WO2018025973A1 - 多能性幹細胞による慢性肺疾患の改善及び治療 - Google Patents
多能性幹細胞による慢性肺疾患の改善及び治療 Download PDFInfo
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
- A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
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- 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 and effective for treating chronic lung disease, and a novel therapeutic method.
- Chronic lung disease especially chronic lung disease in newborns, is a serious and frequent complication of neonatal medical care, and premature infants who have received artificial ventilation due to respiratory disorders such as respiratory distress syndrome Chronic lung lesions found in a group.
- CLD chronic lung disease
- the survival rate of infants with a birth weight of less than 1500 g in advanced countries has increased from less than 70% to more than 80% in the developed countries over the past 20 years. The rate is still high at 53.2% for children under 40% and 40% for children under birth weight 1000g.
- Chronic lung disease For a neonatal chronic lung disease, an internationally proposed definition in 2001 states: “Preterm infants with fewer than 32 weeks of gestation required more than 28% oxygen therapy for more than 28 days by 36 weeks of correction or before discharge. It is classified as “child” and is classified according to its oxygen dependence. Chronic lung disease may require home oxygen therapy even after discharge, and is an important disease in consideration of the medical economy, such as hospitalization for respiratory infections in early childhood. In addition, chronic lung disease has become a major problem not only for newborns and infants but also for the decline in respiratory function that extends to adolescence.
- chronic lung disease in neonates has become emphysematous due to pulmonary immaturity and surfactant deficiency, as well as damage such as infection, patent ductus arteriosus, oxygen toxicity, and artificial ventilation.
- chronic lung disease in newborns is not just a lung injury, but various injuries are added in the process of developing immature lungs out of the womb, It is thought that the development of alveoli and vascular system has stopped.
- Patent Document 1 proposes a pharmaceutical composition applicable to intratracheal administration, which is a combination of a steroid and a lung surfactant, in order to reduce the risk that a patient with respiratory distress syndrome will suffer from neonatal chronic lung disease.
- a pharmaceutical composition applicable to intratracheal administration which is a combination of a steroid and a lung surfactant, in order to reduce the risk that a patient with respiratory distress syndrome will suffer from neonatal chronic lung disease.
- steroids there are concerns about side effects on the central nervous system during the growth phase, and therefore, short-term small doses are administered only to severe cases in a rescue manner.
- chronic lung diseases are treated symptomatically, and there are no reports on therapeutic agents and treatment methods aimed at the cure of the diseases.
- Non-Patent Documents 1 to 3 bone marrow mesenchymal cell fraction (MSC) is isolated from adults and is known to have the ability to differentiate into bone, cartilage, adipocyte, nerve cell, skeletal muscle and the like (Non-patent Document 4). And 5).
- MSC bone marrow mesenchymal cell fraction
- MSC is a group of cells containing various cells, the substance of its differentiation ability is not known, and the therapeutic effect varies greatly.
- iPS cells have been reported as adult-derived pluripotent stem cells.
- a specific gene or a specific gene is identified in the dermal fibroblast fraction, which is a mesenchymal cell.
- iPS cells have a high tumorigenic potential, so there are very high hurdles for clinical application.
- SSEA-3 Stage-Specific Embryonic Antigen-3
- Muse cells Multilineage-differentiating Stress Enduring cells; Muse cells
- Muse cells are responsible for the pluripotency of the mesenchymal cell fraction and can be applied to disease treatment aiming at tissue regeneration It has become.
- Muse cells can be concentrated by stimulating the mesenchymal cell fraction with various stresses (Patent Document 3; Non-Patent Document 6).
- Patent Document 3 Non-Patent Document 6
- the expected therapeutic effect can be obtained by using Muse cells for improvement and / or treatment of chronic lung disease.
- JP 2007-262064 A Japanese Patent No. 4183742 International Publication No. 2011/007900
- An object of the present invention is to provide a new medical use using pluripotent stem cells (Muse cells) in regenerative medicine. More specifically, an object of the present invention is to provide a cell preparation and a pharmaceutical composition containing Muse cells and effective for the treatment of chronic lung disease (CLD), and a novel treatment method.
- CLD chronic lung disease
- chronic lung disease can be improved by preparing a rat model of chronic lung disease caused by high oxygen load and administering Muse cells by intravenous injection, thereby completing the present invention.
- the present invention is as follows.
- a cell preparation for improving and / or treating chronic lung disease comprising SSEA-3-positive pluripotent stem cells isolated from mesenchymal tissue or cultured mesenchymal cells in a living body.
- the cell preparation according to [1] above comprising a cell fraction in which SSEA-3 positive pluripotent stem cells are concentrated by external stress stimulation.
- 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.
- the chronic lung disease is selected from the group consisting of bronchopulmonary dysplasia (BPD), Wilson-Mikity syndrome (WMS), neonatal prolonged pulmonary hypertension (PPHN), and neonatal hypertension ]
- BPD bronchopulmonary dysplasia
- WMS Wilson-Mikity syndrome
- PPHN neonatal prolonged pulmonary hypertension
- neonatal hypertension The cell preparation according to any one of [7] to [7].
- Cells obtained by converting a body weight of about 3 ⁇ 10 4 cells / kg to about 3 ⁇ 10 7 cells / kg per subject, using the pluripotent stem cells as a therapeutically effective amount for a human newborn, infant or infant subject The cell preparation according to any one of [1] to [10] above, wherein an amount is administered.
- the present invention provides an anti-inflammatory action and a tissue repair action to a normal lung tissue by administering Muse cells from a vein or the like to a subject (mainly a newborn) suffering from chronic lung disease. Can do.
- GFP-labeled Muse cells have been engrafted in the lung tissue of 15-day-old chronic lung disease model rats.
- Muse cell administration group (“Muse"), a group (“vehicle”) to which only a cell suspension (HANK'S BALANCED SALT SOLUTION: HBSS) was added, and a sham op group (“Sham”) chronic lung disease model rats The result of evaluating lung tissue is shown.
- the short-term group was a 15-day-old rat, and the long-term group was a 29-day-old rat.
