / immunofluorescence, and the like. Depending on the sensitivity and specificity of the assay, a gene or protein may be considered expressed when at least one molecule is detected, such as in RNA sequencing, or a detection limit above background / noise levels may be defined relative to a control sample, such as flow cytometry. Expression of a marker in a cell or cell population may be determined by the methods described in Example 9.

[0042] As used herein, the term "marker" refers to a naturally occurring identifiable expression produced by a cell, which can be correlated with a certain characteristic of the cell. In a preferred embodiment, the marker is a gene expression or proteomic expression that can be detected and correlated with the identity of the cell. The marker may be referred to as a gene. This can be easily translated into the expression of the corresponding mRNA and protein.

[0043] As used herein, the term "negative" or "-" as used with respect to any marker, such as a surface protein or transcription factor disclosed herein, refers to a marker that is not expressed in a cell or cell population, while the term "weak" or "low" refers to a marker that is expressed at a reduced level in a cell compared to the average expression of the marker in a cell population or compared to a reference sample.

[0044] As used herein, the term "positive" or "+" as used with respect to any marker, such as a surface protein or transcription factor disclosed herein, refers to a marker that is expressed in a cell or cell population, while the term "high" or "strong" refers to a marker that is expressed at an increased level in a cell compared to the average expression of the marker in a cell population or compared to a reference sample.

[0045] As used herein, the term "differentiation" refers broadly to the process by which cells progress from an undifferentiated state or a state different from the intended differentiation state to a specific differentiation state, for example, from an immature state to a less immature state, or from an immature state to a mature state, which may occur continuously during the implementation of the method. The term "differentiation" with respect to pluripotent stem cells refers to the process by which cells progress from an undifferentiated state to a specific differentiation state, i.e., from an immature state to a less immature state or a final state. Changes in cell interactions and maturation occur when cells lose markers of undifferentiated cells or acquire markers of differentiated cells. Loss or acquisition of a single marker may indicate that a cell has become "fully differentiated" or "terminally differentiated". A "terminally differentiated" cell is the final stage of a developmental lineage and cannot be further differentiated.

[0046] As used herein, the term "contacting" in reference to culturing or differentiating cells means exposing the cells to a particular compound, for example, by placing the particular compound in a location that allows it to touch the cells and produce "contacted" cells. Contacting can be accomplished using any suitable means. A non-limiting example of contacting is by adding the compound to the cell culture medium of the cells. Contacting of the cells is assumed to occur as long as the cells and the particular compound are in close proximity, for example, as long as the compound is present in the cell culture medium at a suitable concentration.

[0047] As used herein, the term "inhibitor" refers to a compound that reduces or suppresses or downregulates a process, such as a signaling pathway, that can promote cell differentiation.

[0048] As used herein, the term "active agent" refers to a compound that induces or stimulates or upregulates a process, such as a signaling pathway, that can promote cell differentiation.

[0049] As used herein, when describing the steps of a protocol, a cell may be referred to as a "cell," a "differentiated cell," or, in some cases, a PSC or a "PSC-derived cell." A person skilled in the art will recognize that during a protocol for differentiating a PSC into a specialized cell, the cell will lose pluripotency at some point. Thus, for example, when referring to a "cell" or a "PSC" in a step of contacting a cell with a compound, it refers to a cell that was originally a pluripotent stem cell.

[0050] Stem Cell Products As used herein, the term "differentiated cell" refers to a cell, such as a pluripotent stem cell, that has progressed from an undifferentiated state to a less immature state. A differentiated cell may be a specialized cell at a less immature state, such as a progenitor cell, or may be fully matured into a specialized / final cell type.

[0051] As used herein, the term "cell population" refers to multiple cells in the same culture. A cell population may be a mixture of cells at various developmental stages, such as, for example, cells of different types or at various stages of maturation toward the same or similar specialized characteristics, or may be a more homogenous composition of cells with common markers. As used herein, references to a cell population or percentage of cells, such as "X% of the cell population" and "X% of the cells," may be used interchangeably.

[0052] As used herein, the term "neural cell population" refers to a cell population that comprises neural cells.

[0053] As used herein, the term "genetically modified" in reference to cells refers to cells that have been subjected to gene editing and can no longer be considered as naturally occurring cells. In the case of genetically modified stem cells, even when the stem cells further differentiate into specialized cells, the traits resulting from gene editing persist, thus making the specialized cells genetically modified and artificial, i.e., non-naturally occurring, states. One example of genetically modified stem cells is HLA-deficient stem cells, also called universal donor cells, which are intended to overcome the problem of graft rejection. A method for obtaining HLA-deficient stem cells is disclosed in WO2020 / 260563.

[0054] Neuroectodermal cells As used herein, the term "neural" refers to the nervous system.

[0055] As used herein, the term "nervous system cells" refers to non-naturally occurring cells whose natural counterparts naturally form part of the ectodermal germ layer, more specifically the neuroectoderm, and are meant to encompass cells at any developmental stage within this germ layer, ranging from neural stem cells to neurons and other terminally differentiated cell types (e.g., glial cells), i.e., cell stages such as the neural stem cell stage and the neuroblast stage. Thus, neurons and their precursors are considered a particular type of nervous system cell.

[0056] As used herein, the terms "neuron" and "neuronal cell" may be used interchangeably to refer to a nervous system cell that is postmitotic and has terminally differentiated into specialized cells. Neuronal identity is characterized by expression of one or more of the markers INA, STMN2, TUBB3, MAP2, and RBFOX3.

[0057] As used herein, the term "neuroblast" refers to an intermediate precursor cell that is typically multipotent or simply unipotent and can self-renew only to a limited extent. Neuroblasts eventually give rise to terminally differentiated cell types such as neurons or astrocytes. The terms "neuroblast" and "intermediate precursor cell" and "intermediate progenitor cell" may be used interchangeably. Intermediate precursor cell identity is characterized by the expression of one or more of the markers ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2, and NHLH1.

[0058] As used herein, the terms "neuron progenitor," "precursor of a neuron," and "neuron precursor" may be used interchangeably and refer to a nervous system cell that has the potential or tendency to further specialize into a neuron. The terms "neuron precursor" and "non-native neuronal precursor" may be used interchangeably.

[0059] Neural cells according to the invention may have a particular regional identity, such as cells specific to the forebrain, midbrain, hindbrain, spinal cord, and the like.

[0060] As used herein, the term "forebrain" refers to the rostral region of the CNS and nervous system tracts that give rise to structures including the cerebral cortex and striatum.

[0061] As used herein, the term "midbrain" refers to the nervous system tract (on the rostral-caudal axis) and the medial region of the CNS that gives rise to structures including the substantia nigra.

[0062] As used herein, the terms "hindbrain" and "spinal cord" refer to the caudal region of the nervous system tract that is caudal to the isthmus organizer.

[0063] As used herein, the term "dopaminergic (DA) cell" or "dopaminergic neuron" or "dopamine neuron" refers to a cell capable of synthesizing the neurotransmitter dopamine.

[0064] Methods for enriching a cell population in neurons and / or their precursors - Patents.com One aspect of the invention relates to a method for providing a cell population enriched for neurons and / or their precursors, comprising obtaining a cell population comprising neuronal precursors, and selecting cells positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85, and / or excluding cells positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

[0065] Another aspect of the invention relates to a method for enriching a cell population in neurons and / or their precursors, comprising obtaining a cell population comprising neuronal precursors and selecting cells positive for one or more positive markers and / or excluding cells positive for one or more negative markers.

[0066] As used herein, the term "enriched in" with respect to a cell population means increasing the proportion of a desired cell type in the cell population. Specifically, as used herein, the term "enriching a cell population in neurons and / or their precursors" means increasing the proportion of neurons and / or their precursors in the cell population. Thus, a cell population "enriched in" means that the cell population has an increased proportion of the desired cell type compared to a reference cell population that has not been subjected to a method of enrichment in the desired cell type. In a preferred embodiment, the cell population is enriched by isolating the desired cell type. As used herein, the term "isolation" with respect to a cell population means separating the desired cell type from the undesired cells, either by removing the undesired cells from the cell population or by recovering the desired cell type from the cell population. Thus, a method for enriching a cell population in neurons and / or their precursors may alternatively be expressed as a method for isolating neurons and / or their precursors from the cell population, and the embodiments described herein apply equally to both.

[0067] Thus, the present invention relates to a method for isolating neurons and / or their precursors from a cell population, the method comprising obtaining a cell population comprising neural stem cells and selecting cells positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85, and / or excluding cells positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

[0068] In one embodiment, the neural stem cells are from the forebrain, midbrain, hindbrain, or spinal cord region. In one embodiment, the neural stem cells are ventral midbrain neural stem cells.

[0069] As used herein, the term "obtaining" with respect to a cell population comprising neural stem cells and / or neuronal progenitors means obtaining a batch of obtainable cells according to any method for inducing differentiation of cells, such as PSCs, which differentiate into neural cells, including neural stem cells and / or neuronal progenitors.

[0070] As used herein, the term "selecting" with respect to a cell population that includes cells expressing a particular marker means performing a step that is intended to result in a cell population that has an increased fraction of cells that express the marker compared to the fraction of cells that do not express the marker compared to the initial cell population before performing the step. Any suitable method for selecting cells that express a particular marker may be used, such as isolating cells that express the marker or separating cells.

[0071] As used herein, the term "elimination" with respect to cells expressing a certain marker means performing a process intended to result in a cell population in which the fraction of cells expressing the marker is reduced compared to the fraction of cells not expressing the marker compared to the initial cell population before performing the process. Any suitable method for eliminating cells expressing a certain marker can be used, such as depleting cells expressing the marker or separating cells. Non-limiting examples of methods for selecting and / or eliminating cells expressing a certain marker are antibody-mediated sorting, such as fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), or immunopanning. Thus, in a preferred embodiment, the positive and negative markers are surface markers.

[0072] As used herein, the term "positive marker" refers to a marker that can be used to select cells, and includes the markers PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85.

[0073] As used herein, the term "negative marker" refers to a marker that can be used to exclude cells, and includes the markers SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

[0074] In one embodiment, the method comprises obtaining a cell population comprising neuronal precursors. The cell population comprising neuronal precursors may be obtained by first obtaining a cell population comprising neural stem cells expressing the marker SOX2, and the cell population is further cultured until the expression of SOX2 in the cell population is reduced, thereby indicating that the neural stem cells are further differentiated into intermediate progenitor cells, some of which are neuronal precursors. At this stage, the surface marker according to the method is suitable for cell selection and / or exclusion to isolate cells with a neuronal fate. Thus, in one embodiment, the cell population comprising neuronal precursors is obtained by obtaining a cell population comprising neural stem cells expressing the marker SOX2, and culturing the cell population comprising neural stem cells until the expression of SOX2 in the cell population comprising neural stem cells is reduced. In one embodiment, at least 80%, for example at least 85%, 90%, or 95%, preferably at least 95%, of the obtained cell population comprising neural stem cells express SOX2. In one embodiment, the cell population comprising neural stem cells is cultured until the expression of SOX2 in the cell population is reduced by at least 5%, for example, 10%, 15%, 20%, 25%, 30%, 35% or 40%, preferably at least 20%.In one embodiment, at least 90% of the obtained cell population comprising neural stem cells expresses SOX2, and the cells are further cultured until at least 90% expresses the marker SOX2, preferably until 80% expresses the marker SOX2, more preferably until 70% expresses the marker SOX2.

[0075] In one embodiment, the selection and / or exclusion in step c) is performed within 5 days, e.g., 4 days, 3 days, or 2 days after less than 80% of the cell population comprising neural stem cells expresses the marker SOX2.

[0076] In another embodiment, the timing for selecting and / or excluding cells is determined based on the expression of CD99 instead of SOX2. Thus, in one embodiment, at least 80% of the cell population comprising neural stem cells obtained in step a) expresses CD99. In one embodiment, the cell population is further cultured in step b) at least until the expression of CD99 in the cell population comprising neural stem cells is reduced by 5%, for example, 10%, 15%, 20%, 25%, 30%, 35%, or 40%. In one embodiment, at least 90% of the cell population comprising neural stem cells obtained in step a) expresses CD99, and the cells are further cultured in step b) until at least less than 90% express the marker CD99, preferably less than 80% express the marker CD99, more preferably less than 70% express the marker CD99. In one embodiment, the selection and / or exclusion of step c) is performed within 5 days, e.g., 4 days, 3 days, or 2 days after less than 80% of the cell population comprising neural stem cells expresses the marker CD99.

[0077] In a preferred embodiment, when cells are selected and / or excluded based on positive and negative markers, the cell population comprises intermediate progenitor cells and / or neurons.In one embodiment, when the step of selecting and / or excluding cells is performed, at least 5% of the cell population expresses intermediate progenitor identity or neuronal identity, for example, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40%.In one embodiment, the cell population is cultured until the expression of one or more of the markers ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2, and NHLH1 is at least 5%, for example, at least 10%, at least 15%, or at least 20%. In one embodiment, the cell population is cultured until expression of one or more of the markers INA, STMN2, TUBB3, MAP2, and RBFOX3 is at least 5%, e.g., at least 10%, at least 15%, or at least 20%.

[0078] In one embodiment, when performing the steps of selecting and / or deleting cells, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% of the cell population are positive for one or more of the markers ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2, NHLH1, INA, STMN2, TUBB3, MAP2, and RBFOX3.

[0079] In one embodiment, at the time of cell selection and / or elimination, 10-90%, e.g., 10-80%, 10-70%, 10-60%, or 10-50% of the cell population are positive for SOX2, 5-50%, e.g., 5-40%, 5-30%, 5-20% of the cell population are positive for one or more of the markers ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2, and NHLH1, and 5-20%, e.g., 5-15%, or 5-10% of the cell population are positive for one or more of the markers INA, TUBB3, STMN2, MAP2, and RBFOX3.

[0080] In one embodiment, the neural cells are of ventral midbrain identity and at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% of the cell population are positive for one or both of the markers ASCL1 and INA when performing the steps of selecting and / or excluding the cells.

[0081] In one embodiment, cells positive for the marker CD47 are excluded.In one embodiment, cells positive for the marker CD99 are excluded.

[0082] In one embodiment, the method comprises the further step of maturing the neurons and / or their precursors after the selection and / or elimination step.

[0083] In one embodiment, the method comprises the further step of recovering the neural cell population enriched in neurons and / or their precursors after the selection and / or exclusion step, and optionally after a further step of maturing the neurons and / or their precursors.

[0084] In one embodiment, the recovered cells are negative for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

[0085] In one embodiment, the cell population is a neural cell population. In one embodiment, at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, or more preferably at least 90%, more preferably at least 95%, even more preferably at least 99% of the cell population are neural cells.

[0086] In one embodiment, the enriched cell population has increased tendency to develop into neurons.As used herein, the term "tendency" in relation to cell specialization refers to the possibility that cells develop into a certain cell type.Therefore, increasing the tendency of a cell population to develop into a certain cell type means increasing the proportion of cells that are likely to develop into that cell type.

[0087] In one embodiment, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the neurons and / or their precursors are from the ventral midbrain, ventral spinal cord, dorsal forebrain, or ventral forebrain region.

[0088] In certain embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the neurons and / or their precursors are from the ventral midbrain region.

[0089] In certain embodiments, the cell population of the ventral midbrain region and the cells positive for the marker CD47 are excluded when at least 5%, for example 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the cell population expresses one of the markers ASCL1 and INA. In further embodiments, the cells positive for either the marker CD47 or CD99 are excluded.

[0090] In another embodiment, cell populations of the ventral spinal cord, dorsal forebrain, or ventral forebrain and cells positive for the marker CD99 are excluded.

[0091] In certain embodiments, cell selection and / or exclusion is performed by antibody-mediated cell sorting.In further embodiments, cell selection and / or exclusion is performed using fluorescence-activated cell sorting (FACS), magnetic activated cell sorting (MACS), or immunopanning, or by comparison with a reference sample, and preferably, selection and / or exclusion is performed using MACS.

[0092] In one embodiment, the neural cell population is an in vitro cell culture. In one embodiment, the cells of the neural cell population are non-naturally occurring.

[0093] Those skilled in the art will recognize that the expression of certain markers indicating cell stage may be expressed internally in cells and may be analyzed, for example, by single-cell RNA-seq, while other markers may be expressed on the cell surface and antibody-mediated analysis such as FACS may be used. The timing of cell selection and / or exclusion based on surface markers may be established, for example, by analyzing samples for the expression of SOX2. Typically, for example, during the design of a protocol for obtaining neural stem cells, cell expression during the protocol is analyzed, so that the timing of selection and / or exclusion during the protocol may be determined without the need to further monitor the expression of markers during production settings.

[0094] Methods for obtaining neural cells including neuronal precursors - Patents.com In one embodiment, the cell population comprising neuronal precursors is derived from PSCs, preferably hPSCs. In certain embodiments, the hPSCs are human embryonic stem cells or human induced pluripotent stem cells.

[0095] In one embodiment of the method, obtaining the cell population comprising neuronal precursors comprises allowing the PSCs to differentiate into neural cells comprising neuronal precursors. In certain embodiments, the PSCs are contacted with at least one inhibitor of the SMAD signaling pathway.

[0096] As used herein, the term "SMAD signaling pathway" refers to Small Mothers Against Decapentaplegic (SMAD) protein signaling pathway. In one embodiment, at least one inhibitor of SMAD signaling pathway is selected from Noggin, LY364947, SB431542, and LDN-193189. In one embodiment, PSCs are contacted with two or more inhibitors of SMAD signaling pathway. Those skilled in the art will recognize that contacting PSCs with one or more inhibitors of SMAD signaling pathway is a robust technique for differentiating cells into neural lineages.

[0097] In one embodiment, the PSCs are contacted with one or more inhibitors of the SMAD signaling pathway on day 0. In a further embodiment, the PSCs are contacted with one or more inhibitors of the SMAD signaling pathway for at least 5 days, e.g., at least 7 days, at least 8 days, at least 9 days, or at least 10 days.

[0098] In one embodiment of the method, obtaining a cell population comprising neuronal precursors includes allowing the PSCs to differentiate into neural cells of the forebrain, midbrain, hindbrain, or spinal cord region. In one embodiment in which the cells differentiate into the midbrain, hindbrain, or spinal cord region, the cells are contacted with at least one activator of the WNT pathway. In one embodiment, the cells are contacted with at least one activator of the WNT signaling pathway on days 0 to 5, preferably on day 0. In one embodiment, the cells are contacted with at least one activator of the WNT signaling pathway for at least 5 days, e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days. In one embodiment, the cells are contacted with at least one activator of the WNT signaling pathway simultaneously with contacting the cells with one or more inhibitors of the SMAD signaling pathway.

[0099] As used herein, the term "WNT pathway" refers to the WNT signaling pathway. In one embodiment, at least one activator of the WNT pathway is a GSK3 inhibitor, such as CHIR99021. Thus, in one embodiment, the PSC is contacted with CHIR99021.

[0100] In one embodiment of the method, obtaining a cell population containing neuronal precursors includes a step of allowing PSCs to differentiate into neural cells of the ventral region of the nervous system tube. In such an embodiment, the cells may be contacted with an activator of the SHH signaling pathway. As used herein, the term "SHH" refers to Sonic Hedgehog. In one embodiment, the cells are contacted with a compound selected from Sonic Hedgehog C24II, purmorphamine, and SAG (smoothened agonist). In one embodiment, the cells are contacted with an activator of the SHH signaling pathway from at least day 7, such as at least day 6, at least day 5, at least day 4, at least day 3, at least day 2, at least day 1, or day 0. In a preferred embodiment, the cells are contacted with an activator of the SHH signaling pathway at the same time as contacting the cells with one or more inhibitors of the SMAD signaling pathway.

[0101] In one embodiment of the method, obtaining a cell population comprising neuronal precursors comprises a step of allowing PSCs to differentiate into neural cells of the midbrain or hindbrain region. In such an embodiment, the cells may be contacted with FGF8, preferably FGF8b. As used herein, the term "FGF" refers to fibroblast growth factor. In one embodiment, the cells are contacted with FGF8 on days 5-15, preferably days 8-10, e.g., day 9. In one embodiment, the cells are contacted with FGF8 for at least 2 days, e.g., at least 5 or 6 days. In one embodiment, the cells are contacted with FGF8 when terminating exposure of the cells to one or more inhibitors of the SMAD signaling pathway.

[0102] In one embodiment of the method, obtaining a cell population comprising neuronal precursors comprises allowing PSCs to differentiate into neural cells of the ventral midbrain region.

[0103] In one embodiment for differentiating nervous system cells into ventral midbrain region, cells are contacted with ascorbic acid, such as L-ascorbic acid.In one embodiment, cells are contacted with ascorbic acid when the exposure of cells to one or more inhibitors of SMAD signaling pathway is terminated.In one embodiment, cells are contacted with ascorbic acid from day 9, day 10, day 11, day 12 or day 13, preferably from day 11.In a preferred embodiment, cells are contacted with ascorbic acid until the step of collecting nervous system cells.

[0104] In one embodiment for differentiating nervous system cells into ventral midbrain region, cells are contacted with BDNF. As used herein, the term "BDNF" refers to brain-derived neurotrophic factor. In one embodiment, cells are contacted with BDNF when terminating exposure of cells to one or more inhibitors of SMAD signaling pathway. In one embodiment, cells are contacted with BDNF from day 9, day 10, day 11, day 12, or day 13, preferably from day 11. In a preferred embodiment, cells are contacted with BDNF until the step of recovering nervous system cells. In one embodiment, cells are contacted with ascorbic acid and BDNF at the same time.

[0105] In one embodiment for differentiating nervous system cells into ventral midbrain region, cells are contacted with GDNF. As used herein, the term "GDNF" refers to glial derived neurotrophic factor. In one embodiment, cells are contacted with GDNF when the exposure of cells to one or more inhibitors of SMAD signaling pathway is terminated. In one embodiment, cells are contacted with GDNF from day 11, day 12, day 13, day 14, day 15, day 16, day 17 or day 18, preferably from day 16. In a preferred embodiment, cells are contacted with GDNF until the step of collecting nervous system cells.

[0106] In one embodiment for differentiating nervous system cells into ventral midbrain region, cells are contacted with DAPT or other inhibitors of Notch pathway. As used herein, the term "DAPT" refers to (2S)-N-[(3,5-difluorophenyl)acetyl]-L-alanine-2-phenyl]glycine 1,1-dimethylethyl ester. In one embodiment, cells are contacted with DAPT when terminating exposure of cells to one or more inhibitors of SMAD signaling pathway. In one embodiment, cells are contacted with DAPT from day 11, day 12, day 13, day 14, day 15, day 16, day 17, or day 18, preferably from day 16. In a preferred embodiment, cells are contacted with DAPT until the step of recovering nervous system cells. In one embodiment, cells are contacted with GDNF and DAPT simultaneously.

[0107] In one embodiment, PSCs are cultured and differentiated on a substrate comprising an extracellular matrix such as poly-L-lysine, poly-D-lysine, poly-L-ornithine, laminin, fibronectin, iMatrix and / or collagen, and / or fragments thereof. In one embodiment, the substrate comprises laminin or a fragment thereof, such as laminin-111, laminin-521, and / or laminin-511.

[0108] In one embodiment, the PSCs can be differentiated into neural cells, including neuronal precursors, for at least 10 days, e.g., at least 15 days, at least 18 days, at least 20 days, at least 22 days, at least 25 days, or at least 30 days, preferably at least 20 days, prior to the step of selecting and / or deleting the cells.

[0109] In one embodiment, the PSCs are allowed to differentiate into neural cells, including neuronal precursors, for 15 to 50 days, preferably 18 to 40 days, more preferably 20 to 40 days, more preferably 20 to 30 days prior to the cell selection and / or exclusion step. In one embodiment, the remaining neurons and / or their precursors are allowed to further differentiate / maturate after the cell selection and / or exclusion step.

[0110] In one embodiment, the step of selecting and / or excluding cells is performed between days 15 and 40 from the initiation of differentiation of PSCs into neural cells, including neuronal precursors.

[0111] In one embodiment, the neural cells are differentiated into ventral midbrain neural cells and the step of selecting and / or excluding cells is performed on days 22-25.

[0112] Cell populations enriched for neurons and / or their precursors Another aspect of the invention relates to a cell population of neurons and their precursors, wherein at least 50% of the cells are positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85, and / or less than 50% of the cells are positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

[0113] In one embodiment, at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% of the cells are positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85.

[0114] In one embodiment, less than 50%, preferably less than 45%, more preferably less than 40%, more preferably less than 35%, more preferably less than 30%, more preferably less than 25%, more preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, or even more preferably less than 1% of the cells are positive for one or more of markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

[0115] In one embodiment, less than 50% of the cells are positive for the marker CD47, preferably less than 45%, more preferably less than 40%, more preferably less than 35%, more preferably less than 30%, more preferably less than 25%, more preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, or even more preferably less than 1%.

[0116] In one embodiment, less than 50% of the cells are positive for the marker CD99, preferably less than 45%, more preferably less than 40%, more preferably less than 35%, more preferably less than 30%, more preferably less than 25%, more preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, or even more preferably less than 1%.

[0117] In one embodiment, less than 50% of the cells are positive for the markers CD47 and CD99, preferably less than 45%, more preferably less than 40%, more preferably less than 35%, more preferably less than 30%, more preferably less than 25%, more preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, or even more preferably less than 1%.

[0118] In one embodiment, at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% of the cells are selected from ventral midbrain neurons and / or their precursors, ventral spinal cord neurons and / or their precursors, dorsal forebrain neurons and / or their precursors, and ventral forebrain neurons and / or their precursors.

[0119] In one embodiment, at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% of the cell population is positive for marker FOXA2.

[0120] In one embodiment, at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% of the cell population are ventral midbrain neurons and / or their precursors.

[0121] In certain embodiments, at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% of the cells are neuroblasts and / or neurons.

[0122] In one embodiment, at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% of the cell population is positive for one or both of the markers ASCL1 and INA.

[0123] In one embodiment, the cell population has an increased propensity to develop into neurons.

[0124] In one embodiment, the cell population is in vitro.

[0125] In one embodiment, the cell population is a neural cell population.

[0126] In one embodiment, the cell population is enriched for neurons and / or their precursors.

[0127] In one embodiment, the cells of the cell population are in vitro differentiated cells.

[0128] In one embodiment, the cells of the cell population are non-naturally occurring cells.

[0129] In one embodiment, the cells of the cell population are derived from hPSCs.

[0130] In one embodiment, the cells of the cell population are genetically modified. In a further embodiment, the genetically modified cells are HLA-deficient.

[0131] In one embodiment, the cell population comprises at least 1,000 cells, e.g., at least 100,000 cells, at least 1,000,000 cells, or at least 10,000,000 cells.

[0132] In one embodiment, the cell population is obtained by any of the methods described above.

[0133] Pharmaceutical compositions and medicinal uses One aspect of the present invention relates to a composition comprising the enriched cell population described herein. The composition may include any suitable additive that is pharmacologically acceptable, such as a cryoprotectant and / or a biomaterial, e.g., an extracellular matrix.

[0134] Another aspect relates to a composition or cell population according to the invention for use as a medicament. Thus, in one embodiment, the composition or cell population is for the prevention or treatment of a condition requiring the administration of neurons and / or their precursors.

[0135] In one embodiment, the composition or cell population is for use in the treatment of a neurological condition, such as Parkinson's disease, stroke, traumatic brain injury, spinal cord injury, Huntington's disease, dementia, epilepsy, blindness, Alzheimer's disease, and other neurological conditions in which neurons are lost or dysfunctional.

[0136] In certain embodiments, the cell population comprises ventral midbrain neurons and / or their precursors for the treatment of Parkinson's disease.

[0137] In certain embodiments, the cell population comprises cortical neurons and / or their precursors for the treatment of stroke damage.

[0138] In another aspect, there is provided a method for the treatment or prevention of a neurological condition comprising administering to a patient an effective amount of a cell population or composition according to the invention.

[0139] Specific Embodiments Aspects of the present invention will now be further described by the following non-limiting embodiments. 1. A method for isolating neurons and / or their precursors from a cell population, comprising: a) obtaining a cell population containing neural stem cells expressing SOX2; b) further culturing the cell population comprising neural stem cells; c) selecting cells positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85, and / or excluding cells positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1. 2. The method according to embodiment 1, wherein at least 80% of the cell population comprising neural stem cells obtained in step a) expresses SOX2. 3. The method of embodiment 1 or 2, wherein the cell population is further cultured in step b) at least until the expression of SOX2 in the cell population comprising neural stem cells is reduced by 5%, for example, 10%, 15%, 20%, 25%, 30%, 35%, or 40%. 4. The method according to any one of embodiments 1 to 3, wherein at least 90% of the cell population comprising neural stem cells obtained in step a) expresses SOX2, and the cell population is further cultured in step b) until at least less than 90% expresses the marker SOX2, preferably less than 80% expresses the marker SOX2, more preferably less than 70% expresses the marker SOX2. 5. The method according to any one of embodiments 1 to 4, wherein the selection and / or exclusion in step c) is carried out within 5 days, for example within 4 days, 3 days, or 2 days, after less than 80% of the cell population comprising neural stem cells expresses the marker SOX2. 6. The method according to any one of embodiments 1 to 5, wherein at least 80% of the cell population comprising neural stem cells obtained in step a) expresses CD99. 7. The method according to any one of embodiments 1 to 6, wherein the cell population is further cultured in step b) at least until expression of CD99 in the cell population comprising neural stem cells is reduced by 5%, e.g., 10%, 15%, 20%, 25%, 30%, 35%, or 40%. 8. The method according to any one of embodiments 1 to 7, wherein at least 90% of the cell population comprising neural stem cells obtained in step a) expresses CD99, and the cell population is further cultured in step b) until at least less than 90% expresses the marker CD99, preferably less than 80% expresses the marker CD99, more preferably less than 70% expresses the marker CD99. 9. The method according to any one of embodiments 1 to 8, wherein the selection and / or exclusion in step c) is carried out within 5 days, such as within 4 days, 3 days, or 2 days, after less than 80% of the cell population comprising neural stem cells expresses the marker CD99. 10. The method according to any one of the preceding embodiments, wherein the cell population is further cultured in step b) until the expression of one or more of the markers ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2, and NHLH1 is at least 5%, e.g., 10%, 15%, or 20%. 11. The method according to any one of embodiments 1 to 10, wherein the cell population is further cultured in step b) until the expression of one or more of the markers INA, STMN2, TUBB3, MAP2, and RBFOX3 is at least 5%, for example 10%, 15%, or 20%. 12. The method according to any one of the preceding claims, wherein at the time of selecting and / or excluding cells in step c), 10-90% of the cell population are positive for SOX2, 5-50% of the cell population are positive for one or more of the markers ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2 and NHLH1, and 5-20% of the cell population are positive for one or more of the markers INA, TUBB3, STMN2, MAP2 and RBFOX3. 13. The method of any one of embodiments 1 to 12, wherein the cell population is a neural cell population. 14. The method according to any one of the preceding embodiments, wherein the cell population comprising neural stem cells in step a) is obtained by neural induction of PSCs for at least 14 days. 15. The method of any one of embodiments 1 to 14, wherein the neural stem cells are from the forebrain, midbrain, hindbrain, or spinal cord region. 16. The method of any one of embodiments 1 to 15, wherein the neural stem cells are ventral midbrain neural stem cells. 17. A method for providing a cell population enriched for neurons and / or their precursors, comprising: - obtaining a cell population comprising neuronal precursors; - selecting cells positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85; and / or - Excluding cells positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1. 18. The method according to embodiment 17, wherein the cell population comprising neuronal precursors is obtained by obtaining a cell population comprising neural stem cells expressing the marker SOX2, and culturing the cell population comprising neural stem cells until the expression of the marker SOX2 in the cell population comprising neural stem cells is reduced. 19. The method of embodiment 18, wherein at least 80% of the obtained cell population comprising neural stem cells expresses the marker SOX2. 20. The method of embodiment 19, wherein the cell population is further cultured at least until the expression of SOX2 in the cell population comprising neural stem cells is reduced by 5%, e.g., 10%, 15%, 20%, 25%, 30%, 35%, or 40%. 21. The method according to embodiment 18 or 19, wherein at least 80% of the cell population comprising neural stem cells is positive for SOX2, and the cell population comprising neural stem cells is cultured until the expression of SOX2 is less than 80%. 22. The method according to any one of embodiments 1 to 21, wherein cells positive for the marker CD47 are excluded. 23. The method according to any one of embodiments 1 to 22, wherein cells positive for the marker CD99 are excluded. 24. The method according to any one of the preceding embodiments, wherein cells positive for either of the markers CD99 and CD47 are excluded. 25. The method of any one of embodiments 1 to 24, wherein at least 80% of the cell population are neural cells. 26. The method of any one of embodiments 1 to 25, wherein when selecting and / or excluding cells, at least 5% of the cell population expresses intermediate progenitor identity or neuronal identity. 27. The method of any one of embodiments 1 to 26, wherein when selecting and / or excluding cells, at least 5% of the cell population is positive for one or more of the markers ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2, and NHLH1. 28. The method of any one of embodiments 1 to 27, wherein when selecting and / or excluding cells, at least 5% of the cell population is positive for one or more of the markers INA, TUBB3, STMN2, MAP2, and RBFOX3. 29. The method according to any one of embodiments 1 to 28, wherein the cell population is derived from PSCs, preferably hPSCs. 30. The method of embodiment 29, wherein obtaining the cell population comprises a step of allowing the PSCs to differentiate into neural cells, including neuronal precursors. 31. The method of embodiment 30, wherein the PSCs are differentiated into neural cells of the forebrain, midbrain, hindbrain, or spinal cord region. 32. The method of embodiment 30 or 31, wherein the PSCs can be differentiated into neural cells for 15 to 50 days. 33. The method of embodiment 30 or 31, wherein the PSCs are capable of differentiating into neural cells for at least 20 days. 34. The method of any one of embodiments 30 to 33, wherein the PSC is contacted with at least one inhibitor of the SMAD signaling pathway. 35. The method of any one of embodiments 30 to 34, wherein the cell is contacted with at least one activator of the WNT signaling pathway. 36. The method of any one of embodiments 30 to 35, wherein the cell is contacted with an activator of the SHH signaling pathway. 37. The method of any one of embodiments 30 to 36, wherein the cells are contacted with FGF8. 38. The method according to any one of embodiments 30 to 37, wherein the cells are cultured on a substrate coated with laminin or a fragment thereof, preferably laminin-521 and / or laminin-511 and / or laminin-111. 39. The method of any one of embodiments 30 to 38, wherein the cells are contacted with ascorbic acid. 40. The method of any one of embodiments 30 to 39, wherein the cells are contacted with BDNF and / or GDNF. 41. The method of any one of embodiments 30-40, wherein the cells are contacted with DAPT. 42. The method according to any one of the preceding embodiments, comprising, after the selection and / or exclusion step, a further step of maturing neurons and / or their precursors. 43. The method according to any one of the preceding claims, comprising recovering the cell population enriched for neurons and / or their precursors after the selection and / or exclusion steps, and optionally after a further step of maturing the neurons and / or their precursors. 44. The method of embodiment 43, wherein the harvested cell population enriched for neurons and / or their precursors is cryopreserved. 45. The method of embodiment 43 or 44, wherein the recovered cells are negative for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1. 46. ​​The method of any one of embodiments 1 to 45, wherein the cell population enriched for neurons and / or their precursors has an increased propensity to develop into neurons. 47. The method of any one of embodiments 1-46, wherein at least 50% of the neurons and / or their precursors are from the forebrain, midbrain, hindbrain, or spinal cord region. 48. The method of any one of embodiments 1 to 47, wherein at least 50% of the neurons and / or their precursors are from the ventral midbrain region. 49. The method according to any one of embodiments 31 to 47, wherein at least 50% of the neurons and / or their precursors are from the forebrain region, and the selection and / or exclusion step is carried out on days 30 to 70, preferably days 30 to 40. 50. The method according to any one of embodiments 31 to 47, wherein at least 50% of the neurons and / or their precursors are from the spinal cord region, and the selection and / or exclusion step is carried out on days 20 to 40, preferably days 20 to 30. 51. The method according to any one of embodiments 1 to 50, wherein the selection and / or exclusion of cells is carried out by antibody-mediated cell sorting. 52. The method according to any one of embodiments 1 to 51, wherein cell selection and / or exclusion is carried out using fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), or immunopanning. 53. The method of any one of embodiments 1 to 52, wherein the marker is a surface marker. 54. The method of any one of embodiments 1 to 53, wherein the cell population is in vitro. 55. The method of any one of embodiments 1 to 54, wherein the cells of the cell population are non-naturally occurring. 56. The method of any one of embodiments 1 to 55, wherein the cell population is a neural cell population. 57. A cell population obtained by a method according to any one of embodiments 1 to 56. 58. A cell population of neurons and / or their precursors, wherein at least 50% of the cell population are positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85, and / or less than 50% of the cells are positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1. 59. The cell population of embodiment 58, wherein at least 50% of the cell population is positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85, and negative for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD83, CD99, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1. 60. The cell population of embodiment 58 or 59, wherein at least 50% of the cell population is selected from ventral midbrain neurons and / or their precursors, ventral spinal cord neurons and / or their precursors, dorsal forebrain neurons and / or their precursors, and ventral forebrain neurons and / or their precursors. 61. A cell population according to any one of embodiments 58 to 60, wherein more than 80% of the cell population is negative for the marker CD47. 62. The cell population according to any one of embodiments 58 to 61, wherein more than 80% of the cell population is negative for the marker CD99. 63. A cell population according to any one of embodiments 58 to 62, wherein more than 80% of the cell population is negative for the markers CD47 and CD99. 64. The cell population according to any one of embodiments 58 to 63, wherein at least 60% of the cell population is positive for the marker FOXA2. 65. A cell population according to any one of embodiments 58 to 64, wherein at least 50% of the cell population are ventral midbrain neurons and / or their precursors. 66. A cell population according to any one of embodiments 58 to 65, wherein at least 80% of the cell population is positive for one or both of the markers ASCL1 and INA. 67. A cell population according to any one of embodiments 58 to 66, wherein the cell population has an increased tendency to develop into neurons. 68. A cell population according to any one of embodiments 58 to 67, wherein the cells of the cell population are in vitro differentiated cells. 69. The cell population according to any one of embodiments 58 to 68, wherein the cell population is in vitro. 70. The cell population according to any one of embodiments 58 to 69, wherein the cells of the cell population are non-native cells. 71. A cell population according to any one of embodiments 58 to 70, wherein the cells of the cell population are derived from hPSCs. 72. A cell population according to any one of embodiments 58 to 71, wherein the cells of the cell population are genetically modified. 73. The cell population of embodiment 72, wherein the genetically modified cells are HLA-deficient. 74. The cell population of any one of embodiments 58 to 73, wherein the cell population comprises at least 1,000 cells. 75. The cell population according to any one of embodiments 58 to 74, wherein the cell population is enriched for neurons and / or their precursors. 76. The cell population of any one of embodiments 58 to 75, wherein the cell population is a neural cell population. 77. A cell population according to any one of embodiments 58 to 76, obtained by a method according to any one of embodiments 1 to 56. 78. A composition comprising a cell population according to any one of embodiments 57 to 77. 79. A cell population according to any one of embodiments 57 to 77 or a composition according to embodiment 78 for use as a medicament. 80. A cell population or composition according to embodiment 79 for the prevention or treatment of a condition requiring the administration of neurons and / or their precursors. 81. The population of cells according to any one of embodiments 57-77 or the composition according to embodiment 78 for use in the treatment of a neurological condition selected from Parkinson's disease, stroke, traumatic brain injury, spinal cord injury, Huntington's disease, dementia, epilepsy, blindness, Alzheimer's disease, and other neurological conditions in which neurons are lost or dysfunctional. 82. A method for treating or preventing a neurological condition, comprising administering to a patient an effective amount of a cell population or a composition according to embodiment 79. EXAMPLES

[0140] Below are non-limiting examples for carrying out the present invention.

[0141] Example 1: Differentiation of human pluripotent stem cells into neurons Human embryonic stem cell (hESC) lines RC17 (Roslin CT) and 3053 (Novo Nordisk A / S) were cultured in a 3x10 sucrose-treated 100% ethanol-free medium with human laminin-521 matrix (0.7–1.2 μg / cm 2 Cells were cultured in iPS Brew XF medium (Miltenyi Biotec) supplemented with 60 U / mL penicillin-streptomycin (PS, Thermo Fisher Scientific) on cultureware coated with iPSC (Biolamina). Medium was changed daily and cells were passaged every 4–6 days with EDTA 0.5 mM (Thermo Fisher Scientific). Cultures were maintained at 37 °C, 95% humidity, and 5% CO 2 maintained at the level.

[0142] hESCs were differentiated into ventral midbrain neurons according to established protocols (Nolbrant et al., 2017; Kirkeby et al., 2017). Briefly, hESCs were grown to 70–90% confluency and then dissociated with 0.5 mM EDTA. Cells were cultured in 100% Fibroblast medium containing human laminin-111 (1.2 μg / cm 2 , BioLamina) coated cell culture flasks or plates. 4 Cells / cm 2Cells were exposed to N2-based medium; 50% DMEM / F12 + Glutamax (Gibco), 50% Neurobasal (Gibco), 1% N2 supplement CTS (Thermo Fisher Scientific), 5% GlutaMAX (Thermo Fisher Scientific), 0.2% PS (Thermo Fisher Scientific) supplemented with SMAD inhibitor SB431542 (10 μM, Miltenyi Biotec), Noggin (100 ng / mL, Miltenyi Biotec) for neural induction, Sonic Hedgehog C24II (SHH, 500 ng / mL, Miltenyi Biotec) for ventral fate, and GSK3β inhibitor CHIR99021 (CHIR, 0.5–0.6 μM, Miltenyi Biotec) to promote caudation, from days in vitro (DIV) 0 to 8. N2-based medium supplemented with fibroblast growth factor 8b (FGF8b, 100 ng / mL, Miltenyi Biotec) from DIV9–11. On DIV11, cells were dissociated with Accutase (Thermo Fisher Scientific) and cultured in DIV11–16 medium supplemented with 10 µM Y-27632 (Miltenyi Biotec) (FGF8b (100 ng / mL), L-ascorbic acid (AA, 200 µM, Sigma), human brain-derived neurotrophic factor (BDNF, 20 ng / mL, Miltenyi Biotec) supplemented with Neurobasal, 2% B27 supplement, vitamin A-free CTS (Thermo Fisher Scientific), 5% GlutaMAX, 0.2% PS) supplemented with human laminin-111 (1.2 µg / cm). 2 ) coated cell culture flasks or plates. 6 cells / m 2On day in vitro (DIV) 16, cells were dissociated with Accutase and plated in B27 medium supplemented with poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm) supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 μM), dcAMP (500 μM), DAPT (10 μM), and Y-27632 (10 μM). 2 )-coated cell culture flasks / plates to extend the in vitro culture and allow further differentiation and maturation of the ventral midbrain neural system stem cells into neurons.

[0143] Reference materials: ●Nolbrant S, Heuer A, Parmar M, Kirkeby A. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation.Nat Protoc.2017 Sep;12(9):1962-1979.doi:10.1038 / nprot.2017.078.Epub 2017 Aug 31.PMID:28858290. ●Kirkeby A, Nolbrant S, Tiklova K, Heuer A, Kee N, Cardoso T, Ottosson DR, Lelos MJ, Rifes P, Dunnett SB, Grealish S, Perlmann T, Parmar M. Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease.Cell Stem Cell.2017 Jan 5;20(1):135-148.doi:10.1016 / j.stem.2016.09.004.Epub 2016 Oct 27.PMID:28094017;PMCID:PMC5222722.

[0144] Example 2: scRNA sequencing experimental design to identify novel selection candidates and technical methods To perform single-cell RNA sequencing (scRNA-seq), cell cultures were dissociated into single-cell suspensions with Accutase, and 3,000–10,000 cells were processed using the 10X Genomics Chromium Platform and sequenced on a NextSeq 550. Data were curated to remove apoptosis and stress, as well as multiplets.

[0145] Differentiating human pluripotent stem cells (hPSCs) into ventral midbrain dopamine neurons according to established protocols transitions the cells through several fundamental cellular developmental stages. Initially, hPSCs transition into neural stem cells (NSCs) (Figure 1A-B), then into intermediate progenitor cells (IPs) (Figure 1C), and finally, into a terminally differentiated cell type (Figure 1D). However, this process is not precise, and after the NSC stage, the culture loses its homogeneity and typically begins to differentiate into various cell types, forming a mixed cell culture (Figure 1C). This mixed culture composition persists until differentiation is complete and the cells acquire their final fate (Figure 1D). A combination of differential expression of genes (typically cell fate-instructing transcription factor proteins) is used to specify these various cell types and is illustrated in Figure 1A-D.

[0146] Single-cell RNA-sequencing (scRNA-seq) analysis was applied at several time points along the differentiation, allowing the observation and monitoring of the transition from homogeneous to heterogeneous mixed cultures. The hPSCs used for differentiation and the NSCs they form are highly homogeneous, as seen by their transcriptome profile. hPSCs express markers of their pluripotent identity at high levels, with 99.5% of the analyzed hPSCs expressing the pluripotent marker POU5F1 and the pluripotent / NSC marker SOX2, both of which are expressed at high levels (Figure 2A, 2B). In addition, hPSCs express minimal or low levels of other genes indicative of a differentiated cell fate, such as the intermediate precursor (IP) marker ASCL1 or the neuronal marker INA (Figure 2A, 2B). Similarly, NSCs are highly homogenous, expressing the NSC marker SOX2, but not the pluripotency marker POU5F1, with minimal and low expression of the IP marker ASCL1 and the neuronal marker INA (Figures 3A, 3B).

[0147] The heterogeneity appearing in cultures after the NSC stage is evident at the transcriptional level and is shown as an example of a scRNA-seq analysis at DIV24 (Figure 4A, 4B). At this time point, cell populations express markers of various cell types and stages, e.g., 20.4% NSCs (POU5F1- / SOX2+ / ASCL1- / INA- cells, Figure 4A), as well as 48.2% intermediate progenitors (POU5F1- / SOX2+ or - / ASCL1+ / INA+ or -, Figure 4A), and 16.3% neurons (POU5F1- / SOX2- / ASCL1+ or - / INA+ or -, Figure 4A). Pluripotent stem cells are absent at this stage due to their differentiation into NSCs, IPs, and neurons (POU5F1+ / SOX2+ / ASCL1- / INA-, Figure 4A, 4B).

[0148] Example 3: scRNA sequencing of the mixed culture stage and identification of cell populations and purification candidates An scRNA-seq time course study was performed, sampling every 48 hours, from the time when cells were homogenous (DIV16 NSCs) to the time when they had developed into a mixed culture (DIV26 mixed culture, Figure 1E). By sampling every 48 hours within this window (DIV16, 18, 22, 24, 26), we ensured that a series of data was acquired to allow the trajectory of cell differentiation and diversification into different cell stages and cell types to be observed and to search for markers that may help separate populations of interest or non-interest with surface antigens that are restricted to these populations.

[0149] We identified mixed cultures between DIV22-26 and aligned these datasets to search for subtype-restricted surface antigens (Figure 5). Here, we see that all cells (represented by single dots) express genes corresponding to different cellular stages, such as NSCs and off-targets, which were predominantly present in the lower half of the t-SNE plot (SOX2+, Figure 5), IPs located in the middle of the t-SNE plot (ASCL1+, Figure 5), as well as neurons (INA+, Figure 5), located in the upper part of the t-SNE plot. Further illustrating the heterogeneity of these cultures, a marker corresponding to the ventral midbrain floor plate region of the embryo (LMX1A) was not expressed in all cells and was selectively absent in two clusters located at the bottom of the t-SNE plot (Figure 5, circled). By comparing the clusters corresponding to these different cell types and stages, we were able to identify genes whose expression is restricted to these identities.

[0150] Example 4: Expression profiles of candidate genes for NSC, IP, and neuron isolation The highest scoring differentially expressed surface marker genes in neuronal clusters were confirmed to have a distribution across t-SNE plots similar to the neuronal marker INA (Figures 6-7), and these genes are deemed suitable for the isolation of neurons from mixed cultures. This includes the genes ADGRG1, EPFHB2, GAP43, ADCYAP1, C1QL1, and NRXN1 (Figure 6), as well as FZD1, CACNA2D1, DCC, GPR85, and LRRC4C.

[0151] The highest scoring differentially expressed surface marker genes in the IP cell cluster were confirmed to have a distribution across t-SNE plots similar to IP cell markers of the dopamine neuronal lineage ASCL1 (Figure 8), and these genes are deemed suitable for the isolation of neurons from mixed cultures. This includes the genes TNFRSF21, TRPM8, FREM2, DLL3, DLL1, CHRNA5, and CLDN5.

[0152] The highest scoring differentially expressed surface marker genes in the NSC / off-target cluster were confirmed to have a similar distribution across t-SNE plots as the NSC / off-target marker SOX2 (Figures 9-11), and these genes are deemed suitable for isolation of NSC / off-target cells from mixed cultures. This includes the genes SLIT2, NTN1, CMTM7, CMTM8, CD36, CD47, CD99, TNFRSF1A, TMEM123, LRP10, HLA-A, RAMP1, CMTM6, TNFRSF12A, CRLF1, ITGB8, and CD83.

[0153] Example 5: Fluorescence-activated cell sorting (FACS) of midbrain nervous system cells using antibodies against CD47 and CD99 Ventral midbrain dopaminergic (vmDA) progenitor cells were generated from hESCs in 2D in vitro culture as described in Example 1. 25 days after the initiation of differentiation, cell cultures were dissociated into single cell suspensions using Accutase and collected in N2 medium (CTS™ Neurobasal™ medium supplemented with 1% CTS™ N-2 supplement). Dead cells were labeled using the LIVE / DEAD™ Fixable Near-IR Dead Cell Stain Kit. Cells were then resuspended in B27 medium (CTS™ Neurobasal™ medium supplemented with 1% B-27™ supplement, no vitamin A, 2 mM GlutaMAX™, 60 U / mL penicillin-streptomycin, 10 μM ROCK inhibitor) and stained using an APC-conjugated antibody against CD47 at a concentration of 1:2500. CD47 positive and negative cells were purified by fluorescence-activated cell sorting (FACS) using a BD FACSAria™ Fusion instrument (Figure 12). A "pass-through" control sample was obtained by sorting the total live cell population (Figure 12C). After FACS, purity was confirmed to be >87% and >91% for the CD47 positive and negative cell fractions, respectively (Figure 13). Immediately after FACS, sorted cells were incubated with 10% CO for 1 h in B27 medium supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 μM), dcAMP (500 μM), DAPT (10 μM), and Y-27632 (10 μM) in poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm 2 )-coated 96-well plates at 450,000 cells / cm 2The cells were then reseeded at a density of 100x100x100 / mL. Phase contrast images obtained after 24 hours (Figure 19) and 5 days (Figure 20) showed high viability of the sorted cells, and clear differences in morphology between CD47 positive and negative cells. CD47 positive cells are seen to have large, low contrast cell bodies with little or no processes (Figures 19A-Ai, 20A-Ai), while CD47 negative cells show a typical neuronal morphology characterized by small, high contrast round / oval cell bodies with well-defined processes, which can be seen to increase in number and length over the 5 day culture period (Figures 19B-Bi, 20B-Bi). As expected, the "pass-through" control samples contain a mixture of cells with these two distinct morphologies (Figures 19C-Ci, 20C-Ci). In addition, immediately after cell sorting, samples of sorted cells were processed for intracellular flow cytometry using BD Transcription Factor Buffer Set, stained with antibodies against SOX2 (1:30), Ki-67 (1:100), and FOXA2 (1:320), recorded using a BD FACSymphony™ flow cytometer, and analyzed using FlowJo 10.7.2 software. The purity of the sorted samples was initially confirmed to be >91% (Figures 14, 18). We found that the CD47 positive sorted fraction was enriched for cells expressing the neural stem cell marker SOX2 (71.1%, Figure 15A, 18) as well as the proliferation marker Ki-67 (26.3%, Figure 16A, 18), while the CD47 negative sorted fraction showed significantly lower levels of both SOX2 (16.2%, Figure 15B, 18) and Ki-67 (2.67%, Figure 16B, 18), corresponding to a difference in the percentage of cells expressing SOX2 and Ki67 of 77.2% and 89.8%, respectively. These results support the hypothesis that CD47 antibody-based selection can be used to enrich for neurons, as seen by the percentage of cells expressing FOXA2, a marker of the developing floor plate, as well as to deplete proliferative neural stem cells and their derivatives without depletion of the lineage of interest (Figures 17, 18). These results were supported by immunofluorescence (IF) staining of cells replated in vitro.Five days after sorting, cells were fixed in 4% paraformaldehyde and stained with primary antibodies against Ki-67 (1:250), FOXA2 (1:100), and tyrosine hydroxylase (TH, 1:500), followed by fluorescently labeled secondary antibodies. Images were acquired on an Olympus IX81 microscope using CellSens software. IF staining confirmed the flow cytometry results showing that enrichment of CD47-negative cells reduced the number of Ki67+ proliferative cells (Figure 21, Figure 35) and SOX2+ cells (Figure 36) without depleting the cells of interest, as seen by FOXA2 and the dopamine neuron specific marker TH (Figures 22, 23).

[0154] In a similar experiment, day 25 vmDA cultures were dissociated into single cell suspensions and stained using a PE-conjugated antibody against CD99 at a concentration of 1:500. CD99 positive and negative cells were purified by fluorescence-activated cell sorting (FACS) using a BD FACSAria™ Fusion instrument (Figure 24). A "pass-through" control sample was obtained by sorting the total live cell population (Figure 24C). After FACS, purity was confirmed to be 79% and 100% for the CD99 positive and negative cell fractions, respectively (Figure 25), and high viability (>85%) was observed in all sorted fractions (Figure 26). Sorted cells were cultured in B27 medium supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 μM), dcAMP (500 μM), DAPT (10 μM), and Y-27632 (10 μM) in poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm 2 )-coated 96-well plates at 450,000 cells / cm 2The cells were then reseeded at a density of 100x100 / mL. Phase contrast images obtained after 24 hours (Figure 27), 48 hours (Figure 28), and 5 days (Figure 29) showed good recovery of sorted cells and clear differences in morphology between CD99 positive and negative cells, with the CD99 negative fraction being found to contain non-neuronal cells characterized by large, low contrast cell bodies, and little or no processes (Figures 27A-Ai, 28A-Ai, 29A-Ai). These non-neuronal cells were absent in the CD99 negative sorted fraction, and instead the cells displayed typical neuronal morphology characterized by small, high contrast round / oval cell bodies with well-defined processes, which can be seen to increase in number and length over the 5 day culture period (Figures 27B-Bi, 28B-Bi, 29B-Bi). As expected, the "pass-through" control sample contained a mixture of cells with these two distinct morphologies (Figures 27C-Ci, 28C-Ci).

[0155] 48 hours and 12 days after sorting, cells were qualitatively evaluated by IF using antibodies against Ki-67, FOXA2, and TH. IF staining showed that the CD99-negative sorted fraction contained a reduced number of cells expressing Ki-67 compared to CD99-positive and "pass-through" control cells (Figures 30, 31), but still showed a large amount of cells expressing the dopamine neuron lineage-specific markers FOXA2 and TH (Figures 32, 33). Taken together, these results support the hypothesis that CD99 antibody-based selection can be used to enrich for neurons and deplete proliferative neural stem cells and their non-neuronal derivatives.

[0156] FACS enrichment of CD47-negative or CD99-negative cells allowed for enrichment of neuronal cells and a substantial reduction in the number of proliferative NSC / non-neuronal cell types, whereas neither CD47 nor CD99 alone allowed for complete depletion of these undesirable proliferative cell types. Interestingly, flow cytometry analysis of day in vitro (DIV) 25 vmDA cultures revealed that the majority of cells (34.5%) expressed both CD47 and CD99 at this stage (Figure 34 Q2), but there were also cell populations expressing only CD47 (8.28%, Figure 34 Q3) or CD99 (6.42%, Figure 34 Q1), suggesting that the combination of these markers for enrichment results in an even purer neuronal population than when used by themselves. In one experiment, day 25 vmDA cultures were dissociated into single cell suspensions and stained with antibodies against CD47 alone or in combination with antibodies against CD99, and positive (CD47+) and negative (CD47-) or double positive (CD47+CD99+) and double negative (CD47-CD99-) cell populations, respectively, were purified by FACS and analyzed by intracellular flow cytometry as described previously. First, the percentage of cells expressing CD47 and / or CD99 in the isolated fractions was determined. Unsorted control samples showed expression of CD47 and CD99 in approximately 60% of the cells (Figures 37, 38). As expected, the percentage of cells expressing CD47 was increased in CD47+ and CD47+CD99+ samples (>94%, Figure 37) and was almost completely depleted in CD47- and CD47-CD99- samples (<2.5%, Figure 37), confirming successful separation of the populations. Similarly, the percentage of cells expressing CD99 was increased in both CD47+ and CD47+CD99+ samples (>83%, FIG. 38) and was almost completely depleted in CD47- and CD47-CD99- samples (<3.5%, FIG. 38), indicating that enrichment of CD47- cells by CD47 antibody-based FACS results in depletion of CD99+ cells.Both the CD47- and CD47-CD99- fractions show a significant reduction in the percentage of cells expressing SOX2 (Figure 39) and Ki67 (Figure 40), as well as increased levels of neurons marked by INA (Figure 41). [Table 1]

[0157] Example 6: Magnetic-activated cell sorting (MACS) of midbrain nervous system cells using magnetic sorting antibodies against CD47 Ventral midbrain dopaminergic (vmDA) progenitor cells were generated from hESCs in 2D in vitro culture as described in Example 1. 24 days after the initiation of differentiation, cell cultures were dissociated into single cell suspensions using Accutase and collected in B27 medium (CTS™ Neurobasal™ medium supplemented with 1% B-27™ supplement without vitamin A, 2 mM GlutaMAX™, 60 U / mL penicillin-streptomycin, 10 μM ROCK inhibitor). Cells were then resuspended in Hank's balanced salt solution with 0.5% BSA and stained using an APC-conjugated antibody against CD47 at a concentration of 1:50, followed by labeling with anti-APC microbeads. CD47 positive and negative cells were purified by magnetic activated cell sorting (MACS) using a LS MACS column. The flow-through fraction containing unlabeled cells was collected. Magnetically retained cells were eluted and collected as the positively selected fraction. After MACS, viability was confirmed to be >90% for both CD47 negative and positive cell fractions. Immediately after MACS, sorted cells were cultured in B27 medium supplemented with poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm) supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 μM), dcAMP (500 μM), DAPT (10 μM), and Y-27632 (10 μM). 2 )-coated 96-well plates at 200,000 cells / cm 2The cells were replated at a density of 100-1500 nm. Unsorted cells were used as a control. Phase contrast images obtained after 48 hours (Figure 42) showed high viability of the sorted cells and clear differences in morphology between unsorted and CD47 negative cells. While CD47 negative cells showed typical neuronal morphology characterized by small, high contrast round / oval cell bodies with well-defined processes (Figure 42A), unsorted cells appeared to have a mixture of neuronal cells and cells with large, low contrast cell bodies with little or no processes (Figure 42B). In addition, immediately after cell sorting, a sample of sorted cells was processed for intracellular flow cytometry using BD Transcription Factor Buffer Set, stained with antibodies against SOX2 (1:130), Ki-67 (1:2500), FOXA2 (1:320), LMX1A (1:2500), ASCL1 (1:2500), and INA (1:3000), recorded using a CytoFLEX™ flow cytometer, and analyzed using FlowJo 10.7.2 software. Initially, the purity of the sorted sample was confirmed to be greater than 68% (Figure 43). The CD47 negative fraction was found to express lower levels of the neural stem cell marker SOX2 (52.7%, FIG. 44) as well as the proliferation marker Ki-67 (7.9%, FIG. 44), while the unsorted controls showed higher levels of both SOX2 (78.6%, FIG. 44) and Ki-67 (23.1%, FIG. 44), corresponding to a difference in the percentage of cells expressing SOX2 and Ki67 of 33% and 65.8%, respectively. Furthermore, the CD47 negative fraction was found to maintain a comparable number of cells expressing FOXA2, a marker of the developing floor plate, compared to the unsorted controls (76%, FIG. 44).The CD47-negative fraction was also found to be enriched for cells expressing the early ventral midbrain marker LMX1A (78%, Figure 44), the intermediate progenitor marker ASCL1 (51.2%, Figure 44), and the neuronal marker INA (59.8%, Figure 44), while the unsorted control showed lower levels of all three markers (LMX1A 61%, ASCL1 28.8%, INA 23.3%, Figure 44), corresponding to a difference in the percentage of cells expressing LMX1A, ASCL1, and INA of 21.8%, 43.8%, and 61%, respectively. These results indicate that CD47 antibody-based selection can be used for the enrichment of neurons, as well as the depletion of proliferative neural stem cells and their derivatives. These results were corroborated by immunofluorescence (IF) staining of cells reseeded in vitro. Ten days after sorting, cells were fixed in 4% paraformaldehyde and stained with a primary antibody against the dopaminergic neuron marker tyrosine hydroxylase (TH, 1:500), followed by staining with a fluorescently labeled secondary antibody. Images were obtained on a Zeiss Axio Observer microscope using ZEN 3.2 Pro software. IF staining confirmed that enrichment of CD47-negative cells resulted in a cell population enriched for dopaminergic neurons (Figure 53). [Table 2]

[0158] Example 7: Differentiation of human pluripotent stem cells into forebrain neural system cells and measurement of CD47 and CD99 expression in these cells hESCs were differentiated according to established protocols (Shi et al., 2012(a); Shi et al., 2012(b)). hESCs were seeded and incubated with laminin-521 (1.2 μg / cm 2Cells were cultured on cultureware coated with 100% glycerin (BioLamina) and exposed to differentiation medium once they formed a 95-100% confluent monolayer. From DIV0-10, cells were cultured in N2 / B27-based medium: 50% DMEM / F12 + Glutamax (Gibco), 50% Neurobasal (Gibco), 2% B27 supplement, CTS with vitamin A (Thermo Fisher), 1% N2 supplement CTS (Thermo Fisher), 5% GlutaMAX (Thermo Fisher), 0.2% penicillin streptomycin (P / S, Thermo Fisher), 1% NEAA (Gibco), 0.089% β-mercaptoethanol (Gibco), supplemented with BMP pathway inhibitors LDN-193189 (100 nM, Miltenyi Biotec) for neural induction, and Noggin (100 ng / mL, Miltenyi Biotec). At DIV11, cells were dissociated with 0.5 mM EDTA and passaged at a 1:2 ratio. N2 / B27-based medium was supplemented with fibroblast growth factor b (20 ng / mL, R&D) from DIV11 to 18. At DIV18, cells were dissociated with 0.5 mM EDTA and cryopreserved or passaged at a 1:2 ratio. From DIV20 onwards, cells were cultured in N2 / B27-based medium without supplements and passaged to DIV32. At DIV35, forebrain neural system cells were dissociated into single cell suspensions using Accutase, stained with fluorophore-conjugated antibodies against CD47 and CD99, and analyzed by flow cytometry as described in Example 5. At this stage of differentiation, approximately 50% of the cells expressed the neural stem cell marker SOX2 (Figure 46), while approximately 20% expressed the proliferation marker Ki67 (Figure 46A), indicating that these cells are still mitotic neural stem cells, and approximately 60% expressed the neuronal marker INA (Figure 46B), indicating that they are differentiated or in the process of differentiating into neurons. Analysis showed that 63% of the cells expressed CD99 (Figure 45A) and 78% expressed CD47 (Figure 45B). Furthermore, it was found that the majority of cells expressing CD99 and CD47 co-expressed both SOX2 (Figure 47) and Ki67 (Figure 48).These results strongly support the hypothesis that CD99 and CD47 can be used as antibody targets for enrichment of forebrain neurons and depletion of proliferating cells by FACS or MACS.

[0159] Reference materials: ●Shi Y, Kirwan P, Livesey FJ.Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.Nat Protoc.2012 Oct;7(10):1836-46.doi:10.1038 / nprot.2012.116.Epub 2012 Sep 13.PMID:22976355.(a) ●Shi Y, Kirwan P, Smith J, Robinson HP, Livesey FJ.Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses.Nat Neurosci.2012 Feb 5;15(3):477-86,S1.doi:10.1038 / nn.3041.PMID:22306606;PMCID:PMC3882590.(b)

[0160] Example 8: Differentiation of human pluripotent stem cells into hindbrain / spinal cord nervous system cells and measurement of CD47 and CD99 expression in these cells Undifferentiated hESCs at 80–90% confluence were dissociated with EDTA 0.5 mM (Thermo Fisher) for 5–7 min at room temperature to detach from the flask for reseeding. hESC aggregates were then cultured in a 3x flask with laminin-521 (1.2 μg / cm 2) and evenly distributed onto pre-coated 24-well plates or T25 flasks (Sarstedt). For the first 24 h, cells were maintained in iPS Brew XF (Miltenyi Biotec) supplemented with 10 μM Y27632 (ROCKi, Miltenyi Biotec) to promote cell survival. Medium was changed every 24 h and cells were allowed to grow until 90–100% confluence, at which point differentiation was initiated. During the first 6 days of differentiation, cells were maintained in pan-neuronal basal medium consisting of 45% Neurobasal (Gibco), 45% DMEM / F12+GlutaMAX (Gibco), 5% B27 supplement (Thermo Fisher), 1% N2 supplement (Thermo Fisher), 1% MEM non-essential amino acids (NEAA, Gibco), 0,5% GlutaMAX (Gibco), 0.2% penicillin / streptomycin (P / S, Thermo Fisher), supplemented with BMP pathway inhibitors LDN-193189 (100 nM, Miltenyi Biotec) and Noggin (100 ng / mL, Miltenyi Biotec) for neural induction. For caudation 3 μM CHIR99021 (CHIR, Miltenyi Biotec) was added and the medium was changed daily. On day 6, cells were dissociated using Accutase and passaged at a ratio of 1:6. From DIV6 to 12, cells were grown in pan-neuron basal medium supplemented with BMP pathway inhibitors LDN-193189 (100 nM, Miltenyi Biotec) and Noggin (100 ng / mL, Miltenyi Biotec), as well as 1 μM CHIR, 100 nM purmorphamine (PM, Tocris), and 500 nM retinoic acid (RA, Sigma). From DIV12 to 18, cells were grown in pan-neuron basal medium (without vitamin A) supplemented with 100 nM PM and 500 nM RA. On DIV15, cells were dissociated using Accutase and passaged at a ratio of 1:6. From DIV18 onwards, cultures were maintained in pan-neuron basal medium (-VitA) for maturation and promotion of neurogenesis. Medium was changed daily and cells were washed with DPBS- / - before medium change to remove cell death.At DIV24, hindbrain / spinal cord nervous system cells were dissociated into single cell suspensions using Accutase, stained with fluorophore-conjugated antibodies against CD47 and CD99, and analyzed by flow cytometry as described in Example 5. At this stage of differentiation, approximately 37% of the cells expressed the neural stem cell marker SOX2 (Figure 50), while approximately 15% expressed the proliferation marker Ki67 (Figure 50A), indicating that these cells are still mitotic neural stem cells, and approximately 60% expressed the neuronal marker INA (Figure 50B), indicating that they are differentiated or in the process of differentiating into neurons. Analysis showed that 39% of the cells expressed CD99 (Figure 49A) and 92% expressed CD47 (Figure 49B). Furthermore, it was found that the majority of cells expressing CD99 and CD47 co-expressed both SOX2 (Figure 51) and Ki67 (Figure 52). These results strongly support the hypothesis that CD99 and CD47 can be used as antibody targets for enrichment of hindbrain / spinal cord neurons and depletion of proliferative cells by FACS or MACS.

[0161] Example 9: RNA sequencing methods and experimental design to identify markers of enrichment or depletion To investigate the transcriptomic signature of heterogeneous hPSC-derived ventral midbrain cell cultures with the goal of identifying genes unique or upregulated in desired cell populations (i.e., ventral midbrain intermediate precursors / neuroblasts marked by ASCL1) and undesired populations (i.e., non-ventral midbrain floor plate identity cells, non-neuronal precursors, non-neural cell types) in an unbiased manner, an in vitro time series of hPSC-derived ventral midbrain neural system cells was sampled using single-cell RNA sequencing (Figure 1 and Example 2).

[0162] Desired and undesired populations (or clusters as described in the field of single-cell RNA sequencing and transcriptomics) were identified in the sequencing dataset (i.e., the tSNE plot shown in Figure 5) based on gene expression profiles of known genes. Comparisons between these clusters were performed to identify novel genes / markers for the desired or undesired populations.

[0163] To perform single cell RNA sequencing (scRNA-seq), cell clusters of undifferentiated PSCs as well as cell clusters of differentiated cells were dissociated into single cell suspensions with Accutase, Tryple Select, or other such reagents, and 3000-10000 cells were processed using the 10X Genomics Chromium Platform and sequenced on a NextSeq550. Data were processed in the R programming language using the 10X Cellranger and Seurat analysis packages. After samples were analyzed and filtered for low quality or multiplet cells and analyzed separately for each individual experiment, cells of selected selected differentiated cell lineages as well as hPSCs were combined into one dataset, which was then analyzed using the standard Seurat workflow as outlined for Seurat version 3, i.e., normalization using SCTransform, and finally, the first 29 principal components of the integrated tSNE plot (such as that shown in Figure 5).

[0164] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. A method for providing a cell population enriched with neurons and / or their precursors, comprising: - obtaining a cell population comprising neuronal precursors; - selecting cells that are positive for one or more of the markers selected from PTPRO, ADGRG1, EPHB2, DCC, DLL3, DLL1, ADCYAP1, GPM6A, GAP43, C1QL1, NRXN1, LRRC4C, TNFRSF21, FZD1, FREM2, TRPM8, CACNA2D1, CLDN5, CHRNA5, and GPR85, and / or excluding cells that are positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD99, CD83, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

2. The method according to claim 1, wherein the cell population comprising neuronal precursors is obtained by a process of obtaining a cell population comprising neural stem cells, wherein at least 80% of the cell population comprising neural stem cells expresses the marker SOX2, and culturing the cell population comprising neural stem cells until the expression of SOX2 is reduced by at least 5%.

3. The method according to claim 1, wherein the cell population comprises neural stem cells of the ventral midbrain region, and cells positive for the marker CD47 are excluded.

4. The method according to claim 1, wherein the cell population comprises neural stem cells of the forebrain, midbrain, hindbrain, or spinal cord region, and cells positive for one or both of the markers CD99 and CD47 are excluded.

5. When selecting and / or excluding cells, 10-90% of the cell population is positive for the marker SOX2, 5-50% of the cell population is positive for one or more of the markers ASCL1, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, EOMES, CHX10, ISL1, SOX4, SOX11, NKX2.1, NFE2L2, and NHLH1, and 5-20% of the cell population is positive for one or more of the markers INA, TUBB3, STMN2, MAP2, and RBFOX3. The method according to claim 1.

6. When selecting and / or excluding cells, 10-90% of the cell population is positive for the marker SOX2, 5-50% of the cell population is positive for the marker ASCL1, and 5-20% of the cell population is positive for the marker INA. The method according to claim 3.

7. The step of obtaining the cell population containing neural precursors includes the step of differentiating PSCs into neural stem cells. The method according to claim 1.

8. The PSCs are differentiated into neural stem cells for at least 20 days before the step of selecting and / or excluding cells. The method according to claim 7.

9. The step of selecting and / or excluding cells is carried out by isolating the cells using MACS. The method according to claim 1.

10. An in vitro cell population of neurons and / or their precursors obtained by the method according to any one of claims 1 to 9.

11. An in vitro cell population of neurons and / or their precursors, wherein less than 5% of the cell population is positive for one or more of the markers selected from SLIT2, NTN1, CMTM6, CMTM8, CMTM7, CRLF1, CD36, CD47, CD99, CD83, TNFRSF1A / CD120a, TNFRSF12a / CD266, TMEM123, LRP10, ITGB8, HLA-A, and RAMP1.

12. Less than 5% of the cell population is positive for the marker CD99. The cell population according to claim 11.

13. The cell population according to claim 11, wherein less than 5% of the cell population is positive for the marker CD47 and at least 80% of the cell population is positive for the marker FOXA2.

14. The cell population according to claim 11, wherein at least 80% of the cell population is ventral midbrain neurons and / or their precursors.

15. The cell population according to claim 12, wherein at least 80% of the cell population is forebrain, hindbrain, or spinal cord neurons and / or their precursors.

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