Three-Dimensional Nanostructured Architectures Enable Efficient

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Three-Dimensional Nanostructured Architectures Enable Efficient Neural Differentiation of Mesenchymal Stem Cells via Mechanotransduction Mahla Poudineh, Zongjie Wang, Mahmoud Labib, Moloud Ahmadi, Libing Zhang, Jagotamoy Das, Sharif U. Ahmed, Stéphane Angers, and Shana O. Kelley Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03313 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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Three-Dimensional Nanostructured Architectures Enable Efficient Neural Differentiation of Mesenchymal Stem Cells via Mechanotransduction Mahla Poudineh1‡, Zongjie Wang1‡, Mahmoud Labib1, Moloud Ahmadi1, Libing Zhang1, Jagotamoy Das1, Sharif Ahmed1, Stephane Angers1, and Shana O. Kelley1,2,3 1Department

of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, M5S 3M2, Canada. 2Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, M5S 3M2, Canada. 3Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, M5S 1A8, Canada KEYWORDS: Stem cell differentiation, Nanostructured architectures, Neural differentiation, Cell morphology.

ABSTRACT Cell morphology and geometry affect cellular processes such as stem cell differentiation, suggesting that these parameters serve as fundamental regulators of biological processes within the cell. Hierarchical architectures featuring micro and nanotopographical features therefore offer programmable systems for of stem cell differentiation. However, a limited number of studies have explored the effects of hierarchical architectures, due to the complexity of fabricating structures that with rationally tunable micro- and nanostructuring process. Here, we report threedimensional (3D) nanostructured microarchitectures that efficiently regulate the fate of human mesenchymal stem cells (hMSCs). These nanostructured architectures strongly promote cell alignment and lead to efficient neurogenic differentiation where over 85% of hMSCs express microtubule-associated protein 2 (MAP2), a mature neural marker, after seven days of culture on the nanostructured surface. Remarkably, we found that the surface morphology of nanostructured surface is a key factor that promotes neurogenesis and that highly spiky structures promote more efficient neuronal differentiation. Immunostaining and gene expression profiling revealed significant upregulation of neuronal 1

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markers compared to unpatterned surfaces. These findings suggest that the 3D nanostructured microarchitectures can play a critical role in defining stem cell behavior.

INTRODUCTION Stem cells are undifferentiated cells with self-renewal capacity and ability to differentiate into multiple cell types.1 Because of their multipotency, stem cells hold tremendous promise for the field of regenerative medicine,2 as the tissue regeneration process essentially relies on the differentiation of stem cells into specific cell types.1,3 Hence, precise control over the differentiation process is critical for the development of therapeutic approaches based on stem-cell based treatments. Both in vivo and in vitro, biophysical cues play vital roles in regulating cellular behavior.4–6 Previous studies have shown that stem cell differentiation is closely associated with the stiffness,7,8 elasticity,9,10 and topography11,12 of the extracellular matrix (ECM). Therefore, attaining a detailed understanding of the effects of biophysical parameters on cellular differentiation would help to precisely define the fate of stem cells.13 In living organisms, the ECM is comprised of micro and nanoscaled topographical features that interact with cells and regulate their behavior and fate.4,14 This relationship provides a rationale for developing artificial architectures to induce cellular differentiation through the interaction between stem cells and micro- and nanostructured surfaces to ultimately evoke cell-fate specification. Microscale cues 2


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induce morphological changes, direct the assembly of cytoskeletal proteins, alter the shape of nucleus, and modulate the expression of genes.15 Nanoscale cues can promote the clustering of integrin, a transmembrane receptor protein which triggers a series of dynamic signaling events termed as mechanotransduction.16,17 Integrin clustering activates the integrin-mediated intracellular signaling pathway and in turn regulates stem cell differentiation. Two-dimensional (2D), isotropic geometric templates direct the adipogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs).11 2D micro templates enabling high cytoskeletal tension increase actomyosin contractility, which upregulates osteogenesis but downregulates adipogenesis.11,18 Furthermore, 2D anisotropic geometries, appear to promote differentiation and maturation of stem or progenitor cells into neuronal,19 myogenic20 and myocardial21,22 lineages. Indeed, the differences in feature size appear to be sensed by hMSCs. For instance, compared to 2D microgrooves, 2D nanogrooves more efficiently induce neurogenesis,19,23 even in the absence of soluble differentiation factors.12,24 This is achieved via a focal adhesion kinase (FAK)-mediated signaling pathway which together with actomyosin cytoskeleton contractility induce differential gene expression.24 Nanoscale cues play an important roles in neurogenic differentiation as they reduce

