A METHOD FOR DIRECT REPROGRAMMING OF URINE CELLS INTO NEURAL STEM CELLS USING SYNTHETIC MRNA

Abstract

A method for inducing reprogramming of neural stem cells from urine cells by introducing mRNAs of reprogramming factors Oct4, Sox2, Klf4, and Glis1 is disclosed. A composition for the prevention or treatment of neurological damage diseases with the neural stem cells induced by the method as an active ingredient is disclosed.

Claims

1. A composition for direct reprogramming urine cells into neural stem cells, the composition comprising: (i) an Oct4 protein or a nucleic acid encoding the Oct4 protein; (ii) a Sox2 protein or a nucleic acid encoding the Sox2 protein; (iii) a Klf4 protein or a nucleic acid encoding the Klf4 protein; and (iv) a Glis1 protein or a nucleic acid encoding the Glis1 protein.

2. The composition of claim 1, wherein the urine cells are somatic cells derived from urine.

3. The composition of claim 1, wherein the nucleic acids encoding the Oct4, Sox2, Klf4, and Glis1 proteins are synthetic mRNA.

4. A method for direct reprogramming of urine cells into neural stem cells, the method comprising: (a) isolating urine cells from urine and culturing the urine cells; (b) introducing the composition of claim 1 into the cultured urine cells; (c) inducing direct reprogramming into neural stem cells by culturing urine cells into which the composition has been introduced in a neural stem cell-inducing medium; and (d) selecting a neural stem cell line having characteristics similar to neural stem cell from the cells in which direct reprogramming has been induced by the neural stem cell-inducing medium.

5. The method of claim 4, wherein in step (b), the introduction of synthetic mRNA encoding the Oct4, Sox2, Klf4, and Glis1 is introducing the synthetic mRNA using electroporation.

6. The method of claim 4, wherein in step (c), the culture medium is obtained by mixing a DMEM/F12 medium and a neurobasal medium at a volume ratio of 1:1 and adding 1×N2, 1×B27, human LIF, a TGF-beta inhibitor, and a GSK3-beta inhibitor to the medium mixture.

7. The method of claim 6, wherein the culture medium further comprises one or more of a Shh agonist, an adenylyl cyclase activator, a histone deacetylase inhibitor, and ascorbic acid 2-phosphate.

8. The method of claim 7, wherein the culture in the culture medium is performed under low oxygen (O.sub.2 hypoxia) conditions.

9. A pharmaceutical composition for the prevention or treatment of neurological damage diseases comprising the directly reprogrammed neural stem cells prepared by the method of claim 4 as an active ingredient.

10. The pharmaceutical composition of claim 9, wherein the neurological damage disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, an ischemic brain disease (stroke), a demyelinating disease, multiple sclerosis, epilepsy, a degenerative neurological disease, and spinal cord injury.

11. A method for the prevention or treatment of neurological damage diseases comprising administering the directly reprogrammed neural stem cells prepared by the method of claim 4 as an active ingredient.

12. The method of claim 11, wherein the neurological damage disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, an ischemic brain disease (stroke), a demyelinating disease, multiple sclerosis, epilepsy, a degenerative neurological disease, and spinal cord injury.

Description

DESCRIPTION OF DRAWINGS

[0045] FIG. 1 is a schematic view of a T7-VEE-OKS-iG vector for preparing synthetic mRNAs of OCT4, KLF4, SOX2, and GLIS1 genes.

[0046] FIG. 2 illustrates the results confirming whether synthetic mRNAs of OCT4, KLF4, SOX2, and GLIS1 genes to which GFP (fluorescent gene) is bound are introduced into urine cells through FACS analysis.

[0047] FIG. 3 illustrates the entire process of obtaining a colony of neural stem cells derived from urine cells using a direct reprogramming technique.

[0048] FIG. 4 illustrates the results of confirming whether induced neural stem cells express neural stem cell marker genes such as SOX1, SOX2, PAX6, and PLZF through mRNA level analysis by RT-PCR using neural stem cells derived from H9 embryonic stem cells as a positive control.

[0049] FIG. 5 illustrates the results of confirming whether induced neural stem cells express neural stem cell marker genes such as SOX1, NESTIN, SSEA1, SOX2, PAX6, and PLZF at the protein level through immunoassay.

[0050] FIG. 6 illustrates the results of confirming whether induced neural stem cells have the ability to differentiate through immunostaining of Ki67.

[0051] FIG. 7 illustrates the results of confirming whether induced neural stem cells express neural stem cell marker genes such as SOX1, SOX2, PLZF, and PAX6 compared to neural stem cells derived from H9 embryonic stem cells through quantitative analysis of mRNA by qRT-PCR.

[0052] FIG. 8 illustrates the results of confirming whether induced neural stem cells express developmental forebrain, midbrain, hindbrain, and spinal cord-specific marker genes through mRNA level analysis by RT-PCR.

[0053] FIGS. 9 and 10 illustrate the results of confirming whether the induced neural stem cells exhibit mRNA and long non-coding-RNA (Inc-RNA) expression patterns more similar to those of neural stem cells derived from H9 embryonic stem cells than original urine-derived cells through total RNA sequencing at the global gene expression level.

[0054] FIG. 11 illustrates the results of confirming whether synthetic mRNA introduced into neural stem cells remains in a host and whether the synthetic mRNA is integrated into a host genome, through RT-PCR and genomic DNA PCR analysis of a VEE gene.

[0055] FIG. 12 illustrates the results of confirming whether induced neural stem cells are derived from human urine cells through STR analysis.

[0056] FIG. 13 illustrates the results of confirming whether induced neural stem cells preserve normal chromosomes through karyotype analysis.

[0057] FIG. 14 illustrates the results of confirming the expression of the nerve cell gene markers TUJ1 and MAP2 by an immuno-screening method in order to verify the ability of the induced neural stem cells to differentiate into nerve cells.

[0058] FIG. 15 illustrates the results of confirming through immunostaining that induced neural stem cells can differentiate into GABA nerve cells, motor nerve cells, and dopamine nerve cells.

[0059] FIG. 16 illustrates the results of confirming the expression of S100beta and GFAP, which are markers for verifying the ability of induced neural stem cells to differentiate into astrocytes, which are glial cells, by an immuno-screening method.

[0060] FIG. 17 illustrates the results of confirming the expression of PDGFR, OLIG2, and 04, which are markers for verifying the ability of induced neural stem cells to differentiate into oligodendrocytes, which are glial cells, by an immuno-screening method.

[0061] FIG. 18 illustrates the results of confirming the relative mRNA expression levels of OCT4, REX4, and NANOG by date in the process of inducing from urine cells to neural stem cells.

[0062] FIG. 19 illustrates the results of confirming whether the marker genes of H9 embryonic stem cells and induced neural stem cells at the pluripotent stage are expressed.

[0063] FIG. 20 illustrates the results of comparing the conversion rate when urine-derived cells are induced into neural stem cells under low oxygen conditions (5% hypoxia) using a basic condition (21% 02) as a control.

[0064] FIG. 21 illustrates the results of confirming that the induction efficiency of neural stem cells is increased when one or more of a Shh agonist, an adenylyl cyclase activator, a histone deacetylase inhibitor, and ascorbic acid 2-phosphate are further included in a medium obtained by mixing a DMEMF12 medium and a neurobasal medium at a volume ratio of 1:1, and adding 1×N2, 1×B27, human LIF, a TGF-beta inhibitor, and a GSK3-beta inhibitor to the medium mixture. It was confirmed that the neural stem cell conversion rate was the best in a medium supplemented with all of the Shh agonist, the adenylyl cyclase activator, the histone deacetylase inhibitor, and ascorbic acid 2-phosphate.

MODES OF THE INVENTION

[0065] Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are provided only to more easily understand the present invention, and the present invention is not limited to the following Examples.

Example 1: Isolation of Urine Cells from Urine

[0066] Based on a technique developed by Sutherland and Bain in the United Kingdom in 1972, the specifics are as follows. First, the urine donated by a donor was centrifuged at 1000 g for 10 minutes. After the supernatant was removed, the pellet remaining in the lower layer was diluted with 20 ml of a PBS solution containing 1% Penicillin/Streptomycin/Amphotericin B antibiotics. The diluted PBS+pellet solution was then centrifuged at 1000 g for 10 minutes. After the supernatant was removed again, the pellet remaining in the lower layer was diluted with 1 ml of a basal medium (a medium including 1% Penicillin/Streptomycin Amphotericin B antibiotics, 1% L-glutamine, and 10% FBS based on DMEMF12) and seeded onto a 12-well cell culture dish coated with gelatin. Then, after cells were cultured by adding 1 ml of the basal medium for 3 days, the cells were cultured by changing the medium to a growth medium (a medium including 1% Penicillin/Streptomycin antibiotics, 1% L-glutamine, 5% FBS, 10 ng/ml bFGF, and 10 g/ml EGF based on a medium obtained by mixing DMEM and REGM at 1:1).

Example 2: Introduction of Reprogramming Factor into Urine Cells Using Electroporation

[0067] A method of introducing synthetic mRNA into urine-derived cells cultured in Example 1 by electroporation is specifically as follows. Synthetic mRNA is synthesized through a typical in vitro transcription kit (RiboMAX⊚ Large Scale RNA Production Systems, Promega), and DNA, which becomes a backbone of mRNA to be synthesized, is T7-VEE-OKS-iG (Steven Dowdy, Addgene), which includes genes of OCT4, KLF4, SOX2, and GLIS1 (FIG. 1). As the sequences of the OCT4, KLF4, SOX2, and GLIS1 genes, those disclosed in Nature. 2011 Jun. 8; 474(7350): 225-9. were used, and for the corresponding gene sequence and mRNA application method, Cell Stem Cell. 2013 Aug. 1; 13 (2): 246-54 and U.S. Pat. No. 9,862,930 B2 were referenced.

[0068] The synthesized mRNA was introduced by treating 1×10.sup.6 urine-derived cells pretreated with 0.2 ug/ml of B18R protein with 0.5 μg of the synthesized mRNA 3 times by electroporation at 1600 V and 10 ms for 1 hour. The introduced urine-derived cells were cultured in a growth medium including 0.2 ug/ml of B18R protein for 2 days.

[0069] The present inventors verified the efficiency of human urine-derived cells expressing a GPF fluorescence protein through FACS analysis by simultaneously introducing the synthetic mRNA including the GFP fluorescence protein in order to visualize, determine, and verify the efficiency of introduction through electroporation. By confirming through the verification that the human urine-derived cell introduction efficiency of synthetic mRNA by electroporation reached 72.2% around 48 hours after treatment, it was determined that it was sufficient to perform a neural stem cell induction experiment which is the next step (FIG. 2).

Example 3: Induction of Reprogrammed Neural Stem Cells Derived from Urine

[0070] After mRNA-introduced urine-derived cells were seeded in a cell culture dish coated with Matrigel™, the cells were cultured in a neural stem cell-inducing medium supplemented with 0.5 uM purmorphamine (Shh agonist), 10 uM forskolin (adenylyl cyclase activator), 100 uM sodium butyrate (histone deacetylase inhibitor), and 64 ug/ml ascorbic acid 2-phosphate based on a neural stem cell-inducing medium (a medium obtained by mixing a DMEM/F12 medium and a neurobasal (Gibco) medium at 1:1 and adding 1×N2 (Gibco), 1×B27 (Gibco), 10 ng/ml human LIF, 2 uM SB431542 (TGF-beta inhibitor), and 3 uM CHIR99021 (GSK3-beta inhibitor) to the medium mixture) for 7 to 10 days. The development of neural stem cell colonies could be confirmed when the induction was completed (FIG. 3).

[0071] Further, effects on the induction efficiency of neural stem cells were confirmed when one or more of a Shh agonist, an adenylyl cyclase activator, a histone deacetylase inhibitor, and ascorbic acid 2-phosphate were further included in a medium further supplemented with 1×N2, 1×B27, human LIF, a TGF-beta inhibitor and a GSK3-beta inhibitor. It was confirmed that the neural stem cell conversion rate was the best in a medium supplemented with all of the Shh agonist, the adenylyl cyclase activator, the histone deacetylase inhibitor, and ascorbic acid 2-phosphate (FIG. 21).

[0072] The induced neural stem cell colonies were collected, and cultured by placing the neural stem cell medium in a cell culture dish coated with Matrigel. The induced neural stem cells could then be subcultured using a 0.5 mM EDTA solution or an Accutase solution, and then experiments of verifying the various properties of the induced neural stem cells in the following examples were conducted.

Example 4: Analysis of Molecular Biological Properties of Induced Neural Stem Cells

[0073] Experiments were conducted to verify the molecular biological properties of neural stem cells derived from the present invention.

[0074] <4-1>

[0075] It was confirmed that neural stem cells induced through mRNA level analysis by RT-PCR using neural stem cells derived from H9 embryonic stem cells as a positive control expressed neural stem cell marker genes such as SOX1, SOX2, PAX6, and PLZF (FIG. 4). The sequences of primers used in the present experiment are as follows: SOX1 (forward-ACACTTGAAGCCCAGATGG; SEQ ID NO: 1, reverse-ATAGGCTCACTTTTGGACGG; SEQ ID NO: 2), SOX2(forward-TCAGGAGTTGTCAAGGCAGAGA; SEQ ID NO: 3, reverse-CCGCCGCCGATGATTGTTATTA; SEQ ID NO: 4), PAX6 (forward-GGCTCAAATGCGACTTCAG; SEQ ID NO: 5, reverse-CCCTTCGATTAGAAAACCATACC; SEQ ID NO: 6), PLZF (forward-TATACAGCCACGCTGCAAGCCA; SEQ ID NO: 7, reverse-TGGTCTCCAGCATCTTCAGGCA; SEQ ID NO: 8).

[0076] <4-2>

[0077] It was also confirmed through a protein level analysis using immunostaining that induced neural stem cells expressed neural stem cell marker genes such as SOX1, NESTIN, SSEA1, SOX2, PAX6, and PLZF (FIG. 5). The antibodies used in the present experiment are as follows: SOX1 (AF3369, R&D Systems), NESTIN (MAB5326, Millipore), SSEA1 (MAB4301, Millipore), SOX2 (SC-20088, Santacruz), PAX6 (PAX6, DSHB), PLZF (AF2944, R&D Systems).

[0078] <4-3>

[0079] It was confirmed through immunostaining of Ki67 that neural stem cells induced have a cell division ability (FIG. 6). The antibodies used in the present experiment are as follows: Ki67 (Ab9260, Millipore).

[0080] <4-4>

[0081] It was also confirmed through quantitative analysis of mRNA by qRT-PCR that induced neural stem cells expressed neural stem cell marker genes such as SOX1, SOX2, PLZF, and PAX6, compared to neural stem cells derived from H9 embryonic stem cells (FIG. 7).

