WO2014148646A1 - 神経分化誘導用の多能性幹細胞 - Google Patents
神経分化誘導用の多能性幹細胞 Download PDFInfo
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- WO2014148646A1 WO2014148646A1 PCT/JP2014/058142 JP2014058142W WO2014148646A1 WO 2014148646 A1 WO2014148646 A1 WO 2014148646A1 JP 2014058142 W JP2014058142 W JP 2014058142W WO 2014148646 A1 WO2014148646 A1 WO 2014148646A1
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Definitions
- the present invention relates to a method for producing motor neurons or neurons from pluripotent stem cells. Furthermore, the present invention relates to a pluripotent stem cell that can be rapidly differentiated into a motor nerve cell or a nerve cell by drug treatment.
- Non-patent document 1 a method of inducing differentiation of cells of the nervous system from pluripotent stem cells is formed and differentiated in a serum-free medium (SFEB method) ( Non-patent document 1), a method of culturing embryonic stem cells on stromal cells and differentiating them (SDIA method) (non-patent document 2), a method of adding a drug to Matrigel and culturing (non-patent document 3), cytokines
- a method of fateing a cell by a signal from the outside of the cell such as a method using a low molecular weight compound as an alternative to the above (Patent Document 1), was mainly used.
- Non-Patent Document 4 three types of (motor) neural lineage-specific transcription factors (Ngn2, Lhx3, and Isl1) are applied to neural progenitor cells prepared from human embryonic stem cells or induced pluripotent stem cells by the SFEB method. It has been reported that motor neurons can be obtained 11 days after introduction and expression.
- Non-Patent Document 5 it is shown that a nerve cell can be obtained after about 6 days by introducing and expressing three types of neuronal lineage-specific transcription factors (Ascl1, Brn2, and Mytl1) in human embryonic stem cells. Reporting.
- the nerve cell induced to differentiate by the expression of the foreign gene is distinguished from the nerve cell induced to differentiate by the signal from the outside of the cell as “induced nerve cell (abbreviated as induced neuron, iN)”.
- induced nerve cell abbreviated as induced neuron, iN
- iMN induced motor neuron
- iMN and iN can be obtained in a very short period of time compared to conventional methods for producing motor neurons / neurons, but the differentiation synchrony is not sufficient, and the original (especially disease) There is a harsh opinion as to how far the motor / nerve cell-derived properties (in the patient) are reproduced.
- motor neurons / neurons that have sufficient properties of the original motor neurons / neurons and that can also reproduce the pathology of disease patients are rapidly and synchronized from pluripotent stem cells.
- a method of manufacturing was eagerly desired.
- the present invention has been made in view of the above-mentioned problems of the prior art, and a motor nerve cell or a nerve cell that well reproduces the original (particularly, diseased patient) motor nerve cell / nerve cell properties is pluripotent.
- the object is to provide a method for producing rapidly and synchronously from sex stem cells. It is another object of the present invention to provide a pluripotent stem cell that can be rapidly and synchronously differentiated by a drug treatment into a motor neuron or a neuron that well reproduces the nature of the original motor neuron / nerve cell. Yes.
- the present inventors have introduced Lhx3, Ngn2, and Isl1 genes into pluripotent stem cells and expressed them, rather than expressing the three genes in neural progenitor cells. It was also found that differentiation can be induced into motor neurons in a short period of time and synchronously. In addition, motor neurons prepared from iPS cells derived from amyotrophic lateral sclerosis (ALS) patients using this method not only spontaneously lead to cell death, but the cell death is effective with existing ALS therapeutics. It was confirmed that it was suppressed.
- ALS amyotrophic lateral sclerosis
- Ngn2 of the above three genes when only Ngn2 of the above three genes is introduced into a pluripotent stem cell and expressed, it is rapidly and synchronously differentiated into a neuron, and from an iPS cell derived from an Alzheimer's dementia patient, It was confirmed that the produced nerve cells produced A ⁇ characteristic of the disease.
- the present inventor has completed the present invention based on these findings.
- the present invention includes the following.
- a method for producing motor neurons from pluripotent stem cells comprising the following steps: (1) introducing a nucleic acid encoding Lhx3, Ngn2, and Isl1 into a pluripotent stem cell; (2) maintaining the expression of Lhx3, Ngn2, and Isl1 for 3 days or more; A method for producing motor neurons from pluripotent stem cells, comprising (1)-(2) in the order.
- the transposon is a piggyBac transposon.
- step (2) is a step of expressing the nucleic acid with a drug-responsive promoter.
- step (3) is a step of expressing the nucleic acid with a drug-responsive promoter.
- drug-responsive promoter is a tetracycline-responsive promoter.
- nucleic acid is polycistronically expressed in Lhx3, Ngn2, and Isl1.
- nucleic acid is a nucleic acid in which a nucleic acid encoding Lhx3, Ngn2, and Isl1 is bound by a 2A sequence.
- the method further includes a step of introducing the cell obtained in the step (1) into the animal body, and contacting the drug corresponding to the drug-responsive promoter in the animal body. [6 ] -The method according to any one of [8]. [10] The method according to any one of [1] to [9], further comprising the step of inducing differentiation of the pluripotent stem cells into which the nucleic acid has been introduced into blood cells in the step (1).
- the method according to [10], wherein the blood cells are monocytes and / or macrophages.
- the pluripotent stem cells into which the nucleic acid has been introduced are co-cultured with bone marrow-derived stromal cells, and further stem cell factor, macrophage-colony stimulating factor, and interleukin-3
- a subject that is, a responder who has a therapeutic effect on an investigational drug for motor neurodegenerative disease or nerve injury, comprising the following steps (1) to (3): A method for identifying markers specific to subjects who were not observed (ie, non-responders); (1) a step of producing induced pluripotent stem cells from somatic cells isolated from the responders and non-responders, (2) A step of producing a motor neuron cell from the induced pluripotent stem cell obtained in the step (1) by the method according to any one of [1]-[8], (3) a step of measuring expression of a gene product of a motor neuron derived from a responder and a motor neuron derived from a non-responder obtained in the step (2), and (4) a motion derived from the responder In a neuronal cell, a gene product having a higher expression
- a method for selecting a subject for which a therapeutic agent is effective comprising the following steps (1) to (3); (1) a step of producing induced pluripotent stem cells from somatic cells isolated from a subject, (2) A step of producing a motor neuron cell from the induced pluripotent stem cell obtained in the step (1) by the method according to any one of [1]-[8], (3) A step of detecting a marker specific to the responder and / or non-responder identified by the method according to [15] in the motor neuron obtained in the step (2), (4) A corresponding therapeutic agent is effective for a subject derived from a motor neuron in which a marker specific to the responder is detected or a motor neuron in which a marker specific to the non-responder is not detected.
- a composition for treating motor neurodegenerative disease or nerve injury comprising the pluripotent stem cell according to any one of [17]-[24] as an active ingredient.
- the therapeutic composition according to [28], wherein the motor neurodegenerative disease is amyotrophic lateral sclerosis.
- a composition for treating motor neurodegenerative disease or nerve injury comprising the blood cell according to [26] or [27] as an active ingredient.
- the therapeutic composition according to [30], wherein the motor neurodegenerative disease is amyotrophic lateral sclerosis.
- a method for screening a therapeutic agent for amyotrophic lateral sclerosis comprising the following steps (1) to (5): (1) A step of producing a motor nerve cell by the method according to any one of [1-8, from an induced pluripotent stem cell produced from a somatic cell isolated from a patient with amyotrophic lateral sclerosis, (2) A step of bringing the motor neuron obtained in the step (1) into contact with a test substance, (3) culturing the motor neuron cell contacted with the test substance in the step (2) and the motor neuron cell not contacted with the test substance (that is, a control cell); (4) a step of measuring the number of motor neurons and / or neurite length obtained in the step (3), (5) A step of selecting, as a therapeutic agent for amyotrophic lateral sclerosis, a test substance in which the number of motor neurons and / or neurite length brought into contact with the test substance is higher than that of a control.
- a method for producing nerve cells from pluripotent stem cells comprising the steps of: (1) a step of introducing a nucleic acid encoding Ngn2 into a pluripotent stem cell using a transposon, (2) Inducing activation of the promoter and maintaining Ngn2 expression for 3 days or more, (1)-(2) in the order of production of nerve cells from pluripotent stem cells.
- the transposon is a piggyBac transposon.
- step (2) is a step of expressing the nucleic acid with a drug-responsive promoter.
- the drug-responsive promoter is a tetracycline-responsive promoter.
- the method further comprises a step of introducing the cell obtained in the step (1) into the animal body, and contacting the drug corresponding to the drug-responsive promoter in the animal body.
- step 34 ] -The method according to any one of [38].
- the pluripotent stem cell is a human induced pluripotent stem cell.
- the human induced pluripotent stem cell is an induced pluripotent stem cell produced from a somatic cell isolated from a patient with Alzheimer-type dementia.
- the human induced pluripotent stem cell is a human induced pluripotent stem cell having a mutation in the presenilin 1 gene.
- a subject who has a therapeutic effect (ie, responder) or no therapeutic effect on an investigational drug for Alzheimer's dementia comprising the following steps (1)-(3) A method for identifying markers specific to a subject (ie, non-responder); (1) a step of producing induced pluripotent stem cells from somatic cells isolated from the responders and non-responders, (2) A step of producing a nerve cell from the induced pluripotent stem cell obtained in the step (1) by the method according to any of [34]-[38], (3) a step of measuring the expression of the gene product of the neuron derived from the responder and the non-responder obtained in the step (2), and (4) the motor neuron derived from the responder.
- a gene product having a higher expression level than a non-responder is identified as a marker specific to the responder, or a gene product having a lower expression level than the non-responder is non-responsive in motor neurons derived from the responder. Identifying as a marker specific to a person.
- a method for selecting a subject for which a therapeutic agent is effective comprising the following steps (1) to (3); (1) a step of producing induced pluripotent stem cells from somatic cells isolated from a subject, (2) A step of producing a nerve cell from the induced pluripotent stem cell obtained in the step (1) by the method according to any of [34]-[38], (3) a step of detecting a marker specific to the responder and / or non-responder identified by the method according to [43] in the motor neuron obtained in the step (2), (4) Subjects for which a corresponding therapeutic agent is effective for a subject derived from a nerve cell in which a marker specific to the responder has been detected or a nerve cell in which a marker specific to the non-responder has not been detected The process of sorting as.
- Pluripotent stem cells in which a nucleic acid encoding exogenous Ngn2 is inserted into a chromosome by a transposon.
- the drug-responsive promoter is a tetracycline-responsive promoter.
- the pluripotent stem cell according to any one of [45] to [48], wherein the pluripotent stem cell is a human induced pluripotent stem cell.
- the pluripotent stem cell is a human induced pluripotent stem cell produced from a somatic cell isolated from a patient with Alzheimer-type dementia.
- the method according to [49], wherein the human induced pluripotent stem cell is a human induced pluripotent stem cell having a mutation in the presenilin 1 gene.
- a method for screening for a therapeutic agent for Alzheimer's dementia comprising the following steps (1) to (4): (1) A step of producing a nerve cell from an induced pluripotent stem cell produced from a somatic cell isolated from an Alzheimer-type dementia patient by the method according to any one of [34] to [38], (2) The step of bringing the nerve cell obtained in the step (1) into contact with a test substance, (3) culturing the nerve cells that have been contacted with the test substance in step (2) and the nerve cells that have not been contacted (that is, control cells), and measuring the content of A ⁇ 42 in the medium; (4) A test substance in which the content of A ⁇ 42 in the medium of nerve cells brought into contact with the test substance is lower than the content of A ⁇ 42 in the medium of the control cell is treated with Alzheimer's dementia The process of selecting as a medicine.
- the content of A ⁇ 40 in the medium is further measured, and
- the value obtained by dividing the content of A ⁇ 42 in the medium of nerve cells contacted with the test substance by the content of A ⁇ 40 ie, the content of A ⁇ 42 / the content of A ⁇ 40
- the present invention it is possible to produce a motor nerve cell or a nerve cell having good properties of an original nerve cell in a rapid and synchronized manner from a pluripotent stem cell.
- motor neurons and neurons obtained by this method from pluripotent stem cells derived from patients with neurodegenerative diseases can reproduce well the phenomena characteristic of the diseases, and thus are very suitable as screening systems for therapeutic agents for the diseases.
- the present invention provides pluripotent stem cells and / or blood cells that can be rapidly and synchronously differentiated into motor neurons or neurons by drug treatment or the like. Since the pluripotent stem cells and blood cells can be induced to differentiate into motor neurons or neurons in vivo, they can be suitably used as a composition (transplant therapy) for treating neurodegenerative diseases or nerve damage.
- FIG. 1A shows a schematic diagram of a tetracycline-inducible MNation factor expression vector (part) used in Example 1.
- Tet-O represents a tetracycline-responsive promoter having TRE, Lhx3, Ngn2, Isl1, and mCherry represent the coding sequence of each gene, and Frt represents the target sequence of the recombinant enzyme Flippase.
- Fig. 1B 293T cells transfected with the vector shown in Fig. 1A were cultured with DOX added (DOX +) / non-added (DOX-) for 24 hours, and expression of Lhx3, Ngn2, and Isl1 proteins was analyzed (Western blot) Indicates.
- DOX + DOX +
- DOX- non-added
- FIG. 1C shows the results of immunostaining mouse ES cells introduced with the vector shown in FIG. 1A and examining the expression of undifferentiated markers (Nanog and SSEA1).
- Fig. 1D Addition of DOX (DOX +) / No addition (DOX-) to mouse ES cells (right) and non-introduced cells (left) into which the vector described in Fig. 1A has been introduced, and observed mCherry fluorescence 18 hours later The result of having performed is shown.
- FIG. 1E Mouse ES cells transfected with the vector shown in Figure 1A were added with DOX (DOX +) / not added (DOX-), and the mRNA levels of Lhx3, Ngn2 and Isl1 were measured 18 hours later by real-time PCR. Results are shown.
- FIG. 1F shows a schematic diagram of a process for inducing motor neurons (iMN) from mouse ES cells.
- Figure 1G DOX was added to mouse ES cells into which the vector shown in Figure 1A had been introduced, and the results of immunostaining after 3 days (double immunostaining images of HB9 and ⁇ -III tubulin, MAP2, or ChAT) Show.
- FIG. 1E Mouse ES cells transfected with the vector shown in Figure 1A were added with DOX (DOX +) / not added (DOX-), and the mRNA levels of Lhx3, Ngn2 and Isl1 were measured 18 hours later by real-time PCR. Results are shown.
- FIG. 1H shows the results of measuring the mRNA amounts of HB9 and ChAT by real-time PCR with DOX added (DOX +) / non-added (DOX ⁇ ) to mouse ES cells into which the vector described in FIG. 1A has been introduced.
- FIG. 2A shows the results of examining the conditions of the medium for inducing differentiation of mouse ES cells introduced with the vector described in FIG. 1A into iMN.
- FIG. 2B shows a schematic diagram of the process for examining the DOX addition period.
- FIG. 2C FIG. 2B shows the result of analyzing the expression level of each gene by the real-time PCR method for the cells at Day 7 time point.
- Fig. 3A Phase contrast image of a co-culture obtained by inducing differentiation of mouse ES cells into which the vector described in Fig. 1A has been introduced into iMN under co-culture with C2C12 cells (ie, adding DOX) (left) And the immuno-staining image of (alpha) BTX / SV2 is shown.
- FIG. 3B shows the results of calcium imaging in the co-culture of FIG. 3A.
- FIG. 3C shows potential recording (current clamp) measured by the patch clamp method.
- FIG. 4A Transgenic mouse tissue having wild type human SOD1 gene (SOD1WT) or mutant human SOD1 gene (SOD1G93A) as a foreign gene, MEF prepared from the mouse, and human SOD1 gene in iPS cells established from the MEF The result of having analyzed presence by PCR method is shown.
- FIG. 4B shows a sequence result of the mutation site for the exogenous human SOD1 gene possessed by the established iPS cells.
- FIG. 4C shows the results of measuring the expression level of ES cell marker genes (Eras, Esg1, Rex1, Oct3 / 4, and Sox2) in the established mouse iPS cells by the PCR method.
- FIG. 4D shows the results of quantitative PCR measuring the expression levels of reprogramming factors (Oct3 / 4, Sox2, Klf4 and c-Myc) in the established mouse iPS cells.
- 3 shows immunostained images of three germ layer markers (ectodermal ( ⁇ -III tubulin), mesoderm ( ⁇ -smooth muscle actin) and endoderm ( ⁇ -fetoprotein)) induced in vitro from the established mouse iPS cells. .
- FIG. 6A is a schematic diagram showing a tetracycline-inducible MNation factor expression vector (part) used in Example 2-8.
- TR represents the target sequence of piggyBac transposase (ie, inverted terminal repeat sequence).
- FIG. 6B Undifferentiation markers (Nanog and SSEA1) of the mouse iPS cells (cells abbreviated as mouse WT-derived iPS cells and mouse G93A-derived iPS cells in the specification) into which the vector shown in FIG. 6A has been introduced. An immunostaining image is shown.
- FIG. 6C shows an immunostaining image for ⁇ -III tubulin, or HB9 and ChAT 3 days after adding DOX to mouse iPS cells into which the vector described in FIG.
- FIG. 6D shows immunostained images for misfolded SOD1, Neurofilament (NF-H) and DAPI 3 days after adding DOX to mouse iPS cells into which the vector described in FIG. 6A has been introduced.
- Fig. 6E Survival rate of motor neurons (in the absence of astrocytes) differentiated from mouse iPS cells into which the vector described in Fig. 6A has been introduced (ie, HB9 and ⁇ -III tubulin positive 6 days after addition of DOX) The number of cells / number of HB9 and ⁇ -III tubulin positive cells after 4 days).
- FIG. 6D shows immunostained images for misfolded SOD1, Neurofilament (NF-H) and DAPI 3 days after adding DOX to mouse iPS cells into which the vector described in FIG. 6A has been introduced.
- Fig. 6E Survival rate of motor neurons (in the absence of astrocytes) differentiated from mouse iPS cells into which the vector described in Fig. 6A has been introduced (ie
- FIG. 6F shows the ratio of the LDH amount in the medium 6 days after the addition of DOX to the LDH amount in the medium 4 days after the addition of DOX to mouse iPS cells (SOD1WT and SOD1G93A) into which the vector described in FIG. 6A has been introduced.
- FIG. 6G Survival rate of motor neurons differentiated (in the presence of astrocytes) from mouse iPS cells (SOD1WT and SOD1G93A) introduced with the vector described in FIG. 6A (ie, HB9 and ⁇ 6 days after induction) -III tubulin positive cell count / 4 HB9 and ⁇ -III tubulin positive cell count).
- FIG. 6H shows the average neurite length of motor neurons differentiated from mouse iPS cells (SOD1WT and SOD1G93A) introduced with the vector shown in FIG. 6A at 4 and 6 days after addition of DOX. .
- FIG. 7A shows the result of immunostaining for undifferentiated markers (Nanog and SSEA1) on human normal control-derived iPS cells into which the vector described in FIG. 6A was introduced.
- FIG. 7B shows a schematic diagram of the process of inducing iMN from the iPS cells of FIG. 7A.
- FIG. 7C shows an immunostained image of HB (fluorescence) and ⁇ -III tubulin or ChAT after 7 days after adding DOX to the iPS cells of FIG. 7A.
- FIG. 7D shows the results of measuring the expression levels of HB9 and ChAT after addition of DOX (DOX +) or non-addition (DOX ⁇ ) in human iPS cells into which the vector described in FIG. 6A has been introduced by the PCR method.
- FIG. 7E shows a phase contrast image (left figure) and a stained image of ⁇ BTX / SV2 after co-culture of motor neurons and C2C12 cells derived from human iPS cells into which the vector described in FIG. 6A has been introduced.
- FIG. 7F shows potential recording (current clamp) measured by the patch clamp method.
- FIG. 7G Inward current recording (voltage clamp) when neurotransmitters (glutamic acid, kainic acid and GABA) are added.
- FIG. 8A Mouse ES cells into which the vector described in Fig. 1A has been introduced are cultured on OP9 cells, and an image on day 5 is shown.
- FIG. 8B shows FACS results on the fifth day after culturing mouse ES cells introduced with the vector described in FIG. 1A on OP9 cells.
- FLK-1 is a marker of mesoderm
- SSEA-1 is a marker of undifferentiated cells.
- FIG. 8C shows a stained image of monocytes or macrophages obtained from mouse ES cells introduced with the vector described in FIG. 1A.
- FIG. 9 shows immunostained images of nerve cells derived from blood cells derived from mouse ES cells into which the vector described in FIG. 1A has been introduced.
- FIG. 10A Sequence of SOD1 gene (of the mutation site) against iPS cells established from a human ALS patient with L144FVX mutation (upper figure: SOD1-L144FVX) or G93S mutation (lower figure: SOD1-G93S) in SOD1 gene The result of having performed is shown.
- FIG. 10B shows the relationship between the process of inducing differentiation of motor neurons from the iPS cells established in FIG. 10A and the analysis. Cell survival analysis was performed by analyzing changes in the number of cells from Day 7 to Day 14.
- FIG. 10A Sequence of SOD1 gene (of the mutation site) against iPS cells established from a human ALS patient with L144FVX mutation (upper figure: SOD1-L144FVX) or G93S mutation (lower figure: SOD1-G93S) in SOD1 gene The result of having performed is shown.
- FIG. 10B shows the relationship between the process of inducing differentiation of motor neurons from the iPS cells established in FIG. 10A
- FIG. 10C shows an immunostained image of each iPS cell-derived motor neuron 7 days after induction.
- FIG. 10D shows a stained image of misfolded SOD1.
- FIG. 10E shows the survival rate of motor neurons 7-14 days after differentiation induction (ie, the number of ⁇ -III tubulin positive cells after 14 days / the number of ⁇ -III tubulin positive cells after 7 days).
- FIG. 11A In normal mouse-derived iPS cells into which the vector of FIG. 6A has been introduced, the expression of undifferentiated marker (SSES1), neuron-specific marker (NCAM) after DOX administration, and cell morphology up to 72 hours later It is the result of examining the change.
- FIG. 11B In normal human-derived iPS cells into which the vector of FIG.
- FIG. 12A In iPS cells derived from three types of mice (genetically modified mouse, wild-type SOD1 transgenic mouse, and mutant SOD1 transgenic mouse) into which the vector of FIG. 6A has been introduced, iMN up to 10 days after DOX administration It is the result of measuring the number of cells.
- the vertical axis represents the number of iMN per culture well.
- Fig. 13A In ALS patient-derived iPS cells having the SOD1L144FVX mutation introduced with the vector of Fig.
- FIG. 13B shows the result of counting the number of iMN 14 days after DOX administration in the culture system of FIG. 13A.
- the vertical axis is the relative value of the number of MNs in the well.
- FIG. 14 shows an outline of a drug screening system using iPS cells introduced with MNation factor according to the present invention.
- FIG. 15A is a result of testing the accuracy of the screening system shown in FIG.
- FIG. 15B is a result of examining about 1200 kinds of existing drug compounds using the screening system tested in FIG. 15A.
- FIG. 16A shows the results of examining the expression of undifferentiated markers (SSES1) and nerve cell-specific markers (Tuj1, NCAM) after administration of DOX on NPS-introduced mouse-derived iPS cells.
- FIG. 16B shows a phase contrast image of cells on the 11th day from the start of Ngn2 expression induction. Cell death is not confirmed so much and can be used for analysis.
- FIG. 16C The upper graph shows the contents of A ⁇ 40 and A ⁇ 42 peptides contained in the culture supernatants on days 3-5, 5-7, 7-9 and 9-11 from the start of Ngn2 expression induction. The analysis result of quantity is shown. The middle and lower rows of FIG. 16C show A ⁇ 40 when the control, BSI IV, and Sulfide Slindac were added to the culture supernatant on days 7-9 (middle) or 9-11 (lower) from the start of Ngn2 expression induction. And the analysis result of content of A (beta) 42 peptide is shown.
- FIG. 17 shows an immunostaining image at the administration site 2 weeks after transplantation of MNation factor-introduced mouse ES cells introduced with HB9 :: GFP into the spinal cord of NOG mice and oral administration of DOX. Since the mouse ES cells express GFP in conjunction with the expression of HB9, the GFP positive cells represent transplanted mouse ES cell-derived motor neurons (iMN).
- FIG. 18 shows immunostaining images at the administration site 4 weeks after transplantation of N-factor-introduced mouse iPS cells into the hippocampus of NOG mice and oral administration of DOX. hNCAM-stained cells indicate transplanted human iPS cell-derived neurons (iN).
- FIG. 19 is a diagram for explaining a more accurate clinical test utilizing iPS cells obtained in the present invention.
- motor neurons and neurons obtained by the method according to the present invention are referred to as iMN and iN, respectively, in order to distinguish them from original (ie, native) motor neurons and neurons.
- iMN and iN motor neurons and neurons obtained by the method according to the present invention
- the Lhx3, Ngn2, and Isl1 genes may be collectively referred to as a motor neuronization factor (or MNation factor), and only the Ngn2 gene may be referred to as a neuronalization factor (or National factor).
- a nucleic acid encoding Lhx3, Ngn2, and Isl1 is introduced into a pluripotent stem cell, and the expression of the three genes is maintained for 3 days or more, thereby allowing rapid and synchronized differentiation into motor neurons.
- a method is provided. The induction of the expression of the three genes may be performed either in culture or in an animal body. Furthermore, after the pluripotent stem cell introduced with the expression vector is induced to differentiate into blood cells in culture, the expression of the three genes is induced, and motor neurons can be produced via blood cells.
- a method for rapidly and synchronously differentiating into a neuron by introducing a nucleic acid encoding Ngn2 into a pluripotent stem cell using a transposon and maintaining the expression of the gene for 3 days or more.
- the expression induction of the Ngn2 gene can be performed either in culture or in an animal body. Below, the technology which comprises these methods is explained in full detail.
- a pluripotent stem cell is a stem cell that has pluripotency that can be differentiated into all cells present in a living body and also has proliferative ability. Examples include, but are not limited to, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic germ cells (“EG cells”), induced pluripotent stem (iPS) cells, cultured fibroblasts, bone marrow stem cell-derived pluripotent cells (Muse cells), and the like.
- ES embryonic stem
- ntES embryonic stem
- iPS induced pluripotent stem
- preferred pluripotent stem cells for use in the present invention are ES cells, ntES cells, and iPS cells.
- each stem cell will be described.
- ES cells are stem cells established from the inner cell mass of early embryos (eg, blastocysts) of mammals such as humans and mice and having the pluripotency and the ability to proliferate by self-replication.
- ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication.
- ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were established in primates such as humans and monkeys (JA Thomson et al. al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod ., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).
- ES cells can be established by taking an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. In addition, maintenance of cells by subculture is performed using a culture solution to which substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) are added. It can be carried out.
- LIF leukemia inhibitory factor
- bFGF basic fibroblast growth factor
- DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF is used as the culture medium for ES cell production.
- Human ES cells can be maintained in a humid atmosphere at 37 ° C., 2% CO 2 /98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224).
- ES cells need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl 2 and 20% KSR. Can be used.
- ES cells can be selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index.
- gene markers specific to undifferentiated cells such as OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat Biotechnol., 26: 443-452).
- Human ES cell lines for example, WA01 (H1) and WA09 (H9) are obtained from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from Institute for Regenerative Medicine (Kyoto, Japan), Kyoto University Is possible.
- sperm stem cell is a testis-derived pluripotent stem cell and is a cell that is the origin for spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012).
- GDNF glial cell line-derived neurotrophic factor
- Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, such as LIF, bFGF, stem cell factor, etc. Can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).
- iPS Artificial pluripotent stem cells
- iPS Artificial pluripotent stem cells
- somatic cells in the form of DNA or protein, and are almost equivalent to ES cells
- It is an artificial stem cell derived from a somatic cell having the characteristics of, for example, differentiation pluripotency and proliferation ability by self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol.
- the reprogramming factor is a gene that is specifically expressed in ES cells, its gene product or non-cording RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-cording RNA, or It may be constituted by a low molecular compound.
- genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination.
- the reprogramming factors include histone deacetylase (HDAC) inhibitors [eg, small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg , HDAC1 siRNA Smartpool TM (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene, etc.) and other nucleic acid expression inhibitors etc.], MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase-3 inhibitors (for example, Bio and CHIR99021), DNA methyltransferase inhibitors (for example, 5-azacytidine), histone methyltransferase inhibitors (for example, small molecule inhibitors such as BIX-01294, Suv39hl, Suv39h2, SetDBl And nucleic acid expression inhibitors
- the reprogramming factor may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (for example, HIV-derived TAT and polyarginine), and microinjection.
- a cell membrane-permeable peptide for example, HIV-derived TAT and polyarginine
- Virus vectors include retrovirus vectors, lentivirus vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007. ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054) and the like.
- artificial chromosome vectors examples include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
- HAC human artificial chromosomes
- YAC yeast artificial chromosomes
- BAC bacterial artificial chromosomes
- a plasmid a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008).
- the vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed.
- Selective marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), ⁇ -glucuronidase (GUS), FLAG, etc.
- GFP green fluorescent protein
- GUS ⁇ -glucuronidase
- FLAG FLAG
- the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be.
- RNA it may be introduced into somatic cells by, for example, lipofection or microinjection, and RNA that incorporates 5-methylcytidine and pseudoridine (TriLink Biotechnologies) is used to suppress degradation. (Warren L, (2010) Cell Stem Cell. 7: 618-630).
- Examples of the culture medium for inducing iPS cells include DMEM, DMEM / F12 or DME culture medium containing 10 to 15% FBS (these culture media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, ⁇ -mercaptoethanol, etc.) or a commercially available culture medium [eg, culture medium for mouse ES cell culture (TX-WES culture medium, Thrombo X), primate ES cells Culture medium for culture (primate ES / iPS cell culture medium, Reprocell), serum-free medium (mTESR, Stemcell Technology)] and the like.
- DMEM DMEM / F12 or DME culture medium containing 10 to 15% FBS
- these culture media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, ⁇ -mercaptoethanol, etc.
- a commercially available culture medium eg, culture medium for mouse
- a somatic cell is brought into contact with a reprogramming factor on a DMEM or DMEM / F12 medium containing 10% FBS at 37 ° C. in the presence of 5% CO 2 for about 4 to 7 days. Then, re-spread the cells on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact between the somatic cells and the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.
- feeder cells for example, mitomycin C-treated STO cells, SNL cells, etc.
- 10% FBS-containing DMEM medium including LIF, penicillin / streptomycin, etc.
- feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
- 5% CO 2 at 37 ° C. can be suitably included with puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.
- ES-like colonies after about 25 to about 30 days or more .
- somatic cells to be reprogrammed themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin- 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.
- iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).
- hypoxic conditions oxygen concentration of 0.1% or more and 15% or less
- the culture medium is exchanged with a fresh culture medium once a day from the second day onward.
- the number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
- IPS cells can be selected according to the shape of the formed colonies.
- a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized for example, Oct3 / 4, Nanog
- a culture solution containing the corresponding drug selection The established iPS cells can be selected by culturing with the culture medium.
- the marker gene is a fluorescent protein gene
- iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate can do.
- the term “somatic cell” refers to any animal cell (preferably, a mammalian cell including a human being) excluding germline cells or totipotent cells such as eggs, oocytes, and ES cells. ). Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , Passage cells, and established cell lines are all included.
- somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells such as dental pulp stem cells (somatic stem cells), (2) tissue progenitor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, enterocytes, spleen cells, pancreatic cells (exocrine pancreas cells, etc.), brain cells, lung cells, kidney cells Examples thereof include differentiated cells such as fat cells.
- a diseased somatic cell may be used.
- examples of the disease include neurodegenerative diseases.
- iPS cells may be produced using somatic cells derived from a patient with motor neuron disease.
- motor neuron disease is a disease that causes degeneration of motor neurons, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and bulbar spinal muscular atrophy. Illness and the like are exemplified.
- a somatic cell of a patient with amyotrophic lateral sclerosis is exemplified by a somatic cell having a mutation in the SOD1 gene, and more specifically, a mutation in the SOD1 gene such as G93A, G93S and L144FVX. In particular, it is not limited to these.
- iPS cells may be produced using somatic cells derived from a patient with a neurodegenerative disease.
- neurodegenerative disease refers to a disease caused by degeneration or loss of nerve cells, and examples include Alzheimer-type dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, and spinocerebellar degeneration. Is done.
- a somatic cell of a patient with Alzheimer-type dementia is exemplified by a somatic cell having a mutation in the presenilin 1 gene or the presenilin 2 gene, and more than 30 mutations have been reported so far in the presenilin 1 mutation (Price DL , Sisodia SS., Annu Rev Neurosci. 1998; 21: 479-505.),
- mutations in which D257 or D385 is substituted with alanine or glutamic acid are exemplified, but not limited thereto.
- an ES cell established from an inner cell mass of a clonal embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell.
- nt ES nuclear transfer ES
- nuclear transfer technology JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 47-52).
- Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.
- Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, fibroblasts or bone marrow stromal cells are treated with trypsin for a long time, preferably 8 or 16 hours. It is a pluripotent cell obtained by suspension culture after treatment, and is positive for SSEA-3 and CD105.
- pluripotent stem cells into which a gene causing a disease is introduced can be used from the viewpoint of producing a model cell of a disease state.
- the causative gene is exemplified by SOD1, C9ORF72, TDP43, FUS, PRN1, EPH4N, ANG, UBQLN and HNPNPA, and pluripotent stem cells having these mutant genes Is desirable.
- iPS cells or muscular atrophic lateral cords obtained by introducing mutant SOD1 (A4V, G37R, G41D, H46R, G85R, D90A, G93A, G93S, I112T, I113T, L114F or S134N mutations are exemplified)
- Examples include iPS cells produced by isolating somatic cells of patients with sclerosis.
- somatic cells having mutations in the presenilin 1 gene or the presenilin 2 gene are exemplified, and pluripotent stem cells having these mutant genes are desirable.
- pluripotent stem cells having a mutated gene examples include iPS cells produced by isolating somatic cells of patients with Alzheimer's dementia.
- the amyotrophic lateral sclerosis model cell or Alzheimer-type dementia model cell includes a motor nerve cell or a nerve cell obtained from the pluripotent stem cell by the method described above.
- a model cell having a preferable disease state is a mouse or a human cell.
- the term "motor neuron” refers to a cell that expresses one or more motor neuron marker genes such as HB9 and ChAT (choline acetyltransferase), or one neuron marker gene such as ⁇ -III tubulin, NCAM, and MAP2. It is defined as a cell that is expressed as described above and has a neurite (also called a new light) and a sufficiently thick cell body. This is because it has been confirmed that expression of HB9 or ChAT is observed in cells that express one or more marker genes of the neuronal cells and have neurites and sufficiently thickened cell bodies.
- HB9 and ChAT choline acetyltransferase
- producing motor neurons means obtaining a cell population containing cells satisfying the above definition, preferably a cell population containing 5%, 15%, or 20% or more of the cells. Is to get.
- a nerve cell is defined as a cell that expresses one or more marker genes of nerve cells such as ⁇ -III tubulin, NCAM, and MAP2 and has neurites. Therefore, the criteria for determination of nerve cells (ie, iN) produced by the method according to the present invention also follow this.
- the nerve cells produced in the present invention are preferably glutamatergic.
- producing a nerve cell means obtaining a cell population containing cells satisfying the above definition, and preferably, the cells are 50%, 60%, 70%, 80%, or 90%. It is to obtain a cell population containing the above. Since Tuj1 is an anti- ⁇ -III tubulin, cells expressing the ⁇ -III tubulin may be referred to as Tuj1-positive cells.
- Lhx3 refers to Lhx3 (LIM homeobox 3) gene and Lhx3 protein.
- the nucleic acid encoding Lhx3 is the gene encoded by the polynucleotide represented by NCBI accession number: NM_001039653 (mouse) or NM_014564 or NM_178138 (human), and its transcriptional variants, splicing variants and homologs.
- it may be a nucleic acid having such a complementary relationship that it can hybridize under stringent conditions to the complementary strand sequences of these nucleic acids.
- Ngn2 refers to the Ngn2 (Neurogenin 2) gene and Ngn2 protein.
- the nucleic acid encoding Ngn2 is a polynucleotide represented by NCBI accession number: NM_009718 (mouse) or NM_024019 (human) or a transcriptional variant, splicing variant and homologue thereof, or a complementary strand of these nucleic acids. It may be a nucleic acid having a complementary relationship to the extent that it can hybridize to a sequence under stringent conditions.
- Isl1 is an Isl1 (Islet 1) gene and an Isl1 protein.
- the nucleic acid encoding Isl1 is a polynucleotide represented by NCBI accession number: NM_021459 (mouse) or NM_002202 (human), a transcriptional variant thereof, a splicing variant and a homologue, or a complementary strand of these nucleic acids. It may be a nucleic acid having a complementary relationship to the extent that it can hybridize to a sequence under stringent conditions.
- stringent conditions are nucleic acids that bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). Can be determined based on the melting temperature (Tm). For example, as washing conditions after hybridization, the conditions of about “1 ⁇ SSC, 0.1% SDS, 37 ° C.” can be mentioned.
- the complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions.
- washing is performed under more severe hybridization conditions such as “0.5 ⁇ SSC, 0.1% SDS, 42 ° C.”, more strictly “0.1 ⁇ SSC, 0.1% SDS, 65 ° C.”
- the conditions for maintaining the hybridized state between the positive strand and the complementary strand can be mentioned.
- a complementary strand a strand comprising a base sequence that is completely complementary to the target positive strand base sequence, and at least 90%, preferably 95% or more, more preferably, the strand.
- a chain composed of a base sequence having 97% or more, more preferably 98% or more, particularly preferably 99% or more.
- the nucleic acid encoding Lhx3, the nucleic acid encoding Ngn2, and the nucleic acid encoding Isl1 may be DNA, RNA, or DNA / RNA chimera.
- the nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid. Preferred is double-stranded DNA or single-stranded RNA.
- the nucleic acids encoding Lhx3, Ngn2, and Isl1 are double-stranded DNA (sometimes referred to herein as Lhx3 gene, Ngn2 gene, and Isl1 gene)
- the nucleic acid is inserted into an appropriate expression vector.
- the RNA may be RNA incorporated with 5-methylcytidine and pseudoridine (TriLink Biotechnologies) to suppress degradation.
- the RNA may be modified by phosphatase treatment.
- the Lhx3 encoding RNA and the Lhx3 protein may be collectively referred to as an Lhx3 gene product (the same applies to Ngn2 and Isl1).
- a transcription factor relating to other nerve development is selected as long as it does not inhibit the motor nerve cell induction by the Mn factor or the nerve cell induction by the N factor.
- the encoding nucleic acid may be introduced into the pluripotent stem cell together with the MNation factor or the National factor. Examples of such transcription factors include a nucleic acid encoding Ascl1, a nucleic acid encoding Brn2, a nucleic acid encoding Myt1l, and a nucleic acid encoding HB9.
- a method for introducing a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and a nucleic acid encoding Isl1 into a pluripotent stem cell is not particularly limited. For example, the following method can be used.
- the nucleic acid When the nucleic acid is in the form of DNA, it can be introduced into a pluripotent stem cell by a technique such as lipofection, liposome, or microinjection in a form introduced into a vector such as a virus, plasmid, or artificial chromosome.
- virus vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, and the like.
- a plasmid vector a plasmid for mammalian cells can be used.
- the artificial chromosome vector include human artificial chromosome (HAC), yeast artificial chromosome (YAC), and bacterial artificial chromosome (BAC, PAC).
- plasmid vectors and artificial chromosome vectors are preferred, and plasmid vectors are most preferred.
- These vectors can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc., so that the Lhx3, Ngn2, or Isl1 gene can be expressed.
- Selectable marker sequences such as drug resistance genes (eg, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene), thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as fluorescent protein, ⁇ -glucuronidase (GUS), FLAG, etc. Can be included.
- the expression cassette (promoter, gene sequence) is included in the vector in order to insert a nucleic acid encoding the gene into a chromosome, or to excise the nucleic acid inserted into the chromosome as necessary.
- a gene expression unit including a terminator may have a transposon sequence. Although it does not specifically limit as a transposon arrangement
- a nucleic acid encoding the transposase in order to introduce transposase, may be contained in the aforementioned vector, or a nucleic acid encoding the transposase may be contained in another vector and simultaneously introduced into a cell. May be. Furthermore, a gene product encoding the transposase may be directly introduced.
- a preferred transposase is a transposase corresponding to the above-described transposon sequence, and preferably a piggyBac transposase.
- a mode in which the three genes are expressed polycistronically may be used.
- an IRES or 2A coding sequence between sequences encoding Lhx3, Ngn2, and Isl1, the three genes can be expressed polycistronically (Nat. Biotech., 5, 589). -594, 2004).
- the 2A coding sequence may be derived from any virus (J. General Virology, 89, 1036-1042, 2008), but is preferably the 2A coding sequence of foot-and-mouth disease virus (FMDV).
- the nerve inducer when in the form of RNA, it may be introduced into pluripotent stem cells by techniques such as electroporation, lipofection, and microinjection.
- the nerve inducer when in the form of a protein, it may be introduced into pluripotent stem cells by techniques such as lipofection, fusion with cell membrane permeable peptides (eg, HIV-derived TAT and polyarginine), and microinjection.
- cell membrane permeable peptides eg, HIV-derived TAT and polyarginine
- a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and a nucleic acid encoding Isl1 are functionally joined to an inducible promoter to induce expression of Lhx3, Ngn2, or Isl1 at a desired time.
- inducible promoters include drug-responsive promoters, and preferred examples thereof include a tetracycline-responsive promoter (a CMV minimal promoter having a tetracycline response element (TRE) in which tetO sequences are consecutive seven times). Is mentioned.
- the promoter is a promoter that is activated when tetracycline or a derivative thereof is supplied under the expression of a reverse tetracycline-regulated transactivator (rtTA; a fusion protein composed of reverse tetR (rTetR) and VP16AD). is there. Therefore, when the expression of the gene is induced using a tetracycline-responsive promoter, it is more preferable to use a vector having a mode for expressing the activator.
- rtTA reverse tetracycline-regulated transactivator
- rTetR reverse tetR
- VP16AD reverse tetR
- DOX doxycycline
- expression induction systems using drug responsive promoters other than the above include expression induction systems using estrogen responsive promoters (eg, WO2006 / 129735), RheoSwitch mammalian inducibility using promoters induced by RSL1 Has an expression system (New England Biolabs), a Q-mate system (Krackeler Scientific) or Cumate-inducible expression system (National Research Council (NRC)) using a cumate-inducible promoter, and an ecdysone responsive sequence Examples include a GenoStat inducible expression system (Upstate cell signaling solutions) using a promoter.
- an agent that can induce the activation of the promoter for example, the tetracycline responsiveness
- expression of Lhx3, Ngn2, and Isl1 can be maintained by continuing to add tetracycline or DOX) to the medium for a desired period. Then, it is possible to stop the expression of the gene by removing the drug from the medium (for example, replacing the medium with a medium not containing the drug).
- Lhx3, Ngn2, and Isl1 expression induction is induced by joining the gene in a non-functional form to a constitutive promoter and converting the joined state into a functional joined state at a desired time. May be.
- a specific sequence sandwiched between LoxP sequences between the constitutive promoter and the sequence encoding the gene for example, a sequence encoding a drug resistance gene or a sequence inducing transcription termination
- a method of converting the joined state into a functional joined state by removing Cre and interposing the LoxP sequence at a desired time can be used.
- an FRT sequence or a transposon sequence may be used instead of the LoxP sequence, and FLP (flipase) or the transposon may be used instead of the Cre.
- piggyBac transposon is mentioned as a transposon which can be used suitably for this purpose.
- Constitutive promoters that can be used for the above purpose include SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF- ⁇ promoter, CAG promoter and the like.
- Cre, FLP, and transposon are allowed to act again after a desired period of time to cause the sequence (LoxP sequence, FRT).
- the expression of the gene can also be stopped by removing a sequence sandwiched between sequences or transposon sequences).
- the expression period of the gene is controlled by using a vector that can be easily eliminated from the cell, such as an adenovirus vector, an adeno-associated virus vector, a Sendai virus vector, a plasmid, or an episomal vector. Is also possible.
- the expression of the introduced Lhx3, Ngn2, and Isl1 is preferably maintained for 3 days or more, and the effects of the present invention can be obtained in any of 4, 5, 6, and 7 days. Particularly preferred is 7 days.
- the period of maintaining the expression does not cause a disadvantage in the production of motor neurons, it is preferably 3 days or more and 14 days or less, particularly preferably 7 days or more and 14 days or less.
- the expression of the introduced Ngn2 can exert the effect of the present invention in any of 3, 4, 5, 6, and 7 days.
- it is preferably 3 days or more and 14 days or less, particularly preferably 7 days or more and 14 days or less.
- the pluripotent stem cell introduced with the nucleic acid encoding Lhx3, Ngn2, and Isl1 is used as a medium for inducing differentiation into a motor neuron in culture.
- a medium (culture solution) to which a stimulant and a neurotrophic factor are added can be used.
- Examples of such basic medium include, for example, Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, and Ham's F12 medium. , RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and mixed media thereof.
- the basic medium may contain serum or may be serum-free. If necessary, the medium may be, for example, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acid, May contain one or more serum replacements such as insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and also lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential One or more substances such as amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone and putrescine may also be included.
- KSR Knockout Serum Replacement
- N2 supplement Invitrogen
- B27 supplement Invitrogen
- albumin transferrin
- apotransferrin fatty
- the preferred basic medium in the present invention is a mixed medium of DMEM and F12 containing insulin, apotransferrin, selenate, progesterone and putrescine, and particularly preferably an N3 medium (100 ⁇ g / ml apotransferrin, 5 ⁇ g / DMEM / F12)) supplemented with ml insulin, 30 nM selenite, 20 nM progesterone and 100 nM putrescine.
- N3 medium 100 ⁇ g / ml apotransferrin, 5 ⁇ g / DMEM / F12
- a medium containing 1 ⁇ M retinoic acid (RA), 1 ⁇ M sonic hedgehog (Shh), 10 ng / ml BDNF, 10 ng / ml GDNF, and 10 ng / ml NT3 in the N3 medium is used as a differentiation induction medium for motor neurons. It can be particularly preferably used.
- N2 medium is used instead of the N3 medium, there is a tendency that synchronization of differentiation into iMN and synchronization of cell death of ALS model mouse-derived iMN tend to be reduced.
- an SHH (Sonic hedgehog) signal stimulator is defined as a substance that causes the inhibition of Smoothened (Smo) caused by SHH binding to the receptor Patched (Ptch1) and the subsequent activation of Gli2.
- SHH Smoothened
- Hh-Ag1.5 Li, X., et al., Nature Biotechnology, 23, 215-221, 2005
- Smoothened Agonist SAG (N-Methyl-N- (3-pyridinylbenzyl) ) -N '-(3-chlorobenzo [b] thiophene-2-carbonyl) -1,4-diaminocyclohexane)
- 20a-hydroxycholesterol Purmorphamine and derivatives thereof (Stanton BZ, Peng LF, Mol Biosyst. 6: 44-54, 2010).
- the SHH signal stimulator used in the present invention may preferably be Purmorphamine.
- the concentration of Purmorphamine in the culture solution is not particularly limited as long as it is a concentration that activates Gli2. , 40 ⁇ M or less, 30 ⁇ M or less, 25 ⁇ M or less, 20 ⁇ M or less, 15 ⁇ M or less, or 10 ⁇ M or less can be selected as appropriate. However, it is not limited to these. Preferably, it is 2 ⁇ M.
- a neurotrophic factor is a ligand for a membrane receptor that plays an important role in the survival and function maintenance of motor neurons, such as Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF). , Neurotrophin 3 (NT-3), Neurotrophin 4/5 (NT-4 / 5), Neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor ( HGF), Insulin, Insulin Like Growth Factor 1 (IGF 1), Insulin Like Growth Factor 2 (IGF 2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (6IL) ), Ciliary Neurotrophic Factor (CNTF) and LIF.
- a preferred neurotrophic factor in the present invention is a factor selected from the group consisting of GDNF and BDNF.
- the pluripotent stem cell introduced with the nucleic acid encoding Ngn2 is used as a medium for inducing differentiation into neurons under culture, as a basal medium alone or a basal medium supplemented with a neurotrophic factor.
- a basal medium alone or a basal medium supplemented with a neurotrophic factor.
- Examples of such basic medium include, for example, Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, and Ham's F12 medium. , RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies) and mixed media thereof.
- the basic medium may contain serum or may be serum-free.
- the medium may be, for example, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acid, May contain one or more serum replacements such as insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and also lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential It may also contain one or more substances such as amino acids, vitamins, growth factors, low molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone and putrescine.
- KSR Knockout Serum Replacement
- Neurobasal Medium containing B27 supplement or a mixed medium of DMEM and F12 containing insulin, apotransferrin, selenate, progesterone and putrescine can be suitably used as the basic medium.
- the method of the present invention when differentiation of a nerve cell is induced, it can be induced in a nerve cell without co-culturing with a mouse cell, particularly a mouse glial cell. Therefore, it is desirable not to co-culture with mouse cells for the purpose of preventing contamination.
- the culture temperature for inducing differentiation of the motor nerve cells and nerve cells of the present invention is not particularly limited, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
- the CO 2 concentration is preferably about 2-5%.
- a cell culture having a neuromuscular junction in which myotube cells and motor neurons are adhered is obtained by mixing myotube cells.
- the neuromuscular junction means a structure in which acetylcholine is released from the process end of a motor nerve cell and can be received by a receptor present in a myotube cell.
- the presence of the neuromuscular junction is, for example, by co-localization of synaptic vesicle protein (eg, SV2) expressed by motor neurons and acetylcholine receptor expressed by myotubes by immunostaining and fluorescence microscopy. Can be confirmed.
- the cell culture containing the neuromuscular junction is very useful as a model system for the pathology caused by dysplasia of the neuromuscular junction (for example, myasthenia gravis and Lambert-Eaton myasthenia). is there.
- the present invention provides a pluripotent stem cell in which a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and an exogenous nucleic acid encoding Isl1 are inserted into a chromosome.
- the nucleic acid is preferably under the control of an inducible promoter, more preferably under the control of a drug-responsive promoter.
- the pluripotent stem cell is a cell that can be rapidly and synchronously differentiated into a motor nerve cell by contacting with an agent to which the promoter responds.
- the present invention also provides a pluripotent stem cell in which an exogenous nucleic acid encoding Ngn2 is inserted into the chromosome.
- the nucleic acid is preferably under the control of an inducible promoter, more preferably under the control of a drug-responsive promoter.
- the said pluripotent stem cell is a cell which can be rapidly and synchronously differentiated into a nerve cell by contacting with an agent to which the promoter responds.
- Pluripotent stem cells in which these exogenous nucleic acids are inserted into the chromosome maintain undifferentiated ability and high proliferative ability, and can be proliferated while maintaining the above traits.
- the pluripotent stem cell in which the exogenous nucleic acid is inserted into the chromosome is derived from a patient with a neurodegenerative disease, it can be suitably used as a screening system for a therapeutic agent for the disease. Further, when derived from a healthy person, it can be suitably used as a composition for treating motor neurodegenerative diseases or nerve damage, or for producing a model animal that retains human-derived motor neurons / nerve cells.
- the present invention provides a method for screening for a therapeutic agent for amyotrophic lateral sclerosis comprising the following steps (1) to (5).
- a step of producing motor neurons by the above-described method from an induced pluripotent stem cell produced from a somatic cell isolated from a patient with amyotrophic lateral sclerosis (2) A step of bringing the motor neuron obtained in the step (1) into contact with a test substance, (3) culturing the motor neuron cell contacted with the test substance in the step (2) and the motor neuron cell not contacted with the test substance (that is, a control cell); (4) a step of measuring the number of motor neurons and / or neurite length obtained in the step (3), (5) A step of selecting, as a therapeutic agent for amyotrophic lateral sclerosis, a test substance in which the number of motor neurons and / or neurite length brought into contact with the test substance is higher than that of a control.
- pluripotent stem cells derived from familial and arcuate patients can be used as pluripotent stem cells derived from amyotrophic lateral sclerosis patients.
- pluripotent stem cells derived from familial patients are preferable, and familial patient-derived pluripotent stem cells having a mutation in the SOD1 gene are particularly preferable.
- the test substance (that is, the candidate drug) is, for example, a cell extract, a cell culture supernatant, a microbial fermentation product, an extract derived from marine organisms, a plant extract, a purified protein or a crude protein, a peptide, a non-peptide Examples include compounds, synthetic low molecular weight compounds, and natural compounds.
- the test substance is (1) a biological library, (2) a synthetic library method using deconvolution, and (3) “one-bead one-compound” live. Can be obtained using any of a number of approaches in combinatorial library methods known in the art, including rally methods, and (4) synthetic library methods using affinity chromatography sorting.
- Biological library methods using affinity chromatography sorting are limited to peptide libraries, but other approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67).
- Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al.
- Compound libraries can be either solution (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555- 6), bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos.
- Motor neurons used in the screening method for a therapeutic agent for amyotrophic lateral sclerosis of the present invention may be used in a state of being mixed with other cell types.
- the number of motor neuron cells is immunostained using a method known to those skilled in the art, and is counted as the number of cells expressing a marker gene of neurons such as HB9, ⁇ -III tubulin, ChAT, etc. or motor neurons And may be automatically measured using a cell image analyzer (in-cell analyzer).
- the number of cells may be calculated as the reciprocal of the number of dead cells.
- the number of dead cells can be measured by, for example, a method of measuring the activity of LDH, a method of measuring absorbance using the MTT method, WST-1 method, or WST-8 method, or TO (thiazole orange), PI (propidium iodide), 7AAD, calcein AM, or ethidium homodimer 1 (EthD-1), followed by counting using a flow cytometer, etc., and using a cell image analyzer (in-cell analyzer) Can also be done automatically.
- the cell number and / or neurite length of the motor nerve cell contacted with the test substance is 1.5 times or more, 1.6 times or more compared to the cell number and / or neurite length of the control cell, If it is 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 2.5 times or more, or 3 times or more, it may be determined as “high value”.
- the control cells in the present application include motor neurons that have not been contacted with a test substance and motor neurons that have been contacted with a drug that has been confirmed to be ineffective.
- the culture in the step (3) may be performed in the presence of the test substance.
- the culture period is not particularly limited as long as the number of cells can be measured in the control cells, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days As mentioned above, 8 days or more, 9 days or more, or 10 days or more can be mentioned, and 7 days is particularly preferable.
- the method of measuring the length of the neurite can be performed by visual observation, or may be measured using a cell image analyzer (in-cell analyzer) or the like. At this time, the neurite length may be measured as an area of the neurite on the image.
- Steps (2) to (5) may be performed after introducing a vector for expressing a fluorescent substance (for example, GFP) downstream of the HB9 promoter into the pluripotent stem cells of the step (1) so that motor neurons can be specifically recognized.
- the present invention provides a method for screening for a therapeutic agent for Alzheimer-type dementia, which comprises the following steps.
- a step of producing a nerve cell by the above-described method from an induced pluripotent stem cell produced from a somatic cell isolated from a patient with Alzheimer-type dementia (2) The step of bringing the nerve cell obtained in the step (1) into contact with a test substance, (3) culturing the nerve cells that have been contacted with the test substance in step (2) and the nerve cells that have not been contacted (that is, control cells), and measuring the content of A ⁇ 42 in the medium; (4) A test substance in which the content of A ⁇ 42 in the medium of nerve cells brought into contact with the test substance is lower than the content of A ⁇ 42 in the medium of the control cell is treated with Alzheimer's dementia The process of selecting as a medicine.
- pluripotent stem cells derived from familial and arcuate patients can be used as pluripotent stem cells derived from a patient with Alzheimer-type dementia.
- pluripotent stem cells derived from familial patients are preferred, and familial patient-derived pluripotent stem cells having mutations in the presenilin 1 gene are particularly preferred.
- the method for measuring A ⁇ 42 in the culture supernatant can be performed using a method commonly used by those skilled in the art, and is not particularly limited, but is measured by the ELISA method using the collected culture supernatant.
- a method using MSD Abeta 3 plex (38, 40, 42) assay plate (Meso Scale Discovery), human / rat ⁇ amyloid (42) ELISA kit Wako (WAKO) and the like can be mentioned.
- the measured value of A ⁇ 42 may be used as an index, or a value (A ⁇ 42 / A ⁇ 40) divided by the value of A ⁇ 40 may be used as an index.
- the supernatant cultured for 2 days after the medium exchange may be used.
- the test substance (that is, candidate drug) for the therapeutic agent for Alzheimer-type dementia the same substance as the test substance in the screening method for the therapeutic agent for amyotrophic lateral sclerosis described above can be used.
- composition for the treatment of motor neurodegenerative disease or nerve injury A nucleic acid derived from a healthy person and bound to a foreign nucleic acid (a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and a nucleic acid encoding Isl1 via a 2A sequence by the method according to the present invention, and drug responsiveness
- a pluripotent stem cell in which a foreign nucleic acid operably linked to a promoter) is incorporated in the chromosome, and a motor neuron cell obtained by inducing differentiation of the pluripotent stem cell Can be used as a composition (transplant therapy) for the treatment of motor neurodegenerative diseases or nerve damage.
- Suitable target diseases are those caused by deficient or damaged motor neurons, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and bulbar spinal muscular atrophy. Symptoms and the like.
- the cells used as the therapeutic composition are preferably cells that are in the process of differentiation into the pluripotent stem cells and / or motor neurons, because of the high engraftment rate in the tissue after transplantation. It is a potent stem cell.
- the therapeutic composition according to the present invention is prepared by mixing the pluripotent stem cells and / or motor nerve cells with a pharmaceutically acceptable carrier according to a conventional method, and so on. It can be manufactured as a parenteral preparation.
- pharmaceutically acceptable carriers that can be included in the parenteral preparation include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants.
- An aqueous liquid for injection can be mentioned.
- the preparation includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, polyethylene glycol). Etc.), preservatives, antioxidants and the like.
- a buffer eg, phosphate buffer, sodium acetate buffer
- a soothing agent eg, benzalkonium chloride, procaine hydrochloride, etc.
- a stabilizer eg, human serum albumin, polyethylene glycol). Etc.
- the therapeutic composition can be transplanted, for example, by injecting the aqueous suspension into a lesion site of neurodegeneration or nerve damage.
- the number of cells to be administered can be appropriately changed depending on the degree of lesion, etc. For example, in the case of a human ALS patient, about 1 ⁇ 10 5 to 1 ⁇ 10 8 cells can be administered. Then, by administering a drug to which the drug-responsive promoter responds to the transplanted patient, the pluripotent stem cells and / or motor neurons (while differentiating) are converted into motor neurons at the lesion site. Can be treated.
- Tetracycline or DOX may be administered as parenteral preparations such as injections, suspensions, drops, or oral preparations.
- the above transplantation treatment may be used in combination with drug therapy.
- a concomitant drug for example, when the target disease is ALS, riluzole (trade name: Riltech (registered trademark) (Sanofi)), which is an existing ALS treatment drug, or 1,3-diphenyl described in WO2012 / 029994 Urea derivatives or multikinase inhibitors, HMG-CoA reductase inhibitors described in WO2011 / 074690, or anacadic acid (Egawa, N et al, Sci Transl Med. 4 (145): 145ra104.doi: 10.1126), etc. Can be mentioned.
- These drugs can be used, for example, in dosages and administration routes usually used for the treatment of ALS.
- an exogenous nucleic acid an exogenous nucleic acid in which a nucleic acid encoding Ngn2 is functionally joined to a drug-responsive promoter
- Pluripotent stem cells and nerve cells obtained by inducing differentiation of the pluripotent stem cells can be used as transplantation therapeutic agents for neurodegenerative diseases or nerve damage.
- Suitable target diseases include Alzheimer's dementia and the like.
- the pluripotent stem cell and / or motor neuron is composed of the aforementioned exogenous nucleic acid (the nucleic acid encoding Lhx3, the nucleic acid encoding Ngn2, and the nucleic acid encoding Isl1 via the 2A sequence, and the drug response
- the exogenous nucleic acid the nucleic acid encoding Lhx3, the nucleic acid encoding Ngn2, and the nucleic acid encoding Isl1 via the 2A sequence
- an exogenous nucleic acid operably linked to a sex promoter is incorporated into the chromosome, and can be used as a transplantation therapeutic agent for the disease or nerve injury it can.
- motor neurons and neural cells are differentiated into somatic cells by motoneuron inducers capable of inducing expression with the above-described drug-responsive promoter or pluripotent stem cells introduced with nerve inducers. After induction, motor neurons and neurons may be produced by contacting with a drug corresponding to the drug-responsive promoter.
- the somatic cell to induce differentiation is not particularly limited, but a cell having migration ability such as a microglia cell and a blood cell is desirable. Particularly preferred are monocytes and / or macrophages.
- the exogenous nucleic acid (a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and a nucleic acid encoding Isl1 are bound via a 2A sequence and functionally joined to a drug-responsive promoter.
- a blood cell derived from a pluripotent stem cell containing a sex nucleic acid) in the chromosome is provided.
- the blood cell is a cell that can be converted into a motor nerve cell by contacting with a drug to which the promoter responds.
- Blood cells, especially inflammatory cells have the property of infiltrating lesions of motor neurodegenerative diseases or nerve damage and accumulating in motor nerve cell defect sites. Can be replenished with motor neurons.
- the exogenous nucleic acid (the nucleic acid encoding Lhx3, the nucleic acid encoding Ngn2, and the nucleic acid encoding Isl1 are linked via the 2A sequence, and functions as a drug-responsive promoter.
- Blood cells derived by differentiation from pluripotent stem cells that contain a foreign nucleic acid conjugated to the inside of the chromosome are very useful as transplantation therapeutic agents for motor neurodegenerative diseases or nerve damage.
- the target motor neurodegenerative disease has a pathological condition caused by deficient or damaged motor neurons, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), And bulbospinal muscular atrophy.
- examples of blood cells include monocytes, macrophages, neutrophils, eosinophils, basophils, and lymphocytes. Monocytes or macrophages are preferred.
- the pluripotent stem cells are cultured on OP9 cells (Nakano T, et al. Science 265: 1098-1101, 1994), or embryoid bodies are formed and produced in the culture medium.
- preferable blood cells are monocytes and / or macrophages that express at least CD14, and can be produced by appropriately referring to, for example, the method described in WO2012 / 115276. More preferred is a method in which pluripotent stem cells are co-cultured with bone marrow-derived stromal cells and further cultured in the presence of stem cell factor, macrophage-colony stimulating factor, and interleukin-3.
- the blood cells obtained as described above can be suspended in the aforementioned aqueous liquid and administered as a parenteral preparation, for example, into a patient's body by intravenous injection or intraarterial injection. Then, in the same manner as described above, by administering a drug to which the drug-responsive promoter responds to the administration patient, the blood cells are converted into motor nerve cells and can be treated.
- a drug to which the drug-responsive promoter responds to the administration patient the blood cells are converted into motor nerve cells and can be treated.
- tetracycline-responsive promoter when a tetracycline-responsive promoter is used as a drug-responsive promoter, tetracycline or DOX can be administered in the same manner as described above, but it is expected to replenish motor neurons specifically in the lesion site. Then, it may be administered locally to the site.
- the blood cells administered in the blood may be locally administered to the lesion site after a period required for accumulation in the lesion site (motor neuron cell defect site).
- the therapeutic composition containing blood cells as an active ingredient may be administered a plurality of times as necessary, or may be prepared and used in a form that can be sustainedly released using a technique known per se. Similarly to the above, it may be used in combination with other therapeutic agents for neurodegenerative diseases or nerve damage as appropriate.
- riluzole is the only drug approved as an ALS treatment.
- riluzole has promoted phosphorylation of cAMP response element (CRE) binding protein (CREB), activated the glial cell-derived neurotrophic factor (GDNF) gene promoter (promoter with CRE), and expressed the gene (Tsuchioka, M et al, Brain Res. 1384: 1-8 2011). Therefore, in the present invention, a promoter having one or more CRE is used as a drug-responsive promoter, and an exogenous nucleic acid in which a nucleic acid encoding a MNation factor is functionally joined to the promoter is inserted into the chromosome.
- CRE cAMP response element
- GDNF glial cell-derived neurotrophic factor
- pluripotent stem cells are prepared, it is expected that expression of MNation factor is induced by contacting the pluripotent stem cells with riluzole. Accordingly, if a therapeutic composition containing such pluripotent stem cells and blood cells differentiated from the cells as an active ingredient is prepared, the therapeutic composition and riluzole are used in combination with the therapeutic composition (tetracycline). It can be expected that the motor neurons are supplemented (without the addition of a drug such as) and have a therapeutic effect.
- the therapeutic composition tetracycline
- motor nerve induction factors capable of inducing expression with the drug-responsive promoter described above or pluripotent stem cells into which nerve induction factors have been introduced are introduced into the animal body.
- the method may further comprise the step of producing motor neurons and nerve cells in the animal body by contacting with a drug corresponding to the drug-responsive promoter in the animal body.
- the animal is a mammal, more preferably a mammal such as a human, a mouse, or a rat.
- the pluripotent stem cell in which the exogenous nucleic acid is incorporated into a chromosome, and a cell obtained by inducing differentiation from the pluripotent stem cell are transplanted into an animal, thereby The derived motor neuron or the animal engrafted with the neuron can be produced.
- the animal is a suitable model animal for analyzing the effects of drugs and external stress on human motor neurons or neurons in a more natural state.
- the present invention is specific to a subject with a therapeutic effect (ie, a responder) or a subject with no therapeutic effect (ie, a non-responder) on a study drug for motor neurodegenerative disease or nerve injury.
- a method for identifying or detecting a marker is provided.
- a method including the following steps (1) to (4); (1) a step of producing induced pluripotent stem cells from somatic cells isolated from the responders and non-responders, (2) A step of producing a motor nerve cell from the induced pluripotent stem cell obtained in the step (1) by the method described above, (3) a step of measuring expression of a gene product of a motor neuron derived from a responder and a motor neuron derived from a non-responder obtained in the step (2), and (4) a motion derived from the responder
- a gene product having a higher expression level than that of a non-responder is identified as a marker specific to the responder, or in a motor neuron derived from the responder, a gene product having a lower expression level than that of the non-responder Identifying as a marker specific to non-responders.
- a specific marker is a target cell (in this case, a motor nerve cell derived from a responder) that can distinguish a subject who has a therapeutic effect from a subject who has not had a therapeutic effect.
- examples of the gene product include mRNA, MicroRNA, and protein.
- the gene product may be any gene product, and examples of the mRNA include mRNA mounted on a DNA microarray chip used for gene expression difference analysis. Examples of the DNA microarray chip are those commercially available from Agilent, GE Healthcare Bioscience, and the like.
- the protein include cell surface proteins, and examples of such proteins include proteins contained in BD Lyoplate (registered trademark) sold by BD Japan.
- the investigational drug for motor neurodegenerative disease or nerve injury is any drug expected to have a therapeutic effect on motor neurodegenerative disease or nerve injury, such as riluzole, pentoxifylline, verapamil, azathioprine, Examples include, but are not limited to, topiramate, amantadine, acetylcysteine, physostimine, vitamin C, cyclosporine, celecoxib, guanidine, lamotrigine, minocycline, tyrolone, gabapentin, kenpauron.
- the expression of a gene product can be measured by methods known to those skilled in the art, and examples include PCR, Northern blotting, Western blotting, immunostaining, and microarray methods.
- the present invention further provides a marker specific to a subject who has a therapeutic effect on an investigational drug for Alzheimer-type dementia (ie, a responder) or a subject who has no therapeutic effect (ie, a non-responder).
- a method for identification is provided.
- a method including the following steps (1) to (4); (1) a step of producing induced pluripotent stem cells from somatic cells isolated from the responders and non-responders, (2) A step of producing a nerve cell from the induced pluripotent stem cell obtained in the step (1) by the method described above, (3) a step of measuring the expression of the gene product of the neuron derived from the responder and the non-responder obtained in the step (2), and (4) the motor neuron derived from the responder.
- a gene product having a higher expression level than a non-responder is identified as a marker specific to the responder, or a gene product having a lower expression level than the non-responder is non-responsive in motor neurons derived from the responder. Identifying as a marker specific to a person.
- the investigational drug for Alzheimer-type dementia is any drug that is expected to have a therapeutic effect on Alzheimer-type dementia, and examples include, but are not limited to, Donevecil hydrochloride, memantine, galantamine, etc. .
- the present invention further provides a method for selecting a subject for which the corresponding therapeutic agent is effective using the above-mentioned therapeutic effect-specific marker.
- the method for selecting a subject for which a therapeutic agent is effective includes the following steps (1) to (3); (1) a step of producing induced pluripotent stem cells from somatic cells isolated from a subject, (2) A step of producing a motor nerve cell from the induced pluripotent stem cell obtained in the step (1) by the method described above, (3) detecting a marker specific to the responder and / or non-responder identified by the above method in the motor neuron obtained in the step (2), and (4) to the responder A step of selecting a subject from which a motor neuron in which a specific marker has been detected or a motor neuron in which a specific marker has not been detected in the non-responder is derived as a target for which the corresponding therapeutic agent is effective.
- the present invention further provides a method for selecting a subject for which a therapeutic agent for Alzheimer-type dementia is effective; (1) a step of producing induced pluripotent stem cells from somatic cells isolated from a subject, (2) A step of producing a nerve cell from the induced pluripotent stem cell obtained in the step (1) by the method described above, (3) detecting a marker specific to the responder and / or non-responder identified by the above method in the motor neuron obtained in the step (2), and (4) to the responder A step of selecting a subject from whom a neuron cell in which a specific marker has been detected or a neuron cell in which a non-responder-specific marker has not been detected is derived as a target for which the corresponding therapeutic agent is effective.
- tetracycline-inducible MNation factor expression vector a vector that expresses Lhx3, Ngn2, and Isl1 in response to tetracycline
- tetracycline-inducible Nation a vector that expresses Ngn2 in response to tetracycline
- factor expression vector Sometimes referred to as "factor expression vector”.
- the regions sandwiched between the two Frt sequences or transposon sequences are referred to as the MNation factor expression cassette region and the National factor expression cassette region, respectively, and the cells in which the region is inserted into the chromosome are respectively , May be called MNation factor-introduced cell or N-factor-introduced cell.
- concentration of doxycycline (hereinafter abbreviated as DOX) added to the medium for the purpose of inducing activation of a tetracycline-responsive promoter is 1 ⁇ g / ml at a final concentration unless otherwise specified. is there.
- Example 1 Production of motor neurons (iMN) from mouse-derived pluripotent stem cells
- iMN motor neurons
- Lhx3, Ngn2, and Isl1 genes were introduced into neural progenitor cells differentiated from pluripotent stem cells over about 10 days using an adenovirus vector.
- a method for obtaining iMN from neural progenitor cells in about 11 days by expressing them has been reported (Non-patent Document 4).
- Lhx3 and Isl1 are transcription factors whose expression is induced at the differentiation stage after neural progenitor cells in the process of inducing differentiation of motor neurons from pluripotent stem cells by conventional methods (non-native) Patent Document 4). Therefore, it was thought that these transcription factors could not exhibit their original functions in undifferentiated cells, but we decided to introduce them into mouse-derived ES cells and examine their effects.
- a KH2 strain having a Frt sequence downstream of the ColA1 locus and expressing M2rtTA, a reverse tetracycline-regulated transactivator under the control of the endogenous R26 promoter was used (Beard C, et al., Genesis 2006; 44: 23-28).
- nucleic acids encoding Lhx3, Ngn2, and Isl1 are linked via a 2A sequence, and further nucleic acid linked to mCherry via an IRES sequence is tetracycline.
- a tetracycline-inducible MNation factor expression vector operably linked to a responsive promoter was prepared (only the region sandwiched between FRT sequences is shown in FIG. 1A).
- the vector is a polycistronic vector that expresses Lhx3, Ngn2, Isl1, and mCherry in response to tetracycline. Since the Frt sequence is present before and after the expression cassette containing the 4 genes, the vector is introduced into KH2 cells together with the nucleic acid encoding the flipase, so that the region sandwiched between the two Frt sequences (ie, MNization) A cell in which the factor expression cassette region) is inserted into the chromosome of the cell can be easily obtained.
- This vector was introduced into 293T cells by lipofection, and DOX was added to the medium and analyzed 24 hours later. As a result, it was confirmed that the addition of DOX induced the expression of Lhx3, Ngn2, and Isl1 proteins (FIG. 1B). ).
- the cells were dissociated using 0.25% trypsin, seeded on a Matrigel-coated dish, and medium containing DOX (ie, 1 ⁇ g / ml DOX, 1 ⁇ M retinoic acid (RA), 1 ⁇ M sonic hedgehog (Shh), 10 ng N3 medium containing / ml BDNF, 10ng / ml GDNF and 10ng / ml NT3 (DMEM / F12 supplemented with 100 ⁇ g / ml apotransferrin, 5 ⁇ g / ml insulin, 30 nM selenite, 20 nM progesterone and 100 nM putrescine)
- DOX ie, 1 ⁇ g / ml DOX, 1 ⁇ M retinoic acid (RA), 1 ⁇ M sonic hedgehog (Shh)
- 10 ng N3 medium containing / ml BDNF 10ng / ml GDNF and 10
- the culture medium containing DOX was examined.
- the MNation factor-introduced mouse ES cells were cultured for 3 days with or without the addition of DOX to the following four types of media, and then the number of ⁇ -III tubulin positive cells was counted.
- the period for adding DOX was also examined (FIG. 2B).
- the expression levels of HB9, ChAT, Isl1 are real-time Analysis was performed by PCR.
- Isl1 since only endogenous mRNA cannot be detected, the sum of endogenous mRNA and exogenous mRNA (mRNA transcribed from the Mn factor expression cassette region) (“Isl1” in FIG. 2C), or Only exogenous mRNA (“2A-Isl1” in FIG. 2C) was analyzed.
- C2C12 cells mouse striated muscle-derived cell line, Science, 230, 758-, 1985. The ability to form synapses was analyzed. C2C12 cells were cultured for 7 days in DMEM / F12 containing 0.5% FBS, 10 ⁇ g / ml insulin, 5.5 ⁇ g / ml apotransferrin, 30 nM selenite and 1 mM L-glutamine, and then induced to the myotubes. Using.
- the myotube culture system was seeded with the above-mentioned MNation factor-introduced mouse ES cells (on the myotube) and cultured in a DOX-containing medium to induce differentiation into motor neurons.
- SV2 synaptic vesicle protein 2
- FIG. 3A synaptic vesicle protein 2
- iMN motor neurons prepared from mouse pluripotent stem cells by the method of the present invention can form synapses with muscle cells (ie, form a neuromuscular junction).
- iMN obtained from the mouse-derived pluripotent stem cells by the method according to the present invention has the ability to form functional synapses with muscle cells in the same manner as the original motor neurons. It was done.
- a DNA fragment (HB9 :: GFP, for details, Lee SK, et al., Development, 131, 3295) in which the coding sequence of the GFP gene is operably linked to the promoter of the HB9 gene in the above-mentioned MNation factor-introduced mouse ES cells. , 2004) was introduced using piggyBac transposon to express GFP in conjunction with activation of the HB9 promoter.
- the cells were induced to differentiate by adding DOX, and GFP positive cells emitting GFP fluorescence were observed 3 days after the start of induction (FIG. 1G).
- iMN motor neurons
- iPS cells Mouse embryo fibroblasts (MEF) were prepared from transgenic mice having human SOD1 gene (G93A mutant SOD1 gene or wild type SOD1 gene), Okita K et al., IPS cells were established according to the method described in Nature. 2007, vol.448, pp.313-317. It was confirmed by the sequence of the gene that the iPS cells had the human SOD1 gene (FIGS. 4A and B). These iPS cells express the ES cell marker genes (Eras, Esg1, Rex1, Oct3 / 4 and Sox2), the expression of reprogramming factor is suppressed, and the 3 germ layers It was confirmed that differentiation could be induced (FIGS.
- ES cell marker genes Eras, Esg1, Rex1, Oct3 / 4 and Sox2
- iPS cells established from a transgenic mouse having the G93A mutant SOD1 gene or the wild-type SOD1 gene will be referred to as mouse G93A-derived iPS cells and mouse WT-derived iPS cells, respectively.
- FIG. 6A shows a tetracycline-inducible MNation factor expression vector introduced into the mouse-derived iPS cells. Similar to the vector shown in FIG. 1A, it is a polycistronic expression vector in response to tetracycline, but it has a piggyBAC transposon sequence (TR) instead of the Frt sequence.
- TR piggyBAC transposon sequence
- 6A shows a vector for polycistronic expression of mCherry together with the MNation factor, but in the present application, a vector that does not encode mCherry is also used, and the effect of the present invention can be obtained using this vector. I have confirmed that.
- the vector shown in FIG. 6A was introduced into the mouse G93A-derived iPS cells and mouse WT-derived iPS cells together with the nucleic acid encoding piggyBAC transposase, and the cells in which the MNation factor expression cassette region was inserted into the chromosome were selected. .
- the cells (MNation factor introduced-mouse G93A-derived iPS cells, MNation factor introduced-mouse WT-derived iPS cells) maintain the expression of undifferentiated markers (SSEA1 and NANOG) (FIG. 6B), and the vectors The same high proliferative ability as iPS cells before introduction was maintained.
- MNation factor introduction-mouse G93A-derived iPS cells and MNation factor introduction-mouse WT-derived iPS cells were cultured in a DOX-containing medium, as in the case of the mouse ES cells described above, After 3 days, many HB9, ⁇ -III tubulin and ChAT positive cells were confirmed (FIG. 6C). On the other hand, cells expressing GFAP, an astrocyte marker, were not confirmed. Therefore, even from iPS cells derived from a transgenic mouse having a human SOD1 gene (G93A mutant SOD1 gene or wild-type SOD1 gene), MN can be introduced and induced for expression in only 3 days. It was confirmed that it was obtained.
- MNation factor Introduction-Nothing was recognized in iMN prepared from mouse WT-derived iPS cells, but MNation factor introduction-In iMN prepared from mouse G93A-derived iPS cells, aggregates recognized by the antibody were intracellular. Many were observed (FIG. 6D). Next, the presence or absence of cell death was analyzed.
- FIG. 6E shows the value obtained by dividing the number of iMNs on day 6 by the number of iMNs on day 4 (ie, the number of HB9 and ⁇ -III tubulin positive cells on day 6 / the number of HB9 and ⁇ -III tubulin positive cells on day 4 (Number) is the result of iMN survival.
- iMN produced from iPS cells derived from MNation factor-mouse G93A is a large-scale cell from the start of induction to 4-6. It was revealed that it caused death (Fig. 6E). Furthermore, when the amount of LDH in the culture medium was measured 4 days and 6 days after the start of induction, MNation factor introduction--culture induced from mouse G93A-derived iPS cells is more MNation factor introduction-from mouse WT-derived iPS cells. It was confirmed that the amount of LDH was significantly higher than the induced culture (FIG. 6F).
- MNation factor introduction-iMN produced from mouse WT-derived iPS cells markedly increased. It was also clarified that MNation factor introduction-iMN prepared from mouse G93A-derived iPS cells hardly increased (ie, the protrusion extension stopped) (in contrast to vigorous protrusion extension) (FIG. 6H). ). Therefore, in iMN produced by the method according to the present invention from iPS derived from an ALS model mouse having a mutant human SOD1 gene, misfolding / aggregation of SOD1 protein occurs spontaneously as in motor neurons in the mouse spinal cord. It became clear that it went to cell death. Furthermore, it was shown that the measurement results of LDH value and neurite length in the medium can be used as an indicator of the cell death.
- mice having the G93A mutant human SOD1 gene can reduce the cell death by coexisting with wild-type glial cells (astrocytes). Therefore, the introduction of the MNation factor-mouse G93A-derived iPS cells was induced to differentiate into iMN in the presence of wild-type mouse-derived astrocytes, and the iMN survival rate (ie, HB9 and The number of ⁇ -III tubulin positive cells / the number of HB9 and ⁇ -III tubulin positive cells on day 4) was measured (FIG. 6G). Compared to Fig. 6E (differentiation induction in the absence of astrocytes), Fig.
- 6G differentiate induction in the presence of astrocytes introduced MNation factor-survival rate of iMN obtained from mouse G93A-derived iPS cells Is high.
- MNation factor-survival rate of iMN obtained from mouse G93A-derived iPS cells Is high.
- cell death of iMN obtained from MNation factor-introduced mouse G93A-derived iPS cells was reduced by the coexistence of wild-type astrocytes.
- iMN was prepared from human-derived pluripotent stem cells using the method according to the present invention, and its properties were evaluated.
- Example 3 Preparation of iMN from human (normal control) -derived pluripotent stem cells] ⁇ DNA introduction into human-derived iPS cells
- the vector shown in FIG. 6A was introduced into human-derived iPS cells (Takahashi K, et al., Cell. 2007, vol 131, pp 861-872.).
- IPS cells derived from MNation factor-human normal control were obtained.
- the cells are cultured on SNL cells using a primitive embryonic stem cell medium (ReproCELL) supplemented with 4 ng / ml basic fibroblast growth factor, and undifferentiated markers (NANOG and SSEA4) are expressed (Fig. 7A) and that the same high proliferation ability as iPS cells before introduction of the DNA was maintained.
- ReproCELL primitive embryonic stem cell medium
- NANOG and SSEA4 undifferentiated markers
- a MNation factor-introduced human normal control-derived iPS cell in which HB9 :: GFP is further inserted into the chromosome was prepared, and the cell was cultured in a DOX-containing medium in the presence of mouse-derived primary astrocytes. After culturing for 8-14 days, electrophysiological analysis by patch clamp method with whole cell recording was performed. As a result, Na + / K + current was observed in all analyzed GFP positive cells (10 cells), and action potentials were measured (FIG. 7F). Furthermore, since inward current was induced when glutamic acid or GABA was added to the medium (FIG.
- iMN produced from human-derived pluripotent stem cells by the method according to the present invention has the same electrophysiological characteristics as the original motor nerve cells.
- Example 4 iMN production from human (ALS patient) -derived iPS cells
- iMN was prepared from human ALS patient-derived pluripotent stem cells and analyzed for similarity to the original motor neurons.
- ⁇ Establishment of human-derived iPS cells From two normal human controls (Control1 and Control2) and two ALS patients with mutations in the SOD1 gene, skin-derived fibroblasts collected with the consent of the patients were treated with Okita K. , et al, Nat Methods. 2011, 8: 409-12 by introducing small haipin RNA for OCT3 / 4, SOX2, KLF4, L-MYC, LIN28 and p53 by episomal vectors according to the method described in 8: 409-12.
- FIG. 10A shows that the SOD1 gene of iPS cells established from the ALS patient has the mutation.
- the mutation of SOD1 gene possessed by the two ALS patients is L144FVX mutation (mutation where leucine after the 144th amino acid is substituted with phenylalanine-valine-stop codon, sometimes referred to as SOD1-L144FVX)
- a G93S mutation mutant in which glycine at the 93rd amino acid is substituted with serine, sometimes referred to as SOD1-G93S).
- iPS cells established from patients having the Control 1, Control 2, SOD1-L144FVX mutation or SOD1-G93S mutation are respectively referred to as Control1-derived iPS cells, Control2-derived iPS cells, L144FVX-derived iPS cells, G93S. I will call it an iPS cell.
- DNA introduction into human-derived iPS cells In the same manner as described above, the vector shown in Fig. 6A was introduced into the four types of iPS cells obtained by the above method, and the MNation factor expression cassette region was inserted into the chromosome. Got. Cells derived from the Control1-derived iPS cells, Control2-derived iPS cells, L144FVX-derived iPS cells, and G93S-derived iPS cells were introduced into MNation factor-Control1-derived iPS cells, MNation factor-introduced-Control2-derived iPS cells, and MNization, respectively. Factor introduction—L144FVX-derived iPS cells, MNation factor introduction—G93S-derived iPS cells.
- FIG. 10B shows a schematic diagram of the induction process.
- FIG. 10C shows a schematic diagram of the induction process.
- FIG. 10D the number of ⁇ III Tublin positive cells after 7 days and 14 days was counted as the number of iMN cells, and the value obtained by dividing the number of iMN cells after 14 days by the number of iMN cells after 7 days was graphed as the iMN survival rate (Fig. 10E). From Fig.
- MNation factor introduction-Control1 iPS cells MNation factor introduction-Control2-derived iPS cells have almost the same number of cells after 7 and 14 days, but MNation factor introduction-L144FVX-derived iPS cells It was found that iMN obtained from iPS cells derived from G93S-derived and M93 factor-derived iPS cells significantly decreased in cell number, ie, cell death within 7 to 14 days.
- Example 5 Synchronization of differentiation from pluripotent stem cells to iMN
- IMN production time course from mouse-derived iPS cells From the wild-type mouse-derived iPS cell line (201B7 strain, Takahashi K, et al, Cell. 2007, 131: 861-72.), The same method as in Example 2 was used. Thus, cells in which the MNation factor expression cassette region was inserted into the chromosome were obtained. The obtained cells were seeded in a 96-well plate and cultured in the DOX-containing medium to induce the expression of MNation factor. Immunostaining was performed 10, 24, 36, 48, and 72 hours after the start of induction, and the number of SSES1 or NCAM positive cells was counted.
- FIG. 11A As is clear from FIG. 11A, SSES1-positive cells (that is, undifferentiated cells) rapidly decreased after induction, and disappeared completely after 48 hours. In contrast, NCAM positive cells (that is, cells in the middle of differentiation into nerve cells) appear immediately after the start of induction, and after about 24 hours, cells with NCAM positive and thickened cell bodies and neurites (that is, iMN) appears.
- the pluripotent stem cell containing the Mn factor expression cassette region according to the present invention in the chromosome differentiates rapidly and synchronously to iMN after the induction of Mn factor expression. It was.
- transposon was used as the MNation factor introduction system.
- viral vectors are most preferred in systems aimed at inducing differentiation because they can achieve rapid and high expression of the target gene.
- Ngn2, Lhx3, and / or Isl1 gene was introduced and expressed in pluripotent stem cells, the result was that the synchrony of differentiation was higher when transposon was used ( Data not disclosed).
- the pluripotent stem cell after introducing a vector that expresses a drug-responsive MNation factor into a pluripotent stem cell to obtain a pluripotent stem cell in which the MN factor expression cassette region is inserted into the genome, the pluripotent stem cell is obtained.
- Stem cells were used for each experiment without cloning (ie, a heterogeneous cell population with different insertion sites and inserted copy numbers of the expression cassette). Therefore, in the present method, if the pluripotent stem cell is further cloned and a cell population having a uniform insertion site and number of copies of the foreign DNA is used, the synchronization and differentiation efficiency to iMN are as follows. Expected to be even higher.
- Example 6 Synchrony of cell death of iMN derived from ALS patient / model mouse
- MNation factor introduction-mouse G93A-derived iPS cells or MNation factor introduction-mouse WT-derived iPS cells prepared in Example 2 were seeded in a 96-well plate and cultured in the DOX-containing medium to express the MNation factor. Induced. 4, 6, and 10 days after induction, immunostaining ( ⁇ -III tubulin) and morphological observation were performed, and the number of iMN cells was counted. The results are shown in Table 1 and FIG. 12A.
- a pluripotent stem cell derived from an ALS model mouse having a mutant human SOD1 gene and having an exogenous nucleic acid encoding a MNation factor under the control of an inducible promoter in the chromosome is MNated. It became clear that the number of iMN reached the maximum after 2-3 days from the induction of the expression of the factor, but after that, the cell died rapidly, and most of them died after 8-10 days.
- MNation factor-introduced iPS cells prepared from humans and mice with mutant SOD1 gene are automatically directed to cell death immediately after differentiation into motor neurons (iMN) by induction of MNation factor expression. It was shown that.
- This motor neuron death is not due to an external cell death signal, but is an autonomous cell death resulting from the expression of a mutant SOD1 gene.
- large-scale cell death was not induced autonomously, and some external factors had to be added.
- motor neuron death in these systems cannot be substantially suppressed by riluzole, which is the only approved treatment for ALS, and there are concerns about differences from the original motor neurons (in ALS patients). It had been. Therefore, it was decided to analyze the effect of riluzole on MN cell death according to the present invention. .
- Example 7 Drug sensitivity of iMN derived from ALS patient
- the above-described MNation factor introduction-L144FVX-derived iPS cells were seeded in 96 wells and cultured in a DOX-containing medium to induce the expression of the MNation factor (this day is referred to as Day 0).
- FIG. 13A shows the result of immunostaining the cells after 7 and 14 days with anti- ⁇ -III tubulin antibody.
- iMN cell death was significantly suppressed in wells administered with riluzole compared to wells not administered.
- the results of measuring the number of iMN are shown in Table 3 and FIG. 13B. It was revealed that iMN cell death was suppressed in a concentration-dependent manner up to a riluzole concentration of 50 ⁇ M.
- an exogenous nucleic acid derived from an ALS patient having a SOD1 gene mutation and according to the present invention that is, a nucleic acid in which a nucleic acid encoding a MNation factor is functionally joined to a drug-responsive promoter
- IMN obtained from iPS cells contained in the chromosome is induced by the toxicity itself of the mutant SOD1 gene product in the same manner as the motor neurons in the patient having the mutation, and the cell death is an ALS therapeutic drug.
- RVN was effectively suppressed.
- no motor neuron or iMN cell death system that is effectively suppressed by riluzole has been reported so far. Therefore, the iMN cell death system according to the present invention is very useful as a screening system and evaluation system for ALS therapeutic / prophylactic agents.
- FIG. 14 shows an outline of a screening system for ALS therapeutic / prophylactic drugs using iPS cells introduced with MNation factor having a mutant SOD1 gene according to the present invention.
- a DNA exemplified in FIG. 6A ie, a DNA encoding a MNation factor controlled by an inducible promoter.
- the iPS cells can be stably cultured while maintaining undifferentiated ability, pluripotency, and high proliferation ability.
- this system was used to screen existing drug compounds (about 1200 types).
- the test compound was administered to the well on Day 7, and the number of iMNs on Day 14 was measured.
- the results are shown in FIG. 15B.
- the existing drug compounds included compounds that showed no significant therapeutic effect in clinical trials using humans despite the fact that a therapeutic effect was observed on ALS model mice.
- the results for these compounds are shown in FIG. 15B (15 types with no underline under the compound name). It can be seen that none of the compounds can effectively suppress the cell death of iMN.
- the cell death analysis system using MPS-introduced iPS cells having a mutant SOD1 gene according to the present invention can be a reliable and accurate screening system for ALS treatment / prevention drugs. Indicated. Furthermore, it became clear that the ALS patient-derived iMN prepared by the method according to the present invention exhibits sensitivity correlated with the test results for several types of drugs analyzed in human clinical trials.
- Example 9 Method for producing iMN from pluripotent stem cells via blood cells
- Blood cells are the cells that are most easily introduced into individual animals. In particular, cells having migration ability such as monocytes and macrophages can autonomously move to the deep part of the animal individual. Therefore, the present inventor decided to examine whether pluripotent stem cells into which the DNA according to the present invention was introduced could be differentiated into motor neurons via blood cells.
- ES cells derived from MNation factor-introduced mice prepared in Example 1 were seeded on OP9 cells (day 0) and cultured in ⁇ MEM medium containing 10% FBS. On the fifth day of culture (FIG. 8A), Flk-1-positive and SSEA-1-negative cells were collected using FACS (FIG. 8B).
- the collected cells were seeded on OP9 cells and cultured using ⁇ MEM supplemented with 100 ng / ml mSCF, 20 ng / ml mIL-3 and 10 ng / ml mM-CSF.
- Giemsa staining was performed 7 days later (day 12 after the start of culture), the presence of monocytes / macrophages was confirmed (FIG. 8C), and it was confirmed that the pluripotent stem cells were induced to differentiate into blood cells. .
- the obtained monocytes / macrophages were dissociated with accumax, seeded on a matrigel-coated dish, and cultured in N3 medium. The next day, the cells were cultured in N3 medium supplemented with 1 ⁇ g / ml Doxycycline, 1 ⁇ M retinoic acid and 1 ⁇ M Smoothened Agonist (SAG). Eight days later, neuronal induction was evaluated by immunostaining. As a result, motor neurons were confirmed (FIG. 9). Therefore, it was shown that, after the MN-potent-introduced pluripotent stem cells according to the present invention were differentiated into blood cells, the expression of the MN-expression factor was induced to differentiate into iMN in about 7 days.
- Example 10 iN production from mouse-derived pluripotent stem cells
- iN production from mouse-derived pluripotent stem cells Up to the previous section, attention has been paid to motor neurons that are attracting attention as research subjects of ALS and SMA, and inventions relating to methods for producing such cells from pluripotent stem cells have been described. On the other hand, general nerve cells are also in high demand in research.
- the present inventor has also studied Alzheimer-type dementia using nerve cells obtained from pluripotent stem cells over about 2 months by a conventional method (adhesion method). And in the process of searching for the iMN production method, it was found that if only the Ngn2 gene among the MNation factors is introduced and expressed in pluripotent stem cells, it can be induced to iN very quickly and efficiently. is there.
- the piggybac transposon system includes a cassette linked to Ngn2 so that expression is controlled by a tetracycline-responsive promoter and a cassette linked to a promoter (EF1 ⁇ ) so that rtTA is expressed simultaneously as DNA to be introduced into pluripotent stem cells It was prepared as a construct (see Tanaka A, et al, PLoS One. 2013, 8: e61540) that sandwiched the 5 'side and the 3' side with Terminal repeat (TR) so that it can be introduced in the system.
- this DNA is referred to as DNA encoding the Ngn2 gene under the control of a tetracycline responsive promoter.
- Wild-type mouse-derived iPS cell line (201B7 strain, Takahashi K, et al, Cell. 2007, 131: 861-72.) was added to Ngn2 under the control of the tetracycline-responsive promoter.
- a DNA encoding a gene is introduced together with a nucleic acid encoding a transposase (Woltjen K, et al, Nature. 2009, 458: 766-70) and the DNA is inserted into the genome (hereinafter abbreviated as 201B7_Ngn2). Established).
- this 201B7_Ngn2 was separated into single cells, seeded on a matrigel (BD) -coated dish, and DOX-containing medium (ie, 1 ⁇ g / ml DOX, 10 ng / ml BDNF, 10 ng / ml GDNF and 10 ng / ml NT3) Ngn2 expression induction was started by culturing in N3 medium).
- DOX-containing medium ie, 1 ⁇ g / ml DOX, 10 ng / ml BDNF, 10 ng / ml GDNF and 10 ng / ml NT3
- Ngn2 expression induction was started by culturing in N3 medium).
- a few ⁇ III tublin positive cells were observed 2 days after the start of expression induction, and about 90% of the cells became ⁇ III tublin, MAP2 and vGLT1 positive after 3 days (ie, glutaminergic neurons), and after 4 days.
- Example 11 iN production from human-derived pluripotent stem cells
- iPS cells derived from Alzheimer-type dementia patients.
- N112E14 from normal controls
- AD8K213 from sporadic Alzheimer patients
- AD15E11 presenilin (PS1) mutant
- APP1E211 APP-E693delta
- AD8K213 and APP1E211 are small haipins against OCT3 / 4, SOX2, KLF4, L-MYC, LIN28 and p53 in episomal vectors according to the method described in Okita K, et al, Nat Methods. 2011, 8: 409-12.
- RNA was made by introduction into skin-derived fibroblasts collected with patient consent (see Kondo T, et al, Cell Stem Cell. 2013, 12: 487-96).
- N112E14 and AD15E11 were prepared from epidermal vectors from skin-derived fibroblasts collected with the consent of healthy persons or patients.
- Ngn2 expression induction was initiated by culturing in a DOX-containing medium (ie, Neurobasal medium (Life Technologies) supplemented with 2 ⁇ g / ⁇ l DOX and B-27 Supplement Minus AO (Life Technologies)). After 5 days, the medium was replaced with a culture solution having the same composition. From the start of Ngn2 expression induction to 4 days later, almost no ⁇ III tublin positive cells were observed, but it was observed that the number of positive cells increased rapidly after 5-7 days and hardly increased after 8 days. Moreover, the time course in which ⁇ III tublin-positive cells appear was not significantly different among the four types of stable expression strains. Therefore, it was revealed that iN can be obtained with high synchrony over 5-7 days by introducing and expressing the Ngn2 gene in human-derived pluripotent stem cells.
- DOX-containing medium ie, Neurobasal medium (Life Technologies) supplemented with 2 ⁇ g / ⁇ l DOX and B-27 Supplement Minus AO (
- the four types of stable expression strains were treated with any of the following 1) -3) after 3 days from the start of Ngn2 expression induction, and further cultured for 4 days (cells at this time) Is shown in FIG. 16B).
- the culture supernatant was collected on the 5th, 7th, 9th, and 11th days from the start of Ngn2 expression induction, and the MSD Abeta 3 plex (38, 40, 42) assay plate (Meso Scale Discovery) was used.
- the content of A ⁇ 40 and A ⁇ 42 peptides contained in the culture supernatant was measured (FIG. 16C).
- the change over time of the content of each A ⁇ peptide is shown in the upper part of FIG. 16B.
- the culture supernatant of iN derived from two Alzheimer-type dementia patients (AD8K213, AD15E11) contains a larger amount of A ⁇ 42 peptide than the culture supernatant of iN derived from normal control (N112E14).
- AD15E11-derived iN having a mutation that increases the production amount of A ⁇ 42 peptide resulted in the highest amount of A ⁇ 42 peptide in the culture supernatant.
- iN produced from the pluripotent stem cells derived from Alzheimer-type dementia patients by the method according to the present invention sufficiently produced A ⁇ characteristic of the disease 9 days after induction, and the A ⁇ production pathway It is thought that it can be suitably used as a screening system for inhibitors.
- the conventional method for example, the adhesion method
- the effect of the present invention is very large. I think that the.
- Example 12 In vivo differentiation induction from pluripotent stem cells to iMN]
- an agent that induces activation of the promoter under culture is added to a pluripotent stem cell into which a DNA encoding MNation factor or Ngn2 is introduced under the control of a drug-responsive promoter.
- MNation factor or Ngn2 is introduced under the control of a drug-responsive promoter.
- MNation factor or Ngn2 To induce expression of MNation factor or Ngn2.
- derivation process could be performed in vivo.
- the pluripotent stem cell according to the present invention (that is, a pluripotent stem cell into which a DNA encoding a MNation factor is introduced under the control of a drug-responsive promoter) can be converted into a motor neuron in an animal body. confirmed.
- the pluripotent stem cells according to the present invention can be converted into iMN by inducing the expression of the MNation factor after differentiation into blood cells
- a method may be considered in which blood cells derived from the pluripotent stem cell are transplanted into the animal body and then induced to differentiate into iMN.
- Example 13 In vivo differentiation induction from pluripotent stem cells to iN] Similarly, the N-factor-introduced mouse iPS cells (201B7_Ngn2 cells) prepared in Example 5 were transplanted into the hippocampus of NOG mice one week after drinking DOX, and the cells at the administration site were transplanted 4 weeks after transplantation. It was observed by immunostaining (FIG. 18). It was confirmed that hNCAM positive cells were confirmed, converted into nerve cells in the mouse brain, and could be engrafted.
- ES / iPS cell-derived neurons or motor neurons can be induced in the mouse brain or spinal cord, and a model mouse having human cells for examining candidate drugs in vivo is prepared. It was suggested that it can be done. Furthermore, it was suggested that after transplanting to a desired site in the state of pluripotent stem cells, the cells are more easily engrafted in the site by converting them into motor neurons or neurons in vivo.
- iPS cell technology has been developed mainly for the purpose of creating disease models and transplanted tissues and cells.
- safety tests are conducted using iPS cells derived from healthy individuals, and the effectiveness of the test drug is assessed using iPS cells derived from patients. It is supposed to be produced. That is, it has been envisaged so far to search for and develop candidate drugs in an experimental system using iPS cells, and then conduct clinical trials targeting humans.
- iPS cells derived from different patients Although the disclosure of results was omitted in this application, there were also compounds with significantly different reactivity. It was concluded that the compound has a large variability in reactivity among patients and is not significant. This result suggests that by using the MN analysis system according to the present invention, it is possible to analyze in detail the difference in drug reactivity between cell providers (individuals) and to elucidate the reason for the difference. ing. Furthermore, iPS cells are established from responders and non-responders when the responders and non-responders are found after the actual clinical trials, or after the actual clinical trials.
- marker some characteristic common to each group can be found by inducing differentiation / analysis to MN by the method according to the present invention. And if such a marker is found, it should be possible to conduct a highly accurate clinical trial with a relatively small number of subjects by conducting the second phase test only on the responder having the marker.
- Figure 19 shows a conceptual diagram of a clinical trial using responder / non-responder markers.
- this is a method of using iPS cell technology that is the opposite of the method that has been envisaged so far, in which analysis targets are classified from the results of clinical trials targeting humans and analyzed in an experimental system using iPS cells.
- the present invention since uniform motor neurons or neurons can be efficiently induced from pluripotent stem cells, a cell model excellent in neurodegenerative diseases and nerve damage can be provided. It is useful for searching for therapeutic drugs.
- blood cells derived from pluripotent stem cells that can be induced into motor neurons can be directly induced into motor neurons and accumulate at the defective site of motor neurons. Thus, the blood cells are useful for the treatment of neurodegenerative diseases or nerve damage.
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Abstract
Description
さらに、本発明は、本来の運動神経細胞/神経細胞の性質を良く再現した運動神経細胞又は神経細胞に、薬剤処理によって迅速且つ同調して分化することができる多能性幹細胞の提供を目的としている。
さらに、前記3種類の遺伝子のうちNgn2のみを多能性幹細胞に導入して発現させると迅速且つ同調して神経細胞へと分化すること、及び、アルツハイマー型認知症患者由来iPS細胞から該方法で作製した神経細胞は該疾患に特徴的なAβ産生を行うことが確認された。
本発明者は、これらの知見に基づいて本発明を完成するに至った。
[1] 多能性幹細胞から運動神経細胞を製造する方法であって、下記工程;
(1)多能性幹細胞に、Lhx3、Ngn2、及びIsl1をコードする核酸を導入する工程、
(2)Lhx3、Ngn2、及びIsl1の発現を3日間以上維持する工程、
を(1)-(2)の順番で含む、多能性幹細胞から運動神経細胞を製造する方法。
[2] 前記核酸の導入が、トランスポゾンを用いて導入する、[1]に記載の方法。
[3] 前記トランスポゾンが、piggyBacトランスポゾンである、[2]に記載の方法。
[4] 前記工程(2)が、前記核酸を薬剤応答性プロモーターで発現させる工程である、[1]-[3]のいずれかに記載の方法。
[5] 前記薬剤応答性プロモーターがテトラサイクリン応答性プロモーターである、[4]に記載の方法。
[6] 前記核酸を、Lhx3、Ngn2、及びIsl1をポリシストロニックに発現させる、[1]に記載の方法。
[7] 前記核酸が、Lhx3、Ngn2、及びIsl1をコードする核酸が2A配列により結合された核酸である、[6]に記載の方法。
[8] 前記工程(2)において、Lhx3、Ngn2、及びIsl1の発現を維持する期間が7日間以上である、[1]-[7]のいずれかに記載の方法。
[9] 前記工程(2)において、前記工程(1)で得られた細胞を動物体内に導入する工程をさらに含み、該動物体内で前記薬剤応答性プロモーターに対応する薬剤と接触させる、[6]-[8]のいずれかに記載の方法。
[10] 前記工程(1)において、前記核酸が導入された多能性幹細胞を、血球細胞に分化誘導する工程をさらに含む、[1]-[9]のいずれかに記載の方法。
[11] 前記血球細胞が、単球及び/又はマクロファージである、[10]に記載の方法。
[12] 前記血球細胞に分化誘導する工程が、前記核酸が導入された多能性幹細胞を、骨髄由来間質細胞と共培養し、さらに幹細胞因子、マクロファージ-コロニー刺激因子、及びインターロイキン-3存在下で培養することで血球細胞に分化誘導する工程である、[10]又は[11]に記載の方法。
[13] 前記多能性幹細胞が、ヒト人工多能性幹細胞である、[1]-[12]のいずれかに記載の方法。
[14] 前記多能性幹細胞が、変異型SOD1遺伝子を有する多能性幹細胞である、[1]-[13]のいずれかに記載の方法。
[15] 下記工程(1)-(3)を含むことを特徴とする、運動神経変性疾患又は神経損傷の治験薬に対して治療効果が認められた被験者(すなわち、応答者)又は治療効果が認められなかった被験者(すなわち、非応答者)に特異的なマーカーを同定する方法;
(1)前記応答者及び非応答者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、[1]-[8]のいずれかに記載の方法によって、運動神経細胞を製造する工程、
(3)前記工程(2)で得られた応答者に由来する運動神経細胞と非応答者に由来する運動神経細胞の遺伝子産物の発現を測定する工程、及び
(4)応答者に由来する運動神経細胞において、非応答者より発現量が多い遺伝子産物を応答者に特異的なマーカーとして同定する工程、又は、応答者に由来する運動神経細胞において、非応答者より発現量が少ない遺伝子産物を非応答者に特異的なマーカーとして同定する工程。
[16] 下記工程(1)-(3)を含むことを特徴とする、治療薬が有効である対象を選別する方法;
(1)被験者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、[1]-[8]のいずれかに記載の方法によって、運動神経細胞を製造する工程、
(3)前記工程(2)で得られた運動神経細胞において、[15]に記載の方法によって同定された応答者及び/又は非応答者に特異的なマーカーを検出する工程、
(4)前記応答者に特異的なマーカーが検出された運動神経細胞、又は前記非応答者に特異的なマーカーが検出されなかった運動神経細胞が由来する被験者を、対応する治療薬が有効である対象として選別する工程。
[17] 外来性のLhx3、Ngn2、及びIsl1をコードする核酸が、染色体に挿入された多能性幹細胞。
[18] 前記核酸が、誘導可能なプロモーターの制御下に、Lhx3、Ngn2、及びIsl1をポリシストロニックに発現する核酸である、[17]に記載の多能性幹細胞。
[19] 前記核酸が、トランスポゾンによって染色体に挿入された核酸である、[18]に記載の多能性幹細胞。
[20] 前記トランスポゾンが、piggyBacトランスポゾンである、[19]に記載の多能性幹細胞。
[21] 前記核酸が、薬剤応答性プロモーターに機能的に連結された核酸である、[17]-[20]のいずれかに記載の多能性幹細胞。
[22] 前記薬剤応答性プロモーターがテトラサイクリン応答性プロモーターである、[21]に記載の多能性幹細胞。
[23] 前記核酸が、Lhx3、Ngn2、及びIsl1をコードする配列を2A配列で結合した核酸である、[18]-[22]のいずれかに記載の多能性幹細胞。
[24]
前記多能性幹細胞が、ヒト人工多能性幹細胞である、[17]-[23]のいずれかに記載の多能性幹細胞。
[25] 前記多能性幹細胞が、変異型SOD1遺伝子を有する多能性幹細胞である、[17]-[24]のいずれかに記載の多能性幹細胞。
[26] [17]-[24]のいずれかに記載の多能性幹細胞から分化誘導した血球細胞。
[27] 前記血球細胞が単球及び/又はマクロファージである、[26]に記載の血球細胞。
[28] [17]-[24]のいずれかに記載の多能性幹細胞を有効成分として含む、運動神経変性疾患又は神経損傷の治療用組成物。
[29] 前記運動神経変性疾患が、筋萎縮性側索硬化症である、[28]に記載の治療用組成物。
[30] [26]又は[27]に記載の血球細胞を有効成分として含む、運動神経変性疾患又は神経損傷の治療用組成物。
[31] 前記運動神経変性疾患が、筋萎縮性側索硬化症である、[30]に記載の治療用組成物。
[32] 下記工程(1)-(5)を含む、筋萎縮性側索硬化症の治療薬をスクリーニングする方法;
(1)筋萎縮性側索硬化症患者から単離した体細胞から製造した人工多能性幹細胞から、[1-8のいずれかに記載の方法によって運動神経細胞を製造する工程、
(2)前記工程(1)で得られた運動神経細胞を、被験物質と接触させる工程、
(3)前記工程(2)で前記被験物質と接触させた運動神経細胞、及び前記被験物質を接触させなかった運動神経細胞(すなわち、対照細胞)を培養する工程、
(4)前記工程(3)で得られた運動神経細胞の細胞数及び/又は神経突起長を測定する工程、
(5)前記被験物質と接触させた運動神経細胞の細胞数及び/又は神経突起長が、対照よりも高値であった被験物質を、筋萎縮性側索硬化症の治療薬として選択する工程。
[33] 前記筋萎縮性側索硬化症患者から単離した体細胞が、SOD1変異を有する体細胞である、[32]に記載の方法。
[34] 多能性幹細胞から神経細胞を製造する方法であって、下記工程;
(1)多能性幹細胞に、Ngn2をコードする核酸を、トランスポゾンを用いて導入する工程、
(2)前記プロモーターの活性化を誘導して、Ngn2の発現を3日間以上維持する工程、
を(1)-(2)の順番で含む、多能性幹細胞から神経細胞を製造する方法。
[35]
前記トランスポゾンが、piggyBacトランスポゾンである、[34]に記載の方法。
[36] 前記工程(2)が、前記核酸を、薬剤応答性プロモーターで発現させる工程である、[34]又は[35]に記載の方法。
[37] 前記薬剤応答性プロモーターがテトラサイクリン応答性プロモーターである、[36]に記載の方法。
[38] 前記工程(2)において、Ngn2の発現を維持する期間が7日間以上である、[34]-[37]のいずれかに記載の方法。
[39] 前記工程(2)において、前記工程(1)で得られた細胞を動物体内に導入する工程をさらに含み、該動物体内で前記薬剤応答性プロモーターに対応する薬剤と接触させる、[34]-[38]のいずれかに記載の方法。
[40] 前記多能性幹細胞が、ヒト人工多能性幹細胞である、[34]-[39]のいずれかに記載の方法。
[41] 前記ヒト人工多能性幹細胞が、アルツハイマー型認知症患者から単離した体細胞より製造された人工多能性幹細胞である、[40]に記載の方法。
[42] 前記ヒト人工多能性幹細胞が、プレセニリン1遺伝子に変異を有するヒト人工多能性幹細胞である、[40]に記載の方法。
[43] 下記工程(1)-(3)を含むことを特徴とする、アルツハイマー型認知症の治験薬に対して治療効果が認められた被験者(すなわち、応答者)又は治療効果が認められなかった被験者(すなわち、非応答者)に特異的なマーカーを同定する方法;
(1)前記応答者及び非応答者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、[34]-[38]のいずれかに記載の方法によって、神経細胞を製造する工程、
(3)前記工程(2)で得られた応答者に由来する神経細胞と非応答者に由来する神経細胞の遺伝子産物の発現を測定する工程、及び
(4)応答者に由来する運動神経細胞において、非応答者より発現量が多い遺伝子産物を応答者に特異的なマーカーとして同定する工程、又は、応答者に由来する運動神経細胞において、非応答者より発現量が少ない遺伝子産物を非応答者に特異的なマーカーとして同定する工程。
[44] 下記工程(1)-(3)を含むことを特徴とする、治療薬が有効である対象を選別する方法;
(1)被験者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、[34]-[38]のいずれかに記載の方法によって、神経細胞を製造する工程、
(3)前記工程(2)で得られた運動神経細胞において、[43]に記載の方法によって同定された応答者及び/又は非応答者に特異的なマーカーを検出する工程、
(4)前記応答者に特異的なマーカーが検出された神経細胞、又は前記非応答者に特異的なマーカーが検出されなかった神経細胞が由来する被験者を、対応する治療薬が有効である対象として選別する工程。
[45] 外来性のNgn2をコードする核酸が、トランスポゾンによって染色体に挿入された多能性幹細胞。
[46] 前記トランスポゾンが、piggyBacトランスポゾンである、[44]に記載の多能性幹細胞。
[47] 前記核酸が、薬剤応答性プロモーターに機能的に連結された核酸である、[45]又は[46]に記載の多能性幹細胞。
[48] 前記薬剤応答性プロモーターがテトラサイクリン応答性プロモーターである、[47]に記載の多能性幹細胞。
[49] 前記多能性幹細胞が、ヒト人工多能性幹細胞である、[45]-[48]のいずれかに記載の多能性幹細胞。
[50] 前記ヒト人工多能性幹細胞が、アルツハイマー型認知症患者から単離された体細胞から製造されたヒト人工多能性幹細胞である、[49]に記載の多能性幹細胞。
[51] 前記ヒト人工多能性幹細胞が、プレセニリン1遺伝子に変異を有するヒト人工多能性幹細胞である、[49]に記載の方法。
[52] 下記工程(1)-(4)を含む、アルツハイマー型認知症の治療薬をスクリーニングする方法;
(1)アルツハイマー型認知症患者から単離した体細胞から製造した人工多能性幹細胞から、前記[34]-[38]のいずれかに記載の方法によって神経細胞を製造する工程、
(2)前記工程(1)で得られた神経細胞を、被験物質と接触させる工程、
(3)前記工程(2)で前記被験物質と接触させた神経細胞、及び接触させなかった神経細胞(すなわち、対照細胞)を培養し、培地中のAβ42の含有量を測定する工程、
(4)前記被験物質と接触させた神経細胞の培地中のAβ42の含有量が、前記対照細胞の培地中のAβ42の含有量よりも低値であった被験物質を、アルツハイマー型認知症の治療薬として選択する工程。
[53] [52]に記載された方法において、
前記工程(3)において、培地中のAβ40の含有量をさらに測定し、及び、
前記工程(4)において、
前記被験物質と接触させた神経細胞の培地中のAβ42の含有量をAβ40の含有量で除した値(すなわち、Aβ42の含有量/Aβ40の含有量)が、前記対照細胞の培地中のAβ42の含有量/Aβ40の含有量よりも低値であった被験物質を、アルツハイマー型認知症の治療薬として選択する工程である、
アルツハイマー型認知症の治療薬をスクリーニングする方法
また、本発明により、薬剤処理等によって迅速且つ同調して運動神経細胞又は神経細胞へ分化し得る多能性幹細胞及び/又は血球細胞が提供される。当該多能性幹細胞及び血球細胞は、生体内で運動神経細胞又は神経細胞へ分化誘導できるため、神経変性疾患又は神経損傷の治療用組成物(移植療法剤)として好適に用いることができる。
なお、以降の記載では、本発明に係る方法で得られる運動神経細胞、神経細胞を、本来の(すなわち、生来の)運動神経細胞、神経細胞と区別するために、各々iMN、iNと呼ぶ場合がある。また、前記Lhx3、Ngn2、及びIsl1遺伝子を合わせて運動神経細胞化因子(又は、MN化因子)、Ngn2遺伝子のみを神経細胞化因子(又は、N化因子)と呼ぶ場合がある。
本発明により、多能性幹細胞に、Lhx3、Ngn2、及びIsl1をコードする核酸を導入し、該3遺伝子の発現を3日間以上維持することで、運動神経細胞へと迅速且つ同調して分化させる方法が提供される。前記3遺伝子の発現誘導は、培養下、又は動物体内のいずれで行ってもよい。さらに、前記発現ベクターが導入された多能性幹細胞を、培養下で血球細胞へと分化誘導した後に前記3遺伝子の発現誘導を行い、血球細胞経由で運動神経細胞を製造することもできる。
また、本発明により、多能性幹細胞に、Ngn2をコードする核酸をトランスポゾンを用いて導入し、該遺伝子の発現を3日間以上維持することで、神経細胞へと迅速且つ同調して分化させる方法が提供される。前記Ngn2遺伝子の発現誘導は、培養下、又は動物体内のいずれで行うことができる。
以下に、これらの方法を構成する技術について詳述する。
本発明において、多能性幹細胞とは、生体に存在するすべての細胞に分化可能である多能性を有し、かつ、増殖能をも併せもつ幹細胞のことである。例として、以下に限定するものではないが、胚性幹(ES)細胞、核移植により得られるクローン胚由来の胚性幹(ntES)細胞、精子幹細胞(「GS細胞」)、胚性生殖細胞(「EG細胞」)、人工多能性幹(iPS)細胞、培養線維芽細胞や骨髄幹細胞由来の多能性細胞(Muse細胞)などが含まれる。これらのうち、本発明に用いるのに好ましい多能性幹細胞は、ES細胞、ntES細胞、及びiPS細胞である。以下、各幹細胞について説明する。
ES細胞は、ヒトやマウスなどの哺乳動物の初期胚(例えば胚盤胞)の内部細胞塊から樹立された、多能性と自己複製による増殖能を有する幹細胞である。
ES細胞は、受精卵の8細胞期、桑実胚後の胚である胚盤胞の内部細胞塊に由来する胚由来の幹細胞であり、成体を構成するあらゆる細胞に分化する能力、いわゆる分化多能性と、自己複製による増殖能とを有している。ES細胞は、マウスで1981年に発見され(M.J. Evans and M.H. Kaufman(1981), Nature 292:154-156)、その後、ヒト、サルなどの霊長類でもES細胞株が樹立された(J.A. Thomson et al.(1998), Science 282:1145-1147; J.A. Thomson et al.(1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848;J.A. Thomson et al.(1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall(1998), Curr. Top. Dev. Biol., 38:133-165)。
ES細胞の選択は、一般に、アルカリホスファターゼ、Oct-3/4、Nanogなどの遺伝子マーカーの発現を指標にしてReal-Time PCR法で行うことができる。特に、ヒトES細胞の選択では、OCT-3/4、NANOG、ECADなどの未分化細胞に特異的な遺伝子マーカーの発現を指標とすることができる(E. Kroon et al.(2008), Nat. Biotechnol., 26:443-452)。
精子幹細胞は、精巣由来の多能性幹細胞であり、精子形成のための起源となる細胞である。この細胞は、ES細胞と同様に、種々の系列の細胞に分化誘導可能であり、例えばマウス胚盤胞に移植するとキメラマウスを作出できるなどの性質をもつ(M. Kanatsu-Shinohara et al.(2003) Biol. Reprod., 69:612-616; K. Shinohara et al.(2004), Cell, 119:1001-1012)。神経膠細胞系由来神経栄養因子(glial cell line-derived neurotrophic factor(GDNF))を含む培養液で自己複製可能であるし、またES細胞と同様の培養条件下で継代を繰り返すことによって、精子幹細胞を得ることができる(竹林正則ら(2008),実験医学,26巻,5号(増刊),41~46頁,羊土社(東京、日本))。
胚性生殖細胞は、胎生期の始原生殖細胞から樹立される、ES細胞と同様な多能性をもつ細胞であり、LIF、bFGF、幹細胞因子(stem cell factor)などの物質の存在下で始原生殖細胞を培養することによって樹立しうる(Y. Matsui et al.(1992), Cell, 70:841-847; J.L. Resnick et al.(1992), Nature, 359:550-551)。
人工多能性幹(iPS)細胞は、特定の初期化因子を、DNA又はタンパク質の形態で体細胞に導入することによって作製することができる、ES細胞とほぼ同等の特性、例えば分化多能性と自己複製による増殖能、を有する体細胞由来の人工の幹細胞である(K. Takahashi and S. Yamanaka(2006) Cell, 126:663-676; K. Takahashi et al.(2007), Cell, 131:861-872; J. Yu et al.(2007), Science, 318:1917-1920; Nakagawa, M.ら,Nat. Biotechnol. 26:101-106(2008);国際公開WO 2007/069666)。初期化因子は、ES細胞に特異的に発現している遺伝子、その遺伝子産物もしくはnon-cording RNA又はES細胞の未分化維持に重要な役割を果たす遺伝子、その遺伝子産物もしくはnon-cording RNA、あるいは低分子化合物によって構成されてもよい。初期化因子に含まれる遺伝子として、例えば、Oct3/4、Sox2、Sox1、Sox3、Sox15、Sox17、Klf4、Klf2、c-Myc、N-Myc、L-Myc、Nanog、Lin28、Fbx15、ERas、ECAT15-2、Tcl1、beta-catenin、Lin28b、Sall1、Sall4、Esrrb、Nr5a2、Tbx3又はGlis1等が例示され、これらの初期化因子は、単独で用いても良く、組み合わせて用いても良い。初期化因子の組み合わせとしては、WO2007/069666、WO2008/118820、WO2009/007852、WO2009/032194、WO2009/058413、WO2009/057831、WO2009/075119、WO2009/079007、WO2009/091659、WO2009/101084、WO2009/101407、WO2009/102983、WO2009/114949、WO2009/117439、WO2009/126250、WO2009/126251、WO2009/126655、WO2009/157593、WO2010/009015、WO2010/033906、WO2010/033920、WO2010/042800、WO2010/050626、WO 2010/056831、WO2010/068955、WO2010/098419、WO2010/102267、WO 2010/111409、WO 2010/111422、WO2010/115050、WO2010/124290、WO2010/147395、WO2010/147612、Huangfu D, et al.(2008), Nat. Biotechnol., 26: 795-797、Shi Y, et al.(2008), Cell Stem Cell, 2: 525-528、Eminli S, et al.(2008), Stem Cells. 26:2467-2474、Huangfu D, et al.(2008), Nat Biotechnol. 26:1269-1275、Shi Y, et al.(2008), Cell Stem Cell, 3, 568-574、Zhao Y, et al.(2008), Cell Stem Cell, 3:475-479、Marson A,(2008), Cell Stem Cell, 3, 132-135、Feng B, et al.(2009), Nat Cell Biol. 11:197-203、R.L. Judson et al.,(2009), Nat. Biotech., 27:459-461、Lyssiotis CA, et al.(2009), Proc Natl Acad Sci U S A. 106:8912-8917、Kim JB, et al.(2009), Nature. 461:649-643、Ichida JK, et al.(2009), Cell Stem Cell. 5:491-503、Heng JC, et al.(2010), Cell Stem Cell. 6:167-74、Han J, et al.(2010), Nature. 463:1096-100、Mali P, et al.(2010), Stem Cells. 28:713-720、Maekawa M, et al.(2011), Nature. 474:225-9.に記載の組み合わせが例示される。
上記培養の間には、培養開始2日目以降から毎日1回新鮮な培養液と培養液交換を行う。また、核初期化に使用する体細胞の細胞数は、限定されないが、培養ディッシュ100cm2あたり約5×103~約5×106細胞の範囲である。
nt ES細胞は、核移植技術によって作製されたクローン胚由来のES細胞であり、受精卵由来のES細胞とほぼ同じ特性を有している(T. Wakayama et al.(2001), Science, 292:740-743; S. Wakayama et al.(2005), Biol. Reprod., 72:932-936; J. Byrne et al.(2007), Nature, 450:497-502)。すなわち、未受精卵の核を体細胞の核と置換することによって得られたクローン胚由来の胚盤胞の内部細胞塊から樹立されたES細胞がnt ES(nuclear transfer ES)細胞である。nt ES細胞の作製のためには、核移植技術(J.B. Cibelli et al.(1998), Nature Biotechnol., 16:642-646)とES細胞作製技術(上記)との組み合わせが利用される(若山清香ら(2008),実験医学,26巻,5号(増刊), 47~52頁)。核移植においては、哺乳動物の除核した未受精卵に、体細胞の核を注入し、数時間培養することで初期化することができる。
Muse細胞は、WO2011/007900に記載された方法にて製造された多能性幹細胞であり、詳細には、線維芽細胞又は骨髄間質細胞を長時間トリプシン処理、好ましくは8時間又は16時間トリプシン処理した後、浮遊培養することで得られる多能性を有した細胞であり、SSEA-3及びCD105が陽性である。
本発明において、筋萎縮性側索硬化症モデル細胞又はアルツハイマー型認知症モデル細胞とは、当該多能性幹細胞から上述した方法によって得られる、運動神経細胞又は神経細胞を含む。本発明において、好ましい病態のモデル細胞は、マウス又はヒト細胞である。
本発明において運動神経細胞とは、HB9、ChAT(choline acetyltransferase)等の運動神経細胞のマーカー遺伝子を1以上発現する細胞、あるいは、β-III tubulin、NCAM、MAP2等の神経細胞のマーカー遺伝子を1以上発現し、且つ、神経突起(ニューライトとも呼ばれる)と十分に肥厚した細胞体とを有する細胞、と定義される。前記神経細胞のマーカー遺伝子を1以上発現し、且つ、神経突起と十分に肥厚した細胞体とを有する細胞では、HB9又はChATの発現が認められることを確認しているからである。よって、本発明に係る方法で製造された運動神経細胞(すなわち、iMN)の判定基準もこれに従う。そして、本発明において運動神経細胞を製造するとは、上記定義を満たす細胞を含有する細胞集団を得ることを意味し、好ましくは、該細胞を5%、15%、又は20%以上含有する細胞集団を得ることである。
本発明において神経細胞とは、β-III tubulin、NCAM、MAP2等の神経細胞のマーカー遺伝子を1以上発現し、且つ、神経突起を有する細胞と定義される。よって、本発明に係る方法で製造された神経細胞(すなわち、iN)の判定基準もこれに従う。さらに、本発明において製造される神経細胞は、グルタミン酸作動性であることが好ましい。そして、本発明において神経細胞を製造するとは、上記定義を満たす細胞を含有する細胞集団を得ることを意味し、好ましくは、該細胞を50%、60%、70%、80%、又は90%以上含有する細胞集団を得ることである。
なお、Tuj1は抗β-III tubulinであることから、前記β-III tubulinを発現している細胞を、Tuj1陽性細胞と呼ぶ場合がある。
本発明においてLhx3とは、Lhx3(LIM homeobox 3)遺伝子及びLhx3タンパク質のことである。そして、Lhx3をコードする核酸とは、NCBIのアクッセッション番号:NM_001039653(マウス)もしくはNM_014564又はNM_178138(ヒト)で示されるポリヌクレオチドにコードされる遺伝子、及びその転写変異体、スプライシング変異体及びホモログ、又はこれらの核酸の相補鎖配列にストリンジェントな条件でハイブリダイズすることができる程度の相補関係を有する核酸であってもよい。
また、本発明においてNgn2とは、Ngn2(Neurogenin 2)遺伝子及びNgn2タンパク質のことである。そして、Ngn2をコードする核酸とは、NCBIのアクッセッション番号:NM_009718(マウス)又はNM_024019(ヒト)で示されるポリヌクレオチド又はその転写変異体、スプライシング変異体及びホモログ、又はこれらの核酸の相補鎖配列にストリンジェントな条件でハイブリダイズすることができる程度の相補関係を有する核酸であってもよい。
そして、本発明においてIsl1とは、Isl1(Islet 1)遺伝子及びIsl1タンパク質のことである。そして、Isl1をコードする核酸とは、NCBIのアクッセッション番号:NM_021459(マウス)又はNM_002202(ヒト)で示されるポリヌクレオチド又はその転写変異体、スプライシング変異体及びホモログ、又はこれらの核酸の相補鎖配列にストリンジェントな条件でハイブリダイズすることができる程度の相補関係を有する核酸であってもよい。
Lhx3をコードする核酸、Ngn2をコードする核酸、及びIsl1をコードする核酸を多能性幹細胞に導入する方法は特に限定されないが、例えば、以下の方法を用いることができる。
ウイルスベクターとしては、レトロウイルスベクター、レンチウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、センダイウイルスベクターなどが例示される。また、プラスミドベクターとしては、哺乳動物細胞用プラスミドを使用することができる。そして、人工染色体ベクターとしては、例えばヒト人工染色体(HAC)、酵母人工染色体(YAC)、細菌人工染色体(BAC、PAC)などが挙げられる。このうち、プラスミドベクター、及び人工染色体ベクターが好ましく、最も好ましくはプラスミドベクターである。
これらのベクターには、Lhx3、Ngn2、又はIsl1遺伝子が発現可能なように、プロモーター、エンハンサー、リボゾーム結合配列、ターミネーター、ポリアデニル化サイトなどの制御配列を含むことができ、さらに、必要に応じて、薬剤耐性遺伝子(例えばカナマイシン耐性遺伝子、アンピシリン耐性遺伝子、ピューロマイシン耐性遺伝子など)、チミジンキナーゼ遺伝子、ジフテリアトキシン遺伝子などの選択マーカー配列、蛍光タンパク質、βグルクロニダーゼ(GUS)、FLAGなどのレポーター遺伝子配列などを含むことができる。
Lhx3をコードする核酸、Ngn2をコードする核酸、及びIsl1をコードする核酸は、誘導可能なプロモーターに機能的に接合させることで、所望の時期に、Lhx3、Ngn2、又はIsl1の発現を誘導することができる。そのような誘導可能なプロモーターとしては、薬剤応答性プロモーターを挙げることができ、その好適な例として、テトラサイクリン応答性プロモーター(tetO配列が7回連続したテトラサイクリン応答配列(TRE)を有するCMV最小プロモーター)が挙げられる。該プロモーターは、リバーステトラサイクリン制御性トランス活性化因子(rtTA;reverse tetR(rTetR)とVP16ADから構成される融合タンパク質)の発現下において、テトラサイクリン又はその誘導体が供給されることにより活性化されるプロモーターである。よって、テトラサイクリン応答性プロモーターを用いて前記遺伝子の発現誘導を行う場合には、前記活性化因子を発現する様式を併せ持つベクターを用いるとさらに好適である。前記テトラサイクリンの誘導体としては、ドキシサイクリン(doxycycline、本願では以降、DOXと略記する)を好適に用いることができる。
また、上記以外の薬剤応答性プロモーターを用いた発現誘導系としては、エストロゲン応答性プロモーターを用いた発現誘導システム(例として、WO2006/129735)、RSL1によって誘導されるプロモーターを用いたRheoSwitch哺乳類誘導性発現システム(New England Biolabs社)、cumateによって誘導されるプロモーターを用いたQ-mateシステム(Krackeler Scientific社)又はCumate誘導性発現システム(National Research Council(NRC)社)、及びエクジソン応答性配列を有するプロモーターを用いたGenoStat誘導性発現システム(Upstate cell signaling solutions社)等が挙げられる。
上記目的で用いることができる構成的プロモーターとしては、SV40プロモーター、 LTRプロモーター、CMV(cytomegalovirus)プロモーター、RSV(Rous sarcoma virus)プロモーター、MoMuLV(Moloney mouse leukemia virus) LTR、HSV-TK(herpes simplex virus thymidine kinase)プロモーター、EF-αプロモーター、及びCAGプロモーター等が挙げられる。
上記のようにCre、FLP、トランスポゾンを用いて接合状態を変換することで発現誘導を行った場合には、所望の期間経過後に再度Cre、FLP、トランスポゾンを作用させて前記配列(LoxP配列、FRT配列、又はトランスポゾン配列)で挟まれた配列を除去することで、前記遺伝子の発現を停止させることもできる。
また、別の態様として、アデノウイルスベクター、アデノ随伴ウィルスベクター、センダイウイルスベクターやプラスミド、エピソーマルベクターなどの、容易に細胞内から消失させ得るベクターを用いることで前記遺伝子の発現期間を制御することも可能である。
神経細胞の製造においても同様に、導入したNgn2の発現は、3日間、4日間、5日間、6日間、7日間のいずれにおいても本発明の効果を奏することができ、長期になることで神経細胞の製造に不利益を生じることはないが、好ましくは3日以上14日以下、特に好ましくは7日以上14日以下である。
本発明において、前記Lhx3、Ngn2、及びIsl1をコードする核酸が導入された多能性幹細胞を、培養下で運動神経細胞に分化誘導する際に用いる培地としては、基本培地にレチノイン酸、SHHシグナル刺激剤、及び神経栄養因子を添加した培地(培養液)を用いることができる。そのような基本培地としては、例えば、Glasgow's Minimal Essential Medium(GMEM)培地、IMDM培地、Medium 199培地、Eagle's Minimum Essential Medium(EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium(DMEM)培地、Ham's F12培地、RPMI 1640培地、Fischer's培地、Neurobasal Medium(ライフテクノロジーズ)及びこれらの混合培地などが包含される。基本培地には、血清が含有されていてもよいし、あるいは無血清でもよい。必要に応じて、培地は、例えば、Knockout Serum Replacement(KSR)(ES細胞培養時のFBSの血清代替物)、N2サプリメント(Invitrogen)、B27サプリメント(Invitrogen)、アルブミン、トランスフェリン、アポトランスフェリン、脂肪酸、インスリン、コラーゲン前駆体、微量元素、2-メルカプトエタノール、3'-チオールグリセロールなどの1つ以上の血清代替物を含んでもよく、また、脂質、アミノ酸、L-グルタミン、Glutamax(Invitrogen)、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類、セレン酸、プロゲステロン及びプトレシンなどの1つ以上の物質も含有することができる。
このうち、本発明において好ましい基本培地は、インスリン、アポトランスフェリン、セレン酸、プロゲステロン及びプトレシンを含有するDMEM及びF12の混合培地であり、特に好ましくは、N3培地(100 μg/ml apotransferrin、5 μg/ml insulin、30 nM selenite、20 nM progesterone及び100 nM putrescineを添加したDMEM/F12))である。そして、当該N3培地に、1μMレチノイン酸(RA)、1μM sonic hedgehog(Shh)、10ng/ml BDNF、10ng/ml GDNF、及び10ng/ml NT3を配合した培地を、運動神経細胞の分化誘導培地として特に好適に用いることができる。
なお、上記N3培地の代わりにN2培地を用いた場合には、iMNへの分化の同調性、及びALSモデルマウス由来iMNの細胞死の同調性が低下する傾向が認められる場合がある。
培養液中におけるPurmorphamineの濃度は、Gli2を活性化する濃度であれば特に限定されないが、例えば、1nM以上、10nM以上、50nM以上、100nM以上、500nM以上、750nM以上、又は1μM以上で、50μM以下、40μM以下、30μM以下、25μM以下、20μM以下、15μM以下、又は10μM以下の範囲で適宜選択することができ、通常1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μMなどで使用され得るが、これらに限定されない。好ましくは、2μMである。
このうち、B27サプリメントを含有するNeurobasal Medium、又は、インスリン、アポトランスフェリン、セレン酸、プロゲステロン及びプトレシンを含有するDMEM及びF12の混合培地を基本培地として好適に用いることができる。
本発明の方法においては、神経細胞の分化誘導の際に、マウス細胞、特に、マウスのグリア細胞と共培養しなくとも神経細胞に誘導可能である。よって、夾雑物を混入させないことを目的として、マウス細胞との共培養を行わないことが望ましい。
本発明において、多能性幹細胞から運動神経細胞を製造する工程において、筋管細胞を混在させることで、筋管細胞と運動神経細胞とが接着した神経筋接合部を有する細胞培養物を得ることができる。神経筋接合部とは、運動神経細胞の突起末端よりアセチルコリンが放出され、筋管細胞に存在する受容体が受け取ることができる構造を意味する。神経筋接合部の存在は、例えば、免疫染色や蛍光顕微鏡観察を行い、運動神経細胞が発現するシナプス小胞タンパク質(例えば、SV2)と筋管細胞が発現するアセチルコリン受容体が共局在することによって確認することができる。当該神経筋接合部を含有する細胞培養物は、神経筋接合部の形成不全によって引き起こされる病態(例えば、重症筋無力症及びLambert-Eaton筋無力症)の病態のモデル系として、非常に有用である。
本発明により、Lhx3をコードする核酸、Ngn2をコードする核酸、及びIsl1をコードする外来性の核酸が染色体内に挿入された多能性幹細胞が提供される。当該核酸は誘導可能なプロモーターの制御下にあることが好ましく、より好ましくは、薬剤応答性プロモーターの制御下である。当該多能性幹細胞は、前記プロモーターが応答する薬剤と接触させることにより、迅速且つ同調して運動神経細胞へと分化し得る細胞である。
また、本発明により、Ngn2をコードする外来性の核酸が染色体内に挿入された多能性幹細胞が提供される。当該核酸は誘導可能なプロモーターの制御下にあることが好ましく、より好ましくは、薬剤応答性プロモーターの制御下である。当該当該多能性幹細胞は、前記プロモーターが応答する薬剤と接触させることにより、迅速且つ同調して神経細胞へと分化し得る細胞である。
これらの外来性核酸が染色体内に挿入された多能性幹細胞は、未分化能と高い増殖能を維持しており、前記形質を保持したまま増殖させることできる。さらに凍結保存を行っても前記形質が失われないことから、細胞株として安定に維持することが可能である。
前記外来性核酸が染色体内に挿入された多能性幹細胞が神経変性疾患患者に由来する場合には、該疾患の治療薬のスクリーニング系として好適に用いることができる。また、健常人に由来する場合には、運動神経変性疾患又は神経損傷の治療用組成物として、又はヒト由来運動神経細胞/神経細胞を保持するモデル動物の作製に好適に用いることができる。以下、各用途について詳述する。
本発明では、次の工程(1)-(5)を含む筋萎縮性側索硬化症の治療薬をスクリーニングする方法を提供する。
(1)筋萎縮性側索硬化症患者から単離した体細胞から製造した人工多能性幹細胞から、前述の方法によって運動神経細胞を製造する工程、
(2)前記工程(1)で得られた運動神経細胞を、被験物質と接触させる工程、
(3)前記工程(2)で前記被験物質と接触させた運動神経細胞、及び前記被験物質を接触させなかった運動神経細胞(すなわち、対照細胞)を培養する工程、
(4)前記工程(3)で得られた運動神経細胞の細胞数及び/又は神経突起長を測定する工程、
(5)前記被験物質と接触させた運動神経細胞の細胞数及び/又は神経突起長が、対照よりも高値であった被験物質を、筋萎縮性側索硬化症の治療薬として選択する工程。
また、本発明において、被験物質は、(1)生物学的ライブラリー、(2)デコンヴォルーションを用いる合成ライブラリー法、(3)「1ビーズ1化合物(one-bead one-compound)」ライブラリー法、及び(4)アフィニティクロマトグラフィ選別を使用する合成ライブラリー法を含む当技術分野で公知のコンビナトリアルライブラリー法における多くのアプローチのいずれかを使用して得ることができる。アフィニティクロマトグラフィー選別を使用する生物学的ライブラリー法はペプチドライブラリーに限定されるが、その他のアプローチはペプチド、非ペプチドオリゴマー、又は化合物の低分子化合物ライブラリーに適用できる(Lam(1997) Anticancer Drug Des. 12: 145-67)。分子ライブラリーの合成方法の例は、当技術分野において見出され得る(DeWitt et al.(1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al.(1994) Proc. Natl. Acad. Sci. USA 91: 11422-6;Zuckermann et al.(1994) J. Med. Chem. 37: 2678-85; Cho et al.(1993) Science 261: 1303-5; Carell et al.(1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et.al.(1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al.(1994) J. Med. Chem. 37: 1233-51)。化合物ライブラリーは、溶液(Houghten(1992) Bio/Techniques 13: 412-21を参照のこと)又はビーズ(Lam(1991) Nature 354: 82-4)、チップ(Fodor(1993) Nature 364: 555-6)、細菌(米国特許第5,223,409号)、胞子(米国特許第5,571,698号、同第5,403,484号、及び同第5,223,409号)、プラスミド(Cull et al.(1992) Proc. Natl. Acad. Sci. USA 89: 1865-9)若しくはファージ(Scott and Smith(1990) Science 249: 386-90; Devlin(1990) Science 249: 404-6; Cwirla et al.(1990) Proc. Natl. Acad. Sci. USA 87: 6378-82; Felici(1991) J. Mol. Biol. 222: 301-10; 米国特許出願公開第2002/0103360号)として作製され得る。
運動神経細胞の細胞数は、当業者に公知の方法を用いて免疫染色を行い、HB9、β-III tubulin、ChAT等の神経細胞又は運動神経細胞のマーカー遺伝子を発現する細胞の数として計測することができ、細胞画像解析装置(インセルアナライザー)を用いて自動的に計測してもよい。また、当該細胞数は、死細胞の数の逆数として算出してもよい。死細胞の数の測定は、例えば、LDHの活性を測定する方法、MTT 法、WST-1法、WST-8法を用いて吸光度を測定する方法、又は、TO(thiazole orange) 、PI( propidium iodide)、7AAD、カルセインAM、又はエチジウムホモダイマー1(EthD-1)を用いて染色し、フローサイトメーターを用いて計数する方法等によって行うことができ、さらに細胞画像解析装置(インセルアナライザー)を用いて自動的に行うこともできる。
本発明においては、前記被験物質と接触させた運動神経細胞の細胞数及び/又は神経突起長が、前記対照細胞の細胞数及び/又は神経突起長と比べて、1.5倍以上、1.6倍以上、1.7倍以上、1.8倍以上、1.9倍以上、2倍以上、2.5倍以上又は3倍以上である場合に“高値”と判断してもよい。なお、本願における前記対照細胞には、被験物質と接触させなかった運動神経細胞、及び有効性がないことが確認されている薬剤を接触させた運動神経細胞が包含される。
本発明では、次の工程を含むアルツハイマー型認知症の治療薬をスクリーニングする方法を提供する。
(1)アルツハイマー型認知症患者から単離した体細胞から製造した人工多能性幹細胞から、前述の方法によって神経細胞を製造する工程、
(2)前記工程(1)で得られた神経細胞を、被験物質と接触させる工程、
(3)前記工程(2)で前記被験物質と接触させた神経細胞、及び接触させなかった神経細胞(すなわち、対照細胞)を培養し、培地中のAβ42の含有量を測定する工程、
(4)前記被験物質と接触させた神経細胞の培地中のAβ42の含有量が、前記対照細胞の培地中のAβ42の含有量よりも低値であった被験物質を、アルツハイマー型認知症の治療薬として選択する工程。
その際、Aβ42の測定値を指標として用いてもよく、Aβ40の値で除した値(Aβ42/Aβ40)を指標として用いてもよい。培養上清の回収は、例えば、培地交換後、2日間培養した上清を用いてもよい。
アルツハイマー型認知症の治療剤の被験物質(すなわち、候補薬剤)としては、上述した筋萎縮性側索硬化症治療薬のスクリーニング方法における被験物質と同様の物質を用いることができる。
健常人に由来し、且つ、本発明に係る方法によって外来性核酸(Lhx3をコードする核酸、Ngn2をコードする核酸、及びIsl1をコードする核酸が2A配列を介して結合され、且つ、薬剤応答性プロモーターに機能的に接合された外来性の核酸)が染色体内に組み込まれた多能性幹細胞、及び該多能性幹細胞を分化誘導することで得られる運動神経細胞(途中の分化段階のものも含む)は、運動神経変性疾患又は神経損傷の治療用組成物(移植療法剤)として用いることができる。好適な対象疾患は、運動神経細胞が欠損又は損傷することによって引き起こされる疾患であり、例えば、筋萎縮性側索硬化症(ALS)、脊髄性筋萎縮症(SMA)、及び球脊髄性筋萎縮症等が挙げられる。
上記治療用組成物として用いる細胞は、移植後の組織内での生着率の高さから、前記多能性幹細胞及び/または運動神経細胞への分化途中の細胞が好ましく、特に好ましくは前記多能性幹細胞である。
