Culture media, coating matrices and methods for maturing midbrain dopaminergic progenitor cells - Patent Application 20070122999

Abstract

The disclosure described herein provides, inter alia, a growth method for growing mDAPs and a combination of culture medium and coating matrix for use in the growth method, as well as a maturation method for maturing mDAPs and a culture medium for use in the maturation method. The present disclosure also provides a substantially homogeneous population of mDAPs, and a substantially homogeneous population of mDANs.

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE The present disclosure relates generally to the field of stem cell technology, and in particular to culture media, coating matrices and methods for expanding and maturing midbrain dopaminergic progenitor cells (mDAP). [Background technology]

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disorder. The disease is characterized by the selective loss of dopaminergic neurons (DA neurons) in the substantia nigra of the midbrain. Recent advances in medicine have greatly advanced our general understanding of the pathogenesis of PD, but unfortunately, there is currently no cure for this devastating disease. The primary treatment for PD patients is DA analogs and receptor agonists to counteract the reduction of DA.

[0003] Thus, there remains a need for further research into the mechanisms of PD, its disease progression, and effective clinical intervention approaches to effectively treat PD, and one prerequisite for such research is the availability of midbrain dopaminergic progenitor cells.

[0004] Pluripotent stem cells include human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). These pluripotent stem cells can be propagated in vitro and retain their ability to differentiate into any cell type of the three germ layers, including neurons and tissues. Therefore, these pluripotent stem cells are highly beneficial for studying developmental processes and disease mechanisms, particularly in the brain. In particular, hiPSCs are an unlimited cell source for mechanistic studies, drug screening assays, and ultimately cell replacement therapy for the treatment of neurological disorders such as PD. Numerous protocols have been developed for generating human DA neurons in vitro from hPSCs.

[0005] These protocols typically rely on the directed differentiation of pluripotent stem cells into imDAPs using small molecules and growth factors, followed by proliferation and maturation of imDAPs. They are often laborious, lengthy, highly variable between batches, and result in heterogeneous populations with relatively low numbers of midbrain DA neurons. However, for clinical and therapeutic applications, a homogeneous and robust cell population is highly desirable. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need to provide improved reagents, compositions and methods useful for propagating and / or maturing mDAPs. [Means for solving the problem]

[0007] In a first aspect, the present disclosure provides a culture medium capable of promoting proliferation of midbrain dopaminergic progenitor cells (mDAP), the culture medium comprising: (a) a basal medium; (b) a neural growth supplement; (c) a WNT signaling pathway activator; (d) a Rho kinase (ROCK) inhibitor; and (e) a transforming growth factor β (TGF-β) inhibitor.

[0008] In a second aspect, the present disclosure provides a coating matrix combination capable of promoting the proliferation of midbrain dopaminergic progenitor cells (mDAP), the coating matrix combination comprising: (a) a first coating matrix capable of supporting cell adhesion of mDAP; and (b) a second coating matrix capable of improving expression of mDAP-specific markers during the proliferation and passaging of mDAP, the second coating matrix comprising a Notch agonist.

[0009] In a third aspect, the present disclosure provides a method for expanding midbrain dopaminergic progenitor cells (mDAP), comprising contacting the mDAP with a growth medium on a culture surface coated with a combination of coating matrices of the second aspect of the present disclosure described herein.

[0010] In a fourth aspect, the present disclosure provides a culture medium capable of promoting maturation of mDAP (midbrain dopaminergic progenitor cells), the culture medium comprising: (a) neurobasal medium; (b) human platelet lysate (hPLT); (c) transforming growth factor β (TGF-β); (d) a γ-secretase inhibitor; and (e) a cAMP-based compound or a cyclase activator thereof.

[0011] In a fifth aspect, the present disclosure provides a method for promoting maturation of midbrain dopaminergic progenitor cells (mDAP), the method comprising contacting mDAP with a maturation medium containing a ROCK inhibitor on a culture surface coated with a combination of coating matrices including: (a) a first coating matrix capable of supporting cell adhesion of mDAP, the first coating matrix being other than laminin; and (b) a second coating matrix capable of improving maturation of mDAP, the second coating matrix comprising a polylysine-based compound and / or a polyornithine-based compound.

[0012] In a sixth aspect, the present disclosure provides a substantially homogeneous population of mDAP produced by the method of the third aspect of the present disclosure described herein.

[0013] In a seventh aspect, the present disclosure provides a substantially homogeneous population of midbrain dopaminergic neurons (mDANs) produced by the method of the fifth aspect of the present disclosure described herein.

[0014] In an eighth aspect, the present disclosure provides a kit comprising the culture medium of the first aspect of the present disclosure described herein.

[0015] In certain embodiments, the kit further comprises a coating matrix combination of the second aspect of the present disclosure as described herein.

[0016] In a ninth aspect, the present disclosure provides a kit comprising the culture medium of the fourth aspect of the present disclosure described herein.

[0017] In certain embodiments, the kit further comprises a coating matrix combination of the fifth aspect of the present disclosure described herein.

[0018] Various objects and advantages of the reagents, compositions and methods provided herein will become apparent from the following description, taken in conjunction with the accompanying drawings, which set forth, by way of illustration and example, certain embodiments of the disclosure. [Brief explanation of the drawings]

[0019] [Figure 1]Figure 1 illustrates the development and validation of growth media based on the LDN193189 / CHIR99021 / FGF8b / blebbistatin combination from Examples 1-5 of this disclosure. Figure 1A shows the morphology of cells grown in the absence of blebbistatin (left image), when blebbistatin is removed 24 hours after plating (middle image), and when blebbistatin is present throughout the culture period (right image) (Scale bar: 50 μm). Figure 1B shows the morphology of cells grown without (left image) and with (right image) LDN193189 (Scale bar: 50 μm). Figure 1C shows the effect of CHIR99021 concentration in the growth medium on the expression of the mDAP-specific markers EN1, LMX1A, and FOXA2 (representative markers of mDAP). Figure 1D shows the effect of the presence or absence of FGF8b in the growth medium on the expression of imDAP-specific markers EN1, LMX1A, and FOXA2. Figure 1E shows the typical morphology of expanded imDAP at each passage (P1–P6) 4–5 days after passage (scale bar: 50 µm). Figure 1F shows the representative population doubling time (PDT) of imDAP during passage (P1–P5). Figure 1G shows the results of flow cytometry analysis of FOXA2 expression in imDAP at P0, P1, and P2 (P indicates passage). Figure 1H shows the results of qRT-PCR analysis of the expression of imDAP-specific markers, LMX1A, FOXA2, and EN1, in imDAP at each passage (P0–P6). [Figure 2] FIG. 2 shows a comparison of the effects of Y27632 and blebbistatin on the expression of the imDAP-specific marker FOXA2 according to Example 6 of the present disclosure. [Figure 3] Figure 3 shows the results of single-cell RNA-Seq analysis of imDAP according to Example 7 of the present disclosure, where Figure 3A is a visualization of the clustering results of imDAP single-cell RNA-seq data using UMAP, and Figure 3B is a heatmap of the subset of genes enriched in imDAP (MB-FP: midbrain floor plate; MB-BP: midbrain basal plate; HB: hindbrain; MHB: midbrain-hindbrain boundary). [Figure 4]Figure 4 shows the effects of NOTCH activation and TGF-β inhibition on imDAP proliferation according to Example 8 of the present disclosure. Figure 4A shows the morphology of imDAP grown with and without SB431542 on culture surfaces coated with VTN, VTN + DLL4, or DLL4. Figure 4B shows the effects of DLL4 and SB431542 on imDAP proliferation efficiency. Figure 4C shows the results of FACS analysis of FOXA2 expression in imDAP grown with and without SB431542 on culture surfaces coated with VTN or VTN + DLL4. [Figure 5] Figure 5 shows the effects of LDN193189, FGF8b, and FGF2 on the proliferation of imDAP cells according to Example 9 of the present disclosure. Figure 5A shows the morphology of imDAP cells grown with LDN193189, LDN193189 + FGF8b, LDN193189 + FGF2, and without any of them (scale bar: 50 μm). Figure 5B shows the effect of LDN193189 and / or FGF8b on imDAP proliferation efficiency. Figure 5C shows the results of FACS analysis of FOXA2 expression in imDAP cells grown with LDN193189 or LDN193189 + FGF8b, and without any of them. [Figure 6] Figure 6 shows the effect of NOTCH activation in combination with TGF-β inhibition on the proliferation of imDAP cells according to Example 10 of the present disclosure. Figure 6A shows the typical morphology of P3 imDAP cells on day 5 (scale bar: 50 μm). Figure 6B shows representative PDTs of imDAP cells at each passage (P1-P6) during long-term proliferation. Figure 6C shows the results of FACS analysis of FOXA2 expression in imDAP cells at each passage (P0-P5) during long-term proliferation. Figure 6D shows the results of qRT-PCR analysis of imDAP-specific markers EN1, LMX1A, FOXA2, OTX2, and SOX6 in imDAP cells at each passage (P0-P6) during long-term proliferation. [Figure 7]Figures 7A-C show the effects of human platelet lysate (hPLT) and heat-treated human platelet lysate (HhPLT) in place of B27 on the maturation of imDAPs according to Example 11 of the present disclosure. Figure 7A shows bright-field images of imDAPs cultured with different concentrations of hPLT or HhPLT or with B27 in maturation medium (scale bar: 50 μm). Figure 7B shows the results of FACS analysis of TH (TH (tyrosine hydroxylase) is a representative maturation marker for mDA neurons) expression in imDAPs cultured with different concentrations of hPLT or HhPLT or with B27 in maturation medium. Figure 7C shows the results of immunocytochemical analysis of TH in mDA neurons generated by maturation with 1% HhPLT or B27 (scale bar: 50 μm). [Figure 7] Figures 7D-E show the effect of IWR1 on imDAP maturation according to Example 12 of the present disclosure. Figure 7D shows the morphology of cells matured with or without IWR1 in maturation medium (scale bar: 50 μm). Figure 7E shows the results of qRT-PCR analysis of the expression of markers EN1, LMX1A, FOXA2, NURR1, SOX6, and TH in cells matured with or without IWR1 in maturation medium. [Figure 8] Figures 8A-C show the effects of Y27632 (Y) and PLLH (poly-L-lysine hydrobromide) or PLOH (poly-L-ornithine hydrobromide) on imDAP maturation according to Example 13 of the present disclosure. Figure 8A shows brightfield images of cells matured with VTN+Y, VTN+PLLH, VTN+PLLH+Y, VTN+PLOH, VTN+PLOH+Y, or laminin+PLOH (scale bar: 50 μm). Figure 8B shows the yield of cells matured with VTN+Y, VTN+PLLH+Y, VTN+PLOH+Y, or laminin+PLOH. Figure 8C shows the results of qRT-PCR analysis of the expression of maturation-related markers EN1, LMX1A, FOXA2, SOX6, NURR1, and TH in cells after 7 days of maturation. [Figure 8]FIG. 8D shows the results of immunocytochemical analysis (green light: TH; blue light: DAPI (nuclei)) of frozen sections of neurospheres according to Example 14 of the present disclosure (scale bar: 20 μm). [Figure 9] Figure 9 shows the differentiation potential of expanded imDAPs at early and late passages. Figure 9A shows the results of RT-qPCR analysis of the expression of specific markers EN1, LMX1A, FOXA2, NURR1, SOX6, and TH in early-stage mDA neurons matured from expanded P3 and P6 imDAPs. Figure 9B shows the results of immunocytochemical analysis of the expression of LMX1A and SOX6 in early-stage mDA neurons matured from expanded P3 and P5 imDAPs. Figure 9C shows the quantification of LMX1A- and SOX6-expressing mDA neurons matured from expanded P3 and P5 imDAPs. DETAILED DESCRIPTION OF THE INVENTION

[0020] It will be appreciated that certain aspects, modes, embodiments, variations and features of the present disclosure are described below at varying levels of detail to provide a substantial understanding of the technology.

[0021] Throughout this specification, references to "first," "second," "third," "fourth," "fifth," "sixth," "seventh," "eighth," or "ninth" do not imply any order or sequence of the features, structures (e.g., media or compositions), or properties described with reference to them, but are used for distinguishing purposes only.

[0022] Throughout this specification, reference to a "first aspect," "second aspect," "third aspect," "fourth aspect," "fifth aspect," "sixth aspect," "seventh aspect," "eighth aspect," or "ninth aspect" means that the particular feature, structure, or characteristic described in connection with that aspect is included in at least one or more aspects of the disclosure. Also, particular feature(s), structure(s), characteristic(s) or embodiment(s) in one aspect may be combined in any suitable manner with those in one or more other aspects.

[0023] Throughout this specification, references to "one embodiment," "another embodiment," "preferred embodiment(s)," "some embodiments," or "particular embodiment(s)" mean that the particular feature, structure, or characteristic described in connection with the embodiment(s) is included in at least one or more embodiments of the present disclosure. Also, particular feature(s), structure(s), or characteristic(s) in one embodiment may be combined in any suitable manner with those in one or more other embodiments.

[0024] The present disclosure illustratively described herein may suitably be practiced in the absence of any element(s), limitation(ies) not specifically disclosed herein. As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements essential to the composition or method. "Consisting of" is intended to mean excluding more than trace elements of other components for the claimed composition and substantial method steps. Embodiments defined by each of these transition terms are within the scope of the present disclosure. Thus, the present methods and compositions may include (comprise) additional steps and components, or may include (consist essentially of) insignificant steps and compositions, or are intended to contemplate (consist) only of the recited method steps or compositions. Furthermore, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0025] It is to be understood that this disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0026] 1. Culture medium for growing mDAP In a first aspect, the present disclosure provides a culture medium (e.g., a chemically defined, serum-free growth medium) capable of promoting robust proliferation of midbrain dopaminergic progenitor cells (mDAP), the culture medium comprising: (a) a basal medium; (b) a neural growth supplement; (c) a WNT signaling pathway activator; (d) a Rho kinase (ROCK) inhibitor; and (e) a transforming growth factor β (TGF-β) inhibitor.

[0027] According to a first aspect, by including a TGF-β inhibitor, particularly a ROCK inhibitor and a TGF-β inhibitor, the growth medium of the present disclosure can improve growth efficiency and expression of mDAP-specific markers during the growth and passaging of mDAP.

[0028] The growth medium of the present disclosure can be contacted with mDAP on a cell culture surface.The cell culture surface can be coated with any common growth coating matrix in the art or any other suitable growth coating matrix.Examples of common coating matrices include vitronectin (VTN), matrigel, fibronectin, gelatin, and laminin.

[0029] Any mDAP may be grown using the growth medium of the present disclosure. Examples of mDAP include fetal brain-derived mDAP; hPSC-derived mDAP; and mDAP obtained via transdifferentiation from other cell types. mDAP may be derived (e.g., differentiated) from pluripotent stem cells. Pluripotent stem cells may include induced pluripotent stem cells (e.g., hiPSCs), embryonic stem cells (e.g., hESCs), naive PSCs (NPSCs), and extended pluripotent stem cells (EPSCs). In certain embodiments, the mDAP are ESC-derived midbrain dopaminergic progenitor cells (emDAPs). In certain embodiments, the mDAP are iPSC-derived midbrain dopaminergic progenitor cells (imDAPs). In certain embodiments, the mDAP are NPSC-derived midbrain dopaminergic progenitor cells (nmDAPs). In certain embodiments, the mDAP are EPSC-derived midbrain dopaminergic progenitor cells (epmDAPs).

[0030] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be easily obtained using conventional methods, such as those described in existing technologies, or commercially available products. For example, the CytoTune iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) can be used to reliably generate induced pluripotent stem cells (iPSCs) from somatic cells, including PBMCs and T cells.

[0031] Such iPSCs (e.g., hiPSCs) can be cultured under defined conditions to generate mDAP. For example, iPSCs (e.g., hiPSCs) can be cultured on Matrigel under defined conditions in mTeSR™ medium (Stemcell Technologies, catalog no. 85850). Subconfluent hiPSCs can be passaged onto fresh Matrigel-coated plates and cultured in iPSC medium for an additional period (e.g., 24 hours) to reach 90-100% confluence. Once confluent, the medium can be changed to induce mDAP for an additional period (e.g., 1-3 days), optionally with daily medium changes as needed. See, for example, Fedele et al., Scientific Reports 7:6036 | DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0032] As the name suggests, a basal medium is a medium that can support cell survival, maintenance, growth, and proliferation and is a fundamental component of a growth medium. Generally, a basal medium contains approximately 95% to 99% growth medium by volume. The basal medium used in the growth medium of the present disclosure may be a general basal medium or a basal medium specialized for neural cells, such as a neural basal medium.

[0033] Examples of common basal media include DMEM:F12 (e.g., Gibco catalog number C11330500BT), BME medium (e.g., Gibco catalog number 21010046 or Sigma-Aldrich catalog number B9638), IMDM medium (e.g., Gibco catalog number 12440053; or Sigma-Aldrich catalog number I3390), Eagle's MEM medium (e.g., Minimum Essential Medium (MEM) developed by Harry Eagle, Sigma-Aldrich catalog numbers M2414 / M2279 / M5690), α-MEM medium (e.g., Gibco catalog number 12561056; or Sigma-Aldrich catalog number M0894), DMEM medium (e.g., Gibco catalog number 21068028), RPMI 1640 medium (e.g., Gibco catalog number 11875093), Ham's F12 medium (e.g., Gibco catalog number 11765054), or a mixture thereof.

[0034] Examples of neural basal media include NEUROBASAL™ Basal Medium (e.g., Gibco Catalog No. 21103049), NEUROBASAL-A™ Basal Medium (e.g., Gibco Catalog No. 10888022), NEUROBASAL PLUS™ Basal Medium (e.g., Gibco Catalog No. A3582901), and / or BRAINPHYS™ Basal Medium (e.g., STEMCELL Catalog No. 05790).

[0035] In certain embodiments, the basal medium comprises DMEM:F12 and NEUROBASAL™ Neurobasal Medium, which is present in the basal medium at about 0% to about 100%, about 25% to about 75%, or about 50% by volume.

[0036] In certain embodiments, the nerve growth supplement is selected from the group consisting of B27, N1, N2, and any combination thereof. In certain embodiments, the nerve growth supplement comprises B27 (e.g., B27 from GIBCO BRL catalog number 12587010).

[0037] According to the present disclosure, the concentration of the nerve growth supplement is not particularly limited as long as it does not interfere with the promotion of mDAP proliferation. In certain embodiments, the nerve growth supplement is present in the culture medium at a concentration of about 0.1% to about 20% by volume, preferably about 0.1% to about 10% by volume, and more preferably about 0.5% to about 5% by volume.

[0038] The Wnt signaling pathway is defined by a series of events that occur when Wnt protein ligands bind to cell surface receptors, members of the Frizzled receptor family. This leads to activation of the Dishevelled (Dsh) family of proteins, which inhibit a complex of proteins containing axin, GSK-3, and the protein APC to degrade intracellular β-catenin. The resulting enriched nuclear β-catenin enhances transcription by the TCF / LEF family of transcription factors.

[0039] As used herein, a Wnt signaling pathway activator refers to an agonist of the Wnt signaling pathway (e.g., an agent that can upregulate the activity and / or amount of a component involved in the Wnt signaling pathway) and can be interchangeably referred to as a "Wnt signaling pathway agonist," "Wnt agonist," "Wnt pathway activator," or "Wnt activator." A Wnt signaling pathway activator includes an agent that directly or indirectly activates TCF / LEF-mediated transcription in a cell, for example, by modulating the activity of any one of the proteins / genes in the Wnt signaling cascade (e.g., by enhancing the activity of a positive regulator of the Wnt signaling pathway or inhibiting the activity of a negative regulator of the Wnt signaling pathway).

[0040] The Wnt activator is selected from true Wnt activators that bind to and activate Frizzled receptor family members, including any and all of Wnt family proteins, inhibitors of intracellular β-catenin degradation, and activators of TCF / LEF. The Wnt activator can stimulate Wnt activity in cells by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 90%, at least about 100%, at least about 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold or more, compared to the level of Wnt activity in the absence of the Wnt activator. As known to those skilled in the art, Wnt activity can be determined by measuring Wnt transcriptional activity, for example, using pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (see Korinek et al., Science 275:1784-1787, 1997, incorporated herein by reference).

[0041] Representative Wnt activators may include secreted glycoproteins including Wnt-1 / Int-1, Wnt-2 / Irp (Int-1 related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D systems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi et al., Biochem. Biophys. Res. Com., 283:798-805, 2001), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, Wnt-11, and Wnt-16. An overview of human Wnt proteins is provided in "The Wnt Family of Secreted Proteins," R&D Systems Catalog, 2004 (incorporated herein by reference).

[0042] Additional Wnt activators include the R-spondin family of secreted proteins, which are involved in the activation and regulation of the Wnt signaling pathway and include at least four members: R-spondin 1 (NU206, Nuvelo, San Carlos, CA), R-spondin 2 (R&D systems), R-spondin 3 and R-spondin 4. Wnt activators also include Norrin (also known as Norrie Disease Protein or NDP) (R&D systems), a secreted regulatory protein that functions like Wnt protein in that it binds with high affinity to the Frizzled-4 receptor and induces activation of the Wnt signaling pathway (Kestutis Planutis et al., BMC Cell Biol. 8:12, 2007).

[0043] Wnt activators further include aminopyrimidine derivatives (N4-(benzo[d][1,3]dioxol-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine), which are small molecule agonists of the Wnt signaling pathway, as described by Liu et al. (Angew Chem. Int. Ed. Engl. 44(13):1987-1990, 2005, incorporated herein by reference).

[0044] In certain embodiments, the Wnt signaling pathway activator is a GSK inhibitor, such as a GSK-3β inhibitor. GSK3 inhibitors can include, for example, but are not limited to, polynucleotides, polypeptides, and small molecules.

[0045] GSK inhibitors include small interfering RNA (siRNA, Cell Signaling), lithium (Sigma), Kenpaullone (Biomol International, Leost et al., Eur.J.Biochem.267:5983-5994,2000), 6-bromoindirubin-30-acetoxime (Meyer et al., Chem.Biol.10:1255-1266,2003), SB 216763 and SB 415286 (Sigma-Aldrich), as well as FRAT family members and FRAT-derived peptides that prevent the interaction of GSK-3 with axin. An overview is provided by Meijer et al. (Trends in Pharmacological Sciences 25:471-480,2004 (incorporated herein by reference)). Methods and assays for determining the level of GSK-3 inhibition are known in the art and may include, for example, methods and assays such as those described in Liao et al. (Endocrinology 145(6):2941-2949, 2004, incorporated herein by reference).

[0046] In certain embodiments, the Wnt activator is selected from one or more of a Wnt family member, R-spondins 1-4 (such as R-spondin 1), Norrin, Wnt3a, Wnt-6, and a GSK inhibitor.

[0047] In certain embodiments, any of the specific protein-based Wnt activators mentioned herein, such as R-spondin1 to R-spondin4, any Wnt family member, may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, 99% of the respective Wnt activator activity, and / or a homolog or fragment thereof that shares at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by any art-recognized sequence alignment software based on either global alignment techniques (e.g., Needleman-Wunsch algorithm) or local alignment techniques (e.g., Smith-Waterman algorithm).

[0048] In certain embodiments, exemplary GSK-3β inhibitors include kenpaullone, 1-azakempaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, CT20026, SB216763, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylindole), benzophenone-3-one, ... nazolin-4-yl)amine, pyridocarbazole-cyclopenadienyl ruthenium complex (GSK-3 inhibitor XV), 2-thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole, OTDZT, alpha-4-dibromoacetophenone, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, GF109203X, RO318220 and any combination thereof.

[0049] In certain embodiments, the WNT signaling pathway activator is Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, SB216763, BIO(GSK) 3 inhibitor IX), BIO-acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, 2-thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole, alpha-4-dibromoacetophenone, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, RO318220, GF109203X, and any combination thereof.

[0050] Representative structures of certain WNT signaling pathway activators that may be used in the growth media of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources.

[0051] However, in all instances herein where a given source is provided, it should generally be understood that such source is not limiting. Alternative sources (commercial or non-commercial) of the same or similar materials, chemicals, or compounds can readily be used in light of the exemplary structure of the material, chemical, or compound.

[0052] [ka] Azakenpaullone: ​​MCE, #HY-59090; APExBio, #B3690; Sigma-Aldrich, #A3734; CAS number: 676586-65-9.

[0053] [ka] CHIR99021:APExBio, #A3011.

[0054] [ka] CHIR98014: Sigma-Aldrich, #SML1094; CAS number: 252935-94-7.

[0055] [ka] NP031112 (Tideglusib): Sigma-Aldrich, #SML0339; APExBio, #B1539; MCE, #HY-14872; CAS number: 865854-05-3.

[0056] [ka] TWS119:Sigma-Aldrich, #SML1271;APExBio, #B1540;MCE, #HY-10590;CAS number:601514-19-6.

[0057] [ka] AZD2858:APExBio, #B1537;MCE, #HY-15761;CAS Number: 486424-20-8.

[0058] [ka] AZD1080:APExBio, #B1536;MCE, #HY-13862, CAS number: 612487-72-6.

[0059] [ka] SB415286: Sigma-Aldrich, #S3567; APExBio, #A8241; MCE, #HY-15438; CAS number: 280744-09-4.

[0060]

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[0061]

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[0062]

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[0063]

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[0064]

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[0065]

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[0066] [ka] (5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK-3 inhibitor XIII): MCE, #HY-112392; Absin, #abs819580; Aladdin, #G338805; CAS number: 404828-08-6.

[0067] [ka] Pyridocarbazole-cyclopenadienyl ruthenium complex (GSK-3 inhibitor XV): Sigma-Aldrich, #361558, CAS number: 936112-69-5.

[0068] [ka] 2-Thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole (GSK3 inhibitor II): APExBio, #C4599; CAS number: 478482-75-6.

[0069] [ka] Alpha-4-dibromoacetophenone (2,4'-dibromoacetophenone / 4'-bromophenacyl bromide): Sigma-Aldrich, #D38308; CAS number: 99-73-0.

[0070] [ka] OTDZT (2,4-dibenzyl-5-oxothiadiazolidine-3-thione): CAS number: 373357-10-9.

[0071] [ka] 3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3β inhibitor XI): Sigma-Aldrich, #361553; Aladdin, #G338716-1mg; CAS number: 626604-39-5.

[0072] [ka] L803 H-KEAPPAPPQSpP-NH2: CAS number: 348089-28-1.

[0073] [ka] GF109203X (Bisindolylmaleimide I): Sigma-Aldrich, #G2911; APExBio, #A8342; MCE, #HY-13867; CAS number: 133052-90-1.

[0074] [ka] RO318220:MCE, #HY-13866A;CAS Number:125314-64-9.

[0075] According to the present disclosure, the concentration of the WNT signaling pathway activator is not particularly limited as long as it does not interfere with the promotion of mDAP proliferation. In a specific embodiment, the WNT signaling pathway activator is present in the culture medium at a concentration of about 0.5 μM to about 20 μM, preferably about 0.5 μM to about 10 μM, and more preferably about 0.5 μM to about 5 μM.

[0076] In certain embodiments, the WNT signaling pathway activator comprises CHIR99021. In certain embodiments, the WNT signaling pathway activator (e.g., CHIR99021) is present in the growth medium at about 1 μM to about 5 μM, about 1.25 μM to about 5 μM, about 1.5 μM to about 5 μM, or about 2 μM to about 4 μM.

[0077] ROCK inhibitors include agents that inhibit the binding of ROCK to ROCK receptors. The ROCK inhibitors used in the growth medium of the present disclosure can promote the growth of mDAP, for example, by increasing the growth efficiency and the expression of mDAP-specific markers such as FOXA2.

[0078] In certain embodiments, the ROCK inhibitor is selected from the group consisting of Y27632, HA100, HA1152, HA-1077, and any combination thereof.

[0079] Y-27632 is also known as (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (e.g., Sigma-Aldrich). HA100 is also known as 5-(1-piperazinylsulfonyl)-isoquinoline, dihydrochloride. HA1077 is also known as fasudil hydrochloride or 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline hydrochloride (Cayman Chemical). H-1152 is also known as (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (Tocris Bioscience). Other ROCK inhibitors include N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluoromethyl)phenyl)-1,4,5,6-tetrahydropyridine-3-carboxamide (GSK429286A, Stemgent).

[0080] Representative structures of certain ROCK inhibitors that may be used in the growth media of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources (although such sources are not limiting).

[0081] [ka] Y-27632 (MCE Catalog No. HY-10071, CAS No.: 146986-50-7).

[0082] [ka] HA-100 (hydrochloride salt) (Absin catalog number abs47045575).

[0083] [ka] H1152 (MCE Catalog No. HY-15720, CAS No.: 451462-58-1).

[0084] [ka] HA-1077 (Fasudil / AT877) (MCE Catalog No. HY-10341A, CAS No.: 103745-39-7).

[0085] In certain embodiments, the ROCK inhibitor comprises Y27632. In certain embodiments, the ROCK inhibitor (e.g., Y27632) is present in the growth medium at about 1 μM to about 50 μM, preferably about 1 μM to about 20 μM, or more preferably about 5 μM to about 15 μM.

[0086] TGF-β signaling typically begins with the binding of TGF-β superfamily ligands to type II receptors, which recruit and phosphorylate type I receptors. Type I receptors then phosphorylate SMADs, which act as transcription factors in the nucleus and regulate target gene expression. Alternatively, TGF-β signaling can activate the MAP kinase signaling pathway, for example, via p38 MAP kinase. The TGF-β inhibitors used in the growth medium of the present disclosure can promote the proliferation of mDAPs, for example, by increasing proliferation efficiency and the expression of mDAP-specific markers such as FOXA2.

[0087] TGF-β inhibitors used herein include agents that reduce the activity of TGF-β signaling pathway.There are many different ways to disrupt TGF-β signaling pathway.For example, TGF-β signaling can be disrupted by: inhibiting TGF-β expression with small interfering RNA strategy; inhibiting furin (TGF-β activation protease); inhibiting pathway with physiological inhibitors, for example, inhibiting BMP with Noggin, DAN or DAN-like protein; neutralizing TGF-β with monoclonal antibody; inhibiting TGF-β receptor kinase 1 (also known as activin receptor-like kinase, ALK5), ALK4, ALK6, ALK7 or other TGF-β-related receptor kinase with small molecule inhibitors; inhibiting Smad2 and Smad3 signaling, by overexpressing their physiological inhibitor Smad7, or by using thioredoxin as Smad anchor to disable Smad activation.

[0088] For example, TGF-β inhibitors can target serine / threonine protein kinases selected from TGF-β receptor kinase 1, ALK4, ALK5, ALK7 or p38. ALK4, ALK5 and ALK7 are all closely related receptors of the TGF-β superfamily. The inhibitor of any one of these kinases will reduce the enzymatic activity of any one (or more) of these kinases.

[0089] In certain embodiments, a TGF-β inhibitor can bind to and inhibit the activity of a Smad protein, for example, R-SMAD or SMAD1-5 (ie, SMAD1, SMAD2, SMAD3, SMAD4, or SMAD5).

[0090] In certain embodiments, the TGF-β inhibitor may bind to and reduce the activity of a Ser / Thr protein kinase selected from TGF-β receptor kinase 1, ALK4, ALK5, ALK7, or p38.

[0091] In certain embodiments, the growth medium of the present disclosure comprises an inhibitor of ALK5.

[0092] Various methods are known for determining whether a substance is a TGF-β inhibitor. For example, cell assays can be used, in which cells are stably transfected with a human PAI-1 promoter driving a luciferase reporter gene or a reporter construct containing an Smad binding site. The inhibition of luciferase activity relative to a control group can be used as a measure of compound activity (De Gouville et al., Br. J. Pharmacol. 145(2):166-177, 2005, incorporated herein by reference). Another example is the ALPHASCEEN® phosphosensor assay for measuring kinase activity (Drew et al., J. Biomol. Screen. 16(2):164-173, 2011, incorporated herein by reference).

[0093] The TGF-β inhibitor useful in the present disclosure can be a protein, peptide, small molecule, small interfering RNA, antisense oligonucleotide, aptamer, antibody or its antigen-binding portion.The inhibitor can be naturally occurring or synthetic.Examples of small molecule TGF-β inhibitors that can be used in the context of the present disclosure include but are not limited to RepSox (2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine), SB431542, SB505124, LY36494, SJN-2511, A83-01, D4476, GW788388, LY364947, LY580276, SB525334, SD208, GW6604, and any combination thereof.

[0094] In certain embodiments, the TGF-β inhibitor is selected from the group consisting of RepSox, A83-01, SB431542, D4476, GW788388, LY364947, SB525334, SB505124, SD208, GW6604, and any combination thereof.

[0095] Representative structures of certain TGF-β inhibitors that may be used in the growth media of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources (although such sources are not limiting).

[0096] [ka] RepSox (2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine) (Sigma-Aldrich catalog number R0158; APExBio catalog number A3754; MCE catalog #HY-13012; CAS number: 446859-33-2).

[0097] [ka] A83-01 (Sigma-Aldrich catalog number SML0788; APExBio catalog number A3133; MCE catalog number HY-10432; CAS number: 909910-43-6).

[0098] [ka] SB431542 (APExBio catalog number A8249, CAS number: 301836-41-9).

[0099] [ka] D4476 (Sigma-Aldrich catalog number D1944; APExBio catalog number A3342; MCE catalog number HY-10324; CAS number: 301836-43-1).

[0100] [ka] GW788388 (APExBio catalog number A8301; MCE catalog number HY-10326; CAS number: 452342-67-5).

[0101] [ka] LY364947 (Sigma-Aldrich catalog number L6293; APExBio catalog number B2287; MCE catalog number HY-31462; CAS number: 396129-53-6).

[0102] [ka] SB525334 (Sigma-Aldrich catalog number S8822; APExBio catalog number A5602; MCE catalog number HY-12043; CAS number: 356559-20-1).

[0103] [ka] SB505124 (APExBio catalog number B2289; MCE catalog number HY-13521; CAS number: 694433-59-5).

[0104] [ka] SD208 (Sigma-Aldrich catalog number S7071; APExBio catalog number A3808; MCE catalog number HY-10324; CAS number: 627536-09-8).

[0105] [ka] GW6604 (Absinthe catalogue number abs814099; CAS number: 452342-37-9).

[0106] According to the present disclosure, the concentration of the TGF-β inhibitor is not particularly limited as long as it does not interfere with the promotion of mDAP proliferation. In a specific embodiment, the TGF-β inhibitor is present in the culture medium at a concentration of about 0.5 μM to about 50 μM, preferably about 1 μM to about 20 μM, and more preferably about 1 μM to about 15 μM.

[0107] In certain embodiments, the TGF-β inhibitor comprises SB431542. In certain embodiments, SB431542 is present in the growth medium at about 0.5 μM to about 50 μM, about 1 μM to about 20 μM, or about 1 μM to about 15 μM.

[0108] In certain embodiments, the culture medium of the present disclosure may optionally further comprise glutamine or a derivative thereof.

[0109] In certain embodiments, the glutamine or derivative thereof comprises L-alanyl-L-glutamine dipeptide (e.g., GLUTAMAX™ brand L-alanyl-L-glutamine dipeptide, Gibco catalog number 35050061), L-glutamine (e.g., Sigma-Aldrich catalog numbers G2150 / G7513; APExBio catalog number A8461; MCE catalog number HY-N0390; CAS number: 56-85-9), or a mixture thereof.

[0110] In certain embodiments, glutamine or a derivative thereof is present in the culture medium at a concentration of about 0.5% to 5% by volume, preferably about 0.5% to 2.5% by volume.

[0111] In certain embodiments, the culture media of the present disclosure may optionally further comprise an antioxidant.

[0112] In certain embodiments, the antioxidant comprises ascorbic acid (e.g., Sigma catalog number A8960) or a salt thereof (e.g., Na salt, Mg salt) or an analog or derivative thereof, SOD (e.g., Sigma catalog numbers S7571, S9697, S5395, S8160, S9636, S8409, S7446, CAS number: 9054-89-1), or a mixture thereof.

[0113] In certain embodiments, an antioxidant (e.g., ascorbic acid) is present in the culture medium of the present disclosure at a concentration of about 5 μg / mL to about 200 μg / mL, preferably about 15 μg / mL to about 100 μg / mL, and more preferably about 30 μg / mL to about 80 μg / mL.

[0114] In certain embodiments, the culture medium of the present disclosure further comprises a fibroblast growth factor. In certain embodiments, the fibroblast growth factor comprises FGF2 (e.g., Nuwacell), FGF1 (e.g., MCE Catalog No. HY-P7001), FGF8 (e.g., MCE Catalog No. HY-P7347, MCE Catalog No. HY-P7349, MCE Catalog No. HY-P7350), and / or FGF20 (e.g., R&D Catalog No. 2547-FG).

[0115] In certain embodiments, fibroblast growth factor (e.g., FGF2 or FGF8) is present in the growth medium at about 1 ng / mL to about 2.00 ng / mL, about 5 ng / mL to about 50 ng / mL, or about 5 ng / mL to about 20 ng / mL.

[0116] In certain embodiments, the culture medium of the present disclosure further comprises a BMP4 inhibitor.

[0117] In certain embodiments, the BMP4 inhibitor comprises dorsomorphin, LDN193189, or a combination thereof. Representative structures of certain BMP4 inhibitors that may be used in the culture media of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources.

[0118] [ka] LDN193189 (MCE Catalog No. HY-12071A, CAS No.: 1062368-24-4).

[0119] [ka] Dorsomorphin (Sigma-Aldrich catalog number P5499; APExBio catalog number B3252; MCE catalog number HY-13418A; CAS number: 866405-64-3).

[0120] In certain embodiments, the BMP inhibitor (eg, LDN193189) is present in the culture medium at about 0.05 μM to about 1.0 μM, or about 0.1 μM to about 0.4 μM.

[0121] In certain embodiments, the culture medium of the present disclosure does not include FGF8 and / or FGF2.

[0122] In certain embodiments, the culture medium of the present disclosure comprises (a) about 1 μM to about 10 μM of a WNT signaling pathway activator; (b) about 1 μM to about 20 μM of a ROCK inhibitor; (c) about 1 μM to about 20 μM of a TGF-β inhibitor; and (d) about 0.1% to about 10% by volume of a nerve growth supplement in a basal medium.

[0123] In certain embodiments, the culture medium of the present disclosure comprises (a) about 1 μM to about 10 μM CHIR99021; (b) about 1 μM to about 20 μM Y27632; (c) about 1 μM to about 20 μM SB431542; and (d) about 0.1% to about 10% by volume B27 in a basal medium.

[0124] In certain embodiments, the culture medium of the present disclosure can provide at least a 32-fold, 36-fold, 40-fold, or more expansion fold for each passage of mDAP, which is at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more expansion fold over the previous medium (about 5-10-fold) without a TGF-β inhibitor and a Rock inhibitor. In certain embodiments, the culture medium of the present disclosure can provide at least a 32-fold, 36-fold, 40-fold, or more expansion fold over each passage of mDAP, which is at least 1.6-fold, 1.8-fold, 2-fold, or more expansion fold over the same medium (about 20-fold) without a TGF-β inhibitor.

[0125] A representative example of an mDAP-specific marker is FOXA2. Representative examples of mDAP-specific markers include FOXA2 and one or more selected from LMX1A, EN1, OTX2, and SOX6. In some embodiments, the mDAP-specific markers include FOXA2, LMX1A, EN1, OTX2, and SOX6.

[0126] 2. Combination of coating matrices for growing mDAP In a second aspect, the present disclosure provides a coating matrix combination capable of promoting robust proliferation of midbrain dopaminergic progenitor cells (mDAP), the coating matrix combination comprising: (a) a first coating matrix capable of supporting cell adhesion of mDAP; and (b) a second coating matrix capable of improving expression of mDAP-specific markers during proliferation and passaging of mDAP, the second coating matrix comprising a Notch agonist.

[0127] According to a second aspect, by including a second coating matrix, the combination of the above coating matrices for proliferation can improve proliferation efficiency and expression of mDAP-specific markers during proliferation and passaging of mDAP.

[0128] According to a second aspect, the coating matrix combination of the present disclosure may be used in combination with any common mDAP growth medium in the art or any other suitable mDAP growth medium, such as the Gibco™ PSC Dopaminergic Neuron Differentiation Kit and the STEMdiff™ Dopaminergic Neuron Differentiation Kit / STEMdiff™ (#05835).

[0129] Any mDAP can be grown using the coating matrix combination of the present disclosure. Examples of mDAP include fetal brain-derived mDAP; hPSC-derived mDAP; and mDAP obtained via transdifferentiation from other cell types. mDAP can be derived (e.g., differentiated) from pluripotent stem cells. Pluripotent stem cells can include induced pluripotent stem cells (e.g., hiPSCs), embryonic stem cells (e.g., hESCs), naive PSCs (NPSCs), and extended pluripotent stem cells (EPSCs). In certain embodiments, the mDAP are ESC-derived midbrain dopaminergic progenitor cells (emDAPs). In certain embodiments, the mDAP are iPSC-derived midbrain dopaminergic progenitor cells (imDAPs). In certain embodiments, the mDAP are NPSC-derived midbrain dopaminergic progenitor cells (nmDAPs). In certain embodiments, the mDAP are EPSC-derived midbrain dopaminergic progenitor cells (epmDAPs).

[0130] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be easily obtained using conventional methods, such as those described in existing technologies, or commercially available products. For example, the CytoTune iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) can be used to reliably generate induced pluripotent stem cells (iPSCs) from somatic cells, including PBMCs and T cells.

[0131] Such iPSCs (e.g., hiPSCs) can be cultured under defined conditions to generate mDAP. For example, iPSCs (e.g., hiPSCs) can be cultured on Matrigel under defined conditions in mTeSR™ medium (Stemcell Technologies, catalog no. 85850). Subconfluent hiPSCs can be passaged onto fresh Matrigel-coated plates and cultured in iPSC medium for an additional period (e.g., 24 hours) to reach 90-100% confluence. Once confluent, the medium can be changed to induce mDAP for an additional period (e.g., 1-3 days), optionally with daily medium changes as needed. See, for example, Fedele et al., Scientific Reports 7:6036 | DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0132] In certain embodiments, the first coating matrix is ​​selected from the group consisting of vitronectin (VTN, e.g., Nuwacell® Vitronectin, #RP01002), collagen (e.g., Gibco Cat. Collagen I, #A1048301; Collagen I, #17100017; Type II Collagen, #17101015; Type IV Collagen, #17104019), proteoglycan (e.g., Invitrogen Cat. Nos. RP-77523 / RP-77524; MCE Cat. Nos. HY-P76323 / HY-P71233), fibronectin (e.g., Invitrogen Cat. No. RP-43130; MCE Cat. Nos. HY-P70593 / HY-P70593G / HY-P73063), entactin (e.g., Sigma-Aldrich Cat. No. D89 35; MCE Catalog No. HY-P71763), elastin (e.g., Sigma-Aldrich Catalog Nos. E7402 / E7277 / E6902; CAS No.: 9007-58-3), laminin (e.g., Nuwacell® laminin), functional fragments of any of the above proteins, hyaluronic acid (e.g., MCE Catalog No. HY-B0633A; CAS No.: 9004-61-9), gelatin (e.g., USP, #1288485; MCE Catalog No. HY-Y1365; CAS No.: 9000-70-8), and any combination thereof. In certain embodiments, the first coating matrix comprises VTN.

[0133] According to the present disclosure, the second coating matrix comprises a Notch agonist. As used herein, a Notch agonist refers to an activator of the Notch signaling pathway (e.g., a drug that can upregulate the activity and / or amount of components involved in the Notch signaling pathway). Notch receptor proteins can interact with a number of surface-bound or secreted ligands, including, but not limited to, Jagged-1, Jagged-2, Delta-like 1, Delta-like 3, Delta-like 4, etc. Upon ligand binding, the Notch receptor is activated by sequential cleavage events involving members of the ADAM protease family, as well as intramembrane cleavage regulated by gamma secretase presenilin. As a result, the intracellular domain of Notch translocates to the nucleus, where downstream genes are transcriptionally activated.

[0134] As used herein, "Notch agonist" includes molecules that stimulate the Notch activity in cells by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 90%, at least about 100%, at least about 3 times, 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times or more, compared to the level of Notch activity in the absence of Notch agonist.As known in the art, Notch activity can be determined by measuring the transcriptional activity of Notch, for example, by the 4xwtCBF1-luciferase reporter construct described by Hsieh et al. (Mol.Cell.Biol.16:952-959,1996, incorporated herein by reference).

[0135] In certain embodiments, the Notch agonist is selected from Delta-like 4 (DLL4, Nuwacell®), Delta-like 1 (DLL1, e.g., MCE (HEK293, His), #HY-P7841), Jagged-1 (e.g., MCE catalog number HY-P1846), Jagged-2 (e.g., (JAG2) #BES23045RP), variants thereof, and any combination thereof. In certain embodiments, the Notch agonist is DSL peptide (Dontu et al., Breast Cancer Res., 6:R605-R615, 2004).

[0136] Representative structures of specific Notch agonists that may be used in the coating matrix combinations of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources.

[0137] [ka] Jagged-1(188-204) (e.g., MCE Catalog No. HY-P1846; CAS No.: 219127-21-6)

[0138] In certain embodiments, the Notch agonist comprises DLL4.

[0139] In certain embodiments, any of the specific Notch agonists referred to herein, e.g., Jagged-1, Jagged-2, Delta-1, and Delta-like 4, may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, 99% of the respective Notch agonist, and / or a homolog or fragment thereof that shares at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by any art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0140] In certain embodiments, the coating matrix combination comprises VTN and a Notch agonist. In certain embodiments, the coating matrix combination comprises VTN and DLL4.

[0141] A cell culture surface may be coated with the growth coating matrix combination of the present disclosure by conventional techniques in the art. Generally, the first and second coating matrices may be first mixed in any ratio, and the cell culture surface may be coated with the coating solution. The coating concentrations of the first and second coating matrices may be easily determined by those skilled in the art. For example, the coating concentrations of the first and second coating matrices may be 0.5 to 2 μg / cm, respectively. 2 , e.g., 1 μg / cm 2 It could be.

[0142] In certain embodiments, the coated matrix combinations of the present disclosure can provide at least a 36-fold, 38-fold, 40-fold, or more expansion fold for each passage of mDAP, which is at least 1.8, 1.9, 2-fold, or more than the expansion fold of previous coated matrices such as VTN (approximately 20-fold).

[0143] A representative example of an mDAP-specific marker is FOXA2. Representative examples of mDAP-specific markers include FOXA2 and one or more selected from LMX1A, EN1, OTX2, and SOX6. In some embodiments, the mDAP-specific markers include FOXA2, LMX1A, EN1, OTX2, and SOX6.

[0144] 3. Methods for Propagating mDAP In a third aspect, the present disclosure provides a method for expanding midbrain dopaminergic progenitor cells (mDAP), comprising contacting the mDAP with a growth medium on a culture surface coated with a growth coating matrix combination of the present disclosure.

[0145] According to a third aspect, by using a second coating matrix, the above propagation method can improve the propagation efficiency and the expression of mDAP-specific markers during the propagation and passaging of mDAP.

[0146] Any mDAP may be propagated using the proliferation method of the present disclosure. Examples of mDAP include fetal brain-derived mDAP; hPSC-derived mDAP; and mDAP obtained via transdifferentiation from other cell types. mDAP may be derived (e.g., differentiated) from pluripotent stem cells. Pluripotent stem cells may include induced pluripotent stem cells (e.g., hiPSCs), embryonic stem cells (e.g., hESCs), naive PSCs (NPSCs), and extended pluripotent stem cells (EPSCs). In certain embodiments, the mDAP are ESC-derived midbrain dopaminergic progenitor cells (emDAPs). In certain embodiments, the mDAP are iPSC-derived midbrain dopaminergic progenitor cells (imDAPs). In certain embodiments, the mDAP are NPSC-derived midbrain dopaminergic progenitor cells (nmDAPs). In certain embodiments, the mDAP are EPSC-derived midbrain dopaminergic progenitor cells (epmDAPs).

[0147] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be easily obtained using conventional methods, such as those described in existing technologies, or commercially available products. For example, the CytoTune iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) can be used to reliably generate induced pluripotent stem cells (iPSCs) from somatic cells, including PBMCs and T cells.

[0148] Such iPSCs (e.g., hiPSCs) can be cultured under defined conditions to generate mDAP. For example, iPSCs (e.g., hiPSCs) can be cultured on Matrigel under defined conditions in mTeSR™ medium (Stemcell Technologies, catalog no. 85850). Subconfluent hiPSCs can be passaged onto fresh Matrigel-coated plates and cultured in iPSC medium for an additional period (e.g., 24 hours) to reach 90-100% confluence. Once confluent, the medium can be changed to induce mDAP for an additional period (e.g., 1-3 days), optionally with daily medium changes as needed. See, for example, Fedele et al., Scientific Reports 7:6036 | DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0149] According to the third aspect, any common mDAP growth medium in the art or any other suitable mDAP growth medium can be used in the growth methods of the present disclosure. Examples of common mDAP growth media include Gibco™ PSC Dopaminergic Neuron Differentiation Kit and STEMdiff™ Dopaminergic Neuron Differentiation Kit / STEMdiff™ (#05835).

[0150] The growth coating matrix combinations of the present disclosure are described elsewhere herein (e.g., as described in the coating matrix combinations for growing mDAP or in the second aspect herein), and a description of these same is omitted here for the sake of brevity.

[0151] In certain embodiments, the culture surface is in a culture plate, a culture bottle, a culture flask, or a culture vessel. In certain embodiments, the culture plate comprises a single or multi-layer cell stack, 6 wells, 12 wells, 24 wells, 48 ​​wells, 96 wells, 384 wells, 1536 wells, or more wells. In certain embodiments, the culture plate comprises flat-bottom or round-bottom wells.

[0152] A cell culture surface may be coated with the growth coating matrix combination of the present disclosure by conventional techniques in the art. Generally, the first and second coating matrices may be first mixed in any ratio, and the cell culture surface may be coated with the coating solution. The coating concentrations of the first and second coating matrices may be easily determined by those skilled in the art. For example, the coating concentrations of the first and second coating matrices may be 0.5 to 2 μg / cm, respectively. 2 , e.g., 1 μg / cm 2 It could be.

[0153] In certain embodiments, the method further comprises replacing the growth medium with the same medium every few days during growth, hi certain embodiments, the method further comprises replacing the growth medium with the same medium every three days during growth.

[0154] In certain embodiments, the mDAP is contacted with the growth medium for a total of about 6 days.

[0155] In certain embodiments, the growth medium of the present disclosure is used as the growth medium described above.The growth medium of the present disclosure used in the above embodiments is described elsewhere herein (for example, as the culture medium for growing mDAP herein or as described in the first aspect), and these same descriptions are omitted herein for brevity.According to the above embodiments, the growth method can support the long-term growth of mDAP as a homogeneous population (for example, at least 4, 5, 6 or more passages), or the growth method can maintain or even improve the growth efficiency and the expression of mDAP-specific markers after growth and passage compared to before growth and passage, even if mDAP is grown and passaged once or multiple times.

[0156] In certain embodiments, the mDAP has been propagated and passaged at least 1, 2, 3, 4, 5, or 6 times (e.g., P1, P2, P3, P4, P5, P6 or later passages), preferably at least 4, 5, or 6 times (e.g., P4, P5, P6 or later passages), and most preferably at least 6 times (e.g., P6 or later passages).

[0157] In certain embodiments, the mDAP is about 1×10 3 cells / cm 2 ~Approx. 1×10 5 cells / cm 2 The cells are seeded onto the culture surface at a density of 0.05 to 0.05 μg / ml. Compared to higher seeding densities, such a low seeding density is beneficial for improving the expansion fold of the plated cells.

[0158] In certain embodiments, the expansion method supports the expansion of mDAP with: (1) a population doubling time (PDT) of about 25-28 hours and / or (2) a total cell expansion fold of about 35-40 fold for imDAP cells expanded and passaged at least 1, 2, 3, 4, 5, 6 or more times (e.g., P1-P6).

[0159] In certain embodiments, the expansion methods of the present disclosure can provide at least a 36-fold, 38-fold, 40-fold or more expansion fold for each passage of mDAP, which is at least 1.8, 1.9, 2-fold or more than the expansion fold of previous expansion methods, such as those using VTN (approximately 20-fold).

[0160] A representative example of an mDAP-specific marker is FOXA2. Representative examples of mDAP-specific markers include FOXA2 and one or more selected from LMX1A, EN1, OTX2, and SOX6. In some embodiments, the mDAP-specific markers include FOXA2, LMX1A, EN1, OTX2, and SOX6.

[0161] 4. Culture medium for mDAP maturation In a fourth aspect, the present disclosure provides a culture medium (e.g., a chemically defined, serum-free maturation medium) capable of promoting maturation of mDAP (midbrain dopaminergic progenitor cells), the culture medium comprising: (a) neurobasal medium; (b) human platelet lysate (hPLT); (c) transforming growth factor β (TGF-β); (d) a γ-secretase inhibitor; and (e) a cAMP-based compound or a cyclase activator thereof.

[0162] B27 is commonly used as a medium supplement for neuronal maturation in the prior art. According to the fourth aspect, by replacing B27 with hPLT, the above maturation medium can improve the maturation degree (percentage of TH+ cells) of mDAP.

[0163] According to a fourth aspect, the maturation medium of the present disclosure can be contacted with mDAP on a cell culture surface coated with any common maturation coating matrix in the art or any other suitable maturation coating matrix. Examples of common maturation coating matrices include vitronectin (VTN) and laminin / poly-L-ornithine (PLO).

[0164] Any mDAP may be matured using the maturation medium of the present disclosure. Examples of mDAP include fetal brain-derived mDAP; hPSC-derived mDAP; and mDAP obtained via transdifferentiation from other cell types. mDAP may be derived (e.g., differentiated) from pluripotent stem cells. Pluripotent stem cells may include induced pluripotent stem cells (e.g., hiPSCs), embryonic stem cells (e.g., hESCs), naive PSCs (NPSCs), and extended pluripotent stem cells (EPSCs). In certain embodiments, the mDAP are ESC-derived midbrain dopaminergic progenitor cells (emDAPs). In certain embodiments, the mDAP are iPSC-derived midbrain dopaminergic progenitor cells (imDAPs). In certain embodiments, the mDAP are NPSC-derived midbrain dopaminergic progenitor cells (nmDAPs). In certain embodiments, the mDAP are EPSC-derived midbrain dopaminergic progenitor cells (epmDAPs).

[0165] In certain embodiments, the mDAP is a non-proliferating mDAP. In certain embodiments, the mDAP is a proliferating mDAP, such as a proliferating imDAP. In certain embodiments, the mDAP is a mDAP that has been propagated and passaged one or more times, such as a proliferating P1, P2, P3, P4, P5, or P6 imDAP. In certain embodiments, the mDAP is an immature mDAP. In certain embodiments, the mDAP is a partially mature mDAP.

[0166] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be easily obtained using conventional methods, such as those described in existing technologies, or commercially available products. For example, the CytoTune iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) can be used to reliably generate induced pluripotent stem cells (iPSCs) from somatic cells, including PBMCs and T cells.

[0167] Such iPSCs (e.g., hiPSCs) can be cultured under defined conditions to generate mDAP. For example, iPSCs (e.g., hiPSCs) can be cultured on Matrigel under defined conditions in mTeSR™ medium (Stemcell Technologies, catalog no. 85850). Subconfluent hiPSCs can be passaged onto fresh Matrigel-coated plates and cultured in iPSC medium for an additional period (e.g., 24 hours) to reach 90-100% confluence. Once confluent, the medium can be changed to induce mDAP for an additional period (e.g., 1-3 days), optionally with daily medium changes as needed. See, for example, Fedele et al., Scientific Reports 7:6036 | DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0168] As the name suggests, Neurobasal Medium is a medium that can support the survival, maintenance, growth, and proliferation of neural cells and is a fundamental component of maturation medium. Generally, Neurobasal Medium contains approximately 95% to 99% maturation medium by volume.

[0169] Examples of neural basal media include NEUROBASAL™ Basal Medium (e.g., Gibco Catalog No. 21103049), NEUROBASAL-A™ Basal Medium (e.g., Gibco Catalog No. 10888022), NEUROBASAL PLUS™ Basal Medium (e.g., Gibco Catalog No. A3582901), and / or BRAINPHYS™ Basal Medium (e.g., STEMCELL Catalog No. 05790).

[0170] hPLTs are commercially available, for example, PLTGold Human Platelet Lysate (Biological Industries, #PLTGOLD500R), however, hPLTs from other sources are also available and can be used with the present disclosure.

[0171] According to the present disclosure, the concentration of hPLT is not particularly limited as long as it does not interfere with the promotion of mDAP maturation. In a specific embodiment, hPLT is present in the culture medium at a concentration of about 0.1% to about 5% by volume, preferably about 0.1% to about 2% by volume.

[0172] In certain embodiments, the TGF-β includes TGF-β1 (e.g., APExBio catalog number P1039; MCE catalog number HY-P78168), TGF-β2 (e.g., R&D catalog number 302-B2), TGF-β3 (e.g., Peprotech catalog number 100-36E), and / or TGF-β1β2.

[0173] According to the present disclosure, the concentration of TGF-β is not particularly limited as long as it does not interfere with the promotion of mDAP maturation. In a specific embodiment, TGF-β (e.g., TGF-β3) is present in the culture medium at a concentration of about 0.1 ng / ml to about 10 ng / ml, preferably about 0.5 ng / ml to about 5 ng / ml, and more preferably about 1 ng / ml to about 2 ng / ml.

[0174] In certain embodiments, the gamma-secretase inhibitor comprises or is selected from the group consisting of DAPT, N-[N-(3,5-difluorophenylacetyl)]-L-alanyl-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydro-benzo[E](1,4)diazepin-2-one, LY-411575, dihydroergocristine mesylate, BMS 299897, and any combination thereof.

[0175] Representative structures of certain γ-secretase inhibitors that may be used in the media of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources (although such sources are not limiting).

[0176] [ka] DAPT (MCE catalog number HY-13027, CAS number: 208255-80-5).

[0177] [ka] Compound E (N-[N-(3,5-difluorophenylacetyl)]-L-alanyl-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydro-benzo[E](1,4)diazepin-2-one) (APExBio catalog number C3341; MCE catalog number HY-14176; CAS number: 209986-17-4).

[0178] [ka] LY-411575 (Sigma-Aldrich catalog number SML0506; APExBio catalog number A4019; MCE catalog number HY-50752; CAS number: 209984-57-6).

[0179] [ka] Dihydroergocristine mesylate (APExBio catalog number B6313; MCE catalog number HY-N2319; CAS number: 24730-10-7).

[0180] [ka] BMS 299897 (Sigma-Aldrich catalog number SML0210; APExBio catalog number A4400; MCE catalog number HY-50883; CAS number: 290315-45-6).

[0181] In certain embodiments, the gamma-secretase inhibitor comprises DAPT.

[0182] According to the present disclosure, the concentration of the γ-secretase inhibitor is not particularly limited as long as it does not interfere with the promotion of mDAP maturation. In certain embodiments, the γ-secretase inhibitor (e.g., DAPT) is present in the maturation medium at about 1 μM to about 30 μM, preferably about 5 μM to about 20 μM.

[0183] As used herein, a cAMP-based compound is a cell-permeable cAMP-like compound that directly increases intracellular cAMP content. In certain embodiments, the cAMP-based compound includes cAMP or a derivative thereof or a salt thereof. Examples of cAMP derivatives or salts thereof include Db-cAMP sodium salt, 8-bromo-cAMP sodium salt, 8-chloro-cAMP, 6-Bnz-cAMP sodium salt, and bucladesine calcium salt (dibutyryl cAMP calcium salt). Furthermore, cyclase activators of cAMP-based compounds can also be used in the maturation medium of the present disclosure to generate intracellular cAMP. Examples of cyclase activators include forskolin and NKH477.

[0184] In certain embodiments, the cAMP-based compound or cyclase activator thereof comprises or is selected from the group consisting of Db-cAMP sodium salt, cAMP, forskolin, 8-bromo-cAMP sodium salt, NKH477, 8-chloro-cAMP, 6-Bnz-cAMP sodium salt, bucladesine calcium salt, and any combination thereof.

[0185] Representative structures of certain cAMP-based compounds or cyclase activators thereof that may be used in the maturation media of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources (although such sources are not limiting).

[0186] [ka] cAMP (Sigma-Aldrich catalog number 20-198; MCE catalog number HY-B1511, CAS number: 60-92-4).

[0187] [ka] Db-cAMP sodium salt (Sigma catalog number D0627, CAS number: 16980-89-5).

[0188] [ka] Forskolin (MCE Catalog No. HY-15371; CAS No.: 66575-29-9).

[0189] [ka] 8-Bromo-cAMP sodium salt (Sigma-Aldrich catalog number B7880; APExBio catalog number B9000; MCE catalog number HY-12306; CAS number: 76939-46-3).

[0190] [ka] 8-Chloro-cAMP (MCE Catalog No. HY-123396; CAS No.: 41941-56-4).

[0191] [ka] 6-Bnz-cAMP sodium salt (MCE catalog number HY-103322; CAS number: 1135306-29-4).

[0192] [ka] Bucladesine calcium salt (MCE catalog number HY-B0764A; CAS number: 938448-87-4).

[0193] [ka] NKH477 (Sigma-Aldrich catalog number N3290; MCE catalog number HY-103193; CAS number: 138605-00-2).

[0194] In certain embodiments, the cAMP-based compound or cyclase activator thereof comprises Db-cAMP sodium salt.

[0195] In certain embodiments, the cAMP-based compound (eg, Db-cAMP sodium salt) or cyclase activator thereof is present in the culture medium at a concentration of about 0.1 mM to about 5 mM, preferably about 0.1 mM to about 2 mM.

[0196] Optionally, the culture medium may contain (f) neurotrophic factors, which include ligands for membrane receptors that promote the survival and functional maintenance of neurons.

[0197] In certain embodiments, the neurotrophic factors include nerve growth factor (NGF, e.g., Sigma-Aldrich catalog number N8898; MCE catalog (beta-NGF) #HY-P72488 / HY-P3316 / HY-P70449), brain-derived neurotrophic factor (BDNF), neurotrophin 6 (NT-6), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF, e.g., Sigma-Aldrich catalog number 01-195; MCE catalog (beta-NGF) #HY-P7146 / HY-P7145 / HY-P72943), and / or insulin-like growth factor 2 (IGF2, e.g., APExBio catalog number P1017; MCE catalog number HY-P7019).

[0198] In certain embodiments, the neurotrophic factor is selected from the group including or consisting of BDNF, GDNF, and both.

[0199] According to the present disclosure, the concentration of the neurotrophic factor is not particularly limited as long as it does not interfere with the promotion of mDAP maturation. In certain embodiments, the neurotrophic factor is present in the culture medium at a concentration of about 1 ng / ml to about 100 ng / ml, preferably about 5 ng / ml to about 80 ng / ml, and more preferably about 10 ng / ml to about 50 ng / ml.

[0200] In certain embodiments, BNDF is present in the maturation medium at about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL, or about 10 ng / mL to about 50 ng / mL. In certain embodiments, GDNF is present in the maturation medium at about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL, or about 10 ng / mL to about 50 ng / mL.

[0201] In certain embodiments, the culture medium further comprises (g) a Rho kinase (ROCK) inhibitor.

[0202] ROCK inhibitors include agents that inhibit the binding of ROCK to a ROCK receptor.

[0203] In certain embodiments, the ROCK inhibitor comprises or is selected from the group consisting of Y27632, HA100, HA1152, HA-1077, and any combination thereof.

[0204] Y-27632 is also known as (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (e.g., Sigma-Aldrich). HA100 is also known as 5-(1-piperazinylsulfonyl)-isoquinoline, dihydrochloride. HA1077 is also known as fasudil hydrochloride or 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline hydrochloride (Cayman Chemical). H-1152 is also known as (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (Tocris Bioscience). Other ROCK inhibitors include N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluoromethyl)phenyl)-1,4,5,6-tetrahydropyridine-3-carboxamide (GSK429286A, Stemgent).

[0205] Representative structures of specific ROCK inhibitors that may be used in the maturation media of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources (although such sources are not limiting).

[0206] [ka] Y-27632 (MCE Catalog No. HY-10071, CAS No.: 146986-50-7).

[0207] [ka] HA-100 (hydrochloride salt) (Absin catalog number abs47045575).

[0208] [ka] H1152 (MCE Catalog No. HY-15720, CAS No.: 451462-58-1).

[0209] [ka] HA-1077 (Fasudil / AT877) (MCE Catalog No. HY-10341A, CAS No.: 103745-39-7).

[0210] In certain embodiments, the ROCK inhibitor comprises Y27632. In certain embodiments, the ROCK inhibitor (e.g., Y27632) is present in the maturation medium at about 1 μM to about 50 μM, preferably about 1 μM to about 20 μM, or more preferably about 5 μM to about 15 μM.

[0211] In a specific embodiment, the hPLT is heat-treated human platelet lysate (HhPLT). According to the above embodiment, HhPLT can further improve the maturity of mDAP compared with hPLT. Without wishing to be bound by any particular theory, it is believed that HhPLT can reduce batch-to-batch variation compared with hPLT.

[0212] According to the present disclosure, HhPLT can be produced by subjecting hPLT to heat treatment. In certain embodiments, HhPLT is produced by first centrifuging hPLT, heating the hPLT to approximately 45-60°C, and then centrifuging the hPLT. In certain embodiments, the hPLT is centrifuged at 3000 g for 30 minutes at 4°C to obtain a supernatant, which is then heated to 56°C for 30 minutes. The suspension is finally cooled to 4°C for at least 5 minutes, spun at 3000 g for 30 minutes at 4°C, aliquots are prepared, and stored at -80°C until use.

[0213] According to the present disclosure, the concentration of HhPLT is not particularly limited as long as it does not interfere with the promotion of mDAP maturation. In a specific embodiment, HhPLT is present in the culture medium at a concentration of about 0.1% to about 5% by volume, preferably about 0.1% to about 2% by volume.

[0214] In certain embodiments, the maturation medium of the present disclosure further comprises (h) a WNT signaling pathway inhibitor. According to the above embodiment, the culture medium can further improve the maturation of mDAP compared to a medium that does not contain a WNT signaling pathway inhibitor.

[0215] As used herein, a Wnt signaling pathway inhibitor refers to an antagonist of the Wnt signaling pathway (e.g., an agent that can downregulate the activity and / or amount of a component involved in the Wnt signaling pathway), and can be interchangeably referred to as a "Wnt signaling pathway antagonist," a "Wnt antagonist," a "Wnt pathway inhibitor," or a "Wnt inhibitor." A Wnt signaling pathway inhibitor can include, for example, an agent that antagonizes one or more human FZD proteins, an FZD-binding agent. The FZD-binding agent can be an antibody or a polypeptide.

[0216] Examples of Wnt signaling pathway inhibitors include, but are not limited to, one or more of: a polypeptide comprising the amino acid sequence of a Wnt antagonist; an organic small molecule that inhibits Wnt / β-catenin signaling; an organic small molecule that inhibits the expression or activity of a Wnt agonist; an antibody that binds to and inhibits the activity of a Wnt agonist, preferably an organic small molecule that inhibits Wnt / β-catenin signaling; and an organic small molecule that inhibits the expression or activity of a Wnt agonist.

[0217] In certain embodiments, the WNT signaling pathway inhibitor comprises or is selected from the group consisting of IWR1, iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, ciclopirox, cardamonin, diethylbenzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, XAV-939, fossenvivint (ICG-001), capmatinib, isoquercitrin, gigantol, JW55, MSAB, KY02111, FH535, WIKI4, CCT251545, prodigiosin, KYA1797K, NCB-0846, LF3, iCRT14, adavivint, triptonide, M435-1279, and any combination thereof.

[0218] Representative structures of certain WNT signaling pathway inhibitors that may be used in the maturation media of the present disclosure are provided below, many of which are widely available commercially from multiple sources under the catalog numbers indicated from such selected commercial sources (although such sources are not limiting).

[0219] [ka] IWR1 (MCE catalog number HY-12238, CAS number: 1127442-82-3).

[0220] [ka] iCRT3 (Sigma-Aldrich catalog number SML0211 / 219332; MCE catalog number HY-103705; CAS number: 901751-47-1).

[0221] [ka] IWP-O1 (Sigma-Aldrich catalog number SML1962; MCE catalog number HY-100853; CAS number: 2074607-48-8).

[0222] [ka] IWP-2 (Sigma-Aldrich catalog number I0536; APExBio catalog number A3512; CAS number: 686770-61-6).

[0223] [ka] IWP-3 (APExBio catalog number C3254; MCE catalog number HY-100536; CAS number: 687561-60-0).

[0224] [ka] IWP-4 (Sigma-Aldrich catalog number SML1114; APExBio catalog number B4922; CAS number: 686772-17-8).

[0225] [ka] Ciclopirox (Sigma-Aldrich catalog number SML2011; APExBio catalog number B2087; MCE catalog number HY-B0450; CAS number: 29342-05-0).

[0226] [ka] Cardamonin (Sigma-Aldrich Catalog No. C8249; APExBio Catalog No. B7085; MCE Catalog No. HY-N0279; CAS No.: 18956-16-6).

[0227] [ka] Diethyl benzylphosphonate (Sigma-Aldrich catalog number D91071; CAS number: 1080-32-6).

[0228] [ka] Pamidronate disodium hydrate (Sigma-Aldrich catalog number P2371; APExBio catalog number A2456; MCE catalog number HY-B0012A; CAS number: 57248-88-1.

[0229] [ka] Ginsenoside Rh4 (LeiMeiTian Medicine Catalog No. DR0020; CAS No.: 174721-08-5).

[0230] [ka] KY-05009 (Sigma-Aldrich catalog number SML1506; MCE catalog number HY-05009; CAS number: 1228280-29-2).

[0231] [ka] XAV-939 (Sigma-Aldrich catalog number X3004; APExBio catalog number A1877; MCE catalog number HY-14428; CAS number: 780757-88-2).

[0232] [ka] Fossenvivint (ICG-001) (APExBio Catalog No. A8217; MCE Catalog No. HY-15147; CAS No.: 284028-89-3).

[0233] [ka] Capmatinib (MCE catalog number HY-13404; CAS number: 1029712-80-8).

[0234] [ka] Isoquercitrin (APExBio Catalog No. N1945; MCE Catalog No. HY-N0768; CAS No.: 21637-25-2).

[0235] [ka] Gigantol (Sigma-Aldrich, #SML2036; MCE catalog number HY-N2523; CAS number: 67884-30-4).

[0236] [ka] JW55 (Sigma-Aldrich catalog number SML0630; APExBio catalog number A4529; MCE catalog number HY-13968; CAS number: 664993-53-7).

[0237] [ka] MSAB (Sigma-Aldrich catalog number SML1726; MCE catalog number HY-120697; CAS number: 173436-66-3).

[0238] [ka] KY02111 (Sigma-Aldrich catalog number SML0948; APExBio catalog number A8213; MCE catalog number HY-13815; CAS number: 1118807-13-8).

[0239] [ka] FH535 (Sigma-Aldrich catalog number F5682; APExBio catalog number A3413; MCE catalog number HY-15721; CAS number: 108409-83-2).

[0240] [ka] WIKI4 (APExBio Catalog No. A3413; MCE Catalog No. HY-16910; CAS No.: 838818-26-1).

[0241] [ka] CCT251545 (APExBio catalog number A5979; MCE catalog number HY-12681; CAS number: 1661839-45-7).

[0242] [ka] Prodigiosin (APExBio Catalog No. C3112; MCE Catalog No. HY-100711; CAS No.: 108409-83-2).

[0243] [ka] KYA1797K (Sigma-Aldrich catalog number SML1831; MCE catalog number HY-101090; CAS number: 1956356-56-1).

[0244] [ka] NCB-0846 (MCE catalog number HY-100830; CAS number: 1792999-26-8).

[0245] [ka] LF3 (Sigma-Aldrich catalog number SML1752; MCE catalog number HY-101486; CAS number: 1956356-56-1).

[0246] [ka] iCRT14 (TOCRIS (R&D) catalog number 677331-12-3).

[0247] [ka] Adavivint (MCE catalogue number HY-109049; CAS number: 1467093-03-3).

[0248] [ka] Triptonide (Sigma-Aldrich Catalog No. SMB00325; APExBio Catalog No. A3892; MCE Catalog No. HY-32736; CAS No.: 38647-11-9).

[0249] [ka] M435-1279 (MCE Catalog No. HY-141891; CAS No.: 1359431-16-5).

[0250] In certain embodiments, the Wnt signaling pathway inhibitor comprises IWR1.

[0251] In certain embodiments, a Wnt signaling pathway inhibitor (eg, IWR1) is present in the maturation medium at about 0.25 μM to about 10 μM, preferably about 0.5 μM to about 5 μM.

[0252] In certain embodiments, the maturation culture medium of the present disclosure comprises (a) about 0.1% to about 2% by volume of hPLT or HhPLT; (b) about 0.1 ng / mL to about 5 ng / mL of TGF-β; (d) about 5 μM to about 20 μM of a γ-secretase inhibitor; (d) about 0.1 mM to about 2.5 mM of a cAMP-based compound or a cyclase activator thereof; and (e) about 10 ng / mL to about 80 ng / mL of a neurotrophic factor in a neurobasal medium.

[0253] In certain embodiments, the maturation culture medium of the present disclosure comprises (a) about 0.1% to about 2% by volume of hPLT or HhPLT; (b) about 0.1 ng / mL to about 5 ng / mL of TGF-β; (d) about 5 μM to about 20 μM of a γ-secretase inhibitor; (d) about 0.1 mM to about 2.5 mM of a cAMP-based compound or a cyclase activator thereof; (e) about 10 ng / mL to about 80 ng / mL of a neurotrophic factor; and (f) about 5 μM to about 15 μM of a ROCK inhibitor in a neurobasal medium.

[0254] In certain embodiments, the maturation culture medium of the present disclosure comprises (a) about 0.1% to about 2% by volume of hPLT or HhPLT; (b) about 0.1 ng / mL to about 5 ng / mL of TGF-β; (d) about 5 μM to about 20 μM of a γ-secretase inhibitor; (d) about 0.1 mM to about 2.5 mM of a cAMP-based compound or a cyclase activator thereof; (e) about 10 ng / mL to about 80 ng / mL of a neurotrophic factor; (f) about 5 μM to about 15 μM of a ROCK inhibitor; and (g) about 0.5 μM to about 5 μM of a WNT signaling pathway inhibitor in a neurobasal medium.

[0255] In certain embodiments, the maturation culture medium of the present disclosure comprises (a) about 0.1% to about 2% by volume of hPLT or HhPLT; (b) about 0.1 ng / mL to about 5 ng / mL of TGF-β; (c) about 5 μM to about 20 μM of DAPT; (d) about 0.1 mM to about 2.5 mM of Db-cAMP sodium salt; and (e) about 10 ng / ml to about 80 ng / ml of a combination of BDNF and GDNF in a neurobasal medium.

[0256] In certain embodiments, the maturation culture medium of the present disclosure comprises (a) about 0.1% to about 2% by volume of hPLT or HhPLT; (b) about 0.1 ng / mL to about 5 ng / mL of TGF-β; (c) about 5 μM to about 20 μM of DAPT; (d) about 0.1 mM to about 2.5 mM of Db-cAMP sodium salt; (e) about 10 ng / mL to about 80 ng / mL of a combination of BDNF and GDNF; and (f) about 5 μM to about 15 μM of Y27632 in a neurobasal medium.

[0257] In certain embodiments, the maturation culture medium of the present disclosure comprises (a) about 0.1% to about 2% by volume of hPLT or HhPLT; (b) about 0.1 ng / mL to about 5 ng / mL of TGF-β; (c) about 5 μM to about 20 μM of DAPT; (d) about 0.1 mM to about 2.5 mM of Db-cAMP sodium salt; (e) about 10 ng / mL to about 80 ng / mL of a combination of BDNF and GDNF; (f) about 5 μM to about 15 μM of Y27632; and (g) about 0.5 μM to about 5 μM of IWR1 in a neural basal medium.

[0258] In certain embodiments, the maturation medium of the present disclosure may optionally further comprise glutamine or a derivative thereof.

[0259] In certain embodiments, the glutamine or derivative thereof comprises L-alanyl-L-glutamine dipeptide (e.g., GLUTAMAX™ brand L-alanyl-L-glutamine dipeptide, Gibco catalog number 35050061), L-glutamine (e.g., Sigma-Aldrich catalog numbers G2150 / G7513; APExBio catalog number A8461; MCE catalog number HY-N0390; CAS number: 56-85-9), or a mixture thereof.

[0260] In certain embodiments, glutamine or a derivative thereof is present in the maturation medium of the present disclosure at a concentration of about 0.5% to 5% by volume, preferably about 0.5% to 2.5% by volume.

[0261] In certain embodiments, the maturation medium of the present disclosure may optionally further comprise an antioxidant.

[0262] In certain embodiments, the antioxidant comprises ascorbic acid (e.g., Sigma catalog number A8960) or a salt thereof (e.g., Na salt, Mg salt) or a derivative thereof, SOD (e.g., Sigma catalog numbers S7571, S9697, S5395, S8160, S9636, S8409, S7446, CAS number: 9054-89-1), or a mixture thereof.

[0263] In certain embodiments, an antioxidant (eg, ascorbic acid) is present in the maturation medium of the present disclosure at a concentration of about 50 μM to about 500 μM, preferably about 100 μM to about 400 μM, and more preferably about 100 μM to about 300 μM.

[0264] In certain embodiments, the maturation medium can improve the expression of TH. In certain embodiments, the maturation medium can improve the expression of TH and one or more selected from LMX1A, EN1, OTX2, FOX2, NURR1, and SOX6. In certain embodiments, the maturation medium can improve the expression of TH, LMX1A, EN1, OTX2, FOX2, NURR1, and SOX6.

[0265] 5. Methods for maturing mDAP In a fifth aspect, the present disclosure provides a method for promoting maturation of midbrain dopaminergic progenitor cells (mDAP), the method comprising contacting mDAP with a maturation medium containing a ROCK inhibitor on a culture surface coated with a combination of coating matrices including: (a) a first coating matrix capable of supporting cell adhesion of mDAP, the first coating matrix being other than laminin; and (b) a second coating matrix capable of improving maturation of mDAP, the second coating matrix comprising a polylysine (PL)-based compound and / or a polyornithine (PO)-based compound.

[0266] Existing maturation methods generally use a combination of laminin / PLO as a matrix for mDA neuron maturation, but the cost is too high for large-scale mDA neuron production. According to the fifth aspect, by using a ROCK inhibitor together with the first and second coating matrices, the above maturation method can improve the maturation of mDA neurons (the percentage of TH+ cells) while reducing production costs. Furthermore, the maturation method of the present disclosure can achieve a yield equivalent to that of the maturation method using laminin / PLO. Therefore, the maturation method of the present disclosure is more applicable to large-scale mDA neuron production.

[0267] Any mDAP may be matured using the maturation method of the present disclosure. Examples of mDAP include fetal brain-derived mDAP; hPSC-derived mDAP; and mDAP obtained via transdifferentiation from other cell types. mDAP may be derived (e.g., differentiated) from pluripotent stem cells. Pluripotent stem cells may include induced pluripotent stem cells (e.g., hiPSCs), embryonic stem cells (e.g., hESCs), naive PSCs (NPSCs), and extended pluripotent stem cells (EPSCs). In certain embodiments, the mDAP are ESC-derived midbrain dopaminergic progenitor cells (emDAPs). In certain embodiments, the mDAP are iPSC-derived midbrain dopaminergic progenitor cells (imDAPs). In certain embodiments, the mDAP are NPSC-derived midbrain dopaminergic progenitor cells (nmDAPs). In certain embodiments, the mDAP are EPSC-derived midbrain dopaminergic progenitor cells (epmDAPs).

[0268] In certain embodiments, the mDAP is a non-proliferating mDAP. In certain embodiments, the mDAP is a proliferating mDAP, such as a proliferating imDAP. In certain embodiments, the mDAP is a mDAP that has been propagated and passaged one or more times, such as a proliferating P1, P2, P3, P4, P5, or P6 imDAP. In certain embodiments, the mDAP is an immature mDAP. In certain embodiments, the mDAP is a partially mature mDAP.

[0269] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be easily obtained using conventional methods, such as those described in existing technologies, or commercially available products. For example, the CytoTune iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) can be used to reliably generate induced pluripotent stem cells (iPSCs) from somatic cells, including PBMCs and T cells.

[0270] Such iPSCs (e.g., hiPSCs) can be cultured under defined conditions to generate mDAP. For example, iPSCs (e.g., hiPSCs) can be cultured on Matrigel under defined conditions in mTeSR™ medium (Stemcell Technologies, catalog no. 85850). Subconfluent hiPSCs can be passaged onto fresh Matrigel-coated plates and cultured in iPSC medium for an additional period (e.g., 24 hours) to reach 90-100% confluence. Once confluent, the medium can be changed to induce mDAP for an additional period (e.g., 1-3 days), optionally with daily medium changes as needed. See, for example, Fedele et al., Scientific Reports 7:6036 | DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0271] According to the present disclosure, the ROCK inhibitor-containing maturation medium is not particularly limited as long as it contains a ROCK inhibitor. The above-mentioned maturation medium can be obtained by adding a ROCK inhibitor to a common mDAP maturation medium in the art or any other suitable mDAP maturation medium. Examples of common mDAP maturation media include Gibco™ PSC Dopaminergic Neuron Differentiation Kit and STEMdiff™ Dopaminergic Neuron Differentiation Kit / STEMdiff™ (#08530).

[0272] In certain embodiments, the maturation medium of the present disclosure is used as a ROCK inhibitor-containing maturation medium.The maturation medium of the present disclosure used in the above embodiments is described elsewhere herein (for example, as the culture medium for maturing mDAP herein or described in the fourth aspect), and these same descriptions are omitted herein for brevity.According to the above embodiments, the maturation method can further improve the maturity (the percentage of TH+ cells) of mDAP.

[0273] In certain embodiments, the culture surface is in a culture plate, a culture bottle, a culture flask, or a culture vessel. In certain embodiments, the culture plate comprises a single or multi-layer cell stack, 6 wells, 12 wells, 24 wells, 48 ​​wells, 96 wells, 384 wells, 1536 wells, or more wells. In certain embodiments, the culture plate comprises flat-bottom or round-bottom wells.

[0274] The coating matrix combination used in the fifth embodiment of the present disclosure includes a first coating matrix and a second coating matrix. This first coating matrix is ​​not particularly limited as long as it is not laminin and can support cell adhesion of mDAP. This second coating matrix can improve the maturation of mDAP and includes a polylysine (PL)-based compound and / or a polyornithine (PO)-based compound.

[0275] In certain embodiments, the first coating matrix comprises or is selected from the group consisting of vitronectin (VTN), collagen, proteoglycan, fibronectin, entactin, elastin, functional fragments of any of the foregoing proteins, hyaluronic acid, gelatin, and any combination thereof. In certain embodiments, the first coating matrix comprises VTN.

[0276] According to the present disclosure, polylysine-based compounds may include polylysine, its derivatives, or salts thereof, all of which can function to improve the maturation of mDAP. Examples of polylysine include, but are not limited to, poly-L-lysine and poly-D-lysine. Examples of polylysine derivatives include, but are not limited to, substituted polylysines such as TAMRA-PEG-polylysine and polylysine-PEG-polylysine. Examples of salts of polylysine or its derivatives include, but are not limited to, poly-L-lysine hydrobromide, poly-L-lysine hydrochloride, and salts of substituted polylysines.

[0277] According to the present disclosure, the polyornithine-based compound may include polyornithine, its derivatives, or its salts, all of which can function to improve the maturation of mDAP. Examples of polyornithine include, but are not limited to, poly-L-ornithine and poly-D-ornithine. Examples of polyornithine derivatives include, but are not limited to, substituted polyornithines, such as DBCO-PEG-polyornithine and chitosan-PEG-polyornithine, Con A-PEG-polyornithine, alginate-PEG-polyornithine, and vitamin E-PEG-polyornithine. Examples of polyornithine salts or their derivatives include, but are not limited to, poly-L-ornithine hydrobromide, poly-L-ornithine hydrochloride, and substituted polyornithine salts.

[0278] In certain embodiments, the second coating matrix comprises poly-L-lysine hydrobromide, poly-L-ornithine hydrobromide, or a mixture thereof. Representative structures of second coating matrices that can be used in the maturation methods of the present disclosure are provided below, many of which are widely available commercially from multiple sources, with catalog numbers from selected commercial sources indicated.

[0279] [ka] Poly-L-ornithine hydrobromide (Sigma catalog number P3655, CAS number: 27378-49-0).

[0280] [ka] Poly-L-lysine hydrobromide (Sigma Cat #P2636, CAS number: 25988-63-0).

[0281] In certain embodiments, the coating matrix combination comprises VTN and one or two of poly-L-lysine hydrobromide and poly-L-ornithine hydrobromide.

[0282] A cell culture surface may be coated with the mature coating matrix combination of the present disclosure by conventional techniques in the art. Generally, the cell culture surface may be first coated with a coating solution containing the second coating matrix, followed by a coating solution containing the first coating matrix. Optionally, a washing process (e.g., using DPBS) may be performed after the cell culture surface is coated with the second coating matrix. The coating concentrations of the first and second coating matrices can be easily determined by those skilled in the art. For example, the coating concentration of the first coating matrix may be 0.5 to 2 μg / cm, respectively. 2 , e.g., 1 μg / cm 2 The coating concentration of the first coating matrix may be 1 to 2 μg / cm 2 , e.g., 1 μg / cm 2 may be.

[0283] In certain embodiments, the mDAP is about 1×10 3 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 (For example, about 5 × 10 5 cells / cm 2 ) onto the culture surface at a density of 1000 μg / cm².

[0284] In certain embodiments, the culture surface is in a culture plate, a culture bottle, a culture flask, or a culture vessel. In certain embodiments, the culture plate comprises a single or multi-layer cell stack, 6 wells, 12 wells, 24 wells, 48 ​​wells, 96 wells, 384 wells, 1536 wells, or more wells. In certain embodiments, the culture plate comprises flat-bottom or round-bottom wells.

[0285] In certain embodiments, the mDAP is contacted with the ROCK inhibitor-containing maturation medium for a total of about 6 days.

[0286] In certain embodiments, the ROCK inhibitor-containing maturation medium does not contain a Wnt signaling pathway inhibitor, and the method further comprises replacing the maturation medium with the same medium every few days during maturation. In certain embodiments, the ROCK inhibitor-containing maturation medium does not contain a Wnt signaling pathway inhibitor, and the method further comprises replacing the maturation medium with the same medium every three days during maturation.

[0287] In certain embodiments, the ROCK inhibitor-containing maturation medium comprises a Wnt signaling pathway inhibitor, and the method further comprises replacing the maturation medium every few days with the same medium without the Wnt signaling pathway inhibitor. In certain embodiments, the ROCK inhibitor-containing maturation medium comprises a Wnt signaling pathway inhibitor, and the method further comprises replacing the maturation medium every three days with the same medium without the Wnt signaling pathway inhibitor.

[0288] In certain embodiments, the maturation method can improve the expression of TH. In certain embodiments, the maturation method can improve the expression of TH and one or more selected from LMX1A, EN1, OTX2, FOX2, NURR1, and SOX6. In certain embodiments, the maturation method can improve the expression of TH, LMX1A, EN1, OTX2, FOX2, NURR1, and SOX6.

[0289] 6. Cell Populations By using the proliferation method of the present disclosure, a substantially uniform population of mDAPs can be provided. Thus, in a sixth aspect, the present disclosure provides a substantially uniform population of mDAPs produced by the proliferation method of the present disclosure. The proliferation method of the present disclosure is described elsewhere herein (e.g., in the method for propagating mDAPs or in the third aspect of the present disclosure), and these same descriptions are omitted herein for brevity. The substantially uniform population of mDAPs can be propagated P1, P2, P3, P4, P5, or P6 imDAPs. Proliferated imDAPs at early and late passages can maintain similar high ability to differentiate into mDA neurons.

[0290] In certain embodiments, the mDAPs in the population express FOXA2. In certain embodiments, the mDAPs in the population express FOXA2 and one or more of OTX2, SOX6, LMX1A, and EN1. In certain embodiments, the mDAPs in the population express FOXA2, OTX2, SOX6, LMX1A, and EN1. In certain embodiments, at least about 75%, 80%, 85%, 90%, 95% or more of the mDAPs in the population express FOXA2. In certain embodiments, the substantially homogeneous population of mDAPs is P1, P2, P3, P4, P5, or P6 mDAPs.

[0291] In certain embodiments, populations of mDAPs of the present disclosure can be cryopreserved or preserved for further expansion, maturation and / or differentiation.

[0292] By using the maturation method of the present disclosure from mDAP, a substantially uniform population of mDA neurons (mDAN) can be provided. Thus, in a seventh aspect, the present disclosure provides a substantially uniform population of mDAN produced by the maturation method of the present disclosure. The maturation method of the present disclosure is described elsewhere herein (for example, in the method for maturing mDAP or in the fifth aspect of the present disclosure), and these same descriptions are omitted here for brevity. mDAN can be in early or late stages of maturation. mDAN in the early stage of maturation has the potential for final maturation.

[0293] In certain embodiments, the mDANs in the population express TH and FOXA2. In certain embodiments, the mDANs in the population express TH, FOXA2, and one or more of NURR1, SOX6, LMX1A, and EN1. In certain embodiments, the mDANs in the population express TH, FOXA2, NURR1, SOX6, LMX1A, and EN1. In certain embodiments, about 33%-55%, 33-51%, 33-42%, or 45-51% of the mDANs in the population express TH.

[0294] The population of mDANs of the present disclosure can be cryopreserved or stored prior to use. The mDANs can be used for further drug research, drug screening, and clinical and therapeutic uses.

[0295] 7. Kit In an eighth aspect, the present disclosure provides a kit comprising the growth medium of the present disclosure described herein. The growth medium of the present disclosure is also described elsewhere herein (e.g., as described in the culture medium for growing mDAP herein or in the first aspect), and these same descriptions are omitted herein for brevity. Optionally, the kit may further comprise a combination of the growth coating matrices of the present disclosure described herein. The combination of the growth coating matrices of the present disclosure is described elsewhere herein (e.g., as described in the combination of coating matrices for growing mDAP herein or in the second aspect), and these same descriptions are omitted herein for brevity. When the kit comprises a combination of the growth medium and the growth coating matrix, they are packaged separately.

[0296] In a ninth aspect, the present disclosure provides a kit comprising the maturation medium of the present disclosure described herein. The maturation medium of the present disclosure is described elsewhere herein (e.g., in the culture medium for maturing mDAP herein or in the fourth aspect), and these same descriptions are omitted herein for brevity. Optionally, the kit may further comprise a combination of the maturation coating matrices of the present disclosure described herein. The combination of the maturation coating matrices of the present disclosure is described elsewhere herein (e.g., in the method for maturing mDAP herein or in the fifth aspect), and these same descriptions are omitted herein for brevity. When the kit comprises a maturation medium and a combination of the maturation coating matrices, they are packaged separately.

[0297] It is to be understood that any aspect or embodiment of the disclosure described herein (including those described only in the examples or claims) may be combined with any one or more other aspects and / or embodiments of the disclosure unless such combination is inappropriate or expressly disclaimed. [Example]

[0298] 1.Material All reagents and equipment utilized throughout the examples of this disclosure are commercially available, and sources of these reagents and equipment are described elsewhere herein.

[0299] 2. General test methods (1) Flow cytometry analysis 1. Cells are singulated and collected by either Accutase or TrypLE treatment. 2. Wash the cells with 1 ml of PBS and pellet the cells at 250 x g for 15 seconds. 3. Remove the supernatant, add approximately 200 μl of 4% PFA, mix, and fix at room temperature for 10 minutes. 4. Pellet the cells at 350 x g for 5 minutes. 5. Wash the cells with 1 ml of FACS buffer and pellet the cells at 350 x g for 5 minutes. 6. Remove the supernatant and add 200 μl / tube of FACS buffer / 0.1% TritonX and mix for 10 minutes at room temperature. 7. Wash the cells with 1 ml of FACS buffer and pellet the cells at 350 x g for 5 minutes. 8. Remove the supernatant and add 200 μl / tube of primary antibody diluted in FACS buffer, mix gently and incubate at room temperature for 30 minutes. 9. Wash the cells with 1 ml of FACS buffer and pellet the cells at 350 x g for 5 minutes. 10. Remove the supernatant and add 200 μl of secondary antibody diluted in FACS buffer, mix gently and incubate for 30 minutes at room temperature. 11. Wash the cells with 1 ml of FACS buffer and pellet the cells at 300 x g for 5 minutes. 12. Remove the supernatant, add 200 μl of FACS buffer and run the samples on a flow cytometer. (2) qRT-PCR 1. For qRT-PCR analysis of mRNA gene expression, RNA was collected from at least 2 x 10 6 It should be taken from individual cells. 2. Cells are harvested, 350 μl of Buffer RLT+1% (vol / vol) β-mercaptoethanol is added to the wells, and the lysates are transferred to 1.5 ml tubes and immediately placed on ice. 3. Perform RNA extraction using the Tiangen Kit (DP430) and follow the manufacturer's protocol "Purification of total RNA from animal and human cells." 4. Once the RNA extraction is complete, determine the RNA concentration using the Nanodrop instrument. 5. Perform a first-strand cDNA synthesis reaction with 1 μg of RNA using the HiScript™ II Q RT SuperMIX for qPCR (+ gDNA wiper) kit for RT-qPCR (Vazyme, #R223-01). Follow the manufacturer's "First-Strand cDNA Synthesis" protocol. This reaction will yield 20 μl of product. 6. Dilute 10 μl of the cDNA product into 80 μl of nuclease-free water. 7. Pipette the diluted cDNA product (1 μl), Sybr Green Master Mix (5 μl, Transgen, #AQ131), and 0.95 μM reverse / forward primer mix (4 μl) into a 384-well PCR plate in triplicate using either manual or automated pipetting. The complete primer panel included the target gene and a housekeeping gene (RPL13A). This analysis also included a sample of undifferentiated hPSCs to determine gene expression levels relative to expression in hPSCs. 8. Analyze samples by quantitative PCR on a LightCycler 480 instrument using a two-step protocol with 40 cycles, including a 60-second annealing / extension step at 60°C and a 30-second denaturation step at 95°C. The average CT values ​​from three technical replicates were used to calculate relative gene expression using the △△CT method. For each gene, calculate the FC of the differentiated sample relative to the undifferentiated control sample using a housekeeping gene for normalization. The average FC value is then calculated based on the housekeeping gene. (3) Immunocytochemical analysis 1. Cell fixation: Remove the medium from the cells and wash with PBS. Add 4% (wt / vol) PFA (e.g., 200 μl per well in a 48-well plate) and incubate for 15 minutes at room temperature (RT). After incubation, wash the cells three times in PBS. 2. Blocking: Remove PBS from the wells and add blocking solution to the cells, adding a volume sufficient to cover the cells. Leave the cells in blocking solution for 1 hour at room temperature. 3. Primary antibody incubation: Remove the blocking solution from the cells and incubate with primary antibody solution (approximately 100 μl / cm 2 ) and incubate on a shaker at room temperature for 1 hour. 4. Secondary antibody incubation: Remove the primary antibody solution and wash the cells three times in PBS. Add the secondary antibody solution to the cells (approximately 100 μl / cm 2 ), wrap the plate in aluminum foil to avoid bleaching of the fluorophores, and incubate for 1 h at room temperature on a shaker. 5. DAPI incubation: Remove the secondary antibody solution and wash the cells twice in PBS. Add DAPI solution (1:1000) to the cells (approximately 100 μl / cm 2 ), wrap the plate in aluminum foil to avoid bleaching of the fluorophores, and incubate for 10 min at room temperature on a shaker. 6. Wash cells three times in PBS and keep wrapped in aluminum foil at 4°C until analysis. 7. Analyze immunocytochemically stained cells using a fluorescent microscope and estimate the number of positive cells.

[0300] Examples 1-5: Development and validation of imDAP growth medium based on the combination of LDN193189 / CHIR99021 / FGF8b / blebbistatin We have previously shown that LDN193189, CHIR99021, blebbistatin, and FGF8b are required for the growth of imDAPs as a homogeneous population. However, it was unclear whether imDAPs propagated in a growth medium supplemented with blebbistatin, LDN193189, CHIR99021, and FGF8b retained their properties. This example demonstrates that a growth medium based on this combination can propagate imDAPs with limited growth efficiency and, in particular, cannot support long-term growth of imDAPs while maintaining their specific phenotype.

[0301] Example 1 imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. imDAP (P0) was cultured at 1 × 10 in growth medium (50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 3 μM CHIR99021, 100 ng / mL FGF8b) in the absence or presence of 5 μM blebbistatin. 5 cells / cm 2 VTN coated plates at a density of 1 μg / cm 2 ) were seeded. When blebbistatin was present, it was either removed 24 hours after plating or was present throughout the culture period. The medium was changed daily for 5 days.

[0302] The results are shown in Figure 1A. Figure 1A (left image) shows the morphology of cells grown in the absence of blebbistatin. Figure 1A (center image) shows the morphology of cells grown when blebbistatin was removed 24 hours after plating, and Figure 1A (right image) shows the morphology of cells grown when blebbistatin was present throughout the culture period. Clearly, the addition of blebbistatin throughout the culture supported better imDAP growth compared to medium without blebbistatin or medium to which blebbistatin was added only on the day of plating.

[0303] Example 2 imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. imDAP (P0) was cultured at 1 × 10 in growth medium (50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 3 μM CHIR99021, 100 ng / mL FGF8b, 5 μM blebbistatin) in the absence or presence of 0.2 μM LDN193189. 5 cells / cm 2 VTN coated plates at a density of 1 μg / cm 2 The medium was changed every day for 5 days.

[0304] The results are shown in Figure 1B. It was clear that imDAP cultured without LDN193189 exhibited a flatter morphology compared to those cultured with LDN193189.

[0305] Example 3 imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. 1 × 10 imDAP (P0) was cultured in a growth medium (50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 2% (v / v) B27, 0.2 μM LDN193189, 100 ng / mL FGF8b, 5 μM blebbistatin) containing different concentrations of CHIR99021 (0, 1.25, 3 μM). 5 cells / cm 2 VTN coated plates at a density of 1 μg / cm 2 ) and the medium was changed daily for 5 days. The cells were collected for RT-qPCR assay.

[0306] The results are shown in Figure 1 C. qRT-PCR analysis of expanded imDAPs showed that treatment with 3 μM CHIR99021 during growth allowed better maintenance of mDAP marker expression (EN1, LMX1A, FOXA2).

[0307] Example 4 imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. imDAP (P0) was cultured at 1 × 10 in growth medium (50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 3 μM CHIR99021, 5 μM blebbistatin) in the absence or presence of 100 ng / mL FGF8b. 5 cells / cm 2 VTN coated plates at a density of 1 μg / cm 2 ) and the medium was changed daily for 5 days. The cells were collected for RT-qPCR assay.

[0308] The results are shown in Figure 1 D. qRT-PCR analysis of expanded imDAPs showed that treatment with FGF8b during proliferation allowed for improved mDAP marker expression (EN1, and LMX1A) compared to treatment without FGF8b, but no improvement in FOXA2 expression was observed.

[0309] Example 5 imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. ImDAP (P0) was cultured at 1 × 10 cells / well in growth medium (50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 3 μM CHIR99021, 100 ng / mL FGF8b, 5 μM blebbistatin). 5 cells / cm 2 VTN coated plates at a density of 1 μg / cm 2 ) were seeded onto the imDAP. The medium was changed daily. When the imDAP reached 100% confluence (4–5 days), the cells were enzymatically passaged with 1× TrypLE, centrifuged at 250 g for 5 min, counted, and plated at 1×10 in growth medium. 5 cells / cm 2 New VTN coated plates at a density of 1 μg / cm 2) and replaced the imDAPs. The imDAPs were serially propagated for six passages. At each passage, imDAPs were harvested at a proliferation fold of 5-10 (relative to the initial number of imDAPs plated).

[0310] Typical cell morphology at each passage (P1–P6) is shown in Figure 1E. The population doubling time (PDT) at each passage (P1–P5) is shown in Figure 1F. From P1 to P3, the PDT remained high at approximately 48 h, indicating limited proliferation efficiency. From P3 onward, it was clear that imDAP proliferation began to slow significantly. Figure 1G shows the percentage of Foxa2-expressing cells at P0, P1, and P2. The results showed that the percentage of Foxa2-expressing cells decreased over passages, particularly after two passages using imDAP growth medium based on the combination of LDN193189 / CHIR99021 / FGF8b / blebbistatin. Figure 1H shows the RT-qPCR results for mDAP-specific markers (EN1, LMX1A, and FOXA2) at each passage. Expression of LMX1A / FOXA2 / EN1, especially FOXA, significantly decreased over passages.

[0311] Example 6 Y27632 promoted imDAP proliferation This example demonstrated that growth medium supplemented with Y27632 can improve Foxa2 expression in imDAP cells more than growth medium supplemented with blebbistatin.

[0312] imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. imDAP (P0) was applied to VTN-coated plates (1 μg / cm 2 ) 1×10 on top 4 cells / cm 2Cells were seeded at a low density of 1000 x 1000 x 1000 and cultured in growth medium (50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 3 μM CHIR99021, 100 ng / mL FGF8b) containing either 5 μM blebbistatin or 10 μM Y27632. The medium was replaced every 3 days. On day 6, cells were collected for FACS analysis.

[0313] The results are shown in Figure 2. The data clearly showed that growth medium supplemented with Y27632 could improve the percentage of Foxa2-expressing cells more than blebbistatin. However, on the other hand, even though Y27632 could improve Foxa2 expression compared with blebbistatin, the percentage of Foxa2-expressing cells still decreased during passaging.

[0314] Example 7 NOTCH2 / TGFβ2 Enriched in imDAP This example demonstrated that NOTCH2 / TGFβ2 was enriched in imDAPs. Manipulation of these signaling pathways may enable efficient imDAP expansion while maintaining the mDAP phenotype.

[0315] Single cells were encapsulated in droplets using 10x Genomics Technology and processed according to the following procedure. Briefly, every cell and every transcript was uniquely barcoded using a unique molecular identifier (UMI). Libraries were generated and sequenced from the cDNA, and the 10x barcodes were used to associate individual reads with individual partitions. The cDNA was then pooled together and amplified and sequenced according to standard procedures for Illumina-compatible sequencing libraries.

[0316] For downstream analysis after initial Cell Ranger metric evaluation, cells with low-quality sequencing data were removed. Genes with three or more reads in all cells were considered ubiquitously expressed in all cell types in the sample and were therefore removed before further analysis. Cells from the following brain regions appeared to be absent from the sample: forebrain, diencephalon, posterior midbrain, lateral midbrain, and hindbrain r2 and r3. Cell clustering was performed in the following order: (1) midbrain-hindbrain boundary (MHB, FGF8+), (2) hindbrain r1 (HB r1, GBX2+), (3) midbrain basal plate (MBP, OTX2+ / NKX6.1+, OTX2+ / NKX2.2+, OTX2+ / PITX2+), (4) midbrain caudal basal plate (MFP, EN1+ / LMX1A+ / FOXA2+ / OTX2+), and (5) remaining cells. The remaining cells were then grouped into one of the first four clusters with the most similar overall gene expression pattern. For visualization and clustering, the manifold was calculated using the UMAP method (Figure 3A). Using differentially expressed gene signatures, NOTCH2 / TGFβ2 was found to be enriched in MFP cells (Figure 3B). Manipulation of these signaling pathways may enable efficient imDAP proliferation while maintaining the mDAP phenotype.

[0317] Example 8 NOTCH activation and / or TGF-β inhibition supported better imDAP proliferation This example showed that NOTCH activation (eg, using DLL4) and TGF-β inhibition (eg, using SB431542) supported better proliferation of imDAP.

[0318] imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. imDAP (P0) was prepared by coating DLL4 (1 μg / cm 2 ), VTN coating (1 μg / cm 2 ) and DLL4 / VTN coating (1 μg / cm each) 2 ) plates, 1 x 10 4 cells / cm 2The cells were seeded at a low density of 100 μg / ml and cultured in growth medium in the absence or presence of 5 μM SB431542. The medium was replaced every 3 days until 100% confluence was reached (6 days) for cell harvest. Typical cell morphology was recorded on day 4 (Figure 4A). The number of cells in each group was counted using a Vi-cell cytometer (Figure 4B). Cells in each group were stained with Foxa2-PE antibody (BD, #561589), and the percentage of Foxa2+imDAP was analyzed by flow cytometry (Figure 4C). The growth medium used in this example contained the following components: 50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) B27, 50 μg / ml ascorbic acid, 0.2 μM LDN193189, 100 ng / ml FGF8b, 3 μM CHIR99021 and 10 μM Y27632.

[0319] Here, we investigated the effects of DLL4 and SB431542 on supporting the growth of imDAP as a uniform population. The results are shown in Figure 4. Specifically, Figure 4A shows that DLL4 alone cannot support the attachment of imDAP. imDAP on VTN exhibited a flat morphology. imDAP cultured on surfaces coated with both VTN and DLL4 were more uniform during growth. Figure 4B shows that DLL4 alone, SB431542 alone, or their combination significantly improved imDAP growth efficiency (approximately 32-fold or more) compared to growth without both DLL4 and SB431542 (approximately 20-fold or more). In Figure 4C, comparing the results of -DLL4+VTN / -SB with those of -DLL4+VTN / +SB and +DLL4+VTN / -SB with those of +DLL4+VTN / +SB showed that SB431542 alone could improve FOXA2 expression. Comparing the results of -DLL4+VTN / -SB with those of +DLL4+VTN / -SB and +DLL4+VTN / +SB showed that DLL4 alone could improve FOXA2 expression. Notably, the combination of DLL4 / VTN and SB431542 maintained the best FOXA2 expression during proliferation.

[0320] Example 9 LDN193189, FGF8b and / or FGF2 had no additional effect on imDAP proliferation To further demonstrate whether LDN193189, FGF8b, and FGF2 are essential for imDAP proliferation, in this example, the effects of LDN193189, FGF8b, and FGF2 on imDAP proliferation were examined.

[0321] imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. imDAP (P0) was prepared at 1 × 10 4 cells / cm 2 DLL4 / VTN coating at low density (1 μg / cm each) 2) plates and cultured in growth media (EM1, EM2, EM3, EM4). The medium was replaced every 3 days until 100% confluence was reached (6 days) for cell collection. EM1 used in Example 9 contained the following components: 50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) B27 (50x), 50 μg / ml ascorbic acid, 3 μM CHIR99021, 5 μM SB431542, and 10 μM Y27632. The EM2 used in Example 9 contained the following components: 50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) B27 (50x), 50 μg / ml ascorbic acid, 0.2 μM LDN193189, 3 μM CHIR99021, 5 μM SB431542 and 10 μM Y27632. The EM3 used in Example 9 contained the following components: 50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) B27 (50x), 50 μg / mL ascorbic acid, 0.2 μM LDN193189, 100 ng / ml FGF8b, 3 μM CHIR99021, 5 μM SB431542 and 10 μM Y27632. The EM4 used in Example 9 contained the following components: 50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) B27 (50x), 50 μg / ml ascorbic acid, 0.2 μM LDN193189, 10 ng / ml FGF2, 3 μM CHIR99021, 5 μM SB431542 and 10 μM Y27632.

[0322] Figure 5A shows typical cell morphology on day 5. As shown in Figure 5A, when cells were cultured with FGF2, some obvious heterogeneous cells were observed. The number of cells in each group was counted using a Vi-cell cytometer (Figure 5B). As shown in Figure 5B, the addition of LDN193189 and / or FGF8b had no additional effect on increasing the number of proliferating cells. Cells in each group were stained with Foxa2-PE antibody (BD, #561589), and the percentage of Foxa2+ imDAPs was analyzed by flow cytometry (Figure 5C). As shown in Figure 5C, compared with P0 cells, growth medium without LDN193189 and FGF8b maintained or even improved Foxa2 expression in P1 cells, and growth medium containing LDN193189 and / or FGF8b still maintained or even improved Foxa2 expression in P1 cells, whereas the addition of FGF8b and / or LDN193189 slightly reduced FOXA2 expression in imDAP cells. The results demonstrated that LDN193189, FGF8b, and / or FGF2 had no additional effect on imDAP proliferation and are not essential for imDAP proliferation.

[0323] Example 10 NOTCH activation in combination with TGF-β inhibition supported imDAP long-term proliferation This example demonstrated that the novel combination of CHIR99021, SB431542, and Y27632 supports long-term growth of imDAP on VTN / DLL4-coated surfaces.

[0324] imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0. imDAP (P0) was cultured at 1 × 10 4 cells / cm 2 DLL4 / VTN coating at low density (1 μg / cm each) 2 ) plates. The medium was replaced every 3 days until 100% confluence was reached (6 days) for cell passage. imDAP were enzymatically passaged with 1x TrypLE, centrifuged at 250g for 5 minutes, counted, and cultured at 1x10 in growth medium.4 cells / cm 2 ImDAP cells were plated onto new DLL4 / VTN-coated plates at a density of 1000 μg / ml. The imDAP cells were expanded and maintained in growth medium for six passages. The growth medium used in Example 10 contained the following components: 50% DMEM / F12, 50% Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) B27 (5x), 50 μg / mL ascorbic acid, 3 μM CHIR99021, 5 μM SB431542, and 10 μM Y27632.

[0325] Figure 6A shows typical cell morphology at P3 on day 5. Cells from each passage were collected on day 6. Cell numbers were counted using a Vi-cell cytometer. Figure 6B shows the imDAP PDT at each passage (P1–P6). As shown in Figure 6B, the expanded imDAP maintained a similar PDT at P1–P6, significantly lower than that shown in Figure 1F, at approximately 25–28 h. The shorter PDT at each passage indicates higher proliferation efficiency, and the similar PDT at each passage indicates that the proliferation capacity of cells at each passage is better maintained. Figure 6C shows the percentage of FOXA2+ cells at P0, P1, P2, P3, P4, and P5. As shown in Figure 6C, growth medium containing a combination of CHIR99021, SB431542, and Y27632 can maintain or even improve FOXA2 expression during long-term growth on VTN / DLL4-coated surfaces. Figure 6D shows gene expression analysis of key imDAP markers (P0-P6). As shown in Figure 6D, expression of LMX1A, FOXA2, EN1, OTX2, and SOX6 was substantially maintained during long-term growth. The data indicated that the growth medium containing the above combination could support long-term growth of imDAP on VTN / DLL4-coated surfaces. Not only was growth efficiency significantly improved (expansion fold at each passage was 8-fold higher than that of the growth medium in Example 5), and the improved growth efficiency was substantially maintained at the same or similar level during cell growth and passaging, but the expression of imDAP-specific markers (e.g., Foxa2) or cell purity (e.g., FOXA2+ cells) was also maintained or improved during cell growth and passaging.

[0326] Example 11 Development of imDAP maturation medium B27 is commonly used in the literature as a media supplement for neuronal maturation. This example showed that when B27 was replaced with hPLT or HhPLT, maturation with hPLT or HhPLT resulted in a higher proportion of TH+ neurons compared with maturation with B27.

[0327] imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0 and grown as described in Example 10. The grown imDAP was applied to a VTN coating (1 μg / cm 2 ) 5 x 10 plates 5 cells / cm 2 (day 0) and cultured in maturation medium (MM-P, MM-H, MM-B) supplemented with 1 μM IWR1. MM-P used in Example 11 contained the following components: Neurobasal™ medium, 1% (v / v) Glutamax, 0.5 mM Db-cAMP sodium salt, 200 μM ascorbic acid, 20 ng / ml BDNF, 20 ng / ml GDNF, 1 ng / ml TGF-β3, 10 μM DAPT, and hPLT (0.5%, 1%, 2%). MM-H used in Example 11 contained the following components: Neurobasal™ medium, 1% (v / v) Glutamax, 0.5 mM Db-cAMP sodium salt, 200 μM ascorbic acid, 20 ng / ml BDNF, 20 ng / ml GDNF, 1 ng / ml TGF-β3, 10 μM DAPT, and HhPLT (0.5%, 1%, 2%). MM-B used in Example 11 contained the following components: Neurobasal, 1% (v / v) Glutamax, 0.5 mM Db-cAMP sodium salt, 200 μM ascorbic acid, 20 ng / ml BDNF, 20 ng / ml GDNF, 1 ng / ml TGF-β3, 10 μM DAPT, and 2% (v / v) B27 (50x) (as a control). The above-mentioned HhPLT was produced from hPLT as follows. The hPLTs were centrifuged at 3000 g for 30 min at 4°C to obtain the supernatant, which was then heated to 56°C for 30 min. Finally, the suspension was cooled to 4°C for at least 5 min and spun at 3000 g for 30 min at 4°C to obtain the HhPLT supernatant. Aliquots were prepared and stored at -80°C until use. The medium was changed every 3 days. On day 7, early maturation imDAPs were enzymatically dissociated with 1x TrypLE and centrifuged at 250 g for 5 min.

[0328] Figure 7A shows typical cell morphology on day 7. As shown in Figure 7A, treatment with hPLTs clearly induced imDAP contraction, which increased the difficulty of dissociating and collecting imDAPs after maturation. Also, imDAPs cultured with HhPLTs and B27 appeared less compact, and obvious nerve fibers were easily observed. Cells were counted, stained with TH (1° Ab: TH, Pel freeze, #P40101. 2° Ab: Abcam, #ab130805), and analyzed by flow cytometry (Figure 7B). As shown in Figure 7B, the addition of hPLTs significantly improved imDAP maturation (% TH+ cells) compared with B27, and the addition of HhPLTs further improved imDAP maturation (% TH+ cells) compared with hPLTs. For immunocytochemical analysis, some cells were plated onto laminin / poly-L-ornithine (PLOH)-coated plates for an additional 21 days of maturation before staining with TH antibody (1°Ab: TH, Pel freeze, #P40101. 2°Ab: Invitrogen, #A11012) (Figure 7C). As shown in Figure 7C, immunocytochemical analysis of TH in mDA neurons generated by using 1% HhPLT showed a higher percentage of TH+ neurons compared to B27.

[0329] Example 12 WNT inhibitor-supplemented medium resulted in a higher percentage of TH+ neurons This example demonstrated that IWR1 (a WNT inhibitor) could further improve the maturation of imDAP.

[0330] imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0 and grown as described in Example 10. The grown imDAP was applied to a VTN coating (1 μg / cm 2 ) 5 x 10 plates 5 cells / cm 2ImDAPs were seeded at a density of 1000 kJ / ml (day 0) and cultured in maturation medium supplemented with or without 1 μM IWR1. The medium was changed every 3 days. On day 7, imDAPs in the early maturation stage were enzymatically dissociated with 1× TrypLE and centrifuged at 250 g for 5 minutes. Cells were collected for RT-qPCR analysis of EN1, LMX1A, FOXA2, NURR1, SOX6, and TH gene expression. The maturation medium used in Example 12 contained the following components: Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) HhPLT, 0.5 mM Db-cAMP sodium salt, 200 μM ascorbic acid, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 ng / mL TGF-β3, and 10 μM DAPT.

[0331] Figure 7D shows the effect of IWR1 on cell morphology at day 7 during maturation. As shown in Figure 7D, imDAPs matured for at least 3 days in the presence of IWR1 exhibited more obvious nerve fibers. Furthermore, qRT-PCR analysis of imDAP maturation-related markers, such as NURR1, SOX6, and TH, was performed (Figure 7E). As shown in Figure 7E, the expression of NURR1, SOX6, and TH increased in cells treated with IWR1 compared to cells without IWR1, suggesting better maturation.

[0332] Example 13 Use of a ROCK inhibitor in combination with the coating matrix VTN / PLLH or VTN / PLOH promoted imDAP maturation Laminin / PLOH is commonly used as a matrix for mDA neuron maturation, but its cost is too high for large-scale mDA neuron production. To promote imDAP maturation while reducing costs, several different matrices were tested. This example demonstrated that the use of Y27632 (a ROCK inhibitor in the culture medium) in combination with the coating matrix VTN / PLLH (poly-L-lysine hydrobromide) or VTN / PLOH (poly-L-ornithine hydrobromide) could promote imDAP maturation.

[0333] imDAP was produced according to the method described in Chinese Patent Application Publication No. 201910169525.0 and grown as described in Example 10. The grown imDAP was diluted to 5 × 10 5 cells / cm 2 (day 0) on different matrix-coated plates (VTN, VTN+PLLH, VTN+PLOH, and laminin+PLOH) at a density of 1 μg / cm each. 2 ) and cultured in maturation medium. In addition to containing 10 μM or 0 μM Y27632, the maturation medium used in Example 13 further contained the following components: Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) HhPLT, 1 μM IWR1, 0.5 mM Db-cAMP sodium salt, 200 μM ascorbic acid, 20 ng / ml BDNF, 20 ng / ml GDNF, 1 ng / ml TGF-β3, and 10 μM DAPT. The medium was changed every 3 days. On day 3, IWR1 was removed from the maturation medium. On day 7, imDAPs in the early maturation stage were enzymatically dissociated with 1× TrypLE and centrifuged at 250 g for 5 minutes. Cells were collected and counted using a Vi-cell cytometer. Cells were analyzed for EN1, LMX1A, FOXA2, NURR1, SOX6, and TH gene expression by RT-qPCR. Cells were also cultured at 5 × 10 for immunostaining with LMX1A and SOX6 antibodies. 4 cells / cm 2 The cells were seeded onto cover slips in 48-well plates at a density of 1000 x g (LMX1A: Millipore, #MAB10533. SOX6: Proteintech, #14010-1-AP).

[0334] Figure 8A shows typical cell morphology on day 7 for the combinations VTN+Y (Control 1), VTN+PLLH (Control 2), VTN+PLLH+Y, VTN+PLOH (Control 3), VTN+PLOH+Y, and laminin+PLOH (Control 4). As shown in Figure 8A, cells plated on VTN / PLLH or VTN / PLOH were detached from the dish in maturation medium without Y27632. When the maturation medium was supplemented with Y27632, cells plated on VTN, VTN / PLLH, or VTN / PLOH could grow normally, suggesting that Y27632 can promote cell adhesion to some extent. Figure 8B shows the ratio of imDAP cell number after 7 days of maturation compared to the input cell number. As shown in Figure 8B, there was no significant difference in cell yield among the VTN / PLLH+Y27632, VTN / PLOH+Y27632, and laminin / PLOH combinations, all of which were higher than the VTN+Y27632 combination. Figure 8C shows qRT-PCR analysis of mDA maturation-related markers after 7 days of maturation. As shown in Figure 8C, VTN / PLLH+Y27632 and VTN / PLOH+Y27632 outperformed laminin / PLOH or VTN+Y in gene expression of NURR1, SOX6, and TH at the early stage of maturation. This suggested that VTN / PLLH+Y27632 (or PLLH) and VTN / PLOH+Y27632 (or PLOH) may support better maturation.

[0335] Here, it was shown that the use of Y27632 in the medium in combination with VTN / PLLH or VTN / PLOH not only promoted imDAP maturation but was also found to be less costly and therefore more suitable for large-scale clinical production.

[0336] Example 14 Maturation potential of imDAP cultured by using a ROCK inhibitor in conjunction with the coating matrix VTN / PLLH or VTN / PLOH at the late stage of maturation This example demonstrated that imDAP cultured by using Y27632 in conjunction with VTN / PLLH or VTN / PLOH coated plates at the early stage of maturation had similar potential for final maturation.

[0337] The imDAP cells after 7 days of maturation in Example 13 were enzymatically dissociated with 1x TrypLE and centrifuged at 250g for 5 minutes. The cells were collected and counted using a Vi-cell cytometer. For further maturation, the cells were aggregated into 3D suspension cultures (neurospheres). For this purpose, 5x10 cells were collected. 5 Resuspend cells at a density of 1000 cells / mL on a poly-HEMA coating in 6 mL maturation medium-ND supplemented with 10 μM Y27632 (1 μg / cm). 2) and seeded into T25 flasks. To induce aggregation, the T25 flasks were plated on a Belly Dancer at 15 rpm (day 0). On day 1, the medium was replaced with maturation medium-ND. The next day, half of the medium was replaced with maturation medium-BC. From day 3 onwards, neurospheres were cultured in maturation medium-BC, with the medium being replaced every 3 days. On day 21, neurospheres were collected and fixed in 4% PFA in PBS overnight at 4°C, followed by incubation in 30% sucrose overnight. Next, neurospheres were embedded in OCT blocks and cryosectioned (20 μm) using a cryostat. Cryosections of neurospheres were further immunostained with TH antibody (1° Ab: TH, Millipore, #MAB318.2° Ab: Invitrogen, #A11029). The maturation medium-ND used in Example 14 contained the following components: Neurobasal™ medium, 1% (v / v) Glutamax, 1% HhPLT, 0.5 mM Db-cAMP sodium salt, 200 μM ascorbic acid, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 ng / mL TGF-β3, 10 μM DAPT. The maturation medium-BC used in Example 14 contained the following components: BrainPhys™ medium, 1% (v / v) Glutamax, 1% HhPLT, 0.5 mM Db-cAMP sodium salt, 200 μM ascorbic acid, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 ng / mL TGF-β3, 1 μM Compound E (N-[N-(3,5-difluorophenylacetyl)]-L-alanyl-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydro-benzo[E](1,4)diazepin-2-one).

[0338] Figure 8D shows immunocytochemical analysis of frozen sections of neurospheres further matured from cells after 7 days of maturation using Y27632 in combination with VTN / PLLH or VTN / PLOH. As shown in Figure 8D, imDAPs cultured by using Y27632 in combination with VTN / PLLH or VTN / PLOH at the early stage of maturation had similar final maturation potential.

[0339] Example 15. Proliferated imDAPs at early and late passages maintained similar high capacity to differentiate into mDA neurons at early stages of maturation. This example demonstrated that expanded imDAPs at early and late passages can retain similar high ability to differentiate into mDA neurons at early stages of maturation.

[0340] The grown P3, P5 and P6 imDAPs from Example 10 were plated onto a VTN / PLOH coating (1 μg / cm each). 2 ) 5x10 on the plate 5 cells / cm 2 ImDAPs were seeded at a density of 10 μM (day 0) and cultured in maturation medium supplemented with 10 μM Y27632 and 1 μM IWR1. From day 3 onward, the maturation medium was replaced with maturation medium supplemented with 10 μM Y27632 alone. The medium was changed every 3 days. On day 7, imDAPs in the early maturation stage were enzymatically dissociated with 1x TrypLE and centrifuged at 250 g for 5 minutes. Mature cells from proliferating P3 and P6 imDAPs were collected and counted using a Vi-cell cytometer. Cells were analyzed by RT-qPCR for EN1, LMX1A, FOXA2, NURR1, SOX6, and TH gene expression (Figure 9A). In addition, mature cells from expanded P3 and P5 imDAP were seeded onto glass coverslips in 48-well plates for immunostaining with LMX1A and SOX6 antibodies (LMX1A: Millipore, #MAB10533; SOX6: Proteintech, #14010-1-AP) (Figures 9B and 9C). The maturation medium used in Example 15 contained the following components: Neurobasal™ medium, 1% (v / v) Glutamax, 1% (v / v) HhPLT, 0.5 mM Db-cAMP sodium salt, 200 μM ascorbic acid, 20 ng / ml BDNF, 20 ng / ml GDNF, 1 ng / ml TGF-β3, and 10 μM DAPT.

[0341] Figure 9A shows the results of RT-qPCR analysis of the expression of specific markers, EN1, LMX1A, FOXA2, NURR1, SOX6, and TH, in early-stage mDA neurons matured from expanded P3 and P6 imDAPs. The results showed no significant differences between expanded P3 and P6 imDAPs. Figure 9B shows the results of immunocytochemical analysis of LMX1A and SOX6 expression in early-stage mDA neurons matured from expanded P3 and P5 imDAPs. Figure 9C shows the results of quantification of LMX1A- and SOX6-expressing mDA neurons matured from P3 and P5 imDAPs at early maturation stages. The results showed no differences between expanded P3 and P5 imDAPs. The results indicate that expanded imDAPs generated using this novel expansion method described herein can be passaged several times and retain a high ability to differentiate into mDA neurons.

[0342] Those skilled in the art will readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended as limitations on the scope of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the disclosure disclosed herein without departing from the scope and spirit of the invention.

[0343] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0344] The present disclosure is not limited in terms of the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. Not all of the various embodiments of the present disclosure are described herein. As will be apparent to those skilled in the art, many modifications and variations of the present disclosure can be made without departing from the spirit and scope of the disclosure. Functionally equivalent methods and apparatuses within the scope of the present disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full range of equivalents to which such claims are entitled.

Claims

1. A culture medium comprising: (a) Neural Basal Medium; (b) human platelet lysate (hPLT); (c) transforming growth factor β (TGF-β); (d) a gamma-secretase inhibitor; and (e) cAMP-based compounds or cyclase activators thereof A culture medium comprising:

2. (f) neurotrophic factors 10. The culture medium of claim 1, further comprising:

3. 2. The culture medium of claim 1, wherein the hPLTs are heat-treated human platelet lysates (HhPLTs).

4. (g) WNT signaling pathway inhibitor 10. The culture medium of claim 1, further comprising:

5. 2. The culture medium of claim 1, wherein the hPLTs are present in the culture medium at a concentration of 0.1% to 5% by volume.

6. 2. The culture medium of claim 1, wherein the TGF-β is present in the culture medium at a concentration of 0.1 ng / ml to 10 ng / ml.

7. 2. The culture medium of claim 1, wherein the gamma-secretase inhibitor is present in the culture medium at a concentration of 1 μM to 30 μM.

8. 2. The culture medium of claim 1, wherein the cAMP-based compound or cyclase activator thereof is present in the culture medium at a concentration of 0.1 mM to 5 mM.

9. 3. The culture medium of claim 2, wherein the neurotrophic factor is present in the culture medium at a concentration of 1 ng / ml to 100 ng / ml.

10. 5. The culture medium of claim 4, wherein the WNT signaling pathway inhibitor is present in the culture medium at a concentration of 0.25 μM to 10 μM.

11. 2. The culture medium of claim 1, wherein the gamma-secretase inhibitor is selected from the group consisting of DAPT, N-[N-(3,5-difluorophenylacetyl)]-L-alanyl-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydro-benzo[E](1,4)diazepin-2-one, LY-411575, dihydroergocristine mesylate, BMS 299897, and any combination thereof.

12. 2. The culture medium of claim 1, wherein the cAMP-based compound or cyclase activator thereof is selected from the group consisting of Db-cAMP sodium salt, cAMP, forskolin, 8-bromo-cAMP sodium salt, NKH477, 8-chloro-cAMP, 6-Bnz-cAMP sodium salt, bucladesine calcium salt, and any combination thereof.

13. The culture medium of claim 2 , wherein the neurotrophic factor is selected from the group consisting of BDNF, GDNF, or both.

14. 5. The culture medium of claim 4, wherein the WNT signaling pathway inhibitor is selected from the group consisting of IWR1, iCRT3, IWP-O, IWP-2, IWP-3, IWP-4, ciclopirox, cardamonin, diethylbenzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, XAV-939, fossenvivint (ICG-001), capmatinib, isoquercitrin, gigantol, JW55, MSAB, KY02111, FH535, WIKI4, CCT251545, prodigiosin, KYA1797K, NCB-0846, LF3, iCRT14, adavivint, triptonide, M435-1279, and any combination thereof.

15. (a) 0.1% to 2% by volume of said hPLTs; (b) 0.1 ng / mL to 5 ng / mL of the TGF-β; (d) 5 μM to 20 μM of the γ-secretase inhibitor; (d) 0.1 mM to 2.5 mM of the cAMP compound or cyclase activator thereof; and (e) containing 10 ng / ml to 80 ng / ml of the neurotrophic factor in the neurobasal medium; The culture medium of claim 2.

16. further comprising glutamine or a derivative thereof, 2. The culture medium of claim 1, wherein the glutamine or a derivative thereof is present in the culture medium at a concentration of 0.1% to 5% by volume.

17. further comprising an antioxidant, 2. The culture medium of claim 1, wherein the antioxidant is present in the culture medium at a concentration of 50 μM to 500 μM.

18. 10. The culture medium of claim 1, wherein the culture medium is a chemically defined, serum-free maturation medium.

19. The culture medium according to any one of claims 1 to 18, further comprising a Rho kinase (ROCK) inhibitor.

20. 20. The culture medium of claim 19, wherein the ROCK inhibitor is present in the culture medium at a concentration of 1 μM to 50 μM.

21. 20. The culture medium of claim 19, wherein the ROCK inhibitor is selected from the group consisting of Y27632, HA100, HA1152, HA-1077, and any combination thereof.

22. 20. A kit comprising the culture medium of claim 19.

23. (a) a first coating matrix capable of supporting cell adhesion of the mDAP, the first coating matrix being other than laminin; (b) a second coating matrix capable of improving the maturity of the mDAP, the second coating matrix comprising a polylysine-based compound and / or a polyornithine-based compound; 23. The kit of claim 22, further comprising a coating matrix combination comprising:

24. 1. A method for promoting maturation of midbrain dopaminergic progenitor cells (mDAP), comprising: (a) a first coating matrix capable of supporting cell adhesion of the mDAP, the first coating matrix being other than laminin; (b) a second coating matrix capable of improving the maturation of the mDAP, the second coating matrix comprising a polylysine-based compound and / or a polyornithine-based compound, and contacting the mDAP with a maturation medium containing a ROCK inhibitor on a culture surface coated with a combination of coating matrices comprising the above.

25. 25. The method of claim 24, wherein the ROCK inhibitor-containing maturation medium is the culture medium of claim 19.

26. 25. The method of claim 24, wherein the first coating matrix is ​​selected from the group consisting of vitronectin (VTN), collagen, proteoglycan, fibronectin, entactin, elastin, functional fragments of any of the foregoing proteins, hyaluronic acid, gelatin, and any combination thereof, preferably VTN.

27. 25. The method of claim 24, wherein the second coating matrix comprises poly-L-lysine hydrobromide, poly-L-ornithine hydrobromide, or a mixture thereof.

28. 26. A substantially homogeneous population of midbrain dopaminergic neurons (mDAN) produced by the method of claim 25.

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