Therapeutic agent for epidermolysis bullosa

Abstract

A cell preparation for treating epidermolysis bullosa, comprising SSEA-3-positive pluripotent stem cells (Muse cells) derived from mesenchymal tissue of a living body or cultured mesenchymal cells, wherein the epidermolysis bullosa is preferably simplex epidermolysis bullosa, junctional epidermolysis bullosa, or dystrophic epidermolysis bullosa.

Description

Technical Field

[0001] The present invention relates to cell preparations in regenerative medicine. More specifically, it relates to a cell preparation containing pluripotent stem cells effective for the treatment of epidermolysis bullosa, and a cell preparation containing skin cells differentiated from pluripotent stem cells effective for the treatment of skin diseases such as epidermolysis bullosa.

Background Art

[0002] Epidermolysis bullosa (EB) is a hereditary blistering skin disorder in which the adhesion function between the epidermis and dermis fails due to a gene abnormality of an adhesion structure control protein in the skin basement membrane region, and the epidermis detaches at the basement membrane level with minor external forces in daily life, forming blisters and ulcers similar to burns throughout the body (Table 1). Epidermolysis bullosa is roughly classified into three types: simple type, junctional type, and dystrophic type, depending on the site where blisters form. Blisters and erosions occur due to minor external forces at sites prone to external forces such as the extremities and large joints. Blisters and erosions heal relatively quickly in the simple type and dominant dystrophic type. In the simple type, no scars or skin atrophy remain after healing, but in the dominant dystrophic type, scars remain. In the junctional type and recessive dystrophic type, blisters and erosions are generally intractable, and when healed, skin atrophy remains in the junctional type and scars remain in the recessive dystrophic type. It is difficult to distinguish based on clinical findings at birth, and a comprehensive diagnosis is made in combination with electron microscopy, immunostaining, genetic diagnosis, and clinical findings that change with growth. Early diagnosis provides useful information for the proper management of the skin and the whole body. Since it is a rare and intractable disease, examinations and advice by specialized facilities and specialists are required for diagnosis and treatment.

Table 1

[0003] At present, there is no radical treatment for epidermolysis bullosa, and only symptomatic treatment is available. Since the symptomatic treatment also varies depending on the disease type, accurate disease type diagnosis is essential. In addition, depending on the disease type, this disease may develop various complications, which may worsen the condition and significantly limit the patient's daily life. Therefore, measures for various complications are also necessary. Furthermore, since this disease is a refractory genetic disease, it is necessary to consider the prevention of recurrence in family members.

[0004] <Topical therapy> After washing blisters, erosions, ulcers, etc. with running water, apply petrolatum gauze, etc. to prevent the gauze from sticking to the erosion. At this time, aspirate the blister fluid in advance (do not remove the blister roof). In cases with adhesions between fingers and toes, place petrolatum gauze between the fingers to prevent adhesions between fingers and toes. Since long-term use of antibiotic-containing ointments can cause the emergence of resistant bacteria, except in special cases, there is no need to actively use antibiotic-containing ointments. If deterioration of erosions or ulcers is recognized, since there may be complications of fungal or bacterial infections, especially in dystrophic epidermolysis bullosa, there is a possibility of skin cancer, so actively perform skin biopsy, fungal examination, bacterial culture examination, etc. Basically, perform ointment therapy once a day.

[0005] <Systemic therapy> Nutritional supplementation: Especially in dystrophic epidermolysis bullosa, due to lesions in the oral mucosa and esophagus, it is very often impossible to ingest sufficient nutrients, resulting in chronic malnutrition and anemia. Therefore, oral intake of nutritional agents such as Ensure liquid is useful. In cases where oral intake is difficult, nutritional supplementation may also be done through a nasogastric tube or intravenous drip. If the itching is severe, antihistamines may be effective.

