Method for generating midbrain dopamine neurons, midbrain neurons and their use

Abstract

The present disclosure provides methods for generating midbrain dopamine neurons and their precursors, midbrain dopamine neurons and their precursors generated by such methods, compositions comprising such cells, and uses thereof for preventing, modeling, and / or treating neurological disorders. In certain embodiments, contacting the cells with at least one inhibitor of Wnt signaling begins at least about 5 days after initial contacting of the stem cells with at least one inhibitor of SMAD signaling.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 004,138, filed on 2 April 2020, which is incorporated herein by reference in its entirety and whose priority is claimed.

[0002] 1. Introduction This disclosure provides methods for generating midbrain dopamine (mDA) neurons and their precursors, mDA neurons and their precursors generated by such methods, and compositions comprising such cells. This disclosure also provides the use of mDA neurons and compositions comprising them for the prevention, modeling, and / or treatment of neurological disorders. [Background technology]

[0003] 2.Background Parkinson's disease (PD) is characterized by the loss of mDA neurons, which result in well-known motor symptoms such as tremor, rigidity, and bradykinesia (Lees, et al. Lancet 373, 2055-2066 (2009)). While other cell types such as enteric neurons, olfactory neurons, or cortical neurons are also affected (Del Tredici, et al. Neuropathol Appl Neurobiol 42,33-50 (2016)), mDA neurons are particularly relevant to the development of novel cell-based treatments (Barker, et al. Nature reviews Neurology 11,492-503 (2015); Tabar, et al. Nat Rev Genet 15,82-92 (2014)) and PD disease modeling (Sanchez-Danes, et al. EMBO Mol Med 4,380-395 (2012); Miller, et al. Cell stem cell 13,691-705 (2013); Chung, et al. Stem Cell Reports 7,664-677 (2016); Reinhardt, et al. Cell stem cell 12,354-367 (2013); Chung, et al. al. Science 342,983-987(2013); Cooper, et al. Sci Transl Med 4,141ra190(2012)) remains a key focus. Human pluripotent stem cells (hPSCs), including both human ES cells and iPS cells, have become the cell type of choice for obtaining mDA neurons in vitro. Despite advances in human mDA induction, novel protocols are needed. For cell therapy, there is still no clear consensus on the optimal type and stage of mDA neurons to be used, and considerable molecular and functional differences in the behavior of hPSC-derived DA neurons compared to primary fetal DA neurons have been reported in vitro (La Manno, et al. Cell 167,566-580 e519(2016)) and in vivo (Tiklova, et al. Nature communications 10,581(2019)).Furthermore, there is no reliable cell purification strategy, and the cell viability of hPSC-derived mDA neurons remains low (approximately 10% of transplanted cells) (Sanchez-Danes, et al. EMBO Mol Med 4, 380-395 (2012)). Low mDA viability can lead to variability in clinical cell administration, potentially complicating the routine application of this technology to the broader PD community. These issues are significant for both cell therapy and disease modeling applications.

[0004] In human disease modeling, variability in mDA neuron yield and purity across hPSC strains introduces noise into the detection of disease-related phenotypes, complicating drug discovery efforts. Access to defined mDA neuron subtypes would enable research into cell type-specific susceptibility mechanisms in PD (Surmeier, et al. Cold Spring Harb Perspect Med 2, a009290 (2012); Anderegg, et al. FEBS Lett 589, 3714-3726 (2015); Chung, et al. Hum Mol Genet 14, 1709-1725 (2005); Brichta and Greengard. Front Neuroanat 8, 152 (2014)). Access to defined, more robust mDA neuron cultures and the potential to drive mDA neuron subtypes could significantly accelerate efforts in PD disease modeling and enable improved products for future mDA neuron cell therapies. Therefore, improved methods are still needed to generate mDA neurons that exhibit improved in vivo survival and are suitable for treating neurological disorders such as Parkinson's disease. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Lees,et al.Lancet 373,2055-2066(2009) [Non-Patent Document 2] Del Tredici, et al. Neuropathol Appl Neurobiol 42,33-50(2016) [Non-licensed document 3] Barker, et al. Nature reviews Neurology 11,492-503(2015)

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[0006] 3. Outline of the present invention This disclosure provides methods for generating mDA neurons and their precursors, mDA neurons and their precursors generated by such methods, compositions comprising such cells, and uses of such cells and compositions for preventing and / or treating neurological disorders.

[0007] This disclosure provides an in vitro method for inducing the differentiation of stem cells. In certain embodiments, the method includes the steps of contacting stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator, and contacting cells with at least one fibroblast growth factor (FGF) signaling activator and at least one Wnt signaling inhibitor to obtain a population of differentiated cells expressing at least one marker indicating midbrain dopamine neurons (mDA) or their precursors.

[0008] In certain embodiments, contact between cells and at least one Wnt signaling inhibitor is initiated at least about 5 days after initial contact between stem cells and at least one SMAD signaling inhibitor. In certain embodiments, contact between cells and at least one Wnt signaling inhibitor is initiated within about 15 days after initial contact between stem cells and at least one SMAD signaling inhibitor. In certain embodiments, contact between cells and at least one Wnt signaling inhibitor is initiated about 10 days after initial contact between stem cells and at least one SMAD signaling inhibitor. In certain embodiments, contact between cells and at least one Wnt signaling inhibitor is initiated 10, 11, 12, or 13 days after initial contact between stem cells and at least one SMAD signaling inhibitor.

[0009] In certain embodiments, cells are exposed to at least one Wnt signaling inhibitor for at least about 1 day. In certain embodiments, cells are exposed to at least one Wnt signaling inhibitor for up to about 30 days or up to about 25 days. In certain embodiments, cells are exposed to at least one Wnt signaling inhibitor for about 5 days, about 15 days, or about 20 days. In certain embodiments, cells are exposed to at least one Wnt signaling inhibitor for 4 days, 5 days, 6 days, 7 days, 14 days, 15 days, 19 days, or 20 days.

[0010] In certain embodiments, contact between cells and at least one FGF signaling activator is initiated at least about 5 days after initial contact between cells and at least one SMAD signaling inhibitor. In certain embodiments, contact between cells and at least one FGF signaling activator is initiated at least about 10 days after initial contact between cells and at least one SMAD signaling inhibitor. In certain embodiments, contact between cells and at least one FGF signaling activator is initiated within about 20 days after initial contact between cells and at least one SMAD signaling inhibitor. In certain embodiments, contact between cells and at least one FGF signaling activator is initiated within 18 days after initial contact between cells and at least one SMAD signaling inhibitor. In certain embodiments, contact between cells and at least one FGF signaling activator is initiated before most midbrain dopamine neuron precursors differentiate into postmittal neurons. In certain embodiments, contact between cells and at least one FGF signaling activator is initiated about 10 days after initial contact between cells and at least one SMAD signaling inhibitor. In certain embodiments, contact between cells and at least one FGF signaling activator is initiated 10, 11, 12, or 13 days after initial contact between cells and at least one SMAD signaling inhibitor. In certain embodiments, cells are contacted with at least one FGF signaling activator for at least about 1 day and / or up to about 20 days. In certain embodiments, cells are contacted with at least one FGF signaling activator for at least about 3 days and / or up to about 10 days. In certain embodiments, cells are contacted with at least one FGF signaling activator for at least 4 days and / or up to 7 days. In certain embodiments, cells are contacted with at least one FGF signaling activator for about 5 days. In certain embodiments, cells are contacted with at least one FGF signaling activator for 4, 5, 6, or 7 days.

[0011] In certain embodiments, cells are exposed to at least one SMAD signaling inhibitor for about 5 days. In certain embodiments, cells are exposed to at least one SMAD signaling inhibitor for 6 or 7 days.

[0012] In certain embodiments, cells are exposed to at least one SHH signaling activator for about 5 days. In certain embodiments, cells are exposed to at least one SHH signaling activator for 6 or 7 days.

[0013] In certain embodiments, cells are exposed to at least one Wnt signaling activator for about 15 days. In certain embodiments, cells are exposed to at least one Wnt signaling activator for 16 or 17 days. In specific embodiments, the concentration of at least one Wnt signaling activator is increased for about 4 days from its initial contact with the stem cells. In specific embodiments, the concentration of at least one Wnt signaling activator is increased by about 200% to about 1000% from the initial concentration of at least one Wnt signaling activator. In specific embodiments, the concentration of at least one Wnt signaling activator is increased by about 500% from the initial concentration of at least one Wnt signaling activator. In specific embodiments, the concentration of at least one Wnt signaling activator is increased from about 1 μM to about 5 μM and about 10 μM. In specific embodiments, the concentration of at least one Wnt signaling activator is increased to a concentration of about 6 μM.

[0014] In certain embodiments, at least one Wnt signaling inhibitor can inhibit classical Wnt signaling. In certain embodiments, at least one Wnt signaling inhibitor can inhibit both non-classical and classical Wnt signaling. In certain embodiments, at least one Wnt signaling inhibitor is IWP2, IWR1-endo, XAV939, IWP-O1, IWP12, Wnt-C59, IWP-L6, ICG-001, LGK-974, IWR-1, ETC-159, iCRT3, IWP-4, salinomycin, pyrvinium pamoate, iCRT14, FH535, CCT251545, KYA1797K, Wogonin, NCB-0846 The following are selected from the group consisting of hexachlorophene, PNU-74654, KY02111, SO3031(KY01-I), SO2031(KY02-I), tryptonide, BC2059, PKF115-584, quercetin, NSC668036, G007-LK, MSAB, LF3, JW55, isoquercitrin, WIKI4, their derivatives, and combinations thereof. In certain embodiments, at least one Wnt signaling inhibitor is selected from the group consisting of IWP2, IWR1-endo, IWP-O1, IWP12, Wnt-C59, IWP-L6, LGK-974, IWR-1, ETC-159, iCRT3, IWP-4, salinomycin, pyrvinium pamoate, iCRT14, FH535, CCT251545, Wogonin, NCB-0846, hexachlorophene, KY02111, SO3031(KY01-I), SO2031(KY02-I), BC2059, PKF115-584, quercetin, NSC668036, G007-LK, their derivatives, and combinations thereof. In certain embodiments, at least one Wnt signaling inhibitor is selected from the group consisting of XAV939, ICG-001, PNU-74654, tryptonide, KYA1797K, MSAB, LF3, JW55, isoquercitrin, WIKI4, their derivatives, and combinations thereof.In certain embodiments, at least one Wnt signaling inhibitor comprises IWP2.

[0015] In certain embodiments, at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, FGF8b, FGF4, FGF2, and combinations thereof. In certain embodiments, at least one FGF signaling activator can induce midbrain expansion and upregulate midbrain gene expression. In certain embodiments, at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, FGF4, FGF2, and combinations thereof. In certain embodiments, at least one FGF signaling activator includes FGF18.

[0016] In certain embodiments, at least one SMAD signaling inhibitor includes a TGFβ / Activin-Nodal signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, or a combination thereof. In certain embodiments, at least one TGFβ / Activin-Nodal signaling inhibitor includes an ALK5 inhibitor. In certain embodiments, at least one TGFβ / Activin-Nodal signaling inhibitor is selected from the group consisting of SB431542, derivatives of SB431542, and combinations thereof. In certain embodiments, derivatives of SB431542 include A83-01. In certain embodiments, at least one TGFβ / Activin-Nodal signaling inhibitor includes SB431542. In certain embodiments, at least one BMP signaling inhibitor is selected from the group consisting of LDN193189, Noggin, dorsomorphine, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphine, and combinations thereof. In certain embodiments, at least one BMP inhibitor comprises LDN-193189.

[0017] In certain embodiments, at least one Wnt signaling activator comprises a glycogen synthase kinase 3β (GSK3β) signaling inhibitor. In certain embodiments, at least one Wnt signaling activator is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wnt1, Wnt5a, their derivatives, and combinations thereof. In certain embodiments, at least one Wnt signaling activator comprises CHIR99021.

[0018] In certain embodiments, at least one SHH signaling activator is selected from the group consisting of SHH proteins, smoothed agonists (SAGs), and combinations thereof. In certain embodiments, the SHH protein is selected from the group consisting of recombinant SHH, modified N-terminal SHH, and combinations thereof. In certain embodiments, the modified N-terminal SHH contains two isoleucines at its N-terminus. In certain embodiments, the modified N-terminal SHH has at least about 90% sequence identity with the unmodified N-terminal SHH. In certain embodiments, the unmodified N-terminal SHH is unmodified mouse N-terminal SHH or unmodified human N-terminal SHH. In certain embodiments, the modified N-terminal SHH contains SHH C25II. In certain embodiments, the SAG contains palmorfamine.

[0019] In certain embodiments, at least about 80% of differentiated cells express FOXA2 and EN1 about 15 days after initial contact between stem cells and at least one SMAD signaling inhibitor. In certain embodiments, more than about 80% or more than about 90% of differentiated cells express FOXA2 and EN1 16 days after initial contact between stem cells and at least one SMAD signaling inhibitor.

[0020] In certain embodiments, at least one marker indicating a midbrain dopamine neuron or its precursor is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, GIRK2, ALDH1A1, SOX6, WNT1, VMAT2, DAT(SLC6A3), and combinations thereof. In certain embodiments, differentiated cells do not express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0021] In certain embodiments, the method further includes isolating cells that express at least one positive surface marker and do not express at least one negative surface marker. In certain embodiments, the at least one positive surface marker is selected from the group consisting of CD171, CD184, and combinations thereof. In certain embodiments, the at least one positive surface marker includes CD184. In certain embodiments, the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof. In certain embodiments, the at least one negative surface marker includes CD49e. In certain embodiments, the method includes sorting cells that express CD184 and do not express CD49e.

[0022] In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the stem cells are selected from the group consisting of non-embryonic stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof. In certain embodiments, the stem cells are human stem cells, non-human primate stem cells, or rodent stem cells. In certain embodiments, the stem cells are human stem cells.

[0023] This disclosure provides a cell population of in vitro differentiated cells obtained by the differentiation method disclosed herein.

[0024] This disclosure further provides compositions comprising the cell populations disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0025] Furthermore, this disclosure provides a kit for inducing the differentiation of stem cells into midbrain dopamine neurons or their precursors. In certain embodiments, the kit comprises (a) at least one SMAD signaling inhibitor, (b) at least one SHH signaling activator, (c) at least one Wnt signaling activator, (d) at least one Wnt signaling inhibitor, and (e) at least one FGF signaling activator. In certain embodiments, the kit further comprises (f) instructions for inducing the differentiation of stem cells into a population of differentiated cells expressing at least one marker indicating midbrain dopamine neurons or their precursors.

[0026] This disclosure further provides methods for preventing, modeling, and / or treating neurological disorders in a subject. In certain embodiments, the method comprises administering an effective amount of a cell population or composition disclosed herein to a subject. The cell population or composition disclosed herein can be used for preventing, modeling, and / or treating neurological disorders in a subject. In certain embodiments, the neurological disorder is characterized by a decrease in midbrain dopamine neuronal function. In certain embodiments, the decrease in midbrain dopamine neuronal function is age-related. In certain embodiments, the neurological disorder is selected from the group consisting of parkinsonism, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, and combinations thereof. In certain embodiments, the neurological disorder is selected from the group consisting of parkinsonism, Parkinson's disease, and combinations thereof. In certain embodiments, the signs of the neurological disorder are selected from the group consisting of tremor, bradykinesia, flexion, postural instability, tonicity, dysphagia, and dementia. [Brief explanation of the drawing]

[0027] 4. Brief explanation of the drawing [Figure 1] Figure 1 shows the effect of Wnt signaling on ALDH1A1 induction in mDA cells differentiated using different protocols. mRNA expression levels of FOXA2, LMX1A, EN1, WNT1, OTX2, ALDH1A1, and PAX6 were evaluated in differentiated mDA cells at day 16 produced using the Wnt boost, Wnt boost + IWP2 (days 10-16), or Wnt boost + IWP2 (days 12-16) protocols, with or without FGF18. SMA mRNA expression levels could not be detected.

[0028] [Figure 2-1] Figures 2A and 2B show that the Wnt boost protocol combined with FGF18 and IWP2 produced optimal A / P and D / V patterned mDA precursors. Figure 2A shows FACS analysis of differentiated mDA precursors at day 16 using different protocols. Cells were stained with anti-EN1 and anti-FOXA2 antibodies. Figure 2B shows immunostained images of differentiated mDA at day 16. [Figure 2-2] Same as above.

[0029] [Figure 3] Figure 3 shows the effect of IWP2 on the expression of marker genes in differentiated cells. mRNA expression levels of FOXA2, LMX1A, OTX2, EN1, ALDH1A1, BARHL2, BARHL1, PAX6, ALDH2, and WNT1 were measured in differentiated cells produced using the Wnt boost protocol from day 12 to day 16, with or without the addition of FGF18 and / or IWP2.

[0030] [Figure 4]Figure 4 shows the effect of IWP2 on the expression of marker genes in differentiated cells. mRNA expression levels of FOXA2, LMX1A, OTX2, EN1, ALDH1A1, PAX6, and PITX3 were evaluated in 40-day differentiated cells produced using the Wnt boost protocol, with or without the addition of FGF18 and / or IWP2, from day 12 to day 16.

[0031] [Figure 5] Figure 5 shows the gating paradigm of the double sorting strategy. Differentiated mDA cells were sorted based on the expression of CD49e and CD184 marker proteins.

[0032] [Figure 6] Figure 6 shows the morphology of selected differentiated CD49 weak / CD184 weak and CD49 weak / CD184 strong cells at day 40. Cells were selected at day 25 of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16).

[0033] [Figure 7] Figure 7 shows mRNA expression of dopamine neuron marker genes in selected 40-day differentiated CD49-weak / CD184-weak and CD49-weak / CD184-strong cells. Cells were selected at day 25 of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16).

[0034] [Figure 8] Figure 8 shows mRNA expression of non-dopamine neuron marker genes in selected 40-day differentiated CD49 weak / CD184 weak and CD49 weak / CD184 strong cells. Cells were selected at 25 days of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16).

[0035] [Figure 9-1]Figures 9A-9C show representative immunostained images of selected 40-day differentiated CD49 weak / CD184 weak and CD49 weak / CD184 strong cells. Figure 9A shows the expression of FOXA2, TH, and MAP2. Figure 9B shows the expression of ALDH1A1, EN1, and TH. Figure 9C shows the expression of ALDH1A1, EN1, and TH. Cells were selected on day 25 of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16). [Figure 9-2] Same as above.

[0036] [Figure 10] Figures 10A and 10B show representative immunostained images of differentiated cells after transplantation into mice. Differentiated cells were obtained using the Wnt boost or Wnt boost + FGF18 / IWP2 (days 12-16) protocol and transplanted into mice. Transplanted cells were immunostained one month after transplantation. hNCAM, TH, and ALDH1A1 expression were evaluated (Figure 10A). Ki67 expression was also evaluated (Figure 10B).

