KR20190118031A - Composition of promoting and enhanceing differentiation of urine-derived cells into hematopoietic stem cells and formation rate of embryonic bodies from induced pluripotent stem cells comprising 3,2'- or 3,4'-dihydroxyflavone as active ingredients - Google Patents

Abstract

The present invention relates to a composition for promoting differentiation of stem cells comprising 3,2′-dihydroxyflavone or 3,4′-dihydroxyflavone as an active ingredient.

Description

Differentiation of hematopoietic stem cell line from urine-derived cells and increase in developmental body formation rate in induced pluripotent stem cells comprising 3,2'- or 3,4'-dihydroxy flavone as active ingredients enhanceing differentiation of urine-derived cells into hematopoietic stem cells and formation rate of embryonic bodies from induced pluripotent stem cells comprising 3,2'- or 3,4'-dihydroxyflavone as active ingredients}

The present invention relates to a composition for promoting differentiation of urine-derived pluripotent stem cells comprising 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone as an active ingredient and a method for promoting differentiation.

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. Induced pluripotent stem cells were introduced by Yamanaka's team of professors in 2006 to introduce four factors (Oct3 / 4, Sox2, c-Myc, Klf4) into rat embryonic fibroblasts to produce differentiated pluripotent cells such as ES cells. It is known by publishing a paper on the existence of. 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 are also being 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 suffering from 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 about the current differentiation are still the subject of study, and research to increase the differentiation ability and the accuracy is needed.

Korean Patent Publication No. 10-2017-0126564

An object of the present invention is the differentiation of stem cells comprising 3,2'-dihydroxyflavone (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavone (3,4'-dihydroxyflavone) as an active ingredient It is to provide a composition for promotion.

Still another object of the present invention is to provide a composition for promoting growth of stem cells comprising 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone as an active ingredient.

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 differentiation of cells.

Another object of the present invention is to provide a method for promoting the proliferation of stem cells comprising the step of treating 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones.

In order to achieve the above object, the present invention comprises 3,2'-dihydroxyflavones (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavones (3,4'-dihydroxyflavone) as an active ingredient It provides a composition for promoting differentiation of stem cells.

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

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

In one embodiment of the present invention, the differentiation may be differentiation into 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 one embodiment of the present invention, the 3,2'- dihydroxy flavone or 3,4'- dihydroxy flavone may be to increase the formation rate of EB (Embryoid Bodies).

The present invention also provides a composition for promoting growth of stem cells comprising 3,2'-dihydroxy flavone or 3,4'-dihydroxy flavone as an active ingredient.

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

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) Provide a method to promote differentiation.

The present invention also provides a method for promoting the proliferation of stem cells comprising the step of treating 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones.

3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones according to the present invention promotes the differentiation of urine-derived pluripotent stem cells into hematopoietic stem cells, that is, the effect of increasing the rate of EB formation It can be usefully used for cell therapy by stem cells.

Figure 1 shows the structure of 3,2'-DHF (3,2'-dihydroxyflavone), 3,4'-DHF (3,4'-dihydroxyflavone).
Figure 2 shows the separation of urine-derived cells.
Figure 3 shows the morphology of isolated urine-derived cells (scale bar: 0.2mm).
4 shows the results of confirming the expression of various biomarkers on urine-derived cells.
Figure 5 shows the process of producing induced pluripotent stem cells from urine-derived cells and shows the morphology of the produced induced pluripotent stem cells.
Figure 6 is a result confirming the expression of the markers of pluripotent stem cells Oct4, Sox2 and Nanog for urine-derived pluripotent stem cells.
Figure 7 shows the morphology of cells formed with the same EB 4 days in urine-derived pluripotent stem cells.
FIG. 8 shows the results of comparing the amount of cells settled at the bottom of the tube after separating the EB into single cells and centrifuging (top). The total cell number was measured (bottom right panel).
9 is a result of confirming the differentiation process (upper) and differentiation efficiency into hematopoietic stem cells from urine-derived pluripotent stem cells through FACS analysis.
Figure 10 is a result of comparing the number of cells after differentiation from urine-derived pluripotent stem cells to hematopoietic stem cells (left middle panel: total cell number; right middle panel: cell number expressing CD34; and CD34 + CD45 + and CD34 + As the number of cells, the degree of differentiated cells was inferred from the whole cell population based on FACS analysis information).

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 pluripotent that is cultured in vitro by extracting an inner cell mass from an blastocyst embryo just before the fertilized egg implants in the mother's womb, and can differentiate into cells of all tissues of the individual. It refers to stem cells having self-renewal with pluripotent or totipotent.

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 cells" refers to undifferentiated cells capable of self-reproduction as stem cells originating in differentiated tissues and capable of differentiating into all cell types of the derived tissues. 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 (Mesenchymal Stem Cells), and neural stem cells, stem cells derived from the brain, have subventricular zones ( It is known to be isolated 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" or "hematopoietic stem cell" is a parent cell of lymphocytes forming various blood cells and immune systems, for example, lymphocytes such as B cells, T cells, granulocytes, platelets and erythrocytes. It refers to stem cells that can develop. 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 circulating hematopoietic stem cells.

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" refers to a phenomenon in which a less specialized cell develops into a specific cell so that its size, shape, membrane potential, metabolic activity, or response to a signal changes to a specific type of cell. The differentiation mainly occurs during the process of forming a complex tissue in a single conjugate (zygote), or when adult stem cells repair damaged tissues, adult stem cells differentiate into specific cells.

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. Cell Separation and Culture

To separate cells from urine, between 200-300 ml of middle flow urine from donors was used in a urine collection vessel. The provided urine was transferred to a 50ml tube, centrifuged at 1150 rpm, and PBS (Sigma Aldrich) containing 1% anti-biotics was added to the cell pellet and washed by centrifugation. The culture plate was used after 30 minutes of 0.2% Gelatin coating (Fig. 2). Cells were placed in REBM basal medium (Lonza) to which 10% FBS was added to induce cell attachment by replacing the culture medium by half for the first three days. Then, the cells were grown using REGM SingleQuot kit supplement (Lonza).

1.2. 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.

1.3. Total RNA Isolation and RT-PCR Analysis

According to the manufacturer's manual, Total RNA was extracted using Labozol total RNA extraction kit (Cosmo Genetech). Total RNA concentration was measured by Nanodrop (ND1000) spectrophotometer (Nanodrop Technologies Inc., Wilmington DE, USA), and cDNA was synthesized using M-MuLV Reverse Transcriptase (Cosmo Genetech). After the PCR reaction was analyzed in 1 to 1.5% agarose gel.

1.4. Flow Cytometry (FACS)

For flow cytometry analysis of differentiated hematopoietic stem cells, pluripotent stem cell-derived hematopoietic stem cells in a cell culture plate were treated with 0.25% Trypsin for 3 minutes to float the cells, and 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 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.

1.5. 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 and Morphology of Urine-derived Stem Cells

We collected and separated urine of various ages and successfully isolated and cultured urine-derived cells (FIG. 2). In these isolated cells, both general growth patterns shown in the literature were observed. Cell pellets isolated from the initial urine contained various cell populations (Figure 3, left), of which the cells form type 1 (Figure 3, middle) and colonies that grow into single cells. The cells of type 2 (Fig. 3, right) to grow were confirmed.

Example 3. Characterization of Cells

The present inventors performed experiments to determine the characteristics of the cells using the various biomarkers for the urine-derived cells isolated in Example 2. To date, markers to characterize urine-derived cells have not been clearly identified, so this assay is designed to predict the characteristics of urine-derived cells isolated from a donor using different biomarkers to predict the characteristics of cells from each donor. It was about. As a result, as shown in FIG. 4, E-cadherin and ZO-1, which are markers of Epithelial cells, were not expressed. Vimenetin was not expressed in Vimentin and Fibronetin, which are markers of Fibroblast cells, but Fibronectin was expressed. In addition, expression of SLC2A1 and L1CAM in SLC2A1, L1CAM, and NR3C2 markers of Renal cells was confirmed (FIG. 4). Thus, isolated urine-derived cells were found to have the characteristics of Fibroblast and Renal cells.

Example 4. Preparation of induced pluripotent stem cells

The present inventors performed an experiment to convert the urine-derived cells into induced pluripotent stem cells through adult cell reprogramming. As shown in the upper panel of FIG. 5, the cells were separated from urine samples and formed urine-derived stem cell colonies, and then induced pluripotent stem cells were prepared through a general reprogramming process (FIG. 5, top). As a result, it was confirmed that colonies formed as induced pluripotent stem cells were formed 35 days after separation of urine-derived cells from urine samples (FIG. 5, bottom).

Then, the inventors carried out experiments to determine whether the cells of the formed colonies are induced pluripotent stem cells. First, it was confirmed that the abnormal karyotype on the chromosome at the single cell level by using the Single Cell Capture equipment, the cultured urine-derived pluripotent stem cells were confirmed that there was no problem in the karyotype and produced stably. In addition, experiments were performed to determine whether Oct4, Sox2, and Nanog, which are markers of pluripotent stem cells, were expressed. As a result, it was confirmed that Oct4, Sox2 and Nanog, which were not expressed in urine-derived cells, were expressed in urine-derived pluripotent stem cells (FIG. 6). As a result, it was confirmed that the urine-derived cells were produced or formed into induced pluripotent stem cells.

Example 5 Differentiation of Urine-Induced Pluripotent Stem Cells into Hematopoietic Stem Cells

We attempted to differentiate into several cells in urine-derived pluripotent stem cells (UC-iPSCs). In particular, 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. That is, when the flavonoids were treated, it was confirmed that the size of the colonies was larger than the control group in the form of the cells (FIG. 7), and the number of colonies formed was increased by 1.5 times compared to the control group, and the total cell number was 2 It was confirmed that the fold increased (Fig. 8). EB formation is a step that should be formed upon differentiation into various cells as well as hematopoietic stem 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 an experiment to form EB in urine-derived pluripotent stem cells and induce differentiation into hematopoietic stem cells as described above (Fig. 9, top). In addition, when inducing differentiation of hematopoietic stem cells, experiments were conducted to determine whether the efficiency of differentiation can be increased by adding 3,2'-DHF and 3,4'-DHF as in EB formation. As a result, the present inventors confirmed that differentiation from urine-derived pluripotent stem cells to hematopoietic stem cells can be well induced. However, the addition of 3,2'-DHF and 3,4'-DHF did not affect the differentiation of hematopoietic stem cells (Fig. 9, bottom).

After completing the differentiation into hematopoietic stem cells, the inventors analyzed the total cell number and the number of CD34, CD45 expressing cells 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. 10, left middle 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, it was confirmed that the number of CD34-expressing cells was higher than that of the control group (FIG. 10). , Right middle panel). In addition, the cell number of the cell group (CD34 + CD45 +) expressing CD45, a biomarker for hematopoietic stem cells, was analyzed. As a result, it was confirmed that the addition of 3,2'-DHF increased the cell number of the CD34 + CD45 + compared to the control (Fig. 10, lower panel).

Claims (16)

A composition for promoting differentiation of stem cells comprising 3,2'-dihydroxyflavone (3,2'-dihydroxyflavone) or 3,4'-dihydroxyflavone (3,4'-dihydroxyflavone) as an active ingredient. The method of claim 1,
The stem cell composition, characterized in that the induced pluripotent stem cells. The method of claim 2,
The induced pluripotent stem cells are composition, characterized in that reprogrammed from the urine-derived cells. The method of claim 1,
Wherein said differentiation is differentiation into hematopoietic stem cells. The method of claim 1,
The 3,2'-dihydroxy flavone or 3,4'-dihydroxy flavone is a composition, characterized in that the concentration of 1 to 20 μM. The method of claim 1,
The 3,2'-dihydroxy flavone or 3,4'-dihydroxy flavone is characterized in that to increase the formation rate of EB (Embryoid Bodies). A composition for promoting growth of stem cells comprising 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone as an active ingredient. The method of claim 7, wherein
The stem cell composition, characterized in that the hematopoietic stem cell. A method for promoting differentiation of stem cells comprising treating 3,2'-dihydroxyflavones or 3,4'-dihydroxyflavones . The method of claim 9,
The stem cell is characterized in that the induced pluripotent stem cells. The method of claim 10,
The induced pluripotent stem cells are characterized in that the reprogrammed from the urine-derived cells. The method of claim 9,
Wherein said differentiation is differentiation into hematopoietic stem cells. The method of claim 9,
Wherein said 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone is at a concentration of 1-20 μM. The method of claim 9,
Wherein said 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone increases the formation rate of Embryoid Bodies (EB). A method of promoting proliferation of stem cells comprising the step of treating 3,2'-dihydroxyflavone or 3,4'-dihydroxyflavone. The method of claim 15,
The stem cell composition, characterized in that the hematopoietic stem cell.

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