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

Abstract

The present application provides methods for generating human midbrain immature neurons and mature dopaminergic neurons from neural progenitor cells. In some embodiments, immature and mature midbrain neurons are obtained from neural progenitor cells, such as targeted midbrain neural stem cells (NSCs) and midbrain neural progenitor cells (midbrain NPCs), while they themselves are generated from human pluripotent stem cells. Methods for generating immature and mature neurons of the midbrain use a chemically defined medium that allows for the generation of mature dopaminergic neurons within a short of 23 days. Media, isolated cell populations, and kits are also provided.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 391,207, filed on July 21, 2022, the entire content of which is incorporated herein by reference.

[0003] Government Licensing Rights

[0004] This application was made with government support under Award No. W911NF-17-3-0003, awarded by the U.S. Army ACC-AGP-RTP. The government has certain rights in this application. Background of the Invention

[0006] Parkinson's disease (PD) is the second most common progressive neurodegenerative disease 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 pharmacological approaches aimed at increasing the bioavailability of dopamine by administering the dopamine precursor levodopa (also known as L-dopa). However, the side effects of long-term levodopa treatment pose challenges for its use in the advanced stages of PD. The ability to reconstruct functional dopaminergic neurons in PD patients was first explored by transplanting human fetal midbrain tissue (reviewed in Lindvall et al. (2004) NeuroRx 1:383-393). Results have varied, and this approach has raised ethical concerns regarding the availability and use of fetal tissue, leading to alternative methods for reconstructing dopaminergic neurons in vivo.

[0007] The availability of pluripotent stem cells (PSCs), including embryonic stem (ES) cell lines and induced pluripotent stem cells (iPSCs), has opened the possibility of generating midbrain dopamine (mDA) neuronal progenitors in vitro. Developmental studies have shown that midbrain dopaminergic neurons originate from the ventral midbrain floor plate (mFP), which can be identified by the co-expression of the markers FOXA2 and LMX1A. Early differentiation protocols for deriving midbrain floor plate precursors involve the activation of Sonic hedgehog (SHH) and canonical WNT signaling in PSCs, and dual SMAD inhibition and FGF8 activation, and for a 22-day protocol, 11 days are required to obtain precursors expressing FOXA2 and LMX1A (Kriks et al. (2011) Nature 480:547-551) or involve the activation of SHH, WNT, and FGF8, and the addition of retinoic acid (RA) (Cooper et al. (2010) Mol. Cell. Neurosci. 45:258-266). A similar protocol has been reported in which human iPSC-derived embryoid bodies are exposed to dual SMAD inhibition for five days, followed by SHH and FGF8 activation, generating mDA precursors within 16 days (Hartfield et al. (2014) PLoS One 9:e87388).

[0008] Recently, additional protocols for obtaining midbrain dopamine (MB) dopaminergic progenitors from human pluripotent stem cells have been reported. For example, Nolbrant et al. reported a 16-day protocol that involved exposure to N-2 supplement for the first 11 days, exposure to B27 supplement for the last 5 days, and SHH and WNT activation and ALK inhibition (Nolbrant et al. (2017) Nature Protocols 12:1962-1979). Precious et al. reported a protocol that involved MEK inhibition for two days to block FGF signaling, followed by single SHH activation for 3 days, and SHH and FGF8 activation starting from day 5, generating FOXA2+LMX1A+ progenitors on day 7 (Precious et al. (2020) Front. Neurosci. 14:312). Gartner et al. reported a xeno-free, feeder-free chemically defined protocol that involved incubation in a medium supplemented with (i) LDN193189 and SB431542 from day 0-5 and with LDN193189 only from day 5-10, (ii) CHIR99021 from day 2-13, and (iii) SHH and purmorphamine from day 1-7 (Gartner et al. (2020) Star Protocols 1:100065).

[0009] Human dopaminergic neuron progenitor cells have also been differentiated from human spermatogonial stem cells (hSSCs) using a protocol that involves culturing hSSCs in olfactory ensheathing cell conditioned medium (OECCM) supplemented with RA, SB, VPA, and forskolin for four days and then in OECCM supplemented with SHH, FGF8A, and TFGP3 (Yang et al. (2019) Stem Cell Res. Therap. 10:195).

[0010] Methods for expanding midbrain neural progenitor cells have also been described (Fedele et al. (2017) Sci. Reports 7:6036), as have methods for cryopreserving such progenitor cells (Drummond et al. (2020) Front. Cell. Dev. Biol. 8:578907).

[0011] For example, protocols for differentiating pluripotent stem cells into precursors of midbrain dopaminergic neurons are reviewed in Arenas et al. (2015) Development 142:1918 - 1936 and Wang et al. (2020) Cells 9:1489.

[0012] Thus, while some progress has been made, there remains a need for effective 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. SUMMARY OF THE INVENTION

[0014] The present disclosure provides methods for generating immature and mature dopaminergic neurons from neural progenitor cells such as human directed 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 use chemically defined media and allow for the generation of mature dopaminergic neurons in as little as 23 days of culture starting from human pluripotent stem cells. The media contain small molecule agents that can agonize or antagonize specific signaling pathway activities in pluripotent stem cells, thereby promoting differentiation along the midbrain neural lineage, leading to cell maturation and expression of midbrain neural progenitor cell - related biomarkers, and then further differentiating and maturing into immature midbrain neurons over nine days of culture and into mature dopaminergic neurons over 23 days of culture. The methods of the present disclosure have the advantage that the use of small molecule agents in the media allows for precise control of the culture components and significantly reduces the differentiation time compared to prior art protocols.

[0015] Accordingly, in one aspect, the present disclosure relates to a method for generating human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons, which comprises:

[0016] Culturing human OTX2+FOXA2+LMX1A+ midbrain neural progenitor cells (MB NPCs) in a culture 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 for 3 days (day 0 - 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 culture medium containing a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-α 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.

[0017] In another aspect, the present disclosure relates to a two-stage method for generating mature dopaminergic neurons starting from MB NPCs. Accordingly, in one embodiment, the present disclosure relates to a method for generating human TH+KCNJ6+ mature dopaminergic neurons, which comprises:

[0018] (a) Culturing human OTX2+FOXA2+LMX1A+ midbrain neural progenitor cells (MB NPCs) in a culture 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 for 3 days (day 0 - day 3) to obtain human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons (MB-immature neurons); and

[0019] (b) Culturing the MB-immature neurons in a culture medium containing a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-α pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist for at least 14 days (day 3 - day 17) to obtain TH+KCNJ6+ mature dopaminergic neurons.

[0020] In another aspect, the present disclosure relates to a four-stage method for generating mature dopaminergic neurons starting from pluripotent stem cells, first generating MB NSCs, then generating MB NPCs, then generating immature midbrain neurons, and finally generating mature dopaminergic neurons. Accordingly, in one embodiment, the present disclosure relates to a method for generating human TH+KCNJ6+ mature dopaminergic neurons, which comprises:

[0021] (a) From day 0 to day 3, human pluripotent stem cells are cultured in a medium containing a WNT pathway agonist, an SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist to obtain directed midbrain neural stem cells (MB NSC);

[0022] (b) From day 4 to day 6, the MB NSC are cultured 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 to obtain human OTX2+FOXA2+LMX1A+ midbrain neural progenitor cells (MB NPC);

[0023] (c) From day 6 to day 9, the MB NPC are 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 to obtain human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons (MB-immature neurons); and

[0024] (d) From day 9 to day 23, the MB-immature neurons are 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 to obtain TH+KCNJ6+ mature dopaminergic neurons.

[0025] In one embodiment, the human pluripotent stem cells are induced pluripotent stem cells (iPSC). In one embodiment, the human pluripotent stem cells are embryonic stem cells.

[0026] In one embodiment, the WNT pathway agonist is CHIR99021. Additional exemplary WNT pathway agonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the WNT pathway agonist is present in the medium at a concentration of 0.5 - 2.0 μM. In one embodiment, the WNT pathway agonist is CHIR99021, which is present in the medium at a concentration of 1.0 - 1.1 μM.

[0027] In one embodiment, the mTOR pathway antagonist is AZD3147. Additional exemplary mTOR pathway antagonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the mTOR pathway antagonist is present in the medium at a concentration of 10 - 30 nM. In one embodiment, the mTOR pathway antagonist is AZD3147, which is present in the medium at a concentration of 15 nM.

[0028] In one embodiment, the RAR pathway antagonist is AGN193109. Additional exemplary RAR pathway antagonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the RAR pathway antagonist is present in the culture medium at a concentration of 50 - 250 nM. In one embodiment, the RAR pathway antagonist is AGN193109, which is present in the culture medium at a concentration of 100 nM.

[0029] In one embodiment, the MEK pathway antagonist is PD0325901. Additional exemplary MEK pathway antagonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the MEK pathway antagonist is present in the culture medium at a concentration of 50 - 250 nM. In one embodiment, the MEK pathway antagonist is PD0325901, which is present in the culture medium at a concentration of 100 - 110 nM.

[0030] In one embodiment, the Notch pathway antagonist is DBZ. Additional exemplary Notch pathway antagonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the Notch pathway antagonist is present in the culture medium at a concentration of 50 - 250 nM. In one embodiment, the Notch pathway antagonist is DBZ, which is present in the culture medium at a concentration of 100 nM.

[0031] In one embodiment, the BMP pathway agonist is BMP7. Additional exemplary BMP pathway agonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the BMP pathway agonist is present in the culture medium at a concentration of 5 - 50 ng / ml. In one embodiment, the BMP pathway agonist is BMP7, which is present in the culture medium at a concentration of 10 - 15 ng / ml.

[0032] In one embodiment, the BDNF pathway agonist is BDNF. Additional exemplary BDNF pathway agonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the BDNF pathway agonist is present in the culture medium at a concentration of 5 - 50 ng / ml. In one embodiment, the BDNF pathway agonist is BDNF, which is present in the culture medium at a concentration of 10 ng / ml.

[0033] In one embodiment, the GDNF pathway agonist is GDNF. Additional exemplary GDNF pathway agonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the GDNF pathway agonist is present in the culture medium at a concentration of 5 - 50 ng / ml. In one embodiment, the GDNF pathway agonist is GDNF, which is present in the culture medium at a concentration of 10 ng / ml.

[0034] In one embodiment, the PPAR-a pathway agonist is GW7647. Additional exemplary PPAR-a pathway agonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the PPAR-a pathway agonist is present in the culture medium at a concentration of 200 - 300 nM. In one embodiment, the PPAR-a pathway agonist is GW7647, which is present in the culture medium at a concentration of 250 nM.

[0035] In one embodiment, the heparin or heparin mimetic is heparin. Additional exemplary heparin mimetics, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the heparin or heparin mimetic is present in the culture medium at a concentration of 2 - 8 μg / ml. In one embodiment, the culture medium contains heparin, which is present in the culture medium at a concentration of 5 μg / ml.

[0036] In one embodiment, the dopamine agonist is dopamine. Additional exemplary dopamine agonists, and exemplary concentrations and concentration ranges are disclosed herein. In one embodiment, the dopamine agonist is present in the culture medium at a concentration of 5 - 15 μM. In one embodiment, the dopamine agonist is dopamine, which is present in the culture medium at a concentration of 10 μM.

[0037] In another aspect, the present disclosure relates to a culture medium. In one embodiment, the present disclosure relates to a culture medium for obtaining immature human midbrain neurons, which comprises 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 embodiment, the present disclosure relates to a culture medium for obtaining mature human dopaminergic neurons, which comprises a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-a pathway agonist, a heparin or heparin mimetic, a Notch pathway antagonist, and a dopamine agonist.

[0038] In another aspect, the present disclosure relates to isolated cell cultures. In one embodiment, the present disclosure relates to an isolated cell culture 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. In another embodiment, the present disclosure relates to an isolated cell culture 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.

[0039] Also included are human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons generated by any method of the present disclosure. Also included are human TH+KCNJ6+ mature dopaminergic neurons generated by any method of the present disclosure.

[0040] Other features and advantages of the present application will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shows the results of an HD-DoE model of a 13-factor experiment optimized for maximum expression of OTX2. The upper part of the model shows the prediction of the expression levels of 53 preselected genes when optimized for OTX2. The lower part of the model shows the effectors tested in the model and their contribution to the maximum expression of OTX2. The value column refers to the concentration of each effector required to simulate the model.

