KR20190021885A

KR20190021885A - Method for differentiation of dental epithelium from human induced pluripotent stem cells and use thereof

Info

Publication number: KR20190021885A
Application number: KR1020170107264A
Authority: KR
Inventors: 장미; 이진; 김지혜; 양지혜
Original assignee: 서울대학교산학협력단
Priority date: 2017-08-24
Filing date: 2017-08-24
Publication date: 2019-03-06
Also published as: WO2019039900A1; KR101957034B1

Abstract

The present invention relates to a method for differentiating human dental-derived pluripotent stem cells into dental herpetiform stem cells, dental herpetiform stem cells differentiated by the method, and uses thereof. The method of differentiating into dental herniated stem cells according to the present invention can establish an autologous cell treatment system by using patient-customized inducible pluripotent stem cells which can be established from somatic cells of a patient in need of tooth regeneration, And it is expected to be usefully used in the field of tooth regeneration medicine.

Description

TECHNICAL FIELD The present invention relates to a method for differentiating dental epithelial cells from human derived pluripotent stem cells,

The present invention relates to a method for differentiating human induced pluripotent stem cells into dental heritable stem cells, dental herpetiform stem cells differentiated by the method, and uses thereof.

Teeth are an organ that has not yet been correctly identified for its mechanism of formation and regeneration due to its non-regenerating properties and complicated development processes. However, it has been reported that interactions between mesenchymal stem cells and epithelial stem cells play a major role in tooth development. Therefore, it is considered as a major issue to stably obtain a large amount of the above two cells for various subsequent studies on tooth regeneration. To date, many studies have been carried out by securing the cells from missing or permanent teeth, but there are technical difficulties in securing and culturing the cells. In particular, tooth epithelial stem cells are difficult to isolate and secure, and thus there are fewer studies than mesenchymal stem cells. Previous studies have mainly used epithelial stem cells derived from non-human animals and human-derived epithelial stem cells Even when cells are initially cultured, the cells can not be uniformly supplied to the experiment.

Induced pluripotent stem cells are known to have an advantage in establishing various cell origins as an important material of autoimmune-cell therapy. In addition, it has been reported that the tooth-derived cells have higher efficiency of establishment of induced pluripotent stem cells than other tissues, and since the teeth can be banked, it is possible to obtain induced pluripotent stem cells, It is expected to be used as a material. However, the differentiation of dendritic cells into pluripotent stem cells using inducible pluripotent stem cells has been mainly based on animal model derived cell lines (Tissue Engineering Part A 18.15-16 (2012): 1677-1685) In this study, the culture media derived from animal stem cells were widely used, and the need for research using human - derived cells is emerging.

In order to solve the above conventional problems, the present inventors used human dental-derived pluripotent stem cells to differentiate into dental herpetoid stem cells by using established dental herbal stem cell lines as support cells, The present inventors have completed the present invention by developing a technique capable of stably supplying a human-derived dental implantable stem cell line.

Accordingly, it is an object of the present invention to provide a method of differentiating into dendritic stem cells from the pluripotent stem cells and a dental herpetiform stem cell differentiated by the differentiation method.

It is another object of the present invention to provide a cell therapy agent for regeneration of teeth, which comprises the dental herbal stem cells.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention as described above, the present invention provides a method for inducing pluripotent stem cells in induced pluripotent stem cells, which comprises inducing and culturing induced pluripotent stem cells on support cells, Thereby providing a method of differentiating into stem cells.

In addition, the present invention provides a dental heritable stem cell differentiated by the differentiation method.

In one embodiment of the present invention, the induced pluripotent stem cells may be those prepared from human-derived mesenchymal stem cells.

In another embodiment of the present invention, the supporting cell may be HERS / ERM (Hertwig's Epithelial Rests of Malassez).

In another embodiment of the present invention, the supporting cells may be treated with mitomycin C.

In another embodiment of the present invention, the induced pluripotent stem cells may be cultured on the supporting cells for 7 days to 21 days.

In another embodiment of the present invention, the differentiation method may further include a step of culturing the inducible pluripotent stem cells on support cells, followed by subculturing.

In addition, the present invention provides a cell therapeutic agent for regenerating a tooth, which comprises the differentiated dental heritable stem cell.

The present inventors have established a technique for differentiating human-derived induced pluripotent stem cells into dental stem cells by using a dental herbal stem cell line as a supporting cell as a new method for obtaining dental herbal stem cells which are difficult to separate and secure And differentiated dendritic stem cells were obtained by the above method, and immortalized to obtain dental herniated stem cell lines. Therefore, the method of differentiating into dental blood stem cells according to the present invention can establish an autologous cell treatment system by using patient-customized induced pluripotent stem cells which can be established from somatic cells of a patient in need of dental regeneration, Derived stem cells can be supplied, and thus it is expected to be usefully used in the field of dental regenerative medicine.

FIG. 1 shows a protocol for differentiation of human dental-derived pluripotent stem cells into dental herpetoid stem cells and differentiated iPSCs differentiated according to the protocol and epithelial-like cells obtained by subculturing them ). &Lt; / RTI >
FIG. 2 shows the characteristics of the epithelial-like cells differentiated according to the differentiation protocol. FIG. 2A shows a comparison of the morphology of the epithelial cells before and after subculture, FIG. 2b is a photograph of the epithelial stem cell HSP-SV40 used as a supporting cell in the iPSC-derived Epi-like cell and the differentiated induced pluripotent stem cells, ( Oct4 , Nanog , and Sox2 ), and FIG. 2c shows the results of comparing the expression patterns of the cell-related genes ( E-cadherin , ABCG2 , EpCAM , Bmi1 , p63 , and p75 ) FACS analysis was performed to determine the effects of mesenchymal stem cell markers (CD10 and CD29), vascular endothelial cell markers (CD31), hematopoietic stem cell markers (CD45 and HLA-DR), and human MHC marker (HLA- By comparing the level of expression Nancy is the result.
FIG. 3 shows the results of immortalization of the epithelial-like cells differentiated according to the differentiation protocol of FIG. 1 and the characteristics of the immortalized epithelial-like cell line. FIG. 3 a shows the characteristics of epithelial-like cells differentiated from human tooth- FIG. 3B is a photomicrograph showing the protocol before the transfection and after transfection of the pcDNA 3.1 (+) SV40 into the differentiated epithelial-like cells. Immortalized Epi-like cells and primary cultured mesenchymal stem cells (SHED) cultured in three passages were subjected to immunohistochemical staining to confirm the presence or absence of SV40. Fig. 3c shows the results of immortalization (Passage 1 to 20) of the epithelial-like cell line, and Fig. 3d shows the cell growth ability of the epithelial- All.
FIG. 4 is a result of microsatellite analysis of HERS-SV40 and Immortalized Epi-like cell to verify that the immortalized epithelial-like cell line originated from induced pluripotent stem cells.
Figure 5 is an analysis of the characteristics of the immortalization were epithelium similar cell line results, Figure 5a is HERS-SV40 and the epithelium similar cell lines (Immortalized Epi-like cell) epithelial stem cell gene subjected to RT-PCR with respect to (E -cadherin, ABCG2, EpCAM, Bmi1, p63, and p75) and the stem cells is the result of comparing the expression patterns of genes (Oct4, Nanog, and Sox2), Figure 5b is subjected to FACS analysis for the two cell The expression levels of mesenchymal stem cell markers (CD10 and CD29), vascular endothelial cell markers (CD31), hematopoietic stem cell markers (CD45 and HLA-DR), and human MHC marker (HLA-1) Respectively.
FIG. 6 shows the results of analysis of epithelial-mesenchymal transition (EMT) after treatment of HERS-SV40 and immortalized Epi-like cells with TGF- FIG. 6B is a micrograph showing the changes of cell morphology after 48 hrs treatment with TGF-β1 and E-cadherin , respectively, by real-time PCR on the two cell lines. MRNA expression level of the mesenchymal stem cell marker N-cadherin and Vimentin gene.

The present inventors have completed the present invention by establishing a technique for differentiating human induced pluripotent stem cells into dental herpetiform stem cells as a new method for obtaining dental herbal stem cells which are difficult to separate and secure.

Accordingly, the present invention provides a method for differentiating into pluripotent stem cells in an inducible pluripotent stem cell, wherein the pluripotent stem cells are induced by culturing induced pluripotent stem cells on support cells.

As used herein, the term " differentiation " refers to a process in which stem cells in an undifferentiated state at an early stage have characteristics as respective tissues. For the purpose of the present invention, It has a characteristic as a cell.

As used herein, the term " dental implanted stem cell " refers to a cell capable of differentiating into ameloblast and / or dentate germ cells for tooth formation.

As used herein, the term " induced pluripotent stem cell (iPSC) " refers to an embryonic stem (ES) cell that has been introduced into somatic cells differentiated as degenerated stem cells It is a cell that induces pluripotency like a cell. In the present invention, the type of tissue is not particularly limited as long as the pluripotent pluripotent stem cell is derived from a cell or a human obtained by repopulating the mesenchymal stem cells derived from human-derived tissue.

The term " feeder cells " used in the present invention is known to be associated with undifferentiated maintenance mechanisms that secrete nutritional factors that contribute to maintaining the undifferentiated state of embryonic stem cells and mediate cell contact. The signaling substances secreted by the supporting cells contribute to regulate the undifferentiated maintenance or differentiation initiation of embryonic stem cells. The substances secreted from the supporting cells include Wnt (Wingless-type MMTV integration site family), BMPs (Bone Morphogenetic Proteins), TGF-β (Transforming Growth Factor-beta) and extracellular matrix. There have been reports of differentiation into embryonic stem cells or induced pluripotent stem cells using various supporting cells derived from mouse or human. In the present invention, the cell line obtained by immortalization of HERS / ERM, which is the primary cultured stem cell stem derived from human permanent teeth, established by the inventors of the present invention was used as a supporting cell, and the induced pluripotent stem cells But may be, but not limited to, mitomycin C treated.

In the present invention, the inducible pluripotent stem cells may be cultured on the supporting cells for 7 to 21 days, more preferably for 14 days, to differentiate into epithelial-like cells, and then further subcultured have.

The present inventors induced differentiation into dendritic stem cells from induced pluripotent stem cells according to the above-mentioned differentiation method through the examples.

In one embodiment of the present invention, the induction of differentiation from induced pluripotent stem cells derived from human mesenchymal stem cells into dental heritable stem cells was induced according to the differentiation method (see Example 1) Observation of morphology, gene and protein expression patterns of the cells obtained by differentiation through a microscope confirmed that the cells had epithelial cell characteristics (see Example 2).

In another embodiment of the present invention, the SV40 gene was transfected with pcDNA 3.1 (+) SV40 in order to immortalize the dendritic cells and establish it as a cell line. The SV40 gene was introduced into the cells and immunostained SV40 gene was expressed in all cells. It was experimentally confirmed that the SV40 gene can be proliferated with uniform growth over 20 passages while maintaining the morphology of epithelial cells (see Example 3).

In another embodiment of the present invention, it has been confirmed that the immortalized dental herniated stem cells are derived from pluripotent stem cells through microsomal analysis (see Example 4) (See Examples 5-1 and 5-2). As a result of induction of epithelial-mesenchymal transition (EMT) by treatment with TGF-β1, slight cell morphological changes were observed, and epithelial cells It was confirmed that the EMT was induced by confirming the expression change of the markers of mesenchymal stem cells (see Example 5-3).

