WO2018235834A1 - 表皮水疱症の治療剤 - Google Patents
表皮水疱症の治療剤 Download PDFInfo
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- WO2018235834A1 WO2018235834A1 PCT/JP2018/023352 JP2018023352W WO2018235834A1 WO 2018235834 A1 WO2018235834 A1 WO 2018235834A1 JP 2018023352 W JP2018023352 W JP 2018023352W WO 2018235834 A1 WO2018235834 A1 WO 2018235834A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K35/33—Fibroblasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells
Definitions
- the present invention relates to cell preparations in regenerative medicine. More specifically, a cell preparation containing pluripotent stem cells effective for treating epidermolysis bullosa and skin cells derived from differentiation of pluripotent stem cells effective for treating skin diseases such as epidermolysis bullosa It relates to a cell preparation.
- Epidermolysis bullosa (epidermolysis bullosa, EB) breaks adhesion function between the epidermis and the dermis due to the gene abnormality of adhesion structure regulatory protein in the skin basement membrane region, and the outer skin peels off at the basement membrane level by slight external force of daily life It is a hereditary bullous skin intractable disease that forms a whole body burn-like blisters and ulcers (Table 1). Epidermolysis bullosa is roughly classified into three types of simple type, junction type, and malnutrition type depending on the site where blisters are formed, and minor sites such as extremities of the extremities, large joints, etc. It causes erosion.
- Blisters and erosions cure relatively quickly in the simple and dominant malnutrition types, and after the simple form heals, neither scar nor skin atrophy remains, but the dominant malnutrition forms scars.
- junction type and recessive malnutrition type blisters and erosions are generally incurable, and when cured, skin atrophy is left in junction type and scarring in recessive malnutrition type.
- Clinical findings at birth are difficult to distinguish and comprehensively diagnosed together with electron microscopy, immunostaining, gene diagnosis, and clinical findings that change with growth. Early diagnosis is useful information to manage skin and whole body well. Because of rare and intractable diseases, diagnostic treatment requires examinations and advice from specialized facilities and specialists.
- ⁇ Local treatment> After flushing the skin with blisters, sores, ulcers, etc., apply vaseline gauze etc. to prevent gauze and sores from sticking. At this time, the contents of the blister are punctured and drained in advance (the blister cover is not removed). In cases with adhesion between fingers, Vaseline gauze is put between fingers to prevent adhesion between fingers. It is not necessary to actively use the antibiotic-containing ointment, except in special cases, as long-term use of the antibiotic-containing ointment causes the emergence of resistant bacteria.
- ointment therapy is performed once a day.
- Nutritional supplementation In particular in recessive dystrophic epidermolysis bullosa, the lesions of the oral mucosa and esophagus make it impossible to get enough nutrition, and it is very common for chronic malnutrition and anemia. Therefore, oral intake of nutrients such as Enschiricid is useful. If it is difficult to take orally, nutrition may be provided by a nasal tube or drip infusion. In cases of severe sores, antihistamines may be successful.
- Non-patent document 2 Skin regeneration mechanism by bone marrow-derived cells: Intraepithelial stem cells through peripheral circulation as the epidermal regeneration mechanism of epidermolysis bullosa patient skin which has lost a large amount of epidermal stem cells after repeated extensive exfoliation of the skin for many years It was found that it was mobilized to the injured skin and contributed to the regeneration of the skin on the blisters.
- Non-Patent Document 3 Bone marrow transplantation therapy for epidermolysis bullosa: The research group of the University of Minnesota in the United States implemented the bone marrow transplantation for recessive dystrophic epidermolysis bullosa for the first time in the world, and reported the improvement effect of skin symptoms. However, 2 out of 7 patients die during the course, and development of a safer bone marrow transplantation treatment protocol is essential.
- Non-patent document 5 Bone marrow mesenchymal stem cell transplantation therapy for epidermolysis bullosa: The South American Chile research group subcutaneously transplants healthy human bone marrow-derived cultured mesenchymal stem cells to 2 cases of severe recessive dystrophic epidermolysis bullosa, The effectiveness was clarified (nonpatent literature 6). In addition, groups in the UK and Egyptian administered the bone marrow-derived cultured mesenchymal stem cells in healthy subjects by drip infusion to patients with severe recessive dystrophic epidermolysis bullosa and reported the effectiveness (Non-patent Document 7, Non-patent Literature 8).
- HMGB1 released from exfoliated epidermis exfoliates bone marrow mesenchymal stem cells into exfoliated epidermal skin via peripheral blood It has been found that they are accumulated to strongly induce damaged skin regeneration.
- Non-patent document 4 As described above, basic and clinical researches on gene therapy and regenerative therapy are vigorously promoted for the radical treatment of epidermolysis bullosa. However, even now, there is no known cure that completely cures epidermolysis bullosa, whose safety and efficacy have been confirmed, and the realization of further radical treatment is awaited.
- SSEA-3 Stage-Specific Embryonic Antigen-3
- Muse cells Multilineage-differentiating stress enduring cells; Muse cells
- Patent Document 1 Non-Patent Documents 9 to 11
- Muse cells there is no example which uses Muse cells for treatment of epidermolysis bullosa and reveals that the expected therapeutic effect can be obtained.
- the present invention aims at providing a cell preparation for the treatment of skin diseases such as epidermolysis bullosa.
- the present inventors have found that human collagen is expressed at the wound site by administering human Muse cells from blood vessels to a mouse whose skin has been experimentally damaged, and the repair of the damage is accelerated.
- human type 17 collagen is epidermally formed by forming a blister on the epidermis and then administering human Muse cells from a blood vessel or the like.
- Muse cells can be used for the treatment of epidermolysis bullosa.
- skin cells such as keratinocytes and fibroblasts useful for the treatment of skin diseases can be obtained from Muse cells, and the present invention has been completed.
- the present invention is as follows.
- a cell preparation for treating epidermolysis bullosa comprising SSEA-3 positive pluripotent stem cells derived from mesenchymal tissue or cultured mesenchymal cells of a living body.
- epidermolysis bullosa is simple epidermolysis bullosa.
- epidermolysis bullosa is junction type epidermolysis bullosa.
- epidermolysis bullosa is a malnutrition-type epidermolysis bullosa.
- the pluripotent stem cells are pluripotent stem cells having all of the following properties: (I) low or no telomerase activity; (Ii) have the ability to differentiate into cells of any of the three germ layers; (Iii) show no neoplastic growth; and (iv) have self renewal ability.
- pluripotent stem cells are pluripotent stem cells having all of the following properties: (I) SSEA-3 positive; (Ii) CD105 positive; (Iii) low or no telomerase activity; (Iv) have the ability to differentiate into any of the three germ layers; (V) show no neoplastic growth; and (vi) have self renewal ability.
- a cell preparation for treating a skin disease comprising the skin cell according to [8] or [9].
- a method of treating epidermolysis bullosa comprising the step of administering an effective amount of the cell preparation according to any one of the above [1] to [6] to a patient in need of treatment.
- the injured skin is reconstructed and repaired by administering Muse cells from a blood vessel or the like to a patient with epidermolysis bullosa or directly administering it to and around the target skin area where blisters and erosions were formed. Symptoms can be ameliorated or ameliorated. Therefore, the cell preparation containing Muse cells of the present invention can be used for the treatment of epidermolysis bullosa.
- Muse cells can efficiently migrate to and engraft the skin site where the blisters and erosions have formed, and spontaneously differentiate into epidermal cells at the engrafted site, so they can be transferred to the cells to be treated prior to transplantation. There is no need for differentiation induction. In addition, it is non-tumorigenic and excellent in safety. Furthermore, since Muse cells do not undergo immune rejection, treatment with allogeneic preparations made from donors is also possible. Thus, Muse cells with superior performance as described above can provide an easily viable means for the treatment of patients with epidermolysis bullosa.
- the damaged skin is reconstructed by administering skin cells such as keratinocytes and fibroblasts induced to differentiate from Muse cells to a skin disease site and its periphery for patients with skin diseases such as epidermolysis bullosa. And repair to improve or restore skin symptoms. Therefore, the cell preparation containing skin cells induced to differentiate from Muse cells of the present invention can be used for treatment of skin diseases such as epidermolysis bullosa.
- FIG. 1 is a photograph of wound sites (0, 3, 6, 9 days after administration) of full-thickness wound model mice in each group.
- FIG. 2 is a graph showing the time course of the degree of epithelialization at the wound site of a full thickness wound model mouse of each group.
- FIG. 3 is a photomicrograph showing the results of staining of human nucleus and human type 7 collagen (COL7) in tissue sections of the wound site of a full-thickness wound model mouse of each group.
- FIG. 4 is a photograph showing the skin condition of COL17 knockout mice to which Muse cells (middle) or HBSS (right) were administered. Left indicates pre-dose.
- FIG. 1 is a photograph of wound sites (0, 3, 6, 9 days after administration) of full-thickness wound model mice in each group.
- FIG. 2 is a graph showing the time course of the degree of epithelialization at the wound site of a full thickness wound model mouse of each group.
- FIG. 3 is a photomicrograph showing the
- FIG. 5 is an electrophoresis photograph showing the results of RT-PCR which analyzed the expression of human COL7 gene (A) and human COL17 gene (B) in the skin of COL17 knockout mouse to which Muse cells were administered.
- Lane L electrophoretic marker
- lanes 1-6 results of six COL17 knockout mice to which Muse cells were administered
- lane 7 normal human keratinocytes (positive control)
- lane 8 normal B6 mouse (negative control)
- lane 9 Is water only FIG. 6 is a photomicrograph showing the result of staining human COL7 in a skin tissue section of a COL17 knockout mouse to which Muse cells have been administered.
- FIG. 7 is a photograph showing the differentiation of Muse cells into keratinocytes.
- FIG. 1 is an electrophoresis photograph showing the results of RT-PCR which analyzed the expression of human COL7 gene (A) and human COL17 gene (B) in the skin of COL17 knockout mouse to which Mus
- FIG. 8 is a photograph of immunostaining showing the expression of keratinocyte markers in keratinocytes differentiated from Muse cells.
- FIG. 9 is a photograph of RT-PCR showing the expression of keratinocyte markers in keratinocytes differentiated from Muse cells.
- FIG. 10 is a photograph showing the differentiation of Muse cells into fibroblasts.
- FIG. 11 is a photograph of immunostaining showing expression of fibroblast marker in fibroblasts differentiated from Muse cells.
- FIG. 12 is a photograph of RT-PCR showing the expression of fibroblast markers in fibroblasts differentiated from Muse cells.
- the cell preparation containing ⁇ 1> Muse cell TECHNICAL FIELD This invention relates to the cell preparation for treating epidermolysis bullosa which includes SSEA-3 positive pluripotent stem cells (Muse cell).
- the treatment includes cure of the symptoms, alleviation and prevention of recurrence. The invention is described in detail below.
- the cell preparation containing SSEA-3 positive pluripotent stem cells (Muse cells) of the present invention is used for the treatment of epidermolysis bullosa.