[0006] <Treatment for complications> In dystrophic and junctional types, adhesions between fingers (toes), skin malignancies, esophageal strictures, pyloric strictures, erosions and strictures in the anal area, malnutrition, conjunctival erosions, anemia, etc. are often problematic. In addition, one of the severe complications is secondary systemic amyloidosis. The complications of epidermolysis bullosa often significantly reduce the quality of life, and thus the need for treatment is high. It is important to obtain the cooperation of specialists in various clinical fields, such as plastic surgery, reconstructive surgery, and nutritional management, for diagnosis and treatment. (Non-Patent Document 1)

[0007] However, at present, a safe and reliable methodology for normalizing gene abnormalities has not been established, and there is still no radical treatment for genetic diseases including epidermolysis bullosa. On the other hand, due to the recent progress of research on regenerative medicine, the treatment of epidermolysis bullosa by bone marrow transplantation, bone marrow stem cell transplantation, etc. is being explored.

[0008] For example, the following findings have been published. (Non-Patent Document 2) (1) Skin regeneration mechanism by bone marrow-derived cells: As a result of repeated extensive epidermal exfoliation over the years, it was clarified that as an epidermal regeneration mechanism of the skin of patients with epidermolysis bullosa who have lost a large number of epidermal stem cells, bone marrow stem cells are mobilized to the damaged skin through the peripheral circulation and contribute to the regeneration of the skin in the blister area. (Non-Patent Documents 3 and 4) (2) Bone marrow transplantation therapy for epidermolysis bullosa: The research group at the University of Minnesota in the United States was the first in the world to perform bone marrow transplantation for dystrophic epidermolysis bullosa and reported an improvement effect on skin symptoms. However, 2 out of 7 cases died during the course, and the development of a safer bone marrow transplantation treatment protocol is essential. (Non-Patent Document 5) (3) Bone marrow mesenchymal stem cell transplantation therapy for epidermolysis bullosa: A research group in Chile, South America, subcutaneously transplanted cultured mesenchymal stem cells derived from healthy donors into 2 cases of severe dystrophic epidermolysis bullosa and clarified its effectiveness (Non-Patent Document 6). In addition, groups in the UK and Egypt reported the effectiveness of intravenous administration of cultured mesenchymal stem cells derived from healthy donors to patients with severe dystrophic epidermolysis bullosa (Non-Patent Documents 7 and 8). However, it was also shown that the transplanted mesenchymal stem cells may gradually decrease within a few months. (4) Possibility of regenerative induction therapy using hematopoietic mobilizing factors of bone marrow mesenchymal stem cells for epidermolysis bullosa: It has been found that HMGB1 released from detached epidermis strongly induces damaged skin regeneration by accumulating bone marrow mesenchymal stem cells in the detached epidermis part of the skin via peripheral blood. (Non-Patent Document 4) As described above, basic and clinical research on gene therapy and regenerative therapy is being vigorously promoted aiming at a radical therapy for epidermolysis bullosa. However, at present, no treatment method that has been confirmed to be safe and effective and can completely cure epidermolysis bullosa has been found, and the realization of further radical treatment is awaited.

[0009] On the other hand, according to the research by Izawa et al., pluripotent stem cells (Multilineage-differentiating Stress Enduring cells; Muse cells) that exist in the mesenchymal cell fraction and express SSEA-3 (Stage-Specific Embryonic Antigen-3) as a surface antigen without induction manipulation such as gene introduction or cytokines are responsible for the pluripotency of the mesenchymal cell fraction, and it has been found that they may be applicable to the treatment of diseases aiming at tissue regeneration (for example, Patent Document 1; Non-Patent Documents 9 to 11). However, there is no example that has clarified that using Muse cells for the treatment of epidermolysis bullosa can obtain the expected therapeutic effect.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Summary of the Invention

Problems to be Solved by the Invention

[0012] An object of the present invention is to provide a cell preparation for treating skin diseases such as epidermolysis bullosa.

Means for Solving the Problems

[0013] The present inventors have experimentally found that, by administering human Muse cells from blood vessels to mice with experimentally damaged skin, human-type collagen is expressed at the wound site and the repair of the damage is accelerated. Similarly, in an epidermolysis bullosa model mouse genetically deficient in type XVII collagen (COL17), after forming blisters in the epidermis and administering human Muse cells from blood vessels or the like, it was found that human-type type XVII collagen can be supplied to the epidermis, and thereby, it was found that Muse cells can be used for the treatment of epidermolysis bullosa. Furthermore, the inventors have found that skin cells such as keratinocytes and fibroblasts useful for the treatment of skin diseases can be obtained from Muse cells, and have completed the present invention.

