KR20060110542A - Stem cells derived from normal breast tissue and breast cancer tissue, preparation method thereof and differentiated cells from the stem cell - Google Patents

Abstract

Stem cells derived from the normal breast tissue and breast cancer tissue, a preparation method thereof and differentiated cells from the same stem cells are provided to be differentiated into various cells in human body, so that various diseases are treated. The multi-functional stem cells derived from the normal breast tissue and breast cancer tissue are provided, wherein the stem cells are capable of being differentiated into epidermal cells, nerve cells or bone marrow cells. The method for preparing the multi-functional stem cells derived from the breast tissue comprises the steps of: (a) separating breast cells from the breast tissue; (b) culturing the breast cells in a medium; (c) collecting the floating breast cells from the cultured medium; and (d) subculturing the floating breast cells in a medium and separating the multi-functional stem cells. The differentiated cells are prepared by (a) culturing the multi-functional stem cells derived from the breast tissue in a medium, and (b) separating the target differentiated cells expressing a marker during the cultivation, wherein the target differentiated cell is an epidermal cell, nerve cell or bone marrow cell.

Description

Stem cells derived from breast tissue, methods for preparing the same and cells differentiated therefrom {Stem Cells Derived from Normal Breast Tissue and Breast Cancer Tissue, Preparation Method Thereof and Differentiated Cells from the Stem Cell}

Figure 1 shows the results of observing the change in the form of the passage of breast cells (A-D: abrasion spares derived from normal breast tissue, E-H: abrasion spares derived from breast cancer tissue).

Figure 2a is a graph showing the change in the number of viable cells according to the passage of normal breast cells, Figure 2b is a graph showing the change in the number of viable cells according to the passage of breast cancer cells.

Figure 3 shows the results of morphological changes while differentiating stem cells derived from breast tissue into epithelial cells (A: differentiated stem cells derived from normal breast tissue, B: differentiated stem cells derived from breast cancer tissue) .

Figure 4 shows that the differentiation of stem cells derived from normal breast tissue into epithelial cells, the expression of myoepithelial cells and coronary cell markers.

Figure 5 shows that the differentiation of stem cells derived from breast cancer tissue into epithelial cells, the expression of myoepithelial cells and coronary cell markers.

Figure 6 shows the results of observing changes in morphology while differentiating stem cells derived from breast tissue into nerve cells or bone marrow cells (A: stem cells derived from normal breast tissue, B: stem cells derived from breast cancer tissue) ).

FIG. 7 shows that neuronal or bone marrow cell markers are expressed as a result of differentiation of stem cells derived from normal breast tissue into nerve cells or bone marrow cells.

8 shows that neuronal or bone marrow cell markers are expressed as a result of differentiating stem cells derived from breast cancer tissue into epithelial cells.

TECHNICAL FIELD The present invention relates to stem cells, and more particularly, to stem cells derived from breast tissue and a method for producing the same, and cells differentiated from pluripotent stem cells derived from breast tissue and a method for producing the same.

Embryonic stem cells can be differentiated into all types of cells that make up the body, while adult stem cells mean specific progenitor cells that are more differentiated than embryonic stem cells. do. Adult stem cells have the property that what is found in certain tissues generally has the ability to regenerate on their own, as well as to differentiate into other types of cells.

Such stem cells can be used to treat diseases by differentiating them into cells of interest and transplanting them into a desired site when tissues or organs are damaged or fail to function due to the disease. Embryonic stem cells can be used to treat a variety of diseases because they can differentiate into all types of cells, but are difficult to obtain and have ethical problems that prevent them from being used in clinical practice. In order to replace this, there is an active development of adult stem cells in the art.

International Patent Publication No. WO 2003/027281 relates to pluripotent stem cells derived from the epilepsy of skeletal muscle, wherein the pluripotent stem cells are skeletal muscle cells, smooth muscle cells, cardiomyocytes, blood cells, vascular endothelial cells, fat cells, osteoblasts, It has the ability to differentiate into nerve cells, hepatic cells, pancreatic cells, and can be used for the treatment of tissue or cell regeneration, heart failure, liver failure, renal failure, leukemia, neurodegenerative diseases, arthritis, diabetes, arteriosclerosis, etc. have.

Korean Patent Laid-Open Publication No. 2003-0069115 relates to a method for the isolation and culturing of cord blood-derived mesenchymal stem cells and progenitor cells and to induce differentiation into mesenchymal tissue. After superimposing cord blood in Ficoll-Hypack solution, centrifugation is performed to obtain mononuclear cell layer precipitation. Cells derived from umbilical cord blood, characterized in that the mononuclear cells obtained by monolayer culturing are reacted with antibodies against mesenchymal stem cells and progenitor-specific antigens for a predetermined time and separated and cultured only with the cells bound to the antibody using a cell separator. Disclosed is a method for isolating mesenchymal stem cells and progenitor cells.

Korean Patent Laid-Open Publication No. 2004-0068135 relates to a salivary gland epithelial-derived stem cell and its use, and more particularly derived from salivary gland epithelial cells, and A-fetoprotein positive cells, albumin positive cells, amylase positive by in vitro culture. The present invention relates to stem cells capable of differentiating into cells, insulin-positive cells, and glucagon-positive cells, and a method capable of regenerating organs such as liver by transplanting the stem cells is disclosed.

In addition, adult stem cells, which are known to possess pluripotency to differentiate into several types of cells, include (1) neurons (neurons, oligodentrosites and estrosites), Hematopoietic stem cells that differentiate into muscle cells, cardiomyocytes and hepatocytes, (2) Bone marrow stromal cells, which differentiate into cardiomyocytes and muscle cells, and (3) brain stems that differentiate into blood cells and muscle cells Brain stem cells and the like. To date, no pluripotent stem cells derived from breast tissue and capable of differentiating into cells other than the cells constituting the breast tissue are known.

The present invention provides pluripotent stem cells derived from breast tissue.

In another aspect, (a) separating breast cells from breast tissue; (b) primary culturing of the breast cells in a stem cell culture medium; (c) collecting the suspended cells from the primary culture; And (d) provides a method for producing pluripotent stem cells derived from breast tissue comprising the step of passage the cells in the suspended state in the culture medium for stem cells to separate pluripotent stem cells.

In another aspect, a cell differentiated from the pluripotent stem cells derived from the breast tissue is provided.

As another aspect, breast tissue comprising the steps of: (a) culturing pluripotent stem cells derived from breast tissue in a medium for culturing target differentiated cells, and (b) separating differentiated cells expressing markers of the target differentiated cells during culturing. Provided are methods for producing cells differentiated from derived pluripotent stem cells.

The present invention relates to pluripotent stem cells derived from differentiated breast tissue.

In the present invention, "pluripotent stem cell (or mammoth spare)" refers to a stem cell having the ability to differentiate into not only tissues from which adult stem cells are derived (ie breast tissue) but also other types of cells. Pluripotent stem cells derived from breast tissue according to the present invention can differentiate into epithelial cells, nerve cells or bone marrow cells, but are not limited thereto.

In the present invention, "mamosfair" refers to a spherical cell mass composed of epithelial cells obtained by passage of breast cells isolated from breast tissue under floating culture conditions. Specifically, when breast cells are subcultured in a floating culture condition, most epithelial cells are killed by apoptosis (this process is known as anoikis), but extremely epithelial cells survive and form spheres. These are called "Mamosfair". In this way, at least one passaged at least one passage is made of breast stem / progenitor cells only (Dontu, G. et al. 2003, Cell Prolif. "Stem cells in normal breast development and breast cancer", vol. 36 Suppl 1, pp. 59-72).

Pluripotent stem cells derived from breast tissue according to the present invention comprise the steps of (a) separating breast cells from breast tissue; (b) primary culturing of the breast cells in a stem cell culture medium; (c) collecting the suspended cells from the primary culture; And (d) passaged cells in the suspended state in a culture medium for stem cell culture to separate pluripotent stem cells.

Step (a) of the preparation method of the present invention is a step of finely cutting breast tissue to obtain an isolated, preferably single breast cell. The breast cells may be in a mixed state of differentiated / undifferentiated breast cells. In one aspect, breast tissue can be finely cut by physical means using a homogenizer, mortar, blender, surgical mass, syringe, forceps or ultrasound device. In another embodiment, enzymes may be used, but examples of enzymes that may be used include, but are not limited to, sepine proteases, including neutral proteases, trypsin, chymotrypsin and thermolysine, elastase and collagenase. As another aspect, the above-described physical means and enzyme treatment may be combined.

In step (b) of the preparation method according to the present invention, the previously isolated breast cells are first cultured in a stem cell culture medium. As the medium for stem cell culture, it is suitable to use a medium consisting of DMEM, F12, B27 supplement and growth factors (eg, EGF, PDGF, VEGF, FGF, IGF, LIF, etc.). If necessary, the medium may further include components selected from cytokines (eg, insulin, estradiol, interleukin, corticosterone, etc.) and antibiotics (eg, penicillin, streptomycin, etc.).

In the present invention, it is preferable to use S medium. The "S medium" includes DMEM, F12, B27 supplement, bFGF, hEGF, LIF and antibiotics. Preferred embodiments of the S medium comprises DMEM vs. F12 1 to 5 to 1, preferably 2 to 4 to 1, and B27 supplement 0.1 μl / ml to 1 ml / ml, preferably 0.1 μl / ml to 100 Μl / ml, bFGF 0.1ng / ml to 1mg / ml, preferably 1ng / ml to 100μg / ml, hEGF 0.1ng / ml to 100ng / ml, preferably 1ng / ml To 50 ng / ml, LIF to 0.1 ng / ml to 1 mg / ml, preferably 1 ng / ml to 100 μg / ml, and antibiotics of 0.1 μl / ml to 1 ml / ml, preferably 1 μl / ml to 100µl / ml.

Step (c) of the production method according to the present invention is a step of collecting the suspended cells from the primary culture, the suspended cells are centrifuged or filtered using a cell strainer (cell strainer), etc. It can be carried out according to methods known in the art.

Step (d) of the production method according to the present invention is a step of separating the pluripotent stem cells by passage culture the cells of the suspended state in the culture medium for stem cells, the stem cell culture medium is used in step (a) The method for separating pluripotent stem cells is the same as that for cultured stem cell culture, and can be performed according to methods known in the art. Here, the passage can be performed at least once or more, preferably five or more times, more preferably seven or more times. In each passage, suspended cells are collected and cultured in the culture obtained through the previous culture. Through this passage culture, a wear spare consisting of substantially pure (more than about 95%) pluripotent stem cells can be obtained.

As noted above, stem cells derived from breast tissues according to the present invention are pluripotent and can be differentiated into various types of cells constituting the human body.

Methods of differentiating stem cells into specific types of cells are known in the art and based on such known methods, the stem cells according to the present invention are also referred to as cells of the desired type (hereinafter referred to as target differentiated cells). Can differentiate. Specifically, (a) culturing pluripotent stem cells derived from breast tissues according to the present invention in a medium for culturing target differentiated cells, and (b) separating target differentiated cells expressing markers of target differentiated cells during culturing. Through this, cells differentiated from pluripotent stem cells derived from breast tissue according to the present invention can be produced.

On the other hand, whether the cells in culture express the markers of the differentiated cells of interest can be confirmed using a method known in the art, such as RT-PCR, western blotting (immunohistochemistry), ELISA method Etc., but is not limited thereto.

Stem cells derived from breast tissue according to the present invention can differentiate into epithelial cells. Thus, one aspect of the present invention relates to epithelial cells differentiated from pluripotent stem cells derived from breast tissue characterized in that they are differentiated from the above-mentioned pluripotent stem cells derived from breast tissue and express an epithelial cell marker.

Such epithelial cells are in one embodiment (a) culturing pluripotent stem cells derived from breast tissue according to the present invention in a medium for epithelial cell culture, and (b) isolating cells expressing markers of epithelial cells during culture. It can be manufactured through. The epithelial cell culture medium includes DMEM, F12, growth factors (eg, EGF, PDGF, VEGF, FGF, IGF, LIF, etc.), cytokines (eg, insulin, estradiol, interleukin, corticosterone, etc.) and trace amounts. It is suitable to use a medium consisting of an element (eg transferrin). If desired, the medium may further comprise a component selected from serum (eg FBS) and antibiotics (eg penicillin, streptomycin, etc.).

In the present invention, it is preferable to use SNU medium. The "SNU" medium includes DMEM, F12, EGF, insulin, hydrocortisone, human transferrin, 17-betaestradiol 10 and antibiotics. Preferred embodiments of the SNU medium comprise DMEM vs. F12 1 to 3 to 1, preferably 3 to 1, and EGF 0.01 ng / ml to 1 mg / ml, preferably 0.1 ng / ml to 100 µg / ml And 0.1ng / ml to 1mg / ml, preferably 1ng / ml to 100µg / ml, and 0.1ng / ml to 1mg / ml, preferably 1ng / ml to 100 human transferrin. Μg / ml, 17-betaestradiol 10, 10 ^-12 M / ml to 10 ^-2 M / ml, preferably 10 ^-9 M / ml to 10 ^-3 M / ml, and antibiotics 0.1 μl / ml to 1 ml / ml, preferably 0.1 μl / ml to 10 μl / ml.

Pluripotent stem cells derived from breast tissue according to the present invention can differentiate into neurons or bone marrow cells. Therefore, another aspect of the present invention is a neuronal cell differentiated from pluripotent pluripotent stem cells derived from breast tissue described above and expressing neuronal markers or bone marrow cell markers. Relates to bone marrow cells.

Such nerve cells or bone marrow cells can in one embodiment be (a) culturing pluripotent stem cells derived from breast tissue according to the invention in a medium for culturing nerve cells or bone marrow cells and (b) nerve cell markers or bone marrow during such culture It can be prepared by the step of separating the cells expressing the cell marker. As the medium for culturing nerve cells or bone marrow cells, a medium consisting of DMEM, F12, B27 supplement and serum (eg, FBS) is suitable. If necessary, the medium may contain growth factors (e.g., EGF, PDGF, VEGF, FGF, IGF, LIF, etc.), cytokines (e.g. insulin, estradiol, interleukin, corticosterone and antibiotics (e.g. penicillin, streptomycin, etc.). It may further comprise a component selected from.

In the present invention, it is preferable to use an SD medium. The "SD medium" includes DMEM, F12, B27 supplement, antibiotic and serum. The aforementioned components may be substituted with other substitutes known in the art. Preferred embodiments of the SD medium include DMEM vs. F12 1 to 5 to 1, preferably 2 to 4 to 1, and B27 supplement is 0.1 μl / ml to 1 ml / ml, preferably 0.1 μl / ml to 100 μl / ml, serum from 0.1 μl / ml to 1 ml / l, preferably 0.1 μl / ml to 100 μl / ml, and antibiotics from 0.1 μl / ml to 1 ml / ml, preferably 0.1 It contains 1 μl / ml to 10 μl / ml.

Hereinafter, the examples are only for explaining the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those of ordinary skill in the art to which the present invention pertains. Will be self-evident.

<Example 1>

Medium used in the present invention

Mamospare culture medium consists of DMEM: F12 = 3: 1, B27 supplement, 40ng / ml bFGF, 20ng / ml EGF, 10ng / ml LIF, penicillin and streptomycin (S medium). SNU medium for differentiating stem cells derived from breast tissue into myoepithelial cells and luminal cells was DMEM: F12 = 1: 1, 0.5ng / ml EGF, 5μg / ml insulin, It consists of 0.5 μg / ml hydrocortisone, 4 μg / ml human transferrin, 17-estradiol ^10-8 M, penicillin and streptomycin. DMEM: F12 = 3: 1, B27 supplement, penicillin and streptomycin, and 3% FBS were used as SD medium for differentiating breast cells derived from breast cells into neurons or mesenchymal cells. .

Here, DMEM, F12, trypsin, B27 supplement, penicillin, streptomycin, trypsin and 0.4% trypan blue staining solution were obtained from Gibco-BRL (Grand Island, NY). Poly-L-lysine, type IV collagen and rabbit anti-fibronectin antibodies were obtained from Sigma (St. Louis, MO). Insulin, EGF, bFGF, LIF, hydrocortisone, human transferrin, 17-betaestradiol-10 were purchased from Invitrogen (Carlsbad, Calif.). Collagenase from Roche (Indianapolis, IN), FBS from HyClone (Cramlington, Northumberland, UK), and Vectashield medium with 4 ', 6'-diamidino-2-phenylindole hydrochloride (DAPI) laminated Vector Laboratories (Burlingame, From CA). 40 μm cell strainer, uncoated tissue culture dishes and 24 spheres were all purchased from Falcon (San Jose, Calif.). 18 mm cover slip was obtained from Marienfield (Germany).

<Example 2>

Culture of Stem Cells Derived from Breast Tissue

After removing normal breast tissue / cancerous breast tissue, the tissue was chopped into 1-2 mm fragments and washed three times with phosphate buffer (PBS). The collagenase was then inactivated by digestion with collagenase at 37 ° C. for 1 hour and then treated with FBS. After enzymatic treatment, the tissues were mechanically ground by vortexing at room temperature for several minutes. The cells thus obtained were centrifuged at 2000 RPM for 5 minutes and the pellet was washed twice with PBS. The pellet was resuspended in S medium by pipetting and filtered with a 40 μm cell strainer. At this time, the viability of the single cell suspension was measured using 0.4% trypan blue solution. The cells were seeded in uncoated dishes and primary cultured at 37 ° C. in humid air containing 5% CO ₂ in S medium. The primary cultures were collected by centrifugation for about 5 minutes to collect the suspended cells in the primary culture and then inoculated in an uncoated dish and passaged at 37 ° C. in humid air containing 5% CO ₂ in S medium. . The viable cell number for each passage was measured by 0.4% trypan blue staining. The first isolated breast cells were passaged after 10 days and subsequent cells were passaged every 7 days.

Using an inverted system microscope (IX51 model (Olympus, Tokyo, Japan) equipped with DP50 camera system (Olympus, Tokyo, Japan)), the wear spare was photographed at each passage and stored in a PC and then Photoshop 7.0 software (Adobe Systems, San Jose, CA).

The viable cell number for each passage was measured using 0.4% trypan blue dye in the Neubauer counting chamber. The viable cell number measured at each passage was compared with the initial viable cell number immediately before the initial suspension. As a result, the number of viable cells decreased drastically after the first passage culture and then decreased to less than 10% of the initial viable cells. After that, it gradually decreased to less than 1% of the initial viable cells after the 7th passage. It was. There was no difference between abrasion spares from normal tissues and abrasion spares from cancer tissues until the 10th passage (Fig. 1), and various types of abrasion spares derived from them were observed until the 10th passage. 2a and 2b).

<Example 3>

Differentiation of stem cells derived from breast tissue into epithelial cells

After recovering centrifuged and washed with PBS for 10 passages through Example 2, the abrasion spare was suspended and re-suspended on an 18 mm cover slip coated with 10 μg / ml type IV collagen in a 24-sphere plate. Inoculation. The cells were cultured in SNU medium for 3 days and then examined for differentiation into epithelial cells.

Abrasion spares were attached within 1 hour after inoculation and the initial adherent cells located underneath the abrasion spares moved centrifugally and changed their shape into differentiated cells. After 3 days of attachment, the rapid growth of the cells located at the edges proceeded, but the round shape of the abrasion spares was maintained in the normal cell-derived abrasion spares and the cancer cell-derived abrasion spares (FIG. 3).

In addition, whether stem cells derived from breast tissues differentiate into epithelial cells was confirmed using the following immunostaining chemical analysis. Differentiation into myoepithelial cells was detected with CK14 mouse monoclonal IgG3 antibody (1: 1000; Chemicon, Temecula, Calif.) And differentiation into coronary cells was CK18 rabbit polyclonal IgG antibody (1: 1,000; Calbiochem, Darmstadt , Germany). In addition, secondary antibodies include Rhodamine (TRITC) -conjugated Gout anti-rabbit IgG antibodies (1: 400; ZYMED) and Fluorescein (FITC) -conjugated Gout anti-mouse IgG antibodies (1: 500; Sigma) was used.

Immunohistochemistry proceeded as follows. Cells cultured on 18 mm cover slip were washed with PBS and fixed with 4% formaldehyde solution at room temperature for 15 minutes. After washing three times with PBS, infiltrated with 0.5% Triton X-100 for 15 minutes at room temperature, and then washed three times with PBS. PBS supplemented with 5% FBS was blocked for 1 hour at room temperature and then washed several times with PBS. Cells were allowed to stand for 1 hour with primary antibody. After washing several times with PBS and standing for 1 hour with a secondary antibody and three times with PBS. The immunostained cells were then stored in Vectashield mounting medium.

Confocal microscopy was performed using an Axiovert LSM 510 microscope (Zeiss, Jena, Germany). The obtained images were processed using LSM Pascal version 3.1 and Photoshop 7.0 software (Adobe Systems, San Jose, Calif.). Double labeled samples with FITC- and / or TRITC conjugated secondary antibodies were analyzed simultaneously or sequentially. In each case, FITC was excited with blue beam and detected using an interferential narrow band filter (BP 505-550nm), while TRITC was excited with red beam and detected using a long pass filter (LP 650nm).

Cells were labeled with anti-CK14 (green) and anti-CK18 (red). The nuclei of the cells were stained with DAPI (blue). CK14 and CK18 coexpressing cells were identified in abrasion spares from adherent normal tissue (FIG. 4). The pluripotent stem cells in abrasion spares derived from cancer tissues were identified in this same way (FIG. 5). CK14 and CK18 coexpressing cells were located in abrasion spares, not rapidly growing differentiated cells. From the above results, it was confirmed that pluripotent stem cells derived from breast tissue according to the present invention can be differentiated into epithelial cells.

<Example 4>

Differentiation of stem cells derived from breast tissue into nerve and bone marrow cells

After recovering centrifuged and washed with PBS for 10 passages through Example 2, the abrasion spare was suspended and re-suspended on an 18 mm cover slip coated with 10 μg / ml type IV collagen in a 24-sphere plate. Inoculation. The cells were cultured in SD medium for 3 days and then examined for differentiation into epithelial cells.

Abrasion spares were attached within 1 hour after inoculation and the initial adherent cells located underneath the abrasion spares moved centrifugally and changed their shape into differentiated cells. After 3 days of attachment, the rapid growth of the cells located at the edges proceeded, but the round shape of the abrasion spare was maintained in the abrasion spare derived from the normal tissue and the cancer tissue derived from the attached tissue (FIG. 6).

In addition, whether stem cells derived from breast tissues differentiate into nerve cells or bone marrow cells was confirmed using the following immunostaining chemical analysis. Neural stem cells / progenitor cells were detected using monoclonal IgG1 anti-nestine antibody (1: 100; BD Pharmingen) of mice. Mesenchymal cells were identified with rabbit anti-fibronectin polyclonal antibody (1: 500; Sigma). At this time, the immunochemical analysis was performed according to the same procedure as in Example 3.

Cells were labeled with nuclear staining DAPI (blue), anti-nestine (green), anti-fibronectin (red). Green staining of the nestin, a stem cell marker of neurons, was observed in the undifferentiated portion at the center of the marmosphere, not rapidly growing differentiated cells. Red staining for the bone marrow cell marker, fibronectin, was confirmed in the rapidly growing region as well as the central part. This pattern was similar in abrasion spares from normal tissue (FIG. 7) and abrasion spares from cancer tissue (FIG. 8). From the above results, it was confirmed that pluripotent stem cells derived from breast tissue according to the present invention can differentiate into neurons or bone marrow cells.

Since stem cells derived from breast tissues according to the present invention are pluripotent, they can be differentiated into various cells constituting the human body, and ultimately, they can be used to treat various diseases.

Claims (12)

Pluripotent stem cells derived from breast tissue. The pluripotent stem cell according to claim 1, which possesses the ability to differentiate into epithelial cells, nerve cells or bone marrow cells. (a) separating breast cells from breast tissue; (b) primary culturing of the breast cells in a stem cell culture medium; (c) collecting the suspended cells from the primary culture; And (d) passaging the suspended cells in a stem cell culture medium to separate pluripotent stem cells. The method of claim 3, wherein the stem cell culture medium comprises DMEM vs. F12 1 to 5 to 1, B27 supplement 0.1 μl / ml to 1 ml / ml, bFGF 0.1ng / ml to 1 mg / breast, characterized in that it comprises 0.1 mg / ml to 100 ng / ml of hEGF, 0.1 ng / ml to 1 mg / ml of LIF, and 0.1 μl / ml to 1 ml / ml of antibiotic. Method for producing pluripotent stem cells derived from tissues. Cells differentiated from pluripotent stem cells derived from breast tissue. (a) culturing pluripotent stem cells derived from breast tissue in a medium for culture of target differentiating cells; and (b) separating target differentiated cells expressing markers of the target differentiated cells during the culture. Method for producing cells differentiated from pluripotent stem cells. The method of claim 6, wherein the target differentiated cells are epithelial cells. 8. The method of claim 7, wherein the epithelial cell culture medium comprises DMEM vs. F12 in 1 to 3 to 1, EGF in 0.01ng / ml to 1mg / ml, insulin 0.1ng / ml to 1mg / ml and it includes, human transferrin to 0.1ng / ml to 1mg / ml and including a, 17-beta estradiol 10 to 10 ^-12 M / ml to about 10 ^-2 M to contain / ml, antibiotics 0.1㎕ / ml to a Method for producing cells differentiated from pluripotent stem cells derived from breast tissue, characterized in that containing 1ml / ml. The method for producing cells differentiated from pluripotent stem cells derived from breast tissue according to claim 6, wherein the target differentiated cells are neurons. 10. The method of claim 9, wherein the culture medium for neuronal cells comprises DMEM vs. F12 1 to 5 to 1, B27 supplement 0.1 μl / ml to 1 ml / ml, serum 0.1 μl / ml to 1 ml / A method for producing cells differentiated from pluripotent stem cells derived from breast tissue, comprising l and antibiotics at 0.1 μl / ml to 1ml / ml. The method of producing a differentiated cell from pluripotent stem cells derived from breast tissue, characterized in that the target differentiated cells are bone marrow cells. The method of claim 11, wherein the bone marrow cell culture medium comprises MEM to F12 1 to 5 to 1, B27 supplement 0.1 μl / ml to 1 ml / ml, serum 0.1 μl / ml to 1 ml / A method for producing cells differentiated from pluripotent stem cells derived from breast tissue, comprising l and antibiotics at 0.1 μl / ml to 1ml / ml.

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