Time-Dependent Retention of Nanotopographical Cues in

Letter Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/abseba

Time-Dependent Retention of Nanotopographical Cues in Differentiated Neural Stem Cells Seungwon S. Yang,† Junghwa Cha,†,‡ Seung-Woo Cho,§ and Pilnam Kim*,†,‡ †

Department of Bio and Brain Engineering, KAIST, Daejeon 34141, Korea KAIST Institute for Health Science and Technology, Daejeon 34141, Korea § Department of Biotechnology, Yonsei University, Seoul 03722, Korea ‡

ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/07/19. For personal use only.

S Supporting Information *

ABSTRACT: Exposure time to mechanical cues is important to properly modulating stem cell fate. The phenomenon in which the cells retain information from past stimuli, the so-called “time-retention effect”, has become one of the major factors to modulate stem cell differentiation with different mechanical cues. Using a stress-responsive and tunable nanowrinkle topography, we investigated the effects of timedependent retention of a nanotopographical cue on differentiating the neural stem cells (NSCs). After removing nanotopography used to induce hNSCs neuronal differentiation, we observed that differentiated NSCs exposed to the nanotopography for longer times retained their neural features compared to NSCs exposed shorter. We concluded that the NSCs could retain the nanotopographical stimuli depending on the dosing time during differentiation, suggesting the impact of the timeretention effect in controlling stem cell fate. KEYWORDS: neural stem cells, differentiation, nanotopography, mechanical dosing, time-retention effect, mechanical memory

S

using substrates with anisotropic topography to guide the differentiation of NSCs.12,13 There have been many studies on the modulation of NSC neuronal differentiation using nanotopography of cell culture substrates.14,15 In Yang et al., they used nanogroove patterns to induce morphological changes through contact guidance to enhance neuronal differentiation. However, it has been challenging to assess the presence of mechanical dosing and the time-dependent retention of nanotopography on NSCs differentiation because of technical difficulties found in the conventional methods such as nanogroove patterns and nanofibers, of which topography could not be adjusted in a spontaneous fashion.16−18 The terminology mechanical memory refers to a biological condition where a cell retains information from past mechanical stimuli. To modulate stem cell differentiation using the mechanical cues, it is thus important to understand whether NSCs modulated by nanotopography have developed the time-dependent retention of nanotopography to ensure proper NSCs differentiation. In this study, we examined topographical cue-induced neural differentiation and investigated the optimal temporal guidelines for culture of NSCs to increase their differentiation

tem cells can be influenced by various biochemical and biophysical stimuli from the surrounding microenvironment. As one of the major microenvironmental components, the extracellular matrix (ECM) provides structural and mechanical information, such as stiffness and topography, on stem cells, inducing changes in biological behaviors, including migration, proliferation, and differentiation.1,2 There have been many reports that mechanical stimuli, such as substrate stiffness or substrate topographical cues, can influence the ultimate fate decision of stem cells.3−6 Unlike biochemical cue-mediated differentiation, the dosing of mechanical stimuli is difficult to standardize for manipulation of stem cell differentiation. In this regard, the concept of mechanical dosing and memory has been introduced in the field of stem cell engineering.7 As stem cells are dosed with chemical reagents to modulate differentiation, they can also be dosed with mechanical cues as a function of time.7 Although many groups have subsequently confirmed the concept of mechanical memory, the mechanisms underlying how past mechanical dosing/memory affects specific cell fate decisions have not been elucidated.8,9 Topography plays a key role in guiding neuronal differentiation of neural stem cells (NSCs), which have the capacity to self-renew, proliferate, and differentiate into all brain cell types.10,11 The corpus callosum, often called the “white matter track,” is a topographical feature found in the brain.2 On the basis of the presence of such anatomical features in the brain, many studies have focused on modulating NSC differentiation © XXXX American Chemical Society

Special Issue: Biomaterials for Mechanobiology Received: September 5, 2018 Accepted: January 3, 2019 Published: January 3, 2019 A

DOI: 10.1021/acsbiomaterials.8b01057 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Figure 1. Dynamic nanowrinkle platform. (a) Experimental flow of how to examine the time-dependent retention effect on hNSCs. (b) Fabrication steps of the dynamic nanowrinkle on PDMS substrates. (c) hNSCs cultured on flat and nanowrinkle (NW) substrates. The neurites of hNSCs cultured on the nanowrinkle substrates were more aligned to the direction of the nanowrinkles than those cultured on flat substrates. (d) Distributions of neurites on flat and nanowrinkle substrates. (e) TuJ1-positive cell population on the nanowrinkle substrates was greater than that on flat substrates. (f) Neurite length analysis showing that the neurites on the nanowrinkle substrates were more elongated than those on flat substrates Mean ± SEM is shown (*p < 0.05).

of nanotopography on NSC differentiation in vitro (Figure 1a). Using this approach, we examined topographical cue-induced neural differentiation and investigated the optimal temporal guidelines for in vitro culture of NSCs to increase their differentiation retention. We fabricated structurally tunable nanowrinkle substrates by applying oxygen plasma oxidation to polydimethylsiloxane (PDMS) substrate.21−23 As shown in Figure 1b, a PDMS substrate was uniaxially stretched from l0 = 30 mm to l0(1 + ε) = 36 mm (strain (ε) = 0.2) and followed by oxygen plasma in the stretched state. After plasma treatment, the PDMS substrate was spontaneously released to obtain the wrinkle structure on its surface. We varied the plasma oxidation time

retention by using strain-responsive nanowrinkle substrate. The underlying mechanism for mechanical stimuli-mediated differentiation of neural stem cell has not been fully understood yet. However, based on previous research, it is possible that nanotopography causes changes in NSCs morphology, which induces alteration of focal adhesions sites of NSCs.19,20 The characteristic of the platform suggested here is the tunability of the substrate topography that the substrate topography can be changed from nanowrinkle to flat and vice versa upon the application of strain. The dynamic characteristic of the substrate topography not only enables induction of NSC differentiation, but also allows the assessment of mechanical dosing and induce the time-dependent retention B

DOI: 10.1021/acsbiomaterials.8b01057 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Figure 2. Enhanced neuronal differentiation of hNSCs. (a) hNSCs were fixed on days 1, 5, and 7, and stained for neuronal markers (MAP2 and TuJ1). (b) qRT-PCR results showed that neuronal markers were upregulated when hNSCs were cultured on the nanowrinkle substrates. (c) Mechanical dosing effect and hNSC behavior. (d) Expression levels of early neuronal markers (TuJ1 and Nestin) increased gradually with increasing amounts of mechanical dosing mean ± SEM is shown (*p < 0.05, **p < 0.01, ***p < 0.001 by student t test).

increasing amount of strain (Figure S1c). The nanowrinkle structure of the substrates was flattened upon the application of strain (Figure S1d). These observations confirmed that the topography of the nanowrinkle substrate can be dynamically tuned as needed. We cultured human neural stem cells (hNSCs) on nanowrinkle substrates to examine whether the nanowrinkle topography promotes their neuronal differentiation. We cultured hNSCs for 7 days on nanowrinkle substrates with

from 1 to 10 min to obtain wrinkle substrates of various sizes. The wavelength and amplitude of the wrinkle increase with plasma oxidation time. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to obtain surface images of wrinkled substrates (Figure S1a, b). To investigate whether nanowrinkle substrates can be flattened by stretching, we applied various levels of strain to the nanowrinkle substrates from 0.0 to 0.2. The root-mean-square (RMS) value of the surface of the substrates decreased with C

DOI: 10.1021/acsbiomaterials.8b01057 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Figure 3. Mechanical dosing and memory effect on hNSC differentiation. (a) Schematic of mechanical dosing and memory test. (b) Immunostaining of mechanically dosed hNSCs and hNSCs cultured on flat and nanowrinkle substrates. (c) qRT-PCR analysis. (d) Tuj1 + cell % and neurite length analysis. (e) Distributions of neurite orientation. Mean ± SEM is shown (*p < 0.05).

three different wavelengths, and then collected the cells for further analysis. The morphology of neurites was completely

changed when hNSCs were cultured on nanowrinkle substrates. The neurites of hNSCs cultured on nanowrinkle D

