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US20130108669A1 - Dopaminergic neurons differentiated from pluripotent stem cells and uses of thereof - Google Patents

Dopaminergic neurons differentiated from pluripotent stem cells and uses of thereof Download PDF

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US20130108669A1
US20130108669A1 US13/640,750 US201113640750A US2013108669A1 US 20130108669 A1 US20130108669 A1 US 20130108669A1 US 201113640750 A US201113640750 A US 201113640750A US 2013108669 A1 US2013108669 A1 US 2013108669A1
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Oliver Cooper
Ole Isacson
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Mclean Hospital Corp
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    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • This invention relates to the field of neural cells. Specifically, the invention provides methods for inducing pluripotent cells to differentiate into neuronal phenotypes.
  • Parkinson's disease is a progressive neurodegenerative disease characterized clinically by bradykinesia, rigidity, and resting tremor.
  • the motor abnormalities are associated with a specific loss of dopaminergic neurons in the substantia nigra pars compacta (SN) and depletion of striatal dopamine (DA) levels. While the loss of striatal DA correlates with the severity of clinical disability, clinical manifestations of PD are not apparent until about 80-85% of SN neurons have degenerated and striatal DA levels are depleted by about 60-80%.
  • DA neurons in the ventral midbrain consist of two main groups: the A9 group in the SN, and the A10 group in the medial and ventral tegmentum.
  • Each of these cell groups project to different anatomical structures and is involved in distinct functions.
  • A9 cells mainly project to the dorsolateral striatum, and are involved in the control of motor functions, whereas A10 cells provide connections to the ventromedial striatum, limbic and cortical regions, and are involved in reward and emotional behavior.
  • these groups of DA cells exhibit differences in neurochemistry and electrophysiological properties, illustrating functional differences despite similar neurotransmitter identity.
  • A9 and A10 cells are also reflected in their specific responses to neurodegeneration in PD.
  • Postmortem analyses in human PD brains demonstrate a selective cell loss of the A9 group with a survival rate of about 10% whereas the A10 group is largely spared with a survival rate of about 60%. This indicates that A9 cells are more vulnerable to intrinsic and/or extrinsic factors causing degeneration in PD.
  • three regional gradients of neurodegeneration in the dorso-ventral/rostro-caudal/medio-lateral axis have been reported in PD.
  • Caudally and laterally located ventral DA cells within A9 subgroups are the most vulnerable cells in PD.
  • the medial and rostral part of DA cell subgroups within A10 cells i.e. rostral linear nucleus, RLi
  • the medial and rostral part of DA cell subgroups within A10 cells are the least affected (5-25% cell loss).
  • Cell transplantation therapies have been used to treat neurodegenerative disease, including Parkinson's disease, with moderate success (e.g., Bjorklund et al., Nat. Neurosci., 3:537-544, 2000). However, wide-spread application of cell-based therapies will depend upon the availability of sufficient amounts of neuronal cells.
  • the present invention is based on the discovery, isolation, and characterization of specific neuronal cell populations that are derived in vitro from pluripotent cells, including embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells.
  • the inventive cells are neurons that have the phenotype of dopaminergic neurons and are capable of structurally and functionally integrating into the host brain following transplantation. Accordingly, these cells are useful in cell replacement/transplantation therapies, including therapies designed to treat Parkinson's disease and other conditions caused by a loss of dopaminergic neurons.
  • the present invention provides a substantially homogenous population of cells derived from pluripotent stem cells contacted with an effective amount of retinoic acid, human sonic hedgehog (SHH) protein, and FGF8A protein, wherein the cells have the phenotype of SN-A9 dopaminergic neurons.
  • the pluripotent stem cells are further contacted with a WNT1 protein.
  • the cells are characterized as having a FOXA2 + phenotype, a ⁇ -tubulin + phenotype, and/or expressing tyrosine hydroxylase.
  • the pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells.
  • the induced pluripotent stem cells are Parkinson's disease (PD) patient-specific induced pluripotent stem cells.
  • PD Parkinson's disease
  • the present invention provides a therapeutic composition
  • a therapeutic composition comprising a substantially homogenous population of cells derived from pluripotent stem cells contacted with an effective amount of retinoic acid, human sonic hedgehog (SHH) protein, and FGF8A protein, wherein the cells have the phenotype of SN-A9 dopaminergic neurons, wherein the cell population is in an amount sufficient to treat a disease or condition.
  • the pluripotent stem cells are further contacted with a WNT1 protein.
  • the population of cells is suspended in a physiologically acceptable carrier.
  • the physiologically acceptable carrier is artificial cerebrospinal fluid.
  • the population of cells is encapsulated.
  • the population of cells is contained within an inert biomatrix.
  • the present invention provides a method for treating a disease in a subject, comprising administering to the subject a therapeutic composition comprising a substantially homogenous population of cells derived from pluripotent stem cells contacted with an effective amount of retinoic acid, human sonic hedgehog (SHH) protein, and FGF8A protein, wherein the cells have the phenotype of SN-A9 dopaminergic neurons, wherein the cell population is in an amount sufficient to treat the disease or condition.
  • the pluripotent stem cells are further contacted with a WNT1 protein.
  • the disease is a neurodegenerative disease.
  • the neurodegenerative disease is Parkinson's disease.
  • the administering comprises transplanting the dopaminergic neurons into the brain of the subject.
  • the therapeutic composition is injected into the striatum of the subject.
  • the therapeutic composition is injected into the midbrain of the subject.
  • the present invention provides a method for generating SN-A9 dopaminergic neurons, the method comprising: (a) contacting pluripotent stem cells with an effective amount of retinoic acid to form neural progenitor cells; and (b) contacting the neural progenitor cells with an effective amount of human sonic hedgehog (SHH) protein, and FGF8A protein to stimulate the neural progenitor cells to differentiate into SN-A9 dopaminergic neurons.
  • the pluripotent stem cells are further contacted with a WNT1 protein.
  • the pluripotent stem cells may be contacted with SHH protein, WNT1 protein, and the FGF8A protein either simultaneously with the retinoic acid or after contact with the retinoic acid.