unwanted

astrocytic

surface

coverage25

and

enable

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communication between neurons.26 However, there are limited studies utilizing hierarchical structures with controllable nano- and microstructuring for directing stem cell fate. Indeed, existing work utilizes labor-intensive and time-consuming fabrication processes to create nanostructured architectures (i.e. multiple photolithography27 and dry etching28). In addition, these methods do not finely tune nanoscale spatial cues. In order to precisely direct stem cell differentiation 3


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towards neurogenic lineages, it is essential to introduce a well-controlled manner for fabricating hierarchical architectures with accurately defined nanoscale features. Metal electrodeposition is an ideal approach to generate nanostructured architectures on many different length scales. Our previous studies have demonstrated that metal electrodeposition is a highly tunable process29,30 that leads to the formation of nanostructured architectures with impressive performance in pushing limits of detection for biomolecules into the attomolar to femtomolar range.31,32 The use of these three-dimensional sharp-tipped structures in electrical lysis also facilitates electric field focusing, which is necessary for efficient cell wall rupture and biomarker release.33 Herein, we used these nanostructured architectures as a means to modulate the differentiation of hMSCs. We hypothesized that the enrichment of nanofeatures would facilitate interactions of hMSCs with nanostructured architectures and alter the cell morphology, leading to highly efficient neural differentiation of stem cells.

RESULTS AND DISCUSSION Our approach to exploring the use of nanostructured microarchitectures for regulating stem cell differentiation is shown in Fig. 1A. We generate threedimensional structures using micropatterned linear apertures fabricated using photolithography on the surface of a glass chip. A linear aperture of Au functions as a site for gold electrodeposition and growth of nanostructured architectures. An array of these nanostructures serves as a potential platform for hMSC culture. The large surface areas generated by these structures have been previously employed in electrochemical sensors for detection of biomolecules with ultra-sensitivity.29-33 Here, the nanofeatures of these structures are leveraged as a means to control stem cell differentiation. A bottom-up fabrication strategy is utilized to form an 4


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arrayed architecture, defining a region for elongation of stem cells and induction of neurogenesis (Fig. 1B). Cultured cells were patterned along the linear structures and exhibited an elongated morphology. The morphology of cells was visualized using scanning electron microscopy (SEM). The nanostructured architectures induce efficient neural differentiation in the patterned cells. The presence of neural markers was observed as early as three days after culture. We have shown that the electrodeposition of these structures is highly tunable.30 Different parameters can be optimized to define a desired pattern. For example, by changing the gold ion concentration and deposition voltage, it is possible to preferentially control growth and generate structures with low to high levels of spikiness (Fig. 2). Here, we tested multiple structures with varying levels of spikiness and investigated the effect of nanostructuring on the morphology and fate of cultured stem cells. Application of a 500 mV potential in a solution containing 10 mM of gold ions results in the formation of fairly smooth structures and the overall morphology features low spikiness structure (Fig. 2i). Increasing the overpotential (V=0 mV) and increasing the level of gold ions in the solution (Au=50 mM) leads to an increase in the spikiness of structures and the formation of three dimensional leaves with a smooth surface: intermediate spiky structures (Fig. 2ii). To further promote the formation of the nanostructures and generate finer spiky structures, a second growth process was performed at high overpotential (V=−200 mV), leading to highly spiky structures (Fig. 2iii).34 Electrodeposition conditions for different structures are described in detail in methods section. We tracked the effects of these differently structured surfaces on hMSCs. Nanostructured architectures with different levels of spikiness were prepared and used as a platform for hMSC culture. After 7 days of culture, cells were fixed and their morphology was observed using SEM (Fig. 3.) The presence of microscale 5


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gold apertures guided cell orientation. Regardless of the spikiness level/level of nanostructuring, the hMSCs showed greater elongation along the direction of linear apertures in comparison with flat surface (Fig. 3i). Indeed, various micropatterned linear substrates have been extensively reported to upregulate cell orientation and alter the size of nucleus.35,36 As shown in Fig. 3., increasing the spikiness level subsequently enhances the elongation and orientation of cultured cells. For highly spiky structures, cells were uniformly aligned along the axis of nanostructured architectures. In addition, nanoscale structures promoted the formation of filopodia-like structures in patterned cells. Filopodia are finger-like protrusive structures containing actin bundles and play an essential role in the growth of neurons.37 Previous studies have reported that filopodia are mechanistically related to microspikes.38 As observed in SEM images, highly spiked structures resulted in increased number of filopodia-like junctions formed in the vicinity of the cell-nanostructure interface. The nanoscale structures may work as biomimetic spikes to promote filopodium formation, resulting an efficient neural differentiation process. We characterized the neurogenesis of hMSCs on electrodeposited architectures via immunostaining. The cell morphology, early and mature neurogenesis were examined by analyzing F-actin, neuron-specific class III β-tubulin, Tuj-1, and MAP2 and SYN1 staining, respectively. Representative fluorescence images after 3 and 7 days of culturing cells on nanostructured surfaces are shown in Fig. S2, Fig. 4A and Fig. S8, respectively. The fluorescence intensity of the F-actin filaments within microscale gold apertures was elevated compared to the flat surface, indicating that the presence of microscale gold apertures could improve the orientation and alignment of hMSCs. During the initial time points where cultures were monitored, the hydrophobicity of SU8 also appears to contribute to the cell orientation along the gold apertures (Fig. S5). Furthermore, the hMSCs were moderately attached on 6


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low-spikiness nanostructures and fully attached on intermediate and highly-spiky surfaces. This corroborates with the morphology of cells monitored using SEM (Fig. 3). We did not observe any significant fluorescence signals from Tuj-1 and MAP2 in control samples at day 7. Although the microscale gold apertures significantly alter cell morphology, they appear to promote neurogenesis to a lesser extent. This finding agrees with a previous study12 where a 10 μm micropatterned PDMS did not exert strong neurogenesis on cultured hMSCs. In comparison, enrichment of nanoscale gold structures dramatically boosted the neurogenic differentiation. The majority of the hMSCs cultured on intermediate- and highly-spiky structures expressed Tuj-1 at day 3 and MAP2 at day 7. We further quantified the percentage of differentiated cells at day 7 using co-localization of DAPI-positive and MAP2positive cells. Importantly, in absence of neurogenic chemical cues, 60% and 85% of hMSCs on high-spikiness nanostructures expressed MAP2 at day 3 and day 7, respectively (Fig. 4B). We also monitored the presence of different non-neural markers (endodermal/ectodermal (non-neural)/mesodermal markers) using immunostaining (Fig. S6). Compared to the control sample, no noticeable nonneural marker signals were observed, indicating that the cells did not undergo significant non-neural differentiation. In terms of neurogenic efficiency, our electrodeposited architectures were superior to the previously reported 2D nanogrooves12,24,28 which induce a measurable MAP2 expression after 7 to 14 days of culture. The efficient neural differentiation may be explained by the high density of nanostructures that promotes the formation of filopodia. The filopodium, as part of the actin bundle, can possibly transduce the mechanical stimulus of nanostructures to the cell nucleus via mechanotransduction and activate neurodifferentiation pathways. It is noteworthy that spontaneous electrical activity was not observed in the cells grown on nanostructured substrates (Figure S7). However, 7


