Methods for nociceptor differentiation of human embryonic stem cells and uses thereof

Abstract

To provide a method of nociceptor differentiation of human embryonic stem cells and uses thereof.SOLUTION: The present invention relates to the field of stem cell biology, in particular the linage specific differentiation of pluripotent or multipotent stem cells, which can include, but is not limited to, human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC), somatic stem cells, cancer stem cells, or any other cell capable of lineage specific differentiation. Specifically described are methods to direct the lineage specific differentiation of hESC and / or hiPSC to nociceptors (i.e. nociceptor cells) using novel culture conditions. The nociceptors made using the methods of the present invention are further contemplated for various uses including, but not limited to, use in vitro drug discovery assays, pain research, and as a therapeutic to reverse disease of, or damage to, the peripheral nervous system (PNS).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of stem cell biology, particularly to the lineage-specific differentiation of pluripotent or multipotent stem cells, which may include, but are not limited to, human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), somatic stem cells, cancer stem cells, or any other cells capable of lineage-specific differentiation. In particular, methods are described for directing the lineage-specific differentiation of hESCs and / or hiPSCs into nociceptors (i.e., nociceptor cells) using novel culture conditions. Nociceptors generated using the methods of the present invention are further contemplated for a variety of uses, including, but not limited to, use in in vitro drug discovery assays, pain research, and as therapeutics to reverse diseases or injuries of the peripheral nervous system (PNS). Additionally, compositions and methods are provided for producing melanocytes from human pluripotent stem cells for use in disease modeling. [Background technology]

[0002] Embryonic and somatic stem cells have the potential to differentiate into any cell type: they are therefore highly suitable for cell replacement therapy for diseases that affect or damage / injure characterized cell populations. Beyond their direct therapeutic value, lineage-specific differentiated stem cells are also useful research tools for a variety of purposes, including in vitro screening assays to identify, validate, and test the specification or delivery of therapeutic molecules to treat lineage-specific diseases, further elucidating the complex mechanisms of cell lineage specification and differentiation, and identifying important biochemical differences between normal and diseased or damaged states that can be further considered for use as diagnostic or prognostic markers.

[0003] The potential of embryonic and somatic stem cells as therapeutic agents and model systems for neurodegenerative diseases has been widely explored. However, much of the research and technological development regarding directed differentiation of embryonic and somatic stem cells has been focused on diseases of the central nervous system (CNS), such as Huntington's disease, Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Knowledge regarding directed differentiation of embryonic and somatic stem cells toward peripheral nervous system (PNS) lineages is currently insufficient. The PNS is composed of the somatic nervous system, which integrates musculoskeletal control and perception of external stimuli, and the autonomic nervous system, which regulates visceral organ functions such as heart rate and breathing. Numerous disorders of the PNS exist, including Charcot-Marie-Tooth disease, Guillain-Barré syndrome, and Hirschsprung's disease. Diseases of the peripheral sensory neurons of the PNS, which result in severe pain or an inability to respond to noxious stimuli that cause injury, are particularly burdensome to society and include diseases such as familial dysautonomia, congenital insensitivity to pain, diabetic neuropathy, and damage from chickenpox or shingles infections.

[0004] Understanding the pathology of peripheral sensory neuron diseases and developing treatments are hindered by the difficulty of obtaining human peripheral sensory neurons; current methods are limited to manual isolation from 3- to 5-week-old human embryos or infrequent surgical procedures. Directed differentiation of embryonic or somatic stem cells into specific peripheral sensory neurons, particularly nociceptors, which perceive pain, provides an ideal, renewable source of such cells for both research and therapeutic applications. Recently, attempts have been made to produce peripheral sensory neurons from neuronal intermediates derived from embryonic stem cells. However, these techniques are limited by the need for neuronal intermediates, co-culture with mouse interstitial cells, the length of time required to induce such peripheral sensory neurons, low yields, impure cell populations containing mixed neuronal types, and the limited survival and poor characterization of PNS-generated neurons. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need in the art for a method to produce peripheral sensory neurons, particularly nociceptors, directly from embryonic or somatic stem cells with high purity and yield without the use of contaminating mouse stromal cells. [Means for solving the problem]

[0006] The present invention relates to the field of stem cell biology, particularly to the lineage-specific differentiation of pluripotent or multipotent stem cells, which may include, but are not limited to, human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), somatic stem cells, cancer stem cells, or any other cells capable of lineage-specific differentiation. In particular, methods are described for directing the lineage-specific differentiation of hESCs and / or hiPSCs into nociceptors (i.e., nociceptor cells) using novel culture conditions. Nociceptors generated using the methods of the present invention are further contemplated for a variety of uses, including, but not limited to, use in in vitro drug discovery assays, pain research, and as therapeutics to reverse diseases or injuries of the peripheral nervous system (PNS). Additionally, compositions and methods are provided for producing melanocytes from human pluripotent stem cells for use in disease modeling.

[0007] It is an object of the present invention to overcome limitations and / or alleviate deficiencies in the art. In one embodiment, the present invention provides a method for producing nociceptors, comprising: i) obtaining stem cells (e.g., hESCs, hiPSCs, somatic stem cells, cancer stem cells, human or mammalian pluripotent cells, etc.); ii) culturing the stem cells under conditions that inhibit dual SMAD signaling; and iii) further culturing the cells under conditions that inhibit FGF and Notch signaling and activate Wnt signaling. As used herein, the terms "inhibit" or "block" refer to a decrease in the level of activity of a particular signaling pathway in a cell upon treatment with a compound (i.e., an inhibitor) compared to the activity of the signaling pathway in cells left untreated with such a compound or treated with a control. As used herein, the term "activate" refers to an increase in the level of activity of a particular signaling pathway in a cell upon treatment with a compound (i.e., an activator) compared to the activity of the signaling pathway in cells left untreated with such a compound or treated with a control. Any level of inhibition or activation of a particular signaling pathway is considered an embodiment of the present invention if such inhibition or activation results in directed differentiation of stem cells. In one embodiment, the culture method includes conditions for a feeder-free system. In one embodiment, stem cells are cultured in a monolayer. In a preferred embodiment, the culture method contemplates the use of a medium containing the compounds SB431542, LDN1933189, SU5402, CHIR99021, and DAPT. In one embodiment, the differentiated cells represent at least 10% to 100% of the cultured cell population. In one embodiment, the differentiated cells express one or more markers from the group including ISL1, BRN3A, RET, RUNX1, and NTRK1. In one embodiment, the markers are expressed in at least 10% to 100% of the cultured cell population. In a preferred embodiment, the differentiated cells are nociceptors. In a preferred embodiment, the stem cells are hESCs or hiPSCs.

[0008] In one embodiment, the present invention provides a kit comprising: i) a first inhibitor or combination of inhibitors that blocks both SMAD signaling and TGFβ / Activin-Nodal signaling; ii) a second inhibitor that blocks FGF signaling; iii) a third inhibitor that blocks Notch signaling; and iv) an activator of Wnt signaling. In one embodiment, the first inhibitor is selected from the group consisting of LDN193189 and SB431542, combinations thereof, and mixtures thereof. In one embodiment, the second inhibitor comprises SU5402 and its derivatives. In one embodiment, the third inhibitor comprises DAPT and its derivatives. In one embodiment, the activator comprises CHIR99021 and its derivatives. In one embodiment, the kit further comprises human stem cells. In one embodiment, the kit further provides instructions for practicing the present invention.

[0009] In one embodiment, the present invention provides a kit comprising: i) a first inhibitor or combination of inhibitors that blocks both SMAD signaling and TGFβ / Activin-Nodal signaling; ii) a second inhibitor that blocks FGF signaling; iii) a third inhibitor that blocks Notch signaling; and iv) an activator of Wnt signaling. In one embodiment, the first inhibitor is selected from the group consisting of SB431542, LDN193189, a combination thereof, and a mixture thereof. In one embodiment, the second inhibitor comprises SU5402 and its derivatives. In one embodiment, the third inhibitor comprises DAPT and its derivatives. In one embodiment, the activator comprises CHIR99021 and its derivatives. In one embodiment, the kit further comprises instructions. In one embodiment, the kit further comprises human stem cells. In one embodiment, the human stem cells are human embryonic stem cells. In one embodiment, the human stem cells are human induced pluripotent stem cells.

[0010] The present invention further contemplates a method for evaluating the peripheral sensory neuron subtype of differentiated stem cells. Some embodiments of this method can utilize microscopic analysis, functional assays, or measuring the expression or downregulation of markers associated with specific lineages. In a preferred embodiment, the method comprises measuring markers associated with nociceptor specification selected from the group consisting of ISL1, BRN3A, RET, RUNX1 and NTRK1.

[0011] In one embodiment, the invention provides a method for inducing directed differentiation of stem cells, the method comprising: a) providing: i) a cell culture comprising human stem cells; ii) a first inhibitor or combination of inhibitors that blocks both SMAD signaling and TGFβ / Activin-Nodal signaling; iii) a second inhibitor that blocks FGF signaling; iv) a third inhibitor that blocks Notch signaling; and v) an activator of Wnt signaling; b) contacting the stem cells with the first inhibitor or combination of inhibitors that block both SMAD signaling and TGFβ / Activin-Nodal signaling in vitro for 0 to 48 hours (more typically 1 to 48 hours); and c) further contacting the stem cells with the second inhibitor that blocks FGF signaling; the third inhibitor that blocks Notch signaling; and the activator of Wnt signaling for up to 192 hours (or even up to 240 hours). In one embodiment, the first inhibitor is selected from the group consisting of SB431542, LDN193189, combinations thereof, and mixtures thereof. In one embodiment, the second inhibitor includes SU5402 and its derivatives. In one embodiment, the third inhibitor includes DAPT and its derivatives. In one embodiment, the activator includes CHIR99021 and its derivatives. In one embodiment, the stem cells are human embryonic stem cells. In one embodiment, the stem cells are human induced pluripotent stem cells. In one embodiment, the differentiated cells are neuronal cells. In one embodiment, the neuronal cells are nociceptors. In one embodiment, the differentiated cells express one or more markers from the group consisting of ISL1, BRN3A, RET, RUNX1, and NTRK1. In one embodiment, the differentiated cells respond to external stimuli.

[0012] The present invention further contemplates the use of nociceptors produced by the method of the present invention. In one embodiment, the nociceptors are used in an in vitro assay to identify compounds that can be used as anti-pain therapeutic agents. In one embodiment, the nociceptors are used to study nociceptor function. In one embodiment, the nociceptors are used as in vivo cell replacement therapy in animals suffering from or at risk of PNS injury or disease.

[0013] In one embodiment, the invention provides a method of screening for a biological agent comprising: a) providing i) a nociceptor; and ii) a test compound; b) contacting the nociceptor with the test compound and measuring activation or inhibition of nociceptor function. In one embodiment, the nociceptor is derived from a human stem cell.

[0014] In one embodiment, the present invention provides a kit comprising a first signal transduction inhibitor, a second signal transduction inhibitor, and a third signal transduction inhibitor, wherein the first inhibitor is capable of reducing transforming growth factor β (TGFβ) / activin-nodal signaling, the second inhibitor is capable of reducing small mothers against decapentaplegic (SMAD) signaling, and the third inhibitor is capable of reducing glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling. In one embodiment, the first inhibitor is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. In one embodiment, the second inhibitor is a small molecule selected from the group consisting of LDN193189, derivatives thereof, and mixtures thereof. In one embodiment, the third inhibitor is selected from the group consisting of CHIR99021 and derivatives thereof. In one embodiment, the kit further comprises a fourth inhibitor that reduces fibroblast growth factor (FGF) receptor family signaling, where the FGF receptor family signaling includes vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF) receptor, and platelet-derived growth factor (PDGF) tyrosine kinase receptor. In one embodiment, the fourth inhibitor is selected from the group consisting of SU5402 and its derivatives. In one embodiment, the kit further comprises a fifth inhibitor that can reduce Notch signaling. In one embodiment, the fifth inhibitor is selected from the group consisting of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and its derivatives. In one embodiment, the kit further comprises an inhibitor of nestin,

[0015] [ka]

[0016] In one embodiment, the kit further comprises an antibody used to detect the expression of a protein selected from the group consisting of nestin,

[0017] [ka]

[0018] In one embodiment, the kit further comprises PCR primers for detecting mRNA expression of a gene selected from the group consisting of: Protachykinin-1 (TAC1), vesicular glutamate transporter 2 (VGLUT2), and solute carrier family 15, member 3 (SLC15A3). In one embodiment, the kit further comprises an antibody used for detecting expression of a protein selected from the group consisting of Protachykinin-1 (TAC1), vesicular glutamate transporter 2 (VGLUT2), and solute carrier family 15, member 3 (SLC15A3). In one embodiment, the kit further comprises PCR primers for detecting mRNA expression of a gene selected from the group consisting of Protachykinin-1 (TAC1), vesicular glutamate transporter 2 (VGLUT2), and solute carrier family 15, member 3 (SLC15A3). In one embodiment, the kit further comprises instructions comprising a step for adding the first and second inhibitors two days before adding the third inhibitor. In one embodiment, the kit further comprises instructions including a step for adding the first and second inhibitors two days before adding the combination of the third inhibitor, the fourth inhibitor, and the fifth inhibitor. In one embodiment, the kit further comprises instructions including a step for sequentially administering the inhibitors daily on days 0-10. In one embodiment, the kit further comprises instructions including a step for generating neural stem cell precursors and a step for generating nociceptor cells. In one embodiment, the kit further comprises human stem cells. In one embodiment, the human stem cells are human embryonic stem cells. In one embodiment, the human stem cells are human induced pluripotent stem cells. In one embodiment, the human stem cells are transgenic SOX10::GFP bacterial artificial chromosome (BAC) human puripotent stem cells (hPSCs).

[0019] In one embodiment, the present invention provides a method for inducing directed differentiation of stem cells, comprising: a) a cell culture comprising: i) human stem cells; and ii) a first signaling inhibitor, a second signaling inhibitor, and a third signaling inhibitor, wherein said first inhibitor is capable of reducing transforming growth factor β (TGFβ) / activin-nodal signaling, and said second inhibitor is capable of reducing Small Mothers' signaling. a) providing an inhibitor capable of reducing SMAD signaling and a third inhibitor capable of reducing Glycogen Synthase Kinase 3β (GSK3β) for activation of Wingless (Wnt) signaling; b) contacting the stem cells with the first inhibitor and the second inhibitor in vitro for up to 48 hours (or even up to 96 hours); and c) further contacting the inhibited stem cells with the third inhibitor for up to 192 hours (or even up to 240 hours) to induce directed differentiation of the stem cells, wherein the differentiated stem cells are selected from the group consisting of neural crest stem cells, neural crest lineage cells, and neuronal lineage cells. In one embodiment, the first inhibitor is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. In one embodiment, the second inhibitor is a small molecule selected from the group consisting of LDN193189, derivatives thereof, and mixtures thereof. In one embodiment, the third inhibitor is selected from the group consisting of CHIR99021 and its derivatives. In one embodiment, the kit further comprises a fourth inhibitor that reduces fibroblast growth factor (FGF) receptor family signaling, where the FGF receptor family signaling includes vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF) receptor, and platelet-derived growth factor (PDGF) tyrosine kinase receptor. In one embodiment, the fourth inhibitor is selected from the group consisting of SU5402 and its derivatives. In one embodiment, the kit further comprises a fifth inhibitor that can reduce Notch signaling. In one embodiment, the fifth inhibitor is selected from the group consisting of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and its derivatives.In one embodiment, the kit further comprises a fourth inhibitor and a fifth inhibitor for directed differentiation of neuronal lineage cells into peptidergic nociceptor cells, wherein the fourth inhibitor is selected from the group consisting of SU5402 and its derivatives, and the fifth inhibitor is selected from the group consisting of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and its derivatives. In one embodiment, the peptidergic nociceptor cells express a marker selected from the group consisting of OCT4, DLK1, PAX6, SOX10, POU4F1 (BRN3A), ISL1, NEUROG2, NEUROG1, NTRK1, RET, RUNX1, VGLUT2, TAC1, and TRPV1. In one embodiment, the peptidergic nociceptor cells express a marker selected from the group consisting of ISL1, POU4F1 (BRN3A), RET, RUNX1, and NTRK1. In one embodiment, the marker is selected from the group consisting of proteins and nucleic acids. In one embodiment, the peptidergic nociceptor cells co-express substance P and calcitonin gene-related peptide (CGRP). In one embodiment, the peptidergic nociceptor cells generate action potentials in response to an external stimulus, wherein the external stimulus is an electrical current. In one embodiment, the differentiated peptidergic nociceptor cells are present within a highly enriched population of neurons within 8 to 18 days, more typically within 10 to 15 days, after contacting the stem cells with the first inhibitor and the second inhibitor. In one embodiment, the stem cells are human embryonic stem cells. In one embodiment, the stem cells are human induced pluripotent stem cells.

[0020] In one embodiment, the present invention provides a method for screening biological agents in vitro, comprising: a) providing i) nociceptor cells derived in vitro from directed differentiation of stem cells; and ii) a test compound; and b) contacting the nociceptor cells with the test compound and measuring nociceptor function, wherein the function is a measurement of action potentials. In one embodiment, the nociceptor cells are derived from human stem cells.

[0021] In one embodiment, the present invention provides a kit for directed differentiation of melanocytes.

[0022] In one embodiment, the present invention provides a method for directed differentiation of melanocytes.

[0023] In one embodiment, the present invention provides a method for providing a population of cells of the melanocyte lineage.

[0024] In one embodiment, the present invention provides a method for providing a mature melanocyte cell population.

[0025] definition As used herein, the term "kit" refers to any delivery system for delivering materials. In relation to cell differentiation, kit may refer to a combination of materials for contacting stem cells, and such delivery system includes a system that allows the storage, transport or delivery (from one location to another) of reaction reagents (e.g., compounds, proteins, detection reagents (e.g., PAX6 antibody), etc.) and / or supporting materials (e.g., buffer solutions, instruction manuals for carrying out cell differentiation, etc.) in suitable containers (e.g., tubes, etc.). For example, the kit may include relevant reaction reagents for inhibiting signaling pathways, such as inhibitors for reducing transforming growth factor beta (TGFβ) / activin-nodal signaling, such as SB431542 (or an SB431542 substitute), inhibitors for reducing SMAD signaling, such as LDN193189 (or an LDN193189 substitute), inhibitors for reducing glycogen synthase kinase 3β (GSK3β), such as CHIR99021 (or an CHIR99021 substitute), inhibitors for reducing β-catenin signaling, such as Wingless (Wnt) inhibitors, otherwise known as WNT signaling activators (WNT agonists), for example, for suppressing β-catenin signaling. and Wnts (Wnts) signaling activation inhibitors, inhibitors of fibroblast growth factor (FGF) receptor family signaling, including those that reduce FGF receptor family signaling, such as SU5402 (or an SU5402 substitute), where the FGF receptor family signaling includes vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF) receptor, and platelet-derived growth factor (PDGF) tyrosine kinase receptor signaling, an inhibitor of Notch signaling, such as DAPT (or a DAPT substitute), and / or one or more containers (e.g., boxes or bags, test tubes, Eppendorf tubes, capillary tubes, multi-well plates, etc.) containing support material. In one embodiment, the reagents in the kit may be in solution, frozen, or lyophilized.In one embodiment, the reagents in the kit may be in individual containers or may be provided in a particular combination, such as a combination of LSB, 3i, CHIR, Mel reagents, etc.

[0026] As used herein, the term "signal transduction" with respect to a "signaling protein" refers to a protein that is activated or otherwise affected by ligand binding to a membrane receptor protein or some other stimulus. Examples of signaling proteins include SMADs, WNT complex proteins including β-catenin, Notch, transforming growth factor β (TGFβ), activin, Nodal, and glycogen synthase kinase 3β (GSK3β) proteins. For many cell surface receptors or intracellular receptor proteins, ligand-receptor interactions are not directly linked to cellular responses. Ligand-activated receptors must first interact with other proteins inside the cell before the ligand's ultimate physiological effect on cellular behavior occurs. Often, the sequential behavior of several interacting cellular proteins changes after receptor activation or inhibition. The entire set of cellular changes induced by receptor activation is called a signaling mechanism or pathway.

[0027] As used herein, the term "Notch" refers to a signaling pathway represented by at least five ligands (e.g., designated Jagged-1, -2, and Delta-like (Dll)-1, -3, and -4) that bind to one or more of at least four Notch receptors (designated Notch-1, -2, -3, and -4). Notch signaling is initiated by a receptor-ligand interaction that results in at least one proteolytic cleavage by TACE (TNFα-converting enzyme) and / or the gamma-secretase / presenilin complex. This proteolytic cleavage results in the formation of an intracellular domain protein (N IC This results in the release of Notch (the functionally active form of Notch), which translocates to the nucleus and binds to the DNA-binding protein CBF-1 (also called CSL or RBP-Jκ). ICBinding of Notch to CBF-1 recruits nuclear coactivators, such as MAML1 and histone acetyltransferases, which translocate the repressor complex and convert CBF-1 into a transcriptional activator. CBF-1 / Notch interactions are also involved in the transcription of transcription factors, including, by way of example, Hes (Hairy / Enhancer of Split), Hey (YRPW-associated Hairy / Enhancer of Split) (also known as HesR, HRT, HERP, CHF, and Gridlock), NF-κB and PPAR family transcription factors, and p2l. CIP1 / WAF1 Notch signaling pathways, including the Notch receptor and Hey family members, result in the expression of various target genes, including cell cycle regulators such as cyclin D. Members of the Hes (including Hes-1) and Hey (including Hey1 and Hey2) families are examples of transcription factors that are direct downstream targets of Notch activation. Given the complexity of the Notch signaling pathway, predicting the outcome of Notch activation or inhibition is understandably difficult. Not only are there numerous Notch receptors and ligands (each with its own unique expression pattern), but the numerous target genes and potential crosstalk between Notch and other signaling cascades further complicate this system.

[0028] As used herein, the term "signal" refers to internal and external factors that control changes in cellular structure and function, and can be chemical or physical in nature.

[0029] As used herein, the term "ligand" refers to molecules and proteins that bind to a receptor (R), examples of which include, but are not limited to, transforming growth factor beta, activin, nodal, bone morphogenetic proteins (BMPs), and the like.

[0030] As used herein, the term "inhibitor," in reference to inhibiting a signaling molecule or pathway of a signaling molecule, or the term "signaling inhibitor," such as an inhibitor of SMAD signaling, refers to a compound or molecule (e.g., a small molecule, peptide, peptidomimetic, natural compound, siRNA, antisense nucleic acid, aptamer, or antibody) that interferes with (i.e., reduces or suppresses or eliminates or blocks) the signaling function of a molecule or pathway. In other words, an inhibitor is any compound or molecule that alters the activity of a specified protein (signaling molecule, any molecule involved in a specified signaling molecule, or a specified related molecule such as glycogen synthase kinase 3β (GSK3β)) (including, but not limited to, signaling molecules described herein), for example, by directly contacting SMAD signaling, contacting SMAD mRNA, causing a conformational change in SMAD, reducing SMAD protein levels, or preventing the interaction of SMAD with signaling partners (including, for example, those described herein), and affecting the expression of SMAD target genes (such as those described herein). Inhibitors also include molecules that indirectly regulate the biological activity of SMADs by blocking upstream signaling molecules. For example, an example of a signaling molecule and action is noggin, which sequesteres bone morphogenetic proteins within its extracellular domain and inhibits the activation of ALK receptors 1, 2, 3, and 6, thereby preventing downstream SMAD activation. Similarly, Chordin, Cerberus, and Follistatin similarly sequester extracellular activators of SMAD signaling. The transmembrane protein Bambi also acts as a pseudoreceptor to sequester extracellular TGFβ signaling molecules. Antibodies that block activin, nodal, TGFβ, and BMP are considered for use in neutralizing extracellular activators of SMAD signaling.Thus, in one embodiment, the inhibitors of the present invention induce (alter) or modify differentiation from a default cell type to a non-default cell type. For example, one method of the present invention includes at least three inhibitors that generate non-default neural progenitor cells. In a preferred embodiment, the inhibitors of the present invention "modify," "reduce," or "block" default signaling to direct cell differentiation toward a non-default cell type, as described herein to produce the nociceptor cells of the present invention. Thus, the inhibitors of the present invention are natural compounds or small molecules that increase or decrease the activity of signaling molecules that are useful in producing the nociceptor cells of the present invention. In addition to inhibition induced by binding to and affecting a molecule upstream from a specified signaling molecule (which in turn results in inhibition of the specified molecule), inhibitors are described in terms of competitive inhibition (binding to an active site in a way that eliminates or reduces binding of another known binding compound) and allosteric inhibition (binding to a protein in a way that alters the protein's conformation to prevent a compound from binding to the protein's active site). In some cases, inhibitors are referred to as "direct inhibitors", which refers to the fact that they inhibit signal transduction targets or signal transduction target pathways by actually contacting the signal transduction targets; for example, a direct inhibitor of gamma secretase is a DAPT molecule that binds to gamma secretase protein. Exemplary direct inhibitors include, but are not limited to, lidocaine, myricitrin, chronic capsaicin, camphor, amiloride, capsazepine, linopirdine, and most local anesthetics that block general nerve function.

