WO2019113535A1 - Procédés de production d'astrocytes et utilisations associées - Google Patents

Procédés de production d'astrocytes et utilisations associées Download PDF

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WO2019113535A1
WO2019113535A1 PCT/US2018/064602 US2018064602W WO2019113535A1 WO 2019113535 A1 WO2019113535 A1 WO 2019113535A1 US 2018064602 W US2018064602 W US 2018064602W WO 2019113535 A1 WO2019113535 A1 WO 2019113535A1
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cells
astrocytes
astrocyte
cell
differentiation
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PCT/US2018/064602
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Francesca RAPINO
Lee L. Rubin
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • mouse astrocytes have highlighted their central role in the normal development and function of the central nervous system, as well as their potential participation in many pathological conditions (reviewed in Sofroniew MV, Vinters HV, 2010 and Tyzack G., 2016). Due to the intrinsic differences between rodents and humans, and the limited availability of primary human fetal or mature post-mortem samples, the direct differentiation of human astrocytes from pluripotent stem cells provides an excellent alternative to uncover the complex function of human astrocytes in normal and pathological conditions. Moreover, the differentiation of astrocytes from pluripotent stem cells allows the selection of disease relevant genotypes and the possibility of gene editing to study single mutations in the desired genetic background.
  • a protocol for the rapid generation of a large number of astrocytes amendable for high-throughput screening, as well as for the study of cell autonomous and non-autonomous contributions of glia in physiopathological conditions has been developed and is described herein. Also described herein are screening methods for modulators of C4 secretion in astrocytes. These screening methods highlight the molecular mechanisms underlying the regulation of C4, as well as providing new potentially interesting therapeutic targets.
  • the disclosure provides methods for generating functional astrocytes from stem cells comprising contacting a population of stem cells in spin culture with at least one differentiation medium to induce the differentiation of at least one sphere in the spin culture into an astrocyte sphere; dissociating the astrocyte spheres; and culturing the dissociated spheres in monolayers to obtain functional astrocytes.
  • the functional astrocyte is a spinal cord astrocyte or a brain astrocyte.
  • the differentiation medium includes a KSR medium or a NB medium, and optionally includes a supplemental agent (e.g., SB431542, Dorsomorphin, FGF, EGF, CTNF, and combinations thereof).
  • the differentiation medium includes a KSR medium or a neural induction medium, and optionally includes a supplemental agent (e.g., SB431542, LDN193189, retinoic acid, BDNF, SHH, DAPT and combinations thereof).
  • the disclosure provides methods of generating functional brain astrocytes from stem cells comprising contacting a population of stem cells in spin culture with at least one differentiation medium to induce the differentiation of at least one sphere in the spin culture into a functional brain astrocyte sphere.
  • a first differentiation medium includes a KSR medium, and optionally at least one supplemental agent.
  • the at least one supplemental agent is selected from the group consisting of activin/TGF-b inhibitor (e.g., SB43154), Dorsomorphin, and combinations thereof.
  • a second differentiation medium includes an NB medium, and optionally includes at least one supplemental agent.
  • the at least one supplemental agent is selected from the group consisting of EGF, FGF, CTNF, and combinations thereof.
  • the disclosure provides methods of generating functional spinal cord astrocytes from pluripotent cells comprising contacting a population of pluripotent cells in spin culture with at least one differentiation medium to induce the differentiation of at least one sphere in the spin culture into a functional spinal cord astrocyte sphere.
  • the at least one differentiation medium includes dual SMAD inhibition.
  • a first differentiation medium includes a KSR medium, and optionally includes a supplemental agent.
  • the supplemental agent is selected from the group consisting of SB431542, LDN193189, retinoic acid, BDNF, SHH, DAPT and combinations thereof.
  • a second differentiation medium includes a neural induction medium, and optionally includes a supplemental agent.
  • the supplemental agent is selected from the group consisting of SB431542, LDN193189, retinoic acid, BDNF, SHH, DAPT and combinations thereof.
  • a third differentiation medium includes an astrocyte medium.
  • the disclosure provides methods of screening for modulators of complement component 4 (C4) comprising generating an astrocyte composition comprising a functional spinal cord or brain astrocyte from stem cells; and screening for compounds that decrease C4 secretion from the astrocyte composition.
  • C4 complement component 4
  • the level of C4 secretion is decreased by up to 10% below the standard deviation of a DMSO control.
  • the disclosure provides methods of identifying pathways involved in C4 modulation comprising generating an astrocyte composition comprising a functional spinal cord or brain astrocyte from pluripotent cells; and screening for modulators of C4 secretion from the astrocyte composition, thereby identifying pathways involved in C4 modulation.
  • the disclosure provides an in vz/ro-differentiated astrocyte produced by any of the methods described herein.
  • the disclosure provides a non- naturally occurring astrocyte having astrocyte-like morphology and expressing one or more markers selected from the group consisting of CD44, SI 00b, GFAP, CX43, and ALDH1L1.
  • the astrocyte is produced by differentiation from a pluripotent cell.
  • the disclosure provides methods of modulating C4 secretion by astrocytes comprising contacting one or more astrocytes with a composition identified by a method described herein, thereby modulating C4 secretion by astrocytes.
  • the disclosure provides methods of treating a neuropsychiatric disease comprising administering to an individual in need thereof an agent that modulates C4 secretion by astrocytes.
  • the agent is identified by a method described herein.
  • FIGS. 1A-1E demonstrate differentiation and characterization of pluripotent stem cells derived astrocytes.
  • FIG. 1 A provides a schematic overview of astrocytes differentiation from human pluripotent stem cells.
  • FIG. IB provides representative bright-field image of iPSC-derived astrocytes (1016A HA) (scale bar, 100 pm).
  • FIG. 1C provides representative immunocytochemistry of astrocytes markers CD44, ALDH1L1, GFAP, S 100 b, Aquaporin 4 (AQP4), the gap junction CX43 and the glutamate transporter EAAT1. Blue, dapi staining (scale bar, 100 pm).
  • FIG. 1 A provides a schematic overview of astrocytes differentiation from human pluripotent stem cells.
  • FIG. IB provides representative bright-field image of iPSC-derived astrocytes (1016A HA) (scale bar, 100 pm).
  • FIG. 1C provides representative immunocytochemistry of astrocytes markers CD44, ALDH1L1, GFAP
  • FIG. 1D shows C4 (green), ALDH1L1 (red) and DAPI (blue) staining of 1016A HA, (scale bar, 20 pm).
  • FIG. 1E shows C4 secretion measured by ELISA from 1016A HA astrocytes supernatant treated for 48 hours with DMSO control, Monensin (1 pM) and INFy (250 ng/mL). Data are presented as mean ⁇ SD using Mann Whitney test **** p ⁇ 0 0001
  • FIGS. 2A-2H demonstrate differentiation and characterization of stem cell derived astrocytes compared to primary human fetal astrocytes (primary HA).
  • FIG. 2A provides representative bright-field image of primary HA and HUES8 HA (scale bar, 100 pm).
  • FIG. 2A provides representative bright-field image of primary HA and HUES8 HA (scale bar, 100 pm).
  • FIG. 2B provides representative flow-cytometer analysis of astrocytes stained for the neuronal marker CD200 (red), the astrocyte specific antigen CD44 (green) and relative isotype controls (grey).
  • FIG. 2C provides a graph of flow cytometry analysis on primary HA and stem cell derived astrocytes (1016A HA and Hues8 HA). Data represent mean ⁇ SD of biological differentiations unpaired t-test *p ⁇ 0.05.
  • FIG. 2D provides immunocytochemistry of astrocytes markers and complement component 4 (C4).
  • FIG. 2E provides human cytokine array quantification of secretion comparing primary HA and 1016A HA. Data are represented as technical duplicates.
  • FIG. 2F shows C4 secretion in primary astrocytes and Hues8 HA treated with vehicle (DMSO) Monensin (1 pM) and INFy (250 ng/mL). Data are presented as mean ⁇ SD using Mann Whitney test **** p ⁇ 0.0001.
  • FIG. 2G provides Western blot validation of C4 antibodies in denaturation non-reducing and reducing conditions using human serum enriched for complement components
  • FIG 2H shows C4 secretion in 1016A HA astrocytes cultured in different media with or without fetal bovine serum (FBS)
  • FBS fetal bovine serum
  • AM Astrocytes media
  • NB Neurobasal. Data are represented as mean ⁇ SD. two way Anova, **** p ⁇ 0.0001.
  • FIGS. 3A-3C demonstrate high-throughput small molecules screening to identify modulator of complement component 4
  • FIG. 3A provides a schematic of the screening timeline.
  • FIG. 3A provides a schematic of the screening timeline.
  • FIG. 3B provides representative scatter plot showing the effect of compounds on C4 secretion (black squares represent average of triplicates) compared to DMSO (red squares, average of triplicates) at 1 mM.
  • FIG. 3C provides a bar graph of the pathways involved in the regulation of C4.
  • FIG. 4 shows a screening approach using iPS/ES derived cells.
  • FIG. 5 shows features of organoids and spheroids.
  • FIG. 6 demonstrates 3D differentiation of neuronal subtype and astrocytes.
  • Top panel shows schematic of pluripotent spheres to differentiated cells.
  • Bottom panel shows staining of motor neurons, cortical neurons, dopaminergic neurons, and astrocytes.
  • FIG. 7 demonstrates protocols to generate human neuronal cells from iPS/ES cells using spinner flasks.
  • FIG. 8 demonstrates neuronal precursor spheres express telencephalon markers
  • FIG. 9 shows how differentiated cells can be visualized and studied.
  • FIG. 10 shows 3D imaging of cortical spheroids.
  • FIG. 11 shows 3D imaging of cortical spheroids.
  • FIG. 12 shows cortical spheroids express deep and upper layer cortical markers.
  • FIG. 13 shows dissociated cortical neurons express TBR1, CTIP2, and SATB2.
  • FIG. 14 demonstrates rapid differentiation of a pure population of astrocytes from stem cells.
  • FIG. 15 shows stem cell derived astrocytes are functional.
  • FIG. 16 shows stem cell derived astrocytes express canonical markers.
  • FIG. 17 shows human astrocytes produce and secrete C4.
  • FIG. 18A-18C demonstrate patterning of stem cells and specification of dorsal and ventral fate.
