Methods of in vitro differentiation of mesencephalic dopamine (MDA) neurons

Abstract

To provide a method for invitro differentiation of midbrain dopamine (mDA) neurons.SOLUTION: The presently disclosed subject matter provides invitro methods of inducing differentiation of human stem cells into midbrain dopamine neurons, and precursors thereof, as well as cells produced by such methods. The presently disclosed subject matter also provides uses of such cells for treating a neurodegenerative disorder. In one aspect, an invitro method for differentiating a pluripotent cell is provided, the method comprising contacting a plurality of pluripotent cells with (a) at least one inhibitor of TGF β / activin-Nodal signaling, (b) at least one activator of sonic hedgehog (SHH) signaling, and (c) at least one activator of wingless (Wnt) signaling.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 169,379, filed June 1, 2015, and U.S. Provisional Application No. 62 / 169,444, filed June 1, 2015 (priority is claimed to each, the contents of each of which are incorporated herein by reference in their entirety).

[0002] 1. Introduction The subject matter of this disclosure relates to midbrain dopamine (DA) neurons, and their precursors, derived from human stem cells, and their use for the cell-based treatment of neurological disorders. [Background technology]

[0003] 2. Background of the invention Previously, embryonic stem cells and somatic stem cells have been used as therapeutic agents and model systems for neurodegenerative diseases. Research and development on directed differentiation of embryonic stem cells and somatic stem cells has been conducted in the field of central nervous system (CNS) diseases, such as Huntington's disease, Alzheimer's disease, Parkinson's disease, and multiple sclerosis. However, the results of these studies have shown little potential for restoring neuronal function in vivo and often result in undesirable tumor growth in patients. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is a need for compositions and methods for differentiating neuronal progenitor cells for use in treating neurodegenerative disorders such as Parkinson's disease. [Means for solving the problem]

[0005] 3. Summary of the Invention The subject matter of the present disclosure relates, at least in part, to midbrain dopamine (DA) neurons (mDA), and their precursors, derived from human stem cells by in vitro differentiation.

[0006] The presently disclosed subject matter relates to the discovery that midbrain dopamine (DA) neurons and their precursors can be differentiated from human stem cells by dual inhibition of SMAD signaling (e.g., by inhibiting TGFβ / activin-Nodal signaling and BMP signaling) with activation of Sonic hedgehog (SHH) signaling and activation of Wingless (Wnt) signaling, with the concentration of a Wnt-activating compound increased approximately four days after initial exposure of the cells to the SMAD inhibitor, SHH activator, and Wnt activator. In certain embodiments, the disclosed method provides advantages over methods of differentiating stem cells into midbrain DA cells that do not involve increasing the Wnt-activating compound, for example, by reducing the expression of markers of immature progenitor cells, such as PAX6, and generating cells that differentiate into mDA cells expressing functional tyrosine hydroxylase after engraftment.

[0007] In certain embodiments, the cells are further contacted with DA neuron lineage activators and inhibitors, such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta (TGFβ, e.g., TGFβ3), ascorbic acid (AA), and DAPT (which is also known as N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester; LY-374973, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; or N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester).

[0008] In certain embodiments, the presently disclosed subject matter provides an in vitro method for inducing differentiation of human stem cells into midbrain DA precursors, comprising contacting a population or a plurality of human stem cells with one or more inhibitors of TGFβ / Activin-Nodal signaling (i.e., a first SMAD inhibitor), one or more inhibitors of BMP signaling (i.e., a second SMAD inhibitor), one or more activators of Wingless (Wnt) signaling, and one or more activators of Sonic hedgehog (SHH) signaling. In certain embodiments, the inhibitors and activators are contacted with the cells simultaneously. In certain embodiments, the concentration of the activator of Wnt signaling is increased at least about 2, 3, 4, 5, or 6 days after initial contact of the cells with the Wnt activator. In certain embodiments, the cells are contacted with increasing concentrations of the Wnt activator for at least about 4, 5, 6, 7, 8, 9, or 10 days or more.

[0009] In certain embodiments, the cells are contacted with the agent in an amount effective to increase the detectable level of expression of one or more markers of midbrain DA neurons, or their precursors, such as, but not limited to, engrailed-1 (EN-1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor-associated 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1A).

[0010] In certain embodiments, the cells are contacted with the aforementioned agents in an amount effective to increase a detectable level of expression of one or more of neuron-specific class III beta-tubulin (Tuj1), trefoil factor family 3 (TTF3), paired-like homeodomain 3 (PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT), and G protein-coupled inwardly rectifying potassium channel (Kir3.2 / GIRK2), CD142, DCSM1, CD63, and / or CD99.

[0011] The present disclosure also provides a population of in vitro differentiated cells, which express one or more markers of midbrain DA cells or their precursors, prepared according to the method described herein.In certain embodiments, the differentiated cell population is derived from a population of human stem cells.The subject matter of the present disclosure further provides a composition comprising such differentiated cell population.

[0012] In certain embodiments, the cells are cultured with the aforementioned agents in an amount effective to reduce the detectable level of expression of paired box protein (PAX6) and / or Ki67. In certain embodiments, the cells do not express detectable levels of PAX6 and / or Ki67.

[0013] In certain embodiments, cells prepared according to the methods described herein can be sorted, selected, and isolated based on CD142 expression, and / or cholinergic receptor (CHRNB3) expression, for example, using flow cytometry.

[0014] In certain embodiments, the cells prepared according to the methods described herein are treated with a polysialyltransferase, such as Neisseria meningitidis polysialyltransferase (PST). Nm In certain embodiments, the cell is a recombinant cell expressing a recombinant polysialyltransferase.

[0015] Additionally, the presently disclosed subject matter provides a kit for inducing differentiation of stem cells.

[0016] In certain embodiments, the kit comprises (a) one or more inhibitors of transforming growth factor beta (TGFβ) / activin-Nodal signaling (i.e., a first SMAD inhibitor), (b) one or more inhibitors of bone morphogenetic protein (BMP) signaling (i.e., a second SMAD inhibitor), (c) one or more activators of Wingless (Wnt) signaling, (d) one or more activators of Sonic Hedgehog (SHH) signaling, and (e) instructions for inducing differentiation of stem cells into a population of differentiated cells expressing one or more markers of midbrain DA neurons, or their precursors.

[0017] In certain embodiments, the present disclosure provides kits containing stem cell-derived progenitors prepared according to the methods described herein. In certain embodiments, the stem cell-derived cells are mature differentiated cells, such as midbrain DA cells.

[0018] In certain embodiments, the one or more inhibitors of TGFβ / activin-Nodal signaling are small molecules selected from the group consisting of SB431542, its derivatives, and mixtures thereof. In certain embodiments, the one or more inhibitors of BMP signaling are small molecules selected from the group consisting of LDN193189, its derivatives, and mixtures thereof. In certain embodiments, the one or more activators of Wnt signaling reduce glycogen synthase kinase 3β (GSK3β) to activate Wnt signaling. In certain embodiments, the one or more activators of Wnt signaling are small molecules selected from the group consisting of CHIR99021, WNT3A, their derivatives, and mixtures thereof. In certain embodiments, the activator of SHH signaling is selected from the group consisting of recombinant SHH, purified SHH, C25II, and Smoothened (SMO) receptor agonists such as the small molecule purmorphamine, their derivatives, and mixtures thereof.

[0019] In certain embodiments, the human stem cells are human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, or human induced pluripotent stem cells. Human induced pluripotent stem cells (iPSCs) are selected from the group consisting of human induced pluripotent stem cells (HSCs), epiblast stem cells, and F-class pluripotent stem cells. Human induced pluripotent stem cells (iPSCs) are cells prepared from further differentiated cells, for example, differentiated somatic cells formed by the introduction of embryonic genes (such as, but not limited to, OCT4, SOX2, cMyc, and KLF4 transgenes) into somatic cells (e.g., Takahashi and Yamanaka, Cell 126:663-676 (2006)), which is incorporated herein by reference.

[0020] In certain embodiments, the method comprises subjecting said population of differentiated cells to conditions that favor maturation of said differentiated cells into a population of midbrain DA neurons.

[0021] The presently disclosed subject matter further provides methods of treating a neurodegenerative disorder, e.g., Parkinson's disease, in a subject. In certain embodiments, the method comprises administering to a subject suffering from a neurodegenerative disorder an effective amount of a differentiated cell population described herein.

[0022] The presently disclosed subject matter further provides the differentiated cell populations described herein for treating neurodegenerative disorders.

[0023] The presently disclosed subject matter further provides for the use of a differentiated cell population described herein in the manufacture of a medicament for treating a neurodegenerative disorder. The present invention provides, for example, the following items. (Item 1) An in vitro method for differentiating pluripotent cells, comprising: treating a plurality of pluripotent cells with (a) at least one inhibitor of TGFβ / Activin-Nodal signaling; (b) at least one activator of Sonic Hedgehog (SHH) signaling, and (c) at least one activator of Wingless (Wnt) signaling contacting the Item 2. The in vitro method, wherein the concentration of the at least one activator of Wnt signaling contacted with the plurality of pluripotent cells is increased between about 2 and about 6 days after initial contact of the at least one activator of Wnt signaling with the plurality of pluripotent cells, such that the plurality of cells differentiate and express forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A). 2. The method of claim 1, wherein the cells are contacted with at least one inhibitor of bone morphogenetic protein (BMP) and small mothers against decapentaplegic (SMAD) signaling. (Item 3) 3. The method of claim 2, wherein the at least one inhibitor of TGFβ / Activin-Nodal signaling, the at least one inhibitor of BMP / SMAD signaling, or the at least one activator of Sonic hedgehog (SHH) signaling is contacted with the plurality of pluripotent cells for about 4 days to about 10 days. (Item 4) 3. The method of claim 2, wherein the at least one inhibitor of TGFβ / Activin-Nodal signaling, the at least one inhibitor of BMP / SMAD signaling, or the at least one activator of Sonic hedgehog (SHH) signaling is contacted with the plurality of pluripotent cells for up to about 7 days, or for at least about 7 days. (Item 5) 3. The method of claim 2, wherein the at least one activator of Wingless (Wnt) signaling is contacted with the plurality of pluripotent cells for about 8 days to about 15 days. (Item 6) 3. The method of claim 2, wherein the at least one activator of Wingless (Wnt) signaling is contacted with the plurality of pluripotent cells for about 12 days, up to about 12 days, or at least about 12 days. (Item 7) 3. The method of any preceding item, wherein the concentration of the at least one activator of Wnt signaling is increased about 4 days after initially contacting the plurality of pluripotent cells with the at least one activator of Wnt signaling. (Item 8) 2. The method of any of the preceding items, wherein the increase in concentration of the at least one activator of Wnt signaling is about 400% to 1450% of the initial concentration of the at least one activator of Wnt signaling contacted with the plurality of cells. (Item 9) 2. The method of any preceding item, wherein the increase in concentration of the at least one activator of Wnt signaling is about 700% to 1050% of the initial concentration of the at least one activator of Wnt signaling contacted with the plurality of cells. (Item 10) Item 11. The method of any preceding item, wherein the increase in the concentration of the at least one activator of Wnt signaling is to a concentration of between about 3 and 10 μM. (Item 11) Item 11. The method of any preceding item, wherein the increase in the concentration of the at least one activator of Wnt signaling is to a concentration of about 3 μM. (Item 12) Item 11. The method of any preceding item, wherein the increase in the concentration of the at least one activator of Wnt signaling is to a concentration of about 7.5 μM. (Item 13) 2. The method of any preceding item, wherein the plurality of cells express one or more of tyrosine hydroxylase (TH), engrailed-1 (EN-1), and nuclear receptor-related 1 protein (NURR1). (Item 14) 2. The method of any preceding item, wherein the plurality of cells does not express detectable levels of paired box protein (PAX6) and / or Ki67. (Item 15) 20. The method of any preceding claim, further comprising subjecting the population of differentiated cells to conditions favorable for maturation of said cells into dopamine neurons. (Item 16) 16. The method of claim 15, wherein the conditions favorable for maturation of the cells into dopamine neurons comprise contacting the cells with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta 3 (TGFβ3), ascorbic acid (AA), and / or DAPT. (Item 17) 2. The method according to any preceding item, wherein the pluripotent cells are selected from the group consisting of human, non-human primate, or rodent non-embryonic stem cells, human, non-human primate, or rodent embryonic stem cells, human, non-human primate, or rodent induced pluripotent stem cells, and human, non-human primate, or rodent recombinant pluripotent cells. (Item 18) The method of any of the preceding items, wherein the at least one inhibitor of TGFβ / activin-Nodal signaling comprises 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542). (Item 19) The method of any preceding item, wherein the at least one inhibitor of BMP and SMAD signaling comprises 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (LDN193189). (Item 20) The method of any preceding item, wherein the at least one activator of Sonic Hedgehog (SHH) signaling comprises palmorfamine, recombinant SHH, purified SHH, and / or Smoothened agonist. (Item 21) The method of any preceding item, wherein the at least one activator of Wingless (Wnt) signaling comprises CHIR99021, Wnt3A, and / or Wnt1. (Item 22) The method of any preceding item, wherein the pluripotent cells differentiate into midbrain dopamine neurons within 22 to 27 days after initiation of contact with the at least one inhibitor of TGFβ / Activin-Nodal signaling, the at least one activator of SHH signaling, and the at least one activator of Wnt signaling. (Item 23) 2. The method of any preceding item, wherein the plurality of cells expresses a detectable level of CD142, and the method further comprises selecting a population of midbrain dopamine neurons that express CD142. (Item 24) A midbrain dopamine neuron, or a precursor thereof, differentiated according to the method described in any of the preceding items. (Item 25) 25. The midbrain dopamine neuron, or precursor thereof, of item 24, wherein the cell is contacted with a polysialyltransferase. (Item 26) 25. A kit comprising the midbrain dopamine neuron or a precursor thereof according to Item 24. (Item 27) 25. A method for treating a neurodegenerative disorder in a subject, comprising administering the midbrain dopamine neuron, or a precursor thereof, of item 24 to a subject in need thereof. (Item 28) 28. The method of claim 27, wherein the neurodegenerative disorder is Parkinson's disease and the midbrain dopamine neurons, or precursors thereof, are administered in an amount effective to reduce one or more symptoms of Parkinson's disease. (Item 29) 25. The midbrain dopamine neuron, or a precursor thereof, according to item 24, which is a recombinant cell expressing a detectable marker. (Item 30) 25. The midbrain dopamine neuron, or a precursor thereof, according to item 24, contained in a biocompatible scaffold.

[0024] The foregoing has outlined, rather broadly, the features and technical advantages of the present application in order that the detailed description that follows may be better understood. Additional features and advantages of the present application will be described hereinafter, which form the subject of the claims of the present application. Those skilled in the art should appreciate that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present application. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present application as set forth in the appended claims. The novel features believed characteristic of the present application, both as to its organization and method of operation, together with further objects and advantages, will be better understood from the following description. [Brief explanation of the drawings]

[0025] [Figure 1]Figure 1 shows that hESCs were differentiated for 10 days in KSR medium (non-GMP) or E8 / NB / N2 medium (GMP V1) without Wnt bump-induced midbrain DA neurons and neurons from other brain regions, according to the method described in Kriks et al., Nature. 2011, November 6; vol. 480 (No. 7378): pp. 547-51. Culturing hESCs in E8 / NB / N2 medium according to the method of Example 1 using a 7.5 μM (or 5-10 μM) Wnt bump from D4 to D10 was specific for generating midbrain DA cells.

[0026] [Figure 2] Figure 2 shows the expression of PAX6, TH, NURR1, FOXA2, and LMX1A in hESCs differentiated for 22 to 27 days in KSR medium (Non-GMP) or E8 / NB / N2 medium (GMP V1) without Wnt bump, according to the method described by Kriks et al., Nature. 2011, November 6; 480 (7378):547-51. Cells were harvested and analyzed by qRT-PCR for the indicated genes (n = 3 to 14 per condition). Data are presented as normalized cT values, with lower values ​​indicating higher gene expression.

[0027] [Figure 3]Figure 3 shows the expression of PAX6, TH, NURR1, FOXA2, LMX1A, and En-1 in hESCs differentiated for 22 to 27 days according to the method described in Kriks et al., Nature. 2011, Nov. 6; 480(7378):547-51, without a Wnt bump, in KSR medium (Non-GMP) or E8 / NB / N2 medium (GMP V1), and according to the method described in Example 1, which included culturing hESCs in E8 / NB / N2 medium with a 3 μM (GMP V2A) or 7.5 μM (GMP V2B) Wnt bump. Cells were harvested and analyzed by qRT-PCR for the indicated genes (n = 3 to 14 per condition). Data are presented as normalized cT values, with lower values ​​indicating higher gene expression. Cells cultured according to GMP V2A and GMP V2B expressed high levels of En-1.

[0028] [Figure 4-1] FIG. 4 shows midbrain DA markers that can be used to identify differentiated midbrain DA cells. [Figure 4-2] FIG. 4 shows midbrain DA markers that can be used to identify differentiated midbrain DA cells. [Figure 4-3] FIG. 4 shows midbrain DA markers that can be used to identify differentiated midbrain DA cells. [Figure 4-4] FIG. 4 shows midbrain DA markers that can be used to identify differentiated midbrain DA cells.

[0029] [Figure 5]Figure 5 shows that hESCs maintained under E8 / Matrigel conditions and then differentiated for 25 days in NB / N2 medium with 0.7 μM Wnt but without a Wnt bump, according to the method described by Kriks et al., Nature. 2011; 480(7378):547-51. Following differentiation, the cells were transplanted into uninjured, immunocompromised mice, and grafts were harvested 4 weeks later. Sections were analyzed by ICC for human NCAM, FOXA2, and TH. Numerous TH+ cells expressing FOXA2 were observed, while clusters of hNCAM+ cells were also detected within the graft core. These patches also expressed PAX6, indicating a neural progenitor state.

[0030] [Figure 6A] Figures 6A-B show that (A) hESCs maintained under E8 / Matrigel conditions and then differentiated for 25 days in NB / N2 medium according to the GMP V2A (3 μM Wnt bump in the presence of 0.7 μM Wnt background) and GMP V2B (7.5 μM Wnt bump in the presence of 0.7 μM Wnt background) culture methods described in Example 1 induce the differentiation of mesencephalic DA cells that express high levels of EN-1 and TH. Mesencephalic DA neurons were fixed and stained in vitro for TH and EN-1 (upper panel) and NURR1 and LMX1A (lower panel). (B) Mesencephalic DA cells differentiated according to the GMP V2B protocol were transplanted into the striatum of intact, immunocompromised mice, which showed increased fiber outgrowth and expression of hNCAM and TH 3 weeks after transplantation. [Figure 6B]Figures 6A-B show that (A) hESCs maintained under E8 / Matrigel conditions and then differentiated for 25 days in NB / N2 medium according to the GMP V2A (3 μM Wnt bump in the presence of 0.7 μM Wnt background) and GMP V2B (7.5 μM Wnt bump in the presence of 0.7 μM Wnt background) culture methods described in Example 1 induce the differentiation of mesencephalic DA cells that express high levels of EN-1 and TH. Mesencephalic DA neurons were fixed and stained in vitro for TH and EN-1 (upper panel) and NURR1 and LMX1A (lower panel). (B) Mesencephalic DA cells differentiated according to the GMP V2B protocol were transplanted into the striatum of intact, immunocompromised mice, which showed increased fiber outgrowth and expression of hNCAM and TH 3 weeks after transplantation.

[0031] [Figure 7] Figure 7 shows that hESCs maintained under E8 / Matrigel conditions and then differentiated for 25 days in NB / N2 medium according to the GMP V2B (7.5 μM Wnt bump in the presence of 0.7 μM Wnt background) culture method described in Example 1 eliminated PAX6-expressing cells while maintaining FOXA2-expressing cells. Cells were harvested and analyzed by flow cytometry for the presence of FOXA2 and PAX6.

[0032] [Figure 8] Figure 8 shows that cryopreserved midbrain DA neurons behave similarly in vivo when compared to "fresh" cells. Hncam and TH expression was compared in 3-week grafts of "fresh" cells and 4-week grafts of "frozen" cells transplanted into mice. Both grafts showed early signs of fiber outgrowth, as highlighted in the inset panel on the right.

[0033] [Figure 9A]Figures 9A-B show (A) cell sorting of CD142-expressing midbrain DA cells using MACS flow cytometry. CD142-sorted mDA neurons are shown for the flow-through (negative fraction) and positive fraction at day 24 before sorting ("Pre"). Phycoerythrin was conjugated to CD142, and anti-PE beads were used for isolation to visualize the results. (B) Consistency across six experiments is shown. [Figure 9B] Figures 9A-B show (A) cell sorting of CD142-expressing midbrain DA cells using MACS flow cytometry. CD142-sorted mDA neurons are shown for the flow-through (negative fraction) and positive fraction at day 24 before sorting ("Pre"). Phycoerythrin was conjugated to CD142, and anti-PE beads were used for isolation to visualize the results. (B) Consistency across six experiments is shown.

[0034] [Figure 10A] Figures 10A-B show that mDA neurons survive in uninjured mice and parkinsonian monkeys. (A) CD142-sorted mDA cells survive in vivo. Following the method described by Kirks et al., mDA neurons prepared with KSR were FACS-sorted for CD142 and transplanted into mice. Grafts were harvested 30 days after transplantation and analyzed for TH and hNCAM expression. (B) mDA neurons derived from the KSR protocol (Kriks et al.) survive for 1 year in nonhuman primates (NHPs). Day 25 mDA neurons were transplanted into parkinsonian monkeys, and grafts were harvested 12 months after transplantation. Sections were stained with antibodies detecting human cytoplasm (SC121) and TH. The upper half shows normal and inverted images, while the lower half shows higher magnification. The data showed that MACS-sorted cells exhibited comparable survival to CD142-sorted mDA cells transplanted into mice. [Figure 10B]Figures 10A-B show that mDA neurons survive in uninjured mice and parkinsonian monkeys. (A) CD142-sorted mDA cells survive in vivo. Following the method described by Kirks et al., mDA neurons prepared with KSR were FACS-sorted for CD142 and transplanted into mice. Grafts were harvested 30 days after transplantation and analyzed for TH and hNCAM expression. (B) mDA neurons derived from the KSR protocol (Kriks et al.) survive for 1 year in nonhuman primates (NHPs). Day 25 mDA neurons were transplanted into parkinsonian monkeys, and grafts were harvested 12 months after transplantation. Sections were stained with antibodies detecting human cytoplasm (SC121) and TH. The upper half shows normal and inverted images, while the lower half shows higher magnification. The data showed that MACS-sorted cells exhibited comparable survival to CD142-sorted mDA cells transplanted into mice.

[0035] [Figure 11-1]Figures 11A-D show the effects of polysialyltransferase treatment (Neisseria meningitidis polysialyltransferase (PSTNm)) of mDA neurons in vitro. (A) The effect of cryopreservation on polysialylated (PSA) levels is shown. mDA cells were treated with PSTNm or left unmodified. Half of the cells were fixed, while the other half was cryopreserved. The frozen cells were thawed one week later and also fixed. Cells were immunostained for PSA and analyzed by flow cytometry. The black line represents fresh, unstained cells. PSTNm-induced PSA staining was identical in frozen and unfrozen cells. The blue line represents untreated cells analyzed fresh, while the red and green lines are from PSTNm-treated cells either analyzed fresh (red) or analyzed after cryopreservation (green). (B) The effect of PSTNm treatment on cells sorted by CD142 cytometry is shown. Representative images of TAU-1 staining of CD142-sorted cells, either untreated (left) or treated with PSTNm (right), after 1 day of culture. (C) CD142-sorted cells treated with PSTNm showed increased axon length. Cells were analyzed on days 1 or 4, and images were quantified using NIH's ImageJ software. (D) PSA levels persist in vivo. PSTNm-treated mDA neurons were transplanted into the striatum of mice, and PSA levels were visualized 2 weeks after transplantation. Two fields of view (FOV 1 and FOV 2) are shown for each condition. The graph on the right summarizes the results. [Figure 11-2]Figures 11A-D show the effects of polysialyltransferase treatment (Neisseria meningitidis polysialyltransferase (PSTNm)) of mDA neurons in vitro. (A) The effect of cryopreservation on polysialylated (PSA) levels is shown. mDA cells were treated with PSTNm or left unmodified. Half of the cells were fixed, while the other half was cryopreserved. The frozen cells were thawed one week later and also fixed. Cells were immunostained for PSA and analyzed by flow cytometry. The black line represents fresh, unstained cells. PSTNm-induced PSA staining was identical in frozen and unfrozen cells. The blue line represents untreated cells analyzed fresh, while the red and green lines are from PSTNm-treated cells either analyzed fresh (red) or analyzed after cryopreservation (green). (B) The effect of PSTNm treatment on cells sorted by CD142 cytometry is shown. Representative images of TAU-1 staining of CD142-sorted cells, either untreated (left) or treated with PSTNm (right), after 1 day of culture. (C) CD142-sorted cells treated with PSTNm showed increased axon length. Cells were analyzed on days 1 or 4, and images were quantified using NIH's ImageJ software. (D) PSA levels persist in vivo. PSTNm-treated mDA neurons were transplanted into the striatum of mice, and PSA levels were visualized 2 weeks after transplantation. Two fields of view (FOV 1 and FOV 2) are shown for each condition. The graph on the right summarizes the results. [Figure 11-3]Figures 11A-D show the effects of polysialyltransferase treatment (Neisseria meningitidis polysialyltransferase (PSTNm)) of mDA neurons in vitro. (A) The effect of cryopreservation on polysialylated (PSA) levels is shown. mDA cells were treated with PSTNm or left unmodified. Half of the cells were fixed, while the other half was cryopreserved. The frozen cells were thawed one week later and also fixed. Cells were immunostained for PSA and analyzed by flow cytometry. The black line represents fresh, unstained cells. PSTNm-induced PSA staining was identical in frozen and unfrozen cells. The blue line represents untreated cells analyzed fresh, while the red and green lines are from PSTNm-treated cells either analyzed fresh (red) or analyzed after cryopreservation (green). (B) The effect of PSTNm treatment on cells sorted by CD142 cytometry is shown. Representative images of TAU-1 staining of CD142-sorted cells, either untreated (left) or treated with PSTNm (right), after 1 day of culture. (C) CD142-sorted cells treated with PSTNm showed increased axon length. Cells were analyzed on days 1 or 4, and images were quantified using NIH's ImageJ software. (D) PSA levels persist in vivo. PSTNm-treated mDA neurons were transplanted into the striatum of mice, and PSA levels were visualized 2 weeks after transplantation. Two fields of view (FOV 1 and FOV 2) are shown for each condition. The graph on the right summarizes the results.

[0036] [Figure 12]Figure 12 shows the rotational behavior of injured rats transplanted with mDA precursors differentiated as described in Example 2 and cryopreserved on day 16, compared with sham-transplanted rats. Rats were tested before transplantation and 1, 2, 3, 4, and 5 months after transplantation. Rotational behavior was induced by amphetamine administration. Lesioned rats receiving grafts showed a statistically significant decrease in rotational behavior 4 months after transplantation compared with sham-transplanted rats.

[0037] [Figure 13] Figure 13 shows the in vivo expression of hNCAM, TH, and GIRK2 in injured rats transplanted with mDA precursors differentiated as described in Example 2 and cryopreserved on day 16. The transplanted grafts were examined 5 months after transplantation. TH staining showed typical mDA morphology. TH-positive neurons were also GIRK2-positive.

[0038] [Figure 14] Figure 14 shows that mDA precursors differentiated according to Example 2 and cryopreserved at day 16 survived for 6 weeks after thawing and transplantation into non-human primates. After transplantation, the grafts showed robust fibrous extensions from the transplant core and also displayed typical mDA morphology. DETAILED DESCRIPTION OF THE INVENTION

[0039] 5. Detailed Description of the Invention The subject matter of this disclosure relates to methods for inducing differentiation of human stem cells into cells expressing one or more markers of midbrain dopamine (mDA) cells or their precursors, compositions of cells expressing such markers, and methods for treating neurodegenerative disorders.

[0040] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 5.1. Definition; 5.2. Methods for differentiating stem cells; 5.3. Methods of treating neurodegenerative disorders; and 5.4. Kit.

[0041] 5.1 Definition The terms used in this specification generally have their ordinary meaning in the art, within the context of this invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere herein to provide further guidance to the practitioner in describing the compositions and methods of the invention and how to make and use the compositions of the invention.

[0042] The terms "about" or "approximately" refer to an acceptable range of error for a particular value, as determined by one of ordinary skill in the art, depending in part on how that value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations, depending on the practice in the art. Alternatively, "about" can mean within a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold or within 2-fold of a value.

[0043] As used herein, the term "signal transduction" in reference to a "signal transduction protein" refers to a protein that is activated or otherwise affected by ligand binding to a membrane receptor protein or some other stimulus. Examples of signal transduction proteins include, but are not limited to, SMADs, Wingless (Wnt) complex proteins including beta-catenin, NOTCH, transforming growth factor beta (TGFβ), activin, Nodal, glycogen synthase kinase 3β (GSK3β) protein, bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs). For many cell surface receptors or internal receptor proteins, ligand-receptor interactions do not directly lead to cellular responses. A receptor activated by a ligand may first interact with other proteins inside the cell before the ligand's ultimate physiological effect on cellular behavior occurs. The behavior of several interacting cellular protein chains is often altered after receptor activation or inhibition. The set of cellular changes induced by receptor activation is referred to as a signal transduction mechanism or signal transduction pathway.

[0044] As used herein, the term "signal" refers to internal and external factors that control changes in cellular structure and function. These may be chemical or physical in nature.

[0045] As used herein, the term "ligand" refers to molecules and proteins that bind to receptors, such as transforming growth factor-beta (TFGβ), activin, Nodal, bone morphogenetic proteins (BMPs), and the like.

[0046] As used herein, "inhibitor" refers to a compound or molecule (e.g., a small molecule, peptide, peptidomimetic, natural compound, siRNA, antisense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, diminishes, suppresses, eliminates, or blocks) the signaling function of a molecule or pathway. An inhibitor can be, by way of example, any compound or molecule that alters any activity of a specific protein (signaling molecule, any molecule involved in a specific signaling molecule, or a specific associated molecule, such as glycogen synthase kinase 3β (GSK3β)) (including, but not limited to, signaling molecules described herein) by directly interfering with SMAD signaling, contacting SMAD mRNA, altering SMAD conformation, reducing SMAD protein levels, or interfering with SMAD interaction with signaling partners (e.g., including those described herein), and affecting the expression of SMAD target genes (e.g., those described herein). Inhibitors also include molecules that indirectly regulate biological activity, e.g., SMAD biological activity, by interfering with signaling molecules upstream (e.g., examples of signaling molecules and effects within the extracellular domain include: Noggin, which traps bone morphogenetic proteins, inhibits activation of ALK receptors 1, 2, 3, and 6, thus preventing downstream SMAD activation; and Chordin, Cerberus, and Follistatin similarly trap extracellular activators of SMAD signaling. Bambi, a transmembrane protein, also acts as a pseudoreceptor, trapping extracellular TGFb signaling molecules). Antibodies that block proteins upstream or downstream are contemplated for use in neutralizing, e.g., extracellular activators of signaling proteins.Inhibitors have been described in terms of competitive inhibition (binding to the active site in a way that precludes or reduces binding of another known binding compound) and allosteric inhibition (binding to the protein in a way that alters the protein's conformation, preventing binding of a compound to the protein's active site) in addition to inhibition induced by binding to and affecting a molecule upstream of a specific signaling molecule, which in turn causes inhibition of the specific molecule. Inhibitors can be "direct inhibitors," which inhibit a signaling target or a signaling target pathway by actually contacting the signaling target.

[0047] "Activator," as used herein, refers to a compound that increases, induces, stimulates, activates, promotes, or enhances the signaling function of a molecule or pathway, e.g., activation of Wnt signaling, SHH signaling, etc.

[0048] As used herein, the term "derivative" refers to a chemical compound that has a similar core structure.

[0049] As used herein, the term "cell population" or "cell population" refers to a group of at least two cells. In non-limiting examples, a cell population can 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 can be a pure population containing one cell type, such as a population of midbrain DA precursors or a population of undifferentiated stem cells. Alternatively, a population can include two or more cell types (e.g., a mixed cell population).

[0050] As used herein, the term "stem cell" refers to a cell that has the capacity to divide indefinitely in culture to give rise to specialized cells.

[0051] As used herein, the terms "embryonic stem cells" and "ESCs" refer to primitive (undifferentiated) cells derived from preimplantation stage embryos that can divide in culture without differentiation for extended periods of time and are known to develop into cells and tissues of the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells derived from human embryos. As used herein, the terms "human embryonic stem cells" or "hESCs" refer to a type of pluripotent stem cell derived from early stage human embryos up to and including the blastocyst stage that can divide in culture without differentiation for extended periods of time and are known to develop into cells and tissues of the three primary germ layers.

[0052] 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 periods of days, months, or even years.

[0053] As used herein, the term "totipotency" refers to the ability to give rise to all cell types of the body and all cell types that make up extraembryonic tissues such as the placenta.

[0054] As used herein, the term "pluripotency" refers to the ability to develop into more than one cell type of the body.

[0055] As used herein, the term "pluripotency" refers to the ability to give rise to the three germ layers in the development of an organism, including endoderm, mesoderm, and ectoderm.

[0056] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to a type of pluripotent stem cell formed by introducing certain embryonic genes (e.g., but not limited to, OCT4, SOX2, and KLF4 transgenes) (see, e.g., Takahashi and Yamanaka Cell 126:663-676 (2006)), which is incorporated herein by reference) into somatic cells.

[0057] As used herein, the term "somatic cell" refers to any cell in the body other than a gamete (egg or sperm), sometimes referred to as an "adult" cell.

[0058] As used herein, the term "somatic (adult) stem cells" refers to relatively rare undifferentiated cells found in many organs and differentiated tissues that have a limited capacity for both self-renewal (in the laboratory) and differentiation.

[0059] As used herein, the term "neuron" refers to a nerve cell, the primary functional unit of the nervous system. A neuron consists of a cell body and its processes, i.e., an axon and one or more dendrites. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0060] As used herein, the term "proliferation" refers to an increase in cell number.

[0061] As used herein, the term "undifferentiated" refers to cells that have not yet developed into a specialized cell type.

[0062] As used herein, the term "differentiation" refers to the process by which an unspecialized embryonic cell acquires the characteristics of a specialized cell, such as a neuron, heart, liver, or muscle cell. Differentiation is controlled by the interaction of the cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

[0063] As used herein, the term "directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into specific (e.g., desired) cell types, such as neural, neural crest, cranial placode, and non-neural ectodermal precursors.

[0064] As used herein, the term "induced differentiation" with respect to stem cells refers to the use of small molecules, growth factor proteins, and other growth conditions to promote the transition of stem cells from a pluripotent state to a more mature or specialized cell fate.

[0065] As used herein, the term "inducing differentiation" with respect to a cell refers to changing a default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, "inducing differentiation in a stem cell" refers to inducing a stem cell (e.g., a human stem cell) to divide into progeny cells with characteristics distinct from the stem cell, such as genotype (e.g., changes in gene expression determined by genetic analysis such as microarray) and / or phenotype (e.g., changes in expression of protein markers for midbrain DA cells, or their precursors, e.g., EN-1, OTX2, TH, NURR1, FOXA2, and LLMX1A).

[0066] As used herein, the term "cell culture" refers to the growth of cells in vitro in an artificial medium for research or medical treatment.

[0067] As used herein, the term "culture medium" refers to a nutrient-containing liquid that covers, nourishes, and supports cells in a culture vessel, e.g., a petri dish, a multi-well plate, etc. Culture medium also includes growth factors that are added to effect desired changes in the cells.

[0068] As used herein, the term "contacting" a cell(s) with a compound (e.g., one or more inhibitors, activators, and / or inducers) refers to providing the compound in a location that makes the compound accessible to the cell(s). Contacting can be achieved using any suitable method. For example, contacting can be achieved by adding a concentrated form of the compound to a cell or population of cells to achieve a desired concentration in a cell culture setting. Contacting can also be achieved by including the compound as a component of a formulated culture medium.

[0069] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. Exemplary in vitro environments include, but are not limited to, test tubes and cell cultures.

[0070] As used herein, the term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neurulation, and the like.

[0071] As used herein, the term "expressing" in reference to a gene or protein refers to making mRNA or protein that can be observed using an assay such as a microarray assay, an antibody staining assay, or the like.

[0072] As used herein, the term "marker" or "cell marker" refers to a gene or protein that identifies a particular cell or cell type. A marker for a cell is not limited to one type of marker; a marker can refer to a "pattern" of markers such that a specified group of markers can identify one cell or cell type from another.

[0073] As used herein, the terms "derived from" or "established from" or "differentiated from," when referring to any cell disclosed herein, refer to cells obtained (e.g., isolated, purified, etc.) from the ultimate parent cell in a cell line, tissue (e.g., dissociated embryo), or body fluid using any manipulation, including, but not limited to, single cell isolation, in vitro culture, treatment using, e.g., proteins, chemicals, radiation, viral infection, transfection with DNA sequences, e.g., morphogens, etc., selection (e.g., by continuous culture) of any cells contained in the cultured parent cell, and / or mutagenesis. Derived cells may be selected from a mixed population by response to growth factors, cytokines, selected courses of cytokine treatment, adherence, lack of adherence, sorting procedures, etc.

[0074] As used herein, an "individual" or "subject" refers to a vertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and non-human primates, such as apes and monkeys.

[0075] As used herein, the term "disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0076] As used herein, the term "treating" or "treatment" refers to a clinical intervention that attempts to alter the disease course of the individual or cell being treated, and can be performed either prophylactically or during the course of clinical pathology. The therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, amelioration or reduction of the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can prevent deterioration due to the disorder in affected or diagnosed subjects or subjects suspected of having the disorder, but treatment can also prevent the onset of the disorder or symptoms of the disorder in subjects at risk of the disorder or subjects suspected of having the disorder.

[0077] 5.2 Methods for differentiating stem cells The subject matter of the present disclosure is based, at least in part, on the discovery that midbrain dopamine (mDA) neurons and their precursors can be differentiated from human stem cells by dual inhibition of SMAD signaling (e.g., by inhibiting TGFβ / Activin-Nodal signaling and BMP signaling) with activation of Sonic hedgehog (SHH) signaling and activation of Wingless (Wnt) signaling, and increasing the concentration of a Wnt-activating compound approximately four days after initial exposure of the cells to the SMAD inhibitor, SHH activator, and Wnt activator. In certain non-limiting embodiments, the increase in the concentration of the Wnt-activating compound can be approximately 400% to 1000% of the concentration of the Wnt-activating compound prior to the increase. In certain non-limiting embodiments, the increased concentration of the Wnt activator can be maintained for at least approximately 7 or 8 days. In certain non-limiting embodiments, the increase in Wnt activation is achieved by adding a second or more Wnt activators. Cell can further be contacted with midbrain DA lineage specific activator and inhibitor, for example, BDNF, GDNF, cAMP, TGFβ, AA and DAPT.In certain non-limiting embodiments, the effective amount of the increased concentration of Wnt activator is the concentration that reduces the detectable level of PAX6 expression in the cell population that is contacted with Wnt activator.In certain non-limiting embodiments, PAX6 expression is undetectable in the cell population.

[0078] The subject matter of the present disclosure provides an in vitro method for inducing differentiation of stem cells (e.g., 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 human stem cells are human embryonic stem cells (hESCs).In certain embodiments, the human stem cells are human induced pluripotent stem cells (hiPSCs).

[0079] Non-limiting examples of stem cells that can be used in accordance with the methods described by the present application include human, non-human primate or rodent non-embryonic stem cells, embryonic stem cells, induced non-embryonic pluripotent cells, and engineered pluripotent cells.

[0080] In certain non-limiting embodiments, the stem cells or their progeny contain an introduced heterologous nucleic acid, which may encode a desired nucleic acid or protein product or may be informational (see, e.g., U.S. Patent No. 6,312,911, incorporated by reference in its entirety). Non-limiting examples of desired protein products include markers detectable by in vivo imaging studies, including, but not limited to, receptors or other cell membrane proteins, such as the human sodium-iodide symporter.

[0081] Non-limiting examples of markers further include fluorescent proteins (e.g., green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (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 antigen molecules. As used herein, the term "reporter gene" or "reporter construct" refers to a genetic construct comprising a nucleic acid encoding a readily detectable or readily assayable protein, such as a colored protein, a fluorescent protein such as GFP, or an enzyme such as beta-galactosidase (lacZ gene). In certain embodiments, the reporter may be driven by a recombinant promoter of an mDA marker gene, e.g., TH and / or En-1.

[0082] In certain non-limiting embodiments, stem cells, or progenitor cells thereof, contain introduced heterologous nucleic acids that increase or decrease the metabolic processes of the cells, e.g., glucose metabolism and / or choline metabolism, wherein the cells can be imaged in vivo using positron emission tomography (PET) due to their altered metabolic activity.

[0083] In certain embodiments, the differentiation methods of the present disclosure include contacting a population of human stem cells with one or more inhibitors of transforming growth factor beta (TGFβ) / activin-Nodal signaling, which results in inhibition of small mothers against decapentaplegic (SMAD) signaling. In certain embodiments, the inhibitors of TGFβ / activin-Nodal signaling neutralize ligands, including TGFβ, bone morphogenetic proteins (BMPs), Nodal, and activin, or block their signaling pathways by blocking receptors and downstream effectors. Non-limiting examples of inhibitors of TGFβ / activin-Nodal signaling are disclosed in WO / 2010 / 096496, WO / 2011 / 149762, WO / 2013 / 067362, WO / 2014 / 176606, WO / 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 March;27(3):275-80, Kriks et al., Nature. 2011 November 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 July 1;30(7):715-20 (2012), which are incorporated by reference in their entireties. In certain embodiments, the one or more inhibitors of TGFβ / activin-Nodal signaling are small molecules selected from the group consisting of SB431542, its derivatives, and mixtures thereof. "SB431542" is designated CAS301836-41-9 and has the molecular formula C 22 H 18N4O3, which refers to the molecule named 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide. See, for example, the structure below: [ka]

[0084] The differentiation method of the present disclosure further comprises contacting human stem cells with one or more inhibitors of BMP signaling, which results in the inhibition of SMAD signaling.Non-limiting examples of inhibitors of SMAD signaling are disclosed in WO2011 / 149762, Chambers et al., Nat Biotechnol. March 2009; vol. 27(3): pp. 275-80, Kriks et al., Nature. November 6, 2011; vol. 480(7378): pp. 547-51, and Chambers et al., Nat Biotechnol. July 1, 2012; vol. 30(7): pp. 715-20, the entire contents of which are incorporated herein by reference.In certain embodiments, the one or more inhibitors of BMP / SMAD signaling are small molecules selected from the group consisting of LDN193189, its derivatives, and mixtures thereof. "LDN193189" refers to the small molecule DM-3189, IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, with the chemical formula C 25 H 22 N6 and has the following formula: [ka]

[0085] LDN193189 can function as an SMAD signal transduction inhibitor.LDN193189 is also a highly potent small molecule inhibitor of ALK2, ALK3 and ALK6, protein tyrosine kinase (PTK), and inhibits the signal transduction of ALK1 and ALK3 family members of type I TGFβ receptor, resulting in the inhibition of the transmission of multiple biological signals, including bone morphogenetic protein (BMP) BMP2, BMP4, BMP6, BMP7 and activin cytokine signaling, and the 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, which are incorporated herein by reference).

[0086] The differentiation method of the present disclosure further comprises contacting human stem cells with one or more activators of Wnt signaling. As used herein, the term "WNT" or "wingless" with respect to a ligand refers to a group of secreted proteins (i.e., Intl (integration 1) in humans) that can interact with WNT receptors, such as receptors in the Frizzled and LRPDerailed / RYK receptor families. As used herein, the term "WNT" or "wingless" with respect to a signaling pathway refers to a signaling pathway composed of Wnt family ligands and Wnt family receptors, such as Frizzled and LRPDerailed / RYK receptors that are mediated by β-catenin or do not have β-catenin. For purposes described herein, a preferred WNT signaling pathway includes mediation by β-catenin, such as WNT / -catenin.

[0087] In certain embodiments, one or more activators of Wnt signaling reduce GSK3β for the activation of Wnt signaling. Thus, the activator of Wnt signaling can be a GSK3β inhibitor. GSK3P inhibitors can activate the WNT signaling pathway, see Cadigan et al., J Cell Sci. 2006;119:395-402; Kikuchi et al., Cell Signaling. 2007;19:659-671, the entire contents of which are incorporated herein by reference. As used herein, the term "glycogen synthase kinase 3β inhibitor" refers to a compound that inhibits glycogen synthase kinase 3β enzyme, see Doble et al., J Cell Sci. 2003;116:1175-1186, the entire contents of which are incorporated herein by reference.

[0088] Non-limiting examples of activators of Wnt signaling or GSK3β inhibitors are disclosed in WO2011 / 149762, WO13 / 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. 19; 35(33):11462-81, which are incorporated by reference in their entireties. In certain embodiments, the one or more activators of Wnt signaling are small molecules selected from the group consisting of CHIR99021, derivatives thereof, and mixtures thereof. "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-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile, having the formula: [ka]

[0089] CHIR99021 is highly selective, exhibiting nearly 1000-fold selectivity against a panel of related and unrelated kinases, with an IC50 of 6.7 nM against human GSK3β and nanomolar IC50 values ​​against rodent GSK3β homologues.

[0090] In certain non-limiting embodiments, a population of cells described herein is contacted with an initial concentration of CHIR99021 at a concentration of between about 0.001 and 2 μM, or between about 0.01 and 1.5 μM, or between about 0.1 and 1 μM, or between about 0.5 and 0.8 μM, or about 0.7 μM, and the concentration of CHIR99021 is increased as described herein, for example, to between about 2 and 15 μM, or between about 3 and 14 μM, or between about 4 and 13 μM, or between about 5 and 12 μM, or between about 6 and 11 μM, or between about 7 and 10 μM, or between about 8 and 9 μM, or between about 3 and 10 μM, or between about 5 and 10 μM, or about 3 μM, or about 7.5 μM, about 4 days after initial contact of the cells with CHIR99021.

[0091] The differentiation method of the present disclosure further includes contacting human stem cells with one or more activators of SHH signaling. As used herein, the terms "Sonic hedgehog," "SHH," or "Shh" refer to a protein that is one of at least three proteins in a mammalian signaling pathway family called hedgehog, another being Desert hedgehog (DHH), while the third is 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, which then activates SMO as a signal transducer. In the absence of SHH, PTC typically inhibits SMO, which in turn activates transcriptional repressors to prevent the transcription of certain genes. When Shh is present and bound to PTC, PTC cannot prevent SMO function. When SMO is not inhibited, certain proteins can enter the nucleus and act as transcription factors, enabling the activation of certain genes (see Gilbert, 2000, Developmental Biology (Sunderland, Mass., Sinauer Associates, Inc., Publishers)). In certain embodiments, an activator of Sonic hedgehog (SHH) signaling refers to any molecule or compound that activates the SHH signaling pathway, including molecules or compounds that bind to PTC or Smoothened agonists, etc. Non-limiting examples of activators of Wnt signaling or GSK3β inhibitors are disclosed in WO10 / 096496, WO13 / 067362, Chambers et al., Nat Biotechnol. 2009 March;27(3):275-80, and Kriks et al., Nature. 2011 November 6;480(7378):547-51.Examples of such compounds are recombinant SHH, purified SHH, the protein sonic hedgehog (SHH) C25II (i.e., a recombinant NT-terminal fragment of the full-length mouse sonic hedgehog protein that can bind to the SHH receptor to activate SHH, e.g., R and D Systems catalog number: 464-5H-025 / CF), and small molecule Smoothened agonists, such as parmorphamine.

[0092] In certain embodiments, the inhibitors and activators are added to a cell culture medium containing stem 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 Examples of media include KSR medium, NB medium, N2 medium, B-27 medium, and E8 / E6 medium, as well as combinations thereof. KSR medium is a defined, serum-free formulation optimized for growing and maintaining undifferentiated hESC cells in culture.

[0093] In certain embodiments, the cell culture medium is KSR medium. The components of KSR medium are disclosed in WO2011 / 149762. In certain embodiments, KSR medium comprises Knockout DMEM, Knockout Serum Replacement, L-glutamine, Pen / Strep, MEM, and 13-mercaptoethanol. In certain embodiments, 1 liter of KSR medium comprises 820 mL of Knockout DMEM, 150 mL of Knockout Serum Replacement, 10 mL of 200 mM L-glutamine, 10 mL of Pen / Strep, 10 mL of 10 mM MEM, and 55 μM 13-mercaptoethanol.

[0094] In certain embodiments, stem cells are contacted with one or more inhibitors of TGFβ / activin-Nodal signaling, one or more inhibitors of BMP / SMAD signaling, one or more activators of Wnt signaling, and one or more activators of SHH signaling. In certain embodiments, one or more inhibitors of TGFβ / activin-Nodal signaling, one or more inhibitors of SMAD signaling, one or more activators of Wnt signaling, and one or more activators of SHH signaling are added to a cell culture medium containing stem cells.

[0095] 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 custom medium formulations for the culture of PSCs. An example of E8 / E6 medium is described in Chen et al., Nat Methods 2011 May;8(5):424-9, which is incorporated by reference in its entirety. An example of E8 / E6 medium is disclosed in WO 15 / 077648, which is incorporated by reference in its entirety. In certain embodiments, the E8 / E6 cell culture medium comprises 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 or Wnt components. Thus, in certain embodiments, when E8 / E6 medium is used to culture and differentiate a population of stem cells of the present disclosure into a population of proprioceptors, one or more inhibitors of SMAD signaling (e.g., those that inhibit BMP) do not need to be added to the E8 / E6 medium. In certain embodiments, stem cells are contacted with one or more inhibitors of TGFβ / activin-Nodal signaling, one or more activators of Wnt signaling, and one or more activators of SHH signaling. In certain embodiments, one or more inhibitors of TGFβ / activin-Nodal signaling, one or more activators of Wnt signaling, and one or more activators of SHH signaling are added to a cell culture medium containing stem cells. In certain embodiments, BMP may further be added to the medium.

[0096] In certain embodiments, the subject matter of the present disclosure provides an in vitro method for inducing human stem cells to differentiate into midbrain DA neurons or their precursors.In certain embodiments, stem cells are simultaneously contacted with one or more inhibitors of TGFβ / activin-Nodal signaling, one or more inhibitors of BMP / SMAD signaling, one or more activators of Wnt signaling, and one or more activators of SHH signaling, for example, by adding these inhibitors to the cell culture medium containing stem cells on the same day.In certain embodiments, the concentration of one or more activators of Wnt signaling is increased at least about 2, 3, 4, 5 or 6 days after the first contact of cells with Wnt activators.

[0097] In certain embodiments, the one or more inhibitors of TGFβ / activin-Nodal signaling are contacted with the cells for at least about 4, 5, 6, 7, 8, 9, or 10 or more days, e.g., about 4 to 10 days, or about 5 to 9 days, or about 6 to 8 days. In certain embodiments, the one or more inhibitors of TGFβ / activin-Nodal signaling are contacted with the cells for up to about 4, 5, 6, 7, 8, 9, or 10 or more days. In certain embodiments, the one or more inhibitors of TGFβ / activin-Nodal signaling are contacted with the cells for about 7 days. In certain embodiments, the one or more inhibitors of TGFβ / activin-Nodal signaling are added to the cell culture medium containing the stem cells every day or every other day from day 0 through day 10 (e.g., added on days 0, 2, 4, 6, 8, and 10). In certain embodiments, one or more inhibitors of TGFβ / activin-Nodal signaling are added on days 0, 1, 3, 4, and 6.

[0098] In certain embodiments, the cells are contacted with one or more inhibitors of TGFβ / activin-Nodal signaling at a concentration of about 1 to 20 μM, or about 2 to 19 μM, or about 3 to 18 μM, or about 4 to 17 μM, or about 5 to 16 μM, or about 6 to 15 μM, or about 7 to 14 μM, or about 8 to 13 μM, or about 9 to 12 μM, or about 10 to 11 μM, and values ​​therebetween. In certain embodiments, the cells are contacted with one or more inhibitors of TGFβ / activin-Nodal signaling at a concentration of about 7, 8, 9, 10, 11, 12, or 13 μM. In certain embodiments, the cells are contacted with one or more inhibitors of TGFβ / activin-Nodal signaling at a concentration of about 10.8 μM.

[0099] In certain embodiments, the one or more inhibitors of BMP / SMAD signaling are contacted with the cells for at least about 4, 5, 6, 7, 8, 9, or 10 or more days, e.g., about 4 to 10 days, or about 5 to 9 days, or about 6 to 8 days. In certain embodiments, the one or more inhibitors of BMP / SMAD signaling are contacted with the cells for up to about 4, 5, 6, 7, 8, 9, or 10 or more days. In certain embodiments, the one or more inhibitors of BMP / SMAD signaling are contacted with the cells for about 7 days. In certain embodiments, the one or more inhibitors of BMP / SMAD signaling are added to the cell culture medium containing the stem cells every day or every other day from day 0 through day 10 (e.g., added on days 0, 2, 4, 6, 8, and 10). In certain embodiments, the one or more inhibitors are added on days 0, 1, 3, 4, and 6.

[0100] In certain embodiments, one or more inhibitors of BMP / SMAD signaling are contacted with cells at a concentration of about 50 to 500 nM, or about 75 to 475 nM, or about 100 to 450 nM, or about 125 to 425 nM, or about 150 to 400 nM, or about 175 to 375 nM, or about 200 to 350 nM, or about 225 to 325 nM, or about 250 to 300 nM, and values ​​therebetween. In certain embodiments, one or more inhibitors of BMP / SMAD signaling are contacted with cells at a concentration of about 150, 200, 250, 300, or 350 nM. In certain embodiments, one or more inhibitors of BMP / SMAD signaling are contacted with cells at a concentration of about 250 nM.

[0101] In certain embodiments, the one or more activators of SHH signaling are contacted with the cells for at least about 4, 5, 6, 7, 8, 9, or 10 or more days, e.g., about 4 to 10 days, or about 5 to 9 days, or about 6 to 8 days. In certain embodiments, the one or more activators of SHH signaling are contacted with the cells for up to about 4, 5, 6, 7, 8, 9, or 10 or more days. In certain embodiments, the one or more activators of SHH signaling are contacted with the cells for about 7 days. In certain embodiments, the one or more activators of SHH signaling are added to the cell culture medium containing the stem cells every day or every other day from day 0 to day 10 (e.g., added on days 0, 2, 4, 6, 8, and 10). In certain embodiments, the one or more inhibitors are added on days 0, 1, 3, 4, and 6.

[0102] In certain embodiments, one or more activators of SHH signaling are contacted with cells at a concentration of about 50 to 1000 ng / mL, or about 100 to 950 ng / mL, or about 150 to 900 ng / mL, or about 200 to 850 ng / mL, or about 250 to 800 ng / mL, or about 300 to 750 ng / mL, or about 350 to 700 ng / mL, or about 400 to 650 ng / mL, or about 450 to 600 ng / mL, or about 500 to 550 ng / mL, and values ​​therebetween. In certain embodiments, one or more activators of SHH signaling are contacted with cells at a concentration of about 400, 450, 500, 550, or 600 ng / mL. In certain embodiments, one or more activators of SHH signaling are contacted with cells at a concentration of about 500 ng / mL.

[0103] In certain embodiments, the one or more activators of Wnt signaling are administered for at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more days, e.g., between about 4 and 20 days, or between about 4 and 19 days, or between about 4 and 18 days, or between about 4 and 17 days, or between about 4 and 16 days, or between about 4 and 15 days, or between about 4 and 14 days. In certain embodiments, the cells are contacted with one or more activators of Wnt signaling for up to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more days. In certain embodiments, the cells are contacted with one or more activators of Wnt signaling for about 12 days. In certain embodiments, one or more activators of SHH signaling are added to the cell culture medium containing the stem cells every day or every other day from day 0 to day 12 (e.g., added on days 0, 2, 4, 6, 8, 10, and 12). In certain embodiments, one or more inhibitors are added on days 0, 1, 3, 4, 6, 7, 9, 10, and 11.

[0104] In certain embodiments, the cells are contacted with one or more activators of Wnt signaling at a concentration of between about 0.05 and 15 μM, or between about 0.1 and 14 μM, or between about 0.5 and 13 μM, or between about 1 and 12 μM, or between about 1.5 and 11 μM, or between about 2 and 10 μM, or between about 2.5 and 9.5 μM, or between about 3 and 9 μM, or between about 3.5 and 8.5 μM, or between about 4 and 8 μM, or between about 4.5 and 7.5 μM, or between about 5 and 7 μM, or between about 5.5 and 6.5 μM, and values ​​therebetween. In certain embodiments, the cells are contacted with one or more activators of Wnt signaling at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 μM. In certain embodiments, the one or more activators of Wnt signaling are contacted with the cells at a concentration of about 0.7 μM.

[0105] In certain embodiments, the concentration of the activator of Wnt signaling is increased at least about 2, 3, 4, 5, or 6 days after initial contact of the cells with the Wnt activator, e.g., between about 2 and 10 days, or between about 3 and 9 days, or between about 4 and 8 days, or between about 5 and 7 days. In certain embodiments, the concentration of the activator of Wnt signaling is increased by about 2, 3, 4, 5, or 6 days after initial contact of the cells with the Wnt activator. In certain embodiments, the cells are contacted with the increasing concentrations of Wnt activator for at least about 4, 5, 6, 7, 8, 9, or 10 days or more, e.g., between about 4 and 20 days, or between about 5 and 19 days, or between about 6 and 18 days, or between about 7 and 17 days, or between about 8 and 16 days, or between about 9 and 15 days, or between about 8 and 14 days, or between about 9 and 13 days, or between about 10 and 12 days. In certain embodiments, the cells are contacted with increasing concentrations of a Wnt activator for about 4, 5, 6, 7, 8, 9, or 10 days or more. In certain embodiments, the cells are contacted with increasing concentrations of a Wnt activator for about 5, 6, 7, 8, 9, 10, or 11 days. In certain embodiments, the cells are contacted with increasing concentrations of a Wnt activator for about 8 days.

[0106] In certain embodiments, the concentration of the activator of Wnt signaling is increased to a concentration of about 2 to 15 μM, or about 3 to 14 μM, or about 4 to 13 μM, or about 5 to 12 μM, or about 6 to 11 μM, or about 7 to 10 μM, or about 8 to 9 μM. In certain embodiments, the concentration of the activator of Wnt signaling is increased to a concentration of about 3 to 10 μM, or about 5 to 10 μM. In certain embodiments, the concentration of the activator of Wnt signaling is increased to a concentration of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μM. In certain embodiments, the concentration of the activator of Wnt signaling is increased to a concentration of about 3 μM. In certain embodiments, the concentration of the activator of Wnt signaling is increased to a concentration of about 7.5 μM.

[0107] In certain embodiments, the concentration of the activator of Wnt signaling is increased from the initial concentration contacted with the cells by about 50 to 2000%, or about 100 to 1950%, or about 150 to 1900%, or about 200 to 1850%, or about 250 to 1800%, or about 300 to 1750%, or about 350 to 1700%, or about 400 to 1650%, or about 450 to 1600%, or In certain embodiments, the concentration of the activator of Wnt signaling is increased by about 400 to 1450%, or about 700 to 1050%, or about 700 to 1550%, or about 550 to 1500%, or about 600 to 1450%, or about 650 to 1400%, or about 700 to 1350%, or about 750 to 1300%, or about 800 to 1250%, or about 850 to 1200%, or about 900 to 1150%, or about 950 to 1100%, or about 1000 to 1050%, or values ​​therebetween.

[0108] In certain embodiments, the concentration of the activator of Wnt signaling is increased by about 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1050%, or 1100% or more from the initial concentration contacted with the cells.

[0109] In certain non-limiting embodiments, cells are contacted with one or more inhibitors of TGFβ / Activin-Nodal signaling (i.e., first SMAD inhibitors), such as SB431542 at a concentration of about 10.8 μM, one or more inhibitors of BMP signaling (i.e., second SMAD inhibitors), such as LDN193189 at a concentration of about 250 nM, one or more activators of Wnt signaling, such as CHIR99021 at a concentration of 0.7 μM, and one or more activators of SHH signaling at a concentration of about 500 ng / mL, wherein the cells are contacted with the inhibitors for about 7 days (i.e., from day 0 to day 6 of culture), and the concentration of CHIR99021 is increased to 3 μM or 7.5 μM on day 4 of cell culture.