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upregulation of SYN1 was detected (Figure S8), indicating the progression of these cells towards a neural phenotype. We also examined the change in MAP2 expression using the quantitative polymerase chain reaction (qPCR). MAP2 expression was upregulated 35-fold higher than the control after 7 days of culture on nanostructures. In addition, we also noticed that hMSCs underwent a lower percentage of proliferation at day 3 on nanostructures (data not shown). In summary, we observed a significant contribution of nanoscale gold structures to induce neural differentiation in hMSCs. To gain more insights on possible signaling proteins involved in the mechanotransduction, we examined the expression of focal adhesion kinase (FAK) and c-Jun N-terminal kinase (JNK) both of which have been previously reported to transduce mechanical signals to neuronal differentiation.23 The phosphorylation of FAK and the expression of JNK were enhanced by the degree of nanostructuring (Fig. 5). FAK localizes to sites of transmembrane integrin receptors and facilitates intracellular signaling.39 FAK also works as a switchboard interacting with small GTPase proteins such as RhoA, Rac, and Cdc42.40 FAK phosphorylation turns on these downstream signaling pathways. For instance, Cdc42 further activates the Wiskott-Aldrich-syndrome protein in conjunction with the actin branching complex ARP2/3, which leads to filopodia formation.41 JNK is downstream of FAK and RhoA.40,42 JNK mediates the actin and microtubule dynamics during neuronal migration. We also observed significant JNK upregulation in the presence of nanostructures (Fig. 5), which may be related to elevated MAP2 expression. Taken together, the hMSC interactions with nanostructures enhance FAK phosphorylation, which subsequently promotes the filopodia formation and JNK pathway activation. Moreover, JNK further alters actin and microtubule dynamics to guide the cell orientation and stimulate MAP2 expression.43 8


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CONCLUSIONS In summary, we report a direct link between surface nano/microstructuring and neural differentiation. Nanostructured architectures with different levels of spikiness were utilized as a means to regulate the morphology and fate of human mesenchymal stem cells in the absence of any exogenous factors promoting differentiation. SEM images confirm the role of nanostructured surface on directing the alignment of cultured cells. We showed that highly-spiky structures strongly promote cell alignment and lead to efficient neural differentiation that was monitored via immunostaining and gene expression analysis. This study strongly suggests that the nanoscale mechanical environment can be a defining factor to regulate stem cell differentiation and can be further employed in the design of stem cell-based therapies, drug screening and disease modeling.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed description of the experimental methods and additional data

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Mahla Poudineh: 0000-0001-8684-3102 Zongjie Wang: 0000-0001-9900-7197 9


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Shana O. Kelley: 0000-0003-3360-5359 Present Addresses M. P., Department of Radiology, Stanford University, Stanford, California 94305, USA Author Contributions ‡ M. P. and Z. W. contributed equally to this work. M. P., Z. W., M. L., M. A. and S.O.K conceived and designed the experiments. M. P., Z. W., M. L. and M. A. conducted experiments. M. P., Z. W., M. L. and M. A. analyzed the data. All authors discussed the results and contributed to the preparation and editing of the manuscript.

ACKNOWLEDGMENT Research reported in this publication was supported in part by the Canadian Institutes of Health Research (grant no. FDN-148415), the Natural Sciences and Engineering Research Council of Canada (grant no. RGPIN-2016-06090). This research is part of the University of Toronto’s Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund. Z.W. acknowledges financial support from University of Toronto Connaught Fund.

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(39) Sieg, D. J.; Hauck, C. R.; Schlaepfer, D. D. Required Role of Focal Adhesion Kinase (FAK) for Integrin-Stimulated Cell Migration. J. Cell Sci. 1999, 112 ( Pt 1, 2677–2691. (40) Tomakidi, P.; Schulz, S.; Proksch, S.; Weber, W.; Steinberg, T. Focal Adhesion Kinase (FAK) Perspectives in Mechanobiology: Implications for Cell Behaviour. Cell Tissue Res. 2014, 357 (3), 515–526. (41) Navarro-Garcia, F.; Serapio-Palacios, A.; Ugalde-Silva, P.; Tapia-Pastrana, G.; Chavez-Dueñas, L. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia Coli Pathotypes. Biomed Res. Int. 2013, 2013. (42) Huang, C. MAP Kinases and Cell Migration. J. Cell Sci. 2004, 117 (20), 4619– 4628. (43) Chang, L.; Jones, Y.; Ellisman, M. H.; Goldstein, L. S. B.; Karin, M. JNK1 Is Required for Maintenance of Neuronal Microtubules and Controls Phosphorylation of Microtubule-Associated Proteins. Dev. Cell 2003, 4 (4), 521–533.

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Figure 1. Nanostructured microarchitectures for stem cell diferentiation. A) Fabrication process for nanostructured architectures. A gold coated substrate is passivated with SU8, and 5 μm line apertures are etched at each electrode. Nanostructured architectures are electroplated within each aperture with featuring different morphologies. (i). Human mesenchymal stem cells (hMSCs) seeded on top of the structures are observed to align and undergo neurogenesis after 7 days’ in culture (ii). B) hMSCs were elongated and aligned along the direction of the nanostructures. Cell morphology was visualized by i) immunostaining and ii) SEM imaging.