  • the pluripotent stem cells are cultured in the presence of retinoic acid alone for a period of time and the SHH protein, WNT1 protein, and the FGF8A protein are applied to the cells later without removal of the retinoic acid.
  • Combinations of the foregoing are also contemplated (e.g., in which one or two of the factors are added simultaneously with the retinoic acid and other factor(s) are added later).
  • the pluripotent stem cells may be cultured either in the presence or the absence of feeder cells.
  • the pluripotent stem cells are cultured in the absence of feeder cells.
  • the feeder cells may comprise a monolayer on a solid substrate (e.g., a culture dish).
  • the human SHH protein is activated human SHH protein.
  • the activated human SHH protein is C24II SHH protein.
  • the effective amount of human SHH protein is an amount sufficient to provide a final concentration in the culture media from about 100 to about 1000 ng/ml.
  • the effective amount of human SHH protein is an amount sufficient to provide a final concentration in the culture media of about 500 ng/ml.
  • the effective amount of WNT1 protein is an amount sufficient to provide a final concentration in the culture media of about 100 ng/ml.
  • the WNT1 protein (e.g., human WNT1 protein) is an amount sufficient to provide a final concentration in the culture media from about 100 to about 1000 ng/ml. In a particular embodiment, the effective amount of WNT1 protein is an amount sufficient to provide a final concentration in the culture media of about 500 ng/ml. In yet another particular embodiment, the effective amount of WNT1 protein is an amount sufficient to provide a final concentration in the culture media of about 100 ng/ml.
  • the effective amount of retinoic acid is an amount sufficient to provide a final concentration in the culture media from about 10 ⁇ 9 M to about 10 ⁇ 7 M. In a particular embodiment, the effective amount of retinoic acid is an amount sufficient to provide a final concentration in the culture media of about 10 ⁇ 8 M.
  • the FGF8A is a recombinant human FGF8A protein.
  • the effective amount of human FGF8A protein is an amount sufficient to provide a final concentration in the culture media from about 10 to about 1000 ng/ml. In a particular embodiment, the effective amount of human FGF8A protein is an amount sufficient to provide a final concentration in the culture media of about 100 ng/ml.
  • the present invention provides a kit comprising the agents: (i) retinoic acid, (ii) human sonic hedgehog (SHH) protein, (iii) WNT1 protein, and (iv) FGF8A protein and instructions for contacting the agents to pluripotent stem cells in order to differentiate SN-A9 dopaminergic neurons.
  • the “administration” of an agent to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intracranially, intracerebroventricular, intravenously, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another.
  • differentiation refers to the process whereby an unspecialized stem cell (e.g., PS cells and iPS cells) acquires phenotypic features of a specialized cell or specific cell type, e.g., a neural cell. Differentiation refers to the restriction of the potential of a cell to self-renew and is generally associated with a change in the functional capacity of the cell. Differentiation of a stem cell may be determined by methods well known in the art, including analysis for cell markers or morphological features associated with cells of a defined differentiated state.
  • the term “dopaminergic neuron” refers to a specialized cell that at least partially adopts a neuronal morphology in culture (e.g., develops neurites) and expresses tyrosine hydroxylase (TH).
  • the dopaminergic neuron expresses one or more of neuron-specific enolase (NSE), 1-aromatic amino acid decarboxylase, vesicular monoamine transporter 2, dopamine transporter, Nurr-1, and dopamine-2 receptor (D 2 Receptor).
  • an effective amount refers to a quantity sufficient to achieve a desired physiological and/or therapeutic effect.
  • an effective amount of a substance is any amount of the substance which induces pluripotent cells to differentiate into neurons.
  • Methods of determining an “effective amount” are well known to those skilled in the art and typically involve range-finding studies to assess dose-response relationships between the substance of interest and the desired effect. Such studies may be performed in vitro or in vivo, including in a single individual by titrating the dose of the substance(s) upward until the effect is achieved.
  • isolated when referring to a material, is meant a material that is partially or completely removed from the other material which naturally accompanies it. Therefore, in reference to a cell, the term “isolated” refers to a cell substantially free from other cells accompanying it in vivo.
  • isolated in relation to nucleic acids or polypeptides means that, for example, the nucleic acids or the polypeptides are substantially free from intracellular macromolecules with which it is normally found. Any nucleic acid or polypeptide made by synthetic means (e.g., chemical synthesis and substances recombinant DNA techniques, etc.) is defined as isolated.
  • embryonic stem cells refers to cells derived from the inner cell mass of blastocysts, blastomeres, or morulae that have been serially passaged as cell lines while maintaining an undifferentiated state (e.g. express TERT, OCT4, and/or TRA antigens).
  • the ES cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means to generate hES cells with hemizygosity or homozygosity in the MHC region.
  • pluripotent stem cell refers to a cell capable of self-replication and differentiation into cells of all three germ layers (i.e., ectoderm, mesoderm, and endoderm). Pluripotent stem cells may be, but are not limited to, ESCs and artificially-produced stem cells having characteristics of ESCs but which are not derived from an embryo (e.g., pluripotent stem cells derived from neural progenitor cells and iPS cells). In vitro self-replication, under appropriate conditions, occurs for virtually indefinite period of time and the daughter cells retain the undifferentiated (pluripotent) characteristics of the parent cells.
  • iPS cell refers to pluripotent cells derived from mesenchymal cells through the overexpression of one or more transcription factors.
  • iPS cells are derived from fibroblasts by the overexpression of Oct4, Sox2, c-Myc and Klf4 according to the methods described in Takahashi et al. ( Cell, 126:663-676, 2006), for example.
  • Other methods for producing iPS cells are described, for example, in Takahashi et al. ( Cell, 131:861-872, 2007) and Nakagawa et al. ( Nat. Biotechnol., 26:101-106, 2008).
  • the iPS cells are capable of self-renewal and subsequent differentiation into more than one specialized cell type or cell lineage under appropriate growth conditions either in vitro or in vivo.
  • neural stem cells refers to a subset of pluripotent cells which have partially differentiated along a neural cell pathway and express some neural markers including, for example, nestin. Neural stem cells may differentiate into neurons or glial cells (e.g., astrocytes and oligodendrocytes). Neural stem cells include neural progenitor cells and may be used interchangeably.
  • neural progenitor cells refer to cultured cells derived from pluripotent stem cells (e.g., ES cells and iPS cells) which express FOXA2 and low levels of ⁇ -tubulin, but not tyrosine hydroxylase (i.e., having a FOXA2 + / ⁇ -tubulin LO /TH ⁇ phenotype). These neural progenitor cells have the capacity to differentiate into a variety of neuronal subtypes; particularly a variety of dopaminergic neuronal subtypes, upon culturing the appropriate factors, such as those described herein.
  • pluripotent stem cells e.g., ES cells and iPS cells
  • tyrosine hydroxylase i.e., having a FOXA2 + / ⁇ -tubulin LO /TH ⁇ phenotype.
  • FGF8a refers to the splice variant of the fibroblast growth factor 8 gene product which gives rise to a protein with a predicted molecular mass of 21 kDa.
  • the FGF8a protein is described in Matilla & Harkonnen 2007, Olsen et al., 2006, Ghosh et al 1996, Manila et al., 2001, Valve et al., 2000.
  • the amino acid sequence is provided at GenBank Accession No.: NP — 149355.
  • human sonic hedgehog protein or “(hSHH)” refers to the protein encoded by the human sonic hedgehog gene (GenBank Accession No.: NP — 000184) and having the amino acid sequence proved at GenBank Accession No.: NM — 000193.2, and biologically active variants and fragments thereof.
  • the human sonic hedgehog protein is described in Carpenter et al., 1998, Ingham & McMahon 2001, Mullor et al., 2002, Perrimon 1995 and Taylor et al., 2001.
  • WNT1 refers to the secreted signaling protein encoded by the human WNT1 proto-oncogene and involved in regulation of cell fate and patterning during embryogenesis. WNT1 is described in, for example, Parkin et al., Activity of Wnt-1 as a transmembrane protein, Genes & Development, Vol. 7, 2181-2193, 1993.
  • the term “pharmaceutically acceptable carrier” refers to a carrier that is physiologically acceptable to the treated subject while retaining the therapeutic properties of the compound with which it is administered.
  • exemplary pharmaceutically acceptable carriers include physiological saline and artificial cerebrospinal fluid.
  • Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences , (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.
  • the terms “treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • a subject is successfully “treated” for PD if, after receiving a therapeutic amount of the neural cells according to the methods described herein, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of PD, such as, e.g., bradykinesia, rigidity, and resting tremor.
  • the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
  • the term “subject” refers to any animal, including humans. In some embodiments, the “subject” is a human patient that is to be treated for a disease or condition.
  • substantially homogenous cell population refers to a population or sample of cells which contain a majority (i.e., >50%) of cells having the desired phenotype (i.e., trait(s) of interest).
  • substantially homogenous populations contain at least 60%, at least 70%, at least 80%, at least 90%, or more of the cells having the desired phenotype.
  • FIG. 1 presents data showing early exposure to mShh-N and 10 ⁇ 8 M retinoic acid promotes midbrain regionalization of human ES cell-derived neural progenitor cells.
  • Human ES cells were differentiated by 4 concentrations of RA with mouse Shh for 12 days before exposure to BDNF, ascorbic acid, mouse Shh and Fgf8b for 8 days.
  • B Quantitative PCR determined that 10 ⁇ 8 M retinoic acid with mouse Shh induced a midbrain-like transcriptional profile by significantly increasing the levels of EN1 without changing the levels of OTX2, HOXB1 and PAX2 (*p ⁇ 0.05 ANOVA).
  • FIG. 2 presents data showing forced expression of FoxA2 promotes an A9/A10 DA neuron-like transcriptional profile in 10 ⁇ 8 M RA treated human ES cell-derived neurons.
  • A At Day 28 of differentiation, human ES cell-derived neural progenitor cells were transduced with either a lentivirus overexpressing FoxA2-IRES-zsGreen or solely zsGreen.
  • B-E Immunocytochemistry at Day 42 confirmed transgene expression of FOXA2 and zsGreen, in tyrosine hydroxylase expressing (TH) DA neurons.
  • Quantitative PCR confirmed a significant upregulation of the A9/A10 DA neuron markers SHH, LMX1A and EN1 in response to FoxA2 overexpression (*p ⁇ 0.05 ANOVA).
  • FIG. 3 presents data showing that recombinant activated human SHH promotes the dose-dependent differentiation of FOXA2 expressing neural progenitor cells from 10 ⁇ 8 M RA treated human ES cells.
  • A Human ES cells were exposed for 12 days to an activated form of human SHH and a further 5 days to neural expansion medium.
  • B-E Immunocytochemistry at DIV19 revealed few FOXA2 + / ⁇ -tubulin Low neural progenitor cells that had differentiated from human ES cells after exposure to mouse Fgf8b with (B) or without mouse Shh (E).
  • FIG. 4 presents data showing the phenotypic characterization of FOXA2 expressing neural progenitor cells from RA-treated human ES/iPS cells.
  • A-D Immunocytochemistry at DIV19 revealed that human ES cell-derived neural progenitor cells formed rosette-like structures and coexpressed OTX2 and luminal WNT1 but not FOXA2, ⁇ FP or SOX17 in response to Fgf8b with (A, E) or without mouse Shh (D, H).
  • isolated neural rosette-like structures were observed in cultures exposed to activated human SHH and FGF8A (B, F).
  • FIG. 5 presents data showing the expansion of human ES cell-derived FOXA2 + neural progenitor cells by activated human SHH and human FGF8A for 28 days leads to the generation of FOXA2 expressing DA neurons.
  • A Human ES cell-derived neural progenitor cells were grown in medium supplemented with Shh and FGF8 splice variants before neuronal differentiation.
  • B-E Immunocytochemistry at Day 49 revealed many ⁇ -tubulin + neurons and TH + neurons but very few FOXA2 + cells differentiated from human ES cells after exposure to Fgf8b with (B) or without mouse Shh (E).
  • FIG. 6 presents data showing the phenotypic characterization of dopaminergic neurons generated by activated human SHH and FGF8A from RA-treated human ES/iPS cells.
  • PDC 3F -1 Human PD-iPS cell lines (PDC 3F -1) were competent to generate FOXA2 + dopaminergic neurons (TH; ⁇ -tubulin; arrowheads).
  • B-E In human ES cell-derived cultures at DIV49, clusters of neural progenitor cells were observed. These neural progenitor cells strongly expressed CORIN (B) and FOXA2 (C), E) while adjacent cells coexpressed FOXA2 (C), TH (D) and low levels of CORIN (B, E).
  • FIG. 7 presents data showing that the differentiation of human FOXA2 expressing DA neurons from ES cells requires recombinant WNT1 but not Noggin.
  • A is a diagram illustrating the first 14 days of the protocol;
  • B)-(E) are photographs showing cells after exposure to SHH-C24II, FGF8A, and RA with (B) or without (C) Noggin; and
  • F is a bar graph showing cell counts after treatment with WNT1 with or without Noggin.
  • FIG. 8 presents data showing the feeder cell-free differentiation of stem cell-derived human FOXA2 expressing DA neurons.
  • A is a diagram illustrating the protocol;
  • B)-(D) are photographs showing different magnifications of large clusters of cells that exhibited prominent radial outgrowth after the administration of the protocol.
  • the present inventions are based, in part, on the discovery that when PS cells are exposed to defined amounts of retinoic acid (RA), for specific periods of time and under specific differentiation protocols, the PS cells develop into neural progenitor cells and/or dopaminergic neurons at a higher rate than in the absence of RA. Additionally, the invention is based on the discovery that the PS cells treated with RA further establish a sizeable FOXA2 + neural progenitor cell population in vitro following treatment with an activated form of human sonic hedgehog protein (SHH). Likewise, a similar FOXA2 + neural progenitor cell population can be generated from PD or healthy subject-specific iPS cells using these growth conditions.
  • RA retinoic acid
  • the present inventors also discovered that early exposure to FGF8A, rather than Fgf8b, results in robust differentiation of the FOXA2 + floor plate-like neural progenitor cells into FOXA2 + DA neurons, yielding an about 100,000-fold increase in the number of FOXA2 + DA neurons which resemble SN A9 and/or SN A10 dopaminergic neurons. Further, it has been discovered that human ES cell differentiation into VM DA neurons requires exogenous WNT1 signaling.
  • ES cells derived from the inner cell mass of preimplantation embryos, have been recognized as the most pluripotent stem cell population and are therefore a suitable PS cell for use with the methods and compositions of this invention. These cells are capable of unlimited proliferation ex vivo, while maintaining the capacity for differentiation into a wide variety of somatic and extra-embryonic tissues.
  • ES cells can be male (XY) or female (XX).
  • Stem cells can be derived from any mammal including, but not limited to, mouse, human, and primates. Following acquisition of stem cells, these cells may be used directly in the methods of the invention; for example, umbilical cord blood cells may be acquired in sufficient quantity to use directly for therapeutic purposes.
  • stem cells may first be expanded in order to increase the number of available cells; see, for example, U.S. Pat. No. 6,338,942.
  • Methods for preparing mouse, human, or primate stem cells are known in the art and are described, for example, in Nagy et al., Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (2002); Thomson et al., Science, 282:1145-1147 (1998), Marshall et al., Methods Mol. Biol., 158:11-18 (2001); Thomson et al., Trends Biotechnol., 18:53-57 (2000); Jones et al., Semin. Reprod. Med., 18:219-223 (2000); Voss et al., Exp. Cell Res., 230:45-49 (1997); and Odorico et al., Stem Cells, 19:193-204 (2001).
  • ES cells can be directly derived from the blastocyst or any other early stage of development, or can be a “cloned” stem cell line derived from somatic nuclear transfer and other similar procedures.
  • the inner cell mass of a preimplantation blastocyst is removed from the trophectoderm that surrounds it.
  • the small plastic culture dishes used to grow the cells contain growth medium supplemented with fetal calf serum, and are sometimes coated with a “feeder” layer of nondividing cells.
  • the feeder cells may be mouse embryonic fibroblast (MEF) cells that have been chemically inactivated so they will not divide.
  • MEF mouse embryonic fibroblast
  • cytokine leukemia inhibitory factor LIF
  • LIF cytokine leukemia inhibitory factor
  • Limiting dilution methods can be used to generate a clonal ES cell line.
  • Reagents needed for the culture of stem cells are commercially available, for example, from Invitrogen, Stem Cell Technologies, R&D Systems, and Sigma Aldrich, and are described, for example, in U.S. Patent Publication Nos. 2004/0235159 and 2005/0037492.
  • iPS cells are also a suitable cell for the methods of the invention.
  • iPS cell technology can provide isogenic cells for cell therapy to limit the patient's immune response.
  • the brain is relatively immunoprivileged but activated microglia can compromise the synaptic function of transplanted neurons (Soderstrom et al., 2008).
  • iPS cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell. Yamanaka et al. transfected mouse fibroblasts with four genes (Oct4, Sox2, c-Myc, Klf4) to obtain iPS cells in 2006. Subsequently, iPS cells were created from human adult somatic cells. (Takahashi et al. Cell, 131:861-872 (2007); Yu et al. Science, 318:1917-1920, 2007).
  • the iPS cell can be a mammalian cell, for example a mouse, human, rat, bovine, ovine, horse, hamster, dog, guinea pig, or non-human primate cell.
  • reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells.
  • iPS cells are indistinguishable from ES cells in morphology, proliferation, gene expression, and teratoma formation.
  • Human iPS cells are also expandable and indistinguishable from human embryonic stem (ES) cells in morphology and proliferation. Furthermore, these cells can differentiate into cell types of the three germ layers in vitro and in teratomas.
  • ES embryonic stem
  • the mesenchymal cells useful for creating iPS cells may be obtained from any suitable source and may be any specific mesenchymal cell type. For example, if the ultimate goal is to generate therapeutic cells for transplantation into a patient, mesenchymal cells from that patient are desirably used to generate the iPS cells. Suitable mesenchymal cell types include fibroblasts (e.g., skin fibroblasts), hematopoietic cells, hepatocytes, smooth muscle cells, and endothelial cells. In suitable embodiments, the iPS cells used in the present methods are derived from a PD patient.
  • Methods for cell culturing, developing, and differentiating pluripotent stem cells may be carried out with reference to standard literature in the field. Suitable techniques are described by Wiles et al., Meth. Enzymol., 225:900, 1993; and in Embryonic Stem Cells (Turksen ed., Humana Press, 2002). Established protocols for generation, passaging and preservation of rodent and human pluripotent stem cells are described by, for example, Iannaccone et al. ( Dev. Biol., 163:288, 1994); Matsui et al. ( Cell, 70:841, 1992), Thomson et al., Science, 282:114, 1998; Shamblott et al., Proc. Natl.
  • PS cells include primary tissue and established lines that bear phenotypic characteristics of PS cells, and derivatives of such lines that still have the capacity of producing progeny of each of the three germ layers.
  • PS cell cultures are described as “undifferentiated” or “substantially undifferentiated” when a substantial proportion of stem cells and their derivatives in the population display morphological characteristics of undifferentiated cells, clearly distinguishing them from differentiated cells of embryo or adult origin.
  • Undifferentiated PS cells are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view with high nuclear/cytoplasmic ratios and prominent nucleoli.
  • Cultures that are substantially undifferentiated contain at least 20% undifferentiated PS cells, and may contain at least 40%, 60%, or 80% undifferentiated PS cells.
  • the methods provide contacting PS cells with retinoic acid (RA), an activated form of human SHH, WNT1, and FGF8A in amounts that are sufficient to direct the fate of PS cells towards dopaminergic neurons, including those preferably having a phenotype of SN-A9 and VTA-A10 DA neurons.
  • PS cells are differentiated in the presence of about 10 ⁇ 9 to 10 ⁇ 7 M RA (e.g., about 2 ⁇ 10 ⁇ 9 to about 5 ⁇ 10 ⁇ 8 M or about 5 ⁇ 10 ⁇ 9 to about 2 ⁇ 10 ⁇ 8 M).
  • PS cells are differentiated in the presence of about 10 ⁇ 8 M RA.
  • neuroectodermal differentiation in the presence of RA was allowed to proceed from 10-18 days. In one embodiment, neuroectodermal differentiation in the presence of RA was allowed to proceed 14 days, with the media changed every 2 days. After differentiation, neuroectodermal colonies were picked and replated for differentiation toward the DA neuron phenotype.
  • differentiation toward the DA neuron phenotype occurs by culturing the neuroectodermal colonies with an activated form of human SHH, WNT1, and FGF8A.
  • the cells are differentiated in the presence of human SHH, WNT1, and FGF8A for 18-35 days.
  • the cells are differentiated in the presence of human SHH, WNT1, and FGF8A for 28 days and then differentiated until Day 49 without SHH, WNT1, and FGF8A, but in the presence of one or more of (e.g., 2, 3, 4, or all 5 of) BDFN, AA, cAMP, GDNF, and TGF- ⁇ 3.
  • the human SHH is an activated form of human SHH.
  • the mature biologically active form of SHH molecule is produced by autocatalytic cleavage of its precursor protein and corresponds to the N-terminal domain of the precursor molecule corresponding generally to residues 24-197 of the full-length human SHH protein (Pepinsky et al, 1998; Taylor et al., 2001).
  • the effective amount of human SHH protein is an amount sufficient to provide a final concentration in the culture media from about 100 to about 1000 ng/ml. In a particular embodiment, the effective amount of human SHH protein is an amount sufficient to provide a final concentration in the culture media of about 500 ng/ml.
  • neural progenitor cells are contact with an activated SHH protein.
  • an activated SHH protein In some embodiments, a supra-potent form of the activated SHH protein may be used.
  • modified activated SHH proteins include, for example, activated SHH proteins having N-terminal modifications including, for example, N-terminal acyl amides and thiazolidines, and activated SHH proteins that have been mutagenized to increase biological activity (Taylor et al., 2001).
  • the modified activated SHH is the C24II SHH which is a 20 kDa protein produced from human cells consisting of 175 amino acid residues, including an N-terminal Ile-Ile sequence substituted for the naturally occurring chemically modified Cys residue (see, Taylor et al., 2001).
  • Other modified activated SHH proteins included, for example C24III, C24IIII, C23IIW, C24IW, C24F, C24I, C24FIF, C24W, C24I-G25I, and C24M (Taylor et al., 2001).
  • the amount used may be less than the amount of native activated SHH but sufficient to provide the same biological activity as the amounts of discussed above. Alternatively, greater amounts may be used in order to delivery higher levels of SHH biological activity to the cells.
  • differentiation toward the DA neuron phenotype occurs by culturing the neuroectodermal colonies with FGF8A.
  • FGF8A fibroblast growth factor 8 A.
  • FGF8 is a member of the fibroblast growth factor family that was originally discovered as a growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells. Splicing of mouse FGF8 mRNA generates eight secreted isoforms, designated a-h. Only FGF8a, b, e and f exist in humans.
  • FGF8 contains a 22 amino acid (aa) signal sequence, an N-terminal domain that varies according to the isoform (20 aa for FGF8a, which is the shortest), a 125 aa FGF domain and a 37 aa proline rich C-terminal sequence.
  • the FGF8A is recombinant human FGF8A.
  • the effective amount of human FGF8A protein is an amount sufficient to provide a final concentration in the culture media from about 10 to about 1000 ng/ml human FGF8A protein.
  • the effective amount of human FGF8A protein is an amount sufficient to provide a final concentration in the culture media of about 100 ng/ml.
  • differentiation toward the DA neuron phenotype occurs by culturing the neuroectodermal colonies with human WNT1 protein, either with or without Noggin, and without feeder cells (e.g., MS5 feeder cells).
  • WNT1 has been shown in developmental studies in the embryo to be required to generate appropriately patterned VM neural progenitor cells (Muhr et al., 1999; Nordstrom et al., 2002, 2006). It is also shown herein that neuronal differentiation of FOXA2 + neural progenitor cells without exogenous SHH antagonism can occur.
  • the WNT1 is recombinant human WNT1.
  • the effective amount of human WNT1 protein is an amount sufficient to provide a final concentration in the culture media from about 10 to about 1000 ng/ml human WNT1 protein. In a particular embodiment, the effective amount of human WNT1 protein is an amount sufficient to provide a final concentration in the culture media of about 100 ng/ml.
  • DA dopaminergic
  • SN-A9 specific midbrain
  • the invention provides methods for treating neurodegenerative diseases (e.g., Parkinson's Disease) in a patient by generating dopaminergic neurons, particularly dopaminergic neurons having an A9 phenotype, and transplanting these dopaminergic neurons into the brain of the patient.
  • the dopaminergic neurons are generated from neuronal progenitor cells by contacting those cells with an effective amount of retinoic acid, human sonic hedgehog (SHH) protein, WNT1 protein, and FGF8A protein.
  • the neuronal progenitor cells may be generated by contacting PS cells with RA and other differentiating factors under culture conditions described herein.
  • Transplantation can be allogeneic (between genetically different members of the same species), autologous (transplantation of an organism's own cells or tissues), syngeneic (between genetically identical members of the same species (e.g., identical twins)), or xenogeneic (between members of different species).
  • the DA neurons would be transplanted into the substantia nigra (particularly in or adjacent of the compact region), the ventral tegmental area (VTA), the caudate, the putamen, the nucleus accumbens, the subthalamic nucleus, or any combination thereof, of the brain to replace the DA neurons whose degeneration resulted in PD.
  • Transplantation into the substantia nigra, the caudate, or the putamen is performed because, although the cell bodies of A9 DA neurons are located in the substantia nigra, their axons extend into the forebrain structures where dopamine release occurs.
  • the DA neurons are transplanted into the VTA, the nucleus accumbens, or both regions of the brain.
  • cognitive and behavioral disturbance may be generated from DA loss and synaptic dysfunction in the caudate, cerebral cortex deep layers, nucleus accumbens, and substantia nigra regions of the brain.
  • ventral tegmental DA neuronal phenotype of A10 would be specifically transplanted to these regions to replace lost A10 DA functions.
  • transplantation of A10 DA neurons, or cells primed to differentiate into A10 DA neurons, to the caudate nucleus would be the most effective replacement.
  • Transplantation of the cells of the invention into the brain of the patient with a neurodegenerative disease results in replacement of lost, non-, or dysfunctional DA neurons.
  • the cells are introduced into a subject with a neurodegenerative disease in an amount suitable to replace the dysfunctional DA neurons such that there is an at least partial reduction or alleviation of at least one adverse effect or symptom of the disease.
  • the cells can be administered to a subject by any appropriate route that results in delivery of the cells to a desired location in the subject where at least a portion of the cells remain viable.
  • At least about 5%, preferably at least about 10%, more preferably at least about 20%, yet more preferably at least about 30%, still more preferably at least about 40%, and most preferably at least about 50% or more of the cells remain viable after administration into a subject.
  • the period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as a few weeks to months.
  • One transplantation method that can be used to deliver the cells to a subject is described by Bjorklund et al. ( Proc. Nat. Acad. Sci. USA, 99:2344-2349, 2002).
  • the cells of the invention can be inserted into a delivery device that facilitates introduction by injection or implantation of the cells into the subject.
  • the cells are injected into the target area as a cell suspension.
  • the DA neurons can be embedded in a solid or semisolid support matrix when contained in such a delivery device.
  • Cell transplantation therapies typically involve the intraparenchymal (e.g., intracerebral) grafting of the replacement cell populations into the lesioned region of the nervous system, or at a site adjacent to the site of injury. Most commonly, the therapeutic cells are delivered to a specific site by stereotaxic injection. Conventional techniques for grafting are described, for example, in Bjorklund et al. ( Neural Grafting in the Mammalian CNS , eds. Elsevier, pp. 169-178, 1985), Leksell et al. ( Acta Neurochir., 52:1-7, 1980) and Leksell et al. ( J. Neurosurg., 66:626-629, 1987).
  • intraparenchymal e.g., intracerebral
  • Conventional techniques for grafting are described, for example, in Bjorklund et al. ( Neural Grafting in the Mammalian CNS , eds. Elsevier, pp.
  • Identification and localization of the injection target regions will generally be done using a non-invasive brain imaging technique (e.g., MRI) prior to implantation (see, for example, Leksell et al., J. Neurol. Neurosurg. Psychiatry, 48:14-18, 1985).
  • MRI magnetic resonance imaging
  • administration of cells into selected regions of a patient's brain may be made by drilling a hole and piercing the dura to permit the needle of a microsyringe to be inserted.
  • the cells can be injected into the brain ventricles or intrathecally into a spinal cord region. It also is possible to effect multiple grafting concurrently, at several sites, using the same cell suspension, as well as mixtures of cells. Multiple graftings are particularly useful for administration of cells to larger brain structures such as the caudate and/or putamen.
  • the cells are prepared for implantation.
  • the cells are suspended in a physiologically compatible carrier, such as cell culture medium (e.g., Eagle's minimal essential media), phosphate buffered saline, or artificial cerebrospinal fluid (aCSF).
  • cell culture medium e.g., Eagle's minimal essential media
  • phosphate buffered saline e.g., phosphate buffered saline
  • aCSF artificial cerebrospinal fluid
  • the volume of cell suspension to be implanted will vary depending on the site of implantation, treatment goal, and cell density in the solution.
  • aCSF artificial cerebrospinal fluid
  • about 30-100 ⁇ l of cell suspension will be administered in each intra-nigral or intra-putamenal injection and each patient may receive a single or multiple injections into each of the left and right nigral or putaminal regions.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media.
  • the use of such carriers and diluents is well known in the art.
  • the solution is preferably sterile and fluid.
  • the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, or thimerosal.
  • Solutions of the invention can be prepared by incorporating the cells as described herein in a pharmaceutically acceptable carrier or diluent and, as required, other ingredients.
  • the cells are encapsulated within permeable membranes prior to implantation. Encapsulation provides a barrier to the host's immune system and inhibits graft rejection and inflammation. Several methods of cell encapsulation may be employed. In some instances, cells will be individually encapsulated. In other instances, many cells will be encapsulated within the same membrane. Several methods of cell encapsulation are well known in the art, such as described in European Patent Publication No. 301,777, or U.S. Pat. Nos. 4,353,888, 4,744,933, 4,749,620, 4,814,274, 5,084,350, and 5,089,272.
  • the isolated cells are mixed with sodium alginate and extruded into calcium chloride so as to form gel beads or droplets.
  • the gel beads are incubated with a high molecular weight (e.g., MW 60-500 kDa) concentration (0.03-0.1% w/v) polyamino acid (e.g., poly-L-lysine) to form a membrane.
  • a high molecular weight e.g., MW 60-500 kDa
  • polyamino acid e.g., poly-L-lysine
  • the interior of the formed capsule is re-liquefied using sodium citrate. This creates a single membrane around the cells that is highly permeable to relatively large molecules (MW ⁇ 200-400 kDa), but retains the cells inside.
  • the capsules are incubated in physiologically compatible carrier for several hours in order that the entrapped sodium alginate diffuses out and the capsules expand to an equilibrium state.
  • the resulting alginate-depleted capsules is reacted with a low molecular weight polyamino acid which reduces the membrane permeability (MW cut-off ⁇ 40-80 kDa).
  • the DA neurons Prior to introduction into a subject, the DA neurons can be modified to inhibit immunological rejection.
  • the methods of the invention can include alteration of immunogenic antigens on the surface of the cells prior to introduction into the subject. This step of altering one or more immunogenic antigens on the cells can be performed alone or in combination with administering to the subject an agent that inhibits T cell activity in the subject.
  • inhibition of rejection of the transplanted cells can be accomplished by administering to the subject an agent that inhibits T cell activity in the subject in the absence of prior alteration of an immunogenic antigen on the surface of the transplanted cells.
  • An agent that inhibits T cell activity is defined as an agent which results in removal (e.g., sequestration) or destruction of T cells within a subject or inhibits T cell functions within the subject.
  • T cells may still be present in the subject but are in a non-functional state, such that they are unable to proliferate or elicit or perform effector functions (e.g., cytokine production, cytotoxicity, etc.).
  • the agent that inhibits T cell activity may also inhibit the activity or maturation of immature T cells (e.g., thymocytes).
  • a suitable agent for use in inhibiting T cell activity in a recipient subject is an immunosuppressive drug that inhibits or interferes with normal immune function, e.g., cyclosporin A, FK506, or RS-61443.
  • Additional therapeutic agents that can be administered include steroids (e.g., glucocorticoids such as prednisolone, methyl prednisolone, and dexamethasone).
  • the human ES cell line H9 (WA-09, XX, approximate passage 35) and the human iPS cell lines A6 (XX), PDC 3F -1 (XY), PDB 1lox -17Puro-5 (XY), PDB 1low -21Puro-26 (XY) and PDB 1lox 21Puro-28 (XY) were cultured according to the guidelines established by the National Academy of Sciences.
  • Human ES/iPS cells were propagated as described (Sonntag et al., 2007). The differentiation of human ES/iPS cells was adapted from a published protocol (Sonntag et al., 2007).
  • Neuroectodermal differentiation was achieved using SRM for 11 days, followed by N2 medium (DMEM/F-12; Invitrogen Corporation, Carlsbad, Calif.) (N2-A; Stem Cell Technologies, Vancouver, BC, Canada) for 3 days supplemented with 600 ng/ml Noggin (R&D Systems, Inc., Minneapolis, Minn.), 100 ng/ml human WNT1 (PeproTech EC Ltd., London, UK), 100 ng/ml human FGF8A or mouse Fgf8b (R&D Systems) and all-trans retinoic acid (Sigma-Aldrich, St. Louis, Mo.) to the culture medium. Media were changed every 2 days. At day 14 of differentiation, total human neuroectodermal colonies were manually picked as described (Karki et al., 2006) and replated on polyornithine and laminin-coated culture dishes in N2 medium supplemented with growth factors.
  • N2 medium DMEM/F-12; Invitrogen Corporation,
  • Neural progenitor cells were differentiated toward the DA neuron phenotype with mouse sonic hedgehog (Shh-N), human activated sonic hedgehog (C24II SHH-N), mouse fibroblast growth factor 8b (FGF8b), human FGF8A (all from R&D Systems, Inc.), 20 ng/ml brain derived neurotrophic factor (BDNF) (PeproTech EC Ltd), 1 ng/ml transforming growth factor type ⁇ 3 (TGF-(33) (Calbiochem, San Diego, Calif.), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF), 0.5 mM dibutyryl cAMP, and 0.2 mM ascorbic acid (AA) (all from Sigma-Aldrich).
  • BDNF brain derived neurotrophic factor
  • TGF-(33) transforming growth factor type ⁇ 3
  • GDNF glial cell line-derived neurotrophic factor
  • AA ascorbic acid
  • the mouse FoxA2 cDNA (obtained from Dr. Siew-Lan Ang, MRC National Institute for Medical Research, London, UK) was ligated into the lentiviral vector, pHAGEUBC-MCS-IZsGW (Dr. Jeng-Shin Lee, Harvard Gene Therapy Initiative, Boston, Mass., USA) to generate pHAGE-UBC-mFoxA2-IZsGW.
  • Infectious lentiviral particles were produced by 293T cells after cotransfection with the following constructs, pHDM-Hgpm2 (HIV gag-pol), pMD-tat, pRC/CMV-rev and VSV-G pseudotyped (Env).
  • Culture medium containing virus particles was harvested and filtered through a 0.45 ⁇ m membrane. Viral particles were concentrated by ultracentrifugation and virus titers were determined by Southern blot analysis of infected human U2OS cells.
  • MOI multiplicity of infection
  • PBS phosphate-buffered saline
  • pHAGE-UBC-mFoxA2-IZsGW or phage-UBC-MCS-IZsGW both MOI 5
  • 8 ⁇ g/ml polybrene Sigma-Aldrich
  • mice/sheep anti-tyrosine hydroxylase (1:300, Pel-Freez, Rogers, Ak.)