[0037] [Figure 11-1] Figures 11A to 11C show representative immunostaining images of differentiated cells after transplantation into mice. Differentiated cells were obtained using the Wnt boost or Wnt boost + FGF18 / IWP2 (days 12-16) protocol and transplanted into mice. Transplanted cells were immunostained one month after transplantation. Figure 11A shows SC121 expression. Figure 11B shows TH and Nurr1-GFP expression. Figure 11C shows ALDH1A1 and SOX6-RFP expression. [Figure 11-2] Same as above.

[0038] [Figure 12] Figure 12 shows the effect of IWP2 on the expression of marker genes in differentiated cells. Marker gene mRNA expression levels were measured in differentiated cells produced using the Wnt boost protocol from day 12 to day 16, with or without the addition of FGF18 and / or IWP2.

[0039] [Figure 13] Figure 13 shows the effect of IWP2 on the expression of marker genes in differentiated cells. mRNA expression levels of various genes were evaluated in differentiated cells produced at day 40 using the Wnt boost protocol, with or without the addition of FGF18 and / or IWP2 from day 12 to day 16.

[0040] [Figure 14] Figures 14A and 14B show representative immunofluorescence staining images of differentiated cells at day 60 produced using the Wnt boost protocol, with or without the addition of FGF18 and / or IWP2, from day 12 to day 16. Figure 14A shows immunofluorescence staining images of differentiated cells at day 60 expressing FOXA2, TH, and MAP2 for each condition. Figure 5B shows different staining panels marking EN1 and TH, demonstrating differential expression of EN1 among TH+ dopamine neurons at day 60.

[0041] [Figure 15] Figure 15 shows mRNA expression of marker genes in selected 40-day differentiated CD49-weak / CD184-weak and CD49-weak / CD184-strong cells. Cells were selected at day 25 of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16).

[0042] [Figure 16] Figure 16 shows mRNA expression of marker genes in selected 40-day differentiated CD49-weak / CD184-weak and CD49-weak / CD184-strong cells. Cells were selected at day 25 of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16).

[0043] [Figure 17]Figure 17 shows representative immunostained images of selected differentiated CD49 weak / CD184 weak and CD49 weak / CD184 strong cells at day 60. Cells were selected at day 25 of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16).

[0044] [Figure 18] Figure 18 shows representative immunostained images of selected 60-day differentiated cells with weak CD49 / strong CD184. Cells were selected on day 25 of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16).

[0045] [Figure 19] Figure 19 shows the mRNA expression of marker genes in differentiated cells at day 30 produced using the Wnt boost protocol, with or without the addition of IWP2 and FGF18 from day 12 to day 16, and with or without the addition of IWP2 from day 17 to day 30.

[0046] [Figure 20] Figure 20 shows FACS-mediated sorting of differentiated cells at day 25 produced from the Wnt boost protocol, with or without IWP2 addition from day 12 to day 25, or from day 12 to day 16, and with or without FGF18 addition from day 12 to day 16.

[0047] [Figure 21] Figure 21 shows mRNA expression of marker genes in selected differentiated CD49 weak / CD184 weak and CD49 weak / CD184 strong cells at day 28. Cells were selected at day 25 of in vitro differentiation under Wnt boost, with or without IWP2 added from day 12 to day 25, or from day 12 to day 16, and with or without FGF18 added from day 12 to day 16.

[0048] [Figure 22] Figure 22 shows mRNA expression of non-dopamine neuron marker genes in selected differentiated CD49 weak / CD184 weak and CD49 weak / CD184 strong cells at day 28. Cells were selected at day 25 of in vitro differentiation under Wnt boost, with or without IWP2 added from day 12 to day 25, or from day 12 to day 16, and with or without FGF18 added from day 12 to day 16.

[0049] [Figure 23] Figure 23 shows immunostained images of selected differentiated CD49 weak / CD184 weak and CD49 weak / CD184 strong cells at day 28. Cells were selected at day 25 of in vitro differentiation under Wnt boost, with or without IWP2 added from day 12 to day 25, or from day 12 to day 16, and with or without FGF18 added from day 12 to day 16.

[0050] [Figure 24] Figure 24 shows representative immunostained images of differentiated cells after transplantation into mice. Differentiated cells were obtained using the Wnt boost or Wnt boost + FGF18 / IWP2 (days 12-16) protocol and transplanted into mice. Transplanted cells were immunostained one month after transplantation. The expression of FOXA2, SC121, ALDH1A1, EN1, and Ki67 was evaluated.

[0051] [Figure 25] Figure 25 shows representative immunostained images of differentiated cells after transplantation into mouse brains. Differentiated cells were obtained using the Wnt boost or Wnt boost + FGF18 / IWP2 (days 12-16) protocol and transplanted into mice. The transplanted cells were immunostained one month after transplantation and evaluated for any proliferating cells marked with Ki67.

[0052] [Figure 26]Figure 26 shows representative immunostained images of mouse striatal grafts of a frozen batch of dopamine precursors (differentiation day 16). Differentiated cells at day 16 were obtained using the Wnt boost + FGF18 / IWP2 (days 12-16) protocol and frozen using a controlled-speed freezer. The frozen cells were thawed and directly transplanted into the striatum of NOD-SCID mice. The transplanted cells were immunostained one month after transplantation.

[0053] [Figure 27] Figure 27 shows representative immunostained images of differentiated cells after transplantation into mice. Differentiated cells were obtained using the Wnt boost + FGF18 / IWP2 (days 12-16) protocol and transplanted into mice. The transplanted cells were immunostained 4 months after transplantation.

[0054] [Figure 28] Figure 28 shows representative immunostained images of differentiated cells after transplantation of selected CD49-weak / CD184-strong cells into mice. Cells were selected on day 25 of in vitro differentiation using the Wnt boost or Wnt boost + FGF18 / IWP2 protocol (days 12-16) and transplanted into mice. Transplanted cells were immunostained one month after transplantation.

[0055] [Figure 29] Figure 29 shows representative RNA fluorescence in-situ (FISH) images of PITX3 and NURR1 in TH-positive cells. mRNA signaling was measured by intracellular dots, and the number of dots was quantified at days 35, 59, and 82 during differentiation. Differentiated cells were obtained using the Wnt boost + FGF18 / IWP2 (days 12-16) protocol. [Modes for carrying out the invention]

[0056] 5. Detailed explanation This disclosure provides a method for generating mDA neurons and their precursors, mDA neurons and their precursors generated by such a method, a composition comprising such cells, and its use for preventing and / or treating neurological disorders.

[0057] Wnt signaling is crucial for the specification of mDA neurons. Our previous studies have shown that Wnt boosting results in strong induction of EN1 and suppression of fate in both the hindbrain and subthalamic and forebrain. See, for example, the Wnt boosting method disclosed in International Publication 2016 / 196661, which is incorporated in its entirety by reference. However, prolonged Wnt signaling can interfere with the differentiation and subtype identification of mDA neurons. Furthermore, the expression of PITX3 and ALDH1A1 is suboptimal in previous differentiation protocols. This disclosure is based on the finding that treatment with a Wnt inhibitor can improve mDA neuron induction. Moreover, such treatment with a Wnt inhibitor does not adversely affect the expression of EN1 and other mDA neuron markers; for example, the differentiation method disclosed herein, including a Wnt inhibitor, results in sustained expression of EN1 and other mDA neuron markers. Furthermore, treatment with a Wnt inhibitor does not increase the appearance of contaminating markers (non-mDA neuron markers).

[0058] This disclosure also builds on the finding that Wnt inhibitor treatment results in better separation of A9 subtype neurons and A10 subtype neurons. In certain embodiments, Wnt inhibitor treatment affects (e.g., increases) the mRNA expression of a marker indicating A9 subtype mDA (e.g., ALDH1A1). Non-limiting examples of markers indicating A9 subtype neurons include LMO3, ALDH1A1, SOX6, VGLUT2, and NDNF. In certain embodiments, Wnt inhibitor treatment affects ALDH1A1 (of EN1+) in vitro and in vivo. + Increases the number of cells. ALDH1A1 expression may be elevated without EN1 co-expression. + EN1- The cells are not necessarily A9 subtype neurons and are not clearly defined cells. Differentiation methods disclosed herein, including Wnt inhibitor treatment, result in a high generation of cells expressing both ALDH1A1 and EN1 in vitro and in vivo after transplantation. In certain embodiments, Wnt inhibitor treatment further affects (e.g., increases) the mRNA expression of markers indicating A10 subtype mDA. Non-limiting examples of markers indicating A10 subtype neurons include CALB1, CALB2, OTX2, CCK, VGAT(Slc32a1), and VIP. Increased mRNA expression of A9 and A10 subtype markers supports the appropriate identification of A9 and A10 subtype neurons. For example, certain A10 subtype neuron markers (e.g., CALB1 and CALB2) may only be seen when cells have been appropriately identified as exhibiting an A9 or A10 identity.

[0059] Furthermore, Wnt inhibitor treatment can reduce proliferation and increase the expression of mDA neuron maturation markers. Non-exclusive examples of mDA neuron maturation markers include DAT, VMAT2, PITX3, CHRNA6, and CHRNB3.

[0060] Furthermore, Wnt inhibitor treatment can improve differentiation and reduce the number of remaining Ki67+ proliferating cells, thereby improving the safety profile of DA neurons.

[0061] Furthermore, dopamine neurons derived from stem cells and transplanted by differentiation methods disclosed herein (including, for example, Wnt inhibitor treatment) have improved fibrillation, particularly ALDH1A1 fibers, which are expected to most efficiently induce functional recovery.

[0062] Furthermore, this disclosure is based on the finding that mDA and its precursors produced by the method of this disclosure improve in vivo survival, for example, allowing patients to survive months or years after in vivo transplantation.

[0063] This disclosure provides improved protocols for neuronal induction and mDA neuronal differentiation from stem cells (e.g., human pluripotent stem cells (hPSCs)), including clinical-grade protocols nearing human use. Access to these improved mDA neuronal differentiation protocols allows the field to achieve more complete mDA neuronal recovery using fewer cells and reduce potential side effects. Therefore, the protocols of this disclosure improve safety, as the effects of contaminating cell types in grafts remain unclear. Finally, the protocols of this disclosure enhance the accuracy and reproducibility of mDA neurons in the modeling of human diseases in dishes. The protocols of this disclosure improve the fidelity and robustness of mDA differentiation. The protocols of this disclosure are widely adaptable and widely usable.

[0064] Non-limiting embodiments of the subject matter of this disclosure are described herein and by examples.

[0065] For the purpose of clarifying disclosure rather than limiting it, the detailed explanation is divided into the following subsections. 5.1. Definition; 5.2. Methods for differentiating stem cells; 5.3. Cell populations and compositions; 5.4. Methods for preventing, modeling, and / or treating neurological disorders; and 5.5. Kit.

[0066] 5.1.Definition The terms used herein generally have the common meaning in the art within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners when describing the compositions and methods of this disclosure and how they are prepared and used.

[0067] The terms “about” or “approximately” mean a range of acceptable error for a particular value as determined by those skilled in the art, which depends in part on how the value is measured or determined, i.e., on the limitations of the measuring system. For example, “about” can mean within or beyond three standard deviations, according to convention in the art. Alternatively, “about” can mean a range of up to 20% of a given value, e.g., up to 10%, up to 5%, or up to 1%. Or, particularly with respect to biological systems or processes, the term can mean within one order of magnitude, e.g., within five times or up to twice the value.

[0068] As used herein, the term “signaling” with respect to “signaling proteins” refers to proteins that are activated or otherwise affected by ligand binding to membrane receptor proteins or some other stimulus. Examples of signaling proteins include, but are not limited to, SMAD, Wingless (Wnt) complex proteins including β-catenin, NOTCH, transforming growth factor β (TGFP), Activin, Nodal, glycogen synthase kinase 3β (GSK3β) protein, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF). For many cell surface receptor or internal receptor proteins, the ligand-receptor interaction is not directly related to the cellular response. Ligand-activated receptors can first interact with other proteins within the cell before the final physiological effect of the ligand on the cell's behavior occurs. Often, after the activation or inhibition of a receptor, the behavior of a chain of several interacting cellular proteins changes. The entire set of cellular changes induced by receptor activation is called a signaling mechanism or signaling pathway.

[0069] As used herein, the term “signal” refers to internal and external factors that control changes in the structure and function of cells. These may be chemical or physical in nature.

[0070] As used herein, the term “ligand” refers to molecules and proteins that bind to receptors, such as transforming growth factor β (TFGP), Activin, Nodal, and bone morphogenetic proteins (BMPs).

[0071] As used herein, “inhibitor” refers to a compound or molecule (e.g., small molecule, peptide, peptide mime, native compound, siRNA, antisense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, diminishes, represses, eliminates, or blocks) the signaling function of a molecule or pathway (e.g., the Wnt signaling pathway and SMAD signaling). An inhibitor may be any compound or molecule that alters the activity of any specified protein (signaling molecule, any molecule involved in the specified signaling molecule, a specified related molecule, e.g., glycogen synthase kinase 3β (GSK3β)). (e.g., including, but not limited to, signaling molecules described herein). For example, a SMAD signaling inhibitor may function by, for example, direct contact with SMAD, contact with SMAD mRNA, inducing a conformational change of SMAD, reducing SMAD protein levels, interfering with SMAD interactions with signaling partners, and affecting the expression of SMAD target genes.

[0072] Inhibitors also include molecules that indirectly regulate biological activity (e.g., SMAD biological activity) by blocking upstream signaling molecules (for example, within the extracellular domain, examples of signaling molecules and effects include Noggin, which sequesters bone morphogenetic proteins and inhibits the activation of ALK receptors 1, 2, 3, and 6, thereby preventing downstream SMAD activation. Similarly, Chordin, Cerberus, and Follistatin also sequester extracellular activators of SMAD signaling. The transmembrane protein Bambi also acts as a pseudoreceptor for sequestering extracellular TGFβ signaling molecules). Antibodies that block upstream or downstream proteins are intended for use in neutralizing extracellular activators of protein signaling, etc. The above examples relate to SMAD signaling inhibition, but other signaling molecules can be inhibited using similar or analogous mechanisms. Examples of inhibitors include, but are not limited to, LDN193189 (LDN) and SB431542 (SB) (LSB) for SMAD signaling inhibition, and IWP2 for Wnt inhibition. Inhibitors are described in terms of competitive inhibition (binding to the active site in a manner that eliminates or reduces the binding of another known binding compound) and allosteric inhibition (binding to a protein in a manner that alters the protein's conformation in a manner that prevents the binding of a compound to the protein's active site), in addition to inhibition induced by binding to an upstream molecule of a designated signaling molecule and subsequently causing inhibition of the designated molecule. Inhibitors may be “direct inhibitors” that inhibit a signaling target or signaling target pathway by actually contacting the signaling label.

[0073] As used herein, "activator" refers to a compound that increases, induces, stimulates, activates, promotes, or enhances the signaling function of a molecule or pathway, such as Wnt signaling or SHH signaling.

[0074] As used herein, the terms “Wnt” or “wingless” with respect to ligands refer to a group of secreted proteins (e.g., Integration 1 in humans) that can interact with Wnt receptors, such as receptors of the Frizzled and LRPDerailed / RYK receptor families. As used herein, the terms “Wnt or wingless signaling pathway” refer to a signaling pathway consisting of Wnt family ligands and Wnt family receptors, e.g., Frizzled and LRPDerailed / RYK receptors, mediated with or without β-catenin. Wnt signaling pathways include classical Wnt signaling (e.g., mediated by β-catenin) and non-classical Wnt signaling (mediated without β-catenin).

[0075] As used herein, the term “derivative” refers to a compound having a similar core structure.

[0076] As used herein, the term “population of cells” or “cell population” refers to a group of at least two cells. In non-limiting examples, a cell population may include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 cells. A population may be a pure population containing one cell type, such as a population of midbrain DA precursors, or a population of undifferentiated stem cells, for example, a population of A9 subtype midbrain dopamine neurons. Alternatively, a population may include more than one cell type, for example, a mixed cell population, such as a population of A9 subtype midbrain dopamine neurons and A10 subtype midbrain dopamine neurons.

[0077] As used herein, the term “stem cell” refers to a cell that has the ability to divide indefinitely and produce specialized cells during culture.

[0078] As used herein, the terms “embryonic stem cells” and “ESCs” refer to primitive (undifferentiated) cells derived from a pre-implantation embryo that are known to be able to divide without differentiating for extended periods during culture and to develop into cells and tissues of the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells derived from a human embryo. As used herein, the terms “human embryonic stem cells” or “hESCs” refer to a type of pluripotent stem cell derived from an early stage human embryo up to the blastocyst stage that is known to be able to divide without differentiating for extended periods during culture and to develop into cells and tissues of the three primary germ layers.

[0079] As used herein, the term “embryonic stem cell line” refers to a population of embryonic stem cells cultured under in vitro conditions that allow them to proliferate without differentiation for up to several days, months, or even years.

[0080] As used herein, the term “pluripotency” refers to the ability of an organism to develop into one of the three developmental layers, including the endoderm, mesoderm, and ectoderm.

[0081] As used herein, the term "pluripotency" refers to the ability to produce all cell types of the body, as well as all cell types that make up extraembryonic tissues such as the placenta.

[0082] As used herein, the term “pluripotency” refers to the ability of a body to develop into more than one cell type.

[0083] As used herein, the terms “induced pluripotent stem cell” or “iPSC” refer to a type of pluripotent stem cell formed by introducing specific embryonic genes (e.g., but not limited to OCT4, SOX2, and KLF4 transgenes) (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006) incorporated herein by reference) into somatic cells.

[0084] As used herein, the term “neuron” refers to a nerve cell, which is the primary functional unit of the nervous system. A neuron consists of a cell body and its projections—an axon and at least one dendrite. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0085] As used herein, the term “differentiation” refers to the process by which unspecialized embryonic cells acquire the characteristics of specialized cells, such as neurons, heart cells, liver cells, or muscle cells. Differentiation is typically controlled by the interaction of a cell’s genes with extracellular physical and chemical conditions, usually via signaling pathways involving proteins embedded on the cell surface.

[0086] As used herein, the term “targeted differentiation” refers to the manipulation of stem cell culture conditions to induce differentiation into a specific (e.g., desired) cell type, such as midbrain dopamine neurons or their precursors. With respect to stem cells, “targeted differentiation” refers to the use of small molecules, growth factor proteins, and other growth conditions to facilitate the transition of stem cells from a pluripotent state to a more mature or specialized cell fate.

[0087] As used herein, the term “induce differentiation” with respect to cells means changing the default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Therefore, “induce differentiation in stem cells” means inducing stem cells (e.g., human stem cells) to divide into progeny cells having different characteristics from the stem cell, e.g., genotype (e.g., changes in gene expression determined by genetic analysis such as microarrays) and / or phenotype (e.g., changes in the expression of protein markers of mDA neurons or their precursors, e.g., EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, ALDH1A1, SOX6, WNT1, DAT, VMAT2, and GIRK2).

[0088] As used herein, the term “cell culture” refers to the in vitro growth of cells in an artificial medium for research or medical treatment.

[0089] As used herein, the term “culture medium” refers to a liquid containing nutrients that surrounds and nourishes cells in a culture vessel, such as a petri dish or multiwell plate. Culture medium may also contain growth factors that are added to induce desired changes in the cells.

[0090] As used herein, the term "to bring one or more cells into contact with a compound (e.g., at least one inhibitor, activator, and / or inducer)" means providing the compound at a position that allows one or more cells to access the compound. Contact can be achieved using any suitable method. For example, contact can be achieved by adding a concentrated form of the compound to cells or a population of cells, for example, in the context of a cell culture, to achieve a desired concentration. Contact can also be achieved by including the compound as a component of a formulated culture medium.

[0091] As used herein, the term "in vitro" refers to an artificial environment and any process or reaction occurring within it. Examples of in vitro environments include, but are not limited to, test tubes and cell cultures.

[0092] As used herein, the term “in vivo” refers to the natural environment (e.g., animals or cells), as well as processes or reactions that occur within the natural environment, such as embryonic development, cell differentiation, and neural tube formation.

[0093] As used herein, the term “express” with respect to a gene or protein means producing mRNA or protein that can be observed using an assay such as a microarray assay or an antibody staining assay.

[0094] As used herein, the terms “marker” or “cell marker” refer to a gene or protein that identifies a particular cell or cell type. A cell marker is not limited to a single marker; a marker may refer to a “pattern” of markers such that a given set of markers can distinguish one cell or cell type from another.

[0095] Where used herein, the terms “derived from,” “established from,” or “differentiated from” any cells disclosed herein mean cells obtained from a cell line, tissue (such as a dissociated embryo), or final parental cells in fluid (e.g., isolated, purified, etc.) using any operation, for example, but not limited to, single-cell isolation, in vitro culture, treatment with proteins, chemicals, radiation, viral infection, and / or mutagenesis, such as transfection of DNA sequences with morphogens, or selection of any cells contained in cultured parental cells (e.g., by serial culture). Induced cells may be selected from a mixed population by response to growth factors, cytokines, selected progression of cytokine treatment, adhesion, lack of adhesion, sorting procedures, etc.

[0096] In this specification, “individual” or “subject” means a vertebrate, e.g., human, or a non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, non-human primates, livestock, sport animals, rodents, and pets. Non-exclusive examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates, e.g., apes and monkeys.

[0097] As used herein, the term “disease” means any condition or disorder that impairs or interferes with the normal functioning of a cell, tissue, or organ.

[0098] As used herein, the terms “to treat” or “treatment” refer to a clinical intervention in an attempt to alter the course of a disease in an individual or cell being treated, and may be performed for preventive purposes or in the course of a clinicopathological condition. The therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis. By preventing the progression of disease or disability, treatment can prevent the exacerbation of disability in an affected or diagnosed subject or a subject suspected of having a disability, but treatment can also prevent the onset of disability or signs of disability in a subject at risk of disability or suspected of having a disability.

[0099] 5.2. Methods for differentiating stem cells This disclosure provides a method for inducing differentiation of hepatocytes, comprising the steps of: contacting stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor ("SMAD inhibitor"), at least one Sonic Hedgehog (SHH) signaling activator ("SHH activator"), and at least one Wingless (Wnt) signaling activator ("Wnt activator"); and contacting cells with at least one fibroblast growth factor (FGF) signaling activator ("FGF activator") and at least one Wnt signaling inhibitor to obtain a cell population containing differentiated cells expressing at least one marker indicating mDA neurons or their precursors.

[0100] The use of Wnt signaling inhibitors can improve mDA neuron induction, for example, enabling the induction of a broader set of mDA neurons. Prolonged Wnt signaling can interfere with the differentiation and subtype identification of mDA neurons (Andersson, et al., Proceedings of the National Academy of Sciences of the United States of America (2013); 110, E602-610). For example, inhibiting Wnt signaling by using Wnt signaling inhibitors results in increased expression of mDA neuron markers (A9 subtype mDA neuron markers (e.g., ALDH1A1) and A10 subtype mDA neuron markers (e.g., CALB1) and mDA neuron maturation markers (including, but not limited to, DAT, VMAT2, PITX3, CHRNA6, and CHRNB3)). Wnt signaling inhibitors may affect the expression of A9 subtype mDA neuron markers. Non-exclusive examples of markers indicating A9 subtype midbrain dopamine neurons include LMO3, ALDH1A1, SOX6, VGLUT2, and NDNF. In certain embodiments, Wnt signaling inhibitors increase the expression of A9 subtype mDA neuron markers. In certain embodiments, Wnt signaling inhibitors increase the expression of ALDH1A1. In certain embodiments, Wnt signaling inhibitors increase the expression of A10 subtype mDA neuron markers. In certain embodiments, Wnt signaling inhibitors increase the expression of CALB1.

[0101] Furthermore, mDA neurons or their precursors produced by the methods disclosed herein exhibit improved fiber proliferation and reduced residual Ki67 +The cells proliferate and have improved in vivo survival, making these cells more suitable for therapeutic use. In certain embodiments, mDA neurons or their precursors produced by the methods disclosed herein can survive in vivo for at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, up to about 6 months, up to about 1 year, up to about 2 years, up to about 3 years, up to about 4 years, or up to about 5 years. In certain embodiments, mDA neurons produced by the methods disclosed herein can survive in vivo for at least about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 1 year, up to about 2 years, up to about 3 years, up to about 4 years, or up to about 5 years.

[0102] 5.2.1. Stem cells The subject matter of this disclosure provides an in vitro method for inducing the differentiation of stem cells to produce mDA neurons and their precursors. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and combinations thereof. In certain embodiments, the stem cells are multipotent stem cells. Non-limiting examples of stem cells that can be used with the methods of this disclosure include non-embryonic stem cells, embryonic stem cells, induced non-embryonic pluripotent cells, and engineered pluripotent cells. In certain embodiments, the stem cells are human stem cells. Non-limiting examples of human stem cells include human embryonic stem cells (hESCs), human pluripotent stem cells (hPSCs), human induced pluripotent stem cells (hiPSCs), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, F-class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cells capable of lineage-specific differentiation. In certain embodiments, the stem cells are human embryonic stem cells (hESCs). In certain embodiments, the stem cells are human induced pluripotent stem cells (hiPSCs). In certain embodiments, the stem cells are non-human stem cells. In certain embodiments, the stem cells are non-human primate stem cells. In certain embodiments, the stem cells are rodent stem cells.

[0103] In certain embodiments, stem cells or their progeny cells include introduced heterologous nucleic acids that encode or have informational value for a desired nucleic acid or protein product (see, for example, U.S. Patent No. 6,312,911, which is incorporated in whole by reference). Non-limiting examples of protein products include markers detectable via in vivo imaging studies, such as receptors or other cell membrane proteins. Non-limiting examples of markers include fluorescent proteins (e.g., green fluorescent protein (GFP), blue fluorescent proteins (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent proteins (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), β-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (e.g., oxidases and peroxidases), and antigenic molecules. As used herein, the terms “reporter gene” or “reporter construct” refer to a gene construct comprising nucleic acids encoding readily detectable or readily assayable proteins, such as colored proteins, fluorescent proteins such as GFP, or enzymes such as β-galactosidase (lacZ gene). In certain embodiments, the reporter may be driven by a recombinant promoter of a pre-maturation post-mitotic mDA neuron marker gene, for example, NURR1.

[0104] 5.2.2. SMAD Inhibitors Non-limiting examples of SMAD inhibitors include inhibitors of transforming growth factor β (TGFβ) / Activin-Nodal signaling (referred to as "TGFβ / Activin-Nodal inhibitors") and bone morphogenetic protein (BMP) signaling inhibitors. In certain embodiments, TGFβ / Activin-Nodal inhibitors can neutralize ligands including TGFβ, BMP, Nodal, and activin, and / or block their signaling pathways by blocking receptors and downstream effectors. Non-exclusive examples of TGFβ / Activin-Nodal inhibitors include those described in International Publications 2010 / 096496, 2011 / 149762, 2013 / 067362, 2014 / 176606, 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1;30(7):715-20 (2012), all of which are incorporated herein by reference in their entirety for all purposes. In certain embodiments, at least one TGFβ / Activin-Nodal inhibitor is selected from ALK5 inhibitors, ALK4 inhibitors, ALK7 inhibitors, and combinations thereof. In certain embodiments, the TGFβ / Activin-Nodal inhibitor includes an ALK5 inhibitor. In certain embodiments, the TGFβ / Activin-Nodal inhibitor is a small molecule selected from SB431542, its derivatives, and mixtures thereof. "SB431542" is the CAS number 301836-41-9, C 22 H 18 This refers to a molecule with the molecular formula N4O3 and the name 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide, for example, see the following structure: [ka]

[0105] In certain embodiments, the TGFβ / Activin-Nodal inhibitor includes SB431542. In certain embodiments, the TGFβ / Activin-Nodal inhibitor includes a derivative of SB431542. In certain embodiments, the derivative of SB431542 is A83-01.

[0106] In certain embodiments, at least one SMAD inhibitor comprises a BMP signaling inhibitor (referred to as a "BMP inhibitor"). Non-limiting examples of BMP inhibitors include those described in International Publication 2011 / 149762, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1;30(7):715-20, all of which are incorporated in their entirety by reference. In certain embodiments, the BMP inhibitor is a small molecule selected from LDN193189, Noggin, dorsomorphine, their derivatives, and mixtures thereof. "LDN193189" is a C molecule having the following formula: 25 H 22 This refers to the small molecule DM-3189 with the chemical formula N6, whose IUPAC name is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-yl)quinoline. [ka]

[0107] LDN193189 can function as a SMAD signaling inhibitor. LDN193189 is also a very potent small molecule inhibitor of ALK2, ALK3, and ALK6, protein tyrosine kinases (PTKs), inhibiting signaling of members of the ALK1 and ALK3 families of the type I TGFβ receptor, inhibiting the transmission of multiple biological signals, including bone morphogenetic proteins (BMPs) BMP2, BMP4, BMP6, BMP7, and Activin cytokine signaling, as well as subsequent SMAD phosphorylation of Smad1, Smad5, and Smad8 (Yu et al. (2008) Nat Med 14:1363-1369; Cuny et al. (2008) Bioorg. Med. Chem. Lett. 18:4388-4392, incorporated herein by reference).

[0108] In certain embodiments, the BMP inhibitor includes LDN193189. In certain embodiments, the BMP inhibitor includes Noggin.

[0109] In certain embodiments, stem cells are exposed to one SMAD inhibitor, for example, one TGFβ / Activin-Nodal inhibitor. In certain embodiments, the TGFβ / Activin-Nodal inhibitor is SB431542. In certain embodiments, the TGFβ / Activin-Nodal inhibitor is a derivative of SB431542. In certain embodiments, the TGFβ / Activin-Nodal inhibitor is A83-01.

[0110] In certain embodiments, stem cells are exposed to two SMAD inhibitors. In certain embodiments, the two SMAD inhibitors are a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor. In certain embodiments, stem cells are exposed to SB431542 or A83-01 and LDN193189 or Noggin. In certain embodiments, stem cells are exposed to SB431542 and LDN193189. In certain embodiments, stem cells are exposed to A83-01 and LDN193189. In certain embodiments, stem cells are exposed to SB431542 and Noggin. In certain embodiments, stem cells are exposed to A83-01 and Noggin.

[0111] In certain embodiments, stem cells are exposed to or in contact with at least one SMAD inhibitor for at least about 5 days, or at least about 10 days. In certain embodiments, stem cells are in contact to or exposed to at least one SMAD inhibitor for up to about 5 days, or up to about 10 days. In certain embodiments, stem cells are in contact to or exposed to at least one SMAD inhibitor for about 5 to about 10 days. In certain embodiments, stem cells are in contact to or exposed to at least one SMAD inhibitor for about 5 days. In certain embodiments, stem cells are in contact to or exposed to at least one SMAD inhibitor for 6 days. In certain embodiments, stem cells are in contact to or exposed to at least one SMAD inhibitor for 7 days. In certain embodiments, cells are in contact to or exposed to at least one SMAD inhibitor from day 0 to day 6. In certain embodiments, at least one SMAD inhibitor is added to the cell culture medium containing stem cells from day 0 to day 6, either every day or every other day. In certain embodiments, at least one SMAD inhibitor is added to the cell culture medium containing stem cells every day from day 0 to day 6.

[0112] In certain embodiments, cells are brought into contact with or exposed to a TGFβ / Activin-Nodal inhibitor. In certain embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor brought into contact with or exposed to the cells is approximately 1 μM to 20 μM, approximately 1 μM to 10 μM, approximately 1 μM to 15 μM, approximately 10 μM to 15 μM, approximately 5 μM to 10 μM, approximately 5 μM to 15 μM, approximately 5 μM to 20 μM, or approximately 15 μM to 20 μM. In certain embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor brought into contact with or exposed to the cells is approximately 1 μM to 10 μM. In certain embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor brought into contact with or exposed to the cells is approximately 5 μM. It is approximately 10 μM. In certain embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor brought into contact with or exposed to the cells is approximately 10 μM. In certain embodiments, the TGFβ / Activin-Nodal inhibitor comprises SB431542 or a derivative thereof (e.g., A83-01).

[0113] In certain embodiments, cells are brought into contact with or exposed to a BMP inhibitor. In certain embodiments, the concentration of the BMP inhibitor brought into contact with or exposed to the cells is approximately 50 nM to approximately 500 nM, or approximately 100 nM to approximately 500 nM, or approximately 200 nM to approximately 500 nM, or approximately 200 to approximately 300 nM, or approximately 200 nM to approximately 400 nM, or approximately 100 nM to approximately 250 nM, or approximately 100 nM to approximately 250 nM, or approximately 200 nM to approximately 250 nM, or approximately 250 nM to approximately 300 nM. In certain embodiments, the concentration of the BMP inhibitor brought into contact with or exposed to the cells is approximately 200 nM to approximately 300 mM. In certain embodiments, the concentration of the BMP inhibitor to be contacted or exposed to the cells is about 150 nM, about 200 nM, about 250 nM, about 300 nM, or about 350 nM. In certain embodiments, the concentration of the BMP inhibitor to be contacted or exposed to the cells is about 250 nM. In certain embodiments, the BMP inhibitor comprises LDN193189 or a derivative thereof. In certain embodiments, the BMP inhibitor comprises LDN193189.

[0114] In certain embodiments, cells are simultaneously exposed to or contacted with a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor. In certain embodiments, stem cells are exposed to or contacted with a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor for approximately 5 days. In certain embodiments, stem cells are exposed to or contacted with a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor for 6 days. In certain embodiments, stem cells are exposed to or contacted with a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor for 7 days. In certain embodiments, cells are exposed to or contacted with a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor from day 0 to day 6. In certain embodiments, a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor are added to the cell culture medium containing stem cells every day or every other day from day 0 to day 6. In certain embodiments, a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor are added to the cell culture medium containing stem cells every day (daily) from day 0 to day 6.

[0115] 5.2.3.Wnt activator In certain embodiments, at least one Wnt activator reduces GSK3β for activation of Wnt signaling. Therefore, in certain embodiments, the Wnt activator is a GSK3β inhibitor. GSK3β inhibitors can activate the WNT signaling pathway (see, e.g., Cadigan, et al., J Cell Sci. 2006; 119: 395-402; Kikuchi, et al., Cell Signaling. 2007; 19: 659-671, which is incorporated herein by reference in its entirety). As used herein, the terms “glycogen synthase kinase 3β inhibitor” or “GSK3β inhibitor” refer to compounds that inhibit the glycogen synthase kinase 3β enzyme (see, e.g., Doble, et al., J Cell Sci. 2003; 116: 1175-1186, which is incorporated herein by reference in its entirety). Non-limiting examples of GSK3β inhibitors include CHIR99021, BIO((3E)-6-bromo-3-[3-(hydroxyamino)indole-2-ylidene]-1H-indole-2-one), AMBMP hydrochloride, LP 922056, SB-216763, CHIR98014, lithium, 3F8, deoxycholic acid, and International Publication No. 2011 / 149762, International Publication No. 13 / 067362, Chambers et al., Nat Biotechnol. 2012 Jul 1;30(7):715-20, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Calder et al., J Neurosci. 2015 Aug Examples include those listed in 19;35(33):11462-81 (all of which are incorporated in their entirety by reference).

[0116] Non-limiting examples of Wnt activators include CHIR99021, Wnt3A, Wnt1, Wnt5a, BIO((3E)-6-bromo-3-[3-(hydroxyamino)indole-2-ylidene]-1H-indole-2-one), AMBMP hydrochloride, LP 922056, SB-216763, CHIR98014, lithium, 3F8, deoxycholic acid, and International Publication No. 2011 / 149762, International Publication No. 13 / 067362, Chambers et al., Nat Biotechnol. 2012 Jul 1;30(7):715-20, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Calder et al., J Neurosci. 2015 Aug Examples include those described in 19;35(33):11462-81 (all of which are incorporated in their entirety by reference). In certain embodiments, at least one Wnt activator is a small molecule selected from CHIR99021, Wnt3A, Wnt1, Wnt5a, BIO, CHIR98014, lithium, 3F8, deoxycholic acid, their derivatives, and mixtures thereof. In certain embodiments, at least one Wnt activator comprises CHIR99021 or a derivative thereof. In certain embodiments, at least one Wnt activator comprises CHIR99021. "CHIR99021" (also known as "aminopyrimidine" or "3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylidenyl]-2-indolinone") refers to the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl)pyrimidine-2-ylamino)ethylamino)nicotininonitrile, which has the following formula: [ka]

[0117] CHIR99021 is highly selective, exhibiting nearly 1000-fold selectivity for a panel of related and unrelated kinases, with an IC50 of 6.7 nM for human GSK3β and a nanomolar IC50 for rodent GSK3β homologs.

[0118] In certain embodiments, cells are exposed to or contacted with at least one Wnt activator for at least about 5 days, at least about 10 days, at least about 15 days, or at least about 20 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator for up to about 5 days, up to about 10 days, up to about 15 days, or up to about 20 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator for about 5 to about 20 days, about 5 to about 15 days, about 10 to about 20 days, about 5 to about 15 days, or about 10 to about 15 days. In certain embodiments, cells are exposed to at least one Wnt activator for about 10 to about 20 days. In certain embodiments, cells are exposed to at least one Wnt activator for about 15 days. In certain embodiments, stem cells are exposed to at least one Wnt signaling activator for 16 days. In certain embodiments, stem cells are exposed to at least one Wnt signaling activator for 17 days. In specific embodiments, cells are exposed to at least one Wnt activator from day 0 to day 16. In specific embodiments, at least one Wnt activator is added to the cell culture medium containing cells every day or every other day from day 0 to day 16. In specific embodiments, at least one Wnt activator is added to the cell culture medium containing cells every day (daily) from day 0 to day 16.

[0119] In certain embodiments, the concentration of at least one Wnt activator increases during cell exposure (also known as "Wnt boost"). In certain embodiments, the increase or Wnt boost begins at least about 2 days, at least about 4 days, or at least about 5 days after the initial exposure of the cells to at least one Wnt activator. In certain embodiments, the increase or Wnt boost begins at about 4 days after the initial exposure of the cells to at least one Wnt activator.

