WO2019017691A2 - Method of differentiation of human induced pluripotent stem cell to dermal papilla precursor cell and use thereof - Google Patents

Abstract

The present invention relates to a medium composition for differentiation of a human induced pluripotent stem cell to a dermal papilla precursor cell, a differentiation method, and a use for inducing hair follicle neogenesis using the differentiated dermal papilla precursor cell. The present inventors have developed a method for differentiating a human induced pluripotent stem cell into a dermal papilla precursor cell having hair follicle forming ability and a composition of a dermal papilla precursor cell specific differentiation medium for the above differentiation, and have effectively induced hair follicle neogenesis consisting only of human cells without conventional mouse-human hybrid hair follicles by using the human induced dermal papilla precursor cell and a human induced epidermal precursor cell obtained through the differentiation method. Human hair follicle tissue produced by applying the technique for differentiating the dermal papilla precursor cell according to the present invention is expected to be useful as a therapeutic method for patients suffering from hair loss by overcoming the limitations of hair loss treatments.

Description

METHODS FOR DERIVATIVES FROM HUMAN-GENERATED ALLOCYTE STEM CELLS TO MYULEOID PREGNANCYCULARS

The present invention relates to a culture medium for differentiating human pluripotent stem cells into a papillary precursor cell, a differentiation method using the composition, and a use of inducing a hair follicle precursor cell differentiated by the method.

This application claims priority based on Korean Patent Application No. 10-2017-0091727, filed on July 19, 2017, and Korean Patent Application No. 10-2018-0083386, filed on July 18, 2018, The entire contents of the application and drawings are incorporated herein by reference.

For the treatment of permanent hair loss, hair transplantation can be considered by taking the existing healthy hair that should be used as donor for the hair transplantation in the patient himself. However, patients with intractable permanent hair loss have limited hair follicle transplantation in patients with male hair loss or pediatric cancer survivors because the number of available hair follicles is not sufficient. Therefore, techniques for culturing and using dermal papilla cells (DPCs) and epidermal cells of patients having hair follicle forming ability for hair follicle development have been proposed as long-feasible treatments. Although mouse dermal papilla cells reported to have hair follicle forming ability in some studies, human dermal papilla cells have a limitation in that they can not be used to induce follicular dysgenesis when hair follicle cells are cultured in vitro. Therefore, many studies have been made to improve the hair follicle forming ability of human dermal papilla cells. However, there have not been any successful cases of inducing human hair follicle dermal papilla cells using dermal papilla cells.

The hair follicle-forming ability of human dermal papilla cells or dermal papilla-like cells (DPLCs) has been demonstrated in studies that generally produce mouse-human hybrid hair follicles in combination with neonatal epidermal cells Gnedeva et al., PLoS One 10 , e0116892, 2015). However, there is no report yet on the results of human hair follicle initiation using only human cells without the use of human fetal tissue. Thus, early embryonic-like dermal papilla formation through direct reprogramming from pluripotent stem cells (iPSCs) or from pluripotent stem cells or adult cells can be used to obtain a large number of highly induced dermal papilla cells It has been suggested as a potential strategy. However, due to insufficient understanding of the biological conditions of the human dermal papilla cell line, there is no definitive protocol to differentiate into dermal papilla cells with follicular ability. Recently, studies have been carried out on human pluripotent stem cells to differentiate into epidermal progenitor cells required for hair follicle neogenesis. However, it has been reported that human pluripotent stem cells, including human pluripotent stem cells, Techniques to differentiate into dermal papilla cells have not yet been established.

On the other hand, dermal papilla precursor cells (DPPCs), which are observed in the dermis in the hair follicle stage during embryonic development, exchange signals with epithelial placode cells at the start of embryonic hair follicle development It is known. The first signal from the skin acts on the undifferentiated epidermis to form morphologically recognizable hair placodes, and the stabilized epidermal placodes signal to the sub dermal cells to allow the premature cells to collect. Thereby promoting the formation of dermal condensates. Finally, the papillary precursor cells are known to signal to the epidermis to stimulate the growth and downgrowth of hair germs. After hair follicle formation is complete, the papillary precursor cells become the adjoining dermal sheath adjacent to mature dermal papilla cells and mature hair follicles in the bulb region. Thus, obtaining the hair follicle precursor cells having hair follicle-forming ability as in the embryo is an important step for human hair follicle production. However, little is known about human mammary epithelial cells in the in vivo follicular plaque stage.

As a result of intensive studies to overcome the above-mentioned conventional problems, the inventors of the present invention have developed a specific differentiation medium composition from human-derived pluripotent stem cells to papillary precursor cells and established a differentiation method using the same. Derived mammary epithelial progenitor cells and epidermal precursor cells differentiated from the human-derived pluripotent stem cells, the present inventors have completed the present invention by developing a technique for inducing hair follicle neogenesis composed of only complete human cells, not hybrid.

Accordingly, it is an object of the present invention to provide a medium composition for the differentiation of human pluripotent stem cells into dermal papilla precursor cells.

It is another object of the present invention to provide a method of differentiating human pluripotent stem cells into human papillary precursor cells and to provide a papillary precursor cell differentiated by the above method.

The present invention also provides a hair follicle precursor cell and a skin follicle precursor cell comprising the composition as an effective ingredient, a method for inducing human hair follicle stimulation by delivering the composition to an individual, and a hair follicle neoplasm application for the composition For other purposes.

It is another object of the present invention to provide a method for producing human hair follicles by co-culturing the mammary gland precursor cells and the epidermal progenitor cells.

It is still another object of the present invention to provide a cell therapy agent for treating hair loss, which comprises the mammary gland precursor cells and the epidermal progenitor cells as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a method for producing fibroblast growth factor-2 (FGF2) in DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) glutamax medium, Derived mammalian stem cell, characterized in that it comprises a glycogen synthase kinase-3 (GSK-3) inhibitor, and bone morphogenetic protein 4 (BMP4) dermal papilla precursor cell).

In one embodiment of the invention, the GSK-3 inhibitor is selected from the group consisting of 6-bromoindirubin-3'-oxime (BIO), CHIR-99021 and SB-216763 It can be more than one.

In one embodiment of the present invention, the FGF2 may be included at a concentration of 5 to 30 ng / ml, the GSK-3 inhibitor may be 0.1 to 10 uM, and the BMP4 may be contained at a concentration of 0.5 to 5 ng / ml.

In one embodiment of the present invention, the BIO may be included at a concentration of 0.5 to 5 uM or the CHIR 99021 at a concentration of 0.1 to 10 uM.

In one embodiment of the present invention, the medium for differentiation into the papillary precursor cells is a medium supplemented with DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) glutamax medium, Fetal Bovine Serum (FBS) , Fibroblast growth factor-2 (FGF2), GSK-3 inhibitor, and bone morphogenetic protein 4 (BMP4).

In one embodiment of the present invention, the differentiation medium composition may not contain human neonatal fetal tissue.

In one embodiment of the present invention, the human-derived pluripotent stem cell may be a human embryonic stem cell (ESC) or a human-derived dedifferentiated pluripotent stem cell.

The present invention also provides a method of differentiating human pluripotent stem cells into papillary precursor cells, comprising the steps of:

 (a) culturing an embryoid body of human-derived pluripotent stem cells in a neural crest stem cell-inducing medium to differentiate into neural ridge cells; And

(b) culturing the differentiated neural ridge cells in the differentiation medium composition to differentiate into papillary precursor cells.

In another embodiment of the present invention, it may further comprise the following steps:

(c) matured differentiated precursor cells to obtain precursor cells of a spherical sphere.

In one embodiment of the present invention, the neural ridge cell induction medium is selected from the group consisting of fibroblast growth factor-2 (FGF2), fibronectin, insulin, N2 (S), < / RTI > supplyment, and transferrin.

In another embodiment of the present invention, the step (c) may be performed by culturing the differentiated precursor mammary epithelial cells in a DMEM / F12 glutamax medium supplemented with FGF2 alone.

In one embodiment of the present invention, step (a) is carried out for 5 to 9 days, step (b) is carried out for 11 to 17 days, and step (c) is carried out for 7 to 13 days .

In one embodiment of the present invention, the method of differentiating human pluripotent stem cells into papillary precursor cells may be performed for 26 days or more and 50 days or less.

In addition, the present invention provides human mammary dermat precursor cells differentiated from human pluripotent stem cells by the differentiation method, wherein the human mammary dermat precursor cells have follicle-forming ability.

In one embodiment of the present invention, the papillary precursor cells may be characterized in that they exhibit at least one characteristic selected from the group consisting of the following (a), (b) and (c).

