US20190010451A1 - Chemical reprogramming to generate neuronal cells - Google Patents

Chemical reprogramming to generate neuronal cells Download PDF

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US20190010451A1
US20190010451A1 US15/750,570 US201615750570A US2019010451A1 US 20190010451 A1 US20190010451 A1 US 20190010451A1 US 201615750570 A US201615750570 A US 201615750570A US 2019010451 A1 US2019010451 A1 US 2019010451A1
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cells
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Mingliang Zhang
Sheng Ding
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J David Gladstone Institutes
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
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    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • NSCs neural stem cells
  • pluripotent reprogramming by forced expression of a set of transcription factors (Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors . Cell 131, 861-872 (2007)), somatic cell nuclear transfer (Campbell et al., Sheep cloned by nuclear 20 transfer from a cultured cell line .
  • MEFs mouse embryonic fibroblasts
  • the MEFs are an inherently heterogeneous population containing non-fibroblast precursor cell types that may be specified into neural lineage via processes other than bona fide reprogramming.
  • genetic lineage tracing of the starting fibroblasts would be required, especially for conditions using combinations of small molecules given the indirect induction mechanisms of reprogramming and differentiation.
  • compositions and methods described herein can accomplish reprogramming of differentiated, non-neuronal cells to generate neural progenitor and mature neuronal cells by chemical means and without the need for genetic engineering. Concerns about introduced genetic mutations are obviated when the compositions and methods described herein are employed. Moreover, use of the compositions and methods described herein is less labor intensive, and less time consuming, than previously available methods.
  • compositions that includes at least four of the following active agents: a BMP type I receptor ALK2/3 inhibitor, a TGF-beta inhibitor, a WNT inhibitor, a neuronal differentiation enhancer, a SMO agonist, a retinoic acid receptor ⁇ agonist, a DNA methyltransferase inhibitor, a histone demethylase inhibitor, an autophagy regulator, or any combination thereof.
  • the composition can contain active agents CHIR99021; LDN193189; A83-01; Hh-Ag1.5; retinoic acid; SMER28; RG108, parnate, and bFGF, as well as other ingredients.
  • the other ingredients can be a pharmaceutically acceptable carrier (if the composition is administered to a subject), or cell culture media ingredients, if the composition is part of a cell culture medium.
  • Another aspect is a method of generating a reprogrammed neuronal progenitor cell or a reprogrammed neuronal cell that involves contacting a selected cell with the composition, to thereby generate a reprogrammed neuronal progenitor cell or a reprogrammed neuronal cell.
  • Another aspect is a method that involves administering the composition to a subject.
  • the subject can suffer, or may be suspected of suffering, from Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, multiple sclerosis, Primary lateral sclerosis (PLS), Progressive bulbar palsy, Pseudobulbar palsy, Progressive muscular atrophy, Spinal muscular atrophy (SMA), including Type I (also called Werdnig-Hoffmann disease), Type II, Type III (Kugelberg-Welander disease), Fazio-Londe disease, Huntington's disease, Kennedy's disease also known as progressive spinobulbar muscular atrophy, hereditary spastic paraplegia (HSP), congenital SMA with arthrogryposis, Post-polio syndrome (PPS), traumatic spinal cord injury, progressive pseudobulbar palsy, progressive muscular atrophy, stroke, head trauma, spinal cord injury, or a combination thereof.
  • ALS Amyotrophic lateral sclerosis
  • PLS Primary lateral
  • kits that includes the composition, and instructions for using the composition.
  • a kit can also include components for in vitro cell culture of a selected cell.
  • the kit can contain a diluent, a pharmaceutically acceptable carrier, a syringe, a catheter, or a device for delivery of cells or of the composition to a subject.
  • the kit can also contain antibodies, probes, or primers for detection of one or more neuronal progenitor cell marker or one or more neuronal cell marker.
  • FIG. 1A-11 illustrate conversion of adult cells into chemically induced neural stem cell-like cells (ciNSLCs) using a chemical approach.
  • FIG. 1A shows a schematic diagram (top) illustrating a cell lineage tracing strategy that generates fibroblast cells that express the tdtomato marker and that are converted to ciNSLCs by the chemical reprogramming protocol described herein (summarized below the schematic diagram of chemically induced cell lineage progression).
  • the chemical reprogramming protocol involves culturing cells in medium containing fetal bovine serum (FBS) for about 24 hours, followed by incubation of the cells in medium containing CHIR99021, LDN193189, A83-01, Hh-Ag1.5, retinoic acid, SMER28, RG108, parnate, and bFGF for about ten days.
  • FIG. 1B shows four images of the same ciNSLC colony that was derived from tdtomato-expressing mouse embryonic fibroblasts (tdMEFs).
  • FIG. 1C graphically illustrates expression levels of the neural stem cells genes identified along the x-axis for three independent ciNSLC lines, and two primary neural progenitor cell (NPC) lines.
  • the first bar on the left shows expression of ciNSLC line #1; the second bar from the left shows expression of ciNSLC line #2; the third bar from the left shows expression of ciNSLC line #3; the fourth bar from the left shows expression by a tdtomato-expressing neural progenitor cell (tdNPC); and the rightmost bar shows expression by a Tau-GFP and tdtomato-expressing neural progenitor cell (Tau-GFP tdNPC).
  • ciNSLC lines #1 and #2 were derived from tdMEFs, and ciNSLC line #3 was from tdtomato-expressing mouse tail-tip fibroblasts (tdTTFs).
  • FIG. 1D shows images of expanded ciNSLC colonies derived from tdtomato-expressing mouse embryonic fibroblasts (tdMEFs).
  • the left column of images show the expression of the genes listed to the left (“target”); the middle column of images show expression of tdtomato; and the third column of images show the merged expression of the target genes and tdTomato.
  • Expression is shown of target genes Sox2 (top row of panels), Nestin (second row of panels), Olig2 (third row of panels), N-Cad (fourth row of panels), and BrdU (bottom row of panels).
  • Scale bar is 50 ⁇ m.
  • FIG. 1E shows paired scatter plots comparing the global gene expression pattern of ciNSLC with tdMEF (left) and SCR029 (right).
  • FIG. 1F graphically illustrates the reprogramming efficiency as calculated by Sox2 and Nestin expression of batches tdMEF #1 and tdMEF #2, Tau-GFP MEF, NGFP MEF, and tdTTF. Total cell number was determined by DAPI staining. Error bars, s.d., based on triplicates for FIG. 1C and FIG. 1F .
  • FIGS. 1G-1I illustrate isolation and characterization of tdMEF cells.
  • FIG. 1G illustrates isolation of the tdTomato+/p75 ⁇ cell population by FACS.
  • FIG. 1G illustrates isolation of the tdTomato+/p75 ⁇ cell population by FACS.
  • FIG. 1H shows that the tdTomato+/p75 ⁇ MEF cell population used as a starting cell population does not express neural genes Sox2, Hes5, Ascl1, Gfap, Oli2, or Gaphh as detected by RT-PCR analysis.
  • the neural stem cell (NSC) line SCR029 was used as a positive control.
  • FIG. 1I also shows that the tdTomato+/p75 ⁇ MEF cell population does not express Sox2, Pax6, Olig2, Nestin, GFAP and NG2 as detected by immunostaining.
  • the left column of images show the expression of tdTomato; the middle column of images shows expression of the genes listed to the left (“target”); and the third column of images shows DAPI staining of nuclei. Expression is shown of target genes Sox2 (top row of panels), Pax6 (second row of panels), Olig2 (third row of panels), Nestin (fourth row of panels), GFAP (fifth row of panels), and NG2 (bottom row
  • FIG. 2A-2L illustrate that ciNSLCs are tripotent in vitro and in vivo.
  • FIG. 2A shows images of tdTomato-expressing ciNSLCs that were immunostained for Tuj1, Map2, O4, or GFAP, illustrating that ciNSLCs can differentiate into Tuj1-positive and Map2-positive neurons (top, scale bar is 100 ⁇ m), O4-positive oligodendrocytes (middle, scale bar is 50 ⁇ m), and GFAP-positive astrocytes (bottom, scale bar is 100 m).
  • FIG. 1A shows images of tdTomato-expressing ciNSLCs that were immunostained for Tuj1, Map2, O4, or GFAP, illustrating that ciNSLCs can differentiate into Tuj1-positive and Map2-positive neurons (top, scale bar is 100 ⁇ m), O4-positive oligodendrocytes (middle, scale bar is 50 ⁇ m), and GFAP-positive astrocytes (
  • FIG. 2B graphically illustrates the percentage of vGlut1-positive glutamatergic neurons and GABA-positive GABAergic neurons, normalized to the total number of Tuj1-positive neurons, in ciNSLCs that were further differentiated in vitro under the typical neuronal differentiation conditions (Kim et al. Proc Natl Acad Sci USA 108: 7838-7843 (2011).
  • FIG. 2C graphically illustrates the percentage of Tuj-positive neurons and GFAP-positive astrocytes in the total population of spontaneously differentiated ciNSLCs.
  • FIG. 2D shows representative traces of membrane potentials of ciNSLC-derived neurons in responses to step-current injections.
  • FIG. 2E shows spontaneous synaptic network activities of ciNSLC-derived neurons.
  • FIGS. 2G-2L illustrate further characteristics of the M9 cocktail and of ciNSLCs generated by use of this M9 cocktail.
  • FIG. 2G graphically illustrates the relative reprogramming efficiency of the M9 cocktail without one of its components compared to the M9 cocktail with all components.
  • FIG. 2H shows images of tdMEF-derived small clusters (upper three panels) and tdMEF-derived colonies (bottom three panels) at day 6 of M9 treatment.
  • FIG. 2I shows images of tdMEF colonies at day 6 of treatment with the M9 cocktail.
  • FIG. 2J show images of tdMEF-derived colonies exhibiting alkaline phosphatase (ALP) activity (darker staining).
  • ALP alkaline phosphatase
  • FIG. 2K shows electrophoretically separated products of RT-PCR analysis of the neural gene expression in two independent lines of ciNSLC (#1 and #2), in comparison to parental tdMEF cells and positive control SCR029 cells, illustrating expressing Pax6, Sox2, Asch1, and Olig2.
  • FIG. 2L graphically illustrates the relative reprogramming efficiency for tdMEFs treated with M9, VCR, or DMSO control.
  • VCR is a cocktail of valproic acid, CHIR99021, and RepSox.
  • FIG. 3A-3H illustrate expression patterns of cells during neural reprogramming.
  • FIG. 3A graphically illustrates expression of Sox1, Sox2, Pax6, Olig2, Hes5, and Ascl1 during neural reprogramming of cells on day 0 (D0, first bar to the left with barely discernible expression, day 4 (D4, second bar from the left), day 8 (D8, third bar from the left), and day 12 (D12, fourth bar from the left; highest bar for Pax6).
  • the ciNSLC (fifth bar from the left) exhibit the highest expression of the different cell types for Sox1, Sox2 and Hes1.
  • the NSC line SCR029 (rightmost bar) was used as a positive control.
  • FIG. 3B shows a heatmap of differentially expressed genes at indicated time points (day 0, day 4, day 8, and day 12) of neural reprogramming (left panel) with lists of GO terms (middle panel) and representative genes (right panel) for each block (1 to 5, as labeled). Red and blue colors in the original indicate upregulate and downregulated genes, respectively. In black and white images the maximal expression is shown by the darker shades. For example, ciNSLC and SCR029 exhibit maximal expression at the top right, but the Day 0 (D0) cells exhibit the maximal expression at the bottom left.
  • FIG. 3B shows a heatmap of differentially expressed genes at indicated time points (day 0, day 4, day 8, and day 12) of neural reprogramming (left panel) with lists of GO terms (middle panel) and representative genes (right panel) for each block (1 to 5, as labeled). Red and blue colors in the original indicate upregulate and downregulated genes, respectively. In black and white images the maximal expression is shown by the darker shades. For example, ci
  • FIG. 3C illustrates principle component analysis at the indicated time points (day 0, day 4, day 8, and day 12) of neural reprogramming.
  • FIG. 3D schematically illustrates unsupervised hierarchical clustering of cells undergoing neural reprogramming at the indicated time points (day 0, day 4, day 8, and day 12).
  • FIG. 3E is a heatmap illustrating the expression of neural stem cell-enriched genes (Ascl1, Olig2, Olig1, Sox2, Nrcam, Shh, Grh13, Zic2, and Pax2) and fibroblast-enriched genes (Thy1, S100a4, and Periostin), with lists of representative genes for each group (right panel). Red and blue colors indicate upregulation and downregulated genes, respectively. In black and white images the maximal expression is shown by the darker shades.
  • FIG. 3F illustrates the average transcription activity of genes under GO terms for the ectoderm, mesoderm, endoderm development, stem cell maintenance, and fibroblasts, at day 0 (D0) and day 4 (D4) of reprogramming.
  • FIG. 3G shows images of ciNSLC cells in a monolayer culture (left) of ciNSLC and as ciNSLC neurospheres formed in suspension culture (right) at passage 5. Scale bar is 50 ⁇ m.
  • FIG. 3H shows images of ciNSLC cells in a monolayer culture (left) of ciNSLC and as ciNSLC neurospheres formed in suspension culture (right) at passage 10. Scale bar is 100 ⁇ m.
  • FIG. 4A-4L illustrates establishment of neural fate by chemically inducing an intrinsic neural program.
  • FIG. 4A graphically illustrates the reprogramming efficiency of cells analyzed at day 10 with the M9 composition and when one small molecule inhibitor of the corresponding signaling pathway was added (+) or removed ( ⁇ ). The efficiency of M9-induced reprogramming was assigned as “1”. DMSO was used as negative control.
  • FIG. 4B also graphically illustrates the reprogramming efficiency of cells analyzed at day 10 when a small molecule inhibitor of the corresponding signaling pathway was added (+) or removed ( ⁇ ). The efficiency of M9-induced reprogramming was assigned as “1”. DMSO was used as negative control.
  • FIG. 4A graphically illustrates the reprogramming efficiency of cells analyzed at day 10 with the M9 composition and when one small molecule inhibitor of the corresponding signaling pathway was added (+) or removed ( ⁇ ). The efficiency of M9-induced reprogramming was assigned as “1”. DMSO was used as negative control.
  • FIG. 4C shows a schematic diagram (top panel) of the Sox2 regulatory regions SRR1, SRR2 and promoter (Pro).
  • the bottom left panel of FIG. 4C graphically illustrates the relative binding of Elkl at the Sox2 SRR1, SRR2 and promoter regulatory regions at the indicated time points.
  • the bottom right panel of FIG. 4C shows the relative binding of Gli2 at the Sox2 SRR1, SRR2 and promoter regulatory regions at the indicated time points. Relative binding was relative to binding by an IgG control.
  • TSS refers to transcription starting site
  • Pro refers to promoter.
  • FIG. 4D graphically illustrates the relative reprogramming efficiency assessed when Elkl expression was knocked down by Elkl shRNA at the indicated time points (day 1, day 4, or day 8).
  • FIG. 4E graphically illustrates the relative reprogramming efficiency assessed when Gli2 expression was knocked down by Gli2 shRNA at the indicated time points (day 1, day 4, or day 8).
  • FIG. 4F graphically illustrates the relative reprogramming efficiency assessed when Elkl is over-expressed or when Gli2 is over-expressed.
  • the acronyms have the following meaning: wt, wild type; KD, shRNA-mediated gene knockdown; o/e, overexpression.
  • FIG. 4G is a schematic diagram of a model interpreting M9-induced neural reprogramming through activating intrinsic neural program. Error bars, s.d., based on triplicates for FIG. 4A-F . *, P ⁇ 0.01.
  • FIG. 4G is a schematic diagram of a model interpreting M9-induced neural reprogramming through activating intrinsic neural program. Error bars, s.d., based on triplicates for FIG. 4A-F . *, P ⁇ 0.01.
  • FIG. 4H shows images ciNSLC-derived neurons illustrating the morphology of the neurons at day 3 (left), day 6 (middle), and day 10 (right) of differentiation. Scale bar is 10 ⁇ m.
  • FIG. 4I shows images of images ciNSLC-derived neurons illustrating differentiation into Map-positive and NeuN-positive neurons. Scale bar is 100 ⁇ m.
  • FIG. 4J shows images of images ciNSLC-derived neurons illustrating differentiation into vGLUT 1-positive and Map2-positive neurons. Scale bar is 50 ⁇ m.
  • FIG. 4K shows images of images ciNSLC-derived neurons illustrating differentiation into GABA-positive and Map2-positive neurons. Scale bar is 50 ⁇ m.
  • FIG. 4L shows images of images ciNSLC-derived neurons illustrating differentiation into Synapsin-positive neurons. Scale bar is 50 ⁇ m.
  • FIG. 5A-5E illustrates the membrane properties and synaptic responses of ciNSLC-derived neurons.
  • FIG. 5A shows current traces of ciNSLC-derived neurons in response to 80 pA current injection.
  • FIG. 5B shows representative traces of whole-cell current in the voltage-clamp mode. An inward current was observed that could be blocked by tetrodotoxin (TTX).
  • FIG. 5C shows representative traces of whole-cell current in the voltage-clamp mode. An outward current was observed that could be blocked by tetraethylammonium (TEA).
  • FIG. 5D shows excitatory postsynaptic currents observed in cells clamped at ⁇ 70 mV in response to L-glutamate puffs.
  • FIG. 5E show inhibitory postsynaptic currents in cells clamped at 0 mV in response to GABA puffs.
  • FIG. 6A-6B further illustrate conversion of different populations into ciNSLC by the M9 cocktail composition.
  • FIG. 6A shows that tdTomato-positive and tdTomato-negative populations can be converted into Sox2 + /Nestin + ciNSLC by the M9 cocktail, as detected by immunostaining.
  • FIG. 6B shows pie-charts illustrating the efficiency of conversion of tdTomato-positive and tdTomato-negative populations. The larger top pie-chart shows the tdTomato positive and negative cells in original MEF population, where tdTomato-positive cells constitute 6.92% of the population and tdTomato-negative cells are 90.35% of the population.
  • the two pie charts at the bottom show the reprogramming efficiency for both populations, where 26.7% of tdTomato-positive cells became Sox2 + cells, and where 31% of the tdTomato-negative cells became Sox2 + cells.
  • the expression of Sox2 was used as marker for reprogramming.
  • FIG. 7A-7C illustrate activation of master neural genes by the M9 cocktail composition.
  • FIG. 7A shows the expression of Sox2 as detected by immunostaining and the percentage of cells expressing Sox2 at the indicated times (day 4, day 8, day 12, and passage 1 (P1)). Total cell number was determined by DAPI staining. Scale bar is 20 ⁇ m for day 4 and PI, 50 ⁇ m for day 8, and 100 ⁇ m for day 12. Inset images at the left of most panel are expanded images of the field within the rectangle.
  • FIG. 7B graphically illustrates expression levels of 16 neural genes as determined by RNA-seq analysis at the indicated time points (day 0, day 4, day 8, day 12). The NSC line SCR029 was used as a positive control.
  • FIG. 7A shows the expression of Sox2 as detected by immunostaining and the percentage of cells expressing Sox2 at the indicated times (day 4, day 8, day 12, and passage 1 (P1)). Total cell number was determined by DAPI staining. Scale bar is 20
  • FIG. 8A-8B illustrate the gene ontology of the ciNSLC transcriptome.
  • FIG. 8A graphically illustrates the types of gene transcripts enriched in ciNSLC versus the NSC line SCR029, which was used as a positive control.
  • FIG. 8B graphically illustrates the types of gene upregulated during reprogramming (day 4 versus day 0).
  • differentiated non-neuronal, mammalian cells can be reprogrammed to cross lineage boundaries and to directly convert into neuronal progenitor cells or mature functional neuronal cells, without genetic manipulation. Instead, a differentiated non-neuronal cell can simply be treated with a composition of chemical compounds to change that cell into a neuronal cell.
  • a composition of nine components is described herein that can be used to convert a differentiated non-neuronal cell convert into neuronal progenitor cells or mature functional neuronal cells, without genetic manipulation.
  • the composition includes at least one BMP type I receptor ALK2/3 inhibitor, at least one TGF-beta inhibitor, at least one WNT inhibitor, at least one neuronal differentiation enhancer, at least one SMO agonist, at least one retinoic acid receptor ⁇ agonist, at least one DNA methyltransferase inhibitor, at least one histone demethylase inhibitor, and at least one autophagy regulator.
  • differentiated mammalian cells can be converted into the neuronal cell lineage without such genetic manipulation.
  • the composition of chemical compounds can be administered to a subject, or differentiated (e.g., non-neuronal) cells from the subject can be incubated with such a composition to convert the subject's cells to a neuronal cell type.
  • small molecule-based chemical strategies may have several important advantages. Small molecules are relatively easy to apply, optimize and manufacture, and they can be more readily developed into conventional pharmaceuticals. Unlike the reprogramming mediated by pioneer transcription factors, the chemical-induced cellular reprogramming represents a different process. Mechanistically, small molecules interact with and modulate endogenously expressed proteins of the starting (i.e., selected) cell type, and indirectly and ultimately gain and establish target cell type specificity. Therefore, achieving chemical reprogramming would provide a novel approach and process to investigate the underlying mechanism of cell fate conversion.
  • introduction of expression cassettes encoding the Elk-1 transcription activator and/or the Gli2 zinc-finger transcription factor can increase the proportion of cells that are reprogrammed to cross lineage boundaries and converted into neuronal progenitor cells or mature functional neuronal cells.
  • BMP type I receptor ALK2/3 inhibitors can facilitate conversion of differentiated cells into the neuronal cell lineage.
  • ALK2 activate A receptor type I
  • ALK3 bone morphogenetic protein receptor, type 1A
  • ALK3 is a regulator of cell fate decisions during embryogenesis and tissue homeostasis.
  • BMP type I receptor ALK2/3 inhibitors are available.
  • the methods and compositions described herein can include any of the following:
  • the BMP type I receptor ALK2/3 inhibitor(s) can be 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (LDN-193189).
  • the BMP type I receptor ALK2/3 inhibitor(s) can be used in various concentrations.
  • the BMP type I receptor ALK2/3 inhibitor(s) can be employed at a concentration of about 0.1 nanomolar to about 1 micromolar, or about 1 nanomolar to about 700 nanomolar, or about 10 nanomolar to about 500 nanomolar, or about 50 nanomolar to about 200 nanomolar, or about 100 nanomolar in a solution.
  • BMP type I receptor ALK2/3 inhibitors can be present in amounts of about 0.01 mg to about 2000 mg, or about 0.1 mg to about 1000 mg, or about 1 mg to about 500 mg.
  • Cells can be incubated in a medium containing one or more BMP type I receptor ALK2/3 inhibitor (e.g., LDN-193189) for varying amounts of time.
  • the cells can be incubated in a medium containing a BMP type I receptor ALK2/3 inhibitor until at least some of the cells express neural progenitor markers such as Sox2 + and/or Nkx6.1 + .
  • the incubation time can vary, for example, from about 1 day to about 30 days, or from about 2 days to about 25 days, or from about 4 day to about 20 days, or about 10 days.
  • TGF- ⁇ transforming growth factor-beta
  • TGF- ⁇ transforming growth factor-beta
  • activin activin
  • Nodal Nodal
  • BMPs transforming growth factor-beta
  • a TGF-beta inhibitor can directly or indirectly, negatively regulate TGF-beta signaling.
  • one or more TGF-beta inhibitors binds to and reduces the activity of one or more serine/threonine protein kinases selected from the group consisting of ALK5, ALK4, TGF-beta receptor kinase 1 and ALK7.
  • ALK4, ALK5 and ALK7 are all closely related receptors of the TGF-beta superfamily. Desirable TGF-beta inhibitors can bind to and reduce the activity of ALK4, ALK5 (TGF-beta receptor kinase 1) and/or ALK7.
  • the TGF-beta receptor binds to and reduces the activity of a Smad protein, for example R-SMAD or SMAD1-5 (i.e. SMAD 1, SMAD 2, SMAD 3, SMAD 4 or SMAD 5).
  • a Smad protein for example R-SMAD or SMAD1-5 (i.e. SMAD 1, SMAD 2, SMAD 3, SMAD 4 or SMAD 5).
  • TGF- ⁇ inhibitors include, but are not limited to:
  • a cellular assay may be used in which cells are stably transfected with a reporter construct that includes the human PAI-1 promoter or Smad binding sites, driving a luciferase reporter gene. Inhibition of luciferase activity relative to control groups can be used as a measure of compound activity (De Gouville et al., Br J Pharmacol, 2005 May; 145(2): 166-177).
  • ALPHASCREEN® phosphosensor assay for measurement of kinase activity (Drew A E et al., Comparison of 2 Cell-Based Phosphoprotein Assays to Support Screening and Development of an ALK Inhibitor J Biomol Screen 16(2) 164-173, 2011).
  • the inhibitor that directly or indirectly negatively regulates TGF-beta signaling can, for example, be selected from the group consisting of A83-01, SB-431542, SJN-2511, LY-36494, SB-505124, SB-525334, and SD-208.
  • the inhibitor that directly or indirectly negatively regulates TGF-beta signaling can inhibit ALK4, ALK5 and/or ALK7.
  • the inhibitor that directly or indirectly negatively regulates TGF-beta signaling can be A83-01.
  • the TGF-beta inhibitor can be employed in the compositions and methods described herein in a variety of amounts and/or concentrations.
  • the TGF-beta inhibitor can be employed at a concentration of about 0.001 micromolar to about 20 micromolar, or about 0.01 micromolar to about 10 micromolar, or about 0.05 micromolar to about 5 micromolar, or about 0.1 micromolar to about 1 micromolar, or about 0.5 micromolar in a solution.
  • the TGF-beta inhibitor can be present in amounts of about 0.01 mg to about 1000 mg, or about 0.05 mg to about 500 mg, or about 0.1 mg to about 50 mg.
  • the time of contacting or mixing TGF-beta inhibitor (s) with a population of cells (to generate neural cells) can vary, for example, from about 1 day to about 30 days, or from 2 days to about 25 days, or from 3 days to about 20 days, or from 5 days to about 15 days, or from 7 days to about 14 days, or about 10 days.
  • WNT activator can facilitate conversion of differentiated cells into the neuronal cell lineage.
  • the WNT signaling pathway includes a series of events that occur when a WNT protein binds to a cell-surface receptor of a Frizzled receptor family member. Such events result in the activation of Dishevelled family proteins which inhibit a complex of proteins that includes axin, GSK-3, and the protein APC to degrade intracellular beta-catenin. The resulting enriched nuclear beta-catenin enhances transcription by TCF/LEF family transcription factors.
  • a WNT activator can therefore include an agent that activates TCF/LEF-mediated transcription in a cell.
  • WNT activators can be selected from true WNT agonists that bind and activate a Frizzled receptor family member including any and all of the WNT family proteins, an inhibitor of intracellular beta-catenin degradation, activators of TCF/LEF, and inhibitors of GSK-3.
  • WNT activators examples include one or more of the following compounds:
  • WNT activators can also include small-interfering RNAs (siRNA, Cell Signaling) that act as GSK-inhibitors, lithium (Sigma), kenpaullone (Biomol International, Leost, Metal (2000) Eur J Biochem 267, 5983-5994), 6-Bromoindirubin-30-acetoxime (Meyer, L et al (2003) Chem Biol 10, 1255-1266), SB 216763 and SB 415286 (Sigma-Aldrich), and FRAT-family members and FRAT-derived peptides that prevent interaction of GSK-3 with axin.
  • siRNA small-interfering RNAs
  • Li Li
  • kenpaullone Biomol International, Leost, Metal (2000) Eur J Biochem 267, 5983-5994
  • 6-Bromoindirubin-30-acetoxime Meyer, L et al (2003) Chem Biol 10, 1255-1266
  • SB 216763 and SB 415286 Sigma-Aldrich
  • the WNT activators can, for example, be CHIR99021, SB216763, TWS119, CHIR98014, Tideglusib, SB415286, LY2090314, or any combination thereof.
  • the WNT activators can be CHIR99021, whose structure is shown below.
  • the WNT activators can also be in the form of a salt or hydrate of any of the foregoing compounds.
  • a selected population of cells is contacted or mixed with one or more WNT activators for a time and at a concentration sufficient to differentiate or re-direct the cells to neural lineage.
  • the WNT activators can be employed in the compositions and methods described herein in a variety of amounts and/or concentrations.
  • the WNT activators can be employed at a concentration of about 0.01 micromolar to about 1 millimolar in a solution, or about 0.1 micromolar to about 100 micromolar in a solution, or about 0.5 micromolar to about 10 micromolar in a solution, or about 1 micromolar to about 5 micromolar in a solution.
  • the WNT activators can be present in amounts of about 0.01 mg to about 1000 mg, or about 1 mg to about 100 mg, or about 1 mg to about 10 mg can be present in amounts of about 0.01 mg to about 1000 mg, or about 0.1 mg to about 100 mg, or about 1 mg to about 10 mg.
  • WNT activators can be added to a selected starting cell population during induced pluripotency and while directing the cells into the neural lineage. WNT activators can also be added to a neural cell population to be converted to neural cells.
  • the time of contacting or mixing WNT activator(s) with a population of cells can vary, for example, from about 1 day to about 30 days, or from 2 days to about 25 days, or from 3 days to about 20 days, or from 5 days to about 15 days, or from 7 days to about 14 days, or about 10 days.
  • Methods and assays for determining a level of WNT activation or GSK-3 inhibition are available to a skilled person and include, for example, the methods and assays described in Liao et al., Endocrinology, 145(6): 2941-2949 (2004); and in U.S. Pat. No. 8,323,919, both of which are specifically incorporated by reference herein in their entireties.
  • neuronal differentiation enhancers can facilitate conversion of differentiated cells into the neuronal cell lineage.
  • experimental data described herein shows that neuronal differentiation enhancers can facilitate neuronal conversion of fibroblasts to the neuronal lineage.
  • neuronal differentiation enhancers can be used in the compositions and methods described herein.
  • neuronal differentiation enhancers can include:
  • the neuronal differentiation factor is basic fibroblast growth factor (bFGF).
  • the amounts of neuronal differentiation factors in the compositions and methods provided herein can vary, for example, depending on the number of cells to be treated, the extent of conversion to the neuronal lineage (e.g., as assessed using neuronal cell markers), and the size and duration of the culture. Concentrations can range, for example, between 0.1-200 ng/mL, 0.5-100 ng/mL, 1-50 ng/mL, 2-25 ng/mL, 3-20 ng/mL, 5-15 ng/mL, or 10 ng/mL. In a specific embodiment, 10 ng/mL bFGF is used. Suitable concentrations can be determined by assaying the differentiation potential of cells having undergone the methods described herein.
  • Neuronal differentiation factors can be added to a selected starting cell population while directing the cells into the neural lineage. Neuronal differentiation factors can also be added to a neural progenitor cell population to be converted to neural cells.
  • the time of contacting or mixing neuronal differentiation factors with a population of cells can vary, for example, from about 1 day to about 30 days, or from 2 days to about 25 days, or from 3 days to about 20 days, or from 5 days to about 15 days, or from 7 days to about 14 days, or about 10 days.
  • the 7-pass transmembrane protein Smoothened acts as a positive regulator of Hedgehog signaling.
  • Hedgehog genes encode secreted proteins that undergo post-translational modifications, including autocatalytic cleavage and lipid modification (palmitoylation) at the N-terminus and cholesterol modification of the C-terminus.
  • Patched acts as negative regulator of Hedgehog signaling and at resting state free Ptch (i.e., unbound by Hedgehog) suppresses pathway activity that has been induced by Smo (Taipale et al. (2002) Nature 418: 892).
  • Agonists of Smo include the following:
  • the SMO agonist is Hh-Ag1.5.
  • the amounts of SMO agonist in the compositions and methods provided herein can vary, for example, depending on the number of cells to be treated, the extent of conversion to the neuronal lineage (e.g., as assessed using neuronal cell markers), and the size and duration of the culture.
  • the SMO agonists can be employed at a concentration of about 0.01 micromolar to about 1 millimolar in a solution, or about 0.1 micromolar to about 100 micromolar in a solution, or about 0.5 micromolar to about 10 micromolar in a solution, or about 1 micromolar to about 5 micromolar in a solution.
  • the SMO agonist in a dry formulation, can be present in amounts of about 0.01 mg to about 1000 mg, or about 1 mg to about 100 mg, or about 1 mg to about 10 mg can be present in amounts of about 0.01 mg to about 1000 mg, or about 0.1 mg to about 100 mg, or about 1 mg to about 10 mg.
  • SMO agonists can be added to a selected starting cell population while directing the cells into the neural lineage. SMO agonists can also be added to a neural progenitor cell population to be converted to neural cells.
  • the time of contacting or mixing SMO agonists with a population of cells can vary, for example, from about 1 day to about 30 days, or from 2 days to about 25 days, or from 3 days to about 20 days, or from 5 days to about 15 days, or from 7 days to about 14 days, or about 10 days.
  • retinoic acid receptor-gamma can facilitate conversion of differentiated cells into the neuronal cell lineage.
  • Agonists of RAR ⁇ stimulate the receptor to activate transcription of various genes.
  • RAR ⁇ agonists can be used in the compositions and methods described herein.
  • RAR ⁇ agonists can include:
  • Agonists of RAR ⁇ can be identified or evaluated by transactivation assays.
  • the term “transactivation” refers to the ability of a retinoid to activate the transcription of a gene where the gene transcription is initiated by the binding of a ligand (e.g., agonist) to the RAR ⁇ . Determining the ability of a compound to transactivate a retinoic acid receptor can be performed by methods known to those of skill in the art. Examples of such methods are found in Bernard et al, Biochem. Biophys. Res. Commun., 186: 977-983 (1992) and C. Apfel et al, Proc. Nat. Sci. Acad . ( USA ), 89: 7129-7133 (1992).
  • the RAR ⁇ agonist can, for example, be retinoic acid.
  • the amounts of RAR ⁇ agonists in the compositions and methods provided herein can vary, for example, depending on the number of cells to be treated, the extent of conversion to the neuronal lineage (e.g., as assessed using neuronal cell markers), and the size and duration of the culture.
  • the RAR ⁇ agonists can be employed at a concentration of about 0.01 micromolar to about 1 millimolar in a solution, or about 0.1 micromolar to about 100 micromolar in a solution, or about 0.5 micromolar to about 10 micromolar in a solution, or about 1 micromolar to about 5 micromolar in a solution.
  • the RAR ⁇ agonists can be present in amounts of about 0.01 mg to about 1000 mg, or about 1 mg to about 100 mg, or about 1 mg to about 10 mg can be present in amounts of about 0.01 mg to about 1000 mg, or about 0.1 mg to about 100 mg, or about 1 mg to about 10 mg.
  • RAR ⁇ agonists can be added to a selected starting cell population while directing the cells into the neural lineage.
  • RAR ⁇ agonists can also be added to a neural progenitor cell population to be converted to neural cells.
  • the time of contacting or mixing RAR ⁇ agonists with a population of cells can vary, for example, from about 1 day to about 30 days, or from 2 days to about 25 days, or from 3 days to about 20 days, or from 5 days to about 15 days, or from 7 days to about 14 days, or about 10 days.
  • DNMT DNA Methyltransferase
  • DNMT DNA methyltransferase
  • DNA methyltransferases are enzymes that transfer methyl groups to DNA. Inhibitors of DNA methyltransferases can reactivate the expression of genes that have been repressed by DNA methylation. As illustrated herein, DNA methyltransferase inhibitors can improve the conversion of selected starting cells to neuronal cells.
  • DNA methyltransferase inhibitors can include antibodies that bind to DNA methyltransferases, dominant negative variants of DNA methyltransferases, and siRNA and antisense nucleic acids that suppress expression of DNMT.
  • DNA methyltransferase inhibitors include, but are not limited to, RG108 (N-Phthalyl-L-tryptophan, available, e.g., from Sigma-Aldrich), 5-aza-C (5-azacitidine or azacitidine) (see, e.g., Schermelleh, et al., Nature Methods 2:751-6 (2005)), 5-aza-2′-deoxycytidine (5-aza-CdR) (see, e.g., Zhu, Clinical Medicinal Chemistry 3(3): 187-199 (2003)), decitabine (see, e.g., Gore, Nature Clinical Practice Oncology 2:S30-S35 (2005)), doxorubicin (see, e.g., Le
  • the DNA methyltransferase inhibitor is RG108, which has the following structure.
  • the amounts of DNA methyltransferase inhibitors in the compositions and methods provided herein can vary, for example, depending on the number of cells to be treated, the extent of conversion to the neuronal lineage (e.g., as assessed using neuronal cell markers), and the size and duration of the culture.
  • the DNA methyltransferase inhibitors can be employed at a concentration of about 0.01 micromolar to about 1 millimolar in a solution, or about 0.1 micromolar to about 100 micromolar in a solution, or about 0.5 micromolar to about 50 micromolar in a solution, or about 1 micromolar to about 25 micromolar in a solution, or about 10 micromolar.
  • the DNA methyltransferase inhibitors can be present in amounts of about 0.01 mg to about 1000 mg, or about 1 mg to about 100 mg, or about 1 mg to about 10 mg can be present in amounts of about 0.01 mg to about 1000 mg, or about 0.1 mg to about 100 mg, or about 1 mg to about 10 mg.
  • DNA methyltransferase inhibitors can be added to a selected starting cell population while directing the cells into the neural lineage. DNA methyltransferase inhibitors can also be added to a neural progenitor cell population to be converted to neural cells.
  • the time of contacting or mixing DNA methyltransferase inhibitors with a population of cells can vary, for example, from about 1 day to about 30 days, or from 2 days to about 25 days, or from 3 days to about 20 days, or from 5 days to about 15 days, or from 7 days to about 14 days, or about 10 days.
  • histone demethylase inhibitors can facilitate conversion of differentiated cells into the neuronal cell lineage.
  • Histone demethylases remove methyl groups from histone.
  • the lysine-specific demethylase 1 (LSD1, also called KDM1, AOF2, or BHC110) is a histone demethylase that suppresses gene expression by converting di-methylated lysines on histone H3 to monomethylated and unmethylated lysines.
  • Histone methylation can influence epigenetic patterns of gene expression due to association with active promoters.
  • use of one or more inhibitors of histone demethylase enzymes can facilitate conversion of differentiated cells into the neural lineage.
  • Exemplary inhibitors of histone demethylase include, but are not limited to, parnate (also called tranylcypromine sulfate) or an equivalent salt of parnate, and phenelzine (Nardil, 2-phenylethylhydrazine). See, also, Huang et al., Proc Natl Acad Sci USA. 104(19): 8023-8028 (2007); Bi, X. et al., Bioorg. Med. Chem. Lett. 16:3229-3232 (2006); International Patent Application Nos. WO2007/021839 and WO2008/127734.
  • MAO inhibitors can also serve as epigenetic modulators.
  • the histone demethylase inhibitor is parnate.
  • histone demethylase inhibitors in the compositions and methods provided herein can vary, for example, depending on the number of cells to be treated, the extent of conversion to the neuronal lineage (e.g., as assessed using neuronal cell markers), and the size and duration of the culture.
  • the histone demethylase inhibitors can be employed at a concentration of about 0.01 micromolar to about 1 millimolar in a solution, or about 0.01 micromolar to about 50 micromolar in a solution, or about 0.1 micromolar to about 25 micromolar in a solution, or about 0.5 micromolar to about 10 micromolar in a solution, or about 2 micromolar.
  • the histone demethylase inhibitors can be present in amounts of about 0.01 mg to about 1000 mg, or about 1 mg to about 100 mg, or about 1 mg to about 10 mg can be present in amounts of about 0.01 mg to about 1000 mg, or about 0.1 mg to about 100 mg, or about 1 mg to about 10 mg.
  • Histone demethylase inhibitors can be added to a selected starting cell population while directing the cells into the neural lineage. Histone demethylase inhibitors can also be added to a neural progenitor cell population to be converted to neural cells.
  • the time of contacting or mixing histone demethylase inhibitors with a population of cells can vary, for example, from about 1 day to about 30 days, or from 2 days to about 25 days, or from 3 days to about 20 days, or from 5 days to about 15 days, or from 7 days to about 14 days, or about 10 days.
  • use of one or more autophagy regulators can facilitate conversion of differentiated cells into the neuronal cell lineage.
  • autophagy regulators examples include SMER28, apigenin, berberine, beta-elemene, capsaicin, curcumin, genistein, kaempferol, oridonin, paclitaxel, quercetin, resveratrol, silybin, triptolide, and ursolic acid.
  • the autophagy regulator is SMER28.
  • SMER28 (6-bromo-N-2-propenyl-4-quinazolinamine) enhances A53T alpha-synuclein clearance in PC-12 cells independent of rapamycin treatment. It appears to act independent of the mTOR pathway, but combined treatment with saturating rapamycin concentration enhances the effect of either compound alone on A53T alpha-synuclein clearance; autophagy inducers may prove useful in the treatment of neurodegenerative and infectious diseases and cancer.
  • the amounts of autophagy regulators in the compositions and methods provided herein can vary, for example, depending on the number of cells to be treated, the extent of conversion to the neuronal lineage (e.g., as assessed using neuronal cell markers), and the size and duration of the culture.
  • the autophagy regulators can be employed at a concentration of about 0.01 micromolar to about 500 micromolar in a solution, or about 0.1 micromolar to about 100 micromolar in a solution, or about 0.5 micromolar to about 50 micromolar in a solution, or about 1 micromolar to about 25 micromolar in a solution, or about 10 micromolar.
  • the autophagy regulators can be present in amounts of about 0.01 mg to about 1000 mg, or about 1 mg to about 100 mg, or about 1 mg to about 10 mg can be present in amounts of about 0.01 mg to about 1000 mg, or about 0.1 mg to about 100 mg, or about 1 mg to about 10 mg.
  • Autophagy regulators can be added to a selected starting cell population while directing the cells into the neural lineage. Autophagy regulators can also be added to a neural progenitor cell population to be converted to neural cells.
  • the time of contacting or mixing autophagy regulators with a population of cells can vary, for example, from about 1 day to about 30 days, or from 2 days to about 25 days, or from 3 days to about 20 days, or from 5 days to about 15 days, or from 7 days to about 14 days, or about 10 days.
  • Gli2 and/or Elk2 transcription factors/activators increases the proportion of cells that are reprogrammed to cross lineage boundaries and converted into neuronal progenitor cells or mature functional neuronal cells (see e.g., FIGS. 4D-4F ).
  • the methods described herein can include expression of the Gli2 and/or Elk1 protein with treatment that includes the compositions described herein.
  • Such proteins can be expressed from a transgene or expression cassette that includes a promoter that is operably linked to a nucleic acid segment that encodes the Gli2 or Elk1 protein.
  • the promoter can be a heterologous promoter, meaning that the promoter is not the promoter that naturally drives expression of the Gli2 or Elk1 protein.
  • the promoter can be the natural promoter that does drive expression of the Gli2 or Elk1 protein.
  • Expression vectors for overexpression of Elkl (e.g., addgene 27156) or Gli2 (e.g., addgene 37671) can also be obtained from Addgene and used for increasing the expression of the Gli2 and/or Elk1 proteins.
  • Elkl e.g., addgene 27156
  • Gli2 e.g., addgene 37671
  • the transcription factors/activators that can increase the proportion of cells that are reprogrammed to cross lineage boundaries and that are converted into neuronal progenitor cells or mature functional neuronal cells includes related transcription factors with at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97% sequence identity to SEQ ID NO:1 or 2.
  • a selected starting population of cells may be derived from essentially any source, and may be heterogeneous or homogeneous.
  • the term “selected cell” or “selected cells” is also used to refer to starting cells.
  • the selected starting cells to be treated as described herein are adult cells, including essentially any accessible adult cell type(s).
  • the selected starting cells treated according to the invention are adult stem cells, progenitor cells, or somatic cells.
  • the selected starting cells treated with any of the compositions and/or methods described herein include any type of cell from a newborn, including, but not limited to newborn cord blood, newborn stem cells, progenitor cells, and tissue-derived cells (e.g., somatic cells).
  • the starting population of cells does not include pluripotent stem cells. In other embodiments, the starting population of cells can include pluripotent stem cells. Accordingly, a starting population of cells that is reprogrammed by the compositions and/or methods described herein, can be essentially any live cell type, particularly a somatic cell type.
  • fibroblasts can be reprogrammed to cross lineage boundaries and to be directly converted to other cell types such as neuronal progenitor or mature functional neuronal cell types.
  • Various cell types from all three germ layers have been shown to be suitable for somatic cell reprogramming by genetic manipulation, including, but not limited, to liver and stomach (Aoi et al., Science 321(5889):699-702 (2008); pancreatic ⁇ cells (Stadtfeld et al., Cell Stem Cell 2: 230-40 (2008); mature B lymphocytes (Hanna et al., Cell 133: 250-264 (2008); human dermal fibroblasts (Takahashi et al., Cell 131, 861-72 (2007); Yu et al., Science 318(5854) (2007); Lowry et al., Proc Natl Acad Sci USA 105, 2883-2888 (2008); Aasen et al., Nat Biotechnol 26(11):
  • neural progenitor cells (Eminli et al., Stem Cells 26(10): 2467-74 (2008). Any such cells can be reprogrammed and/or programmed by use of the compositions and methods described herein.
  • the cells can be autologous or allogeneic cells (relative to a subject to be treated or who may receive the cells).
  • Selected starting cells are treated for a time and under conditions sufficient to convert the starting cells across lineage and/or differentiation boundaries to form neuronal progenitor cells or mature functional neuronal cells.
  • Selected starting cells can be incubated with a composition that includes at least one BMP type I receptor ALK2/3 inhibitors, at least one TGF-beta inhibitor, at least one WNT inhibitor, at least one neuronal differentiation enhancer, at least one SMO agonist, at least one retinoic acid receptor ⁇ agonist, at least one DNA methyltransferase inhibitor, at least one histone demethylase inhibitor, at least one autophagy regulator, or any combination thereof.
  • the composition can contain at least two of the agents, or at least three of the agents, or at least four of the agents, or at least five of the agents, or at least six of the agents, or at least seven of the agents, or at least eight of the agents. This composition is referred to herein as the M9 cocktail.
  • the time for conversion of starting cells into neuronal progenitor and mature neuronal cells can vary.
  • the starting cells can be incubated with the M9 reprogramming cocktail until neuronal cell markers are expressed.
  • neuronal cell markers can include Tuj1, Map2, NeuN, Sox2, synapsins (e.g., Synl and Syn2), synaptophysin, synaptotagmins (e.g., Sytl, Syt4, Sytl3, Syt16), NeuroD, Is1l, and cholineacetyltransferase (ChAT, e.g., vascular ChAT (VChAT)).
  • ChAT e.g., vascular ChAT (VChAT)
  • the starting cells can be incubated with the M9 reprogramming cocktail until oligodendrocyte markers are expressed, such as O4. In some cases, starting cells can be incubated with the M9 reprogramming cocktail until astrocyte markers are expressed, such as GFAP. In other cases, starting cells can be incubated with the M9 reprogramming cocktail until vGlut1-positive glutamatergic neurons and/or GABA-positive GABAergic neurons are detected Neuronal progenitor cells can, for example, be detected by observing expression of Tuj1, a neuron-specific class III beta-tubulin. Human ⁇ -Tubulin 3 is a 50,432 Dalton structural protein (450 amino acid) expressed in neurons of the peripheral and central nervous systems. It contributes to microtubule stability in neuronal cell bodies and axons, and plays a role in axonal transport.
  • the starting cell(s) can also be incubated with the reprogramming composition until a more mature neuronal cell marker is expressed by the cells.
  • the starting cell(s) can be incubated with the reprogramming composition until expression of the Tau marker is observed.
  • TAU is a neuronal microtubule-associated protein found predominantly on axons.
  • the starting cell(s) can be incubated with the reprogramming composition until expression of the NeuN marker is observed.
  • NeuN neuroneuronal nuclei
  • MAP2 is expressed by the cells.
  • MAP2 is also a microtubule-associated protein with a role in neurogenesis.
  • the starting cell(s) can also be incubated with the reprogramming composition until the more mature neuronal cell marker Synapsin I is expressed by the cells.
  • Synapsin I is a major phosphoprotein in synaptic terminals.
  • the time for conversion of starting cells into neuronal progenitor and mature neuronal cells can therefore vary.
  • the starting cells can be incubated with the composition under cell culture conditions for at least about 3 days, or for at least about 4 days, or for at least about 5 days, or for at least about 6 days, or for at least about 7 days, or for at least about 8 days, or for at least about 9 days, or for at least about 10 days, or for at least about 11 days, or for at least about 12 days, or for at least about 13 days, or for at least about 14 days, or for at least about 15 days, or for at least about 16 days, or for at least about 17 days, or for at least about 18 days, or for at least about 19 days.
  • the starting cells can be incubated with the composition under cell culture conditions for about 3 days to about 30 days, or about 4 days to about 25 days, or about 5 days to about 20 days, or about 7 days to about 15 days, or about 8 days to about 12 days, or about 10 days.
  • a composition of nine chemical compounds (at least one BMP type I receptor ALK2/3 inhibitor, at least one TGF-beta inhibitor, at least one WNT inhibitor, at least one neuronal differentiation enhancer, at least one SMO agonist, at least one retinoic acid receptor ⁇ agonist, at least one DNA methyltransferase inhibitor, at least one histone demethylase inhibitor, at least one autophagy regulator) was sufficient to efficiently induce over 25% human fibroblasts to form Sox2/Nestin double-positive cells after ten days of incubation.
  • Semi-quantitative RT-PCR confirmed the expression of other neural stem cell genes, including Pax6, Sox2, Ascl1, and Olig2.
  • the starting cells can be incubated with the M9 cocktail that is combined with a cell culture medium.
  • cell culture medium (also referred to herein as a “culture medium” or “medium”) as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation.
  • the cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc.
  • Cell culture media ordinarily used for particular cell types are available to those skilled in the art.
  • cell culture media that can be employed Dulbecco's modified Eagle's medium (e.g., supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, and 2 mM Glutamax), N2B27 medium (e.g., containing 50% Neural basal medium and 50% DMEM/F12 medium supplemented with 1% GlutaMax, 1% N2 (Life Technologies), 2% B27 (Gibco), and 0.1% BSA)), or neural stem cell medium (NSC medium, 50% Neural basal, 50% DMEM/F12/Glutamax, 1 ⁇ N2, 1 ⁇ B27 without vitamin A, 0.075% BSA, 0.1 mM nonessential amino acids, 20 ng/ml bFGF, 20 ng/ml EGF).
  • Dulbecco's modified Eagle's medium e.g., supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, and 2 mM Glutamax
  • the composition can include 50% Neural basal, 50% DMEM/F12/Glutamax, 1 ⁇ N2, 1 ⁇ B27 without vitamin A, 0.075% BSA, 0.1 mM nonessential amino acids, CHIR99021 at 3 ⁇ M, LDN193189 at 100 nM, A83-01 at 0.5 ⁇ M, Hh-Ag1.5 at 0.5 ⁇ M, retinoic acid at 1 ⁇ M, SMER28 at 10 ⁇ M, RG108 at 10 ⁇ M, Parnate at 2 ⁇ M, and bFGF at 10 ng/ml.
  • the cells can be cultured within, or transferred after culture in the DMEM or N2B27 medium to a maturation medium.
  • a maturation medium is the N2B27 medium that contains 0.5% Albumin, plus 20 ng ml ⁇ 1 GDNF (R&D Systems), 10 ng ml ⁇ 1 BDNF (R&D Systems), 10 ng ml-1 NT3 (R&D Systems), and 3 ⁇ M Forskolin (Tocris).
  • Examples of commercially available media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPM1 1640, Ham's F-10, Ham's F-12, a-Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium, or a general purpose media modified for use with pluripotent cells, such as X-VIVO (Lonza) or a hematopoietic base media.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • RPM1 1640 Ham's F-10
  • Ham's F-12 Ham's F-12
  • aMEM a-Minimal Essential Medium
  • G-MEM Glasgow's Minimal Essential Medium
  • Iscove's Modified Dulbecco's Medium or a general
  • the starting cells can be dispersed in a cell culture medium that contains the M9 cocktail at a density that permits cell expansion.
  • a cell culture medium that contains the M9 cocktail at a density that permits cell expansion.
  • about 1 to 10 10 cells can be contacted with the M9 cocktail in a selected cell culture medium, especially when the cells are maintained at a cell density of about 1 to about 10 8 cells per milliliter, or at a density of about 100 to about 10 7 cells per milliliter, or at a density of about 1000 to about 10 6 cells per milliliter.
  • Such methods can therefore be used to generate a population of neuronal cells that can be transplanted into a subject or used for experimentation.
  • a reprogrammed population of cells can be frozen at liquid nitrogen temperatures, stored for periods of time, and then thawed for use at a later date. If frozen, a population of reprogrammed cells can be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Once thawed, the cells can be expanded by culturing the cells in an appropriate medium that can contain selected growth factors, vitamins, feeder cells, and other components selected by a person of skill in the art.
  • the reprogrammed cells and/or compositions containing the M9 cocktail (with or without reprogrammed cells) described herein can also be employed in a method of treating a subject with a neuronal disease, condition, or injury.
  • the M9 cocktail can be administered to a patient to treat a neuronal disease, condition, or injury.
  • Examples of diseases, conditions, and injuries that can be treated using the reprogrammed cells and compositions include Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, multiple sclerosis, Primary lateral sclerosis (PLS), Progressive bulbar palsy, Pseudobulbar palsy, Progressive muscular atrophy, Spinal muscular atrophy (SMA), including Type I (also called Werdnig-Hoffmann disease), Type II, Type III (Kugelberg-Welander disease), Fazio-Londe disease, Huntington's disease, Kennedy's disease also known as progressive spinobulbar muscular atrophy, hereditary spastic paraplegia (HSP), congenital SMA with arthrogryposis, Post-polio syndrome (PPS), traumatic spinal cord injury, progressive pseudobulbar palsy, progressive muscular atrophy, post-polio syndrome, stroke, head trauma, spinal cord injury, and the like.
  • ALS Amyotrophic lateral sclerosis
  • PLS Primary lateral sclerosis
  • Diseases and conditions that can be treated include those that occur as a consequence of genetic defect, physical injury, environmental insult or conditioning, bad health, obesity and other disease risk factors commonly known by a person of ordinary skill in the art.
  • EMG electromyography
  • MRI magnetic resonance imaging
  • efficacy of treatment can also be assessed by a muscle or nerve biopsy, which can help confirm nerve disease and nerve regeneration.
  • a small sample of the muscle or nerve is removed under local anesthetic and studied under a microscope. The sample may be removed either surgically, through a slit made in the skin, or by needle biopsy, in which a thin hollow needle is inserted through the skin and into the muscle. A small piece of muscle remains in the hollow needle when it is removed from the body.
  • efficacy of treatment can also be monitored by a transcranial magnetic stimulation to study areas of the brain related to motor activity.
  • Reprogrammed cells generated as described herein can be employed for tissue reconstitution or regeneration in a human patient or other subjects in need of such treatment.
  • the cells are administered in a manner that permits them to graft or migrate to a diseased or injured tissue site and to reconstitute or regenerate the functionally deficient area.
  • Devices are available that can be adapted for administering cells, for example, into the spinal cord or other parts of the central or peripheral nervous system.
  • Reprogrammed cells can be administered to reconstitute the neuronal cell population in the spinal cord, brain, or at an alternative desired location.
  • the cells may be administered to a recipient by local injection, or by systemic injection.
  • the cells can be administered parenterally by injection into a convenient cavity or by intramuscular injection.
  • cell types are capable of migrating to an appropriate site for regeneration and differentiation within a subject.
  • the cells can first be tested in a suitable animal model. At one level, cells are assessed for their ability to survive and maintain their phenotype in vivo. Cells can also be assessed to ascertain whether they migrate to diseased or injured sites in vivo, or to determine an appropriate number of cells to be administered.
  • Cell compositions can be administered to immunodeficient animals (such as nude mice, or animals rendered immunodeficient chemically or by irradiation). Tissues can be harvested after a period of regrowth, and assessed as to whether the administered cells or progeny thereof are still present, are alive, and/or have migrated to desired or undesired locations.
  • Injected cells can be traced by a variety of methods. For example, cells containing or expressing a detectable label (such as green fluorescent protein, or beta-galactosidase) can readily be detected.
  • the cells can be pre-labeled, for example, with tdTomato, BrdU or [ 3 H] thymidine, or by introduction of an expression cassette that can express green fluorescent protein, or beta-galactosidase.
  • the reprogrammed cells can be detected by their expression of a cell marker that is not expressed by the animal employed for testing (for example, a human-specific antigen or tdTomato).
  • the presence and phenotype of the administered population of reprogrammed cells can be assessed by fluorescence microscopy (e.g., for green fluorescent protein, or beta-galactosidase), by immunohistochemistry (e.g., using an antibody against a human antigen), by ELISA (using an antibody against a human antigen), or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for human polynucleotides.
  • fluorescence microscopy e.g., for green fluorescent protein, or beta-galactosidase
  • immunohistochemistry e.g., using an antibody against a human antigen
  • ELISA using an antibody against a human antigen
  • RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for human polynucleotides.
  • a number of animal models of motor neuron diseases are available for such testing, for example as the S0D1(G93A) mutant mouse and SMA (B6.129-Smn1 tm1Jme J ) mouse models from Jackson laboratories.
  • a reprogrammed population of cells can be introduced by injection, catheter, implantable device, or the like.
  • a population of reprogrammed cells can be administered in any physiologically acceptable excipient or carrier that does not adversely affect the cells.
  • a population reprogrammed cells can be supplied in the form of a pharmaceutical composition.
  • a composition can include an isotonic excipient prepared under sufficiently sterile conditions for human administration.
  • Cell Therapy Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy , by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy , E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.
  • the choice of the cellular excipient and any accompanying constituents of the composition that includes a population of reprogrammed cells can be adapted to optimize administration by the route and/or device employed.
  • a composition that includes a population of reprogrammed cells can also include or be accompanied by one or more other ingredients that facilitate engraftment or functional mobilization of the reprogrammed cells.
  • Suitable ingredients include matrix proteins that support or promote adhesion of the reprogrammed cells, or complementary cell types, such as glial and/or muscle cells.
  • the composition may include physiologically acceptable matrix scaffolds. Such physiologically acceptable matrix scaffolds can be resorbable and/or biodegradable.
  • the population of reprogrammed cells generated by the methods described herein can include low percentages of non-neuronal cells (e.g., fibroblasts).
  • a population of reprogrammed cells for use in compositions and for administration to subjects can have less than about 90% non-neuronal cells, less than about 85% non-neuronal cells, less than about 80% non-neuronal cells, less than about 75% non-neuronal cells, less than about 70% non-neuronal cells, less than about 65% non-neuronal cells, less than about 60% non-neuronal cells, less than about 55% non-neuronal cells, less than about 50% non-neuronal cells, less than about 45% non-neuronal cells, less than about 40% non-neuronal cells, less than about 35% non-neuronal cells, less than about 30% non-neuronal cells, less than about 25% non-neuronal cells, less than about 20% non-neuronal cells, less than about 15% non-neuronal cells, less than about 12% non-n
  • the invention also relates to compositions containing a selection of some of the following chemical agents: at least one BMP type I receptor ALK2/3 inhibitor, at least one TGF-beta inhibitor, at least one WNT inhibitor, at least one neuronal differentiation enhancer, at least one SMO agonist, at least one retinoic acid receptor ⁇ agonist, at least one DNA methyltransferase inhibitor, at least one histone demethylase inhibitor, and/or at least one autophagy regulator.
  • the composition can contain at least two of the agents, or at least three of the agents, or at least four of the agents, or at least five of the agents, or at least six of the agents, or at least seven of the agents, or at least eight of the agents.
  • the compositions can also contain reprogrammed cells.
  • compositions of the invention can be pharmaceutical compositions.
  • the compositions can include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • the composition is a cell reprogramming composition.
  • compositions can contain any of the agent(s) or compound(s) described herein in an amount sufficient to reprogram a cell into a neuronal cell type.
  • the compositions can contain any of the agent(s) or compound(s) described herein in an amount sufficient to induce a cell to express Sox2, Nestin, Pax6, Sox2, Ascl1, Olig2, or Tuj1, and/or in an amount sufficient to induce a cell to express Tau, and/or in an amount sufficient to induce a cell to express NeuN, and/or in an amount sufficient to induce a cell to express MAP2, and/or in an amount sufficient to induce a cell to express Synapsin.
  • the cell contacted or treated by the compositions can be any of the starting cells described herein.
  • the cell can be a non-neuronal cell and/or a differentiated cell.
  • the therapeutic compositions are administered in a “therapeutically effective amount.”
  • a therapeutically effective amount is an amount sufficient to obtain the desired physiological effect, e.g., treatment of a condition, disorder, disease and the like or reduction in symptoms of the condition, disorder, disease and the like.
  • the therapeutic agents can be administered to treat a condition, disorder, or disease such Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), multiple sclerosis, hereditary spastic paraplegia (HSP), primary lateral sclerosis, Huntington's disease, progressive pseudobulbar palsy, progressive muscular atrophy, progressive bulbar palsy, post-polio syndrome, stroke, head trauma, spinal cord injury, and the like.
  • ALS amyotrophic lateral sclerosis
  • HSP hereditary spastic paraplegia
  • the composition can be formulated in single or divided dosages.
  • at least one BMP type I receptor ALK2/3 inhibitor, at least one TGF-beta inhibitor, at least one WNT inhibitor, at least one neuronal differentiation enhancer, at least one SMO agonist, at least one retinoic acid receptor ⁇ agonist, at least one DNA methyltransferase inhibitor, at least one histone demethylase inhibitor, and/or at least one autophagy regulator can be present in the composition in amounts specified above or in dosages of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages may provide beneficial results.
  • the amount administered will vary depending on various factors including, but not limited to the combination of compounds chosen for administration, the disease, the weight, the physical condition, the health, and the age of the mammal. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.
  • Reprogrammed cells can be included in the compositions in varying amounts depending upon the disease or injury to be treated.
  • the compositions can be prepared in liquid form for local or systemic administration containing about 10 3 to about 10 12 reprogrammed cells, or about 10 4 to about 10 10 reprogrammed cells, or about 10 5 to about 10 8 reprogrammed cells.
  • One or more of the following types of compounds can also be present in the composition with the cells: at least one BMP type I receptor ALK2/3 inhibitor, at least one TGF-beta inhibitor, at least one WNT inhibitor, at least one neuronal differentiation enhancer, at least one SMO agonist, at least one retinoic acid receptor ⁇ agonist, at least one DNA methyltransferase inhibitor, at least one histone demethylase inhibitor, and/or at least one autophagy regulator.
  • Administration of the composition, or contacting cell(s) with the composition may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is in response to traumatic injury or for more sustained therapeutic purposes, and other factors known to skilled practitioners.
  • the administration or contacting of the compounds and compositions of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • the compounds are synthesized and/or the cells are generated, and the components are purified as necessary or desired.
  • the compounds, cells, and/or other agents can be suspended in a pharmaceutically acceptable carrier. If the composition contains only compounds, without cells, the composition can be lyophilized. These compounds and cells can be adjusted to an appropriate concentration, and optionally combined with other agents.
  • the absolute weight of a given compound and/or other agent included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least one compound can be administered.
  • the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.
  • Daily doses of the compounds can vary as well. Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day.
  • a pharmaceutical composition may be formulated with the appropriate ratio of each compound in a single unit dosage form for administration with or without cells.
  • Cells can be separately provided and either mixed with a liquid solution of the compound composition, or administered separately.
  • the compounds can also be formulated for sustained release (for example, using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091).
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • One or more suitable unit dosage forms containing the compounds and/or the reprogrammed cells can be administered by a variety of routes including parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), intracranial, intraspinal, oral, rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes.
  • parenteral including subcutaneous, intravenous, intramuscular and intraperitoneal
  • compositions of the invention may be prepared in many forms that include aqueous solutions, suspensions, tablets, hard or soft gelatin capsules, and liposomes and other slow-release formulations, such as shaped polymeric gels.
  • administration of cells often involves parenteral or local administration in an aqueous solution.
  • compositions containing cells and/or compounds can be administered in a device, scaffold, or as a sustained release formulation.
  • compositions containing only compounds can be administered in an oral dosage form
  • compositions containing cells are administered locally or systemically as non-oral formulations.
  • those compositions can be formulated as an oral dosage form so that the compounds are released into the stomach for quick absorption or in the intestine after passing through the stomach.
  • Different types of formulating procedures are described in U.S. Pat. No. 6,306,434 and in the references contained therein.
  • Liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, dry powders for constitution with water or other suitable vehicles before use.
  • Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
  • Compounds and/or cells can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative.
  • the pharmaceutical compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Suitable carriers include saline solution, phosphate buffered saline, and other materials commonly used in the art.
  • compositions can also contain other ingredients such as agents useful for treatment of neuronal diseases and injuries, such as, for example, riluzole, ceftriaxone, lithium, xaliproden, pioglitazone, pyridostigmine, seligiline, RNA interference (RNAi) nucleic acids for reducing ALS susceptibility, Alzheimer's symptoms, or for reducing expression of mutated genes (e.g., RNAi of mutant SOD1 genes, or RNAi for any of the mutant NFH, dynactin, vesicular binding protein or ALSIN genes), neurotrophic factors (e.g., IGF-1, EPO, CTNF, BDNF, VEGF), anti-oxidative agents such as HIF-loc, amino acids, creatine, and other agents or stem cells, e.g, for the treatment of motor neuron diseases.
  • agents useful for treatment of neuronal diseases and injuries such as, for example, riluzole, ceftriaxone, lithium, x
  • compositions of the invention may also be used in conjunction with other forms of therapy.
  • Supplementary factors can be included in the compositions and/or in a cell culture media containing any of the compositions, compounds or agents described herein.
  • supplementary factors include bone morphogenic protein (BMP)-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, brain derived neurotrophic factor, ciliary neutrophic factor, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2 ⁇ , cytokine-induced neutrophil chemotactic factor 2 ⁇ , ⁇ endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor (
  • cytokines can be included such as interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN), IFN- ⁇ , tumor necrosis factor (TNF), TNF1, TNF2, TNF- ⁇ , macrophage colony stimulating factor (M-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), megakaryocyte colony stimulating factor (Meg-CSF)-thrombopoietin, stem cell factor, and erythropoietin.
  • Chemokines can also be included such as IP-10 and Stromal Cell-Derived Factor 1 ⁇ .
  • Exemplary hormones contemplated for inclusion in the compositions and/or cell culture media described herein can include, but are not limited to, steroid hormones and peptide hormones, such as insulin, somatostatin, growth hormone, hydrocortisone, dexamethasone, 3,3′,5-Triiodo-L-thyronine, and L-Thyroxine.
  • steroid hormones and peptide hormones such as insulin, somatostatin, growth hormone, hydrocortisone, dexamethasone, 3,3′,5-Triiodo-L-thyronine, and L-Thyroxine.
  • kits are described herein that include any of the compositions, compounds and/or agents described herein.
  • the compounds and/or agents described herein can be packaged separately into discrete vials, bottles or other containers.
  • any of the compounds and/or agents described herein can be packaged together as a single composition, or as two or more compositions that can be used together or separately.
  • the compounds and/or agents described herein can be packaged in appropriate ratios and/or amounts to facilitate conversion of selected cells across differentiation boundaries to form neuronal cells.
  • kits for culture of cells in vitro that can include any of the compositions, compounds and/or agents described herein, as well as instructions for using those compositions, compounds and/or agents.
  • Some kits can include a cell culture medium or a variety of cell culture media that includes any of the compositions, compounds and/or agents described herein.
  • the kits can include one or more sterile cell collection devices such as a swab, skin scrapping device, a needle, a syringe, and/or a scalpel.
  • the kits can also include antibodies for detection of neuronal cell markers such as antibodies against Sox2, Pax6, Tuj1, Tau, NeuN, MAP2, Ascl1, Olig2, Synapsin, or any combination thereof.
  • the antibodies can be labeled so that a detectable signal can be observed when the antibodies form a complex with the neuronal cell marker(s).
  • the instructions can include guidance for culturing cells for a time and under conditions sufficient to convert a selected cell across differentiation boundaries and into the neuronal lineage.
  • the instructions can describe amounts of the compositions, compounds and/or agents described herein to add to cell culture media, times sufficient to convert cells to the neuronal lineage, maintenance of appropriate cell densities for optimal conversion, and the like.
  • the instructions can describe procedures for rehydration or dilution of the compositions, compounds and/or agents described herein.
  • the instructions can describe how to add other compounds and/agents.
  • the instructions can also describe how to convert the selected cells to neuronal progenitor cells or to mature neuronal cells.
  • the instructions can also describe procedures for detecting neuronal cell markers by use of the antibodies against those markers so that the extent of conversion and/or differentiation can be assessed.
  • kits are also described herein that includes any of the compositions, compounds and/or agents described herein for therapeutic treatment of a subject.
  • the kit can include the M9 cocktail.
  • the kit can include any of the compositions, compounds and/or agents described herein, as well as instructions for administering those compositions, compounds and/or agents. Such instructions can provide the information described throughout this application.
  • the kit can also include cells.
  • the kit can include chemically induced neuronal cells that have been treated by the methods described herein and that are ready for administration.
  • the cells, compositions and/or compounds can be provided within any of the kits in a delivery device.
  • a delivery device can be separately included in the kit(s), and the instructions can describe how to assemble the delivery device prior to administration to a subject.
  • the delivery device can provide a scaffold for cell growth and/or a matrix for controlled release of any of the compositions, compounds or agents described herein.
  • kits can also include syringes, catheters, scalpels, sterile containers for sample or cell collection, diluents, pharmaceutically acceptable carriers, and the like.
  • kits can provide other factors such as any of the supplementary factors described herein for the compositions in the preceding section.
  • neuronal cell refers to a cell of a neuronal lineage.
  • Examples of neuronal cells include, but are not limited to, neurons, astrocytes, oligodendrocytes, and neural precursor cells.
  • mature neuron refers to a differentiated neuron.
  • a neuron is said to be a mature neuron if it expresses one or more markers of mature neurons, e.g., microtubule-associated protein 2 (MAP2) and Neuronal Nuclei (NeuN).
  • MAP2 microtubule-associated protein 2
  • NeuN Neuronal Nuclei
  • a neuron refers to a differentiated neuron that is able to send or receive electrical signals.
  • a neuron is said to be a functional neuron if it exhibits electrophysiological properties (e.g., if the neuron produces excitatory postsynaptic currents, which are indicative of functional synapses, and/or produces whole-cell currents and/or neurotransmitter receptor-mediated currents) and/or if it expresses one or more markers of functional neurons, e.g., Synapsin, vesicular GABA transporter (VGAT), vesicular glutamate transporter (VGLUT), and gamma-aminobutyric acid (GABA).
  • VGAT vesicular GABA transporter
  • VGLUT vesicular glutamate transporter
  • GABA gamma-aminobutyric acid
  • a “differentiated non-neuronal cell” may refer to a cell that is not able to differentiate into all cell types of an adult organism (i.e., is not a pluripotent cell), and which is of a cellular lineage other than a neuronal lineage (e.g., a hematopoietic lineage or a connective tissue lineage).
  • Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In particular embodiments, a less potent cell is considered “differentiated” in reference to a more potent cell.
  • a cell that differentiates into a mesodermal, ectodermal or endodermal lineage defines a cell that becomes committed to a specific mesodermal, ectodermal or endodermal lineage, respectively.
  • Examples of cells that differentiate into a mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, chondrogenic, cardiogenic, dermatogenic, hematopoietic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or stromal.
  • Examples of cells that differentiate into ectodermal lineage include, but are not limited to epidermal cells, neurogenic cells, and neurogliagenic cells.
  • Examples of cells that differentiate into endodermal lineage include, but are not limited to pleurigenic cells, and hepatogenic cells, that give rise to the lining of the intestine, and cells that give rise to pancreogenic and splanchogenic cells.
  • a “somatic cell” is a cell forming the body of an organism. Somatic cells include cells making up organs, skin, blood, bones and connective tissue in an organism, but not germ cells.
  • Cells can be from, e.g., human or non-human mammals.
  • exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, and non-human primates.
  • a cell is from an adult human or non-human mammal.
  • a cell is from a neonatal human, an adult human, or non-human mammal.
  • totipotent means the ability of a cell to form all cell lineages of an organism. For example, in mammals, only the zygote and the first cleavage stage blastomeres are totipotent.
  • embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm.
  • multipotent refers to the ability of an adult stem cell to form multiple cell types of one lineage.
  • hematopoietic stem cells are capable of forming all cells of the blood cell lineage, e.g., lymphoid and myeloid cells.
  • oligopotent refers to the ability of an adult stem cell to differentiate into only a few different cell types.
  • lymphoid or myeloid stem cells are capable of forming cells of either the lymphoid or myeloid lineages, respectively.
  • spermatogonial stem cells are only capable of forming sperm cells.
  • direct reprogramming or “transdifferentiation” refers to the generation of a cell of a certain lineage (e.g., a neuronal cell) from a different type of cell (e.g., a fibroblast cell) without an intermediate process of de-differentiating the cell into a cell exhibiting pluripotent stem cell characteristics.
  • a cell of a certain lineage e.g., a neuronal cell
  • a different type of cell e.g., a fibroblast cell
  • the terms “subject” or “patient” refers to any animal, such as a domesticated animal, a zoo animal, or a human.
  • the “subject” or “patient” can be a mammal like a dog, cat, bird, livestock, zoo animal, endangered species animal, or a human.
  • Specific examples of“subjects” and “patients” include, but are not limited to, individuals with a neuronal disease or disorder, and individuals with neuronal disorder-related characteristics or symptoms.
  • neuroneuronal disorder refers to disorders of the nerves of the brain, spinal cord, or peripheral nervous system, including, but not limited to neurodegenerative/neurological disorders such as progressive deterioration of the nerves in the spinal cord and/or brain.
  • neuron disorders include, but are not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), multiple sclerosis, hereditary spastic paraplegia (HSP), primary lateral sclerosis, Huntington's disease, progressive pseudobulbar palsy, progressive muscular atrophy, progressive bulbar palsy, post-polio syndrome, stroke, head trauma, spinal cord injury, and the like.
  • symptoms of neuron disorder and “characteristics of neuron disorder” include, but are not limited to, lower extremity weakness, bladder disturbance, impaired position sense in the legs, and neurologic deficits, such as a decrease in the function of the brain, spinal cord, muscles, and/or nerves, for example, inability to speak, decreased sensation, loss of balance, weakness, cognitive dysfunction, visual changes, abnormal reflexes, and problems walking.
  • This Example describes some of the materials and methods employed in the devilment of the invention.
  • mice were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco. Homozygous Tau-EGFP knock-in mice were purchased from the 6 Jackson Laboratory (Mapt tm1(EGFP)klt , stock number 004779); Fsp1-Cre mice were from the Jackson Laboratory (BALB/c-Tg (S100a4-cre) 1Egn/YunkJ) and were kindly provided by Dr. Deepak Srivastava at the Gladstone Institute of Cardiovascular Disease; ROSA26-tdTomato mice were from the Jackson Laboratory (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze) and were kindly provided by Dr. Ken Nakamura at Gladstone Institute of Neurological Disease.
  • Mapt tm1(EGFP)klt stock number 004779
  • Fsp1-Cre mice were from the Jackson Laboratory (BALB/c-Tg (S100a4-cre) 1Egn/YunkJ) and were kindly provided
  • the chimera mouse embryos with Nanog-GFP were generated as described by Wernig et al. ( Nat Biotechnol 26: 916-924 (2008)), with some modification.
  • the NGFP iPSCs cells were injected into blastocyst and transferred into recipient CD1 females.
  • the genotype of all mouse lines was validated before breeding.
  • the Tau-GFP mice were maintained as a homozygous line.
  • the Fsp1-Cre/ROSA26 tdTomato mice were obtained by crossing Fsp 1-Cre mice with ROASA26-tdTomato mice.
  • Mouse embryonic fibroblasts (MEF) and mouse tail-tip fibroblast (TTF) preparations were obtained using methods similar to those described by Kim et al. ( Proc Natl Acad Sci USA 108: 7838-7843 (2011)), with some modifications. Briefly, the mouse embryonic fibroblasts and mouse tail-tip fibroblasts of the desired genotype were obtained from E13.5 mouse embryos, or from 3-week postnatal mouse tail-tip tissues, respectively. To obtain fibroblasts, the E13.5 embryos or tail-tip tissues were sliced into small pieces, these small pieces were trypsinized, and the cells were plated in MEF medium. All fibroblasts were expanded for two passages before being used for experiments. To prepare the tdMEF or tdTTF, the resulting fibroblasts were sorted for tdTomato+/p75 ⁇ by FACS.
  • All fibroblasts were cultured in MEF medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, and 2 mM Glutamax) at 37° C. Medium was refreshed every other day. All neural stem cells, including primary neural stem cells and chemical-induced neural stem cells, were cultured in neural stem cell medium (NSC medium, 50% Neural basal, 50% DMEM/F12/Glutamax, 1 ⁇ N2, 1 ⁇ B27 without vitamin A, 0.075% BSA, 0.1 mM nonessential amino acids, 20 ng/ml bFGF, 20 ng/ml EGF). Medium was refreshed daily.
  • NSC medium 50% Neural basal, 50% DMEM/F12/Glutamax, 1 ⁇ N2, 1 ⁇ B27 without vitamin A, 0.075% BSA, 0.1 mM nonessential amino acids, 20 ng/ml bFGF, 20 ng/ml EGF.
  • NSC medium 50% Neural basal
  • MEFs were seeded into Matrigel (Corning, 1:40 dilution)-coated 24-well plates at 15,000 per well, or 1-1.5 million in 15-cm dishes in MEF medium (Dulbecco's modified Eagle's medium supplemented with 10% FBS, 0.1 mM nonessential amino acids, and 2 mM Glutamax) for the first 24 hours.
  • MEF medium Dulbecco's modified Eagle's medium supplemented with 10% FBS, 0.1 mM nonessential amino acids, and 2 mM Glutamax
  • MEF cells were washed twice with 1 ⁇ PBS before being cultured into medium containing the M9 mixture of compounds (50% Neural basal, 50% DMEM/F12/Glutamax, 1 ⁇ N2, 1 ⁇ B27 without vitamin A, 0.075% BSA, 0.1 mM nonessential amino acids, where the M9 mixture was: CHIR99021 at 3 ⁇ M, LDN193189 at 100 nM, A83-01 at 0.5 ⁇ M, Hh-Ag1.5 at 0.5 ⁇ M, retinoic acid at 1 ⁇ M, SMER28 at 10 ⁇ M, RG108 at 10 ⁇ M, Parnate 2 ⁇ M, bFGF 10 ng/ml) at 5% O 2 and 5% CO 2 incubator at 37° C. M9 medium was refreshed every other day. After a 10-day induction, cells were cultured in neural stem cells medium (NSC medium). Afterwards, the cells could be repeatedly propagated in NSC medium.
  • NSC medium neural stem cells medium
  • Alkaline phosphatase activity was evaluated according to the Alkaline Phosphatase Detection Kit (Sigma).
  • ciNSLCs were seeded onto the laminine/poly-orthinine-coated glass cover slips in 24-well plates containing NSC medium for first 24 hours. After 24 hours, for neuron differentiation, the medium was switched to neuron differentiation medium (NSC medium without bFGF and EGF, with addition of 200 ⁇ M ascorbic acid, 2 ⁇ M db-cAMP, 25 ng/ml BDNF, 25 ng/ml NT3 and 50 ng/ml GDNF). Half of the medium was changed every 2 days. Specific neuron markers were analyzed by day 10 to day 20 after differentiation using methods described by 2, 4-6 Kim et al.
  • the differentiation medium contained 10 ng/ml PDGF-AA, 10 ng/ml bFGF, 10 ng/nl SHH, and 40 ng/ml T3, and the cells were cultured for 8-12 days (Najm et al., Nat. Biotechnol. 31: 426-433 (2013)).
  • BMP4 50 ng/ml was applied into differentiation medium for 8-12 days (Kim et al., Proc Natl Acad Sci USA 108: 7838-7843 (2011); Najm et al., Nat. Biotechnol. 31: 426-433 (2013); Ring et al., Cell Stem Cell 11, 100-109 (2012); Zhu et al., Cell Res 24: 126-129 (2014)).
  • Monolayer cultured ciNSLCs were trypsinized to generate single cells and incubated at a density of 10,000 cells per ml in neural stem cell medium.
  • Cells were cultured within ultralow attachment 6-well plates. The medium was changed every day by spinning down the cells at 1,000 rpm for 5 min and re-suspending the cells into fresh neural stem cell medium (Ring et al., Cell Stem Cell 11, 100-109 (2012)).
  • the artificial cerebral spinal fluid was bubbled with 95% O 2 and 5% CO 2 throughout the recordings.
  • Data were gathered through a MultiClamp 700B amplifier (Axon Instruments), filtered at 2 kHz, and digitized at 10 kHz.
  • Offline analysis was carried out in Igor Pro (Wavemetrics). Action potentials were recorded under the current-clamp whole-cell configuration.
  • the pipette solution for current-clamp experiments contained (in mM): K-gluconate at 123 mM, KCl at 10 mM, MgCl 2 at 1 mM, HEPES at 10 mM, EGTA at 1 mM, CaCl 2 at 0.1 mM, K 2 ATP at 1 mM, Na 4 GTP at 0.2 mM, and glucose at 4 mM, with the pH adjusted to 7.2 using KOH.
  • Membrane potential was held around ⁇ 70 mV and step currents of ⁇ 20 to 50 pA were introduced at 10-pA intervals.
  • Whole-cell currents were recorded at a holding potential of ⁇ 70 mV with voltage steps ranging from ⁇ 70 mV to +30 mV that were delivered at 20-mV increments.
  • Spontaneous postsynaptic currents were recorded in the whole-cell voltage-clamp mode.
  • the whole-cell pipette solution for synaptic current recordings contained (in mM): CsCl at 135 mM, HEPES at 10 mM, EGTA at 1 mM, Mg-ATP at 4 mM, Na 4 GTP at 0.4 mM, and QX-314 at 10 mM, pH 7.4.
  • RNA-seq libraries were prepared with ovation RNA-seq system v2 kit (NuGEN). In this method, the total RNA (50 ng) is reverse-transcribed to synthesize the first-strand cDNA with a combination of random hexamers and a poly-T chimeric primer. The RNA template is then partially degraded by heating and the second-strand cDNA is synthesized using DNA polymerase. The double-stranded DNA is then amplified using single primer isothermal amplification (SPIA).
  • SPIA single primer isothermal amplification
  • SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′-end of the double-stranded DNA, after which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′-end of the primer by displacement of the existing forward strand. Random hexamers are then used to amplify the second-strand cDNA linearly. Finally, libraries from the SPIA amplified cDNA were made using an Ultralow V2 library kit (NuGEN). The RNA-seq libraries were analyzed by Bioanalyzer and quantified by QPCR (KAPA). Three RNA-seq libraries were pooled per lane of paired-end 100 bp sequencing on HiSeq 2500 instrument (Illumina).
  • Chromatin immunoprecipitation was performed according to the instructions of an EZ-ChiP kit. In brief, 1-10 million cells were fixed with 1% paraformaldehyde at room temperature for 10 min. The fixation reaction was then quenched by incubation in 0.125 M glycine for 5 min at room temperature. After twice washing with pre-chilled 1 ⁇ PBS, the cells were suspended into 1 ml of chilled nuclei buffer (5 mM PIPES (pH 8), 85 mM KCl, 1% NP-40, with freshly added Protease Inhibitor Cocktail II, Millipore Cat#20-283).
  • nuclei buffer 5 mM PIPES (pH 8), 85 mM KCl, 1% NP-40
  • Cell nuclei were released by homogenizing the tissue in a Dounce Homogenizer (Wheaton, 1 ml, tight, Cat#357538) for 20 strokes and centrifugation at 430 rcf for 5 min. Nuclei were resuspended into cell lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris pH 8.1). The released chromatin was sheared by Covaris S2 sonicator into fragment sizes of about 200-500 bp. For each sample, 20 ⁇ l of solubilized chromatin was used as input DNA to normalize sequencing results and the remaining chromatin was pre-cleaned with 50 ⁇ l Protein-G beads at 4° C.
  • ciNSLCs labeled with tdTomato were microinjected into the cortices of P4-P5 pups with a beveled micropipette and a Nanoject by Drummond on a stereotaxic frame. Two injections of about 20,000 cells per injection site were made in the right hemisphere between bregma and lambda at a 250 angle towards the midline to a depth of about 0.30 mm. The pups were anesthetized on ice and held stable in a head mold during the procedure.
  • mice were perfused with saline and 4% paraformaldehyde.
  • the brains were fixed for 24-48 h, washed with PBS, cryopreserved in 30% sucrose, and sectioned using a sliding microtome into 30- ⁇ m coronal sections, and immunostained with the following primary antibodies: monoclonal mouse anti-NeuN (1:2000; Millipore MAB377), polyclonal rabbit anti-GFAP (1:500; Dako), and polyclonal rabbit anti-Olig2 (1:500; Millipore AB9610). Alexa-488 or -647 (1:1000; Life Technology) secondary antibodies were used for immunofluorescence. Sections were mounted in Prolong Gold anti-fade reagent with DAPI (Life Technology). Images were taken on Zeiss LSM 700 confocal laser scanning microscope.
  • the fibroblasts with desired genotype were culture in MEF medium until 80% confluence.
  • the cells were washed with 1 ⁇ PBS and de-attached by Accutase treatment at 37° C. for 5 min. After harvesting, the cells were passed through 70- ⁇ m filter, washed twice with and resuspended into pre-cold F ACS buffer (1 ⁇ PBS, 1.5% FBS, 0.5% BSA).
  • the cells were incubated with either FITC-conjugated p75 antibody (abeam) or isotype control (BD) with suggested concentrations on ice for 45 min, followed by six washes with FACS buffer. Cells were then resuspended into FACS buffer and sorted by BD FACSAris II.
  • small molecule inhibitors including 0.5 ⁇ M PD0325901, 10 ⁇ M SP 600125, 5 ⁇ M SB203580, 5 ⁇ M LY294002, or 2 ⁇ M LDE 225, was added in the freshly made M9 culture medium.
  • DMSO was used in basal medium as control. The medium was changed every 2 days, and the Sox2+/Nestin+ cells were analyzed by immunostaining by day 10 after treatment.
  • the shRNA constructs targeting Elk1 or Gli2 were purchased from Origene (cat# TR500594 for mouse Elkl shRNA constructs in retroviral untagged vector pRS, and TR517874 for mouse Gli2 shRNA constructs in retroviral untagged vector pRS).
  • the virus was packed and collected according to manual.
  • the tdMEFs treated with M9 were infected by the retrovirus at the indicated time points twice, and immunostaining was performed at day 10 after chemical treatment to analyze the Sox2+/Nestin+ cells by InCell Analyzer 2000, and quantified by In Cell Developer.
  • the overexpression plasmid carrying Elkl (addgene 27156) or Gli2 (addgene 37671) was purchased from Addgene.
  • the plasmid was transfected into tdMEF cells with Neon transfection system (Invitrogen) at 1450 V, 20 ms, and two pulses.
  • This Example describes how fibroblasts can be identified and traced using a genetic lineage tracing strategy.
  • Primary MEFs contain heterogeneous populations of non-fibroblast precursor cells.
  • the origin/identity of the starting fibroblasts can unambiguously be defined using fibroblast-specific protein 1 (Fsp1, also known as S100a4), which have been validated as specific markers tor fibroblasts, and Fsp1-Cre, which has been used to lineage-trace fibroblast origin (Qian et al., Nature 485, 593-598 (2012)).
  • Fsp1 also known as S100a4
  • tdMEF neural crest progenitors
  • the tdMEF cells were negative for GFAP and NG2, markers for astrocytes/radial glia cells and oligodendrocyte progenitor cells, respectively, that have the potential to become NSCs ( FIG. 1I ).
  • the absence of several neural gene transcripts was also confirmed, including Pax6, Sox2, Hes5, Aashl, Gfap and Olig2, in tdMEF by RT-PCR ( FIG. 1H ).
  • the tdMEF cells were cultured under extended neuronal differentiation condition. No Tuj1-positive neurons were detected from 105 starting tdMEFs after a 15-day differentiation (data not shown). Consequently, these tdMEF cells were established as suitable starting cells for chemical-based neural reprogramming studies.
  • This Example describes experiments designed to define small molecules that target and modulate epigenetic functions and neuro-developmental signaling to induce a neural transcriptional program in fibroblasts.
  • LDN193189 LDN, an inhibitor of BMP type I receptor ALK2/3) and A83-01 (A83, an inhibitor of TGF-type I receptor ALK4/5/7), which inhibit mesoderm and endoderm specification
  • A83 an inhibitor of TGF-type I receptor ALK4/5/7
  • CHIR99021 CHIR99021
  • bFGF basic fibroblasts growth factor
  • tdMEFs were plated into neural reprogramming basal medium at 15,000 cells per well in 24-well plate, and after an overnight culture, individual small molecules from a focused chemical collection, including epigenetic modifiers, metabolism regulators, and signaling modulators, were added.
  • individual small molecules from a focused chemical collection including epigenetic modifiers, metabolism regulators, and signaling modulators, were added.
  • Ten days post-treatment cells were fixed, immune-stained, and analyzed for co-expression of Sox2 and Nestin, two typical NSC markers ( FIG. 1A-1B ).
  • Hh-Ag 1.5 Hh, a potent Smo agonist
  • RA retinoic acid
  • RG108 RG, a DNA methyltransferase inhibitor
  • Parnate Par, a histone demethylase inhibitor
  • SMER28 SR, an autophagy modulator
  • These three small molecules were included in the neural induction cocktail, resulting in a nine-molecule combination of CHIR, LDN, A83, RA, Hh, RG, Par, SR and bFGF shown in Table 3 below that robustly induced Sox2/Nestin double-positive cells at percentage of about 25.62% to 30.04% ( FIG. 1F ).
  • each component was individually removed from the cocktail and the neural reprogramming efficiency was evaluated ( FIG. 2G ). Removal of any of the nine molecules significantly compromised the reprogramming efficiency ( FIG. 2G ). This combination of nine molecules (named as M9 hereafter) was used in subsequent assays after optimization of dosage.
  • tdTomato+/Sox2+/Nestin+ cells were isolated and serially passaged in conventional NSC expansion medium containing bFGF and epidermal growth factor (EGF). Notably, they could robustly proliferate and form neurospheres over ten passages ( FIG. 3G-3H ), maintained typical morphology of NSCs, and expressed multiple NSC markers, including Sox2, Nestin, Olig2, and N-cadherin, by quantitative PCR and by immunostaining ( FIG. 1C-1D ). The expression levels of these genes were comparable to those in primary NSCs and a NSC line SCR029 (Ring et al., Cell Stem Cell 11, 100-109 (2012).
  • ciNSLC fibroblast-originated, highly proliferative, and self-renewable Sox2 and Nestin double-positive cells are thereafter referred to as chemical induced neural stem cell-like cells (ciNSLCs).
  • Example 4 ciNSLC are Tripotent In Vitro and In Vivo
  • FIG. 4H To characterize their differentiation potential, expanded ciNSLCs were first differentiated in vitro under the typical neuronal differentiation condition (Kim et al. Proc Natl Acad Sci USA 108: 7838-7843 (2011). The tdTomato-positive cells with immature neuronal morphology were observed as early as day 3 ( FIG. 4H ). Upon further differentiation, mature neurons with elaborate processes that expressed a panel of typical neuronal markers, including P-III tubulin, Map2, NeuN, and Synapsin I, were readily detected at day 10 onwards ( FIG. 2A , and FIG. 4I-L ). As illustrated in FIG.
  • ciNSLCs can differentiate into neurons that express Tuj1 and Map2, as well as into oligodendrocytes that express O4, and astrocytes that express GFAP. Subsequent analysis revealed that the majority of the neurons were vGlut1-positive, indicating an excitatory glutamatergic phenotype, while the percentage of GABA-positive inhibitory neurons was about 8.6 ⁇ 14.7% ( FIG. 2B and FIG. 4J-4K ). To more rigorously characterize these differentiated neurons, their electrophysiological properties were examined. As expected, the neurons generated repetitive trains of action potentials elicited by depolarizing the membrane in the current-clamp mode ( FIG. 2D and FIG. 5A ).
  • ciNSLC can differentiate into O4-positive oligodendrocytes with typical multi-branching morphology with the treatment of PDGF-AA, Shh, bFGF and T3, and characteristic GFAP-positive astrocytes when treated with BMP422 ( FIG. 2A ).
  • the expanded ciNSLCs are tripotent and can robustly generate neurons, astrocytes, and oligodendrocytes under appropriate differentiation cues in vitro.
  • ciNSLCs were micro-injected into the cortex of postnatal mouse pups, and their survival and differentiation were evaluated 2-4 weeks after injection. Remarkably, tdTomato-marked ciNSLCs survived in the mouse cortex and efficiently differentiated into NeuN-positive mature neuronal cells, Olig2-positive oligodendrocytes, and GF AP-positive astrocytes ( FIG. 2F ). No tumors were found in the animals at fourweeks post-injection (data not shown). The ciNSLC in vivo differentiation potential was validated by two independent lines. Taken together, these data show that the expanded ciNSLCs resemble primary NSCs by marker expression and in by their abilities to self-renew and differentiate in vitro and in vivo.
  • Fsp1-Cre/ROSA26 tdTomato MEFs were sorted by FACS into p75 ⁇ /tdTomato+ and p75 ⁇ /tdTomato ⁇ populations, and the cells were treated with the M9 condition.
  • Sox2 was initially examined by immunostaining ( FIG. 7A ). Induction of endogenous Sox2 protein expression in individual cells could be observed as early as day 4 after M9 treatment, at which time 1.29% of the cells exhibited Sox2 expression, and the number of Sox2-positive cells increased significantly by around day 8 in those that had undergone mesenchymal-to-epithelial transition and formed small clusters/colonies (7.32%). By day 12, robust expression of Sox2 was detected in cells within the large and flat colonies (30.04%).
  • transcriptome analysis of the ciNSLC induction process was performed by RNA-seq ( FIG. 3B ).
  • Principle components analysis revealed that all intermediates, upon M9 treatment, were distinct from starting tdMEFs (D0), indicating M9 treatment led to dramatic transcriptional changes ( FIG. 3C ).
  • Paired scatter plots and hierarchical cluster analysis showed that ciNSLCs and control NSCs were similar in transcriptional profile, but clearly differed from tdMEF ( FIG. 1E , FIG. 3D ), demonstrating a NSC identity.
  • genes differentially expressed in both ciNSLCs and control NSCs fell into the categories of neural development related biological processes, confirmed an acquisition of NSCs identity ( FIG. 3B and FIG. 8A ), whereas genes that were down-regulated from day 4 onwards were involved in fibroblast function, such as extracellular matrix/structure organization and cell adhesion.
  • genes showing transient activation in reprogramming intermediates were determined to be involved in inflammatory responses/wound healing, as well as epithelium development, which is consistent with previous studies showing that the activation of innate immunity and the mesenchymal-to-epithelial transition process are important for efficient reprogramming (Li et al., Cell Stem Cell 7: 51-63 (2010); Lee et al., Cell 151, 547-558 (2012)).
  • fibroblast genes such as Thy1, S100a4, Periostin, and Snail
  • fibroblast genes such as Thy1, S100a4, Periostin, and Snail
  • a panel of NSC genes was gradually upregulated during the ciNSLC induction process.
  • FIG. 8B although there was a gradual acquisition of neural genes, the transcripts of other lineage-specific genes, such as Nanog and Oct4 (pluripotency), T and Mesp1 (mesoderm), and Sox17 and Foxa2 (endoderm) could not be detected.
  • the expression of a cohort of genes was analyzed, where the genes were under the GO terms of ectoderm development, mesoderm development, endoderm development, fibroblasts, and stem cell maintenance, at day 4 after M9 treatment. As shown in FIG. 3F , the observed transcriptional activation was predominantly towards ectoderm, rather than other lineages.
  • bFGF mitogen-activated protein kinase
  • PI3K phosphatidylinositol 3-kinase pathway
  • bFGF and Hg—Ag 1.5 may directly participate in transcriptional activation of endogenous master NSC genes, such as Sox2.
  • Elkl and Gli2 are the major direct transcription factors downstream of the bFGF pathway and the Hg—Ag 1.5 pathway, respectively.
  • Sox2 is expressed at high levels in neuroepithelial stem cells and persists in neural stem/progenitor cells throughout adulthood. Sox2 has at least two regulatory regions in addition to the core promoter, the Sox2 regulatory region 1 (SRR1) and the Sox2 regulatory region 2 (SRR2; FIG. 4C ), which drive strong expression in these cells.
  • SRR1 Sox2 regulatory region 1
  • SRR2 Sox2 regulatory region 2
  • M9 treatment significantly triggered the recruitment and binding of Elkl and Gli2 to the Sox2 promoter by as early as day 4 of M9 treatment ( FIG. 4C ), confirming that Elk1 and Gli2 participate in Sox2 gene activation.
  • This recruitment of Elk 1 and Gli2 to Sox2 promoter also coincided with the acquisition of active chromatin status ( FIG. 7C ) and the activation of neural program ( FIG. 3 ).
  • the foregoing experiments demonstrate that the M9 mixture of small molecules enables efficient and specific reprogramming of mouse fibroblasts into ciNSLCs.
  • the ciNSLCs resemble primary NSCs in several key aspects, including their molecular profiles, their ability to self-renew long-term, and their ability to differentiate into functional neurons, astrocytes, and oligodendrocytes in vitro and in vivo.

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US9909104B2 (en) * 2013-07-26 2018-03-06 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Generating definitive endoderm and pancreatic progenitor cells
WO2015041809A2 (fr) * 2013-08-26 2015-03-26 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Reprogrammation cellulaire par petites molécules pour générer des cellules neuronales
US9512406B2 (en) * 2013-12-20 2016-12-06 The J. David Gladstone Institute, a testamentary trust established under the Will of J. David Gladstone Generating hepatocytes
US20190010451A1 (en) 2015-08-07 2019-01-10 The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Glads Chemical reprogramming to generate neuronal cells

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US20180094241A1 (en) * 2015-04-14 2018-04-05 Kyoto University Method for inducing cerebral cortex neurons
US10626368B2 (en) * 2015-04-14 2020-04-21 Kyoto University Method for inducing cerebral cortex neurons
US12104169B2 (en) 2015-08-07 2024-10-01 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Chemical reprogramming to generate neuronal cells
US11413286B2 (en) * 2016-06-22 2022-08-16 City Of Hope Producing astrocytes using small molecules

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US20220243173A1 (en) 2022-08-04
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EP3331988A4 (fr) 2019-01-16
EP3331988B1 (fr) 2023-09-20

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