JP6604492B2 - Pluripotent stem cells for cerebral infarction treatment - Google Patents

Description

  The present invention relates to a cell preparation in regenerative medicine. More specifically, the present invention relates to a cell preparation containing pluripotent stem cells effective for repair and regeneration of brain tissue damaged by cerebral infarction.

  Cerebral infarction refers to cerebral dysfunction caused by ischemic necrosis in the brain, is a disease requiring emergency treatment, and is one of the three leading causes of death along with cancer and heart disease. Cerebral infarction is classified into thrombotic, embolic, and hemodynamic in terms of mechanism of action, and is classified into atherothrombotic cerebral infarction, cardiogenic cerebral embolism, lacunar infarction in terms of clinical findings. The

  Ischemia occurs when the local cerebral blood flow is blocked by a cerebrovascular lesion such as arteriosclerosis or a cardiogenic thrombus, and neuronal cell death due to energy depletion is caused at the central part of the ischemia. In the vicinity of the ischemic center, blood flow through the accessory blood circulation remains, and the nerve cells are alive but not functioning electrophysiologically. Unless this part is treated, this part of the nerve cell will die in the future, and pathologically, it becomes a disorder of cerebral infarction, and clinically, it is impaired as a malfunction. Therefore, if the function of the nerve cells in this part can be restored as early as possible, the dysfunction can be treated. This reversible incomplete ischemic region is called a penumbra. The purpose of treatment in the acute phase of cerebral infarction is to restore the function of neurons in this penumbra region, the outcome of which depends on the extent of ischemia and its duration. That is, how quickly blood flow is resumed in the penumbra region determines the outcome. It is said that nerve cells in this penumbra region can survive for 3 to 6 hours after onset. In addition, an allowable time during which the nerve cells in the penampula region can recover their function by treatment is referred to as “treatable time” (Non-patent Document 1).

  Currently, thrombolytic therapy using recombinant human tissue plasminogen activator (rt-PA), which is approved in the United States as a therapeutic agent for acute cerebral infarction, is achieved by dissolving thrombus causing ischemia. It was developed to restore blood flow to the penumbra region. In an rt-PA intravenous therapy study in patients with cerebral infarction within 3 hours after onset, a US clinical study examined by placebo double-blind showed that the outcome after 3 months was significantly higher in the rt-PA administration group It was good. It is believed that rt-PA dissolves the thrombus to resume blood supply to the ischemic region, suppresses the extension of cerebral infarction, and improves dysfunction caused by cerebral infarction. This result showed that early resumption of cerebral blood flow by thrombolytic action improves long-term prognosis (Non-patent Document 2). In addition to using thrombolytic therapy as described above, stem cell therapy has been expected as a new therapeutic method in the treatment of cerebral infarction, but it has not yet produced a sufficient therapeutic effect and has been established as a therapeutic method. There is no current situation (Non-Patent Document 3).

  According to research by Dezawa, one of the present inventors, SSEA-3 (Stage-Specific Embryonic Antigen-3), which is present in the mesenchymal cell fraction and is obtained without induction, is expressed as a surface antigen. It has been found that pluripotent stem cells (Multilineage-differentiating Stress Ending cells; Muse cells) are responsible for the pluripotency of the mesenchymal cell fraction and can be applied to disease treatment aimed at tissue regeneration. (Patent Document 1; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6). However, there is no example that shows that the expected therapeutic effect can be obtained by using Muse cells for the treatment of cerebral infarction.

International Publication No. WO2011 / 007900

Stroke, vol. 21, p. 637-676 (1990) N. Eng. J. et al. Med. , Vol. 333, p. 1581-1587 (1995) Sinden, J .; D. & Muir, K .; W. , Vol. 7, p. 426-434 (2012) Kuroda, Y. et al. , Et al. , Proc. Natl. Acad. Sci. USA, Vol. 107, p. 8639-8643 (2010) Wakao, S, et al. , Proc. Natl. Acad. Sci. USA, Vol. 108, p. 9875-9880 (2011) Kuroda, Y. et al. , Et al. Nat. Protoco. , Vol. 8, p. 1391-1415 (2013)

  An object of the present invention is to provide a new medical use using pluripotent stem cells (Muse cells) in regenerative medicine. More specifically, the present invention provides a cell preparation for treatment of cerebral infarction and prevention and / or treatment of sequelae (motor disorders, sensory disorders, speech disorders, etc.) accompanying the treatment, including Muse cells. The purpose is to do.

  The present inventors inserted an embolus into a blood vessel in the brain, and injected the Muse cell into the brain parenchyma in a rat cerebral infarction model caused by ischemia reperfusion, whereby the Muse cell was damaged brain tissue. It has been found that survival after several months after engraftment and spontaneous differentiation into brain cells leads to reduction of infarct size and improvement or recovery of brain function, thereby completing the present invention.

That is, the present invention is as follows.
[1] A cell preparation for treating cerebral infarction comprising SSEA-3 positive pluripotent stem cells isolated from mesenchymal tissue or cultured mesenchymal cells in a living body.
[2] The cell preparation according to [1] above for preventing and / or treating sequelae after cerebral infarction.
[3] The cell preparation according to [1] and [2] above, wherein the cell fraction enriched by SSEA-3 positive pluripotent stem cells by external stress stimulation.
[4] The cell preparation according to [1] to [3] above, wherein the pluripotent stem cell is CD105 positive.
[5] The cell preparation according to [1] to [4], wherein the pluripotent stem cells are CD117 negative and CD146 negative.
[6] The cell preparation according to any one of claims 1 to 5, wherein the pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative, and CD271 negative.
[7] The above [1], wherein the pluripotent stem cells are CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snai1 negative, Slug negative, Tyrp1 negative, and Dct negative. The cell preparation according to [6].
[8] The cell preparation according to [1] to [7] above, wherein the pluripotent stem cell is a pluripotent stem cell having all of the following properties:
(I) low or no telomerase activity;
(Ii) has the ability to differentiate into cells of any germ layer of the three germ layers;
(Iii) no neoplastic growth; and (iv) self-renewal ability.
[9] The above [1] to [8], wherein the pluripotent stem cell has the ability to differentiate into one or more cells selected from the group consisting of nerve cells, glial cells, vascular endothelial cells, and / or microglia. ] The cell preparation as described in.

  The present invention is directed to a subject suffering from cerebral infarction by administering a Muse cell in the brain parenchyma, whereby the Muse cell differentiates into a cell constituting the brain tissue in the damaged brain tissue, Cerebral infarct size can be dramatically reduced.

After injecting human skin fibroblast-derived Muse cells, human skin fibroblasts excluding Muse cells (ie, non-Muse cells), or phosphate buffered saline (PBS) into the brain parenchyma of a rat cerebral infarction model, Results shown by neurological severity score (NSS) over 3 months are shown. A decrease in the score value on the vertical axis corresponds to recovery of brain function. The result of a rotarod test is shown about the motor function of the rat cerebral infarction model inject | poured the Muse cell, the non-Muse cell, or phosphate buffered saline (PBS). The recovery of brain function was observed over time by the percentage of the average value of the measurement values (twice) on each measurement day based on the measurement values of the Muse cells and the like before the transplantation. The result of measuring the somatosensory evoked potential test (SEP) of a rat cerebral infarction model 85 days after injection of Muse cells and the like is shown. It is a fluorescence image which shows engraftment and differentiation of Muse cell in a brain tissue. Human-derived Muse cells were labeled green with a human mitochondrial marker, and neurons were labeled red using β-tubulin III as a marker. It was suggested that Muse cells injected into the brain parenchyma accumulated in the infarct boundary region and differentiated into neurons after engraftment. On the other hand, engraftment and differentiation of non-Muse cells were not observed. The result is obtained by observing human mitochondrial marker positive cells under a fluorescence microscope and counting the number of cells contained in 10 fields. In the infarct border region, non-Muse cells were hardly engrafted, but many Muse cells were present.

  The present invention relates to a cell preparation for treating cerebral infarction comprising SSEA-3 positive pluripotent stem cells (Muse cells). The present invention is described in detail below.

