JP2007267672A - Differentiation inducting method in stem cell of mammal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology effectively differentiation inducing biofunctional cells for tissue reproduction or restoration by using a stem cell separated form a biomedical tissue of mammal. <P>SOLUTION: The technology comprises preparation of biofunctional cells for transplantation used for reproduction medicine with high taking rate by using stem cells separated from a biomedical tissue of mammal and combining an adhesion culture with a suspension culture. The cell aggregate (sphere body) with a size of 50-500 μm diameter on the suspension culture process is continuously kept in an undifferentiated state of the stem cells, and manifested an excellent improvement in differentiation inducing rate to biofunctional cells of the skin and taking rate after transplantation. The invention relates to the technology continuously keeping undifferentiated state of stem cells and then effectively inducing differentiation of biofunctional cells for reproduction or restoration of tissue with a high taking rate. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to the regeneration and repair of tissue with a very high engraftment rate by combining stem cells isolated from mammalian biological tissue with one or more subculture (adhesion culture) and suspension culture processes. The present invention relates to an efficient differentiation-inducing method for obtaining functional cells for use in the present invention, and uses of the cells.

  In the case of vertebrate, particularly mammalian tissue, when cell or organ damage occurs due to injury or disease, or aging, the regeneration system works to try to recover the damage of the cell or organ. The pluripotent cells (hereinafter referred to as stem cells) provided in the tissue and surrounding cells play a major role in this action. In particular, stem cells have pluripotency to differentiate into all cells and organs, and it is considered that this property leads to recovery by supplementing damaged portions of cells and tissues. Expectations are gathered for regenerative medicine, which is the next generation of medicine using such stem cells.

  The most advanced tissue in stem cell research in mammals is the bone marrow. Living bone-forming hematopoietic stem cells exist in the bone marrow and are considered to be the main source of blood cells. Furthermore, it has been reported that the bone marrow includes stem cells that can be differentiated into other organs (eg, bone, cartilage, muscle, fat, etc.) in addition to hematopoietic stem cells (Non-patent Document 1).

Pittenger M.M. F. , Et al. , Science, 1999, 284, 143-147.

  Furthermore, in recent years, in addition to bone marrow, it has been clarified that stem cells exist in all organs such as liver, pancreas, fat and the like, and it is considered that they are greatly involved in maintaining homeostasis of each organ (non-patent literature). 2-5). In order to use these stem cells for regenerative medicine, it is necessary to separate the stem cells from the living tissue and efficiently induce differentiation into the intended functional cells of interest.

Goodell M.M. F. , Et al. Nat. Med. 1997, 3, 1337-1345. Zulewski H. , Et al. Diabetes, 2001, 50, 521-533. Suzuki A. , Et al. , Hepatology, 2000, 32, 1230-1239 Zuk P.M. A. , Et al. , Tissue Engineering, 2001, 7, 211-228.

  To date, there are several reports that isolate stem cells from bone marrow, blood, liver, pancreas, fat, and the like. These methods are mainly methods in which cells are dispersed from each organ by enzyme treatment or the like, and stem cells are separated using a flow cytometer or the like using centrifugation or a stem cell marker (protein or gene) as an index.

  For example, Zuk et al. Have reported a method of separating stem cells from fat using a centrifugation method (Non-patent Document 6). In addition, Reyes et al. Have reported a technique for isolating a stem cell by using a marker protein (AC133) of a stem cell (AC133) existing in the bone marrow and culturing it using a special culture solution (non-contained). Patent Document 7). Similarly, regarding a method for separating stem cells from the pancreas, a patent relating to a selective separation method using marker proteins (c-Met, c-Kit, CD45 and TER119) has been published (Patent Document 1). Furthermore, in another method, an invention relating to a method for separating stem cells by using mRNA encoding a stem cell marker protein (Ttm2A) in the liver is disclosed (Patent Document 2). As described above, there are reports or disclosures on techniques for selectively separating stem cells from each organ.

Zuk P.M. A. , Et al. , Molecular Biology of the Cell, 2002, 13, 4279-4295. Reyes M.M. , Et al. , J .; Clin. Invest. , 2002, 109, 337-346 WO2002 / 088335 JP 2004-187679 A

  In addition, studies on the induction of differentiation of stem cells isolated from each tissue are also underway.