[0014] That is, the present invention is as follows. [1] A cell preparation for treating epidermolysis bullosa, comprising SSEA-3-positive pluripotent stem cells derived from a mesenchymal tissue of a living body or cultured mesenchymal cells. [2] The cell preparation according to [1], wherein the epidermolysis bullosa is simplex epidermolysis bullosa. [3] The cell preparation according to [1], wherein the epidermolysis bullosa is junctional epidermolysis bullosa. [4] The cell preparation according to [1], wherein the epidermolysis bullosa is dystrophic epidermolysis bullosa. [5] The cell preparation according to [4], wherein the dystrophic epidermolysis bullosa is dominant dystrophic epidermolysis bullosa or recessive dystrophic epidermolysis bullosa. [6] The cell preparation according to any one of [1] to [5] above, wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties: (i) Having low or no telomerase activity; (ii) Having the ability to differentiate into cells of any of the three germ layers; (iii) Not showing tumorous growth; and (iv) Having self-renewal ability. [7] The cell preparation according to any one of [1] to [5] above, wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties: (i) SSEA-3 positive; (ii) CD105 positive; (iii) having low or no telomerase activity; (iv) having the ability to differentiate into any of the three germ layers; (v) not showing tumorous growth; and (vi) having self-renewal ability. [8] Skin cells induced to differentiate from SSEA-3-positive pluripotent stem cells derived from mesenchymal tissues or cultured mesenchymal cells of a living body. [9] The skin cells according to [8], wherein the skin cells are keratinocytes and / or fibroblasts.

[10] A cell preparation for treating skin diseases, comprising the skin cells according to [8] or [9].

[11] The cell preparation according to

[10] , wherein the skin disease is epidermolysis bullosa.

[12] A method for treating epidermolysis bullosa, comprising the step of administering an effective amount of the cell preparation according to any one of [1] to [6] to a patient in need of treatment. [Effect of the Invention]

[0015] In the present invention, for patients with epidermolysis bullosa, by administering Muse cells from blood vessels or the like, or directly administering them to the skin site where blisters and erosions have formed in the subject and its surroundings, damaged skin can be reconstructed and repaired, and skin symptoms can be improved or restored. Therefore, the cell preparation containing Muse cells of the present invention can be used for the treatment of epidermolysis bullosa.

[0016] Muse cells can efficiently migrate and engraft into the skin site where blisters and erosions have formed, and spontaneously differentiate into epidermal cells at the engrafted site, so differentiation induction into target cells for treatment is not required prior to transplantation. In addition, they are non-tumorigenic and have excellent safety. Furthermore, since Muse cells are not subject to immune rejection, treatment with allogeneic preparations produced from donors is also possible. Therefore, Muse cells having the excellent performance shown above can provide an easily practicable means for the treatment of patients with epidermolysis bullosa.

[0017] In the present invention, skin cells such as keratinocytes and fibroblasts induced to differentiate from Muse cells are administered to patients with skin diseases such as epidermolysis bullosa to the affected area of the skin disease and its periphery, whereby damaged skin can be reconstructed and repaired, and skin symptoms can be improved or restored. Therefore, the cell preparation containing skin cells induced to differentiate from the Muse cells of the present invention can be used for the treatment of skin diseases such as epidermolysis bullosa.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0019] <1>Cell preparation containing Muse cells The present invention relates to a cell preparation for treating epidermolysis bullosa, which contains SSEA-3 positive pluripotent stem cells (Muse cells). In addition, treatment includes cure of symptoms, alleviation, prevention of recurrence, etc. The present invention will be described in detail below.

[0020] 1. Applicable diseases The cell preparation containing SSEA-3 positive pluripotent stem cells (Muse cells) of the present invention is used for the treatment of epidermolysis bullosa. In the present invention, "epidermolysis bullosa" refers to a hereditary blistering skin disease in which the adhesion function between the epidermis and the dermis fails due to genetic abnormalities of adhesion structure control proteins in the skin basement membrane region, etc., and the epidermis peels off at the basement membrane level by minor external forces in daily life, forming blisters, ulcers and / or erosions.

