KR101982801B1 - Composition for inducing differentiation to retinal pigment epithelium - Google Patents
Composition for inducing differentiation to retinal pigment epithelium Download PDFInfo
- Publication number
- KR101982801B1 KR101982801B1 KR1020150150366A KR20150150366A KR101982801B1 KR 101982801 B1 KR101982801 B1 KR 101982801B1 KR 1020150150366 A KR1020150150366 A KR 1020150150366A KR 20150150366 A KR20150150366 A KR 20150150366A KR 101982801 B1 KR101982801 B1 KR 101982801B1
- Authority
- KR
- South Korea
- Prior art keywords
- cells
- mir
- rpe
- pigment epithelium
- mesenchymal stem
- Prior art date
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/65—MicroRNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
Abstract
The present invention relates to a composition for inducing differentiation of retinal pigment epithelium comprising a miR-410 inhibitor, a kit for inducing differentiation of retinal pigment epithelium containing the composition, a method for differentiating mesenchymal stem cells into retinal pigment epithelium using the composition, The present invention relates to a pharmaceutical composition for treating a retinal disease comprising a mesenchymal stem cell treated with a composition. The composition comprising the miR-410 inhibitor according to the present invention can differentiate mesenchymal stem cells into retinal pigment epithelium without special conditions, and thus can be widely used for the treatment of various retinal diseases due to damage of the retinal pigment epithelium .
Description
More particularly, the present invention relates to a composition for inducing differentiation of retinal pigment epithelium comprising a miR-410 inhibitor, a kit for inducing differentiation of retinal pigment epithelium comprising the composition, , A method for differentiating mesenchymal stem cells into retinal pigment epithelium, a method for treating retinal pigment epithelium differentiated by the above method and a mesenchymal stem cell treated with the composition.
Blindness refers to the absence of a wide range of medical conditions, and is currently suffering from millions of patients globally, 0.2-0.5% of the world's population, resulting in huge personal, social and economic losses. Globally, one of the major causes of blindness is the degeneration of retinal photoreceptor cells and retinal pigment epithelial cells, which are caused by congenital or other various causes. Retinopathy, retinal degeneration, aged macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital retinal atrophy, Leber congenital amaurosis ), Retinal detachment, glaucoma, optic neuropathy, and trauma. To date, there has been no pharmacotherapy for radical treatment of these diseases. Regenerating and transplanting photoreceptor cells and retinal pigment epithelial cells, which are the cause and result of these diseases, as impaired functional cells into new functional cells is considered to be the most promising therapy have. Photoreceptor cells and retinal pigment epithelial cell transplantation methods have been postulated to delay or inhibit retinal degeneration and to regenerate retinal degeneration or degeneration of the retina and improve retinal function to prevent blindness or to regenerate damaged vision .
(BMS), retinal stem cell (RSC), embryonic stem cell (ESC), induced pluripotent stem cell (iPSC), and somatic cell nucleus (SCNT), and the like. However, the results of research on the differentiation of stem cells into retinal cells and cell therapy using them have been in a poor state. When stem cells can be differentiated into retinal cells, it is possible to supply infinite cells for effective cell therapy, 2) retinal cells from previously unknown embryonic cells and retinal progenitors 3) identification of the genes and molecules involved in differentiation of the retina, and 4) identification of the mechanism of development of retinal degenerative diseases, and 5) development of drugs for preventing retinal degeneration and retinal nerve protection Lt; / RTI >
Since the establishment of the first human embryonic stem cell line, human embryonic stem cells have the ability to differentiate into a wide variety of cells, and the differentiated cells can be used for human cell therapy It has been proposed that the present invention can be applied. In other words, human embryonic stem cells can be identified as a powerful candidate for abundant supply of cells for replacement of dysfunctional cells with new cells in clinical treatment, which can accurately identify the features. Differentiated cells constituting various organs derived from human embryonic stem cells have been supposed to have the same properties and functions as the corresponding cells formed through normal differentiation process. Based on this possibility, differentiation-inducing methods that provide an environment similar to that of developmental stage are known as pancreatic hormone-expressing endocrine cells (D'Amour, et al., Nat. Biotechnol., 2006; 24: 1392-401) , et al., Stem Cells 2007; 25: 1511-20), muscle cells (Barberi et al., Nat. Med., 2007; 13: 642-8), vascular endothelial cells (Wang, et al., Nat. Biotechnol., 2007; 25: 317-8). There have been many attempts to produce photoreceptor cells and retinal pigment epithelial cells, which are representative cells of retinal cells, differentiated from human embryonic stem cells in retinal diseases, but the results are very poor.
