Three-Dimensional Nanofibrillar Surfaces Promote Self-Renewal in Mouse Embryonic

江边孤钓

作者：Alam Nur-E-Kamala, Ijaz Ahmeda, Jabeen Kamala, Melvin Schindlerb, Sally Meinersa作者单位：a Department of Pharmacology, Robert Wood Johnson Medical School-University of Medicine and Dentistry, New Jersey, Piscataway, New Jersey, USA;b Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA
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          【摘要】" L3 A! B. y0 T% ?
      The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self-renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self-renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts. We have recently used a synthetic polyamide matrix (Ultra-Web) whose three-dimensional (3D) nanofibrillar organization resembles the ECM/basement membrane. Growth of mESCs on this nanofibrillar surface greatly enhanced proliferation and self-renewal in comparison with growth on tissue culture surfaces without nanofibers, despite the presence of LIF in both systems. Enhanced proliferation and self-renewal of the stem cells on nanofibrillar surfaces were correlated with the activation of the small GTPase Rac, the activation of phosphoinositide 3-kinase (PI3K) pathway, and the enhanced expression of Nanog, a homeoprotein required for maintenance of pluripotency. Inhibitors of PI3K reduced the expression level of Nanog in mESCs cultured on 3D nanofibrillar surfaces. These results provide support for the view that the three-dimensionality of the culture surface may function as a cue for the activation of Rac and PI3K signaling pathways, resulting in stem cell proliferation and self-renewal.   G( \5 d" z: S* L6 v# R7 w! m" v
          【关键词】 Mouse embryonic stem cells Three-dimensional nanofibrillar surfaces Nanofiber Rac Phosphoinositide -kinase Nanog Signaling Proliferation Differentiation
4 Y- z1 F9 c& F' E                  INTRODUCTION
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Embryonic stem cells, derived from the inner cell mass of the blastocyst, can be maintained in culture under conditions that retain their pluripotency .8 f  i0 a6 B" t7 t% @3 q% s
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Extensive efforts have been made to selectively regulate these pathways in vitro to amplify the number of pluripotent embryonic stem cells or to regulate their differentiation for applications in regenerative medicine .
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Recently, several investigations have provided evidence that components of the extracellular matrix (ECM) such as fibronectin and laminin-1 may substitute for feeder layers to promote the self-renewal pathway in stem cells .2 A- L+ U* d9 U# N% I1 ]

$ u) G. N* J% m6 PIt has been suggested that presentation of ECM/basement membrane proteins and their conformational flexibility within a three-dimensional (3D) matrix may be critical parameters for promoting in vivo-like functional and structural changes between and within cells , we hypothesized that Ultra-Web might also provide important topological cues for the growth and/or differentiation of embryonic stem cells.
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' v+ u) w0 j: R( ^! i9 uAlthough several reports , and the activation of the phosphoinositide 3-kinase (PI3K)/ AKT, components of the PI3K signaling pathway. Moreover, results from this work suggest that the three-dimensionality of the ECM/basement membrane may be as important as the chemistry in regulation of stem cell proliferation.
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MATERIALS AND METHODS+ w* r2 |% D2 W% n5 r& v  r/ B0 O, t
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Materials2 D7 c: k7 g* b( O
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mESCs (E14.5) were obtained from Dr. S. Jin (Department of Pharmacology, University of Medicine and Dentistry New Jersey-Robert Wood Johnson Medical School)  were obtained from Donaldson Co., Inc. (Minneapolis, www.synthetic-ecm.com).
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Mouse Embryonic Stem Cell Culture
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mESCs were cultured as described previously . Briefly, gelatin (1%) in phosphate-buffered saline (PBS) was poured onto 6-, 12-, or 24-well culture plates and incubated for 30 minutes at room temperature. Excess gelatin solution was removed by aspiration and rinsing with PBS. Mititocally inactivated MEFs (5 x 104/ml) (2 ml/well in a six-well plate) were seeded in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) containing 10% calf serum and incubated in 95% air and 5% CO2 at 37¡ãC overnight. mESCs were suspended at a density of 105/ml (2 ml/well in a 6-well plate) in DMEM containing fetal calf serum (5%), 2-mercaptoethanol (10 mM), nonessential amino acids (10 mM), nucleotides (2.5 mM), and LIF (10 ng/ml) (mESC cell medium) and plated onto the feeder layer. Cells were incubated under conditions described above with change of media every 48 hours until the stem cells were approximately 80% confluent. The stem cells were then trypsinized (0.05% trypsin) and suspended at a density of 105 cells/ml (0.5 ml/well) in the stem cell medium described above. Glass coverslips (12 mm) with and without Ultra-Web were placed into individual wells of a 12-well plate, sterilized under ultraviolet light for 15 minutes, and coated with gelatin. The trypsinized stem cells (with the contaminating MEFs) (0.5 ml) were then seeded on feeder layer-free coverslips with and without Ultra-Web previously placed in 12-well plates for subsequent experiments.