[0082] <4-5>

[0083] It was also confirmed through mRNA level analysis by RT-PCR that induced neural stem cells expressed developmental forebrain, midbrain, hindbrain, and spinal cord-specific marker genes (FIG. 8). The sequences of primers used in the present experiment are as follows: EMX1 (forward-CAGGCCCAGGTAGTTCAATGGG; SEQ ID NO 9, reverse-GCTCAGCCTTAAGCCCTGTCTC; SEQ ID NO: 10), FOXG1 (forward-ACCCGTCAATGACTTCGCAGAG; SEQ ID NO: 11, reverse-AGGGTTGGAAGAAGACCCCTGA; SEQ ID NO: 12), OTX1 (forward-CTCAAACAACCCCCATACGGCA; SEQ ID NO: 13, reverse-GGTAGCGAGTCTTGGCGAAGAG; SEQ ID NO: 14), OTX2 (forward-TTCAGGGCTGTGTGAATTGTGTGA; SEQ ID NO: 15, reverse-CAGAGGTGGAGTTCAAGGTTGCAT; SEQ ID NO: 16), EN1 (forward-GAGTTCCAGGCAAACCGCTACA; SEQ ID NO: 17, reverse-GTTGTACAGTCCCTGGGCCATG; SEQ ID NO: 18), GBX2 (forward-CGGTTAGCAGCCACCTTTCCAT; SEQ ID NO: 19, reverse-GACGTGCTTCACATGGCTCAGA; SEQ ID NO: 20), HOXB2 (forward-GAGACCCAGGAGCCAAAAGC; SEQ ID NO: 21, reverse-GAAGGAGACGTGGCGGATTG; SEQ ID NO: 22), HOXB4 (forward-CTGAGGGCCAGAATGACTGCTC; SEQ ID NO: 23, reverse-CAGAACTCAACTGGCCCCTCAC; SEQ ID NO: 24).

[0084] <4-6>

[0085] It was confirmed through total RNA sequencing at the global gene expression level that the induced neural stem cells exhibited mRNA and Inc-RNA expression patterns more similar to those of neural stem cells derived from H9 embryonic stem cells than original urine-derived cells (FIGS. 9 and 10).

[0086] <4-7>

[0087] It was confirmed through RT-PCR and genomic DNA PCR analysis of a VEE gene that synthetic mRNA introduced into neural stem cells no longer remained in a host, and was not integrated into a host genome (FIG. 11). The sequences of primers used in the present experiment are as follows: VEE (forward-ACGAAGGGCAAGTCGCTGTT; SEQ ID NO: 25, reverse-TTTCGTCGGCCCAGTTGGTA; SEQ ID NO: 26).

[0088] <4-8>

[0089] It was confirmed through STR analysis that human urine cells and induced neural stem cells were derived from the same individual (FIG. 12).

[0090] <4-9>

[0091] It was confirmed through karyotype analysis that induced neural stem cells preserved normal chromosomes (FIG. 13).

Example 5: Analysis of Ability of Induced Neural Stem Cells to Differentiate

[0092] <5-1> Analysis of Ability to Differentiate into Nerve Cells

[0093] It was confirmed through immunostaining that neural cell marker genes TUJ1 and MAP2 were expressed when the induced neural stem cells were differentiated into nerve cells (FIG. 14). The nerve cell differentiation method is as follows. Neural stem cells are analyzed after being cultured in a nerve cell differentiation medium obtained by adding 1×N2, 1×B27, 300 ng/ml cAMP, 0.2 mM vitamin C, 10 ng/ml BDNF, and 10 ng/ml GDNF to a DMEM/F12 medium for 14 days. The antibodies used in the present experiment are as follows. Tuj1 (801202, BioLegend), MAP2 (AB5622, Millipore)

[0094] Further, it could be confirmed that the induced neural stem cells could differentiate into GABA nerve cells, motor nerve cells, and dopamine nerve cells by staining GABA, HB8, and TH, respectively through immunostaining (FIG. 15). The antibodies used in the present experiment are as follows: GABA (A2052, Sigma), HB9 (81.5C10, DSHB), TH (AB152, Millipore).

[0095] <5-2> Analysis of Ability to Differentiate into Glial Cells

[0096] It was confirmed that the induced neural stem cells could differentiate into astrocytes, which are one of the glial cells, through S100beta and GFAP staining through immunostaining (FIG. 16). The astrocyte differentiation method is as follows. Induced neural stem cells are cultured in an astrocyte differentiation medium supplemented with 2.5% FBS or 20 ng/ml CNTF, and 10 ng/ml BMP4 in a medium containing 1×N2 and 1×B27 in a DMEM/F12 medium for 14 days or more, and then analyzed. The antibodies used in the present experiment are as follows: S100beta (S2535, Sigma), GFAP (SAB4501162, Sigma).

[0097] It was confirmed that the induced neural stem cells could differentiate into oligodendrocytes, which are one of the glial cells, through PDGFR, OLIG2, and O4 staining through immunostaining (FIG. 17). The oligodendrocyte differentiation method is as follows. After induced neural stem cells are cultured in a medium obtained by adding 1×N2, 1×B27, 25 ug/ml insulin, 1 uM Shh agonist, and 100 nM retinoic acid to a DMEM/F12 medium for 5 days or more, the cells are cultured in a medium obtained by adding 1×N2, 1×B27, 25 ug/ml insulin, 100 ng/ml biotin, 60 ng/ml T3, 10 ng/ml PDGF-AA, 10 ng/ml IGF-1, 5 ng/ml HGF, 10 ng/ml NT-3, and 1 uM cAMP to a DMEM/F12 medium for 7 days or more, and then analyzed. The antibodies used in the present experiment are as follows: PDGFR (SC338, Santa Cruz), OLIG2 (AB9610, Millipore), 04 (MAB345, Millipore).

Example 6: Verification and Analysis of Neural Stem Cell Induction Method that does not go Through Pluripotent Stage

[0098] It is known that the combination of the reprogramming factors OCT4, SOX2, KLF4, and GLIS1 genes used in the present invention can establish induced pluripotent stem cells under specific conditions (U.S. patent Ser. No. 09/862,930).

[0099] However, it was verified that the neural stem cells induced in the present invention do not go through the pluripotent stage for the following reasons.

[0100] First, a neural stem cell-inducing medium excluding bFGF, which is essential for establishing human-induced pluripotent stem cells, and a B18R protein, which is necessary for the expression of mRNA of an endogenous gene, was used, so that it is not possible to establish human-induced pluripotent stem cells according to the research results reported to date.

[0101] Second, the time when neural stem cells derived from the present invention are formed is around day 8 after the induction process according to the protocol (FIG. 3), and known human-induced pluripotent stem cells require a period of 3 weeks or more after the induction process to form, so that it is too short of a time to have gone through the pluripotent stage.

[0102] Third, when viewed by a date-based qRT-PCR analysis, the expression of the pluripotent stage marker genes OCT4, REX1 and NANOG maintained low expression from day 0 to day 12 (FIG. 18). The sequences of primers used in the present experiment are as follows. OCT4 (forward-GACAGGGGGAGGGGAGGAGCTAGG; SEQ ID NO: 27, reverse-CTTCCCTCCAACCAGTTGCCCCAAAC; SEQ ID NO: 28), REX1 (forward-CTGAAGAAACGGGCAAAGAC; SEQ ID NO: 29, reverse-GAACATTCAAGGGAGCTTGC; SEQ ID NO: 30), NANOG (forward-CAGCCCTGATTCTTCCACCAGTCCC; SEQ ID NO: 31, reverse-GGAAGGTTCCCAGTCGGGTTCACC; SEQ ID NO: 32).

[0103] Fourth, it was confirmed that when induced neural stem cells were verified through immunostaining, the expression of OCT4 and NANOG was not detected unlike the H9 embryonic stem cells (FIG. 19). The antibodies used in the present experiment are as follows: OCT4 (SC5279, Santa Cruz), NANOG (AF1997, R&D Systems).

[0104] Based on the points as described above, it could be confirmed that the method of the present invention could immediately induce urine cells into neural stem cells without going through the pluripotent stem cell stage.

Example 7: Experiment to Enhance Efficiency of Induction into Neural Stem Cells

[0105] Culture conditions capable of improving the efficiency of conversion to neural stem cells from urine cells, and the like were further confirmed.

[0106] Induction was performed by adjusting the oxygen concentration using general oxygen conditions (21% O.sub.2) as a control. Specifically, after a neural stem cell induction method was performed on 1×10.sup.5 urine-derived cells under basic conditions (21% O.sub.2) and low oxygen conditions (5% hypoxia), respectively, it was confirmed by counting the number of colonies positive for neural stem cell marker genes SOX1 and PLZF using immunostaining that an approximate 2-fold increase in conversion efficiency was exhibited under low oxygen conditions (FIG. 20).