KR20160035920A - Method for inducing differentiation of human embryonic stem cells into mesodermal lineage cells - Google Patents

Abstract

The present invention relates to a method for inducing differentiation from mesenchymal stem cells into mesoderm cells and a container for inducing differentiation. In the method for inducing differentiation from mesenchymal stem cells into human embryonic stem cells, a plurality of nanopores And culturing the human embryonic stem cells in a culture container. One aspect of the present invention may be useful for differentiating into mesodermal cells from human embryonic stem cells.

Description

[0001] The present invention relates to a method for inducing differentiation of human embryonic stem cells into mesodermal lineage cells

The present invention relates to a method for inducing differentiation into mesodermal cells from human embryonic stem cells and a vessel for inducing differentiation into mesodermal cells in human embryonic stem cells.

Embryonic stem cells (ESCs) have the ability to self-proliferate continuously while maintaining normal karyotype without differentiation in in vitro culture conditions. In addition, almost all cells that theoretically constitute living organisms Type. ≪ / RTI > Because of these characteristics, human embryo stem cells have recently been recognized as a potential therapeutic agent in the field of regenerative medicine and medical industry development. Therefore, it is important to develop the technology for inducing differentiation from human embryonic stem cells into cells having specific functions, . In particular, in the field of regenerative medicine, the importance of producing high yields of certain types of cells is becoming more apparent in order to increase the applicability and applicability of embryonic stem cells. Thus, various studies have been conducted to induce differentiation of embryonic stem cells into specific cells, but most of them are based on chemical approaches such as soluble chemicals, growth factor treatment, or genetic manipulation. However, these cell growth factors have disadvantages that they are very expensive, and they also have various limitations such as cell infectivity, gene mutation, and possibility of cell mutation. In addition, when such a chemical approach is used, there is a problem that the efficiency of differentiation into a specific cell is not sufficient to be used for cell therapy. Therefore, a method for inducing differentiation of embryonic stem cells into specific cells or a method for increasing the efficiency of differentiation is further required.

In recent years, a growing body of evidence demonstrates that, in addition to solubility factors, physical interactions between cells and underlying surfaces play an important role in cell culture and cell differentiation. Cellular actions such as cell attachment, migration, growth and differentiation are affected by the size and spacing of nanoscale surface features of various cell types, from fibroblasts to stem cells. Thus, the present inventors have completed the present invention by confirming that the culture container in which the nanopore is formed enhances the differentiation efficiency of mesenchymal stem cells into human embryonic stem cells.

It is an object of the present invention to provide a method for inducing differentiation into mesodermal cells from an effective human embryonic stem cell.

It is another object of the present invention to provide a container for inducing the differentiation of human embryonic stem cells into mesodermal cells.

One aspect of the present invention provides a method for inducing differentiation from mesenchymal stem cells into human embryonic stem cells, comprising culturing human embryonic stem cells in a culture container containing a nanostructure.

In the present invention, the term "human embryonic stem cell (hESC)" refers to a cell obtained by extracting the inner cell mass from a blastocyst embryo immediately before fertilization of the embryo into the uterus of the mother, Which is a cell with a pluripotency that can be made. However, the present invention is not limited thereto and broadly includes embryonic stem cells derived from human embryonic stem cells or similar stem cells such as induced pluripotent stem cells (iPS cells).

In the present invention, the term "mesoderm" means an intermediate layer present between the ectoderm and the endoderm in the early development of most multicellular animals. The mesoderm is then differentiated into tissues that specifically form the heart, blood vessels, muscles, bones, reproductive organs, and several secretions. The term "mesodermal cell" means a cell capable of differentiating into muscle cells, bone cells, cartilage cells, adipocytes, reticular cells or other connective tissue cells including blood cells, vascular endothelial cells, smooth muscle and myocardium.

