Media, coating matrices and methods for maturing midbrain dopaminergic progenitor cells

Abstract

The disclosure described herein provides an amplification method for amplifying mDAP and a culture medium and coating matrix combination for use in the amplification method, as well as a maturation method for maturation of mDAP and a culture medium for use in the maturation method. The present disclosure also provides a population of substantially homogeneous mDAPs and a population of substantially homogeneous mDANs.

Description

Technical Field

[0001] The present disclosure generally relates to the field of stem cell technology, and more particularly to media, coating matrices, and methods for expanding and maturing midbrain dopaminergic progenitor cells (mDAP). Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disorder. The hallmark of the disease is the selective loss of dopaminergic neurons (DA neurons) in the substantia nigra of the midbrain. Despite recent advances in medical science that have greatly enhanced the general understanding of the pathogenesis of PD, unfortunately, there is currently no cure for this devastating disease. The main treatment for PD patients is DA analogs and receptor agonists to counteract the reduction of DA.

[0003] Therefore, there is still a need to further advance research on PD mechanisms, its disease progression, and effective clinical intervention methods for the effective treatment of PD, and a prerequisite for such research is the availability of midbrain dopaminergic progenitor cells.

[0004] Pluripotent stem cells include human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC). These pluripotent stem cells can be expanded in vitro and retain their ability to differentiate into any cell type of the three germ layers (including neuronal cells and tissues). Therefore, these pluripotent stem cells are very valuable for studying developmental processes and disease mechanisms (especially in the brain). Specifically, hiPSC represents an unlimited source of cells for applications such as mechanism research, drug screening assays, and ultimately cell replacement therapies for treating neurological disorders such as PD. A variety of protocols have been developed to generate human DA neurons in vitro from hPSC.

[0005] These protocols generally rely on the use of small molecules and growth factors to direct the differentiation of pluripotent stem cells into imDAP, as well as the expansion and maturation of imDAP. They are generally laborious, time-consuming, and vary greatly between batches, producing a heterogeneous population with a relatively low number of midbrain DA neurons. However, in clinical and therapeutic applications, there is a strong need for homogeneous and robust cell populations.

[0006] Therefore, there is still a need to provide improved reagents, compositions, and methods for expanding and / or maturing mDAP.

[0007] Summary

[0008] In a first aspect, the present disclosure provides a culture medium capable of promoting the expansion of midbrain dopaminergic progenitor cells (mDAP), the culture medium comprising: (a) a basal medium; (b) a nerve growth supplement; (c) a WNT signaling pathway activator; (d) a Rho kinase (ROCK) inhibitor; and (e) a transforming growth factor β (TGF-β) inhibitor.

[0009] In a second aspect, the present disclosure provides a coated matrix combination capable of promoting the expansion of midbrain dopaminergic progenitor cells (mDAP), the coated matrix combination comprising: (a) a first coated matrix capable of supporting the adhesion of mDAP cells; and (b) a second coated matrix capable of enhancing the expression of mDAP-specific markers during the expansion and passage of mDAP, wherein the second coated matrix comprises a Notch agonist.

[0010] In a third aspect, the present disclosure provides a method for expanding midbrain dopaminergic progenitor cells (mDAP), which comprises contacting mDAP with an expansion culture medium on a culture surface coated with the coated matrix combination of the second aspect of the present disclosure described herein.

[0011] In a fourth aspect, the present disclosure provides a culture medium capable of promoting the maturation of mDAP (midbrain dopaminergic progenitor cells), the culture medium comprising: (a) a neurobasal medium; (b) human platelet lysate (hPLT); (c) transforming growth factor β (TGF-β); (d) a γ-secretase inhibitor; and (e) a cyclic adenosine phosphate compound or its cyclase activator.

[0012] In a fifth aspect, the present disclosure provides a method for promoting the maturation of midbrain dopaminergic progenitor cells (mDAP), which comprises contacting mDAP with a maturation culture medium containing a ROCK inhibitor on a culture surface coated with a coated matrix combination, the coated matrix combination comprising: (a) a first coated matrix capable of supporting the adhesion of mDAP cells, wherein the first coated matrix is not laminin; and (b) a second coated matrix capable of enhancing the maturity of mDAP, wherein the second coated matrix comprises a polylysine compound and / or a polyornithine compound.

[0013] 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.

[0014] In a seventh aspect, the present disclosure provides a substantially homogeneous population of midbrain dopaminergic neurons (mDAN) produced by the method of the fifth aspect of the present disclosure described herein.

[0015] In an eighth aspect, the present disclosure provides a kit comprising the culture medium of the first aspect of the present disclosure described herein.

[0016] In certain embodiments, the kit further comprises the coated matrix combination of the second aspect of the present disclosure described herein.

[0017] In a ninth aspect, the present disclosure provides a kit comprising the culture medium of the fourth aspect of the present disclosure described herein.

[0018] In certain embodiments, the kit further comprises the coated matrix combination of the fifth aspect of the present disclosure described herein.

[0019] Various objects and advantages of the reagents, compositions, and methods provided herein will become apparent from the following description in conjunction with the accompanying drawings, in which certain embodiments of the present disclosure are illustrated by way of illustration and example. Brief Description of the Drawings

[0021] Figure 1 shows the development and validation of an amplification culture medium based on the LDN193189 / CHIR99021 / FGF8b / Blebbistatin combination according to Examples 1-5 of the present disclosure, wherein Figure 1A the morphology of the amplified cells is shown in the absence of Blebbistatin (left panel), in the case where Blebbistatin is removed 24 hours after plating (middle panel), and in the presence of Blebbistatin throughout the culture period (right panel) (scale bar: 50 μM); Figure 1B the morphology of the amplified cells is shown in the absence of LDN193189 (left panel) and in the presence of LDN193189 (right panel) (scale bar: 50 μM); Figure 1C the effect of the concentration of CHIR99021 in the amplification culture medium on the expression of the imDAP-specific markers EN1, LMX1A, and FOXA2 (representative markers of mDAP) is shown; Figure 1D the effect of the presence or absence of FGF8b in the amplification culture medium on the expression of the imDAP-specific markers EN1, LMX1A, and FOXA2 is shown; Figure 1E the typical morphology of the amplified imDAP at each passage (P1 - P6) 4 - 5 days after passage is shown (scale bar: 50 μm); Figure 1F the representative population doubling time (PDT) of imDAP during passage (P1 - P5) is shown; Figure 1G the flow cytometry analysis results of FOXA2 expression in P0, P1, P2 (P represents passage number) imDAP are shown; and Figure 1H the qRT-PCR analysis results of the expression of the imDAP-specific markers LMX1A, FOXA2, and EN1 in imDAP at each passage (P0 - P6) are shown.

[0022] Figure 2Shows the comparison of the effects of Y27632 and Bretastatin on the expression of imDAP-specific marker FOXA2 according to Example 6 of the present disclosure.

[0023] 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 the visualization of the clustering results of imDAP single-cell RNA sequencing data using UMAP; and Figure 3B is the heatmap of a subset of genes enriched in imDAP, where MB-FP: midbrain floor plate; MB-BP: midbrain basal plate; HB: hindbrain; MHB: midbrain-hindbrain boundary.

[0024] Figure 4 shows the effects of NOTCH activation and TGF-β inhibition on the expansion of imDAP according to Example 8 of the present disclosure, where Figure 4A shows the morphology of imDAP expanded with and without SB431542 on culture surfaces coated with VTN, VTN+DLL4, or DLL4; Figure 4B shows the effects of DLL4 and SB431542 on the expansion efficiency of imDAP; and Figure 4C shows the FACS analysis results of FOXA2 expression in imDAP expanded with and without SB431542 on culture surfaces coated with VTN or VTN+DLL4.

[0025] Figure 5 shows the effects of LDN193189, FGF8b, and FGF2 on the expansion of imDAPs according to Example 9 of the present disclosure, where Figure 5A shows the morphology of imDAP expanded with LDN193189, LDN193189+FGF8b, LDN193189+FGF2, and without any of them (scale bar: 50 μM); Figure 5B shows the effects of LDN193189 and / or FGF8b on the expansion efficiency of imDAP; and Figure 5C shows the FACS analysis results of FOXA2 expression in imDAP expanded with LDN193189 or LDN193189+FGF8b and without any of them.

[0026] Figure 6 shows the effects of NOTCH activation combined with TGF-β inhibition on the expansion of imDAP according to Example 10 of the present disclosure, where Figure 6A shows the typical morphology of P3 imDAP on day 5 (scale bar: 50 μm); Figure 6B shows the representative PDT of imDAP in each generation (P1 - P6) during long-term expansion; Figure 6Cshows the FACS analysis results of FOXA2 expression in imDAPs at each passage (P0 - P5) during long-term expansion; and Figure 6D shows the qRT-PCR analysis results of imDAP-specific markers EN1, LMX1A, FOXA2, OTX2, and SOX6 in imDAPs at each passage (P0 - P6) during long-term expansion.

[0027] Figure 7A -C shows the effect of human platelet lysate (hPLT) and heat-treated human platelet lysate (HhPLT) according to Example 11 of the present disclosure in place of B27 on imDAP maturation, where Figure 7A shows bright-field images of imDAPs cultured in maturation medium with different concentrations of hPLT or HhPLT, or with B27 (scale bar: 50 μM); Figure 7B shows the FACS analysis results of TH (TH (tyrosine hydroxylase) is a representative maturation marker of mDA neurons) expression in imDAPs cultured in maturation medium with different concentrations of hPLT or HhPLT, or with B27; and Figure 7C shows the immunocytochemical analysis results of TH in mDA neurons matured with 1% HhPLT or B27 (scale bar: 50 μM).

[0028] Figure 7D -E shows the effect of IWR1 on imDAP maturation according to Example 12 of the present disclosure, where Figure 7D shows the cell morphology of cells matured in maturation medium with or without IWR1 (scale bar: 50 μm); and Figure 7E shows the qRT-PCR analysis results of the expression of markers EN1, LMX1A, FOXA2, NURR1, SOX6, and TH in cells matured in maturation medium with or without IWR1.

[0029] Figure 8A -C shows the effect of Y27632 (Y) and PLLH (poly-L-lysine hydrobromide) or PLOH (poly-L-ornithine hydrobromide) according to Example 13 of the present disclosure on imDAP maturation, where Figure 8A shows bright-field images of cells matured using 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 using VTN + Y, VTN + PLLH + Y, VTN + PLOH + Y, or laminin + PLOH; and Figure 8CShows the qRT-PCR analysis results of the expression of maturation-related markers EN1, LMX1A, FOXA2, SOX6, NURR1, and TH in cells after 7 days of maturation.

[0030] Figure 8D Shows the immunocytochemical analysis results of cryosections of neurospheres according to Example 14 of the present disclosure (green light: TH; blue light: DAPI (cell nucleus)) (scale bar: 20 μm).

[0031] Figure 9 shows the differentiation ability of amplified imDAPs at early and late passages, where Figure 9A Shows the RT-qPCR analysis results of the expression of specific markers EN1, LMX1A, FOXA2, NURR1, SOX6, and TH in early-stage mDA neurons matured from amplified P3 and P6 imDAPs; Figure 9B Shows the immunocytochemical analysis results of the expression of LMX1A and SOX6 in early-stage mDA neurons matured from amplified P3 and P5 imDAPs; and Figure 9C Shows the quantification results of mDA neurons expressing LMX1A and expressing SOX6 matured from amplified P3 and P5 imDAPs.

[0032] Details

[0033] It should be understood that certain aspects, modes, embodiments, variations, and features of the present disclosure are described below with different levels of detail in order to provide an understanding of the essence of the technology.

[0034] The "first", "second", "third", "fourth", "fifth", "sixth", "seventh", "eighth", or "ninth" mentioned throughout this specification does not denote the order or sequence of the features, structures (e.g., culture media or compositions), or properties related to the mentioned description, but may be for the purpose of distinction only.

[0035] The "first aspect", "second aspect", "third aspect", "fourth aspect", "fifth aspect", "sixth aspect", "seventh aspect", "eighth aspect", or "ninth aspect" mentioned throughout this specification indicates that the specific features, structures, or properties described in relation to that aspect are included in at least one or more aspects of the present disclosure. In addition, the specific features, structures, properties, or embodiments in one aspect may be combined with the specific features, structures, properties, or embodiments in one or more other aspects in any appropriate manner.

[0036] As used throughout this specification, the terms "one embodiment", "another embodiment", "a preferred embodiment", "some embodiments" or "certain embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one or more embodiments of the present disclosure. In addition, the particular features, structures, or characteristics in one embodiment may be combined with the particular features, structures, or characteristics in one or more other embodiments in any suitable manner.

[0037] The present disclosure described illustratively herein may be suitably practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. As used herein, the term "comprising / including" 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 shall mean excluding other elements of any substantial significance to the said compositions or methods. "Consisting of" shall mean excluding other ingredients in excess of trace amounts for the claimed compositions and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of the present disclosure. Thus, it is intended that the methods and compositions may include additional steps and components (comprising / including), or alternatively may include insignificant steps and compositions (consisting essentially of), or alternatively are intended to be only the recited method steps or compositions (consisting of). In addition, in each case herein, any one of the terms "comprising / including", "consisting essentially of" and "consisting of" may be replaced by any one of the other two terms.

[0038] It should be understood that the present disclosure is not limited to a particular use, method, reagent, compound, composition or biological system, although these may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0039] 1. Medium for amplifying mDAP

[0040] In a first aspect, the present disclosure provides a medium (e.g., a chemically defined serum-free amplification medium) capable of promoting robust amplification of midbrain dopaminergic progenitor cells (mDAP), the medium comprising: (a) a basal medium; (b) a nerve growth supplement; (c) a WNT signaling pathway activator; (d) a Rho kinase (ROCK) inhibitor; and (e) a transforming growth factor β (TGF-β) inhibitor.

[0041] According to a first aspect, due to the inclusion of a TGF-β inhibitor, particularly an inclusion of a ROCK inhibitor and a TGF-β inhibitor, the amplification medium of the present disclosure is capable of enhancing the amplification efficiency and the expression of mDAP-specific markers during mDAP amplification and passage.

[0042] The amplification 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 amplification coating matrix in the art or any other suitable amplification coating matrix. Examples of common coating matrices include vitronectin (VTN), Matrigel, fibronectin, gelatin, and laminin.

[0043] Any mDAP can be amplified using the amplification medium of the present disclosure. Examples of mDAP include fetal brain-derived mDAP, hPSC-derived mDAP; and mDAP obtained by transdifferentiation from other cell types. mDAP can be derived from (e.g., differentiated from) pluripotent stem cells. Pluripotent stem cells can include induced pluripotent stem cells (e.g., hiPSC), embryonic stem cells (e.g., hESC), naive PSC (NPSC), and extended pluripotent stem cells (EPSC). In certain embodiments, mDAP is ESC-derived midbrain dopaminergic progenitor cells (emDAP). In certain embodiments, mDAP is iPSC-derived midbrain dopaminergic progenitor cells (imDAP). In certain embodiments, mDAP is NPSC-derived midbrain dopaminergic progenitor cells (nmDAP). In certain embodiments, mDAP is EPSC-derived midbrain dopaminergic progenitor cells (epmDAP).

[0044] ESC (e.g., hESC) and iPSC (e.g., hiPSC) are known in the art and can be readily obtained using conventional methods (e.g., those described in the prior art) 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 (iPSC) from somatic cells including PBMC and T cells.

[0045] Such iPSC (e.g., hiPSC) can be cultured under defined conditions to generate mDAP. For example, it can be cultured in mTeSR TMiPSCs (such as hiPSCs) are cultured on Matrigel in a medium (Stemcell Technologies, catalog number 85850) under defined conditions, and sub-confluent hiPSCs are passaged onto fresh Matrigel-coated plates and cultured in iPSC medium for an additional time (such as 24 hours) to reach 90 - 100% confluence. Once confluent, the medium can be changed to induce mDAP for more time (such as 1 - 3 days), and the medium can be changed daily if desired. For example, Fedele et al., Scientific Reports 7:6036|DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0046] As the name implies, a basal medium can support the survival, maintenance, growth, and proliferation of cells as a culture medium and is a basic component of an expansion medium. Generally, the basal medium accounts for about 95% - 99% of the expansion medium by volume. The basal medium used in the expansion medium of the present disclosure can be a common basal medium or a basal medium specifically for nerve cells, such as Neurobasal medium.

[0047] 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 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.

[0048] Examples of Neurobasal media include NEUROBASAL TM basal medium (e.g., Gibco catalog number 21103049), NEUROBASAL-A TM basal medium (e.g., Gibco catalog number 10888022), NEUROBASAL PLUSTM Basal medium (e.g., Gibco catalog number A3582901), and / or BRAINPHYS TM Basal medium (e.g., STEMCELL catalog number 05790).

[0049] In certain embodiments, the basal medium comprises DMEM:F12 and NEUROBASAL TM Neurobasal medium. In certain embodiments, the neurobasal medium is present in the basal medium at about 0% to about 100%, about 25% to about 75%, or about 50% by volume.

[0050] In certain embodiments, the neurotrophic supplement is selected from B27, N1, N2, and any combination thereof. In certain embodiments, the neurotrophic supplement comprises B27 (e.g., B27 from GIBCO BRL catalog number 12587010).

[0051] According to the present disclosure, the concentration of the neurotrophic supplement is not particularly limited as long as it does not prevent the promotion of the expansion of mDAP. In certain embodiments, the neurotrophic supplement is present in the culture medium at about 0.1% to about 20%, preferably about 0.1% to about 10%, and more preferably about 0.5% to about 5% by volume.

[0052] The Wnt signaling pathway is defined by a series of events that occur when a Wnt protein ligand binds to a cell surface receptor of the Frizzled receptor family member. This leads to the activation of the Dishevelled (Dsh) family of proteins, which inhibits the degradation of the complex of proteins including axin, GSK-3, and protein APC, intracellularly, of β-catenin. The resulting enriched nuclear β-catenin enhances transcription through the TCF / LEF family of transcription factors.

[0053] As used herein, a Wnt signaling pathway activator refers to an agonist of the Wnt signaling pathway (e.g., a reagent capable of upregulating the activity and / or amount of components involved in the Wnt signaling pathway), and can be interchanged with "Wnt signaling pathway agonist", "Wnt agonist", "Wnt pathway activator", or "Wnt activator". Wnt signaling pathway activators include reagents that directly or indirectly activate TCF / LEF-mediated transcription in cells, such as by modulating the activity of any one protein / gene in the Wnt signaling cascade (e.g., enhancing the activity of positive regulators of the Wnt signaling pathway, or inhibiting the activity of negative regulators of the Wnt signaling pathway).

[0054] The Wnt activator is selected from true Wnt activators that bind to and activate members of the Frizzled receptor family, which include 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 relative to the level of Wnt activity in the absence of the Wnt activator. As is known to those skilled in the art, Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example, by pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (see Korinek et al., Science 275:1784-1787, 1997, incorporated herein by reference).

[0055] Representative Wnt activators can 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 “Wnt Family of Secreted Proteins,” R&D Systems Catalog, 2004 (incorporated herein by reference).

[0056] Further 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 4 members, namely 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), which is a secreted regulatory protein that functions similarly to Wnt proteins, i.e., it binds to the Frizzled-4 receptor with high affinity and induces activation of the Wnt signaling pathway (Kestutis Planutis et al., BMC Cell Biol. 8:12, 2007).

[0057] Wnt activators also include small molecule agonists of the Wnt signaling pathway, aminopyrimidine derivatives (N4-(benzo[d][1,3]dioxol-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine), as described in Liu et al. (Angew Chem. Int. Ed. Engl. 44(13):1987-1990, 2005, incorporated herein by reference).

[0058] In certain embodiments, the Wnt signaling pathway activator is a GSK inhibitor, such as a GSK-3β inhibitor. GSK3 inhibitors can include (by way of example and not limitation) polynucleotides, polypeptides, and small molecules.

[0059] GSK inhibitors include small interfering RNAs (siRNAs, 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), and FRAT family members and FRAT-derived peptides that block the interaction of GSK-3 with axin. An overview is provided in 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 can include, for example, those described in Liao et al. (Endocrinology 145(6):2941-2949, 2004, incorporated herein by reference).

[0060] In certain embodiments, the Wnt activator is selected from one or more of: a Wnt family member, R-spondin 1-4 (e.g., R-spondin 1), Norrin, Wnt3a, Wnt-6, and a GSK inhibitor.

[0061] In certain embodiments, any protein-based Wnt activator mentioned herein, such as R-spondin 1 to R-spondin 4, any Wnt family member, etc., can be replaced by a homolog or a fragment thereof that is naturally, synthetically, or recombinantly produced, the homolog or fragment thereof retaining at least about 80%, 85%, 90%, 95%, 99%, and / or being replaced by a homolog or a fragment thereof having 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 global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0062] In certain embodiments, examples of GSK-3β inhibitors include: Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, CT20026, SB216763, TDZD-8, BIO, BIO-Acetoxime, (5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, Pyridocarbazole-cyclopenadienylruthenium complex (GSK-3 inhibitor XV), 2-Thio(3-iodobenzyl)-5-(1-pyridinyl)[1,3,4]oxadiazole, OTDZT, α-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.

[0063] In certain embodiments, the Wnt signaling pathway activator is selected from 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-pyridinyl)[1,3,4]oxadiazole, α-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.

[0064] Representative structures of certain Wnt signaling pathway activators that can be used in the amplification media of the present disclosure are provided below, many of which are generally commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources.

[0065] However, it should generally be understood that in all cases where a given source is provided herein, such sources are not restrictive. Given the exemplary structures of materials, chemicals, or compounds, alternative sources (commercial or non-commercial) of the same or similar materials, chemicals, or compounds can be readily used.

[0066] Aza Kempaloxone: MCE, #HY-59090; APExBio, #B3690; Sigma-Aldrich, #A3734; CAS No.: 676586-65-9.

[0067] CHIR99021: APExBio, #A3011.

[0068] CHIR98014: Sigma-Aldrich, #SML1094; CAS No.: 252935-94-7.

[0069] NP031112 (Tigulixib): Sigma-Aldrich, #SML0339; APExBio, #B1539; MCE, #HY-14872; CAS No.: 865854-05-3.

[0070] TWS119: Sigma-Aldrich, #SML1271; APExBio, #B1540; MCE, #HY-10590; CAS No.: 601514-19-6.

[0071] AZD2858: APExBio, #B1537; MCE, #HY-15761; CAS No.: 486424-20-8.

[0072] AZD1080: APExBio, #B1536; MCE, #HY-13862, CAS No.: 612487-72-6.

[0073] SB415286: Sigma-Aldrich, #S3567; APExBio, #A8241; MCE, #HY-15438; CAS No.: 280744-09-4.

[0074] LY2090314: Sigma-Aldrich, #SML1438; APExBio, #A3570; MCE, #HY-16294; CAS No.: 603288-22-8.

[0075] AR-A014418: Sigma-Aldrich, #A3230; APExBio, #A3184; MCE, #HY-10512; CAS No.: 487021-53-3.

[0076] SB216763: Sigma-Aldrich, #S3442; APExBio, #A8240; MCE, #HY-12012; CAS No.: 280744-09-4.

[0077] TDZD-8: Sigma-Aldrich, #T8325; APExBio, #B1249; MCE, #HY-11012; CAS No.: 327036-89-5.

[0078] BIO (GSK 3 Inhibitor IX): Sigma-Aldrich, #B1686; APExBio, #B1538; MCE, #HY-10580; CAS No.: 667463-62-9.

[0079] BIO-Acetoxime; Sigma-Aldrich, #SML0531; APExBio, #B5488; MCE, #HY-15356; CAS No.: 667463-85-6.

[0080] (5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK-3 Inhibitor XIII): MCE, #HY-112392; absin, #abs819580; aladdin, #G338805; CAS No.: 404828-08-6.

[0081] Pyrido-carbazole-cyclopentadienyl ruthenium complex (GSK-3 Inhibitor XV): Sigma-Aldrich, #361558, CAS No.: 936112-69-5.

[0082] 2-Thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]oxadiazole (GSK3 Inhibitor II): APExBio, #C4599; CAS No.: 478482-75-6.

[0083] α-4-Dibromoacetophenone (2,4'-Dibromoacetophenone / 4'-Bromobenzoylmethyl Bromide): Sigma-Aldrich, #D38308; CAS No.: 99-73-0.

[0084] OTDZT (2,4-Dibenzyl-5-oxothiazolidine-3-thione): CAS No.: 373357-10-9.

[0085] 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 No.: 626604-39-5.

[0086] L803H-KEAPPAPPQSpP-NH2: CAS No.: 348089-28-1.

[0087] GF109203X (Bisindolylmaleimide I): Sigma-Aldrich, #G2911; APExBio, #A8342; MCE, #HY-13867; CAS No.: 133052-90-1.

[0088] RO318220: MCE, #HY-13866A; CAS No.: 125314-64-9.

[0089] According to the present disclosure, the concentration of the WNT signaling pathway activator is not particularly limited as long as it does not prevent the promotion of mDAP amplification. In certain embodiments, 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.

[0090] In certain embodiments, the WNT signaling pathway activator includes CHIR99021. In certain embodiments, the WNT signaling pathway activator (e.g., CHIR99021) is present in the expansion 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.

[0091] ROCK inhibitors include reagents that inhibit the binding between ROCK and the ROCK receptor. The ROCK inhibitors used in the expansion medium of the present disclosure can promote the expansion of mDAPs, such as increasing the expansion efficiency and the expression of mDAP-specific markers such as FOXA2.

[0092] In certain embodiments, the ROCK inhibitors are selected from: Y27632, HA100, HA1152, HA-1077, and any combination thereof.

[0093] 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-piperazinesulfonyl)-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).

[0094] Representative structures of certain ROCK inhibitors that can be used in the expansion medium of the present disclosure are provided below, many of which are generally commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources (although these sources are not limiting).

[0095] Y-27632 (MCE catalog number HY-10071, CAS number: 146986-50-7).

[0096] HA-100 (hydrochloride) (absin catalog number abs47045575).

[0097] H1152 (MCE Catalog No. HY-15720, CAS No.: 451462-58-1).

[0098] HA-1077 (Fasudil / AT877) (MCE Catalog No. HY-10341A, CAS No.: 103745-39-7).

[0099] In certain embodiments, the ROCK inhibitor includes Y27632. In certain embodiments, the ROCK inhibitor (e.g., Y27632) is present in the expansion 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.

[0100] TGF-β signaling generally begins with the binding of a TGF-β superfamily ligand to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates SMAD, which acts as a transcription factor in the nucleus and regulates target gene expression. Optionally, TGF-β signaling can activate the MAP kinase signaling pathway, e.g., via p38 MAP kinase. The TGF-β inhibitor used in the expansion medium of the present disclosure can promote the expansion of mDAP, e.g., by increasing the expansion efficiency and the expression of mDAP-specific markers such as FOXA2.

[0101] As used herein, TGF-β inhibitors include reagents that reduce the activity of the TGF-β signaling pathway. There are many different ways to interfere with the TGF-β signaling pathway. For example, TGF-β signaling can be interfered with by: inhibiting TGF-β expression by small interfering RNA strategies; inhibiting furin protease (a TGF-β activating protease); inhibiting the pathway by physiological inhibitors, e.g., inhibiting BMP by Noggin, DAN or DAN-like proteins; neutralizing TGF-β with monoclonal antibodies; inhibiting with small molecule inhibitors of TGF-β receptor kinase 1 (also known as activin receptor-like kinase, ALK5), ALK4, ALK6, ALK7 or other TGF-β related receptor kinases; inhibiting Smad 2 and Smad 3 signaling by overexpressing its physiological inhibitor Smad 7, or by using thioredoxin as a Smad anchor to render Smad inactive.

[0102] 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 in the TGF-β superfamily. An inhibitor of any one of these kinases is an inhibitor that causes a reduction in the enzymatic activity of any one (or more) of these kinases.

[0103] In certain embodiments, the TGF-β inhibitor can bind to and inhibit the activity of Smad proteins such as R-SMAD or SMAD1-5 (i.e., SMAD1, SMAD2, SMAD3, SMAD4, or SMAD5).

[0104] In certain embodiments, the TGF-β inhibitor can bind to and reduce the activity of a Ser / Thr protein kinase selected from the group consisting of TGF-β receptor kinase 1, ALK4, ALK5, ALK7, or p38.

[0105] In certain embodiments, the amplification medium of the present disclosure comprises an inhibitor of ALK5.

[0106] There are known a variety of methods for determining whether a substance is a TGF-β inhibitor. For example, a cell assay can be employed, in which cells are stably transfected with a reporter construct comprising a human PAI-1 promoter or a Smad binding site driving a luciferase reporter gene. 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 a phospho-sensor assay (Drew et al., J. Biomol. Screen. 16(2):164-173, 2011, incorporated herein by reference).

[0107] The TGF-β inhibitors useful in the present disclosure can be proteins, peptides, small molecules, small interfering RNAs, antisense oligonucleotides, aptamers, antibodies, or antigen-binding portions thereof. The inhibitors can be naturally occurring or synthetic. Examples of small molecule TGF-β inhibitors useful 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.

[0108] In certain embodiments, the TGF-β inhibitor is selected from RepSox, A83-01, SB431542, D4476, GW788388, LY364947, SB525334, SB505124, SD208, GW6604, and any combination thereof.

[0109] Representative structures of certain TGF-β inhibitors that can be used in the amplification media of the present disclosure are provided below, many of which are commonly commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources (although these sources are not limiting).

[0110] 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 number HY-13012; CAS number: 446859-33-2).

[0111] A83-01 (Sigma-Aldrich catalog number SML0788; APExBio catalog number A3133; MCE catalog number HY-10432; CAS number: 909910-43-6).

[0112] SB431542 (APExBio catalog number A8249, CAS number: 301836-41-9).

[0113] D4476 (Sigma-Aldrich catalog number D1944; APExBio catalog number A3342; MCE catalog number HY-10324; CAS number: 301836-43-1).

[0114] GW788388 (APExBio catalog number A8301; MCE catalog number HY-10326; CAS number: 452342-67-5).

[0115] LY364947 (Sigma-Aldrich catalog number L6293; APExBio catalog number B2287; MCE catalog number HY-31462; CAS number: 396129-53-6).

[0116] SB525334 (Sigma-Aldrich catalog number S8822; APExBio catalog number A5602; MCE catalog number HY-12043; CAS number: 356559-20-1).

[0117] SB505124 (APExBio catalog number B2289; MCE catalog number HY-13521; CAS number: 694433-59-5).

[0118] SD208 (Sigma-Aldrich Catalog No. S7071; APExBio Catalog No. A3808; MCE Catalog No. HY-10324; CAS No.: 627536-09-8).

[0119] GW6604 (absin Catalog No. abs814099; CAS No.: 452342-37-9).

[0120] According to the present disclosure, the concentration of the TGF-β inhibitor is not particularly limited as long as it does not prevent the promotion of mDAP amplification. In certain embodiments, 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.

[0121] In certain embodiments, the TGF-β inhibitor includes SB431542. In certain embodiments, SB431542 is present in the amplification 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.

[0122] In certain embodiments, the culture medium of the present disclosure may optionally further comprise glutamine or its derivatives.

[0123] In certain embodiments, the glutamine or its derivatives include L-alanyl-L-glutamine dipeptide (e.g., GLUTAMAX TM brand L-alanyl-L-glutamine dipeptide, Gibco Catalog No. 35050061), L-glutamine (e.g., Sigma-Aldrich Catalog No. G2150 / G7513; APExBio Catalog No. A8461; MCE Catalog No. HY-N0390; CAS No.: 56-85-9), or a mixture thereof.

[0124] In certain embodiments, the glutamine or its derivatives are present in the culture medium at a concentration of about 0.5% to 5% by volume, and preferably about 0.5% to 2.5% by volume.

[0125] In certain embodiments, the culture medium of the present disclosure may optionally further comprise an antioxidant.

[0126] 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 analogue 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.

[0127] In certain embodiments, the 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.

[0128] In certain embodiments, the culture medium of the present disclosure further comprises fibroblast growth factor. In certain embodiments, the fibroblast growth factor comprises FGF2 (e.g., Nuwacell), FGF1 (e.g., MCE catalog number HY-P7001), FGF8 (e.g., MCE catalog numbers HY-P7347, MCE catalog number HY-P7349, MCE catalog number HY-P7350), and / or FGF20 (e.g., R&D catalog number 2547-FG).

[0129] In certain embodiments, the fibroblast growth factor (e.g., FGF2 or FGF8) is present in the expansion 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.

[0130] In certain embodiments, the culture medium of the present disclosure further comprises a BMP4 inhibitor.

[0131] In certain embodiments, the BMP4 inhibitor comprises dorsomorphin, LDN193189, or a combination thereof. Representative structures of certain BMP4 inhibitors that can be used in the culture medium of the present disclosure are provided below, many of which are generally commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources.

[0132] LDN193189 (MCE catalog number HY-12071A, CAS number: 1062368-24-4).

[0133] Dorsomorphin (Sigma-Aldrich catalog number P5499; APExBio catalog number B3252; MCE catalog number HY-13418A; CAS number: 866405-64-3).

[0134] In certain embodiments, a BMP inhibitor (e.g., 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.

[0135] In certain embodiments, the culture medium of the present disclosure does not contain FGF8 and / or FGF2.

[0136] In certain embodiments, the culture medium of the present disclosure comprises in a basal medium: (a) a WNT signaling pathway activator at about 1 μM to about 10 μM; (b) a ROCK inhibitor at about 1 μM to about 20 μM; (c) a TGF-β inhibitor at about 1 μM to about 20 μM; and (d) a nerve growth supplement at about 0.1% to about 10% by volume.

[0137] In certain embodiments, the culture medium of the present disclosure comprises in a basal medium: (a) CHIR99021 at about 1 μM to about 10 μM; (b) Y27632 at about 1 μM to about 20 μM; (c) SB431542 at about 1 μM to about 20 μM; and (d) B27 at about 0.1% to about 10% by volume.

[0138] In certain embodiments, the culture medium of the present disclosure can provide at least 32-fold, 36-fold, 40-fold or more amplification for each generation of mDAP, which is at least 3, 4, 5, 6, 7, 8 or more times the amplification (about 5-10 fold) of the previous culture medium without TGF-β inhibitor and Rock inhibitor. In certain embodiments, the culture medium of the present disclosure can provide at least 32-fold, 36-fold, 40-fold or more amplification for each generation of mDAP, which is at least 1.6-fold, 1.8-fold, 2-fold or more times the amplification (about 20 fold) of the same culture medium without TGF-β inhibitor.

[0139] 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.

[0140] 2. Coated matrix combination for amplifying mDAP

[0141] In a second aspect, the present disclosure provides a coated matrix combination capable of promoting robust expansion of midbrain dopaminergic progenitor cells (mDAPs), the coated matrix combination comprising: (a) a first coated matrix capable of supporting the attachment of mDAP cells; and (b) a second coated matrix capable of enhancing the expression of mDAP-specific markers during mDAP expansion and passage, wherein the second coated matrix comprises a Notch agonist.

[0142] According to the second aspect, due to the inclusion of the second coated matrix, the coated matrix combination for expansion can enhance the expansion efficiency and the expression of mDAP-specific markers during mDAP expansion and passage.

[0143] According to the second aspect, the coated matrix combination of the present disclosure can be used in combination with any conventional mDAP expansion medium or any other suitable mDAP expansion medium in the art. Examples of conventional mDAP expansion media include Gibco TM PSC Dopaminergic Neuron Differentiation Kit and STEMdiff TM Dopaminergic Neuron Differentiation Kit / STEMdiff TM (#05835).

[0144] Any mDAP can be expanded using the coated matrix combination of the present disclosure. Examples of mDAPs include fetal brain-derived mDAPs; hPSC-derived mDAPs; and mDAPs obtained by transdifferentiation from other cell types. mDAPs can be derived from (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 is an ESC-derived midbrain dopaminergic progenitor cell (emDAP). In certain embodiments, the mDAP is an iPSC-derived midbrain dopaminergic progenitor cell (imDAP). In certain embodiments, the mDAP is an NPSC-derived midbrain dopaminergic progenitor cell (nmDAP). In certain embodiments, the mDAP is an EPSC-derived midbrain dopaminergic progenitor cell (epmDAP).

[0145] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be readily obtained using conventional methods (e.g., those described in the prior art) 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.

[0146] 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 TM medium (Stemcell Technologies, catalog number 85850), and sub-confluent hiPSCs are passaged onto fresh Matrigel-coated plates and cultured for an additional time (e.g., 24 hours) in iPSC medium to reach 90 - 100% confluence. Once confluent, the medium can be changed to induce mDAP for a further period (e.g., 1 - 3 days), optionally changing the medium daily as required. For example, Fedele et al., Scientific Reports 7:6036|DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0147] In certain embodiments, the first coating matrix is selected from vitronectin (VTN, e.g., Vitronectin, #RP01002), collagen (e.g., Gibco Cat. Collagen I, #A1048301; Collagen I, #17100017; Collagen type II, #17101015; Collagen type IV, #17104019), proteoglycan (e.g., Invitrogen catalog number RP-77523 / RP-77524; MCE catalog number HY-P76323 / HY-P71233), fibronectin (e.g., Invitrogen catalog number RP-43130; MCE catalog number HY-P70593 / HY-P70593G / HY-P73063), entactin (e.g., Sigma-Aldrich catalog number D8935; MCE catalog number HY-P71763), elastin (e.g., Sigma-Aldrich catalog number E7402 / E7277 / E6902; CAS number: 9007-58-3), laminin (e.g., Laminin), functional fragments of any of the foregoing 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.

[0148] 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 reagent capable of upregulating the activity and / or amount of components involved in the Notch signaling pathway). Notch receptor proteins can interact with a variety of surface-bound or secreted ligands, including but not limited to Jagged-1, Jagged-2, Delta-like ligand 1, Delta-like 3, Delta-like ligand 4, etc. After ligand binding, the Notch receptor is activated through a series of cleavage events involving members of the ADAM protease family and intramembrane cleavage regulated by the γ-secretase presinilin. As a result, the intracellular domain of Notch translocates to the nucleus, where it transcriptionally activates downstream genes.

[0149] As used herein, "Notch agonist" includes molecules that stimulate 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-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more relative to the level of Notch activity in the absence of the Notch agonist. As is known in the art, Notch activity can be measured, for example, by measuring the transcriptional activity of Notch using the 4xwtCBF1-luciferase reporter construct described by Hsieh et al. (Mol. Cell. Biol. 16:952-959, 1996, incorporated herein by reference).

[0150] In certain embodiments, the Notch agonist is selected from: Delta-like ligand 4 (DLL4, )), Delta-like ligand 1 (DLL1, e.g., MCE (HEK293, His), #HY-P7841), Jagged-1 (e.g., MCE Catalog No. HY-P1846), Jagged-2 (e.g., (JAG2) #BES23045RP), variants thereof, and any combination thereof. In certain embodiments, the Notch agonist is a DSL peptide (Dontu et al., Breast Cancer Res., 6:R605-R615, 2004).

[0151] Representative structures of certain Notch agonists that can be used in the coating matrix combinations of the present disclosure are provided below, many of which are commonly commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources.

[0152]

[0153] Jagged-1(188 - 204) (e.g., MCE Catalog No. HY-P1846; CAS No.: 219127-21-6)

[0154] In certain embodiments, the Notch agonist includes DLL4.

[0155] In certain embodiments, any particular Notch agonist mentioned herein, such as Jagged-1, Jagged-2, Delta-1, and Delta-like ligand 4, can be replaced by a homolog or a fragment thereof that is naturally, synthetically, or recombinantly produced, wherein the homolog or the fragment retains at least about 80%, 85%, 90%, 95%, 99%, and / or is replaced by a homolog or a fragment thereof having at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by any art-recognized sequence alignment software such as by global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0156] In certain embodiments, the coating matrix combination comprises VTN and a Notch agonist. In certain embodiments, the coating matrix combination comprises VTN and DLL4.

[0157] The cell culture surface can be coated with the amplification-coated matrix combination of the present disclosure using conventional techniques in the art. Generally, the first coated matrix and the second coated matrix can be first mixed in any ratio, and then the cell culture surface can be coated with the coating solution. Those skilled in the art can easily determine the coating concentrations of the first coated matrix and the second coated matrix. For example, the coating concentrations of the first coated matrix and the second coated matrix can each be 0.5 to 2 μg / cm 2 , such as 1 μg / cm 2 .

[0158] In certain embodiments, the coated matrix combination of the present disclosure can provide an amplification fold of at least 36-fold, 38-fold, 40-fold or more for each generation of mDAP, which is at least 1.8-fold, 1.9-fold, 2-fold or more of the amplification fold of a previous coated matrix such as VTN (about 20-fold).

[0159] 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.

[0160] 3. Method for amplifying mDAP

[0161] In a third aspect, the present disclosure provides a method for amplifying midbrain dopaminergic progenitor cells (mDAP), which includes contacting mDAP with an amplification culture medium on a culture surface coated with the amplification-coated matrix combination of the present disclosure.

[0162] According to the third aspect, due to the use of the second coated matrix, the above amplification method can improve the amplification efficiency and the expression of mDAP-specific markers during the amplification and passage of mDAP.

[0163] Any mDAP can be amplified using the amplification method of the present disclosure. Examples of mDAP include fetal brain-derived mDAP; hPSC-derived mDAP; and mDAP obtained by transdifferentiation from other cell types. mDAP can be derived from (e.g., differentiated from) pluripotent stem cells. Pluripotent stem cells can include induced pluripotent stem cells (e.g., hiPSC), embryonic stem cells (e.g., hESC), naive PSC (NPSC), and extended pluripotent stem cells (EPSC). In certain embodiments, mDAP is ESC-derived midbrain dopaminergic progenitor cells (emDAP). In certain embodiments, mDAP is iPSC-derived midbrain dopaminergic progenitor cells (imDAP). In certain embodiments, mDAP is NPSC-derived midbrain dopaminergic progenitor cells (nmDAP). In certain embodiments, mDAP is EPSC-derived midbrain dopaminergic progenitor cells (epmDAP).

[0164] ESC (e.g., hESC) and iPSC (e.g., hiPSC) are known in the art and can be readily obtained using conventional methods (e.g., those described in the prior art) 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 (iPSC) from somatic cells including PBMC and T cells.

[0165] Such iPSC (e.g., hiPSC) can be cultured under defined conditions to generate mDAP. For example, iPSC (e.g., hiPSC) can be cultured on Matrigel under defined conditions in mTeSR TM medium (Stemcell Technologies, catalog number 85850), and sub-confluent hiPSC can be passaged onto fresh Matrigel-coated plates and cultured for an additional time (e.g., 24 hours) in iPSC medium to reach 90 - 100% confluence. Once confluent, the medium can be changed to induce mDAP for more time (e.g., 1 - 3 days), optionally changing the medium daily as needed. For example, Fedele et al., Scientific Reports 7:6036|DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0166] According to a third aspect, any ordinary mDAP amplification medium in the art or any other suitable mDAP amplification medium can be used in the amplification method of the present disclosure. Examples of ordinary mDAP amplification media include Gibco TMPSC Dopaminergic Neuron Differentiation Kit and STEMdiff TM Dopaminergic Neuron Differentiation Kit) / STEMdiff TM (#05835).

[0167] The amplification-coated matrix combinations of the present disclosure have been described elsewhere herein (e.g., as described for the amplification-coated matrix combination of mDAP or in the second aspect herein), and for the purpose of simplification, these same descriptions are omitted herein.

[0168] In certain embodiments, the culture surface is in a culture plate, culture flask, culture bottle, or culture vessel. In certain embodiments, the culture plate includes a single layer or multi-layer cell stack, 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, 1536-well, or more wells. In certain embodiments, the culture plate includes flat-bottomed wells or round-bottomed wells.

[0169] The cell culture surface can be coated with the amplification-coated matrix combinations of the present disclosure by conventional techniques in the art. Generally, the first coated matrix and the second coated matrix can be mixed in any ratio first, and then the cell culture surface is coated with the coating solution. Those skilled in the art can easily determine the coating concentrations of the first coated matrix and the second coated matrix. For example, the coating concentrations of the first coated matrix and the second coated matrix can each be 0.5 to 2 μg / cm 2 , such as 1 μg / cm 2 .

[0170] In certain embodiments, the method further includes replacing the amplification medium with the same medium every few days during amplification. In certain embodiments, the method further includes replacing the amplification medium with the same medium every 3 days during the amplification process.

[0171] In certain embodiments, mDAP is contacted with the amplification medium for a total of about 6 days.

[0172] In some embodiments, the amplification medium of the present disclosure is used as the above-mentioned amplification medium. The amplification medium of the present disclosure used in the above embodiments has been described elsewhere herein (e.g., as the medium for amplifying mDAP or as described in the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein. According to the above embodiments, the amplification method can support the long-term amplification of mDAP in a homogeneous population (such as at least 4, 5, 6 generations or more), or even if mDAP is amplified and passaged one or more times, after amplification and passage, the amplification method can still maintain or even improve the amplification efficiency and the expression of mDAP-specific markers compared to before amplification and passage.

[0173] In certain embodiments, mDAP is amplified and passaged at least 1, 2, 3, 4, 5, or 6 times (e.g., P1, P2, P3, P4, P5, P6, or subsequent generations), preferably at least 4, 5, or 6 times (e.g., P4, P5, P6, or subsequent generations), and most preferably at least 6 times (e.g., P6, or subsequent generations).

[0174] In certain embodiments, mDAP is seeded at a density of about 1×10 3 cells / cm 2 to about 1×10 5 cells / cm 2 onto the culture surface. Compared with a higher seeding density, such a low seeding density is beneficial for increasing the amplification fold of the plated cells.

[0175] In certain embodiments, the amplification method supports the amplification of mDAP and: (1) the population doubling time (PDT) is about 25 - 28 hours and / or (2) for imDAP cells that are amplified and passaged at least 1, 2, 3, 4, 5, 6 times or more (e.g., P1 - P6), the total cell amplification fold is about 35 - 40.

[0176] In certain embodiments, the amplification method of the present disclosure can provide an amplification fold of at least 36-fold, 38-fold, 40-fold or more for each generation of mDAP, which is at least 1.8-fold, 1.9-fold, 2-fold or more of the amplification fold of the previous amplification method, such as the method using VTN (about 20-fold).

[0177] A representative example of the mDAP-specific marker is FOXA2. Representative examples of the mDAP-specific marker include FOXA2, and one or more selected from LMX1A, EN1, OTX2, and SOX6. In some embodiments, the mDAP-specific marker includes FOXA2, LMX1A, EN1, OTX2, and SOX6.

[0178] 4. Medium for maturing mDAP

[0179] In a fourth aspect, the present disclosure provides a medium (e.g., a chemically defined serum-free maturation medium) capable of promoting the maturation of mDAP (midbrain dopaminergic progenitor cells), the medium comprising: (a) a neurobasal medium; (b) human platelet lysate (hPLT); (c) transforming growth factor β (TGF-β); (d) a γ-secretase inhibitor; and (e) a cyclic adenosine compound or its cyclase activator.

[0180] In the prior art, B27 is commonly used as a medium supplement for neuronal maturation. According to the fourth aspect, by replacing B27 with hPLT, the above-mentioned maturation medium can improve the maturity of mDAP (percentage of TH+ cells).

[0181] According to the fourth aspect, the maturation medium of the present disclosure can be contacted with mDAP on a cell culture surface coated with any ordinary maturation coating matrix or any other suitable maturation coating matrix in the art. Examples of ordinary maturation coating matrices include vitronectin (VTN) and laminin / poly-L-ornithine (PLO).

[0182] Any mDAP can be matured using the maturation medium of the present disclosure. Examples of mDAP include fetal brain-derived mDAP; hPSC-derived mDAP; and mDAP obtained by transdifferentiation from other cell types. mDAP can be derived from (e.g., differentiated from) pluripotent stem cells. Pluripotent stem cells can include induced pluripotent stem cells (e.g., hiPSC), embryonic stem cells (e.g., hESC), naive PSC (NPSC), and extended pluripotent stem cells (EPSC). In certain embodiments, mDAP is ESC-derived midbrain dopaminergic progenitor cells (emDAP). In certain embodiments, mDAP is iPSC-derived midbrain dopaminergic progenitor cells (imDAP). In certain embodiments, mDAP is NPSC-derived midbrain dopaminergic progenitor cells (nmDAP). In certain embodiments, mDAP is EPSC-derived midbrain dopaminergic progenitor cells (epmDAP).

[0183] In certain embodiments, mDAP is non-amplified mDAP. In certain embodiments, mDAP is amplified mDAP, such as, for example, amplified imDAP. In certain embodiments, mDAP is mDAP that has been amplified and passaged one or more times, such as, for example, amplified P1, P2, P3, P4, P5, or P6 imDAP. In certain embodiments, mDAP is immature mDAP. In certain embodiments, mDAP is partially mature mDAP.

[0184] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be readily obtained using conventional methods (e.g., those described in the prior art) 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).

[0185] 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 TM medium (Stemcell Technologies, catalog number 85850), and sub-confluent hiPSCs are passaged onto fresh Matrigel-coated plates and cultured in iPSC medium for an additional time (e.g., 24 hours) to reach 90 - 100% confluence. Once confluent, the medium can be changed to induce mDAP for more time (e.g., 1 - 3 days), optionally changing the medium daily as needed. For example, Fedele et al., Scientific Reports 7:6036|DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0186] As the name implies, neural basal medium can support the survival, maintenance, growth, and proliferation of neural cells as a culture medium and is a basal component of a maturation medium. Generally, neural basal medium accounts for approximately 95% to 99% by volume of the maturation medium.

[0187] Examples of neural basal media include NEUROBASAL TM basal medium (e.g., Gibco catalog number 21103049), NEUROBASAL-A TM basal medium (e.g., Gibco catalog number 10888022), NEUROBASAL PLUS TM basal medium (e.g., Gibco catalog number A3582901) and / or BRAINPHYS TM basal medium (e.g., STEMCELL catalog number 05790).

[0188] hPLT is commercially available, e.g., PLTGold Human Platelet Lysate (Biological Industries, #PLTGOLD500R). However, hPLT from other sources is also available and can be used in conjunction with the present disclosure.

[0189] According to the present disclosure, the concentration of hPLT is not particularly limited as long as it does not prevent the promotion of the maturation of mDAP. In certain embodiments, hPLT is present in the culture medium at a concentration of about 0.1% to about 5% by volume, and preferably about 0.1% to about 2% by volume.

[0190] In certain embodiments, TGF-β includes TGF-β1 (e.g., APExBio Catalog No. P1039; MCE Catalog No. HY-P78168), TGF-β2 (e.g., R&D Catalog No. 302-B2), TGF-β3 (e.g., Peprotech Catalog No. 100-36E), and / or TGF-β1β2.

[0191] According to the present disclosure, the concentration of TGF-β is not particularly limited as long as it does not prevent the promotion of the maturation of mDAP. In certain embodiments, 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.

[0192] In certain embodiments, the γ-secretase inhibitor includes or is selected from the following: DAPT, N-[N-(3,5-difluorobenzoyl)]-L-alanyl-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydro-benzo[E](1,4)diazepin -2-one, LY-411575, Dihydroergocristine mesylate, BMS299897, and any combination thereof.

[0193] Representative structures of certain γ-secretase inhibitors that can be used in the culture medium of the present disclosure are provided below, many of which are generally commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources (although these sources are not limiting).

[0194] DAPT (MCE Catalog No. HY-13027, CAS No.: 208255-80-5).

[0195] Compound E (N-[N-(3,5-difluorobenzoyl)]-L-alanyl-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydro-benzo[E](1,4)diazepin -2-one) (APExBio Catalog No. C3341; MCE Catalog No. HY-14176; CAS No.: 209986-17-4).

[0196] LY-411575 (Sigma-Aldrich catalog number SML0506; APExBio catalog number A4019; MCE catalog number HY-50752; CAS number: 209984-57-6).

[0197] Dihydroergocristine mesylate (APExBio catalog number B6313; MCE catalog number HY-N2319; CAS number: 24730-10-7).

[0198] BMS299897 (Sigma-Aldrich catalog number SML0210; APExBio catalog number A4400; MCE catalog number HY-50883; CAS number: 290315-45-6).

[0199] In certain embodiments, the γ-secretase inhibitor includes DAPT.

[0200] According to the present disclosure, the concentration of the γ-secretase inhibitor is not particularly limited as long as it does not prevent 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, and preferably about 5 μM to about 20 μM.

[0201] As used herein, a cyclic adenosine monophosphate compound is a cell-permeable cAMP compound that directly increases the intracellular cAMP content. In certain embodiments, the cyclic adenosine monophosphate compound includes cAMP, or a derivative or salt thereof. Examples of derivatives or salts of cAMP include sodium Db-cAMP, sodium 8-bromo-cAMP, 8-chloro-cAMP, sodium 6-Bnz-cAMP, and the calcium salt of Bucladesine (calcium dibutyryl cAMP). In addition, a cyclase activator of the cyclic adenosine monophosphate compound can also be used in the maturation medium of the present disclosure to produce intracellular c-AMP. Examples of cyclase activators include forskolin and NKH477.

[0202] In certain embodiments, the cyclic adenosine monophosphate compound or its cyclase activator includes or is selected from sodium Db-cAMP, cAMP, forskolin, sodium 8-bromo-cAMP, NKH477, 8-chloro-cAMP, sodium 6-Bnz-cAMP, the calcium salt of Bucladesine, and any combination thereof.

[0203] Representative structures of certain cyclic adenosine monophosphate compounds or their cyclase activators in the maturation medium useful for the present disclosure are provided below, many of which are generally commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources (although these sources are not limiting).

[0204] cAMP (Sigma-Aldrich catalog number 20-198; MCE catalog number HY-B1511, CAS number: 60-92-4).

[0205] Db-cAMP sodium salt (Sigma catalog number D0627, CAS number: 16980-89-5).

[0206] Forskolin (MCE catalog number HY-15371; CAS number: 66575-29-9).

[0207] 8-Bromo-cAMP sodium salt (Sigma-Aldrich catalog number B7880; APExBio catalog number B9000; MCE catalog number HY-12306; CAS number: 76939-46-3).

[0208] 8-Chloro-cAMP (MCE catalog number HY-123396; CAS number: 41941-56-4).

[0209] 6-Bnz-cAMP sodium salt (MCE catalog number HY-103322; CAS number: 1135306-29-4).

[0210] Bucladesine calcium salt (MCE catalog number HY-B0764A; CAS number: 938448-87-4).

[0211] NKH477 (Sigma-Aldrich catalog number N3290; MCE catalog number HY-103193; CAS number: 138605-00-2).

[0212] In certain embodiments, the cyclic adenosine monophosphate compound or its cyclase activator includes Db-cAMP sodium salt.

[0213] In certain embodiments, the cyclic adenosine monophosphate compound (e.g., Db-cAMP sodium salt) or its cyclase activator is present in the medium at a concentration of about 0.1 mM to about 5 mM, and preferably about 0.1 mM to about 2 mM.

[0214] Optionally, the culture medium may contain (f) neurotrophic factors. Neurotrophic factors contain ligands for membrane receptors that promote neuronal survival and maintenance of function.

[0215] In certain embodiments, the neurotrophic factors include nerve growth factor (NGF, e.g., Sigma - Aldrich catalog number N8898; MCE catalog (Beta - NGF) numbers 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) numbers 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).

[0216] In certain embodiments, the neurotrophic factors include or are selected from BDNF, GDNF, and both.

[0217] According to the present disclosure, the concentration of the neurotrophic factor is not particularly limited as long as it does not prevent the promotion of the maturation of mDAP. 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.

[0218] In certain embodiments, BDNF 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.

[0219] In certain embodiments, the culture medium further contains (g) Rho - kinase (ROCK) inhibitor.

[0220] ROCK inhibitors include reagents that inhibit the binding between ROCK and its receptor.

[0221] In certain embodiments, the ROCK inhibitors include or are selected from: Y27632, HA100, HA1152, HA - 1077, and any combination thereof.

[0222] 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-piperazinesulfonyl)-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-diaz 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).

[0223] Representative structures of certain ROCK inhibitors that can be used in the maturation media of the present disclosure are provided below, many of which are generally commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources (although these sources are not limiting).

[0224] Y-27632 (MCE catalog number HY-10071, CAS number: 146986-50-7).

[0225] HA-100 (hydrochloride) (absin catalog number abs47045575).

[0226] H1152 (MCE catalog number HY-15720, CAS number: 451462-58-1).

[0227] HA-1077 (fasudil / AT877) (MCE catalog number HY-10341A, CAS number: 103745-39-7).

[0228] In certain embodiments, the ROCK inhibitor includes Y27632. In certain embodiments, the ROCK inhibitor (e.g., Y27632) is present in the maturation media 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.

[0229] In certain embodiments, hPLT is heat-treated human platelet lysate (HhPLT). According to the above embodiments, HhPLT can further improve the maturation of mDAP compared to hPLT. While not wishing to be bound by any particular theory, it is believed that HhPLT can reduce batch-to-batch variability compared to hPLT.

[0230] According to the present disclosure, HhPLT can be prepared by heat-treating hPLT. In certain embodiments, HhPLT is produced by first centrifuging hPLT, heating hPLT at about 45 - 60 °C, and then centrifuging hPLT. In certain embodiments, hPLT is centrifuged at 3000 g for 30 minutes at 4 °C to obtain a supernatant, which is heated to 56 °C for 30 minutes, and finally the suspension is cooled to 4 °C for at least 5 minutes and centrifuged at 3000 g for 30 minutes at 4 °C, and aliquots are prepared and stored at -80 °C until use.

[0231] According to the present disclosure, the concentration of HhPLT is not particularly limited as long as it does not prevent the promotion of mDAP maturation. In certain embodiments, HhPLT is present in the culture medium at a concentration of about 0.1% to about 5% by volume, and preferably about 0.1% to about 2% by volume.

[0232] In certain embodiments, the maturation medium of the present disclosure further comprises a (h)WNT signaling pathway inhibitor. According to the above embodiments, the medium can further improve the maturation of mDAP compared to a medium without a WNT signaling pathway inhibitor.

[0233] As used herein, a Wnt signaling pathway inhibitor refers to an antagonist of the Wnt signaling pathway (e.g., a reagent capable of downregulating the activity and / or amount of components involved in the Wnt signaling pathway), and can be interchangeable with "Wnt signaling pathway antagonist", "Wnt antagonist", "Wnt pathway inhibitor", or "Wnt inhibitor". Wnt signaling pathway inhibitors can include, for example, reagents that antagonize one or more human FZD proteins, FZD binders. The FZD binder can be an antibody or a polypeptide.

[0234] Examples of Wnt signaling pathway inhibitors include, but are not limited to, one or more of the following: polypeptides comprising the amino acid sequence of a Wnt antagonist, small organic molecules that inhibit Wnt / β-catenin signaling, small organic molecules that inhibit the expression or activity of a Wnt agonist, antibodies that bind and inhibit the activity of a Wnt agonist, and preferably small organic molecules that inhibit Wnt / β-catenin signaling, and small organic molecules that inhibit the expression or activity of a Wnt agonist.

[0235] In certain embodiments, the WNT signaling pathway inhibitor comprises or is selected from IWR1, iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, Ciclopirox, Cardamonin, diethyl benzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, XAV-939, Foscenvivint (ICG-001), crizotinib, isoquercitrin, Gigantol, JW55, MSAB, KY02111, FH535, WIKI4, CCT251545, Prodigiosin, KYA1797K, NCB-0846, LF3, iCRT14, Adavivint, Triptonide, M435-1279, and any combination thereof.

[0236] Representative structures of certain Wnt signaling pathway inhibitors that can be used in the maturation media of the present disclosure are provided below, many of which are commonly commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources (although these sources are not limiting).

[0237]

[0238] IWR1 (MCE catalog number HY-12238, CAS number: 1127442-82-3).

[0239] iCRT3 (Sigma-Aldrich catalog number SML0211 / 219332; MCE catalog number HY-103705; CAS number: 901751-47-1).

[0240] IWP-O1 (Sigma-Aldrich catalog number SML1962; MCE catalog number HY-100853; CAS number: 2074607-48-8).

[0241] IWP-2 (Sigma-Aldrich catalog number I0536; APExBio catalog number A3512; CAS number: 686770-61-6).

[0242] IWP-3 (APExBio catalog number C3254; MCE catalog number HY-100536; CAS number: 687561-60-0).

[0243] IWP-4 (Sigma-Aldrich Catalog No. SML1114; APExBio Catalog No. B4922; CAS No.: 686772-17-8).

[0244] Ciclopirox (Sigma-Aldrich Catalog No. SML2011; APExBio Catalog No. B2087; MCE Catalog No. HY-B0450; CAS No.: 29342-05-0).

[0245] Cardamonin (Sigma-Aldrich Catalog No. C8249; APExBio Catalog No. B7085; MCE Catalog No. HY-N0279; CAS No.: 18956-16-6).

[0246] Diethyl benzylphosphonate (Sigma-Aldrich Catalog No. D91071; CAS No.: 1080-32-6).

[0247] Pamidronate disodium hydrate (Sigma-Aldrich Catalog No. P2371; APExBio Catalog No. A2456; MCE Catalog No. HY-B0012A; CAS No.: 57248-88-1

[0248] Ginsenoside Rh4 (LeiMeiTian Medicine Catalog No. DR0020; CAS No.: 174721-08-5).

[0249] KY-05009 (Sigma-Aldrich Catalog No. SML1506; MCE Catalog No. HY-05009; CAS No.: 1228280-29-2).

[0250] XAV-939 (Sigma-Aldrich Catalog No. X3004; APExBio Catalog No. A1877; MCE Catalog No. HY-14428; CAS No.: 780757-88-2).

[0251] Foscenvivint (ICG-001) (APExBio Catalog No. A8217; MCE Catalog No. HY-15147; CAS No.: 284028-89-3).

[0252] Cabozantinib (MCE Catalog No. HY-13404; CAS No.: 1029712-80-8).

[0253] Isoquercitrin (APExBio Catalog No. N1945; MCE Catalog No. HY-N0768; CAS No.: 21637-25-2).

[0254] Gigantol (Sigma-Aldrich, #SML2036; MCE Catalog No. HY-N2523; CAS No.: 67884-30-4).

[0255] JW55 (Sigma-Aldrich Catalog No. SML0630; APExBio Catalog No. A4529; MCE Catalog No. HY-13968; CAS No.: 664993-53-7).

[0256] MSAB (Sigma-Aldrich Catalog No. SML1726; MCE Catalog No. HY-120697; CAS No.: 173436-66-3).

[0257] KY02111 (Sigma-Aldrich Catalog No. SML0948; APExBio Catalog No. A8213; MCE Catalog No. HY-13815; CAS No.: 1118807-13-8).

[0258] FH535 (Sigma-Aldrich Catalog No. F5682; APExBio Catalog No. A3413; MCE Catalog No. HY-15721; CAS No.: 108409-83-2).

[0259] WIKI4 (APExBio Catalog No. A3413; MCE Catalog No. HY-16910; CAS No.: 838818-26-1).

[0260] CCT251545 (APExBio Catalog No. A5979; MCE Catalog No. HY-12681; CAS No.: 1661839-45-7).

[0261] Prodigiosin (APExBio Catalog No. C3112; MCE Catalog No. HY-100711; CAS No.: 108409-83-2).

[0262] KYA1797K (Sigma-Aldrich Catalog No. SML1831; MCE Catalog No. HY-101090; CAS No.: 1956356-56-1).

[0263] NCB-0846 (MCE Catalog No. HY-100830; CAS No.: 1792999-26-8).

[0264] LF3 (Sigma-Aldrich Catalog No. SML1752; MCE Catalog No. HY-101486; CAS No.: 1956356-56-1).

[0265] iCRT14 (TOCRIS (R&D) Catalog No. 677331-12-3).

[0266] Adavivint (MCE Catalog No. HY-109049; CAS No.: 1467093-03-3).

[0267] Triptonide (Sigma-Aldrich Catalog No. SMB00325; APExBio Catalog No. A3892; MCE Catalog No. HY-32736; CAS No.: 38647-11-9).

[0268] M435-1279 (MCE Catalog No. HY-141891; CAS No.: 1359431-16-5).

[0269] In certain embodiments, the Wnt signaling pathway inhibitor includes IWR1.

[0270] In certain embodiments, the Wnt signaling pathway inhibitor (e.g., IWR1) is present in the maturation medium at about 0.25 μM to about 10 μM, and preferably about 0.5 μM to about 5 μM.

[0271] In certain embodiments, the maturation medium of the present disclosure comprises, in neural basal medium: (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 cyclic adenosine compound or its cyclase activator; and (e) about 10 ng / ml to about 80 ng / ml of a neurotrophic factor.

[0272] In certain embodiments, the maturation medium of the present disclosure comprises, in a neural basal medium: (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 cyclic adenosine phosphate compound or its cyclase activator; (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.

[0273] In certain embodiments, the maturation medium of the present disclosure comprises, in a neural basal medium: (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 cyclic adenosine phosphate compound or its cyclase activator; (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.

[0274] In certain embodiments, the maturation medium of the present disclosure comprises, in a neural basal medium: (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) a combination of about 10 ng / ml to about 80 ng / ml of BDNF and GDNF.

[0275] In certain embodiments, the maturation medium of the present disclosure comprises, in a neural basal medium: (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) a combination of about 10 ng / ml to about 80 ng / ml of BDNF and GDNF; and (f) about 5 μM to about 15 μM of Y27632.

[0276] In certain embodiments, the maturation medium of the present disclosure comprises, in a neural basal medium: (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) a combination of about 10 ng / ml to about 80 ng / ml 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.

[0277] In certain embodiments, the maturation medium of the present disclosure may optionally further comprise glutamine or a derivative thereof.

[0278] In certain embodiments, glutamine or a derivative thereof includes the dipeptide L-alanyl-L-glutamine (e.g., GLUTAMAX TM 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.

[0279] 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, and preferably about 0.5% to 2.5% by volume.

[0280] In certain embodiments, the maturation medium of the present disclosure may optionally further comprise an antioxidant.

[0281] In certain embodiments, the antioxidant includes ascorbic acid (e.g., Sigma catalog number A8960) or a salt thereof (e.g., Na salt, Mg salt) or a derivative SOD (e.g., Sigma catalog numbers S7571, S9697, S5395, S8160, S9636, S8409, S7446, CAS number: 9054-89-1), or a mixture thereof.

[0282] In certain embodiments, the antioxidant (e.g., 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.

[0283] In certain embodiments, the maturation medium is capable of enhancing the expression of TH. In certain embodiments, the maturation medium is capable of enhancing the expression of TH and one or more selected from LMX1A, EN1, OTX2, FOX2, NURR1, and SOX6. In certain embodiments, the maturation medium is capable of enhancing the expression of TH, LMX1A, EN1, OTX2, FOX2, NURR1, and SOX6.

[0284] 5. Method for maturing mDAP

[0285] In a fifth aspect, the present disclosure provides a method for promoting the maturation of midbrain dopaminergic progenitor cells (mDAPs), which comprises contacting the mDAPs with a maturation medium containing a ROCK inhibitor on a culture surface coated with a coating matrix combination, the coating matrix combination comprising: (a) a first coating matrix capable of supporting the adhesion of mDAP cells, wherein the first coating matrix is not laminin; and (b) a second coating matrix capable of enhancing the maturity of mDAPs, wherein the second coating matrix comprises a polylysine (PL)-like compound and / or a polyornithine (PO)-like compound.

[0286] Existing maturation methods generally use a combination of laminin / PLO as the matrix for mDA neuron maturation, but it is too costly for large-scale mDA neuron production. According to the fifth aspect, by using a ROCK inhibitor in combination with the first coating matrix and the second coating matrix, the above-mentioned maturation method can enhance the maturity (percentage of TH+ cells) of mDAPs while reducing production costs. In addition, the maturation method of the present disclosure can achieve a yield comparable to that of the maturation method using laminin / PLO. Therefore, the maturation method of the present disclosure is more suitable for large-scale mDA neuron production.

[0287] Any mDAP can be matured using the maturation method of the present disclosure. Examples of mDAPs include fetal brain-derived mDAPs; hPSC-derived mDAPs; and mDAPs obtained by transdifferentiation from other cell types. mDAPs can be derived from (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 is an ESC-derived midbrain dopaminergic progenitor cell (emDAP). In certain embodiments, the mDAP is an iPSC-derived midbrain dopaminergic progenitor cell (imDAP). In certain embodiments, the mDAP is an NPSC-derived midbrain dopaminergic progenitor cell (nmDAP). In certain embodiments, the mDAP is an EPSC-derived midbrain dopaminergic progenitor cell (epmDAP).

[0288] In certain embodiments, the mDAP is non-amplified mDAP. In certain embodiments, the mDAP is amplified mDAP, such as, for example, amplified imDAP. In certain embodiments, the mDAP is mDAP that has been amplified and passaged one or more times, such as, for example, amplified P1, P2, P3, P4, P5, or P6 imDAP. In certain embodiments, the mDAP is immature mDAP. In certain embodiments, the mDAP is partially mature mDAP.

[0289] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be readily obtained using conventional methods (e.g., those described in the prior art) 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.

[0290] 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 TM medium (Stemcell Technologies, catalog number 85850), and sub-confluent hiPSCs are passaged onto fresh Matrigel-coated plates and cultured in iPSC medium for an additional time (e.g., 24 hours) to reach 90 - 100% confluence. Once confluent, the medium can be changed to induce mDAP for a further time (e.g., 1 - 3 days), optionally changing the medium daily as needed. See, for example, Fedele et al., Scientific Reports 7:6036 | DOI:10.1038 / s41598-017-05633-1 (2017, incorporated by reference).

[0291] According to the present disclosure, the maturation medium containing a ROCK inhibitor is not particularly limited as long as the maturation medium 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 TM PSC Dopaminergic Neuron Differentiation Kit and STEMdiff TMDopaminergic Neuron Differentiation Kit) / STEMdiff TM (#08530).

[0292] In certain embodiments, the maturation medium of the present disclosure is used as a maturation medium containing a ROCK inhibitor. The maturation medium of the present disclosure used in the above embodiments has been described elsewhere herein (e.g., as the medium for mature mDAP or as described in the fourth aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein. According to the above embodiments, the maturation method can further improve the maturity of mDAP (percentage of TH+ cells).

[0293] In certain embodiments, the culture surface is located in a culture plate, culture flask, culture bottle, or culture container. In certain embodiments, the culture plate includes a single layer or multiple layers of cell stacks, 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, 1536-well, or more wells. In certain embodiments, the culture plate includes flat-bottomed wells or round-bottomed wells.

[0294] The coating matrix combination used in the fifth aspect of the present disclosure comprises a first coating matrix and a second coating matrix. The first coating matrix is not particularly limited as long as it is not laminin and can support the adhesion of mDAP cells. The second coating matrix can improve the maturity of mDAP and comprises a polylysine (PL)-like compound and / or a polyornithine (PO)-like compound.

[0295] In certain embodiments, the first coating matrix comprises or is selected from: vitronectin (VTN), collagen, proteoglycan, fibronectin, nestin, elastin, a functional fragment of any of the foregoing proteins, hyaluronic acid, gelatin, and any combination thereof. In certain embodiments, the first coating matrix comprises VTN.

[0296] According to the present disclosure, the polylysine-like compound can include polylysine, its derivatives, or its salts, all of which can play a role in improving the maturity of mDAP. Examples of polylysine include but are not limited to poly-L-lysine and poly-D-lysine. Examples of derivatives of polylysine include but are not limited to substituted polylysines, such as, for example, 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.

[0297] According to the present disclosure, polyornithine compounds can include polyornithine, its derivatives, or its salts, all of which can play a role in improving the maturation of mDAP. Examples of polyornithine include, but are not limited to, poly-L-ornithine and poly-D-ornithine. Examples of derivatives of polyornithine include, but are not limited to, substituted polyornithine, such as, for example, DBCO-PEG-polyornithine and chitosan-PEG-polyornithine, Con A-PEG-polyornithine, alginate-PEG-polyornithine, and vitamin E-PEG-polyornithine. Examples of salts of polyornithine or its derivatives include, but are not limited to, poly-L-ornithine hydrobromide, poly-L-ornithine hydrochloride, and salts of substituted polyornithine.

[0298] In certain embodiments, the second coating matrix comprises poly-L-lysine hydrobromide, poly-L-ornithine hydrobromide, or a mixture thereof. Representative structures of the second coating matrix for use in the maturation methods of the present disclosure are provided below, many of which are commonly commercially available from multiple sources and are labeled with catalog numbers from such selected commercial sources.

[0299] Poly-L-ornithine hydrobromide (Sigma catalog number P3655, CAS number: 27378-49-0).

[0300] Poly-L-lysine hydrobromide (Sigma catalog number P2636, CAS number: 25988-63-0).

[0301] In certain embodiments, the coating matrix combination comprises VTN, and one or both of poly-L-lysine hydrobromide and poly-L-ornithine hydrobromide.

[0302] The cell culture surface can be coated with the maturation coating matrix combination of the present disclosure by conventional techniques in the art. Generally, the cell culture surface can be coated first with a coating solution containing the second coating matrix and then with a coating solution containing the first coating matrix. Optionally, a washing treatment (e.g., using DPBS) can be performed after coating the cell culture surface with the second coating matrix. Those skilled in the art can easily determine the coating concentrations of the first coating matrix and the second coating matrix. For example, the coating concentrations of the first coating matrix can each be 0.5 to 2 μg / cm 2 , such as 1 μg / cm 2 , and the coating concentrations of the first coating matrix can each be 1 to 2 μg / cm 2 , such as 1 μg / cm 2 .

[0303] In certain embodiments, mDAP is at about 1×10 3 cells / cm2 to about 1 × 10 6 cells / cm 2 (e.g., about 5 × 10 5 cells / cm 2 ) are seeded on the culture surface.

[0304] In certain embodiments, the culture surface is located in a culture plate, culture flask, culture bottle, or culture vessel. In certain embodiments, the culture plate includes a single layer or multiple layers of cell stacks, 6 - well, 12 - well, 24 - well, 48 - well, 96 - well, 384 - well, 1536 - well, or more wells. In certain embodiments, the culture plate includes flat - bottomed wells or round - bottomed wells.

[0305] In certain embodiments, mDAP is contacted with a maturation medium containing a ROCK inhibitor for a total of about 6 days.

[0306] In certain embodiments, the maturation medium containing a ROCK inhibitor does not contain a Wnt signaling pathway inhibitor, and the method further includes replacing the maturation medium with the same medium every few days during maturation. In certain embodiments, the maturation medium containing a ROCK inhibitor does not contain a Wnt signaling pathway inhibitor, and the method further includes replacing the maturation medium with the same medium every 3 days during maturation.

[0307] In certain embodiments, the maturation medium containing a ROCK inhibitor contains a Wnt signaling pathway inhibitor, and the method further includes replacing the maturation medium with the same medium without any Wnt signaling pathway inhibitor every few days during maturation. In certain embodiments, the maturation medium containing a ROCK inhibitor contains a Wnt signaling pathway inhibitor, and the method further includes replacing the maturation medium with the same medium without any Wnt signaling pathway inhibitor every 3 days during maturation.

[0308] In certain embodiments, the maturation method can increase the expression of TH. In certain embodiments, the maturation method can increase the expression of TH and one or more selected from LMX1A, EN1, OTX2, FOX2, NURR1, and SOX6. In certain embodiments, the maturation method can increase the expression of TH, LMX1A, EN1, OTX2, FOX2, NURR1, and SOX6.

[0309] 6. Cell population

[0310] A substantially homogeneous population of mDAPs can be provided by using the amplification methods of the present disclosure. Thus, in a sixth aspect, the present disclosure provides a substantially homogeneous population of mDAPs produced by the amplification methods of the present disclosure. The amplification methods of the present disclosure have been described elsewhere herein (e.g., as described in the method for amplifying mDAPs herein or in the third aspect), and for the purpose of simplicity, these same descriptions are omitted herein. The substantially homogeneous population of mDAPs can be amplified P1, P2, P3, P4, P5, or P6 imDAPs. The amplified imDAPs at early and late passages can maintain a similar high ability to differentiate into mDA neurons.

[0311] 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.

[0312] In certain embodiments, the mDAP population of the present disclosure can be cryopreserved or stored for further amplification, maturation, and / or differentiation.

[0313] A substantially homogeneous population of mDA neurons (mDANs) can be provided from mDAPs by using the maturation methods of the present disclosure. Thus, in a seventh aspect, the present disclosure provides a substantially homogeneous population of mDANs produced by the maturation methods of the present disclosure. The maturation methods of the present disclosure have been described elsewhere herein (e.g., as described in the method for maturing mDAPs herein or in the fifth aspect), and for the purpose of simplicity, these same descriptions are omitted herein. The mDANs can be in an early or late stage of maturation. The mDANs in the early stage of maturation have the potential for terminal maturation.

[0314] 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.

[0315] The mDAN population of the present disclosure can be cryopreserved or stored before use. The mDAN can be used for further drug research, drug screening, and clinical and therapeutic applications.

[0316] 7. Kit

[0317] In an eighth aspect, the present disclosure provides a kit comprising the amplification medium of the present disclosure described herein. The amplification medium of the present disclosure has also been described elsewhere herein (e.g., as the medium for amplifying mDAP or as described in the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein. Optionally, the kit further comprises the amplification coated matrix combination of the present disclosure described herein. The amplification coated matrix combination of the present disclosure has been described elsewhere herein (e.g., as the coated matrix combination for amplifying mDAP or as described in the second aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein. When the kit comprises the amplification medium and the amplification coated matrix combination, they are packaged separately.

[0318] 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 has been described elsewhere herein (e.g., as the medium for maturing mDAP or as described in the fourth aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein. Optionally, the kit further comprises the maturation coated matrix combination of the present disclosure described herein. The maturation coated matrix combination of the present disclosure has been described elsewhere herein (e.g., as the method for maturing mDAP or as described in the fifth aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein. When the kit comprises the maturation medium and the maturation coated matrix combination, they are packaged separately.

[0319] It should be understood that any aspect or embodiment of the present disclosure described herein, including those described only in the examples or claims, can be combined with any one or more other aspects and / or embodiments of the present disclosure, unless such combination is inappropriate or expressly denied. Examples

[0320] 1. Materials

[0321] All reagents and instruments used in the examples throughout the present disclosure are commercially available. The sources of these reagents and instruments have also been described elsewhere herein.

[0322] 2. General test methods

[0323] (1) Flow cytometry analysis

[0324] 1. Dissociate and collect cells by treatment with Accutase or TrypLE.

[0325] 2. Wash the cells with 1 ml PBS and pellet the cells at 250 x g for 15 seconds.

[0326] 3. Remove the supernatant, add ~200 μl of 4% PFA, mix and fix at RT for 10 minutes.

[0327] 4. Pellet the cells at 350 x g for 5 minutes.

[0328] 5. Wash the cells with 1 ml of FACS buffer and pellet the cells at 350 x g for 5 minutes.

[0329] 6. Remove the supernatant, add 200 μl / tube of FACS buffer / 0.1% Triton X, mix at RT for 10 minutes.

[0330] 7. Wash the cells with 1 ml of FACS buffer and pellet the cells at 350 x g for 5 minutes.

[0331] 8. Remove the supernatant, add 200 μl / tube of primary antibody diluted in FACS buffer, mix gently and incubate at RT for 30 minutes.

[0332] 9. Wash the cells with 1 ml of FACS buffer and pellet the cells at 350 x g for 5 minutes.

[0333] 10. Remove the supernatant, add 200 μl of secondary antibody diluted in FACS buffer, mix gently and incubate at RT for 30 minutes.

[0334] 11. Wash the cells with 1 ml of FACS buffer and pellet the cells at 300 x g for 5 minutes.

[0335] 12. Remove the supernatant, add 200 μl of FACS buffer and run the sample on a flow cytometer.

[0336] (2) qRT-PCR

[0337] 1. For qRT-PCR analysis of mRNA gene expression, RNA should be harvested from at least 2 x 10 6 cells.

[0338] 2. Harvest the cells, add 350 μl of buffer RLT + 1% (vol / vol) β-mercaptoethanol to the wells, transfer the lysate to a 1.5 ml tube and immediately place on ice.

[0339] 3. RNA extraction was performed using the Tiangen Kit (DP430), following the manufacturer's protocol "Purification of total RNA from animal and human cells".

[0340] 4. After RNA extraction was completed, the RNA concentration was measured using a Nanodrop instrument.

[0341] 5. The HiScript TMII Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, #R223-01) was used for the first-strand cDNA synthesis reaction with 1 μg of RNA for RT-qPCR. The manufacturer's "First-Strand cDNA Synthesis" protocol was followed. This reaction would produce a 20 μl product.

[0342] 6. 10 μl of the cDNA product was diluted in 80 μl of nuclease-free water.

[0343] 7. The diluted cDNA product (1 μl), Sybr green master mix (5 μl, Transgen, #AQ131), and 0.95 μM reverse / forward primer mix (4 μl) were pipetted into a 384-well PCR plate in triplicate, either manually or using an automated pipetting robot. The complete primer set included the target gene and the housekeeping gene (RPL13A). Undifferentiated hPSC samples were also included in the analysis to determine the gene expression levels relative to the expression in hPSCs.

[0344] 8. The samples were analyzed by running quantitative PCR on a LightCycler 480 instrument using a 40x cycle two-step protocol (60 °C, 60 s annealing / extension step and 95 °C, 30 s denaturation step). The ΔΔCT method was used, and the relative gene expression was calculated using the average CT values from three technical replicates. For each gene, the housekeeping gene was used for normalization to calculate the fold change (FC) of the differentiated samples relative to the undifferentiated control samples. Subsequently, the average value of the FC was calculated based on the housekeeping gene.

[0345] (3) Immunocytochemistry analysis

[0346] 1. Cell fixation: The medium was removed from the cells and the cells were washed with PBS. 4% (wt / vol) PFA was added (e.g., 200 μl to one well in a 48-well plate) and incubated at room temperature (RT) for 15 minutes. After incubation, the cells were washed three times in PBS.

[0347] 2. Blocking: Remove PBS from the wells and add blocking solution to the cells, adding sufficient volume to cover the cells. Incubate the cells in the blocking solution for 1 hour at RT.

[0348] 3. Primary antibody incubation: Remove the blocking solution from the cells and add the primary antibody solution (~100 μl / cm 2 ). Incubate on an orbital shaker for 1 hour at RT.

[0349] 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 (~100 μl / cm 2 ), wrap the plate with aluminum foil to avoid fluorophore bleaching, and incubate on an orbital shaker for 1 hour at RT.

[0350] 5. DAPI incubation: Remove the secondary antibody solution and wash the cells twice in PBS. Add the DAPI solution (1:1000) to the cells (~100 μl / cm 2 ), wrap the plate with aluminum foil to avoid fluorophore bleaching, and incubate on an orbital shaker for 10 minutes at RT.

[0351] 6. Wash the cells three times in PBS and keep the plate wrapped with aluminum foil at 4 °C until analysis.

[0352] 7. Analyze the immunocytochemically stained cells using a fluorescence microscope and evaluate the number of positive cells.

[0353] Examples 1 - 5: Development and validation of an imDAP expansion medium based on the LDN193189 / CHIR99021 / FGF8b / Brefeldin combination

[0354] It has been previously shown that LDN193189, CHIR99021, Brefeldin, and FGF8b are necessary to expand mDAP into a homogeneous population. However, it was unclear whether the expanded imDAP retained its properties in an expansion medium supplemented with Brefeldin, LDN193189, CHIR99021, and FGF8b. This example shows that the expansion medium based on this combination can only expand imDAP with limited expansion efficiency, especially it cannot support long-term expansion of imDAP while maintaining the imDAP-specific phenotype.

[0355] Example 1

[0356] Generate imDAP according to the method described in CN201910169525.0. In the absence or presence of 5 μM Brefeldin, seed imDAP (P0) at a density of 1×10 5 cells / cm 2 onto VTN-coated plates (1 μg / cm 2)In the upper amplification medium (50% DMEM / F12, 50% Neurobasal TM medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 3 μM CHIR99021, 100 ng / mL FGF8b). In the presence of Bredinin, Bredinin was removed 24 hours after plating or was present throughout the culture period. The medium was changed daily for 5 days.

[0357] The results are as Figure 1A shown. Figure 1A (Left panel) shows the morphology of the amplified cells in the absence of Bredinin. Figure 1A (Middle panel) shows the morphology of the amplified cells when Bredinin was removed 24 hours after plating, Figure 1A (Right panel) shows the morphology of the amplified cells in the presence of Bredinin throughout the culture period. Clearly, compared to the medium without Bredinin or with Bredinin added only on the plating day, addition of Bredinin throughout the culture process supports better imDAP amplification.

[0358] Example 2

[0359] imDAP was generated according to the method described in CN201910169525.0. imDAP (P0) was seeded at a density of 1×10 5 cells / cm 2 onto VTN-coated plates (1 μg / cm 2 ) in the upper amplification medium (50% DMEM / F12, 50% Neurobasal TM medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 3 μM CHIR99021, 100 ng / mL FGF8b, 5 μM Bredinin) in the absence or presence of 0.2 μM LDN193189. The medium was changed daily for 5 days.

[0360] The results are as Figure 1B shown. Clearly, compared to imDAP cultured with LDN193189, imDAP cultured without LDN193189 showed a flatter morphology.

[0361] Example 3

[0362] imDAP was generated according to the method described in CN201910169525.0. imDAP (P0) was seeded at a density of 1×10 5 cells / cm 2 onto VTN-coated plates (1 μg / cm2 ) in amplification medium (50% DMEM / F12, 50% Neurobasal TM medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 100 ng / mL FGF8b, 5 μM Brefeldin) with different concentrations of CHIR99021 (0, 1.25, 3 μM). The medium was changed daily for 5 days. Cells were collected for RT-qPCR analysis.

[0363] The results are as Figure 1C shown. qRT-PCR analysis of amplified imDAPs showed that treatment with 3 μM CHIR99021 during amplification allowed better maintenance of mDAP marker expression (EN1, LMX1A, FOXA2).

[0364] Example 4

[0365] imDAPs were generated according to the method described in CN201910169525.0. imDAPs (P0) were seeded at a density of 1×10 5 cells / cm 2 onto VTN-coated plates (1 μg / cm 2 ) in amplification medium (50% DMEM / F12, 50% Neurobasal TM medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 3 μM CHIR99021, 5 μM Brefeldin) in the absence or presence of 100 ng / mL FGF8b. The medium was changed daily for 5 days. Cells were collected for RT-qPCR analysis.

[0366] The results are as Figure 1D shown. qRT-PCR analysis of amplified imDAPs showed that treatment with FGF8b during amplification allowed increased mDAP marker expression (EN1 and LMX1A) compared to treatment without FGF8b, but no increase in FOXA2 expression was observed.

[0367] Example 5

[0368] imDAPs were generated according to the method described in CN201910169525.0. imDAPs (P0) were seeded at a density of 1×10 5 cells / cm 2 onto VTN-coated plates (1 μg / cm 2 ) in amplification medium (50% DMEM / F12, 50% Neurobasal TMIn a culture medium containing 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 3 μM CHIR99021, 100 ng / mL FGF8b, and 5 μM Blebbistatin. The culture medium was changed daily. When imDAP reached 100% confluence (4 - 5 days), the cells were enzymatically passaged with 1×TrypLE, centrifuged at 250 g for 5 minutes, counted, and replated at a density of 1×10 5 cells / cm 2 onto a new VTN-coated plate (1 μg / cm 2 ) in the expansion medium. imDAP was expanded for 6 consecutive passages. At each passage, imDAP was harvested at an expansion fold of 5 - 10 (relative to the initial number of imDAP seeded).

[0369] The typical cell morphologies of each passage (P1 - P6) are shown as Figure 1E indicated. The population doubling times (PDTs) of each passage (P1 - P5) are shown as Figure 1F indicated. Apparently, from P1 to P3, the PDT remained at a high value of about 48 hours, which means limited expansion efficiency, and starting from P3, the proliferation of imDAP began to slow down significantly. Figure 1G The percentages of cells expressing FOXA2 at P0, P1, and P2 are shown in Figure 1H . The results showed that using the imDAP expansion medium based on the combination of LDN193189 / CHIR99021 / FGF8b / Blebbistatin, the percentage of cells expressing Foxa2 decreased with passage, especially significantly after 2 passages.

[0370] Example 6 Y27632 Promotes imDAP Expansion

[0371] This example demonstrates that the expansion medium supplemented with Y27632 can increase the expression of FOXA2 in imDAP cells compared to the expansion medium supplemented with Blebbistatin.

[0372] imDAP was generated according to the method described in CN201910169525.0. imDAP (P0) was seeded at a low density of 1×10 4 cells / cm 2 onto a VTN-coated plate (1 μg / cm 2 ) and cultured in an expansion medium containing 5 μM Blebbistatin or 10 μM Y27632 (50% DMEM / F12, 50% Neurobasal TMCultured in medium, 1% (v / v) Glutamax, 2% (v / v) B27 (50x), 0.2 μM LDN193189, 3 μM CHIR99021, 100 ng / mL FGF8b). The medium was changed every three days. On day 6, cells were collected for FACS analysis.

[0373] The results are as Figure 2 shown. The data clearly show that the amplified medium supplemented with Y27632 can increase the percentage of cells expressing FOXA2 compared to the medium supplemented with Bredinin. However, on the other hand, although Y27632 can increase FOXA2 expression compared to Bredinin, the percentage of cells expressing FOXA2 still decreases during passage.

[0374] Example 7 Enrichment of NOTCH2 / TGFβ2 in imDAP

[0375] The said example shows the enrichment of NOTCH2 / TGFβ2 in imDAP. Manipulating these signaling pathways may allow for effective imDAP amplification while maintaining the mDAP phenotype.

[0376] Single cells were encapsulated in droplets using 10× Genomics Technology and processed according to the following procedure. Briefly, each cell and each transcript were uniquely barcoded with unique molecular identifiers (UMIs). Libraries were generated from cDNA and sequenced, and individual reads were associated back to individual partitions using 10× barcodes. Then, according to the standard procedure for Illumina-ready sequencing libraries, the cDNA was collected together and amplified and sequenced.

[0377] For downstream analysis after initial Cell Ranger metric assessment, cells with low-quality sequencing data were removed. Genes with reads greater than or equal to 3 in all cells were considered to be ubiquitously expressed in all cell types of the sample and were thus removed before further analysis. Cells in the following brain regions did not appear to be present in the sample: forebrain, diencephalon, dorsal midbrain, lateral midbrain, hindbrain r2,3. Cell clustering was performed in the following order: (1) midbrain-hindbrain boundary (MHB, FGF8+), (2) hindbrain r1 (HBr1, GBX2+), (3) midbrain floor plate (MBP, OTX2+ / NKX6.1+, OTX2+ / NKX2.2+, OTX2+ / PITX2+), (4) midbrain caudal floor plate (MFP, EN1+ / LMX1A+ / FOXA2+ / OTX2+), (5) remaining cells. Then the remaining cells were grouped into one of the top 4 clusters with the closest total gene expression profile. For visualization and clustering, the manifold was calculated using the UMAP method ( Figure 3A)。Using differential expression gene markers, NOTCH2 / TGFβ2 was found to be enriched in MFP cells ( Figure 3B )。Manipulating these signaling pathways allows for efficient imDAP expansion while maintaining the mDAP phenotype.

[0378] Example 8 NOTCH activation and / or TGF-β inhibition supports better imDAP expansion

[0379] The examples show that Notch activation (e.g., using DLL4) and TGF-β inhibition (e.g., using SB431542) support better expansion of imDAP.

[0380] imDAP was generated according to the method described in CN201910169525.0. imDAP (P0) was seeded at a low density of 1×10 4 cells / cm 2 onto plates coated with DLL4 (1 μg / cm 2 ), VTN (1 μg / cm 2 ), and DLL4 / VTN (1 μg / cm each 2 ), and cultured in expansion medium in the absence or presence of 5 μM SB431542. The medium was changed every 3 days until 100% confluence (6 days) for cell collection. Representative cell morphology on day 4 was recorded ( Figure 4A ). The cell number of each group was counted using a Vi-cell cell counter ( 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 expansion medium used in this example contained the following components: 50% DMEM / F12, 50% Neurobasal TM 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.

[0381] Here, the effects of DLL4 and SB431542 on supporting the expansion of imDAP into a homogeneous population were examined. The results are shown in Figure 4. Specifically, Figure 4A it was shown that DLL4 alone could not support the adhesion of imDAP. imDAP on VTN presented a flat morphology. imDAP cultured on surfaces coated with VTN and DLL4 was more homogeneous during expansion. Figure 4BIt is shown that compared with the amplification (about 20 - fold or more) without DLL4 and SB431542, either DLL4 alone, SB431542 alone, or their combination can greatly improve the imDAP amplification efficiency (about 32 - fold or more). In Figure 4C it, by comparing the results of - DLL4 + VTN / -SB with those of - DLL4 + VTN / +SB and by comparing the results of + DLL4 + VTN / -SB with those of + DLL4 + VTN / +SB, it is shown that SB431542 alone can improve FOXA2 expression; and by comparing the results of - DLL4 + VTN / -SB with those of + DLL4 + VTN / -SB and comparing the results of - DLL4 + VTN / +SB with those of + DLL4 + VTN / +SB, it is shown that DLL4 alone can improve the expression of FOXA2. In particular, the combination of DLL4 / VTN and SB431542 maintained the optimal FOXA2 expression during amplification.

[0382] Example 9: LDN193189, FGF8b, and / or FGF2 have no additional effect on imDAP amplification

[0383] To further demonstrate whether LDN193189, FGF8b, and FGF2 are necessary for imDAP amplification, this example examined the effects of LDN193189, FGF8b, and FGF2 on imDAP amplification.

[0384] imDAP was generated according to the method described in CN201910169525.0. imDAP (P0) was seeded at a low density of 1×10 4 cells / cm 2 onto plates coated with DLL4 / VTN (1 μg / cm 2 each) and cultured in amplification media (EM1, EM2, EM3, EM4). The medium was changed every 3 days until 100% confluence (6 days) for cell collection. EM1 used in Example 9 contained the following components: 50% DMEM / F12, 50% Neurobasal TM 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. EM2 used in Example 9 contained the following components: 50% DMEM / F12, 50% Neurobasal TMMedium, 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. EM3 used in Example 9 contains the following components: 50% DMEM / F12, 50% Neurobasal TM 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. EM4 used in Example 9 contains the following components: 50% DMEM / F12, 50% Neurobasal TM 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.

[0385] Figure 5A The typical cell morphology on day 5 is shown. As Figure 5A shown, when culturing cells with FGF2, some obvious heterogeneous cells were observed. The cell numbers of each group were counted by a Vi-cell cell counter ( Figure 5B ). As Figure 5B shown, adding LDN193189 and / or FGF8b had no additional effect on increasing the cell number during the amplification process. The cells of each group were stained with Foxa2-PE antibody (BD, #561589), and the percentage of Foxa2+ imDAP was analyzed by flow cytometry ( Figure 5C ). As Figure 5C shown, compared with P0 cells, the amplification medium without LDN193189 and FGF8b could maintain or even increase the FOXA2 expression of P1 cells. Although the amplification medium with LDN193189 and / or FGF8b could still maintain or even increase the FOXA2 expression of P1 cells, adding FGF8b and / or LDN193189 slightly decreased the FOXA2 expression of imDAP. The results indicate that LDN193189, FGF8b, and / or FGF2 have no additional effect on imDAP amplification and are not essential for imDAP amplification.

[0386] Example 10 NOTCH activation combined with TGF-β inhibition supports long-term amplification of imDAP

[0387] This example shows that the new combination of CHIR99021, SB431542 and Y27632 supports the long-term expansion of imDAP on VTN / DLL4-coated surfaces.

[0388] imDAP was generated according to the method described in CN201910169525.0. imDAP (P0) was seeded at a low density of 1×10 4 cells / cm 2 in expansion medium on DLL4 / VTN-coated (1 μg / cm 2 each) plates. The medium was changed every 3 days until 100% confluence (6 days) for cell passage. imDAP was enzymatically passaged with 1×TrypLE, centrifuged at 250 g for 5 minutes, counted and seeded at a density of 1×10 4 cells / cm 2 in fresh expansion medium on new DLL4 / VTN-coated plates. imDAP was expanded and maintained for 6 passages in expansion medium. The expansion medium used in Example 10 contained the following components: 50% DMEM / F12, 50% Neurobasal TM 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.

[0389] Figure 6A Shows the typical cell morphology of P3 on day 5. Cells of each passage were collected on day 6. The cell number was counted by a Vi-cell cell counter, and Figure 6B shows the imDAP PDT of each passage (P1 - P6). As Figure 6B shown, compared with Figure 1F , the expanded imDAP maintained a similar PDT with a significantly lower value of approximately 25 - 28 hours from P1 to P6. A shorter PDT per passage indicates a higher expansion efficiency per passage, and a similar PDT per passage indicates that the expansion ability of the cells per passage can be better maintained. Figure 6C Shows the percentage of FOXA2+ cells at P0, P1, P2, P3, P4 and P5. As Figure 6C shown, the expansion medium with the combination of CHIR99021, SB431542 and Y27632 was able to maintain and even increase FOXA2 expression during the long-term expansion on VTN / DLL4-coated surfaces. Figure 6D Shows the gene expression analysis of key mDAP markers (P0 - P6). As Figure 6DAs shown, the expression of LMX1A, FOXA2, EN1, OTX2, and SOX6 was basically maintained during long-term expansion. The data indicate that the expansion medium with the above combination can support the long-term expansion of imDAP on the VTN / DLL4-coated surface. It not only significantly improves the expansion efficiency during cell expansion and passage (the expansion multiple per generation is 8 times that of the expansion medium in Example 5) and basically maintains the improved expansion efficiency at the same or similar level, but also maintains or increases the expression of imDAP-specific markers (such as Foxa2) or cell purity (such as FOXA2+ cells) during cell expansion and passage.

[0390] Example 11 Development of imDAP Maturation Medium

[0391] In the literature, B27 is usually used as a medium supplement for neuron maturation. This example shows that when B27 is replaced with hPLT or HhPLT, maturation with hPLT or HhPLT produces a higher proportion of TH+ neurons compared to maturation with B27.

[0392] imDAP was generated according to the method described in CN201910169525.0 and expanded as described in Example 10. The expanded imDAP was seeded at a density of 5×10 5 cells / cm 2 (day 0) on VTN-coated (1 μg / cm 2 ) plates and cultured in maturation medium (MM-P, MM-H, MM-B) supplemented with 1 μM IWR1. MM-P used in Example 11 contains the following components: Neurobasal TM 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, hPLT (0.5%, 1%, 2%). MM-H used in Example 11 contains the following components: Neurobasal TMCulture 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, 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, 2% (v / v) B27 (50x) (as a control). The above HhPLT was produced from hPLT as follows. hPLT was centrifuged at 3000 g for 30 minutes at 4°C to obtain the supernatant, and the supernatant was heated to 56°C for 30 minutes. Finally, the suspension was cooled to 4°C for at least 5 minutes and centrifuged at 3000 g for 30 minutes at 4°C to obtain the HhPLT supernatant. Aliquots were prepared and stored at -80°C until use. The above culture medium was changed every 3 days. On day 7, early mature stage imDAP was enzymatically dissociated with 1×TrypLE and centrifuged at 250 g for 5 minutes.

[0393] Figure 7A The typical cell morphology on day 7 is shown. As Figure 7A shown, treatment with hPLT induced significant contraction of imDAP, which increased the difficulty of post-mature imDAP dissociation and collection, while imDAP cultured with HhPLT and B27 appeared less dense and it was easier to observe obvious nerve fibers. Cells were counted and stained with TH (1°Ab: TH, Pel freez, #P40101. 2°Ab: Abcam, #ab130805) for flow cytometry analysis ( Figure 7B ). As Figure 7B shown, compared with B27, the addition of hPLT greatly improved the maturity of imDAP (TH+ cell %), while compared with hPLT, the addition of HhPLT was able to further improve the maturity of imDAP (TH+ cell %). Some cells were also plated on laminin / poly-L-ornithine (PLOH)-coated plates for an additional 21 days of maturation, and then stained with TH antibody (1°Ab: TH, Pel freez, #P40101. 2°Ab: Invitrogen, #A11012) for immunocytochemistry analysis ( Figure 7C ). As Figure 7C shown, compared with B27, immunocytochemistry analysis of TH in mDA neurons generated by using 1% HhPLT showed a higher proportion of TH+ neurons.

[0394] Example 12: Wnt inhibitor-supplemented medium results in a higher proportion of TH+ neurons

[0395] This example shows that IWR1 (a Wnt inhibitor) can further improve the maturity of imDAPs.

[0396] imDAPs were generated according to the method described in CN201910169525.0 and amplified as described in Example 10. The amplified imDAPs were seeded at a density of 5×10 5 cells / cm 2 onto VTN-coated (1 μg / cm 2 ) plates on day 0 and cultured in maturation medium with or without 1 μM IWR1. The medium was changed every 3 days. On day 7, early maturation-stage imDAPs 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 TM 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, 10 μM DAPT.

[0397] Figure 7D Shows the effect of IWR1 on cell morphology on day 7 during maturation. As Figure 7D shown, imDAPs matured in the presence of IWR1 for at least 3 days showed more prominent neurites. In addition, qRT-PCR analysis of mDA maturation-related markers such as NURR1, SOX6, and TH was performed ( Figure 7E ). As Figure 7E shown, the expression of NURR1, SOX6, and TH was increased in cells treated with IWR1 compared to cells not treated with IWR1, indicating better maturation.

[0398] Example 13: ROCK inhibitor combined with coating matrices VTN / PLLH or VTN / PLOH promotes imDAP maturation

[0399] Laminin / PLOH is commonly used as a substrate for mDA neuron maturation, but it is too costly for large-scale mDA neuron production. To promote imDAP maturation and reduce costs, several different substrates were tested. This example shows that Y27632 (a ROCK inhibitor in the medium) in combination with the coated substrates VTN / PLLH (poly-L-lysine hydrobromide) or VTN / PLOH (poly-L-ornithine hydrobromide) can promote imDAP maturation.

[0400] imDAPs were generated according to the method described in CN201910169525.0 and amplified as described in Example 10. The amplified imDAPs were seeded at a density of 5×10 5 cells / cm 2 (day 0) onto plates coated with different substrates (VTN, VTN+PLLH, VTN+PLOH, and laminin+PLOH) (1 μg / cm 2 each) and cultured in maturation medium. The maturation medium used in Example 13 further contained the following components in addition to 10 μM or 0 μM of Y27632: Neurobasal TM 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, 10 μM DAPT. The medium was changed every 3 days. At day 3, IWR1 was removed from the maturation medium. At day 7, the early maturation stage imDAPs were enzymatically dissociated with 1×TrypLE and centrifuged at 250 g for 5 minutes. The cells were collected and counted using a Vi-cell cell counter. The EN1, LMX1A, FOXA2, NURR1, SOX6, and TH gene expressions of the cells were analyzed by RT-qPCR. And the cells were also seeded at a density of 5×10 4 cells / cm 2 onto coverslips in 48-well plates for immunostaining with LMX1A and SOX6 antibodies (LMX1A: Millipore, #MAB10533. SOX6: Proteintech, #14010-1-AP).

[0401] Figure 8A The typical cell morphologies of the combinations of VTN+Y (control 1), VTN+PLLH (control 2), VTN+PLLH+Y, VTN+PLOH (control 3), VTN+PLOH+Y, and laminin+PLOH (control 4) at day 7 are shown. As Figure 8AAs shown, in the maturation medium without Y27632, the cells plated on VTN / PLLH or VTN / PLOH detached from the culture dish. When the maturation medium was supplemented with Y27632, the cells plated on VTN, VTN / PLLH, or VTN / PLOH were all able to grow normally, indicating that Y27632 can promote cell adhesion to a certain extent. Figure 8B Showing the ratio of the number of imDAP cells after 7-day maturation to the number of input cells. As Figure 8B shown, there was no significant difference in cell yield among the combinations of VTN / PLLH + Y27632, VTN / PLOH + Y27632, and laminin / PLOH, and they were all higher than the combination of VTN + Y27632. Figure 8C Showing the qRT-PCR analysis of mDA maturation-related markers after 7-day maturation. As Figure 8C shown, in the early stage of maturation, VTN / PLLH + Y27632 and VTN / PLOH + Y27632 were superior to laminin / PLOH or VTN + Y in the expression of NURR1, SOX6, and TH genes, indicating that VTN / PLLH + Y27632 (or PLLH) and VTN / PLOH + Y27632 (or PLOH) can support better maturation.

[0402] It is shown here that the combined use of Y27632 with VTN / PLLH or VTN / PLOH in the medium not only promotes imDAP maturation but also has a lower cost, so it is more suitable for large-scale clinical production.

[0403] Example 14 Maturation potential of imDAP cultured with ROCK inhibitor in combination with coated matrix VTN / PLLH or VTN / PLOH in the late stage of maturation

[0404] This example shows that imDAP cultured on plates coated with VTN / PLLH or VTN / PLOH in combination with Y27632 in the early stage of maturation has similar terminal maturation potential.

[0405] The imDAP after 7-day maturation in Example 13 was enzymatically dissociated with 1×TrypLE and centrifuged at 250g for 5 minutes. The cells were collected and counted using a Vi-cell cell counter. The cells were aggregated into 3D suspension cultures (neurospheres) for further maturation. For this purpose, the cells were resuspended at a density of 5×10 5 cells / mL and seeded into poly-HEMA-coated (1 μg / cm 2)6 mL of maturation medium - ND supplemented with 10 μM Y27632 was placed in a T25 flask. To induce aggregation, the T25 flask was placed on a Belly dancer at 15 rpm (day 0). On day 1, the medium was changed to maturation medium - ND. On the second day, half of the medium was changed to maturation medium - BC. From day 3 onwards, the neurospheres were cultured in maturation medium - BC and the medium was changed every 3 days. On day 21, the neurospheres were collected and fixed overnight at 4 °C in 4% PFA in PBS, and then incubated overnight in 30% sucrose. Next, the neurospheres were embedded in OCT blocks and cryosectioned (20 μm) using a Cryostat. The cryosections of the neurospheres were further immunostained with TH antibodies (1° Ab: TH, Millipore, #MAB318. 2° Ab: Invitrogen, #A11029). The maturation medium - ND used in Example 14 contained the following components: Neurobasal TM 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 TM 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 - dihydrobenzo[E](1,4)diazepin -2 - one).

[0406] Figure 8D Immunocytochemical analysis of cryosections of neurospheres showing further maturation from cells matured for 7 days using Y27632 in combination with VTN / PLLH or VTN / PLOH. As Figure 8D shown, imDAPs cultured with Y27632 in combination with VTN / PLLH or VTN / PLOH in the early stage of maturation had similar terminal maturation potential.

[0407] Expanded imDAPs at early and late passages in Example 15 maintained a similar high ability to differentiate into early - mature mDA neurons

[0408] This example shows that expanded imDAPs at early and late passages are able to retain a similar high ability to differentiate into early mature mDA neurons.

[0409] The expanded P3, P5, and P6 imDAPs from Example 10 were seeded at a density of 5x10 5 cells / cm 2 on VTN / PLOH-coated (1 μg / cm each) 2 ) plates and cultured in maturation medium supplemented with 10 μM Y27632 and 1 μM IWR1. Starting from day 3, the above maturation medium was replaced with maturation medium supplemented only with 10 μM Y27632. The medium was changed every 3 days. On day 7, imDAPs at the early maturation stage were enzymatically dissociated with 1x TrypLE and centrifuged at 250 g for 5 minutes. Cells matured from the expanded P3 and P6 imDAPs were collected and counted using a Vi-cell cell counter. The EN1, LMX1A, FOXA2, NURR1, SOX6, and TH gene expressions of the cells were analyzed by RT-qPCR ( Figure 9A ). And cells matured from the expanded P3 and P5 imDAPs were also seeded on coverslips in 48-well plates for immunostaining with LMX1A and SOX6 antibodies (LMX1A: Millipore, #MAB10533. SOX6: Proteintech, #14010-1-AP) ( Figure 9B and 9C ). The maturation medium used in Example 15 contained the following components: Neurobasal TM 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, 10 μM DAPT.

[0410] Figure 9A RT-qPCR analysis results showing the expression of specific markers EN1, LMX1A, FOXA2, NURR1, SOX6, and TH in early mDA neurons matured from the expanded P3 and P6 imDAPs. The results showed no significant difference between the expanded P3 and P6 imDAPs. Figure 9B Immunocytochemical analysis results showing the expression of LMX1A and SOX6 in early mDA neurons matured from the expanded P3 and P5 imDAPs. Figure 9CShow the quantitative results of mDA neurons expressing LMX1A and expressing SOX6 matured by P3 and P5 imDAP at early maturation. The results show no difference between the amplified P3 and P5 imDAP. The results show that the amplified imDAP generated by the novel amplification method described herein can be passaged several times and still retain a high ability to differentiate into mDA neurons.

[0411] Those skilled in the art will readily understand that the methods, compositions, and products described herein represent exemplary embodiments and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various alternatives and modifications can be made to the present disclosure without departing from the scope and spirit of the invention.

[0412] All patents and publications mentioned in the specification indicate the level of those skilled in the art to which the present disclosure pertains. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0413] The present disclosure is not limited to the specific embodiments described in this application, which are intended to be illustrative of various aspects of the present disclosure. All various embodiments of the present disclosure will not be described herein. It will be apparent to those skilled in the art that many modifications and variations can be made without departing from the spirit and scope of the present disclosure. In addition to the methods and apparatuses enumerated herein, those skilled in the art will also be clear about functionally equivalent methods and apparatuses within the scope of the present disclosure based on the above description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is limited only by the terms of the appended claims and the full scope of the equivalents of those claims.

Claims

1. A culture medium capable of promoting the 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) γ-secretase inhibitor; and (e) Cyclic adenosine phosphate compounds or their cyclase activators.

2. The culture medium of claim 1, further comprising: (f) Neurotrophic factor.

3. The culture medium of claim 1 or 2, further comprising: (g) Rho kinase (ROCK) inhibitor.

4. The culture medium of any one of claims 1-3, wherein the hPLT is heat-treated human platelet lysate (HhPLT).

5. The culture medium of any one of claims 1-4, further comprising: (h) WNT signaling pathway inhibitor.

6. The culture medium of claim 1, wherein the hPLT is present in the culture medium at a concentration of about 0.1% to about 5% by volume.

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

8. The culture medium of claim 1, wherein the γ-secretase inhibitor is present in the culture medium at a concentration of about 1 μM to about 30 μM.

9. The culture medium of claim 1, wherein the cyclic adenosine phosphate compounds or their cyclase activators are present in the culture medium at a concentration of about 0.1 mM to about 5 mM.

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

11. The culture medium of claim 3, wherein the ROCK inhibitor is present in the culture medium at a concentration of about 1 μM to about 50 μM.

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

13. The culture medium according to claim 1, wherein the γ-secretase inhibitor is selected from DAPT, N-[N-(3,5-difluorophenylacetyl)]-L-alanyl-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydrobenzo[E](1,4)diazepin -2-one, LY-411575, dihydroergocristine mesylate, BMS299897, and any combination thereof.

14. The culture medium of claim 1, wherein the cyclic adenosine phosphate compounds or their cyclase activators are selected from 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.

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

16. The culture medium of claim 3, wherein the ROCK inhibitor is selected from Y27632, HA100, HA1152, HA-1077, and any combination thereof.

17. The culture medium of claim 5, wherein the WNT signaling pathway inhibitor is selected from IWR1, iCRT3, IWP-O, IWP-2, IWP-3, IWP-4, ciclopirox, myristicin, diethyl benzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, XAV-939, Foscenvivint (ICG-001), crizotinib, isoquercitrin, Gigantol, JW55, MSAB, KY02111, FH535, WIKI4, CCT251545, prodigiosin, KYA1797K, NCB-0846, LF3, iCRT14, Adavivint, triptolide, M435-1279, and any combination thereof.

18. The culture medium of claim 2 or 4, which comprises in a neural basal medium: (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 cyclic adenosine phosphate compound or its cyclase activator; and (e) about 10 ng / ml to about 80 ng / ml of a neurotrophic factor.

19. The culture medium of claim 18, which comprises in a neural basal medium: (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) a combination of about 10 ng / ml to about 80 ng / ml of BDNF and GDNF.

20. The culture medium of any one of claims 1-19, which further comprises glutamine or its derivative.

21. The culture medium of claim 20, wherein the glutamine or its derivative is present in the culture medium at a concentration of about 0.1% to about 5% by volume.

22. The culture medium of any one of claims 1-21, which further comprises an antioxidant.

23. The culture medium of claim 22, wherein the antioxidant is present in the culture medium at a concentration of about 50 μM to about 500 μM.

24. The culture medium of any one of claims 1-23, wherein the mDAP is an amplified and / or passaged iPSC-derived midbrain dopaminergic progenitor cell (imDAP).

25. The culture medium of any one of claims 1-24, wherein the culture medium is a chemically defined serum-free maturation medium.

26. The culture medium of any one of claims 1-25, which is capable of improving the maturity of mDAP.

27. A method for promoting the maturation of midbrain dopaminergic progenitor cells (mDAP), which comprises contacting mDAP with a maturation medium containing a ROCK inhibitor on a culture surface coated with a coating matrix combination, the coating matrix combination comprising: (a) A first coating matrix capable of supporting the adhesion of mDAP cells, wherein the first coating matrix is not laminin; and (b) A second coating matrix capable of enhancing the maturity of mDAP, wherein the second coating matrix comprises a polylysine compound and / or a polyornithine compound.

28. The method of claim 27, wherein the maturation medium containing a ROCK inhibitor is the medium of any one of claims 1-26.

29. The method of claim 27 or 28, wherein the first coating matrix is selected from vitronectin (VTN), collagen, proteoglycan, fibronectin, nestin, elastin, a functional fragment of any of the foregoing proteins, hyaluronic acid, gelatin, and any combination thereof, and is preferably VTN.

30. The method of any one of claims 27-29, wherein the second coating matrix comprises poly-L-lysine hydrobromide, poly-L-ornithine hydrobromide, or a mixture thereof.

31. The method of any one of claims 27-30, wherein the mDAP is an amplified and / or passaged iPSC-derived midbrain dopaminergic progenitor cell (imDAP).

32. The method of any one of claims 27-31, which is capable of enhancing the maturity of mDAP.

33. A substantially homogeneous population of midbrain dopaminergic neurons (mDAN) produced by the method of any one of claims 27-32.

34. A kit comprising the medium of any one of claims 1-26.

35. The kit of claim 34, which further comprises the coating matrix combination of any one of claims 27-32.

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