[0021] In the present invention, epidermolysis bullosa is roughly classified into three types: simplex epidermolysis bullosa, junctional epidermolysis bullosa, and dystrophic epidermolysis bullosa (e.g., dominant dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, etc.) according to the site where blisters form. For example, it is a condition in which blisters or erosions occur due to minor external force at sites that are easily subjected to external force, such as the extremities and large joints.

[0022] 2. Cell preparation (1) Multipotent stem cells (Muse cells) The pluripotent stem cells used in the cell preparation of the present invention are cells that Izawa et al. discovered their existence in the human body 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 the skin, etc., and are also known to widely exist in the connective tissues of tissues and organs. In addition, this cell is a cell having the properties of both pluripotent stem cells and mesenchymal stem cells, and is identified, for example, as a "SSEA-3 (Stage-specific embryonic antigen-3)" positive cell, preferably a double positive cell that is SSEA-3 positive and CD-105 positive, which are cell surface markers. Therefore, Muse cells or a cell population containing Muse cells can be separated from a biological tissue using, for example, the expression of SSEA-3 alone or SSEA-3 and CD-105 as an index. Details such as the separation method, identification method, and characteristics of Muse cells are disclosed in International Publication No. WO2011 / 007900. In addition, by utilizing the high resistance of Muse cells to various external stresses, Muse cells can be selectively concentrated by culturing under various external stress conditions such as proteolytic enzyme treatment, hypoxic conditions, low phosphate conditions, low serum concentration, low nutrient conditions, exposure to heat shock, in the presence of harmful substances, in the presence of reactive oxygen species, under mechanical stimulation, and under pressure treatment. In this specification, as a cell preparation for treating epidermolysis bullosa, pluripotent stem cells (Muse cells) or a cell population containing Muse cells prepared from a biological mesenchymal tissue or a cultured mesenchymal tissue using SSEA-3 as an index may be simply described as "SSEA-3 positive cells". In this specification, "non-Muse cells" may refer to cells contained in a biological mesenchymal tissue or a cultured mesenchymal cell that are other than "SSEA-3 positive cells".

[0023] Muse cells or a cell population containing Muse cells can be prepared from a biological tissue (e.g., a mesenchymal tissue) using the cell surface markers SSEA-3 or SSEA-3 and CD-105 as indicators. Here, "biological" refers to the biological body of a mammal. In the present invention, the biological body does not include a fertilized egg or an embryo at a developmental stage prior to the blastocyst stage, but includes an embryo at a developmental stage after the blastocyst stage, including a fetus or a blastocyst. Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, and guinea pigs, and cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets, etc. The Muse cells used in the cell preparation of the present invention are clearly distinguished from embryonic stem cells (ES cells) and induced pluripotent stem (iPS) cells in that they are directly separated from biological tissues with markers. Also, "mesenchymal tissue" refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, umbilical cord blood, amnion, and tissues present in various organs. For example, Muse cells can be obtained from bone marrow, skin, adipose tissue, blood, dental pulp, umbilical cord, umbilical cord blood, amnion, etc. For example, it is preferable to collect a biological mesenchymal tissue, prepare Muse cells from this tissue, and use them. Also, Muse cells may be prepared from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells using the above preparation means.

[0024] Also, a cell population containing Muse cells used in the cell preparation of the present invention can also be prepared by a method including selectively proliferating cells resistant to external stress by applying an external stress stimulus to a biological mesenchymal tissue or cultured mesenchymal cells and collecting cells with an increased abundance ratio of such cells. The external stress may be any one or a combination of 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, culture at low temperature, freezing treatment, culture in the presence of harmful substances, culture in the presence of reactive oxygen species, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment, or physical shock. The treatment time with the protease is preferably 0.5 to 36 hours in total to apply external stress to the cells. Also, the protease concentration may be any concentration used when detaching the cells adhered to the culture vessel, separating the cell mass into single cells, or recovering single cells from the tissue. The protease is preferably a serine protease, aspartic protease, cysteine protease, metalloprotease, glutamic protease, or N-terminal threonine protease. Further, the protease is preferably trypsin, collagenase, or dispase.

[0025] In the cell preparation of the present invention, the Muse cells used may be autologous or allogeneic to the recipient who receives the cell transplantation.

[0026] As described above, Muse cells or a cell population containing Muse cells can be prepared from a biological tissue using, for example, SSEA-3 positivity or double positivity for SSEA-3 and CD-105 as an index. However, it is known that human adult skin contains various types of stem cells and progenitor cells. However, Muse cells are not the same as these cells. Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural crest stem cells (NCSC), melanoblasts (MB), perivascular cells (PC), endothelial progenitor cells (EP), and adipose-derived stem cells (ADSC). Muse cells can be prepared using as an index the "non-expression" of markers specific to these cells. More specifically, Muse cells can be separated using as an index the non-expression of at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 markers selected from the group consisting of CD34 (a marker for EP and ADSC), CD117 (c-kit) (a marker for MB), CD146 (a marker for PC and ADSC), CD271 (NGFR) (a marker for NCSC), NG2 (a marker for PC), von Willebrand factor (vWF factor) (a marker for EP), Sox10 (a marker for NCSC), Snai1 (a marker for SKP), Slug (a marker for SKP), Tyrp1 (a marker for MB), and Dct (a marker for MB). For example, but not limited to, they can be prepared using as an index the non-expression of CD117 and CD146, and further, they can be prepared using as an index the non-expression of CD117, CD146, NG2, CD34, vWF, and CD271, and further, they can be prepared using as an index the non-expression of the above 11 markers.

[0027] In addition, the Muse cells having the above characteristics used in the cell preparation of the present invention are as follows: (i) Have low or no telomerase activity; (ii) Have the ability to differentiate into cells of any of the three germ layers; (iii) Do not show tumorous growth; and (iv) Have self-renewal ability It may have at least one property selected from the group consisting of. Preferably, the Muse cells used in the cell preparation of the present invention have all of the above properties. Here, regarding the above (i), "low or no telomerase activity" means, for example, that when telomerase activity is detected using the TRAPEZE XL telomerase detection kit (Millipore), it is low or undetectable. "Low" telomerase activity means, for example, having telomerase activity comparable to that of human fibroblasts, which are somatic cells, or having telomerase activity that is 1 / 5 or less, preferably 1 / 10 or less, compared to Hela cells. Regarding the above (ii), Muse cells have the ability to differentiate into the three germ layers (endoderm, mesoderm, and ectoderm) in vitro and in vivo. For example, by induction culture in vitro, they can differentiate into hepatocytes (including hepatoblasts or cells expressing hepatocyte markers), nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, adipocytes, etc. Also, when transplanted into the testis in vivo, they may show the ability to differentiate into the three germ layers. Furthermore, by transplantation into the living body by intravenous injection, they can migrate and engraft into damaged organs (heart, skin, spinal cord, liver, muscle, etc.) and have the ability to differentiate into cells according to the tissue. Regarding the above (iii), Muse cells have the property of growing at a growth rate of about 1.3 days. In suspension culture, they grow from a single cell, form embryoid body-like cell masses, and stop growing after reaching a certain size in about 14 days. However, when these embryoid body-like cell masses are transferred to adherent culture, cell growth starts again, and the cells growing from the cell masses spread at a growth rate of about 1.3 days. Furthermore, when transplanted into the testis, they have the property of not becoming cancerous for at least six months. Regarding the above (iv), Muse cells have self-renewal (self-replication) ability. Here, "self-renewal" means that when culturing in suspension from a single Muse cell, it is possible to confirm the differentiation of cells contained in the embryoid body-like cell mass obtained thereby into cells of the three germ layers. At the same time, by taking the cells of the embryoid body-like cell mass back to a single cell again for suspension culture, a next-generation embryoid body-like cell mass is formed, and from which the differentiation into cells of the three germ layers and the embryoid body-like cell mass in suspension culture can be confirmed again. Self-renewal may be repeated one or more cycles.