- "epidermis bullosa” means that the adhesion function between the epidermis and the dermis breaks down due to a gene abnormality of the adhesion structural control protein in the skin basement membrane region, and the epidermis is a basement membrane with a slight external force of daily life.
- a hereditary bullous intractable disease that exfoliates at the level to form blisters, ulcers and / or erosions.
- epidermolysis bullosa is a simple type epidermolysis bullosa, junctional epidermolysis bullosa, junctional epidermolysis bullosa, and malnutrition-type epidermolysis bullosa (eg, dominant malnutrition epidermolysis bullosa, recessive malnutrition depending on the site where the blisters are formed). It is divided roughly into three types of epidermolysis bullosa etc.), and for example, blisters and erosions are caused by slight external force at a site susceptible to external forces such as extremities and large joints.
- Pluripotent stem cells The pluripotent stem cells used in the cell preparation of the present invention are cells that Dezawa et al. Found their presence in human beings and named "Muse (Multilineage-differentiating Stress Enduring) cells". Muse cells can be obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)), dermal connective tissue of skin, etc. It is known to be widely present in connective tissues of tissues and organs.
- this cell is a cell having both the properties of pluripotent stem cells and mesenchymal stem cells, and for example, cell surface marker “SSEA-3 (Stage-specific embryonic antigen-3)” positive cells, preferably Are identified as SSEA-3 positive and CD-105 positive double positive cells. Therefore, Muse cells or cell populations containing Muse cells can be separated from living tissues, for example, using SSEA-3 alone or expression of SSEA-3 and CD-105 as an indicator. Details of methods for separation, identification and characteristics of Muse cells are disclosed in WO 2011/07900.
- Muse cells taking advantage of the high resistance of Muse cells to various external stresses, proteolytic enzyme treatment, hypoxic conditions, low phosphate conditions, low serum concentrations, low nutrient conditions, exposure to heat shock, harmful Muse cells can be selectively concentrated by culture under various external stress conditions such as in the presence of a substance, in the presence of active oxygen, under mechanical stimulation and under pressure treatment.
- a cell population comprising Muse cells) or Muse cells may be simply referred to as "SSEA-3 positive cells”.
- “non-Muse cell” refers to a cell contained in mesenchymal tissue or cultured mesenchymal cell of a living body and is a cell other than “SSEA-3 positive cell”. is there.
- Muse cells or cell populations containing Muse cells can be prepared from living tissues (eg, mesenchymal tissues) using cell surface markers SSEA-3 or SSEA-3 and CD-105 as indexes.
- a living body means a living body of a mammal. In the present invention, the living body does not include embryos whose developmental stage precedes the fertilized egg or blastocyst stage, but includes embryos of developmental stages after 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 and guinea pigs, cats, dogs, sheep, pigs, cattle, horses, donkeys, goats, ferrets etc.
- ES cells embryonic stem cells
- iPS induced pluripotent stem
- mesenchymal tissue refers to tissues present in various organs and tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, cord blood, amniotic membrane and the like
- Muse cells can be obtained from bone marrow, skin, adipose tissue, blood, dental pulp, umbilical cord, cord blood, amniotic membrane, and the like.
- Muse cells may be prepared from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells by using the above preparation means.
- a cell population containing Muse cells used in the cell preparation of the present invention can be resistant to external stress by applying external stress to mesenchymal tissue or cultured mesenchymal cells of a living body. It can also be prepared by a method comprising recovering cells which have been selectively grown to increase their abundance.
- the external stress may be protease treatment, culture under low oxygen concentration, culture under low phosphate conditions, culture under low serum concentration, culture under low nutrient conditions, culture under exposure to heat shock, low temperature Culture, freezing, culture in the presence of harmful substances, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking, culture under pressure, or physical shock It may be any or a plurality of combinations.
- the treatment time with the protease is preferably a total of 0.5 to 36 hours to apply external stress to the cells.
- the protease concentration may be a concentration used when detaching cells adhered to the culture vessel, breaking up the cell mass into single cells, or recovering single cells from tissue.
- the protease is preferably a serine protease, an aspartic protease, a cysteine protease, a metalloprotease, a glutamic acid protease or an N-terminal threonine protease.
- the protease is trypsin, collagenase or dispase.
- the Muse cells used may be autologous or allogeneic to the recipient receiving the cell transplantation.
- Muse cells or cell populations containing Muse cells can be prepared from living tissue, for example, using SSEA-3 positive or SSEA-3 and CD-105 double positive as an indicator.
- the skin is known to contain various types of stem 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), melanoblasts (MB), perivascular cells (PC), endothelial progenitor cells (EP), adipose-derived stem cells (ADSC) Can be mentioned.
- Muse cells can be prepared using the “non-expression” of the marker unique to these cells as an index.
- Muse cells include CD34 (EP and ADSC markers), CD117 (c-kit) (MB markers), CD146 (PC and ADSC markers), CD271 (NGFR) (NCSC markers), NG2 (marker of PC), vWF factor (von Willebrand factor) (marker of EP), Sox10 (marker of NCSC), Snai1 (marker of SKP), Slug (marker of SKP), Tyrp1 (marker of MB), 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 It is possible to separate non-expression of single or eleven markers as an index.
- non-expression of CD117 and CD146 can be prepared as an indicator, and furthermore, non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be prepared as an indicator, and further, as described above
- the non-expression of 11 markers can be prepared as an indicator.
- Muse cells having the above-mentioned characteristics used in the cell preparation of the present invention are as follows: (I) low or no telomerase activity; (Ii) have the ability to differentiate into cells of any of the three germ layers; (Iii) does not exhibit neoplastic growth; and (iv) may have at least one property selected from the group consisting of having self renewal ability.
- the Muse cells used in the cell preparation of the present invention have all the above properties.
- "low or no telomerase activity” means that low or undetectable when telomerase activity is detected using, for example, TRAPEZE XL telomerase detection kit (Millipore). Say.
- the "low" telomerase activity is, for example, a telomerase activity similar to that of human somatic cells, or 1/5 or less, preferably 1/10 or less of that of Hela cells. It means having the activity.
- Muse cells have the ability to differentiate into three germ layers (endodermal, mesodermal and ectodermal) in vitro and in vivo, eg, induction culture in vitro
- the cells can be differentiated into hepatocytes (including hepatoblasts or cells expressing hepatocyte markers), neurons, skeletal muscle cells, smooth muscle cells, bone cells, adipocytes and the like. It may also show the ability to differentiate into three germ layers when transplanted to the testis in vivo.
- Muse cells proliferate at a growth rate of about 1.3 days, but proliferate from one cell in suspension culture, and proliferate in about 14 days when they become embryoid-like cell masses and become a certain size.
- cell growth is started again, and the cells grown from the cell clusters spread at a growth rate of about 1.3 days. .
- Muse cells have the ability to self renew (self-renew).
- self renewal means that differentiation of cells contained in an embryoid-like cell mass obtained by culturing from one Muse cell in suspension culture into tridermal cells can be confirmed at the same time. By bringing the cells of the embryoid-like cell mass into suspension culture again with one cell, the next generation embryoid-like cell mass is formed, and from there, the embryo in tridermal differentiation and suspension culture again. It means that a body-like cell mass can be confirmed.
- the self renewal may be repeated one or more cycles.
- the cell preparation containing Muse cells of the present invention is not limited, but the Muse cells obtained in the above (1) or the cell population containing Muse cells It is obtained by suspending in an appropriate buffer (eg, phosphate buffered saline).
- an appropriate buffer eg, phosphate buffered saline
- the cells may be cultured prior to cell transplantation and allowed to grow until a predetermined number of cells is obtained.
- Muse cells do not become tumorous, and therefore, there is a possibility of canceration even if cells recovered from living tissues are contained undifferentiated. Is low and safe.
- the culture of the recovered Muse cells can be performed in a general growth medium (eg, ⁇ -minimum essential medium ( ⁇ -MEM) containing 10% calf serum), although not particularly limited. More specifically, referring to the above-mentioned WO 2011/07900 pamphlet, in the culture and growth of Muse cells, a medium, additives (eg, antibiotics, serum) etc. are appropriately selected, and Muse cells of a predetermined concentration are selected. Can be prepared. When a cell preparation containing Muse cells of the present invention is administered to a human subject, bone marrow fluid is collected from human iliac bone, and for example, bone marrow mesenchymal stem cells are effectively cultured as adherent cells from bone marrow fluid.
- a general growth medium eg, ⁇ -minimum essential medium ( ⁇ -MEM) containing 10% calf serum
- a medium, additives eg, antibiotics, serum
- Muse cells of a predetermined concentration are selected.
- Muse cells can be separated using the SSEA-3 antigen marker as an indicator, and autologous or allogeneic Muse cells can be prepared as a cell preparation.
- autologous or allogeneic Muse cells can be used as cells. It can be prepared as a formulation.
- Muse cells in cell preparations, dimethylsulfoxide (DMSO), serum albumin, etc. are protected to protect the cells, and antibiotics etc. are contained in the cell preparation to prevent bacterial contamination and proliferation. May be
- other pharmaceutically acceptable ingredients eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
- DMSO dimethylsulfoxide
- serum albumin etc.
- antibiotics etc. are contained in the cell preparation to prevent bacterial contamination and proliferation.
- other pharmaceutically acceptable ingredients eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
- Muse cells can also be used as a pharmaceutical composition containing various additives.
- the number of Muse cells contained in the cell preparation prepared above is such as the sex, age, weight of the subject, the condition of the affected area, the condition of the cells used, etc., so as to obtain the desired effect in the treatment of epidermolysis bullosa. It can be adjusted appropriately in consideration.
- the target individuals include, but are not limited to, mammals such as humans.
- the cell preparation containing Muse cells of the present invention may be repeated several times, as needed, at intervals (for example, twice a day, once a day, twice a week, etc.) until the desired therapeutic effect is obtained. Once a week, once every two weeks, once a month, once every two months, once every three months, once every six months).
- a therapeutically effective dose may be, for example, 1 ⁇ 10 3 to 1 ⁇ 10 10 cells per animal at a dose of 1 to 10 times in a year.
- the total administration amount in one individual is not limited, but is 1 ⁇ 10 3 cells to 1 ⁇ 10 11 cells, preferably 1 ⁇ 10 4 cells to 1 ⁇ 10 10 cells, more preferably 1 ⁇ 10 5 cells to 1 ⁇ 10 10 9 cells and the like.
- Muse cells used in the cell preparation of the present invention have the property of migrating to and engrafting to the damaged skin of epidermolysis bullosa. Therefore, in the administration of the cell preparation, the administration site and administration method of the cell preparation are not limited, and may be locally administered to the affected area or may be administered to a vein or the like.
- the cell preparation containing Muse cells of the present invention can repair and regenerate damaged skin of patients with epidermolysis bullosa. Repair and regeneration of damaged skin can be confirmed by, for example, restoration of expression and / or expression of collagen proteins such as COL7 and COL17. That is, the cell preparation containing Muse cells of the present invention has an effect of restoring expression and / or expression of collagen protein.
