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  • G01N2800/2821—Alzheimer

Definitions

• the present invention relates to a method for diagnosing the onset of a protein misfolding disease and a risk of development thereof, which method uses nerve cells derived from induced pluripotent stem (iPS) cells, and to a kit to be used in this method.
• the present invention further relates to a method for prediction of the age of the onset of a protein misfolding disease and a kit to be used in this method.
• Protein misfolding diseases are known as neurodegenerative diseases caused by cytotoxicity due to abnormally aggregated proteins (misfolded proteins) (1).
• Alzheimer's disease is a representative protein misfolding disease, which causes deposition of the amyloid ⁇ -protein ( ⁇ ) outside nerve cells in brain. Since initiation of therapy as early as possible leads to effective therapy of Alzheimer's disease, development of a method of early diagnosis of the disease is an important task in an aging society.
• NINCDS-ADRDA and DSM-IV which are excellent for diagnosis of positivity of Alzheimer's disease, are employed as clinical diagnostic criteria, but with these criteria, the possibility that a patient is diagnosed as negative for the disease at an early stage of onset cannot be eliminated. Needless to say, it is impossible to give a diagnosis before onset.
• Alzheimer's disease is caused by accumulation of ⁇ , and there have been reports wherein decrease in the ⁇ 42/ ⁇ 40 ratio and increase in phosphorylated tau (p-tau) protein in the cerebrospinal fluid, and their combination ⁇ - ⁇ 3 ⁇ /( ⁇ 42/ ⁇ 40), were used as diagnostic indices.
• p-tau phosphorylated tau
• nerve cell death has already been progressed in the period wherein these values as diagnostic indices increase. Therefore, even with these indices, early diagnosis and prediction of onset of Alzheimer's disease are difficult.
• nepnlysin functions as a protease that degrades ⁇ in brain (2), and it has been reported that early
• nepnlysin is decreased in hippocampus and temporal lobe gyri, wherein senile plaques are observed, so that the relationship between neprilysin and Alzheimer's disease has been suggested also in human (5).
• iPS cells that are obtained in such a process may be produced using cells derived from a patient to be treated and then allowed to differentiate into cells of various tissues, they are considered to enable reproduction of the diseased state in vitro.
• iPS cells derived from a patient suffering from amyotrophic lateral sclerosis which is a neurodegenerative disease, were produced, and the cells were successfully induced to differentiate into nerve cells (8).
• nerve cells derived from iPS cells are used to analyze expression and functional change of a molecule involved in development of a protein misfolding disease, especially Alzheimer's disease, and the diseased state is reproduced, thereby carrying out early diagnosis of Alzheimer's disease.
• the present invention aims to diagnose the onset of a protein misfolding disease and a risk of development thereof, or to predict the age of the onset of a protein misfolding disease, using nerve cells derived from iPS cells produced from somatic cells of a protein misfolding disease patient. Therefore, the objects of the present invention are to provide a method for diagnosing the onset of and/or a risk of development of, a protein misfolding disease using cells derived from a test subject, and a kit used in the method; and to provide a method for predicting the age of the onset of a protein misfolding disease using cells derived from a test subject, and a kit using the method.
• the protein misfolding disease is preferably Alzheimer's disease.
• the simplest way of diagnosis of a protein misfolding disease is to compare the amount of a causative protein or a function to degrade the protein (hereinafter referred to as the amount of a causative protein or the like) in a control subject where onset of the disease is known, with the amount of the causative protein or the like in the test subject. Since, in such a case, it is sometimes difficult to distinguish between progression of the disease condition and aging, it is necessary, for example, to compare a control subject whose onset of the disease is known with a test subject at the same age. However, since the amount of the protein at the desired age cannot be measured after confirming the onset of the disease, it is very difficult to
• the amount of the causative protein or the like needs to be preliminarily measured at the same age as the test subject in control subjects with a wide range of ages of onset, and, needless to say, it is difficult.
• the area mainly affected by protein misfolding diseases is nerve cells
• measurement of the amount of the causative protein or the like is preferably carried out for nerve cells. Because biopsy of nerve cells imposes a heavy burden to a living body, preliminary preparation of control subjects at the respective ages for diagnosis of a protein misfolding disease is problematic from an ethical point of view.
• iPS cells can differentiate into any tissue by returning them to a blastocyst, they have properties similar to those of cells at an early stage of development. Therefore, nerve cells induced from iPS cells are considered, irrespective of the age of the individual from whom the somatic cells were obtained, to be in the state of nerve cells at a certain age of the individual. Thus, by comparing nerve cells obtained from iPS cells under the same conditions including differentiation induction, days of culture and the like, individual differences in nerve cells at an identical age among the individuals from whom the cells were obtained can be known.
• the inventors of the present invention focused attention on Alzheimer's disease as an example of protein misfolding diseases, and established iPS cells from somatic cells derived from a test subject. Nerve cells were obtained by differentiation induction of the iPS cells, and the content of ⁇ in the culture supernatant of the obtained nerve cells was measured.
• the inventors of the present invention focused on activity of neprilysin, which degrades ⁇ , and the activity of neprilysin in the cell lysate was measured. As a result, it was confirmed that these indices of Alzheimer's disease can be measured also in nerve cells produced by differentiation induction of the iPS cells.
• the inventors of the present invention discovered that the onset of, or the risk of development of, Alzheimer's disease can be diagnosed by using nerve cells produced by differentiation induction of iPS cells derived from a test subject to measure the amount of ⁇ or the activity or the expression level of neprilysin, followed by comparing the obtained value with a measured value in such nerve cells from a control subject wherein the age of the onset of Alzheimer's disease is known.
• the inventors of the present invention discovered that it is useful for prediction of the age of onset of Alzheimer's disease in a test subject to compare the amount of ⁇ or an activity index or the expression level of neprilysin in cells derived from a control subject wherein the age onset of Alzheimer's disease is known with the amount of ⁇ or the activity index or the expression level of neprilysin in nerve cells produced by differentiation induction of iPS cells derived from the test subject, thereby completed the present invention.
• the present invention is as follows.
• a method for diagnosing whether a test subject has developed a protein misfolding disease or whether a test subject has a risk of developing it comprising the steps of:
• control cells are nerve cells obtained by differentiation induction of iPS cells produced from somatic cells derived from a control subject who has not developed the protein misfolding disease at the same age as the test subject, and when the amount of said causative protein in the test subject is higher than that in the control subject, or when the activity or the expression level of the enzyme involved in degradation of said causative protein in the test subject is lower than that in the control subject, it is indicated that the test subject has developed the protein misfolding disease or has a risk of developing it.
• control cells are nerve cells obtained by differentiation induction of iPS cells produced from somatic cells derived from a control subject who has already developed the protein misfolding disease at the same age as the test subject, and when the amount of said causative protein in the test subject is higher than or equivalent to that in the control subject, or when the activity or the expression level of the enzyme involved in degradation of said causative protein in the test subject is lower than or equivalent to that in the control subject, it is indicated that the test subject has developed the protein misfolding disease or has a risk of developing it.
