WO2017122829A1 - 虚血性脳梗塞への多能性幹細胞の動員 - Google Patents
虚血性脳梗塞への多能性幹細胞の動員 Download PDFInfo
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- G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
Definitions
- the present invention relates to a prognosis of cerebral infarction in a subject, a test method for predicting or diagnosing cerebral infarction risk or asymptomatic cerebral infarction after a transient ischemic attack, and a kit used in the test method.
- Stroke is one of the main causes of human death. Of all stroke patients, more than 80% have ischemic stroke [1]. Despite many studies, stroke treatment is still limited to thrombolytic therapy with tissue plasminogen activator (tPA) within 4.5 hours after onset. Only supportive care and rehabilitation has been established for patients with ischemic stroke in the chronic phase. Therefore, there is a need for alternative treatment approaches to improve patient functional outcomes.
- tPA tissue plasminogen activator
- the cells include endothelial progenitor cells (EPC), hematopoietic stem cells, CD31 positive cells (vascular progenitor cells) and the like, and contribute to angiogenesis in the brain.
- EPC endothelial progenitor cells
- CD31 positive cells vascular progenitor cells
- MSCs mesenchymal stem cells
- Muse cells Multilineage-differentiating Stress Ending cells [4]. They can be efficiently isolated as cells positive for the Stage Specific Embryonic Antigen (SSEA) -3, a well-known human embryonic stem (ES) cell marker. Using SSEA-3 antibody, MUSE cells can be efficiently separated from human bone marrow and fibroblasts by FACS [4].
- SSEA Stage Specific Embryonic Antigen
- ES human embryonic stem
- Muse cells are capable of self-replication, express genes related to pluripotency such as Nanog, Oct3 / 4 and Sox2, and differentiate from single cells into cells of endoderm, ectoderm and mesoderm lineages Can do. Under cytokine induction, Muse cells differentiate into neuronal marker positive cells at a very high rate of about 90% [5]. In animal experiments, they act as tissue repair cells when transplanted in vivo; they migrate towards the damaged tissue and in some animal models of injury, spontaneously to cells that are compatible with the homed tissue Differentiate [4].
- Muse cells when injected directly into the infarcted brain of mice, they engraft in the host brain and then integrate into the tissue, expressing neuronal markers and significantly enhancing functional recovery [6].
- pluripotent stem cells such as ES cells and induced pluripotent stem cells (iPS cells)
- Muse cells have low telomerase activity and do not form malformations in immunodeficient mouse testis [5, 7].
- SSEA-3-positive pluripotent stem cells are mobilized from bone marrow to peripheral blood in patients with ischemic cerebral infarction in the acute phase, thereby completing the present invention. It came to.
- the present invention is as follows.
- the test method according to [1] wherein the blood sample collected from the subject is collected during a period from immediately after the onset of cerebral infarction-like symptoms to 60 days.
- test according to any one of [1] to [5] above, comprising a reagent capable of measuring the number of SSEA-3-positive pluripotent stem cells present in a blood sample collected from a subject Kit for use in the method.
- a therapeutic agent for cerebral infarction comprising the step of contacting a subject with a candidate compound for a therapeutic agent for cerebral infarction and then measuring the number of SSEA-3-positive pluripotent stem cells in a blood sample derived from the subject How to screen.
- the screening method according to [7] further comprising a step of comparing with a cutoff value.
- the prognosis of cerebral infarction can be predicted, the risk of cerebral infarction caused by a transient ischemic attack can be diagnosed, and further, in the diagnosis of asymptomatic cerebral infarction Useful.
- FIG. 1 shows representative FLAIR images of small, medium and large cerebral infarctions (arrows).
- FIG. 2 shows representative FACS data in healthy individuals (A) and ischemic cerebral infarction (B).
- the upper panel represents control cell count data
- the middle panel represents cell count data with only secondary antibodies
- the lower panel represents cell count data with primary and secondary antibodies.
- SSEA-3 positive cells are identified in the lower panel (arrow).
- FIG. 3 shows the temporal profile of SSEA-3 + cells 30 days after the onset of ischemic cerebral infarction. A) reduced group, B) unchanged group, and C) increased group.
- FIG. 5 shows the relationship between age and the percentage of SSEA-3 + cells in the bone marrow. White circles represent data for patients who did not receive chemotherapy, and black circles represent data for patients who received chemotherapy.
- the present invention is used in a test method and a test method for predicting the prognosis of cerebral infarction in a subject based on measuring the number of pluripotent stem cells (Muse cells) mobilized from bone marrow to peripheral blood in the subject.
- Target Disease The present invention measures the prognosis of cerebral infarction, the risk of cerebral infarction based on transient ischemic attack, and the absence of SSEA-3-positive pluripotent stem cells (Muse cells) in blood.
- Cerebral infarction refers to a state in which local ischemia occurs in the brain due to cerebral blood vessel occlusion or reduction in perfusion pressure, resulting in irreversible cell death of nerve cells.
- cerebral infarction includes any of acute phase, subacute phase, recovery phase (chronic phase) and the like.
- onset is defined as the bedtime when the cerebral infarction occurs when the normal state of the patient is seen last time or when the witness is absent.
- Cerebral infarction is classified into cerebral thrombosis and cerebral embolism according to the origin of the thrombus, and the present invention is useful for diagnosis of cerebral thrombosis and cerebral embolism, and further for prognosis of cerebral infarction.
- Transient ischemic attack refers to transient acute neurological dysfunction resulting from thromboembolism in the cerebral circulation. According to the present invention, it is possible to diagnose the risk of whether or not a cerebral infarction can develop after such a transient ischemic attack.
- asymptomatic cerebral infarction refers to, for example, in brain tissue that is free of symptoms characterizing acute and overt stroke, such as unilateral paralysis, hypoperception, and / or aphasia Refers to an ischemic condition.
