Tethered Epidermal Growth Factor Provides a Survival Advantage to Mesenchymal St - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:54 |只看该作者 |倒序浏览 |打印

作者：Vivian H. Fana,b, Ada Aua, Kenichi Tamamac, Romie Littrella, Llewellyn B. Richardsond, John W. Wrighta, Alan Wellsc, Linda G. Griffitha,e

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      【摘要】
   MSC can act as a pluripotent source of reparative cells during injury and therefore have great potential in regenerative medicine and tissue engineering. However, the response of MSC to many growth factors and cytokines is unknown. Many envisioned applications of MSC, such as treating large defects in bone, involve in vivo implantation of MSC attached to a scaffold, a process that creates an acute inflammatory environment that may be hostile to MSC survival. Here, we investigated cellular responses of MSC on a biomaterial surface covalently modified with epidermal growth factor (EGF). We found that surface-tethered EGF promotes both cell spreading and survival more strongly than saturating concentrations of soluble EGF. By sustaining mitogen-activated protein kinase kinase-extracellular-regulated kinase signaling, tethered EGF increases the contact of MSC with an otherwise moderately adhesive synthetic polymer and confers resistance to cell death induced by the proinflammatory cytokine, Fas ligand. We concluded that tethered EGF may offer a protective advantage to MSC in vivo during acute inflammatory reactions to tissue engineering scaffolds. The tethered EGF-modified polymers described here could be used together with structural materials to construct MSC scaffolds for the treatment of hard-tissue lesions, such as large bony defects.; l+ t5 C: r: G8 g- h+ T, h
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Disclosure of potential conflicts of interest is found at the end of this article. ( e3 [1 f) P. u' E
      【关键词】 Mesenchymal stem cells Epidermal growth factor Extracellular signal-regulated protein kinase Fas ligand Cell death Cell spreading Bone graft
   INTRODUCTION

MSC were first described 40 years ago as bone-forming progenitors isolated from marrow . Despite recent clinical and scientific advances in stem-cell research, there is limited understanding of how survival and function of MSC are governed by interactions with scaffolds used for MSC delivery. Furthermore, it is unclear how MSC fate may be influenced by cytokines present during the inflammatory response to biomaterials used for implantation, and to injury itself.

The use of progrowth and prosurvival cytokines as adjuvants to MSC application may improve implant outcomes. Epidermal growth factor (EGF) is a well characterized cytokine involved in the growth and repair of various tissues. Upon binding to the EGF receptor (EGFR), EGF activates intracellular signals via the extracellular-regulated kinase (ERK) and Akt pathways, among others . We hypothesized, however, that MSC could respond differently to tethered EGF compared with soluble EGF as a result of binding the ligand on a surface rather than in solution and thus preferentially activate surface-associated signaling pathways.7 s4 j8 ]; {2 y! E7 q

In addition to prosurvival growth factors, multiple prodeath ligands are upregulated during inflammation of bone tissue, including Fas ligand (FasL), tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), interferon  (IFN-), and interleukin 1 (IL-1) .0 S8 L; L, x7 R
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Here, we show that surface-tethered EGF improved MSC survival upon stimulation with a prodeath stimulus. We used a polymer substrate developed to present clusters of closely spaced ligands on short poly(ethylene oxide) (PEO) tethers  to covalently tether EGF via the N terminus, so that EGF is bioactive but restricted to the material surface. Surprisingly, we found that tethered EGF exceeded the ability of soluble EGF to promote cell spreading and survival in the presence of FasL, which we showed to be a potent death factor for human MSC. This study suggests that MSC-implant interactions can be improved by providing local, spatially controlled growth factors to the biomaterial surface.+ Q* ~! Q  `% ]7 h" X

MATERIALS AND METHODS& ]3 f2 P, _4 b2 J. z
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Growth Factors, Antibodies, and Signaling Reagents
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Murine EGF and human EGF were purchased from Peprotech (Rocky Hill, NJ, http://www.peprotech.com), and porcine EGF was purchased from GroPep Limited (Thebarton, Australia, http://www.gropep.com.au). EGFR inhibitors AG1478 and PD153035 and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 were from Calbiochem (San Diego, http://www.emdbiosciences.com). The EGFR ligand binding site-blocking antibody C225 was a gift from H.S. Wiley (Pacific Northwest National Laboratory, Richland, WA). Rabbit polyclonal anti-phospho-p44/42 Map kinase, rabbit polyclonal anti-EGF receptor, and rabbit polyclonal anti-phospho-Akt were purchased from Cell Signaling Technology (Beverly, MA, http://www.cellsignal.com); rabbit polyclonal anti-MAP kinase/ERK1/2-CT was from Upstate (Charlottesville, VA, http://www.upstate.com). Mouse monoclonal anti--tubulin was from Calbiochem. Donkey anti-rabbit and sheep anti-mouse IgG peroxidase-linked secondary antibodies were from Amersham Biosciences (Piscataway, NJ, http://www.amersham.com). Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) or ECL Advanced Western Blotting Detection Kit (Amersham Biosciences) was used to visualize the protein bands with Kodak ISO1000 Image Station (Rochester, NY, http://www.kodak.com). Densitometry was performed with Kodak software." E3 o- f; c0 M  \- n6 N
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Cell Culture

Human telomerase reverse transcriptase (hTERT)-immortalized human MSC (hTMSC) were a gift from Dr. Junya Toguchida (Kyoto University, Kyoto, Japan) . Cells were obtained and maintained in an osteogenic medium, minimal essential medium- buffered with bicarbonate, 10% FBS, 1% penicillin-streptomycin, 10¨C8 M dexamethasone (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and 50 µg/ml ascorbate (Sigma-Aldrich) at 37¡ãC in 5% CO2/95% air. Cells were cultured for approximately 10 days (85% confluent within the colonies) before assays. Poietics primary human MSC (phMSC) were purchased from Cambrex (Walkersville, MD, http://www.cambrex.com) and maintained according to the supplier's instructions. phMSC were cultured in MSC growth medium containing MSC growth supplement, L-glutamine, and penicillin-streptomycin (Cambrex). Growth media were changed every 2¨C3 days, and the cells were split at 5,000¨C6,000 cells per cm2 every 5¨C6 days. Cells up to the fifth passage were used in this study.' @5 c7 {5 Z7 s) y, |
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hCTP Selection
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Bone marrow aspirates from human subjects were collected at the Muschler laboratory . A medium change was performed on every third day until the colonies reached sufficient confluence for cytokine stimulation.; r3 i6 `: p+ c& N; ?3 Y" X
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Polymeric Substrate Preparation( t+ ~  x7 ?; H) n
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Two different poly(methyl methacrylate)-graft-poly(ethylene oxide) (PMMA-g-PEO) comb polymers differing in the total weight percent (wt%) PEO (and consequently, the spacing between PEO side chains emanating from the culture surface) were synthesized using general protocols described previously . This tEGF-polymer has a weight-average molecular weight and polydispersity index (PDI) of 96,000 and 3.2, respectively, obtained by gel permeation chromatography using polystyrene standards. It comprises 33 wt% PEO and has 20 PEO side chains per chain on average (each side chain of 10 EO repeating units, spaced less than 2 nm apart along the backbone). For this PEO content, PMMA-g-PEO is highly resistant to cell adhesion unless covalently modified with cell adhesion ligands. To create cell-adhesive regions on the surface, the tEGF-polymer was diluted with a second PMMA-g-PEO comb polymer (mol. wt., 45,000; PDI, 1.8) comprising only 20 wt% PEO, a composition known to be cell-adhesive in the presence of serum or cell-secreted extracellular matrix (ECM).
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Specifically, glass coverslips (12 mm in diameter) were silanized with Siliclad (Gelest Inc., Morrisville, PA, http://www.gelest.com) prior to polymer thin film preparation by spin coating. The tEGF-polymer was activated on the hydroxyl chain ends with 4-nitrophenyl chloroformate, mixed with diluent polymer (40:60 tEGF-polymer:diluent), and spin-coated to form an 100-nm thin transparent film on the substrate (as measured by ellipsometry). Murine EGF was surface-coupled to the activated side chains of the spin-coated polymer by incubation in 25 µg/ml EGF in phosphate-buffered saline (PBS) (100 mM, pH 8¨C8.5) at room temperature for 22¨C24 hours followed by PBS washes. The remaining active groups were blocked in 100 mM Tris buffer (pH 9) at room temperature for 2 hours, followed by PBS washes to achieve a surface density of approximately 5,000¨C7,000 tethered EGF cells per µm2. Substrates were stored in PBS at 4¡ãC until use. For in vitro experiments, each substrate was placed in individual wells of a 24-well plate and secured with a section of silicone rubber tubing, providing 0.64 cm2 of available surface area.
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Activation of EGFR and EGFR Signaling Pathways

Prior to growth factor stimulation, the cells were serum-starved in a serum-free medium (Advanced DMEM , supplemented with 1 mM pyruvate, 1 mM L-glutamine, 1 µM nonessential amino acids, and 100 units/ml penicillin-streptomycin) for 14¨C16 hours. The cells were detached with Versene (Invitrogen) and plated in the serum-free medium with the appropriate stimuli at a density of 25 x 103 cells per cm2 on the polymeric substrates and incubated at 37¡ãC. The cells were pretreated for an hour with inhibitors when needed. At the indicated time points after plating, all adherent and nonadherent cells were pooled from two coverslips and incubated on ice for at least 15 minutes in 20 µl of lysis buffer (50 mM ¦Â-glycerophosphate at pH 7.3, 10 mM Na-pyrophosphate, 30 mM NaF, 50 mM Tris base at pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM benazmidine, 2 mM EGTA, 100 µM Na3VO4, 1 mM dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml microcystin-LR, and 1 mM phenylmethylsulfonyl fluoride). The supernatants were collected following 15 minutes of centrifugation at 16,000g and stored at ¨C80¡ãC. For the 0-hour time point, the cell mixtures were spun down at 450g for 10 minutes at 4¡ãC. Medium was aspirated, and the cell pellet was lysed. The protein content of the cell lysates was determined using the Micro BCA protein assay (Pierce, Rockford, IL, http://www.piercenet.com). The supernatants were collected and stored at ¨C80¡ãC.
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Cell Death Quantification Assay$ R6 e5 _( G8 z5 l* R' F

Serum-starved (24 hours) hTMSC were plated in serum-free media on polymer substrates at 25 x 103 cells per cm2, allowed to attach for 4¨C8 hours (as indicated in figure legends), and then stimulated with prodeath cytokines at 100 ng/ml by addition to the medium. phMSC cultured to 80% confluence in serum-containing culture medium were detached with trypsin and plated on polymer substrates in standard (serum-containing medium) at 25 x 103 cells per cm2. The culture medium was changed to a serum-free medium (Cambrex Mesenchymal Stem Cell Basal Medium) 4 or 5 hours after plating (as indicated in figure legends 5 and 6), and FasL was added 1 hour later (5 or 6 hours after plating). Human recombinant superFasL and TRAIL were from Alexis Biochemical (Lausen, Switzerland, http://www.alexis-corp.com). In some experiments with phMSC, substrates were coated with human plasma fibronectin (Sigma-Aldrich) by incubating comb copolymer (22 wt% PEO) substrates with 0.1 µg/ml fibronectin in PBS for 24 hours at room temperature followed by two gentle PBS washes. All adherent and nonadherent cells, detached with trypsin and collected from two coverslips, were spun down at 450g and stained in 10 µg/ml propidium iodide (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) for 15 minutes. The positive staining population was quantified by direct fluorescence using a FACSCalibur flow cytometer.
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Cell Morphology Measurements! d" I9 A* w1 W# N, P/ L- X3 p

Static images of cells were captured under differential interference contrast (DIC) optics with a x10 or x20 lens after at least 4 hours of incubation on at least three independent samples to represent the average cell morphology. The acquired images of hTMSC were imported to ImageJ (Version 1.36b) to quantify the spread area per cell on the polymeric substrates. At least 20 cells per field were traced to calculate the average area per cell.

Statistical Analyses

Western blot densitometry data and propidium iodide (PI)  staining population data were analyzed with analysis of variance and the t test. Cell spread area data were analyzed with Randsum and Kruskal-Wallis tests. Significance was set at p

RESULTS
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Kinetics of EGFR Signaling Pathways in MSC Stimulated by Soluble and Tethered EGF

Tethered EGF substrates were prepared with murine EGF, which has a single primary amine at the N terminus. This allows it to be precisely linked to the surface via amine-targeting chemistry, tethering the EGF molecules in a configuration competent to bind and activate EGFR and creating a locally high concentration of EGF in the vicinity of the cell-substrate interface .9 m- ^; t- d, E+ d5 z

We first investigated the kinetics of hTMSC ERK and Akt/PKB . Therefore, murine EGF was comparable to human EGF for hTMSC, similar to other cells types responding to EGFR ligands from other mammalian species. Murine EGF was used for all subsequent experiments as its single primary amine allows for more specific covalent coupling to the substratum.

Figure 1. Soluble EGF transiently activated EGF receptor signaling pathways in human MSC. Human telomerase reverse transcriptase-immortalized human MSC (hTMSC) were plated on tissue culture-grade polystyrene to subconfluence, serum-starved for 14¨C16 hours, and then stimulated with 100 ng/ml soluble EGF of human (A) or murine (B) origin. hTMSC lysates at the indicated times were analyzed for ERK and Akt phosphorylation by Western blotting and quantified by densitometry. Data indicate the ratio of phosphorylated ERK to total ERK and the ratio of phosphorylated Akt to tubulin upon the stimulation of soluble human EGF (A) and murine EGF (B), respectively. Shown are the average ¡À SEM of three independent replicates for each comparison; there are no significant differences between the ligands. Abbreviations: EGF, epidermal growth factor; ERK, extracellular-regulated kinase; min, minutes; pAkt, phospho-Akt; pERK, phospho-extracellular-regulated kinase.

To exclude the possibility that the EGF response of hTMSC was an artifact of the hTERT immortalization procedure, we also investigated ppMSC, as they are readily obtainable and have metabolic rates comparable to human MSC . In response to either porcine or murine soluble EGF stimulation, ERK phosphorylation in pMSC peaked at 5 minutes and gradually diminished (supplemental online Fig. 1). This indicated that both immortalized hTMSC and primary pMSC were responsive to murine soluble EGF.4 N+ K) f! _; Q! p

As the hTMSC were similar to the porcine ones (Fig. 1), we chose the immortalized human cell line to conduct most of the remaining intracellular signaling experiments because these cells were easier to maintain and their behavior was more consistent compared with ppMSC. To investigate hTMSC responses to tethered EGF, we defined three experimental conditions. The first condition (control) was the unmodified comb copolymer surface, which served as a control substrate to study MSC behavior on the inert biomaterial (i.e., signals generated by adhesion) under EGF-free conditions. Second was the tEGF substrate, obtained by covalently attaching murine EGF to the control substrate to the free ends of PEO side chains. The tethering chemistry yields an average surface ligand density of 5,000 EGF molecules/µm2, theoretically sufficient to saturate endogenous EGF receptors (estimated to be fewer than 100 EGFR per µm2 cell surface area for cells expressing EGFR at 10,000 EGFR per cell . Last, to distinguish cellular responses specifically conferred by the tEGF substrate from those induced by EGF generally, we plated hTMSC on the control substrate in the presence of saturating murine soluble EGF (sEGF) (100 ng/ml). The sEGF condition thus acted as a more stringent comparison for characterizing MSC behavior unique to tEGF substrates.4 d- R2 V6 U: p& M
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The tEGF is covalently linked to the substratum; this is postulated not only to prevent ligand-receptor internalization but also to compartmentalize EGFR signaling to the cell surface . As survival and proliferation signals come from both surface and internalized EGFR-induced cascades, it was unclear how tEGF would modify these cascades. To examine the bioactivity of tEGF, we investigated ERK phosphorylation as a recognized intracellular signal downstream of EGFR that also effects transcriptional changes. At 8 hours, ERK phosphorylation in the sEGF group was only slightly elevated compared with the control group, consistent with the rapid adaptation of ERK signaling observed previously in hTMSC treated with EGF on culture-grade polystyrene (Fig. 1A). sEGF-induced ERK phosphorylation continued to decline, such that it was indistinguishable from the control by 24 hours (p

Figure 2. ERK phosphorylation in human telomerase reverse transcriptase-immortalized human MSC (hTMSC) was stronger and more sustained with tEGF compared with sEGF. Serum-starved hTMSC were plated at a density of 25 x 103 cells per cm2 on the control substrates with or without sEGF (100 ng/ml) and the tEGF substrates, and cells were lysed at the indicated times. pERK was normalized by densitometry relative to total ERK. Error bars represent SEM of quadruplet independent lysates. *, p
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Interestingly, the activation of EGF signaling on tEGF substrate appeared to be pathway specific, since Akt phosphorylation was similar across the three experimental groups (data not shown). Significant Akt phosphorylation in the control group indicated that the Akt signaling in hTMSC might be a nonspecific response to plating and binding through adhesion receptors. Alternatively, Akt could be downregulated by mechanisms distinct from that of ERK . Nevertheless, the clear and persistent ERK activation indicated that tEGF was functional as a bioactive ligand for hTMSC.
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MSC Morphology on Polymeric Substrates Modified with tEGF. ?% L  M, \: x6 G

Tethering EGF to the substratum should enhance plasma membrane signaling, with pronounced effects on cell attachment and spreading, as this cellular process proceeds mainly from periplasma membrane epigenetic events. When hTMSC were plated under control, tEGF, and sEGF conditions, we observed morphological differences among the groups, starting as early as 8 hours after plating and persisting for at least 24¨C48 hours. On the control substrate, hTMSC attached but remained rounded with little spreading after 24 hours of incubation (Fig. 3A). By contrast, hTMSC on tEGF substrate exhibited a clear adhesive phenotype, evidenced by the presence of lamellipodia and increased cell area (Fig. 3C). Importantly, addition of sEGF to control substrates could not produce the same spreading observed on the tEGF substrate (Fig. 3B). When the average cell area was quantified for the three conditions, we found that the tEGF condition caused a three- to fourfold increase in spreading compared with either the control or sEGF groups (p " S) f) d9 x( ^) Q# S3 K

Figure 3. tEGF causes spreading of human MSC on polymeric substrates. Light microscopy images of human telomerase reverse transcriptase-immortalized human MSC plated and maintained in serum-free medium were taken at a magnification of x10 at 24 hours on control substrates (without sEGF ) and tethered EGF substrates (G, J). Scale bar = 100 µM. Results are representative of three biological replicates. Abbreviations: sEGF, soluble epidermal growth factor; tEGF, tethered epidermal growth factor.9 I  c: z8 z9 L; p! l$ S4 N

The presentation mode of EGF also influenced the morphology of low-passage phMSC. phMSC were allowed to attach to the synthetic polymer substrates for 4 hours in the presence of serum, which contains fibronectin, vitronectin, and other molecules that are known to promote cell attachment and spreading. Images acquired 1 hour after changing to serum-free medium (5 hours after plating) show that although phMSC had begun to spread under all conditions, they were far more extensively spread on tEGF compared with control substrates (Fig. 3E¨C3G). Specifically, most phMSC on control substrates in the absence of EGF exhibited numerous filipodia and lamellipodia emanating from a rounded central cell body (Fig. 3E). In the presence of soluble EGF, far fewer cells appeared attached to control substrates, and soluble EGF seemed to slightly inhibit spreading of attached phMSC (Fig. 3F) compared with behavior of cells on control substrates in the absence of EGF (Fig. 3E). In contrast, most cells on tEGF were already extremely spread with flattened cell bodies at 5 hours (Fig. 3G). The trends observed at 5 hours were accentuated by 24 hours: phMSC on control substrates were moderately spread and highly elongated in the absence of EGF (Fig. 3H); phMSC were fewer in number and appeared more spindly on control substrates in the presence of soluble EGF (Fig. 3I); and phMSC on tEGF substrates, although still elongated, appeared flatter and more spread (Fig. 3J) than under other conditions. Thus, presentation of EGF in a mode that prevents internalization promoted attachment and spreading of phMSC, whereas soluble EGF appeared inhibitory compared with control.! `! Q# ^! f1 C  X2 V: j6 B: Z
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Cellular Signaling Events Controlling tEGF-Induced MSC Spreading

Although both cell spreading and sustained ERK phosphorylation were apparent on tEGF substrates, it was possible that ERK phosphorylation was not directly induced by tEGF binding to EGFR. Furthermore, it was unclear whether EGFR and ERK signaling were required for the observed differences in morphology. To determine whether ERK activation was directly induced by tEGF and involved in cell spreading, we used various EGF pathway inhibitors to probe the upstream signaling requirements for ERK phosphorylation (Fig. 4). Specifically, we selected an EGF-blocking antibody, C225  to specifically inhibit the MEK-ERK signaling pathway. Through these inhibitors, we were able to examine the dependence of tEGF-induced morphology on EGF binding, EGFR tyrosine kinase activity, and MEK-ERK signaling., A/ i9 o/ D0 k! x
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Figure 4. MSC spreading on tEGF was blocked by inhibitors of epidermal growth factor receptor (EGFR) signaling. Serum-starved human telomerase reverse transcriptase-immortalized human MSC were plated on control and tEGF substrates at a density of 25 x 103 cells per cm2 (light microscopy images at a magnification of x10 taken at 8 hours , respectively). DMSO was included as a carrier control (C) for AG1478, PD153035, and U0126. (H): Average cell spreading quantified from (A¨CG). Error bars indicate SEM of 80 individual cells. *, p

Phase-contrast images of serum-starved hTMSC on tEGF substrates 8 hours after plating showed early increases in spreading, with noticeably more prominent and extended lamellipodia compared with the control group (Fig. 4A, 4B). hTMSC on tEGF substrates showed no reduction of cell spreading when a fixed amount of 0.1% vol/vol dimethyl sulfoxide (DMSO) was added to the medium to account for any possible effects of the inhibitor diluent (Fig. 4C). The quantified average cell spreading area was approximately twofold increased on tEGF (with or without DMSO) compared with the control (p & K  D( `6 I0 |/ V

The effectiveness of the MEK inhibitor U0126 in blocking the hTMSC morphology raised the possibility that all of the inhibitors were perturbing MEK-ERK as a common mediator of tEGF-induced cell spreading. To examine this, we prepared lysates from each of the conditions in Figure 4A¨C4G and measured ERK phosphorylation by Western blotting. At 8 hours, ERK phosphorylation from the tEGF groups (with and without carrier control) was elevated in comparison with the control (Fig. 4I). The EGFR-blocking antibody (C225) effectively suppressed downstream ERK activation in the hTMSC, indicating that ERK phosphorylation in the tEGF group was initiated by EGF receptor binding. The EGF receptor kinase inhibitors (PD153035 and AG1478) and the MEK inhibitor (U0216) also blocked ERK activation at 8 hours to a similar extent. Since perturbation of either the initial tEGF-EGFR binding event or the MEK relay point was sufficient to block ERK activation (Fig. 4I) as well as cell spreading (Fig. 4A¨C4G), we concluded that sustained signaling via ERK (Fig. 2) was responsible for tEGF-induced spreading.+ S$ S, b, G8 e0 D- c8 e7 |, ~
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MSC Death Induced by TNF-Family Cytokines6 H( }4 J5 i5 q5 F6 A

The differential cell spreading on the tEGF substrates provided the first evidence that new MSC responses could be achieved by tethering EGF to the culture substrate. Although cell morphology itself does not directly translate to improved cellular function in vivo for scaffolds bearing tEGF, we reasoned that the increased cell spreading might indicate the existence of other tEGF-induced phenotypes that are relevant for in vivo performance of a tissue engineering scaffolds such as those used for bone grafts. Such a cell response would be important during wound healing when proinflammatory cytokines are prevalent during the acute inflammatory phase when healing initiates . These cytokines activate immune cells to help clear dying and infected tissue, but they can also cause collateral damage to reparative cells at the implant site. We therefore asked whether such cytokines could stimulate cell death in hTMSC. If so, then tEGF on the surface of a bone graft scaffold might help these progenitors resist death and thus promote tissue regeneration.

We screened FasL ; accordingly, FasL- and TRAIL-induced cell death was dramatically reduced without CHX on the highly adhesive tissue culture polystyrene surfaces. RankL, with or without CHX, did not significantly influence cell death, as expected. These results implicated FasL and TRAIL as relevant prodeath ligands for hTMSC.1 G% m" d5 t! j+ c5 D

Figure 5. FasL and TRAIL induced cell death in hTMSC, hCTP, and phMSC. (A): PI  stained population percentage quantified in hTMSC. Serum-starved hTMSC on tissue culture polystyrene at subconfluence were stimulated with various tumor necrosis factor-family cytokines in the presence or absence of the protein synthesis inhibitor CHX. Cells were stained with PI at 24 hours and analyzed via fluorescence-activated cell sorting. (B): PI  stained population percentage quantified in hCTP. Error bars represent SEM of triplicate independent cell samples. *, p 3 R! R5 w9 k% K; r1 i  y

To determine whether the sensitivity to FasL and TRAIL could be extended beyond the immortalized hTMSC line, we replicated the experiment in a primary hCTP population (as described in Materials and Methods). As with the hTMSC, both FasL and TRAIL, but not RankL, induced cell death significantly above the control level in hCTP (p

Finally, we confirmed that FasL induced cell death in primary human cells conforming to conventional definitions of mesenchymal stem cells by treating phMSC obtained from a commercial source with FasL. Even in the absence of the protein synthesis inhibitor CHX, or in the presence of the adhesion-promoting protein fibronectin on the substrate, FasL induced cell death significantly above controls in phMSC (p

hTMSC Resistance to FasL-Induced Cell Death on tEGF

Having found that TNF-family cytokines can drive hTMSC death, we next investigated whether tEGF provided any protection. To be in line with the duration of acute inflammation, we extended the hTMSC death experiment to 48 hours after plating on the polymeric substrates. In the absence of cytokines, we observed a subtle decrease in cell death in the tEGF group compared with both the control and sEGF groups (p
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Figure 6. tEGF conferred resistance to FasL-induced death via epidermal growth factor receptor (EGFR) and extracellular-regulated kinase. (A): Serum-starved human telomerase reverse transcriptase-immortalized human MSC (hTMSC) were plated on control substrates, with and without sEGF, and tEGF substrates. Prodeath stimuli were added at 8 hours. Cells were stained with PI at 24 hours and analyzed via fluorescence-activated cell sorting (FACS). Error bars represent SEM of triplicate independent cell samples. (B): Primary human MSC were plated in serum-containing media for 5 hours and then exchanged to serum-free media. FasL was added at 6 hours. Cells were stained with PI at 24 hours and analyzed via FACS. Error bars represent SEM of triplicate independent cell samples. *, p / @4 ?) a5 h: t& t6 r' K  r6 M: H
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We next examined the % PI response of phMSC to FasL in the presence of soluble and tethered EGF to determine whether the protective effects of tEGF extended to primary cells. Even in the absence of FasL, soluble EGF induced phMSC death significantly more than controls (p

It was likely that the tEGF-mediated protection to FasL-induced cell death was occurring via differential signaling from the tethered EGF ligand (Fig. 2). To probe the intracellular basis of the antideath function provided by tEGF, we first used the MEK inhibitor U0126, since this small molecule suppressed both ERK activation and tEGF-induced cell spreading (Fig. 4G¨C4I). When U0126 was added to hTMSC, we found that the PI  population on tEGF substrates was raised by threefold, to a level comparable to the control and sEGF groups, indicating that the suppression of ERK activation was sufficient to abolish the antideath function by tEGF (Fig. 6C). Interestingly, direct inhibition of EGF receptor signaling using the EGF receptor kinase inhibitor PD153035 1 hour before addition of FasL resulted in a doubling of the % PI  population for cells on tEGF (p ) o+ K- X: U  O. E

Whereas MEK inhibition completely abolished the protective effect of tEGF compared with controls (Fig. 6C), inhibition of EGF receptor signaling did not completely abolish the protective effect of tEGF compared with controls when cells were challenged with the prodeath stimulus FasL (Fig. 6D). For both the control and tEGF conditions, the % PI  population increased about twofold in the presence of PD153035 and FasL (Fig. 6D), thus maintaining a significant difference between the two conditions. FasL was added 8 hours after the initial switch to various EGF conditions (inhibitor PD153035 was added 7 hours after medium switch), and during this interval, tEGF may initiate prosurvival adhesion signals or autocrine loops that persist during the FasL stimulation period. Integrins and other adhesion receptors signal through ERK . Taken together, we conclude that both spreading and anti-death responses mediated by tEGF occured predominantly via potentiated ERK signaling in hTMSC.

DISCUSSION

In this study, we found that tEGF induced elevated ERK signaling in MSC to cause increased cell spreading and increased resistance to FasL-mediated cell death. Importantly, these responses were unique to tEGF, because saturating concentrations of sEGF did not reproduce the same morphological or functional phenotypes. Our group has reported elsewhere that tEGF inhibits both EGFR internalization and degradation compared with sEGF. EGF tethering thus prevents two recognized mechanisms for downregulating EGFR signaling , these mechanisms allow for potentiated and sustained EGFR signaling via ERK, at least in MSC.2 v+ W- o" k& s1 \2 F5 M3 T0 K

ERK is activated by growth factors to affect a diverse array of cellular events, including cell proliferation, migration, and survival . Therefore, the implication of ERK in spreading and survival of tEGF-stimulated hTMSC is consistent with published mechanisms.- a: @( a6 u' ?

It is interesting to consider whether the tethering of other EGFR ligands would induce cellular responses similar to tEGF. There are several EGF-family growth factors that bind to EGFR, including transforming growth factor- (TGF-), amphiregulin, betacellulin, epiregulin, and heparin-binding EGF-like growth factor . Tethering of other EGF-family or growth-factor ligands may promote healing and tissue regeneration for other therapeutic applications.
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EGF-like repeats are contained in various ECM molecules, including tenascin, laminin, thrombospodin, and versican . Although these results have been attributed to the integrin-binding motifs of laminin 5 (rather than the EGF repeats), taken together with the results reported here, they suggest that the observed difference between soluble EGF and tethered EGF may reflect the ability of MSC to discriminate between naturally occurring molecules., H/ g; }: u% ^- v' v! N
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We further explored MSC physiology by perturbing these cells with cytokines relevant to inflammation, as these might lead to MSC death during the initial phases of wound repair .

Importantly, FasL-induced cell death was significantly reduced by adhesion to tEGF surfaces. Specifically, we showed that tEGF-induced sustained ERK phosphorylation provided a critical antideath signal to MSC stimulated with FasL. These data agree with various studies implicating ERK in survival . The involvement of FasL in MSC biology is likely to be physiologically important, as is the possible antagonism between FasL and EGF-like repeat-containing ECM proteins in the wound-healing milieu.* W- n& e0 J/ R1 ?, @6 G
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Most tissue-engineering efforts to date have focused on modifying polymer surfaces with adhesion ligands or fragments (e.g., fibronectin, arginine-glycine-aspartate, laminin)  but has only limited effects on hTMSC that are plated on polymer surfaces. By tethering EGF, we increased and sustained ERK activation to promote hTMSC spreading and survival.

The introduction of an implant device initiates an array of tissue responses beginning with an acute inflammatory phase within minutes to days after implantation, followed by a chronic inflammatory phase. Inflammation serves to eliminate any infectious causes of injury, to remove dead cells and debris, and to initiate healing that would restore tissue structure. For an implant, however, many inflammatory functions are deleterious, causing foreign-body reactions and premature implant failure. In particular, inflammation may damage progenitor cells while repopulating an implant surface . A crucial challenge for inductive biomaterials is therefore to promote the regenerative functions of progenitors such as MSC despite inflammation.6 |8 `- m. X, V/ h3 @
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The tEGF surface modification we describe provides two key features favoring MSC repopulation. First, the tethering of EGF promoted hTMSC spreading. Progenitors such as MSC are found locally in the marrow, periosteum, and surrounding tissues. The value of tEGF modification would thus be to retain the MSC that interact with the implant surface. Second, tEGF provides a clear prosurvival stimulus to hTMSC. In the current study, we found that tEGF protected MSC for at least 40 hours from the types of apoptotic stimuli that are present during inflammation. This suggests that MSC retained on an implant surface will be more likely to survive against an inflammatory insult.. {( p' m1 ]6 ~$ m& A, ]8 m

Although not investigated explicitly here, we speculate that tEGF should augment other MSC functions that are important for bone healing. Previously, we demonstrated that EGF promotes MSC proliferation while preserving pluripotency ) to treat small- to medium-sized bony defects such as periodontal lesions suggests a more immediate clinical application. The tEGF surface-modification protocol could easily be adapted for bioglass to improve cell survival of progenitors in the grafted surroundings. Overall, we expect that the presentation of inductive MSC ligands through tethering will synergize with advances in materials to provide viable therapies for large bony defects.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST- B: j% T# W0 d6 H+ ^, V) E

The authors indicate no potential conflicts of interest.& D* p7 ^1 Y. o! y, n
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ACKNOWLEDGMENTS1 _% F$ E. ]4 W6 J1 f1 A5 h% b

We thank Dr. J. Toguchida (Kyoto University, Kyoto, Japan) for providing the immortalized human MSC, Dr. H. Abukawa from the Vacanti Laboratory (Massachusetts General Hospital, Boston, MA) for harvesting the primary porcine MSC, Dr. G. Muschler's laboratory (Cleveland Clinic, Cleveland, OH) for supplying the primary hCTP, and Prof. A. Mayes (Massachusetts Institute of Technology, Cambridge, MA) for guidance on the polymer chemistry. We also thank Ikuo Taniguchi (Massachusetts Institute of Technology) for preparing and characterizing activated polymers and Nicholas Marcantonio (Massachusetts Institute of Technology) for preparing polymer substrates for some experiments. This work was supported by NIH Grants R01-GM59870, R01-AR42997, and U54-GM064346 and by a the Harvard School of Dental Medicine NIDCR Training Grant.
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