WO2021241781A1

WO2021241781A1 - Recombinant-phage-based container for stem cell culture, and use thereof

Info

Publication number: WO2021241781A1
Application number: PCT/KR2020/006920
Authority: WO
Inventors: 권상모; 장웅비; 임혜지
Original assignee: 부산대학교 산학협력단
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2021-12-02

Abstract

The present invention relates to a recombinant-phage-based container for a stem cell culture, and a use thereof. More specifically, provided are: a recombinant-phage-based container for a stem cell culture, on which cysteamine and a recombinant phage displaying a cell delivery peptide on the coat protein are coated; and a preparation method therefor. According to the present invention, a recombinant-phage-based container for a stem cell culture, of the present invention, provides a physical and mechanical niche environment created by the formation of a nanofibrous structure of the phage and promotes an increase in the physiological activity of target cells so as to help in tissue regeneration, and thus can be usable as an excellent therapeutic agent for corresponding diseases.

Description

Recombinant phage-based vessel for stem cell culture and use thereof

The present invention relates to a recombinant phage-based container for culturing stem cells and uses thereof.

Cardiac progenitor cells are stem cells that exist in the heart and are involved in tissue regeneration such as angiogenesis and myocardial regeneration. Cardiovascular disease is one of the diseases with the highest mortality rate in the world, and the incidence of such heart disease is increasing due to various causes such as stress, bad eating habits, smoking, lack of exercise, and diabetes.

As a treatment for this, various surgical therapies such as drug therapy, heart transplantation, and stent surgery have been developed.

Therefore, as an alternative to such low-efficiency treatment methods, cell therapy using cells in the ischemic region has been proposed.

In the case of cell therapy using such stem cells, the use as a therapeutic agent is limited due to the low proliferative capacity and cell function that occur during in vitro culture. Accordingly, a new culture method for securing the number of stem cells and enhancing their functions is required.

As a conventional therapy for cardiovascular regeneration, there are methods such as administration of stem cells alone or growth factors, but there is a functional limitation in actually proliferating and differentiating vascular cells. When stem cells are used alone, the engraftment and survival rates of the administered stem cells are low, there is a limit in controlling proliferation and differentiation even when the stem cells are alive, and single administration of a growth factor provides only a one-time effect. There is a limit to producing a lasting effect.

On the other hand, in order to enhance the function of stem cells, there are methods for enhancing the function of stem cells by treating growth factors, synthetic compounds, compounds derived from natural products, etc. There is a limit to enhancing adhesion and regulating cell proliferation and function.

In order to solve this problem, it is considered important to implement a cell microenvironment capable of enhancing the adhesion and function of stem cells during in vitro culture and to develop a plate that mimics the cell microenvironment.

There are several methods for implementing a cell microenvironment in a cell culture medium. Among them, M13 phage display expresses various peptides in genes based on M13 bacteriophage that specifically infects bacteria. It is a technology for arranging on a substrate using recombinant bacteriophages. In the case of such phage display technology, it is harmless to the human body and has already been verified based on peptide delivery technology for enhancing the function of stem cells.

According to recent research results in the field of tissue regeneration and tissue engineering, the importance of the cell microenvironment, that is, the “niche”, which corresponds to the external environment that can control the proliferation and differentiation of cells, is emerging. It is considered important to construct a simulated environment with biochemical and structural characteristics that can simulate the red varnish.

Phage display that effectively implements such an intracellular environment and promotes proliferation and function of cardiac progenitor cells may be used.

There are several techniques for exploring protein-protein interaction, among which phage display is a parasite that specifically infects bacteria, bacteriophage genes. It is a technology that selects an unknown amino acid sequence capable of binding to a specific protein using recombinant bacteriophage produced by artificially introducing a gene that produces various amino acid sequences. Identification of antigenic determinants (epitope mapping), vaccine development, effector-receptor Its utility has been verified in various fields such as ligand-receptor affinity research and selection of bioactive peptides. In the case of the representative M13 phage display system, the gene sequence is artificially expressed so that peptides of 7 to 15 random amino acid sequences are expressed at the end of the gene that produces pIII or pVIII, a kind of coat protein, in the genome of M13 bacteriophage. After insertion, the recombinant bacteriophage expressing more than hundreds of millions of different peptides obtained by infection with E. coli goes through a series of processes called biopanning to select phages showing strong binding ability with specific proteins. has been devised By extracting genomic DNA from the selected bacteriophage and analyzing the DNA sequence expressing the artificially inserted specific peptide, the desired functional peptide can be obtained.

That is, the phage display uses the genetic information of the phage to express a specific envelope protein on the surface of the phage, and uses the surface protein of the phage surface to recognize the cell and the cell proliferation and differentiation into blood vessels through the surface protein. In addition to being able to induce and control, it is possible to provide a physical and mechanical varnish environment created by the formation of nanofibrous structures of phages.

Therefore, if cardiovascular regeneration is induced by supplying cardiac progenitor cells cultured by phage-based plates with enhanced physiological activity of stem cells to places where irreversible tissue necrosis has occurred in ischemic diseases, etc., it would be possible to overcome the limitations of current cardiovascular therapeutics. will be able

The present inventors made diligent efforts to develop a therapeutic agent for cardiovascular disease and a treatment method. As a result, stem cells were cultured by coating M13 phage expressing cell transduction signal peptide on the surface by recombinant genetic engineering technique using structural characteristics of M13 phage, which is non-toxic to human tissue, coated with antioxidant cysteamine. When, the stem cells cultured by the phage-based plate have completed the present invention by confirming that adhesion, proliferation, migration and angiogenesis are excellently enhanced while maintaining the intrinsic properties of stem cells.

Accordingly, an object of the present invention is cysteamine (Cysteamine); And to provide a recombinant phage-based container for culturing stem cells coated with a recombinant phage in which a cell delivery peptide is displayed on a coat protein.

In addition, another object of the present invention is to provide a method for manufacturing a recombinant phage-based vessel for culturing the stem cells.

In addition, another object of the present invention is to provide a method for improving the adhesion, proliferation, migration and angiogenesis of stem cells, comprising the step of culturing the stem cells in the recombinant phage-based vessel for culturing the stem cells. there is

In addition, another object of the present invention is to provide a method for producing stem cells for cardiovascular regeneration, comprising the step of culturing the stem cells in the recombinant phage-based vessel for culturing the stem cells.

The terminology used herein is used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention is cysteamine (Cysteamine); And it provides a recombinant phage (Recombinant Phage) for displaying a cell delivery peptide on the coat protein; a recombinant phage-based container for culturing stem cells is coated.

Cysteamine used in the phage-based stem cell culture plate of the present invention is an antioxidant, and is a compound identified as CAS No. 156-57-0.

In the present invention, the cysteamine concentration is not limited as long as the desired effect is achieved in the present invention, but is preferably 1 mM to 1 M, more preferably 10 mM to 100 mM, and most preferably 50 mM.

The term "recombinant phage-based container for stem cell culture" used in the present invention refers to a recombinant phage in which a cell delivery peptide is displayed on a coat protein after coating with cysteamine. ) means a coated cell culture vessel, and is used interchangeably with “phage-based culture (for) plate” herein.

The vessel may be in the form of a cell culture plate, a Petri dish, a culture flask, a chamber slide, a chamber, or a tube, but is not limited thereto, but in an embodiment of the present invention, a plate is used.

In addition, the cell culture vessel may be at least one material selected from the group consisting of glass, polystyrene, polycaprolactone and polypropylene, but any material suitable for attachment of target cells may be used without limitation.

As used herein, the term "cell transduction peptide" is used interchangeably with "signal peptide" and "signal sequence", and corresponds to a cell adhesion motif. Accordingly, longer amino acid sequences including the cell adhesion amino acid sequence as an essential sequence are also included in the scope of the present invention.

According to a preferred embodiment of the present invention, the cell transfer peptide of the present invention is RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser), RGDC (Arg-Gly-Asp-Cys), RGDV (Arg -Gly-Asp-Val), RGES (Arg-Gly-Glu-Ser), RGDSPASSKP (Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), GRGDS (Gly-Arg-Gly- Asp-Ser), GRADSP (Gly-Arg-Ala-Asp-Ser-Pro), KGDS (Lys-Gly-Asp-Ser), GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP (Gly- Arg-Gly-Asp-Thr-Pro), GRGES (Gly-Arg-Gly-Glu-Ser), GRGDSPC (Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP (Gly-Arg-Gly-Glu) -Ser-Pro), SDGR (Ser-Asp-Gly-Arg), YRGDS (Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala- Lys-Gln-Ala-Gly-Asp-Val), GPR (Gly-Pro-Arg), GHK (Gly-His-Lys), YIGSR (Tyr-Ile-Gly-Ser-Arg), PDSGR (Pro-Asp- Ser-Gly-Arg), CDPGYIGSR (Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR (Leu-Cys-Phe-Arg), EIL (Glu-Ile-Leu), EILDV ( Glu-Ile-Leu-Asp-Val), EILDVPST (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST (Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV (Leu-Asp-Val) and LDVPS (Leu-Asp-Val-Pro-Ser), more preferably RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser) , RGDC(Arg-G ly-Asp-Cys), RGDV (Arg-Gly-Asp-Val) and RGES (Arg-Gly-Glu-Ser), most preferably RGD (Arg-Gly-Asp).

Said "Arg-Gly-Asp peptide" or "RGD peptide" refers to one or more Arg- containing sequences capable of serving as a binding site for the "Arg-Gly-Asp family of receptors", eg, integrins, for receptors. It is intended to refer to a peptide having Gly-Asp. RGD peptides also include peptides having amino acids that are functional equivalents of RGD peptides when interacting with the same RGD receptor.

Peptides containing the RGD sequence of the present invention can be synthesized from amino acids by means well known in the art.

As used herein, the term “phage” or “bacteriophage” is a kind of virus that infects bacteria and is also called phage. A phage is an organism with a simple structure in which a protein coat surrounds the center of a genetic material composed of nucleic acids. Nucleic acids are composed of single-stranded or double-stranded DNA or RNA. This nucleic acid has a simple structure surrounded by a protein coat, and is divided into three basic structures: an icosahedral head with a tail, an icosahedral head without a tail, and a filamentous type. The most common icosahedral head-tailed bacteriophages are Myoviridae with contractile tails, Siphoviridae with long, non-contractile tails, and grapes with short tails, depending on their tail characteristics. It can be subdivided into Podoviridae. Bacteriophages without an icosahedral head and no tail are subdivided according to the shape of the head, the composition of the head, and the presence or absence of an integument. Finally, filamentous bacteriophages are subdivided according to size, shape, integument and filament composition.

The type of the phage is not limited, and may be T1, T2, T4, T6 or λ (lambda), μ (mu), M13, and the like, and M13 is used in the present invention.

The recombinant phage used in the present invention expresses the target peptide by artificially inserting the gene sequence so that the target peptide of 3 to 15 amino acid sequence is expressed in the gene for producing the envelope protein in the genome of the M13 phage.

The phage is composed of various coat proteins, and may be composed of, for example, P3(PIII), P6(PVI), P7(VII), P8(VIII), P9(IX), and the like.

According to a preferred embodiment of the present invention, the envelope protein is at least one selected from the group consisting of P3, P6, P7, P8 and P9.

In the present invention, the recombinant phage includes a peptide in one or more proteins selected from the group consisting of P3, P8 and P9.

In addition, the recombinant phage used in the present invention may include a cell delivery peptide consisting of 5 to 10 amino acid sequences including RGD in P8, a major coat protein, and most preferably includes RGD. .

That is, in the recombinant phage, RGD having cell affinity is expressed in the major coat protein P8, and has a genome composed of the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the concentration of the recombinant phage (Recombinant Phage) is not limited as long as it achieves the desired effect in the present invention, but is preferably 0.01 mg/ml to 10 mg/ml, more preferably 0.1 mg/ml to 1 mg/ml, and in the present invention, 0.25 mg/ml concentration was used.

In addition, according to a preferred embodiment of the present invention, the stem cells cultured by the recombinant phage coated on the plate for stem cell culture of the present invention maintain adhesion, proliferation, migration and angiogenesis ( angiogenesis) is significantly improved.

Preferably, the stem cells are cardiac progenitor cells (CPC), Endothelial Progenitor Cells (EPCs), Endothelial Colony Forming Cells (ECFCs), Vasculogenic Progenitor Cells (VPCs), Mesenchymal Stem Cells. , is selected from the group consisting of embryonic stem cells (Embryonic Stem Cells) and myoblasts (Myoblasts), more preferably CPC (cardiac progenitor cell), EPCs (Endothelial Progenitor Cells), ECFCs (Endothelial Colony Forming Cells) and VPCs ( Vasculogenic Progenitor Cells), most preferably a cardiac progenitor cell (CPC).

The recombinant phage-based container for stem cell culture is characterized in that it does not contain feeder cells.

That is, the recombinant phage-based container for stem cell culture of the present invention uses phage recombined so that a specific envelope protein is expressed on the surface of the phage by coating cysteamine, an antioxidant, without a feeder cell. By using the envelope protein on the surface of the phage, the function of the cell can be improved through the surface protein that recognizes the target cell, the stem cell (preferably, CPC), as well as by the formation of the nanofibrous structure of the phage. By providing the generated physical and mechanical nish environment, it is possible to promote adhesion, proliferation, migration and angiogenesis while maintaining the intrinsic properties of stem cells of cardiac progenitor cells.

Accordingly, the present invention provides the advantage of a plate for culturing stem cells capable of culturing a large amount of stem cells while increasing the physiological activity of the above-described target stem cells at an economical cost in vitro.

In addition, according to another aspect of the present invention, the present invention is cysteamine (Cysteamine); and a recombinant phage displaying a cell delivery peptide on a coat protein; and coating a cell culture vessel with a recombinant phage-based vessel for stem cell culture. do.

Since the method of the present invention is to prepare a recombinant phage-based vessel for stem cell culture using the above-described cysteamine and recombinant phage, the common content is omitted in order to avoid excessive complexity of the present specification according to repeated description. do.

In addition, the present invention provides a method for culturing stem cells using the above-described recombinant phage-based vessel for culturing stem cells.

In addition, according to another aspect of the present invention, the present invention improves the adhesion, proliferation, migration and angiogenesis of stem cells, comprising the step of culturing the stem cells in the recombinant phage-based vessel for culturing the stem cells. provides a way to do it.

In addition, according to another aspect of the present invention, the present invention provides a method for producing stem cells for cardiovascular regeneration, comprising the step of culturing the stem cells in a recombinant phage-based vessel for culturing the stem cells, wherein the stem cells are It is characterized in that it is a stem cell with improved adhesion, proliferation, migration and angiogenesis.

Since the cultured stem cells, preferably cardiac progenitor cells, have improved adhesion, proliferation, mobility and angiogenesis while maintaining the intrinsic properties of cardiac progenitor cells, they can be applied for treatment of ischemic diseases. .

The ischemic disease may be selected from the group consisting of ischemic heart disease, ischemic brain disease, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic baldness and ischemic lower extremity disease.

The ischemic heart disease may be myocardial infarction, heart failure or angina pectoris.

The recombinant phage-based container for stem cell culture of the present invention provides a physical and mechanical nish environment generated by the formation of a nanofibrous structure of phage to enhance the physiological activity of target cells, and thus ultimately helps specific tissue regeneration It can be applied to the manufacture of excellent therapeutic agents for target diseases, and through this, stem cell therapeutics with improved functions can be effectively produced.

1 is a genetic schematic diagram of a recombinant M13 bacteriophage. The results of sequencing of the phage including the RGD sequence in the P8 region, which is the outer part of the phage, are shown.

Figure 2 schematically shows the manufacturing process of the plate for phage-based culture of the present invention.

Figure 3a shows the results of CCK assay confirming the adhesion ability of cardiac progenitor cells cultured in the phage-based culture plate of the present invention. Figure 3b is the result of confirming the proliferative capacity of cardiac progenitor cells cultured in the phage-based culture plate of the present invention (day 1, day 3). Figure 3c shows the morphology of cells cultured in the phage-based culture plate of the present invention.

4 shows the FACS results confirming the expression of markers of cardiac progenitor cells cultured in the phage-based culture plate of the present invention.

Figure 5a shows the results of the scratch wound healing assay confirming the cell migration ability of cardiac progenitor cells cultured in the phage-based culture plate of the present invention. 5B is a graph showing the quantification of the above results.

6A shows the results of a tube-formation assay confirming the angiogenic performance of cardiac progenitor cells cultured in the phage-based culture plate of the present invention. 6B is a graph quantifying the results.

Hereinafter, the examples are only for explaining the present invention in more detail, and those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention It will be self-evident for

실시예 1. 파지-기반 배양 플레이트의 제작 및 배양Example 1. Fabrication and Culture of Phage-Based Culture Plates

The present inventors prepared a functional M13 phage-based stem cell culture plate as follows to obtain stem cells with improved physiological activity, necessary for application to ischemic cardiovascular disease.

Briefly, as shown in Figure 2, the plate was coated with cysteamine (Cysteamine, 50 mM) for 4 hours, washed, and the M13 bacteriophage was suspended in PBS and coated by concentration to proceed with the experiment.

At this time, M13 phage (New England Biolabs) is a M13-RGD phage (SEQ ID NO: 1; MLSFFAGGRGDSDDYDPAK) and wild-type phage (SEQ ID NO: 2) expressing RGD having cell affinity in the p8 region, the main envelope protein, based on the genetic recombination technique. ; MLSFAAEGDDPAKAAFN) was used (FIG. 1).

실시예 2. 본 발명의 재조합 파지-기반플레이트에 의한 줄기세포 활성 증진 확인Example 2. Recombinant phage of the present invention - Confirmation of enhancement of stem cell activity by the based plate

The present inventors confirmed in vitro the physiological and functional activity level of stem cells obtained by the recombinant phage-based plate culture system constructed in Example 1 above.

2-1. Increased adhesion and proliferation capacity of stem cells by the recombinant phage-based plate of the present invention

To evaluate the function of stem cells improved by the recombinant phage-based plate culture system of the present invention, CCK assay is performed, and the recombinant phage-based plate culture system cultured, cardiac stem cells, cardiac progenitor cells (cardiac progenitor cells) , CPC) was checked for adhesion and proliferation rate.

Briefly, cell proliferation was performed according to the manufacturer's protocol using a CCK8-assay kit (CCK-3000, Donginbio tech). 10000 cells were cultured in a 96-well plate for one day, and the cell proliferation ability was verified the next day using CCK8-kit.

At this time, phage concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg/ml were used, and as a control, cardiac progenitor cells obtained by culturing on a plate coated with wild type phage (WT) were used for comparison. .

As a result, as shown in FIG. 3a , compared with the control group of wild type phage, cardiac progenitor cells cultured on a plate coated with RGD phage significantly increased their adhesion in a RGD phage-concentration-dependent manner. confirmed that.

In addition, as shown in FIG. 3b , it was confirmed that the proliferative capacity of cardiac progenitor cells cultured on a plate coated with RGD phage was significantly improved when compared with the control group, a wild type phage group.

Figure 3c shows the morphology of cells in the phage-based culture plate of the present invention.

2-2. Maintaining the intrinsic properties of stem cells by the recombinant phage-based plate of the present invention

In order to evaluate whether the intrinsic properties of stem cells are maintained by the recombinant phage-based plate culture system of the present invention, the recombinant phage-based plate culture system is expressed in cardiac progenitor cells (CPC). Markers were confirmed by FACS .

At this time, FACS dispensed 200,000 cells into each group on a 60mm² plate, removed the cells from the plate, and treated fluorescence-conjugated antibody for flow cytometry in FACS buffer at a concentration of 1:100, followed by 30 minutes at 4°C. Block the light and react. Then, after washing in FACS buffer, it was measured by a flow cytometry machine.

In this case, a phage concentration of 0.25 mg/ml was used, and as a control, cardiac progenitor cells obtained by culturing on a plate coated with wild type phage (WT) were used for comparison.

As a result, as shown in FIG. 4, when compared with the control group, a wild type phage group, positive for CD44, CD105 and CD29, which are known markers of CPC; It was shown to be negative for CD34 and CD45, which means that the stem cells cultured by the recombinant phage-based plate culture system of the present invention maintain their intrinsic capacity.

2-3. Recombinant phage-based plate of the present invention to increase the mobility of stem cells

To evaluate the function of stem cells improved by the recombinant phage-based plate culture system of the present invention, a scratch wound healing assay was performed, and cardiac progenitor cells (CPCs) cultured by the recombinant phage-based plate culture system were performed. ) was confirmed.

Briefly, in order to compare and verify cell migration ability, 200,000 cells were dispensed in a 12-well plate, incubated until the cells were filled in the wells, and scratched using a yellow tip, and then washed with PBS. After incubation for 3 hours, the wound distance of the cells was imaged on a microscope.

As a result, as shown in FIGS. 5A and 5B , it was confirmed that the migration ability of cardiac progenitor cells cultured on a plate coated with RGD phage was significantly improved as compared to the control group, a wild type phage group. .

2-4. Increased angiogenic ability of stem cells by the recombinant phage-based plate of the present invention

The recombinant phage-based plate culture system of the present invention conducted a tube-formation assay to evaluate the improved stem cell function, and the recombinant phage-based plate culture system of cardiac progenitor cells (CPC). Mobility was confirmed.

Briefly, after coating a 96-well plate with 55 μl growth factor-reduced matrigel (BD bioscience), 8000 cells were aliquoted, and _{cultured at 5% CO 2} , 37° C. for 6 hours. The resulting tube was observed and imaged under a microscope. The number and length of all formed tubes were quantified using Image J.

As a result, as shown in FIGS. 6A and 6B , it was confirmed that the angiogenic performance of cardiac progenitor cells cultured on a plate coated with RGD phage was significantly improved when compared to a control group of wild type phage. did

In conclusion, the method for culturing stem cells using the phage-based culture plate coated with cysteamine and RGD phage of the present invention is optimized to increase the efficacy of stem cells compared to the case of using wild-type phage. provide a way

It can be seen that the culture of stem cells using such a phage-based culture plate is not only economically very useful, but also very suitable for large-scale culture.

Claims

Cysteamine (Cysteamine); And Recombinant Phage (Recombinant Phage) for displaying the cell delivery peptide on the coat protein (coat protein); Recombinant phage-based container for stem cell culture is coated.
According to claim 1,

The recombinant phage-based vessel for stem cell culture is characterized in that it is selected from the group consisting of a culture plate, a Petri dish, a culture flask, a chamber slide, a chamber and a tube form, a recombinant phage-based vessel for stem cell culture.
According to claim 1,

The cell delivery peptide of the recombinant phage is RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser), RGDC (Arg-Gly-Asp-Cys), RGDV (Arg-Gly-Asp-Val), RGES (Arg-Gly-Glu-Ser), RGDSPASSKP (Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), GRGDS (Gly-Arg-Gly-Asp-Ser), GRADSP (Gly -Arg-Ala-Asp-Ser-Pro), KGDS (Lys-Gly-Asp-Ser), GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP (Gly-Arg-Gly-Asp-Thr- Pro), GRGES (Gly-Arg-Gly-Glu-Ser), GRGDSPC (Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP (Gly-Arg-Gly-Glu-Ser-Pro), SDGR ( Ser-Asp-Gly-Arg),YRGDS(Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly- Asp-Val), GPR (Gly-Pro-Arg), GHK (Gly-His-Lys), YIGSR (Tyr-Ile-Gly-Ser-Arg), PDSGR (Pro-Asp-Ser-Gly-Arg), CDPGYIGSR (Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR (Leu-Cys-Phe-Arg), EIL (Glu-Ile-Leu), EILDV (Glu-Ile-Leu-Asp- Val), EILDVPST (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST (Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV (Leu-Asp-Val) and LDVPS (Leu-Asp-Val-Pro-Ser) characterized in that selected from the group consisting of,

Recombinant phage-based vessel for stem cell culture.
According to claim 1,

The recombinant phage is characterized in that it has a genome consisting of the nucleotide sequence of SEQ ID NO: 1,

Recombinant phage-based vessel for stem cell culture.
According to claim 1,

Recombinant phage-based vessel for stem cell culture, characterized in that the adhesion, proliferation, migration and angiogenesis of stem cells are improved,

Recombinant phage-based vessel for stem cell culture.
According to claim 1,

The stem cells include cardiac progenitor cells (CPCs), Endothelial Progenitor Cells (EPCs), Endothelial Colony Forming Cells (ECFCs), Vasculogenic Progenitor Cells (VPCs), Mesenchymal Stem Cells, and embryonic stem cells. (Embryonic Stem Cells) and myoblasts (Myoblasts) characterized in that selected from the group consisting of,

Recombinant phage-based vessel for stem cell culture.
According to claim 1,

The recombinant phage-based container for stem cell culture is characterized in that it does not contain a feeder cell,

Recombinant phage-based vessel for stem cell culture.
According to claim 1,

The cysteamine concentration is 1 mM to 1 M; The concentration of the recombinant phage (Recombinant Phage) is characterized in that 0.01 mg / ml to 10 mg / ml,

Recombinant phage-based vessel for stem cell culture.
Cysteamine (Cysteamine); And Recombinant phage (Recombinant Phage) displaying a cell delivery peptide on a coat protein; A method of manufacturing a recombinant phage-based vessel for stem cell culture comprising the step of coating the cell culture vessel.
According to any one of claims 1 to 8, wherein any one of the recombinant phage for culturing stem cells - A method of improving the adhesion, proliferation, migration and angiogenesis of stem cells, comprising the step of culturing the stem cells in a vessel.
Claims 1 to 8 as a method for producing stem cells for cardiovascular regeneration, comprising the step of culturing the stem cells in a recombinant phage-based vessel for stem cell culture of any one of claims 1 to 8,

The stem cell is a method, characterized in that the adhesion, proliferation, migration and angiogenesis are improved stem cells.