WO2022014681A1 - 運動ニューロン疾患(mnd)の治療に有効な多能性幹細胞 - Google Patents
運動ニューロン疾患(mnd)の治療に有効な多能性幹細胞 Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Definitions
- the present invention relates to a cell preparation for regenerative medicine. More specifically, the invention relates to cell formulations that are effective for the treatment, prevention, alleviation and / or delay of onset of motor neuron disease (MND) in a subject, including pluripotent stem cells.
- MND motor neuron disease
- ALS Amyotrophic lateral sclerosis
- SOD1 Cu / Zn superoxide dismutase
- TDP-43 TAR DNA-binding protein 43
- Non-Patent Documents 4 and 5 [ 4, 5]
- hexanucleotide repeated extension of the C9orf72 gene [Non-Patent Documents 6 and 7] [6, 7] have a genetic type associated with the mutation.
- Non-Patent Documents 8 and 9 [8, 9].
- the therapeutic effects of these treatments are still very limited and new therapeutic strategies for ALS are needed.
- Muse (Multilineage-diffrentiating stress ending) cells are endogenous pluripotent-like stem cells that can be recovered as stage-specific embryonic antigen (SSEA) -3 positive cells, which are pluripotent stem cell surface markers. These are usually localized in the connective tissues of bone marrow, peripheral blood and organs and are non-neoplastic [Non-Patent Documents 10-13] [10-13]. Muse cells are venous because they express sphingosine-1-phosphate receptors (S1PR2) that recognize damaged tissue and recognize sphingosine-1-phosphate (S1P) produced by damaged cells / apoptotic cells. It selectively accumulates at the site of injury by injection.
- S1PR2 sphingosine-1-phosphate receptors
- S1P sphingosine-1-phosphate
- Muse cells After homing to the site of injury, Muse cells can replace damaged / apoptotic cells by spontaneous differentiation into damaged / apoptotic cell types and contribute to tissue repair, including stroke, acute myocardial infarction, epidermal vesicular disease, chronic renal disease, etc. And animal models of liver cirrhosis have been reported [Non-Patent Documents 14-18] [14-18]. In addition to tissue remodeling, Muse cells have multifaceted effects such as angiogenesis, immunomodulation, nutrition, anti-apoptosis, and anti-fibrosis [Non-Patent Documents 18 and 19] [18, 19].
- Non-Patent Document 20 Muse cells can target injured tissue, the number of cells required for treatment is an order of magnitude less than the number of mesenchymal stem cells (MSCs) [Non-Patent Document 21] [21]. For these properties, we investigated the possible therapeutic potential of Muse cells for ALS animal models.
- the present invention provides a novel medical application using pluripotent stem cells (for example, Muse cells) in regenerative medicine. More specifically, the invention is a cellular formulation and / or pharmaceutical composition effective for treating, preventing, alleviating and / or delaying the onset of motor neuron disease (MND) in a subject, including Muse cells. It provides objects, as well as methods of using them to treat subjects with the above disorders.
- pluripotent stem cells for example, Muse cells
- MND motor neuron disease
- the present invention is as follows.
- Treat, prevent, alleviate and / or treat motor neuron disease (MND) in a subject including SSEA-3 positive pluripotent stem cells isolated from living mesenchymal tissue or cultured mesenchymal cells. Or a cell preparation to delay the onset.
- MND motor neuron disease
- the cell preparation according to the above [1] which comprises a cell fraction in which SSEA-3 positive pluripotent stem cells are concentrated by external stress stimulation.
- pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative, and CD271 negative.
- Pluripotent stem cells are CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snai1 negative, Slug negative, Tyrp1 negative, and Dct negative.
- the 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 of the three germ layers; (Iii) show no neoplastic growth; and (iv) have self-renewal ability.
- MND is amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), progressive muscular atrophy (PMA), or spinal and bulbar muscularis.
- SBMA atrophy
- Muse cells by administering Muse cells directly to an injured site or from a vein or the like to a subject suffering from motor neuron disease (MND), the Muse cells constitute normal tissue around the injured site.
- Motor neurons (MNDs) can be repaired by a tissue regeneration mechanism that differentiates into cells.
- A The distribution of GFP-labeled Muse cells in the spinal cord 7 days after intravenous (iv) or intrathecal (it) injection is shown.
- the dotted box on the panel indicates the high magnification of each panel. Notably, i. v. Only injection delivered a large number of GFP-labeled Muse cells to the pia mater and the lower white matter of both the cervical and lumbar spinal cords.
- B The number of GFP-labeled Muse cells is i. t. From injection i. v. It was expensive later.
- C Shows the distribution of nano-lantern-labeled human MSC and Muse cells in spinal cord, brain, muscle, lung, and leg bone 7 days after intravenous administration.
- Group C was compared with groups A and D ($$: p ⁇ 0.01 group D vs. C group, repeated measures ANOVA, factor: group).
- Body weight The graph shows changes in body weight [g] per group (B, C, and D) measured once a week over the entire dosing period. Each point represents the mean ⁇ SEM of all animals in each group and each week.
- Group C was compared with groups B and D ($$: p ⁇ 0.01 group D vs. C group, repeated measures ANOVA, factor: group).
- Hanging wire The graph shows the course of wire suspension time [seconds] per group (A, C, and D) measured after 1 week and every 2 weeks throughout the treatment period. Each point represents the mean ⁇ SEM of all animals in each group and each week.
- Group C was compared with groups A and D (&; p ⁇ 0.05 group C vs. A group, $$: p ⁇ 0.01 group D vs. C group, repeated measures ANOVA, factor: group).
- Hanging wire The graph shows the passage of time [seconds] from wire to fall per group (B, C, and D) measured at week 1 and is measured every 2 weeks over the entire treatment period. .. Each point represents the mean ⁇ SEM of all animals in each group and each week.
- Group C was compared with groups B and D ($$: p ⁇ 0.01 group D vs. C group, repeated measures ANOVA, factor: group).
- Rotorrod The graph represents the time [seconds] to average fall of each group at test 1, 2, 3, and baseline inspection and at week 12.
- Group C was compared with group D, and groups A to B were compared with group C (##: p ⁇ 0.01, t-test). The data are displayed as a bar graph of mean ⁇ standard error for all animals in each group.
- Clasping The graph represents the mean classification score [n] for each group at baseline and at week 12. Group C was compared with group D, and groups A to B were compared with group C (##: p ⁇ 0.01, t-test). The data are displayed as a bar graph of mean ⁇ standard error for all animals in each group.
- the present invention is a cell containing SSEA-3 positive pluripotent stem cells (eg, Muse cells) for treating, preventing, alleviating and / or delaying the onset of motor neuron disease (MND) in a subject.
- SSEA-3 positive pluripotent stem cells eg, Muse cells
- the present invention relates to a preparation and a pharmaceutical composition, and a method for treating MND using the cell preparation and the like.
- a detailed description of the present invention will be described.
- Ang1 Angiopoietin-1 ANOVA: ANOVA BW: Body weight BRET: Bioluminescence resonance EGF: Energy transfer of epidermal growth factor; FACS: Fluorescence-activated cell fractionation GFAP: Glia fibrous acidic protein GFP: Green fluorescent protein HBSS: Hanks equilibrium salt solution HE: Hematoxillin and eosin HGF: Hepatocyte growth factor Iba1: Ionized calcium binding adapter molecule 1 IGF-1: Insulin-like growth factor 1 i. t. : Intramedullary cavity i. v.
- Intravenous MAP2 Microtubule-associated protein 2 MSC: Mesenchymal stem cells NMJ: Neuromuscular junction NeuN: Neuron nucleus PGE2: Prostaglandin E2 SOD1: Superoxide dismutase S1P: Sphingosine-1-phosphate S1PR2: Sphingosine-1-phosphate receptor 2 TDP-43: TAR DNA binding protein 43 Tg: Transgenic VAChT: Vesicular acetylcholine transporter VEGF: Vascular endothelial growth factor WT: Wild type
- the present invention uses a cell preparation or a pharmaceutical composition containing SSEA-3 positive pluripotent stem cells (Muse cells) for amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS). ), Spinal muscular atrophy (SMA), progressive muscular atrophy (PMA), or motor neuron disease such as spinal and bulbar muscular atrophy (SBMA) (hereinafter, may be simply abbreviated as "MND”). It can be used for treatment, prevention, mitigation and / or delay of its onset.
- the present invention uses a cell preparation or pharmaceutical composition containing SSEA-3 positive pluripotent stem cells (Muse cells) to TDP-43 in frontotemporal lobar degeneration (FTLD) in nerve cells.
- FTLD frontotemporal lobar degeneration
- motor neuron disease refers to a neurological disorder that selectively destroys motor neurons.
- motor neuron disease also referred to as motor neuron degenerative disease
- ICD-10 International Structural Classification of Disease and Reserved Health Products
- WHO World Health Organization
- MND typically amyotrophic lateral sclerosis (including familial amyotrophic lateral sclerosis), primary muscular sclerosis, and spinal muscular atrophy (distal spinal muscular atrophy).
- Muscle atrophy familial spinal muscular atrophy, including scapulohumeral spinal muscular atrophy), progressive muscular atrophy (juvenile progressive muscular atrophy, pediatric progressive muscular atrophy, infant progressive (Spinal muscular atrophy, progressive spinal muscular atrophy, etc.), spinal muscular atrophy, Weldnig-Hoffmann disease, diffuse atrophic palsy, motor neuron disease, pseudosphere palsy, sphere palsy, juvenile unilateral upper limb muscle atrophy , Progressive bulbar palsy, traumatic bulbar palsy, cervical spondylotic muscular atrophy.
- MND is a neurological disorder that selectively destroys motor neurons, which can degenerate and lead to death.
- Motor neurons including primary (upper) and secondary (lower) motor neurons
- Primary motor neurons originate from the cerebral cortex, send fibers through the brainstem and spinal cord, and are involved in the control of secondary motor neurons.
- Secondary motor neurons are located in the brainstem and spinal cord and send fibers to muscles.
- Secondary motor neuron disease is a disease that involves degeneration of secondary motor neurons. When a secondary motor neuron degenerates, it usually cuts off the activating muscle fibers and ceases to contract, causing muscle weakness and hyporeflexia. Loss of either type of neuron leads to weakness, and muscular atrophy (muscle wasting) and painless weakness are clinical features of MND.
- ALS Amyotrophic lateral sclerosis
- SOD1 Cu / Zn superoxide dismutase 1 enzyme
- TDP-43 abnormal accumulation of phosphorylated or ubiquitinated TDP-43 in nerve cells is involved in the onset of ALS, and abnormalities in the gene encoding TDP-43 and the mechanism of TDP-43 degradation are known. Abnormalities have been reported and are found in some familial ALS (with mutations in the ALS10, TARDBP gene) and in about 90% of sporadic ALS. In the present specification, an ALS mouse model overexpressing TDP-43 was prepared, and the therapeutic efficacy of the cell preparation of the present invention was examined (see Example 2).
- PLS Primary lateral sclerosis
- FTLD frontotemporal lobar degeneration
- SMA Spinal muscular atrophy
- Type I severe type (also known as Weldnig-Hoffmann disease)
- type II intermediate type (also known as Dubowitz disease)
- type III mild type (also known as Kugelberg-Welander disease), which develops in childhood, in adulthood It is classified as type IV that develops.
- PMA Progressive muscular atrophy
- SBMA Spinal and bulbar muscular atrophy
- FTLD Frontotemporal lobar degeneration
- FTLD-TDP accumulation of TDP-43
- FTLD-TAU accumulation of TAU
- FTLD-FUS accumulation of FUS
- FTLD-others accumulation of other proteins
- FTLD-TDP accounts for about 45% of FTLD and is classified into types A to D based on histopathological characteristics.
- Pluripotent stem cells The pluripotent stem cell used in the cell preparation of the present invention is a cell that Izuzawa, one of the present inventors, discovered its existence in a human body and named it "Muse (Multipleage-differentiation stress ending) cell".
- Is. Muse cells are obtained from skin tissues such as bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)) and dermal connective tissue. It can also be scattered in the connective tissue of each organ.
- these cells are cells having the properties of both pluripotent stem cells and mesenchymal stem cells, and are, for example, the respective cell surface markers "SSEA-3 (Stage-specific embryonic antigen-3)" and " Identified as a double positive for "CD105". Therefore, a Muse cell or a cell population containing a Muse cell can be separated from a living tissue using these antigen markers as an index, for example. Details such as Muse cell separation methods, identification methods, and characteristics are disclosed in International Publication No. WO2011 / 007900. Also, as reported by Wakao et al. (S. Wakao, et al., Proc. Natl. Acad. Sci. USA, Vol.
- Muse cells can be simply purified and used using SSEA-3 as an antigen marker. ..
- SSEA-3 as an antigen marker, which can be used in a cell preparation (including a pharmaceutical composition) for treating a motor neuron disease, a living mesophyll tissue or a cultured mesenchymal tissue.
- a cell population containing pluripotent stem cells (Muse cells) or Muse cells isolated from the cells may be simply referred to as "SSEA-3 positive cells”.
- the “non-Muse cell” refers to a cell contained in a living mesenchymal tissue or a cultured mesenchymal tissue, and is a cell other than the “SSEA-3 positive cell”.
- a Muse cell or a cell population containing a Muse cell is a living tissue (eg, using an antibody against the cell surface marker SSEA-3 alone, or with both antibodies against SSEA-3 and CD105, respectively). , Can be separated from the mesenchymal tissue).
- the "living body” means a living body of a mammal. In the present invention, the living body does not include a fertilized egg or an embryo in the developmental stage before the blastogenic stage, but includes an embryo in the developmental stage after the blastogenic stage including a foetation or a blastoblast.
- Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits and guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets and the like. Be done.
- the Muse cells used in the cell preparation of the present invention are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are directly separated from living tissues with markers.
- ES cells embryonic stem cells
- iPS cells embryonic stem cells
- the "mesophageal tissue” refers to tissues existing in various organs such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermatitis, ligament, tendon, dental pulp, umbilical cord, and cord blood.
- Muse cells can be obtained from bone marrow, skin and adipose tissue. For example, it is preferable to collect mesenchymal tissue of a living body, separate Muse cells from this tissue, and utilize it. In addition, Muse cells may be separated from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells by using the above-mentioned separation means. In the cell preparation of the present invention, the Muse cell used may be autologous or allogeneic to the recipient receiving the cell transplant.
- Muse cells or cell populations containing Muse cells can be separated from living tissues using, for example, SSEA-3 positive and double positive of SSEA-3 and CD105 as indicators, but human adult skin. Is known to include various types of stem cells and progenitor cells. However, Muse cells are not the same as these cells.
- Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural ridge stem cells (NCSC), melanoblasts (MB), perivascular cells (PC), endothelial progenitor cells (EP), and adipose-derived stem cells (ADSC). ).
- SSEA-3 positive and double positive of SSEA-3 and CD105 as indicators, but human adult skin.
- Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural ridge stem cells (NCSC), melanoblasts (MB), perivascular cells (PC), endothelial progenitor cells (EP), and adipose-derived stem cells (ADSC).
- Muse cells include CD34 (marker of EP and ADSC), CD117 (c-kit) (marker of MB), CD146 (marker of PC and ADSC), CD271 (marker of NCFR), NG2 (PC marker), vWF factor (Fonville brand factor) (EP marker), Sox10 (NCSC marker), Sai1 (SKP marker), Slug (SKP marker), Typ1 (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 11 or 11 markers can be used as an index for separation.
- the non-expression of CD117 and CD146 can be separated as an index, and further, the non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be separated as an index, and further, the above-mentioned The non-expression of 11 markers can be separated as an index.
- the Muse cells having the above characteristics used in the cell preparation of the present invention are as follows: (I) Low or no telomerase activity; (Ii) Has the ability to differentiate into cells of any of the three germ layers; It may have at least one property selected from the group consisting of (iii) no neoplastic growth; and (iv) self-renewal ability.
- the Muse cells used in the cell preparation of the present invention have all the above-mentioned properties.
- "low or no telomerase activity” means that, for example, when telomerase activity is detected using TRAPEZE XL telomerase detection kit (Millipore), it is low or cannot be detected.
- telomerase activity means, for example, telomerase having the same level of telomerase activity as somatic human fibroblasts, or telomerase of 1/5 or less, preferably 1/10 or less of that of Hela cells. It means having activity.
- Muse cells have the ability to differentiate into three germ layers (endoderm lineage, mesodermal lineage, and ectodermal lineage) in vitro and in vivo, and are, for example, induced and cultured in vitro. Can differentiate into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, fat cells and the like. It may also show the ability to differentiate into three germ layers when transplanted in vivo into the testis.
- Muse cells proliferate at a growth rate of about 1.3 days in suspension culture, but grow from one cell in suspension culture to form embryo-like cell clusters and stop growing in about 14 days. However, when these embryo-like cell masses are brought into the adhesive culture, cell proliferation is started again, and the cells proliferated from the cell mass spread. Furthermore, when transplanted into the testis, it has the property of not becoming cancerous for at least half a year.
- Muse cells have a self-renewal (self-renewal) ability.
- self-renewal means that the differentiation of cells contained in embryo-like cell mass obtained by culturing from one Muse cell in suspension culture to trigermoid cells can be confirmed at the same time. By bringing the cells of the embryo-like cell mass into the suspension culture again with one cell, the next generation embryo-like cell mass is formed, and from there, the embryos in the three germ layer differentiation and suspension culture are again formed. It means that an adenomorphic cell mass can be confirmed. Self-renewal may be repeated one or more cycles.
- the cell fraction containing Muse cells used in the cell preparation of the present invention gives an external stress stimulus to the mesenchymal tissue or cultured mesenchymal cells of a living body, and cells other than cells resistant to the external stress.
- Cells enriched with SSEA-3 positive and CD105 positive pluripotent stem cells having at least one, preferably all of the following properties, obtained by methods involving killing cells and recovering surviving cells: It may be a fraction.
- Iii Low or no telomerase activity;
- Iv Has the ability to differentiate into three germ layers;
- V) show no neoplastic growth; and (vi) have self-renewal ability.
- the above external stresses are protease treatment, culture at low oxygen concentration, culture under low phosphoric acid condition, culture at low serum concentration, culture under low nutritional condition, culture under heat shock exposure, low temperature. Culturing in, freezing treatment, culturing in the presence of harmful substances, culturing in the presence of active oxygen, culturing under mechanical stimulation, culturing under shaking treatment, culturing under pressure treatment or physical impact. It may be any or a combination of two or more.
- the treatment time with the protease is preferably 0.5 to 36 hours in total in order to give external stress to the cells.
- the protease concentration may be any concentration used when peeling off the cells adhered to the culture vessel, breaking up the cell mass into a single cell, or recovering a single cell from the tissue.
- the protease is preferably serine protease, aspartic protease, cysteine protease, metal protease, glutamate protease or N-terminal threonine protease. Further, it is preferable that the protease is trypsin, collagenase or dispase.
- the cell preparation of the present invention is not limited to the Muse cells obtained in (1) above, or a cell population containing Muse cells in a physiological saline solution or an appropriate buffer solution (for example, phosphorus). Obtained by suspending in acid buffered saline). In this case, if the number of Muse cells separated from autologous or allogeneic tissues is small, the cells may be cultured before cell transplantation and proliferated until a predetermined cell concentration is obtained. As already reported (Pamphlet of International Publication No. WO2011 / 007900), Muse cells do not become tumors, so even if cells recovered from living tissues are contained in an undifferentiated state, they may become cancerous. Is low and safe.
- the culture of the recovered Muse cells is not particularly limited, but can be carried out in a normal growth medium (for example, ⁇ -minimum essential medium ( ⁇ -MEM) containing 10% calf serum). More specifically, with reference to the above-mentioned 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 having a predetermined concentration are selected. A solution containing the above can be prepared.
- a normal growth medium for example, ⁇ -minimum essential medium ( ⁇ -MEM) containing 10% calf serum.
- the cell preparation of the present invention When administering the cell preparation of the present invention to a human subject, it is effective to collect about several ml of bone marrow fluid from human intestinal bone and, for example, cultivate bone marrow mesenchymal stem cells as adherent cells from the bone marrow fluid.
- Muse cells After increasing the therapeutic amount of Muse cells until they reach a cell volume that can be separated, Muse cells can be separated using the antigen marker of SSEA-3 as an index, and autologous or allogeneic Muse cells can be prepared as a cell preparation.
- the cells are cultured and expanded until an effective therapeutic amount is reached, and then autologous or allogeneic Muse cells can be prepared as a cell preparation. can.
- DMSO dimethylsulfoxide
- serum albumin etc.
- antibiotics etc. are contained in the cell preparations to prevent bacterial contamination and proliferation. May be.
- other pharmaceutically acceptable ingredients eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
- the cell preparation may contain cells or components other than Muse cells contained in mesenchymal stem cells. Those skilled in the art can add these factors and agents to the cell preparation at appropriate concentrations.
- Muse cells can also be used as a pharmaceutical composition containing various additives.
- the number of Muse cells contained in the cell preparation or pharmaceutical composition prepared above is such that the desired effect in motor neuron disease (eg, increase in muscle strength, suppression of progression of muscle atrophy, etc.) can be obtained. It can be appropriately adjusted in consideration of gender, age, weight, condition of affected area, condition of cells to be used, and the like.
- the target individuals include, but are not limited to, mammals such as humans.
- the cell preparation of the present invention may be administered once, but may be administered multiple times (for example, 2 to 10 times or more) at appropriate intervals (for example, 1 day) until the desired therapeutic effect is obtained.
- the administration may be in the early, middle or late stage of onset as long as the therapeutic effect can be obtained.
- the therapeutically effective amount depends on the condition of the subject, but for example, 1 ⁇ 10 3 cells to 1 ⁇ 10 11 cells, preferably 1 ⁇ 10 4 cells to 1 ⁇ 10 10 cells per individual, and further. Preferred examples thereof include 1 ⁇ 10 5 cells to 1 ⁇ 10 9 cells.
- the cell preparation of the present invention is not particularly limited, but may be administered directly to the brain (brain stem, cerebral cortex) or spinal cord, or may be administered intravenously. Further, from Examples and the like, it is particularly preferable to administer intravenously.
- Human-derived Muse cells can be used for the cell preparations and pharmaceutical compositions of the present invention, but for subjects to which the subject is heterologous to the cells (eg, mice, rats), the heterologous cells are used.
- immunosuppressive agents such as cyclosporin
- an immunosuppressive agent may or may not be used in combination.
- the cell preparation and the pharmaceutical composition of the present invention are administered to a patient, it is particularly preferable not to use an immunosuppressant in combination.
- the cell formulations and pharmaceutical compositions of the invention can treat, prevent, alleviate and / or delay the onset of motor neuron disease (MND). More specifically, according to the present invention, it is possible to recover the muscle strength of the trunk and limbs caused by the motor neuron disease, or to suppress the progression of muscular atrophy.
- treatment means suppressing or completely eliminating various symptoms caused by MND.
- alleviation means alleviation of various symptoms caused by MND and suppression of progression, and preferably means alleviation of symptoms to the extent that they do not interfere with daily life. The term “alleviation” may also include the meaning of "delayed symptoms”.
- the cells of the present invention are obtained by measuring the motor ability of a mouse using an ALS model mutant SOD1 (G93A) Tg mouse or TDP-43 Tg mouse.
- the effectiveness of the formulation or pharmaceutical composition can be evaluated.
- the evaluation method may be, but is not limited to, a commonly used method such as a hanging wire test, a rotor rod, a clasping, a grip strength test, and a beam balance test.
- mice carrying the G93A human SOD1 mutation were obtained from Jackson Laboratories (Bar Harbor, ME, USA) [2] and were obtained from Tg males and C57BL. It was maintained as a hemi-joint by mating 6J females. Three animals in each group were used in the comparative experiment of intravenous (iv) injection and intrathecal (it.) Injection, and two animals were used in the exvivo imaging using nano-lantern. , Medium, MSC and Muse cells were used.
- GFP-labeled MSCs are prepared by labeling human bone marrow-MSCs (Lonza, Basel, Switzerland) with lentivirus GFP as previously reported [22]. did.
- GFP-labeled Muse cells as SSEA-3 positive cells were isolated from GFP-MSCs by fluorescence activated cell selection (FACS). The GFP-MSC and Muse cells were frozen in a Beissel freezing container (Nihon Freezer, Tokyo, Japan) and kept in liquid nitrogen until use.
- Nano-lantern labels with bioluminescence resonance energy transfer (BRET) potency are bright photoproteins that can be detected even in a small number of transplanted cells [25].
- Human bone marrow-MSCs (Lonza) were labeled with nano-lanterns [25] as previously reported [25].
- Nano-lantern-labeled Muse cells were isolated from nano-lantern-labeled MSCs as SSEA-3 positive cells by FACS. i. v.
- HBSS injection medium
- mice were euthanized when they were unable to stand up within 15 seconds of falling to one side and recorded as the time of death. Each animal was injected intraperitoneally with pentobarbital (20 mg / kg) for deep anesthesia and sampling as previously reported [26].
- the primary antibodies used were goat anti-GFP antibody (1: 500, Abcam, Cambridge, UK); rabbit anti-GFP antibody (1: 500, MBL, Woburn, USA); rabbit anti-Iba1 antibody (1: 500, Osaka, Japan).
- Mouse anti- ⁇ III tubulin (Tuji1) antibody (1: 100, Santa Cruz Biotechnology); Rabbit anti-glial fibrous acid protein (GFAP) antibody (1: 500, Dako, Glostrup, Denmark); Anti-NeuN antibody (1: 100, Millipore, MA, USA); goat anti-vesicular acetylcholine transporter (VAChT) antibody (1: 200, Thermo Scientific).
- the secondary antibody used was either Alexa 488 or 546 conjugated anti-goat, rabbit or mouse IgG (1: 500, Alexa Fluor TM, Invitrogen, Carlsbad, CA, USA).
- ⁇ -Bungarotoxin conjugated with Alexa 594 (1: 500, Millipore) was also used to detect the acetylcholine receptor.
- NMJs neuromuscular junctions
- Nano-lantern-labeled cells were used to investigate later in vivo kinetics of MSCs and Muse cells.
- MSC group on day 7, a strong signal was detected in the lungs and a trace signal was detected in the femur, but no signal was detected in other organs including the brain, cervical cord and lumbar spinal cord.
- Muse group signals were detected in the cervical and lumbar spinal cords (Fig. 1c, upper right), as well as in the lungs, while not in the brain (Fig. 1c, middle). The femoral signal was higher in the Muse group than in the MSC group.
- Immunohistological analysis confirmed the presence of nano-lantern-Muse cells in the pulmonary vascular lumen and bone marrow (Fig. 1d). This signal was consistently below the detection limit for all organs examined in the vehicle group (Fig. 1c).
- the average survival time was not significantly different between the 3 groups (medium; 144.4 ⁇ 8.0 days, MSC; 143.9 ⁇ 6.9 days, Muse cells; 142.6 ⁇ 6.7 days).
- there was no statistical difference in body weight between the three groups over the entire period (Fig. 2a).
- the rotarod test showed reduction in the Muse group on days 67 and 70, and statistical significance in the medium group (Fig. 2b).
- the hanging wire scores on days 84, 112, and 133, as well as the muscle strength of the lower extremities on days 126 and 140 were also significantly improved only in the Muse cell group compared to the vehicle group (FIGS. 2c, d). ).
- GFP-positive cells could not be detected in the medium group (Fig. 2e), but a small number of GFP-positive cells were observed in the MSC group (Fig. 2f).
- Muse cell population GFP-positive cells were found in the spinal cord soft membrane (Fig. 2 g, solid line box), lower white matter, and anterior horn (Fig. 2 g, dotted box, 2h), and 85.7% (201) of these cells. (180 of the GFP-positive cells) co-expressed the astrocyte marker GFAP located in both the soft membrane (FIG. 2i) and the anterior horn (FIG. 2j). The remaining GFP-positive cells (14.3%) were microglia marker Iba-1 (Fig.
- the number of survival motor neurons in the anterior horn was significantly lower in the media, MSC and Muse groups when compared to the wild-type (WT) group (WT, FIGS. 3a, b, * p ⁇ 0.05).
- WT wild-type
- FIGS. 3a, b, * p ⁇ 0.05 The number of survival motor neurons in the anterior horn was significantly lower in the media, MSC and Muse groups when compared to the wild-type (WT) group.
- WT wild-type
- FIGS. 3a, b, * p ⁇ 0.05 The number of survival motor neurons in the anterior horn was significantly lower in the media, MSC and Muse groups when compared to the wild-type (WT, FIGS. 3a, b, * p ⁇ 0.05.
- the Muse group had a larger number of motor neurons than the medium group and was statistically significant (FIGS. 3a, b, # p ⁇ 0.05). No statistical significance was found between the vehicle group and the MSC group (FIGS. 3
- ALS model mice i.e. in that Muse cells are delivered to the spinal cord, which is an important therapeutic target.
- the injection is i. t. It was superior to injection (Table 1, FIGS. 1a, b).
- i. v. The administration is easy to access, less invasive, and less burdensome to the patient. t. It has superior advantages over administration. This allows repeated administration of Muse cells to ALS patients.
- Muse cells expressing sphingosine-1-phosphate receptor 2 (S1PR2) are specific for the site of injury by sensing sphingosine-1-phosphate (S1P) produced by the injured cells. It has been shown that it can be homing to [18].
- Muse cells are i. v.
- the spinal cord of ALS mice can be reached by injection.
- Nano-lantern imaging showed a strong signal of Muse cells in the femur rather than the MSC (Fig. 1c). Bone marrow abnormalities have been reported in ALS [32]. Therefore, Muse cells selectively and actively accumulated in the femoral bone marrow and spinal cord in a lesion-dependent manner, in contrast to passive confinement in pulmonary capillaries. Another interesting point is i. v. The injected Muse cells migrated to the pia mater, lower white matter, and abdominal horn (FIGS. 2 g-j), suggesting that Muse cells home to the spinal cord via the pia mater perforated artery. On the other hand, i. t. The injected Muse cells were rarely detected in the spinal cord (FIGS. 1a, 1b). These results suggest that Muse cell homing factors are endocrine into the circulatory system rather than paracrine primarily into the spinal subarachnoid space [32-35].
- ALS In the animal disease model, i. v.
- the injected Muse cells spontaneously differentiated into tissue-constituting cells after homing specifically to the injured tissue; that is, in the stroke model, they differentiated into neurons and oligodendrocytes [16].
- ALS In contrast to stroke, ALS is chronic and progressive, and its pathology is quite different from stroke.
- the injected Muse cells differentiated predominantly into the astroglial lineage rather than the neuronal lineage in the spinal cord of ALS mice. Reactive astrocytes are important therapeutic targets for ALS [36].
- Muse cells include hepatocyte growth factor (HGF), intravascular growth factor (VEGF), insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), prostaglandin E2 (PGE2) and angio. It produces various neurotrophic factors such as poietin-1 (Ang1) [17-19]. Therefore, they may provide beneficial factors for motor neurons and astrocytes and prevent myofiber atrophy in the ALS model.
- HGF hepatocyte growth factor
- VEGF intravascular growth factor
- IGF-1 insulin-like growth factor 1
- EGF epidermal growth factor
- PGE2 prostaglandin E2
- angio angiogenesis
- Muse cells can be a promising cell resource for the treatment of ALS patients.
- ALS Amyotrophic lateral sclerosis
- TAR human transgenic response
- TDP-43 DNA-binding protein 43 kDa
- Muse cells were isolated and identified according to the method described in WO2011 / 007900. More specifically, Muse cells were obtained by expanded concentration culture of mesenchymal stem cells under stress conditions. Animals were dosed with 5 ml / kg of 2 ⁇ 10 6 viable cells / ml working fluid.
- Animal management 5-1 Animals were housed in individual ventilation cages using standard rodent bedding supplied by the breeding facility Rettenmaier. Up to 5 animals in the same group were housed in one cage. Room temperature was maintained at about 21 ° C. and relative humidity was maintained at 40-70%. Animals were bred with a constant light-dark cycle (12 hours / 12 hours). Animals were given free access to dry pelletized standard rodent feed (altromin) and regular tap water.
- the identified animals were numbered consecutively by the classical ear punch method. Each cage was identified by a color card indicating test number, gender, individual registration number (IRN), date of birth, genotyping date, and dosing group allocation.
- IRN individual registration number
- each animal was determined by a polymerase chain reaction specific for the transgenic construct. Prior to the start of the test, each mouse was genotyped using DNA isolated from ear tissue collected from ear punches for animal identification.
- mice were allowed access to food pellets and water at the bottom of the cage when animals showed severe motor dysfunction and could no longer reach food / water bottles. We also used a drinking bottle with a long cap.
- a reflection test and a modified Irwin test including hanging wires were performed once for all animals. Coordination hanging wires were evaluated biweekly during the test (6 times in total), rotor rods twice (at baseline and at the end of the test), and clasping behavior twice (at baseline and at the end of the test).
- 6-3 As a behavioral baseline test, a reflex test and a modified Irwin test including hanging wires were performed once on all animals. Coordination hanging wires were evaluated biweekly during the test (6 times in total), rotor rods twice (at baseline and at the end of the test), and clasping behavior twice (at baseline and at the end of the test). The test was conducted at random.
- the hanging wire test is a test for evaluating neuromuscular abnormalities in exercise intensity, and is performed during the test period (starting from the second week of administration) and as part of the Irwin test (as behavior at baseline). Conducted every other week.
- This test used a wire cage lid with duct tape around it to prevent the mouse from coming off the edge. The animal was placed on top of the cage lid. The lid was lightly shaken three times to force the mouse to grip the wire and then turned upside down. The lid was held at a height of about 55-60 cm above the soft underlay, which was high enough to prevent the mouse from jumping down, but not high enough to harm it if it fell. The incubation time until the fall was quantified. A cutoff time of 300 seconds was used. Normal mice may hang upside down for several minutes.
- Rotarod test The rotarod test was conducted twice at the start of the test (at baseline) and at the end of the test. In this test, motor coordination is evaluated by placing the animal on a rotating rod (4 lane rotor rod; Ugo Basile) traveling at a constant speed or acceleration speed. If the mouse loses balance and falls to the underlying platform, Red automatically stops and records fall latency measurements as well as fall speed.
- a rotating rod (4 lane rotor rod; Ugo Basile
- mice Prior to the first test session, the mice were acclimatized to the test system so that they could stay on the rod at a constant speed of 2 rpm for about 1 minute. During the test, one animal was exposed to the device three times for the test for 180 seconds. The initial speed increased from 2 rpm to 20 rpm with an acceleration time of 180 seconds. If the mouse fell, the session ended and the Ugo Basile Program stopped the timer.
- PB phosphate buffer
- the entire spinal column (n 8 per group) was fixed by immersing it in freshly prepared 4% paraformaldehyde in PB (pH 7.4) for 24 hours at 4 ° C. The next day, an incision was made from the fixed spinal column of the spinal cord and transferred to a 15% sucrose-PBS solution overnight for cryoprotection. Then, the tissue block was cut into 8 pieces, placed on a cryomold as a tissue array, and embedded in OCT medium. The samples were then snap-frozen in dry ice-cooled liquid isopentane and stored in an ultra-deep freezer (set to ⁇ 80 ° C. target temperature) until sectioning.
- Experiment II-MBP Iba1, MAP-2
- myelin basic protein (MBP) ionized calcium-associated adapter molecule 1 (Iba1) and microtubule-associated protein 2 (MAP-2) were added to five fragments (L2, L4, L6, L8 and L10) per mouse brain.
- MBP myelin basic protein
- Iba1 ionized calcium-associated adapter molecule 1
- MAP-2 microtubule-associated protein 2
- ⁇ Immune response area Percentage of ROI covered by objects above the threshold (eg, cell body cells); this is the most comprehensive parameter indicating whether there is an overall difference in immune response. be.
- ⁇ Object density [number of objects / mm 2 ]: The number of objects that exceed the threshold standardized for the size of the target area. This is particularly useful for detecting changes in the density of immunoreactive objects.
- ⁇ Object strength [a. u. ]: Average brightness of pixels of immunoreactive objects above the threshold; this indicates whether there is a difference in the cellular expression level of the target protein.
- ⁇ Object size [ ⁇ m 2 ]: Size of immunoreactive object above threshold; this is particularly useful for detecting changes in the size of immunoreactive object.
- ⁇ ROI strength [a. u. ]: Average brightness of pixels within the ROI; this indicates whether there is an overall difference in immunofluorescent signals.
- ⁇ GFP average object size [ ⁇ m 2 ], average object strength [a. u. ], Object density [n / mm 2 ], Immune active region [%]
- Iba1 Average object size [ ⁇ m 2 ], average object strength [a. u. ], Object density [n / mm 2 ], Immune active region [%]
- hTDP-43 average object size [ ⁇ m 2 ], average object strength [a. u. ], Object density [n / mm 2 ], Immune active region [%]
- MBP Average ROI [a. u.
- TDP-43 levels determined by Western blot were quantified and statistically evaluated in the spinal cord and hippocampus.
- TDP-43 / ⁇ -tubulin isotype III showed a significant increase in group C compared to group D.
- Groups A to B showed no significant difference compared to Group C (Fig. 20).
- Arai, T. et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 351, 602-611, doi: 10.1016 / j.bbrc .2006.10.093 (2006).
- Alpha1-syntrophin-deficient skeletal muscle exhibits hypertrophy and aberrant formation of neuromuscular junctions during regeneration. J Cell Biol 158, 1097-1107, doi: 10.1083 / jcb.200204076 (2002).
- Nagano, I. et al. Therapeutic benefit of intrathecal injection of insulin-like growth factor-1 in a mouse model of Amyotrophic Lateral Sclerosis. J Neurol Sci 235, 61-68, doi: 10.1016 / j.jns. 2005.04 .011 (2005).
- Glial cell line-derived neurotrophic factor protein prevents motor neuron loss of transgenic model mice for amyotrophic lateral sclerosis.
- Bossolasco P. et al. Metalloproteinase alterations in the bone marrow of ALS patients.
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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、対象における運動ニューロン疾患(MND)を治療し、予防し、軽減し及び/又は発症を遅延させるための細胞製剤。
[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]多能性幹細胞がCD105陽性である、上記[1]又は[2]記載の細胞製剤。
[4]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1つに記載の細胞製剤。
[5]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1つに記載の細胞製剤。
[6]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1つに記載の細胞製剤。
[7]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1つに記載の細胞製剤:
(i)テロメラーゼ活性が低いか又は無い;
(ii)三胚葉のいずれか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]MNDが、筋萎縮性側索硬化症(ALS)、原発性側索硬化症(PLS)、脊髄性筋萎縮症(SMA)、進行性筋萎縮症(PMA)、又は球脊髄性筋萎縮症(SBMA)である、上記[1]~[7]のいずれか1つに記載の細胞製剤。
[9]多能性幹細胞が脊髄内に生着する能力を有する、上記[1]~[8]のいずれか1つに記載の細胞製剤。
Ang1:アンジオポイエチン-1
ANOVA:分散分析
BW:体重
BRET:生物発光共鳴
EGF:上皮細胞増殖因子のエネルギー移動;
FACS:蛍光活性化細胞分別
GFAP:グリア線維性酸性タンパク質
GFP:緑色蛍光タンパク質
HBSS:ハンクス平衡塩類溶液
HE:ヘマトキシリン及びエオシン
HGF:肝細胞増殖因子
Iba1:イオン化カルシウム結合アダプター分子1
IGF-1:インスリン様増殖因子1
i.t.:髄腔内
i.v.:静脈内
MAP2:微小管関連タンパク質2
MSC:間葉系幹細胞
NMJ:神経筋接合部
NeuN:ニューロン核
PGE2:プロスタグランジンE2
SOD1:スーパーオキシドジスムターゼ
S1P:スフィンゴシン-1-リン酸
S1PR2:スフィンゴシン-1-リン酸受容体2
TDP-43:TAR DNA結合タンパク質43
Tg:トランスジェニック
VAChT:小胞アセチルコリントランスポーター
VEGF:血管内皮増殖因子
WT:野生型
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、筋萎縮性側索硬化症(ALS)、原発性側索硬化症(PLS)、脊髄性筋萎縮症(SMA)、進行性筋萎縮症(PMA)、又は球脊髄性筋萎縮症(SBMA)などの運動ニューロン疾患(以下、単に「MND」と略することがある)の治療、予防、軽減及び/又はその発症の遅延に使用することができる。
(1)多能性幹細胞(Muse細胞)
本発明の細胞製剤に使用される多能性幹細胞は、本発明者らの一人である出澤が、ヒト生体内にその存在を見出し、「Muse(Multilineage-differentiating stress enduring)細胞」と命名した細胞である。Muse細胞は、骨髄液、脂肪組織(Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17,2014))や真皮結合組織等の皮膚組織から得ることができ、各臓器の結合組織にも散在する。また、この細胞は、多能性幹細胞と間葉系幹細胞の両方の性質を有する細胞であり、例えば、それぞれの細胞表面マーカーである「SSEA-3(Stage-specific embryonic antigen-3)」と「CD105」のダブル陽性として同定される。したがって、Muse細胞又はMuse細胞を含む細胞集団は、例えば、これらの抗原マーカーを指標として生体組織から分離することができる。Muse細胞の分離法、同定法、及び特徴などの詳細は、国際公開第WO2011/007900号に開示されている。また、Wakaoら(S. Wakao, et al., Proc. Natl. Acad. Sci. USA, Vol. 108, p.9875-9880 (2011))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においては、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、運動ニューロン疾患を治療するための細胞製剤(医薬組成物を含む)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)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、細胞移植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号パンフレットを参照して、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、運動ニューロン疾患(MND)の治療し、予防し、軽減し及び/又は発症を遅延させることができる。より具体的には、本発明により、運動ニューロン疾患に起因する体幹や四肢の筋力を回復し、又は筋萎縮の進行を抑制することができる。ここで、「治療」とは、MNDに起因する各種の症状を抑制すること又は完全に消失させることをいう。また、「軽減」とは、MNDに起因する各種の症状の緩和及び進行の抑制を意味し、好ましくは、日常生活に差し支えない程度にまでに症状を緩和することを意味する。この「軽減」なる用語には、「症状の遅延」の意味も含み得る。
動物及び実験群
G93AヒトSOD1変異(G1H/+)を有するトランスジェニック(Tg)マウスをJackson Laboratories(Bar Harbor、ME、USA)[2]から入手し、Tg雄とC57BL/6J雌を交配することによりヘミ接合体として維持した。静脈内(i.v.)注射と髄腔内(i.t.)注射の比較実験では、各群3匹を使用し、ナノ・ランタンを用いたエクスビボイメージングでは、2匹の動物を使用し、媒体、MSC及びMuse細胞を使用した。静脈内注射による処置効果の評価には、媒体群で10匹(雄5匹及び雌5匹)、MSC群で9匹(雄4匹及び雌5匹)、Muse細胞群で9匹(雄5匹及び雌4匹)を用いた。
GFP標識されたMSCは、以前に報告されているように[22]、ヒト骨髄-MSC(Lonza、Basel、Switzerland)をレンチウイルスGFPで標識することによって調製した。報告されるように[10、11]、蛍光活性化細胞選別(FACS)によりGFP-MSCからSSEA-3陽性細胞としてGFP標識されたMuse細胞を単離した。上記GFP-MSC及びMuse細胞を、Beissel凍結コンテナ(Nihon Freezer、Tokyo、Japan)中で凍結し、使用するまで液体窒素中に保持した。
250μlのHank平衡塩類溶液(HBSS、pH7.4)中の上記GFP標識されたMuse細胞(2.0×105個)を、以前に報告されるように[23、24]、i.v.用の尾静脈、又は髄腔内注射用の大槽に注入した。7日目に、全ての動物を屠殺した。
生物発光共鳴エネルギー移動(BRET)効力を有するナノ・ランタン標識は、少数の移植細胞でさえも検出が可能な明るい発光タンパク質である[25]。ヒト骨髄-MSC(Lonza)を、以前に報告されるように[25]、ナノ・ランタンで標識した[25]。ナノ・ランタン標識されたMuse細胞をFACSによりSSEA-3陽性細胞としてナノ・ランタン標識されたMSCから単離した。i.v.注射のエクスビボ動態のために、媒体(HBSS)、ナノ・ランタン標識されたMSC及びMuse細胞(1.0×105細胞/250μl)を14週齢で静脈内注射した。7日後、動物を深麻酔下で屠殺し、以前に報告されるように分析した[26]。
溶媒、GFP-MSC及びGFP-Muse細胞(両方とも5.0×104細胞/250μl)を各動物の尾静脈に1分間にわたって注射した。本発明者ら及び他のグループは、G93A Tgマウスが約56日齢で早期発症を示すことが報告されている[27]ことを確認したため、本発明者らは56日齢で細胞投与を開始し、同数の細胞を用いて119日目(計10回の注射)まで週1回投与(計10回の注射)を継続することを決定した。免疫抑制剤であるシクロスポリンA(10mg/kg/日、Novartis International、Basel、Switzerland)もまた、媒体、MSC、又はMuse細胞群での投与直後に、屠殺まで隔日に全ての動物に腹腔内(i.p.)投与された。生存を毎日チェックし、体重(BW)とロータロッドスコアを56日齢から週2回測定した。本発明者らの以前の方法[24、28]に従って、ロータロッド試験及び車輪走行活動を行った。ハンギングワイヤ試験は、以前に報告されるように、週1回、筋力及び協調運動の測定として評価された[29]。精密ばねスケール(Ooba Keiki Co.、Tokyo、Japan)を用いて、下肢の筋力を週1回測定した。
マウスが片側に倒れてから15秒以内に立ち上がれない状態になった時点で安楽死させ、死亡時点として記録した。各動物にペントバルビタール(20mg/kg)を腹腔内注射して深麻酔し、既報のようにサンプリングを行った[26]。使用した一次抗体は、ヤギ抗GFP抗体(1:500、Abcam、Cambridge、UK);ウサギ抗GFP抗体(1:500、MBL、Woburn、USA);ウサギ抗Iba1抗体(1:500、Osaka、Japan);マウス鵜抗βIIIチューブリン(Tuj1)抗体(1:100、Santa Cruz Biotechnology);ウサギ抗グリア線維酸性タンパク質(GFAP)抗体(1:500、Dako、Glostrup、Denmark);抗NeuN抗体(1:100、Millipore、MA、USA);ヤギ抗小胞アセチルコリントランスポーター(VAChT)抗体(1:200、Thermo Scienctific)であった。使用した二次抗体は、Alexa 488又は546でコンジュゲートした抗ヤギ、ウサギ又はマウスIgG(1:500、Alexa Fluor(商標)、Invitrogen、Carlsbad、CA、USA)のいずれかであった。Alexa 594(1:500、Millipore)でコンジュゲートしたα-ブンガロトキシンもまた、アセチルコリン受容体を検出するために使用された。
研究者は、データ収集と分析の間、実験群に対して盲検化された。本研究の所見を裏付けるデータは、合理的な要請があれば、担当者から入手することができる。データは、SPSSバージョン22.0.0.0(IBM Corp.、Armonk、New York、USA)で分析し、平均値±SDで表した。治療効果は、非反復測定の分散分析(ANOVA)及びDunnett’t検定によって評価された。免疫組織学的データをKruskall-Wallisを用いて分析し、続いてBonferroni補正を伴うMann-Whitney U検定を行った。すべての統計解析において、有意性はp<0.05であるとみなした。
投与経路を決定するために、GFP-Muse細胞のホーミングを7日目にG93Aマウス脊髄の免疫組織学的分析によりi.v.注射とi.t.注射で比較した。予備研究は、GFP-Muse細胞数がi.t.注射群では頸部、胸部及び腰髄で一貫して低く又は無視することができたが、一方、i.v.注射群ではi.t.注射群と比較して頚部及び腰髄で高く、GFP-Muse細胞は主に軟膜及び下部白質に位置することが示された。GFP-Muse細胞は、i.v.注射によっても胸髄ではほとんど検出されなかった(表1、図1a、b)。したがって、以下の実験では、i.v.注射を投与経路として選択した。
本研究は、ALSモデルG93A Tgマウスにおいて、Muse細胞の多重静脈内投与がロータロッド、ハンギングワイヤ及び下肢の筋力を改善することを示した最初の報告である。i.v.注射したMuse細胞は、腰髄、肺及び骨にホーミングした(図1c、d)。腰髄では、GFP陽性Muse細胞は、主にアストログリアマーカーGFAPを発現し、末期にグリア様形態を示した(154日齢、図2g~j)。さらに、腰髄における生存運動ニューロンの数は、媒体群に比べて統計的有意に多かった(図3a、b)。これは、下肢筋肉の除神経と筋線維萎縮の両方の軽減をもたらした可能性がある(図3c~f)。
本研究の目的は、TDP-43 Tgマウスの運動機能に対するMuse細胞の効果を評価することである。
筋萎縮性側索硬化症(ALS)は、脳と脊髄の運動ニューロンの変性を特徴とする神経変性疾患である。ヒトトランスアクティブ応答(TAR)DNA結合タンパク質43kDa(TDP-43)と呼ばれるユビキチン化タンパク質がALSの病因において中心的役割を果たすという証拠がある。TDP-43 Tgマウスは、TDP-43産生及び細胞内沈着を研究するための適切なモデルである。ALSの臨床的特徴を模倣したこのモデルは、ALSに対する薬物の効果を試験するのに有用である。提案された研究では、ヒト野生型TDP-43(hTDP-43)を過剰発現し、ニューロンマウスThy-1プロモーターの制御下にあるマウスを使用する。ホモ接合マウスでは、ヒトTDP-43タンパク質は、非トランスジェニック同腹仔における内因性タンパク質よりも3.8倍高い。
WO2011/007900に記載されている方法に従って、ヒトMuse細胞の単離及び同定を行った。より具体的には、ストレス条件下での間葉系幹細胞の拡大濃縮培養によって、Muse細胞を得た。動物には、2×106生存細胞/ml作用液の5ml/kgを投与した。
ハンクス平衡塩類溶液、カルシウム不含、マグネシウム不含、フェノールレッド不含(HBSS;Gibco 14175-046)を媒体として使用した。
5-1.飼育設備
Rettenmaierから供給された標準的なげっ歯類用床敷を用いて、個別換気ケージに動物を収容した。同群の最大5匹を1ケージに収容した。室温を約21℃に維持し、相対湿度を40~70%に維持した。動物は一定の明暗周期(12時間/12時間)で飼育した。動物には、乾燥したペレット状の標準げっ歯類用飼料(アルトロミン)と、通常の水道水を自由に摂取させた。
最初の処置の開始時から、マウスイグルー及びFAST Trac(PLEXX由来)を環境エンリッチメントとして用いて、全ての動物を飼育した。
動物には古典的なイヤーパンチ法により連続的に番号を付けた。各ケージは、試験番号、性別、個体登録番号(IRN)、生年月日、遺伝子型決定日、投与群割り付けを示すカラーカードで識別した。
健康状態が明らかに良好な動物のみを対象とした。全ての動物を(全群及びコホートの平均体重及び性別が十分に分布していることを考慮して)全投与群の動物からなるコホートに無作為に割り付けた。最初のコホートの動物数は、同年齢及び均一な取扱いを確保するために制限した。
試験に割り付ける前に、各個体の健康状態を評価した。試験期間中、ケージ外側からの特筆すべき観察結果を記録し、その後の処置(安楽死など)を決定した。
早期に死亡した動物(IRN 5921)を剖検した。死亡率は、これ以外の安楽処置動物、又は早期に死亡した動物がいなかったため評価しなかった。
15匹ずつ3群に分けた45匹のホモ接合TDP-43 Tgマウス、及び8週齢の15匹の非トランスジェニック同腹仔群を本研究に用いた。動物には、Muse細胞又は媒体のいずれかを静脈内(i.v.)投与により2回投与した。
本研究では、15匹ずつ3群に分けた45匹のホモ接合TDP-43 Tgマウスと、8週齢の15匹の非トランスジェニック同腹仔群を非疾患対照として用いた。12週間の試験では、各動物に、Muse細胞と媒体、媒体とMuse細胞、又は媒体と媒体のいずれかをそれぞれ1週目及び8週目に2回静脈内(i.v.)注射した。すなわち、Muse細胞は第1週(A群)又は第8週(B群)のいずれかで1回のみ投与された。以下の群を用いた。
ベースライン試験として、反射試験及びハンギングワイヤを含む改変Irwin試験を全ての動物に1回実施した。運動協調性ハンギングワイヤを試験期間中(計6回)、ロータロッドを2回(ベースライン時及び試験終了時)、クラスピング行動を2回(ベースライン時及び試験終了時)隔週で評価した。試験は無作為に実施した。
ハンギングワイヤ試験は、運動強度の神経筋異常を評価する試験であり、試験期間中(投与2週目から開始する)及びIrwin試験の一環として(ベースライン時の行動として)隔週に実施した。この試験では、マウスがエッジから外れるのを防ぐために、ダクトテープを周囲に配置したワイヤケージ蓋を使用した。動物をケージ蓋の上部に置いた。蓋を3回軽く振り、マウスに強制的にワイヤを握らせた後、上下反転させた。蓋は、マウスが飛び降りるのを防ぐのに十分な高さであるが、落ちた場合に危害を与えるほどの高さではない、柔らかい下敷きの上方約55~60cmの高さに保持した。落下までの潜伏時間を定量化した。300秒のカットオフ時間を使用した。正常なマウスは数分間、逆さまの状態でぶら下がることがある。
試験開始時(ベースライン時)及び試験終了時にロータロッド試験を2回実施した。この試験では、動物を一定速度又は加速速度で走行する回転ロッド(4レーンのロータロッド;Ugo Basile)上に置くことによって運動協調性を評価する。マウスがバランスを失い、下にあるプラットフォームに落下した場合、赤は自動的に停止し、落下速度と同様に落下潜時の測定値を記録した。
クラスピング行動は試験開始時(ベースライン)及び試験終了時に2回評価した。
4点:マウスを尾で懸垂させたときに、外側正中線から離れる方向に後肢が完全に伸長する状態になりこれを2秒間保持できる。数秒間、2~3回懸垂したとき、マウスは懸垂中に少なくとも2回、両後肢で動きを示す。
3点:尾懸垂時に、外側正中線に向かっての脚伸長が屈折するか若しくは部分的に屈折する状態、又は後肢が震える状態。
2点:30cm(12インチ)の歩行中に少なくとも2回、つま先が下方に曲がるか、又は足のいずれかの部分をケージの底部/テーブルに沿って引きずっている状態。
1点:硬質麻痺又は最小限の関節の動きであり、足が前進運動に使用されていない。
0点:マウスは、片側に倒れて、(15~)30秒以内に起き上がることができない。
試験終了時、全動物をペントバルビタール注射(600mg/kg)で安楽死させ、半脳及び脊髄を採取した。
マウスを経心臓的に0.9%生理食塩水で潅流した。この目的のために、0.9%生理食塩水ボトルに接続した23ゲージ針を左心室に挿入した。右心室をハサミで開放した。溶液ボトルを圧力計制御空気圧縮機に接続することにより、潅流溶液上に100~120mm Hgの一定圧力を維持した。灌流は、肝臓が青白くなり、血液の代わりに灌流液のみが右心室から出るまで継続した。
潅流後、頭蓋骨を開き、その後、脳を慎重に除去し、冷却した表面上で片側を切断した。右半脳を、室温で2時間、新しく調製した4%パラホルムアルデヒド/PB(pH=7.4)に浸漬することにより固定した。左半脳をさらに海馬とそれ以外の部分に分けた。全ての部品を秤量し、ドライアイス上でスナップ凍結し、-80℃で保存した。
免疫組織学的検査(群あたりn=8)
免疫組織学的分析のために、脊柱を動物から切開し、後に浸漬固定を容易にするために筋肉組織の多くを除去した。頸部、胸部及び腰部を含む脊柱全体を、新たに調製した4%パラホルムアルデヒド/PBを含む15mlのチューブに、固定のために直立位置で移した。脊柱を4℃で24時間固定した。
ウエスタンブロット法によるタンパク質発現解析のために、脊柱を動物から切開し、脊髄を除去し、秤量し、ドライアイス上で凍結し、-80℃で保存した。
(1)組織の調製
右半脳(群あたりn=15)を室温で2時間、リン酸緩衝液(PB;pH7.4)中に新しく調製した4%パラホルムアルデヒド中に浸漬することにより固定した。次に、凍結保護のために、試料を15%ショ糖-PBS溶液に一晩かけて移した。その後、組織ブロックを必要に応じてトリミングし、クライオモールドに移し、OCT培地に包埋し、次に、ドライアイス冷却液体イソペンタン中でスナップ凍結し、切片化するまで超深冷凍庫(-80℃目標温度に設定)に保存した。
右半脳(群あたりn=8)
12の中外側レベルからの5つの凍結切片(合計60切片)を、Leicaクライオトーム上で10μm厚で矢状に切断した。次の23切片をレベルごとに廃棄した。Paxinos and Franklin(「The Mouse Brain in Stereotaxic Coordinates」(第2版)2001年)に従って切片化レベルを選択した。切片の採取は、標的領域を通した系統的なランダムサンプリングを確実にするために、正中線から外側約0.2mmのレベルで開始し、半球を通して行った(図4)。ディープフリーザー(目標温度設定-20℃)に切片を保管した。
脊髄組織アレイからの5つの凍結切片をLeicaクライオトーム上で10μm厚で切断した。次の15切片を廃棄した。この回収スキームを5レベル(計30切片)で繰り返した。その結果、頸部、胸部、腰部、仙骨の各部位から切片を採取し、脊髄を通して系統的に無作為にサンプリングすることができた(図5)。ディープフリーザー(目標温度設定-20℃)に切片を保管した。
実験I-GFAP、NeuN、及びhTDP-43
実験Iでは、グリア線維性酸性タンパク質(GFAP)、ニューロン核(NeuN)及びhTDP-43をマウス脳(L2、L4、L6、L8及びL10の1切片;合計n=160切片)あたり5切片の均一系統的ランダムセット、及び脊髄組織アレイのレベル3の1切片(合計n=32切片)で評価した。
実験IIでは、ミエリン塩基性タンパク質(MBP)、イオン化カルシウム結合アダプター分子1(Iba1)及び微小管関連タンパク質2(MAP-2)をマウス脳あたり5つの断片(L2、L4、L6、L8及びL10の1断片;合計n=160断片)の均一系統的無作為セット、及び脊髄組織アレイのレベル3の1断片(合計n=32断片)で評価した。
免疫蛍光染色切片の全スライドスキャンをZeiss Axiocam 506モノ及び日立3CCD HV-F202SCLカメラ及びZeiss ZEN 2.3ソフトウェアを備えた、高開口レンズを有するZeiss自動顕微鏡AxioScan Z1上に記録した。
●GFAP、
●Iba1、
●hTDP-43、
●MBP、
●MAP2、
●NeuN
●免疫反応領域[%]:閾値以上の物体(例えば、細胞体細胞)によってカバーされるROIのパーセンテージ;これは、免疫反応性に全体的な差があるかどうかを示す最も包括的なパラメータである。
●物体密度[物体の数/mm2]:標的領域の大きさに規格化された閾値を超える物体の数。これは、免疫反応性物体の密度の変化を検出するのに特に有効である。
●物体強度[a.u.]:閾値を超える免疫反応性物体の画素の平均輝度;これは標的タンパク質の細胞発現レベルに差があるかどうかを示す。
●物体の大きさ[μm2]:閾値以上の免疫反応性物体の大きさ;これは免疫反応性物体の大きさの変化を検出するのに特に役立つ。
●ROI強度[a.u.]:ROI内の画素の平均輝度;これは、免疫蛍光シグナルに全体的な差があるかどうかを示す。
標的物体のパラメータがテスト回で定義されると、定量的画像解析は自動的に実行され、結果はオペレータに依存せず、完全に再現可能である。
●GFAP:平均物体サイズ[μm2]、平均物体強度[a.u.]、物体密度[n/mm2]、免疫活性領域[%]
●Iba1:平均物体サイズ[μm2]、平均物体強度[a.u.]、物体密度[n/mm2]、免疫活性領域[%]
●hTDP-43:平均物体サイズ[μm2]、平均物体強度[a.u.]、物体密度[n/mm2]、免疫活性領域[%]
●MBP:ROIの平均値[a.u.]、免疫反応領域[%]
●MAP2:ROIの平均値[a.u.]、免疫反応領域[%]
●NeuN:平均物体サイズ[μm2]、平均物体強度[a.u.]、物体密度[n/mm2]、免疫活性領域[%]
(1)試料調製
凍結された固定されていない組織試料(群あたり15匹の全動物の海馬及び群あたり7匹の脊髄)を重量あたり5容量のRIPA緩衝液(50mMのTris-HCl、pH7.4、1mMのEDTA、150mMのNaCl、1%NP-40又は0.1%Triton-X、2%SDS、1μMのNaF、0.2mMデオキシコール酸ナトリウム、80μMグリセロリン酸1×プロテアーゼ阻害剤(Calbiochem、Germany)、1×ホスファターゼ阻害剤)中でホモジナイズした。タンパク質濃度をBCAアッセイにより測定した。ホモジネートをさらに使用するまで-80℃で保存した。
各試料の等量のタンパク質を、SDS-PAGE(ドデシル硫酸ナトリウム-ポリアクリルアミドゲル電気泳動;例えば、Any kD(商標)Mini-PROTEAN(登録商標)TGX(商標)プレキャストタンパク質ゲル、BioRad)を介して分子量によって分離した。各ゲル上の予め染色されたタンパク質マーカーは、タンパク質の正しい分離を可視化し、後にウエスタンブロットフィルム上で正しいタンパク質バンドサイズを確認した。その後、タンパク質をニトロセルロース膜上に移し、1×TBS(トリス緩衝生理食塩水)中の5%非脂肪乾燥ミルクでブロックし、ヒト-TDP-43抗体(1:1000、Abnova、Germany、H00023435-M01)及びβ-チューブリンアイソタイプIII(1:5000、Sigma-Aldrich、USA、T8660)を用いて特異的抗体とインキュベートした。一次抗体、ECL(増強化学ルミネセンス)及びC-digitブロットスキャナー(Licor)の種に対して生じたHPR(西洋ワサビペルオキシダーゼ)結合二次抗体を所望のタンパク質バンドの可視化及び半定量化のために使用した。
7-1.体重
全実験期間にわたって群を比較することにより統計解析を行い、体重に対するMuse細胞の影響を評価した。反復測定ANOVAを行い、群間因子の有意水準を評価した。結果を図6及び7に示した。
(1)D群とC群、及びC群とA群の比較
1~12週目でC群はD群と比較して有意差を示し、A群はC群と比較して有意差を示さなかった。
(2)D群とC群、及びC群とB群の比較
1~7週目、及び8~12週目で、C群はD群と比較して有意差を示し、B群は各期間においてC群と比較して有意差を示さなかった。
ハンギングワイヤ試験におけるMuse細胞の効果を評価するために、全実験期間にわたって群を比較することにより統計解析を行った。反復測定ANOVAを行い、群間因子の有意水準を評価した。結果を図8及び9に示した。
(1)D群とC群、及びC群とA群の比較
0~12週目でC群はD群と比較して有意差が認められ、A群はC群と比較して有意差が認められた。
(2)D群とC群、及びC群とB群の比較
0~6週目、及び第8~12週目で、C群はD群と比較して有意差を示し、B群は各期間においてC群と比較して有意差を示さなかった。
結果を図10に示した。試験1~3及びベースライン時及び12週目の平均値では、C群はD群と比較して有意な低下を示し、A及びB群はC群と比較して有意差を示さなかった。
結果を図11に示した。ベースライン時及び12週目に、C群はD群と比較して有意な低下を示し、A及びB群はC群と比較して有意差を示さなかった。
免疫蛍光シグナルを脊髄(頸部、胸部及び腰部)で定量し、統計学的に評価した。
結果を図12に示した。
(1)D群とC群の比較
頸部及び胸部の平均物体強度、物体密度、免疫反応領域、全腰部の読み出しにおいて、C群はD群と比較して有意な増加を示し、2元配置ANOVAの結果、因子:群間に有意差を示した。
(2)C群とA~B群の比較
A群は、胸部及び腰部の平均物体強度、免疫反応領域において、C群と比較して有意な減少を示したが、2元配置ANOVAの結果、平均物体強度、物体密度、免疫反応領域において、因子:群間に有意差が認められたことから、Dunnett検定を実施したところ、A~B群はC群と比較して有意差が認められた。
結果を図13に示した。
(1)D群とC群の比較
頸部の平均物体サイズ、物体密度及び免疫反応領域、ならびに胸部及び腰部の平均物体強度、物体密度及び免疫反応領域において、C群はD群と比較して有意な増加を示した。2元配置ANOVAの結果として、全ての読み出しにおいて、因子:群は有意差を示した。
(2)C群とA~B群の比較
頸部の平均物体サイズにおいて、A~B群はC群と比較して有意な減少を示したが、2元配置ANOVAの結果として、平均物体サイズにおいて、因子:群は有意差を示した。したがって、Dunnett検定を行ったところ、A~B群はC群と比較して有意差を示した。
結果を図14に示した。
(1)D群とC群の比較
胸部の免疫反応領域において、C群はD群と比較して有意な増加を示した。2元配置ANOVAの結果として、平均ROI強度及び免疫反応領域において、因子:群は有意差を示した。
(2)C群とA~B群の比較
全ての読み出しにおいて、A~B群はC群と比較して有意差を示さなかったが、2元配置ANOVAの結果として、ROI強度の平均値と免疫反応領域において、因子:群は有意差を示した。したがって、Dunnett検定を行ったところ、平均ROI強度において、A~B群はC群と比較して有意差を示し、免疫反応領域おいて、A群はC群と比較して有意差を示した。
結果を図15に示した。
(1)D群とC群の比較
頸部の平均物体サイズ、胸部の物体密度、並びに腰部の物体密度及び免疫反応領域において、C群はD群と比較して有意な増加を示した。2元配置ANOVAの結果として、全ての読み出しにおいて、因子:群は有意差を示した。
(2)C群とA~B群の比較
全ての読み出しにおいて、A~B群はC群と比較して有意差を示さなかった。2元配置ANOVAの結果として、平均物体強度において、因子:群は有意差を示した。したがって、Dunnett検定を行ったところ、A群はC群と比較して有意差を示した。
頚部の物体密度において、C群はD群と比較して有意な増加を示した。2元配置ANOVAの結果として、物体密度において、因子:群は有意差を示した。A群及びB群は、これらの変化において何ら有意な影響を示さなかった(データを示さず)。
全ての読み出しにおいて、C群はD群と比較して有意差を示さなかった。2元配置ANOVAの結果として、因子:群は有意差を示さなかった(データを示さず)。
免疫蛍光シグナルを脳(大脳皮質及び脳幹)において定量化し、統計学的に評価した。
結果を図16に示した。脳幹及び皮質の全ての読み取りにおいて、C群はD群と比較して有意な増加を示した。A~B群はC群と比較して有意差を示さなかった。
結果を図17に示した。脳幹の物体密度及び免疫反応領域、ならびに大脳皮質の全ての読み取りにおいて、C群はD群と比較して有意な増加を示した。A~B群はC群と比較して有意差を示さなかった。
脳幹及び皮質の全ての読み取りにおいて、C群はD群と比較して有意差を示さなかった(データを示さず)。
結果を図18に示した。脳幹の全ての読み取り、及び皮質の平均物体強度において、C群はD群と比較して有意な増加を示した。A~B群はC群と比較して有意差を示さなかった。
結果を図19に示した。脳幹の平均物体サイズにおいて、C群はD群と比較して有意な減少を示し、皮質の物体密度において、C群はD群と比較して有意な増加を示した。全ての読み取りにおいて、A~B群はC群と比較して有意差を示さなかった。
脳幹及び皮質の全ての読み取りにおいて、C群はD群と比較して有意差を示さなかった(データを示さず)。
ウエスタンブロットにより決定したTDP-43レベルを脊髄及び海馬において定量し、統計学的に評価した。脊髄及び海馬において、TDP-43/β-チューブリンアイソタイプIIIについて、C群はD群と比較して有意な増加を示した。A~B群はC群と比較して有意差を示さなかった(図20)。
本研究は、ALSモデル hTDP-43 Tgマウスにおいて、Muse細胞の8週齢時の単回静脈内投与が、ハンギングワイヤ試験の成績を改善することを示した最初の報告である。免疫組織学的評価を行ったところ、脊髄において、hTDP-43 Tgマウスで上昇したhTDP-43の陽性反応が低下したことから、Muse細胞の投与により脊髄の神経細胞の細胞質におけるhTDP-43の凝集が抑制されて、運動機能が改善した可能性が考えられる。
6-5(1)で調製した右半脳のうち、上述の免疫組織学的分析に用いなかった7例の脳から、(2)と同様に凍結切片を作製する。また、脊髄組織アレイ(群あたりn=8)の残存サンプルを用いて、同様に凍結切片を作製する。作製した切片を用いて、ユビキチン(細胞質内でのTDP-43タンパク質蓄積の指標)、トランスロケータータンパク質(神経変性疾患における神経炎症の指標)及びコリンアセチルトランスフェラーゼ(一次及び二次運動ニューロンのマーカー)について、上述の方法と同様に蛍光免疫染色による評価を行う。
6-4(2)で調製した左半脳のうち、上述のウエスタンブロット法によるタンパク質発現解析で使用しなかった海馬以外の部分(15例)を用い、核分画と細胞質分画を分離し、それぞれの分画において、ヒト-TDP-43抗体を用いてウエスタンブロットを行う。TDP-43による神経変性は、核分画から細胞質分画へTDP-43が移行することにより惹起されることから、それぞれの分画におけるTDP-43の分布に対するMuse細胞投与の影響を調べることにより、Muse細胞のALSモデル hTDP-43 Tgマウスの病態に対する改善作用の特徴をさらに明らかにすることができる。
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Claims (9)
- 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、対象における運動ニューロン疾患(MND)を治療し、予防し、軽減し及び/又は発症を遅延させるための細胞製剤。
- 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。
- 多能性幹細胞がCD105陽性である、請求項1又は2記載の細胞製剤。
- 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。
- 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。
- 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。
- 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)三胚葉のいずれか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。 - MNDが、筋萎縮性側索硬化症(ALS)、原発性側索硬化症(PLS)、脊髄性筋萎縮症(SMA)、進行性筋萎縮症(PMA)、又は球脊髄性筋萎縮症(SBMA)である、請求項1~7のいずれか1項に記載の細胞製剤。
- 多能性幹細胞が脊髄内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。
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WO2024020587A2 (en) | 2022-07-22 | 2024-01-25 | Tome Biosciences, Inc. | Pleiopluripotent stem cell programmable gene insertion |
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