[0028] (2) Preparation and Use of a Cell Preparation Containing Muse Cells The cell preparation containing the Muse cells of the present invention can be obtained, without limitation, by suspending the Muse cells or the cell population containing the Muse cells obtained in the above (1) in physiological saline or an appropriate buffer (for example, phosphate buffered saline). In this case, when the number of Muse cells isolated from autologous or allogeneic tissues is small, the cells may be cultured before cell transplantation to proliferate until a predetermined number of cells is obtained. As already reported (International Publication No. WO2011 / 007900 pamphlet), since Muse cells do not form tumors, even if the cells recovered from a biological tissue remain undifferentiated, the possibility of canceration is low and they are safe. In addition, the culture of the recovered Muse cells is not particularly limited, but can be carried out in a normal growth medium (for example, α-minimum essential medium (α-MEM) containing 10% fetal bovine serum, etc.). More specifically, referring to the above International Publication No. WO2011 / 007900 pamphlet, in the culture and proliferation of Muse cells, a medium, additives (for example, antibiotics, serum), etc. can be appropriately selected to prepare a solution containing Muse cells at a predetermined concentration. When administering the cell preparation containing the Muse cells of the present invention to a human subject, bone marrow fluid is collected from the ilium of the human, and for example, mesenchymal stem cells are cultured as adherent cells from the bone marrow fluid until the cell amount reaches the amount of cells from which an effective therapeutic amount of Muse cells can be obtained. Then, the Muse cells are separated using the antigen marker of SSEA-3 as an index, and autologous or allogeneic Muse cells can be prepared as a cell preparation. Alternatively, for example, mesenchymal stem cells obtained from bone marrow fluid are cultured under external stress conditions to proliferate and concentrate the Muse cells until an effective therapeutic amount is reached, and then autologous or allogeneic Muse cells can be prepared as a cell preparation.

[0029] In addition, in the use of Muse cells in cell preparations, dimethyl sulfoxide (DMSO), serum albumin, etc. may be contained in the cell preparation to protect the cells, and antibiotics, etc. may be contained in the cell preparation to prevent contamination and growth of bacteria. Furthermore, other pharmaceutically acceptable components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) may be contained in the cell preparation. Those skilled in the art can add these factors and drugs to the cell preparation at appropriate concentrations. Thus, Muse cells can also be used as a pharmaceutical composition containing various additives.

[0030] The number of Muse cells contained in the cell preparation prepared above can be appropriately adjusted in consideration of the sex, age, weight, condition of the affected area, condition of the cells to be used, etc. of the subject so that a desired effect can be obtained in the treatment of epidermolysis bullosa. The subject individuals include, but are not limited to, mammals such as humans. In addition, the cell preparation containing the Muse cells of the present invention may be administered a plurality of times at appropriate intervals (for example, twice a day, once a day, twice a week, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months) until a desired therapeutic effect is obtained. Therefore, depending on the condition of the subject, the therapeutically effective amount is, for example, 1×10 3 cells~1×10 10 cells, and a dosage of 1 to 10 times a year is preferable. The total dosage per individual is not limited, but 1×10 3 cells~1×10 11 cells, preferably 1×10 4 cells~1×10 10 cells, more preferably 1×10 5 cells~1×10 9 cells, etc. can be mentioned.

[0031] The Muse cells used in the cell preparation of the present invention have the property of migrating to and engrafting in the damaged skin of epidermolysis bullosa. Therefore, in the administration of the cell preparation, the administration site and method of the cell preparation are not limited, and local administration to the affected area or administration to a vein, etc. may be used.

[0032] The cell preparation containing the Muse cells of the present invention can repair and regenerate damaged skin of patients with epidermolysis bullosa. The repair and regeneration of damaged skin can be confirmed, for example, by the restoration and / or upregulation of the expression of collagen proteins such as COL7 and COL17. That is, the cell preparation containing the Muse cells of the present invention has an action of restoring and / or upregulating the expression of collagen proteins.

[0033] <2>Skin cells differentiated from Muse cells and cell preparations containing the same In the present invention, skin cells such as keratinocytes and / or fibroblasts differentiated from Muse cells can also be used as a cell preparation. Keratinocytes are cells that produce keratin and are also called epidermal cells or keratinized cells. Inducing differentiation of Muse cells into keratinocytes can be achieved, for example, by culturing Muse cells in a medium containing keratinocyte growth factor (KGF) and epidermal growth factor (EGF). Preferably, after culturing Muse cells in a medium containing KGF and EGF, it can be carried out by culturing in a medium containing KGF, EGF, hepatocyte growth factor (HGF), and insulin-like growth factor 2 (IGF2). Here, the preferred concentration range of KGF is, for example, 5 - 20 ng / ml, the preferred concentration range of EGF is, for example, 20 - 40 ng / ml, and the preferred concentration range of IGF2 is, for example, 40 - 80 ng / ml. The preferred culture period is, for example, 7 - 28 days. Fibroblasts are cells that produce dermal components such as collagen and elastin. Inducing differentiation of Muse cells into fibroblasts can be achieved, for example, by culturing Muse cells in a medium containing transforming growth factor-β2 (TGF-β2) and ascorbic acid (AA). Preferably, after culturing Muse cells in a medium containing TGF-β2 and AA, it can be carried out by culturing in a medium containing AA. Here, the preferred concentration range of TGF-β2 is, for example, 30 - 60 μg / ml, and the preferred concentration range of AA is, for example, 20 - 80 mmol / l. The preferred culture period is, for example, 7 - 28 days.

