阅读说明：本技术 大疱性表皮松解症的治疗剂 (Therapeutic agent for epidermolysis bullosa ) 是由 清水宏 藤田靖幸 桝富直哉 于 2018-06-19 设计创作，主要内容包括：一种用于治疗大疱性表皮松解症的细胞制剂，其含有来源于活体的间充质组织或培养的间充质细胞的SSEA?3阳性多能干细胞(Muse细胞)。大疱性表皮松解症优选为单纯型大疱性表皮松解症、交界型大疱性表皮松解症或营养不良型大疱性表皮松解症。(A cell preparation for the treatment of epidermolysis bullosa, comprising SSEA-3 positive pluripotent stem cells (Muse cells) derived from living mesenchymal tissue or cultured mesenchymal cells. The epidermolysis bullosa is preferably simple epidermolysis bullosa, junctional epidermolysis bullosa, or dystrophic epidermolysis bullosa.)

1. A cell preparation for the treatment of epidermolysis bullosa, comprising SSEA-3 positive pluripotent stem cells derived from living mesenchymal tissue or cultured mesenchymal cells.

2. The cell preparation of claim 1, wherein epidermolysis bullosa is epidermolysis bullosa simplex.

3. The cell preparation of claim 1, wherein epidermolysis bullosa is junctional epidermolysis bullosa.

4. The cell preparation of claim 1, wherein epidermolysis bullosa is dystrophic epidermolysis bullosa.

5. The cell preparation of claim 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 claims 1 to 5, wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties:

(i) low or no telomerase activity;

(ii) has the ability to differentiate into cells of any of the three germ layers;

(iii) does not show tumorous proliferation; and

(iv) has self-renewal capability.

7. The cell preparation according to any one of claims 1 to 5, wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties:

(i) SSEA-3 positive;

(ii) positive for CD 105;

(iii) low or no telomerase activity;

(iv) has the ability to differentiate into any of the three germ layers;

(v) does not show tumorous proliferation; and

(vi) has self-renewal capability.

8. A skin cell induced from the differentiation of SSEA-3 positive pluripotent stem cells derived from living mesenchymal tissue or cultured mesenchymal cells.

9. The skin cell according to claim 8, wherein the skin cell is a keratinocyte and/or a fibroblast.

10. A cell preparation for treating skin diseases, which contains the skin cells according to claim 8 or 9.

11. The cell preparation according to claim 10, wherein the skin disease is epidermolysis bullosa.

Technical Field

The present invention relates to a cell preparation for regenerative medicine. More specifically, the present invention relates to a cell preparation containing pluripotent stem cells effective for the treatment of epidermolysis bullosa and a cell preparation containing skin cells obtained by inducing differentiation of pluripotent stem cells effective for the treatment of skin diseases such as epidermolysis bullosa.

Background

Epidermolysis Bullosa (EB) is an inherited blistering skin disease in which the adhesion function between the epidermis and the dermis is disrupted by gene abnormality of adhesion structure control proteins in the basal membrane region of the skin, and the epidermis is peeled off at the basal membrane level by a slight external force in daily life to form a systemic scald-like blister or ulcer (table 1). Epidermolysis bullosa is classified into three types, namely simple type, borderline type and malnutrition type, according to the formation site of the blister, and blisters and erosion are generated by slight external force at the parts such as extremities and large joints which are easily subjected to external force. In the simple type and the dominant malnutrition type, the blisters and erosion can be cured quickly, the simple type can not leave scars and skin atrophy after being cured, but the dominant malnutrition type can leave scars. In the borderline type and the recessive malnutrition type, blisters and erosion are generally difficult to cure, and after curing, skin atrophy is left in the borderline type, and scars are left in the recessive malnutrition type. It is difficult to identify the clinical manifestations at birth, and a comprehensive diagnosis is required together with electron microscopy, immunostaining, genetic diagnosis, and clinical manifestations that change with growth. Early diagnosis is useful information in managing the skin and the whole body well. Because of the rare intractable diseases, examination and advice by professional facilities and specialists are required in the diagnosis and treatment.

[ Table 1]

< disease type, causative Gene and protein >

Homepage of research group on rare intractable skin diseases, epidermolysis bullosa "

The treatment of epidermolysis bullosa is not curative but symptomatic treatment at the present stage. Since symptomatic therapy varies depending on the disease type, accurate diagnosis of the disease type is essential. In addition, since this disease causes various complications depending on the type of disease, and sometimes causes deterioration of the disease state, and significantly restricts the daily life of the patient, it is also necessary to treat various complications. Furthermore, since this disease is a intractable genetic disease, it is also necessary to consider prevention of recurrence in patients in the family.