- the pluripotent stem cell used in the test method of the present invention was found by Dezawa, one of the inventors of the present invention, in the human body, and "Muse (Multilineage-differentiating Stress Ending) cell” It may be a cell named “.”
- Muse cells are used for bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)), dermal connective tissue, etc. It can be obtained from skin tissue and is also scattered in the connective tissue of each organ.
- this cell is a cell having the properties of both pluripotent stem cells and mesenchymal stem cells.
- each cell surface marker “SSEA-3 (Stage-specific embryonic antigen-3)” is positive. Or identified as “SSEA-3” and “CD105” double positive. Accordingly, Muse cells or cell populations containing Muse cells can be separated from living tissues using, for example, these antigen markers as indicators. Details such as a method for separating Muse cells, an identification method, and characteristics are disclosed in International Publication No. WO2011 / 007900. Also, as reported by Wakao et al. (2011, supra), when mesenchymal cells are cultured from bone marrow, skin, etc. and used as the population of Muse cells, all SSEA-3 positive cells are CD105 It is known to be a positive cell.
- the Muse cells when separating Muse cells from living mesenchymal tissue or cultured mesenchymal stem cells, the Muse cells can be purified and used simply by using SSEA-3 as an antigen marker.
- pluripotent isolated from living mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an antigen marker which can be used in a method for diagnosing cerebral infarction (including sequelae) Sexual stem cells (Muse cells) or a cell population containing Muse cells may be simply referred to as “SSEA-3 positive cells”.
- “non-Muse cells” refer to cells other than “SSEA-3-positive cells”, which are cells contained in a mesenchymal tissue or cultured mesenchymal tissue in a living body.
- Muse cells or cell populations containing Muse cells can be obtained from living tissue (eg, using antibodies against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively) , Mesenchymal tissue).
- living tissue eg, using antibodies against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively
- Mesenchymal tissue e.g., Mesenchymal tissue.
- “living body” means a living body of a mammal. In the present invention, the living body does not include embryos whose developmental stage is earlier than the fertilized egg or blastocyst stage, but includes embryos in the developmental stage after the blastocyst stage including the fetus and blastocyst.
- Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets, etc. It is done. Muse cells are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are separated from living tissues with a marker directly. “Mesenchymal tissue” refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, umbilical cord blood, and tissues present in various organs.
- Muse cells can be obtained from bone marrow, skin, or adipose tissue.
- Muse cells may be separated from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells using the separation means.
- a Muse cell or a cell population containing a Muse cell can be separated from a living tissue using, for example, SSEA-3 positive and SSEA-3 and CD105 double positive as an index.
- SSEA-3 positive and SSEA-3 and CD105 double positive are known to include various types of stem cells and progenitor cells.
- Muse cells are not the same as these cells.
- Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural crest stem cells (NCSC), melanoblast (MB), perivascular cells (PC), endothelial progenitor cells (EP), adipose-derived stem cells (ADSC). ).
- Muse cells can be isolated using “non-expression” of a marker unique to these cells as an index.
- Muse cells are CD34 (EP and ADSC markers), CD117 (c-kit) (MB markers), CD146 (PC and ADSC markers), CD271 (NGFR) (NCSC markers), NG2 (PC marker), vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB marker), and At least one of 11 markers selected from the group consisting of Dct (MB marker), for example 2, 3, 4, 5, 6, 7, 8, 9, 10 The non-expression of individual or eleven markers can be separated into indicators.
- non-expression of CD117 and CD146 can be separated as an index
- non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be separated as an index
- the non-expression of 11 markers can be separated as an index.
- Muse cells having the above characteristics are: (I) low or no telomerase activity; (Ii) has the ability to differentiate into cells of any germ layer of the three germ layers; It may have at least one property selected from the group consisting of (iii) showing no neoplastic growth; and / or (iv) having a self-renewal capability.
- Muse cells have all of the above properties.
- telomerase activity is low or absent means that, for example, when telomerase activity is detected using TRAPEZE XL telomerase detection kit (Millipore), it is low or cannot be detected. Say.
- “Low” telomerase activity means, for example, telomerase having a telomerase activity comparable to that of somatic human fibroblasts, or 1/5 or less, preferably 1/10 or less compared to Hela cells. It means having activity.
- the Muse cell has the ability to differentiate into three germ layers (endoderm, mesodermal, and ectoderm) in vitro and in vivo, for example, induction culture in vitro Can be differentiated into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, fat cells and the like. In addition, when transplanted to the testis in vivo, it may show the ability to differentiate into three germ layers.
- Muse cells grow at a growth rate of about 1.3 days in suspension culture, but grow from 1 cell in suspension culture to form an embryoid body-like cell mass and stop growing in about 14 days. However, when these embryoid body-like cell masses are brought into an adhesion culture, cell proliferation is started again, and the proliferated cells spread from the cell masses. Furthermore, when transplanted to the testis, it has the property of not becoming cancerous for at least half a year.
- Muse cells typically have a self-renewal (self-renewal) ability.
- self-renewal means that differentiation from cells contained in embryoid body-like cell clusters obtained by culturing in suspension culture from one Muse cell to trioderm cells can be confirmed, Bring the cells of embryoid body-like cell mass to suspension culture with one cell again to form the next generation embryoid body-like cell mass, from which again embryos in trioderm differentiation and suspension culture This means that a clot-like cell mass can be confirmed.
- the self-renewal may be repeated once or multiple times.
- the method for predicting the prognosis of cerebral infarction in a subject as an index of the number of Muse cells comprising the step of measuring the number of Muse cells present in a blood sample collected from the subject. An inspection method is provided.
- the “subject” may be any animal that may cause cerebral infarction, and more specifically includes rodents such as humans, monkeys, or rats. .
- the method for examining cerebral infarction of the present invention is particularly preferably performed in a human suspected of having a cerebral infarction or a human after onset of cerebral infarction.
- the “blood sample” collected from the subject is not particularly limited as long as it contains Muse cells mobilized from bone marrow to peripheral blood and can measure the number thereof.
- EDTA Any of plasma, plasma such as citrate plasma, serum, and whole blood may be used. Among these, EDTA plasma can be easily collected, is easily stored, and is preferably used because it has a large amount of collection.
- the timing of collecting the blood sample from the subject may be any timing as long as the diagnosis of cerebral infarction is performed, for example, a period from immediately after the onset of cerebral infarction-like symptoms to 60 days. It is preferable.
- Muse cells collected in a EDTA-containing tube or contained in a solution stored therein are easily performed using a fluorescence activated cell sorting (FACS) technique. be able to. Since the collected EDTA solution contains mononuclear cells in addition to Muse cells, the ratio of SSEA-3-positive cells in the mononuclear cells can be calculated to determine the number of Muse cells.
- FACS fluorescence activated cell sorting
- an anti-SSEA-3 antibody can be reacted as a primary antibody to isolated mononuclear cells, and then a fluorescently labeled secondary antibody can be reacted, and the number of cells can be specified by a FACS device.
- the content of Muse in a blood sample collected from a subject is measured, Using this as an index, cerebral infarction or its prognosis can be examined.
- the contents in specimens such as CRP and D-dimer, which are existing cerebral infarction markers, may be combined, or the observation of clinical symptoms and the results of echocardiography, MRI, MRA, cervical vascular echo, etc. By using a composite index, it is possible to inspect more accurately.
- the pattern can be classified mainly into three patterns by increasing or decreasing the number of Muse cells in the blood mobilized from the bone marrow. Specifically, (i) a pattern in which the number of cells significantly decreases after onset (FIG. 3A), (ii) a pattern in which no significant change in the number of cells is observed after onset (FIG. 3B), and (iii) It is divided into a pattern (FIG. 3B) in which the cell nest increases significantly after onset.
- test method of the present invention can correlate the prognosis of cerebral infarction with the presence or absence of smoking and / or drinking in the subject.
- smoking cessation in a subject refers to tobacco smoking on a daily basis within 3 months before onset or hospitalization.
- Alcohol intake means that the amount of alcohol consumed by a subject consumes more than 150 g of alcohol per week within 3 months.
- the present invention can provide a test method for predicting the prognosis of cerebral infarction.
- the prognosis of cerebral infarction is indicated by the degree of damage caused by cerebral infarction of the subject after a certain period of time after the onset of cerebral infarction.
- the degree of failure is known per se and can be evaluated using an already established index.
- Indicators include, for example, the Japanese Modified Rankin Score (mRS) criteria (Yukito Shinohara et al., MRS Reliability Research Group, modified Rankin Scale Reliability Study-Introduction of Japanese Version Criteria and Questionnaire, Stroke 2007; 29: 6-13) and the like are used.
- mRS Japanese Modified Rankin Score
- “after a certain period of time” may be any time after the onset, but from the viewpoint of evaluating the state of rehabilitation of the subject, for example, it is evaluated between 3 months and 1 year after the onset It is common.
- the number of Muse cells in a blood sample obtained from a subject from immediately after onset to 60 days is not limited. A method for predicting the state of the subject is preferred.
- the prognosis of the cerebral infarction is examined using the number of Muse cells present in the blood sample collected from the subject as an index, it may be compared with the number of Muse cells in the blood sample of a healthy person.
- a method of inspecting by specifying an appropriate cut-off value may be used.
- the number of Muse cells in a healthy person can be obtained by collecting blood from a healthy person who has been clinically confirmed in advance to have no cerebral infarction, and performing the same treatment and measurement as the blood sample collected from the subject, and quantifying it. Can do.
- the “cut-off value” generally refers to a value determined when a target disease group and a non-disease group are determined by paying attention to a certain substance.
- Example 2 As shown in Example 2 described later, in healthy individuals, the number of Muse cells in the blood was found to be an average of 3.5 ⁇ 4.3 / ⁇ l by measurement. Can be used.
- FIG. 3 shows that all cerebral infarction patients have higher values than the above values, and that Muse cells are present in the blood of the patients. Therefore, by using such a cut-off value, for example, it can be applied to a diagnosis that a patient has asymptomatic cerebral infarction, and the effect of a therapeutic agent for cerebral infarction can be evaluated.
- a subject who has been diagnosed with cerebral infarction by the test method of the present invention has a very good prognostic course and therapeutic effect by receiving a treatment suitable for each disease.
- the kit for measuring cerebral infarction further includes a kit for use in the above-described testing method, which includes a reagent capable of measuring the number of Muse cells present in a blood sample collected from a subject, prevention of cerebral infarction Alternatively, a kit for evaluating a therapeutic effect or a method for screening for a cerebral infarction prevention or treatment drug is provided.
- the contents of the kit are composed of a combination of reagents or measuring instruments, but if the substance is essentially the same as each component described below or contains essentially the same substance, And different forms are included in the kit of the present invention.
- the reagent include an anti-SSEA-3 antibody when the number of Muse cells is measured by an immunoassay.
- a biological sample diluent an antibody-immobilized solid phase, a reaction buffer, a washing solution, a labeled secondary antibody, a labeled detection reagent, a standard substance, and the like are also included as necessary.
- the diluted solution of the biological sample include an aqueous solution containing a protein such as BSA or casein in an EDTA solution, a surfactant, a buffering agent, or the like.
- the reaction buffer may be any one as long as it provides a solvent environment for the binding reaction between the surface antigen of Muse cells and the primary antibody thereto.
- the labeled secondary antibody is an antibody against the primary antibody, and an antibody labeled with FITC, horseradish peroxidase (HRP), bovine intestinal alkaline phosphatase, ⁇ -galactosidase, or the like is used.
- Method for confirming therapeutic effect of cerebral infarction includes the step of measuring the number of Muse cells in a blood sample collected from a subject to whom a therapeutic agent for cerebral infarction has been administered. This is a method for confirming the therapeutic effect of infarction.
- the therapeutic agent for cerebral infarction may be any one as long as it is used as a drug capable of treating cerebral infarction.
- thrombolytic agents such as urokinase and tissue plasminogen activator, antiparin such as heparin, etc.
- anticoagulants such as coagulants, cyclooxygenase inhibitors, phosphodiesterase inhibitors, thromboxen A2 (TAX2) synthesis inhibitors, and brain protective agents such as free radical scavengers.
- the number of Muse cells in a blood sample taken from a subject to whom a therapeutic agent for cerebral infarction has been administered is increased or decreased compared to the amount before administration, or a control subject who has not developed cerebral infarction (for example, When the number of Muse cells in a healthy person is approached, it can be determined that there was a therapeutic effect.
- Method for screening cerebral infarction preventive or therapeutic agent includes a step of contacting a subject with a candidate compound for a therapeutic agent for cerebral infarction and then measuring the number of Muse cells in the blood sample of the subject.
- a method for screening a therapeutic agent for cerebral infarction may be a non-human animal individual in which the number of Muse cells in the blood sample exhibits an abnormality similar to that of a cerebral infarction state.
- a cerebral infarction model non-human animal in which a cerebral infarction state has been formed by a surgical technique or the like Yuji Kuge, Kazuo Minematsu et al.
- candidate therapeutic agents for cerebral infarction to be brought into contact with these subjects include peptides, proteins, non-peptide compounds, low-molecular synthetic compounds, and the like. These compounds may be novel compounds or known compounds. If the number of Muse cells in a subject contacted with a candidate compound increases or decreases compared to the number before contact, or approaches the number of Muse cells in a control subject, the candidate compound becomes cerebral infarction. It can be determined that it has a therapeutic effect.
- Subjects The study included 29 adult patients admitted to the hospital because of ischemic cerebral infarction in the upper tent area within 24 hours after onset. Patients with lacunar infarction were excluded. There were 16 men and 13 women. The average age was 71.4 ⁇ 13.3 years, from 41 to 93 years. This study was approved by the Ethics Review Committee of Toyama University Hospital and Saiseikai Toyama Hospital, and informed consent was obtained from each participant.
- peripheral blood was obtained total 3ml from all patients on admission and 7 days and 30 days. Blood was stored in tubes containing ethylenediaminetetraacetic acid (EDTA). To isolate mononuclear cells, the blood was diluted with an equal volume of saline, overlaid on 2 ml Lymphoprep (Axis-Shield Diagnostics Ltd., Scotland) and centrifuged at 800 g for 15 minutes at room temperature. . Next, the ratio of SSEA-3 positive cells in mononuclear cells was determined using fluorescence activated cell sorting (FACS) technology.
- FACS fluorescence activated cell sorting
- isolated mononuclear cells (approximately 1 ⁇ 10 6 ) are placed in 100 ⁇ l ice-cold phosphate buffered saline (PBS) containing 0.5% bovine serum albumin and 2 mM EDTA (FACS buffer). Resuspended. Primary antibody against SSEA-3 (1:50 dilution, Millipore, MAB4303) was added and incubated for 60 minutes at 4 ° C. with gentle rocking. After primary antibody binding, cells were washed twice with FACS buffer and resuspended in 100 ⁇ l FACS buffer containing 1: 100 FITC-conjugated goat anti-rat IgM (Jackson ImmunoResearch Laboratories, Inc., Baltimore, PA).
- peripheral blood was obtained to determine control values for SSEA-3 + cells of 5 healthy individuals with no history of cardiovascular disorders. There were 2 males and 3 females, with an average age of 56.2 ⁇ 4.2 years old.
- the absolute number of SSEA-3 + cells was judged to increase when it increased more than twice the control at admission on day 7 or 30, and at admission on day 7 or 30 A decrease was judged to be less than half that of the control.
- each section was treated with a primary antibody to SSEA-3 (rat monoclonal, 1: 100 dilution, Millipore, MAB4303) overnight at 4 ° C. and then fluorescein (FITC) Sfini Pure (goat anti-rat IgM , 1:50 dilution, Jackson Immunoresearch) for 1 hour at room temperature. Finally, sections were stained for 24 hours at room temperature with ProLong Gold Antifade reagent containing DAPI. The percentage of total bone marrow cells, SSEA-3 + cells, was calculated in 5 fields randomly under a microscope (BZ9000, Keyence Co., Osaka, Japan) at 20 ⁇ magnification.
- Example 1 Clinical features The average NIHSS at admission was 8.6 ⁇ 7.4, ranging from 0-24.
- Clinical diagnoses included 17 cardiac emboli, 7 atherothrombotic strokes, 3 aortic emboli, and 2 other patients.
- Past medical history included hypertension in 16 people, diabetes in 3 people, hyperlipidemia in 4 people, smoking in 7 people, and alcohol consumption in 10 people.
- the average mRS on the 30th day was 2.3 ⁇ 2.2, which was in the range of 0-6.
- Two patients (6.9%) died between 8-30 days after onset, and data on admission and only on day 7 were analyzed in this study.
- the size of cerebral infarction was divided into small in 13 patients, medium in 10 and large in 6 people.
- Example 2 Circulating SSEA-3 + cells
- Figure 2 shows representative results of FACS analysis in controls and patients with ischemic cerebral infarction.
- the number of SSEA-3 + cells was very low in the range of 0-10 ⁇ l. The average value was 3.5 ⁇ 4.3 / ⁇ l.
- the baseline number of SSEA-3 + cells at admission varied greatly among patients.
- the average number of SSEA-3 + cells was 81.9 ⁇ 78.0 / ⁇ l, ranging from 4.7 to 249.1 / ⁇ l.
- 22 of 29 subjects (75.9%) had a marked increase in the number of SSEA-3 + cells within 24 hours of the onset of ischemic stroke.
- FIG. 3 shows the temporal profile of the number of circulating SSEA-3 + cells.
- the kinetics could be divided into three patterns; in 8 of 29 patients (27.6%), the number of circulating SSEA-3 + cells was significantly reduced on day 7. Those numbers did not recover on day 30 in 7 out of 8 patients. In 29 of 29 patients (44.8%), the number did not change significantly within 30 days after onset and remained higher than controls.
- Example 3 Distribution of SSEA-3 + cells in human bone marrow A small fraction of bone marrow cells was positive for SSEA-3. As shown in FIG. 4, the distribution of SSEA-3 + cells was not uniform. They were distributed like clusters. The percentage of SSEA-3 + cells varied from 0% to 0.5% with an average value of 0.2 ⁇ 0.17%. As shown in FIG. 5, the percentage of SSEA-3 + cells was less than 0.1% in 3 patients treated with chemotherapy for malignancy. Excluding these three patients, although not statistically significant, there tended to be a negative correlation between the patient's age and the percentage of SSEA-3 + cells in the bone marrow.
- Non-hematopoietic stem cells expressing early progenitor markers are mobilized from bone marrow to peripheral blood after acute myocardial infarction [9, 10] and ischemic cerebral infarction [11].
- Yu et al. isolated peripheral blood mononuclear cells on days 0, 1 and 7 from patients with acute myocardial infarction and measured mRNA expression of embryonic stem cell markers. They report that Oct4, Nanog, CD31, and VE-cadherin mRNA levels were significantly higher in peripheral blood on days 0 and 1 [12].
- similar phenomena have been observed in patients with ischemic cerebral infarction [13, 14]. Current knowledge mimics these previous data. Therefore, the number of circulating SSEA-3 + cells was much higher at admission than in controls in 22 of 29 ischemic stroke patients.
- stem / progenitor cell mobilization including endothelial progenitor cells
- Taguchi et al. (2004) also report that CD34 positive cells continue to increase for 7 days and then decrease to baseline levels on day 30 [13].
- the temporal profile of stem / progenitor cell mobilization can be highly dependent on the patient's condition and the type of cell mobilized.
- Alcohol abuse also adversely affects human health.
- epidemiological studies have shown that moderate consumption of ethanol reduces the risk of coronary heart disease, sudden heart death, and ischemic stroke. Indeed, moderate amounts of ethanol enhance angiogenesis in cultured cells [23].
- Chiva-Blanch et al. (2014) report that the non-alcoholic fraction of beer increases the number of circulating endothelial cells in patients at high cardiovascular risk [24].
- Medium intake of red wine also improves cell mobilization in diabetic mice [25].
- some components contained in alcohol can contribute to the recruitment of stem / progenitor cells as well as ethanol itself.
- Vasa M Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: E1-7.
- Liu Z Ding X, Fang F, Wang R, Chen Y, Ma Y, et al. Higher numbers of circulating endothelial progenitor cells in stroke patients with intracranial arterial stenosis. BMC neurology. 2013; 13: 161.
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Abstract
Description
[1]被験者から採取された血液試料中に存在するSSEA-3陽性の多能性幹細胞の数を測定する工程を含む、多能性幹細胞の数を指標とした、被験者における脳梗塞の予後、一過性虚血発作後の脳梗塞リスク若しくは無症候性脳梗塞を予測又は診断するための検査方法。
[2]被験者から採取された血液試料が、脳梗塞様症状の発症直後から60日までの期間に採取されたものである、上記[1]に記載の検査方法。
[3]多能性幹細胞が、虚血性脳梗塞の急性期において骨髄から末梢血に動員されたものである、上記[1]又は[2]に記載の検査方法。
[4]脳梗塞の予後と被験者の喫煙及び/又は飲酒の有無とを関連付けることを特徴とする、上記[1]~[3]のいずれかに記載の検査方法。
[5]カットオフ値と比較する工程をさらに含む、上記[1]~[4]のいずれかに記載の検査方法。
[6]被験者から採取された血液試料中に存在するSSEA-3陽性の多能性幹細胞の数を測定することができる試薬を含む、上記[1]~[5]のいずれかに記載の検査方法に使用するためのキット。
[7]被験者と脳梗塞の治療薬の候補化合物を接触させた後、該被験者由来の血液試料中のSSEA-3陽性の多能性幹細胞の数を測定する工程を含む、脳梗塞の治療薬をスクリーニングする方法。
[8]カットオフ値と比較する工程をさらに含む、上記[7]に記載のスクリーニング方法。
本発明は、SSEA-3陽性の多能性幹細胞(Muse細胞)の血中の数を測定することにより、脳梗塞の予後、一過性虚血発作に基づく脳梗塞のリスク、及び無症候性脳梗塞の診断を目的とする。ここで、「脳梗塞」とは、脳血管の閉塞や灌流圧低下により、脳に局所的な虚血部分が生じ、神経細胞の不可逆的細胞死を呈した状態をいう。より具体的には、脳内小動脈が閉塞して発症するラクナ梗塞、脳内大動脈が粥腫で閉塞して発症するアテローム血栓性脳梗塞、心臓内の血栓が栓子となり脳内動脈を閉塞して発症する心原性脳塞栓症のいずれをも含む。また、脳梗塞でも急性期、亜急性期、回復期(慢性期)等のいずれをも含む。本発明においては、発症直後から60日までの被験者の血液試料を測定対象とする。ここで、「発症」とは、患者の正常な状態を最後に見たとき、又は目撃者のいない就寝中に脳梗塞が起こった際の就寝時と定義される。脳梗塞には、血栓の由来により脳血栓と脳塞栓に分類され、本発明は、脳血栓及び脳塞栓の診断、さらには、脳梗塞の予後の診断に有用である。「一過性虚血発作」とは、脳循環における血栓塞栓症に起因する一過性の急性神経機能障害を指す。本発明によれば、このような一過性虚血発作後に脳梗塞を発症し得るかどうかのリスクを診断することができる。また、本明細書において使用するとき、「無症候性脳梗塞」とは、例えば、急性かつ顕性の脳卒中を特徴付ける症状、例えば片側不全麻痺、知覚減退、及び/又は失語症のない、脳組織における虚血の状態を指す。
本発明の検査方法等に使用される多能性幹細胞は、本発明者らの一人である出澤が、ヒト生体内にその存在を見出し、「Muse(Multilineage-differentiating Stress Enduring)細胞」と命名した細胞であってもよい。一般的に、Muse細胞は、骨髄液、脂肪組織(Ogura,F.,et al.,Stem Cells Dev.,Nov 20,2013(Epub)(published on Jan 17,2014))や真皮結合組織等の皮膚組織から得ることができ、各臓器の結合組織にも散在する。また、この細胞は、多能性幹細胞と間葉系幹細胞の両方の性質を有する細胞であり、例えば、それぞれの細胞表面マーカーである「SSEA-3(Stage-specific embryonic antigen-3)」陽性として、又は「SSEA-3」と「CD105」のダブル陽性として同定される。したがって、Muse細胞又はMuse細胞を含む細胞集団は、例えば、これらの抗原マーカーを指標として生体組織から分離することができる。Muse細胞の分離法、同定法、及び特徴などの詳細は、国際公開第WO2011/007900号に開示されている。また、Wakaoら(2011、上述)によって報告されているように、骨髄、皮膚などから間葉系細胞を培養し、それをMuse細胞の母集団として用いる場合、SSEA-3陽性細胞の全てがCD105陽性細胞であることが分かっている。したがって生体の間葉系組織又は培養間葉系幹細胞からMuse細胞を分離する場合は、単にSSEA-3を抗原マーカーとしてMuse細胞を精製し、使用することができる。なお、本明細書においては、脳梗塞(後遺症を含む)を診断する方法に使用され得る、SSEA-3を抗原マーカーとして、生体の間葉系組織又は培養間葉系組織から分離された多能性幹細胞(Muse細胞)又はMuse細胞を含む細胞集団を単に「SSEA-3陽性細胞」と記載することがある。また、本明細書においては、「非Muse細胞」とは、生体の間葉系組織又は培養間葉系組織に含まれる細胞であって、「SSEA-3陽性細胞」以外の細胞を指す。
(i)テロメラーゼ活性が低いか又は無い;
(ii)三胚葉のいずれの胚葉の細胞に分化する能力を持つ;
(iii)腫瘍性増殖を示さない;及び/又は
(iv)セルフリニューアル能を持つ
からなる群から選択される少なくとも1つの性質を有してもよい。本発明の一局面では、Muse細胞は、上記性質を全て有する。ここで、上記(i)について、「テロメラーゼ活性が低いか又は無い」とは、例えば、TRAPEZE XL telomerase detection kit(Millipore社)を用いてテロメラーゼ活性を検出した場合に、低いか又は検出できないことをいう。テロメラーゼ活性が「低い」とは、例えば、体細胞であるヒト線維芽細胞と同程度のテロメラーゼ活性を有しているか、又はHela細胞に比べて1/5以下、好ましくは1/10以下のテロメラーゼ活性を有していることをいう。上記(ii)について、Muse細胞は、in vitro及びin vivoにおいて、三胚葉(内胚葉系、中胚葉系、及び外胚葉系)に分化する能力を有し、例えば、in vitroで誘導培養することにより、肝細胞、神経細胞、骨格筋細胞、平滑筋細胞、骨細胞、脂肪細胞等に分化し得る。また、in vivoで精巣に移植した場合にも三胚葉に分化する能力を示す場合がある。さらに、静注により生体に移植することで損傷を受けた臓器(心臓、皮膚、脊髄、肝、筋肉等)に遊走及び生着し、組織に応じた細胞に分化する能力を有する。上記(iii)について、Muse細胞は、浮遊培養では増殖速度約1.3日で増殖するが、浮遊培養では1細胞から増殖し、胚様体様細胞塊を作り14日間程度で増殖が止まる、という性質を有するが、これらの胚様体様細胞塊を接着培養に持っていくと、再び細胞増殖が開始され、細胞塊から増殖した細胞が広がっていく。さらに精巣に移植した場合、少なくとも半年間は癌化しないという性質を有する。また、上記(iv)について、Muse細胞は、典型的には、セルフリニューアル(自己複製)能を有する。ここで、「セルフリニューアル」とは、1個のMuse細胞から浮遊培養で培養することにより得られる胚様体様細胞塊に含まれる細胞から3胚葉性の細胞への分化が確認できると同時に、胚様体様細胞塊の細胞を再び1細胞で浮遊培養に持っていくことにより、次の世代の胚様体様細胞塊を形成させ、そこから再び3胚葉性の分化と浮遊培養での胚様体様細胞塊が確認できることをいう。セルフリニューアルは1回又は複数回のサイクルを繰り返せばよい。
