Methods and compositions for generating human midbrain dopamine neurons from neural progenitor cells

Abstract

A method for generating human midbrain immature neurons and mature dopaminergic neurons from neural progenitor cells is provided. The immature and mature midbrain neurons are obtained from neural progenitor cells such as committed midbrain neural stem cells (NSCs) and midbrain neural progenitor cells (midbrain NPCs), which are themselves generated from human pluripotent stem cells. The method for generating midbrain immature neurons and mature neurons uses a chemically defined medium that enables the generation of mature dopaminergic neurons in just 23 days. Media, isolated cell populations, and kits are also provided.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 391,207, filed on Jul. 21, 2022, the entire contents of which are incorporated herein by reference.

[0002] Government Licensing Rights This invention was made with government support under grant number: W911NF-17-3-0003 awarded by the U.S. Army ACC-AGP-RTP. The U.S. government has certain rights in this invention.

Background Art

[0003] Background of the Invention Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder after Alzheimer's disease and is characterized by the degeneration of midbrain dopamine (mDA) neurons in the substantia nigra pars compacta. Current treatments typically employ a pharmacological approach aimed at enhancing the bioavailability of dopamine by administering levodopa (also known as L-dopa), a precursor of dopamine. However, the side effects of long-term treatment with levodopa are an issue regarding the use of levodopa in the later stages of PD. The ability to reconstitute functional dopaminergic neurons in vivo in PD patients was first explored by transplanting human fetal midbrain tissue (reviewed in Lindvall et al. (2004) NeuroRx 1:383-393 (Non-Patent Document 1)). The outcomes were variable, and this approach raised ethical concerns regarding the availability and use of fetal tissue, which led to the development of alternative approaches for the reconstitution of dopaminergic neurons in vivo.

[0004] The availability of pluripotent stem cells (PSCs), including embryonic stem (ES) cell lines and induced pluripotent stem cells (iPSCs), has opened up the possibility of generating precursors of mDA neurons in vitro. Developmental studies have demonstrated that midbrain dopaminergic neurons are derived from the ventral midbrain floor plate (mFP), which can be identified by the co-expression of the markers FOXA2 and LMX1A. Initial differentiation protocols for inducing midbrain floor plate precursors required activation of sonic hedgehog (SHH) and classical WNT signaling in PSCs, as well as dual SMAD inhibition and FGF8 activation, and took 11 days to obtain precursors expressing FOXA2 and LMX1A (Kriks et al. (2011) Nature 480:547-551 (Non-Patent Document 2)), or required activation of SHH, WNT, and FGF8, as well as the addition of retinoic acid (RA) for a 22-day protocol (Cooper et al. (2010) Mol. Cell. Neurosci. 45:258-266 (Non-Patent Document 3)). Similar protocols have also been reported in which human iPSC-derived embryoid bodies were exposed to dual SMAD inhibition for 5 days, followed by activation of SHH and FGF8, thereby yielding mDA precursors in 16 days (Hartfield et al. (2014) PLoS One 92:e87388 (Non-Patent Document 4)).

[0005] More recently, additional protocols for obtaining MB dopamine - competent precursors from human pluripotent stem cells have been reported. For example, Nolbrant et al. reported a 16 - day protocol that requires exposure to N - 2 supplement for the first 11 days and B27 supplement for the last 5 days, in addition to SHH and WNT activation and ALK inhibition (Nolbrant et al. (2017) Nature Protocols 12:1962 - 1979 (Non - Patent Document 5)). Precious et al. reported a protocol that requires 2 - day MEK inhibition to block FGF signaling, followed by 3 - day activation with SHH alone and activation with SHH and FGF8 from day 5 onwards, which resulted in FOXA2+ LMX1A+ precursors by day 7 (Precious et al. (2020) Front. Neurosci. 14:312 (Non - Patent Document 6)). Gartner et al. reported a xeno - free, feeder - free, chemically defined protocol that requires incubation in a medium supplemented with (i) LDN193189 and SB431542 from day 0 to 5 and LDN193189 only from day 5 to 10, (ii) CHIR99021 from day 2 to 13, and (iii) SHH and purmorphamine from day 1 to 7 (Gartner et al. (2020) Star Protocols 1:100065 (Non - Patent Document 7)).

[0006] Human dopamine - competent neuron precursors were also differentiated from human spermatogonial stem cells (hSSC) using a protocol that requires culture in SHH, FGF8A, and TFGβ3 - supplemented olfactory ensheathing cell - conditioned medium (OECCM) after 4 - day culture in hSSC - supplemented OECCM containing RA, SB, VPA, and forskolin (Yang et al. (2019) Stem Cell Res. Therap. 10:195 (Non - Patent Document 8)).

[0007] Methods for expanding midbrain neural progenitor cells (Fedele et al. (2017) Sci. Reports 7:6036 (Non-Patent Document 9)), and methods for cryopreserving such precursors have also been reported (Drummond et al. (2020) Front. Cell. Dev. Biol. 8:578907 (Non-Patent Document 10)).

[0008] Protocols for differentiating pluripotent stem cells into precursors of midbrain dopaminergic neurons have been outlined, for example, in Arenas et al. (2015) Development 142:1918-1936 (Non-Patent Document 11) and Wang et al. (2020) Cells 9:1489 (Non-Patent Document 12).

[0009] Thus, although some progress has been made, there is still a need for efficient and robust methods and compositions for generating midbrain neural progenitor cells from human pluripotent stem cells, and for generating immature and mature dopaminergic neurons from midbrain neural progenitor cells.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Summary of the Invention

[0011] The present disclosure provides a method for generating immature dopaminergic neurons and mature dopaminergic neurons from neural progenitor cells, such as human committed midbrain (MB) neural stem cells (NSCs) and midbrain neural progenitor cells (NPCs). The neural progenitor cells are obtained from pluripotent stem cells. The culture methods provided herein using a chemically defined medium enable the generation of mature dopaminergic neurons from human pluripotent stem cells in as little as 23 days of culture. The medium contains small molecule agents that stimulate or attenuate specific signaling pathway activities in pluripotent stem cells such that differentiation along the midbrain neural lineage is promoted, cell maturation and expression of midbrain neural progenitor cell-related biomarkers occur, and thereafter further differentiation and maturation into immature midbrain neurons by 9 days of culture and into mature dopaminergic neurons by 23 days of culture occur. The methods of the present disclosure have the advantage that precise control of the culture components is enabled by using small molecule agents in the medium, and the differentiation time is significantly reduced compared to prior art protocols.

[0012] Accordingly, in one aspect, the present disclosure relates to a method for generating human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons, the method comprising culturing human OTX2+ FOXA2+ LMX1A+ midbrain neural progenitor cells (MB NPCs) in a medium comprising a WNT pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist for 3 days (day 0 to day 3) to obtain human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons (MB immature neurons). The method may further comprise culturing the MB immature neurons in a medium comprising a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist for at least 14 days to obtain TH+ KCNJ6+ mature dopaminergic neurons.

[0013] In another aspect, the present disclosure relates to a two-step method for generating mature dopaminergic neurons from MB NPCs. Thus, in one aspect, the present disclosure provides a method for generating human TH+ KCNJ6+ mature dopaminergic neurons, comprising: (a) culturing human OTX2+ FOXA2+ LMX1A+ midbrain neural progenitor cells (MB NPCs) in a medium comprising a WNT pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist for 3 days (from day 0 to day 3) to obtain human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons (MB immature neurons); and (b) culturing the MB immature neurons in a medium comprising a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist for at least 14 days (from day 3 to day 17) to obtain TH+ KCNJ6+ mature dopaminergic neurons. The present disclosure also relates to a method comprising the above steps.

[0014] In yet another aspect, the present disclosure relates to a four-step method for generating mature dopaminergic neurons from pluripotent stem cells, which first generates MB NSCs, then generates MB NPCs, then generates immature midbrain neurons, and finally generates mature dopaminergic neurons. Thus, in one aspect, the present disclosure provides a method for generating human TH+ KCNJ6+ mature dopaminergic neurons, comprising: (a) culturing human pluripotent stem cells in a medium comprising a WNT pathway agonist, an SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist from day 0 to day 3 to obtain committed midbrain neural stem cells (MB NSCs); (b) To obtain human OTX2+ FOXA2+ LMX1A+ midbrain neural progenitor cells (MB NPCs), on days 4 to 6, culturing MB NSCs in a medium containing a BMP pathway agonist, a RA pathway agonist, an LXR pathway agonist, an AKT pathway antagonist, an mTOR pathway antagonist, and a TGFβ pathway antagonist; (c) To obtain human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons (MB immature neurons), on days 6 to 9, culturing MB NPCs in a medium containing a WNT pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist; and (d) To obtain TH+ KCNJ6+ mature dopaminergic neurons, on days 9 to 23, culturing MB immature neurons in a medium containing a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist relates to a method comprising the above.

[0015] In one aspect, the human pluripotent stem cells are induced pluripotent stem cells (iPSCs). In one aspect, the human pluripotent stem cells are embryonic stem cells.

[0016] In one aspect, the WNT pathway agonist is CHIR99021. Further exemplary WNT pathway agonists are disclosed herein together with exemplary concentrations and concentration ranges. In one aspect, the WNT pathway agonist is present in the medium at a concentration within the range of 0.5 to 2.0 μM. In one aspect, the WNT pathway agonist is CHIR99021 and CHIR99021 is present in the medium at a concentration of 1.0 to 1.1 μM.

[0017] In one aspect, the mTOR pathway antagonist is AZD3147. Additional exemplary mTOR pathway antagonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the mTOR pathway antagonist is present in the medium at a concentration within the range of 10 - 30 nM. In one aspect, the mTOR pathway antagonist is AZD3147, and AZD3147 is present in the medium at a concentration of 15 nM.

[0018] In one aspect, the RAR pathway antagonist is AGN193109. Additional exemplary RAR pathway antagonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the RAR pathway antagonist is present in the medium at a concentration within the range of 50 - 250 nM. In one aspect, the RAR pathway antagonist is AGN193109, and AGN193109 is present in the medium at a concentration of 100 nM.

[0019] In one aspect, the MEK pathway antagonist is PD0325901. Additional exemplary MEK pathway antagonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the MEK pathway antagonist is present in the medium at a concentration within the range of 50 - 250 nM. In one aspect, the MEK pathway antagonist is PD0325901, and PD0325901 is present in the medium at a concentration of 100 - 110 nM.

[0020] In one aspect, the Notch pathway antagonist is DBZ. Additional exemplary Notch pathway antagonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the Notch pathway antagonist is present in the medium at a concentration within the range of 50 - 250 nM. In one aspect, the Notch pathway antagonist is DBZ, and DBZ is present in the medium at a concentration of 100 nM.

[0021] In one aspect, the BMP pathway agonist is BMP7. Additional exemplary BMP pathway agonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the BMP pathway agonist is present in the medium at a concentration within the range of 5 - 50 ng / ml. In one aspect, the BMP pathway agonist is BMP7 and BMP7 is present in the medium at a concentration of 10 - 15 ng / ml.

[0022] In one aspect, the BDNF pathway agonist is BDNF. Additional exemplary BDNF pathway agonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the BDNF pathway agonist is present in the medium at a concentration within the range of 5 - 50 ng / ml. In one aspect, the BDNF pathway agonist is BDNF and BDNF is present in the medium at a concentration of 10 ng / ml.

[0023] In one aspect, the GDNF pathway agonist is GDNF. Additional exemplary GDNF pathway agonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the GDNF pathway agonist is present in the medium at a concentration within the range of 5 - 50 ng / ml. In one aspect, the GDNF pathway agonist is GDNF and GDNF is present in the medium at a concentration of 10 ng / ml.

[0024] In one aspect, the PPAR-a pathway agonist is GW7647. Additional exemplary PPAR-a pathway agonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the PPAR-a pathway agonist is present in the medium at a concentration within the range of 200 - 300 nM. In one aspect, the PPAR-a pathway agonist is GW7647 and GW7647 is present in the medium at a concentration of 250 nM.

[0025] In one aspect, the heparin or heparin mimetic is heparin. Additional exemplary heparin mimetics are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the heparin or heparin mimetic is present in the medium at a concentration within the range of 2 - 8 μg / ml. In one aspect, the medium contains heparin and the heparin is present in the medium at a concentration of 5 μg / ml.

[0026] In one aspect, the dopamine agonist is dopamine. Additional exemplary dopamine agonists are disclosed herein along with exemplary concentrations and concentration ranges. In one aspect, the dopamine agonist is present in the medium at a concentration within the range of 5 - 15 μM. In one aspect, the dopamine agonist is dopamine and the dopamine is present in the medium at a concentration of 10 μM.

[0027] In another aspect, the present disclosure relates to a medium. In one aspect, the present disclosure relates to a medium for obtaining human midbrain immature neurons, comprising a WNT pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist. In another aspect, the present disclosure relates to a medium for obtaining human mature dopaminergic neurons, comprising a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist.

[0028] In another aspect, the present disclosure relates to a culture of isolated cells. In one aspect, the present disclosure is a culture of isolated cells of human midbrain immature neurons, which are human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons cultured in a medium containing a WNT pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist. In another aspect, the present disclosure is a culture of isolated cells of human mature dopaminergic neurons, which are human TH+ KCNJ6+ mature dopaminergic neurons cultured in a medium containing a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist.

[0029] Also included are human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons produced by any method of the present disclosure. Also included are human TH+ KCNJ6+ mature dopaminergic neurons produced by any method of the present disclosure.

[0030] Other features and advantages of the present invention will become apparent from the following detailed description and the appended claims.

Brief Description of the Drawings

[0031]

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[0032] Detailed Description of the Invention Methods and compositions are described herein that enable the generation of mature dopaminergic neurons from midbrain neural progenitor cells (the midbrain neural progenitor cells themselves are generated from human pluripotent stem cells) under chemically defined culture conditions using a low-molecular-weight-based approach. The methods of the disclosure generate midbrain neural progenitor cells in a two-step protocol. In this protocol, OTX2+ LMX1A+ committed MB neural stem cells (NSCs) are generated in 3 days, and then OTX2+ LMX1A+ FOX2A+ MB neural progenitor cells (NPCs) are generated by day 6 of culture (referred to herein as stage 1 and 2 recipes). The MB-NPCs are then further differentiated using another two-step protocol (referred to herein as stage 3 and 4 recipes) to yield immature midbrain neurons by day 9 of culture and mature dopaminergic neurons by day 23 of culture. Thus, the disclosure enables the availability of mature dopaminergic neurons in a much shorter time than prior art protocols using chemically defined culture conditions.

[0033] As described in Example 1, a High-Dimensional Design of Experiments (HD-DoE) approach was used to simultaneously test multiple process inputs (e.g., small molecule agonists or antagonists) for output responses such as gene expression. These experiments enabled the identification of a chemically defined medium containing agonists and / or antagonists of specific signaling pathways sufficient to generate committed midbrain pluripotent stem cells and midbrain progenitor cells in a very short time. The optimized medium was further verified by factor criticality analysis, which examines the effect of removing one agonist or antagonist agent at a time, as described in Example 2. As described in Example 3, the phenotype of the cells generated by the differentiation protocol was further confirmed by immunohistochemistry. Furthermore, as described in Example 4, the expression of MB progenitor cell genes was also confirmed by RNA-seq analysis of cells cultured according to the differentiation protocol.

[0034] Figure 16 schematically shows aspects of the method of the present disclosure for generating MB NSCs and MB NPCs.

[0035] Figure 18 schematically shows aspects of the method of the present disclosure for generating immature midbrain neurons and mature dopaminergic neurons.

[0036] Various aspects of the invention are described in more detail in the following subsections.

[0037] I. Cells The starting cells used in the cultures of the present disclosure are human pluripotent stem cells. As used herein, the term "human pluripotent stem cell" (abbreviated as hPSC) refers to human stem cells having the ability to differentiate into various different cell types. As used herein, the term "pluripotency" refers to cells that have the ability to differentiate into cell types characterized by all three germ cell layers (endoderm, mesoderm, and ectoderm) under different conditions. For example, nude mouse and teratoma formation assays are used to characterize pluripotent cells, primarily by their ability to differentiate into all three germ layers. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, but the preferred test for pluripotency is demonstration of the ability to differentiate into cells of each of the three germ layers.

[0038] Human pluripotent stem cells include, for example, induced pluripotent stem cells (iPSCs) and human embryonic stem cells such as ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSCs) include 19-11-1, 19-9-7, or 6-9-9 cells (such as those described in Yu, J. et al. (2009) Science 324:797-801). Non-limiting examples of human embryonic stem cell lines include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, J.A. et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cellular markers that can be used to identify the cells as PSCs. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2. The methods of the present disclosure for generating committed midbrain neural stem cells and midbrain neural progenitor cells are used to differentiate (mature) a starting pluripotent stem cell population. Thus, in various embodiments, the midbrain-committed neural cell populations generated by the methods of the present disclosure lack the expression of one or more stem cell markers, such as one or more stem cell markers selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2.

[0039] Pluripotent stem cells are subjected to culture conditions that induce cellular differentiation as described herein. As used herein, the term "differentiation" refers to the development of a cell toward a more mature (i.e., less primitive) cell that typically exhibits phenotypic characteristics of commitment to a particular cell lineage from a more primitive stage.

[0040] As used herein, "neural stem cell" refers to a cell that is more differentiated than a pluripotent stem cell in that it is committed to the neural lineage, but still has the ability to differentiate into various types of cells along the neural lineage.

[0041] As used herein, "neural progenitor cell" refers to a cell that is more differentiated than a neural stem cell and can further differentiate into a specific type of neural cell.

[0042] In multiple aspects, cells can be identified and characterized based on the expression of one or more biomarkers, such as specific biomarkers of neural precursors or neural cells committed to the midbrain region. Non-limiting examples of biomarkers whose expression can be evaluated in the characterization of cells of interest include the following: OTX2, a midbrain marker involved in midbrain positioning and maintenance of the midbrain-hindbrain boundary (Vernay et al. (2005) J. Neurosci. 25:4856-4867); LMX1A, involved in the generation and differentiation of midbrain dopaminergic precursors (Yan et al. (2011) J. Neurosci. 31:12413-12425); FOXA2, which regulates the generation of midbrain dopaminergic neurons in the early and late stages of development (Ferri et al. (2007) Development 134:2761-2769); PAX2, expressed in the midbrain and in the anterior hindbrain (Urbanek et al. (1997) Proc. Natl. Acad. Sci. USA 94:5703-5708); nestin, a marker of early neurons; KI67, a proliferation marker; and GBX2, a hindbrain marker.

[0043] As used herein, an "extremely 'low'" level of expression of a biomarker of interest by a cell is intended to refer to a level that exceeds the background level by at most 20%, more preferably less than 20%, less than 15%, less than 10%, or less than 5% (where the background level corresponds, for example, to the expression level of a negative control marker that is considered not to be expressed by the cell).

[0044] In a plurality of aspects, the cells produced by the methods of the present disclosure are committed midbrain (MB) neural stem cells (NSCs). As used herein, "committed midbrain neural stem cells" or "committed MB NSCs" refer to stem cell-derived neural stem cells that express the biomarkers OTX2 and LMX1A. In one aspect, the committed MB NSCs do not express or express only low levels of the biomarker FOXA2. In one aspect, the committed MB NSCs do not express or express only low levels of the biomarker GBX2. In addition to OTX2 and LMX1A, the committed MB NSCs may also express additional biomarkers including, but not limited to, PAX2, nestin, and / or KI67.

[0045] In a plurality of aspects, the cells produced by the methods of the present disclosure are midbrain neural progenitor cells, which are more differentiated (more mature) cells than the committed MB NSCs. As used herein, "midbrain neural progenitor cells" or "MB NPCs" refer to stem cell-derived progenitor cells that express the biomarkers OTX2, LMX1A, and FOXA2. In one aspect, the MB NPCs do not express or express only low levels of the biomarker GBX2. In addition to OTX2, LMX1A, and FOXA2, the MB NPCs may also express additional biomarkers including, but not limited to, PAX2, nestin, and / or KI67.

[0046] The committed MB NSCs and MB NPCs produced by the methods of the present disclosure can be further cultured in vitro according to the culture protocols described herein to produce mature dopaminergic neurons. As used herein, "immature midbrain neurons" or "MB immature neurons" refer to neuron-derived cells that express the biomarkers FOXA2, LMX1A, MSX1, PITX3, and DCX. As used herein, mature dopaminergic neurons refer to neuron-derived cells that express the biomarkers TH and KCNJ6, and may also express TUBB3, MAP2, SYN1, and NF.

[0047] II. Medium Components The methods of the present disclosure for producing mature dopaminergic neurons, MB immature neurons, MB NSCs, or MB NPCs often lack exogenously-added growth factors and include culturing human pluripotent stem cells in a medium containing specific agonists and / or antagonists of cell signaling pathways.

[0048] As described herein, a medium containing a WNT pathway agonist, an SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist was sufficient to generate MB NSCs expressing OTX2 and LMX1A in just 3 days (referred to herein as "stage 1" of the differentiation protocol). Further differentiating the MB NSCs in a medium containing a BMP pathway agonist, a RA pathway agonist, an LXR pathway agonist, an AKT pathway antagonist, an mTOR pathway antagonist, and a TGF-β pathway antagonist was sufficient to generate OTX2+FOXA2+LMX1A+ MB NPCs in an additional 3 days for the 2-stage protocol over the full 6 days for generating MB NPCs (referred to herein as "stage 2"). Further differentiating the MB NPCs in a medium containing a Wnt pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist for an additional 3 days was sufficient to generate FOXA2+, LMX1A+, MSX1+, PITX3+, DCX+ immature MB neurons by day 9 of culture (referred to herein as "stage 3"). Finally, further differentiating the immature MB neurons in a medium containing a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist for an additional 14 days was sufficient to generate TH / KCNJ6+ mature dopaminergic neurons by day 23 of culture (referred to herein as "stage 4").

[0049] As used herein, an "agonist" of a cellular signaling pathway is intended to refer to an agent that stimulates (upregulates) the cellular signaling pathway. Stimulation of a cellular signaling pathway can be initiated extracellularly, for example, by use of an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., the agonist may be a receptor ligand). Additionally or alternatively, stimulation of cellular signaling can be initiated intracellularly, for example, by use of a small molecule agonist that interacts intracellularly with a component(s) of the signaling pathway.

[0050] As used herein, an "antagonist" of a cellular signaling pathway is intended to refer to an agent that inhibits (downregulates) the cellular signaling pathway. Inhibition of a cellular signaling pathway can be initiated extracellularly, for example, by use of an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of cellular signaling can be initiated intracellularly, for example, by use of a small molecule antagonist that interacts intracellularly with a component of the signaling pathway.

[0051] The agonists and antagonists used in the methods of the present disclosure are known in the art and are commercially available. These are used in the culture medium at effective concentrations to achieve a desired outcome, such as the generation of midbrain NSCs and / or midbrain NPCs that express a midbrain marker of interest. Non-limiting examples of suitable agonist and antagonist agents, as well as effective concentration ranges, are further described below.

[0052] WNT pathway agonists include agents, molecules, compounds, or substances that can stimulate (upregulate) the classical Wnt / β-catenin signaling pathway, which is biologically activated by the binding of Wnt-protein ligands to Frizzled family receptors. In one aspect, the WNT pathway agonist is a glycogen synthase kinase 3 (Gsk3) inhibitor. In one aspect, the WNT pathway agonist is selected from the group consisting of CHIR99021, CHIR98014, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, AZD1080, WNT3A, alsterpaullone, indirubin-3-oxime, 1-azakenpaullone, kenpaullone, TC-G24, TDZD 8, TWS 119, NP 031112, AT 7519, KY 19382, AZD2858, and combinations thereof. In one aspect, the WNT pathway agonist is present in the medium at a concentration within the range of 0.3 to 3.0 μM, 0.5 to 2.0 μM, 0.75 to 1.5 μM, or 1.0 to 1.2 μM. In one aspect, the WNT pathway agonist is CHIR99021. In one aspect, the WNT pathway agonist is CHIR99021 and CHIR99021 is present in the medium at a concentration within the range of 0.3 to 3.0 μM, 0.5 to 2.0 μM, 0.75 to 1.5 μM, or 1.0 to 1.2 μM. In one aspect, the WNT pathway agonist is CHIR99021 and CHIR99021 is present in the medium (e.g., in stage 1 medium) at a concentration of 1.1 μM. In one aspect, the WNT pathway agonist is CHIR99021 and CHIR99021 is present in the medium (e.g., in stage 3 medium) at a concentration of 1.0 mM.

[0053] Agonists of the SHH (Sonic Hedgehog) pathway include agents, molecules, compounds, or substances that can stimulate (activate) signal transduction via the SHH pathway, which biologically requires the binding of SHH to the Patched-1 (PTCH1) receptor and transmission via the Smoothened (SMO) transmembrane protein. In one aspect, the SHH pathway agonist is selected from the group consisting of purmorphamine, GSA 10, SAG, and combinations thereof. In one aspect, the SHH pathway agonist is present in the medium at a concentration within the range of 100-1000 nM, 200-800 nM, 250-750 nM, or 500-600 nM. In one aspect, the SHH pathway agonist is purmorphamine. In one aspect, the SHH pathway agonist is purmorphamine and purmorphamine is present in the medium at a concentration of 100-1000 nM, 200-800 nM, 250-750 nM, or 500-600 nM. In one aspect, the SHH pathway agonist is purmorphamine and purmorphamine is present in the medium at a concentration of 550 nM.

[0054] Antagonists of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) the BMP signaling pathway, which is biologically activated by the binding of BMP to BMP receptors that are activin receptor-like kinases (ALKs) (type I BMP receptors including, without limitation, ALK2 and ALK3). In one aspect, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof. In one aspect, the BMP pathway antagonist is present in the culture medium at a concentration in the range of 100-500 nM, 100-400 nM, 150-350 nM, or 200-300 nM. In one aspect, the BMP pathway antagonist is LDN193189. In one aspect, the BMP pathway antagonist is LDN193189 and LDN193189 is present in the culture medium at a concentration in the range of 100-500 nM, 100-400 nM, 150-350 nM, or 200-300 nM. In one aspect, the BMP pathway antagonist is LDN193189 and LDN193189 is present in the culture medium at a concentration of 275 nM.

[0055] Antagonists of the AKT pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) the signaling pathway of one or more serine / threonine kinases of the AKT family members, and the pathway includes AKT1 (also known as PKB or RacPK), AKT2 (also known as PKBβ or RacPK-β), and AKT3 (also known as PKBγ or thymoma viral proto-oncogene 3). In one aspect, the AKT pathway antagonist is selected from the group consisting of MK2206, GSK690693, perifosine (KRX-0401), ipatasertib (GDC-0068), capivasertib (AZD5363), PF-04691502, AT7867, triciribine (NSC154020), ARQ751, miransertib (ab235550), borussertib, cerisertib, and combinations thereof. In one aspect, the AKT pathway antagonist is present in the medium at a concentration within the range of 25 to 300 nM, 50 to 250 nM, 75 to 200 nM, or 125 to 150 nM. In one aspect, the AKT pathway antagonist is MK2206. In one aspect, the AKT pathway antagonist is MK2206 and MK2206 is present in the medium at a concentration within the range of 25 to 300 nM, 50 to 250 nM, 75 to 200 nM, or 125 to 150 nM. In one aspect, the AKT pathway antagonist is MK2206 and MK2206 is present in the medium at a concentration of 138 nM.

[0056] In one aspect, the AKT pathway antagonist present in the medium in step (a) is the same as the AKT pathway antagonist present in the medium in step (b). In one aspect, the AKT pathway antagonist present in the medium in step (a) is a different AKT pathway antagonist from the AKT pathway antagonist present in the medium in step (b). In one aspect, the AKT pathway antagonist present in the medium in both step (a) and step (b) is MK2206, and MK2206 is present in the medium in both steps, for example, at a concentration within the range of 25 to 300 nM, 50 to 250 nM, 75 to 200 nM, or 125 to 150 nM, for example, at 138 nM in both steps.

[0057] Antagonists of the MEK pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) the signaling pathway of one or more components of the MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway). In one aspect, the MEK pathway antagonist is selected from the group consisting of PD0325901, binimetinib (MEK162), cobimetinib (XL518), selumetinib, trametinib (GSK1120212), CI-1040 (PD-184352), refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO 5126766, BIX 02189, pimasertib, TAK 733, AZD8330, PD318088, SL 327, GDC 0623, RO5126766, myricetin, and combinations thereof. In one aspect, the MEK pathway antagonist is present in the medium at a concentration within the range of 25 to 300 nM, 50 to 250 nM, 75 to 200 nM, or 100 to 120 nM. In one aspect, the MEK pathway antagonist is PD0325901. In one aspect, the MEK pathway antagonist is PD0325901 and PD0325901 is present in the medium at a concentration within the range of 25 to 300 nM, 50 to 250 nM, 75 to 200 nM, or 100 to 120 nM. In one aspect, the MEK pathway antagonist is PD0325901 and PD0325901 is present in the medium at a concentration of 110 nM (e.g., in the stage 1 protocol). In one aspect, the MEK pathway agonist is PD0325901 and PD0325901 is present in the medium at a concentration of 100 nM (e.g., in the stage 3 protocol).

[0058] Agonists of the RA pathway include agents, molecules, compounds, or substances that can stimulate retinoic acid receptors (RARs) activated by both all-trans retinoic acid and 9-cis retinoic acid. There are three RARs, RAR-α, RAR-β, and RAR-γ, which are encoded by the RARA, RARB, and RARG genes, respectively. A variety of retinoic acid analogs that can activate the retinoic acid pathway have been synthesized. Non-limiting examples of such compounds include TTNPB (an agonist of RAR-α, β, and γ), AM 580 (an RARα agonist), CD 1530 (a potent and selective RARγ agonist), CD 2314 (a selective RARβ agonist), Ch 55 (a potent RAR agonist), BMS 753 (an RARα-selective agonist), tazarotene (a receptor-selective retinoid; binds to RAR-β and γ), isotretinoin (an endogenous agonist for retinoic acid receptors; an inducer of neuronal differentiation), and AC 261066 (an RARβ2 agonist). In some embodiments, the RA signaling pathway agonist is selected from the group consisting of (i) a retinoid compound, (ii) a retinoid X receptor (RXR) agonist, and (iii) a 25 retinoic acid receptor (RAR) agonist. In certain embodiments, the RA pathway agonist is selected from the group consisting of retinoic acid, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxoretinoic acid, and all-trans retinoic acid (ATRA).

[0059] Thus, in one aspect, the RA pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD 2314, Ch 55, BMS 753, tazarotene, isotretinoin, AC 261066, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxoretinoic acid, and all-trans retinoic acid (ATRA), or combinations thereof. In one aspect, the RA pathway agonist is present in the medium at a concentration within the range of 5 - 500 nM, 25 - 250 nM, 10 - 100 nM, or 25 - 75 nM. In one aspect, the RA pathway agonist is TTNPB. In one aspect, the RA pathway agonist is TTNPB and TTNPB is present in the medium at a concentration within the range of 5 - 500 nM, 25 - 250 nM, 10 - 100 nM, or 25 - 75 nM. In one aspect, the RA pathway agonist is TTNPB and TTNPB is present in the medium at a concentration of 50 nM.

[0060] Retinoic acid receptor pathway antagonists include agents, molecules, compounds, or substances that can inhibit the retinoic acid receptor (RAR) (i.e., the receptor activated by retinoic acid). In one aspect, the RAR pathway antagonist is selected from the group consisting of AGN193109, BMS 195614, CD 2665, ER 50891, LE 135, LY 2955303, MM11253, and combinations thereof. In one aspect, the RAR pathway antagonist is present in the medium at a concentration within the range of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM, or 100 - 120 nM. In one aspect, the RAR pathway antagonist is AGN193109. In one aspect, the RAR pathway antagonist is AGN193109 and AGN193109 is present in the medium at a concentration within the range of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM, or 100 - 120 nM. In one aspect, the RAR pathway antagonist is AGN193109 and AGN193109 is present in the medium at a concentration of 100 nM (e.g., in a stage 3 protocol).

[0061] Agonists of the LXR (liver X receptor) pathway include substances, molecules, compounds, or agents that can stimulate (activate) signal transduction via the LXR pathway, which biologically requires heterodimerization of LXR and retinoid X receptor (RXR) and activation by oxysterols. In one aspect, the LXR pathway agonist is selected from the group consisting of GW3965, T0901317, DMHCA, AZ876, and combinations thereof. In one aspect, the LXR pathway agonist is present in the medium at a concentration in the range of 100 - 1000 nM, 200 - 800 nM, 250 - 750 nM, or 550 - 650 nM. In one aspect, the LXR pathway agonist is GW3965. In one aspect, the LXR pathway agonist is GW3965 and GW3965 is present in the medium at a concentration of 100 - 1000 nM, 200 - 800 nM, 250 - 750 nM, or 550 - 650 nM. In one aspect, the LXR pathway agonist is GW3965 and GW3965 is present in the medium at a concentration of 500 nM.

[0062] Agonists of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the BMP signaling pathway, which is biologically activated by the binding of BMP to BMP receptors that are activin receptor-like kinases (ALKs) (type I BMP receptors, including, without limitation, ALK2 and ALK3). In one aspect, the BMP pathway agonist is selected from the group consisting of BMP, sb4, ventromorphin (e.g., as described in Genthe et al. (2017) ACS Chem. Biol. 12:2436-2447), and combinations thereof. In one aspect, the BMP pathway agonist is present in the medium at a concentration in the range of 1 to 100 ng / ml, 5 to 50 ng / ml, 10 to 25 ng / ml, or 12.5 to 17.5 ng / ml. In one aspect, the BMP pathway agonist is BMP7. In one aspect, the BMP pathway agonist is BMP7 and BMP7 is present in the medium at a concentration of 1 to 100 ng / ml, 5 to 50 ng / ml, 10 to 25 ng / ml, or 12.5 to 17.5 ng / ml. In one aspect, the BMP pathway agonist is BMP7 and BMP7 is present in the medium at a concentration of 15 ng / ml in step (b) (i.e., stage 2) of the method. In one aspect, the BMP pathway agonist is BMP7 and BMP7 is present in the medium at a concentration of 10 ng / ml in stage 3.

[0063] Antagonists of the TGFβ (transforming growth factor β) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) signaling through TGFβ receptor family members, which are a family of serine / threonine kinase receptors. In one aspect, this includes those selected from the group consisting of A 83-01, SB-431542, GW788388, SB525334, TP0427736, RepSox, SD-208, and combinations thereof. In one aspect, the TGFβ pathway antagonist is present in the medium at a concentration within the range of 100 - 500 nM, 200 - 400 nM, 250 - 350 nM, or 275 - 325 nM. In one aspect, the TGFβ pathway antagonist is A 83-01. In one aspect, the TGFβ pathway antagonist is A 83-01 and A 83-01 is present in the medium at a concentration of 100 - 500 nM, 200 - 400 nM, 250 - 350 nM, or 275 - 325 nM. In one aspect, the TGFβ pathway antagonist is A 83-01 and A 83-01 is present in the medium at a concentration of 300 nM in step (b) (i.e., stage 2) of the method.