[0120] In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for at least about 5 days, or at least about 10 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for at least about 5 days. In certain embodiments, cells are exposed to at least one Wnt activator at an increased concentration for up to about 5 days, up to about 10 days, or up to about 15 days. In certain embodiments, cells are exposed to at least one Wnt activator at an increased concentration for up to about 10 days.

[0121] In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for about 5 to 15 days, or about 5 to 10 days, or about 10 to 15 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for about 5 to 10 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for about 5 days, about 10 days, or about 15 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for about 5 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for 5 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for 6 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration from day 4 to day 9. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for about 10 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for 12 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration for 13 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt activator at an increased concentration from day 4 to day 16.

[0122] In certain embodiments, the initial concentration of at least one Wnt activator that contacts or exposes the cells before Wnt boosting is less than about 5 μM, less than about 3 μM, or less than about 1.5 μM (including, but not limited to, about 0.01 μM to about 5 μM, about 0.01 μM to about 3 μM, about 0.05 μM to about 3 μM, about 0.1 μM to about 3 μM, about 0.5 μM to about 3 μM, about 0.5 μM to about 2 μM, about 0.5 μM to about 1 μM, or about 0.5 μM to about 1.5 μM). In certain embodiments, the initial concentration of at least one Wnt activator that contacts or exposes the cells before Wnt boosting is about 1 μM. In certain embodiments, the initial concentration of at least one Wnt activator that is contacted or exposed to the cells before Wnt boosting is less than about 1.5 μM, for example, about 1 μM, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, or about 0.9 μM. In certain embodiments, the initial concentration of at least one Wnt activator that is contacted or exposed to the cells before Wnt boosting is about 1 μM. In certain embodiments, the initial concentration of at least one Wnt activator that is contacted or exposed to the cells before Wnt boosting is about 0.5 μM. In certain embodiments, the initial concentration of at least one Wnt activator that is contacted or exposed to the cells before Wnt boosting is about 0.7 μM.

[0123] In certain embodiments, the increased concentration of at least one Wnt activator after Wnt boosting is about 3 μM or greater, about 5 μM or greater, about 10 μM or greater, about 15 μM or greater, or about 20 μM or greater. In certain embodiments, the increased concentration of at least one Wnt activator after Wnt boosting is about 3 μM to about 15 μM, about 3 μM to about 10 μM, or about 5 μM to about 10 μM. In certain embodiments, the increased concentration of at least one Wnt activator after Wnt boosting is about 5 μM to about 10 μM. In certain embodiments, the increased concentration of at least one Wnt activator after Wnt boosting is approximately 3 μM, approximately 3.5 μM, approximately 4 μM, approximately 4.5 μM, approximately 5 μM, approximately 5.5 μM, approximately 6 μM, approximately 6.5 μM, approximately 7 μM, approximately 7.5 μM, approximately 8 μM, approximately 8.5 μM, approximately 9 μM, approximately 9.5 μM, or approximately 10 μM. In certain embodiments, the increased concentration of at least one Wnt activator after Wnt boosting is approximately 3 μM. In certain embodiments, the increased concentration of at least one Wnt activator after Wnt boosting is approximately 6 μM. In certain embodiments, the increased concentration of at least one Wnt activator after Wnt boosting is approximately 7 μM. In certain embodiments, the increased concentration of at least one Wnt activator after Wnt boosting is approximately 7.5 μM.

[0124] In certain embodiments, the concentration of at least one Wnt activator increases from about 50% to about 2000%, or about 100% to about 1500%, or about 150% to about 1500%, or about 200% to about 1500%, or about 250% to about 1500%, or about 300% to about 1500%, or about 300% to about 1000%, or about 300% to about 1000%, or about 300% to about 400%, or about 500% to about 1000%, or about 800% to about 1000%, or about 900% to about 1000%, or about 950% to about 1000%. In certain embodiments, the concentration of at least one Wnt activator increases from about 300% to about 1000% from the initial concentration to which the cells were exposed. In certain embodiments, the concentration of at least one Wnt activator is increased by about 300% to about 500% from the initial concentration to which the cells were exposed or contacted. In certain embodiments, the concentration of at least one Wnt activator is increased by about 900% to about 1000% from the initial concentration to which the cells were exposed or contacted. In certain embodiments, the concentration of at least one Wnt activator is increased by about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1050%, or about 1100% from the initial concentration to which the cells were exposed or contacted. In certain embodiments, the concentration of at least one Wnt activator is increased by about 200% from the initial concentration to which the cells were exposed or contacted. In certain embodiments, the concentration of at least one Wnt activator is increased by about 300% from the initial concentration to which the cells were exposed or contacted. In certain embodiments, the concentration of at least one Wnt activator is increased by about 350% from the initial concentration to which the cells were exposed or contacted. In certain embodiments, the concentration of at least one Wnt activator is increased by about 500% from the initial concentration to which the cells were exposed or contacted. In certain embodiments, the concentration of at least one Wnt activator is increased by about 950% from the initial concentration to which the cells were exposed or contacted. In certain embodiments, the concentration of at least one Wnt activator is increased by about 1000% from the initial concentration to which the cells were exposed or contacted.

[0125] In certain embodiments, the concentration of at least one Wnt activator is increased from about 1 μM to about 5 μM to about 10 μM. In certain embodiments, the concentration of at least one Wnt activator is increased from about 1 μM to about 6 μM. In certain embodiments, the concentration of at least one Wnt activator is increased from about 1 μM to about 3 μM to about 5 μM. In certain embodiments, the concentration of at least one Wnt activator is increased from about 1 μM to about 3 μM.

[0126] In certain embodiments, at least one Wnt activator comprises a GSK3β inhibitor. In certain embodiments, at least one Wnt activator comprises CHIR99021 or a derivative thereof. In certain embodiments, at least one Wnt activator comprises CHIR99021.

[0127] 5.2.4. SHH Activators As used herein, the terms “Sonic Hedgehog,” “SHH,” or “Shh” refer to a protein that is one of at least three proteins in the mammalian signaling pathway family called hedgehog, another being desert hedgehog (DHH), and a third being Indian hedgehog (IHH). SHH interacts with at least two transmembrane proteins by interacting with the transmembrane molecules Patched (PTC) and Smoothened (SMO). SHH typically binds to PTC and then activates SMO as a signaling molecule. In the absence of SHH, PTC typically inhibits SMO, which then activates transcriptional repressors, so that transcription of certain genes does not occur. When SHH is present and binds to PTC, PTC cannot interfere with the function of SMO. If SMO is not inhibited, certain proteins can act as transcription factors that allow them to enter the nucleus and activate specific genes (see Gilbert, 2000 Developmental Biology (Sunderland, Mass., Sinauer Associates, Inc., Publishers). In certain embodiments, an SHH activator refers to any molecule or compound that can activate the SHH signaling pathway, including molecules or compounds that can bind to PTC or SMO. In certain embodiments, at least one SHH activator is selected from the group consisting of molecules that bind to PCT, molecules that bind to SMO, and combinations thereof. Non-limiting examples of SHH activators include International Publication No. 10 / 096496, International Publication No. 13 / 067362, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, and Kriks et al., Nature. 2011 Nov. Examples include those described in 6;480(7378):547-51. In certain embodiments, at least one SHH activator is selected from the group consisting of SHH proteins, SMO agonists, or combinations thereof.In certain embodiments, the SHH protein is selected from the group consisting of recombinant SHH, modified N-terminal SHH, or a combination thereof. In certain embodiments, recombinant SHH comprises an N-terminal fragment and a C-terminal fragment. In certain embodiments, modified N-terminal SHH comprises two isoleucines at the N-terminus. In certain embodiments, modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity with unmodified N-terminal SHH. In certain embodiments, modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity with unmodified human N-terminal SHH. In certain embodiments, modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity with unmodified mouse N-terminal SHH. In certain embodiments, modified N-terminal SHH comprises SHH C25II. In certain embodiments, the modified N-terminal SHH contains SHH C24II.

[0128] Non-limiting examples of SMO agonists (SAGs) include palmorfamine, GSA10, and 20(S)-hydroxycholesterol. In certain embodiments, the SAG includes palmorfamine.

[0129] In certain embodiments, cells are exposed to or in contact with at least one SHH activator for at least about 5 days, or at least about 10 days. In certain embodiments, cells are exposed to or in contact with at least one SHH activator for up to about 5 days, or up to about 10 days. In certain embodiments, cells are exposed to or in contact with at least one SHH activator for about 5 to about 10 days. In certain embodiments, cells are exposed to or in contact with at least one SHH activator for about 5 days. In certain embodiments, cells are exposed to or in contact with at least one SHH activator for 6 days. In certain embodiments, cells are exposed to or in contact with at least one SHH activator for 7 days. In certain embodiments, cells are exposed to or in contact with at least one SHH activator from day 0 to day 6. In certain embodiments, at least one SHH activator is added to the cell culture medium containing the cells every day, or every other day, from day 0 to day 6. In certain embodiments, at least one SHH activator is added to the cell culture medium containing the cells every day from day 0 to day 6.

[0130] In certain embodiments, the concentration of at least one SHH activator that comes into contact with or is exposed to cells is about 50 ng / mL to about 1000 ng / mL, about 100 ng / mL to about 1000 ng / mL, about 20 ng / mL to about 1000 ng / mL, about 300 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 1000 ng / mL, about 500 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 800 ng / mL, about 400 ng / mL to about 700 ng / mL, about 400 ng / mL to about 600 ng / mL, or about 500 ng / mL to about 600 ng / mL. In certain embodiments, the concentration of at least one SHH activator that comes into contact with or is exposed to cells is about 400 ng / mL to about 600 ng / mL. In certain embodiments, the concentration of at least one SHH activator that comes into contact with or is exposed to the cells is about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, or about 600 ng / mL. In certain embodiments, the concentration of at least one SHH activator that comes into contact with or is exposed to the cells is about 500 ng / mL.

[0131] In certain embodiments, at least one SHH signaling activator comprises SHH C25II.

[0132] 5.2.5. FGF Activators The FGF family includes secretory signaling proteins (secretory FGFs) that signal to receptor tyrosine kinases. Phylogenetic analysis suggests that the 22 FGF genes can be arranged into seven subfamilies, each containing 2 to 4 members. The length of the branching is proportional to the evolutionary distance between each gene.

[0133] In certain embodiments, at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF8b, FGF2, FGF4, and their derivatives. In certain embodiments, at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF2, FGF4, and their derivatives. In certain embodiments, at least one FGF activator is selected from the group consisting of FGF8a, FGF17, and FGF18.

[0134] The FGF8 subfamily consists of FGF8a, FGF8b, FGF17, and FGF18. Early patterning of the midbrain and cerebellum in vertebrates is regulated by midbrain / hindbrain organizers that produce FGF8a, FGF8b, FGF17, and FGF18. FGF8b has been shown to function differently from FGF8a, FGF17, and FGF18 (Liu et al., Development. 2003 Dec;130(25):6175-85). FGF8b is the only protein that can induce the r1 gene Gbx2, strongly activate the pathway inhibitor Spry1 / 2, and repress the midbrain gene Otx2 (Liu 2003). Furthermore, FGF8b extends organizers along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellar development (Liu 2003). In contrast, FGF8a, FGF17, and FGF18 induce midbrain expansion and upregulation of midbrain gene expression (Liu 2003).

[0135] In certain embodiments, at least one FGF activator can induce midbrain enlargement and upregulate midbrain gene expression. In certain embodiments, at least one FGF activator can promote midbrain development. In certain embodiments, at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF2, FGF4, their derivatives, and combinations thereof. In certain embodiments, at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, and combinations thereof. In certain embodiments, at least one FGF activator includes or is FGF18.

[0136] In certain embodiments, cells are exposed to or contacted with at least one FGF activator for at least about 1 day, at least about 3 days, at least about 5 days, at least about 8 days, or at least about 10 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for at least about 5 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for at least 4 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for up to about 5 days (e.g., up to 5 days, up to 6 days, or up to 7 days), or up to about 10 days (e.g., up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days), or up to about 15 days, or up to about 20 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for at least 4 days and / or up to 7 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for about 1 to 20 days, about 1 to 15 days, about 1 to 5 days, about 5 to 20 days, about 5 to 15 days, or about 5 to 10 days, or about 10 to 20 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for about 1 to 10 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for about 3 days, about 5 days, or about 8 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for about 1 to 5 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for about 5 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for about 4 days. In certain embodiments, cells are exposed to or contacted with at least one FGF activator for 5 days.

[0137] In certain embodiments, cell contact or exposure to at least one FGF activator is initiated at least about 5 days or at least about 10 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one FGF activator is initiated within about 15 days or within about 20 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one FGF activator is initiated within 18 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one FGF activator is initiated at least about 5 to about 20 days, about 5 to about 20 days, about 10 to about 15 days, about 10 to about 18 days, about 5 to about 15 days, or about 10 to about 20 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one FGF activator is initiated about 5 to 10 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one FGF activator is initiated about 10 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one FGF activator is initiated 12 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one FGF activator is initiated 13 days after the initial cell contact or exposure to at least one SMAD inhibitor.

[0138] In certain embodiments, cell contact or exposure to at least one FGF activator is initiated about 10 days after the initial cell contact or exposure to at least one SMAD inhibitor, and the cells are in contact with the above-mentioned at least FGF activator for about 5 days. In certain embodiments, cell contact or exposure to at least one FGF activator is initiated 12 or 13 days after the initial cell contact or exposure to at least one SMAD inhibitor, and the cells are in contact with the above-mentioned at least one FGF activator for 4 or 5 days. In certain embodiments, cells are in contact with or exposed to at least one FGF activator from day 12 to day 16. In certain embodiments, at least one FGF activator is added to the cell culture medium containing the cells every day or every other day from day 12 to day 16. In certain embodiments, at least one FGF activator is added to the cell culture medium containing the cells every day (daily) from day 12 to day 16.

[0139] In certain embodiments, the concentration of at least one FGF activator that comes into contact with or is exposed to cells is about 10 ng / mL to about 500 ng / mL, about 50 ng / mL to about 500 ng / mL, about 100 ng / mL to about 500 ng / mL, about 100 ng / mL to about 400 ng / mL, about 100 ng / mL to about 300 ng / mL, about 100 ng / mL to about 200 ng / mL, or about 100 ng / mL to about 250 ng / mL. In certain embodiments, the concentration of at least one FGF activator that comes into contact with or is exposed to cells is about 100 ng / mL to about 200 ng / mL. In certain embodiments, the concentration of at least one FGF activator that comes into contact with or is exposed to cells is about 100 ng / mL. In certain embodiments, the concentration of at least one FGF activator that comes into contact with or is exposed to cells is about 200 ng / mL.

[0140] In certain embodiments, at least one FGF activator comprises FGF18.

[0141] 5.2.6. Wnt inhibitors Wnt signaling includes classical Wnt signaling and non-classical Wnt signaling. In certain embodiments, at least one Wnt inhibitor can inhibit classical Wnt signaling. In certain embodiments, at least one Wnt inhibitor can inhibit both classical and non-classical Wnt signaling. Non-exclusive examples of Wnt inhibitors that can inhibit both classical and non-classical Wnt signaling include IWP2, IWR1-endo, IWP-O1, Wnt-C59, IWP-L6, IWP12, LGK-974, IWR-1, ETC-159, iCRT3, IWP-4, salinomycin, pyrvinium pamoate, iCRT14, FH535, CCT251545, Wogonin, NCB-0846, hexachlorophene, KY02111, SO3031 (KY01-I), SO2031 (KY02-I), BC2059, PKF115-584, quercetin, NSC668036, G007-LK, and their derivatives. In certain embodiments, at least one Wnt inhibitor is IWP2, IWR1-endo, XAV939, IWP-O1, Wnt-C59, IWP-L6, LGK-974, IWR-1, Wnt-C59, ETC-159, iCRT3, IWP-4, ICG-001, salinomycin, pyrvinium pamoate, iCRT14, FH535, CCT251545, KYA1797K, Wogonin, NCB-0846, hexachlorophene, PN The following are selected from the group consisting of U-74654, KY02111, SO3031 (KY01-I), SO2031 (KY02-I), tryptonide, IWP12, BC2059, PKF115-584, quercetin, NSC668036, G007-LK, MSAB, LF3, JW55, isoquercitrin, WIKI4 (Wnt inhibitor kinase inhibitor 4), their derivatives, and combinations thereof.In certain embodiments, at least one Wnt signaling inhibitor is selected from the group consisting of IWP2, IWR1-endo, IWP-O1, IWP12, Wnt-C59, IWP-L6, LGK-974, IWR-1, ETC-159, iCRT3, IWP-4, salinomycin, pyrvinium pamoate, iCRT14, FH535, CCT251545, Wogonin, NCB-0846, hexachlorophene, KY02111, SO3031(KY01-I), SO2031(KY02-I), BC2059, PKF115-584, quercetin, NSC668036, G007-LK, their derivatives, and combinations thereof. In certain embodiments, at least one Wnt signaling inhibitor is selected from the group consisting of XAV939, ICG-001, PNU-74654, tryptonide, KYA1797K, MSAB, LF3, JW55, isoquercitrin, WIKI4, their derivatives, and combinations thereof. In certain embodiments, at least one Wnt inhibitor includes IWP2 or a derivative thereof.

[0142] In certain embodiments, cells are exposed to or contacted with at least one Wnt inhibitor for at least about 1 day, at least about 3 days, at least about 5 days, at least about 8 days, at least about 10 days, at least about 15 days, or at least about 20 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt inhibitor for up to about 5 days, up to about 10 days, up to about 15 days, up to about 20 days, up to about 25 days, or up to about 30 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt inhibitor for about 1 to 20 days, about 1 to 15 days, about 1 to 5 days, about 5 to 20 days, about 5 to 15 days, or about 5 to 10 days, about 10 to 20 days, about 10 to 15 days, or about 15 to 20 days, about 10 to 30 days, about 10 to 25 days, about 15 to 30 days, about 15 to 25 days, about 20 to 30 days, about 20 to 25 days, or about 25 to 30 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt inhibitor for about 1 to 10 days. In certain embodiments, cells are exposed to or contacted with at least one Wnt inhibitor for about 10 to 15 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for about 15 to about 20 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for about 5 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for about 15 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for about 20 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 4 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 5 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 6 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 7 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 14 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 15 days.In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 19 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 20 days. In certain embodiments, cells are exposed to or contact with at least one Wnt inhibitor for 16, 17, 18, 21, 22, or 23 days.

[0143] In certain embodiments, the cells brought into contact with at least one Wnt inhibitor include mDA neuron precursors and mDA neurons.

[0144] In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated at least about 5 days or at least about 10 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated within about 15 days or within about 20 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated at least about 5 to about 20 days, about 5 to about 20 days, about 10 to about 15 days, about 5 to about 15 days, or about 10 to about 20 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated at least 5 to about 10 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated about 10 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated 10 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated 11 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated 12 days after the initial cell contact or exposure to at least one SMAD inhibitor. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated 13 days after the initial cell contact or exposure to at least one SMAD inhibitor.

[0145] In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated about 10 days after the initial cell contact or exposure to at least one SMAD inhibitor, and the cells are in contact with the at least Wnt inhibitor for about 5 days. In certain embodiments, cell contact or exposure to at least one Wnt inhibitor is initiated 12 or 13 days after the initial cell contact or exposure to at least one SMAD inhibitor, and the cells are in contact with the at least one Wnt inhibitor for 4 or 5 days. In certain embodiments, cells are exposed to at least one Wnt inhibitor from day 12 to day 16. In certain embodiments, at least one Wnt inhibitor is added to the cell culture medium containing the cells every day or every other day from day 12 to day 16. In certain embodiments, at least one Wnt inhibitor is added to the cell culture medium containing the cells every day (daily) from day 12 to day 16. In certain embodiments, cells are exposed to or contacted with at least one Wnt inhibitor from day 12 to day 25. In certain embodiments, at least one Wnt inhibitor is added to the cell culture medium containing the cells every day or every other day from day 12 to day 25. In certain embodiments, at least one Wnt inhibitor is added to the cell culture medium containing the cells every day (daily) from day 12 to day 25. In certain embodiments, cells are exposed to or contacted with at least one Wnt inhibitor from day 12 to day 30. In certain embodiments, at least one Wnt inhibitor is added to the cell culture medium containing the cells every day or every other day from day 12 to day 30. In certain embodiments, at least one Wnt inhibitor is added to the cell culture medium containing the cells every day (daily) from day 12 to day 30. In certain embodiments, cells are exposed to or contacted simultaneously with at least one Wnt inhibitor and at least one FGF activator. In certain embodiments, at least one Wnt inhibitor and at least one FGF activator are added together to a cell culture medium containing cells.

[0146] In certain embodiments, the concentration of at least one Wnt inhibitor to be brought into contact with or exposed to the cells is approximately 0.5 μM to approximately 20 μM, approximately 0.5 μM to approximately 10 μM, approximately 0.5 μM to approximately 5 μM, approximately 0.5 μM to approximately 1 μM, approximately 0.5 μM to approximately 2 μM, approximately 5 μM to approximately 10 μM, approximately 10 μM to approximately 20 μM, approximately 1 μM to approximately 2 μM, or approximately 1 μM to approximately 5 μM. In certain embodiments, the concentration of at least one Wnt inhibitor to be brought into contact with or exposed to the cells is approximately 0.5 μM to approximately 2 μM. In certain embodiments, the concentration of at least one Wnt inhibitor to be brought into contact with or exposed to the cells is approximately 1 μM.

[0147] In certain embodiments, at least one Wnt inhibitor comprises IWP2.

[0148] 5.2.7. Illustrative Methods In certain embodiments, stem cells are treated with at least one TGFβ / Activin-Nodal inhibitor (e.g., SB431542, e.g., at a concentration of about 10 μM), at least one BMP inhibitor (e.g., LDN193189, e.g., at a concentration of about 250 nM), and at least one SHH activator (e.g., SHH The cells are then exposed to or in contact with C25II (e.g., at a concentration of approximately 500 ng / mL) for approximately 5 days (e.g., 7 days, e.g., from day 0 to day 6), and then exposed to at least one Wnt activator (e.g., CHIR99021, e.g., at a concentration of approximately 1 μM for approximately 5 days (e.g., 4 days, e.g., from day 0 to day 3), and at a concentration of approximately 6 μM for approximately 5 days (e.g., 6 days, e.g., from day 4 to day 9), and at a concentration of approximately 3 μM for approximately 5 days (e.g., 7 days, e.g., from day 10 to day 16). The cells are then exposed to or in contact with at least one FGF activator (e.g., FGF18, e.g., at a concentration of approximately 100 ng / mL), where the contact between the cells and at least one FGF activator is approximately 10 days (e.g., 10 days or) from the initial contact between the cells and at least one SMAD inhibitor. The contact between cells and at least one FGF activator is initiated on day 12, and the cells are exposed to at least one FGF activator for approximately 5 days (e.g., 5 days (from day 12 to day 16) or 7 days (e.g., from day 10 to day 16). The cells are then exposed to at least one Wnt inhibitor (e.g., IWP2, at a concentration of approximately 1 μM), where the contact between cells and at least one Wnt inhibitor is initiated approximately 10 days (e.g., day 10 or 12) from the initial contact between cells and at least one SMAD inhibitor, and the cells are exposed to at least one Wnt inhibitor for approximately 5 days (e.g., 5 days (from day 12 to day 16), 7 days (e.g., from day 10 to day 16), approximately 15 days (e.g., 14 days (from day 12 to day 25)), or approximately 20 days (e.g., 19 days (from day 12 to day 30)).

[0149] 5.2.8. Cell Culture Medium In certain embodiments, the above-mentioned inhibitors and activators are added to a cell culture medium containing cells. Suitable cell culture media include, but are not limited to, Knockout® Serum Replacement ("KSR") medium, Neurobasal® medium (NB), N2 medium, B-27 medium, and Essential 8® / Essential 6® ("E8 / E6") medium, as well as combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium, and E8 / E6 medium are commercially available. KSR medium is a prescribed serum-free formulation optimized for growing and maintaining undifferentiated hESCs in culture.

[0150] In certain embodiments, the cell culture medium is KSR medium. The components of KSR medium are disclosed in International Publication No. 2011 / 149762. In certain embodiments, KSR medium comprises knockout DMEM, knockout serum substitute, L-glutamine, Pen / Strep, MEM, and 13-mercaptoethanol. In certain embodiments, 1 liter of KSR medium contains 820 mL of knockout DMEM, 150 mL of knockout serum substitute, 10 mL of 200 mM L-glutamine, 10 mL of Pen / Strep, 10 mL of 10 mM MEM, and 55 μM of 13-mercaptoethanol.

[0151] In certain embodiments, the cell culture medium is E8 / E6 medium. E8 / E6 medium is a feeder-free and xeno-free medium that supports the growth and proliferation of human pluripotent stem cells. E8 / E6 medium has been shown to support somatic cell reprogramming. Furthermore, E8 / E6 medium can be used as a base for formulating custom media for PSC culture. An example of E8 / E6 medium is described in Chen et al., Nat Methods 2011 May;8(5):424-9, which is incorporated in its entirety by reference. Another example of E8 / E6 medium is disclosed in International Publication No. 15 / 077648, which is incorporated in its entirety by reference. In certain embodiments, the E8 / E6 cell culture medium contains DMEM / F12, ascorbic acid, selenium, insulin, NaHCO3, transferrin, FGF2, and TGFβ. E8 / E6 medium differs from KSR medium in that it does not contain active BMP components. Therefore, in certain embodiments, when culturing the stem cells of the present disclosure using E8 / E6 medium to differentiate them into mDA neurons or their precursors, it is not necessary to add at least one BMP inhibitor to the E8 / E6 medium. In certain embodiments, when culturing the stem cells of the present disclosure using E8 / E6 medium to differentiate them into mDA neurons or their precursors, at least one BMP inhibitor is added to the E8 / E6 medium.

[0152] 5.2.9. Differentiated cells In certain embodiments, the method involves obtaining a population of differentiated cells in which at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the differentiated cells express at least one marker indicating mDA neurons or their precursors. Non-limiting examples of markers indicating mDA neurons or their precursors include engrailed-1 (EN1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor-associated protein 1 (NURR1), forkheadbox protein A2 (FOXA2), and LIM homeobox transcription factor 1α (LMX1A), PITX3, LMO3, SNCA, ADCAP1, CHRNA4, ALDH1A1, DAT, VMAT1, SOX6, WNT1, and GIRK2.

[0153] In certain embodiments, differentiated cells express at least one marker indicating an mDA neuron or its precursor at least about 10 days (e.g., about 15, 20, 30, 40, or 50 days) after initial contact between the cell and at least one SMAD inhibitor. In certain embodiments, differentiated cells express at least one marker indicating an mDA neuron or its precursor at about 15 days (e.g., 15, 16, or 17 days) after initial contact between the cell and at least one SMAD inhibitor.

[0154] Treatment of cells with at least a Wnt inhibitor can improve mDA neuron induction. In certain embodiments, treatment of cells with at least a Wnt inhibitor increases the expression of at least one of the A9 subtype mDA neuron marker, the A10 subtype mDA neuron marker, and the mDA neuron maturation marker. In certain embodiments, treatment of cells with at least a Wnt inhibitor increases the expression of ALDH1A1. In certain embodiments, treatment of cells with at least a Wnt inhibitor increases the expression of CALB1. In certain embodiments, treatment of cells with at least a Wnt inhibitor increases the expression of DAT. In certain embodiments, treatment of cells with at least a Wnt inhibitor increases the expression of VMAT2. In certain embodiments, treatment of cells with at least a Wnt inhibitor increases the expression of both DAT and VMAT2.

[0155] In certain embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%) of differentiated cells express ALDH1A1 about 15 days after initial contact between stem cells and at least one SMAD signaling inhibitor. In certain embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%) of differentiated cells express ALDH1A1 about 16 days after initial contact between stem cells and at least one SMAD signaling inhibitor. In certain embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%) of differentiated cells express ALDH1A1 about 25 days after initial contact between stem cells and at least one SMAD signaling inhibitor. In certain embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%) of the differentiated cells express ALDH1A1 about 30 days after initial contact between the stem cells and at least one SMAD signaling inhibitor.

[0156] Furthermore, the mDA neurons or their precursors generated by the methods disclosed herein exhibit improved fiber growth and decreased residual Ki67 +The cells proliferate and have improved in vivo survival, making these cells more suitable for therapeutic use. In certain embodiments, mDA neurons or their precursors produced by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker for at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after in vivo transplantation. In certain embodiments, mDA neurons or their precursors produced by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker for at least about 2 weeks after in vivo transplantation. In certain embodiments, mDA neurons or their precursors produced by the methods disclosed herein may have a detectable expression level of at least one mDA neuron marker for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 1 year, up to about 2 years, up to about 3 years, up to about 4 years, or up to about 5 years after in vivo implantation. In certain embodiments, mDA neurons or their precursors produced by the methods disclosed herein may have a detectable expression level of at least one mDA neuron marker for about 1 month after in vivo implantation. In certain embodiments, mDA neurons or their precursors produced by the methods disclosed herein may have a detectable expression level of at least one mDA neuron marker for about 2 months after in vivo implantation. In certain embodiments, mDA neurons or their precursors produced by the methods disclosed herein may have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1A1 for at least about 1 month after in vivo implantation. In certain embodiments, mDA neurons or their precursors generated by the methods disclosed herein have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1A1 for about two months after in vivo implantation.In certain embodiments, mDA neurons or their precursors generated by the methods disclosed herein have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1A1 for at least about two months after in vivo implantation.

[0157] In certain embodiments, differentiated cells derived from the method of the present disclosure do not express or have low expression of at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0158] In certain embodiments, cells are contacted with the activators and inhibitors described herein at concentrations and for durations effective in reducing the expression of SMA, SIX1, PITX2, SIM1, POU4F1, and / or PHOX2A. In certain embodiments, cells are contacted with the activators and inhibitors described herein at concentrations and for durations effective in reducing the expression of PAX6, BARHL1, and / or BARHL2.

[0159] In certain embodiments, at least about 80% of differentiated cells express FOXA2 and EN1 about 15 days after initial contact between stem cells and at least one SMAD signaling inhibitor. In certain embodiments, more than about 80% (e.g., more than about 85% or more than about 90%) of differentiated cells express FOXA2 and EN1 16 days after initial contact between stem cells and at least one SMAD signaling inhibitor.

[0160] 5.2.10. Selection Method In certain embodiments, the differentiation method disclosed herein further comprises isolating mDA neurons and their precursors based on at least one or at least two surface markers. In certain embodiments, the surface marker is a negative surface marker, and the cells do not express detectable levels of the negative surface marker. In certain embodiments, the surface marker is a positive surface marker, and the cells express detectable levels of the positive surface marker.

[0161] In certain embodiments, the differentiation method disclosed herein further includes isolating cells that do not express at least one negative surface marker at a detectable level. In certain embodiments, the differentiation method disclosed herein further includes isolating cells that express at least one positive surface marker at a detectable level. In certain embodiments, the differentiation method disclosed herein further includes isolating cells that do not express at least one negative surface marker at a detectable level and express at least one positive surface marker at a detectable level.

[0162] In certain embodiments, at least one negative surface marker is selected from the group consisting of CD49e, CD99, CD340, and combinations thereof. In certain embodiments, at least one negative surface marker includes CD49e. In certain embodiments, at least one positive surface marker is selected from the group consisting of CD171, CD184, and combinations thereof. In certain embodiments, at least one positive surface marker includes CD184.

[0163] In certain embodiments, the differentiation method disclosed herein further includes isolating cells that do not express detectable levels of CD49e and express detectable levels of CD184.

[0164] Any cell isolation technique based on any surface marker known in the art can be used in the method of this disclosure. In certain embodiments, flow cytometry is used in the isolation method of this disclosure.

[0165] 5.2.11. Differentiation of mDA precursors into mDA neurons In certain embodiments, cells (e.g., mDA precursors) are further contacted with DA neuronal lineage-specific activators and inhibitors, such as L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor β (TGFβ, e.g., TGFβ3), ascorbic acid (AA), and DAPT (also known as N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-l,l-dimethylethyl ester; LY-374973,N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; or N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). In certain embodiments, cells are exposed to the aforementioned DA neuron system-specific activators and inhibitors for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days or more, for example, about 2 to about 20 days, about 3 to about 19 days, about 4 to about 18 days, about 5 to about 17 days, about 6 to about 16 days, about 7 to about 15 days, about 8 to about 15 days, about 9 to about 14 days, or about 10 to about 13 days. In certain embodiments, cells are exposed to the aforementioned DA neuron system-specific activators and inhibitors for a maximum of about 2 days, a maximum of about 3 days, a maximum of about 4 days, a maximum of about 5 days, a maximum of about 6 days, a maximum of about 7 days, a maximum of about 8 days, a maximum of about 9 days, or a maximum of about 10 days or more. In certain embodiments, cells are exposed to the aforementioned DA neuron system-specific activators and inhibitors for approximately 4, 5, 6, 7, or 8 days.

[0166] In certain embodiments, cells are contacted with L-glutamine at concentrations of approximately 0.5 mM to 5 mM, or approximately 1 mM to 5 mM, or approximately 1.5 mM to 2.5 mM, or approximately 1 mM to 2 mM. In certain embodiments, cells are contacted with L-glutamine at a concentration of approximately 2 mM.

[0167] In certain embodiments, cells are contacted with BDNF at concentrations of approximately 5 ng / ml to 50 ng / mL, or approximately 10 ng / ml to 50 ng / mL, or approximately 10 ng / ml to 40 ng / mL, or approximately 20 ng / ml to 50 ng / mL, or approximately 20 ng / ml to 40 ng / mL, or approximately 10 ng / ml to 30 ng / mL, or approximately 10 ng / ml to 20 ng / mL, or approximately 20 ng / ml to 30 ng / mL. In certain embodiments, cells are contacted with BDNF at a concentration of approximately 20 ng / mL.

[0168] In certain embodiments, cells are contacted with ascorbic acid (AA) at concentrations of approximately 50 nM to 500 nM, or approximately 100 nM to 500 nM, or approximately 100 nM to 400 nM, or approximately 200 nM to 400 nM, or approximately 200 nM to 300 nM, or approximately 100 nM to 300 nM. In certain embodiments, cells are contacted with AA at a concentration of approximately 200 nM.

[0169] In certain embodiments, cells are contacted with GDNF at concentrations of approximately 5 ng / ml to 50 ng / mL, or approximately 10 ng / ml to 50 ng / mL, or approximately 10 ng / ml to 40 ng / mL, or approximately 20 ng / ml to 50 ng / mL, or approximately 20 ng / ml to 40 ng / mL, or approximately 10 ng / ml to 30 ng / mL, or approximately 10 ng / ml to 20 ng / mL, or approximately 20 ng / ml to 30 ng / mL. In certain embodiments, cells are contacted with GDNF at a concentration of approximately 20 ng / mL.

[0170] In certain embodiments, cells are contacted with cAMP at concentrations of approximately 200 nM to 800 nM, or approximately 200 nM to 700 nM, or approximately 300 nM to 700 nM, or approximately 300 nM to 600 nM, or approximately 400 nM to 600 nM, or approximately 450 nM to 550 nM. In certain embodiments, cells are contacted with cAMP at a concentration of approximately 500 nM.

[0171] In certain embodiments, cells are contacted with TGFβ3 at concentrations of approximately 0.01 ng / ml to 5 ng / mL, or approximately 0.1 ng / ml to 4 ng / mL, or approximately 0.5 ng / ml to 5 ng / mL, or approximately 1 ng / ml to 3 ng / mL, or approximately 1 ng / ml to 2 ng / mL. In certain embodiments, cells are contacted with TGFβ3 at a concentration of approximately 1 ng / mL.

[0172] In certain embodiments, cells are brought into contact with DAPT at concentrations of approximately 1 nM to approximately 50 nM, or approximately 5 nM to approximately 50 nM, or approximately 1 nM to approximately 20 nM, or approximately 5 nM to approximately 20 nM, or approximately 1 nM to approximately 10 nM, or approximately 5 nM to approximately 10 nM, or approximately 5 nM to approximately 15 nM, or approximately 10 nM to approximately 20 nM, or approximately 10 nM to approximately 30 nM, or approximately 30 nM to approximately 50 nM. In certain embodiments, cells are brought into contact with DAPT at a concentration of approximately 10 nM.

[0173] In certain embodiments, the differentiated midbrain DA precursor is further cultured as described in U.S. Patent Application Publication No. 2015 / 0010514, which is incorporated in whole by reference.

[0174] 5.3. Cell populations and compositions This disclosure provides a cell population of in vitro differentiated cells obtained by the methods disclosed herein, for example, in Section 5.2.

[0175] This disclosure provides a population of in vitro differentiated cells in which at least about 50% (e.g., at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of the cells express at least one marker indicating mDA neurons or their precursors. Non-limiting examples of markers indicating mDA neurons or their precursors include EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, SOX6, ALDH1A1, WNT1, DAT, VMAT1, and GIRK2. This disclosure also provides compositions comprising such a cell population. In certain embodiments, the in vitro differentiated cells are obtained by a differentiation method described herein, for example, in Section 5.2.

[0176] In certain embodiments, less than 50% of differentiated cells (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%) express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0177] Furthermore, this disclosure provides compositions comprising any of the cell populations disclosed herein.

[0178] In certain embodiments, the cells are included in a composition further comprising a biocompatible scaffold or matrix, for example, a biocompatible three-dimensional scaffold that promotes tissue regeneration when the cells are implanted or transplanted into a target. In certain embodiments, the biocompatible scaffold comprises extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogels. (See, for example, U.S. Patent Application Publications 2015 / 0159135, 2011 / 0296542, 2009 / 0123433 and 2008 / 0268019. The contents of each of these are incorporated in whole by reference.) In certain embodiments, the composition further comprises growth factors for promoting the maturation of cells implanted / transplanted into midbrain DA cells.