(a) exhibits positive immunological properties for ALP (alkaline phosphatase), alpha SMA (alpha smooth muscle actin), versican (VCAN), and nestin;

(b) demonstrates the structural characteristics of dermal papilla cells that spontaneously form spheres; And

(b), (b), (c), and (c), the genes for human breastpox signature genes ALX3 (ALX Homeobox 3), SOX2 (SRY-Box 2), HEY1 (Hes Related Family BHLH Transcription Factor With YRPW Motif 1), BMP4 (Bone Morphogenetic Protein 4) and Lymphoid Enhancer Binding Factor 1), WIF1 (WNT inhibitory factor 1), and VCAN (Versican).

In one embodiment of the present invention, the precancerous precursor cells may be CD133-negative.

In addition, the present invention provides a subcultured human dermal papillary precursor cell having a folliculating ability differentiated from human pluripotent stem cells by a differentiation method.

The present invention also relates to a method for the treatment and prophylactic treatment of follicular neoplasia comprising an epithelial stem cell differentiated from the human pluripotent stem cell and a human derived mammary epithelial progenitor cell having a hair follicle forming ability differentiated from the human pluripotent stem cell Thereby providing a composition for induction.

The present invention also relates to a method for inducing human hair follicle neovascularization by delivering epithelial stem cells differentiated from human pluripotent stem cells and human precursor cells derived from human pluripotent stem cells, . ≪ / RTI >

The present invention also relates to a method for producing a stem cell, comprising the steps of transplanting an epithelial stem cell differentiated from human pluripotent stem cells and a human-derived mammary epithelial progenitor cell having a hair follicle-forming ability differentiated from the human-derived pluripotent stem cell, Lt; RTI ID = 0.0 > follicle < / RTI >

The present invention also provides a method for producing a human hair follicle by co-culturing a human-derived mammary epithelial precursor cell having a hair follicle-forming ability differentiated from the human-derived pluripotent stem cell and an epithelial stem cell differentiated from human-derived pluripotent stem cells .

The present invention also relates to a composition comprising an epithelial stem cell differentiated from a human-derived pluripotent stem cell and a human-derived mammary epithelial precursor cell having a hair follicle-forming ability differentiated from the human-derived pluripotent stem cell as an active ingredient, Provide a new use.

The present invention also relates to a method for treating hair loss, comprising an epithelial stem cell differentiated from human pluripotent stem cells and a human derived mammary epithelial precursor cell having hair follicle forming ability differentiated from the human pluripotent stem cell, Thereby providing a cell therapeutic agent.

The present invention also relates to a cell therapy agent comprising, as an active ingredient, an epithelial stem cell differentiated from human pluripotent stem cells and a human-derived mammary epithelial progenitor cell having a hair follicle forming ability differentiated from the human pluripotent stem cell, To a subject in need thereof.

In addition, the present invention provides an epithelial stem cell differentiated from human pluripotent stem cells and a human hair papilla precursor cell having hair follicle forming ability differentiated from the human pluripotent stem cell, for alopecia treatment.

In one embodiment of the present invention, the papillary precursor cells may be differentiated for 30 days to 50 days from the pluripotent stem cells.

In another embodiment of the present invention, the epidermal progenitor cells may be differentiated from the pluripotent stem cells for 15 to 21 days.

The present inventors have developed a method of differentiating human pluripotent stem cells into hair follicle precursor cells having hair follicle forming ability and a composition of the hair follicle precursor cell-specific differentiation medium for the above differentiation. The human-derived papillary precursor cells and human- As described above, the present invention provides an in vitro model for the differentiation of human papilloma progenitor cells according to the present invention. The present invention relates to a method for producing human papillomavirus And human hair follicle tissue produced by applying the present method of the present invention can be used as a therapeutic method for patients suffering from hair loss by overcoming the limitation of hair loss treatment It is expected.

Figure 1 shows the results of immunostaining for the expression of SDC1 and CD133 in hair placode, hair germ, and hair peg stage, respectively, in the scalp of the embryo.

FIGS. 2A to 2F are the results of examining the differentiation process and characteristics of the human papillary precursor cells. FIG. 2A is a graph illustrating the process of differentiating human pre-differentiation pluripotent stem cells into human papillary precursor cells according to time, (Hi-NCSC), and HNK1 and p75NTR proteins. FIG. 2C shows the morphology of the differentiated papillary precursor cells (hi-DPPC) Alkaline phosphatase activity (ALP activity). Figure 2d shows the results of flow cytometric analysis to determine whether SDC1 + CD133 cells can be differentiated according to various factor combinations. Figure 2e shows the results of Flow cytometric analysis of SDC1 + CD133 cells after treatment with CHIR-99021 as an alternative to BIO. FIG. 2f shows the results that various human-derived pluripotent stem cells can be differentiated into SDC1 + CD133 DPPCs.

FIGS. 3A to 3C show the expression patterns of various genes in the differentiation stage from human de-differentiating pluripotent stem cells to papillary progenitor cells. FIG. 3 A shows the results of flow cytometry analysis of differentiated cells (iPSC, NCSC , DPPC D25, DPPC D40, and DPPC P5). Fig. 3B shows the results of stepwise gene expression changes in the cells of each differentiation stage. Fig. 3C shows the changes in the expression of SDC1 and CD133 in the differentiation step. After isolating CD133 + and CD133- from the cells, genes expressing in the dermal papilla, ALX3 , CTNNB1 , SOX2 , LEF1 , and Of VCAN And the level of expression was compared.

FIGS. 4A to 4C show the results of comparing the characteristics of human dermal papillary precursor cells (hi-DPPC) according to the present invention with human dermal papilla cells (hDPC). FIG. 4A shows the results of ALP activity activity and immune cell chemistry Figure 4b shows the results of spontaneous sphere formation, Figure 4c shows the expression of the mammary epitope genes by quantitative RT-PCR analysis, The results are compared.

5A and 5B are graphs showing results of patch assay using hi-DPPC 3D or hDPC 3D in order to examine the follicle-forming ability of the papillary precursor cells according to the present invention, FIG. 5A is a result of hypodermic transplantation of the two cells with the epidermal cells derived from C57BL / 6 mice, and FIG. 5B shows the results of induction of hybridization with the cell tracker (CM-DiI) In the presence of the dermal papilla cells.

6A and 6B are diagrams for explaining the gene expression profile of the human papillary progenitor cells. FIGS. 6A and 6B are graphs showing the expression profiles of human degenerating pluripotent stem cell-derived neural ridge cells (hi-NCSCs) (Fig. 6A) and a distance matrix (Fig. 6B) by performing RNA sequence analysis on human dermal papilla cells (cDPC 2D) and papillary precursor cell spheres (hi-DPPC 3D) FIG. 6c is a result of analyzing the results of the RNA sequence analysis by the dimensional reduction approach, and FIG. 6D is a result of comparing the expression levels of the mammalian signature gene and the stem cell marker genes in the cells.

FIGS. 7A and 7B are results of in vitro follicular neoplasia analysis according to co-culture of human dermal papillary precursor cells and human epidermal progenitor cells. FIG. 7A is a result of microscopic observation of morphological changes due to co-cultivation of the two cells, (SDC1, LEF1, SOX2, Nestin, VCAN, and WIF1) and epidermal markers (CD133, BMP4, K15, K14, and WIF1) proteins in the hair follicles and human fetal scalp This is a result showing the expression.

8A to 8C show the results of a hair follicle neogenesis according to a hippocampal progenitor cell (hi-DPPC) and a hippocampal progenitor cell (hi-EpSC) transplantation differentiated from human de-differentiated pluripotent stem cells in a mouse body through a chamber assay, FIG. 8A is a micrograph showing the multiple root sheath and the papilla structure as a result of H & E staining for the formed follicular tissue, and FIG. 8B is a micrograph showing human COX4 (hCOX4) and human pancreatic epithelial cells in the hair follicles and human fetal Immunofluorescent staining for human mitochondria, and FIG. 8c is the result of immunofluorescent staining for K14, K15, K17, and K75 in the hair follicles formed.

The present invention relates to a culture medium for differentiation from human-derived pluripotent stem cells to a papillary precursor cell, a differentiation method, and a use of inducing a hair follicle precursor cell using the differentiated papilloma precursor cells.

Accordingly, the present invention relates to a method for the treatment and prophylaxis of fibroblast growth factor-2 (FGF2), glycogen synthase kinase-3 (DMEM / F12, Dulbecco's Modified Eagle's Medium / Nutrient Mixture F- Wherein the culture medium for the differentiation from human-derived pluripotent stem cells into dermal papilla precursor cells is characterized by comprising an inhibitor of GSK-3: GSK-3, and bone morphogenetic protein 4 (BMP4) to provide.

In this medium composition, the inhibitor of glycogen synthase kinase-3 (GSK-3) is 6-bromoindirubin-3'-oxime (BIO), CHIR- 99021 and SB-216763, but are not limited thereto, and any of those known in the art as glycogen synthesis canine-3 inhibitors can be used without limitation.