  • FIG. 18A provides a general scheme of the differentiation of astrocyte subtypes using spinner flask.
  • FIG. 18B provides a schematic representation of the culture conditions for the differentiation of brain astrocytes.
  • FIG. 18C provides a schematic representation of the culture conditions for the differentiation of spinal cord astrocytes
  • FIGS. 19A-19B demonstrate that iPSC derived cells are patterned towards ventral or dorsal fates.
  • FIG. 19A provides time course expression of pluripotent, progenitors and ventral or dorsal genes after patterning of stem cells.
  • FIG. 19B shows representative immunofluorescence for pluripotent genes in spheres sections. Nuclei are stained with DAPI (blue). Top panels expression of pluripotent genes: Oct4 and Nanog (red) and Tral-60 (green). Middle panels expression of neural progenitors markers Pax6 (red) and Sox2 (green). Lower panel, expression of region specific gene. For ventral patterning HOXB4 (green) and for dorsal patterning OTX1/2 (green).
  • FIGS. 20A-20B demonstrate that iPSC derived cells express markers of astrocytes.
  • FIG. 20A provides Bright field images of dissociated bA and spA astrocytes.
  • FIG. 20B provides representative immunofluorescence of astrocytes specific markers such as slOOb, ALDH1L1, CD44, GFAP and the gap junction connexin 43 (CX43).
  • FIGS. 21A-21B demonstrate iPSC-derived astrocytes are functional.
  • FIG. 21A shows spA can propagate calcium waves to adjacent cells upon mechanical stimuli.
  • Doxy inducible Gcamp6 lentiviral vector was used to infect primary and spin derived astrocytes.
  • FIG. 2 IB provides MEA measurement for NGN2-induced cortical neurons co-cultured with different glial cells.
  • FIG. 22 demonstrate spin derived spA and bA respond to pro-inflamatory stimuli and become reactive. qPCR analysis of genes typical of the reactive astrocytes state upon treatment with pro-inflammatory cytokines (ILlp and TNFa) for 7 days.
  • pro-inflammatory cytokines ILlp and TNFa
  • FIGS. 23A-23G demonstrate screening for modulators of complement component 4 (C4)
  • FIG. 23A shows C4 expression in iPSC derived astrocytes.
  • FIG. 23B provides schematic representation of the screening workflow.
  • FIG. 23 C shows raw data from a primary screen.
  • FIGS. 23D-23E provide pipeline analysis for hit selection.
  • FIG. 23F identifies pathways potentially involved in C4 regulation.
  • FIG. 23 G provides a schematic of JAK/STAT and NFkB pathways.
  • FIG. 24 provides a schematic for the protocol for the generation of iPS/ES cell- derived astrocytes in 3D culture.
  • FIGS. 25A-25C demonstrates patterning of iPS/ES cells generates a population of cells that express astrocytes markers.
  • FIG. 25 A shows time course analysis of gene expression during differentiation.
  • FIG. 25B shows immunostaining for astrocyte markers.
  • FIG. 25C provides FACS analysis of the percentage of CD44 positive cells.
  • FIGS. 26A-26E demonstrate the iPS/ES cell-derived astrocytes support neuronal growth and secret the complement component 4 (C4).
  • FIGS. 26A-26B shows co-culturing of NGN2-derived neurons and astrocytes.
  • FIG. 26A provides MEA recording of ngn-2 derived cortical neurons in co-culture with mouse or spin derived bA. The graph show weighted mean firing rate (Hz) over 30 days of co-culture.
  • FIG. 26B provides a graph presenting immunofluorescence quantification of astrocytes-neurons co-culture stained for the synaptic marker Synapsin I.
  • FIGS 26C-26E show astrocytes express and secrete C4.
  • FIG. 26C shows protein expression by immunostaining.
  • FIG. 26D shows protein secretion measured by ELISA.
  • FIG. 26E shows astrocyte conditioned medium (ACM) influences the amount of neuronal C4.
  • ACM astrocyte conditioned medium
  • FIGS. 27A-27C provides pipeline analysis for hit selection.
  • FIG. 27A shows a threshold for nuclei selection.
  • FIG. 27B shows an overview of the compounds.
  • FIG 27C shows selection of compounds with decreased C4 secretion.
  • FIGS. 28A-28F shows pathways potentially involved in the modulation of C4.
  • FIG. 28A shows annotation of the pathways.
  • FIG. 28B provides a schematic of the IAK/STAT pathways (left panel) and shows that inhibitors of the JAK/STAT pathway decrease the secretion of C4 (right panel).
  • FIGS. 28C-28F shows epigenetic modifier inhibitor decreases the secretion of C4 in a dose dependent manner and it is able to block the IFNy mediated response
  • FIGS. 29A-29B indicate the relationship between a complement component and synaptic pruning.
  • FIG. 29A demonstrates that complement component 4 (C4) is associated with a high risk of schizophrenia.
  • FIG. 29B demonstrates that schizophrenic patients have less synapses. See Glantz et al., Arch Gen Psychiatry (2000) 57(l):65-73.
  • FIGS. 30A-30B demonstrate the biological function of C4.
  • FIG. 30A shows the complement activation pathways of the complement system, which is an essential component of innate immunity. See Wagner et al. Nature Reviews Drug Discovery (2010) 9:43-56
  • FIG. 30B demonstrates synapse pruning during development and shows that Clq-/-C3-/-C4- /- mice have less synaptic pruning compared to wile type mice. See Stephan et al. Annu.
  • FIGS. 31A-31B demonstrate the structure expression and association of C4 with schizophrenia.
  • FIG. 31 A shows the functional specialization of C4 into C4A and C4B and indicates the sequences differences between C4A and C4B.
  • FIG. 3 IB shows the structural variation of C4.
  • FIG. 31C provides the measure copy number of each C4 gene type.
  • 3 ID shows the schizophrenia risk associated with various structural forms of C4 (left panel) and brain mRNA expression levels associated with various structural forms of C4 (right panel). See Sekar et al. Nature (2016) 530(7589):177-183.
  • FIG. 32 demonstrates that reduced synapses number in schizophrenia patients may be explained by excessive synaptic pruning due to increased C4 expression, and that compounds that reduce C4 levels might then rescue the over-pruning phenotype.
  • FIGS. 33A-33E show where the complement components are produced in the CNS.
  • FIG. 33A shows that astrocytes express and secrete C3.
  • C3 mRNA levels for wild type and IkBa knockout (KO) primary neurons or astroglia are provided, as is ELISA quantification of C3 protein levels in conditioned media of WT or IkBa KO astroglial cultures See Lian et al. Neuron. (2015) 85(1): 101-115.
  • FIG. 33B shows that astrocytes upregulate C lq expression for all three chains (A, B, and C) by neurons. See Stevens et al. , Cell (2007) 131(6): 1164- 1178.
  • FIG. 33C shows that astrocytes express C4.
  • FIG. 33D shows genome-wide distributions of expression fidelity for astrocytes (A), oligodendrocytes (O), microglia (M), and neurons (N). See Kelley el at. (2016) oldhamlab.ctec.ucsf.ed. In the CNS neurons, astrocytes, microglia, and oligodendrocytes can synthetize complement components. In fact, astrocytes are able to synthetize as many complement components as the liver.
  • FIG. 33E shows a mixed population of iPSC-derived neurons and astrocytes by immunostaining.
  • FIGS. 34A-34B demonstrate that human astrocytes produce and secrete C4.
  • FIG. 34A shows protein expression in 1016A cells by immunostaining. C4 (green), ALDH1L1 (red) and DAPI (blue) staining of 1016A HA, (scale bar, 20 pm)
  • FIG. 34B shows protein secretion by ELISA. C4 secretion measured by ELISA from 1016A HA astrocytes supernatant treated for 48 hours with DMSO control, Monensin (1 mM) and INFy (250 ng/mL). Data are presented as mean ⁇ SD using Mann Whitney test **** p ⁇ 0.0001 (left panel).
  • Astrocytes are crucial for the formation and remodeling of synapses. As disclosed herein, it is shown that astrocytes may be obtained through a large scale and rapid differentiation protocol, and that these differentiated astrocytes are functional (e.g., express markers of astrocytes, secrete C4, and/or exhibit functional characteristics). Also disclosed herein are screens for identifying modulators of C4 secretion, as well as identifying pathways involved in C4 modulation. Also disclosed herein are methods of modulating C4 secretion by administering an agent or compound. Further disclosed herein are methods of treating a neurodegenerative or neuropsychiatric disease by administering an agent that downregulates C4 expression.
  • differentiated cell is meant to include any primary cell that is not, in its native form, pluripotent as that term is defined herein. Stated another way, the term “differentiated cell” refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) in a cellular differentiation process.
  • a stem cell such as an induced pluripotent stem cell
  • germline cells also known as “gametes” are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops.
  • the somatic cell is a“non-embryonic somatic cell,” by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro.
  • the somatic cell is an“adult somatic cell,” by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro.
  • adult cell refers to a cell found throughout the body after embryonic development.
  • progenitor or“precursor” cell are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive (i.e., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the
  • phenotype refers to one or a number of total biological characteristics that define the cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.
  • pluripotent refers to a cell with the capacity to differentiate to more than one differentiated cell type, and preferably to differentiate to cell types characteristic of all three germ cell layers.
  • Pluripotent cells are characterized primarily by their ability to differentiate to more than one cell type, preferably to all three germ layers, using, for example, a nude mouse teratoma formation assay.
  • Pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not, on its own, render them pluripotent.
  • ES embryonic stem
  • Reprogrammed pluripotent cells e.g., iPS cells as that term is defined herein
  • iPS cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture.
  • iPS cell and“induced pluripotent stem cell” are used interchangeably and refers to a pluripotent stem cell artificially derived (e g , induced or by complete reversal) from a non-pluripotent cell, typically an adult somatic cell, for example, by inducing a forced expression of one or more genes.
  • stem cell refers to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential.
  • stem cell refers to a subset of progenitors that have the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating.
  • the term stem cell refers generally to a naturally occurring mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues.
  • Cellular differentiation is a complex process typically occurring through many cell divisions.
  • a differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably Some differentiated cells also have the capacity to give rise to cells of greater developmental potential Such capacity may be natural or may be induced artificially upon treatment with various factors.
