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Enhanced Survival of Neural Cells Embedded in Hydrogels Composed of Collagen and Laminin-Derived Cell Adhesive Peptide Makiko Hiraoka, Koichi Kato, Tadashi Nakaji-Hirabayashi, and Hiroo Iwata* Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Received January 7, 2009; Revised Manuscript Received February 20, 2009
To develop biomaterials that serve to improve the survival of neural cells transplanted into central nervous tissues, type I collagen-based hydrogels were prepared as a cell carrier. The hydrogels were modified with a laminin-derived peptide that is known to have an affinity for alpha3beta1 integrin, to transduce antiapoptotic signaling in embedded cells. For the modification of collagen, the peptide was fused to the N- or C-terminus, or both termini of a collagen-binding polypeptide domain by means of recombinant DNA technology. The chimeric proteins were characterized by polyacrylamide gel electrophoresis and circular dichroism spectroscopy, while binding of chimeric proteins to collagencoated substrates was verified by surface plasmon resonance analysis under physiological conditions. Cell culture assays revealed that the adhesion of neurosphere-forming cells to collagen-coated polystyrene surfaces was significantly promoted by the incorporation of the chimeric proteins in a peptide-density dependent manner. The live/dead assays for cells cultured for 24 or 48 h in the hydrogels revealed that peptide incorporation improved the survival of cells embedded in collagen hydrogels. These results suggest that collagen hydrogel containing the laminin-derived peptide provides microenvironments suitable for the survival of neural cells.
INTRODUCTION Recently, stem cell transplantation has received much attention as potential treatments for neurodegenerative diseases and traumatic injuries of the central nervous system (1). Neural stem/ progenitor cells derived from brain tissues or embryonic stem cells have been transplanted after appropriate processing in vitro. Many studies demonstrated the engraftment of neural stem/ progenitor cells directly infused into the brain of model animals (2). However, the number of cells surviving initial few days in the host tissues is limited, which reduces therapeutic effects of cell transplantation (3). One of the most critical causes of this problem may be cellular apoptosis that is triggered by disruption of cell-cell and cell-extracellular matrix interactions upon cell harvesting for transplantation (4, 5). In addition, infiltration of inflammatory microglia into transplanted sites is considered as another cause for limited engraftment (6, 7). In an attempt to promote cell survival, we have studied the development of collagen-based hydrogels to be used as a carrier for neural cells (8, 9). We expect that the form of hydrogels serves as a temporal barrier against infiltrating microglia, reducing adverse effects due to acute inflammation. To enhance cell survival, attempts were made to incorporate epidermal growth factor to the collagen matrix (8). That study led us to suppose as well that, in addition to growth factor stimulation, signaling through cell adhesion molecules would have additional effects for promoting cell survival. In practice, hippocampus neurons were shown to adhere and extend neurites on collagen that incorporated a peptide mimetic of the neural cell adhesion molecule (9). In contrast, here we examined the effects of an integrin-binding peptide on the survival of neural cells. It has been reported that neural stem cells express various integrin complexes (10) whose interactions with extracellular matrix play important roles in the development of the central nervous system (11, 12). Among integrins expressed on neural stem cells, R3β1 integrin is known to interact with laminin-5 and has been * To whom correspondence should be addressed. E-mail: iwata@ frontier.kyoto-u.ac.jp, tel and fax: +81-75-751-4119.
