WO2019198962A1 - Method for promoting proliferation and differentiation of induced pluripotent stem cells prepared from urine-derived stem cells - Google Patents

Abstract

The present invention relates to: a method for isolating and proliferating urine-derived stem cells; a method for promoting the proliferation or differentiation of stem cells differentiated from induced pluripotent stem cells which are reprogrammed from the urine-derived stem cells; and a method for increasing the formation rate of EB from the induced pluripotent stem cells.

Description

Method for promoting proliferation and differentiation of induced pluripotent stem cells prepared from urine-derived stem cells

The present invention is a method for isolation and proliferation of urine-derived stem cells; Methods for promoting proliferation or differentiation of stem cells; And it relates to a method of increasing the formation rate of EB (Embryoid Bodies) from induced pluripotent stem cells.

Stem cell research is an explosive and exciting study with the potential to improve therapies for humans. Such stem cell research is being carried out by mesenchymal stem cells and pluripotent stem cells.

For mesenchymal stem cells, bone marrow derived stem cells, adipose derived stem cells, and umbilical cord derived stem cells have been studied for a long time. Attempts have been made to experimental studies and preclinical studies. In addition, autologous stem cells are clinically useful because they do not induce immune and rejection responses. However, stem cells, which can be separated from tissues and organs, make up a very small cell population in the body and have difficulty in separating cells because a special tissue separation technique is required. Urine derived stem cells used in the present invention can be easily separated from human urine, and has the ability to differentiate into various tissues and organs like other somatic stem cells. In recent decades, urine-derived stem cells have emerged as promising cellular resources for cell therapy and tissue engineering, and play an important role in stem cell biology based on these applications.

Pluripotent stem cells are included in embryonic stem cells and induced pluripotent stem cells. Embryonic stem cells are derived from the inner cell mass of early embryos and can differentiate into three types of germ layer cells in vitro and in vivo. In addition, induced pluripotent stem cells were reprogrammed similar to embryonic stem cells by introducing the Oct3 / 4, Sox2, c-Myc, and Klf4 genes into mouse somatic cells using a retrovirus in 2006 by Yamanaka's team at Kyoto University. Reprogrammed cells were generated, which were termed induced pluripotent stem cells. Induced pluripotent stem cells can generate cell lines of various lines in vitro by circumventing the ethical problems of embryonic stem cells resulting from the destruction of early embryos. Induced pluripotent stem cells are cells that can solve the ethical issues of embryonic stem cells, and have a pluripotency that can be differentiated into all the cells of the human body. Is attracting attention.

In order to make human induced pluripotent stem cells, adult cells (somatic cells) are collected from a donor and manufactured. A process called reprogramming is performed by collecting cells from a donor's fibroblast and blood. Through. Recently, researches to find a safer and more efficient method for obtaining cells from donors for the reprogramming process have been conducted. In particular, invasive methods such as the collection of blood, etc. may be used to improve problems related to hematoma that may occur during blood collection, concern about infection, and blood collection from patients with blood-borne diseases. Methods of separating cells from the donor's urine have been invented. In addition, in attempting to differentiate into various human constituent cells using induced pluripotent stem cells, the parts to be improved on the current differentiation are still the subject of study, and research to increase the differentiation ability and the accuracy is necessary.

An object of the present invention is to provide a method for separating and propagating urine-derived stem cells comprising the step of culturing in a cell culture dish coated with a coating material by separating the cells from the urine collected from the individual.

Still another object of the present invention is a stem comprising treating 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone. It is to provide a method for promoting the proliferation or differentiation of cells.

Another object of the present invention is an induction comprising treating 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones. It is to provide a method for increasing the formation rate of EB (Embryoid Bodies) from pluripotent stem cells.

In order to achieve the above object, the present invention provides a method for separating and propagating urine-derived stem cells comprising the step of culturing in a cell culture dish coated with a coating material by separating the cells from the urine collected from the individual.

In one embodiment of the present invention, the urine may be collected from the middle urine of the urine.

In one embodiment of the present invention, the cells may be isolated within 2 hours by collecting from the urine of the individual.

In one embodiment of the present invention, the separation may be performed by centrifugation for 5 to 15 minutes at 300g to 500g, preferably at 400g may be performed for 10 minutes.

In one embodiment of the invention, the method may further comprise the step of washing with PBS (phosphate buffered saline) after separating the cells.

In one embodiment of the present invention, the coating material may be Matrigel.

In one embodiment of the present invention, the culturing may be to culture by adding Y-27632.

In addition, the present invention is a stem cell comprising the step of treating 3,2'-dihydroxyflavones (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavones (3,4'-dihydroxyflavone) Provided are methods for promoting proliferation or differentiation.

In one embodiment of the present invention, the stem cells may be stem cells differentiated from induced pluripotent stem cells, the induced pluripotent stem cells may be reprogrammed from urine-derived stem cells.

In one embodiment of the present invention, the stem cells may be hematopoietic stem cells.

In one embodiment of the present invention, the 3,2'- dihydroxy flavone or 3,4'- dihydroxy flavone may be a concentration of 1 to 20 μM, preferably a concentration of 5 to 15 μM It may be, and more preferably may be a concentration of 10 μM.

In addition, the present invention is the induced pluripotent stem comprising the step of treating 3,2'-dihydroxyflavones (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavones (3,4'-dihydroxyflavone) Provided are methods for increasing the formation rate of Embryoid Bodies (EB) from cells.

In one embodiment of the present invention, the induced pluripotent stem cells may be reprogrammed from urine-derived stem cells.

In one embodiment of the present invention, the 3,2'- dihydroxy flavone or 3,4'- dihydroxy flavone may be a concentration of 1 to 20 μM, preferably a concentration of 5 to 15 μM It may be, and more preferably may be a concentration of 10 μM.

The method for isolating and propagating urine-derived stem cells according to the present invention can efficiently separate and proliferate stem cells from urine, and can be utilized in various fields using stem cells.

In addition, induced pluripotent stem cells can be prepared from urine-derived stem cells, and treated with 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones to increase the EB formation rate in induced pluripotent stem cells, Proliferation and differentiation of differentiated stem cells can be further promoted, and can be utilized in various fields using stem cells.

1 is a schematic diagram showing a method for separating urine-derived stem cells.

Figure 2 shows the results of analyzing the characteristics of the urine-derived stem cells.

3 is a Gelatin coating group; Gelatin coating + Y-27632 treatment group; Matrigel coating group; And the number of colony-forming cells on the 5th day of isolation and culture of urine-derived stem cells according to Matrigel coating + Y-27632 treatment group.

4 is a Gelatin coating group; Gelatin coating + Y-27632 treatment group; Matrigel coating group; And 14 days after the separation of urine-derived stem cells in Matrigel coating + Y-27632 treatment group by measuring the number of cells is the result of comparing the separation efficiency of urine-derived stem cells.

5 is a Gelatin coating group; Gelatin coating + Y-27632 treatment group; Matrigel coating group; And Matrigel-coated + Y-27632 treatment group to compare the proliferation efficiency of urine-derived stem cells.

6 is a Gelatin coating group; Gelatin coating + Y-27632 treatment group; Matrigel coating group; And cell migration ability of urine-derived stem cells in Matrigel coating + Y-27632 treatment group.

7 is a Gelatin coating group; Gelatin coating + Y-27632 treatment group; Matrigel coating group; And Matrigel coating + Y-27632 treatment group to compare the colony forming ability of urine derived stem cells.

8 is a result of comparing the time-specific proliferation rate of adipose derived stem cells, umbilical cord derived stem cells and urine derived stem cells.

9 is a result of comparing the amount of cells obtained from adipose derived stem cells, umbilical cord derived stem cells and urine derived stem cells.

10 is a result of comparing the colony forming ability of adipose derived stem cells, umbilical cord derived stem cells and urine derived stem cells.

Figure 11 shows a schematic diagram (top) showing the production of induced pluripotent stem cells from urine-derived stem cells (top) and produced induced pluripotent stem cells (bottom).

12 is a result of confirming whether the expression of pluripotent stem cells Oct4, Sox2 and Nanog in induced pluripotent stem cells prepared from urine-derived stem cells and chromosomal analysis results.

Figure 13 shows a schematic diagram (top) and the prepared kidney organoid (bottom) showing the production process of kidney organoid in the induced pluripotent stem cells.

Figure 14 shows the EB formation rate in induced pluripotent stem cells following treatment with 3,2'-dihydroxyflavone (3,2'-DHF) or 3,4'-dihydroxyflavone (3,4'-DHF). The result is a comparison.

15 is a schematic diagram showing the differentiation process of the induced pluripotent stem cells into hematopoietic stem cells (top) and hematopoietic stems according to treatment of 3,2'-DHF or 3,4'-DHF through markers of hematopoietic stem cells This is a result of comparing the differentiation efficiency of the cells.

Figure 16 is a result of comparing the total cell number and CD34 + CD45 + cell number after treatment with 3,2'-DHF or 3,4'-DHF in hematopoietic stem cells differentiated from induced pluripotent stem cells.

17 is a result of analyzing CFU (Colony forming unit) according to the treatment of 3,2'-DHF or 3,4'-DHF in hematopoietic stem cells differentiated from induced pluripotent stem cells.

As used herein, the term “stem cell” refers to a cell having pluripotent or totipotent self-renewal capable of differentiating into cells of all tissues of an individual, Embryonic stem cells, induced pluripotent stem cells and adult stem cells.

The term "embryonic stem cell" refers to a cell cultured in vitro by extracting an inner cell mass from an blastocyst embryo just before the fertilized egg implants in the mother's uterus, which can differentiate into cells of all tissues of the individual. It refers to a stem cell having a pluripotent or totipotent self-renewal.

The term "induced pluripotent stem cells (iPSC)" refers to pluripotent pluripotent stem cells, which have been differentiated into somatic cells or cells with limited differentiation, and then reprogrammed to regenerate into pluripotent cells through reprogramming. Refers to the cells that induced sex. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically showing similar cell shapes, gene and protein expression patterns are similar, and have the characteristics of differentiation ability in and outside the body. It is not derived from female eggs, such as human embryonic stem cells, but is derived from already differentiated somatic cells, so it does not involve ethical problems such as human embryonic stem cells, and can be converted from a patient's somatic cells to stem cells. Therefore, the problem of immune rejection is less. However, despite such usefulness, there is a disadvantage in that a sufficient number of induced pluripotent stem cells cannot be obtained due to low dedifferentiation efficiency.

The term "reprogramming" refers to a process by which differentiated cells can be restored or converted into a pluripotent state. In the present invention, the reprogramming includes any process of returning differentiated cells having a differentiation capacity of 0% to less than 100% to an undifferentiated state, preferably differentiated cells having a differentiation capacity of 0% or more than 0%. It includes both restoring or converting partially differentiated cells having a differentiation capacity of less than 100% into cells having 100% differentiation ability. Such reprogramming may be performed by a reprogramming reprogramming factor.

The term "reprogramming inducer" refers to a substance that, when processed or expressed in differentiated cells, induces iPSC-specific gene expression and induces induced pluripotent stem cells having pluripotency. The reprogramming inducer may be included without limitation in the present invention as long as it is a substance that induces reprogramming of differentiated cells, and examples thereof include Oct3 / 4, sox-2, c-Myc, and Klf-4. It can select according to the kind of cell to do, and is not limited to the said example.

The term "adult stem cell" refers to an undifferentiated cell capable of self-reproduction as a stem cell generated in differentiated tissue and capable of differentiating into all cell types of the derived tissue. Specifically, the adult stem cells are cord blood (umbilical cord blood), adult bone marrow, spleen, ovary, testis, peripheral blood, amniotic fluid, brain, blood vessels, skeletal muscle, skin or gastrointestinal epithelium, cornea, tooth dimensions, retina, liver or It can be extracted from the pancreas, and refers to primitive cells just before differentiation into cells of specific organs such as bone, liver, and blood. For example, the spinal cord has been reported to have hematopoietic stem cells (HSCs) and mesenchymal stem cells, and neural stem cells, stem cells derived from the brain, have subventricular zones. It is known to separate from the subventricular zone, ventricular zone and hippocampus of the central nervous system (CNS). In general, adult stem cells are known to be difficult to proliferate, but have a strong tendency to differentiate. As a result, adult stem cells can be used not only to regenerate organs required by actual medicine but also to characterize each organ after transplantation. It has characteristics that can be differentiated accordingly.

As used herein, the term "hematopoietic stem cell" refers to a variety of blood cells and parental cells of lymphocytes that form the immune system, for example stems that can develop into lymphocytes, granulocytes, platelets, and red blood cells, such as B cells and T cells. Says a cell. It is an essential cell for bone marrow transplantation. In normal bone marrow blood, about 1% of cells (CD34 positive cells) that have the ability to produce all blood cells are called hematopoietic stem cells. Found throughout the body, including cord blood and umbilical cord blood, but is produced in large quantities in the bone marrow, and also has the ability to replicate itself. Peripheral blood hematopoietic stem cells are derived from bone marrow and have the property of self-replicating and matured cells as hematopoietic stem cells circulating in the bloodstream.

The term "proliferation" of the stem cell in the present invention is a concept that is differentiated from the differentiation (differentiation) of stem cells, the stem cells are not differentiated into specific cells, the cells are divided while maintaining the characteristics of the stem cells intact It means that the total number of cells is increased.

As used herein, the term "differentiation" of stem cells refers to a phenomenon in which less specialized cells develop into specific cells so that the size or shape of the cells, membrane potential, metabolic activity, and response to a signal change to a specific type of cell. Adult stem cells differentiate into specific cells when multicellular organisms occur during the formation of complex tissues in a single zygote, or when they repair damaged tissues as adults.

As used herein, the term "medium" refers to a composition comprising nutrients necessary to maintain the growth and survival of cells in vitro .

As used herein, the term "passage culture" means a method of continually culturing the cell band while transferring a portion of the cell to a new culture vessel periodically to continuously culture the cell in a healthy state for a long time. do. As the number of cells increases in a culture vessel with a limited space, proliferation of nutrients is consumed or contaminants accumulate, causing the cells to die naturally, and thus used as a method for increasing the number of healthy cells. Replacing a culture vessel or culturing a cell group is called one passage.

The active ingredient of the present invention is 3,2'-dihydroxyflavone (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavone (3,4'-dihydroxyflavone), each structure is represented by the following formula .

[Formula]

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1 Experimental Materials and Methods

1.1. Human Urine-derived Stem Cell Isolation and Culture

Urine-derived stem cells were obtained from six or more different sexes and ages. The urine-derived stem cells were collected from urine intermediate urine and separated within 2 hours at room temperature. A total of 100-200 mL of urine was put in a urine container, divided into four 50 mL tubes, and then centrifuged at 400 g for 10 minutes using a centrifuge at room temperature, and then the supernatant was removed. To wash the cells, 15 mL of PBS containing Antibiotic-Antimycotic (Gibco) was placed in a 50 mL tube, centrifuged at 400 g for 10 minutes, seeded in a cell culture dish coated with Gelatin, Matrigel, and Y-27632 was added to the cell culture medium. It was. For 3 days after cell separation, 37 ° C, 5% CO ₂ in a 1: 1 mixture of DMEM / high glucose and Ham's F12 nutrient mix containing 10% FBS, Antibiotic-Antimycotic (Gibco), and REGM SingleQuot kit supplement (Lonza) The cells were cultured in a cell culture incubator under the conditions, and after 3 days, the cells were replaced with REGM BulletKit (Lonza) culture medium.

1.2. Total RNA Isolation and RT-PCR Analysis

According to the manufacturer's method, Total RNA was extracted using an Easy-Blue total RNA extraction kit (iNtRON Biotechnology, Republic of Korea). The concentration of total RNA was measured with a Nanodrop (ND1000) spectrophotometer (Nanodrop Technologies Inc., Wilmington DE, USA). CDNA was synthesized using 2 μg total RNA and M-MLV reverse transcriptase, and analyzed after completion of the PCR reaction in 1% or 1.5% agarose gel. qPCR was analyzed using SYBR Green master mix and mRNA expression was calculated using House keeping gene (GAPDH) as a reference value.

1.3. Western blot

Cell lysis buffer [1% Triton X-100 (Sigma-Aldrich), 100 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10% glycerol (Amresco, Solon OH, USA), 50 mM sodium fluoride (Sigma-Aldrich ), 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), 1 mM p-nitrophenyl phosphate (Sigma-Aldrich), and 1 mM sodium orthovanadate (Sigma-Aldrich)] at 13,000 rpm for 15 minutes at 4 ° C. Centrifuged as. Protein supernatants were quantified with Bradford protein reagents and proteins were subjected to SDS-PAGE (10% or 12%). The isolated protein was transferred to a nitro-ceulocellulose membrane and blocked for 1 hour with 5% skim milk powder dissolved in a tris-buffered saline (TBS) solution, followed by anti-OCT4, anti-SOX2, anti-NANOG and anti Primary antibodies of -ACTIN (Santa Cruz Biotechnology, Dallas, TX, USA) were reacted at 4 ° C. for 12-18 hours. Then, HRP-tagged anti-mouse, anti-goat or anti-rabbit secondary antibody was reacted, and the protein was identified using an ECL kit (Amersham Bioscience, Piscataway NJ, USA).

1.4. Fibroblast colony-forming unit (CFU-F) assay

Urine-derived stem cells, adipose derived stem cells, umbilical stem cells in culture were counted and laid on a cell culture plate by 50 / cm ² , and cultured for 10-14 days to confirm colony formation of cells. When the colonies were formed after incubation of the cells, the cells were washed with PBS, stained with crystal violet reagent for 20 minutes, and then washed with PBS again to select colonies.

1.5. Flow cytometry

For flow cytometry of urine-derived stem cells, the urine-derived stem cells in the cell culture plate were treated with 0.25% Trypsin for 3 minutes to float the cells, and then centrifuged at 1,000 rpm. The supernatant was then discarded, washed with 1X PBS and centrifuged again. Blocking Buffer (0.5% BSA, 2% FBS in PBS) was then reacted on ice for 30 minutes and each primary antibody (CD34, CD45, containing FACS Buffer (0.05% sodium azide, 0.5% BSA in PBS)) was added. CD105, CD73, CD90) were reacted on ice for 1 hour. After washing, secondary antibodies (FITC, PE) were reacted on ice for 30 minutes, and the cells were washed twice with 1X PBS, then resuspended in 1X PBS and analyzed by FACS Calibur.

1.6. Cell Reprogramming and Hematopoietic Stem Cell Differentiation

Proliferated cells were reprogrammed using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) according to the manufacturer's manual. Reprogramming induced pluripotent stem cell colonies were transferred to Matrigel (BD Biosciences) coated culture plates and incubated with mTeSR ™ 1 (STEMCELL Technologies) and 10 μM of Y-27632 (STEMCELL Technologies).

EB formation was induced for 5 days to differentiate into hematopoietic stem cells using proliferated cultured induced pluripotent stem cells. Then, in IMDM medium containing 15% FBS, SCF (40ng / mL), FLT-3L (20ng / mL), IL-3 (10ng / mL), IL-6 (10ng / mL), BMP4 (20ng / mL) Differentiation was induced by the addition of the medium, and half of the culture medium was changed every day for a total of 21 days of differentiation.

1.7. Embryoid Bodies (EB) Formation Rate

EB formation rate was measured as an average of three times by counting the number of EB through the microscope at the end of EB formation. In addition, EB was treated with StemPro Accutase (Thermo Fisher Scientific) at 37 ° C. for 10 minutes, the cells were slowly pipetted to separate into single cells, and the total cell number was measured.

Example 2. Isolation of Urine-derived Stem Cells

In order to separate urine-derived stem cells, 100-200 mL of intermediate urine was received in a sterile urine tube, and then centrifuged at 400 g for 10 minutes using a centrifuge using a 50 mL conical tube. After centrifugation, the cells were collected in one 50mL conical tube, PBS was added, and centrifugation was further performed at 400g for 10 minutes. The isolated urine-derived stem cells were cultured for up to 14 days, the cell number was measured, and then passaged (Fig. 1).

The inventors performed RT-PCR to characterize the isolated urine-derived stem cells. As a result, the expression of epithelial markers E-cadherin, Claudin 1, Occludin in urine-derived stem cells was confirmed, and the expression of Vimentin, Twist1, Fibronetin, fibroblast markers, and specific expression of Renal epithelial markers SLC2A1 and L1CAM. (Fig. 2 top panel).

In addition, we confirmed the expression of the adult stem cell positive markers CD73, CD90, CD105 in the urine-derived stem cells through flow cytometry, and confirmed the non-expression of the negative markers CD34, CD45 by the urine-derived stem cells known adult stem It was confirmed that the same expression pattern as the cell marker (Fig. 2 lower panel). As a result, it was confirmed that the isolated cells are urine-derived stem cells having characteristics similar to that of adult stem cells.

Example 3 Increased Separation Efficiency of Urine-derived Stem Cells

To separate urine-derived stem cells, middle urine was centrifuged and the cells were seeded on cell culture plates. The cell culture plate was divided into Gelatin-coated groups, Matrigel-coated groups, and Y-27632-treated and untreated groups on cell culture plates, respectively, to confirm cell adhesion efficiency (top panel of FIG. 3).

3 to 5 days after the cell separation, it was confirmed that the urine stem cells grow to form colonies by adhering to the culture dish. The urine stem cells isolated from the Gelatin-coated control group were found to have approximately 6 colonies formed. It was confirmed that 15 colonies were formed in the Y-27632 treatment group, and approximately 15 colonies were formed in the Matrigel-coated group, and 25 or more colonies were formed in the Matrigel-coated and Y-27632 treatment groups. It could be confirmed (Figure 3 bottom panel).

After 14 days of urine-derived stem cell separation, the number of cells was confirmed. In the group treated with Gelatin and Y-27632 (Gelatin + Y-27632), the cell separation efficiency was about 10 times higher than that of the group coated with Gelatin alone. In the group coated with Matrigel, the cell separation efficiency was about 6 times, and in the group treated with Matrigel and Y-27632 (Matrigel + Y-27632), about 60 times higher than the group coated with Gelatin alone. Showed cell separation efficiency (FIG. 4).

Therefore, when Matrigel was coated on the plate and treated with Y-27632, it was confirmed that the separation efficiency of urine-derived stem cells was high.

Example 4 Comparison of Proliferation Rates of Urine-derived Stem Cells

In the process of culturing cells in vitro, if the proliferation rate of urine-derived stem cells is higher than that of using a conventionally commercialized medium, the time to mass cultivation can be shortened. Thus, the early cultured urine-derived stem cells can be used. By differentiating into cells such as bone forming cells, chondrocytes and adipocytes can be used as a cell therapy can have many advantages.

In order to increase the proliferation rate of urine-derived stem cells, stem cells were cultured by adding Y-27632 and Matrigel in addition to Gelatin. As a result, the cell proliferation rate was increased in the group treated with Y-27632 together with Gelatin alone, and the cell proliferation rate was increased in the group treated with Matrigel rather than Gelatin, and treated with Matrigel and Y-27632 together. In the group, it was confirmed that the proliferation rate of the cells increased about 4 times compared to the group treated with Gelatin alone (FIG. 5).

Therefore, Matrigel coated on the plate, and treated with Y-27632 was confirmed that can increase the proliferation of urine-derived stem cells.

Example 5. Comparison of Cell Mobility of Urine-derived Stem Cells

The present inventors performed cell migration experiments to confirm the effective regeneration of damaged urine-derived stem cells into damaged tissues after transplantation.

As a result, as shown in Figure 6, the urine-derived stem cells isolated from the general gelatin showed a cell migration capacity of 50% at 24 h, 60% cell migration capacity at 48 h. Urine-derived stem cells isolated from the Y-27632 group showed 60% cell migration at 24 h and about 80% cell migration at 48 h. In addition, urine-derived stem cells isolated from Matrigel-coated petri dishes showed 40% cell migration at 24 h and 70% at 48 h, and approximately 65%, 48 at 24 h in the Matrigel-coated and Y27632 treated groups. It showed cell migration capacity of about 90% at h.

Therefore, Matrigel coated on the plate, and treated with Y-27632 was confirmed that can increase the cell migration capacity of the urine-derived stem cells.

Example 6 Comparison of Colony Forming Capacity of Urine-derived Stem Cells

The inventors performed CFU-F (colony-forming unit fibroblast) analysis to compare the self-renewal ability which is one of the characteristics of adult stem cells. The size of colonies is different depending on the self-proliferative capacity of the colony, that is, small colony shows low self-proliferative power, and large colony shows high self-proliferative power.

As a result of CFU-F analysis, more colonies were formed in Y-27632 plate than Gelatin-coated plate, and more colonies in Matrigel plate. In particular, the plate treated with Matrigel and Y-27632 at the same time confirmed that the formation of a larger number of colonies than the group treated with Gelatin alone (Fig. 7).

Therefore, Matrigel coated on the plate, and treated with Y-27632 was confirmed that can increase the proliferation of urine-derived stem cells.

Example 7 Comparison of Adipose-derived Stem Cells, Umbilical Cord-derived Stem Cells, and Urine-derived Stem Cells

The present inventors performed experiments comparing the time-dependent proliferation rate of the isolated adipose derived stem cells, umbilical cord derived stem cells and urine derived stem cells. As a result, the urine-derived stem cells showed a faster proliferation rate than the adipose derived stem cells or umbilical stem-derived stem cells (FIG. 8). In addition, it was confirmed that the urine-derived stem cells can secure a larger amount of cells than adipose derived stem cells or umbilical cord stem cells (FIG. 9).

In addition, it was confirmed that the urine-derived stem cells have a similar level of colony forming ability as the adipose derived stem cells or the umbilical cord derived stem cells (FIG. 10).

Example 8. Preparation of induced pluripotent stem cells (USC-iPSCs) from urine-derived stem cells

The present inventors have produced induced pluripotent stem cells through the reprogramming process shown in the upper panel of FIG. 11 to reprogram the urine-derived stem cells (reprogramming) to produce induced pluripotent stem cells. As a result, it was confirmed that colonies were formed from urine-derived stem cells to be seen as induced pluripotent stem cells (Fig. 11 lower panel).

Then, the inventors carried out experiments to determine whether the cells of the formed colonies are induced pluripotent stem cells. First, experiments were performed to determine whether Oct4, Sox2, and Nanog were expressed as markers of pluripotent stem cells. As a result, it was confirmed that Oct4, Sox2 and Nanog, which were not expressed in urine derived cells, were expressed in the induced pluripotent stem cells (FIG. 12 left panel).

In addition, the present inventors confirmed that abnormal karyotypes on chromosomes were generated at the level of single cells using a single cell capture device, and cultured urine-derived pluripotent stem cells were confirmed to be stably produced as normal cell lines without karyotype problems. (Figure 12 right panel).

Example 9 Induction of Differentiation in Induced Pluripotent Stem Cells (USC-iPSCs)

The present inventors attempted to differentiate into several cells from induced pluripotent stem cells (USC-iPSCs) produced from urine-derived stem cells. First, as shown in FIG. 13, differentiation into kidney organoid was induced through known differentiation methods. As a result, it was confirmed that differentiation into kidney organoid was well induced on the induced pluripotent stem cells at 16 days (Fig. 13 lower panel).

In addition, the present inventors compared with the control group when flavonoid-based 3,2'-DHF (3,2'-dihydroxyflavone) and 3,4'-DHF (3,4'-dihydroxyflavone) were added during differentiation. It was confirmed that the formation rate of EB (Embryoid Bodies) increased in induced pluripotent stem cells (FIG. 14). EB formation is a step that should be formed upon differentiation into not only hematopoietic stem cells but also various cells, and a relatively large and large number of EB formation within the same time may be advantageous for further differentiation.

The present inventors performed experiments to form EB in induced pluripotent stem cells (USC-iPSCs) prepared as described above and induce differentiation into hematopoietic stem cells (Fig. 15 top panel). In addition, when inducing differentiation of hematopoietic stem cells, experiments were conducted to determine whether the efficiency of differentiation can be enhanced by adding 3,2'-DHF and 3,4'-DHF as in EB formation. As a result, the present inventors confirmed that differentiation of hematopoietic stem cells from induced pluripotent stem cells can be induced well. However, it was confirmed that addition of 3,2'-DHF and 3,4'-DHF did not affect the differentiation of hematopoietic stem cells (Fig. 15 lower panel).

Example 10. Effect of Promoting Proliferation or Differentiation of Differentiated Hematopoietic Stem Cells

After completing the differentiation into hematopoietic stem cells, the present inventors analyzed the total cell number and the distribution of cells with high CD34 +, CD45 + expression by flow cytometry. As a result, it was confirmed that the addition of 3,2'-DHF and 3,4'-DHF increased the total cell number compared to the control (Fig. 16 left panel). CD34 is a positive marker for hematopoietic stem cells isolated from peripheral blood or bone marrow, and when 3,2'-DHF and 3,4'-DHF were added, the number of CD34-expressing cells was higher than that of the control group. In addition, the cell number of the cell group (CD34 + CD45 +) expressing CD45, which is a biomarker for hematopoietic stem cells, with CD34 was analyzed. As a result, the addition of 3,2'-DHF and 3,4'-DHF increased the cell number of the CD34 + CD45 + compared to the control group, in particular, only markedly CD34 + CD45 + cells of the 3,2'-DHF It was confirmed that the number was increased (FIG. 16 right panel).

In addition, the present inventors performed CFU (Colony forming unit) analysis of hematopoietic stem cells in order to measure the proliferation and differentiation capacity of individual cells in hematopoietic stem cell specimens. The number of granulocytes (Granulocyte, CFU-G), macrophages (Macrophage, CFU-M), red blood cells (Erythroid, CFU-E) and mixed colonies (CFU-GM) formed by HSC-CFU medium was measured. It was confirmed that the formation rate of CFU-G was significantly increased in the group treated with, 2′-DHF, and the formation rate of CFU-E was also increased by 2 times (FIG. 17).

Claims (15)

1. Separating and proliferating the urine-derived stem cells comprising the step of culturing in a cell culture dish coated with a coating material by separating the cells from the urine collected from the individual.
2. The method of claim 1,

   The urine is characterized in that the urine is taken from the middle urine.
3. The method of claim 1,

   Wherein said cells are harvested from the subject's urine and separated within 2 hours.
4. The method of claim 1,

   The separation is characterized in that it is carried out by centrifugation for 5 to 15 minutes at 300g to 500g.
5. The method of claim 1,

   The method further comprises the step of washing with phosphate buffered saline (PBS) after separating the cells.
6. The method of claim 1,

   The coating material is Matrigel.
7. The method of claim 1,

   The culture is characterized in that the cultivation by the addition of Y-27632.
8. Promotes the proliferation or differentiation of stem cells comprising treating 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones How to let.
9. The method of claim 8,

   The stem cell is characterized in that the stem cells differentiated from induced pluripotent stem cells.
10. The method of claim 9,

    The induced pluripotent stem cells are characterized in that the reprogrammed from the urine-derived stem cells.
11. The method of claim 8,

    The stem cell is characterized in that hematopoietic stem cells.
12. The method of claim 8,

    Wherein said 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone is at a concentration of 1-20 μM.
13. Embryoid from induced pluripotent stem cells comprising treating 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones Bodies) How to increase the formation rate.
14. The method of claim 13,

    The induced pluripotent stem cells are characterized in that the reprogrammed from the urine-derived stem cells.
15. The method of claim 13,

    Wherein said 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone is at a concentration of 1-20 μM.

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