1. Applicable diseases The present invention aims to treat cerebral infarction using a cell preparation containing SSEA-3 positive pluripotent stem cells (Muse cells). Here, “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. In the present invention, it is an acute phase of cerebral infarction within 48 hours after onset, preferably within 24 hours after onset, more preferably within 6 hours, and most preferably within 3 hours. To do. Here, “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 thrombus, and the present invention is useful for treatment of cerebral thrombosis and cerebral embolism. “Treatment of cerebral infarction” means the effect of preventing the progression of infarct in the acute phase of cerebral infarction, the effect of improving dysfunction or subjective symptoms associated with cerebral infarction, and / or the suppression of the development of mental symptoms and seizures in the chronic phase Means. Furthermore, prevention of recurrence of stroke is also included. In addition, according to CT findings before administration, the degree of cerebral infarction is as follows: infarct size, infarct spread (penetrating branches, cortical branches), infarct side (left, right, both sides), infarct area (pre-cerebral artery area) And middle cerebral artery region, posterior cerebral artery region, watershed region, brain stem, cerebellum, etc.) and edema. “Suppressing the development of the cerebral infarction lesion” refers to an effect of suppressing the expansion of the infarction lesion over time after the onset of the ischemic event as compared with the case of no treatment. “The reduction effect of cerebral infarction volume” means that the volume of the infarct caused by cerebral infarction measured before administration of the cell preparation of the present invention is measured at the time of evaluation after a certain period after drug administration. It means to be smaller than before administration. Moreover, according to the present invention, the cell preparation of the present invention can also be used in the prevention and / or treatment of sequelae remaining after cerebral infarction. Here, “sequelae” includes language disorders, sensory disturbances such as numbness, movement disorders such as limbs, headache, vomiting, loss of vision, dysphagia, dysarthria, dementia and the like.

2. Cell preparation (1) Pluripotent stem cells (Muse cells)
The pluripotent stem cell used in the cell preparation of the present invention was found by Dezawa, one of the present inventors, in the human body and named as “Muse (Multilineage-differentiating Stress Ending) cell”. It is. Muse cells are obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)) and dermal connective tissue. Can also be scattered in the connective tissue of each organ. In addition, this cell is a cell having the properties of both pluripotent stem cells and mesenchymal stem cells. For example, each cell surface marker “SSEA-3 (Stage-specific embryonic antigen-3)” and “ CD105 "is identified as a 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. Accordingly, in the cell preparation of the present invention, when separating Muse cells from living mesenchymal tissue or cultured mesenchymal stem cells, the Muse cells can be purified and used simply with SSEA-3 as an antigen marker. . In the present specification, SSEA-3, which can be used in a cell preparation for treating cerebral infarction (including sequelae), is isolated from mesenchymal tissue or cultured mesenchymal tissue in vivo using SSEA-3 as an antigen marker. A pluripotent stem cell (Muse cell) or a cell population containing Muse cells may be simply referred to as “SSEA-3 positive cells”. In the present specification, “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.

  Briefly, Muse cells or cell populations containing Muse cells can be obtained from living tissue (e.g., using antibodies against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively). , Mesenchymal tissue). Here, “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 used in the cell preparation of the present invention 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. For example, Muse cells can be obtained from bone marrow, skin, or adipose tissue. For example, it is preferable to collect a mesenchymal tissue of a living body and separate and use Muse cells from this tissue. Alternatively, Muse cells may be separated from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells using the separation means. In the cell preparation of the present invention, the Muse cell used may be autologous to the recipient who receives the cell transplant, or may be another family.

  As described above, 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. Are known to include various types of stem cells and progenitor cells. However, 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. More specifically, 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. For example, without limitation, non-expression of CD117 and CD146 can be separated as an index, and non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be separated as an index, and The non-expression of 11 markers can be separated as an index.

Further, the Muse cell having the above characteristics used in the cell preparation of the present invention is as follows:
(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 (iv) having a self-renewal capability. In one aspect of the present invention, the Muse cell used in the cell preparation of the present invention has all the above properties. Here, with regard to (i) above, “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. Regarding (ii) above, 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. Furthermore, it has the ability to migrate and engraft in organs (heart, skin, spinal cord, liver, muscle, etc.) damaged by implantation into a living body by intravenous injection and differentiate into cells according to the tissue. As for (iii) above, 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. Moreover, about said (iv), a Muse cell has self-renewal (self-replication) ability. Here, “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, By bringing the cells of embryoid body-like cell mass to one cell again in suspension culture, the next generation embryoid body-like cell mass is formed, from which embryos in 3 germ layer differentiation and suspension culture are again formed. This means that a clot-like cell mass can be confirmed. The self-renewal may be repeated once or multiple times.