- shaft shows the ratio of a structure
- Non-Muse chronic of Muse cell administration group
- Non-Muse non-Muse cell administration group
- HBSS group to which only cell suspension
- Vehicle group to which only cell suspension
- Sham sham-operated group
- the result of having evaluated the lung tissue of a lung disease model rat is shown.
- the short-term group was a 15-day-old rat
- the long-term group was a 29-day-old rat.
- shaft shows the ratio of a structure
- a higher alveolar wall ratio indicates that alveolar walls are densely formed and lung tissue is repaired.
- shaft shows the relative expression level which set the sham operation group to 1.
- Muse cell administration group (“Muse group”), the group to which only the cell suspension (HBSS) was added (“Vehicle group”), and the Sham op group (“Sham group”) The results are shown.
- the vertical axis shows the right ventricular wall divided by the left ventricular wall and septum weight.
- Muse cell administration group (“Muse group”), non-Muse cell administration group (“Non-Muse cell”), a group to which only a cell suspension (HBSS) is added (“vehicle group”), and a sham operation group (“sham group”
- the vertical axis shows the right ventricular wall divided by the left ventricular wall and septum weight.
- decrease of the thickening of the pulmonary artery blood vessel wall in various administration groups using the change of the inner wall rate of the pulmonary artery blood vessel as an index is shown.
- FIG. 8A is a view of a tissue obtained by cross-sectioning a pulmonary artery blood vessel stained with an anti- ⁇ -SMA antibody.
- FIG. 8B shows the results of measuring the inner wall rate of pulmonary artery cells in the short-term group and the long-term group. The result of having examined the suppression of the neoplasia of the pulmonary artery cell promoted with a chronic lung disease in various administration groups is shown.
- FIG. 9 (A) shows the result of histological staining. The nucleus of proliferating pulmonary artery cells is stained with anti-Ki-67 antibody (green), and ⁇ -smooth muscle actin is stained with anti- ⁇ -SMA antibody.
- FIG. 9B shows the positive rate with anti-Ki-67 antibody. The result of having examined the number of the inflammatory cells in an alveolar lavage fluid is shown.
- the present invention relates to a cell preparation and a pharmaceutical composition for improving and / or treating chronic pulmonary disease, including SSEA-3-positive pluripotent stem cells (Muse cells), and a novel therapeutic method.
- SSEA-3-positive pluripotent stem cells Muse cells
- the present invention is described in detail below.
- the present invention aims to improve and treat chronic lung diseases using cell preparations or pharmaceutical compositions containing SSEA-3 positive pluripotent stem cells (Muse cells).
- SSEA-3 positive pluripotent stem cells Muse cells.
- chronic lung injury a study group of the Ministry of Health, Labor and Welfare in Japan
- chronic lung disease is Based on background factors and chest X-ray findings, it is classified into I-VI disease types as shown in Table 1 below.
- Diagnosis of chronic lung disease is diagnosed based on symptoms of polypnea in respiratory distress syndrome and chest X-ray as described in the above criteria, but after denying the possibility of other respiratory diseases Exclusion diagnosis is the basis.
- neonatal chronic lung disease is known as risk factors such as lung immaturity, oxygen toxicity, artificial ventilation, inflammation, infection, and patent ductus arteriosus, and development of alveoli and vascular system is suppressed.
- it is not limited to chronic lung disease as a disease to which the present invention is applied, but includes bronchopulmonary dysplasia (BPD), Wilson-Mikity syndrome (WMS), neonatal prolongation Pulmonary hypertension (PPHN), neonatal hypertension and the like can also be included.
- BPD Bronchopulmonary dysplasia
- BPD was first reported by Northway et al. In 1967, and is generally an alias for chronic lung disease in the West.
- Neonatal chronic lung disease is a disease that is common in premature infants, and is a disease in which breathing disorders requiring oxygen persist even after the age of 28 days or a corrected 36 weeks.
- the etiology of chronic lung disease is as described above. In particular, it is largely caused by high oxygen therapy (long-term high-concentration oxygen absorption, high-pressure ventilator management, etc.) or inflammation.
- prenatal infections such as chorioamnionitis and umbilitis cause the pathogenesis of chronic lung injury, which can also be the subject of the present invention.
- Chorioamnionitis is one of the infections in which inflammation has spread to the amniotic membrane surrounding the fetus, resulting in placental inflammation.
- transplantation in order to treat the above-mentioned applicable diseases, cell preparations and pharmaceutical compositions described later are administered to the subject (hereinafter sometimes collectively referred to as “transplantation”) to improve the applied diseases and / Or allows treatment.
- “improvement” means alleviation of various symptoms associated with chronic lung disease and suppression of progression, and preferably means alleviation of symptoms to such an extent that it does not interfere with daily life.
- treatment refers to suppressing or completely eliminating various symptoms associated with chronic lung disease.
- Pluripotent stem cell The pluripotent stem cell used in the cell preparation and pharmaceutical composition of the present invention is typically produced by Mr. Dezawa, one of the present inventors. It is a cell that has been found in vivo and named as “Muse (Multilineage-differentiating Stress Ending) cell”. Muse cells can be obtained from bone tissue, skin tissue such as adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (published on Jan 17, 2014)) and dermal connective tissue. Also 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.
- Muse cells or cell populations containing Muse cells can be separated from living tissues using, for example, these antigen markers as indicators.
- Muse cells are stress-resistant and can be concentrated from mesenchymal tissues or cultured mesenchymal cells by various stress stimuli.
- a cell fraction in which Muse cells are concentrated by stress stimulation can also be used. 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.
- a pluripotent stem cell (Muse cell) or a cell population containing Muse cells isolated from a tissue 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 used in the cell preparation and pharmaceutical composition of the present invention are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are isolated from living tissues using markers directly.
- ES cells embryonic stem cells
- iPS cells embryonic stem cells
- “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.
- the Muse cells used may be autologous to the recipient or may be allogeneic.
- 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.
- the Muse cell having the above-described characteristics used in the cell preparation and pharmaceutical composition 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.
- Muse cells used in the cell preparation and pharmaceutical composition of the present invention have all 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.
- 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.
- induction culture in vitro Can be differentiated into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, fat cells and the like.