  • stem cells are also“multipotent” because they can produce progeny of more than one distinct cell type, but this is not required for“stern ness.”
  • Self-renewal is the other classical part of the stem cell definition, and it is essential as used in this document. In theory, self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only.
  • pluripotent stem cell includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, etc.
  • Functional astrocyte As used herein“functional astrocyte,”“non-naturally occurring astrocyte,” and“non native astrocyte,” all refer to a functional astrocyte that is generated via the differentiation of a stem cell. Functional astrocytes may exhibit one or more features which may be shared with endogenous astrocytes, including, but not limited to, capacity to propagate calcium waves, ability to increase neuron firing potential and synapsis number when co-cultured with neurons (e.g., cortical neurons), exhibit appropriate expression of gene markers, and capacity to produce and secrete complement component 4 (C4). However non-naturally occurring astrocytes are not identical to and distinguishable from endogenous astrocytes.
  • stem cells can differentiate to lineage-restricted precursor cells (such as an ectodermal stem cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as a neural ectodermal cell), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • precursor cells such as an ectodermal stem cell
  • end-stage differentiated cell which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • embryonic stem cell is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see U.S. Pat. Nos. 5,843,780, 6,200,806). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U. S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970).
  • the distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.
  • adult stem cell or“ASC” is used to refer to any multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue.
  • Stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture.
  • Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As indicated above, stem cells have been found resident in virtually every tissue. Accordingly, the present invention appreciates that stem cell populations can be isolated from virtually any animal tissue
  • reprogramming refers to the process that alters or reverses the differentiation state of a somatic cell.
  • the cell can either be partially or terminally differentiated prior to the reprogramming.
  • Reprogramming encompasses complete reversion of the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation produces an induced pluripotent (iPS) cell.
  • iPS induced pluripotent
  • Reprogramming as used herein also encompasses partial reversion of a cells differentiation state, for example to a multipotent state or to a somatic cell that is neither pluripotent or multipotent, but is a cell that has lost one or more specific characteristics of the differentiated cell from which it arises, e.g. direct reprogramming of a differentiated cell to a different somatic cell type.
  • Reprogramming generally involves alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation as a zygote develops into an adult.
  • nucleic acid modification e.g., methylation
  • chromatin condensation e.g., chromatin condensation
  • epigenetic changes e.g., genomic imprinting, etc.
  • agent means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc.
  • An“agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally- occurring proteinaceous and non-proteinaceous entities.
  • an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc.
  • agents are small molecule having a chemical moiety.
  • chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof.
  • Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • the term“contacting” is intended to include incubating the differentiation medium and/or agent and the cell together in vitro (e g., adding the differentiation medium or agent to cells in culture). In some embodiments, the term “contacting” is not intended to include the in vivo exposure of cells to the compounds as disclosed herein that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).
  • the step of contacting at least one pluripotent sphere or a precursor thereof with a differentiation medium or agent as in the embodiments related to the production of functional astrocytes can be conducted in any suitable manner.
  • the cells may be treated in adherent culture, or in suspension culture.
  • the cells are treated in conditions that promote cell clustering.
  • the disclosure contemplates any conditions which promote cell clustering. Examples of conditions that promote cell clustering include, without limitation, suspension culture in low attachment tissue culture plates, spinner flasks, aggrewell plates.
  • the inventors have observed that clusters have remained stable in media containing 10% serum.
  • the conditions that promote clustering include a low serum medium.
  • the cells contacted with a differentiation medium and/or agent can also be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further.
  • another agent such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further.
  • At least one pluripotent sphere of cells or a precursor thereof can be contacted with at least one differentiation medium or agent and then contacted with at least another differentiation medium or agent.
  • a cell is contacted with at least one differentiation medium or agent, and the contact is temporally separated, and in some embodiments, a cell is contacted with at least one differentiation medium substantially simultaneously
  • a cell is contacted with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at leastlO differentiation mediums or agents.
  • cell culture medium (also referred to herein as a“culture medium” or “medium”) as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation.
  • the cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc.
  • Cell culture media ordinarily used for particular cell types are known to those skilled in the art.
  • cell line refers to a population of largely or substantially identical cells that has typically been derived from a single ancestor cell or from a defined and/or substantially identical population of ancestor cells.
  • the cell line may have been or may be capable of being maintained in culture for an extended period (e.g., months, years, for an unlimited period of time). It may have undergone a spontaneous or induced process of transformation conferring an unlimited culture lifespan on the cells.
  • Cell lines include all those cell lines recognized in the art as such. It will be appreciated that cells acquire mutations and possibly epigenetic changes over time such that at least some properties of individual cells of a cell line may differ with respect to each other.
  • a cell line comprises an astrocyte described herein.
  • feeder cells refer to cells of one type that are co-cultured with cells of another type, to provide an environment in which the cells of the second type can grow.
  • the feeder cells are optionally from a different species as the cells they are supporting.
  • a culture or cell population may be referred to as“feeder free”, meaning the composition is essentially free of feeder cells.
  • growth environment refers to an environment in which cells of interest will proliferate or differentiate in vitro.
  • the term“growth environment” refers to an environment in which cells of interest will proliferate or differentiate in vitro.
  • the environment include the medium in which the cells are cultured, the temperature, the partial pressure of 02 and C02, and a supporting structure (such as a substrate on a solid surface) if present.
  • the term“nutrient medium” refers to a medium for culturing cells containing nutrients that promote proliferation.
  • the nutrient medium may contain any of the following in an appropriate combination: isotonic saline, buffer, amino acids, antibiotics, serum or serum replacement, and exogenously added factors.
  • A“conditioned medium” is prepared by culturing a first population of cells in a medium, and then harvesting the medium. The conditioned medium (along with anything secreted into the medium by the cells) may then be used to support the growth of a second population of cells.
  • exogenous refers to a substance present in a cell or organism other than its native source.
  • exogenous nucleic acid or“exogenous protein” refer to a nucleic acid or protein that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found or in which it is found in lower amounts.
  • a substance will be considered exogenous if it is introduced into a cell or an ancestor of the cell that inherits the substance
  • endogenous refers to a substance that is native to the biological system.
  • “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, translation, folding, modification and processing.“Expression products” include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.
  • the terms“genetically modified” or“engineered” cell as used herein refers to a cell into which an exogenous nucleic acid has been introduced by a process involving the hand of man (or a descendant of such a cell that has inherited at least a portion of the nucleic acid).
  • the nucleic acid may, for example, contain a sequence that is exogenous to the cell, it may contain native sequences (i.e , sequences naturally found in the cells) but in a non-naturally occurring arrangement (e.g., a coding region linked to a promoter from a different gene), or altered versions of native sequences, etc.
  • the process of transferring the nucleic acid into the cell can be achieved by any suitable technique.
  • Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector.
  • the polynucleotide or a portion thereof is integrated into the genome of the cell.
  • the nucleic acid may have subsequently been removed or excised from the genome, provided that such removal or excision results in a detectable alteration in the cell relative to an unmodified but otherwise equivalent cell.
  • the term genetically modified is intended to include the introduction of a modified RNA directly into a cell (e g., a synthetic, modified RNA).
  • Such synthetic modified RNAs include modifications to prevent rapid degradation by endo- and exo-nucleases and to avoid or reduce the cell's innate immune or interferon response to the RNA.
  • Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5 end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3 ' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5 end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.
  • identity refers to the extent to which the sequence of two or more nucleic acids or polypeptides is the same.
  • the percent identity between a sequence of interest and a second sequence over a window of evaluation may be computed by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the window of evaluation that are opposite an identical residue allowing the introduction of gaps to maximize identity, dividing by the total number of residues of the sequence of interest or the second sequence (whichever is greater) that fall within the window, and multiplying by 100.
  • Percent identity can be calculated with the use of a variety of computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments and provide percent identity between sequences of interest.
  • the algorithm of Karlin and Altschul Karlin and Altschul (Karlin and Altschul, Proc Natl. Acad. Sci. USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc. Natl. Acad. ScL USA 90:5873-5877, 1993 is incorporated into the NBLAST and XBLAST programs of Altschul et al.
  • isolated refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and/or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides.
  • a chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation is considered“isolated”.
  • isolated cell refers to a cell that has been removed from an organism in which it was originally found or a descendant of such a cell.
  • the cell has been cultured in vitro, e g., in the presence of other cells.
  • the cell is later introduced into a second organism or re-introduced into the organism from which it (or the cell from which it is descended) was isolated.
  • isolated population refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells.
  • an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched from.
  • enriching or“enriched” are used interchangeably herein and mean that the yield (fraction) of cells of one type is increased by at least 10% over the fraction of cells of that type in the starting culture or preparation.
  • proliferation refers to the expansion of cells by the repeated division of single cells into two identical daughter cells.
  • linearages describes a cell with a common ancestry or cells with a common developmental fate.
  • a cell that is of ectoderm origin or is“ectodermal linage” this means the cell was derived from an ectoderm cell and can differentiate along the ectoderm lineage restricted pathways.
  • xenogeneic refers to cells that are derived from different species.
  • A“marker” as used herein is used to describe the characteristics and/or phenotype of a cell. Markers can be used for selection of cells comprising characteristics of interests Markers will vary with specific cells. Markers are characteristics, whether morphological, functional or biochemical (enzymatic) characteristics of the cell of a particular cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, possess an epitope for antibodies or other binding molecules available in the art. However, a marker may consist of any molecule found in a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids.
  • morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio.
  • functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers may be detected by any method available to one of skill in the art. Markers can also be the absence of a morphological characteristic or absence of proteins, lipids etc Markers can be a combination of a panel of unique characteristics of the presence and absence of polypeptides and other morphological characteristics.
  • module is used consistently with its use in the art, i.e., meaning to cause or facilitate a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, or change in relative strength or activity of different components or branches of the process, pathway, or phenomenon.
  • A“modulator” is an agent that causes or facilitates a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest
  • polynucleotide is used herein interchangeably with“nucleic acid” to indicate a polymer of nucleosides.
  • a polynucleotide of this invention is composed of nucleosides that are naturally found in DNA or RNA (e g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycyti dine) joined by phosphodiester bonds.
  • nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications.
  • this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double- stranded forms (and complements of each single-stranded molecule) are provided.
  • Polynucleotide sequence as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e. the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.
  • polypeptide refers to a polymer of amino acids.
  • protein and“polypeptide” are used interchangeably herein.
  • a peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length.
  • Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used.
  • One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc.
  • a polypeptide that has a non polypeptide moiety covalently or non-covalently associated therewith is still considered a “polypeptide”. Exemplary modifications include glycosylation and palmitoylation.
  • Polypeptides may be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid phase peptide synthesis, etc.
  • the term“polypeptide sequence” or“amino acid sequence” as used herein can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide.
  • a polypeptide sequence presented herein is presented in an N- terminal to C-terminal direction unless otherwise indicated.
  • the term“functional fragments” as used herein is a polypeptide having amino acid sequence which is smaller in size than, but substantially homologous to the polypeptide it is a fragment of, and where the functional fragment polypeptide sequence is about at least 50%, or 60% or 70% or at 80% or 90% or 100% or greater than 100%, for example 1.5-fold, 2- fold, 3 -fold, 4-fold or greater than 4-fold effective biological action as the polypeptide from which it is a fragment of.
  • Functional fragment polypeptides may have additional functions that can include decreased antigenicity, increased DNA binding (as in transcription factors), or altered RNA binding (as in regulating RNA stability or degradation).
  • vector refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as“expression vectors”.
  • an“expression vector” is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell
  • the gene of interest is operably linked to another sequence in the vector.
  • Vectors can be viral vectors or non-viral vectors.
  • viral vectors are replication defective, which can be achieved for example by removing all viral nucleic acids that encode for replication.
  • a replication defective viral vector will still retain its infective properties and enters the cells in a similar manner as a replicating adenoviral vector, however once admitted to the cell a replication defective viral vector does not reproduce or multiply.
  • Vectors also encompass liposomes and nanoparticles and other means to deliver DNA molecule to a cell.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector.
  • operatively linked includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence
  • viral vectors refers to the use of viruses, or virus-associated vectors as carriers of a nucleic acid construct into a cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including reteroviral and lentiviral vectors, for infection or transduction into cells.
  • AAV Adeno-associated virus
  • HSV Herpes simplex virus
  • the vector may or may not be incorporated into the cell's genome.
  • the constructs may include viral sequences for transfection, if desired.
  • the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors.
  • regulatory sequence and“promoter” are used interchangeably herein, and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operatively linked.
  • transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended
  • the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of a protein.
  • the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required for initiating transcription of a specific gene.
  • transcription factor refers to a protein that binds to specific parts of DNA using DNA binding domains and is part of the system that controls the transfer (or transcription) of genetic information from DNA to RNA.
  • proliferating and proliferation refer to an increase in the number of cells in a population (growth) by means of cell division.
  • Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, including growth factors and other mitogens. Cell proliferation may also be promoted by release from the actions of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.
  • selectable marker refers to a gene, RNA, or protein that when expressed, confers upon cells a selectable phenotype, such as resistance to a cytotoxic or cytostatic agent (e.g., antibiotic resistance), nutritional prototrophy, or expression of a particular protein that can be used as a basis to distinguish cells that express the protein from cells that do not.
  • cytotoxic or cytostatic agent e.g., antibiotic resistance
  • Proteins whose expression can be readily detected such as a fluorescent or luminescent protein or an enzyme that acts on a substrate to produce a colored, fluorescent, or luminescent substance (“detectable markers”) constitute a subset of selectable markers.
  • selectable marker genes can be used, such as neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyl transferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK),
  • neomycin resistance gene neo
  • puro puro
  • DHFR dihydrofolate reductase
  • ada puromycin-N-acetyltransferase
  • PAC hygromycin resistance gene
  • midr multidrug resistance gene
  • TK thymidine kinase
  • Detectable markers include green fluorescent protein (GFP) blue, sapphire, yellow, red, orange, and cyan fluorescent proteins and variants of any of these. Luminescent proteins such as luciferase (e.g., firefly or Renilla luciferase) are also of use.
  • GFP green fluorescent protein
  • Luminescent proteins such as luciferase (e.g., firefly or Renilla luciferase) are also of use.
  • the term“selectable marker” as used herein can refer to a gene or to an expression product of the gene, e.g., an encoded protein.
  • the selectable marker confers a proliferation and/or survival advantage on cells that express it relative to cells that do not express it or that express it at significantly lower levels.
  • proliferation and/or survival advantage typically occurs when the cells are maintained under certain conditions, i.e.,“selective conditions.”
  • a population of cells can be maintained under conditions and for a sufficient period of time such that cells that do not express the marker do not proliferate and/or do not survive and are eliminated from the population or their number is reduced to only a very small fraction of the population.
  • the process of selecting cells that express a marker that confers a proliferation and/or survival advantage by maintaining a population of cells under selective conditions so as to largely or completely eliminate cells that do not express the marker is referred to herein as“positive selection”, and the marker is said to be “useful for positive selection”.
  • Negative selection and markers useful for negative selection are also of interest in certain of the methods described herein. Expression of such markers confers a proliferation and/or survival disadvantage on cells that express the marker relative to cells that do not express the marker or express it at significantly lower levels (or, considered another way, cells that do not express the marker have a proliferation and/or survival advantage relative to cells that express the marker). Cells that express the marker can therefore be largely or completely eliminated from a population of cells when maintained in selective conditions for a sufficient period of time.
  • A“reporter gene” as used herein encompasses any gene that is genetically introduced into a cell that adds to the phenotype of the stem cell. Reporter genes as disclosed in this invention are intended to encompass fluorescent, luminescent, enzymatic and resistance genes, but also other genes which can easily be detected by persons of ordinary skill in the art. In some embodiments of the invention, reporter genes are used as markers for the identification of particular stem cells, cardiovascular stem cells and their differentiated progeny. A reporter gene is generally operatively linked to sequences that regulate its expression in a manner dependent upon one or more conditions which are monitored by measuring expression of the reporter gene. In some cases, expression of the reporter gene may be determined in live cells.
  • reporter gene expression may be monitored at multiple time points, e g., 2, 3, 4, 5, 6, 8, or 10 or more time points.
  • reporter gene expression is monitored with a frequency of at least about 10 minutes to about 24 hours, e.g., 20 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, or another frequency from any integer between about 10 minutes to about 24 hours.
  • “decrease”,“reduced”,“reduction”,“decrease” or“inhibit” are all used herein generally to mean a decrease by a statistically significant amount.
  • “reduced”,“reduction” or“decrease” or“inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10- 100% as compared to a reference level.
  • the terms“increased”,“increase” or“enhance” or“activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms“increased”,“increase” or“enhance” or“activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • conditionally significant or“significantly” refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker.
  • 2SD two standard deviation
  • the term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.
  • Stem cells are cells that retain the ability to renew themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types.
  • the two broad types of mammalian stem cells are: embryonic stem (ES) cells that are found in blastocysts, and adult stem cells that are found in adult tissues.
  • ES embryonic stem
  • stem cells can differentiate into all of the specialized embryonic tissues.
  • stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.
  • Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.
  • germ cells may be used in place of, or with, the stem cells to provide at least one astrocyte, using similar protocols as the illustrative protocols described herein.
  • Suitable germ cells can be prepared, for example, from primordial germ cells present in human fetal material taken about 8-11 weeks after the last menstrual period.
  • Illustrative germ cell preparation methods are described, for example, in Shamblott et alirri Proc. Natl. Acad. Sci. USA 95: 13726, 1998 and U. S. Pat. No. 6,090,622.
  • ES cells e.g., human embryonic stem cells (hESCs) or mouse embryonic stem cells (mESCs)
  • hESCs human embryonic stem cells
  • mESCs mouse embryonic stem cells
  • hESC cells are described, for example, by Cowan et al. (N Engl. J. Med. 350: 1353, 2004) and Thomson et al. (Science 282: 1145, 1998); embryonic stem cells from other primates, Rhesus stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol. Reprod. 55:254, 1996) and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95 :13726, 1998) may also be used in the methods disclosed herein.
  • the stem cells may be, for example, unipotent, totipotent, multipotent, or pluripotent.
  • any cells of primate origin that are capable of producing progeny that are derivatives of at least one germinal layer, or all three germinal layers, may be used in the methods disclosed herein.
  • ES cells may be isolated, for example, as described in Cowan et al. (N Engl. J. Med. 350: 1353, 2004) and U S Pat. No. 5,843,780 and Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995
  • hESCs cells can be prepared from human blastocyst cells using the techniques described by Thomson et al. (U.S. Pat. No. 6,200,806; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133 ff, 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000.
  • Equivalent cell types to hESCs include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as outlined, for example, in WO 01/51610 (Bresagen). hESCs can also be obtained from human pre-implantation embryos.
  • EPL ectoderm-like
  • in vitro fertilized (IVF) embryos can be used, or one-cell human embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma).
  • the inner cell masses can be isolated by immunosurgery, in which blastocysts are exposed to a 1 :50 dilution of rabbit anti-human spleen cell antiserum for 30 min, then washed for 5 min three times in DMEM, and exposed to a 1 :5 dilution of Guinea pig complement (Gibco) for 3 min (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers.
  • immunosurgery in which blastocysts are exposed to a 1 :50 dilution of rabbit anti-human spleen cell antiserum for 30 min, then washed for 5 min three times in DMEM, and exposed to a 1 :5 dilution of Guinea pig complement (
  • inner cell mass-derived outgrowths can be dissociated into clumps, either by exposure to calcium and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then replated on mEF in fresh medium.
  • PBS calcium and magnesium-free phosphate-buffered saline
  • ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli.
  • hESCs can then be routinely split every 1-2 weeks, for example, by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (about 200 U/mL; Gibco) or by selection of individual colonies by micropipette. In some examples, clump sizes of about 50 to 100 cells are optimal.
  • mESCs cells can be prepared from using the techniques described by e.g., Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).
  • Embryonic stem cells can be isolated from blastocysts of members of the primate species (U.S. Pat. No. 5,843,780; Thomson et al., Proc. Natl. Acad Sci. USA 92:7844, 1995).
  • Human embryonic stem (hES) cells can be prepared from human blastocyst cells using the techniques described by Thomson et al. (U.S. Pat. No. 6,200,806; Science 282: 1 145, 1998; Curr Top. Dev. Biol. 38: 133 ff, 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000.
  • Equivalent cell types to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as outlined in WO 01/51610 (Bresagen).
  • hES cells can be obtained from human preimplantation embryos.
  • in vitro fertilized (IVF) embryos can be used, or one cell human embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma).
  • the inner cell masses are isolated by immunosurgery, in which blastocysts are exposed to a 1 :50 dilution of rabbit anti -human spleen cell antiserum for 30 min, then washed for 5 min three times in DMEM, and exposed to a 1 :5 dilution of Guinea pig complement (Gibco) for 3 min (Sober et al., Proc. Natl Acad. Sci USA 72:5099, 1975). After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers.
  • ICM inner cell mass
  • inner cell mass-derived outgrowths are dissociated into clumps, either by exposure to calcium and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then replated on mEF in fresh medium.
  • PBS calcium and magnesium-free phosphate-buffered saline
  • EDTA calcium and magnesium-free phosphate-buffered saline
  • ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells are then routinely split every 1-2 weeks by brief trypsinization, exposure to
  • human Embryonic Germ (hEG) cells are pluripotent stem cells which can be used in the methods as disclosed herein to differentiate into primitive endoderm cells hEG cells can be prepared from primordial germ cells present in human fetal material taken about 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95 : 13726, 1998 and U.S. Pat No. 6,090,622, which is incorporated herein in its entirety by reference
  • genital ridges processed to form disaggregated cells EG growth medium is DMEM, 4500 mg/L D-glucose, 2200 mg/L mM NaHCOy 15% ES qualified fetal calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/mL human recombinant leukemia inhibitory factor (LIF, Genzyme); 1-2 ng/mL human recombinant bFGF (Genzyme); and 10 mM forskolin (in 10% DMSO).
  • fetal calf serum BRL
  • BBL 2 mM glutamine
  • LIF human recombinant leukemia inhibitory factor
  • Genzyme 1-2 ng/mL human recombinant bFGF
  • 10 mM forskolin in 10% DMSO.
  • Ninety-six well tissue culture plates are prepared with a
  • CRL 1503 cultured for 3 days in modified EG growth medium free of LIF, bFGF or forskolin, inactivated with 5000 rad g-irradiation ⁇ 0.2 mL of primary germ cell (PGC) suspension is added to each of the wells.
  • PPC primary germ cell
  • the first passage is done after 7-10 days in EG growth medium, transferring each well to one well of a 24-well culture dish previously prepared with irradiated STO mouse fibroblasts.
  • the cells are cultured with daily replacement of medium until cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages
  • the stem cells can be undifferentiated (e g. a cell not committed to a specific linage) prior to exposure to at least one differentiation medium and/or agent according to the methods as disclosed herein, whereas in other examples it may be desirable to differentiate the stem cells to one or more intermediate cell types prior to exposure of the at least one differentiation medium or agent described herein.
  • the stems cells may display morphological, biological or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryo or adult origin.
  • undifferentiated cells may appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli.
  • the stem cells may be themselves (for example, without substantially any undifferentiated cells being present) or may be used in the presence of differentiated cells
  • the stem cells may be cultured in the presence of suitable nutrients and optionally other cells such that the stem cells can grow and optionally differentiate.
  • suitable nutrients and optionally other cells such that the stem cells can grow and optionally differentiate.
  • embryonic fibroblasts or fibroblast-like cells may be present in the culture to assist in the growth of the stem cells.
  • the fibroblast may be present during one stage of stem cell growth but not necessarily at all stages.
  • the fibroblast may be added to stem cell cultures in a first culturing stage and not added to the stem cell cultures in one or more subsequent culturing stages.
  • Stem cells used in all aspects of the present invention can be any cells derived from any kind of tissue (for example embryonic tissue such as fetal or pre-fetal tissue, or adult tissue), which stem cells have the characteristic of being capable under appropriate conditions of producing progeny of different cell types, e.g. derivatives of at least one of the 3 germinal layers (endoderm, mesoderm, and ectoderm). These cell types may be provided in the form of an established cell line, or they may be obtained directly from primary embryonic tissue and used immediately for differentiation. Included are cells listed in the NEH Human Embryonic Stem Cell Registry, e g.
  • hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc ); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hESl (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and HI, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)).
  • the source of human stem cells or pluripotent stem cells used for chemically-induced differentiation into astrocytes did not involve destroying a human embryo.
  • the stem cells can be isolated from tissue including solid tissue
  • the tissue is skin, fat tissue (e.g. adipose tissue), muscle tissue, heart or cardiac tissue.
  • the tissue is for example but not limited to, umbilical cord blood, placenta, bone marrow, or chondral
  • Stem cells of interest also include embryonic cells of various types, exemplified by human embryonic stem (hES) cells, described by Thomson et al. (1998) Science 282: 1145; embryonic stem cells from other primates, such as Rhesus stem cells (Thomson et al. (1995) Proc Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al, Proc. Natl Acad. Sci. USA 95 : 13726, 1998).
  • hES human embryonic stem
  • stem cells such as mesodermal stem cells and other early cardiogenic cells
  • the stem cells may be obtained from any mammalian species, e.g. human, equine, bovine, porcine, canine, feline, rodent, e g. mice, rats, hamster, primate, etc.
  • a human embryo was not destroyed for the source of pluripotent cell used on the methods and compositions as disclosed herein.
  • ES cells are considered to be undifferentiated when they have not committed to a specific differentiation lineage. Such cells display morphological characteristics that distinguish them from differentiated cells of embryo or adult origin. Undifferentiated ES cells are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli. Undifferentiated ES cells express genes that may be used as markers to detect the presence of undifferentiated cells, and whose polypeptide products may be used as markers for negative selection. For example, see U.S. application Ser. No. 2003/0224411 Al;
  • Human ES cell lines express cell surface markers that characterize undifferentiated nonhuman primate ES and human EC cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase.
  • SSEA stage-specific embryonic antigen
  • the globo-series glycolipid GL7, which carries the SSEA-4 epitope, is formed by the addition of sialic acid to the globo-series glycolipid GbS, which carries the SSEA-3 epitope.
  • GbS which carries the SSEA-3 epitope.
  • the undifferentiated human ES cell lines did not stain for SSEA-1, but differentiated cells stained strongly for SSEA-I. Methods for proliferating hES cells in the undifferentiated form are described in WO 99/20741, WO 01/51616, and WO 03/020920.
  • a mixture of cells from a suitable source of endothelial, muscle, and/or neural stem cells can be harvested from a mammalian donor by methods known in the art.
  • a suitable source is the hematopoietic microenvironment.
  • circulating peripheral blood preferably mobilized (i.e., recruited) may be removed from a subject.
  • bone marrow may be obtained from a mammal, such as a human patient, undergoing an autologous transplant.
  • stem cells can be obtained from the subjects adipose tissue, for example using the CELUTIONTM SYSTEM from Cytori, as disclosed in U. S. Pat. Nos. 7,390,484 and 7,429,488 which is incorporated herein in its entirety by reference.
  • human umbilical cord blood cells are useful in the methods as disclosed herein.
  • Human UBC cells are recognized as a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113).
  • umbilical cord and placental blood were considered a waste product normally discarded at the birth of an infant.
  • Cord blood cells are used as a source of transplantable stem and progenitor cells and as a source of marrow repopulating cells for the treatment of malignant diseases (i.e.
  • HUCBC human umbilical cord blood contains mesenchymal and hematopoietic progenitor cells, and endothelial cell precursors that can be expanded in tissue culture (Broxmeyer et al., 1992 Proc. Natl. Acad Sci. USA 89:4109-41 13; Kohli-Kumar et al., 1993 Br. J. Haematol.
  • the total content of hematopoietic progenitor cells in umbilical cord blood equals or exceeds bone marrow, and in addition, the highly proliferative hematopoietic cells are eightfold higher in HUCBC than in bone marrow and express hematopoietic markers such as CD14, CD34, and CD45 (Sanchez-Ramos et al., 2001 Exp. Neur. 171 : 109-1 15; Bicknese et al., 2002 Cell Transplantation 11 :261-264; Lu et al., 1993 J. Exp Med. 178:2089-2096).
  • pluripotent cells are cells in the hematopoietic micro environment, such as the circulating peripheral blood, preferably from the mononuclear fraction of peripheral blood, umbilical cord blood, bone marrow, fetal liver, or yolk sac of a mammal
  • the stem cells especially neural stem cells, may also be derived from the central nervous system, including the meninges.
  • pluripotent cells are present in embryoid bodies are formed by harvesting ES cells with brief protease digestion, and allowing small clumps of undifferentiated human ESCs to grow in suspension culture. Differentiation is induced by withdrawal of conditioned medium. The resulting embryoid bodies are plated onto semi-solid substrates. Formation of differentiated cells may be observed after around about 7 days to around about 4 weeks. Viable differentiating cells from in vitro cultures of stem cells are selected for by partially dissociating embryoid bodies or similar structures to provide cell aggregates Aggregates comprising cells of interest are selected for phenotypic features using methods that substantially maintain the cell to cell contacts in the aggregate.
  • the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic or differentiated cells.
  • the de differentiated stem cells can be for example, but not limited to, neoplastic cells, tumor cells and cancer cells or alternatively induced reprogrammed cells such as induced pluripotent stem cells or iPS cells.
  • Illustrative reagents, cloning vectors, and kits for genetic manipulation may be commercially obtained, for example, from BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.
  • Suitable cell culture methods may be found in the current edition of Culture of Animal Cells: A Manual of Basic Technique (R. I. Freshney ed., Wiley & Sons); General Techniques of Cell Culture (M. A. Harrison & I. F. Rae, Cambridge Univ. Press), and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press).
  • Suitable tissue culture supplies and reagents are commercially available, for example, from
  • Pluripotent stem cells can be propagated by one of ordinary skill in the art and continuously in culture, using culture conditions that promote proliferation without promoting differentiation.
  • Exemplary serum-containing ES medium is made with 80% DMEM (such as Knock-Out DMEM, Gibco), 20% of either defined fetal bovine serum (FBS, Hyclone) or serum replacement (WO 98/30679), 1% non-essential amino acids, 1 mM L- glutamine, and 0.1 mM b-mercaptoethanol.
  • human bFGF is added to 4 ng/mL (WO 99/20741, Geron Corp.).
  • ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue.
  • the environment for feeder-free cultures includes a suitable culture substrate, particularly an extracellular matrix such as
  • MATRIGEL® gelatinous protein mixture
  • laminin laminin
  • Feeder-free cultures are supported by a nutrient medium containing factors that support proliferation of the cells without differentiation.
  • factors may be introduced into the medium by culturing the medium with cells secreting such factors, such as irradiated ( ⁇ 4,000 rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells.
  • Medium can be conditioned by plating the feeders at a density of ⁇ 5-6x 10 4 cm -2 in a serum free medium such as KO DMEM
  • ES cells Under the microscope, ES cells appear with high nuclear/cytoplasmic ratios, prominent nucleoli, and compact colony formation with poorly discernable cell junctions. Primate ES cells express stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282: 1145, 1998). Mouse ES cells can be used as a positive control for SSEA-l, and as a negative control for SSEA-4, Tra-l-60, and Tra-l-8l . SSEA-4 is consistently present human embryonal carcinoma (hEC) cells. Differentiation of pluripotent SCs in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression, and increased expression of SSEA-l, which is also found on undifferentiated hEG cells.
  • SSEA stage-specific embryonic antigens
  • aspects of the disclosure relate to generating functional astrocytes (e.g., brain astrocytes, spinal cord astrocytes, and the like)
  • functional astrocytes e.g., brain astrocytes, spinal cord astrocytes, and the like
  • the functional astrocytes produced according to the methods disclosed herein demonstrate several hallmarks of functional astrocytes, including, but not limited to, ability to propagate calcium waves, expression of canonical markers, and produce and secrete C4.
  • the functional astrocytes can be produced according to any suitable culturing protocol to differentiate a stem cell or pluripotent cell to a desired stage of differentiation.
  • the functional astrocytes are produced by culturing at least one stem cell for a period of time and under conditions suitable for the at least one stem cell to differentiate into the astrocyte or a precursor thereof.
  • the functional astrocytes or precursor thereof is a substantially pure population of functional astrocytes or precursors thereof
  • a population of functional astrocytes or precursors thereof comprises a mixture of pluripotent cells or differentiated cells.
  • a population of functional astrocytes or precursors thereof is substantially free or devoid of embryonic stem cells or pluripotent cells.
  • the functional astrocytes or precursors thereof are maintained in culture by methods known by one of ordinary skill in the art, and in some embodiments, propagated prior to being converted into functional astrocytes by the methods as disclosed herein.
  • stem cells e.g , hESCs or iPSCs
  • the stem cells may be grown in mTeSR medium or StemFlexTM medium.
  • the pluripotent spheres may express appropriate markers, including OCT4, NANOG, and/or Tral-60.
  • mTeSR medium is supplemented with a ROCK inhibitor.
  • the ROCK inhibitor is Y-27632.
  • neutralization is initiated by supplementing the mTESR medium with an activin/TGF-b inhibitor and a BMP inhibitor.
  • the activin/TGF-b inhibitor is SB431542.
  • the BMP inhibitor is LDN193189.
  • the combination of the activin/TGF-b inhibitor and BMP inhibitor may be referred to as dual SMAD inhibition.
  • the stem cells may be differentiated to form brain astrocytes.
  • the pluripotent spheres were cultured in at least one differentiation medium. The culturing of the pluripotent spheres in the differentiation medium induces the differentiation of at least one sphere in the spin culture into an astrocyte sphere.
  • a first differentiation medium is a KSR medium.
  • KSR media includes a supplemental agent.
  • the supplemental agent may be selected from the group consisting of activin/TGF-b inhibitor, Dorsomorphin, and combinations thereof.
  • the activin/TGF-b inhibitor is SB431542.
  • a second differentiation medium is a neurobasal (NB) medium.
  • the NB medium includes a supplemental agent.
  • the supplemental agent may be selected from the group consisting of EGF, FGF, CTNF, and combinations thereof.
  • the spheres subjected to differentiation may be maintained in KSR for a period of 1 to 5 days, and in certain embodiments for a period of 5 days.
  • the KSR medium includes a supplemental agent.
  • differentiated spheres may be maintained in nuerobasal medium.
  • differentiated spheres may be maintained in nuerobasal medium for a period of 1 to 10 days, 1 to 15 days, or 1 to 20 days, and in certain embodiments for a period of 15 days.
  • the NB medium includes a supplemental agent.
  • the stem cells may be differentiated to form spinal cord astrocytes.
  • the pluripotent spheres were gradually adapted to neural induction medium (NIM).
  • the pluripotent spheres may be gradually adapted to NIM through a dilution series of KSR and NIM.
  • the dilution series of KSR and NIM includes dual SMAD inhibition.
  • the pluripotent spheres were cultured in at least one differentiation medium. The culturing of the pluripotent spheres in the
  • a first differentiation medium is a KSR medium.
  • the KSR medium includes a supplemental agent.
  • the supplemental agent may be selected from the group consisting of SB431542, LDN193189, retinoic acid, BDNF, SHH, DAPT, and combinations thereof.
  • a second differentiation medium is a neural induction medium.
  • the neural induction medium (NIM) includes a supplemental agent.
  • the supplemental agent may be selected from the group consisting of SB431542, LDN193189, retinoic acid, BDNF, SHH, DAPT, and combinations thereof
  • a third differentiation medium is an astrocyte medium.
  • the spheres subjected to differentiation may be maintained in KSR for a period of 1 to 5 days, and in certain embodiments for a period of 5 days.
  • the KSR medium includes a supplemental agent.
  • the KSR medium includes dual SMAD inhibition.
  • the spheres subjected to differentiation may be maintained in NIM for a period for 1 to 15 days, and in certain embodiments for a period of 5 days.
  • spheres subjected to differentiation are gradually adapted to NIM through a dilution series of KSR and NIM, optionally with dual SMAD inhibition.
  • the dilution series of KSR and NIM media may include 75% KSR and 25% NIM, 50% KSR and 50% NIM, and 25% KSR and NIM 75% media.
  • the dilution series occurs over a period of 1, 2, 3, 4, 5, 6, or 7 days.
  • the spheres subjected to differentiation may be maintained in NIM.
  • NIM includes a supplemental agent.
  • the spheres subjected to differentiation may be maintained in NIM for a period of 1 to 10 days, and in certain embodiments for a period of 5 days.
  • the differentiated spheres are maintained in astrocyte media for a period of 1 to 15 days, and in certain embodiments for a period of 15 days.
  • astrocyte spheres are dissociated and plated. In some aspects astrocyte spheres are dissociated 30 days after culture and differentiation begin. Functional Astrocytes
  • the disclosure provides functional astrocytes.
  • the functional astrocytes include functional brain astrocytes.
  • the functional astrocytes include functional spinal cord astrocytes.
  • the method can include detecting the positive expression (e g the presence) of a marker for astrocytes (e g., brain astrocytes and/or spinal cord astrocytes).
  • a marker for astrocytes e g., brain astrocytes and/or spinal cord astrocytes.
  • the marker can be detected using a reagent.
  • a reagent for a marker can be, for example, an antibody against the marker or primers for a RT- PCR or PCR reaction, e.g , a semi-quantitative or quantitative RT-PCR or PCR reaction.
  • the antibody or other detection reagent can be linked to a label, e.g., a radiological, fluorescent (e.g., GFP) or colorimetric label for use in detection. If the detection reagent is a primer, it can be supplied in dry preparation, e.g., lyophilized, or in a solution.
  • the progression of at least one stem cell or precursor thereof to a functional astrocyte can be monitored by determining the expression of markers characteristic of astrocytes (e.g., brain astrocytes and/or spinal cord astrocytes). In some processes, the expression of certain markers is determined by detecting the presence or absence of the marker. Alternatively, the expression of certain markers can be determined by measuring the level at which the marker is present in the cells of the cell culture or cell population. In certain processes, the expression of markers characteristic of functional astrocytes, as well as the lack of significant expression of markers characteristic of the stem cells or precursors thereof is determined.
  • markers characteristic of astrocytes e.g., brain astrocytes and/or spinal cord astrocytes.
  • the expression of certain markers is determined by detecting the presence or absence of the marker. Alternatively, the expression of certain markers can be determined by measuring the level at which the marker is present in the cells of the cell culture or cell population. In certain processes, the expression of markers characteristic of functional astrocytes, as well as the lack of significant expression of
  • markers of astrocytes are secreted proteins (e.g., complement protein C4).
  • techniques for measuring extracellular marker content such as ELISA, may be utilized.
  • cells of the inventive culture express one or more gene expression markers selected from CD44, SlOOb, GFAP, CX43, and ALDH1L1.
  • cells of the inventive culture produce and secrete C4.
  • the present invention is not limited to those markers listed as functional astrocyte markers herein, and the present invention also encompasses markers such as cell surface markers, antigens, and other gene products including ESTs, RNA (including microRNAs and antisense RNA), DNA (including genes and cDNAs), and portions thereof.
  • the methods of the invention allow for the generation of functional astrocytes that exhibit one or more features.
  • the one or more features include the ability to propagate calcium waves.
  • the one or more features include the ability to increase the firing potential and synapsis number of cortical neurons (e.g., ngn2-derived cortical neurons) when co-cultured with astrocytes.
  • the methods of the invention allow for the generation of functional spinal cord astrocytes.
  • the astrocytes may maintain expression of spinal cord astrocyte markers.
  • the spinal cord astrocytes propagate calcium waves.
  • the methods of the invention allow for the generation of functional brain astrocytes.
  • the astrocytes may maintain expression of brain astrocyte markers.
  • the brain astrocytes when co-cultured with neurons (e.g., ngn2- derived cortical neurons) increase the firing potential and synapsis number of the neurons.
  • astrocytes are treated with a test agent.
  • the astrocytes are functional spinal cord astrocytes and/or functional brain astrocytes produced by the differentiation protocols disclosed herein.
  • test agents that may be screened include those contained within a Target Selective Inhibitor Library (Sellckchem (Catalog No. L3500)), incorporated herein by reference.
  • the level of secreted C4 from the treated astrocytes may be assessed and measured, for example, by using an ELISA.
  • the total number of nuclei in the treated cells is counted and used to normalize the C4 secretion levels.