• said subject is human.
• a method for predicting the age of onset of a protein misfolding disease of a test subject comprising the steps of:
• kits for diagnosing whether a test subject has developed a protein misfolding disease or whether a test subject has a risk of developing it, or for predicting the age of onset of a protein misfolding disease in a test subject comprising
• a reagent for measurement of the amount of a causative protein or a reagent for measurement of the activity or the expression level of an enzyme involved in degradation of a causative protein (c) a reagent for measurement of the amount of a causative protein or a reagent for measurement of the activity or the expression level of an enzyme involved in degradation of a causative protein.
• kits according to [ 17] wherein said reprogramming substance(s) comprise(s) at least one factor selected from the group consisting of the OCT family, MYC family, LF family and SOX family.
• kits according to [17], wherein said reagent(s) for differentiation induction into nerve cells comprise(s) at least one factor selected from the group consisting of BDNF, GDNF and neurotensin-3.
• kit according to [22] wherein said reagent for measurement of the activity of the enzyme involved in degradation of said causative protein contains a substrate of neprilysin.
• said substrate of neprilysin is succinyl- alanyl-alanyl-phenylalanine-4-methylcoumarin-7-amide.
• Fig. 1 shows the results of measurement of the contents of ⁇ 40 (A) and ⁇ 42 (B) in the culture supernatant of nerve cells derived from iPS cells, and the ⁇ 42/ ⁇ 40 ratio (C).
• D shows the values obtained by measuring the neprilysin activity in iPS cell-derived nerve cells in the presence and absence of a neprilysin (NEP)-specific inhibitor thiorphan, thereby calculating the neprilysin-specific enzyme activity.
• the abscissa indicates the days after allowing the formed neurosphere to adhere to a culture dish and inducing its differentiation into nerve cells. In this figure, N.D. indicates that the obtained value was below the detection limit.
• Each experiment was carried out twice using 3 lots, and the mean ⁇ SE among the lots is shown.
• Fig. 2 shows the protocol of differentiation induction to nerve cells.
• N2B27 means the medium containing N2 and B27 supplement.
• Fig. 3 shows the image of immunostaining for Foxgl (green), Cuxl (red), Satb2(red), Tbrl (green) and Ctip2 (green) at 52days after differentiation induction (upper; photograph), and the results of measurement of relative expression amount of Foxgl, Cuxl, Satb2, Tbrl and Ctip2 mRNA (lower) in nerve cells derived from iPS cells (253G4).
• the value at 38days is used as standard.
• the mean ⁇ SE among three times is shown.
• Fig. 4 shows the image of immunostaining for Tuj 1 (red), GFAP (green) and Tau (green) at 25, 38, 45, and 52days after differentiation induction (photograph).
• Fig. 5 shows the results of measurement of relative expression amount for Tuj 1 and Synapsin mRNA in nerve cells derived from iPS cells (253G4) at each term. The value at Oday after differentiation induction is used as standard.
• Fig. 6 shows the image of immunostaining for Glutaminase, GAD, vGultl, Tuj 1 , GABA at 52days from differentiation induction (photograph).
• Glutaminase and GAD are shows by red and green respectively (left).
• vGultl and Tuj 1 are shows by green and red respectively (middle).
• GABA and Tuj 1 are shows by green and red respectively (right).
• Scale bar in each figure means 50 ⁇ .
• Fig. 7 shows the results of measurement of relative expression amount of full- length APP (FL-APP), a soluble form of APP a (APPsa) and a soluble form of APP ⁇ (APPsP), and the results of mRNA expression ratio of APP splicing variant (APP770, APP751 and APP695) in nerve cells derived from iPS cells (253G4) at each term.
• FL-APP full- length APP
• APPsa soluble form of APP a
• APPsP soluble form of APP ⁇
• mRNA expression ratio of APP splicing variant APP770, APP751 and APP695
• Fig. 8 shows the results of measurement of relative expression amount of
• BACE1 protein in nerve cells derived from iPS cells (253G4) at each term is used as standard.
• Fig. 9 shows the results of measurement of relative expression amount of Presenilin 1, Nicastrin and Pen2 protein, and relative expression amount of Aph-IA and Aph- 1 B mRNA in nerve cells derived from iPS cells (253G4) at each term. The value at 38days is used as standard.
• Fig. 10 shows the results of measurement of relative expression amount of Neprilysin mRNA in nerve cells derived from iPS cells (253G4) at each term. The value at 38days is used as standard.
• Fig. 11 shows the results of measurement of the contents of ⁇ 40 (left)
• ⁇ 42 (middle) in the culture media of nerve cells derived from iPS cells (253G4), and the ⁇ 42/ ⁇ 40 ratio (right).
• Fig. 12 shows the results of measurement of the contents of ⁇ 40 (left) and ⁇ 42 (right) in the culture media of nerve cells derived from iPS cells (253G4) at 38 and 52 days.
• the abscissa indicates concentration of ⁇ -secretase inhibitor, Compound E.
• DMSO means absence of Compound E.
• Fig. 13 shows the image of immunostaining for Tuj 1 (red) and Tau (green) at 38, 45, and 52days after differentiation induction (photograph).
• Fig. 14 shows the image of immunostaining for SATB2 (green), CUX1 (red), CTIP2 (green) and TBR1 (green) at 52days from differentiation induction (photograph).
• Fig. 15 shows the results of mRNA expression ratio of APP splicing variant (APP770, APP751 and APP695) in nerve cells derived from Alzheimer's disease (AD)-iPS cells at each term.
• Fig. 16 shows the results of measurement of the contents of ⁇ 40 and ⁇ 42 in the culture supernatant of nerve cells derived from AD-iPS cells, the ⁇ 42/ ⁇ 40 ratio and relative expression amount of FL-APP (the value of FL-APP at 38days is used as standard).
• Fig. 17 shows the results of Ap O/FL-APP ratio, Ap42/FL-APP ratio, Ap40/p-actin, A 42/p-actin and FL- ⁇ / ⁇ -actin.
• the values of FL-APP and ⁇ - actin at 38days are used as standard.
• Fig. 18 shows the results of measurement of the contents of ⁇ 40 (left) and ⁇ 42 (right) in the culture media of nerve cells derived from AD-iPS cells at 38 and 52 days.
• the abscissa indicates concentration of ⁇ -secretase inhibitor, Compound E.
• DMSO means absence of Compound E.
• the present invention provides a method for diagnosing whether a test subject has developed a protein misfolding disease or whether a test subject has a risk of developing it, the method comprising the steps of:
• the present invention also provides a method for predicting the age of the onset of a protein misfolding disease of a test subject, the method comprising the steps of:
• the measured value is equivalent to the amount of the causative protein, or the activity or the expression level of the enzyme involved in degradation of the causative protein in nerve cells obtained by differentiation induction of iPS cells produced from somatic cells of a control subject whose age of onset of the protein misfolding disease is known, it is indicated that the test subject develops the protein misfolding disease at the age when the control subject developed the protein misfolding disease.
• the present invention also provides a kit to be used for these methods.
• protein misfolding disease means a group of diseases caused when a protein is not normally folded or the original structure of a protein is degenerated (misfolding).
• diseases conventionally called neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, dementia with Lewy Bodies and multiple system atrophy.
• the causative protein may be either a protein that is deposited outside the cells or a protein that accumulates inside the cells.
• examples of the causative protein include, but are not limited to, ⁇ and tau.
• examples of the causative protein include, but are not limited to, a-synuclein.
• the enzyme involved in degradation of the causative protein is preferably a protease.
• the causative protein is amyloid ⁇
• examples of the enzyme include, but are not limited to, neprilysin.
• iPS cells can be produced by introducing certain specific nuclear reprogramming substances to somatic cells, which nuclear reprogramming substances may be in the forms of DNAs or proteins.
• iPS cells are somatic cell-derived artificial stem cells having properties almost equivalent to those of ES cells, such as pluripotency of differentiation and growth ability by self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al.
• the nuclear reprogramming substances are not restricted as long as these are genes specifically expressed in ES cells, or genes playing important roles in maintenance of the undifferentiated state of ES cells, or gene products thereof.
• nuclear reprogramming substances examples include Oct3/4, Klf4, Klfl, Klf2, Klf5, Sox2, Soxl, Sox3, Soxl5, Soxl7, Soxl8, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb and Esrrg.
• reprogramming substances may be used in combination when iPS cells are to be established.
• the above reprogramming substance(s) may be used solely or as a combination of 2 or 3 reprogramming substances, preferably as a combination of 4 reprogramming substances.
• nucleotide sequences of the mouse and human cDNAs of the respective nuclear reprogramming substances described above, and the amino acid sequence information of the proteins encoded by the cDNAs can be obtained by reference to the NCBI accession numbers described in WO 2007/069666, and the mouse and human cDNA sequences and the amino acid sequence information of L-Myc, Lin28, Lin28b, Esrrb and Esrrg can be obtained by reference to the respective following NCBI accession numbers.
• Those skilled in the art can prepare a desired nuclear reprogramming substance based on the cDNA sequence or the amino acid sequence information, according to a conventional method.
• nuclear reprogramming substances may be introduced into somatic cells in the form of proteins by a method such as lipofection, linking to a cell- permeable peptide, or microinjection.
• the nuclear reprogramming substances may be introduced into somatic cells in the form of DNAs by a method such as usage of a vector including virus, plasmid and artificial chromosome vectors; lipofection; usage of liposome; or microinjection.
• virus vectors include retrovirus vectors, lentivirus vectors (these are described in Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; and Science, 318, pp.
• adenovirus vectors examples include human artificial chromosome (HAC), yeast artificial chromosome (YAC), and bacterial artificial chromosome (BAC, PAC).
• HAC human artificial chromosome
• YAC yeast artificial chromosome
• BAC bacterial artificial chromosome
• plasmids examples include plasmids for mammalian cells (Science, 322: 949-953, 2008).
• the vectors may comprise a regulatory sequence(s) such as a promoter, enhancer, ribosome binding sequence, terminator and/or polyadenylation site in order to allow expression of the nuclear reprogramming substance(s); and, as required, a sequence of a selection marker such as a drug resistance gene (e.g., kanamycin-resistant gene, ampicillin- resistant gene or puromycin-resistant gene), thymidine kinase gene or diphtheria toxin gene; and a gene sequence of a reporter such as the green-fluorescent protein (GFP), ⁇ -glucuronidase (GUS) or FLAG.
• a regulatory sequence(s) such as a promoter, enhancer, ribosome binding sequence, terminator and/or polyadenylation site in order to allow expression of the nuclear reprogramming substance(s); and, as required, a sequence of a selection marker such as a drug resistance gene (e.g., kanamycin-resistant gene, ampicillin- resistant gene
• the vector may have loxP sequences in the upstream and the downstream of these sequences. Further, the above vector may contain EBNA-1 and oriP, or Large T and SV40ori sequences.
• HDAC histone deacetylase
• VPA valproic acid
• trichostatin A sodium butyrate
• MCI 293 and M344 nucleic acid-type expression inhibitors
• siRNAs and shRNAs against HDAC e.g., HDAC1 siRNA Smartpool (registered trademark) (Millipore) and HuSH 29mer shRNA Constructs against FID AC 1 (OriGene)
• DNA methyltransferase inhibitors e.g., 5'- azacytidine
• methyltransferase inhibitors for example, low molecular inhibitors such as ⁇ - 01294 (Cell Stem Cell, 2: 525-528 (2008)); and nucleic acid-type expression inhibitors such as siRNAs and shRNAs against G9a (e.g., G9a siRNA (human) (Santa Cruz Biotechnology))], L-channel calcium agonists (e.g., Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors [e.g., siRNAs and shRNAs against p53 (Cell Stem Cell, 3, 475-479 (2008))], Wnt Signaling (e.g., soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), cytokines such as LIF an bFGF, ALK5 inhibitors (e.g., SB431542) (Nat Methods, 6: 805-8 (2009)), mitogen-activated protein kinase signaling inhibitors, glycogen synthase
• Examples of the culture medium for induction of the iPS cells include (1) DMEM, DMEM/F 12 and DME media supplemented with 10 to 15% FBS (these media may further contain LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol and/or the like, as appropriate); (2) culture media for ES cells containing bFGF or SCF, for example, culture media for mouse ES cells (e.g., TX-WES medium, Thromb-X) and culture media for primate ES cells (e.g., culture medium for primate (human and monkey) ES cells,
• Examples of the culture method include a method wherein somatic cells and nuclear reprogramming substances (DNAs or proteins) are brought into contact with each other at 37°C in the presence of 5% C0 2 in DMEM or DMEM/F 12 medium supplemented with 10% FBS, and the cells are cultured for about 4 to 7 days, followed by plating the cells on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells or the like) and starting culture in a bFGF-containing culture medium for primate ES cells about 10 days after the contact between the somatic cells and the reprogramming substances, thereby allowing iPS-like colonies to appear about 30 to about 45 days after the contact, or later.
• feeder cells e.g., mitomycin C-treated STO cells, SNL cells or the like
• a bFGF-containing culture medium for primate ES cells about 10 days after the contact between the somatic cells and the reprogramming substances, thereby allowing iPS-like colonies to appear about 30 to
• the somatic cells may be cultured on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells or the like) in DMEM medium supplemented with 10% FBS (which may further contain LIF,
• feeder cells e.g., mitomycin C-treated STO cells, SNL cells or the like
• FBS which may further contain LIF
• the culture medium is replaced with a fresh culture medium once every day from Day 2 of the culture.
• the number of the somatic cells used for nuclear reprogramming is not restricted, and usually within the range of about 5> ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100-cm 2 area on the culture dish.
• cells expressing the marker gene can be selected by culturing the cells in a culture medium (selection medium) containing the corresponding drug.