DOI: 10.1021/acsbiomaterials.8b01057 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

cultured on flat substrates for 7 days. There was no difference in the gene expression level of astrocyte marker GFAP, which indicates that the nanowrinkle substrates are associated in neuronal differentiation of hNSCs. Moreover, the gene expression level of focal adhesion kinase was measured to investigate whether mechanical dosing and memory are related to focal adhesion. The gene expression level was slightly increased as hNSCs mechanically dosed. However, there was no significant difference between the groups (Figure 3c). The increased glial gene expression on the flat substrate has been found in other literature.24 Taken together, these results indicate that mechanical dosing affects hNSC differentiation and induces the time-retention effect on hNSC differentiation and phenotype retention. The results of this study confirmed that hNSCs with greater mechanical dosing had increased neuronal gene expression levels, suggesting that mechanical dosing may be closely related to the differentiation of hNSCs. Additionally, by changing the substrate topography in situ, we showed that hNSCs with greater mechanical dosing retained their differentiated morphology, whereas those exposed to less mechanical dosing did not. These observations also indicate that mechanical dosing creates a threshold that causes hNSCs to respond to the external mechanical cues. Previously, it was very difficult to assess these effects in vitro due to technical limitations. Typically, enzymatic approach, called trypsin-EDTA, has been widely used to collect NSCs to investigate the concept of the time-retention effect in other research.25 However, the time-retention effect could not be elucidated clearly because of cell loss, signal noises, and the loss of mechanical signal due to the enzymatic effect. This made it difficult to assess the mechanical dosing effect without the artifacts. However, the dynamic nanowrinkle platform allowed us to elucidate the mechanical dosing effect on hNSC differentiation and the time-retention effect on differentiated NSCs without recollecting the cells. Therefore, our system can be used in stem cell engineering to determine the optimal amounts of topographical stimuli to induce stem cell differentiation and increase the retention of differentiated phenotypical characteristics by stem cells.

substrates were elongated and highly aligned in the direction of nanowrinkles (Figure 1d). We investigated neuronal gene expression of hNSCs, neurite length, and orientation as physical markers for neuronal differentiation of hNSCs. hNSCs cultured on the nanowrinkle substrates showed neuronal morphology that the hNSCs were higher in the number of Tuj1 positive and more elongated (Figure 1e, f). hNSCs were collected after 7 days of culture on flat and nanowrinkle substrates with wavelengths of 800, 1200, and 1400 nm (Figure S2). We measured the number of Tuj1 positive hNSCs and the neurite length. The results indicated that the nanowrinkle with 800 nm promotes neuronal differentiation better than the other conditions with macroscale topography features. We observed significantly enhanced gene expression of neuronal markers (TuJ1, MAP2, and Nestin) in hNSCs on the nanowrinkle substrates compared to those on flat substrates (Figure 2a, b). However, there were no differences in gene expression levels of the astrocyte marker, GFAP, between hNSCs cultured on flat and nanowrinkle substrates (Figure 2b). We confirmed that nanowrinkles with a wavelength of 800 nm were adequate to induce neuronal differentiation, and the wavelength of 800 nm was more efficient in inducing differentiation than the other wavelengths examined. These findings support those of previous studies indicating that nanoscale topography is preferred over macroscale topography for inducing neuronal differentiation.12,13,24 Therefore, our results showed that nanowrinkle topography led to enhanced neuronal differentiation of hNSCs. Additionally, to investigate whether mechanical dosing is related to neuronal differentiation, we measured the neuronal gene markers at different time points (i.e., 3, 5, 7, and 10 days) (Figure 2 c). Longer periods of exposure of hNSCs to the nanowrinkle substrates were associated with higher levels of neuronal gene expression (Figure 2d). This suggested that mechanical dosing by culturing hNSCs on the nanowrinkle substrates affected their neuronal differentiation. To elucidate the mechanical dosing and the time-dependent retention of nanowrinkle on hNSC, we dosed hNSCs mechanically by culturing the cells on the nanowrinkle substrates for various periods. The nanowrinkle substrates were removed in situ after application of a sufficient amount of mechanical dosing (MD) under three different conditions: MD1, hNSCs on the nanowrinkle patterns for 1 day; MD3, hNSCs on nanowrinkle patterns for 3 days; and MD5, hNSCs on the nanowrinkle patterns for 5 days (Figure 3a). To identify the time-retention effect on morphology, we immunostained hNSCs mechanically dosed for 1, 3, or 5 days for the early neuronal marker, TuJ1 (Figure 3b). The direction of neurites from hNSCs mechanically dosed for 5 days retained the direction of the wrinkles, whereas those of hNSCs mechanically dosed for 1 or 3 days showed changes in direction (Figure 3b, e). Moreover, hNSCs mechanically dosed for longer times had a greater percentage of aligned neurites. These observations indicated that mechanical dosing affects the orientation of neurites to a sufficient or better degree, and they tended to retain their morphology even when the nanowrinkle topography was removed (Figure 3d). The same trends were observed in neuronal gene expression levels. hNSCs mechanically dosed for longer than 5 days had increased neuronal gene expression levels (TuJ1 and MAP2) (Figure 3c). However, hNSCs mechanically dosed for less than 3 days had similar neuronal gene expression patterns to those

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b01057. Detailed information on the nanowrinkle pattern analysis including AFM and SEM images when the nanowrinkle PDMS substrates were released and stretched. Line profiles of the nanowrinkle patterns with respect to oxygen plasma exposure time. And the surface roughness to identify the effective amount of strain for the flattened nanowrinkle patterns. Nanowrinkle size optimization data. Neurite analysis, Immunostain, PCR analysis to fine the most effective size of nanowrinkle patterns for enhancing neuronal differentiation (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. E

DOI: 10.1021/acsbiomaterials.8b01057 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering ORCID

to Enhance Neuronal Differentiation. Sci. Rep. 2013, 3, No. 1553, DOI: 10.1038/srep01553. (15) Yang, K.; Park, E.; Lee, J. S.; Kim, I. S.; Hong, K.; Park, K. I.; Cho, S. W.; Yang, H. S. Biodegradable Nanotopography Combined with Neurotrophic Signals Enhances Contact Guidance and Neuronal Differentiation of Human Neural Stem Cells. Macromol. Biosci. 2015, 15 (10), 1348−1356. (16) Tan, K. K. B.; Tann, J. Y.; Sathe, S. R.; Goh, S. H.; Ma, D.; Goh, E. L. K.; Yim, E. K. F. Enhanced Differentiation of Neural Progenitor Cells into Neurons of the Mesencephalic Dopaminergic Subtype on Topographical Patterns. Biomaterials 2015, 43, 32−43. (17) Chua, J. S.; Chng, C. P.; Moe, A. A.; Tann, J. Y.; Goh, E. L.; Chiam, K. H.; Yim, E. K. Extending Neurites Sense the Depth of the Underlying Topography During Neuronal Differentiation and Contact Guidance. Biomaterials 2014, 35 (27), 7750−7761. (18) Yang, K.; Lee, J. S.; Kim, J.; Lee, Y. B.; Shin, H.; Um, S. H.; Kim, J. B.; Park, K. I.; Lee, H.; Cho, S. W. Polydopamine-Mediated Surface Modification of Scaffold Materials for Human Neural Stem Cell Engineering. Biomaterials 2012, 33 (29), 6952−6964. (19) Teo, B. K.; Wong, S. T.; Lim, C. K.; Kung, T. Y.; Yap, C. H.; Ramagopal, Y.; Romer, L. H.; Yim, E. K. Nanotopography Modulates Mechanotransduction of Stem Cells and Induces Differentiation through Focal Adhesion Kinase. ACS Nano 2013, 7 (6), 4785−4798. (20) Yim, E. K.; Darling, E. M.; Kulangara, K.; Guilak, F.; Leong, K. W. Nanotopography-Induced Changes in Focal Adhesions, Cytoskeletal Organization, and Mechanical Properties of Human Mesenchymal Stem Cells. Biomaterials 2010, 31 (6), 1299−1306. (21) Yin, J.; Yague, J. L.; Boyce, M. C.; Gleason, K. K. Biaxially Mechanical Tuning of 2-D Reversible and Irreversible Surface Topologies through Simultaneous and Sequential Wrinkling. ACS Appl. Mater. Interfaces 2014, 6 (4), 2850−2857. (22) Cha, J.; Shin, H.; Kim, P. Crack/Fold Hybrid Structure-Based Fluidic Networks Inspired by the Epidermis of Desert Lizards. ACS Appl. Mater. Interfaces 2016, 8 (42), 28418−28423. (23) Shin, H.; Choi, Y.; Cha, J.; Kim, P. Spatially Controlled Folding Instability of Moduli-Patterned and Bilayered Membrane under Compressive Stresses. Adv. Mater. Interfaces 2016, 3 (17), 1600105. (24) Ankam, S.; Suryana, M.; Chan, L. Y.; Moe, A. A. K.; Teo, B. K. K.; Law, J. B. K.; Sheetz, M. P.; Low, H. Y.; Yim, E. K. F. Substrate Topography and Size Determine the Fate of Human Embryonic Stem Cells to Neuronal or Glial Lineage. Acta Biomater. 2013, 9 (1), 4535− 4545. (25) Yang, K.; Jung, H.; Lee, H. R.; Lee, J. S.; Kim, S. R.; Song, K. Y.; Cheong, E.; Bang, J.; Im, S. G.; Cho, S. W. Multiscale, Hierarchically Patterned Topography for Directing Human Neural Stem Cells into Functional Neurons. ACS Nano 2014, 8 (8), 7809−7822.