[0042] Figure 2 Shows the results of an HD-DoE model of a 13-factor experiment optimized for maximum expression of FOXA2. The upper and lower parts are as Figure 1 described. This condition highlights the effector purinamid, with a factor contribution of 22.2, which is an important input for high expression of FOXA2.

[0043] Figure 3 Shows the dynamic curves of the expression levels of OTX2, LMX1A, DMBX1, and FOXA2 genes relative to the concentrations of the 13 effectors tested. The positive effects of purinamid, CHIR99021, and LDN193189 on FOXA2 expression and their factor contributions are shown by the slopes of the graphs of each effector.

[0044] Figure 4 Display the dynamic curves of the expression levels of OTX2, LMX1A, DMBX1, and FOXA2 genes relative to the concentrations of 13 tested effectors. The positive effects of MK2206, PD0325901, LDN193189, and CHIR99021 on OTX2 expression and their factor contributions are shown by the slopes of the graphs for each effector.

[0045] Figure 5 Display the results of the HD-DoE model for a 12-factor experiment applied to stage 1 neural stem cells to generate a stage 2 differentiation recipe. Optimize the model for the maximum expression of LMX1A. This setting highlights the role of TTNPB in LMX1A expression, with a factor contribution of 19.5.

[0046] Figure 6 Display the results of the HD-DoE model for a 12-factor experiment applied to stage 1 neural stem cells to generate a stage 2 differentiation recipe. This setting highlights the positive effects of purinamine and CHIR99021 on FOXA2 expression, with factor contributions of 15.3 and 10.7, respectively.

[0047] Figure 7 Display the dynamic curves of the expression levels of LMX1A, FOXA2, and GBX2 genes relative to the concentrations of 12 tested effectors. The positive effects of TTNPB, CHIR99021, and GW3965 on LMX1A expression and the positive effects of purinamine, MK2206, and GW3965 on FOXA2 expression and their factor contributions are shown by the slopes of the graphs for each effector.

[0048] Figure 8 Display the results of the HD-DoE model for a 12-factor experiment applied to stage 1 neural stem cells to generate a stage 2 differentiation recipe. This setting highlights the positive effects of BMP7 and MK2206 on FOXA2 expression, with factor contributions of 13.7 and 14.2, respectively.

[0049] Figure 9 Display the results of the HD-DoE model for a 12-factor experiment applied to stage 1 neural stem cells to generate a stage 2 differentiation recipe. This setting highlights the positive effect of MK2206 on LMX1A expression and the negative effect of FGF8b on LMX1A expression, with factor contributions of 12 and 16.9, respectively.

[0050] Figure 10Show the dynamic curves of the expression levels of LMX1A, FOXA2, and GBX2 genes relative to the concentrations of 12 tested effectors. The positive effects of BMP7 and MK2206 on FOXA2 expression and the positive effect of AZD 3147 on LMX1A expression and their factor contributions are shown by the slopes of the graphs for each effector.

[0051] Figures 11A - 11B Show the dynamic curves of the expression levels of OTX2, DMBX1, FOXA2, and LMX1A genes tested in the stage 1 differentiation formulation relative to the concentrations of 5 validated effectors. Figure 11A Show the expression levels of genes of interest in the presence of all five finalized effectors. Figure 11B Show the expression levels of genes of interest in the presence of other finalized effectors at a moment when one finalized effector is absent.

[0052] Figures 12A - 12B Show the dynamic curves of the expression levels of LMX1A, FOXA2, and GBX2 genes tested in the stage 2 differentiation formulation relative to the concentrations of 3 validated effectors (TTNPB, A 83-01, and GW3965). Figure 12A Show the expression levels of genes of interest in the presence of all three finalized effectors. Figure 12B Show the expression levels of genes of interest in the presence of other finalized effectors at a moment when one finalized effector is absent.

[0053] Figures 13A - 13B Show the dynamic curves of the expression levels of LMX1A, FOXA2, and GBX2 genes tested in the stage 2 differentiation formulation relative to the concentrations of 3 validated effectors (MK2206, AZD 3147, and BMP7). Figure 13A Show the expression levels of genes of interest in the presence of all three finalized effectors. Figure 13B Show the expression levels of genes of interest in the presence of other finalized effectors at a moment when one finalized effector is absent.

[0054] Figure 14 Show photos of fluorescence images of MB-derived neural stem cells at the end of the stage 1 treatment. The cells are stained with midbrain biomarkers including FOXA2, LMX1A, OTX2, mid-hindbrain boundary biomarker PAX2, and early neuron biomarker nestin and hindbrain biomarker GBX2. At this stage, the cells are positive for all biomarkers except FOXA2.

[0055] Figure 15Photograph showing the fluorescence image of neural stem cells of MB origin at the end of the two-stage process. The cells were stained with midbrain biomarkers including FOXA2, LMX1A, OTX2 and hindbrain biomarker GBX2. At this stage, the cells were positive for all midbrain biomarkers and very low expression of GBX2 was observed.

[0056] Figure 16 Schematic diagram showing a two-stage culture protocol for generating midbrain neural progenitor cells from hiPCs within six days, including a one-stage medium used from day 0 to day 3 for generating midbrain neural stem cells (MB-NSCs) from iPSCs and a two-stage medium used from day 3 to day 6 for generating midbrain neural progenitor cells (MB-NPCs) from MB-NSCs.

[0057] Figures 17A - 17C RNA-seq data of cells after culturing in MB differentiation medium for 3 days (one-stage) and 6 days (two-stage). Figure 17A Bar graph showing the differential expression of selected genes at the one-stage. At the end of the one-stage, the expression levels of stem cell genes NANOG and POU5F1 decreased, while the expression levels of genes involved in the early development of the midbrain region and neuronal identity increased. Figure 17B Bar graph showing the differential expression of selected genes at the two-stage. At the end of the two-stage, the expression levels of MB progenitor cell genes including DDC, LMX1B, SOX6 and EN1 increased. Figure 17C Heatmap showing the gene profiles of MB neural progenitor cells on day 6 compared to the gene profiles of hiPSCs on day 0.

[0058] Figure 18 Schematic diagram showing a four-stage culture protocol for generating dopaminergic neurons, including a three-stage medium used from day 6 to day 9 for generating midbrain immature neurons (MB-immature neurons) from MB-NPCs and a four-stage medium used from day 9 to day 23 for generating dopaminergic neurons from MB-immature neurons.

[0059] Figure 19 Results of the HD-DoE model of the 12-factor experiment optimized for the maximum expression of CORIN. The upper part of the model shows the prediction of the expression levels of 53 preselected genes when optimizing for CORIN. The lower part of the model shows the effectors tested in this model and their contribution to the maximum expression of CORIN. The value column refers to the concentration of each effector required to simulate the model.

[0060] Figure 20Show the results of the HD-DoE model of the 12-factor experiment optimized for the maximum expression of MSX1. The upper part of the model shows the prediction of the expression levels of 53 preselected genes when optimized for MSX1. The lower part of the model shows the effectors tested in this model and their contributions to the maximum expression of MSX1. The value column refers to the concentration of each effector required for the simulation model. This condition highlights effector BMP7, with a factor contribution of 11.06, as an important input for the high expression of MSX1.

[0061] Figure 21 Show the results of the HD-DoE model of the 12-factor experiment optimized for the maximum expression of SOX6. The upper part of the model shows the prediction of the expression levels of 53 preselected genes when optimized for SOX6. The lower part of the model shows the effectors tested in this model and their contributions to the maximum expression of SOX6. The value column refers to the concentration of each effector required for the simulation model. This condition highlights effectors AGN193109 (factor contribution of 26.1) and PD0325901 (factor contribution of 13.5), as two important inputs for the high expression of SOX6.

[0062] Figure 22 Show the results of the HD-DoE model of the 12-factor experiment applied to 2-stage neural progenitor cells to generate a 3-stage differentiation formula. The model is optimized for the maximum expression of KCNJ6. The upper part of the model shows the prediction of the expression levels of 53 preselected genes when optimized for KCNJ6. The lower part of the model shows the effectors tested in this model and their contributions to the maximum expression of KCNJ6. The value column refers to the concentration of each effector required for the simulation model. This setting highlights the role of AGN193109 in the expression of KCNJ6, with a factor contribution of 17.

[0063] Figure 23 Show the dynamic curve analysis of the expression levels of CORIN, MSX1, SOX6, and KCNJ6 relative to the concentrations of 12 effectors. The positive effects of AGN193109 and CHIR99021 on KCNJ6 expression and their factor contributions are shown by the slopes of the graphs of each effector.

[0064] Figure 24 Show the results of the HD-DoE model of the 12-factor experiment applied to 2-stage neural progenitor cells to generate a 3-stage differentiation formula. This setting highlights the positive roles of PDBZ and AZD3147 in SOX6 expression, with factor contributions of 21.5 and 20.2, respectively.

[0065] Figure 25Dynamic curve analysis showing the expression levels of SOX6 and MSX1 relative to the concentrations of 12 effectors. The positive effects of DBZ, AZD3147, and PD0325901 on SOX6 expression and the negative effects of activin A and Takinib on MSX1 expression and their factor contributions are shown by the slopes of the graphs for each effector.

[0066] Figure 26 Dynamic curve analysis showing the expression levels of CORIN, KCNJ6, SOX6, and MSX1 relative to the concentrations of DBZ and AZD3147. The positive effects of DBZ and AZD3147 on the expression of all selected genes and their factor contributions are shown by the slopes of the graphs for each effector.

[0067] Figure 27 Showing the results of the HD-DoE model for an 8-factor experiment applied to stage 3 immature neurons to generate a stage 4 differentiation formulation. This setup highlights the positive effect of BDNF on NR4A2 expression and the negative effect of CHIR99021 on NR4A2 expression, with factor contributions of 17.1 and 20.7, respectively.

[0068] Figure 28 Dynamic curve analysis showing the expression levels of NR4A2 and PITX3 relative to the concentrations of 8 effectors. The positive effects of BDNF on the expression of both genes and the negative effect of rosiglitazone on their expression levels and their factor contributions are shown by the slopes of the graphs for each effector.

[0069] Figures 29A - 29B Showing the dynamic curves of the expression levels of CORIN, KCNJ6, MSX1, and SOX6 in a stage 3 differentiation formulation relative to the concentrations of four validated effectors. Figure 29A Showing the expression levels of genes of interest in the presence of the finalized effectors. Figure 29B Showing the expression levels of genes of interest in the presence of other finalized effectors at a moment when one finalized effector is absent.

[0070] Figures 30A - 30B Showing the dynamic curves of the expression levels of SOX6 and MSX1 in a stage 3 differentiation medium formulation relative to the concentrations of four validated effectors. Figure 30A Showing the expression levels of genes of interest in the presence of the finalized effectors. Figure 30B Showing the expression levels of genes of interest in the presence of other finalized effectors at a moment when one finalized effector is absent.

[0071] Figures 31A - 31BShow the dynamic curves of the expression levels of NR4A2 and PITX3 in the 4-stage differentiation medium formulation relative to the concentrations of all six finalized effectors. Figure 31A Show the expression levels of genes of interest in the presence of all finalized effectors. Figure 31B Show the expression levels of genes of interest in the presence of other finalized effectors while lacking one finalized effector at a certain time point.

[0072] Figure 32 Show a photograph of the fluorescence image of MB-derived immature neurons at the end of the 3-stage. The cells were stained with midbrain biomarkers including LMX1A, FOXA2, MSX1, S0X6, and PITX3, and the pan-neuronal biomarker TUBB3 and the immature neuron biomarker DCX. At this stage, the cells were positive for all biomarkers expected in the population of immature midbrain neurons.

[0073] Figure 33 Show a photograph of the fluorescence image of SNc dopaminergic neurons at the end of the 4-stage. The cells were stained with midbrain biomarkers including TH, KCNJ6, and CALB1, and the mature neuron biomarkers MAP2 and SYN1. At this stage, the cells were positive for all expected biomarkers, and very low expression of CALB1 was observed. Detailed Description of the Invention

[0075] Methods and compositions for generating mature dopaminergic neurons from midbrain neural progenitor cells (which are themselves generated from human pluripotent stem cells) under chemically defined culture conditions using a small molecule-based approach are described herein. The methods of the present disclosure generate midbrain neural progenitor cells in a two-stage protocol, where OTX2+LMX1A+ committed MB neural stem cells (NSCs) are generated within 3 days, and then OTX2+LMX1A+FOX2A+ MB neural progenitor cells (NPCs) (referred to herein as the 1-stage and 2-stage formulations) are generated on the sixth day of culture. The MB-NPCs are then further differentiated using another two-stage protocol (referred to herein as the 3-stage and 4-stage protocols) to generate immature midbrain neurons on day 9 of culture and mature dopaminergic neurons on day 23 of culture. Thus, compared to existing art protocols using chemically defined culture conditions, the present disclosure allows for the obtaining of mature dopaminergic neurons in a significantly shorter time.

[0076] As described in Example 1, a high-dimensional experimental design (HD-DoE) method is used to simultaneously test the effects of multiple process inputs (e.g., small molecule agonists or antagonists) on an output response (such as gene expression). These experiments allow for the identification of chemically defined media, including agonists and / or antagonists of specific signaling pathways, that are sufficient to generate directed midbrain pluripotent stem cells and midbrain progenitor cells in a very short period of time. The optimized media are further validated by factor criticality analysis, which examines the effect of eliminating individual agonists or antagonists, as described in Example 2. Immunohistochemistry further confirms the phenotype of the cells generated by the differentiation protocol, as described in Example 3. In addition, RNA-seq analysis of the cells cultured according to the differentiation protocol also confirms the expression of MB progenitor cell genes, as described in Example 4.

[0077] Figure 16 An embodiment of the method of the present disclosure for generating MB NSCs and MB NPCs is schematically illustrated.

[0078] Figure 18 An embodiment of the method of the present disclosure for generating immature midbrain neurons and mature dopaminergic neurons is schematically illustrated.

[0079] Multiple aspects of the present application will be described in further detail in the following subsections.

[0080] I. Cell

[0081] 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 that have the ability to differentiate into multiple different cell types. The term "pluripotent" as used herein refers to cells that have the ability to differentiate into cell types characteristic of all three germ cell layers (endoderm, mesoderm, and ectoderm) under different conditions. The main characteristic of pluripotent cells is that they can differentiate into all three germ layers, for example, using nude mouse and teratoma formation assays. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is to demonstrate the ability to differentiate into cells of each of the three germ layers.

[0082] 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 (e.g., as 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 cell 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. Since the methods of generating directed midbrain neural stem cells and midbrain neural progenitor cells of the present disclosure are used to differentiate (mature) starting pluripotent stem cell populations, in various embodiments, the midbrain-directed 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.

[0083] As described herein, pluripotent stem cells are subjected to culture conditions that induce cell differentiation. As used herein, the term "differentiation" refers to the development of a cell from a more primitive stage to a more mature (i.e., less primitive) cell, typically exhibiting phenotypic characteristics of a specific cell lineage.

[0084] As used herein, a "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 different types of cells along the neural lineage.

[0085] As used herein, a "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.

[0086] In embodiments, cells can be identified and characterized based on the expression of one or more biomarkers, such as specific biomarkers of neural progenitor cells or midbrain region-directed neural cells. Non-limiting examples of biomarkers whose expression can be evaluated when characterizing cells of interest include OTX2, which is a midbrain biomarker involved in midbrain positioning and maintenance of the mid-hindbrain boundary (Vernay et al. (2005) J. Neurosci. 25:4856-4867); LMX1A, which is involved in the generation and differentiation of midbrain dopaminergic progenitor cells (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, which is expressed in the midbrain and the anterior and posterior brains (Urbanek et al. (1997) Proc. Natl. Acad. Sci. USA 94:5703-5708); nestin, which is an early neuronal biomarker; KI67, which is a proliferation biomarker; and GBX2, which is a hindbrain biomarker.

[0087] As used herein, a cell expressing a biomarker of interest at only low levels is intended to mean a level that is at most 20% higher than the background level, and more preferably, a level that is less than 20%, less than 15%, less than 10%, or less than 5% higher than the background level (where the background level corresponds to, for example, the expression level of a negative control biomarker that is not considered to be expressed by the cell).

[0088] In embodiments, the cells generated by the methods of the present disclosure are directed midbrain (MB) neural stem cells (NSCs). As used herein, "directed midbrain neural stem cell" or "directed MB NSC" refers to a stem cell-derived neural stem cell that expresses the biomarkers OTX2 and LMX1A. In one embodiment, the directed MB NSCs do not express or express only low levels of the biomarker FOXA2. In one embodiment, the directed MB NSCs do not express or express only low levels of the biomarker GBX2. In addition to OTX2 and LMX1A, the directed MB NSCs may also express other biomarkers, including but not limited to PAX2, nestin, and / or KI67.

[0089] In an embodiment, the cells generated by the methods of the present disclosure are midbrain neural progenitor cells, which are more differentiated (more mature) cells than directed MBNSCs. As used herein, "midbrain neural progenitor cell" or "MB NPC" refers to a stem cell-derived progenitor cell that expresses the biomarkers OTX2, LMX1A, and FOXA2. In an embodiment, 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 other biomarkers, including but not limited to PAX2, nestin, and / or KI67.

[0090] According to the culture protocols described herein, the directed MB NSCs and MB NPCs generated by the methods of the present disclosure can be further cultured in vitro to generate mature dopaminergic neurons. As used herein, "immature midbrain neuron" or "MB-immature neuron" refers to a neuron-derived cell that expresses the biomarkers FOXA2, LMX1A, MSX1, PITX3, and DCX. As used herein, a mature dopaminergic neuron refers to a neuron-derived cell that expresses the biomarkers TH and KCNJ6 and may also express TUBB3, MAP2, SYN1, and NF.

[0091] II. Culture medium components

[0092] The methods of the present disclosure for generating mature dopaminergic neurons, MB-immature neurons, MB NSCs, or MB NPCs include culturing human pluripotent stem cells in a medium that is generally devoid of exogenously added growth factors and that contains specific agonists and / or antagonists of cell signaling pathways.

[0093] As described herein, a culture medium containing a WNT pathway agonist, a SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist is sufficient to generate OTX2- and LMX1A-expressing MB NSCs within just 3 days (referred to herein as the "stage 1" of the differentiation protocol). Further differentiation of MB NSCs in a culture 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 is sufficient to generate OTX2+FOXA2+LMX1A+ MB NPCs within an additional 3 days (referred to herein as "stage 2"), forming an overall two-stage six-day protocol for generating MB NPCs. Further differentiation of MB NPCs in a culture 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 is sufficient to generate FOXA2+, LMX1A+, MSX1+, PITX3+, DCX+ immature MB neurons on the ninth day of culture (referred to herein as "stage 3"). Finally, further differentiation of immature MB neurons in a culture 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 14 days is sufficient to generate TH / KCNJ6+ mature dopaminergic neurons on the 23rd day of culture (referred to herein as "stage 4").

[0094] As used herein, a cell signaling pathway "agonist" is intended to refer to an agent that stimulates (upregulates) a cell signaling pathway. Stimulation of a cell signaling pathway can be initiated extracellularly, for example, by using an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., the agonist can be a receptor ligand). Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, for example, by using a small molecule agonist that interacts with components of the signaling pathway within the cell.

[0095] As used herein, a cell signaling pathway "antagonist" is intended to refer to an agent that inhibits (downregulates) a cell signaling pathway. Inhibition of a cell signaling pathway can be initiated extracellularly, for example, by using an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated intracellularly, for example, by using a small molecule antagonist that interacts with components of the signaling pathway within the cell.

[0096] The agonists and antagonists used in the methods of the present disclosure are known in the art and are commercially available. They are used in the culture medium at a concentration effective to achieve the desired result, such as generating midbrain NSCs of interest and / or midbrain NPCs expressing midbrain markers. Non-limiting examples of suitable agonists and antagonists and effective concentration ranges are further described below.

[0097] Agonists of the WNT pathway include agents, molecules, compounds or substances capable of stimulating (upregulating) the canonical Wnt / β-catenin signaling pathway, which is biologically activated by the binding of a Wnt-protein ligand to a Frizzled family receptor. In one embodiment, the WNT pathway agonist is a glycogen synthase kinase 3 (Gsk3) inhibitor. In one embodiment, the WNT pathway agonist is selected from the group consisting of: CHIR99021, CHIR98014, SB 216763, SB 415286, LY2090314, 3F8, A1070722, AR-A 014418, BIO, BIO-acetoxime, AZD1080, WNT3A, atiprimod, indirubin-3'-oxime, 1-azakenpaullone, kenpaullone, TC-G 24, TDZD 8, TWS119, NP031112, AT 7519, KY 19382, AZD2858, and combinations thereof. In one embodiment, the WNT pathway agonist is present in the culture medium at a concentration of 0.3 - 3.0 μM, 0.5 - 2.0 μM, 0.75 - 1.5 μM or 1.0 - 1.2 μM. In one embodiment, the WNT pathway agonist is CHIR99021. In one embodiment, the WNT pathway agonist is CHIR99021, which is present in the culture medium at a concentration of 0.3 - 3.0 μM, 0.5 - 2.0 μM, 0.75 - 1.5 μM or 1.0 - 1.2 μM. In one embodiment, the WNT pathway agonist is CHIR99021, which is present in the culture medium at a concentration of 1.1 μM (e.g., in stage 1 medium). In one embodiment, the WNT pathway agonist is CHIR99021, which is present in the culture medium at a concentration of 1.0 μM (e.g., in stage 3 medium).

[0098] Agonists of the SHH (Sonic Hedgehog) pathway include agents, molecules, compounds or substances that can stimulate (activate) signal transduction through the SHH pathway, which is biologically involved in the binding of SHH to the Patched-1 (PTCH1) receptor and transduction through the Smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway agonist is selected from the group consisting of purmorphamine, GSA 10, SAG, and combinations thereof. In one embodiment, the SHH pathway agonist is present in the culture medium at a concentration of 100 - 1000 nM, 200 - 800 nM, 250 - 750 nM or 500 - 600 nM. In one embodiment, the SHH pathway agonist is purmorphamine. In one embodiment, the SHH pathway agonist is purmorphamine, which is present in the culture medium at a concentration of 100 - 1000 nM, 200 - 800 nM, 50 - 750 nM or 500 - 600 nM. In one embodiment, the SHH pathway agonist is purmorphamine, which is present in the culture medium at a concentration of 550 nM.

[0099] 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, which are activin receptor-like kinases (ALKs) (e.g., type I BMP receptors, including but not limited to ALK2 and ALK3). In one embodiment, 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 embodiment, the BMP pathway antagonist is present in the culture medium at a concentration of 100 - 500 nM, 100 - 400 nM, 150 - 350 nM or 200 - 300 nM. In one embodiment, the BMP pathway antagonist is LDN193189. In one embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture medium at a concentration of 100 - 500 nM, 100 - 400 nM, 150 - 350 nM or 200 - 300 nM. In one embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture medium at a concentration of 275 nM.

[0100] AKT pathway antagonists include agents, molecules, compounds or substances that can inhibit (downregulate) the signaling pathways of one or more serine / threonine kinase AKT family members, including 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 embodiment, 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 embodiment, the AKT pathway antagonist is present in the culture medium at a concentration of 25-300 nM, 50-250 nM, 75-200 nM, or 125-150 nM. In one embodiment, the AKT pathway antagonist is MK2206. In one embodiment, the AKT pathway antagonist is MK2206, which is present in the culture medium at a concentration of 25-300 nM, 50-250 nM, 75-200 nM, or 125-150 nM. In one embodiment, the AKT pathway antagonist is MK2206, which is present in the culture medium at a concentration of 138 nM.

[0101] In one embodiment, the AKT pathway antagonist present in the culture medium in step (a) is the same as the AKT pathway antagonist present in the culture medium in step (b). In one embodiment, the AKT pathway antagonist present in the culture medium in step (a) is a different AKT pathway antagonist from the AKT pathway antagonist present in the culture medium in step (b). In one embodiment, the AKT pathway antagonists present in the culture media in steps (a) and (b) are MK2206, for example, which are present in the culture medium at 25-300 nM, 50-250 nM, 75-200 nM, or 125-150 nM in both steps, such as at a concentration of 138 nM in both steps.

[0102] MEK pathway antagonists 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 embodiment, the MEK pathway antagonists are 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 embodiment, the MEK pathway antagonist is present in the culture medium at a concentration of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM or 100 - 120 nM. In one embodiment, the MEK pathway antagonist is PD0325901. In one embodiment, the MEK pathway antagonist is PD0325901, which is present in the culture medium at a concentration of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM or 100 - 120 nM. In one embodiment, the MEK pathway antagonist is PD0325901, which is present in the culture medium at a concentration of 110 nM (e.g., in a phase 1 protocol). In one embodiment, the MEK pathway antagonist is PD0325901, which is present in the culture medium at a concentration of 100 nM (e.g., in a phase 3 protocol).