One of the greatest successes reported so far is that retinal-derived cells are effectively induced from human embryonic stem cells, but the differentiation of photoreceptor cells from induced retinal origin has failed (less than 0.01% differentiation) (Lamba , Proc Natl Acad Sci USA, 2006, 103: 12769-74). Another study reported that successfully differentiating photoreceptor cells from human embryonic stem cells resulted in a total induction period of more than 200 days and a differentiation rate of 8% (Osakada et al., Nat. Biotechnol., 2008; 26: 215-24).
Under these circumstances, the present inventors have made extensive efforts to develop a technique capable of more effectively differentiating mesenchymal stem cells into retinal pigment epithelium (RPE), and as a result, it has been found that UCB-MSC It was confirmed that when the activity of miR-410 is inhibited, it can be effectively differentiated into retinal cell epithelial-like cells, and the present invention has been completed.
It is an object of the present invention to provide a composition for inducing differentiation of retinal pigment epithelium comprising a miR-410 inhibitor.
Another object of the present invention is to provide a kit for inducing differentiation of retinal pigment epithelium comprising the above composition.
It is still another object of the present invention to provide a method for differentiating mesenchymal stem cells into retinal pigment epithelium using the composition.
It is yet another object of the present invention to provide a retinal pigment epithelium differentiated by the above method.
It is still another object of the present invention to provide a cell therapy agent for treating retinal diseases, which comprises mesenchymal stem cells treated with the composition.
The present inventors have focused on microRNAs while carrying out various studies to develop a method that can more effectively differentiate mesenchymal stem cells into RPEs. (UCB-MSC), which is a type of mesenchymal stem cell, is highly expressed in human RPE-derived cell line ARPE-19 cells and retinal tissue cells. As a result, 21 kinds of microRNAs were selected. MicroRNAs that serve as common targets of RPE-specific transcription factors and visual circuit-specific genes in the selected microRNAs were selected and ultimately miR-410 was selected. As a result of analysis of the characteristics of miR-410, the expression level of miR-410 was significantly reduced in the undifferentiated stem cells but the expression level of miR-410 was significantly reduced in UCB-MSC, The expression level of RPE-specific marker gene and protein was increased in the cultured cells, and it was confirmed that the specific cell activity was increased in RPE cells. Therefore, it was found that the differentiated cells were similar to RPE-like cells.
As described above, a technique for differentiating the stem cells into RPE cells by inhibiting the activity of miR-410 in mesenchymal stem cells has not been reported at all and has been developed for the first time by the present inventors.
In one embodiment of the present invention, there is provided a composition for inducing differentiation of retinal pigment epithelium comprising a miR-410 inhibitor.
The term " miR-410 " of the present invention means a microRNA having the nucleotide sequence of SEQ ID NO: 1 (5'-AAUAUAACACAGAUGGCCUGU-3 ').
The term " miR-410 inhibitor " of the present invention means a substance capable of inhibiting the activity of miR-410. The inhibitor is not particularly limited as long as it can inhibit the activity of miR-410. For example, the polynucleotide may be a polynucleotide, a peptide, a protein, a compound, a polymer, or the like. As another example, the polynucleotide may be a polynucleotide capable of specifically binding miR-410 to inhibit its function, , a single-stranded polynucleotide consisting of a nucleotide sequence complementary to miR-410.
The term " retinal pigment epithelium (RPE) " of the present invention means a monolayer of pigment cells forming part of the outer blood vessel retinal barrier. These cells are characterized by their important role in maintaining retinal function and are designed to absorb light energy, interact with the photoreceptor to maintain the visual cycle, maintain the binding structure of the photoreceptor and the capillary vascular layer It is known to secrete a variety of growth factors, to transport ions, water, and metabolites from the subretinal space to the blood, and to perform the phagocytosis of the outer segment of the photoreceptor.
The term " differentiation " of the present invention generally means a phenomenon in which a relatively simple system is separated into two or more qualitatively different partial systems (partial systems). Specifically, It means a phenomenon in which structure or function is specialized in each other, that is, a cell or tissue of a living organism changes shape or function in order to perform a given task. For example, there is a qualitative difference between the parts of a biological system that were almost homogeneous at first, such as the distinction of heads or trunks between the eggs that were homogeneous at first in the development of an individual, or the distinction of cells such as muscle cells or nerve cells Or a phenomenon that is divided into a partial or partial system that can be distinguished qualitatively as a result. Relatively, " undifferentiated " refers to a state in which the above-mentioned differentiation does not yet occur and contains characteristics as stem cells.
For the purpose of the present invention, the differentiation may be interpreted as a differentiation of mesenchymal stem cells into retinal pigment epithelial cells by the composition for inducing differentiation of retinal pigment epithelium.
According to one embodiment of the present invention, microarray analysis of microRNAs that affect the induction of RPE differentiation expressed in UCB-MSC revealed low expression level in retinal tissue cells while high expression in UCB-
On the other hand, according to a known method, UCB-MSC is treated with Actin A and nicotinamide to obtain RPE-like cells differentiated or treated with miR-410 inhibitor to obtain differentiated RPE-like cells, As a result of analysis of RPE marker expression levels in the cells, RPE markers were found to be expressed in gene level (Fig. 2b) and protein level (Fig. 2c). In particular, the expression level of EMMPRIN or bestropine among the RPE markers was confirmed by immunofluorescence staining analysis (Figs. 3A to 3C). Finally, analysis of the phagocyte activity confirmed in the RPE cells revealed that the RPE-like cells exhibited a high level of phagocytic activity as compared to the undifferentiated UCB-MSC (FIGS. 4A and 4B).
Therefore, it is possible to differentiate mesenchymal stem cells into retinal pigment epithelium by treating miR-410 inhibitor with mesenchymal stem cells.
In another embodiment, the present invention provides a kit for inducing differentiation of retinal pigment epithelium comprising the composition for inducing differentiation of retinal pigment epithelium.
The kit of the present invention can induce differentiation of mesenchymal stem cells into retinal pigment epithelium. The kit for inducing differentiation of retinal pigment epithelium of the present invention may contain, in addition to the miR-410 inhibitor, one or more other component compositions, solutions or devices suitable for the treatment of the inhibitor and the differentiation process of mesenchymal stem cells.
As a specific example, the kit of the present invention may be a kit containing essential elements necessary for effectively treating miR-410 inhibitor to mesenchymal stem cells. For example, a test tube or other suitable container, a reaction buffer (varying in pH and buffer concentration), and the like.
As another example, the kit of the present invention may be a kit containing essential elements necessary for effectively performing differentiation of mesenchymal stem cells. For example, a culture container, a culture medium, sterilized water, and the like.
In another embodiment, the present invention provides a method for differentiating mesenchymal stem cells into retinal pigment epithelium comprising treating the mesenchymal stem cell with a composition for inducing differentiation of retinal pigment epithelium, and a method for differentiating the differentiated retinal pigment epithelium Lt; / RTI >
The term "mesenchymal stem cells" (MSCs) of the present invention refers to undifferentiated stem cells that can differentiate into bone, cartilage, fat, bone marrow, muscle, nerve, etc. In adults, But it also exists in cord blood, peripheral blood, and other tissues. For the purpose of the present invention, the mesenchymal stem cells are not particularly limited, but may be cells capable of mixed culturing with respective mesenchymal stem cells derived from different origins derived from human tonsil, Cells can be differentiated into mesodermal tissue cells such as chondrocytes, as well as endodermal tissue cells such as parathyroid tissue cells. In addition, the mesenchymal stem cells used in the present invention may be mesenchymal stem cells expressing ZNF281. In the present invention, UCB-MSC, which is a mesenchymal stem cell derived from cord blood, was used as the mesenchymal stem cell.
The method for treating the mesenchymal stem cells with the miR-410 inhibitor contained in the composition for inducing the differentiation of retinal pigment epithelium is not particularly limited, but the mesenchymal stem cells can be treated once or several times, For example, in the present invention, the UCB-MSCs were treated 4 times at 0, 3, 10, and 17 days after culturing, and 21 days after the UCB-MSC treatment, differentiation into retinal pigment epithelial cells was induced.
On the other hand, the retinal pigment epithelium differentiated from mesenchymal stem cells according to the above method exhibits a high level of EMMPRIN or vestropine, which is a marker of the retinal pigment epithelium, and exhibits a high level of phagocytotic activity , Different from pre-differentiation mesenchymal stem cells and similar to conventional retinal pigment epithelium.
In another embodiment, the present invention provides a cell therapy agent for treating retinal disease, wherein the composition for treating retinal pigment epithelium differentiation comprises mesenchymal stem cells treated with the composition.
The miR-410 inhibitor or the composition comprising the miR-410 inhibitor according to the present invention can differentiate mesenchymal stem cells into retinal pigment epithelial-like cells. Therefore, the miR-410 inhibitor or a composition containing the miR- It may have an effect of treating a disease caused by abnormalities or damages of the pigment epithelium.
The term " retinal disease " of the present invention means a disease caused by abnormal or damaged retinas due to external, inherent or genetic causes.
In the present invention, the retinal disease can be interpreted as a disease caused by abnormalities or damage of the retinal pigment epithelium. The retinal diseases can be alleviated, alleviated, improved or treated by the pharmaceutical composition provided by the present invention Examples include, but are not limited to, retinal degeneration, retinal degeneration, aged macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital retinopathy, Atrophy, Leber congenital amaurosis, retinal detachment, glaucoma, optic neuropathy and trauma.
The term " cellular therapeutic agent " of the present invention refers to a medicament (US FDA regulation) used for the purpose of treatment, diagnosis and prevention of cells and tissues prepared by isolation, cultivation and special manipulation from an individual, Diagnosis, and prevention through a series of actions, such as selecting a living individual, allogeneic or xenogeneic cell in vitro, or otherwise altering the biological characteristics of the cell, in order to restore the function of the tissue .
In the present invention, the cell therapy agent can be interpreted as a cell therapy agent capable of regenerating the retinal pigment epithelium which is a cause of the above-mentioned retinal disease, including mesenchymal stem cells treated with the composition for inducing differentiation of retinal pigment epithelium have.
The cell treatment agent of the present invention may further comprise an appropriate carrier, excipient or diluent conventionally used for maintaining the activity of the mesenchymal stem cell, which is a non-naturally occuring carrier, . ≪ / RTI > Specifically, the cell therapeutic agent can be formulated and used in the form of a composition for implantation for treating a retinal disease. In the present invention, the carrier, excipient and diluent which can be contained in the cell therapeutic agent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
The composition comprising the miR-410 inhibitor according to the present invention can differentiate mesenchymal stem cells into retinal pigment epithelium without special conditions, and thus can be widely used for the treatment of various retinal diseases due to damage of the retinal pigment epithelium .
FIG. 1A is a diagram showing the results of uncovering microRNAs showing different expression levels for each tissue. FIG.
FIG. 1B shows the result of performing target prediction analysis using three microarray target prediction programs (TargetScan, miRanda and DIANA) targeting 21 kinds of microRNAs and 14 genes found in Example 1-1 Graph.
FIG. 1C is a graph showing the results of quantitative analysis of expression levels of miR-410 expressed from UCB-MSC, miR-410 inhibitor-treated UCB-MSC and positive control group ARPE-19 cells in the negative control group to be.
2A is a schematic diagram illustrating the overall flow of a method for inducing RPE differentiation using a UCB-MSC.
FIG. 2B is a photograph showing the results of real-time RT-PCR analysis of expression levels of RPE-specific markers expressed in RPE-like cells differentiated from UCB-MSCs.
FIG. 2C is a photograph showing the results of Western blot analysis of expression levels of RPE-specific markers expressed in RPE-like cells differentiated from UCB-MSCs.
FIG. 3A shows the results of immunofluorescence staining for the level of EMMPRIN or vestropine, an RPE marker expressed in RPE-like cells and ARPE-19 cells obtained by culturing for 9 weeks using UCB-MSC and two factors It is a photograph which shows.
FIG. 3B is a photograph showing the results of immunoprophoresis analysis of the level of EMMPRIN or vestropine, an RPE-like RPE marker expressed in RPE-like cells obtained by treating miR control and miR-410 inhibitor and inducing differentiation to be.
FIG. 3C shows the ratio of positive cells expressing EMMPRIN or vestropin in RPE-like cells (anti-miR-410) and ARPE-19 cells obtained by treating and differentiating UCB-MSC and miR-410 inhibitor FIG.
FIG. 4A is a photograph showing the results of analysis of phagocytic activity of RPE-like cells (anti-miR-410) obtained by treating and inducing differentiation of UCB-MSC or miR-410 inhibitor. The size bar of the top photograph shows 50 탆 and the bottom bar shows 5 탆.
4B is a graph showing the results of quantitative analysis of the phagocytic activity of RPE-like cells (anti-miR-410) or ARPE-19 cells obtained by treating and differentiating UCB-MSC, miR-410 inhibitor.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
Example One: UCB - At MSC Expressed RPE Analysis of microRNAs affecting differentiation induction
Example 1-1: UCB - At MSC Expressed RPE Identification of microRNAs that influence differentiation induction
In order to identify candidate microRNAs expressed at a high level in UCB-MSC and targeting RPE development related genes, expression level is decreased in human retinal tissue cells and human RPE cell line ARPE-19 cells, and UCB Microarray analysis was performed to identify microRNAs with increased expression levels in MSCs. Approximately, each of the above cells was subjected to microarray analysis using TRIzol wool to obtain respective total RNAs, and the obtained total RNAs were applied to a Human microRNA Microarray (G4851a; Agilent Technologies, USA). The obtained data was analyzed using GeneSpring GX software (version 11.5.1). As a result, 51 microRNAs expressing low expression levels in retinal tissue cells and high expression levels in UCB-MSC were found, while low expression levels were found in ARPE-19 cells and high expression levels in UCB-MSC Twenty-eight microRNAs were isolated, and 21 microRNAs expressing low expression levels in retinal tissue and ARPE-19 cells, but high expression levels in UCB-MSC were excavated (FIG. 1A and Table 1). FIG. 1A is a diagram showing the results of uncovering microRNAs showing different expression levels for each tissue. FIG.
hsa-miR-127-3p
hsa-miR-136
hsa-miR-146a
hsa-miR-199a-5p
hsa-miR-214
hsa-miR-224
hsa-miR-299-5p
hsa-miR-337-5p
hsa-miR-34a
hsa-miR-376a
hsa-miR-376c
hsa-miR-377
hsa-miR-379
hsa-miR-381
hsa-miR-409-3p
hsa-miR-410
hsa-miR-424
hsa-miR-654-3p
hsa-miR-758
hsa-miR-762
2.29
5.64
4.27
4.86
2.17
3.94
2.09
3.21
56.10
5.05
6.35
4.02
3.16
3.00
2.46
2.06
6.44
2.84
2.18
5.00
3.96
5.49
11.59
8.63
10.55
2.22
3.71
3.24
2.39
6.10
8.13
5.72
3.53
3.05
2.81
2.57
7.20
3.56
2.32
6.27
As shown in Table 1, it was confirmed that all of the 21 microRNAs that were uncovered were expressed at a higher level in UCB-MSC than in retinal cells and ARPE-19 cells. For example, it was found that miR-410 is expressed in UCB-MSC at a level of about two times that expressed in retinal cells and about 2.5 times that expressed in ARPE-19 cells.
Example 1-2: Target Prediction Analysis of Excised MicroRNAs
In order to determine which protein acts as a target of the 21 kinds of microRNAs discovered in Example 1-1, the 21 microRNAs and 14 genes were subjected to three microarray target prediction programs TargetScan, miRanda, and DIANA) (Fig. 1B). Three types of RPE-specific transcription factors (OTX2, MITF and PAX6), three visual circuit-specific genes (RPE65, LRAT and CRALBP), three membrane-associated protein genes (BEST1, PEDF and ZO-1), four predation-specific genes (MERTK, GULP1, LAMP2 and VDP) and one pigment synthesis-specific gene were used.
FIG. 1B shows the result of performing target prediction analysis using three microarray target prediction programs (TargetScan, miRanda and DIANA) targeting 21 kinds of microRNAs and 14 genes found in Example 1-1 Graph. As shown in FIG. 1B, it was found that 21 kinds of microRNAs act on different genes as targets.
Among the 14 genes, in order to select microRNAs that serve as a common target of RPE-specific transcription factors and visual circuit-specific genes, OTX2, an RPE-specific transcription factor, and a visual cycle-specific gene Target prediction analysis was performed on RPE65 (Table 2).
hsa-miR-127-3p
hsa-miR-136
hsa-miR-146a
hsa-miR-199a-5p
hsa-miR-214
hsa-miR-224
hsa-miR-299-5p
hsa-miR-337-5p
hsa-miR-34a
hsa-miR-376a
hsa-miR-376c
hsa-miR-377
hsa-miR-379
hsa-miR-381
hsa-miR-409-3p
hsa-miR-410
hsa-miR-424
hsa-miR-654-3p
hsa-miR-758
hsa-miR-762
×
×
×
×
×
×
×
×
×
×
×
○
×
×
×
○
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
○
×
×
×
×
As shown in Table 2, miR-410 was selected as a microRNA that serves as a common target of RPE-specific transcription factor OTX2 and visual circuit-specific gene RPE65.
Therefore, it was analyzed that miR-410 is closely related to RPE differentiation induction.
Example 1-3: In various cells miR Analysis of expression level of -410
MSC, miR-410 inhibitor treated UCB-MSC, and positive control, ARPE-19 cells were tested in order to confirm whether the expression level of miR-410 was associated with RPE differentiation in UCB-MSC. Expression levels of miR-410 expressed from these were analyzed by quantitative RT-PCR (Fig. 1C). At this time, as a miR-410 inhibitor, a single-stranded polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence constituting miR-410 was used.
FIG. 1C is a graph showing the results of quantitative analysis of expression levels of miR-410 expressed from UCB-MSC, miR-410 inhibitor-treated UCB-MSC and positive control group ARPE-19 cells in the negative control group to be. As shown in FIG. 1C, miR-410 is expressed at a relatively low level in ARPE-19 cells, which are a kind of RPE cells, and miR-410 is expressed at a relatively high level in undifferentiated UCB-MSCs. In UCB-MSC treated with inhibitor, miR-410 was expressed at a level higher than that of UCB-MSC but higher than that of ARPE-19 cells.
From these results, it was analyzed that the expression level of miR-410 was suppressed when differentiation to RPE was induced.
Example 2: UCB - At the MSC miR -410 inhibition of expression RPE Analysis of effects on differentiation
According to the results of the above Example 1 and a known method, UCB-MSCs were differentiated into RPE cells in two ways.
One was to cultivate UCB-MSC at 80% saturation, then treat 100 ng / ml Actin A and 10 mM nicotinamide in medium and change media every 3 days for 9 weeks (2F-RPE-like cells). On the other hand, UCB-MSCs were cultured at 80% saturation and cultured for 3 weeks to obtain 0, 3, 10 and 17 At the time point, the miR-410 inhibitor was treated and differentiated into RPE-like cells (miR-RPE-like cells) (Fig. 2a).
2A is a schematic diagram illustrating the overall flow of a method for inducing RPE differentiation using a UCB-MSC.
Next, RPE-specific marker analysis was performed by real-time RT-PCR on RPE-like cells induced by the above two methods (FIG. 2B). RPE-like, miR-410 inhibitors obtained by using UCB-MSCs, miR control and ARPE-19 cells as the control group and culturing for 9 weeks using the two factors as experimental groups RPE-like (2 days), RPE-like (21 days) obtained by treating miR-410
FIG. 2B is a photograph showing the results of real-time RT-PCR analysis of expression levels of RPE-specific markers expressed in RPE-like cells differentiated from UCB-MSCs. As shown in FIG. 2B, RPE-like cells differentiated from UCB-MSC showed increased expression levels of RPE-specific markers such as MITF, bestropine and EMMPRIN.
RPE-specific marker analysis was also performed by Western blot analysis on RPE-like cells induced by the above two methods (FIG. 2C). RPE-like cells (2F-RPE-like) and miR-410 inhibitors obtained by using UCB-MSCs and ARPE-19 cells as control groups and culturing for 9 weeks using two factors were treated and induced to differentiate The obtained RPE-like cells (miR-RPE-like) were used. Best RPE and EMMPRIN were used as RPE-specific markers and GAPDH was used as an internal control.
FIG. 2C is a photograph showing the results of Western blot analysis of expression levels of RPE-specific markers expressed in RPE-like cells differentiated from UCB-MSCs. As shown in FIG. 2C, RPE-specific RPE-like cells differentiated from UCB-MSC showed increased levels of RPE-specific markers such as bestropine and EMMPRIN.
Thus, when miC-410 inhibitor was treated with UCB-MSC, differentiation into RPE was induced.
Example 3: UCB - From MSC Differentiated RPE Characterization of pseudo-cells
Example 3-1: Immunofluorescent staining analysis
First, RPE-like cells and ARPE-19 cells obtained by culturing for 9 weeks using the two factors of the cultured UCB-MSC were treated with 4% formaldehyde for 10 minutes to fix the cells. Then, the cells were fixed with 0.05% Triton X-100 was treated for 10 minutes to perforate cell membranes of each fixed cell. Then, each perforated cell was treated with 5% normal goat serum at room temperature for 1 hour to block, treated with antibody to EMMPRIN or antibody to bestropine, and reacted at 4 캜. After completion of the reaction, the fluorescence-stained EMMPRIN or the level of the bestropine was measured after treatment with a fluorescently labeled secondary antibody (goat anti-mouse Alexa fluor 488; Invitrogen) diluted 1: 1000 3a). At this time, nuclei were counterstained with DAPI.
FIG. 3A shows the results of immunofluorescence staining for the level of EMMPRIN or vestropine, an RPE marker expressed in RPE-like cells and ARPE-19 cells obtained by culturing for 9 weeks using UCB-MSC and two factors It is a photograph which shows. As shown in FIG. 3A, the fluorescence staining level of EMMPRIN or bestropine was lowest in UCB-MSC, highest in ARPE-19 cells, and intermediate in RPE-like cells.
Next, RPE-like cells and ARPE-19 cells obtained by culturing the cultured UCB-MSC using the two factors for 9 weeks, RPE-like cells obtained by treating miR control and miR-410 inhibitor and inducing differentiation RPE-like) was used (Fig. 3B).
FIG. 3B is a photograph showing the results of immunoprophoresis analysis of the level of EMMPRIN or vestropine, an RPE-like RPE marker expressed in RPE-like cells obtained by treating miR control and miR-410 inhibitor and inducing differentiation to be. As shown in FIG. 3B, the fluorescence staining level of EMMPRIN or bestropine was lowest in UCB-MSC, highest in ARPE-19 cells, and intermediate in RPE-like cells.
Finally, the proportion of positive cells expressing EMMPRIN or vestropin in RPE-like cells (anti-miR-410) and ARPE-19 cells obtained by culturing and inducing differentiation of cultured UCB-MSC and miR- (Fig. 3C).
FIG. 3C shows the ratio of positive cells expressing EMMPRIN or vestropin in RPE-like cells (anti-miR-410) and ARPE-19 cells obtained by treating and differentiating UCB-MSC and miR-410 inhibitor FIG. As shown in FIG. 3C, EMMPRIN and bestropine showed different patterns. The ratio of EMMPRIN-positive cells was the lowest in UCB-MSC, the highest in ARPE-19 cells and the median value of RPE-like cells, while the bestropine was highest in RPE-like cells Respectively.
Example 3-2: Phytogenic activity analysis
Since RPE cells are known to exhibit excellent phytotoxic activity, in order to confirm whether the RPE-like cells differentiated in the present invention exhibit phagocytosis activity, phagocyte activity analysis was performed using fluorescence labeled microspheres.
Specifically, RPE-like cells (anti-miR-410) obtained by culturing and inducing differentiation of cultured UCB-MSC or miR-410 inhibitor were coated with 1 X 10 9 FluoSpheres Carboxylate-Modified Microspheres , red fluorescent 580/605 (Invitrogen)) was added and reacted at 37 ° C for 18 hours. After completion of the reaction, the fluorescent microspheres were removed, and 1 ml of FBS was added to the cells, followed by reaction at 37 ° C for 15 minutes. After the reaction was completed, each cell was washed with PBS, the nuclei were counterstained with Hoechst for 10 minutes, and then photographed with a confocal microscope and quantitatively analyzed (FIGS. 4A and 4B). At this time, ARPE-19 cells were used as a control group for quantitative analysis.
FIG. 4A is a photograph showing the results of analysis of phagocytic activity of RPE-like cells (anti-miR-410) obtained by treating and inducing differentiation of UCB-MSC or miR-410 inhibitor. The size bar of the top photograph shows 50 탆 and the bottom bar shows 5 탆. As shown in FIG. 4A, the undifferentiated UCB-MSC absorbed the fluorescent microspheres to a low level, but the RPE-like cells (anti-miR-410) showed high absorption of the fluorescent microspheres.
4B is a graph showing the results of quantitative analysis of the phagocytic activity of RPE-like cells (anti-miR-410) or ARPE-19 cells obtained by treating and differentiating UCB-MSC, miR-410 inhibitor. As shown in FIG. 4B, the UCB-MSC exhibited a low absorption rate of the fluorescent microspheres, whereas the RPE-like cells (anti-miR-410) exhibited the absorption rate of the fluorescent microspheres in the ARPE- It was found that the level was the same.
Therefore, when miC-410 inhibitor was treated with UCB-MSC through phylogenetic analysis, it was found that RPE differentiation was induced.
<110> SNU R & DB FOUNDATION
Kang Stem Biotech CO., LTD.
<120> Composition for inducing differentiation to retinal pigment
epithelium
<130> KPA151096-KR
<160> 1
<170> Kopatentin 2.0
<210> 1
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<223> miR-410
<400> 1
Claims (12)
Wherein said miR-410 is a microRNA consisting of the nucleotide sequence of SEQ ID NO: 1.
Wherein said composition is cultured in mesenchymal stem cells and treated at a time when 0, 3, 10 and 17 days have elapsed.
Wherein said mesenchymal stem cells are cord blood-derived mesenchymal stem cells.
Wherein the retinal pigment epithelium expresses a relatively high level of EMMPRIN or bestropine, which is a marker of the retinal pigment epithelium, and a relatively high level of phagocytosis, compared to mesenchymal stem cells.
Wherein the retinal pigment epithelium is formed at 21 days after inducing differentiation from mesenchymal stem cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150150366A KR101982801B1 (en) | 2015-10-28 | 2015-10-28 | Composition for inducing differentiation to retinal pigment epithelium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150150366A KR101982801B1 (en) | 2015-10-28 | 2015-10-28 | Composition for inducing differentiation to retinal pigment epithelium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20170049775A KR20170049775A (en) | 2017-05-11 |
| KR101982801B1 true KR101982801B1 (en) | 2019-05-30 |
Family
ID=58740994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150150366A KR101982801B1 (en) | 2015-10-28 | 2015-10-28 | Composition for inducing differentiation to retinal pigment epithelium |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101982801B1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL305070A (en) | 2014-12-30 | 2023-10-01 | Cell Cure Neurosciences Ltd | Rpe cell populations and methods of generating same |
| BR112017014332A2 (en) * | 2014-12-30 | 2018-01-02 | Cell Cure Neurosciences Ltd | retinal disease treatment methods |
| JP7264382B2 (en) * | 2018-05-17 | 2023-04-25 | オリーブス バイオセラピューティクス インコーポレイテッド | Pharmaceutical composition containing CCN5 as an active ingredient for preventing or treating retinal diseases |
| KR102262206B1 (en) | 2019-01-16 | 2021-06-07 | 전북대학교산학협력단 | Hydrogel composition comprising retinal pigment epithelium cells and use of the same |
| KR20220051791A (en) | 2020-10-19 | 2022-04-26 | 주식회사 시그넷바이오텍 | Composition for preventing or treating macular degeneration comprising Fas signaling-blocking peptide |
| KR20230129667A (en) * | 2022-03-02 | 2023-09-11 | 서울대학교병원 | Composition containing exosomes extracted from mesenchymal stem cells for preventing or treating retinal degeneration disease |
-
2015
- 2015-10-28 KR KR1020150150366A patent/KR101982801B1/en active IP Right Grant
Non-Patent Citations (1)
| Title |
|---|
| Choi SW, et al. Stem Cell Rev. 2015 Jun* |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20170049775A (en) | 2017-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101982801B1 (en) | Composition for inducing differentiation to retinal pigment epithelium | |
| Catala et al. | Approaches for corneal endothelium regenerative medicine | |
| CN101583371B (en) | RNAi methods and compositions for stimulating proliferation of cells with adherent junctions | |
| CN104039954B (en) | The anti-tumor method of inducing differentiation of iPS cells can be suppressed | |
| He et al. | Stem cell-based therapy for treating limbal stem cells deficiency: A review of different strategies | |
| Wongvisavavit et al. | Challenges in corneal endothelial cell culture | |
| Maqueda et al. | Repopulation of decellularized retinas with hiPSC-derived retinal pigment epithelial and ocular progenitor cells shows cell engraftment, organization and differentiation | |
| Li et al. | Long-term corneal recovery by simultaneous delivery of hPSC-derived corneal endothelial precursors and nicotinamide | |
| CN111500578A (en) | Circ RNA-FTO for regulating and controlling osteogenic differentiation and tissue regeneration of ADSCs and application thereof | |
| WO2018235899A1 (en) | Method for culturing muscle satellite cells | |
| CN109486766B (en) | Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell | |
| Yam et al. | Human corneal stromal stem cells express anti-fibrotic microRNA-29a and 381-5p–A robust cell selection tool for stem cell therapy of corneal scarring | |
| KR20120002134A (en) | Method for dedifferentiations of adipose tissue stromal cells | |
| Kharitonov et al. | Possibilities for using pluripotent stem cells for restoring damaged eye retinal pigment epithelium | |
| Zhang et al. | Effects of RPE‑conditioned medium on the differentiation of hADSCs into RPE cells, and their proliferation and migration | |
| US9896657B2 (en) | Method of inducing differentiation of stem cell into corneal limbal stem cell | |
| Singh et al. | Recent and evolving therapies in the management of endothelial diseases | |
| KR102417981B1 (en) | Artificial scaffold for corneal endothelial cell transplantation, manufacturing method thereof, and ultra-thin descemet stripping endothelial graft using the same | |
| Hills et al. | Neurite Outgrowth and Gene Expression Profile Correlate with Efficacy of Human Induced Pluripotent Stem Cell-Derived Dopamine Neuron Grafts | |
| US20230092155A1 (en) | Chondrocyte culture with high tissue regeneration ability | |
| CN111019884B (en) | Method for inhibiting epithelial-mesenchymal transition of human amniotic epithelial cells | |
| JP2022042998A (en) | Cell sheet pieces, syringe containing cell sheet pieces, method for producing cell sheet pieces, and method for using the same | |
| JP2022550911A (en) | Chondrogenic human mesenchymal stem cell (MSC) sheet | |
| Mingo-Botín et al. | Corneal endothelium: isolation and cultivation methods | |
| US20220025325A1 (en) | Generation and cryopreservation of pluripotent stem cell-derived clinical grade corneal endothelial cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant |