KR101853477B1 - Methods for Regulating Differentiation of kidney precursor Cells from Embryonic Stem Cells Through modulation of SIRT1 expression - Google Patents

Abstract

The present invention relates to a method for regulating the differentiation of progenitor precursor cells from embryonic stem cells by controlling the expression of SIRT1 (silent mating type information regulation 2 homolog: sirtuin 1) The kidney cells or the kidney precursor cells produced by the differentiation induction method can be utilized for tissue regeneration and can be used as a composition for regulating differentiation of progenitor precursor cells by administration of SIRT1 inhibitor or promoter.

Description

[0002] Methods for regulating the differentiation of embryonic stem cells into the kidney precursor cells through the modulation of SIRT1 expression [

The present invention relates to a method of regulating the differentiation of progenitor precursor cells from embryonic stem cells by controlling expression of SIRT1 (silent mating type information regulation 2 homolog: sirtuin 1), more particularly, to a method of regulating the expression of silent mating type information regulation A method for inhibiting the differentiation of embryonic stem cells into a kidney precursor cell comprising the step of suppressing the expression of the sirtuin 1 homolog, 2) a method for promoting differentiation from embryonic stem cells into a kidney precursor cell including a step of promoting SIRT1 expression, A kidney cell, an artificial kidney, a renal function remedy including a kidney precursor cell produced by the above method, and a therapeutic agent.

SIRT1 (sirtuin 1) is a NAD ⁺ -dependent deacetylase that is known to be an enzyme that deacetylates lysine residues of various proteins and regulates protein function (Aging Res, Vol.1, p. 313 -326, (2002)) and is most similar to Sir2 of yeast with NAD ⁺ dependent class III histone deacetylase activity. In particular, it is known that the acetyl groups attached to transcription factors such as Nuclear factor-kB and p53 are cleaved to regulate their function (Cancer Res, Vol. 64, pp. 7513-7525, (2004); Cell, Vol.107 149-159, (2001); Trends Endocrinol Metab, Vol.17, pages 186-191, (2006)).

SIRT1 is involved in chromatin reorganization, DNA damage response, and prolonged lifespan associated with dietary restriction (Chen et al., Science, 310: 1641, 2005). In other words, SIRT1 reconstitutes chromatin through histone deacetylation, inhibits gene expression, and induces deacetylation of various transcription factors involved in cell growth, stress response, and endocrine regulation in addition to histone protein. Further, according to recent studies, there has been reported a technique of increasing the deacetylation activity of SIRT1 to apply it to diabetes, obesity, neurodegenerative diseases or aging-related diseases, and SIRT1 inhibits PPAR- (Frederic Picard et al., Nature, 429: 771, 2004). However, no study has been conducted to control the differentiation of SIRT1 into progenitor progenitor cells in embryonic stem cells.

The inventors of the present invention found that SIRT1 (silent mating type information regulation 2 homologue: sirtuin 1) regulates the expression of SIRT1 in the kidney embryonic stem cells It was confirmed that the differentiation into the renal cell was inhibited when the SIRT1 expression was inhibited and the differentiation into the renal cell was promoted during SIRT1 activation. Thus, the present invention was completed.

The present invention has been made in view of the fact that the method of regulating differentiation from embryonic stem cells into adipocytes is known, but the method of regulating differentiation into renal precursor cells is unknown. The present invention provides a method for regulating differentiation from embryonic stem cells And a method for inhibiting differentiation into a cell.

It is yet another object of the present invention to provide a method for promoting differentiation of embryonic stem cells into progenitor progenitor cells by controlling SIRT1 expression.

It is still another object of the present invention to provide a kidney cell produced by the above method.

It is still another object of the present invention to provide a renal function remedy and therapeutic agent comprising the renal progenitor cells produced by the above method.

The present invention provides a method of regulating the proliferation of embryonic stem cells (ES cells), comprising the step of regulating expression of SIRT1 (silent mating type information regulation 2 homolog: sirtuin 1) of embryonic stem cells .

Specifically, as a preferable differentiation control method, the inhibition of the expression of SIRT1 gene inhibits proliferative progenitor cell differentiation, and the proliferation of SIRT1 gene promotes differentiation of progenitor progenitor cells.

The SIRT1 gene expression inhibitor may be a known inhibitor, and specifically, Sirtinol (2-Hydroxynaphthalen-1-ylmethylene) amino] -N- (1-phenethyl) benzamide, Nicotinamide -3-carboxamide) and EX527 (6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide).

The present invention also includes a method for inhibiting differentiation of progenitor progenitor cells of embryonic stem cells, which comprises culturing embryonic stem cells in a medium containing SIRT1 expression inhibitor to inhibit differentiation into progenitor precursor cells.

According to a preferred embodiment of the present invention, the SIRT1 expression inhibitor may be treated for 1 to 7 days to induce differentiation.

According to another preferred embodiment of the present invention, the SIRT1 expression inhibitor is selected from the group consisting of Sirtinol (2-Hydroxynaphthalen-1-ylmethylene) amino] -N- (1-phenethyl) benzamide, Nicotinamide 3-carboxamide) or EX527 (6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide) and may be added to the SIRT1 expression inhibitor medium at a concentration of 4 to 25 μM.

According to another preferred embodiment of the present invention, mRNA expression level of SIX2 (sine oculis homeobox homolog 2) and WT1 (wilms tumor 1) of embryonic stem cells is decreased due to suppression of SIRT1 expression, It can be seen that the differentiation is suppressed (see Fig. 3).

The present invention also encompasses a method for promoting differentiation of progenitor progenitor cells of embryonic stem cells, which comprises culturing embryonic stem cells in a medium containing SIRT1 active substance to promote differentiation into progenitor progenitor cells.

According to a preferred embodiment of the present invention, the SIRT1 expression promoting agent may be treated for 1 to 21 days, preferably 1 to 7 days, for inducing differentiation.

According to another preferred embodiment of the present invention, the SIRT1 expression promoting agent is selected from the group consisting of SRT1270 (ChemCruz Biochemicals), adenovirus, lentivirus, BML-278 (ChemCruz Biochemicals), and resveratrol And may be preferably resveratrol. The SIRT1 expression promoter may be added to the medium at a concentration of 10 to 50 μM.

According to another preferred embodiment of the present invention, mRNA expression levels of SIX2 (sine oculis homeobox homolog 2) and WT1 (wilms tumor 1) of embryonic stem cells are increased due to SIRT1 expression promoter, (Fig. 3 and Fig. 4).

According to another preferred embodiment of the present invention, the medium is a medium for inducing differentiation of progenitor progenitor cells, and the retinoic acid ((2E, 4E, 6E, 8E) -3,7-dimethyl- -trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid) and actin A,

In order to solve still another problem of the present invention, the present invention provides a kidney cell.

In order to solve still another problem of the present invention, there is provided a renal function remedy and therapeutic agent comprising renal precursor cells.

The method of the present invention for regulating the differentiation of progenitor progenitor cells from embryonic stem cells by controlling the expression of SIRT1 can control the differentiation of progenitor progenitor cells by treatment with SIRT1 expression inhibitor or activator, In addition to being used as a study model of kidney injury disease involved in the renal cell renewal process, it is also possible to select a SIRT1 inhibitor to reduce renal cell damage during kidney injury or to improve kidney function after renal injury And so on.

1 is a schematic diagram showing a culture protocol for differentiating into embryonic stem cells into renal precursor cells (RA: retinoic acid).
(A) and WT1 (B) after differentiation of SIRT1 protein expressing cells (SIRT1 + / +; WT) and SIRT1 protein expressing cells (SIRT1 - / - Of the mRNA expression level.
FIG. 3 shows mRNA expression levels of WT1 (A) and Six2 (B) in the kidney precursor cell markers when the SIRT1 inhibitor EX527 (5-10 uM) and the SIRT1 promoter resveratrol (10-20 uM) .
FIG. 4 shows the results of a comparison between the expression of SIRT1 in conditionally knockout mice (Sirt1 co / co; Hoxb7-Cre (+)) and wild type mice (Sirt1 co / co; Hoxb7-Cre Immunofluorescence staining data measuring the expression positions of Six2 (FIG. 4A) and WT1 (FIG. 4B) at the first day of life.
FIG. 5 shows the results of a comparison between the expression of Sirt1 in Sirt1 co / co and Hoxb7-Cre (-) in wild type mice and Sirt1 co / co (Hoxb7- Quantitative RT-PCR data measuring the mRNA expression levels of Six2 (FIG. 4A) and WT1 (FIG. 4B) at the first day of life.

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

As described above, various studies on the function of the SIRT1 gene have been made, and it has been reported that when the expression of SIRT1 is increased, the expression of PPAR-γ is inhibited to inhibit differentiation into adipocytes. However, There have been no studies on the regulation of differentiation into renal progenitor cells according to the expression control.

The present invention sought to solve the above-mentioned problem by providing a method of regulating the proliferation of ES cells of embryonic stem cells by regulating the expression of SIRT1. It was confirmed that SIRT1 (silent mating type information regulation 2 homolog: sirtuin 1) is involved in the differentiation of embryonic stem cells into progenitor progenitor cells and promotes differentiation into progenitor progenitor cells by inhibition of SIRT1 expression. The present invention can be used as a study model of renal diseases according to the regulation of SIRT1 expression in embryo through the method of regulating differentiation of progenitor cells of embryonic stem cells.

Accordingly, the present invention provides a method for regulating the proliferation of embryonic stem cells, comprising the step of controlling the expression of SIRT1 (silent mating type information regulation 2 homolog: sirtuin 1) in embryonic stem cells, To inhibit the differentiation into progenitor progenitor cells by treating the SIRT1 expression inhibitor and a method of promoting differentiation into progenitor precursor cells by treating the SIRT1 expression promoter.

SIRT1 of the present invention is silent mating type information regulation 2 homolog; The nucleotide sequence of the SIRT1 gene can be represented by SEQ ID NO: 1.

The term "stem cell" of the present invention refers to a cell having an ability to differentiate into various cells and having a self-proliferative capacity. The term " embryonic stem cell (ES cell) Three types of embryonic germ cells (EG cells) isolated from cells and multipotent adult progenitor cells (MAPC cells) isolated from adult bone marrow are most well known.

In the present invention, mouse embryonic stem cells are used as embryonic stem cells but the present invention is not limited thereto. Specific examples of mouse derived embryonic stem cells include EB3 cells, E14 cells, D3 cells, CCE cells, R1 cells, 129SV cells and J1 Cells, and the like. In addition, standard protocols (Matsui et al., Cell, 70: 841, 1992; Shamblott et al., Proc . ... Natl Acad Sci USA, 95:. 13726, 1998; U.S. Patent No. 6,090,622; Jiang et al, Nature, 418 :. 41, 2002; in reference to International Patent Publication 01/11011) make these stem cells facilitate And can be cultured using various feeder cells and embryonic stem cell culture media already known.

Cells usable in the present invention are not limited to embryonic stem cells but include embryos of mammals or stem cells having similar characteristics to embryonic stem cells. In this case, the term "a trait similar to embryonic stem cells" means that a surface (antigen) marker specific to embryonic stem cells is present, an embryonic stem cell-specific gene is expressed, a teratoma- Or a chimeric mouse capable of forming an embryonic stem cell.

The SIRT1 gene expression inhibitor may be a known inhibitor, and specifically, Sirtinol (2-Hydroxynaphthalen-1-ylmethylene) amino] -N- (1-phenethyl) benzamide, Nicotinamide -3-carboxamide) and EX527 (6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide).

According to one embodiment of the present invention, when EX527 (6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide) was treated on embryonic stem cells, expression of Six2 and WT1 was observed in the control (See Fig. 3). Although EX527 is preferably used in the present invention, known SIRT1 expression inhibitors capable of inhibiting SIRT1 expression may be used without limitation, and SIRT1 specific sequences such as siRNA which inhibits protein synthesis of SIRT1 may be used .

The SIRT1 expression inhibitor may be added to the medium at a concentration of 4 to 25 μM, preferably at a concentration of 5 to 10 μM, and when added at a concentration of 5 μM or less, there may be no inhibitory effect on the differentiation into progenitor cells, When added at a concentration of more than 1 μM, the cytotoxicity may be exhibited or the efficiency may not be improved due to the increase of the concentration due to the increase of the concentration.

In one embodiment of the present invention, mouse embryonic stem cells, R1 cells, were used. In order to determine whether the differentiation into the progenitor progenitor cells of embryonic stem cells depends on the expression of SIRT1 protein, the mouse embryonic stem (SIRT1 + / +; WT) and mouse embryonic stem cells (SIRT1 - / -; KO) lacking the SIRT1 gene were prepared. SIRT1 gene-expressing mouse embryonic stem cells were treated with SIRT1 inhibitor, EX527, Inhibition of renal progenitor cell differentiation.

FIG. 1 is a schematic diagram showing a culture protocol for differentiating embryonic stem cells into progenitor progenitor cells. In one embodiment of the present invention, retinoic acid (hereinafter, referred to as " retinoic acid ") is added to a cell culture medium except Leukemia Inhibitory Factor (LIF; Milipore, USA) Retinoic acid (100 nmol / L) and actin A (10 ng / mL) were treated for 7 days to induce differentiation into progenitor progenitor cells.

As described above, the induction of differentiation of embryonic stem cells into progenitor progenitor cells was carried out using mouse embryonic stem cell (SIRT1 + / +; WT) group expressing SIRT1 gene, mouse embryonic stem cell (SIRT1 - / -; KO) to determine the effect of SIRT1 on differentiation into renal progenitor cells.

In the present invention, suppression of SIRT1 expression inhibits SIRT1 expression in embryonic stem cells, thereby suppressing mRNA expression levels of SIX2 (sine oculis homeobox homolog 2) and WT1 (wilms tumor 1) in embryonic stem cells, May be suppressed.

In one embodiment of the present invention, in order to investigate inhibition of proliferative progenitor cell differentiation of SIRT1, the amount of mRNA expression of intracellular Six2 and WT1 was measured by a PCR method, and the result is shown in FIG. FIG. 2 shows data of mRNA expression of Six2 and WT1 according to the presence or absence of SIRT1 expression in the differentiation of embryonic stem cells into progenitor progenitor cells. In SIRT1 + / + cells, the expression of Six2 and WT1 MRNA expression of SIRT1 - / - cells was significantly increased, while that of SIRT1 - / - cells was significantly inhibited by mRNA expression of Six2 and WT1.

The expression promoter of the SIRT1 gene may be a known promoter and may be specifically SRT1270 (ChemCruz Biochemicals), Sirt1 overexpressing virus (adenovirus or lentivirus), BML-278 (ChemCruz Biochemicals), or resveratrol , And most preferably resveratrol.

According to one embodiment of the present invention, when resveratrol was treated at 20 μM, the expression of Six2 and WT1 was significantly promoted as compared with that of the control, thereby confirming that differentiation into progenitor precursor cells was promoted (see FIG. 3). Although resveratrol is preferably used in the present invention, known SIRT1 expression promoting agents capable of promoting SIRT1 expression may be used without limitation, and SIRT1-specific sequences such as RNA promoting protein synthesis of SIRT1 may be synthesized and used .

The SIRT1 expression promoter may be added to the medium at a concentration of 5 to 50 μM, preferably 10 to 20 μM, and when added at a concentration of 5 μM or less, there may be no inhibitory effect on the differentiation into progenitor cells, When added at a concentration of more than 1 μM, the cytotoxicity may be exhibited or the efficiency may not be improved due to the increase of the concentration due to the increase of the concentration.

The SIRT1 gene expression promoter or inhibitor may be treated for 1 to 21 days of inducing differentiation of embryonic stem cells, preferably for 1 to 7 days. If treated for a period shorter than 1 day, the effect of promoting or inhibiting the differentiation of progenitor progenitor cells may not be effective, and when treated for a period longer than 21 days, the cells may be cytotoxic and cause apoptosis.

FIG. 3 shows the mRNA expression levels of WT1 (A) and Six2 (B), which are progenitor precursor cell indicators, when injected with SIRT1 inhibitor EX527 (5-10 uM) and SIRT1 promoter resveratrol (10-20 uM) . As described above, when SIRT1 inhibitor was administered, the amount of Six2 and WT1 mRNA expression was significantly decreased, and when SIRT1 expression promoter resveratrol was administered, the expression of Six2 and WT1 mRNA was significantly increased.

In FIGS. 4A and 4B, expression of Six2 and WT1 was increased at 1 day of age in wild-type mouse (Sirt1 co / co; Hoxb7-Cre (-)), but the expression of Sirt1 knockout mouse: Sirt1 co / co; Hoxb7-Cre (+)) decreased expression of Six2 and WT1. This means that SIRT1 inhibition can inhibit differentiation of progenitor progenitor cells.

The method of the present invention for regulating progenitor progenitor cell differentiation of embryonic stem cells further comprises inducing differentiation into progenitor precursor cells in a medium containing a SIRT1 expression regulator, Wherein the SIRT1 expression regulator is a SIRT1 expression inhibitor or an expression promoter and may be treated for 1 to 21 days for inducing differentiation, and preferably for 1 to 7 days.

The composition for tissue regeneration comprising the extracellular precursor cells derived from embryonic stem cells of the present invention as an active ingredient can be formulated by a known method in the pharmaceutical field and mixed with the structure itself or a pharmaceutically acceptable carrier, And can be formulated into various pharmaceutical formulations such as conventional pharmaceutical preparations such as liquid preparations, ointments, emulsions, gels, creams, pastes, and the like. There is no particular limitation on the dosage of the therapeutic agent for tissue regeneration according to the present invention, but the preferable dosage may be appropriately selected by those skilled in the art, depending on the condition and weight of the patient, the degree of disease or condition,

The present invention provides kidney cells derived from the kidney precursor cells produced by the method.

The present invention provides a renal function remedy or therapeutic agent containing kidney precursor cells produced by the above method.

The renal function remedy or therapeutic agent of the present invention replenishes (reconstructs) or reconstructs (renames) damaged kidney cells. The therapeutic agent may preferably be a cytotherapeutic agent.

"Regeneration" is a phenomenon in which a portion of a formed organs or an individual is lost when it is lost, and "restoration" is sometimes referred to as "reconstitution" It refers to reconstituting tissues or organs from dissociated cells or tissues.

This can be advantageously performed by direct implantation into the lesion site in the form of kidney cells or the composition containing them (cell therapy agent). Methods of neural transplantation and cell culture can be performed using known methods well known to those skilled in the art or those described in the examples of the present invention.

The term " treating "or" treating "as used herein is a therapeutic treatment and prevention or prevention measure. In other words, those in need of treatment already have those with renal dysfunction. The methods of the invention can be used to treat any mammal in need of treatment, including, but not limited to, humans, primates and livestock, raising, pet or sports animals such as dogs, horses, cats, sheep, pigs, Can be used. As used herein, a "therapeutically effective amount" of a cell is an amount sufficient to stop or alleviate the physiological effect of the patient caused by loss, damage or denaturation of the differentiated kidney cells.

The therapeutically effective amount of the employed cell will depend on the needs of the patient, the age, physiological condition and health of the patient, the desired therapeutic effect, the size and area of the tissue to be targeted for treatment, the severity of the lesion and the selected delivery route. For example, treatment of a disorder affecting a larger area of the brain may require more cells to achieve a therapeutic effect when compared to a smaller target area. In addition, the cells can be administered at one or more sites within a given target tissue with multiple sub-doses of a low cell dose. The cells of the invention can be completely isolated prior to transplantation, for example, to form a suspension of single cells, or to be almost completely isolated prior to transplantation, e.g., to form small aggregates of cells. The cells may be administered in a manner such that they are transplanted or transferred to a predetermined tissue site and the functionally deficient region is reconstituted or regenerated.

The appropriate range of cells that can be administered therapeutically effective can be suitably used for the patient within the ordinary knowledge of those skilled in the art.

It should be understood, however, that the actual dosage should be determined in light of various relevant factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease, And are not intended to limit the scope of the invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Secretion and culture of mouse embryonic stem cells

In the present invention, mouse embryonic stem cells R1 cells were purchased from the ATCC (American Type Culture Collection, ATCC SCRC-1011) and used to determine whether or not the differentiation into progenitor cells of embryonic stem cells depends on the expression of SIRT1 protein (SIRT1 + / +; WT) and mouse embryonic stem cells (SIRT1 - / -; KO) lacking the SIRT1 gene were prepared (Han MK et al. Cell Stem Cell., Mar 6: 2 (3): 241, 2008).

Mouse embryonic stem cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium supplemented with 2 mM GlutaMAX ™ (Gibco, USA), 1X Non-essential amino acid (1X NEAA; Gibco, USA), 0.1 mM beta-mercaptoethanol (β-mercaptoethanol; Invitrogen, USA) and 1,000 unit / ml Leukemia Inhibitory Factor (LIF; Milipore, USA).

Of mouse embryonic stem cells Into renal progenitor cells  Induction of differentiation

In the present invention, in order to examine whether the differentiation into the progenitor cells of embryonic stem cells depends on the expression of SIRT1 protein, SIRT1 gene-expressing mouse embryonic stem cells (SIRT1 + / +; WT) prepared in Example 1 and SIRT1 (SIRT1 - / -; KO), which induced the differentiation of mouse embryonic stem cells (SIRT1 - / -; KO) lacking the gene into progenitor progenitor cells.

As shown in FIG. 1, experiments were conducted according to a culture protocol for differentiating into embryonic stem cells into kidney precursor cells. After treatment with 100 nmol / L of Retinoic acid and 10 ng / mL of Actin A (10 ng / mL) in cell culture medium except Leukemia Inhibitory Factor (LIF; Milipore, USA) And WT1 mRNA expression levels were measured. The process of induction into renal precursor cells was carried out by inoculating embryonic stem cells into each well using a 6-well plate.

The medium for differentiation of progenitor progenitor cells was prepared by adding 2 mM Gluta MAX (Gibco, USA), 1X non-essential aminoacid (1X NEAA; Gibco, USA), 0.1 mM beta- Mercaptoethanol (Invitrogen, USA) and 1,000 unit / ml Leukemia Inhibitory Factor (LIF; Milipore, USA).

mRNA  Expression levels of embryonic stem cells Into renal progenitor cells  Differentiation confirmation

In order to confirm the differentiation into embryonic stem cell progenitor cells according to the presence or absence of SIRT1 protein expression, mouse embryonic stem cells (SIRT1 + / +; WT) expressing SIRT1 gene prepared in Example 1 and mouse embryos lacking SIRT1 gene Differentiation of stem cells (SIRT1 - / -; KO) into progenitor progenitor cells was confirmed. Differentiation was confirmed by the amount of mRNA expression of the elongated progenitor cell markers, Six2 and WT1.

In the differentiation stage into renal progenitor cells, mRNA expression levels of the elongated progenitor cells, Six2 and WT1, were measured by PCR. The primers used in the PCR were as shown in Table 1 below and were prepared by Biona (Korea). GAPDH was used as a control for the reliability of PCR.

Primer base sequence primer The base sequence (5 '-> 3') Tm (占 폚) Six2 Forward CAAGGAAAGGGAGAACAGCGA SEQ ID NO: 2 60 Reverse GCGTCTTCTCATCCTCGGAA SEQ ID NO: 3 WT1 Forward ATCCCAGGCAGGAAAGTGTG SEQ ID NO: 4 60 Reverse GTGCTGTCTTGGAAGTCGGA SEQ ID NO: 5 GAPDH Forward AGGTCGGTGTGAACGGATTTG SEQ ID NO: 6 60 Reverse GGGGTCGTTGATGGCAACA SEQ ID NO: 7

Differentiation was induced from embryonic stem cells into kidney precursor cells in the same manner as in Example 2. After harvesting each cell on the 11th day of differentiation induction, total RNA was isolated using TRIzol reagent (Life Technology, USA) And extracted. The extracted RNA was synthesized by using a cDNA synthesis kit (Roche, Germany) and PCR was carried out using the primers shown in Table 1.

As a result, as shown in FIG. 2, SIRT1 - / - cells showed inhibition of mRNA expression of Six2 and WT1 after induction of differentiation into progenitor progenitor cells, whereas SIRT1 + / + cells showed mRNA expression of Six2 and WT1 .

In other words, suppression of SIRT1 expression inhibited the expression of intracellular Six2 and WT1 mRNA in the differentiation of embryonic stem cells into progenitor progenitor cells, thereby inhibiting the differentiation into progenitor progenitor cells. This inhibited the expression of SIRT1 , Suggesting that the differentiation of embryonic stem cells into progenitor progenitor cells is inhibited.

With SIRT1 inhibition  Activation of Embryonic Stem Cells Into renal progenitor cells  Confirmation of differentiation

In order to confirm the differentiation into embryonic stem cell progenitor cells according to the suppression or stimulation of the SIRT1 protein expression, the SIRT1 gene expressing mouse embryonic stem cells (SIRT1 + / +; WT) prepared in Example 1 Differentiation was confirmed. The differentiation was confirmed by the amount of protein expression of the elongated progenitor cell markers, Six2 and WT1.

In the stage of differentiation into renal progenitor cells, the amount of mRNA expression of renal precursor cells, Six2 and WT1, was measured by quantitative PCR.

As a result, as shown in FIG. 3, it can be seen that WT1 mRNA expression level (A) and Six2 mRNA expression level (B) decrease during administration of SIRT1 inhibitor, and that when Resveratrol stimulates SIRT1 expression, WT1 mRNA expression is increased. This suggests that the expression of SIRT1 protein is promoted to differentiate into progenitor progenitor cells.

SIRT1  Upon inhibition of expression, Into renal progenitor cells  Differentiation confirmation

The expression of Six2 and WT1 protein was observed in the conditional knockout mouse (Sirt1 co / co; Hoxb7-Cre (+)) on the first day of birth by specifically inhibiting SIRT1 protein in kidney collecting cell.

Changes in protein expression of Six2 and WT1 were measured by immunofluorescence staining. In order to perform immunochemical staining, kidneys of the pregnant rats or mice of the 0th day of birth were obtained, fixed with 4% paraformaldehyde for 6 hours, and then cut to a thickness of 10 mm and used for staining. The Zeiss Z1 microscope and a Zeiss LSM 510 confocal microscope (Carl Zeiss, Germany) were used for the anti-Six2 (Abcam; ab68908, Cambridge, UK) and Anti- WT1 (Abcam;

As a result, as shown in FIG. 4 and FIG. 5, it was confirmed that the group that specifically inhibited the SIRT1 protein only in the collecting duct cells of the kidney was different from the group that did not inhibit the differentiation into the progenitor precursor cells. This means that even when mRNA or protein is not expressed due to deficiency of SIRT1, differentiation into kidney precursor cells can be suppressed.

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Methods for Regulating Differentiation of Kidney Precursor Cells          from Embryonic Stem Cells Through modulation of SIRT1 expression <130> 1042837 <150> KR 2015/0089783 <151> 2015-06-24 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 2214 <212> DNA <213> Artificial Sequence <220> <223> sirt1 gene <400> 1 atggcggacg aggtggcgct cgcccttcag gccgccggct ccccttccgc ggcggccgcc 60 atggaggccg cgtcgcagcc ggcggacgag ccgctccgca agaggccccg ccgagacggg 120 cctggcctcg ggcgcagccc gggcgagccg agcgcagcag tggcgccggc ggccgcgggg 180 tgtgaggcgg cgagcgccgc ggccccggcg gcgctgtggc gggaggcggc aggggcggcg 240 gcgagcgcgg agcgggaggc cccggcgacg gccgtggccg gggacggaga caatgggtcc 300 ggcctgcggc gggagccgag ggcggctgac gacttcgacg acgacgaggg cgaggaggag 360 gcgaggcgg cggcggcagc ggcggcggca gcgatcggct accgagacaa cctcctgttg 420 accgatggac tcctcactaa tggctttcat tcctgtgaaa gtgatgacga tgacagaacg 480 tcacacgcca gctctagtga ctggactccg cggccgcgga taggtccata tacttttgtt 540 cagcaacatc tcatgattgg caccgatcct cgaacaattc ttaaagattt attaccagaa 600 acaattcctc cacctgagct ggatgatatg acgctgtggc agattgttat taatatcctt 660 tcagaaccac caaagcggaa aaaaagaaaa gatatcaata caattgaaga tgctgtgaag 720 ttactgcagg agtgtaaaaa gataatagtt ctgactggag ctggggtttc tgtctcctgt 780 gggattcctg acttcagatc aagagacggt atctatgctc gccttgcggt ggacttccca 840 gacctcccag accctcaagc catgtttgat attgagtatt ttagaaaaga cccaagacca 900 ttcttcaagt ttgcaaagga aatatatccc ggacagttcc agccgtctct gtgtcacaaa 960 ttcatagctt tgtcagataa ggaaggaaaa ctacttcgaa attatactca aaatatagat 1020 accttggagc aggttgcagg aatccaaagg atccttcagt gtcatggttc ctttgcaaca 1080 gcatcttgcc tgatttgtaa atacaaagtt gattgtgaag ctgttcgtgg agacattttt 1140 aatcaggtag ttcctcggtg ccctaggtgc ccagctgatg agccacttgc catcatgaag 1200 ccagagattg tcttctttgg tgaaaactta ccagaacagt ttcatagagc catgaagtat 1260 gacaaagatg aagttgacct cctcattgtt attggatctt ctctgaaagt gagaccagta 1320 gcactaattc caagttctat accccatgaa gtgcctcaaa tattaataaa tagggaacct 1380 ttgcctcatc tacattttga tgtagagctc cttggagact gcgatgttat aattaatgag 1440 ttgtgtcata ggctaggtgg tgaatatgcc aaactttgtt gtaaccctgt aaagctttca 1500 gaaattactg aaaaacctcc acgcccacaa aaggaattgg ttcatttatc agagttgcca 1560 ccaacacctc ttcatatttc ggaagactca agttcacctg aaagaactgt accacaagac 1620 tcttctgtga ttgctacact tgtagaccaa gcaacaaaca acaatgttaa tgatttagaa 1680 gtatctgaat caagttgtgt ggaagaaaaa ccacaagaag tacagactag taggaatgtt 1740 gagaacatta atgtggaaaa tccagatttt aaggctgttg gttccagtac tgcagacaaa 1800 aatgaaagaa cttcagttgc agaaacagtg agaaaatgct ggcctaatag acttgcaaag 1860 gagcagatta gtaagcggct tgagggtaat caatacctgt ttgtaccacc aaatcgttac 1920 atattccacg gtgctgaggt atactcagac tctgaagatg acgtcttgtc ctctagttcc 1980 tgtggcagta acagtgacag tggcacatgc cagagtccaa gtttagaaga acccttggaa 2040 gatgaaagtg aaattgaaga attctacaat ggcttggaag atgatacgga gaggcccgaa 2100 tgtgctggag gatctggatt tggagctgat ggaggggatc aagaggttgt taatgaagct 2160 atagctacaa gacaggaatt gacagatgta aactatccat cagacaaatc ataa 2214 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> six2 forward primer <400> 2 caaggaaagg gagaacagcg a 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> six2 reverse primer <400> 3 gcgtcttctc atcctcggaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WT1 forward primer <400> 4 atcccaggca ggaaagtgtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WT1 reverse primer <400> 5 gtgctgtctt ggaagtcgga 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 6 aggtcggtgt gaacggattt g 21 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 7 ggggtcgttg atggcaaca 19

Claims (10)

A method for regulating differentiation of progenitor cell proliferation in embryonic stem cells, comprising the step of regulating the expression of SIRT1 (silent mating type information regulation 2 homolog: sirtuin 1) of embryonic stem cells.
A method for inhibiting differentiation of progenitor precursor cells of embryonic stem cells, which comprises treating embryonic stem cells with a SIRT1 expression inhibitor to inhibit differentiation into progenitor precursor cells.
[3] The method according to claim 2, wherein the SIRT1 expression inhibitor is treated for 1 to 21 days in the early stage of culturing.
3. The method of claim 2, wherein the SIRT1 expression inhibitor is selected from the group consisting of Sirtinol (2 - [(2-Hydroxynaphthalen-1-ylmethylene) amino] -N- (1-phenethyl) benzamide, Nicotinamide EX527 (6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide).
3. The method according to claim 2, wherein the mRNA expression level of SIX2 (sine oculis homeobox homolog 2) and WT1 (Wilms tumor 1) of embryonic stem cells is decreased due to inhibition of SIRT1 expression, Way.
A method for promoting differentiation of progenitor progenitor cells in embryonic stem cells, characterized in that embryonic stem cells are treated with a promoter of SIRT1 expression to promote differentiation into progenitor progenitor cells.
[Claim 7] The method according to claim 6, wherein the SIRT1 expression promoter is at least one selected from the group consisting of SRT1270, BML-278, and resveratrol.
[Claim 7] The method according to claim 6, wherein the SIRT1 expression promoter is treated for 1 to 21 days to induce differentiation.
delete delete

Images