  • the number of counted nuclei may be compared to a standard deviation of a control (e.g., a DMSO control). In some aspects, the number of nuclei, and thus the level of C4 secretion, is below the standard deviation of the control. In some aspects agents are identified as being of interest as a modulator of C4 secretion if the level of C4 secretion is decreased by up to 10%. In some aspects identified modulators of C4 secretion are used to identify pathways involved in C4 modulation. In some aspects screening for modulators of C4 secretion results in identifying pathways potentially involved in C4 regulation.
  • a control e.g., a DMSO control
  • agents are identified as being of interest as a modulator of C4 secretion if the level of C4 secretion is decreased by up to 10%.
  • identified modulators of C4 secretion are used to identify pathways involved in C4 modulation. In some aspects screening for modulators of C4 secretion results in identifying pathways potentially involved in C4 regulation
  • Pathways that may be involved in C4 regulation include, but are not limited to, angiogenesis, MAPK, JAK/STAT, epigenetics, transmembrane transporters, protein tyrosine kinase, proteases, metabolism, GPCR & G protein, cell cycle, apoptosis, NF-kB, MAPK, ubiquitin, endocrinology and hormones, PBK/Akt/mTOR, neuronal signaling, TGF-p/Smad, microbiology, DNA damage, and cytoskeletal signaling.
  • test agent identified as downregulating C4 secretion may exhibit beneficial effects on a disease (e.g., a neurodegenerative, neuropsychiatric or
  • test agent may reduce excessive synaptic pruning.
  • the invention provides methods of treating or preventing a disease characterized by over secretion of complement component 4 (C4) comprising administering to the subject an effective amount of at least one agent which modulates C4 secretion in a subject.
  • the at least one agent is an agent which decreases or reduces C4 secretion.
  • the at least one agent is an agent which modulates (e.g., decreases) the expression of C4.
  • the disease characterized by over secretion of C4 is a neurodegenerative, neuropsychiatric, or neurodevelopmental disease.
  • the disease characterized by over secretion of C4 exhibits excessive synaptic pruning.
  • the disease is schizophrenia.
  • the disease is Alzheimer’s disease.
  • the disease is Rett Syndrome.
  • the disease is Huntington Disease.
  • the disease is multiple sclerosis.
  • the at least one agent which downregulates the over secretion of C4 is an agent which targets one or more pathways involved in C4 secretion.
  • the at least one agent targets an epigenetic pathway, a JAK/STAT pathway, a PBK/Akt/mTOR pathway, a MAPK pathway, a metabolism pathway, an angiogenesis pathway, a GPCR and/or G protein pathway, a NF-kB pathway, a proteases pathway, a protein tyrosine kinase pathway, an ubiquitin pathway, an apoptosis pathway, a cell cycle pathway, a DNA damage pathway, a transmembrane transporter pathway, an endocrinology and hormone pathway, a neuronal signaling pathway, a TGF-Beta/SMAD pathway, a microbiology pathway, or a cytoskeletal signaling pathway.
  • the at least one agent targets an epigenetic pathway
  • the at least one agent targets a JAK/STAT pathway, a PBK/Akt
  • the at least one agent targets a PBK/Akt/mTOR pathway. In some aspects, the at least one agent targets a MAPK pathway. In some aspects, the at least one agent targets a metabolism pathway. In some aspects, the at least one agent targets an angiogenesis pathway. In some aspects, the at least one agent targets a GPCR and/or G protein pathway In some aspects, the at least one agent targets a NF-kB pathway.
  • the at least one agent targets a proteases pathway. In some aspects, the at least one agent targets a protein tyrosine kinase pathway. In some aspects, the at least one agent targets an ubiquitin pathway. In some aspects, the at least one agent targets an apoptosis pathway. In some aspects, the at least one agent targets a cell cycle pathway In some aspects, the at least one agent targets a DNA damage pathway. In some aspects, the at least one agent targets a transmembrane transporter pathway. In some aspects, the at least one agent targets an endocrinology and hormone pathway. In some aspects, the at least one agent targets a neuronal signaling pathway.
  • the at least one agent targets a TGF-beta/SMAD pathway In some aspects, the at least one agent targets a microbiology pathway. In some aspects, the at least one agent targets a cytoskeletal signaling pathway. Targeting of a pathway is used to describe an agent that acts as an agonist or antagonist of a pathway.
  • beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment.
  • treatment includes prophylaxis.
  • treatment is “effective” if the progression of a disease is reduced or halted.“Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • the symptom of a disorder characterized by over secretion of C4 is alleviated by at least 20%, at least 30%, at least 40%, or at least 50%. In one embodiment, the symptom of a disorder characterized by over secretion of C4 is alleviated by more than 50%. In one embodiment, the symptom of a disorder characterized by over secretion of C4 is alleviated by 80%, 90%, or greater. In one embodiment, the symptom of a neurodegenerative disorder is alleviated by at least 20%, at least 30%, at least 40%, or at least 50%. In one embodiment, the symptom of a
  • neurodegenerative disease is alleviated by more than 50%.
  • the symptom of a neurodegenerative disorder is alleviated by 80%, 90%, or greater.
  • treatment also includes improvements in synaptic function.
  • synaptic function improves by at least about 10%, 20%, 30%, 40%, 50% or more.
  • treatment includes downregulating the secretion of C4.
  • the secretion of C4 is reduced by at least about 10%, 20%, 30%, 40%, 50% or more.
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “patient” and“subject” are used interchangeably herein.
  • the subject is a human.
  • test compound refers to any of a small molecule, nucleic acid, amino acid, polypeptide, antibody and antibody-like molecules, aptamers, macrocycles, or other molecules.
  • a test compound is a small organic molecule.
  • the small organic molecule has a molecular weight of less than about 5,000 daltons.
  • neurodegenerative disorder or“neuro disease” refer to neurodegenerative disorders, neuropsychiatric disorders and/or neurodevelopmental disorders. Neuro disorders may be any disease affecting neuronal network connectivity, synaptic function and activity. “Neurodegenerative disorder” refers to a disease or disorder caused by or associated with the deterioration of cells or tissues of the nervous system.
  • Non-limiting examples of neurodegenerative disorders include polyglutamine expansion disorders (e g., HD, dentatorubropallidoluysian atrophy, Kennedy's disease (also referred to as spinobulbar muscular atrophy), and spinocerebellar ataxia (e.g., type 1, type 2, type 3 (also referred to as Machado-Joseph disease), type 6, type 7, and type 17)), other trinucleotide repeat expansion disorders (e g., fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8, and spinocerebellar ataxia type 12), Alexander disease, Alper's disease, Alzheimer disease, amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, Batten disease (also referred to as Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockayne syndrome, corticobas
  • neurodegenerative disorders encompass neurological injuries or damages to the CNS or the PNS associated with physical injury (e.g., head trauma, mild to severe traumatic brain injury (TBI), spinal cord injury, diffuse axonal injury, craniocerebral trauma, cranial nerve injuries, cerebral contusion, intracerebral haemorrhage and acute brain swelling), ischemia (e.g., resulting from spinal cord infarction or ischemia, ischemic infarction, stroke, cardiac insufficiency or arrest, atherosclerotic thrombosis, ruptured aneurysm, embolism or haemorrhage), certain medical procedures or exposure to biological or chemic toxins or poisons (e.g., surgery, coronary artery bypass graft (CABG), electroconvulsive therapy, radiation therapy, chemotherapy, anti-neoplastic drugs, immunosuppressive agents, psychoactive, sedative or hypnotic drugs, alcohol, bacterial or industrial toxins, plant poisons, and venom
  • physical injury
  • the subject suffers from a disorder or disease characterized by over secretion of C4. In some aspects the subject suffers from a disorder or disease characterized by excessive synaptic pruning. In some aspects the subject suffers from a neurodegenerative disease. In some aspects the neurodegenerative disease is characterized by over secretion of C4 or excessive synaptic pruning.
  • the methods described herein further comprise selecting a subject diagnosed with a disorder characterized by over secretion of C4. In some aspects the methods described herein further comprise selecting a subject diagnosed with a disorder characterized by excessive synaptic pruning. In some aspects the methods described herein further comprise selecting a subject diagnosed with a neurodegenerative disease. A subject suffering from a neurodegenerative disease can be selected based on the symptoms presented.
  • the methods described herein further comprise diagnosing a subject for a neurodegenerative disease or disorder. In some embodiments, the methods described herein further comprise diagnosing a subject for schizophrenia. In some embodiments, the methods described herein further comprise diagnosing a subject for Alzheimer’s disease.
  • the methods further comprises co-administering an additional pharmaceutically active agent approved for treatment of the neurodegenerative disorder or alleviating a symptom thereof.
  • iPSCs Induced pluripotent stem cells
  • ESCs embryonic stem cells
  • ES and iPS cells into brain (bA) or spinal cord astrocytes (spA).
  • stem cells acquire a neuroepithelial fate expressing neural stem cells markers such as Pax6 and Sox2 and express markers appropriate to of either dorsal or ventral identity (OTX1/2 and HoxB4).
  • OTX1/2 and HoxB4 dorsal or ventral identity
  • These populations of astrocytes can be expanded and/or cryopreserved, surviving freeze-thaw cycles.
  • the stem cell- derived astrocytes exhibit the ability to propagate calcium waves.
  • Co-culture of ngn2-derived cortical neurons with spin produced bA increases they firing potential as well the synapsis number, pointing toward a physiological role of the differentiated astrocytes in supporting neuronal growth and maturation.
  • pro-inflammatory stimuli such as TNFa and IL 1 b both spinal cord and brain astrocytes become reactive, a hallmark of traumatic and pathological conditions.
  • astrocytes from patient specific cells and the capability of modulating their phenotype in a biologically relevant manner, provide an opportunity to unravel the potential contribution of astrocytes in neurodegenerative and neuropsychiatric diseases. Furthermore the large production of iPS/ES- deived astrocytes provide a unique source for high-throughput screening
  • astrocytes Compounds, shRNAs library or CRISPR/Cas9 library.
  • the generation of these astrocytes may lead to a better understanding of astrocyte-neuron interaction, as well as cell autonomous disease phenotypes.