In the above-mentioned medium composition, preferably, the FGF2 may be contained at a concentration of 5 to 30 ng / ml, the GSK-3 inhibitor may be 0.1 to 10 uM, and the BMP4 may be contained at a concentration of 0.5 to 5 ng / ml. In this concentration range, differentiation from human-derived stem cells to papillary precursor cells may be most effective.

In the above-mentioned culture medium composition, preferably, the BIO may be contained at a concentration of 0.5 to 5 uM, and the CHIR 99021 may be contained at a concentration of 0.1 to 10 uM. In this concentration range, differentiation from human-derived stem cells to papillary precursor cells may be most effective.

In the above-mentioned medium composition, the human-derived pluripotent stem cell may be a human-derived embryonic stem cell or a human-derived re-pluripotent pluripotent stem cell.

The medium composition may preferably be one in which no human tissues are incorporated. The term "human tissue not incorporated" means that the content of human tissue incorporated into the media composition is less than 5%, 3%, 1%, most preferably 0%, relative to the total media composition. This 0% incorporation means that no human tissue is incorporated into the medium composition.

The present invention also provides a method of differentiating human pluripotent stem cells into papillary precursor cells, comprising the steps of:

(a) culturing an embryoid body of human-derived pluripotent stem cells in a neural crest stem cell-inducing medium to differentiate into neural ridge cells; And

(b) culturing the differentiated neural ridge cells in the differentiation medium composition of claim 1 to differentiate into pre-papillary precursor cells.

Hereinafter, each step of the above-mentioned culture medium composition or method of differentiation will be described in detail.

As used herein, the term " pluripotent stem cell " (PSC) refers to pluripotent stem cells, which may include various stem cells known in the art, such as embryonic stem cells, Stem cells may be included, but are not limited thereto.

The term " human-derived pluripotent stem cells " used in the present invention means that the source of pluripotent stem cells such as human tissues, tissues and blood is a human. Examples of such pluripotent stem cells include human-derived embryonic stem cells, Human pluripotent stem cells may include human fibroblast-derived hiPSC (FB-derived hiPSC) or dermal papilla cell-derived hiPSC (DPC-derived hiPSC) .

The term " induced pluripotent stem cell (iPSC) " used in the present invention refers to a pluripotent stem cell that is pluripotent, such as an embryonic stem cell, which has been transferred to a differentiated somatic cell, ≪ / RTI > In 2006, Professor Shinya Yamanaka of Kyoto University introduced several genes into the skin fibroblasts of mice to create pluripotent stem cells as embryonic stem cells. In 2007, we introduced genes into adult skin cells to induce pluripotent stem cells It succeeded in making. These genes are SOX2, c-MYC, OCT4, and KLF4, also called Yamanaka factors. In the present invention, the four genes were introduced to produce the dedifferentiated pluripotent stem cells from human fibroblasts.

Although the DEA pluripotent stem cells according to the present invention are prepared from human-derived dermal fibroblasts, the types of the pluripotent pluripotent pluripotent stem cells are not limited as long as they are human-derived adult cells capable of producing the reprogrammed pluripotent stem cells.

The step (a) may further include culturing the human-derived pluripotent stem cells into an embryo. This step is preferably carried out for about 3 to 7 days.

The step (a) is a step of differentiating the human pluripotent stem cell into an embryoid body into neural ridge cells. The neuronal ridge cell induction medium was supplemented with DMEM / F12 glutamax, fibroblast growth factor-2 (FGF2), fibronectin, insulin, N2 supplement, and transferrin- But is not limited thereto.

In one embodiment of the present invention, the characteristics of the neural ridge cells differentiated through the step (a) were examined. As a result, the morphology of the neural ridge cells was observed under a microscope and the expression of the neural ridge markers HNK1 and p75NTR Confirming that the differentiation from the pluripotent stem cells into neural ridge cells was well performed.

The step (b) is a step of differentiating the precursor cells from the neural ridge cells differentiated in the step (a). In order to differentiate the mesenchymal precursor cells from the neural ridge cells, the inventors of the present invention have found that, in a general DMEM / F12 glutamax culture medium used for differentiation from conventional neural ridge cells to papillary-like cells, As a result, it was found that the addition of FGF2, GSK-3 inhibitor, and BMP4 as differentiation factors ultimately leads to the differentiation into the precursor cells of the mammary gland. As a result, Respectively.

Thus, the neural ridge cells differentiated in step (a) may be cultured in the culture medium for differentiating the precursor cells for 12 to 40 days to differentiate into precursor cells of the mammary epithelium.

Further, after step (b), the following steps may be further included:

(c) matured differentiated precursor cells to obtain precursor cells of a spherical sphere.

The step (c) is a step of obtaining the precursor cells of a spherical sphere by propagating the precursor cells differentiated in the step (b) while subculturing.

The step (c) may be performed by culturing the differentiated precursor cells in a DMEM / F12 glutamax medium supplemented with FGF2 alone.

In an embodiment of the present invention, in order to verify whether the differentiation into the precursor cells of the mammary epithelium was accomplished through the above-described method, it was confirmed by microscopic observation that the morphology changed into a fusiform polygonal shape similar to that of the primary dermal papilla cells, And showed alkaline phosphatase activity.

In the differentiation method of the present invention, preferably, step (a) is carried out for 5 to 9 days, step (b) is carried out for 11 to 17 days, or step (c) . In this case, differentiation from human-derived pluripotent stem cells to papillary precursor cells can be performed most effectively.

In the differentiation method of the present invention, preferably, the method of differentiating human pluripotent stem cells into papillary precursor cells is performed for 26 days or more and 50 days or less, more preferably 30 days or more and 45 days or less, still more preferably And can be performed for about 40 days. At this time, differentiation from human-derived pluripotent stem cells to papillary precursor cells can be performed most effectively. The point of origin of "above 26 days and less than 50 days" is the time point when the human-derived pluripotent stem cells are started to be applied to the differentiation method of the present invention. For example, in applying the human-derived pluripotent stem cells to the differentiation method of the present invention, If the embryoid body formation process of human-derived pluripotent stem cells is included before step a), the embryoid body formation process of human-derived pluripotent stem cells may be included in the period from 26 days to 50 days.

In addition, the present invention provides human mammary dermat precursor cells differentiated from human pluripotent stem cells by the differentiation method, wherein the human mammary dermat precursor cells have follicle-forming ability.

The papillary precursor cells are characterized by exhibiting at least one characteristic selected from the group consisting of (a), (b) and (c) below.

(a) exhibits positive immunological properties for ALP (alkaline phosphatase), alpha SMA (alpha smooth muscle actin), versican (VCAN), and nestin;

(b) demonstrates the structural characteristics of dermal papilla cells that spontaneously form spheres; And

(b), (b), (c), and (c), the genes for human breastpox signature genes ALX3 (ALX Homeobox 3), SOX2 (SRY-Box 2), HEY1 (Hes Related Family BHLH Transcription Factor With YRPW Motif 1), BMP4 (Bone Morphogenetic Protein 4) and Lymphoid Enhancer Binding Factor 1), WIF1 (WNT inhibitory factor 1), and VCAN (Versican).

The prefrontal precursor cells express CD133 negative. Or CD133 negative and SDC1 positive.

CD133 is known to be a surface marker of hair-inducing skin cells in mice. However, in the case of the papillary precursor cells according to the present invention, as shown in Fig. 3C, the CD133 negative The expression of CD133 ( ALX3 ), catenin beta 1 ( CTNNB1 ), SOX2 , and lymphoid enhancer binding factor 1 ( LEF1 ) was significantly increased in the cells, positivity And the results were not shown in the cells.

In addition, the present invention provides a human mammary dermal precursor cell differentiated from human pluripotent stem cells by a differentiation method, wherein the human mammary dermal progenitor cell provides a subcultured human mammary dermal precursor cell having a hair follicle forming ability.

In one embodiment of the present invention, the present inventors examined whether the epidermal cells (mEPI) of C57BL / 6 neonatal mouse and the papillary progenitor cells were subcutaneously injected into immunodeficient mice in order to examine whether the differentiated papillary precursor cells had hair follicle forming ability And transplantation was performed. As a result, it was confirmed that mouse-human hybrid hair follicle development was induced two weeks later.

In another embodiment of the present invention, gene expression profiling was analyzed after RNA sequencing was performed on the induction neural crest cell, inducible papillary precursor cell corpuscles, cultured dermal papilla cell protoplasts, and cultured dermal papilla cells. As a result, the dermal papilla cells exhibited a gene expression pattern very different from that of neural ridge cells, and they were similar to human dermal papilla cells through high expression of core genes of dermal papilla cells and showed early-stage molecular signatures.

Based on the above results, in another embodiment of the present invention, when the epidermal progenitor cells and the papillary precursor cells are co-cultured in vitro, a circular structure very similar to that of the hair follicle formation is formed and a gene expression pattern similar to that of the human fetal scalp is observed Respectively.

In another embodiment of the present invention, a chamber assay is performed using the papillary precursor cells and epidermal progenitor cells to verify that human hair follicle neogenesis is induced in the mouse. As a result, it was confirmed that the morphological and structural characteristics of human fetal hair follicles were similar to that of human fetal hair follicles when they were transplanted into mouse together with 40-day-old and 18-day-old epidermal progenitor cells after the start of differentiation, It was confirmed that human hair follicle development could be induced by using precursor cells.

Accordingly, the present invention provides a composition for inducing human hair folliculogenesis, comprising an epithelial stem cell differentiated from the human pluripotent stem cell and the human-derived mammary epithelial progenitor cell as an active ingredient.

In addition, a method for inducing human hair follicle development by transferring epithelial stem cells differentiated from human pluripotent stem cells and human-derived mammary epithelial progenitor cells having hair follicle-forming ability differentiated from the human pluripotent stem cells to the individual to provide.

Also provided is a method for producing a human hair follicle by co-culturing human-derived mammary epithelial precursor cells having hair follicle-forming ability differentiated from the human-derived pluripotent stem cells and epithelial stem cells differentiated from human-derived pluripotent stem cells . In the present method, preferably, said " co-cultivation " can be carried out in vitro .

In addition, a human hair follicle neovasic application of a composition comprising an epithelial stem cell differentiated from a human-derived pluripotent stem cell and a human-derived mammary epithelial progenitor cell having a hair follicle-forming ability differentiated from the human pluripotent stem cell as an effective ingredient to provide.

The term " human hair follicle " as used in the present invention means a human hair follicle, but does not mean that the individual to be follicularized is limited to a human. May be included in the present invention. For example, various animals known in the art that can have such hair follicles can include mammals, and the mammals can include mice and the like. According to embodiments of the present invention, human hair follicle neogenesis was induced in mice.

Also provided is a cell therapy agent for treating hair loss, comprising epidermal progenitor cells differentiated from human pluripotent stem cells and the human derived mammary prefrontal cells as an effective ingredient.

In addition, it is possible to administer to a subject a cell treatment agent containing, as an active ingredient, an epithelial stem cell differentiated from a human-derived pluripotent stem cell and a human-derived mammary epithelial precursor cell having a hair follicle-forming ability differentiated from the human-derived pluripotent stem cell A method of treating hair loss.

Further, there is provided an application for treating hair loss in an epithelial stem cell differentiated from a human-derived pluripotent stem cell and a composition comprising as an active ingredient a human-derived mammary epithelial precursor cell having a hair follicle-forming ability differentiated from the human-derived pluripotent stem cell do.

The present invention also provides a method for inducing follicular neoplasia comprising the step of transplanting the human-derived epidermal progenitor cells and the human-derived epidermal precursor cells into a tissue other than the human body.

The mammary epithelial progenitor cells may be differentiated from the human-derived pluripotent stem cells for 30 days to 50 days, and the epidermal progenitor cells may be differentiated from the human-derived pluripotent stem cells for 15 days to 21 days.

The prefrontal precursor cells may be CD133 negative and the epidermal progenitor cells may be CD133 positive.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1. Experimental Preparation and Experimental Methods

1-1. From human skin fibroblasts Of differentiated pluripotent stem cells  Produce

Human dermal fibroblasts (ATCC PCS-201-012) were subcultured three times and then reprogramming factors OCT4, SOX2, KLF4 and c-MYC were transduced with retrovirus. Twenty-four hours after transfection, the medium containing the virus was replaced with a new growth medium, and iPSC-like colonies were observed in transformed human dermal fibroblasts two weeks after transfection. Was removed and transferred to a new STO feeder. After 2 days of colonization, the culture medium was replaced with fresh embryonic stem cell culture medium supplemented with 10 ng / ml bFGF.

The embryonic stem cell culture medium was then changed daily and the cells were cultured in DMEM / F12 glutamax (Thermo Fisher, catalog No. 10565042) supplemented with 20% knockout serum (Thermo Fisher), 1% nonessential amino acid (Invitrogen), 2 mM glutamine Inactivated mouse STO feeder in human embryonic stem cell culture medium containing 0.1 mM [beta] -mercaptoethanol (Thermo Fisher), and 10 ng / ml bFGF (Thermo Fisher) O ₂ conditions. Subculture was carried out every 5-7 days using the usual method or 1 mg / ml collagenase type IV (Thermo Fisher). All the cell lines maintained more than 50 passages, and OCT3 / 4, NANOG, and SSEA4 were stably expressed.

1-2. human From the differentiated pluripotent stem cells Into neural ridge cells  differentiation

In order to make an embryonic body (EB), 2 mg / ml collagenase IV was cultured at 37 ° C for 1 hour when the colonies of the DEA pluripotent stem cells reached 80-90% And the differentiated colonies were removed with a pipette tip, and the loose colonies were gently recovered. The supernatant was discarded and resuspended in the culture medium of bFGF-deficient pluripotent stem cells. Then, the embryoid body was transferred to a 10 cm culture dish and cultured in a culture medium of 8 ml per culture dish. The next day, the medium was changed, the agglutinates were further cultured for 5 days, and the regenerated pluripotent stem cell culture medium was supplemented every other day.

After incubation, the embryoid bodies were dispensed on a cell culture dish coated with 15 ug / ml poly-L-ornithine (Sigma-Aldrich) and 10 / / ml fibronectin (BD) and incubated overnight at room temperature in N2 medium Lt; / RTI > N2 medium Composition: DMEM / F12 Glutamax (Thermo Fisher, catalog no. 10565042), 1% N2 supply (Thermo Fisher), 20 ug / ml insulin (Sigma-Aldrich), 0.1 mg / ml transferrin , 20 ng / ml bFGF (Thermo Fisher), 2.5 ug / ml fibronectin (Sigma-Aldrich), 1X penicillin / streptomycin. The medium was changed daily.

1-3. In neural ridge cells To breast milk precursor cells  differentiation

(One) Development of medium composition

In order to develop the precursor cell culture media for inducing the differentiation into mesodermal precursor cells from the neural ridge cells, various factors known to be important for the differentiation of the dermal papilla cells were isolated and combined to confirm the possibility of differentiation into the precursor cells of the dermal papilla. To differentiate into pre-papillary progenitor cells, the cells were stained with PBS containing 2% FBS in mouse anti-human SDC1, CD133 / 2 antibody, and then stained cells were analyzed using a flow cytometer (BD Biosciences) Respectively.

The neural ridge cells induced differentiation in human pluripotent stem cells were cultured in DMEM / F12 glutamax (Thermo Fisher, catalog No. 10565042), 6-Bromoindirubin-3'-oxime (6-Bromoindubin-3'-oxime (BIO)), bFGF, and BMP4 in combination with GSK-3 alone or in combination with GSK 3 cells were treated with 3 inhibitors (6-Bromoindirubin-3'-oxime (BIO)), bFGF and BMP4, and about 2 weeks later, differentiated cells were analyzed using SDC1 and CD133, (6-Bromoindirubin-3'-oxime (BIO)), bFGF, and BMP4.

(One) In neural ridge cells To breast milk precursor cells  differentiation

In order to induce differentiation from neural ridge cells to papillary progenitor cells, neural ridge cells were derived from embryonic bodies at 50-60% of the cultured area for 5 days. The differentiation-induced neural ridge cells were treated with an inhibitor of DMEM / F12 glutamax (Thermo Fisher, catalog no. 10565042), 10% FBS, GSK-3 (1 uM of 6-Bromoindirubin-3'- Sigma-Aldrich), 20 ng / ml bFGF, 1X penicillin / streptomycin, and the medium was replaced every other day. Fusiform neural ridge cells were changed when 1 ng / ml human recombinant BMP4 (bone morphogenetic protein 4) (R & D Systems) was added to the differentiation medium of the papillary progenitor cells at the point where they converted to fibroblast-like morphology for 2 days.

After about 2 weeks, the separated preimplanted cells were washed with PBS, 1 ml of Tryple express (Life technologies) was added, and incubated at 37 ° C for 5 minutes. The preimplantation cells were recovered with PBS and incubated at room temperature with 1200 rpm Gt; min. ≪ / RTI > The initial passages were 1: 2 or 1: 3 cultures in uncoated culture dishes using a papillary precursor cell differentiation medium without BIO (Sigma-Aldrich) and human recombinant BMP4 (R & D Systems). After passage, most undifferentiated cells were not adhered and only the differentiated preformed mammary gland precursor cells survived.

(One) GSK - 3 of  As an inhibitor CHIR Use of -99021

6-bromoindirubin-3'-oxime was used as an inhibitor of GSK-3 as a WNT signaling agent in the formation of a precursor cell culture medium for inducing differentiation from neural ridge cells to papillary precursor cells. 3'-oxime (BIO) as well as CHIR-99021 at 0.1 uM, 1 uM, and 10 uM, respectively. To differentiate into pre-papillary progenitor cells, the cells were stained with PBS containing 2% FBS in mouse anti-human SDC1, CD133 / 2 antibody, and then stained cells were analyzed using a flow cytometer (BD Biosciences) Respectively.