- Skin cells differentiated from Muse cells and cell preparation containing the same skin cells such as keratinocytes and / or fibroblasts differentiated from Muse cells can also be used as cell preparations .
- Keratinocytes are cells that produce keratin and are also called epidermal cells or keratinocytes.
- Muse cells are cultured in a medium containing keratinocyte growth factor (KGF) and epidermal growth factor (EGF), preferably after Muse cells are cultured in a medium containing KGF and EGF It can be carried out by culturing in a medium containing KGF and EGF, hepatocyte growth factor (HGF) and insulin-like growth factor 2 (IGF2).
- KGF keratinocyte growth factor
- EGF epidermal growth factor
- IGF2 insulin-like growth factor 2
- the preferable culture period is, for example, 7 to 28 days.
- Fibroblasts are cells that produce dermal components such as collagen and elastin, and differentiation of Muse cells to fibroblasts can be induced, for example, by transforming Muse cells with transforming growth factor- ⁇ 2 (TGF- ⁇ 2) and ascorbic acid. It can be carried out by culturing in a medium containing acid (AA), preferably culturing Muse cells in a medium containing TGF- ⁇ 2 and AA, and then culturing in a medium containing AA.
- the preferred concentration range of TGF- ⁇ 2 is, for example, 30 to 60 ⁇ g / ml
- the preferred concentration range of AA is, for example, 20 to 80 mmol / l.
- the preferable culture period is, for example, 7 to 28 days.
- Cell preparations containing skin cells such as keratinocytes derived from Muse cells and / or fibroblasts can be used not only for epidermolysis bullosa but also for treatment of skin diseases in general that can be treated by skin cell replacement therapy.
- DMSO dimethylsulfoxide
- serum albumin serum albumin or the like to protect the cells and an antibiotic to prevent bacterial contamination and growth Etc.
- other pharmaceutically acceptable ingredients eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
- DMSO dimethylsulfoxide
- emulsifiers e.g., emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
- the dosage of the cell preparation can be appropriately adjusted in consideration of the sex, age, body weight of the subject, the condition of the affected area, the condition of the cells to be used, etc., so as to obtain the desired effects in the treatment of the skin disease.
- the target individuals include, but are not limited to, mammals such as humans.
- the cell preparation may be repeated several times, as needed, at intervals (for example, twice a day, once a day, twice a week, once a week, two weeks) until the desired therapeutic effect is obtained. Once a month, once every two months, once every three months, once every six months).
- a therapeutically effective dose may be, for example, 1 ⁇ 10 3 to 1 ⁇ 10 10 cells per animal at a dose of 1 to 10 times in a year.
- the total administration amount in one individual is not limited, but is 1 ⁇ 10 3 cells to 1 ⁇ 10 11 cells, preferably 1 ⁇ 10 4 cells to 1 ⁇ 10 10 cells, more preferably 1 ⁇ 10 5 cells to 1 ⁇ 10 10 9 cells and the like.
- the method of administration is not particularly limited, it is preferable to administer locally at or around the skin disease site.
- the skin cells may be sheeted and applied to the affected area.
- Muse cells were obtained according to the method described in International Publication No. WO 2011/07900 for separation and identification of human Muse cells. Muse cells were expanded and enriched by culturing mesenchymal stem cells under stress conditions. In addition, commercially available MSCs were purchased and used as MSCs.
- Example 1 Evaluation in full-thickness wound model mice A full-thickness wound was given to the back of adult C57BL / 6 mice and used as a model.
- the epithelialization rate was calculated as follows. The back skin at the time of wound creation and on days 3, 6, 9 and 11 was photographed with a digital camera with a ruler, and the skin ulcer area (mm 2 ) of each was calculated using Image J software (version 1.50i) did. The reduction rate of the area was calculated in%, using the area at the time of wound preparation as a reference value.
- FIG. 1 and FIG. Wounds healed with the passage of time in any group, but at the third day, Muse cell (3 ⁇ 10 5 / mouse) administration group had significantly faster healing and higher healing compared to MSC administration group and HBSS administration group An epithelialization rate was observed, and it was shown that the administration of Muse cells is effective for the treatment of epidermolysis bullosa.
- Example 3 Induction of Differentiation of Muse Cells into Keratinocytes The differentiation of keratinocytes into keratinocytes was carried out by culturing Muse cells according to the following procedure. Day 0 Muse cell plating Day 1 DMEM low glucose medium + 10% FBS + KGF (10 ng / ml) + EGF (20-30 ng / ml) culture for 3 days Day 4 DMEM low glucose medium + 10% FBS + KGF (10 ng / ml) Culture for 8-14 days while changing the medium every other day with + EGF (20-30 ng / ml) + HGF (10 ng / ml) + IGF2 (60 ng / ml)
- FIGS. 7-9 The results are shown in FIGS. 7-9. As shown in FIG. 7, cells at day 8 of differentiation exhibited keratinocyte-like morphology. Also, as shown in FIGS. 8 and 9, differentiation-induced cells showed expression of keratinocyte marker at protein level and mRNA level.
- Example 4 Induction of differentiation of Muse cells into fibroblasts The differentiation of fibroblasts was induced by culturing Muse cells according to the following procedure.
- Day 0 Muse cell plating Day 1 10 ml DMEM low glucose medium + 2 ⁇ l TGF- ⁇ 2 (50 ⁇ g / ml) + 60 ⁇ l AA (50 mM) + 100 ⁇ l ITS-A (insulin, transferrin, sodium selenite) medium every other day
- ITS-A insulin, transferrin, sodium selenite
- FIGS. 10-12 The results are shown in FIGS. 10-12. As shown in FIG. 10, the cells at day 10 of differentiation exhibited fibroblast-like morphology. Also, as shown in FIGS. 11 and 12, the differentiation-induced cells showed the expression of fibroblast markers at the protein level and the mRNA level.
- the cell preparation of the present invention can be applied to a patient suffering from epidermolysis bullosa, to reconstruct and repair the damaged skin, to ameliorate or restore skin symptoms, and can be applied to the treatment of epidermolysis bullosa.
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Abstract
Description
水疱、びらん、潰瘍などを流水洗浄したのち、ガーゼとびらんが固着することを防ぐため、ワセリンガーゼなどを塗布する。この際、水疱内容はあらかじめ穿刺排液しておく(水疱蓋は除去しない)。指趾間の癒着を伴う症例では、指間にワセリンガーゼを挟むなどして、指趾間の癒着を予防する。抗生物質含有軟膏の長期間にわたる使用は耐性菌の出現の原因となるため、特別な場合を除き、抗生物質含有軟膏を積極的に使用する必要はない。びらんや潰瘍の悪化が認められた場合は、真菌や細菌感染の合併、特に劣性栄養障害型表皮水疱症では皮膚癌の出現の可能性があるため、皮膚生検や真菌検査、細菌培養検査などを積極的に施行する。基本的には軟膏療法を1日1回実施する。
栄養補給:特に劣性栄養障害型表皮水疱症では口腔粘膜や食道の病変により、栄養を十分摂取できず、慢性的な栄養不良、貧血になっていることが非常に多い。そのためエンシュアリキッドなどの栄養剤の経口摂取が有用である。経口摂取が困難な場合は経鼻チューブや点滴で、栄養を補給することもある。そう痒がはげしい場合は抗ヒスタミン剤が奏功することもある。
劣性栄養障害型と接合部型において、指(趾)間癒着、皮膚悪性腫瘍、食道狭窄、幽門狭窄、肛門部のびらん・狭窄、栄養不良、結膜びらん、貧血などが問題になることが多い。また、重篤な合併症のひとつに続発性全身性アミロイドーシスがある。
表皮水疱症の合併症はQOLを著しく低下させるものが多くそれだけに、治療の必要性が高い。形成術や拡張術、栄養管理など臨床各分野の専門医の協力を得て、診断治療にあたることが重要である。(非特許文献1)
一方、近年の再生医療の研究の進展により、骨髄移植、骨髄幹細胞移植等による表皮水疱症の治療が探索されつつある。
(1)骨髄由来細胞による皮膚再生メカニズム:長年広範囲の表皮剥離を繰り返す結果、大量の表皮幹細胞を喪失している表皮水疱症患者皮膚の表皮再生機序として、骨髄内幹細胞が末梢循環を介して損傷部皮膚に動員され、水疱部皮膚の再生に寄与していることを明らかにした。(非特許文献3、非特許文献4)
(2) 表皮水疱症に対する骨髄移植療法:米国ミネソタ大学の研究グループは世界で初めて劣性栄養障害型表皮水疱症に対する骨髄移植を実施し、皮膚症状の改善効果を報告した。しかし、7 例中2 例が経過中に死亡しており、より安全な骨髄移植治療プロトコール開発が必要不可欠である。(非特許文献5)
(3) 表皮水疱症に対する骨髄間葉系幹細胞移植療法:南米チリの研究グループは、重症劣性栄養障害型表皮水疱症の2 症例に対して健常者骨髄由来培養間葉系幹細胞を皮下移植し、その有効性を明らかにした(非特許文献6)。また、英国やエジプトのグループは、重症劣性栄養障害型表皮水疱症の患者に対して健常者骨髄由来培養間葉系幹細胞を点滴投与し、その有効性を報告した(非特許文献7、非特許文献8)。しかし移植した間葉系幹細胞は数ヶ月で次第に減少する可能性も併せて示された。
(4)表皮水疱症に対する骨髄間葉系幹細胞血中動員因子を利用した再生誘導医療の可能性:剥離表皮から放出されるHMGB1 が末梢血液を介して骨髄間葉系幹細胞を剥離表皮部皮膚に集積させて損傷皮膚再生を強く誘導していることが見出された。(非特許文献4)
上記の通り、表皮水疱症の根治的療法を目指し、遺伝子治療や再生治療の基礎的、臨床的研究が精力的に進められている。しかし、現在も、安全性と有効性が確認された、表皮水疱症を完全に治癒させる治療法は見いだされておらず、さらなる根治的治療の実現が待たれている。
[1]生体の間葉系組織又は培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞を含む、表皮水疱症を治療するための細胞製剤。
[2]表皮水疱症が、単純型表皮水疱症である、[1]に記載の細胞製剤。
[3]表皮水疱症が、接合部型表皮水疱症である、[1]に記載の細胞製剤。
[4]表皮水疱症が、栄養障害型表皮水疱症である、[1]に記載の細胞製剤。
[5]栄養障害型表皮水疱症が、優性栄養障害型表皮水疱症又は劣性栄養障害型表皮水疱症である、[4]に記載の細胞製剤。
[6]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]~[5]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。
[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]~[5]のいずれかに記載の細胞製剤:
(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)三胚葉のいずれかの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。
[8]生体の間葉系組織又は培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞から分化誘導された皮膚細胞。
[9]皮膚細胞がケラチノサイトおよび/または線維芽細胞である、[8]に記載の皮膚細胞。
[10][8]または[9]に記載の皮膚細胞を含む、皮膚疾患を治療するための細胞製剤。