[0103] RA pathway agonists include agents, molecules, compounds or substances that can stimulate retinoic acid receptors (RARs) activated by all-trans retinoic acid and 9-cis retinoic acid. There are three types of RARs: RAR-α, RAR-β and RAR-γ, which are encoded by the RARA, RARB, and RARG genes, respectively. Different retinoic acid analogs have been synthesized that can activate the retinoic acid pathway. 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 of 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) retinoid compounds, ii) retinoid X receptor (RXR) agonists, and iii) 25 retinoic acid receptor (RAR) agonists. In a specific embodiment, 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).

[0104] Thus, in one embodiment, the RA pathway agonist is selected from the group consisting of: TTNPB, AM 580, CD 1530, CD2314, 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 a combination thereof. In one embodiment, the RA pathway agonist is present in the culture medium at a concentration of 5 - 500 nM, 25 - 250 nM, 10 - 100 nM, or 25 - 75 nM. In one embodiment, the RA pathway agonist is TTNPB. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture medium at a concentration of 5 - 500 nM, 25 - 250 nM, 10 - 100 nM, or 25 - 75 nM. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture medium at a concentration of 50 nM.

[0105] Antagonists of the retinoic acid receptor pathway include agents, molecules, compounds or substances that are capable of inhibiting the retinoic acid receptor (RAR) (i.e., the receptor activated by retinoic acid). In one embodiment, the RAR pathway antagonists are selected from the group consisting of: AGN193109, BMS195614, CD 2665, ER 50891, LE 135, LY 2955303, MM11253, and combinations thereof. In one embodiment, the RAR pathway antagonist is present in the culture medium at a concentration of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM or 100 - 120 nM. In one embodiment, the RAR pathway antagonist is AGN193109. In one embodiment, the RAR pathway antagonist is AGN193109, which is present in the culture medium at a concentration of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM or 100 - 120 nM. In one embodiment, the RAR pathway antagonist is AGN193109, which is present in the culture medium at a concentration of 100 nM (e.g., in a three - stage protocol).

[0106] Agonists of the LXR (liver X receptor) pathway include agents, molecules, compounds or substances that are capable of stimulating (activating) signal transduction through the LXR pathway, which biologically involves the heterodimerization of LXR with the retinoid X receptor (RXR) and activation by oxysterols. In one embodiment, the LXR pathway agonists are selected from the group consisting of: GW3965, T0901317, DMHCA, AZ876, and combinations thereof. In one embodiment, the LXR pathway agonist is present in the culture medium at a concentration of 100 - 1000 nM, 200 - 800 nM, 250 - 750 nM or 550 - 650 nM. In one embodiment, the LXR pathway agonist is GW3965. In one embodiment, the LXR pathway agonist is GW3965, which is present in the culture medium at a concentration of 100 - 1000 nM, 200 - 800 nM, 250 - 750 nM or 550 - 650 nM. In one embodiment, the LXR pathway agonist is GW3965, which is present in the culture medium at a concentration of 500 nM.

[0107] 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, which are activin receptor-like kinases (ALKs) (e.g., type I BMP receptors, including but not limited to ALK2 and ALK3). In one embodiment, the BMP pathway agonist is selected from the group consisting of: BMP, sb4, ventromorphins (e.g., as described in Genthe et al. (2017) ACS Chem. Biol. 12:2436-2447), and combinations thereof. In one embodiment, the BMP pathway agonist is present in the culture medium at a concentration of 1-100 ng / ml, 5-50 ng / ml, 10-25 ng / ml or 12.5-17.5 ng / ml. In one embodiment, the BMP pathway agonist is BMP7. In one embodiment, the BMP pathway agonist is BMP7, which is present in the culture medium at a concentration of 1-100 ng / ml, 5-50 ng / ml, 10-25 ng / ml or 12.5-17.5 ng / ml. In one embodiment, the BMP pathway agonist is BMP7, which is present in the culture medium at a concentration of 15 ng / ml in step (b) (i.e., the 2nd stage) of the method. In one embodiment, the BMP pathway agonist is BMP7, which is present in the culture medium at a concentration of 10 ng / ml in the 3rd stage.

[0108] Antagonists of the TGFβ (transforming growth factor β) pathway include agents, molecules, compounds or substances that can inhibit (downregulate) signal transduction through members of the TGFP receptor family (a family of serine / threonine kinase receptors). In one embodiment, the TGFβ pathway antagonist is selected from the group consisting of: A 83-01, SB-431542, GW788388, SB525334, TP0427736, RepSox, SD-208, and combinations thereof. In one embodiment, the TGFβ pathway antagonist is present in the culture medium at a concentration of 100-500 nM, 200-400 nM, 250-350 nM or 275-325 nM. In one embodiment, the TGFβ pathway antagonist is A83-01. In one embodiment, the TGFβ pathway antagonist is A 83-01, which is present in the culture medium at a concentration of 100-500 nM, 200-400 nM, 250-350 nM or 275-325 nM. In one embodiment, the TGFβ pathway antagonist is A 83-01, which is present in the culture medium at a concentration of 300 nM in step (b) (i.e., the 2nd stage) of the method.

[0109] mTOR (mammalian target of rapamycin) pathway antagonists include agents, molecules, compounds or substances that can inhibit (downregulate) signal transduction through mTOR, which is a member of the PI3K-related kinase family and a core component of the mTORC1 and mTORC2 complexes. In one embodiment, the mTOR pathway antagonist is selected from the group consisting of: AZD3147, rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, umirolimus, zotarolimus, torin-1, torin-2, vistusertib, AZD8055, dactolisib, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, niclosamide, omipalisib, PF04691502, PF 05212384, WYE 687, XE 388, STK16-IN-1, PP 242, torkinib, sapanisertib, voxtalisib, and combinations thereof. In one embodiment, the mTOR pathway antagonist is present in the culture medium at a concentration of 5-100 nM, 5-50 nM, 10-30 nM or 10-20 nM. In one embodiment, the mTOR pathway antagonist is AZD3147. In one embodiment, the mTOR pathway antagonist is AZD3147, which is present in the culture medium at a concentration of 5-100 nM, 5-50 nM, 10-30 nM or 10-20 nM. In one embodiment, the mTOR pathway antagonist is AZD3147, which is present in the culture medium at a concentration of 15 nM in step (b) (i.e., the two-stage) of the method. In one embodiment, the mTOR pathway antagonist is AZD3147, which is present in the culture medium at a concentration of 15 nM in the three-stage.

[0110] Antagonists of the Notch pathway include agents, molecules, compounds or substances that are capable of inhibiting (downregulating) signal transduction through the Notch receptor. In one embodiment, the Notch pathway antagonist is selected from the group consisting of: DBZ, avagacestat, begacestat, BMS299897, Compound E, DAPT, JLK6, L-685,458, LY450139, 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 culture medium at a concentration 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 is present in the culture medium at a concentration of 25 - 300 nM, 50 - 250 nM, 75 - 200 nM or 100 - 120 nM. In one embodiment, the Notch pathway antagonist is DBZ and is present in the culture medium at a concentration of 100 nM (e.g., in a three-stage protocol).

[0111] Agonists of the brain-derived neurotrophic factor (BDNF) pathway include agents, molecules, compounds or substances that are capable of stimulating (upregulating) the BDNF signaling pathway. In one embodiment, the BDNF pathway agonist is selected from the group consisting of: BDNF, rotigotine, 7,8-DHF, ketamine, tricyclic dimer peptide-6 (TDP6), LM22A-4, and combinations thereof. In one embodiment, the BDNF pathway agonist is present in the culture medium at a concentration of 1 - 100 ng / ml, 5 - 50 ng / ml, 10 - 25 ng / ml or 12.5 - 17.5 ng / ml. In one embodiment, the BDNF pathway agonist is BDNF. In one embodiment, the BDNF pathway agonist is BDNF and is present in the culture medium at a concentration of 1 - 100 ng / ml, 5 - 50 ng / ml, 10 - 25 ng / ml or 12.5 - 17.5 ng / ml. In one embodiment, the BDNF pathway agonist is BDNF and is present in the culture medium of the fourth stage of the method at a concentration of 10 ng / ml.

[0112] 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 embodiment, the GDNF pathway agonist is selected from the group consisting of GDNF, BT13, BT44, and combinations thereof. In one embodiment, the GDNF pathway agonist is present in the culture medium at a concentration of 1-100 ng / ml, 5-50 ng / ml, 10-25 ng / ml, or 12.5-17.5 ng / ml. In one embodiment, the GDNF pathway agonist is GDNF. In one embodiment, the GDNF pathway agonist is GDNF, which is present in the culture medium at a concentration of 1-100 ng / ml, 5-50 ng / ml, 10-25 ng / ml, or 12.5-17.5 ng / ml. In one embodiment, the GDNF pathway agonist is GDNF, which is present at a concentration of 10 ng / ml in the culture medium in the 4th stage of the method.

[0113] Agonists of the PPAR-α (peroxisome proliferator-activated receptor α) pathway include agents, molecules, compounds or substances that can stimulate (upregulate) the PPAR-α signaling pathway. In one embodiment, the PPAR-α pathway agonist is selected from the group consisting of: 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, Indeglitazar, Netoglitazone, GW0742, Bezafibrate, Bezafibrate-d4, Chiglitazar, BMS 687453, Lanifibranor, Saroglitazar, Saroglitazar magnesium, Saroglitazar-d5, Imiglitazar, AVE-8143, GW590735, Ertiprotafib, LJ 570, Seladelpar Sodium Salt, Edaglitazone, Muraglitazar, Ragaglitazar, GW9578, MHY 908, KRP-297, Elafibranor, Aleglitazar, AM3102, Eupatilin, Clofibric acid, Clofibric acid-d4, Naveglitazar, Naveglitazar racemate, and combinations thereof. In one embodiment, the PPAR-α pathway agonist is present in the culture medium at a concentration of 50 - 500 nM, 100 - 400 nM, 200 - 300 nM, or 225 - 275 nM. In one embodiment, the PPAR-α pathway agonist is GW7647. In one embodiment, the PPAR-α pathway agonist is GW7647, which is present in the culture medium at a concentration of 100 - 500 nM, 200 - 400 nM, 250 - 350 nM, or 275 - 325 nM. In one embodiment, the PPAR-α pathway agonist is GW7647, which is present in the culture medium at a concentration of 250 nM in the 4th stage of the method.

[0114] Heparin is an anticoagulant that has long been known in the art, and heparin mimetics are highly sulfated synthetic and semi-synthetic compounds that are structurally distinct glycosaminoglycan analogs. In an embodiment, the culture medium comprises heparin or a heparin mimetic, such as a heparin mimetic selected from the group consisting of heparan sulfate, enoxaparin, low molecular weight heparin, AV5026, M402, and combinations thereof. In one embodiment, the heparin or heparin mimetic is present in the culture medium at a concentration of 1-10 μg / ml, 2-8 μg / ml, 3-7 μg / ml, or 4-6 μg / ml. In one embodiment, the culture medium comprises heparin, which is present in the culture medium at a concentration of 1-10 μg / ml, 2-8 μg / ml, 3-7 μg / ml, or 4-6 μg / ml. In one embodiment, the culture medium comprises heparin, which is present in the culture medium at a concentration of 5 μg / ml.

[0115] Dopamine agonists include agents, molecules, compounds or substances that can stimulate (upregulate) the dopamine signaling pathway. In an embodiment, the dopamine agonist is selected from the group consisting of: dopamine, dopamine hydrochloride, (R)-(-)-apomorphine hydrochloride, bromocriptine mesylate, bromocriptine-13C,d3, CNV dopamine, dihydroergotamine mesylate, lisuride, lisuride maleate, mesulergine hydrochloride, piribedil dihydrochloride, piribedil, quinelorane hydrochloride, (-)-quinpirole hydrochloride, ropinirole, Tau-aggregation-IN-1, NMI 8739, U91356, carbidopa phosphate, quinagolide hydrochloride, pramipexole dihydrochloride, PD-168077 maleate, rotigotine hydrochloride, PF592379, rotigotine, (Rac)-rotigotine hydrochloride, (Rac)-rotigotine-d7 hydrochloride, Ro 10-5824 dihydrochloride, (+)-dihydroxytryptamine hydrochloride, talipexole, PF2562, neuromedin N, A68930, A68930 hydrochloride, A77636 dihydrochloride, PD 119819, PD 128907 hydrochloride, CY 208-243, pramipexole, pramipexole-d3 dihydrochloride, pramipexole-d7 dihydrochloride, SKF 38393 hydrochloride, SKF 38393 hydrobromide, SKF 82958, ABT 670, ABT-724, ABT-724 trihydrochloride, (R)-PF-06256142, talipexole dihydrochloride, R-procaterol, padovanol hydrochloride, padovanol, pergolide mesylate, BP897, BP897 hydrochloride, LY3154207, tavapadon, roxindole, UNC994, cabergoline, brexpiprazole, pergolide-d7 mesylate, cabergoline-d6, roxindole hydrochloride, WAY-100635 maleate, selegiline hydrochloride, dihydroxytryptamine, dihydroxytryptamine hydrochloride, biphenylnolol, OS-3-106, brilaroxazine, pramipexole dihydrochloride hydrate, pramipexole, pramipexole dihydrochloride, cabergoline-d5, perospirone, brexpiprazole-d8, (Rac)-tavapadon, ML417, brexpiprazole S-oxide, pramipexole-d7 dihydrochloride, pramipexole-d5 dihydrochloride, UCSF924, WAY-100635, SKF83959, pramipexole (N-propyl-3,3,3-d3) (dihydrochloride), sarizotan, brexpiprazole S-oxide D8, SKF 83959 hydrobromide, and combinations thereof. In one embodiment, the dopamine agonist is present in the culture 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. In one embodiment, the dopamine agonist is dopamine, which is present in the culture 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, which is present in the culture medium at a concentration of 10 μM in stage 4 of the method.

[0116] III. Culture Conditions

[0117] In combination with the chemical definitions and optimized culture medium described in Section II above, the methods of the present disclosure for generating mature dopaminergic neurons, immature midbrain neurons, committed MB NSCs, and MB NPCs utilize standard culture conditions established in the art for cell culture. For example, the cells can be cultured at 37 °C, 5% O 2 and 5% CO 2 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 adhered to the plate, preferably 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).

[0118] The pluripotent stem cells can be cultured in a commercially available culture medium prior to differentiation. For example, the stem cells can be cultured in Essential 8Flex medium (Thermo Fisher #A2858501) for at least one day before starting the differentiation protocol. In a non-limiting exemplary embodiment, the stem cells are passaged at a density of 150,000 cells / cm2 onto a vitronectin (ThermoFisher #A14700)-coated 96-well plate and cultured in Essential 8Flex medium for one day before differentiation.

[0119] When initiating the differentiation protocol, the culture medium for the stem cells is changed to a basal differentiation medium supplemented with a signaling pathway agonist and / or antagonist, as described in Section II above. The basal differentiation medium can include, for example, a commercially available basal medium supplemented with additional standard medium components required to maintain cell viability and growth, but lacking serum (the basal differentiation medium is a serum-free medium) or any other exogenously added growth factors such as FGF2, PDGF, IGF, or HGF. In a non-limiting exemplary embodiment, the basal differentiation medium contains 1x IMDM (Thermo Fisher #12440046), 1x F12 (Thermo Fisher #11765047), 1 mg / ml polyvinyl alcohol (Sigma #p8136), 1% chemically defined lipid concentrate (Thermo Fisher #11905031), 450 uM 1-thioglycerol (Sigma #M6145), 0.7 ug / ml insulin (Sigma #11376497001), and 15 ug / ml transferrin (Sigma #10652202001) (also referred to herein as "CDM2" medium, as Figure 16 and Figure 18 used in the exemplary differentiation protocol shown).

[0120] The medium is typically changed regularly to fresh medium. For example, in one embodiment, the medium is changed every 24 hours.

[0121] To generate mature dopaminergic neurons, immature midbrain neurons, committed MB NSCs, and MB NPCs, the starting pluripotent stem cells are cultured in an optimized medium for a sufficient time for cell differentiation and expression of markers associated with committed MB NSCs or MBNPCs. As described in the Examples, it has been found that culturing pluripotent stem cells using a two-stage method, one stage optimized for the generation of MB NSCs and the other stage optimized for the generation of MB NPCs, can result in the production of MB NPCs in as little as six days of culture, where the culture period for the first stage ("Stage 1", generating MB NSCs) is 0 - 3 days and the culture period for the second stage ("Stage 2", generating MB NPCs) is 4 - 6 days. The MBNPCs are further cultured in a Stage 3 medium for 6 - 9 days to generate immature midbrain neurons (MB-immature neurons), and the MB-immature neurons are further cultured in a Stage 4 medium for 9 - 23 days to generate mature dopaminergic neurons.

[0122] Thus, in the first stage of the method for generating MB NSCs (also referred to herein as "step (a)" or "stage 1"), the pluripotent stem cells are cultured in a stage 1 optimized medium from day 0 to day 3, or starting from day 0 and continuing until day 3, or for 72 hours (3 days), or for at least 60 hours, or for at least 64 hours, or for at least 68 hours, or for at least 70 hours, or for at least 72 hours, or for 60 hours, or for 64 hours, or for 68 hours, or for 70 hours, or for 72 hours.

[0123] Thus, in the second stage of the method for generating MB NPCs (also referred to herein as "step (b)" or "stage 2"), the MB NSCs generated in step (a) are further cultured in a stage 2 optimized medium from day 4 to day 6, or starting from day 4 and continuing until day 6, or starting from day 4 and continuing for 72 hours (3 days), or starting from day 4 and continuing 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 starting from day 4 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours or 72 hours.

[0124] Thus, in the third stage of the method for generating immature midbrain neurons (also referred to herein as "step (c)" or "stage 3"), the MB NPCs generated in step (b) are further cultured in a stage 3 optimized medium from day 6 to day 9, or starting from day 6 and continuing until day 9, or starting from day 6 and continuing for 72 hours (3 days), or starting from day 6 and continuing 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 starting from day 6 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours or 72 hours.

[0125] Thus, in the fourth stage of the method for generating mature dopaminergic neurons (also referred to herein as "step (d)" or "stage 4"), the immature midbrain neurons generated in step (c) are further cultured in a stage 4 optimized medium from day 9 to day 23, or starting from day 9 and continuing until day 23, or starting from day 9 and continuing for a sufficient time to generate TH+KCNJ6+ mature dopaminergic neurons (e.g., cultured for 14 days or two weeks in a stage 4 medium).

[0126] IV. Use

[0127] The methods and compositions of the present disclosure for generating mature dopaminergic neurons, immature midbrain neurons, committed MB NSCs, and MBNPCs allow for the effective and robust availability of these cell populations for a variety of uses. For example, these methods and compositions can be used to study the development and biology of midbrain neural progenitor cells, including their differentiation into dopaminergic neurons, to assist in understanding and potentially treating 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 using agents that bind to surface markers expressed on the cells according to methods established in the art. Thus, in one embodiment, the present disclosure provides a method for isolating mature dopaminergic neurons or immature midbrain neurons, the method comprising:

[0128] contacting mature dopaminergic neurons or immature midbrain neurons generated by the methods of the present disclosure with at least one binding agent that binds to a cell surface marker expressed on the mature dopaminergic neurons or immature midbrain neurons; and isolating the cells that bind to the binding agent to thereby isolate mature dopaminergic neurons or immature midbrain neurons.

[0129] In one embodiment, the binding agent is an antibody, e.g., a monoclonal antibody (mAb) that binds to a cell surface marker. Cells that bind the antibody can be isolated by methods known in the art, including but not limited to fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS).

[0130] Progenitor cells of the midbrain dopaminergic neural lineage are also contemplated for treating neurological diseases and disorders by delivering the cells to a subject having a disease or disorder including but not limited to Parkinson's disease, which would benefit from enhanced dopaminergic neuron function.

[0131] The cells of the present disclosure can also be used to screen for potential drugs or develop novel cell therapies for treating diseases or disorders involving dysfunction of dopaminergic neurons.

[0132] V. Compositions

[0133] In other aspects, the present disclosure provides compositions related to the methods for generating mature dopaminergic neurons, immature midbrain neurons, committed MB NSCs, and MBNPCs, including culture media and cell cultures, as well as isolated progenitor cells and cell populations.

[0134] In one aspect, the present disclosure provides a culture medium for obtaining human-directed midbrain neural stem cells, which comprises a WNT pathway agonist, a SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist. In one embodiment, the culture medium lacks exogenously added growth factors.

[0135] In another aspect, the present disclosure provides a culture medium for obtaining human midbrain neural progenitor cells, which comprises a BMP pathway agonist, a RA pathway agonist, a LXR pathway agonist, an AKT pathway antagonist, an mTOR pathway antagonist, and a TGF-β pathway antagonist. In one embodiment, the culture medium lacks exogenously added growth factors.

[0136] In another aspect, the present invention provides a culture medium for obtaining human midbrain immature neurons, which comprises 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 culture medium lacks exogenously added growth factors.

[0137] In another aspect, the present disclosure provides a culture medium for obtaining human mature dopaminergic neurons, which comprises a BDNF pathway agonist, a GDNF pathway agonist, a PPAR-α pathway agonist, heparin or a heparin mimetic, a Notch pathway antagonist, and a dopamine agonist. In one embodiment, the culture medium lacks exogenously added growth factors.

[0138] In another aspect, the present disclosure provides an isolated cell culture of human-directed midbrain neural stem cells, the culture comprising: human OTX2+LMX1A+ directed midbrain neural stem cells cultured in a culture medium comprising a WNT pathway agonist, a SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist and lacking exogenously added growth factors.

[0139] In another aspect, the present disclosure provides an isolated cell culture of human midbrain neural progenitor cells, the culture comprising: human OTX2+FOXA2+LMX1A+ midbrain neural progenitor cells cultured in a culture medium comprising a BMP pathway agonist, a RA pathway agonist, a LXR pathway agonist, an AKT pathway antagonist, an mTOR pathway antagonist, and a TGF-β pathway antagonist and lacking exogenously added growth factors.

[0140] In another aspect, the present disclosure provides an isolated cell culture 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.

[0141] In another aspect, the present disclosure provides an isolated cell culture 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.

[0142] In another aspect, the present disclosure provides human OTX2+FOXA2+LMX1A+ midbrain neural progenitor cells generated by the methods of the present disclosure. In one embodiment, 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 the expression of GBX2 or have only low levels of GBX2 expression. In one embodiment, the present disclosure relates to an isolated cell population of human midbrain neural progenitor cells (NPCs) that comprises at least 1x 10 6 OTX2+FOXA2+LMX1A+ human midbrain NPCs, wherein the cell population lacks neural stem cells that express GBX2. In one embodiment of the isolated cell population, the human midbrain NPCs are bound to at least one antibody that binds to at least one marker expressed by the human midbrain NPCs.

[0143] In another aspect, the present disclosure provides human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons generated by the methods described herein. In another aspect, the present disclosure provides human TH+KCNJ6+ mature dopaminergic neurons generated by the methods described herein.

[0144] This application is further illustrated by the following examples, which should not be construed as further limitations. The content of the figures cited in this application and all references, patents, and published patent applications are hereby expressly incorporated herein by reference. Examples

[0145] Example 1 : Development of a culture protocol for generating stem cell-derived midbrain neural progenitor cells expressing FOXA2 and LMX1A

[0146] In this example, a two-stage culture protocol for generating midbrain-derived neural progenitor cells was developed, which can direct human pluripotent stem cells to become progenitor cells expressing FOXA2 and LMX1A after 6 days of culture. These cells can further differentiate into mature dopaminergic neurons.

[0147] This example adopted the high-dimensional experimental design (HD-DoE) method previously described in Bukys et al. (2020) Iscience 23:101346. This method uses computer-designed geometry to simultaneously test multiple process inputs and provides a mathematical model of the depth effect / response space. This method allows for finding the combined signal inputs that control complex processes such as the cell differentiation process. It allows testing multiple reasonable key process parameters because such parameters affect output responses such as gene expression. Since gene expression provides an iconic feature of the phenotype of, for example, human cells, this method can be used to identify and understand which signaling pathways control cell fate. In the current example, the purpose of applying the HD-DOE method is to directly find the conditions for inducing the expression of genes in midbrain neural progenitor cells from the pluripotent stem cell state.

[0148] To formulate the protocol for each stage, we tested and simulated the effects of agonists and antagonists (referred to herein as "effectors") of multiple signaling pathways on the expression of 53 preselected genes in two groups after 3 days of treatment. These effectors are small molecules or proteins commonly used in the process of stepwise differentiation of stem cells into specific fates. The selection of the effectors to be tested was based on the current literature on neural induction in the midbrain region of the developing brain and the differentiation of stem cells into neural progenitor cells.

[0149] To test the effectors, an experiment with at least 8 factors was designed, which can evaluate the cell's response to 48 or more different combinations of effectors within different concentration ranges. To analyze the model, we focused on the gene expression in the midbrain region, including the expression of OTX2, DMBX1, FOXA2, LMX1A, and the absence of the hindbrain marker GBX2. The effect of each effector on the gene expression level was defined by a parameter called factor contribution, which was calculated for each effector during the modeling process.

[0150] To identify the formulation for the first stage of differentiation, cells were treated with multiple effectors for 3 days, and the gene expression of the cells was modeled. Specifically, when optimizing the maximum expression of OTX2 at 12760.1, a model showed promising results in the upregulation of DMBX1, LMX1A, and OTX2 and the downregulation of GBX2. The model consisted of 13 factors, including LDN193189, PD173074, BLU9931, puromycin, SC79, MK2206, ZM336372, PD0325901, CHIR99021, XAV939, UCLA-gp130, tofacitinib, and GO 6983. Four effectors, the AKT signaling pathway antagonist MK2206, the MEK signaling pathway antagonist PD0325901, the WNT signaling pathway agonist CHIR99021, and the BMP signaling pathway antagonist LDN193189, had significant positive effects on the expression of the genes of interest, with factor contributions of 22.3, 18.1, 13.5, and 11.9( Figure 1 ).

[0151] Since FOXA2 was not upregulated with the optimization of OTX2, we next optimized the model for the maximum expression of FOXA2 at 1581. Three effectors with significant positive effects on FOXA2 expression were identified, including LDN193189, CHIR99021, and puromycin, with factor contributions of 13.6, 15.6, and 22.2( Figure 2 ). LDN193189 and CHIR99021 were common in both optimization settings, and the remaining factors other than puromycin had factor contributions of less than 10. Therefore, we focused on the effect of the addition of puromycin on the original 4 effectors.

[0152] This assessment was made through dynamic curve analysis of the model, focusing on the expression of OTX2, DMBX1, LMX1A, and FOXA2( Figure 3 ). Since puromycin had no positive effect on the expression of OTX2, DMBX1, and LMX1A in the previous settings, we expected a decrease in the expression levels of these genes. However, it was observed that their expression levels remained within the same range as the previous conditions, 3000, 450, and 11000 for DMBX1, LMX1A, and OTX2, respectively( Figure 4 ).

[0153] Table 1 below summarizes the effectors verified for the first stage of this protocol (generation of midbrain-directed neural stem cells):

[0154] Table 1: Effectors verified in the first stage of the protocol

[0155] Effector Function Concentration LDN193189 BMP antagonist 275 nM PD0325901 MEK antagonist 110 nM CHIR99021 WNT agonist 1.1 μM MK2206 AKT antagonist 138 nM Puramine SHH agonist 550 nM

[0156] To further guide the differentiation of midbrain-directed neural stem cells into neural progenitor cells at stage 2, we conducted additional HD-DoE experiments. From this, we obtained additional gene regulatory models for preparing the differentiation protocol. The above was based on a 12-factor HD-DoE experiment, focusing on initiating the differentiation of cells into midbrain neural progenitor cells within an additional 3 days after the end of the treatment at stage 1. Here, we focused on the expression of LMX1A and FOXA2 in neural progenitor cells with low or zero GBX2 expression. LMX1A had a significantly high expression level in the model, with a value of 47888. Therefore, we used 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, puromycinamine, and GSI-XX. When optimized for LMX1A, one factor, TTNPB (a small molecule agonist of the RA signaling pathway), had a significantly 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), while the positive factor contribution of AGN193109 was less than 1( Figure 5 ).

[0157] When the same experiment was optimized to achieve the maximum expression of FOXA2 at 33193, three effectors were identified to have a significantly positive effect on the expression of FOXA2, including TTNPB, A 83-01, and puromycinamine, with factor contributions of 10.7, 6.7, and 15.3 respectively( Figure 6 ).

[0158] Similar to the experimental model at stage 1, the analysis again showed that puromycinamine had a positive effect on the expression level of FOXA2 and a negative effect on the expression level of LMX1A, with a factor contribution of 26.2. The model also showed the same trend for A 83-01 and CHIR99021, having positive effects only on FOXA2 and LMX1A respectively. Therefore, we used dynamic curve analysis to adjust the formulation to optimize the expression of the LMX1A and FOXA2 genes and minimize the expression of GBX2( Figure 7)。It was observed that even without adding purine amine, the expression level of FOXA2 reached 5000. Therefore, including this effector is not necessary, but it can increase the expression of FOXA2. Based on the dynamic profile, it was also concluded that although CHIR99021 has a positive effect on LMX1A, it also increases the expression level of GBX2 and decreases the relative expression of FOXA2. Therefore, this factor was eliminated from the final formulation. A 83-01 is another factor included in the final formulation that has opposite effects on FOXA2 and LMX1A. Dynamic curve analysis showed that A 83-01 added at a medium concentration (300 nM) can increase the expression of FOXA2 and help reduce the level of GBX2 to almost 0.

[0159] To test other factors commonly used in the midbrain differentiation protocol (such as FGF8), we conducted another 12-factor experiment, which consisted of LDN193189, BMP7, A 83-01, activin A, Takinib, PD0325901, MK2206, FGF8b, AZD3147, MHY1485, GSI-XX, and Yhhu 3792. Similar to the previous experiment, hiPSCs were treated with stage 1 medium for 3 days and then with 96 conditions of factor combinations for another 3 days. When the model was optimized for the maximum expression of FOXA2, BMP7 with a factor contribution of 13.7 and MK2206 with a factor contribution of 14.2 had the greatest impact on its expression, followed by AZD 3147 with a factor contribution of 10.9. Yhhu 3792 and Takinib also had positive effects, but the factor contributions were less than 10. Surprisingly, FGF8 had a negative effect, with a factor contribution of 12.8( Figure 8 )。

[0160] The model was also optimized for the maximum expression of LMX1A, and MK2206 with a factor contribution of 12 had the highest positive impact. AZD 3147, GSI-XX, activin A, and Takinib also had positive effects on its expression, but the factor contributions were all less than 10( Figure 9 )。The model showed that Yhhu 3792 had a negative effect on the expression of LMX1A, with a factor contribution of 11.7, which was the opposite of the case of FOXA2. Another difference was BMP7, which had a negative effect on LMX1A. However, the factor contribution was less than 10. Therefore, to evaluate the effects of the interactions between factors and the optimal conditions for the expression of FOXA2 and LMX1A, we used dynamic curve analysis( Figure 10 )。

[0161] Using dynamic curve analysis, we excluded GSI-XX, activin A, and Takinib because they did not produce a meaningful positive change in the levels of both FOXA2 and LMX1A expression. Yhhu 3792 was also removed because it had a substantial negative impact on LMX1A while having a positive impact on GBX2. BMP7 and AZD 3147 had significant positive effects on FOXA2 and LMX1A respectively, and they did not reduce the expression of the other gene, so they were included in the final formulation. It was also shown that although MK2206 reduced the level of LMX1A, it had a promising effect on FOXA2 and GBX2, so it was included in the final formulation at a moderate level.

[0162] Table 2 below summarizes the effectors verified for the second stage of this protocol (generation of midbrain-derived neural progenitor cells):

[0163] Table 2: Effectors verified in the second stage of the protocol

[0164]

[0165]

[0166] Considering the two models, the conditions for maximizing the differentiation of cells into the midbrain region with neural progenitor cell identity were associated with strong and elevated expression of OTX2, FOXA2, and LMX1A, including the following effector inputs: TTNPB, BMP7, A 83-01, GW3965, AZD 3147, and MK2206.

[0167] The criticality of each individually verified effector for the stage 1 and stage 2 protocols was further evaluated as described in Example 2.

[0168] Example 2 : Factor criticality analysis of the induction culture conditions for stem cell-derived midbrain neural progenitor cells

[0169] To evaluate the impact of eliminating each verified factor, we used dynamic curve analysis and compared the expression levels of the genes of interest in the absence of each finally determined factor but in the presence of the other factors. Since the expression levels of the genes of interest revealed whether the desired results could be achieved, this factor criticality analysis revealed the degree of importance of each input effector.

[0170] In the stage 1 formulation, each of the five finally determined factors was removed while the other four factors were present, and the expression levels of OTX2, DMBX1, FOXA2, and LMX1A were evaluated and compared with the levels when all five factors were present ( Figure 11A-B). When MK2206 was removed, the values of OTX2 and DMBX1 decreased from 12,000 and 3,000 to 9,500 and 1,500 respectively, while FOXA2 and LMX1A remained unchanged. The absence of PD0325901 led to a decrease in DMBX1 expression and it 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, however, the values of OTX2, FOXA2 and LMX1A decreased. After removing CHIR99021, the values of all genes of interest decreased, which further demonstrated its importance in the stage 1 formulation. As expected, the absence of purine amine led to a decrease in FOXA2, while it was beneficial to other genes.

[0171] In the stage 2 formulation, each of the six finalized factors was removed while the other five factors were present, and the expression levels of FOXA2, LMX1A and GBX2 were evaluated in comparison with the levels when all factors were present. According to the first experimental model, the absence of TTNPB led to an increase in GBX2 expression, while the values of FOXA2 and LMX1A decreased dramatically from 10,000 to 0 and from 30,000 to 15,000 respectively. The absence of A 83-01 led to a decrease in FOXA2 expression from 10,000 to 7,000. However, as expected, the value of LMX1A increased from 30,000 to 40,000. The deletion of GW3965 dramatically decreased the value of LMX1A from 30,000 to 10,000 and increased the value of FOXA2 to 17,000( Figure 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, with BMP7 as the main effector leading to 0 expression of FOXA2. 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( Figure 13A -B), and thus these factors were added to the final formulation.

[0172] Example 3 : Immunocytochemical verification of FOXA2 and LMX1A expression in stem cell-derived midbrain neural progenitor cells

[0173] To further validate the developed culture protocol as described in Example 1, cells were treated with stage 1 and stage 2 differentiation media, and immunocytochemistry was used to evaluate the expression of biomarkers in the midbrain region and neural progenitor cells at the end of each stage. The biomarkers tested included: OTX2, a midbrain marker involved in midbrain positioning and maintenance of the mid-hindbrain boundary (Vernay et al. (2005) J. Neurosci. 25:4856-4867); LMX1A, which is involved in the generation and differentiation of midbrain dopaminergic progenitor cells (Yan et al. (2011) J. Neurosci. 31:12413-12425); FOXA2, which regulates the generation of midbrain dopaminergic neurons in early and late stages of development (Ferri et al. (2007) Development 134:2761-2769); PAX2, which is expressed in the midbrain and fore- and hindbrains (Urbanek et al. (1997) Proc. Natl. Acad. Sci. USA 94:5703-5708); nestin, which is an early neuronal marker; KI67, which is a proliferation marker; and GBX2, which is a hindbrain marker.

[0174] Immunocytochemistry images confirmed that at the end of treatment with stage 1 media, more than 90% of the cells expressed OTX2 and LMX1A. Some expression of the biomarkers PAX2, nestin, and KI67, and some expression of GBX2 were observed in some cells. However, FOXA2 was not expressed ( Figure 14 ). After treatment with stage 2 media, we observed that the expression of LMX1A and OTX2 was maintained, while the expression of FOXA2 increased significantly, and FOXA2 was detected in more than 90% of the cells. By the end of stage 2, GBX2 expression was also almost eliminated ( Figure 15 ). The detection of OTX2, LMX1A, and FOXA2, and the non-detection of GBX2 at the end of the 2 stages of differentiation confirmed the differentiation of human induced pluripotent stem cells into midbrain neural progenitor cells after 6 days of treatment with the stage 1 and stage 2 formulations.

[0175] Example 4 : RNA-seq validation of LMX1A and FOXA2 expression in stem cell-derived midbrain neural progenitor cells

[0176] RNA sequencing was used to obtain the gene profiles of cultured cells in the candidate formulations. Human iPSCs were cultured in stage 1 and stage 2 media for a total of 6 days, and RNA sequencing of the generated cells was performed at the end of each stage. 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, S0X1, S0X2, vimentin), and stem cell state (NANOG, P0U5F1) in triplicates at day 0, day 3, and day 6. As Figure 17A and Figure 17B shown, the stem cell gene levels decreased in neural progenitor cells, while the levels of neuron genes derived from the midbrain region increased; this verified the differentiation of hiPSCs into neural lineages with midbrain identity. Figure 17A shows the fold change of 19 selected genes after treatment with stage 1 medium for 3 days compared to stage 0; compared to hiPSCs, FOXA2, LMX1A, and SOX1 had the highest positive differential expression levels (10.6, 9.5, and 9.1, respectively). The sternness genes NANOG and POU5F1 and the hindbrain gene GBX2 were 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 had the highest positive differential expression, 11.3, 10, and 7.7, respectively. Similar to stage 1, the lowest differential expression of NANOG, POUF51, and GBX2 was observed. A heatmap of the scaled gene profiles of 18 selected genes in hiPSCs at day 0 and MB neural progenitor cells at day 6 ( Figure 17C ) shows that the expression of midbrain progenitor cell genes including DDC, LMX1A / B, SOX6, NEUROG2, FOXA2, and EN1 increased after treatment with stage 1 and stage 2 media. The expression level of the gene GFAP expressed in glial cells was also observed and remained unchanged during the 6-day differentiation, indicating that the culture was mainly neuronal. These data demonstrate the ability of the stage 1 and stage 2 formulations to direct cell differentiation into midbrain neuron identity as a staged differentiation medium.

[0177] Example 5 : Development of a culture protocol for generating dopaminergic neurons expressing TH and KCNJ6

[0178] After treating cells with 1-stage and 2-stage media to generate MB neural progenitor cells, as described in Examples 1-4, according to the culture protocol developed in this example, the MB neural progenitor cells were treated with 3-stage media for 3 days and then with 4-stage media for 14 days to differentiate them into dopaminergic neurons expressing TH and KCNJ6.

[0179] To develop a neuronal differentiation protocol, the effects of various agonists and antagonists (effectors) on the maturation of MB neural progenitor cells were studied using the HD-DoE method described in Example 1. These effectors were selected based on the available literature on developmental biology and stem cell differentiation at that time, as well as mouse and human single-cell RNA-seq data from midbrain neurons.

[0180] As further described below, these experiments led to the generation of a 3-stage protocol shown in Table 3 below and a 4-stage protocol shown in Table 4 below.

[0181] Table 3 . Factors validated in the 3-stage protocol

[0182] Effector Function Concentration BMP7 BMP agonist 15 ng / ml AGN193109 RAR antagonist 100 nM CHIR99021 WNT agonist 1 μM PD0325901 MEK antagonist 100 nM AZD3147 mTOR antagonist 15 nM DBZ Notch antagonist 100 nM

[0183] Table 4 . Factors validated in the 4-stage protocol

[0184]

[0185]

[0186] To design a protocol for manufacturing the 3-stage of differentiation, cells were first cultured in the 1-stage and 2-stage media described herein and then treated with combinations of 8 or 12 factors for 3 days, and the gene expression of cells in each condition was modeled. To direct the cells to differentiate into the midbrain dopaminergic neuron subtype located in the substantia nigra pars compacta (SNc) region of the brain, the neural progenitor cells need to be SOX6 +(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 additional markers involved in the neurogenesis of dopaminergic neurons originating from the developing midbrain floor plate region (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.575:403-410).

[0187] Therefore, we focused on maximizing the expression of SOX6, CORIN, NGN2, and MSX1 in the modeling experiments. One of the 12-factor models (the results of which are as Figure 19 shown) included LDN193189, AGN193109, BMP7, TTNPB, PD0325901, A8301, MHY1485, CHIR99021, XAV939, SANT-1, puromycin, and MK2206, providing a set of combinations that could maximize the expression level of CORIN 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 high 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 expression level of CORIN, with factor contributions of 13.5, 10.9, and 9.9, respectively. Within the specification range of achieving 80% of the maximum expression of CORIN, the Cpk value (process capability index) of this complex medium composition was 0.54, corresponding to a failure risk of 4.9%.

[0188] When the same model was optimized to achieve the maximum expression of MSX1 at 179, the factor with the highest positive effect was BMP7 at 11.1( Figure 20)。The negative regulatory effects of LDN193189, the RA agonist TTNPB, and XAV939 on its expression were the highest, being 19.9, 15.5, and 11.2 respectively. Within the specification of achieving 80% of the maximum expression of MSX1, the Cpk value (process capability index) of this complex medium composition was 0.62, corresponding to a failure risk of 3.1%.

[0189] This model was also optimized for the maximum expression of SOX6 at 296, which led to the identification of two factors, AGN193109 and PD0325901, that showed significant positive effects on its expression, with factor contributions of 26.1 and 13.5 ( 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 specification of achieving 80% of the maximum expression of SOX6, the Cpk value (process capability index) of this complex medium composition was 0.9, corresponding to a failure risk of 0.44%.

[0190] This 12-factor model was the first to result in an increase in the expression level of KCNJ6 in a differentiation experiment. KCNJ6, a G protein-activated potassium channel, is a terminal differentiation marker expressed by all human SNc and some VTA dopaminergic neurons (Reyes et al. (2012) J. Comp. Neurol. 520:2591 - 607). Therefore, we also optimized the model to achieve the maximum expression of KCNJ6 at 47. The positive regulators of this gene were identified as AGN193109 at 17, PD0325901 at 10.9, CHIR99021 at 10.7, and BMP7 and DBZ with low factor contributions ( Figure 22 )。

[0191] Next, we used dynamic curve analysis to find a set of combinations that could optimize the expression of all four genes. Among all the effectors that showed high positive factor contributions to the selected genes, including AGN193109, PD0325901, BMP7, LDN193189, and CHIR99021, one factor, LDN193189, had to be excluded because it had a relatively high negative effect on the expression level of MSX1 ( Figure 23 )。

[0192] In another 12-factor model, other factors including activin A, Takinib (TAK inhibitor), FGF8b, AZD3147 (mTOR antagonist), MHY1485 (mTOR agonist), and Yhhu-3792 (Notch agonist) were tested. Some factors from the previous model (BMP7, A 8301, LDN193189, PD0325901, MK2206, and DBZ) were also included. When optimized for SOX6 at 250, the two new factors DBZ and AZD3147 had the highest positive impact, with factor contributions of 21.5 and 20.2( Figure 24 ). Within the specification of achieving 80% of the maximum expression of SOX6, the Cpk value (process capability index) of this complex medium composition was 0.60, corresponding to a failure risk of 3.6%.

[0193] Since the expression level of CORIN in this model was significantly lower (20 compared to 2000), we did not optimize the model to achieve its maximum expression. However, we used dynamic curve analysis to analyze the effect of these inputs on the expression of MSX1( Figure 25 ). According to this model, activin A and Takinib, which have a positive effect on SOX6, have a negative regulatory effect on MSX1, so they were excluded from the final formulation. AZD3147 and DBZ did not show any significant effect on the expression level of MSX1.

[0194] We also observed the positive regulatory effect of DBZ and AZD3147 on the genes of interest in the 8-factor model, further confirming the analysis results of the previous model( Figure 26 ).

[0195] Therefore, considering these models, a 3-stage candidate formulation consisting of BMP7, PD0325901, AGN193109, CHIR99021, AZD3147, and DBZ was developed. This formulation was selected to maximize cell differentiation, which is related to the strong and elevated expression of SOX6, KCNJ6, CORIN, and MSX1. This formulation was further verified by immunocytochemistry assay (see Example 7).

[0196] To further differentiate the cells into a mature neuronal identity, additional HD-DoE experiments were performed on cells cultured in the differentiation media of stage 1, stage 2, and stage 3. After 4 days, the gene expression of the cells under different combined conditions was studied. At this time, we focused on the maximum expression of more mature dopaminergic genes such as NR4A2, PITX3, and TH (Tiklová et al. (2020) Nat Commun. 11:2434; Fiorenzano et al. (2021) Nat Commun. 12:7302). In one model, the cells were treated with a combined matrix with eight inputs, including CHIR99021, BDNF, GDNF, Rosiglitazone, GW7647, DBZ, and heparin. When the maximum expression level of NR4A2 reached 337.8, six factors showed a positive regulatory effect on its expression, including BDNF (the highest factor contribution was 17), dopamine, DBZ, GW7647, GDNF, and heparin( Figure 27 ). While CHIR99021 and Rosiglitazone had a significant negative impact on NR4A2, with factor contributions of 20.7 and 13.6, respectively. Within the specification of reaching 80% of the maximum expression of NR4A2, the Cpk value (process capability index) of this complex culture medium composition was 0.35, corresponding to a failure risk of 8.9%.

[0197] The effect of the compounds on the expression level of PITX3 was studied using dynamic curve analysis, and a similar trend of the two genes was observed, except for CHIR99021, which had no significant effect on the expression level of P1TX3( Figure 28 ).

[0198] Considering this model, the conditions for maximizing the differentiation of cells into the SNc region with the identity of dopaminergic neuronal cells are related to the strong and elevated expression of NR4A2 and PITX3, including the following effector inputs: BDNF, GDNF, dopamine, GW7647, DBZ, and heparin. This stage 4 formulation was further verified by immunocytochemistry assay (see Example 7).

[0199] Example 6: Factor Criticality Analysis of the Culture Conditions for Inducing Dopaminergic Neurons

[0200] To evaluate the effect of eliminating each of the verified factors identified in Example 5 on the stage 3 and stage 4 formulations, we used dynamic curve analysis and compared the expression levels of the genes of interest in the absence of each finally determined factor while other factors were present. Since the expression levels of the genes of interest reveal whether the desired results can be achieved, this factor criticality analysis reveals the degree of importance of each input effector.

[0201] In the 3-stage formulation, each finalized factor was removed from its respective model while the other factors were present, and the expression levels of the genes of interest were evaluated compared to the presence of all factors. The results are as Figure 29A and Figure 29B shown. When AGN193109 was excluded, the levels of all CORIN, KCNJ6, MSX1, and SOX6 decreased from 900 to 450, from 50 to 35, from 140 to 110, and from 105 to 60, respectively. When BMP7 was removed, the expression levels of CORIN and MSX1 decreased to 500 and 100, whereas 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 was the highest, and even with the negative regulatory effect brought by PD0325901, it still remained at a significant high level (900), while if PD0325901 was removed, the expression level of SOX6 decreased significantly from 105 to 20. Therefore, it was decided to add this compound to the final formulation. CHIR99021 had a similar effect on CORIN, but its absence significantly affected MSX1 in a negative manner. Therefore, CHIR99021 was also included in the final formulation.

[0202] In another model, to determine the combined effects of AZD3147 and DBZ on the cellular gene profile in the presence of the other factors in the final formulation, the expression levels of SOX6 and MSX1 were compared in the absence of each factor in the presence of PD0325901 and BMP7. These two compounds were the only final compounds in the input of this experimental model, and the results are as Figure 30A and Figure 30B shown. 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 SOX6 and MSX1 decreased to 140 and 35, respectively.

[0203] As a validation analysis, we also analyzed the expression levels of these two genes in the absence of PD0325901 and confirmed that it led to a decrease in the expression of S0X6 from 185 to 165 and a decrease in the expression of MSX1 from 45 to 40.

[0204] In the 4-stage formulation, each of the six finalized factors was excluded while the other factors were present, and the expression levels of NR4A2 and PITX3 were evaluated in comparison to the presence of all factors. The results are as Figure 31A and Figure 31B shown. In the absence of BDNF, the expression of PITX3 and NR4A2 decreased from 40 to 10 and from 400 to 100, respectively. When dopamine was excluded from the formulation, the expression level of NR4A2 almost reached 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 NR4A2 level decreased again to 200, and PITX3 reached 30. A similar trend was observed when GW7647 was excluded, resulting in lower expression levels of both genes. Only in the absence of heparin, the expression level of NR4A2 did not change significantly, but still led to a decrease in the expression of PITX3 to 30.

[0205] Factor criticality analysis demonstrated the importance of adding each compound to the 3-stage and 4-stage differentiation medium formulations.

[0206] Example 7: Immunocytochemical Verification of Midbrain Dopaminergic Neurons Expressing TH and KCNJ6

[0207] To further verify the formulation developed in Example 5, cells were first treated with the 1-stage and 2-stage differentiation media in Example 1, then treated with the 3-stage differentiation medium for 3 days each and the 4-stage medium for 14 days, and then standard immunocytochemical assays were used to evaluate the expression of biomarkers of immature neurons in the ventral midbrain region at the end of the 3-stage and biomarkers of mature neurons at the end of the 4-stage. Biomarkers include midbrain neuron-specific markers such as SOX6, ALDH1A1, MSX1, TH, NURR1 (NR4A2), PITX3, and KCNJ6, and mature pan-neuronal markers such as TUBB3, MAP2, neurofilament (NF), and SYN1.

[0208] Immunocytochemical images confirmed that at the end of the 3-stage, SOX6 and MSXI were expressed in more than 90% of the cells ( Figure 32 ). We also detected the initial expression of the neuronal marker β-tubulin III (TUBB3), the immature neuron marker Doublecortin (DCX), and the post-mitotic dopaminergic neurons PITX3 and NURR1, as early as day 9 in vitro.

[0209] Images of mature neurons at the end of stage 4 confirmed that the cells in culture expressed mature dopaminergic markers such as KCNJ6 and TH. We also observed that most cells in culture expressed mature pan-neuronal markers, including MAP2, NF, and Synl( Figure 33 ). We performed competitive fate mapping of dopaminergic neuronal cells in the ventral tegmental area (VTA) expressing CALB1 (Brignani et al. (2017) Front. Neuroanat. 11:55) and detected less than 10% of the cells expressing it. Expression of NURR1 and ALDH1A1 was observed in most cells at this time.

[0210] TH and KCNJ6 were detected at the end of stage 4 of differentiation, while CALB1 was not detected, confirming the robustness and high conversion potential of the staged formulation described herein for the differentiation of human induced pluripotent stem cells into SNc dopaminergic neurons after 23 days in vitro.

[0211] Equivalent

[0212] 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 present application described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for generating human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons, which comprises: culturing human OTX2+FOXA2+LMX1A+ midbrain neural progenitor cells (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 for 3 days (day 0 - day 3) to obtain human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons (MB-immature neurons).

2. The method according to claim 1, which further comprises: culturing the 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 for at least 14 days to obtain TH+KCNJ6+ mature dopaminergic neurons.

3. A method for generating human TH+KCNJ6+ mature dopaminergic neurons, which comprises: (a) culturing human OTX2+FOXA2+LMX1A+ midbrain neural progenitor cells (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 for 3 days (day 0 - 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 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 at least 14 days (day 3 - day 17) to obtain TH+KCNJ6+ mature dopaminergic neurons.

4. A method for generating human TH+KCNJ6+ mature dopaminergic neurons, which comprises: (a) culturing human pluripotent stem cells in a medium containing a WNT pathway agonist, an SHH pathway agonist, a BMP pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist for 0 - 3 days to obtain directed midbrain neural stem cells (MB NSCs); (b) culturing the MB NSCs in a medium containing a BMP pathway agonist, an RA pathway agonist, an LXR pathway agonist, an AKT pathway antagonist, an mTOR pathway antagonist, and a TGFβ pathway antagonist for 4 - 6 days to obtain human OTX2+FOXA2+LMX1A+ midbrain neural progenitor cells (MB NPCs); (c) Culturing the 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 for 6 - 9 days to obtain human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons (MB-immature neurons); and (d) Culturing the 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 for 9 - 23 days to obtain TH+KCNJ6+ mature dopaminergic neurons.

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

6. The method according to claim 4, wherein the human pluripotent stem cells are embryonic stem cells.

7. The method according to any one of claims 1 - 6, wherein the WNT pathway agonist is selected from the group consisting of: CHIR99021, CHIR98014, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A014418, BIO, BIO-acetoxime, AZD1080, WNT3A, atiprimod, indirubin-3'-oxime, 1-azakenpaullone, kenpaullone, TC-G 24, TDZD 8, TWS119, NP 031112, AT 7519, KY 19382, AZD2858, and combinations thereof.

8. The method according to claim 7, wherein the WNT pathway agonist is present in the medium at a concentration of 0.5 - 2.0 μM.

9. The method according to claim 7, wherein the WNT pathway agonist is CHIR99021, which is present in the medium at a concentration of 1.0 - 1.1 μM.

10. The method according to any one of claims 1 - 6, wherein the mTOR pathway antagonist is selected from the group consisting of: AZD3147, rapamycin, sirolimus, temsirolimus, everolimus, deforolimus, biolimus, zotarolimus, torin-1, torin-2, vistusertib, MHY1485, AZD8055, datolisib, 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, tofacitinib, sapitinib, volasertib, and combinations thereof.

11. The method according to claim 10, wherein the mTOR pathway antagonist is present in the medium at a concentration of 10 - 30 nM.

12. The method according to claim 10, wherein the mTOR pathway antagonist is AZD3147, which is present in the culture medium at a concentration of 15 nM.

13. The method according to any one of claims 1-6, wherein the RAR pathway antagonist is selected from the group consisting of: AGN193109, BMS195614, CD 2665, ER 50891, LE 135, LY 2955303, MM11253, and combinations thereof.

14. The method according to claim 13, wherein the RAR pathway antagonist is present in the culture medium at a concentration of 50-250 nM.

15. The method according to claim 13, wherein the RAR pathway antagonist is AGN193109, which is present in the culture medium at a concentration of 100 nM.

16. The method according to any one of claims 1-6, wherein 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, RO5126766, BIX 02189, pimasertib, TAK 733, AZD8330, PD318088, SL 327, GDC 0623, RO5126766, myricetin, and combinations thereof.

17. The method according to claim 16, wherein the MEK pathway antagonist is present in the culture medium at a concentration of 50-250 nM.

18. The method according to claim 16, wherein the MEK pathway antagonist is PD0325901, which is present in the culture medium at a concentration of 100-110 nM.

19. The method according to any one of claims 1-6, wherein the Notch pathway antagonist is selected from the group consisting of: DBZ, celecoxib, bexarotene, BMS299897, Compound E, DAPT, JLK6, L-685,458, LY 450139, MRK 560, PF3084014 hydrobromide, LY 3039478, LY 411575, RO 4929097, and combinations thereof.

20. The method according to claim 19, wherein the Notch pathway antagonist is present in the culture medium at a concentration of 50-250 nM.

21. The method according to claim 19, wherein the Notch pathway antagonist is DBZ, which is present in the culture medium at a concentration of 100 nM.

22. The method according to any one of claims 1-6, wherein the BMP pathway agonist is selected from the group consisting of: BMP, sb4, endomorphin, and combinations thereof.

23. The method according to claim 22, wherein the BMP pathway agonist is present in the culture medium at a concentration of 5-50 ng / ml.

24. The method according to claim 22, wherein the BMP pathway agonist is BMP7, which is present in the culture medium at a concentration of 10-15 ng / ml.

25. The method according to any one of claims 2-6, wherein the BDNF pathway agonist is selected from the group consisting of: BDNF, rotigotine, 7,8-DHF, ketamine, tricyclic dimer peptide-6 (TDP6), LM22A-4, and combinations thereof.

26. The method according to claim 25, wherein the BDNF pathway agonist is present in the culture medium at a concentration of 5-50 ng / ml.

27. The method according to claim 25, wherein the BDNF pathway agonist is BDNF, which is present in the culture medium at a concentration of 10 ng / ml.

28. The method according to any one of claims 2-6, wherein the GDNF pathway agonist is selected from the group consisting of: GDNF, BT13, BT44, and combinations thereof.

29. The method according to claim 28, wherein the GDNF pathway agonist is present in the culture medium at a concentration of 5-50 ng / ml.

30. The method according to claim 28, wherein the GDNF pathway agonist is GDNF, which is present in the culture medium at a concentration of 10 ng / ml.

31. The method according to any one of claims 2-6, wherein the PPAR-α pathway agonist is selected from the group consisting of: GW7647, fenofibrate, fenofibrate-d6, WY 14643, CP 775146, CP 868388 free base, tigliptin, oleoylethanolamide, oleoylethanolamide-d2, oleoylethanolamide-d4, PPAR agonist 1, clofibrate, clofibrate-d4, wisterin, englitazide, troglitazone, GW0742, bezafibrate, bezafibrate-d4, ciglitazone, BMS 687453, ranilazine, saroglitazar, saroglitazar magnesium, saroglitazar-d5, iglitazone, AVE-8143, GW 590735, ertiprotafib, LJ570, sladenafil sodium, aleglitazar, moglitazar, roglitazar, GW 9578, MHY 908, KRP-297, elafibranor, aleglitazar, AM3102, eupatilin, clofibric acid, clofibric acid-d4, nateglitazar, nateglitazar racemate, and combinations thereof.

32. The method according to claim 31, wherein the PPAR-α pathway agonist is present in the culture medium at a concentration of 200-300 nM.

33. The method according to claim 31, wherein the PPAR-α pathway agonist is GW7647, which is present in the culture medium at a concentration of 250 nM.

34. The method according to any one of claims 2-6, wherein the heparin or heparin mimetic is selected from the group consisting of: heparin, heparan sulfate, enoxaparin, low molecular weight heparin, AV5026, M402, and combinations thereof.

35. The method according to claim 34, wherein the heparin or heparin mimetic is present in the culture medium at a concentration of 2-8 μg / ml.

36. The method according to claim 34, wherein the culture medium contains heparin, which is present in the culture medium at a concentration of 5 μg / ml.

37. The method according to any one of claims 2-6, wherein the dopamine agonist is selected from the group consisting of: dopamine, dopamine hydrochloride, (R)-(-)-apomorphine hydrochloride, bromocriptine mesylate, bromocriptine-13C,d3, CNV dopamine, dihydroergotamine mesylate, ergoloid mesylate, ergoloid mesylate maleate, mesulergine hydrochloride, piribedil dihydrochloride, piribedil, quinelorane hydrochloride, (-)-quinpirole hydrochloride, ropinirole, Tau-aggregation-IN-1, NMI 8739, U91356, carbidopa phosphate, quinagolide hydrochloride, pramipexole dihydrochloride, PD-168077 maleate, rotigotine hydrochloride, PF592379, rotigotine, (Rac)-rotigotine hydrochloride, (Rac)-rotigotine-d7 hydrochloride, Ro 10-5824 dihydrochloride, (+)-dihydroxytryptamine hydrochloride, talipexole, PF2562, neuromedin N, A68930, A68930 hydrochloride, A77636 dihydrochloride, PD 119819, PD 128907 hydrochloride, CY 208-243, pramipexole, pramipexole-d3 dihydrochloride, pramipexole-d7 dihydrochloride, SKF 38393 hydrochloride, SKF 38393 hydrobromide, SKF 82958, ABT 670, ABT-724, ABT-724 trihydrochloride, (R)-PF-06256142, talipexole dihydrochloride, R-proclorperazine, padovanol hydrochloride, padovanol, pergolide mesylate, BP897, BP897 hydrochloride, LY 3154207, talapodone, rocindole, UNC994, cabergoline, brexpiprazole, pergolide mesylate-d7, cabergoline-d6, rocindole hydrochloride, WAY-100635 maleate, sibutramine hydrochloride, dihydroxytryptamine, dihydroxytryptamine hydrochloride, biphenylalunol, OS-3-106, brexanolone, pramipexole dihydrochloride hydrate, pramipexole, pramipexole dihydrochloride, cabergoline-d5, perospirone, brexpiprazole-d8, (Rac)-talapodone, ML417, brexpiprazole S-oxide, pramipexole-d7 dihydrochloride, pramipexole-d5 dihydrochloride, UCSF924, WAY-100635, SKF83959, pramipexole (N-propyl-3,3,3-d3)(dihydrochloride), sarizotan, brexpiprazole S-oxide D8, SKF 83959 hydrobromide, and combinations thereof.

38. The method according to claim 37, wherein the dopamine agonist is present in the culture medium at a concentration of 5-15 μM.

39. The method according to claim 37, wherein the dopamine agonist is dopamine, which is present in the culture medium at a concentration of 10 μM.

40. A culture medium for obtaining immature neurons of the human midbrain, 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.

41. A culture medium for obtaining mature dopaminergic neurons of the human, 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.

42. An isolated cell culture of immature neurons of the human midbrain, the culture comprising: human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons cultured in a culture 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.

43. An isolated cell culture of mature dopaminergic neurons of the human, the culture comprising: human TH+KCNJ6+ mature dopaminergic neurons cultured in a culture 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.

44. Human FOXA2+LMX1A+MSX1+PITX3+DCX+ immature midbrain neurons produced by the method according to claim 1.

45. Human TH+KCNJ6+ mature dopaminergic neurons produced by the method according to any one of claims 2-4.

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