  • astrocytes are crucial for the formation and remodeling of synapses, and based on recent literature proposing the complement component and the synaptic pruning as a hallmark of schizophrenia, it was hypothesized that astrocytes might play a role in this process. It was shown that astrocytes are able to secrete the complement component 4 (C4) and this secretion can be modulated. Taking advantage of the capability to produce large amount of astrocytes, a screening of compounds was performed to find modulators of C4 secretion in astrocytes. The screening was performed seeding iPSC-derived astrocytes (bA) into 96 well plates (30.000 cells per well). The day after medium was removed and fresh medium containing the compounds was added to the cells.
  • bA iPSC-derived astrocytes
  • the compound library used was a kinase inhibitor library containing 464 different compounds (Selleckchem).
  • the library was screened in triplicate plates at two different concentrations (1 and 0.3 uM). Two days after the addition of compounds supernatant was used to assess the level of secreted C4 by home made ELISA. Plates with cells were stained with DAPI and acquired on the Operetta High- content Imaging System (PerkinElmer). Nuclei were counted using the Columbus software. The total number of nuclei was used to normalize the C4 secretion levels. For the analysis of the screening all nuclei that were below the standard deviation of the average of nuclei in a specific plate were excluded (to exclude false positive due to compounds toxicity).
  • iPSCs or ESCs were maintained in mTeSR (STEM CELL Technologies) or
  • StemFlexTM Medium (Thermo-Fisher Scientific). Pluripotent stem cells were cultured in 10 or 15-cm dishes (Coming) and passaged when reaching 80% confluency in colonies using 0.5 mM EDTA at room temperature on Matrigel (Corning) coated plates. Human brain astrocytes media
  • Neurobasal (Gibco), 2X N2 supplement 100X (Gibco), Glutamax (Gibco), NEAA (Gibco), and penicillin-streptomycin (Gibco).
  • NCM Neural induction media
  • DMEM/F12 (Gibco), N2 supplement (Gibco), Glutamax (Gibco), NEAA (Millipore), penicillin-streptomycin (Gibco), glucose, and ascorbic acid to a final concentration of 0.4 ug/mL (Sigma).
  • medium was changed to KSR with activin/TGF-b inhibitor SB431542 (R&D Systems) and Dorsomorphin (stemgent) to a final concentration of 10 uM and 1 uM respectively.
  • Media is changed every day for the first 5 days. From day 6 to day 12 media is changed every 2 days NB media 2X N2 with dorsomorphin and supplement with different cytokines addition as specified. From day 6 and day 8 FGF2 and EGF (10 ng/mL) are added to the media. On day 10 and 12 FGF2 , EGF and CTNF 20ng/mL are added. On day 14 NB 2 x N2 media contain CTNF and FGF 20ng/mL. From day 16 on media is changed every 2 days (NB 2x N2 CTNF 20 ng/mL).
  • astrocytes spheres were dissociated and plated. Spheres were collected in a 15 mL tube and let settle. The media was removed and spheres were washed with IX PBS. After the spheres settled and the PBS is removed . Double of the volume of 0.25% trypsin is added to the spheres and the tube is incubated in a water bath at 37 °C for 5-10 minutes. Spheres are shacked every couple of minutes until the suspension look cloudy. An equal amount of the sphere volume of FBS is added to quench the trypsin. Cells are spun for 3 minutes at 300g.
  • a 3 mL of dissociation buffer is added to tube and spheres are mechanically dissociated using a 5 mL pipette. This operation is repeated until the spheres are completely dissociated.
  • Single cells are filtered using a 40 uM filter and centrifuged at 300g for 3 minutes. Cells are resuspended at the desired concentration and plated on Poly-L-Lysine coated plates.
  • PBS-Glucose buffer 250 mL
  • lx PBS 5% FBS (12.5 mL)
  • 25mM Glucose 6.25 mL of 1M
  • 5 mM MgCl 2 1.25 ml of 1M
  • iPSCs and ESCs were cultured in StemFlex medium (ThermoFisher A3349401). When pluripotent stem cells reached 80-85% of confluency, colonies were dissociated using 0.5 mM EDTA in calcium/magnesium-free PBS at room temperature and passaged on Matrigel (Coming 354234) coated 10 or l5-cm 2 tissue culture dishes (Coming). All human pluripotent stem cells used were maintained below passage 40 and confirmed to be karyotypically normal and mycoplasma negative
  • Pluripotent stem cells were single-cell dissociated using ACCUTASE as previously described [1] Briefly cells were seeded into a 125 mL spinner flask in 100 mL of mTeSR medium supplemented with 10 mM ROCK inhibitor Y-27632 (STEMCELL) at a concentration of lxlO 6 cells/ mL. Spinner flask was placed on a nine-position stir plate (Dura-Mag) at a speed of 55 rpm, in a 37°C incubator with 5% C0 2 . Under this condition cells spontaneously aggregate forming pluripotent spheres.
  • Dura-Mag nine-position stir plate
  • astrocytes spheres were dissociated using 0.25% trypsin (Gibco 25200056) and plated on overnight poly-L lysine (PLL) (MP BIOMEDICALS 02194544) coated plates.
  • PLL poly-L lysine
  • spheres were collected in a 15 mL tube and let settled down by gravity. The media was removed and spheres were washed with IX PBS. After the spheres settled down, the PBS was removed. Double of the volume of 0.25% trypsin is added to the spheres and the tube is incubated in a water bath at 37 °C for 5-10 minutes. Spheres were shaken every couple of minutes until the suspension looked cloudy.
  • Astrocytes were plated on PLL coated plates 6 wells or 96 wells at a density of 5X10 5 cells and 3X10 4 cells per well respectively. The next day cells were fixed using 4% PFA for 15 minutes and washed with PBS three times. The cells were blocked in 10% horse serum 0.01% Triton X-100 in PBS (for CD44 staining only) or 5% horse serum 0.3% PBS Triton X- 100 for 1 hour a room temperature. Primary antibodies were diluted in 5% horse serum at 4°C overnight followed by washes in PBS and incubation with secondary antibodies (diluted 1 : 1000) and Hoechst (1 :5000) for 1 hour a room temperature.
  • Fluorescently conjugated antibodies used were goat anti-mouse IgG Alexa Fluor 488 (Life Technologies A11001) goat anti-rabbit IgG Alexa Fluor 546 (Life Technologies A11010). Bright field images were acquired using an inverted Eclipse Ti microscope (Nikon). Immunofluorescences were acquired either using a ImageXpress Micro Confocal (Molecular device) or Opera High Content Screening System (Perkin Elmer). All images were processed with Adobe Photoshop software.
  • Freshly dissociated or frozen astrocytes were cultured as previously described until they reached 80% confluency. Cells were detached using Trypsin-EDTA solution (Sigma, T3924). lxlO 6 cells were staining following the manufacturer's instruction for cell surface antigens using directly conjugated antibodies against FITC CD44,
  • Astrocytes were plated in 6 well plates at a density of 5xl0 5 cells per well in complete AM media (Science Cell) the next day cells were treated with compound or mock and after 48 hours supernatant was collected and stored at-80 °C
  • Proteome ProfilerTM Human Cytokine Array (R&D Systems, #ARY005B) was used according to manufacturer's guidelines. Proteome profiler intensity dot blots were quantified using Adobe Photoshop software and were normalized to mean intensities of reference spots.
  • the membrane was washed once with TBST and incubated with antibodies against C4 (Dako F 0169 1 : 1000 or Quidel A305 1 : 1000 in 5% TBST overnight at 4°C.
  • Membranes were washed three times for 10 minutes and incubated with a 1 : 1000 dilution of horseradish peroxidase-conjugated anti-rabbit antibodies for 1 h. Blots were washed with TBST three times and developed with the SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific 34075)
  • Sofroniew MV Vinters HV. Astrocytes: biology and pathology. Act Neuropathol 2010 Jan; 119(l):7-35.
  • Tyzack G Lakatos A, Patani R. Human stem cell-derived astrocytes: specification and relevance for neurological disorders. Curr Stem Cell Rep. 2016; 2:236-247. Krencik R, Weick JP, Liu Y, Zhang ZJ, Zhang SC. Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nat Biotechnol. 2011 May 22;29(6):528-34.

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Abstract

L'invention concerne des astrocytes n'existant pas à l'état naturel et des procédés de production d'astrocytes n'existant pas à l'état naturel à partir de cellules souches. L'invention concerne également des procédés de criblage à la recherche de modulateurs du constituant 4 du complément (C4). L'invention concerne également des méthodes de traitement d'un trouble neurodégénératif ou neuropsychiatrique par la modulation de la sécrétion de C4.
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CN113337469A (zh) * 2021-07-16 2021-09-03 广州市妇女儿童医疗中心 星形胶质细胞亚群及其分离纯化方法和应用
WO2021231204A1 (fr) * 2020-05-11 2021-11-18 Genentech, Inc. Inhibiteurs du composant 4 du complément pour le traitement de maladies neurologiques, et compositions associées, systèmes et procédés d'utilisation de ceux-ci
US11989960B2 (en) 2019-02-20 2024-05-21 Bluerock Therapeutics Lp Detecting cells of interest in large image datasets using artificial intelligence

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KRENCIK, R ET AL.: "Directed Differentiation of Functional Astroglial Subtypes from Human Pluripotent Stem Cells", NATURAL PROTOCOLS, vol. 6, no. 11, 13 October 2011 (2011-10-13), pages 1710 - 1717, XP055615854 *
RIGAMONTI, A ET AL.: "Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System", STEM CELL REPORTS, vol. 6, no. 6, 14 June 2016 (2016-06-14), pages 993 - 1008, XP055395234 *
WASIAK, S ET AL.: "Downregulation of the Complement Cascade In Vitro, in Mice and in Patients with Cardiovascular Disease by the BET Protein Inhibitor Apabetalone (RVX-208", JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH, vol. 10, no. 4, 31 May 2017 (2017-05-31), pages 337 - 347, XP036313993 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11989960B2 (en) 2019-02-20 2024-05-21 Bluerock Therapeutics Lp Detecting cells of interest in large image datasets using artificial intelligence
WO2021231204A1 (fr) * 2020-05-11 2021-11-18 Genentech, Inc. Inhibiteurs du composant 4 du complément pour le traitement de maladies neurologiques, et compositions associées, systèmes et procédés d'utilisation de ceux-ci
CN113337469A (zh) * 2021-07-16 2021-09-03 广州市妇女儿童医疗中心 星形胶质细胞亚群及其分离纯化方法和应用

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