The neural ridge cells induced differentiation in human pluripotent stem cells were cultured in DMEM / F12 glutamax (Thermo Fisher, catalog no. 10565042), 10% FBS basic medium with inhibitor of GSK-3 (CHIR-99021), bFGF, BMP4 Approximately two weeks after the treatment of all three, the differentiation efficiency was increased according to the concentration of GSK-3 inhibitor (CHIR-99021) when the differentiated cells were analyzed by flow cytometry using SDC1 and CD133, markers of papillary precursor cells In addition, we confirmed the significance of WNT signaling in inducing differentiation into papillary precursor cells as well as the induction of concentration - dependent differentiation.

1-4. human From the differentiated pluripotent stem cells To epidermal progenitor cells  differentiation

According to a conventionally known differentiation protocol, the differentiation from pluripotent pluripotent stem cells obtained by dediffering from human dermal fibroblasts into the epithelial stem cells (EpSCs) was carried out in Example 1-1. Briefly, pre-differentiation pluripotent stem cells were isolated by treatment with 2 mg / ml collagenase IV (Thermo Fisher) for 1 hour at 37 ° C prior to differentiation, and cell aggregates were treated with 1 ng / ml of human recombinant BMP4 Grown embryonic stem cell culture for 24 hours. On the second day, the embryoid bodies were collected, the supernatant was discarded, and the cultures were dispensed into mitomycin-C treated STO cells. The cells were grown in a differentiation medium containing 1 μM of all-trans RA (Sigma-Aldrich). After 2 days, the cells of the ectoderm lineage migrated from the embryoid body, and 25 ng / ml of human recombinant BMP4, 20 ng / ml human EGF (Sigma-Aldrich), 1 uM all-trans RA, and cells were cultured in the differentiation medium until clones of epidermal cells were observed. The amount of CD200 + / ITGA6 + cells reached a maximum at about 18 days after culturing in the differentiated epidermal cells. The CD200 + / ITGA6 + cells were separated by MACS analysis and used for the hair follicle development experiment.

1-5. human's Dermal papilla cells  Isolation and Culture

To obtain human dermal papilla cells (hDPCs), a skin biopsy sample (a 1.5 × 1.0 cm scalp tissue sample at the larynx) was taken from healthy male volunteers without a scalp disease (mean age 36.6 ± 9.4 years). Human dermal papilla cells were isolated from each hair follicle, which was considered to be morphologically growing. Separated dermal papilla cells were suspended in DMEM (Dulbecco ' s modified Eagle ' s medium) supplemented with 10% FBS (Thermo Fisher), 1X penicillin / streptomycin ) (cultured under Welgene) 37 ℃, 5% CO 2 conditions.

1-6. Histology and immunochemical staining immunofluorescence )

The human fetal scalp tissue was mounted on a Tissue-Tek cryo-OCT (Fisher Scientific) and frozen using methyl butane / dry ice. The tissue block was cut to a thickness of 10 μm at 25 ° C and stored at 80 ° C until immunochemical staining.

1-7. Antibody staining for cell membrane protein

The tissue embedded in paraffin was cut to a thickness of 4 μm, and the cut sections were stained with hematoxylin and eosin. In other cases, citrate buffer (DAKO) at pH 9.0 was treated at 120 ° C for 15 min on sections of 4 μm thick microtitrated tissue for antigen detection.

On the other hand, for the antibody staining, the cut frozen tissue was fixed with 4% paraformaldehyde for 20 minutes, and the fixed tissue was incubated with the Ultravision protein blocking solution (Thermo Fisher) for 30 minutes. Next, the primary antibody was treated and cultured, and the secondary antibody was diluted with antibody dilution solution (Life technologies) at a ratio of 1: 200, followed by incubation for 1 hour and washing. The cells were then counterstained with DAPI (4 ', 6-diamidino-2-phenylindole) (Thermo Fisher) and mounted with an immuno-mount (Thermo Fisher).

1-8. Antibody staining for transcription factor

Treatment of the blocking solution containing 0.01% saponin (Sigma-Aldrich), 0.25% fish gelatin (Sigma-Aldrich) in frozen section tissue or human hair follicles fixed with 4% paraformaldehyde Thereby increasing the permeability of the cell membrane. The primary antibody and the secondary antibody were stained using blocking solution. Isotype control was performed to confirm background staining in each experiment.

1-9. Flow cell  Analysis and cell sorting

(IPSCs), neural ridge cells (NCSCs), and papillary precursor cells (DPPCs) ( Day 25, 40, Passage 5) were cultured with mouse anti-human SDC1, CD133 / 2 and SSEA3, 2% FBS in PBS. The stained cells were then analyzed using an LSRII flow cytometer (BD Biosciences).

For cell sorting, the cells were separated with a AriaTMII cell separator (BD Biosciences) at a concentration of 10 ⁶ cells / ml in PBS. For magnetic bead separation, a Miltenyi MACS bead separation system was used according to the manufacturer's instructions and separation conditions. The results were analyzed using FlowJo software (FlowJo, LLC).

1-10. patch The patch assay  through Follicular newborn  analysis

Prior to the analysis of the hair follicle, SSEA3-cells were isolated from human demineralized pluripotent stem cells (hiPSC-derived DPPCs) by MACS analysis to remove undifferentiated cells. 20 [mu] l of 10% DMEM containing 1 x 10 < ⁴ > cells of selected human papilloma precursor cells or cultured human dermal papilla cells were dropped one by one over the lid of the Petri dish. Temporal dermal papilla cell or human de-differentiated pluripotent stem cell derived dermal papilloma precursor cell spheroids were obtained at 48 hours after dosing. Cultured human dermal papilla cells or human deregulated pluripotent stem cell derived mammary epithelial progenitor cells were cultured with epidermal cells of C57BL / 6 neonatal mice or with CD200 + / ITGA6 + / SSEA3 cells derived from human degenerated pluripotent stem cells. In all experiments, human dermal papilla cells (1 × 10 ⁶ total, 100 spherical bodies) or human demyelinated pluripotent stem cell derived papillary precursor cells (total 1 × ^{10 6} , 100 spheres) and epidermal cells ( ⁵ × 10 ⁵ ) (SHO) female mice (18-20 g; Jackson Laboratory) which were mixed in F12 Glutamax medium and fed with 7 weeks of severe combined immunodeficiency. To confirm the origin of human dermal papillary precursor cells in the formed hair follicle, the dermal papillary precursor cells were labeled with a fluorescent dye reagent, CM-DiI (invitrogen). Host mice were sacrificed at 5-6 weeks post-transplant to obtain regenerated hair follicular structure. This experiment was approved by Seoul National University Hospital Clinical Trials Committee (Approval No. 15-0178-C1A0).

1-11. chamber Chamber assay.  through Follicular newborn  analysis

Cell preparation was performed as described in the patch assay. (HiPSC-DPPC) (total 5X10 ⁶ , 500 spheres) and human degenerated pluripotent stem cell-derived epidermal progenitor cells (DMEM / F12 glutamax) 5 x 10 < ⁶ >) were transferred to a silicon chamber implanted in the back skin of SHO mice. After 2 weeks, the chamber was removed and the implanted area was dressed and after 5 weeks the implanted area was obtained and histological analysis was performed.

1-12. Quantitative Reverse transcription  Quantitative reverse transcription polymerase chain reaction (PCR)

Total RNA was isolated from degenerate pluripotent stem cells, neural stem cells, papillary precursor cells, 2D cultured dermal papilla cells, and 3D cultured dermal papilla cells using RNAiso Plus (Takara Bio). CDNA was synthesized using Revert First strand cDNA synthesis kit (Thermo Fisher) using the separated RNA as a template. Then, quantitative RT-PCR analysis was carried out using SYBR premix Ex Taq II (Takara Bio). The primer sequences used were shown in Table 1 below.

1-13. Alkaline  Phosphatase activity

Alkaline phosphatase activity was assayed on cells plated on plates. More specifically, the cells were fixed with 4% paraformaldehyde for 20 minutes and NTMT buffer was diluted with NBT / BCIP (Roche) for 20 minutes.

1-14. RNA sequence analysis

Total RNA was isolated from human neural ridge cells, human dermal papilla cells, cultured dermal papilla cells, and cultured dermal papilla cell sphere using RNAiso Plus (Takara Bio). Next, a transcript library was prepared using Illumina's TruSeq stranded mRNA kit for 1 RNA of the RNA isolated from the above. Poly (A) + RNA was isolated using AMPure XP beads (Beckman Coulter) and fragmented using the Ambion Fragmentation Reagents kit (Ambion, Austin, TX, USA). cDNA synthesis, tail repair, base addition, and ligation of Illumina indexed adapters were all performed according to Illumina's protocol. The library was selected with a size of 250,300 bp using BluePipin (Sage Science, MA, USA) and amplified by PCR using 14 cycles of Phusion DNA polymerase (New England Biolabs). The amplified library was amplified using AMPure XP beads ≪ / RTI > Library quality was assessed by measuring the size and concentration using an Agilent 2100 Bioanalyzer.