From the results of the above examples, it was confirmed that the differentiation from human-derived induced pluripotent stem cells into dendritic stem cells was accomplished through the differentiation method according to the present invention.

Accordingly, as another aspect of the present invention, the present invention provides a dental heritable stem cell differentiated by the above differentiation method.

As another embodiment of the present invention, the present invention provides a cell therapy agent for regenerating a tooth comprising the differentiated dental heritable stem cells.

Dental caries, dental caries, attrition, abrasion, erosion, and perioperis are examples of diseases that can be used for the cell therapy agent. However, There is no limit to the type of disease that requires tooth regeneration.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1: Induction of differentiation from human tooth-derived pluripotent stem cells into dendritic stem cells

The present inventors have produced induced pluripotent stem cells from mesenchymal stem cells derived from human dentition to differentiate human dental-derived pluripotent stem cells into dental pulp stem cells. More specifically, Oct-4 , Sox-2 , Klf-4 , L-myc and Lin28 genes were prepared as three episomal vectors in the mesenchymal stem cells and electroporation (Neon-invitrogen) And incubated for 3 days. Then, the cells were placed on SNL-supporting cells and cultured in ESC medium. Subsequently, the colony formed was subcultured on supporting cells to produce an induced pluripotent stem cell line.

The induced pluripotent stem cells were used to induce differentiation into dendritic cells according to the procedure shown in FIG. More specifically, 10 μg / ml of mitomycin C (MMC) (Sigma Aldrich, MO, USA, M4287) was inoculated into the HERS / ERM cell line HERS-SV40, a primary cultured epithelial stem cell derived from permanent teeth, (Gibco, CA, USA, 25200) to separate into single cells and then cultured in a 35 mm culture dish at a density of 1 × 10 ⁶ cells / ml. After 24 hours, iPSC clumps isolated from the undifferentiated state by physical methods were placed on the cultured supporter cell layer and co-cultured for 14 days in KGM-2 (Lonza, MD, USA, CC-3107) Induced differentiation into stem cells. Subsequently, differentiated epithelial cells were treated with Accutase (Merck Millipore, MA, USA, SCR005) to separate into single cells and transferred to a new culture dish and subcultured.

Example 2. Identification of human dental-derived pluripotent stem cells from induced pluripotent stem cells

2-1. Cell morphology observation

In order to observe the morphology of epithelial-like cells differentiated according to the method of Example 1, before and after subculture of subculture and subculture were observed by optical microscope, As shown in FIG. 2A, it was confirmed that the inducible pluripotent pluripotent stem cell clumps were differentiated into epithelial-like cells through co-cultivation with the supporting cells, and the cell morphology was maintained even after the subculture.

2-2. Analysis of gene expression patterns of differentiated epithelial-like cells

In addition to the results of Example 2-1, reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine whether epithelial-like cells differentiated by the method of Example 1 had epithelial characteristics, Respectively. For this, HERS-SV40 supporting cells and the differentiated epithelial-like cells cultured for 14 days after passage were treated with trypsin-EDTA to remove the cells, and total RNA was extracted using RNeasy Mini Kit (Qiagen, Germany, 74106) Respectively. Next, cDNA was synthesized using the extracted RNA as a template using amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, TX, USA, R5600), and the respective genes shown in the following Table 1, specifically the epithelial stem cell- ( E-cadherin , ABCG2 , EpCAM , Bmi1 , p63 , and p75 ) and stem cell-related genes ( Oct4 , Nanog , and Sox2 ). The amplification was carried out at 94 ° C for 2 minutes (T100TM Thermal Cycler, Bio-Rad, CA, each E-cadherin, p63, Nanog, and Sox2 has Tm = from 55 ℃ 35 cycles, Oct4 is Tm = from 57 ℃ 35 cycles, ABCG2, EpCAM , Bmi1, and p75 is Tm = from 60 ℃ 40 cycles, GAPDH is Tm = 60 占 폚. PCR amplification products were then electrophoresed and confirmed on an electrophoretic gel recording apparatus (Bio-profil X-press zoom2000, Germany, Vilber Lourmat).

gene direction The sequence (5'-3 ') SEQ ID NO: E-cadherin Forward TGC CCA GAA AAT GAA AAA GG One Reverse GTG TAT GTG GCA ATG CGT TC 2 ABCG2 Forward CCA CAG GTG GAG GCA AAT CT 3 Reverse TCG CGG TGC TCC ATT TATCA 4 EpCAM Forward GCT GGC CGT AAA CTG CTT TG 5 Reverse ACA TTT GGC AGC CAG CTT TG 6 Bmi1 Forward CAG CCC AGC AGG AGG TAT TC 7 Reverse GGA TGA GGA GAC TGC ACT GG 8 p63 Forward ATG TTG TAC CTG GAA AAC AAT GC 9 Reverse GTG ATG GAG AGA GAG CAT CGAE 10 p75 Forward ACC GAG CTG GAA GTC GAG 11 Reverse CTC ACC GCT GTG TGT GTA C 12 Oct4 Forward ACC CCT GGT GCC GTG AA 13 Reverse GGC TGA ATA CCT TCC CAA ATA 14 Nanog Forward CCT ATG CCT GTG ATT TGT GG 15 Reverse TTC TCT GCA GAA GTG GGT TG 16 Sox2 Forward GAC TTC ACA TGT CCC AGC AC 17 Reverse GGG TTT TCT CCA TGC TGT TT 18 GAPDH Forward GAT GCT GGC GCT GAG TAC G 19 Reverse GCT AAG CAG TTG GTG GTG C 20

As shown in FIG. 2B, the HERS-SV40 cell line and the differentiated iPSC-derived Epi-like cells differentiated from the epithelial stem cell-associated genes E-cadherin , ABCG2 , EpCAM , Bmi1 , p63 , and p75, and stem cell related genes, Oct4 , Nanog , and Sox2 . It was confirmed that the epithelial-like cells differentiated from the above-described results exhibited epithelial cell characteristics.

2-3. Analysis of protein expression patterns of differentiated epithelial-like cells

In order to analyze the protein expression pattern in addition to the gene expression analysis result of Example 2-2, FACS analysis was performed to observe the expression level of the cell marker. To this end, the HERS-SV40 cell line and iPSC-derived Epi-like cells (iPSC-derived Epi-like cells) were treated with trypsin-EDTA to separate into single cells, Formaldehyde (Junsei, Japan, 69360-0380) was treated and cells were fixed at room temperature for 10 minutes. (BD Bioscience, CA, USA, 555443). The antibody and the treatment ratio used in the above were as follows: < tb > (BD Biosciences, 559866) 1: 100, CD45-FITC (eBioscience, CA, USA, 11-0459-73) 1: 100, HLA-DR-APC : 100, CD31-FITC (BD Bioscience, 555445) 1: 100, HLA-I-FITC (eBioscience, 11-9983-42) 1:50. Cell surface antigen analysis was performed on 10,000 cells per each antibody using FACS Callibur (Becton Dickson, CA, USA) and the results were analyzed using BD CellQuest Pro software.

As a result, as shown in FIG. 2C, the two cells showed the same protein expression pattern. More specifically, CD29 was mostly expressed in the mesenchymal stem cell marker but CD10 was not expressed. The hematopoietic stem cell markers CD45 and HLA -DR and CD31, a vascular endothelial cell marker, were not expressed, whereas HLA-I, a human MHC marker, was expressed in most of the cells. It was confirmed that the epithelial-like cells differentiated from the above-described results exhibited epithelial cell characteristics.

Example 3 Induction and Identification of Dendritic Blood Stem Cells Differentiated from Human Dental Derived Derivative Stem Cells

3-1. Immortalization induction experiment of differentiated stromal stem cells

The present inventors intend to immortalize dental stem cells obtained by differentiating from human dental-derived pluripotent stem cells through the method of Example 1 to establish them as cell lines. For this, as shown in FIG. 3A, the inducible pluripotent stem cells were differentiated into epithelial-like cells in the same manner as in Example 1, and then 1.5 × 10 ⁵ cells were seeded on a 35 mm culture dish and 50% confluency . The X-tremeGENE 9 DNA transfection reagent (Roche, IN, USA, 06365787001) was then mixed with pcDNA 3.1 (+) SV40 and transfected into the epithelial-like cells to introduce the SV40 gene. Cells transfected with pcDNA3.1 (+) SV40 were treated for 14 days after treatment with 100 μg / ml G418 (Cellgro Mediatech, DC, USA, 61-234-RF) As a result of the microscopic observation, it was confirmed that the shape of the cells before and after After transfection was not changed as shown in Fig. 3A.

3-2. Immortalization of epithelial-like cells

Immunostaining was carried out to examine whether the epithelial-like cells immortalized by introducing the SV40 gene by the method of Example 3-1 express the transgene. To this end, the epithelial-like cell line that had been passaged three times (Passage 3) was treated with 4% paraformaldehyde (Tech & Innovation, Korea, BPP-9004) to fix the cells for 10 minutes. At this time, SHED (Stem Cells from Human Exfoliated Deciduous Teeth), a primary cultured mesenchymal stem cell derived from Passage 3, was used as a comparative group. Rabbit polyclonal SV40 primary antibody (Santa Cruz Biotechnology, CA, USA, SC-20800) was diluted at a ratio of 1:50 and treated at room temperature for 1 hour. The secondary antibody, Alexa Fluor 488 Goat (Sigma-Aldrich, D9542) at a ratio of 1: 700, and stained with 1: 1000. The cells were stained with an inverted fluorescence microscope (Nikon Eclipse , Tokyo, Japan, TE2000-U) were used for immunofluorescence staining.

As a result, as shown in FIG. 3B, the SV40 was not expressed in the comparative group (SHED), whereas the immortalized epithelial-like cell line showed SV40 expression in all cells.

Based on the above results, long-term culture was attempted to confirm the characteristics of the immortalized epithelial-like cell line. As a result, it was confirmed that the epithelial cell shape was maintained and proliferated stably over 20 passages (P20) as shown in Fig. 3C.

In addition, the number of cells was measured by subculture every 3 days to analyze the cell growth potential of the immortalized epithelial-like cell lines. Specifically, the cells were treated with trypsin-EDTA on the third day of subculture, and subcultured in a 35 mm culture dish at a density of 1.5 × 10 ⁴ cells / cm ² . After 24 hours, the medium was replaced with fresh medium. After 2 days, the above procedure was repeated to analyze the growth curve using cell numbers. As a result, it was confirmed that the immortalized epithelial-like cell line showed uniform growth over 50 days as shown in Fig.

Example 4. Identification of Immortalized Epithelial Cell Lines

The HERS-SV40 cell line, induced pluripotent stem cell (iPSC), and immortalized epi-like cell line were used to verify that the origin of the immortalized epithelial-like cell line was the inducible pluripotent stem cell according to the method of Example 3 above. Microsatellite analysis was performed for each. More specifically, genomic DNA was extracted from each of the above three cells using a LaboPass ^™ Tissue mini prep kit (COSMO genetech, Korea, CME0112), and then microsphage analysis was performed on Macrogen. The microsomes were analyzed using Hi-Di formamide (Applied Biosystems, CA, USA) and GeneScan ^TM 500 LIZ ^® Size Standard (Applied Biosystems) and analyzed using a 3730XL DNA analyzer (Applied Biosystems).

As a result, as shown in Fig. 4, 15 microsatellite loci (chromosome 8 (D8S1179), 21q11.2 to q21 (D21S11), 7q11.21 to 22 (D7S820), 5q33.3 to 34 (13), 12p12 ~ 13 (D13S317), 16q24 ~ qter (D16S539), 2q35 ~ 37.1 (D2S13380, 19q12 ~ 13.1 (D19S433), 12p12 ~ pter ) And amelogenin loci on the sex chromosome (X, Y) were identified from the 2p23 to 2per (TPOX), 19q21.3 (D18S51), 5q21 to 31 (D5S818), and 4q281 . The result of the HPS-SV40 cell line used as a supporting cell in the present invention was inconsistent with the results of the iPSC and the epithelial cell line immortalized in all the loci confirmed. .

Example 5. Characterization of Immortalized Epithelial Cell Lines

5-1. Analysis of gene expression patterns of immortalized epithelial-like cell lines

In order to verify whether the immortalized epithelial-like cell line of the inducible pluripotent stem cell origin examined in Examples 3 and 4 exhibited epithelial cell characteristics, RT-PCR was carried out in the same manner as in Example 2-2, The expression pattern of the gene was observed.

As shown in FIG. 5A, the HERS-SV40 cell line and the immortalized epi-like cell line derived from the inducible pluripotent stem cell ( ESC ) cell line express E-cadherin , ABCG2 , EpCAM , Bmi1 , p63 , And p75 and the stem cell related genes Oct4 , Nanog , and Sox2 showed similar expression patterns, indicating that the immortalized epithelial cell line exhibited epithelial cell characteristics.

5-2. Protein expression pattern of immortalized epithelial-like cell line

In order to analyze the protein expression pattern in addition to the results of Example 5-1 at all times, the HERS-SV40 cell line and the immortalized epi-like cell line were subjected to FACS Analysis.

As a result, as shown in FIG. 5B, the two cells showed the same protein expression patterns. More specifically, CD29 was mostly expressed in the mesenchymal stem cell marker, but CD10 was not expressed, and hematopoietic stem cell markers CD45 HLA-DR and CD31, a vascular endothelial cell marker, were not expressed while HLA-I, a human MHC marker, was expressed in most of the cells. It was confirmed that the epithelial-like cells differentiated from the above-described results exhibited epithelial cell characteristics.

5-3. EMT analysis of immortalized epithelial-like cell lines

In addition to the analysis of the gene expression and protein expression pattern of the immortalized epithelial-like cell line, the epithelial-mesenchymal transition (EMT) was performed by treating TGF-β1 (transfroming growth factor- Respectively. As a control, HERS-SV40, a permanent-derived epithelial cell line used as a supporting cell in the present invention, was used. More specifically, TGF-beta1 (Peprotech, NJ, USA, 100-21C-10) was treated with 20 ng / ml of each of the above two cell lines for 48 hours.

As a result, as shown in FIG. 6A, although the cell morphology was not completely changed in both of the above two cell groups, it was presumed that the process of transition to the mesenchymal cells was observed when the cell end became slightly sharp.

In addition, HERS-SV40 and immortalized epithelial-like cell lines were stored in RNALater (Invitrogen, CA, USA, AM7021) at -20 ° C, RNA was isolated using RNeasy mini kit (Qiagen, Germany, 74106) amfiRivert cDNA synthesis Reverse transcription with cDNA using Platinum Master Mix (GenDEPOT, TX, USA, R5600). The thus synthesized cDNA was used as a template, THUNDERBIRD SYBR qPCR Mix (Toyobo, Japan), E-cadherin , the epithelial stem cell marker described in Table 1 of Example 2-2, and the mesenchymal stem cell marker PCR was performed using N-cadherin and Vimentin specific primers. The Tm of GAPDH was set at 60 ℃ and the Tm of E-cadherin , N-cadherin , and Vimentin was set at 55 ℃. To perform real-time PCR, CFX Connect Real-Time PCR (94 ° C for 10 seconds, Tm for 30 seconds, and 72 ° C for 33 seconds) x 40 cycles, 94 ° C for 10 seconds, and 65 ° C for 5 seconds using the Detection System (1855201, Bio-Rad, CA, USA) And 94 ° C for 5 minutes. The relative quantification values were quantified using the 2 ^{- △ ΔCT} method.

gene direction The sequence (5'-3 ') SEQ ID NO: N-cadherin Forward ACA GTG GCC ACC TAC AAA GG 21 Reverse CCG AGA TGG GGT TGA TAA TG 22 Vimentin Forward TCT ACG AGG AGG AGA TGC GG 23 Reverse GGT CAA GAC GTG CCA GAG AC 24

As a result, expression of E-cadherin , an epithelial cell marker, and N-cadherin , a mesenchymal stem cell marker, were increased after TGF-β1 treatment in both cell lines. However, the expression level of Vimentin , a marker of mesenchymal stem cells, was increased in HERS-SV40 but in the immortalized cell line, expression level was similar before and after TGF-β1 treatment.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> SNU R & DB FOUNDATION <120> Method for differentiation of dental epithelium from human induced pluripotent stem cells and use thereof <130> PD17-068 <160> 24 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin_Forward <400> 1 tgcccagaaa atgaaaaagg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin_Reverse <400> 2 gtgtatgtgg caatgcgttc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ABCG2_Forward <400> 3 ccacaggtgg aggcaaatct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ABCG2_Reverse <400> 4 tcgcggtgct ccatttatca 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EpCAM_Forward <400> 5 gctggccgta aactgctttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EpCAM_Reverse <400> 6 acatttggca gccagctttg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bmi1_Forward <400> 7 cagcccagca ggaggtattc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bmi1_Reverse <400> 8 ggatgaggag actgcactgg 20 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p63_Forward <400> 9 atgttgtacc tggaaaacaa tgc 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> p63_Reverse <400> 10 gtgatggaga gagagcatcg aa 22 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> p75_Forward <400> 11 accgagctgg aagtcgag 18 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> p75_Reverse <400> 12 ctcaccgctg tgtgtgtac 19 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Oct4_Forward <400> 13 acccctggtg ccgtgaa 17 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Oct4_Reverse <400> 14 ggctgaatac cttcccaaat a 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog_Forward <400> 15 cctatgcctg tgatttgtgg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog_Reverse <400> 16 ttctctgcag aagtgggttg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2_Forward <400> 17 gacttcacat gtcccagcac 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2_Reverse <400> 18 gggttttctc catgctgttt 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_Forward <400> 19 gatgctggcg ctgagtacg 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_Reverse <400> 20 gctaagcagt tggtggtgc 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> N-cadherin_Forward <400> 21 acagtggcca cctacaaagg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> N-cadherin_Reverse <400> 22 ccgagatggg gttgataatg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Vimentin_Forward <400> 23 tctacgagga ggagatgcgg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Vimentin_Reverse <400> 24 ggtcaagacg tgccagagac 20

Claims

Inducing pluripotent stem cells are cultured on support cells, and the supporting cells are dental herpetiform stem cells.

The method according to claim 1,
Wherein the inducible pluripotent stem cells are prepared from human-derived mesenchymal stem cells.

The method according to claim 1,
Characterized in that the supporting cells are HERS / ERM (Hertwig's Epithelial Root Sheath / Epithelial Rests of Malassez).

The method according to claim 1,
Wherein said supporting cells are treated with mitomycin C (Mitomycin C).

The method according to claim 1,
Wherein said induced pluripotent stem cells are cultured on said support cells for 7 to 21 days.

The method according to claim 1,
Wherein the differentiation method further comprises the step of culturing the inducible pluripotent stem cells on support cells and then subculturing.

A dental herbal stem cell produced by the differentiating method of claim 1.

A cell therapeutic agent for regeneration of teeth, comprising the dental herpetiform stem cell of claim 7.