WO2015131797A1 - Method for inducing the transdifferentiation of somatic cells into neural stem cells and application for same - Google Patents

Method for inducing the transdifferentiation of somatic cells into neural stem cells and application for same Download PDF

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WO2015131797A1
WO2015131797A1 PCT/CN2015/073549 CN2015073549W WO2015131797A1 WO 2015131797 A1 WO2015131797 A1 WO 2015131797A1 CN 2015073549 W CN2015073549 W CN 2015073549W WO 2015131797 A1 WO2015131797 A1 WO 2015131797A1
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neural stem
cells
stem cells
induced
cell
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裴钢
赵简
程林
胡文祥
裘斌龙
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中国科学院上海生命科学研究院
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    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5026—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell morphology
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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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

Definitions

  • the present invention belongs to the field of biotechnology and neurodevelopment, and in particular, to a method for inducing somatic cell transduction into neural stem cells and an application thereof.
  • Terminally differentiated cells are considered to be a class of cells with specific functions and phenotypes that lose further developmental potential.
  • early studies have found that the nuclei of terminally differentiated cells can be used to clone animals.
  • in vitro cell fusion can also lead to reprogramming of cell lineages.
  • epigenetic modifications during development are reversible.
  • a large number of recent studies have found that the combination of specific transcription factors can not only induce somatic cells to be differentiated into pluripotent stem cells by reprogramming, but also directly transdifferentiate into specific somatic cells of other lineages, thus providing a new source of cells for personalized treatment of patients. .
  • Neural stem cells are a kind of cells that can self-proliferate, renew and differentiate into different neuronal cells, and have great research and clinical application value.
  • methods for extracting neural stem cells from brain tissue and differentiating embryonic stem cells and induced pluripotent stem cells into neural stem cells have matured.
  • methods for inducing somatic cell transdifferentiation into neural stem cells by different factor combinations are also gradually improved; Transdifferentiation methods involve the involvement of foreign genes and have great clinical safety risks.
  • the present invention provides a method for inducing somatic cell transdifferentiation into neural stem cells in a hypoxic (especially normal physiological hypoxia) environment to be a neural stem cell.
  • HDACs histone deacetylase
  • GSK-3 glycogen synthase kinase
  • TGF-beta transforming growth factor beta
  • a small molecule compound composition wherein the small molecule compound consists of the following group Sub-composition:
  • HDACs histone deacetylase
  • GSK-3 glycogen synthase kinase
  • TGF-beta Transforming growth factor beta
  • composition of the first or second aspect for inducing somatic transdifferentiation into neural stem cells in a hypoxic environment.
  • the hypoxic environment comprises a normal physiological hypoxic environment.
  • the hypoxic environment is an environment having an oxygen concentration of 3-8%, preferably 4-6%.
  • the somatic cells include fibroblasts, epithelial cells.
  • the somatic cells are derived from a mammal, preferably a human, a rodent (mouse, rat).
  • the fibroblasts include mouse embryonic fibroblasts, mouse tail tip fibroblasts, and human dermal fibroblasts.
  • the epithelial cells are isolated from human urine.
  • a method for inducing somatic cell transdifferentiation into neural stem cells in vitro wherein the culture body is cultured under a hypoxic environment and a combination of the small molecule compound according to the first or second aspect of the present invention cell.
  • the culture conditions further include a neural stem cell culture medium.
  • the neural stem cell culture medium contains epidermal growth factor EGF, basic fibroblast growth factor bFGF, heparin, or a combination thereof.
  • the culture is cultured for at least 4 passages, preferably at least 5-8 passages, more preferably at least 10-15 passages.
  • the HDACs inhibitor in the small molecule compound combination comprises sodium valproate (VPA), sodium butyrate (NaB), or trichostatin A (TSA); and/or
  • the GSK-3 inhibitor comprises CHIR99021, lithium chloride (LiCl), or lithium carbonate (Li 2 CO 3 ); and/or
  • the TGF- ⁇ signaling pathway inhibitors include Repx, SB431542, or Tranilast.
  • the minimum effective concentration of each component in the small molecule compound combination is as follows:
  • HDACs inhibitor 0.2-1 mM, preferably 0.3-0.8 mM, more preferably 0.4-0.6 mM; NaB 0.2-1 mM, preferably 0.3-0.8 mM, more preferably 0.4-0.6 mM; TSA 5-20nM, 8-15nM, more Good place, 10-12nM;
  • GSK-3 inhibitor CHIR990211-5 ⁇ M, preferably 2-4 ⁇ M; LiCl 0.5-3 ⁇ M, preferably 1-2 ⁇ M; Li 2 CO 3 0.05-1 mM, preferably 0.1-0.8 mM, more preferably, 0.2-0.5 mM;
  • TGF- ⁇ inhibitor signaling pathway Repsox 0.2-3 ⁇ M, preferably 0.5-2 ⁇ M; SB4315420.2-3 ⁇ M, preferably 0.5-2 ⁇ M; Tranilast 10-50 ⁇ M, preferably 20-40 ⁇ M.
  • a neural stem cell prepared by the method of the fourth aspect of the invention.
  • the neural stem cell has one or more of the following characteristics:
  • Neural stem cells have differentiated pluripotency.
  • the neural stem cell-specific genes include Nestin, Sox2, Blpp, Pax6, and Ascl1.
  • the neural stem cell pluripotency genes include Nestin, Sox2, Blpp, and Pax6.
  • a use of the neural stem cell of the fifth aspect of the invention for the preparation of a pharmaceutical composition for preventing or treating a nervous system disease.
  • the neurological diseases include neurodegenerative diseases, neurological diseases caused by genetic mutations, and neurological diseases caused by brain trauma or cerebral hemorrhage.
  • the neurological condition comprises Alzheimer's disease, Parkinson's disease, or Huntington's disease.
  • composition comprising: the neural stem cell of the fifth aspect of the invention.
  • the composition comprises a pharmaceutical composition, a food composition, a health care composition.
  • FIG. 1 VCRP induces the formation of dense cell clones of mouse embryonic fibroblasts (MEFs) under normal physiological hypoxia.
  • Figure 1A shows the different oxygen concentrations (21%, 3% and 5%) after 15 days of VCRP treatment. Morphological changes in MEFs. 20,000 cells were seeded in 6-well plates and cultured for 24 hours at 21% oxygen concentration and replaced with KSR medium containing small molecule compound combination VCRP (0.5 mM VPA, 3 ⁇ M CHIR99021, 1 ⁇ M Repsox and 2 ⁇ M Parnate) for 5 The culture medium was changed once a day for 20 days; on the 10th day, only the densely treated cell clones began to appear in the drug-treated group under normal physiological hypoxia.
  • KSR medium containing small molecule compound combination VCRP 0.5 mM VPA, 3 ⁇ M CHIR99021, 1 ⁇ M Repsox and 2 ⁇ M Parnate
  • FIG. 1B shows that the expression of alkaline phosphatase (AP) in the dense cell clone of the VCR-treated group was significantly increased under normal physiological hypoxia.
  • AP alkaline phosphatase
  • the scale is 200 ⁇ m; all data are taken with mean ⁇ SEM; representative images are from at least three independent experiments.
  • Figure 2A shows the number of clones produced by different compound combinations induced by MEFs under normal physiological hypoxic conditions.
  • Figure 2B shows the addition of other compounds (1 ⁇ M OAC1(O), 7.5 ⁇ M Luteolin (L), 300 ng/mL poly I:C) based on VCR (0.5 mM VPA, 3 ⁇ M CHIR99021 and 1 ⁇ M Repsox) under normal physiological hypoxia.
  • I Number of clones induced by the induced cells (Day 15).
  • Figure 2C shows the detection of Sox2 expression levels of MEFs after 10 days of VCR treatment at different oxygen concentrations (21% and 5%).
  • Figure 2D shows the detection of Sox2 expression levels after treatment of cells with different compounds and their combinations for 10 days under normal physiological hypoxia. All data were taken with mean ⁇ SEM; representative images were from at least three independent experiments.
  • Figure 3 Compound combination VCR induces mouse embryonic fibroblasts to neural stem cells under physiologically normal physiological hypoxia conditions.
  • Figure 3A shows a dense clone of a compound-combined VCR that induces alkaline phosphatase AP-positive under physiologically normal physiological hypoxic conditions.
  • Mouse embryonic fibroblasts were treated with compound combination VCR (0.5 mM VPA, 3 ⁇ M CHIR99021 and 1 ⁇ M Repsox) under 21% (normal oxygen pressure) or 5% (normal physiological hypoxia) O2 culture conditions. Clones were counted 15 days after VCR treatment.
  • the bar graph represents the number of clones induced by the initial 200,000 cells.
  • Figure 3B shows the relative expression levels of pluripotency-related genes detected by quantitative chain polymerase reaction. All samples were normalized to day 0 with a value of 1 on day 0.
  • Figure 3C shows a VCR-induced cell population that produces neural stem cells. VCR-treated mouse embryonic fibroblasts were digested and cultured in neural embryonic stem cell culture medium (mouse embryonic fibroblasts and passages 1, 5 and 13).
  • Figure 3D shows the relative expression levels of neural stem cell-specific genes detected by quantitative chain polymerase reaction. All samples were normalized to the expression level of mouse embryonic fibroblasts, and the expression level of mouse embryonic fibroblasts was 1.
  • Figure 3E shows immunofluorescence staining of Nestin, Pax6 and Sox2.
  • Figure 3F shows a flow chart of an experimental strategy for inducing neural stem cells from mouse embryonic fibroblasts. Data are mean ⁇ standard error and at least three replicates were performed, ***P ⁇ 0.001, **P ⁇ 0.01.
  • Figure 4 Induction of MEFs transdifferentiation into neural stem cells (ciNPCs).
  • Figure 4A shows the morphology of the first generation ciNPCs, the expression levels of Nestin, Sox2 and Pax6.
  • VCR-treated MEFs were further cultured for 7-10 days in a culture medium containing growth factors, and similar neural stem cell morphology was observed.
  • the expression of neural stem cell marker proteins Nestin, Sox2 and Pax6 was detected by immunofluorescence staining, and positive cells were counted. Such cells undergo further suspension culture to form Sox2 and Nestin-positive neurospheres.
  • Figure 4B shows the proportion of positive cells such as Nestin, Sox2 and Pax6 that can be enriched by suspension culture of primary neurospheres over a longer generation.
  • the scale is 200 ⁇ m; all data are taken with mean ⁇ SEM; representative images are from at least three independent experiments.
  • Figure 5 shows immunofluorescence staining of the 13th generation ciNPCs (Nestin and Sox2).
  • Figure 5B shows the statistics of the number of Nestin and Sox2 positive cells in Figure 5A.
  • Figure 5C shows immunofluorescence staining of the 13th generation ciNPCs (Nestin and Pax6).
  • Figure 5D shows the statistics of the number of Nestin and Pax6 positive cells in Figure 5C. Representative pictures are from at least three independent experiments.
  • Figure 6 Compound-induced proliferation and self-renewal of neural stem cells.
  • Figure 6A shows a representative picture of the 13th generation of compound-induced immunofluorescence staining of neural stem cells Ki67 and Nestin.
  • Figure 6B shows the neurospheres of the 13th passage of the compound-induced neural stem cells and the 5th generation of control neural stem cells in suspension culture.
  • Figure 6C shows immunofluorescence staining of Nestin, Pax6 and Sox2 of neural stem cells induced by the 23rd generation compound in adherent monolayer culture.
  • Figure 6D shows immunofluorescence staining of neurospheres Nestin, Pax6 and Sox2 induced by the 23rd generation compound-induced neural stem cells. The nuclei were stained with DAPI. The picture scale is 50 ⁇ m.
  • Figure 7 Genomic transcriptional profiling of compound-induced neural stem cells.
  • Figure 7A shows clustering and heat map analysis chip data. The neural stem cells induced by the compounds from the 5th and 13th generations of mouse embryonic fibroblasts were compared. In the heat map, red indicates increased expression relative to mouse embryonic fibroblasts and green indicates decreased expression.
  • Figure 7B shows a paired scatter plot analysis of 251 chip database genes by "neuro" search. Red dot expression is up-regulated, green indicates down-regulated expression, and gray indicates no significant change.
  • Figure 7C shows that the Venn diagram shows the induction of neural stem cells and control stem cells from the fifth and third generations of the gene with high expression ( ⁇ 10-fold, P ⁇ 0.05) relative to mouse embryonic fibroblasts. Overlap.
  • Figure 7D shows the gene-local analysis (GO analysis) of 774 shared genes in Figure 7C. The P value represents the EASE score.
  • Figure 8 Whole genome expression profiling of starting cell MEFs, control neural stem cells (NPCs), passage 5 and passage 31 ciNPC.
  • Figure 8A shows NPCs, ciNPC 5th and 31st compared to MEFs Gene-based analysis (GO) of up-regulated genes in generations.
  • Figure 8B shows a heat map of a portion of a neural gene, a pluripotency-related gene, and a fibroblast-specific gene.
  • Figure 8C shows functional annotations of genes (233) that are gradually down-regulated from MEFs to 5th and 31st generation ciNPCs.
  • Figure 8D shows gene-based analysis (GO) of down-regulated genes in NPCs, 5th and 31st generation ciNPCs compared to MEFs.
  • Figure 8E shows a heat map of genes expressed in specific regions of brain tissue. The P value represents the EASE value; red represents high expression and green represents low expression.
  • FIG. 9A shows immunofluorescence staining of neural stem cell marker proteins.
  • Primary MEFs were cultured to the third passage and cultured in DMEM or neural stem cell culture medium (NEM) containing 10% serum for 7 days for later detection. The data indicated that the initial MEFs did not contain Sox2, Pax6 or Nestin-positive neural stem cells, and the primary neural stem cells isolated from mouse brain tissue served as a positive control.
  • Figure 9B shows qRT-PCR further analysis of the amount of expression of a specifically overexpressed gene in neural stem cells. The scale is 200 ⁇ m; all data are taken with mean ⁇ SEM; representative images are from at least three independent experiments.
  • Figure 10 Analysis of genes associated with HDACs, TGF- ⁇ , GSK-3, and normal physiological hypoxia-related signaling pathways.
  • Figure 10A shows cluster analysis and heat map of 196 genes associated with HDACs.
  • Figure 10B shows cluster analysis and heat map of 74 genes associated with TGF- ⁇ .
  • Figure 10C shows cluster analysis and heat map of 114 genes associated with GSK-3.
  • Figure 10D shows cluster analysis and heat map of 71 genes associated with normal physiological hypoxia.
  • Figure 11A shows that 5th generation ciNPCs differentiated in growth factor-containing neural basal medium for 7 days, and detect GFAP-positive glial cells and Tuj1-positive immature neurons (left panel) and MAP2-positive mature nerves. Yuan (right).
  • Figure 11B shows mature neurons differentiated from passage 13 ciNPCs. After the 13th generation ciNPCs were cultured for 4 weeks in the neuron differentiation specific medium, GAD67-positive and Synapsin-positive cells were detected. DAPI labeled nuclei; the scale is 50 ⁇ m (left), and the right panel is 8 times larger than the Synapsin/Tuj1 immunofluorescence stain in Figure 12B.
  • Figure 12 Pluripotency of compound-induced differentiation of neural stem cells in vitro and in vivo.
  • Figure 12A shows the expression of the astrocyte marker gene GFAP, the neuronal marker genes Tuj1 and Map2, and the oligodendrocyte marker genes Olig2 and Mbp after culture of the 13th generation compound-induced neural stem cells in a differentiation medium.
  • Figure 12B shows immunofluorescence staining of NeuN, Glutamate and Synapsin after passage of the 13th generation of compound-induced neural stem cells in neuron differentiation medium for 4 weeks. The nuclei were stained with DAPI. The picture scale is 50 ⁇ m. Representative pictures of three replicate experiments were shown.
  • Figure 12C shows the current clamp recording Representative Action Potentials of Neurons Induced by Neural Stem Cell Differentiation
  • Figure 12D shows representative spontaneous post-synaptic currents of neurons induced by compound-induced neural stem cell differentiation.
  • Figure 12E shows a representative Na+ current of a voltage clamp recording neuron-differentiated neuron-differentiated neurons.
  • Figure 12F shows the oligodendrocyte marker gene Olig2, the astrocyte marker gene GFAP and the neuronal marker gene NeuN detected one month after GFP-labeled compound-induced neural stem cell transplantation.
  • GFP-labeled compound-induced neural stem cells were transplanted to E13.5 embryos one month later, and the mouse brains were sectioned and immunofluorescently stained, and the arrows indicated GFP-positive cells expressing Olig2, GFAP or NeuN, respectively. The nuclei were stained with DAPI.
  • the scales of Figures 12A and 12B are 50 ⁇ m.
  • Figure 12F scale is a 15 ⁇ m. 24 mouse embryos transplanted with GFP-labeled compounds for neural stem cell transplantation.
  • Figure 13 Identification of GFP-tagged ciNPCs.
  • Figure 13A shows that the 17th generation ciNPC still has the ability to form neurospheres after infection with a GFP-expressing lentivirus.
  • Figure 13B shows that GFP-ciNPCs express neural stem cell marker proteins Nestin and Sox2.
  • Figure 13C shows that GFP-ciNPCs still have the pluripotency of differentiation into GFAP-positive glial cells, Tujl-positive neurons, and Olig2-positive oligodendrocytes. Arrows indicate GFP positive cells expressing Tuj1 or Olig2. The scale is 50 ⁇ m.
  • Figure 14 Transplantation of GFP-expressing ciNPCs in vivo.
  • Figure 14A shows the in vivo distribution of GFP-positive cells after 1 week of inoculation of GFP-ciNPCs in E13.5 day mouse embryonic brain tissue.
  • Figure 14B shows that GFP-ciNPCs still express Ki67 after 1 week of transplantation in vivo.
  • Figure 14C shows that GFP-ciNPCs differentiate into Olig2-positive or GFAP-positive cells after 1 week of transplantation in vivo.
  • Figure 14D shows that GFP-ciNPCs differentiate into Mbp-positive oligodendrocytes or Tuj1-positive neurons after 1 month of transplantation in vivo.
  • Figure 14E shows that Ki67 expression was not detected after 1 month of transplantation of GFP-ciNPCs in vivo.
  • the scale is 50 ⁇ m; representative pictures are from brain tissue of at least 10 transplanted mice.
  • Figure 15 Compounds combined with NLS or TLT induced MEFs transdifferentiation into neural stem cells (ciNPCs) under normal physiological hypoxic conditions.
  • Figure 15A shows the adherent morphology of the 5th generation ciNPCs induced by NLS, neurospheres, and immunofluorescence staining for Sox2, Pax6, and Nestin.
  • Figure 15B shows the adherent morphology, neurospheres, and immunofluorescence staining for Sox2, Pax6, and Nestin of the 5th generation ciNPCs obtained by TLT induction.
  • the scale is 200 ⁇ m; representative images are from at least three independent experiments.
  • Figure 16A shows quantitative chain polymerase reaction analysis of NLS (0.5 mM NaB, 1 mM LiCl and 1 ⁇ M SB431542) (left panel) or TLT (10 nM TSA, 0.3 mM Li 2 CO 3 and 30 ⁇ M Tranilast) under 5% O 2 conditions Sox2 expression levels of treated mouse fibroblasts. All samples were normalized to the DMSO group on day 0 with a value of 1.
  • Figure 16B shows the morphology of neural stem cells obtained by NLS or TLT treatment and the immunofluorescence staining of Nestin, Sox2 and Pax6. The nuclei were stained with DAPI. The picture scale is 50 ⁇ m.
  • Figure 16C shows the quantitative chain polymerase reaction analysis of the expression level of neural stem cell-specific genes. All samples were normalized to the MEF group with a value of 1. Data are mean ⁇ standard error.
  • Figure 17 Induction of neural stem cells from mouse tail-tip fibroblasts and human urine-derived epithelial cells (abbreviated as urine cells) and qualitative.
  • Figure 17A shows quantitative chain polymerase reaction analysis of VCR expression levels of pluripotency-related genes after treatment of mouse tail tip fibroblasts under normal physiological hypoxic conditions. All samples were normalized to group d0 with a value of one.
  • Figure 17B shows a morphological map of mouse tail tip fibroblasts and the first generation of neural stem cells produced from mouse tail tip fibroblasts.
  • Figure 17C shows the morphology of neural stem cells from the 13th generation of mouse tail tip fibroblasts and immunofluorescence staining of Nestin, Sox2 and Pax6.
  • Figure 17D shows the expression level of neural stem cell-specific genes of neural stem cells produced from mouse tail-tip fibroblasts in the 16th generation by quantitative chain polymerase reaction analysis. All samples were normalized to the TTF group with a value of 1.
  • Figure 17E shows the astrocyte marker gene GFAP, the neuronal marker genes Tuj1 and Map2, the oligodendrocyte marker gene Olig2 and Mbp in mouse tail-tip fibroblast-derived 13th-generation compound-induced neural stem cells Expression after culture in differentiation medium.
  • Figure 17F shows a phase contrast picture of human urine cells before and after normal physiological hypoxic treatment of VCR.
  • Figure 17G shows quantitative chain polymerase reaction analysis of VCR expression levels of pluripotency-related genes after treatment of human urine cells under normal physiological hypoxic conditions.
  • Fig. 17H is a view showing the morphology of the adherent monolayer culture and suspension culture of the neural stem cells induced by the fifth generation of neural stem cells and control humans derived from human urine cells. Human induced neural stem cells are induced from specific transcription factors.
  • Figure 17I shows the quantitative chain polymerase reaction analysis of the expression level of neural stem cell-specific genes. All samples were normalized to the hUCs group with a value of 1.
  • Figure 17J shows the expression of the astrocyte marker gene GFAP, neuronal marker genes Tuj1 and Map2, after cultured in differentiation medium, induced by human urine cell-derived 5th generation compound. The nuclei were stained with DAPI. The picture scale is 50 ⁇ m. Data are mean ⁇ standard error.
  • FIG. 1 VCR induced mouse tail tip fibroblasts to differentiate into neural stem cells under normal physiological hypoxia (5th generation). Immunofluorescence staining of Nestin, Sox2 and Pax6 (left panel) and corresponding statistical map (right panel). The scale is 50 ⁇ m; representative images are from at least three independent experiments.
  • Figure 19 VCR induces transdifferentiation of cells in human urine to neural stem cells under normal physiological hypoxic conditions.
  • Figure 19A shows immunofluorescence staining (Nestin and Sox2) of cell-derived ciNPCs in human urine from passages 11 and 22.
  • Figure 19B shows the growth curves of cell-derived ciNPCs and positive control iNPCs in human urine.
  • HDACs histone deacetylase
  • GSK-3 glycogen synthase kinase
  • TGF- ⁇ transforming growth factor beta
  • HDACs Histone deacetylase
  • Histone deacetylase is a class of proteases that play an important role in the structural modification of chromosomes and the regulation of gene expression.
  • histone acetylation is in a dynamic equilibrium with histone deacetylation and is regulated by histone acetyltransferase and histone deacetylase.
  • Histone deacetylase inhibitors can change the chromatin structure by increasing the degree of histone acetylation in specific regions of chromatin, thereby regulating the expression and stability of apoptosis and differentiation-related proteins; by tissue type, histone deacetylation
  • the enzyme inhibitors can be roughly classified into: hydroxamic acids (such as trichostatin A), cyclic tetrapeptides (such as Trapoxin, etc.), fatty acid salts (such as sodium valproate, butyl) Sodium benzoate or the like, a benzamide compound (such as MS275), and an electrophilic ketone compound (such as trifluoromethyl ketone).
  • Glycogen synthase kinase is a multifunctional serine/threonine protein kinase that is involved not only in hepatic glucose metabolism but also in Wnt and Hedgehog signaling pathways, which regulates cellular processes by phosphorylating multiple substrate proteins.
  • glycogen synthase kinase inhibitors have potential therapeutic effects on the treatment of neurodegenerative diseases, cancer and type II diabetes; they can be divided into ATP competitive inhibitors and ATP competitive inhibitors.
  • the former includes Paullones, Indirubin, Maleimides, Pyrimidines, Pyridines, and Aloisines; the latter includes Li ions and TDZD derivatives.
  • TGF- ⁇ Transforming growth factor beta
  • TGF- ⁇ belongs to a class of cytokine superfamilies that promote cell growth and transformation.
  • Type its intracytoplasmic signaling pathway mainly includes membrane receptor serine/threonine kinase system and Smad protein signaling system.
  • TGF- ⁇ inhibitor research mainly includes inhibition of TGF- ⁇ and its receptor expression (such as tranilast, etc.), blocking the binding of TGF- ⁇ and receptor (such as SB-431542, LY2157299, etc.), interfering with receptor kinase Signal transmission (such as SIS3, etc.).
  • small molecule compound combination refers to a combination comprising: (a) a histone deacetylase (HDACs) inhibitor; (b) glycogen synthase kinase (GSK-3) inhibition. (c) Transforming growth factor beta (TGF- ⁇ ) signaling pathway inhibitor.
  • HDACs histone deacetylase
  • GSK-3 inhibition glycogen synthase kinase
  • TGF- ⁇ Transforming growth factor beta
  • the small molecule compound combination may further contain a pharmaceutically acceptable carrier, and in this case, the small molecule compound combination is a pharmaceutical composition having an activity of inducing somatic cell transdifferentiation into neural stem cells.
  • the HDACs inhibitor comprises VPA (sodium valproate), NaB (sodium butyrate), or TSA (triccobacterin A);
  • the GSK-3 inhibitor comprises CHIR99021, LiCl (lithium chloride), or Li 2 CO 3 (lithium carbonate);
  • the TGF-beta inhibitor signaling pathway includes Repox, SB431542, or Tranilast (Trnilast).
  • each component should meet its lowest effective concentration.
  • the minimum effective concentration of each component in the small molecule compound combination is as follows:
  • HDACs inhibitor VPA: 0.2-1 mM, preferably 0.3-0.8 mM, more preferably 0.4-0.6 mM; NaB 0.2-1 mM, preferably 0.3-0.8 mM, more preferably 0.4-0.6 mM; TSA 5-20 nM, 8-15 nM, more preferably, 10-12 nM;
  • GSK-3 inhibitor CHIR990211-5 ⁇ M, preferably 2-4 ⁇ M; LiCl 0.5-3 ⁇ M, preferably 1-2 ⁇ M; Li 2 CO 3 0.05-1 mM, preferably 0.1-0.8 mM, more preferably, 0.2-0.5 mM;
  • TGF- ⁇ inhibitor signaling pathway Repsox 0.2-3 ⁇ M, preferably 0.5-2 ⁇ M; SB4315420.2-3 ⁇ M, preferably 0.5-2 ⁇ M; Tranilast 10-50 ⁇ M, preferably 20-40 ⁇ M.
  • VCR VCR
  • CHIR99021, Repox NLS
  • NLS NaB, LiCl, and SB431542
  • TLT TSA, Li 2 CO 3 , and Tranilast
  • Neural stem cells have the ability to differentiate into neuronal neurons, astrocytes, and oligodendrocytes, self-renewing, and sufficient to provide a large number of brain tissue cells. It can produce various types of cells of nerve tissue through unequal division. In all neural tissues such as the cerebrospinal and spinal cord, different neural stem cell types produce different types of progeny cells and different distributions.
  • ciNPCs compound-induced neural stem cells
  • compound-induced neural stem cells refer to neural stem cells produced after induction of somatic cells by a combination of small molecule compounds (pharmaceutical compositions) of the invention.
  • neural stem cells are used interchangeably and refer to neural stem cells from different parts of a mammal, such as a human or mouse, and are commonly used herein for ciNPC. Control.
  • neural stem cell-specific gene refers to a gene (or protein thereof) that is highly expressed in neural stem cells as compared to non-neuronal stem cells.
  • the neural stem cell-specific genes include Sox2, Nestin, Pax6, Ascl1, and Blbp.
  • neural stem cell pluripotency gene refers to a gene (or protein thereof) that is highly expressed in neural stem cells and is associated with pluripotent differentiation properties of neural stem cells compared to non-neuronal stem cells.
  • the neural stem cell pluripotency genes include Sox2, Nestin, Pax6, Ascl1, and Blbp.
  • hypoxic environment refers to the in vitro or in vivo simulation of the cellular environment of the body.
  • the hypoxic environment refers to a normal physiological hypoxic environment, such as an oxygen concentration (or oxygen pressure) boundary.
  • the environment between % and 8%, preferably, the normal physiological hypoxic environment refers to an environment having an oxygen concentration of 4% to 6%, more preferably 5%.
  • the experiments of the present invention prove that 3%-5% (especially 5%) of the environment is the best environment for obtaining neural stem cells, and compared with the environment of normal oxygen concentration (about 21%), hypoxia (especially normal) Physiological hypoxic conditions are necessary for somatic cell transdifferentiation into neural stem cells.
  • the method for inducing somatic cell transdifferentiation into neural stem cells of the present invention generally refers to an in vitro induction method, and of course, further in vivo induction can be carried out according to an in vitro induction experiment, which can be obtained according to a conventional technique or method in the art.
  • somatic cells can be cultured in the presence of a combination of small molecule compounds of the present invention.
  • the somatic cells can be further cultured using neural stem cells or neural cell culture media conventional in the art.
  • the neural stem cell or nerve cell culture medium may contain epidermal growth factor EGF, basic fibroblast growth factor bFGF, heparin, or a combination thereof.
  • the present invention provides a composition comprising the neural stem cells of the present invention.
  • the composition is a pharmaceutical composition, a food composition, a nutraceutical composition, or the like.
  • the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient: a neural stem cell according to the present invention.
  • the term "effective amount” or “effective amount” refers to an amount that can produce a function or activity on a human and/or animal and that can be accepted by a human and/or animal.
  • a component of a “pharmaceutically acceptable carrier” is a substance that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, a substance having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise a safe and effective amount of the active ingredient of the present invention together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched with the administration mode, and the pharmaceutical composition of the present invention is in the form of an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent.
  • it is prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc. Usually, when the active ingredient of the present invention is about daily A satisfactory effect can be obtained by administering a dose of 0.00001 mg to 50 mg/kg of animal body weight (preferably 0.0001 mg to 10 mg/kg of animal body weight). For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • Pharmaceutically acceptable carriers of the invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide materials, cellulose, nanogels, or Its combination.
  • the choice of carrier should be compatible with the mode of administration, which are well known to those of ordinary skill in the art.
  • the invention also provides the use of the pharmaceutical composition for the prevention or treatment of a neurological condition.
  • the method of the present invention can successfully utilize a combination of inhibitors of specific signaling pathways to induce transdifferentiation of various somatic cells into neural stem cells without introducing an exogenous gene, and the prepared stem cell shape is very similar to that of neural stem cells and has Good pluripotent differentiation
  • current methods for obtaining patient-specific iNPCs include induction of transdifferentiation by transfection of a set of transcription factors or differentiation of iPCCs derived from human fibroblasts into iNPCs. According to existing methods, it takes at least three months from obtaining iPSCs to getting iNPCs. According to the induction method of the present invention, a similar amount of ciNPCs can be obtained in a shorter time.
  • the present invention provides a better alternative strategy for studying patient-specific neuronal and related cell therapies.
  • Mouse tail tip fibroblasts were isolated from newborn C57BL/6 mice for 3 days. Simply put, the first 1/5 of the tail is cut into small pieces and cultured for 6 days. It migrated from the tip of the tail to the third generation and can be used in other experiments.
  • Mouse MEFs and TTFs were cultured in DMEM (Life Technologies, C11965) at 37 ° C, 5% CO 2 supplemented with 10% FBS (PAA Laboratories, A15-101), 1 mM GlutaMAX (Life Technologies, 35050-061) And 0.1 mM non-essential amino acids (NEAA, Millipore, TMS-001-C).
  • Mouse neural stem cells were obtained from E12.5 day mouse embryos and cultured in neuron expansion culture (NEM) with addition of 30 ng/ml heprin, 20 ng/ml EGF and 20 ng/ml bFGF.
  • Human urine cells epidermal cells isolated from human urine
  • REGM Longza, CC-4127
  • the starting cells were cultured in DMEM for 24 hours and then replaced with KSR medium containing knockout DMEM (Life Technologies, 10829-018) 15% serum replacement, 1% NEAA (Life Technologies) , 35050), 1% Glutamax (Life Technologies, 35050-061), 1% sodium pyruvate (Life Technologies, 11360), 0.1 mM ⁇ -mercaptoethanol (Life Technologies, 21985-023) and 1000 U/ml of leukemia inhibitory factor (LIF) ) (Chemicon, ESG1107).
  • the cells were cultured at 37 ° C, 5% O 2 (hypoxia) and 5% CO 2 .
  • the culture solution containing the compound is changed every five days.
  • ciNPCs are further enriched during multiple rounds of neurosphere suspension culture.
  • Mouse fibroblast-derived ciNPCs were cultured in a neuronal expansion medium in which EGF (20 ng/ml) and bFGF (20 ng/ml) were added.
  • EGF 20 ng/ml
  • bFGF 20 ng/ml
  • 20,000 ciNPCs were plated on PDL/Laminin-coated 24-well plates and cultured in N2B27 (DMEM: F12, 1% N2, 2% B27) medium containing no EGF and bFGF.
  • Stable astrocyte production induction was the addition of BMP4 (50 ng/ml; R&D Biosystems) and 1% FBS in growth factor-free N2B27.
  • Neuronal differentiation was performed by placing ciNPCs on PDL/Laminin-coated coverslips in Neural basal medium (2% B27, 1% N2, 10 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml). IGF-1, 1 ⁇ M cAMP, 200 Culture in ⁇ M Ascorbic acid). Neuronal molecular marker gene expression and electrophysiological analysis were detected at specific time points, respectively.
  • oligodendrocyte differentiation 20,000 cells were plated on PDL/Laminin-coated coverslips and cultured in N2B27 containing bFGF (10 ng/mL; Invitrogen) and PDGF-AA (10 ng/mL; Peprotech). The medium was cultured for 7 days, and then differentiated by adding T3 (100 ng/mL; 20 Sigma-Aldrich) for 5 days.
  • neurospheres were digested with accutase (Life Technologies) and 10,000 cells were plated onto Poly-L-ornithine/laminin coated slides.
  • EGF and bFGF were removed from the culture medium, and neurotrophic factors including BDNF, GDNF, IGF (both 10 ng/mL) and 100 mM cAMP, 200 ng/mL of ascorbic acid were added.
  • Neuron differentiation culture was changed once every two days. After 2 weeks, the expression of neuronal molecular markers was detected. After about 50 days, the expression of astrocyte molecular markers was detected.
  • Alkaline phosphatase (AP) staining was stained with an alkaline phosphatase kit (Sigma-Aldrich, 85L3R) according to the manufacturer's protocol. The image was acquired using the Zeiss Observer Z1.
  • the neurosphere is stained, and the neurosphere suspension is first transferred to a 15 ml tube to allow the neurosphere to settle naturally.
  • the neurospheres were then fixed in 4% PFA for 15 minutes at room temperature and incubated overnight in 5 ml of 30% sucrose at 4 °C until stable.
  • Neurosphere pellets were transferred to tissue cryopreservation on cryostat chuck (Leica, 020108926).
  • Neurosphere slices of 10 ⁇ m thickness were prepared and embedded in foil and stored at -80 ° C for analysis.
  • Mouse brain sections were prepared as previously described. Briefly, the mouse brain was perfused with a 4% PFA PBS heart. After freezing in 30% sucrose, the mouse brain was cut with 20 ⁇ m thick ice and analyzed by immunofluorescence staining.
  • RNA samples were extracted with TRIzol (Sigma-Aldrich, T9424) according to the manufacturer's instructions, and RNA integrity was analyzed using an Agilent 2100 bioanalyzer. 200 ng of total RNA per sample was subjected to labeling reaction by a single color rapid label amplification kit (Agilent, 5190-2305). The amplified RNA was purified using the RNeasy mini kit (Qiagen, 74104).
  • the cDNA chip for 8X60array was from Agilent Technologies and used the Agilent Gene Expression Hybridization Kit (catalog number: G4852A). After 17 hours of hybridization at 65 ° C and washing, the chips were scanned with an Agilent chip scanner (Agilent Technologies, USA). Image extraction software (version 10.7.1.1, Agilent Technologies) was used to analyze the chip image to obtain raw data. GeneSpring software is used to perform basic analysis of raw data. First, the raw data is normalized using a quantile algorithm. The differentially expressed genes were identified by a fold change and a threshold of 10 was set. Subsequent Gene Ontology (GO) analysis and KEGG analysis were applied to determine the effect of these differentially expressed mRNAs. A total of 55,821 probes from 39,430 genes from the entrenza-gene database were detected.
  • Agilent Gene Expression Hybridization Kit catalog number: G4852A
  • Image extraction software version 10.7.1.1, Agilent Technologies
  • GeneSpring software is used to perform
  • the intracellular fluid contained 93 mM K-gluconate, 16 mM KCl, 2 mM MgCl2, 10 mM HEPES 4 mM ATP-Mg, 0.3 mM GTP-Na 2 , 10 mM creatine phosphate, 0.5% Alexa Fluor 568 hydrazide (Invitrogen) (pH 7.25, 290/300 mOsm), 0.4% neurobiotin (Invitrogen).
  • the membrane potential was stabilized at approximately -70 mV, and the action potential was excited by the input of a repeated current of 2 pA increments. A potential difference of -70 to +70 mV is used to activate the sodium ion inward current and the potassium ion outward current. Data were analyzed using pClamp (Clampfit).
  • In vivo transplantation is performed according to the literature. Briefly, the uterine horn of pregnant mice C57BL/6 at E13.5 days of gestation was cultured in a sterile environment. PBS containing approximately 20 GFP-ciNPC neurospheres was injected into the embryonic ventricle through obliquely calibrated glass microtubules, wherein the diameter of the neurospheres must not exceed 80 ⁇ m. Thereafter, the uterine horn was replaced, the peritoneal cavity was lavaged with 10 mL of warm PBS, the PBS contained antibiotics, and the wound was sutured. After 1 week or 1 month, the mice were anesthetized on ice or sodium pentobarbital. The preparation of the brain slices was as described above and can be used for subsequent analysis.
  • mice can receive food and water at any time. All experiments were performed in accordance with animal care and the use of national research institute health guidelines and approved by the Biological Research Ethics Committee of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences.
  • Example 1 Screening of a combination of compounds that induce somatic cells to produce neural stem cells under normal physiological hypoxia conditions
  • a dense clone could be produced by treating a mouse fibroblast with a compound combination VPCR (VPA, CHIR99021, Repox, and Parnate) under 3% or 5% O 2 for about 10 days, while at 21 No dense clones were generated under %O2 conditions (Fig. 1A). Approximately 40 dense clones can appear in 200,000 starting cells. The induction efficiency of the intermediate clone was slightly higher under the 5% O 2 condition than the 3% O 2 condition, and therefore, the culture condition of 5% O 2 was taken in the subsequent induction experiment.
  • Alkaline phosphatase AP staining of these intermediate clones revealed that approximately 3/4 of the dense clones were highly expressed.
  • Alkaline phosphatase AP (Fig. 1B, Fig. 3A), and neither dense clone nor AP positive cells were found in VPCR-treated mouse fibroblasts under normal oxygen pressure conditions.
  • VPA VPA
  • CHIR99021 Repox and Parnate are all essential components for inducing the acquisition of dense clones.
  • mouse fibroblasts were treated with a combination of any three compounds in the VPCR to check for the formation of dense clones.
  • VCR compound combination ie, VPA, CHIR99021, Repox
  • the cells treated with VCR combination for about 10 days were digested and re-planted and cultured under the condition of neural stem cell medium containing heparin, epidermal growth factor EGF and basic fibroblast growth factor bFGF. After about 7-10 days, a neural stem cell-like bipolar morphology appeared in the cultured cells (Fig. 3C).
  • the neural stem cell marker genes Nestin, Sox2 and Pax6 can be detected by immunofluorescence staining (Fig. 4A). Further, reverse transcription polymerase chain reaction detected that expression levels of neural stem cell-specific genes including Sox2, Pax6, Blpp, Ascl1, and Brn2 were also enhanced (Fig. 3D, ciNPCp1).
  • Neural stem cell-like cells appear in the culture system. When these cells were cultured in suspension, floating clusters of cells were formed, and immunofluorescence staining of the cell clusters showed that they were positive for Sox2 and Nestin, and had neurosphere properties (Fig. 4A). These floating clusters of cells were collected and named as compound-induced neural stem cell/neurosphere (ciNPC) algebra 1 (p1).
  • ciNPC compound-induced neural stem cell/neurosphere
  • the 13th generation (p13) of the compound induced neural stem cell adherent monolayer culture exhibited a bipolar morphology similar to mouse embryonic neural stem cells (Fig. 3C, ciNPC p5). Quantitative chain polymerase enzymatic reaction was used to detect the expression levels of Sox2, Pax6, Blbp, Ascl1 and Brn2 in neural stem cells induced by different algebraic compounds. It was found that suspension culture can well enrich neural stem cells in the original induced mixed cells (Fig. 3D, ciNPC p13).
  • VCR treatment of mouse embryonic fibroblasts under normal physiological hypoxic conditions can obtain relatively pure expandable neural stem cells.
  • Neural stem cells induced by mouse embryo-derived neural stem cells (control NPCs), mouse embryonic fibroblasts, and 5th and 13th generation compounds were extracted and subjected to genome-wide expression type analysis using a chip.
  • FIG. 7A Whole genome clustering and heat map analysis (Figure 7A) and scatter plot analysis (Figure 8B) revealed that compound-induced neural stem cells and mouse embryonic fibroblasts are very different, but compound-induced neural stem cells and mouse heads Derived neural stem cells have great similarities.
  • Fig. 7C There are 774 core target genes in neural stem cells and control neural stem cells induced by different algebraic compounds (Fig. 7C). These genes are mainly related to processes such as neurogenesis and cell morphology by gene ontology analysis (GO analysis) (Fig. 7D and Fig. 7D) 8A).
  • Neural stem cell-specific genes such as Sox2, Pax6, Ncan, Tox3, Hes5, Gpm6a, Nes, Bmi1, Zbtb16, Rfx4, Gpm6a and Slc1a3 are significantly up-regulated in compound-induced neural stem cells and are comparable to mouse embryonic neural stem cells.
  • the expression level of the pluripotency-related genes Pof5f1 and Nanog was not up-regulated, indicating that the induced neural stem cells did not have the characteristics of pluripotent stem cells. (Fig. 8B).
  • the expression profile of biological processes such as the skeletal system is the gene that is most down-regulated by the compound-induced neural stem cells relative to mouse fibroblasts ( Figures 8C and 8D).
  • Figures 8C and 8D the expression levels of 424 genes such as Col3a1, DKK3, Thy1, Snail1 and other fibroblast-specific genes were gradually down-regulated from the 5th generation to the 13th generation.
  • Both compound-induced neural stem cells and mouse brain-derived neural stem cells have higher expression levels of ventral brain-specific genes such as Oligo2 and Nkx2.2, while no detectable genes in the dorsal brain region such as Pax3 and Pax7 were detected. expression.
  • the experiment also found high expression of the forebrain specific genes Emx2, Foxg1 and Nr2e1 as well as the midbrain specific genes Gbx2 and En1, but there was no high expression of hindbrain specific genes such as Hoxa7 and Hoxb7.
  • the compound-induced neural stem cells obtained by the present invention have the characteristics of the ventral anterior midbrain region, but do not well have the properties of other brain regions.
  • HDACs histone deacetylases
  • GSK-3 glycogen synthase kinase 3 ⁇
  • TGF- ⁇ transforming growth factor beta
  • Compound-induced neural stem cells and control neural stem cells have similar expression types in these signaling pathways, and there is a large difference in mouse embryonic fibroblasts (Fig. 10).
  • the neural stem cells induced by the compounds of the present invention have great similarities with mouse neural stem cells, but are quite different from mouse fibroblasts. Furthermore, the neural stem cells induced by the compounds of the present invention are also characterized by the ventral anterior midbrain region, and histone deacetylases (HDACs), glycogen synthase kinase 3 ⁇ (GSK-3), transforming growth factor beta (TGF) - ⁇ ) and the conduction pathway of normal physiological hypoxia signals are necessary in the process of transforming somatic cells into neural stem cells.