implicated in brain development (13) through the modulation of neuronal migration and neuron-glial interactions (14, 15). In addition, cell adhesion to laminin-5 via R3β1 integrin was reported to mediate survival signaling in a variety of epithelial cells (16, 17). For these reasons, we sought a peptide sequence capable of specific binding to R3β1 integrin and incorporated such a peptide into collagen hydrogels to promote the survival of embedded neural cells on account of integrin signaling. We employed here a small peptide, PPFLMLLKGSTR, derived from the G3 domain of a laminin R3 chain of laminin-5 (designated this peptide as G3P hereafter). Kim et al. (18) demonstrated that this sequence has a strong affinity for R3β1 integrin expressed on epithelial cells. To incorporate the peptide into collagen hydrogels, we adopted a similar strategy as in our previous studies (8, 9): Chimeric proteins consisting of G3P and the A3 domain of von Willebrand factor (vWF) that has an affinity for collagen R chains (designated this domain as CBD hereafter) were prepared by recombinant DNA technology. It was shown that (8) CBD contained in the chimeric proteins specifically bound to type I collagen under physiological conditions, presenting its fusion partners on collagen. In the present study, chimeric proteins with different domain structures were prepared. The G3P was fused to the C- or N-terminus, or both termini of CBD, which allows us to study the effects of molecular architecture. Another polypeptide that contained no G3P was prepared and used as a control. These proteins were bound to a collagen-coated polystyrene surface to evaluate the effect of chimeric proteins on the adhesion and neurite extension of the neural stem cell-containing population. Further investigation was conducted to determine the viability of neural cells embedded in collagen hydrogels that incorporated G3P-containing chimeric proteins. In this article, we report that the incorporation of G3P significantly promotes cell survival in collagen hydrogels.
EXPERIMENTAL PROCEDURES Genetic Engineering. A bacterial expression system was used to synthesize three types of chimeric proteins consisting of G3P
10.1021/bc9000068 CCC: $40.75 2009 American Chemical Society Published on Web 04/07/2009
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Figure 1. (A) Schematic representation of a chimeric protein consisting of a laminin-derived cell adhesive peptide (G3P) and a collagen binding domain (CBD) derived from vWF. (B) Structure of chimeric proteins synthesized in this study. His: hexahistidine sequence for affinity purification. The dipeptide, L-E, inserted upstream of His comes from the plasmid. The number of amino acid residues: CBD, 189; G3P, 12; His, 6.
and CBD. The structures of these proteins are shown in Figure 1. Chimeric proteins fused with G3P at the N-, C-, and both termini are designated as nG3P-CBD, cG3P-CBD, and dG3PCBD, respectively. A control protein lacking G3P is designated as CBD. The complete sequence of chimeric proteins is provided in Supporting Information. For the preparation of chimeric genes, polymerase chain reaction (PCR) was performed using primers shown in Table 1. A DNA sequence encoding GP3 was incorporated in forward and/or reverse primers depending on the structure of chimeric proteins. Forward primers contained an Nde I restriction site, while reverse primers contained an Xho I site. The chimeric genes for four proteins are separately amplified from the EGFCBD expression vector (8). PCR was performed using a thermal cycler (Gene Amp PCR 9700G, Applied Biosystems) under the following conditions: 35 cycles, denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 30 s. DNA fragments thus obtained were separated by agarose gel electrophoresis and digested with restriction enzymes, Nde I and Xho I. The fragments were inserted into the upstream of 6 × histadine-tag in pET-22b(+) plasmid (Novagen) and transformed to Escherichia coli (E. coli) DH5R. After overnight culture, colonies were randomly picked up, and the presence of the plasmid was verified by colony PCR using primers specific for T7 promoter and T7 terminator. The correctness of the plasmids was verified by sequencing. Cells harboring the correct plasmid were cultured in LB medium to prepare glycerol stocks. Protein Expression. E. coli strain BL21-CodonPlus (Stratagene) was transformed with the plasmid and cultured in a medium (Overnight Express Autoinduction System, Novagen) at 37 °C for 12 h. Proteins expressed as inclusion bodies were extracted with 20 mM phosphate buffer (pH 7.4) containing 8 M urea and 5 mM 2-mercaptoethanol and purified by Ni-chelate ¨ KTA system (GE affinity chromatography using a Prime A Healthcare) equipped with a His Trap HP column (GE Healthcare). The purity and the molecular size of the proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were refolded by stepwise dialysis against the series of buffers (see Supporting Information for buffer compositions). Finally, the solutions of refolded proteins in 5 mM 2-amino-2-hydroxymethyl-1,3propanediol (Tris)-HCl buffer were stored at -80 °C until use. Circular Dichroism (CD) Spectroscopy. CD spectra were recorded using JASCO J-805 spectropolarimeter. Stock solutions of proteins were diluted with 5 mM Tris-HCl (pH 8.9 for CBD, nG3P-CBD, and cG3P-CBD; pH 9.5 for dG3P-CBD) to the final concentration of 150 µg/mL. Far-UV CD spectra were recorded in a 0.1 cm path length cell at 20 °C, a response time of 0.5 s, a bandwidth of 1 nm, and a scan speed of 100 nm/min with an accumulation of eight scans. Pure Tris-HCl buffer (5 mM, pH 8.9 or 9.5) was used as reference. Surface Plasmon Resonance (SPR) Analysis. The binding of chimeric proteins to collagen was analyzed by SPR as
reported before (9, 19). In brief, a glass plate bearing a thin gold layer and a carboxylic acid-terminated self-assembled monolayer was mounted to the hemicylinderical glass prism of an SPR sensor equipped with an He-Ne laser tube. Light reflectance was continuously monitored at a fixed incident angle, while protein or pure buffer solution was sequentially circulated over the surface of the plate in a flow cell at 37 °C and a flow rate of 3 mL/min. After equilibrating with phosphate-buffered saline (PBS), the surface was exposed to 100 µg/mL type I collagen solution (Sigma) in 0.1 M acetic acid buffer (pH 4.9) for 30 min, followed by circulation of PBS for 60 min to wash the surface. Then, chimeric protein solution (diluted with PBS to a final concentration of 50 µg/mL) was circulated for 60 min to bind the protein to preadsorbed collagen, followed by washing with pure PBS for 60 min. The reflectance changes were used to determine a resonance angular shift and then converted to the surface density of protein assuming the unit angular shift of 0.5 µg/cm2/DA (20). Cell Isolation and Adhesion Assay. Chimeric protein was diluted with PBS to a concentration of 50 µg/mL. The solution was added to a collagen-coated polystyrene dish (Asahi Techno Glass Corp., Tokyo, Japan) and incubated at room temperature for 2 h to bind chimeric protein onto collagen. The plate was washed with PBS to remove unbound protein and immediately used for cell adhesion assays. As reported previously (21), the striatum was isolated from rat fetuses (embryonic day 16) and dissociated into single cells, according to the guideline of the institutional Animal Welfare Committee. The cells were cultured in suspension to form neurospheres (22) that contained neural stem cells (50-60% of total cells) as well as more differentiated cells (21). Neurospheres at passage 2 were dissociated into single cells by trypsin treatment. This cell population is referred to as neurosphere-forming cells in this paper. The cells were suspended in DMEM/F12 (1:1) (Gibco) containing 2% B27 supplements (Gibco), 5 µg/mL heparin, 100 unit/mL penicillin, and 100 µg/mL streptomycin. The cells were seeded to the protein-bound dish at a density of 5.0 × 104 cells/cm2 and incubated at 37 °C under 5% CO2 atmosphere. In separate experiments, soluble peptide (PPFLMLLKGSTR, Invitrogen) was dissolved in a cell suspension to a concentration of 25 µg/ mL, and the cells were incubated at 37 °C for 30 min prior to seeding. The cells were then cultured on the protein-bound dish under the same condition as described above to confirm the specific effect of surface-bound peptide on cell adhesion. After incubation for 6 h, cells were washed gently with the same medium as mentioned above to remove weakly attaching cells and observed with a phase-contrast optical microscope (DP51, Olympus Optical CO., Ltd., Tokyo, Japan). The number of cells that adhered to the dish was determined on the microphotographs acquired at five different sights (area: 0.33 mm2) on the same sample and averaged. The data are expressed in this paper as mean ( standard deviation for four independent samples. Cell Culture in Collagen Hydrogel. Type I collagen solution (3 mg/mL, Cellmatrix type I-A, Nitta Gelatin Inc., Osaka, Japan)
Restriction sites are underlined. Italics represent the sequence encoding G3P. Boldface represents the sequence encoding the 5′ or 3′ region of CBD.
dG3P-CBD
cG3P-CBD
nG3P-CBD
Hiraoka et al.
a
5′-GGTCGTCATATGGACTGCAGCCAGCCCCTGGAC-3′ 5′-TCCTCGAGAGAGCACAGTTTGTGGAGGAAGG-3′ 5′-GGTCGTCATATGCCACCTTTTCTAATGTTGCTTAAAGGTTCTACCAGGGACTGCAGCCAGCCCCTGGAC-3′ 5′-TCCTCGAGAGAGCACAGTTTGTGGAGGAAGG-3′ 5′-GGTCGTCATATGGACTGCAGCCAGCCCCTGGAC-3′ 5′-TCCTCGAGCCTGGTAGAACCTTTAAGCAACATTAGAAAAGGTGGAGAGCACAGTTTGTGGAGGAAGG-3′ 5′-GGTCGTCATATGCCACCTTTTCTAATGTTGCTTAAAGGTTCTACCAGGGACTGCAGCCAGCCCCTGGAC-3′ 5′-TCCTCGAGCCTGGTAGAACCTTTAAGCAACATTAGAAAAGGTGGAGAGCACAGTTTGTGGAGGAAGG-3′ CBD
chimeric proteins
Table 1. Primers Used for PCR
FW RV FW RV FW RV FW RV
DNA sequence of forward (FW) and reverse (RV) primersa
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was mixed on ice with 5-fold concentrated culture medium and reconstitution buffer (50 mM NaOH, 260 mM NaHCO3, 200 mM HEPES) at a volume ratio of 7:2:1. Then, nG3P-CBD solution was added to the mixture to an nG3P-CBD concentration of 300 µg/mg collagen. An aliquot of the mixture (500 µL) was added into each well of a 12-well tissue culture plate and incubated at 37 °C for 90 min to allow formation of hydrogels with a thickness of approximately 1 mm. The neurosphere-forming cells obtained as above were seeded onto the collagen hydrogel at a density of 1.0 × 105 cells/cm2 and incubated for 4 h at 37 °C under 5% CO2 and subsequently overlaid with 500 µL of a chimeric protein-containing collagen hydrogel of the same composition as described above to embed the cells in a hydrogel. The embedded cells were additionally cultured for 24 and 48 h in an incubator at 37 °C under 5% CO2 atmosphere. Cell Viability. The live/dead assay (23) was performed by staining cells with 3′,6′-di(O-acetyl)-4′,5′-bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, tetraacetoxymethyl ester (calcein-AM), and propidium iodide (PI). Cells cultured in a collagen gel for 24 and 48 h were exposed to PBS containing 1 µg/mL calcein-AM, 1 µg/mL PI, 0.05 mM MgCl2, and 0.9 mM CaCl2 for 30 min and then washed with PBS containing 0.05 mM MgCl2 and 0.9 mM CaCl2. In this procedure, calceinAM penetrates into the cytosol of living cells and fluorescently stains them in green, while PI fluorescently stains the nucleus of dead cells in red. The microphotographs of cells stained in green and red were recorded using the epifluorescent microscope (DP70, Olympus). For the quantification of living cells, the number of cells stained in red (dead cells) was determined on five different microphotographs (area: 0.54 mm2) recorded for each sample, while total cell numbers were determined by Hoechst staining. Cell viability was determined as percent living cells to the total cells and presented as mean ( standard deviation for four independent samples. Statistical Analysis. The results of cell proliferation and viability assays were statistically analyzed using a software (JMP5.0.1a, SAS Institute Inc., Cary, NC). One-way analysis of variance (ANOVA) was performed for assessing significant differences. Tukey’s honestly significant difference (HSD) test was performed for multiple comparisons at a significance level set of p < 0.05.
RESULTS Preparation of Chimeric Proteins. Figure 2 shows the result of SDS-PAGE analysis for the chimeric and control proteins. These proteins were separated as single bands. The molecular weight estimated from the mobility of protein bands are as follows: CBD 22 kDa, nG3P-CBD 23 kDa, cG3P-CBD 23 kDa, and dG3P-CBD 24 kDa. These results are consistent with the molecular weights predicted from their amino acid compositions. As shown in Figure 3, all the proteins gave CD spectra with minima at 208 and 222 nm for the R-helix and at 216 nm for the β-sheet (24). Compared with CBD, nG3P-CBD and cG3PCBD exhibited slightly decreased Cotton effects, indicating lower contents of secondary structure. dG3P-CBD exhibited much more reduction in negative Cotton effects at 208, 216, and 222 nm. The R-helix contents of these proteins were determined from mean residue molar ellipticity at 222 nm (25) and compared with those calculated based on crystallographic data (26). The results are shown in Table 2. The experimentally determined R-helix contents are in good agreement with predictions for CBD, nG3P-CBD, and cG3P-CBD, suggesting the structural integrity of these proteins. In the case of dG3PCBD, a relatively large difference was observed between experimental and predicted values. This is probably due to the fact that proper refolding is hindered by hydrophobic amino
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Bioconjugate Chem., Vol. 20, No. 5, 2009 979 Table 3. Amount of Chimeric Proteins Bound to the Collagen-Coated Surface Determined by SPR Analysisa chimeric protein
chimeric protein bound (ng/cm2)b
surface density of G3P (104 molecules/µm2)
CBD nG3P-CBD cG3P-CBD dG3P-CBD
47 ( 6 57 ( 16 61 ( 11 131 ( 27
s 1.5 ( 0.4 1.6 ( 0.3 6.6 ( 1.4
a The amount of collagen preadsorbed to the substrate: 120 ( 30 ng/ cm2 (mean ( standard deviation for n ) 12). b Mean ( standard deviation for n ) 3.
Figure 2. The result of SDS-PAGE analysis. Chimeric proteins were electrophoresed in 12.5% polyacrylamide gel and visualized by coomassie brilliant blue staining. Molecular weight standard was electrophoresed in two lanes at both sides.
Figure 3. Far UV circular dichroism spectra of chimeric proteins recorded at 20 °C. Mean residue molar ellipticity [θ] is shown as a function of wavelength. Table 2. r-Helix Content Determined from CD Spectra R-helix content (%) protein
determined from CD spectra
predicted from structural dataa
CBD nG3P-CBD cG3P-CBD dG3P-CBD
28.4 26.2 26.2 19.5
28.9 27.3 27.3 25.8
a Calculated under the assumption that G3P, hexahistidine, and Leu-Glu sequence have no R-helical structure. R-Helix content of the wild-type A3 domain is 30.2% when calculated from structural data (23).
acids rich in G3P. In practice, dG3P-CBD had poor solubility in buffer solutions compared with other chimeric proteins. This appeared remarkably when the solutions at relatively high concentration were dialyzed against neutral buffer solutions. Binding of Chemic Proteins to Collagen. Figure 4 shows SPR sensorgrams recorded for the binding of chimeric proteins to collagen. The amounts of bound proteins determined from these sensorgrams are shown in Table 3. As is seen, CBD, nG3P-CBD, and cG3P-CBD bound to collagen at a similar density to each other. On the other hand, more than 2-fold larger amount of dG3P-CBD bound to the collagen compared with
nG3P-CBD and cG3P-CBD. Accordingly, the surface density of G3P is approximately 4-fold higher with dG3P-CBD than with nG3P-CBD and cG3P-CBD. It seems that the hydrophobic nature of dG3P-CBD has an additional contribution to collagen binding. Cell Adhesion to Collagen-Coated Surfaces. Reverse transcriptase-PCR (RT-PCR) was performed to analyze the expression of mRNA for R3 and β1 integrins in neurosphere-forming cells. As shown in Supporting Information Figure S1, these cells certainly express both R3 and β1 integrins, although the presence of an R3β1 complex is not identified. These cells were cultured for 6 h on the collagen-coated surfaces bound with chimeric proteins. Figure 5 shows the phase-contrast images of these cells. It is clearly seen that few cells adhered to the collagen-coated dish bearing no chimeric proteins (Figure 5A). Similarly, poor cell adhesion was observed on a collagen-coated surface bound with CBD (Figure 5B). In contrast, a substantial number of cells are seen on surfaces with nG3P-CBD, cG3P-CBD, and dG3PCBD (Figure 5C-E). These cells extended dendrites, producing cellular networks. The addition of soluble G3P to the medium largely blocked cell adhesion to the collagen-coated surface with chimeric proteins (Figure 5F), suggesting that the observed cell adhesion (Figure 5C-E) is mediated by specific interaction of cells with the surface-bound G3P. The number of adhering cells are shown in Figure 6. The data show that cell adhesion to collagen was significantly promoted by nG3P-CBD and cG3PCBD, and much more by dG3P-CBD. It seems that cell differentiation was not effectively induced by the chimeric proteins because of the short period (6 h) after seeding. The formation of dendrites (Figure 5C-E) is not indicative of cell differentiation. In fact, as shown in Supporting Information Figure S2, the fraction of neural stem cells was rather higher in the cells adhering to the G3P-bearing surface (70%) than that in neurosphere-forming cells (50-60%) (21), indicating somewhat preferential adhesion of neural stem cells to the G3P-bearing surface. Cell Survival in Collagen Hydrogels. Figure 7 shows the phase-contrast images of cells cultured in a collagen hydrogel with or without incorporated nG3P-CBD. Although cell adhesion was most effectively promoted by dG3P-CBD on polystyrene-based substrates (Figures 5 and 6), we used nG3P-CBD in this experiment. This is because the handling of nG3P-CBD was much easier because of its moderate hydrophilicity. As shown in Figure 7, observation of cells with an optical microscope was difficult because cells were not distributed in the same plane. However, careful inspection of cells revealed that most of the cells had round shapes in a pure collagen hydrogel. On the other hand, many cells are seen to flatten and extend dendrites in the collagen hydrogel with incorporated nG3P-CBD. Compared to the cells on rigid polystyrene substrates (Figure 5), cell aggregation is noticeable in the hydrogels. Figure 8 shows the results of live/dead assays performed for cells cultured in collagen hydrogels with or without incorporated nG3P-CBD. In pure collagen hydrogels, the fraction of living cells decreased rapidly likely due to an anoikis effect, and only
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Figure 4. SPR sensorgrams for the binding of chimeric proteins onto collagen. a-g represent the times when the following solutions were injected to the gold surface bearing a self-assembled monolayer of COOH-terminated alkanethiol, initially equilibrated with PBS: a, acetic acid buffer (pH 4.9); b, 100 µg/mL collagen in sodium acetate buffer (pH 4.9); c, sodium acetate buffer (pH 4.9); d, PBS (pH 7.4); e, PBS (pH 7.4) mixed with pure Tris-HCl buffer at the same mixing ratio as in f; f, 50 µg/mL chimeric protein diluted with PBS (pH 7.4); g, PBS (pH 7.4) mixed with pure Tris-HCl buffer at the same mixing ratio as in f. All measurements were carried out at 37 °C.
Figure 5. Phase-contrast microphotographs of cells cultured for 6 h on the collagen-coated polystyrene surfaces bound with or without chimeric proteins. (A) No chimeric proteins were bound (collagen-coated surface). (B-E) Collagen-coated surfaces bound with (B) CBD, (C) nG3P-CBD, (D) cG3P-CBD, and (E) dG3P-CBD. (F) Cells were cultured on the collagen-coated surface bound with dG3P-CBD in a medium containing 25 µg/mL soluble G3P. Bars: 100 µm.
21.0 ( 5.6% of cells survived 2 days. On the other hand, a significantly larger number of cells survived 24 h (54.0 ( 4.8%) or 48 h (45.5 ( 3.7%) in collagen hydrogels with incorporated nG3P-CBD. These results indicate that cell death was delayed by incorporating nG3P-CBD.
DISCUSSION This study demonstrates that the viability of neurosphereforming cells embedded in a collagen hydrogel is improved by incorporating G3P to the hydrogel (Figures 7 and 8). Because the viability of most cells largely relies on adhesion signaling (27), the improved viability is conceivably the consequence of enhanced cell adhesion in the hydrogel networks. In fact, neurosphere-forming cells cultured in the G3P-incorporating collagen hydrogel exhibited more extended forms than in the
pure collagen hydrogel. The continued existence of an increased number of living cells for 2 days will be greatly beneficial to cell transplantation therapy in the brain. In this study, the peptide G3P was attached to collagen networks through specific interactions of the fusion partner CBD with collagen. To date, a variety of conjugation methods have been reported for the modification of collagen with various peptides (28, 29) including G3P as well (30). These methods often require severe reaction conditions. In addition, it is not always straightforward to modulate the orientation and density of peptides with conventional chemical methods. Our approach using biospecific interactions allows us to densely immobilize a peptide on collagen under physiological conditions. This is exemplified by the data presented in Table 3 as well as our previous studies (8, 9). In addition, one can easily design
Collagen Hydrogel with Laminin-Derived Peptide
Figure 6. The number of cells adhered to collagen-coated polystyrene surfaces bound with chimeric proteins. Data are expressed as mean ( standard deviation for n ) 4. *Statistically significant (p < 0.05, Tukey’s HSD test).
collagen-binding chimeric proteins bearing different peptides using the CBD as a universal coupler. The laminin-derived cell-adhesive peptide, G3P, was originally identified by Kim et al. as a ligand for R3β1 integrin expressed on epithelial cells (18). This peptide is contained in a G3 domain of a laminin R3 chain, a component of a laminin-5 isoform. The interaction between laminin-5 and R3β1 integrin was reported to have an antiapoptotic effect in a variety of epithelial cells (16, 17). Laminin-5 is abundantly found in epithelial basement membranes (31) and also expressed in developing brain tissues (32). Therefore, the collagen matrix bound with G3P can be regarded as a biomaterial that mimics in part the native basement membrane.
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We examined the effects of chimeric proteins with three different structures: G3P was fused at the N- or C-terminus, or both termini of CBD (Figure 1). It was shown that nG3P-CBD and cG3P-CBD had comparable properties with regard to solubility, collagen binding (Table 3), and cell adhesion (Figure 5). This finding suggests the structural and functional similarities between G3Ps in nG3P-CBD and cG3P-CBD. A CBD contains a disulfide bond between two cysteine residues located at the N- and C-terminal regions (26), forming a globular domain with both termini close each other. Accordingly, it is expected that G3Ps in nG3P-CBD and cG3P-CBD have minor differences with regard to their arrangements in the proteins, although the peptides are tethered to CBD in an opposite orientation. Among chimeric proteins studied, dG3P-CBD having G3P at the both ends exhibited the highest binding capacity for collagen (Table 3). As described earlier, dG3P-CBD exhibited low solubility due in part to the high occupancy of amino acids with nonpolar side chains (P, F, M, L, and G). Presumably, two G3Ps raise the hydrophobicity of dG3P-CBD. In addition, the slightly disordered structure of this protein, as demonstrated by CD spectroscopy (Figure 3), suggests the enhanced exposure of hydrophobic amino acids that would be located in the core region in the native state. It is likely that such structural alteration enhances the nonspecific adsorption of dG3P-CBD to collagen and hence gives rise to elevated surface density. The highest cell density observed for the dG3P-CBD-collagen surface (Figures 5 and 6) may be directly related to exceptionally high surface density of G3P (Table 3). The probability of interaction between G3P and integrin may be enhanced on this surface. The range of the G3P density that has influence on the number of adhering cells (15 000-66 000 molecules/µm2) is similar to the dynamic range of ligand density reported by Maheshwari et al. (33) for the adhesion of fibroblasts via interaction between integrins and surface-immobilized RGD peptide. On the other hand, it may not be the case that two G3Ps in the same molecule simultaneously engage in binding to two proximal integrins, contributing to the clustering of
Figure 7. Results of cell culture assays in collagen hydrogels with or without incorporated nG3P-CBD. (A, B) Phase-contrast microphotographs of cells cultured for 48 h in (A) a pure collagen hydrogel and (B) a collagen hydrogel containing nG3P-CBD. Bars: 100 µm. (C, D) Fluorescent microphotographs of cells cultured for 48 h in (C) a pure collagen hydrogel and (D) a collagen hydrogel containing nG3P-CBD. Cells were stained with calcein-AM (live cells in green) and propidium iodide (dead cells in red). Bars: 100 µm.
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Figure 8. The viability of cells cultured for 24 and 48 h in (O) collagen hydrogels containing nG3P-CBD or (b) pure collagen hydrogels. Data are expressed as mean ( standard deviation for n ) 4. *Statistically significant (p < 0.05, Tukey’s HSD test).
integrins and the formation of focal contacts. This is because an R3β1 integrin complex is much larger than the spun of two G3Ps contained in a single dG3P-CBD molecule. As we observed in the live/dead assays (Figures 7 and 8), a larger number of neurosphere-forming cells survived in collagen hydrogels containing nG3P-CBD than in pure collagen hydrogels. We were unable to distinguish necrotic cells from apoptotic ones by PI staining alone, and possibly both mechanisms were involved in observed cell death. Regarding necrotic conditions, if any, cells were equally effected in both hydrogels, and cell viability may be improved by optimizing culture conditions such as oxygen and nutrient supply. For the observed difference in viability, we assume that this is due to antiapoptotic signaling cascades activated in the cells in consequence of an interaction between G3P and R3β1 integrin. Actually, integrin subunits R3 and β1 are both expressed on the neurosphere-forming cells (Supporting Information Figure S1), being in agreement with reports for neural stem cells (10), cerebral cortex neurons (34), and differentiating neuroblastoma cells (35). However, we need further mechanistic study on intracellular signaling to understand more deeply the effect of the incorporated G3P.
CONCLUSION The chimeric proteins consisting of the laminin-derived G3P and the vWF-derived CBD bind to collagen under physiological conditions, providing collagen-based substrates to which neurosphere-forming cells are able to adhere. When incorporated in collagen hydrogels, the chimeric protein serves to enhance the survival of neurosphere-forming cells in the hydrogels. Although further mechanistic studies are needed, the promoted cell adhesion and survival may be ascribed to signaling mediated by the specific interaction of the G3P with R3β1 integrin. The composite hydrogels reported here have the potential to improve the survival and engraftment of neural cells transplanted into central nervous tissues.
ACKNOWLEDGMENT This work was supported by Grants-in-Aid for Scientific Research (No. 19659364), MEXT, Japan. T.N.-H. acknowledges JSPS for a research fellowship. Supporting Information Available: Amino acid sequences of chimeric proteins, dialysis conditions, results of RT-PCR, and immunocytochemistry. This material is available free of charge via the Internet at http://pubs.acs.org.
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