KR102444801B1 - A Cross-linked extracellular matrix and a Culturing method of human pluripotent stem cell using thereof - Google Patents

Abstract

The present invention relates to a cross-linked extracellular matrix matrix obtained by crosslinking a fibroblast-derived extracellular matrix matrix, and to a method for preparing the same. In addition, there is provided a method of culturing human embryonic stem cells using the cross-linked extracellular matrix matrix.
The extracellular matrix matrix prepared through the present invention can have increased strength compared to that without crosslinking, and can induce cell proliferation while maintaining an undifferentiated state when culturing human embryonic stem cells. In addition, the period during which human embryonic stem cells can be cultured using the same substrate is significantly increased, thereby reducing the cost and time required for cell culture. Therefore, the cross-linked fibroblast-derived extracellular matrix matrix prepared by the method of the present invention can be effectively used for culturing human embryonic stem cells.

Description

A Cross-linked extracellular matrix and a Culturing method of human pluripotent stem cell using the same

The present invention relates to a cross-linked extracellular matrix matrix obtained by crosslinking a fibroblast-derived extracellular matrix matrix, and to a method for preparing the same. In addition, there is provided a method of culturing human embryonic stem cells using the cross-linked extracellular matrix matrix.

Human pluripotent stem cells (hPSCs; human embryonic stem cells, hESC) are stem cells having pluripotency capable of differentiating into endoderm, mesenchymal and ectoderm. These hPSCs are well known to be cultured on Sandos inbred mouse (SIM)-derived 6-thioguanine and ouabain-resistant cells (STO cells) or mouse embryonic fibroblasts (MEFs), and the STO cells or MEFs are cultured on stromal cells ( referred to as feeder cells). It is important to use appropriate stromal cells for culturing or maintaining hPSCs, genetic manipulation, and cloning of single cell units. It is judged that the culture method is not suitable for clinical application.

A substrate developed as an alternative for this is Matrigel ^® (Matrigel; Corning, Corning, NY). Matrigel does not use stromal cells in the culture of hPSCs, but uses only extracellular structural materials, and is one of the most used types. However, this Matrigel has limitations in controlling and using it in a desired form, and since it is a material derived from the breeding of Engelbre-Holm-Swarm mice, it contains heterogeneous substances, so there is a possibility that undesired exogenous substances may be included. have For this reason, there is a need to prepare a platform that can effectively culture hPSCs without using stromal cells, and research aimed at using them to develop cell therapeutics that can be applied to the human body continues. have.

The extracellular matrix (ECM) is a non-cellular structure of a tissue or organ, composed of various polymers secreted by cells. ECM is structurally intact and can be used as a natural scaffold material that can be used for biological purposes. Their specific composition and function may vary depending on the type of specific tissue from which they are derived, but the two most well-known types are: one is glycosaminoglycan (GAG), and one is proteoglycan. It is a known chain structure polymer, and the other is a fibrous protein such as collagen, vitronectin, elastin, fibronectin and laminin. Because they have very diverse structures, they can be structurally altered and can play a pivotal role in biophysical and biochemical interactions between various cells. Although the range of these characteristics varies depending on the cells to be cultured, it is known that essential characteristics of ECM such as homeostasis, cell adhesion, cell-cell interaction and differentiation capacity may be affected.

Accordingly, in order to create a biologically suitable environment by appropriately using the starting cells of the ECM, attention is focused on a technology for controlling the characteristics of the ECM. In early studies related to this, it was possible to generate a matrix of naturally derived culture substrates suitable for stem cell adhesion, proliferation and differentiation, aimed at controlling the biophysical and biochemical characteristics appearing after the decellularization process. Therefore, studies have been conducted for the purpose of properly controlling the characteristics of an appropriate matrix according to the lineage characteristics of stem cells. For example, in order for cells to be cultured to be influenced by an external force to give a method for culturing in an appropriate environment, the stiffness of the substrate may be an essential characteristic.

To this end, studies have been made on a method for regulating mechanical properties (mechanotransduction) in culturing human mesenchymal stem cells (hMSC), and in this case, the importance of cell-ECM interaction to control the characteristics of stem cells is significant. It was highlighted (Non-Patent Document 1, Non-Patent Document 2). In addition, in Korean Patent Registration No. 10-1528909, a method for producing ECM using osteoblasts or fibroblasts and inducing differentiation of chondrocytes using the same is known (Patent Document 1).

However, this may not be suitable for culturing hPSCs, because although hPSCs have various applications in regenerative medicine, there have been no reports of studies to fundamentally confirm the interaction between hPSC-ECM. Although a method for culturing hPSCs on a synthetic substrate or a biological substrate has been reported through various studies (Non-Patent Document 3), research on an appropriate method for a culture environment for regulating the pluripotency of hPSCs should be continued.

Accordingly, the present inventors made efforts to develop a culture substrate of hPSC suitable for culturing as a cell therapeutic agent. As a result, ECM (FDM) prepared by culturing and decellularizing fibroblasts was cross-linked as a cross-linking agent. ECM was prepared to control The invention was completed.

An object of the present invention is to provide a method for preparing an extracellular matrix matrix suitable for culturing human embryonic stem cells.

Another object of the present invention is to provide a method for culturing human embryonic stem cells using the prepared extracellular matrix matrix.

In order to achieve the above object, the present invention provides a method for preparing an extracellular matrix for culturing human embryonic stem cells (hPSC), comprising the steps of:

i) culturing fibroblasts in a saturated state;

ii) decellularizing the cells cultured in step i) to obtain an extra-cellular matrix (ECM);

iii) treating the ECM obtained in step ii) with a cross-linker to induce cross-linking between substrates; and

iv) obtaining the cross-linked ECM in step iv).

In a specific embodiment of the present invention, the crosslinking agent in step iii) may be genipin, wherein the genipin is treated at a concentration of 0.5% (w/v) to 2.0% (w/v). it could be

In addition, the present invention provides a method for culturing human embryonic stem cells, including; inoculating and culturing human embryonic stem cells in the ECM prepared by the above method.

In a specific embodiment of the present invention, the culturing method may be a method of inducing proliferation of human embryonic stem cells while maintaining the undifferentiated state.

Accordingly, the present invention provides a method for preparing ECM (X-FDM) so that physical properties can be controlled by cross-linking ECM (FDM) prepared by culturing and decellularizing fibroblasts as a cross-linking agent. At this time, when treating Jennifin as the crosslinking agent, it is most preferable to treat it at a concentration of 0.5% (w/v), and the X-FDM prepared through this may have increased strength compared to the non-crosslinked FDM. And, when culturing human embryonic stem cells, it is possible to induce cell proliferation while maintaining the undifferentiated state. In addition, the period during which human embryonic stem cells can be cultured using the same substrate is significantly increased, thereby reducing the cost and time required for cell culture. Therefore, the cross-linked fibroblast-derived extracellular matrix matrix prepared by the method of the present invention can be effectively used for culturing human embryonic stem cells. In addition, human embryonic stem cells cultured through this can be used as a stem cell therapeutic agent.

1 is a schematic diagram showing the manufacturing process of the fibroblast-derived extracellular matrix (FDM) and its cross-linked extracellular matrix (X-FDM) of the present invention.
Figure 2 shows the results of confirming the morphological characteristics of the FDM and X-FDM prepared in the present invention.
3 shows the results of observing the surface morphology of the FDM and X-FDM prepared in the present invention with a scanning electron microscope (SEM) and a bio-atomic force microscope (bio-AFM).
4 shows the cell morphology after inoculation and culturing of hPSCs in FDM and X-FDM.
5 shows the results of confirming the colony form and maintenance degree when culturing hPSCs in FDM and X-FDM.
6 shows the results of confirming the colony morphology of cells cultured in FDM (Gx0) over time.
7 shows the results of confirming the colony form and matrix strength of hPSC cultured in FDM and X-FDM.
8 shows the results of confirming the adhesion ability to the colonies of hPSC cultured in X-FDM (Gx2.0).
9 shows the results of observing the change in colony morphology of hPSCs cultured in X-FDM (Gx0.5) over time.
10 shows the results of confirming colony stability and analysis of gene expression levels of pluripotency or differentiation markers.
11 shows the results of confirming the colony morphology of hPSCs cultured for 5 days in mTeSR ^TM -1-Matrigel, FDM and X-FDM.
12 shows the results of comparing the expression levels of epithelial-mesenchymal metastasis (EMT)-related genes in hPSCs cultured in FDM or X-FDM.
13 shows the results of confirming the correlation between the genes whose expression level is changed in hPSC cultured in FDM or X-FDM and the expression level of the protein expressed from the gene.
14 shows the results of confirming the change in the expression level of local adhesion-related genes in hPSC cultured in FDM or X-FDM.
15 shows a list of genes whose expression level is changed in hPSCs cultured in X-FDM compared to hPSCs cultured in FDM.
16 shows a list of genes whose expression level changes more than twofold in hPSCs cultured in X-FDM compared to hPSCs cultured in FDM.
FIG. 17 shows a schematic diagram in which the interaction between genes whose expression level is changed in a network of hPSCs cultured in X-FDM compared to hPSCs cultured in FDM is expressed as a network.
18 shows the results of confirming the colony morphology change according to the subculture order of hPSC cultured in X-FDM (Gx0.5).
19 shows the results of confirming the pluripotency of hPSCs cultured in X-FDM (Gx0.5).
20 is a result confirming the expression level of pluripotency marker protein in hPSC cultured in X-FDM (Gx0.5).

Hereinafter, the present invention will be described in detail.

The present invention describes a method for preparing an extracellular matrix for culturing human embryonic stem cells (hPSC), comprising the steps of:

i) culturing fibroblasts in a saturated state;

ii) decellularizing the cells cultured in step i) to obtain an extra-cellular matrix (ECM);

iii) treating the ECM obtained in step ii) with a cross-linker to induce cross-linking between substrates; and

iv) obtaining the cross-linked ECM in step iv).

In the "method for preparing an extracellular matrix for human embryonic stem cell culture" of the present invention, the cross-linking agent of step iii) is in the art for cross-linking, such as genipine or glutaraldehyde. Any crosslinking agent to be used can be used without limitation, but it is more preferable to use Jennipin from the viewpoint of using a biosafety natural material. The Jennipin may be added by appropriately adjusting the concentration for the purpose of controlling the physical properties of the extracellular matrix, and it is more preferable to treat at a concentration of 0.5% (w/v) to 2.0% (w/v). . In the case of cross-linking by treatment at a concentration of less than 0.5% (w/v), the strength of the extracellular matrix does not appear properly, so differentiation of stem cells can be made, and it is wasteful because subculture must be performed at intervals of 5 to 7 days. It may be undesirable. In addition, when the cross-linking agent is treated in excess of 2.0% (w/v), the cross-linking may occur excessively and the strength of the substrate may be excessively increased, thereby adversely affecting the adhesion and proliferation of stem cells. It is not recommended because it can go crazy. Therefore, the most preferable concentration range of Jennifin corresponds to 0.5% (w/v) to 2.0% (w/v), and from the viewpoint of inducing proliferation while maintaining the cultured human embryonic stem cells in an undifferentiated state, 0.5% (w/v) to 2.0% (w/v) It is most preferable to treat genifin in % (w/v). FDM cross-linked by treatment with 0.5% (w/v) genifin not only gives an advantage to cell culture in that human embryonic stem cells are maintained in an undifferentiated state, but also can be cultured for 7 to 14 days. Therefore, the unit period required for subculture can be extended, which is effective in terms of cost of cell culture.

In addition, the present invention comprises the steps of inoculating and culturing human embryonic stem cells in the cross-linked extracellular matrix (X-FDM) prepared by the above method, specifically, the fibroblast-derived extracellular matrix; It provides a method for culturing human embryonic stem cells, comprising a.

In the "culture method of human embryonic stem cells" of the present invention, the culture method can induce the proliferation of cells while maintaining the undifferentiated state without the differentiation of human embryonic stem cells. Therefore, it is possible to manage the cultured human embryonic stem cells according to the purpose, and through this, it is possible to develop a stem cell therapeutic agent in an appropriate direction.

Hereinafter, the present invention will be described in more detail through preparation examples and examples. These preparations and examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these preparations and examples. .

[Preparation Example 1]

Preparation of fibroblast derived extra-cellular matrix (FDM)

The fibroblast-derived extracellular matrix matrix of the present invention and the cross-linked extracellular matrix matrix were prepared according to the schematic procedure of FIG. 1 . To prepare fibroblast-derived extra-cellular matrix (ECM), NIH3T3 mouse fibroblast cell line (ATCC) or human foreskin-derived BJ1 fibroblast cell line (ATCC) was used. . Fibroblast lines were inoculated in plastic dishes or 12-well plates of gelatin-coated glass at a density of 2×10 ⁴ cells/cm ² , followed by 10% fetal bovine serum (FBS) and 1% antibiotic (penicillin streptomycin). It was cultured for 1 week in DMEM medium containing (Dulbecco's modified Eagle's medium). After incubation, the confluent cells were washed with PBS, immersed in a decellularization solution, and decellularized at 37° C. for 2 minutes. As the decellularization solution, a PBS solution containing 0.25% Triton X-100 and 10 mM NH ₄ OH was used. Then, the sample was again treated with 50 U/ml of DNase I and 2.5 μl/ml of RNase A and reacted at 37°C for 2 hours. The reaction solution was removed from the decellularized sample, washed carefully 3 times with PBS, and then FDM was obtained and stored at 4°C.

[Preparation Example 2]

Preparation of cross-linked FDM

The prepared FDM was cross-linked by treating various concentrations of genipin (GN; Wako Chemicals USA) as a cross-linking agent. Specifically, 0.5%, 1.0%, and 2.0% Jennipin solutions were prepared by diluting 5% (w/v) DMSO (dimethyl sulfoxide) of Jennipin with a stock solution of a stock solution with a PBS solution. . The jenipin solution was added to the FDM and incubated for 4 hours at room temperature to induce cross-linking of the FDM. After crosslinking, it was washed 5 times with PBS solution and then stored at 4°C.

Through this, cross-linked FDM (X-FDM) of the present invention was prepared. FDM without cross-linking is Gx0; X-FDM cross-linked with 0.5% genipine was Gx0.5; X-FDM cross-linked with 1.0% genipine was Gx1.0; and X-FDM cross-linked with 2.0% genipine was named Gx2.0.

[Experimental example]

First, an experimental method for carrying out an embodiment of the present invention will be described.

1. Quantitative Analysis of ECM-Containing Proteins

In order to quantify the amount of protein constituting the ECM of the present invention, that is, FDM and X-FDM of Comparative Examples and Examples, fibronectin (FN), laminin (LN), type 1 collagen (cellagen) as the corresponding proteins type I, ColI) and vitronectin (vitronectin, VN) were selected.

Each sample was prepared in a 6-well plate, and the following primary antibodies were added and incubated overnight: mouse-derived anti-FN antibody (Santa Cruz Biotechnology, Inc.), mouse-derived anti-LN antibody (Santa Cruz). Biotechnology, Inc.), anti-Col I antibody from rabbit (Abcam, Cambridge, UK) and anti-VN antibody from rabbit (Santa Cruz Biotechnology, Inc.). Then, as a secondary antibody, a goose-derived anti-mouse IgG antibody or anti-rabbit IgG antibody bound to HRP (horseradish peroxidase) was treated and reacted. After the reaction, the sample was washed several times, and then 1 ml of 3,3',5,5'-TMB (3,3',5,5'-tetramethylbenzidine) substrate was added and incubated for 20 minutes. After incubation, 0.16 M sulfuric acid was added to terminate the color reaction, and 100 μl was dispensed and absorbance was measured at 405 nm. In order to compare each experimental group with each other, the expression level of the protein was calculated by leveling it to 9.6 cm ² , which is one well surface area per each sample.

2. Observation of ECM morphology

The observation of the surface morphology of FDM and X-FDM was analyzed using a scanning electron microscope (SEM). In addition, the surface morphology of FDM and X-FDM in liquid medium was observed using a bio-atomic force microscope (bio-AFM) equipped with an inverted optical microscope. In addition, the Young's modulus for each sample was measured using the AFM-nano indentation technique. ★1

3. Cell Culture

In the present invention, the following cell lines were used: a mouse embryonic fibroblast (MEF) H9 cell line and a primary MEF (Orient Bio Inc.). H9-hPSCs were cultured in 80% DMEM medium, which contained 20% KnockOut ^™ serum supplement, 1% antibiotic (P/S; ThermoFisher Scientific), and 1% minimal nutrient medium non-essential amino acids (MEM-NEAA; ThermoFisher Scientific). Fisher Scientific), 0.1% beta-mercaptoethanol (β-ME; Thermo Fisher Scientific) and 4 ng/ml recombinant human basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ) Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, Waltham, Mass.) was used. The hPSC culture medium was replaced with fresh medium every day, and each culture was subcultured every 5 to 7 days. Primary CF1 MEFs were cultured by adding mitomycin C (MMC) (M-MEF; Sigma-Aldrich Co.), and used to serve as a base culture layer for hPSCs. M-MEF was cultured in DMEM medium containing high concentration of 90% glucose. The DMEM medium containing high concentration of glucose was used by adding 10% fetal bovine serum, 1% antiseptic (P/S), 1% MEM-NEAA and 0.1% β-ME. Cell medium was changed every 2 days. In order to subculture hPSCs onto FDM and X-FDM, hPSCs adhered to the culture medium were dissociated by treatment with Dispase® (Thermo Fisher Scientific) or physically dissociated using a glass Pasteur pipette (Corning). All cells were cultured in a 5% CO ₂ incubator at 37°C.

4. Alkaline phosphatase staining (AP staining) and immunocytochemistry analysis

To determine whether the hPSC colonies were in an undifferentiated state, AP expression was confirmed. AP staining was performed using the ES Cell Characterization Kit (EMD Millipore, Billerica, MA). For immunostaining, cells were first treated with 4% paraformaldehyde (PFA) and fixed by incubation at room temperature for 20 minutes, and then treated with PBS solution containing 0.03% Triton X-100 for 5 minutes to give permeability to the cell membrane. Then, 5% normal goose serum was treated for 30 minutes, and each of the following primary antibodies was treated and incubated overnight at 4°C: anti-OCT4 antibody, anti-SOX2 antibody, anti-SSEA-4 antibody and anti- TRA-1-60 antibody (EMD Millipore). Then, Alexa Fluor ^® 488, 555 or 594-conjugated secondary antibody was used as a secondary antibody to confirm the expression of each hPSC-specific marker. Cell nuclei were stained using DAPI (DAKO, Carpentaria, CA), and all images were observed using a fluorescence microscope.

5. Quantitative PCR analysis (qPCR)

To confirm the mRNA expression level, qPCR was performed. First, intracellular total RNA was extracted using TRIzol ^® reagent. Then, the concentration of extracted total RNA was confirmed using a NanoDrop ^® ND-1000 spectrophotometer. Using 1 μg of the extracted total RNA as a template, reverse transcription was performed using Maxim RT premix kit (iNtRon Biotechnology, Sungnam, Korea) to synthesize cDNA. Then, each marker gene expression level was confirmed using the cDNA as a template and specific primers for confirming the expression level of the target gene. The sequences of the primers were those described in [Table 1] below.

Sequence of primers used in the PCR process of the present invention target gene direction order SEQ ID NO: OCT4
forward 5'-AAC TCG AGC AAT TTG CCA AGC TCC-3' SEQ ID NO: 1 reverse 5'-TTC GGG CAC TGC AGG AAC AAA TTC-3' SEQ ID NO: 2 E-cadherin
forward 5'-CGA GAG CTA CAC GTT CAC GG-3' SEQ ID NO: 3 reverse 5'-GGG TGT CGA GGG AAA AAT AGG-3' SEQ ID NO: 4 N-cadherin
forward 5'-TCA CAG ATT CGG GTA ATC CTC-3' SEQ ID NO: 5 reverse 5'-TGC AGC TGG CTC AAG TCA TA-3' SEQ ID NO: 6 Vimentin
forward 5'-AAG TTT GCT GAC CTC TCT GAG GCT-3' SEQ ID NO: 7 reverse 5'-TTC CAT TTT CAC GCA TCT GGC GTT TC-3' SEQ ID NO: 8 GAPDH
forward 5'-GTG GGG CGC CCC AGG CAC CAG GGC-3' SEQ ID NO: 9 reverse 5'-CTC CTT AAT GTC ACG CAC GAT TTC-3' SEQ ID NO: 10

6. Transmission electron microscope

For image analysis of the intracellular level of hPSCs, transmission electron microscopy (TEM) analysis was performed. Cells were inoculated on FDM or X-FDM substrate, and 2.5% glutaraldehyde/0.1 M PBS was added and the cells were cultured overnight at 4° C. and fixed. Then, it was treated with 1% osmium tetraoxide and incubated for 2 hours. After incubation, the sample was dehydrated by sequentially adding 70% to 100% ethanol, and then soaked with a 1:1 mixture of epoxy resin (Polysciences, Inc., Warrington, PA) and propylene oxide and treated for 2 hours. did. The sample was again placed on a fresh epoxy resin, dried in vacuum for 3 hours, and then placed at 60° C. for polymerization with an embedding medium. To observe the longitudinal cross-section of each sample, a thin-film section was prepared using an ultra-microtome, and the TEM image was observed through a TEM microscope (TEM; Tecnai F20; FEI, Hillsboro, OR).

7. Quantitative PCR and RT ² Profile PCR analysis

When hPSCs were cultured in FDM (Gx0) and X-FDM (Gx0.5), a total of 84 gene expression patterns related to epithelial-mesenchymal transition (EMT) and focal adhesion were analyzed. Gene expression patterns were analyzed using EMT and Focal Adhesion RT ² Profiler PCR Array kit (Qiagen, Hilden, Germany). Target genes were screened using the LightCycler ^® 96 Real-Time PCR system (F. Hoffmann-La Roche Ltd., Basel, Switzerland) according to the protocol provided by the manufacturer. As a sample for analysis, cells were cultured and then total RNA was extracted using TRIzol ^® reagent, and the RNA concentration was measured. Using the measured RNA as a template, cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). By using the synthesized cDNA as a sample, the expression pattern of the target gene was confirmed, and among the genes known to be expressed during differentiation, genes whose expression level changed more than twice were selected. The results of all experiments were normalized based on the average expression levels of five essential genes, ACTB, B2M, GAPDH, HPRT1 and RPLP0. Result analysis was performed using SA Biosciences and Cluster software (RT2 profiler PCR array data analysis version 3.5, Qiagen). A reciprocal network of genes related to EMT and FA was constructed using Gene Network Central Pro™ (Qiagen).

8. Karyotype analysis

The karyotype analysis of hPSC was performed by partially modifying the known method [★13]. Specifically, after replacing the plate 3 days after the initiation of hPSC culture, 100 μl of colcemid (Thermo Fisher Scienti.c) was treated and cultured at 37° C. for 3 hours, and then detached using 0.25% trypsin-EDTA. ) was obtained. The obtained cells were lysed using 1% citric acid buffer as a hypotonic solution, and then the cell lysate was mixed in a fixing solution in which ethanol:glacial acetic acid was mixed in a ratio of 3:1 and fixed. After fixation, chromosomes were identified using G-banding, and karyotype analysis was performed using a genome diagnosis company (GenDix Inc., Seoul, Korea).

9. Teratoma formation and histochemical analysis

In order to confirm the pluripotency of hPSCs cultured in cross-linked X-FDM (Gx0.5), hPSC 3×10 ⁶ cells cultured 10 times were used in 6-week-old, non-obese, diabetic/severe immunodeficient mice. (6-week-old, non-obese, diabetic/severe combined immunodeficiency; NOD/SCID). After injection, rats were bred for 12 weeks, and then obtained by removing the formed teratomas, paraffin-embedded, and then sequential sections were prepared in a size of 5 μm using a microtome (Leica Microsystems, Wetzlar, Germany). Three-type germ layers were identified by histochemical staining. For histochemical staining, the epithelium (gut epithelium) is stained with hematoxylin and eosin, the secretory epithelium is stained with periodic acid shiff (PAS), and the cartilage tissue is alcian blue. was stained with, and muscle fibers were stained with Masson's trichrome staining method. Each image was analyzed using an inverted microscope (Nikon, Chiyoda-ku, Japan).

Morphological characterization of fibroblast-derived extracellular matrix (FDM) and its cross-linked extracellular matrix (X-FDM)

First, the shape of the prepared FDM was observed. When the matrix before and after the decellularization process was optically imaged, it was confirmed that the cell structure was almost removed from the cells in a saturated state through the surface shape of the matrix. Accordingly, when fibronectin and laminin, which are major ECM proteins, were confirmed through immunofluorescence staining, it was confirmed that the structures were maintained even after decellularization in FDM ( FIG. 2a ). In addition, when major ECM proteins were quantitatively analyzed through ELISA analysis, it was confirmed that ColI was maintained before and after decellularization, but other ECM proteins showed a somewhat decreased level of protein concentration after than before decellularization (Fig. 2b). ). The new unused FDM (Gx0) showed a mean root mean square (RMS) of 135±45 nm, whereas the roughness of the surface increased at Gx0.5, Gx1.0 and Gx2.0, It was confirmed that the average RMS of 189±35 nm, 228±27 nm, and 344±35 nm, respectively, was shown ( FIG. 2c ).

In addition, the same experiment was performed through X-FDM, and it was confirmed that FN, LN, Col I and VN were well preserved even after treatment with genipine. When the surface shape of both the FDM and X-FDM substrates was magnified through SEM images, it was confirmed that the fine fibers were randomly distributed regardless of the concentration of Jennifin to show a self-assembled shape (Fig. 3a). ). When the fiber structure of FDM was observed through AFM, it was confirmed that the topographic difference between each group of FDM and X-FDM was observed (FIG. 3b).

In addition, as a result of measuring the Young's modulus (E) to confirm the strength of the substrate, the Young's modulus increased as the concentration of jennypin increased, so Gx0 represents a Young's modulus of 118±51 Pa, and Gx0.5, Gx1.0 and Gx2 It was confirmed that .0 represents the Young's modulus of 800±180, 5600±1100, and 8900±1500 Pa, respectively (FIG. 2d).

It was confirmed that the force-displacement curve also showed a significantly different pattern between Gx0, Gx0.5, Gx1.0 and Gx2.0. From the indentation force of Gx0, the strength gradually increased as the concentration of jennifin increased, whereas a more rigid matrix (eg, Gx2.0) had a rigid surface at a specific point representing a rigid surface. It was confirmed that the indentation strength rapidly increased (FIG. 2e).

Through these results, it was confirmed that the crosslinking of FDM with genipine is directly involved in the roughness and strength of the matrix, which can be changed depending on the concentration of genipine.

Identification of culture characteristics of human pluripotent stem cells cultured in FDM and X-FDM

In order to confirm the cell adhesion ability in the FDM and X-FDM of the present invention, the cells were cultured by inoculating hPSCs at a density of 1×10 ⁵ cells/cm ² ( FIG. 4 ). While it was confirmed that cell adhesion was most desirable in Gx0, it was confirmed that the number of cells adhered to the matrix decreased sharply as the strength of the matrix increased (FIG. 5a). When Gx0, Gx0.5, Gx1.0 and Gx2.0 were AP-stained and live cells were stained to confirm cell adhesion and initial pluripotency, the number of AP-positive colonies was inversely proportional to the matrix strength. was confirmed to be indicated. The cell adhesion rate was confirmed by the number of AP-positive colonies, and as a result, it was confirmed that the cell adhesion rates of Gx0, Gx0.5, Gx1.0 and Gx2.0 were 85%, 80%, 55% and 43%, respectively. (Fig. 3b).

When the cells were grown and expanded by inoculating hPSCs in the medium and culturing for 5 days, Gx0 showed the most favorable degree of cell proliferation in terms of colony formation (Fig. 6), in contrast to the colonies of cells cultured in X-FDM substrate. It was confirmed that the form was shown (Fig. 5c). After 1 to 5 days of initiation of culture, the size of the colonies changed significantly, and the cell proliferation rate was the highest in Gx0, which increased by about 5.7 times, and the cell colonies cultured in Gx2.0 increased by 2.3 times for 5 days, resulting in the lowest proliferation rate. was confirmed to represent (Fig. 5d).

To confirm that the differentiation capacity of hPSCs cultured on FDM or X-FDM is maintained, AP staining was performed 7 days after initiation of culture. As a result, it was confirmed that AP expression was gradually decreased in the experimental group of Gx0 (black arrow in FIG. 5e ), whereas it was confirmed that such a decrease in AP expression did not appear in X-FDM ( FIG. 5e ). Although the colony size of the Gx0.5 experimental group was smaller than the colony size of the Gx0 experimental group, AP expression was maintained for most hPSCs, and it was confirmed that the growth stagnant phenomenon did not appear in the Gx1.0 and Gx2.0 experimental groups (Fig. 5e). indicated by the white arrow). As a result of quantifying AP-positive hPSCs to more specifically confirm the degree of pluripotency maintenance, it was confirmed that the number of AP-positive colonies was related to the concentration of genipin added. AP expression significantly increased as the concentration of jenipin in the culture substrate increased, indicating 65%, 85%, 90% and 95% in Gx0, Gx0.5, Gx1.0 and Gx2.0, respectively (Fig. 5f).

In addition, it was confirmed that hPSC cultured in Gx0 not only exhibited migration activity of peripheral cells on the 8th day of culture (red arrow in FIG. blue arrow). In contrast, cells cultured in Gx0.5 maintained a fixed state, and it was confirmed that the morphology was also consistently maintained. When hPSCs were cultured at Gx0.5 for 14 days, it was confirmed that hPSCs could be cultured while maintaining the same substrate, indicating that as the strength of the matrix increased, the maintenance period of the matrix could also be significantly increased. It was confirmed that (Fig. 7b). When culturing hPSCs, it was confirmed that the period of culture in Gx2.0 could be maintained up to 18 days, but it was confirmed that most hPSCs did not adhere or showed a tendency to attach loosely after attaching to the Gx2.0 substrate ( Fig. 8).

Through this, in maintaining cell growth and undifferentiated state, it was confirmed that the Gx0.5 matrix could be the most desirable platform for hPSC preservation, expansion, and long-term culture ( FIG. 9 ).

Characterization of epithelial-mesenchymal transition (EMT) of human pluripotent stem cells cultured in FDM and X-FDM

In order to determine how the strength of FDM affects hPSC culture, hPSCs were cultured in Gx0, Gx0.5, Gx1.0 and Gx2.0 matrices for 7 days, then cells were obtained, and epithelial and mesenchymal characteristics The relative expression levels for four genes known to be related to (OCT4, E-cadherin, N-cadherin and vimentin) were confirmed through qPCR analysis. The expression levels of OCT4 and E-cadherin, which are pluripotency and epithelial markers, were significantly increased in both the Gx0.5 and Gx2.0 experimental groups, whereas the expression levels were decreased in the Gx0 experimental group. In contrast, the mesenchymal markers, N-cadherin and vimentin, significantly increased their expression levels in the Gx0 experimental group, whereas their expression levels were relatively low in hPSC cultured in X-FDM, indicating a similar expression level to that of the control cells. was confirmed (FIG. 10a).

In order to once again confirm the change in the gene expression level, immunostaining was performed for E-cadherin and DAPI in each experimental group to confirm the expression level change at the cellular level. In the Gx0 experimental group, the expression level of E-cadherin was decreased, but it was confirmed that the migration ability was increased in the area around the hPSC colonies ( FIGS. 10b and 10c ). On the other hand, it was confirmed that the Gx0.5 experimental group showed a tendency to slightly decrease the expression level of E-cadherin compared to the Gx2.0 experimental group, but E-cadherin expression in hPSC colonies was localized in both the Gx0.5 and Gx2.0 experimental groups. It was confirmed that the expression is maintained in an aggressive manner, and migration ability does not appear ( FIGS. 10b and 10c ).

In order to analyze the degree of cell migration in more depth, the hPSC colonies were enlarged through SEM analysis. As a result of observing at the micro level, it was observed that ECM cleavage clearly appeared in the periphery of Gx0 (white arrow in Fig. 10c). On the other hand, it was confirmed that damage to the ECM did not appear in both Gx0.5 and Gx2.0 (black arrow in FIG. 10c ). These results were confirmed to be consistent with the TEM image analysis results confirmed at the cellular level. In the Gx0 experimental group, it was confirmed that the function as a barrier of the epithelium was lost (white arrow in Fig. 10d), as intercellular separation frequently appeared at the edge of the colony. However, in the hPSC cultured in X-FDM, it was confirmed that cells were well connected to each other through dense junctions between cells in both the periphery and the center (black arrow in Fig. 10d).

Based on these results, it is shown that the new unused FDM can induce cell migration and cadherin switch, whereas in X-FDM, E-cadherin expression is maintained to maintain colony integration. Confirmed.

Analysis of genes whose expression level changes in the epithelial-mesenchymal transition program

In order to more specifically confirm the mechanism specifically shown in hPSCs cultured on FDM and X-FDM, the expression patterns of 84 genes known to be changed or regulated by EMT were confirmed by qRT-PCR array analysis. To confirm the change in gene expression level in hPSCs, hPSCs were cultured at Gx0 and Gx0.5 for 5 days, and hPSCs were cultured in mTeSR ^TM -1-Matrigel as a control ( FIG. 11 ).

As a result of analyzing the difference in expression level change and creating a scatter plot, the expression level is gene-dependent, but most of the genes related to EMT showed an increase in expression level in Gx0 compared to that of mTeSRTM1. . These patterns were confirmed to be similar to each other between Gx0 and Gx0.5.

However, it was confirmed that the expression levels of these genes were similar in mTeSR ^TM -1 and Gx0.5 ( FIG. 12a ). When these genes with changed expression levels were analyzed by dividing them into functional groups, the expression levels of genes related to EMT induction and cell migration, such as VIM, TGFB, SLUG (SNAI2) and EGFR, were increased in the Gx0 group, whereas CDH1 and Tumor suppressor genes such as CAV2 were confirmed to exhibit a tendency to be down-regulated (Fig. 12b). In addition, when looking at the list of all genes whose expression level is changed more than two-fold, it was confirmed that the EMT-induced transcription factor ZEB1 and its analog ZEB2 also increased in Gx0 compared to the Gx0.5 experimental group (Fig. 12c). ).

In order to more specifically confirm the interaction between genes based on previously known gene expression patterns, the EMT-gene regulatory network was constructed in silico . was confirmed to be co-expressed with (Fig. 13a). ZEB1 is known to act as a regulator in relation to TGF-β signaling and thus is involved in numerous cellular processes including EMT. In addition, when the expression levels of ZEB1, E-cadherin and vimentin in the Gx0 experimental group and the Gx0.5 experimental group were confirmed by immunofluorescence staining, it was confirmed that the expression levels were increased in the early stages of cell migration and EMT. In the Gx0 experimental group, the expression level of vimentin was stronger in the periphery, and it was confirmed that the expression level of ZEB1 was not only stronger in migratory cells (white arrow in FIG. 13b) but also weaker in the expression level of E-cadherin. . In contrast, in the Gx0.5 experimental group, ZEB1 expression sites were dispersed (white arrow in Fig. 13c), but it was confirmed that E-cadherin was intensively expressed inside the colony (Figs. 13b and 13c).

These results were consistent with the PCR results, and it was confirmed that both ZEB1, a major gene inducing EMT progression, and the SLUG gene, which suppresses E-cadherin transcription, were downregulated in the Gx0.5 experimental group. These results indicate that the induction of matrix-related cell migration and EMT increased in the Gx0 experimental group, which could be inhibited as the strength of FDM increased as confirmed in Gx0.5.

Identification of changes in gene expression levels related to focal adhesion

Cell adhesion and signal transmitters such as integrin and protein kinase are known as major regulators in local adhesion and behavior of hPSCs, so the present inventors further selected 84 genes related to local adhesion and confirmed their expression patterns. . Similar to the EMT array, the expression of most genes associated with local adhesion was increased in hPSC cultured in Gx0, and the difference in expression level showed a significantly increased difference compared to the control, mTeSR ^TM -1 (Fig. 14). . By analyzing the difference in gene expression, it was confirmed that various genes related to integrin, such as ITGAV, as well as its subunits ITGB1 (fibronectin) and ITGB3 (vitronectin) were strongly expressed in the Gx0 experimental group. In addition, focal adhesion kinase (FAK) signal It was confirmed that the expression of the transduction pathway and its sub-target gene, AKT, was also upregulated ( FIG. 15 ). In addition, it was confirmed that the expression level of the CRK gene, which is a gene known to play various roles in cell migration and differentiation, among the genes showing a 2-fold or more difference, was strongly increased in the Gx0 experimental group ( FIG. 16 ).

Although the mechanisms regulating signal transduction are not fully understood to date, the gene regulatory network involved in local adherence can be achieved through the accumulation of responses related to cell adhesion and motility in hPSCs cultured on Gx0, expression levels of related genes. It can be understood as indicating this change, and in the present invention, this was confirmed through functional analysis and EMT analysis (FIG. 17). Through these results, it was found that hPSC cultured in Gx0 increased the expression of any signal substances that induce cell adhesion, migration and differentiation, whereas in the Gx0.5 experimental group, signal transduction related to cell adhesion and migration ability was significantly changed. Confirmed.

Characterization of long-term cultured hPSCs on Gx0.5

Among the FDMs and X-FDMs prepared in the present invention, it was confirmed that Gx0.5 could be used as the most optimized matrix for hPSC culture. This is because, even in hPSCs cultured on Gx0.5 for 5 days or more, the morphological characteristics of undifferentiated hPSCs can be maintained ( FIG. 18 ). Accordingly, the present inventors performed immunostaining to confirm the expression levels of pluripotency markers OCT4, SSEA4, TRA-1-60 and SOX2. As a result, it was confirmed that the expression level of OCT4 was strongly expressed even in colonies that were subcultured 5 times (FIG. 19a). As a result of quantitatively analyzing the OCT4 expression level, the expression level of OCT4 in hPSC cultured on Gx0.5 decreased slightly from the time the subculture was repeated 3 times. recovery was confirmed. This trend was not observed in the case of Gx0, and it was confirmed that the expression level of OCT4 decreased sharply with each subculture order ( FIG. 19b ). In the case of other pluripotency markers SSEA4, TRA-1-60, and SOX2, it was confirmed that they were strongly expressed in the Gx0.5 experimental group even when subcultures were performed 10 times (FIG. 20).

Immunofluorescence staining of the embryoidbody (EB) formed by culturing hPSCs was performed to confirm the expression of AFP, TuJ1, and PECAM, and it was confirmed whether the EB maintains the differentiation ability to differentiate into three types of lineages: endoderm, mesenchymal and ectoderm. (Fig. 19c). In addition, it was confirmed that the cells maintained the karyotype of normal cells through chromosome analysis (FIG. 19d). In addition, the result of inducing teratoma formation in immunodeficient mice and performing histochemical immunoassay on the formed teratoma, and maintaining the pluripotency to differentiate into endoderm, mesenchymal and ectoderm lineages in the Gx0.5 experimental group was confirmed (FIG. 19e).

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> A Cross-linked extracellular matrix and a Culturing method of human pluripotent stem cells using them <130> 1062881 <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_OCT4_F <400> 1 aactcgagca atttgccaag ctcc 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_OCT4_R <400> 2 ttcgggcact gcaggaacaa attc 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_E-cadherin_F <400> 3 cgagagctac acgttcacgg 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer_E-cadherin_R <400> 4 gggtgtcgag ggaaaaatag g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer_N-cadherin_F <400> 5 tcacagattc gggtaatcct c 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_N-cadherin_R <400> 6 tgcagctggc tcaagtcata 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_Vimentin_F <400> 7 aagtttgctg acctctctga ggct 24 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer_Vimentin_R <400> 8 ttccattttc acgcatctgg cgtttc 26 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_GAPDH_F <400> 9 gtggggcgcc ccaggcacca gggc 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_GAPDH_R <400> 10 ctccttaatg tcacgcacga tttc 24

Claims (5)

i) culturing fibroblasts in a saturated state;
ii) decellularizing the cells cultured in step i) to obtain an extra-cellular matrix (ECM);
iii) Treating the ECM obtained in step ii) with genipin at a concentration of 0.5% (w/v) to 2.0% (w/v) as a cross-linker to prevent cross-linking between substrates inducing; and
iv) obtaining the cross-linked ECM in step iv);
delete delete A method of culturing human embryonic stem cells, including; inoculating and culturing human embryonic stem cells in the ECM prepared by the method of claim 1 .
The method of claim 4, wherein the culture method is a method of inducing proliferation of human embryonic stem cells while maintaining them in an undifferentiated state.

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