[11]皮膚疾患が表皮水疱症である、[10]に記載の細胞製剤。
[12]上記[1]~[6]のいずれかに記載の細胞製剤の有効量を、治療を必要とする患者に投与する工程を含む、表皮水疱症の治療方法。
本発明は、SSEA-3陽性の多能性幹細胞(Muse細胞)を含む、表皮水疱症を治療するための細胞製剤に関する。なお、治療には症状の治癒、緩和、再発防止などが含まれる。本発明を以下に詳細に説明する。
本発明のSSEA-3陽性の多能性幹細胞(Muse細胞)を含む細胞製剤は、表皮水疱症の治療に使用される。
本発明において、「表皮水疱症」とは、皮膚基底膜領域における接着構造制御蛋白質の遺伝子異常等の原因により表皮・真皮間の接着機能が破綻し、日常生活の軽微な外力で表皮が基底膜レベルで剥離して水疱、潰瘍および/またはびらんを形成する遺伝性水疱性皮膚難病をいう。
(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、出澤らが、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))や皮膚の真皮結合組織等から得ることができるほか、広く組織や臓器の結合組織に存在することが知られている。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、細胞表面マーカーである「SSEA-3(Stage-specific embryonic antigen-3)」陽性細胞、好ましくはSSEA-3陽性かつCD-105陽性の二重陽性細胞として同定される。したがって、Muse細胞又はMuse細胞を含む細胞集団は、例えば、SSEA-3単独又はSSEA-3及びCD-105の発現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、Muse細胞が様々な外的ストレスに対する耐性が高いことを利用して、蛋白質分解酵素処理や、低酸素条件、低リン酸条件、低血清濃度、低栄養条件、熱ショックへの暴露、有害物質存在下、活性酸素存在下、機械的刺激下、圧力処理下など各種外的ストレス条件下での培養によりMuse細胞を選択的に濃縮することができる。なお、本明細書においては、表皮水疱症を治療するための細胞製剤として、SSEA-3を指標として用いて、生体の間葉系組織又は培養間葉系組織から調製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細胞に含まれる細胞であって、「SSEA-3陽性細胞」以外の細胞を指すことがある。
前記外的ストレスは、プロテアーゼ処理、低酸素濃度での培養、低リン酸条件下での培養、低血清濃度での培養、低栄養条件での培養、熱ショックへの暴露下での培養、低温での培養、凍結処理、有害物質存在下での培養、活性酸素存在下での培養、機械的刺激下での培養、振とう処理下での培養、圧力処理下での培養又は物理的衝撃のいずれか又は複数の組み合わせであってもよい。
前記プロテアーゼによる処理時間は、細胞に外的ストレスを与えるために合計0.5~36時間行うことが好ましい。また、プロテアーゼ濃度は、培養容器に接着した細胞を剥がすとき、細胞塊を単一細胞にばらばらにするとき、又は組織から単一細胞を回収するときに用いられる濃度であればよい。
前記プロテアーゼは、セリンプロテアーゼ、アスパラギン酸プロテアーゼ、システインプロテアーゼ、金属プロテアーゼ、グルタミン酸プロテアーゼ又はN末端スレオニンプロテアーゼであることが好ましい。更に、前記プロテアーゼがトリプシン、コラゲナーゼ又はジスパーゼであることが好ましい。
(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。好ましくは、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(肝芽細胞又は肝細胞マーカーを発現する細胞を含む)、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傷害を受けた臓器(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、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一定の大きさになると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移行すると、再び細胞増殖が開始され、細胞塊から増殖した細胞が約1.3日の増殖速度で広がっていく。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。
本発明のMuse細胞を含む細胞製剤は、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、所定の細胞数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)など)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Muse細胞を含む細胞製剤を投与する場合には、ヒトの腸骨から骨髄液を採取し、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細胞が得られ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、骨髄液から得られた骨髄間葉系幹細胞を外的ストレス条件下で培養して有効な治療量に達するまでMuse細胞を増殖、濃縮した後、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。
本発明においては、Muse細胞から分化誘導されたケラチノサイトおよび/または線維芽細胞などの皮膚細胞を細胞製剤として使用することもできる。
ケラチノサイトとはケラチンを産生する細胞であり、表皮細胞または角化細胞ともよばれる。Muse細胞からケラチノサイトへの分化誘導は、例えば、Muse細胞をケラチノサイト増殖因子(KGF)と上皮増殖因子(EGF)を含む培地で培養する、好ましくはMuse細胞をKGFとEGFを含む培地で培養した後に、KGFとEGFと肝細胞増殖因子(HGF)とインスリン様増殖因子2(IGF2)を含む培地で培養することによって行うことができる。ここで、KGFの好ましい濃度範囲は例えば5~20ng/mlであり、EGFの好ましい濃度範囲は例えば20~40ng/mlであり、IGF2の好ましい濃度範囲は例えば40~80ng/mlである。好ましい培養期間は例えば7~28日である。
線維芽細胞とは、コラーゲンやエラスチンなどの真皮成分を産生する細胞であり、Muse細胞から線維芽細胞への分化誘導は、例えば、Muse細胞をトランスフォーミング増殖因子-β2(TGF-β2)とアスコルビン酸(AA)を含む培地で培養する、好ましくはMuse細胞をTGF-β2とAAを含む培地で培養した後に、AAを含む培地で培養することによって行うことができる。ここで、TGF-β2の好ましい濃度範囲は例えば30~60μg/mlであり、AAの好ましい濃度範囲は例えば20~80mmol/lである。好ましい培養期間は例えば7~28日である。