[0034] Cell preparations containing skin cells such as keratinocytes and / or fibroblasts differentiated from Muse cells can be used not only for the treatment of epidermolysis bullosa, but also for the treatment of all skin diseases that can be treated by replacement therapy of skin cells.

[0035] In the use of a cell preparation containing skin cells differentiated from Muse cells, dimethyl sulfoxide (DMSO), serum albumin, etc. may be contained in the cell preparation to protect the cells, and antibiotics, etc. may also be contained to prevent contamination and growth of bacteria. Furthermore, other pharmaceutically acceptable components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) may be contained in the cell preparation.

[0036] The dosage of the cell preparation can be appropriately adjusted in consideration of the sex, age, weight, condition of the affected area, condition of the cells to be used, etc. of the subject so as to obtain a desired effect in the treatment of skin diseases. The subject individuals include, but are not limited to, mammals such as humans. Also, the cell preparation may be administered a plurality of times at appropriate intervals (for example, twice a day, once a day, twice a week, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months) until a desired therapeutic effect is obtained. Therefore, depending on the condition of the subject, as a therapeutically effective amount, for example, 1×10 3 cells to 1×10 10 cells per administration per individual is preferred for one year, and the total dosage per individual is not limited, but 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 9 cells, etc. can be mentioned. The administration method is not particularly limited, but local administration to the skin disease site or its vicinity is preferred. The skin cells may be sheeted and applied to the affected area.

[0037] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples at all.

Example

[0038] Preparation of human Muse cells Muse cells were obtained according to the method described in International Publication No. WO2011 / 007900 regarding the isolation and identification of human Muse cells. The Muse cells were expanded and enriched by culturing mesenchymal stem cells under stress conditions. In addition, commercially available MSCs were purchased and used as an MSC group.

[0039] Example 1. Evaluation in a full-thickness wound model mouse A full-thickness wound was made on the back of adult C57BL / 6 mice and used as a model. Within 30 minutes after making the full-thickness wound, the Muse cells (3×10 5 / mouse or 3×10 4 / mouse), MSCs (3×10 5 / mouse) or 200 μl of HBSS prepared above were injected into the tail vein, and the therapeutic effect was examined. Note that the epithelialization rate was calculated as follows. The back skin at the time of wound creation and on the 3rd, 6th, 9th, and 11th days after creation was photographed with a digital camera together with a ruler, and the area of each skin ulcer (mm 2 ) was calculated using Image J software (version 1.50i). Based on the area at the time of wound creation as a reference value, the reduction rate of the area was calculated in %.

Number

[0040] The results are shown in FIGS. 1 and 2. In all groups, the wounds healed over time. However, at the 3rd day, in the Muse cell (3×10 5 / mouse) administration group, significantly faster healing and a higher epithelialization rate were observed compared to the MSC administration group and the HBSS administration group, indicating that the administration of Muse cells is effective for the treatment of epidermal blistering.

[0041] On the 14th day, the skin tissue at the wound site was isolated, sections were prepared, and nuclear staining and human COL7 staining were performed. As shown in Fig. 3, the presence of human COL7 was confirmed in the epidermis and dermis in the Muse cell administration group, and it was confirmed that the administered Muse cells migrated to the skin and produced molecules necessary for epidermal-dermal adhesion.