[0031] As used herein, the term "extracellular signaling effect" refers to the effect of an extracellular signaling molecule (e.g., a test substance such as a small molecule described herein, a pharmaceutical agent, a ligand for a receptor, a cytokine, a chemokine, a soluble factor, an adhesion molecule, or other signaling molecule) on a cell (e.g., a eukaryotic cell). In some embodiments, the extracellular signaling reduces signaling activity, such as SMAD activity, alters SMAD activation kinetics, or alters the expression pattern of SMAD target genes.

[0032] As used herein, the term "Small Mothers Against Decapentaplegic" or "Small Mothers Against Decapentaplegic" or "SMAD" refers to signaling molecules.

[0033] As used herein, the terms "activator" and "activating" refer to compounds for activating molecules that result in directed differentiation of the cells of the present invention. Exemplary activators include, but are not limited to, noxious heat / cold, mechanical stimuli, and chemical stimuli (menthol, piperine, acute capsaicin, cinnamaldehyde, bradykinin, ATP, prostaglandins, inflammatory cytokines, acidic saline, fibroblast growth factor (FGF), etc.).

[0034] As used herein, the term "LSB" refers to a combination of two compounds, LDN-193189 and SB431542, that can reduce or block signal transduction consisting of transforming growth factor beta (TGFβ) / activin-nodal signaling and small mothers against decapentaplegic (SMAD) signaling in cells.

[0035] As used herein, the term "SB431542" refers to a molecule capable of reducing or blocking transforming growth factor β (TGFβ) / activin-nodal signaling, and has CAS number 301836-41-9, molecular formula C 22 H 18 It refers to a molecule having the name N4O3, and 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, see, for example, the structure below.

[0036] [ka]

[0037] As used herein, the term "LDN193189" refers to C 25 H 22 It refers to the small molecule DM-3189, IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, with the chemical formula N6. LDN193189 can function as a SMAD signaling inhibitor. LDN193189 is also a highly potent small molecule inhibitor of ALK2, ALK3, and ALK6, protein tyrosine kinases (PTKs), and inhibits signaling of members of the ALK1 and ALK3 family of type I TGFβ receptors, resulting in inhibition of the transduction of multiple biological signals, including bone morphogenetic protein (BMP) BMP2, BMP4, BMP6, BMP7, and activin cytokine signals, and subsequent SMAD phosphorylation of Smad1, Smad5, and Smad8 (Yu et al. (2008) Nat Med 14:1363-1369; Cuny et al. (2008) Bioorg. Med. Chem. Lett. 18:4388-4392, incorporated herein by reference).

[0038] [ka]

[0039] As used herein, the term "dorsomorphin" refers to Dorsomorphin, CAS number 866405-64-3, molecular formula C 24 H 25 It refers to a molecule having the name NO and 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride, see, for example, the structure below.

[0040] [ka]

[0041] As used herein, the term "LSB / C" or "LSB-C" refers to a combination of two compounds, such as LDN-193189 and SB431542, that can combine to reduce or block signal transduction consisting of cellular transforming growth factor β (TGFβ) / activin-nodal signaling and Small Mothers Against Decapentaplegic (SMAD) signaling, in addition to a glycogen synthase kinase 3β inhibitor, e.g., CHIR99021, that acts as a WNT agonist.

[0042] As used herein, the term "glycogen synthase kinase 3β inhibitor" or "GSK3β inhibitor" refers to a compound that inhibits the glycogen synthase kinase 3β enzyme, see, e.g., Doble et al., J Cell Sci. 2003;116:1175-1186, which is incorporated herein by reference. For purposes of the present invention, a GSK3β inhibitor can activate the WNT signaling pathway, see, e.g., Cadigan et al., J Cell Sci. 2006;119:395-402; Kikuchi et al., Cell Signalling. 2007;19:659-671, which are incorporated herein by reference.

[0043] As used herein, the term "CHIR99021" or "aminopyrimidine" or "3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylidenyl]-2-indolinone" refers to the IUPAC designation 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile. CHIR99021 is an example of a small molecule chemical inhibitor of glycogen synthase kinase 3β (GSK3β) / activator of the WNT signaling pathway that is highly selective, exhibiting nearly thousand-fold selectivity against a panel of related and unrelated kinases, with an IC50 of 100 for human GSK3β. 50 = 6.7 nM and nanomolar IC for the rodent GSK3β homologue 50 It has a value.

[0044] [ka]

[0045] As used herein, the term "triple inhibitor" or "3i" refers to the combination of three small molecules: CHIR99021, SU5402, and DAPT. In other embodiments, triple inhibitor refers to a combination of three compounds (i.e., small molecules) capable of combined inhibition of glycogen synthase kinase 3β (GSK3β) / activator of WNT signaling (i.e., a WNT agonist), a Notch signaling inhibitor, i.e., a gamma-secretase inhibitor that can reduce Notch signaling, and inhibition of fibroblast growth factor receptor (i.e., an indolinone derivative is an example of a fibroblast growth factor receptor inhibitor).

[0046] [ka]

[0047] As used herein, the term "Notch inhibitor" or "Notch signaling inhibitor" refers to any compound that has the ability to inhibit Notch activation, such as DAPT, gamma secretase inhibitors (GS1s), e.g., tripeptide aldehyde inhibitor, gamma secretase inhibitor XII, and peptidomimetic inhibitor (LY-411,575).

[0048] As used herein, the term "gamma secretase inhibitors" or "GSIs" refers to a novel class of agents that prevent the generation of the active domain of the Notch molecule, resulting in the suppression of downstream Notch signaling.

[0049] As used herein, the term "γ-secretase inhibitor" refers to a compound capable of inhibiting γ-secretase, a multi-subunit transmembrane protease. One example of a target (i.e., substrate) of γ-secretase is, for example, Notch signaling, and other γ-secretase substrates include low-density lipoprotein (LDL) receptor-associated protein, E-cadherin, and ErbB-4. γ-secretase inhibitors, such as DAPT and γ-secretase inhibitor XII, therefore block the proteolysis of such γ-secretase substrates, including Notch.

[0050] As used herein, the term "DAPT" refers to 23 H 26 N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester, having the chemical formula F2N2O4; also known as LY-374973, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, is an example of a gamma-secretase inhibitor that inhibits Notch and is designated as a dipeptide gamma-secretase-specific inhibitor.

[0051] [ka]

[0052] One example of a DAPT derivative is the photoactivatable DAPT derivative, DAP-BpB (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide).

[0053] As used herein, the term "fibroblast growth factor receptor inhibitor" or "FGFR inhibitor" refers to small molecules such as SU5402, PD173074, etc. One example of an FGFR inhibitor is the indolinone derivative SU5402, an exemplary structure of which is shown below.

[0054] [ka]

[0055] As used herein, the term "SU5402" refers to C 17 H 16N2O3 refers to a small molecule having the chemical formula and chemical name: 2-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-4-methyl-1H-pyrrole-3-propanoic acid (incorporated herein by reference; Sun et al. (1999) Design, synthesis, and evaluations of substituted 3-[(3- or 4-carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of VEGF, FGF, and PDGF receptor tyrosine kinases. J. Med. Chem. 42 5120; Paterson et al. (2004) Preclinical studies of fibroblast growth factor receptor 3 as a therapeutic target in multiple myeloma. Br. J. Haematol. 124 595; Tanaka et al. (2005) FGF-induced vesicular release of sonic hedgehog and retinoic acid in leftward nodal flow is critical for left-right determination.Nature 435:172).

[0056] As used herein, the term "derivative" refers to a chemical compound that has a similar core structure.

[0057] As used herein, the terms "WNT" or "Wingless" with respect to ligands refer to a group of secreted proteins that can interact with WNT receptors, such as those in the Frizzled and LRPDerailed / RYK receptor families (i.e., Int1 (integration 1) in humans).

[0058] As used herein, the terms "WNT" or "Wingless" with respect to a signaling pathway refer to a signaling pathway consisting of Wnt family ligands and Wnt family receptors (such as Frizzled and LRPDerailed / RYK receptors) that is mediated by β-catenin or does not involve β-catenin. For purposes herein, a preferred WNT signaling pathway involves β-catenin mediation, i.e., WNT / β-catenin.

[0059] [ka]

[0060] As used herein, the term "PD 173074" refers to a small molecule having the chemical name: N-[2-[[4-(diethylamino)butyl]amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea (Bansal et al. (2003) Specific inhibitor of FGF receptor signaling: FGF-2-mediated effects on proliferation, differentiation, and MAPK activation are inhibited by PD 173074 in oligodendrocyte-lineage cells. J. Neurosci. Res. 74:486, incorporated herein by reference).

[0061] As used herein, the term "LSB-3i" or "LSB3i" with respect to compositions and methods of using the compositions refers to a combination of an LSB molecule (or equivalent) capable of producing neuronal lineage cells and a 3i molecule (or equivalent) capable of directed differentiation of neuronal lineage cells, as used in the exemplary methods described herein for directed differentiation of neuronal lineage cells that give rise to nociceptors.

[0062] As used herein, the term "bone morphogenetic protein" or "BMP" refers to proteins and corresponding genes that are members of the BMP subfamily, which includes GDFs (growth / differentiation factors) based on sequence homology within the TGFβ superfamily of proteins (see, e.g., Yamashita et al. (1996) Bone 19:569, incorporated herein by reference). Examples of BMPs include BMP1, BMP2, etc. BMPs / GDFs are classified into subsets based on amino acid sequence homology. The proposed classification is: 1) BMP-2 and BMP-4; 2) BMP-3 and BMP-3b; 3) BMP-5, BMP-6, BMP-7, and BMP-8; 4) BMP-9 and BMP-10; 5) BMP-12, BMP-13, and BMP-14; and 6) BMP-11 and GDF-8 (see, e.g., Yamashita et al. (1996) Bone 19:569, Hogan (1996) Genes Dev. 10:1580, Mehler et al. (1997) Trends Neurosci. 20:309, Ebendal et al. (1998) J. Neurosci. Res. 51:139, all of which are incorporated herein by reference). Ligands of the TGFβ superfamily include molecules such as bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin, nodal, TGFβ, etc. The TGFβ family includes: TGFβ1, TGFβ2, and TGFβ3. Like BMPs, TGFβs are involved in embryogenesis and cell differentiation, but they are also involved in apoptosis and other functions. They bind to the TGFβ receptor type 2 (TGFBR2).

[0063] As used herein, the term "bone morphogenetic protein receptor" or "bone morphogenetic protein receptor type II" or "BMPR2" refers to a serine / threonine kinase receptor that binds to bone morphogenetic proteins.

[0064] As used herein, the term "LSB-Mel" refers to directed differentiation compositions and methods involving LSB / C treatment of cells followed by contact with BMP4 and endothelin-3 (EDN3) to produce melanocyte precursor cells (melanocyte progenitors), which are identified and isolated based on the expression of a specific marker, i.e., c-kit.

[0065] As used herein, the term "mature pigmented melanocyte" refers to a pigment cell that produces pigmented melanosomes, such as a melanocyte precursor cell of the present invention that has been contacted with BMP4 and cAMP.

[0066] As used herein, the term "embryonic stem cell" refers to a primitive (undifferentiated) cell derived from a preimplantation stage embryo, which can divide in culture for a long period without differentiation, and is known to develop into the cells and tissues of the three primary germ layers.Human embryonic stem cells refer to human embryonic stem cells, such as WA-09.

[0067] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells cultured under in vitro conditions that allow them to proliferate for days, months, or even years without differentiation.

[0068] As used herein, the term "stem cell" refers to a cell that has the ability to divide indefinitely in culture and give rise to specialized cells. Human stem cells refer to stem cells that are human.

[0069] As used herein, the term "human embryonic stem cell" or "hESC" refers to a type of pluripotent stem cell derived from the early human embryo (up to and including the blastocyst stage) that is capable of dividing in culture for extended periods without differentiation and is known to develop into cells and tissues of the three primary germ layers.

[0070] As used herein, the term "totipotent" refers to the ability to give rise to all cell types of the body and all cell types that make up extraembryonic tissues such as the placenta. (See also pluripotency and multipotency).

[0071] As used herein, the term "multipotent" refers to the ability to develop into more than one cell type of the body. See also pluripotent and totipotent.

[0072] As used herein, the term "pluripotent" refers to the capacity to develop into an organism's three developmental germ layers, including endoderm, mesoderm, and ectoderm.

[0073] As used herein, the term "somatic (adult) stem cells" refers to relatively rare undifferentiated cells found in many organs and differentiated tissues that have limited capacity for both self-renewal (in the laboratory) and differentiation. The differentiation potential of such cells varies but is usually restricted to cell types in the organ of origin.

[0074] As used herein, the term "somatic cell" refers to any cell in the body other than a gamete (egg or sperm), sometimes referred to as an "adult" cell.

[0075] As used herein, the term "neuron lineage cells" refers to cells that contribute to the nervous system (both central and peripheral) or neural crest cell fate during development or in adults. The nervous system includes the brain, spinal cord, and peripheral nervous system. Neural crest cell fate includes cranial nerves, trunk nerves, vagus nerves, sacral nerves, and cardiac nerves, and gives rise to parts of the mesectoderm, head cartilage, skull, thymus, teeth, melanocytes, iris pigment cells, cranial ganglia, spinal ganglia, sympathetic / parasympathetic ganglia, endocrine cells, the enteric nervous system, and the heart.

[0076] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to a type of pluripotent stem cell, similar to an embryonic stem cell, that is formed by introducing certain embryonic genes (such as the OCT4, SOX2, and KLF4 transgenes) (see, e.g., Takahashi and Yamanaka, Cell 126, 663-676 (2006), incorporated herein by reference) into somatic cells, such as C14, C72, etc.

[0077] As used herein, the term "specialized cell" refers to a type of cell that performs a specific function in a multicellular organism. For example, groups of specialized cells, such as neurons, cooperate to form a system, such as the nervous system.

[0078] As used herein, the term "nociceptor" with respect to the cells of the present invention refers to a neuron capable of generating action potentials and sensing noxious stimuli (responsible for the perception of pain). Stimuli include, but are not limited to, thermal stimuli (heat and cold), mechanical stimuli, chemical stimuli, and inflammation. Nociceptors are cells that express specific genes and proteins, such as BRN3A, ISL1, TAC1, VGLUT2, and SLC15A3, and contain a morphology described as a cell body and two distinct processes along an axon-like structure. A "functional nociceptor" with respect to the cells of the present invention refers to a cell resulting from directed differentiation, characterized by the expression of genes and proteins described herein, the morphology described herein, and the ability to generate action potentials as described herein.

[0079] As used herein, the term "peptidergic neurons" generally refers to neurons that are identified by the expression of different classes of ion channels and by the expression of small peptides such as tachykinins. For example, peptidergic nociceptors express NTRK1 and the tachykinin substance P.

[0080] In contrast, "non-peptidergic" neurons refer to neurons that do not express NTRK1 or substance P.

[0081] As used herein, the term "neurectoderm" refers to cells or cell fates observed early in development or during pluripotent stem cell differentiation that can give rise to cells of the neuronal lineage.

[0082] As used herein, the term "marker of cell proliferation" refers to the expression of a molecule associated with fast-cycling cells, which is typically absent in mature slow-cycling or noncycling cells (i.e., present in actively dividing cells but absent in cells with long cycle times or noncycling cells). Examples of such markers include the Ki67 marker of cell proliferation (Gerdes et al., Int J Cancer 31:13-20 (1983), incorporated herein by reference) and the phosphohistone H3 marker of the G2 / M phase of mitosis (Hendzel et al., Chromosoma 106:348-360 (1997), incorporated herein by reference).

[0083] As used herein, the term "proliferation" refers to an increase in cell number.

[0084] As used herein, the term "differentiation" refers to the process by which an unspecialized embryonic cell acquires the characteristics of a specialized cell, such as a heart, liver, or muscle cell. Differentiation is controlled by the interaction of the cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

[0085] As used herein, the term "directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a particular (e.g., desired) cell type, such as the nociceptor cells of the present invention.

[0086] As used herein, the term "directed differentiation" with respect to stem cells refers to the use of small molecules, growth factor proteins and other growth conditions to promote the transition of stem cells from a pluripotent state to a more mature or specialized cell fate (e.g., central nervous system cells, neurons, nociceptors, etc.).

[0087] As used herein, the term "inducing differentiation" with respect to a cell refers to changing a default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, "inducing differentiation in a stem cell" refers to inducing a cell to divide into progeny cells that have properties that differ from the stem cell, such as genotype (i.e., changes in gene expression as determined by genetic analysis such as microarray) and / or phenotype (i.e., changes in expression of a protein such as PAX6 or a set of proteins such as being positive (+) for HMB45 but negative (-) for SOX10).

[0088] As used herein, the term "transdifferentiation" refers to the process by which stem cells or mature cells from one tissue differentiate into cells of another tissue.

[0089] As used herein, the term "undifferentiated" refers to cells that have not yet developed into a specialized cell type.

[0090] As used herein, the term "cell differentiation" refers to the pathway by which unspecialized cells (i.e., stem cells) develop or mature to have a more defined morphology and function (e.g., iPSCs progressing to neural crest precursors, to cells of a neuronal lineage, to neural crest cells, to neurons, to nociceptor cells, to peptidergic nociceptors or neuroectoderm, to cells of the central nervous system).

[0091] As used herein, the term "differentiation," when used with respect to cells in a differentiating cell line, refers to the process by which a cell differentiates from one cell type (e.g., a multipotent, totipotent, or pluripotent differentiable cell) to another cell type, such as a target differentiated cell.

[0092] As used herein, the terms "default" or "passive" with respect to a cell differentiation pathway refer to a pathway by which unspecialized cells become specific differentiated cell types in culture when not treated with a specific compound, i.e., under normal cell culture conditions. In other words, default cells arise when cells are not contacted with molecules (i.e., morphogens) that can change the differentiated cell type, such as the nestin+TUJ1- cells of the present invention. In contrast, "non-default" with respect to a cell refers to a differentiated cell type that gives rise to a cell type different from the default cell; i.e., non-default cells are differentiated cell types that arise from non-default conditions, such as the cells of the present invention, including TUJ1+Nestin- neuronal cells, sensory neuronal cells, peptidergic nociceptors, melanocytes, etc. Default cells can also be default cells in the absence of subsequent morphogenic compounds after cells have become non-default cells through contact with morphogens, such as non-default TUJ1+Nestin- cells that subsequently become default non-peptidergic nociceptors.

[0093] As used herein, the term "fate" with respect to a cell, such as "cell fate commitment," generally refers to a cell with a genetically determined lineage whose progeny can become a variety of cell types or a few specific cell types depending on in vivo or in vitro culture conditions. In other words, a cell's predetermined fate is determined by its environment, which directs the cell down a specific differentiation pathway to become one cell type rather than another; for example, the progeny of a stem cell with a "neural fate" will become a neuron rather than a muscle or skin cell. Typically, a cell's "fate" is irreversible except under highly specific conditions. In another example, "CNS fate" refers to a cell that can become a cell related to the central nervous system. Conversely, a cell destined to become a neuron can be called a "neural progenitor cell."

[0094] As used herein, the term "neurite outgrowth" refers to the observation of elongated, membrane-enclosed protrusions of cytoplasm from a cell.

[0095] As used herein, the term "dopamine neurons" or "dopaminergic neurons" generally refers to cells capable of expressing dopamine. "Midbrain dopamine neurons" or "mDA" refer to putative dopamine-expressing cells in forebrain structures and dopamine-expressing cells in forebrain structures.

[0096] As used herein, the term "neural stem cell" refers to stem cells found in adult nervous tissue that can give rise to neurons and glial (supporting) cells. Examples of glial cells include astrocytes and oligodendrocytes.

[0097] As used herein, the term "neuron" refers to a nerve cell, the primary functional unit of the nervous system. A neuron consists of a cell body and its processes—an axon and one or more dendrites. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0098] As used herein, the term "cell culture" refers to the growth of cells in vitro in an artificial medium for research or medical treatment.

[0099] As used herein, the term "medium" refers to the liquid that covers the cells in a culture vessel such as a Petri dish, multi-well plate, etc., and contains nutrients that nourish and support the cells. The medium may also contain growth factors that are added to produce desired changes in the cells.

[0100] As used herein, the term "feeder layer" refers to the cells used in co-culture to maintain pluripotent stem cells.For human embryonic stem cell culture, typical feeder layers include mouse embryonic fibroblasts (MEFs) or human embryonic fibroblasts that are treated to prevent them from dividing under culture.

[0101] As used herein, the term "passage" in reference to cell culture refers to the process of separating, washing, and inoculating new culture vessels with cells after successive rounds of cell growth and proliferation. The number of successive passages a cultured cell has passed through is an indication of its age and expected stability.

[0102] As used herein, the term "express" in reference to a gene or protein refers to the production of mRNA or protein that can be observed using assays such as microarray assays, antibody staining assays, and the like.

[0103] As used herein, the term "paired box gene 6" or "PAX6" refers to a marker of non-default neural progenitor cells.

[0104] As used herein, the term "TUJ1" or "neuron-specific class III beta tubulin" with respect to differentiating cells of the present invention refers to a marker of early neural human cell differentiation, such as neural progenitor cells, and is expressed in neurons of the PNS and CNS.

[0105] As used herein, the term "nestin" with respect to differentiating cells of the present invention refers to an intermediate filament-associated protein that is a marker for neural crest stem cells and CNS neural stem cells.

[0106] As used herein, the term "homodimer" with respect to a SMAD molecule refers to at least two molecules of a SMAD linked together, such as by a disulfide bond.

[0107] As used herein, the term "EDN3" refers to a secreted peptide from an endothelial-derived protein of the endothelin family that binds to the cell surface receptor EDNRB, which is commonly found on neural crest-derived cell lineages such as bipotential glial-melanocyte stem cells. An example of an EDN3 amino acid sequence is endothelin 3 (SEQ ID NO: 1) in Accession No. NP_000105; Accession PI14138 (EDN3 HUMAN):

[0108] [ka]

[0109] As used herein, the term "noggin" refers to a secreted homodimeric glycoprotein that binds to and inactivates members of the transforming growth factor beta (TGFβ) superfamily of signaling proteins, such as bone morphogenetic protein 4 (BMP4).

[0110] Noggin is a 65 kDa protein that is typically expressed in human cells as a glycosylated, disulfide-linked dimer (Groppe et al. (2002). Nature 420, 636-642; Xu et al. (2005) Nat Methods 2, 185-190; Wang et al. (2005) Biochem Biophys Res Commun 330:934-942). An example of a Noggin amino acid sequence is Accession No. U79163, the single amino acid mouse Noggin (SEQ ID NO: 2):

[0111] [ka]

[0112] As used herein, the term "lefty" refers to novel members of the transforming growth factor-β superfamily, including but not limited to LEFTY1, LEFTY2, LEFTYA, etc., also known as "EBAF" or "endometrial bleeding-associated factor" or "left-right axis determining factor A," that inhibit TGFβ. Lefty proteins are required for left-right asymmetry determination of organ systems in mammals.

[0113] As used herein, the term "activin" refers to members of the transforming growth factor beta (TGFβ) superfamily, such as activin A, activin B, and the like.

[0114] As used herein, the term "transforming growth factor beta" or "TGFβ" refers to a cytokine that regulates the proliferation and differentiation of a variety of cell types.

[0115] As used herein, the term "Nodal" refers to a member of the TGFβ family of signaling molecules. Nodal signaling inhibits the differentiation of human embryonic stem cells along the neuroectodermal default pathway (Vallier et al., Dev. Biol. 275, 403-421).

[0116] As used herein, the term "ALK" or "anaplastic lymphoma kinase" or "anaplastic lymphoma receptor tyrosine kinase" or "Ki-1" refers to a membrane-associated tyrosine kinase receptor.

[0117] As used herein, the term "ALK5," in reference to type I serine / threonine kinase receptor, refers to the anaplastic lymphoma receptor tyrosine kinase 5 receptor, which binds to TGFβ1 and functions as a TGFβ1 receptor.

[0118] As used herein, the term "ALK7," in reference to type I serine / threonine kinase receptor, refers to the anaplastic lymphoma receptor tyrosine kinase 7 receptor, which binds to Nodal and Nodal-related proteins and functions as a Nodal and Nodal-related protein receptor.

[0119] As used herein, the term "contacting" a cell with a compound of the present invention refers to placing the compound in a position that allows it to come into contact with the cell, resulting in a "contacted" cell. Contacting can be achieved using any suitable method. For example, in one embodiment, contacting is achieved by adding the compound to a tube of cells. Contacting can also be achieved by adding the compound to a culture of cells.