- it may show the ability to differentiate into three germ layers.
- 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.
- 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.
- 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 cell fraction containing Muse cells used in the cell preparation of the present invention gives external stress stimulation to mesenchymal tissue or cultured mesenchymal cells in a living body, and collects cells resistant to the external stress.
- a cell fraction enriched with SSEA-3 positive and CD105 positive pluripotent stem cells having at least one, preferably all of the following properties, obtained by a method comprising: (I) SSEA-3 positive; (Ii) CD105 positive; (Iii) low or no telomerase activity; (Iv) has the ability to differentiate into three germ layers; (V) exhibits no neoplastic growth; and (vi) has the ability to self-renew.
- the above external stress includes protease treatment, culture at low oxygen concentration, culture under low phosphate conditions, culture at low serum concentration, culture under low nutrient conditions, culture under exposure to heat shock, low temperature Incubation in freezing, culture in the presence of harmful substances, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment or physical impact Any one or a plurality of combinations may be used.
- the protease treatment time is preferably 0.5 to 36 hours in total in order to give external stress to the cells.
- the protease concentration may be a concentration used when peeling cells adhered to the culture vessel, separating the cell mass into single cells, or collecting single cells from the tissue.
- the protease is preferably a serine protease, aspartic protease, cysteine protease, metalloprotease, glutamic acid protease or N-terminal threonine protease. Further, the protease is preferably trypsin, collagenase or dispase.
- the Muse cell having the above characteristics used for the cell preparation of the present invention is engrafted in lung tissue of chronic lung disease after being administered to a living body by intravenous administration or the like, and then the Muse cell constitutes the tissue. It is thought to differentiate into cells and ameliorate and / or treat chronic lung disease.
- the cell preparation and pharmaceutical composition of the present invention are not limited, but the Muse cell or the cell population containing Muse cell obtained in (1) above is treated with physiological saline or It is obtained by suspending in an appropriate buffer (for example, phosphate buffered saline, HBSS).
- an appropriate buffer for example, phosphate buffered saline, HBSS.
- the cells may be cultured before administration and grown until a predetermined cell concentration is obtained.
- HBSS phosphate buffered saline
- the culture of the collected Muse cells is not particularly limited, but can be performed in a normal growth medium (for example, ⁇ -minimal essential medium ( ⁇ -MEM) containing 10% calf serum). More specifically, referring to the above 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 are selected. A solution containing can be prepared.
- a normal growth medium for example, ⁇ -minimal essential medium ( ⁇ -MEM) containing 10% calf serum.
- the cell preparation or pharmaceutical composition of the present invention When the cell preparation or pharmaceutical composition 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.
- the effective therapeutic amount of Muse cells is increased until reaching a cell amount that can be separated, and then the Muse cells are separated using the SSEA-3 antigen marker as an index, and self or other Muse cells are prepared as cell preparations.
- the cells are cultured and expanded until an effective therapeutic amount is reached, and then autologous or allogeneic Muse cells can be prepared as cell preparations. it can.
- Muse cells when using Muse cells in cell preparations and pharmaceutical compositions, cells such as dimethyl sulfoxide (DMSO) and serum albumin are used to protect the cells, and antibiotics are used to prevent bacterial contamination and growth. You may make it contain in a formulation and a pharmaceutical composition.
- DMSO dimethyl sulfoxide
- 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 cell preparations and pharmaceutical compositions. Those skilled in the art can add these factors and drugs to cell preparations and pharmaceutical compositions at appropriate concentrations.
- the number of Muse cells contained in the cell preparation and the pharmaceutical composition prepared above is the desired effect in the improvement and / or treatment of chronic lung disease (for example, mortality reduction, shortening of artificial respiration management days, oxygen administration days Can be appropriately adjusted in consideration of the sex, age, weight, state of the affected area, state of cells to be used, and the like.
- chronic lung disease for example, mortality reduction, shortening of artificial respiration management days, oxygen administration days
- various effects of Muse cell transplantation were examined using model rats with chronic lung disease caused by high oxygen load, but SSEA3 positive was observed for the model rats weighing about 5 to 15 g. A very good effect was obtained by administering the cells at 1 ⁇ 10 4 cells / head (per individual).
- the individual includes a rat and a human, but is not limited thereto.
- the cell preparation and the pharmaceutical composition of the present invention may be used a plurality of times (for example, 2 to 10 times) at appropriate intervals (for example, twice a day, once a day) until a desired therapeutic effect is obtained. Twice a week, once a week, once every two weeks). Therefore, although depending on the condition of the subject, the therapeutically effective dose is preferably, for example, a dose of about 1 ⁇ 10 4 cells to about 3 ⁇ 10 8 cells per individual 1 to 10 times.
- the total dose in one individual is not limited, but 1 ⁇ 10 4 cells to 3 ⁇ 10 9 cells, 1 ⁇ 10 4 cells to 1 ⁇ 10 9 cells, 1 ⁇ 10 4 cells to 5 ⁇ 10 8 cells, 1 ⁇ 10 4 to 3 ⁇ 10 8 cells, 1 ⁇ 10 4 to 1 ⁇ 10 8 cells, 1 ⁇ 10 4 to 5 ⁇ 10 7 cells, 1 ⁇ 10 4 to 3 ⁇ 10 7 cells, 1 ⁇ 10 4 Cells to 1 ⁇ 10 7 cells, 1 ⁇ 10 4 cells to 5 ⁇ 10 6 cells, 1 ⁇ 10 4 cells to 3 ⁇ 10 6 cells, 1 ⁇ 10 4 cells to 1 ⁇ 10 6 cells, 1 ⁇ 10 4 cells to 5 ⁇ 10 5 cells, 1 ⁇ 10 4 cells to 3 ⁇ 10 5 cells, 1 ⁇ 10 4 cells to 1 ⁇ 10 5 cells, 1 ⁇ 10 5 cells to 3 ⁇ 10 9 cells, 1 ⁇ 10 5 cells to 1 ⁇ 109 cells, 1 ⁇ 10 5 cells ⁇ 5 ⁇ 10 8 cells, 1 ⁇ 10 5 cells
- the cell preparation and pharmaceutical composition of the present invention are intended to ameliorate and treat chronic lung disease, but the administration time was diagnosed as chronic lung disease by postnatal pediatric findings, chest radiographs, CT, etc. It may be within several months after the diagnosis. Alternatively, it may be early in life and when it is determined that the respiratory condition is poor and there is a high possibility of chronic lung disease (for example, the first week after birth).
- the cell preparation or the like is preferably administered immediately after the injury, but at a later time after the injury, for example, 1 week, 1 month, 3 months, 6 months after the injury, Even after 12 months, the effects of the cell preparation of the present invention can be expected.
- the Muse cell used does not elicit an immune response even when derived from another family, until a desired effect is obtained in the improvement and treatment of chronic lung disease. It may be administered as appropriate.
- lung tissue evaluation and cardiac evaluation are performed on improvement of the disease by Muse cells using chronic lung disease model rats, the therapeutic effect in the short term (15 days after birth) There is a tendency that a long-term treatment effect (29 days after birth) is more prominent.
- a chronic lung disease model rat can be constructed and used in order to examine the improvement and therapeutic effect of chronic lung disease by the cell preparation of the present invention.
- the rats used as the model include, but are not limited to, Wistar / ST rats and Sprague Dawley (SD) rats.
- Methods for producing chronic lung disease model rats are known, for example, see Lu, A. et al. (Pediatr. Res., 77, 784-792 (2015)) can be used to prepare chronic lung disease model rats.
- it can be confirmed by evaluation of lung tissue whether the model rat produced by the above method has chronic lung disease.
- Muse cells used in the cell preparation and pharmaceutical composition of the present invention have a property of accumulating at a disease site. Therefore, in administration of cell preparations or pharmaceutical compositions, their administration site (for example, intraperitoneal, intramuscular, disease site), the type of blood vessel to be administered (vein and artery), etc. are not limited.
- a Muse gene-transferred in advance so as to express a fluorescent protein eg, green fluorescent protein (GFP)
- the Muse cell used for the cell formulation and pharmaceutical composition of the present invention is derived from a human, it has a heterogeneous relationship with the rat.
- an immunosuppressant such as cyclosporine
- the cell preparation and pharmaceutical composition of the present invention improve and / or improve chronic lung disease and various symptoms associated therewith in mammals including humans. Can be treated.
- the chronic lung disease model rat prepared as described above it is possible to experimentally study the improvement of symptoms by Muse cells in rats with chronic lung diseases and to evaluate the effect of the Muse cells.
- the evaluation method can be performed using a general measurement system for evaluating pulmonary function using rats, such as FinePoint (trademark) -respiratory / pulmonary function evaluation system Noninactive Airways Mechanics (NAM).
- a respiratory and pulmonary function assessment system can be used.
- lung tissue extracted from model rats for example, measurement of alveolar wall (expansion of alveolar space), measurement of mRNA expression level such as inflammatory cytokine, or right ventricular myocardial evaluation associated with chronic lung disease It can be evaluated by performing pulmonary blood vessel evaluation.
- tissue volume density tissue Volume Density
- lung tissue removed from a test subject is fixed with 4% paraformaldehyde, a paraffin block section is prepared, and then stained with hematoxylin and eosin. Under the microscope, the proportion of the lung tissue can be counted in a predetermined number of grids to evaluate the lesion of the lung tissue (see Example 4 described later). When lung tissue is damaged, the alveolar rate increases.
- the effectiveness of the cell preparation or pharmaceutical composition of the present invention against chronic lung disease can be evaluated by measuring the expression level of inflammatory cytokines expressed in lung tissue.
- inflammatory cytokines include IL-1 ⁇ , IL-1 ⁇ , IL-6, CCL2 (MCP-1), TNF- ⁇ , TGF- ⁇ , VEGF and the like.
- the expression level of the inflammatory cytokine expressed in the tissue can be measured by a conventional method, for example, using RT-PCR. As described in Example 5 described later, in the group administered with Muse cells, the expression level of inflammatory cytokines can be significantly reduced compared to the control group administered with vehicle. It can be expected to treat chronic lung disease.
- Heart assessment (pulmonary hypertension assessment) In chronic lung disease caused by high oxygen load, thickening of the pulmonary blood vessels occurs due to the applied high oxygen concentration, and the thickening of the heart is confirmed accordingly.
- Neonatal chronic lung disease is complicated by secondary pulmonary hypertension (as severe as possible). This secondary pulmonary hypertension is also an important factor that determines the prognosis of life. Since continuous pulmonary hypertension leads to thickening of the right ventricular wall due to right heart load, pulmonary hypertension can be evaluated using an evaluation of the heart (myocardium). As shown in Example 6 to be described later, right ventricular wall thickening, which is considered to be due to right heart load, is also observed in chronic lung disease model rats, and this thickening can be reduced by administration of Muse cells.
- Example 1 Preparation of Muse cells Muse cells were obtained according to the method described in International Publication No. WO2011 / 007900 concerning the separation and identification of human Muse cells.
- Example 2 Production of rat model of chronic lung disease and administration of Muse cells
- the protocol for experimental animals used in this study was approved by the Animal Experiment Committee of Nagoya University School of Medicine. Pregnant SD rats were obtained from SLC Japan (Shizuoka Prefecture, Japan). Immediately after birth (within 24 hours), mother and pups are allowed to receive food and water freely throughout the experiment and are subjected to high oxygen loads (animals with oxygen controllers and sensor adapters). In the chamber) under a 12 hour light / dark cycle. The animal room and cage were always maintained at 23 ° C. Since the mother rats were also injured by hyperoxia, surrogate mother rats were replaced every two days.
- the above chronic lung disease model rats were anesthetized by inhalation of isoflurane, and to the treatment group, Muse cells (1 ⁇ 10 4 cells / individual) were administered from the right external jugular vein (treatment group). In the control group, only the same volume of HBSS was administered instead of Muse cells. Rats that were not provided with Muse cells and HBSS and were not subjected to a high oxygen load were taken as a sham group. In the following experiments, rats of day 15 and day 29 were used for various evaluations as a short-term group and a long-term group, respectively. The rats were exposed to a high oxygen concentration (80%) until day 15 when the short-term evaluation was performed, and then were exposed to the normal oxygen concentration (21%) until day 29 when the long-term evaluation was performed.
- Example 3 Confirmation of Muse Cell Engraftment in Lung Tissue
- Muse cells used for transplantation had engrafted in lung tissue.
- a lentivirus-GFP gene was introduced into Muse cells in advance so that green fluorescent protein (GFP) was expressed and Muse cells were labeled thereby.
- Muse cells labeled with GFP were separated by FACS as double positive cells of GFP and SSEA-3. Thereafter, Muse cells were administered as described in Example 2.
- the Muse cells were engrafted in the lung tissue as follows in the lungs of 15-day-old rats (14 days after high oxygen load) to which the Muse cells were administered.
- the lung was excised and a section of lung tissue was prepared according to a conventional method. A frozen section was prepared, and antigen activation was performed with HistoVT one (Nacalai Tesque, Inc.). After blocking with donkey serum in order to prevent non-specific binding of the antibody, it was incubated with anti-GFP antibody at 4 ° C. overnight. Subsequently, further incubation with an appropriate secondary antibody was performed. Thereafter, it was encapsulated with an encapsulant containing DAPI for staining DNA. From this result, it was shown that Muse cells transplanted from the right external jugular vein of a rat were engrafted in lung tissue which suffered from lung injury (FIG. 1).
- Example 4 Lung tissue evaluation Evaluation of lung tissue of chronic lung disease model rats was performed as follows. The model rats of the short-term group and the long-term group were euthanized, physiological saline was injected from the right ventricle to perfuse the pulmonary blood vessels, and then the lungs were inflated with 4% paraformaldehyde aqueous solution via a tracheal catheter ( 20 cm H 2 O, 20 minutes). The lungs were removed and fixed in 4% paraformaldehyde solution for 18-24 hours (4 ° C.), and then cut into each lobe.
- the cut lung lobe was dehydrated with an aqueous ethanol solution and xylene, embedded in paraffin, the lung lobe was cut into 5 ⁇ m thick sections, and a specimen was prepared by staining with hematoxylin-eosin (HE staining).
- HE staining hematoxylin-eosin
- the non-Muse group observed an effect of reducing lung tissue damage compared to the vehicle group, but the effect was smaller than that of the Muse group.
- rats administered Muse cells it was shown that lung tissue damage was relieved to near normal as in previous results.
- lung tissue was evaluated by measuring the alveolar wall ratio in 29-day-old rats.
- the prepared specimen was photographed with a digital camera for microscope (manufactured by Olympus, model number DP73).
- the photographed image was analyzed using imaging software (manufactured by Olympus Corporation, trade name cellSens).
- the area of the alveoli was obtained by excluding the areas of the trachea, blood vessels, and damaged site from the area of the entire specimen section, and the ratio of the area occupied by the alveolar wall (alveolar wall ratio) was calculated by the following formula.
- Alveolar wall ratio (%) alveolar wall area ⁇ ⁇ total lung specimen area ⁇ (tracheal area + blood vessel area + lesion area) ⁇ ⁇ 100
- the results are shown in FIG. In the sham group, the alveolar wall ratio was about 22-23%, and in the group in which Muse cells were administered to chronic lung disease model rats, the destroyed alveolar wall recovered to around 21%. Moreover, although the recovery effect was seen also in the non-Muse cell group, it did not reach as much as the Muse cell group. As described above, the above lung tissue evaluation showed a significant effect in the lung Muse group, and it was shown to be relieved to a normal state.
- Example 5 Measurement of mRNA expression of inflammatory cytokines In chronic lung disease, the expression of various inflammatory cytokines increases in lung tissue. It was investigated whether the expression level of these could be reduced by transplantation of Muse cells. Lung tissues were collected from 15-day-old rats, and after total RNA was extracted according to a conventional method, mRNA expression levels of inflammatory cytokines CCL2 and VEGF were compared between the treatment group and the vehicle group. As a specific method, TRI reagent was added to a container from which lung tissue was quickly removed, and homogenized with a Dounce homogenizer and a syringe with a 21G needle.
- the vertical axis shows the relative expression level of each cytokine mRNA when the expression level of the sham group is 1.
- the treatment group (Muse group) to which the Muse cells were administered was able to significantly reduce the mRNA expression level of any inflammatory cytokine as compared to the treatment group (vehicle group) to which HBSS was administered.
- Paraffin sections were prepared in the same manner as in the above examples, and then antigen activation was performed using a citrate buffer. After blocking with donkey serum to prevent non-specific binding of the antibody, incubation was performed overnight at 4 ° C. with anti- ⁇ -SMA antibody and anti-Ki-67 antibody. After further incubation with an appropriate secondary antibody, it was encapsulated with an encapsulant containing DAPI that stains the DNA.
- Inner wall ratio (%) ⁇ (vascular outer diameter ⁇ vascular inner diameter) / vascular outer diameter ⁇ ⁇ 100
- the Muse cell group was able to remarkably suppress the thickening of the pulmonary artery blood vessel wall caused by pulmonary hypertension.
- the inhibitory effect was observed also in the non-Muse cell group, the effect was small compared with the Muse cell group.
- FIG. 9A shows the results of triple staining with anti- ⁇ -SMA antibody, anti-Ki-67 antibody, and DAPI.
- the pulmonary artery specimens shown in the figure belong to the group administered with non-Muse cells, and proliferating pulmonary artery cells stained with anti-Ki-67 antibody were observed.
- Example 7 Measurement of the number of inflammatory cells in the alveolar lavage fluid In chronic lung disease, an increase in the number of inflammatory cells is observed. Whether or not the number of these cells decreases by administration of Muse cells. investigated. After euthanizing the rats in the short-term group, the blood vessels were perfused with physiological saline from the pulmonary artery, and 0.4 ml (0.2 ml ⁇ 2 times) of physiological saline was injected from the intubated tracheal cannula. Washing (BAL) was performed, and the BAL solution (BALF) was recovered. The number of leukocytes, macrophages, neutrophils, and lymphocytes in BALF was measured by the following method.
