KR20140056014A - Polydopamine-peptide-immobilized substrates for feeder-free maintenance and enhanced differentiation of stem cells and methods for preparing the substrates - Google Patents

Abstract

The present invention relates to a tissue engineering polymer supporting body in which protein/peptide is immobilized based on polydopamin coating and a manufacturing method thereof. The polymer supporting body according to the present invention is a polymer supporting body which peptide derived from proteins having cell adhesion properties or biological activity and polydopamin are connected, wherein protein/peptide having cell adhesion properties is immobilized as compared with convention supporting body so that proliferation of human pluripotent stem cells can be enhanced and protein/peptide having biological activity is effectively immobilized on the surface so that excellent differentiation to osteocytes of mesenchyma stem cells can be achieved. In addition, by stably immobilizing various bioactive substances on the surface of the polymer supporting body, there is possibility of cellular transplantation therapy using differentiation of stem cells and differentiated cells.

Description

[0001] The present invention relates to a polydodipine-peptide immobilized surface substrate for the undifferentiated culture and differentiation of stem cells, and a method for preparing the same,

The present invention relates to a polydodamine-peptide immobilized surface substrate for the undifferentiated culture and differentiation of stem cells, and a method for producing the same.

Conventional methods for undifferentiated growth proliferation of human pre-differentiating stem cells, including human embryonic stem cells and human induced pluripotent stem cells, are mostly based on animal-derived supportive cells such as mouse embryonic fibroblast or Matrigel Matrix.

The use of animal-derived materials in conventional methods makes culturing processes difficult and difficult, and has the problem of scaling up for mass production. In addition, when infectious viruses / bacteria derived from animals are infected into stem cells during culturing and applied to humans as a therapeutic agent, there is a problem that such pathogenic viruses or bacteria may be transmitted to the human body. To solve this problem, extracellular matrix-derived peptides were immobilized on a polymer surface such as polystyrene and used for culturing stem cells. In order to immobilize a peptide, a chemical modification method using various chemical substances or a physical adsorption method was applied . Most of the chemical modification methods require complicated pretreatment of the polymer surface, and there is a problem that the immobilization efficiency of the peptide on the polymer surface is low. On the other hand, the physical adsorption method is simple, but the efficiency is very low and the specific reactivity is poor.

It is an object of the present invention to provide a biomimetic method of easily and easily applying a cell adhesive peptide to a surface of a polymer matrix using a polydodamine coating method which is an adhesive substance derived from a mussel.

Another object of the present invention is to cultivate human pre-differentiating stem cells under the conditions that are chemically proven without using existing animal-derived materials for the development of clinically applicable stem cell therapeutic agents, and to differentiate stem cells into bone cells And to provide a culture substrate which can be cultured in a medium.

The present invention relates to a polymeric scaffold for culturing human pre-differentiative stem cells and a polymer scaffold for bone cell differentiation of mesenchymal stem cells, wherein a cell adhesion peptide is bound to polydodamine.

In one embodiment of the present invention, there is provided a stem cell culturing or stem cell differentiation complex immobilized with a peptide to which polydodamine is bound. In the above embodiment, the peptide is bitonectin, which is a cell adhesion peptide, the bitronectin is a peptide represented by SEQ ID NO: 1 or 2, the stem cell is a human pre-differentiating stem cell, Wherein the peptide is bound to the polydodamine as a dimer and the bitonectin improves the culture of human induced pluripotent stem cells and the culture of the human pluripotent stem cells is cultured without supporting cells Wherein the polymer is any one selected from the group consisting of polystyrene, polycaprolactone, polydimethylsiloxane and polylactic-glycolic acid (PLGA), and a method for culturing human induced pluripotent stem cells.

In one embodiment of the present invention, there is provided a polymer scaffold for differentiating stem cells immobilized with a peptide to which polydodamine is bound. In the above embodiment, the peptide is a physiologically active peptide BMP-2, the BMP-2 is a peptide represented by SEQ ID NO: 3, and the BMP-2 peptide induces the differentiation of mesenchymal stem cells into bone cells Wherein the surface of the support is hydrophilic and the polymer is any one selected from the group consisting of polystyrene, polycaprolactone, polydimethylsiloxane, and polylactic-glycolic acid (PLGA) . ≪ / RTI >

In one embodiment of the present invention, there is provided a process for preparing a polymeric material comprising: (a) coating a polymeric support with polypodamine; And (b) binding the cell adhesion peptide to the polydodamine. The present invention also provides a method for producing a supporter for stem cell culture. In the above embodiment, the stem cell is a human induced pluripotent stem cell, the cell adhesion peptide is bitonectin, and the bitronectin is a peptide represented by SEQ ID NO: 1 or 2. do.

In one embodiment of the present invention, there is provided a process for preparing a polymeric material comprising: (a) coating a polymeric support with polypodamine; And (b) immobilizing a peptide having physiological activity on the surface of the supporter. In the above embodiment, the peptide having the physiological activity is BMP-2, the BMP-2 is a peptide represented by SEQ ID NO: 3, and the peptide is a stem cell A method for producing a support for differentiation is provided.

(PS), polycaprolactone, polydimethylsiloxane, or polylactic-glycolic acid (PLGA), but not limited thereto. The term " polymer scaffold " . In addition, the polymeric scaffold may include a "biodegradable" support, which is degradable when exposed to a physiological solution at pH 6-8 in clinical use, including, but not limited to, polylactic-glycolic acid (PLGA) can be used. It is a characteristic that the transplanted cells can be completely degraded and disappeared in vivo after being maintained until a desired function and role as a tissue is sufficiently performed.

In the present invention, "stem cell" means a kind of parent cell or omnipotent cell capable of growing into different cells or organs constituting a living body, and includes embryonic stem cells and adult stem cells.

In the present invention, "differentiation" refers to a phenomenon in which a structure or function is specialized with each other while a cell is growing by proliferation and proliferation, that is, a cell or tissue of an organism is changed in shape or function to perform a task given to each.

In the present invention, "cell adhesion peptide" is a protein capable of adhering cells, including, but not limited to, laminin, vitronectin and fibronectin.

In the present invention, the term "physiologically active peptide" means a peptide exhibiting useful biological activity when administered to a mammal including a human.

The surface of the polydodamine-peptide polymer according to the present invention was successfully applied to the culture of human pre-differentiation stem cells (human induced pluripotent stem cells) together with the culture medium (mTeSR1) developed for use under feeder-free conditions, Induce stable adherence and proliferation of human induced pluripotent stem cells. In addition, it is possible to subculture the human pre-differentiating stem cells to efficiently propagate the clinically applicable undifferentiated pluripotent stem cells and improve the induction of osteogenic differentiation of mesenchymal stem cells to be used for bone tissue transplantation and regeneration treatment .

FIG. 1 schematically shows a process for preparing a polymer scaffold on which a cell-adhesive peptide of the present invention is immobilized.
Fig. 2 shows the results of the analysis of the surface of the polymer scaffold on which the cell-adhesive peptide of the present invention is immobilized and the predicted structure.
FIG. 3 shows the results of confirming the attachment efficiency of human induced pluripotent stem cells to the surface of the polymer scaffold on which the cell adhesion peptide of the present invention is immobilized.
FIG. 4 shows the results of analysis of the characteristics of human induced pluripotent stem cells adhered to the surface of the polymer scaffold immobilized with the cell adhesion peptide of the present invention through cell immuno staining, AP staining, RT-PCR, and qPCR.
FIG. 5 shows the results of analysis of the characteristics of subcultured and subcultured human induced pluripotent stem cells of human induced pluripotent stem cells adhered on the surface of a polymer scaffold immobilized with the cell adhesion peptide of the present invention.
FIG. 6 schematically shows the preparation of a polymer scaffold on which the physiologically active peptide of the present invention is immobilized and the procedure of transplantation experiment.
FIG. 7 is a result of analyzing that the physiologically active peptide of the present invention is stably immobilized on a polymer scaffold.
8 is a result of analysis of bone cell differentiation of mesenchymal stem cells on a polymer scaffold on which the physiologically active peptide of the present invention is immobilized.
FIG. 9 shows osteoblast differentiation of mesenchymal stem cells and transplantation of mesenchymal stem cells on a polymer scaffold on which the physiologically active peptide of the present invention is immobilized.

Hereinafter, the components and technical features of the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention and are not intended to limit the scope of the invention.

Total differentiation ability  Stem cell Feeder - free  Undifferentiated growth culture

1.1 Fabrication of Polymer Surface Immobilized with Vitronectin Peptide via Polydodamine

1.1.1 Polydodamine Coating

To test the adhesion of human induced pluripotent stem cells, polydopamine was coated on the polystyrene surface by adding 2 mg / ml of 10 mM Tris-HCl dopamine solution to a 24-well polystyrene plate and adjusting the pH to 8.5 to induce polymerization of dopamine Respectively. After the polymerization was performed for about 4 hours, the solution was removed and washed with distilled water to remove uncoated dopamine (FIG. 1).

1.1.2 Combination of bitonectin

After the polydodamine coating, two kinds of peptide solutions derived from vitronectin represented by SEQ ID NO: 1 (VN1): CGGPQVTRGDVFTMP and SEQ ID NO: 2 (VN2): CGGGKKQRFRHRNRKG dissolved in 10 mM Tris-HCl buffer at pH 8.5 1 mg / ml, 2 mg / ml or 4 mg / ml and reacted for 6 hours to immobilize the peptide on polydodamine. The unattached peptides were then removed by washing with distilled water (Figure 1).

1.1.3 Analysis of Polymer Surface

XPS surface analysis was performed to confirm whether the process of coating polydodamine and immobilizing bitronectin was performed efficiently according to the above example.

XPS surface analysis division C N O S PS 98.07 0.00 1.93 0.00 PS-PD 65.24 9.92 24.84 0.00 PS-PD-VN1 63.20 12.58 22.80 1.42 PS-PD-VN2 61.97 13.90 23.01 1.12

The nitrogen (N) component, which was not observed on the surface of polystyrene (PS), was detected in 9-10% of the surface coated with polydodamine (PD) (PS-PD) and the surface on which the peptide was immobilized (PS-PD-VN1 / VN2 ), The elemental nitrogen (N) component was detected by 12 to 14%. The sulfur component (S) was detected on the PS-PD-VN1 / VN2 surface by 1 to 2% due to the thiol group (-SH) of the cysteine amino acid present in the immobilized peptide (Table 1).

(PD) film was well coated on the surface of polystyrene (PS) for cell culture through the surface element quantitative data obtained by XPS surface analysis, and it was confirmed that VN1 peptide and VN2 peptide were well immobilized on the polydopamine (PD) film (FIGS. 2A and 2B) . Further, through the HPLC (FIG. 2C) and mass spectrum analysis (FIG. 2D), two molecules of VN2 peptide having a terminal cysteine residue were synthesized in the process of preparing a polydodamine- peptide surface for culturing human pre- It was confirmed that the dimer formed first through the bond formation and the dimer formed thereafter adhered to the catechol group of the polypdopamine.

The structure of the surface of the polydodamine-VN peptide according to the present invention for culturing human induced pluripotent stem cells without supporting cells was expected to be as shown in FIG. 2E.

1.1.4 Analysis of Polymer Surface (Fluorescamine Analysis)

VN2 peptide was immobilized on poly (lactic-co-glycolic acid) (PLGA) polymer which is widely used as cell culture polystyrene (PS) polymer or cell scaffold scaffold and polydopamine (PD) The amount of VN2 peptide desorbed from the polymer surface was quantitated by fluorescence analysis (peptide quantitation) while culturing in ˚C, PBS buffer. The peptide (D0) that could not be immobilized on the surface of PD and PLGA was calculated to be about 4 to 5% of the applied peptide, and no further peptide desorption was observed during the subsequent incubation period (FIG. 2f). This result shows that more than 95% of the peptides are efficiently and continuously immobilized on the surface of the polymer under the cell culture conditions through the polydodamine (PD) coating.

1.2 Human induced pluripotent stem cell attachment

When human induced pluripotent stem cells (human iPSC) were cultured on polystyrene surface immobilized with VN1 or VN2 peptide using polydodamine without animal-derived support cells, stem cells were observed to have similar or improved efficiency as Matrigel Respectively. VN1 peptide showed the highest stem cell attachment efficiency at 4X concentration (4 mg / ml) and VN2 peptide showed the highest stem cell attachment efficiency at 2X concentration (2 mg / ml) (Fig. 3a).

In addition to two types of VN peptides (VN1 and VN2), peptides derived from extracellular matrix proteins (fibronectin: FN, laminin: LM) were immobilized on polystyrene surface using polydopamine coating, Respectively. Unlike the VN peptide, it was confirmed that the FN peptide and the LM peptide did not induce adhesion of human induced pluripotent stem cells (FIG. 3B).

Human embryonic stem cells (SNU-HES30), another type of differentiable stem cells, were attached to the polystyrene (PS) surface on which VN1 peptide was immobilized by polydodamine (PD) coating, and AP (Alkaline Phosphatase) staining. It was confirmed that the cells were well adhered and formed colonies like human induced pluripotent stem cells (IPSC), and the undifferentiated proliferation was well performed (FIG. 3C). This is a result showing that it is possible to adhere and undifferentiate various kinds of human pre-differentiating stem cells without supporting cells derived from animals using the immobilized surface of the polydodamine-based bitronectin peptide according to the present invention.

1.3 Characterization of human induced pluripotent stem cells

Expression of OCT4, TRA1-60 expression (Fig. 4A), SSEA-4 (Fig. 4A), which is a precursor cell stem cell marker for human induced pluripotent stem cells attached to polystyrene surface immobilized with VN1 peptide and VN2 peptide via polydodamine (Fig. 4B) and expression of AP (Alkaline phosphatase) (Fig. 4C) were observed. This indicates that the induced pluripotent stem cells adhered and cultured on the surface of PD-VN1 / PD-VN2 retain the characteristics of the pluripotent stem cells.

In addition, RT-PCR analysis of human induced pluripotent stem cells adhered and cultured on the surface of polystyrene immobilized with VN1 / VN2 peptide through polydodamine was performed to further observe the expression of the pluripotent stem cell markers. Human induced pluripotent stem cells (MAT-iPSC, STN-iPSC) cultured on matrigel or STO support cells as positive controls, human induced pluripotent stem cells (VN1-iPSC, VN2-iPSC) cultured on VN1 / VN2 peptide- (Fig. 4d; RT-PCR, Fig. 4e: qPCR), similar to that of iPSC.

1.4 Passage of human induced pluripotent stem cells

VN1 / VN2 peptide subculture was carried out on the same surface (PD-VN1, PD-VN2) of human induced pluripotent stem cell (iPSC) cultured on polystyrene (PS) surface immobilized with polydodamine (PD) Respectively. In the case of subculturing on STO-supporting cells or Matrigel, iPSC attachment was required for more than 1 day, whereas iPSCs were attached within 12 hours when subcultured on PD-VN1 and PD-VN2 surfaces (Fig. 5A). This shows that efficient subculture of human induced pluripotent stem cells on the surface of PD-VN1 and PD-VN2 is possible. In addition, it was confirmed that human-induced pluripotent stem cells expressing the sub-cultured human embryonic stem cells were expressed by immunocyte staining. Thus, it was confirmed that the characteristics of the pluripotent stem cells were maintained even after the subculture (FIG. 5B).

Osteogenic protein ( BMP -2) The peptide  The immobilized polymer ( PLGA ) Osteoblast differentiation of stem cells applied in supporter

2.1 Identification of Polydodamine / BMP-2 Peptide Immobilization

According to the procedure described in FIG. 6, the BMP-2 peptide (SEQ ID NO: 3: KIPKASSVPTELSAISTLYL) having physiological activity after application of polydodamine (PD) was immobilized on a PLGA support and the immobilization of the peptide was analyzed by XPS surface analysis. 7A). It was confirmed that the attached peptide was not desorbed during cultivation and maintained well on the surface (FIG. 7A), and it was confirmed that the peptides on the surface of other types of polymers (EPTFE, PUA) The fluorescence microscope analysis confirmed that the peptide was immobilized very efficiently through dopamine (Fig. 7A, B). XPS surface peak analysis and XPS surface element quantitative analysis were carried out to observe immobilization of BMP-2 peptide by increasing N element content after PD-BMP-2 immobilization (FIG. 7B and Table 2).

XPS surface element quantitative analysis division C O N PLGA 62.8 37.2 0 PD-PLGA 72.1 21.6 6.3 PD-BMP-2-PLGA 68.7 20.4 10.9 BMP-2-PLGA 63.6 33.3 3.1

(A and B in FIG. 7C) after PD-BMP-2 immobilization by the AFM surface analysis, the water contact angle of the polymer surface was measured to determine the surface roughness after PD-BMP- By confirming that the property of the PLGA surface changes to hydrophilic (C, D in Fig. 7C), immobilization of BMP-2 peptide through PD on the surface of PLGA was confirmed. In addition, immobilization of BMP-2 peptide on poly (3-D) porous PLGA polymer substrate via polydodamine (PD) followed by observation of immobilization of peptide by scanning electron microscopy (SEM) PD-BMP-2 < / RTI > coating and PD-BMP-2 aggregation on the PLGA support surface (FIG.

2.2 Osteoblast differentiation and stem cell transplantation of mesenchymal stem cells on PD-BMP-2-PLGA scaffolds

On the surface of the PD-BMP-2-PLGA scaffold, human adipose-derived mesenchymal stem cells were cultured under a medium medium containing no bone-cell differentiation medium or osteocyte differentiation factor. After incubation for 3 weeks, staining of calcium deposition staining (alizarin red S staining, A in Fig. 8A and A in Fig. 8B), bone cell markers (OPN, Col I) (B in Fig. 8A and B in Fig. The quantitative PCR results (C.D in FIG. 8A and C and D in FIG. 8B) were used to analyze the differentiation of mesenchymal stem cells into bone cells. The stem cells cultured on the surface of PD-BMP-2-PLGA showed improved osteoclast differentiation ability than the stem cells cultured on the surface of PLGA lacking PD or BMP-2 (Fig. 8A) , Fig. 8b-use of a medium not containing osteoclast differentiation factor).

Human adipose-derived mesenchymal stem cells were incubated with PD-BMP-2 immobilized porous PLGA scaffolds and transplanted into the defect sites of the skull-deficient mouse model. After 8 weeks, micro-CT (Fig. 9a), Goldner ' s staining of collagen in bone tissue. The degree of regeneration of bone tissue in the cranial defect area was analyzed by trichrome staining (Fig. 9b) and immunostaining for bone tissue specific protein (OPN, Col I) (Fig. 9c). When the PD-BMP-2 peptide-loaded PLGA scaffold was applied, it was confirmed that the bone tissue regeneration effect by the stem cell transplantation was the highest.

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

<110> YONSEI UNIVERSITY <120> Polydopamine-peptide-immobilized Substrates for Feeder-free          Maintenance and Enhanced Differentiation of Stem Cells and          Methods for Preparing the Substrates <130> IPDB50839 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 15 <212> PRT <213> HUMAN <400> 1 Cys Gly Gly Pro Gln Val Thr Arg Gly Asp Val Phe Thr Met Pro   1 5 10 15 <210> 2 <211> 16 <212> PRT <213> HUMAN <400> 2 Cys Gly Gly Gly Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly   1 5 10 15 <210> 3 <211> 20 <212> PRT <213> HUMAN <400> 3 Lys Ile Pro Lys Ala Ser Ser Val Pro Thr Glu Leu Ser Ala Ile Ser   1 5 10 15 Thr Leu Tyr Leu              20

Claims (26)

A complex for culturing stem cells or for stem cell differentiation immobilized with a peptide conjugated with polydodamine.
The method according to claim 1,
Wherein said peptide is a cell adhesion peptide, vitronectin.
3. The method of claim 2,
Wherein the bitronectin is a peptide represented by SEQ ID NO: 1 or 2.
The method according to claim 1,
Wherein said stem cells are human induced pluripotent stem cells.
3. The method of claim 2,
Wherein the cell adhesion peptide is chemically bound to the catechol group of the polydodamine.
3. The method of claim 2,
Wherein said cell adhesion peptide is bound to said polydodamine as a dimer.
3. The method of claim 2,
Wherein said bitronectin enhances the culture of human induced pluripotent stem cells.
8. The method of claim 7,
Wherein said human induced pluripotent stem cells are cultured without supporting cells.
A method for culturing a human induced pluripotent stem cell on a complex of any one of claims 1 to 8 coated on a polymer scaffold.
10. The method of claim 9,
Wherein the polymer is any one selected from the group consisting of polystyrene, polycaprolactone, polydimethylsiloxane and polylactic-glycolic acid (PLGA).
The method according to claim 1,
Wherein said peptide is a physiologically active peptide, BMP-2.
12. The method of claim 11,
Wherein said BMP-2 is a peptide represented by SEQ ID NO: 3.
13. The method of claim 12,
Wherein said BMP-2 promotes differentiation of mesenchymal stem cells into bone cells.
14. The method of claim 13,
Wherein the surface of the composite is hydrophilic.
A method for differentiating mesenchymal stem cells into osteocytes on the complex of any one of claims 1 and 11 to 14 coated on a polymer scaffold.
16. The method of claim 15,
Wherein the polymer is any one selected from the group consisting of polystyrene, polycaprolactone, polydimethylsiloxane, and polylactic-glycolic acid (PLGA).
A film comprising the composite of any one of claims 1, 2 to 8 and 12 to 16.
(a) coating the polymeric support with polydodamine; And
(b) binding the peptide to the polydodamine;
Wherein the stem cells are cultured in a culture medium.
19. The method of claim 18,
Wherein the peptide is vitronectin, which is a cell adhesive peptide.
20. The method of claim 19,
Wherein the bitronectin is a peptide represented by SEQ ID NO: 1 or 2.
20. The method of claim 19,
Wherein the stem cell is a human induced pluripotent stem cell.
19. The method of claim 18,
Wherein the peptide is BMP-2, a peptide having physiological activity.
23. The method of claim 22,
Wherein the BMP-2 is a peptide of SEQ ID NO: 3.
24. The method of claim 23,
Wherein the peptide is used to differentiate mesenchymal stem cells into osteocytes.
19. The method of claim 18,
Wherein the polymer is any one selected from the group consisting of polystyrene, polycaprolactone, polydimethylsiloxane, and polylactic-glycolic acid (PLGA).
19. The method of claim 18,
Wherein the polydodamine coating is a polymerization reaction carried out under weakly basic conditions.

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