WO2015195780A1 - Assay for cell specification in self-renewing human pluripotent stem cells - Google Patents

Assay for cell specification in self-renewing human pluripotent stem cells Download PDF

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WO2015195780A1
WO2015195780A1 PCT/US2015/036196 US2015036196W WO2015195780A1 WO 2015195780 A1 WO2015195780 A1 WO 2015195780A1 US 2015036196 W US2015036196 W US 2015036196W WO 2015195780 A1 WO2015195780 A1 WO 2015195780A1
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cells
cell
population
expression
cultured
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French (fr)
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Ronald D.G. MCKAY
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Lieber Institute For Brain Development
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics

Definitions

  • the invention provides assays and systems using pluripotent cells for patient stratification, target identification and drug screening.
  • the present invention provides cells and in vitro assay systems useful for simulating and modeling early processes in cell specification and differentiation and morphogenesis as well as cell self-renewal and pluripotency.
  • the inventors have surprisingly found that these initial steps in cell specification and differentiation and morphogenesis may significantly depend upon the genomic and epigenetic context of the cells.
  • the invention provides in vitro assay systems for analyzing the early steps of cell specification, differentiation and self-renewal to permit the identification of pharmacological targets involved in these processes and as well as providing systems for the screening of agents that can modulate, i.e., inhibit or promote these processes.
  • the invention further provides assay systems and methods that exploit the genome and epigenetic specificity of the behavior of cells, including patient specific cells, in these systems to analyze and compare these early cell specification, morphogenesis and self-renewal processes in pluripotent cells derived from specific patients, for example, patients known to have one or more genetic polymorphisms associated with a particular disease or disorder.
  • the results of these assays inform disease mechanisms, revealing potential pharmacological targets, and are useful in drug discovery.
  • the behavior of patient specific cells in the assays of the invention may also be characteristic or diagnostic of a disease state, predisposition to a disease state or of patient and genome specific cell specification.
  • pluripotent stem cell cultures for example human embryonic stem cells or induced pluripotent stem cells (iPS cells)
  • iPS cells induced pluripotent stem cells
  • An "equivalence group” is a population of cells that have been set to an equivalent state of pluripotency and earliest stage of cell specification, in which the cells are synchronized to initiate processes of cell specification and morphogenesis.
  • the cells may become lineage primed, as evidenced through changes in gene expression, but remain pluripotent during the cycle of the assay system of the invention.
  • the equivalence group cells can be characterized by the expression of particular molecular markers, for example, but not limited to, expression of pluripotentcy factors, such as, but not limited to, NANOG and OCT4, or lack of protein expression of one or more markers of cell differentiation, such as but not limited to, SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN.
  • the equivalence group may also be defined functionally, for example as described in Example 2 herein.
  • the equivalence group establishes a baseline for the temporal processes of cell specification, particularly neuronal specification (although the system may also be used to simulate the processes of specification to other cell fates such as mesodermal or endodermal cell fates), morphogenesis and self-renewal.
  • the equivalence group of cells is then cultured for a period of time during which the cell specification is reversible, that is the cells may be reset to the original state of potency and cell specification, i.e., not beyond the point at which the cells are committed to a particular cell fate such that they cannot be reset to the equivalence group at a particular state of potency and cell specification by cell dissociation. This period of time may be anywhere from 1 to 10 days and may be determined experimentally.
  • the cells form epithelial cell monolayers that simulate cell specification, particularly neuronal specification and morphogenesis, and self renewal.
  • molecular markers for pluripotency such as NANOG and OCT4
  • molecular markers for cell specification to a particular lineage such as, neurectodermal, mesodermal or endodermal lineages, for example, the proteins SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and/or EOMESODERMIN, increase.
  • the epithelial sheet of cells becomes progressively more organized into zones of cells that represent spatially restricted expression of specific markers of cell specification and morphological characteristics, resulting in a specific laminar organization of the epithelial sheet or colony.
  • the cells may be organized into edge, intermediate and core zones (EZ, IZ, and CZ, respectively) of the epithelial sheet (see FIG 2).
  • the zones may be characterized by expression of different levels of molecular markers, or different molecular markers, or by phenotypic or morphological differences, such as nuclear area.
  • the EZ may be characterized by EpCAM expression, which is reduced or not present in the IZ and/or CZ.
  • Nanog, SOX2 and SOX21 may also be used to identify and delineate the zones.
  • the zones may also be delineated by differences in nuclear area. Nuclear area may be assessed in cells stained with DAPI. Characteristically, the inner zone (IZ) has low nuclear area while the edge zone (EZ) has a larger nuclear area. A combination of nuclear area and molecular marker visualization may be used to define the different zones. Such cells may be lineage primed, i.e., they are on a pathway towards that fate specification, but they are still able to be reset to the equivalence group by cell dissociation.
  • the cultured cells may be analyzed, assayed and/or compared during this culture period.
  • the cells may be monitored continually or at specific timepoints during the culture period.
  • the cells may be assayed for changes in expression of particular markers, for changes to gene expression fingerprints, for morphological and/or phenotypic changes.
  • the pattern of gene expression within the epithelial sheet, including the laminar organization of the sheet, particularly, the spatially distinct expression of molecular markers of cell specification may be assayed, characterized and compared.
  • Such analysis may be carried out, for example, by high content analysis of the colony organization, for example, as described herein.
  • Comparison of these molecular and phenotypic changes across cells derived from different sources can reveal genomic and epigenetic differences and the physiological, morphological, phenotypic or gene expression impact of those differences.
  • the entire cultured cell population may be assayed together or the different zones, or combinations of zones, may be assayed separately.
  • the dynamic distribution of these markers, indicative of steps in cell specification and morphogenesis may be monitored and visualized.
  • Characteristic patterns of gene expression within the laminar organization of the colony may be assayed and defined by any method known in the art, for example, high content analysis or any other analytical method.
  • the gene expression, phenotypic and/or morphological read outs from the cells may be diagnostic and/or prognostic of a disease state or susceptibility to a disease state, or susceptibility to treatment of a disease state.
  • the cultured cells may be useful to screen for and identity therapeutic and diagnostic targets or be useful for screening for potential therapeutic agents.
  • the cells may be dissociated to single cells to reset to an equivalence group with characteristic pluripotency and gene expression.
  • the invention provides methods for synchronizing and resetting the potency of a population of pluripotent cells, preferably human, pluripotent cells, where the method comprises the steps of (1) dissociating the population of human pluripotent cells such at least 95%, and preferably, 99%, 99.9% or even 100% of the cells are single cells; (2) culturing said dissociated cells in the absence of feeder cells (generally, non-pluripotent cells that are adhered to the culture substrate), where the culturing may be in the absence of a survival factor or, to improve the survivability of the cells, in the presence of a survival factor presence of a survival factor for a time sufficient to reset the cells to an equivalence group; and (3) culturing the cells of the equivalence group in the absence of a survival factor for a period of time during which the cells can still be reset to the equivalence group, i.e., the cells are still capable of being made pluripotent by dissoci
  • the cells may be dissociated as in step (1) and then cultured as in step (2) to reset them to an equivalence group. See, for example, FIG. 6. In certain embodiments, this process is repeated once, twice, three times, 4, 5, 6 7, 8, 9 or 10 times.
  • the population of cells may be identified as reset to the equivalence group by expression or absence of expression of certain molecular markers.
  • cells may be considered to be reset as an equivalence group if at least 90%, 95%, 99% or even 99.9% of the cells express one or more pluripotency markers, for example, NANOG or OCT4 and/or where less than 10%, 5%, 1% or 0.1% of the cells express detectable levels of a protein marker of cell specification or differentiation, such as, but not limited to SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN.
  • Function assays such as those described in Example 2, may be used to assess whether the cells form an equivalence group or not.
  • the cells may be dissociated by any method known in the art for detaching and dissociating cultured mammalian, particularly, pluripotent, cells from standard tissue culture plasticware, for example, using enzymes or combination of enzymes such as Accutase ® .
  • the cells are cultured in the absence of any survival factor or, alternatively, are cultured in the presence of a survival factor.
  • the survival factor may be any factor that promotes the survivability of the dissociated cells, for example, but not limited to, a RHO-associated kinase (ROCK) inhibitor, such as Y27632, myosin II inhibitors such as blebbistatin, JAK1 inhibitors or NRG1B.
  • the cells are cultured in the presence of the survival factor for a period sufficient for them to be reset to the equivalence group as determined by expression of molecular markers, gene expression profiling, etc.
  • the period of time is preferably 24 hours, but may be anywhere from 10-15 hours, 15 to 25 hours, 12-15 hours, 18 to 22 hours, or 22 to 30 hours.
  • the pluripotent cells are embryonic stem cells, preferably human embryonic stem cells.
  • the pluripotent cells are induced pluripotent stem (iPS) cells, preferably human.
  • the iPS cells may be generated by any method known in the art, for example, by introducing pluripotency factors, such as one or more of OCT4, KLF4, SOX2, cMYC and LIN28, into somatic cells, for example by retrovirus, plasmid or as synthetic mRNAs, or any other method known in the art.
  • the pluripotent cells prior to the cell dissociation step, are cultured on feeder cells, particularly mouse fibroblast feeder cells.
  • the pluripotent cells are cultured in the absence of feeder cells.
  • the cells prior to and/or during step (2) and/or step (3), the cells are cultured in serum free media, or in media having no animal-derived products.
  • the iPS cells are derived from tissue obtained from a human subject.
  • the tissue may be of any source, including any tissue that is a source of fibroblasts, including dermal cells, tissue obtained by biopsy, or post mortem tissue.
  • iPS cells may be obtained from subjects, preferably human, for patient specific assays and to compare the behavior (including changes in gene expression, particularly the pattern of spatially restricted gene expression within the epithelial sheet of cells, i.e., the laminar organization of the colony, morphology, phenotype, etc.) among cells derived from different patients.
  • the subject has been diagnosed with a particular disease or disorder, or the subject has one or more genetic polymorphisms associated with a disease or disorder, including with predisposition to a particular disease or disorder, or that renders a patient with a particular disease or disorder more or less susceptible to treatment with a particular therapeutic.
  • the disease or disorder may be, schizophrenia and related neurodevelopmental disorders, autism or cancer.
  • the subject is healthy and cells are assayed to assess normal variation.
  • the cells are, in one embodiment, cultured for a period such that the cells can still be reset to an equivalence group, i.e., to a set of pluripotent cells, by dissociation of the cultured cells to single cells.
  • the cells are preferably cultured for 6 days prior to cell dissociation, but may also be cultured for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days during step (3) and prior to cell dissociation.
  • the cells may be cultured until they reach at least 90%, 95% or 100% confluence and then are subcultured by dissociation of the cells into single cells, repeating steps (1), (2) and (3) of the method of the invention.
  • the cells are cultured in the absence of any morphogen or factor that modulates cell specification (renewal conditions).
  • the cells are cultured in the presence of a factor or combination of factors that modulate cell specification and/or act as morphgens, for example, the factor may guide the cells to a mesodermal fate, an endodermal fate or a neurectodermal fate. These cells may be exposed to one or more of these factors during the entire culture period or only at specific points during the culture period.
  • the factor may be one or more of BMP4, Noggin, neuregulin, or Noggin and SB431542.
  • the cells are exposed to a morphogen factor, such as a protein from the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin.
  • a morphogen factor such as a protein from the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin.
  • the cells may also be treated with an agent, such as a protein, small molecule, antibody, soluble receptor, siRNA, antisense or any other agent, that modulates, either inhibiting or enhancing the activity of an endogenous morphogen, including one or more of the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin.
  • Cells may also be subjected to other treatments that inhibit expression of certain genes involved in cell specification and/or self-renewal, such as, but not limited to, Nanog, Oct-4, and Sox2. Inhibition may be effected by any method well known in the art, for example, by siRNA.
  • the cells form an epithelial sheet of cells.
  • Cells may undergo lineage priming where the cells develop towards a particular cell fate, for example, neurectodermal, mesodermal or endodermal cell fate, but are not committed to that fate, i.e., may be reset to pluripotency by dissociation to single cells.
  • the epithelial sheet of cells may form spatial zones of cells in a laminar organization with different cell specifications as determined, for example, by expression of certain molecular markers, cell morphology and/or cell phenotype. As exemplified in FIGS. 2A-D, these zones include an Edge Zone (EZ), an Intermediate Zone (IZ) or a Core Zone (CZ).
  • EZ Edge Zone
  • IZ Intermediate Zone
  • CZ Core Zone
  • the epithelial sheet of cells may recapitulate early cell specification events and morphogenesis permitting the interrogation and analysis of these events in a robust system where the specification process is reproducible and temporally synchronized.
  • the cells during step (3) are analyzed for one or more traits, including expression of molecular markers, for example, by probing with labeled antibodies against proteins that serve as molecular markers, bioinformatics analysis, for example, gene expression analysis using mRNA analysis, including RT-PCR or Next-generation sequencing or other high throughput sequencing for analysis of all or a portion of the transcriptome, microscopic analysis of cell phenotype, morphology, molecular markers on live cells, including real time monitoring by video.
  • the pattern of expression of molecular markers of cell specification and self-renewal may be assayed, characterized and quantified using methods well known in the art and described herein.
  • the pattern of expression is characterized by high content analysis as described and taught herein.
  • the cells may be analyzed at any time during step (3). They may be assayed continuously, for example, by video through a microscope, or hourly, daily, every other day, on days 2, 4 and 6 or on days 1, 3, and 5, or at any time during the course of step (3).
  • the invention further provides methods of assaying and characterizing patient specific cells.
  • the methods of the invention may be used to stratify a patient within a patient population based upon the behavior of the pluripotent cells derived from the patient in the assay methods of the invention, for example, by the pattern or laminar organization of certain markers of cell specification or self-renewal within the epithelial sheet of cells, including changes of that pattern over the culture period.
  • cells derived from patients having a particular disease state may display a characteristic behavior in the assay system of the invention, in that the cells may be lineage primed for a particular fate, may have a particular gene expression fingerprint or exhibit a particular cell morphology, epithelial sheet pattern or laminar organization or cell phenotype at a particular time during the step (3) culture period or may exhibit a characteristic dynamic change in expression of certain markers of cell specification or self-renewal or dynamic alteration in the laminar organization of the colonies formed.
  • the cells being assayed may be cultured in the absence of any exogenous cell specification factor (renewal conditions) or may be cultured in the presence of such a factor, such as BMP4, Noggin, neuregulin, or Noggin and SB431542 or any morphogen, such as, factors from the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin.
  • Cells may be subjected to other treatments that inhibit expression of certain genes involved in cell specification and/or self- renewal, such as, but not limited to, Nanog, Oct-4, or Sox2. Inhibition may be effected by any method well known in the art, for example, by siRNA.
  • Patterns of expression in the presence or absence of these treatments or between cells cultured in the presence of different factors may be compared.
  • the results may be diagnostic of a particular disease or disorder in the patient, or may be indicative of susceptibility of a disease or disorder to a particular treatment.
  • the methods of the invention may also be useful to compare the impact of different genomes or epigenetic backgrounds on cell specification and morphogenesis, for example, comparing two or more pluripotent cell populations derived from different subjects having different genomes and/or epigenetic backgrounds with respect to lineage priming, gene expression, particularly, the spatially and temporally dynamic pattern of gene expression within the epithelial sheet of cells, cell morphology or phenotype, or any other trait at any time point during the culture period in step (3).
  • the subjects from which the cells were derived may have known genetic polymorphisms, for example, polymorphisms that pre-dispose a subject to a particular disease or disorder. Alternatively, cells derived from healthy normal subjects may be used to assess normal variation. Comparisons with pluripotent cells derived from a number of patient sources may be used to identify targets for drug discovery.
  • the invention further provides methods for screening agents for activity in modulating cell specification, the methods comprising the steps of (1) dissociating a population of human pluripotent cells such that at least 95% (or 98% or 99% or 99.9% or 100%) are single cells; (2) culturing the dissociated cells, preferably but not necessarily, in the presence of a survival factor, and in the absence of feeder cells, for a time sufficient for the cells to be reset to an equivalence group; (3) culturing the population of cells from step (2) in the absence of said survival factor and in the absence of feeder cells for a period of time during with the cells remain reversibly specified, that is, the cells may still be returned to a pluripotent state by dissociating the cultured cells to single cells, and during step (3), contacting the cells with the agent to be tested in a first cell population; and comparing an assayable trait of the cells contacted with said agent with said assayable train of cells from step (3) not contacted with said agent.
  • step (3) the steps of (1) dissociating
  • the invention further provides cell populations obtained by the methods of the invention.
  • the invention provides cell populations that result from two or more cycles of dissociating pluripotent cells to single cells and then culturing those cells until a time at which their specification is reversible, i.e., they may be reset to pluripotency by dissociation to single cells.
  • the cell population may be an equivalence group of cells that have been reset through the process of the invention to the same state of potency and specification.
  • the cells are cultured after dissociation and form an epithelial sheet of cells.
  • the epithelial sheet of cells may have different zones containing different cell types in the process of acquiring different cell fates or primed to a particular cell lineage.
  • FIGS. 1A-E RNA-seq data in differentiating pluripotent cell lines across time.
  • a Principal component analysis (PCA) of poly-A RNA-seq data (gene-level log2[RPKM]) across all lines, conditions, and time. Colors indicate differentiation conditions. Individual cell lines are represented by different plotting symbols. The size of plotting symbols indicates days of differentiation. Dotted lines connect individual cell line observations within a condition across time. SA01 is represented by the round plotting symbol and is highlighted in green.
  • PCA within the Noggin-SB431542 condition PC#1, upper panel). Self-renewal data projected into Noggin-SB431542 PC#1 (lower panel).
  • FIGS. 2A-D Distinct spatial zones in self-renewing feeder-free cultured hPSC colonies.
  • FIGS 3A-D Distinct cell states are in an equivalence group.
  • B. TaqMan analysis of pluripotency and lineage marker mRNA demonstrates resetting of cell state at each passage.
  • C. Lineage of example cells expressing low or high levels of EpCAM after passage at t 0 give rise to a range of EpCAM-expressing cells after 48 hours. anti-EpCAM immunofluorescence expressed as pseudocolor in fire plot.
  • D Schematic representation of additional complete lineages demonstrating EpCAM expression in the founder cell (left side) and EpCAM signal in all derivative cells to the right. The low and high lineages from (C.) are marked.
  • FIGS 4A-C A. Lineage responsiveness changes through time in culture. A.
  • FIGS 5A-D Genotype-specific developmental bias.
  • B Immunofluorescence images of SA01 colony edge demonstrate localized expression of ERBB2 and ERBB3 in the EZ under control conditions.
  • FIG. 6 Schematic of cell dissociation and cell culture assay cycle.
  • FIGS. 7A-B PCA in cells treated with BMP4.
  • FIGS. 8A-F PCA for each of six cell lines.
  • FIGS. 9A-D PCA in cell line SA01 of OTX2(A), SOX21(B), FGFR3(C), and
  • FIGS. lOA-C The transcriptional signature defined by the neurectoderm- specific PCI starts high in early fetal development and disappears over late fetal and early postnatal life. This signature parallels closely the expression of Nestin, a widely used marker of neural stem cells.
  • FIG. 11A-C CoGAPS analysis of iPS cells derived from WS patients.
  • FIG. 12A-B Changes in nuclear area during formation of three distinct domains in PSC colonies.
  • A Box plot of nuclear area measurement between CON, NSB and BMP4 conditions. *, Significantly different from the CON (p ⁇ 0.001).
  • FIG. 13A-C Distinct cell states show different lineage bias.
  • FIG. 14A-C SOX21 and OTX2 are required for neuroectoderm differentiation.
  • the invention provides in vitro assay systems for analyzing the early steps of cell specification, differentiation and self-renewal to permit the identification of pharmacological targets involved in these processes and as well as providing systems for the screening of agents that can modulate, i.e., inhibit or promote these processes.
  • the assay systems and methods of the invention may exploit the genomic and epigenetic specificity of the behavior of cells, including patient specific cells, in these systems to analyze and compare these early cell specification and morphogenesis processes in pluripotent cells derived from specific patients, for example, patients known to have one or more genetic polymorphisms associated with a particular disease or disorder.
  • the cells form epithelial sheets in which the cells spatially organize into zones that have distinct gene expression, simulating and recapitulating the early stages of cell specification.
  • Patterns of gene expression within and between the zones of the epithelial sheet e.g., the laminar organization of the colony, provide a readout for identifying and characterizing the differences among different genetic and epigenetic contexts.
  • the results of these assays inform disease mechanisms, revealing potential pharmacological targets, and are useful in drug discovery.
  • the behavior of patient specific cells in the assays of the invention may also be characteristic or diagnostic of a disease state, predisposition to a disease state or of patient and genome specific cell specification.
  • the invention provides methods for synchronizing and resetting the potency of a population of pluripotent cells, preferably human, pluripotent cells, where the method comprises the steps of (1) dissociating the population of human pluripotent cells such at least 95% (or 98% or 99% or 99.9% or 100%) of the cells are single cells; (2) culturing said dissociated cells in the absence of feeder cells (generally, non-pluripotent cells that are adhered to the culture substrate), where the culturing may be in the absence of a survival factor or, to improve the survivability of the cells, in the presence of a survival factor presence of a survival factor for a time sufficient to reset the cells to an equivalence group; and (3) culturing the cells of the equivalence group in the absence of a survival factor for until a time at which the cells still be reset to the equivalence group, i.e., the cells are still capable of being made pluripotent by dissociation to
  • step (3) the cells are dissociated as in step (1) and then cultured as in step (2) to reset them to an equivalence group.
  • this process is repeated once, twice, three times, 4, 5, 6 7, 8, 9 or 10 times. See, for example, the schematic in FIG. 6.
  • the cells are cultured under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group
  • Pluripotent cells means cells that can differentiate into cell types from the three different germ layers— ectoderm, mesoderm and endoderm— for example, when injected in SCID mice, and can undergo many passages (20, 30, 50, 100 or even more) in in vitro culture and retain the ability to differentiate into cells of the three different germ layers after each division.
  • the cells may be cultured for a period such that the cells can still be reset to an equivalence group, i.e., to a set of pluripotent cells, by dissociation of the cultured cells to single cells.
  • the cells are preferably cultured for 6 days prior to cell dissociation, but may also be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days during step (3) and prior to cell dissociation.
  • the cells are cultured in the presence of a factor or combination of factors that modulate cell specification, for example, the factor may guide the cells to a mesodermal fate, an endodermal fate or a neurectodermal fate.
  • the factor may be one or more of BMP4, Noggin, neuregulin, or Noggin and SB431542.
  • the factor may be a protein of the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin, or is an agent that modulates, either inhibits or enhances, the activity of one or more these factors.
  • the cells are cultured in the absence of any factors that modulate cell specification (renewal conditions).
  • the cells may form an epithelial sheet of cells.
  • Cells may undergo lineage priming where the cells develop towards a particular cell fate, for example, neurectodermal, mesodermal or endodermal cell fate, but are not committed to that fate, i.e., may be reset to pluripotency by dissociation to single cells or, alternatively, the cells are cultured under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group.
  • the epithelial sheet of cells may form spatial zones of cells with different cell specifications as determined, for example, by expression of certain molecular markers, cell morphology and/or cell phenotype.
  • the epithelial sheet of cells becomes progressively more organized into these zones of cells that represent spatially restricted expression of specific markers of cell specification and morphological characteristics.
  • the cells may be organized into edge, intermediate and core zones (EZ, IZ, and CZ, respectively) of the epithelial sheet.
  • the epithelial sheet of cells may recapitulate early cell specification events permitting the interrogation and analysis of these events in a robust system where the specification process is reproducibly and temporally synchronized.
  • the cultured cells during step (3) provide a reproducible and robust system for assaying morphogenetic events as the cells in the epithelial sheet undergo organization into zones of different cell types as takes place during morphogenesis.
  • the cells during step (3) are analyzed for one or more traits, including expression of molecular markers, for example, by probing with labeled antibodies against proteins that serve as molecular markers, bioinformatics analysis, for example, gene expression analysis using mR A analysis, including Next generation sequencing or other high throughput sequencing, particularly to generate RNASeq data, microscopic analysis of cell phenotype, morphology, molecular markers on live cells, monitoring by video. Any protein, mRNA or any other marker may be analyzed.
  • markers include, but are not limited to, Brachyury (T), CDX2, Eomesoderm, ERBB2, ERBB3, Nanog, Oct4, OTX2, Soxl, Sox2, or Sox21, to name a few.
  • the cells may be analyzed at any time during step (3). They may be assayed continuously, for example, by video through a microscope, or hourly, daily, every other day, on days 2, 4 and 6 or on days 1, 3, and 5, or at any time during the course of step (3).
  • the invention further provides methods of assaying and characterizing patient specific cells.
  • the methods of the invention may be used to stratify a patient within a patient population based upon the behavior of the pluripotent cells derived from the patient in the assay methods of the invention.
  • cells derived from patients having a particular disease state may display a characteristic behavior in the assay system of the invention, in that the cells may be lineage primed for a particular fate, may have a particular gene expression fingerprint or exhibit a particular cell morphology, epithelial sheet pattern (laminar organization of the colony) or cell phenotype at a particular time during the step (3) culture period.
  • the results may be diagnostic of a particular disease or disorder in the patient, or may be indicative of susceptibility of a disease or disorder to a particular treatment.
  • the methods of the invention may also be useful to compare the impact of different genomes or epigenetic backgrounds on cell specification, for example, comparing two or more pluripotent cell populations derived from different subjects having different genomes and/or epigenetic backgrounds with respect to lineage priming, gene expression, cell morphology or phenotype, or any other trait at any time point during the culture period in step (3).
  • the subjects from which the cells were derived may have known genetic polymorphisms, for example, polymorphisms that pre-dispose a subject to a particular disease or disorder. Comparisons with pluripotent cells derived from a number of patient sources may be used to identify targets for drug discovery.
  • the invention further provides methods for screening agents for activity in modulating cell specification, the methods comprising the steps of (1) dissociating a population of human pluripotent cells such that at least 95% (or 98% or 99% or 99.9%) are single cells; (2) culturing the dissociated cells, preferably but not necessarily, in the presence of a survival factor, and in the absence of feeder cells for a time sufficient for the cells to be reset to an equivalence group; (3) culturing the population of cells from step (2) in the absence of said survival factor and in the absence of feeder cells for a period of time during with the cells remain reversibly specified, that is, the cells may still be returned to a pluripotent state by dissociating the cultured cells to single cells (or, alternatively, culturing the cells under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group), and during step (3), contacting the cells with the agent to be tested in a first cell population
  • the invention further provides cell populations obtained by the methods of the invention.
  • the invention provides cell populations that result from two or more cycles of dissociating pluripotent cells to single cells and then culturing those cells until a time at which their specification is reversible, i.e., they may be reset to pluripotency by dissociation to single cells.
  • the cell population may be an equivalence group of cells that have been reset through the process of the invention to the same state of potency and specification.
  • the cells are cultured after dissociation and form an epithelial sheet of cells.
  • the epithelial sheet of cells may have different zones containing different cell types in the process of acquiring different cell fates or primed to a particular cell lineage.
  • Pluripotent cells may be generated using any methods known in the art.
  • the pluripotent cells may be of any species, particularly vertebrate, and more particularly mammalian.
  • the cells are preferably from humans but may also be from non-human mammalian species, for example, but not limited to, non-human primates (such as, apes, monkeys, etc.), rodents (such as mice or rats), cows, pigs, sheep, horses, rabbits, or any other species.
  • Pluripotent cells may be embryonic stem cells (ESCs), particularly human embryonic stem cells (HESCs), induced pluripotent stem (iPS) cells, or any other pluripotent cell.
  • the cells may be totipotent.
  • ESCs and particularly HESCs may be prepared according to methods well known in the art.
  • ESCs are generally derived from pre-implantation embryos. These cells are pluripotent and can be cultured and retain pluripotency for extended periods of time over multiple passages, for example, one year or more. See, e.g., Thomson, US Patent No. 6,200,806, which is incorporated by reference herein in its entirety.
  • iPS cells may be generated using any method known in the art. iPS cells are generally prepared by introducing a combination of so-called "reprogramming factors" into a differentiated somatic cell, such as a fibroblast. The expression or presence of the reprogramming factors in a cell leads to dedifferentiation, resulting in a pluripotent cell line. See, e.g., Yamanaka et al, US 7,964,401; Thomson et al, US 8,440,461; Jaenish et al, US 7,682,828, all of which are incorporated herein by reference in their entireties. Reprogramming factors include, but are not limited to Oct-3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog.
  • the reprogramming factors may be introduced by any known method, including, by expression from a retroviral vector, non-integrating viral vectors or non-viral vectors or episomes.
  • the reprogramming factors are introduced into the somatic cell as modified mRNAs, for example, having modified nucleosides such as 5-methylcytidine, 2-thiouridine, pseudoruidine, or 2'-0— methyladenosine. See, for example, Rossi et al, US patent application publication 2012/130624, which is incorporated herein by reference in its entirety.
  • HESCs and iPS cell lines are also available from a variety of cell banks and commercial sources.
  • Pluripotent cells may be identified by their ability to differentiate into cells of all three germ layers, for example, by injection into SCID mice.
  • the pluripotent cells may express certain markers associated with pluripotency, for example, SSEA3, SSEA4, Oct4, Nanog, EpCAM, and/or Sox 2.
  • Any type of somatic cell may be used to generate the iPS cells.
  • skin, lung, heart, brain or other tissue of the nervous system, liver, blood, kidney or muscle cells can be used.
  • the subject can be of any age.
  • the subject is preferably human. In certain embodiments, the subject suffers from a particular disease or disorder.
  • Samples may be fresh or may be any tissue sample, such as biopsy or other pathology samples or may be post-mortem samples, including preserved samples.
  • the human subject may also have one or more genetic polymorphisms that are associated with a particular disease or disorder, predisposition to a particular disease or disorder or that are associated with susceptibility or lack of susceptibility to a particular treatment regimen for a disease or disorder.
  • the disease or disorder is schizophrenia or any neurodevelopmental disorder, cancer, diabetes, cardiovascular disease, or autism.
  • the human subject has not been diagnosed with or does not have a polymorphism associated with a particular disease or disorder and iPS cells may be isolated from such subjects to analyze and characterize differences revealed in the assays of the invention due to normal variation among healthy subjects.
  • the pluripotent cells are cultured by methods well known in the art. In certain embodiments they are cultured on feeder cells, which are any cells that promote the growth and maintain the pluripotency of the pluripotent cells. Feeder cells are generally fibroblasts, particularly murine fibroblasts. The pluripotent cells may also be cultured in the absence of feeder cells. The cells are cultured under conditions known for the culture of mammalian cells, particularly, pluripotent cells. In certain embodiments, the cells are grown in cell culture media, for example hES media, the components of which are provided in Table 1 in Example 1 herein. The media may contain serum, but is preferably serum free. The media may be supplemented with growth factors, for example bFGF.
  • feeder cells are any cells that promote the growth and maintain the pluripotency of the pluripotent cells.
  • Feeder cells are generally fibroblasts, particularly murine fibroblasts.
  • the pluripotent cells may also be cultured in the absence of feeder cells.
  • the cells are cultured
  • the cells may be cultured under conditions suitable for culturing pluripotent cells.
  • the cells are preferably cultured in the absence of feeder cells.
  • the media may be mTeSR®l medium, supplied by StemCell Technologies (Vancouver, Canada) (comprised of 400 mL mTeSR®l basal medium (Catalog #05851) and 100 mL mTeSR®l 5X supplement (Catalog #05852)).
  • the medium used to culture the cells in the methods of the invention are preferably serum free
  • colonies are dislodged by any method known in the art, for example by incubation with an enzyme such as collagenase IV. The colonies are then dissociated, preferably enzymatically.
  • Any enzyme known to be useful to dissociate cultured mammalian cells may be used, for example, the Accutase enzyme (StemPro® Accutase® Cell Dissociation Reagent; Invitrogen Cat# Al l 1105-01).
  • the cells are incubated in the presence of the enzyme for sufficient time and under conditions that promote cell dissociation and to achieve dissociation almost entirely to single cells.
  • the cells may be incubated for 3 minutes, 5 minutes, 7 minute or 10 minutes depending upon the cells and conditions.
  • the cells may be agitated or aspirated, for example, by pipetting the cell solution with an automatic micro pipettor.
  • the enzymatic action may be stopped, for example by addition of serum to the cells.
  • at least 80%, 85%, 90%, 95%, 99% or 100% of the cells harvested are dissociated to single cells.
  • the cells may be incubated in the presence or absence of a growth factor, such as FGF-2 or bFGF, and, optionally a survival factor.
  • the survival factor may be any factor that promotes the survivability of the dissociated cells, for example, but not limited to, a RHO-associated kinase (ROCK) inhibitor, such as Y27632, a myosin II inhibitor, such as blebbistatin, a JAK1 inhibitor, or NRG IB.
  • a RHO-associated kinase (ROCK) inhibitor such as Y27632
  • a myosin II inhibitor such as blebbistatin
  • a JAK1 inhibitor or NRG IB.
  • the dissociated cells are incubated with Y27632.
  • the cells may be incubated with the survival factor during the plating process.
  • the dissociated cells may then be plated and cultured using any method known in the art.
  • the cells may be cultured in any cell culture vessel, for example a dish, a flask, a roller bottle or any other vessel for mammalian cell culture.
  • the cell culture vessel is preferably coated with a coating such as, but not limited to, Matrigel ® .
  • the cells are plated and cultured in the cell culture vessel, preferably in the presence of the survival factor, specifically Y27632.
  • the cells may be maintained in the presence of the survival factor for a period of time, for example, 24 hours, or for a shorter period of time, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 15 hours or even 20 or 24 hours.
  • the "equivalence group” is a population of cells that are all pluripotent and are in the same state of cell specification. As such, the population of cells begin the program towards lineage priming and then lineage specification at the same time and can be analyzed temporally and monitored as the cells undergo cell specification and the population of cells go through morphogenesis. The cells may become lineage primed, as evidenced through changes in gene expression, but remain pluripotent during the cycle of the assay system of the invention.
  • the equivalence group cells can be characterized by the expression of particular molecular markers, for example, but not limited to, expression of pluripotentcy factors, such as, but not limited to, NANOG and OCT4, or lack of protein expression of one or more markers of cell differentiation, such as but not limited to SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN.
  • the equivalence group may also be defined functionally, for example as described in Example 2 herein.
  • the equivalence group of cells when treated with BMP4 at day 0 of the dissociation and culture cycle described herein, may induce the expression of certain lineage specific markers, such as CDX2 and/or Brachyury (T), in most cells of the equivalence group, but by day 2, expression of the markers is spatially restricted.
  • lineage specific markers such as CDX2 and/or Brachyury (T)
  • T Brachyury
  • inhibition of Oct4 on day 0 may also result in induction of markers for multiple lineages while inhibition at day 2 leads to expression of markers for only a specific lineage or to more restricted expression.
  • the population of cells may be identified as reset to the equivalence group by expression or absence of expression of certain molecular markers.
  • cells may be considered to be reset as an equivalence group if at least 90%, 95%, 99% or even 99.9% of the cells express one or more of the markers NANOG or OCT4 and/or where less than 10%, 5%, 1% or 0.1% of the cells express detectable levels of a marker of cell specification or differentiation, such as, but not limited to SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN.
  • a marker of cell specification or differentiation such as, but not limited to SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN.
  • the cells may be cultured in the absence of the survival factor under conditions appropriate for pluripotent mammalian cells as known in the art, for example, as described in Example 1.
  • the media may be exchanged as generally routine in the art, for example, every 24 hours.
  • the cells are cultured and allowed to form and epithelial monolayer of cells.
  • the cells are subcultured once they reach 90% to 100% confluence and prior to achieving a point in cell specification where the cells cannot be reset to the equivalence group by cell dissociation.
  • the cells are generally cultured for approximately 4, 5, 6, 7, 8, 9 or 10 days.
  • the cells may be cultured under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group
  • the cells may be incubated with one or more factors that promote or direct a particular cell fate or cell specification factors.
  • the cells may be exposed to one or more of the factors either throughout the culture period or at particular times and for particular periods during the culture period, for example from days 1-6, 1-2, 2-3, 3- 4, 4-5, 5-6, 1-3, 2-4, 3-5, 4-6, 1-3, 4-6, 1-4, or 2-6, or whatever period of time is useful for the desired analysis.
  • factors include, but are not limited to Noggin, BMP4, neuregulin, orSB431542.
  • the factor may also be one or more of a protein of the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin, or is an agent that modulates, either inhibits or enhances, the activity of one or more these factors.
  • the cells will generally tend to develop towards a neurectodermal fate. Noggin exposure promotes a neurectodermal cell specification while BMP4 promotes a mesodermal cell specification.
  • the behavior of the cells either without any exogenous factors or in response to one or more of these factors may depend upon the genomic and/or epigenetic context of the cell.
  • the cultured cells form an epithelial sheet, which recapitulates or simulates cell specification and morphogenesis.
  • Cells may undergo lineage priming where the cells develop towards a particular cell fate, for example, neurectodermal, mesodermal or endodermal cell fate, but are not committed to that fate, i.e., may be reset to pluripotency by dissociation to single cells.
  • molecular markers for pluripotency decrease while molecular markers for cell specification to a particular lineage, such as, neurectodermal, mesodermal or endodermal lineages, including markers such as, SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN, increase.
  • the epithelial sheet of cells becomes, during the culture period, progressively more organized into zones of cells that represent spatially restricted expression of specific markers of cell specification and morphological characteristics.
  • the cells may be organized into edge, intermediate and core zones (EZ, IZ, and CZ, respectively) of the epithelial sheet.
  • the zones may be characterized by expression of different levels of molecular markers, or different molecular markers, or by phenotypic or morphological differences, such as nuclear area.
  • the cells during step (3) are analyzed for one or more traits.
  • traits include expression of particular genes, either at the mR A level or protein level, cell morphology, such as nuclear size and other aspects of cell morphology and cell phenotype.
  • cell morphology such as nuclear size and other aspects of cell morphology and cell phenotype.
  • the spatial and temporal distribution of these traits within the epithelial sheet of cells formed in step (3), and the zones, EZ, IZ and/or CZ may be assayed, including specific mRNA levels, protein expression, and cell morphology, and characterized using methods for assessing patterns of gene expression.
  • Gene expression may be analyzed by any method known in the art.
  • the presence or absence of specific mRNAs or levels of specific mRNAs can be assayed, for example, by northern analysis or RT-PCR or any other method known in the art for detecting the presence or amount of specific mRNA species.
  • bioinformatics analysis such as sequencing of all or a portion of mRNAs expressed in a cell population (e.g., the transcriptome) may be used to identify and analyze gene expression in a cell population.
  • the mRNAs may be sequenced by any method known in the art, such as Next Generation sequencing, or any other high throughput sequencing platform, or by microarray analysis.
  • RNASeq data may be analyzed to identify gene expression profiles.
  • gene expression is analyzed at the protein level.
  • Antibodies against specific proteins may be used in western analysis, immunoassays, and the like to detect the presence of specific proteins in a cell population.
  • gene expression can be detected, analyzed and monitored in live cells using labeled antibodies against specific cell surface protein markers (for example, fluorescently labeled antibodies— see Table 2 herein).
  • labeled antibodies against specific cell surface protein markers for example, fluorescently labeled antibodies— see Table 2 herein.
  • the labeled proteins can be visualized on the cells.
  • Such labeled antibodies are useful to assess the spatial and temporal pattern of expression of different molecular makers within the epithelial sheet of cells.
  • the patterns of expression can be captured, visualized and monitored by microscopy, such as a confocal microscope, including quantitative fluorescence microscopy.
  • Nuclear markers such as DAPI staining, may be used to identify and mark individual cells.
  • Molecular markers such as EpCAM, Nanog, Sox21 and Sox2, may be used as reference points, for example, EpCAM, may be used to delineate the Edge Zone or EZ of the epithelial sheet.
  • EpCAM may be used to delineate the Edge Zone or EZ of the epithelial sheet.
  • the different zones may also be distinguished based upon differences in nuclear area.
  • DAPI may be used to label nuclei so they can be measured and quantitated.
  • the inner zone correlates with low nuclear area while the edge zone correlates with a larger nuclear area.
  • a combination of nuclear area and molecular markers may be used to characterized and identify the different zones within the epithelial sheet. Alternatively, different zones or regions of the epithelial sheet may be isolated and analyzed for m NA or protein expression.
  • the cells may be analyzed at any time during step (3). They may be assayed continuously, for example, by video through a microscope, or hourly, daily, every other day, on days 2, 4 and 6 or on days 1, 3, and 5, or at any time during the course of step (3).
  • Data obtained from analysis of the cells can be processed and analyzed by any method available in the art.
  • gene expression patterns may be analyzed by principal component analysis (PCA) or any similar type of analysis, or any non-negative matrix factorization (NMF), such as but not limited to CoGAPS (see Fertig et al., 2010).
  • PCA principal component analysis
  • NMF non-negative matrix factorization
  • the pattern of gene expression within the epithelial sheet may be analyzed using a high content analysis, for example, as described in Example 1 herein.
  • the output of the data analysis may be used to compare results across cells derived from different sources, e.g., from patients of different genetic or epigenetic backgrounds, which can be used to identify patterns, leading to understanding of mechanisms of cell differentiation, morphogenesis and disease.
  • the data output may also be compared to that in available clinical or other databases to identify and understand patterns of gene expression associated with particular disease state.
  • the output may be compared to known standards, for example, to use the output from cells of a particular patient to diagnose or prognose a particular disease or disorder or to assays whether a patient is amenable to a particular treatment for a disease or disorder.
  • Such analysis may be used to stratify patients having a particular disease or disorder according to likelihood of developing the disease or disorder, severity of the disease or disorder, whether patients are amenable to certain treatments or not.
  • hES medium a human ES cells medium supplemented with 4 ng/mL recombinant human FGF-2.
  • Table I shows the components used to prepare about 500 mL of the hES medium.
  • mTeSR®l medium a commercial medium supplied by StemCell Technologies (Vancouver, Canada), mTeSR®l medium, is used to serially subculture human pluripotent stem cells in feeder-free monolayer culture on Matrigel coated dishes. This medium is comprised of 400 mL mTeSR®l basal medium (Catalog #05851) and 100 mL mTeSR®l 5X supplement (Catalog #05852).
  • BD Matrigel® is diluted by adding 60 Matrigel in 12 mL DMEM:F12 (Invitrogen 11330-032) and the solution gently mixed.
  • Sufficient Matrigel® solution is added to cover the surface of the tissue culture vessel. For instance, 1 mL of the solution can be used for each well of a 6-well plate. The vessel is incubated at room temperature for at least 1 hr.
  • MONOLAYERS (1) After reaching appropriate size colonies in feeder culture, the tissue culture vessel containing human pluripotent stem cells is removed from the humidified incubator into the biocabinet. The vessel is tilted and the cell culture medium gently removed using a plastic pipette.
  • Each well is washed with 3 mL of the medium to ensure detachment of all colonies, and the content of each well is transferred into respective 15 mL centrifuge tubes.
  • the cell suspension is centrifuged for 2 min at 200 g.
  • the colonies are dissociated into single cells by gently aspirating (10-15 times) the cell suspension with a PI 000 Pipetman set to a volume of 850 ⁇ ,.
  • the cell suspension is centrifuged for 5 min at 200 g, the pellet resuspended in 1 mL hES medium supplemented with 4 ng/mL FGF-2 and 5 ⁇ Y27632.
  • (14) Upon acquiring a single cell suspension, cell counts are performed using Trypan-blue exclusion method.
  • the culture vessel (plate) is incubated at 37oC in a humidified chamber containing 5% C02, and 95% air saturation.
  • each well is washed with 1 mL pre -warmed mTeSR®l medium. Then, the washing medium is exchanged with 2.5 mL of the pre-warmed mTeSR®l medium.
  • the matrigel-coated tissue culture vessel containing human pluripotent stem cells is removed from the humidified incubator into the biocabinet.
  • the cell culture medium is removed from the culture by tilting the vessel and using a plastic pipette.
  • the cell suspension is centrifuged for 5 min at 200 g, and the pellet washed with lOmls of mTeSR® 1 medium supplemented with 5 ⁇ Y27632.
  • the cell suspension is centrifuged for 5 min at 200 g, and the pellet resuspended in 1 mL of the mTeSR® 1 medium supplemented with 5 ⁇ Y27632.
  • cell counts are performed using Trypan-blue exclusion method.
  • 750,000 cells per well (equivalent of 300,000 Cells/mL) are inoculated into each well of a matrigel-coated 6-well plate, which has been preincubated with prewarmed 2.5 mL of the mTeSR® 1 medium supplemented with 5 ⁇ Y27632.
  • the culture vessel is incubated at 37oC in a humidified chamber containing 5% C02, and 95% air saturation.
  • Table 1 Medium components to prepare 500.0 L of hES growth medium for expansion in feeder culture.
  • siRNA SOX21 (sc-38433, Santa Cruz), OTX2 (EHU129881, Sigma Aldrich),
  • OCT4 (sl0873, Life technologies), NANOG (s36649, Life technologies), SOX2 (sc-38408, Santa Cruz).
  • ESC culture For maintenance of human PSCs in feeder-free condition, cells are dissociated to single cell populations with Accutase® (Al l 105, Life technologies), plated at a density of 1X106 cells in a Matrigel® (BD)-coated 6-well plate and cultured with mTeSRl (Stem Cell Technology, #05850, www.stemcell.com/en/Products/All-Products/mTeSRl .aspx). The cells are cultured with 5 ⁇ Y27632, ROCK inhibitor (Y0503, Sigma-Aldrich) to increase the single cell survival upon dissociation. At 24 hours after plating, Y27632 was removed from the medium and cells cultured for another 4 days before the next passaging.
  • Noggin 500 ng/ml, 719-NG, R&D
  • SB431542 2 ⁇
  • mTeSRl medium for neuroectodermal differentiation and BMP4 (100 ng/ml, 314-BP, R&D) for mesendodermal differentiation upon Y27632 withdrawal.
  • BMP4 100 ng/ml, 314-BP, R&D
  • mesendodermal differentiation upon Y27632 withdrawal.
  • recombinant human NRGip 100 ng/ml, #396-HB, R&D
  • siRNA treatment Silencing endogenous genes is performed by transfection using
  • DharmaFECT 1 reagent (Thermo Scientific).
  • Cells are transfected with non-targeting negative control siRNA (#4390843, Life technologies) or siRNA targeting SOX21 and OTX2 at a final concentration of 50 nM for 4 days after removal of Y27632.
  • the siRNAs of OCT4, NANOG and SOX21 are treated for 48 hours after removal of Y27632 on either day 0 or day 2.
  • Immunofluorescence Cells are fixed with 4% paraformaldehyde for 10 min and permeabilized for 30 min using 0.1% Triton X-100 (Sigma- Aldrich) in PBS. Subsequently, cells are blocked with 10% donkey serum (Sigma- Aldrich) and incubated with primary antibodies overnight. Secondary antibody staining is performed with Alexa Fluor®-conjugated antibodies (Life technologies). For direct immunostaining, primary antibodies are conjugated using Alexa Fluor® antibody labeling kit (Life Technologies). Nuclei are counterstained with DAPI and imaged with a confocal laser-scanning microscope.
  • DuplexDish 35 (Zeiss cat no. 415101-4400-551) in the presence of the ROCK inhibitor Y27632. After 24 hours, Y27632 is removed and colonies are allowed to develop for an additional 72 hours under control conditions of mTeSRl culture medium only.
  • EZ Edge Zone
  • the live culture is incubated with anti-EpCAM antibody after direct conjugation to Alexa647 (Molecular Probes Cat No. A-20186) for 15 min in the tissue-culture incubator then washed twice with pre-equilibrated media without antibody.
  • LCM was performed using the Zeiss PALM microbeam system in non-contact mode on live culture. Briefly, fluorescence illumination of Alexa647 enabled visualization of the EZ. Due to the high-contrast of the anti- EpCAM staining at the edge compared to the core, histogram-based thresholding is used to segment the EZ from the IZ.
  • Quantitative RT-PCR Reverse transcription was performed in the sample lysate using Superscript III Reverse Transcriptase (Invitrogen). Relative RNA levels were calculated using the AACt method with human GAPDH as reference genes. Measuring passage efficiency
  • SA01 cells were grown to confluence on monolayer conditions (4-days) in a 6- well plate as described earlier. Cells were passaged using enzymatic digestion (acutase). Viability was quantified automatically using the Countess (LifeTechnologies C 10310) with trypan blue exclusion. 1,000,000 cells were plated on each well of a 6-well plate in the presence of rock inhibitor for one hour then imaged. Single fields were counted from each of six wells manually to distinguish live from dead (pyknotic) cells. Survival is represented as the number of live/dead cells counted per area extrapolated to the entire area of the well divided by 1,000,000 (the number of cells plated per well).
  • SA01 cells were passaged using Accutase. While in suspension, cells were incubated with anti-epCAM antibody (RnD systems) directly conjugated to Alexa 647 for 10 min at room temp. Cells were pelleted once then resuspended in media lacking antibody then counted with the Countess. 1X10 6 live cells/well were plated on 35mm plates (Greiner Advanced TC) coated with Matrigel in mTeSRl in the presence of Y27632. Imaging began less than 5 minutes after the EpCAM wash. At the beginning of the recording, many cells can be observed attaching to the surface.
  • RnD systems anti-epCAM antibody
  • An image area was chosen that includes two non-contiguous "edge zones”.
  • the poly-A containing mRNA molecules are purified from 1 ⁇ g DNAse treated total RNA. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Reverse transcriptase and random primers are used to copy the cleaved RNA fragments into first strand cDNA. The second strand cDNA is synthesized using DNA Polymerase I and RNaseH.
  • Ribosomal RNA depletion with strand-specific library preparation and Q/C
  • RNA-seq libraries were constructed using Illumina TruSeq Stranded Total RNA
  • Ribo-Zero sample Prep Kit following the manufacturer's protocol.
  • the ribosome RNAs were removed using Ribo-zero beads from ⁇ 800 ng DNAse treated total RNA.
  • the total RNA without Ribosome RNA was fragmented into small pieces using divalent cations under elevated temperature (94 degree) for 2 minutes. Under this condition, the range of the fragments length is from 130-290 bp with a median length of 185 bp.
  • Reverse transcriptase and random primers were used to copy the cleaved RNA fragments into first strand cDNA.
  • the second strand cDNA was synthesized using DNA Polymerase I and RNaseH, dUTP in place of dTTP These cDNA fragments then went through an end repair process using T4 DNA polymerase, T4 PNK and Klenow DNA polymerase, and the addition of a single 'A' base using Klenow exo (3' to 5' exo minus), then ligation of the Illumina PE adapters using T4 DNA Ligase. An index (96 unique dual-index pairs) was inserted into Illumina adapters so that multiple samples can be sequenced in one lane of 8-lane flow cell if necessary. These products were then purified and enriched with 15 cycles of PCR to create the final cDNA library for high through put DNA sequencing using Highseq2000. The concentration of R A libraries was measured by Qubit (Invitrogen, CA). The quality of R A-seq library was measured by LabChipGX (Caliper, MA) using HT DNA lK/12K/HiSens Labchip.
  • the Illumina Real Time Analysis (RTA) module performed image analysis, base calling, and the BCL Converter (CASAVA vl .8.2) generated the sequence reads in FASTQ file format. lOObp reads with a targeted coverage over 80-100 million sequencing reads are used per sample.
  • the Illumina Real Time Analysis (RTA) module was run to perform image analysis, base calling, and the BCL Converter (CASAVA vl .8.2) were followed to generate FASTQ files which contain the sequence reads.
  • the current sequencing depth is 80-120 million (40-60M paired-end) mappable sequencing reads.
  • Read-level Q/C was performed by FastQC (vO.10.1). Pair-end reads of cDNA sequences obtained by the Highseq 2000 are aligned back to the human genome (UCSC hgl9) by the spliced-read mapper TopHat (v2.0.4) based on known transcripts of Ensembl Build GRCh37.67.
  • the alignment statistics and Q/C was achieved by samtools (v0.1.18) and RSeQC (v2.3.5) to calculate quality control metrics on the resulting aligned reads, which provides useful information on mappability, uniformity of gene body coverage, insert length distributions and junction annotation, respectively.
  • samtools v0.1.18
  • RSeQC v2.3.5
  • quality control metrics on the resulting aligned reads, which provides useful information on mappability, uniformity of gene body coverage, insert length distributions and junction annotation, respectively.
  • To obtain gene-level expression the properly-paried and mapped reads are only counted by htseq-count vO.5.3 (with intersection-strict mode) and RPKM is calculated.
  • CoGAPS decomposes a matrix of experimental observations— in this case, log2 ratios from two color expression arrays and RNAseq RPKMs— with genes as rows and samples as columns, into two matrices, the pattern matrix P defining relationships (i.e. patterns) between samples and the amplitude matrix A indicating the strength of involvement of a given gene in each pattern.
  • a Markov Chain Monte Carlo algorithm with sparse atomic prior is used to compute the optimal solutions for A and P given the experimental data and a user defined error model ⁇ — in this case, a constant 0.1/ln(2) for array data and 10 percent of the signal for RNAseq.
  • the final number of patterns used in the decomposition was determined using the ⁇ 2 fit of the recomposed AP to the original data and the persistence of patterns across multiple simulations and gene sets.
  • nuclei were segmented in the DAPI channel using the
  • each nucleus perimeter from segmentation, is known and the location of each colony perimeter is known also from colony segmentation.
  • the minimum distance between the nucleus edge and colony edge was calculated using the ' Measure Distance' function in Volocity. Thus each cell has a value reflecting its minimum distance to the nearest colony edge.
  • iPS stem cell lines were grown in a simple cycle where they were dissociated to single cells and placed in new tissue culture plates every 5-7 days (FIG. 6).
  • iPS induced pluripotent stem cell lines
  • PC2 separates out the different days of treatment for all three conditions. Combined, PCI and PC2 reveal global transcriptional trajectories for early neuronal and mesendodermal differentiation followed by all six cell-lines. The iPS line i04 moves most quickly along the mesendodermal trajectory while human ES line SA01 leads the neurectodermal trajectory. This indicates that cell lines show distinct differentiation efficiencies.
  • CoGAPS uses computational power to iteratively decompose high-dimensional data into any number of component patterns specified by the investigator (Fertig et al, 2010).
  • Fertig et al, 2010 When tasked with identifying 20 patterns, CoGAPS identified 14 that define distinct components of gene expression change during differentiation exhibited by all the lines (FIG. ID), and 6 patterns that define high levels of transcription that are linked to a specific genome spanning all three conditions (FIG. ID).
  • the weights generated by CoGAPS were used to interrogate the behavior of these 6 cell lines in a micro-array analysis of gene expression that included 21 pluripotent human cell lines (Mallon et al., 2013). The same pattern of variant gene expression in individual lines was seen in the context of this larger panel of pluripotent human cell lines analyzed by a different method at a different time (FIG. 10). This result demonstrates that the cell line signatures are stable traits. [0099] To rigorously confirm a genetic origin for cell line specific transcriptional signatures, iPS cells that were derived from two William's Syndrome (WS) patients that carry different defined deletions on chromosome 7 were analyzed.
  • WS William's Syndrome
  • Replicate lines were generated from individual genomes by mRNA transfection and RNAseq was conducted by the Ribo-Zero method instead of the Oligo-dT isolation of polyA+ RNA previously used.
  • Replicate iPS lines from the same patient show the same CoGAPS pattern, but the transcriptome from the two William's patients was defined by different patterns (FIG. IE). These two William's patients carry deletions of different sizes that were detected by CoGAPS (FIG. 11). Importantly, these patterns also reproducibly identify genes in other regions of the genome that contribute to the transcriptional identity of both patients and not a control iPS cell line (FIG. 11). These data show that CoGAPS is a diagnostic tool to measure the consequences of genetic variation on the phenotype of human pluripotent cells.
  • the CoGAPS pattern 15 predicts that dynamic change in expression of SOX21 and OTX2 would be required for the emergence of the later step in neurectodermal differentiation defined by pattern 12.
  • the expression of the SOX21 protein was defined using confocal microscopy to measure signal from specific antibodies directly conjugated to different fluorescent reporters in the SA01 cell line. Under control conditions on day 4, few SOX21 positive cells were present (FIGS. 2A and 2B). Under Noggin-SB conditions, SOX21 was strongly induced. A striking inverse correlation was seen between SOX21 and NANOG, a transcription factor that promotes pluripotency (FIGS. 2 A and 2B).
  • EPCAM was used to specifically identify cells at the edge of cell sheets defining three morphological domains; the edge-, intermediate- and core-zones (EZ, IZ and CZ) (FIG. 2D).
  • mR A levels for OCT4, SOX2, NANOG, ESRRB, EOMESODERMIN, SOX21 and OTX2 were assessed by quantitative RT-PCR (FIG. 2E).
  • SOX21 and OTX2 message levels were reduced in the EZ/IZ domain while the mesendodermal regulator EOMESODERMIN was enriched in this domain. Consistent with the CoGAPS analysis, these results demonstrate that expression of the OTX2 and SOX21 genes is regulated by morphogenic mechanisms.
  • NANOG and OCT4 levels were high early and declined at later times when expression of the SOX genes increased. 98% of cells survived the initial dissociation and 75% of these cells were present 24 hours after replating. Consistent with previous reports showing regulation of gene expression and developmental potential in pluripotent cells (Chambers et al., 2007; Macfarlan et al., 2012; Surani and Tischler, 2012), our data suggest that neurectodermal fate regulators are dynamically controlled in the pluripotent state.
  • EOMESODERMIN EOMES
  • BRACHYURY T
  • CDX2 homeobox transcription factor
  • Immunological analysis of protein expression shows that 24 hours after treatment with BMP4, CDX2 and T are expressed in many cells at day 0 before the emergence of distinct zones (FIG 4B).
  • BMP4 When exposure to BMP4 was delayed by 48 hours, CDX2 and T expression was restricted to cells in the IZ and EZ.
  • This effect of BMP4 is mediated by phosphorylation of SMAD proteins, a family of signal transducing transcriptional regulators. Nuclear localization of phosphorylated SMADs following the delayed BMP4 treatment was restricted to the zone where CDX2 and T expression was observed (FIG. 13).
  • the imaging analysis extends the RNASeq analysis by showing that fate bias was regulated by a morphogenic mechanism that organizes developmentally equivalent cells into distinct zones biased towards mesendodermal or neurectodermal fates.
  • SOX21 expression during self-renewal leads to efficient neurectodermal specification.
  • SOX1 and PAX6, two well-characterized regulators of neuronal development were used to monitor neurectodermal differentiation (Osumi et al, 2008; Pevny et al, 1998; Wood and Episkopou, 1999).
  • SOX1 contributes to the neurectodermal differentiation patterns 15 and 12, but PAX6 expression only contributes to pattern 12. Because neither gene was associated with a cell line transcriptional signature, SOX1 and PAX6 expression define the early and late steps of neurectodermal differentiation independent of the initial state of the self-renewing cell.
  • OTX2 levels in these 5 samples showed a small change in response to NRGl treatment under NOGGIN-SB conditions.
  • the line carrying the protective allele responded to NRGl treatment (blue circle).
  • NOGGIN-SB conditions on day 6 the lines carrying the non-risk allele now show elevated OTX2 expression and all of these samples showed reduced OTX2 expression when treated with NRGl .
  • multi-way ANOVA analysis shows that cells carrying the AKT1 risk alleles already show a significant elevation of OTX2 expression.
  • RNASeq data show that NRGl and AKT1 are strongly expressed in pluripotent human stem cells throughout the 6 day period of this assay.
  • RNAseq data also shows that both, ERBB2 and ERBB3, the genes encoding the dimeric neuregulin receptor were present in human pluripotent cells.
  • Immunostaining shows that ERBB2/3 expression is restricted to the EZ and IZ (FIG. 5B).
  • NRGl down regulates SOX21 and OTX2 while promoting expression of NANOG (FIG. 5C).
  • the effect of NRGl on the level of mRNA for OCT4, NANOG and SOX21 suggests that NRGl exposure sustains the EZ/IZ state while blocking formation of the CZ (FIG. 5D).
  • Otx2 is an intrinsic determinant of the embryonic stem cell state and is required for transition to a stable epiblast stem cell condition. Development 140, 43-55.
  • Adorno M., Sikandar, S., Mitra, S.S., Kuo, A., Nicolis Di Robilant, B., Haro-
  • Colantuoni C, Lipska, B.K., Ye, T., Hyde, T.M., Tao, R., Leek, J.T., Colantuoni,
  • CoGAPS an R/C++ package to identify patterns and biological process activity in transcriptomic data. Bioinformatics 26, 2792-2793.
  • mouse epiblast stem cells resemble the anterior primitive streak.
  • Sox2 acts through Sox21 to regulate transcription in pluripotent and differentiated cells. Current biology : CB 22, 1705-1710.
  • CNS stem cells express a new class of intermediate filament protein. Cell 60, 585-595.
  • StemCellDB the human pluripotent stem cell database at the National Institutes of Health. Stem cell research 10, 57-66.
  • Sox21 promotes hippocampal adult neurogenesis via the transcriptional repression of the Hes5 gene.
  • the Journal of neuroscience the official journal of the Society for Neuroscience 32, 12543-12557.
  • Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator. Stem cells 26, 1663-1672.
  • Singh A.M., Reynolds, D., Cliff, T., Ohtsuka, S., Mattheyses, A.L., Sun, Y.,
  • Soxl, Sox2 and Sox3 genes from pre-gastrulation to early somite stages. Mechanisms of development 86, 197-201.

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Abstract

Provided are cells and in vitro assay systems useful for simulating and modeling early processes in cell specification and differentiation and morphogenesis as well as cell self-renewal and pluripotency. Pluripotent stem cell cultures, for example human embryonic stem cells or induced pluripotent stem cells (iPS cells), are dissociated to single cells and then cultured, thereby resetting and synchronizing the cells to a population in an equivalent state of pluripotency and earliest stage of cell specification. These cells then are cultured and simulate in vitro cell specification, differentiation and morphogenesis. These synchronized and cultured cells are useful in assays for identifitying and investigating disease mechanisms, pharmacological targets, and in drug discovery. The assays may also be diagnostic of a disease state, predisposition to a disease state or of patient and genome specific cell specification.

Description

ASSAY FOR CELL SPECIFICATION IN SELF-RENEWING HUMAN PLURIPOTENT
STEM CELLS
FIELD OF THE INVENTION
[0001] The invention provides assays and systems using pluripotent cells for patient stratification, target identification and drug screening.
BACKGROUND OF THE INVENTION
[0002] Contemporary stem cell research shows that the molecular pathways regulating function in development play key roles in many tissues throughout life and disease. For example, a recent report in a mouse model for Down's Syndrome defines clear effects on multiple somatic cell types, including hematopoietic and neuronal stem cells (Adorno et al., 2013). Recent papers mapping the chromatin landscape also demonstrate the power of stem cell technologies to systematically define the critical events in tissue development (Hnisz et al., 2013; Stergachis et al, 2013). Thus, human pluripotent cells are valuable in defining disease mechanisms and identifying new pharmacological targets (Tabar and Studer, 2014) (Bellin et al., 2012). However, the variation between different human pluripotent cells has not been fully defined and the differentiation procedures required to generate neurons are complex. There is a need for assays and systems to assess the variability and impact of genetic and epigenetic differences of pluripotent cells on developmental pathways.
SUMMARY OF THE INVENTION
[0003] The present invention provides cells and in vitro assay systems useful for simulating and modeling early processes in cell specification and differentiation and morphogenesis as well as cell self-renewal and pluripotency. The inventors have surprisingly found that these initial steps in cell specification and differentiation and morphogenesis may significantly depend upon the genomic and epigenetic context of the cells. Thus, the invention provides in vitro assay systems for analyzing the early steps of cell specification, differentiation and self-renewal to permit the identification of pharmacological targets involved in these processes and as well as providing systems for the screening of agents that can modulate, i.e., inhibit or promote these processes. The invention further provides assay systems and methods that exploit the genome and epigenetic specificity of the behavior of cells, including patient specific cells, in these systems to analyze and compare these early cell specification, morphogenesis and self-renewal processes in pluripotent cells derived from specific patients, for example, patients known to have one or more genetic polymorphisms associated with a particular disease or disorder. The results of these assays inform disease mechanisms, revealing potential pharmacological targets, and are useful in drug discovery. The behavior of patient specific cells in the assays of the invention may also be characteristic or diagnostic of a disease state, predisposition to a disease state or of patient and genome specific cell specification.
[0004] The inventors have found that pluripotent stem cell cultures, for example human embryonic stem cells or induced pluripotent stem cells (iPS cells), may be dissociated to single cells and then cultured, thereby resetting the cells to an equivalence group of cells. An "equivalence group" is a population of cells that have been set to an equivalent state of pluripotency and earliest stage of cell specification, in which the cells are synchronized to initiate processes of cell specification and morphogenesis. The cells may become lineage primed, as evidenced through changes in gene expression, but remain pluripotent during the cycle of the assay system of the invention. In particular, the equivalence group cells can be characterized by the expression of particular molecular markers, for example, but not limited to, expression of pluripotentcy factors, such as, but not limited to, NANOG and OCT4, or lack of protein expression of one or more markers of cell differentiation, such as but not limited to, SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN. The equivalence group may also be defined functionally, for example as described in Example 2 herein. The equivalence group of cells, when treated with BMP4 at day 0 of the dissociation and culture cycle described herein (FIG. 6), may induce the expression of certain lineage specific markers, such as CDX2 and/or Brachyury (T), in most cells of the equivalence group, but by day 2, expression of the markers is spatially restricted. In addition, inhibition of Oct4 on day 0 may also result in induction of markers for multiple lineages while inhibition at day 2 leads to expression of markers for only a specific lineage or to more restricted expression.
[0005] The equivalence group establishes a baseline for the temporal processes of cell specification, particularly neuronal specification (although the system may also be used to simulate the processes of specification to other cell fates such as mesodermal or endodermal cell fates), morphogenesis and self-renewal. The equivalence group of cells is then cultured for a period of time during which the cell specification is reversible, that is the cells may be reset to the original state of potency and cell specification, i.e., not beyond the point at which the cells are committed to a particular cell fate such that they cannot be reset to the equivalence group at a particular state of potency and cell specification by cell dissociation. This period of time may be anywhere from 1 to 10 days and may be determined experimentally.
[0006] During the culturing of the equivalence group cells in a controlled, feeder-free environment, the cells form epithelial cell monolayers that simulate cell specification, particularly neuronal specification and morphogenesis, and self renewal. During the culture period, molecular markers for pluripotency, such as NANOG and OCT4, decrease while molecular markers for cell specification to a particular lineage, such as, neurectodermal, mesodermal or endodermal lineages, for example, the proteins SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and/or EOMESODERMIN, increase. The epithelial sheet of cells becomes progressively more organized into zones of cells that represent spatially restricted expression of specific markers of cell specification and morphological characteristics, resulting in a specific laminar organization of the epithelial sheet or colony. For example, the cells may be organized into edge, intermediate and core zones (EZ, IZ, and CZ, respectively) of the epithelial sheet (see FIG 2). The zones may be characterized by expression of different levels of molecular markers, or different molecular markers, or by phenotypic or morphological differences, such as nuclear area. The EZ may be characterized by EpCAM expression, which is reduced or not present in the IZ and/or CZ. Nanog, SOX2 and SOX21, or any other marker of cell specification and renewal, may also be used to identify and delineate the zones. The zones may also be delineated by differences in nuclear area. Nuclear area may be assessed in cells stained with DAPI. Characteristically, the inner zone (IZ) has low nuclear area while the edge zone (EZ) has a larger nuclear area. A combination of nuclear area and molecular marker visualization may be used to define the different zones. Such cells may be lineage primed, i.e., they are on a pathway towards that fate specification, but they are still able to be reset to the equivalence group by cell dissociation.
[0007] The cultured cells may be analyzed, assayed and/or compared during this culture period. The cells may be monitored continually or at specific timepoints during the culture period. The cells may be assayed for changes in expression of particular markers, for changes to gene expression fingerprints, for morphological and/or phenotypic changes. In particular the pattern of gene expression within the epithelial sheet, including the laminar organization of the sheet, particularly, the spatially distinct expression of molecular markers of cell specification, may be assayed, characterized and compared. Such analysis may be carried out, for example, by high content analysis of the colony organization, for example, as described herein. Comparison of these molecular and phenotypic changes across cells derived from different sources can reveal genomic and epigenetic differences and the physiological, morphological, phenotypic or gene expression impact of those differences. In the assays of the invention, the entire cultured cell population may be assayed together or the different zones, or combinations of zones, may be assayed separately. Using microscopic techniques with labeled probes against particular molecular markers, for example, fluorescently labeled antibodies against specific markers, the dynamic distribution of these markers, indicative of steps in cell specification and morphogenesis, may be monitored and visualized. Characteristic patterns of gene expression within the laminar organization of the colony may be assayed and defined by any method known in the art, for example, high content analysis or any other analytical method. The gene expression, phenotypic and/or morphological read outs from the cells may be diagnostic and/or prognostic of a disease state or susceptibility to a disease state, or susceptibility to treatment of a disease state. And, the cultured cells may be useful to screen for and identity therapeutic and diagnostic targets or be useful for screening for potential therapeutic agents.
[0008] At the end of the culture period, the cells may be dissociated to single cells to reset to an equivalence group with characteristic pluripotency and gene expression.
[0009] In specific embodiments, the invention provides methods for synchronizing and resetting the potency of a population of pluripotent cells, preferably human, pluripotent cells, where the method comprises the steps of (1) dissociating the population of human pluripotent cells such at least 95%, and preferably, 99%, 99.9% or even 100% of the cells are single cells; (2) culturing said dissociated cells in the absence of feeder cells (generally, non-pluripotent cells that are adhered to the culture substrate), where the culturing may be in the absence of a survival factor or, to improve the survivability of the cells, in the presence of a survival factor presence of a survival factor for a time sufficient to reset the cells to an equivalence group; and (3) culturing the cells of the equivalence group in the absence of a survival factor for a period of time during which the cells can still be reset to the equivalence group, i.e., the cells are still capable of being made pluripotent by dissociation to single cells or, alternatively, culturing the cells under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group. After step (3), the cells may be dissociated as in step (1) and then cultured as in step (2) to reset them to an equivalence group. See, for example, FIG. 6. In certain embodiments, this process is repeated once, twice, three times, 4, 5, 6 7, 8, 9 or 10 times.
[0010] The population of cells may be identified as reset to the equivalence group by expression or absence of expression of certain molecular markers. For example, cells may be considered to be reset as an equivalence group if at least 90%, 95%, 99% or even 99.9% of the cells express one or more pluripotency markers, for example, NANOG or OCT4 and/or where less than 10%, 5%, 1% or 0.1% of the cells express detectable levels of a protein marker of cell specification or differentiation, such as, but not limited to SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN. Function assays, such as those described in Example 2, may be used to assess whether the cells form an equivalence group or not.
[0011] In step (1), the cells may be dissociated by any method known in the art for detaching and dissociating cultured mammalian, particularly, pluripotent, cells from standard tissue culture plasticware, for example, using enzymes or combination of enzymes such as Accutase®.
[0012] In step (2), the cells are cultured in the absence of any survival factor or, alternatively, are cultured in the presence of a survival factor. The survival factor may be any factor that promotes the survivability of the dissociated cells, for example, but not limited to, a RHO-associated kinase (ROCK) inhibitor, such as Y27632, myosin II inhibitors such as blebbistatin, JAK1 inhibitors or NRG1B. The cells are cultured in the presence of the survival factor for a period sufficient for them to be reset to the equivalence group as determined by expression of molecular markers, gene expression profiling, etc. The period of time is preferably 24 hours, but may be anywhere from 10-15 hours, 15 to 25 hours, 12-15 hours, 18 to 22 hours, or 22 to 30 hours.
[0013] In certain embodiments, the pluripotent cells are embryonic stem cells, preferably human embryonic stem cells. In other embodiments, the pluripotent cells are induced pluripotent stem (iPS) cells, preferably human. The iPS cells may be generated by any method known in the art, for example, by introducing pluripotency factors, such as one or more of OCT4, KLF4, SOX2, cMYC and LIN28, into somatic cells, for example by retrovirus, plasmid or as synthetic mRNAs, or any other method known in the art. In certain embodiments, prior to the cell dissociation step, the pluripotent cells are cultured on feeder cells, particularly mouse fibroblast feeder cells. In other embodiments, the pluripotent cells are cultured in the absence of feeder cells. In other embodiments, prior to and/or during step (2) and/or step (3), the cells are cultured in serum free media, or in media having no animal-derived products.
[0014] In specific embodiments, the iPS cells are derived from tissue obtained from a human subject. The tissue may be of any source, including any tissue that is a source of fibroblasts, including dermal cells, tissue obtained by biopsy, or post mortem tissue. iPS cells may be obtained from subjects, preferably human, for patient specific assays and to compare the behavior (including changes in gene expression, particularly the pattern of spatially restricted gene expression within the epithelial sheet of cells, i.e., the laminar organization of the colony, morphology, phenotype, etc.) among cells derived from different patients. In specific embodiments, the subject has been diagnosed with a particular disease or disorder, or the subject has one or more genetic polymorphisms associated with a disease or disorder, including with predisposition to a particular disease or disorder, or that renders a patient with a particular disease or disorder more or less susceptible to treatment with a particular therapeutic. For example, but not by way of limitation, the disease or disorder may be, schizophrenia and related neurodevelopmental disorders, autism or cancer. Alternatively, the subject is healthy and cells are assayed to assess normal variation.
[0015] In step (3), the cells are, in one embodiment, cultured for a period such that the cells can still be reset to an equivalence group, i.e., to a set of pluripotent cells, by dissociation of the cultured cells to single cells. The cells are preferably cultured for 6 days prior to cell dissociation, but may also be cultured for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days during step (3) and prior to cell dissociation. The cells may be cultured until they reach at least 90%, 95% or 100% confluence and then are subcultured by dissociation of the cells into single cells, repeating steps (1), (2) and (3) of the method of the invention. In a specific embodiment, during step (3) the cells are cultured in the absence of any morphogen or factor that modulates cell specification (renewal conditions). In specific embodiments, during step (3), the cells are cultured in the presence of a factor or combination of factors that modulate cell specification and/or act as morphgens, for example, the factor may guide the cells to a mesodermal fate, an endodermal fate or a neurectodermal fate. These cells may be exposed to one or more of these factors during the entire culture period or only at specific points during the culture period. The factor may be one or more of BMP4, Noggin, neuregulin, or Noggin and SB431542. In particular embodiments, the cells are exposed to a morphogen factor, such as a protein from the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin. The cells may also be treated with an agent, such as a protein, small molecule, antibody, soluble receptor, siRNA, antisense or any other agent, that modulates, either inhibiting or enhancing the activity of an endogenous morphogen, including one or more of the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin. Cells may also be subjected to other treatments that inhibit expression of certain genes involved in cell specification and/or self-renewal, such as, but not limited to, Nanog, Oct-4, and Sox2. Inhibition may be effected by any method well known in the art, for example, by siRNA.
[0016] During step (3), the cells form an epithelial sheet of cells. Cells may undergo lineage priming where the cells develop towards a particular cell fate, for example, neurectodermal, mesodermal or endodermal cell fate, but are not committed to that fate, i.e., may be reset to pluripotency by dissociation to single cells. The epithelial sheet of cells may form spatial zones of cells in a laminar organization with different cell specifications as determined, for example, by expression of certain molecular markers, cell morphology and/or cell phenotype. As exemplified in FIGS. 2A-D, these zones include an Edge Zone (EZ), an Intermediate Zone (IZ) or a Core Zone (CZ). The epithelial sheet of cells may recapitulate early cell specification events and morphogenesis permitting the interrogation and analysis of these events in a robust system where the specification process is reproducible and temporally synchronized.
[0017] In particular embodiments, the cells during step (3) are analyzed for one or more traits, including expression of molecular markers, for example, by probing with labeled antibodies against proteins that serve as molecular markers, bioinformatics analysis, for example, gene expression analysis using mRNA analysis, including RT-PCR or Next-generation sequencing or other high throughput sequencing for analysis of all or a portion of the transcriptome, microscopic analysis of cell phenotype, morphology, molecular markers on live cells, including real time monitoring by video. In particular, the pattern of expression of molecular markers of cell specification and self-renewal may be assayed, characterized and quantified using methods well known in the art and described herein. In specific embodiments, the pattern of expression is characterized by high content analysis as described and taught herein. The cells may be analyzed at any time during step (3). They may be assayed continuously, for example, by video through a microscope, or hourly, daily, every other day, on days 2, 4 and 6 or on days 1, 3, and 5, or at any time during the course of step (3).
[0018] The invention further provides methods of assaying and characterizing patient specific cells. In particular, the methods of the invention may be used to stratify a patient within a patient population based upon the behavior of the pluripotent cells derived from the patient in the assay methods of the invention, for example, by the pattern or laminar organization of certain markers of cell specification or self-renewal within the epithelial sheet of cells, including changes of that pattern over the culture period. For example, cells derived from patients having a particular disease state may display a characteristic behavior in the assay system of the invention, in that the cells may be lineage primed for a particular fate, may have a particular gene expression fingerprint or exhibit a particular cell morphology, epithelial sheet pattern or laminar organization or cell phenotype at a particular time during the step (3) culture period or may exhibit a characteristic dynamic change in expression of certain markers of cell specification or self-renewal or dynamic alteration in the laminar organization of the colonies formed. The cells being assayed may be cultured in the absence of any exogenous cell specification factor (renewal conditions) or may be cultured in the presence of such a factor, such as BMP4, Noggin, neuregulin, or Noggin and SB431542 or any morphogen, such as, factors from the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin. Cells may be subjected to other treatments that inhibit expression of certain genes involved in cell specification and/or self- renewal, such as, but not limited to, Nanog, Oct-4, or Sox2. Inhibition may be effected by any method well known in the art, for example, by siRNA. Patterns of expression in the presence or absence of these treatments or between cells cultured in the presence of different factors may be compared. The results may be diagnostic of a particular disease or disorder in the patient, or may be indicative of susceptibility of a disease or disorder to a particular treatment.
[0019] The methods of the invention may also be useful to compare the impact of different genomes or epigenetic backgrounds on cell specification and morphogenesis, for example, comparing two or more pluripotent cell populations derived from different subjects having different genomes and/or epigenetic backgrounds with respect to lineage priming, gene expression, particularly, the spatially and temporally dynamic pattern of gene expression within the epithelial sheet of cells, cell morphology or phenotype, or any other trait at any time point during the culture period in step (3). The subjects from which the cells were derived may have known genetic polymorphisms, for example, polymorphisms that pre-dispose a subject to a particular disease or disorder. Alternatively, cells derived from healthy normal subjects may be used to assess normal variation. Comparisons with pluripotent cells derived from a number of patient sources may be used to identify targets for drug discovery.
[0020] The invention further provides methods for screening agents for activity in modulating cell specification, the methods comprising the steps of (1) dissociating a population of human pluripotent cells such that at least 95% (or 98% or 99% or 99.9% or 100%) are single cells; (2) culturing the dissociated cells, preferably but not necessarily, in the presence of a survival factor, and in the absence of feeder cells, for a time sufficient for the cells to be reset to an equivalence group; (3) culturing the population of cells from step (2) in the absence of said survival factor and in the absence of feeder cells for a period of time during with the cells remain reversibly specified, that is, the cells may still be returned to a pluripotent state by dissociating the cultured cells to single cells, and during step (3), contacting the cells with the agent to be tested in a first cell population; and comparing an assayable trait of the cells contacted with said agent with said assayable train of cells from step (3) not contacted with said agent. Alternatively, in step (3), the cells may be cultured under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group
[0021] The invention further provides cell populations obtained by the methods of the invention. In particular, the invention provides cell populations that result from two or more cycles of dissociating pluripotent cells to single cells and then culturing those cells until a time at which their specification is reversible, i.e., they may be reset to pluripotency by dissociation to single cells. The cell population may be an equivalence group of cells that have been reset through the process of the invention to the same state of potency and specification. In other aspects, the cells are cultured after dissociation and form an epithelial sheet of cells. The epithelial sheet of cells may have different zones containing different cell types in the process of acquiring different cell fates or primed to a particular cell lineage. BRIEF DESCRIPTION OF THE FIGURES
[0022] FIGS. 1A-E. RNA-seq data in differentiating pluripotent cell lines across time. A Principal component analysis (PCA) of poly-A RNA-seq data (gene-level log2[RPKM]) across all lines, conditions, and time. Colors indicate differentiation conditions. Individual cell lines are represented by different plotting symbols. The size of plotting symbols indicates days of differentiation. Dotted lines connect individual cell line observations within a condition across time. SA01 is represented by the round plotting symbol and is highlighted in green. B) PCA within the Noggin-SB431542 condition (PC#1, upper panel). Self-renewal data projected into Noggin-SB431542 PC#1 (lower panel). C) PCA within self-renewing pluripotent cells (PC#3, upper panel). SOX21 expression in self-renewal (ranked #10 in PC#3 weights, lower panel). D) Two of 22 patterns generated by the CoGAPS algorithm. Pattern 12 represents early steps toward telencephalic neural precursor cells (left panel). Pattern 16 is one of 6 patterns that each identify a single cell line (SA01) across conditions and time (right panel). E) Two replicate iPS cell lines from William's patients (Right: patient 306, Left: patient 192) have reproducible, but distinct CoGAPS patterns.
[0023] FIGS. 2A-D. Distinct spatial zones in self-renewing feeder-free cultured hPSC colonies. A) Spatial expression of SOX21, NANOG and SOX2 on day 4. Maximum Z projections of confocal series from the edge. Scale bar, 100 μιη. B) Scatter plot representing all three measurements from (A). Points colored to reflect SOX2 expression: green=low, yellow=intermediate, red=high C) (left) High-content montage from 64 imaged fields including 4 xlO4 cells (right) Cartesian representation of cells from left colored by distance from the edge: min=grey; max=green. (bottom) Scatter plot relating mean nuclear expression of SOX2, SOX21, NANOG and nuclear area to distance from edge. Vertical lines demarcate zones. D) (top) Spatial expression of SOX21 and EpCAM on day 4. Scale bar, 100 μιη. (middle) Scatter plot of relative expression of Sox21 and EpCAM coloured by three zones: EZ=EpCAMhigh/SOX21low, IZ=EpCAMlow/SOX21low, CZ= EpCAMlow/Sox21high (bottom) Projection of scatter plot quadrants onto cellular position in image above. Notice the high green EpCAM signal at the edges. E) Laser capture microdissection of EpCAMhlgh (Edge) and EpCAMlow (Core) domains under control growth conditions analyzed by Taqman pPCR analysis of select genes from each domain normalized to EpCAMlow cells. F) 2-hour EdU pulse demonstrates an anti-correlation between EdU incorporation and SOX21 labeling. [0024] FIGS 3A-D. Distinct cell states are in an equivalence group. A. Homogeneous to localized expression of EpCAM and pluripotency factors through time. B. TaqMan analysis of pluripotency and lineage marker mRNA demonstrates resetting of cell state at each passage. C. Lineage of example cells expressing low or high levels of EpCAM after passage at t=0 give rise to a range of EpCAM-expressing cells after 48 hours. anti-EpCAM immunofluorescence expressed as pseudocolor in fire plot. D. Schematic representation of additional complete lineages demonstrating EpCAM expression in the founder cell (left side) and EpCAM signal in all derivative cells to the right. The low and high lineages from (C.) are marked.
[0025] FIGS 4A-C. A. Lineage responsiveness changes through time in culture. A.
SA01 cells treated with indicated siRNAs against pluripotency factors at Day 0 (top) and Day 2 (bottom) de-repress different lineages 48 hours after treatment under control conditions. NCR = negative control siRNA B. Cells treated with BMP4 at Day 0 or Day 2 then stained after 24 hours with antibodies against indicated targets. Note that all cells are BMP -responsive at Day 0 while only the edge is responsive two days later. C. qPCR of cultures grown under conditions indicated below bar charts for 6 days. Cultures were exposed to siRNAs for the 1st 4 days. qPCR probes are indicated above figure. All values are normalized to respective NCR levels in each condition (*,# indicate p<0.05 relative to NCR values within each condition).
[0026] FIGS 5A-D. Genotype-specific developmental bias. A. Scatter plot emphasizing genome similarity and cell line difference in average nuclear area and OTX2 expression. Colors reflect donor genomes. Triangle = AKT1 risk allele; circle = AKT1 protective allele. Note that visible differences in this display are highly significant as even the blue and red circles in NSB+NRG1B at Day6 have an anova p<10~16 for both nuclear area and OTX2 level. B. Immunofluorescence images of SA01 colony edge demonstrate localized expression of ERBB2 and ERBB3 in the EZ under control conditions. C. Six day treatment of SA01 line in NSB or NRG IB. D. qPCR analysis of mRNA levels normalized to expression in control condition for each mRNA.
[0027] FIG. 6. Schematic of cell dissociation and cell culture assay cycle.
[0028] FIGS. 7A-B. PCA in cells treated with BMP4.
[0029] FIGS. 8A-F. PCA for each of six cell lines. [0030] FIGS. 9A-D. PCA in cell line SA01 of OTX2(A), SOX21(B), FGFR3(C), and
EGFRI(D).
[0031] FIGS. lOA-C. The transcriptional signature defined by the neurectoderm- specific PCI starts high in early fetal development and disappears over late fetal and early postnatal life. This signature parallels closely the expression of Nestin, a widely used marker of neural stem cells.
[0032] FIG. 11A-C. CoGAPS analysis of iPS cells derived from WS patients.
[0033] FIG. 12A-B. Changes in nuclear area during formation of three distinct domains in PSC colonies. A. Box plot of nuclear area measurement between CON, NSB and BMP4 conditions. *, Significantly different from the CON (p < 0.001). B. Scatter plot relating mean nuclear area (um2) and mean nuclear SOX21 and SOX2. Data points are coloured with mean nuclear expression of SOX2: min =green; average =yellow; max = red.
[0034] FIG. 13A-C. Distinct cell states show different lineage bias. A. Expression of
SOX21 and EOMES after treatment of NANOG siRNA in combination with or without OCT4 siRNA for 48 hours. The siRNAs were treated at day 0 or day 2. B. Percentage of number of SOX21high- and EOMEShigh-cells after OCT4-, SOX2-, and NANOG-KD. The siRNAs for each factor were treated for 48 hour. C. Nuclear localization of phospho-Smadl/5 after BMP4 treatment at day 0 or day 2.
[0035] FIG. 14A-C. SOX21 and OTX2 are required for neuroectoderm differentiation. A. Expression of SOX1, NANOG and OCT4A at day 6 in control and Noggin- SB conditions after treatment of SOX21 and OTX2 siRNAs. Cells were exposed to each siRNA for the first 4 days. B. The relative niRNA expression of SOX1 and OCT4 (POU5F1) after the SOX21- and OTX2-KD. All values are normalized to respective NCR levels in each condition (*, # indicate p<0.05 relative to NCR values within each condition) C. Scatter plot of relative expression levels between SOX1 and NANOG and between SOX1 and OCT4A after the SOX21 and OTX2-KD.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The invention provides in vitro assay systems for analyzing the early steps of cell specification, differentiation and self-renewal to permit the identification of pharmacological targets involved in these processes and as well as providing systems for the screening of agents that can modulate, i.e., inhibit or promote these processes. The assay systems and methods of the invention may exploit the genomic and epigenetic specificity of the behavior of cells, including patient specific cells, in these systems to analyze and compare these early cell specification and morphogenesis processes in pluripotent cells derived from specific patients, for example, patients known to have one or more genetic polymorphisms associated with a particular disease or disorder. In particular, the cells form epithelial sheets in which the cells spatially organize into zones that have distinct gene expression, simulating and recapitulating the early stages of cell specification. Patterns of gene expression within and between the zones of the epithelial sheet, e.g., the laminar organization of the colony, provide a readout for identifying and characterizing the differences among different genetic and epigenetic contexts. The results of these assays inform disease mechanisms, revealing potential pharmacological targets, and are useful in drug discovery. The behavior of patient specific cells in the assays of the invention may also be characteristic or diagnostic of a disease state, predisposition to a disease state or of patient and genome specific cell specification.
[0037] In specific embodiments, the invention provides methods for synchronizing and resetting the potency of a population of pluripotent cells, preferably human, pluripotent cells, where the method comprises the steps of (1) dissociating the population of human pluripotent cells such at least 95% (or 98% or 99% or 99.9% or 100%) of the cells are single cells; (2) culturing said dissociated cells in the absence of feeder cells (generally, non-pluripotent cells that are adhered to the culture substrate), where the culturing may be in the absence of a survival factor or, to improve the survivability of the cells, in the presence of a survival factor presence of a survival factor for a time sufficient to reset the cells to an equivalence group; and (3) culturing the cells of the equivalence group in the absence of a survival factor for until a time at which the cells still be reset to the equivalence group, i.e., the cells are still capable of being made pluripotent by dissociation to single cells. After step (3), the cells are dissociated as in step (1) and then cultured as in step (2) to reset them to an equivalence group. In certain embodiments, this process is repeated once, twice, three times, 4, 5, 6 7, 8, 9 or 10 times. See, for example, the schematic in FIG. 6. Alternatively, in step (3), the cells are cultured under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group "Pluripotent cells" means cells that can differentiate into cell types from the three different germ layers— ectoderm, mesoderm and endoderm— for example, when injected in SCID mice, and can undergo many passages (20, 30, 50, 100 or even more) in in vitro culture and retain the ability to differentiate into cells of the three different germ layers after each division.
[0038] In step (3), the cells may be cultured for a period such that the cells can still be reset to an equivalence group, i.e., to a set of pluripotent cells, by dissociation of the cultured cells to single cells. The cells are preferably cultured for 6 days prior to cell dissociation, but may also be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days during step (3) and prior to cell dissociation. In specific embodiments, during step (3), the cells are cultured in the presence of a factor or combination of factors that modulate cell specification, for example, the factor may guide the cells to a mesodermal fate, an endodermal fate or a neurectodermal fate. The factor may be one or more of BMP4, Noggin, neuregulin, or Noggin and SB431542. In other embodiments, the factor may be a protein of the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin, or is an agent that modulates, either inhibits or enhances, the activity of one or more these factors. Alternatively, in step (3), the cells are cultured in the absence of any factors that modulate cell specification (renewal conditions).
[0039] During step (3), the cells may form an epithelial sheet of cells. Cells may undergo lineage priming where the cells develop towards a particular cell fate, for example, neurectodermal, mesodermal or endodermal cell fate, but are not committed to that fate, i.e., may be reset to pluripotency by dissociation to single cells or, alternatively, the cells are cultured under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group. The epithelial sheet of cells may form spatial zones of cells with different cell specifications as determined, for example, by expression of certain molecular markers, cell morphology and/or cell phenotype. The epithelial sheet of cells becomes progressively more organized into these zones of cells that represent spatially restricted expression of specific markers of cell specification and morphological characteristics. For example, the cells may be organized into edge, intermediate and core zones (EZ, IZ, and CZ, respectively) of the epithelial sheet. The epithelial sheet of cells may recapitulate early cell specification events permitting the interrogation and analysis of these events in a robust system where the specification process is reproducibly and temporally synchronized. In particular, the cultured cells during step (3) provide a reproducible and robust system for assaying morphogenetic events as the cells in the epithelial sheet undergo organization into zones of different cell types as takes place during morphogenesis.
[0040] In particular embodiments, the cells during step (3) are analyzed for one or more traits, including expression of molecular markers, for example, by probing with labeled antibodies against proteins that serve as molecular markers, bioinformatics analysis, for example, gene expression analysis using mR A analysis, including Next generation sequencing or other high throughput sequencing, particularly to generate RNASeq data, microscopic analysis of cell phenotype, morphology, molecular markers on live cells, monitoring by video. Any protein, mRNA or any other marker may be analyzed. For example, such markers include, but are not limited to, Brachyury (T), CDX2, Eomesoderm, ERBB2, ERBB3, Nanog, Oct4, OTX2, Soxl, Sox2, or Sox21, to name a few. The cells may be analyzed at any time during step (3). They may be assayed continuously, for example, by video through a microscope, or hourly, daily, every other day, on days 2, 4 and 6 or on days 1, 3, and 5, or at any time during the course of step (3).
[0041] The invention further provides methods of assaying and characterizing patient specific cells. In particular, the methods of the invention may be used to stratify a patient within a patient population based upon the behavior of the pluripotent cells derived from the patient in the assay methods of the invention. For example, cells derived from patients having a particular disease state may display a characteristic behavior in the assay system of the invention, in that the cells may be lineage primed for a particular fate, may have a particular gene expression fingerprint or exhibit a particular cell morphology, epithelial sheet pattern (laminar organization of the colony) or cell phenotype at a particular time during the step (3) culture period. The results may be diagnostic of a particular disease or disorder in the patient, or may be indicative of susceptibility of a disease or disorder to a particular treatment.
[0042] The methods of the invention may also be useful to compare the impact of different genomes or epigenetic backgrounds on cell specification, for example, comparing two or more pluripotent cell populations derived from different subjects having different genomes and/or epigenetic backgrounds with respect to lineage priming, gene expression, cell morphology or phenotype, or any other trait at any time point during the culture period in step (3). The subjects from which the cells were derived may have known genetic polymorphisms, for example, polymorphisms that pre-dispose a subject to a particular disease or disorder. Comparisons with pluripotent cells derived from a number of patient sources may be used to identify targets for drug discovery.
[0043] The invention further provides methods for screening agents for activity in modulating cell specification, the methods comprising the steps of (1) dissociating a population of human pluripotent cells such that at least 95% (or 98% or 99% or 99.9%) are single cells; (2) culturing the dissociated cells, preferably but not necessarily, in the presence of a survival factor, and in the absence of feeder cells for a time sufficient for the cells to be reset to an equivalence group; (3) culturing the population of cells from step (2) in the absence of said survival factor and in the absence of feeder cells for a period of time during with the cells remain reversibly specified, that is, the cells may still be returned to a pluripotent state by dissociating the cultured cells to single cells (or, alternatively, culturing the cells under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group), and during step (3), contacting the cells with the agent to be tested in a first cell population; and comparing an assayable trait of the cells contacted with said agent with said assayable train of cells from step (3) not contacted with said agent.
[0044] The invention further provides cell populations obtained by the methods of the invention. In particular, the invention provides cell populations that result from two or more cycles of dissociating pluripotent cells to single cells and then culturing those cells until a time at which their specification is reversible, i.e., they may be reset to pluripotency by dissociation to single cells. The cell population may be an equivalence group of cells that have been reset through the process of the invention to the same state of potency and specification. In other aspects, the cells are cultured after dissociation and form an epithelial sheet of cells. The epithelial sheet of cells may have different zones containing different cell types in the process of acquiring different cell fates or primed to a particular cell lineage.
Preparation of pluripotent cells
[0045] Pluripotent cells may be generated using any methods known in the art. The pluripotent cells may be of any species, particularly vertebrate, and more particularly mammalian. The cells are preferably from humans but may also be from non-human mammalian species, for example, but not limited to, non-human primates (such as, apes, monkeys, etc.), rodents (such as mice or rats), cows, pigs, sheep, horses, rabbits, or any other species. Pluripotent cells may be embryonic stem cells (ESCs), particularly human embryonic stem cells (HESCs), induced pluripotent stem (iPS) cells, or any other pluripotent cell. In certain embodiments, the cells may be totipotent.
[0046] ESCs, and particularly HESCs may be prepared according to methods well known in the art. ESCs are generally derived from pre-implantation embryos. These cells are pluripotent and can be cultured and retain pluripotency for extended periods of time over multiple passages, for example, one year or more. See, e.g., Thomson, US Patent No. 6,200,806, which is incorporated by reference herein in its entirety.
[0047] iPS cells may be generated using any method known in the art. iPS cells are generally prepared by introducing a combination of so-called "reprogramming factors" into a differentiated somatic cell, such as a fibroblast. The expression or presence of the reprogramming factors in a cell leads to dedifferentiation, resulting in a pluripotent cell line. See, e.g., Yamanaka et al, US 7,964,401; Thomson et al, US 8,440,461; Jaenish et al, US 7,682,828, all of which are incorporated herein by reference in their entireties. Reprogramming factors include, but are not limited to Oct-3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog. The reprogramming factors may be introduced by any known method, including, by expression from a retroviral vector, non-integrating viral vectors or non-viral vectors or episomes. In one embodiment, the reprogramming factors are introduced into the somatic cell as modified mRNAs, for example, having modified nucleosides such as 5-methylcytidine, 2-thiouridine, pseudoruidine, or 2'-0— methyladenosine. See, for example, Rossi et al, US patent application publication 2012/130624, which is incorporated herein by reference in its entirety.
[0048] HESCs and iPS cell lines are also available from a variety of cell banks and commercial sources.
[0049] Pluripotent cells may be identified by their ability to differentiate into cells of all three germ layers, for example, by injection into SCID mice. The pluripotent cells may express certain markers associated with pluripotency, for example, SSEA3, SSEA4, Oct4, Nanog, EpCAM, and/or Sox 2. [0050] Any type of somatic cell may be used to generate the iPS cells. For example, skin, lung, heart, brain or other tissue of the nervous system, liver, blood, kidney or muscle cells can be used. The subject can be of any age. The subject is preferably human. In certain embodiments, the subject suffers from a particular disease or disorder. Samples may be fresh or may be any tissue sample, such as biopsy or other pathology samples or may be post-mortem samples, including preserved samples. In addition, the human subject may also have one or more genetic polymorphisms that are associated with a particular disease or disorder, predisposition to a particular disease or disorder or that are associated with susceptibility or lack of susceptibility to a particular treatment regimen for a disease or disorder. In specific embodiments, the disease or disorder is schizophrenia or any neurodevelopmental disorder, cancer, diabetes, cardiovascular disease, or autism. In other embodiments, the human subject has not been diagnosed with or does not have a polymorphism associated with a particular disease or disorder and iPS cells may be isolated from such subjects to analyze and characterize differences revealed in the assays of the invention due to normal variation among healthy subjects.
[0051] Prior to using the cells in the methods of the invention, the pluripotent cells are cultured by methods well known in the art. In certain embodiments they are cultured on feeder cells, which are any cells that promote the growth and maintain the pluripotency of the pluripotent cells. Feeder cells are generally fibroblasts, particularly murine fibroblasts. The pluripotent cells may also be cultured in the absence of feeder cells. The cells are cultured under conditions known for the culture of mammalian cells, particularly, pluripotent cells. In certain embodiments, the cells are grown in cell culture media, for example hES media, the components of which are provided in Table 1 in Example 1 herein. The media may contain serum, but is preferably serum free. The media may be supplemented with growth factors, for example bFGF.
[0052] In culturing the cells after the cell dissociation step, the cells may be cultured under conditions suitable for culturing pluripotent cells. The cells are preferably cultured in the absence of feeder cells. The media may be mTeSR®l medium, supplied by StemCell Technologies (Vancouver, Canada) (comprised of 400 mL mTeSR®l basal medium (Catalog #05851) and 100 mL mTeSR®l 5X supplement (Catalog #05852)). The medium used to culture the cells in the methods of the invention are preferably serum free
Cell Dissociation [0053] For pluripotent cells that are being cultured as colonies in feeder cell cultures or in a feeder free culture, colonies are dislodged by any method known in the art, for example by incubation with an enzyme such as collagenase IV. The colonies are then dissociated, preferably enzymatically. Any enzyme known to be useful to dissociate cultured mammalian cells may be used, for example, the Accutase enzyme (StemPro® Accutase® Cell Dissociation Reagent; Invitrogen Cat# Al l 1105-01). The cells are incubated in the presence of the enzyme for sufficient time and under conditions that promote cell dissociation and to achieve dissociation almost entirely to single cells. For example, the cells may be incubated for 3 minutes, 5 minutes, 7 minute or 10 minutes depending upon the cells and conditions. To promote and facilitate cell dissociation, the cells may be agitated or aspirated, for example, by pipetting the cell solution with an automatic micro pipettor. Once the cells have been incubated for a suffient amount of time, the enzymatic action may be stopped, for example by addition of serum to the cells. Preferably, at least 80%, 85%, 90%, 95%, 99% or 100% of the cells harvested are dissociated to single cells.
[0054] Once the cells have been dissociated, the cells may be incubated in the presence or absence of a growth factor, such as FGF-2 or bFGF, and, optionally a survival factor. The survival factor may be any factor that promotes the survivability of the dissociated cells, for example, but not limited to, a RHO-associated kinase (ROCK) inhibitor, such as Y27632, a myosin II inhibitor, such as blebbistatin, a JAK1 inhibitor, or NRG IB. In a preferred embodiment, the dissociated cells are incubated with Y27632. The cells may be incubated with the survival factor during the plating process.
[0055] The dissociated cells may then be plated and cultured using any method known in the art. The cells may be cultured in any cell culture vessel, for example a dish, a flask, a roller bottle or any other vessel for mammalian cell culture. The cell culture vessel is preferably coated with a coating such as, but not limited to, Matrigel®. The cells are plated and cultured in the cell culture vessel, preferably in the presence of the survival factor, specifically Y27632. The cells may be maintained in the presence of the survival factor for a period of time, for example, 24 hours, or for a shorter period of time, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 15 hours or even 20 or 24 hours. [0056] After incubation in the survival factor, the cells have formed an equivalence group. The "equivalence group" is a population of cells that are all pluripotent and are in the same state of cell specification. As such, the population of cells begin the program towards lineage priming and then lineage specification at the same time and can be analyzed temporally and monitored as the cells undergo cell specification and the population of cells go through morphogenesis. The cells may become lineage primed, as evidenced through changes in gene expression, but remain pluripotent during the cycle of the assay system of the invention. In particular, the equivalence group cells can be characterized by the expression of particular molecular markers, for example, but not limited to, expression of pluripotentcy factors, such as, but not limited to, NANOG and OCT4, or lack of protein expression of one or more markers of cell differentiation, such as but not limited to SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN. The equivalence group may also be defined functionally, for example as described in Example 2 herein. The equivalence group of cells, when treated with BMP4 at day 0 of the dissociation and culture cycle described herein, may induce the expression of certain lineage specific markers, such as CDX2 and/or Brachyury (T), in most cells of the equivalence group, but by day 2, expression of the markers is spatially restricted. In addition, inhibition of Oct4 on day 0 may also result in induction of markers for multiple lineages while inhibition at day 2 leads to expression of markers for only a specific lineage or to more restricted expression.
[0057] The population of cells may be identified as reset to the equivalence group by expression or absence of expression of certain molecular markers. For example, cells may be considered to be reset as an equivalence group if at least 90%, 95%, 99% or even 99.9% of the cells express one or more of the markers NANOG or OCT4 and/or where less than 10%, 5%, 1% or 0.1% of the cells express detectable levels of a marker of cell specification or differentiation, such as, but not limited to SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN.
Cell Culture Period
[0058] Once the cells have been set or reset to an equivalence group of cells, the cells may be cultured in the absence of the survival factor under conditions appropriate for pluripotent mammalian cells as known in the art, for example, as described in Example 1. The media may be exchanged as generally routine in the art, for example, every 24 hours. The cells are cultured and allowed to form and epithelial monolayer of cells. The cells are subcultured once they reach 90% to 100% confluence and prior to achieving a point in cell specification where the cells cannot be reset to the equivalence group by cell dissociation. The cells are generally cultured for approximately 4, 5, 6, 7, 8, 9 or 10 days. Alternatively, the cells may be cultured under conditions and for a period of tine to achieve irreversible cell specification such that the cells are not capable of being reset to the equivalence group
[0059] During the culture period, the cells may be incubated with one or more factors that promote or direct a particular cell fate or cell specification factors. For, example, the cells may be exposed to one or more of the factors either throughout the culture period or at particular times and for particular periods during the culture period, for example from days 1-6, 1-2, 2-3, 3- 4, 4-5, 5-6, 1-3, 2-4, 3-5, 4-6, 1-3, 4-6, 1-4, or 2-6, or whatever period of time is useful for the desired analysis. Such factors, include, but are not limited to Noggin, BMP4, neuregulin, orSB431542. The factor may also be one or more of a protein of the TGFbeta superfamily, WNT superfamily, NOTCH superfamily, FGF superfamily, EGF superfamily, Hedgehog superfamily, or IGF superfamily or insulin, or is an agent that modulates, either inhibits or enhances, the activity of one or more these factors. In the absence of any of these cell specification factors (renewal conditions), the cells will generally tend to develop towards a neurectodermal fate. Noggin exposure promotes a neurectodermal cell specification while BMP4 promotes a mesodermal cell specification. The behavior of the cells either without any exogenous factors or in response to one or more of these factors may depend upon the genomic and/or epigenetic context of the cell.
[0060] During the culture period, the cultured cells form an epithelial sheet, which recapitulates or simulates cell specification and morphogenesis. Cells may undergo lineage priming where the cells develop towards a particular cell fate, for example, neurectodermal, mesodermal or endodermal cell fate, but are not committed to that fate, i.e., may be reset to pluripotency by dissociation to single cells. During the culture period, molecular markers for pluripotency, such as NANOG and OCT4, decrease while molecular markers for cell specification to a particular lineage, such as, neurectodermal, mesodermal or endodermal lineages, including markers such as, SOX21, Brachyury (T), CDX2, SOX1, PAX6, SOX17, and EOMESODERMIN, increase. The epithelial sheet of cells becomes, during the culture period, progressively more organized into zones of cells that represent spatially restricted expression of specific markers of cell specification and morphological characteristics. For example, the cells may be organized into edge, intermediate and core zones (EZ, IZ, and CZ, respectively) of the epithelial sheet. The zones may be characterized by expression of different levels of molecular markers, or different molecular markers, or by phenotypic or morphological differences, such as nuclear area.
Cell Assays
[0061] In particular embodiments, the cells during step (3) are analyzed for one or more traits. These traits include expression of particular genes, either at the mR A level or protein level, cell morphology, such as nuclear size and other aspects of cell morphology and cell phenotype. In addition, the spatial and temporal distribution of these traits within the epithelial sheet of cells formed in step (3), and the zones, EZ, IZ and/or CZ, may be assayed, including specific mRNA levels, protein expression, and cell morphology, and characterized using methods for assessing patterns of gene expression.
[0062] Gene expression may be analyzed by any method known in the art. The presence or absence of specific mRNAs or levels of specific mRNAs can be assayed, for example, by northern analysis or RT-PCR or any other method known in the art for detecting the presence or amount of specific mRNA species. In addition, bioinformatics analysis, such as sequencing of all or a portion of mRNAs expressed in a cell population (e.g., the transcriptome) may be used to identify and analyze gene expression in a cell population. The mRNAs may be sequenced by any method known in the art, such as Next Generation sequencing, or any other high throughput sequencing platform, or by microarray analysis. RNASeq data may be analyzed to identify gene expression profiles.
[0063] In other embodiments, gene expression is analyzed at the protein level.
Antibodies against specific proteins may be used in western analysis, immunoassays, and the like to detect the presence of specific proteins in a cell population. For cell surface proteins, gene expression can be detected, analyzed and monitored in live cells using labeled antibodies against specific cell surface protein markers (for example, fluorescently labeled antibodies— see Table 2 herein). Contacting cells with one or more of these labeled antibodies with specificity for one or more particular targets, the labeled proteins can be visualized on the cells. Such labeled antibodies are useful to assess the spatial and temporal pattern of expression of different molecular makers within the epithelial sheet of cells. The patterns of expression can be captured, visualized and monitored by microscopy, such as a confocal microscope, including quantitative fluorescence microscopy. Nuclear markers, such as DAPI staining, may be used to identify and mark individual cells. Molecular markers, such as EpCAM, Nanog, Sox21 and Sox2, may be used as reference points, for example, EpCAM, may be used to delineate the Edge Zone or EZ of the epithelial sheet. The different zones may also be distinguished based upon differences in nuclear area. DAPI may be used to label nuclei so they can be measured and quantitated. The inner zone correlates with low nuclear area while the edge zone correlates with a larger nuclear area. A combination of nuclear area and molecular markers may be used to characterized and identify the different zones within the epithelial sheet. Alternatively, different zones or regions of the epithelial sheet may be isolated and analyzed for m NA or protein expression.
[0064] The cells may be analyzed at any time during step (3). They may be assayed continuously, for example, by video through a microscope, or hourly, daily, every other day, on days 2, 4 and 6 or on days 1, 3, and 5, or at any time during the course of step (3).
[0065] Data obtained from analysis of the cells can be processed and analyzed by any method available in the art. For example, gene expression patterns may be analyzed by principal component analysis (PCA) or any similar type of analysis, or any non-negative matrix factorization (NMF), such as but not limited to CoGAPS (see Fertig et al., 2010). The pattern of gene expression within the epithelial sheet may be analyzed using a high content analysis, for example, as described in Example 1 herein. The output of the data analysis may be used to compare results across cells derived from different sources, e.g., from patients of different genetic or epigenetic backgrounds, which can be used to identify patterns, leading to understanding of mechanisms of cell differentiation, morphogenesis and disease. The data output may also be compared to that in available clinical or other databases to identify and understand patterns of gene expression associated with particular disease state. In addition, the output may be compared to known standards, for example, to use the output from cells of a particular patient to diagnose or prognose a particular disease or disorder or to assays whether a patient is amenable to a particular treatment for a disease or disorder. Such analysis may be used to stratify patients having a particular disease or disorder according to likelihood of developing the disease or disorder, severity of the disease or disorder, whether patients are amenable to certain treatments or not.
EXAMPLES
Example 1
[0066] The following methods provide exemplary methodology and reagents for carrying out the assays of the invention. Prior to establishing the monolayer feeder-free cultures, human pluripotent stem cells (both hES and iPS cell lines) are serially subcultured on mouse embryonic fibroblasts using a human ES cells medium, hereafter termed hES medium supplemented with 4 ng/mL recombinant human FGF-2. Table I shows the components used to prepare about 500 mL of the hES medium. Then, a commercial medium supplied by StemCell Technologies (Vancouver, Canada), mTeSR®l medium, is used to serially subculture human pluripotent stem cells in feeder-free monolayer culture on Matrigel coated dishes. This medium is comprised of 400 mL mTeSR®l basal medium (Catalog #05851) and 100 mL mTeSR®l 5X supplement (Catalog #05852).
[0067] COATING TISSUE CULTURE VESSELS
(1) BD Matrigel® is diluted by adding 60 Matrigel in 12 mL DMEM:F12 (Invitrogen 11330-032) and the solution gently mixed.
(2) Sufficient Matrigel® solution is added to cover the surface of the tissue culture vessel. For instance, 1 mL of the solution can be used for each well of a 6-well plate. The vessel is incubated at room temperature for at least 1 hr.
(3) The Matrigel® solution is removed and the surface of the vessel rinsed with sterile IX PBS once.
(4) 2.5 mL (or respective working volume for alternative tissue culture flasks) is placed in each well of 6-well plates and incubate the plates at 37oC in a humidified chamber containing 5% C02, and 95% air saturation.
[0068] CELL HARVESTING FROM FEEDER CULTURE TO DERIVE
MONOLAYERS (1) After reaching appropriate size colonies in feeder culture, the tissue culture vessel containing human pluripotent stem cells is removed from the humidified incubator into the biocabinet. The vessel is tilted and the cell culture medium gently removed using a plastic pipette.
(2) 1 mL of 1 mg/mL (IX) collagenase IV is added gently to each well and incubated for 30 min at 37oC in a humidified chamber containing 5% C02, and 95% air saturation. During incubation time, each culture flask is tapped 2-3 times to help dislodge the colonies.
(3) The collagenase solution is replaced with 1 mL hES medium (Table 1) supplemented with 4 ng/mL FGF-2 and 5 μΜ Y27632.
(4) The colonies are detached by washing them using a PI 000 Pipetman, and the content of each well is transferred into a 15 mL centrifuge tube.
(5) Each well is washed with 3 mL of the medium to ensure detachment of all colonies, and the content of each well is transferred into respective 15 mL centrifuge tubes.
(6) The cell suspension is centrifuged for 2 min at 200 g.
(7) The supernatant is removed and 5.0 mL pre-warmed hES cell medium added to the pellet.
(8) The cells are resuspended and the cell suspension centrifuged two more times for 2 min at 200 g.
(9) The supernatant is removed, and 1.0 mL pre-warmed Accutase® enzyme (StemPro® Accutase® Cell Dissociation Reagent; Invitrogen Cat# Al l 1105-01) is added to the pellet and the pellet is gently resuspended into Accutase solution.
(10) The cell suspension is incubated in the enzyme solution for 5-7 min at 37oC in a humidified chamber.
(11) The colonies are dissociated into single cells by gently aspirating (10-15 times) the cell suspension with a PI 000 Pipetman set to a volume of 850 μΐ,.
(12) An equal volume of FBS (ES cell grade) and 4 mL of the hES medium supplemented with 4 ng/mL FGF-2 and 5 μΜ Y27632 is added to the cell suspension to block the action of the enzyme.
(13) The cell suspension is centrifuged for 5 min at 200 g, the pellet resuspended in 1 mL hES medium supplemented with 4 ng/mL FGF-2 and 5 μΜ Y27632. (14) Upon acquiring a single cell suspension, cell counts are performed using Trypan-blue exclusion method.
(15) 1,000,000 cells per well are inoculated onto a matrigel-coated 6-well plate, which has been preincubated with prewarmed 2.5 mL of mTeSR® 1 medium supplemented with 5 μΜ Y27632.
(16) The culture vessel (plate) is incubated at 37oC in a humidified chamber containing 5% C02, and 95% air saturation.
(17) After 24 h and every day thereafter (until subsequent passaging), each well is washed with 1 mL pre -warmed mTeSR®l medium. Then, the washing medium is exchanged with 2.5 mL of the pre-warmed mTeSR®l medium.
(18) Upon reaching 90-100% confluency, the cells are subcultured using the Cell Harvesting and Passage of Monolayers protocol below.
[0069] CELL HARVESTING AND PASSAGE OF MONOLAYERS
(1) Upon reaching 90-100%) confluency, the matrigel-coated tissue culture vessel containing human pluripotent stem cells is removed from the humidified incubator into the biocabinet. The cell culture medium is removed from the culture by tilting the vessel and using a plastic pipette.
(2) 1.0 mL pre-warmed Accutase® enzyme is gently added to each well of 6-well plate, and the plate incubated for 5-7 min at 37oC in a humidified chamber.
(3) The cells are dislodged from each well by gently aspirating the enzyme solution, and the cell suspension is then transferred into a 15 mL centrifuge tube.
(4) The cell suspension is centrifuged for 5 min at 200 g, and the pellet washed with lOmls of mTeSR® 1 medium supplemented with 5 μΜ Y27632.
(5) The cell suspension is centrifuged for 5 min at 200 g, and the pellet resuspended in 1 mL of the mTeSR® 1 medium supplemented with 5 μΜ Y27632.
(6) Upon acquiring a single cell suspension, cell counts are performed using Trypan-blue exclusion method.
(7) 750,000 cells per well (equivalent of 300,000 Cells/mL) are inoculated into each well of a matrigel-coated 6-well plate, which has been preincubated with prewarmed 2.5 mL of the mTeSR® 1 medium supplemented with 5 μΜ Y27632. (8) The culture vessel is incubated at 37oC in a humidified chamber containing 5% C02, and 95% air saturation.
(9) After 24 h and every day until subsequent passaging, each well is washed with 1 mL of the pre-warmed mTeSR®l medium. Then, the washing medium is exchanged with 2.5 mL of the pre-warmed fresh mTeSR®l medium.
(10) Upon reaching 90-100 % confluency, serially subculture the cells using enzymatic treatment with Accutase® into fresh feeder- free culture as described in this protocol.
Table 1. Medium components to prepare 500.0 L of hES growth medium for expansion in feeder culture.
Figure imgf000028_0001
REAGENTS AND ANTIBODIES
Catalog
Primary antibodies Supplier Species Type Dilution number
Cell Signaling
AKT, phospho (pSer473) (D9E) 4060 Rabbit Monoclonal 1 :200
Technology
Cell Signaling
Phospho-Akt (Thr308) (C31 E5E) 2965 Rabbit Monoclonal 1 :200
Technology
Brachyury R&D AF2085 Goat Polyclonal 1 :500
CDX2 Biogenex AM392 Mouse Monoclonal 1 : 1 Eomesoderm Abeam ab23345 Rabbit Polyclonal 1 :200
ERBB2 R&D MAB1 129 Mouse Monoclonal 1 :200
ERBB3 R&D MAB3481 Mouse Monoclonal 1 :200
Fibronectin R&D MAB1918 Mouse Monoclonal 1 :200
NANOG R&D AF1997 Goat Polyclonal 1 :200
RCAB004
NANOG ReproCell Rabbit Polyclonal 1 :200
P-F
MAB1759
OCT4A R&D Mouse Monoclonal 1 :200
1
OTX2 R&D AF1979 Goat Polyclonal 1 :200
SMAD1/5, phospho (Ser463/465) Cell Signaling
9516 Rabbit Monoclonal 1 :200 (41 D10) Technology
SMAD2 (Ser465/467)/SMAD3 Cell Signaling
8828 Rabbit Monoclonal 1 :200 (Ser423/425), phosphor (D27F4) Technology
SOX1 R&D AF3369 Goat Polyclonal 1 :400
SOX2 R&D AF2018 Goat Polyclonal 1 :200
SOX2 R&D MAB2018 Mouse Monoclonal 1 :200
SOX21 R&D AF3538 Goat Polyclonal 1 :200
SOX21 Neuromics GT15209 Goat Polyclonal 1 :200
[0071] siRNA: SOX21 (sc-38433, Santa Cruz), OTX2 (EHU129881, Sigma Aldrich),
OCT4 (sl0873, Life technologies), NANOG (s36649, Life technologies), SOX2 (sc-38408, Santa Cruz).
[0072] ESC culture: For maintenance of human PSCs in feeder-free condition, cells are dissociated to single cell populations with Accutase® (Al l 105, Life technologies), plated at a density of 1X106 cells in a Matrigel® (BD)-coated 6-well plate and cultured with mTeSRl (Stem Cell Technology, #05850, www.stemcell.com/en/Products/All-Products/mTeSRl .aspx). The cells are cultured with 5 μΜ Y27632, ROCK inhibitor (Y0503, Sigma-Aldrich) to increase the single cell survival upon dissociation. At 24 hours after plating, Y27632 was removed from the medium and cells cultured for another 4 days before the next passaging.
[0073] For differentiation, Noggin (500 ng/ml, 719-NG, R&D) plus SB431542 (2 μΜ,
S4317, Sigma-Aldrich) are added to mTeSRl medium for neuroectodermal differentiation and BMP4 (100 ng/ml, 314-BP, R&D) for mesendodermal differentiation upon Y27632 withdrawal. [0074] For the controlling the pluripotency states, recombinant human NRGip (100 ng/ml, #396-HB, R&D) is added for 6 days in mTeSRl medium.
[0075] siRNA treatment: Silencing endogenous genes is performed by transfection using
DharmaFECT 1 reagent (Thermo Scientific). Cells are transfected with non-targeting negative control siRNA (#4390843, Life technologies) or siRNA targeting SOX21 and OTX2 at a final concentration of 50 nM for 4 days after removal of Y27632. The siRNAs of OCT4, NANOG and SOX21 are treated for 48 hours after removal of Y27632 on either day 0 or day 2.
[0076] Immunofluorescence: Cells are fixed with 4% paraformaldehyde for 10 min and permeabilized for 30 min using 0.1% Triton X-100 (Sigma- Aldrich) in PBS. Subsequently, cells are blocked with 10% donkey serum (Sigma- Aldrich) and incubated with primary antibodies overnight. Secondary antibody staining is performed with Alexa Fluor®-conjugated antibodies (Life technologies). For direct immunostaining, primary antibodies are conjugated using Alexa Fluor® antibody labeling kit (Life Technologies). Nuclei are counterstained with DAPI and imaged with a confocal laser-scanning microscope.
[0077] Laser Capture Microdissection (LCM): SA01 ES cells are passaged onto Zeiss
DuplexDish 35 (Zeiss cat no. 415101-4400-551) in the presence of the ROCK inhibitor Y27632. After 24 hours, Y27632 is removed and colonies are allowed to develop for an additional 72 hours under control conditions of mTeSRl culture medium only. To identify the Edge Zone (EZ), the live culture is incubated with anti-EpCAM antibody after direct conjugation to Alexa647 (Molecular Probes Cat No. A-20186) for 15 min in the tissue-culture incubator then washed twice with pre-equilibrated media without antibody. LCM was performed using the Zeiss PALM microbeam system in non-contact mode on live culture. Briefly, fluorescence illumination of Alexa647 enabled visualization of the EZ. Due to the high-contrast of the anti- EpCAM staining at the edge compared to the core, histogram-based thresholding is used to segment the EZ from the IZ.
[0078] Quantitative RT-PCR: Reverse transcription was performed in the sample lysate using Superscript III Reverse Transcriptase (Invitrogen). Relative RNA levels were calculated using the AACt method with human GAPDH as reference genes. Measuring passage efficiency
[0079] SA01 cells were grown to confluence on monolayer conditions (4-days) in a 6- well plate as described earlier. Cells were passaged using enzymatic digestion (acutase). Viability was quantified automatically using the Countess (LifeTechnologies C 10310) with trypan blue exclusion. 1,000,000 cells were plated on each well of a 6-well plate in the presence of rock inhibitor for one hour then imaged. Single fields were counted from each of six wells manually to distinguish live from dead (pyknotic) cells. Survival is represented as the number of live/dead cells counted per area extrapolated to the entire area of the well divided by 1,000,000 (the number of cells plated per well).
Time-Lapse recording
[0080] After 4 days in culture, SA01 cells were passaged using Accutase. While in suspension, cells were incubated with anti-epCAM antibody (RnD systems) directly conjugated to Alexa 647 for 10 min at room temp. Cells were pelleted once then resuspended in media lacking antibody then counted with the Countess. 1X106 live cells/well were plated on 35mm plates (Greiner Advanced TC) coated with Matrigel in mTeSRl in the presence of Y27632. Imaging began less than 5 minutes after the EpCAM wash. At the beginning of the recording, many cells can be observed attaching to the surface. One day prior to passage, and on one day after the passage, cells were transfected with 1 μg mRNA encoding nuclear-localized GFP (Stemgent) to aid in single-cell visualization and manual tracking. After 48 hours, cells were fixed then stained with EpCAM directly conjugated to Alexa 488, and DAPI. Imaging was performed using a Zeiss 780 confocal microscope with a 20x 0.8NA objective at full resolution at 2048 x 2048 px/field. Every three minutes a 2x2 mosaic was captured using a 633 nm laser to excite Alexa647 and also provide transmitted light for concurrent DIC imaging. The gas environment was maintained at 5% C02 with a Zeiss SI gas control system. The microscope body and stage was heated to 37 with a Zeiss heating unit and environmental enclosure (PECAM).
Lineage Analysis
[0081] An image area was chosen that includes two non-contiguous "edge zones".
Complete lineage analysis was achieved using manual tracking and SIMI BioCell to record lineage structures. Complete lineages are presented either intact or in schematic with the founder cell on the left showing the EpCAM signal at time t=0 and the EpCAM signal in all the derivatives at t=48 hrs.
RNAseq
Poly-A selection library preparation and O/C
[0082] The poly-A containing mRNA molecules are purified from 1 μg DNAse treated total RNA. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Reverse transcriptase and random primers are used to copy the cleaved RNA fragments into first strand cDNA. The second strand cDNA is synthesized using DNA Polymerase I and RNaseH. These cDNA fragments after 2 minutes under 94 degree then go through an end repair process using T4 DNA polymerase, T4 PNK and Klenow DNA polymerase, and the addition of a single Ά base using Klenow exo (3' to 5' exo minus), then ligation of the illumina PE adapters using T4 DNA Ligase. An index (up to 12) is inserted into Illumina adapters so that multiple samples can be sequenced in one lane of an 8-lane flow cell if necessary. These products are then purified and enriched with 15 cycles of PCR to create the final cDNA library for high throughput DNA sequencing using an Illumina Highseq 2000.
Ribosomal RNA depletion (RiboZero) with strand-specific library preparation and Q/C
[0083] RNA-seq libraries were constructed using Illumina TruSeq Stranded Total RNA
Ribo-Zero sample Prep Kit following the manufacturer's protocol. The ribosome RNAs were removed using Ribo-zero beads from ~ 800 ng DNAse treated total RNA. Following the purification, the total RNA without Ribosome RNA was fragmented into small pieces using divalent cations under elevated temperature (94 degree) for 2 minutes. Under this condition, the range of the fragments length is from 130-290 bp with a median length of 185 bp. Reverse transcriptase and random primers were used to copy the cleaved RNA fragments into first strand cDNA. The second strand cDNA was synthesized using DNA Polymerase I and RNaseH, dUTP in place of dTTP These cDNA fragments then went through an end repair process using T4 DNA polymerase, T4 PNK and Klenow DNA polymerase, and the addition of a single 'A' base using Klenow exo (3' to 5' exo minus), then ligation of the Illumina PE adapters using T4 DNA Ligase. An index (96 unique dual-index pairs) was inserted into Illumina adapters so that multiple samples can be sequenced in one lane of 8-lane flow cell if necessary. These products were then purified and enriched with 15 cycles of PCR to create the final cDNA library for high through put DNA sequencing using Highseq2000. The concentration of R A libraries was measured by Qubit (Invitrogen, CA). The quality of R A-seq library was measured by LabChipGX (Caliper, MA) using HT DNA lK/12K/HiSens Labchip.
[0084] The Illumina Real Time Analysis (RTA) module performed image analysis, base calling, and the BCL Converter (CASAVA vl .8.2) generated the sequence reads in FASTQ file format. lOObp reads with a targeted coverage over 80-100 million sequencing reads are used per sample.
[0085] For PolyA samples, 4 out of 54 samples have two independent library preparations and run different sequencing batches. Those samples were used to check the transcriptome reproducibility for different sequencing batches. Also, current indexes of barcodes make possible that multiple samples can be sequenced in one lane. 28 samples (PolyA 13, RiboZero 15) were multiplexed into several lanes of the same batch. Lane effects was also checked.
Data analysis
[0086] After sequencing, the Illumina Real Time Analysis (RTA) module was run to perform image analysis, base calling, and the BCL Converter (CASAVA vl .8.2) were followed to generate FASTQ files which contain the sequence reads. The current sequencing depth is 80-120 million (40-60M paired-end) mappable sequencing reads. Read-level Q/C was performed by FastQC (vO.10.1). Pair-end reads of cDNA sequences obtained by the Highseq 2000 are aligned back to the human genome (UCSC hgl9) by the spliced-read mapper TopHat (v2.0.4) based on known transcripts of Ensembl Build GRCh37.67. The alignment statistics and Q/C was achieved by samtools (v0.1.18) and RSeQC (v2.3.5) to calculate quality control metrics on the resulting aligned reads, which provides useful information on mappability, uniformity of gene body coverage, insert length distributions and junction annotation, respectively. To obtain gene-level expression, the properly-paried and mapped reads are only counted by htseq-count vO.5.3 (with intersection-strict mode) and RPKM is calculated.
CoGAPS
[0087] All 30176, 21278 genes where randomly distributed in to seven sets upon which the CoGAPS nonnegative matrix factorization was performed. Briefly, CoGAPS decomposes a matrix of experimental observations— in this case, log2 ratios from two color expression arrays and RNAseq RPKMs— with genes as rows and samples as columns, into two matrices, the pattern matrix P defining relationships (i.e. patterns) between samples and the amplitude matrix A indicating the strength of involvement of a given gene in each pattern. A Markov Chain Monte Carlo algorithm with sparse atomic prior is used to compute the optimal solutions for A and P given the experimental data and a user defined error model∑ — in this case, a constant 0.1/ln(2) for array data and 10 percent of the signal for RNAseq. The final number of patterns used in the decomposition was determined using the χ2 fit of the recomposed AP to the original data and the persistence of patterns across multiple simulations and gene sets.
[0088] To account for the probabilistic nature of the CoGAPS nonnegative matrix factorization across sets, the following pipeline was developed and is now available as part of the CoGAPS R package. The resulting patterns from the initial CoGAPS factorization where matched across sets by hierarchical clustering. To ensure robustness, clusters where required to contain patterns from at least half and no more than 1.5x the total number of sets. Those clusters failing to meet these criteria where dropped or subjected to an additional round of hierarchical clustering, respectively. Patterns within each cluster were averaged using the cube of the correlation to the within cluster mean as a weight. To ease across pattern comparison, the resulting representative patterns were scaled by their individual maxima and then fixed. CoGAPS nonnegative matrix factorization was rerun using these fixed patterns for all sets, thus, enforcing reciprocity across the resulting amplitude matrixes.
Time-Lapse Recording
[0089] After 4 days in culture, SA01 cells were passaged using Accutase. While in suspension, cells were incubated with anti-epCAM antibody (RnD systems) directly conjugated to Alexa 647 for 10 min at room temp. Cells were pelleted once then resuspended in media lacking antibody then counted with the Countess. 1X106 live cells/well were plated on 35mm plates (Greiner Advanced TC) coated with matrigel in mTeSRl in the presence of Y27632. Imaging began less than 5 minutes after the EpCAM wash. At the beginning of the recording, many cells can be observed attaching to the surface. One day prior to passage, and on one day after the passage, cells were transfected with 1 μg mRNA encoding nuclear-localized GFP (Stemgent) to aid in single-cell visualization and manual tracking. After 48 hours, cells were fixed then stained with EpCAM directly conjugated to Alexa 488, and DAPI. Imaging was performed using a Zeiss 780 confocal microscope with a 20x 0.8NA objective at full resolution at 2048 x 2048 px/field. Every three minutes a 2x2 mosaic was captured using a 633 nm laser to excite Alexa647 and also provide transmitted light for concurrent DIC imaging. The gas environment was maintained at 5% C02 with a Zeiss SI gas control system. The microscope body and stage was heated to 37 with a Zeiss heating unit and environmental enclosure (PECAM).
High-content Analysis of Colony Morphology
[0090] Cells were cultured on 96-well TC-treated plates (Perkin Elmer), fixed 20 min with PFA then stained using indicated antibodies. Images were acquired with the Operetta (Perkin Elmer), analyzed in batch mode with custom building blocks on a Columbus server (Perkin Elmer) and visualized with Spotfire (Perkin Elmer). Colony morphology analysis ('distance from the edge' measurement) was achieved using a custom Acapella script to montage data from 49 individual Columbus fields into a single image (Perkin Elmer) then a custom set of building blocks in Volocity that reports the minimum distance between the edge of each cell and the colony perimeter (Perkin Elmer).
[0091] Specifically, after montage, nuclei were segmented in the DAPI channel using the
MidGray algorithm within the auto-local threshold function on ImageJ. The binary segmented channel was merged into the original files as an additional channel and used to find nuclei with Volocity (Perkin Elmer). To identify colonies, segmented nuclei were dilated 8 times to the point where gaps between cells were eliminated except those resulting from recent fusion of two colonies where a large gap remains.
[0092] The X-Y locations of each nucleus perimeter, from segmentation, is known and the location of each colony perimeter is known also from colony segmentation. The minimum distance between the nucleus edge and colony edge was calculated using the ' Measure Distance' function in Volocity. Thus each cell has a value reflecting its minimum distance to the nearest colony edge.
[0093] Using nuclear segmentation, the fluorescent signals from each nucleus in other channels was measured. Mean signal for each nucleus in each channel is plotted as a function of the cell's distance from the edge, creating the clear relationships in figure 2C. Example 2
[0094] To define variation across different human stem cell lines, 3 embryonic (ES) and
3 induced pluripotent (iPS) stem cell lines were grown in a simple cycle where they were dissociated to single cells and placed in new tissue culture plates every 5-7 days (FIG. 6). Using Next-generation sequencing technologies, gene expression patterns were assessed at days 2, 4 and 6 in untreated control conditions and in conditions that induce differentiation towards mesendoderm (BMP4) or neurectoderm (NOGGIN + SB431542) (Chambers et al, 2009). The sequence data was used to generate expression levels for 23,368 RefSeq annotated genes. The overall pattern of gene expression was assessed by principal component analysis (PCA; FIG. 1A). The first principal component (PCI) gives the direction of the maximal spread of the data and clearly separates the BMP4 treated samples from all the others. PC2 separates out the different days of treatment for all three conditions. Combined, PCI and PC2 reveal global transcriptional trajectories for early neuronal and mesendodermal differentiation followed by all six cell-lines. The iPS line i04 moves most quickly along the mesendodermal trajectory while human ES line SA01 leads the neurectodermal trajectory. This indicates that cell lines show distinct differentiation efficiencies.
[0095] PCA was conducted on data from each of the three conditions separately (FIGS.
IB and 1C, upper panels, FIG. 7). Consistent with the PCA conducted with all three conditions, the greatest variation in NOGGIN-SB PCA captured by PCI demonstrated separation of SA01 samples above all other cell lines (FIG. IB). A simple extension of this analysis was used to ask if the cell lines show similar ranking during self-renewal. In this treatment, the PC weights generated under NOGGIN-SB conditions were multiplied by the gene expression values obtained during self-renewal. Although the dynamics are of lesser magnitude (note scale differences in FIG. IB upper and lower panels), a highly similar progression and ranking of cell lines was obtained. This result suggests that the dynamic of gene expression during self-renewal predicts how individual cell lines will respond to differentiation inducing conditions.
[0096] The differences between cell lines during self-renewal were assessed by PCA only using self-renewal data. This analysis shows that each of the first six PC's separates a single cell line from all others (FIG. 1C and FIG. 8). In the SA01 specific PC, the neurectodermal fate regulators SOX21 and OTX2 genes are among the top 15 genes (Acampora et al., 2013; Matsuda et al, 2012; Simeone et al, 1994) (FIG. 9). The level of expression of SOX21 shows, as expected from the PCA, that this gene is expressed more highly in SA01 than other cell lines (FIG. 1C lower panel). This analysis shows that individual cell lines have genome-specific transcriptional signatures during self-renewal that include genes likely to influence their differentiation.
[0097] To test the in vivo significance of the transcriptional signature associated with efficient differentiation in vitro, the weights generated from the PCA obtained under NOGGIN- SB conditions were used to probe the gene expression pattern in the developing human prefrontal cortex (Colantuoni et al., 2011). The transcriptional signature defined by the neurectoderm- specific PCI starts high in early fetal development and disappears over late fetal and early postnatal life (FIG. 10). This signature parallels closely the expression of Nestin, a widely used marker of neural stem cells (Lendahl et al., 1990). The transcriptional dynamics defined by PCA of the i04 cell line under BMP conditions, defines a distinct pattern of gene expression in the human brain (Data not shown). This result suggests that the transcriptional dynamics most clearly seen in the SA01 line in the NOGGIN-SB condition identifies a pattern of gene expression found in stem cells in the developing human forebrain.
[0098] Based upon the identification of cell identity and differentiation signatures by
PCA, a more precise analysis of the dynamics of gene expression was carried out. To achieve this, a Markov Chain Monte Carlo (MCMC) non-negative matrix factorization (NNMF) method, CoGAPS, was used. Unlike PCA that achieves computational simplicity by imposing an orthogonality constraint on the data analysis, CoGAPS uses computational power to iteratively decompose high-dimensional data into any number of component patterns specified by the investigator (Fertig et al, 2010). When tasked with identifying 20 patterns, CoGAPS identified 14 that define distinct components of gene expression change during differentiation exhibited by all the lines (FIG. ID), and 6 patterns that define high levels of transcription that are linked to a specific genome spanning all three conditions (FIG. ID). The weights generated by CoGAPS were used to interrogate the behavior of these 6 cell lines in a micro-array analysis of gene expression that included 21 pluripotent human cell lines (Mallon et al., 2013). The same pattern of variant gene expression in individual lines was seen in the context of this larger panel of pluripotent human cell lines analyzed by a different method at a different time (FIG. 10). This result demonstrates that the cell line signatures are stable traits. [0099] To rigorously confirm a genetic origin for cell line specific transcriptional signatures, iPS cells that were derived from two William's Syndrome (WS) patients that carry different defined deletions on chromosome 7 were analyzed. Replicate lines were generated from individual genomes by mRNA transfection and RNAseq was conducted by the Ribo-Zero method instead of the Oligo-dT isolation of polyA+ RNA previously used. Replicate iPS lines from the same patient show the same CoGAPS pattern, but the transcriptome from the two William's patients was defined by different patterns (FIG. IE). These two William's patients carry deletions of different sizes that were detected by CoGAPS (FIG. 11). Importantly, these patterns also reproducibly identify genes in other regions of the genome that contribute to the transcriptional identity of both patients and not a control iPS cell line (FIG. 11). These data show that CoGAPS is a diagnostic tool to measure the consequences of genetic variation on the phenotype of human pluripotent cells.
Dynamics of fate bias in self-renewing cells
[0100] The use of computational tools to define the effects of cell line specific transcriptional variation on human pluripotent cell lines is an exciting prospect. As a first step in assessing the developmental impact of patterns defined by CoGAPS analysis, we chose to focus on the 2 patterns that define neurectodermal differentiation. Of the 14 CoGAPS patterns that define groups of genes that change with differentiation, 2 identify early and later steps in the differentiation of neurectoderm. SOX21 and OTX2 contribute strongly to pattern 15. Many of the transcription factors known to regulate differentiation of the mouse cerebral cortex are strongly represented in pattern 12 (Pattabiraman et al., 2014).
[0101] The CoGAPS pattern 15 predicts that dynamic change in expression of SOX21 and OTX2 would be required for the emergence of the later step in neurectodermal differentiation defined by pattern 12. The expression of the SOX21 protein was defined using confocal microscopy to measure signal from specific antibodies directly conjugated to different fluorescent reporters in the SA01 cell line. Under control conditions on day 4, few SOX21 positive cells were present (FIGS. 2A and 2B). Under Noggin-SB conditions, SOX21 was strongly induced. A striking inverse correlation was seen between SOX21 and NANOG, a transcription factor that promotes pluripotency (FIGS. 2 A and 2B). This analysis suggests that the induction of SOX21 and down regulation of NANOG were regulated by a spatial mechanism established in the epithelial sheets characteristically formed by human pluripotent stem cells and the equivalent mouse epiblast stem cell (EpiSC) (Tesar et al, 2007). An image analysis method that defines the distance of all cells from the edge shows that this measurement quantitatively defines a positional relationship between NANOG, SOX2, SOX21 and nuclear area (FIG. 2C and FIG 12). A similar progressive restriction in nuclear area has been reported as epithelial cells set up local micro-environments that inhibit growth through contact inhibition (Puliafito et al, 2012). The imaging of gene expression and morphological patterns suggests that a self- organizing mechanism generates distinct spatial domains in pluripotent stem cells.
[0102] To define these domains, a monoclonal antibody against the cell surface molecule
EPCAM was used to specifically identify cells at the edge of cell sheets defining three morphological domains; the edge-, intermediate- and core-zones (EZ, IZ and CZ) (FIG. 2D). The expression of EPCAM ws used to guide laser capture microdissection of the EZ/IZ and CZ domains in self-renewing conditions. mR A levels for OCT4, SOX2, NANOG, ESRRB, EOMESODERMIN, SOX21 and OTX2 were assessed by quantitative RT-PCR (FIG. 2E). SOX21 and OTX2 message levels were reduced in the EZ/IZ domain while the mesendodermal regulator EOMESODERMIN was enriched in this domain. Consistent with the CoGAPS analysis, these results demonstrate that expression of the OTX2 and SOX21 genes is regulated by morphogenic mechanisms.
[0103] The reduction of nuclear area that occurs as cells become more distant from an edge suggests that spatial location may regulate cell growth (FIG. 2C). To determine when and where pluripotent cells divide, proliferating cells were labeled by a short pulse of the nucleic acid analogue EdU and co-expression with SOX21 established. This analysis shows that dividing cells were present at days 2 and 4 in SOX21 negative cells (FIG. 2F). This observation suggests that cell proliferation and a bias towards neurectodermal differentiation occur in mutually exclusive domains.
[0104] The rapid generation of distinct spatial zones during normal growth of pluripotent stem cells raises the fundamentally important question whether these zones are generated by cells that are distinct when the cells are passaged, or whether these differences emerge following passage. To explore how cells change through time, antibodies against OCT4, NANOG and EPCAM were applied on different days following passage. All three antigens were initially expressed widely and then increasingly restricted to the IZ or EZ (FIG. 3A). Dynamic temporal alterations in gene expression were also seen using quantitative RT-PCR to define changes in gene expression through two cycles of self-renewal (FIG. 3B). In this analysis, the mR A expression of [SOX2, SOX3, SOX21] and [NANOG, OCT4] cycled with different periodicities. Consistent with the data on cell proliferation, NANOG and OCT4 levels were high early and declined at later times when expression of the SOX genes increased. 98% of cells survived the initial dissociation and 75% of these cells were present 24 hours after replating. Consistent with previous reports showing regulation of gene expression and developmental potential in pluripotent cells (Chambers et al., 2007; Macfarlan et al., 2012; Surani and Tischler, 2012), our data suggest that neurectodermal fate regulators are dynamically controlled in the pluripotent state.
[0105] By coupling a monoclonal antibody against EPCAM to two different fluorophores, the expression of EPCAM immediately after passage and at later times was directly measured by live cell imaging. Quantitative fluorescence microscopy immediately after passage shows that both low-EPCAM and high-EPCAM cells at t=0 give rise to progeny with a range of EpCAM expression 48 hours later (FIG. 3C and Supp Mov 1). 14 lineages of single founder cells with different levels of initial EPCAM expression show that individual cells give rise to progeny with a range of EPCAM levels (FIG. 3D). These data show that the EpCAM level in founder cells does not define the formation of these zones with different lineage potentials. Similar to results using live-cell imaging to define the potential of neural stem cells, cell passage is required to regenerate the multi-potent state (Ravin et al, 2008). The cell survival and live cell imaging data suggest that, in spite of their molecular differences, the cells are a developmental equivalence group where local signals rather than lineage constraints regulate the temporal and spatial expression of OTX2 and SOX21.
Distinct cell states show lineage bias
[0106] The observations reported here suggest that the control of pluripotency and lineage bias by these core transcriptional regulators may vary in different spatial domains. To examine whether the spatially dynamic expression of OCT4, NANOG, and SOX2 regulate lineage bias in these regions, siRNA knockdown (KD) was used on different days of culture and monitored SOX21 and EOMES expression patterns (FIG. 4A and FIG. 13). OCT4-KD initiated on Day 0 induced SOX21 expression in the CZ and EOMES expression in the EZ of the colonies 48 hours later. However, when OCT4-KD was initiated on Day 2, only SOX21 was induced and no induction of EOMES was observed. Early SOX2-KD resulted in EOMES induction while its KD later showed no effect (FIG. 4A). Induction of EOMES was also observed when both OCT4 and SOX2 down regulation were initiated on Day 2 (FIG. 13). These results suggest induction of EOMES can be achieved when most cells exhibit NANOGhigh/SOX21lowEZ/IZ states by OCT4 and SOX2-KD. These studies suggest that pluripotent stem cells under self-renewing conditions rapidly generate distinct spatial zones where the interaction between core pluripotency genes and fate regulators generates a bias towards different fates.
[0107] Certain genes are rapidly induced with mesendodermal differentiation when cells were treated with BMP4. They include EOMESODERMIN (EOMES), BRACHYURY (T) and the homeobox transcription factor CDX2. There is strong evidence for the functional role of these genes in early development of the mouse (Arnold and Robertson, 2009) and SOX21 inhibits expression of CDX2 both in adult intestine and in differentiating pluripotent stem cells (Kuzmichev et al., 2012). In the CoGAPS analysis these genes strongly contribute to patterns 8 and 9, identifying genes that show rapid changes in gene expression levels on days 2 and 4 of BMP4 treatment. Immunological analysis of protein expression shows that 24 hours after treatment with BMP4, CDX2 and T are expressed in many cells at day 0 before the emergence of distinct zones (FIG 4B). When exposure to BMP4 was delayed by 48 hours, CDX2 and T expression was restricted to cells in the IZ and EZ. This effect of BMP4 is mediated by phosphorylation of SMAD proteins, a family of signal transducing transcriptional regulators. Nuclear localization of phosphorylated SMADs following the delayed BMP4 treatment was restricted to the zone where CDX2 and T expression was observed (FIG. 13). The imaging analysis extends the RNASeq analysis by showing that fate bias was regulated by a morphogenic mechanism that organizes developmentally equivalent cells into distinct zones biased towards mesendodermal or neurectodermal fates.
[0108] The two neurectodermal patterns defined by CoGAPS suggest that OTX2 and
SOX21 expression during self-renewal leads to efficient neurectodermal specification. Expression of SOX1 and PAX6, two well-characterized regulators of neuronal development were used to monitor neurectodermal differentiation (Osumi et al, 2008; Pevny et al, 1998; Wood and Episkopou, 1999). In the CoGAPS analysis, SOX1 contributes to the neurectodermal differentiation patterns 15 and 12, but PAX6 expression only contributes to pattern 12. Because neither gene was associated with a cell line transcriptional signature, SOX1 and PAX6 expression define the early and late steps of neurectodermal differentiation independent of the initial state of the self-renewing cell. Both OTX2-siRNA and SOX21-siRNA prevented SOX1 and PAX6 induction and immunostaining also confirmed that OTX2- and SOX21-KD prevents induction of SOX1 and PAX6 protein upon NOGGIN-SB treatment (FIG. 4C and FIG. 14). These data show that OTX2 and SOX21 act co-ordinately to induce later neurectodermal fates as predicted by the CoGAPS analysis.
Variation between genomes in early morphogenesis
[0109] In principle, imaging of early stages of iPS cell growth and differentiation may make a powerful contribution to understanding the genetic basis of disease mechanisms. To determine if there is a genomic origin of differences in early morphogenesis, a set of iPS cells carrying alleles of the serine/threonine kinase AKT1 associated with schizophrenia and working memory were developed (Emamian et al., 2004; Tan et al., 2012; Tan et al., 2008). Epistatic effects between AKT1 and the growth factor neuregulin 1 (NRG1) have also been biologically validated via functional neuroimaging (Nicodemus et al, 2010). A role for neuregulin signaling through AKT activation in the self-renewal of human pluripotent cells has been established (Singh et al, 2012).
[0110] Human fibroblasts carrying risk and protective alleles of AKT1 were reprogrammed using RNA transfection, a method that leaves no genetic trace of the reprogramming procedure (Warren et al, 2010). Because gene expression data suggests that male genomes exhibit less variability in gene expression and more rapid differentiation, systematic protocols were used to derive banks of male iPS cell lines from Caucasian donors homozygous for the risk and protective alleles of AKT1. High-content imaging was used to monitor OTX2 expression and nuclear area in the newly generated iPS cell lines on days 3 and 6 of differentiation in control and NOGGIN-SB conditions (FIG. 5A; note that each data point represents >40,000 measurements of individual cells). In control and NOGGIN-SB conditions, the anticipated inverse relationship between OTX2 levels and large nuclear area was observed. A one-way ANOVA test was used to determine the significance of differences between cell lines generated from different genomes. This analysis shows that each genome is distinct with a P value of <2.2.e-16. The extraordinarily high confidence of this statistical measure is a consequence of the large number of individual cells that have been analyzed by the imaging tools employed.
[0111] When the cell lines were annotated by AKT1 genotype, they divide into two groups. Cells lines carrying two copies of the risk allele (triangle) showed higher OTX2 levels than the lines with a high protected status (circle) in both control and NOGGIN-SB conditions on day 6 (FIG. 5A). Under control conditions at day 6, the 3 samples showing the highest OTX2 levels were homozygous for the AKT1 risk allele. Continuous NRGl treatment during self- renewal led to a reduction in OTX2 levels in these samples. In the NOGGIN-SB condition that drives neurectodermal differentiation, 5 of the 6 samples showing the highest OTX2 levels carried the AKT1 risk genotype. OTX2 levels in these 5 samples (green and yellow triangles) showed a small change in response to NRGl treatment under NOGGIN-SB conditions. In contrast, the line carrying the protective allele responded to NRGl treatment (blue circle). In NOGGIN-SB conditions on day 6 the lines carrying the non-risk allele now show elevated OTX2 expression and all of these samples showed reduced OTX2 expression when treated with NRGl . Even at day 3, multi-way ANOVA analysis shows that cells carrying the AKT1 risk alleles already show a significant elevation of OTX2 expression.
[0112] RNASeq data show that NRGl and AKT1 are strongly expressed in pluripotent human stem cells throughout the 6 day period of this assay. RNAseq data also shows that both, ERBB2 and ERBB3, the genes encoding the dimeric neuregulin receptor were present in human pluripotent cells. Immunostaining shows that ERBB2/3 expression is restricted to the EZ and IZ (FIG. 5B). NRGl down regulates SOX21 and OTX2 while promoting expression of NANOG (FIG. 5C). The effect of NRGl on the level of mRNA for OCT4, NANOG and SOX21 suggests that NRGl exposure sustains the EZ/IZ state while blocking formation of the CZ (FIG. 5D). The accelerated neurectodermal differentiation of the iPS lines carrying the risk allele of the AKT1 gene (Tan et al., 2008 and 2012) is consistent with NRGl promoting the EZ/IZ state where proliferation occurs and inhibiting the neurectodermal CZ state regulated by SOX21 and OTX2. These observations show that the effect of the widely studied AKT1 cell signaling pathway on morphogenesis is consistent with its action on Schizophrenia risk in adult life. [0113] All patents, patent applications and publications referred to or cited herein are hereby incorporated by reference in their entireties for all purposes.
[0114] It is understood that the embodiments and examples described herein are for illustrative purposes and that various modifications or changes in light thereof will be suggested to persons skilled in the pertinent art and are to be included within the spirit and purview of this application and scope of the appended claims. It is to be understood that suitable methods and materials are described herein for the practice of the embodiments; however, methods and materials that are similar or equivalent to those described herein can be used in the practice or testing of the invention and described embodiments.
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Claims

What is claimed is:
1. A method for synchronizing and resetting the potency of a population of human pluripotent cells said method comprising the steps of a. dissociating a population of human pluripotent cells; b. culturing said dissociated cells in the presence of a survival factor and in the absence of feeder cells for a time sufficient to produce an equivalence group of cells; and c. culturing the population of cells from step (b) in the absence of said survival factor and in the absence of feeder cells for a period of time sufficient to produce lineage primed cells within the population but wherein the cells in said population can be reset to pluripotency by cell dissociation.
2. The method of claim 1 which further comprises repeating steps (a) through (c) at least one time.
3. The method of claims 1 or 2 wherein said human pluripotent cells are human embryonic stem cells.
4. The method of claims 1 or 2 wherein said human pluripotent cells are human induced pluripotent stem (iPS) cells.
5. The method of claim 4 wherein the iPS cells are derived from a human subject that has been diagnosed with a schizophrenia, neurodegenerative disease, cancer, or autism.
6. The method of claim 4 or 5 wherein the human subject has a genetic polymorphism associated with a disease or disorder, predisposition to a disease or disorder or with a disease susceptible to a particular treatment.
7. The method of any of claims 1 to 7 wherein prior to step (a) said population of cells is cultured on mouse embryonic fibroblasts.
8. The method of any of claims 1 to 7 wherein the cells are dissociated with Accutase®.
9. The method of any of claims 1 to 8 wherein the survival factor is a RHO-associated kinase (ROCK) inhibitor.
10. The method of claim 10, wherein the ROCK inhibitor is Y27632.
11. The method of any of claims 1 to 10 wherein said dissociated cells are cultured in the presence of said survival factor for 24 hours.
12. The method of any of claims 1 to 11 wherein the equivalence group of cells is characterized by expression of Nanog or Oct4.
13. The method of any of claims 1 to 12 wherein, in step (c), said population of cells is cultured for 6 days.
14. The method of any of claims 1 to 13 wherein, in step (c), said cells are cultured in the presence of a factor or combination of factors that modulate cell specification.
15. The method of claim 14, wherein said factor is BMP4, Noggin, neuregulin, or Noggin and SB431542.
16. The method of any of claims 1 to 15, wherein during step (c), said population of cells is assayed for gene expression, cell phenotype, cell morphology or laminar structure of colony organization.
17. The method of claim 16, wherein the spatial distribution of expression of one or more genes in an epithelial sheet of said population of cells is determined.
18. The method of claim 17, wherein the spatial distribution of expression is analyzed by high content analysis.
19. A method for analyzing patient specific cells, said method comprising a. obtaining iPS cells from a human patient; b. dissociating a population of said iPS cells; c. culturing said dissociated cells in the presence of a survival factor and in the absence of feeder cells for a time sufficient to produce an equivalence group of cells; d. culturing the population of cells from step (c) in the absence of said survival factor and in the absence of feeder cells for a period of time sufficient to produce lineage primed cells within the population but wherein the cells in said population can be reset to pluripotency by cell dissociation; and e. analyzing one or more traits of the cells of step (d).
20. The method of claim 19 wherein prior to step (e), steps (b) through (d) are repeated at least one time prior to step (e).
21. The method of either of claims 19 or 20 wherein the human subject has been diagnosed with schizophrenia, neurodegenerative disease, cancer, diabetes, cardiovascular disease or autism.
22. The method of either of claims 19 or 20 wherein the human subject is a healthy human subject.
23. The method of any of claims 19 to 22 wherein prior to step (a) said population of cells is cultured on mouse embryonic fibroblasts.
24. The methods of any of claims 19 to 23 wherein the cells are dissociated with Accutase®.
25. The methods of any of claims 19 to 24 wherein the survival factor is a Rho-associated kinase (ROCK) inhibitor.
26. The method of claim 25, wherein the ROCK inhibitor is Y27632.
27. The method of any of claims 19 to 26 wherein said dissociated cells are cultured in the presence of said survival factor for 24 hours.
28. The method of any of claims 19 to 27 wherein the equivalence group of cells is characterized by expression of Nanog or Oct4.
29. The method of any of claims 19 to 28 wherein, in step (c), said population of cells is cultured for 6 days.
30. The method of any of claims 19 to 29 wherein, in step (d), said cells are cultured in the presence of a factor or combination of factors that modulates cell specification.
31. The method of claim 30, wherein said factor is BMP4, neuregulin, Noggin or Noggin and SB431542.
32. The method of any of claims 19 to 31 wherein said analyzing is analysis of cell morphology, gene expression, transcriptome, cell surface markers, or laminar organization of a colony.
33. The method of claim 32, wherein the spatial distribution of expression of one or more genes in an epithelial sheet of said population of cells is determined.
34. The method of claim 33, wherein the spatial distribution of expression is analyzed by high content analysis.
35. The method of any of claims 19 to 34 wherein said cells are analyzed continuously or at day 1, 2, 3, 4, 5, or 6 of step (d).
36. The method of any of claims 19 to 35 wherein the results of said analysis are compared to results from comparable cells derived from a second human subject.
37. The method of claim 33 wherein said second human subject has been diagnosed with schizophrenia, neurodegenerative disease, cancer, diabetes, cardiovascular disease or autism.
38. The method of claim 33 wherein said second human subject has not been diagnosed with the disease of the human subject.
39. The method of any of claims 19 to 38, wherein the results of said analysis are used to diagnose the patient with a disease or disorder, determine whether said patient is predisposed to a disease or disorder, or determine whether the patient is amenable to a particular treatment for a disease or disorder.
40. A method for screening an agent for an effect on reversibly differentiated cells, said method comprising the steps of a. dissociating a population of said iPS cells; b. culturing said dissociated cells in the presence of a survival factor and in the absence of feeder cells for a time sufficient to produce an equivalence group of cells; c. culturing the population of cells from step (b) in the absence of said survival factor and in the absence of feeder cells for a period of time sufficient to produce lineage primed cells within the population but wherein the cells in said population can be reset to pluripotency by cell dissociation; d. contacting cells from step (c) with said agent; and e. comparing an assayable trait of said cells contacted with said agent with said assayable train of cells from step (c) not contacted with said agent.
41. The method of claim 40 which further comprises repeating steps (a) through (c) at least one time prior to step (d).
42. The method of claims 40 or 41 wherein said human pluripotent cells are human embryonic stem cells.
43. The method of claims 40 or 41 wherein said human pluripotent cells are human induced pluripotent stem (iPS) cells.
44. The method of claim 43 wherein the iPS cells are derived from a human subject that has been diagnosed with schizophrenia, neurodegenerative disease, cancer, diabetes, cardiovascular disease or autism.
45. The method of claim 43 or 44 wherein the human subject has a genetic polymorphism associated with a disease or disorder, predisposition to a disease or disorder or with a disease susceptible to a particular treatment.
46. The methods of any of claims 40 to 45 wherein the cells are dissociated with Accutase®.
47. The methods of any of claims 40 to 45 wherein the survival factor is a RHO-associated kinase (ROCK) inhibitor.
48. The method of claim 47, wherein the ROCK inhibitor is Y27632.
49. The method of any of claims 40 to 48 wherein said dissociated cells are cultured in the presence of said survival factor for 24 hours.
50. The method of any of claims 40 to 49 wherein the equivalence group of cells is characterized by expression of Nanog or Oct4.
51. The method of any of claims 40 to 50 wherein, in step (c), said population of cells is cultured for 6 days.
52. The method of any of claims 40 to 51 wherein, in step (c), said cells are cultured in the presence of a factor or combination of factors that modulate cell specification.
53. The method of claim 52, wherein said factor is BMP4, Noggin, neuregulin, or Noggin and SB431542.
54. The method of any of claims 40 to 53 wherein said assayable trait is cell morphology, gene expression, transcriptome, cell surface markers, or laminar organization of a colony.
55. The method of claim 54, wherein the spatial distribution of expression of one or more genes in an epithelial sheet of said population of cells is determined.
56. The method of claim 55, wherein the spatial distribution of expression is analyzed by high content analysis.
57. The method of any of claims 40 to 56 wherein said agent is a small molecule, a siR A, an antibody, a peptide, a biologic.
58. The method of any of claims 40 to 57 wherein said cells are analyzed continuously or at day 1, 2, 3, 4, 5, or 6 of step (e).
59. A homogeneous population of cultured cells produced according to the method of any of claims 1 to 18.
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