• Cells expressing a marker gene can be detected by observation under a fluorescence microscope in cases where the marker gene is the gene of a fluorescent protein; by adding a luminescent substrate in cases where the marker gene is the gene of luciferase; or by adding a coloring substrate in cases where the marker gene is the gene of a coloring enzyme.
• somatic cells used in the present specification means any cells, excluding germ cells, derived from a mammal (e.g., human, mouse, monkey, pig or rat).
• somatic cells include epithelial cells which are keratinized (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the lingual surface), epithelial cells of exocrine glands (e.g., mammary cells), hormone- secreting cells (e.g., adrenomedullary cells), cells for metabolism and storage (e.g., hepatic cells), luminal epithelial cells constituting boundary surfaces (e.g., type I alveolar cells), luminal epithelial cells in the closed circulatory system (e.g., vascular endothelial cells), ciliated cells having a carrying capacity (e.g., tracheal epithelial cells), extracellular matrix-secreting cells (
• undifferentiated progenitor cells including somatic stem cells
• terminally-differentiated mature cells may be used as the source of the somatic cells in the present invention.
• undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells and dental pulp stem cells.
• the mammalian subject from which the somatic cells are collected is not restricted, and preferably human. More preferably, the somatic cells are collected from a patient whose age of onset of the protein misfolding disease is known, and from a test subject.
• neural stem cells means cells having an ability to differentiate into nerve cells, astrocytes and oligodendrocytes and capable of self- renewal.
• nerve cell means cells having an ability to differentiate into nerve cells, astrocytes and oligodendrocytes and capable of self- renewal.
• nerve cell means cells having an ability to differentiate into nerve cells, astrocytes and oligodendrocytes and capable of self- renewal.
• nerve cell is not distinguished with the terms of "neuron” or "neural cell”
• the method of differentiation induction of the above-mentioned iPS cells into neural stem cells is not restricted, and examples of the method which may be used include a differentiation induction method by high-density culture on a fibroblast feeder layer (JP 2008-201792 A), a differentiation induction method by co-culturing with stromal cells (SDIA method) (e.g., WO 2001/088100 and WO 2003/042384) and a differentiation induction method by suspension culture (SFEB method) (WO 2005/123902), and methods by combination of these methods.
• a differentiation induction method by high-density culture on a fibroblast feeder layer JP 2008-201792 A
• SDIA method differentiation induction method by co-culturing with stromal cells
• SFEB method suspension culture
• the differentiation induction may be carried out by forming a neurosphere by suspension culture.
• Preferred examples of the method include a method wherein the cells to be used for the suspension culture is prepared by adherent culture on a culture dish preliminarily subjected to coating treatment, which contains an arbitrary medium.
• the differentiation induction may be carried out by adhesion culture using coated dish to substitute for feeder cells.
• Examples of the coating agent to be used in the adherent culture before the formation of a neurosphere include collagen, gelatin, poly-L-lysine, poly-D-lysine, fibronectin, lamimn, entactin, and collagen IV, and combinations thereof.
• the coating agent is preferably the combination of poly-L-lysine and lamimn, or entactin, collagen IV and laminin.
• the combination of entactin, collagen IV and laminin can be purchased from Millipore as "ECL Cell Attachment Matrix"
• the medium to be used may be prepared by adding an additive(s) to a basal medium.
• the basal medium is not restricted as long as it can be used for culture of animal cells, and examples thereof include Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, aMEM medium, DMEM medium, DMEM/F12 medium, Ham's medium, RPMI 1640 medium and Fischer's medium, and mixed media thereof.
• the basal medium is more preferably a mixture of Neurobasal medium and DMEM/F12.
• additive(s) examples include serum, retinoic acid, BMP inhibitors, TGFP family inhibitors, bFGF, EGF, HGF, LIF, amino acids, vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS- supplement and antibiotics.
• Preferred additives are BMP inhibitor, glutamine as an amino acid, B27-supplement and N2-supplement.
• BMP inhibitor involved in inhibition of the BMP signaling that is mediated by binding of BMP to a BMP receptor (type I or type II).
• BMP inhibitor having such properties include a compound that inhibits BMP2, BMP4, BMP6 or BMP7 capable of activating a transcription factor SMAD1, SMAD5, or SMAD8, such as Noggin, chordin, follistatin, Dorsomorphin (that is, 6-[4-(2-piperidin-l-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[l,5- ajpyrimidine) and a derivative thereof (P. B. Yu et al. (2007), Circulation, 116:
• BMP inhibitor is Noggin.
• TGFP family inhibitor is a compound which interferes with the signaling of the TGFp family.
• examples of such TGFP family inhibitor include SB431542, SB202190 (R. K. Lindemann et al., Mol. Cancer 2: 20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, and LY580276 (Lilly Research Laboratories), and A-83- 01(WO 2009146408).
• SB431542 is preferred.
• the concentration of the iPS cells at the beginning of the culture may be set appropriately to allow efficient formation of neural stem cells.
• the concentration of the iPS cells at the beginning of the culture is not restricted, and, for example, about l xlO 3 to about 1 *10 6 cells/ml, preferably about 1 *10 4 to about 5*10 5 cells/ml.
• the culture temperature is not restricted, and, for example, about 30 to 40°C, preferably about 37°C.
• the C0 2 concentration is, for example, about 1 to 10%, preferably about 5%.
• the neurosphere may be formed by suspension culture using the above basal medium and additive(s).
• Preferred examples of the medium include a mixture of Neurobasal medium and DMEM/F 12.
• Preferred examples of the additive(s) include serum, Noggin as a BMP inhibitor, bFGF, EGF, heparin, B27-supplement and N2- supplement
• the cell concentration at the beginning of formation of the neurosphere may be set appropriately to allow efficient formation of the neurosphere.
• the cell concentration at the beginning of the culture is not restricted, and, for example, about lxlO 4 to about 5> ⁇ 10 6 cells/ml, preferably about 5 ⁇ 10 5 to about 2> ⁇ 10 6 cells/ml.
• the cells may be subcultured when the size of the neurosphere becomes appropriate.
• the days of formation of the neurosphere is not restricted, and preferably every 15 days to 45 days, more preferably every 30 days.
• the cells may not necessarily be completely separated from each other, and separation of the cells may be carried out either by a mechanical method or by using a separation solution having a protease activity and collagenase activity.
• the number of times of subculture is not restricted, and preferably not less than once. Preferably, the number of times of subculture is twice before differentiation induction into nerve cells.
• the culture vessel is preferably non-cell- adhesive.
• the non-cell-adhesive culture vessel one that has not been artificially treated for the purpose of enhancement of adhesiveness to cells (e.g., not coated with an extracellular matrix or the like), or one that has been artificially treated such that adhesion of cells is suppressed (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).
• poly-HEMA polyhydroxyethyl methacrylate
• the culture temperature is not restricted, and, for example, about 30 to 40°C, preferably about 37°C.
• the C0 2 concentration is, for example, about 1 to 10%, preferably about 5%.
• iPS cells may be used, whose size of the colony becomes appropriate.
• the above coating agent, basal medium and additive(s) are used for culture.
• Preferred examples of the medium include a mixture of Neurobasal medium and DMEM/F12.
• Preferred examples of the additive(s) include serum, Noggin as a BMP inhibitor, SB431542 as a TGFP family inhibitor, B27-supplement and N2-supplement.
• the days of using Noggin and SB431542 as additive is not restricted, and preferably 10 days to 24 days, more preferably every 17 days. After that, the additives may be changed to B27-supplement and N2-supplement without Noggin and SB431542.
• the days of adhesion culture in total for inducing neural stem cells are preferably 15 days to 30 days, more preferably 24 days.
• the coating agent may be changed at passage.
• Preferable coating agent is poly-L-lysine and laminin at first.
• coating agent may be changed to poly-L-lysine laminin, entactin, and collagen IV.
• the days of using poly-L-lysine and laminin as coating agents is not restricted, and preferably 7 days to 14 days, more preferably every 10 days, and the days of using poly-L-lysine laminin, entactin, and collagen IV as coating agents is not restricted, and preferably 4 days to 10 days, more preferably every 7 days.
• the thus induced neural stem cells can be identified by intermediate filament proteins such as N-CAM, polysialylated N-CAM, A2B5, nestin and vimentin; and expression markers for primitive neuroectoderm and neural stem cells, such as a transcription factor Pax-6.
• the cells are preferably confirmed by expression of nestin.
• the neural stem cells induced by the above-mentioned method can be induced to differentiate into nerve cells by separating the neural stem cells by an arbitrary method and culturing them on a culture dish subjected to coating treatment, which contains an arbitrary medium.
• the nerve cells are preferably cerebrocortical neurons.
• the separation of the cells may be carried out either by a mechanical method or by using a separation solution having a protease activity and collagenase activity (e.g., Accutase (TM) or Accumax (TM)).
• a separation solution having a protease activity and collagenase activity e.g., Accutase (TM) or Accumax (TM)
• the coating agent examples include collagen, gelatin, poly-L-lysine, poly- D-lysine, fibronectin and laminin, and combinations thereof.
• the coating agent is preferably the combination of poly-L-lysine, fibronectin and laminin.
• the medium to be used may be prepared by adding an additive(s) to a basal medium.
• the basal medium is not restricted as long as it can be used for culture of animal cells, and examples thereof include Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, aMEM medium, DMEM medium, DMEM/F12 medium, Ham's medium, RPMI 1640 medium and Fischer's medium, and mixed media thereof.
• the basal medium is preferably a mixture of Neurobasal medium and DMEM/F12.
• additive(s) examples include serum, retinoic acid, Wnt, BMP, bFGF, EGF, HGF, Sonic hedgehog (Shh), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), insulin-like growth factor (IGF1), amino acids, vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement and antibiotics.
• BDNF brain-derived neurotrophic factor
• GDNF glial cell line-derived neurotrophic factor
• NT-3 neurotrophin-3
• IGF1 insulin-like growth factor
• Preferred additives are retinoic acid, Shh, BDNF, GDNF, NT-3, B27-supplement and N2-supplement.
• Combination of the additives may be changed in a stepwise manner, and the culture is preferably first carried out in a medium supplemented with retinoic acid, Shh, B27-supplement and N2-supplement, and then in a medium supplemented with BDNF, GDNF, NT-3, B27-supplement and N2-supplement.
• the concentration of the neural stem cells may be set appropriately to allow efficient formation of nerve cells.
• the concentration of the neural stem cells at the beginning of the culture is not restricted, and, for example, about 1 x 10 3 to about l x lO 6 cells/ml, preferably about l x lO 4 to about 5*10 5 cells/ml.
• the culture temperature is not restricted, and, for example, about 30 to 40°C, preferably about 37°C.
• the C0 2 concentration is, for example, about 1 to 10%, preferably about 5%.
• the 0 2 concentration is 1 to 20%.
• the culturing period after separation of a neurosphere and adhesion thereof on a culture dish is not restricted, and preferably not less than 5 days, more preferably 14, 21, 22 or 28 days.
• the nerve cells can be characterized by their ability to express BF1, ⁇ tubulin, TuJl, NeuN, 160kDa neurofilament protein, MAP2ab, glutamate, synaptophysin, glutamic acid decarboxylase (GAD), tyrosine hydroxylase, GABA, serotonin, GFAP, Foxgl, Cuxl, Satb2, Tbrl, Ctip2 and Synapsin, but the indices for characterization of the nerve cells is not restricted thereto.
• the amount of the protein may be measured using a lysate of nerve cells derived from iPS cells.
• the causative protein is a secretory protein
• the amount of the protein contained in the culture liquid may be measured.
• a per se known method may be used for the measurement. Examples of the measurement method in cases where the causative protein is ⁇ are described below.
• ⁇ means amyloid ⁇ -peptide, which is a protein fragment produced by cleavage of amyloid precursor protein (APP) by ⁇ - and ⁇ - secretase, and the amyloid ⁇ -peptide is preferably constituted by 40 amino acid residues ( ⁇ 40) or 42 amino acid residues ( ⁇ 42). Therefore, the amount of ⁇ used in the present invention may be the concentration of ⁇ 40 or ⁇ 42 secreted into the culture liquid, and may preferably be the ratio of abundance of ⁇ 42 with respect to ⁇ 40 ( ⁇ 42/ ⁇ 40), full-length amyloid precursor proteins (FL-APP), housekeeping gene (ex. ⁇ -actin, HPRT, GAPDH, and HSP90) or nerve cell marker (ex.
• APP amyloid precursor protein
• tubulin TuJl, NeuN, 160kDa neurofilament protein, MAP2ab, glutamate, synaptophysin, GAD, tyrosine hydroxylase, GABA, serotonin, GFAP, Foxgl, Cuxl, Satb2, Tbrl , Ctip2 and Synapsin).
• the diagnosis or prediction of the age of onset of Alzheimer's disease of the present invention may be carried out by measuring the concentration of ⁇ in the culture liquid of nerve cells derived from iPS cells obtained as described above.
• the measurement of the concentration of ⁇ can be carried out by using a per se known method. Examples of the method include ELISA, Western blotting, immunoprecipitation, slot or dot blot assay, immunohistostaining, radioimmunoassay (RIA), fluoroimmunoassay, and immunoassay using the avidin-biotin or streptavidin- biotin system.
• the amount of ⁇ obtained as described above can be used as intermediate data for diagnosis of Alzheimer's disease by a physician or the like.
• measurement of the activity of the enzyme involved in degradation of the causative protein can be carried out by measuring the amount of degradation of the causative protein or a fragment thereof as a substrate of the enzyme.
• the enzyme involved in degradation of the causative protein is an intracellular protein
• a lysate of nerve cells derived from iPS cells may be brought into contact with the substrate, followed by measuring the amount of degradation of the substrate.
• the culture liquid may be brought into contact with the substrate, followed by measuring the amount of degradation of the substrate.