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Assay for Pluripotency of Mouse Embryonic Stem Cells
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Pluripotency of the mESCs was determined by an alkaline phosphatase assay. mESCs were cultured for 3 to 5 days in mESC medium to obtain spheroidal colonies. Cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 5 minutes. After washing, the cells were incubated in alkaline phosphatase staining mixture (a mixture of fast red violet solution with naphthol AS-BI phosphate solution and water in a 2:1:1 ratio) at room temperature for 15 minutes in the dark. Cells were again washed with PBS, and colonies were counted using a Zeiss Axioplan Epi-Fluorescent Microscope. Pluripotent colonies expressing alkaline phosphatase appeared red, whereas differentiated colonies were colorless.
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Proliferation Assay0 f, l7 @$ ]$ X/ ]! v+ ~
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mESCs were seeded onto coverslips (12 mm) with and without Ultra-Web as described above at a density of 105 cells/ml (0.5 ml/well). Cell growth was evaluated every 24 hours. Cell viability was determined by using a modification of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay . Alkaline phosphatase staining was performed as described above. The number of alkaline phosphatase-positive colonies was counted using a Zeiss Axioplan Epi-Fluorescent Microscope. Colony areas were determined for 50 colonies using the NIH Image J program.
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Rho/Rac/Cdc42 Pull-Down Assay
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Separate cultures of mESCs (with contaminating MEFs) and MEFs were trypsinized and suspended at a density of 5 x 105/ml and 5 x 104/ml, respectively, in mESC medium. The mESCs (106 cells) or MEFs (105 cells) were plated onto coverslips with and without Ultra-Web. Cells were incubated under standard cell culture conditions at 37¡ãC overnight. Activation of Rho and Rac/Cdc42 was determined using kits purchased from Cytoskeleton. Briefly, cells were washed with PBS and suspended in lysis buffer provided by the supplier. Aliquots (100 µg) were taken from each cell lysate to confirm equal loading of the amount of total Rho, Rac, and Cdc42 present in the lysate. GTP-bound forms of Rho, Rac, and Cdc42 were pulled down using instructions and reagents provided by the supplier. Proteins present in total cell lysates and Rho/Rac/Cdc42 pulled-down samples were separated by SDS-PAGE (12%) and transferred onto a nylon membrane. Western blotting was performed according to the ECL protocol provided by the suppliers using Rac, Rho, or Cdc42 antibodies. Rho, Rac, and Cdc42 bands were quantified using the Kodak Imaging Station 2000R.
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Transfection of Rac Mutants. _0 a3 F' t8 ^
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mESCs were seeded onto coverslips (in triplicate) with and without Ultra-Web at a density of 5 x 104 cells/ml (1 ml/well). Each coverslip was placed into an individual well of a 12-well cell culture plate as described above. After overnight incubation, mESCs were transfected with a plasmid vector (alone), Rac dominant-negative mutant (dnRacN17), or Rac constitutively active mutant (caRacQL). Two micrograms of each vector complexed with Cellfectin (Invitrogen) was used. Transfection was performed according to the manufacturer¡¯s instructions. Medium was replaced after 16 hours, and incubation was continued for another 72 hours. mESCs were then fixed with 4% paraformaldehyde and stained for alkaline phosphatase as described above. The number of alkaline phosphatase-positive colonies was counted in 25 random fields using a Zeiss Axioplan Epi-Fluorescent Microscope. The experiment was repeated three times.