< local therapy >

After washing blisters, erosion, ulcers and the like with running water, Vaseline and the like are smeared on the gauze to prevent the gauze from adhering to the erosion. At this time, the blister contents were subjected to puncture drainage in advance (without removing the blister skin). In the case of inter-digital adhesion, vaseline gauze or the like is sandwiched between the fingers to prevent the inter-digital adhesion. The long-term use of the ointment containing antibiotics is responsible for the appearance of drug-resistant bacteria, and therefore, except for special cases, the active use of the ointment containing antibiotics is not required. When it is confirmed that erosion or ulcer is worsened, there is a possibility that complication of fungal or bacterial infection may occur, and particularly, skin cancer may occur in latent dystrophy type epidermolysis bullosa, and therefore, skin biopsy, fungal test, bacterial culture test, and the like are actively performed. Ointment therapy was performed essentially 1 time per day.

< systemic therapy >

And (3) nutrition supply: particularly, in the latent malnutrition-type epidermolysis bullosa, sufficient nutrition cannot be taken due to lesions of the oral mucosa and esophagus, and chronic malnutrition and anemia often occur. Therefore, oral ingestion of a nutritional agent such as Ensure Liquid (trade name) is useful. In the case where oral intake is difficult, nutrition may be supplemented by nasal catheter or drip. In cases where itching is severe, antihistamines sometimes also become effective.

< treatment of complications >

In the recessive malnutrition type and the borderline type, adhesion between fingers (toes), malignant skin tumor, esophageal stenosis, pyloric stenosis, erosion and stenosis of the anus, malnutrition, conjunctival erosion, anemia and the like are frequently problematic. Furthermore, as one of the serious complications, there is secondary systemic amyloidosis.

Since complications of epidermolysis bullosa significantly reduce QOL in many cases, the necessity of treatment is high. It is important to perform diagnosis and treatment in cooperation with specialists in various clinical fields such as angioplasty, dilation surgery, and nutrition management. (non-patent document 1)

However, at the present time, no safe and reliable method 0 for normalizing gene abnormalities has been established, and there is no radical therapy for genetic diseases represented by epidermolysis bullosa.

Meanwhile, with the progress of research on regenerative medicine in recent years, the treatment of epidermolysis bullosa by bone marrow transplantation, bone marrow stem cell transplantation, and the like has been studied.

For example, the following findings are disclosed. (non-patent document 2)

(1) Skin regeneration mechanism based on bone marrow derived cells: as a mechanism of epidermal regeneration in the skin of a patient with epidermolysis bullosa, in which extensive exfoliation occurs over a long period of time and a large amount of epidermal stem cells are lost, stem cells in bone marrow are mobilized to the damaged skin via the peripheral circulation, and it has been clarified that these stem cells contribute to regeneration of the skin of the blistered part. (non-patent document 3 and 4)

(2) Bone marrow transplantation therapy for epidermolysis bullosa: a research group of the university of minnesota in the united states performed bone marrow transplantation for the first time in the world against recessive dystrophic epidermolysis bullosa and reported an improvement effect of skin symptoms. However, 2 of 7 died during the course of the procedure, and development of a safer bone marrow transplant treatment regimen was indispensable. (non-patent document 5)

(3) Bone marrow mesenchymal stem cell transplantation therapy for epidermolysis bullosa: the research group of south america chile subcutaneously transplanted cultured mesenchymal stem cells derived from the bone marrow of healthy subjects to 2 cases of severe recessive dystrophy type epidermolysis bullosa and clarified its effectiveness (non-patent document 6). Further, the uk and egyptian groups administered cultured mesenchymal stem cells derived from the bone marrow of healthy subjects to patients with severe recessive dystrophy type epidermolysis bullosa by drip administration and reported their effectiveness (non-patent documents 7 and 8). However, the possibility that the transplanted mesenchymal stem cells gradually decreased within several months is also shown.

