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WO2014163380A1 - Procédé de criblage d'agents thérapeutiques pour la maladie de charcot-marie-tooth et neurones moteurs auto-différenciés utilisés pour celui-ci - Google Patents

Procédé de criblage d'agents thérapeutiques pour la maladie de charcot-marie-tooth et neurones moteurs auto-différenciés utilisés pour celui-ci Download PDF

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Publication number
WO2014163380A1
WO2014163380A1 PCT/KR2014/002794 KR2014002794W WO2014163380A1 WO 2014163380 A1 WO2014163380 A1 WO 2014163380A1 KR 2014002794 W KR2014002794 W KR 2014002794W WO 2014163380 A1 WO2014163380 A1 WO 2014163380A1
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Prior art keywords
cmt
cells
patient
derived
screening
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Korean (ko)
Inventor
김윤태
최병옥
우소연
김지연
정성철
홍영빈
박진모
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Chong Kun Dang Corp
Samsung Life Public Welfare Foundation
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Chong Kun Dang Corp
Samsung Life Public Welfare Foundation
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Priority to CN201480031644.5A priority Critical patent/CN105264068A/zh
Priority claimed from KR20140038467A external-priority patent/KR20140120834A/ko
Publication of WO2014163380A1 publication Critical patent/WO2014163380A1/fr
Priority to US14/873,125 priority patent/US20160011177A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells

Definitions

  • the present invention relates to a method for preparing dedifferentiation-induced pluripotent stem cells and a method for screening an agent for Charco-Marie-Tooth disease using autologous cells differentiated from dedifferentiation-induced pluripotent stem cells.
  • Charcot® Marie-Tooth disease or hereditary motor sensory neuropathy refers to a condition in which motor and sensory nerves are damaged by specific gene mutations.
  • Hereditary peripheral neuropathies are classified into three classes: Hereditary motor and sensory neuropathies (HMSN), Hereditary motor neuropathies (HMN), and Hereditary sensory neuropathies (HSN). Neuropathy is one of them.
  • HMSN Hereditary motor and sensory neuropathies
  • HSN Hereditary motor neuropathies
  • HSN Hereditary motor neuropathies
  • Sharco-Marietus disease is divided into type 1, type 2 with autosomal dominant genetics, type 4 with autosomal recessive genetics, and type CMTX with X-chromosome associated genes.
  • type 1 type 2 with autosomal dominant genetics
  • type 4 with autosomal recessive genetics
  • type CMTX with X-chromosome associated genes.
  • gene mutations were named in order of 1A, IB, 1C, etc., in the order in which they were reported.
  • the targeted therapy according to the change in the molecular technique of the rare disease which is classified by the change of the diagnostic technique according to the development of molecular biology and the individualization, that is, the tai lored therapy.
  • the same drug may be a molecular genetic era in which the treatment may be significantly different even in the same stage due to the development of pharmacogenetics according to the genetic characteristics of the patient or the rare disease.
  • CMT is the most diverse of rare diseases, ranging from drug therapy to symptomatic treatment aimed at alleviating symptoms with additional treatment, and supportive therapy to control and alleviate various side effects and complications.
  • Therapeutic choice and prognosis are possible, especially since CMT is caused by abnormalities of the symptoms, so the symptoms continue to progress and are not cured. The purpose was to delay and improve the quality of life.
  • genetic and molecular biological researches have attempted biological treatment methods, and possible results have been reported.
  • due to the nature of regressive diseases the number of affected patients is limited and the research interest is limited. This is not established and the absence of clinicians and researchers who diagnose and treat centering on rare diseases is a reality (Acta Paediatri, 2012).
  • the therapeutic agents are limited to CMT type 1
  • CMT is caused by dozens of genetic mutations
  • development of a customized treatment technology for each gene defect and a model that can verify the same Introduction is urgent and there is a significant difference in the effects of CMT patients on drugs, and there are limitations in drug selection according to CMT patients and symptoms.
  • Stem cells obtained through the skin tissue obtained from the patient retains the characteristics of the patient's genetic mutation, so when differentiated into neurons, neurons having the same characteristics as the patient can be obtained. It can be used to.
  • the typical Charco-Marie-Tooth disease (CMT) of hereditary peripheral neuropathy is a monogenetic disease that regenerates in vitro differentiation of dedifferentiated stem cells derived from skin cells of the patient to reproduce the disease.
  • Models can be used to treat diseases using these disease models. You can make a new drug. Initiation muscular dystrophy ⁇ Reproduced in vitro the same symptoms as those of patients with abnormal cell differentiation using dedifferentiated stem cells derived from patients suffering from familial dysautonomia or LEOPARD syndrome. Cultured cells showed improvement when treated with experimental drugs for these diseases (Ebert AD. Et al, Nature, 2009, 457: 277-280, Lee G. et al, Nature, 2009, 461: 402-). 406, Cavajal-Vergara X.
  • inverted pluripotent stem cells and differentiated autologous cells may be useful for patients with intractable rare diseases as a method of developing patient-specific drugs in many other diseases without therapeutic drugs.
  • the present inventors have prepared a retrodifferentiation-induced pluripotent stem cells in human fibroblasts derived from CMT patients who have been studying patient-specific treatments for Charcot-Marie-Tooth disease (CMT).
  • CMT Charcot-Marie-Tooth disease
  • CMT Charcot-Marie-Tooth disease
  • Still another object of the present invention is to provide a customized therapeutic screening method for treating CMT type patients using automotor neurons of a CMT patient prepared by the method of the present invention.
  • the present invention provides a method for producing motor neurons in somatic cells derived from a Charcot-Marie-Tooth disease (CMT) patient.
  • CMT Charcot-Marie-Tooth disease
  • the present invention also provides a method for screening a CMT therapeutic agent candidate.
  • the present invention also provides autologous motor neurons of CMT patients differentiated from pluripotent stem cells produced by the method of the present invention.
  • the present invention provides a method for screening a customized therapeutic agent for treating CMT type patients using automotor neurons of a CMT patient prepared by the method of the present invention.
  • pluripotent stem cells from human fibroblasts derived from Char cot -Marie-Tooth disease (CMT) patients of the present invention, and differentiated from the pluripotent stem cells It is possible to confirm the efficacy of the drug through the screening method of the drug candidate Charcopomarimaritus jajahwan drug using the motor neuron, and the autologous motor neuron (moter) through the method of producing the pluripotent stem cell neurons) can be used for screening patient-specific treatments and for patient-specific treatment.
  • CMT Char cot -Marie-Tooth disease
  • FIG. 1 is a diagram showing the shape of human fibroblasts used in the present invention.
  • 2 is a diagram illustrating a method of differentiating CMT-derived pluri potent stem cells (iPSCs) into motor neurons.
  • iPSCs CMT-derived pluri potent stem cells
  • Figure 3 shows the identification of 5 / ⁇ 7 gene mutation that is the cause of CMT in CMT-derived differentiation induced pluripotent stem cells (CMT 2F-iPSC).
  • FIG. 4 is a diagram showing the form of colonies (colony) of CMT 2F PSC. The photograph was observed on the 20th day of culture, and the pluripotent fungal cell colonies of the differentiation showed dense cells.
  • Figure 6 is a diagram confirming the expression of the multipotency marker (sternness maeker) protein of CMT 2F-iPSC.
  • FIG. 7 shows the in vitro differentiation ability of embryonic bodies (EBs) derived from CMT 2F-iPSC, and the smooth muscles of nestin and mesoderm markers, which are markers of ectoderm. Confirmation of alpha-fetoprotein (AFP) expression, which is a smooth muscle actin (SMA) and endoderm marker.
  • EBs embryonic bodies
  • AFP alpha-fetoprotein
  • SMA smooth muscle actin
  • 9 shows marker protein expression and neuromuscular junction formation of motor neuron cells CMT 2F-MN differentiated from CMT2F patients;
  • 9A is a diagram confirming the expression of the marker proteins HB9, ISL1, SMI32, Tujl, MAP2 Synapsin and ChAT of CMT 2F-MN;
  • 9B is a diagram confirming the ratio of SMI32 and MPA2 positive proteins of CMT 2F-MN.
  • 9C is a diagram measuring the length of the axon projection of CMT 2F-MN.
  • FIG. 10 is a diagram confirming the formation of neuromuscular junction of CMT 2F- ⁇ .
  • Figure 11 shows the expression of acetylated a-tubulin (acetylatkm) as a CMT indicator for confirming the axonal transport efficiency with or without treatment of tuberstatin ACtubastatin A) in CMT 2F-MN Confirmed;
  • FIG. 11A is a diagram confirming the acetylation of ⁇ -tubulin in a CMT 2F-MN;
  • FIG. 11C is a diagram comparing quantification of acetylation of a-tubulin in CMT 2F- ⁇ with and without treatment of tubastatin A.
  • FIG. 12 is a diagram showing the mitochondria transport (moving mitochondria) as a CMT indicator for confirming the axon transport efficiency according to the treatment of tubastatin A in CMT 2F- ⁇ ;
  • 12A is a diagram illustrating axon mitochondria of motor neurons through mito-RED2 transformed with CMT-2F- ⁇ ;
  • FIG. 12B is a diagram comparing mitochondrial migration rate in CMT 2F-MN with or without treatment with tubastatin A; FIG. And
  • Figure 12c is a diagram showing the percentage of mitochondria migrated in CMT 2F-VIII with or without treatment with tubastatin A.
  • FIG. 13 is a view showing microfluidic cukiture for analyzing axon transport efficiency of motor neurons of CMT 2F-M by analyzing mitochondrial transport.
  • the present invention provides a method for producing motor neurons in somatic cells derived from a Char cot-Marie-Tooth disease (CMT) patient comprising the following steps. 1) obtaining human somatic cells from a Charcot® Marie-Tooth disease (CMT) patient;
  • step 3 inducing motor neurons by culturing in the presence of retinoic acid and sonic hedgehog in the reverse differentiation induced pluripotent stem cells of step 3).
  • Charcot-Marie-Tooth disease of step 1) is CMT type 1, CMT type 2, CMT type 4 or CMTX type, preferably CMT 2F.
  • the CMT 2F type is characterized in that the genes of the heat-shock potein (HSPs) 27 404 th and 545 th cytosine (cytosin) is substituted with thymin.
  • HSPs heat-shock potein
  • cytosin cytosine
  • the protein synthesized by the mutation is a variant protein in which the 135th amino acid is replaced with phenylalanine in serine from wild type HSP27 protein, or the 182th amino acid is substituted with Leucine in Proline. It is characterized by being.
  • Human somatic cells of step 1) are preferably fibroblasts, but are not limited thereto.
  • the vector used in step 2) may use a virus-mediated or non-viral vector using Sendai virus, retrovirus and lentivirus, and more preferably, Sendai virus is used.
  • a medium used for culturing human somatic cells to obtain the dedifferentiation-induced pluripotent stem cells after transformation may use a conventional medium.
  • a conventional medium for example, Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), a-MEM (Stanner, CP. Et al., Nat. New Biol. 230: 52 (1971)), Iscove's MEMdscove, N. et al. , J. Exp. Med. 147: 923 (1978), 199 Badge (Morgan) et al., Proc. Soc. Exp. Bio. Med.
  • Induced pluri potent stem cell refers to a cell induced to have pluripotent differentiation ability through artificial dedifferentiation from already differentiated cells. Also known as pluripotent cell.
  • the dedifferentiation-induced pluripotent stem cells have almost the same characteristics as embryonic enjoyment cells, specifically show similar cell shapes, and gene and protein expression patterns are similar.
  • iPSCs with pluripotency differentiation can confirm the expression of pluripotency marker proteins in vitro and show teratoma formation in vivo. In particular, when the iPSC is inserted into a mouse blastocyst, chimera mice are formed, and germline transmission of genes is possible.
  • the iPSC of the present invention includes all iPSCs derived from humans, pigs, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, and the like, preferably human-derived iPSCs, most preferably CMT patients IPSC derived from.
  • the transgene of the present invention refers to a gene or genetic material that is transferred by natural migration or by genetic engineering technology from one organism to another. Specifically, it is a DNA segment containing a gene sequence that is separated from one organism and introduced into another organism.
  • the gene sequence used as the trans gene uses 0CT4, S0X2, KLF4, and c_MYC as transgenes in the vector, which are originally derived from differentiated cells to induction of pluripotent stem cells. It is necessary to dedifferentiate.
  • 'de-differentiation' refers to epigenetic retrograde processes that allow the regeneration of existing .differentiated cells to an undifferentiated state, thereby enabling the formation of new differentiated tissues.
  • the reverse differentiation is a process for returning differentiated cells having differentiation capacity of 0% or more to less than 100% to an undifferentiated state, which are all included therein, for example, differentiated cells having 0% differentiation capacity. It may also include the process of undifferentiating the into differentiated cells having a differentiation capacity of 1%.
  • step 3) is preferably the following (3-1) to (3-2) to differentiate the prepared differentiated induced pluripotent stem cells into motor neurons, but is not limited thereto;
  • the neurotrophin (neurotrophin) of step 4) is the neuronal factor (Nerve growth factor; NGF), brain-derived neurotrophic factor (BDNF), Neutrophin (neurotrophin-3; NT-3) And a glia cell-derived neurotrophic factor (Glial eel 1 -derived neurotrophic factor; GDNF), but is not limited thereto.
  • the inventor's stone has four transcription factors (Klf4, 0ct3 / 4, from fibroblasts obtained from a skin biopsy of a CMT 2F patient comprising a mutation of S135F or P182L of the HSP27 gene).
  • Sox2 and c-Myc were used to produce induced pur i potent stem cells (iPSCs) and embryoid bodies (see FIG. 4), wherein the iPSCs included mutations in S135F or P182L.
  • Figure 3 Since it expresses multipotent marker genes and proteins, it was confirmed that it can be used as a pluripotent stem cell model of CMT disease (see FIGS.
  • CMT 2F-iPSCs into motor neurons from CMT 2F-iPSCs based on known methods to use CMT 2F-iPSCs as a model of peripheral neuropathy (Amoroso ⁇ , et al, JNeurosci 2013; 33: 574-586 (see FIG. 2), the efficiency of differentiation into motor neurons was confirmed by confirming marker protein expression and neuromuscular junction formation of the differentiated motor neurons (see FIGS. 9 and 10). ).
  • CPS-derived iPSCs model of the present invention exhibits the same mutations as the genetic mutations of CMT patients and not only shows multipotency, but also effectively differentiates into autologous motor neurons through neurospheres.
  • the method of manufacturing iPSCs model can be usefully used in the method used for analytical research for CMT.
  • the present invention provides a method for screening a composition for preventing and treating Sharco-Marie-Tooth disease, which comprises the following steps.
  • step 3 selecting a candidate for increase or decrease by comparing the level of the CMT indicator of step 2) with the control group.
  • the present invention also provides a customized therapeutic screening method for treating CMT type patients.
  • Cells differentiated from dedifferentiation-induced pluripotent stem cells prepared by dedifferentiating from the cells of the CMT patient can be prepared by the method of steps 1) to 2) and (3-1) to (3-2).
  • Motor neurons differentiated from CMT-derived iPSCs of the invention can be used to screen for candidate drugs for CMT disease.
  • the candidate drug preferably includes, but is not limited to, histone deacetylase 6 (HDAC6) inhibitors Trichostatin, Tuvacin and Tuvastatin A.
  • HDAC6 histone deacetylase 6
  • the drug concentration of dose-dependent treatment of the candidate drug in the normal control group and CMT-derived neurons can be determined to determine the drug concentration at which the survival of the cell is not inhibited. have.
  • the CMT indicator is preferably a marker of axonal movement (axonal transport), specifically, the acetylation of alpha-tubulin (ace tylated a -tubulin), mitochondrial unsongryang (moving mitochondria) or the electrical physiological indicator of the action potential amplitude (action potential ampHtude) is more preferably any one or more selected from the group consisting of, more specifically, it is most preferably either or both of acetylated a (tubulin) or mitochondria (moving mitochondria), It is not limited to this.
  • the amount of acetylated alpha-tubulin (acetylated ⁇ -Uibulin) is increased in the cells treated with the CMT drug candidate, it may be confirmed that the drug candidate is effective for treating CMT. At this time, the amount of acetylated alpha-tubulin was higher than without treatment with the drug candidate. When it is increased by 20% or more, preferably 30% or more, more preferably 35% or more can be confirmed that the therapeutic effect.
  • the candidate drug is said to be effective in treating CMT when the mitochondria and act ion potent ial amplitudes in the cells treated with the candidate CMT drug have been restored to the level of neurons derived from the normal control group. Can be.
  • the measurement of the protein expression change can be carried out through various methods known in the art.
  • enzyme immunoassay ELISA
  • western blot ICC
  • the measurement of gene expression change is RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press), cDNA microarrays (Sambrook et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (200D), etc.) Can be.
  • Charcoal-Maritus disease of step 1) is CMT type 1, CMT2 type, CMT4 type or CMTX type, preferably CMT 2F type.
  • the CMT 2F type is characterized in that the 404 th and 545 th cytosine gene of the heat-activated protein 27 is mutated substituted with thymine.
  • the protein synthesized due to the mutation is characterized in that the variant protein in which the 135th amino acid is substituted from serine to phenylalanine from the wild-type HSP27 protein or the 182th amino acid is substituted for leucine in plin.
  • the present inventors have identified neurons differentiated from iPSCs derived from CMT patients through micro-leubulin track and function identification associated with dysfunction of the axonal transport system, which is a major symptom in CMT 2F.
  • the acetylation level of ⁇ -leuulin and the mitochondria transport amount (moving mitochondria) As a result of confirming the axon transport efficiency of the motor neurons of CMT 2F- ⁇ , CMT 2F- ⁇ was confirmed to decrease the acetylation level of ⁇ -tubulin compared to the normal control WA09_MN (see Fig.
  • iPSCs derived from CMT patients of the present invention can produce automotor neurons via neurospheres while maintaining mutations in the CMT-causing gene, and the prepared autologous motor neurons do not directly administer CMT therapeutic candidates to patients.
  • patients can choose the patient-specific drug that is the most effective drug regardless of the efficacy of the drug. You can determine in advance which drug is least cytotoxic.
  • the present invention will be described in detail by way of examples.
  • Skin biopsy is a safe, low-invasive and economical method for the pathological diagnosis of skin lesions.
  • CMT 2F patients and normal groups including mutations of S135F or P182L of the HSP27 gene were linked (Ewha Mans University, Mokdong Hospital, Korea).
  • Skin biopsies were performed using a group of 4 mm diameter circular blades after local anesthesia for normal and CMT patients. 10 mg / collagenase type of skin tissue obtained by the above method IVClnvitrogen, USA), 50 M / mi di spase (Roche), 0.05% tr ipsin / EDTA containing in the reaction was added to DMEM medium for 40 minutes at 37 ° C.
  • the cell suspension thus obtained was filtered through a nylon cell strainer having a size of 70! M to obtain cells.
  • the obtained fibroblasts were cultured in DMEM medium containing 20% fetal bovine serum (FBS) and 100 ug / n penicillin / streptomycin. Each sample was classified as shown in Table 1 below.
  • iPSCs for neuronal differentiation from fibroblasts of CMT patient skin biopsies obtained in Example 1.
  • Transfection was performed using Sendai virus system (Cell biolabs, USA) containing Klf4, 0ct3 / 4, Sox2, c-Myc).
  • the vector of the Sendai virus used will not be inserted into the host genome and will disappear after several passages, resulting in more stable iPSCs.
  • the amount of sendaivirus was set to M0I (multiplicity of infection) 3 and after overnight infection with Sendaivirus, the skin biopsy of the CMT patient was performed.
  • Fibroblast culture medium was replaced with DMEM medium containing 10% fetal calf serum and incubated for 6 eggs to stabilize.
  • the cells were then transferred to SNL feeder cells (mouse embryonic fibroblasts (MEF) treated with mitomycin C (Invitrogen), SNL feeder cells (Cell Biolads, USA) and added 4 ng / m £ bFGF. / iPSC medium (KnockOut TM, USA) and incubated with fresh medium every day. About 30 days after Sendaivirus infection, cell colonies were selected and isolated according to the type of iPSC. The isolated iPSCs were analyzed for nucleotide sequences to determine whether they maintained the causal mutation of CMT.
  • iPSCs derived from CMT patients showed 404OT or 5450T mutations in 5 7 genes, and the HSP27 protein synthesized therefrom. It was confirmed to form variants substituted with S135F or P182L (Table 1 and FIG. 3).
  • iPSCs induced pluripotent stem cells
  • fibroblasts fibroblasts isolated from normal control and CMT patients were formed of normal human pluripotent cells with individual cells showing flat pebble shapes and sharp edges. It was confirmed that the form (Fig. 4).
  • CMT 2F-iPSCs show multipotency
  • expression of endogenous KLF4, 0CT4, S0X2 and c-Myc genes was confirmed in developmentally induced CMT 2F-iPSCs.
  • CMT 2F-iPSC or the normal control M09_hESC generated in ⁇ Example 2> was repeatedly cultured with 10 cell passages (eel hilar passage), and then obtained to obtain a trizol (TRIzol; Gibco, USA) )
  • total RNA of CMT 2F-iPSC or WA09_hESC was extracted according to the manufacturer's protocol.
  • RNA l ⁇ g and AMV reverse transcriptase ( ⁇ reverse transcriptase; Promega Inc., USA) using oligonucleotides (oliT dT), the forward primer and reverse primer described in Table 2 below CDNAs of KLF4, 0CT4, 56? and c-Myc genes were synthesized and amplified The expression of the gene was confirmed at the mRNA level by electrophresis.
  • Table 2 Table 2
  • stemEA marker proteins SSEA4 and NAN0G proteins was further confirmed.
  • Anti-SSEA4 antibody mouse IgG3, 1: 100 dilution; MC-813-70, DSHB, USA
  • anti-NAN0G antibody mouse IgGl, 1: 500 dilution; NNG-811, Abeam, USA
  • Cy3-linked goat-derived anti-mouse IgG secondary antibody and DAPI control chromosome were used for visualization.
  • the differentiation was induced to form an embryoid body (EB) from CMT 2F-iPSC, followed by ectoderm and mesoderm. And induced differentiation into tissues of endoderm origin.
  • EB embryoid body
  • the cell colonies of the CMT 2F-iPSC or the normal control WA09_hESC generated or induced in ⁇ Example 2> were transferred to an uncoated Petri dish where cells did not adhere well to the bottom of the ESC / iPSC medium (KnockOut TM, Gibco). Inc., USA) was incubated for 8 days with replacing it once to obtain floating cells as embryoid bodies (EB). Transfer the obtained embryoid body to a gelatin-coated slide culture vessel (chamber slide, Lab— Tek) and incubated for additional 8 days in 10% FBS / DMEM medium to induce differentiation into cells of ectoderm, mesoderm and endoderm origin. It was.
  • EB embryoid bodies
  • Primary antibodies include anti-alpha fetoprotein At, ant i -AFP Ab, mouse IgG2b, 1: 100; 2A9, Abeam, USA), anti-alpha smooth muscle actin Ab (mouse IgG2a, 1: 100 dilution; 1A4, Abeam, USA) and anti-Nestine antibodies (anti-Nest in Ab, mouse IgGl, 1: 1000 dilution; 10C2, Abeam, USA)
  • the secondary antibody was reacted with a goat-derived anti-mouse IgG antibody conjugated with FTTC, and then mounted with a solution containing chromosomes relative to DAPr and analyzed by confocal microscopy.
  • the compensation body is eu alpha fetoprotein (a -fetoprotein; AFP) (endoderm (endoderm)), smooth muscle actin ( It was confirmed that smooth muscle act in; SMA (mesoderm) and Nestin (Ectoderm) differentiated without abnormalities in both normal and CMT patient group cells (FIG. 7).
  • AFP alpha fetoprotein
  • SMA meoderm
  • Nestin Ectoderm
  • the CMT 2F-iPSC (S135F and P182L) or the normal control WA09 ⁇ hESC induced by the same method as described in Example 2 was detached into a small cell group, and then 1.0X10 6
  • the cells were counted and mixed at a ratio of matrigel and l: l (v: v).
  • the mixed Matrigel-cell complex was injected subcutaneously into the dorsal side of 5 week old female immunodeficient mice (N0D / SCID mice), and then xenograft mice were bred for 8 weeks.
  • mice were regenerated, explanted the teratoma formed, fixed in 10% Natural buffered forma ldehyde (10% NBF) overnight, and then made paraffin blotting, cut to 0.4 ⁇ ⁇ thickness hemaroxylin & Eosin (Hematoxylin and Eos in, H & E) staining was observed.
  • CMT 2F-iPSCs As a model of peripheral neuropathy, they were differentiated from CMT 2F-iPSCs into motor neurons according to known methods as described in FIG. 2 (Amoroso MW, et al, J Neurosci 2013 33: 574-586) (FIG. 2).
  • basal medium was used as ESC / iPSCs medium, Rho-associated kinase inhibitor 10 ⁇ Y27632 (Tocris Bioscience, UK), 20 ng / ml bFGF (Invitrogen, USA), 10 ⁇ M SB435142 (Stemgent) , USA), 0.2 ⁇ LDN193189 (Stemgent, USA), and penicillin with penicillin / streptomycin added were cultured in suspension for 2 days to form an embryoid body. Three days after the start of the culture, the basal medium was replaced with Neural stem cell media (Stemline;
  • the basal medium was changed to Neurobasal (Neurobasal; Invitrogen, USA) and maintained at 10 ng / m «IGF-1, 10 ng / mi GDNF, 10 ng / mi while maintaining all the added factors.
  • Neurobasal Neurobasal
  • CNTF R & D, USA
  • B27 supplement Gibco, USA
  • neurospheres were cultured to differentiate into motor neuron cells. Cells were maintained in suspension culture and cultured.
  • the cells were treated with accutase (accutase, PAA Laboratories) to disperse the cells, and then poly-L-lysine / laminin (poly Motor neurons differentiated from CMT-2F iPSCs or WA09_hESCs (CMT-2F-MN or WA09 ⁇ ⁇ ) in L-lysine / laminin-coated culture vessels or slide chambers (Nalgene Nunc, USA) Obtained.
  • accutase accutase, PAA Laboratories
  • Example ⁇ 3-1> CMT-2F- ⁇ or WA09J obtained in Example ⁇ 3-1> was performed by immunocytostaining in the same manner as in Example ⁇ 2-3> to confirm the expression of the motor neuron marker protein.
  • Primary antibodies used were anti-HB9 antibody (mouse IgGl, 1: 100 dilution; 81.5C10, DSHB, USA), anti-Islet-1 / 2 antibody (mouse IgG2b, 1:50 antibody; 39.4DS, DSHB, USA ), An anti-SMI32 antibody (mouse IgGl, 1: 500 dilution; Covance, USA), an anti-neuronal specific beta III tubulin (Tujl), which is an anti-H-non-phosphorylated neurofiber Antibodies (Rabbit IgG, 1: 1000); Abeam, USA; Anti-microtubule-associated protein 2, anti-MAP2 Antibody (Rabbit IgG, 1: 200 dilution; Millipore, Inc
  • DAPI stand Non chromosomes were used for visualization. The degree of development of motor neurons was compared by measuring the percentage of immunocytostained SMI32 / DAPI or MAP2 / DAPI positive cells and measuring the length of axons.
  • C2C12 mouse myoblasts (CRL-1772, purchased from ATCC) were cultured in DMEM medium containing 10% FBS, 1 mM glutamine and penicillin / streptomycin. When the cultured cells became 70% confluence, 1% insulin-transferrin-selenium (ITS) additive (Sigma, USA) was added to the medium to myotubes. Differentiation was induced. After 2 days of incubation, 10 ⁇ M cytosine arabinoside was added to remove the dividing cells and incubated for 2 to 4 days.
  • ITS insulin-transferrin-selenium
  • CMT-2F- or 09_MN obtained in Example ⁇ 3-1> was co-cultured with the inoculated myotubes at a ratio of 10: 1 and MN differentiation medium.
  • the co-cultured motor neurons and myotubes were stained with Alexa 488-linked ⁇ -Bungarotoxin ( ⁇ -bungarotoxin, ⁇ - ⁇ ; Invitrogen, USA) to confirm the formation of neuromuscular joints. .
  • CMT-2F- ⁇ co-cultured with the root canal cells confirmed that the neuromuscular joint was normally formed (FIG. 10).
  • Example ⁇ 3-1> After the differentiation-induced CMT-2F-MN or WA09_VIII was obtained by the method of Example ⁇ 3-1>, 5 ⁇ tuberstatin ⁇ was treated in the medium and incubated for 12 hours. Then, a-tubulin and acetylated a-leuublin were immunocytostained in the same manner as in Example ⁇ 2-3>.
  • Primary antibodies include anti-a-leuublin antibody (rabbit IgG, 1: 500 dilution; Abeam, USA) and anti-acetylated ⁇ -tubulin antibody (mouse IgG, 1: 200 dilution; Abeam, USA ) And Alexa 488-linked goat anti-rabbit IgG and Cy3-linked goat anti-mouse IgG antibodies were used as secondary antibodies.
  • CMT-2F- or 09_MN treated with 5 ⁇ tovastatin ⁇ was lSOmMNaCl
  • the membrane was anti-acetylated with ⁇ -tubulin antibody (mouse IgG2b, 1: 1000 dilution; 6-11B-1, Abeam) and anti- ⁇ -tubulin antibody (rabbit, mouse IgGl, 1: 1000 dilution; immunoblotting (i ⁇ unoblotting) using DM1A, Sigma, USA), followed by UN-SCAN-IT gel software (Silk Scientific, USA) to determine the acetylation level of ⁇ ⁇ tubulin by analyzing the band density.
  • immunocytostaining and immunoblot were performed in the same manner as above using CMT-2F-VIII or M09_MN not treated with 5 ⁇ tubastatin A.
  • CMT 2F-MN showed no decrease in the acetylation level of ⁇ -tubulin compared to the normal control group 09_MN when the tubevastatin A was not treated, but treated with 5 ⁇ tubastatin ⁇ .
  • the acetylation level of CMT2F- ⁇ ⁇ -tubulin increased to recover to a level similar to the normal control (Figs. 11a, lib and 11c).
  • microchannel plates Provided by Mok Professor, Seoul National University, Korea) Park JW et al., Nat Protoc 2006; 1: 2128-2136
  • axons grow perfectly through a micrometer-si zed groove and extend into the opposite compartment, using lipofectamine 2000 (1 ipofectamine 2000; Invitrogen, USA)
  • Mito-dsRED2 Mito-dsRED2 was transformed into the processed motor neurons.
  • tubastatin A was treated in the medium and incubated for 6 hours, and then mitochondrial images were taken using a fluorescence microscope at a rate of 121 snaps / 2 minutes, ImageJ and Kymograph was used to measure the moving velocity of the motor neuron.
  • the degree of migration is expressed as the percentage of the mitochondrial number that migrates to the total number of mitochondria.

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Abstract

La présente invention concerne des cellules souches pluripotentes induites et un procédé pour cribler un agent thérapeutique pour la maladie de Charcot-Marie-Tooth (CMT) en utilisant des neurones moteurs différenciés à partir des cellules souches pluripotentes induites. En particulier, la présente invention permet que les cellules souches pluripotentes induites soient préparées à partir d'un fibroblaste humain dérivé d'un patient CMT, que l'efficacité d'un médicament soit déterminée par l'intermédiaire du criblage d'un matériau candidat pour un agent thérapeutique pour CMT en utilisant des neurones moteurs différenciés à partir des cellules souches pluripotentes induites, et que des neurones moteurs autologues soient préparés par le procédé pour préparer la cellule souche pluripotente induite, de manière à être utilisée pour cribler un médicament de traitement personnalisé pour un patient et un traitement personnalisé pour un patient.
PCT/KR2014/002794 2013-04-02 2014-04-01 Procédé de criblage d'agents thérapeutiques pour la maladie de charcot-marie-tooth et neurones moteurs auto-différenciés utilisés pour celui-ci Ceased WO2014163380A1 (fr)

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CN201480031644.5A CN105264068A (zh) 2013-04-02 2014-04-01 Charcot-Marie-Tooth病的治疗剂的筛选方法以及为此所使用的自分化运动神经元
US14/873,125 US20160011177A1 (en) 2013-04-02 2015-10-01 Screening method for therapeutic agents for charcot-marie-tooth disease and self-differentiation motor neurons used therefor

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120027353A (ko) * 2009-06-02 2012-03-21 파넥스트 Ｃｍｔ 및 관련 장애를 치료하기 위한 신규 조성물
WO2012045804A1 (fr) * 2010-10-08 2012-04-12 Vib Vzw Inhibiteurs des hdac pour traiter l'amyotrophie péronière de charcot-marie-tooth

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120027353A (ko) * 2009-06-02 2012-03-21 파넥스트 Ｃｍｔ 및 관련 장애를 치료하기 위한 신규 조성물
WO2012045804A1 (fr) * 2010-10-08 2012-04-12 Vib Vzw Inhibiteurs des hdac pour traiter l'amyotrophie péronière de charcot-marie-tooth

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
OKADA, Y. ET AL.: "Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells", DEVELOPMENTAL BIOLOGY, vol. 275, no. 1, 2004, pages 124 - 142 *
SAPORTA, MARIO A ET AL.: "Induced pluripotent stem cells in the study of neurological diseases", STEM CELL RESEARCH & THERAPY, vol. 2, no. 5, 2011, pages 1 - 9 *
SAPORTA, MARIO ET AL.: "Establishing a Charcot-Marie-Tooth (CMT) induced pluripotent stem cell bank: Towards a human disease model for CMT", 2012 AMERICAN ACADEMY OF NEUROLOGY MEETING, 25 April 2012 (2012-04-25) *

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