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. P( i& V* `, f, M4 Y4 I' KInhibitor Experiments% i2 f0 E: N$ O* e" |, E8 y
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mESCs or MEFs were seeded onto coverslips with and without Ultra-Web at a density of 105 cells/ml (1 ml/well). Cells were incubated in stem cell medium at 37¡ãC for 24 hours. For inhibitor experiments, Wortmannin (0, 50, or 100 nM), caffeine (0, 0.25, 0.5, or 1 mM), or retinoic acid (0, 17, 35, or 70 nM) was added to the medium, and cells were incubated for 24 hours. Cells were lysed in Lammeli SDS sample buffer. Proteins were separated by SDS-PAGE (12%) and transferred onto a nylon membrane. Western blotting was performed according to the ECL protocol using specific antibodies.
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# y4 ?' d/ P! VInduction of Differentiation
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mESCs (105/ml) (1 ml/well) were seeded onto coverslips with and without Ultra-Web and incubated for 24 hours as described above. Cells were treated with dimethylsulfoxide, Wortmannin (100 nM), caffeine (1 mM), or retinoic acid (70 nM) for another 72 hours. Induction of differentiation-inducible gene expression was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Cells were trypsinized and collected by centrifugation at 5,000 rpm for 5 minutes. The cell pellet was lysed, and total RNAs were isolated using the TRIzol kit, followed by cleanup with the RNeasy Mini Kit according to the instructions provided by the suppliers. Expression of mRNA was determined using the Superscript III First-Strand Synthesis System with platinum Taq DNA polymerase according to the supplier¡¯s instructions. For semiquantitation, RT-PCR was programmed for 28 cycles of amplification. The following primers were used for RT-PCR: glial fibrillary acidic protein (GFAP) (forward: 5' tct ccc tgt ctc gaa tga ct 3'; reverse: 5' gcc ggg cac tgt tgg ccg ta 3'); nestin (forward: 5' atg ggg acg agg atc aag 3'; reverse: 5' gtg agc cac aga aga aag 3'); nanog (forward: 5' ctc aag gac agg ttt cag a 3'; reverse: 5' ggt gct gag ccc ttc tga a 3'); and actin (forward: 5' cgg ctt cgc ggg cga cga tg; reverse: 5' tct tgc tct ggg cct cgt c 3'). The PCR products were characterized by 1.5% agarose gel electrophoresis.
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3 Y8 N5 R! ~* k; d  f# IRESULTS2 D5 D3 h2 t, y% l. X9 G. V. `
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Colony Size, Cell Number, and Alkaline Phosphatase Expression for Mouse Embryonic Stem Cells Cultured on Ultra-Web 3D Nanofibrillar Surfaces
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We studied the pluripotency of mESCs cultured for 3 days on Ultra-Web 3D nanofibrillar surfaces by determining the expression of alkaline phosphatase, a marker for undifferentiated stem cells. As shown in Figure 1A, mESCs cultured on glass coverslips had small colonies of undifferentiated cells. In contrast, mESCs cultured on 3D nanofibrillar surfaces had significantly larger colonies of undifferentiated cells (Fig. 1B). Significantly, cells did not adhere to 2D films composed of polyamide (data not shown), demonstrating the importance of the nanofibrillar geometry for stem cell proliferation. Measurements of colony area for 50 colonies after 3 days of culture showed a mean projected colony area of 4,590 ¡À 730 µm2 on glass coverslips and 9,930 ¡À 1035 µm2 on 3D nanofibrillar surfaces. We determined cell proliferation every 24 hours for 3 days for mESCs cultured on glass coverslips with and without Ultra-Web coatings. It was found that proliferation of mESCs was stimulated when cultured on the 3D nanofibrillar surfaces (Fig. 1C). There was little stimulation in growth of mESCs that were cultured on coverslips in the absence of Ultra-Web (Fig. 1C).
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9 J, S9 N  P6 F3 j1 n! u- v& s& A# nFigure 1. Growth of mouse embryonic stem cells (mESCs) in the presence and absence of 3D nanofibrillar surfaces (Ultra-Web). (A, B): Alkaline phosphatase staining of mESC colonies after 3 days of culture in the absence (A) and presence (B) of the 3D nanofibrillar surface (Ultra-Web). Colonies were significantly larger for mESCs cultured on Ultra-Web than for mESCs cultured on plain coverslips. Scale bar = 10 µm. (C): Proliferation of mESCs was determined at 24-hour intervals for 3 days in the presence and absence of Ultra-Web, as described in Materials and Methods. The rate of proliferation was enhanced for cells cultured on Ultra-Web compared with cells cultured on glass. Data are shown as mean ¡À standard deviation (n = 3).