(4) Possibility of regeneration-induced therapy using mobilizing factors in bone marrow mesenchymal stem cell blood against epidermolysis bullosa: it was found that HMGB1 released from the exfoliated epidermis causes mesenchymal stem cells to aggregate on the skin of the exfoliated epidermis portion via peripheral blood, and strongly induces regeneration of the damaged skin. (non-patent document 4)

As described above, the basic and clinical studies of gene therapy and regenerative therapy are being actively promoted with the aim of radical therapy for epidermolysis bullosa. However, no treatment method has been found that can completely cure epidermolysis bullosa with safety and effectiveness confirmed, and further radical treatment is expected.

On the other hand, it has been found from studies by Taozu et al that pluripotent stem cells (Multi-lineage-differentiating pluripotent Stem cells) expressing the surface Antigen SSEA-3(Stage-specific extracellular Antigen-3) are carriers of pluripotency of mesenchymal cell fractions, and that such cells are present in mesenchymal cell fractions, can be obtained without gene transfer or induction with cytokines or the like, and are potentially useful for treating diseases for the purpose of tissue regeneration (for example, patent document 1; non-patent documents 9 to 11). However, no example is disclosed in which Muse cells are used for the treatment of epidermolysis bullosa to obtain a desired therapeutic effect.

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a cell preparation for treating a skin disease such as epidermolysis bullosa.

Means for solving the problems

The present inventors have found that, by experimentally applying human Muse cells to a mouse whose skin has been damaged from a blood vessel, human collagen is expressed at a wound site, and the repair of the damage is advanced. Similarly, it was found that human type 17 collagen can be supplied to the epidermis by administering human Muse cells from blood vessels or the like after forming blisters on the epidermis in a model mouse of epidermolysis bullosa genetically lacking type 17 collagen (COL17), and that Muse cells can be used for the treatment of epidermolysis bullosa. Furthermore, they 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.

Namely, the present invention is as follows.

[1] A cell preparation for the treatment of epidermolysis bullosa, comprising SSEA-3 positive pluripotent stem cells derived from living mesenchymal tissue or cultured mesenchymal cells.

[2] The cell preparation according to [1], wherein the epidermolysis bullosa is simple 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], wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties:

(i) low or no telomerase activity;

(ii) has the ability to differentiate into cells of any of the three germ layers;

(iii) does not show tumorous proliferation; and

(iv) has self-renewal capability.

[7] The cell preparation according to any one of [1] to [5], wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties:

(i) SSEA-3 positive;

(ii) positive for CD 105;

(iii) low or no telomerase activity;

(iv) has the ability to differentiate into any of the three germ layers;

(v) does not show tumorous proliferation; and

(vi) has self-renewal capability.

[8] A skin cell induced from the differentiation of SSEA-3 positive pluripotent stem cells derived from living mesenchymal tissue or cultured mesenchymal cells.

[9] The skin cell according to [8], wherein the skin cell is a keratinocyte and/or a fibroblast.

[10] A cell preparation for treating skin diseases, which comprises 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, which comprises 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.

Effects of the invention

In the present invention, it is possible to reconstruct and repair damaged skin and improve or restore skin symptoms by administering Muse cells from blood vessels or the like to a patient suffering from epidermolysis bullosa or by directly administering Muse cells to a skin site where blisters and erosion are formed and the periphery of the skin site in a subject. Therefore, the Muse cell preparation of the present invention can be used for the treatment of epidermolysis bullosa.

Muse cells can migrate and engraft efficiently to skin sites where blisters and erosion are formed, and spontaneously differentiate into epidermal cells at the site of engraftment, so that it is not necessary to perform differentiation induction into cells to be treated prior to transplantation. In addition, it is non-tumorigenic and very safe. Furthermore, because Muse cells are not subject to immunological rejection, they can also be used in the treatment of xenogeneic preparations made from donors. Therefore, an easily practiced method for treating a patient with epidermolysis bullosa can be provided by using Muse cells having the excellent properties shown above.

In the present invention, by applying skin cells such as keratinocytes and fibroblasts obtained by inducing differentiation of Muse cells to the skin disease site and the periphery of the skin disease site of a patient suffering from a skin disease such as epidermolysis bullosa, it is possible to reconstruct and repair damaged skin and improve or restore skin symptoms. Therefore, the cell preparation of the present invention containing skin cells derived from Muse cell differentiation induction can be used for the treatment of skin diseases such as epidermolysis bullosa.

Drawings

FIG. 1 is a photograph of wound sites (0, 3, 6, 9 days after application) of each group of full-thickness wound model mice.