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。Muse細胞は間葉系幹細胞をストレス条件下で培養することにより拡大富化培養した。また、市販のMSCを購入してMSC群として使用した。
成体C57BL/6マウスの背面に全層創傷を施してモデルとして用いた。全層創傷を施してから30分以内に、上記で調製したMuse細胞 (3×105/マウスまたは3×104/マウス)、MSC (3×105/mouse)またはHBSS 200μlを尾静脈注射し、治療効果を調べた。
なお、上皮化率は以下のようにして算出した。
創傷作成時および作成後3、6、9、11日目の背部皮膚を、定規とともにデジタルカメラで撮影し、それぞれの皮膚潰瘍面積(mm2)をImage Jソフトウェア(バージョン1.50i)を用いて算出した。創傷作成時の面積を基準値として、面積の縮小率を%で算出した。
COL17遺伝子ノックアウトマウス(参考文献Nat Med. 2007 Mar;13(3):378-83.)(3~4週齢)において、表皮を摩擦して水疱を形成させ、水疱形成から30分以内にMuse細胞 (3 ×105/マウス)を尾静脈より注射した。1ヶ月後に皮膚の状態を観察した結果、図4に示すように、HBSSを投与したコントロールマウスでは被毛状態が悪く、傷や粘膜びらんの形成が広範に認められたが、Muse細胞を投与したマウスでは被毛状態、傷の形成ともに軽度であった。また、投与1ヶ月後の皮膚組織を単離し、そこからRNAを抽出し、RT-PCRにより、ヒト由来COL7遺伝子およびCOL17遺伝子の発現を調べた。結果を図5に示す。その結果、Muse細胞投与マウスではヒトCOL7およびヒトCOL17の存在が確認され、投与されたMuse細胞が接着因子を供給していることが確認できた。ヒトCOL7の発現はタンパク質レベルでも確認できた(図6)。
以下の手順でMuse細胞を培養することにより、ケラチノサイトへの分化誘導を行った。
0日目 Muse細胞のプレーティング
1日目 DMEM lowグルコース培地+10%FBS+KGF(10ng/ml)+EGF(20~30ng/ml)で3日間培養
4日目 DMEM lowグルコース培地+10%FBS+KGF(10ng/ml)+EGF(20~30ng/ml)+HGF(10ng/ml)+IGF2(60ng/ml)で1日おきに培地を交換しながら8~14日間培養
以下の手順でMuse細胞を培養することにより、線維芽細胞への分化誘導を行った。
0日目 Muse細胞のプレーティング
1日目 10ml DMEM lowグルコース培地+2μl TGF-β2(50μg/ml)+60μl AA(50mM)+100μl ITS-A(インスリン、トランスフェリン、亜セレン酸ナトリウム)で1日おきに培地を交換しながら6日間培養
7日目 DMEM lowグルコース培地+20%FBS+60μl AA(50mM)で1日おきに培地を交換しながら10日間培養
17日目 DMEM lowグルコース培地+10%FBSで培養
Claims (11)
- 生体の間葉系組織又は培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞を含む、表皮水疱症を治療するための細胞製剤。
- 表皮水疱症が、単純型表皮水疱症である、請求項1に記載の細胞製剤。
- 表皮水疱症が、接合部型表皮水疱症である、請求項1に記載の細胞製剤。
- 表皮水疱症が、栄養障害型表皮水疱症である、請求項1に記載の細胞製剤。
- 栄養障害型表皮水疱症が、優性栄養障害型表皮水疱症又は劣性栄養型表皮水疱症である、請求項4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1~5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。 - 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1~5のいずれかに記載の細胞製剤:
(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)三胚葉のいずれかの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。 - 生体の間葉系組織又は培養間葉系細胞に由来するSSEA-3陽性の多能性幹細胞から分化誘導された皮膚細胞。
- 皮膚細胞がケラチノサイトおよび/または線維芽細胞である、請求項8に記載の皮膚細胞。
- 請求項8または9に記載の皮膚細胞を含む、皮膚疾患を治療するための細胞製剤。
- 皮膚疾患が表皮水疱症である、請求項10に記載の細胞製剤。
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JP2019525640A JPWO2018235834A1 (ja) | 2017-06-19 | 2018-06-19 | 表皮水疱症の治療剤 |
KR1020197038843A KR20200016298A (ko) | 2017-06-19 | 2018-06-19 | 표피 수포증 치료제 |
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WO2021201286A1 (ja) | 2020-04-02 | 2021-10-07 | 国立大学法人東北大学 | ハイポテンシャル多能性幹細胞 |
WO2022018884A1 (ja) * | 2020-07-22 | 2022-01-27 | 国立大学法人大阪大学 | 栄養障害型表皮水疱症の治療薬 |
WO2022019325A1 (ja) * | 2020-07-22 | 2022-01-27 | 国立大学法人大阪大学 | 栄養障害型表皮水疱症の治療薬 |
WO2023164241A1 (en) * | 2022-02-28 | 2023-08-31 | Jlm Exograde, Llc | Method for enriching muse cells and obtaining exosomes, microvesicles or the secretome therefrom |
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US20200206272A1 (en) | 2020-07-02 |
EP3643316A1 (en) | 2020-04-29 |
AU2018289935A1 (en) | 2020-01-23 |
JP2023060125A (ja) | 2023-04-27 |
KR20200016298A (ko) | 2020-02-14 |
CN110831607A (zh) | 2020-02-21 |
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