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Biomimetic Interfacial Interpenetrating Polymer Networks Control Neural Stem Cell Behavior

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Biomimetic Interfacial Interpenetrating Polymer Networks Control Neural Stem Cell Behavior Biomimetic interfacial interpenetrating polymer networks control neural stem cell behavior Krishanu Saha,1 Elizabeth F. Irwin,2 Julia Kozhukh,1 David V. Schaffer,1,3 Kevin E. Healy2,4 1Department of Chemical Engineering, University of California at Berkeley, Berkeley, California 2Department of Bioengineering, University of California at Berkeley, Berkeley, California 3The Helen Wills Neuroscience Institute, University of California at Berkeley, Berkeley, California 4Department of Materials Science and Engineering, University of California at Berkeley, Berkeley, California Received 7 April 2006; revised 21 June 2006; accepted 31 July 2006 Published online 18 January 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30986 Abstract: Highly-regulated signals surrounding stem cells, fied with two cell-binding ligands, CGGNGEPRGDTYRAY such as growth factors at specific concentrations and matrix from bone sialoprotein [bsp-RGD(15)] and CSRARKQAA- mechanical stiffness, have been implicated in modulating SIKVAVSADR from laminin [lam-IKVAV(19)], were assayed stem cell proliferation and maturation. However, tight con- for their ability to regulate self-renewal and differentiation trol of proliferation and lineage commitment signals is in a dose-dependent manner. IPNs with >5.3 pmol/cm2 bsp- rarely achieved during growth outside the body, since the RGD(15) supported both self-renewal and differentiation, spectrum of biochemical and mechanical signals that govern whereas IPNs with lam-IKVAV(19) failed to support stem stem cell renewal and maturation are not fully understood. cell adhesion and did not influence differentiation. The Therefore, stem cell control can potentially be enhanced IPN platform is highly tunable to probe stem cell signal through the development of material platforms that more transduction mechanisms and to control stem cell behav- precisely orchestrate signal presentation to stem cells. Using ior in vitro. Ó 2007 Wiley Periodicals, Inc. J Biomed Mater a biomimetic interfacial interpenetrating polymer network Res 81A: 240–249, 2007 (IPN), we define a robust synthetic and highly-defined plat- a b form for the culture of adult neural stem cells. IPNs modi- Key words: hydrogel; RGD; laminin; IKVAV; v 3 integrin INTRODUCTION mal product impurities and genetic or epigenetic instability), or harsh microenvironments at the im- 2 For stem cells to function therapeutically in the plantation site. A rational strategy to address these body, stringent temporal and spatial requirements on problems is to tailor the cellular microenvironment self-renewal, differentiation, and migration must be during harvest, culture, and/or implantation to met.1 However, such control can potentially be com- enhance control over stem cell function. Employing a promised by heterogeneity in harvested cell popula- synthetic materials approach, we engineered a system tions, artifacts introduced by ex vivo culture (e.g., ani- to present well-characterized signals to stem cells. This tunable and modular microenvironment enables control over stem cell self-renewal and differentiation Correspondence to: D.V. Schaffer; e-mail: schaffer@cchem. signals in vitro, and can enable quantitative analysis of berkeley.edu or K.E. Healy; e-mail: [email protected] Contract grant sponsor: National Institute of Health; mechanisms that regulate these processes in vitro and contract grant number: AR43187-07 in vivo. Contract grant sponsor: Berkeley Futures Grant Considerable work on controlling the microenvir- Contract grant sponsor: NINDS grants; contract grant onment around stem cells has focused on defining number: NS048248 soluble media components for many stem cell lines3–5; Contract grant sponsor: National Science Foundation Graduate Fellowship (to KS) however, stem cells are most often propagated on sur- Contract grant sponsor: Whitaker Graduate Fellowship faces that contain animal-derived components with Award ambiguous signaling properties. Typical culture sur- Contract grant sponsor: Graduate Research and Education faces consist of poly(styrene) with either a passively in Adaptive bio-Technology (GREAT) Training Program, UC adsorbed animal-derived extracellular matrix (ECM) Systemwide Biotechnology Research and Education Program; contract grant number: 2005-280 protein or a ‘‘feeder’’ layer of mammalian cells. Ani- mal-derived ECM or protein molecules, in either two- ' 2007 Wiley Periodicals, Inc. or three-dimensional supports, likely contain variable SYNTHETIC IPNs FOR STEM CELL CONTROL 241 splice and glycoforms, offer limited micro- and nano- 2-(N-morpholino) ethanesulfonic acid, 0.9 % sodium chlo- scale control of solid-phase signaling, and otherwise ride buffer (MES; ‘‘BupH MES Buffered Saline Pack’’), and present problems for therapeutic application.6 In par- sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1- ticular, the ECM component laminin is a large, post- carboxylate (sulfo-SMCC) were acquired from Pierce (Rock- translationally modified protein (850 kDa) that is diffi- ford, IL). [3-(3,4-Dimethyl-9-oxo-9H-thioxanthen-2-yloxy)- 2-hydroxypropyl] trimethylammonium chloride (QTX) cult to produce via recombinant expression systems was obtained from Aldrich (Milwaukee, WI). Allyltrichloro- and thus is commonly purified from mammalian cell silane (ATC) was obtained from Gelest (Morrisville, PA). lines. In addition, human laminin, such as for human Diamino-poly(ethylene glycol) [PEG(NH2)2; 3400 g/mol, embryonic stem cell propagation, is particularly prob- chromatographically pure] was purchased from Nektar lematic due to significant source variability. Finally, (Huntsville, AL). All water used in this study was ultrapure the functions of different cell-binding motifs of lami- ASTM Type I reagent grade water (18.2 MO cm, pyrogen nin and other ECM components have only been par- free, endotoxin < 0.03 EU/mL). RGD and RGE peptides tially characterized in select cell lines and are largely were synthesized by American Peptide (Sunnyvale, CA) unknown for many stem cell lines. and characterized using mass spectrometry and high-per- Multipotent neural stem cells (NSCs) derived formance liquid chromatography (purity >95%). RGD and from the adult hippocampus are potentially attractive RGE peptides were based on the integrin-binding sequence 1,6–8 from rat bone sialoprotein: bsp-RGD(15) peptide, Ace- for neuroregeneration therapies. Natural ECM ÀÀ ÀÀ tyl CGGNGEPRGDTYRAY NH2; bsp-RGE(15) peptide, molecules such as laminin, collagen, and vitronectin ÀÀ ÀÀ ÀÀ Acetyl CGGNGEPRGETYRAY NH2; bsp-RGD(15) FI modulate NSC growth rates in vitro (data not shown) ÀÀ ÀÀ TC, Acetyl CGGNGEPRGDTYRAYK(FITC)GG NH2 [note and likely engage various integrin adhesion receptors. that bsp-RGD(15) peptide is the same as l-RGD, as described Integrin signaling has previously been implicated in previously].15,16 The lam-IKVAV(19) peptide was purchased the survival, proliferation, and differentiation mecha- from Sigma-Aldrich (St. Louis, MO) and was chosen from nisms of fetal and postnatal neural progenitors.9 the laminin A chain (amino acids 2091–2108, i.e., laminin In vitro, NSCs are often propagated in adherent culture peptide PA22-2): CSRARKQAASIKVAVSADR. Poly(styrene) on surfaces coated with laminin-1,10 which engages a 8-well strips (Costar #2580), 35-mm tissue culture poly(styrene) a b a b dishes, and paraformaldehyde (reagent grade) were pur- number of integrin receptors, including v 3, 1 1, a b , a b , a b , a b , and a b , and other unknown chased from Fisher Scientific (Santa Clara, CA). Dulbecco’s IIb 3 2 1 6 1 7 1 6 4 phosphate-buffered saline (DPBS, without calcium chloride, cellular receptors.11 Rather than using a ligand that without magnesium chloride, pH ¼ 7.2 6 0.1), Dulbecco’s engages multiple cellular receptors with numerous, modified eagle medium (F-12/DMEM, high glucose, with undetermined downstream signal transducers, this l-glutamine, with pyridoxine hydrochloride, without sodium initial attempt to control NSC behavior employed a pyruvate), N-2 supplement, 40,6-diamidino-2-phenylindole single well-characterized ECM motif common to lami- (DAPI), polyornithine, natural mouse laminin, and recombi- nin, vitronectin, and fibronectin: an Arg–Gly–Asp nant basic fibroblast growth factor-2 (bFGF) were purchased (RGD) adhesion motif. This motif was grafted onto from Invitrogen (Carlsbad, CA). Retinoic acid and forskolin a synthetic interpenetrating polymer network (IPN) were both obtained from BioMol (Plymouth Meeting, PA). For to create a highly mechanically and chemically defined immunofluorescent staining, primary antibodies of mouse NSC culturing environment. Additionally, we explored anti-nestin were purchased from Becton Dickinson (Franklin the function of a laminin motif, lam-IKVAV(19), since Lakes, New Jersey), mouse anti-microtubule associated protein 2ab (Map2ab) from Sigma-Aldrich (St. Louis, MO), and guinea both two- and three-dimensional presentation of vari- pig anti-glial fibrillary acidic protein (GFAP) from Advanced ous laminin domains such as IKVAV influence neurite 12,13 Immunochemical (Long Beach, CA). Alexa fluorochrome-con- extension of adult whole hippocampal explants jugated secondary antibodies, CyQUANT dye, and Syber- 14 and fetal neural progenitor differentiation. Such pre- green were purchased from Molecular Probes (Eugene, Ore- cise control of particular ECM motifs is not possible in gon). For characterization by quartz crystal microbalance traditional cell culture materials, and our results on with dissipation monitoring (QCM-D), quartz
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