(2) Preparation and use of cell preparation The cell preparation of the present invention is not limited, but the Muse cell or the cell population containing Muse cell obtained in (1) above is treated with physiological saline or an appropriate buffer (for example, phosphorous). Acid buffered saline). In this case, when the number of Muse cells isolated from the tissue of the home or other family is small, the cells may be cultured before cell transplantation and grown until a predetermined cell concentration is obtained. As already reported (International Publication No. WO2011 / 007900 pamphlet), since Muse cells do not become tumors, even if cells collected from living tissue remain undifferentiated, they may become cancerous. Is low and safe. The culture of the collected Muse cells is not particularly limited, but can be performed in a normal growth medium (for example, α-minimum essential medium (α-MEM) containing 10% calf serum). In more detail, referring to the above-mentioned pamphlet of International Publication No. WO2011 / 007900, in the culture and proliferation of Muse cells, a medium, additives (for example, antibiotics, serum) and the like are appropriately selected, and Muse cells at a predetermined concentration. Can be prepared. When the cell preparation of the present invention is administered to a human subject, about several ml of bone marrow fluid is collected from human iliac bone, and for example, bone marrow mesenchymal stem cells are cultured as adherent cells from the bone marrow fluid and effective. After the therapeutic amount of Muse cells has been increased to reach the amount of cells that can be separated, the Muse cells can be isolated using the SSEA-3 antigen marker as an indicator, and autologous or other Muse cells can be prepared as cell preparations. Alternatively, for example, after separating Muse cells using SSEA-3 antigen marker as an indicator, and then cultivating and increasing the cells until an effective therapeutic amount is reached, autologous or other-use Muse cells may be prepared as cell preparations. it can.

  In addition, when using Muse cells in cell preparations, dimethyl sulfoxide (DMSO), serum albumin, etc. are included in the cell preparations to protect the cells, and antibiotics, etc., are included in the cell preparations to prevent bacterial contamination and growth. May be. Furthermore, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) Cells or components other than Muse cells contained in mesenchymal stem cells may be contained in the cell preparation. One skilled in the art can add these factors and agents to the cell preparation at appropriate concentrations. Thus, Muse cells can also be used as pharmaceutical compositions containing various additives.

The number of Muse cells contained in the cell preparation prepared above depends on the desired effect on cerebral infarction and sequelae (for example, suppression of cerebral infarction lesion development, reduction of cerebral infarct volume, recovery of motor function, recovery of language function) In other words, the target gender, age, weight, state of the affected area, state of cells to be used, and the like can be appropriately adjusted so that the recovery of the sensory function can be obtained. In Examples 3 and 4 to be described later, various effects of Muse cell transplantation were examined on a rat cerebral infarction model in which cerebral infarction was caused by an embolus. About 200 to 300 g of Wistar rats, an excellent effect was obtained by administering SSEA3 positive cells at 3 × 10 ⁴ cells / head. From this result, it is expected that an excellent effect can be obtained by administering a cell amount in terms of body weight of 1 to 1.5 × 10 ⁵ cells / kg per mammal. The target individuals include rats and humans, but are not limited thereto. In addition, the cell preparation of the present invention can be used a plurality of times (for example, 2 to 10 times) at appropriate intervals (for example, twice a day, once a day, once a week) until a desired therapeutic effect is obtained. 2 times, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months). Therefore, although depending on the condition of the subject, the therapeutically effective dose is preferably 1 to 10 doses of 1 × 10 ³ cells to 2 × 10 ⁷ cells per individual, for example. The total dose in one individual is not limited, but 1 × 10 ³ cells to 2 × 10 ⁸ cells, 1 × 10 ⁴ cells to 1 × 10 ⁸ cells, 2 × 10 ⁴ cells to 5 × 10 ⁷ cells, 5 × Examples include 10 ⁴ cells to 2 × 10 ⁷ cells, 1 × 10 ⁵ cells to 1 × 10 ⁷ cells, and the like.

3. Preparation of rat cerebral infarction model In the present specification, a rat cerebral infarction model can be constructed and used in order to examine the therapeutic effect of cerebral infarction (including sequelae) by the cell preparation of the present invention. The rats used as the model include, but are not limited to, Wistar rats and Sprague Dawley (SD) rats. In the cerebral infarction model, an embolus is inserted from the carotid artery of a rat to promote a symptom close to that of a human cerebral infarction, and an artery connected to the brain tissue causing the cerebral infarction (for example, middle cerebral artery (MCA)) Is embolized for a predetermined time (ischemic state), and then the embolus is withdrawn. The state of cerebral infarction can be confirmed by a brain tissue section (TTC staining). In addition, since the cell preparation of the present invention is human-derived Muse cells, it has a heterogeneous relationship with the rat to which the preparation is administered. Usually, in an experiment in which a heterogeneous cell or the like is administered in a model animal, an immunosuppressant (such as cyclosporine) is administered before or simultaneously with the administration of the heterologous cell in order to suppress the rejection reaction in the living body of the heterologous cell.

4). Therapeutic effect by Muse cells In the embodiments of the present invention, the cell preparation of the present invention can recover or restore the brain function of a patient with cerebral infarction or a patient suffering from sequelae. As used herein, “recovery” of brain function means alleviation of various dysfunctions (including sequelae) associated with cerebral infarction and suppression of progression, preferably to the extent that it does not interfere with daily life. It means to alleviate dysfunction. Further, “normally recovering” the brain function means that the dysfunction caused by cerebral infarction (including sequelae) returns to the state before cerebral infarction. In addition, although evaluation of recovery of brain function is not limited, evaluation by electrophysiological examination, neurological severity score (NSS), imaging examination, and pathological examination is common. Here, “electrophysiological examination” refers to various organs including the brain by observing potentials (waveforms of electrical signals) obtained by electrical stimulation of functions of the central nerve, peripheral nerves, muscles, etc. with a predetermined device. This is done to evaluate the functions. For example, the examination of the central nervous system (spinal cord) is called “Somatosensory Evoked Potential (SEP)”, and the reaction by sensory stimulation of the extremities passes through the sensory conduction pathway of the spinal cord to the cerebral cortex. A test that measures the potential evoked when delivered. Thereby, after administering the cell preparation of this invention to a patient, the extent of the functional recovery of a patient's central nerve can be confirmed objectively. Further, the “neurological severity score” (NSS) is evaluated by scoring the degree of function of the damaged brain for each item. NSS for rats has been described by Chen, J. et al. (Stroke, Vol. 32, p. 1005-1111 (2001)).

  The following examples further illustrate the present invention, but the present invention is not limited to these examples.

Example 1: Preparation of a rat cerebral infarction model The experimental protocol using rats in this example complies with the “Regulations for Animal Experiments, etc. of Tohoku University”, and the experimental animals are supervised by the Animal Experiment Center of Tohoku University. In accordance with the regulations. More specifically, in the rat cerebral infarction model, an embolus was inserted from the carotid artery of a Wistar rat (male 10 weeks old) to occlude a part of the cerebral blood vessel (for example, the middle cerebral artery (MCA)). Thereafter, the embolus was withdrawn and reperfused, and the rat was used as a cerebral infarction model in the following experiment. The state of cerebral infarction was confirmed by a brain tissue section (TTC staining). Moreover, in order to transplant the human Muse cell which becomes heterogeneous with respect to a rat, the immunosuppressant (FK506) was administered to the cerebral infarction rat before transplantation.

Example 2: Preparation of Muse cells Mice cells derived from human fibroblasts were prepared according to the method described in International Publication No. WO2011 / 007900. More specifically, mesenchymal cells having adhesiveness were cultured from human bone marrow fluid, and after proliferation, Muse cells or a cell population containing Muse cells were separated as SSEA-3 positive cells by FACS. Non-Muse cells are a group of SSEA-3 negative cells among the above mesenchymal cells and used as a control. Then, it adjusted to the predetermined density | concentration using the phosphate buffered saline or a culture solution, and used for the brain function evaluation by Muse cell in the following rat cerebral infarction model. When cells obtained by culturing mesenchymal cells such as bone marrow mesenchymal cells are used as the population of Muse cells, as reported by Wakao et al. (2011, supra), SSEA-3 positive cells Are all known to be CD105 positive cells.

Example 3 Evaluation of Brain Function by Muse Cell Transplantation Cerebral infarction rats prepared in Example 1 were divided into three groups, and on the second day after reperfusion, human fibroblast-derived Muse cells (1 × 10 ⁴ cells / 2 μl PBS × 3 sites), non-Muse cells (1 × 10 ⁴ cells / 2 μl PBS × 3 sites), or physiological saline (6 μl) was injected directly into the brain parenchyma of each group of rats. Thereafter, the improvement of the motor function of the rat was evaluated over time, and the cell dynamics analysis was performed after a predetermined time.

(1) Comprehensive evaluation based on neurological severity score (NSS) In the 3 months after transplantation, various brain dysfunctions (paralysis, sensory impairment, visual impairment, etc.) Severity Severity Score (NSS) (Chen, J., Stroke, Vol. 32, p. 1005-1111 (2001)). In this NSS assessment, points are assigned for motor function and behavioral changes, so that a maximum score of 18 represents severe neurological dysfunction, while a score of 0 indicates a normal neurological condition. . Specifically, the following items were evaluated: Raising the body with the tail (1 point for each item (maximum 3 points)); State when placed on the floor (0 to 3 points); Sensory test (1 Or 2 points); beam balance test (0-6 points); and lack of reflexes and movement abnormalities (1 point for each item (up to 4 points)). The result of NSS evaluation of each rat group (n = 10) is shown in FIG. In the non-Muse cell administration group and the physiological saline administration group, the score was lowered to about the first 10 days, and thereafter, the score tended to be maintained at 6 to 8 points. In contrast, in the Muse cell administration group, the score was significantly lowered on the 20th day compared to the other groups, and the score tends to be further lowered at the time when the experiment was terminated (after 80 days). There was a significant difference compared to the other groups.

(2) Rotarod test The recovery of brain dysfunction caused by transplantation of Muse cells and the like was examined using a device generally known as a device for measuring the coordination and sense of balance of motor functions of experimental animals. In this test, the time until the rat falls on the rotating table was measured twice a day at a frequency of once a week (0 to 84 days), and the average value was obtained to determine the cerebral infarction. The calculation was performed by calculating a score (%) based on the average value of two times before onset. The results are shown in FIG. In the non-Muse cell administration group and the physiological saline administration group, recovery of motor function was observed up to about 70% from the 21st to 28th days, but after that, motor function was not recovered to 100%. It was. On the other hand, in the Muse cell administration group, after recovering to 90% once on the 28th day, the score temporarily decreases to 70%, but from 56th day the motor function recovered to almost 100%. It was. From the results of the above comprehensive evaluation by NSS and the rotarod test, it was suggested that Muse cells significantly improve the brain function of cerebral infarction rats.

(3) Electrophysiological examination Somatosensory evoked potential examination (SEP) of a rat cerebral infarction model 85 days after injection of Muse cells and the like was measured (FIG. 3). The rectus femoris muscle was stimulated at 10 mA, 1 Hz × 100 times (1 second interval), and the potential measurement points were 2.5 mm lateral, 2.5 mm backward, and 1 mm deep from the previous term (bregma). The right brain-left foot (rt-lt) indicates the latency of the stimulus transmitted to the impaired side, and the left brain-left foot (lt-lt) indicates the stimulus transmitted to the brain on the same side, that is, the latency on the healthy side. The shorter the latency, the faster the recovery. In both the right brain-left foot (rt-lt) and the left brain-left foot (lt-lt), the group to which Muse cells were administered had a shorter latency than PBS or non-Muse cells, and no statistically significant difference was observed. However, the measured values suggested that the neural network recovered.