  For example, an invention that induces differentiation into nerve cells by regulating the expression of octamer binding transcription factor-3 / 4 (Oct-3 / 4) in stem cells is disclosed (Patent Document 3). Also disclosed are methods for inducing differentiation into cartilage using mesenchymal stem cells (Non-patent Documents 8 and 4) and methods for inducing differentiation into white blood cells by causing Notch ligand protein to act on hematopoietic stem cells. There is also (patent document 5). Furthermore, differentiation induction techniques for various cells such as differentiation induction into insulin-producing cells (β cells) (Non-patent Document 9) and differentiation induction into cardiomyocytes (Non-patent Document 10) have been reported.

JP-A-2004-236607 Grigoriadis A. E. , Et al. , Journal of Cell Biology, 1988, 106, 2139-2151. JP 2004-254655 A JP 2005-13059 A Lumelsky N.M. , Et al. , Science, 2001, 292, 1389-1394. Makino S. , Et al. , Journal of Clinical Investology, 1999, 103, 697-705.

  Furthermore, in recent years, there are reports or disclosures on special culture techniques that perform these differentiation induction techniques more effectively and increase differentiation induction efficiency.

  For example, an invention (Patent Document 6) that efficiently induces vascular cells by applying electrical stimulation of 1 to 5000 mV at the time of inducing differentiation of stem cells, or after putting stem cells into the lumen of a hollow fiber, centrifugal force or pressure There is also a disclosure of an invention that enhances the efficiency of inducing differentiation into chondrocytes by culturing stem cells in an undifferentiated state by forming an aggregate by applying (Patent Document 7). In addition, there is a report on a suspension culture method that specifically guides stem cells obtained from the brain to nerve cells (Non-patent Document 11).

JP 2004-129603 A JP 2004-166604 A Reynolds B.M. A. , Journal of Neuroscience, 1992, 12, 4565-4574.

  As described above, there are several prior arts related to the present invention. Considering application to regenerative medicine, in order to regenerate a complex tissue, functional cells adapted to the damage of the tissue are used. It is necessary to prepare it efficiently.

  In particular, the skin is a complex tissue composed of many functional cells such as keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and hair follicle cells. It is necessary to efficiently prepare these functional cells and use them for transplantation.

  In recent years, in skin regenerative medicine, clinical applications such as cultured skin sheets and three-dimensional cultured skin have been promoted. However, since these cultured skins are composed only of keratinocytes or fibroblasts, they do not contain other functional cells (pigment cells, sebaceous gland cells, adipocytes, or hair follicle cells) and incorporate these cells. However, regeneration of highly functional skin has been desired (Non-patent Documents 12 and 13).

Sadaki Higuchi, History of Medicine, 2001, 199, 1142-1146 Yuji Shirakata, Journal of the Japanese Society of Skin, 1999, 109, 1301-1307

  In addition, until now, the more complex tissues, the lower the survival rate after cell transplantation, which requires repeated cell transplantation, and a satisfactory therapeutic effect has not been obtained. In the future, in order to stably supply functional cells for transplantation with a high engraftment rate in regenerative medicine, a simpler method for inducing differentiation of functional cells with a high engraftment rate has been desired.

  In view of such a situation, the present invention solves the problems in the prior art as described above, and provides functional cells used for tissue regeneration and repair, which have an extremely high engraftment rate, from stem cells separated from living tissue. An object of the present invention is to provide an efficient differentiation induction method for obtaining the above.

  Under such circumstances, as a result of intensive research, the present inventors have combined stem cells isolated from mammalian biological tissue with one or more adhesion culture and suspension culture steps, and thus the undifferentiated state of the stem cells. Culturing was carried out while maintaining the above, and a technique for inducing differentiation into functional cells with an extremely high engraftment rate was found, and the present invention was completed.

  Therefore, the present invention is a culture technique for inducing differentiation of the cells into functional cells step by step in the process of culturing the stem cells, and the culture stage of the cells is 1) a process by adhesion culture, 2) suspension culture 3) It is carried out in three stages including a process by differentiation-inducing culture. By culturing by this process, culturing is performed while the undifferentiated state of the stem cells is continuously maintained. A very high functional cell for transplantation is prepared.

  Hereinafter, the present invention will be described in more detail.