As used herein, the term "differentiation " has the meaning of an evolving generalized differentiation in which the cell increases to the level of a specific cell or tissue complex or individual with a particular function. The term "induction of differentiation from mesenchymal stem cells into human embryonic stem cells " refers not only to the case where complete differentiation of mesenchymal stem cells into human embryonic stem cells is induced, but also when mesenchymal stem cells are differentiated into cells expressing a specific protein The specific protein may be, for example, brachyury.

In the differentiation induction method of the present invention, the nanostructure may be a plurality of nanopores formed at regular intervals.

The term "nano pore" in the present invention means a structure formed concavely in the lower part of a cell culture surface in a cell culture container, and can be used in combination with "nano pore or nano hole ". The nanopore is elongated in a tubular shape having a constant width, and the lower and upper portions of the nanopore may be round semicircular. The upper round semicircle may be formed to have a wider diameter than the lower semicircle, and the cross section between the nanopores and the nano pores may have a spiral structure with a pointed end.

The nanopore may have a diameter of, for example, 30 to 500 nm, 50 to 450 nm, 70 to 400 nm, 90 to 350 nm, 100 to 300 nm, 110 to 330 nm, 120 to 300 nm, To 250 nm. Also, for example, the diameter of the nanopore may be about 200 nm.

The depth of the nanopore may be in the range of, for example, 10 to 1000 nm, 50 to 900 nm, 100 to 800 nm, 150 to 700 nm, 200 to 750 nm, 250 to 700 nm, or 300 to 600 nm . In this case, the aspect ratio of the nanopores may be 1 to 5, 1 to 4, or 1 to 3. Further, for example, the depth of the nanopore may be about 500 nm.

The spacing of the nanopores may be, for example, 80 to 1500 nm, 100 to 1300 nm, 150 to 1000 nm, 200 to 900 nm, 250 to 800 nm, 300 to 700 nm, or 400 to 600 nm. The distance between the nanopores is based on the distance between the centers of the nanopores. For example, the nanopores may be arranged at a constant interval of about 500 nm.

The nanopore is formed on the cell culture surface in the culture container. The term "culture surface" refers to a portion in a culture vessel in which cells are attached to multiply or differentiate. The material of the cell culture surface may be, for example, polystyrene (PS), which is a thermoplastic resin, and thermoplastic resins such as polymethyl methacrylate (PMMA) and polycarbonate (PC) or thermosetting resins may be used. It may also be formed of an elastomer such as polydimethylsiloxane. In one embodiment of the present invention, the culture surface may be formed of polystyrene.

Further, an additional treatment such as surface plasma treatment, ozone treatment or coating with a cell adhesion promoting substance to improve the adhesion of cells on the cell culture surface may be performed. The cell culture container used in the differentiation induction method of the present invention can be coated with fibronectin, for example, on the cell culture surface on which the nanopore is formed. As the fibronectin coating method, a method commonly known in stem cell culture can be used.

The method of inducing differentiation of human embryonic stem cells according to the present invention affects the adhesion, proliferation and differentiation of human embryonic stem cells by using a cell culture container in which a plurality of nanopores are uniformly formed and arranged at regular intervals , And improves the efficiency of differentiation into mesodermal cells.

In the method of inducing differentiation according to the present invention, the cultured human embryonic stem cells in the culture container containing the nanopore may be a human embryonic stem cell derived recombinant cultured in a culture medium containing a mesodermal inducer. In the present specification, the term "embryoid body (EB)" refers to a cell mass in which embryonic stem cells are naturally differentiated, and can be differentiated into three germ layers of endoderm, mesoderm and ectoderm Lt; / RTI > cell aggregates. Cell aggregation occurs by droplet culture, untreated cell culture dish culture or agitation culture to prevent cells from attaching to the surface to form a typical colony proliferation. Differentiation begins with aggregation, and cells begin to repeat a limited degree of embryogenesis. The somata are derived from multipotent cells and thus can be composed of a wide variety of differentiated cell types.

The mesodermal inducer may be used as long as it is a substance known to induce differentiation of mesenchymal stem cells into mesoderm in the technical field of the present invention. The mesodermal inducer may be, for example, BMP4 or Activin A, but is not limited thereto. Other known mesodermal inducers may also be used. When using the culture medium cultured in the presence medium of the mesodermal inducer, the differentiation efficiency of the method for inducing differentiation of mesenchymal stem cells into mesodermal cells according to the present invention can be further enhanced.

In one embodiment, the present invention provides a method for inducing differentiation into mesodermal cells from human embryonic stem cells, comprising the steps of: (a) forming an amphora from human embryonic stem cells in a medium comprising BMP4 and actin A; And (b) culturing the retentate obtained from step (a) in a culture vessel containing a plurality of nanopores having a diameter of 30 to 500 nm.

According to one embodiment, the method for inducing differentiation of human embryonic stem cells comprises the step of forming an amphora derived from human embryonic stem cells in a medium containing BMP4 and actin A. And separating the colony of human embryonic stem cells cultured in the nutrient cell layer from the culture dish prior to the formation of the entrapment body. This may be specifically performed by culturing human embryonic stem cells in a culture medium together with a nutrient cell layer for 1 day and then dissecting the resulting colonies. The resulting colonies are then cultured in a culture medium containing BMP4 and actin A to form an ampoule. In the step of forming an amorphous body in the culture medium containing BMP4 and Actibin A, the early mesoderm differentiation of the human embryonic stem cell-derived body can proceed due to the presence of BMP4 and actin A. Finally, the differentiation induction method comprises culturing in an incubator having a plurality of nanopores having a diameter of 30 to 500 nm by obtaining a culture that is cultured in a medium containing BMP4 and actin A,

The differentiation induction method of the present invention may be up-regulated the expression of integrin alpha 5 gene in a human embryonic stem cell-derived soma. The integrin alpha 5 is a fibronectin receptor and interacts with fibronectin. Regarding upregulation of gene expression of integrin alpha 5, the culture surface surface containing the nanopore of the present invention stimulates the integrin alpha 5 receptor of human embryonic stem cells to promote differentiation of mesenchymal stem cells into mesodermal . In addition, the differentiation method of the present invention may be to up-regulate T and MixL expression in a human embryonic stem cell-derived soma. The expression of T and MixL is a marker representative of hypogastric differentiation. In one embodiment, the human embryonic stem cells differentiated according to the differentiation inducing method of the present invention increased expression of integrin alpha 5 by 4 times or more as compared with human embryonic stem cells cultured and differentiated in a flat culture dish, and T and MixL Was increased by about 2 to 3 times.

Another aspect of the present invention provides a container for inducing differentiation into mesodermal cells from human embryonic stem cells comprising a plurality of nanopores arranged at regular intervals.

In the vessel for inducing differentiation of the present invention, the nanopore is as described above.

In the container, the diameter of the nanopore may be, for example, 30 to 500 nm, 50 to 450 nm, 70 to 400 nm, 90 to 350 nm, 100 to 300 nm, 110 to 330 nm, To 270 nm or from 150 to 250 nm. In one embodiment of the present invention, the diameter of the nanopore may be about 200 nm.

The depth of the nanopore may be in the range of, for example, 10 to 1000 nm, 50 to 900 nm, 100 to 800 nm, 150 to 700 nm, 200 to 750 nm, 250 to 700 nm, or 300 to 600 nm. At this time, the aspect ratio of the nanopores may be 1 to 5, 1 to 4, or 1 to 3. Further, in one embodiment of the present invention, the depth of the nanopore may be about 500 nm.

The distance between the nanopores may be, for example, 80 to 1500 nm, 100 to 1300 nm, 150 to 1000 nm, 200 to 900 nm, 250 to 800 nm, 300 to 300 nm, 700 nm, or 400 to 600 nm. In addition, in one embodiment of the present invention, the nanopores can be arranged at regular intervals of about 500 nm.

The nanopore is formed on a cell culture surface where human embryonic stem cells adhere and proliferate and differentiate. The material of the cell culture surface may be, for example, polystyrene (PS), which is a thermoplastic resin, and thermoplastic resins such as polymethyl methacrylate (PMMA) and polycarbonate (PC) or thermosetting resins may be used. It may also be formed of an elastomer such as polydimethylsiloxane. In one embodiment of the present invention, the culture surface may be formed of polystyrene.

Further, an additional treatment such as surface plasma treatment, ozone treatment or coating with a cell adhesion promoting substance to improve the adhesion of cells on the cell culture surface may be performed.

In one embodiment of the present invention, the cell culture container may be prepared by separately manufacturing a substrate including a cell culture surface made of a polymer material for cell culture and attaching the substrate to the container, or may be integrally formed.

The container for inducing the differentiation of mesenchymal stem cells into human embryonic stem cells according to the present invention exhibits a very high efficiency for differentiating human embryonic stem cells into mesodermal cells.

The method of differentiating human embryonic stem cells according to an embodiment of the present invention can improve the efficiency of differentiation from human embryonic stem cells into mesodermal cells to a greater extent than conventionally, and is useful for inducing differentiation of human embryonic stem cells. Accordingly, the present invention can be applied to the development of new drugs or the development of therapeutic methods using mesodermal cells.

FIG. 1 shows the results of differentiation of human embryonic stem cells into Brachyury expressing cells. (A) is a schematic view of the whole differentiation process, and (B) is a photomicrograph of EB (indwelling material) formed in the presence of BMP4 and Actibin A. FIG. (C) and (D) are SEM images of a flat dish and a nanopore dish. (E) and (F) are SEM images of EBs formed in flat dishes and nanopore dishes in the absence of BMP4 and Activin A, indicating that all of the Brachythry expression was not induced (G) and F) is an SEM image of EB formed in flat dishes and nanopore dishes in the absence of BMP4 and Activin A. BMP4 and Actin A-treated EB (BA-treated) induce Burkiewicz expression, And increased by more than 50% in the forage dish.
Figure 2 shows the analysis of qPCR results for genes representative of (A) pluripotency, (B) early mesoderm differentiation, and (C) EMT (epithelial-mesodermal metastasis).
Figure 3 shows the results of Focal adhesion (FA) analysis. (A) and (B) visualization of FA in EB cultured in flat dishes and nanopore dishes. The red dot indicates empty coolin-positive FA (white arrow). (C) FA quantitative result graph. It can be seen that more FA is present in cells cultured in flat dishes than in nanofoam dishes. (D) and (E) are the results of our colocalization analysis. Vincurin-positive FA was not present in the brackish expressing cells of all dishes. (F) and (G) show the results of phalloidin staining of the cytosceletal organization in cells attached to flat dishes and nanopore dishes.
Figure 4 shows human FAK signaling PCR analysis. (A) is a scatter diagram of the PCR analysis result, the upward-regulated gene is shown in red, and the down-regulated gene is shown in green. Genes with no differences are shown in black. (B) is a list of up-regulated and down-regulated genes. The gene highlighted in blue is the gene associated with local attachment, and the one highlighted in yellow is the integrin.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Production of vessel for differentiation of human embryonic stem cells

One) Nanopore  Manufacture of nickel nano-mold inserts for molding

Biofabrication, 2013. 5 (2): p. 025007, or Macromol Biosci, 2012. 12 (11): p. In order to obtain a durable nano-metal insert at high pressure and temperature during the nano-injection molding process, a two-step anodization process of an aluminum substrate is used to manufacture a nano-template and manufacture Metallic nano-mold inserts were fabricated by nickel electroforming process on the nano-templates. Specifically, an anodic aluminum oxide (AAO) nanopore array (diameter: ~ 200 nm, depth: ~ 500 nm, and inter-center distance: ~ 500 nm) Nano-templates were formed. The nano-templates were fabricated by adjusting the size and spacing of the desired nanopores by adjusting the parameters of the two-step anodization process, for example, the electrolyte, anodization time, applied voltage, and extension time. Next, a 20 nm nickel seed layer was formed on the AAO nano-template with an electron beam evaporator to make the surface electrically conductive for electroplating fixation. The electroplating process of the AAO nano-template was performed by controlling the applied voltage and time in a nickel sulfamate bath (Bacher, W., et al., Microsyst. Technol. 117-119). Finally, the electroplated nickel nano-mold insert was detached from the AAO nano-template and then cut and polished (size: 20 mm x 20 mm, thickness: 1 mm).

2) Nano-injection molding

Nano-injection molding was performed through an injection molding machine (SE50D; Sumitomo) using a mold base composed of three plates equipped with a nano-mold insert. The mold base is designed so that the mold insert can be replaced at the center of the plate. In addition, a petri dish (hereinafter referred to as a nano-dish) having a nano-pore formed to have a rectangular-shaped bottom surface was designed (area: 30 mm x 30 mm). The nanopore array was located in the 20 mm x 20 mm area of the bottom of the cell culture container, and the height of the dish side wall was 10 mm. In addition, a mold insert having a mirror-like surface was used to fabricate a flat petridish (hereinafter referred to as a 'flat dish') as a control. (Cha, KJ, et al., Journal of Micromechanics and Microengineering, 2014. 24). The nano- The flat dish was made of polystyrene (GP125EI; Kumho Chemical Co., Ltd.).

Example  2. induction of differentiation from human embryonic stem cell into mesodermal cell

1) Human embryonic stem cell maintenance ( maintenance )

H9 human embryonic stem cell lines purchased from WICELL Institute were maintained by known methods. Briefly, mitomycin (MMC, Sigma-Aldrich, MO) -treated STO cells were cultured in a gelatin-coated culture vessel with 10% fetal bovine serum (FBS) 1% nonessential amino acids, 1% Anti-Anti and 0.1% -Mercaptoethanol (all from Gibco) at 15,000 cells / cm < ² > in DMEM (Gibco BRL, MD). One day after cell inoculation, the hESC colonies were dissociated into smaller clumps using a glass Pasteur pipette and washed with 20% knockout serum replacement (Gibco), 1% non-essential amino acid, 1% Anti-Anti, 0.1% transferred to a MMC-treated STO (1: 2 split) non-natriuretic cell layer in DMEM / F12 (Gibco) supplemented with 10 ng / ml MW-β-mercaptoethanol and 4 ng / ml bFGF (Invitrogen, NY). The medium was replaced every day, and after 5 days of culture, the cells were transferred to freshly prepared MMC-treated STO cells. Cell culture was maintained at 37 ° C, 5% carbon dioxide, and 95% humidity.

2) Amphibian ( EB ) Formation

The hESCs cultured for 5 days were washed once with PBS and treated with Gibco for 10 minutes on a warm plate (37 ° C) to isolate colonies from the feeder layer. The separated colonies were then washed three times with DMEM / F12 and gently pipetted to induce aggregation. Aggregates were then centrifuged at 1000 rpm for 2 min and resuspended in RPMI-1600, Gibco (Invitrogen) supplemented with N2 supplement (Invitrogen), BMP4 and Activin A (both R & D, Systems, MN at 25 ng / )). ≪ / RTI > Two days after suspension culture, the resulting EBs were plated in the same differentiation medium supplemented with fibronectin-coated (5 [mu] g / ml) flat dishes and 0.5% FBS in nanodisperse. Additionally, the aggregates were cultured in RPMI-1600 (Gibco) medium without BMP4 and Actibin A under the same conditions, and the results were observed.

Example  2. Differentiation induced in human embryonic stem cells Amphibious  Identify characteristics

Way

Immunostaining ( Immunocytochemistry )

The samples were washed with PBS and fixed with 4% paraformaldehyde for 5 minutes at room temperature. The fixed sample was permeabilized with 0.3% Triton 100X (for nuclear transfer factor) or 0.03% Triton 100X (for surface marker) at room temperature for 10 minutes and then blocked with the same solution with 5% goat serum for 30 minutes . Each primary antibody of Brachyar (Abcam, MA, 1: 400), vinculin (Sigma-Aldrich, 1: 100) and Paloidine (Invitrogen, 1: 200) was incubated overnight at 4 ° C in blocking solution. Samples were washed 3 times for 5 minutes each in PBS and then incubated for 1 minute at room temperature with secondary antibody (Alexa Flour 488 or 594, all from Invitrogen). The samples were washed three times with PBS (Sigma-Aldrich) and stained for 2 minutes in PBS with DAPI (Sigma-Aldrich, 1: 1000).

Quantitative polymerase chain reaction qPCR )

Trisol was used to perform RNA extraction according to the manufacturer's instructions. The extracted RNA was quantified using a spectrophotometer (NanoDrop ND-1000; Thermo Scientific), and 1 쨉 g of RNA was used to synthesize cDNA using a commercial kit (SuperScript II reverse transcriptase kit, Invitrogen). For each PCR reaction, the following components were added to give a total reaction volume of 20 [mu] l; SYBR Green PCR Master Mix (10 μl), primers (2 μl), cDNA (1 μl) and water for PCR (7 μl). The reaction was carried out using the ABI 7300 qRT-PCR system and the measurement was performed three times. The values were generalized to endogenous housekeeping genes (b-actin and GAPDH) and expression of the associated genes was calculated using the delta-delta CT method. All primers were purchased from Macrogen (Korea), and the sequences of the primers are shown in Table 1.

primer The sequence (5'-3 ') SEQ ID NO: Hs GAPDH Sense ACATCAAGAAGGTGGTGAAG One anti sense GTTGAAGTCAGAGGAGACCA 2 Hs beta-actin Sense CATTAAGGAGAAGCTGTGCT 3 anti sense GCTCGTAGCTCTTCTCCAG 4 Hs OCT4 Sense AGGAGATATGCAAAGCAGAA 5 anti sense AGGAACAAATTCTCCAGGTT 6 Hs NANOG Sense CTATCCATCCTTGCAAATGT 7 anti sense TCCTGAATAAGCAGATCCAT 8 Hs SOX2 Sense TTGTTTAAAAAGGGCAAAAG 9 anti sense AAATTACCAACGGTGTCAAC 10 Hs Brachyury Sense TATTCTGAATTCCATGCACA 11 anti sense AAGTTCTCCTCGGCATATTT 12 Hs Mixl1 Sense CTTTCAAAACACTCGAGGAC 13 anti sense GGAGTGATCGAAGTAACAGG 14 Hs WNT3A Sense CCCACTCGGATACTTCTTACT 15 anti sense GGAATACTGTGGCCCAAC 16 Hs E-cadherin Sense CACCACACTGAAAGTGACTG 17 anti sense AATTGTCCACCATCATCATT 18 Hs N-cadherin Sense ACATGTATGGTGGAGGTGAT 19 anti sense TGCTTTTTGGGAATATCAGT 20 Hs Fibronectin Sense CTGGGTATGACACTGGAAAT 21 anti sense CCTAAAACCATGTTCCTCAA 22 Hs ALK Sense ATGGACATTGATGTGATCCT 23 anti sense ACCTTGGCTGTAGTCATCTG 24 Hs SP7 / OSX Sense CCACACCAACACACTTTCTA 25 anti sense GGAAATAAGCTTGAGAAGCA 26 Hs Osteopontin Sense CAGGAACTTTCCAAAGTCAG 27 anti sense CTTCCTTACTTTTGGGGTCT 28 Hs COL1A1 Sense AAAAATGGGAGACAATTTCA 29 anti sense GTTCGGTTGGTCAAAGATAA 30

result

Nanopore In the dish Increased Braki us  Expression was observed.

FIG. 1A shows a process of total differentiation using the present invention. HESC colonies stoichiometrically cultured in the presence of the mesoderm inducers BMP4 and actin A formed spherical EB within 2 days (Fig. 1B). The EBs were then plated in fibronectin-coated flat dishes and nano-dishes in the same medium supplemented with 0.5% FBS. Plating of EB formed in the absence of BMP4 and actin A failed to induce differentiation into BrkW positive cells in both flat dish and nano-dish (Fig. 1E and Fig. 1F). This indicates that early mesodermal induction during EB formation is required. On the other hand, it was confirmed that BMP4 and actin A treated EB cultured on flat dish and nano-dish were all expressed in Brachy. Especially, in nano-dish (about 42 ± 3.55%), 2-fold increase (Fig. 1G and Fig. 1H).

Nano In the dish  Attached EB Up-regulation of mesoderm-associated genes in

One day after plate plating on each dish, the samples were harvested and the mesodermal-associated gene cluster was analyzed using real-time PCR. No significant changes were observed in Oct4 expression, a typical pluripotency marker in EBs attached to flat dishes and nanodisks (Figure 2A). Markers T and MixL, representing early midsagittal differentiation, were up-regulated by 2 to 3 fold compared to flat dishes for EBs attached to nanodisks. This is consistent with an immunocytochemical analysis (Fig. 1G and Fig. 1H) in which the expression of BrkYi in EBs attached to nanodisks is approximately twice as high as that in flat dishes. The epithelial-mesenchymal transition (EMT) is a characteristic biologic process that occurs during the acquisition of mesodermal and endodermal characteristics of the hESC (Hay, ED, Acta Anat (Basel), 1995. 154 p. E-cadherin expression reduction and concomitant N-cadherin expression increase are hallmarks of EMT. This change in cadherin expression was observed in all dishes, but there was no significant difference between the two dishes (Fig. 2C and D). However, another marker for fibronectin, another marker for EMT, fibronectin was highly expressed in EBs attached to nanodigles (about 4-fold increase, Fig. 2E) when compared to flat dishes. Finally, real-time PCR suggests that nanofoil topography is favorable to some degree of early mesenchymal differentiation when compared to a flat surface.

Local attachment Braki us  Unnecessary for expression

Increased Browrie in Nano-Dishes To determine whether our expression is due to some change in focal adhesion (FA), immunocytochemistry with antibodies to VINCURIN, one of the many proteins used during FA formation (Ezzell, RM, et al., Exp Cell Res 1997, 231 (1): 14-26). In the EBs attached to the flat dish, vincculin was expressed in the terminal region of the attached EB (mean ~ 7 per cell), whereas in the nanodish the expression of vincculin was minimal in both the onset and center of the attached EB Average ~ 1) (Figure 3A-C). Interestingly, the expression of vinculin was absent in BrkW positive cells of all EBs attached to both dishes (Figs. 3D and E). This indicates that FA formation can play an irrelevant role in inducing Brackweill positive cells from hESCs. Cytoskeletogenesis is also not affected by differences in topography.

Local attachment Kinase ( FAK ) Signaling pathway PCR  analysis

To further investigate the signaling pathways that may contribute to increased Brackilian expression, PCR analysis (Qiagen) for FAK signaling was performed. FAK is one of many proteins that are used when stable FA is formed in cell attachment and causes downstream signal transduction cascades (Zachary, I., Focal adhesion kinase . Int J Biochem Cell Biol, 1997. 29 (7): p. 929-34). Since we observed differences in the number of FAs, we hypothesized that FAK signaling would also be affected. Results show that about nine times the genes from the 84 genes analyzed were about two-fold increased compared to the flat dish in EBs cultured in nano-dishes, whereas one gene was down-regulated about two-fold (Figures 4A and B) . Interestingly, the fibronectin receptor integrin alpha 5 was upregulated 4-fold in EBs attached to nanodigles as compared to flat dishes (Fig. 4B). Integrin alpha 5 is involved in mesodermal formation during early embryonic development and endodermal differentiation. It is presumed that the nanopore enhances the deposition of fibronectin and, in turn, increases the expression of integrin alpha 5, inducing mesenchymal differentiation of human embryonic stem cells.

<110> CHABIOTECH CO., LTD. <120> Method for inducing differentiation of human embryonic stem cells          into mesodermal lineage cells <130> PN107150 <160> 30 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs GAPDH sense <400> 1 acatcaagaa ggtggtgaag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs GAPDH anti sense <400> 2 gttgaagtca gaggagacca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs beta-actin sense <400> 3 cattaaggag aagctgtgct 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Hs beta-actin anti sense <400> 4 gctcgtagct cttctccag 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs OCT4 sense <400> 5 aggagatatg caaagcagaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs OCT4 anti sense <400> 6 aggaacaaat tctccaggtt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs NANOG sense <400> 7 ctatccatcc ttgcaaatgt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs NANOG anti sense <400> 8 tcctgaataa gcagatccat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs SOX2 sense <400> 9 ttgtttaaaa agggcaaaag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs SOX2 anti sense <400> 10 aaattaccaa cggtgtcaac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs Brachyury sense <400> 11 tattctgaat tccatgcaca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs Brachyury anti sense <400> 12 aagttctcct cggcatattt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs Mixl1 sense <400> 13 ctttcaaaac actcgaggac 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs Mixl1 anti sense <400> 14 ggagtgatcg aagtaacagg 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Hs WNT3A sense <400> 15 cccactcgga tacttcttac t 21 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Hs WNT3A anti sense <400> 16 ggaatactgt ggcccaac 18 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs E-cadherin sense <400> 17 caccacactg aaagtgactg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs E-cadherin anti sense <400> 18 aattgtccac catcatcatt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs N-cadherin sense <400> 19 acatgtatgg tggaggtgat 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs N-cadherin anti sense <400> 20 tgctttttgg gaatatcagt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs Fibronectin sense <400> 21 ctgggtatga cactggaaat 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs Fibronectin anti sense <400> 22 cctaaaacca tgttcctcaa 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs ALK sense <400> 23 atggacattg atgtgatcct 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs ALK anti sense <400> 24 accttggctg tagtcatctg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs SP7 OSX sense <400> 25 ccacaccaac acactttcta 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs SP7 OSX anti sense <400> 26 ggaaataagc ttgagaagca 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs Osteopontin sense <400> 27 caggaacttt ccaaagtcag 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs Osteopontin anti sense <400> 28 cttccttact tttggggtct 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs COL1A1 sense <400> 29 aaaaatggga gacaatttca 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hs COL1A1 anti sense <400> 30 gttcggttgg tcaaagataa 20

Claims (8)

A method for inducing differentiation from mesenchymal stem cells into human embryonic stem cells, comprising culturing human embryonic stem cells in a culture container containing a plurality of nanopores formed at regular intervals. The method according to claim 1, wherein the diameter of the nanopore is 30 to 500 nm. The mesenchymal stem cell according to claim 1, wherein the interval of the nanopores is 80 to 1500 nm based on the distance between the centers of the respective nanopores, and the depth of the nanopore is 10 nm to 1 탆. Lt; / RTI &gt; The method according to claim 1, wherein the nanopore is formed on a cell culture surface in a culture container, and the culture surface is made of polystyrene. [Claim 2] The method according to claim 1, wherein the cultured human embryonic stem cells in the culture container containing the nanopore are human embryonic stem cell derived cultures cultured in a culture medium containing mesodermal inducer, Lt; / RTI &gt; The method according to claim 5, wherein the mesodermal inducer is BMP4 and actin A, wherein the mesodermal inducer is BMP4 and actin A. The method of claim 5,
(a) forming an amphora from human embryonic stem cells in a medium comprising BMP4 and actin A; And
(b) culturing the soma obtained from step (a) in a culture container containing a plurality of nanopores having a diameter of 30 to 500 nm, to induce differentiation into mesodermal cells from human embryonic stem cells. [Claim 6] The method according to claim 5, wherein the gene expression of integrin alpha 5 is upregulated in a somatic cell derived from human embryonic stem cells.

Images