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It is important to note that these proliferation measurements were performed in the presence of less than 5% of the original feeder MEFs, which remained during passage. Since MEFs normally provide cues that promote stem cell proliferation, these results suggest that the 3D nanofibrillar surfaces can compensate, at least in part, for the absence of MEFs while standard tissue culture surfaces cannot perform the same synergistic or replacement function. The enhanced proliferation of mESCs on the 3D nanofibrillar surface suggests that the increase in colony size was due to an enhanced number of cells per colony rather than a change in the geometry of the cells.6 C: `& F, G4 Z1 Q5 m! p- Z

, X( [: B# o" D/ o! lPreferential Activation of the Small GTPase Rac in mESCs Cultured on Ultra-Web 3D Nanofibrillar Surfaces
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To examine the possibility that the changes in the proliferation and colony size of mESCs induced by the 3D nanofibrillar surface were mediated by alterations in the signaling pathways initiated by the activation of Rho, Rac, and/or Cdc42, the most well-characterized members of the Rho family of small GTPases, we performed GTPase activation pull-down assays. As shown in Figure 2Ai and quantitatively in Figure 2Aii, Rac activity was significantly higher in mESCs cultured on surfaces coated with Ultra-Web. In contrast, there was only a mild activation of Rho and Cdc42 in mESCs cultured on Ultra-Web (Figs. 2Ai, 2Aii). We attempted to determine the activation of Rac, Rho, and Cdc42 in MEFs alone (approximately equivalent to the number of MEFs transferred during serial passage). However, due to the small number of mitotically inactivated MEFs, the amounts of Rac, Rho, and Cdc42 were below the levels of detection (Figs. 2Ai, 2Aii). This suggests that the observed activation of Rac was a result of activation within the stem cells rather than activation within the feeder fibroblasts. However, it is also possible that the effect of the 3D nanofibrillar surfaces on the stem cells was indirect (i.e., mediated by changes in fibroblast phenotype and function), as we previously showed for NIH 3T3 fibroblasts cultured on Ultra-Web , as opposed to direct (i.e., mediated by the 3D nanofibrillar surface itself). Although these possibilities cannot be distinguished in the coculture system currently required for stem cell growth, the practical implication is that the 3D nanofibrillar surface/feeder fibroblast combination influences stem cell signaling in ways not observed for culture on standard tissue culture surfaces.
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# V6 ^! ^. C" \  b: XFigure 2. An essential role for Rac in mouse embryonic stem cells (mESCs) cultured on Ultra-Web. (A): Western blot analysis (Ai) and quantitation (Aii) of Rho, Rac, and Cdc42 activation were performed as described in Materials and Methods. The activation (fold) (Ai) is defined as the ratio between activation of GTPases on Ultra-Web versus uncoated surfaces. Shown in (Ai) as controls (C) are the equivalent number of mouse embryonic fibroblasts normally used as a feeder layer cultured on Ultra-Web. The cell number was too low to allow detection of Rac, Cdc42, and Rho. Rac was selectively activated in mESCs cultured on Ultra-Web. (B): The number of colonies for mESCs cultured on Ultra-Web and uncoated surfaces. The numbers of colonies are shown for mESCs transfected with plasmid vector (control), dominant-negative Rac (dnRac), and constitutively activated Rac (caRac) construct. A large enhancement in colony number was observed for mESCs transfected with constitutively activated Rac but not for cells transfected with dominant-negative Rac. Data in (Aii) and (B) are shown as mean ¡À standard deviation (n = 3).. t5 R! U4 ?+ j0 r' t
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To determine whether the increase in Rac activation was causally related to the enhanced rate of proliferation and self-renewal in mESCs, we transfected the mESCs with a dominant-negative Rac (dnRac) and a constitutively active Rac (caRac). Changes were then determined in colony number (Fig. 2B). As shown in Figure 2B, the number of colonies was enhanced for control cells grown in the presence of 3D nanofibrillar surfaces compared with mESCs grown on uncoated surfaces. In contrast, the number of colonies was reduced for the mESCs transfected with dnRac and slightly enhanced for mESCs transfected with caRac and cultured on 3D nanofibrillar surfaces. Both dnRac and caRac had little effect when expressed in mESCs cultured on surfaces not containing Ultra-Web (Fig. 2B). These results indicate an essential role for Rac on the proliferation of mESCs cultured on 3D nanofibrillar surfaces. Consistent with our studies, Rac1 has recently been shown to regulate survival of epidermal stem cells .