[0110] In certain embodiments, the cells are contacted with one or more activators of Wnt signaling for approximately 11 days (i.e., from day 0 to day 11 of culture), and the cells are contacted with 7.5 μM CHIR99021 from day 4 to day 11 of cell culture, or the cells are contacted with 7.5 μM CHIR99021 from day 4 to day 9 of culture, and then with 3 μM CHIR99021 from day 10 to day 11 of cell culture.

[0111] In certain embodiments, cells are contacted with activators and inhibitors described herein at concentrations and for times effective to increase detectable levels of expression of one or more of engrailed-1 (EN-1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor-associated 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1A).

[0112] In certain embodiments, cells are contacted with the activators and inhibitors described herein at concentrations and for a time effective to increase detectable levels of expression of one or more of neuron-specific class III beta-tubulin (Tuj1), trefoil factor family 3 (TTF3), paired-like homeodomain 3 (PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT), and G-protein-coupled inwardly rectifying potassium channel (Kir3.2 / GIRK2), CD142, DCSM1, CD63, and / or CD99.

[0113] In certain embodiments, cells are contacted with the activators and inhibitors described herein at concentrations and for a time effective to increase a detectable level of expression of one or more markers of DA neurons, e.g., CD142, wherein the cells are type A9 neuronal cells.

[0114] In certain embodiments, cells are contacted with the activators and inhibitors described herein at concentrations and for a time effective to reduce the expression of paired box protein (PAX6) and Ki67.

[0115] In certain embodiments, the cells are further contacted with DA neuron lineage-specific activators and inhibitors, such as L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta (TGFβ, e.g., TGFβ3), ascorbic acid (AA), and DAPT (N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl)glycine-1,1-dimethylenyl ester; also known as LY-374973, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; or N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). In certain embodiments, cells are contacted with the above-mentioned DA neuron lineage-specific activators and inhibitors for at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days, e.g., about 2 to 20 days, about 3 to 19 days, about 4 to 18 days, about 5 to 17 days, about 6 to 16 days, about 7 to 15 days, about 8 to 15 days, about 9 to 14 days, or about 10 to 13 days. In certain embodiments, cells are contacted with the above-mentioned DA neuron lineage-specific activators and inhibitors for up to about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days. In certain embodiments, cells are contacted with the above-mentioned DA neuron lineage-specific activators and inhibitors for about 4, 5, 6, 7, or 8 days.

[0116] In certain embodiments, the cells are contacted with L-glutamine at a concentration of about 0.5 to 5 mM, or about 1 to 4 mM, or about 1.5 to 3 mM, hi certain embodiments, the cells are contacted with L-glutamine at a concentration of about 2 mM.

[0117] In certain embodiments, cells are contacted with BDNF at a concentration of about 5 to 50 ng / mL, or about 10 to 40 ng / mL, or about 15 to 30 ng / mL, or about 18 to 25 ng / mL, hi certain embodiments, cells are contacted with BDNF at a concentration of about 20 ng / mL.

[0118] In certain embodiments, cells are contacted with AA at a concentration of between about 50 and 500 nM, or between about 100 and 400 nM, or between about 150 and 300 nM, or between about 180 and 250 nM. In certain embodiments, cells are contacted with AA at a concentration of about 200 nM.

[0119] In certain embodiments, the cells are contacted with GDNF at a concentration of between about 5 and 50 ng / mL, or between about 10 and 40 ng / mL, or between about 15 and 30 ng / mL, or between about 18 and 25 ng / mL, hi certain embodiments, the cells are contacted with GDNF at a concentration of about 20 ng / mL.

[0120] In certain embodiments, the cells are contacted with cAMP at a concentration of about 200 to 800 nM, or about 250 to 750 nM, or about 300 to 700 nM, or about 350 to 650 nM, or about 400 to 600 nM, or about 450 to 550 nM. In certain embodiments, the cells are contacted with cAMP at a concentration of about 500 nM.

[0121] In certain embodiments, the cells are contacted with TGFβ3 at a concentration of between about 0.01 and 5 ng / mL, or between about 0.05 and 4 ng / mL, or between about 0.1 and 3 ng / mL, or between about 0.5 and 2 ng / mL. In certain embodiments, the cells are contacted with TGFβ3 at a concentration of about 1 ng / mL.

[0122] In certain embodiments, differentiated midbrain DA precursors are further cultured as described in U.S. Patent Application Publication No. 2015 / 0010514, which is incorporated by reference in its entirety.

[0123] In certain embodiments, cells prepared according to the methods described herein can be sorted, selected, and isolated based on CD142 expression, or cholinergic receptor (CHRNB3) expression, for example, using flow cytometry.

[0124] In certain embodiments, the cells prepared according to the methods described herein are treated with a polysialyltransferase, such as a bacterial polysialyltransferase, e.g., Neisseria meningitidis polysialyltransferase (PST). Nm In certain embodiments, the cell is a recombinant cell expressing a recombinant polysialyltransferase.

[0125] 5.3 Methods of Treating Neurodegenerative Disorders In vitro differentiated cells expressing one or more markers of midbrain DA neurons, or their precursors (also referred to as "stem cell-derived midbrain DA precursors" or "mDA" precursors), can be used to treat neurodegenerative disorders. The presently disclosed subject matter provides a method of treating a neurodegenerative disorder, comprising administering an effective amount of the disclosed stem cell-derived precursors to a subject suffering from a neurodegenerative disorder.

[0126] Non-limiting examples of neurodegenerative disorders include Parkinson's disease, Huntington's disease, Alzheimer's disease, and multiple sclerosis.

[0127] In certain embodiments, the neurodegenerative disorder is Parkinson's disease. Cardinal motor symptoms of Parkinson's disease include, but are not limited to, tremors in the hands, arms, legs, jaw, and face, bradykinesia or slowness of movement, stiffness or rigidity of the limbs and trunk, and postural instability or impaired balance and coordination.

[0128] In certain embodiments, neurodegenerative disease is parkinsonism disease, which refers to the disease associated with the lack of dopamine in the basal ganglia, the part of the brain that controls movement.Symptoms include tremor, bradykinesia (extremely slow movement), flexed posture, unstable posture and rigidity.Non-limiting examples of parkinsonism disease include corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy and progressive supranuclear palsy.

[0129] The stem cell-derived precursors of the present disclosure can be administered or provided to a subject systemically or directly to treat or prevent neurodegenerative disorders.In certain embodiments, the stem cell-derived precursors of the present disclosure are directly injected into target organs (for example, central nervous system (CNS) or peripheral nervous system (PNS)).In certain embodiments, the stem cell-derived precursors of the present disclosure are directly injected into the striatum.

[0130] The stem cell-derived precursors of the present disclosure can be administered in any physiologically acceptable vehicle. Pharmaceutical compositions comprising the stem cell-derived precursors of the present disclosure and a pharmaceutically acceptable vehicle are also provided. The stem cell-derived precursors of the present disclosure and pharmaceutical compositions comprising the cells can be administered by local injection, orthotopic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In certain embodiments, the stem cell-derived precursors of the present disclosure can be administered to patients suffering from neurodegenerative disorders by orthotopic (OT) injection.

[0131] Pharmaceutical compositions comprising the stem cell-derived precursors and cells of the present disclosure can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which 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 viscosity range appropriate for longer contact periods with specific tissues. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium, including, 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 presently disclosed subject matter, such as a composition comprising the stem cell-derived precursors of the present disclosure, into the required amount of an appropriate solvent containing various desired amounts of other ingredients. Such compositions may be present in a mixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, etc. The composition may also be lyophilized. Depending on the desired route of administration and preparation, the compositions can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents, etc. Standard textbooks, such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th Edition, 1985, incorporated herein by reference, can be consulted to prepare suitable preparations without undue experimentation.

[0132] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial activity is ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.Prolonged absorption of injectable pharmaceutical forms can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used should be compatible with the stem cell-derived precursor of the present disclosure.

[0133] If necessary, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The concentration of the thickening agent can vary depending on the drug selected. The important point is to use an amount that achieves the selected viscosity. The selection of appropriate carriers and other additives will vary depending on the exact route of administration and the nature of the specific dosage form, for example, a liquid dosage form (e.g., whether the composition will be formulated into a liquid, suspension, gel, or other liquid form, such as a time-release form or liquid-filled form).

[0134] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and not affect the viability or efficacy of the stem cell-derived precursors of the present disclosure. This does not present a challenge to those skilled in chemical and pharmaceutical principles, and can be easily avoided from this disclosure and the documents cited herein by reference to standard textbooks or by simple experimentation (without undue experimentation).

[0135] In certain non-limiting embodiments, the cells and precursors described herein are included in a composition that further comprises a biocompatible scaffold or matrix, e.g., a biocompatible three-dimensional scaffold that promotes tissue regeneration when the cells are implanted or transplanted into a subject. In certain non-limiting embodiments, the biocompatible scaffold comprises an extracellular matrix material, a synthetic polymer, a cytokine, a collagen, a polypeptide or protein, a polysaccharide (including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or a hydrogel). (See, e.g., U.S. Patent Application Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entirety.) In certain embodiments, the composition further comprises growth factors to promote maturation of the implanted / transplanted midbrain DA cells.

[0136] One consideration regarding therapeutic applications of the stem cell-derived progenitors of the present disclosure is the quantity of cells needed to achieve an optimal effect, including but not limited to, repopulation of CNS and / or PNS regions of a subject suffering from a neurodegenerative disorder and / or improvement in the function of the CNS and / or PNS of a subject.

[0137] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to affect a beneficial or desired clinical outcome upon treatment. An effective amount can be administered to a subject in one or more doses. For treatment, an effective amount is an amount sufficient to palliate, improve, stabilize, reverse, or slow the progression of a neurodegenerative or pituitary disorder, or alternatively reduce the pathological consequences of a neurodegenerative disorder. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one of ordinary skill in the art. Several factors are typically taken into consideration when determining an appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells administered.

[0138] In certain embodiments, an effective amount of stem cell-derived progenitors of the present disclosure is an amount sufficient to repopulate CNS and / or PNS regions in a subject suffering from a neurodegenerative disorder. In certain embodiments, an effective amount of stem cell-derived progenitors of the present disclosure is an amount sufficient to improve CNS and / or PNS function in a subject suffering from a neurodegenerative disorder, for example, improved function can be about 1%, 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 normal human CNS and / or PNS function.

[0139] The amount of cells administered will vary depending on the subject being treated. In certain embodiments, about 1 x 10 4 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 6 , about 1×10 5 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 8, about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 8 ~Approx. 1×10 10 , or approximately 1 × 10 9 ~Approx. 1×10 10 of stem cell-derived progenitors of the present disclosure are administered to a subject. In certain embodiments, about 1 x 10 5 ~Approx. 1×10 7 of stem cell-derived progenitors of the present disclosure are administered to a subject suffering from a neurodegenerative disorder. In certain embodiments, about 1 x 10 6 ~Approx. 1×10 7 of stem cell-derived progenitors of the present disclosure are administered to a subject suffering from a neurodegenerative disorder. In certain embodiments, about 1 x 10 6 ~Approx. 4×10 6 The stem cell-derived precursors of the present disclosure are administered to a subject suffering from a neurodegenerative disorder.The precise determination of the effective dose to be considered may be based on individual factors for each subject, including the size, age, sex, weight and condition of the specific subject.Dosage can be easily ascertained by those skilled in the art from this disclosure and knowledge in the art.

[0140] In certain embodiments, the cells administered to a subject suffering from a neurodegenerative disorder to treat the neurodegenerative disorder are a population of neurons differentiated / mature from stem cell-derived midbrain DA precursors of the present disclosure.

[0141] 5.4 Kits The subject matter of the present disclosure provides a kit for inducing stem cell differentiation. In certain embodiments, the kit includes: (a) one or more inhibitors of transforming growth factor beta (TGFβ) / activin-Nodal signaling, (b) one or more inhibitors of BMP / SMAD signaling, (c) one or more activators of Wnt signaling, (d) one or more activators of SHH signaling, and (e) instructions for inducing stem cell differentiation into a population of differentiated cells that express one or more markers of midbrain DA neurons or their precursors.

[0142] In certain embodiments, the kit does not include one or more inhibitors of BMP / SMAD signaling.

[0143] In certain embodiments, the kit further comprises one or more activators of BMP signaling.

[0144] In certain embodiments, the instructions include contacting the stem cells with the inhibitor, activator, and molecule in a specific order, which order can be determined by the cell culture medium used to culture the stem cells.

[0145] In certain embodiments, the instructions include contacting the stem cells with inhibitors, activators, and molecules as described by the methods of the present disclosure (see section 5.2, supra).

[0146] In certain embodiments, the present disclosure provides a kit comprising an effective amount of a population of stem cell-derived progenitors of the present disclosure or a composition comprising the progenitors in a unit dosage form.In certain embodiments, the stem cell-derived cells are mature differentiated cells, such as midbrain DA neurons.In certain embodiments, the kit comprises a sterile container containing a therapeutic composition, and such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a 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 materials suitable for holding medicines.

[0147] In certain embodiments, the kit includes instructions for administering a population of stem cell-derived progenitors of the present disclosure or a composition comprising the same to a subject suffering from a neurodegenerative disorder. The instructions can include information about using the cells or composition to treat or prevent the neurodegenerative disorder. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent, dosing schedule and administration for treating or preventing the neurodegenerative disorder or its symptoms, precautions, warnings, indications, counter-indications, overdose information, adverse reactions, animal pharmacology, clinical trials, and / or references. The instructions can be printed directly on the container (if present), as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. [Example]

[0148] 6. Working Example The presently disclosed subject matter will be better understood with reference to the following examples, which are provided by way of illustration of the presently disclosed subject matter, and not by way of limitation.

[0149] 6.1 Example 1: Method for preparing stem cell-derived midbrain dopamine (DA) progenitor cells

[0150] overview Human embryonic stem cells (hESCs) can potentially give rise to any cell type in the body. A long-term goal is to develop a strategy to recapitulate the complete human lineage tree in vitro. This example describes a strategy for differentiating hESCs into midbrain dopamine neuron precursor cells. Cells were differentiated in neurobasal (NB) / N2 medium (optionally containing E6 medium) supplemented with SB431542 (a TGFβ / activin-Nodal signaling inhibitor), LDN193189 (a BMP / SMAD signaling inhibitor), sonic hedgehog (SHH), and CHIR99021 (a GSK3β inhibitor that enhances Wingless (Wnt) signaling). The concentration of CHIR99021 was increased after initial incubation with growth factors for 4 or 5 days ("bump"). Cells were then cultured with increasing concentrations of CHIR99021 for 7 or 8 days. The cells were then cultured with the DA neuron growth factors brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta 3 (TGFβ3), ascorbic acid (AA), and DAPT to generate midbrain DA precursors.

[0151] method hESCs were maintained in E8 / Matrigel medium prior to differentiation. Cells were differentiated in growth factor-supplemented NB / N2 medium according to the following culture strategy (cells were cultured with either a 3 μM bump on day 4 or a 7.5 μM bump on day 5).

