Selective Differentiation of Neural Progenitor Cells by High–Epitope

R EPORTS sequence glutamic acid–glutamine–serine (EQS) (36). As discussed below, these mole- Selective Differentiation of cules form physically similar scaffolds by self- assembly, but cells encapsulated within the Neural Progenitor Cells by EQS gels did not sprout neurites or differentiate morphologically or histologically. High–Epitope Density Nanofibers The chemical structure of the IKVAV- containing peptide amphiphile (IKVAV- Gabriel A. Silva,1*† Catherine Czeisler,2* Krista L. Niece,3 PA) and a molecular graphics illustration of Elia Beniash,3 Daniel A. Harrington,3 John A. Kessler,2 its self-assembly are shown in Fig. 1A, and Samuel I. Stupp1,3,4‡ a scanning electron micrograph of the scaffold it forms is shown in Fig. 1B. In addi- Neural progenitor cells were encapsulated in vitro within a three-dimensional tion to the neurite-sprouting epitope, the network of nanofibers formed by self-assembly of peptide amphiphile mole- molecules contain a Glu residue that gives cules. The self-assembly is triggered by mixing cell suspensions in media with them a net negative charge at pH 7.4 so that dilute aqueous solutions of the molecules, and cells survive the growth of the cations in the cell culture medium can nanofibers around them. These nanofibers were designed to present to cells the screen electrostatic repulsion among them neurite-promoting laminin epitope IKVAV at nearly van der Waals density. and promote self-assembly when cell sus- Relative to laminin or soluble peptide, the artificial nanofiber scaffold induced pensions are added. The rest of the se- very rapid differentiation of cells into neurons, while discouraging the devel- quence consists of four Ala and three Gly opment of astrocytes. This rapid selective differentiation is linked to the am- residues (A4G3), followed by an alkyl tail plification of bioactive epitope presentation to cells by the nanofibers. of 16 carbons. The A4G3 and alkyl segments create an increasingly hydrophobic Artificial three-dimensional (3D) scaffolds formed by these systems could be delivered sequence away from the epitope. Thus, that store or attract cells, and then direct cell to living tissues by simply injecting a liquid once electrostatic repulsions are screened proliferation and differentiation, are of criti- (i.e., peptide amphiphile solutions that self- by electrolytes, the molecules are driven to cal importance in regenerative medicine. Ear- assemble in vivo). We show that an artificial assemble by hydrogen bond formation and lier work demonstrated that tissue regenera- scaffold can direct the differentiation of neu- by the unfavorable contact among hydro- tion using cell-seeded artificial scaffolds is ral progenitor cells largely into neurons while phobic segments and water molecules. possible, either by implanting the scaffolds in suppressing astrocyte differentiation. The nanofibers that self-assemble in aque- vivo or maintaining them in a bioreactor fol- We used murine neural progenitor cells ous media place the bioactive epitopes on their lowed by transplantation (1–9). The scaffold (NPCs) to study in vitro the use of a self- surfaces at van der Waals packing distances materials used in most previous work have assembling artificial scaffold to direct cell dif- (37, 38). These nanofibers bundle to form 3D been biodegradable, nonbioactive polymers ferentiation (22). The choice of cell was moti- networks and produce a gel-like solid (Fig. 1, C such as poly(L-lactic acid) and poly(glycolic vated by the potential advantages of using to E). The nanofibers have high aspect ratio and acid) (10, 11), as well as biopolymers such as NPCs to replace lost central nervous system high surface areas, 5 to 8 nm in diameter and collagen, fibrin, and alginate (12–18). The cells after degenerative or traumatic insults (23– with lengths of hundreds of nanometers to a polymer scaffolds are typically prefabricated 26). The molecular design of the scaffold in- few micrometers. Thus, the nanofibers that porous objects, fabrics, or films that are seed- corporated the pentapeptide epitope isolucine- form around cells in 3D present the epitopes at ed with cells of the tissue to be regenerated. lysine-valine-alanine-valine (IKVAV), which is an artificially high density relative to a natural In the case of biopolymers, a common form found in laminin and is known to promote extracellular matrix. Although we do not expect of the scaffold is an amorphous gel in which neurite sprouting and to direct neurite growth all of the epitopes to be available for receptor cells can be encapsulated (19–21). (27–35). As a control for bioactivity we synthe- binding, we expect the molecularly designed We report here on solid scaffolds that sized a similar molecule lacking the natural scaffold to be a good vehicle for intense signal incorporate peptide sequences known to di- epitope, replacing it with the nonphysiological presentation to cells in 3D. rect cell differentiation and to form by self- assembly from aqueous solutions of peptide amphiphiles. The scaffolds consist of nano- Fig. 1. (A) Molecular fiber networks formed by the aggregation of graphics illustration the amphiphilic molecules, and this process is of an IKVAV-containing peptide amphi- triggered by the addition of cell suspensions phile molecule and its to the aqueous solutions. The nanofibers can self-assembly into nano- be customized through the peptide sequence fibers. (B) Scanning elec- for a specific cell response, and the scaffolds tron micrograph of an IKVAV nanofiber network formed by adding 1Institute for Bioengineering and Nanoscience in Ad- cell media (DMEM) to vanced Medicine, 2Department of Neurology, 3De- a peptide amphiphile partment of Materials Science and Engineering, 4De- aqueous solution. The partment of Chemistry, Northwestern University, sample in the image was Chicago, IL 60611, USA. obtained by network de- *These authors contributed equally to this work. hydration and critical- †Present address: Jacobs Retina Center, University of point drying of samples California, San Diego, La Jolla, CA 92093–0946, USA. caged in a metal grid to prevent network collapse (samples were sputtered with gold-palladium films and E-mail: [email protected] imaged at 10 kV ). (C and D) Micrographs of the gel formed by adding to IKVAV peptide amphiphile solutions ‡To whom correspondence should be addressed. E- (C) cell culture media and (D) cerebral spinal fluid. (E) Micrograph of an IKVAV nanofiber gel surgically mail: [email protected] extracted from an enucleated rat eye after intraocular injection of the peptide amphiphile solution. 1352 27 FEBRUARY 2004VOL 303 SCIENCE www.sciencemag.org R EPORTS When 1 weight % (wt %) peptide amphi- Fig. 2. Cell survival and morphol- phile aqueous solution was mixed in a 1:1 ogy of NPCs encapsulated in volume ratio with suspensions of NPCs in me- IKVAV-PA gels or cultured on dia or physiological fluids, we obtained within poly-(D-lysine) (PDL)-coated cover slips. Cell survival of encapsulated seconds the transparent gel-like solid shown in NPCs was determined by a fluores- Fig. 1, C and D (39). This solid contained cent viability/cytotoxicity assay. encapsulated dissociated NPCs or clusters of Live cells fluoresce green due to the cells known as neurospheres (22). The cells the uptake and fluorescence of survived the self-assembly process and re- calcein in response to intracellular mained viable during the time of observation esterase activity; dead cells fluoresce red as a result of the entry of (22 days) (Fig. 2, A to D) (40). There was no ethidium homodimer-1 through significant difference in viability between cells damaged cell membranes and cultured on poly(D-lysine) (PDL, a standard subsequent binding to nucleic ac- substrate used to culture many cell types) rela- ids. Cell survival was determined tive to cells encapsulated in the nanofiber net- at (A) 1 day, (B), 7 days, and (C) work (Fig. 2D). These results suggest that dif- 22 days in vitro. (D) Quantifica- tion of cell survival expressed as a fusion of nutrients, bioactive factors, and oxy- percentage of total cells. There gen through these highly hydrated networks is was no difference in survival rates sufficient for survival of large numbers of cells between experimental IKVAV-PA for extended periods of time. The artificial scaf- gels and PDL controls at any of the folds formed by the self-assembling molecules time points indicated. (E) Cell contain 99.5 wt % water, and it is the high body areas of differentiated neurons in the IKVAV-PA gels were aspect ratio of the nanofibers that allows a significantly larger than those of mechanically supportive matrix to form at such controls at both 1 and 7 days low concentrations of the peptide amphiphiles. (*P Ͻ 0.05, **P Ͻ 0.01). (F) TEM Thus, the artificial extracellular matrix not only of NPC encapsulated in an IKVAV- provides mechanical support for cells but also PA gel at 7 days. The cell has a normal ultrastructural morphology (N, nucleus; arrow, mitochondria). In serves as a medium through which diffusion of addition, numerous processes can be seen in cross section (red asterisks) within the gel, surrounded by PA nanofibers (NF). soluble factors and migration of cells can occur. In the bioactive scaffolds, cell body areas and neurite lengths of NPCs that had differentiated into neurons as determined by immuno- cytochemistry (see below) showed statistically significant differences with respect to cells cultured on PDL- or laminin-coated substrates. Neurons within the nanofiber networks were noticeably larger than neurons in control cul- tures. The average cell body area of encapsulated progenitor cells in the networks was significantly greater after 1 and 7 days (Fig. 2E). Encapsulation in the nanofiber scaffold led to the formation of large neurites after only 1 day (about 57 Ϯ 26 ␮m, mean Ϯ SD), whereas cells cultured on PDL and laminin had not developed neurites at this early time.

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