Phage-based matrix for inducing stem cell differentiation and method for preparing the same

Abstract

The present disclosure relates to a phage-based matrix for inducing stem cell differentiation and a method for preparing the same. More specifically, the present disclosure relates to a composition for inducing differentiation of stem cells, which includes a phage-based matrix in which a gradient of stiffness is controlled by crosslinking a recombinant phage with a polymer, and a method for preparing a phage-based matrix for stem cell differentiation. According to the present invention, the method of the present disclosure provides a physical and mechanical niche environment created by the formation of a nanofibrous structure of the phage whose stiffness is controlled, thereby promoting the differentiation of stem cells into target cells. Therefore, it can be applied to a tissue matrix platform as a variety of conventional tissue engineering materials.

Claims

1. A composition for inducing differentiation of stem cells into osteocytes or endothelial cells (EC), comprising a phage-based matrix in which a gradient of stiffness is controlled by crosslinking a recombinant phage with a polymer, wherein the recombinant phage is a recombinant phage displaying a cell delivery peptide on a major coat protein and displaying HPQ on a minor coat protein; wherein the recombinant phage comprises a genome consisting of the nucleotide sequence represented by SEQ ID NO: 2 or 3; wherein the polymer is at least one selected from the group consisting of streptavidin, poly(diallyldimethylammonium chloride) (PDDA), polyacrylamide and bisacrylamide; and wherein the stiffness generated is 1 kPa to 1 MPa 8 to 10 kPa for differentiation of the stem cells into endothelial cells or 80 kPa to 90 kPa for differentiation of the stem cells into osteocytes.

2. The composition according to claim 1, wherein the stem cell is selected from the group consisting of Mesenchymal Stein Cells (MSC), Adipose Stein Cells (ASC), Endothelial Progenitor Cells (EPC), Cardiac Progenitor Cells (CPC), Endothelial Colony Forming Cells (ECFC), Vasculogenic Progenitor Cells (VPC) and embryonic Stem Cells.

3. The composition according to claim 1, wherein the recombinant phage comprises a genome consisting of the nucleotide sequence represented by SEQ ID NO: 2.

4. The composition according to claim 1, wherein the recombinant phage comprises a genome consisting of the nucleotide sequence represented by SEQ ID NO: 3.

5. The composition according to claim 1, wherein the recombinant phage comprises a genome consisting of the nucleotide sequence represented by SEQ ID NO: 2 and wherein the stiffness generated is 8 to 10 kPa for differentiation of stem cells into endothelial cells or 80 kPa to 90 kPa for differentiation of stem cells into osteocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A illustrates a cleavage map of recombinant M13. FIG. 1B illustrates the results of sequencing of a phage (p7HPQp8RGDp3HPQ) containing RGD in the major coat p8 region of the phage and expressing HPQ in the minor coat p3 and p7 regions. FIG. 1C illustrates the results of sequencing of a phage (p9HPQp8RGDp3HPQ) containing RGD in the major coat p8 region of the phage and expressing HPQ in the minor coat p3 and p9 regions.

(2) FIG. 2 schematically illustrates a process of forming a stiffness-graded phage matrix according to recombination phage pulling (patterning).

(3) FIG. 3 illustrates the difference in cell response due to interaction with a recombinant phage in recombinant phage pulling (patterning).

(4) FIG. 4 illustrates the difference in cell differentiation due to interaction with a recombinant phage in recombinant phage pulling (patterning).

(5) FIG. 5 illustrates the results of osteogenic differentiation of cells by recombinant phage-based matrix (PhaTch) in recombinant phage pulling (patterning).

(6) FIG. 6 illustrates AFM (atomic force microscopy) results obtained by pulling the modified M13 phage on a gold panel.

(7) FIGS. 7A-7B illustrate the results of induction of differentiation (bone formation) of stem cells into bone cells according to the stiffness of a recombinant phage pulling matrix.

(8) FIG. 8 schematically illustrates stiffness-graded phage matrix formation process according to a recombinant phage gel system (PhaGel).

(9) FIG. 9 illustrates the difference in cell response due to interaction with a recombinant phage in a recombinant phage gel system (PhaGel).

(10) FIG. 10 illustrates the results of induction of differentiation (angiogenesis) of stem cells into vascular cells according to the stiffness of a recombinant phage gel matrix.

(11) FIGS. 11A-11C illustrate the morphology of cells on day 2 in a recombinant phage gel matrix.

(12) FIGS. 12A-12B illustrate the expression levels of CD34, the EPC marker and CD31, the EC marker on day 2 in a recombinant phage gel matrix.

(13) FIGS. 13A-13D illustrate the stiffness according to a recombinant phage gel matrix prepared at various concentrations.

(14) FIG. 14 illustrates the expression level of CD34, the EPC marker on day 7, when stem cells were differentiated in recombinant phage gel matrices having different stiffness.

DETAILED DESCRIPTION

(15) Hereinafter, it will be apparent to a person having ordinary skill in the technical field to which the present disclosure pertains that the examples are for illustrative purposes only in more details and that the scope of the present disclosure is not construed as being limited by these examples without departing from gist of the present disclosure.

Example 1. Preparation of Novel Recombinant Phage

(16) For the preparation of functional M13 phage that can control stiffness for application to tissue matrix platforms that provide differentiation and proliferation of stem cells into specific cells, the present inventors prepared and constructed M13 phages p7HPQp8RGDp3HPQ (SEQ ID NO: 2) and p9HPQp8RGDp3HPQ (SEQ ID NO: 3), which expresse RGD having cell affinity on their p8 major coat protein, and expresse HPQ which can be used for fixing various growth factors and cytokines with a medium of streptavidin or implementing stiffness, on p3, p7, and/or p9 minor coat proteins.

(17) The structure of the newly constructed M13 phage (FIG. 1A) and the sequencing confirmation result thereof (FIG. 1B) are shown in FIG. 1.

(18) In addition, the types of phage constructed are shown in Table 1, and the primer sequences used for phage construction are shown in Table 2.

(19) TABLE-US-00001 TABLE1 p7 p9 p8 p3 Name Sequence sequence sequence sequence comments RDD8H3H MEQV TSHPQS* AGGRGD SHSACHPQGPLC C-terminusHPQengineering 9 SDDYDP GGGAET onp9 RDD8H3H MEQV TSA(Q)H AGGRGD SHSACHPQGPLC C-terminusHPQengineering X9 PQHRS* SDDYDP GGGAET onp9 RDGDY8H MEQV TSA(Q)H AGGRGD SHSACHPQGPLC C-terminusHPQengineering 3HX9 PQHRS* SDDYDP GGGAET onp9 RDGDY8H MEQV TSA(Q)H AGGRGD SHSACHPQGPLC C-terminusHPQengineering 3HX9 PQHRS* SDDYDP GGGAET onp9 RGD8HPQ MEQV TSHPQS* ADLGRG SHSACHPQGPLC C-terminusHPQengineering 3HPQ9 DTEDP GGGAET onp9 RGD8HLQ MEQV TSHPQS* ADLGRG SHSACHLQGPLC C-terminusHPQengineering 3HPQ9 DTEDP GGGAET onp9,HPQonp3mutated RGE8HPQ MEQV TSPQHP ADSGRG SHSACHPQGPLC C-terminusHPQengineering 3HPQ9 QNKS* ETEDP GGGAET onp9 RD8H3H7 ME-HPQ- MSV AGGRGD SHSACHPQGPLC N-terminusHPQengineering V SDGYDP GGGAET onp7 RD8H3H9 QRDP* MSHPQV AGGRGD SHSACHPQGPLC N-terminusHPQengineering GGGAET onp9,c-teminusof SDGYDP p7willbechanged RD8H3cH7- MECLHP MSV AGGRGD SHSACHPQGPLC N-terminuscircularHPQ 1 QTCV SDGYDP GGGAET engineeringonp7 RD8H3cH7- MECWH MSV AGGRGD SHSACHPQGPLC N-terminuscircularHPQ 2 PQMCV SDGYDP GGGAET engineeringonp7

(20) TABLE-US-00002 TABLE2 p8-RDDD 5- Fw ATATATCTGCAGNNGGCCGTGGCGATTCTGATGACG ATGATCCCGCAAAAGCGGCCTTTAATCCC-3 (SEQID:4) p8- 5-CCTCTGCAGCGAAAGACAGCATCGG-3 rev1376 (SEQID:5) (rev1376) p3- 5-AAACACTCGGCCGAAACTGTTGAAAGT Fwd1626 TGTTTAGC-3 (SEQID:6) p3-rev 5-TATATACGGCCGATCCACCGCCGCAGC RGD TATCGCCACGGCCGCACGC CGAGTGAGAATAGAAAGGAACCACTAAAG GAATTGCG-3 (SEQID:7) Fw- 5-AAACACTCATGAAAAAGTCTTTAGTCC BspHI- TCAAAGCCTCTGTAG-3 p9 (SEQID:8) Re-BspHI- 5-TATATATCATGANTCAGCTCTGCGGATGGGAAG HPQ-p9 TTTCCATTAAACG-3 (SEQID:9) Re-BspHI- 5- XXHPQXXS- TATATATCATGANTCAGCTMNNMNNCTGCGGATGMN p9 NMNNGGAAGTTTCCATTAACG-3 (SEQID:10) BamHI- 5- N-HPQFw ATATATGGATCCATGGAGCATCCGCAGGTCGCGGAT p7 TTCGACACAATTTATCAG-3 (SEQID:11) BamHI-N- 5-AAACACGGATCCGTTACTTAGCCGGAACGAGGC HPQ GCAGACGGT-3 Rep7 (SEQID:12) BamHI- 5- N-HPQFw ATATATGGATCCATGAGTCATCCGCAGGTTTTAGTG p9 TATTCTTTTGCCTCTTTCGTT-3 (SEQID:13) BamHI- 5-AAACACGGATCCCTTTGACCCCCAGCGATTATA N-HPQRe CCAAGCGC-3 (SEQID:14) p9 BamHI- 5- cir ATATATGGATCCATGGAGTGCNNKCATCCGCAGNNK N-HPQ TGTGTCGCGGATTTCGACACAATTTATCAG-3 Fwp7 (SEQID:15) BamHI- 5- cir ATATATGGATCCATGAGTTGCNNKCATCCGCAGNNK N-HPQ TGTGTTTTAGTGTATTCTTTTGCCTCTTTCGTT-3 Fwp9 (SEQID:16)

(21) More specifically, as wild-type phage M13WT (SEQ ID NO: 1), M13KE (New England Biolabs, Ipswich, Mass.; N0316S) was purchased to use. The phage M13s (SEQ ID NOS: 2 and 3) having the vector map of FIG. 1A used in the present disclosure were produced by a gene recombinant technique known in the pertinent art.

Example 2: Achievement of a Phage Matrix with Different Stiffness Regulated by a Novel Recombinant Phage and Induction Effect on Stem Cell Differentiation Thereby

(22) The present inventors formed the phage matrix, which is a nanofibrous structure, using the phage constructed in Example 1, and confirmed the reaction and differentiation of cells by controlling the stiffness thereof.

(23) 2-1. Phage Engineering and Formation of Phage Matrix by Pulling (Patterning)

(24) In order to form a phage matrix (Phage based pulling patterned patch: PhaTch) by pulling (patterning) the phage expressing the RGD-peptide and HPQ-peptide of the present disclosure constructed in Example 1 above, the present inventors combined streptavidin or PDDA with a recombinant phage (FIG. 2).

(25) First, a hydrophilic treatment was performed on a glass slide glass, and then the prepared phage matrix was adhered.

(26) The phage matrix was crosslinked with glutaraldehyde vapor.

(27) When osteocytes were cultured on glass slide glass coated with phage matrices (10.sup.12 phages/mL) expressing DGEA, DGDA, EGEA, RGD, RGE peptides (DGEA is used as a collagen functional peptide. DGDA and EGEA are used as comparative peptides to identify DGEA-specific functions by substituting amino acids (D and E) having a similar property to DGEA. RGD is a fibronectin and ECM-like functional peptide and is used as a cell-affinity peptide. RGE is used as a comparative peptide to identify RGD-specific functions by substituting amino acids (D and E) having a similar property to RGD), the difference in the reaction of the osteocytes by the interaction with the recombinant phage was confirmed.

(28) As a result, as shown in FIG. 3, it can be seen that osteoblasts respond very specifically to DGEA. When osteoblasts were cultured on a DGEA phage, the area of osteoblasts was larger in DGEA than in the other peptide expression phages.

(29) In addition, the difference in cell differentiation due to interaction with the recombinant phage was confirmed.

(30) As a result, as shown in FIG. 4, it can be seen that the ALP activation reaction on the DGEA phage is high. This indicates that the degree of differentiation is different due to the interaction with the peptide expressed on the DGEA phage.

(31) In addition, as shown in FIG. 5, the expressions of bone cell differentiation markers (COL1, OP, ALP, OCN and Dmp1) are highly expressed in osteoblasts cultured on a DGEA phage. It can be also seen that the interaction of peptides expressed on the DEGA phage makes the cells react, and that these interactions influence the differentiation. As such, DGEA is an osteogenic specific peptide, so that a specific reaction of osteoblasts can be observed.

(32) As such, it was found that the reaction and differentiation of cells can be controlled depending on the peptides displayed in recombinant phages.

(33) On the other hand, modified M13 phage, streptavidin and PDDA were pulled on a gold panel and images were confirmed for the achievement of a matrix suitable for cell culture with various stiffness prepared using streptavidin and PDDA (FIG. 6, top panel)

(34) As a result, as shown in FIG. 6, the thickness of the implemented matrix was not related to the stiffness (FIG. 6, lower left panel), and the difference in stiffness with or without PDDA or streptavidin was clearly noted (FIG. 6, lower right panel).

(35) Accordingly, the RGD-peptide and the HPQ-peptide of the nanofiber were mixed with streptavidin or PDDA to control the stiffness of the phage matrix using a recombinant phage, and the stiffness was controlled based on the concentration of the phage and the mixing substance. That is, the stiffness can be controlled according to the concentrations of phage, streptavidin and PDDA, or according to the matrix achievement methods (pulling order, pulling rate, mixing ratio and method with streptavidin and PDDA, etc.). The stiffness of 20 to 120 kPa, the averagely high stiffness of 120 to 2900 kPa of the phage pulled by using streptavidin, and the low stiffness of 8 to 20 kPa of the phage pulled by using PDDA can be implemented by the concentration of the phage, etc.

(36) 2-2. Induction of Differentiation of Stem Cells into Osteoblasts (Osteogenesis) According to Phage Matrix Stiffness

(37) The present inventors evaluated morphological changes and gene expression during bone differentiation in vitro by controlling the stiffness of the constructed phage matrix to various sizes.

(38) As a result, as shown in FIG. 7A, it can be understood that the stiffness can be controlled according to a ratio and a way of pulling.

(39) In addition, as shown in FIG. 7B, the higher the stiffness was, the higher the expression of osteogenic marker was observed, which proved to induce bone differentiation.

Example 3: Achievement of a Phage Gel System (PhaGel) with a Controlled Stiffness Using a Novel Recombinant Phage and its Induction Effect on Stem Cell Differentiation

(40) The present inventors formed a phage gel, which is composed of the nanofibrous structured phage constructed in Example 1, and confirmed the reaction and differentiation of cells by controlling the stiffness thereof.

(41) 3-1. Phage Engineering and Formation of Phage Matrices According to a Gel System (PhaGel)

(42) The present inventors formed a phage matrix (Phage based hydrogel: PhaGel) by combining the phage with the RGD-peptide and HPQ-peptide of the present disclosure constructed in Example 1 with the gel system, and in order to control the stiffness, constructed the PhaGel system by mixing polyacrylamide and bisacrylamide at various ratios together with the recombinant phage (FIG. 8).

(43) First, a hydrophilic treatment was performed on a glass slide glass, and then the prepared phage hydrogel was attached.

(44) ASC (Adipose Stem Cell; ATCC, PCS-500-011) was cultured on a glass slide glass coated with the PhaGel. On an ASC medium, for differentiation into each differentiation medium (for differentiation into vascular endothelial cells, a vascular endothelial cell differentiation medium was used, and for differentiation into osteoblasts, an osteoblast differentiation medium was used) vascular endothelial cell, a medium containing VEGF and/or IGF can be used. In this experiment, Endothelial basal medium-2 (LONZA, USA) supplemented with EGM-2MV SingleQuots kit (E-media, LONZA, USA) was used. ASC medium (ATCC PCS-500-030) may be used for comparison. The difference in cell response due to the interaction with a recombinant phage was confirmed.

(45) As a result, as shown in FIG. 9, as the content of bisacrylamide increased, the stiffness became stronger (FIG. 9, left panel). On the 7.sup.th day of culture, ASC cultured in PhaGel was attached, whereas ASC cultured in hydrogel, which is a control group, was not attached (FIG. 9, middle panel). In addition, there was a significant difference in the aspect ratios between all the experimental groups at each time point (FIG. 9. upper right panel), and ASCs cultured in PhaGel showed significantly increased cell area on the 7.sup.th day of culture (FIG. 9, lower right panel).

(46) That is, the control of the stiffness can be controlled by the mixing ratio of polyacrylamide and bisacrylamide. The present inventors observed that the stiffness was significantly changed when the wild-type phage or the recombinant phage was mixed, and that the cells were grown only in the gel mixed with the phage.

(47) 3-2. Induction of Differentiation (Vascularization) of Stem Cells into Vascular Cells According to Stiffness of Phage Gel (PhaGel)

(48) The present inventors adjusted the stiffness of the constructed PhaGel in various sizes to evaluate morphological changes and gene expression during angiogenesis differentiation in vitro and in vivo.

(49) As a result, as shown in FIG. 10, it can be understood that when ASCs at 2 kPa and 16 kPa were grown in an EPC medium and an ASC medium, more angiogenic markers were expressed at 16 kPa than 2 kPa when grown on an EPC medium. On the other hand, there was no expression of angiogenic markers on an ASC medium. In other words, it was found that the environment inducing the differentiation of vascular endothelial cells was established at 16 kPa rather than 2 kPa.

(50) In addition, as shown in FIGS. 11A-11C, the morphology of the cells was confirmed on day 2.

(51) In the undifferentiated EPC and ASC cultures, round-shaped cells were observed up to 0.2 to 2 kPa, and the changes from a round-shape to an elongated shape at 8 to 16 kPa, and relatively high cell affinity (cell distribution and number) were observed. Therefrom, it is expected that the induction of differentiation such as adipogenic cells (adipocytes) will be advantageous at 2 kPa or less, and for affinity and differentiation of vascular endothelial cells, it will be advantageous at about 8 to 16 kPa.

(52) In addition, the expressions of EPC marker CD34 and EC marker CD31 were confirmed at day 2.

(53) As a result, as shown in FIGS. 12A and 12B, it was found that the differentiation toward EC was promoted around 8 kPa.

(54) Further, as shown in FIG. 13, when wild type phage (WT) or recombinant phage 184 (YSY184; hereinafter referred to as 184) according to the mixing ratio (FIGS. 13A and 13C) of acrylamide and bisacrylamide for producing a polyacrylamide hydrogel substrate was added, it was confirmed that the stiffness was significantly changed (FIGS. 13B and 13D). For example, when the acrylamide is 8% and the bisacrylamide is 0.06%, the stiffness is 8 to 10 kPa when the recombinant phage (denoted as 6_184 in FIG. 13B) and the wild type phage (denoted as 6_WT in FIG. 13B) were added. On the other hand, the stiffness was 5 kPa or less when the recombinant phage (denoted as 6_184 in FIG. 13D) and the wild type phage (denoted as 6_WT in FIG. 13D) were added if the acrylamide was 6% and the bisacrylamide was 0.06%.

(55) In addition, when the recombinant phage (184) or the wild type phage (WT) was added to the above-mentioned various concentrations of gel, the expression level of CD34 was confirmed when the EPC and ASC were cultured on the 7.sup.th day in a gel having a stiffness with various gradients.

(56) As a result, as shown in FIG. 14, the expression level of CD34 was the highest at a stiffness of 8 to 10 kPa. PhaGel with recombinant phage 184 contributes to the regulation of vascular differentiation, while having a stiffness of 8 to 10 kPa, whereas the hydrogel without phage but with the same stiffness does not exhibit cell-affinity and differentiation control ability. It can be seen that the proper differentiation of cells can be controlled through the interaction between the stiffness realized by the matrix using a phage and the cell affinity peptides of the constituting phage.

(57) Accordingly, it can be seen that the above results can realize various stiffness with the Phage based Matrix (PhaTch or PhaGel) of the present disclosure, and that the addition of phage contributes to the stiffness, and in particular, when the stiffness is 8 to 10 kPa, it was confirmed that EPC and EC differentiation were optimum conditions and that the optimum condition of bone differentiation was obtained at a stiffness of 80 kPa to 90 kPa.

(58) In conclusion, the present disclosure provides a method of regulating the degree of differentiation of stem cells by controlling the stiffness formed according to the functions of the phage, and the matrix concentration and structure.