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Analysis of Chirality Effects on Stem Cell Fate Using Three-dimensional Fibrous Peptide Hydrogels Hangyu Zheng, Toru Yoshitomi, and Keitaro Yoshimoto ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00123 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 4, 2018

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ACS Applied Bio Materials

Analysis of Chirality Effects on Stem Cell Fate Using Three-dimensional Fibrous Peptide Hydrogels Hangyu Zhenga, Toru Yoshitomia, and Keitaro Yoshimotoa, b* a

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan b JST, PRESTO, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan *

Corresponding authors: Keitaro Yoshimoto Phone and FAX: +81-3-5454-6583; E-mail: [email protected]

ABSTRACT: Chirality effects on stem cell fate were investigated in three-dimensional culture using soft fibrous hydrogels consisting of self-assembled l- and d-form Fmoc-Phe-Phe-Cys networks photo-crosslinked by poly(ethylene glycol) (l- and d-gel, respectively). Encapsulated human bone marrow-derived mesenchymal stem cells were all alive, spread, and grew in both hydrogels. Interestingly, the cells preferably spread and grew in l-gel compared to in d-gel under mixed induction, cell osteogenesis and adipogenesis differentiation in d-gel were suppressed compared to in l-gel. These results revealed that stem cell function and fate can be regulated in three-dimensional hydrogel culture systems with chiral motifs.

KEYWORDS: chirality, 3D culture, stem cell fate, peptide self-assembly, fibrous hydrogels, polymer crosslinker, cell spreading, cell proliferation

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Stem cells can self-renew and differentiate into many types of specific cells, giving them the potential to greatly drive biological research and improve disease treatment. Stem cell behavior is considered to be regulated by the microenvironment in which the major component is the extracellular matrix (ECM).1, 2 To develop stem cell-based applications, it is essential to understand how stem cells respond to culture matrices.3 Over the last decade, various properties of culture substrates and scaffolds have been explored to study their effects on stem cell functions.4-11 Chirality is a universal property of many molecules in the biological world. For example, natural proteins consist of left-handed (L-) amino acids. It is unclear why natural proteins choose single chirality and selective interactions occur between homochiral molecules. Previous reports of two dimensional culture systems showed different cell adhesion behaviors on low-molecular weight enantiomorphous moiety-based matrices.12-18 Ding et al. revealed that chirality also influenced stem cell fate using L- or D-amino acid-based monolayers. On these surfaces under mixed adipogenic and osteogenic induction, higher cell adhesion and adipocyte differentiation occurred on the l-form surface, while more efficient osteoblast differentiation was observed on the d-form surface because of lower cell adhesion.19 However, because cells in the body are surrounded by the ECM in all directions, the development of more physiologically relevant three-dimensional (3D) culture systems with different chirality is needed to better understand the effects of chirality on stem cell fate. Here, we utilized hydrogel systems to investigate how chirality affects stem cell behavior in 3D culture. The hydrogels were constructed based on an ultra-short peptide, Fmoc-Phe-Phe (Fmoc-FF), which is the shortest peptide that can self-assemble into nanofiber hydrogels in aqueous solution at pH 7.20 However, these self-assembled hydrogels are composed of fibers without crosslinking and tend to disintegrate in culture media, making them unsuitable for long-term cell culture. To overcome this issue, fibrous networks of cystatin-ended Fmoc-FF, Fmoc-Phe-Phe-Cys (Fmoc-FFC, Scheme S1A and Figure S1), photocrosslinked by poly(ethylene glycol) dimethacrylate (PEGDMA, Scheme S1B and Figure S2) were developed, as shown in Figure 1A. First, l-Fmoc-FFC and d-Fmoc-FFC consisting of L-amino acids and D-amino acids, respectively, were dissolved in PBS by adjusting the pH value. Next, PEGDMA and a photoinitiator were added to the peptide solution. The mixture was kept at room temperature for several minutes to form self-assembled hydrogels. After crosslinking with UV light, the obtained hydrogels containing L-amino acids or D-amino acids were referred to as l-gel or d-gel, respectively. Human bone marrow-derived mesenchymal stem cells, UE7T-13 cells, were encapsulated in the hydrogels (see Supporting Information). The properties of l-gel and d-gel were measured without cell encapsulation. Circular dichroism spectra of the two diluted hydrogels showed opposite signal peaks at 210 nm, which are characteristic of the π–π interactions in Fmoc-FF assemblies, indicating that each hydrogel consisted of different chiral amino acids (Figure 1B). Both l- and d-Fmoc-FFC formed transparent fibrous hydrogels within several minutes (Figure S3). Transmission electron microscopy images revealed that similar fiber structures were constructed and that post-photo-crosslinking by PEG chains between fibers did not destroy the fibrous structure (Figure 1C, upper). The crosslinked hydrogels maintained their integrity when rinsed with 2


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medium after preparation (Figure 1C, lower), while the non-crosslinked blend hydrogels were broken (data not shown). The l-gel and d-gel showed similar stiffness (approximately 200 Pa), which is classified as a soft hydrogel (Figure 1D). These results indicate that constructed chiral fibrous networks have nearly the same properties except chirality. Using these hydrogels, we investigated chirality effects on stem cell behaviors such as cell proliferation, spreading, and differentiation.

Figure 1. (A) Schematic illustration of the formation of Fmoc-FFC self-assembled fibrous hydrogels cross-linked by PEG chains via thiol-ene photopolymerization. (B) Circular dichroism spectra of diluted hydrogels in water. (C) TEM images (upper; scale bar, 50 nm) and the pictures of the hydrogels after rinsing in DMEM (lower; scale bar, 5 mm). (D) Shear storage modulus (G′) of the hydrogels measured with a rheometer. First, both l-gel and d-gel encapsulating UE7T-13 cells, which is cell line of human bone marrow-derived mesenchymal stem cells, were cultured in growth medium (GM). As expected, cross-linking effectively maintained the gel structures (Figure S4). These gels were stable during long-term cell culture including during analytical procedures such as staining and extraction. The viability of cells encapsulated in the hydrogels was examined by calcein-AM staining for live cells and propidium iodide staining for dead cells, which were detected as green and red colors, respectively. After 4 weeks of culture GM, nearly all cells were alive in both l-gel and d-gel (Figure 2A). Next, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to measure cell proliferation in mixed adipogenic and osteogenic induction media (1:1 v/v). As shown in Figure 2B, after 3 days of culture in GM, which was the starting point for culture in induction medium (IM), the cell growth in l-gel and d-gel were nearly the same. After 2 weeks of culture in IM, the cell growth doubled in both hydrogels. However, after 4 3



weeks of culture in IM, the proliferation rate of UE7T-13 cells in l-gel was larger than that in d-gel, and the cell growth in l-gel was approximately 2.5-fold higher than that in d-gel. Because culture environments in hydrogels, including chemical composition, structure, and stiffness, were nearly the same between l-gel and d-gel, the difference in cell proliferation can be attributed to the different chirality of the scaffold molecules. Additionally, to investigate the changes in the number of cell-cell contacts in both gels, the gene expression level of N-cadherin was measured (Figure 2C).21,22 The N-cadherin gene expression level in cells in l-gel was similar to that in d-gel after 2 weeks of culture in IM, while a higher expression level in l-gel than in d-gel was observed after 4 weeks of culture in IM. These results indicate that the number of cell-cell contacts was increased in both gels, with a larger number of cell-cell contacts generated in l-gel at 4 weeks, which agrees with the results obtained from the MTT assay.