[0152] 3μMbump protocol (GMP V2A) Day 0 (D0): hESCs were transferred from E8 / Matrigel medium to differentiation medium containing neurobasal (NB) / N2 medium supplemented with the following differentiation factors (D0 medium also contained 10 μM Y-27632 ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor to enhance cell survival). Cells were at a concentration of approximately 2 million cells per mL: 250nM LDN193189 10 μM SB431542 500ng / mL SHH 0.7 μM CHIR99021

[0153] - D1: The differentiation medium was replaced with fresh differentiation medium without Y-27632.

[0154] - D2: No medium change.

[0155] - D3: medium changed as described in D1.

[0156] - D4: The differentiation medium was replaced with fresh differentiation medium containing 3 μM CHIR99021 (without Y-27632).

[0157] - D5: No medium change.

[0158] - D6: medium changed as described in D4.

[0159] - D7: Differentiation medium was replaced with fresh medium containing NB / N2 (without LDN193189, SB431542, SHH or Y-27632) supplemented with 3 μM CHIR99021.

[0160] - D8: No medium change.

[0161] - D9: medium changed as described in D7.

[0162] - D10: The medium was replaced with fresh NB / B27 medium containing 3 μM CHIR99021, BDNF, GDNF, cAMP, TGFβ3, and AA.

[0163] - D11: medium changed as described in D10.

[0164] - D12: Medium changed as described in D10, except that the medium additionally contained DAPT and did not contain CHIR99021.

[0165] - D13 to D19: Medium changed as described in D12. (Culture period is up to D27 or beyond, and may include at least one passage on D17 or D18.)

[0166] - D20: The medium was aspirated from the cells, the cells were washed and suspended in NB medium and then plated.

[0167] - Cells were then tested for expression of a combination of early midbrain DA markers: 1) OTX2 / EN / LMX1A and 2) PAX6 / FOXA2 / EN / and NKX2.2 or NKX6.1, or a combination of late midbrain DA markers: 1) TH / EN / FOXA2 and 2) LMX1A / OTX2 / NURR1.

[0168] - The cells were subjected to cryopreservation or transplantation into mice.

[0169] - For transplantation into mice, cells were suspended in HBSS+HEPPES at a concentration of 150,000 cells / microliter, where 5-6 million cells were transplanted into immunodeficient mice.

[0170] 7.5μMbump protocol (GMP V2B) Day 0 (D0): hESCs were transferred from E8 / Matrigel medium to differentiation medium containing neurobasal (NB) / N2 medium supplemented with the following differentiation factors (D0 medium also contained 10 μM Y-27632 ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor to enhance cell survival). Cells were at a concentration of approximately 2 million cells per mL: 250nM LDN193189 10 μM SB431542 500ng / mL SHH 0.7 μM CHIR99021

[0171] - D1: The differentiation medium was replaced with fresh differentiation medium without Y-27632.

[0172] - D2: No medium change.

[0173] - D3: medium changed as described in D1.

[0174] - D4: No medium change.

[0175] - D5: Differentiation medium was replaced with fresh differentiation medium containing 7.5 μM CHIR99021 (without Y-27632).

[0176] - D6: No medium change.

[0177] - D7: Differentiation medium was replaced with fresh medium containing NB / N2 (without LDN193189, SB431542, SHH or Y-27632) supplemented with 7.5 μM CHIR99021.

[0178] - D8: No medium change.

[0179] - D9: medium changed as described in D7.

[0180] - D10: The medium was replaced with fresh NB / B27 medium containing 3 μM CHIR99021, BDNF, GDNF, cAMP, TGFβ3, and AA.

[0181] - D11: medium changed as described in D10.

[0182] - D12: Medium changed as described in D10, except that the medium additionally contained DAPT and did not contain CHIR99021.

[0183] - D13 to D19: Medium changed as described in D12. (Culture period is up to D27 or beyond, and may include at least one passage on D17 or D18.)

[0184] - D20: The medium was aspirated from the cells, the cells were washed and suspended in NB medium and then plated.

[0185] - Cells were tested for expression of a combination of early midbrain DA markers: 1) OTX2 / EN / LMX1A and 2) PAX6 / FOXA2 / EN / and NKX2.2 or NKX6.1, or a combination of late midbrain DA markers: 1) TH / EN / FOXA2 and 2) LMX1A / OTX2 / NURR1.

[0186] - The cells were subjected to cryopreservation or transplantation into mice.

[0187] - For transplantation into mice, cells were suspended in HBSS+HEPPES at a concentration of 150,000 cells / microliter, where 5-6 million cells were transplanted into immunodeficient mice.

[0188] result Kriks et al., Nature. 2011, November 6; Vol. 480 (No. 7378): 547-51, describes a protocol for differentiation of hESCs into midbrain DA cells by culturing the cells in KSR medium containing dual SMAD inhibition, SHH activation, and Wnt activation (without the "bump" described below). However, KSR medium is not defined, and cells generated using this differentiation protocol perform poorly in vivo after transplantation. Cells differentiated according to this example utilize a culture protocol containing dual SMAD inhibition (with SB431542 and LDN193189), SHH activation, and Wnt activation in NB / N2 medium, in which the concentration of Wnt is increased from a baseline concentration of 0.7 μM to between 5 and 10 μM on day 4 or day 5 of culture (i.e., the Wnt "bump").

[0189] As shown in Figure 1, differentiation of hESCs in KSR medium (non-GMP) or E8 / NB / N2 medium (GMP V1) according to the method described by Kriks et al. generated midbrain DA neurons, but also neurons from other brain regions. When cells were cultured in E8 / NB / N2 medium using the method of this example, which utilizes a 7.5 μM (or 5-10 μM) Wnt bump from D4 to D10, midbrain DA cells were specifically generated.

[0190] As shown in Figure 2, differentiation of hESCs in KSR medium (Non-GMP) or E8 / NB / N2 medium (GMP V1) according to the method described in Kriks et al. generated cells that expressed similar levels of PAX6, TH, NURR1, FOXA2, and LMX1A. However, cells generated using either medium showed poor survival when transplanted into rodents.

[0191] When hESCs were differentiated using E8 / NB / N2 medium and a 3 μM (GMP V2A) or 7.5 μM (GMP V2B) Wnt bump, the cells also expressed similar levels of PAX6, TH, NURR1, FOXA2, and LMX1A as compared with the Kriks et al. protocol using KSR (non-GMP) or E8 / NB / N2 (GMP V1). However, cells differentiated according to the GMP V2A or GMP V2B protocols expressed higher levels of the midbrain DA marker EN-1 (Figure 3). Figure 4 describes other midbrain DA markers that can be used to identify differentiated cells.

[0192] hESCs differentiated using the protocol of Kriks et al. in E8 / NB / N2 medium resulted in good survival of DA cells expressing Hncam, FOXA2, and TH in vivo after 25 days of differentiation and transplantation into uninjured, immunocompromised mice. Grafts were examined 4 weeks after transplantation. However, cells were also positive for PAX6, showing dense patches of Hncam expression, indicative of a neural progenitor state (Figure 5). hESCs cultured according to GMP V2A (3 μM bump) or GMP V2B (7.5 μM bump) generated DA cells in vitro that expressed increased levels of NURR1, LMX1A, EN-1, and TH (Figure 6A), but did not express PAX6 (Figure 7). Furthermore, mesencephalic DA cells differentiated using the GMP V2B (7.5 μM bump) protocol and transplanted into the striatum of intact, immunocompromised mice showed increased fiber outgrowth and hNCAM and TH expression 3 weeks after transplantation, without hNCAM patches (Figure 6B). Furthermore, when cells were cryopreserved before transplantation, they exhibited fiber outgrowth similar to that of cells transplanted into mice that had not previously been cryopreserved (Figure 8).

[0193] As shown in Figure 9A-B, cells prepared according to the GMP V2A or GMP V2B protocol can be successfully sorted based on CD142 expression. Furthermore, mesencephalic DA cells cultured in KSR medium according to the method described by Kriks et al. and sorted based on CD142 expression were transplanted into mice and non-human primates. As shown in Figure 10A-B, these cells survived for a short period of time and contained many TH-expressing neurons when transplanted into mice (30 days after transplantation) and non-human primates (1 year after transplantation).

[0194] Furthermore, differentiated mesencephalic DA cells were treated with polysialyltransferase (Polysialyltransferase of Neisseria meningitidis (PST)). NmThe increased levels of polysialylation induced by polysialyltransferase treatment were stable after freeze-thaw cycles and after in vivo transplantation into mice. Furthermore, sorting of CD142 cells was not affected by polysialyltransferase treatment (Figures 11A, B, and D). Furthermore, while untreated cells had approximately 10% neurons with no noticeable outgrowth in vivo after transplantation into mice, none of the polysialyltransferase-treated cells had axons less than 100 μm long 2 weeks after transplantation of mDA cells into the mouse striatum (Figure 11C).

[0195] 6.2 Example 2: Method for preparing stem cell-derived midbrain dopamine (DA) progenitor cells

[0196] overview This example provides a modified version of the GMP V2A differentiation protocol described in Example 1.

[0197] hESCs were maintained in E8 / Matrigel medium prior to differentiation. Cells were differentiated in NB / N2 / B27 medium supplemented with growth factors according to the following culture strategy, in which cells were cultured with a 7.5 μM bump on day 4.

[0198] 7.5μMbump protocol (modified from GMP V2B) Day 0 (D0): hESCs were transferred from E8 / Matrigel medium to differentiation medium containing neurobasal (NB) / N2 / B27 medium supplemented with the following differentiation factors (D0 medium also contained 10 μM Y-27632 ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor to enhance cell survival). Cells were at a concentration of approximately 750 million cells per liter: 2mM L-glutamine 250nM LDN193189 10.8 μM SB431542 500ng / mL SHH 0.7 μM CHIR99021

[0199] - D1: The differentiation medium was replaced with fresh differentiation medium without Y-27632.

[0200] - D2: No medium change.

[0201] - D3: medium changed as described in D1.

[0202] - D4: The differentiation medium was replaced with fresh differentiation medium containing 7.5 μM CHIR99021 (without Y-27632).

[0203] - D5: No medium change.

[0204] - D6: medium changed as described in D4.

[0205] - D7: Differentiation medium was replaced with fresh medium containing NB / N2 / B27 (without LDN193189, SB431542, SHH or Y-27632) supplemented with 2 mM L-glutamine and 7.5 μM CHIR99021.

[0206] - D8: No medium change.

[0207] - D9: medium changed as described in D7.

[0208] - D10: The medium was replaced with fresh NB / B27 medium containing 2 mM L-glutamine, 3 μM CHIR99021, 20 ng / mL BDNF, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 200 nM AA.

[0209] - D11: Passage of cells and medium changed as described in D10 to achieve a concentration of 1.5 billion cells per liter.

[0210] - D12: Medium changed as described in D10, except that the medium additionally contained 10 μM DAPT and did not contain CHIR99021.

[0211] - D13-D15: Maturation medium. Medium changed as described on D12.

[0212] - D16: Cells were harvested and cryopreserved in NB.

[0213] method 5.0 Reagents and Materials 5.1 Texwipe Sterile TechniSat Presaturated Wipers (Fisher, Cat. No. 19-003-239) or equivalent 5.2 Centrifuge tubes (Fisher, Cat. No. 05-538-51 for 15 mL; Fisher, Cat. No. 05-538-49 for 50 mL) or equivalent 5.3 Tissue culture plates (Fisher, Cat. No. 08-772-24 15cm) or equivalent 5.4 Tissue culture flasks (Fisher, Cat. No. 13-680-65, 75 cm 2 ;Fisher, Cat Number 12-565-221 225cm 2 ) or equivalent 5.5 Sterile polystyrene serological pipettes (Fisher, Cat. No. 13-675-47, 2 mL; Fisher, Cat. No. 13-678-11D, 5 mL; Fisher, Cat. No. 13-678-11E, 10 mL; Fisher, Cat. No. 13-678-11, 25 mL; Fisher, Cat. No. 13-678-11F; Fisher, Cat. No. 13-675-73, 100 mL) or equivalent 5.6 Sterile aspirating pipette (Fisher, Cat. No. 13-678-20D) or equivalent 5.7 zCellometer counting slides (Nexcelom, Cat. No. CHT4-PD-100-003) or equivalent 5.8 Sterile pipette tips (Fisher, Cat. No. 21-403-00 for 20 μL; Fisher, Cat. No. 21-403-01 for 200 μL; Fisher, Cat. No. 21-403-02 for 1000 μL) or equivalent 5.9 Cryopreservation vials (Fisher, Cat. No. 12-565-164N) or equivalent 5.10 CryoColor Code Cap Inserts (Fisher, Cat. No. 12-565-242 for white; Fisher, Cat. No. 12-565-180 for various colors) or equivalent 5.11 Cell strainer (Fisher, Cat. No. 08-771-1 for 40 μM) or equivalent 5.12 AOPI (Nexcelom, Cat. No. CS2-0106-5 mL) 5.13 DMEM / F-12 (Life Technologies, Cat. No. 11320) 5.14 Accutase® Cell Detachment Solution (Innovative Cell Technology, Cat. No. AT104) 5.15 Geltrex™ LDEV-free reduced growth factor (Life Technologies, Cat. No. A14132-01) 5.16 CTS™ (Cell Therapy Systems) DPBS without calcium chloride and without magnesium chloride (Life Technologies, Cat. No. A1285601) 5.17 B27 (without vitamin A) (Life Technologies, Cat. No. 12587-010) 5.18 N2 Supplement B (Stem Cell Technologies, Cat. No. 07156) 5.19 Neurobasal Medium (Life Technologies, Cat. No. 21103049) 5.20 50 mg / mL Gentamicin [Optional] (Life Technologies, Cat. No. 15750078) 5.21 500 μM LDN 193189 (SSCRF-RP-011) 5.22 10.8 mM SB431542 (SSCRF-RP-013) 5.23 100 μg / mL Shh (SSCRF-RP-014) 5.24 15 mM CHIR99021 (SSCRF-RP-004) 5.25 10 μg / mL BDNF (SSCRF-RP-003) 5.26 10mM Y-27632 (SSCRF-RP-016) 5.27 100mM Ascorbic Acid (SSCRF-RP-002) 5.28 10 μg / mL GDNF (SSCRF-RP-007) 5.29 100 mM dbcAMP (SSCRF-RP-006) 5.30 2 μg / mL TGF-beta 3 (SSCRF-RP-015) 5.31 10 mM DAPT (SSCRF-RP-005) 5.32 4 mM HCl (SSCRF-RP-008) 5.33 15 mg / mL Poly-L-ornithine (SSCRF-RP-012) 5.34 1 mg / mL human fibronectin (SSCRF-RP-001) 5.35 mg / ml Cultrex Mouse Laminin I (SSCRF-RP-010) 5.36 Essential 8™ Medium (SSCRF-FR-002) 5.37 200mM L-glutamine (SSCRF-RP-009)

[0214] 6.0 Equipment

[0215] 6.1 Gilson adjustable pipettors (Pipetman-p1000, p200, p20, p10, p2) or equivalent 6.2 Integra Pipetteboy Pro Pipetaid Device or equivalent 6.3 Cellometer® Vision System 6.4 CO2 incubator (Nuaire IR Autoflow CO2 water-jacked incubator) or equivalent 6.5 Sorvall Legend XTR Centrifuge (Thermo Scientific, Cat. No. 75004520) or equivalent 6.6 CryoMed Controlled-Rate Freezer (Thermo Scientific, Cat. No. 7450) or equivalent 6.7 Inverted microscope (Olympus CK2) or equivalent 6.8 Biological Safety Cabinet (Baker SterileGARD Class II) or equivalent

[0216] 7.0 Procedure

[0217] 7.1 The day before differentiation begins: Thawing Geltrex 7.1.1 Thaw 6 x 5 mL frozen Geltrex™ vials at 4°C overnight or until ice crystals are free.

[0218] 7.2 Day 0: hESC feeding and Geltrex coating 7.2.1 Replenish all WA09 hESC cultures with fresh E8 medium. 7.2.2 48 x T75 flasks were labeled with increasing batch record numbers. Each task will be performed sequentially later. 7.2.3 Make a 1:30 mixture of Geltrex™:DMEM / F12. 7.2.3.1 Add 870 mL of chilled DMEM / F12 to a 1 L bottle. 7.2.3.2 Carefully add 30 mL of Geltrex™. Wash each Geltrex™ vial once with DMEM / F12 from the bottle to recover most of the product. 7.2.3.3 Tighten the cap and mix the Geltrex and medium by inverting once, carefully swirling occasionally between uses to prevent settling. 7.2.4 Record the time the Geltrex™ coating started. 7.2.5 Coat each T75 vessel sequentially with 15 mL of Geltrex™. 7.2.6 Record the time the Geltrex™ incubation began. 7.2.7 Incubate in the hood at room temperature for 2-3 hours. 7.2.8 Record the time when suction starts. 7.2.9 Following incubation, aspirate the Geltrex™ and add 15 mL of plain Neurobasal. Allow the vessel to stand at room temperature until ready to plate the cells. 7.2.10 Record the time the Geltrex™ is removed from the container.

[0219] 7.3 Day 0: Cell preparation and induction

[0220] NOTE: 7.3 can be performed simultaneously by two independent operators per production set. The two sets can be processed simultaneously to reduce processing time. The products should be pooled before inoculation to ensure homogeneity.

[0221] NOTE: If the yield is higher than required, only 48 flasks may be seeded. If the yield is less than 48 flasks, the trial is aborted.

[0222] 7.3.1 A single container of Accutase® to use for OCT4+ QC (SSCRF-SOP-107) and cDNA generation (SSCRF-SOP-109) and to assess yield before starting. 7.3.1.1 Identify a representative plate based on density, colony size, and distribution of colonies over the surface. 7.3.1.2 Aspirate the media from the cells and add Accutase®. 7.3.1.2.1 15 mL per T225 flask 7.3.1.2.2 10 mL per 15 cm dish 7.3.1.3 Incubate the cells at 37°C for 20-30 minutes. 7.3.1.4 Using a 10 mL pipette, transfer and triturate the cells to form a homogenous suspension. 7.3.1.5 Pipette into a 50 mL conical and add 10 mL of E8 medium (20 mL total). 7.3.1.6 Centrifuge at 200 x g for 5 minutes at room temperature. 7.3.1.7 Aspirate the medium and add 5 mL of E8 medium. 7.3.1.8 Perform cell count (see 7.3.20) 7.3.1.9 3×10 6 Aliquot cells into cryopreservation tubes labeled SSCRF-SOP-109 (cDNA generation for QC). 7.3.1.10 3×10 6 Aliquots of cells are placed into cryopreservation tubes labeled SSCRF-SOP-107 (OCT4 measurement). 7.3.1.11 Submit the QC tube to another operator to take to the QC laboratory.

[0223] 7.3.2 Prepare 2.5 L of NB / N2 / B27 containing 2 mM L-glut, 250 nM LDN193189, 10.8 μM SB431542, 500 ng / mL Shh, 0.7 μM CHIR, and 10 μM Y-27632.

[0224] 7.3.3 Record the Input WA09 number (batch number) and associated data in the batch record.

[0225] 7.3.4 Work sequentially in batches of 12 containers, one at a time. Note: This is done on a per-operator basis.

[0226] 7.3.5 Record the time when suction begins.

[0227] 7.3.6 Remove 12 vessels of WA09 hESCs from the incubator, aspirate the media from the cells and add Accutase®. 7.3.6.1 15 mL per T225 flask 7.3.6.2 10 mL per 15 cm dish

[0228] 7.3.7 Incubate the cells at 37°C for 20-30 minutes.

[0229] 7.3.8 For batch records, record the start time of the Accutase® incubation.

[0230] 7.3.9 Begin assembling 12 [T225] or 6 [15 cm] x 50 mL conical tubes.

[0231] 7.3.10 For batch records, record the end time of the Accutase® incubation.

[0232] 7.3.11 Using a 10 mL pipette, wash the surface of each vessel approximately 5-10 times with Accutase® to transfer the cells and triturate until no clumps are visible.

[0233] 7.3.12 Transfer dissociated cells in Accutase® to a 50 mL conical.

[0234] 7.3.13 Wash the surface of the dried vessel with fresh E8 medium (using a volume equal to the Accutase®) to collect any remaining cells and add to the conical with the cell-Accutase®. NOTE: For example, a T225 flask that had 15 mL of Accutase® is transferred to a conical. The dried flask is then washed with 15 mL of E8 medium, and the recovered cell-E8 is added to the conical containing 15 mL of Accutase®-cells (30 mL total). A 15 cm dish containing 10 mL of Accutase® is transferred to the conical and then washed with 10 mL of fresh E8 (20 mL total). Two 15 cm dishes can be pooled into one conical (40 mL total). NOTE: If possible, live samples should also be cryopreserved for archival purposes. Live samples can be frozen as single cells using the cryopreservation protocol "USER1" in the FreSR-S and QC laboratories.

[0235] 7.3.14 Centrifuge the cells for 5 minutes at 200 x g at room temperature.

[0236] 7.3.15 Aspirate the medium and gently resuspend each pellet in 10 mL of fresh plain Neurobasal.

[0237] 7.3.16 Pool the three tubes into a fresh 50 mL conical (giving a total of 2 x 50 mL conicals per batch).

[0238] 7.3.17 Centrifuge the cells for 5 minutes at 200 x g at room temperature.

[0239] 7.3.18 Aspirate the medium and gently resuspend in a total of 5 ml of NB / N2 / B27 containing 2 mM L-glutamic acid, 250 nM LDN193189, 10.8 μM SB431542, 500 ng / mL Shh, 0.7 μM CHIR, and 10 μM Y-27632. Place at 4°C until all vessels have been processed.

[0240] 7.3.19 Once all vessels have been processed, pool all cells and bring to 200 mL.

[0241] 7.3.20 Count the pooled cells: 7.3.20.1 Make known dilutions of cells in plain Neurobasal (without growth factors). 7.3.20.2 Mix 20uL of diluted cells with 20uL of AOPI. 7.3.20.3 Load 20 uL of the cell / AOPI mixture onto a Cellometer® slide. 7.3.20.4 Insert the slide into the slot. 7.3.20.5 Select the program file “hES Cells AOPI.” 7.3.20.6 Provide a file name using the following format: "DA01 Lot#MMDDYY d0". 7.3.20.7 Enter the dilution value. 7.3.20.8 Using the F1 channel, focus on the cells using the knob on the right side of the machine. 7.3.20.9 Once focus is achieved, press "Count."

[0242] 7.3.21 Record the total number of visible and non-visible cells, total number of cells, and % viability per mL.

[0243] 7.3.22 3×10 6 Aliquot 3 x 10 cells into a sterile cryopreservation tube labeled MICR-CULT-SOP-1634. 6 Aliquot cells into cryopreservation tubes labeled PT-OP-7020 (Mycoplasma). Adjust each to 1 mL before submission and send to another operator for processing.

[0244] 7.3.23 Adjust cell concentration to 750 million cells per liter (2 liters total required). 7.3.23.1 Calculate the volume required to achieve 750 million cells. 7.3.23.2 Remove the calculated volume from each 1 L bottle of NB / N2 / B27 containing 2 mM L-glut, 250 nM LDN193189, 10.8 μM SB431542, 500 ng / mL Shh, 0.7 μM CHIR, and 10 μM Y-27632. 7.3.23.3 Add the calculated volume containing 750 million cells to each 1 L bottle.

[0245] 7.3.24 Sequentially work on each T75 flask one at a time.

[0246] 7.3.25 Aspirate the plain Neurobasal medium from the Geltrex™ coated flasks.

[0247] 7.3.26 Carefully add 40 mL of cell suspension to each T75. NOTE: Make sure to suspend the cells carefully before pipetting.

[0248] 7.3.27 Gently rock the flask to evenly coat the surface with cells. NOTE: Cell distribution can be verified microscopically.

[0249] 7.3.28 Allow to stand in the hood without disturbance for 10-15 minutes.

[0250] 7.3.29 Carefully transfer the flask to the incubator.

[0251] 7.3.30 Incubate overnight at 37°C with 5% CO2.

[0252] 7.4 Day 1: Feeding (0.7 μM CHIR) 7.4.1 Control point: Check the confluence of the flasks indicated by BR. The culture should be 100% confluent. Note the confluence of the designated flask. 7.4.2 Sequentially work on each T75 flask one at a time. 7.4.3 Aspirate the existing medium and gently add 40 mL of NB / N2 / B27 medium containing 2 mM L-glut, 250 nM LDN193189, and 10.8 μM SB431542, 500 ng / mL SHH, and 0.7 μM CHIR99021 per T75.

[0253] 7.5 Day 2: No medium change

[0254] 7.6 Day 3: Feeding (0.7 μM CHIR) 7.6.1 Sequentially work on each T75 flask one at a time. 7.6.2 Aspirate the existing medium and gently add 40 mL of NB / N2 / B27 medium containing 2 mM L-glut, 250 nM LDN193189 and 10.8 μM SB431542, 500 ng / mL SHH and 0.7 μM CHIR99021.

[0255] 7.7 Day 4: Feeding (7.5 μM CHIR BUMP) 7.7.1 Sequentially work on each T75 flask one at a time. 7.7.2 Aspirate the existing medium and gently add 40 mL of NB / N2 / B27 medium containing 2 mM L-glut, 250 nM LDN193189 and 10.8 μM SB431542, 500 ng / mL SHH and 7.5 μM CHIR.

[0256] 7.8 Day 5: No medium change

[0257] 7.9 Day 6: Feeding (7.5 μM CHIR) 7.9.1 Sequentially work on each T75 flask one at a time. 7.9.2 Aspirate the existing medium and gently add 40 mL of NB / N2 / B27 medium containing 2 mM L-glut, 250 nM LDN193189 and 10.8 μM SB431542, 500 ng / mL SHH and 7.5 μM CHIR99021.

[0258] 7.10 Day 7: Removal of LDN, SB, and SHH 7.10.1 Sequentially work on each T75 flask one at a time. 7.10.2 Aspirate the existing medium and gently add 40 mL of NB / N2 / B27 medium containing 2 mM L-glut and 7.5 μM CHIR99021.

[0259] 7.11 Day 8: No medium change

[0260] 7.12 Day 9: Feeding (7.5 μM CHIR) and PO coating 7.12.1 Supply 7.5 μM CHIR: 7.12.1.1 Sequentially work on each T75 flask one at a time. 7.12.1.2 Aspirate the existing medium and gently add 40 mL of NB / N2 / B27 medium containing 2 mM L-glut and 7.5 μM CHIR99021. 7.12.2 PO coating 7.12.2.1 Each T75 flask was labeled with an incrementing batch record number. Each task was performed sequentially. 7.12.2.2 Coat 48 x T75 flasks with 15 mL of 15 μg / mL poly-L-ornithine in DPBS per flask. Note: The required volume is 720 mL, but make an excess of 800 mL. 7.12.2.3 Incubate overnight at 37°C with 5% CO2.

[0261] 7.13 Day 10: Feeding (3 μM CHIR, no DAPT) and F / L coating 7.13.1 Add 3 μM CHIR to the plate. 7.13.1.1 Sequentially work on each T75 flask one at a time. 7.13.1.2 Aspirate the existing medium and gently add 40 mL of NB / B27 medium containing 2 mM L-glut, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 3 μM CHIR99021. 7.13.2 F / L Coating 7.13.2.1 Flasks can be processed in batches of four. 7.13.2.2 Aspirate and then add 15 mL of DPBS. 7.13.2.3 Wash with gentle rocking, then repeat two more times for a total of 3x DPBS washes. 7.13.2.4 Aspirate and then add 15 mL of 2 μg / mL fibronectin / laminin in DPBS. Note: The required volume is 720 mL, but make an excess of 800 mL. 7.13.2.5 Incubate overnight at 37°C with 5% CO2.

[0262] 7.14 Day 11: Passaging NOTE: 7.14 can be performed simultaneously by two independent operators per production set. The two sets can process flasks simultaneously, reducing processing time. The product should be pooled before inoculation to ensure homogeneity. NOTE: If the yield is greater than required, only 48 flasks can be passaged. 9 If there are fewer than 100 cells, the batch may be aborted.

[0263] 7.14.1 Prepare 2.5 L of NB / B27 medium containing 2 mM L-glutamic acid, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 3 μM CHIR99021.

[0264] 7.14.2 Work sequentially in batches of 12 containers, one at a time.

[0265] 7.14.3 Carefully remove the flasks from the hood and arrange them in a sequence, noting when the aspiration begins.

[0266] 7.14.4 Aspirate the existing media and add 5 mL of Accutase® per T75.

[0267] 7.14.5 After the final addition of Accutase®, record the start time of the incubation.

[0268] 7.14.6 Incubate at 37°C for 30-40 minutes.

[0269] 7.14.7 To dry as thoroughly as possible, aspirate the fibronectin / laminin coating and leave the flask open under the hood until dry.

[0270] 7.14.8 Sequentially label 50 mL conicals to match the T75 flasks being passaged.

[0271] 7.14.9 In the hood, using careful sterile technique, place a 40 μm blue cell strainer into one of the labeled 50 mL conicals for each flask.

[0272] 7.14.10 Once the incubation time has elapsed, record the incubation stop time.

[0273] 7.14.11 Remove the plate from the incubator.

[0274] 7.14.12 Using a 10 mL pipette, pipette the cells vigorously approximately 5-10 times to disrupt clusters.

[0275] 7.14.13 Carefully pipette the disrupted cells through a 40 μM filter.

[0276] 7.14.14 Add 15 mL of plain Neurobasal medium to the dried flask and collect the settled cells.

[0277] 7.14.15 Add the Neurobasal containing the harvested cells to the 40 μM filter. NOTE: Two vessels can be processed at this step and contained within the same tube.

[0278] 7.14.16 Carefully remove and discard the filter. Cap the tube.

[0279] 7.14.17 Repeat the process for each T75 flask.

[0280] 7.14.18 Centrifuge the cells for 5 minutes at 200 x g at room temperature.

[0281] 7.14.19 Aspirate the medium and resuspend each pellet in 10 mL of plain Neurobasal.

[0282] 7.14.20 Combine the pellets from no more than four tubes and place into a 50 mL conical tube.

[0283] 7.14.21 Centrifuge the cells for 5 minutes at 200 x g at room temperature.

[0284] 7.14.22 Aspirate the medium and resuspend each pellet in 10 mL of NB / B27 medium containing 2 mM L-glut, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 3 μM CHIR99021. NOTE: If further vessel processing is required, cells can be stored at 4 °C.