[0179] In a particular embodiment, the composition is approximately 1 x 10 4 ~approximately 1x10 10 Approximately 1x10 4 ~approximately 1x10 5 Approximately 1x10 5 ~approximately 1x10 9 Approximately 1x10 5 ~approximately 1x10 6 Approximately 1x10 5 ~approximately 1x10 7 Approximately 1x10 6 ~approximately 1x10 7 Approximately 1x10 6 ~approximately 1x10 8 Approximately 1x10 7 ~approximately 1x10 8 Approximately 1x10 8 ~approximately 1x10 9 Approximately 1x10 8 ~approximately 1x10 10 , or approximately 1x10 9 ~approximately 1x10 10 It includes a population of cells, and the cells are administered to the target. In a particular embodiment, approximately 1 × 10 5 ~Approx. 1×10 7 Each of these cells is administered to the target.

[0180] In certain embodiments, the composition is frozen. In certain embodiments, the composition further comprises, but is not limited to, at least one cryoprotectant, such as dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or a combination thereof.

[0181] In certain embodiments, the composition further comprises a biocompatible scaffold or matrix, for example, a biocompatible three-dimensional scaffold that promotes tissue regeneration when cells are implanted or transplanted into a target. In certain embodiments, the biocompatible scaffold comprises extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, polysaccharides, and / or hydrogels. (See, for example, U.S. Patent Application Publications 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of these incorporated in whole by reference).

[0182] In certain embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition can be used to prevent and / or treat neurodegenerative disorders, including Parkinson's disease, Huntington's disease, Alzheimer's disease, and multiple sclerosis.

[0183] The subject matter of this disclosure also provides devices comprising differentiated cells or compositions thereof as disclosed herein. Non-limiting examples of devices include syringes, microglass tubes, stereotactic needles, and cannulas.

[0184] 5.4 Methods for preventing, modeling, and / or treating neurological disorders Cell populations and compositions disclosed herein (e.g., those disclosed in Section 5.3) can be used to prevent, model and / or treat at least the signs of neurological disorders in subjects. The subject matter of this disclosure provides methods for preventing, modeling and / or treating at least the signs of neurological disorders in subjects. In certain embodiments, the method comprises administering an effective amount of stem cell-derived mDA neurons or compositions thereof of this disclosure to a subject suffering from a neurological disorder. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0185] In certain embodiments, the neurological disorder is characterized by a decrease in midbrain dopamine neuron function. This decrease in midbrain dopamine neuron function may be age-related.

[0186] In certain embodiments, signs of neurological disorders are selected from the group consisting of tremor, bradykinesia, flexion, postural instability, tonicity, dysphagia, and dementia.

[0187] Non-specific examples of neurological disorders include parkinsonism, Parkinson's disease, Huntington's disease, Alzheimer's disease, and multiple sclerosis. In certain embodiments, the neurological disorder is parkinsonism or Parkinson's disease.

[0188] In certain embodiments, the neurological disorder is Parkinson's disease. The main motor signs of Parkinson's disease include, but are not limited to, tremors of the hands, arms, legs, jaw, and face; bradykinesia or slowness of movement; rigidity or stiffness of the limbs and trunk; and postural instability or impairment of balance and coordination.

[0189] In certain embodiments, the neurological disorder is a parkinsonist disorder, which refers to a disorder associated with a deficiency of dopamine in the basal ganglia, the part of the brain that controls movement. Signs include tremor, bradykinesia (extremely slow movement), flexion, postural instability, and tonicity. Non-exclusive examples of parkinsonist disorders include corticobasal degeneration, Lewy body dementia, multiple system atrophy, and progressive supranuclear palsy.

[0190] Cells or compositions may be administered or provided systemically or directly to a subject to prevent, model, and / or treat neurological disorders. In certain embodiments, cells or compositions are injected directly into the organ of interest (e.g., the central nervous system (CNS)). In certain embodiments, cells or compositions are injected directly into the striatum.

[0191] Cells or compositions can be administered in any physiologically acceptable vehicle. Cells or compositions can be administered by local injection, orthotopic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In certain embodiments, cells or compositions are administered to subjects suffering from neurodegenerative disorders via orthotopic (OT) injection.

[0192] Cells or compositions may be provided as appropriate as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions that can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient for administration, particularly by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide a longer contact period with specific tissues. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating a composition of the subject matter of this disclosure, for example, a composition containing the stem cell-derived precursor of this disclosure, into the required amount of a suitable solvent, along with various amounts of other components as needed. Such compositions may be mixed with suitable carriers, diluents, or excipients such as sterile water, saline, glucose, or dextrose. The compositions may also be lyophilized. Depending on the route of administration and the desired preparation, the composition may contain auxiliary substances such as wetting agents, dispersants or emulsifiers (e.g., methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavoring agents, and coloring agents. Suitable preparations can be prepared without excessive experimentation by referring to standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition (1985), which is incorporated herein by reference.

[0193] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of microbial action can be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Long-term absorption of injectable pharmaceutical forms can be achieved by using absorption-delaying agents, such as aluminum inurn monostearate and gelatin.

[0194] The viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickener, if desired. Methylcellulose can be used because it is readily and economically available and easy to handle. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer. The concentration of the thickener may depend on the selected agent. The important point is to use an amount that achieves the desired viscosity. The selection of a suitable carrier and other additives depends on the exact route of administration and the specific dosage form, for example, the properties of the liquid dosage form (e.g., whether the composition is formulated in a solution, suspension, gel or other liquid form, e.g., time-release or liquid-filled form).

[0195] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and will not affect the viability or efficacy of the stem cell-derived precursors of this disclosure. This does not present a problem for those skilled in the chemical and pharmaceutical principles, or the problem can be easily avoided by referring to standard texts from this disclosure and the literature cited herein, or by simple experiments (not excessive experiments).

[0196] One consideration regarding the therapeutic use of cells is the quantity of cells required to achieve the optimal effect. Optimal effects include, but are not limited to, regrowth of the CNS region affected by neurodegenerative disorder in the target and / or improvement of the function of the target CNS.

[0197] An effective dose (or therapeutic effective dose) is the amount sufficient to produce a beneficial or desired clinical outcome at the time of treatment. An effective dose can be administered to a subject in at least one dose. With respect to treatment, an effective dose is the amount sufficient to alleviate, improve, stabilize, reverse or delay the progression of neurodegenerative disease, or to mitigate the pathological consequences of neurodegenerative disease in other ways. The effective dose is generally determined by the physician on a case-by-case basis and is within the scope of the skill of those skilled in the art. Several factors are usually considered when determining the appropriate dose to achieve an effective dose. These factors include the subject's age, sex, and weight, the symptoms being treated, the severity of the symptoms, and the morphology and effective concentration of the cells being administered.

[0198] In certain embodiments, the effective amount of cells is sufficient to regrow the CNS region of the subject suffering from neurodegenerative disease. In certain embodiments, the effective amount of cells is sufficient to improve the function of the CNS of the subject suffering from neurodegenerative disease, for example, the improvement in function may be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the function of the CNS of a normal person.

[0199] The amount of cells administered varies depending on the target being treated. In certain embodiments, approximately 1 x 10⁻⁶ cells are administered. 4 ~approximately 1x10 10 Approximately 1x10 4 ~approximately 1x10 5 Approximately 1x10 5 ~approximately 1x10 9 Approximately 1x10 5 ~approximately 1x10 6 Approximately 1x10 5 ~approximately 1x10 7 Approximately 1x10 6 ~approximately 1x10 7 Approximately 1x10 6 ~approximately 1x10 8 Approximately 1x10 7 ~approximately 1x10 8 Approximately 1x10 8 ~approximately 1x10 9 Approximately 1x10 8 ~approximately 1x1010 , or approximately 1x10 9 ~approximately 1x10 10 Individual cells are administered to the target. In a particular embodiment, approximately 1 × 10⁶ cells are administered. 5 pieces~approx. 1×10 7 Individual cells are administered to subjects suffering from neurological disorders. In certain embodiments, approximately 1 × 10⁶ cells are administered. 6 pieces~approx. 1×10 7 Individual cells are administered to subjects suffering from neurological disorders. In certain embodiments, approximately 1 × 10⁶ cells are administered. 6 pieces~approx. 4×10 6 Individual cells are administered to subjects suffering from neurological disorders. The precise determination of what is considered an effective dose may be based on individual factors for each subject, including the size, age, sex, weight, and symptoms of the particular subject. The dosage can be readily determined by those skilled in the art from this disclosure and knowledge of the art.

[0200] 5.5. Kit The subject of this disclosure is a kit for inducing the differentiation of stem cells into mDA neurons or their precursors. In certain embodiments, the kit comprises (a) at least one SMAD signaling inhibitor, (b) at least one Wnt signaling activator, (c) at least one SHH signaling activator, (d) at least one FGF signaling activator, and (e) at least one Wnt signaling inhibitor. In certain embodiments, the kit further comprises (f) instructions for inducing the differentiation of stem cells into a population of differentiated cells expressing at least one marker indicating mDA neurons or their precursors.

[0201] In certain embodiments, the description includes contacting stem cells with inhibitors and activators in a specific order. The order in which the stem cells are contacted with inhibitors and activators can be determined by the cell culture medium used for culturing the stem cells.

[0202] In certain embodiments, the description includes contacting stem cells with an inhibitor(s) and an activator(s) as described by the methods of the present disclosure (see Section 5.2).

[0203] In certain embodiments, the Disclosure provides a kit comprising an effective amount of a cell population or composition disclosed herein in unit dosage form. In certain embodiments, the kit comprises a sterile container containing the therapeutic composition. Such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the Art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding the drug.

[0204] In certain embodiments, the kit includes instructions for administering a population of cells or a composition to a subject suffering from a neurological disorder. The instructions may include information on the use of the cells or composition to prevent, model and / or treat at least the signs of the neurological disorder in a subject. In certain embodiments, the instructions may include at least one of the following: a description of the therapeutic agent; a dosing schedule and administration for preventing, modeling and / or treating at least the signs of the neurological disorder in a subject suffering from or suffering from a neurological disorder; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if any), as a label affixed to the container, or as a separate sheet, brochure, card, or folder supplied inside or with the container. [Examples]

[0205] 6. Examples The subject matter of this disclosure will be better understood by referring to the following examples provided as illustrations of the subject matter of this disclosure, rather than being limited thereto.

[0206] Example 1: Exemplary Midbrain DA Neuron Differentiation Protocol The following is an exemplary protocol of the method of the present disclosure according to a specific embodiment.

[0207] Day 0: The cells were supplied with Accutase derived from hPSC / hiPSC as single cells and plated at a density of 400,000 cells / cm 2 onto Geltrex-coated plates in medium 1 containing Y-drug.

[0208] Days 1 - 2: The cells should have reached 100% confluence. The cells were supplied with medium 1 in duplicate.

[0209] Day 3: The cells were supplied with medium 1.

[0210] Day 4: The cells were supplied with medium 2. For the CHIR boost protocol, the CHIR concentration was changed from 1 μM to 6 μM for WA-09 hESC line-mediated differentiation (this may vary slightly depending on the hPSC / hiPSC line).

[0211] Days 5 - 6: The cells were supplied with medium 2 in duplicate.

[0212] Day 7: The cells were supplied with medium 3.

[0213] Days 8 - 9: The cells were supplied with medium 3 daily.

[0214] Day 10: The cells were supplied with medium 4.

[0215] Day 11: The cells were incubated with Accutase at 37°C for 30 minutes. The cells were plated at a density of 800,000 cells / cm 2 in medium 4.

[0216] Day 12: The cells should have reached 100% confluence. The cells were supplied with medium 5.

[0217] Days 12-16: Cells were supplied with culture medium 5 daily. On day 16, FACS analysis revealed that over 90% of the cells were FOXA2-positive. + / EN + That was the case.

[0218] Days 16-100: Cells were supplied with culture medium 6 daily.

[0219] Composition of medium 1: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 μM SB, 250 nM LDN, 500 ng / ml SHH C5II, and 1 μM CHIR.

[0220] Composition of medium 2: Neuronal basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C5II, and 6 μM CHIR.

[0221] Composition of culture medium 3: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, and 6 μM CHIR.

[0222] Composition of medium 4: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 3 μM CHIR, 20 ng / ml BDNF, 0.2 μM ascorbic acid (AA), 20 ng / ml GSNF, 0.5 mM dcAMP, and 1 ng / ml TGF-β3.

[0223] Composition of culture medium 5: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 3 μM CHIR, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, 1 μM IWP2, and 100 ng / ml FGF18.

[0224] Composition of Medium 6: Neural basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, and 10 nM DAPT.

[0225] The protocol described in this example is denoted as "Wnt boost + FGF18 (days 12 - 16) + IWP2 (days 12 - 16)" in Example 3.

[0226] Example 2: Exemplary Midbrain DA Neuron Differentiation Protocol The following is an exemplary protocol of the method of the present disclosure according to a specific embodiment.

[0227] Day 0: Cells were supplied with Accutase derived from hPSC / hiPSC as single cells and plated at a density of 400,000 cells / cm 2 on Geltrex coating in Medium 1 containing Y-drug.

[0228] Days 1 - 2: The cells should have reached 100% confluence. The cells were supplied with Medium 1 in duplicate.

[0229] Day 3: The cells were supplied with Medium 1.

[0230] Day 4: The cells were supplied with Medium 2. For the CHIR boost protocol, the CHIR concentration was changed from 1 μM to 6 μM for WA-09 hESC line-mediated differentiation (this may vary slightly depending on the hPSC / hiPSC line).

[0231] Days 5 - 6: The cells were supplied with Medium 2 in duplicate.

[0232] Day 7: The cells were supplied with Medium 3.

[0233] Days 8 - 9: The cells were supplied with Medium 3 daily.

[0234] Day 10: The cells were supplied with culture medium 4.

[0235] Day 11: Cells were incubated with Accutase at 37°C for 30 minutes. 800,000 cells / cm³ in medium 4. 2 Plate the cells at this density.

[0236] Day 12: The cells should have reached 100% confluence. Medium 5 was supplied to the cells.

[0237] Days 12-16: Cells were supplied with culture medium 5 daily. On day 16, FACS analysis revealed that over 90% of the cells were FOXA2-positive. + / EN + That was the case.

[0238] Days 16-100: Cells were supplied with culture medium 6 daily.

[0239] Composition of medium 1: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 μM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 1 μM CHIR.

[0240] Composition of medium 2: Neuronal basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 6 μM CHIR.

[0241] Composition of culture medium 3: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, and 6 μM CHIR.

[0242] Composition of medium 4: Neurological basal medium, B27 supplement, Pen / Strep, L-glutamine, 1 μM IWP2, 20 ng / ml BDNF, 0.2 μM ascorbic acid (AA), 20 ng / ml GDNF, 0.5 mM dcAMP, and 1 ng / ml TGF-β3.

[0243] Composition of culture medium 5: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, 1 μM IWP2, and 100 ng / ml FGF18.

[0244] Composition of medium 6: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, and DAPT (10 nM).

[0245] In Example 3, the protocol described in this example will be referred to as "Wnt Boost + FGF18 (Day 12-16) + IWP2 (Day 10-16)".

[0246] Example 3: Treatment with Wnt inhibitor during mDA neuron differentiation Different WNT signaling genes are associated with subtypes of dopamine neurons. Aldehyde dehydrogenase 1 family member A1 (ALDH1A1) is a marker of the hDA2 subtype (A9 type) during mouse and human mDA neuron development ((La Manno, et al. Cell 167, 566-580 e519 (2016); Toledo, et al. Br J Pharmacol 174 (24), 4716-4724 (2017)). ALDH1A1 belongs to the aldehyde dehydrogenase family of proteins and is the second enzyme in the major oxidative pathway of alcohol metabolism.

[0247] hPSCs and hiPSCs were used as differentiation methods. First, the effect of Wnt signaling on ALDH1A1 induction in mDA cells differentiated using different protocols was evaluated. mRNA expression levels of FOXA2, LMX1A, EN1, WNT1, OTX2, ALDH1A1, and PAX6 were evaluated in differentiated mDA cells at day 16 produced using the Wnt boost, Wnt boost + IWP2 (days 10-16), and Wnt boost + IWP2 (days 12-16) protocols, with or without FGF18 (Figure 1).

[0248] The "Wnt Boost + FGF18 (Day 12-16) + IWP2 (Day 12-16)" protocol is described in Example 1.

[0249] The "Wnt Boost + FGF18 (Day 12-16) + IWP2 (Day 10-16)" protocol is described in Example 2.

[0250] The "Wnt Boost" protocol referred to in this embodiment is provided below.

[0251] Day 0: Cells were supplied with hPSC / hiPSC-derived Accutase in single cells, and 400,000 cells / cm³ were placed on a Geltrex coating plated in medium 1 containing Y-drug. 2 The plates were plated at this density.

[0252] Days 1-2: The cells should have reached 100% confluence. The cells were supplied with a double dose of culture medium 1.

[0253] Day 3: The cells were supplied with culture medium 1.

[0254] Day 4: Cells were supplied with medium 2. For the CHIR boost protocol, the CHIR concentration was changed from 1 μM to 6 μM for WA-09 hESC strain-mediated differentiation (this may vary slightly depending on the hPSC / hiPSC strain).

[0255] Days 5-6: The cells were supplied with a double dose of culture medium 2.

[0256] Day 7: The cells were supplied with culture medium 3.

[0257] Days 8-9: The cells were supplied with culture medium 3 daily.

[0258] Day 10: The cells were supplied with culture medium 4.

[0259] Day 11: Cells were incubated with Accutase at 37°C for 30 minutes. 800,000 cells / cm³ in medium 4. 2 Plate the cells at this density.

[0260] Day 12: The cells should have reached 100% confluence. Medium 5 was supplied to the cells.

[0261] Days 12-16: Cells were supplied with culture medium 5 daily. On day 16, FACS analysis revealed that over 90% of the cells were FOXA2-positive. + That was the case.

[0262] Days 16-100: Cells were supplied with culture medium 6 daily.

[0263] Composition of medium 1: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 μM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 1 μM CHIR.

[0264] Composition of medium 2: Neuronal basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 6 μM CHIR.

[0265] Composition of culture medium 3: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, and 6 μM CHIR.

[0266] Composition of medium 4: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 3 μM CHIR, 20 ng / ml BDNF, 0.2 μM ascorbic acid (AA), 20 ng / ml GDNF, 0.5 mM dcAMP, and 1 ng / ml TGF-β3.

[0267] Composition of culture medium 5: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3.

[0268] Composition of medium 6: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, and 10 nM DAPT.

[0269] SMA mRNA expression levels were not detected. The Wnt-boost protocol combined with FGF18 and IWP2 produced optimal A / P and D / V patterned precursors, with over 90% of cells being FOXA2 / EN1 double-positive (Figure 2A). Figure 2A shows FACS analysis of differentiated mDA precursors at day 16 using different protocols. Immunostaining images of differentiated mDA at day 16 using different protocols are shown in Figure 2B. Furthermore, mRNA expression levels of FOXA2, LMX1A, OTX2, EN1, ALDH1A1, BARHL2, BARHL1, PAX6, ALDH2, and WNT1 were evaluated at day 16 in differentiated mDA cells produced using the Wnt-boost and Wnt-boost + IWP2 (days 12-16) protocols with or without FGF18 (Figure 3). The effect of IWP2 on marker gene expression in differentiated cells was determined. As shown in Figure 4, mRNA expression levels of FOXA2, LMX1A, OTX2, EN1, ALDH1A1, PAX6, and PITX3 were evaluated in differentiated cells at day 40 using the Wnt boost protocol, with or without the addition of FGF18 and / or IWP2, from day 12 to day 16. This disclosure observed the presence of very high quadrupole-positive cells at day 16 (FOXA2 / LMX1A, OTX2 / EN1). Furthermore, high expression of EN1 was driven by FGF18. In addition, the expression of ALDH1A1, WNT1, PITX3, DAT, DDC, and VMTA2 increased with the addition of IWP2 and FGF18, while IWP2 decreased the expression of Ki67, SMA, and SIX1. Immunostaining images of differentiated cells at day 60 were collected and showed the expression of FOXA2, TH, and MAP2 (Figure 14A), as well as EN1 and TH (Figure 14B).

[0270] Differentiated cells at day 25 were sorted using the Wnt boost protocol with or without the addition of FGF18 and IWP2 (Figure 5). Differentiated mDA cells were sorted based on the expression of CD49e marker protein and CD184 protein marker. Sorted differentiated CD49e cells at day 40 弱 / CD184 弱 Cells and CD49弱 / CD184 強 The morphology of the cells is shown in Figure 6. Selected differentiated CD49 cells at 40 days. 弱 / CD184 弱 Cells and CD49 弱 / CD184 強 In cells, mRNA expression levels of FOXA2, LMX1A, EN1, NURR1, ALDH1A1, PITX3, DAT, VMAT2, CALB1, CALB2, PITX2, BARHL1, SIM1, PHOX2A, and POU4F1 were evaluated and analyzed (Figures 7 and 8). Selected differentiated CD49 cells at day 40. 弱 / CD184 弱 Cells and CD49 弱 / CD184 強 Immunostaining images of cells were collected, showing the expression of FOXA2, TH, and MAP2 (Figure 9A), as well as ALDHA1A1, EN1, and TH (Figures 9B-9C). Furthermore, differentiated CD49 cells selected at day 60 were also examined. 弱 / CD184 弱 Cells and CD49 弱 / CD184 強 Immunostaining images of cells were collected, showing the expression of TH and EN1 (Figure 17), as well as ALDHA1A1, EN1, and TH (Figure 18).

[0271] Next, cells were differentiated using the "Wnt Boost" protocol or the "Wnt Boost + FGF18 (days 12-16) + IWP2 (days 12-16)" protocol and transplanted into mice. The transplanted cells were immunostained one month after transplantation. The expression of hNCAM, TH, ALDH1A1, FOXA2, SC121, EN1, and Ki67 was evaluated (Figures 10A, 10B, and 24). Furthermore, the transplanted cells were immunostained two months after transplantation. The expression levels of SC121, TH, Nurr1, ALDH1A1, and SOX6 were determined (Figures 11A-11C).

[0272] Example 4: Optimization of WNT inhibition This embodiment is designed to optimize the time window and concentration of WNT treatment and to test whether reactivation of non-classical signaling is necessary for optimal levels of differentiation or maturation of mDA neurons. Preliminary data (not shown) suggest that inhibition of classical signaling only (e.g., by using a selective inhibitor of classical signaling (e.g., XAV939; tankirase inhibitor-stabilized AXIN)) may not be sufficient to obtain results comparable to IWP2 treatment. IWP2 inhibits both non-classical and classical signaling. Expression of PITX3, DAT, and VMAT2 is quantified by qRT-PCR and immunocytochemistry (ICC). It is confirmed that IWP2 (or other candidate WNT inhibitors) does not adversely affect EN1 expression or the appearance of contamination markers (SIX1 and SMA). The optimized conditions will be validated using hPSC strains including male and females (three hESC strains and three iPSC strains; each independently differentiated at least three times) (Zimmer, et al. Proceedings of the National Academy of Sciences of the United States of America 115, E8775-E8782 (2018)).

[0273] Example 5: Detailed in vitro molecular and functional evaluation of generated mDA neurons The identity of mDA neurons generated by the differentiation method of this disclosure is verified. This verification includes: i) detailed transient characterization of marker expression (including ALDH1A1 and PITX3) by ICC and in situ expression; ii) analysis of bulk RNA sequencing over time (days 0, 11, 16, 25, 40, and 60); iii) use of a set of 42 arrayed qRT-PCR markers developed to optimize clinical-grade mDA neuron differentiation to assess whether the differentiation method of this disclosure conforms to or exceeds the previously established QC ("release criteria") for clinical-grade mDA neurons; and iv) at days 30, 50, and 70 of differentiation, as previously described (Kriks, et al. Nature). 480, 547-551 (2011)), evaluation of the biochemical maturity of mDA neurons by measuring DA release by HPLC (electrochemical detection); v) determination of differences in maturity levels (e.g., resting membrane potential, input resistance) in mDA neuron-specific parameters, including the presence of an autonomous pacemaker or Sag current, by in vitro electrophysiological testing. KCL-induced DA release in mDA neurons developed by the differentiation method of this disclosure is expected to occur earlier and at higher levels per cell than by conventional methods. The emergence of spontaneous in vitro network activity is verified using a high-density microelectrode array system (MEA).

[0274] Example 6: In vivo functional evaluation of generated mDA neurons To evaluate in vivo survival and function, cells will be transplanted on day 16. Before commencing functional studies, a short-term transplant (1 month) into the striatum of non-lesion NSG mice will be performed to confirm robust short-term survival for each treatment group (n=5 / group). For functional studies, a 6-month transplant study will be conducted in 6OHDA-lesioned rat hosts (nu / nu rats). The groups are i) saline control, ii) Wnt boost, iii) Wnt boost + FGF18, iv) Wnt boost + FGF18 / IWP2 (n=10 / group). As previously described (Kriks, et al. Nature 480, 547-551 (2011)), rats will be subjected to unilateral 6OHDA lesions targeting the medial forebrain bundle (MFB) before transplantation. Only animals with stable rotational behavior (>6 rotations / min; consecutive tests twice weekly) will be included. In addition to amphetamine-induced rotation (monthly), several non-drug-induced assays (pre-transplant and 3 and 6 months post-transplant), including stepping and cylinder tests (Kriks, et al. Nature 480, 547-551 (2011)), will be monitored. Transplantation will be performed via stereotactic surgery and 200x10 to the host striatum. 3 This is performed via injection of cells (2 μl volume) as described above (Kriks, et al. Nature 480, 547-551 (2011)). All conditions induce a significant recovery of amphetamine-induced behavior (compared to saline controls), but the FGF18 and FGF18 / IWP2 protocols induce a more rapid recovery and are expected to show overcompensation (negative scores) in the rotation assay at a later stage. Furthermore, grafts of mDA neurons generated by the differentiation methods of this disclosure show improved recovery in the stepping and cylinder tests, which is typically more difficult to recover than amphetamine-induced rotation.

[0275] Example 7: Histological analysis i) Total number of viable mDA neurons (stereometric count of TH+ cells in the graft); ii) Human identity of TH+ cells confirmed by co-expression with human nuclear antigen (hNA); iii) Markers of mDA neuron identity, subtype, and biochemical maturation (TH / EN1 / FOXA2, TH / DAT / VMAT2, TH / GIRK2 / CALB); iv) Degree of fibrillary proliferation (percentage of striatal reinnervation by TH / hNCAM and / or TH / SC121); v) Percentage of non-dopaminergic neurons (GABA, serotonin, glutamate)¹¹ and vi) Percentage of glial cells (GFAP, oligo2) and other proliferating (Ki67) cells to determine if there are differences in these percentages. Histological analysis will be performed to determine if there are differences in these percentages.

[0276] Example 8: Treatment with Wnt inhibitor during mDA neuron differentiation This example demonstrates an updated experiment of Example 3. mRNA expression levels of FOXA2, LMX1A, OTX2, EN1, ALDH1A1, WNT1, BARHL1, PAX6, OTX2, and NKX2-2 were evaluated on day 16 in differentiated mDA cells produced using the Wnt boost and Wnt boost + IWP2 (days 12-16) protocols with or without FGF18. This disclosure finds that IWP2 exposure resulted in increased ALDH1A1 and high endogenous WNT1 expression on day 16 in both the Wnt boost and Wnt boost + FGF18 conditions. Furthermore, IWP2 exposure reduced PAX6 and NKX2-2 expression (Figure 12). Similar changes were observed in differentiated cells on day 40 (Figure 13). Immunostaining of differentiated cells at day 60 produced using the Wnt boost protocol, with or without the addition of FGF18 and / or IWP2 from day 12 to day 16, confirmed that FGF18 and IWP2 exposure maintained high levels of FOXA2 and TH expression (Figure 14A) and increased EN1 and TH expression in cells (Figure 14B).

[0277] FACS-mediated sorting of differentiated cells on day 25 was performed with or without the addition of FGF18 and IWP2 using the Wnt boost protocol. Differentiated mDA cells were sorted based on the expression of the CD49e marker protein and the CD184 protein marker. The sorted differentiated CD49 弱 / CD184 弱 cells and CD49 弱 / CD184 強 cells were evaluated for the mRNA expression levels of FOXA2, LMX1A, EN1, NURR1, ALDH1A1, PITX3, DAT, VMAT2, CALB1, PITX2, BARHL1, SIM1, and PHOX2A (Figures 15 and 16). The immunostaining images of the sorted differentiated CD49 弱 / CD184 弱 cells and CD49 弱 / CD184 強 cells showed the expression of ALDH1A1, EN1, and TH (Figures 17 and 18).

[0278] Example 9: Increased exposure to Wnt inhibitors Increased exposure to Wnt inhibitors was tested. An exemplary midbrain DA neuron differentiation protocol using Wnt inhibitor exposure from day 12 to day 25 is as follows.

[0279] Day 0: Cells were supplied with Accutase derived from hPSC / hiPSC as single cells and plated at a density of 400,000 cells / cm 2 on Geltrex coating plated in medium 1 containing Y-drug.

[0280] Days 1 - 2: Cells should have reached 100% confluence. Cells were supplied with medium 1 in duplicate.

[0281] Day 3: Cells were supplied with medium 1.

[0282] Day 4: Cells were supplied with medium 2. For the CHIR boost protocol, the CHIR concentration was changed from 1 μM to 6 μM for WA-09 hESC strain-mediated differentiation (this may vary slightly depending on the hPSC / hiPSC strain).

[0283] Days 5-6: The cells were supplied with a double dose of culture medium 2.

[0284] Day 7: The cells were supplied with culture medium 3.

[0285] Days 8-9: The cells were supplied with culture medium 3 daily.

[0286] Day 10: The cells were supplied with culture medium 4.

[0287] Day 11: Cells were incubated with Accutase at 37°C for 30 minutes. 800,000 cells / cm³ in medium 4. 2 Plate the cells at this density.

[0288] Day 12: The cells should have reached 100% confluence. Medium 5 was supplied to the cells.

[0289] Days 12-16: Cells were supplied with culture medium 5 daily. On day 16, FACS analysis revealed that over 90% of the cells were FOXA2-positive. + / EN + That was the case.

[0290] Days 16-25: The cells were supplied with culture medium 6 daily.

[0291] Days 25-100: Cells were supplied with culture medium 7 daily.

[0292] Composition of medium 1: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 μM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 1 μM CHIR.

[0293] Composition of medium 2: Neuronal basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 6 μM CHIR.

[0294] Composition of culture medium 3: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, and 6 μM CHIR.

[0295] Composition of medium 4: Neurological basal medium, B27 supplement, Pen / Strep, L-glutamine, 1 μM IWP2, 20 ng / ml BDNF, 0.2 μM ascorbic acid (AA), 20 ng / ml GDNF, 0.5 mM dcAMP, and 1 ng / ml TGF-β3.

[0296] Composition of culture medium 5: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, 1 μM IWP2, and 100 ng / ml FGF18.

[0297] Composition of medium 6: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, 1 μM IWP2, and DAPT (10 nM).

[0298] Composition of medium 7: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, and DAPT (10 nM). An exemplary midbrain DA neuron differentiation protocol using Wnt inhibitor exposure from day 12 to day 30 is as follows:

[0299] Day 0: Cells were supplied with hPSC / hiPSC-derived Accutase in single cells, and 400,000 cells / cm³ were placed on a Geltrex coating plated in medium 1 containing Y-drug. 2 The plates were plated at this density.

[0300] Days 1-2: The cells should have reached 100% confluence. The cells were supplied with a double dose of culture medium 1.

[0301] Day 3: The cells were supplied with culture medium 1.

[0302] Day 4: Cells were supplied with medium 2. For the CHIR boost protocol, the CHIR concentration was changed from 1 μM to 6 μM for WA-09 hESC strain-mediated differentiation (this may vary slightly depending on the hPSC / hiPSC strain).

[0303] Days 5-6: The cells were supplied with a double dose of culture medium 2.

[0304] Day 7: The cells were supplied with culture medium 3.

[0305] Days 8-9: The cells were supplied with culture medium 3 daily.

[0306] Day 10: The cells were supplied with culture medium 4.

[0307] Day 11: Cells were incubated with Accutase at 37°C for 30 minutes. 800,000 cells / cm³ in medium 4. 2 Plate the cells at this density.

[0308] Day 12: The cells should have reached 100% confluence. Medium 5 was supplied to the cells.

[0309] Days 12-16: Cells were supplied with culture medium 5 daily. On day 16, FACS analysis revealed that over 90% of the cells were FOXA2-positive. + / EN + That was the case.

[0310] Days 16-30: Cells were supplied with culture medium 6 daily.

[0311] Days 30-100: Cells were supplied with culture medium 7 daily.

[0312] Composition of medium 1: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 μM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 1 μM CHIR.

[0313] Composition of medium 2: Neuronal basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 6 μM CHIR.

[0314] Composition of culture medium 3: Neurological basal medium, N2 supplement, B27 supplement, Pen / Strep, L-glutamine, and 6 μM CHIR.

[0315] Composition of medium 4: Neurological basal medium, B27 supplement, Pen / Strep, L-glutamine, 1 μM IWP2, 20 ng / ml BDNF, 0.2 μM ascorbic acid (AA), 20 ng / ml GDNF, 0.5 mM dcAMP, and 1 ng / ml TGF-β3.

[0316] Composition of culture medium 5: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, 1 μM IWP2, and 100 ng / ml FGF18.

[0317] Composition of medium 6: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, 1 μM IWP2, and DAPT (10 nM).

[0318] Composition of medium 7: Neuronal basal medium, B27 supplement, Pen / Strep, L-glutamine, 20 ng / ml BDNF, 0.2 μM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-β3, and DAPT (10 nM).

[0319] This disclosure found that continued exposure to IWP2 up to day 30 further induced ALDH1A1 expression (Figure 19). Figure 20 shows the FACS-mediated sorting strategy of differentiated cells at day 25 produced from the Wnt boost protocol, with or without IWP2 addition from day 12 to day 25, or from day 12 to day 16, and with or without FGF18 addition from day 12 to day 16. Sorted differentiated CD49 at day 28. 弱 / CD184 弱 Cells and CD49 弱 / CD184 強 mRNA expression of marker genes in cells was measured (Figures 21 and 22). Consistent with the results in Figure 19, exposure to IWP2 from day 12 to day 25 further induced ALDH1A1 expression. This result was confirmed using immunofluorescence staining (Figure 23).

[0320] Example 10: In vivo transplantation of differentiated cells Repeated in vivo transplantation experiments were performed in Example 3. Differentiated cells generated according to Wnt boosting using the IWP2 and FGF18 protocols showed improved striatal innervation, maintenance of EN1 expression, and A9 type ALDH1A1 + Increase in cells, and proliferating cells (Ki67 + The graft had many advantages, such as a reduction in the number of cells (Figures 24 and 25).

[0321] Four months after transplantation, transplanted cells generated according to Wnt boosting using the IWP2 and FGF18 protocols possessed axonal projections of type A9 DA neurons and covered almost the entire striatum (Figure 27).

[0322] Next, the selected CD49弱 / CD184 強 Cells were transplanted into mice. Cells were sorted on day 25 of in vitro differentiation using either the Wnt boost or the Wnt boost + FGF18 / IWP2 protocol (days 12-16). Transplanted cells were immunostained one month after transplantation. Transplanted cells showed good viability and had a homogeneous DA population expressing TH and FOXA2 under both conditions (Figure 28). PITX3 expression was also measured in in vitro differentiated cells under Wnt boost with / without IWP2 and FGF18 (days 12-16) using an RNA in situ assay (Figure 29).

[0323] Example 11: In vivo transplantation of differentiated cells The clinical validity of cells generated by the protocol described herein was tested by examining the transplantation of frozen differentiated cells (off-the-shelf cell sources). Two frozen batches of cells were transplanted, immunostained, and evaluated by TH and HNA one month after transplantation (Figure 26). The transplanted cells showed superior graft survival by expressing mDA markers such as TH and FOXA2 in two different batches, demonstrating the clinical validity of the method described herein (Figure 26).

[0324] While the subject matter and its merits have been described in detail, it should be understood that various modifications, substitutions, and changes can be made herein without departing from the spirit and scope of this disclosure. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, manufactures, and compositions, means, methods, and steps described herein. Those skilled in the art will readily understand from this disclosure that existing or future-developed subject matter, processes, machines, manufactures, compositions, means, methods, or steps of this disclosure can be utilized in accordance with the subject matter of this disclosure, performing substantially the same functions or achieving substantially the same results as the corresponding embodiments described herein. Accordingly, the appended claims are intended to include such processes, machines, manufactures, compositions, means, methods, or steps within their scope.