Sequence analysis was then performed on the bidirectional end libraries using Illumina HiSeq 2000 (2 x 100 nucleotides read length) and the readings passed through the purity filter of the Illumina BaseCall software were used for subsequent analysis. The R package "Cuffdiff" was used for the differential expression of the genes and a hierarchical clustering analysis was performed on genes with a q value of less than 0.05 in order to produce a total of 3065 genes. In addition, distance matrix analysis, multidimensional scaling plot, and gene cluster analysis were performed using R package "CummeRbund". To generate 50 clusters out of a total of 3471 genes, gene clustering analysis was performed on important genes with an alpha value of less than 0.01.

1-15. Accession codes

Raw data for RNA sequencing were obtained from accession number GSE100793 and NCBI Gene Expression Omnibus.

1-16. Statistical analysis

Gene expression and flow cytometry were analyzed using Student's t test or ANOVA, and the qPCR results were analyzed after correction with β-actin. Statistical significance was considered as p <0.05.

Example  2. In vivo human hair follicles Placod  In step SDC1  And CD133 Expression Analysis

To induce human hair follicle development, the inventors have established a simple strategy to mimic the biological state of the hair placode period based on the morphogenesis of hair follicles occurring in human embryos. More specifically, the focus was on obtaining skin precursor cells in the dermal papillary precursor cells and the hair fracode stage.

In order to monitor the differentiation process, a specific surface marker for human mammary dermal progenitor cells was required. SDC1 (Syndecan-1) is a well-known surface marker that is strongly expressed not only in mouse skin but also in the scalp of human fetus in the hair follicle stage, and is also known as a human breastmilk signature gene. On the other hand, CD133 was originally known as a surface marker of hair-derived dermal papilla cells in mice, but it has been reported that expression pattern similar to that of mouse is not observed in CD133 in human dermal papilla cells. Therefore, the present inventors first examined the expression of SDC1 and CD133 in the scalp skin of a human 16-week embryo.

As a result, as shown in Fig. 1, it was confirmed that SDC1 was predominantly expressed in the dermis at the time when the epidermal pleocade and the dermis were formed. In addition, CD133 expression was not observed in the very early stage dermis, but this result is similar to that observed in morphogenesis in mouse embryos. In epidermal plicated cells, CD133 was expressed along the apical and lateral sides of cells and cells consistent with the reports in human scalp.

Example  3. Human Papillary progenitor cells Of the differentiation medium  Produce

(One) Human origin Of differentiated pluripotent stem cells (hiPSC)  Produce

According to a known method (Cell 131 , 861-872.), The present inventors have discovered that four genes called Yamanaka factor: OCT4 (octamer-binding transcription factor 4), SOX2 (sex determining region Y-box 2), KLF4 Depleted pluripotent stem cells (hiPSC) were prepared from human dermal fibroblasts isolated using Kruppel-like factor 4, and c-MYC. The dedifferentiated pluripotent stem cell clone showed a hESC form and exhibited a high level of alkaline phosphatase (ALP) activity. In addition to OCT3 / 4 and NANOG, SSEA4 (surface antigen stage-specific embryonic antigen 4). In addition, the expression of endogenous stem cell potential genes including OCT3 / 4, SOX2, NANOG, REX1 and telomerase reverse transcriptase (TERT) is increased through quantitative real-time PCR (qPCR) Respectively. In addition, the pluripotent pluripotent stem cell clones were verified for pluripotency by performing a teratoma formation assay, and the clones were used as pluripotent stem cells after 100 or more consecutive subcultures.

(One) From human-derived pluripotent stem cells To breast milk precursor cells  differentiation

Next, based on the results that the precursor cells of the mammary gland originate from the neural ridge cells in the embryonic development stage, the present inventors tried to differentiate the precursor cells from the demodified pluripotential stem cells using the neural ridge cells as the intermediate. For this purpose, in order to prepare neural ridge cells from the human de-differentiating pluripotent stem cells, the embryoid bodies of the reprogramming pluripotent stem cells are cultured in the presence of fibroblast growth factor-2 (FGF2), fibronectin Lt; RTI ID = 0.0 &gt; N2 &lt; / RTI &gt; differentiation medium. After incubation for 1 week, the cells cultured through immunocytochemistry were examined for the expression of the neural ridge markers HNK1 and p75NTR. As a result, it was confirmed that the HNK1 and p75NTR proteins were expressed as shown in FIG. 2B Respectively.

(2-1) In the breast milk precursor cells  Specific for Differentiation medium  Development of composition

Next, in order to develop a specific differentiation medium composition for the papillary precursor cells, the inventors of the present invention used a DMEM (Dulbecco's modified Eagle's medium) / F12 glutamax supplemented with 10% fetal bovine serum (FBS) And essential factors for cell differentiation were screened. In particular, the focus was on identifying essential factors that can maintain the inherent and endogenous molecular signatures of human dermal papilla cells or recover the inherent dermal papilla characteristics during in vitro culture. The results showed that WNT, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), sonic hedgehog ) And endothelin 3 (EDN3), and eventually received three essential factors, FGF2, 6-bromoindirubin-3'-oxime, 3'-oxime (BIO), and BMP4 were used to induce differentiation of papillary precursor cells.

More specifically, neural ridge cells induced differentiation in human pluripotent stem cells were cultured in DMEM / F12 glutamax (Thermo Fisher, catalog No. 10565042), 10% FBS basic medium with 6-Bromoindirubin-3 (6-Bromoindirubin-3'-oxime (BIO)), bFGF, and BMP4 in combination with GSK-3 inhibitor , Or inhibitors of GSK-3 (6-Bromoindirubin-3'-oxime (BIO)), bFGF, and BMP4. After about two weeks, the differentiated cells were analyzed using SDC1 and CD133. As a result, SDC1 + / CD133- cell fraction, which is a marker of papillary precursor cells, was significantly increased only in the group treated with all three inhibitors of GSK-3 (6-Bromoindirubin-3'-oxime (BIO)), bFGF and BMP4 Respectively. (Figure 2d)

(2-2) Use of CHIR-99021 as an inhibitor of GSK-3 (Figure 2e)

To confirm the role of the inhibitor of GSK-3 as a WNT signaling agent in the formation of a precursor cell precursor cell culture medium, CHIR-99021 as well as 6-bromoindirubin-3'-oxime (BIO) were added at 0.1 uM, 1 uM, 10 uM To confirm the possibility of differentiation into the precursor cells of the mammary gland. The neural ridge cells induced differentiation in human pluripotent stem cells were cultured in DMEM / F12 glutamax (Thermo Fisher, catalog no. 10565042), 10% FBS basic medium with inhibitor of GSK-3 (CHIR-99021), bFGF, BMP4 Approximately two weeks after the treatment of all three, the differentiation efficiency was increased according to the concentration of GSK-3 inhibitor (CHIR-99021) when the differentiated cells were analyzed by flow cytometry using SDC1 and CD133, markers of papillary precursor cells In addition, we confirmed the significance of WNT signaling in inducing differentiation into papillary precursor cells as well as the induction of concentration - dependent differentiation. (Figure 2E)

(2-3) Degenerate pluripotent stem cells  From a variety of pluripotent stem cells Papillary progenitor cells  Differentiation (Figure 2f)

In order to confirm whether the composition of the differentiation medium of the precursor cells of the mammary gland is universally applicable to various human cell-derived pluripotent stem cells including human-derived embryonic stem cells, one human-derived embryonic stem cell, a human fibroblast-derived pluripotent stem cell Two weeks, the ability to differentiate into human papillary progenitor cells was confirmed by using one human pluripotent stem cell-derived pluripotent stem cell as a source of human pluripotent stem cells. The neural ridge cells induced differentiation in human pluripotent stem cells were cultured in DMEM / F12 glutamax (Thermo Fisher, catalog no. 10565042), 10% FBS basic medium with inhibitor of GSK-3 (CHIR-99021), bFGF, BMP4 After about two weeks of treatment, the differentiated cells were analyzed by flow cytometry using SDC1 and CD133, the markers of the papillary progenitor cells, and there was a difference in the differentiation efficiencies of all pluripotent stem cell origin. However, And a significant increase in the SDC1 + / CD133- cell fraction, which is a marker of the cell, was confirmed. (Figure 2f)

(2-4) Papillary progenitor cells  Induction of differentiation

As shown in FIG. 2A, in order to induce the differentiation of the precursor cells of the mammary gland using the essential factors selected above, human pre-differentiation pluripotent stem cells were differentiated into neural ridge cells (STAGE 1) , The differentiated cells were cultured in DMEM / F12 glutamax medium supplemented with FGF2 (10 ng / mL), GSK-g inhibitor and BMP4 (1 ng / mL) for a short period of time STAGE 2), followed by subculture of surrounding dermal papilla cells with DMEM / F12 glutamax medium supplemented with FGF2 (10 ng / mL) alone (STAGE 3).

