Methods for the generation of functional neurons

Abstract

Provided herein is a cell culture medium containing a GDNF receptor RET agonist and its use for generating various functional neuronal cell types from pluripotent cells.Provided herein is an in vitro method for generating various functional neuronal cell types by culturing in a differentiation cell culture medium containing a glial cell line-derived neurotrophic factor (GDNF) receptor RET (transmembrane receptor tyrosine kinase rearranged in transfection) agonist, preferably, the differentiation cell culture medium is essentially protein-free.

Description

[Technical field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 278,902, filed November 12, 2021, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] BACKGROUND OF THEINVENTION Human pluripotent stem cells (hPSCs), including embryonic and induced subtypes, are useful for a variety of applications, including modeling human development or disease. 1 hPSCs have been used extensively as source cells to derive functional end cell types (e.g., mature neurons) for clinical applications, drug screening, and cell-based therapeutics. Derivation of functional cell types from hPSCs in vitro involves mimicking the natural developmental process of progenitor cells from the blastocyst environment, where many biochemical signals (e.g., growth factors morphogens and small molecules) are presented to the cells in precise amounts, timing, and order to progressively specify the cell's fate.

[0003] Current methods for inducing functional neuronal subtypes from hPSCs in vitro rely heavily on the use of recombinant proteins supplemented to cell culture media to recapitulate endogenous signaling processes. For example, glial derived neurotrophic factor (GDNF) is prevalent in many protocols for neuronal differentiation from hPSCs to activate GDNF-mediated RET signaling, which plays a key role in neuronal maintenance. Protocols for inducing dopaminergic neuronal precursors or neuroblasts from hPSCs that have shown efficacy in animal models of Parkinson's disease require the addition of GDNF as frequently as 75% of the duration of the differentiation protocol. The generation of hPSC-derived striatal neurons, which has shown efficacy in animal models of Huntington's disease, also requires substantial amounts of GDNF during the maturation stage of the differentiation process. Furthermore, the use of GDNF has also been prevalent in the generation of several additional hPSC-derived neuronal subtypes (e.g., neuroepithelial stem cells, interneurons, cholinergic neurons, and serotonin neurons) that are cell therapy candidates for various neurological disorders, including stroke, neuropathic pain, schizophrenia, autism, epilepsy, and learning / memory disorders.

[0004] Cerebellar neurons that extend beyond the cerebrum and have been derived from hPSCs to model and potentially treat cerebellar degeneration require substantial amounts of GDNF to reach a mature state, in some cases over 100 days of exposure. Peripheral sensory neurons (including nociceptors, mechanoreceptors, and proprioceptors) derived from hPSCs also rely heavily on the use of GDNF to generate functional neurons for personalized neuropathic treatment modeling. Differentiation and maturation of motor neurons from hPSCs for modeling or treating degenerative conditions (including SMA and ALS) requires GDNF for nearly 50% of their differentiation time.

[0005] Although the majority of these neural cell types are candidates for cell replacement therapies for a range of neurological indications, including neural tissue regeneration, generating clinical-grade cells for transplantation in humans has emerged as a significant bottleneck in translating these candidate therapies into the clinic. Depending on the indication, millions of cells may be required for a single administration to a patient, which could amount to as much as 10 cells per year for a single allogeneic product. 14 The estimated manufacturing burden is up to 100 cells. For current Good Manufacturing Process (cGMP) grade cells required for clinical development and commercialization of these cell therapy candidates, recombinant proteins are among the most expensive raw materials. For example, in a 40-day protocol for the generation of dopaminergic neurons from hPSCs, GDNF, BDNF, and TGF-β are supplemented in the cell culture medium for 30 days, which accounts for 50% of the total cost of the reagents. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention Provided herein is an in vitro method for generating various functional neural cell types by culturing in a differentiation cell culture medium containing a glial cell line-derived neurotrophic factor (GDNF) receptor RET (transmembrane receptor tyrosine kinase rearranged in transfection) agonist, preferably the differentiation cell culture medium is essentially protein-free. In some embodiments, functional neural cells are generated from induced (stem cell-derived) neural progenitor cells according to the methods described herein. In other embodiments, functional neural cells are generated from mammalian pluripotent cells according to the methods described herein.

[0007] Also provided herein is a cell culture medium useful for generating various functional neuronal cell types, the cell culture medium comprises one or more GDNF receptor RET agonists.In a preferred embodiment, the cell culture medium does not contain GNDF, BDNF (brain-derived neurotrophic factor) and TGFβ, and preferably is essentially protein-free. Description of the drawings [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows a comparison between the standard protocol (i) for differentiation of hPSCs into dopaminergic neurons and the process (ii) described herein. In contrast to this standard protocol, dopaminergic neurons are generated according to the present method in induction medium (between days 0 and 10) that does not contain FGF8, and differentiation medium (from days 11 to 20 onwards) that does not contain GDNF, BDNF or TGFβ3.

[0009] [Diagram 2] FIG. 2 shows routine imaging of growing and differentiating neural aggregates in differentiation medium containing BT-13 and DAPT but not GDNF, BDNF and TGFβ3.