[0325] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are referenced throughout this application, and their disclosures are incorporated herein by reference in their entirety for any purpose. The present invention provides, for example, the following items: (Item 1) An in vitro method for inducing the differentiation of stem cells, The steps include contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator, and The process involves contacting the aforementioned cells with at least one fibroblast growth factor (FGF) signaling activator and at least one Wnt signaling inhibitor to obtain a population of differentiated cells expressing at least one marker indicating midbrain dopamine neurons or their precursors. Methods that include... (Item 2) The method according to item 1, wherein the contact between the cells and the at least one Wnt signaling inhibitor is initiated at least about 5 days after the initial contact between the stem cells and the at least one SMAD signaling inhibitor. (Item 3) The method according to item 1 or 2, wherein the contact between the cells and the at least one Wnt signaling inhibitor is initiated within approximately 15 days of the initial contact between the stem cells and the at least one SMAD signaling inhibitor. (Item 4) The method according to any one of items 1 to 3, wherein the contact between the cells and the at least one Wnt signaling inhibitor is initiated about 10 days after the initial contact between the stem cells and the at least one SMAD signaling inhibitor. (Item 5) The method according to any one of items 1 to 4, wherein the contact between the cells and the at least one Wnt signaling inhibitor is initiated 10, 11, 12, or 13 days after the initial contact between the stem cells and the at least one SMAD signaling inhibitor. (Item 6) The method according to any one of items 1 to 5, wherein the cells are brought into contact with the at least one Wnt signaling inhibitor for at least about 1 day. (Item 7) The method according to any one of items 1 to 6, wherein the cells are brought into contact with the at least one Wnt signaling inhibitor for a maximum of approximately 30 days or a maximum of approximately 25 days. (Item 8) The method according to any one of items 1 to 7, wherein the cells are brought into contact with the at least one Wnt signaling inhibitor for about 5 days, about 15 days, or about 20 days. (Item 9) The method according to any one of items 1 to 8, wherein the cells are brought into contact with the at least one Wnt signaling inhibitor for 4, 5, 6, 7, 14, 15, 19, or 20 days. (Item 10) The method according to any one of items 1 to 9, wherein the contact between the cells and the at least one FGF signaling activator is initiated at least about 5 days or at least about 10 days after the initial contact between the cells and the at least one SMAD signaling inhibitor. (Item 11) The method according to any one of items 1 to 10, wherein the contact between the cells and the at least one FGF signaling activator is initiated within approximately 20 days or 18 days from the initial contact between the cells and the at least one SMAD signaling inhibitor. (Item 12) The method according to any one of items 1 to 11, wherein the contact between the cells and the at least one FGF signaling activator is initiated about 10 days after the initial contact between the cells and the at least one SMAD signaling inhibitor. (Item 13) The method according to any one of items 1 to 12, wherein the contact between the cells and the at least one FGF signaling activator is initiated 10, 11, 12, or 13 days after the initial contact between the cells and the at least one SMAD signaling inhibitor. (Item 14) The method according to any one of items 1 to 13, wherein the cells are brought into contact with the at least one FGF signaling activator for at least about 1 day and / or up to about 20 days, at least about 3 days and / or up to about 10 days, or at least 4 days and / or up to 7 days. (Item 15) The method according to any one of items 1 to 14, wherein the cells are brought into contact with the at least one FGF signaling activator for about 5 days. (Item 16) The method according to any one of items 1 to 15, wherein the cells are brought into contact with the at least one FGF signaling activator for 4, 5, 6, or 7 days. (Item 17) The method according to any one of items 1 to 16, wherein the cells are brought into contact with the at least one SMAD signaling inhibitor for about 5 days. (Item 18) The method according to any one of items 1 to 17, wherein the cells are brought into contact with the at least one SMAD signaling inhibitor for 6 or 7 days. (Item 19) The method according to any one of items 1 to 18, wherein the cells are brought into contact with the at least one SHH signaling activator for about 5 days. (Item 20) The method according to any one of items 1 to 19, wherein the cells are brought into contact with the at least one SHH signaling activator for 6 or 7 days. (Item 21) The method according to any one of items 1 to 20, wherein the cells are brought into contact with the at least one Wnt signaling activator for about 15 days. (Item 22) The method according to any one of items 1 to 21, wherein the cells are brought into contact with the at least one Wnt signaling activator for 16 or 17 days. (Item 23) The method according to any one of items 1 to 22, wherein the concentration of the at least one Wnt signaling activator increases approximately 4 days from its first contact with the stem cell. (Item 24) The method according to item 23, wherein the concentration of the at least one Wnt signaling activator increases by about 200% to about 1000% from the initial concentration of the at least one Wnt signaling activator. (Item 25) The method according to item 23 or 24, wherein the concentration of the at least one Wnt signaling activator increases by about 500% from the initial concentration of the at least one Wnt signaling activator. (Item 26) The method according to any one of items 23 to 25, wherein the concentration of the at least one Wnt signaling activator increases from about 1 μM to about 5 μM and about 10 μM. (Item 27) The method according to any one of items 23 to 26, wherein the concentration of the at least one Wnt signaling activator is increased to a concentration of about 6 μM. (Item 28) The method according to any one of items 1 to 27, wherein the at least one Wnt signaling inhibitor can inhibit non-classical Wnt signaling and classical Wnt signaling. (Item 29) The method according to any one of items 1 to 28, wherein the at least one Wnt signaling inhibitor is selected from the group consisting of IWP2, IWR1-endo, XAV939, IWP-O1, Wnt-C59, IWP-L6, and ICG-001, and combinations thereof. (Item 30) The method according to any one of items 1 to 29, wherein the at least one Wnt signaling inhibitor comprises IWP2. (Item 31) The method according to any one of items 1 to 30, wherein the at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, FGF8b, FGF4, FGF2, and combinations thereof. (Item 32) The method according to any one of items 1 to 31, wherein the at least one FGF signaling activator can cause midbrain enlargement and upregulate midbrain gene expression. (Item 33) The method according to item 32, wherein the at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, FGF4, FGF2, and combinations thereof. (Item 34) The method according to item 33, wherein the at least one FGF signaling activator comprises FGF18. (Item 35) The method according to any one of items 1 to 34, wherein the at least one SMAD signaling inhibitor includes a TGFβ / Activin-Nodal signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, or a combination thereof. (Item 36) The method according to item 35, wherein the at least one TGFβ / Activin-Nodal signaling inhibitor comprises an ALK5 inhibitor. (Item 37) The method according to item 35 or 36, wherein the at least one TGFβ / Activin-Nodal signaling inhibitor is selected from the group consisting of SB431542, derivatives of SB431542, and combinations thereof. (Item 38) The method according to item 37, wherein the derivative of SB431542 includes A83-01. (Item 39) The method according to any one of items 35 to 38, wherein the at least one TGFβ / Activin-Nodal signaling inhibitor comprises SB431542. (Item 40) The method according to item 35, wherein the at least one BMP signaling inhibitor is selected from the group consisting of LDN193189, Noggin, dorsomorphine, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphine, and combinations thereof. (Item 41) The method according to item 35 or 40, wherein the at least one BMP inhibitor comprises LDN-193189. (Item 42) The method according to any one of items 1 to 41, wherein the at least one Wnt signaling activator comprises a glycogen synthase kinase 3β (GSK3β) signaling inhibitor. (Item 43) The method according to any one of items 1 to 42, wherein the at least one Wnt signaling activator is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wnt1, Wnt5a, their derivatives, and combinations thereof. (Item 44) The method according to any one of items 1 to 43, wherein the at least one Wnt signaling activator comprises CHIR99021. (Item 45) The method according to any one of items 1 to 44, wherein the at least one SHH signaling activator is selected from the group consisting of SHH proteins, smoothed agonists (SAGs), and combinations thereof. (Item 46) The method according to item 45, wherein the SHH protein is selected from the group consisting of recombinant SHH, modified N-terminal SHH, and combinations thereof. (Item 47) The method according to item 46, wherein the modified N-terminal SHH contains two isoleucines at its N-terminus. (Item 48) The method according to item 46 or 47, wherein the modified N-terminal SHH has at least about 90% sequence identity with the unmodified N-terminal SHH. (Item 49) The method according to item 48, wherein the unmodified N-terminal SHH is unmodified mouse N-terminal SHH or unmodified human N-terminal SHH. (Item 50) The method according to any one of items 46 to 49, wherein the modified N-terminal SHH contains SHH C25II. (Item 51) The method according to item 45, wherein the SAG contains palmorfamine. (Item 52) The method according to any one of items 1 to 51, wherein at least about 80% of the differentiated cells express FOXA2 and EN1 about 15 days after initial contact between the stem cells and the at least one SMAD signaling inhibitor. (Item 53) The method according to any one of items 1 to 52, wherein more than 80% or more than 90% of the differentiated cells express FOXA2 and EN1 16 days after initial contact between the stem cells and the at least one SMAD signaling inhibitor. (Item 54) The method according to any one of items 1 to 53, wherein at least one marker indicating the midbrain dopamine neuron or its precursor is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof. (Item 55) The method according to any one of items 1 to 54, wherein the differentiated cells do not express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof. (Item 56) The method according to any one of items 1 to 55, further comprising isolating cells that express at least one positive surface marker and do not express at least one negative surface marker. (Item 57) The method according to item 56, wherein the at least one positive surface marker is selected from the group consisting of CD171, CD184, and combinations thereof. (Item 58) The method according to item 56 or 57, wherein the at least one positive surface marker comprises CD184. (Item 59) The method according to any one of items 56 to 58, wherein the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof. (Item 60) The method according to any one of items 56 to 59, wherein the at least one negative surface marker comprises CD49e. (Item 61) The method according to any one of items 56 to 60, comprising selecting cells that express CD184 but do not express CD49e. (Item 62) The method according to any one of items 1 to 61, wherein the stem cells are pluripotent stem cells. (Item 63) The method according to any one of items 1 to 62, wherein the stem cells are selected from the group consisting of non-embryonic stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof. (Item 64) The method according to any one of items 1 to 63, wherein the stem cells are human stem cells, non-human primate stem cells, or rodent stem cells. (Item 65) The method according to any one of items 1 to 64, wherein the stem cells are human stem cells. (Item 66) A cell population of in vitro differentiated cells obtained by the method described in any one of items 1 to 65. (Item 67) A composition comprising the cell population described in item 66. (Item 68) The composition according to item 67, which further comprises a pharmaceutically acceptable carrier. (Item 69) A kit for inducing the differentiation of stem cells into midbrain dopamine neurons or their precursors, (a) at least one SMAD signaling inhibitor, (b) at least one SHH signaling activator, (c) at least one Wnt signaling activator, (d) at least one Wnt signaling inhibitor, and (e) at least one FGF signaling activator A kit that includes this. (Item 70) (f) The kit described in item 69, further comprising instructions for inducing the differentiation of stem cells into a population of differentiated cells expressing at least one marker indicating midbrain dopamine neurons or their precursors. (Item 71) A method for preventing, modeling and / or treating at least one sign in a subject with neurological disorders, wherein an effective amount of the following: (a) Cell populations as described in item 66; or (b) Compositions described in item 67 or 68 A method comprising administering one of the above to the subject. (Item 72) The method according to item 71, wherein the aforementioned neurological disorder is characterized by a decrease in midbrain dopamine neuron function. (Item 73) The method described in item 72, wherein the decline in midbrain dopamine neuron function is age-related. (Item 74) The method according to any one of items 71 to 73, wherein the neurological disorder is selected from the group consisting of parkinsonism, parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, and combinations thereof. (Item 75) The method according to any one of items 71 to 74, wherein the neurological disorder is selected from the group consisting of parkinsonism, parkinson's disease, and combinations thereof. (Item 76) The method according to any one of items 71 to 75, wherein the aforementioned signs of neurological disorder are selected from the group consisting of tremor, bradykinesia, flexion, postural instability, tonicity, dysphagia, and dementia. (Item 77) A cell population as described in item 66 or a composition as described in item 67 or 68 for use in subjects having neurological disorders, for use in preventing, modeling and / or treating at least one sign in subjects having neurological disorders. (Item 78) A cell population or composition for use as described in item 77, wherein the neurological disorder is characterized by a decrease in midbrain dopamine neuron function. (Item 79) A cell population or composition for use as described in item 78, wherein the decline in midbrain dopamine neuron function is age-related. (Item 80) A cell population or composition for use according to any one of items 77 to 79, wherein the neurological disorder is selected from the group consisting of parkinsonism, parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, and combinations thereof. (Item 81) A cell population or composition for use according to any one of items 77 to 80, wherein the neurological disorder is selected from the group consisting of parkinsonism, parkinson's disease, and combinations thereof. (Item 82) A cell population or composition for use according to any one of items 77 to 81, wherein the aforementioned signs of neurological disorder are selected from the group consisting of tremor, bradykinesia, flexion, postural instability, tonicity, dysphagia, and dementia.

Claims

1. An in vitro method for inducing the differentiation of stem cells, The process involves contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator, and The process involves contacting the aforementioned cells with at least one fibroblast growth factor (FGF) signaling activator and at least one Wnt signaling inhibitor to obtain a population of differentiated cells expressing at least one marker indicating midbrain dopamine neurons or their precursors. Includes, The contact between the cells and the at least one Wnt signaling inhibitor is initiated at least 10 days after the initial contact between the stem cells and the at least one SMAD signaling inhibitor. A method wherein the contact between the cells and the at least one FGF signaling activator is initiated at least 10 days after the initial contact between the cells and the at least one SMAD signaling inhibitor.

2. The method according to claim 1, wherein the contact between the cells and the at least one Wnt signaling inhibitor is initiated within 15 days of the initial contact between the stem cell and the at least one SMAD signaling inhibitor.

3. The method according to claim 1 or 2, wherein the contact between the cells and the at least one Wnt signaling inhibitor is initiated 10, 11, 12, or 13 days after the initial contact between the stem cells and the at least one SMAD signaling inhibitor.

4. The method according to any one of claims 1 to 3, wherein the cells are brought into contact with the at least one Wnt signaling inhibitor for at least one day.

5. The method according to any one of claims 1 to 4, wherein the cells are contacted with the at least one Wnt signaling inhibitor for a maximum of 30 days or a maximum of 25 days.

6. The method according to any one of claims 1 to 5, wherein the cells are contacted with the at least one Wnt signaling inhibitor for 5 days, 15 days, or 20 days.

7. The method according to any one of claims 1 to 6, wherein the cells are contacted with the at least one Wnt signaling inhibitor for 4, 5, 6, 7, 14, 15, 19, or 20 days.

8. The method according to any one of claims 1 to 7, wherein the contact between the cells and the at least one FGF signaling activator is initiated within 20 days or within 18 days of the initial contact between the cells and the at least one SMAD signaling inhibitor.

9. The method according to any one of claims 1 to 8, wherein the contact between the cells and the at least one FGF signaling activator is initiated 10, 11, 12, or 13 days after the initial contact between the cells and the at least one SMAD signaling inhibitor.

10. The method according to any one of claims 1 to 9, wherein the cells are brought into contact with the at least one FGF signaling activator for at least one day and / or up to 20 days, at least three days and / or up to 10 days, or at least four days and / or up to 7 days.

11. The method according to any one of claims 1 to 10, wherein the cells are brought into contact with the at least one FGF signaling activator for four, five, six, or seven days.

12. The method according to any one of claims 1 to 11, wherein the cells are contacted with the at least one SMAD signaling inhibitor for five, six, or seven days.

13. The method according to any one of claims 1 to 12, wherein the cells are brought into contact with the at least one SHH signaling activator for five, six, or seven days.

14. The method according to any one of claims 1 to 13, wherein the cells are brought into contact with the at least one Wnt signaling activator for 15, 16, or 17 days.

15. The method according to any one of claims 1 to 14, wherein the concentration of the at least one Wnt signaling activator increases for 4 days from its first contact with the stem cell.

16. The method according to claim 15, wherein the concentration of the at least one Wnt signaling activator increases by 200% to 1000% from the initial concentration of the at least one Wnt signaling activator.

17. The method according to claim 15 or 16, wherein the concentration of the at least one Wnt signaling activator is increased by 500% from the initial concentration of the at least one Wnt signaling activator.

18. The method according to any one of claims 15 to 17, wherein the concentration of the at least one Wnt signaling activator increases from 1 μM to between 5 μM and 10 μM.

19. The method according to any one of claims 15 to 18, wherein the concentration of the at least one Wnt signaling activator is increased to a concentration of 6 μM.

20. The method according to any one of claims 1 to 19, wherein the at least one Wnt signaling inhibitor can inhibit non-classical Wnt signaling and classical Wnt signaling.

21. (a) The at least one Wnt signaling inhibitor is selected from the group consisting of IWP2, IWR1-endo, XAV939, IWP-O1, Wnt-C59, IWP-L6, and ICG-001, and combinations thereof, and / or (b) The at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, FGF8b, FGF4, FGF2, and combinations thereof, and / or (c) The at least one SMAD signaling inhibitor comprises a TGFβ / Activin-Nodal signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, or a combination thereof, and / or (d) The at least one Wnt signaling activator comprises a glycogen synthase kinase 3β (GSK3β) signaling inhibitor, and / or (e) The at least one SHH signaling activator is selected from the group consisting of SHH protein, smoothed agonist (SAG), recombinant SHH, modified N-terminal SHH, SHH C25II, palmorfamine, and combinations thereof. The method according to any one of claims 1 to 20.

22. The method according to claim 21, wherein the at least one TGFβ / Activin-Nodal signaling inhibitor is selected from the group consisting of ALK5 inhibitors, SB431542, derivatives of SB431542, A83-01, and combinations thereof.

23. The method according to claim 21, wherein the at least one BMP signaling inhibitor is selected from the group consisting of LDN193189, Noggin, dorsomorphine, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphine, and combinations thereof.

24. The method according to any one of claims 1 to 21, wherein the at least one Wnt signaling activator is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wnt1, Wnt5a, derivatives thereof, and combinations thereof.

25. The method according to claim 21, wherein the SHH protein is selected from the group consisting of recombinant SHH, modified N-terminal SHH, and combinations thereof.

26. The method according to any one of claims 1 to 25, wherein at least 80% of the differentiated cells express FOXA2 and EN1 15 days after the initial contact between the stem cell and the at least one SMAD signaling inhibitor.

27. The method according to any one of claims 1 to 26, wherein more than 80% or more than 90% of the differentiated cells express FOXA2 and EN1 16 days after the first contact between the stem cell and the at least one SMAD signaling inhibitor.

28. The method according to any one of claims 1 to 27, wherein at least one marker indicating the midbrain dopamine neuron or its precursor is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof.

29. The method according to any one of claims 1 to 28, wherein the differentiated cells do not express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

30. The method according to any one of claims 1 to 29, further comprising isolating cells that express at least one positive surface marker and do not express at least one negative surface marker, wherein the at least one positive surface marker is selected from the group consisting of CD171, CD184, and combinations thereof, and the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof.

31. The method according to any one of claims 1 to 30, wherein the stem cells are pluripotent stem cells, non-embryonic stem cells, embryonic stem cells, induced pluripotent stem cells, human stem cells, non-human primate stem cells, rodent stem cells, and combinations thereof.

32. A cell population of in vitro differentiated cells obtained by the method described in any one of claims 1 to 31.

33. A composition comprising the cell population described in claim 32.

34. The composition according to claim 33, further comprising a pharmaceutically acceptable carrier.

35. A kit for inducing the differentiation of stem cells into midbrain dopamine neurons or their precursors, (a) at least one SMAD signaling inhibitor, (b) at least one SHH signaling activator, (c) at least one Wnt signaling activator, (d) at least one Wnt signaling inhibitor (e) at least one FGF signaling activator, and (f) A description for inducing the differentiation of the stem cells into a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or its precursor, the description relating to the method of claim 1. A kit that includes this.

36. A cell population according to claim 32 or a composition according to claim 33 or 34 for use in preventing, modeling and / or treating at least one sign in a subject having neurological disorders.

37. The cell population or composition for use according to claim 36, wherein the neurological disorder is characterized by a decrease in midbrain dopamine neuron function.

38. The cell population or composition for use according to claim 37, wherein the decline in midbrain dopamine neuron function is age-related.

39. The cell population or composition for use according to any one of claims 36 to 38, wherein the neurological disorder is selected from the group consisting of parkinsonism, parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, and combinations thereof.

40. A cell population or composition for use according to any one of claims 36 to 39, wherein the aforementioned signs of neurological disorder are selected from the group consisting of tremor, bradykinesia, flexion, postural instability, tonicity, dysphagia, and dementia.

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