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# x8 A+ ~3 p7 O9 ^! ZRequirement of Phosphoinositide 3-Kinase Signaling Pathway in the Expression of Nanog in mESCs
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Several investigations have demonstrated a role for PI3K as a downstream effector in the LIF/gp130 pathway for self-renewal in mESCs . We found that retinoic acid inhibits expression of Nanog in mESCs cultured on Ultra-Web (Fig. 3Ciii). Using RT-PCR, it was found that PI3K inhibitors (Wortmannin and caffeine) as well as retinoic acid inhibited transcription of the Nanog gene (Fig. 3D). These data suggest a link between Rac activation, PI3K activity, and the expression level of Nanog in mESCs cultured on Ultra-Web., z1 T" h& }3 g) ]- h" P3 _& v

# \0 y& d) Z/ ^8 q; z8 J( fFigure 3. Nanog expression in mouse embryonic stem cells (mESCs) cultured on 3D nanofibrillar surfaces (Ultra-Web) involves signaling through phosphoinositide 3-kinase. Phosphorylation of AKT (A) and expression of Nanog (B) in mESCs cultured on Ultra-Web and uncoated surfaces. Enhanced levels of both were seen for mESCs cultured on Ultra-Web compared with mESCs cultured on uncoated coverslips. Shown as controls (mouse embryonic fibroblasts ) in (A) and (B) are the equivalent number of MEFs normally used as feeder layers cultured on Ultra-Web. (C): Inhibition of Nanog expression by Wortmannin (Ci), caffeine (Cii), and retinoic acid (Ciii). (D): Inhibition of Nanog transcription by dimethylsulfoxide (control), retinoic acid (70 nM), caffeine (1 mM), and Wortmannin (100 nM). Expression of Nanog mRNAs was determined by reverse transcription¨Cpolymerase chain reaction as described in Material and Methods. (E): Western blot analysis of the expression of c-Fos in feeder cells (MEFs) alone and mESCs (with contaminating MEFs) cultured with and without Ultra-Web. mESCs expressed more c-Fos when cultured on Ultra-Web than when cultured on uncoated surface. Expression levels of P-AKT (A), Nanog (B), and c-Fos (E) were normalized to ß-actin expression levels.
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7 M0 `5 x: R) @9 ?$ K# l: wAlthough Nanog expression is essential for maintaining the undifferentiated state of stem cells, it apparently is not involved in the proliferation of stem cells with self-renewal , we hypothesized that the level of c-Fos would be higher for cells grown on 3D nanofibrillar surfaces as opposed to standard tissue culture surfaces. As shown in Figure 3E, c-Fos expression was indeed enhanced in mESCs cultured in the presence of Ultra-Web (Fig. 3E), whereas this enhancement was not observed for feeder fibroblasts alone (Fig. 3E).
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Retinoic Acid Induced Differentiation of mESCs Cultured on Ultra-Web 3D Nanofibrillar Surfaces( h, }1 N( E7 ^& f
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The enhanced proliferation and self-renewal of mESCs cultured on 3D nanofibrillar surfaces raise the question of whether these cells retain their ability to differentiate when exposed to differentiating factors. Previous studies have shown that treatment of mESCs with retinoic acid caused induction of differentiation of embryonic stem cells into neuronal precursor cells as evidenced by upregulation of GFAP and nestin , we found that treatment with retinoic acid induced the expression of GFAP and nestin (Fig. 4) in mESCs cultured in the presence and absence of Ultra-Web, whereas GFAP and nestin were not detected in mESCs not treated with retinoic acid (Fig. 4). Hence, stem cells cultured on the 3D nanofibrillar surface maintain their ability to differentiate in the presence of differentiating factors.
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) Y6 D" |7 ?  j2 t) kFigure 4. Induction of glial fibrillary acidic protein (GFAP) and nestin expression by retinoic acid in mouse embryonic stem cells (mESCs) cultured on 3D nanofibrillar surfaces (Ultra-Web). Expression of GFAP and nestin in feeder cells alone (C) and mESCs (with contaminating mouse embryonic fibroblasts) cultured on Ultra-Web and uncoated surfaces after treatment with retinoic acid (70 nM). Reverse transcription-free (C-) acts as a negative control. Levels of GFAP and Nestin transcripts were normalized to ß-actin transcript levels. Detection of GFAP and nestin indicates that mESCs are capable of differentiation on Ultra-Web in response to outside cues./ g- L, X! j* M% L  J
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DISCUSSION! w7 N2 \  _7 v- i$ S* T. D

0 Z) e" ~% _5 c$ t# t) l5 R& J6 TRecent experiments using mesenchymal stem cells have demonstrated an important role for mechanical cues in regulating stem cell fate .9 f) N: B" }! i! k7 X8 N, [

# M+ w; |1 U/ V8 G) s$ l4 RWork with ECM components has demonstrated that the physical presentation of these molecules strikingly affects morphology, proliferation, and morphogenesis of differentiated cells . These observations were made in the absence of added ECM, suggesting that the geometry of the ECM can influence cellular phenotype and function even in the absence of chemistry.+ I. `( {& j2 d& I' x- m* ^3 Y# @

0 X! R( u4 a% {) s8 G! ]% S2 rBecause nanofibers influence cellular parameters such as cell shape, actin cytoskeleton, and fibronectin deposition , it is quite possible that they influence stem cells both directly and indirectly by altering the phenotype of the feeder cells. However, large differences in the fibronectin deposition were not observed in mitotically arrested MEFs used as a feeder layer (data not shown). Nonetheless, cytokine production or other soluble factors by feeder cells might be altered by culture on 3D nanofibrillar surfaces. We cannot currently completely eliminate the possibility of an indirect effect of 3D nanofibrillar surfaces on mESCs.; F; L7 M8 E7 J
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In work to examine the role of integrin activation on signaling pathways downstream of 3D surfaces, it was shown that ligation of ß1 integrin by type I collagen (in a 3D collagen gel) promoted the phosphorylation of FAK, the p85 subunit of PI3K and AKT, both components of the PI3K/AKT signal pathway . These observations are most relevant for the interpretation of our results with mESCs in that a 3D nanofibrillar surface induced activation of Rac (Fig. 2), the phosphorylation of AKT (Fig. 3), the expression of both Nanog and c-Fos (Fig. 3), and the requirement for PI3K activity to maintain stem cell pluripotency and proliferation (Fig. 3). Accordingly, we hypothesize that the resultant mechanical interactions between mESCs and the 3D nanofibrillar surface trigger signaling cascades that lead to the activation of Rac, PI3K, and downstream effector molecules.
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4 e7 g& n  t1 q% k# [( S( NConsistent with these reports, we demonstrated that inhibition of PI3K causes downregulation of Nanog (Figs. 3C, 3D). It is possible that the activation of PI3K has a more dominant role than Rac in maintaining stem cell pluripotency; inhibition of PI3K causes downregulation of Nanog in human ES cells .
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% w% x. M( I: t) J1 \% P" n3 l6 B/ XCONCLUSION
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7 ]" I  T7 u/ ]) P) z3 v0 XThe results reported in this study constitute the first demonstration that a 3D nanofibrillar surface composed of polyamide nanofibers (Ultra-Web) can promote the proliferation and self-renewal of mESCs through mechanisms involving Rac, PI3K/ AKT signaling, and upregulation of Nanog and c-Fos. The data provide evidence for a role for dimensionality in maintaining stemness in proliferating mESCs and suggest that nanofibers may provide an important new tool for promoting stem cell proliferation for applications in regenerative medicine.) S. B# J# ~: r0 }9 X8 _
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ACKNOWLEDGMENTS; z6 c  d5 k3 |# |. l* n

; k, O! t) e9 M. f5 M$ L+ UThis work was supported by National Institutes of Health grant R01 NS40394 and New Jersey Commission on Spinal Cord Research grant 04¨C3034 SCR-E-O to S.M. The authors are grateful to Dr. S. Jin (RWJMS-UMDNJ) for kindly providing the mouse embryonic stem cell line and Dr. L.-H. Wang (Mount Sinai School of Medicine, New York) for providing the plasmid containing Rac GTPase constructs.; j# C8 m/ ], k$ b5 _5 E

, j5 P+ A6 }# U1 [2 ?DISCLOSURES
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" G2 d- l  _2 Z* K/ bI.A., J.K., S.M., A.N., and M.S. have acted as consultants within the last 2 years for Donaldson Co.- F  d& p0 S0 P8 X8 F. g* F5 x
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( f/ |# x5 P1 V, C  UBurdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol 2002;12:432¨C438.8 p1 b8 ]: ~6 T; g- K9 s
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