• the measurement of degradation of the substrate may be carried out by using a per se known method.
• the expression level of the enzyme may also be used as an index.
• the expression level of the gene encoding the enzyme can be measured by RT-PCR, Northern blotting or the like, and the amount of protein translated from the gene can be measured by ELISA, Western blotting or the like.
• the enzyme activity may preferably be represented as the amount of the substrate degraded during a certain period of time per unit number of cells.
• the total protein mass in the cell lysate may be used as an alternative to the number of cells. Examples of the method for measuring the activity in cases where the enzyme involved in degradation of the causative protein is neprilysin are described below.
• neprilysin (EC 3.4.24.11) as an example of the enzyme involved in degradation of the causative protein is a membrane-bound neutral endopeptidase that exists in various animal tissues and whose active site is oriented toward the outside of the cells.
• neprilysin was discovered as an enkephalin-degrading peptidase, and it is also called enkephalinase.
• peptides that may be substrates of neprilysin has been revealed by in vitro experiments, and examples of the peptides include enkephalin, substance P, atrial natriuretic peptide (ANP), gastrin releasing peptide (GRP) and endothelin.
• the activity of neprilysin can be measured by, for example, the method described in JP 2002-34596 A or JP 2004-151079 A.
• the activity of neprilysin is preferably represented by the amount of an artificial substrate cleaved by a specific amount of the protein in a nerve cell extract in a given period of time. More particularly, a substrate of neprilysin is allowed to react with the nerve cells, the nerve cells that was fixed, a cell lysate of the nerve cells, neprilysin purified from the nerve cells, or the like, followed by calculation based on the amount of degradation of the substrate.
• examples of the substrate of neprilysin include, in addition to the substrates mentioned above, synthetic substrates such as benzyloxycarbonyl-alanyl- alanyl-leucyl-paranitroanilide, benzyloxycarbonyl-alanyl-alanyl-phenylalanyl- paranitroanilide, benzyloxycarbonyl-glycyl-glycyl-leucyl-paranitroanilide, benzyloxycarbonyl-glycyl-glycyl-phenylalanyl-paranitroanilide, glutaryl-alanyl- alanyl-phenylalanyl-4-methoxy-2-naphthylamide, glutaryl-alanyl-alanyl- phenylalanyl-2-naphthylamide, and succinyl-alanyl-alanyl-phenylalanine-4- methylcoumarin-7-amide, but the substrate is not limited
• the reaction conditions may be selected appropriately by those skilled in the art depending on the substrate used.
• concentration of the substrate varies depending on the type of the substrate to be used, the reaction may be carried out with a substrate at a concentration of 0.1 to 1000 ⁇ g/ml.
• concentration of the substrate is preferably 1 to 100 ⁇ g ml, more preferably 3 to 30 ⁇ g/ml in the reaction system.
• the reaction temperature is preferably 20°C to 45°C, more preferably 20°C to 40°C.
• the reaction time varies depending on the conditions of the reaction system such as the type of the substrate to be used and the concentration thereof.
• the reaction system is preferably designed such that the measurement can be carried out in a short period of time such as 5 minutes to 60 minutes.
• the reaction is preferably carried out at a neutral pH, that is, pH 6 to 9, more preferably pH 7 to 8.
• the amount of degradation of the substrate may be determined by measuring the concentration of a compound obtained after the degradation.
• the method of measurement is not restricted, and, in cases where the compound obtained by the degradation emits fluorescence or in cases where the compound obtained by the degradation reacts with a reagent to emit fluorescence, the amount of degradation can be measured by measuring the fluorescence intensity.
• the amount of degradation of the substrate may be analyzed by thin layer
• the amount of degradation may be corrected by a measured value obtained after addition of an inhibitor (e.g., thiorphan).
• the activity of neprilysin may be represented by the amount of degradation of a substrate per unit number of cells.
• neprilysin obtained as mentioned above can be used as intermediate data for diagnosis of Alzheimer's disease by a physician or the like.
• control cells examples include nerve cells produced by differentiation induction of iPS cells obtained from somatic cells derived from a control subject (e.g. normal subject) who has not developed a protein misfolding disease, and nerve cells produced by differentiation induction of iPS cells obtained from somatic cells derived from a control subject (patient) who has developed a protein misfolding disease.
• control cells are nerve cells produced by differentiation induction of iPS cells obtained from somatic cells derived from a control subject who has not developed a protein misfolding disease
• a test subject can be judged as having developed the protein misfolding disease and/or having a risk of developing it when the amount of a causative protein in the cells derived from the test subject is higher than the amount of the causative protein in the control cells, or when the activity or the expression level of an enzyme involved in degradation of a causative protein in the cells derived from the test subject is lower than the activity or the expression level of the enzyme in the control cells.
• a test subject in cases where the control cells are nerve cells produced by differentiation induction of iPS cells obtained from somatic cells derived from a control subject who has developed a protein misfolding disease, a test subject can be judged as having developed the protein misfolding disease or having a risk of developing it when the amount of a causative protein in the cells derived from the test subject is equivalent to or higher than the amount of the causative protein in the control cells, or when the activity or the expression level of an enzyme involved in degradation of a causative protein in the cells derived from the test subject is equivalent to or lower than the activity or the expression level of the enzyme in the control cells.
• the protein misfolding disease of which onset and/or the risk of development are to be diagnosed, is Alzheimer's disease as an example, and the diagnosis may be carried out for either familial or sporadic Alzheimer's disease. Familial Alzheimer's disease is exemplified as the Alzheimer's disease. In a more preferred embodiment, the Alzheimer's disease is early-onset Alzheimer's disease.
• the amount of ⁇ as a causative protein is preferably measured as the amount thereof in the culture supernatant of nerve cells produced by
• neprilysin as an enzyme involved in degradation of the causative protein is preferably measured using a cell lysate or the like.
• the amount of a causative protein, or the activity or the expression level of an enzyme involved in degradation of a causative protein in control cells may be preliminarily measured to prepare Table 1 or Table 2, and the judgment may be made using a reference value determined such that both the sensitivity and the specificity shown in Table 1 or Table 2 are not less than 0.9, preferably not less than 0.95, more preferably not less than 0.99. Especially preferably, both the sensitivity and the specificity are 1.
• the fact that both the sensitivity and the specificity are 1 means that the reference value is ideal, producing neither false-positive nor false-negative result at all.
• the test subject in cases where the amount of a causative protein such as ⁇ in the culture supernatant of the cells derived from a test subject is higher than the reference value, the test subject can be judged as having developed a protein misfolding disease or having a risk of development thereof. Similarly, in cases where the activity or the expression level of an enzyme involved in degradation of a causative protein in the cells derived from a test subject is lower than the reference value, the test subject can be judged as having developed a protein misfolding disease or having a risk of development thereof.
• the diagnosis of a protein misfolding disease may be carried out in combination with another diagnostic method such as a method based on findings from the PET or MRI exam of the patient, or a neuropsychological test for the cognitive function including WMS-R logical memory score, MMSE or ADAS-Jcog.
• another diagnostic method such as a method based on findings from the PET or MRI exam of the patient, or a neuropsychological test for the cognitive function including WMS-R logical memory score, MMSE or ADAS-Jcog.
• the test subject By comparing the amount of a causative protein in nerve cells produced by differentiation induction of iPS cells established from somatic cells of a test subject with the amount of the protein in control cells, or by comparing the activity or the expression level of an enzyme involved in degradation of a causative protein in such nerve cells derived from a test subject with the activity or the expression level of the enzyme in control cells, and when the compared values are equivalent between the nerve cells derived from the test subject and the control cells, it is indicated that the test subject develops the protein misfolding disease at the same age as the age when the control subject from whom the control cells were derived has developed the disease.
• the protein misfolding disease for which the age of onset is to be predicted is Alzheimer's disease as an example, and the prediction may be carried out for either familial or sporadic Alzheimer's disease.
• the familial Alzheimer's disease is exemplified as the Alzheimer's disease.
• the Alzheimer's disease is early-onset Alzheimer's disease (juvenile Alzheimer' s disease).
• the amount of ⁇ as a causative protein is preferably measured as the amount thereof in the culture supernatant of nerve cells produced by
• neprilysin as an enzyme involved in degradation of the causative protein is preferably measured using a cell lysate or the like.
• control cells are preferably nerve cells produced by differentiation induction of iPS cells established from somatic cells of a control subject (patient) whose age of onset of the protein misfolding disease is known.
• equivalent includes a case where the value is strictly identical as well as a case where an error preferably within the range of ⁇ 5% exists with respect to the measured value.
• the same age may include an error within the range of ⁇ 5% with respect to the age or age within decade.
• the kit of the present invention for diagnosis of, or for prediction of the age of onset of, a protein misfolding disease comprises: (a) a reprogramming
• substance(s) for production of iPS cells (b) a reagent(s) for differentiation induction into nerve cells, and (c) a reagent for measurement of the amount of a causative protein or a reagent for measurement of the activity or the expression level of an enzyme involved in degradation of a causative protein.
• the reprogramming substances for production of iPS cells may be used.
• another factor may also be included in the reprogramming substances.
• the reprogramming substances preferably contain at least one factor selected from the group consisting of the OCT family, MYC family, KLF family and
• the reagent(s) for differentiation induction into nerve cells may be used as the reagent(s) for differentiation induction into nerve cells.
• the reagents are preferably those described in the method for differentiation induction into nerve cells, and more preferably contain at least one factor selected from the group consisting of BDNF, GDNF and neurotensin-3.
• the reagent for measurement of the amount of a causative protein is preferably a reagent for measurement of the amount of ⁇ , and, as the reagent, the reagents described in the above-mentioned method for measuring the amount of ⁇ may be used.
• the reagent is preferably an antibody specific to ⁇ 40 and/or ⁇ 42 used for the ELIS A method.
• the reagent for measurement of the activity of an enzyme involved in degradation of a causative protein is preferably a reagent for measuring the activity of a protease, and more preferably contains a causative protein or a fragment thereof as a substrate of the protease.
• the reagent for measurement of activity of an enzyme involved in degradation of a causative protein is a reagent for measuring activity of neprilysin, and the reagents described in the above-mentioned method for measuring the activity of neprilysin may be used.
• the reagent preferably contains a substrate of neprilysin, more preferably succinyl- alanyl-alanyl-phenylalanine-4-methylcoumarin-7-amide.
• the reagent for measuring activity of neprilysin may further contain an inhibitor or activator of neprilysin for measurement of a control value, and the inhibitor is preferably thiorphan.
• the reagent for measuring the expression level of an enzyme involved in degradation of a causative protein preferably contains an antibody against the enzyme, PCR primers or a hybridization probe specific to the gene of the enzyme, or the like.
• the kit of the present invention for diagnosis of, or prediction of the age of onset of, a protein misfolding disease may comprise means of screening such as a document or instruction wherein a procedure of the screening is described; a program(s) for execution of a procedure of the screening on a computer; a computer- readable storage medium wherein a list of the program(s) and the program(s) are recorded (e.g., flexible disk, optical disk, CD-ROM, CD-R, CD-RW or the like); and/or a device or system (e.g., computer) for carrying out the screening.
• means of screening such as a document or instruction wherein a procedure of the screening is described
• a program(s) for execution of a procedure of the screening on a computer e.g., flexible disk, optical disk, CD-ROM, CD-R, CD-RW or the like
• a device or system e.g., computer
• iPS cell 26(1): 101-6. was used as the iPS cell,. Briefly, the iPS cells were established by introducing Oct3/4, Sox2 and Klf4 to fibroblasts derived from a Caucasian female of 36 years old.
• a neurosphere was carried out by the method described in Wada T, et al, PLoS ONE 4(8), e6722, 2009 with a slight modification. More particularly, the iPS cells were divided into small clusters and cultured using N2B27 medium (Gibco) prepared by mixing DMEM/F 12 and Neurobasal medium A together at a volume ratio of 1:1 and adding 1% N2, 2% B27 and 200 ⁇ glutamine thereto, in a dish coated with poly-L-lysine/laminin (PLL/LM) (Sigma-Aldrich). Further, to this medium, Noggin (R&D systems) was added at 100 ng/ml. The medium was replaced with a medium containing 100 ng/ml Noggin every 3 days, and, after 10 days of culture, the cells were subcultured to a PLL LM-coated dish.
• N2B27 medium Gibco
• Noggin R&D systems
• the medium was replaced with a medium containing 100 ng/ml Noggin every other day, followed by plating the cells in a 2-hydroxyethylmetacrylate (HEMA)-coated dish at a concentration of 1 ,000,000 cells/ml on Day 7.
• HEMA 2-hydroxyethylmetacrylate
• N2B27 medium supplemented with 20 ng/ml EGF (R&D systems), 20 ng/ml bFGF and 50 ng/ml heparin (Sigma-Aldrich) was used. All these cultures were carried out by incubation at 37°C under a humidified atmosphere of 5% C0 2 . The subculturing was carried out every 30 days with pipetting, and the medium was replaced every 7 days.
• a secondary neurosphere which was prepared by subculturing the above- mentioned neurosphere to allow formation of a neurosphere again, was separated using Accutase (Innovative Cell Technologies), and plated at a concentration of 15,000 cells/ml on a PLL/LM/ fibronectin-coated dish containing N2B27 medium supplemented with 1 ⁇ retinoic acid (Sigma-Aldrich) and 100 ng/ml sonic hedgehog (R&D systems). The medium was replaced every other day.
• N2B27 medium supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF) (R&D systems), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF) (R&D systems) and 10 ng/ml neurotensin-3 (NT-3) (R&D systems).