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1 2 3 4 5 6 7 8 i) Low 9 10 11 12 13 14 15 16 5 µm 20 µm 1 µm 17 18 19 ii) Intermediate 20 21 22 23 24 25 26 27 28 29 30 iii) High 31 32 33 34 35 36 37 38 39 40 41 42 43Figure 2. SEM images of the nanostructured architectures with different levels 44of spikiness. i) Architectures with low levels of spikiness exhibit small granules on 45 46their surfaces. ii) Intermediate spiky structures display 3D leaf structures with smooth 47surfaces. iii) Highly spiky structures feature 3D leaf structures at the microscale with 48nanostructured needles. 49 50 51 52 53 54 55 56 57 58 59 ACS Paragon Plus Environment 60

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1 2 3 4 5 6 i) Control 7 8 9 10 11 12 13 5 µm 2 µm 14 10 µm 20 µm 15 16 ii) Low 17 18 19 20 21 22 23 24 25 26 iii) Intermediate 27 28 29 30 31 32 33 34 35 36 iv) High 37 38 39 40 41 42 43 44 45 46 47 48Figure 3. SEM images of hMSCs cultured on nanostructured surface. Significant differences in cell morphology 49were observed for different architectures. For surface without nanostructures, hMSCs were randomly attached to 50the surface (i). For structures with a low level of spikiness (ii), hMSCs were partially attached and aligned. For 51intermediate (iii) and highly spiky (iv) structures, hMSCs were fully attached and well-aligned. Green color represent 52 cells seeded on nanostructure devices. 53 54 55 56 57 58 59 ACS Paragon Plus Environment 60

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1 2 3 4 5 6 Tuj-1 (early MAP2 (mature DAPI Actin 7 (nucleus) (morphology) neurogenesis) neurogenesis) 8 9 10 Control 11 12 13 14 15 16 17 Low 18 19 20 21 22 23 24 Intermediate 25 26 27 28 29 30 High 31 32 33 34 35 36 100 60 37 ■ Low ■ Intermediate 38 80 ■ High 39 40 40 60 41 42 40 43 20 44 20 45 46 0 0 Positive Low Intermediate High 47 Day 3 Day 7 control 48 49 Figure 4. Nanostructured surfaces enables neurogenesis of hMSCs. A) Representative fluorescence images of 50 hMSCs after seven days of culture. Cells were cultured in proliferation medium and immunostained for DAPI/Actin/Tuj51 1/MAP2. All images were taken with a 20X objective lens. (Scale bar = 100 μm). B) Quantification of MAP2 positive cells 52 (mature neural cells) on nanostructured surface, n = 5, *p < 0.05, ***p < 0.001, C) Quantification of MAP2 gene 53 expression of hMSCs cultured on the nanostructured surface at day 7 (n = 3, ***p < 0.001). For positive control, SH-SY5 54 cells were treated with 1 ! M retinoic acid which induces their neuronal differentiation. 55 56 57 58 59 ACS Paragon Plus Environment 60

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1 2 3 4 5 6 JNK (Cell A Actin pFAK (focal DAPI 7 migration & (morphology) adhesion) 8 (nucleus) Neural growth) 9 10 11 Control 12 13 14 15 16 17 18 Low 19 20 21 22 23 24 Intermediate 25 26 27 28 29 30 31 High 32 33 34 35 36 B 37 38 39 40 41 42 43 44 45 46 47 48 49Figure 5. Nanostructured architectures enhance the neurogenesis of hMSCs via 50mechanotransduction. (A) Representative fluorescence images of hMSCs at day 7. Cells were 51cultured in proliferation medium and immunostained for DAPI/Actin/pFAK/JNK. All images were taken 52 with a 50X objective lens. (Scale bar = 50 μm). (B) Signaling pathway for neurogenesis mediated by 53 54nanostructured surface via FAK/JNK. 55 56 57 58 59 ACS Paragon Plus Environment 60

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Three-Dimensional Nanostructured Architectures Enable Efficient

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