  Stem cells that can be used in the present invention are embryonic stem cells or bone marrow, blood, skin, fat, brain, liver, pancreas, kidney, muscle, and other tissues as long as they meet the purpose of the present invention. Somatic stem cells obtained, and further, primary cultured cells, subcultured cells, or frozen cells of the cells may be used. However, preferably, stem cells derived from bone marrow, blood, skin, or adipose tissue can be used. Moreover, if it has the same characteristic about the direction of differentiation of a stem cell in a mammal, the process of differentiation, etc., it can apply to all mammals. For example, stem cells obtained from mammals such as human, monkey, mouse, rat, guinea pig, rabbit, cat, dog, horse, cow, sheep, goat and pig can be used.

  Moreover, as a method of obtaining a mammalian stem cell, it isolate | separates using apparatuses, such as the method described in said nonpatent literature 1-11, the method by centrifugation, and a stem cell marker as a parameter | index, such as a flow cytometer. A method is mentioned.

  With respect to the stem cells obtained from these mammals, the stem cells are cultured by a culture method including the following three stages, and then differentiation induction is performed.

  1) Step by adhesion culture: The first step of culturing stem cells obtained from a mammal in an in vitro environment. Specifically, a step of mainly cultivating stem cells by adhesion-culturing stem cells using a culture solution for adhesion culture. This step is preferably performed at least once.

  2) Step by suspension culture: 1) The second step of culturing in an in vitro environment after the step by adhesion culture. Specifically, a step of suspension-culturing stem cells using a culture solution for suspension culture, mainly improving the pluripotency of stem cells and the survival rate at the time of transplantation. Preferably, a step of forming a single cell and / or an aggregate (sphere body) of cells by floating cells. This step is preferably performed at least once.

  The cell aggregate (sphere body) in the above-mentioned 2) suspension culture step is a stem cell proliferated by suspension culture and is a real cell aggregate of mulberry. Confirmation of the aggregate is performed with a microscope. At this time, the size of the aggregate is preferably 50 to 500 μm in diameter. Moreover, it is more preferable that a diameter is 100-300 micrometers.

  3) Step by differentiation induction culture: 2) Third step of culturing in an in vitro environment after the step by suspension culture. Specifically, a step of inducing differentiation of stem cells using a culture medium for differentiation induction culture, and mainly inducing differentiation of the stem cells into target functional cells.

  1) As a culture solution for adhesion culture used in the process by adhesion culture, the following can be used.

  Specifically, a basic medium (for example, Dulbecco's Modified Eagle Medium (D-MEM), Minimum) containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins) necessary for viable growth of cells. Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), GlasgowM And at least one of basic fibroblast growth factor (bFGF) and leukocyte migration inhibitory factor (LIF) Medium is used, preferably those that all of these growth factors is contained. In order to increase the growth rate, epithelial cell growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, Progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement may be contained. Moreover, antibiotics may be contained as needed.

  Moreover, it is preferable that serum is contained in the culture solution at a content of 1 to 20%. However, since serum has different components depending on the lot and there are variations in the effects, it is preferable to use the serum after a lot check.

  As commercially available products, mesenchymal stem cell basal medium manufactured by Invitrogen, mesenchymal stem cell basal medium manufactured by Sanko Junyaku, etc. can be used.

  2) As the culture solution for suspension culture used in the suspension culture step, the following can be used.

  Specifically, a basic medium (for example, Dulbecco's Modified Eagle Medium (D-MEM), Minimum) containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins) necessary for viable growth of cells. Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), GlasgowM Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), leukocyte migration inhibitory factor (LIF), Is B27-medium supplemented with at least one kind of supplement is used, it is preferably, that all of these additives factors is contained. In order to maintain the undifferentiated state of stem cells and to improve the engraftment rate at the time of transplantation, tumor necrosis factor (TNF), insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, Progesterone, selenite, N2-supplement, ITS-supplement may be contained. Moreover, antibiotics may be contained as needed.

  Moreover, it is preferable that the said culture solution does not contain serum. This is because serum has different components depending on lots and may contain unknown components that affect stem cell differentiation.

  As commercial products, Neurobasal Medium manufactured by Invitrogen, neural progenitor cell culture medium kit manufactured by Takara, and the like can be used.

  3) As the culture medium for differentiation induction used in the step of differentiation induction culture, the following can be used.

  Specifically, a basic medium (for example, Dulbecco's Modified Eagle Medium (D-MEM), Minimum) containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins) necessary for viable growth of cells. Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), GlasgowM As factors, cell basic fibroblast growth factor (bFGF), epithelial cell growth factor (EGF) according to each functional cell Keratinocyte growth factor (KGF), nerve cell growth factor (NGF), stem cell factor (SCF), transforming growth factor (TGF-β), tumor necrosis factor (TNF), endothelins, vitamins, cytokines, inter A leukin, insulin, transferrin, heparin, heparan sulfate, retinoic acid, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement are contained and used as necessary. Moreover, antibiotics may be contained as needed.