Fig. 2 is a graph showing the change with time of the epithelialization rate of the wound site in each group of full-thickness wound model mice.

Fig. 3 is a micrograph showing the results of staining human nuclei and human type 7 collagen (COL7) in tissue sections of the wound sites of the full-thickness wound model mice of each group.

Fig. 4 is a photograph showing the skin condition of COL17 knockout mice administered with Muse cells (middle) or HBSS (right). The left side indicates before administration.

FIG. 5 is an electrophoretogram showing the results of RT-PCR analyzing the expression of human COL7 gene (A) and human COL17 gene (B) in the skin of a COL17 knockout mouse to which Muse cells were applied. Lane L is the electrophoretic marker, lanes 1-6 are the results of 6 COL17 knockout mice administered Muse cells, lane 7 is normal human keratinocytes (positive control), lane 8 is normal B6 mice (negative control), and lane 9 is water only.

FIG. 6 is a photomicrograph showing the results of staining human COL7 in skin tissue sections of COL17 knockout mice administered Muse cells.

FIG. 7 is a photograph showing the differentiation of Muse cells into keratinocytes.

Fig. 8 is a photograph showing immunostaining of 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 differentiation of Muse cells into fibroblasts.

Fig. 11 is a photograph showing immunostaining of expression of fibroblast markers 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.

Detailed Description

<1> Muse cell-containing cell preparation

The invention relates to a cell preparation for the treatment of epidermolysis bullosa, which contains SSEA-3-positive pluripotent stem cells (Muse cells). In addition, treatment includes cure, alleviation of symptoms, prevention of relapse, and the like. The present invention is explained in detail below.

1. Is suitable for diseases

The cell preparation of the invention containing SSEA-3 positive pluripotent stem cells (Muse cells) is used for the treatment of epidermolysis bullosa.

In the present invention, the term "epidermolysis bullosa" refers to a hereditary blistering skin disease in which the adhesion function between the epidermis and the dermis is disrupted by gene abnormality of an adhesion structure controlling protein in the basal membrane region of the skin, and the epidermis is peeled off at the basal membrane level by a slight external force in daily life to form blisters, ulcers and/or erosion.

In the present invention, epidermolysis bullosa is classified into three types, roughly, of simple epidermolysis bullosa, junctional epidermolysis bullosa, and dystrophic epidermolysis bullosa (e.g., dominant dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, etc.), depending on the site where the blister is formed, and blisters and erosion may occur due to a slight external force, for example, at the extremities, the great joints, etc., where the external force is easily applied.

2. Cell preparation

(1) Pluripotent stem cells (Muse cells)

The pluripotent stem cells used in the cell preparation of the present invention are cells that are found in the human body by Takara et al and are named "Muse (multiple-differentiating Stress reducing) cells". It is known that Muse Cells are widely present in connective tissues of tissues and organs, in addition to being obtained from bone marrow fluid, adipose tissues (Ogura, F., et al., Stem Cells Dev., Nov20, 2013(Epub) (published on Jan 17(2014)), dermal connective tissues of the skin, and the like, and that the Cells are Cells having properties of both pluripotent Stem Cells and mesenchymal Stem Cells, and are identified, for example, as "SSEA-3 (staged-specific cytotoxic-3)" positive Cells, preferably as double positive Cells which are SSEA-3 positive and CD-105 positive, and thus, for example, the isolation, identification, characterization, and the like of Muse Cells or a cell population containing Muse Cells from tissue can be performed using, as an index, SSEA-3 alone, or SSEA-3 and CD-105 as well as published International publication No. WO 007900, muse cells can be selectively concentrated by culturing under various external stress conditions such as protease treatment, hypoxic conditions, low phosphate conditions, low serum concentration, low nutrient conditions, exposure to heat shock, presence of harmful substances, presence of active oxygen, mechanical stimulation, and pressure treatment, taking advantage of the fact that Muse cells have high resistance to various external stresses. In the present specification, as a cell preparation for the treatment of epidermolysis bullosa, SSEA-3 may be used as an index, and a pluripotent stem cell (Muse cell) or a cell population containing Muse cell prepared from a living mesenchymal tissue or a cultured mesenchymal tissue may be simply referred to as "SSEA-3 positive cell". In the present specification, the term "non-Muse cell" may mean a cell contained in a living mesenchymal tissue or a cultured mesenchymal cell, that is, a cell other than the "SSEA-3 positive cell".