  • HDACs histone deacetylases
  • GSK-3 glycogen synthase kinase 3 ⁇
  • TGF transforming growth factor beta
  • 80% of the cells have neuronal morphology and are Tuj1 positive, after 10-14 days of culture. It is a morphology with mature neurons and a double positive for Map2/Tuj1 (Fig. 11A and Fig. 12A). Map2 or Tuj1 is not expressed in GFAP-positive cells, which means that the differentiated cells are functionally specific.
  • the compound-induced neural stem cells were transplanted into embryonic mice, and the neural stem cells induced by the 17th generation compound were labeled with lentiviral GFP.
  • GFP-tagged compounds-induced neural stem cells still have neural stem cell-related properties, including proliferative capacity, neurosphere formation ability, neural stem cell-specific gene expression, and in vitro differentiation ability (Fig. 13).
  • GFP-labeled compound-induced neural stem cells were injected into embryos of E13.5, and immunofluorescence staining showed that GFP-labeled compound-induced neural stem cells could survive in different brain regions of mice 1 week after transplantation (Fig. 14A). Furthermore, this GFP-tagged compound-induced neural stem cell can be labeled with Ki67, Olig2 or GFAP, but cannot be labeled with Tuj1 (Fig. 14B and Fig. 14C), which means that the induced neural stem cells are more easily differentiated into glial cells in vivo and Less glial cells.
  • Olig2 + or Mbp + oligodendrocytes, GFAP + astrocytes and mature neurons of NeuN + or Tuj1 + can still be found by GFP-labeled compound-induced differentiation of neural stem cells.
  • Fig. 12F, Fig. 14D Ki67-positive GFP-labeled cells were not found (Fig. 14E), and no tumor formation was found in the transplanted brain region.
  • Compound-induced neuronal cells can differentiate into major neural lineages in vitro, including astrocytes, neurons, and oligodendrocytes.
  • the transplanted compound-induced neural stem cells can differentiate into different neural lineages in vivo and do not form tumors in the transplanted brain regions, so the compound-induced neural stem cells have potential clinical application prospects.
  • VPA, CHIR99021 and Repox are histone deacetylases (HDACs) and glycogen synthase, respectively.
  • HDACs histone deacetylases
  • the method was the same as the VCR-induced experimental protocol. It was found that under the same experimental conditions, the compound combination NLS (NaB, LiCl and SB431542) and TLT (TSA, Li 2 CO 3 and Tranilast) can be treated under 5% O 2 conditions. Murine embryonic fibroblasts can obtain dense clones and activate Sox2 expression. Further, these intermediate clones were further cultured in suspension to produce Nestin + /Pax6 + or Nestin + /Sox2 + + neural stem cells (Fig. 15).
  • These purified compound-induced neural stem cells can have classical neural stem cell morphology and neurosphere forming ability after passage 13 passages (Fig. 16B).
  • NLS and TLT compounds can induce the same effect of producing neural stem cells under the conditions of normal physiological hypoxic culture, and further supports the conclusion of chip analysis that activation of a series of signal transduction pathways can be coordinated. Promotes transdifferentiation of mouse embryonic fibroblasts to neural stem cells.
  • Example 8 Mouse tail tip fibroblasts and human urine cells induce ciNPCs
  • Neonatal mouse tail tip fibroblasts were treated using the same method and compound combination VCR.
  • Fig. 17A the expression of Sox2 was up-regulated under normal physiological hypoxic conditions for 10 days of VCR treatment. Further culture was carried out for 7 to 10 days in neuroblast-enhanced culture medium to which heparin, EGF and bFGF were added, and the TCRs after VCR treatment had the same morphological changes as the VCR-treated MEF (Fig. 17B). In the process of passage, homogeneous ciNPCs can be obtained step by step (Fig. 18).
  • the 16th generation ciNPCs derived from TTFs have typical neural stem cell morphology and neurosphere forming ability (Fig. 17C). Both immunofluorescence staining and real-time quantitative PCR analysis were able to detect the expression of neural stem cell molecular marker genes Nestin, Sox2, Pax6 and Blbp (Fig. 17D).
  • TTFs-derived ciNPCs were able to induce GFAP-positive astrocytes, Tuj1/MAP2 double-positive neurons, and Olig2/MBP double-positive oligodendrocytes under specific differentiation conditions (Fig. 17E).
  • VPA VPA
  • CHIR99021 Repox
  • normal physiological hypoxia can directly convert mouse fibroblasts from different sources into ciNPCs.
  • VCR-induced intermediate cells After culturing for 5 generations or more in the neuron expansion medium, these VCR-induced intermediate cells began to exhibit the same morphology as the control iNPCs (as previously obtained by introducing a gene into hUCs) (Fig. 17H).
  • hUCs-derived ciNPCs By detecting qRT-PCR (Fig. 17I) and immunofluorescence staining (Fig. 19A), these hUCs-derived ciNPCs express neural stem cell-specific genes, including Sox2, Nestin, Sox1, and Pax6. More importantly, hUCs-derived ciNPCs have similar proliferative capacity to control iNPCs (Fig. 19B), and it can differentiate into Tuj1/MAP2 double positive neurons and GFAP positive astrocytes in neural differentiation medium ( Figure 17J).
  • the study of the present invention shows for the first time that a pure compound group is utilized under conditions mediated by non-foreign genes.
  • the combination can completely induce somatic cell reprogramming and direct transdifferentiation into neural stem stem cells.
  • the induction strategy of the invention mainly comprises two aspects: 1. Under normal physiological hypoxia conditions, the compound combination induces the cells to enter the reprogramming stage, and the induced intermediate cells are transdifferentiated under lineage-specific induction conditions. In view of other lineage cells, for example, cardiomyocytes, vascular endothelial cells, etc., can also be obtained by a trans-factor method, or by stem cell in vitro differentiation method. Therefore, other pedigrees induced by purification can be obtained by using the induction strategy of the present invention. Specific cells.
  • HDACs inhibitors can induce terminally differentiated somatic cells to enter a reprogramming state, and The reprogramming state is accompanied by activation of the expression of the Sox2 gene. Therefore, the above three signaling pathways are likely to promote cell reprogramming by regulating the expression of Sox2-related genes.
  • cDNA chip data also indicated that the changes in the genes involved in the above three signaling pathways were very similar in compound-induced neural stem cells and in control neural stem cells, and were significantly different from the starting fibroblasts.
  • HDACs, GSK-3 and TGF- ⁇ signaling pathways regulated by small molecule compounds are crucial for inducing transdifferentiation of fibroblasts into neural stem cells, but the specific molecular mechanism remains to be further studied.
  • normal physiological hypoxic conditions are necessary for purifying cells to induce reprogramming, but normal physiological hypoxia mimicking compounds, such as cobalt chloride, do not replace normal physiological hypoxic conditions to induce cell development.
  • normal physiological hypoxia mimicking compounds such as cobalt chloride
  • the standard mammalian cells are cultured in an oxygen concentration of 21% in vitro, the actual oxygen concentration in the body tissues is 1% to 5%, and under normal physiological conditions, the microenvironment of stem cells is also a normal physiological hypoxic condition, so further detection is performed. Whether a combination of small molecule compounds that induce cell reprogramming in vitro can also promote transdifferentiation in vivo is of great significance.
  • the combination of compounds can also induce direct transdifferentiation of cells in human urine into neural stem cells.
  • the present invention provides a new, convenient, and safe method for obtaining patient-specific neural stem cells, for further treatment of nerves.
  • Diseases such as Alzheimer's disease and Parkinson's disease offer new therapeutic avenues.

Abstract

Provided are a method for inducing the transdifferentiation of somatic cells into neural stem cells and application for same. Using a combination of a histone deacetylase (HDAC) inhibitor, a glycogen synthase kinase (GSK-3) inhibitor, and a transforming growth factor β (TGF-β) signal pathway inhibitor, in a low-oxygen normal physiological environment, induce somatic cells such as fibroblasts and epithelial cells to form into neural stem cells having good pluripotency and passage stability.

Description

诱导体细胞转分化为神经干细胞的方法及其应用Method for inducing somatic cell transdifferentiation into neural stem cells and application thereof 技术领域Technical field
本发明属于生物技术和神经发育领域，具体地，本发明涉及一种诱导体细胞转转分化为神经干细胞的方法及其应用。The present invention belongs to the field of biotechnology and neurodevelopment, and in particular, to a method for inducing somatic cell transduction into neural stem cells and an application thereof.
背景技术Background technique
终末分化细胞被认为是一类具有特定功能和表型，并丧失进一步发育潜能的细胞。但是，早期研究发现终末分化细胞的细胞核可被用于克隆动物，此外，体外细胞融合也可以导致细胞谱系的重编程，以上结果表明发育过程中的表观遗传学修饰是可逆的。近期大量研究发现，通过特异转录因子组合不但可以诱导体细胞通过重编程去分化为多潜能干细胞，也可以直接转分化为其他谱系的特定体细胞，从而为病人的个性化治疗提供新的细胞来源。Terminally differentiated cells are considered to be a class of cells with specific functions and phenotypes that lose further developmental potential. However, early studies have found that the nuclei of terminally differentiated cells can be used to clone animals. In addition, in vitro cell fusion can also lead to reprogramming of cell lineages. The above results indicate that epigenetic modifications during development are reversible. A large number of recent studies have found that the combination of specific transcription factors can not only induce somatic cells to be differentiated into pluripotent stem cells by reprogramming, but also directly transdifferentiate into specific somatic cells of other lineages, thus providing a new source of cells for personalized treatment of patients. .
神经干细胞是一类能够自我增殖、更新和分化为不同神经类细胞的细胞，具有巨大的研究和临床应用价值。目前，从脑组织提取神经干细胞和从胚胎干细胞和诱导性多能干细胞分化为神经干细胞的方法已经成熟，此外，不同因子组合诱导体细胞转分化为神经干细胞的方法也日渐完善；但是现有的转分化方法涉及到外源基因的介入，具有很大的临床安全隐患。Neural stem cells are a kind of cells that can self-proliferate, renew and differentiate into different neuronal cells, and have great research and clinical application value. At present, methods for extracting neural stem cells from brain tissue and differentiating embryonic stem cells and induced pluripotent stem cells into neural stem cells have matured. In addition, methods for inducing somatic cell transdifferentiation into neural stem cells by different factor combinations are also gradually improved; Transdifferentiation methods involve the involvement of foreign genes and have great clinical safety risks.
因此，本领域迫切需要开发不需要外源基因介入的诱导体细胞转分化为神经干细胞将为神经干细胞的方法。Therefore, there is an urgent need in the art to develop a method for inducing somatic cell transdifferentiation into a neural stem cell which does not require exogenous gene intervention.
发明内容Summary of the invention
本发明提供了一种在低氧(尤其是正常生理低氧)环境下诱导体细胞转分化为神经干细胞将为神经干细胞。The present invention provides a method for inducing somatic cell transdifferentiation into neural stem cells in a hypoxic (especially normal physiological hypoxia) environment to be a neural stem cell.
本发明第一方面，提供了一种小分子化合物组合，所述的小分子化合物包括以下组分：In a first aspect of the invention, there is provided a combination of small molecule compounds comprising the following components:
(a)组蛋白去乙酰化酶(HDACs)抑制剂；(a) histone deacetylase (HDACs) inhibitors;
(b)糖原合成酶激酶(GSK-3)抑制剂；(b) a glycogen synthase kinase (GSK-3) inhibitor;
(c)转化生长因子β(TGF-β)信号通路抑制剂；和(c) a transforming growth factor beta (TGF-beta) signaling pathway inhibitor;
(d)任选的药学上可接受的载体。(d) an optional pharmaceutically acceptable carrier.
本发明第二方面，提供了一种小分子化合物组合，所述的小分子化合物由以下组 分构成：According to a second aspect of the present invention, there is provided a small molecule compound composition, wherein the small molecule compound consists of the following group Sub-composition:
(a)组蛋白去乙酰化酶(HDACs)抑制剂；(a) histone deacetylase (HDACs) inhibitors;
(b)糖原合成酶激酶(GSK-3)抑制剂；(b) a glycogen synthase kinase (GSK-3) inhibitor;
(c)转化生长因子β(TGF-β)信号通路抑制剂。(c) Transforming growth factor beta (TGF-beta) signaling pathway inhibitor.
本发明第三方面，提供了第一或第二方面所述的组合物的用途，用于在低氧环境下诱导体细胞转分化为神经干细胞。In a third aspect of the invention, there is provided the use of the composition of the first or second aspect for inducing somatic transdifferentiation into neural stem cells in a hypoxic environment.
在另一优选例中，所述的低氧环境包括正常生理低氧环境。In another preferred embodiment, the hypoxic environment comprises a normal physiological hypoxic environment.
在另一优选例中，所述的低氧环境为氧浓度3-8％的环境，较佳地，为4-6％。In another preferred embodiment, the hypoxic environment is an environment having an oxygen concentration of 3-8%, preferably 4-6%.
在另一优选例中，所述的体细胞包括成纤维细胞、上皮细胞。In another preferred embodiment, the somatic cells include fibroblasts, epithelial cells.
在另一优选例中，所述的体细胞来源于哺乳动物，较佳地为人、啮齿动物(小鼠、大鼠)。In another preferred embodiment, the somatic cells are derived from a mammal, preferably a human, a rodent (mouse, rat).
在另一优选例中，所述的成纤维细胞包括小鼠胚胎成纤维细胞、小鼠尾尖成纤维细胞、人皮肤成纤维细胞。In another preferred embodiment, the fibroblasts include mouse embryonic fibroblasts, mouse tail tip fibroblasts, and human dermal fibroblasts.
在另一优选例中，所述的上皮细胞分离自人尿液。In another preferred embodiment, the epithelial cells are isolated from human urine.
本发明第四方面，提供了一种体外诱导体细胞转分化为神经干细胞的方法，在低氧环境以及本发明第一或第二方面所述的小分子化合物组合存在的培养条件下，培养体细胞。According to a fourth aspect of the present invention, a method for inducing somatic cell transdifferentiation into neural stem cells in vitro is provided, wherein the culture body is cultured under a hypoxic environment and a combination of the small molecule compound according to the first or second aspect of the present invention cell.
在另一优选例中，所述的培养条件还包括神经干细胞培养基。In another preferred embodiment, the culture conditions further include a neural stem cell culture medium.
在另一优选例中，所述的神经干细胞培养基含有表皮生长因子EGF、碱性成纤维细胞生长因子bFGF、肝素、或其组合。In another preferred embodiment, the neural stem cell culture medium contains epidermal growth factor EGF, basic fibroblast growth factor bFGF, heparin, or a combination thereof.
在另一优选例中，所述的培养为至少培养4代，较佳地，至少5-8代，更佳地，至少10-15代。In another preferred embodiment, the culture is cultured for at least 4 passages, preferably at least 5-8 passages, more preferably at least 10-15 passages.
在另一优选例中，所述的小分子化合物组合中HDACs抑制剂包括丙戊酸钠(VPA)、丁酸钠(NaB)、或曲古抑菌素A(TSA)；和/或In another preferred embodiment, the HDACs inhibitor in the small molecule compound combination comprises sodium valproate (VPA), sodium butyrate (NaB), or trichostatin A (TSA); and/or
所述的GSK-3抑制剂包括CHIR99021、氯化锂(LiCl)、或碳酸锂(Li2CO3)；和/或The GSK-3 inhibitor comprises CHIR99021, lithium chloride (LiCl), or lithium carbonate (Li 2 CO 3 ); and/or
所述的TGF-β信号通路抑制剂包括Repsox、SB431542、或曲尼司特(Tranilast)。The TGF-β signaling pathway inhibitors include Repx, SB431542, or Tranilast.
在另一优选例中，所述小分子化合物组合中各组分的最低有效浓度如下所示：In another preferred embodiment, the minimum effective concentration of each component in the small molecule compound combination is as follows:
HDACs抑制剂：VPA：0.2-1mM，较佳地0.3-0.8mM，更佳地，0.4-0.6mM；NaB0.2-1mM，较佳地0.3-0.8mM，更佳地，0.4-0.6mM；TSA 5-20nM，8-15nM，更 佳地，10-12nM；HDACs inhibitor: VPA: 0.2-1 mM, preferably 0.3-0.8 mM, more preferably 0.4-0.6 mM; NaB 0.2-1 mM, preferably 0.3-0.8 mM, more preferably 0.4-0.6 mM; TSA 5-20nM, 8-15nM, more Good place, 10-12nM;
GSK-3抑制剂：CHIR990211-5μM，较佳地2-4μM；LiCl 0.5-3μM，较佳地1-2μM；Li2CO30.05-1mM，较佳地，0.1-0.8mM，更佳地，0.2-0.5mM；GSK-3 inhibitor: CHIR990211-5 μM, preferably 2-4 μM; LiCl 0.5-3 μM, preferably 1-2 μM; Li 2 CO 3 0.05-1 mM, preferably 0.1-0.8 mM, more preferably, 0.2-0.5 mM;
TGF-β抑制剂信号通路：Repsox 0.2-3μM，较佳地，0.5-2μM；SB4315420.2-3μM，较佳地0.5-2μM；Tranilast 10-50μM，较佳地，20-40μM。TGF-β inhibitor signaling pathway: Repsox 0.2-3 μM, preferably 0.5-2 μM; SB4315420.2-3 μM, preferably 0.5-2 μM; Tranilast 10-50 μM, preferably 20-40 μM.
本发明第五方面，提供了一种神经干细胞，所述的神经干细胞是由本发明第四方面所述的方法制备的。According to a fifth aspect of the invention, there is provided a neural stem cell prepared by the method of the fourth aspect of the invention.
在另一优选例中，所述的神经干细胞具有以下一个或多个特征：In another preferred embodiment, the neural stem cell has one or more of the following characteristics:
(i)神经干细胞特异性基因高表达；(i) high expression of neural stem cell-specific genes;
(ii)神经干细胞多能性基因高表达；(ii) high expression of neural stem cell pluripotency genes;
(iii)神经干细胞具有分化多潜能性。(iii) Neural stem cells have differentiated pluripotency.
在另一优选例中，所述的神经干细胞特异性基因包括Nestin、Sox2、Blbp、Pax6和Ascl1。In another preferred embodiment, the neural stem cell-specific genes include Nestin, Sox2, Blpp, Pax6, and Ascl1.
在另一优选例中，所述的神经干细胞多能性基因包括Nestin、Sox2、Blbp和Pax6。In another preferred embodiment, the neural stem cell pluripotency genes include Nestin, Sox2, Blpp, and Pax6.
本发明第六方面，提供了本发明第五方面神经干细胞的用途，用于制备预防或治疗神经***疾病的药物组合物。According to a sixth aspect of the invention, there is provided a use of the neural stem cell of the fifth aspect of the invention for the preparation of a pharmaceutical composition for preventing or treating a nervous system disease.
在另一优选例中，所述的神经***疾病包括神经退行性病变、由于基因突变引起的神经***疾病，以及因脑外伤或脑溢血等导致的神经***病变。In another preferred embodiment, the neurological diseases include neurodegenerative diseases, neurological diseases caused by genetic mutations, and neurological diseases caused by brain trauma or cerebral hemorrhage.
在另一优选例中，所述的神经***疾病包括阿尔兹海默病、帕金森症、或亨廷顿舞蹈症。In another preferred embodiment, the neurological condition comprises Alzheimer's disease, Parkinson's disease, or Huntington's disease.
本发明第七方面，提供了一种组合物，所述的组合物包括：本发明第五方面所述的神经干细胞。According to a seventh aspect of the invention, there is provided a composition comprising: the neural stem cell of the fifth aspect of the invention.
在另一优选例中，所述的组合物包括药物组合物、食品组合物、保健品组合物。In another preferred embodiment, the composition comprises a pharmaceutical composition, a food composition, a health care composition.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.
附图说明DRAWINGS
图1、VCRP在正常生理低氧条件下诱导小鼠胚胎成纤维细胞(MEFs)形成致密细胞克隆。图1A显示了VCRP处理15天后，不同氧浓度(21％、3％和5％)条件下 MEFs的形态学变化。20,0000细胞种植于6孔板并在21％氧浓度下培养24小时后更换为包含有小分子化合物组合VCRP(0.5mM VPA、3μM CHIR99021、1μM Repsox和2μM Parnate)的KSR培养液，每5天更换一次培养液直至20天；处理至第10天，仅正常生理低氧条件下的药物处理组开始出现致密细胞克隆。右图为细胞克隆数目统计。图1B显示了正常生理低氧条件下VCR处理组的致密细胞克隆的碱性磷酸酶(AP)表达量显著增高。每20,0000细胞中约有40个克隆，其中3/4的克隆表达碱性磷酸酶。标尺为200μm；所有数据采用mean±SEM；代表性图片来自于至少三次的独立实验。Figure 1. VCRP induces the formation of dense cell clones of mouse embryonic fibroblasts (MEFs) under normal physiological hypoxia. Figure 1A shows the different oxygen concentrations (21%, 3% and 5%) after 15 days of VCRP treatment. Morphological changes in MEFs. 20,000 cells were seeded in 6-well plates and cultured for 24 hours at 21% oxygen concentration and replaced with KSR medium containing small molecule compound combination VCRP (0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox and 2 μM Parnate) for 5 The culture medium was changed once a day for 20 days; on the 10th day, only the densely treated cell clones began to appear in the drug-treated group under normal physiological hypoxia. The picture on the right shows the number of cell clones. Figure 1B shows that the expression of alkaline phosphatase (AP) in the dense cell clone of the VCR-treated group was significantly increased under normal physiological hypoxia. There are about 40 clones per 20,000 cells, of which 3/4 clones express alkaline phosphatase. The scale is 200 μm; all data are taken with mean ± SEM; representative images are from at least three independent experiments.
图2、筛选VCRP组合中的必要化合物。图2A显示了正常生理低氧条件下，不同化合物组合诱导MEFs产生的克隆数目。图2B显示了正常生理低氧条件下，VCR(0.5mM VPA、3μM CHIR99021和1μM Repsox)基础之上添加其他化合物(1μM OAC1(O),7.5μM Luteolin(L),300ng/mL poly I:C(I))诱导细胞产生的克隆数目(第15天)。图2C显示了MEFs在不同氧浓度(21％和5％)下经VCR处理10天后的Sox2表达量检测。图2D显示了正常生理低氧条件下，不同化合物及其组合处理细胞10天后的Sox2表达量检测。所有数据采用mean±SEM；代表性图片来自于至少三次的独立实验。Figure 2. Screening for essential compounds in a VCRP combination. Figure 2A shows the number of clones produced by different compound combinations induced by MEFs under normal physiological hypoxic conditions. Figure 2B shows the addition of other compounds (1 μM OAC1(O), 7.5 μM Luteolin (L), 300 ng/mL poly I:C) based on VCR (0.5 mM VPA, 3 μM CHIR99021 and 1 μM Repsox) under normal physiological hypoxia. (I)) Number of clones induced by the induced cells (Day 15). Figure 2C shows the detection of Sox2 expression levels of MEFs after 10 days of VCR treatment at different oxygen concentrations (21% and 5%). Figure 2D shows the detection of Sox2 expression levels after treatment of cells with different compounds and their combinations for 10 days under normal physiological hypoxia. All data were taken with mean ± SEM; representative images were from at least three independent experiments.
图3、化合物组合VCR在生理正常生理低氧条件下诱导小鼠胚胎成纤维细胞到神经干细胞。图3A显示了化合物组合VCR在生理正常生理低氧条件下诱导碱性磷酸酶AP阳性的致密克隆。小鼠胚胎成纤维细胞在21％(正常氧压)或5％(正常生理低氧)O2培养条件下，用化合物组合VCR(0.5mM VPA、3μM CHIR99021和1μM Repsox)处理。克隆是在VCR处理15天后计数的。柱形图代表起始的20万细胞诱导产生的克隆数目。图3B显示了定量链式聚合酶反应检测多能性相关基因的相对表达水平。所有的样品都归一化到第0天，第0天的值为1。图3C显示了VCR诱导产生神经干细胞状的细胞群。VCR处理的小鼠胚胎成纤维细胞经消化后在神经胚胎干细胞培养基中培养(小鼠胚胎成纤维细胞和第1、5和13代)。图3D显示了定量链式聚合酶反应检测神经干细胞特异基因的相对表达水平。所有的样品都归一化到小鼠胚胎成纤维细胞的表达水平，小鼠胚胎成纤维细胞的表达水平值为1。图3E显示了免疫荧光染色Nestin、Pax6和Sox2。细胞核用DAPI染色。Nestin/Pax6和Nestin/Sox2双阳性的细胞在右边的通道展示。图片标尺为50μm。图3F显示了从小鼠胚胎成纤维细胞诱导神经干细胞的实验策略流程图。数据是平均值±标准误，且至少进行三次重复实验，***P<0.001,**P<0.01。 Figure 3. Compound combination VCR induces mouse embryonic fibroblasts to neural stem cells under physiologically normal physiological hypoxia conditions. Figure 3A shows a dense clone of a compound-combined VCR that induces alkaline phosphatase AP-positive under physiologically normal physiological hypoxic conditions. Mouse embryonic fibroblasts were treated with compound combination VCR (0.5 mM VPA, 3 μM CHIR99021 and 1 μM Repsox) under 21% (normal oxygen pressure) or 5% (normal physiological hypoxia) O2 culture conditions. Clones were counted 15 days after VCR treatment. The bar graph represents the number of clones induced by the initial 200,000 cells. Figure 3B shows the relative expression levels of pluripotency-related genes detected by quantitative chain polymerase reaction. All samples were normalized to day 0 with a value of 1 on day 0. Figure 3C shows a VCR-induced cell population that produces neural stem cells. VCR-treated mouse embryonic fibroblasts were digested and cultured in neural embryonic stem cell culture medium (mouse embryonic fibroblasts and passages 1, 5 and 13). Figure 3D shows the relative expression levels of neural stem cell-specific genes detected by quantitative chain polymerase reaction. All samples were normalized to the expression level of mouse embryonic fibroblasts, and the expression level of mouse embryonic fibroblasts was 1. Figure 3E shows immunofluorescence staining of Nestin, Pax6 and Sox2. The nuclei were stained with DAPI. Nestin/Pax6 and Nestin/Sox2 double positive cells are displayed in the channel on the right. The picture scale is 50 μm. Figure 3F shows a flow chart of an experimental strategy for inducing neural stem cells from mouse embryonic fibroblasts. Data are mean ± standard error and at least three replicates were performed, ***P < 0.001, **P < 0.01.
图4、诱导MEFs转分化为神经干细胞(ciNPCs)。图4A显示了第1代ciNPCs的形态，Nestin、Sox2和Pax6的表达量检测。VCR处理的MEFs在包含有生长因子的神经培养液中继续培养7-10天，可观察到类似神经干细胞形态出现。通过免疫荧光染色检测了神经干细胞标记蛋白Nestin、Sox2和Pax6等的表达，并对阳性细胞进行统计。这类细胞经过进一步悬浮培养可形成Sox2和Nestin阳性的神经球。图4B显示了原代神经球经过更长代数的悬浮培养可以富集Nestin、Sox2和Pax6等阳性细胞的比例。标尺为200μm；所有数据采用mean±SEM；代表性图片来自于至少三次的独立实验。Figure 4. Induction of MEFs transdifferentiation into neural stem cells (ciNPCs). Figure 4A shows the morphology of the first generation ciNPCs, the expression levels of Nestin, Sox2 and Pax6. VCR-treated MEFs were further cultured for 7-10 days in a culture medium containing growth factors, and similar neural stem cell morphology was observed. The expression of neural stem cell marker proteins Nestin, Sox2 and Pax6 was detected by immunofluorescence staining, and positive cells were counted. Such cells undergo further suspension culture to form Sox2 and Nestin-positive neurospheres. Figure 4B shows the proportion of positive cells such as Nestin, Sox2 and Pax6 that can be enriched by suspension culture of primary neurospheres over a longer generation. The scale is 200 μm; all data are taken with mean ± SEM; representative images are from at least three independent experiments.
图5、纯化合物诱导的神经干细胞比例统计。图5A显示了第13代ciNPCs的免疫荧光染色(Nestin和Sox2)。图5B显示了图5A中Nestin和Sox2阳性细胞数目统计。图5C显示了第13代ciNPCs的免疫荧光染色(Nestin和Pax6)。图5D显示了图5C中Nestin和Pax6阳性细胞数目统计。代表性图片来自于至少三次的独立实验。Figure 5. Statistics of the proportion of neural stem cells induced by pure compounds. Figure 5A shows immunofluorescence staining of the 13th generation ciNPCs (Nestin and Sox2). Figure 5B shows the statistics of the number of Nestin and Sox2 positive cells in Figure 5A. Figure 5C shows immunofluorescence staining of the 13th generation ciNPCs (Nestin and Pax6). Figure 5D shows the statistics of the number of Nestin and Pax6 positive cells in Figure 5C. Representative pictures are from at least three independent experiments.
图6、化合物诱导的神经干细胞的增殖和自我更新。图6A显示了第13代的化合物诱导的神经干细胞Ki67和Nestin免疫荧光染色代表性图片。图6B显示了悬浮培养的第13代的化合物诱导的神经干细胞和第5代的对照神经干细胞的神经球。图6C显示了贴壁单层培养的第23代化合物诱导的神经干细胞的Nestin、Pax6和Sox2的免疫荧光染色。图6D显示了第23代化合物诱导的神经干细胞形成的神经球Nestin、Pax6和Sox2的免疫荧光染色。细胞核用DAPI染色。图片标尺为50μm。Figure 6. Compound-induced proliferation and self-renewal of neural stem cells. Figure 6A shows a representative picture of the 13th generation of compound-induced immunofluorescence staining of neural stem cells Ki67 and Nestin. Figure 6B shows the neurospheres of the 13th passage of the compound-induced neural stem cells and the 5th generation of control neural stem cells in suspension culture. Figure 6C shows immunofluorescence staining of Nestin, Pax6 and Sox2 of neural stem cells induced by the 23rd generation compound in adherent monolayer culture. Figure 6D shows immunofluorescence staining of neurospheres Nestin, Pax6 and Sox2 induced by the 23rd generation compound-induced neural stem cells. The nuclei were stained with DAPI. The picture scale is 50 μm.
图7、化合物诱导的神经干细胞的基因组转录谱分析。图7A显示了聚类和热图分析芯片数据。比较来自小鼠胚胎成纤维细胞核第5及13代的化合物诱导的神经干细胞。在热图中，红色表示相对于小鼠胚胎成纤维细胞增加表达而绿色表示减少表达。图7B显示了配对散点图分析251个通过“neuro”搜索来的芯片数据库基因。红色点表达上调表达，绿色表示下调表达，而灰色表示无明显变化。图7C显示了韦恩图展示相对于小鼠胚胎成纤维细胞高表达(≥10-fold,P<0.05)的基因中，第5代的和第13代的化合物诱导的神经干细胞和对照干细胞的重叠情况。图7D显示了基因本土分析(GO分析)774个图7C中的共用基因。P值代表EASE打分。Figure 7. Genomic transcriptional profiling of compound-induced neural stem cells. Figure 7A shows clustering and heat map analysis chip data. The neural stem cells induced by the compounds from the 5th and 13th generations of mouse embryonic fibroblasts were compared. In the heat map, red indicates increased expression relative to mouse embryonic fibroblasts and green indicates decreased expression. Figure 7B shows a paired scatter plot analysis of 251 chip database genes by "neuro" search. Red dot expression is up-regulated, green indicates down-regulated expression, and gray indicates no significant change. Figure 7C shows that the Venn diagram shows the induction of neural stem cells and control stem cells from the fifth and third generations of the gene with high expression (≥10-fold, P<0.05) relative to mouse embryonic fibroblasts. Overlap. Figure 7D shows the gene-local analysis (GO analysis) of 774 shared genes in Figure 7C. The P value represents the EASE score.
图8、起始细胞MEFs、对照组神经干细胞(NPCs)、第5代和第31代ciNPC的全基因组表达谱分析。图8A显示了与MEFs比较，NPCs、ciNPC第5代和第31 代中表达上调基因的基因本位分析(GO)。图8B显示了部分神经类基因、多潜能相关基因和成纤维细胞特异表达基因的热图。图8C显示了从MEFs到第5代和第31代ciNPC，表达量逐渐下调的基因(233条)的功能注释。图8D显示了与MEFs比较，NPCs、第5代和第31代ciNPC中表达下调基因的基因本位分析(GO)。图8E显示了脑组织特定区域表达基因的热图。P值代表EASE值；红色代表高表达，绿色代表低表达。Figure 8. Whole genome expression profiling of starting cell MEFs, control neural stem cells (NPCs), passage 5 and passage 31 ciNPC. Figure 8A shows NPCs, ciNPC 5th and 31st compared to MEFs Gene-based analysis (GO) of up-regulated genes in generations. Figure 8B shows a heat map of a portion of a neural gene, a pluripotency-related gene, and a fibroblast-specific gene. Figure 8C shows functional annotations of genes (233) that are gradually down-regulated from MEFs to 5th and 31st generation ciNPCs. Figure 8D shows gene-based analysis (GO) of down-regulated genes in NPCs, 5th and 31st generation ciNPCs compared to MEFs. Figure 8E shows a heat map of genes expressed in specific regions of brain tissue. The P value represents the EASE value; red represents high expression and green represents low expression.
图9、起始MEFs中不包含有神经干细胞(NPCs)。图9A显示了神经干细胞标记蛋白的免疫荧光染色。原代MEFs培养至第3代后分别培养于包含有10％血清的DMEM或神经干细胞培养液(NEM)中7天用于后期检测。数据表明起始MEFs中不包含有Sox2、Pax6或Nestin阳性的神经干细胞，小鼠脑组织分离得到的原代神经干细胞作为阳性对照。图9B显示了qRT-PCR进一步分析在神经干细胞中特异性高表达基因的表达量。标尺为200μm；所有数据采用mean±SEM；代表性图片来自于至少三次的独立实验。Figure 9. Neural stem cells (NPCs) are not included in the starting MEFs. Figure 9A shows immunofluorescence staining of neural stem cell marker proteins. Primary MEFs were cultured to the third passage and cultured in DMEM or neural stem cell culture medium (NEM) containing 10% serum for 7 days for later detection. The data indicated that the initial MEFs did not contain Sox2, Pax6 or Nestin-positive neural stem cells, and the primary neural stem cells isolated from mouse brain tissue served as a positive control. Figure 9B shows qRT-PCR further analysis of the amount of expression of a specifically overexpressed gene in neural stem cells. The scale is 200 μm; all data are taken with mean ± SEM; representative images are from at least three independent experiments.
图10、与HDACs、TGF-β、GSK-3和正常生理低氧相关信号通路基因的分析。图10A显示了与HDACs相关联的196个基因的聚类分析及热图。图10B显示了与TGF-β相关联的74个基因的聚类分析及热图。图10C显示了与GSK-3相关联的114个基因的聚类分析及热图。图10D显示了与正常生理低氧相关联的71个基因的聚类分析及热图。Figure 10. Analysis of genes associated with HDACs, TGF-β, GSK-3, and normal physiological hypoxia-related signaling pathways. Figure 10A shows cluster analysis and heat map of 196 genes associated with HDACs. Figure 10B shows cluster analysis and heat map of 74 genes associated with TGF-β. Figure 10C shows cluster analysis and heat map of 114 genes associated with GSK-3. Figure 10D shows cluster analysis and heat map of 71 genes associated with normal physiological hypoxia.
图11、ciNPCs体外分化的多潜能性。图11A显示了第5代ciNPCs在包含有生长因子的神经基础培养液中分化7天，可检测到GFAP阳性的胶质细胞和Tuj1阳性的非成熟神经元(左图)和MAP2阳性的成熟神经元(右图)。图11B显示了第13代ciNPCs分化得到的成熟神经元。第13代ciNPCs于神经元分化特异培养液中培养4周后，可检测到GAD67阳性和Synapsin阳性细胞。DAPI标记细胞核；标尺为50μm(左图)，右图为图12B中Synapsin/Tuj1免疫荧光染色图放大8倍。Figure 11. Multipotency of ciNPCs in vitro differentiation. Figure 11A shows that 5th generation ciNPCs differentiated in growth factor-containing neural basal medium for 7 days, and detect GFAP-positive glial cells and Tuj1-positive immature neurons (left panel) and MAP2-positive mature nerves. Yuan (right). Figure 11B shows mature neurons differentiated from passage 13 ciNPCs. After the 13th generation ciNPCs were cultured for 4 weeks in the neuron differentiation specific medium, GAD67-positive and Synapsin-positive cells were detected. DAPI labeled nuclei; the scale is 50 μm (left), and the right panel is 8 times larger than the Synapsin/Tuj1 immunofluorescence stain in Figure 12B.
图12、化合物诱导的神经干细胞的体外和体内分化的多能性。图12A显示了星形胶质细胞标记基因GFAP，神经元标记基因Tuj1和Map2，少突胶质细胞标记基因Olig2和Mbp在第13代化合物诱导的神经干细胞在分化培养基中培养后的表达。图12B显示了第13代化合物诱导的神经干细胞在神经元分化培养基中培养4周后，免疫荧光染色NeuN、Glutamate和Synapsin。细胞核用DAPI染色。图片标尺为50μm。三次重复实验的代表性图片被展示。图12C显示了电流钳记录化 合物诱导的神经干细胞分化的神经元的代表性的动作电位。图12D显示了化合物诱导的神经干细胞分化的神经元的代表性的自发突触后电流。图12E显示了电压钳记录化合物诱导的神经干细胞分化的神经元的代表性的Na+电流。图12F显示了GFP标记的化合物诱导的神经干细胞移植一个月后，检测少突胶质细胞标记基因Olig2，星形胶质细胞标记基因GFAP和神经元标记基因NeuN。GFP标记的化合物诱导的神经干细胞移植E13.5胚胎一个月后，切片小鼠脑袋并免疫荧光染色，箭头指示分别表达Olig2、GFAP或NeuN的GFP阳性细胞。细胞核用DAPI染色。图片图12A和图12B标尺为50μm。图片图12F标尺为15μm.24个小鼠胚胎用GFP标记的化合物诱导的神经干细胞移植。Figure 12. Pluripotency of compound-induced differentiation of neural stem cells in vitro and in vivo. Figure 12A shows the expression of the astrocyte marker gene GFAP, the neuronal marker genes Tuj1 and Map2, and the oligodendrocyte marker genes Olig2 and Mbp after culture of the 13th generation compound-induced neural stem cells in a differentiation medium. Figure 12B shows immunofluorescence staining of NeuN, Glutamate and Synapsin after passage of the 13th generation of compound-induced neural stem cells in neuron differentiation medium for 4 weeks. The nuclei were stained with DAPI. The picture scale is 50 μm. Representative pictures of three replicate experiments were shown. Figure 12C shows the current clamp recording Representative Action Potentials of Neurons Induced by Neural Stem Cell Differentiation Figure 12D shows representative spontaneous post-synaptic currents of neurons induced by compound-induced neural stem cell differentiation. Figure 12E shows a representative Na+ current of a voltage clamp recording neuron-differentiated neuron-differentiated neurons. Figure 12F shows the oligodendrocyte marker gene Olig2, the astrocyte marker gene GFAP and the neuronal marker gene NeuN detected one month after GFP-labeled compound-induced neural stem cell transplantation. GFP-labeled compound-induced neural stem cells were transplanted to E13.5 embryos one month later, and the mouse brains were sectioned and immunofluorescently stained, and the arrows indicated GFP-positive cells expressing Olig2, GFAP or NeuN, respectively. The nuclei were stained with DAPI. The scales of Figures 12A and 12B are 50 μm. Figure 12F scale is a 15 μm. 24 mouse embryos transplanted with GFP-labeled compounds for neural stem cell transplantation.
图13、GFP标记的ciNPCs鉴定。图13A显示了第17代ciNPC被表达GFP的慢病毒感染后仍旧具有形成神经球能力。图13B显示了GFP-ciNPCs表达神经干细胞标记蛋白Nestin和Sox2。图13C显示了GFP-ciNPCs仍具有分化为GFAP阳性胶质细胞、Tuj1阳性神经元和Olig2阳性少突状胶质细胞的多潜能性。箭头指示为表达Tuj1或Olig2的GFP阳性细胞。标尺为50μm。Figure 13. Identification of GFP-tagged ciNPCs. Figure 13A shows that the 17th generation ciNPC still has the ability to form neurospheres after infection with a GFP-expressing lentivirus. Figure 13B shows that GFP-ciNPCs express neural stem cell marker proteins Nestin and Sox2. Figure 13C shows that GFP-ciNPCs still have the pluripotency of differentiation into GFAP-positive glial cells, Tujl-positive neurons, and Olig2-positive oligodendrocytes. Arrows indicate GFP positive cells expressing Tuj1 or Olig2. The scale is 50 μm.
图14、体内移植表达GFP的ciNPCs。图14A显示了E13.5天小鼠胚胎脑组织接种GFP-ciNPCs 1周后，GFP阳性细胞的体内分布。图14B显示了GFP-ciNPCs在体内移植1周后仍旧表达Ki67。图14C显示了GFP-ciNPCs在体内移植1周后分化为Olig2阳性或GFAP阳性细胞。图14D显示了GFP-ciNPCs在体内移植1个月后分化为Mbp阳性少突状胶质细胞或Tuj1阳性神经元。图14E显示了GFP-ciNPCs在体内移植1个月后未检测到Ki67表达。标尺为50μm；代表性图片来自于至少10只移植后小鼠的脑组织。Figure 14. Transplantation of GFP-expressing ciNPCs in vivo. Figure 14A shows the in vivo distribution of GFP-positive cells after 1 week of inoculation of GFP-ciNPCs in E13.5 day mouse embryonic brain tissue. Figure 14B shows that GFP-ciNPCs still express Ki67 after 1 week of transplantation in vivo. Figure 14C shows that GFP-ciNPCs differentiate into Olig2-positive or GFAP-positive cells after 1 week of transplantation in vivo. Figure 14D shows that GFP-ciNPCs differentiate into Mbp-positive oligodendrocytes or Tuj1-positive neurons after 1 month of transplantation in vivo. Figure 14E shows that Ki67 expression was not detected after 1 month of transplantation of GFP-ciNPCs in vivo. The scale is 50 μm; representative pictures are from brain tissue of at least 10 transplanted mice.
图15、正常生理低氧条件下，化合物组合NLS或TLT诱导MEFs转分化为神经干细胞(ciNPCs)。图15A显示了NLS诱导获得的第5代ciNPCs的贴壁形态、神经球和针对Sox2、Pax6以及Nestin的免疫荧光染色。图15B显示了TLT诱导获得的第5代ciNPCs的贴壁形态、神经球和针对Sox2、Pax6以及Nestin的免疫荧光染色。标尺为200μm；代表性图片来自于至少三次的独立实验。Figure 15. Compounds combined with NLS or TLT induced MEFs transdifferentiation into neural stem cells (ciNPCs) under normal physiological hypoxic conditions. Figure 15A shows the adherent morphology of the 5th generation ciNPCs induced by NLS, neurospheres, and immunofluorescence staining for Sox2, Pax6, and Nestin. Figure 15B shows the adherent morphology, neurospheres, and immunofluorescence staining for Sox2, Pax6, and Nestin of the 5th generation ciNPCs obtained by TLT induction. The scale is 200 μm; representative images are from at least three independent experiments.
图16、其它化合物组合诱导神经干细胞。图16A显示了定量链式聚合酶反应分析NLS(0.5mM NaB,1mM LiCl和1μM SB431542)(左图)或TLT(10nM TSA,0.3mM Li2CO3和30μM Tranilast)在5％O2条件下处理的小鼠成纤维细胞的Sox2表达水平。所有的样品都归一化到第0天的DMSO组，它的值为1。图16B显示了NLS或TLT处理获得的神经干细胞的形态图和Nestin,Sox2及Pax6免疫荧光染 色。细胞核用DAPI染色。图片标尺为50μm。图16C显示了定量链式聚合酶反应分析神经干细胞特异基因的表达水平。所有的样品都归一化到MEF组，它的值为1。数据是平均值±标准误。Figure 16. Combination of other compounds induces neural stem cells. Figure 16A shows quantitative chain polymerase reaction analysis of NLS (0.5 mM NaB, 1 mM LiCl and 1 μM SB431542) (left panel) or TLT (10 nM TSA, 0.3 mM Li 2 CO 3 and 30 μM Tranilast) under 5% O 2 conditions Sox2 expression levels of treated mouse fibroblasts. All samples were normalized to the DMSO group on day 0 with a value of 1. Figure 16B shows the morphology of neural stem cells obtained by NLS or TLT treatment and the immunofluorescence staining of Nestin, Sox2 and Pax6. The nuclei were stained with DAPI. The picture scale is 50 μm. Figure 16C shows the quantitative chain polymerase reaction analysis of the expression level of neural stem cell-specific genes. All samples were normalized to the MEF group with a value of 1. Data are mean ± standard error.
图17、从小鼠尾尖成纤维细胞和人的尿液来源的上皮细胞(简称尿细胞)诱导产生神经干细胞并定性。图17A显示了定量链式聚合酶反应分析VCR在正常生理低氧条件下处理小鼠尾尖成纤维细胞后多能性相关基因的表达水平。所有的样品都归一化到d0组，它的值为1。图17B显示了小鼠尾尖成纤维细胞和第1代从小鼠尾尖成纤维细胞产生的神经干细胞的形态图。图17C显示了第13代从小鼠尾尖成纤维细胞产生的神经干细胞的形态图和Nestin,Sox2及Pax6免疫荧光染色。图17D显示了定量链式聚合酶反应分析第16代从小鼠尾尖成纤维细胞产生的神经干细胞的神经干细胞特异基因的表达水平。所有的样品都归一化到TTF组，它的值为1。图17E显示了星形胶质细胞标记基因GFAP，神经元标记基因Tuj1和Map2，少突胶质细胞标记基因Olig2和Mbp在小鼠尾尖成纤维细胞衍生的第13代化合物诱导的神经干细胞在分化培养基中培养后的表达。图17F显示了人的尿细胞在正常生理低氧处理VCR前后的相差图片。图17G显示了定量链式聚合酶反应分析VCR在正常生理低氧条件下处理人尿细胞后多能性相关基因的表达水平。所有的样品都归一化到d0组，它的值为1。图17H显示了第5代从人尿细胞产生的神经干细胞和对照人的诱导神经干细胞的贴壁单层培养和悬浮培养的形态图。人的诱导神经干细胞从特定的转录因子诱导而来。图17I显示了定量链式聚合酶反应分析神经干细胞特异基因的表达水平。所有的样品都归一化到hUCs组，它的值为1。图17J显示了星形胶质细胞标记基因GFAP，神经元标记基因Tuj1和Map2在人的尿细胞衍生的第5代化合物诱导的神经干细胞在分化培养基中培养后的表达。细胞核用DAPI染色。图片标尺为50μm。数据是平均值±标准误。Figure 17. Induction of neural stem cells from mouse tail-tip fibroblasts and human urine-derived epithelial cells (abbreviated as urine cells) and qualitative. Figure 17A shows quantitative chain polymerase reaction analysis of VCR expression levels of pluripotency-related genes after treatment of mouse tail tip fibroblasts under normal physiological hypoxic conditions. All samples were normalized to group d0 with a value of one. Figure 17B shows a morphological map of mouse tail tip fibroblasts and the first generation of neural stem cells produced from mouse tail tip fibroblasts. Figure 17C shows the morphology of neural stem cells from the 13th generation of mouse tail tip fibroblasts and immunofluorescence staining of Nestin, Sox2 and Pax6. Figure 17D shows the expression level of neural stem cell-specific genes of neural stem cells produced from mouse tail-tip fibroblasts in the 16th generation by quantitative chain polymerase reaction analysis. All samples were normalized to the TTF group with a value of 1. Figure 17E shows the astrocyte marker gene GFAP, the neuronal marker genes Tuj1 and Map2, the oligodendrocyte marker gene Olig2 and Mbp in mouse tail-tip fibroblast-derived 13th-generation compound-induced neural stem cells Expression after culture in differentiation medium. Figure 17F shows a phase contrast picture of human urine cells before and after normal physiological hypoxic treatment of VCR. Figure 17G shows quantitative chain polymerase reaction analysis of VCR expression levels of pluripotency-related genes after treatment of human urine cells under normal physiological hypoxic conditions. All samples were normalized to group d0 with a value of one. Fig. 17H is a view showing the morphology of the adherent monolayer culture and suspension culture of the neural stem cells induced by the fifth generation of neural stem cells and control humans derived from human urine cells. Human induced neural stem cells are induced from specific transcription factors. Figure 17I shows the quantitative chain polymerase reaction analysis of the expression level of neural stem cell-specific genes. All samples were normalized to the hUCs group with a value of 1. Figure 17J shows the expression of the astrocyte marker gene GFAP, neuronal marker genes Tuj1 and Map2, after cultured in differentiation medium, induced by human urine cell-derived 5th generation compound. The nuclei were stained with DAPI. The picture scale is 50 μm. Data are mean ± standard error.
三次重复实验的代表性图片被展示。Representative pictures of three replicate experiments were shown.
图18、正常生理低氧条件下，VCR诱导小鼠尾尖成纤维细胞转分化为神经干细胞(第5代)。Nestin、Sox2和Pax6的免疫荧光染色(左图)及对应的统计图(右图)。标尺为50μm；代表性图片来自于至少三次的独立实验。Figure 18. VCR induced mouse tail tip fibroblasts to differentiate into neural stem cells under normal physiological hypoxia (5th generation). Immunofluorescence staining of Nestin, Sox2 and Pax6 (left panel) and corresponding statistical map (right panel). The scale is 50 μm; representative images are from at least three independent experiments.
图19、正常生理低氧条件下，VCR诱导人尿液中的细胞转分化为神经干细胞。图19A显示了第11代和第22代的人尿液中的细胞来源ciNPCs的免疫荧光染色(Nestin和Sox2)。图19B显示了人尿液中的细胞来源ciNPCs与阳性对照iNPCs的生长曲线。 Figure 19. VCR induces transdifferentiation of cells in human urine to neural stem cells under normal physiological hypoxic conditions. Figure 19A shows immunofluorescence staining (Nestin and Sox2) of cell-derived ciNPCs in human urine from passages 11 and 22. Figure 19B shows the growth curves of cell-derived ciNPCs and positive control iNPCs in human urine.
具体实施方式detailed description
本发明人经过广泛而深入的研究，经过大量的化合物筛选，首次意外地发现了特定小分子化合物的组合能够诱导体细胞转分化为神经干细胞。实验表明，将组蛋白去乙酰化酶(HDACs)抑制剂、糖原合成酶激酶(GSK-3)抑制剂、转化生长因子β(TGF-β)信号通路抑制剂这三类化合物联合应用于体细胞(如成纤维细胞)时，能够使体细胞进入重编程并转分化为与神经干细胞外形、性能特征(如良好的多能分化性)极其相似的，并具有稳定传代功能的神经干细胞，从而摆脱了只有引入外源基因才能够诱导体细胞分化为神经干细胞的方法。在此基础上，完成了本发明。The inventors have conducted extensive and intensive research, and for the first time, through a large number of compound screenings, it has been unexpectedly discovered that a combination of specific small molecule compounds can induce somatic cell transdifferentiation into neural stem cells. Experiments have shown that the combination of histone deacetylase (HDACs) inhibitors, glycogen synthase kinase (GSK-3) inhibitors, and transforming growth factor beta (TGF-β) signaling pathway inhibitors is applied to the body. When cells (such as fibroblasts), the somatic cells can be reprogrammed and transdifferentiated into neural stem cells that are very similar to neural stem cell shape and performance characteristics (such as good pluripotent differentiation) and have stable passage function. It is possible to get rid of the method of inducing somatic cell differentiation into neural stem cells only by introducing foreign genes. On the basis of this, the present invention has been completed.
组蛋白去乙酰化酶(HDACs)抑制剂Histone deacetylase (HDACs) inhibitor
组蛋白去乙酰化酶是一类蛋白酶,对染色体的结构修饰和基因表达调控发挥着重要的作用。在细胞核内，组蛋白乙酰化与组蛋白去乙酰化过程处于动态平衡，并由组蛋白乙酰化转移酶和组蛋白去乙酰化酶共同调控。组蛋白去乙酰化酶抑制剂则可通过提高染色质特定区域组蛋白乙酰化程度来改变染色质结构，从而调控细胞凋亡及分化相关蛋白的表达和稳定性；按结构类型,组蛋白去乙酰化酶抑制剂大致可以分为:异羟肟酸类化合物(如曲古抑菌素A等)、环状四肽类化合物(如Trapoxin等)、脂肪酸盐类化合物(如丙戊酸钠、丁酸钠等)、苯甲酰胺类化合物(如MS275等)和亲电酮类化合物(如三氟甲基酮等)等。Histone deacetylase is a class of proteases that play an important role in the structural modification of chromosomes and the regulation of gene expression. In the nucleus, histone acetylation is in a dynamic equilibrium with histone deacetylation and is regulated by histone acetyltransferase and histone deacetylase. Histone deacetylase inhibitors can change the chromatin structure by increasing the degree of histone acetylation in specific regions of chromatin, thereby regulating the expression and stability of apoptosis and differentiation-related proteins; by tissue type, histone deacetylation The enzyme inhibitors can be roughly classified into: hydroxamic acids (such as trichostatin A), cyclic tetrapeptides (such as Trapoxin, etc.), fatty acid salts (such as sodium valproate, butyl) Sodium benzoate or the like, a benzamide compound (such as MS275), and an electrophilic ketone compound (such as trifluoromethyl ketone).
糖原合成酶激酶(GSK-3)抑制剂Glycogen synthase kinase (GSK-3) inhibitor
糖原合成酶激酶是一个多功能的丝氨酸/苏氨酸蛋白激酶,不仅参与肝糖代谢过程,而且还参与Wnt和Hedgehog信号通路,通过磷酸化多种底物蛋白来调节细胞的生理过程。糖原合成酶激酶抑制剂作为目前备受关注的小分子抑制剂,对治疗神经退化性疾病、癌症、Ⅱ型糖尿病具有潜在的疗效；可分为ATP竞争性抑制剂和ATP竞争性抑制剂，前者包括Paullones、靛玉红类(Indirubin)、马来酰胺类(Maleimides)、嘧啶类(Pyrimidines)、吡啶类(Pyridines)和吡嗪(Aloisines)等；后者包括Li离子和TDZD衍生物。Glycogen synthase kinase is a multifunctional serine/threonine protein kinase that is involved not only in hepatic glucose metabolism but also in Wnt and Hedgehog signaling pathways, which regulates cellular processes by phosphorylating multiple substrate proteins. As a small molecule inhibitor of current interest, glycogen synthase kinase inhibitors have potential therapeutic effects on the treatment of neurodegenerative diseases, cancer and type II diabetes; they can be divided into ATP competitive inhibitors and ATP competitive inhibitors. The former includes Paullones, Indirubin, Maleimides, Pyrimidines, Pyridines, and Aloisines; the latter includes Li ions and TDZD derivatives.
转化生长因子β(TGF-β)信号通路抑制剂Transforming growth factor beta (TGF-β) signaling pathway inhibitor
TGF-β属于一类促进细胞生长和转化的细胞因子超家族，目前共发现5种亚 型，其胞浆内信号传导通路主要包括膜受体丝氨酸/苏氨酸激酶***和Smad蛋白信号传递***。TGF-β抑制剂研究主要包括抑制TGF-β及其受体的表达(如曲尼司特等)，阻断TGF-β和受体的结合(如SB-431542、LY2157299等)，干扰受体激酶信号传递(如SIS3等)。TGF-β belongs to a class of cytokine superfamilies that promote cell growth and transformation. Type, its intracytoplasmic signaling pathway mainly includes membrane receptor serine/threonine kinase system and Smad protein signaling system. TGF-β inhibitor research mainly includes inhibition of TGF-β and its receptor expression (such as tranilast, etc.), blocking the binding of TGF-β and receptor (such as SB-431542, LY2157299, etc.), interfering with receptor kinase Signal transmission (such as SIS3, etc.).
小分子化合物组合Small molecule compound combination
如本文所用，术语“小分子化合物组合”指的是含有以下组分的组合：(a)组蛋白去乙酰化酶(HDACs)抑制剂；(b)糖原合成酶激酶(GSK-3)抑制剂；(c)转化生长因子β(TGF-β)信号通路抑制剂。此外，所述的小分子化合物组合还可以含有药学上可接受的载体，在这样的情况下，所述的小分子化合物组合即为具有诱导体细胞转分化为神经干细胞活性的药物组合物。As used herein, the term "small molecule compound combination" refers to a combination comprising: (a) a histone deacetylase (HDACs) inhibitor; (b) glycogen synthase kinase (GSK-3) inhibition. (c) Transforming growth factor beta (TGF-β) signaling pathway inhibitor. Further, the small molecule compound combination may further contain a pharmaceutically acceptable carrier, and in this case, the small molecule compound combination is a pharmaceutical composition having an activity of inducing somatic cell transdifferentiation into neural stem cells.
其中，所述的HDACs抑制剂包括VPA(丙戊酸钠)、NaB(丁酸钠)、或TSA(曲古抑菌素A)；Wherein, the HDACs inhibitor comprises VPA (sodium valproate), NaB (sodium butyrate), or TSA (triccobacterin A);
所述的GSK-3抑制剂包括CHIR99021、LiCl(氯化锂)、或Li2CO3(碳酸锂)；The GSK-3 inhibitor comprises CHIR99021, LiCl (lithium chloride), or Li 2 CO 3 (lithium carbonate);
所述的TGF-β抑制剂信号通路包括Repsox、SB431542、或Tranilast(曲尼司特)。The TGF-beta inhibitor signaling pathway includes Repox, SB431542, or Tranilast (Trnilast).
可用于本发明小分子组合物的各组分之间的比例没有任何限制。通常，各组分应当满足其最低的有效浓度。在一优选例中，所述小分子化合物组合中各组分的最低有效浓度如下所示：There is no limitation on the ratio between the components which can be used in the small molecule composition of the present invention. Generally, each component should meet its lowest effective concentration. In a preferred embodiment, the minimum effective concentration of each component in the small molecule compound combination is as follows:
HDACs抑制剂：VPA：0.2-1mM，较佳地0.3-0.8mM，更佳地，0.4-0.6mM；NaB0.2-1mM，较佳地0.3-0.8mM，更佳地，0.4-0.6mM；TSA 5-20nM，8-15nM，更佳地，10-12nM；HDACs inhibitor: VPA: 0.2-1 mM, preferably 0.3-0.8 mM, more preferably 0.4-0.6 mM; NaB 0.2-1 mM, preferably 0.3-0.8 mM, more preferably 0.4-0.6 mM; TSA 5-20 nM, 8-15 nM, more preferably, 10-12 nM;
GSK-3抑制剂：CHIR990211-5μM，较佳地2-4μM；LiCl 0.5-3μM，较佳地1-2μM；Li2CO30.05-1mM，较佳地，0.1-0.8mM，更佳地，0.2-0.5mM；GSK-3 inhibitor: CHIR990211-5 μM, preferably 2-4 μM; LiCl 0.5-3 μM, preferably 1-2 μM; Li 2 CO 3 0.05-1 mM, preferably 0.1-0.8 mM, more preferably, 0.2-0.5 mM;
TGF-β抑制剂信号通路：Repsox 0.2-3μM，较佳地，0.5-2μM；SB4315420.2-3μM，较佳地0.5-2μM；Tranilast 10-50μM，较佳地，20-40μM。TGF-β inhibitor signaling pathway: Repsox 0.2-3 μM, preferably 0.5-2 μM; SB4315420.2-3 μM, preferably 0.5-2 μM; Tranilast 10-50 μM, preferably 20-40 μM.
在本发明中，验证了VCR(VPA、CHIR99021、Repsox)、NLS(NaB、LiCl和SB431542)和TLT(TSA、Li2CO3和Tranilast)的组合具有良好的诱导体细胞分化神经干细胞的活性。当然，本领域技术人员也可以根据本发明的启示，对以上三类抑制剂进行任意的组合，开发新的具有诱导体细胞转分化神经干细胞活性小分子化合物组合。 In the present invention, it was verified that a combination of VCR (VPA, CHIR99021, Repox), NLS (NaB, LiCl, and SB431542) and TLT (TSA, Li 2 CO 3 , and Tranilast) has a good activity of inducing somatic cell differentiation of neural stem cells. Of course, those skilled in the art can also arbitrarily combine the above three types of inhibitors according to the teachings of the present invention to develop a novel small molecule compound combination having induced somatic cell transdifferentiation neural stem cell activity.
神经干细胞Neural stem cell
神经干细胞(Neural Stem Cells，NSCs或Neural progenitor Cells，NPCs)具有分化为神经神经元、星形胶质细胞和少突胶质细胞的能力，能自我更新，并足以提供大量脑组织细胞的细胞群，它可以通过不对等的***方式产生神经组织的各类细胞。在脑脊髓等所有神经组织中，不同的神经干细胞类型产生的子代细胞种类不同，分布也不同。Neural stem cells (NSCs or Neural progenitor cells, NPCs) have the ability to differentiate into neuronal neurons, astrocytes, and oligodendrocytes, self-renewing, and sufficient to provide a large number of brain tissue cells. It can produce various types of cells of nerve tissue through unequal division. In all neural tissues such as the cerebrospinal and spinal cord, different neural stem cell types produce different types of progeny cells and different distributions.
如本文所用，术语“ciNPCs”、“化合物诱导的神经干细胞”可互换使用，指的是体细胞经本发明小分子化合物组合(药物组合物)诱导后，产生的神经干细胞。As used herein, the terms "ciNPCs", "compound-induced neural stem cells" are used interchangeably and refer to neural stem cells produced after induction of somatic cells by a combination of small molecule compounds (pharmaceutical compositions) of the invention.
如本文所用，术语“NSCs”、“NPCs”、“神经干细胞”可互换使用，指的是来自哺乳动物(如人或小鼠)的不同部位的神经干细胞，在本文中，通常用于ciNPC的对照。As used herein, the terms "NSCs", "NPCs", "neural stem cells" are used interchangeably and refer to neural stem cells from different parts of a mammal, such as a human or mouse, and are commonly used herein for ciNPC. Control.
神经干细胞特异性基因Neural stem cell specific gene
如本文所用，术语“神经干细胞特异性基因”指的是较非神经干细胞而言，在神经干细胞中高表达的基因(或其蛋白)。通常，所述的神经干细胞特异性基因包括Sox2、Nestin、Pax6、Ascl1和Blbp等。As used herein, the term "neural stem cell-specific gene" refers to a gene (or protein thereof) that is highly expressed in neural stem cells as compared to non-neuronal stem cells. Generally, the neural stem cell-specific genes include Sox2, Nestin, Pax6, Ascl1, and Blbp.
如本文所用，术语“神经干细胞多能性基因高表达”指的是较非神经干细胞而言，在神经干细胞中高表达的，并与神经干细胞多能分化性能相关的基因(或其蛋白)。通常，所述的神经干细胞多能性基因包括Sox2、Nestin、Pax6、Ascl1和Blbp等。As used herein, the term "high expression of neural stem cell pluripotency gene" refers to a gene (or protein thereof) that is highly expressed in neural stem cells and is associated with pluripotent differentiation properties of neural stem cells compared to non-neuronal stem cells. Generally, the neural stem cell pluripotency genes include Sox2, Nestin, Pax6, Ascl1, and Blbp.
低氧环境Hypoxic environment
如本文所用，术语“低氧环境”指的是模拟体内细胞环境的体外或体内，通常，所述的低氧环境指的是正常生理低氧环境，例如氧浓度(或氧压)界于3％-8％之间的环境，较佳地，所述的正常生理低氧环境指的是氧浓度为4％-6％的环境，更佳地，为5％。As used herein, the term "hypoxic environment" refers to the in vitro or in vivo simulation of the cellular environment of the body. Generally, the hypoxic environment refers to a normal physiological hypoxic environment, such as an oxygen concentration (or oxygen pressure) boundary. The environment between % and 8%, preferably, the normal physiological hypoxic environment refers to an environment having an oxygen concentration of 4% to 6%, more preferably 5%.
本发明实验证明，3％-5％(尤其是5％)的环境是获得神经干细胞效果最佳的环境，且对比于正常氧浓度(约21％)的环境而言，低氧(尤其是正常生理低氧环境)对体细胞转分化为神经干细胞是必须的。 The experiments of the present invention prove that 3%-5% (especially 5%) of the environment is the best environment for obtaining neural stem cells, and compared with the environment of normal oxygen concentration (about 21%), hypoxia (especially normal) Physiological hypoxic conditions are necessary for somatic cell transdifferentiation into neural stem cells.
诱导方法Induction method
本发明诱导体细胞转分化为神经干细胞的方法通常指的是体外的诱导方法，当然也可以根据体外诱导实验进行进一步的体内诱导，这可以根据本领域常规技术或方法进行研究获得。The method for inducing somatic cell transdifferentiation into neural stem cells of the present invention generally refers to an in vitro induction method, and of course, further in vivo induction can be carried out according to an in vitro induction experiment, which can be obtained according to a conventional technique or method in the art.
通常，可在本发明小分子化合物组合存在的条件下，培养体细胞。In general, somatic cells can be cultured in the presence of a combination of small molecule compounds of the present invention.
此外，还可以采用本领域常规的神经干细胞或神经细胞培养基对所述的体细胞进行进一步的培养。优选的，所述的神经干细胞或神经细胞培养基中可以含有表皮生长因子EGF、碱性成纤维细胞生长因子bFGF、肝素、或其组合。Furthermore, the somatic cells can be further cultured using neural stem cells or neural cell culture media conventional in the art. Preferably, the neural stem cell or nerve cell culture medium may contain epidermal growth factor EGF, basic fibroblast growth factor bFGF, heparin, or a combination thereof.
药物组合物Pharmaceutical composition
本发明提供了一种包括本发明所述神经干细胞的组合物。The present invention provides a composition comprising the neural stem cells of the present invention.
优选地，所述的组合物为药物组合物、食品组合物、保健品组合物等。Preferably, the composition is a pharmaceutical composition, a food composition, a nutraceutical composition, or the like.
本发明的药物组合物，包括药学上可接受的载体和有效量活性成分：本发明所述的神经干细胞。The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient: a neural stem cell according to the present invention.
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。As used herein, the term "effective amount" or "effective amount" refers to an amount that can produce a function or activity on a human and/or animal and that can be accepted by a human and/or animal.
如本文所用，“药学上可接受的载体”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和***反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。As used herein, a component of a "pharmaceutically acceptable carrier" is a substance that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, a substance having a reasonable benefit/risk ratio. . The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。The pharmaceutical compositions of the present invention comprise a safe and effective amount of the active ingredient of the present invention together with a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, and the pharmaceutical composition of the present invention is in the form of an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent. For example, it is prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的活性成分每天以约 0.00001mg-50mg/kg动物体重(较佳的0.0001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc. Usually, when the active ingredient of the present invention is about daily A satisfactory effect can be obtained by administering a dose of 0.00001 mg to 50 mg/kg of animal body weight (preferably 0.0001 mg to 10 mg/kg of animal body weight). For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
本发明所述的药学上可接受的载体包括(但不限于)：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。Pharmaceutically acceptable carriers of the invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide materials, cellulose, nanogels, or Its combination. The choice of carrier should be compatible with the mode of administration, which are well known to those of ordinary skill in the art.
本发明还提供了所述药物组合物的用途，用于预防或治疗神经***疾病。The invention also provides the use of the pharmaceutical composition for the prevention or treatment of a neurological condition.
本发明有益效果Advantageous effects of the invention
本发明方法能够在不引入外源性基因的情况下，成功利用特定信号通路的抑制剂的组合，诱导多种体细胞转分化为神经干细胞，且所制备的干细胞外形与神经干细胞极其相似并具有良好的多能分化性能The method of the present invention can successfully utilize a combination of inhibitors of specific signaling pathways to induce transdifferentiation of various somatic cells into neural stem cells without introducing an exogenous gene, and the prepared stem cell shape is very similar to that of neural stem cells and has Good pluripotent differentiation
此外，目前现有的获得病人特异的iNPCs细胞的方法包括通过转一组转录因子诱导转分化或者通过人成纤维细胞来源的iPSCs分化为iNPC获得。根据现有的方法，从获得iPSCs到得到iNPCs将耗费至少3个月的时间。而按照本发明诱导方法，能够在更短的时间能获得相近数量的ciNPCs。In addition, current methods for obtaining patient-specific iNPCs include induction of transdifferentiation by transfection of a set of transcription factors or differentiation of iPCCs derived from human fibroblasts into iNPCs. According to existing methods, it takes at least three months from obtaining iPSCs to getting iNPCs. According to the induction method of the present invention, a similar amount of ciNPCs can be obtained in a shorter time.
因此，本发明为研究病人特异的神经细胞及相关细胞治疗提供了一个更好的备选策略。Thus, the present invention provides a better alternative strategy for studying patient-specific neuronal and related cell therapies.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.
通用方法General method
细胞培养Cell culture
原代小鼠胚胎成纤维细胞(MEFs)分离于E13.5天的小鼠胚胎，如文献所述。简单来说，剪除头部，四肢，内脏组织，生殖腺和脊柱；将剩余的部分剪成小块，再用胰酶消化。分离获得的MEFs传至第3代，然后即可用于其他实验。小鼠尾尖成纤维细胞(TTFs)分离于新生3天的C57BL/6小鼠。简单来说，尾部的前1/5 部分切成小块并且培养6天。从尾尖小块迁移出来的传至第3代，然后就可以用于其他实验。小鼠的MEFs和TTFs在DMEM(Life Technologies,C11965)，37℃，5％CO2中培养，其中添加了10％FBS(PAA Laboratories,A15-101)，1mM GlutaMAX(Life Technologies,35050-061)和0.1mM非必须氨基酸(NEAA,Millipore,TMS-001-C)。小鼠神经干细胞取自E12.5天的小鼠胚胎，在神经细胞扩大培养液(NEM)中培养，添加30ng/ml heprin，20ng/ml EGF和20ng/ml bFGF。人尿细胞(分离自人尿液的上皮细胞)收集和培养在REGM(Lonza,CC-4127)培液中。Primary mouse embryonic fibroblasts (MEFs) were isolated from E13.5 day mouse embryos as described in the literature. Briefly, cut the head, limbs, visceral tissue, gonads and spine; cut the rest into small pieces and digest it with trypsin. The MEFs obtained by the separation were passed to the third generation and then used in other experiments. Mouse tail tip fibroblasts (TTFs) were isolated from newborn C57BL/6 mice for 3 days. Simply put, the first 1/5 of the tail is cut into small pieces and cultured for 6 days. It migrated from the tip of the tail to the third generation and can be used in other experiments. Mouse MEFs and TTFs were cultured in DMEM (Life Technologies, C11965) at 37 ° C, 5% CO 2 supplemented with 10% FBS (PAA Laboratories, A15-101), 1 mM GlutaMAX (Life Technologies, 35050-061) And 0.1 mM non-essential amino acids (NEAA, Millipore, TMS-001-C). Mouse neural stem cells were obtained from E12.5 day mouse embryos and cultured in neuron expansion culture (NEM) with addition of 30 ng/ml heprin, 20 ng/ml EGF and 20 ng/ml bFGF. Human urine cells (epithelial cells isolated from human urine) were collected and cultured in REGM (Lonza, CC-4127) culture medium.
ciNPCs的诱导Induction of ciNPCs
对于MEFs和TTFs的神经干细胞诱导，起始细胞在DMEM中培养24小时后换成KSR培液，其中包含knockout DMEM(Life Technologies,10829-018)15％去除血清替代物，1％NEAA(Life Technologies,35050)，1％Glutamax(Life Technologies,35050-061)，1％丙酮酸钠(Life Technologies,11360)，0.1mMβ巯基乙醇(Life Technologies,21985-023)和1000U/ml的白血病抑制因子(LIF)(Chemicon,ESG1107)。细胞培养在37℃，5％O2(hypoxia)和5％CO2条件下。含有化合物的培液每五天换一次。对于人细胞的ciNPCs诱导，尿细胞铺在Matrigel包被的6孔板培养在RGEM培养液。2天后，培液换成mTeSR(Stem Cell Technologies,05850/05896)并包含化合物组合并且在37℃，5％O2(hypoxia)，5％CO2条件下培养。培液每5天换一次。当小鼠成纤维细胞或人尿细胞培养过程中形成紧密的细胞克隆，包含克隆的细胞混液在添加了生长因子的NEM中进一步培养。ciNPCs在多轮神经球悬浮培养过程中进一步富集。For neural stem cell induction of MEFs and TTFs, the starting cells were cultured in DMEM for 24 hours and then replaced with KSR medium containing knockout DMEM (Life Technologies, 10829-018) 15% serum replacement, 1% NEAA (Life Technologies) , 35050), 1% Glutamax (Life Technologies, 35050-061), 1% sodium pyruvate (Life Technologies, 11360), 0.1 mM β-mercaptoethanol (Life Technologies, 21985-023) and 1000 U/ml of leukemia inhibitory factor (LIF) ) (Chemicon, ESG1107). The cells were cultured at 37 ° C, 5% O 2 (hypoxia) and 5% CO 2 . The culture solution containing the compound is changed every five days. For induction of ciNPCs in human cells, urinary cells were plated in Matrigel-coated 6-well plates in RGEM cultures. After 2 days, the culture solution was changed to mTeSR (Stem Cell Technologies, 05850/05896) and contained a combination of compounds and cultured at 37 ° C, 5% O 2 (hypoxia), 5% CO 2 . The culture solution is changed every 5 days. When a tight cell clone is formed during mouse fibroblast or human urine cell culture, the cell mixture containing the clone is further cultured in NEM supplemented with growth factor. ciNPCs are further enriched during multiple rounds of neurosphere suspension culture.
ciNPCs的体外分化In vitro differentiation of ciNPCs
小鼠成纤维细胞来源的ciNPCs培养在神经细胞扩大培养液中，其中添加EGF(20ng/ml)和bFGF(20ng/ml)。对于通用的神经分化方法，20000ciNPCs铺到PDL/Laminin包被的24孔板上，在不含EGF和bFGF的N2B27(DMEM:F12,1％N2,2％B27)培液中培养。稳定的星形胶质细胞生成诱导是在无生长因子的N2B27中添加BMP4(50ng/ml；R&D Biosystems)和1％FBS。神经元分化则是将ciNPCs铺在PDL/Laminin包被的盖玻片上，在神经分化培养基(Neural basal medium,2％B27,1％N2,10ng/ml BDNF,10ng/ml GDNF,10ng/ml IGF-1,1μM cAMP,200 μM Ascorbic acid)中培养。神经元分子标记基因表达和电生理分析分别在特定的时间点进行检测。而少突胶质细胞的分化，则是将20000细胞铺在PDL/Laminin包被的盖玻片上，在包含bFGF(10ng/mL；Invitrogen)和PDGF-AA(10ng/mL；Peprotech)的N2B27培养中培养7天，然后加入T3(100ng/mL；20Sigma-Aldrich)后分化5天。Mouse fibroblast-derived ciNPCs were cultured in a neuronal expansion medium in which EGF (20 ng/ml) and bFGF (20 ng/ml) were added. For the general neural differentiation method, 20,000 ciNPCs were plated on PDL/Laminin-coated 24-well plates and cultured in N2B27 (DMEM: F12, 1% N2, 2% B27) medium containing no EGF and bFGF. Stable astrocyte production induction was the addition of BMP4 (50 ng/ml; R&D Biosystems) and 1% FBS in growth factor-free N2B27. Neuronal differentiation was performed by placing ciNPCs on PDL/Laminin-coated coverslips in Neural basal medium (2% B27, 1% N2, 10 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml). IGF-1, 1μM cAMP, 200 Culture in μM Ascorbic acid). Neuronal molecular marker gene expression and electrophysiological analysis were detected at specific time points, respectively. For oligodendrocyte differentiation, 20,000 cells were plated on PDL/Laminin-coated coverslips and cultured in N2B27 containing bFGF (10 ng/mL; Invitrogen) and PDGF-AA (10 ng/mL; Peprotech). The medium was cultured for 7 days, and then differentiated by adding T3 (100 ng/mL; 20 Sigma-Aldrich) for 5 days.
对于hUC来源的ciNPC的神经元分化，神经球用accutase(Life Technologies)消化以后，10000细胞铺至Poly-L-ornithine/laminin包被的玻片上。第二天，培养液中撤去EGF和bFGF，加入神经营养因子，其中包括BDNF，GDNF，IGF(均为10ng/mL)和100mM cAMP，200ng/mL的ascorbic acid。神经元分化培液两天换一次，2周以后，检测神经元分子标记的表达，约50天以后，检测星形胶质细胞分子标记的表达。For neuronal differentiation of hUC-derived ciNPCs, neurospheres were digested with accutase (Life Technologies) and 10,000 cells were plated onto Poly-L-ornithine/laminin coated slides. On the next day, EGF and bFGF were removed from the culture medium, and neurotrophic factors including BDNF, GDNF, IGF (both 10 ng/mL) and 100 mM cAMP, 200 ng/mL of ascorbic acid were added. Neuron differentiation culture was changed once every two days. After 2 weeks, the expression of neuronal molecular markers was detected. After about 50 days, the expression of astrocyte molecular markers was detected.
碱性磷酸酶分析Alkaline phosphatase assay
染色之前，细胞用4％多聚甲醛固定2分钟。碱性磷酸酶(AP)染色参照生产商的操作步骤用碱性磷酸酶试剂盒(Sigma-Aldrich,85L3R)进行染色。图像获得采用Zeiss Observer Z1。Cells were fixed with 4% paraformaldehyde for 2 minutes prior to staining. Alkaline phosphatase (AP) staining was stained with an alkaline phosphatase kit (Sigma-Aldrich, 85L3R) according to the manufacturer's protocol. The image was acquired using the Zeiss Observer Z1.
免疫荧光染色Immunofluorescence staining
培养于玻片上的细胞先用4％PFA溶液固定10分钟，然后在包含或不包含0.5％TritonX-100的封闭缓冲液(1％bovine serum albumin in PBS)中室温(RT)封闭30分钟。随后，样品孵育一抗4℃过夜，而后和适合的荧光二抗室温孵育1小时。细胞核用DAPI进行染色。图片分别用荧光显微镜(Olympus IX71)和Leica Sp-8共聚焦显微镜拍照。采用的特异性一抗包括Nestin(1:1000,Millipore,MAB5326)，Sox2(1:200,R&D,AF2018)，Pax6(1:500,Covance,RPB-278P)，Ki67(1:500,Abcam,ab15580)，GFAP(1:1000,Dako,Z0334)，Tuj(1:500,Covance,MMS435P)，MAP2(1:250,Millipore,AB5622)，MBP(1:250,Covance,SMI94)，Oligo2(1:400,Santa Cruz,sc-19969)，NeuN(1:200,Millipore,MAB377)，GAD67(1:200,Millipore,MAB5406)，Synapsin(1:200,Millipore,AB1543)，Glutamate(1:200,Millipore,MAB5304)。Cells cultured on slides were first fixed with 4% PFA solution for 10 minutes and then blocked for 30 minutes at room temperature (RT) in blocking buffer (1% bovine serum albumin in PBS) with or without 0.5% Triton X-100. Subsequently, the samples were incubated with a primary antibody at 4 ° C overnight and then incubated with a suitable fluorescent secondary antibody for 1 hour at room temperature. The nuclei were stained with DAPI. Images were taken with a fluorescence microscope (Olympus IX71) and a Leica Sp-8 confocal microscope. The specific primary antibodies used included Nestin (1:1000, Millipore, MAB5326), Sox2 (1:200, R&D, AF2018), Pax6 (1:500, Covance, RPB-278P), Ki67 (1:500, Abcam, Ab15580), GFAP (1:1000, Dako, Z0334), Tuj (1:500, Covance, MMS435P), MAP2 (1:250, Millipore, AB5622), MBP (1:250, Covance, SMI94), Oligo2 (1 : 400, Santa Cruz, sc-19969), NeuN (1:200, Millipore, MAB377), GAD67 (1:200, Millipore, MAB5406), Synapsin (1:200, Millipore, AB1543), Glutamate (1:200, Millipore, MAB5304).
神经球的染色，神经球悬液先转移至15ml管，使神经球自然沉降。然后将神经球于4％PFA中室温固定15分钟，于4℃5ml 30％蔗糖中孵育过夜直到稳定。 神经球沉淀转移至cryostat chuck上的组织冻存液中(Leica,020108926)。10μm厚度的神经球切片准备并包埋在箔中，保存于-80℃待分析。The neurosphere is stained, and the neurosphere suspension is first transferred to a 15 ml tube to allow the neurosphere to settle naturally. The neurospheres were then fixed in 4% PFA for 15 minutes at room temperature and incubated overnight in 5 ml of 30% sucrose at 4 °C until stable. Neurosphere pellets were transferred to tissue cryopreservation on cryostat chuck (Leica, 020108926). Neurosphere slices of 10 μm thickness were prepared and embedded in foil and stored at -80 ° C for analysis.
小鼠大脑切片制备如前所述。简单来说，用4％PFA PBS心脏灌流小鼠大脑。冷冻于30％蔗糖后，小鼠大脑以20μm厚冰切并做免疫荧光染色分析。Mouse brain sections were prepared as previously described. Briefly, the mouse brain was perfused with a 4% PFA PBS heart. After freezing in 30% sucrose, the mouse brain was cut with 20 μm thick ice and analyzed by immunofluorescence staining.
基因芯片分析Gene chip analysis
全基因组表达分析由Shanghai OE Biotech.Co.,Ltd公司根据安捷伦科技基于单色芯片分析的操作方法进行。简单来说，RNA样品根据生产商说明书用TRIzol(Sigma-Aldrich,T9424)进行抽提，并且RNA完整性用Agilent 2100bioanalyzer进行分析。每个样品的200ng总RNA通过单色快速标记扩增试剂盒(Agilent,5190-2305)进行标记反应。标记扩增好的RNA用RNeasy mini试剂盒(Qiagen,74104)进行纯化。用于8X60array的cDNA芯片来自安捷伦科技，并且采用安捷伦基因表达杂交试剂盒(catalog number:G4852A)。于65℃杂交17小时并清洗后，芯片用安捷伦芯片扫描仪(Agilent Technologies,USA)进行扫描。图像提取软件(version10.7.1.1,Agilent Technologies)用于分析芯片图像来获得原始数据。GeneSpring软件用于完成对原始数据的基础分析。首先，采用分位数算法对原始数据进行归一化。差异表达的基因通过倍数变化并设置10的阈值来进行鉴别。随后Gene Ontology(GO)分析和KEGG分析应用于确定这些差异表达mRNAs的作用。来自entrenz-gene数据库的39430个基因的55821个探针被检测。Whole genome expression analysis was performed by Shanghai OE Biotech. Co., Ltd. according to Agilent Technologies' monochromatic chip analysis based method of operation. Briefly, RNA samples were extracted with TRIzol (Sigma-Aldrich, T9424) according to the manufacturer's instructions, and RNA integrity was analyzed using an Agilent 2100 bioanalyzer. 200 ng of total RNA per sample was subjected to labeling reaction by a single color rapid label amplification kit (Agilent, 5190-2305). The amplified RNA was purified using the RNeasy mini kit (Qiagen, 74104). The cDNA chip for 8X60array was from Agilent Technologies and used the Agilent Gene Expression Hybridization Kit (catalog number: G4852A). After 17 hours of hybridization at 65 ° C and washing, the chips were scanned with an Agilent chip scanner (Agilent Technologies, USA). Image extraction software (version 10.7.1.1, Agilent Technologies) was used to analyze the chip image to obtain raw data. GeneSpring software is used to perform basic analysis of raw data. First, the raw data is normalized using a quantile algorithm. The differentially expressed genes were identified by a fold change and a threshold of 10 was set. Subsequent Gene Ontology (GO) analysis and KEGG analysis were applied to determine the effect of these differentially expressed mRNAs. A total of 55,821 probes from 39,430 genes from the entrenza-gene database were detected.
定量实时PCRQuantitative real-time PCR
细胞总RNA根据生产商说明书(Sigma-Aldrich,T9424)用Trizol试剂进行抽提。抽提的RNA采用随机六聚体引物和M-MLV反转录酶(Promega)反转为cDNA。cDNA样品与2XPCR mix(Qiagen)和Eva Green(Biotium)混合后置于MX3000P Stratagene PCR仪进行实时定量PCR分析。相对表达量通过与内参(HPRT)比较进行归一化处理。PCR所用引物的序列如下： Total cellular RNA was extracted with Trizol reagent according to the manufacturer's instructions (Sigma-Aldrich, T9424). The extracted RNA was inverted into cDNA using random hexamer primers and M-MLV reverse transcriptase (Promega). The cDNA samples were mixed with 2X PCR mix (Qiagen) and Eva Green (Biotium) and placed in a MX3000P Stratagene PCR machine for real-time quantitative PCR analysis. The relative expression levels were normalized by comparison with internal reference (HPRT). The sequences of the primers used in PCR are as follows:
Figure PCTCN2015073549-appb-000001
Figure PCTCN2015073549-appb-000001
电生理分析Electrophysiological analysis
对ciNPC分化获得的neurons进行全细胞膜片钳记录。采用Multiclamp 700B amplifier(Molecular Devices)进行记录。包含神经元的玻片始终保持在常温且有新鲜的人工脑脊液(ACSF)中。ACSF包含126mM NaCl，3mM KCl，1.25mM KH2PO41.3mM MgSO4，3.2mM CaCl2，26mM NaHCO3和10mM的葡萄糖，用95％O2和5％CO2吹出气泡。信号在10kHz with a 2kHz low-pass过滤器中进行采样.全细胞电容被全补偿。Ra>50M或信号波动>20％的信号被排除。细胞内液包含93mM K-gluconate，16mM KCl，2mM MgCl2，10mM HEPES 4mM ATP-Mg，0.3mM GTP-Na2，10mM creatine phosphate,0.5％Alexa Fluor 568hydrazide(Invitrogen) (pH 7.25,290/300mOsm)，0.4％neurobiotin(Invitrogen)。膜电位稳定在约-70mV,采用2pA增量的反复电流的输入来激发动作电位。采用-70至+70mV的电位差来激活钠离子内向电流和钾离子外向电流。数据均用pClamp(Clampfit)进行分析。Whole cell patch clamp recording of neurons obtained from ciNPC differentiation. Recording was performed using a Multiclamp 700B amplifier (Molecular Devices). Slides containing neurons are always kept at room temperature and fresh in artificial cerebrospinal fluid (ACSF). ACSF contained 126 mM NaCl, 3 mM KCl, 1.25 mM KH 2 PO 4 1.3 mM MgSO 4 , 3.2 mM CaCl 2 , 26 mM NaHCO 3 and 10 mM glucose, and air bubbles were blown with 95% O 2 and 5% CO 2 . The signal is sampled in a 10 kHz with a 2 kHz low-pass filter. The whole cell capacitance is fully compensated. Signals with Ra>50M or signal fluctuation >20% are excluded. The intracellular fluid contained 93 mM K-gluconate, 16 mM KCl, 2 mM MgCl2, 10 mM HEPES 4 mM ATP-Mg, 0.3 mM GTP-Na 2 , 10 mM creatine phosphate, 0.5% Alexa Fluor 568 hydrazide (Invitrogen) (pH 7.25, 290/300 mOsm), 0.4% neurobiotin (Invitrogen). The membrane potential was stabilized at approximately -70 mV, and the action potential was excited by the input of a repeated current of 2 pA increments. A potential difference of -70 to +70 mV is used to activate the sodium ion inward current and the potassium ion outward current. Data were analyzed using pClamp (Clampfit).
体内移植In vivo transplantation
体内移植按照文献进行操作。简单来说，E13.5天孕期的孕鼠C57BL/6的子宫角培养在无菌的环境中。包含约20个GFP-ciNPC神经球的PBS通过斜角校准的玻璃微管注入到胚胎脑室，其中神经球的直径不得超过80μm。此后，子宫角被替代，腹膜腔用10mL温热PBS进行灌洗，PBS含有抗生素，然后缝合伤口。1周或1月之后，小鼠在冰上麻醉或者戊巴比妥钠麻醉，脑片制备如前所述并可用于后续分析。In vivo transplantation is performed according to the literature. Briefly, the uterine horn of pregnant mice C57BL/6 at E13.5 days of gestation was cultured in a sterile environment. PBS containing approximately 20 GFP-ciNPC neurospheres was injected into the embryonic ventricle through obliquely calibrated glass microtubules, wherein the diameter of the neurospheres must not exceed 80 μm. Thereafter, the uterine horn was replaced, the peritoneal cavity was lavaged with 10 mL of warm PBS, the PBS contained antibiotics, and the wound was sutured. After 1 week or 1 month, the mice were anesthetized on ice or sodium pentobarbital. The preparation of the brain slices was as described above and can be used for subsequent analysis.
动物饲养Animal breeding
所有小鼠可以随时获得给予的食物和水。所有实验都遵守动物关爱与利用国家研究机构健康指南进行操作并由中科院上海生命科学研究院生物研究伦理委员会批准。All mice can receive food and water at any time. All experiments were performed in accordance with animal care and the use of national research institute health guidelines and approved by the Biological Research Ethics Committee of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences.
数据分析data analysis
所有定量数据按照期望标准误来进行统计分析。除非有其他说明，统计显著性差异均由单向方差分析完成，并以P值的形式具体在文章和图片叙述中阐述。All quantitative data were statistically analyzed according to expected standard errors. Unless otherwise stated, statistically significant differences are made by one-way analysis of variance and are described in the form of P values in the article and picture narrative.
实施例1 正常生理低氧条件下筛选诱导体细胞产生神经干细胞的化合物组合Example 1 Screening of a combination of compounds that induce somatic cells to produce neural stem cells under normal physiological hypoxia conditions
1.1 筛选VPCR组合在正常生理低氧情况下培养体细胞形成的克隆情况1.1 Screening of clones of VPCRs in cultured somatic cells under normal physiological hypoxia
通过大量组合物筛选，基本确定采用化合物组合VPCR(VPA、CHIR99021、Repsox和Parnate)在3％或者5％O2条件下处理小鼠成纤维细胞10天左右就可以出现致密的克隆，而在21％O2条件下则无致密克隆生成(图1A)。20万个起始细胞可以出现大约40个致密的克隆。中间态克隆的诱导效率在5％O2条件下相对于3％O2条件略高，因此，在后续的诱导实验中采取5％O2的培养条件。Through a large number of composition screenings, it was basically determined that a dense clone could be produced by treating a mouse fibroblast with a compound combination VPCR (VPA, CHIR99021, Repox, and Parnate) under 3% or 5% O 2 for about 10 days, while at 21 No dense clones were generated under %O2 conditions (Fig. 1A). Approximately 40 dense clones can appear in 200,000 starting cells. The induction efficiency of the intermediate clone was slightly higher under the 5% O 2 condition than the 3% O 2 condition, and therefore, the culture condition of 5% O 2 was taken in the subsequent induction experiment.
1.2 对1.1中的细胞克隆进行AP染色1.2 AP staining for cell clones in 1.1
对这些中间态克隆进行碱性磷酸酶AP染色发现，约有3/4的致密克隆高表达 碱性磷酸酶AP(图1B、图3A)，而在正常氧压条件下VPCR处理的小鼠成纤维细胞中既未发现致密克隆也未发现AP阳性的细胞。Alkaline phosphatase AP staining of these intermediate clones revealed that approximately 3/4 of the dense clones were highly expressed. Alkaline phosphatase AP (Fig. 1B, Fig. 3A), and neither dense clone nor AP positive cells were found in VPCR-treated mouse fibroblasts under normal oxygen pressure conditions.
1.3 对1.1中的VPCR的组分进行进一步筛选1.3 Further screening of the components of the VPCR in 1.1
检查VPA、CHIR99021、Repsox和Parnate都是诱导获得致密克隆的必要成分。为此，采用VPCR中的任三种化合物的组合处理小鼠成纤维细胞，检查能否获得致密的克隆形成。Examination of VPA, CHIR99021, Repox and Parnate are all essential components for inducing the acquisition of dense clones. To this end, mouse fibroblasts were treated with a combination of any three compounds in the VPCR to check for the formation of dense clones.
结果如图2所示，Parnate对于致密克隆的形成是可有可无的，而另外三个化合物则是必需的成分，不用这三个化合物中的任一个则大大地减少诱导形成的致密克隆的数目(图2A)。在5％O2条件下用VCR(VCR、CHIR99021和Repsox)处理获得的致密克隆中有90％是AP阳性的。进一步实验表明，再添加一些促重编程化合物31-33到VCR组合中也无进一步的提升效果(图2B)。As a result, as shown in Figure 2, Parnate is dispensable for the formation of dense clones, while the other three compounds are essential components, and the use of any of these three compounds greatly reduces the formation of dense clones. Number (Figure 2A). 90% of the dense clones obtained by treatment with VCR (VCR, CHIR99021 and Repox) under 5% O 2 were AP positive. Further experiments showed that there was no further improvement in the addition of some of the reprogramming compounds 31-33 to the VCR combination (Fig. 2B).
结论：VCR化合物组合中的任一组分(即VPA、CHIR99021、Repsox)对体细胞在正常生理低氧条件下转分化为神经干细胞是必须的。Conclusion: Any component of the VCR compound combination (ie, VPA, CHIR99021, Repox) is essential for somatic cells to transdifferentiate into neural stem cells under normal physiological hypoxia.
实施例2 VCR处理细胞克隆的特性检测Example 2 Characterization of VCR-treated cell clones
2.1 Sox2在不同氧压下的表达2.1 Sox2 expression under different oxygen pressures
在正常氧压条件下或用其它缺失Repsox、CHIR99021或者VPA的化合物组合则不能有效的诱导Sox2表达(图2C和2D)Under normal oxygen pressure conditions or with other combinations of compounds lacking Repsox, CHIR99021 or VPA, it is not effective to induce Sox2 expression (Figures 2C and 2D).
2.2 不同神经干细胞相关基因在细胞克隆中的表达2.2 Expression of different neural stem cell related genes in cell cloning
如图3B所示，在正常生理低氧条件下用VCR处理小鼠成纤维细胞，发现Sox2的表达在第5天明显提高，在第10天达到顶峰,在第15天略有回落；而Oct4和Nanog的表达则只是在第10天略有提高表达。As shown in Figure 3B, treatment of mouse fibroblasts with VCR under normal physiological hypoxic conditions revealed that Sox2 expression was significantly increased on day 5, peaked on day 10, and slightly decreased on day 15; The expression of Nanog and the expression of Nanog only slightly increased on the 10th day.
结论：小分子化合物组合VCR在5％O2正常生理低氧条件下有利于小鼠胚胎成纤维细胞转分化到中间态的致密克隆。Conclusion: Small molecule compound combination VCR is beneficial to the transdifferentiation of mouse embryonic fibroblasts to intermediate dense clones under normal physiological hypoxia conditions of 5% O 2 .
实施例3 VCR处理细胞克隆的神经干细胞分化Example 3 VCR treatment of cell clones for neural stem cell differentiation
3.1 培养克隆的形态观察3.1 Morphological observation of cultured clones
在正常生理低氧条件下，将VCR组合处理10天左右的细胞进行消化重新铺种并在含肝素heparin，表皮生长因子EGF，碱性成纤维细胞生长因子bFGF的神经干细胞培养基条件下培养，大约7-10天后，培养的细胞中出现神经干细胞状的双极性形态(图3C)。 Under normal physiological hypoxia, the cells treated with VCR combination for about 10 days were digested and re-planted and cultured under the condition of neural stem cell medium containing heparin, epidermal growth factor EGF and basic fibroblast growth factor bFGF. After about 7-10 days, a neural stem cell-like bipolar morphology appeared in the cultured cells (Fig. 3C).
3.2 神经干细胞特异性基因检测3.2 Neural stem cell specific gene detection
神经干细胞标记基因Nestin、Sox2和Pax6可以用免疫荧光染色方法检测到(图4A)。进一步地，逆转录聚合酶链式反应检测到神经干细胞特异基因包括Sox2、Pax6、Blbp、Ascl1和Brn2的表达水平也是增强的(图3D，ciNPCp1)。The neural stem cell marker genes Nestin, Sox2 and Pax6 can be detected by immunofluorescence staining (Fig. 4A). Further, reverse transcription polymerase chain reaction detected that expression levels of neural stem cell-specific genes including Sox2, Pax6, Blpp, Ascl1, and Brn2 were also enhanced (Fig. 3D, ciNPCp1).
结论：神经干细胞状的细胞出现在培养体系中。当这些细胞悬浮培养时形成了漂浮的细胞簇，细胞簇免疫荧光染色显示是Sox2和Nestin阳性的，具有神经球的性质(图4A)。收集这些漂浮的细胞簇并把它们命名为化合物诱导的神经干细胞/神经球(ciNPC)代数1(p1)。Conclusion: Neural stem cell-like cells appear in the culture system. When these cells were cultured in suspension, floating clusters of cells were formed, and immunofluorescence staining of the cell clusters showed that they were positive for Sox2 and Nestin, and had neurosphere properties (Fig. 4A). These floating clusters of cells were collected and named as compound-induced neural stem cell/neurosphere (ciNPC) algebra 1 (p1).
实施例4 化合物诱导的神经干细胞(ciNPC)的增殖和自我更新特性鉴定Example 4 Characterization of Compound-induced Proliferation and Self-renewal of Neural Stem Cells (ciNPC)
4.1 悬浮培养神经球细胞并测定其是否具有神经干细胞的基本性质(增殖和自我更新)4.1 Suspension culture of neurosphere cells and determination of their basic properties of neural stem cells (proliferation and self-renewal)
培养4代(p5)后，大约50％的化合物诱导的神经干细胞免疫荧光染色是Sox2阳性的，60％多的细胞室Pax6阳性的，大约40％的细胞是Nestin阳性的，而有30％的细胞是Nestin/Pax6或者Nestin/Sox2双阳性的(图4B)。After 4 passages (p5), approximately 50% of the compound-induced neural stem cell immunofluorescence staining was Sox2-positive, more than 60% of the cell compartment was Pax6 positive, and approximately 40% of the cells were Nestin positive, while 30% were The cells were either Nestin/Pax6 or Nestin/Sox2 double positive (Fig. 4B).
13代(p13)的化合物诱导的神经干细胞贴壁单层培养时展示了类似于小鼠胚胎神经干细胞的双极形态(图3C，ciNPC p5)。定量链式聚合酶酶反应检测不同代数的化合物诱导的神经干细胞中Sox2、Pax6、Blbp、Ascl1和Brn2的表达水平，发现悬浮培养可以很好地富集原始诱导的混合细胞中的神经干细胞(图3D，ciNPC p13)。The 13th generation (p13) of the compound induced neural stem cell adherent monolayer culture exhibited a bipolar morphology similar to mouse embryonic neural stem cells (Fig. 3C, ciNPC p5). Quantitative chain polymerase enzymatic reaction was used to detect the expression levels of Sox2, Pax6, Blbp, Ascl1 and Brn2 in neural stem cells induced by different algebraic compounds. It was found that suspension culture can well enrich neural stem cells in the original induced mixed cells (Fig. 3D, ciNPC p13).
在细胞代数13代时，超过96％的化合物诱导的神经干细胞呈Nestin、Sox2或Pax6单阳性，大约有93％的化合物诱导的神经干细胞呈Nestin/Sox2或Nestin/Pax6双阳性(图3E，图5),提示已经形成较纯的神经干细胞群体(图3F)。并且不仅仅这些13代的化合物诱导的神经干细胞呈增殖标记物Ki67阳性(图6A),而且当这些神经球在低密度铺种时展示出类似于小鼠脑袋衍生的神经干细胞第5代的大小和数量(图6B)。其中，Nestin、Sox2或Pax6单阳性代表具有类似神经干细胞的细胞的生成；Nestin/Sox2或Nestin/Pax6双阳性代表神经干细胞的生成。In the 13th generation of cell algebra, more than 96% of the compound-induced neural stem cells were single-positive with Nestin, Sox2 or Pax6, and about 93% of the compound-induced neural stem cells were double positive for Nestin/Sox2 or Nestin/Pax6 (Fig. 3E, Fig. 3E 5), suggesting that a relatively pure population of neural stem cells has been formed (Fig. 3F). And not only these 13-generation compounds induced neural stem cells were positive for the proliferation marker Ki67 (Fig. 6A), and when these neurospheres were seeded at low density, they exhibited a size similar to that of the 5th generation of mouse brain-derived neural stem cells. And quantity (Figure 6B). Among them, Nestin, Sox2 or Pax6 single positive represents the generation of cells with similar neural stem cells; Nestin/Sox2 or Nestin/Pax6 double positive represents the generation of neural stem cells.
以上结果表明，这些化合物诱导的神经干细胞具有小鼠脑袋衍生的神经干细胞类似的增殖和自我更新能力。The above results indicate that these compound-induced neural stem cells have similar proliferation and self-renewal ability to mouse brain-derived neural stem cells.
4.2 神经球形成能力的传代稳定性测定 4.2 Determination of the stability of the generation of neurospheres
对这些化合物诱导的神经干细胞的增殖能力进行进一步的检测，发现这类神经干细胞的神经干细胞标记基因表达水平和悬浮培养时的神经球形成能力直至25代仍没有改变(图6C和6D)。Further examination of the proliferative capacity of neural stem cells induced by these compounds revealed that the expression levels of neural stem cell marker genes of such neural stem cells and the ability of neurosphere formation in suspension culture remained unchanged until the 25th generation (Fig. 6C and 6D).
结论：在正常生理低氧条件下VCR处理小鼠胚胎成纤维细胞可以获得较纯的可以扩增的神经干细胞。Conclusion: VCR treatment of mouse embryonic fibroblasts under normal physiological hypoxic conditions can obtain relatively pure expandable neural stem cells.
实施例5 化合物诱导的神经干细胞的转录谱研究Example 5 Transcriptional study of compound-induced neural stem cells
采用小鼠胚胎衍生的神经干细胞(对照NPCs)、小鼠胚胎成纤维细胞和第5代及13代的化合物诱导的神经干细胞抽提mRNA并运用芯片对这些细胞进行全基因组表达类型分析。Neural stem cells induced by mouse embryo-derived neural stem cells (control NPCs), mouse embryonic fibroblasts, and 5th and 13th generation compounds were extracted and subjected to genome-wide expression type analysis using a chip.
5.1 化合物诱导的神经干细胞与小鼠神经干细胞的相似性5.1 Compound-induced similarity between neural stem cells and mouse neural stem cells
全基因组聚类和热图分析(图7A)和散点图分析(图8B)揭示化合物诱导的神经干细胞和小鼠胚胎成纤维细胞具有很大的不同，但是化合物诱导的神经干细胞和小鼠脑袋衍生的神经干细胞具有很大的相似性。不同代数的化合物诱导的神经干细胞和对照的神经干细胞共有774个核心靶基因(图7C)，通过基因本体分析(GO分析)发现这些基因主要与神经发生和细胞形态等过程相关(图7D和图8A)。Whole genome clustering and heat map analysis (Figure 7A) and scatter plot analysis (Figure 8B) revealed that compound-induced neural stem cells and mouse embryonic fibroblasts are very different, but compound-induced neural stem cells and mouse heads Derived neural stem cells have great similarities. There are 774 core target genes in neural stem cells and control neural stem cells induced by different algebraic compounds (Fig. 7C). These genes are mainly related to processes such as neurogenesis and cell morphology by gene ontology analysis (GO analysis) (Fig. 7D and Fig. 7D) 8A).
神经干细胞特异基因比如Sox2、Pax6、Ncan、Tox3,、Hes5、Gpm6a、Nes,、Bmi1、Zbtb16、Rfx4、Gpm6a和Slc1a3在化合物诱导的神经干细胞中表达明显上调，并具有和小鼠胚胎神经干细胞相当的表达水平；然而多能性相关基因Pof5f1和Nanog的表达并未上调,说明诱导获得的神经干细胞并不具有多能干细胞的特性。(图8B)。Neural stem cell-specific genes such as Sox2, Pax6, Ncan, Tox3, Hes5, Gpm6a, Nes, Bmi1, Zbtb16, Rfx4, Gpm6a and Slc1a3 are significantly up-regulated in compound-induced neural stem cells and are comparable to mouse embryonic neural stem cells. The expression level of the pluripotency-related genes Pof5f1 and Nanog was not up-regulated, indicating that the induced neural stem cells did not have the characteristics of pluripotent stem cells. (Fig. 8B).
5.2 化合物诱导的神经干细胞与小鼠成纤维细胞的区别5.2 Compound-induced difference between neural stem cells and mouse fibroblasts
生物学过程比如骨架***的表达谱是化合物诱导的神经干细胞相对于小鼠成纤维细胞最明显下调表达的基因(图8C和8D)。其中，424个基因比如Col3a1、DKK3、Thy1、Snail1和其它成纤维特异的基因的表达水平从第5代至第13代逐渐下调。这些发现表明化合物诱导的神经干细胞保存部分的成纤维细胞的表观遗传记忆，可以排除起始的小鼠胚胎成纤维细胞中可能的神经干细胞污染。另一方面，小鼠成纤维细胞直接在DMEM或神经干细胞培养基中培养并没有检测到Nestin、Sox2或者Pax6的表达(图9)。The expression profile of biological processes such as the skeletal system is the gene that is most down-regulated by the compound-induced neural stem cells relative to mouse fibroblasts (Figures 8C and 8D). Among them, the expression levels of 424 genes such as Col3a1, DKK3, Thy1, Snail1 and other fibroblast-specific genes were gradually down-regulated from the 5th generation to the 13th generation. These findings indicate that the compound-induced neuronal stem cell preserves part of the epigenetic memory of fibroblasts and can rule out possible neural stem cell contamination in the starting mouse embryonic fibroblasts. On the other hand, mouse fibroblasts were cultured directly in DMEM or neural stem cell culture medium and no expression of Nestin, Sox2 or Pax6 was detected (Fig. 9).
5.3 芯片数据中不同脑区特异的基因表达(图8E) 5.3 Gene expression in different brain regions in the chip data (Fig. 8E)
化合物诱导的神经干细胞和小鼠脑袋衍生的神经干细胞都具有较高的腹侧脑区特异的基因如Oligo2和Nkx2.2的表达水平，而没有检测到背侧脑区特异的基因如Pax3和Pax7的表达。Both compound-induced neural stem cells and mouse brain-derived neural stem cells have higher expression levels of ventral brain-specific genes such as Oligo2 and Nkx2.2, while no detectable genes in the dorsal brain region such as Pax3 and Pax7 were detected. expression.
同时，实验还发现前脑特异基因Emx2、Foxg1和Nr2e1以及中脑特异基因Gbx2和En1的高表达，但是，并没有后脑特异基因如Hoxa7和Hoxb7的高表达。At the same time, the experiment also found high expression of the forebrain specific genes Emx2, Foxg1 and Nr2e1 as well as the midbrain specific genes Gbx2 and En1, but there was no high expression of hindbrain specific genes such as Hoxa7 and Hoxb7.
综上，本发明所获得的化合物诱导的神经干细胞具有腹侧的前中脑区的特性，但不是很好地具有其它脑区的性质。In summary, the compound-induced neural stem cells obtained by the present invention have the characteristics of the ventral anterior midbrain region, but do not well have the properties of other brain regions.
5.4 芯片中ciNPCs和NPCs的组蛋白去乙酰化酶(HDACs)、糖原合成酶激酶3β(GSK-3)、转化生长因子β(TGF-β)和正常生理低氧信号传导通路的表达类型研究5.4 Expression patterns of histone deacetylases (HDACs), glycogen synthase kinase 3β (GSK-3), transforming growth factor beta (TGF-β) and normal physiological hypoxic signaling pathways in ciNPCs and NPCs
化合物诱导的神经干细胞和对照神经干细胞中在这些信号传导通路具有相似的表达类型，并有小鼠胚胎成纤维细胞具有很大的区别(图10)。Compound-induced neural stem cells and control neural stem cells have similar expression types in these signaling pathways, and there is a large difference in mouse embryonic fibroblasts (Fig. 10).
这些数据揭示激活这几条信号传导通路对于成功的转分化小鼠胚胎成纤维细胞到神经干细胞是必需的。These data reveal that activation of these signaling pathways is essential for successful transdifferentiation of mouse embryonic fibroblasts to neural stem cells.
结论：从基因表达谱来看，本发明化合物诱导的神经干细胞与小鼠神经干细胞具有很大的相似性，但与小鼠成纤维细胞则区别很大。此外，本发明化合物诱导的神经干细胞还具有腹侧的前中脑区的特征，且组蛋白去乙酰化酶(HDACs)、糖原合成酶激酶3β(GSK-3)、转化生长因子β(TGF-β)和正常生理低氧信号的传导通路在体细胞转化为神经干细胞的过程中是必须的。Conclusion: From the gene expression profile, the neural stem cells induced by the compounds of the present invention have great similarities with mouse neural stem cells, but are quite different from mouse fibroblasts. Furthermore, the neural stem cells induced by the compounds of the present invention are also characterized by the ventral anterior midbrain region, and histone deacetylases (HDACs), glycogen synthase kinase 3β (GSK-3), transforming growth factor beta (TGF) -β) and the conduction pathway of normal physiological hypoxia signals are necessary in the process of transforming somatic cells into neural stem cells.
实施例6 化合物诱导的神经干细胞的多能性鉴定Example 6 Identification of pluripotency of neural stem cells induced by compounds
6.1 体外分化实验检测化合物诱导的神经干细胞的分化能力6.1 In vitro differentiation assay to detect the differentiation ability of compound-induced neural stem cells
6.11 实验发现，第5代或13代的化合物诱导的神经干细胞在添加了BMP4和1％FBS并且撤掉生长因子的N2B27培养基中培养7天后，约有90％的细胞具有星形胶质细胞的形态并且免疫荧光染色是GFAP阳性的。6.11 It was found that about 7th or 13th generation of compound-induced neural stem cells were cultured in N2B27 medium supplemented with BMP4 and 1% FBS and with growth factor removed for 7 days, and about 90% of cells had astrocytes. The morphology and immunofluorescence staining were GFAP positive.
而在添加了B27、N2、BDNF、GDNF、IGF、cAMP和Ascorbic acid的神经基础培养基中培养7天则有80％的细胞具有神经元的形态且是Tuj1阳性的，在培养10-14天后则是具有成熟神经元的形态和Map2/Tuj1双阳性的(图11A和图12A)。Map2或Tuj1在GFAP阳性的细胞中不表达，这代表分化的细胞具有功能特异性。On the 7th day of culture in the basal medium supplemented with B27, N2, BDNF, GDNF, IGF, cAMP and Ascorbic acid, 80% of the cells have neuronal morphology and are Tuj1 positive, after 10-14 days of culture. It is a morphology with mature neurons and a double positive for Map2/Tuj1 (Fig. 11A and Fig. 12A). Map2 or Tuj1 is not expressed in GFAP-positive cells, which means that the differentiated cells are functionally specific.
当采用更细致的诱导分化条件时发现第13代的化合物诱导的神经干细胞在含bFGF,PDGF-AA和T3的培养基中培养12天后，可以观察到Olig2/Mbp双阳性 的细胞并且具有少突胶质细胞的形态(图12A，分化效率为25％左右)。进一步地，分化四周后可以发现成熟神经元如NeuN、Synapsin和GAD67的表达(图11B和图12B)。When using the more detailed conditions for inducing differentiation, it was found that the 13th generation of compound-induced neural stem cells were observed for 12 days in culture medium containing bFGF, PDGF-AA and T3, and Olig2/Mbp double positive was observed. The cells also have the morphology of oligodendrocytes (Fig. 12A, differentiation efficiency is about 25%). Further, expression of mature neurons such as NeuN, Synapsin and GAD67 can be found four weeks after differentiation (Fig. 11B and Fig. 12B).
6.12 进而用全细胞膜片钳实验检测化合物诱导的神经干细胞的成熟度和功能6.12 Further, the whole cell patch clamp test was used to detect the maturity and function of the compound-induced neural stem cells.
实验发现，化合物诱导的神经干细胞分化的神经元可以产生重复记录的动作电位(图12C)和自发突触后电流(图12D)。此外，Na+电流也可以在这些分化得到的神经元中记录到(图12E)。It has been found that compound-induced neural stem cell differentiation of neurons can produce repeatedly recorded action potentials (Fig. 12C) and spontaneous postsynaptic currents (Fig. 12D). In addition, Na + current can also be recorded in these differentiated neurons (Fig. 12E).
6.2 体内分化实验检测化合物诱导的神经干细胞的分化能力6.2 In vivo differentiation assay to detect the differentiation ability of compound-induced neural stem cells
将化合物诱导的神经干细胞移植到胚鼠体内，并用慢病毒GFP标记第17代的化合物诱导的神经干细胞。The compound-induced neural stem cells were transplanted into embryonic mice, and the neural stem cells induced by the 17th generation compound were labeled with lentiviral GFP.
实验显示GFP标记的化合物诱导的神经干细胞仍具有神经干细胞相关的性质，包括增殖能力、神经球形成能力、神经干细胞特异基因表达和体外分化能力都没有改变(图13)。Experiments have shown that GFP-tagged compounds-induced neural stem cells still have neural stem cell-related properties, including proliferative capacity, neurosphere formation ability, neural stem cell-specific gene expression, and in vitro differentiation ability (Fig. 13).
GFP标记的化合物诱导的神经干细胞被注射到E13.5的胚胎中，免疫荧光染色显示移植1周后GFP标记的化合物诱导的神经干细胞可以在小鼠不同脑区存活(图14A)。此外，这种GFP标记的化合物诱导的神经干细胞能够被Ki67、Olig2或GFAP标记，但是不能被Tuj1标记(图14B和图14C),这代表诱导获得的神经干细胞在体内更易分化成胶质细胞和少突状胶质细胞。GFP-labeled compound-induced neural stem cells were injected into embryos of E13.5, and immunofluorescence staining showed that GFP-labeled compound-induced neural stem cells could survive in different brain regions of mice 1 week after transplantation (Fig. 14A). Furthermore, this GFP-tagged compound-induced neural stem cell can be labeled with Ki67, Olig2 or GFAP, but cannot be labeled with Tuj1 (Fig. 14B and Fig. 14C), which means that the induced neural stem cells are more easily differentiated into glial cells in vivo and Less glial cells.
移植1个月后，仍可以发现GFP标记的化合物诱导的神经干细胞分化得到的Olig2+或Mbp+的少突胶质细胞,GFAP+的星形胶质细胞和NeuN+或Tuj1+的成熟神经元(图12F，图14D)。但是并没有发现Ki67阳性的GFP标记的细胞(图14E)，也没有在移植的脑区发现肿瘤形成。After 1 month of transplantation, Olig2 + or Mbp + oligodendrocytes, GFAP + astrocytes and mature neurons of NeuN + or Tuj1 + can still be found by GFP-labeled compound-induced differentiation of neural stem cells. (Fig. 12F, Fig. 14D). However, Ki67-positive GFP-labeled cells were not found (Fig. 14E), and no tumor formation was found in the transplanted brain region.
结论：化合物诱导的神经元细胞能够在体外分化到主要的神经谱系，包括星形胶质细胞、神经元和少突胶质细胞。Conclusion: Compound-induced neuronal cells can differentiate into major neural lineages in vitro, including astrocytes, neurons, and oligodendrocytes.
而移植的化合物诱导的神经干细胞可以在体内分化为不同的神经谱系，且不会在移植的脑区形成肿瘤，因此化合物诱导的神经干细胞具有潜在的临床应用前景。The transplanted compound-induced neural stem cells can differentiate into different neural lineages in vivo and do not form tumors in the transplanted brain regions, so the compound-induced neural stem cells have potential clinical application prospects.
实施例7 其它化合物组合诱导神经干细胞Example 7 Combination of other compounds induces neural stem cells
VPA、CHIR99021和Repsox分别是组蛋白去乙酰化酶(HDACs)、糖原合成酶 激酶3β(GSK-3)、转化生长因子β(TGF-β)信号通路的抑制剂。因此本发明还检测了是否这些信号传导通路的其它抑制剂组合也可以诱导神经干细胞的产生，比如是否NaB或者TSA可以取代VPA，LiCl或者Li2CO3可以取代CHIR99021,SB431542或者Tranilast可以取代Repsox，其中，各组抑制剂的化学结构式如表1所示：VPA, CHIR99021 and Repox are histone deacetylases (HDACs) and glycogen synthase, respectively. Inhibitors of kinase 3β (GSK-3) and transforming growth factor beta (TGF-β) signaling pathways. Therefore, the present invention also detects whether other inhibitor combinations of these signaling pathways can also induce neural stem cell production, such as whether NaB or TSA can replace VPA, LiCl or Li2CO3 can replace CHIR99021, SB431542 or Tranilast can replace Repox, wherein each The chemical structural formula of the group inhibitor is shown in Table 1:
表1Table 1
Figure PCTCN2015073549-appb-000002
Figure PCTCN2015073549-appb-000002
方法同VCR诱导的实验方案，结果发现，在同样的实验条件下，化合物组合NLS(NaB、LiCl和SB431542)和TLT(TSA、Li2CO3和Tranilast)能够在5％O2条件下处理小鼠胚胎成纤维细胞可以获得致密克隆和激活Sox2表达。并且这些中间态的克隆进一步地悬浮培养后可以产生Nestin+/Pax6+或Nestin+/Sox2+的神经干细胞(图15)。The method was the same as the VCR-induced experimental protocol. It was found that under the same experimental conditions, the compound combination NLS (NaB, LiCl and SB431542) and TLT (TSA, Li 2 CO 3 and Tranilast) can be treated under 5% O 2 conditions. Murine embryonic fibroblasts can obtain dense clones and activate Sox2 expression. Further, these intermediate clones were further cultured in suspension to produce Nestin + /Pax6 + or Nestin + /Sox2 + + neural stem cells (Fig. 15).
这些纯化的化合物诱导的神经干细胞在传代13代后可以具有经典的神经干细胞的形态和神经球形成能力(图16B)。These purified compound-induced neural stem cells can have classical neural stem cell morphology and neurosphere forming ability after passage 13 passages (Fig. 16B).
免疫荧光染色发现这两中化合物组合诱导获得的神经干细胞高表达神经干细胞基因Nesting、Sox2和Pax6。逆转录聚合酶链式反应同样确认了这些神经干细胞标记基因的高表达(图16C)。Immunofluorescence staining revealed that the neural stem cells induced by the combination of the two compounds highly expressed the neural stem cell genes Nesting, Sox2 and Pax6. Reverse transcription polymerase chain reaction also confirmed high expression of these neural stem cell marker genes (Fig. 16C).
结论：NLS和TLT化合物组合可以在正常生理低氧培养条件下具有和VCR化合物组合同样的诱导产生神经干细胞的效果，进一步地支持芯片分析得到的结论，即激活一系列的信号传导通路可以协调地促进小鼠胚胎成纤维细胞到神经干细胞的转分化。 Conclusion: The combination of NLS and TLT compounds can induce the same effect of producing neural stem cells under the conditions of normal physiological hypoxic culture, and further supports the conclusion of chip analysis that activation of a series of signal transduction pathways can be coordinated. Promotes transdifferentiation of mouse embryonic fibroblasts to neural stem cells.
实施例8 小鼠尾尖成纤维细胞和人尿细胞诱导ciNPCsExample 8 Mouse tail tip fibroblasts and human urine cells induce ciNPCs
8.1 采用相同的方法以及化合物组合VCR，处理新生小鼠尾尖成纤维细胞(TTFs)。8.1 Neonatal mouse tail tip fibroblasts (TTFs) were treated using the same method and compound combination VCR.
结果如图17A所示，在正常生理低氧条件VCR处理10天时，Sox2的表达上调。在加了heparin，EGF和bFGF的神经细胞扩大培养液里进一步培养7到10天，VCR处理后的TTFs与VCR处理后的MEF一样，具有相同的形态变化(图17B)。在传代过程中，能逐步获得均质的ciNPCs(图18)。As a result, as shown in Fig. 17A, the expression of Sox2 was up-regulated under normal physiological hypoxic conditions for 10 days of VCR treatment. Further culture was carried out for 7 to 10 days in neuroblast-enhanced culture medium to which heparin, EGF and bFGF were added, and the TCRs after VCR treatment had the same morphological changes as the VCR-treated MEF (Fig. 17B). In the process of passage, homogeneous ciNPCs can be obtained step by step (Fig. 18).
TTFs来源的第16代的ciNPC具有典型的神经干细胞的形态和神经球形成能力(图17C)。免疫荧光染色和实时定量PCR分析都能够检测到神经干细胞分子标记基因Nestin，Sox2，Pax6和Blbp的表达(图17D)。The 16th generation ciNPCs derived from TTFs have typical neural stem cell morphology and neurosphere forming ability (Fig. 17C). Both immunofluorescence staining and real-time quantitative PCR analysis were able to detect the expression of neural stem cell molecular marker genes Nestin, Sox2, Pax6 and Blbp (Fig. 17D).
此外，TTFs来源的ciNPCs在特定分化条件下还能够诱导出GFAP阳性的星形胶质细胞，Tuj1/MAP2双阳性的神经元以及Olig2/MBP双阳性的少突胶质细胞(图17E)。In addition, TTFs-derived ciNPCs were able to induce GFAP-positive astrocytes, Tuj1/MAP2 double-positive neurons, and Olig2/MBP double-positive oligodendrocytes under specific differentiation conditions (Fig. 17E).
因此，VPA，CHIR99021，Repsox和正常生理低氧的结合能够直接使不同来源的小鼠成纤维细胞转化为ciNPCs。Thus, the combination of VPA, CHIR99021, Repox and normal physiological hypoxia can directly convert mouse fibroblasts from different sources into ciNPCs.
8.2 采用药物组合VCR来诱导hUCs为ciNPCs。5％O2，VCR处理20天后，在hUC培养中类似于VCR处理MEF所获得的紧密细胞克隆开始出现(图17F)。第15天的时候Sox2的表达上调(图17G)。8.2 The drug combination VCR was used to induce hUCs as ciNPCs. After 5% O 2 , 20 days after VCR treatment, tight cell clones obtained similar to VCR-treated MEF in hUC culture began to appear (Fig. 17F). The expression of Sox2 was up-regulated on day 15 (Fig. 17G).
在神经细胞扩大培养液里培养5代以上，这些VCR诱导的中间态细胞开始展现出与对照组iNPCs相同的形态(如前所述，通过在hUCs中导入基因诱导获得)(图17H)。After culturing for 5 generations or more in the neuron expansion medium, these VCR-induced intermediate cells began to exhibit the same morphology as the control iNPCs (as previously obtained by introducing a gene into hUCs) (Fig. 17H).
通过检测qRT-PCR(图17I)和免疫荧光染色(图19A)，这些hUCs来源的ciNPCs表达神经干细胞特异的基因，包括Sox2、Nestin、Sox1和Pax6。更重要的是hUCs来源的ciNPCs具有与对照iNPCs相近的增殖能力(图19B)，并且它能在神经分化培养基里分化为Tuj1/MAP2双阳性的神经元和GFAP阳性的星形胶质细胞(图17J)。By detecting qRT-PCR (Fig. 17I) and immunofluorescence staining (Fig. 19A), these hUCs-derived ciNPCs express neural stem cell-specific genes, including Sox2, Nestin, Sox1, and Pax6. More importantly, hUCs-derived ciNPCs have similar proliferative capacity to control iNPCs (Fig. 19B), and it can differentiate into Tuj1/MAP2 double positive neurons and GFAP positive astrocytes in neural differentiation medium ( Figure 17J).
以上结果表明人尿细胞在药物组合VCR处理后，可以诱导成为神经干细胞。The above results indicate that human urine cells can be induced to become neural stem cells after treatment with a drug combination VCR.
讨论：discuss:
首先，本发明的研究首次表明，在非外源基因介导的条件下，利用纯化合物组 合完全可以诱导体细胞发生重编程并直接转分化为神经干干细胞。First, the study of the present invention shows for the first time that a pure compound group is utilized under conditions mediated by non-foreign genes. The combination can completely induce somatic cell reprogramming and direct transdifferentiation into neural stem stem cells.
本发明诱导策略主要包括两个方面：一、在正常生理低氧条件下，化合物组合诱导细胞进入重编程阶段，二、诱导获得的中间态细胞在谱系特异的诱导条件下进行转分化。鉴于其他谱系细胞，例如：心肌细胞、血管内皮细胞等，也可以通过转因子方法诱导获得，或者通过干细胞体外分化法获得，因此，应用本发明的诱导策略可以获得纯化何物法诱导的其他谱系特定细胞。The induction strategy of the invention mainly comprises two aspects: 1. Under normal physiological hypoxia conditions, the compound combination induces the cells to enter the reprogramming stage, and the induced intermediate cells are transdifferentiated under lineage-specific induction conditions. In view of other lineage cells, for example, cardiomyocytes, vascular endothelial cells, etc., can also be obtained by a trans-factor method, or by stem cell in vitro differentiation method. Therefore, other pedigrees induced by purification can be obtained by using the induction strategy of the present invention. Specific cells.
其次，本发明发现在正常生理低氧条件下，不同的HDACs抑制剂、GSK-3激酶抑制剂和TGF-β信号通路抑制剂组合均可以诱导终末分化的体细胞进入重编程状态，并且该重编程状态伴随Sox2基因的表达激活。因此，上述三个信号通路很有可能通过调节Sox2相关基因的表达从而促进细胞重编程。此外，cDNA芯片数据也表明上述三个信号通路相关基因的变化在化合物诱导获得神经干细胞与对照神经干细胞中非常类似，且显著区别于起始成纤维细胞。综上，小分子化合物调节的HDACs、GSK-3和TGF-β信号通路对于诱导成纤维细胞转分化为神经干细胞是至关重要的，但是具体的分子机制还有待深入研究。Secondly, the present inventors have found that under normal physiological hypoxic conditions, different HDACs inhibitors, GSK-3 kinase inhibitors and TGF-β signaling pathway inhibitor combinations can induce terminally differentiated somatic cells to enter a reprogramming state, and The reprogramming state is accompanied by activation of the expression of the Sox2 gene. Therefore, the above three signaling pathways are likely to promote cell reprogramming by regulating the expression of Sox2-related genes. In addition, cDNA chip data also indicated that the changes in the genes involved in the above three signaling pathways were very similar in compound-induced neural stem cells and in control neural stem cells, and were significantly different from the starting fibroblasts. In conclusion, HDACs, GSK-3 and TGF-β signaling pathways regulated by small molecule compounds are crucial for inducing transdifferentiation of fibroblasts into neural stem cells, but the specific molecular mechanism remains to be further studied.
第三，本发明发现正常生理低氧条件对于纯化何物诱导细胞进入重编程状态是必须的，但是正常生理低氧模拟化合物，如氯化钴等，并不能取代正常生理低氧条件诱导细胞发生重编程。尽管标准的哺乳动物细胞体外培养的氧气浓度度为21％，但是体内组织的实际氧气浓度为1％到5％，而且正常生理条件下，干细胞的微环境也是正常生理低氧条件，因此进一步检测体外诱导细胞发生重编程的小分子化合物组合在体内是否也能促进转分化具有重要的意义。Third, the present inventors have found that normal physiological hypoxic conditions are necessary for purifying cells to induce reprogramming, but normal physiological hypoxia mimicking compounds, such as cobalt chloride, do not replace normal physiological hypoxic conditions to induce cell development. Reprogramming. Although the standard mammalian cells are cultured in an oxygen concentration of 21% in vitro, the actual oxygen concentration in the body tissues is 1% to 5%, and under normal physiological conditions, the microenvironment of stem cells is also a normal physiological hypoxic condition, so further detection is performed. Whether a combination of small molecule compounds that induce cell reprogramming in vitro can also promote transdifferentiation in vivo is of great significance.
最后，利用的化合物组合也可以诱导人尿液中的细胞直接转分化为神经干细胞，本发明为获得病人的特异性神经干细胞提供了全新的、便捷的、安全的可行性方法，为进一步治疗神经类疾病，如阿尔茨海默病和帕金森症等提供了新的治疗途径。Finally, the combination of compounds can also induce direct transdifferentiation of cells in human urine into neural stem cells. The present invention provides a new, convenient, and safe method for obtaining patient-specific neural stem cells, for further treatment of nerves. Diseases such as Alzheimer's disease and Parkinson's disease offer new therapeutic avenues.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (10)

  1. 一种小分子化合物组合，其特征在于，所述的小分子化合物包括以下组分：A combination of small molecule compounds, characterized in that the small molecule compound comprises the following components:
    (a)组蛋白去乙酰化酶(HDACs)抑制剂；(a) histone deacetylase (HDACs) inhibitors;
    (b)糖原合成酶激酶(GSK-3)抑制剂；(b) a glycogen synthase kinase (GSK-3) inhibitor;
    (c)转化生长因子β(TGF-β)信号通路抑制剂；和(c) a transforming growth factor beta (TGF-beta) signaling pathway inhibitor;
    (d)任选的药学上可接受的载体。(d) an optional pharmaceutically acceptable carrier.
  2. 一种小分子化合物组合，其特征在于，所述的小分子化合物由以下组分构成：A combination of small molecule compounds, characterized in that the small molecule compound consists of the following components:
    (a)组蛋白去乙酰化酶(HDACs)抑制剂；(a) histone deacetylase (HDACs) inhibitors;
    (b)糖原合成酶激酶(GSK-3)抑制剂；(b) a glycogen synthase kinase (GSK-3) inhibitor;
    (c)转化生长因子β(TGF-β)信号通路抑制剂。(c) Transforming growth factor beta (TGF-beta) signaling pathway inhibitor.
  3. 如权利要求1或2所述的组合物的用途，其特征在于，用于在低氧环境下诱导体细胞转分化为神经干细胞。Use of a composition according to claim 1 or 2 for inducing somatic transdifferentiation into neural stem cells in a hypoxic environment.
  4. 如权利要求3所述的用途，其特征在于，所述的低氧环境为氧浓度3-8％的环境，较佳地，为4-6％。The use according to claim 3, characterized in that the hypoxic environment is an environment having an oxygen concentration of from 3 to 8%, preferably from 4 to 6%.
  5. 如权利要求3所述的用途，其特征在于，所述的体细胞包括成纤维细胞、上皮细胞。The use according to claim 3, wherein said somatic cells comprise fibroblasts, epithelial cells.
  6. 一种体外诱导体细胞转分化为神经干细胞的方法，其特征在于，在低氧环境以及权利要求1或2所述的小分子化合物组合存在的培养条件下，培养体细胞。A method for inducing somatic cell transdifferentiation into neural stem cells in vitro, wherein the somatic cells are cultured under a culture condition in which a hypoxic environment and the small molecule compound according to claim 1 or 2 are combined.
  7. 如权利要求6所述的方法，其特征在于，所述的小分子化合物组合中，HDACs抑制剂包括丙戊酸钠(VPA)、丁酸钠(NaB)、或曲古抑菌素A(TSA)；和/或The method according to claim 6, wherein in the small molecule compound combination, the HDACs inhibitor comprises sodium valproate (VPA), sodium butyrate (NaB), or trichostatin A (TSA). );and / or
    所述的GSK-3抑制剂包括CHIR99021、氯化锂(LiCl)、或碳酸锂(Li2CO3)；和/或The GSK-3 inhibitor comprises CHIR99021, lithium chloride (LiCl), or lithium carbonate (Li 2 CO 3 ); and/or
    所述的TGF-β信号通路抑制剂包括Repsox、SB431542、或曲尼司特(Trani last)。The TGF-β signaling pathway inhibitors include Repx, SB431542, or Trani last.
  8. 一种神经干细胞，其特征在于，所述的神经干细胞是由权利要求6所述的方法制备的。A neural stem cell characterized in that the neural stem cell is produced by the method of claim 6.
  9. 权利要求8所述神经干细胞的用途，其特征在于，用于制备预防或治疗神经***疾病的药物组合物。Use of a neural stem cell according to claim 8, characterized in that it is used for the preparation of a pharmaceutical composition for preventing or treating a nervous system disease.
  10. 一种组合物，其特征在于，所述的组合物包括：权利要求8所述的神经干细胞。 A composition comprising: the neural stem cell of claim 8.
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