[0120] As used herein, the term "adherent cells" refers to cells that grow in vitro by adhering to the bottom or sides of a culture vessel, and may contact the vessel via extracellular matrix molecules or the like, and require the use of enzymes, i.e., trypsin, dispase, etc., to detach the cells from the culture dish / vessel, unlike cells in suspension culture, which are not adherent and do not require the use of enzymes to remove the cells from the culture vessel.

[0121] As used herein, the term "marker" or "cellular marker" refers to a gene or protein that identifies a particular cell or cell type. A cellular marker need not be limited to one marker; multiple markers can represent a "pattern" of markers, such that a specified group of markers can identify one cell or cell type from another. For example, the nociceptor cells of the present invention express one or more markers that distinguish nociceptor cells from less differentiated progenitor cells, i.e., TUJ1-positive and nestin-negative nociceptors, from non-nociceptor or precursor cells, i.e., TUJ1-negative and nestin-positive cells.

[0122] As used herein, the term "positive cells" with respect to staining refers to cells that express a marker and thus "stain" for that marker in a detectable quantitative and / or qualitative amount above control or comparison cells. Positive cells can also refer to cells that stain for molecules such as nestin, etc.

[0123] As used herein, the term "negative cells" refers to cells that have no detectable signal for a marker, such as cells that do not stain after contact with, for example, a nestin antibody detection method.

[0124] As used herein, the term "DAPI" refers to 4',6-diamidino-2-phenylindole·2HCl fluorescent staining. DAPI fluorescent staining is well known, and one of many examples is DAPI Nucleic Acid Stain, 2006, Molecular Probes, Inc., Eugene, Oregon, 97402, USA.

[0125] As used herein, the term "reporter gene" or "reporter construct" refers to a genetic construct that includes a nucleic acid that encodes a readily detectable or readily assayable protein, such as a chromogenic protein, a fluorescent protein such as GFP, or an enzyme such as β-galactosidase (lacZ gene).

[0126] As used herein, the term "GFP" refers to any green fluorescent protein DNA sequence that can produce a fluorescent protein when expressed in a cell, typically used as an indicator marker for the expression of a target gene. Examples of GFP include the GFP sequence isolated from coelenterates such as Pacific jellyfish (Aequoria Victoria), and its synthetic sequence derivatives such as "eGFP".

[0127] The term "sample" is used in its broadest sense. In one sense, it can refer to cells or tissues. In another sense, it is intended to include specimens or cultures obtained from any source, encompassing liquids, solids, and tissues. Environmental samples include environmental materials such as surface matter, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0128] As used herein, the terms "purified," "to purify," "purified," "isolated," "isolating," and grammatical equivalents refer to the reduction of the amount of at least one contaminant from a sample. For example, a desired cell type, such as differentiated neuronal cells isolated from non-neuronal cells, is purified by at least 10%, preferably at least 30%, more preferably at least 50%, even more preferably at least 75%, and most preferably at least 90%, with a corresponding reduction in the amount of undesired cell types. In other words, "purify" and its equivalents refer to the removal of specific cells (e.g., undesired cells) from a sample. For example, to provide a purified population of TUJ1+ neuronal cells of the present invention, TUJ1+ nestin- neuronal cells are purified by the removal of contaminating nestin+ TUJ1- neuronal cells by sorting a mixed cell population into NTRK1+ and NTRK1- cells by flow cytometry, as described herein; neuronal nociceptor cells are also purified or "selected" from non-nociceptor cells (default cells) using specified methods of cell culture, including the compositions and methods of the present invention. Removal or selection of non-nociceptor cells results in an increase in the proportion of desired nociceptor cells in the sample.

[0129] Purification of a cell type therefore results in an "enrichment" or increase in the amount of desired cells, i.e. nociceptors, in the sample.

[0130] As used herein, the term "naturally occurring" when applied to objects (e.g., cells, tissues, etc.) and / or chemicals (e.g., proteins, amino acid sequences, nucleic acid sequences, codons, etc.) means that the objects and / or compounds are / are found in nature. For example, a naturally occurring cell refers to a cell present in an organism that can be isolated from a natural source, such as an embryonic cell, where the cell has not been intentionally modified by humans in a laboratory.

[0131] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments are exemplified, but not limited to, test tubes and cell cultures.

[0132] As used herein, the term "in vivo" refers to the natural environment (eg, an animal or cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neurulation, and the like.

[0133] The terms "derived from" or "established from" or "differentiated from," when used with respect to any cell disclosed herein, refer to cells obtained (e.g., isolated, purified, etc.) from parent cells in a cell line, tissue (e.g., embryo or fluid separated using any manipulation, such as, but not limited to, single cell isolation, in vivo culture, treatment and / or mutagenesis with, for example, proteins, chemicals, radiation, infection with viruses, transfection with DNA sequences such as morphogens, etc., selection (such as by continuous culture) of any cells contained in the cultured parent cells, etc.). Derived cells can be selected from a mixed population by response to growth factors, cytokines, selective progression of cytokine treatment, adherence, lack of adherence, sorting procedures, etc.

[0134] As used herein, the term "cell" refers to a single cell as well as a population (i.e., two or more) of cells. A population may be a pure population containing one cell type, such as a population of neuronal cells or a population of blastocysts. Alternatively, a population may contain two or more cell types, for example, a mixed cell population. It is not intended to limit the number of cells in a population, for example, a mixed population of cells may contain at least one differentiated cell. In one embodiment, the mixed population may contain at least one differentiated cell. The present invention does not limit the number of cell types that a cell population may contain.

[0135] As used herein, the term "highly enriched population" refers to a population of cells, such as a population of cells in a culture dish, that express a marker at a higher percentage or amount than a comparison population; for example, treatment of a cell culture contacted with LSB with CHIR / SU or CHIR / DAPT on day 2 results in a highly enriched population compared to treatment with SU / DAPT.

[0136] The term "cell biology" or "cellular biology" refers to the study of living cells, including their anatomy and function, such as their physiology, structure, organelles, and interactions with their environment, their life cycle, division, and death.

[0137] The term "nucleotide sequence of interest" refers to any nucleotide sequence (e.g., RNA or DNA) the manipulation of which may be determined by one of skill in the art to be desirable for any reason (e.g., treating disease, conferring improved quality, expression of a protein of interest in a host cell, expression of a ribozyme, etc.). Such nucleotide sequences include, but are not limited to, coding sequences for structural genes (e.g., reporter genes, selectable marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences that do not encode an mRNA or protein product (e.g., promoter sequences, polyadenylation sequences, termination sequences, enhancer sequences, etc.).

[0138] As used herein, the term "protein of interest" refers to a protein encoded by a nucleic acid of interest.

[0139] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor (e.g., proinsulin). A polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or functional property (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full length or fragment is retained. The term also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region at both the 5' and 3' ends (over a distance of about 1 kb or more on the ends, whereby the gene corresponds to the length of the full-length mRNA). Sequences located 5' to the coding region and present on the mRNA are referred to as 5' untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain the coding region interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript; introns are therefore absent in the messenger RNA (mRNA) transcript. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0140] As used herein, the term "gene expression" refers to the process by which genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., by the enzymatic action of RNA polymerase) and, for protein-coding genes, into protein through "translation" of the mRNA. Gene expression can be regulated at many stages in this process. "Up-regulation" or "activation" refers to regulation that increases production of the gene expression product (i.e., RNA or protein), while "down-regulation" or "repression" refers to regulation that decreases production. Molecules (e.g., transcription factors) involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively.

[0141] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA sequence encoding," and "RNA encoding" refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA or RNA sequence therefore encodes an amino acid sequence.

[0142] The term "isolated," when used with reference to a nucleic acid, as in "isolated oligonucleotide" or "isolated polynucleotide," refers to a nucleic acid sequence that has been identified and separated from at least one component or contaminant that is normally associated with it in its natural source. An isolated nucleic acid exists in a form or setting that is different from that in which it is found in nature. In contrast, a non-isolated nucleic acid is a nucleic acid, such as DNA or RNA, that is found in the state in which it exists in nature. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome adjacent to neighboring genes; an RNA sequence, such as a particular mRNA sequence that encodes a particular protein, is found in the cell as a mixture with many other mRNAs that encode many proteins. However, an isolated nucleic acid encoding a given protein includes, by way of example, a nucleic acid in a cell that normally expresses the given protein, where the nucleic acid is in a different chromosomal location than in natural cells or is flanked by nucleic acid sequences that are different from those found in nature. An isolated nucleic acid, oligonucleotide, or polynucleotide can exist in single-stranded or double-stranded form. When the isolated nucleic acid, oligonucleotide, or polynucleotide is utilized to express a protein, the oligonucleotide or polynucleotide will minimally contain the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and antisense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0143] As used herein, the term "melanocyte" with respect to the cells of the present invention generally refers to cells derived from PSCs, including early melanocytes, which express a group of markers including Sox10, HMB45, c-kit, the essential melanocyte transcription factors MITF-M or MITFM, isoforms of microphthalmia-associated transcription factor (MITF), members of the basic helix-loop-helix leucine zipper transcription factor family expressed in melanocytes, tyrosinase (TYR), tyrosinase-related protein 1 (TYR-1), TYR-related protein 2 / dopachrome tautomerase (DCT), etc., and contain premelosomes and / or melanosomes with or without apparent pigment (observed macroscopically or by microscopy). Mature melanocytes typically contain pigmented melanosomes, are tyrosinase-positive, and express melanocyte proteins such as tyrosinase-related protein 1 (TRP1).

[0144] As used herein, the terms "early melanocytes" or "melanoblasts" or "melanocyte precursors" or "melanocyte progenitor cells" with respect to the cells of the present invention refer to cells that co-express Sox10::GFP and MITF and c-kit, which can further differentiate into mature melanocytes. In one embodiment, Sox10::GFP is a marker for putative melanocyte precursors. In another embodiment, c-kit is a marker for putative melanocyte precursors. In a further embodiment, Sox10::GFP / c-kit double-positive cells are putative melanocyte precursor cells.

[0145] As used herein, the term "HMB45+" with respect to the cells of the present invention refers to cells that express the pre-melanosomal glycoprotein, i.e., human Pmel17 (Theos et al., Pigment Cell Res. 2005.18(5):322-36, incorporated herein by reference), such as early (stage) melanocytes (i.e., immature melanocytes), cells that can differentiate into pigment cells of the retinal pigment epithelium, and mature melanocytes containing immature melanosomes.

[0146] As used herein, the term "disease modeling" refers to the process of using experimental organisms or in vitro cell cultures to mimic specific signs or symptoms seen in humans as a result of a disorder. In one embodiment, human pluripotent stem cells derived from a human with a genetic mutation that causes a neurological disorder can be propagated and differentiated into neural cells that retain defects similar to those seen in the human. The present invention therefore provides the following: (Item 1) A kit comprising a first signal transduction inhibitor, a second signal transduction inhibitor, and a third signal transduction inhibitor, wherein the first inhibitor is capable of reducing transforming growth factor beta (TGFβ) / activin-nodal signaling, the second inhibitor is capable of reducing small mothers against decapentaplegic (SMAD) signaling, and the third inhibitor is capable of reducing glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling. (Item 2) The kit according to Item 1, wherein the first inhibitor is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. (Item 3) The kit according to Item 1, wherein the second inhibitor is a small molecule selected from the group consisting of LDN193189, derivatives thereof, and mixtures thereof. (Item 4) The kit according to Item 1, wherein the third inhibitor is selected from the group consisting of CHIR99021 and its derivatives. (Item 5) The kit according to Item 1, further comprising a fourth inhibitor that reduces fibroblast growth factor (FGF) receptor family signaling, wherein the FGF receptor family signaling includes vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF) receptor, and platelet-derived growth factor (PDGF) tyrosine kinase receptor. (Item 6) The kit according to Item 5, wherein the fourth inhibitor is selected from the group consisting of SU5402 and derivatives thereof. (Item 7) The kit described in Item 1, further comprising a fifth inhibitor capable of reducing Notch signaling. (Item 8) The kit according to Item 7, wherein the fifth inhibitor is selected from the group consisting of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and derivatives thereof. (Item 9) Nestin, [ka] 2. The kit of claim 1, further comprising an antibody used for detecting the expression of a protein selected from the group consisting of: (Item 10) Nestin, [ka] 2. The kit of item 1, further comprising PCR primers for detecting mRNA expression of a gene selected from the group consisting of: (Item 11) The kit described in Item 1, further comprising an antibody used for detecting the expression of a protein selected from the group consisting of protachykinin-1 (TAC1), vesicular glutamate transporter 2 (VGLUT2), and solute carrier family 15 member 3 (SLC15A3). (Item 12) The kit described in Item 1, further comprising PCR primers for detecting mRNA expression of a gene selected from the group consisting of protachykinin-1 (TAC1), vesicular glutamate transporter 2 (VGLUT2), and solute carrier family 15 member 3 (SLC15A3). (Item 13) The kit according to Item 1, further comprising instructions, the instructions including a step for adding the first inhibitor and the second inhibitor two days before adding the third inhibitor. (Item 14) The kit according to item 1, further comprising instructions, the instructions including steps for producing neural stem cell precursors and steps for producing nociceptor cells. (Item 15) The kit according to Item 1, further comprising human stem cells. (Item 16) The kit according to Item 15, wherein the human stem cells are human embryonic stem cells. (Item 17) The kit according to Item 15, wherein the human stem cells are human induced pluripotent stem cells. (Item 18) The kit described in Item 13, wherein the human stem cells are transgenic SOX10::GFP bacterial artificial chromosome (BAC) human pluripotent stem cells (hPSCs). (Item 19) A method for inducing directed differentiation of stem cells, comprising: a) i) a cell culture comprising human stem cells; and ii) a first signal transduction inhibitor, a second signal transduction inhibitor, and a third signal transduction inhibitor, wherein the first inhibitor is capable of reducing transforming growth factor beta (TGFβ) / activin-nodal signaling, the second inhibitor is capable of reducing small mothers against decapentaplegic (SMAD) signaling, and the third inhibitor is capable of reducing glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling; providing; b) contacting the stem cells with the first inhibitor and the second inhibitor in vitro for up to 48 hours; and c) further contacting the inhibited stem cells with the third inhibitor for up to an additional 192 hours to induce directed differentiation of the stem cells. wherein the differentiated stem cells are neural crest stem cells. (Item 20) The method according to Item 19, wherein the first inhibitor is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. (Item 21) The method according to Item 19, wherein the second inhibitor is a small molecule selected from the group consisting of LDN193189, derivatives thereof, and mixtures thereof. (Item 22) The method according to Item 19, wherein the third inhibitor is selected from the group consisting of CHIR99021 and its derivatives. (Item 23) The method described in Item 19, further comprising a fourth inhibitor that reduces fibroblast growth factor (FGF) receptor family signaling, wherein the FGF receptor family signaling includes vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF) receptor, and platelet-derived growth factor (PDGF) tyrosine kinase receptor. (Item 24) The method according to Item 23, wherein the fourth inhibitor is selected from the group consisting of SU5402 and derivatives thereof. (Item 25) The method of Item 19, further comprising a fifth inhibitor capable of reducing Notch signaling. (Item 26) The method according to Item 25, wherein the fifth inhibitor is selected from the group consisting of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and derivatives thereof. (Item 27) The method described in Item 19, further comprising a fourth inhibitor and a fifth inhibitor for the directed differentiation of neuronal lineage cells into peptidergic nociceptor cells, wherein the fourth inhibitor is selected from the group consisting of SU5402 and its derivatives, and the fifth inhibitor is selected from the group consisting of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and its derivatives. (Item 28) The method described in Item 27, wherein the peptidergic nociceptor cells express a marker selected from the group consisting of OCT4, DLK1, PAX6, SOX10, POU4F1 (BRN3A), ISL1, NEUROG2, NEUROG1, NTRK1, RET, RUNX1, VGLUT2, TAC1 and TRPV1. (Item 29) The method described in Item 27, wherein the peptidergic nociceptor cells express a marker selected from the group consisting of ISL1, POU4F1 (BRN3A), RET, RUNX1 and NTRK1. (Item 30) The method according to Item 27, wherein the marker is selected from the group consisting of proteins and nucleic acids. (Item 31) The method described in Item 27, wherein the peptidergic nociceptor cells co-express substance P and calcitonin gene-related peptide (CGRP). (Item 32) The method described in Item 27, wherein the peptidergic nociceptor cells generate action potentials in response to an external stimulus, and the external stimulus is an electric current. (Item 33) The method described in Item 27, wherein the differentiated peptidergic nociceptor cells are present within a highly enriched population of neurons within 10 to 15 days after contacting the stem cells with the first inhibitor and the second inhibitor. (Item 34) The method described in Item 19, wherein the stem cells are human embryonic stem cells. (Item 35) The method described in Item 19, wherein the stem cells are human induced pluripotent stem cells. (Item 36) A method for screening biological agents in vitro, comprising: a) i) nociceptor cells derived in vitro from directed differentiation of stem cells; and ii) Test Compound providing b) contacting said nociceptor cells with said test compound and measuring nociceptor function, said function being a measure of action potentials; A method comprising: (Item 37) The method described in Item 36, wherein the nociceptor cells are derived from human stem cells. [Brief explanation of the drawings]

[0147] [Figure 1] Figure 1 shows an exemplary LSB3i differentiation scheme, a preferred embodiment of the method of the present invention. When dual SMAD inhibition was induced using LDN-193189 and SB431542 (LSB), optimal neuronal differentiation was observed when CHIR99021, SU5402, and DAPT (3i) were added on day 2 of differentiation. Starting on day 4, N2 medium was added in incremental 25% increments to replace KSR over the following period. hESCs were plated as single-cell monolayers without an interstitial feeder layer. For the first five days, combined inhibition of BMP (exemplified by LDN193189) and TGF / Nodal / Activin (exemplified by SB431542) (LSB) was used to limit differentiation and promote neural differentiation. Forty-eight hours after initial induction, three additional inhibitors (exemplified by CHIR99021, DAPT, and SU5402; collectively referred to as 3i) were applied. There is no difference in differentiation between the inclusion and exclusion of LDN193189 from days 5 to 10. In summary, cells are fed daily and the medium is transitioned from KSR to N2 to support the emerging neuronal population. ---+L--- indicates duplicate cultures with LDN added from days 5 to 10. [Figure 2]Figure 2 shows the exemplary efficiency of LSB3i and demonstrates that a preferred embodiment of the present invention, using a combination of LSB and 3i treatments, efficiently promotes the generation of neuron-like cell populations compared to LSB treatment alone (A, B). Staining for the neuronal marker TUJ1 revealed a significantly higher number of positive cells when 3i (CHIR99021, DAPT, SU5402) was added 48 hours after hPSC treatment compared to LSB alone, whereas LSB alone showed a large number of PAX6 green / dark cells and a small number of TUJ1+ (red / bright) cells (C, D). Cells were respotted, and Ki67 expression (pink / bright cells) was used to quantify the number of cells in the cell cycle. With 3i addition, nearly one-third fewer Ki67(+) cells were observed compared to LSB alone, indicating postmitotic neurons. Comparison of Ki67 (pink / clear cells) and (E, F) phosphohistone H3 (PPH3) (red / clear cells) expression in hPSCs treated with LSB (E) and LSB3i (F) showed a significant decrease in proliferation by day 12. (G) Intracellular FACS was used to measure the number of progenitor cells (nestin-positive cells; gray bars) and neurons (TUJ1-positive cells; white bars). While only approximately 5% of cells were TUJ1-positive with LSB treatment alone, over 75% of cells were TUJ1-positive with the addition of 3i. The same level of TUJ1 cells was not achieved with the addition of one or two of the three inhibitors compared to the preferred embodiment of the 3i composition. However, treatment of LSB cells with CHIR in addition to either SU5402 or DAPT (C) achieved greater than 53% neuronal differentiation, indicating that CHIR, a glycogen synthase kinase 3β (GSK3β) inhibitor / activator of WNT signaling (i.e., a WNT agonist), in combination with at least one inhibitor selected from a gamma-secretase inhibitor and a fibroblast growth factor receptor inhibitor is required for the formation of TUJ1+ neurons. Scale bars for (A, B) indicate 200 μm, and for (C, D) indicate 100 μm. [Figure 3]Figure 3 shows that the exemplary LSB3i neurons were nociceptors, demonstrating that a preferred embodiment of the present invention using a combination of LSB and 3i treatments efficiently promotes the generation of nociceptors compared to LSB treatment alone. TUJ1-positive neurons from the combination of LSB and 3i treatments express (A) ISL1, (B) BRN3A, (C) RET, and (D) RUNX1, as measured by immunofluorescence at day 12. (E) More than 61% of cells express NTRK1 at day 10, and hiPSCs treated with LSB3i simultaneously form neurons with moderate efficiency (measured by FACS). Taken together, these results indicate that the overwhelming majority of neurons generated using the combination of LSB and 3i treatments are nociceptors. Scale bar indicates 100 μm. [Figure 4] Figure 4 shows that exemplary iPSCs were induced to develop into nociceptors using LSB3i. When hiPSC lines (e.g., C14) were treated with LSB3i, neurons similar to those shown in Figure 3 were observed. Figure 4 demonstrates that a preferred embodiment of the present invention using a combination of LSB and 3i treatments efficiently promotes the generation of nociceptors from hiPSC populations compared to LSB treatment alone. TUJ1-positive neurons from a preferred embodiment of the present invention using a combination of LSB and 3i treatments of the hiPSC line (C14) express (A) ISL1, (B) BRN3A, (C) RET, and (D) RUNX1, as measured by immunofluorescence at day 12. Two separate hiPSC lines (C14 and C72) can also generate nociceptors. (E) Intracellular FACS was used to measure the number of progenitor cells (nestin-positive cells; gray bars) and neurons (TUJ1-positive cells; white bars) for the indicated treatments. Scale bars indicate 100 μm. [Figure 5]Figure 5 shows exemplary SOX10 expression and demonstrates that a preferred embodiment of the present invention, using a combination of LSB and 3i treatment, drives nociceptor differentiation through a pathway involving a neural crest stem cell-like state. To observe the emergence of neural crest stem cells, transgenic SOX10::GFP BAC hESC cell lines were treated with A) LSB, B) LSB and CHIR99021 (LSB / C), and C) LSB3i. Fluorescence microscopy revealed numerous green (bright GFP+ cells) in B) and C). D) and E) show quantitative expression of GFP in the treated cell populations after flow cytometry analysis. Using the transgenic SOX10::GFP BAC hESC line, expression of SOX10, a marker of neural crest stem cells, can be detected in more than 64% of cells by day 8. As shown in D) and E), SOX10::GFP+ expression was accelerated, with maximal expression (large GFP+ population) occurring earlier (80% GFP+ by day 12) compared to LSB and CHIR99021 (LSB / C) or LSB treatment alone. LSB values ​​are just above baseline, LSB / C points are the black line (between LSB and LSB3i), and LSB3i values ​​are connected by a red (light) line. Scale bar = 50 μm. [Figure 6]Figure 6 shows an exemplary representation of 3i added when cells still retain OCT4 expression, demonstrating that a preferred embodiment of the present invention, using a combination of LSB and 3i treatment, drives nociceptor differentiation beginning very early in the differentiation pathway (when hESC populations still retain pluripotent properties). (A) After LSB induction, CHIR99021, DAPT, and SU5402 were added to seven replicate cultures on various days (i.e., one culture on each day from day 1 through day 7). Cells were fixed on day 11. Maximum cell viability and most uniform TUJ1 expression were observed on day 2. Therefore, day 2 was found to be the optimal time point for 3i addition. (B) This corresponds to the time point at which cells cultured in noggin and SB431542 (NSB) continue to express OCT4, a marker of pluripotency, but have not yet expressed PAX6, a marker of neuronal fate (see lack of staining indicated by an asterisk). Addition of 3i on days 5–7, when cells are committed to the neuronal lineage as indicated by PAX6 expression, results in significant cell death. See A) 3i added on days 5, 6, and 7, and B) DAPI staining on day 6 of culture. Cells were stained to identify antibodies bound to OCT4 (red / dark) and PAX6 (green / light) in addition to nuclear 4',6-diamidino-2-phenylindole (DAPI) staining (light blue). [Figure 7]Figure 7 shows that exemplary LSB3i-treated induced hPSCs (SOX10::GFP cells) exhibited the development of neural crest intermediate cells, which mature at an accelerated rate into bipolar nociceptors capable of firing action potentials. SOX10::GFP+ cells were sorted from SOX10::GFP-negative cells using flow cytometry. When SOX10::GFP+ cells were treated with LSB3i, they gave rise to (A) ISL1- and (B) BRN3A-positive neurons. LSB3i neurons stained for (C) glutamate and (D) TRPV1. (E) Each TUJ1-positive neuron exhibited a bipolar morphology with two distinct growth cones and (F) expressed MAP2 with polarity. After one month, (G) neuronal cell bodies clustered to form ganglia positive for (H) substance P and (I) CGRP. (J) 95 pA (red trace) is sufficient to elicit a mature single action potential from an LSB3i nociceptor. Scale bars indicate 100 µm (A–D and F–I) and 50 µm (E). [Figure 8] Figure 8 shows that an exemplary LSB3i-treated iPSC clone, C72, rapidly acquired a nociceptor phenotype. TUJ1-positive clones (green / bright axonal staining) from LSB3i-treated iPSC clone C72 cells expressed A) ISL1, B) BRN3A, C) RET, and D) RUNX1 (red / pink staining of cell bodies). [Figure 9] Figure 9 shows exemplary NTRK1 FACS sorting enriched hiPSC-derived LSB3i neurons. NTRK1 FACS sorting on day 10 of differentiation enriched TUJ1-positive (green / bright axonal staining) neurons in NTRK1+ cells and depleted nestin (red)-positive precursor (NTRK1-) cell populations from both C14 and C72 cell lines. Cells were immunostained for TUJ1 and nestin in addition to DAPI 24 hours after plating on Matrigel-coated culture vessels. [Figure 10]Figure 10 shows exemplary gene expression in LSB3i nociceptors. Gene expression analysis was performed on days 2, 3, 5, 7, 9, and 15 for both LSB- and LSB3i-treated cells. (a) Different stages of differentiation are observed when examining markers of neuroectoderm, neural crest, neurons, and nociceptors (NE, NC, Nn, and Noci, respectively). (b) The top 20 significantly up-regulated (red) and down-regulated (blue) genes by fold change were compared with LSB-treated cells at day 15. (c) Expression of OCT4, DLK1, PAX6, SOX10, POU4F1 (BRN3A), ISL1, NEUROG2, NEUROG1, NTRK1, VGLUT2, TAC1, and TRPV1 coincides with the emergence of peptidergic nociceptors. [Figure 11] Figure 11 shows exemplary qRT-PCR validation of induced genes in the SOX10::GFP BAC cell line. Compared with hPSCs sorted on SSEA-4 and a previous method for enriching neural crest stem cells by sorting HNK1+ from neural cultures (Lee et al., Nat Biotechnol 25, 1468-1475, incorporated herein by reference), GFP+ cells sorted using the SOX10::GFP BAC were significantly enriched for cells expressing the neural crest genes SOX10, p75, and AP2B, as measured by qRT-PCR. [Figure 12] Figure 12 shows that exemplary LSB3i nociceptors have two distinct growth cones. When passaged 12 days after initial contact with LSB, LSB3i nociceptors were fixed and stained with TUJ1 antibody (green; light area) and DAPI (blue / dark nuclei), two distinct growth cones could be seen in these representative cells, between which was the cell body indicated by the DAPI (blue / darker oval area) nuclear region, with various axon-like shapes and sizes. One end showed complex branches resembling dendrites (top), and the other end showed a spherical shape resembling a synaptic terminal (bottom). In general, the morphology of the peptidergic nociceptors of the present invention matched that of sensory neurons. [Figure 13]Figure 13 shows exemplary specification and isolation of melanocyte precursors / melanoblasts. The LSB-C protocol on day 11 supported the induction of melanocyte precursors co-expressing Sox10::GFP and MITF (A, right panel). A MITF mono-positive population was also observed (A, left panel). c-Kit was identified as a potential marker of melanocyte precursors. A low percentage of Sox10::GFP and c-kit co-expressing cells was observed after LSB-C differentiation (B, orange population). qRT-PCR analysis confirmed enrichment of the melanocyte markers MITFM and Dct in the double-positive population (C). Treatment with BMP4 and EDN3 ("LSB-Mel") enhanced the induction of a Sox10::GFP, c-kit double-positive putative melanocyte precursor population (D). Sox10::GFP, c-kit double-positive cells isolated after LSB-Mel treatment showed significantly higher levels of the melanocyte markers MITFM and Dct (E). All error bars represent the standard error of the mean (sem). *p<0.05. [Figure 14] Figure 14 shows exemplary proliferation and maturation of melanocyte precursors. Summary of differentiation conditions (A). After specification in LSB-C (LSB-Mel) conditions with BMP4 and EDN3, cells were sorted and replated on day 11. Post-sorted (PS) cells were maintained in maturation medium containing c-kit ligand (SCF), endothelin 3 (EDN3), fibroblast growth factor (FGF), and CHIR. Pigment cells identified by brightfield microscopy on day 6 of PS were positive for the melanocyte marker MITF but appeared to down-regulate the Sox10::GFP reporter (B). All populations, except for the Sox10::GFP, c-kit double-negative population, eventually gave rise to MITF-expressing cells and macroscopically visible pigment clusters, although at different rates (C). Treatment with BMP4 and cAMP enhanced differentiation into pigment cells exhibiting the spindle-like morphology characteristic of melanocytes (D). [Figure 15]Figure 15 shows exemplary characterization of mature melanocytes. A pure population of mature melanocytes derived with the LSB-Mel protocol maintains expression of common melanocyte markers, including MITF, Sox10, Tyrp1, and HMB45, after more than 8 weeks in culture (A). Melanocytes retain their darkly pigmented phenotype over several weeks of passage (B). 1 x 10 cells were pelleted and photographed to assess pigmentation levels. Electron microscopic ultrastructural characterization of mature melanocytes (C, D). The presence of numerous darkly pigmented melanosomes in the cytoplasm of LSB-Mel-derived melanocytes can be observed by TEM (C). Note the presence and progressive deposition of melanin pigment as melanosome vesicles mature from stage I to stage IV (D). [Figure 16] Figure 16 shows that an exemplary LSB-Mel medium formulation required linoleic acid for melanocyte proliferation and shows a schematic diagram of the melanocyte lineage. Media components shown above the micrograph are those excluded from the formulation; Ph = phase contrast; BF = bright field. The exemplary diagram shows melanocyte precursor markers used to identify cells of the melanocyte lineage generated in the present invention. [Figure 17] Figure 17 shows an exemplary differentiation model. Early LSB treatment of pluripotent embryonic human stem cells inhibits trophectoderm, mesendoderm, and non-neurectodermal cell fates, resulting in cells with a neuroectodermal fate. Addition of CHIR99021, SU5402, and DAPT (3i) 2 days after initial LSB treatment induced neural crest stem cell-identifying markers by day 8 and accelerated (compared to LSB-C and LSB treatments) the rapid differentiation of neural crest stem cells into peptidergic nociceptors by day 10. DETAILED DESCRIPTION OF THE INVENTION

[0148] Detailed Description of the Invention The present invention relates to the field of stem cell biology, particularly to the lineage-specific differentiation of pluripotent or multipotent stem cells, which may include, but are not limited to, human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), somatic stem cells, cancer stem cells, or any other cells capable of lineage-specific differentiation. In particular, methods are described for directing the lineage-specific differentiation of hESCs and / or hiPSCs into nociceptors (i.e., nociceptor cells) using novel culture conditions. Nociceptors generated using the methods of the present invention are further contemplated for a variety of uses, including, but not limited to, use in in vitro drug discovery assays, pain research, and as therapeutics to reverse diseases or injuries of the peripheral nervous system (PNS). Additionally, compositions and methods are provided for producing melanocytes from human pluripotent stem cells for use in disease modeling.

[0149] From the description contained in this specification, those skilled in the art can easily ascertain the essential characteristics of the present invention, and can make changes and modifications to the present invention to adapt it to various applications and conditions and to make the most of the present invention without departing from the spirit and scope thereof. The embodiments and examples described below should be construed as merely illustrative and should not be construed as limiting the scope of the present invention in any way.

[0150] The inventors have previously disclosed the use of dual SMAD inhibition to direct stem cell differentiation toward neural cell populations, and have disclosed that the ratio of CNS to neural crest progeny depends on the confluency of the cells at the time of treatment initiation; high plating densities, i.e., high confluency, give rise to CNS progeny, while low plating densities, i.e., low confluency, give rise to neural crest progeny. The inventors have further disclosed that patterning of differentiated CNS neuronal progeny into functional dopaminergic neurons can be achieved.

[0151] The invention described herein discloses the novel and unexpected observation that functional nociceptors, which are neural crest-derived cell lines, can be differentiated directly from high-density plated embryonic or somatic stem cells in approximately 10 days by sequentially inhibiting SMAD signaling, followed by inhibition of FGF and Notch signaling and activation of Wnt signaling, and that such functional nociceptors can be maintained in vitro for more than 7 days.

[0152] In particular, we conducted a combinatorial small molecule screen to discover compounds for use in directed differentiation of human pluripotent stem cells. During this screen, we found small molecules that convert PSCs into postmitotic neurons. Specifically, we discovered that a combination of five pathway inhibitor small molecules, namely SB431542, LDN-193189, CHIR99021, SU5402, and DAPT, was sufficient to generate neurons from hPSCs with >75% efficiency within 10 days of differentiation, without the presence of any recombinant growth factors, under the specific test conditions described herein. Thus, use of the compositions (including kits) and methods of the present invention results in at least a 50% yield, or at least a 60%, or at least a 70%, or at least a 75% efficiency, of peptidergic nociceptors from hPSCs. These resulting human neurons expressed canonical markers of nociceptive sensory neuron fate, including NTRK1, BRN3A, ISL1, NEUROG1, substance P, and CGRP. This small-molecule-based acceleration of neuronal fate acquisition occurred within a timeframe 3-5 times faster than normal in vivo development (Bystron et al., Nat Neurosci 9:880-886 (2006), incorporated herein by reference), demonstrating that inhibition of specific signaling pathways is sufficient to accelerate the timing of human neuronal cell development. This rapid, potentially scalable (i.e., batch processing to produce large numbers of mature sensory peptidergic nociceptor neurons), and highly efficient induction of peptidergic nociceptors has enabled unprecedented access to this novel method for producing medically relevant cell types for use in the study of human pain perception. Combinatorial small-molecule screening is a powerful methodological tool for a new generation of directed differentiation strategies in hPSC biology.

[0153] The discovery of a composition and method for the in vitro production of mature sensory peptidergic nociceptor neurons within 10 days represents a significantly shorter time than current methods for producing mature sensory neurons. Prior to this discovery, in vitro induction of postmitotic neurons from hPSCs required a long culture period, typically lasting 30 days or more (Zhang et al., Methods Mol Biol 584:355-366 (2010); Elkabetz et al., Genes Dev 22:152-165 (2008)). This time-consuming in vitro differentiation of hPSCs was thought to reflect the timeline of human development in vivo (Perrier, Proc Natl Acad Sci USA 101:12543-12548 (2004)). Thus, in one embodiment, compositions and methods for producing mature peptidergic nociceptor neurons include culturing for less than 30 days after initial contact with at least one of the five compounds, i.e., SB431542, LDN-19318, or an equivalent. Thus, peptidergic nociceptors can be obtained in less than 29 days, less than 25 days, less than 20 days, less than 15 days, less than 12 days, and less than 10 days after initial contact with at least one of the five compounds.

[0154] Identifying in vitro strategies that overcome the slow rate of human development is a key challenge for realizing the full potential of hPSCs in basic biology and human disease modeling (Saha, Cell Stem Cell 5, 584-595 (2009), incorporated herein by reference). The inventors describe herein the discovery of a novel, small-molecule-based method for converting pluripotent cells into mature neurons. Thus, in one embodiment, pluripotent cells are directed toward differentiation into mature nociceptor cells. Furthermore, the inventors describe materials and methods for producing mature neurons, i.e., nociceptor cells, in various morphologies and in high numbers.

[0155] I. Cell culture method for inducing neuronal precursor (lineage) cells: Neuronal lineage cells were produced by contacting human pluripotent stem cells with SB431542 and LDN-193189.

[0156] The following examples set forth exemplary methods for providing cells of neuronal lineage for use during the development of the present invention.

[0157] Dual SMAD inhibition has previously been used as a rapid and highly effective method to induce certain neuronal lineage cells from hPSCs (Chambers et al., Nat Biotechnol 27 (2009), incorporated herein by reference). These neuronal lineage cells induced by molecules including noggin had a default pathway that allowed development into central nervous system cells, i.e., neuronal cell fates. A supplementary study reported that using the small molecule dorsomorphin (DM) instead of noggin produced at least partially similar cells, although there were differences in culture consistency (Kim et al., Robust enhancement of neural differentiation from human psoriasis, incorporated herein by reference). ES and iPS cells regardless of their innate difference in differentiation propensity.Stem Cell Rev 6,270-281(2010);Zhou et al., High-Efficiency Induction of Neural Conversion in hESCs and hiPSCs with a Single Chemical Inhibitor of TGF-beta Superfamily Receptors.Stem Cells,504(2010)).

[0158] The inventors observed that, despite exhibiting the same developmental stage, expression of most of the same markers, and similar developmental potential to generate various neuronal lineages as LDN-treated cells, cells generated using Noggin also exhibit differences compared to neurons induced using LDN, such as being more anterior in the anterior-posterior axis (i.e., more forebrain, more cells expressing FOXG1, etc.) Thus, although LDN was used instead of Noggin to inhibit BMP, among other signaling pathways, Noggin and LDN are likely to have different types of activities other than inhibiting BMP.

[0159] Due in part to the high cost of using noggin, the inventors considered the use of a BMP inhibitor to be a substitute for noggin in producing cells with a neural fate. Therefore, to generate primitive neuroectoderm, cells with a neural fate, i.e., CNS cells, from hPSCs, the small molecule BMP inhibitor, LDN-193189 (Yu et al., Nat Med 14, 1363-1369 (2008), incorporated herein by reference), was used in combination with SB431542 and found to replace noggin during the development of the present invention (Figure 2A). This combined treatment was designated LSB, referring to the combination of these two inhibitors, LDN-193189 and SB431542.

[0160] Typically, cell differentiation is initiated by treating highly confluent monolayers of hES or hiPS cells with dual inhibition of SMAD signaling. Preferred embodiments utilize a confluency percentage of 50%-100%, with the most preferred embodiment utilizing 70%-80% confluency. It will be apparent to those skilled in the art that the initial plating density required to achieve the preferred confluency of the present invention will depend on cell type, size, plating efficiency, viability, adhesion, and other parameters that can be empirically determined without undue experimentation on the part of those skilled in the art. Dual inhibition of SMADs can be achieved using a variety of compounds, including noggin, SB431542, LDN193189, dorsomorphin, or other molecules that block TGFβ, BMP, and activin / nodal signaling. A preferred embodiment utilizes a composition comprising SB431542 and LDN193189 (collectively LSB) at a concentration of 0.1 μM to 250 μM, more preferably 1 to 25 μM, and most preferably 10 μM SB431542 and 10 to 5000 nM, and most preferably 100 to 500 nM LDN193189.

[0161] II. Compounds for Use in Directed Differentiation: Screening small molecules using the neuronal lineage cells of the present invention yielded compounds that yield low PAX6 and high TUJ1 neuronal cells for use in directed differentiation.

[0162] The following example describes the use of exemplary cells of a neuronal lineage from Example II to screen small molecule candidate compounds for use in directed differentiation.

[0163] Specifically, in the context of dual SMAD inhibition (LSB), human ES cells were first treated with LSB (LDN-193189 and SB431542) to screen candidate compounds (i.e., small molecules) under approximately 400 conditions (to identify combinations of small molecules that could accelerate the acquisition of postmitotic neuronal markers from human ES cells). Candidate compounds were selected from molecules that target (alter) cell signaling pathways (e.g., FGF, Notch, WNT, SHH (Sonic hedgehog), etc.) known to be important in developmental studies and frequently used to determine cell fate, which determines which cells can develop into the CNS. As an example, four inhibitors (i.e., SU / DAPT / CHIR / cyclopamine) were tested in different combinations (supplied to cells in cell culture medium) on different days after LSB treatment. Each treatment was then screened for TUJ1 / PAX6 expression on day 10. As an example of treatment conditions, LSB was supplied daily, and CHIR and SU were added to the medium and supplied to the cells daily from day 4 to day 10.

[0164] In general, the screening process resulted in a large number of cultures containing dead cells. In other words, viable culture conditions in this screen were found to occur much less frequently than non-viable conditions (i.e., cell death) (e.g., when SU / DAPT was added to the initial culture, i.e., before day 2). The inventors believe that CNS stem cells depend on FGF signaling and gamma-secretase activity / Notch signaling for survival, and therefore, in the absence of CHIR, when SU / DAPT induced cells to switch from CNS to neural crest, the cells died rather than switching.

[0165] Ten days after the addition of LSB, cells that survived the screening were observed for the loss of the human neuroectoderm marker PAX6 (Zhang et al., Cell Stem Cell 7, 90-100 (2010), incorporated herein by reference) and the onset of neuronal differentiation by TUJ1 expression (Lee et al., Cell Motil Cytoskeleton 17, 118-132 (1990), incorporated herein by reference). Cells were stained for neurons (TUJ1+) by immunofluorescence (immunoF) and for the loss of neuroectoderm (observation of fewer PAX6+ cells) using an antibody that binds to the C-terminus of PX6. This screening was performed with numerous combinations of inhibitors (e.g., SU, SU / DAPT, SU / DAPT / CHIR, DAPT / CHIR, SU / CHIR, SU / cyclopamine, etc.), which were added to various daily feeds on the days of combination (e.g., days 0-10, days 1-10, days 2-10, days 3-10, etc.). Results were generally determined by observing the relative amount of TUJ1 / PAX6 staining in cells generated by each treatment, with the condition and compound showing the highest amount of TUJ1 / PAX6 staining selected as successful for providing cells for further analysis. One example of a small molecule that failed in the screening test for producing TUJ1 / PAX6 cells by immunostaining was cyclopamine. Cyclopamine did not appear to affect cells in terms of producing TUJ1 / PAX6 staining, regardless of when it was added. In other words, cell morphology remained similar to cells treated with LSB alone at day 10 by immunofluorescence (i.e., >90% PAX6+ and <10% TUJ1+).

[0166] However, during screening, the inventors discovered that a specific combination of three small molecules (SU5402, CHIR99021, and DAPT; referred to as 3i for the three inhibitors) added on day 2 of LSB treatment (Figures 6A and B) abolished PAX6 expression and induced TUJ1 in hPSCs on day 10 of differentiation (Figures 2A and B). This was a surprising finding because on day 2 of LSB treatment, the treated cells were still unclear regarding their neuronal fate or their ability to develop into a neuronal fate. Instead, 3i treatment directed cells away from a neuronal fate toward neural crest cells, which further differentiated into the nociceptor cells of the present invention.

[0167] Next, we investigated the function of each of these small molecules to determine which signaling pathways are thought to be involved in converting the PAX6+TUJ1- human ES cell population into the PAX6-TUJ1+ population. First, SU5402 was reported as a potent inhibitor of VEGF, FGF, and PDGF tyrosine kinase signaling (Sun et al., J Med Chem 42, 5120-5130 (1999)). Therefore, it was generally believed that at least one of the small molecules was involved in inhibiting the FGFR signaling pathway. Second, CHIR99021 was reported as a WNT agonist by selectively inhibiting GSK-3β (stabilizing β-catenin) (Bennett et al., J Biol Chem 277, 30998-31004 (2002)). Thus, generally, at least one of the small molecules was considered to be involved in inhibiting glycogen synthase kinase 3β (GSK3β). In one embodiment, the small molecule can selectively activate at least one WNT signaling pathway, such as through glycogen synthase kinase 3β (GSK3β) inhibition. And third, DAPT was reported as a gamma-secretase inhibitor capable of blocking Notch signaling (Dovey et al., J Neurochem 76, 173-181 (2001), incorporated herein by reference). Thus, generally, at least one of the small molecules was considered to be involved in inhibiting at least one Notch signaling pathway. Thus, in one embodiment, one of the small molecules was considered to be a non-selective or pan-Notch inhibitor. In another embodiment, one of the inhibitors is an inhibitor of a gamma-secretase molecule capable of blocking at least one Notch signaling pathway.Thus, in one exemplary embodiment, a combination of inhibitors includes at least one small molecule involved in inhibiting the FGFR signaling pathway, at least one small molecule involved in inhibiting at least one Notch signaling pathway, and at least one small molecule involved in inhibiting GSK3β while activating at least one WNT signaling pathway to produce the PAX6-TUJ1+ human neuronal cells of the present invention. In a further embodiment, one of the inhibitors could block at least one gamma-secretase molecule in the Notch signaling pathway.

[0168] A. LSB-3i: The combination of two inhibitors of FGF and Notch signaling with an activator of Wnt signaling produced TUJ1+ neuronal cells.

[0169] Inhibitors of FGF and Notch signaling and activators of Wnt signaling were added approximately 2, 3, 4, 5, 6, or 7 days after the initiation of LSB treatment. Inhibition of FGF signaling can be achieved using various compounds, including SU5402, PD-161570, PD-173074, suramin, or other molecules that block the FGF signaling pathway. Inhibition of Notch signaling can be achieved using various compounds, including DAPT, L-685,458, Compound E, MK0752, or other molecules that block the Notch signaling pathway.

[0170] Activation of Wnt signaling can be achieved using a variety of compounds, including CHIR99021, LiCl, TDZD-8, recombinant Wnt, or other molecules that activate the Wnt signaling pathway. A preferred embodiment utilizes a composition comprising CHIR99021, DAPT, and SU5402 (collectively 3i) at concentrations of 0.3-100 μM, more preferably 3-10 μM, and most preferably 3 μM CHIR99021; 1-100 μM, and most preferably 10 μM DAPT; and 0.5-200 μM, more preferably 5-20 μM, and most preferably 10 μM SU5402.

[0171] Stem cells treated with the combination of LSB and 3i were fixed on day 11 and examined for viability and expression of the neuronal marker TUJ1. The population treated with 3i on day 2 of LSB treatment yielded the highest viability and high expression of the neuronal marker TUJ1, whereas the population treated with 3i on day 5 of LSB treatment exhibited cytotoxicity and cell death (Figure 6A). Surprisingly, at day 2 after LSB treatment, the cell population still remained progenitor-like, as evidenced by high expression of Oct4 (Figure 6B). It wasn't until day 6 after LSB treatment that the neural commitment marker Pax6 was expressed; however, treatment with 3i on day 6 resulted in cytotoxicity, thereby indicating that the neuronal population induced by the combination of LSB and 3i treatment was directly differentiated from pluripotent stem cells and not from a neuronal intermediate. Therefore, the preferred embodiment of 3i treatment is between days 1 and 4 after LSB treatment, and the most preferred embodiment of 3i treatment is on day 2 after LSB treatment. In addition, all three components of the 3i composition are necessary for maximal yield of differentiated neurons (Figure 2E).

[0172] TUJ1+ neuronal cells show a loss of expression of cell proliferation markers.

[0173] The following example describes an exemplary method for determining the maturation (cell cycle) stage of TUJ1+ neuronal cells.

[0174] After maturation, neurons produced in culture ceased to undergo mitosis and lost Ki67 and phosphohistone H3 (PHH3), markers of cell proliferation (Gerdes et al., Int J Cancer 31, 13-20 (1983), incorporated herein by reference) and the G2 / M phase of mitosis (Hendzel et al., Chromosoma 106, 348-360 (1997), incorporated herein by reference), respectively. Therefore, cells produced using LSB in combination with 3i (i.e., LSB3i) were grown at a lower density, approximately 10-100,000 cells / cm. 2The cells were passaged and fixed to better assess the expression of individual cells, which were then tested for the cell proliferation markers Ki67 and phosphohistone H3 (PHH3). In particular, Ki67 expression is known to be a good predictor of proliferation. Thus, after 12 days, fewer cells (50% and 16%, respectively) exhibited Ki67 and pHH3 expression in cells cultured in the presence of 3i compared with cells cultured in LSB without 3i compounds (Figure 2C-F).

[0175] To quantify the efficiency (percentage) of neuronal differentiation using LSB3i compared with LSB / CHIR (CHIR99021:C), SU / DAPT (SU5402 / DAPT), SU / CHIR (SU5402 / CHIR99021), DAPT, SU (SU5402), and CHIR, plus LSB alone as a control, intercellular FACS staining for nestin, a marker of neural precursors, and β3-tubulin (TUJ1), a marker of neuronal differentiation, was performed (Figure 2G). In the presence of LSB, SU / DAPT, DAPT, SU, and CHIR, the majority of cells expressed nestin. Notably, >95% of the LSB cell population was nestin+. Numerous cells showed nestin staining after dual SMAD inhibition but were not quantified, whereas cells cultured for longer periods, i.e., 19 days, exhibited TUJ1+ neurons, and the majority of these cells co-expressed tyrosine hydroxylase (TH), identifying potential dopaminergic neurons (Chambers et al., Nat Biotechnol 27 (2009), incorporated herein by reference). Conversely, when cells exposed to LSB were exposed to 3i compounds after 2 days, approximately 25% of the cells expressed nestin and approximately 75% of the cells expressed TUJ1 after 10 days, demonstrating efficient conversion to a neuronal cell fate after a short period, i.e., less than 19 days, of cell culture.