[0064] Antagonists of the mTOR (mammalian target of rapamycin) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) signaling through mTOR, where mTOR is a member of the PI3K-related kinase family and is a core component of the mTORC1 and mTORC2 complexes. In one aspect, the mTOR pathway antagonist is selected from the group consisting of AZD3147, rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, umirolimus, zotarolimus, Torin-1, Torin-2, bisindolylmaleimide, AZD8055, dactolisib, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, niclosamide, omipalisib, PF 04691502, PF 05212384, WYE 687, XL 388, STK16-IN-1, PP 242, torquinib, sapitinib, bicalutamide, and combinations thereof. In one aspect, the mTOR pathway antagonist is present in the medium at a concentration within the range of 5 to 100 nM, 5 to 50 nM, 10 to 30 nM, or 10 to 20 nM. In one aspect, the mTOR pathway antagonist is AZD3147. In one aspect, the mTOR pathway antagonist is AZD3147 and AZD3147 is present in the medium at a concentration of 5 to 100 nM, 5 to 50 nM, 10 to 30 nM, or 10 to 20 nM. In one aspect, the mTOR pathway antagonist is AZD3147 and AZD3147 is present in the medium at a concentration of 15 nM in step (b) (i.e., stage 2) of the method. In one aspect, the mTOR pathway agonist is AZD3147 and AZD3147 is present in the medium at a concentration of 15 nM in stage 3.

[0065] Notch pathway antagonists include agents, molecules, compounds, or substances that can inhibit (downregulate) signal transduction via the Notch receptor. In one embodiment, the Notch pathway antagonist is selected from the group consisting of DBZ, avagacestat, bevasestat, BMS 299897, Compound E, DAPT, JLK6, L-685,458, LY 450139, MRK 560, PF 3084014 hydrobromide, LY 3039478, LY 411575, RO 4929097, and combinations thereof. In one embodiment, the Notch pathway antagonist is present in the medium at a concentration within the range of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM, or 100 - 120 nM. In one embodiment, the Notch pathway antagonist is DBZ. In one embodiment, the Notch pathway antagonist is DBZ and DBZ is present in the medium at a concentration within the range of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM, or 100 - 120 nM. In one embodiment, the Notch pathway antagonist is DBZ and DBZ is present in the medium at a concentration of 100 nM (e.g., in a stage 3 protocol).

[0066] Agonists of the brain-derived neurotrophic factor (BDNF) pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the BDNF signaling pathway. In one aspect, the BDNF pathway agonist is selected from the group consisting of BDNF, ropinirole, 7,8-DHF, ketamine, tricyclic dimeric peptide-6 (TDP6), LM22A-4, and combinations thereof. In one aspect, the BDNF pathway agonist is present in the medium at a concentration in the range of 1 to 100 ng / ml, 5 to 50 ng / ml, 10 to 25 ng / ml, or 12.5 to 17.5 ng / ml. In one aspect, the BDNF pathway agonist is BDNF. In one aspect, the BDNF pathway agonist is BDNF, and BDNF is present in the medium at a concentration of 1 to 100 ng / ml, 5 to 50 ng / ml, 10 to 25 ng / ml, or 12.5 to 17.5 ng / ml. In one aspect, the BDNF pathway agonist is BDNF, and BDNF is present in the medium at a concentration of 10 ng / ml at stage 4 of the method.

[0067] Agonists of the glial cell line-derived neurotrophic factor (GDNF) pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the GDNF signaling pathway. In one aspect, the GDNF pathway agonist is selected from the group consisting of GDNF, BT13, BT44, and combinations thereof. In one aspect, the GDNF pathway agonist is present in the medium at a concentration in the range of 1 to 100 ng / ml, 5 to 50 ng / ml, 10 to 25 ng / ml, or 12.5 to 17.5 ng / ml. In one aspect, the GDNF pathway agonist is GDNF. In one aspect, the GDNF pathway agonist is GDNF, and GDNF is present in the medium at a concentration of 1 to 100 ng / ml, 5 to 50 ng / ml, 10 to 25 ng / ml, or 12.5 to 17.5 ng / ml. In one aspect, the GDNF pathway agonist is GDNF, and GDNF is present in the medium at a concentration of 10 ng / ml at stage 4 of the method.

[0068] Agonists of the PPAR-α (peroxisome proliferator-activated receptor α) pathway include substances, molecules, compounds, or agents that can stimulate (upregulate) the PPAR-α signaling pathway. In one aspect, PPAR-α pathway agonists include GW7647, fenofibrate, fenofibrate-d6, WY 14643, CP 775146, CP 868388 free base, tesaglitazar, oleylethanolamide, oleylethanolamide-d2, oleylethanolamide-d4, PPAR agonist 1, clofibrate, clofibrate-d4, Wistin, indiglitazar, netoglitazone, GW0742, bezafibrate, bezafibrate-d4, chiglitazar, BMS 687453, lanifibranor, saroglitazar, saroglitazar magnesium, saroglitazar-d5, imiglitazar, AVE-8143, GW 590735, ertiprotafib, LJ570, ceradelparna sodium salt, edaglitazone, muraglitazar, lagaglitazar, GW 9578, MHY 908, KRP-297, elafibranor, aleglitazar, AM3102, eupatilin, clofibric acid, clofibric acid-d4, naveglitazar, naveglitazar racemate, and combinations thereof. In one aspect, the PPAR-α pathway agonist is present in the medium at a concentration within the range of 50 - 500 nM, 100 - 400 nM, 200 - 300 nM, or 225 - 275 nM. In one aspect, the PPAR-α pathway agonist is GW7647. In one aspect, the PPAR-α pathway agonist is GW7647 and GW7647 is present in the medium at a concentration of 100 - 500 nM, 200 - 400 nM, 250 - 350 nM, or 275 - 325 nM. In one aspect, the PPAR-α pathway agonist is GW7647 and GW7647 is present in the medium at a concentration of 250 nM at stage 4 of the method.

[0069] Heparin is an anticoagulant long known in the art, and heparin mimetics are synthetic and semi-synthetic compounds that are highly sulfated and structurally distinct analogs of glycosaminoglycans. In multiple embodiments, the medium comprises a heparin mimetic selected from the group consisting of heparin or a heparin mimetic such as heparan sulfate, enoxaparin, low molecular weight heparin, AV5026, M402, and combinations thereof. In one embodiment, the heparin or heparin mimetic is present in the medium at a concentration within the range of 1-10 μg / ml, 2-8 μg / ml, 3-7 μg / ml, or 4-6 μg / ml. In one embodiment, the medium comprises heparin and the heparin is present in the medium at a concentration within the range of 1-10 μg / ml, 2-8 μg / ml, 3-7 μg / ml, or 4-6 μg / ml. In one embodiment, the medium comprises heparin and the heparin is present in the medium at a concentration of 5 μg / ml.

[0070] Dopamine agonists include agents, molecules, compounds, or substances that can stimulate (upregulate) the dopamine signaling pathway. In multiple aspects, the dopamine agonist is dopamine, dopamine hydrochloride, (R)-(-)-apomorphine hydrochloride, bromocriptine mesylate, bromocriptine-13C,d3, CNV dopamine, dihydroergotamine mesylate, lisuride, lisuride maleate, methysergide hydrochloride, piribedil dihydrochloride, piribedil, quinelorane hydrochloride, (-)-quinpirole hydrochloride, ropinirole, tau-aggregation-IN-1, NMI 8739, U91356, phosphacarbidopa, quinagolide hydrochloride, dexfamipexole dihydrochloride, PD-168077 maleate, rotigotine hydrochloride, PF592379, rotigotine, (Rac)-rotigotine hydrochloride, (Rac)-rotigotine-d7 hydrochloride, Ro 10-5824 dihydrochloride, (+)-dihydroxidine hydrochloride, talipexole, PF2562, neuromedin N, A68930, A68930 hydrochloride, A77636 dihydrochloride, PD 119819, PD 128907 hydrochloride, CY 208-243, dexfamipexole, dexfamipexole-d3 dihydrochloride, dexfamipexole-d7 dihydrochloride, SKF 38393 hydrochloride, SKF 38393 hydrobromide, SKF 82958, ABT 670, ABT-724, ABT-724 trihydrochloride, (R)-PF-06256142, talipexole dihydrochloride, R-preclamol, pardoprunox hydrochloride, pardoprunox, pergolide mesylate, BP897, BP897 hydrochloride, LY 3154207, tabapadon, roxindole, UNC994, cabergoline, brexpiprazole, pergolide-d7 mesylate, cabergoline-d6, roxindole hydrochloride, WAY-100635 maleate, cibenadet hydrochloride, dihydroxidine, dihydroxidine hydrochloride, biphenprinox, OS-3-106, brilaroxazine, pramipexole dihydrochloride hydrate, pramipexole, pramipexole dihydrochloride, cabergoline-d5, perospirone, brexpiprazole-d8, (Rac)-tabapadon, ML417, brexpiprazole S-oxide, pramipexole-d7 dihydrochloride, pramipexole-d5 dihydrochloride, UCSF924, WAY-100635, SKF83959, pramipexole (N-propyl-3,3,(3-d3) (dihydrochloride), salizotan, brexpiprazole S-oxide D8, SKF 83959 hydrobromide, and combinations thereof. In one embodiment, the dopamine agonist is present in the medium at a concentration in the range of 5-15 μM, 7.5-12.5 μM, or 9-11 μM. In one embodiment, the dopamine agonist is dopamine. In one embodiment, the dopamine agonist is dopamine and dopamine is present in the medium at a concentration of 5-15 μM, 7.5-12.5 μM, or 9-11 μM. In one embodiment, the dopamine agonist is dopamine and dopamine is present in the medium at a concentration of 10 μM in step 4 of the method.,

[0071] III. Culture Conditions The method of making the mature dopamine - operative neurons, immature midbrain neurons, committed MB NSCs, and MB NPCs of the present disclosure, in combination with the chemically - defined and optimized medium described in subsection II above, utilizes standard culture conditions established in the art for cell culture. For example, the cells can be cultured at 37 °C under 5% O2 and 5% CO2 conditions. The cells can be cultured in standard culture vessels or plates such as 96 - well plates. In certain embodiments, the starting pluripotent stem cells are attached to the plate, preferably a plate coated with an extracellular matrix material such as vitronectin. In one embodiment, the stem cells are cultured on a vitronectin - coated culture surface (e.g., a vitronectin - coated 96 - well plate).

[0072] Pluripotent stem cells can be cultured in commercially available media before differentiation. For example, before the differentiation protocol begins, the stem cells can be cultured in Essential 8 Flex Medium (Thermo Fisher # A2858501) for at least one day. In a non-limiting exemplary embodiment, the stem cells are passaged onto a vitronectin (Thermo Fisher # A14700)-coated 96-well plate at a density of 150,000 cells / cm2 and cultured in Essential 8 Flex Medium for one day before differentiation.

[0073] To initiate the differentiation protocol, the medium containing the cultured stem cells is replaced with a basal differentiation medium to which a signal transduction pathway agonist and / or the antagonists described above in Subsection II are added. The basal differentiation medium may contain, for example, additional standard medium components necessary to maintain cell survival and proliferation, but lacks serum (the basal differentiation medium is a serum-free medium) or may contain a commercially available base lacking other externally added growth factors such as FGF2, PDGF, IGF, or HGF. In a non-limiting exemplary embodiment, the basal differentiation medium contains 1×IMDM (Thermo Fisher #12440046), 1×F12 (Thermo Fisher #11765047), 1 mg / ml poly(vinyl alcohol) (Sigma #p8136), 1% chemically defined lipid concentrate (Thermo Fisher #11905031), 450 μM 1-thioglycerol (Sigma #M6145), 0.7 μg / ml insulin (Sigma #11376497001), and 15 μg / ml transferrin (Sigma #10652202001) (also referred to herein as "CDM2" medium as used in the exemplary differentiation protocols shown in FIGS. 16 and 18).

[0074] The medium is typically replaced regularly with fresh medium. For example, in one embodiment, the medium is replaced every 24 hours.

[0075] To generate mature dopaminergic neurons, immature midbrain neurons, committed MB NSCs, and MB NPCs, starting pluripotent stem cells are cultured in an optimized medium for a time sufficient for cell differentiation and expression of markers associated with committed MB NSCs or MB NPCs. As described in the Examples, it was discovered that when pluripotent stem cells are cultured in a two-step method, one step optimized for generation of MB NSCs and the other step optimized for generation of MB NPCs, MB NPCs can be generated in just 6 days of culture. The culture period for the first step (“Step 1”, leading to MB NSCs) is from day 0 to day 3, and the culture period for the second step (“Step 2”, leading to MB NPCs) is from day 4 to day 6. Immature midbrain neurons (MB immature neurons) were generated by further culturing MB NPCs in Step 3 medium from day 6 to day 9, whereas mature dopaminergic neurons were generated by further culturing MB immature neurons in Step 4 medium from day 9 to day 23.

[0076] Accordingly, in the first step of the method for generating MB NSCs, also referred to herein as “step (a)” or “Step 1”, pluripotent stem cells are cultured in Step 1 optimized medium from day 0 to day 3, or starting on day 0 and continuing through day 3, or for 72 hours (3 days), or for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours.

[0077] Therefore, in the second stage of the method for producing MB NPCs, also referred to herein as "step (b)" or "stage 2", the MB NSCs produced in step (a) are cultured further in the stage 2 optimization medium on the 4th to 6th day, or continuously starting from the 4th day until the 6th day, or continuously starting from the 4th day for 72 hours (3 days), or continuously starting from the 4th day for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or continuously starting from the 4th day for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours.

[0078] Therefore, in the third stage of the method for producing immature midbrain neurons, also referred to herein as "step (c)" or "stage 3", the MB NPCs produced in step (b) are cultured further in the stage 3 optimization medium on the 6th to 9th day, or continuously starting from the 6th day until the 9th day, or continuously starting from the 6th day for 72 hours (3 days), or continuously starting from the 6th day for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or continuously starting from the 6th day for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours.

[0079] Therefore, in the fourth stage of the method for producing mature dopaminergic neurons, also referred to herein as "step (d)" or "stage 4", the immature midbrain neurons produced in step (c) are cultured further in the stage 4 optimization medium on the 9th to 23rd day, or continuously starting from the 9th day until the 23rd day, or continuously starting from the 9th day for a time sufficient to produce TH+ KCNJ6+ mature dopaminergic neurons (e.g., cultured in the stage 4 medium for 14 days, i.e., 2 weeks).

[0080] IV. Use The methods and compositions of the present disclosure for generating mature dopaminergic neurons, immature midbrain neurons, committed MB NSCs, and MB NPCs enable the efficient and robust procurement of these cell populations for various uses. For example, the methods and compositions can be used in the study of the development and biology of midbrain neural progenitor cells, including differentiation into dopaminergic neurons, to aid in the understanding and potential treatment of neuronal diseases and disorders such as Parkinson's disease. For example, mature dopaminergic neurons, immature midbrain neurons, committed MB NSCs, and / or MB NPCs generated using the methods of the present disclosure can be further purified according to methods established in the art using agents that bind to cell surface markers expressed on said cells. Thus, in one aspect, the present disclosure provides a method of isolating a mature dopaminergic neuron or an immature midbrain neuron, the method comprising contacting a mature dopaminergic neuron or an immature midbrain neuron generated by the methods of the present disclosure with at least one binding agent that binds to a cell surface marker expressed by the mature dopaminergic neuron or the immature midbrain neuron, and isolating the cells bound to the binding agent, thereby isolating the mature dopaminergic neuron or the immature midbrain neuron.

[0081] In one aspect, the binding agent is an antibody, such as a monoclonal antibody (mAb) that binds to a cell surface marker. Cells bound to the antibody can be isolated by methods known in the art including, but not limited to, fluorescent activated cell-sorting (FACS) and magnetic activated cell sorting (MACS).

[0082] The precursor cells of the mesencephalic dopaminergic nervous system lineage are also intended for use in the treatment of neurological diseases and disorders that benefit from the enhancement of dopaminergic neuron function by delivery of said cells to a subject having said disease or disorder, including but not limited to Parkinson's disease.

[0083] The cells of the present disclosure are also useful for screening potential drugs for treating a disease or disorder associated with dopaminergic neuron dysfunction, or for developing novel cell therapies for treating a disease or disorder associated with dopaminergic neuron dysfunction.

[0084] V. Composition In another aspect, the present disclosure provides compositions related to methods of making mature dopaminergic neurons, immature mesencephalic neurons, committed MB NSCs, and MB NPCs, including media and cell cultures, as well as isolated precursor cells and cell populations.

[0085] In one aspect, the present disclosure provides a medium for obtaining human committed mesencephalic neural stem cells, comprising a WNT pathway agonist, an SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist. In one embodiment, the medium lacks exogenously added growth factors.

[0086] In another aspect, the present disclosure provides a medium for obtaining human mesencephalic neural progenitor cells, comprising a BMP pathway agonist, an RA pathway agonist, an LXR pathway agonist, an AKT pathway antagonist, an mTOR pathway antagonist, and a TGF-β pathway antagonist. In one embodiment, the medium lacks exogenously added growth factors.

[0087] In another aspect, the present disclosure provides a medium for obtaining human midbrain immature neurons, comprising a WNT pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist. In one embodiment, the medium lacks exogenously added growth factors.

[0088] In another aspect, the present disclosure provides a medium for obtaining human mature dopaminergic neurons, comprising a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist. In one embodiment, the medium lacks exogenously added growth factors.

[0089] In another aspect, the present disclosure provides a culture of isolated cells of human committed midbrain neural stem cells, comprising human OTX2+ LMX1A+ committed midbrain neural stem cells cultured in a medium lacking exogenously added growth factors and comprising a WNT pathway agonist, an SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist.

[0090] In another aspect, the present disclosure provides a culture of isolated cells of human midbrain neural progenitor cells, comprising human OTX2+ FOXA2+ LMX1A+ midbrain neural progenitor cells cultured in a medium lacking exogenously added growth factors and comprising a BMP pathway agonist, a RA pathway agonist, an LXR pathway agonist, an AKT pathway antagonist, an mTOR pathway antagonist, and a TGF-β pathway antagonist.

[0091] In another aspect, the present disclosure provides a culture of isolated cells of human midbrain immature neurons, the culture comprising human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons cultured in a medium comprising a WNT pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist.

[0092] In another aspect, the present disclosure provides a culture of isolated cells of human mature dopaminergic neurons, the culture comprising human TH+ KCNJ6+ mature dopaminergic neurons cultured in a medium comprising a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist.

[0093] In another aspect, the present disclosure provides human OTX2+ FOXA2+ LMX1A+ midbrain neural progenitor cells produced by the method of the present disclosure. In one aspect, the present disclosure relates to a composition comprising human midbrain neural progenitor cells (NPCs), wherein the human midbrain NPCs express OTX2, FOXA2, and LMX1A and lack GBX2 expression or have only low levels of GBX2 expression. In one aspect, the present disclosure provides an isolated cell population of human midbrain neural progenitor cells (NPCs) comprising at least 1×10 6 individuals of OTX2+ FOXA2+ LMX1A+ human midbrain NPCs, the isolated cell population being devoid of neural stem cells expressing GBX2. In an aspect of the isolated cell population, the human midbrain NPCs bind to at least one antibody that binds to at least one marker expressed by the human midbrain NPCs.

[0094] In other aspects, the present disclosure provides human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons produced by the methods described herein. In another aspect, the present disclosure provides human TH+ KCNJ6+ mature dopaminergic neurons produced by the methods described herein.

[0095] The present invention is further illustrated by the following examples. These should not be construed as further limitations. The contents of the drawings and all references, patents, and published patent applications cited throughout this application are hereby expressly incorporated herein by reference.

Examples

[0096] Example 1 : Development of a culture protocol for the generation of stem cell-derived midbrain neural precursors expressing FOXA2 and LMX1A In this example, a two-step culture protocol for the generation of midbrain-derived neural precursors that can direct human pluripotent stem cells to precursors expressing FOXA2 and LMX1A after 6 days of culture was developed. These cells are further differentiable into mature dopaminergic neurons.

[0097] In this example, the method of high-dimensional design of experiments (HD-DoE) described in Bukys et al. (2020) Iscience 23:101346 is utilized. This method simultaneously tests multiple process inputs using computer-aided design geometry and provides a mathematical modeling of the deep effector / response space. By this method, it becomes possible to find combinatorial signaling inputs that control complex processes, such as during cell differentiation. This enables testing multiple promising important process parameters because such parameters affect output responses such as gene expression. Since gene expression results in significant characteristics of the phenotype of human cells, the method can be applied to identify and understand which signaling pathways control cell fate. In this example, the HD-DOE method was applied with the aim of finding conditions for directly inducing genes expressed in midbrain neural precursors from a pluripotent stem cell state.

[0098] To develop a recipe for each stage, the effects of agonists and antagonists (referred to herein as "effectors") of multiple signaling pathways were examined and modeled after 3 days of treatment on the expression of two sets of 53 pre-selected genes. These effectors are small molecules or proteins commonly used when stepwise differentiating stem cells into specific fates. The selection of effectors for testing was based on the latest literature on neural induction in the midbrain region of the developing brain and the differentiation of stem cells into neural precursors.

[0099] To test the effectors, an experiment was designed using at least 8 factors capable of evaluating the response of cells to various combinations of over 48 effectors in a certain concentration range. To analyze the model, the inventors focused on the expression of genes expressed in the midbrain region, including OTX2, DMBX1, FOXA2, LMX1A, and the absence of GBX2, a hindbrain marker. The effect of each effector on the gene expression level was defined by a parameter called the factor contribution calculated for each effector during modeling.

[0100] To identify the recipe for stage 1 of differentiation, cells were treated with various effectors for 3 days to model the gene expression of the cells. When optimized for the maximum expression at 12760.1 of OTX2, one model showed promising results especially for the upregulation of DMBX1, LMX1A, and OTX2 and the downregulation of GBX2. This model consisted of 13 factors including LDN193189, PD173074, BLU9931, purmorphamine, SC79, MK2206, ZM336372, PD0325901, CHIR99021, XAV939, UCLA-gp130, tofacitinib, and GO 6983. Four of the effectors, namely MK2206, an antagonist of the AKT signaling pathway, PD0325901, an antagonist of the MEK signaling pathway, CHIR99021, an agonist of the WNT signaling pathway, and LDN193189, an antagonist of the BMP signaling pathway, had a significant positive effect on the expression of the genes of interest, and the factor contribution degrees were 22.3, 18.1, 13.5, and 11.9 respectively (Figure 1).

[0101] Since FOXA2 was not upregulated by the optimization of OTX2, the inventors then optimized the model for the maximum expression at 1581 of FOXA2. Three effectors having a significant positive effect on the expression of FOXA2 were identified, including LDN193189, CHIR99021, and purmorphamine, and the factor contribution degrees were 13.6, 15.6, and 22.2 respectively (Figure 2). LDN193189 and CHIR99021 were common to both optimization settings, and the remaining factors other than purmorphamine had a factor contribution degree of less than 10. Therefore, the inventors focused on the effect of adding purmorphamine to the original four effectors.

[0102] This evaluation was performed by dynamic profile analysis of the model focusing on the expression of OTX2, DMBX1, LMX1A, and FOXA2 (Figure 3). Since pulmorphamine had no positive effect on the expression of OTX2, DMBX1, and LMX1A in the previous setting, the inventors expected a decrease in the expression levels of these genes, but it was observed that these expression levels remained almost the same as the previous conditions, being 3000, 450, and 11000 for DMBX1, LMX1A, and OTX2, respectively (Figure 4).

[0103] The effectors verified for stage 1 of the protocol (generating neural stem cells committed to the midbrain) are summarized in Table 1 below.

[0104] (Table 1) Effectors verified in stage 1 of the protocol TIFF2025524856000001.tif30128

[0105] In order to further induce the differentiation of neural stem cells committed to the midbrain into neural progenitor cells in stage 2, the inventors conducted additional HD-DoE experiments. By doing so, the inventors obtained a further gene regulatory model, which was used for the preparation of the differentiation protocol. This basis was an HD-DoE experiment with 12 factors, focusing on the initiation of cell differentiation towards midbrain neural progenitor cells during an additional three days after the end of the stage 1 treatment. Here, the inventors focused on the expression of LMX1A and FOXA2 in neural progenitor cells with low to zero expression of GBX2. LMX1A had a significantly high expression level in this model, with a value of 47888. Therefore, the inventors used the optimized settings for this gene to identify positive factors. The factors in this experiment included SC79, MK2206, ZM336372, PD0325901, CHIR99021, A 83-01, TTNPB, AGN193109, GW3965, SR9243, purmorphamine, and GSI-XX. When optimized for LMX1A, one factor, TTNPB, a small molecule agonist of the RA signaling pathway, had a significant positive effect, with a factor contribution of 19.5. CHIR99021, SC79, and GW3965 also had positive effects, but their factor contributions were less than 10 (8.3, 7.3, and 5.4 respectively), and the positive factor contribution of AGN193109 was <1 (Figure 5).

[0106] When the same experiment was optimized for the maximum expression of FOXA2 at 33193, three effectors with significant positive effects on the expression of FOXA2 were identified, including TTNPB, A 83-01, and purmorphamine, with factor contributions of 10.7, 6.7, and 15.3 respectively (Figure 6).

[0107] Similar to the experimental model of stage 1, the analysis also showed that purmorphamine had a positive effect on the FOXA2 expression level and a negative effect on the LMX1A expression level, with a factor contribution of 26.2. The model also showed the same trend for A 83-01 and CHIR99021, having a positive effect only on FOXA2 and LMX1A, respectively. Therefore, the inventors used dynamic profile analysis to adjust the recipe for the optimized expression of both LMX1A and FOXA2 genes and the minimal expression of GBX2 (Figure 7). It was observed that the expression level of FOXA2 was 5000 even without the addition of purmorphamine. Thus, although it is not essential to include this effector, it can improve the expression of FOXA2. Based on the dynamic profile plot, it was also concluded that CHIR99021, even though it had a positive effect on LMX1A, increased the expression level of GBX2 and decreased the relative expression of FOXA2. Therefore, this factor was excluded from the final recipe. A 83-01 is another factor that has opposite effects on FOXA2 and LMX1A, and this was included in the final recipe. Dynamic profile analysis showed that the addition of A 83-01 at a moderate concentration (300 nM) could help improve the expression of FOXA2 and reduce the level of GBX2 to almost zero.

[0108] To further test additional factors, such as FGF8, commonly used in midbrain differentiation protocols, we performed another 12-factor experiment consisting of LDN193189, BMP7, A 83-01, activin A, takinib, PD0325901, MK2206, FGF8b, AZD3147, MHY1485, GSI-XX, and Yhhu 3792. As in the previous experiment, hiPSCs were treated with stage 1 medium for 3 days and then with 96 different factor combinations for an additional 3 days. When this model was optimized for maximum FOXA2 expression, BMP7 (factor contribution 13.7) and MK2206 (factor contribution 14.2) had the greatest effect on FOXA2 expression, followed by AZD 3147 (factor contribution 10.9). Yhhu 3792 and takinib also had a positive effect, but their factor contributions were less than 10. Surprisingly, FGF8 had a negative effect with a factor contribution of 12.8 (FIG. 8).

[0109] This model was also optimized for maximum LMX1A expression, and MK2206, with a factor contribution of 12, had the highest positive effect. AZD 3147, GSI-XX, activin A, and takinib also had positive effects on LMX1A expression, but their factor contributions were less than 10 (Figure 9). The model also showed that Yhhu 3792 had a negative effect on LMX1A expression, with a factor contribution of 11.7, the opposite of the FOXA2 condition. Another difference was BMP7, which had a negative effect on LMX1A. However, its factor contribution was less than 10. Therefore, we used dynamic profile analysis to evaluate the interaction effects and optimal conditions for both FOXA2 and LMX1A expression (Figure 10).

[0110] Using dynamic profile analysis, we eliminated GSI-XX, activin A, and takinib because they did not significantly positively change the expression levels of both FOXA2 and LMX1A. Yhhu 3792 was also eliminated because it had a significant negative effect on LMX1A and a significant positive effect on GBX2. BMP7 and AZD 3147 had a significant positive effect on FOXA2 and LMX1A, respectively, while not reducing the expression of the remaining genes, and therefore were included in the final recipe. MK2206 also reduced the level of LMX1A, but was shown to have the desired effect on FOXA2 and GBX2, and therefore was included in the final recipe at a moderate level.

[0111] The effectors that were tested for step 2 of the protocol (generating midbrain-derived neural progenitor cells) are summarized in Table 2 below.

[0112] Table 2. Effectors validated in step 2 of the protocol TIFF2025524856000002.tif35128

[0113] Considering both models, conditions that maximize differentiation of cells into midbrain regions with neural progenitor identity, as associated with robust and elevated expression of OTX2, FOXA2, and LMX1A, included the following effector inputs: TTNPB, BMP7, A 83-01, GW3965, AZD 3147, and MK2206.

[0114] The importance of each individual validated effector to the Phase 1 and Phase 2 protocols was further evaluated as described in Example 2.

[0115] Example 2 : Factor importance analysis of culture conditions for inducing stem cell-derived midbrain neural precursors To evaluate the impact of the exclusion of each verified factor, the inventors used dynamic profile analysis to compare the expression levels of the genes of interest in the absence of each finally determined factor while the rest were present. Since the expression levels of the genes of interest clarify whether the desired outcome is achievable, this factor importance analysis revealed the degree of importance of each input effector.

[0116] In the stage 1 recipe, while excluding each of the 5 finally determined factors and having the remaining 4 factors present, the expression levels of OTX2, DMBX1, FOXA2, and LMX1A were evaluated by comparing them with the levels in the presence of all 5 factors (Figs. 11A - B). When MK2206 was excluded, the values of OTX2 and DMBX1 decreased from 12000 and 3000 to 9500 and 1500 respectively, while FOXA2 and LMX1A remained at the same values. The absence of PD0325901 resulted in a decrease in the expression of DMBX1 which reached 900, while the expression of FOXA2 and LMX1A increased from 600 and 300 to 700 and 500. In the absence of LDN193189, the expression level of DMBX1 increased, while the values of OTX2, FOXA2, and LMX1A decreased. After excluding CHIR99021, the values of all genes of interest decreased, which further demonstrated its importance in the stage 1 recipe, and, as expected, the absence of purmorphamine resulted in a reduction of FOXA2 while being beneficial to the other genes.

[0117] In the stage 2 recipe, while excluding each of the six final determinants and leaving the remaining five determinants present, the expression levels of FOXA2, LMX1A, and GBX2 were evaluated by comparing them with the levels in the presence of all determinants. According to the first experimental model, the absence of TTNPB resulted in an increase in GBX2 expression, while the values of FOXA2 and LMX1A decreased drastically from 10,000 to 0 and from 30,000 to 15,000, respectively. The absence of A 83-01 caused the expression of FOXA2 to decrease from 10,000 to 7,000, while as expected, the value of LMX1A increased from 30,000 to 40,000. The deletion of GW3965 caused the value of LMX1A to decrease drastically from 30,000 to 10,000, and the value of FOXA2 to increase to 17,000 (Figs. 12A - B). According to the second experimental model, the absence of both BMP7 and MK2206 decreased the level of FOXA2 while increasing the value of LMX1A, and the absence of BMP7 as the main effector resulted in an expression of FOXA2 of 0. The absence of AZD 3147 decreased the level of LMX1A from 4,000 to 2,000 while having little effect on the value of FOXA2 (Figs. 13A - B), and thus these factors were added to the final recipe.

[0118] Example 3 : Immunocytochemical verification of stem cell-derived midbrain neural precursors expressing FOXA2 and LMX1A To further validate the culture protocol developed as described in Example 1, cells were treated with the stage 1 and stage 2 differentiation media, and at the end of each stage, immunocytochemistry was used to evaluate the expression of biomarkers of the midbrain region and neural precursors. The test biomarkers included OTX2 (Vernay et al. (2005) J. Neurosci. 25:4856-4867), a midbrain marker involved in midbrain positioning and maintenance of the midbrain-hindbrain boundary, LMX1A (Yan et al. (2011) J. Neurosci. 31:12413-12425), which is involved in the generation and differentiation of midbrain dopaminergic precursors, FOXA2 (Ferri et al. (2007) Development 134:2761-2769), which regulates the generation of midbrain dopaminergic neurons in the early and late stages of development, PAX2 (Urbanek et al. (1997) Proc. Natl. Acad. Sci. USA 94:5703-5708), which is expressed in the midbrain and fore- and hindbrains, nestin, a marker of early neurons, KI67, a proliferation marker, and GBX2, a hindbrain marker.

[0119] By immunocytochemical imaging, expression of OTX2 and LMX1A was confirmed in over 90% of cells by the end of the treatment with the stage 1 medium. Similar to the somewhat expression of GBX2 being observed, the markers PAX2, nestin, and KI67 were also observed in some cells. However, FOXA2 was not expressed (Figure 14). After treatment with the stage 2 medium, the inventors observed that while the expression of LMX1A and OTX2 was maintained, the expression of FOXA2 increased significantly and FOXA2 was detected in over 90% of cells. Also, GBX2 expression had almost disappeared by the end of stage 2 (Figure 15). The detection of OTX2, LMX1A, and FOXA2 but not GBX2 by the end of stage 2 of differentiation confirmed the recipe for stages 1 and 2 for differentiating human induced pluripotent stem cells into midbrain neural precursors after 6 days of treatment.

[0120] Example 4RNA-seq Validation of Stem Cell-Derived Midbrain Neural Progenitors Expressing LMX1A and FOXA2 RNA sequencing was used to obtain the gene profiles of cultured cells in the candidate recipe. Human iPSCs were cultured in stage 1 and stage 2 media for a total of 6 days, and at the end of each stage, the RNA of the generated cells was sequenced. Figure 17 shows the normalized expression levels of selected genes representing the midbrain region (OTX2, DMBX1, FOXA2, LMX1A) of the developing brain, early neural identity (nestin, SOX1, SOX2, vimentin), and stem cell state (NANOG, POU5F1) in three replicate experiments at day 0, day 3, and day 6. As shown in Figures 17A and 17B, the levels of stem cell genes decreased in neural precursors, while the levels of genes of neurons derived from the midbrain region increased, demonstrating the differentiation of hiPSCs into a neural lineage with midbrain identity. Figure 17A shows the fold change of 19 selected genes after 3 days of treatment with stage 1 medium compared to stage 0, and FOXA2, LMX1A, and SOX1 have the highest positive differential expression levels compared to hiPSCs (10.6, 9.5, and 9.1, respectively). The stem cell genes NANOG and POU5F1, as well as the hindbrain gene GBX2, are at the lowest levels (-8.7, -3.5, and -3.8, respectively). Figure 17B shows the fold change of 21 selected genes in cells treated with stage 1 and stage 2 media compared to hiPSCs. FOXA2, SOX1, and DDC have the highest positive differential expression at 11.3, 10, and 7.7. Similar to stage 1, the lowest differential expression was observed for NANOG, POUF51, and GBX2. The heatmap of the scaled gene profiles of 18 selected genes in hiPSCs at day 0 and MB neural precursors at day 6 (Figure 17C) shows the expression of midbrain precursor genes including DDC, LMX1A / B, SOX6, NEUROG2, FOXA2, and EN1, and their expression increased after treatment with stage 1 and stage 2 media. The expression level of the gene GFAP, which is expressed in glial cells, was also observed and remained the same during the 6-day differentiation, indicating that the culture is mainly neuronal.This data demonstrates the ability of the stage 1 and stage 2 recipes as stepwise differentiation media when inducing cells towards midbrain neuron identity.

[0121] Example 5 : Development of a culture protocol for generating dopamine - active neurons expressing TH and KCNJ6 As described in Examples 1 - 4, after treating cells with the stage 1 and stage 2 media to generate MB neural progenitor cells, based on the culture protocol developed in this example, the MB neural progenitor cells were treated with the stage 3 media for 3 days and then with the stage 4 media for 14 days to differentiate into dopamine - active neurons expressing TH and KCNJ6.

[0122] To develop a neuron differentiation recipe, the effects of various agonists and antagonists (effectors) on the maturation of MB neural progenitor cells were investigated using the HD - DoE method described in Example 1. These effectors were selected based on available literature on developmental biology, stem cell differentiation, and mouse and human single - cell RNA - seq data from midbrain neurons at that time.

[0123] As further explained below, these experiments led to the creation of the stage 3 recipe shown in Table 3 below and the stage 4 recipe shown in Table 4 below.

[0124] (Table 3) Verified factors in the stage 3 recipe TIFF2025524856000003.tif48128

[0125] (Table 4) Verified factors in the stage 4 recipe TIFF2025524856000004.tif50148

[0126] To manipulate the recipe for stage 3 of differentiation, first, cells were cultured in stage 1 and stage 2 media as described herein, then treated for 3 days with a combination of 8 or 12 factors, and the gene expression of cells under each condition was modeled. Neural progenitor cells need to be SOX6 + for cells to be directed to the subtype of midbrain dopaminergic neurons in the substantia nigra compacta (SNc) region of the brain (Pereira et al. (2021) Cell Rep. 37:109975; Oosterveen et al. (2021) Stem Cell Reports 16:2718-2735; Poulin et al. (2020) Trends Neurosci. 43:155-169). CORIN, NGN2, and MSX1 are other markers involved in the neurogenesis of dopaminergic neurons arising from the floor plate region of the developing midbrain (Wang et al. (2020) Cells 9:1489; Samata et al. (2016) Nat Commun. 7:13097; Ono et al. (2007) Development 134:3213-3225; Prakash et al. (2006) J. Physiol. :403-410).

[0127] Therefore, the inventors focused on maximizing the expression of SOX6, CORIN, NGN2, and MSX1 when modeling the experiment. One of the 12-factor models whose results are shown in Figure 19, including LDN193189, AGN193109, BMP7, TTNPB, PD0325901, A8301, MHY1485, CHIR99021, XAV939, SANT-1, pulmorphamine, and MK2206, resulted in a combination set that could maximize the CORIN expression level to over 2000. In this model, the RA antagonist AGN193109 had the most positive regulatory effect with a factor contribution of 20.3. The next two effectors with large positive factor contributions were LDN193189, an inhibitor of ALK1, ALK2, ALK3, and ALK6, and BMP7. The WNT inhibitor XAV939, the WNT activator CHIR99021, and the SHH antagonist SANT-1 had the most negative effects on the CORIN expression level with factor contributions of 13.5, 10.9, and 9.9, respectively. Within the range of the standard for achieving 80% of the maximum expression of CORIN, the Cpk value (process capability index) of this complex medium composition was 0.54, and the corresponding risk rate of failure was 4.9%.

[0128] When the same model was optimized for the maximum expression of 179 MSX1, the factor with the most significant positive effect was BMP7 at 11.1 (Figure 20). LDN193189, the RA agonist TTNPB, and XAV939 had the most significant negative regulatory effects on MSX1 expression at 19.9, 15.5, and 11.2, respectively. Within the range of the standard for achieving 80% of the maximum expression of MSX1, the Cpk value (process capability index) of this complex medium composition was 0.62, and the corresponding risk rate of failure was 3.1%.

[0129] This model was also optimized for maximum SOX6 expression of 296, which led to the identification of two factors, AGN193109 and PD0325901, that showed a significant positive effect on SOX6 expression with factor contributions of 26.1 and 13.5, respectively (Figure 21). XAV939 and TTNPB had the greatest negative effects on the optimization of this gene, with factor contributions of 10.8 and 9.5, respectively. Within the range of criteria to achieve 80% of the maximum SOX6 expression, the Cpk value (process capability index) of this complex medium composition was 0.9, and the corresponding risk of failure was 0.44%.

[0130] This 12-factor model first increased the KCNJ6 expression level during the differentiation experiment. KCNJ6, a G protein-activated potassium channel, is a terminal differentiation marker expressed by all human SNc dopaminergic neurons and some VTA dopaminergic neurons (Reyes et al. (2012) J. Comp. Neurol. 520:2591-607). Therefore, the inventors also optimized the model for maximum KCNJ6 expression of 47. The positive regulators of this gene were found to be 17 of AGN193109, 10.9 of PD0325901, 10.7 of CHIR99021, and BMP7 and DBZ with small factor contributions (Figure 22).

[0131] Next, the inventors utilized dynamic profile analysis to discover a combination set that could optimize the expression of all four genes. One factor, LDN193189, among all the effectors that demonstrated a large positive factor contribution to the selected genes, including AGN193109, PD0325901, BMP7, LDN193189, and CHIR99021, had to be excluded due to its large negative effect on the MSX1 expression level (Figure 23).

[0132] In another 12-factor model, additional factors including activin A, tazobutide (TAK inhibitor), FGF8b, AZD3147 (mTOR antagonist), MHY1485 (mTOR agonist), and Yhhu-3792 (Notch agonist) were tested. Some of the factors from the previous model (BMP7, A8301, LDN193189, PD0325901, MK2206, and DBZ) were also included. When optimized for 250 of SOX6, the two new factors, DBZ and AZD3147, had the greatest positive impact with factor contributions of 21.5 and 20.2 (Figure 24). Within the range of criteria to achieve 80% of the maximum expression of SOX6, the Cpk value (process capability index) of this complex medium composition was 0.60, and the corresponding risk of failure was 3.6%.

[0133] In this model, the CORIN expression level was quite low at 20 compared to 2000, so the inventors did not optimize the model for its maximum expression. However, the inventors analyzed the effects of these inputs on MSX1 expression using dynamic profile analysis (Figure 25). According to this model, activin A and tazobutide, which had a positive effect on SOX6, negatively regulated MSX1, so these were excluded from the final recipe. AZD3147 and DBZ showed no significant effect on the MSX1 expression level.

[0134] The inventors also observed the positive regulatory effects of DBZ and AZD3147 on the gene of interest in the 8-factor model. This further confirmed the analysis results of the previous model (Figure 26).

[0135] Therefore, considering these models, a candidate recipe for stage 3 consisting of BMP7, PD0325901, AGN193109, CHIR99021, AZD3147, and DBZ was created. This recipe was selected to maximize the differentiation of the cells themselves, which is related to the robust and high expression of SOX6, KCNJ6, CORIN, and MSX1. This recipe was further verified by immunocytochemistry assay (see Example 7).

[0136] To further differentiate the cells into mature neuron identity, additional HD-DoE experiments were performed on the cells cultured in the differentiation media of stage 1, stage 2, and stage 3. Four days later, the gene expression of the cells was examined under different combination conditions. At this time, the inventors focused on the maximum expression of more mature dopaminergic genes such as NR4A2, PITX3, and TH (Tiklova et al. (2020) Nat Commun. 11:2434; Fiorenzano et al. (2021) Nat Commun. 12:7302). In one model, the cells were treated with a combinatorial matrix of 8 inputs including CHIR99021, BDNF, GDNF, rosiglitazone, GW7647, DBZ, and heparin. When maximized for NR4A2 with an expression level of 337.8, 6 factors including BDNF, dopamine, DBZ, GW7647, GDNF, and heparin with the 17 largest factor contributions showed a positive regulatory effect on NR4A2 expression (Figure 27). In contrast, CHIR99021 and rosiglitazone had significant negative effects on NR4A2 with factor contributions of 20.7 and .6 respectively. Within the range of the standard to obtain 80% of the maximum expression of NR4A2, the Cpk value (process capability index) of this complex medium composition was 0.35, and the corresponding risk rate of failure was 8.9%.

[0137] The effect of the compound on the expression level of PITX3 was examined using dynamic profile analysis. A similar trend was observed for both genes, except for CHIR99021, which had no significant effect on the PITX3 expression level (Figure 28).

[0138] Considering this model, the conditions that maximize cell differentiation into the SNc region, which has the dopamineergic neuron cell identity itself associated with robust and high expression of NR4A2 and PITX3, included the following effector inputs: BDNF, GDNF, dopamine, GW7647, DBZ, and heparin. This stage 4 recipe was further verified by immunocytochemistry assays (see Example 7).

[0139] Example 6 : Factor Importance Analysis of Culture Conditions for Inducing Dopamineergic Neurons To evaluate the effect of removing each of the validated factors identified in Example 5 for the stage 3 and stage 4 recipes, the inventors used dynamic profile analysis to compare the expression levels of the genes of interest in the absence of each of the final determined factors while the other factors remained present. This factor importance analysis revealed the degree of importance of each input effector, as it became clear whether a desirable outcome was achievable from the expression levels of the genes of interest.

[0140] In the stage 3 recipe, each of the finally determined factors was removed in each model while the other factors remained present, and the expression level of the gene of interest was evaluated by comparing it with the presence of all factors. The results are shown in FIGS. 29A and 29B. When AGN193109 was excluded, all levels of CORIN, KCNJ6, MSX1, and SOX6 decreased from 900 to 450, 50 to 35, 140 to 110, and 105 to 60, respectively. When BMP7 was removed, the expression levels of CORIN and MSX1 decreased to 500 and 100. However, the levels of KCNJ6 and SOX6 did not change significantly. When PD0325901 was removed, the expression levels of CORIN and KCNJ6 increased to 1200 and 70, while the levels of MSX1 and SOX6 decreased to 105 and 20. In the absence of CHIR99021, the expression level of CORIN increased again to 1200, while the levels of KCNJ6 and MSX1 decreased to 50 and 115. The level of SOX6 also increased to 125. Among the selected genes, the expression level of CORIN is the highest, and even under the negative regulatory influence from PD0325901, it is still at a significantly high level (900), while SOX6 significantly decreases from 105 to 20 when PD0325901 is removed. Therefore, it was decided to include this compound in the final recipe. CHIR99021 had a similar effect on CORIN, but the absence of CHIR99021 had a significantly negative impact on MSX1. Therefore, CHIR99021 was also included in the final recipe.

[0141] In another model, to confirm the combined effect of AZD3147 and DBZ on the gene profile of cells in the presence of other factors of the final recipe, the expression levels of SOX6 and MSX1 were compared in the absence of each factor and in the presence of PD0325901 and BMP7. These two compounds were the only final compounds included in the input of this experimental model. The results are shown in FIGS. 30A and 30B. When AZD3147 was excluded from the combination set, the level of SOX6 decreased from 185 to 150, while MSX1 did not change. When DBZ was removed, the levels of both SOX6 and MSX1 decreased to 140 and 35, respectively.

[0142] As a verification analysis, the inventors also analyzed the expression levels of these two genes in the absence of PD0325901 and confirmed that the SOX6 expression decreased from 185 to 165 and the MSX1 expression decreased from 45 to 40.

[0143] In the stage 4 recipe, with other factors remaining present, each of the six finally determined factors was excluded, and the expression levels of NR4A2 and PITX3 were evaluated in comparison with the presence of all factors. The results are shown in FIGS. 31A and 31B. In the absence of BDNF, the expression of PITX3 decreased from 40 to 10, and the expression of NR4A2 decreased from 400 to 100. When dopamine was excluded from the recipe, the expression level of NR4A2 reached almost 0, and PITX3 decreased to 20. When GDNF was removed, the expression level of NR4A2 decreased to 300, and the expression level of PITX3 decreased to 25. When DBZ was removed, the level of NR4A2 decreased again to 200, and PITX3 reached 30. A similar trend was observed when GW7647 was excluded, and the expression levels of both genes became low. Only in the absence of heparin, the expression level of NR4A2 did not change significantly, but the PITX3 expression decreased to 30.

[0144] Factor importance analysis demonstrated the importance of including each of the above compounds in the recipes of the stage 3 and stage 4 differentiation media.

[0145] Example 7 :Immunocytochemical verification of midbrain dopaminergic neurons expressing TH and KCNJ6 To further verify the developed recipe of Example 5, first, cells were treated with the differentiation media of stage 1 and stage 2 according to Example 1, then treated with the differentiation media of stage 3 for 3 days, treated with the media of stage 4 for 14 days, and then, using a standard immunocytochemical assay, the biomarker expression of immature neurons in the ventral midbrain region was evaluated at the end of stage 3, and the biomarker expression of mature neurons was evaluated at the end of stage 4. Biomarkers included midbrain neuron-specific markers such as SOX6, ALDH1A1, MSX1, TH, NURR1 (NR4A2), PITX3, and KCNJ6, together with mature pan-neuron markers such as TUBB3, MAP2, neurofilament (NF), and SYN1.