本発明によれば、被験者から採取された血液試料中に存在するMuse細胞の数を測定する工程を含む、Muse細胞の数の指標とした、被験者における脳梗塞の予後を予測するための検査方法が提供される。
本発明は、さらに、被験者から採取された血液試料中に存在するMuse細胞の数を測定することができる試薬を含む、上記の検査方法で使用されるキット、脳梗塞の予防若しくは治療効果の評価、又は脳梗塞予防若しくは治療薬のスクリーニング方法を行うためのキットが提供される。キットの内容は、試薬又は測定機器の組み合わせにより構成されるが、後述する各構成要素と本質的に同一であるか、又はその一部と本質的に同一な物質が含まれていれば、構成及び形態が異なっていても、本発明のキットに包含される。試薬としては、例えば、免疫測定法によりMuse細胞の数を測定する場合には、抗SSEA-3抗体を含む。また、必要に応じ、生体試料の希釈液、抗体固定化固相、反応緩衝液、洗浄液、標識された二次抗体、標識体の検出用試薬、標準物質なども含まれる。生体試料の希釈液としては、EDTA溶液、界面活性剤、緩衝剤などにBSAやカゼインなどのタンパク質を含む水溶液などが挙げられる。
本発明の別の態様は、脳梗塞の治療薬が投与された被験者から採取された血液試料中のMuse細胞の数を測定する工程を含む、該被験者における脳梗塞の治療効果を確認する方法である。脳梗塞の治療薬とは、脳梗塞を治療し得る薬として用いられているものであればいずれのものでもよいが、例えば、ウロキナーゼ、組織プラスミノゲンアクチベーターなどの血栓溶解剤、へパリンなどの抗凝固剤、サイクロオキシゲナーゼ阻害薬、フォスフォリジエステラーゼ阻害薬、スロンボキセンA2(TAX2)合成阻害薬などの抗血小板剤、フリーラジカルスカベンジャーなどの脳保護薬等が挙げられる。
本発明のさらに別の態様は、被験者と脳梗塞の治療薬の候補化合物を接触させた後、該被験者の血液試料中のMuse細胞の数を測定する工程を含む、脳梗塞の治療薬のスクリーニング方法である。このスクリーニング方法における被験者とは、血液試料におけるMuse細胞の数が、脳梗塞状態と同様の異常を示す非ヒト動物個体であり得る。脳梗塞の症状を有する動物としては、例えば、外科的手法等で脳梗塞状態を形成させた脳梗塞モデル非ヒト動物(Yuji Kuge,Kazuo Minematsu et al.Nylon Monofilament for Intraluminal Middle Cerebral Artery Occlusion in Rats.Stroke,26,1655(1995))などが挙げられる。血液試料中のMuse細胞の数をもとに、脳梗塞状態の非ヒト動物個体(例えば、マウスやラット)でヒトにおけるある病型に類似した病態を構築できれば、ある病型の脳梗塞患者における治療薬を開発するための実験系を確立することができる。また、非ヒト動物個体の予後をMuse細胞の数をモニタリングすることにより、ヒトの脳梗塞の予後に有効な治療薬を開発することが可能となる。
(1)被験者
本研究には、発症後、24時間以内にテント上領域の虚血性脳梗塞を理由に病院に入院した29人の成人患者が含まれた。ラクナ梗塞の患者は除外された。男性16名、女性13名であった。平均年齢は71.4±13.3歳であり、41歳から93歳であった。本研究は、富山大学病院及び済生会富山病院の倫理審査委員会により承認され、各参加者からインフォームドコンセントを得た。
入院時には、すべての患者において、血圧、ECG、及び実験データが記録された。これらの試験は、発症の7日後及び30日後に繰り返された。
入院時に、1.5テスラのMR装置を用いて、拡散強調画像、T2強調画像、及び流体減衰反転回復(FLAIR)画像、並びにMR血管造影がすべての患者において得られた。脳梗塞の大きさを小、中、及び大の3グループに分けられた。脳梗塞の大きさは、2つを超える皮質枝の領域に病変が位置する場合に大、病変が1つの皮質枝の領域に位置する場合に中、及び病変がより小さくなる場合に小として等級分けした(図1)。
循環SSEA-3+細胞を定量するために、入院時及び7日目と30日目にすべての患者から末梢血を全3ml得た。血液をエチレンジアミン四酢酸(EDTA)含有チューブ中で保存した。単核細胞を単離するために、血液を等容量の生理食塩水で希釈し、2mlのLymphoprep(Axis-Shield Diagnostics Ltd.、Scotland)上に重層し、室温で15分間、800gで遠心分離した。次に、蛍光活性化セルソーティング(FACS)技術を用いて、単核細胞におけるSSEA-3陽性細胞の割合を決定した。簡潔には、単離された単核細胞(約1×106)を0.5%ウシ血清アルブミン及び2mM EDTA(FACS緩衝液)を含有する100μlの氷冷リン酸緩衝食塩水(PBS)に再懸濁した。SSEA-3(1:50希釈、Millipore、MAB4303)に対する一次抗体を添加し、穏やかに揺動しながら4℃で60分間インキュベートした。一次抗体結合後、細胞をFACS緩衝液で2回洗浄し、1:100のFITC結合ヤギ抗ラットIgM(Jackson ImmunoResearch Laboratories、Inc.、Baltimore、PA)を含有する100μlのFACS緩衝液に再懸濁し、穏やかな揺動しながら4℃の暗所にて60分間インキュベートした。二次抗体結合後、細胞を氷冷FACS緩衝液で3回洗浄し、1mlの氷冷FACS緩衝液に再懸濁し、細胞ストレーナーチューブ(No.352235、BD Falcon)に通し、すぐにFACSCanto TM II(BD Biosciences)で分析した。すべてのFACS分析において、非特異的結合及び/又は自家蛍光の可能性を排除するために、細胞の対照試料(二次抗体のみに曝露された)を使用した。BD FACSDivaソフトウェア(BD Biosciences)を用いて、FITC陽性細胞の数をカウントした。SSEA-3+細胞の絶対数は、以下の式に従って計算された:
FACS上のSSEA-3+細胞数(/μl)=(全WBCの数-PMNの数)(/μl)×SSEA-3+細胞(%)(式中、PMNは多形核細胞を表す)
ヒト骨髄におけるSSEA-3+細胞の割合及び分布を明らかにするために、脳血管疾患の既往のない8人の剖検患者から検体を得た。男性は5人、女性は3人であった。平均年齢は63.9±9.0歳であり、57歳から73歳までの範囲であった。彼らは、心筋梗塞、悪性腫瘍、心不全などの様々な疾患のために死亡した。検体を緩衝化されたホルマリン(4%)中で固定し、パラフィンに包埋した。その後、厚さ4μmの切片を次の染色のために調製した。脱パラフィン化された切片を圧力ポットで2分間、抗原回収することによって処理した。免疫組織化学を用いて、ヒト骨髄中のSSEA-3+細胞を同定した。簡単には、各切片を4℃で一晩、SSEA-3に対する一次抗体(ラットモノクローナル、1:100希釈、Millipore、MAB4303)で処理し、次に、フルオレセイン(FITC)Sffini Pure(ヤギ抗ラットIgM、1:50希釈、Jackson Immunoresearch)とともに、室温にて1時間インキュベートした。最後に、DAPIを含むProLong Gold Antifade試薬を用いて室温で24時間、切片を染色した。骨髄細胞全体のSSEA-3+細胞の割合を20×の倍率で顕微鏡(BZ9000、Keyence Co.、Osaka、Japan)下で無作為に5視野において計算した。
データを平均±SDとして表した。χ2検定を用いて分類変数を比較した。連続変数は、2つのグループ間の両側不対t検定及び3つのグループ間の1因子ANOVAを用いて比較された。P値が0.05未満では、差異は統計的に有意であるとみなされた。
入院時の平均NIHSSは8.6±7.4であり、0~24の範囲であった。臨床診断には、17人の心臓塞栓症、7人のアテローム血栓性脳卒中、3人の大動脈塞栓症、及び2人の他の患者が含まれた。過去の病歴には、16人において高血圧症、3人において糖尿病、4人において高脂血症、7人において喫煙、及び10人においてアルコール摂取であった。
図2は、対照及び虚血性脳梗塞を有する患者におけるFACS分析の代表的な結果を示す。健常対照において、SSEA-3+細胞の数は、0~10μlの範囲で非常に少なかった。平均値は3.5±4.3/μlであった。一方、入院時のSSEA-3+細胞のベースライン数は、患者間で大きく異なっていた。SSEA-3+細胞の平均数は、81.9±78.0/μlであり、4.7~249.1/μlの範囲であった。したがって、29人の被験者のうち22人(75.9%)において、虚血性脳梗塞が発症してから24時間以内にSSEA-3+細胞の数が顕著に増加した。単変量解析では、入院時のSSEA-3+細胞の数と、年齢、性別、入院時のNIHSS、過去の履歴、虚血性脳梗塞のサブタイプ、脳梗塞のサイズ、及び30日目のmRSの間に有意な関係がないことを検出した。多重線形回帰分析はまた、入院時に循環するSSEA-3+細胞の絶対数を予測する重要な因子を同定しなかった。
OR:オッズ比、CI:信頼区間。
骨髄細胞の小画分はSSEA-3に対して陽性であった。図4に示されるように、SSEA-3+細胞の分布は均一ではなかった。それらはクラスターのように分布していた。SSEA-3+細胞の割合は、0%から0.5%まで変化し、平均値は0.2±0.17%であった。図5に示されるように、SSEA-3+細胞の割合は、悪性腫瘍のために化学療法を処置された3人の患者において0.1%未満であった。これらの3人の患者を除くと、統計学的に有意ではないが、患者の年齢と骨髄中のSSEA-3+細胞の割合との間に負の相関が生じる傾向にあった。
この研究では、SSEA-3に対する一次抗体を用いて骨髄の検体を染色した。これは、以前の研究では、骨髄におけるMuse細胞の局在を示していないためである。結果として、この研究は、SSEA-3+細胞が骨髄細胞全体の約0.2%に相当することを示している。以前の研究によれば、Muse細胞は、ヒト骨髄の吸引液中に0.03%の割合で同定され、MSCでは5~6%の割合で同定される[5]。それらの集団は、年齢とともに徐々に減少する傾向があるが、おそらく試料のサイズが小さいために統計的有意性が得られない。興味深いことに、化学療法と放射線照射が、幹細胞/前駆細胞及びMSCを含む骨髄細胞の長期的な損傷を引き起こす可能性があるため、悪性腫瘍に対する化学療法で処置された患者では、Muse細胞の集団は非常に小さかった[8]。したがって、化学療法後に、SSEA-3+細胞でさえ不可逆的又は持続的な損傷を被る場合がある。
本研究は、虚血性脳梗塞の急性期に、多能性のMuse細胞が骨髄から末梢血に動員されていることを明確に示している。喫煙及びアルコール摂取は、Muse細胞の時間的プロファイルに有意に影響を及ぼす。この研究では、SSEA-3+細胞のベースライン数及び動態は、おそらく末梢血中の量が少ないため、機能的転帰と関連していない。しかしながら、内因性のMuse細胞を増加させる治療的介入、又はMuse細胞の外因的投与は、虚血性脳梗塞後の機能的転帰を改善するための新規な治療戦略となる。
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Claims (8)
- 被験者から採取された血液試料中に存在するSSEA-3陽性の多能性幹細胞の数を測定する工程を含む、多能性幹細胞の数を指標とした、被験者における脳梗塞の予後、一過性虚血発作後の脳梗塞リスク若しくは無症候性脳梗塞を予測又は診断するための検査方法。
- 被験者から採取された血液試料が、脳梗塞様症状の発症直後から60日までの期間に採取されたものである、請求項1に記載の検査方法。
- 多能性幹細胞が、虚血性脳梗塞の急性期において骨髄から末梢血に動員されたものである、請求項1又は2に記載の検査方法。
- 脳梗塞の予後と被験者の喫煙及び/又は飲酒の有無とを関連付けることを特徴とする、請求項1~3のいずれか1項に記載の検査方法。
- カットオフ値と比較する工程をさらに含む、請求項1~4のいずれか1項に記載の検査方法。
- 被験者から採取された血液試料中に存在するSSEA-3陽性の多能性幹細胞の数を測定することができる試薬を含む、請求項1~5のいずれか1項に記載の検査方法に使用するためのキット。
- 被験者と脳梗塞の治療薬の候補化合物を接触させた後、該被験者由来の血液試料中のSSEA-3陽性の多能性幹細胞の数を測定する工程を含む、脳梗塞の治療薬をスクリーニングする方法。
- カットオフ値と比較する工程をさらに含む、請求項7に記載のスクリーニング方法。
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