- the cell preparation and pharmaceutical composition of the present invention can be applied to the improvement and treatment of neonatal chronic lung disease.
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Abstract
Description
[1]生体の間葉系組織又は培養間葉系細胞から分離されたSSEA-3陽性の多能性幹細胞を含む、慢性肺疾患を改善及び/又は治療するための細胞製剤。
[2]外部ストレス刺激によりSSEA-3陽性の多能性幹細胞が濃縮された細胞画分を含む、上記[1]に記載の細胞製剤。
[3]前記多能性幹細胞が、CD105陽性である、上記[1]又は[2]に記載の細胞製剤。
[4]前記多能性幹細胞が、CD117陰性及びCD146陰性である、上記[1]~[3]のいずれかに記載の細胞製剤。
[5]前記多能性幹細胞が、CD117陰性、CD146陰性、NG2陰性、CD34陰性、vWF陰性、及びCD271陰性である、上記[1]~[4]のいずれかに記載の細胞製剤。
[6]前記多能性幹細胞が、CD34陰性、CD117陰性、CD146陰性、CD271陰性、NG2陰性、vWF陰性、Sox10陰性、Snai1陰性、Slug陰性、Tyrp1陰性、及びDct陰性である、上記[1]~[5]のいずれかに記載の細胞製剤。
[7]前記多能性幹細胞が、以下の性質の全てを有する多能性幹細胞である、上記[1]~[6]のいずれかに記載の細胞製剤:
(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
(iv)セルフリニューアル能を持つ。
[8]慢性肺疾患が、気管支肺異形成症(BPD)、ウィルソン・ミキティ症候群(WMS)、新生児遷延性肺高血圧症(PPHN)、及び新生児高血圧症からなる群から選択される、上記[1]~[7]のいずれかに記載の細胞製剤。
[9]前記多能性幹細胞が肺組織に生着する能力を有する、上記[1]~[8]のいずれかに記載の細胞製剤。
[10]ヒト新生児、乳児又は幼児対象に前記多能性幹細胞を治療上有効量として約1×104細胞/個体~約3×108細胞/個体で投与する、上記[1]~[9]のいずれかに記載の細胞製剤。
[11]ヒト新生児、乳児又は幼児対象に前記多能性幹細胞を治療上有効量として、該対象一個体あたり約3×104細胞/kg~約3×107細胞/kgを体重換算した細胞量を投与する、上記[1]~[10]のいずれかに記載の細胞製剤。
本発明は、SSEA-3陽性の多能性幹細胞(Muse細胞)を含む細胞製剤又は医薬組成物を用いて、慢性肺疾患の改善及び治療を目指す。わが国では1996年に厚生労働省研究班により、新生児の「慢性肺障害」を「先天奇形を除く肺の異常により、酸素投与を必要とするような呼吸窮迫症状が新生児期に始まり、日齢28日を越えて続くもの」と定義し、さらに、肺障害のうち大部分を占める低出生体重児の慢性肺障害を疾患として特徴付けるために、「慢性肺疾患」を「胸部X線写真でびまん性不透亮像、泡沫状陰影など明らかな異常所見を伴う慢性肺障害のある場合」と定め、背景因子及び胸部X線所見から以下の表1に示すようにI~VIの病型に分類される。
(1)多能性幹細胞
本発明の細胞製剤及び医薬組成物に使用される多能性幹細胞は、典型的には、本発明者らの一人である出澤氏が、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る。Muse細胞は、骨髄液、脂肪組織(Ogura,F.,et al.,Stem Cells Dev.,Nov 20,2013(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)」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。また、Muse細胞はストレス耐性であり、間葉系組織又は培養間葉系細胞から種々のストレス刺激により濃縮することができる。本発明の細胞製剤には、ストレス刺激によりMuse細胞が濃縮された細胞画分を用いることもできる。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、本発明における細胞製剤及び医薬組成物において、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、慢性肺疾患を改善及び/又は治療するための細胞製剤及び医薬組成物において使用され得る、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
(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、本発明の細胞製剤及び医薬組成物に使用される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、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。
(i)SSEA-3陽性;
(ii)CD105陽性;
(iii)テロメラーゼ活性が低いか又は無い;
(iv)三胚葉に分化する能力を持つ;
(v)腫瘍性増殖を示さない;及び
(vi)セルフリニューアル能を持つ。
本発明の細胞製剤及び医薬組成物は、限定されないが、上記(1)で得られたMuse細胞又はMuse細胞を含む細胞集団を生理食塩水や適切な緩衝液(例えば、リン酸緩衝生理食塩水、HBSS)に懸濁させることによって得られる。この場合、自家又は他家の組織から分離したMuse細胞数が少ない場合には、投与前に細胞を培養して、所定の細胞濃度が得られるまで増殖させてもよい。なお、すでに報告されているように(国際公開第WO2011/007900号パンフレット)、Muse細胞は、腫瘍化しないため、生体組織から回収した細胞が未分化のまま含まれていても癌化の可能性が低く安全である。また、回収したMuse細胞の培養は、特に限定されないが、通常の増殖培地(例えば、10%仔牛血清を含むα-最少必須培地(α-MEM))において行うことができる。より詳しくは、上記国際公開第WO2011/007900号を参照して、Muse細胞の培養及び増殖において、適宜、培地、添加物(例えば、抗生物質、血清)等を選択し、所定濃度のMuse細胞を含む溶液を調製することができる。ヒト対象に本発明の細胞製剤又は医薬組成物を投与する場合には、ヒトの腸骨から数mL程度の骨髄液を採取し、例えば、骨髄液からの接着細胞として骨髄間葉系幹細胞を培養して有効な治療量のMuse細胞を分離できる細胞量に達するまで増やした後、Muse細胞をSSEA-3の抗原マーカーを指標として分離し、自家又は他家のMuse細胞を細胞製剤として調製することができる。あるいは、例えば、Muse細胞をSSEA-3の抗原マーカーを指標として分離後、有効な治療量に達するまで細胞を培養して増やした後、自家又は他家のMuse細胞を細胞製剤として調製することができる。
本明細書においては、本発明の細胞製剤による慢性肺疾患の改善及び治療効果を検討するために慢性肺疾患モデルラットを構築し、使用することができる。該モデルとして使用されるラットには、限定されないが、一般的に、Wistar/ST系ラット、スプラーグドーリー(SD)系ラットが挙げられる。慢性肺疾患モデルラットを作製する方法は公知であり、例えば、Lu,A.ら(Pediatr.Res.,77,784-792(2015))の方法に従って慢性肺疾患モデルラットを作製することができる。また、上記方法によって作製されたモデルラットが、慢性肺疾患を有するかどうかを肺組織の評価により確認することができる。
本発明の実施形態では、本発明の細胞製剤及び医薬組成物は、ヒトを含む哺乳動物における慢性肺疾患及びそれに伴う各種症状を改善及び/又は治療することができる。本発明によれば、上記で作製した慢性肺疾患モデルラットを用いて、実験的に慢性肺疾患のラットにおけるMuse細胞による症状の改善等を検討し、該Muse細胞の効果を評価することができる。評価方法としては、ラットを用いた肺機能を評価する一般的な測定系を用いて行うことができ、例えば、FinePointe(商標)-呼吸・肺機能評価システム Noninvasive Airway Mechanics(NAM)などのラットの呼吸及び肺機能評価システムを利用することができる。また、モデルラットから取り出した肺組織について、例えば、肺胞壁の測定(肺胞間腔の拡大)、炎症性サイトカインなどのmRNA発現量の測定、あるいは、慢性肺疾患と関連付けられる右室心筋評価、肺血管評価を行うことにより評価することができる。
本発明によれば、肺組織の評価の1つのとして、組織体積密度(Tissue Volume Density)を測定することにより、慢性肺疾患の治療改善効果を評価することができる。簡単には、被験対象から取り出した肺組織を4%パラフォルムアルデヒドで固定し、パラフィンブロック切片を作成後、ヘマトキシリン・エオジンで染色する。顕微鏡下で所定数のグリッドのうち肺組織の割合をカウントし、肺組織の病変を評価することができる(後述する実施例4を参照)。肺組織に損傷があると、肺胞腔率が高くなる。
高酸素負荷に起因した慢性肺疾患では、適用される高酸素濃度により、肺血管の肥厚が生じ、これに伴い心臓の肥厚が確認される。新生児の慢性肺疾患では、(重症になるほど)2次的な肺高血圧症を合併する。この2次的な肺高血圧症も生命予後を決める重要な要因である。持続的な肺高血圧は、右心負荷による右心室壁肥厚につながるため、心臓(心筋)の評価を用いて肺高血圧を評価することができる。後述する実施例6に示されるように、慢性肺疾患モデルラットにおいても右心負荷によると考えられる右室壁肥厚が観察され、この肥厚をMuse細胞の投与により低減させることができる。
慢性肺疾患においては、炎症性細胞数の増加が観察されるが、各種処置による慢性肺疾患の治療効果を肺胞からの洗浄液中の炎症性細胞数を測定することにより評価することができる。後述する実施例7に示されるように、Muse細胞は、慢性肺疾患モデルラットにおける炎症性細胞数を有意に減少させることができる。
ヒトMuse細胞の分離及び同定に関する国際公開第WO2011/007900号に記載された方法に準じて、Muse細胞を得た。
本研究に使用された実験動物に関するプロトコールは、名古屋大学医学部動物実験委員会によって承認されたものである。妊娠SDラットを日本エスエルシー株式会社(日本、静岡県)より入手した。出生後すぐ(24時間以内)より、母ラットと仔ラットは、実験期間中、食餌と水を自由に摂取できるようにされ、高酸素負荷がかけられるケージ(酸素コントローラーとセンサーアダプターを備えたアニマルチャンバー)内で、12時間の明暗サイクル下で飼育された。動物室とケージ内を常に23℃に維持した。母ラットもまた高酸素化で受傷するため、代理母ラットを2日毎に交替させた。
移植に使用されたMuse細胞が、肺組織に生着したことを確認する実験を行った。まず、緑色蛍光タンパク質(GFP)を発現し、Muse細胞がこれにより標識されるように、予めレンチウイルス-GFP遺伝子をMuse細胞に導入した。GFPで標識されたMuse細胞をGFPとSSEA-3の二重の陽性細胞としてFACSにて分離した。その後、実施例2に記載した通りに、Muse細胞を投与した。
慢性肺疾患モデルラットの肺組織の評価を以下のように行った。上記の短期群と長期群の該モデルラットを安楽死させ、右心室から生理食塩水を注入し肺血管を灌流した後、気管カテーテルを介して4%パラフォルムアルデヒド水溶液で肺を膨張させた(20cmH2O、20分間)。肺を摘出し、4%パラフォルムアルデヒド溶液中で18~24時間(4℃)固定した後、各肺葉に切り分けた。切り分けた肺葉をエタノール水溶液、キシレンで脱水し、パラフィン包埋後、肺葉を厚さ5μmの切片とし、ヘマトキシリン-エオジン染色(HE染色)して標本を作製した。倒立顕微鏡(オリンパス社製、型番IX83)を使用し、顕微鏡ソフトウェア(Stereo Investigator)上で100のグリッド(×3カ所×6切片)を置き、それぞれのグリッド下が空間又は肺組織のいずれかであるかを確認した。100(計1,800)のグリッドのうち何%が肺組織であるかを評価した。通常、正常な肺組織では40%程度を占め、肺組織の損傷に伴って肺胞腔が大きくなる。図2に示されるように、短期群(日齢15)のラットにおいて、Muse細胞が投与された処置群(Muse群)及びHBSSが投与された処置群(ベヒクル群)は、正常状態のシャム群と比較して同程度であったが、長期群(日齢29)のラットでは、ベヒクル群と比較して、Muse群では有意な効果を示し、正常状態に近くに軽快することが示された。
肺胞壁割合(%)=肺胞壁面積÷{肺標本全体面積-(気管部位面積+血管部位面積+障害部位面積)}×100
この肺胞壁割合が高いほど、肺胞壁が密に形成され肺組織が修復されていることを表わす。結果を図4に示す。シャム群では、肺胞壁割合は約22~23%であり、慢性肺疾患モデルラットにMuse細胞を投与した群では、破壊された肺胞壁が21%前後までに回復した。また、非Muse細胞群においても回復の効果が見られたが、Muse細胞群ほどには至らなかった。このように、上記の肺組織評価から、肺Muse群では有意な効果を示し、正常状態に近くに軽快することが示された。
慢性肺疾患では、肺組織において各種炎症性サイトカインの発現が増大するが、Muse細胞の移植によりこれらの発現量を低減させ得るのかを検討した。日齢15のラットから肺組織を採取し、常法に従って総RNAを抽出後、炎症性サイトカインであるCCL2及びVEGFのmRNA発現量を処置群とベヒクル群で比較した。具体的な手法としては、肺組織を素早く取り出した容器にTRI試薬を添加し、Dounceホモゲナイザー、21G針付きシリンジにてホモゲナイズした。クロロホルムを添加後、遠心分離し、RNAを含む水層を得た。イソプロパノールにてRNAを沈殿させ、指摘濃度にて溶解させたRNAをSuperScript(登録商標)VILO(商標)cDNA Synthesis KitにてcDNAを合成した。その後、SYBR Greenを用いて、リアルタイム定量RT-PCRを行った。結果を図5に示す。縦軸は、シャム群の発現量を1とした時の各サイトカインのmRNAの相対的な発現量を示す。Muse細胞を投与した処置群(Muse群)は、HBSSが投与された処置群(ベヒクル群)と比較して、いずれの炎症性サイトカインのmRNA発現量を有意に低下させることができた。
Muse細胞による慢性肺疾患の治療効果として、ラットの心臓を用いた評価を行った。上記の短期群及び長期群のモデルラットから心臓を取り出し、右室壁(RV)と中隔+左室壁(IVS+LV)の2つに分離後、充分(60℃、48時間)に乾燥機にて乾燥させ、それぞれの重量を測定した。持続的な肺高血圧があると右室壁が肥厚(=重くなる)する。図6のシャム群は正常値であるが、慢性肺疾患(ベヒクル群)になると、0.4程度まで上昇した。これは、右室が肥厚したことを示す。これに対して、Muse細胞を投与した群(Muse群)では、ほぼ正常化することが分かった。
内壁率(%)={(血管外径-血管内径)/血管外径}×100
に従って算出し、結果を図8(B)に示す。この図に示される通り、短期群及び長期群はともに類似した結果であり、Muse細胞群では、肺高血圧に起因する肺動脈血管壁の肥厚を顕著に抑制することができた。また、非Muse細胞群においても抑制効果は観察されたが、Muse細胞群と比較してその効果は小さかった。
慢性肺疾患においては、炎症性細胞数の増加が観察されるが、Muse細胞の投与により、これらの細胞数が減少するか否かを検討した。短期群のラットを安楽死させた後、肺動脈より血管を生理食塩水で灌流し、挿管されている気管カニューレから生理食塩水0.4ml(0.2ml×2回)を注入して、肺胞洗浄(BAL)を行い、BAL液(BALF)を回収した。BALF中の白血球、マクロファージ、好中球、及びリンパ球の数を以下の手法により計測した。具体的には、気管支肺胞洗浄液10μLにTurk溶液20μLを加えて染色し、Burker-Turk血球計算盤を用いて総細胞数を測定した。次いで、Cytospin4(登録商標)を使用して、気管支肺胞洗浄液100μLの塗抹標本を作製し、作製した塗抹標本をMay-Giemsa染色で染色した。顕微鏡下に、1標本あたり少なくとも200個の細胞をカウントして白血球分画を測定し、マクロファージ数、好中球数、及びリンパ球数を算出した。結果を図10に示す。慢性肺疾患に伴い増加した各種炎症性細胞の数が、Muse細胞の投与により軽減した。この結果から、Muse細胞は、慢性肺疾患の治療において有意な効果を奏することが示された。
Claims (11)
- 生体の間葉系組織又は培養間葉系細胞から分離されたSSEA-3陽性の多能性幹細胞を含む、慢性肺疾患を改善及び/又は治療するための細胞製剤。
- 外部ストレス刺激によりSSEA-3陽性の多能性幹細胞が濃縮された細胞画分を含む、請求項1に記載の細胞製剤。
- 前記多能性幹細胞が、CD105陽性である、請求項1又は2に記載の細胞製剤。
- 前記多能性幹細胞が、CD117陰性及びCD146陰性である、請求項1~3のいずれか1項に記載の細胞製剤。
- 前記多能性幹細胞が、CD117陰性、CD146陰性、NG2陰性、CD34陰性、vWF陰性、及びCD271陰性である、請求項1~4のいずれか1項に記載の細胞製剤。
- 前記多能性幹細胞が、CD34陰性、CD117陰性、CD146陰性、CD271陰性、NG2陰性、vWF陰性、Sox10陰性、Snai1陰性、Slug陰性、Tyrp1陰性、及びDct陰性である、請求項1~5のいずれか1項に記載の細胞製剤。
- 前記多能性幹細胞が、以下の性質の全てを有する多能性幹細胞である、請求項1~6のいずれか1項に記載の細胞製剤:
(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
(iv)セルフリニューアル能を持つ。 - 慢性肺疾患が、気管支肺異形成症(BPD)、ウィルソン・ミキティ症候群(WMS)、新生児遷延性肺高血圧症(PPHN)、及び新生児高血圧症からなる群から選択される、請求項1~7のいずれか1項に記載の細胞製剤。
- 前記多能性幹細胞が肺組織に生着する能力を有する、請求項1~8のいずれか1項に記載の細胞製剤。
- ヒト新生児、乳児又は幼児対象に前記多能性幹細胞を治療上有効量として約1×104細胞/個体~約3×108細胞/個体で投与する、請求項1~9のいずれか1項に記載の細胞製剤。
- ヒト新生児、乳児又は幼児対象に前記多能性幹細胞を治療上有効量として、該対象一個体あたり約3×104細胞/kg~約3×107細胞/kgを体重換算した細胞量を投与する、請求項1~10のいずれか1項に記載の細胞製剤。
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