[0285] 7.14.23 Once all vessels have been treated, pool all cells and bring to a final volume of 200 mL using NB / B27 medium containing 2 mM L-glut, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3 and 3 μM CHIR99021. 7.14.23.1 Make known dilutions of cells in plain Neurobasal medium. 7.14.23.2 Mix 20uL of diluted cells with 20uL of AOPI. 7.14.23.3 Load 20 uL of the cell / AOPI mixture onto a Cellometer® slide. 7.14.23.4 Insert the slide into the Cellometer®. 7.14.23.5 Select the program file “mDA Neurons AOPI.” 7.14.23.6 Provide a file name using the following format: "DA01 Lot#MMDDYY d11". 7.14.23.7 ​​Enter the dilution value. 7.14.23.8 Using the F1 channel, focus on the cells using the knob on the right side of the machine. 7.14.23.9 Once focus is achieved, press "Count."

[0286] 7.14.24 Record the total number of visible and non-visible cells, total number of cells, and % viability per mL.

[0287] 7.14.25 3×10 6 Aliquot cells into cryopreservation tubes labeled SSCRF-SOP-109 (cDNA generation for QC).

[0288] 7.14.26 3×10 6 Aliquot cells into a cryopreservation tube labeled PT-OP-7020 (Mycoplasma). Adjust to 1 mL before submission.

[0289] 7.14.27 3×10 6 Aliquot cells into a cryopreservation tube labeled MICR-CULT-SOP-1634 (sterile). Adjust to 1 mL before submission. NOTE: If possible, samples should also be submitted for cryopreservation of live samples for archival purposes. Live samples can be frozen as single cells using the cryopreservation protocol "USER1" in Stem-Cellbanker® and QC laboratories.

[0290] 7.14.28 Send all QC tubes to another operator for processing as per relevant SOP.

[0291] 7.14.29 Adjust cell concentration to 1.5 billion cells per liter (2 liters total required). 7.14.29.1 Calculate the volume required to achieve 1.5 billion cells. 7.14.29.2 Remove the calculated volume from each 1 L bottle of NB / B27 medium containing 2 mM L-glut, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 3 μM CHIR99021. 7.14.29.3 Add the calculated volume containing 1.5 billion cells to each 1 L bottle. NOTE: Cell yield is 3 x 10 9 Less than 1 x 10 9 If more than 1.5 x 10 cells per mL 6 Calculate the number of mL needed to achieve a final concentration of cells and seed as many flasks as possible.

[0292] 7.14.30 Add 40 mL of cell suspension to each T75 (60 x 10 total cells). 6 cells). NOTE: Make sure to suspend the cells carefully before pipetting.

[0293] 7.14.31 Carefully rock the plate to ensure a uniform cell suspension.

[0294] 7.14.32 Allow the plate to incubate at room temperature for 10 minutes.

[0295] 7.14.33 Carefully transfer the flask to the incubator. Note: Spot inspect flasks under a microscope to ensure uniform coating.

[0296] 7.14.34 Incubate the plate at 37°C and 5% CO2 overnight.

[0297] 7.15 Day 12 of feeding (CHIR removed, DAPT added [maturation medium]) 7.15.1 Control Points: Spot check flask confluence and record in BR. Cultures should be 100% confluent. Note the confluence of designated flasks. NOTE: If the maximum number of flasks is not achieved during passaging, maturation medium may be used the following day. 7.15.2 Sequentially work on each T75 flask one at a time. 7.15.3 Aspirate the existing medium and gently add NB / B27 medium containing 2 mM L-glut, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 10 μM DAPT to each T75 flask.

[0298] 7.16 Day 13 (Maturation medium) 7.16.1 Sequentially work on each T75 flask one at a time. 7.16.2 Aspirate the existing medium and gently add NB / B27 medium containing 2 mM L-glut, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 10 μM DAPT to each T75 flask.

[0299] 7.17 Day 14 (Maturation medium) 7.17.1 Sequentially work on each T75 flask one at a time. 7.17.2 Aspirate the existing medium and gently add NB / B27 medium containing 2 mM L-glut, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 10 μM DAPT to each T75 flask.

[0300] 7.18 Day 15 (Maturation medium) 7.18.1 Sequentially work on each T75 flask one at a time. 7.18.2 Aspirate the existing medium and gently add NB / B27 medium containing 2 mM L-glut, 20 ng / mL BDNF, 200 nM AA, 20 ng / mL GDNF, 500 nM cAMP, 1 ng / mL TGFβ3, and 10 μM DAPT to each T75 flask.

[0301] 7.19 Day 16: Cell harvest and cryopreservation NOTE: 7.14 can be performed simultaneously by two independent operators per production set. The two sets can process flasks simultaneously, reducing processing time. The product should be pooled before inoculation to ensure homogeneity.

[0302] 7.19.1 Sequentially label all cryopreservation tubes and chill them to 4°C.

[0303] 7.19.2 Label boxes sequentially with lot number and tube range.

[0304] 7.19.3 Note the time before starting suction.

[0305] 7.19.4 Working sequentially in batches of 12, aspirate the existing medium from the flasks and add 5 mL of Accutase® per T75.

[0306] 7.19.5 Record the start time of the incubation.

[0307] 7.19.6 Incubate at 37°C for 30-40 minutes.

[0308] 7.19.7 In the hood, install a 40 μm blue cell strainer into a 50 mL conical (one conical for every two T75 flasks).

[0309] 7.19.8 After incubation, remove the flask from the incubator and record the time in the batch record.

[0310] 7.19.9 Using a 10 mL pipette, pipette approximately 5-10 times until clusters are no longer visible.

[0311] 7.19.10 Carefully transfer the cell suspension through a 40 μM filter.

[0312] 7.19.11 Add 15 mL of Neurobasal medium to the dried flask and collect the cells.

[0313] 7.19.12 Transfer the Neurobasal containing the harvested cells through the same 40 μM filter.

[0314] 7.19.13 Repeat for each flask in turn.

[0315] 7.19.14 Centrifuge the cells for 5 minutes at 200 x g at room temperature.

[0316] 7.19.15 Aspirate the medium and resuspend each pellet in 5 mL of Neurobasal medium. NOTE: Store the conical at 4°C until all vessels have been processed.

[0317] 7.19.16 Once all vessels have been processed, pool all cells and adjust to a total volume of 200 mL.

[0318] 7.19.17 Perform cell counts: 7.19.17.1 Make known dilutions of cells in plain Neurobasal. 7.19.17.2 Mix 20uL of diluted cells with 20uL of AOPI. 7.19.17.3 Load 20 uL of the cell / AOPI mixture onto a Cellometer® slide. 7.19.17.4 Insert the slide into the Cellometer®. 7.19.17.5 Select the program file “mDA Neurons AOPI.” 7.19.17.6 Provide a file name using the following format: "DA01 Lot#MMDDYY d16 cryo". 7.19.17.7 Enter the dilution value. 7.19.17.8 Using the F1 channel, focus on the cells using the knob on the right side of the machine. 7.19.17.9 Once focus is achieved, press "Count."

[0319] 7.19.18 Record the total number of visible and non-visible cells, total number of cells, and % viability per mL.

[0320] 7.19.19 3×10 6 Aliquot cells into cryopreservation tubes labeled SSCRF-SOP-109 (cDNA generation for QC).

[0321] 7.19.20 3×10 6 Place an aliquot of cells into a cryopreservation tube labeled PT-OP-7020 (Mycoplasma). Adjust the volume to 1 mL using plain Neurobasal.

[0322] 7.19.21 3×10 6Aliquot 100 cells into a cryopreservation tube labeled MICR-CULT-SOP-1634 (sterile). Adjust the volume to 1 mL using plain Neurobasal.

[0323] 7.19.22 Send the tube to another operator for processing as per relevant SOP.

[0324] 7.19.23 Cryopreservation of remaining cells according to SOP-SSCRF-105 (Cryopreservation of Final Product on Day 16).

[0325] 7.19.24 Once completed, the cells should be promptly removed from the freezer, placed in pre-labeled boxes, and loaded onto the transporter.

[0326] 7.19.25 Transfer the box to liquid nitrogen.

[0327] 7.19.26 Record information in the batch record.

[0328] On day 16, cryopreserved cells were thawed and transplanted into injured rats (NIH nude, Taconic Biosciences, Inc.) (i.e., a parkinsonian rat model). Amphetamine-induced rotational behavior was examined in transplanted and sham-transplanted rats before transplantation and at 1, 2, 3, 4, and 5 months after transplantation. In vivo expression of hNCAM and mDA markers TH (tyrosine hydroxylase) and GIRK2 (G protein-activated inwardly rectifying potassium channel 2) was examined 5 months after transplantation.

[0329] On day 16, the cryopreserved cells were thawed and transplanted into non-human primates. Fiber outgrowth from the transplanted grafts and cell morphology were examined 6 weeks after transplantation.

[0330] result As shown in Figure 12, 4 months after transplantation of mDA precursors into injured rats, rats receiving the grafts showed fewer rotations per minute compared to sham-treated rats. Five months after transplantation, immunocytochemistry of the transplanted grafts showed that the grafts showed typical TH staining of mDA morphology, and the TH-positive neurons were also positive for GIRK2 expression (Figure 13).

[0331] The morphology of mDA precursor grafts implanted in non-human primates was examined 6 weeks after implantation. The grafts showed robust fibrous extensions from the graft core and also displayed typical mDA morphology (Figure 14).

[0332] While the subject matter and advantages of the present disclosure have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods, and steps described herein. As one skilled in the art will readily appreciate from this disclosure of the subject matter of the present disclosure, existing or later-developed processes, machines, manufacture, compositions of matter, means, methods, or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the subject matter of the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0333] Throughout this application, patents, patent applications, publications, product descriptions, and protocols are cited, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Claims

1. An in vitro method for differentiating pluripotent stem cells, said method comprising: (a) contacting a plurality of pluripotent stem cells with at least one inhibitor of TGFβ / Activin-Nodal signaling; (b) contacting the cell with at least one activator of Sonic Hedgehog (SHH) signaling and at least one activator of Wingless (Wnt) signaling; 1. An in vitro method comprising: (a) increasing the concentration of the at least one activator of Wnt signaling contacted with the cells between 2 and 6 days after initial contact of the cells with the at least one activator of Wnt signaling; and (b) increasing the concentration of the at least one activator of Wnt signaling contacted with the cells by 50% to 2000% from the initial concentration of the at least one activator of Wnt signaling contacted with the cells, thereby obtaining a population of differentiated cells that express forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A).

2. The method further comprising contacting the cells with at least one inhibitor of bone morphogenetic protein (BMP).

3. The method described in claim 2, wherein the cells are contacted with at least one inhibitor of TGFβ / activin-Nodal signaling, at least one inhibitor of BMP, and at least one activator of sonic hedgehog (SHH) signaling for a period of 4 to 10 days.

4. A method according to claim 2 or 3, wherein the cells are contacted with at least one inhibitor of TGFβ / activin-Nodal signaling, at least one inhibitor of BMP, and at least one activator of sonic hedgehog (SHH) signaling for up to 7 days or for at least 7 days.

5. A method described in any one of claims 1 to 4, wherein the cells are contacted with at least one activator of Wingless (Wnt) signaling for a period of 8 to 15 days.

6. A method according to any one of claims 1 to 5, wherein the cells are contacted with the at least one activator of Wingless (Wnt) signalling for up to 12 days or for at least 12 days.

7. A method described in any one of claims 1 to 6, wherein the concentration of the at least one activator of Wnt signaling is increased 4 days after initial contact of the cells with the at least one activator of Wnt signaling.

8. A method according to any one of claims 1 to 7, wherein the increase in concentration of the at least one activator of Wnt signaling is between 100% and 1950% of the initial concentration of the at least one activator of Wnt signaling contacted with the cells.

9. A method according to any one of claims 1 to 8, wherein the increase in concentration of the at least one activator of Wnt signaling is 150% to 1900% of the initial concentration of the at least one activator of Wnt signaling contacted with the cells.

10. A method according to any one of claims 1 to 9, wherein the increase in concentration of the at least one activator of Wnt signalling is 200% to 1850% of the initial concentration of the at least one activator of Wnt signalling contacted with the cells.

11. A method according to any one of claims 1 to 10, wherein the increase in concentration of the at least one activator of Wnt signalling is to a concentration between 3 μM and 10 μM.

12. A method according to any one of claims 1 to 10, wherein the increase in the concentration of the at least one activator of Wnt signalling is up to a concentration of 3 μM.

13. A method according to any one of claims 1 to 10, wherein the increase in the concentration of the at least one activator of Wnt signalling is an increase to a concentration of 7.5 μM.

14. A method described in any one of claims 1 to 13, wherein the differentiated cells express one or more of tyrosine hydroxylase (TH), engrailed-1 (EN-1), and nuclear receptor-related 1 protein (NURR1).

15. A method described in any one of claims 1 to 14, wherein the differentiated cells do not express detectable levels of paired box protein (PAX6) and / or Ki67.

16. A method according to any one of claims 1 to 15, further comprising the step of subjecting the population of differentiated cells to conditions favourable for the maturation of said cells into dopamine neurons.

17. The method of claim 16, wherein the conditions favorable for maturation of the cells into dopamine neurons include contacting the cells with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta 3 (TGFβ3), ascorbic acid (AA), and / or DAPT.

18. The method of any one of claims 1 to 17, wherein the pluripotent cells are selected from the group consisting of human non-embryonic stem cells, non-human primate non-embryonic stem cells, rodent non-embryonic stem cells, human embryonic stem cells, non-human primate embryonic stem cells, rodent embryonic stem cells, human induced pluripotent stem cells, non-human primate induced pluripotent stem cells, rodent induced pluripotent stem cells, human recombinant pluripotent cells, non-human primate recombinant pluripotent cells, and rodent recombinant pluripotent cells.

19. The method of claim 1, wherein the at least one inhibitor of TGFβ / activin-Nodal signaling comprises an inhibitor of a TGFβ receptor.

20. The method of claim 19, wherein the inhibitor of the TGFβ receptor comprises 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542).

21. The method of any one of claims 2 to 20, wherein the at least one inhibitor of BMP comprises 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (LDN193189), noggin, or a combination thereof.

22. A method according to any one of claims 1 to 21, wherein the at least one activator of sonic hedgehog (SHH) signalling comprises an SHH protein, a smoothened agonist, or a combination thereof.

23. The method of claim 22, wherein the SHH protein comprises recombinant SHH, purified SHH, or a combination thereof.

24. The method of claim 23, wherein the recombinant SHH comprises SHH C25II.

25. The method of claim 22, wherein the smoothened agonist comprises palmorfamine.

26. The method of any one of claims 1 to 25, wherein the at least one activator of Wingless (Wnt) signaling comprises CHIR99021, Wnt3A, Wnt1, or a combination thereof.

27. ​​A method described in any one of claims 1 to 26, wherein the differentiated cells expressing FOXA2 and LMX1A are midbrain dopamine neurons.

28. A method according to any one of claims 1 to 27, wherein the pluripotent stem cells are differentiated into differentiated cells that express FOXA2 and LMX1A within 22 to 27 days of initial contact of the pluripotent stem cells with the at least one inhibitor of TGFβ / activin-Nodal signalling.

29. A method described in any one of claims 1 to 28, wherein the differentiated cells express a detectable level of CD142.

30. The method described in claim 29, further comprising a step of selecting cells that express CD142.

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