In accordance with the above steps, the cells were differentiated into human pre-differentiated pluripotent stem cells, and then morphological changes were observed with a microscope and ALP activity was measured . As a result, as shown in FIG. 2C, it was observed that the cell morphology changed into a fusiform polygonal shape similar to that of human primary dermal papilla cells in a typical state, and the proliferated dermal papillary precursor cells were isolated, and the important characteristic of the inherent dermal papilla cells It was confirmed that a three-dimensional spherical body was produced.

Example  4. Hair follicles Placod  In step Degenerate pluripotent stem cells  origin Premature oocyte (hiPSC-derived DPPCs )

The present inventors observed flow cytometric analysis of the SDC1 and CD133 transient levels during the differentiation steps from the human de-differentiating pluripotent stem cells of Example 3 and Fig. 2A to the pre-papillary progenitor cells. As a result, as shown in FIG. 3A, the expression of SDC1 gradually increased with the progress of differentiation into the precursor cells of the papilla, while the expression of the stem cell marker CD133 decreased. From the above results, it can be seen that for the SDC1 + / CD133- cell population, the period from the 25th day to the 40th day after the initiation of differentiation reaches a peak of 99% of the cells. Consistent with the above results, the pluripotent stem cell (SSEA3 +) population also gradually decreased during the differentiation process.

In addition to the above results, the present inventors tried to analyze transient gene expression profiles in cells of differentiation stage (iPSC, NCSC, DPPC D25, DPPC D40, DPPC P5). To this end, neuronal stem cells (expressing NCSC, p75NTR, HNK1, and SOX10) were transfected into reprogrammed pluripotent stem cells (expressing iPSC, OCT3 / 4 and NANOG) approximately 7 days after induction of differentiation, , Hs-related family BHLH transcription factor [HEY1] with SDC1, biglycan [BGN], WNT5A, BMP4, and YRPW motif 1). As a result, as shown in FIG. 3B, the expression of human breast markers genes increased from about 25 days after differentiation and showed the highest expression level at 40 days, and was similar to that of human in vitro cultured dermal papilla cells And the level of expression was decreased. This expression pattern is consistent with SDC1 expression by flow cytometry.

On the other hand, CD133 is known as a surface marker of hair-inducing skin cells in mice. Thus, the present inventors have used CD133 ⁺ and CD133 ^{&lt; RTI ID = 0.0 &gt;} The cells were separated and analyzed for their characteristics. As a result, as shown in FIG. 3C, CD133 Expression of ALX homeobox3 ( ALX3 ), catenin beta 1 ( CTNNB1 ), SOX2 , and lymphoid enhancer binding factor 1 ( LEF1 ) was significantly increased in the cells expressing the typical dermal papilla. On the other hand, it was confirmed that the above results were not observed in CD133 ⁺ cells.

Example  5. Human Degenerate pluripotent stem cells  origin Premature oocyte ( hiPSC -derived DPPCs) and Hair follicle formation ability  analysis

(HDPC) of human preform precursor cells differentiated according to the method of Example 3 described above. For this purpose, immunocytochemistry was performed on human dermal papillary precursor cells (hi-DPPC) to determine the activity of alkaline phosphatase (ALP), α-smooth muscle actin, versican (VCAN), and nestin gene Was expressed. As a result, as shown in FIG. 4A, the activity of alkaline phosphatase was also observed in the papillary precursor cells (hi-DPPC) differentiated to human dermal papilla cells (hDPC), and it was confirmed that αSMA, VCAN and nestin were also strongly expressed Respectively.

In addition, spontaneous sphere formation similar to the in vivo papilla structure is an inherent characteristic of the papilla, which is related to the restoration of the hair-inducing ability and the inherent characteristics of human papilla cells. . As a result, as shown in FIG. 4B, it was confirmed that the mammary goblet precursor cells effectively form a 3D spherical body structure at very low attachment conditions reflecting the characteristics of human dermal papilla cells.

As a result of analysis of transcripts by quantitative PCR, human papillary precursor cell sphincters (hi-DPPC 3D) were compared with human dermal papilla cells (cDPC 2D) and dermal papilla cell dendritic cells (cDPC 3D) Signaling genes ALX3 , SOX2 , HEY1 , BMP4 , LEF1 , WNT inhibitory factor 1 [ WIF1 ], and VCAN were increased. In particular, the expression of papillary transcription factor was rapidly decreased in the one-dimensional culture of dermal papilla cells, whereas the expression of ALX3, SOX2 and HEY1 was significantly increased in the hi-DPPC spheres compared to the other. In addition, the factors involved in the WNT signaling pathway, including LEF1 and WIF1, and the factors involved in the support matrix during follicular morphogenesis, such as VCAN, also increased significantly in the papillary precursor cell populations. These results indicate that the prefrontal cells reflect the early precursor cell stage of human dermal papilla cells.

Furthermore, the inventors of the present invention conducted experiments to determine whether the hair follicle precursor cells according to the present invention can induce hair follicle formation when they are transplanted into a hairless hairless (SHO) mouse deficient in immunity, . Specifically, the human dermal papilla cell spheroid (cDPD 3D) or the 40 day dermal papillary precursor cell spheroid (hiDPPC 3D) was subcutaneously transplanted together with the epidermal cell (mEPI) of C57BL / 6 neonatal mouse. Since the SHO mouse has no hair follicle and an albino genetic background, it can easily distinguish black-colored follicles newly formed by C57BL / 6 mouse-derived epidermal cells.

As a result, as shown in FIG. 5A, a large number of new hybrid follicles were observed at the site where the papillary precursor cell sphere and the papilla cell were transplanted two weeks after the experiment. In addition, in order to determine whether or not the transplanted mammary epithelial cell spheroid contributes to the dermal papilla of the neonatal hair follicle, the papillary precursor cells were labeled with a cell-tracer (CM-DiI) showing red fluorescence. As a result, The presence of red fluorescence in the dermal papilla of the hair follicle confirmed the presence of the dermal papillary precursor cells and contributed to the hair follicle neogenesis. The quantitative results of the hybrid follicular neoplasms are shown in Table 2 below.

Example  6. Human Degenerate pluripotent stem cells  origin Premature oocyte ( hiPSC Analysis of gene expression profiling of total RNA isolated from -derived DPPCs

To confirm the gene expression profile of the papillary precursor cells according to the present invention, the inventors of the present invention used human papillomavirus-derived pluripotent stem cell-derived neural ridge cells (hi-NCSCs), human dermal papilla cell spheroids (cDPC 3D) 2D) was performed to analyze the RNA sequence of the human dermal papilla cell spheroid. As a result, a heat map was generated for a total of 3065 genes and hierarchical clustering was performed using a distance matrix. As shown in FIGS. 6A and 6B, the papillary precursor cells were clustered with papillary cell sphincters and papillary cells, Which is a very different gene expression pattern. Also, as can be seen in FIG. 6C, based on the diminishing approach, the group of mammary epithelial cells was observed as a distinct intermediate in the differentiation from neural ridge cells to the papillary cell sphere.

In particular, as shown in FIG. 6D, many of the mammalian signature genes such as WNT5A , SHH , transcriptional repressor GATA binding 1 ( TRPS1 ), HEY1 , LEF1 , and FOXO1 were observed in the dermal papilla cell and dermal papilla cells Compared to the control group. In contrast, expression of stem cell markers such as NANOG and LIN28A was rarely found in differentiated papillary progenitor cells. This result implies that, in contrast to the neural ridge cells before the differentiation, the precursor cells of the mammary epithelium share a more similar or early precursor molecule signature than the human dermal papilla cell protoplasts rather than the dermal papilla cells.

Example  7. Formation and characterization of human neonatal hair follicles in vitro

To induce human hair follicle development in vitro, we induced differentiation from human de-differentiating pluripotent stem cells into folliculogenic EpSCs according to the known methods described in Examples 1-4 above. The hair follicle epidermal precursor cells (hi-EpSCs) differentiated according to the above method continuously express keratin 14 (keratin 14; K14), keratin 15 (K15) and integrin 6 (integrin 6) And hybrid follicles were induced upon binding to mouse neonatal skin cells (Figure S5). Surprisingly, the human epidermal progenitor cells expressed CD133 in the plasma membrane at the time of hair fracode with the accumulation of beta-catenin (beta-catenin) similar to that of human epidermal plastocytes.

Furthermore, in order to examine the hair follicle-forming ability of human dermal papillary precursor cells, in vitro hair follicle formation analysis was performed by incubation with hi-EpSCs before analysis of body hair follicle formation. First, the precursor cells of the mammary gland were divided into 10,000 cells / well on plates with very low adherence to form small spherical bodies having their basal membranes with recombinant human extracellular space proteins.

Next, the epidermal progenitor cells were co-cultured in a number of 25,000 cells / well in a preform of premolar cell. As a result of the cultivation, as shown in FIG. 7A, surprisingly, a mixture of the two cells produced a round structure (yield ~ 65%) which is very similar to that of hair follicular morphogenesis showing flat polarity and proliferation in the surrounding layer.

Next, the characteristics of the skin and epidermal constituents were compared with in vivo steps of the human fetal scalp. As a result, hair follicles showed expression patterns of skin markers (SDC1, LEF1, SOX2, Nestin, VCAN and WIF1) and epidermal markers (CD133, BMP4, K15, K14 and WIF1) Respectively. Interestingly, CD133 expression was slightly increased in skin components of co-cultured hair follicles similar to hair germ or hair peg stage.

Example  8. Human Degenerate pluripotent stem cells  origin Premature oocyte ( iPSC -derived DPPCs) and human Degenerate pluripotent stem cells  origin Epidermal progenitor cells (hiPSC-derived EpSC) Human by Follicular newborn  Confirm

In the present invention, human hair follicle progenitor cells and epidermal progenitor cells differentiated from human reprogramming pluripotent stem cells were used to examine human hair follicle development. For this purpose, a chamber assay was carried out according to the method of Example 1-11 to perform in vivo follicular neoplasia analysis, and the differentiation of prehyperidone precursor cell spheres (hi-DPPC D25, hi-DPPC D40, and hi- DPPC P25) was transplanted with 18-day-old epidermal progenitor cells (hi-EpSC D18) after the onset of differentiation and histological analysis was performed.

As a result, it was observed that a new hair follicle having several layers of epidermis, a papilla structure, and a pigment-free morgan was formed in Fig. 8A. The hair follicle showed a structure similar to the human fetal hair follicle, and clearly different from that of the incomplete hair follicle produced in the SHO mouse.

In addition, as shown in FIG. 8B, an interesting human hair follicle (hi-DPPC + hi-EpSC) is produced in the cytoplasm similar to the human fetal hair follicle (COC4) (anti-human cytochrome c oxidase subunit 4) And it was confirmed to be positive by antibody and anti-human mitochondrial antibody.

Furthermore, it was observed that hair-specific keratin markers (K14, K15, K17, and K75) were expressed multilayered in the center circle of newly formed hair follicles. K14 represents the outer root sheath, K15 represents the hair follicle stem cell, K17 represents the maternal quality and hair follicle quality, and K75 reflects the presence of a companion layer in the new hair follicle. In addition, quantitative results of follicular neoplasia through the chamber assay are shown in Table 3, and it was found that the hair follicle progenitor cells on the 40th day of differentiation and the epidermal progenitor cells on the 18th day were highly transfected, have.

From the above results, it was confirmed that human hair follicle neogenesis could be induced in mouse by using both pre-papillary precursor cells and epidermal progenitor cells differentiated from human reprogramming pluripotent stem cells.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (25)

1. Fibroblast growth factor-2 (FGF2), glycogen synthase kinase-3 (GSK-3) were added to DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F- 3) inhibitor, and bone morphogenetic protein 4 (BMP4). 2. The composition according to claim 1, wherein the bone morphogenetic protein is BMP4.
2. The method according to claim 1,

   The inhibitor of glycogen synthase kinase-3 (GSK-3) is 6-bromoindirubin-3'-oxime (BIO), CHIR-99021 and SB-216763 &Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
3. The differentiation medium composition according to claim 1, wherein the FGF2 is contained at a concentration of 5 to 30 ng / ml, the GSK-3 inhibitor is 0.1 to 10 uM, and the BMP4 is contained at a concentration of 0.5 to 5 ng / ml.
4. The culture medium for differentiation according to claim 2, wherein the BIO is contained at a concentration of 0.5 to 5 uM, or the CHIR 99021 is contained at a concentration of 0.1 to 10 uM.
5. The method according to claim 1,

   Wherein said human-derived pluripotent stem cells are human-derived embryonic stem cells or human-derived pluripotent pluripotent stem cells.
6. The differentiation medium composition according to claim 1, wherein the differentiation medium composition is free of human neonatal fetal tissue.
7. A method of differentiating human pluripotent stem cells into papillary progenitor cells, comprising the steps of:

   (a) culturing an embryoid body of human-derived pluripotent stem cells in a neural crest stem cell-inducing medium to differentiate into neural ridge cells; And

   (b) culturing the differentiated neural ridge cells in the differentiation medium composition of claim 1 to differentiate into pre-papillary precursor cells.
8. 8. The method according to claim 7, further comprising the step of:

   (c) matured differentiated precursor cells to obtain precursor cells of a spherical sphere.
9. 8. The method of claim 7,

   The neural ridge cell induction medium was supplemented with DMEM / F12 glutamax medium supplemented with fibroblast growth factor-2 (FGF2), fibronectin, insulin, N2 supplementation, and transferrin transferrin). &lt; / RTI &gt;
10. 9. The method of claim 8,

    Wherein the step (c) comprises culturing the differentiated precursor cells in a DMEM / F12 glutamax medium to which FGF2 alone is added.
11. The method according to claim 7 or 8, wherein (a) is performed for 5 to 9 days, (b) is performed for 11 to 17 days, or (c) Wherein said method comprises the steps of:
12. 8. The differentiation method according to claim 7, wherein the method of differentiating human pluripotent stem cells into a mammary gland precursor cell is performed for 26 days or more and 50 days or less.
13. 13. A human mammary dermal progenitor cell differentiated from human pluripotent stem cells by the differentiation method according to any one of claims 7 to 12, wherein the human dermal papilla precursor cell has follicle-forming ability.
14. 14. The method of claim 13,

    Wherein said papillary precursor cells exhibit at least one characteristic selected from the group consisting of (a), (b) and (c):

    (a) exhibits positive immunological properties for ALP (alkaline phosphatase), alpha SMA (alpha smooth muscle actin), versican (VCAN), and nestin;

    (b) demonstrates the structural characteristics of dermal papilla cells that spontaneously form spheres; And

    (b), (b), (c), and (c), the genes for human breastpox signature genes ALX3 (ALX Homeobox 3), SOX2 (SRY-Box 2), HEY1 (Hes Related Family BHLH Transcription Factor With YRPW Motif 1), BMP4 (Bone Morphogenetic Protein 4) and Lymphoid Enhancer Binding Factor 1), WIF1 (WNT inhibitory factor 1), and VCAN (Versican).
15. 14. The method of claim 13, wherein the papillary precursor cells are CD133 negative.
16. A subculture human mammary papillary precursor cell having a hair follicle-forming ability differentiated from human-derived pluripotent stem cells by the differentiation method according to any one of claims 7 to 12.
17. A composition for inducing human hair follicular growth comprising an epithelial stem cell differentiated from human pluripotent stem cells and a human derived mammary epithelial precursor cell having a hair follicle forming ability differentiated from the human pluripotent stem cell of paragraph 13 as an active ingredient .
18. 18. The method of claim 17,

    Wherein the mammary gland precursor cells are differentiated from the human-derived pluripotent stem cells for 30 days to 50 days.
19. 18. The method of claim 17,

    Wherein the epidermal progenitor cells are differentiated from the human-derived pluripotent stem cells for 15 days to 21 days.
20. A method for inducing human hair follicular neoplasia by transferring epithelial stem cells differentiated from human pluripotent stem cells and human precursor cells derived from human pluripotent stem cells differentiated from the human pluripotent stem cell of claim 13 to a subject.
21. A method for producing a human hair follicle by co-culturing a human-derived mammary epithelial progenitor cell having a hair follicle-forming ability differentiated from the human-derived pluripotent stem cell of claim 13 and an epithelial stem cell differentiated from human-derived pluripotent stem cell.
22. An epithelial stem cell differentiated from human pluripotent stem cells and a human hair follicle precursor cell having a hair follicle forming ability differentiated from the human pluripotent stem cell of claim 13 as an active ingredient.
23. An epithelial stem cell differentiated from human pluripotent stem cells and a human derived mammalian precursor cell of paragraph 13 as an active ingredient.
24. An epithelial stem cell differentiated from human pluripotent stem cells and a human cell precursor cell having hair follicle forming ability differentiated from the human pluripotent stem cell of item 13 above as an active ingredient are administered to an individual &Lt; / RTI &gt;
25. An epithelial stem cell differentiated from human pluripotent stem cells and a human hairpin progenitor cell having hair follicle forming ability differentiated from the human pluripotent stem cell of claim 13 as an active ingredient.

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