KR101439074B1 - Method for recovering stemness of adult stem cells comprising increase of PPAR expression - Google Patents

Abstract

The present invention relates to a method for restoring the stemness of senescent adult stem cells, and more particularly, to a method for regenerating stem cells by increasing the expression of peroxisome proliferator-activated receptor gamma (PPARγ) Stem cell function and a method of improving the ability to differentiate into an insulin producing cell.

Description

[0001] The present invention relates to a method for recovering the stem cell function of adult stem cells comprising increasing the expression of peroxisome proliferator activated receptor gamma,

The present invention relates to a method for restoring the stemness of senescent adult stem cells, and more particularly, to a method for regenerating stem cells by increasing the expression of peroxisome proliferator-activated receptor gamma (PPARγ) Stem cell function and a method of improving the ability to differentiate into an insulin producing cell.

Adult stem cells are undifferentiated cells that are found between differentiated cells in tissues or organs. They can proliferate on their own, and have somatic cells capable of differentiating into cells with specific functions of tissues or organs. stem cells. The main role of adult stem cells is to maintain the cells of tissues or organs where adult stem cells are present and to treat any damaged cells.

The important thing in understanding adult stem cells is that adult stem cells are present in small amounts in each tissue. Until the tissue becomes diseased or damaged and adult stem cells are activated, they remain silent for years without splitting or multiplying. The organs that have been reported to have adult stem cells are the brain, bone marrow, peripheral blood, blood vessels, muscles, skin and liver. Scholars are studying how to propagate these adult stem cells through cell culture and induce differentiation into specific cells to use when their bodies are hurt or become sick.

Mesenchymal stem cells (MSCs), which are representative adult stem cells, have advantages in that they can be easily obtained from adipose tissue obtained from liposuction, which is a cosmetic technique in the case of adipose derived mesenchymal stem cells, It is advantageous to obtain the mesenchymal stem cells from adipose tissue and easily utilize them again. These isolated cells are known to be able to attach to plastic cell culture dishes and to form fibroblast-like colony. In addition, it is known to have multipotent ability to differentiate into osteoblasts, adipocytes, chondrocytes, and neurons. Despite the many advantages of these mesenchymal stem cells, the number of cells required to pass through a subculture in a plastic culture dish is needed to increase the number of grafted cells.

However, it is known that the mesenchymal stem cells have a prolonged incubation period, and their ability to multiply and to differentiate into various systems is decreased through several subculture cultures. In a study by Prockop et al. ( Brit J Haematol , 107: 275-281, 1999), colony forming ability in mesenchymal stem cells of the late passage was markedly decreased as compared to the mesenchymal stem cells of the early passage, But the differentiation into adipocytes was suppressed. In addition, senescence of mesenchymal stem cells is related to the weakened proliferative capacity over a longer incubation period than the age of the bone marrow donor (Kassem, Bone , 33: 919-926, 2003), telomerase activity (Kassem, Nat . Biotechnol , 20: 592-596, 2002).

Thus, the amount of mesenchymal stem cells available from humans is limited, and in order to induce the differentiation of the thus-obtained cells into a specific system, problems such as reduction of the differentiation ability due to subculture are inevitable. These disadvantages are impractical in inducing differentiation into precise target cells and in clinical applications where large amounts of suitable stem cells are required for the induction. Therefore, there is a need for a new method for culturing mesenchymal stem cells capable of maintaining the proliferation rate and maintaining the pluripotency of various lines for a long period of time, as well as reactivating the stem cells of senescent mesenchymal stem cells.

It is an object of the present invention to provide a method for the treatment of stem cells of adult stem cells comprising the step of increasing the expression of peroxisome proliferator-activated receptor gamma (PPAR?) In adult stem cells. Of the patient.

Another object of the present invention is to provide a composition for restoring stem cell function of adult stem cells, comprising peroxisome proliferator activated receptor gamma as an active ingredient.

In one aspect of the present invention, the present invention provides a method for the proliferation of adult stem cells, comprising the step of increasing the expression of peroxisome proliferator-activated receptor gamma (PPARγ) in adult stem cells It provides a way to restore stem cell function.

In order to solve the conventional problem that the stemness of the stem cells is decreased as the number of passages increases in the passage culture of mesenchymal stem cells, the present inventors have found that when the PPARγ is activated or overexpressed, And the decreased expression of PDX1, which is a typical marker of insulin-secreting cells, is increased. As a result, PPARγ of the present invention restores the stemness of adult stem cells and the ability to differentiate into insulin-producing cells .

As used herein, the term "Peroxisome proliferator-activated receptor" is a member of the nuclear receptor giant family of transcription factors including steroids, thyroid and vitamin D receptors. They control the expression of proteins that regulate lipid metabolism and are activated by fatty acids and fatty acid metabolites. Three PPAR subtypes, PPARa, PPAR [beta] and PPAR [gamma], are known, each of which exhibits a different pattern of tissue expression and can be distinguished in their activation by various structurally diverse compounds. Among these, PPARγ is an important factor in the cell signaling pathway, and it is known to regulate the transcription factors associated with adipocyte and adipocyte formation. Therefore, PPARγ is most expressed in adipocytes and is expressed at low levels in skeletal muscle, heart, liver, intestine, kidney, endothelial and smooth muscle cells and macrophages. PPARγ is known to play a multifaceted role in vascular cell proliferation, migration and differentiation, macrophage activation, and inflammation.

In the present invention, the term "adult stem cell" means a cell immediately before being differentiated into cells of a specific organ, which is extracted from a bone marrow or blood of an umbilical cord blood or a multiparous adult. Lt; / RTI > The adult stem cells may be selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta, although the present invention is not limited thereto. The adult stem cells include neural stem cells capable of differentiating into neurons, hematopoietic cells capable of differentiating into blood cells, mesenchymal stem cells capable of differentiating into bone, cartilage, fat, muscle, etc., liver cells capable of differentiating into hepatocytes A stem cell, preferably a mesenchymal stem cell, more preferably a mesenchymal stem cell derived from an adipocyte or adipose tissue, but is not limited thereto. The method of obtaining stem cells from each of the strains can be performed by a method known in the art and is not limited to the method of the embodiment of the present invention.

In the present invention, the term "stemness" refers to pluripotency, which is capable of producing all cells such as embryonic stem cells, and self-renewal (self- -renewal) is commonly used in the industry. Therefore, it is meant that the undifferentiated cells increase proliferation while maintaining undifferentiated state, increase telomerase activity, increase the expression of stemness acting signals or increase cell migration activity, (Pittenger, MF, Mackay, AM, Beck, SC, Jaiswal, RK, Douglas, R., Mosca, JD, Moorman, Simonetti, DW, Craig, S., and Marshak , DR 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284 (5411), 143-147).

Increasing or activating the expression of PPARy in stem cells through the method of the present invention may increase expression of Oct4 (octamer-binding transcription factor 4) reduced by subculture.

Oct4, which is known to express undifferentiated stem cells, is a transcription factor that is expressed mainly in embryonic stem cells. It plays a role in preventing cell differentiation and disappears when natural differentiation of cells begins It is known. Therefore, the degree of differentiation of stem cells can be predicted according to the expression level of Oct4.

In one embodiment of the present invention, the expression level of Oct4 was decreased as the number of passages in subculturing adipose tissue-derived stem cells was increased (Experimental Example 2), but when PPARγ was overexpressed in the above-mentioned aged stem cells , Oct4 expression level was significantly increased (Example 7). This suggests that stem cell function was restored through increased expression or activation of PPARγ in adult stem cells.

When the expression of PPARy in the stem cells is increased or activated by the method of the present invention, it is possible to increase insulin production by decreasing insulin and / or expression of? -Cell differentiation transcription factors such as PDX1 and NGN3, It can promote the differentiation into cells and promote the secretion of insulin.

The term "insulin (INS) " in the present invention is a hormone that regulates carbohydrate and fat metabolism in the body. It is secreted from β-cells of islets of Langerhans and plays a role in maintaining a constant blood glucose level in the blood. That is, when the concentration of blood glucose is higher than a certain level, insulin is secreted, and glucose is injected into the cells to promote the action of storing glucose and polysaccharide (eg, glycogen) in the liver and muscle. People who do not have normal insulin secretion are prone to diabetes.

In the present invention, the term "insulin producing cell (IPC)" refers to a cell capable of producing insulin such as pancreatic cells. Thus, it is possible to include, without limitation, genetically engineered cells that can produce insulin through processes such as transfection, regardless of the origin of the cells, and further that "functional" insulin producing cells secrete insulin under high glucose conditions Cells that can be treated.

The term "PDX-1 (pancreatic duodenal homeobox-1)" in the present invention is a transcription factor, also known as insulin promoter factor 1, which is encoded by PDX- It is a transcription factor essential for interest development and β-cell maturation by binding to the A-box of the insulin promoter.

In one embodiment of the present invention, the expression level of insulin and / or β-cell differentiation transcription factors such as PDX1 and NGN3 decreased in the subculturing of adipose tissue-derived stem cells (Experimental Example 3) When overexpressing PPARγ in the above-mentioned aged stem cells, it was confirmed that the expression levels of insulin and PDX1 were significantly increased (Example 8).

In the present invention, the step of increasing the expression of PPARy to restore the stemness of adult stem cells can be carried out by a method known in the art. Specifically, a method of introducing the PPARγ into adult stem cells, a method of increasing the number of intracellular copies of the gene encoding the PPARγ, a method of introducing a mutation into the expression control sequence of the gene on the chromosome encoding the PPARγ, A method for replacing the regulatory sequence for expression of a gene on the chromosome with a sequence having a stronger activity than that for the same, a method for replacing the gene encoding the PPARγ on the chromosome with a gene mutated to increase the activity of the PPARγ, A method of introducing a mutation into a gene on the chromosome encoding the PPAR gamma, but the present invention is not limited thereto.

As another embodiment, the present invention provides a composition for restoring stem cell function of adult stem cells, comprising a substance which activates peroxisome proliferator-activated receptor gamma or peroxisome proliferator-activated receptor gamma as an active ingredient .

In the present invention, peroxisome proliferator-activated receptor gamma, adult stem cells, and stem cell function are as described above. The composition contains peroxisome proliferator-activated receptor gamma as an active ingredient, thereby increasing the activation or expression of PPARy in stem cells, restoring the stemness of adult stem cells, Can be promoted.

The present invention relates to a method for restoring the stemness of senescent adult stem cells by repeated subculture by increasing the expression of PPARγ in adult stem cells, By not using the substance, it is possible to restrain the adverse effect and to restore the property of the adult stem cells in the early passages.

In addition, by restoring the stem cell function of aged adult stem cells, it is possible to mass-produce adult stem cells having the same differentiation power and proliferative power as the early sub-cultured cells even with a very small amount of adult stem cells without collecting additional adult stem cells from the patient Furthermore, by stimulating the differentiation of human adipose tissue-derived stem cells into insulin-producing cells, the secretion amount of insulin can be improved even in a high number of adult stem cells, and thus the potency as a cell therapy agent can be improved.

In addition, the present invention can be used in a wide variety of clinical fields by improving utilization of adult stem cells, which are superior in terms of safety to embryonic stem cells.

FIG. 1A is a graph showing a decrease in proliferative capacity according to continuous passaging (passages 4, 7, 12 and 20) of human adipose-derived mesenchymal stem cells. FIG. 1B shows an increase in aging markers, FIG. 1C shows multipotency and differentiation Lt; RTI ID = 0.0 > expression < / RTI >
FIG. 2A is a graph showing the expression of insulin-producing cell differentiation markers after differentiation into adipocyte-producing cells by subculturing the adipose-derived mesenchymal stem cells. FIG. 2B is a graph showing the amount of insulin secreted after differentiating into insulin-producing cells by passage.
FIG. 3 is a graph showing changes in expression of expression factors of nuclear receptor transcription factors by lineage after subculture. FIG.
FIG. 4 is a graph showing that expression of Oct4, a key marker of stem cell function, is increased when the expression or activity of reduced PPARγ is increased. In particular, when agonist-treated cells are treated with an activator of PPARγ or overexpressed, This shows that the ability to differentiate into cells is restored.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

Experimental material

STEMPRO ^® hADCSs were purchased from Life Technologies (Cat.Co.R7788-115), were cultured in medium MesenPRO ^TM. The reagents used were each purchased from the named supplier; Troglitazone from Enzo LifeScience; Activin A, basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) are found in Peprotech; B27 serum-free supplenment and N2 supplement were purchased from Life Technologies; And 15-deoxy-Δ ^12,14 -prostaglandin J ₂ (15PGJ ₂ ), valproic acid, exendin-4, nicotinamide, dexamethasone, 3-isobutyl-1-methylxanthine, insulin, ascorbate-2 phosphate,? -Glycerol phosphate, GW9662, and 3, 3 ', 5-triido-L-tyrosine (T3) was purchased from Sigma-Aldrich.

Example  1: Cell culture

In the present invention, human adipose tissue-derived stem cells (hADSCs) in the 4th, 7th, 12th and 20th passages were used. Growth driving force of stem cells according to each passage was measured by cell proliferation for 1 week. That is, the cells in each passage were seeded on a 6-well plate at a density of 10 ⁴ cells / cm 2 and allowed to divide for 7 days. The badge was changed twice a week. One medium from each group was used to determine the number of cells. For the chemical treatment group, the cells were seeded to a 6-well plate at a density of 10 ⁴ cells / cm 2 and allowed to attach to the plate overnight. And then replaced with fresh medium containing troglitazone or 15PGJ ₂ . Samples were extracted at various time points between cultures.

Example  2: RNA  Separation and quantitative PCR

For analysis of cell mRNA expression, total RNA was isolated from the cells using Trizol reagent. To remove the possibility of contamination by genomic DNA, RNA samples were treated with DNase. RNA was quantified using ND-1000 spectrophotometer (Thermo Fisher Scientific), and cDNA was synthesized from PrimeScript ^™ 1st strand cDNA synthesis kit (Takara). Quantitative PCR was performed with a reaction mixture containing 1 × Power SYBR-Green Master-mix (Applied Biosystems), 600-750 nM forward and reverse primers and 50 ng of cDNA template (Xie CQ meat al ., Mol Endocrinol 23: 724-733 (2009)). PCR amplification was performed using an Applied-Biosystem Prism 7900HT Real-Time PCR Sequence Detection System and stopped at 40 cycles. Relative gene expression levels were calibrated to human cyclophilin using the relative C _T method. The primer sequences used in the present invention are shown in Table 1 below.

Target Forward primer Reverse primer Cyclophilin TGCCATCGCCAAGGAGTAG
(SEQ ID NO: 1) TGCACAGACGGTCACTCAAA
(SEQ ID NO: 2) Insulin GCAGCCTTTGTGAACCAACA
(SEQ ID NO: 3) TTCCCCGCACACTAGGTAGAGA
(SEQ ID NO: 4) Nanog CCAACATCCTGAACCTCAGC
(SEQ ID NO: 5) GCTATTCTTCGGCCAGTTG
(SEQ ID NO: 6) Ngn3 CGGAGTCGGCGAAAGAAG
(SEQ ID NO: 7) CGTCCAGTGCCGAGTTGAG
(SEQ ID NO: 8) Oct4 ACATCAAAGCTCTGCAGAAAGAACT
(SEQ ID NO: 9) CTGAATACCTTCCCAAATAGAACCC
(SEQ ID NO: 10) p16 ^INK4A GAAGGTCCCTCAGACATCCCC
(SEQ ID NO: 11) CCCTGTAGGACCTTCGGTGAC
(SEQ ID NO: 12) PDX1 GAACTTGACCGAGAGACACATCAA
(SEQ ID NO: 13) TTGTCCTCCTCCTTTTTCCACTT
(SEQ ID NO: 14) PPARg AGATCCAGTGGTTGCAGATT
(SEQ ID NO: 15) GGAGATGCAGGCTCCACTTT
(SEQ ID NO: 16) Rex1 GAAGAGGCCTTCACTCTAGTAGTG
(SEQ ID NO: 17) TTTCTGGTGTCTTGTCTTTGCCCG
(SEQ ID NO: 18) Sox2 TGCGAGCGCTGCACAT
(SEQ ID NO: 19) GCAGCGTGTACTTATCCTTCTTCA
(SEQ ID NO: 20)

Experimental Example  One: In subculture  Derived fatty tissue Intermediate lobe  Aging and stem cell function of stem cells stemness  Change observation

In order to observe the morphology, stem cell function (growth rate) and degree of senescence of adipose tissue-derived mesenchymal stem cells in different passages, total cell extracts extracted from undifferentiated cells in the 4th, 7th, 12th and 20th passages Prepared. After the Bradford assay, 40 μg of protein per lane was separated by electrophoresis on a 15% SDS (sodium dodecyl sulphate) polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane and the membrane was blocked with 5% skim milk in Tris-buffered saline (TBS) with 0.05% Tween 20 for 1 hour. And then incubated overnight with primary antibody against p16 ^INK4A (1: 500; Upstate). The secondary antibody was then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz) for 1 hour at room temperature and visualized using a Fujifilm luminescent image analyzer LAS4000. On the other hand, beta-actin was used as a loading control.

As a result, in the morphological changes of the cells, the low passage number (P4 and P7) cells had a spindle shape, and they were found to be firmly attached to the plate at high passage number as they became larger and flattened.

On the other hand, the growth rate of cells gradually decreased as the number of passages increased, and the increase of cell number also remarkably decreased as the number of passages increased.

Regarding cell senescence, mRNA and protein expression of p16 ^INK4A was increased in cells with a higher number of passages than in the fourth passage. p16 ^INK4A is known to be involved in cellular senescence and stem cell senescence and is thought to be a biomarker of aging as it accumulates in many aging cells and tissues.

Experimental Example  2: Stem cells in proliferation medium Marker Expression  Confirm

hADSCs are also known to express several markers of pluripotent stem cells. In order to confirm the expression of several important universal markers, FACS analysis was performed to investigate the expression patterns of hADSC mesenchymal surface markers in different subtypes Respectively. Specifically, single-cell suspensions were obtained in the 4th, 7th, 12th and 20th passages and washed twice with FACS buffer containing 1% fetal bovine serum (FBS) in PBS (phosphate-buffered saline) Respectively. The number of cells was counted and suspended at a concentration of 5 x 10 ⁵ cells in 100 μL FACS buffer. And incubated with fluorescent antibodies of CD73-PE, CD90-FITC and CD105-FITC (BD Biosciences) under dark conditions at 4 ° C for 30 minutes. Cells were washed twice with FACS buffer and analyzed using a flow cytometer (FACS Calibur System; Becton Dickinson), after which data was collected and analyzed (Cell Questsoftware; Becton Dickinson).

As a result, there was no difference between the cells expressing CD73, CD90 or CD105, but Oct4, an important transcription factor for maintaining the undifferentiated state of embryonic stem cells, was remarkably decreased in the 7th, 12th, and 20th passages Respectively. A similar pattern was observed at the Nanog level. On the other hand, the expression of Sox2 (sex determining region Y-box 2) and Rex1 (reduced expression 1) did not change significantly.

Experimental Example  3: In subculture  Derived fatty tissue Intermediate lobe  Stem Cells to Insulin-Producing Cells Multipotential  Change observation

To measure the ability of hADSCs to differentiate into insulin-producing cells in different passages, trypsinize 4, 7, 12, and 20 passages of cells to induce differentiation into insulin-producing cells, And centrifuged. Cells were counted and seeded at a density of 10 ⁴ cells / cm 2 in a 12-well low adhesion plate (SPL 30212). The cells were differentiated into insulin-producing cells using a basic differentiation medium. The basic differentiation medium is serum-free DMEM / F12 (1: 1) containing 1% B27 serum-free supplement, 1% N2 supplement and 1% penicillin / streptomycin. In order to differentiate hADSCs into insulin-producing cells, the following three-step 10-day protocol was used: (1) in a basal medium supplemented with 50 ng / mL of actin A and 2 mM valproic acid for 3 days culture; (2) culturing for 3 days in a basal medium supplemented with 10 nM exendin-4 and 10 ng / mL bFGF; And (3) incubated for 4 days in a basal medium supplemented with 10 nM exendin-4, 50 ng / mL HGF, and 10 mM nicotinamide.

As a result, in all the passages, during the 10 day incubation period, the cells aggregated with each other, formed oval clusters, and showed an islet-like structure.

However, hADSCs in the 4, 7, and 12 passages formed clusters of smooth surfaces, while cells in the 20 passages formed wrinkled, rough surface clusters.

In order to confirm whether the morphological changes reflected the successful differentiation of hADSCs, it was shown that in differentiated cells of insulin and beta-cell differentiation transcription factors PDX1 (pancreatic and duodenal homeobox 1) and NGN3 (neurogenin-3) mRNA expression was analyzed by quantitative PCR as described in Example 2 above. As a result, the expression level of insulin and the transcription factor significantly decreased in the 12th and 20th passages compared to the fourth passages.

On the other hand, insulin secretion amount of differentiated cells was measured using a human insulin ELISA kit (Mercodia) in order to confirm the function of the differentiated cells by measuring the amount of secreted insulin. As a result, the amount of insulin secreted from differentiated cells in the 7th, 12th, and 20th passages was significantly reduced. Insulin secretion in the 20th passage was reduced by about 3 times compared to the fourth passage.

Experimental Example  4: In vitro  In the medium hADSC of Nuclear receptor ( NR ) Expression pattern analysis

Stemness can be maintained by a number of key regulatory transcription factors and signaling molecules that establish an accurate pattern of gene expression that characterizes undifferentiated phenotypes. Nuclear receptors (NRs) are involved in the pathway of tissue-specific stem cell regulation, and some NRs play an important role in maintaining embryonic stem cells in cooperation with Oct4 and Nanog. In order to observe the change in the NR expression profile of hADSCs between cultures, the expression of 48 human NRs in the 4th, 7th, 12th and 20th passages was screened by the method described in Example 2 above. Analysis of NR mRNA expression level revealed that 24 NRs were expressed in all the passages. These 24 NRs can be distinguished into three groups according to their expression patterns in four subcategories: NR (13/24) retaining consistently, NR (10/24) decreasing expression, and increased expression NR (1/24).

Only two of them showed statistically significant values. PPARγ and thyroid hormone receptor (TR) β were significantly decreased in the 12th and 20th passages compared to the 4th passages.

Experimental Example  5: PPAR by γ Oct4  Regulation of gene expression levels

12 and 20, respectively. Thus, we investigated whether PPARγ plays an important role in the regulation of the stem cell marker Oct4. Specifically, the Luciferase reporter construct (phOct4-Luc), including the upstream region of human Oct4, was provided from Shinya Yamanaka (Addgene plasmid No. 17221). To examine the effect of PPARy overexpression, 20 passages of hADSCs were infected with pcDNA flag PPARg plasmid or pcDNA plasmid (250 ng each) and then infected with phOct4-Luc plasmid (500 ng) and? -Galactosidase plasmid . Two electrical pulses were injected at 1,005 V for 35 ms duration using a Neon transfection system. Cells were harvested 36 hours after infection using 1 x passive lysis buffer (Promega). The intensity of the luciferase activity was normalized to < RTI ID = 0.0 > and-galactosidase < / RTI > As a result, the treatment of troclitazone, a PPARgamma activator in the hADSCs of the 20th passage, increased the expression of PPARγ mRNA in a dose - and time - dependent manner, and the expression of Oct4 mRNA increased in a similar pattern.

In order to confirm whether troglitazone treatment increased Oct4 gene expression, Oct4 promoter activity in hADSCs transiently infected with phOct4-luc plasmid was measured, and as a result, troglitazone (20 μM) significantly decreased luciferase activity Respectively. Furthermore, it was confirmed that treatment with another PPARγ activator, 15PGJ ₂ , increased PPARγ and Oct4 mRNA levels in a time-dependent manner.

Subsequently, it was examined whether PPAR [gamma] overexpression increases Oct4 promoter activity. As a result, it was confirmed that the overexpression of hADSCs in the 20th passage in PPAR [gamma] increases Oct4 luciferase activity.

Additional experiments were performed using GW9662, a PPAR [gamma] -specific antagonist, to determine whether modulation of Oct4 expression is affected by PPAR [gamma]. As a result, troclitazone in combination with GW9662 (10 μM) did not increase Oct4 mRNA levels in hADSCs. Thus, this means that PPAR gamma directly increases the transcription of the Oct4 gene.

TRβ expression was also significantly decreased in the 20th passage, so we examined whether Oct4 expression was affected by TRβ activation. As a result, treatment with T3 (10-40 ng / mL) in 20 passages of hADSC showed no effect on Oct4 or TRβ mRNA levels.

Experimental Example  6: PPAR by γ Eruptive talent  possesion

PPAR [gamma] activating factor treatment or PPAR [gamma] overexpression increased Oct4 mRNA expression, confirming whether overexpression of PPAR [gamma] could sustain the differentiation potential of hADSCs. HADSCs in the 20th passage were infected with the PPARy plasmid and then differentiated into insulin producing cells.

As a result, insulin and PDX1 mRNA levels were increased in PPARγ-overexpressing cells compared to pcDNA-infected cells.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

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Claims (11)

Comprising increasing the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in adult stem cells. &Lt; Desc / Clms Page number 20 &gt;
2. The method of claim 1, wherein the method increases the ability of adult stem cells to differentiate into insulin producing cells.
2. The method of claim 1, wherein the method increases expression of Oct4 (octamer-binding transcription factor 4).
2. The method of claim 1, wherein the method increases the expression of PDX1 (Pancreatic and duodenal homeobox 1) in insulin producing cells differentiated from adult stem cells.
The method according to claim 1, wherein said adult stem cells are mesenchymal stem cells.
6. The method of claim 5, wherein the mesenchymal stem cells are derived from adipocytes or adipose tissue.
The method according to claim 1, wherein the step of increasing the expression of PPARγ comprises the steps of introducing the PPARγ into adult stem cells, increasing the number of intracellular copies of the gene encoding the PPARγ, regulating the expression of the gene on the chromosome encoding the PPARγ A method of introducing a mutation into the sequence, a method of replacing the regulatory sequence for regulating the expression of a gene on the chromosome encoding the PPARγ with a sequence having a stronger activity than that, a method of encoding the PPARγ on the chromosome, And a method of introducing a mutation into a gene on a chromosome encoding the PPAR gamma to enhance the activity of the PPAR gamma.
A composition for restoring stem cell function in adult stem cells, comprising peroxisome proliferator activated receptor gamma as an active ingredient.
9. The composition of claim 8, wherein the composition enhances the ability of adult stem cells to differentiate into insulin producing cells.
9. The composition according to claim 8, wherein said adult stem cells are mesenchymal stem cells.
11. The composition of claim 10, wherein the mesenchymal stem cells are derived from adipocytes or adipose tissue.

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