Figure 2. (A) Live/dead assay of UE7T-13 cells encapsulated in l-gel and d-gel cultured in growth medium (GM) for 4 weeks. (B) Cell proliferation evaluated by MTT assay after culture in GM after 3 days (starting time of induction), 2 and 4 weeks in induction medium (IM). Absorbance was measured at 560 nm. (C) RT-PCR analysis for N-cadherin gene expression cultured after 2 and 4 weeks in IM. Values are the mean ± SE of three independent experiments. *P < 0.01. Scale bar: 50 µm. To understand the changes in cell morphology during culture, F-actin of the encapsulated cells was stained (Figure 3). After 3 days of culture in GM, most cells maintained a rounded shape in both gels. Interestingly, after 2 weeks of culture in IM, connecting actin filaments of stretched cells were formed in l-gel, whereas most of cells showed poor spreading in d-gel. After 4 weeks of culture in IM, cells in l-gel contained actin filament bundles with high density, while cells in d-gel possessed less connected actin filaments of elongated cells. These results suggest that UE7T-13 cells can spread and 4


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proliferate more easily in l-gel than in d-gel. In addition, UE7T-13 cells did not spread and grow in PEG hydrogel (10 wt%) without Fmoc-FFC fiber, as shown in Figure S5, indicating that the cells adhered to and spread along the nanofibers in these soft gels. More importantly, cells in the two fibrous hydrogels with distinct chirality showed different spreading and proliferation rates during culture, offering an appropriate platform for investigating the chirality effects on stem cell differentiation in 3D culture.

Figure 3. Alexa Fluor 568 phalloidin staining for F-actin of UE7T-13 cells encapsulated in land d-gels at 3 days after cultured in GM (left) and 2 weeks (center) and 4 weeks (right) after cultured in IM. Scale bar: 50 µm. The differentiation of cells encapsulated in hydrogels was assessed by RT-PCR analysis. The gene expression levels of osteogenic and adipogenic biomarkers, RUNX2 and PPARγ, were measured, respectively. In addition, Oil Red O (ORO) and Alizarin Red S (ARS) staining were conducted to quantify adipogenesis and osteogenesis, respectively (Figure 4 and S6). Figure 4A and B show the gene expression levels of PPARγ and ORO values for different culture periods. After 2 weeks of induction, PPARγ expression levels of cells in l-gel and d-gel were similar at lower levels, while after 4 weeks of induction, the expression level of PPARγ in l-gel was higher than that in d-gel. The ORO values, which were normalized by the absorbance of MTT assay, suggested that the extent of lipid accumulation in l-gel was similar to that in d-gel at extremely low levels after 2 weeks, but higher after 4 weeks. These results indicate that sufficient differentiation of encapsulated UE7T-13 cells to adipogenesis requires more than 2 weeks, which is correlated with the PPARγ expression levels of UE7T-13 cells after 2 and 4 weeks. The MTT assay and gene expression analysis of N-cadherin in both gels revealed nearly no difference after 2 weeks of culture in IM, but a significant difference was observed after 4 weeks of culture in IM (Figure 2B and C). These data suggest that the increase and difference in cell-cell contact amount in the gels triggered 5



and caused differences in adipogenesis differentiation, respectively. In a previous study, a similar mesenchymal stem cell (MSC) differentiation trend, showing that cell-cell contact plays an important role in adipose differentiation was reported in a two-dimensional (2D) culture system.23 It is worth to note that the cells in the hydrogels also spread well even at high density compared to those on the culture surface. In contrast, differentiation behavior of encapsulated UE7T-13 cells to osteogenesis differed from that of adipogenesis. The results of osteogenic differentiation analysis in both gels are shown in Figure 4C and D. These results indicate that the differentiation of encapsulated UE7T-13 cells to osteogenesis started within 2 weeks of culture in IM. Furthermore, RUNX2 expression level in cells in l-gel was higher than those in d-gel after both 2 and 4 weeks. Consistent with the RUNX2 data, ARS quantification showed that the extent of calcium deposition in l-gel was higher than that in d-gel during these culture periods. As shown in Figure 3, the cells spread wider in l-gel than d-gel after 2 and 4 weeks of culture in IM, which may have caused this difference in the differentiation level to osteogenesis between the two gels. These results are consistent with those of previous studies in 2D culture, where the size of a single cell plays an important role in differentiation and larger-size MSCs tend to differentiate to osteogenesis effectively.23,24 The effect of chiral molecules on MSC fate was studied using a 2D culture system,19 which demonstrated that under co-induction, MSCs prefer to adhere to an L-amino acid-modified surface, where MSCs showed restrained spreading because of high cell density, resulting in more efficient differentiation to adipogenesis. In contrast, less cell adhesion occurs for MSCs on a D-amino acid-modified surface, which increases cell spreading, resulting in enhanced osteogenesis differentiation. In 2D culture, stem cell differentiation is mainly affected by cell shape, which is determined by the initial cell density on the culture substrate. However, in 3D culture, as in this study, the cell growth and spreading changed during culture time and influenced UE7T-13 cell differentiation. Thus, in our hydrogels, evaluation of MSC fate can be performed in the culture system that more faithfully imitates the in vivo environment. In the present study, we found that, as an overall trend in stem cell fate, the differentiation rates to both osteogenesis and adipogenesis in d-gel were suppressed compared to those in l-gel. These results indicate that the chirality effect on the stem cell behavior in 3D culture differs from that in 2D culture. Dissimilar stem cell fate decision between 2D and 3D cultures was also observed in previous studies.25, 26

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Figure 4. Differentiation analysis of UE7T-13 cells in the hydrogels after 2 and 4 weeks of culture in IM. (A) Real-time RT-PCR analysis of expression of PPARγ in encapsulated MSCs under co-induction conditions. (B) Quantification of ORO staining. Absorbance was measured at 500 nm. (C) Real-time RT-PCR analysis of expression of RUNX2 in encapsulated MSCs under co-induction conditions. (D) Quantification of ARS staining. Absorbance was measured at 405 nm. Absorbance ratios in B and D were calculated using absorbances of ORO and ARS staining after normalization by absorbances obtained from MTT assay. Error bars represent the standard error of the mean where n = 3. * P < 0.05; * * P < 0.01; * * * P < 0.001. Taken together, in this 3D soft fibrous hydrogel culture system, UE7T-13 cells favorably spread, grew, and differentiated in l-gel compared to in d-gel. As described above, the tendency for differentiation of UE7T-13 in our 3D gels with different chirality was associated with the results of previous studies of MSC fate in 2D culture without chiral molecules,23, 24, 27 while the reasons for differences in cell spreading and proliferation caused by chirality in the hydrogels in 3D culture require further studies. In conclusion, by utilizing self-assembling ultra-short peptides with photo-cross-linking by polymer chains, stable, soft fibrous hydrogels that permit long-term cell culture, cell spreading, and proliferation were constructed. Using this novel scaffold, we investigated the effects of chirality on stem cell behavior in 3D culture. Cell attachment, spreading level, and differentiation rate were controlled by changing the chirality of molecules in the 3D microenvironment. The d-form hydrogel may help to avoid tumorigenesis in stem cell transplant by suppressing cell growth and maintain stem cell pluripotency by restraining differentiation.28, 29 Furthermore, the hydrogel culture system synthesized here provides an ideal platform for examining stiffness effects on cellular behaviors, as matrix elasticity can be modulated by changing the light exposure time rather than peptide concentration. Understanding the interplay between chirality and stiffness would not only help us 7



understand chirality selection in nature, but also offer biomaterials that benefit stem cell-based therapies.

Supporting Information Synthesis and characterization of l-, d-Fmoc-FFC and PEGDMA, methods of hydrogel preparation and cell encapsulation, procedures of analysis of cell growth, spreading and differentiation

ORCID Keitaro Yoshimoto: 0000-0002-6052-1399

Notes The authors declare no competing financial interest

Acknowledgements This work was also supported by the Japan Science and Technology Agency, PRESTO [grant number: JPMJPR16FB (to K.Y.)] and in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science KAKENHI [grant number: 16K16398 (to T.Y.)].

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