  • mouse/goat anti-FoxA2/HNF3 ⁇ (1:100, Santa Cruz Biotechnology, Santa Cruz, Calif.
  • mouse/rabbit anti- ⁇ -III-tubulin (TuJ1) (1:500, Covance, Berkeley, Calif.)
  • mouse anti-3CB2 mouse anti-Islet1
  • mouse anti-SOX17 both 1:100, Chemicon International, Temecula, Calif.
  • rat anti-CORIN (1:100, R & D Systems).
  • the appropriate fluorescent-labeled secondary antibodies (1:500. Alexa Fluor goat or donkey anti-rabbit, -mouse, -rat or -goat 488, 568, 594, 647; Invitrogen Corporation) were applied for visualization, and nuclei were counterstained with Hoechst 33342 (5 ⁇ g/ml; Invitrogen Corporation). On selected coverslips, the primary antibody was omitted to verify specificity of staining.
  • Quantitative analysis of immunocytochemistry was performed on randomly selected confocal fields from at least two independent differentiation experiments.
  • images of separate channels (Hoechst, 488, 568, 594) were acquired at 40 ⁇ magnification on an integrated confocal microscope (LSM510/Meta, Carl Zeiss) and stereology workstation (StereoInvestigator, MBF Bioscience, Inc., Williston, Vt.), where images of cells in independent channels and merged images were counted.
  • LSM510/Meta Carl Zeiss
  • stereology workstation StereoInvestigator, MBF Bioscience, Inc., Williston, Vt.
  • tyrosine hydroxylase TH
  • ⁇ -tubulin was used to identify SN-A9/VTA-A10 DA neurons which were differentiated according to a previously published protocol (Sonntag et al., 2007). While the percentage of TH + / ⁇ -tubulin + cells within the total cell population (Hoechst labeled) was 5.16 ⁇ 2.6%, surprisingly few of the TH + / ⁇ -tubulin + cells coexpressed FOXA2 (approximately 0.00001%).
  • RA retinoic acid
  • 10 ⁇ 8 M RA increased the expression levels of the midbrain transcription factor, EN1 by more than 2000-fold.
  • expression levels of the ventral neural transcription factor, FOXA2 were consistently low.
  • 10 ⁇ 8 M retinoic acid (RA) generated a human neural progenitor cell that exhibited a transcriptional profile consistent with midbrain regionalization without the appropriate ventralization signal to generate SN-A9 DA neurons.
  • FOXA2 Due to the lack of FOXA2 expressing human neurons in the cultures, a FOXA2 transgene was forcibly expressed in human neural progenitor cells to examine whether this gene can improve the ventral midbrain-like transcriptional profile of our cultures.
  • Lentiviral forced expression of FoxA2 in RA-treated cells increased the levels of SN-A9/VTA-A10 DA neuron-associated transcripts, SHH, LMX1A, and EN1 ( FIG. 2 ). Under the control of the human ubiquitin promoter, FoxA2 was overexpressed in neural progenitor cells ( FIG. 2A ).
  • the role of the recombinant Shh protein used in previous differentiation protocols was investigated.
  • the concentration of the recombinant N-terminus truncated form of mouse Shh was increased from 200 ng/ml to 500 ng/ml.
  • 500 ng/ml few FOXA2 expressing progenitor cells were observed (data not shown).
  • the neural progenitor cells were exposed to Shh for an additional 7 days without increasing the yield of FOXA2 expressing neural progenitor cells (data not shown).
  • neural progenitor cells exposed to 10 ⁇ 8 M RA activated human SHH and FGF8A organized into rosette-like structures and coexpressed OTX2, FOXA2 and WNT1, indicative of a ventral midbrain-like neural progenitor cell phenotype ( FIG. 4B ).
  • many of the neural progenitor cells exposed to 10 ⁇ 8 M RA, activated human SHH and Fgf8b coexpressed OTX2 and FOXA2 FIG. 4C ).
  • these FOXA2 + neural progenitor cells neither organized into rosette-like structures nor expressed WNT1.
  • FOXA2 expressing progenitor cells induced by 10 ⁇ 8 M RA, FGF8A and activated human SHH organized into rosette-like structures and did not coexpress ⁇ FP or SOX17 ( FIG. 4F ).
  • many FOXA2 expressing progenitor cells exposed to 10 ⁇ 8 M RA, Fgf8b and activated human SHH coexpressed ⁇ FP and SOX17 ( FIG. 4G ), indicative of an endodermal lineage.
  • FOXA2 expressing progenitor cells generated by exposure to 10 ⁇ 8 M RA, FGF8A and activated human SHH coexpressed nestin ( FIG. 41 ), the radial glial marker 3CB2 ( FIG. 4J ) and NCAM (data not shown).
  • FOXA2 expressing neural progenitor cells expressed the floor plate marker, CORIN, only after exposure to 500 ng/ml of activated human SHH ( FIG. 41 ).
  • high yields of FOXA2 expressing progenitor cells were obtained from RA-treated PD and healthy control iPS cell lines after exposure to 500 ng/ml activated human SHH and FGF8A ( FIG. 4K , PDC 3F -1).
  • human ES cells were cultured with or without WNT1 and/or Noggin, with Noggin alone or without growth factors during the first 14 days of differentiation before exposure to the previously described recombinant protein supplements, including SHH-C24II, as showing in FIG. 7(A) .
  • cultures grown without Noggin contained a few clusters of cells exhibiting neural differentiation.
  • human ES cell-derived cultures grown with or without Noggin contained cells that coexpressed FOXA2, TH and ⁇ -tubulin, as shown in FIG. 7 (B)-(C).
  • a combination of RA, an activated form of human SHH and exposure to FGF8A was sufficient to direct the fate of human ES/iPS cells towards SN-A9 and VTA-A10 DA neurons.
  • the cellular yield of SN-A9/VTA-A10 DA neurons was improved by targeting both early posteriorizing and ventralizing neural patterning pathways. Although isolated non-neural cell types may remain in these cultures, enriched populations (even purified populations) of SN-A9/VTA-A10 DA neurons may be separated from these mixed cultures using techniques such as flow cytometry.

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