• BDNF brain-derived neurotrophic factor
• GDNF glial cell line-derived neurotrophic factor
• NT-3 10 ng/ml neurotensin-3
• the secondary neurosphere was allowed to adhere to a dish, followed by culturing it for 4 days or 22 days, after which the culture supernatant of the nerve cells was collected. The culture supernatant was centrifuged at 4°C at 3,000 rpm for 5 minutes to remove the precipitate before use.
• ELISA using a combination of a capture antibody that recognizes an internal sequence (17-24) of the ⁇ peptide and a detection antibody that recognizes the carboxyl terminus (Wako Pure Chemical Industries, Ltd.), the contents of ⁇ (1-40) and ⁇ (1-42) were measured by duplicate assayfor each of 3 lots.
• the concentration of ⁇ was calculated based on a calibration curve prepared using synthetic peptides of ⁇ (1-40) and ⁇ (1-42).
• the measurement results represented as the mean ⁇ SE among the lots of nerve cells are shown in panels A to C in Fig. 1. The obtained values were as follows.
• the secondary neurosphere was allowed to adhere to a dish, followed by culturing it for 4 days or 22 days, after which the cells were collected.
• the collected cells were lysed using 50 of 1 % triton/50 mM Tris-HCl, 150 mM NaCl, Complete (EDTA free), Z-Leu-Leu-Leu-H (aldehyde) [MG132], pepstatin A, pH 7.4.
• the cell lysate was mixed with an artificial substrate (Succinyl-Ala-Ala-Phe-MCA, Bachem AG.), and was incubated for 1 h at 37 °C.
• leucine aminopeptidase L-5006; Sigma, St Louis, MO, USA
• phosphoramidon Peptide Institute, Osaka, Japan
• the fluorescence intensity of the liberated AMC (7-amino-4- methylcoumarin) was measured.
• the amount of the degradation product was determined based on a calibration curve of the fluorescence intensity-concentration prepared by using a dilution series of a standard product AMC.
• the measurement was carried out by duplicate assay for each of 3 lots, and the results are shown in panel D in Fig. 1. The value was below the detection limit on Day 4, but the mean on Day 22 was 1.831 (nmol/mg protein/min). Thus, since the number of differentiated nerve cells increased as the days of culture increased, higher values were obtained on Day 22 than on Day 4.
• the amount of secretion of ⁇ from nerve cells produced by differentiation induction of iPS cells and the neprilysin activity can be measured as described above. Further, since it can be easily understood that the measured values may change when the genetic background is different, it is suggested that the onset of Alzheimer's disease can be correlated with the measured values of the respective markers in nerve cells produced by differentiation induction of iPS cells.
• Human iPS cell line 253G4 were cultured on mitomycin C-treated mouse embryonic fibroblasts in primate ES medium (ReproCELL) supplemented with bFGF (Wako). To obtain neurons derived from the iPS cell line, the reported method was partially modified (Wada, T. et al 2009, Chambers, S. M. et al 2009).
• small clumps of iPS cell colonies (40 - 100 um in diameter) were selected with Cell Strainer (BD Falcon) and plated on poly-L-lysine (Sigma)/ Laminin (BD Falcon) (PLL/LM) coated dishes in N2B27 medium [DMEM/F 12, Neurobasal, N2 supplement, B27 supplement, L-Gln], supplemented 100ng/ ml human recombinant Noggin (R&D systems) and 1 ⁇ of SB431542 (Sigma) for 10 days. Then the colonies were dissociated into small clumps by 200 U/ml Collagenase with CaC12 and plated into PLL/ECL (Millipore) coated dishes.
• N2B27 medium [DMEM/F 12, Neurobasal, N2 supplement, B27 supplement, L-Gln]
• the cells were dissociated by Accutase (Innovative Cell Technologies) and cultured on PLL/ECL coated dishes for 7days (24d). Finally, the cells dissociated by Accutase and selected with 40 ⁇ cell strainer (BD Falcon) were counted and cultured on PLL/LM/Fibronectin (Millipore) coated dishes in N2B27 medium supplemented with 10 ng/ml BDNF, GDNF, and NT-3 (R&D systems) for 14days(38d), 21 days
• the nerve cells obtained with above differentiated method were evaluated by immunocytochemistry and quantitative real-time PCR (q-PCR). These methods are well known in the art. Primer pairs used in q-PCR are listed in the Table 3.
• Glutaminase, GAD, vGultl, GAB A and Tau were shown in Fig. 3, 4, 5 and 6.
• the cells differentiated from iPS cells (52d) were confirmed to be neurons including Glutamatergic and GABAergic cells.
• the level of FL-APP (full length amyloid ⁇ protein precursor), APPs alpha (APPsa) and APPs beta (APPsP) in the differentiated cell at 38d, 45d and 52d were quantified by western blot analysis using 6E10 or 22C11 antibody (Fig. 7).
• APP splicing variant 3 types were estimated from a difference of molecular size of each variant on the same Western blot membrane.
• the neuron-dominant type (APP695) was major splicing variant in the neuron derived iPS cells.
• the expression of beta-secretase (BACEl) and gamma-secretase component genes were measured by western blotting, and other gamma-secretase component genes (Aph-IA and Aph-IB) and neprilysin were measured by q-PCR with primer pairs listed in Table 4.
• ⁇ ( 1 -40) and ⁇ (1-42) in the nerve cells derived from iPS cells were measured with the ELIS A method described in Example 1.
• the concentration of ⁇ 40 and ⁇ 42 and ratio ( ⁇ 42/ ⁇ 40) were shown in Fig.11.
• ⁇ - Secretase inhibitor compound E was tested for confirmation whether secretion of ⁇ was produced from APP C-terminal fragment by ⁇ -secretase-dependent cleavage (Fig. 12). Consequently, it was confirmed that ⁇ -secretase also played an important role in secretion of ⁇ in the case of neurons derived from iPS cells.
• AD-iPS cell line was established by using the method described in Takahashi K, et al, Cell. 131 :861-72, 2007. Briefly, the iPS cells were established by introducing Oct3/4, Sox2, Klf4 and c-Myc to fibroblasts derived from Sporadic AD patient who has the ApoE 3/4 isoform and has an onset of the disease in 50's.
• Example 2 To obtain neurons derived from AD-iPS cell line, the method described in Example 2 was used. The neurons were evaluated with expression of Tuj 1 , Tau, SATB2, CUX1, CTIP2 and TBR1 (Fig. 13 and 14). It was observed that expression of the neuron related genes in AD-iPS cell line was indistinct from that of normal iPS cell line described in Example 2.
• ⁇ 40 and ⁇ 42 in the nerve cells derived from AD-iPS cells were measured with the ELISA method described in Example 1.
• concentration of ⁇ 40 and ⁇ 42, ratio ( ⁇ 42/ ⁇ 40) and relative amount of FL-APP were shown in Fig.16.
• the value of ⁇ 40 and ⁇ 42 corrected with amount of FL-APP or ⁇ -actin was shown in Fig. 17. These values can be used for assessing for the risk of onset or predicting the age of onset of Alzheimer's disease at 50's.
• the present invention is very useful for early treatment or prophylaxis of protein misfolding diseases.

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