  Moreover, it is preferable that serum is contained in the culture solution at a content of 0.1 to 20%. However, since serum has different components depending on the lot and there are variations in the effects, it is preferable to use the serum after a lot check.

  As a commercially available product, a differentiation induction culture solution manufactured by Sanko Junyaku, a differentiation culture kit manufactured by TOYOBO, or the like can be used.

  In any of the culturing steps, it is preferable to perform the culture in the environment of 37 ° C. and 5% carbon dioxide gas and replace the medium every 1 to 3 days using the above culture solution.

  By performing the above culturing steps 1) to 3), culturing while maintaining the undifferentiated ability of the stem cells, and then regenerating and / or regenerating tissue having a very high engraftment rate by inducing differentiation. Functional cells used for repair can be obtained.

  The present invention uses stem cells isolated from mammalian tissue such as bone marrow, blood, fat, or skin tissue, and maintains the undifferentiated state of the stem cells by the culture steps of 1) to 3) above. It is a differentiation induction method characterized in that functional cells used for regeneration and / or repair of a tissue having an excellent engraftment rate are obtained simply and efficiently by culturing and then differentiation induction. In particular, when the functional cells used for transplantation are keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and / or hair follicle cells that constitute the skin, a significant improvement in engraftment is achieved. It is a differentiation induction method that can be seen. From the above, the present invention is expected to contribute greatly in the field of tissue regenerative medicine.

  Hereinafter, specific and detailed examples will be given to describe the present invention in detail, but the present invention is not limited thereto.

  Stem cells are separated from bone marrow, blood, fat, or skin tissue by centrifugation according to the following method, followed by one or more adhesion cultures, which are the culture steps of 1) to 2) above, and then transferred to suspension culture. Stem cell state (undifferentiated state) in a conventional method of culturing only by adhesion culture, a method of culturing only by suspension culture, and a method combining the adhesion culture and suspension culture of the present invention Was evaluated by the following method.

Preparation of culture fluid 1 (adhesive culture fluid)
As an adhesion culture medium, a 1: 1 mixed culture medium (Gibco) of Dulbecco's Modified Eagle Medium and F-12 culture medium (Febco), fetal bovine serum (FBS, 10%), non-essential amino acid solution (Gibco), nucleoside solution ( Gibco), a culture solution containing 10 ³ U ESGRO (CHEMICON), 100 unit / mL penicillin (Sigma) and 100 μg / mL streptomycin (Boehringer) was used (hereinafter referred to as culture solution 1).

Preparation of culture solution 2 (floating culture solution)
As a suspension culture solution, 1: 1 mixed culture solution (manufactured by Gibco) of Dulbecco's Modified Eagle Medium and F-12 culture medium (manufactured by Gibco), basic fibroblast growth factor (bFGF, 10 ng / mL, manufactured by Sigma), epithelial cells Growth factor (EGF, 10 ng / mL, manufactured by Sigma), non-essential amino acid solution (manufactured by Gibco), nucleoside solution (manufactured by Gibco), B27 supplement (manufactured by Invitrogen), 10 ³ U ESGRO (manufactured by CHEMICON), 100 unit / mL A serum-free culture medium supplemented with penicillin (manufactured by Sigma) and 100 μg / mL streptomycin (manufactured by Boehringer) was used (hereinafter referred to as culture medium 2).

Preparation of Bone Marrow-Derived Stem Cells The femurs of C57BL / 6 mice (male, 4 weeks old) were aseptically removed, and the peripheral connective tissue was removed as much as possible. Thereafter, a syringe with a 25G needle was inserted into one of the bone ends, PBS (−) was injected, and the bone marrow was pushed out into a 50 mL centrifuge tube (Falcon). Then, it was transferred to another 50 mL centrifuge tube while passing through a cell strainer (Falcon) and centrifuged. The supernatant was removed, and the culture medium 2 was newly added to disperse and wash the cells. This washing operation was repeated twice. After washing, stem cells were separated by centrifugation.

Preparation of blood-derived stem cells Blood was collected from C57BL / 6 mice (male, 4 weeks old) and treated with erythrocyte blood (Miltiny Bio) for 7 minutes. Then, after centrifuging and removing the supernatant, the culture medium 2 was newly added to disperse and wash the cells. This washing operation was repeated twice. After washing, stem cells were separated by centrifugation.

Preparation of skin and adipose-derived stem cells After C57BL / 6 (male, 4 weeks old) was shaved, the skin and abdominal subcutaneous adipose tissue were separately aseptically removed and washed three times with PBS (−) And transferred to a tissue culture dish (Falcon) having a diameter of 6 cm. Each tissue was chopped into approximately 2 mm squares with a pointed blade, 0.2% collagenase solution (manufactured by Nitta Gelatin) was added, and the plastic dish was shaken up and down and left and right to diffuse into the solution. These were incubated at 37 ° C. for 30 minutes to digest the extracellular matrix and then gently pipet to disperse the cells. This cell dispersion was transferred to a 50 mL centrifuge tube (Falcon) while passing through a cell strainer (Falcon). Further, an appropriate amount of the culture solution 1 was added, pipetted well, and then centrifuged for 5 minutes. After centrifugation, the supernatant fraction was removed, and the culture medium 1 was newly added to disperse and wash the cells. This washing operation was repeated twice. After washing, stem cells were separated by centrifugation.

  Using stem cells obtained from bone marrow, blood, skin, and adipose tissue by the above method, first, adhesion culture was performed for 3 days with culture medium 1, and then transferred to suspension culture with culture medium 2 for 14 days. Culture was carried out (culture method for transferring to suspension culture after adhesion culture; hereinafter referred to as Example 1). In addition, the cell aggregate (sphere body) obtained by suspension culture was 100-300 micrometers (it applies also to subsequent tests).

Comparative Example 1
Similarly to Example 1, each cell prepared from bone marrow, blood, skin, and adipose tissue was used for continuous culture for 17 days with culture medium 1 without performing suspension culture with culture medium 2. (Culture method by adhesion culture. Hereinafter, referred to as Comparative Example 1).

Comparative Example 2
In the same manner as in Example 1, each cell prepared from bone marrow, blood, skin and adipose tissue was used for continuous culture for 17 days in culture medium 2 without performing adhesion culture in culture medium 1. (Culture method by suspension culture. Hereinafter, referred to as Comparative Example 2). In addition, the cell aggregate (sphere body) obtained by suspension culture was 100-300 micrometers similarly to Example 1 (it applies also to subsequent tests).

Comparison of undifferentiated state of stem cells by measurement of alkaline phosphatase activity The undifferentiated state of stem cells in Comparative Example 1, Comparative Example 1 and Comparative Example 2 were compared by the following method.

  Alkaline phosphatase activity was measured as an indicator of the undifferentiated state of stem cells (Reference: Hirofumi Suemori, separate volume on experimental medicine, stem cell / clone research protocol, 2001, 10-16). Specifically, using an alkaline phosphatase detection kit (manufactured by CHEMICON), the alkaline phosphatase activity at the start of the culture in Example 1, Comparative Example 1 and Comparative Example 2 was taken as 100%, after 3 days, 7 days and 14 days after the culture. The alkaline phosphatase activity (%) was measured and the results are shown in 1-4.

  From the results shown in Tables 1 to 4 above, the method of Example 1 was compared with Comparative Example 1 and Comparative Example 2 in all bone marrow, blood, skin and adipose-derived stem cells. Even after 14 days, the undifferentiated state of the stem cells was remarkably maintained.

  In addition, by adjusting the period of suspension culture, the same test was performed on cell aggregates (sphere bodies) having a diameter of 50 μm or less and a diameter of 500 μm or more, compared with those having a diameter of 100 to 300 μm. Thus, the undifferentiated state (alkaline phosphatase activity%) decreased by about 20%.

  From the above results, it was confirmed that the cell aggregate (sphere body) having a diameter of 100 to 300 μm in the culture method according to Example 1 continuously maintained the undifferentiated state of the stem cells.

  Using each cell cultured in Example 1, to the keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and hair follicle cells, which are functional cells constituting the skin by the following method. The differentiation induction efficiency (%) was compared.

Differentiation induction and confirming the embodiment of the keratinocytes 1, each of the cells cultured in Comparative Example 1 or Comparative Example 2, keratinocyte growth factor keratinocyte culture medium for (manufactured by TOYOBO) (KGF, 10ng / mL And cultured at 37 ° C. and 5% CO ₂ for 14 days. The culture medium was replaced with a fresh culture medium for keratinocytes every 3 days. Confirmation of differentiation induction into keratinocytes was performed using cells stained with keratin as an index by immunostaining (keratin) after fixing the cells with a 4% paraformaldehyde solution.

Differentiation induction and confirmation into fibroblasts Each cell cultured in Example 1, Comparative Example 1 or Comparative Example 2 was added to fibroblast growth medium (manufactured by TOYOBO) with fibroblast growth factor (FGF, 10 ng / mL). And cultured at 37 ° C. and 5% CO ₂ for 14 days. The culture medium was replaced with a fresh fibroblast culture medium every 3 days. Confirmation of differentiation induction into fibroblasts was performed using cells stained with fibronectin as an index by immunostaining (fibronectin) after fixing the cells with a 4% paraformaldehyde solution.

Differentiation induction into pigment cells and confirmation Each cell cultured in Example 1, Comparative Example 1 or Comparative Example 2 was treated with stem cell factor (SCF, 10 ng / mL, manufactured by Sigma) and endothelin in pigment cell culture medium (TOYOBO). In a medium supplemented with (10 ng / mL, manufactured by Sigma), the cells were cultured for 14 days under conditions of 37 ° C. and 5% CO ₂ . The culture medium was replaced with a fresh pigment cell culture medium every 3 days. Confirmation of differentiation induction into pigment cells was performed using cells stained with Fontana-Masson staining (melanin) after fixing the cells with a 4% paraformaldehyde solution as an index.

Differentiation induction and confirmation into sebaceous gland cells Each cell cultured in Example 1, Comparative Example 1 or Comparative Example 2 was mixed with a culture solution for sebaceous gland cells (Dulbecco's Modified Eagle Medium and F-12 culture solution in 1: 1 mixture). In a medium in which fetal bovine serum (FBS, 10%), epidermal growth factor (EGF, 10 ng / mL, Sigma) and testosterone (Sigma) are added to a culture solution (Gibco)) at 37 ° C., 5 ° C. The cells were cultured for 14 days under the condition of% CO ₂ . The culture solution was replaced with a fresh adipocyte culture solution every 3 days. Confirmation of differentiation induction into sebaceous gland cells was performed by fixing cells with 4% paraformaldehyde solution and then immunostaining (PPARγ) using the cells stained with PPARγ as an index.

Differentiation induction into adipocytes and confirmation Each cell cultured in Example 1, Comparative Example 1 or Comparative Example 2 was cultured in a medium in which insulin (Sigma) was added to a culture medium for fat cells (TOYOBO). ° C., and cultured for 14 days in 5% CO ₂ conditions. The culture solution was replaced with a fresh adipocyte culture solution every 3 days. Confirmation of differentiation into adipocytes was performed by fixing cells with a 4% paraformaldehyde solution and then using oil red O staining as a marker for cells in which fat droplets were stained red.

Differentiation induction and confirming the embodiment of the hair follicle cells 1, the medium of each of cells cultured in Comparative Example 1 or Comparative Example 2, with the addition of magnesium ascorbate (Sigma) in hair follicle cells for culture (manufactured by TOYOBO) And cultured at 37 ° C. under 5% CO ₂ for 14 days. The culture medium was replaced with a fresh culture medium for hair follicle cells every 3 days. Confirmation of differentiation induction into hair follicle cells was carried out by fixing cells with a 4% paraformaldehyde solution and then immunostaining (hair keratin) using the cells stained with hair keratin as an index.

Comparative Example 3
Using each cell cultured in Comparative Example 1 (adhesion culture), the same as in Example 2, the above keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and hair follicle cells. Differentiation induction and confirmation were performed.

Comparative Example 4
Using each cell cultured in Comparative Example 2 (suspension culture), in the same manner as in Example 2, the keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and hair follicle cells Differentiation induction and confirmation were performed.

The cells cultured in Comparative Example 2, Comparative Example 3 or Comparative Example 4 in differentiation induction efficiency (%) of Example 2, Comparative Example 3 and Comparative Example 4 were keratinocytes, fibroblasts, pigment cells, and sebaceous glands, respectively. After induction of differentiation into cells, adipocytes, and hair follicle cells, each cell was confirmed and stained, and the number of differentiated cells per single cell (500 cells) was counted by microscopic observation. Based on the result, the differentiation induction efficiency (ratio% of the number of differentiated cells in the measured cells (500)) was calculated, and the differentiation induction efficiency in Example 2, Comparative Example 3 and Comparative Example 4 were compared.

  From the results shown in Tables 5 to 8, in the method of Example 2, the functional cells (corner) constituting the skin in all stem cells derived from bone marrow, blood, skin, and fat, compared to Comparative Example 3 and Comparative Example 4. Differentiation induction efficiency into cells, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and / or hair follicle cells) significantly increased.

  Moreover, when the same test was performed on the cell aggregate (sphere body) having a diameter of 50 μm or less and a diameter of 500 μm or more, differentiation induction efficiency (%) was higher than that of 100 to 300 μm in diameter. It decreased by about 20%.

  From the above results, it was confirmed that the differentiation induction efficiency into each functional cell was remarkably improved by the culture method according to Example 2.

  Using the cells induced to differentiate in Example 2, Comparative Example 3 and Comparative Example 4, cell transplantation was performed by the following method, and the respective engraftment ratios (%) after transplantation were compared.

Confirmation of engraftment rate (%) After separating stem cells from bone marrow, blood, fat or skin tissue, each cell (keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and cells induced to differentiate in Example 2) (Or hair follicle cells) was subjected to skin transplantation by the following method, and the survival rate (%) was measured.

Preparation of functional cells constituting skin for transplantation Each of the cells (keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and / or hair follicle cells) induced to differentiate in Example 2 was used. Functional cells that were dispersed in Hank's balanced salt solution with% FBS, differentiated with the following markers for each cell, were labeled and separated and collected with a flow cytometer. Specifically, an anti-keratin antibody (manufactured by CHEMICON) as a marker for separating keratinocytes, an anti-fibroblast antibody (manufactured by R & D) as a marker for separating fibroblasts, and an anti-MITF antibody (Santa) as a marker for separating pigment cells Cruz), anti-PPARγ antibody (manufactured by Baybio) as a sebaceous gland cell separation marker, Nile Red staining (manufactured by Aldrich) as a fat cell separation marker, anti-hair keratin antibody (manufactured by Santa Cruz) as a marker for hair follicle cell separation ) For 30 minutes in ice and then washed 3 times with 5% FBS-added Hanks solution. Subsequently, it was reacted with Alexa Fruo 488-labeled anti-IgG monoclonal secondary antibody (manufactured by Invitrogen) for 30 minutes in ice. NillRed staining was omitted because the secondary antibody reaction step was not necessary. After completion of the reaction, the cells were washed 3 times with Hanks solution containing 5% FBS, and then the cells were suspended in PBS. Each of these fluorescently labeled functional cells was separated and collected by a flow cytometer (manufactured by Beckton Dickinson), and used as a cell for transplantation.

Measurement of functional cell transplantation and engraftment rate (%) Each functional cell (keratinocyte, fibroblast, pigment cell, sebaceous gland cell, adipocyte and / or hair follicle cell) separated and collected by a flow cytometer 10 ⁶ were implanted subcutaneously in nude mice (male, 4 weeks old). Specifically, 10 ⁶ cells to be transplanted, and fluorescently labeled in advance Cell Tracker (Molecular Probe) was 100% by measuring the fluorescence intensity at that time. Mark the transplant site with magic, remove the transplant site 1 week, 2 weeks and 4 weeks after transplantation, disperse the cells by enzyme treatment, measure the fluorescence intensity, and compare it with the fluorescence intensity at the time of transplantation (100%) Thus, the survival rate (%) was calculated.

Comparative Example 5
Using each cell (keratinocyte, fibroblast, pigment cell, sebaceous gland cell, adipocyte and / or hair follicle cell) induced to differentiate in Comparative Example 3, nude mice (male, 4 weeks) (Age) was transplanted subcutaneously, and the survival rate (%) was measured.

Comparative Example 6
Using each cell (keratinocyte, fibroblast, pigment cell, sebaceous gland cell, adipocyte and / or hair follicle cell) induced to be differentiated in Comparative Example 4, nude mice (male, 4 weeks) (Age) was transplanted subcutaneously, and the survival rate (%) was measured.

Functional cells (keratinocytes, fibers) constituting the skin in Comparative Example 3, Comparative Example 5 or Comparative Example 6 in engraftment rate (%) after cell transplantation according to Example 3, Comparative Example 5 or Comparative Example 6 The engraftment rate (%) after 1 week, 2 weeks and 4 weeks of transplantation of blast cells, pigment cells, sebaceous gland cells, adipocytes and / or hair follicle cells) was compared, and the results are shown in 9-14.

  From the results shown in Tables 9 to 14, Example 3 showed an extremely high engraftment rate after 1 week, 2 weeks, and 4 weeks after transplantation, as compared with Comparative Example 5 or Comparative Example 6.

  Further, cells other than those in Tables 9 to 14 (fibroblasts, pigment cells, sebaceous gland cells, adipocytes or hair follicle cells, blood-derived stem cells derived from bone marrow-derived stem cells in Example 3, Comparative Example 5 and Comparative Example 6) Differentiation induced from keratinocytes, pigment cells, sebaceous gland cells, adipocytes or hair follicle cells, fibroblasts differentiated from skin-derived stem cells, pigment cells, sebaceous gland cells, adipocytes or hair follicle cells, differentiation from adipose-derived stem cells When the same test was performed on induced keratinocytes, fibroblasts, pigment cells), all of the methods of Example 3 were compared with Comparative Example 5 and Comparative Example 6 for 1 week, 2 weeks of transplantation, All four weeks later, a very high survival rate was confirmed.

  In addition, when the same test was performed with the cell aggregate (sphere body) having a diameter of 50 μm or less and a diameter of 500 μm or more, the engraftment rate (%) was higher than that of 100 to 300 μm in diameter. It decreased by about 30%.

  From the above results, it was confirmed that the method according to Example 3 significantly improved the engraftment rate in cell transplantation.

  From the above results, it is possible to combine adhesion culture and suspension culture using stem cells obtained from bone marrow, blood, fat or skin tissue, compared to conventional culture using only adhesion culture or culture using suspension culture alone. It was revealed that the differentiation induction efficiency into functional cells with a high engraftment rate was significantly improved by maintaining the undifferentiated state of stem cells. At this time, regarding the size of the cell aggregate (sphere body) in the floating cell having a diameter of 50 to 500 μm, maintenance of the undifferentiated state of the stem cell and subsequent differentiation induction efficiency into the functional cell, and further transplantation The survival rate at the time was very good. In particular, in the differentiation induction method of the present invention, the differentiation induction rate is synergistically used as functional cells in keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and / or hair follicle cells constituting the skin. It was clarified to improve. According to the present invention, it is possible to easily prepare functional cells (keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and / or hair follicle cells) constituting the skin, It is considered possible to prepare necessary functional cells from stem cells according to the damage. Therefore, it is considered that the present invention can greatly contribute to a cell preparation method and a transplantation method for transplantation in future regenerative medicine.

As an application example of the present invention, application to regenerative medicine is expected. For example, it has become possible to efficiently prepare transplant cells with a high engraftment rate in regenerative medicine from bone marrow, blood, skin and adipose tissue stem cells. In particular, it is possible to easily prepare functional cells (keratinocytes, fibroblasts, pigment cells, sebaceous gland cells, adipocytes and / or hair follicle cells) constituting the skin, and further, engraftment after transplantation. Since the rate is extremely high, it is expected as a useful cell preparation technique in future regenerative medicine. That is, the present invention is an excellent stem cell that efficiently induces differentiation of a stem cell obtained from a living tissue into a functional cell for regeneration and / or repair of tissue such as skin, which has a much higher engraftment rate than before. It can be said that this is a differentiation induction method.

Claims (7)

Stem cells isolated from mammalian biological tissue are performed in three stages including 1) adhesion culture, 2) suspension culture, and 3) differentiation induction culture. A method for inducing differentiation into sex cells. The method for inducing differentiation of a stem cell into a functional cell according to claim 1, wherein the living tissue is derived from bone marrow, blood, skin and / or adipose tissue. The method for inducing differentiation of a stem cell into a functional cell according to claim 1, wherein single cells and / or cell aggregates (sphere bodies) are formed as suspension culture. The method for inducing differentiation of a stem cell into a functional cell according to claim 1, wherein the cell aggregate (sphere body) is 50 to 500 μm. 5. The stem cell according to claim 1, wherein the functional cell is a keratinocyte, fibroblast, pigment cell, sebaceous gland cell, adipocyte and / or hair follicle cell constituting the skin. A method for inducing differentiation into functional cells. A functional cell for tissue regeneration and / or repair, prepared by the method for inducing differentiation of a stem cell according to claim 1. A stem cell undifferentiated maintenance culture method characterized in that stem cells isolated from mammalian biological tissue are performed in two stages including 1) a process by adhesion culture and 2) a process by suspension culture.