Muse cells or a cell population containing Muse cells can be prepared from living tissue (e.g., mesenchymal tissue) using SSEA-3 or SSEA-3 and CD-105 as markers on the cell surface. Here, "living body" refers to a living body of a mammal. In the present invention, the living body does not include a fertilized egg, an embryo at a development stage before the blastocyst stage, but includes an embryo at a development stage after the blastocyst stage, including a fetus and a blastocyst. The mammal is not limited, and may be a primate such as a human, monkey; rodents such as mice, rats, rabbits, guinea pigs, and the like; cats, dogs, sheep, pigs, cattle, 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 artificial pluripotent stem (iPS) cells in that the carrying label is directly isolated from a living tissue. Further, "mesenchymal tissue" refers to a tissue present in tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord blood, amniotic membrane, and various organs. For example, Muse cells can be obtained from bone marrow, skin, adipose tissue, blood, dental pulp, umbilical cord blood, amniotic membrane, and like tissues. For example, it is preferable to select mesenchymal tissue of a living body, prepare Muse cells from the tissue, and use the Muse cells. Furthermore, Muse cells can also be prepared from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells using the above preparation method.

Furthermore, the Muse cell-containing cell population used in the cell preparation of the present invention can also be prepared by a method comprising the steps of: a method for producing a cell having an increased presence ratio of cells in a living body by applying an external stress stimulus to mesenchymal tissue of the living body or cultured mesenchymal cells, selectively proliferating the cells resistant to the external stress, and recovering the cells.

The external stress may be a combination of any one or more of the following: protease treatment, culture under low oxygen concentration, culture under low phosphate condition, culture under low serum concentration, culture under low nutrient condition, culture under exposure to heat shock, culture under low temperature, freezing treatment, culture in the presence of harmful substances, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment, or physical shock.

The protease treatment time is preferably 0.5 to 36 hours in total to apply external stress to the cells. The protease concentration may be a concentration used for peeling off cells adhering to the culture vessel, dispersing cell aggregates into single cells, or collecting single cells from a tissue.

The protease is preferably: a serine protease, an aspartic protease, a cysteine protease, a metalloprotease, a glutamic protease, or an N-terminal threonine protease. The protease is further preferably trypsin, collagenase or dispase.

The Muse cells used in the cell preparation of the present invention may be autologous or allogeneic to the recipient to whom the cells are transplanted.

As described above, Muse cells or a cell population containing Muse cells can be prepared from living tissues using, for example, SSEA-3 positive or double positive of SSEA-3 and CD105 as an indicator, but it is known that adult skin contains various types of stem cells and precursor cells. However, Muse cells are not identical to these cells. Examples of such stem cells and precursor cells include skin-derived precursor cells (SKP), Neural Crest Stem Cells (NCSC), Melanoblasts (MB), Perivascular Cells (PC), endothelial precursor cells (EP), and adipose-derived stem cells (ADSC). Muse cells can be prepared by using as an indicator that markers inherent to these cells are "not expressed". More specifically, Muse cells can be isolated with no expression of at least 1 of 11 markers, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 markers selected from the group consisting of CD34 (markers for EP and ADSC), CD117(c-kit) (markers for MB), CD146 (markers for PC and ADSC), CD271(NGFR) (markers for NCSC), NG2 (markers for PC), vWF factor (von Willebrand factor) (markers for EP), Sox10 (markers for NCSC), Snai1 (markers for SKP), Slug (markers for SKP), Tyrp1 (markers for MB), and Dct (markers for MB), as an index. For example, although not limited thereto, the preparation may be performed using a marker that does not express CD117 and CD146, further, using a marker that does not express CD117, CD146, NG2, CD34, vWF, and CD271 as an indicator, and further, using a marker that does not express the above 11 markers as an indicator.

Furthermore, the Muse cells used in the cell preparation of the present invention having the above-described characteristics may also have at least one property selected from the group consisting of:

(i) low or no telomerase activity;

(ii) has the ability to differentiate into cells of any of the three germ layers;

(iii) does not show tumorous proliferation; and

(iv) has self-renewal capability.

Preferably, the Muse cells used in the cell preparation of the invention have all of the properties described above.

Here, with respect to the above (i), "telomerase activity is low or no telomerase activity" means that, for example, in the case of detecting telomerase activity using the trap XLtelomerase detection kit (Millipore corporation), the activity is low or undetectable. "low" telomerase activity means, for example, that the telomerase activity is comparable to that of human fibroblasts which are somatic cells, or that the telomerase activity is 1/5 or less, preferably 1/10 or less, as compared with that of Hela cells.

Regarding (ii) above, Muse cells have the ability to differentiate into the three germ layers (inner germ layer system, mesogerm layer system and outer germ layer system) in vitro and in vivo, and can be differentiated into hepatocytes (including cells expressing hepatic fibroblasts or hepatocyte markers), nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, adipocytes, and the like, for example, by in vitro induction culture. In addition, in the case of in vivo transplantation into a testis, the ability to differentiate into the three germ layers is also exhibited in some cases. Further, it has an ability to migrate and engraft into damaged organs (heart, skin, spinal cord, liver, muscle, etc.) and differentiate into cells corresponding to tissues by being transplanted into a living body by intravenous injection.

With regard to (iii) above, Muse cells have the following properties: the cells were proliferated at a proliferation rate of about 1.3 days, and proliferated from 1 cell in suspension culture to form an embryoid body-like cell mass, and when the cell mass reached a certain size, the proliferation was stopped by about 14 days, but when the embryoid body-like cell mass was transferred to adherent culture, the cell proliferation was restarted, and the cells proliferated from the cell mass gradually diffused at a proliferation rate of about 1.3 days. Further, when transplanted into a testis, the graft has a property of not being cancerous for at least half a year.

In addition, regarding (iv) above, Muse cells have a self-renewal (self-replication) ability. Here, "self-refresh" means: differentiation into a triblast cell was confirmed from the cells contained in the embryoid body-like cell mass obtained by suspension culture of 1 Muse cell, and formation of a next generation embryoid body-like cell mass by subjecting the cells of the embryoid body-like cell mass to suspension culture of 1 cell again was confirmed, whereby it was confirmed that the triblast differentiation was again caused from the cell mass and the embryoid body-like cell mass obtained by suspension culture was confirmed. Self-renewal may be 1 or repeated multiple cycles.

(2) Preparation and use of cell preparations containing Muse cells

The Muse cells-containing cell preparation of the present invention is not limited, but can be obtained by suspending the Muse cells obtained in (1) above or a cell population containing Muse cells in a physiological saline solution or an appropriate buffer solution (e.g., phosphate buffered physiological saline), in which case, when the number of Muse cells isolated from autologous or allogeneic tissues is small, the cells can be cultured before cell transplantation and expanded to obtain a predetermined number of cells, and further, as has been reported (International publication No. WO 2011/007900), because Muse cells do not undergo tumorigenesis, the possibility of canceration is low even if undifferentiated cells recovered from living tissues are included, and the cells are safe, and further, the culture of the recovered Muse cells is not particularly limited, and can be performed in a normal expansion medium (e.g., α -minimal medium (e.g., MEM) containing 10% fetal bovine serum, and the culture medium can be used in international publication No. WO 007900, and the culture of Muse cells can be prepared in a medium, and the Muse cells can be used as an effective therapeutic agent after the culture of autologous or autologous bone marrow cells can be prepared, and the Muse cells can be obtained as an effective supplement under the conditions for the preparation of a therapeutic agent, for example, and the present invention.

In addition, in the use of a cell preparation of Muse cells, dimethyl sulfoxide (DMSO), serum albumin, or the like may be contained in the cell preparation in order to protect the cells, or antibiotics or the like may be contained in the cell preparation in order to prevent bacterial contamination and proliferation. The cell preparation may further contain other pharmaceutically acceptable components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, physiological saline, and the like). Those skilled in the art can add these factors and agents to the cell preparation at appropriate concentrations. Thus, Muse cells can also be used as pharmaceutical compositions containing various additives.

The number of Muse cells contained in the cell preparation prepared above can be appropriately adjusted in consideration of sex, age, body weight, condition of affected part, condition of cells used, and the like of the subject so as to obtain a desired effect in the treatment of epidermolysis bullosa. In addition, the subject includes, but is not limited to, a humanAnd the like. Furthermore, the Muse cell preparation of the present invention containing Muse cells may be administered multiple times at appropriate intervals (e.g., 1 day 2, 1 day 1, 1 week 2, 1 week 1, 2 weeks 1, 1 month 1, 2 months 1, 3 months 1 and 6 months 1) until the desired therapeutic effect is obtained. Therefore, the therapeutically effective amount, although varying depending on the state of the subject, may preferably be, for example: 1 × 10 each time for one individual³Cell 1X 10¹⁰1-10 application rates of cells in 1 year. The total amount of administration to an individual is not limited and may include 1X 10³Cell 1X 10¹¹Cells, preferably 1X 10⁴Cell 1X 10¹⁰The cells are more preferably 1X 10⁵Cell 1X 10⁹Cells, and the like.

Muse cells used in the cell preparation of the present invention have the property of migrating and surviving to damaged skin of epidermolysis bullosa. Therefore, in the administration of the cell preparation, the site or method of administration of the cell preparation is not limited, and the cell preparation may be administered locally to the affected part or may be administered intravenously.

The Muse cell preparation of the present invention can repair and regenerate damaged skin of a patient with epidermolysis bullosa. The repair and regeneration of the damaged skin can be confirmed by, for example, recovery of expression and/or increase in expression of collagen proteins such as COL7 and COL 17. That is, the Muse cell-containing cell preparation of the present invention has an effect of restoring and/or increasing the expression of collagen protein.

<2> skin cells induced from Muse cell differentiation and cell preparations containing the same in the present invention, skin cells such as keratinocytes and/or fibroblasts induced from Muse cell differentiation can also be used as cell preparations.

Keratinocytes are keratin-producing cells, also known as epidermal cells or keratinocytes. Differentiation induction from Muse cells into keratinocytes can be performed by culturing: for example, 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, the Muse cells are further cultured in a medium containing KGF, EGF, Hepatocyte Growth Factor (HGF) and insulin-like growth factor 2(IGF 2). Here, the preferable concentration range of KGF is, for example, 5 to 20ng/ml, the preferable concentration range of EGF is, for example, 20 to 40ng/ml, and the preferable concentration range of IGF2 is, for example, 40 to 80 ng/ml. The preferable culture time is, for example, 7 to 28 days.

Fibroblast is a cell that produces dermal components such as collagen and elastin, and differentiation induction from Muse cells to fibroblasts can be performed by culturing Muse cells in a medium containing, for example, transforming growth factor- β 2(TGF- β 2) and Ascorbic Acid (AA), preferably culturing Muse cells in a medium containing TGF- β 2 and AA, and then culturing the Muse cells in a medium containing AA, wherein TGF- β 2 is preferably used at a concentration of, for example, 30 to 60. mu.g/ml, AA at a concentration of, for example, 20 to 80mmol/l, and the culture time is preferably 7 to 28 days.

Cell preparations containing skin cells such as keratinocytes and/or fibroblasts obtained by inducing differentiation of Muse cells are useful not only for the treatment of epidermolysis bullosa but also for the treatment of all skin diseases that can be treated by a skin cell-supplementing therapy.

In the use of a cell preparation containing skin cells induced from Muse cell differentiation, dimethyl sulfoxide (DMSO), serum albumin, or the like may be contained in the cell preparation in order to protect the cells, or antibiotics or the like may be contained in the cell preparation in order to prevent bacterial contamination and proliferation. Further, the cell preparation may contain other pharmaceutically acceptable components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, physiological saline, and the like).

The amount of the cell preparation to be administered can be appropriately adjusted in consideration of the sex, age, body weight, state of an affected part, state of cells to be used, and the like of a subject so as to obtain a desired effect in the treatment of skin diseases. In addition, the subject includes, but is not limited to, a human or the likeA mammal. In addition, the cell preparation may be administered multiple times at appropriate intervals (e.g., 1 day 2, 1 day 1, 1 week 2, 1 week 1, 2 weeks 1, 1 month 1, 2 months 1, 3 months 1 and 6 months 1) until the desired therapeutic effect is obtained. Therefore, the therapeutically effective amount, although varying depending on the state of the subject, may preferably be, for example: 1 × 10 each time for one individual³Cell 1X 10¹⁰1-10 application rates of cells in 1 year. The total amount of administration to an individual is not limited and may include 1X 10³Cell 1X 10¹¹Cells, preferably 1X 10⁴Cell 1X 10¹⁰The cells are more preferably 1X 10⁵Cell 1X 10⁹Cells, and the like. The application method is not particularly limited, and topical application to the skin disease site or its surroundings is preferred. The skin cells may be flaked and applied to the affected part.

The present invention will be further specifically described with reference to the following examples, but the present invention is not limited to these examples.