Amelioration and treatment of brain damage with fetal growth retardation using pluripotent stem cells
阅读说明：本技术 利用多能干细胞进行的伴随胎儿生长迟缓的脑损伤的改善和治疗 (Amelioration and treatment of brain damage with fetal growth retardation using pluripotent stem cells ) 是由 佐藤义朗 北濑悠磨 清水忍 水野正明 早川昌弘 出泽真理 辻雅弘 于 2018-06-20 设计创作，主要内容包括：本发明的目的在于,提供一种在再生医疗中使用多能干细胞(Muse细胞)的新的医疗用途。本发明提供一种含有从生物体的间充质组织或培养间充质细胞分离的SSEA－3阳性多能干细胞、用于对运动质量异常、神经发展异常等伴随胎儿生长迟缓的脑损伤进行改善和治疗的细胞制剂和医药组合物。本发明的细胞制剂是基于下述机制假设的：通过对具有上述障碍的对象用Muse细胞给药而植入受损脑组织,对上述障碍进行改善和治疗。(The purpose of the present invention is to provide a novel medical use of pluripotent stem cells (Muse cells) for regenerative medicine. The present invention provides a cell preparation and a pharmaceutical composition for improving and treating brain damage accompanied by fetal growth retardation, such as abnormal motor quality and abnormal nerve development, which contain SSEA-3-positive pluripotent stem cells isolated from mesenchymal tissue or cultured mesenchymal cells of an organism. The cell preparation of the present invention is based on the following mechanism hypothesis: the above-mentioned disorders are ameliorated and treated by implanting damaged brain tissue with Muse cell administration to a subject having the disorder.)
1. A cell preparation for ameliorating and/or treating brain damage associated with retarded fetal growth, comprising SSEA-3 positive pluripotent stem cells isolated from mesenchymal tissue or cultured mesenchymal cells of an organism.
2. The cell preparation of claim 1, comprising a cell fraction in which SSEA-3 positive pluripotent stem cells are concentrated due to external stress stimulation.
3. The cell preparation of claim 1 or 2, wherein said pluripotent stem cells are CD105 positive.
4. The cell preparation of any one of claims 1 to 3, wherein said pluripotent stem cells are CD117 negative and CD146 negative.
5. The cell preparation of any one of claims 1 to 4, wherein said pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative and CD271 negative.
6. The cell preparation of any one of claims 1 to 5, 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.
7. The cell preparation according to any one of claims 1 to 6, wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties:
(i) low or no telomerase activity;
(ii) has the ability to differentiate into cells of any of the three germ layers;
(iii) does not show tumorous proliferation; and
(iv) has self-renewal capability.
8. The cell preparation of any one of claims 1 to 7, wherein the brain injury accompanied by retarded fetal growth is selected from the group consisting of abnormal motor quality, abnormal neural development, cerebral palsy, cognitive disorders, and behavioral disorders.
9. The cell preparation according to any one of claims 1 to 8, wherein said pluripotent stem cells have an ability to be implanted into brain tissue.
10. The cell preparation of any one of claims 1 to 9, wherein said pluripotent stem cells are administered as a therapeutically effective amount of about 1 x10 to a human neonatal, infant or young child subject5Cell/subject to about 1X 108Cells/subject were dosed.
11. The cell preparation according to any one of claims 1 to 10, wherein the pluripotent stem cells are administered as a therapeutically effective amount of about 1 x10 per individual to a human neonatal, infant or young child subject5Cells/kg to about 1X 108The cells were administered in a cell amount per kg of body weight.
Technical Field
The present invention relates to a cell preparation in regenerative medicine. More particularly, it relates to a cell preparation containing pluripotent stem cells, which is effective for the treatment of brain damage accompanying retarded fetal growth, and a novel therapeutic method.
Background
FGR (fetal growth retardation) or IUGR (intrinsic growth retardation) occurs due to various causes, and the combined effects of FGR caused by hypertensive pregnancy syndrome occurring in the middle of gestation and chronic hypoxemia, circulatory failure, malnutrition and the like mediated by the placenta are causes of neurological sequelae such as mental retardation, cognitive impairment and the like, and no treatment method thereof has been established. Currently, fetal growth retardation is found in about 8 to 10% of all pregnancies, and is confirmed in about 18% of perinatal deaths and about 31% of fetal deaths. In addition, fetal growth retardation is also a cause of small-for-gettionational age (SGA) in which the height and weight of a newborn are lower than the reference values, and is a group of high risk groups of neurological sequelae. Various therapeutic interventions have been attempted to prevent the neurological sequelae, but none of them have a significant effect (non-patent document 1).
In recent years, in the field of regenerative medicine, cell therapy using stem cells has been studied for various diseases, and embryonic stem cells (ES cells), neural stem/progenitor cells (NSPC), artificial pluripotent stem cells (iPS cells), and umbilical cord blood stem cells (UCBC) are known as stem cells expected to be used in clinical applications.
Furthermore, bone marrow mesenchymal cell fractions (MSCs) are isolated from adults and are known to have the ability to differentiate into, for example, bone, cartilage, adipocytes, nerve cells, skeletal muscle, and the like (
As a result of studies conducted by the present inventors, it was found that pluripotent stem cells (Muse cells) expressing SSEA-3 (Stage-Specific embryo Antigen-3) as a surface Antigen, which are present in a mesenchymal cell fraction and obtained without induction, have the pluripotency possessed by the mesenchymal cell fraction and are likely to be applicable to the treatment of diseases aimed at tissue regeneration. Further, it is also known that Muse cells can be concentrated by stimulating the mesenchymal cell fraction with various stresses (
Summary of The Invention
Problems to be solved by the invention
The purpose of the present invention is to provide a novel medical use of pluripotent stem cells (Muse cells) for regenerative medicine. More specifically, the present invention aims to provide a cell preparation and a pharmaceutical composition containing Muse cells, which are effective for the treatment of brain injury accompanying fetal growth retardation, and a novel therapeutic method.
Means for solving the problems
The present inventors have found that brain damage (e.g., learning disorders and dyskinesia) associated with growth retardation can be ameliorated by preparing a chronic ischemia model by installing a vasoconstrictor (AC) in the uterine artery of a pregnant rat and administering the model with Muse cells for intravenous injection, thereby completing the present invention.
Namely, the present invention is as follows.
[1] A cell preparation for ameliorating and/or treating brain damage associated with retarded fetal growth, comprising SSEA-3 positive pluripotent stem cells isolated from mesenchymal tissue or cultured mesenchymal cells of an organism.
[2] The cell preparation according to [1] above, which comprises a cell fraction in which SSEA-3-positive pluripotent stem cells are concentrated by external stress stimulation.
[3] The cell preparation according to [1] or [2], wherein the pluripotent stem cells are CD 105-positive.
[4] The cell preparation according to any one of the above [1] to [3], wherein the pluripotent stem cells are CD 117-negative and CD 146-negative.
[5] The cell preparation according to any one of the above [1] to [4], wherein the pluripotent stem cells are CD 117-negative, CD 146-negative, NG 2-negative, CD 34-negative, vWF-negative and CD 271-negative.
[6] The cell preparation according to any one of [1] to [5], wherein the pluripotent stem cells are CD 34-negative, CD 117-negative, CD 146-negative, CD 271-negative, NG 2-negative, vWF-negative, Sox 10-negative, Snai 1-negative, Slug-negative, Tyrp 1-negative, and Dct-negative.
[7] The cell preparation according to any one of [1] to [6], wherein the pluripotent stem cells are all pluripotent stem cells having the following properties:
(i) low or no telomerase activity;
(ii) has the ability to differentiate into cells of any of the three germ layers;
(iii) does not show tumorous proliferation; and
(iv) has self-renewal capability.
[8] The cell preparation according to any one of the above [1] to [7], wherein the brain injury accompanied by retarded fetal growth is selected from the group consisting of abnormal motor quality, abnormal neural development, cerebral palsy, cognitive disorder, abnormal behavior and dyskinesia.
[9] The cell preparation according to any one of the above [1] to [8], wherein the pluripotent stem cells have an ability to engraft into a brain tissue.
[10]Above-mentioned [1]～[9]The cell preparation of any one of the above, wherein the pluripotent stem cells are administered to a human neonatal, infant or young child subject in a therapeutically effective amount of about
[11]Above-mentioned [1]～[10]The cell preparation according to any one of the preceding claims, which is administered to a human neonatal, infant or young child subject in a therapeutically effective amount of about
Effects of the invention
The invention can make use of the following brain tissue regeneration mechanism to reduce brain damage significantly: in a subject suffering from brain damage accompanied by retarded fetal growth, Muse cells are selectively accumulated in damaged brain tissue by administering the Muse cells from a vein or the like, and the Muse cells are differentiated into cells constituting the brain tissue in the tissue.
Drawings
Fig. 1A shows the results of negative tendency tests on the behavioral improvement of fetal growth retardation model rats of the Muse cell administration group ("Muse"), the non-Muse cell administration group ("nonMuse"), the group ("vehicle") to which only a cell preservation solution (STEM-cellbank (registered trademark)) was added, and the sham group ("sham"). Shown are the results of 1 experiment performed for each group using 8 to 11-day-old rats for 4
Fig. 1B shows the result of performing a negative trend test in the same manner as fig. 1A, and shows the result of summing up the measurement times (seconds) for all 4 days in each rat and dividing by 4 to obtain an average value.
Fig. 2 shows the results of the rotarod test on the improvement of motor function of the fetal growth retardation model rats in the Muse cell administration group ("Muse"), the non-Muse cell administration group ("nonMuse") and the group ("vehicle") to which only the cell preservation solution (STEM-CELLBANKER (registered trademark)) was added.