[0176] Surprisingly, LSB treatment followed by contacting the cells with CHIR99021 and either DAPT or SU two days later differentiated 50% of the cell population into TUJ1+ cells. When each of the three inhibitors was used alone after LSB treatment, less than 20% of the cells were TUJ1+. Therefore, CHIR99021 was found to be a key contributor to the directed differentiation of this cell population into TUJ1+ neuronal cells. The inventors hypothesized that the directed differentiation of nestin+TUJ1- cells into nestin-TUJ1+ neuronal cells depends on the inhibition of GSK3β while activating at least one of the WNT signaling pathways, in addition to inhibiting either γ-secretase in the FGF receptor pathway or the Notch signaling pathway. Furthermore, the addition of 3i compounds resulted in the conversion of an additional 25% of nestin-TUJ1+ neuronal cells. See Figure 2G.

[0177] In summary, we further investigated the neuronal population derived from the preferred embodiment of LSB treatment followed by 3i treatment two days later. This population showed higher expression of the neuronal marker TUJ1 (Figure 2A, B) as well as loss of Ki67 (Figure 2C, D) compared to cells treated with LSB alone. Loss of Ki67 indicates a decrease in cells in the cell cycle, a hallmark of postmitotically differentiated neurons. In addition, FACS analysis revealed that over 75% of the cell population treated with the preferred composition consisting of LSB and 3i expressed TUJ1, whereas 99% of the population treated with LSB alone expressed the progenitor marker nestin (Figure 2G).

[0178] We further investigated the neuronal population derived from the preferred embodiment of LSB treatment followed by 3i treatment two days later. This population showed higher expression of the neuronal marker TUJ1 (Figure 2A, B) as well as a loss of Ki67 (Figure 2C, D) compared to cells treated with LSB alone. The loss of Ki67 indicates a decrease in cells in the cell cycle, a hallmark of postmitotically differentiated neurons. In addition, FACS analysis revealed that over 75% of the cell population treated with the preferred composition of LSB and 3i expressed TUJ1, whereas 99% of the population treated with LSB alone expressed the progenitor marker nestin (Figure 2E).

[0179] B. TUJ1+ neuronal cells expressed PNS cell markers but not CNS cell markers.

[0180] The following examples describe exemplary methods for identifying the types of TUJ1-positive neurons produced during development of the present invention.

[0181] To further characterize the neuronal subtypes resulting from the preferred embodiment of 3i treatment after 2 days of LSB treatment, the TUJ1-positive population was stained for markers of various neuronal subtypes. Specifically, the dual SMAD inhibition protocol was known to generate PAX6+ neuroepithelial cells biased toward an anterior forebrain identity that expresses FOXG1 (forkhead box protein G1) (Chambers et al., Nat Biotechnol 27 (2009)). Therefore, to determine the neuronal subtype identity after LSB3i treatment, cells were cultured at a lower density of approximately 10-100,000 cells / cm on day 10. 2 and assessed for expression of a range of markers on day 12.

[0182] Because the expected neuronal type was CNS-fate, most of the initial markers tested were for the identification of CNS-type cells. Indeed, CNS forebrain neurons were expected, since LSB cells are by default of this subtype (PAX6, FOXG1 positive). Surprisingly, at least 12 negative results (10 exemplary results are shown below) were obtained for CNS markers before staining for ISL1, a marker for PNS cells, was found. ISL1 is expressed by motor neurons and peripheral sensory neurons. BRN3A expression was tested and found to be expressed by LSB / 3i cells. Therefore, the inventors discovered BRN3A+ / ISL1+ neurons, indicating the development of peripheral sensory neurons. See Table A below.

[0183] Table A: The following list of genes / proteins shows numerous CNS fate molecules predicted to be positive (expressed) for cells using the LDN / 3i-induced differentiation described herein. However, these results show a lack of exemplary CNS markers, a result supported by the subsequent observation of potential markers for PNS lineages, namely ISL1 and BRN3A.

[0184]

number

[0185] Surprisingly, uniform expression of ISL1 and BRN3A (red / darker areas within the cells) (Figures 3A and B) was observed in our TUJ1+ cells (green / brighter cell bodies compared to red staining). ISL1 and BRN3A are key markers for sensory neurons (ISL1: Sun et al., Nat Neurosci 11, 1283-1293 (2008); BRN3A: Gerrero et al., Proc Natl Acad Sci USA 90, 10841-10845 (1993)), all of which are incorporated herein by reference. This finding indicated that the neurons resulting from LSB3i treatment were PNS rather than CNS cells. These results contrast with LSB cells, which are by default CNS forebrain neuron subtypes (PAX6+, FOXG1 positive). This is a highly unexpected observation, since, according to the teachings of the prior art, high confluency of stem cells at the initiation of treatment, as indicated by plating density, should give rise to a CNS-derived neuronal population. However, nociceptors are derived from neural crest cell populations, which, according to the teachings of the prior art, are derived from low confluency of stem cells at the initiation of treatment, as indicated by plating density. In other words, the expectation was >20,000 cells / cm at the initiation of LSB treatment. 2 High initial plating densities of pluripotent stem cells give rise to committed CNS neuronal populations, whereas neural crest cells give rise to approximately 10,000 cells / cm. 2 It was known that a low initial plating density of 100 ng / ml was required (Chambers et al., Nature Biotech, 2009 (see bottom half of Figure 4), which is incorporated herein by reference in its entirety).

[0186] To further characterize the neuronal subtypes obtained from the preferred embodiment of 3i treatment after 2 days of LSB treatment, the TUJ1-positive population was stained for markers of various neuronal subtypes. This population was positive for the expression of ISL1, BRN3A, RET, and RUNX1 (Figure 3A-D). FACS analysis revealed that over 60% of these neurons were positive for NTRK1 (Figure 3E).

[0187] These markers collectively indicate that the neuronal population is a peripheral sensory neuron, specifically a nociceptor. This is a highly unexpected observation, since, according to prior art teachings, high stem cell confluency at the onset of treatment, as indicated by plating density, should result in a CNS-derived neuronal population. However, nociceptors are derived from neural crest cell populations, which, according to prior art teachings, are derived from low stem cell confluency at the onset of treatment, as indicated by plating density. Therefore, the preferred embodiment of combining 3i treatment with LSB on day 2 results in the unexpected formation of a neural crest-derived population, namely, nociceptors. To establish the generality of the present invention, the inventors repeated the preferred embodiment of the present invention, combining 3i treatment two days after LSB treatment, using hiPSCs as a stem cell source. The current art describes many methods for producing hiPSCs, which are known to those skilled in the art. Highly confluent hiPSC cells plated with LSB, followed by 3i on day 2, resulted in the formation of neuronal cells positive for the nociceptor markers ISL1, BRN3A, RET, and RUNX1 (Figure 4A-D).

[0188] C. PNS TUJ1+ neuronal cells expressed nociceptor-peptidergic cell markers.

[0189] The following examples describe the use of exemplary methods to determine what types of peripheral nervous system (PNS) neurons have been produced using the methods described herein.

[0190] It was unclear what type of PNS neuron would be produced by the methods described herein, as there are several types of candidate neurons, such as sensory and motor neurons, and at least three major subsets of known sensory neurons in the PNS, including proprioceptor cells, mechanoceptor cells, and nociceptor cells.

[0191] During development, early stage nociceptors are both peptidergic and non-peptidergic and uniquely express NTRK1, RUNX1, and then RET (for example information regarding RET, see Woolf et al., Neuron 2004, 14, 111-113, which is incorporated herein by reference). 55, 353-364 (2007). Duplicate early-stage LSB3i cultures containing TUJ1+ neurons were tested for RET expression (Fig. 3C) and found to be positive for this marker (TUJ1+ staining (green / lighter cell bodies compared to RET staining) and red / darker areas within the cells in the larger box compared to the lighter staining area in the inset RET box) (Fig. 3D), and more than 60% of all cells in culture expressed NTRK1 as measured by FACS at day 10 (Fig. 3E).

[0192] In summary, this population was positive for the expression of ISL1, BRN3A, RET, and RUNX1 (Figure 3A-D), which indicate the generation of early-stage nociceptors (both peptidergic and non-peptidergic). FACS analysis revealed that over 60% of these neurons were positive for NTRK1 (Figure 3E). Collectively, these markers indicate that this population of neurons represents peripheral sensory neurons, specifically nociceptors.

[0193] Therefore, the preferred embodiment of the combination of 3i treatment and LSB on day 2 results in the unexpected formation of a neural crest-derived population, namely nociceptors.

[0194] Furthermore, the inventors combined information from several studies, including the observations described herein that cells derived from LSB / 3i treatment transiently express neurogenin 1 (NEUROG1) instead of migrating to neural crest and differentiating into a CNS fate, initial immunofluorescence results (i.e., BRN3A+, ISL1+), array data (i.e., TAC1 (Substance P) expression), and then selected the NTRK1 marker to find NTRK1+ cells, leading to the conclusion that the resulting PNS cells are likely peptidergic nociceptors.

[0195] D.LSB3i reproducibly induced PNS TUJ1+ nociceptor-peptidergic neuronal cells.

[0196] The following example describes the use of an exemplary method of the present invention to determine reproducibility.

[0197] To establish the generality of the present invention, the inventors repeated a preferred embodiment of the present invention, combining LSB treatment with 3i treatment two days later, using hiPSCs as a stem cell source. The reproducibility of LSB3i treatment was evaluated across additional induced pluripotent stem cell (hPSC) lines, including hiPSC lines. Current technology describes many methods for producing hiPSCs, known to those skilled in the art. In particular, two hiPSC lines (C14 and C72) created by inserting genes such as Oct4 (octamer-binding transcription factor 4), Sox2 (SRY (sex-determining region Y) box 2), Klf4 (Krüppel-like factor 4), and c-Myc (transcription factor p64) have been shown to be capable of efficient neuralization (see Papapetrou et al., Proc Natl Acad Sci., USA 106 (2009)).

[0198] PAX6 expression was then examined by ImmunoF. LSB and LSB3i treatment of C14 and C72 cell lines showed similar neuronal staining results when compared to the human cell lines shown in Figures 3A-D. As mentioned above, exemplary C14 staining results are shown in Figures 4A-D, and exemplary C72 staining results are shown in Figures 8A-D for ISL1, BRN3A, RET, RUNX1, and TUJ1.

[0199] LSB treatment of the C14 and C72 cell lines uniformly gave rise to nestin-positive cells (>95% of the treated cell population), which were able to form TUJ1+ cells when treated with the LSB3i combination as measured by FACS (40% for C14 and 33% for C72; Figure 4E). These results were compared to the H9 cell line (i.e., an hESC line) treated with LSB and LSB3i, as shown for the LSB and LSB3i results in Figure 4E. Even higher neuronal yields (>90% of the nuclear staining when sorted for NTRK1, from 40% and 33%, as measured by FACS, were obtained in these two hiPSC lines when bulk cultures were passaged into Matrigel™-coated culture vessels containing N2 medium after sorting for NTRK1 (neurotrophin tyrosine kinase receptor type 1) marker expression. Cells were dissociated with Accutase, resuspended in N2, and incubated with APC-conjugated NTRK1 antibody (R&D) on ice for 15 minutes, washed, and resuspended in N2 for FACS. After sorting, cells were cultured in N2 medium for 24 hours and fixed appropriately. Cells were harvested and stained for BRN3A, ISL1, TUJ1, and DAPI. Notably, LSB3i-treated cells exhibited numerous nestin+ cells (red / dark staining) in both C14 and C72 NTRK1- cells, compared with the few nestin+ cells in the representative NTRK1+ LSB3i-treated cell population (Figure 9). Furthermore, while few C14 NTRK1- cells expressed TUJ1, C27 exhibited a higher number of NTRK1-TUJ1+ (green; bright staining). Both cell lines exhibited a high number of nestin-TUJ1+ cells, as seen relative to cell bodies identified by DAPI (blue; bright nuclei) staining.

[0200] In summary, hiPSC cells treated with LSB followed by 3i on day 2 and plated at high confluency resulted in the formation of neuronal cells positive for the nociceptor markers ISL1, BRN3A, RET, and RUNX1 (Figures 4A-D, 8A-D, and 9).

[0201] The most striking observation was the rapidity with which stable, mature neuronal cell fates could be differentiated from hPSCs using this small molecule combinatorial approach (Figure 4). The 10-15 day time frame for generation of a mature neuronal phenotype is significantly accelerated compared to the estimated 30-50 days for nociceptor emergence during human development (Kitao et al., J Comp Neurol 371, 249-257 (1996), incorporated herein by reference). Upregulation of ISL1 and BRN3A coexists with SOX10 expression, beginning between days 5 and 7. The optimal time point for 3i addition is day 2 of dual SMAD inhibition, reflecting previous observations from our laboratory that treatment with Sonic hedgehog on day 2 is most effective in promoting FOXA2 expression and human floor plate differentiation (Fasano et al., Cell Stem Cell 6, 336-347 (2010), incorporated herein by reference). This suggests that neural patterning can occur prior to the loss of OCT4 protein expression, and that the presence of OCT4 protein does not appear to limit pre-patterning events. The important role of CHIR99021 in inducing neural crest-derived sensory neurons is likely related to the activation of canonical WNT signaling, which is known to be essential in early neural crest specification (Dorsky et al., Nature 396, 370-373 (1998), incorporated herein by reference) and can direct naive neural crest precursors toward the sensory neuron lineage (Lee et al., Science 303, 1020-1023 (2004), incorporated herein by reference).

[0202] Transcription factor-based lineage reprogramming of mouse cells has received much deserved attention as a means of directly inducing neurons from fibroblasts (Vierbuchen et al., Nature 463, 1035-1041 (2010), incorporated herein by reference), and this method may one day be used in human cells. The data presented here demonstrate that LSB3i can rapidly induce human postmitotic neurons. Some of the key advantages of using methods involving LSB3i were the speed and efficiency of producing human postmitotic neurons from human precursor cells, i.e., PSCs. Furthermore, this protocol did not require any mechanical interventions such as genetic manipulation or passaging, and yielded highly enriched neuronal populations within 10 days of single culture.

[0203] LSB3i also represents one of the first examples of the use of combinatorial small molecule screening to drive lineage specification in hPSCs. Given the limited number of developmental pathways that are repeatedly used at developmental decision points (Brivanlou et al., Science 295, 813-818 (2002), incorporated herein by reference), the approach described herein should be generally applicable to specifying human pluripotent lineages. Most of the five small molecules used in LSB3i are known signaling pathway inhibitors, indicating that suppression of endogenous signaling pathways is particularly effective in directing hPSC fate. While off-target effects, in which small molecules often produce unintended or unexpected results, are an important consideration when using small molecules, data obtained during the development of this invention revealed that in this particular invention, small molecule combinatorial inhibition of endogenous signaling pathways provides an efficient, non-genetic (no alterations in DNA coding sequences), cross-species, cost-effective, rapid, and reversible means to regulate hPSC cell fate.

[0204] III. LSB-C: CHIR99021 was found to be required for the generation of LSB3i nociceptors and direct their differentiation towards neural crest stem cells.

[0205] During development of the present invention, the inventors discovered that LSB-contacted cells could be instructed to differentiate into nociceptors in high numbers when contacted with CHR / SU or CHR / DAPT on day 2 after LSB treatment, but not with SU / DAPT. Upon further investigation, the inventors surprisingly discovered that LSB-treated cells contacted with LSB-C, CHIR, gave rise to a neural crest stem cell population.

[0206] A. CHIR99021(C) is a key factor for inducing neuronal differentiation from LSB cultured cells (i.e., LSB-C).

[0207] The following examples describe the use of exemplary methods to test the efficacy of each compound for inducing directed neuronal differentiation.

[0208] To gain mechanistic insight into the sufficiency of each compound found to be associated with the induction of TUJ1+ cells in Example III, specific combinations of 3i compounds were tested for inducing cellular expression of nestin and TUJ1 as measured using intracellular FACS (shown in Figure 1G). Nestin was used as a marker for LSB neuronal lineage cells, and TUJ1 was used to identify downstream (i.e., more differentiated) neuronal cells.

[0209] Although none of the individual factors produced high numbers (>60%) of TUJ1+ neurons, CHIR99021 in combination with either one of the other two signal inhibitors was able to generate moderate numbers of TUJ1+ neurons (53% for DAPT and 58% for SU5402). These data indicate that under the test conditions used here, CHIR99021 was a key factor for accelerating neuronal differentiation, with SU5402 and DAPT providing important, but additional, stimuli.

[0210] In addition, all three components of the 3i composition are necessary for maximal yield of differentiated neurons (Figure 2G).

[0211] B. Neural crest stem cells were induced from LSB-contacted cells (D0) that were further contacted with CHIR (D2).

[0212] Through experiments using early withdrawal of these respective inhibitors, the inventors found that BMP signaling and TGFβ signaling were optimized for neural crest induction. Next, Wnt signaling was activated in conjunction with GSK3β inhibition using a small molecule GSK3β inhibitor (CHIR99021). The inventors then found that a narrow window (day 2) of Wnt signaling governs neural crest induction in conjunction with the dual SMAD inhibition protocol. A modified dual SMAD inhibition protocol (LSB-C), combining optimized signaling for these three pathways, enhanced the induction of neural crests expressing Sox10::GFP in up to 65% of the population.

[0213] IV. LSB3i and LSB-C derived artificial SOX10+ cells can produce nociceptor cells.

[0214] The following example describes the use of an exemplary method of the invention for directed differentiation of engineered Sox10+GFP-expressing human cells.

[0215] Nociceptor cells are thought to arise from two types of cellular intermediates during human development: Specifically, it has been shown that SOX10+ chick embryonic neural crest cells can generate trunk nociceptor cells adjacent to the spinal cord (George et al., Nat. Neurosci. 2004, 103:111-114, which is incorporated herein by reference). Neurosci 10:1287-1293 (2007)). In addition, head placode tissue of the African clawed frog (Xenopus laevis) contributed to the trigeminal nociceptor cell population in facial tissue (Schlosser et al., J Comp Neurol 418:121-146 (2000); Schlosser et al., Dev Biol 294:303-351 (2006), which are incorporated herein by reference).

[0216] To determine whether the neural crest intermediate cell fate indicated by SOX10 in human cells (Aoki et al., Dev Biol 259, 19-33 (2003); Lee et al., Nat Biotechnol 25, 1468-1475 (2007)), which is incorporated herein by reference, could be observed during differentiation using a transgenic SOX10::GFP bacterial artificial chromosome (BAC) hPSC line, we generated this SOX10::GFP(BAC) cell line, which co-expresses enriched neural crest gene markers with the GFP gene, using a previously reported method (Placantonakis et al., Stem Cells 27:521-532 (2009)). The SOX10:GFP cell line was a subclone of the H9 hESC line. Cells were dissociated, and gene delivery was performed using reagents (Solution V), protocols (B-16), and equipment from Amaxa. The nucleofected DNA (transfected into the nucleus) was a bacterial artificial chromosome (BAC) containing inserted GFP and SOX10 genes, obtained from the Gene Expression Nervous System Atlas [GENSAT] (accession number: GENSAT1-BX1086). The BAC was then modified to contain a neomycin resistance gene for selection using cre / LoxP recombination from a selection cassette excised from the pL452 plasmid into the GENSAT BAC (see Tomishima et al., Stem Cells 25(1):39-45. Epub 2006 Sep 21 (2007), incorporated herein by reference). After gene delivery, hESCs were seeded as single cells in the presence of G418 for neomycin resistance selection, and clones were manually picked and screened for the presence of GFP after differentiation. GFP cells were sorted by qRT-PCR to confirm the expression of SOX10 and other neural crest markers.

[0217] Two additional duplicate samples were each contacted with one of the LSBs and then with CHIR99021 (LSB / C) or LSB and 3i, and GFP expression was measured by FACS identification and sorting of SOX10::GFP+ cells at 4, 8, 12, and 16 days after initiation of differentiation in LSB.

[0218] In the presence of CHIR99021, over 70% of these treated cells in culture became SOX10::GFP+ by day 12 of differentiation, depending on the culture conditions (70% for LSB / C and 80% for LSB3i; Figure 5D and E). This result indicated that the majority of cells expressed neural crest markers, supporting the inventors' observation that CHIR99021 is required for the generation of LSB3i nociceptor cells. Therefore, because LSB3i-treated cells acquired neural crest fate more rapidly compared to LSB / C-treated hPSCs (Figure 5D and E), combined inhibition of these small molecules, which inhibit tyrosine receptor kinase receptors and Notch signaling, in addition to contact with SU5402 and DAPT, accelerated neural crest cell fate. The inventors hypothesized that CHIR induces neural crest and sensory neurons, while SU accelerates neural crest marker expression and neuronal differentiation. Finally, the inventors hypothesized that DAPT in combination with CHIR and SU accelerates neuronal differentiation. Furthermore, the use of CHIR99021 in combination with LSB, i.e., LSB / C, resulted in a slower conversion rate of over 60% nestin-TUJl+ neuronal cells compared to LSB3i between days 12 and 16 when engineered SOX::GFP cells were used as a readout.

[0219] V. NTRK1+ human nociceptor cells produced by the methods described herein exhibited electrophysiological responses similar to in situ rat nociceptor cells.

[0220] The following example describes the use of an exemplary method of the invention to determine the functional capacity of nociceptor cells produced by the methods described herein.

[0221] To confirm that the LSB3i-derived neurons were genuine nociceptor neuron cells, LSB3i-treated cells were examined for function, maturation stage, and behavior. After LSB3i treatment, pluripotent stem cells that give rise to nociceptor cells were obtained at 10–100,000 cells / cm. 2 Long-term cultures were established from plating densities of 1000 and subcultured at days 10 and 30 in N2 medium supplemented with human βNGF, BDNF, and GDNF (see Example I for further details). The viability of these cells under longer-term culture conditions was found to be NGF-dependent, corresponding to the NTRK1+ nociceptor status. LSB3i nociceptors expressed high levels of TUJ1, ISL1, and BRN3A (Figures 7A-C), as previously shown, in addition to glutamate (Figure 7C). Glutamate production was consistent with excitatory glutamatergic neurons, i.e., nociceptive afferent fibers that release glutamate, and the capsaicin receptor TRPV1, a key ion channel for noxious stimuli (Figure 7D). At 15 days in culture, two distinct outgrowth processes could be identified for each neuron (Figures 7E and 12).

[0222] The dendritic marker MAP2 was expressed primarily in one of the two processes in a polarized fashion (Figure 7F). The bipolarity of the neurons is consistent with their role as sensory neurons in peripheral ganglia, with cell bodies located in dorsal root ganglia and projecting processes toward both the spinal cord and the periphery (Woolf et al., Neuron 55, 353-364 (2007); George et al., Nat Neurosci 10, 1287-1293 (2007), incorporated herein by reference).

[0223] Neurons were cultured long-term in the presence of nerve growth factor (NGF) (e.g., cells were passaged on day 10 and cultured until day 30). LSB was harvested from the cells on day 5, and 3i was harvested on day 10, on which day NGF / GDNF / BDNF was added to the culture medium. Neurons were supplied with NGF / GDNF / BDNF from day 10 to day 30. At day 30, days after initial LSB treatment, neurons were observed to begin self-organizing into ganglion-like structures. This type of morphology is common in peripheral sensory neurons (Marmigere et al., Nat Rev Neurosci 8, 114-127 (2007)), which is incorporated herein by reference (Figures 7G, ​​H, and I).

[0224] Mature nociceptors are typically either peptidergic or non-peptidergic, depending on the expression of neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P (neuropeptide) expressed by peptidergic sensory neurons (Woolf et al., Neuron 55, 353-364 (2007), incorporated herein by reference). In contrast, non-peptidergic neurons express neither CGRP nor substance P and have other markers, such as binding to the lectin IB4.

[0225] Therefore, LSB3i-induced neurons were sorted by FACS for NTRK1 expression (see methods described above) into NTRK1+ and NTRK1- populations (see Figure 7G for an example of sorted cells). NTRK1+ cells were positive for both substance P and CGRP and exhibited a predominantly peptidergic nociceptor phenotype (Figures 7H and 7I; day 30 of differentiation).

[0226] A key functional characteristic (i.e., function) of sensory neuron identity is their electrophysiological properties (Fang et al., J Physiol 565, 927-943 (2005), incorporated herein by reference). NTRK1+ sorted neurons were also tested using standard electrophysiological techniques for cultured neurons (an example is shown in Placantonakis et al., Stem Cells. 2009, Figure 5, incorporated herein by reference in its entirety).

[0227] NTRK1+ cells exhibited electrophysiological features, including a characteristic single action potential (AP) firing pattern, and an average membrane resting potential of 67±4 mV by 21 days after initial LSB3i treatment. The timing of APs and the shape of the activity curves generated in LSB3i human neurons are shown in Figure 7J (see thick red line) and Table 1 below. These results were similar to those previously reported in an electrophysiological study of primary anesthetized adult rat nociceptors (Fang et al., J Physiol 565, 927-943 (2005)).

[0228] [Table 1-1]

[0229] VI. Global gene expression analysis shows exemplary timing of gene expression.

[0230] The following examples describe the use of exemplary methods for determining global gene expression of nociceptor cells and other cell types produced by the methods described herein.

[0231] To further characterize the timing of events (i.e., marker expression) during the induced differentiation process, global gene expression analysis was performed with fine temporal resolution on both LSB- and LSB3i-treated hPSCs (days 2, 3, 5, 7, 9, and 15, NCBI Gene Expression Omnibus (GEO) accession number GSE26867). When selected markers for neuroectoderm, neural crest, neurons, and nociceptors were analyzed (see Table 2 below), distinct stages of differentiation could be observed for each (Figure 10).

[0232] [Table 2-1]

[0233] This gene expression analysis (Figure 10B, C and Table 2 above) was largely consistent with the immunofluorescence results. For example, gene analysis showed that ISL1, POU4F1 (BRN3A), SOX10, TAC1 (propeptide of substance P), NTRK1, and the glutamate vesicular transporter VGLUT2 genes were all upregulated in maturing neurons (i.e., many cells in culture increased their expression of these markers over time). Concurrently, these markers were found to increase in induced cells, whereas markers for hESC-derived primitive neuroectoderm, particularly DLK1, LHX2, OTX2, LEFTY2, PAX6, and HES5, were found to be downregulated (i.e., expressed by fewer cells in culture).

[0234] However, expression of somatostatin (SST) and SOX10, which are expected to be expressed in mature nociceptors, was observed in LSB3i-treated cell cultures at day 15. However, SST has also been shown to be expressed in developing sensory neurons. Therefore, the inventors considered this marker to indicate the presence of immature cells at day 15. Although somewhat downregulated, SOX10 expression was also observed at a time point when the majority of cells appeared to be neurons. This observation was unexpected, as SOX10 was expected to be downregulated as cells differentiate into neurons. This unexpected finding of SST and SOX10 expression in cells from day 15 cultures suggested that not all cells would become nociceptor cells, approximately 20–30%. This indicated that other mature cell types (e.g., Schwann cells) continue to express SOX10.

[0235] hESC-derived primitive neuroectodermal cell cultures produced by dual SMAD inhibition (Chambers et al., Nat Biotechnol 27 (2009); Fasano et al., Cell Stem Cell 6, 336-347 (2010)), each incorporated herein by reference, showed high expression of the DLK1, LHX2, OTX2, LEFTY2, PAX6, and HES5 genes. Similarly, when hESC-derived primitive neuroectodermal cell cultures were produced by dual SMAD inhibition with LSB, similar high expression of these genes was observed (see Figures 10B and 10C and Table 3 below). These genes were reduced during LSB3i treatment, during which nociceptors were produced in the development of the present invention.

[0236] [Table 3-1]

[0237] Additionally, temporal transcriptome analysis provided further evidence for a nociceptor intermediate cell fate distinct from mechanoceptors and proprioceptors. The neurogenin basic helix-loop-helix protein mediates two sequential waves of neurogenesis in the dorsal root ganglion during mouse development (Marmigere et al., Nat Rev Neurosci 8, 114-127 (2007); Ma et al., Genes Dev 13, 1717-1728 (1999)). The first wave, represented by NEUROG2 (neurogenin 2), gives rise to mechanoceptors and proprioceptors, while the second wave, represented by NEUROG1 (neurogenin 1), gives rise to nociceptors. When hPSCs were treated with LSB, NEUROG2 expression was strongly induced by day 7 (Figure 10C and Table 4 below). In contrast, hPSCs treated with LSB3i show less pronounced induction of NEUROG2 by day 7, but selective induction of NEUROG1 by day 9 (FIG. 10C).

[0238] [Table 4-1]

[0239] VII. Large-scale cultures contemplated using the compositions and methods of the present invention to provide exemplary nociceptor cells.

[0240] The following contemplated description sets forth exemplary methods for the large-scale production and use of nociceptor cells produced by the methods described herein.

[0241] Scalable production (i.e., relatively small numbers of cells, e.g., 1.5 x 10 cells in a 48-well plate as described in the Examples, supra) using LSB3i 4 cells / well, and 5 x 10 in a 96-well plate is contemplated. 3 From both cultures containing cells / well, large-scale batch culture of hPSC-derived nociceptors (e.g., 1 x 10 cells in a batch of eighteen 15 cm dishes) 7 ~1×108 cells (approx. 5.5 x 10 7 The present inventors have proposed a method for generating nociceptor cells, which is believed to be successful. These methods are contemplated to provide hPSC-derived nociceptor cells for use in testing compounds for use in basic biology studies and for drug discovery applicable to medical applications in humans and animals. In particular, the inventors contemplate using the compositions and methods of the present invention for the treatment of acute and chronic pain relief in humans and animals.

[0242] In particular, exemplary nociceptor cells, e.g., peptidergic nociceptor cells, are cultured at 7×10 7 ~7×10 8 (70% efficiency of nociceptor cell collection is contemplated). 8 ~1×10 9 Large-scale batch culture is contemplated, in which hPSC cells are grown in batch embryoid body culture.Exemplary nociceptor cells will express the genes (i.e., mRNA and protein) that identify nociceptor cells, such as TAC1, VGLUT2 and SLC15A3.Exemplary nociceptor cells will express the identification markers, such as ISL1, BRN3A, RET, RUNX1, substance P, CGRP, etc.

[0243] In summary, the inventors contemplate using the compositions and methods of the present invention to provide a novel platform in the basic biology and drug discovery for the testing and treatment of conditions associated with nociceptor cells, particularly pain, in humans and animals.

[0244] VIII. Induction of melanocytes from human pluripotent stem cells: LSB-Mel, LDN-193189, SB431542, CHIR99021, EDNR3 and BMP.

[0245] Melanocytes are pigment-producing cells found primarily in the epidermis, where they establish a photoprotective barrier against UV radiation-induced DNA damage. Defects in melanocyte biology are associated with many pigmentation disorders, including albinism, vitiligo, and pididymides. Melanocytes are the cell of origin for malignant melanoma. However, understanding and treatment of these disorders is limited by the lack of suitable experimental systems for studying human melanocytes in vitro.

[0246] During the development of this invention, a protocol was discovered that results in the rapid and highly efficient differentiation of human pluripotent cells into both neural cell precursors and neural crest (NC) precursors. Because skin melanocytes are derived from neural crest precursors, the inventors discovered a method for using LSB-C-derived neural crest-lineage cells to direct differentiation along the melanocyte lineage into mature melanocytes. In other words, pluripotent embryonic stem cells (ESCs) were induced to become neural crest precursor cells (LSB-Cs), which were then induced to become melanocyte precursors, and then differentiated melanocytes. This process was modeled as progressive specification along the melanocyte lineage, with pluripotent ESCs passing through neural crest precursors to more committed melanocyte precursors, before finally establishing a differentiated state (see Figure 16 for a schematic showing exemplary markers for each of these stages). The inventors contemplate using these directed differentiation melanocytes in novel assays to identify the molecular mechanisms of melanocyte development. In particular, the inventors contemplate assays using these directed differentiated melanocytes in combination with recently established approaches for deriving patient-specific induced pluripotent stem cells (iPSCs), which are contemplated to generate assays for melanocyte-related models of human diseases, including albinism, vitiligo, pied skin, melanoma, and malignant melanoma.

[0247] A. Induction of neural crest from human ESCs (first step in directed differentiation to produce melanocytes).

[0248] Melanocytes arise from a transient migratory population of vertebrate-specific cells known as the neural crest (NC), which arises during gastrulation at the boundary between the neuroectoderm and non-neural ectoderm. The multipotent neural crest differentiates into a wide range of derivatives, determined in part by the anatomical location (axial level) of the NC cells.

[0249] A large body of evidence in the literature has identified Wnt, BMP, and TGFβ signaling as key requirements for early neural crest specification. Of these, the latter two pathways are actively inhibited by small-molecule treatment in a dual SMAD inhibition protocol. As described herein, the inventors discovered that BMP and TGFβ signaling are optimized for neural crest induction through early withdrawal of their respective inhibitors. Furthermore, as described herein, the use of a small-molecule GSK3β inhibitor (CHIR99021), which later activates Wnt signaling, was found to produce a population expressing neural crest stem cell markers when added to LSB-treated cells on day 2 of treatment. Therefore, a modified dual SMAD inhibition protocol combining optimized signaling for all three pathways was used with the Sox10::GFP cell line, and we observed enhanced induction of neural crest cells expressing Sox10::GFP to 65% of the population (LSB-C treatment).

[0250] B. Lineage specification and isolation of neural crest-derived melanoblasts.

[0251] LSB-C-induced Sox10::GFP-expressing NCs were then tested for their ability to differentiate along the melanocyte lineage. The presence of putative melanocyte precursors was confirmed at day 11 of the modified differentiation protocol (LSB-C) through the identification of cells co-expressing Sox10::GFP and MITF, a marker expressed in, but not specific to, the melanocyte lineage (Figure 13A).

[0252] To further optimize the induction of these cell populations and subsequently isolate or purify specific types of melanocyte precursors, a cell surface marker that would allow identification of the melanocyte lineage was required. After literature review, c-kit was identified as a candidate marker for putative melanocyte precursors. Testing of c-kit markers in Sox10::GFP+ cells confirmed the presence of a low percentage (approximately 9%) of Sox10::GFP / c-kit co-expressing cells (Figure 13B), while the expression of early melanocyte markers was greatly enriched (Figure 13C). Further optimization of the differentiation protocol revealed that the abundance of Sox10::GFP / c-kit double-positive cells was increased nearly fourfold through additional treatment with BMP4 and endothelin-3 (LSB-Mel, Figure 13D-E), two factors involved in melanocyte specification.

[0253] C. Melanocyte proliferation and maturation.

[0254] We found that putative melanocyte precursors could mature to a pigmented state after only six additional days in culture after sorting (Figure 14A-B). Surprisingly, we observed that both the Sox10::GFP / c-kit double-positive population and the single-positive populations for each of the two markers gave rise to pigment cells, albeit with different kinetics (Figure 14C), demonstrating a lineage hierarchy among the three populations (cKit+ / SOX10-, cKit- / SOX10+, cKit+ / SOX10+). The identification of these three melanocyte-lineage cells would allow for the isolation of differentiation intermediates along the melanocyte lineage.

[0255] Using these melanocyte precursor cells, optimal maturation conditions capable of inducing and supporting cells with a mature melanocyte phenotype were identified using a number of compounds believed to support such maturation. Melanocyte characteristics evaluated included the induction of spindle morphology, pigmentation, and melanosome formation.

[0256] We found that adding BMP4 and cAMP to the culture medium promoted mature spindle-like morphology and pigmentation (Figure 14D). Based on the expression of mature melanocyte markers MITF, SOX10, Tyrp1, and HMB45, pure cultures of melanocytes were obtained when cells were grown for 8 weeks (long-term) in medium containing SCF, EDN3, FGF, Wnt (CHIR), BMP4, and cAMP (Figure 15A). When long-term LSB-MEL cells were centrifuged to assess pigment concentration, a dark pellet was observed (Figure 15B). Electron microscopic ultrastructural characterization of mature melanocytes revealed the presence of numerous darkly pigmented melanosomes in the cytoplasm of LSB-Mel-derived melanocytes (Figure 15C) at various developmental stages (Figure 15D).

[0257] D. Melanocytes derived from human pluripotent stem cells: LSB-melanocytes (LSB-Mel).

[0258] The following describes exemplary compositions and methods for providing melanocytes for use in relevant disease modeling.

[0259] We generated a SOX10::GFP bacterial artificial chromosome (BAC) human embryonic stem cell (hESC) reporter system, which allowed us to observe neural crest cell induction in vitro as this cell line responded to contact with small molecules. Sox10 is the most robust early marker of multipotent neural crest stem cells and was also found to be expressed in some neural crest derivatives, including melanocyte precursors. We used this reporter system to prospectively identify and isolate neural crest populations in the development of a directed differentiation scheme to produce melanocyte cultures with higher purity and numbers than previously achieved with maturation schemes (Figure 14, LSB-C).

[0260] In a dual SMAD inhibition protocol (Chambers et al., Nat. Biotech. (2009), incorporated herein by reference), human pluripotent stem cells (hPSCs) treated with two small molecules that inhibit SMAD signaling efficiently produced CNS neural tissue. Additionally, when hESCs were plated at lower densities, low levels of spontaneous neural crest cell induction were observed (e.g., approximately 3% Soxl0::GFP+ neural crest cells were observed). However, for use in research and medical testing, more neural crest cells were needed. Furthermore, for melanocyte studies, a purer population with a larger number of cells was needed, which low levels of spontaneous differentiation did not provide.

[0261] During the development of the present invention, the inventors discovered ways to optimize the dual SMAD inhibition protocol for neural crest induction to produce a highly pure product of melanocyte precursors, maturing melanocytes, and mature melanocytes.

[0262] Specifically, the following time series of culture conditions were developed to produce melanocytes of the present invention: on days 0 and 1, LDN and SB (using the same concentration range as LDN and SB in the 3i-inclusive methods); on day 2, LDN, SB, and CHIR (using the same concentration range as LDN, SB, and CHIR in the 3i-inclusive methods described herein); in one embodiment, on day 3, SB, and CHIR (using the same concentration range as SB and CHIR in the 3i-inclusive methods described herein); and in another embodiment, on day 3, LDN, SB, and CHIR (using the same concentration range as LDN, SB, and CHIR in the 3i-inclusive methods described herein); and on days 4 and 5, CHIR. (using the same concentration range as CHIR in the 3i-containing methods described herein); from day 6 to day 11, CHIR, BMP4, and EDN3 (using the same concentration range as CHIR in the 3i-containing methods described herein, see concentration ranges below for BMP4 and EDN3) were fed. Cells were passaged on day 11 and fed with MEL medium (containing CHIR) for up to 8 weeks.

[0263] The MEL medium was enriched for melanocytes, such that by 8 weeks the cell cultures exhibited up to 100% pure populations. Thus, this LSB-MEL method / protocol had a high efficiency of melanocyte production. The inventors also discovered that linoleic acid is at least one necessary component in the MEL medium during melanocyte development (see Figure 16).

[0264] During melanocyte development, multiple precursor stages are recognized in the following order: neural crest stem cells, embryonic glial-melanoblast stem cells, adult melanocyte stem cells, and melanocytes. See exemplary schematic diagram in Figure 13.

[0265] Figure 13. Specification and isolation of melanocyte precursors / melanoblasts.

[0266] The LSB-C protocol on day 11 supported the induction of melanocyte precursors co-expressing Sox10::GFP and MITF (A, right panel). A single MITF positive population was also observed (A, left panel). c-Kit was identified as a potential marker of melanocyte precursors. A low percentage of Sox10::GFP and c-kit co-expressing cells was observed after LSB-C differentiation (B, orange population). qRT-PCR analysis confirmed enrichment of the melanocyte markers MITFM (basic helix-loop-helix leucine zipper protein) and Dct (dopachrome tautomerase (dopachrome delta isomerase, tyrosine-linked protein 2)) in the double-positive population (C). Treatment with BMP4 and EDN3 ("LSB-Mel") enhanced the induction of a Sox10::GFP and c-kit double-positive putative melanocyte precursor population (D). Sox10::GFP, c-kit double-positive cells isolated after LSB-Mel treatment showed significantly higher levels of the melanocyte markers MITFM and Dct (E). Error bars indicate s.e.m. * p<0.05.

[0267] Figure 14 Proliferation and maturation of melanocyte precursors.

[0268] Summary of differentiation conditions (A). After differentiation in LSB-C (LSB-Mel) conditions with BMP4 and EDN3, cells were sorted and replated on day 11. Post-sorted (PS) cells were maintained in maturation medium containing c-kit ligand (SCF), endothelin 3 (EDN3), fibroblast growth factor (FGF), and Wnt activators. Pigment cells identified by brightfield microscopy on day 6 of PS were positive for the melanocyte marker MITF but appeared to down-regulate the Sox10::GFP reporter (B). All populations, except for the Sox10::GFP, c-kit double-negative population, eventually gave rise to MITF-expressing cells and macroscopically visible pigment clusters, although at different rates (C). Treatment with BMP4 and cAMP enhanced differentiation into pigment cells exhibiting the spindle-like morphology characteristic of melanocytes (D).

[0269] Figure 15. Characterization of mature melanocytes.

[0270] A pure population of mature melanocytes derived with the LSB-Mel protocol maintains expression of common melanocyte markers, including MITF, Sox10, Tyrp1 (tyrosinase-related protein 1), and HMB45, after more than 8 weeks in culture (A). Melanocytes retain their darkly pigmented phenotype over several weeks of passage (B). 1 × 10 cells were used to assess pigmentation levels. 6 Cells were pelleted and photographed. Electron microscopic ultrastructural characterization of mature melanocytes (C, D). The presence of numerous darkly pigmented melanosomes in the cytoplasm of LSB-Mel-derived melanocytes was observed by TEM (C). Note the presence and progressive deposition of melanin pigment as melanosome vesicles mature from stage I to stage IV (D).

[0271] Therefore, we demonstrated that the dual SMAD inhibition protocol, LSB, rapidly and efficiently generates neural crest populations expressing Soxl0::GFP from human embryonic stem cells. This modified protocol supported the induction of low levels of melanocyte precursors, which were prospectively identified and isolated by c-kit expression. The induction of these cells was further enhanced through treatment with BMP4 and EDN3. The melanocyte precursors then matured to a pigmented state after further in vitro culture in the presence of BMP4 and cAMP.

[0272]

number

[0273] A concentration range of 10 ng / ml to 100 ng / ml (25 ng / ml in one embodiment) is used for BMP4 from R&D, and 25 to 300 nM (100 nM in one embodiment) for EDN from American Peptide Company.

[0274] Figure 16 shows an exemplary LSB-MEL medium formulation that requires linoleic acid for melanocyte proliferation. Media components shown above the micrograph are those excluded from the formulation; Ph = phase contrast; BF = bright field. An exemplary schematic shows melanocyte precursor markers used to identify the cells of the invention.

[0275] Thus, the inventors have discovered and developed a rapid and well-defined protocol for the induction of neural crest cells in vitro. Furthermore, the inventors have used this rapid and well-defined protocol for the induction of neural crest cells in vitro to develop compositions and methods for the directed differentiation of these cells into melanocytes. These melanocytes are unique in their capacity for long-term culture and sustained production of eumelanin.

[0276] Therefore, the derivation of melanocytes from human embryonic stem cells (hESCs) may provide a valuable tool for further exploration of melanocyte disease biology.

[0277] experiment The following examples are intended to help illustrate certain embodiments and aspects of the present invention and should not be construed as limiting its scope. In the following experimental disclosure, the following abbreviations are used: N (normality); M (molarity); mM (millimolarity); μM (micromolar concentration); mol (mole); mmol (millimolar); μmol (micromole); nmol (nanomole); pmol (picomole); g (gram); mg (milligram); μg (microgram); ng (nanogram); pg (picogram); L (liter); ml (milliliter); μl (microliter); cm (centimeter); mm (millimeter); μm (micrometer); nm (nanometer); U (unit); min (minute); s and sec (second); deg (degree); pen (penicillin), streptomycin, and ° C 10 (degrees Celsius).

[0278] The following formulations set forth exemplary cell culture media for use in developing embodiments of the present invention.

[0279] hESC medium for maintenance (1 liter): 800 mL of DMEM / F12, 200 mL of knockout serum replacement, 5 mL of 200 mM L-glutamine, 5 mL of Pen / Strep, 10 mL of 10 mM MEM minimal non-essential 15 amino acid solution, 55 µM 13-mercaptoethanol and bFGF (final concentration is 4 ng / mL).

[0280] KSR medium for hESC differentiation (1 liter): 820 mL of Knockout DMEM, 150 mL of Knockout Serum Replacement, 10 mL of 200 mM L-glutamine, 10 mL of Pen / Strep, 10 mL of 10 mM MEM and 55 µM 13-mercaptoethanol.

[0281] N2 medium for hESC differentiation (1 liter): 985 ml of distilled HO with DMEM / F12 powder, 1.55 g of glucose (Sigma, catalog number G7021), 2.00 g of sodium bicarbonate (Sigma, catalog number S5761), putrescine (100 µL aliquot of 1.61 g dissolved in 100 mL of distilled water; Sigma, catalog number P5780), progesterone (20 µL aliquot of 0.032 g dissolved in 100 mL of 100% ethanol; Sigma, catalog number P8783), sodium selenite (60 µL aliquot of a 0.5 mM solution in distilled water; Bioshop Canada, catalog number SEL888), and 100 mg of transferrin (Celliance / Millipore, catalog number 4452-01), and 25 mg of insulin (Sigma, catalog number 16634) in 10 mL of 5 mM NaOH.

[0282] Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS for preparing PMEFs (primary mouse embryonic fibroblast (PMEF) feeder cells) (1 liter): 885 mL of DMEM, 100 mL of FBS, 10 mL of Pen / Strep, and 5 mL of L-glutamine.

[0283] α-Minimum essential medium (MEM) with 10% FBS to prepare MS-5 feeder cell medium (1 liter): 890 mL of α-MEM, 100 mL of FBS, 10 mL of Pen / Strep gelatin solution (500 ml): Dissolve 0.5 g of gelatin in 500 ml of warm (50–60 °C) Milli-Q water. Cool to room temperature. [Example]

[0284] Example I Experimental Materials and Methods The following examples set forth exemplary materials and methods used during the development of the present invention.

[0285] Cells and culture conditions. Human embryonic stem cell (hESC) cells (WA-09; passages 32–50) and hiPSC lines (C14, C72; passages 10–20) were cultured at 12–15,000 cells / cm. 2 The cells were cultured with pre-plated mouse embryonic fibroblasts (MEFs, Globalstem, Rockville, State of Maryland, United States of America (USA)) at 27°C. A human induced pluripotent stem cell (hiPSC) line was cultured as described (Papapetrou et al., Proc Natl Acad Sci (2016) 113:131-132, which is incorporated herein by reference). The cells were generated using a 2009 (USA 106 (2009)) medium containing Dulbecco's Modified Eagle Medium (DMEM) / F12, 20% knockout serum replacement, 1 mM L-glutamine (Invitrogen, Carlsbad, California, USA), 100 μM MEM non-essential amino acids (Invitrogen), and 0.1 mM β-mercaptoethanol (Invitrogen). Six ng / ml of fibroblast growth factor 2 (FGF-2, R&D Systems, Minneapolis, Minnesota) was added after sterile filtration, and the cells were fed daily and passaged weekly using 6 U / ml dispase (Worthington Biochemical, Lakewood, New Jersey, USA). The SOX10::GFP bacterial artificial chromosome cell line was generated as described (Placantonakis et al., Stem Cells 27, 521-532 (2009), incorporated herein by reference).

[0286] Nerve and Nociceptor Induction. Nerve induction was performed as previously reported (Chambers et al., Nat Biotechnol 27 (2009), incorporated herein by reference). Briefly, cells were collected, then conditioned into a single-cell suspension using ACCUTASE (Sigma-Aldrich Corp. St. Louis, Missouri, USA) and plated on gelatin for 30 minutes to remove mouse embryonic fibroblast (MEF) feeder cells (MEFs adhere to gelatin-coated plates). Nonadherent cells were collected and cultured at 20–40,000 cells / cm in the presence of MEF-conditioned hESC medium containing 10 ng / ml FGF-2 and 10 μM Y-27632 (a rho kinase inhibitor—Tocris Bioscience). 2 Cells were plated into Matrigel-treated dishes at a density of 1000 μg / ml. Neural differentiation was initiated when cells reached confluence using Knockout Serum Replacement (KSR) medium containing 820 ml of Knockout DMEM, 150 ml of Knockout Serum Replacement, 1 mM L-glutamine, 100 μM MEM non-essential amino acids, and 0.1 mM β-mercaptoethanol. To inhibit SMAD signaling, 100 nM LDN-193189 and 10 μM SB431542 were added daily from day 0 (the day the SMAD signaling inhibitor LSB was added) through day 5. Cells were fed daily (i.e., six feedings of inhibitor: D0, D1, D2, D3, D4, and D5). Starting on day 4, N2 medium was added to the initial medium in incremental 25% increments every other day (until 100% N2 on day 10). Nociceptor induction was initiated by adding three inhibitors (unless otherwise indicated) at 3 μM CHIR99021, 10 μM SU5402, and 10 μM DAPT daily from day 2 to day 10. From day 10 onwards, long-term culture medium consisted of N2 medium containing 10–100 ng / ml human β-nerve growth factor (NGF), 10–100 ng / ml brain-derived neurotrophic factor (BDNF), and 10–100 ng / ml glial cell line-derived neurotrophic factor (GDNF).

[0287] Microscopy, antibodies, and flow cytometry (FACS). Cells were fixed with 4% paraformaldehyde for 20 min, washed with phosphate-buffered saline (PBS), permeabilized with 0.5% Triton X in PBS, and blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). For glutamate staining, 0.05% glutaraldehyde was added to the fixative. The primary antibodies used for microscopy were PAX6; paired box gene 6 (aniridia, keratitis) (Covance, Princeton, New Jersey, USA), TUJ1; neuron-specific class III β-tubulin (Covance, Princeton, New Jersey, USA), Ki67; antigen KI-67; MKI67 (Sigma-Aldrich Corp. St. Louis, Missouri, USA), ISL1 (Developmental Studies Hybridoma Bank; DSHB), BRN3A; brain-specific homeobox / POU domain protein 3A (Chemicon, Billerica, Massachusetts, USA), RET; proto-oncogene tyrosine protein kinase receptor (R&D), RUNX1; Runt-related transcription factor 1 (Sigma-Aldrich Corp. St. Louis, Missouri, USA), MAP2; microtubule-associated protein 2 (Sigma-Aldrich Corp. St. Louis, Missouri, United States of America), TRPV1; transient receptor potential cation channel subfamily V member 1 (Neuromics Inc., Minneapolis, United States of America), substance P (Neuromics Inc., Minneapolis, United States of America), CGRP; calcitonin gene-related peptide (Neuromics Inc., Minneapolis, United States of America).For flow cytometry, cells were fixed using the BD Cytofix / Cytoperm Kit (BD Biosciences Pharmingen), and in one embodiment, cells were further fixed in 4% paraformaldehyde. The primary conjugated antibodies for flow cytometry were NTRK1 (neurotrophic tyrosine kinase receptor, type 1)-APC (R&D Systems, Inc., Minneapolis, Minnesota, USA), nestin-Alexa647 (BD Biosciences Pharmingen, San Diego, California, USA), and TUJ1-Alexa488 (BD Biosciences Pharmingen, San Diego, California, USA).

[0288] Electrophysiology. Neurotrophic tyrosine kinase receptor, type 1 (NTRK1)+ sorted cells were plated on polyornithine / laminin / fibronectin-treated coverslips on days 10–12 and allowed to mature for an additional 3 weeks in long-term culture medium. Coverslips were transferred to artificial cerebrospinal fluid containing (mM): 125 NaCl, 2.5 KCl, 1.25 KH2PO4, 1 MgCl2, 2 CaCl2, 25 NaHCO3, 1.3 ascorbic acid, 2.4 pyruvate, and 25 glucose (aerated with 95% O2 and 5% CO2, room temperature). Cells were visualized using an infrared differential interference contrast (DIC) microscope (Olympus) equipped with epifluorescence illumination, a charge-coupled device camera, and two water-immersion lenses (x10 and x60) to target recording electrodes to the cells. Glass recording electrodes (7-9 MΩ resistance) were filled with an intracellular solution consisting of (in mM, pH 7.25) 130 mM potassium gluconate, 16 mM KCl, 2 mM MgCl2, 0.2 mM EGTA, 10 mM HEPES, 4 mM Na2ATP, 0.4 mM Na3GTP, and 0.2% Alexa-568. Action potential characteristics at threshold current were determined from intracellular recordings after application of a series of increasing 25 pA 300 ms current steps. Recordings were collected and analyzed using an Axopatch 700B amplifier and pCLAMP10 software (Molecular Devices, Sunnyvale, California, United States).

[0289] Gene expression profiling. Total RNA was isolated from LSB- or LSB3i-treated hPSCs on days 2, 3, 5, 7, 9, and 15 of differentiation using Trizol LS. Samples were collected at Memorial Sloan-Kettering Cancer Center. (MSKCC) Genomics Core Facility and hybridized to Illumina Human HT-12 v4 Expression BeadChips. Normalized and model-based expression measures were calculated using the Illumina analysis package (LUMI) from the Bioconductor project (www.bioconductor.org) in conjunction with the statistical programming language R (http: / / cran.r-project.org / ). Expression values ​​are the log2 of fold changes. The cutoff for pairwise comparisons was significance when the p-value corrected for multiple testing was <0.05.

[0290] Quantitative real-time PCR. Total RNA was extracted using the RNeasy kit (Qiagen). For each sample, 1 μg of total RNA was treated for DNA contamination and reverse transcribed using the Quantitect RT kit (Qiagen). Amplified material was detected using the Quantitect SYBR green probe and PCR kit (Qiagen) on a Mastercycler RealPlex2 (Eppendorf). All results were normalized to the HPRT control, and each data point represents the results from 4–6 technical replicates of 2–3 independent biological samples.

[0291] Example II Neuronal lineage cells were produced by contacting human pluripotent stem cells with SB431542 and LDN-193189 (LSB).

[0292] The following examples set forth exemplary methods for providing cells of neuronal lineage for use during the development of the present invention.

[0293] Dual SMAD inhibition has previously been used as a rapid and highly effective method to induce certain neuronal lineage cells from hPSCs (Chambers et al., Nat Biotechnol 27 (2009), incorporated herein by reference). These neuronal lineage cells induced by molecules including noggin had a default pathway that allowed development into central nervous system cells, i.e., neuronal cell fates. A supplementary study reported that using the small molecule dorsomorphin (DM) instead of noggin produced at least partially similar cells, although there were differences in culture consistency (Kim et al., Robust enhancement of neural differentiation from human psoriasis, incorporated herein by reference). ES and iPS cells regardless of their innate difference in differentiation propensity.Stem Cell Rev 6,270-281(2010);Zhou et al., High-Efficiency Induction of Neural Conversion in hESCs and hiPSCs with a Single Chemical Inhibitor of TGF-beta Superfamily Receptors.Stem Cells,504(2010)).

[0294] The inventors observed that, despite exhibiting the same developmental stage, expression of most of the same markers, and similar developmental potential to generate various neuronal lineages as LDN-treated cells, cells generated using Noggin also exhibit differences compared to neurons induced using LDN, such as being more anterior in the anterior-posterior axis (i.e., more forebrain, more cells expressing FOXG1, etc.) Thus, although LDN was used instead of Noggin to inhibit BMP, among other signaling pathways, Noggin and LDN are likely to have different types of activities other than inhibiting BMP.

[0295] Due in part to the high cost of using noggin, the inventors considered the use of a BMP inhibitor to be a substitute for noggin in producing cells with a neural fate. Therefore, to generate primitive neuroectoderm, cells with a neural fate, i.e., CNS cells, from hPSCs, the small molecule BMP inhibitor, LDN-193189 (Yu et al., Nat Med 14, 1363-1369 (2008), incorporated herein by reference), was used in combination with SB431542 and found to replace noggin during the development of the present invention (Figure 2A). This combined treatment was designated LSB, referring to the combination of these two inhibitors, LDN-193189 and SB431542.

[0296] Example III Screening small molecules using the neuronal lineage cells of the present invention resulted in compounds that produced low PAX6 and high TUJ1 neuronal cells.

[0297] The following example describes the use of exemplary cells of a neuronal lineage from Example II to screen small molecule candidate compounds for use in directed differentiation.

[0298] Specifically, in the context of dual SMAD inhibition (LSB), human ES cells were first treated with LSB (LDN-193189 and SB431542) to screen candidate compounds (i.e., small molecules) under approximately 400 conditions (to identify combinations of small molecules that could accelerate the acquisition of postmitotic neuronal markers from human ES cells). Candidate compounds were selected from molecules that target (alter) cell signaling pathways (e.g., FGF, Notch, WNT, SHH (Sonic hedgehog), etc.) known to be important in developmental studies and frequently used to determine cell fate, which determines which cells can develop into the CNS. As an example, four inhibitors (i.e., SU / DAPT / CHIR / cyclopamine) were tested in different combinations (supplied to cells in cell culture medium) on different days after LSB treatment. Each treatment was then screened for TUJ1 / PAX6 expression on day 10. As an example of treatment conditions, LSB was supplied daily, and CHIR and SU were added to the medium and supplied to the cells daily from day 4 to day 10.

[0299] In general, the screening process resulted in a large number of cultures containing dead cells. In other words, viable culture conditions in this screen were found to occur much less frequently than non-viable conditions (i.e., cell death) (e.g., when SU / DAPT was added to the initial culture, i.e., before day 2). The inventors believe that CNS stem cells depend on FGF signaling and gamma-secretase activity / Notch signaling for survival, and therefore, in the absence of CHIR, when SU / DAPT induced cells to switch from CNS to neural crest, the cells died rather than switching.

[0300] Ten days after the addition of LSB, cells that survived the screening were observed for the loss of the human neuroectoderm marker PAX6 (Zhang et al., Cell Stem Cell 7, 90-100 (2010), incorporated herein by reference) and the onset of neuronal differentiation by TUJ1 expression (Lee et al., Cell Motil Cytoskeleton 17, 118-132 (1990), incorporated herein by reference). Cells were stained for neurons (TUJ1+) by immunofluorescence (immunoF) and for the loss of neuroectoderm (observation of fewer PAX6+ cells) using an antibody that binds to the C-terminus of PX6. This screening was performed with numerous combinations of inhibitors (e.g., SU, SU / DAPT, SU / DAPT / CHIR, DAPT / CHIR, SU / CHIR, SU / cyclopamine, etc.), which were added to various daily feeds on the days of combination (e.g., days 0-10, days 1-10, days 2-10, days 3-10, etc.). Results were generally determined by observing the relative amount of TUJ1 / PAX6 staining in cells generated by each treatment, with the condition and compound showing the highest amount of TUJ1 / PAX6 staining selected as successful for providing cells for further analysis. One example of a small molecule that failed in the screening test for producing TUJ1 / PAX6 cells by immunostaining was cyclopamine. Cyclopamine did not appear to affect cells in terms of producing TUJ1 / PAX6 staining, regardless of when it was added. In other words, cell morphology remained similar to cells treated with LSB alone at day 10 by immunofluorescence (i.e., >90% PAX6+ and <10% TUJ1+).

[0301] However, during screening, the inventors discovered that a specific combination of three small molecules (SU5402, CHIR99021, and DAPT; referred to as 3i for the three inhibitors) added on day 2 of LSB treatment (Figures 6A and B) abolished PAX6 expression and induced TUJ1 in hPSCs on day 10 of differentiation (Figures 2A and B). This was a surprising finding because on day 2 of LSB treatment, the treated cells were still unclear regarding their neuronal fate or their ability to develop into a neuronal fate. Instead, 3i treatment directed cells away from a neuronal fate toward neural crest cells, which further differentiated into the nociceptor cells of the present invention.

[0302] Next, we investigated the function of each of these small molecules to determine which signaling pathways are thought to be involved in converting the PAX6+TUJ1- human ES cell population into the PAX6-TUJ1+ population. First, SU5402 was reported as a potent inhibitor of VEGF, FGF, and PDGF tyrosine kinase signaling (Sun et al., J Med Chem 42, 5120-5130 (1999)). Therefore, it was generally believed that at least one of the small molecules was involved in inhibiting the FGFR signaling pathway. Second, CHIR99021 was reported as a WNT agonist by selectively inhibiting GSK-3β (stabilizing β-catenin) (Bennett et al., J Biol Chem 277, 30998-31004 (2002)). Thus, generally, at least one of the small molecules was considered to be involved in activating at least one WNT signaling pathway through the inhibition of glycogen synthase kinase 3β (GSK3β). And thirdly, DAPT was reported as a gamma-secretase inhibitor that can block Notch signaling (Dovey et al., J Neurochem 76, 173-181 (2001), incorporated herein by reference). Thus, generally, at least one of the small molecules was considered to be involved in inhibiting at least one Notch signaling pathway. Thus, in one embodiment, one of the small molecules was considered to be a non-selective or pan-Notch inhibitor. In another embodiment, one of the inhibitors is an inhibitor of gamma-secretase molecules that can block at least one Notch signaling pathway. Thus, in one exemplary embodiment, the combination of inhibitors includes at least one small molecule involved in inhibiting the FGFR signaling pathway, at least one small molecule involved in inhibiting at least one Notch signaling pathway, and at least one small molecule involved in inhibiting GSK3β while activating at least one of the WNT signaling pathways to produce the PAX6-TUJ1+ human neuronal cells of the present invention.In a further embodiment, one of the inhibitors was capable of blocking at least one gamma-secretase molecule in the Notch signaling pathway.

[0303] Example IV TUJ1+ neuronal cells show a loss of expression of cell proliferation markers.

[0304] The following example describes an exemplary method for determining the maturation (cell cycle) stage of TUJ1+ neuronal cells.

[0305] After maturation, neurons produced in culture ceased to undergo mitosis and lost Ki67 and phosphohistone H3 (PHH3), markers of cell proliferation (Gerdes et al., Int J Cancer 31, 13-20 (1983), incorporated herein by reference) and the G2 / M phase of mitosis (Hendzel et al., Chromosoma 106, 348-360 (1997), incorporated herein by reference), respectively. Therefore, cells produced using LSB in combination with 3i (i.e., LSB3i) were grown at a lower density, approximately 10-100,000 cells / cm. 2 The cells were passaged and fixed to better assess the expression of individual cells, which were then tested for the cell proliferation markers Ki67 and phosphohistone H3 (PHH3). In particular, Ki67 expression is known to be a good predictor of proliferation. Thus, after 12 days, fewer cells (50% and 16%, respectively) exhibited Ki67 and pHH3 expression in cells cultured in the presence of 3i compared with cells cultured in LSB without 3i compounds (Figure 2C-F).

[0306] To quantify the efficiency (percentage) of neuronal differentiation using LSB3i compared with LSB / CHIR (CHIR99021:C), SU / DAPT (SU5402 / DAPT), SU / CHIR (SU5402 / CHIR99021), DAPT, SU (SU5402), and CHIR, plus LSB alone as a control, intercellular FACS staining for nestin, a marker of neural precursors, and β3-tubulin (TUJ1), a marker of neuronal differentiation, was performed (Figure 2G). In the presence of LSB, SU / DAPT, DAPT, SU, and CHIR, the majority of cells expressed nestin. Notably, >95% of the LSB cell population was nestin+. Numerous cells showed nestin staining after dual SMAD inhibition but were not quantified, whereas cells cultured for longer periods, i.e., 19 days, exhibited TUJ1+ neurons, and the majority of these cells co-expressed tyrosine hydroxylase (TH), identifying potential dopaminergic neurons (Chambers et al., Nat Biotechnol 27 (2009), incorporated herein by reference). Conversely, when cells exposed to LSB were exposed to 3i compounds after 2 days, approximately 25% of the cells expressed nestin and approximately 75% of the cells expressed TUJ1 after 10 days, demonstrating efficient conversion to a neuronal cell fate after a short period, i.e., less than 19 days, of cell culture.

[0307] Surprisingly, LSB treatment followed by contacting the cells with CHIR99021 and either DAPT or SU two days later differentiated 50% of the cell population into TUJ1+ cells. When each of the three inhibitors was used alone after LSB treatment, less than 20% of the cells were TUJ1+. Therefore, CHIR99021 was found to be a key contributor to the directed differentiation of this cell population into TUJ1+ neuronal cells. The inventors hypothesized that the directed differentiation of nestin+TUJ1- cells into nestin-TUJ1+ neuronal cells depends on the inhibition of GSK3β while activating at least one of the WNT signaling pathways, in addition to inhibiting either γ-secretase in the FGF receptor pathway or the Notch signaling pathway. Furthermore, the addition of 3i compounds resulted in the conversion of an additional 25% of nestin-TUJ1+ neuronal cells. See Figure 2G.

[0308] In summary, we further investigated the neuronal population derived from the preferred embodiment of LSB treatment followed by 3i treatment two days later. This population showed higher expression of the neuronal marker TUJ1 (Figure 2A, B) as well as loss of Ki67 (Figure 2C, D) compared to cells treated with LSB alone. Loss of Ki67 indicates a decrease in cells in the cell cycle, a hallmark of postmitotically differentiated neurons. In addition, FACS analysis revealed that over 75% of the cell population treated with the preferred composition consisting of LSB and 3i expressed TUJ1, whereas 99% of the population treated with LSB alone expressed the progenitor marker nestin (Figure 2G).

[0309] Example V The TUJ1+ neurons were surprisingly peripheral nervous system (PNS) cells rather than the expected central nervous system (CNS) cells.

[0310] The following examples describe exemplary methods for identifying the types of TUJ1-positive neurons produced during development of the present invention.

[0311] To further characterize the neuronal subtypes resulting from the preferred embodiment of 3i treatment after 2 days of LSB treatment, the TUJ1-positive population was stained for markers of various neuronal subtypes. Specifically, the dual SMAD inhibition protocol was known to generate PAX6+ neuroepithelial cells biased toward an anterior forebrain identity that expresses FOXG1 (forkhead box protein G1) (Chambers et al., Nat Biotechnol 27 (2009)). Therefore, to determine the neuronal subtype identity after LSB3i treatment, cells were cultured at a lower density of approximately 10-100,000 cells / cm on day 10. 2 and assessed for expression of a range of markers on day 12.

[0312] Because the expected neuronal type was CNS-fate, most of the initial markers tested were for the identification of CNS-type cells. Indeed, CNS forebrain neurons were expected, since LSB cells are by default of this subtype (PAX6, FOXG1 positive). Surprisingly, at least 12 negative results (10 exemplary results are shown below) were obtained for CNS markers before staining for ISL1, a marker for PNS cells, was found. ISL1 is expressed by motor neurons and peripheral sensory neurons. BRN3A expression was tested and found to be expressed by LSB / 3i cells. Therefore, the inventors discovered BRN3A+ / ISL1+ neurons, indicating the development of peripheral sensory neurons. See Table A below.

[0313] Table A: The following list of genes / proteins shows numerous CNS fate molecules predicted to be positive (expressed) for cells using the LDN / 3i-induced differentiation described herein. However, these results show a lack of exemplary CNS markers, a result supported by the subsequent observation of potential markers for PNS lineages, namely ISL1 and BRN3A.

[0314]

number

[0315] Surprisingly, uniform expression of ISL1 and BRN3A (red / darker areas within the cells) (Figures 3A and B) was observed in our TUJ1+ cells (green / brighter cell bodies compared to red staining). ISL1 and BRN3A are key markers for sensory neurons (ISL1: Sun et al., Nat Neurosci 11, 1283-1293 (2008); BRN3A: Gerrero et al., Proc Natl Acad Sci USA 90, 10841-10845 (1993)), all of which are incorporated herein by reference. This finding indicated that the neurons resulting from LSB3i treatment were PNS rather than CNS cells. These results contrast with LSB cells, which are by default CNS forebrain neuron subtypes (PAX6+, FOXG1 positive). This is a highly unexpected observation, since, according to the teachings of the prior art, high confluency of stem cells at the initiation of treatment, as indicated by plating density, should give rise to a CNS-derived neuronal population. However, nociceptors are derived from neural crest cell populations, which, according to the teachings of the prior art, are derived from low confluency of stem cells at the initiation of treatment, as indicated by plating density. In other words, the expectation was >20,000 cells / cm at the initiation of LSB treatment. 2 High initial plating densities of pluripotent stem cells give rise to committed CNS neuronal populations, whereas neural crest cells give rise to approximately 10,000 cells / cm. 2 It was known that a low initial plating density of 100 ng / ml was required (Chambers et al., Nature Biotech, 2009 (see bottom half of Figure 4), which is incorporated herein by reference in its entirety).

[0316] Example VI Peripheral nervous system (PNS) neurons have been found to be early stage nociceptor cells.

[0317] The following examples describe the use of exemplary methods to determine what types of peripheral nervous system (PNS) neurons have been produced using the methods described herein.

[0318] It was unclear what type of PNS neuron would be produced by the methods described herein, as there are several types of candidate neurons, such as sensory and motor neurons, and at least three major subsets of known sensory neurons in the PNS, including proprioceptor cells, mechanoceptor cells, and nociceptor cells.

[0319] During development, early stage nociceptors are both peptidergic and non-peptidergic and uniquely express NTRK1, RUNX1, and then RET (for example information regarding RET, see Woolf et al., Neuron 2004, 14, 111-113, which is incorporated herein by reference). 55, 353-364 (2007). Duplicate early-stage LSB3i cultures containing TUJ1+ neurons were tested for RET expression (Fig. 3C) and found to be positive for this marker (TUJ1+ staining (green / lighter cell bodies compared to RET staining) and red / darker areas within the cells in the larger box compared to the lighter staining area in the inset RET box) (Fig. 3D), and more than 60% of all cells in culture expressed NTRK1 as measured by FACS at day 10 (Fig. 3E).

[0320] In summary, this population was positive for the expression of ISL1, BRN3A, RET, and RUNX1 (Figure 3A-D), which indicate the generation of early-stage nociceptors (both peptidergic and non-peptidergic). FACS analysis revealed that over 60% of these neurons were positive for NTRK1 (Figure 3E). Collectively, these markers indicate that this population of neurons represents peripheral sensory neurons, specifically nociceptors.

[0321] Therefore, the preferred embodiment of the combination of 3i treatment and LSB on day 2 results in the unexpected formation of a neural crest-derived population, namely nociceptors.

[0322] Furthermore, the inventors combined information from several studies, including the observations described herein that cells derived from LSB / 3i treatment transiently express neurogenin 1 (NEUROG1) instead of migrating to neural crest and differentiating into a CNS fate, initial immunofluorescence results (i.e., BRN3A+, ISL1+), array data (i.e., TAC1 (Substance P) expression), and then selected the NTRK1 marker to find NTRK1+ cells, leading to the conclusion that the resulting PNS cells are likely peptidergic nociceptors.

[0323] Example VII The LSB3i process is reproducible.

[0324] The following example describes the use of an exemplary method of the present invention to determine reproducibility.

[0325] To establish the generality of the present invention, the inventors repeated a preferred embodiment of the present invention, combining LSB treatment with 3i treatment two days later, using hiPSCs as a stem cell source. The reproducibility of LSB3i treatment was evaluated across additional induced pluripotent stem cell (hPSC) lines, including hiPSC lines. Current technology describes many methods for producing hiPSCs, known to those skilled in the art. In particular, two hiPSC lines (C14 and C72) created by inserting genes such as Oct4 (octamer-binding transcription factor 4), Sox2 (SRY (sex-determining region Y) box 2), Klf4 (Krüppel-like factor 4), and c-Myc (transcription factor p64) have been shown to be capable of efficient neuralization (see Papapetrou et al., Proc Natl Acad Sci., USA 106 (2009)).

[0326] PAX6 expression was then examined by ImmunoF. LSB and LSB3i treatment of C14 and C72 cell lines showed similar neuronal staining results when compared to the human cell lines shown in Figures 3A-D. As mentioned above, exemplary C14 staining results are shown in Figures 4A-D, and exemplary C72 staining results are shown in Figures 8A-D for ISL1, BRN3A, RET, RUNX1, and TUJ1.

[0327] LSB treatment of the C14 and C72 cell lines uniformly gave rise to nestin-positive cells (>95% of the treated cell population), which were able to form TUJ1+ cells when treated with the LSB3i combination as measured by FACS (40% for C14 and 33% for C72; Figure 4E). These results were compared to the H9 cell line (i.e., an hESC line) treated with LSB and LSB3i, as shown for the LSB and LSB3i results in Figure 4E. Even higher neuronal yields (>90% of the nuclear staining when sorted for NTRK1, from 40% and 33%, as measured by FACS, were obtained in these two hiPSC lines when bulk cultures were passaged into Matrigel™-coated culture vessels containing N2 medium after sorting for NTRK1 (neurotrophin tyrosine kinase receptor type 1) marker expression. Cells were dissociated with Accutase, resuspended in N2, and incubated with APC-conjugated NTRK1 antibody (R&D) on ice for 15 minutes, washed, and resuspended in N2 for FACS. After sorting, cells were cultured in N2 medium for 24 hours and fixed appropriately. Cells were harvested and stained for BRN3A, ISL1, TUJ1, and DAPI. Notably, LSB3i-treated cells exhibited numerous nestin+ cells (red / dark staining) in both C14 and C72 NTRK1- cells, compared with the few nestin+ cells in the representative NTRK1+ LSB3i-treated cell population (Figure 9). Furthermore, while few C14 NTRK1- cells expressed TUJ1, C27 exhibited a higher number of NTRK1-TUJ1+ (green; bright staining). Both cell lines exhibited a high number of nestin-TUJ1+ cells, as seen relative to cell bodies identified by DAPI (blue; bright nuclei) staining.

[0328] In summary, hiPSC cells treated with LSB followed by 3i on day 2 and plated at high confluency resulted in the formation of neuronal cells positive for the nociceptor markers ISL1, BRN3A, RET, and RUNX1 (Figures 4A-D, 8A-D, and 9).