[0146] By immunocytochemical images, the expression of SOX6 and MSX1 was confirmed in more than 90% of the cells by the end of stage 3 (Figure 32). The inventors also detected the early expression of the neuron marker β-tubulin III (TUBB3), the immature neuron marker doublecortin (DCX), and the postmitotic dopaminergic neuron PITX3 and NURR1 as early as day 9 in vitro.

[0147] By images of mature neurons at the end of stage 4, cells in culture expressing mature dopaminergic markers such as KCNJ6 and TH were confirmed. The inventors also observed that most cells in culture expressed mature pan-neuron markers including MAP2, NF, and Syn1 (Figure 33). The inventors stained the cells to determine whether there were competing fate ventral tegmental area (VTA) dopaminergic neurons expressing CALB1 (Brignani et al. (2017) Front. Neuroanat. 11:55) and detected that less than 10% of the cells expressed CALB1. At this point, the expression of NURR1 and ALDH1A1 was observed in most cells.

[0148] TH and KCNJ6 were detected until the end of differentiation stage 4, but CALB1 was not detected. This confirmed the robustness and high conversion ability of the stepwise recipe described herein for differentiating human induced pluripotent stem cells into SNc dopaminergic neurons after 23 days in vitro.

[0149] Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for creating human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons (MB immature neurons), To obtain human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ MB immature neurons, human OTX2+ FOXA2+ LMX1A+ midbrain neural progenitor cells (MB NPCs) are cultured for at least 60 hours in a medium containing a Wingless-related integration site (WNT) pathway agonist, a mammalian target of rapamycin (mTOR) pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a mitogen-activated protein kinase kinase (MEK) pathway antagonist, a Notch pathway antagonist, and a bone morphogenetic protein (BMP) pathway agonist. The method, including the method.

2. The method according to claim 1, further comprising the step of culturing the MB immature neurons in a medium comprising a brain-derived neurotrophic factor (BDNF) pathway agonist, a glial cell line-derived neurotrophic factor (GDNF) pathway agonist, a peroxisome proliferator-activated receptor α (PPAR-α) pathway agonist, heparin or heparin mimetic, a Notch pathway antagonist, and a dopamine agonist for a period of time sufficient to obtain TH+ KCNJ6+ mature dopaminergic neurons.

3. A method for producing human TH+ KCNJ6+ mature dopaminergic neurons, (a) A step of culturing human OTX2+ FOXA2+ LMX1A+ MB NPCs for at least 60 hours in a medium containing a WNT pathway agonist, an mTOR pathway antagonist, a RAR pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist in order to obtain human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ MB immature neurons; and (b) The process of culturing the MB immature neurons in a medium containing a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-α pathway agonist, heparin or heparin mimetic, a Notch pathway antagonist, and a dopamine agonist for a sufficient amount of time to obtain TH+ KCNJ6+ mature dopaminergic neurons. The method, including the method.

4. A method for producing human TH+ KCNJ6+ mature dopaminergic neurons, (a) A step of culturing human pluripotent stem cells for at least 60 hours in a medium containing a WNT pathway agonist, a Sonic Hedgehog (SHH) pathway agonist, a BMP pathway antagonist, an Akt pathway antagonist, and a MEK pathway antagonist in order to obtain committed midbrain neural stem cells (MB NSCs); (b) A step of culturing the MB NSCs for at least 60 hours in a medium containing a BMP pathway agonist, a retinoic acid (RA) pathway agonist, a liver X receptor (LXR) agonist, an Akt pathway antagonist, an mTOR pathway antagonist, and a transforming growth factor β (TGFβ) pathway antagonist in order to obtain human OTX2+ FOXA2+ LMX1A+ MB NPCs; (c) A step of culturing human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ MB immature neurons in a medium containing a WNT pathway agonist, an mTOR pathway antagonist, a RAR pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist for at least 60 hours in order to obtain human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ MB immature neurons; and (d) The process of culturing the MB immature neurons in a medium containing a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-α pathway agonist, heparin or heparin mimetic, a Notch pathway antagonist, and a dopamine agonist for a sufficient amount of time to obtain TH+ KCNJ6+ mature dopaminergic neurons. The method, including the method.

5. The method according to claim 4, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells.

6. (a) The WNT pathway agonist is selected from the group consisting of CHIR99021, CHIR98014, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, AZD1080, WNT3A, Alsterpaulon, Indirubin-3-oxime, 1-Azakenpaulon, Kenpaulon, TC-G 24, TDZD 8, TWS 119, NP 031112, AT 7519, KY 19382, AZD2858, and combinations thereof; (b) The mTOR pathway antagonist is selected from the group consisting of AZD3147, rapamycin, sirolimus, temsirolimus, everolimus, ridafololimus, umilolimus, zotarolimus, torin-1, torin-2, bisutucertib, MHY1485, AZD8055, dactricib, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, niclosamide, omiparisib, PF 04691502, PF 05212384, WYE 687, XL 388, STK16-IN-1, PP 242, tolkinib, sapanicertib, voxtalisib, and combinations thereof; (c) The RAR pathway antagonist is selected from the group consisting of AGN193109, BMS 195614, CD 2665, ER 50891, LE 135, LY 2955303, MM11253, and combinations thereof; (d) The MEK pathway antagonist is selected from the group consisting of PD0325901, binimetinib (MEK162), cobimetinib (XL518), selumetinib, trametinib (GSK1120212), CI-1040 (PD-184352), refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO 5126766, BIX 02189, pimacertib, TAK 733, AZD8330, PD318088, SL 327, GDC 0623, RO5126766, myricetin, and combinations thereof; (e) The Notch pathway antagonist is selected from the group consisting of DBZ, abagacestat, begacestat, BMS 299897, compound E, DAPT, JLK6, L-685,458, LY 450139, MRK 560, PF 3084014 hydrobromide, LY 3039478, LY 411575, RO 4929097, and combinations thereof; and / or (f) The BMP pathway agonist is selected from the group consisting of BMP, sb4, ventromorphine, and combinations thereof. The method according to any one of claims 1 to 5.

7. (a) The WNT pathway agonist is present in the culture medium at a concentration in the range of 0.3 to 3.0 μM; (b) The mTOR pathway antagonist is present in the culture medium at a concentration in the range of 5 to 100 nM; (c) The RAR pathway antagonist is present in the culture medium at a concentration in the range of 25 to 300 nM; (d) The MEK pathway antagonist is present in the culture medium at a concentration in the range of 25 to 300 nM; (e) The Notch pathway antagonist is present in the culture medium at a concentration in the range of 25 to 300 nM; and / or (f) The BMP pathway agonist is present in the culture medium at a concentration in the range of 1 to 100 ng / ml. The method according to claim 6.

8. (a) The BDNF pathway agonist is selected from the group consisting of BDNF, rotigotine, 7,8-DHF, ketamine, tricyclic dimeric peptide-6 (TDP6), LM22A-4, and combinations thereof; (b) The GDNF agonist is selected from the group consisting of GDNF, BT13, BT44, and combinations thereof; (c) The PPAR-α pathway agonists include GW7647, fenofibrate, fenofibrate-d6, WY 14643, CP 775146, CP 868388 free base, tesaglitazal, oleylethanolamide, oleylethanolamide-d2, oleylethanolamide-d4, PPAR agonist 1, clofibrate, clofibrate-d4, wistin, indeglitazar, netoglitazone, GW0742, bezafibrate, bezafibrate-d4, tiglitazar, BMS 687453, ranifibranol, saroglitazal, saroglitazal magnesium, saroglitazal-d5, imiglitazal, AVE-8143, GW 590735, ertiprotafib, LJ Selected from the group consisting of 570, selladelpar sodium salt, edaglitazone, mulaglitazal, laglitazal, GW 9578, MHY 908, KRP-297, ellafibranolic acid, alleglitazal, AM3102, eupatilin, clofibrine, clofibrine-d4, nabeglitazar, nabeglitazar racemic mixture, and combinations thereof; (d) Heparin or heparin mimetic is selected from the group consisting of heparin, heparan sulfate, enoxaparin, low molecular weight heparin, AV5026, M402, and combinations thereof; and / or (e) The dopamine agonist is dopamine, dopamine hydrochloride, (R)-(-)-apomorphine hydrochloride, bromocriptine mesylate, bromocriptine-13C,d3, CNV dopamine, dehydroergotamine mesylate, rithlid, rithlid maleate, meslergin hydrochloride, pyribezil dihydrochloride, pyribezil, quinerolan hydrochloride, (-)-quinpyrrole hydrochloride, ropinirole, tau aggregation-IN-1, NMI 8739, U91356, foscarbidopa, quinagolide hydrochloride, dexpramipexole dihydrochloride, PD-168077 maleate, rotigotine hydrochloride, PF592379, rotigotine, (Rac)-rotigotine hydrochloride, (Rac)-rotigotine-d7 hydrochloride, Ro 10-5824 dihydrochloride, (+)-dihydrolexidine hydrochloride, talipexole, PF2562, Neuromedin N, A68930, A68930 hydrochloride, A77636 dihydrochloride, PD 119819, PD 128907 hydrochloride, CY 208-243, dexpramipexole, dexpramipexole-d3 dihydrochloride, dexpramipexole-d7 dihydrochloride, SKF 38393 hydrochloride, SKF 38393 hydrobromide, SKF 82958, ABT 670, ABT-724, ABT-724 trihydrochloride, (R)-PF-06256142, talipexole dihydrochloride, R-precramol, paldoprnox hydrochloride, paldoprnox, pergolide mesylate, BP897, BP897 hydrochloride, LY 3154207, tabapadone, roxindol, UNC994, cabergoline, brexpiprazole, pergolide-d7 mesylate, cabergoline-d6, roxindol hydrochloride, WAY-100635 maleate, cibenadeto hydrochloride, dihydrolexidine, dihydrolexidine hydrochloride, bifeprnox, OS-3-106, brillaroxazine, pramipexole dihydrochloride hydrate Product, Pramipexole, Pramipexole dihydrochloride, Cabergoline-d5, Perospirone, Brexpiprazole-d8, (Rac)-Tabapadone, ML417, Brexpiprazole S-oxide, Pramipexole-d7 dihydrochloride, Pramipexole-d5 dihydrochloride, UCSF924, WAY-100635, SKF83959, Pramipexole (N-propyl-3,3,Selected from the group consisting of 3-d3) (dihydrochloride), salizotan, brexpiprazole S-oxide D8, SKF 83959 hydrobromide, and combinations thereof, The method according to any one of claims 2 to 5.

9. (a) The BDNF pathway agonist is present in the culture medium at a concentration in the range of 1 to 100 ng / ml; (b) The GDNF agonist is present in the culture medium at a concentration in the range of 1 to 100 ng / ml; (c) The PPAR-a pathway agonist is present in the culture medium at a concentration in the range of 50 to 500 nM; (d) Heparin or heparin mimetic is present in the culture medium at a concentration in the range of 1 to 10 μg / ml; and / or (e) The dopamine agonist is present in the culture medium at a concentration in the range of 5 to 15 μM. The method according to claim 8.

10. A culture medium for obtaining human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ MB immature neurons, containing WNT pathway agonists, mTOR pathway antagonists, RAR pathway antagonists, MEK pathway antagonists, Notch pathway antagonists, and BMP pathway agonists.

11. A culture medium for obtaining human TH+ KCNJ6+ mature dopaminergic neurons, including BDNF pathway agonists, GDNF pathway agonists, PPAR-α pathway agonists, heparin or heparin mimetic, Notch pathway antagonists, and dopamine agonists.

12. A culture of isolated human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ MB immature neurons, comprising human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ MB immature neurons cultured in a medium containing a WNT pathway agonist, an mTOR pathway antagonist, a RAR pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist.

13. A culture of isolated human TH+ KCNJ6+ mature dopaminergic neurons, comprising human TH+ KCNJ6+ mature dopaminergic neurons cultured in a medium containing a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-α pathway agonist, heparin or heparin mimetic, a Notch pathway antagonist, and a dopamine agonist.

14. Human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ MB immature neurons prepared by the method described in claim 1.

15. Human TH+ KCNJ6+ mature dopaminergic neurons prepared by the method described in any one of claims 2 to 4.

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