本発明において、運動神経細胞及び神経細胞を製造するにあたり、上述した薬剤応答性プロモーターにて発現を誘導することができる運動神経誘導因子又は神経誘導因子を導入した多能性幹細胞を体細胞に分化誘導した後、前記薬剤応答性プロモーターに対応する薬剤と接触させることによって、運動神経細胞及び神経細胞を製造しても良い。分化誘導させる体細胞は特に限定されないが、マイクログリア細胞、血球細胞など遊走能を有する細胞であることが望ましい。特に好ましくは、単球及び/又はマクロファージである。
本発明では、前記外来性核酸(Lhx3をコードする核酸、Ngn2をコードする核酸、及びIsl1をコードする核酸が2A配列を介して結合され、且つ、薬剤応答性プロモーターに機能的に接合された外来性の核酸)を染色体内に含む多能性幹細胞から誘導された血球細胞を提供する。当該血球細胞は、前記プロモーターが応答する薬剤と接触させることにより、運動神経細胞へと変換することができる細胞である。血球細胞、特に炎症細胞は、運動神経変性疾患又は神経損傷の病変部に浸潤し、運動神経細胞の欠損部位に集積する性質があるので、当該欠損部位で運動神経に変換させることにより、効果的に運動神経細胞を補充することができる。
よって、健常人に由来し、且つ、前記外来性核酸(Lhx3をコードする核酸、Ngn2をコードする核酸、及びIsl1をコードする核酸が2A配列を介して結合され、且つ、薬剤応答性プロモーターに機能的に接合された外来性の核酸)を染色体内に含む多能性幹細胞から分化誘導された血球細胞は、運動神経変性疾患又は神経損傷の移植療法剤として、非常に有益である。対象となる運動神経変性疾患は、運動神経細胞が欠損又は損傷することによって引き起こされる病態を有するものであり、例えば、筋萎縮性側索硬化症(ALS)、脊髄性筋萎縮症(SMA)、及び球脊髄性筋萎縮症が挙げられる。
また、特に限定されないが、OP9細胞(Nakano T, et al. Science 265:1098-1101, 1994)上で当該多能性幹細胞を培養する、又は、胚様体を形成し、培養液中へ産出された血球細胞を採取し、各マーカー(例、CD68、CD14、CD11c、CD11b、CD32、CD43、CD69、CD44、CD154、CD19、CD20、CD4、CD8等)を指標として所望する血球細胞を単離することによって得ることができる。本発明において、好ましい血球細胞は、くは、少なくともCD14を発現する単球及び/又はマクロファージであり、例えば、WO2012/115276に記載の方法を適宜参照して、製造することができる。より好ましくは、多能性幹細胞を骨髄由来間質細胞と共培養し、さらに幹細胞因子、マクロファージ-コロニー刺激因子、及びインターロイキン-3存在下で培養する方法である。
前記血球細胞を有効成分として含む治療用組成物は、必要に応じて複数回投与してもよく、あるいは自体公知の手法を用いて、持続放出可能な製剤形態に調製して用いることもできる。また、前述と同様に、他の神経変性疾患又は神経損傷の治療薬と適宜併用してもよい。
本発明では、運動神経細胞及び神経細胞を製造するにあたり、上述した薬剤応答性プロモーターにて発現を誘導することができる運動神経誘導因子又は神経誘導因子を導入した多能性幹細胞を動物体内に導入する工程をさらに含み、該動物体内で前記薬剤応答性プロモーターに対応する薬剤と接触させることによって、動物体内で運動神経細胞及び神経細胞を製造しても良い。本発明において、動物とは、哺乳動物であり、より好ましくは、ヒト、マウス、ラット等の哺乳動物である。
そして、本発明では、前述した手法に従って、前記外来性核酸が染色体内に組み込まれた多能性幹細胞、及び該多能性幹細胞から分化誘導して得られる細胞を動物に移植することにより、ヒト由来運動神経細胞又は神経細胞が生着した動物を作製することができる。当該動物は、薬物や外的ストレスがヒト運動神経細胞又は神経細胞に及ぼす影響をより自然な状態で解析するための好適なモデル動物である。
本発明は、運動神経変性疾患又は神経損傷の治験薬に対して治療効果が認められた被験者(すなわち、応答者)又は治療効果が認められなかった被験者(すなわち、非応答者)に特異的なマーカーを同定する方法または検出する方法を提供する。
(1)前記応答者及び非応答者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、上述の方法によって、運動神経細胞を製造する工程、
(3)前記工程(2)で得られた応答者に由来する運動神経細胞と非応答者に由来する運動神経細胞の遺伝子産物の発現を測定する工程、及び
(4)応答者に由来する運動神経細胞において、非応答者より発現量が多い遺伝子産物を応答者に特異的なマーカーとして同定する工程、又は、応答者に由来する運動神経細胞において、非応答者より発現量が少ない遺伝子産物を非応答者に特異的なマーカーとして同定する工程。
(1)前記応答者及び非応答者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、上述の方法によって、神経細胞を製造する工程、
(3)前記工程(2)で得られた応答者に由来する神経細胞と非応答者に由来する神経細胞の遺伝子産物の発現を測定する工程、及び
(4)応答者に由来する運動神経細胞において、非応答者より発現量が多い遺伝子産物を応答者に特異的なマーカーとして同定する工程、又は、応答者に由来する運動神経細胞において、非応答者より発現量が少ない遺伝子産物を非応答者に特異的なマーカーとして同定する工程。
本発明は、さらに、前述の治療効果特異的マーカーを用いて当該対応する治療薬が有効である対象を選別する方法を提供する。
(1)被験者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、上述の方法によって、運動神経細胞を製造する工程、
(3)前記工程(2)で得られた運動神経細胞において、上述の方法によって同定された応答者及び/又は非応答者に特異的なマーカーを検出する工程、及び
(4)前記応答者に特異的なマーカーが検出された運動神経細胞、又は前記非応答者に特異的なマーカーが検出されなかった運動神経細胞が由来する被験者を、対応する治療薬が有効である対象として選別する工程。
(1)被験者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、上述の方法によって、神経細胞を製造する工程、
(3)前記工程(2)で得られた運動神経細胞において、上述の方法によって同定された応答者及び/又は非応答者に特異的なマーカーを検出する工程、及び
(4)前記応答者に特異的なマーカーが検出された神経細胞、又は前記非応答者に特異的なマーカーが検出されなかった神経細胞が由来する被験者を、対応する治療薬が有効である対象として選別する工程。
また、以下の実施例において、テトラサイクリン応答性プロモータの活性化を誘導することを目的として培地に添加したdoxycycline(以降、DOXと略記する)の濃度は、特に断りがない限り終濃度1μg/mlである。
前述したように、多能性幹細胞からiMNを製造する方法としては、多能性幹細胞から約10日かけて分化させた神経前駆細胞に、Lhx3、Ngn2、及びIsl1遺伝子をアデノウィルスベクターを用いて導入・発現させることで、神経前駆細胞から約11日でiMNを得る方法が報告されている(非特許文献4)。上記3種類の遺伝子のうちLhx3及びIsl1は、従来法で多能性幹細胞から運動神経細胞を分化誘導する過程では、神経前駆細胞より後の分化段階で発現が誘導される転写因子である(非特許文献4)。
よって、未分化な細胞内ではこれらの転写因子が本来の機能を発揮できない可能性が十分考えられたが、マウス由来ES細胞に導入してその効果を調べてみることにした。
このベクターを293T細胞にリポフェクション法で導入し、DOXを培地に添加して24時間後に解析した結果、DOX添加によってLhx3、Ngn2、及びIsl1タンパク質の発現が誘導されることが確認された(図1B)。
前記ベクターをflipaseをコードする核酸とともにKH2細胞にエレクトロポレーション法で導入し、前記MN化因子発現カセット領域が染色体内に組み込まれたKH2細胞(すなわち、MN化因子導入マウスES細胞)を選別した。当該細胞をMEF(マウス胎児繊維芽細胞)フィーダー細胞上でマウスES細胞用培地(15%FBS、LIF、β-mercaptoethanol、L-glutamine、nonessential amino acids及びpenicillin/streptomycinを添加したDMEM)を用いて培養し、免疫染色を行ったところ、未分化マーカー遺伝子(NANOG及びSSEA1)の発現が観察され(図1C)、多能性を保持していることが確認された。また、DOX添加群特異的にmCherryの蛍光が観察され(図1D)、DOX添加に遺伝子発現が誘導されることが確認された。さらに、DOX添加によってLhx3、Ngn2、及びIsl1のmRNA合成が誘導されることも確認された(図1E)。
前記MN化因子導入マウスES細胞に対し、図1Fに示した工程に従って、Lhx3、Ngn2、及びIsl1の発現誘導を行った。当該細胞を0.25% trypsinを用いて解離させた後、マトリゲルコートディッシュ上へ播種し、DOXを含有する培地(すなわち、1μg/ml DOX、1μMレチノイン酸(RA)、1μM sonic hedgehog(Shh)、10ng/ml BDNF、10ng/ml GDNF及び10ng/ml NT3を含有するN3培地(100 μg/ml apotransferrin、5 μg/ml insulin、30 nM selenite、20 nM progesterone及び100 nM putrescineを添加したDMEM/F12))中で培養することで前記3遺伝子の発現を誘導した。誘導開始から36時間後には、神経細胞様に形態変化した細胞が認められた。そして、72時後には、神経細胞マーカー(β-III tubulin及びMAP2)に加えて、運動神経細胞マーカー(HB9及びChAT)を発現し、且つ、形態的にも成熟した細胞が多数観察された(図1G)。HB9及びChATの発現をリアルタイムPCR法で解析したところ、DOX添加によってそれらの発現量が増大することが確認された(図1H)。
従って、マウス由来多能性幹細胞に、Lhx3、Ngn2、及びIsl1遺伝子を導入して発現を誘導すると、僅か3日で、運動神経細胞特異的なタンパク質を発現し、形態的にも成熟した神経細胞(iMN)が得られることが明らかとなった。
本発明において運動神経細胞の誘導により適した条件を探すため、DOXを含有させる培地の検討を行った。前記MN化因子導入マウスES細胞を、下記4種類の培地にDOXを添加又は非添加して3日間培養した後、β-III tubulin陽性細胞数を計測した。
(1)10% KSRを含有するDMEM/F12培地
(2)10% KSR、1μM RA及び1μM Shhを含有するDMEM/F12培地
(3)N3培地
(4)1μM RA及び1μM Shhを含有するN3培地
その結果、(4)の培地にDOXを添加した場合に、β-III tubulin陽性細胞数が最も多くなることが明らかとなった(図2A)。よって、以降の実施例において運動神経細胞を培養下で分化誘導する場合には、特に断りがない限り、当該(4)の培地に10ng/ml BDNF、10ng/ml GDNF、及び10ng/ml NT3を添加した培地を用いることとし、さらにDOX(特に断りが無い限り、1μg/ml)を添加した培地を“DOX含有培地”と呼ぶことにした。
よって、多能性幹細胞を運動神経細胞へ分化誘導するためにLhx3、Ngn2、及びIsl1遺伝子の発現を誘導する期間は、3日で十分であることがわかった。
上記方法によってマウスES細胞から作製した運動神経細胞(iMN)の機能を評価するために、C2C12細胞(マウス横紋筋由来細胞株、Science, 230, 758-, 1985)とのシナプス形成能を解析した。C2C12細胞は、0.5% FBS、10 μg/ml insulin、5.5 μg/ml apotransferrin、30 nM selenite及び1mM L-glutamineを含有するDMEM/F12中で7日間培養することで、筋管へ誘導したものを用いた。当該筋管の培養系に、前記MN化因子導入マウスES細胞を(該筋管の上に)播種し、DOX含有培地で培養して運動神経細胞への分化誘導を行った。免疫染色の結果、iMNから伸びたsynaptic vesicle protein 2(SV2)陽性の神経突起が、α-bungarotoxinで標識されたアセチルコリン受容体と共局在することが確認された(図3A)。このことは、本発明の方法によってマウス多能性幹細胞から作製した運動神経細胞(iMN)が、筋肉細胞とシナプスを形成(すなわち、神経筋接合部を形成)できることを示す結果である。さらに、カルシウムイメージング法を用いてC2C12細胞のカルシウム流入量を測定したところ、iMNと共培養させた場合にのみ、カルシウム流入量が有意に増加することが確認された(図3B)。
よって、マウス由来多能性幹細胞から本発明に係る方法で得られるiMNは、本来の運動神経細胞と同様に、筋肉細胞との間で機能的なシナプスを形成する能力を備えていることが示された。
よって、本発明に係る方法でマウス由来多能性幹細胞から製造したiMNは、本来の運動神経細胞と同様の電気生理学的特性を備えていることが示された。
次に、ALSモデルマウス由来多能性幹細胞からiMNを作製し、本来の運動神経細胞との類似性について解析することにした。
ヒトSOD1遺伝子(G93A変異型SOD1遺伝子、又は野生型SOD1遺伝子)を有するトランスジェニックマウスからマウス胎児繊維芽細胞(MEF)を調製し、Okita K et al., Nature. 2007, vol.448, pp.313-317に記載の方法に従ってiPS細胞を樹立した。当該iPS細胞が前記ヒトSOD1遺伝子を有していることを、当該遺伝子のシークエンスによって確認した(図4A及びB)。そして、これらのiPS細胞が、ES細胞のマーカー遺伝子(Eras、Esg1、Rex1、Oct3/4及びSox2)を発現していること、初期化因子の発現が抑制されていること、及び、3胚葉へ分化誘導され得ることを確認した(図4C、4D及び図5)。本願では以降、前記G93A変異型SOD1遺伝子、又は野生型SOD1遺伝子を有するトランスジェニックマウスから樹立したiPS細胞をそれぞれ、マウスG93A由来iPS細胞、マウスWT由来iPS細胞と呼ぶことにする。
前記マウスG93A由来iPS細胞、マウスWT由来iPS細胞に、非特許文献4と同様にアデノウィルスベクターを用いてLhx3、Ngn2及びIsl1遺伝子を導入したところ、細胞死が高い頻度で起こり、さらにiMNへの分化の同調性も十分ではなかった。そこで、本発明者は、上記以外の方法でiPS細胞に遺伝子導入を行う方法を検討した。試行錯誤の末、トランスポゾン、特にpiggyBacトランスポゾン(Woltjen K, et al, Nature. 2009, 458:766-70を参照)を用いると、細胞死の程度も低く、さらに、iMNへ同調して分化させられることを見出した。なお、ヒト由来iPS細胞においても同様の結果が得られたので、本願では、MN化因子の導入には主にトランスポゾンを用いることにした。
前記MN化因子導入-マウスG93A由来iPS細胞、及びMN化因子導入-マウスWT由来iPS細胞をDOX含有培地で培養したところ、前述のマウスES細胞の場合と同様に、3日後には多数のHB9、β-III tubulin及びChAT陽性細胞が確認された(図6C)。一方、アストロサイトのマーカーであるGFAPを発現する細胞は確認されなかった。
よって、ヒトSOD1遺伝子(G93A変異型SOD1遺伝子、又は野生型SOD1遺伝子)を有するトランスジェニックマウス由来iPS細胞からも、MN化因子を導入して発現誘導することで、僅か3日で運動神経細胞が得られることが確認された。
G93A変異型ヒトSOD1遺伝子を有するトランスジェニックマウスでは、運動神経細胞内で変異型SOD1タンパク質がミスフォールド及び凝集することが知られており、そのことが運動神経細胞死と密接に関わると考えられている。そこで、当該トランスジェニックマウスに由来するiPS細胞から本発明に係る方法で作製したiMNについて、SOD1タンパク質のミスフォールド/凝集の有無を調べることにした。抗ミスフォールドSOD1抗体(A5C3、B8H10及びC4F6、いずれもMEDIMABS社製、野生型・変異型を問わずミスフォールドしたSOD1タンパク質を認識し得る抗体)を用いて免疫染色を行ったところ、MN化因子導入-マウスWT由来iPS細胞から作製されたiMNでは何も認識されなかったが、MN化因子導入-マウスG93A由来iPS細胞から作製されたiMNでは、該抗体で認識される凝集体が細胞内に多数観察された(図6D)。
次に、細胞死の有無について解析した。前記細胞をDOX含有培地で4日間培養した後にDOX不含培地でさらに2日間培養し、DOXによる誘導開始から4日後と6日後におけるHB9及びβ-III tubulin陽性細胞数(すなわち、iMN数)を測定した。図6Eに、6日目のiMN数を4日目のiMN数で除した値(すなわち、6日目のHB9及びβ-III tubulin陽性細胞数/4日目のHB9及びβ-III tubulin陽性細胞数)をiMNの生存率として示した結果を示す。該図より、MN化因子導入-マウスWT由来iPS細胞から作製されたiMNと異なり、MN化因子導入-マウスG93A由来iPS細胞から作製されたiMNは、誘導開始から4-6にかけて大規模な細胞死を起こすことが明らかとなった(図6E)。さらに、誘導開始から4日後と6日後における培地中のLDH量を測定したところ、MN化因子導入-マウスG93A由来iPS細胞から誘導した培養の方が、MN化因子導入-マウスWT由来iPS細胞から誘導した培養よりもLDH量が有意に高いことが確認された(図6F)。そして、前記誘導開始から4日後と6日後におけるHB9及びβ-III tubulin陽性細胞の神経突起の長さを測定したところ、MN化因子導入-マウスWT由来iPS細胞から作製したiMNでは顕著に増加する(すなわち、盛んな突起伸展が起こる)のに対し、MN化因子導入-マウスG93A由来iPS細胞から作製したiMNではほとんど増加しない(すなわち、突起伸展が停止する)ことも明らかとなった(図6H)。
従って、変異型ヒトSOD1遺伝子を有するALSモデルマウス由来iPSから本発明に係る方法で作製したiMNでは、当該マウス脊髄内の運動神経細胞と同様に、SOD1タンパク質のミスフォールド・凝集が自発的に起こり、細胞死へと向かうことが明らかとなった。さらに、当該細胞死の指標として、培地中のLDH値や神経突起長の測定結果を使用できることが示された。
・ヒト由来iPS細胞へのDNA導入
ヒト由来iPS細胞(Takahashi K, et al., Cell. 2007, vol 131, pp 861-872.)に、前述と同様に図6Aに示したベクターを導入して、MN化因子導入-ヒト正常対照由来iPS細胞を得た。そして、当該細胞をSNL細胞上で4 ng/ml basic fibroblast growth factorを添加したprimate embryonic stem cell medium(ReproCELL)を用いて培養し、未分化マーカー(NANOG及びSSEA4)を発現していること(図7A)、及び、前記DNA導入前のiPS細胞と同等の高い増殖能を維持していることを確認した。
前記MN化因子導入-ヒト正常対照由来iPS細胞をAcutaseを用いて解離し、マトリゲルをコートしたディッシュへ移し、DOX含有培地で培養して運動神経細胞への分化誘導を行った。(図7B)。誘導開始から7日後には、HB9、β-III tubulin、及びChAT陽性で、形態的にも成熟した神経細胞様細胞が多数観察された(図7C)。さらにHB9及びChATの発現量をPCRによって測定したところ、DOX添加によってそれらの発現量が増大することが確認された(図7D)。
従って、ヒト由来多能性幹細胞にMN化因子を導入して発現させると、運動神経細胞特異的なタンパク質を発現し、形態的にも成熟したiMNが約7日で得られることが明らかとなった。
前記MN化因子導入-ヒト正常対照由来iPS細胞から得られたiMNに対し、前述の方法を用いて、C2C12細胞とのシナプス形成能を解析した。すると、分化誘導開始から10日後に、前記iMNから伸びたSV2陽性の神経突起がα-bungarotoxinで標識されたアセチルコリン受容体と共局在することが確認された(図7E)。また、前述と同様に、さらに前記HB9::GFPが染色体に挿入されたMN化因子導入-ヒト正常対照由来iPS細胞を作製し、該細胞をマウス由来初代アストロサイトの共存下DOX含有培地中で8-14日間培養した後、全細胞記録によるパッチクランプ法による電気生理学的解析を行った。その結果、解析したすべてのGFP陽性細胞(10細胞)においてNa+/K+電流が認められ、且つ、活動電位が測定された(図7F)。さらに、グルタミン酸又はGABAを培地に添加すると内向き電流が誘導されることから(図3D)、当該興奮性及び抑制性の神経伝達物質に対する受容体を発現していることが確認された(図7G)。
よって、本発明に係る方法でヒト由来多能性幹細胞から製造したiMNは、本来の運動神経細胞と同様の電気生理学的特性を備えていることが確認された。
次に、ヒトALS患者由来多能性幹細胞からiMNを作製し、本来の運動神経細胞との類似性について解析することにした。
・ヒト由来iPS細胞の樹立
ヒト正常対照2名(Control1及びControl2)、及び、SOD1遺伝子に変異を有するALS患者2名から、患者の同意を得て採取した皮膚由来の線維芽細胞に、Okita K, et al, Nat Methods. 2011, 8:409-12に記載の方法に従って、エピソーマルベクターにてOCT3/4、SOX2、KLF4、L-MYC、LIN28及びp53に対するsmall haipin RNAを導入することによってiPS細胞を樹立した(Kondo T, et al, Cell Stem Cell. 2013, 12:487-96参照のこと)。図10Aに、前記ALS患者から樹立したiPS細胞のSOD1遺伝子が当該変異を有していることを示す。
なお、前記2名のALS患者が有するSOD1遺伝子の変異は、L144FVX変異(144番目のアミノ酸であるロイシン以降が、フェニルアラニン-バリン-終止コドンに置換する変異、SOD1-L144FVXと記載することもある)、又はG93S変異(93番目のアミノ酸であるグリシンがセリンに置換する変異、SOD1-G93Sと記載することもある)である。そして、本願では以降、前記Control 1、Control 2、SOD1-L144FVX変異又はSOD1-G93S変異を有する患者から樹立されたiPS細胞を各々、Control1来iPS細胞、Control2由来iPS細胞、L144FVX由来iPS細胞、G93S由来iPS細胞、と呼ぶことにする。
上記方法で得られた4種類のiPS細胞に、前述と同様に図6Aに示したベクターを導入し、前記MN化因子発現カセット領域が染色体に挿入された細胞を得た。前記Control1由来iPS細胞、Control2由来iPS細胞、L144FVX由来iPS細胞、G93S由来iPS細胞から得られた細胞を各々、MN化因子導入-Control1来iPS細胞、MN化因子導入-Control2由来iPS細胞、MN化因子導入-L144FVX由来iPS細胞、MN化因子導入-G93S由来由来iPS細胞、と呼ぶことにする。
上記方法で作製された4種類のMN化因子導入iPS細胞をDOX含有培地で培養してMN化因子の発現誘導を行った。図10Bに当該誘導工程の模式図を示す。誘導開始から7日後に免疫染色を行ったところ、HB9及びβ-III tubulin陽性細胞が約50%程度認められた(図10C)。また、前述の抗ミスフォールドSOD1抗体を用いた免疫染色では、MN化因子導入-L144FVX由来iPS細胞、及びMN化因子導入-G93S由来由来iPS細胞から得られたiMNでのみ、該抗体で認識される凝集体が観察された(図10D)。そして、7日後と14日後のβIII Tublin陽性細胞数をiMNの細胞数として計測し、14日後のiMN細胞数を7日後のiMN細胞数で除した値をiMNの生存率としてグラフ化した(図10E)。図10Eより、MN化因子導入-Control1来iPS細胞、MN化因子導入-Control2由来iPS細胞から得られるiMNでは7日後と14日後の細胞数はほぼ一定だが、MN化因子導入-L144FVX由来iPS細胞、及びMN化因子導入-G93S由来由来iPS細胞から得られるiMNは、7-14日後の間に細胞数が大幅に減少すること、すなわち細胞死を起こすことがわかった。
次に、本発明に係る方法でiMNが産生されるタイムコースについて詳細に解析した。
野生型マウス由来iPS細胞株(201B7株、Takahashi K, et al, Cell. 2007, 131:861-72.参照)から、実施例2と同じ方法を用いて、前記MN化因子発現カセット領域が染色体内に挿入された細胞を得た。得られた細胞を96ウェルプレートに播種し、前記DOX含有培地で培養してMN化因子の発現を誘導した。誘導開始から、10、24、36、48、72時間後に免疫染色を行い、SSES1又はNCAM陽性細胞数を計測した。そして、NCAM陽性で、且つ、神経突起と十分に肥厚した細胞体を有する細胞をiMNとして当該細胞数を計測した。さらに、DAPI染色も行って総細胞数を計測した。結果を図11Aに示す。
図11Aから明らかなように、誘導開始後、SSES1陽性細胞(すなわち、未分化能を有する細胞)は速やかに減少し、48時間後には完全に消失した。これに対し、誘導開始直後からNCAM陽性細胞(すなわち、神経細胞への分化途中の細胞)が現れ、その後24時間後くらいから、NCAM陽性で且つ肥厚した細胞体と神経突起を有する細胞(すなわち、iMN)が現れることがわかる。そして、24-72時間後にかけてiMNの数は急増し、72時間後には約50%の細胞がiMNとなり、以降iMN数はほとんど増えないことが明らかとなった。なお、72時間後にiMNと判定されない細胞は、SSES1、NCAMともに陰性であり、神経細胞以外に変化した細胞と考えられる。
よって、本発明に係るMN化因子導入多能性幹細胞からiMNへの分化誘導は同調性が非常に高く、マウス由来多能性幹細胞では、MN化因子の発現誘導後速やかにiPS細胞としての性質を失い、約50%が2-3日後にかけてiMNになることが明らかとなった。
同様の解析を、実施例3で作製したMN化因子導入-ヒト正常対照由来iPS細胞に対して行った。図11Bに示されるように、神経突起と十分に肥厚した細胞体を有するNCAM陽性細胞(すなわち、iMN)は、誘導開始から5日後ではほとんど認めらなかったが、その後急激に増加し、7日後には約25%に達していた。よって、ヒト由来多能性幹細胞では、MN化因子の発現誘導から5-7日後の間に同調してiMNになることが明らかとなった。なお、5日後の時点でもNCAMやβ-III tubulin陽性で長い突起を有する細胞は多数認められたが、細胞体の厚みが不十分(比較的扁平)であり、そのような細胞ではHB9の発現が不十分であることを確認している。
なお、本願実施例では、多能性幹細胞に薬剤応答性MN化因子発現するベクターを導入して当該MN因子発現カセット領域がゲノムに挿入された多能性幹細胞を得た後、該多能性幹細胞をクローン化することなく(すなわち、当該発現カセットの挿入部位や挿入されたコピー数が異なるヘテロな細胞集団のまま)、各実験に使用した。よって、本方法において、さらに、前記多能性幹細胞のクローン化を行い、前記外来性DNAの挿入部位や挿入コピー数が揃った細胞集団を用いれば、iMNへの分化の同調性及び分化効率はより一層高くなることが期待される。
次に、ALSモデルマウス由来iPS細胞から誘導されるiMNが細胞死に至るタイムコースについて詳細に解析した。
実施例2で作製したMN化因子導入-マウスG93A由来iPS細胞、又は、MN化因子導入-マウスWT由来iPS細胞を96ウェルプレートに播種し、前記DOX含有培地で培養してMN化因子の発現を誘導した。誘導から4、6、10日後に免疫染色(β-III tubulin)と形態観察を行い、iMNの細胞数を計測した。結果を表1及び図12Aに示す。
なお、本願では割愛したが、SOD1-G93S変異を有するALS患者に由来するMN化因子導入iPS細胞を用いた場合にも、上記SOD1-L144FVX変異を有するALS患者に由来する細胞を用いた場合と同様の結果を得ている。
そこで、本発明に係るMN細胞死に対するリルゾールの効果を解析することにした。
。
実施例6と同様に、前述のMN化因子導入-L144FVX由来iPS細胞を96ウェルに播種し、DOX含有培地で培養してMN化因子の発現を誘導した(この日をDay0とする)。7日後(=Day7)に種々の濃度のリルゾールを投与し(0, 12.5, 25, 50, 100μM)、14日後(=Day14)に前述の方法に従ってiMN数を測定した。図13Aに、7日後と14日後の細胞を抗β-III tubulin抗体で免疫染色した結果を示す。リルゾールを投与したウェルでは、非投与ウェルに比べてiMNの細胞死が有意に抑制されていることがわかる。
iMN数を測定した結果を表3及び図13Bに示す。リルゾールの濃度が50μMまでは濃度依存的にiMNの細胞死が抑制されることが明らかとなった。
よって、本発明に係るiMN細胞死の系は、ALS治療・予防薬のスクリーニング系及び評価系として非常に有益と思われる。
本発明に係る変異型SOD1遺伝子を有するMN化因子導入iPS細胞を用いたALS治療・予防薬のスクリーニング系の概要を図14に示す。
まず、本解析系の精度を評価した。前述のSOD1-L144FVX変異を有するALS患者由来のMN化因子導入iPS細胞を96ウェルに播種し、前記DOX含有培地で培養してMN化因子の発現を誘導した(=Day0)。7日後(=Day7)、陰性コントロールとしてDMSO、陽性コントロールとしてSOD1変異を有するALS患者由来iPS細胞から誘導したMNの生存率を上げることが報告されているケンパウロン(50μM、Cell Stem Cell, vol. 12, pp. 1-14, 2013)をコントロールウェルに投与し、14日目に細胞を固定してβ-III tubulinの免疫染色を行った。染色像をIN Cell Analyzer 6000(GEヘルスケア)を用いて解析し、iMN数を測定した。結果を図15Aに示す。本解析におけるZ’値は0.65で、0.5以上であることから、当該実験系によって得られる値はばらつきが非常に小さく、精度が非常に高い実験系であることが示された。
また、当該既存薬化合物には、ALSモデルマウスに対して治療効果が認められたにも関わらず、ヒトを用いた臨床試験では有意な治療効果が認められなかった化合物が含まれていた。これらの化合物の結果を図15Bに示す(化合物名の下にアンダーラインの無い15種類)。いずれの化合物も、前記iMNの細胞死を効果的には抑制できないことがわかる。
血球細胞は、動物個体に最も導入し易い細胞である。特に、単球やマクロファージのような遊走能を有する細胞は、動物個体の深部まで自律的に移動することが可能である。そこで、本発明者は、本発明に係るDNAを導入した多能性幹細胞を、血球細胞経由で運動神経細胞に分化させることができないか、検討することにした。
実施例1で作製したMN化因子導入マウス由来ES細胞をOP9細胞上に播種し(0日目)、10%FBSを含有するαMEM培地中で培養した。培養開始5日目(図8A)に、FACSを用いて、Flk-1陽性かつSSEA-1陰性の細胞を収集した(図8B)。収集した細胞をOP9細胞上に播種し、100ng/ml mSCF、20ng/ml mIL-3及び10ng/ml mM-CSFを添加したαMEMを用いて培養した。7日後(培養開始から12日目)にギムザ染色を行ったところ、単球/マクロファージの存在が確認され(図8C)、前記多能性幹細胞が血球細胞に分化誘導されることが確認された。
よって、本発明に係るMN化因子導入多能性幹細胞を血球細胞に分化させた後に、該MN化因子の発現を誘導することで、7日程度でiMNに分化させられることが示された。
前項までは、神経細胞の中でもALSやSMAの研究対象として注目される運動神経細胞に注目し、多能性幹細胞から該細胞を製造する方法に係る発明を記載した。一方で、一般的な神経細胞も、研究上大変需要の高い細胞である。本発明者も、これまでは、従来法(接着法)によって多能性幹細胞から約2ヶ月かけて得られる神経細胞を用いて、アルツハイマー型痴呆症の研究を行っていた。そして、前記iMNの製造方法を模索する過程で、MN化因子のうち、Ngn2遺伝子のみを多能性幹細胞に導入・発現させると、非常に迅速且つ高効率でiNへと誘導できることを見出したのである。
多能性幹細胞に導入するDNAとして、テトラサイクリン応答性プロモーターによって発現が制御されるようにNgn2と連結されたカセット、及び同時にrtTAを発現するようにプロモーター(EF1α)と連結させたカセットをpiggybacトランスポゾンシステムで導入できるようにTerminal repeat(TR)で5’側と3’側を挟んだコンストラクト(Tanaka A,et al, PLoS One. 2013, 8:e61540参照のこと)として作製した。以下、当該DNAを、テトラサイクリン応答性プロモーターの制御下にNgn2遺伝子をコードするDNAと呼ぶことにする。
野生型マウス由来iPS細胞株(201B7株、Takahashi K, et al, Cell. 2007, 131:861-72.参照)に、前記テトラサイクリン応答性プロモーターの制御下にNgn2遺伝子をコードするDNAを、トランスポゼース(Woltjen K, et al, Nature. 2009, 458:766-70)をコードする核酸とともに導入し、前記DNAがゲノムに挿入された安定発現株(以下、201B7_Ngn2と略記する)を樹立した。続いて、この201B7_Ngn2をシングルセルへ分離し、マトリゲル(BD)をコートしたディッシュへ播種し、DOX含有培地(すなわち、1μg/ml DOX、10ng/ml BDNF、10ng/ml GDNF及び10ng/ml NT3を含有するN3培地)で培養することでNgn2の発現誘導を開始した。発現誘導開始から2日後にはβIII tublin陽性細胞が僅かに認められ、3日後には約90%の細胞がβIII tublin、MAP2及びvGLT1陽性となり(すなわち、グルタミン作動性神経細胞となり)、4日後以降にはβIII tublin陽性細胞数はほとんど増加しないことが観察された(図16A)。
よって、マウス由来多能性幹細胞にNgn2遺伝子を導入して発現させることにより、ほぼすべての細胞が2-3日後にかけて同調してiNになることが明らかとなった。
次に、アルツハイマー型痴呆症患者に由来する多能性幹細胞からiNを作製することを試みた。
・アルツハイマー型痴呆症患者からのiPS細胞の樹立
ヒトiPS細胞(N112E14(正常対照由来)、AD8K213(孤発型アルツハイマー患者由来)、AD15E11(プレセニリン(PS1)変異体)、APP1E211(APP-E693delta))は、次の方法を用いて製造した。AD8K213及びAPP1E211は、Okita K, et al, Nat Methods. 2011, 8:409-12に記載の方法に従って、エピソーマルベクターにてOCT3/4、SOX2、KLF4、L-MYC、LIN28及びp53に対するsmall haipin RNAを、患者の同意を得て採取した皮膚由来の線維芽細胞へ導入することによって作製した(Kondo T, et al, Cell Stem Cell. 2013, 12:487-96参照のこと)。N112E14及び AD15E11についても同様に、健常者又は患者の同意を得て採取した皮膚由来の線維芽細胞よりエピソーマルベクターを用いて作製した。
上記方法で得られた4種類のヒトiPS細胞に、前記テトラサイクリン応答性プロモーターの制御下にNgn2遺伝子をコードするDNAを前記方法に従って導入し、該DNAがゲノムに挿入された安定発現株(すなわち、N化因子導入細胞株)を樹立した。得られた4種類の安定発現株をAccutase(Thermo)を用いて分離し、マトリゲル(BD)及び0.01mg/ml human fibronectin(BD)をコートした12well plateへ30×104/wellを播種した。DOX含有培地(すなわち、2 μg/μl DOX及びB-27 Supplement Minus AO(Life Technologies)を添加したNeurobasal medium(Life Technologies))中で培養することにより、Ngn2の発現誘導を開始した。5日後に同じ組成の培養液にて培地交換した。Ngn2の発現誘導開始から4日後までは、βIII tublin陽性細胞はほとんど認められなかったが、5-7日後にかけて当該陽性細胞数が急増し、8日後以降はほとんど増えないことが観察された。また、βIII tublin陽性細胞が出現するタイムコースは、前記4種類の安定発現株間で有意な差は認められなかった。
よって、ヒト由来多能性幹細胞にNgn2遺伝子を導入して発現させることにより、5-7日後にかけて高い同調性でiNが得られることが明らかとなった。
アルツハイマー型痴呆症患者の脳では、APPタンパクのプロセシングが変化して、Aβ40ペプチド及びAβ42ペプチド産生が増加する傾向があることが報告されている。特にAβ42ペプチドは凝集性が高く細胞毒性を有するので、該疾患の治療薬(候補)として、Aβ42ペプチドの産生を抑制し得る化合物の探索が精力的に行われている。
そこで、前記患者由来多能性幹細胞から得られたiNについて、前記2種類のAβペプチドの分泌量を解析することにした。
1)DMSO添加群(陰性対照)
2)1 μM BSI IV添加群
3)100 μM Sulindac sulfide添加群
(N112E14(正常対照由来)、AD8K213(孤発型アルツハイマー患者由来)、AD15E11(プレセニリン(PS1)変異体)、APP1E211(APP-E693delta))
そして、Ngn2の発現誘導開始から5日目、7日目、9日目、11日目に培養上清を回収し、MSD Abeta 3 plex(38, 40, 42)assay plate(Meso Scale Discovery)を用いて当該培養上清中に含まれるAβ40及びAβ42ペプチドの含有量を測定した(図16C)。図16Bの上段に、各Aβペプチドの含有量の経時変化を示す。これより、2名のアルツハイマー型痴呆症患者(AD8K213、AD15E11)に由来するiNの培養上清には、正常対照(N112E14)由来iNの培養上清よりも、多量のAβ42ペプチドが含まれていることがわかる。特に、Aβ42ペプチドの産生量を増加させる変異を有するAD15E11由来iNでは、培養上清中のAβ42ペプチド量が最も高い結果となった。これに対し、APP1E211由来iNの培養上清には、患者由来であるにも関わらずAβ42ペプチドがほとんど検出されないが、当該変異を有する神経細胞ではAβ42ペプチドが細胞外に分泌されずに細胞内に蓄積することが報告されている。よって、これら3名の患者に由来するiNは、いずれも本来の神経細胞の性質を良く再現していると考えることができる。また、Aβ42ペプチドをほとんど分泌しないAPP1E211由来iN以外のiNではいずれも、Ngn2の発現誘導開始から9日目の培養上清(すなわち、7日目から9日目の間に培地に分泌されたペプチド量)でAβの測定量が最大になることが明らかとなった。
本願実施例1-11では、薬剤応答性プロモーターの制御下にMN化因子又はNgn2をコードするDNAを導入した多能性幹細胞に対し、培養下で、当該プロモーターの活性化を誘導する薬剤を培地に添加することで、MN化因子又はNgn2の発現を誘導した。そこで、次に、当該誘導工程を生体内で行えないか、検討することにした。
実施例1において、iMNに分化した細胞を視覚化するために作製したマウスES細胞(すなわち、図1Aに示したDNA、及び、HB9遺伝子のプロモーターにGFP遺伝子のコード配列を連結させたDNA断片(HB9::GFP)がゲノムに挿入されたKH2細胞)を、NOGマウスの脊髄に移植した。そして、当該マウスにDOXの飲水投与とレチノイン酸の腹腔内投与を行い、移植から2週間後に投与部位の免疫染色を行った。その結果、移植の1週間前からDOXとレチノイン酸の投与を行ったマウスで、最も多くのGFP陽性細胞(すなわち、iMN)が確認された(図17)。さらに、当該GFP陽性細胞はTuj1陽性で、且つ、神経突起を伸展させており、移植したマウスの脊髄内で生着していることが確認された(図17)。
よって、本発明に係る多能性幹細胞(すなわち、薬剤応答性プロモーターの制御下にMN化因子をコードするDNAが導入された多能性幹細胞)は、動物体内においても運動神経細胞へ変換できることが確認された。実施例9の結果(すなわち、本発明に係る多能性幹細胞を血球細胞に分化させた後に、MN化因子の発現を誘導することで、iMNへと変換できること)を参酌すると、本発明に係る多能性幹細胞自体を動物体内に直接導入するのではなく、該多能性幹細胞から誘導した血球細胞を動物体内に移植し、それからiMNに分化誘導させる方法も可能と思われる。
同様に、実施例5において作製したN化因子導入マウスiPS細胞(201B7_Ngn2細胞)を、DOXを飲水投与して1週後のNOGマウスの海馬へ移植し、移植後4週間後に投与部位の細胞を免疫染色にて観察した(図18)。hNCAM陽性細胞が確認されマウスの脳内で神経細胞へと変換され、且つ、生着できることが確認された。
iPS細胞技術はこれまで、疾患モデルの作製や移植用組織・細胞の作製を主目的として開発が進められている。そして、創薬・治験の分野では、健常人由来のiPS細胞を用いて安全性試験を行い、患者由来のiPS細胞を用いて試験薬の有効性を評価することで、新たな候補治療薬を生み出すことが想定されている。すなわち、iPS細胞を用いた実験系で候補薬を探索・開発し、その後ヒトを対象とした臨床試験を行う、という流れがこれまで想定されている。
この結果は、本発明に係るMN解析系を用いれば、細胞提供者(個体)間の薬剤反応性の違いを詳細に解析し、その違いをもたらす理由を解明できる可能性があることを示唆している。さらに、特定の薬剤が有効な被験者群(=レスポンダー)と有効でない被験者群(=非レスポンダー)、あるいは実際の臨床試験後に、レスポンダーと非レスポンダーが判明した場合、レスポンダー、非レスポンダーからiPS細胞を樹立し、本発明に係る方法でMNに分化誘導・解析することで、各群に共通する何らかの特徴(=マーカー)を見出せる可能性も考えられる。そして、このようなマーカーが見つかれば、当該マーカーを有するレスポンダーに対してのみ第二相試験を行うことで、比較的少数の被験者数で精度の高い治験が行えるはずである。
Claims (53)
- 多能性幹細胞から運動神経細胞を製造する方法であって、下記工程;
(1)多能性幹細胞に、Lhx3、Ngn2、及びIsl1をコードする核酸を導入する工程、
(2)Lhx3、Ngn2、及びIsl1の発現を3日間以上維持する工程、
を(1)-(2)の順番で含む、多能性幹細胞から運動神経細胞を製造する方法。 - 前記核酸の導入が、トランスポゾンを用いて導入する、
請求項1に記載の方法。 - 前記トランスポゾンが、piggyBacトランスポゾンである、
請求項2に記載の方法。 - 前記工程(2)が、前記核酸を薬剤応答性プロモーターで発現させる工程である、
請求項1-3のいずれかに記載の方法。 - 前記薬剤応答性プロモーターがテトラサイクリン応答性プロモーターである、
請求項4に記載の方法。 - 前記核酸を、Lhx3、Ngn2、及びIsl1をポリシストロニックに発現させる、
請求項1に記載の方法。 - 前記核酸が、Lhx3、Ngn2、及びIsl1をコードする核酸が2A配列により結合された核酸である、
請求項6に記載の方法。 - 前記工程(2)において、Lhx3、Ngn2、及びIsl1の発現を維持する期間が7日間以上である、
請求項1-7のいずれかに記載の方法。 - 前記工程(2)において、前記工程(1)で得られた細胞を動物体内に導入する工程をさらに含み、該動物体内で前記薬剤応答性プロモーターに対応する薬剤と接触させる、
請求項6-8のいずれかに記載の方法。 - 前記工程(1)において、前記核酸が導入された多能性幹細胞を、血球細胞に分化誘導する工程をさらに含む、
請求項1-9のいずれかに記載の方法。 - 前記血球細胞が、単球及び/又はマクロファージである、
請求項10に記載の方法。 - 前記血球細胞に分化誘導する工程が、前記核酸が導入された多能性幹細胞を、骨髄由来間質細胞と共培養し、さらに幹細胞因子、マクロファージ-コロニー刺激因子、及びインターロイキン-3存在下で培養することで血球細胞に分化誘導する工程である、
請求項10又は11に記載の方法。 - 前記多能性幹細胞が、ヒト人工多能性幹細胞である、
請求項1-12のいずれかに記載の方法。 - 前記多能性幹細胞が、変異型SOD1遺伝子を有する多能性幹細胞である、
請求項1-13のいずれかに記載の方法。 - 下記工程(1)-(3)を含むことを特徴とする、運動神経変性疾患又は神経損傷の治験薬に対して治療効果が認められた被験者(すなわち、応答者)又は治療効果が認められなかった被験者(すなわち、非応答者)に特異的なマーカーを同定する方法;
(1)前記応答者及び非応答者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、請求項1-8のいずれかに記載の方法によって、運動神経細胞を製造する工程、
(3)前記工程(2)で得られた応答者に由来する運動神経細胞と非応答者に由来する運動神経細胞の遺伝子産物の発現を測定する工程、及び
(4)応答者に由来する運動神経細胞において、非応答者より発現量が多い遺伝子産物を応答者に特異的なマーカーとして同定する工程、又は、応答者に由来する運動神経細胞において、非応答者より発現量が少ない遺伝子産物を非応答者に特異的なマーカーとして同定する工程。 - 下記工程(1)-(3)を含むことを特徴とする、治療薬が有効である対象を選別する方法;
(1)被験者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、請求項1-8のいずれかに記載の方法によって、運動神経細胞を製造する工程、
(3)前記工程(2)で得られた運動神経細胞において、請求項15に記載の方法によって同定された応答者及び/又は非応答者に特異的なマーカーを検出する工程、
(4)前記応答者に特異的なマーカーが検出された運動神経細胞、又は前記非応答者に特異的なマーカーが検出されなかった運動神経細胞が由来する被験者を、対応する治療薬が有効である対象として選別する工程。 - 外来性のLhx3、Ngn2、及びIsl1をコードする核酸が、染色体に挿入された多能性幹細胞。
- 前記核酸が、誘導可能なプロモーターの制御下に、Lhx3、Ngn2、及びIsl1をポリシストロニックに発現する核酸である、
請求項17に記載の多能性幹細胞。 - 前記核酸が、トランスポゾンによって染色体に挿入された核酸である、
請求項18に記載の多能性幹細胞。 - 前記トランスポゾンが、piggyBacトランスポゾンである、
請求項19に記載の多能性幹細胞。 - 前記核酸が、薬剤応答性プロモーターに機能的に連結された核酸である、
請求項17-20のいずれかに記載の多能性幹細胞。 - 前記薬剤応答性プロモーターがテトラサイクリン応答性プロモーターである、
請求項21に記載の多能性幹細胞。 - 前記核酸が、Lhx3、Ngn2、及びIsl1をコードする配列を2A配列で結合した核酸である、
請求項18-22のいずれかに記載の多能性幹細胞。 - 前記多能性幹細胞が、ヒト人工多能性幹細胞である、
請求項17-23のいずれかに記載の多能性幹細胞。 - 前記多能性幹細胞が、変異型SOD1遺伝子を有する多能性幹細胞である、
請求項17-24のいずれかに記載の多能性幹細胞。 - 請求項17-24のいずれかに記載の多能性幹細胞から分化誘導した血球細胞。
- 前記血球細胞が単球及び/又はマクロファージである、
請求項26に記載の血球細胞。 - 請求項17-24のいずれかに記載の多能性幹細胞を有効成分として含む、運動神経変性疾患又は神経損傷の治療用組成物。
- 前記運動神経変性疾患が、筋萎縮性側索硬化症である、
請求項28に記載の治療用組成物。 - 請求項26又は27に記載の血球細胞を有効成分として含む、運動神経変性疾患又は神経損傷の治療用組成物。
- 前記運動神経変性疾患が、筋萎縮性側索硬化症である、
請求項30に記載の治療用組成物。 - 下記工程(1)-(5)を含む、筋萎縮性側索硬化症の治療薬をスクリーニングする方法;
(1)筋萎縮性側索硬化症患者から単離した体細胞から製造した人工多能性幹細胞から、請求項1-8のいずれかに記載の方法によって運動神経細胞を製造する工程、
(2)前記工程(1)で得られた運動神経細胞を、被験物質と接触させる工程、
(3)前記工程(2)で前記被験物質と接触させた運動神経細胞、及び前記被験物質を接触させなかった運動神経細胞(すなわち、対照細胞)を培養する工程、
(4)前記工程(3)で得られた運動神経細胞の細胞数及び/又は神経突起長を測定する工程、
(5)前記被験物質と接触させた運動神経細胞の細胞数及び/又は神経突起長が、対照よりも高値であった被験物質を、筋萎縮性側索硬化症の治療薬として選択する工程。 - 前記筋萎縮性側索硬化症患者から単離した体細胞が、SOD1変異を有する体細胞である、
請求項32に記載の方法。 - 多能性幹細胞から神経細胞を製造する方法であって、下記工程;
(1)多能性幹細胞に、Ngn2をコードする核酸を、トランスポゾンを用いて導入する工程、
(2)前記プロモーターの活性化を誘導して、Ngn2の発現を3日間以上維持する工程、
を(1)-(2)の順番で含む、多能性幹細胞から神経細胞を製造する方法。 - 前記トランスポゾンが、piggyBacトランスポゾンである、
請求項34に記載の方法。 - 前記工程(2)が、前記核酸を、薬剤応答性プロモーターで発現させる工程である、
請求項34又は35に記載の方法。 - 前記薬剤応答性プロモーターがテトラサイクリン応答性プロモーターである、
請求項36に記載の方法。 - 前記工程(2)において、Ngn2の発現を維持する期間が7日間以上である、
請求項34-37のいずれかに記載の方法。 - 前記工程(2)において、前記工程(1)で得られた細胞を動物体内に導入する工程をさらに含み、該動物体内で前記薬剤応答性プロモーターに対応する薬剤と接触させる、
請求項34-38のいずれかに記載の方法。 - 前記多能性幹細胞が、ヒト人工多能性幹細胞である、
請求項34-39のいずれかに記載の方法。 - 前記ヒト人工多能性幹細胞が、アルツハイマー型認知症患者から単離した体細胞より製造された人工多能性幹細胞である、
請求項40に記載の方法。 - 前記ヒト人工多能性幹細胞が、プレセニリン1遺伝子に変異を有するヒト人工多能性幹細胞である、
請求項40に記載の方法。 - 下記工程(1)-(3)を含むことを特徴とする、アルツハイマー型認知症の治験薬に対して治療効果が認められた被験者(すなわち、応答者)又は治療効果が認められなかった被験者(すなわち、非応答者)に特異的なマーカーを同定する方法;
(1)前記応答者及び非応答者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、請求項34-38のいずれかに記載の方法によって、神経細胞を製造する工程、
(3)前記工程(2)で得られた応答者に由来する神経細胞と非応答者に由来する神経細胞の遺伝子産物の発現を測定する工程、及び
(4)応答者に由来する運動神経細胞において、非応答者より発現量が多い遺伝子産物を応答者に特異的なマーカーとして同定する工程、又は、応答者に由来する運動神経細胞において、非応答者より発現量が少ない遺伝子産物を非応答者に特異的なマーカーとして同定する工程。 - 下記工程(1)-(3)を含むことを特徴とする、治療薬が有効である対象を選別する方法;
(1)被験者から単離した体細胞から、人工多能性幹細胞を製造する工程、
(2)前記工程(1)で得られた人工多能性幹細胞から、請求項34-38のいずれかに記載の方法によって、神経細胞を製造する工程、
(3)前記工程(2)で得られた運動神経細胞において、請求項43に記載の方法によって同定された応答者及び/又は非応答者に特異的なマーカーを検出する工程、
(4)前記応答者に特異的なマーカーが検出された神経細胞、又は前記非応答者に特異的なマーカーが検出されなかった神経細胞が由来する被験者を、対応する治療薬が有効である対象として選別する工程。 - 外来性のNgn2をコードする核酸が、トランスポゾンによって染色体に挿入された多能性幹細胞。
- 前記トランスポゾンが、piggyBacトランスポゾンである、
請求項44に記載の多能性幹細胞。 - 前記核酸が、薬剤応答性プロモーターに機能的に連結された核酸である、
請求項45又は46に記載の多能性幹細胞。 - 前記薬剤応答性プロモーターがテトラサイクリン応答性プロモーターである、
請求項47に記載の多能性幹細胞。 - 前記多能性幹細胞が、ヒト人工多能性幹細胞である、
請求項45-48のいずれかに記載の多能性幹細胞。 - 前記ヒト人工多能性幹細胞が、アルツハイマー型認知症患者から単離された体細胞から製造されたヒト人工多能性幹細胞である、
請求項49に記載の多能性幹細胞。 - 前記ヒト人工多能性幹細胞が、プレセニリン1遺伝子に変異を有するヒト人工多能性幹細胞である、
請求項49に記載の方法。 - 下記工程(1)-(4)を含む、アルツハイマー型認知症の治療薬をスクリーニングする方法;
(1)アルツハイマー型認知症患者から単離した体細胞から製造した人工多能性幹細胞から、前記請求項34-38のいずれかに記載の方法によって神経細胞を製造する工程、
(2)前記工程(1)で得られた神経細胞を、被験物質と接触させる工程、
(3)前記工程(2)で前記被験物質と接触させた神経細胞、及び接触させなかった神経細胞(すなわち、対照細胞)を培養し、培地中のAβ42の含有量を測定する工程、
(4)前記被験物質と接触させた神経細胞の培地中のAβ42の含有量が、前記対照細胞の培地中のAβ42の含有量よりも低値であった被験物質を、アルツハイマー型認知症の治療薬として選択する工程。 - 請求項52に記載された方法において、
前記工程(3)において、培地中のAβ40の含有量をさらに測定し、及び、
前記工程(4)において、
前記被験物質と接触させた神経細胞の培地中のAβ42の含有量をAβ40の含有量で除した値(すなわち、Aβ42の含有量/Aβ40の含有量)が、前記対照細胞の培地中のAβ42の含有量/Aβ40の含有量よりも低値であった被験物質を、アルツハイマー型認知症の治療薬として選択する工程である、
アルツハイマー型認知症の治療薬をスクリーニングする方法。
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EP2977449B1 (en) | 2020-02-26 |
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