Fig. 3 shows the results of the open field test on the emotional behavior (hypermobility, etc.) of the fetal growth retardation model rats in the Muse cell administration group ("Muse"), non-Muse cell administration group ("nonMuse") and group ("vehicle") to which only the cell preservation solution (STEM-cellbank (registered trademark)) was added. A shows the result of the distance traveled (distance) of the animal to be tested over a certain period of time, B shows the result of the time of immobility (time period), and C shows the result of the number of spontaneous locomotion starts (mobile epidemiods).
Fig. 4A shows the results of negative tendency tests on the behavioral improvement of fetal growth retardation model rats of the Muse cell administration group ("Muse"), the non-Muse cell administration group ("nonMuse"), the group ("vehicle") to which only a cell preservation solution (STEM-cellbank (registered trademark)) was added, and the sham group ("sham"). A is the result of a geotaxis test using 8 to 11 day-old rats for each group, and the results of 1 test performed 1 time for 4 days.
Fig. 4B shows the result of summing up the measurement times (seconds) for all 4 days in each rat and dividing by 4, which is the same as fig. 1A.
Fig. 5 shows the results of negative tendency tests on the behavioral improvement of fetal growth retardation model rats of the Muse cell administration group ("Muse"), the non-Muse cell administration group ("nonMuse"), the group ("vehicle") to which only a cell preservation solution (STEM-cellbank (registered trademark)) was added, and the sham group ("sham").
Fig. 6 shows the results of a new object recognition test on the improvement of memory learning and visual cognitive memory of the fetal growth retardation model rats of the Muse cell administration group ("Muse"), the non-Muse cell administration group ("nonuse"), the group ("vehicle") to which only the cell preservation solution (STEM-cellbank (registered trademark)) was added, and the sham operation group ("sham"). The recognition rate of the novel object was evaluated using rats.
Fig. 7 shows the results of negative geotaxic tests on the behavioral improvement of fetal growth retardation model rats (day-old 9) of the Muse cell administration group ("Muse") (n-21), the non-Muse cell administration group ("nonMuse") (n-20), the group to which only basal fluid (HBSS fluid) is added ("vehicle") (n-18), and the sham operation group ("sham") (n-25). The results of the geotaxis test using 8 to 11-day-old rats in each group are shown as the results of 1 test performed 1 time for 4 days.
Fig. 8 shows the results of rotarod test on improvement of motor function of fetal growth retardation model rats (1 month after birth) of the Muse cell administration group ("Muse") (n-12), non-Muse cell administration group ("non-12), group to which only basal fluid (HBSS fluid) is added (" vehicle ") (n-9), and sham operation group (" sham ") (n-16).
Fig. 9 shows the results of Y maze tests on spontaneous motor activity and spatial working memory of fetal growth retardation model rats (1 month after birth) of the Muse cell administration group ("Muse") (n-12), non-Muse cell administration group ("nonMuse") (n-12), group to which only basal fluid (HBSS fluid) is added ("vehicle") (n-11), and sham group ("sham") (n-15).
Fig. 10 shows the results of rotarod test on improvement of motor function of fetal growth retardation model rats (5 months after birth) of the Muse cell administration group ("Muse") (n-12), non-Muse cell administration group ("non-12), group to which only basal fluid (HBSS fluid) is added (" vehicle ") (n-11), and sham operation group (" sham ") (n-16).
Fig. 11 shows the results of the open field tests performed on the emotional behavior (hypermobility, etc.) of the fetal growth retardation model rats (5 months after birth) of the Muse cell administration group ("Muse") (n-12), the non-Muse cell administration group ("nonMuse") (n-12), the group to which only the base fluid (HBSS fluid) was added ("vehicle") (n-11), and the sham operation group ("sham") (n-16). The left panel shows the result of the moving distance of the tested animal within a certain time, the center panel shows the result of the motionless time, and the right panel shows the result of the number of spontaneous movement starts (the number of movements between a bright place and a dark place).
Fig. 12 shows the results of rotarod test on improvement of motor function of fetal growth retardation model rats (1 month after birth) of the Muse cell administration group ("Muse") (n-10), the non-Muse cell administration group ("non-11), the group to which only the base fluid (HBSS fluid) is added (" vehicle ") (n-9), and the sham operation group (" sham ") (n-16).
Fig. 13 shows the results of rotarod test on improvement of motor function of fetal growth retardation model rats (5 months after birth) of the Muse cell administration group ("Muse") (n-10), the non-Muse cell administration group ("non-11), the group to which only basal fluid (HBSS fluid) is added (" vehicle ") (n-9), and the sham operation group (" sham ") (n-16).
Detailed Description
The present invention relates to a cell preparation and a pharmaceutical composition for ameliorating and/or treating brain damage accompanied by fetal growth retardation, which contain SSEA-3-positive pluripotent stem cells (Muse cells), and a novel therapeutic method. The present invention will be described in detail below.
1. Applicable diseases and diagnosis thereof
The present invention aims to improve and treat brain damage accompanied by fetal growth retardation using a cell preparation or a pharmaceutical composition containing SSEA-3 positive pluripotent stem cells (Muse cells). Generally, perinatal (in humans, the period from the beginning of 22 weeks of gestation to less than 7 days after birth) growth retardation includes intrauterine (fetal) growth retardation caused by the fetal period and extrauterine growth retardation in which the physical development is inhibited after birth. Here, "fetal growth retardation" (or "intrauterine growth retardation") is a state in which fetal development is inhibited for various reasons. Fetal factors include multiple fetuses, fetal malformation, chromosomal abnormalities, congenital infections (congenital rubella, congenital cytomegalovirus infection, congenital toxoplasma infection, congenital herpes infection), etc.), and placental/umbilical cord factors include placental dysplasia, subpial hematoma, pre-placenta, chorioamnionitis, umbilical cord adhesion abnormality, umbilical cord torsion, and umbilical cord nodules. The maternal factors are not only basic diseases of the mother, such as pregnancy-induced hypertension syndrome, heart disease, hypertension, nephropathy, diabetes, collagen diseases, respiratory diseases, autoimmune diseases, etc., but also lifestyle habits such as smoking, drinking, taking medicines, etc. In addition to cases in which fetal abnormalities, chromosomal abnormalities, congenital infections, and the like clearly affect the central nervous system of the fetus, the prognosis of the development of intrauterine dysplasia is inferior to that of normal-developing infants. This tendency is known to be significant particularly in cases where the head circumference development is inhibited.
According to the time when the development of the fetus is hindered, the fetal growth retardation is roughly classified into "balanced type" and "unbalanced type". In the "balanced type", the cell growth is inhibited, and therefore, the cell is insufficient, and the development of the neck, trunk, and limbs is suppressed to the same extent, and the body shape is balanced, but the characteristics are small as a whole. In addition, with regard to postnatal prognosis, since the fetus itself has a growth-hindering factor, there is a tendency for poor prognosis. The main causes of the fetus in the early stage of pregnancy include chromosomal abnormality, congenital malformation, intra-fetal infection, alcoholism, and the like. On the other hand, the "unbalanced type" is characterized in that the size of local (mainly trunk) cells is small, and the development of the head is observed, but the development of the trunk is suppressed, and thus, the body becomes thin. The disease frequency is about 2-3 times of the 'balanced type'. With regard to postnatal prognosis, the prognosis is better because the factor of stunting development is placental blood flow disturbance (fetal malnutrition).
Here, as the definition of "fetal growth retardation", it is generally known that "height and weight average at birth is 10% lower than a reference value" (SGA), "10% lower than a development estimated fetal weight based on the number of gestational weeks (days)", or "1.5 SD or less of a standard value in the case of using a standardized fetal weight estimation equation". Here, the diagnosis of fetal growth retardation is initially asymptomatic, and is often found in ultrasonography, and more preferably performed using a standardized fetal weight estimation equation (APES method) as described above. Specifically, the following equation is used.
EFW(g)＝1.07×BPD3+0.30×AC2×FL
(in the formula, EFW is the estimated fetal weight (estimated fetal weight), BPD is the biparietaldimeter, AC is the abdominal circumference (approximate circumference), FL is the femoral length (femor length))
As a clinical measure, in general, a fetus diagnosed as a fetal growth retardation is subjected to a delivery at an appropriate time and is transferred to an extrafetal treatment while evaluating the state of the fetus using a biophysical characteristic score (BPS). After birth, in a newborn, death, hyperemia, thrombocytopenia, leukopenia, cholestasis, digestive tract abnormality, hypoglycemia, heart failure, pulmonary hemorrhage, abnormal exercise quality, cerebral palsy, and the like are observed; as for infants, low height, abnormal nerve development (cognitive impairment, behavioral abnormalities), etc. are observed; furthermore, in adults, abnormal glucose tolerance, abnormal heart and blood vessels, and abnormal glycolipids are observed. Thus, various sequelae are produced in association with the retarded growth of the fetus.
The present invention aims to improve and treat sequelae accompanying retarded fetal growth, particularly sequelae caused by complex and slow factors such as hypoxemia, circulatory failure, malnutrition, and the like due to placenta and umbilical cord factors, particularly motor quality abnormality (for example, decrease in motor localization and order and decrease in motor ability), and nerve development abnormality (for example, decrease in intelligence and cognition, academic problems, behavioral problems, decrease in adaptability to the society). As described above, the present invention is applicable to a disease in which a brain injury accompanied by retarded fetal growth is a subject to be improved or treated, particularly a brain injury caused by chronic hypoperfusion (ischemia), which is distinguished from a perinatal brain injury caused by hypoxic ischemia accompanied by any event before or at birth.
According to the present invention, in order to treat the applicable disease, improvement and/or treatment of the applicable disease can be achieved by administering a cell preparation and a pharmaceutical composition (hereinafter, sometimes collectively referred to as "transplantation") to a subject, which will be described later. Here, "improvement" means alleviation of brain damage and suppression of deterioration accompanying fetal growth retardation, and preferably means alleviation of symptoms to such an extent that does not interfere with daily life. Furthermore, "treatment" refers to inhibiting or completely eliminating brain damage associated with retarded fetal growth.
2. Cell preparation and pharmaceutical composition
(1) Pluripotent stem cells
The pluripotent stem cells used in the cell preparation and the pharmaceutical composition of the present invention are typically cells that have been found to be present in a human living body by one of the present inventors and are named "Muse (multiple-differentiated stress) cells". Muse cells can be obtained from bone marrow fluid, adipose tissue (Ogura, F. et al, Stemcells Dev., 11/20/2013, (Epub) (published 1/17/2014)), dermal connective tissue, and the like, and are present in the connective tissue of each organ in a scattered manner. The cells are cells having properties of both pluripotent stem cells and mesenchymal stem cells, and are identified as being double positive for "SSEA-3 (Stage-specific antigen-3)" and "CD 105", for example, as cell surface markers thereof. Therefore, Muse cells or a cell population containing Muse cells can be isolated from a biological tissue using these antigen markers as indicators, for example. Furthermore, Muse cells are stress tolerant and can be concentrated from mesenchymal tissue or cultured mesenchymal cells using various stress stimuli. The cell preparation of the present invention may also use a cell fraction in which Muse cells are concentrated due to stress stimulation. Details of the method of isolating Muse cells, the method of identifying Muse cells, the characteristics of Muse cells, and the like are disclosed in International publication No. WO 2011/007900. Furthermore, as reported by Wakao et al (2011, the above-mentioned document), it is known that when mesenchymal cells are cultured from bone marrow, skin, or the like and used as a mother group of Muse cells, the SSEA-3 positive cells are all CD105 positive cells. Therefore, in the cell preparation and the pharmaceutical composition of the present invention, when Muse cells are isolated from mesenchymal tissues or cultured mesenchymal stem cells of a living body, the Muse cells can be easily purified and used by using SSEA-3 as an antigen marker. In the present specification, a pluripotent stem cell (Muse cell) or a cell population containing Muse cells isolated from a mesenchymal tissue or a cultured mesenchymal tissue of a living body, which is labeled with SSEA-3 as an antigen and used in a cell preparation or a pharmaceutical composition for improving and/or treating brain damage associated with fetal growth retardation, may be simply referred to as "SSEA-3 positive cell". In the present specification, the term "non-Muse cell" refers to a cell other than the "SSEA-3 positive cell" which is a mesenchymal tissue of a living body or a cell contained in a cultured mesenchymal tissue. In the examples described later, cell populations from which SSEA-3 and CD105 positive cells were removed from MSCs were used as non-Muse cells according to the method described in international publication No. WO2011/007900, which relates to isolation and identification of human Muse cells.
Briefly, Muse cells or cell populations containing Muse cells can be isolated from biological tissues (e.g., mesenchymal tissues) using antibodies to SSEA-3 as a cell surface marker, either alone or both, respectively, to SSEA-3 and CD 105. Herein, "organism" refers to a mammalian organism. In the present invention, the living body does not include a fertilized egg and an embryo at a development stage before the blastocyst stage, but includes a fetus and an embryo at a development stage after the blastocyst stage including the blastocyst. The mammal is not limited, and examples thereof include primates such as humans and monkeys, rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, and ferrets. The Muse cells used in the cell preparation and the pharmaceutical composition of the present invention are clearly different from embryonic stem cells (ES cells) and iPS cells in that they are isolated from the tissues of the living body directly using a label. The term "mesenchymal tissue" refers to tissues present in bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord blood, and other tissues and various organs. For example, Muse cells can be obtained from bone marrow, skin, adipose tissue. For example, it is preferable to collect mesenchymal tissue of a living body and isolate Muse cells from the tissue for use. Furthermore, Muse cells can also be isolated from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells using the above isolation method. In the cell preparations and pharmaceutical compositions of the present invention, the Muse cells used may be autologous to the recipient or may be autologous to another individual.
As described above, Muse cells or cell populations containing Muse cells can be isolated from biological tissues using, for example, SSEA-3-positive and SSEA-3-and CD 105-double-positive as indicators, and it is known that human adult skin contains various types of stem cells and progenitor cells. However, Muse cells are different from these cells. Examples of such stem cells and progenitor cells include skin-derived progenitor cells (SKP), Neural Crest Stem Cells (NCSC), Melanocytes (MB), Perivascular Cells (PC), endothelial progenitor cells (EP), and adipose-derived stem cells (ADSC). Muse cells can be isolated using "no expression" of markers inherent to these cells as an indicator. More specifically, Muse cells can be isolated with the non-expression of at least 1, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 markers among 11 markers selected from the group consisting of CD34 (markers for EP and ADSC), CD117 (c-kit) (marker for MB), CD146 (markers for PC and ADSC), CD271 (marker for NGFR) (marker for NCSC), NG2 (marker for PC), vWF factor (marker for von Willebrand factor) (marker for EP), Sox10 (marker for NCSC), Snai1 (marker for SKP), Slug (marker for SKP), Tyrp1 (marker for MB), and Dct (marker for MB) as an index. For example, although not limited thereto, separation may be performed using, as an index, the non-expression of CD117 and CD146, further, separation may be performed using, as an index, the non-expression of CD117, CD146, NG2, CD34, vWF, and CD271, and further, separation may be performed using, as an index, the non-expression of the 11 markers.
Furthermore, Muse cells having the above characteristics used in the cell preparations and pharmaceutical compositions of the present invention may have a sequence selected from the group consisting of:
(i) low or no telomerase activity;
(ii) has the ability to differentiate into cells of any of the three germ layers;
(iii) does not show tumorous proliferation; and
(iv) has self-refresh capability
At least 1 property of the group. In one aspect of the invention, Muse cells used in the cell preparations and pharmaceutical compositions of the invention have all of the above properties. Here, the phrase "having low or no telomerase activity" as used in the above-mentioned (i) means that, for example, when the telomerase activity is detected using the TRAPEZE XL telomerase detection kit (Millipore Co.), the telomerase activity is low or undetectable. "low" telomerase activity means, for example, that it has a telomerase activity comparable to that of human fibroblasts which are somatic cells, or that it has a telomerase activity of 1/5 or less, preferably 1/10 or less, as compared with Hela cells. Regarding (ii) above, Muse cells have the ability to differentiate into three germ layers (endoderm, mesoderm and ectoderm) in vitro (in vitro) and in vivo (in vivo), and can differentiate into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, adipocytes, and the like, for example, by in vitro induction culture. In addition, the ability to differentiate into three germ layers is also shown in some cases when transplanted into a testis in vivo. Further, the cells have the ability to migrate, implant, and differentiate into cells corresponding to tissues in damaged organs (heart, skin, spinal cord, liver, muscle, etc.) by transplantation into living bodies by intravenous injection. With regard to (iii) above, Muse cells have the following properties: proliferating at a proliferation rate of about 1.3 days in suspension culture, proliferating from 1 cell in suspension culture to prepare an embryoid body-like cell mass, and stopping proliferating for about 14 days; when these embryoid body-like cell masses are cultured by adhesion, cell proliferation starts again, and the cells proliferated from the cell mass expand. Further, it has a property of not cancerating for at least half a year in the case of transplantation to the testis. In addition, regarding (iv) above, Muse cells have a self-renewal (self-replication) ability. Here, "self-renewal" means that differentiation of cells contained in an embryoid body-like cell mass obtained by culturing 1 Muse cell by suspension culture into 3 germ layer cells can be confirmed, and at the same time, a next generation embryoid body-like cell mass can be formed by suspension culturing the cells of the embryoid body-like cell mass again in the form of 1 cell, and thus, the re-differentiation of 3 germ layer and the embryoid body-like cell mass in suspension culture can be confirmed. The self-update may be repeated for 1 or more cycles.
The Muse cell-containing cell fraction used in the cell preparation of the present invention may be a cell fraction obtained by concentrating SSEA-3-positive and CD 105-positive pluripotent stem cells, which have at least 1 of the following properties, preferably all of the following properties, and which is obtained by a method comprising applying an external stress stimulus to mesenchymal tissue or cultured mesenchymal cells of an organism, thereby killing cells other than the cells which are resistant to the external stress, and collecting the surviving cells.
(i) SSEA-3 positive;
(ii) positive for CD 105;
(iii) low or no telomerase activity;
(iv) has the ability to differentiate into three germ layers;
(v) does not show tumorous proliferation; and
(vi) has self-renewal capability.
The external stress may be any one or a combination of more of protease treatment, culture under low oxygen concentration, culture under low phosphoric acid condition, culture under low serum concentration, culture under low nutrient condition, culture under exposure to heat shock, culture under low temperature, freezing treatment, culture in the presence of harmful substances, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under stress treatment, or physical shock. For example, the treatment time with the protease is preferably 0.5 to 36 hours in total in order to apply an external stress to the cells. The protease concentration may be a concentration used when cells adhering to a culture container are peeled off, when a cell mass is dispersed into single cells, or when single cells are collected from a tissue. The protease is preferably a serine protease, an aspartic protease, a cysteine protease, a metalloprotease, a glutamine protease or an N-terminal threonine protease. Further, the protease is preferably trypsin, collagenase or neutral protease.
Furthermore, as described later, the Muse cells having the above-described characteristics used in the cell preparation of the present invention are implanted into damaged brain tissue after administration to an organism by intravenous administration or the like. Muse cells are then thought to differentiate into the cells that make up the tissue, ameliorating and/or treating brain damage that accompanies retarded fetal growth.
(2) Preparation and use of cell preparations and pharmaceutical compositions
The cell preparation and pharmaceutical composition of the present invention are not limited, and can be obtained by suspending the Muse cells obtained in (1) above or a cell population containing Muse cells in physiological saline or an appropriate buffer (e.g., phosphate buffered physiological saline), in which case, when the number of Muse cells isolated from a tissue of an individual or other individual is small, the cells can be cultured before administration and proliferated to obtain a predetermined cell concentration, it is noted that, as described in the report (International publication No. WO 2011/007900), Muse cells are not tumorigenic, and therefore, even if cells recovered from a biological tissue are contained in an undifferentiated state, the possibility of being cancerous is low, and it is safe, and, in addition, without particular limitation, the culture of the recovered Muse cells can be performed in a normal proliferation medium (e.g., α -minimum essential (α -MEM) containing 10% calf serum, and the culture medium can be used as an effective indicator for the preparation of cells isolated from a normal proliferation medium (e.g., Muse cells obtained after preparation of Muse cells, preparation of Muse cells from International publication No. WO 007900, and culture medium, and when the Muse cells isolated from a serum-containing a predetermined amount of a cell preparation, or when the Muse cells isolated from a serum-enriched medium is used as an effective indicator, for example, or when the preparation of a cell preparation of a bone marrow cell is prepared, or when the present invention, the cell preparation is prepared, and the cell preparation is obtained from a cell preparation, and the cell preparation is prepared from a cell is prepared by culturing the culture medium, and the culture medium can be used as an effective indicator, and the invention.
In addition, Muse cells can be used in cell preparations and pharmaceutical compositions containing dimethyl sulfoxide (DMSO), serum albumin, and the like for protecting the cells, and antibiotics for preventing bacterial contamination and proliferation. The cell preparation and the pharmaceutical composition may further contain other components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, physiological saline, etc.) acceptable for the preparation, and cells or components other than Muse cells contained in the mesenchymal stem cells. One skilled in the art can add these factors and agents at appropriate concentrations in cell preparations and pharmaceutical compositions.
The number of Muse cells contained in the cell preparations and pharmaceutical compositions prepared as described above can be appropriately adjusted in consideration of sex, age, body weight, condition of affected part, condition of cells used, and the like of the subject to improve brain damage associated with fetal growth retardation and/or to treat metaphaseThe desired effect (e.g., improvement in motor quality, improvement in nerve development). In example 1 described later, an intrauterine growth model was prepared by installing a vasoconstrictor (AC) in the uterus of a pregnant rat to cause chronic ischemia of a fetus. The therapeutic effect of Muse cell transplantation was investigated using a model rat produced from the parent and involved in fetal growth retardation as a treatment target. For the model rat with the weight of about 15-20 g, the weight is measured by 1 × 104Very good results were obtained with cells/head (per individual) dosed with SSEA3 positive cells. From these results, it is expected that the human neonates (within 28 days after birth), infants (less than 1 year after birth) or infants (1 to 6 years after birth) will have an average of about
The cell preparation and the pharmaceutical composition of the present invention are intended to improve or treat brain damage associated with retarded fetal growth, and may be administered immediately after birth, when the neurological symptoms and growth retardation are confirmed and the fetal growth retardation is strong. That is, it is preferably administered immediately after the injury, and the effect of the cell preparation of the present invention and the like can be expected even after a certain period of time has passed after the injury, for example, 1 hour after the injury, 1 day after the injury, 1 week after the injury, 1 month after the injury, 3 months after the injury, and 6 months after the injury. Furthermore, it was confirmed by experiments of the present inventors that even when Muse cells used are derived from another person, they do not cause immune response, and therefore, they can be appropriately administered until desired effects are obtained in the improvement and treatment of brain damage accompanied by retarded fetal growth. In examples 2 to 4 described below, if a model rat produced from the intrauterine growth model (hereinafter, sometimes referred to as "fetal growth retardation model rat") is used to conduct a behavioral evaluation for improvement of brain injury accompanied by fetal growth retardation using Muse cells, the improvement of the brain injury is remarkably observed.
The Muse cells used in the cell preparation and the pharmaceutical composition of the present invention have a property of accumulating at a disease site. Therefore, in the administration of the cell preparation or the pharmaceutical composition, the site of administration (for example, intraperitoneal, intramuscular, or disease site), the type of blood vessel to be administered (vein and artery), and the like are not limited. Further, as a method for confirming that administered Muse cells reach and are implanted into a disease site, for example, Muse cells into which genes have been introduced so as to express a fluorescent protein (e.g., Green Fluorescent Protein (GFP)) are prepared in advance, and after administration to an organism, the Muse cells can be observed using a system capable of detecting fluorescence (e.g., IVIS (registered trademark) imaging System (Kyowa Kagaku Co., Ltd.)), whereby the dynamics of the Muse cells can be confirmed. The Muse cells used in the cell preparations and pharmaceutical compositions of the present invention are of human origin and are therefore xenogeneic with rats. In experiments in which administration is carried out using xenogenic cells or the like in model animals, in order to suppress rejection of xenogenic cells in vivo, an immunosuppressive agent (e.g., cyclosporin) may be administered before or simultaneously with administration of xenogenic cells.
3. Making of intrauterine development model for inducing slow growth of fetus
In the present specification, in order to study the improvement and therapeutic effect of the cell preparation and pharmaceutical composition of the present invention on brain damage (e.g., abnormal motor quality, abnormal neural development) accompanied by fetal growth retardation, an intrauterine growth model causing growth retardation in pregnant rats can be constructed, and rats having brain damage produced from the rats can be used. The intrauterine development model can be produced by, for example, the following method without limitation: vasoconstrictors (AC) (inner diameter: 0.45mm, 0.40mm, etc.), microcoils (inner diameter: 0.24mm, etc.) were installed at four locations of the uterine artery of the rat on day 17 of pregnancy, causing chronic ischemia of the fetus. The AC used contained casein in the interior, was notched in the interior, had a hole in the center, and was covered with a wide titanium ring. As a mechanism of action to cause chronic ischemia, as casein contained in the AC slowly swells due to water absorption, pores become smaller, and the diameter of a uterine artery portion to which the AC is attached becomes narrower, thereby causing ischemia. The rats that can be used in the intrauterine growth model include, but are not limited to, Wistar/ST-series rats and Sprague Dawley (SD) -series rats.
4. Improvement and therapeutic effects brought by Muse cells in fetal growth retardation model rat
The cell preparation and the pharmaceutical composition of the present invention can improve and/or treat brain damage accompanied with fetal growth retardation in mammals including humans. According to the present invention, using a rat model with fetal growth retardation produced from the intrauterine growth model prepared as described above, the improvement of symptoms and the like by Muse cells in rats with brain damage associated with fetal growth retardation can be experimentally investigated to evaluate the effect of the Muse cells. Specific evaluation methods can be performed using a general experimental system for evaluating brain function using rats, and examples of the behavioral evaluation include a negative tendency Test (negative geotaxis), a rotarod Test (Rota Rod Test), an Open Field Test (Open Field Test), a Shuttle Avoidance Test (trajectory assessment Test), a new Object Recognition Test (Novel Object Recognition Test), a gait Test (Cat Walk Test), and a Morris Water Maze Test (Morris Water Maze Test).
"negative going tests" are methods that measure and evaluate the time before a test animal begins to move away from a stimulus. For example, the time until the reflex is completed can be used as an index of improvement of brain function by placing the rat head down on an inclined plate and measuring the time of the motor reaction adjusted to be upward.
The "rotarod test" is a test using an apparatus for measuring the sense of coordination and balance of the movement function possessed by an animal to be tested. Specifically, the animal to be tested is placed on a device having a rotating bar capable of a certain acceleration, and is gradually accelerated from the start of slow rotation. The time during which the animal to be tested could travel in accordance with the rotation without falling from the rotating rod in this state was investigated. By repeated testing, the motor learning function can also be detected.
The "open field test" is based on observing emotional behaviors such as a search behavior of a test animal by placing the test animal in a new predetermined space (for example, a box of 60(W) × 60(D) × 40(H) cm). The observer can take a picture with a camera while actually recording the behavior of the animal to be tested, extract data, and evaluate an index of activity/emotion (for example, moving distance, stationary time, staying rate at the center).
The "shuttle avoidance test" is a test using an apparatus for observing the conditioned reflex behavior of a test animal with respect to sound and light, and can evaluate learning failure. This is to sound or light a room, and then apply a current to the floor, and to avoid the electrical stimulation, the animal to be tested escapes to another room. By repeating this operation, the presence or absence of learning failure in the test animal can be evaluated.
The "new object recognition test" is a test in which a test animal freely searches a space containing two objects, then changes one of the objects to a new object, and measures the increase and decrease in the search time for the new object by the test animal, thereby making it possible to evaluate memory learning and visual cognitive memory.
The gait test is to make the tested animal walk on a transparent glass plate with LED irradiation inside, shoot the footprints of only the contact surface luminescence due to the principle of total internal reflection from the lower part, and analyze by computer software by using the images, so as to evaluate the limb movement and walking state of the tested animal.
The "morris water maze test" is a test in which a rat swims in a water tank having a platform under the water surface, and the time, distance, and the like until reaching the platform are evaluated, whereby spatial recognition can be evaluated.
The present invention will be further specifically illustrated by the following examples, but the present invention is not limited to these examples.