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WO2018160028A1 - Composition de milieu pour la différenciation neuronale et procédé de différenciation de cellules somatiques en neurones en utilisant la même composition de milieu - Google Patents

Composition de milieu pour la différenciation neuronale et procédé de différenciation de cellules somatiques en neurones en utilisant la même composition de milieu Download PDF

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WO2018160028A1
WO2018160028A1 PCT/KR2018/002520 KR2018002520W WO2018160028A1 WO 2018160028 A1 WO2018160028 A1 WO 2018160028A1 KR 2018002520 W KR2018002520 W KR 2018002520W WO 2018160028 A1 WO2018160028 A1 WO 2018160028A1
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neurons
inhibitor
differentiation
composition
inhibitors
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Korean (ko)
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김경규
데보죠티데
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Cellatoz Therapeutics Inc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/727—Kinases (EC 2.7.)
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts

Definitions

  • the present invention relates to a medium composition for neuron differentiation and a method for differentiation from somatic cells to neurons using the medium composition, and more specifically, chemically induced nerves from somatic cells with low molecular weight material without gene introduction in a safe and high efficiency.
  • the present invention relates to a medium composition for differentiating neurons for differentiating cells (CiN, chemically induced neuron) and a method for differentiating somatic cells to neurons using the medium composition.
  • Physiologically active neurons can be obtained through differentiation of human embryonic stem cells (ES cells, Embryonic stem cells), but are problematic in terms of solubility, immune sensitivity, functional integrity, tumor development risk and efficiency, In addition, it is difficult to transplant neurons or neural progenitor cells derived from appropriate immune-type matched donors, which have various limitations in their use as cell therapeutics. Embryonic stem cells also pose ethical problems.
  • a method of obtaining pluripotent stem cells by dedifferentiating somatic cells of a patient has been developed, and neuronal and neural stem cells are differentiated by differentiating induced pluripotent stem cells (iPSCs). I can make it.
  • these neurons also have the disadvantage that there is a risk of tumor development.
  • the method of making somatic cells through the dedifferentiated stem cells has a disadvantage that it takes a lot of time and money.
  • a technique for directly dividing somatic cells into cells of various strains without reverse differentiation has been developed.
  • iPSC the differentiation of dedifferentiated stem cells made using somatic cells to differentiate into various neurons through neural stem cells and neural progenitor cells, while direct differentiation directly converts somatic cells into neural stem cells or functional neurons.
  • neural cell differentiation by direct differentiation has a great advantage in terms of time, cost, and effectiveness compared to the method of reverse differentiation.
  • direct differentiation is characterized by the ability to produce patient-specific cells using somatic cells of the patient, thus enabling autograft treatment without an immune response.
  • Many direct differentiations known to date have been achieved by introducing foreign genes directly into cells using viruses or plasmids.
  • Direct differentiation using small molecules has advantages in terms of research method in addition to the clinical aspects described above compared to other differentiation methods, including ease of introduction of molecules, reproducibility of results and efficient scalability. Therefore, developing a method of directly differentiating nerve cells from somatic cells in a safer and more convenient way without the problems of conventional cell therapy products is necessary to make a neuronal cell therapy necessary to treat neuronal damage-related diseases. .
  • the technical problem to be achieved by the present invention is to provide a neuronal cell differentiation medium composition that enables the direct differentiation of somatic cells into neurons in a relatively fast time.
  • Another object of the present invention to provide a neuronal differentiation method for inducing neurons from somatic cells using the media composition for neuronal differentiation.
  • the present invention is a histone deacetylase inhibitor (Histone deacetylase inhibitor) ,; GSK inhibitors (Glycogen synthease kinase inhibitors); ALK-5 inhibitors; cAMP signaling activator; And it provides a composition for neuronal differentiation medium comprising a bromodomain inhibitor (bromodomain inhibitor).
  • the present invention also provides a media composition for neuronal differentiation further comprising a ROCK inhibitor.
  • the present invention is at least one selected from the group consisting of the cytokine NT3, BDNF, GDNF and bFGF; BMP4 inhibitors; And it provides a neuronal differentiation medium composition further comprising one or more selected from the group consisting of supplements B27 and N2.
  • the present invention provides a neuronal cell differentiation medium composition further comprising a histone dimethylase inhibitor.
  • the present invention also provides a neuronal cell differentiation medium composition comprising a histone acetyltransferase inhibitor (Histone Acetyltransferase inhibitor).
  • a histone acetyltransferase inhibitor Histone Acetyltransferase inhibitor
  • the present invention is the histone deacetylase inhibitor trichostatin A (Vrichostatin A), Valproic acid (Valproic acid), suberoylanilide hydroxamic acid (Suberoylanilide hydroxamic acid), hydroxamic acid (hydroxamic acid), between Click Tetrapeptide (cyclic tetrapeptide), depsipeptides, vorinostat, Velinostat, Belinostat, Panobinostat, Benzamide, Entinostat, and Butylate It provides a neuronal differentiation medium composition, characterized in that at least one member selected from the group consisting of (butyrate).
  • the GSK inhibitor is Chir99021 (6- (2- (4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino) ethylamino) nicotinonitrile); 1-azakenpaullone (9-Bromo-7,12-dihydro-pyrido [3 ', 2': 2,3] azepino [4,5-b] indol-6 (5H) -one); BIO ((2'Z, 3'E) -6-Bromoindirubin-3'-oxime); ARA014418 (N- (4-Methoxybenzyl) -N '-(5-nitro-l, 3-thiazol-2-yl) urea); Indirubin-3'-monoxime; 5-Iodo-indirubin-3'-monoxime; kenpaullone (9-Bromo-7,12-dihydroindolo- [
  • the ALK-5 inhibitor is RepSox (1,5-Naphthyridine, 2- [3- (6-methyl-2-pyridinyl) -1 H-pyrazol-4-yl]); SB525334 (6- (2-tert-butyl-4- (6-methylpyridin-2-yl) -1H-imidazol-5-yl) quinoxaline); GW788388 (4- (4- (3- (pyridin-2-yl) -1H-pyrazol-4-yl) pyridin-2-yl) -N- (tetrahydro-2H-pyran-4-yl) benzamide); SD-208 (2- (5-chloro-2-fluorophenyl) -N- (pyridin-4-yl) pteridin-4-amine); Galunisertib (LY2157299, 4- (2- (6-methylpyridin-2-yl) -5,6-dihydro-4H-pyrrolo [1,2-b] pyrazol
  • the present invention is characterized in that the cAMP signaling activator is at least one selected from the group consisting of Forskolin, isoproterenol, NKH 477 isoprotereno (Chemical based), PACAP 1-27 and PACAP 1-38 (peptide based) Provided is a medium composition for neuronal differentiation.
  • the present invention is the histone acetyltransferase inhibitor Garcinol (Garcinol), C646 (4-[(4Z) -4-[[5- (4,5-dimethyl-2-nitrophenyl) furan-2-yl ] methylidene] -3-methyl-5-oxopyrazol-1-yl] benzoic acid), MG 149 (2- (4-heptylphenethyl) -6-hydroxybenzoic acid), NU 9056 (1,2-di (isothiazol-5- yl) disulfane) and Anacardic acid (2-hydroxy-6-pentadecylbenzoic acid) provides a neuronal differentiation medium composition, characterized in that at least one member selected from the group consisting of.
  • the bromoregion inhibitor is (+)-JQ1, ARV825, Bromosporine (ethyl (3-methyl-6- (4-methyl-3- (methylsulfonamido) phenyl)-[1,2,4] triazolo [4,3-b] pyridazin-8-yl) carbamate), GW841819X ((R) -benzyl (6- (4-chlorophenyl) -8-methoxy-1-methyl-4H-benzo [f] [1,2 , 4] triazolo [4,3-a] [1,4] diazepin-4-yl) carbamate), CPI-203 ((S) -2- (4- (4-chlorophenyl) -2,3,9- trimethyl-6H-thieno [3,2-f] [1,2,4] triazolo [4,3-a] [1,4] diazepin-6-yl) acetamide), and RVX-208 (2- [4 -(2-
  • the present invention is the ROCK inhibitor is Y-27632 (4-[(1R) -1-aminoethyl] -N-pyridin-4-ylcyclohexane-1-carboxamide), Y-33075 (4- (1-aminoethyl)- N- (1H-pyrrolo [2,3-b] pyridin-4-yl) benzamide), Y-39983 dihydrochloride (4-[(1R) -1-aminoethyl] -N- (1H-pyrrolo [2,3- b] pyridin-4-yl) benzamide; dihydrochlorid), SR-3677 (N- [2- [2- (dimethylamino) ethoxy] -4- (1H-pyrazol-4-yl) phenyl] -2,3-dihydro -1,4-benzodioxine-3-carboxamide) and AS1892802 ((S, Z) -N '
  • the present invention provides a composition comprising: i) a histone deacetylase inhibitor; GSK inhibitors (Glycogen synthease kinase inhibitors); ALK-5 inhibitors; cAMP signaling activator; And culturing somatic cells in the presence of a neuronal cell differentiation medium composition comprising a bromodomain inhibitor and inducing them into neurons; And ii) aging the induced neurons in a medium comprising a GSK inhibitor, an ALK-5 inhibitor, a cAMP signaling activator and a ROCK inhibitor.
  • the present invention provides a method for differentiating somatic cells to nerve cells, characterized in that the medium composition for neuronal differentiation further comprises a ROCK inhibitor.
  • the present invention provides a method of differentiating somatic cells to neurons, characterized in that step i) the culture is carried out in the range of 2 hours to 10 days, step ii) is carried out in the range of 2 to 10 days. do.
  • the present invention also provides a method for differentiating somatic cells into nerve cells, wherein the somatic cells are fibroblasts.
  • the present invention provides a pharmaceutical composition comprising neurons differentiated by the differentiation method.
  • the present invention provides the pharmaceutical composition of the carrier, excipient, diluent, antioxidant, preservative, colorant, flavor and diluent, emulsifier, suspending agent, solvent, filler, bulking agent, buffer, delivery vehicle, isotonic agent, cosolvent It provides a pharmaceutical composition characterized in that it further comprises one or more pharmaceutically acceptable salts, excipients or vehicles selected from the group consisting of wetting agents, complexing agents, buffers, antibacterial agents and surfactants.
  • the present invention was directly differentiated from somatic cells to neurons using only low molecular weight material without gene introduction, and it was confirmed that direct differentiation with high yield in a very fast time.
  • a simple method of culturing cells in a medium containing low molecular weight material By showing a technique of directly differentiating fibroblasts into nerve cells, it is useful as a safe cell therapy using autologous cells without genetic modification, and the nerve cells produced by the present invention are nerves such as stroke and cerebral palsy.
  • FIG. 1 is a schematic diagram illustrating the differentiation process of human epidermal fibroblasts into neurons during chemical treatment of the present invention according to Example 1 of the present invention.
  • FIG. 2 shows the expression of neuronal markers differentiated according to Example 1 of the present invention
  • Figure 3 is a diagram comparing the expression of neuronal-specific and fibroblast specific transcripts
  • Figure 4 is a diagram showing the results of differentiation by excluding individual components from the total combination in order to confirm the essential composition of the neuronal cell differentiation medium composition according to the present invention
  • Example 5 is a schematic diagram illustrating the differentiation process from human epidermal fibroblasts to neurons during chemical treatment of the present invention using the composition of Example 3 of the present invention.
  • Example 6 is a result of observing neuronal differentiation with the composition of Example 3 according to the present invention over time
  • Figure 7 is a neuronal marker confirming results for confirming the expression of differentiated neurons in Example 3.
  • 8A and 8B show the results of differentiating neurons using the compositions of Examples 3 and 4, respectively.
  • FIG. 9 (A) shows the results obtained after 3 hours of application of the composition of Example 4 to fibroblasts
  • FIG. 9 (B) shows the results observed after 6 hours of application of the composition of Example 5
  • FIG. 10 is a micrograph of neurons made by direct differentiation of mouse embryonic fibroblasts into neurons using the media composition for neuron differentiation of the present invention.
  • neuronal cell includes a neuron having the above-described neural cell and a similar neural cell having the ability to become a neural cell through a maturation process, and identify a cell that can be identified by at least one neuron specific marker or criteria. It means.
  • Expression of various neuron-specific markers can be detected by known biochemical or immunochemical methods, and such methods can be used without limitation.
  • marker specific polyclonal antibodies or monoclonal antibodies that bind to neuronal progenitor cells or neurons can be used.
  • Antibodies that target individual specific markers can be used commercially or without limitation, those prepared by known methods.
  • neural progenitor or neuron specific markers is not limited to specific methods, but may be used to amplify, detect, or encode mRNA encoding any marker protein, such as reverse transcriptase mediated polymerase chain reaction (RT-PCR) or hybridization assays. It can be confirmed by molecular biological methods commonly used in the art for interpretation. Nucleic acid sequences encoding marker proteins specific for neurons are already known and can be obtained from public databases such as GenBank and can readily determine the marker specific sequences required for use as primers or probes. In addition, in order to confirm the differentiation of somatic cells into neurons, physiological criteria may additionally be used. In other words, cells derived from pluripotent cells may be used as useful indicators, such as having independent pulsatile rhythms, expressing various ion channels, and responding to electrophysiological stimuli.
  • RT-PCR reverse transcriptase mediated polymerase chain reaction
  • the type of starting somatic cell is not particularly limited, and any somatic cell can be used.
  • mature somatic cells may be used in addition to somatic cells of the embryonic period.
  • somatic cells separated from the patient it is preferable to use somatic cells separated from the patient.
  • somatic cells involved in the disease or somatic cells involved in the disease treatment can be used.
  • the fibroblasts were used as the somatic cells.
  • the fibroblasts include all fibroblasts derived from mammals such as humans and mice, horses, sheep, pigs, goats, camels, antelopes, and dogs. .
  • Neuron differentiation medium composition of the present invention is a histone deacetylase inhibitor (Histone deacetylase inhibitor, HDAC inhibitor) ,; GSK inhibitors (Glycogen synthease kinase inhibitors); ALK-5 inhibitors; cAMP signaling activator; And essentially five basic components of bromodomain inhibitors, optionally ROCK inhibitors (ROCK inhibitors, Rho Kinase inhibitors), histone dimethylase inhibitors and / or histone acetyltransferases. It may further include an inhibitor (Histone Acetyltransferase inhibitor).
  • the media composition for neuronal differentiation of the present invention is at least one selected from the group consisting of cytokines NT3, BDNF, GDNF and bFGF; BMP4 inhibitors; And it may further comprise one or more selected from the group consisting of supplements B27 and N2.
  • histone deacetylase inhibitor refers to substances that inhibit the activity of an enzyme that removes an acetyl group from histones.
  • the histone deacetylase inhibitors include Trichostatin A, Valproic acid, Suberoylanilide hydroxamic acid, Hydroxamic acid, Cyclic tetrapeptide Consisting of tetrapeptide, depsipeptides, verinostat, verinostat, belinostat, panobinostat, benzamide, benzamide, entinostat and butyrate It may include one or more selected from the group, preferably Trichostatin A, but is not limited thereto.
  • the histone deacetylase inhibitor is preferably included in the range of 0.5 to 1,000 ⁇ M based on the total composition. If the content of the histone deacetylase inhibitor is out of the above range is because there is a problem such as a low conversion rate to neurons or the generation of abnormal cells.
  • GSK synthease kinase inhibitor refers to a substance that activates the Wnt signal by inhibiting GSK activity by targeting GSK1 / 2 involved in the Wnt signaling process.
  • the GSK inhibitors include: Chir99021 (6- (2- (4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino) ethylamino) nicotinonitrile); 1-azakenpaullone (9-Bromo-7,12-dihydro-pyrido [3 ', 2': 2,3] azepino [4,5-b] indol-6 (5H) -one); BIO ((2'Z, 3'E) -6-Bromoindirubin-3'-oxime); ARA014418 (N- (4-Methoxybenzyl) -N '-(5-nitro-l, 3-thiazol-2-yl) ure
  • ALK-5 inhibitor is in the present invention
  • ALK-5 inhibitor (ALK-5 inhibitor)" is bound to TGF by binding to ALK5 (activin A receptor type II-like kinase 5)
  • ALK5 activin A receptor type II-like kinase 5
  • ALK5 activin A receptor type II-like kinase 5
  • TGF- ⁇ type I TGF- ⁇ type I receptor
  • TGF- ⁇ type I TGF- ⁇ type I is a cell proliferation, differentiation and As a multifunctional peptide that has a variety of functions on a variety of cells, this multifunctionality plays a pivotal role in the growth and differentiation of various tissues.
  • the ALK-5 inhibitors include RepSox (1,5-Naphthyridine, 2- [3- (6-methyl-2-pyridinyl) -1H-pyrazol-4-yl]); SB525334 (6- (2-tert-butyl-4- (6-methylpyridin-2-yl) -1H-imidazol-5-yl) quinoxaline); GW788388 (4- (4- (3- (pyridin-2-yl) -1H-pyrazol-4-yl) pyridin-2-yl) -N- (tetrahydro-2H-pyran-4-yl) benzamide); SD-208 (2- (5-chloro-2-fluorophenyl) -N- (pyridin-4-yl) pteridin-4-amine); Galunisertib (LY2157299, 4- (2- (6-methylpyridin-2-yl) -5,6-dihydro-4H-pyrrolo
  • cAMP signaling activator means a substance that activates a cAMP signal.
  • the cAMP signaling activator may include one or more selected from the group consisting of Forskolin, isoproterenol, NKH 477 isoprotereno (Chemical based), PACAP 1-27, and PACAP 1-38 (peptide based), preferably May be, but is not limited to, Forskolin.
  • the cAMP signaling activator is preferably included in the range of 0.5 to 100 ⁇ M based on the total composition.
  • the "bromodomain” is a region for recognizing acetylated lysine of histones, and 100 to 110 amino acids present in proteins in the nucleus such as histone acetyltransferase (HAT) or chromatin reassortant factor. It means the area that consists of.
  • the bromoregion inhibitors are (+)-JQ1, ARV825, Bromosporine (ethyl (3-methyl-6- (4-methyl-3- (methylsulfonamido) phenyl)-[1,2,4] triazolo [4,3- b] pyridazin-8-yl) carbamate), GW841819X ((R) -benzyl (6- (4-chlorophenyl) -8-methoxy-1-methyl-4H-benzo [f] [1,2,4] triazolo [ 4,3-a] [1,4] diazepin-4-yl) carbamate), CPI-203 ((S) -2- (4- (4-chlorophenyl) -2,3,9-trimethyl-6H-thieno [3,2-f] [1,2,4] triazolo [4,3-a] [1,4] diazepin-6-yl) acetamide) and RVX-208 (2- [4- (2-hydroxyethoxy) -3,
  • histone acetyltransferase inhibitor means a substance that inhibits histone acetylation in normal cells together with histone deacetylase.
  • the histone acetyltransferase inhibitor may include one or more selected from the group consisting of,
  • ROC Kinase inhibitor is Y-27632 (4-[(1R) -1-aminoethyl] -N-pyridin-4-ylcyclohexane-1-carboxamide), Y-33075 (4- (1-aminoethyl) -N- (1H-pyrrolo [2,3-b] pyridin-4-yl) benzamide), Y-39983 dihydrochloride (4-[(1R) -1-aminoethyl] -N- (1H- pyrrolo [2,3-b] pyridin-4-yl) benzamide; dihydrochlorid), SR-3677 (N- [2- [2- (dimethylamino) ethoxy] -4- (1H-pyrazol-4-yl) phenyl] -2,3-dihydro-1,4-benzodioxine-3-carboxamide) and AS1892802 ((S, Z) -N
  • the neuronal differentiation medium composition of the present invention may further comprise a histone dimethylase inhibitor.
  • Preferred examples of the histone dimethylase include 2,4-Pyridinedicarboxylic acid, the content of which is preferably included in the range of 0.5 to 1,000 ⁇ M based on the total composition.
  • the media composition for neuronal differentiation of the present invention is at least one selected from the group consisting of cytokines NT3, BDNF, GDNF and bFGF; At least one selected from the group consisting of GSK inhibitors, cAMP signaling activators, BMP4 inhibitors (BMP4 inhibitors) and ROCK inhibitors which are small molecular compounds; And it may further include one or more selected from the group consisting of supplements B27 and N2, the type is not limited.
  • the "BMP4 inhibitor” is DM3189 (Quinoline, 4- [6- [4- (1-piperazinyl) phenyl] pyrazolo [1,5-a] pyrimidin-3-yl]-), Dorsomorphin 2HCl (6 -[4- (2-piperidin-1-ylethoxy) phenyl] -3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine; dihydrochloride) or MP470 (N- (1,3-benzodioxol-5-ylmethyl) -4-([1] benzofuro [3,2-d] pyrimidin-4-yl) piperazine-1-carbothioamide), preferably DM3189, but is not limited thereto.
  • the culture medium for culturing the somatic cells includes all of the medium culture medium commonly used in the fibroblast culture in the art.
  • the culture medium used for the culture generally contains a carbon source, a nitrogen source and a trace element component.
  • the medium preferably includes DMEM / F12, N2, B27, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).
  • the medium for culturing of the present invention can be used without limitation a basal medium known in the art.
  • the basal medium may be prepared by artificially synthesizing, or a commercially prepared medium may be used.
  • commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, ⁇ -MEM ( ⁇ -Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium), Isocove's Modified Dulbecco's Medium, and Neurobasal Plus media containing 1% B27 supplement, but are not limited thereto, and may be DMEM medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basic Medium Eagle
  • RPMI 1640 F-10, F-12
  • ⁇ -MEM ⁇ -Minimal
  • essential medium G-MEM (Glas
  • the present invention also provides a method of culturing somatic cells in the presence of the media composition for neuronal differentiation to differentiate into neurons.
  • most of the methods of directing somatic cells into neurons are performed by introducing external genes.
  • introducing a gene using a virus causes genetic instability due to random integration of an external gene, which may cause cancer in future clinical applications.
  • methods for using small molecules without gradually injecting external genes have been proposed.
  • at least one gene has been used, and without the introduction of genes, it is still unable to switch from human somatic cells to desired neurons.
  • the present invention is a method of ensuring genetic stability of inducing neurons from somatic cells without introducing an external gene, and is designed to solve a method of inducing genetic defects using existing genes.
  • somatic cells were directly differentiated into neurons using only a combination of low molecular weight substances.
  • the present invention has been made to solve the problems of the prior art as described above, the inventors of the present invention to find a method for transforming somatic cells directly into neurons in a fast and time-efficient cell differentiation-related signal regulators known as follows Combined; (1) glycogen synthase kinase inhibitors that activate Wnt / beta-catenin signaling, (2) ALK-5 kinase inhibitors that inhibit TFG-beta signaling, and (3) cyclic AMP (cAMP), a cell signaling agent cAMP signaling activator.
  • cAMP cyclic AMP
  • ROCK inhibitor can increase neuronal differentiation efficiency by reducing the death of differentiated neurons and accelerating the growth of neurite.
  • the differentiation method of the somatic cells from the present invention to nerve cells may be performed in a single step, but may be performed in two steps for efficiency.
  • the culture of step i) may be performed without limitation as long as it can induce differentiation into neurons, but preferably may be performed for 2 hours to 10 days, more preferably for 3 hours to 3 days Can be.
  • the culture of step ii) may be performed without limitation as long as the differentiation-induced cells can mature, but preferably 2 to 10 days, more preferably 3 to 8 days. May be performed. If it is out of this period is due to the lack of conversion to neurons or side effects of apoptosis.
  • the culture period is not limited thereto.
  • the differentiation method of the present invention there is an advantage that the differentiation to the desired cell can be efficiently induced only with a shorter time as compared with the known chemically induced cell differentiation method.
  • the neuronal differentiation medium composition of the present invention can be used as a cell therapy (pharmaceutical composition) for treating neurological diseases by differentiating neurons using the medium composition.
  • the neurological disorders include nerve damage caused by accidents including stroke, neurological diseases such as cerebral palsy, spinal cord injury, nerves caused by genetic abnormalities including battens disease.
  • Diseases Alzheimer's disease (Alzheimer's disease), Parkinson's disease (Parkinson's disease), a neurodegenerative disease, including Lou Gehrig, Huntington, and the like, but is not limited thereto.
  • the term “cellular therapeutic agent” refers to a medicinal product (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special manipulation from humans. Or through a series of actions such as proliferating and screening living autologous, allogeneic, or heterologous cells in vitro or otherwise altering the biological properties of a cell to restore tissue function. Means the drug used for the purpose.
  • treatment means any action that improves or benefits the condition of the disease by administration of the cell therapy agent.
  • the route of administration of the cell therapy composition of the present invention may be administered via any general route as long as it can reach the desired tissue.
  • Parenteral administration for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration may be, but is not limited thereto.
  • composition may be formulated in a suitable form with a pharmaceutical carrier generally used for cell therapy.
  • a pharmaceutical carrier generally used for cell therapy.
  • 'Pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not cause an allergic or similar reaction, such as gastrointestinal disorders, dizziness or the like, when administered to a human.
  • Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, carriers for parenteral administration such as aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • Other pharmaceutically acceptable carriers may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
  • composition may also be administered by any device in which the cell therapy agent can migrate to the target cell.
  • the cell therapy composition of the present invention may include a therapeutically effective amount of cell therapy for the treatment of a disease.
  • ⁇ therapeutically effective amount '' is meant the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician. This includes the amount that induces alleviation of the symptoms of the disease or disorder being treated.
  • the optimal cell therapy content can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of other components contained in the composition, the type of formulation, and the age, weight, general health, sex and diet of the patient. It can be adjusted according to various factors including the time of administration, the route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously. In consideration of all the above factors, it is important to include an amount that can achieve the maximum effect in a minimum amount without side effects.
  • the daily dose of nerve cells of the present invention is 1.0 ⁇ 10 4 to 1.0 ⁇ 10 10 cells / kg body weight, preferably 1.0 ⁇ 10 5 to 1.0 ⁇ 10 9 cells / kg body weight administered once or divided into several can do.
  • the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore the dosage should It does not limit the scope of the present invention in terms of aspects.
  • composition comprising the cell therapy of the present invention as an active ingredient in the treatment method of the present invention is rectal, intravenous (intravenous therapy, iv), intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular Or via the intradermal route.
  • the present invention provides a method of treatment comprising administering to a mammal a therapeutically effective amount of said cell therapy composition of the invention.
  • mammal refers to a mammal that is the subject of treatment, observation or experiment, preferably human.
  • Human epidermal fibroblasts Human foreskin fibroblasts, SCC058, Millipore
  • Human foreskin fibroblasts, SCC058, Millipore were cultured in high-glucose DMEM (Welgene) added with 10% FBS, 1% penicillin and streptomycin (Welgene).
  • HFF Human epidermal fibroblasts
  • the CRFPGTJ combination was 5 ⁇ M CHIR99021 (C), 5 ⁇ M RepSox (R), 50 ⁇ M Forskolin (F), 3.5 ⁇ M 2,4-Pyridinedicarboxylic Acid (P), 3.5 ⁇ M Garcinol (G), 3.5 ⁇ M Trichostatin A ( T) and 1 ⁇ M (+)-JQ-1 (J) (Medchemexpress). Fibroblasts were induction phase for 3 days.
  • Human fibroblast-derived neurons were harvested on day 5 or 10 after induction or maturation, respectively, washed twice with 1X PBS (Welgene) and then fixed for 10 minutes with 4% paraformaldehyde (Sigma-Aldrich).
  • PBS containing 0.25% Triton X-100 USB Corporation was treated at 22 ° C. for 10 minutes and then washed twice with PBS for 5 minutes each. Block for 60 minutes with blocking solution containing 1% BSA (Amresco), 22.52 mg / ml glycine (Affymetrix), and 0.1% Tween 20 (Affymetrix) in PBS and overnight at 4 ° C. with appropriate primary antibody diluted with blocking solution. Stained.
  • Antibodies used were rabbit anti-Tubulin ⁇ -3 (TUBB3, Biolegend, 802001, dilution 1: 100), rabbit Doublecortin (ab18723, Abcam, dilution 1: 100) and mouse monoclonal NeuN, clone A60 (MAB377, Millipore, dilution) 1: 100). After incubation with primary antibody, cells were washed three times in PBST and diluted 1: 100 to Alexa-488-conjugated goat secondary anti-mouse antibody (A11001, Invitrogen) or Alexa-563-conjugated goat secondary anti-rabbit antibody ( A21428, Invitrogen) was stirred at room temperature for 2 hours.
  • Alexa-488-conjugated goat secondary anti-mouse antibody A11001, Invitrogen
  • Alexa-563-conjugated goat secondary anti-rabbit antibody A21428, Invitrogen
  • Example 1 Based on the experimental results of Example 1 above, the conditions for promoting neuronal growth and reducing neuronal death of differentiated neurons were further investigated, and when ROCK inhibitor (Y-27632) was added It was confirmed that cell survival and maturation are promoted. Therefore, a combination of eight compounds (CRFPGTJY) adding Y27632 10 ⁇ M to the medium composition of Example 1 was used as an efficient neuronal differentiation condition.
  • ROCK inhibitor Y-27632
  • FIG. 4 In order to further investigate the role of the eight compounds and to further verify the necessity of each substance, neuron differentiation experiments were conducted under the condition that the eight compounds were removed one by one, and the results are shown in FIG. 4. As can be seen in Figure 4, components shown in red letters are histone deacetylase inhibitors; GSK inhibitor (Glycogen synthease kinase inhibitor) ,; ALK-5 inhibitors (ALK-5 kinase inhibitors) ,; cAMP signaling activator; And bromodomain inhibitors are essential for neuronal differentiation. Histone demethylase inhibitors, histone acetyltransferase inhibitors, and ROCK inhibitors are indicated in blue. It was determined that it is not essential.
  • the present inventors confirmed that the differentiation occurs even under the condition of Y, that is, a ROCK inhibitor, but the efficiency of differentiation is reduced in terms of neuronal cell differentiation time and number of differentiated neurons. Therefore, it was confirmed that neuronal differentiation of somatic cells was induced with the highest efficiency by the composition consisting of six compounds.
  • FIG. 5 is a schematic diagram illustrating the differentiation process of human epidermal fibroblasts into neurons during chemical treatment of the present invention in accordance with Example 3 of the present invention.
  • FIG. 6 After treating the neuronal differentiation medium composition of the present invention to somatic cells, the degree of cell conversion was observed with time, and the results are summarized in FIG. 6.
  • Figure 6 As can be seen in Figure 6, as a result of treating the neuronal differentiation medium composition according to the present invention to the fibroblasts can be seen that neuronal differentiation occurs within only 6 hours, that synapses are formed in 4 days of aging Observation was possible (white arrow in the lower photo of Figure 6).
  • Figure 7 is a result of analyzing the cell characteristics after the conversion to the neurons of the fibroblasts treated using the neuronal differentiation medium composition of Example 3 with a plurality of markers. As can be seen in Figure 7, the neurons differentiated using the neuronal cell differentiation medium composition of the present invention can be seen that the characteristics of the neurons well even though the differentiation time is very short.
  • Example 4 when applying the composition of Example 4, it was confirmed that the differentiation of neurons occurs more quickly under the same conditions than the composition of Example 3. That is, in the composition of Example 3 of Table 2, it took 6 hours for the fibroblasts to differentiate into neurons, but the experiments with the composition of Example 4 confirmed that the fibroblasts were converted into neurons in 3 hours. In order to confirm which of the replaced components have such an effect, the present inventors replaced the six components one by one and observed the results. As a result, it was confirmed that the ROCK inhibitor component had the greatest influence on the rate of neuronal differentiation among the neuronal cell differentiation media compositions. Table 3 below summarizes the composition of Example 5 in which only the ROCK inhibitor was changed to Y27632 under the conditions of Examples 4 and 4.
  • FIG. 9 (A) shows the results obtained after 3 hours of application of the composition of Example 4 to fibroblasts
  • FIG. 9 (B) shows the results observed after 6 hours of application of the composition of Example 5.
  • FIG. 9 (A) shows the results obtained after 3 hours of application of the composition of Example 4 to fibroblasts
  • FIG. 9 (B) shows the results observed after 6 hours of application of the composition of Example 5.
  • FIG. 9 As can be seen in Figure 9, the same ROCK inhibitor was used, but when using Y-33075 it was found that the differentiation of neurons is completed in 3 hours.
  • Mouse embryo fibroblasts were isolated from C57BL.6 mouse embryos. In addition, the head, spinal cord and internal organs were carefully removed from mouse embryos to eliminate the possibility of neuronal cell contamination. Single cell suspensions were prepared by cutting the remaining tissue with 0.25% trypsin-EDTA (GIBCO) and trypsinizing. MEFs were then incubated in high glucose DMEM (Welgene) supplemented with 10% FBS and 1% penicillin and streptomycin (Welgene).
  • MEF cells were seeded at a seeding density of 40000 cells / well in a 24-well culture dish, and 18 hours after sowing, the induction medium containing the neuronal differentiation medium composition of the present invention (Neurobasal with 1% B27 added) ) was added.
  • the chemical cocktail composition used the composition of Example 4.
  • FIG. 10 is a micrograph of neurons made by directly differentiating mouse embryonic fibroblasts into neurons using the media composition for neuronal cell differentiation of the present invention.
  • the neuronal differentiation medium composition of the present invention was found to act on the somatic cells of mice as well as human somatic cells to differentiate into neurons.

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Abstract

La présente invention concerne une composition de milieu pour la différenciation neuronale et un procédé de différenciation de cellules somatiques en neurones en utilisant la composition de milieu. Plus particulièrement, la présente invention concerne une composition de milieu pour la différenciation neuronale qui convertit directement les cellules somatiques en neurones en utilisant uniquement une substance de bas poids moléculaire sans introduire de gène, et un procédé de différenciation de cellules somatiques en neurones en utilisant la composition de milieu. Les présents inventeurs ont mis au point une composition de milieu pour la différenciation neuronale composée d'une substance de bas poids moléculaire qui est prévue pour induire plus efficacement les neurones parmi les substances de bas poids moléculaire telles que différentes substances d'induction de différenciation, et ont tenté une conversion directe. En résultat, il était confirmé que lorsque les neurones étaient induits à partir de cellules somatiques, les neurones étaient directement convertis sous un rendement élevé sur une très courte période allant de 3 heures à 6 heures. Par conséquent, la composition de milieu de la présente invention peut être efficacement utilisée pour le développement d'agents thérapeutiques cellulaires plus sûrs en utilisant des cellules auto-somatiques sans manipulation de gène.
PCT/KR2018/002520 2017-03-02 2018-03-02 Composition de milieu pour la différenciation neuronale et procédé de différenciation de cellules somatiques en neurones en utilisant la même composition de milieu Ceased WO2018160028A1 (fr)

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CN110972481A (zh) * 2018-09-17 2020-04-07 诺未科技（北京）有限公司 一种组合物及其用途
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CN112805015A (zh) * 2018-10-12 2021-05-14 生命技术公司 造血干细胞和造血祖细胞扩增系统
CN112055746A (zh) * 2020-05-19 2020-12-08 武汉睿健医药科技有限公司 一种感觉神经元细胞的无血清诱导方法
CN113278585A (zh) * 2021-03-26 2021-08-20 广西大学 一种高效诱导人体细胞重编程为神经元细胞的方法
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