WO2015133879A1 - Composition for inducing direct transdifferentiation into oligodendrocyte progenitor cells from somatic cells and use thereof - Google Patents

Composition for inducing direct transdifferentiation into oligodendrocyte progenitor cells from somatic cells and use thereof Download PDF

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WO2015133879A1
WO2015133879A1 PCT/KR2015/002253 KR2015002253W WO2015133879A1 WO 2015133879 A1 WO2015133879 A1 WO 2015133879A1 KR 2015002253 W KR2015002253 W KR 2015002253W WO 2015133879 A1 WO2015133879 A1 WO 2015133879A1
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cells
vector
nucleic acid
differentiation
direct cross
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PCT/KR2015/002253
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French (fr)
Korean (ko)
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김정범
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국립대학법인 울산과학기술대학교 산학협력단
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Priority to CN201580012322.0A priority Critical patent/CN106170543B/en
Priority to US15/120,192 priority patent/US10801010B2/en
Priority to EP15758550.6A priority patent/EP3118306A4/en
Priority claimed from KR1020150032322A external-priority patent/KR101731068B1/en
Publication of WO2015133879A1 publication Critical patent/WO2015133879A1/en

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Definitions

  • a composition for inducing direct cross-differentiation from induced somatic cells to induced Oligodendrocyte Progenitor Cells (iOPCs) and a method for direct cross-differentiation from somatic cells to oligodendrocyte precursors comprising the step of treating the composition. will be.
  • the present invention is a pharmaceutical composition, cell therapy, drug for the prevention or treatment of spinal cord injury or myelin deprivation disease, including rare oligodendrocyte progenitor cells differentiated by the method of direct cross-differentiation from the somatic cells to oligodendrocyte progenitor cells
  • the present invention relates to a composition for screening, or a 3D printed biomaterial composition for artificial fabrication.
  • Oligodendrocytes are derived from neuroepithelial cells during the developmental differentiation of the glial lineage of defined precursors known as Oligodendrocyte Progenitor Cells (OPCs). Rare oligodendrocytes are one of the types of glial cells that play an essential role in the production of myelin sheath in the central nervous system (CNS). Myelination allows neurons to maintain electrical impulses of action potential through disruption of axons.
  • OPCs Oligodendrocyte Progenitor Cells
  • Dysfunction of oligodendrocytes is a disorder caused by the loss of myelin sheath, multiple sclerosis, cerebral palsy, leukodystrophy, neuropathy, central limb myelination and myelination It causes many neurodegenerative diseases, including hypomyelination.
  • transplanting oligodendrocyte progenitor cells, which have the function of restoring endogenous cells or producing myelin, into the central nervous system is the most fundamental method of treating myelin damage.
  • the myeloma was treated by transplanting oligodendrocytes, but the effective differentiation method of oligodendrocytes was not clear, and it was very difficult to obtain a large amount of oligodendrocytes.
  • oligodendrocytes from embryonic stem cells (ESCs) and induced pluripotent stem cells
  • ESCs embryonic stem cells
  • induced pluripotent stem cells the induction efficiency into the desired cells is low and when the embryonic stem cells or pluripotent stem cells are differentiated into specific cells This is problematic because there is a risk of tumor formation from cells.
  • the present inventors have transdifferentiation factor for OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, precursor cells of oligodendrocytes using any one or more of the gene NKX6.2 from somatic cells without the risk of tumorigenesis
  • the present invention was completed by confirming that it can be prepared by a direct cross-differentiation method.
  • An object of the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced
  • the present invention provides a composition for inducing direct cross-differentiation from somatic cells comprising a vector as an active ingredient to oligodendrocyte progenitor cells.
  • Another object of the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced
  • Pharmaceutical composition for the prevention or treatment of spinal cord injury or myelin deprivation disease comprising a vector or the direct cross-differentiation-induced oligodendrocyte progenitor cells, cell therapy for the prevention or treatment of spinal cord injury or myelinated disease, spinal cord injury Or to provide a composition for screening drug treatment for myelin deprivation disease and 3D printing biomaterial composition for the manufacture of artificial tissue for the treatment of spinal cord injury or myelin deprivation disease.
  • Another object of the present invention is to provide a method for direct cross-differentiation from somatic cells to oligodendrocyte progenitor cells.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the Provided is a composition for inducing direct cross-differentiation from somatic cells comprising a vector into which a nucleic acid molecule has been introduced as an oligodendrocyte progenitor cell.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced
  • Vectors are introduced into somatic cells to provide direct cross-differentiation-induced oligodendrocyte progenitor cells.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced It provides a pharmaceutical composition for the prevention or treatment of spinal cord injury or demyelination disease comprising a vector or the direct cross-differentiation-induced oligodendrocyte progenitor cells as an active ingredient.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced
  • a cell therapy agent for the prevention or treatment of spinal cord injury or myelin deprivation disease comprising a vector or the above-described direct cross-differentiation-induced oligodendrocyte progenitor cells as an active ingredient.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced
  • a composition for screening a therapeutic drug for spinal cord injury or myelin deprivation disease comprising a vector or the direct cross-differentiation induced oligodendrocyte progenitor cells as an active ingredient.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced
  • a 3D printing biomaterial composition for the manufacture of artificial tissue for the treatment of spinal cord injury or myelin deprivation disease comprising a vector or the direct cross-differentiation induced oligodendrocyte progenitor cells as an active ingredient.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced
  • a method of directly cross-differentiating somatic cells into oligodendrocyte progenitor cells comprising introducing a vector into somatic cells.
  • Composition in the present invention is able to induce transdifferentiation of a oligodendrocyte progenitor cells from somatic cells by directly using the cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, any one or more of the gene NKX6.2
  • OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 any one or more of the gene NKX6.2
  • IOPC-C1 and "iOPC-C2" in the Figures represent clone 1 and clone 2 of oligodendrocyte precursor cells prepared according to the present invention, respectively.
  • Figure 1a from fibroblasts OCT4 - is a schematic view showing a manufacturing process of the induced iOPC Fig.
  • Figure 1b shows an image of a phase contrast microscope to confirm the change as the differentiation from fibroblasts to iOPCs gradually progress.
  • "B” is uninfected fibroblast
  • C is control
  • D is fibroblast- OCT4 on day 25
  • "E” is OCT4 -induced fibroblast-forming aggregate
  • "F” is iOPC-like population
  • G represents the bipolar form of iOPCs.
  • the scale bars of E and F are 500 um
  • the scale bars of B to D are 200 um
  • the scale bars of G are 100 um.
  • Figure 1c is PDGFR- ⁇ , NG2, A2B5, and iOPC a specific double staining marker of the OCT4 Olig2 - shows an immunofluorescence analysis of the clones derived iOPC. Scale bar is 125um.
  • 1D shows phase contrast microscopy images of self-dividing iOPCs. Scale bar is 100um.
  • Figure 1e shows the growth rate of iOPCs with time in the initial passage (P3) and late passage (P31), respectively. Data are expressed as mean ⁇ standard error.
  • 2A shows immunofluorescence images of undifferentiated iOPCs.
  • A shows images stained with PDGFR- ⁇ , GFAP,
  • B shows images stained with NG2, Tuj1, and
  • C shows images stained with A2B5 and RIP. Scale bar is 250um.
  • Figure 2b shows a phase change microscopy image of the shape change during in vitro differentiation of iOPCs.
  • D represents Day 1
  • E represents Day 5
  • F represents Day 16.
  • the green arrow points to oligodendrocytes and the red arrow points to astrocytes. Scale bar is 100um.
  • FIG. 2C shows the presence of O4 + oligodendrocytes (green) and GFAP + astrocytes (red) together, indicating the multipotency of iOPCs after differentiation. Scale bar is 125um.
  • 2D shows immunofluorescence images of oligodendrocytes expressing mature oligodendrocyte markers 30 days after differentiation.
  • M and P represent expression of RIP
  • N and “Q” represent GalC
  • O and “R” represent MBP expression.
  • Scale bar is 125um.
  • FIG. 3a is OCT4 - shows a heat map (Heatmap) the result of microarray analysis for the overall expression of genes induced iOPCs.
  • fibroblasts are fibroblasts
  • OCT4 fib- D3 is differentiation-induced by OCT4 3 il Me fibroblasts
  • OCT4 fib- D10 is differentiation-induced by OCT4 10 il Me fibroblasts
  • mat is a control
  • wtOPC ′′ refers to OPC derived from pluripotent stem cells.
  • Figure 3b shows the overall gene expression showing the relationship between fibroblasts and OPC, fibroblasts and iOPCs-C1, fibroblasts and iOPCs-C2, iOPC-C1 and OPC, iOPC-C2 and OPC, iOPC-C1 and iOPC-C2 The scatter plot is compared. Black lines represent borderline with 2 fold change in gene expression between paired samples. Gene expression levels are expressed as log2 values.
  • FIG. 3c OCT4 expressing the OPC marker gene sequence and oligodendrocytes marker gene indicates a quantitative RT-PCR results of the induced OPCs.
  • the graph is shown as log2 fold change and normalized to GAPDH .
  • 3D shows the results of 3D principal component analysis.
  • the first principal component (PC1) accounted for 64% of gene expression variability.
  • the second principal component (PC2) accounted for 14% of volatility and the third principal component (PC3) accounted for 8.3% of volatility.
  • 3E shows the hierarchical clustering results of overall gene expression.
  • the results of microarray analysis confirmed changes in gene expression after OCT4 induction. It was confirmed that oligodendrocytes and neural development were related to genes expressed highly in iOPCs compared to fibroblasts.
  • FIG. 4A shows images of spinal cord sections of H & E stained mice 12 weeks after injury and transplantation. Scale bar is 200um.
  • Figure 4b shows the results of immunohistochemistry analysis on the spinal cord of the damaged part. Stain with “C” and “F” O4, “D” and “G” with MBP, "E and H” with GFAP.
  • 4C shows the results of differentiating iOPCs transplanted into O4, MBP and GFAP positive cells in the rat spinal cord and immature markers (A and I ') of A2B5 of undifferentiated iOPCs.
  • the white arrowheads at J ' indicate O4 positive and MBP positive cells. Scale bars are 100um and 50um.
  • FIG. 5 shows a cleavage map of the pMX retroviral vector.
  • FIG. 6 shows quantitative RT-PCR results of iOPCs derived from SOX1 , SOX2, SOX10, OLIG2, NKX2.2 or NKX6.2 expressing OPC family marker genes and oligodendrocyte marker genes.
  • the graph is shown as log2 fold change and normalized to GAPDH .
  • iOPCs induced oligodendrocyte progenitor cells using any one or more genes of the direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 , NKX6.2.
  • OCT4 direct cross-differentiation factor 4
  • SOX1, SOX2, SOX10 OLIG2, NKX2.2 , NKX6.2.
  • OCT4 direct cross-differentiation factor
  • iOPCs were transplanted into spinal cord-injured rats in vivo to confirm the effect on spinal cord injury. As a result, it was confirmed that the injured spinal cord tissue was significantly reduced, and that the myelin formation was restored without the occurrence of a tumor.
  • OCT4 octamer-binding transcription factor 4
  • POU5F1 protein
  • OCT4 is one of the homeodomain transcription factors of the POU family.
  • OCT4 protein has been known to be involved in autologous division of undifferentiated embryonic stem cells, but there is no known information on direct cross-differentiation from somatic cells to oligodendrocyte progenitor cells. Gene sequence of OCT4 is registered in NCBI Registration No. NM_014209, NM_013633.3.
  • the SOX1 is one of a transfer of personnel SOX protein family, is registered in NCBI Registration No. NM_005986.2, NM_009233.3.
  • SOX2 is one of the transcription factors of the SOX protein family and is registered under NCBI Accession Nos. NM_003106.3, NM_011443.3.
  • SOX10 is one of the transcription factors of the SOX protein family and is registered under NCBI Accession Nos. NM_006941.3, NM_011437.1.
  • OLIG2 is one of the transcription factors of the OLIG protein family and is registered under NCBI accession numbers NM_005806.3, _NM_016967.2.
  • NKX2.2 is one of the homeodomain transcription factors of the NK2 family and is registered under NCBI registration numbers NM_002509.3 and NM_001077632.1.
  • NKX6.2 is one of the homeodomain transcription factors of the NK2 family and is registered under NCBI accession numbers NM_177400.2 and NM_183248.3.
  • the OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 genes of the present invention may be provided in the form of a protein or nucleic acid encoding the protein, wherein the protein is human, mouse, horse, sheep, pig, It can include all proteins from animals such as goats, camels, antelopes, dogs, and the like.
  • the OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, and NKX6.2 proteins used in the present invention include protein variants of each gene as well as proteins having their wild type amino acid sequences.
  • a variant of the protein means a protein in which the natural amino acid sequence of the protein and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
  • the variant may be a functional equivalent that exhibits the same biological activity as a natural protein or a variant in which the physicochemical properties of the protein are modified as necessary, and may be a variant in which structural stability to physical and chemical environments is increased or physiological activity is increased. have.
  • nucleic acid encoding the protein is a nucleotide sequence encoding a protein of the wild type or a variant form as described above, one or more bases may be mutated by substitution, deletion, insertion or combination thereof, or is isolated from nature It may be prepared using chemical synthesis.
  • the nucleic acid having a nucleotide sequence encoding the above protein may be a single chain or a double chain, and may be a DNA molecule (genomic DNA, cDNA) or RNA molecule.
  • the nucleic acid molecule encoding the OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 protein in the present invention is a vector expressing a protein comprising a nucleic acid encoding each protein Can be.
  • vector is an expression vector capable of expressing a target protein in a host cell, and may refer to a gene construct including essential regulatory elements operably linked to express a gene insert.
  • the 'vector' in the present invention includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, and variously prepared according to the purpose.
  • the promoter of the vector may be constitutive or inducible.
  • the expression vector includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. Vectors can self replicate or integrate into host DNA.
  • Vectors include plasmid vectors, cosmid vectors, viral vectors and epismal vectors and the like. Preferably, it may be a viral vector.
  • Viral vectors are lentiviral vectors, retroviruses, such as Human immunodeficiency virus (HIV), Murineleukemia virus (MLV), Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), and Rous sarcoma virus (RSV).
  • HIV Human immunodeficiency virus
  • MMV Murineleukemia virus
  • ASLV Avian sarcoma / leukosis
  • SNV Spleen necrosis virus
  • RSV Rous sarcoma virus
  • vectors derived from Mouse mammary tumor virus (MMTV) Adenovirus, Adeno-associated virus, Herpes simplex virus, and the like.
  • the nucleic acid molecule encoding the protein is included in the vector and virus produced to express each gene by transforming and infecting a viral vector containing the nucleic acid encoding the protein with packaging cells.
  • viruses include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and the like.
  • the term "somatic cell” may mean any cell except germ cells.
  • fibroblasts, muscle cells, neurons, gastric mucosa, goblet cells, G cells, pericyte, astrocytes, B cells, blood cells, epithelial neural stem cells, hematopoietic stem cells, intermediate Leaf stem cells or umbilical cord blood stem cells may be used.
  • the direct cross-differentiation is not limited to the above, because if the starting cells are somatic cells can be applied regardless of the specific tissue cells.
  • fibroblasts of the skin were used.
  • oligodendrocyte progenitor cell refers to a cell of a subtype of a glial cell in the central nervous system. It is a precursor of oligodendrocytes and can differentiate into neurons and astrocytes.
  • iOPCs refers to induced oligodendrocyte progenitor cells, and for example, may refer to oligodendrocyte progenitor cells derived from somatic cells through direct differentiation according to the method of the present invention.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced
  • Vectors are introduced into somatic cells to provide direct cross-differentiation-induced oligodendrocyte progenitor cells.
  • the spinal cord injury includes all cases in which the central nervous system, the spinal cord, is damaged due to external or internal factors and fails to function. For example, concussion, compression, contusion, etc. Damage may be due to trauma, but is not limited thereto.
  • the spinal cord which is part of the central nervous system, fails to perform its function, thermoregulation disorders, diaphragmatic paralysis, intercostal paralysis, sweating, exercise paralysis, perception paralysis, urination disorder, pressure sores, ectopic dysfunction.
  • Various symptoms such as ossification and convulsions, can occur.
  • the "cell therapeutic agent” is a medicine (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special chewing from humans. It refers to a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise changing the biological characteristics of a living autologous, allogeneic, or heterologous cell in vitro.
  • Evaluation of the toxicity is based on the differentiation-induced oligodendrocyte progenitor cells of the present invention in the presence or absence of a therapeutic candidate substance of the present invention to inhibit the differentiation into oligodendrocytes or to differentiate the induced oligodendrocyte progenitor cells of the present invention.
  • a therapeutic candidate substance of the present invention such as IC 50
  • the evaluation of the efficacy of the drug in the art such as in the presence and absence of the therapeutic candidate substance of the present invention, the differentiation-induced oligodendrocyte progenitor cells promote differentiation into oligodendrocytes or promote myelin production. It can be evaluated according to the method to confirm that it is effective in the treatment of a dropout disease.
  • the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced
  • a method of directly cross-differentiating somatic cells into oligodendrocyte progenitor cells comprising introducing a vector into somatic cells.
  • ectopic expression of the direct cross-differentiation factor can be induced.
  • Ectopic expression means that a certain gene is expressed in tissues or extracellularly expressed originally, or is expressed at a different time than originally expressed.
  • the present invention may be to induce the expression of direct cross-differentiation factor in somatic cells by introducing the composition for induction into somatic cells.
  • oligodendrocyte progenitor cells can be produced from somatic cells.
  • Rare oligodendrocyte progenitor cells prepared according to the present invention play an essential role in myelin production in the central nervous system, and can be differentiated into oligodendrocytes, neurons or astroglia, and thus can be applied to the prevention or treatment of diseases caused by myeloma loss. .
  • Dermal fibroblasts were dispensed into 3 ⁇ 10 4 cells on gelatin coated 6well plates in 10% FBS culture medium. After one day, the infected fibroblasts into pMX retroviral vector expressing OCT4 in medium containing protamine sulfate of 6 ⁇ g / ml. 24 hours after infection, virus supernatants were removed and replaced with fresh medium. On day 3 of infection, cells were chemically modified in OPC medium (DMEM / F12 supplemented with N2, penicillin / streptomycin), PDGF- ⁇ 20ng / ml (Peprotech), FGF-2 10ng / ml (Peprotech Changed to)). After 12 days of further incubation in OPC medium, colonies such as OPC were mechanically isolated and subcultured by picking or trypsinizing mature iOPC on gelatin coated plates.
  • OPC medium DMEM / F12 supplemented with N2, penicillin / streptomycin
  • oligodendrocyte differentiation medium 1 DMEM / F12 supplemented with N2, penicillin / streptomycin, FGF-2 10ng / ml (Peprotech), forskolin 10mM (Sigma)
  • oligodendrocyte differentiation medium 2 30 ng / ml 3, 3, 5-tri-iodothyronine (T3; Sigma), 20 ng / ml ascorbic acid (AA; Sigma)).
  • RNA without DNA was extracted using RNeasy mini kit (Qiagen).
  • CDNA was synthesized with SuperScript® III reverse transcriptase (Invitrogen) using a total of 500 ng of RNA per reaction.
  • the synthesized cDNA was used as a template using a LightCycler 480 SYBR Green I Mastermix (Roche) with a total volume of 20ul.
  • OPCs marker genes, pluripotent stem cells and neural markers were repeated three times and normalized to the housekeeping gene Gapdh. Gene expression was measured by Ct value calculation method. All experiments were performed according to the manufacturer's instructions.
  • the cells were fixed in 4% para-formaldehyde for 10 minutes and treated with 0.1% Triton X-100 for 10 minutes to make them permeable. Cells were incubated for 30 minutes in 4% FBS blocking solution and then incubated for 1 hour at room temperature in primary antibody diluted with blocking solution. Oligo2 (Santacruz, 1: 200), PDGF- ⁇ (Abcam, 1: 500), A2B5 (Millipore, 1: 500), NG2 (Millipore, 1: 200), O4 (Millipore, 1: 400), RIP (DSHB, 1: 200), GalC (Chemicon, 1: 200), GFAP (Sigma, 1: 500).
  • Dermal fibroblasts were isolated and incubated in MEF medium (Dullbecco's modified Eagle's medium supplemented with 10% FBS, nonessential amino acids, L-glutamine, penicillin / streptomycin, mercaptoethanol) at 37 °C, 5% CO 2 incubator.
  • MEF medium Dullbecco's modified Eagle's medium supplemented with 10% FBS, nonessential amino acids, L-glutamine, penicillin / streptomycin, mercaptoethanol
  • RT-PCR was performed for 35 cycles using Taq DNA polymerase recombinant (Invitrogen).
  • cells were fixed in para-formaldehyde in 4% PBS (pH 7.4) for 10 minutes and treated with 0.1% Triton X-100 for 10 minutes to make them permeable. Cells were incubated for 30 min in 4% FBS blocking solution and then incubated for 1 hour at room temperature in the primary antibody diluted with blocking solution.
  • Oct4 (Santacruz, 1: 200), Sox2 (Santacruz, 1: 400), Olig2 (Santacruz, 1: 200), Pax6 (DSHB, 1: 200), O4 (Millipore, 1: 400), RIP (DSHB, 1 : 200), GFAP (Sigma, 1: 500).
  • primary antibody culture cells were washed and incubated for 1 hour in secondary antibody (Alexa Fluor 488/568 anti-mouse IgG1, IgG3, IgM, anti-goat IgG (Invitrogen, 1: 1000)). Nuclei were stained for 10 seconds using Hoechst 33342 (Thermo).
  • UPMA unweighted average distance
  • Spinal cord injury model was constructed using an air punch device to damage the spinal cord spine level 9 (T9) in 6-week-old male rats. After 1 week of injury, harvested iOPCs were labeled with DiI and injected into the spinal cord level 8 (T8) and thoracic spine level 10 (T10), respectively, by stereotaxic at a concentration of 1 ⁇ 10 5 iOPCs / rat. . Rats were given water and food regularly and massaged bladder for urination. The animal testing process was approved by the animal facility and the IBR Committee of Ulsan National University of Science and Technology. The animal testing process was based on guidelines from the National Institutes of Health on animal studies.
  • mice Twelve weeks after transplantation, mice were anesthetized and perfuse PBS.
  • the spinal cord was fixed overnight with 4% paraformaldehyde and dehydrated via staged concentrations of alcohol and xylene.
  • the tissues were placed in paraffin blocks and subsequently divided into 4um thickness in the sagittal and coronal directions. The divided portions were permeable with 0.25% Triton X-100 and blocked with 10% blocking solution for nonspecific binding sites.
  • the divided sections were incubated with primary antibody PDGF- ⁇ (Abcam, 1: 100), O4 (Millipore, 1: 100), RIP (DSHB, 1: 100), GFAP (Sigma, 1 : 100), MBP (Millipore, 1: 100).
  • Spinal cord sections were then stained with secondary antibodies. Fluorescence images were visualized with Olympus microscope equipment IX81-ZDC.
  • iOPC iOPC aggregates
  • iOPC-Ag iOPC aggregates
  • OCT4 Using PCR with the DNA of host iOPC it was confirmed that the OCT4 gene is integrated transition. In order to confirm the silencing of the transferred gene, the mRNA level of OCT4 was confirmed by real-time PCR. In addition, the iOPCs maintained the normal karyotype of the chromosome after cell fate transition to viral induction. Through this, OCT4 demonstrated from fibroblasts that sufficient conversion of the early fate iOPCs.
  • oligodendrocytes By day 16, the mature morphology of oligodendrocytes was confirmed more and was present with differentiated astrocytes (F in FIG. 2B). After differentiation, staining with oligodendrocyte marker O4 and astrocyte marker GFAP confirmed that oligodendrocytes and astroglia were present together (G to L in FIG. 2C). However, the possibility of oligodendrocyte differentiation of these iNSCs depends on the transcription factor used, and the efficiency is relatively low. In order to evaluate the differentiation efficiency of OCT4-iOPC, staining with O4, GFAP, Tuj1, and A2B5 was analyzed for the ratio of iOPCs after differentiation to confirm the progression of differentiation during maturation.
  • iOPCs were injected into the injured spinal cord and observed up to 12 weeks.
  • H & E staining was performed on the spinal cord area.
  • the group injected with iOPCs confirmed that the damaged tissue of the damaged part was significantly reduced compared to the control group (FIG. 4A).
  • Immunohistochemical staining was performed to confirm the engraftment of the transplanted cells.
  • iOPCs were injected into mice, and tumors were confirmed until 8 months after injection.
  • iOPCs transplantation has been demonstrated to restore myelination in the spinal cord and to recover damaged areas in spinal cord injury model mice without tumor development.

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Abstract

The present invention relates to a composition for inducing direct transdifferentiation into oligodendrocyte progenitor cells (OPCs) from somatic cells, the composition containing at least one protein selected from the group consisting of direct transdifferentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, and NKX6.2, a nucleic acid molecule coding the protein, or a vector including the nucleic acid molecule introduced thereinto; a pharmaceutical composition for preventing or treating spinal cord injuries or demyelination diseases; a cell therapy agent for preventing or treating spinal cord injuries or demyelination diseases; a cell therapy agent for treating spinal cord injuries or demyelination diseases; a composition for screening drugs for the treatment of spinal cord injuries or demyelination diseases; a 3D printing biomaterial composition for manufacturing artificial tissues for the treatment of spinal cord injuries or demyelination diseases; and a method for direct transdifferentiation into oligodendrocyte progenitor cells from somatic cells. According to the present invention, the oligodendrocyte progenitor cells are prepared from somatic cells through direct transdifferentiation, and thus can be favorably utilized for the treatment of spinal cord injuries and demyelination diseases.

Description

체세포로부터 희소돌기아교 전구세포로의 직접교차분화 유도용 조성물 및 이의 이용Composition for inducing direct cross-differentiation from somatic cells to oligodendrocyte progenitor cells and use thereof
직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 유효성분으로 포함하는 체세포로부터 유도희소돌기아교 전구세포(induced Oligodendrocyte Progenitor Cell; iOPCs)로의 직접교차분화 유도용 조성물 및 상기 조성물을 처리하는 단계를 포함하는 체세포로부터 희소돌기아교 전구세포로 직접교차분화하는 방법에 관한 것이다. 또한, 본 발명은 상기 체세포로부터 희소돌기아교 전구세포로 직접교차분화하는 방법에 의해 분화 유도된 희소돌기아교 전구세포를 포함하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 약학 조성물, 세포치료제, 약물 스크리닝용 조성물, 또는 인공조작 제작을 위한 3D 프린팅 생체 소재 조성물에 관한 것이다.Direct cross-differentiation factor OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , any one or more proteins selected from the group consisting of, a nucleic acid molecule encoding the protein or a vector into which the nucleic acid molecules are introduced as an active ingredient A composition for inducing direct cross-differentiation from induced somatic cells to induced Oligodendrocyte Progenitor Cells (iOPCs) and a method for direct cross-differentiation from somatic cells to oligodendrocyte precursors comprising the step of treating the composition. will be. In addition, the present invention is a pharmaceutical composition, cell therapy, drug for the prevention or treatment of spinal cord injury or myelin deprivation disease, including rare oligodendrocyte progenitor cells differentiated by the method of direct cross-differentiation from the somatic cells to oligodendrocyte progenitor cells The present invention relates to a composition for screening, or a 3D printed biomaterial composition for artificial fabrication.
희소돌기아교세포(Oligodendrocyte)는 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cells, OPCs)로 알려진 한정된 전구체의 신경교 세포 계통(glial lineage)의 발달 분화 중의 신경상피로부터 유래한다. 희소돌기아교 전구세포는 중추신경계(central nervous system, CNS)에서 수초(myelin sheath) 생성에 필수적인 역할을 하는 신경아교세포 유형 중 하나이다. 수초형성(Myelination)은 뉴런이 축삭의 단절을 통하여 활동전위(action potential)의 전기적 자극(electrical impulse)을 유지하게 한다.Oligodendrocytes are derived from neuroepithelial cells during the developmental differentiation of the glial lineage of defined precursors known as Oligodendrocyte Progenitor Cells (OPCs). Rare oligodendrocytes are one of the types of glial cells that play an essential role in the production of myelin sheath in the central nervous system (CNS). Myelination allows neurons to maintain electrical impulses of action potential through disruption of axons.
희소돌기아교세포의 기능장애(dysfunction)는 수초의 손실로 인한 장애로, 다발성 경화증(multiple sclerosis), 뇌성마비(cerebral palsy), 백질이영양증(leukodystrophy), 신경병질, 중심다리뇌 수초용해 및 수초형성부전증(hypomyelination)을 포함하는 많은 신경퇴행성(neurodegenerative) 질환을 유발한다. 손상된 희소돌기아교세포의 치료하기 위해서 내생(endogenous) 세포를 재생(restoration)하거나 수초를 생성하는 기능을 가진 희소돌기아교 전구세포를 중추신경계로 이식하는 것이, 수초 손상을 치료하는 가장 근본적인 방법이다.Dysfunction of oligodendrocytes is a disorder caused by the loss of myelin sheath, multiple sclerosis, cerebral palsy, leukodystrophy, neuropathy, central limb myelination and myelination It causes many neurodegenerative diseases, including hypomyelination. In order to treat damaged oligodendrocytes, transplanting oligodendrocyte progenitor cells, which have the function of restoring endogenous cells or producing myelin, into the central nervous system is the most fundamental method of treating myelin damage.
이와 관련하여, 희소돌기아교세포를 이식하여 수초 손상을 치료하였으나, 희소돌기아교세포의 효과적인 분화방법이 명확하지 않았고, 많은 양의 희소돌기아교세포를 얻는 것이 매우 어려워 문제점이 있었다.In this regard, the myeloma was treated by transplanting oligodendrocytes, but the effective differentiation method of oligodendrocytes was not clear, and it was very difficult to obtain a large amount of oligodendrocytes.
또한, 배아줄기세포(embryonic stem cells, ESCs) 및 유도만능줄기세포로부터 희소돌기아교세포를 분화하는 방법도 있으나, 목적하는 세포로의 유도효율이 낮고 특정 세포로 분화 시 상기 배아줄기세포 또는 만능줄기세포로부터 종양이 형성 될 수 있는 위험성이 있기 때문에 문제가 있다.In addition, there is a method of differentiating oligodendrocytes from embryonic stem cells (ESCs) and induced pluripotent stem cells, but the induction efficiency into the desired cells is low and when the embryonic stem cells or pluripotent stem cells are differentiated into specific cells This is problematic because there is a risk of tumor formation from cells.
상기 종양형성 위험성을 없애기 위하여, 만능줄기세포 상태를 통하지 않고 체세포를 다른 계통의 체세포로 또는 다분화능 줄기세포로 직접교차분화하기 위한 연구가 진행되고 있으나, 이러한 세포 리프로그래밍과정의 기작은 유도를 위해 사용된 유전자의 수가 너무 많아 자세한 기작을 규명하기 어렵기 때문에 현재까지 밝혀진 수준이 미미한 상태이다. 또한, 역분화기술이 확립이 되었음에도 실제 치료적용을 위해서는 기술적으로 간편하고 효율이 높은 방법을 계속해서 개발해야만 하고 이렇게 개발된 기술들은 모두 효율과 안전성 측면에서 평가되어야 하므로 제한적이다. 아울러, 아직까지 체세포로부터 단일 유전자만을 발현시켜 희소돌기아교 전구세포를 제조하는 방법은 알려진 바가 없다.In order to eliminate the risk of tumor formation, research is being conducted to directly cross-differentiate somatic cells into somatic cells or multipotent stem cells of other lineages without passing through the pluripotent stem cell state. The number of genes used is so high that it is difficult to elucidate detailed mechanisms. In addition, despite the fact that de-differentiation technology has been established, it is limited because it is necessary to continuously develop technically simple and high-efficiency methods for actual therapeutic application and all the developed technologies must be evaluated in terms of efficiency and safety. In addition, there is no known method for preparing oligodendrocyte progenitor cells by expressing only a single gene from somatic cells.
이러한 배경 하에, 본 발명자들은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 중 어느 하나 이상의 유전자를 사용하여 종양형성 위험성 없이 체세포로부터 희소돌기아교세포의 전구세포를 직접교차분화 방법으로 제조할 수 있음을 확인하여 본 발명을 완성하였다. Under this background, the present inventors have transdifferentiation factor for OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, precursor cells of oligodendrocytes using any one or more of the gene NKX6.2 from somatic cells without the risk of tumorigenesis The present invention was completed by confirming that it can be prepared by a direct cross-differentiation method.
본 발명의 목적은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 유효성분으로 포함하는 체세포로부터 희소돌기아교 전구세포로의 직접교차분화 유도용 조성물을 제공하는데 있다.An object of the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced The present invention provides a composition for inducing direct cross-differentiation from somatic cells comprising a vector as an active ingredient to oligodendrocyte progenitor cells.
본 발명의 다른 목적은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 약학조성물, 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 세포치료제, 척수 손상 또는 수초탈락 질환 치료 약물 스크리닝용 조성물 및 척수 손상 또는 수초탈락 질환 치료용 인공조직 제작을 위한 3D 프린팅 생체 소재 조성물을 제공하는데 있다.Another object of the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced Pharmaceutical composition for the prevention or treatment of spinal cord injury or myelin deprivation disease, comprising a vector or the direct cross-differentiation-induced oligodendrocyte progenitor cells, cell therapy for the prevention or treatment of spinal cord injury or myelinated disease, spinal cord injury Or to provide a composition for screening drug treatment for myelin deprivation disease and 3D printing biomaterial composition for the manufacture of artificial tissue for the treatment of spinal cord injury or myelin deprivation disease.
본 발명의 또 다른 목적은 체세포로부터 희소돌기아교 전구세포로 직접교차분화하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for direct cross-differentiation from somatic cells to oligodendrocyte progenitor cells.
상기 목적을 달성하기 위해, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 유효성분으로 포함하는 체세포로부터 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cell)로의 직접교차분화 유도용 조성물을 제공한다.In order to achieve the above object, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the Provided is a composition for inducing direct cross-differentiation from somatic cells comprising a vector into which a nucleic acid molecule has been introduced as an oligodendrocyte progenitor cell.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 체세포에 도입하여 직접교차분화 유도된 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cell)를 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced Vectors are introduced into somatic cells to provide direct cross-differentiation-induced oligodendrocyte progenitor cells.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced It provides a pharmaceutical composition for the prevention or treatment of spinal cord injury or demyelination disease comprising a vector or the direct cross-differentiation-induced oligodendrocyte progenitor cells as an active ingredient.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 세포치료제를 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced Provided is a cell therapy agent for the prevention or treatment of spinal cord injury or myelin deprivation disease, comprising a vector or the above-described direct cross-differentiation-induced oligodendrocyte progenitor cells as an active ingredient.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환 치료 약물 스크리닝용 조성물을 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced Provided is a composition for screening a therapeutic drug for spinal cord injury or myelin deprivation disease, comprising a vector or the direct cross-differentiation induced oligodendrocyte progenitor cells as an active ingredient.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환 치료용 인공조직 제작을 위한 3D 프린팅 생체 소재 조성물을 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced Provided is a 3D printing biomaterial composition for the manufacture of artificial tissue for the treatment of spinal cord injury or myelin deprivation disease comprising a vector or the direct cross-differentiation induced oligodendrocyte progenitor cells as an active ingredient.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 체세포에 도입하는 단계를 포함하는 체세포를 희소돌기아교 전구세포로 직접교차분화하는 방법을 제공한다. In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced Provided is a method of directly cross-differentiating somatic cells into oligodendrocyte progenitor cells comprising introducing a vector into somatic cells.
본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 중 어느 하나 이상의 유전자의 사용으로 체세포로부터 희소돌기아교 전구세포로의 직접교차분화를 유도할 수 있는 조성물을 제공하고, 상기 조성물을 이용하여 체세포로부터 희소돌기아교 전구세포를 제조할 수 있고, 이를 통하여 척수 손상 및 수초탈락 질환 등의 치료에 우수하게 활용할 수 있다.Composition in the present invention is able to induce transdifferentiation of a oligodendrocyte progenitor cells from somatic cells by directly using the cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, any one or more of the gene NKX6.2 To provide a rare oligodendrocyte progenitor cells from the somatic cells using the composition, it can be excellently used in the treatment of spinal cord injury and demyelination diseases.
도면에서 "iOPC-C1" 및 "iOPC-C2"는 본 발명에 따라 제조된 희소돌기아교세포 전구체 세포의 클론 1 및 클론 2를 각각 나타낸다."IOPC-C1" and "iOPC-C2" in the Figures represent clone 1 and clone 2 of oligodendrocyte precursor cells prepared according to the present invention, respectively.
도 1a는 섬유아세포로부터 OCT4-유도된 iOPC의 제조과정을 도식화한 그림이다.Figure 1a from fibroblasts OCT4 - is a schematic view showing a manufacturing process of the induced iOPC Fig.
도 1b는 섬유아세포로부터 iOPCs로의 분화가 점진적으로 진행됨에 따른 변화를 확인한 위상차-현미경의 이미지를 나타낸다. "B"는 감염되지 않은 섬유아세포, "C"는 대조군, "D"는 섬유아세포-OCT4 25일 째, "E"는 OCT4-유도된 섬유아세포 형성 집합체, "F"는 iOPC 같은 군집, 및 "G"는 iOPCs의 양극성 형태를 나타낸다. E 및 F의 스케일 바(Scale bars)는 500um, B 내지 D의 스케일 바는 200um, G의 스케일 바는 100um이다.Figure 1b shows an image of a phase contrast microscope to confirm the change as the differentiation from fibroblasts to iOPCs gradually progress. "B" is uninfected fibroblast, "C" is control, "D" is fibroblast- OCT4 on day 25, "E" is OCT4 -induced fibroblast-forming aggregate, "F" is iOPC-like population, and "G" represents the bipolar form of iOPCs. The scale bars of E and F are 500 um, the scale bars of B to D are 200 um, and the scale bars of G are 100 um.
도 1c는 PDGFR-α, NG2, A2B5, 및 Olig2의 iOPC 특이적인 마커로 이중 염색된 OCT4-유도된 iOPC 클론의 면역형광분석 결과를 나타낸다. 스케일 바는 125um이다.Figure 1c is PDGFR-α, NG2, A2B5, and iOPC a specific double staining marker of the OCT4 Olig2 - shows an immunofluorescence analysis of the clones derived iOPC. Scale bar is 125um.
도 1d는 자가분열하는 iOPCs의 위상차 현미경 이미지를 나타낸다. 스케일 바는 100um 이다.1D shows phase contrast microscopy images of self-dividing iOPCs. Scale bar is 100um.
도 1e는 초기 패시지(P3)와 늦은 패시지(P31)에서 시간에 따른 iOPCs의 증식률을 각각 나타낸 것이다. 데이터는 평균 ± 표준오차로 나타내었다.Figure 1e shows the growth rate of iOPCs with time in the initial passage (P3) and late passage (P31), respectively. Data are expressed as mean ± standard error.
도 2a는 분화되지 않은 iOPCs의 면역형광 이미지를 나타낸다. "A"는 PDGFR-α, GFAP로 염색한 이미지, "B"는 NG2, Tuj1로 염색한 이미지, "C"는 A2B5 and RIP로 염색한 이미지를 나타낸다. 스케일 바는 250um 이다.2A shows immunofluorescence images of undifferentiated iOPCs. "A" shows images stained with PDGFR-α, GFAP, "B" shows images stained with NG2, Tuj1, and "C" shows images stained with A2B5 and RIP. Scale bar is 250um.
도 2b는 iOPCs의 in vitro 분화 중 형상 변화를 위상차 현미경 이미지로 나타낸 것이다. "D"는 1일차, "E"는 5일 차, "F"는 16일 차의 결과를 나타낸다. 녹색화살표는 희소돌기아교세포를 가리키고 빨간색 화살표는 성상교세포를 가리킨다. 스케일 바는 100um 이다.Figure 2b shows a phase change microscopy image of the shape change during in vitro differentiation of iOPCs. "D" represents Day 1, "E" represents Day 5, and "F" represents Day 16. The green arrow points to oligodendrocytes and the red arrow points to astrocytes. Scale bar is 100um.
도 2c는 분화 후에 iOPCs의 다분화능을 나타내는 O4+ 희소돌기아교세포(녹색) 및 GFAP+ 성상교세포(빨간색)가 함께 존재하는 것을 나타낸다. 스케일 바는 125um이다.FIG. 2C shows the presence of O4 + oligodendrocytes (green) and GFAP + astrocytes (red) together, indicating the multipotency of iOPCs after differentiation. Scale bar is 125um.
도 2d는 분화 30일 후에 성숙한 희소돌기아교세포 마커를 발현하는 희소돌기아교세포의 면역형광 이미지를 나타낸다. "M" 및 "P"는 RIP의 발현, "N" 및 "Q"는 GalC의 발현, "O" 및 "R"은 MBP 발현을 나타낸다. 스케일 바는 125um이다.2D shows immunofluorescence images of oligodendrocytes expressing mature oligodendrocyte markers 30 days after differentiation. "M" and "P" represent expression of RIP, "N" and "Q" represent GalC, and "O" and "R" represent MBP expression. Scale bar is 125um.
도 3a은 OCT4-유도된 iOPCs의 전체적인 유전자 발현에 대한 마이크로어레이 분석의 열지도(Heatmap) 결과를 나타낸 것이다. "fibroblasts"는 섬유아세포, "fib-OCT4 D3"는 OCT4에 의한 분화 유도 3일 째 섬유아세포, "fib-OCT4 D10"은 OCT4에 의한 분화 유도 10일 째 섬유아세포, "mock"은 대조군, "wtOPC"는 만능 줄기 세포로부터 유래된 OPC를 나타낸다.Figure 3a is OCT4 - shows a heat map (Heatmap) the result of microarray analysis for the overall expression of genes induced iOPCs. "fibroblasts" are fibroblasts, "OCT4 fib- D3" is differentiation-induced by OCT4 3 il Me fibroblasts, "OCT4 fib- D10" is differentiation-induced by OCT4 10 il Me fibroblasts, "mock" is a control, " wtOPC ″ refers to OPC derived from pluripotent stem cells.
도 3b는 섬유아세포와 OPC, 섬유아세포와 iOPCs-C1, 섬유아세포와 iOPCs-C2, iOPC-C1과 OPC, iOPC-C2와 OPC, iOPC-C1과 iOPC-C2의 상관관계를 보여주는 전체적인 유전자 발현과 비교한 산포도(Scatter plot)를 나타낸다. 검은 선은 짝지은 샘플 사이에 2배의 유전자 발현 변화가 있는 경계선을 나타낸다. 유전자 발현 수준은 log2 값으로 나타내었다.Figure 3b shows the overall gene expression showing the relationship between fibroblasts and OPC, fibroblasts and iOPCs-C1, fibroblasts and iOPCs-C2, iOPC-C1 and OPC, iOPC-C2 and OPC, iOPC-C1 and iOPC-C2 The scatter plot is compared. Black lines represent borderline with 2 fold change in gene expression between paired samples. Gene expression levels are expressed as log2 values.
도 3c는 OPC 계열 마커 유전자와 희소돌기아교세포 마커 유전자를 발현하는 OCT4-유도된 OPCs의 정량 RT-PCR 결과를 나타낸다. 그래프는 log2 배 변화(log2 fold change)로 나타내었고 GAPDH 로 정상화(normalize)하였다.Figure 3c OCT4 expressing the OPC marker gene sequence and oligodendrocytes marker gene indicates a quantitative RT-PCR results of the induced OPCs. The graph is shown as log2 fold change and normalized to GAPDH .
도 3d는 3D 주성분 분석 결과를 나타낸다. 1차 주요 성분은(first principal component, PC1) 유전자 발현 변동성의 64%를 차지했다. 2차 주요 성분(second principal component, PC2)은 변동성의 14%를 차지했고 3차 주요 성분(third principal component, PC3)은 변동성의 8.3%를 차지했다.3D shows the results of 3D principal component analysis. The first principal component (PC1) accounted for 64% of gene expression variability. The second principal component (PC2) accounted for 14% of volatility and the third principal component (PC3) accounted for 8.3% of volatility.
도 3e는 전체적인 유전자 발현의 계층 클러스터링 결과를 나타낸다. 마이크로어레이 분석 결과를 통하여 OCT4 유도 후에 유전자 발현의 변화를 확인하였다. 희소돌기아교세포와 신경 발달은 섬유아세포와 비교하여 iOPCs에서 매우 높게 발현되는 유전자와 관계있음을 확인하였다.3E shows the hierarchical clustering results of overall gene expression. The results of microarray analysis confirmed changes in gene expression after OCT4 induction. It was confirmed that oligodendrocytes and neural development were related to genes expressed highly in iOPCs compared to fibroblasts.
도 4a는 손상 및 이식 12주 후에 H&E 염색된 쥐의 척수 부분의 이미지를 나타낸다. 스케일 바는 200um이다.4A shows images of spinal cord sections of H & E stained mice 12 weeks after injury and transplantation. Scale bar is 200um.
도 4b는 손상 받은 부분의 척수에 대한 면역조직화학 분석 결과를 나타낸다. "C" 및 "F" O4로 염색, "D" 및 "G"는 MBP로 염색, "E 및 H"는 GFAP로 염색한 결과를 나타낸다.Figure 4b shows the results of immunohistochemistry analysis on the spinal cord of the damaged part. Stain with "C" and "F" O4, "D" and "G" with MBP, "E and H" with GFAP.
도 4c는 쥐의 척수에서 이식된 iOPCs의 O4, MBP 및 GFAP 양성 세포로의 분화 및 분화되지 않은 iOPCs의 A2B5의 미성숙 마커(I 및 I') 발현을 확인한 결과를 나타낸다. J'에서 하얀색 화살표 머리는 O4 양성 및 MBP 양성 세포를 가리킨다. 스케일 바는 100um, 50um이다.4C shows the results of differentiating iOPCs transplanted into O4, MBP and GFAP positive cells in the rat spinal cord and immature markers (A and I ') of A2B5 of undifferentiated iOPCs. The white arrowheads at J 'indicate O4 positive and MBP positive cells. Scale bars are 100um and 50um.
도 5는 pMX 레트로바이러스 벡터의 개열지도를 나타낸다.5 shows a cleavage map of the pMX retroviral vector.
도 6은 OPC 계열 마커 유전자와 희소돌기아교세포 마커 유전자를 발현하는 SOX1, SOX2, SOX10, OLIG2, NKX2.2 또는 NKX6.2로 유도된 iOPCs의 정량 RT-PCR 결과를 나타낸다. 그래프는 log2 배 변화(log2 fold change)로 나타내었고 GAPDH 로 정상화(normalize)하였다.6 shows quantitative RT-PCR results of iOPCs derived from SOX1 , SOX2, SOX10, OLIG2, NKX2.2 or NKX6.2 expressing OPC family marker genes and oligodendrocyte marker genes. The graph is shown as log2 fold change and normalized to GAPDH .
본 발명의 구체적인 실시예에서는 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 중 어느 하나 이상의 유전자를 이용하여 유도희소돌기아교 전구세포(induced Oligodendrocyte Progenitor Cells, iOPCs)를 제조할 수 있는지 확인하기 위해서, 체세포를 분리하고 직접교차분화인자 유전자 전사 인자를 암호화하는 레트로바이러스(retrovirus)를 체세포에 감염시켜 iOPCs를 제조하였다(실험결과 1).In a specific embodiment of the present invention , induced oligodendrocyte progenitor cells (iOPCs) using any one or more genes of the direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 , NKX6.2. In order to confirm that it can be prepared, iOPCs were prepared by isolating somatic cells and infecting somatic cells with a retrovirus encoding a direct cross-differentiation factor gene transcription factor (Experiment 1).
또 다른 실시예에서는, 제조된 iOPCs가 전구체로서의 미성숙 상태(immature state)임을 면역세포화학 분석 및 이중염색을 통하여 확인하였고, 그 결과, 전구체로서의 자가분열 능력 및 31 패시지 이상까지 생장률을 확인하였다(실험결과 2).In another embodiment, it was confirmed through immunocytochemical analysis and double staining that the prepared iOPCs are immature state as a precursor, as a result, the ability to self-dividing as a precursor and growth rate up to 31 passages (experimental) Result 2).
또한, 제조한 iOPCs의 분화능을 확인하기 위하여 분화를 유도하였다. 그 결과, 성숙한 희소돌기아교세포 또는 성상교세포로 분화할 수 있음을 확인하였다(실험결과 3).In addition, differentiation was induced to confirm the differentiation capacity of the prepared iOPCs. As a result, it was confirmed that they could be differentiated into mature oligodendrocytes or astrocytes (Experiment 3).
본 발명의 또 다른 실시예에서는 제조된 iOPCs와 OPCs의 전체적인 유전자 프로필 발현을 비교하였다. 그 결과, iOPCs와 OPCs의 전체적인 유전자 프로필 발현이 유사함을 확인하여, 제조된 iOPCs가 OPCs의 기능을 수행할 수 있음을 다시 한 번 확인하였다(실험결과 4).In another embodiment of the present invention was compared the overall gene profile expression of the prepared iOPCs and OPCs. As a result, it was confirmed that the expression of the overall gene profile of the iOPCs and OPCs are similar, and once again confirmed that the prepared iOPCs can perform the function of the OPCs (Experiment 4).
나아가, 본 발명에서는 in vivo 상에서 척수 손상된 쥐에 iOPCs를 이식하여 척수 손상에 미치는 영향을 확인하였다. 그 결과, 손상된 척수 조직이 현저히 감소한 것을 확인하였고, 종양의 발생 없이 수초형성을 회복시키는 것을 입증하였다.Furthermore, in the present invention, iOPCs were transplanted into spinal cord-injured rats in vivo to confirm the effect on spinal cord injury. As a result, it was confirmed that the injured spinal cord tissue was significantly reduced, and that the myelin formation was restored without the occurrence of a tumor.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 유효성분으로 포함하는 체세포로부터 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cell)로의 직접교차분화 유도용 조성물을 제공한다. The present invention is directed to any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or a vector into which the nucleic acid molecule is introduced. Provided is a composition for inducing direct cross-differentiation from somatic cells comprising an active ingredient into oligodendrocyte progenitor cells.
상기 OCT4 (octamer-binding transcription factor 4) 단백질은 POU5F1 단백질으로도 알려져 있으며, POU5F1 유전자에 의하여 코딩된다. OCT4는 POU 패밀리의 호메오도메인(homeodomain) 전사 인자 중 하나이다. OCT4 단백질은 분화되지 않은 배아줄기세포의 자가분열과 관계되어 있음은 알려져 있으나, 체세포로부터 희소돌기아교 전구세포로의 직접교차분화와 관계된 내용은 전혀 알려진 바가 없다. OCT4의 유전자 서열은 NCBI 등록번호 NM_014209, NM_013633.3으로 등록되어 있다.The OCT4 (octamer-binding transcription factor 4 ) protein is also known as protein POU5F1, POU5F1 is coded by the gene. OCT4 is one of the homeodomain transcription factors of the POU family. OCT4 protein has been known to be involved in autologous division of undifferentiated embryonic stem cells, but there is no known information on direct cross-differentiation from somatic cells to oligodendrocyte progenitor cells. Gene sequence of OCT4 is registered in NCBI Registration No. NM_014209, NM_013633.3.
상기 SOX1는 SOX 단백질 패밀리의 전사인사 중 하나이고, NCBI 등록번호 NM_005986.2, NM_009233.3으로 등록되어 있다.The SOX1 is one of a transfer of personnel SOX protein family, is registered in NCBI Registration No. NM_005986.2, NM_009233.3.
상기 SOX2는 SOX 단백질 패밀리의 전사인사 중 하나이고, NCBI 등록번호 NM_003106.3, NM_011443.3으로 등록되어 있다. SOX2 is one of the transcription factors of the SOX protein family and is registered under NCBI Accession Nos. NM_003106.3, NM_011443.3.
상기 SOX10는 SOX 단백질 패밀리의 전사인사 중 하나이고, NCBI 등록번호 NM_006941.3, NM_011437.1로 등록되어 있다. SOX10 is one of the transcription factors of the SOX protein family and is registered under NCBI Accession Nos. NM_006941.3, NM_011437.1.
상기 OLIG2는 OLIG 단백질 패밀리의 전사인사 중 하나이고, NCBI 등록번호 NM_005806.3,_NM_016967.2로 등록되어 있다. OLIG2 is one of the transcription factors of the OLIG protein family and is registered under NCBI accession numbers NM_005806.3, _NM_016967.2.
상기 NKX2.2는 NK2 패밀리의 호메오도메인(homeodomain) 전사 인자 중 하나이고, NCBI 등록번호 NM_002509.3, NM_001077632.1로 등록되어 있다. NKX2.2 is one of the homeodomain transcription factors of the NK2 family and is registered under NCBI registration numbers NM_002509.3 and NM_001077632.1.
상기 NKX6.2는 NK2 패밀리의 호메오도메인(homeodomain) 전사 인자 중 하나이고, NCBI 등록번호 NM_177400.2, NM_183248.3으로 등록되어 있다. NKX6.2 is one of the homeodomain transcription factors of the NK2 family and is registered under NCBI accession numbers NM_177400.2 and NM_183248.3.
본 발명의 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 유전자는 단백질 또는 이의 단백질을 코딩하는 핵산의 형태로 제공될 수 있는데, 단백질은 인간과 마우스, 말, 양, 돼지, 염소, 낙타, 영양, 개 등의 동물 유래의 모든 단백질을 포함할 수 있다. 또한, 본 발명에 사용되는 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 단백질은 이의 야생형(wild type)의 아미노산 서열을 갖는 단백질뿐만 아니라 각 유전자의 단백질 변이체를 포함한다.The OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 genes of the present invention may be provided in the form of a protein or nucleic acid encoding the protein, wherein the protein is human, mouse, horse, sheep, pig, It can include all proteins from animals such as goats, camels, antelopes, dogs, and the like. In addition, the OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, and NKX6.2 proteins used in the present invention include protein variants of each gene as well as proteins having their wild type amino acid sequences.
상기 단백질의 변이체란, 상기 단백질의 천연 아미노산 서열과 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 단백질을 의미한다. 상기 변이체는 천연 단백질과 동일한 생물학적 활성을 나타내는 기능적 등가물이거나 필요에 의해서 단백질의 물리 화학적 성질이 변형된 변이체일 수 있고, 물리, 화학적 환경에 대한 구조적 안정성이 증대되거나 생리학적 활성이 증대된 변이체일 수 있다.A variant of the protein means a protein in which the natural amino acid sequence of the protein and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. The variant may be a functional equivalent that exhibits the same biological activity as a natural protein or a variant in which the physicochemical properties of the protein are modified as necessary, and may be a variant in which structural stability to physical and chemical environments is increased or physiological activity is increased. have.
또한, 상기 단백질을 코딩하는 핵산은 야생형 또는 상기한 바와 같은 변이체 형태의 단백질을 코딩하는 염기서열로서, 하나 이상의 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 천연에서 분리되거나 화학적 합성법을 이용하여 제조할 수 있다. 상기한 단백질을 코딩하는 염기서열을 갖는 핵산은 단쇄 또는 이중쇄일 수 있으며, DNA 분자(genomic DNA, cDNA) 또는 RNA 분자일 수 있다.In addition, the nucleic acid encoding the protein is a nucleotide sequence encoding a protein of the wild type or a variant form as described above, one or more bases may be mutated by substitution, deletion, insertion or combination thereof, or is isolated from nature It may be prepared using chemical synthesis. The nucleic acid having a nucleotide sequence encoding the above protein may be a single chain or a double chain, and may be a DNA molecule (genomic DNA, cDNA) or RNA molecule.
본 발명의 일 구현예로, 본 발명에서 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 단백질을 코딩하는 핵산분자는 각 단백질을 코딩하는 핵산을 포함하는 단백질을 발현하는 벡터일 수 있다.In one embodiment of the invention, the nucleic acid molecule encoding the OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 protein in the present invention is a vector expressing a protein comprising a nucleic acid encoding each protein Can be.
본 발명에서 ‘벡터’란 숙주세포에서 목표 단백질을 발현할 수 있는 발현 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 의미할 수 있다. In the present invention, "vector" is an expression vector capable of expressing a target protein in a host cell, and may refer to a gene construct including essential regulatory elements operably linked to express a gene insert.
또한, 본 발명에서 ‘벡터’는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널, 인핸서 같은 발현 조절 요소 외에도 막 표적화 또는 분비를 위한 신호 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 또한, 발현벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택성 마커를 포함하고, 복제 가능한 발현벡터인 경우 복제 기원을 포함한다. 벡터는 자가 복제하거나 숙주 DNA에 통합될 수 있다.In addition, the 'vector' in the present invention includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, and variously prepared according to the purpose. Can be. The promoter of the vector may be constitutive or inducible. In addition, the expression vector includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. Vectors can self replicate or integrate into host DNA.
벡터는 플라스미드 벡터, 코즈미드 벡터, 바이러스 벡터 및 에피조말(episomal) 벡터 등을 포함한다. 바람직하게는, 바이러스 벡터일 수 있다. 바이러스 벡터는 렌티바이러스 벡터, 레트로바이러스(Retrovirus), 예를 들어 HIV(Human immunodeficiency virus), MLV(Murineleukemia virus), ASLV(Avian sarcoma/leukosis), SNV(Spleen necrosis virus), RSV(Rous sarcoma virus), MMTV(Mouse mammary tumor virus) 등, 아데노바이러스(Adenovirus), 아데노 관련 바이러스(Adeno-associatedvirus), 헤르페스 심플렉스 바이러스(Herpes simplex virus) 등에서 유래한 벡터를 포함하나, 이에 제한되지 않는다. 이러한 벡터 시스템은 특정 세포에 관련된 유전자를 체세포에서 과발현시켜 직접교차분화를 유도하는 목적을 위해 사용하는것으로서 어떠한 벡터 시스템을 사용하더라도 본 발명의 효과를 나타낼 수 있다. 본 발명의 구체적인 실시예로서, OCT4를 발현하는 pMX 기반의 레트로바이러스 벡터일 수 있다 (도 5).Vectors include plasmid vectors, cosmid vectors, viral vectors and epismal vectors and the like. Preferably, it may be a viral vector. Viral vectors are lentiviral vectors, retroviruses, such as Human immunodeficiency virus (HIV), Murineleukemia virus (MLV), Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), and Rous sarcoma virus (RSV). Including, but not limited to, vectors derived from Mouse mammary tumor virus (MMTV), Adenovirus, Adeno-associated virus, Herpes simplex virus, and the like. Such a vector system is used for the purpose of inducing direct cross-differentiation by overexpressing a gene related to a specific cell in a somatic cell, and may exhibit the effects of the present invention regardless of which vector system is used. As a specific embodiment of the invention, it may be a retrovirus vector based on pMX expressing OCT4 (Fig. 5).
또한, 상기 단백질을 암호화하는 핵산은 당 분야의 공지 방법, 예를 들어 벡터 형태의 네이키드 DNA로 세포내로 전달하거나, 리포좀(Liposome), 양이온성 고분자(Cationic polymer)등을 이용하여 세포 내로 도입할 수 있다. 상기 리포좀은 유전자 전달을 위하여 DOTMA나 DOTAP 등의 양이온성 인지질을 혼합하여 제조한 인지질 막으로, 양이온성의 리포좀과 음이온성의 핵산이 일정 비율로 혼합하면 핵산-리포좀 복합체를 형성하여 세포 내로 도입될 수 있다.In addition, the nucleic acid encoding the protein can be introduced into the cell using a known method in the art, for example, naked DNA in the form of a vector, or using a liposome, a cationic polymer, or the like. Can be. The liposomes are phospholipid membranes prepared by mixing cationic phospholipids such as DOTMA or DOTAP for gene transfer. When liposomes and anionic nucleic acids are mixed at a predetermined ratio, the liposomes can be introduced into cells by forming a nucleic acid-liposomal complex. .
구체적으로, 본 발명에서 상기 단백질을 암호화하는 핵산분자는, 상기 단백질을 암호화하는 핵산을 포함하는 바이러스 벡터를 패키징(packaging) 세포로 형질전환 및 감염시켜 각 유전자를 발현하도록 제작한 벡터 및 바이러스에 포함되어 체세포 내로 도입될 수 있다. 상기 바이러스는 레트로바이러스, 아데노바이러스, 아데노 관련 바이러스, 헤르페스 심플렉스 바이러스 등을 포함하며 이에 제한되지 않는다.Specifically, in the present invention, the nucleic acid molecule encoding the protein is included in the vector and virus produced to express each gene by transforming and infecting a viral vector containing the nucleic acid encoding the protein with packaging cells. Can be introduced into somatic cells. Such viruses include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and the like.
본 발명에서 용어 “체세포”는, 생식세포를 제외한 모든 세포를 의미할 수 있다. 예를 들어, 섬유아세포, 근육세포, 신경세포, 위점막세포, 배상세포, G세포, 주피세포(pericyte), 성상교세포(astrocyte), B세포, 혈액세포, 상피세포 신경줄기세포, 조혈모세포, 중간엽줄기세포 또는 제대혈 줄기세포 등을 사용할 수 있다. 그러나, 직접교차분화는 시작세포가 체세포이면 특정 조직세포 여부에 상관없이 적용할 수 있으므로, 상기에 제한되지 않는다. 본 발명의 구체적인 실시예에서는 피부의 섬유아세포(fibroblast)를 사용하였다.In the present invention, the term "somatic cell" may mean any cell except germ cells. For example, fibroblasts, muscle cells, neurons, gastric mucosa, goblet cells, G cells, pericyte, astrocytes, B cells, blood cells, epithelial neural stem cells, hematopoietic stem cells, intermediate Leaf stem cells or umbilical cord blood stem cells may be used. However, the direct cross-differentiation is not limited to the above, because if the starting cells are somatic cells can be applied regardless of the specific tissue cells. In a specific embodiment of the present invention, fibroblasts of the skin were used.
본 발명에서 용어 “희소돌기아교 전구세포”는 중추신경계에 있는 신경교 세포(glial cell)의 아류(subtype)의 세포를 의미한다. 이는 희소돌기아교세포의 전구체이고 뉴런과 성상교세포로 분화할 수 있다. 또한 본 발명에서 “iOPCs”는 유도된 희소돌기아교 전구세포를 말하고, 예를 들어, 본 발명의 방법에 따른 직접분화를 통하여 체세포로부터 유도된 희소돌기아교 전구세포를 의미할 수 있다.As used herein, the term “oligodendrocyte progenitor cell” refers to a cell of a subtype of a glial cell in the central nervous system. It is a precursor of oligodendrocytes and can differentiate into neurons and astrocytes. In addition, in the present invention, "iOPCs" refers to induced oligodendrocyte progenitor cells, and for example, may refer to oligodendrocyte progenitor cells derived from somatic cells through direct differentiation according to the method of the present invention.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 체세포에 도입하여 직접교차분화 유도된 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cell)를 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced Vectors are introduced into somatic cells to provide direct cross-differentiation-induced oligodendrocyte progenitor cells.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 약학 조성물을 제공한다. In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced It provides a pharmaceutical composition for the prevention or treatment of spinal cord injury or demyelination disease comprising a vector or the direct cross-differentiation-induced oligodendrocyte progenitor cells as an active ingredient.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 세포치료제를 제공한다.In addition, the present invention is any one protein selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced Provided is a cell therapy agent for the prevention or treatment of spinal cord injury or myelin deprivation disease, comprising a vector or the above-described direct cross-differentiation-induced oligodendrocyte progenitor cells as an active ingredient.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환 치료 약물 스크리닝용 조성물을 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced Provided is a composition for screening a therapeutic drug for spinal cord injury or myelin deprivation disease, comprising a vector or the direct cross-differentiation induced oligodendrocyte progenitor cells as an active ingredient.
상기 척수 손상은 중추신경계인 척수가 외부적 요인 또는 내부적 요인으로 인하여 손상되어 제 기능을 하지 못하는 모든 경우를 포함하며, 예를 들어, 진탕(commotion), 압박(compression), 좌상(contusion) 등의 외상에 의한 손상일 수 있으나 이에 제한되지 않고, 중추신경계의 일부인 척수가 자신의 기능을 수행하지 못함에 따라 체온조절 장애, 횡경막마비, 늑간근마비, 발한, 운동마비, 지각마비, 배뇨장애, 욕창, 이소성골화, 경축 등의 다양한 증상이 나타날 수 있다.The spinal cord injury includes all cases in which the central nervous system, the spinal cord, is damaged due to external or internal factors and fails to function. For example, concussion, compression, contusion, etc. Injury may be due to trauma, but is not limited thereto. As the spinal cord, which is part of the central nervous system, fails to perform its function, thermoregulation disorders, diaphragmatic paralysis, intercostal paralysis, sweating, exercise paralysis, perception paralysis, urination disorder, pressure sores, ectopic dysfunction. Various symptoms, such as ossification and convulsions, can occur.
또한, 상기 수초탈락 질환은 신경에서 수초와 관련된 모든 질환을 의미하고, 예를 들어, 다발성 경화증, 뇌성마비, 백질이영양증, 신경병질, 중심다리뇌 수초용해 또는 수초형성부전증 등 일 수 있으나, 이에 제한되지 않는다.In addition, the myelin deprivation disease refers to all diseases related to myelin sheath in the nerve, and may be, for example, multiple sclerosis, cerebral palsy, white matter dystrophy, neuropathy, central limb myelination or myelin dysplasia, and the like. It doesn't work.
본 발명에서 있어서, "세포치료제"는 사람으로부터 분리, 배양 및 특수한 저작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식, 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 지칭한다. In the present invention, the "cell therapeutic agent" is a medicine (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special chewing from humans. It refers to a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise changing the biological characteristics of a living autologous, allogeneic, or heterologous cell in vitro.
즉, 척수 손상 또는 수초탈락 질환의 치료 후보 물질의 존재 및 부재하에서 본 발명의 분화 유도된 희소돌기아교 전구세포의 반응성을 확인하는 방법으로 척수 손상 또는 수초탈락 질환의 치료제를 스크리닝하는데 유용하게 사용할 수 있다. 예를 들어, 본 발명의 분화 유도된 희소돌기아교 전구세포는 수초탈락질환의 회복 또는 치료에 중요한 신경세포로서 후보 물질에 대한 독성 또는 약효를 평가하는데 사용할 수 있다.That is, it can be usefully used for screening a therapeutic agent for spinal cord injury or myelin deprivation disease by confirming the reactivity of the differentiation-induced oligodendrocyte progenitor cells of the present invention in the presence and absence of a candidate substance for treatment of spinal cord injury or myelin deprivation disease. have. For example, the differentiation-induced oligodendrocyte progenitor cells of the present invention can be used to evaluate the toxicity or efficacy of a candidate substance as neurons important for the recovery or treatment of myelin deprivation disease.
상기 독성의 평가는 본 발명의 치료 후보 물질의 존재 및 부재하에서 본 발명의 분화 유도된 희소돌기아교 전구세포가 희소돌기아교세포로 분화를 억제하거나 또는 본 발명의 분화 유도된 희소돌기아교 전구세포의 IC50 등 당업계에서 통상적으로 독성을 판단하는 방법에 따라 평가할 수 있다. 또한, 상기 약효의 평가는 본 발명의 치료 후보 물질의 존재 및 부재하에서 본 발명의 분화 유도된 희소돌기아교 전구세포가 희소돌기아교세포로 분화를 촉진하거나 또는 수초 생성을 촉진하는 등 당업계에서 수초탈락질환의 치료에 효과가 있음을 확인할 수 있는 방법에 따라 평가할 수 있다.Evaluation of the toxicity is based on the differentiation-induced oligodendrocyte progenitor cells of the present invention in the presence or absence of a therapeutic candidate substance of the present invention to inhibit the differentiation into oligodendrocytes or to differentiate the induced oligodendrocyte progenitor cells of the present invention. In the art, such as IC 50 , can be evaluated according to the method for determining toxicity. In addition, the evaluation of the efficacy of the drug in the art, such as in the presence and absence of the therapeutic candidate substance of the present invention, the differentiation-induced oligodendrocyte progenitor cells promote differentiation into oligodendrocytes or promote myelin production. It can be evaluated according to the method to confirm that it is effective in the treatment of a dropout disease.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 상기 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환 치료용 인공조직 제작을 위한 3D 프린팅 생체 소재 조성물을 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , nucleic acid molecules encoding the protein, the nucleic acid molecule is introduced Provided is a 3D printing biomaterial composition for the manufacture of artificial tissue for the treatment of spinal cord injury or myelin deprivation disease comprising a vector or the direct cross-differentiation induced oligodendrocyte progenitor cells as an active ingredient.
또한, 본 발명은 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 체세포에 도입하는 단계를 포함하는 체세포를 희소돌기아교 전구세포로 직접교차분화하는 방법을 제공한다.In addition, the present invention is any one or more proteins selected from the group consisting of direct cross-differentiation factors OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , a nucleic acid molecule encoding the protein or the nucleic acid molecule is introduced Provided is a method of directly cross-differentiating somatic cells into oligodendrocyte progenitor cells comprising introducing a vector into somatic cells.
보다 구체적으로, 체세포를 배지에서 배양하는 단계, 상기 배양한 체세포에 상기 유전자를 삽입한 벡터로 형질감염(transfection) 시키는 단계, 및 상기 감염된 체세포를 직접교차분화를 유도할 수 있는 배양조건에서 배양하는 단계를 포함한다.More specifically, culturing the somatic cells in the medium, transfecting (transfection) with the vector inserting the gene into the cultured somatic cells, and culturing in the culture conditions that can induce direct cross-differentiation of the infected somatic cells Steps.
상기 체세포의 배양에 사용되는 배지는 당해 분야에서 체세포 배양에 통상적으로 사용되는 배지를 모두 포함한다. 배양에 사용되는 배지는 일반적으로 탄소원, 질소원 및 미량원소 성분을 포함한다. 본 발명의 구체적인 실시예에서는 프로타민 설페이트(protamine sulphate)를 함유하는 배지를 사용하였다.The medium used for culturing the somatic cells includes all mediums commonly used for culturing somatic cells in the art. The medium used for culturing generally contains a carbon source, a nitrogen source and a trace element component. In a specific embodiment of the present invention, a medium containing protamine sulphate was used.
또한, 상기 체세포를 직접교차분화를 유도할 수 있는 배양조건은 당해 분야에서 체세포에 대하여 직접교차분화를 유도하는 데 통상적으로 사용되는 배지를 포함할 수 있다. 본 발명의 구체적인 실시예에서는 N2가 보충된 DMEM/F12, 페니실린/스트렙토마이신(penicillin/streptomycin), PDGF-α 20ng/ml, FGF-2 10ng/ml를 포함하는 배지를 사용하였다.In addition, the culture conditions that can induce direct cross-differentiation of the somatic cells may comprise a medium commonly used to induce direct cross-differentiation for somatic cells in the art. In a specific embodiment of the present invention, a medium containing DMEM / F12, penicillin / streptomycin supplemented with N2, 20 ng / ml PDGF-α, and 10 ng / ml FGF-2 was used.
상기 본 발명의 직접교차분화 유도용 조성물을 체세포에 도입하는 단계를 통하여, 직접교차분화인자의 이소성 발현을 유도할 수 있다. 이소성 발현이란, 어떤 유전자가 원래 발현하는 조직이나 세포 외에서 발현하는 것, 또는 원래 발현하는 시기와 다른 시기에 발현하는 것을 의미한다. 본 발명의 구체적인 실시예에서는 유도용 조성물을 체세포에 도입함으로써 체세포에서 직접교차분화인자의 발현을 유도하는 것일 수 있다. 그에 따라, 체세포로부터 희소돌기아교 전구세포가 제조될 수 있다.Through the step of introducing the composition for inducing direct cross-differentiation of the present invention into somatic cells, ectopic expression of the direct cross-differentiation factor can be induced. Ectopic expression means that a certain gene is expressed in tissues or extracellularly expressed originally, or is expressed at a different time than originally expressed. In a specific embodiment of the present invention may be to induce the expression of direct cross-differentiation factor in somatic cells by introducing the composition for induction into somatic cells. Thus, oligodendrocyte progenitor cells can be produced from somatic cells.
본 발명에 따라 제조된 희소돌기아교 전구세포는 중추신경계에서 수초 생성에 필수적인 역할을 하고, 희소돌기아교세포, 뉴런 또는 성상교세포로 분화가 가능하므로 수초 손실로 인한 질병의 예방 또는 치료에 적용될 수 있다.Rare oligodendrocyte progenitor cells prepared according to the present invention play an essential role in myelin production in the central nervous system, and can be differentiated into oligodendrocytes, neurons or astroglia, and thus can be applied to the prevention or treatment of diseases caused by myeloma loss. .
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
실험예 1. iOPCs의 제조Experimental Example 1. Preparation of iOPCs
피부 섬유아세포를 10% FBS 배양 배지 내 젤라틴 코팅된 6well 플레이트 위에 3×104 cells로 분주하였다. 하루 후에, 섬유아세포를 6μg/ml의 프로타민 설페이트를 함유하는 배지에서 OCT4를 발현하는 pMX 레트로바이러스 벡터로 감염시켰다. 감염 24시간 후에, 바이러스 상층액을 제거하고 새로운 배지로 대체하였다. 감염 3일 째, 세포들을 화학적으로 변형된 OPC 배지(N2가 보충된 DMEM/F12, 페니실린/스트렙토마이신 (penicillin/streptomycin), PDGF-α 20ng/ml (Peprotech), FGF-2 10ng/ml (Peprotech))로 바꾸어주었다. OPC 배지에서 추가 배양 12일 후에, OPC 같은 군집을 기계적으로 분리하고 젤라틴 코팅된 플레이트에서 성숙한 iOPC를 선별(picking)하거나 트립신처리하여 계대배양(subculture) 하였다.Dermal fibroblasts were dispensed into 3 × 10 4 cells on gelatin coated 6well plates in 10% FBS culture medium. After one day, the infected fibroblasts into pMX retroviral vector expressing OCT4 in medium containing protamine sulfate of 6μg / ml. 24 hours after infection, virus supernatants were removed and replaced with fresh medium. On day 3 of infection, cells were chemically modified in OPC medium (DMEM / F12 supplemented with N2, penicillin / streptomycin), PDGF-α 20ng / ml (Peprotech), FGF-2 10ng / ml (Peprotech Changed to)). After 12 days of further incubation in OPC medium, colonies such as OPC were mechanically isolated and subcultured by picking or trypsinizing mature iOPC on gelatin coated plates.
실험예 2. iOPCs의 분화(in vitro)Experimental Example 2 Differentiation of iOPCs (in vitro)
성숙한 희소돌기아교세포의 제조를 위해서, iOPCs를 OPC 배지의 PDL/laminin-coated 4-well 플레이트에 플레이팅하였다. 그 다음날, 배지를 희소돌기아교세포 분화 배지 1(N2가 보충된 DMEM/F12, 페니실린/스트렙토마이신, FGF-2 10ng/ml(Peprotech), forskolin 10mM(Sigma))로 교체하고 4-5일간 유지하였다. 첫 번째 분화단계 이후에, 세포를 희소돌기아교세포 분화 배지 2(30ng/ml 3, 3, 5-tri-iodothyronine(T3; Sigma), 20ng/ml ascorbic acid (AA; Sigma))로 처리하였다.For the preparation of mature oligodendrocytes, iOPCs were plated in PDL / laminin-coated 4-well plates of OPC medium. The next day, replace the medium with oligodendrocyte differentiation medium 1 (DMEM / F12 supplemented with N2, penicillin / streptomycin, FGF-2 10ng / ml (Peprotech), forskolin 10mM (Sigma)) and hold for 4-5 days. It was. After the first differentiation step, cells were treated with oligodendrocyte differentiation medium 2 (30 ng / ml 3, 3, 5-tri-iodothyronine (T3; Sigma), 20 ng / ml ascorbic acid (AA; Sigma)).
실험예 3. 레트로바이러스 생산Experimental Example 3. Retrovirus Production
OCT4를 발현하는 pMX 기반의 레트로바이러스 벡터를 VSV-G 유사유형 바이러스의 패키지인 X-tremeGENE9 DNA 형질감염 시약(Roche)을 이용하여 293T 세포(ATCC, CAT #. CRL-3216)로 함께 형질감염시켰다. 형질감염 48시간 후에, 바이러스를 함유하는 상층액을 수집하고 0.45μm 주사기 필터(syringe filter)에 여과하여 바이러스를 수확하였다.To the pMX-based retroviral vector of expressing OCT4 using a VSV-G similar to the type of the virus package X-tremeGENE9 DNA transfection reagent (Roche) were transfected together into 293T cells (ATCC, CAT #. CRL- 3216) . 48 hours after transfection, the virus-containing supernatants were collected and filtered on a 0.45 μm syringe filter to harvest the virus.
실험예 4. 정량 실시간 PCRExperimental Example 4. Quantitative Real-Time PCR
DNA가 없는 전체 RNA를 RNeasy mini kit (Qiagen)을 이용하여 추출하였다. 반응 당 총 500ng의 RNA를 이용하여 SuperScript® III 역전사효소(Invitrogen)로 cDNA를 합성하였다. 합성된 cDNA는 총 부피 20ul로 LightCycler 480 SYBR Green I Mastermix (Roche)을 이용하여 주형으로 사용하였다. OPCs 마커 유전자와 만능줄기세포 및 신경 마커에 대하여 실험은 3회 반복하여 실시하였고 항존유전자(housekeeping gene) Gapdh로 정상화하였다. 유전자 발현은 Ct 값 계산 방법으로 측정하였다. 모든 실험은 제조업체의 설명에 따라서 수행하였다. Total RNA without DNA was extracted using RNeasy mini kit (Qiagen). CDNA was synthesized with SuperScript® III reverse transcriptase (Invitrogen) using a total of 500 ng of RNA per reaction. The synthesized cDNA was used as a template using a LightCycler 480 SYBR Green I Mastermix (Roche) with a total volume of 20ul. OPCs marker genes, pluripotent stem cells and neural markers were repeated three times and normalized to the housekeeping gene Gapdh. Gene expression was measured by Ct value calculation method. All experiments were performed according to the manufacturer's instructions.
실험예 5. 면역세포화학 분석Experimental Example 5. Immunocytochemical Analysis
세포를 4% 파라-포름알데하이드(para-formaldehyde)에서 10분간 고정하고 0.1% Triton X-100으로 10분동안 처리하여 투과성으로 만들었다. 세포는 4% FBS 블라킹(blocking) 용액에서 30분간 배양하고 그 뒤에 블라킹 용액으로 희석시킨 1차 항체에서 상온으로 1시간동안 배양하였다 Oligo2 (Santacruz, 1:200), PDGF-α (Abcam, 1:500), A2B5 (Millipore, 1:500), NG2 (Millipore, 1:200), O4 (Millipore, 1:400), RIP (DSHB, 1:200), GalC (Chemicon, 1:200), GFAP (Sigma, 1:500). 1차 항체 배양 후에, 세포는 PBST(0.05% tween20)로 3회 세척하였다. 2차 항체를 PBS로 희석하고 1시간동안 세포와 함께 배양하였다(Alexa Fluor 488/568 anti-mouse IgG1, IgG3, IgM, anti-goat IgG (Invitrogen, 1:1000)). 핵은 Hoechst 33342 (Thermo)을 이용하여 10초간 염색하였다. 실험에서 사용한 1차 항체는 표 1에 정리하였다.The cells were fixed in 4% para-formaldehyde for 10 minutes and treated with 0.1% Triton X-100 for 10 minutes to make them permeable. Cells were incubated for 30 minutes in 4% FBS blocking solution and then incubated for 1 hour at room temperature in primary antibody diluted with blocking solution. Oligo2 (Santacruz, 1: 200), PDGF-α (Abcam, 1: 500), A2B5 (Millipore, 1: 500), NG2 (Millipore, 1: 200), O4 (Millipore, 1: 400), RIP (DSHB, 1: 200), GalC (Chemicon, 1: 200), GFAP (Sigma, 1: 500). After primary antibody culture, cells were washed three times with PBST (0.05% tween20). Secondary antibodies were diluted in PBS and incubated with cells for 1 hour (Alexa Fluor 488/568 anti-mouse IgG1, IgG3, IgM, anti-goat IgG (Invitrogen, 1: 1000)). Nuclei were stained for 10 seconds using Hoechst 33342 (Thermo). The primary antibody used in the experiment is summarized in Table 1.
표 1
Antibody Source Isotype Dilution Localization
Olig2 Santacruz Goat IgG 1:200 Nucleus
PDGFR-α Abcam Mouse IgG 1:200 Receptor
A2B5 Millipore Mouse IgM 1:500 Cell surface
NG2 Millipore Mouse IgG 1:100 Cell surface
O4 Millipore Mouse IgM 1:200 Cell surface
RIP DSHB Mouse IgG1 1:200 Cell surface
MBP Millipore Mosue IgG2a 1:100 Myelin membrane
GalC Millipore Mosue IgG3 1:200 Cytoplasm
GFAP SIGMA Mouse IgG1 1:500 Cytoplasm
Table 1
Antibody Source Isotype Dilution Localization
Olig2 Santacruz Goat IgG 1: 200 Nucleus
PDGFR-α Abcam Mouse IgG 1: 200 Receptor
A2B5 Millipore Mouse igm 1: 500 Cell surface
NG2 Millipore Mouse IgG 1: 100 Cell surface
O4 Millipore Mouse igm 1: 200 Cell surface
RIP DSHB Mouse IgG1 1: 200 Cell surface
MBP Millipore Mosue IgG2a 1: 100 Myelin membrane
GalC Millipore Mosue IgG3 1: 200 Cytoplasm
GFAP SIGMA Mouse IgG1 1: 500 Cytoplasm
실험예 6. 피부 섬유아세포의 준비Experimental Example 6. Preparation of Skin Fibroblasts
피부 섬유아세포를 분리하고 MEF 배지(Dullbecco's modified Eagle's medium supplemented with 10% FBS, nonessential amino acids, L-glutamine, penicillin/streptomycin, mercaptoethanol)에서 37℃, 5% CO2 incubator에서 배양하였다.Dermal fibroblasts were isolated and incubated in MEF medium (Dullbecco's modified Eagle's medium supplemented with 10% FBS, nonessential amino acids, L-glutamine, penicillin / streptomycin, mercaptoethanol) at 37 ℃, 5% CO 2 incubator.
실험예 7. RT-PCR 및 면역세포화학에 의한 섬유아세포의 특성 확인Experimental Example 7. Characterization of fibroblasts by RT-PCR and immunocytochemistry
Taq DNA 중합효소 재조합체(Invitrogen)를 이용하여 35사이클 동안 RT-PCR을 수행하였다. 면역세포화학을 위해서, 세포를 4% PBS(pH 7.4) 내 파라-폼알데하이드(para-formaldehyde)에서 10분간 고정하고 0.1% Triton X-100으로 10분동안 처리하여 투과성으로 만들었다. 세포는 4% FBS 블라킹(blocking) 용액에서 30분간 배양하고 그 뒤에 블라킹 용액으로 희석시킨 1차 항체에서 상온으로 1시간 동안 배양하였다. Oct4 (Santacruz, 1:200), Sox2 (Santacruz, 1:400), Olig2 (Santacruz, 1:200), Pax6 (DSHB, 1:200), O4 (Millipore, 1:400), RIP (DSHB, 1:200), GFAP (Sigma, 1:500). 1차 항체 배양 후에, 세포를 세척하고 2차 항체에서 1시간 동안 배양하였다(Alexa Fluor 488/568 anti-mouse IgG1, IgG3, IgM, anti-goat IgG (Invitrogen, 1:1000)). 핵은 Hoechst 33342 (Thermo)을 이용하여 10초간 염색하였다.RT-PCR was performed for 35 cycles using Taq DNA polymerase recombinant (Invitrogen). For immunocytochemistry, cells were fixed in para-formaldehyde in 4% PBS (pH 7.4) for 10 minutes and treated with 0.1% Triton X-100 for 10 minutes to make them permeable. Cells were incubated for 30 min in 4% FBS blocking solution and then incubated for 1 hour at room temperature in the primary antibody diluted with blocking solution. Oct4 (Santacruz, 1: 200), Sox2 (Santacruz, 1: 400), Olig2 (Santacruz, 1: 200), Pax6 (DSHB, 1: 200), O4 (Millipore, 1: 400), RIP (DSHB, 1 : 200), GFAP (Sigma, 1: 500). After primary antibody culture, cells were washed and incubated for 1 hour in secondary antibody (Alexa Fluor 488/568 anti-mouse IgG1, IgG3, IgM, anti-goat IgG (Invitrogen, 1: 1000)). Nuclei were stained for 10 seconds using Hoechst 33342 (Thermo).
실험예 8. 정량적인 실시간 PCR을 통한 Experimental Example 8 Through Quantitative Real-Time PCR OCT4OCT4 이식유전자의 사일런싱 확인 Confirmation of Silencing of Transgenes
전체 RNA를 RNeasy mini kit (Qiagen)을 이용하여 추출하였다. 반응 당 총 500ng의 RNA를 SuperScript® III 역전사효소(Invitrogen)를 이용하여 cDNA를 합성하였다. 실시간 PCR 분석은 총 부피 20ul로 LightCycler 480 SYBR Green I Mastermix (Roche)을 이용하여 수행하였다. 실험은 3회 반복하여 실시하였고 항존유전자(housekeeping gene) GAPDH로 정상화(normalize)하였다. 유전자 발현은 Ct 값 계산 방법으로 측정하였다. Total RNA was extracted using RNeasy mini kit (Qiagen). A total of 500 ng of RNA per reaction was synthesized cDNA using SuperScript® III reverse transcriptase (Invitrogen). Real-time PCR analysis was performed using LightCycler 480 SYBR Green I Mastermix (Roche) with a total volume of 20ul. The experiment was repeated three times and normalized to housekeeping gene GAPDH . Gene expression was measured by Ct value calculation method.
실험예 9. 마이크러어레이 분석Experimental Example 9. Microarray Analysis
마이크로어레이 분석을 이용하여 섬유아세포, OCT4 감염 3일 후의 섬유아세포 (fibroblasts-day3), OCT4 감염 10일 후의 섬유아세포 (fibroblasts-day10), 대조군(감염엾이 OPC 배지에서 배양한 섬유아세포), iOPC-C1, iOPC-C2의 전체적인 유전바 발현을 프로파일 하고 이전에 발표된 데이터의 bona fide OPC 샘플과 비교하였다. 전체 RNA는 제조업체의 지시에 따라 RNeasy mini kit (Qiagen)을 이용하여 분리하였다. 샘플은 Affymetrix array와 혼성화 하였다. RMA (Robust Multi-array Analysis) 알고리즘으로 계산하여 정상화하였다. 데이터 처리 및 그래픽은 내부에 개발된 Matlab으로 수행하였다. 유전자 및 샘플의 계층 클러스터링은 one minus correlation metric and the unweighted average distance (UPGMA) linkage 방법으로 수행하였다.Microarray analysis using the fibroblasts, OCT4 infected 3 days fibroblast cells (fibroblasts-day3) after, OCT4 infection 10 days fibroblast cells (fibroblasts-day10) after the control group (infected pitiful one fibroblasts cultured in OPC medium), iOPC Overall gene bar expression of -C1, iOPC-C2 was profiled and compared with bona fide OPC samples of previously published data. Total RNA was isolated using RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Samples were hybridized with Affymetrix array. Normalized by calculation with RMA (Robust Multi-array Analysis) algorithm. Data processing and graphics were performed with Matlab developed internally. Hierarchical clustering of genes and samples was performed by one minus correlation metric and the unweighted average distance (UPGMA) linkage method.
실험예 10. 척수 손상 및 이식Experimental Example 10 Spinal Cord Injury and Transplantation
척수 손상 모델은 6주령의 남성 쥐에 척수의 가슴등뼈 level 9(T9)에 손상을 주기 위한 공기 펀치 장치를 이용하여 제작하였다. 손상 1주 후에, 수확한 iOPCs를 DiI으로 표지하고 1×105 iOPCs/rat 농도로 정위 고정방법(stereotaxic)으로 척수의 가슴등뼈 level 8(T8) 및 가슴등뼈 level 10(T10)에 각각 주입하였다. 쥐에게 규칙적으로 물과 음식을 제공하고 배뇨 작용을 위해 방광을 마사지 해주었다. 동물 실험 과정은 동물 시설(animal facility) 및 울산과학기술대학교의 IBR 위원회에서 승인받았다. 동물 실험 과정은 동물연구에 대한 미국국립보건원의 지침을 기반으로 하였다.Spinal cord injury model was constructed using an air punch device to damage the spinal cord spine level 9 (T9) in 6-week-old male rats. After 1 week of injury, harvested iOPCs were labeled with DiI and injected into the spinal cord level 8 (T8) and thoracic spine level 10 (T10), respectively, by stereotaxic at a concentration of 1 × 10 5 iOPCs / rat. . Rats were given water and food regularly and massaged bladder for urination. The animal testing process was approved by the animal facility and the IBR Committee of Ulsan National University of Science and Technology. The animal testing process was based on guidelines from the National Institutes of Health on animal studies.
실험예 11. 조직학적 과정 및 면역조직화학 분석Experimental Example 11. Histological Processes and Immunohistochemical Analysis
이식 12주 후에 쥐를 마취하고 PBS를 관류(perfuse)시켰다. 척수는 4% 파라폼알데하이드로 밤새 고정시키고 알코올 및 자일렌의 단계적 농도를 거쳐 탈수시켰다. 조직은 파라핀 블록에 넣고 사상면(sagital) 및 관상면(coronal) 방향으로 4um 두께로 연속하여 분할하였다. 분할된 부분은 0.25% Triton X-100로 투과성으로 만들고 비특이적인 결합 부위에 대하여 10% 블라킹 용액으로 블라킹하였다. 면역조직화학 분석을 위해, 분할된 부분은 일차 항체로 배양하였다 PDGF-α (Abcam, 1:100), O4 (Millipore, 1:100), RIP (DSHB, 1:100), GFAP (Sigma, 1:100), MBP (Millipore, 1:100). 그 후 척수 부분은 이차 항체로 염색하였다. 형광 이미지는 Olympus microscope equipment IX81-ZDC로 시각화 하였다.Twelve weeks after transplantation, mice were anesthetized and perfuse PBS. The spinal cord was fixed overnight with 4% paraformaldehyde and dehydrated via staged concentrations of alcohol and xylene. The tissues were placed in paraffin blocks and subsequently divided into 4um thickness in the sagittal and coronal directions. The divided portions were permeable with 0.25% Triton X-100 and blocked with 10% blocking solution for nonspecific binding sites. For immunohistochemical analysis, the divided sections were incubated with primary antibody PDGF-α (Abcam, 1: 100), O4 (Millipore, 1: 100), RIP (DSHB, 1: 100), GFAP (Sigma, 1 : 100), MBP (Millipore, 1: 100). Spinal cord sections were then stained with secondary antibodies. Fluorescence images were visualized with Olympus microscope equipment IX81-ZDC.
실험결과Experiment result
1. One. OCT4OCT4 단독의 성체 섬유아세포로부터 iOPCs 제조 확인 Confirmation of iOPCs Production from Isolated Adult Fibroblasts
도 1a의 분화 유도 과정에 따라 OPCs로의 운명 변화를 유도하기 위해 OCT4 전사 인자를 암호화하는 레트로바이러스(retrovirus)를 피부 섬유아세포에 감염시켰다. 도 1b에서 확인할 수 있는 바와 같이 감염되지 않은 섬유아세포는 유도 전까지 전형적인 섬유아세포의 형태를 나타내었다. 분리된 섬유아세포는 만능분화세포 유전자 및 신경 계통 유전자를 발현하지 못하였고, 면역염색을 통하여도 만능줄기세포 또는 신경 세포 군집을 확인할 수 없었다. 섬유아세포의 형태는 신경교세포 유도 배지에 OCT4 투입 후 25일부터 더 작아지고 신경 세포 같은 모양으로 변화하였으나, OCT4를 투입하지 않은 대조군 세포는 변화가 없었다(도 1b의 D 및 C). iOPC 같은 세포의 발생을 증가시키기 위해, OPCs에 필수적인 생장 인자인 PDGF-AA가 보충된 특정 배양 배지에 배양하였다. 투입 30일 후에, 신경 세포 같은 형태의 세포를 기계적으로 분리하고 특정 OPC 배지에서 신경교세포 유도를 계속하였다. 그 결과 OPC 배지에서 배양 20일 후에 iOPC 집합체(iOPC aggregates, iOPC-Ag)를 확인하였다(도 1b의 E). 다음, iOPC-Ag를 젤라틴으로 코팅된 플레이트로 옮겼을 때 iOPC 같은 무리(cluster)를 형성하였고(도 1b의 F), iOPC 같은 세포는 부착 후에 상기 무리로부터 벗어났다(도 1b의 G). PCR을 이용하여 iOPC의 기주 DNA로 OCT4 유전자가 전이되어 통합되었음을 확인하였다. 상기 전이된 유전자의 침묵(silencing)을 확인하기 위하여, 실시간 PCR을 이용하여 OCT4의 mRNA 수준을 확인하였다. 게다가, 상기 iOPCs는 바이러스 감염(viral induction)으로 세포 운명 전환 후에 염색체의 정상 핵형(karyotype)을 유지하였다. 이를 통하여, OCT4는 섬유아세포로부터 iOPCs의 초기 운명 전환에 충분한 것을 입증하였다.Retroviruses (retrovirus) coding for the transcription factors OCT4 to derive a change to the OPCs fate in accordance with the differentiation induction process of Figure 1a and infected skin fibroblasts. As can be seen in Figure 1b uninfected fibroblasts showed the typical morphology of fibroblasts before induction. The isolated fibroblasts did not express pluripotent differentiation genes and neural lineage genes and could not identify pluripotent stem cells or neuronal cell populations through immunostaining. The morphology of fibroblasts was smaller from 25 days after OCT4 injection into glial cell induction medium and changed to a neuron-like shape, but control cells without OCT4 were unchanged (D and C of FIG. 1B). To increase the incidence of cells such as iOPC, they were cultured in specific culture media supplemented with PDGF-AA, a growth factor essential for OPCs. Thirty days after the dosing, cells of neuron-like form were mechanically isolated and glial cell induction was continued in specific OPC medium. As a result, iOPC aggregates (iOPC aggregates, iOPC-Ag) were confirmed after 20 days of culture in OPC medium (E of FIG. 1B). Next, when iOPC-Ag was transferred to a plate coated with gelatin, iOPC-like clusters were formed (F in FIG. 1B), and cells such as iOPC were released from the group after attachment (G in FIG. 1B). Using PCR with the DNA of host iOPC it was confirmed that the OCT4 gene is integrated transition. In order to confirm the silencing of the transferred gene, the mRNA level of OCT4 was confirmed by real-time PCR. In addition, the iOPCs maintained the normal karyotype of the chromosome after cell fate transition to viral induction. Through this, OCT4 demonstrated from fibroblasts that sufficient conversion of the early fate iOPCs.
2. 2. OCT4OCT4 에 의해 유도된 iOPCs의 자가 분열 및 OPCs 특성 확인Characterization of OPCs and OPCs by Induced iOPCs
OCT4에 의해 유도된 iOPCs의 계통 및 전구체로서의 미성숙 상태를 확인하기 위하여, PDGFR-α, NG2, A2B5 및 Olig2의 OPC 마커로 면역세포화학(immunocytochemical) 분석을 수행하였다. 상기 마커들은 모든 iOPCs에서 발현하였고, 이중 면역염색(double-immunostaining)을 통하여 상기 마커들이 공통적으로 발현되는 것을 확인하였다. In order to confirm the immature status of iOPCs induced by OCT4 as a lineage and precursor, immunocytochemical analysis was performed with OPC markers of PDGFR-α, NG2, A2B5 and Olig2. The markers were expressed in all iOPCs, and double-immunostaining confirmed that the markers were expressed in common.
안정적으로 균일하게 증식하는 2개의 iOPC 클론을 얻었다. 2개의 클론 모두 자신의 특성 및 형태의 변화없이 31 패시지 이상까지 확장가능하였다(도 1d). 생장 특성을 평가하기 위해, iOPCs의 평균 배가 기간(doubling time)을 측정하였다(도 1e). 생장 곡선(growth curve)은 iOPCs의 자가분열 능력을 나타낸다. 생장률(Growth rate)은 31 패시지 이상의 늦은 패시지까지 유지되었다.Two iOPC clones were obtained that stably and uniformly proliferated. Both clones were expandable up to 31 passages without changing their properties and morphology (FIG. 1D). To assess growth characteristics, the average doubling time of iOPCs was measured (FIG. 1E). Growth curves show the self-dividing capacity of iOPCs. Growth rate was maintained up to late passages above 31 passages.
3. 다분화능 iOPCs의 신경교 제한적 체세포의 줄기세포로서 성숙한 희소돌기아교세포 및 성상교세포로의 분화3. Differentiation of pluripotent iOPCs into mature oligodendrocytes and astrocytes as stem cells of glial restricted somatic cells
iOPC 클론이 신경교 계통 제한적이고 균질의 전구체임을 평가하기 위하여, iOPCs에서 GFAP (성상교세포), Tuj1 (미성숙 뉴런), RIP (성숙한 희소돌기아교세포) 마커 발현을 확인하였다. GFAP 양성 성상교세포는 검출되지 않거나 분화되지 않은 iOPCs 사이에는 거의 존재하지 않았다(도 2a의 A). iOPCs가 신경교 계통에 제한적인지 확인하기 위하여, 세포를 분화되지 않은 iOPCs에서는 발현되지 않는 신경 마커인 Tuj1으로 염색하였다(도 2a의 B). 게다가, 분화되지 않은 iOPC에서 늦은 단계의 희소돌기아교세포 마커인 RIP는 확인되지 않았다. 이러한 결과를 통하여 iOPCs는 균일한 개체군임을 확인하였다(도 2a의 C).To evaluate that iOPC clones are glial lineage-restricted and homogeneous precursors, expression of GFAP (astroglia), Tuj1 (maturated neurons), and RIP (matured oligodendrocyte) markers were identified in iOPCs. GFAP positive astrocytes were rarely present between undetected or undifferentiated iOPCs (A in FIG. 2A). To confirm that iOPCs are restricted to glial lineages, cells were stained with Tuj1, a neural marker that was not expressed in undifferentiated iOPCs (FIG. 2A). In addition, RIP, a late oligodendrocyte marker, was not identified in undifferentiated iOPCs. These results confirmed that iOPCs are a uniform population (C in FIG. 2A).
iOPCs는 신경교 계통의 세포 유형으로 분화할 수 있는 다분화능 줄기세포이다. iOPCs의 다분화능을 확인하기 위하여, iOPCs에 분화를 유도하여 iOPCs가 성숙한 희소돌기아교세포 또는 다른 신경교 세포 유형으로 분화하는 능력이 있는지 알아보았다. 분화되지 않은 iOPCs는 PDL/Laminin-coated 플레이트에 희소돌기아교세포의 성숙을 촉진하는 forskolin, ascorbic acid 및 T3가 보충된 분화 유도 배지와 함께 플레이팅한 후에 즉시 변화하였다(도 2b의 D). 분화 5일간 작은 가지와 함께 점차적으로 납작해지는 형상으로 변화하였다(도 2b의 E). 16일까지, 희소돌기아교세포의 성숙한 형상을 더 많이 확인하였고, 분화된 성상교세포와 함께 존재하였다(도 2b의 F). 분화 후에 희소돌기아교세포 마커인 O4 및 성상교세포 마커인 GFAP로 염색하여, 희소돌기아교세포와 성상교세포가 함께 존재하는 것을 확인하였다(도 2c의 G 내지 L). 그러나, 이러한 iNSCs의 희소돌기아교세포 분화 가능성은 사용한 전사인자에 따라 달라지고, 효율은 상대적으로 낮다. OCT4-iOPC의 분화 효율을 평가하기 위하여, O4, GFAP, Tuj1, 및 A2B5으로 염색하여 분화 후에 iOPCs의 비율을 분석하여, 성숙하는 동안에 분화 진행을 확인하였다. 게다가, iOPCs는 성상교세포 및 뉴런 조건으로 유도할 때 신경교 제한적 특성을 나타내었다. iOPCs는 성상교세포로 효율적으로 분화하는 것에 반하여 Tuj1 양성 뉴런으로는 분화하지 못하였다. 이러한 결과를 통하여 신경교 계통 제한적 iOPCs는 성상교세포 뿐만 아니라 희소돌기아교세포로의 분화할 수 있는 다분화능이 있다는 것을 입증하였다.iOPCs are multipotent stem cells that can differentiate into cell types of the glial lineage. To confirm the multipotentiality of iOPCs, we examined whether iOPCs were capable of differentiating into mature oligodendrocytes or other glial cell types by inducing differentiation in iOPCs. Undifferentiated iOPCs changed immediately after plating with differentiation induction medium supplemented with forskolin, ascorbic acid and T3 to promote maturation of oligodendrocytes in PDL / Laminin-coated plates (FIG. 2B). It changed into a shape gradually flattening with a twig for 5 days of differentiation (E of FIG. 2B). By day 16, the mature morphology of oligodendrocytes was confirmed more and was present with differentiated astrocytes (F in FIG. 2B). After differentiation, staining with oligodendrocyte marker O4 and astrocyte marker GFAP confirmed that oligodendrocytes and astroglia were present together (G to L in FIG. 2C). However, the possibility of oligodendrocyte differentiation of these iNSCs depends on the transcription factor used, and the efficiency is relatively low. In order to evaluate the differentiation efficiency of OCT4-iOPC, staining with O4, GFAP, Tuj1, and A2B5 was analyzed for the ratio of iOPCs after differentiation to confirm the progression of differentiation during maturation. In addition, iOPCs exhibited glial restrictive properties when induced into astrocyte and neuronal conditions. iOPCs did not differentiate into Tuj1-positive neurons, whereas they effectively differentiated into astrocytes. These results demonstrate that glial lineage-restricted iOPCs are capable of differentiation into oligodendrocytes as well as astroglia.
iOPCs가 늦은 단계의 성숙한 희소돌기아교세포로 완전히 분화하였을 때, 분화되지 않은 iOPCs는 RIP를 발현하지 못하는데(도 2a의 C) 반하여, RIP, GalC, 및 MBP와 같은 단백질 마커를 발현하였다(도 2d의 M 내지 R). 게다가, 이러한 결과를 통하여 균일한 OCT4-iOPCs는 희소돌기아교세포로의 분화할 수 있는 다분화능이 있다는 것을 입증하였다.When iOPCs completely differentiated into late stage mature oligodendrocytes, undifferentiated iOPCs did not express RIP (FIG. 2C), whereas they expressed protein markers such as RIP, GalC, and MBP (FIG. 2D). M to R). Furthermore, OCT4 -iOPCs uniform through These results demonstrated that there are multipotential capable of differentiating into oligodendrocytes.
4. iOPCs의 OPCs 전체적인 유전자 프로필 발현 확인4. Confirmation of Overall Gene Profile Expression in OOPs of iOPCs
섬유아세포와 iOPCs의 전체적인 유전자 발현 양상을 평가하기 위하여, 마이크로어레이 분석을 수행하였다. 2개의 확립된(established) iOPC 클론의 유전자 발현 패턴은 만능줄기세포에서 유도된 OPCs와 매우 유사하였다(도 3a). 이와 대조적으로, 섬유아세포와 대조군(Mock)(OCT4 유도 없이 OPC 배지에서 배양된 섬유아세포)에서는 전체적인 유전자 발현이 반대 패턴임을 확인하였다. 게다가, OCT4 유도 없이 OPC 배지에서만 배양한 섬유아세포에서 특별한 변화를 확인할 수 없었다. 이러한 결과를 통하여, OCT4가 iOPC로의 세포 운명 전환에 필수적인 것을 확인하였다. 마이크로어레이 데이터의 Pairwise scatter plot을 통하여 섬유아세포와 iOPCs 사이의 유사성, OPC(bona fide)와 iOPCs사이의 유사성을 확인하였다(도 3b). 3D PCA 분석 및 계층 클러스터링(hierarchical clustering)를 통하여, iOPC 및 OPCs(bona fide)는 섬유아세포와 비교하여 밀접한 연관성이 있다는 것을 입증하였다(도 3d 및 3e). 다음으로, 실시간 PCR(real-time PCR)을 이용하여 OPC 특이적인 유전자 발현을 확인하였다(도 3c). 마이크로어레이 데이터와 일치하게, iOPCs는 PTPRZ1, SIAT8A, OLIG2, OLIG1, SOX10, NKX2.2, MAG, MYRF 및 CNP 와 같은 OPC 특이적인 유전자의 mRNA를 높은 수준으로 발현하였다. 전환 후에 발현이 증가한 유전자를 확인하기 위하여, 섬유아세포와 비교하여 iOPCs에서 발현이 증가한 희소돌기아교세포 및 신경계 발달과 관련된 68개의 유전자를 확인하였다.In order to evaluate the overall gene expression patterns of fibroblasts and iOPCs, microarray analysis was performed. The gene expression pattern of the two established iOPC clones was very similar to OPCs derived from pluripotent stem cells (FIG. 3A). In contrast, in the fibroblasts and control (Mock) (fibroblasts cultured in OPC medium without OCT4 induction) it was confirmed that the overall gene expression is the opposite pattern. In addition, no specific changes could be identified in fibroblasts cultured only in OPC medium without OCT4 induction. Through these results, it was confirmed that OCT4 is essential for cell fate transition to iOPC. The pairwise scatter plot of the microarray data confirmed the similarity between fibroblasts and iOPCs and the similarity between OPC (bona fide) and iOPCs (FIG. 3B). 3D PCA analysis and hierarchical clustering demonstrated that iOPC and OPCs (bona fide) were closely related compared to fibroblasts (FIGS. 3D and 3E). Next, OPC-specific gene expression was confirmed using real-time PCR (FIG. 3C). Consistent with microarray data, iOPCs expressed high levels of mRNA of OPC specific genes such as PTPRZ1 , SIAT8A , OLIG2 , OLIG1 , SOX10 , NKX2.2 , MAG, MYRF and CNP . To identify genes with increased expression after conversion, 68 genes associated with oligodendrocytes with increased expression in iOPCs and nervous system development were compared with fibroblasts.
5. iOPCs에 의한 척수 손상된 쥐의 회복력 증가 확인(in vivo)5. Confirmation of increased resilience of spinal cord-injured rats by in vivo
In vivo에서 iOPCs의 기능적인 특성을 확인하기 위하여, 척수 손상 모델인 성체 쥐에 세포 이식을 수행하였다. 척수 손상 1주 후까지, iOPCs를 손상받은 척수에 주입하였고 12주까지 관찰하였다. 손상 부위의 회복을 확인하기 위하여, 척수 부분에 H&E 염색을 수행하였다. iOPCs가 주입된 군은 대조군과 비교하여 손상된 부분의 손상된 조직이 현저히 감소한 것을 확인하였다(도 4a). 면역조직화학 염색을 수행하여 이식된 세포의 생착(engraftment)를 확인하였다. 이식된 골수에서 O4, MBP 및 GFAP 양성 세포가 많이 확인된 반면, 섬유아세포를 이식한 쥐에서는 거의 확인할 수 없었다(도 4b의 C 내지 H). 회복된 척수 세포에서, 손상 부위를 A2B5, O4, MBP및 GFAP와 같은 희소돌기아교세포 마커로 염색하였다(도 4c의 I 내지 K'). A2B5 및 O4 양성 세포는 MBP를 공통 발현하였으나 GFAP 양성 세포는 MBP를 발현하지 못하였다. 이를 통하여, 이식된 iOPCs는 수초-형성 희소돌기아교세포 또는 GFAP 양성 성상교세포 중 하나로 분화할 수 있다는 것을 입증하였다. 종양유전자의 우려를 배제하기 위하여, 마우스에 iOPCs를 주입하고, 주입 후 8개월까지 종양이 발생하는지 확인하였다. 그 결과, iOPCs 이식은 종양의 발생 없이 척수에서 수초형성을 회복시키고 척수 손상 모델 쥐에서 손상된 부분을 회복시키는 것을 입증하였다.To confirm the functional properties of iOPCs in vivo, cell transplantation was performed in adult rats, a spinal cord injury model. Until one week after spinal cord injury, iOPCs were injected into the injured spinal cord and observed up to 12 weeks. In order to confirm the recovery of the injury site, H & E staining was performed on the spinal cord area. The group injected with iOPCs confirmed that the damaged tissue of the damaged part was significantly reduced compared to the control group (FIG. 4A). Immunohistochemical staining was performed to confirm the engraftment of the transplanted cells. While many O4, MBP and GFAP positive cells were found in the transplanted bone marrow, they were hardly confirmed in the mice transplanted with fibroblasts (C to H of FIG. 4B). In recovered spinal cord cells, the injury site was stained with oligodendrocyte markers such as A2B5, O4, MBP and GFAP (I-K ′ in FIG. 4C). A2B5 and O4 positive cells co-expressed MBP, but GFAP positive cells did not express MBP. This demonstrated that transplanted iOPCs could differentiate into either myeloid-forming oligodendrocytes or GFAP positive astrocytes. In order to rule out oncogene concerns, iOPCs were injected into mice, and tumors were confirmed until 8 months after injection. As a result, iOPCs transplantation has been demonstrated to restore myelination in the spinal cord and to recover damaged areas in spinal cord injury model mice without tumor development.
6. 직접교차분화인자 6. Direct Cross Differentiation Factor SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 중 단일유전자 하나만으로 제작한 iOPCs의 유전자발현 확인 Gene expression of iOPCs produced using only one single gene
신경계발달에 중요한 유전자 SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 중 하나의 단일유전자만을 이용하여 iPSCs를 유도하였다. 각 유전자로 제작한 iOPCs의 OPC 특이적인 유전자의 mRNA 발현을 확인한 결과 PTPRZ1, NKX2.2, OLIG2, OLIG1 및 SOX10의 발현이 높게 발현하는 것을 확인하였다(도 6). Genes important for nervous system development SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 IPSCs were induced using only one single gene. Of iOPCs produced by each gene As a result of confirming the mRNA expression of OPC-specific genes, it was confirmed that the expression of PTPRZ1 , NKX2.2 , OLIG2 , OLIG1 and SOX10 was highly expressed (FIG. 6).

Claims (10)

  1. 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 유효성분으로 포함하는 체세포로부터 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cell)로의 직접교차분화 유도용 조성물.Direct cross-differentiation factor OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , any one or more proteins selected from the group consisting of, a nucleic acid molecule encoding the protein or a vector into which the nucleic acid molecules are introduced as an active ingredient Composition for inducing direct cross-differentiation from somatic cells containing oligodendrocyte progenitor cells (Oligodendrocyte Progenitor Cell).
  2. 제1항에 있어서, 상기 벡터는 플라스미드 벡터, 코즈미드 벡터, 바이러스 벡터, 렌티바이러스 벡터, 레트로바이러스(Retrovirus) 벡터, HIV(Human immunodeficiency virus) 벡터, MLV(Murineleukemia virus)벡터, ASLV(Avian sarcoma/leukosis) 벡터, SNV(Spleen necrosis virus)벡터, RSV(Rous sarcoma virus)벡터, MMTV(Mouse mammary tumor virus) 벡터, 아데노바이러스(Adenovirus) 벡터, 아데노 관련 바이러스(Adeno-associatedvirus) 벡터, 헤르페스 심플렉스 바이러스(Herpes simplex virus) 벡터 및 에피조말 (episomal) 벡터로 이루어진 군에서 선택된 어느 하나 이상의 벡터인 것을 특징으로 하는 체세포로부터 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cell)로의 직접교차분화 유도용 조성물.The method of claim 1, wherein the vector is a plasmid vector, a cosmid vector, a viral vector, a lentiviral vector, a retrovirus vector, a human immunodeficiency virus vector, a Murineleukemia virus vector, or an ASLV (Avian sarcoma /). leukosis vector, SNV (Spleen necrosis virus) vector, RSV (Rous sarcoma virus) vector, MMTV (Mouse mammary tumor virus) vector, Adenovirus vector, Adeno-associated virus vector, herpes simplex virus (Herpes simplex virus) A composition for inducing direct cross-differentiation from somatic cells to oligodendrocyte progenitor cells, characterized in that at least one vector selected from the group consisting of a vector and an epizomal vector.
  3. 제1항에 있어서, 상기 체세포는 섬유아세포(fibroblst), 상피세포, 근육세포, 신경세포, 위점막세포, 배상세포, G세포, B세포, 주피세포, 성상교세포 (astrocyte) 혈액세포, 신경줄기세포, 조혈모줄기세포, 제대혈 줄기세포 및 중간엽 줄기세포로 이루어진 군에서 선택된 어느 하나의 세포인 것을 특징으로 하는 체세포로부터 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cell)로의 직접교차분화 유도용 조성물.According to claim 1, wherein the somatic cells are fibroblst, epithelial cells, muscle cells, nerve cells, gastric mucosa cells, goblet cells, G cells, B cells, epidermal cells, astrocyte blood cells, neural stem cells , Hematopoietic stem cells, umbilical cord blood stem cells and mesenchymal stem cells, characterized in that any one selected from the group consisting of cells for direct cross-differentiation induction to oligodendrocyte progenitor cells (Oligodendrocyte Progenitor Cell).
  4. 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 체세포에 도입하여 직접교차분화 유도된 희소돌기아교 전구세포(Oligodendrocyte Progenitor Cell).Direct cross-differentiation factor OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , any one or more proteins selected from the group consisting of, a nucleic acid molecule encoding the protein or a vector into which the nucleic acid molecules are introduced into somatic cells Direct cross-differentiation induced oligodendrocyte progenitor cells (Oligodendrocyte Progenitor Cell).
  5. 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 제4항에 따른 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 약학 조성물.Direct cross-differentiation factor OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 Any one or more proteins selected from the group consisting of, nucleic acid molecules encoding the protein, vector or the nucleic acid molecule introduced therein Pharmaceutical composition for the prevention or treatment of spinal cord injury or myelin deprivation disease comprising a direct cross-differentiation induced oligodendrocyte precursor cells as an active ingredient.
  6. 제5항에 있어서, 상기 수초탈락 질환은 다발성 경화증(multiple sclerosis), 뇌성마비(cerebral palsy), 백질이영양증(leukodystrophy), 신경병질, 중심다리뇌 수초용해 또는 수초형성부전증(hypomyelination)인 것을 특징으로 하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 약학 조성물.The method of claim 5, wherein the myelin deprivation disease (multiple sclerosis), cerebral palsy (cerebral palsy), leukodystrophy, neuropathy, central limb myelolysis or hypomyelination, characterized in that Pharmaceutical composition for the prevention or treatment of spinal cord injury or myelin deprivation disease.
  7. 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 제4항에 따른 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환의 예방 또는 치료용 세포치료제.Direct cross-differentiation factor OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 Any one or more proteins selected from the group consisting of, nucleic acid molecules encoding the protein, vector or the nucleic acid molecule introduced therein Cell therapy for the prevention or treatment of spinal cord injury or myelin deprivation disease comprising a direct cross-differentiation induced oligodendrocyte progenitor cells as an active ingredient.
  8. 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 제4항에 따른 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환 치료 약물 스크리닝용 조성물.Direct cross-differentiation factor OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 Any one or more proteins selected from the group consisting of, nucleic acid molecules encoding the protein, vector or the nucleic acid molecule introduced therein Direct cross-differentiation induced oligodendrocyte progenitor cells according to the composition for screening a spinal cord injury or myelin deprivation disease treatment drug comprising as an active ingredient.
  9. 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자, 상기 핵산분자가 도입된 벡터 또는 제4항에 따른 직접교차분화 유도된 희소돌기아교 전구세포를 유효성분으로 포함하는 척수 손상 또는 수초탈락 질환 치료용 인공조직 제작을 위한 3D 프린팅 생체 소재 조성물.Direct cross-differentiation factor OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 Any one or more proteins selected from the group consisting of, nucleic acid molecules encoding the protein, vector or the nucleic acid molecule introduced therein 3D printing biomaterial composition for manufacturing artificial tissue for the treatment of spinal cord injury or myelin deprivation disease comprising direct cross-differentiation induced oligodendrocyte progenitor cells as an active ingredient.
  10. 직접교차분화인자 OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 및 NKX6.2 로 이루어진 군에서 선택된 어느 하나 이상의 단백질, 상기 단백질을 코딩하는 핵산분자 또는 상기 핵산분자가 도입된 벡터를 체세포에 도입하는 단계를 포함하는 체세포를 희소돌기아교 전구세포로 직접교차분화하는 방법.Direct cross-differentiation factor OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2 and NKX6.2 , any one or more proteins selected from the group consisting of, a nucleic acid molecule encoding the protein or a vector into which the nucleic acid molecules are introduced into somatic cells Direct cross-differentiation of somatic cells comprising oligodendrocyte progenitor cells comprising the step of.
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