Example 4: Engraftment and differentiation of Muse cells in brain tissue In order to investigate the behavior of Muse cells and non-Muse cells injected into the brain parenchyma, it was examined whether these cells were engrafted and differentiated in brain tissue. did. 85 days after administration of these cells, brain tissue sections were prepared and observed under a fluorescence microscope (FIG. 4). In any section, cell nuclei were stained with DAPI, and further double-stained with human mitochondrial marker and nerve cell marker β-tubulin III. As a result, in brain slices of rats injected with Muse cells, the fluorescence of the human mitochondrial marker (green) and the fluorescence of the β-tubulin III marker (red) indicating neuronal cells were observed in the same cell group. It was suggested that Muse cells were engrafted in brain tissue and differentiated into nerve cells. On the other hand, no engraftment of non-Muse cells was observed in brain tissue sections when non-Muse cells were injected. Further, regarding the engraftment of Muse cells and non-Muse cells in the infarct boundary region, human mitochondrial marker positive cells were observed under a fluorescence microscope, and the number of each cell contained in 10 fields was counted (FIG. 5). In the infarct border region, non-Muse cells were hardly engrafted, but many Muse cells were present. From these results, it was suggested that Muse cells engraft in the infarct boundary region and differentiate into neurons as compared to non-Muse cells.

  The cell preparation of the present invention can regenerate brain cells (neuronal cells, glial cells, etc.) at the infarct site by administering it into the brain parenchyma of a cerebral infarction model, thereby reducing the infarct size and improving the brain function. It can be applied to the treatment of cerebral infarction and the prevention and / or treatment of sequelae after cerebral infarction.

  All publications and patent documents cited herein are hereby incorporated by reference in their entirety. While specific embodiments of the invention have been described herein for purposes of illustration, it will be apparent to those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims (8)

A cell preparation for treating cerebral infarction comprising SSEA-3 positive pluripotent stem cells isolated from living mesenchymal tissue or cultured mesenchymal cells as an active ingredient,
Wherein the pluripotent stem cells are:
(I) CD105 positive;
(Ii) low or no telomerase activity;
(Iii) has the ability to differentiate into any of the three germ layers;
(Iv) exhibits no neoplastic growth; and (v) has all the properties of having a self-renewal ability,
Here, the treatment of cerebral infarction is treatment of cerebral thrombosis and cerebral embolism, suppression of infarct focus development, improvement of dysfunction or subjective symptoms associated with cerebral infarction, suppression of chronic psychiatric symptoms or expression of seizures , relapse prevention of cerebral infarction, or Ri reduction der cerebral infarct volume,
As a therapeutically effective amount of the pluripotent stem cells, per mammal solid,
(A) 1 to 1.5 × 10 ⁵ cells / kg body weight converted cell weight,
(B) 1 to 10 doses of 1 × 10 ³ cells to 2 × 10 ⁷ cells, or
(C) 1 × 10 ³ cells to 2 × 10 ⁸ cells, 1 × 10 ⁴ cells to 1 × 10 ⁸ cells, 2 × 10 ⁴ cells to 5 × 10 ⁷ cells, or 5 × 10 ⁴ cells to 2 × 10 ⁷ cells Total dose of cells,
A cell preparation as described above.   The cell preparation according to claim 1, for preventing and / or treating sequelae after cerebral infarction.   The cell preparation according to claim 2, wherein the sequelae after cerebral infarction are speech disorder, sensory disorder, movement disorder, paralysis, headache, vomiting, visual disorder, dysphagia, articulation disorder, or dementia.   The cell preparation according to any one of claims 1 to 3, comprising a cell fraction in which SSEA-3 positive pluripotent stem cells are concentrated by external stress stimulation.   The cell preparation according to any one of claims 1 to 4, wherein the pluripotent stem cells are CD117 negative and CD146 negative.   The cell preparation according to any one of claims 1 to 5, wherein the pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative, and CD271 negative.   The pluripotent stem cell is CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snai1 negative, Slug negative, Tyrp1 negative, and Dct negative. The cell preparation according to claim 1.   The pluripotent stem cell has the ability to differentiate into one or more cells selected from the group consisting of nerve cells, glial cells, vascular endothelial cells, and / or microglia. The cell preparation described in 1.