[0329] Example VIII CHIR99021(C) is a key factor for inducing neuronal differentiation from LSB cultured cells (i.e., LSB-C).

[0330] The following examples describe the use of exemplary methods to test the efficacy of each compound for inducing directed neuronal differentiation.

[0331] To gain mechanistic insight into the sufficiency of each compound found to be associated with the induction of TUJ1+ cells in Example III, specific combinations of 3i compounds were tested for inducing cellular expression of nestin and TUJ1 as measured using intracellular FACS (shown in Figure 1G). Nestin was used as a marker for LSB neuronal lineage cells, and TUJ1 was used to identify downstream (i.e., more differentiated) neuronal cells.

[0332] Although none of the individual factors produced high numbers (>60%) of TUJ1+ neurons, CHIR99021 in combination with either one of the other two signal inhibitors was able to generate moderate numbers of TUJ1+ neurons (53% for DAPT and 58% for SU5402). These data indicate that under the test conditions used here, CHIR99021 was a key factor for accelerating neuronal differentiation, with SU5402 and DAPT providing important, but additional, stimuli.

[0333] In addition, all three components of the 3i composition are necessary for maximal yield of differentiated neurons (Figure 2G).

[0334] Example IX Artificial SOX10+ cells can produce nociceptor cells.

[0335] The following example describes the use of an exemplary method of the invention for directed differentiation of engineered Sox10+GFP-expressing human cells.

[0336] Nociceptor cells are thought to arise from two types of cellular intermediates during human development: Specifically, it has been shown that SOX10+ chick embryonic neural crest cells can generate trunk nociceptor cells adjacent to the spinal cord (George et al., Nat. Neurosci. 2004, 103:111-114, which is incorporated herein by reference). Neurosci 10:1287-1293 (2007)). In addition, head placode tissue of the African clawed frog (Xenopus laevis) contributed to the trigeminal nociceptor cell population in facial tissue (Schlosser et al., J Comp Neurol 418:121-146 (2000); Schlosser et al., Dev Biol 294:303-351 (2006), which are incorporated herein by reference).

[0337] To determine whether the neural crest intermediate cell fate indicated by SOX10 in human cells (Aoki et al., Dev Biol 259, 19-33 (2003); Lee et al., Nat Biotechnol 25, 1468-1475 (2007)), which is incorporated herein by reference, could be observed during differentiation using a transgenic SOX10::GFP bacterial artificial chromosome (BAC) hPSC line, we generated this SOX10::GFP(BAC) cell line, which co-expresses enriched neural crest gene markers with the GFP gene, using a previously reported method (Placantonakis et al., Stem Cells 27:521-532 (2009)). The SOX10:GFP cell line was a subclone of the H9 hESC line. Cells were dissociated, and gene delivery was performed using reagents (Solution V), protocols (B-16), and equipment from Amaxa. The nucleofected DNA (transfected into the nucleus) was a bacterial artificial chromosome (BAC) containing inserted GFP and SOX10 genes, obtained from the Gene Expression Nervous System Atlas [GENSAT] (accession number: GENSAT1-BX1086). The BAC was then modified to contain a neomycin resistance gene for selection using cre / LoxP recombination from a selection cassette excised from the pL452 plasmid into the GENSAT BAC (see Tomishima et al., Stem Cells 25(1):39-45. Epub 2006 Sep 21 (2007), incorporated herein by reference). After gene delivery, hESCs were seeded as single cells in the presence of G418 for neomycin resistance selection, and clones were manually picked and screened for the presence of GFP after differentiation. GFP cells were sorted by qRT-PCR to confirm the expression of SOX10 and other neural crest markers.

[0338] Two additional duplicate samples were each contacted with one of the LSBs and then with CHIR99021 (LSB / C) or LSB and 3i, and GFP expression was measured by FACS identification and sorting of SOX10::GFP+ cells at 4, 8, 12, and 16 days after initiation of differentiation in LSB.

[0339] In the presence of CHIR99021, over 70% of these treated cells in culture became SOX10::GFP+ by day 12 of differentiation, depending on the culture conditions (70% for LSB / C and 80% for LSB3i; Figure 5D and E). This result indicated that the majority of cells expressed neural crest markers, supporting the inventors' observation that CHIR99021 is required for the generation of LSB3i nociceptor cells. Therefore, because LSB3i-treated cells acquired neural crest fate more rapidly compared to LSB / C-treated hPSCs (Figure 5D and E), combined inhibition of these small molecules, which inhibit tyrosine receptor kinase receptors and Notch signaling, in addition to contact with SU5402 and DAPT, accelerated neural crest cell fate. The inventors hypothesized that CHIR induces neural crest and sensory neurons, while SU accelerates neural crest marker expression and neuronal differentiation. Finally, the inventors hypothesized that DAPT in combination with CHIR and SU accelerates neuronal differentiation. Furthermore, the use of CHIR99021 in combination with LSB, i.e., LSB / C, resulted in a slower conversion rate of over 60% nestin-TUJl+ neuronal cells compared to LSB3i between days 12 and 16 when engineered SOX::GFP cells were used as a readout.

[0340] Example X NTRK1+ human nociceptor cells produced by the methods described herein exhibited gene expression consistent with peptidergic cells and electrophysiological responses similar to in situ rat nociceptor cells.

[0341] The following example describes the use of an exemplary method of the invention to determine the functional capacity of nociceptor cells produced by the methods described herein.

[0342] To confirm that the LSB3i-derived neurons were genuine nociceptor neuron cells, LSB3i-treated cells were examined for function, maturation stage, and behavior. After LSB3i treatment, pluripotent stem cells that give rise to nociceptor cells were obtained at 10–100,000 cells / cm. 2 Long-term cultures were established from plating densities of 1000 and subcultured at days 10 and 30 in N2 medium supplemented with human βNGF, BDNF, and GDNF (see Example I for further details). The viability of these cells under longer-term culture conditions was found to be NGF-dependent, corresponding to the NTRK1+ nociceptor status. LSB3i nociceptors expressed high levels of TUJ1, ISL1, and BRN3A (Figures 7A-C), as previously shown, in addition to glutamate (Figure 7C). Glutamate production was consistent with excitatory glutamatergic neurons, i.e., nociceptive afferent fibers that release glutamate, and the capsaicin receptor TRPV1, a key ion channel for noxious stimuli (Figure 7D). At 15 days in culture, two distinct outgrowth processes could be identified for each neuron (Figures 7E and 12).

[0343] The dendritic marker MAP2 was expressed primarily in one of the two processes in a polarized fashion (Figure 7F). The bipolarity of the neurons is consistent with their role as sensory neurons in peripheral ganglia, with cell bodies located in dorsal root ganglia and projecting processes toward both the spinal cord and the periphery (Woolf et al., Neuron 55, 353-364 (2007); George et al., Nat Neurosci 10, 1287-1293 (2007), incorporated herein by reference).

[0344] Neurons were cultured long-term in the presence of nerve growth factor (NGF) (e.g., cells were passaged on day 10 and cultured until day 30). LSB was harvested from the cells on day 5, and 3i was harvested on day 10, on which day NGF / GDNF / BDNF was added to the culture medium. Neurons were supplied with NGF / GDNF / BDNF from day 10 to day 30. At day 30, days after initial LSB treatment, neurons were observed to begin self-organizing into ganglion-like structures. This type of morphology is common in peripheral sensory neurons (Marmigere et al., Nat Rev Neurosci 8, 114-127 (2007)), which is incorporated herein by reference (Figures 7G, ​​H, and I).

[0345] Mature nociceptors are typically either peptidergic or non-peptidergic, depending on the expression of neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P (neuropeptide) expressed by peptidergic sensory neurons (Woolf et al., Neuron 55, 353-364 (2007), incorporated herein by reference). In contrast, non-peptidergic neurons express neither CGRP nor substance P and have other markers, such as binding to the lectin IB4.

[0346] Therefore, LSB3i-induced neurons were sorted by FACS for NTRK1 expression (see methods described above) into NTRK1+ and NTRK1- populations (see Figure 7G for an example of sorted cells). NTRK1+ cells were positive for both substance P and CGRP and exhibited a predominantly peptidergic nociceptor phenotype (Figures 7H and 7I; day 30 of differentiation).

[0347] A key functional characteristic (i.e., function) of sensory neuron identity is their electrophysiological properties (Fang et al., J Physiol 565, 927-943 (2005), incorporated herein by reference). NTRK1+ sorted neurons were also tested using standard electrophysiological techniques for cultured neurons (an example is shown in Placantonakis et al., Stem Cells. 2009, Figure 5, incorporated herein by reference in its entirety).

[0348] NTRK1+ cells exhibited electrophysiological features, including a characteristic single action potential (AP) firing pattern, and an average membrane resting potential of 67±4 mV by 21 days after initial LSB3i treatment. The timing of APs and the shape of the activity curves generated in LSB3i human neurons are shown in Figure 7J (see thick red line) and Table 1 below. These results were similar to those previously reported in an electrophysiological study of primary anesthetized adult rat nociceptors (Fang et al., J Physiol 565, 927-943 (2005)).

[0349] [Table 1-2]

[0350] Example XI Gene expression in cells produced by the compositions and methods described herein.

[0351] The following examples describe the use of exemplary methods for determining global gene expression of nociceptor cells and other cell types produced by the methods described herein.

[0352] To further characterize the timing of events (i.e., marker expression) during the induced differentiation process, global gene expression analysis was performed with fine temporal resolution on both LSB- and LSB3i-treated hPSCs (days 2, 3, 5, 7, 9, and 15, NCBI Gene Expression Omnibus (GEO) accession number GSE26867). When selected markers for neuroectoderm, neural crest, neurons, and nociceptors were analyzed (see Table 2 below), distinct stages of differentiation could be observed for each (Figure 10).

[0353] [Table 2-2]

[0354] This gene expression analysis (Figure 10B, C and Table 2 above) was largely consistent with the immunofluorescence results. For example, gene analysis showed that ISL1, POU4F1 (BRN3A), SOX10, TAC1 (propeptide of substance P), NTRK1, and the glutamate vesicular transporter VGLUT2 genes were all upregulated in maturing neurons (i.e., many cells in culture increased their expression of these markers over time). Concurrently, these markers were found to increase in induced cells, whereas markers for hESC-derived primitive neuroectoderm, particularly DLK1, LHX2, OTX2, LEFTY2, PAX6, and HES5, were found to be downregulated (i.e., expressed by fewer cells in culture).

[0355] However, expression of somatostatin (SST) and SOX10, which are expected to be expressed in mature nociceptors, was observed in LSB3i-treated cell cultures at day 15. However, SST has also been shown to be expressed in developing sensory neurons. Therefore, the inventors considered this marker to indicate the presence of immature cells at day 15. Although somewhat downregulated, SOX10 expression was also observed at a time point when the majority of cells appeared to be neurons. This observation was unexpected, as SOX10 was expected to be downregulated as cells differentiate into neurons. This unexpected finding of SST and SOX10 expression in cells from day 15 cultures suggested that not all cells would become nociceptor cells, approximately 20–30%. This indicated that other mature cell types (e.g., Schwann cells) continue to express SOX10.

[0356] hESC-derived primitive neuroectodermal cell cultures produced by dual SMAD inhibition (Chambers et al., Nat Biotechnol 27 (2009); Fasano et al., Cell Stem Cell 6, 336-347 (2010)), each incorporated herein by reference, showed high expression of the DLK1, LHX2, OTX2, LEFTY2, PAX6, and HES5 genes. Similarly, when hESC-derived primitive neuroectodermal cell cultures were produced by dual SMAD inhibition with LSB, similar high expression of these genes was observed (see Figures 10B and 10C and Table 3 below). These genes were reduced during LSB3i treatment, during which nociceptors were produced in the development of the present invention.

[0357] [Table 3-2]

[0358] Additionally, temporal transcriptome analysis provided further evidence for a nociceptor intermediate cell fate distinct from mechanoceptors and proprioceptors. The neurogenin basic helix-loop-helix protein mediates two sequential waves of neurogenesis in the dorsal root ganglion during mouse development (Marmigere et al., Nat Rev Neurosci 8, 114-127 (2007); Ma et al., Genes Dev 13, 1717-1728 (1999)). The first wave, represented by NEUROG2 (neurogenin 2), gives rise to mechanoceptors and proprioceptors, while the second wave, represented by NEUROG1 (neurogenin 1), gives rise to nociceptors. When hPSCs were treated with LSB, NEUROG2 expression was strongly induced by day 7 (Figure 10C and Table 4 below). In contrast, hPSCs treated with LSB3i show less pronounced induction of NEUROG2 by day 7, but selective induction of NEUROG1 by day 9 (FIG. 10C).

[0359] [Table 4-2]

[0360] Example XII Exemplary large-scale cultures contemplated using the compositions and methods of the present invention to provide nociceptor cells.

[0361] The following contemplated description sets forth exemplary methods for the large-scale production and use of nociceptor cells produced by the methods described herein.

[0362] Scalable production (i.e., relatively small numbers of cells, e.g., 1.5 x 10 cells in a 48-well plate as described in the Examples, supra) using LSB3i 4 cells / well, and 5 x 10 in a 96-well plate is contemplated. 3 From both cultures containing cells / well, large-scale batch culture of hPSC-derived nociceptors (e.g., 1 x 10 cells in a batch of eighteen 15 cm dishes)7 ~1×10 8 cells (approx. 5.5 x 10 7 The present inventors have proposed a method for generating nociceptor cells, which is believed to be successful. These methods are contemplated to provide hPSC-derived nociceptor cells for use in testing compounds for use in basic biology studies and for drug discovery applicable to medical applications in humans and animals. In particular, the inventors contemplate using the compositions and methods of the present invention for the treatment of acute and chronic pain relief in humans and animals.

[0363] In particular, exemplary nociceptor cells, e.g., peptidergic nociceptor cells, are cultured at 7×10 7 ~7×10 8 (70% efficiency of nociceptor cell collection is contemplated). 8 ~1×10 9 Large-scale batch culture is contemplated, in which hPSC cells are grown in batch embryoid body culture.Exemplary nociceptor cells will express the genes (i.e., mRNA and protein) that identify nociceptor cells, such as TAC1, VGLUT2 and SLC15A3.Exemplary nociceptor cells will express the identification markers, such as ISL1, BRN3A, RET, RUNX1, substance P, CGRP, etc.

[0364] In summary, the inventors contemplate using the compositions and methods of the present invention to provide a novel platform in the basic biology and drug discovery for the testing and treatment of conditions associated with nociceptor cells, particularly pain, in humans and animals.

[0365] [Table 5-1]

[0366] [Table 5-2]

[0367] [Table 5-3]

[0368] The following references are incorporated herein in their entirety:

[0369] [ka]

[0370] Example XIII Melanocytes are derived from human pluripotent stem cells: LSB-melanocytes (LSB-Mel).

[0371] The following describes exemplary compositions and methods for providing melanocytes for use in relevant disease modeling.

[0372] We generated a SOX10::GFP bacterial artificial chromosome (BAC) human embryonic stem cell (hESC) reporter system, which allowed us to observe neural crest cell induction in vitro as this cell line responded to contact with small molecules. Sox10 is the most robust early marker of multipotent neural crest stem cells and was also found to be expressed in some neural crest derivatives, including melanocyte precursors. We used this reporter system to prospectively identify and isolate neural crest populations in the development of a directed differentiation scheme to produce melanocyte cultures with higher purity and numbers than previously achieved with maturation schemes (Figure 14, LSB-C).

[0373] In a dual SMAD inhibition protocol (Chambers et al., Nat. Biotech. (2009), incorporated herein by reference), human pluripotent stem cells (hPSCs) treated with two small molecules that inhibit SMAD signaling efficiently produced CNS neural tissue. Additionally, when hESCs were plated at lower densities, low levels of spontaneous neural crest cell induction were observed (e.g., approximately 3% Soxl0::GFP+ neural crest cells were observed). However, for use in research and medical testing, more neural crest cells were needed. Furthermore, for melanocyte studies, a purer population with a larger number of cells was needed, which low levels of spontaneous differentiation did not provide.

[0374] During the development of the present invention, the inventors discovered ways to optimize the dual SMAD inhibition protocol for neural crest induction to produce a highly pure product of melanocyte precursors, maturing melanocytes, and mature melanocytes.

[0375] Specifically, the following time series of culture conditions were developed to produce melanocytes of the present invention: on days 0 and 1, LDN and SB (using the same concentration range as LDN and SB in the 3i-inclusive methods); on day 2, LDN, SB, and CHIR (using the same concentration range as LDN, SB, and CHIR in the 3i-inclusive methods described herein); in one embodiment, on day 3, SB, and CHIR (using the same concentration range as SB and CHIR in the 3i-inclusive methods described herein); and in another embodiment, on day 3, LDN, SB, and CHIR (using the same concentration range as LDN, SB, and CHIR in the 3i-inclusive methods described herein); and on days 4 and 5, CHIR. (using the same concentration range as CHIR in the 3i-containing methods described herein); from day 6 to day 11, CHIR, BMP4, and EDN3 (using the same concentration range as CHIR in the 3i-containing methods described herein, see concentration ranges below for BMP4 and EDN3) were fed. Cells were passaged on day 11 and fed with MEL medium (containing CHIR) for up to 8 weeks.

[0376] The MEL medium was enriched for melanocytes, such that by 8 weeks the cell cultures exhibited up to 100% pure populations. Thus, this LSB-MEL method / protocol had a high efficiency of melanocyte production. The inventors also discovered that linoleic acid is at least one necessary component in the MEL medium during melanocyte development (see Figure 16).

[0377] During melanocyte development, multiple precursor stages are recognized in the following order: neural crest stem cells, embryonic glial-melanoblast stem cells, adult melanocyte stem cells, and melanocytes. See exemplary schematic diagram in Figure 13.

[0378] Figure 13. Specification and isolation of melanocyte precursors / melanoblasts.

[0379] The LSB-C protocol on day 11 supported the induction of melanocyte precursors co-expressing Sox10::GFP and MITF (A, right panel). A single MITF positive population was also observed (A, left panel). c-Kit was identified as a potential marker of melanocyte precursors. A low percentage of Sox10::GFP and c-kit co-expressing cells was observed after LSB-C differentiation (B, orange population). qRT-PCR analysis confirmed enrichment of the melanocyte markers MITFM (basic helix-loop-helix leucine zipper protein) and Dct (dopachrome tautomerase (dopachrome delta isomerase, tyrosine-linked protein 2)) in the double-positive population (C). Treatment with BMP4 and EDN3 ("LSB-Mel") enhanced the induction of a Sox10::GFP and c-kit double-positive putative melanocyte precursor population (D). Sox10::GFP, c-kit double-positive cells isolated after LSB-Mel treatment showed significantly higher levels of the melanocyte markers MITFM and Dct (E). Error bars indicate s.e.m. * p<0.05.

[0380] Figure 14 Proliferation and maturation of melanocyte precursors.

[0381] Summary of differentiation conditions (A). After differentiation in LSB-C (LSB-Mel) conditions with BMP4 and EDN3, cells were sorted and replated on day 11. Post-sorted (PS) cells were maintained in maturation medium containing c-kit ligand (SCF), endothelin 3 (EDN3), fibroblast growth factor (FGF), and Wnt activators. Pigment cells identified by brightfield microscopy on day 6 of PS were positive for the melanocyte marker MITF but appeared to down-regulate the Sox10::GFP reporter (B). All populations, except for the Sox10::GFP, c-kit double-negative population, eventually gave rise to MITF-expressing cells and macroscopically visible pigment clusters, although at different rates (C). Treatment with BMP4 and cAMP enhanced differentiation into pigment cells exhibiting the spindle-like morphology characteristic of melanocytes (D).

[0382] Figure 15. Characterization of mature melanocytes.

[0383] A pure population of mature melanocytes derived with the LSB-Mel protocol maintains expression of common melanocyte markers, including MITF, Sox10, Tyrp1 (tyrosinase-related protein 1), and HMB45, after more than 8 weeks in culture (A). Melanocytes retain their darkly pigmented phenotype over several weeks of passage (B). 1 × 10 cells were used to assess pigmentation levels. 6 Cells were pelleted and photographed. Electron microscopic ultrastructural characterization of mature melanocytes (C, D). The presence of numerous darkly pigmented melanosomes in the cytoplasm of LSB-Mel-derived melanocytes was observed by TEM (C). Note the presence and progressive deposition of melanin pigment as melanosome vesicles mature from stage I to stage IV (D).

[0384] Therefore, we demonstrated that the dual SMAD inhibition protocol, LSB, rapidly and efficiently generates neural crest populations expressing Soxl0::GFP from human embryonic stem cells. This modified protocol supported the induction of low levels of melanocyte precursors, which were prospectively identified and isolated by c-kit expression. The induction of these cells was further enhanced through treatment with BMP4 and EDN3. The melanocyte precursors then matured to a pigmented state after further in vitro culture in the presence of BMP4 and cAMP.

[0385]

number

[0386] A concentration range of 10 ng / ml to 100 ng / ml (25 ng / ml in one embodiment) is used for BMP4 from R&D, and 25 to 300 nM (100 nM in one embodiment) for EDN from American Peptide Company.

[0387] Figure 16 shows an exemplary LSB-MEL medium formulation that requires linoleic acid for melanocyte proliferation. Media components shown above the micrograph are those excluded from the formulation; Ph = phase contrast; BF = bright field. An exemplary schematic shows melanocyte precursor markers used to identify the cells of the invention.

[0388] Thus, the inventors have discovered and developed a rapid and well-defined protocol for the induction of neural crest cells in vitro. Furthermore, the inventors have used this rapid and well-defined protocol for the induction of neural crest cells in vitro to develop compositions and methods for the directed differentiation of these cells into melanocytes. These melanocytes are unique in their capacity for long-term culture and sustained production of eumelanin.

[0389] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are obvious to those skilled in the art of cell biology, neurobiology, cancer cell biology, molecular biology, biochemistry, chemistry, organic synthesis, or related fields are intended to be within the scope of the following claims.

Claims

1. 1. A method for inducing directed differentiation of stem cells into neural crest intermediate cells that express SOX10, said method comprising: a) inducing inhibition of SMAD signaling in a cell culture comprising stem cells for up to four days, wherein inducing inhibition of SMAD signaling comprises contacting the cells with an inhibitor of TGFβ signaling and an inhibitor of BMP signaling; b) inducing activation of Wingless (Wnt) signaling in the cell culture, inducing inhibition of FGF receptor family signaling in the cell culture, and inducing inhibition of Notch signaling in the cell culture for up to 192 hours, wherein inducing activation of Wnt signaling comprises contacting the cells with an inhibitor of glycogen synthase kinase 3β (GSK3β). Including, wherein step a) is carried out before step b), wherein step b) is performed within 4 days of the initial induction of said inhibition of SMAD signaling.

2. 2. The method of claim 1, wherein the inhibitor of TGFβ signaling is SB431542.

3. 2. The method of claim 1, wherein the inhibitor of TGFβ signaling comprises SB431542.

4. The method of any one of claims 1 to 3, wherein the inhibitor of BMP signaling is selected from LDN193189 and noggin.

5. The method of any one of claims 1 to 3, wherein the inhibitor of BMP signaling comprises LDN193189.

6. The method of any one of claims 1 to 5, wherein the inhibitor of GSK3β is CHIR99021.

7. 6. The method of any one of claims 1 to 5, wherein the inhibitor of GSK3β comprises CHIR99021.

8. The method of any one of claims 1 to 7, wherein the initial induction of activation of Wnt signaling is 2 days after the initial induction of inhibition of SMAD signaling.

9. The method according to any one of claims 1 to 8, wherein the FGF receptor family comprises a VEGF receptor, an FGF receptor, and a PDGF tyrosine kinase receptor.

10. The method of any one of claims 1 to 9, wherein inducing the inhibition of FGF receptor family signaling comprises contacting the cell with an inhibitor of FGF receptor family signaling.

11. The method of claim 10, wherein the inhibitor of FGF receptor family signaling is SU5402.

12. The method of claim 10, wherein the inhibitor of FGF receptor family signaling comprises SU5402.

13. The method of any one of claims 1 to 12, wherein inducing inhibition of Notch signalling comprises contacting the cell with an inhibitor of Notch signalling.

14. The method of claim 13, wherein the inhibitor of Notch signaling is selected from DAPT and DAP-BpB.

15. The method of claim 13, wherein the inhibitor of Notch signaling comprises DAPT or DAP-BpB.

16. The method of any one of claims 1 to 15, wherein the neural crest intermediate cells further express at least one marker selected from BRN3A and ISL1.

17. The method of claim 16 , wherein the marker is a protein or a nucleic acid.

18. The method according to any one of claims 1 to 17, wherein the stem cells are pluripotent stem cells.

19. The method of claim 18, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

20. 20. The method of claim 18 or 19, wherein the stem cells are human stem cells.

21. The method of any one of claims 1 to 20, wherein the inhibition of SMAD signaling in the cell culture is induced for 2 days.

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