[0010] [Diagram 3] FIG. 3 shows immunocytochemical staining for DAPI (blue) and tyrosine hydroxylase (TH) at 20 days of representative aggregates differentiated using differentiation medium containing BT-13 and DAPT but not GDNF, BDNF, or TGFβ3.

[0011] [Figure 4] FIG. 4 shows the results of a cost analysis comparing the standard protocol for differentiation of human pluripotent stem cells (hPSCs) into dopaminergic neurons with the present method.

[0012] [Diagram 5]Figure 5 shows tyrosine hydroxylase positive neurons at day 18 that were generated by culturing hPSCs in induction medium without FGF8 (between days 0 and 10) and differentiation medium containing 10 nM Q525 (which does not contain GDNF, BDNF and TGFβ3) (from days 11 to 20 onwards). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description of the Invention (definition)

[0014] As used herein, "activator" refers to a compound that increases, induces, stimulates, activates, promotes, or enhances the activation of the signaling function of a molecule or pathway (e.g., Wnt signaling, SHH signaling, etc.).

[0015] As used herein, the term "cell population" or "cell population" refers to a group of at least two cells.In a non-limiting example, cell population can comprise at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells.The population can be a pure population that comprises one type of cell (e.g., a population of dopaminergic neurons, or a population of undifferentiated stem cells).Alternatively, the population can comprise more than one type of cell (e.g., a mixed cell population).

[0016] As used herein, the term "stem cell" refers to a cell that has the capacity to divide in culture for an indefinite period of time to give rise to specialized cells.

[0017] As used herein, the terms "embryonic stem cell" and "ESC" refer to primitive (undifferentiated) cells derived from preimplantation embryos, which can divide for long periods without differentiation in culture, and are known to develop into cells and tissues of the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells derived from human embryos. As used herein, the terms "human embryonic stem cell" or "hESC" refer to a type of pluripotent stem cell derived from early human embryos (up to blastocyst stage), which can divide for long periods without differentiation in culture, and are known to develop into cells and tissues of the three primary germ layers.

[0018] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells that have been cultured under in vitro conditions that allow proliferation for days, months or even years without differentiation.

[0019] As used herein, the term "GDNF receptor RET agonist" includes molecules that indirectly or directly activate transmembrane receptor tyrosine kinase RET. GDNF receptor RET agonist can indirectly activate RET by increasing the activity of GDNF family ligand (GFL), selected from glial cell line-derived neurotrophic factor (GDNF), artemin (ARTN), neurturin (NRTN) and persephin (PSPN), all of which signal through transmembrane receptor tyrosine kinase RET. Direct activation of RET by GDNF receptor RET agonist occurs independently of GFL protein. For example, BT-13 and BT-18 directly activate RET, while XIB4035 indirectly activates RET by increasing the activation of GDNF or ARTN.

[0020] The term "dopaminergic neuron" is intended to encompass neural cells that express tyrosine hydroxylase; in particular, the term is intended to include neural cells that express tyrosine hydroxylase, including dopamine precursor cells and dopamine neuroblasts.

[0021] As used herein, the term "pluripotency" refers to the capacity to develop into the three developmental germ layers of an organism, including endoderm, mesoderm, and ectoderm.

[0022] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to a type of pluripotent stem cell formed by the introduction of certain embryonic genes (e.g., but not limited to, the OCT4 transgene, the SOX2 transgene, and the KLF4 transgene) into somatic cells (see, e.g., Takahashi and Yamanaka, Cell, 126, 663-676 (2006), incorporated herein by reference).

[0023] As used herein, the term "neuron" refers to a nerve cell, which is the main functional unit of the nervous system. A neuron consists of a cell body and its processes (axon and one or more dendrites). A neuron transmits information to other neurons or cells by releasing neurotransmitters at synapses.

[0024] As used herein, the term "undifferentiated" refers to cells that have not yet developed into a specialized cell type.

[0025] As used herein, the term "differentiation" refers to the process by which an unspecialized embryonic cell acquires the characteristics of a specialized cell (e.g., a nerve cell, a heart cell, a liver cell, or a muscle cell). Differentiation is controlled by the interaction of the cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

[0026] As used herein, the term "inducing differentiation" in reference to a cell refers to changing from its default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, "inducing differentiation in a stem cell" refers to inducing the stem cell (e.g., a human stem cell) to divide into progeny cells that have characteristics (e.g., genotype (e.g., changes in gene expression determined by genetic analysis (e.g., microarray)) and / or phenotype (e.g., changes in expression of protein markers of midbrain dopamine (DA) cells or their precursors (e.g., EN-1, OTX2, TH, NURR1, FOXA2, and LLMX1A)) that are different from the stem cell.

[0027] As used herein, the term "marker" or "cell marker" or "biomarker" refers to a gene or protein that identifies a particular cell or cell type. A marker for a cell may not be limited to one marker, and may refer to a "pattern" of markers, such that a specified group of markers can distinguish one cell or cell type from another cell or cell type.

[0028] Provided herein is the use of GDNF receptor RET agonists as cell culture medium additives that eliminate the requirement for GDNF, BDNF and TGFβ in the generation of induced neuronal precursor cells or functional neurons from mammalian pluripotent cells.

[0029] In some embodiments, an in vitro method is provided for generating neurons from induced neuronal precursor cells (iNPCs), comprising culturing the iNPCs in a differentiation cell culture medium comprising a GDNF receptor RET agonist. In some embodiments, the iNPCs for use according to the method express the markers Pax6, Nestin, and CD133.

[0030] In a preferred embodiment, the GDNF receptor RET agonist has the structure: [ka] BT-13 (N,N-diethyl-3-[4-[4-fluoro-2-(trifluoromethyl)-benzoyl]piperazin-1-yl]-4-methoxybenzenesulfonamide) having the structure: [ka] and / or BT-44. In some preferred embodiments, the differentiation cell culture medium comprises BT-13, BT-18 and / or BT-44 at a concentration of about 2 μM to about 20 μM. In other embodiments, the differentiation cell culture medium comprises BT-13, BT-18 and / or BT-44 at a concentration of about 2 μM to about 10 μM or about 5 μM.

[0031] In a related embodiment, the GDNF receptor RET agonist is XIB4035 (aminoquinol; [ka] ), and preferably, the differentiation cell culture medium contains about 10 nM to about 1000 nM of XIB4035.

[0032] In another embodiment, the GDNF receptor RET agonist is Q525 [ka] and preferably, the differentiation cell culture medium contains about 1 nM to about 100 nM Q525.

[0033] In other embodiments, the GDNF receptor RET agonist is selected from those listed in Table 3 of Jmaeff et al., JBC, 295(19):6532-6542 (2020), the structure of each of which is identified in Table 1 of that reference, the contents of which are incorporated herein by reference in their entirety. In some preferred embodiments, the differentiation cell culture medium comprises about 1 nM to about 100 nM Q525 or Q508.

[0034] In another embodiment, the GDNF receptor RET agonist is dopamine neuron stimulating peptide-11 (DNSP-11; PPEAPAEDRSL (SEQ ID NO:1)).

[0035] In other embodiments, the GDNF receptor RET agonist is selected from those described in Runeberg-Roos et al., Neurobiol. Dis., 96:335-345 (2016); Jmaeff et al., Mol. Pharmacol., 98:1-12 (2020); Mahato et al., Mov. Disord., 35:245-255 (2020); Sidorova et al., Front. Pharmacology, 8:365 (2017); and Sidorova et al., Int. J. Mol. Sci., 21(18)6575 (2020), the contents of each of which are incorporated by reference in their entirety. Other GDNF receptor RET agonists useful according to the present methods include those described in US Pat. No. 8,901,129, the contents of which are incorporated herein by reference in their entirety (eg, BT10, BT16, BT17, or BT292651).

[0036] In some preferred embodiments, the differentiation cell culture medium comprises a GDNF receptor RET agonist (e.g., BT-13 or Q525) and further comprises a notch pathway inhibitor (e.g., DAPT (N-[2S-(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl ester-glycine) and / or dibutyryl cAMP (db-cAMP), RO4929097, BMS-906024, YO-01027, LY-411575, or tangeretin). In some preferred embodiments, the differentiation cell culture medium comprises DAPT (e.g., at a concentration of 5 μM to 20 μM, preferably about 10 μM).

[0037] In some embodiments, the differentiation cell culture medium is serum-free and comprises (i) neurobasal medium (e.g., Thermo Fisher Scientific, 11320033) (optionally supplemented with glutamine (e.g., glutamax) and / or N2 supplement (e.g., Thermo Fisher Scientific, 17502048) and / or B27 supplement (e.g., Thermo Fisher Scientific, 17504044) and / or ascorbic acid), (ii) a GDNF receptor RET agonist (e.g., BT13 or Q525), and (iii) a notch pathway inhibitor (preferably, DAPT and / or db-cAMP). The presence of ascorbic acid is not necessary to generate functional neurons according to the present methods, but may contribute to cell health and maintenance.

[0038] In some embodiments, the B-27 and N-2 supplements in the differentiation cell culture medium are replaced with one or more insulin receptor activator molecules, preferably a selective insulin receptor activator (e.g., demethylasteriquinone B1 (DMAQ-B1, also known as DAQB1) (preferably at a concentration between 10 μM and 100 μM), or 5,8-diacetyloxy-2,3-dichloro-1,4-naphthoquinone (DDN)). Thus, in some preferred embodiments, the differentiation cell culture medium is serum-free and includes (i) a neurobasal medium (e.g., Thermo Fisher Scientific, 11320033), (ii) an insulin receptor activator (preferably, DAQB1), (iii) a GDNF receptor RET agonist (e.g., BT13 or Q525), and (iv) a notch pathway inhibitor (preferably, DAPT and / or db-cAMP), and the medium does not include B-27 supplement and N-2 supplement. Optionally, the differentiation cell culture medium includes an iron transport molecule, such as hinokitiol (5 μM to 50 μM) and / or a substitute for BSA (e.g., recombinant human serum albumin (HSA)) (10 μg / mL to 100 μg / mL). In some embodiments, the differentiation cell culture medium is a completely chemically defined serum-free and xeno-free medium (e.g., CTS KnockOut SR XenoFree supplement (12618012)). By xeno-free, it is intended that the culture medium does not contain bovine or other non-human animal-derived components.

[0039] In particularly preferred embodiments, the differentiation cell culture medium is free of one or more of GDNF, BDNF and TGFβ. In particularly preferred embodiments, the differentiation cell culture medium is substantially free of GDNF, BDNF and TGFβ. In other preferred embodiments, the differentiation cell culture medium is essentially free of proteins.

[0040] In some embodiments, the induced neural progenitor cells for use according to the methods described herein are obtained by culturing mammalian pluripotent cells in neural induction medium for a suitable time to induce the induced neural progenitor cells.Typically, the pluripotent cells express the following markers: Oct4, SOX2, Nanog, SSEA3, SSEA4, TRA 1 / 81.

[0041] In some embodiments, the pluripotent cell is a human pluripotent cell. In another embodiment, the pluripotent cell is a non-human mammalian pluripotent cell. In a preferred embodiment, the pluripotent cell is a stem cell. In some aspects, the stem cell is an embryonic stem cell, preferably a human embryonic stem cell (e.g., human embryonic stem cell lines SA01, VUB01, HUES 24, H1, H9, WT3, HUES1). In other aspects, the stem cell is a non-human (e.g., mouse, rodent or primate) embryonic stem cell. In other aspects, the stem cell is an adult human stem cell. In other preferred embodiments, the stem cell is an induced pluripotent stem cell (iPSC). An induced pluripotent stem cell is a type of pluripotent stem cell that is artificially induced from a non-pluripotent, typically adult, somatic cell by inducing the forced expression of certain genes. For example, human skin fibroblasts can be reprogrammed into pluripotent stem cells using four Yamanaka factors (Oct3 / 4, Sox2, Klf4 and cMyc). See, e.g., Takahashi K, Yamanaka S., Cell, 2006;126(4):663-676, the entire contents of which are incorporated herein by reference.

[0042] In some embodiments, to generate induced neuronal precuorsor cells from mammalian pluripotent stem cells, neuronal induction of the pluripotent stem cells is initiated by culturing the stem cells in the presence of a dual inhibitor of the SMAD pathway (generally by inhibiting the bone morphogenetic protein (BMP) signaling pathway and the TGFβ signaling pathway) without the need for feeder cells.

[0043] Culturing stem cells in the presence of BMP inhibitor includes any culture condition that can inhibit BMP signaling pathway, whether by direct action on BMP and its receptor or by inhibiting their expression.Suitable inhibitors of BMP signaling pathway include, but are not limited to, LDN193189, DMH1, Noggin, Chordin, Follistatin, Dorsomorphin (6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), K02288, LDN212854, and ML347, LDN214117.In a preferred embodiment, the BMP inhibitor is LDN193189.The concentration of the BMP inhibitor in the culture is effective to inhibit BMP signaling pathway.

[0044] Culturing stem cells in the presence of TGFβ inhibitor includes any culture condition that can inhibit TGFβ, whether by directly acting on TGFβ to inhibit its function or by inhibiting the production of TGFβ itself.Suitable inhibitors of TGFβ signaling pathway include, but are not limited to, A83-01, SB-431542, LY364947, SB-525334, SD208, LY2157299, LY2109761, SB-505124, GW788388 and EW-7197.In a preferred embodiment, the TGFβ inhibitor is SB-431542.The concentration of the TGFβ inhibitor in the culture is an effective concentration for inhibiting TGFβ.

[0045] In a preferred embodiment, the induced neuronal precursor cell is a floor plate-based precursor cell (e.g., midbrain floor plate cell). In some embodiments, for patterning into midbrain fate, stem cells are cultured in induction medium that includes an inhibitor of SMAD pathway, (i) an activator of Sonic Hedgehog (SHH), (ii) an activator of WNT signaling pathway, and optionally (iii) an agonist of FGF receptor (FGFR). Representative methods for generating midbrain precursor include those described in U.S. Patent No. 10,858,625 (the entire contents of which are incorporated herein by reference).

[0046] The term "sonic hedgehog agonist" or "SHH agonist" as used herein includes recombinant sonic hedgehog, purmorphamine and SAG (which stands for smoothened agonist, a chlorobenzothiophene-containing compound). Shh can also be replaced with recombinant mammalian desert hedgehog (Dhh) or recombinant mammalian Indian hedgehog (Ihh). Activated smoothened (SMO) can also be used. In a preferred embodiment, the SHH activator is SAG.

[0047] Suitable activator of WNT signaling pathway includes GSK-3β inhibitor, for example CHIR99021, LiCl, BIO((2'Z,3'E)-6-bromoindirubin-3'-oxime), Kenpaullone, A1070722, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, Bikinin, IM-12, 1-Azakenpaullone, LY2090314, AZD1080, AZD2858, AR-A014418, TDZD-8 and Indirubin.In a preferred embodiment, the WNT activator is CHIR99021.

[0048] As used herein, "FGF receptor (FGFR) agonist" refers to a molecule that can activate FGFR (e.g., a molecule that binds to FGFR, induces dimerization of the receptor, and activates the signal transduction P13K pathway and Ras / ERK pathway). Non-limiting examples of FGFR agonists include FGF2, FGF8, and SUN11602. In a preferred embodiment, the FGFR agonist is FGF8 (e.g., recombinantly produced FGF8).

[0049] In some preferred embodiments, the neural induction medium comprises (i) LDN193189 (LDN) for inhibiting BMP signaling, (ii) SB-431542 (SB) (e.g., 10 mM) for inhibiting TGFβ signaling, (iii) recombinant FGF8, (iv) smoothand agonist (SAG; 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzyl]-1-benzothiophene-2-carboxamide) for activating sonic hedgehog signaling, and (iv) CHIR99021 (CHIR, e.g., 10 mM) for activating WNT signaling. Alternatively, CT99021 (GSK3 inhibitor) can be used to activate WNT signaling.

[0050] In other embodiments, the neural induction medium comprises (i) LDN193189 (LDN) for inhibiting BMP signaling, (ii) Smoothened agonist (SAG; 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzyl]-1-benzothiophene-2-carboxamide) for activating Sonic Hedgehog signaling, and (iii) CHIR99021 (CHIR, e.g., 10 mM) for activating WNT signaling, the neural induction medium does not comprise an inhibitor of TGFβ signaling (e.g., SB-431542 (SB)), and / or the neural induction medium does not comprise FGF8. In some embodiments, iPSCs or ESCs are identified by the expression of Oct4 / POU5F1, Nanog, and Sox2.

[0051] In some embodiments, neural progenitor cells are identified by expression of Pax6, Nestin and CD133.

[0052] In some embodiments, midbrain progenitor cells are identified by expression of one or more (e.g., all) of the following markers: FOXA2, LMX1A, OXT2, EN1 / 2, GBX2, Wnt1, CNPY1, SPRY1, and Pax8.

[0053] In some embodiments, mature dopaminergic neurons are identified by expression of one or more (e.g., all) of the following markers: tyrosine hydroxylase, CORIN, Nurr1, GRK2, Pitx3, DAT, LRTM1, ALCAM, DRD2, DBH, CHRNB3.

[0054] In some aspects, a method for generating differentiated neural cells from mammalian stem cells is provided, the method comprising the steps of (i) culturing the stem cells in a neural induction medium, which results in the generation of induced neuronal precursor cells; and (ii) culturing the induced neuronal precursor cells in a differentiation medium comprising a GDNF receptor RET agonist (preferably BT13), which results in the generation of a population of differentiated neural cells. In some embodiments, the stem cells are cultured in the neural induction medium according to step (i) for about 10 to about 12 days, 9 to 11 days, or about 10 days.

[0055] Advantageously, the generation of functional neurons from mammalian stem cells according to the method does not require the presence of FGFR agonist (e.g., FGF8) in the neural induction medium of step (i).Therefore, in some preferred embodiments, a method for generating neural cells from mammalian stem cells is provided, which comprises the steps of: (i) culturing the stem cells in a neural induction medium comprising (a) an inhibitor of BMP signaling, (b) an inhibitor of TGFβ signaling, (c) an activator of Sonic Hedgehog (SHH), and (d) an activator of WNT, wherein the neural induction medium does not comprise FGFR agonist; and (ii) culturing the neural progenitor cells in a differentiation medium comprising a GDNF receptor RET agonist (preferably BT13), thereby generating neural cells.

[0056] In another preferred embodiment, the generation of functional neurons from mammalian stem cells according to the method is carried out without the inclusion of an inhibitor of TGFβ signaling in the neural induction medium of step (i).Therefore, a method for generating neural cells from mammalian stem cells is provided, which comprises the steps of: (i) culturing the stem cells in a neural induction medium comprising (a) an inhibitor of BMP signaling, (b) an activator of Sonic Hedgehog (SHH), and (c) an activator of WNT, wherein the neural induction medium does not comprise an inhibitor of TGFβ signaling; and (ii) culturing the neural precursor cells in a differentiation medium comprising a GDNF receptor RET agonist (e.g., BT13 or Q525), thereby generating neural cells.In a related embodiment, the neural induction medium of step (i) comprises an inhibitor of TGFβ signaling and does not comprise an FGFR agonist.

[0057] A variety of differentiated neural cells can be generated according to the method, for example, by varying the number of days neural precursor cells are cultured in differentiation medium containing GDNF receptor RET agonist (e.g., BT13 or Q525) according to step (ii). In some embodiments, the differentiated neural cells generated according to the method described herein are selected from dopamine precursor cells, dopamine neuroblasts, striatal neurons, neuroepithelial stem cells, GABAergic interneurons, cortical interneurons, cholinergic neurons, serotonin interneurons, cerebellar neurons, sensory neurons, and motor neurons.

[0058] Table 1 below shows various neural cell types that can be generated from mammalian pluripotent cells according to the present methods, along with their potential medical uses, the number of days of GDNF required to generate each cell type according to prior art methods, markers for identifying that neural cell type, and references describing the prior art methods, the contents of each of which are incorporated herein by reference. For each of the protocols below, a GDNF receptor RET agonist (e.g., BT13 or Q525) is used in place of GDNF according to the present methods (in place of BDNF, TGFβ, and other proteins in some protocols).

[0059] [Table 1-1] [Table 1-2]

[0060] The type of neuron produced according to this method can be identified by the expression of one or more surface markers.In some embodiments, dopaminergic neurons are produced.Dopaminergic neurons can be identified by the expression of tyrosine hydroxylase, and optionally one or more of DAT, CORIN, GIRK2, PITX3 and NURR1.In other embodiments, striatal neurons are produced.Striatal neurons can be identified by the expression of one or more of DARPP32, CITP2, CALBINDIN and GABA.

[0061] The ability to generate action potentials is a hallmark of neuronal maturity and function, with different neuronal phenotypes exhibiting distinct and specific firing patterns. Thus, the functionality of neurons generated according to the present method can be confirmed by assessing the ability of the neurons to generate action potentials, in addition to the expression of one or more surface markers, for example, using patch clamp electrophysiology or using membrane potential imaging methods described in Adil, M. et al., Sci.Rep., 7, 40573 (2017), the entire contents of which are incorporated herein by reference. Functional midbrain dopaminergic neurons exhibit firing patterns of periodic spikes, for example, at 2Hz-5Hz. Neurons generated according to the present method can also be transplanted (e.g., into the striatum) into animal models (e.g., Fisher 344 rats), and the survival of the transplanted neurons can be evaluated at a later time point (e.g., 6 weeks after transplantation).

[0062] In some embodiments (e.g., using neurosphere-based induction from embryoid bodies), after step (i), the cell clusters are separated into single cells (e.g., on day 11) for culturing in differentiation medium according to step (ii). In other embodiments, a 2D monolayer-based method (e.g., a 2D surface coated with Matrigel) is used. In other embodiments, a 3D cell culture-based method is used, for example, in which cells are embedded in a biomaterial (e.g., alginate, collagen, hyaluronic acid, or materials described in Adil, M. et al., Sci. Rep., 7, 40573 (2017), the entire contents of which are incorporated herein by reference).

[0063] In some embodiments, culturing in differentiation medium as described herein is performed for a time sufficient to generate desired neurons (see, e.g., Table 1). Generally, culturing in differentiation medium as described herein is performed for a period of about 4 days to about 110 days. In some embodiments, culturing in differentiation medium as described herein is performed for a period of about 4 days to about 60 days, or about 5 days to about 40 days, or about 16 days to about 32 days.

[0064] In a related aspect, the cell population produced according to the method is provided.The cell population produced according to the method can typically include other cell types in addition to differentiated neural cells.In one embodiment, the population of the present invention is characterized in that it comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, preferably at least 90% or at least 95% of cells that show high expression of at least one biomarker (e.g., TH gene product) characteristic of differentiated neurons.

[0065] Other biomarkers characteristic of differentiated neural cells will depend on the type of neuron being generated, but may include, but are not limited to, one or more of the markers listed in Table 1.

[0066] Any method known in the art for measuring gene expression may be used, in particular quantitative methods (e.g., using real-time quantitative PCR or microarrays, or gene reporter expression) or qualitative methods (e.g., immunostaining or cell sorting methods that identify cells that display particular biomarkers, including cell surface markers).

[0067] In some embodiments, a cell culture differentiation medium useful for generating functional neurons from mammalian pluripotent stem cells or from induced neuronal precuorsor cells is provided, the differentiation medium comprising a GDNF receptor RET agonist (e.g., BT-13 or Q525). In some embodiments, the differentiation medium is useful for generating tyrosine hydroxylase positive dopaminergic neurons. In some embodiments, the culture medium further comprises a notch pathway inhibitor (e.g., DAPT and / or db-cAMP). In a preferred embodiment, the culture medium does not comprise GDNF, BDNF and TGF-β. In another preferred embodiment, the culture medium is essentially protein-free. In some embodiments, the cell culture medium comprises a neurobasal medium supplemented with an N2 supplement and a B17 supplement. In another embodiment, the cell culture medium comprises a neurobasal medium and an insulin receptor activator (preferably, DMAQ-B1), does not comprise an N2 supplement, and does not comprise a B17 supplement. EXAMPLES

[0068] The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. Although the present invention has been described in relation to its preferred embodiments, various modifications thereof will become apparent to those skilled in the art upon reading this application.

[0069] Example 1 (method)

[0070] Human pluripotent stem cell cultures. Human induced pluripotent stem cells (hPSCs) (ThermoFisher A18945) were subcultured in monolayer form on a layer of 1% Matrigel and maintained in Essential 8 medium during expansion. At 80% confluency, H9 were passaged using Versene solution and re-seeded at a 1:8 split.

[0071] Seeding of 3D hPSC cultures hPSCs were dissociated into single cells using Accutase solution and resuspended in Essential 8 (E8) medium containing 10 μM Y-27632 (Rock inhibitor, RI). hPSCs were counted and resuspended in 11% AXgel on ice at a defined density. Cells suspended in AXgel were distributed into multi-well tissue culture plates and heated to 37°C for 15 min, after which pre-warmed E8 medium containing 10 μM RI was added to each well. 3D cell suspensions were maintained in E8 containing RI for 2 days (day -2 to day 0).

[0072] 3D Dopaminergic Neuronal Differentiation Starting from day 0, hPSCs in AXgel were transferred to differentiation medium to induce neural lineage commitment and subsequent specialization into midbrain dopaminergic neurons. Starting from day 11, neural precursors were transferred to maturation medium containing GDNF agonists BT-13 (5 μM) or Q525 (5 nM) instead of the proteins GDNF, BDNF, and TGF-β. Media formulations were according to Table 2:

[0073] [Table 2] Further reagents are listed in Table 3:

[0074] [Table 3]

[0075] Immunocytochemistry At the end of the experiment, cell aggregates were collected from AXgel by replacing the warm medium with cooled medium, plated on 8-chamber culture slides coated with 10 μg / mL laminin, and cultured overnight in an incubator to allow the aggregates to adhere to the surface. The aggregates attached to the culture slides were then fixed using 4% paraformaldehyde (PFA) for 15 minutes. The aggregates were washed twice in PBS for 5 minutes each, and incubated in 0.25% Triton®-X+5% donkey serum in PBS for 10 minutes to permeabilize the cells. After permeabilization, the aggregates were washed five times in 5% donkey serum for 5 minutes each, incubated with the primary antibody of interest diluted in PBS+donkey serum (dilution details in Table 3) and stored overnight at 4°C. After primary staining, the aggregates were washed twice in PBS for 5 minutes each, incubated in a solution containing the corresponding secondary antibody (dilution details in Table 3) and incubated at 37°C for 2 hours. After secondary staining, aggregates were washed twice for 5 min each in PBS and culture slides were mounted on cover slips for imaging.

[0076] Microscopy: Live cell aggregates suspended in AXgel were imaged periodically throughout the experiment using an EVOS XL Core Imaging System for transmitted light microscopy available at the Cell and Tissue Analysis Facility through QB3-Berkeley. Fixed, stained, and mounted cell aggregates were imaged with a 20x or 40x objective using a Perkin Elmer Opera Phenix automated confocal fluorescence microscope available at the High-Throughput Screening Facility through QB3-Berkeley. Laser exposure times and powers were kept constant for the fluorescent channels within the imaging set.

[0077] (Results and Discussion)

[0078] Substitution of GDNF, BDNF, and TGF-β with BT-13 or Q525 is sufficient to generate hPSC-derived neurons expressing the functional biomarker tyrosine hydroxylase.

[0079] hPSC-derived dopaminergic neurons have demonstrated safety and efficacy as a cell therapy for Parkinson's disease in many rodent and non-human primate studies, and human trials have been initiated. Due to their remarkable progress and promise of translation to the clinic, we chose the generation of hPSC-derived dopaminergic neurons as an important use case for a minimal protein medium formulation in which recombinant proteins FGF8, TGF-β, BDNF, and GDNF were removed and replaced with BT-13 or Q525 starting from day 10 (Figure 1 (BT-13) and Figure 5 (Q525)) in a 3D differentiation system described in Adil et al., Sci Rep, 7:40573 (2017). Removal of FGF8 for the first 6 days of differentiation did not affect the proliferation or patterning of the 3D aggregates (Figure 2). By day 20, expression of tyrosine hydroxylase, a rate-limiting enzyme in dopamine production and a functional biomarker for dopaminergic neurons, was detected in neuronal cell bodies within all differentiating aggregates (Figures 3 and 5). Substitution of GDNF, BDNF, and TGF-β with BT-13 or Q525 is applicable to all neuronal subtypes that require GDNF for differentiation (see Table 1). The results presented herein show that two structurally unrelated RET agonists can each substitute for GDNF, BDNF, and TGF-β in generating functional dopaminergic neurons from pluripotent cells, thus supporting the use of any GDNF receptor RET agonist in the methods described herein.

[0080] We then modeled the cost of scaling up to a 1-liter bioreactor and employing cGMP to produce hPSC-derived dopaminergic neurons for clinical development and commercialization, and compared the cost of using the original media formulation compared to the minimal protein BT-13 replacement media formulation (Figure 4). The stage of differentiation for optimal engraftment and efficacy remains an area of ​​active investigation. Shorter differentiation times risk unpurified cell populations containing uncommitted proliferative precursors that may result in off-target differentiation or undesirable cell overgrowth after transplantation, while longer differentiation times risk more mature committed cells that are less robust to the stresses of transplantation and increased cell death during transplantation. Thus, we included two scenarios in this cost analysis: 25 days of differentiation for transplantation of dopamine precursors, and 40 days of differentiation for transplantation of dopamine neuroblasts (Figure 4). The minimal protein formulation with BT-13 can reduce the cost of materials by more than 50% in longer differentiation protocols. The use of small molecule substitutes for GDNF reduces the cost of media for generating hPSC-derived neurons by more than 50%.

[0081] While the materials and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

Claims

1. 1. A method for generating a cell population comprising functional neural cells, comprising: Culturing induced neural progenitor cells in a differentiation cell culture medium containing one or more glial cell line-derived neurotrophic factor (GDNF) receptor transmembrane receptor tyrosine kinase REarranged during Transfection (RET) agonists. A method comprising:

2. 2. The method of claim 1, wherein the GDNF receptor RET agonist is selected from DNSP-11, BT-18, BT-44, XIB4035, BT-13 and Q525.

3. 3. The method of claim 2, wherein the GDNF receptor RET agonist is BT-13 and / or Q525, and BT-13 is present in the cell culture medium at a concentration of about 2 μM to about 20 μM, preferably about 5 μM, and / or Q525 is present in the culture medium at a concentration of about 1 nM to about 100 nM.

4. 2. The method of claim 1, wherein the differentiation cell culture medium does not contain one or more of GDNF, brain-derived neurotrophic factor (BDNF) and TGFβ, preferably does not contain GDNF, BDNF and TGFβ.

5. 5. The method of claim 4, wherein the differentiation cell culture medium is essentially protein-free.

6. 2. The method of claim 1, wherein the differentiation cell culture medium comprises neurobasal medium supplemented with N-2 supplement and / or B-27 supplement.

7. 2. The method of claim 1, wherein the differentiation cell culture medium comprises a neurobasal medium and an insulin receptor activator, but does not contain an N-2 supplement and / or a B-27 supplement.

8. 8. The method of claim 7, wherein the insulin receptor activator is demethylasteriquinone B1 (DMAQ-B1).

9. The method of claim 1 , wherein the differentiation cell culture medium comprises a Notch pathway inhibitor.

10. 10. The method of claim 9, wherein the Notch pathway inhibitor is DAPT (N-[2S-(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl ester-glycine) or dibutyryl cAMP (db-cAMP).

11. 2. The method of claim 1, wherein the induced neural progenitor cells are cultured in differentiation medium for a period of about 4 days to about 110 days, about 4 days to about 60 days, about 5 days to about 40 days, or about 16 days to about 32 days.

12. 2. The method of claim 1, wherein the functional neural cells are selected from dopamine progenitor cells, dopamine neuroblasts, striatal neurons, neuroepithelial stem cells, GABAergic interneurons, cortical interneurons, cholinergic neurons, serotonin interneurons, cerebellar neurons, sensory neurons, and motor neurons.

13. 13. The method of claim 12, wherein the functional neuronal cells are dopaminergic neurons.

14. 14. The method of claim 13, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the cells in the cell population express tyrosine hydroxylase (TH).

15. 2. The method of claim 1, wherein the induced neural progenitor cells are generated by culturing mammalian pluripotent cells in an induction medium containing an effective amount of an inhibitor of the bone morphogenetic protein (BMP) signaling pathway, and optionally containing an effective amount of an inhibitor of the TGFβ signaling pathway.

16. 16. The method of claim 15, wherein the inhibitor of the bone morphogenetic protein (BMP) signaling pathway is LDN193189 and / or the inhibitor of the TGFβ signaling pathway is SB-431542.

17. The method of claim 15, wherein the induction medium further comprises an activator of the WNT signaling pathway and / or an activator of Sonic Hedgehog (SHH).

18. 18. The method of claim 17, wherein the activator of the WNT signaling pathway is a GSK3 inhibitor, preferably CHIR99021, and / or the activator of SHH is smoothened agonist (SAG).

19. 16. The method of claim 15, wherein the induction medium does not contain an FGF receptor (FGFR) agonist and / or contains an inhibitor of TGFβ signaling.

20. 20. The method of claim 19, wherein the induction medium does not contain an FGF receptor (FGFR) agonist and contains an inhibitor of TGFβ signaling.

21. 1. A method for generating a population comprising functional neural cells, comprising: (i) culturing mammalian pluripotent cells in an induction medium under conditions sufficient to generate induced neural progenitor cells; and (ii) culturing the induced neural progenitor cells in a differentiation medium containing an effective amount of a GDNF receptor RET agonist; A method comprising:

22. 22. The method of claim 21, wherein the mammalian pluripotent cells are human embryonic stem cells or human induced pluripotent stem cells.

23. 1. A cell culture medium useful for generating functional neurons from neural progenitor cells, comprising: the cell culture medium is a protein-free neurobasal medium containing a GDNF receptor RET agonist, preferably BT-13, and a Notch pathway inhibitor, preferably DAPT and / or db-cAMP; The culture medium does not contain GDNF, BDNF, and TGFβ. Cell culture medium.

24. 24. The cell culture medium of claim 23, wherein the neurobasal medium is supplemented with N2 supplement and / or B27 supplement.

25. 24. The cell culture medium of claim 23, wherein the neurobasal medium comprises an insulin receptor activator, preferably DAQ-B1, and does not comprise an N2 supplement and / or a B27 supplement.

26. 2. The method of claim 1, wherein the GDNF receptor RET agonist selectively activates RET independently of GFRα1.

27. A method for generating a cell population comprising functional neurons. Incubating human pluripotent stem cells in neurobasal cell culture medium under conditions suitable for generating a neural progenitor cell population, preferably the neurobasal cell culture medium comprising LDN193189, CHIR99021 and a smoothoid agonist, and not comprising FGF8; and incubating the generated neural progenitor cell population in a neurobasal cell culture medium comprising one or more GNDF receptor RET agonists and a Notch pathway inhibitor, preferably DAPT and / or db-cAMP, for a time sufficient to generate a population of functional neurons, wherein the neurobasal cell culture medium does not comprise GDNF, BDNF, or TGF-β. A method comprising:

28. 28. The method of claim 27, wherein the neurobasal cell culture medium comprises N-2 and B-27.

29. 28. The method of claim 27, wherein the neurobasal cell culture medium comprises an insulin receptor activator molecule, preferably DMAQ-B1, and is free of N-2 and B-27.

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