[0042] Example 2. Evaluation in a COL17 knockout epidermolysis bullosa model mouse In COL17 gene knockout mice (reference: Nat Med. 2007 Mar;13(3):378-83.) (3 - 4 weeks old), the epidermis was rubbed to form blisters, and Muse cells (3 × 10 5 / mouse) were injected via the tail vein within 30 minutes after blister formation. One month later, the skin condition was observed. As shown in Fig. 4, in the control mice administered with HBSS, the hair condition was poor, and extensive formation of wounds and mucosal erosions was observed. However, in the mice administered with Muse cells, both the hair condition and the formation of wounds were mild. Also, one month after administration, the skin tissue was isolated, RNA was extracted from it, and the expression of human-derived COL7 gene and COL17 gene was examined by RT-PCR. The results are shown in Fig. 5. As a result, the presence of human COL7 and human COL17 was confirmed in the Muse cell-administered mice, and it was confirmed that the administered Muse cells were supplying adhesion factors. The expression of human COL7 was also confirmed at the protein level (Fig. 6).

[0043] Example 3. Induction of differentiation of Muse cells into keratinocytes Differentiation of Muse cells into keratinocytes was induced by culturing Muse cells according to the following procedure. Day 0 Seeding of Muse cells Day 1 Culture for 3 days in DMEM low glucose medium + 10% FBS + KGF (10 ng / ml) + EGF (20 - 30 ng / ml) Day 4 Culture for 8 - 14 days while changing the medium every other day in DMEM low glucose medium + 10% FBS + KGF (10 ng / ml) + EGF (20 - 30 ng / ml) + HGF (10 ng / ml) + IGF2 (60 ng / ml)

[0044] The results are shown in Figs. 7 to 9. As shown in Fig. 7, the cells on the 8th day of differentiation showed a keratinocyte-like morphology. Also, as shown in Figs. 8 and 9, the induced differentiated cells showed the expression of keratinocyte markers at the protein level and mRNA level.

[0045] Example 4. Induction of differentiation of Muse cells into fibroblasts Differentiation of Muse cells into fibroblasts was induced by culturing Muse cells according to the following procedure. Day 0 Seeding of Muse cells Day 1 Cultured for 6 days while changing the medium every other day with 10 ml of DMEM low glucose medium + 2 μl of TGF-β2 (50 μg / ml) + 60 μl of AA (50 mM) + 100 μl of ITS-A (insulin, transferrin, sodium selenite) Day 7 Cultured for 10 days while changing the medium every other day with DMEM low glucose medium + 20% FBS + 60 μl of AA (50 mM) Day 17 Cultured with DMEM low glucose medium + 10% FBS

[0046] The results are shown in Figs. 10 to 12. As shown in Fig. 10, the cells on the 10th day of differentiation showed a fibroblast-like morphology. Also, as shown in Figs. 11 and 12, the induced differentiated cells showed the expression of fibroblast markers at the protein level and mRNA level.

Industrial Applicability

[0047] The cell preparation of the present invention can reconstruct and repair damaged skin and improve or recover skin symptoms by administering it to a patient with epidermolysis bullosa, and can be applied to the treatment of epidermolysis bullosa.

Claims

1. A cell preparation for intravenous injection containing, as an active ingredient, pluripotent stem cells positive for SSEA-3 and including a cell fraction enriched with the pluripotent stem cells, for restoring and / or increasing the expression of keratin, type VII collagen, and / or type XVII collagen in a patient with epidermolysis bullosa, wherein the pluripotent stem cells are pluripotent stem cells isolated from a mesenchymal tissue or cultured mesenchymal cells of a living body using the antigen marker of SSEA-3 as an index, and the pluripotent stem cells have all of the following properties: (i) Positive for SSEA-3; (ii) Positive for CD105; (iii) Having low or no telomerase activity; (iv) Having the ability to differentiate into any of the three germ layers; (v) Not showing tumorous growth; and (vi) Having self-renewal ability.

2. The cell preparation according to Claim 1, wherein the epidermolysis bullosa is simplex epidermolysis bullosa.

3. The cell preparation according to Claim 1, wherein the epidermolysis bullosa is junctional epidermolysis bullosa.

4. The cell preparation according to Claim 1, wherein the epidermolysis bullosa is dystrophic epidermolysis bullosa.

5. The cell preparation according to Claim 4, wherein the dystrophic epidermolysis bullosa is dominant dystrophic epidermolysis bullosa or recessive dystrophic epidermolysis bullosa.

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