Seung-Woo Cho: 0000-0001-8058-332X Pilnam Kim: 0000-0003-0611-6525 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was funded by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) & funded by the Korean government (MSIP&MOHW) [NRF-2015M3A9B3028685], and Ministry of Science, ICT, and Future Planning [2016M3A9B4915823]. And this research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea [HI14C1324].

■

REFERENCES

(1) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 2006, 126 (4), 677−689. (2) Kim, D. H.; Provenzano, P. P.; Smith, C. L.; Levchenko, A. Matrix Nanotopography as a Regulator of Cell Function. J. Cell Biol. 2012, 197 (3), 351−360. (3) Turner, L.-A.; J. Dalby, M. Nanotopography - Potential Relevance in the Stem Cell Niche. Biomater. Sci. 2014, 2 (11), 1574−1594. (4) Rosa, A. L.; Kato, R. B.; Castro Raucci, L. M.; Teixeira, L. N.; de Oliveira, F. S.; Bellesini, L. S.; de Oliveira, P. T.; Hassan, M. Q.; Beloti, M. M. Nanotopography Drives Stem Cell Fate toward Osteoblast Differentiation through Alpha1beta1 Integrin Signaling Pathway. J. Cell. Biochem. 2014, 115 (3), 540−548. (5) Solanki, A.; Shah, S.; Yin, P. T.; Lee, K. B. NanotopographyMediated Reverse Uptake for Sirna Delivery into Neural Stem Cells to Enhance Neuronal Differentiation. Sci. Rep. 2013, 3, 1553. (6) Kshitiz; Park, J.; Kim, P.; Helen, W.; Engler, A. J.; Levchenko, A.; Kim, D. H. Control of Stem Cell Fate and Function by Engineering Physical Microenvironments. Integr Biol. (Camb) 2012, 4 (9), 1008− 1018. (7) Yang, C.; Tibbitt, M. W.; Basta, L.; Anseth, K. S. Mechanical Memory and Dosing Influence Stem Cell Fate. Nat. Mater. 2014, 13 (6), 645−652. (8) Murphy, W. L.; McDevitt, T. C.; Engler, A. J. Materials as Stem Cell Regulators. Nat. Mater. 2014, 13, 547. (9) Rosales, A. M.; Anseth, K. S. The Design of Reversible Hydrogels to Capture Extracellular Matrix Dynamics. Nature Reviews Materials 2016, 1, 15012. (10) Recknor, J. B.; Sakaguchi, D. S.; Mallapragada, S. K. Directed Growth and Selective Differentiation of Neural Progenitor Cells on Micropatterned Polymer Substrates. Biomaterials 2006, 27 (22), 4098−4108. (11) Yim, E. K.; Pang, S. W.; Leong, K. W. Synthetic Nanostructures Inducing Differentiation of Human Mesenchymal Stem Cells into Neuronal Lineage. Exp. Cell Res. 2007, 313 (9), 1820−1829. (12) Moe, A. A.; Suryana, M.; Marcy, G.; Lim, S. K.; Ankam, S.; Goh, J. Z.; Jin, J.; Teo, B. K.; Law, J. B.; Low, H. Y.; Goh, E. L.; Sheetz, M. P.; Yim, E. K. Microarray with Micro- and NanoTopographies Enables Identification of the Optimal Topography for Directing the Differentiation of Primary Murine Neural Progenitor Cells. Small 2012, 8 (19), 3050−3061. (13) Yang, K.; Jung, K.; Ko, E.; Kim, J.; Park, K. I.; Kim, J.; Cho, S. W. Nanotopographical Manipulation of Focal Adhesion Formation for Enhanced Differentiation of Human Neural Stem Cells. ACS Appl. Mater. Interfaces 2013, 5 (21), 10529−10540. (14) Solanki, A.; Shah, S.; Yin, P. T.; Lee, K. B. NanotopographyMediated Reverse Uptake for Sirna Delivery into Neural Stem Cells F

DOI: 10.1021/acsbiomaterials.8b01057 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX