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Mechanotransduction of human pluripotent stem cells cultivated on tunable cell-derived extracellular matrix
Biomaterials 150 (2018) 100e111
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Mechanotransduction of human pluripotent stem cells cultivated on tunable cell-derived extracellular matrix In Gul Kim a, 1, Chang-Hyun Gil b, 1, Joseph Seo b, 1, Soon-Jung Park b, Ramesh Subbiah a, Taek-Hee Jung b, Jong Soo Kim b, Young-Hoon Jeong b, Hyung-Min Chung b, Jong Ho Lee a, Man Ryul Lee d, Sung-Hwan Moon b, *, 1, Kwideok Park a, c, **, 1 a
Center for Biomaterials, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea Department of Stem Cell Biology, Konkuk University, School of Medicine, Seoul, 05029, Republic of Korea Biomedical Engineering Major, Korea University of Science and Technology (UST), Daejeon, 34113, Republic of Korea d Soonchunhyang Institute of Medi-bio Science (SIMS), Institute of Tissue Regeneration, College of Medicine, Soon Chun Hyang University, Cheonan, 31151, South Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2017 Accepted 7 October 2017 Available online 9 October 2017
Cell-derived matrices (CDM) are becoming an attractive alternative to conventional biological scaffolding platforms due to its unique ability to closely recapitulate a native extracellular matrix (ECM) de novo. Although cell-substrate interactions are recognized to be principal in regulating stem cell behavior, very few studies have documented the acclimation of human pluripotent stem cells (hPSCs) on pristine and altered cell-derived matrices. Here, we investigate crosslink-induced mechanotransduction of hPSCs cultivated on decellularized fibroblast-derived matrices (FDM) to explore cell adhesion, growth, migration, and pluripotency in various biological landscapes. The results showed either substrate-mediated induction or inhibition of the Epithelial-Mesenchymal-Transition (EMT) program, strongly suggesting that FDM stiffness can be a dominant factor in mediating hPSC plasticity. We further propose an optimal FDM substratum intended for long-term hPSC cultivation in a feeder-free niche-like microenvironment. This study carries significant implications for hPSC cultivation and encourages more in-depth studies towards the fundamentals of hPSC-CDM interactions. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Human pluripotent stem cells (hPSCs) are traditionally cultivated on feeder cells such as mouse embryonic fibroblasts (MEFs) but there has been a progressive move away from the use of feeders because they can introduce xenogeneic contaminants or other undefined factors into the culture systems [1]. For this reason, Matrigel® (Corning, Corning, NY) is one of the most widely used extracellular components for feeder-free culture of hPSCs but it suffers from limited customization and inherent heterogeneity as it is derived from Engelbreth-Holm-Swarm mouse sarcomas which
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (S.-H. Moon), [email protected] (K. Park). 1 These authors contributed equally to this work as first and corresponding authors, respectively. https://doi.org/10.1016/j.biomaterials.2017.10.016 0142-9612/© 2017 Elsevier Ltd. All rights reserved.
may also contain unwanted xenogeneic materials [2]. While recent advancements continue to demonstrate synthetic and biological alternatives ranging from nanopatterns [3], hydrogels [4], and single ECM components such as vitronectin [5], another caveat is that unlike cellular secretions, these platforms are unable to recapitulate a complex, organized, and heterogeneous mixture of macromolecules reminiscent of native microenvironments. The extracellular matrix (ECM) is the non-cellular component of tissues and organs composed of various macromolecules secreted by cells to serve as a natural scaffold with structural integrity and biological cues. While its specific composition and function may vary tremendously depending on the type of particular tissue, it is mainly composed of two interlocking macromolecules known as proteoglycans, such as glycosaminoglycans (GAGs), and fibrous proteins, such as collagen, vitronectin, elastin, fibronectin, and laminin [6]. In essence, it is a highly dynamic structure that is constantly being remodeled and subjected to a myriad of posttranslational modifications as it plays a pivotal role in the
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biophysical and biochemical dialogue between various cells [7]. It possesses a wide range of distinct properties that depend on the inhabitant cells but it is widely known that homeostasis, cell adhesion, cell-to-cell communication, and differentiation are fundamental functions of the ECM [8]. There has been a growing interest in engineering cell-derived matrices (CDM) to create a biomimetic microenvironment that resembles a native niche. Early studies have shown that the biophysical and biochemical cues can be reserved after the decellularization process, which makes the generated matrix a naturally derived culture substratum for stem cell adhesion, proliferation, and differentiation [9,10]. Substrate features are of particular interest because it has a profound impact on stem cell lineage specification. For example, substrate stiffness is an essential characteristic by which cells sense the external forces and subsequently respond to the environment in an appropriate manner. Our previous study demonstrated the effects of mechanotransduction on the fate of human mesenchymal stem cells (hMSCs) and recent reports continue to highlight the importance of cell-ECM interactions in regulating stem cell fate [11,12]. However, even though hPSCs possess enormous potential in regenerative therapy, little attention has been paid to some fundamental aspects of hPSC-CDM interactions partly due to the lack of proper tools. Hence, we prepared a natural ECM analog that is engineered de novo from in vitro cultured fibroblasts to explore the acclimation of hPSCs cultivated on pristine and altered decellularized fibroblastderived matrices (FDM). Mechanotransduction was conducted via non-cytotoxic crosslinking to modulate FDM rigidity to various degrees. Both substrate types were utilized to investigate cell adhesion, growth, migration, and pluripotency. This study shows that simple yet effective physiological controls can dictate hPSCECM dynamics in a closely recapitulated natural environment. Based on our findings, we further propose an optimal FDM platform intended for feeder-free and long-term hPSC cultivation (up to 14 days).
2. Materials and methods 2.1. Preparation of fibroblast-derived extracellular matrix (FDM) To prepare FDM, NIH3T3 mouse fibroblasts (ATCC, Manassas, VA) or human foreskin BJ1 fibroblasts (ATCC) were seeded at the density of 2 104 cells/cm2 on either plastic dishes or gelatincoated glass in 12-well plates, and cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin streptomycin (P/S; Thermo Fisher Scientific). After 1 week, confluent cells were washed twice with phosphate buffered saline (PBS; Thermo Fisher Scientific), then decellularized by incubation with a detergent solution containing 0.25% Triton X-100 and 10 mM NH4OH (SigmaAldrich Co., St. Louis, MO) in PBS at 37 C. The samples were then treated with 50 U/ml DNase I and 2.5 mL/ml RNase A (Thermo Fisher Scientific) for 2 h at 37 C. The decellularized samples were gently washed with PBS three times and the resulting FDM was used immediately or stored at 4 C prior to use. Major FDM components are identified by immunofluorescence of mouse anti-fibronectin (FN; Santa Cruz Biotechnology, Inc., Dallas, Texas) and mouse anti-laminin (LN; Santa Cruz Biotechnology, Inc.). Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) and Alexa Fluor 594-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Inc., West Grove, PA) were used as a secondary antibody.
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2.2. Preparation of crosslinked FDMs The prepared FDM was then treated with different amounts of genipin (GN; Wako Chemicals USA, Richmond, VA) to induce a crosslinking of the FDM. Briefly, a stock solution of 5% (w/v) GN was prepared in dimethyl sulfoxide (DMSO) and diluted to different concentrations (0.5, 1, and 2%) with PBS. GN solution was added to FDM, and the samples were incubated at room temperature for 4 h to allow the crosslinking of FDM itself. The crosslinked FDM (XFDM) was then washed five times with PBS and stored at 4 C before use. The X-FDM was named as follows: Gx0.5, Gx1.0 and Gx2.0, respectively. Gx0 represents the non-crosslinked or pristine FDM. 2.3. Quantitative analysis of ECM proteins To quantify the ECM constituents of the FDM and X-FDMs, fibronectin (FN), laminin (LN), collagen type I (Col I), and vitronectin (VN) were selected. Each sample in 6-well plates was incubated overnight with the following primary antibodies: mouse anti-FN (Santa Cruz Biotechnology, Inc.), mouse anti-LN (Santa Cruz Biotechnology, Inc.), rabbit anti-Col I (Abcam, Cambridge, UK), and rabbit anti-VN (Santa Cruz Biotechnology, Inc.), respectively and followed by the treatment of goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (HRP; Santa Cruz Biotechnology, Inc.). After several washing, the samples were added with 1 ml of 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) and incubated for 20 min. The reaction was stopped using 0.16 M sulfuric acid, after which 100 mL aliquots were taken for the absorbance measurements at 405 nm (Multiskan Microplate Reader, Thermo Fisher Scientific). For the comparison, the fluorometric readouts are converted to the protein levels that are normalized to the surface area (9.6 cm2) of one well per each sample. 2.4. Characterization of FDM and X-FDMs: SEM and AFM The surface morphology of FDM and X-FDM was observed using a scanning electron microscope (SEM; Model S-3000 N, Hitachi). Bio-atomic force microscope (Bio-AFM; NanoWizard II, JPK Instruments, Germany) equipped with an inverted optical microscope (Nikon) was also used in a liquid contact mode to investigate the surface topography and morphology of FDM and X-FDM, respectively. In addition, the AFM-nano indentation technique was employed to measure the Young's modulus (E) of each sample, as described previously [13]. A 10 mm diameter SiO2 particle attached to PT.SiO2.AU.SN10 cantilevers (Novascan Technologies, Ames, USA) with a spring constant of 0.01 N/m was utilized. Based on the force spectra curves, E was calculated using Hertz's contact model in JPK data processing software (v3.3.25). The Poisson ratio of cells was set to 0.5. Approximately 5 indentations on each sample (n ¼ 3, each group) at different regions were carried out for E measurement. 2.5. Cell culture Various cell types are used: human pluripotent stem cell line; H9 (WiCell Research Institute, Madison, WI) [14], mouse embryonic fibroblasts (MEFs), and primary MEFs (Orient Bio Inc., Seongnam, Korea). H9-hPSCs were cultured in 80% DMEM: Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, Waltham, MA) supplemented with 20% KnockOut™ Serum Replacement, 1% P/S (Thermo Fisher Scientific), 1% Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA; Thermo Fisher Scientific), 0.1% beta-mercaptoethanol (b-ME;
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Thermo Fisher Scientific and 4 ng/ml recombinant human basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ). hPSCs culture media was changed daily and cultures were passaged every 5e7 days. The primary CF1 MEFs were treated with Mitomycin C (MMC; M-MEF; Sigma-Aldrich Co.) and served as a feeder layer for hPSCs. M-MEFs were cultured in 90% high-glucose DMEM (Thermo Fisher Scientific supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 1% P/S, 1% MEM-NEAA, and 0.1% b-ME. The cell culture media was changed every 2 days. For subculture of hPSCs onto FDM and X-FDM, hPSCs were treated with Dispase® (Thermo Fisher Scientific) or mechanically dissected with a glass Pasteur pipette (Corning). All the cells were cultivated in a thermostat incubator at 37 C under 5% CO2. 2.6. Alkaline phosphatase staining and immunocytochemistry To confirm alkaline phosphatase (AP) expression in the hPSC colonies, AP staining was performed with an ES Cell Characterization Kit (EMD Millipore, Billerica, MA). For immunostaining, the cells were fixed for 20 min at room temperature in 4% paraformaldehyde (PFA) and permeabilized with 0.03% Triton X-100 in PBS for 5 min. After the treatment with 5% normal goat serum for 30 min, they were incubated overnight at 4 C with primary antibodies; OCT4, SOX2, SSEA-4, and TRA-1-60 (EMD Millipore). Alexa Fluor® 488, 555 or 594 secondary antibodies (Molecular Probes Inc., Sunnyvale, CA) were used to visualize the hPSCs-specific markers. Cell nuclei were stained with DAPI (DAKO, Carpentaria, CA). All images were acquired using a fluorescence microscope (Nikon, Chiyoda-ku, Japan). 2.7. Quantitative polymerase chain reaction (qPCR) For analysis of messenger ribonucleic acid (mRNA) expression, total RNA was extracted using TRIzol® Reagent (Thermo Fisher Scientific). The quantity and purity of extracted RNA were examined via a NanoDrop® ND-1000 spectrophotometer (NanoDrop Products, Wilmington, DE). Total RNA (1 mg) was reversetranscribed into complementary deoxyribonucleic acid (cDNA) using a Maxim RT premix kit (iNtRon Biotechnology, Sungnam, Korea). The target genes and associated primers are as follows: OCT4 (forward) 50 -AAC TCG AGC AAT TTG CCA AGC TCC-30 and (reverse) 50 -TTC GGG CAC TGC AGG AAC AAA TTC-30 , E-cadherin (forward) 50 -CGA GAG CTA CAC GTT CAC GG-30 and (reverse) 50 GGG TGT CGA GGG AAA AAT AGG-30 , N-cadherin (forward) 50 -TCA CAG ATT CGG GTA ATC CTC-30 and (reverse) 50 -TGC AGC TGG CTC AAG TCA TA-30 , Vimentin (forward) 50 -AAG TTT GCT GAC CTC TCT GAG GCT-30 and (reverse) 50 -TTC CAT TTT CAC GCA TCT GGC GTT TC30 , and GAPDH (forward) 50 -GTG GGG CGC CCC AGG CAC CAG GGC30 and (reverse) 50 -CTC CTT AAT GTC ACG CAC GAT TTC-3’. 2.8. Transmission electron microscope For the sub-cellular level examination of hPSCs, cells cultured on the non-crosslinked FDM (Gx0) and crosslinked FDMs (Gx0.5, Gx1.0, Gx2.0), respectively were fixed overnight at 4 C in 2.5% glutaraldehyde/0.1 M PBS and then treated with 1% osmium tetroxide for 2 h. Samples were dehydrated using increased concentrations of ethanol (70%e100%) and infiltrated with a 1:1 mixture of epoxy resin (Polysciences, Inc., Warrington, PA) and propylene oxide for 2 h. Samples were desiccated under a vacuum for 3 h, put in fresh Epoxy resin, and then kept at 60 C overnight for polymerization of the embedding medium. Vertical sections were prepared by thin-sectioning of each sample using an ultramicrotome (Ultracut S;Reichert Technologies, Depew, NY) and observed via transmission electron microscope (TEM; Tecnai F20;
FEI, Hillsboro, OR). 2.9. Quantitative PCR and RT2 profiler PCR array Upon the cultivation of hPSCs on FDM (Gx 0) and X-FDM (Gx 0.5), the expression of 84 genes associated with the epithelialmesenchymal transition (EMT) and focal adhesion (FA) were analyzed via EMT and Focal Adhesion RT2 Profiler PCR Array kit (Qiagen, Hilden, Germany). Target genes were screened using a LightCycler® 96 Real-Time PCR System (F. Hoffmann-La Roche Ltd., Basel, Switzerland) following the manufacturer's instructions. Total RNA was isolated using Invitrogen™ TRIzol™ reagent (Thermo Fisher Scientific) and the RNA concentrations were determined using a spectrophotometer (NanoDrop One; Thermo Fisher Scientific). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Data are representative of the means of two separate experiments. The genes with a fold change more than two were identified amongst the differentially expressed genes. All the data were normalized to the average values of five housekeeping genes, ACTB, B2M, GAPDH, HPRT1, and RPLP0. Data analysis was performed using SA Biosciences and Cluster software (RT2 profiler PCR array data analysis version 3.5, Qiagen). The interactive network during EMT and FA was constructed using Gene Network Central Pro™ (Qiagen). 2.10. Karyotype analysis Chromosome analysis of hPSCs was performed according to the previously described method with slight modifications [15]. Briefly, hPSCs were re-plated and after 3 days, the cells were incubated with 100 ml of colcemid (Thermo Fisher Scientific) for 3 h at 37 C, and then detached using 0.25% trypsin-EDTA. Cells were treated with a hypotonic solution (1% citrate buffer) and the cell lysates were fixed in a fixation solution (ethanol: glacial acetic acid ¼ 3:1). The chromosomes were identified through G-banding and karyotype analysis was performed by a genome diagnosis company (GenDix Inc., Seoul, Korea). 2.11. Teratoma formation and histochemical analysis To confirm the pluripotency of hPSCs cultivated on the crosslinked FDM (Gx0.5), passage 10 hPSCs (3 106) were injected into the dorsal region of 6-week-old, non-obese, diabetic/severe combined immunodeficiency (NOD/SCID) mice (The Jackson Laboratory, Bar Harbor, ME). When the resulting teratomas were removed 12 weeks later, the samples were paraffin-embedded, then serially sectioned (5 mm) using a microtome (Leica Microsystems, Wetzlar, Germany). The three-germ layers were confirmed by histochemical staining via hematoxylin and eosin for the gut epithelium and by some special stains as follows: Periodic acid shiff (PAS) stain for secretory epithelium, Alcian blue stain for cartilage, and Masson's trichrome stain for muscle fibers. Images were analyzed using an inverted microscope (Nikon, Chiyoda-ku, Japan). 2.12. Statistical analysis Data are represented as mean ± standard deviation. Statistical analysis was performed using student t-tests or one-way analysis of variance (ANOVA), with Tukey's post-hoc multiple comparisons (GraphPad Prism 5). Statistically significant difference is marked as *(p < 0.05), **(p < 0.01), or ***(p < 0.001). Histograms of the obtained data were generated using Microsoft Excel 2010 software (Microsoft Corporation, Redmond, WA).
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3. Results
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matrix roughness and stiffness is directly influenced by genipin concentration.
3.1. Preparation and characterization of pristine and altered FDMs FDM was generated by cultivating fibroblasts which were subsequently decellularized through a chemical approach. FDM was further treated with a natural crosslinking agent (genipin) in different concentrations and the resultant FDMs (X-FDMs) with distinct rigidity were prepared accordingly (Fig. 1). Optical images taken before or after the decellularization process showed distinct surface morphologies that suggest the near removal of the cellular components out of confluent cells. Major ECM proteins (fibronectin and laminin) were found in the FDM via immunofluorescence (Fig. 2A). When the amount of ECM proteins in the FDM before or after decellularization was quantified using ELISA, Col I remained unchanged but other ECM components showed reduced levels in comparison to that of native cells (Fig. 2B). In addition, the same experiment using X-FDMs exhibited that FN, LN, Col I, and VN were well conserved even after genipin treatment. The surface texture of both substrate types were highly magnified by SEM which showed self-assembled fine fibrils randomly dispersed across the entire surface irrespective of genipin concentrations (Fig. S1A). The fibrous structure of FDM was also visualized via AFM which revealed topographic differences between each group (Fig. S1B). The pristine FDM (Gx0) showed an average root mean square (RMS) of 135 ± 45 nm but X-FDMs with 0.5, 1, or 2% genipin treatment had increased roughness values of 189 ± 35, 228 ± 27, and 344 ± 35 nm, respectively (Fig. 2C). Furthermore, the Young's modulus (E) measurements found a trend of increasing matrix stiffness in genipin concentrationdependent manner; 118 ± 51 Pa for Gx0 and 800 ± 180, 5600 ± 1100, and 8900 ± 1500 Pa for Gx0.5, Gx1.0, and Gx2.0 respectively (Fig. 2D). There were statistically significant differences among the four groups. The force-displacement curves also present significantly different patterns; the indentation force of Gx0 s gradually increased with displacement of AFM probe, whereas the stiffer matrix (e.g., Gx2.0) exhibited very sharp increase of indentation force at specific points which is indicative of a more rigid surface (Fig. 2E). These results show that genipininduced crosslinking of FDM is effective because the degree of
3.2. Cell adhesion, colony formation, and preservation of hPSCs on FDMs Cell adhesion was examined with FDM and crosslinked FDMs by seeding hPSCs at a cell density of 1 105/cm2 (Fig. S2). Gx0 appeared most favorable for cell adhesion, whereas the number of cells attached to the matrix significantly declined with increasing matrix stiffness (Fig. 3A). When each group was live-stained with alkaline phosphatase (AP) to quantify cell adhesion and to confirm early pluripotency, an inverse relationship was clear between the number of AP-positive colonies and the degree of matrix stiffness. The number of positively stained colonies determined the cell attachment rate, which was 85%, 80%, 55%, and 43% for the Gx0, 0.5, 1.0, and 2.0, respectively (Fig. 3B). As hPSCs grow and expand in defined medium for 5 days, Gx0 favored cell proliferation the most in terms of colony formation (Fig. S3, Supplementary live image 1). This is sharply contrasted with the images of colony size on the XFDMs (Fig. 3C). A fold difference in colony size from day 1e5 showed that Gx0 had the highest growth rate, with a 5.7-fold increase, but that of Gx2.0 had the lowest, with a 2.3-fold increase on day 5 (Fig. 3D). Supplementary video related to this article can be found at https://doi.org/10.1016/j.biomaterials.2017.10.016. To investigate the maintenance potential of hPSCs cultivated on FDM or X-FDMs, AP staining was performed on day 7. Interestingly, the Gx0 group began to show a loss of AP expression as indicated by black arrows, which was less apparent for X-FDMs (Fig. 3E). Although the colony size on Gx0.5 was smaller than that of Gx0, AP expression was maintained for most hPSCs and stagnant growth was not observed as seen in Gx1.0 and Gx2.0 (Fig. 3E, white arrows). Quantification of AP-positive hPSCs revealed that the number of APþ colonies correlated with genipin concentration; AP expression increased to 65%, 85%, 90%, and 95% for the Gx0, 0.5, 1.0, and 2.0 group, respectively (Fig. 3F). In addition, hPSCs on Gx0 began to exhibit not only migratory activity of peripheral cells on day 8 (Fig. S4A, red arrows) but also morphological changes within the colony by day 10 (Fig. S4A, blue arrows). In contrast, cells on Gx0.5
Fig. 1. Schematic of FDM-based platform for hPSCs cultivation in a feeder-free system. Fibroblast-derived extracellular matrix (FDM) is prepared via decellularization of in vitrocultured fibroblasts. The decellularized matrix is subsequently treated using a natural cross-linker, genipin at different concentrations (0.5, 1.0, and 2.0%, w/v), respectively. hPSCs are then seeded and cultivated on the non-crosslinked, natural FDM (Gx0) and crosslinked FDMs, respectively.
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Fig. 2. Characterization of FDM and crosslinked FDMs. (A) ECM components (e.g., fibronectin and laminin) are examined before or after decellularization via immunofluorescence, along with DAPI staining. Scale bars, 50 mm. (B) Various ECM proteins (FN, LN, VN, Col I) from FDM (Gx0) and X-FDMs (Gx0.5, Gx2.0) are quantitatively determined and compared to those of native cells (before decellularization). (C) Average root mean square for roughness measurement by AFM and (D) matrix elasticity (stiffness) as indicated by Young's modulus respectively (at least 20 independent sites probed from n ¼ 5 FDM and X-FDM). (E) Representative of force-displacement curves of each sample. Statistically significant difference is indicated as *(p < 0.05), **(p < 0.01), or ***(p < 0.001).
remained stationary and maintained morphologic consistency. Continuous hPSC cultivation on Gx0.5 lasted until day 14 which suggested that matrix stiffness could significantly extend the maintenance period (Fig. S4B). Even though hPSCs exhibited prolonged maintenance on Gx2.0 (up to day 18), most hPSCs were unable to adhere or slackly attached to Gx2.0 post plating (Fig. S5). Taken together, when accounting for both cell growth and maintenance, Gx0.5 was the most favorable platform for hPSC preservation, expansion and long-term culture (Fig. S6).
3.3. Epithelial-mesenchymal transition analysis by microscopy In order to determine whether FDM stiffness can alter the characteristics of hPSCs, we examined the relative expression levels of four specific genes related to epithelial and mesenchymal properties (OCT4, E-cadherin, N-cadherin, and vimentin) via quantitative PCR on day 7. Both OCT4 and E-cadherin were highly expressed on both Gx0.5 and Gx2.0 but Gx0 showed a downregulation of the pluripotent and epithelial markers. Interestingly mesenchymal markers, N-cadherin and vimentin were significantly upregulated on Gx0 while their expression levels were relatively much weaker on X-FDMs that were similar to those of the control (Fig. 4A). This shift in gene expression was also examined at the cellular level by immunostaining of each group for E-cadherin and DAPI. For Gx0, downregulated E-cadherin but upregulated migratory activities were apparently observed at the marginal areas of the colony (Fig. 4B and C). While the Gx0.5 group presented a slight loss of E-cadherin expression in comparison to Gx2.0, both Gx0.5 and Gx2.0 would maintain localized expression of E-cadherin within the hPSC colony and exhibit little to no migratory activity (Fig. 4B and C). For a more in-depth analysis of cell migration, a
closer inspection of the hPSCs colony was taken by SEM. At the micro level, it was clear that ECM cleavage occurred at the marginal domain of Gx0 (Fig. 4C, white arrows) while it remained intact for both Gx0.5 and 2.0 (Fig. 4C, black arrows). This observation was consistent with the TEM images at the sub-cellular level. The Gx0 group experienced a loss of epithelial barrier function, as evidenced by cell-cell detachment mostly at the margins of the colony (Fig. 4D, white arrows). However the cells on X-FDMs were closely bound to each other via tight junctions in both the marginal and central domain (Fig. 4D, black arrows). These results demonstrate that the pristine FDM induced cell migration and a cadherin switch, whereas X-FDMs preserved E-cadherin expression and colony integration.
3.4. Differential gene expression analysis of the epithelialmesenchymal transition program To further investigate the mechanisms behind the unique hPSC behavior on FDM and X-FDM, qRT-PCR array was conducted to profile 84 genes that either change or regulate the EMT. Differential gene expression of hPSCs was examined on mTeSR™1- Matrigel®, Gx0, and Gx0.5 at 5 day (Fig. S7). A scatter plot analysis of foldchange differences showed that the expression levels were genedependent but the majority of EMT-related genes were upregulated in Gx0 when compared to those of mTeSR™1. This pattern was similar between Gx0 and Gx0.5 but lesser variance and lower degrees of fold-changes were reflected. Interestingly, expression levels remained analogous between mTeSR™1 and Gx0.5 (Fig. 5a). When we analyzed differential gene expression categorized under functional groupings, many regulatory factors that are often involved in EMT induction and cell migration, such as VIM, TGFB,
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Fig. 3. Colony formation and maintenance of hPSCs on various FDMs. (A) AP staining of adherent colonies and (B) Quantitative comparison of cell attachment rate. (C) Morphology of hPSCs colony on day 5. Scale bars, 500 mm. (D) Comparison of colony size with time for up to 5 days as indicated by fold difference. (E) AP staining of hPSCs colony on day 7 shows the sign of differentiation (black arrows) and poor cell growth (white arrows). Scale bars, 1000 mm. (F) Maintenance potential as determined by the ratio between APþ and AP colony. Statistically significant difference is indicated as **(p < 0.01) or ***(p < 0.001).
SLUG (SNAI2), and EGFR were upregulated in Gx0 while potent tumor suppression genes, such as CDH1 and CAV2 were downregulated (Fig. 5B) [16e18]. Furthermore, a list of all genes that exhibited greater than 2-fold difference found that the EMTinducing transcription factor ZEB1 and its paralog ZEB2 were also upregulated in Gx0 as compared to Gx0.5 (Fig. 5C) [19]. In order to better understand gene interactions based on a predetermined list of known associations, an EMT-gene regulatory network was constructed in silico and it showed that ZEB1 was coexpressed either directly or indirectly with other EMT-inducing genes (Fig. 6A). ZEB1 is involved in regulatory roles with respect to TGF-b signaling, which is involved in numerous cellular processes including EMT. In addition, cell migration and the early stage of EMT were also confirmed by immunostaining of ZEB1, E-cadherin, and vimentin for the Gx0 and Gx0.5 group. Gx0 revealed stronger expression of vimentin in the periphery and ZEB1in migrating cells (Fig. 6B, white arrows) as well as weaker expression of E-cadherin. In contrast, Gx0.5 showed sparse ZEB1 (Fig. 6C, white arrows) localization and condensed E-cadherin expression within the colony (Fig. 6B and C). This was consistent with the PCR data, as
the master gene (ZEB1) that drives EMT and SLUG gene that represses E-cadherin transcription were both downregulated in the Gx0.5 group. These results are indicative of substrate-mediated cell migration and EMT induction inGx0, which can be suppressed by increasing FDM stiffness as evidenced by Gx0.5. 3.5. Differential gene expression analysis of focal adhesion Since cell adhesion and signal transduction molecules, such as integrins and protein kinases are major regulators of hPSCs focal adhesion and behavior, we further profiled 84 additional genes involved in focal adhesion. Similar to that of EMT array, the scatter plot highlighted upregulation of the majority of focal adhesionrelated genes in Gx0 with amplified fold-change differences relative to mTeSR™1 (Fig. S8). Differential gene expression analysis revealed that many integrin-related genes including ITGAV as well as its subunits ITGB1 (fibronectin) and ITGB3 (vitronectin) were strongly expressed in Gx0, and genes associated with focal adhesion kinase (FAK) signaling and its downstream target AKT were also upregulated (Fig. S9) [20].
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Fig. 4. Analysis of pluripotent or differentiation genes and colony stability. (A) Differentiation related genes in hPSCs are analyzed via q-PCR as they are cultured on MEF, control, and FDMs, respectively. (B) Immunostaining of E-cadherin shows decreased expression at the marginal region of colony. Scale bars, 100 mm (C) SEM images exhibit significantly different colony pattern, either separated (white arrows) or integrated (black arrows) on specific matrix microenvironment. The area marked in white box is enlarged. Scale bars, 200 mm (D) TEM images at the subcellular level display either loose or stable cell-cell tight junctions at the margin and center of hPSCs colony. Scale bars, 500 nm. Statistically significant difference is indicated as *(p < 0.05), **(p < 0.01), or ***(p < 0.001).
In addition, among the genes that exhibited greater than 2-fold difference, CRK, which is known for its diverse roles in cell motility and differentiation, was identified as strongly expressed in Gx0 (Fig. S10). Although the exact mechanism that governs signal transduction is not fully understood at this time, the focal adhesion-gene regulatory network suggests the dynamics of associated genes for which the cumulative responses contributed to the adhesive and migratory ability of cells on Gx0, which is consistent with our functional assays and EMT analysis. This was further evidenced upon staining prominent focal adhesion markers phalloidin and vinculin, which showed proper anchorage of actin stress fibers to focal adhesions, but only Gx0 exhibited branching focal adhesion behavior (Fig. S11). Current data suggest that Gx0 possesses some signaling cues that are oriented towards cell adhesion, migration, and differentiation, whereas Gx0.5 holds significantly altered signaling outputs with respect to cell adhesion
and motility. 3.6. Characterization of hPSCs long-term cultured on Gx0.5 Among the tested FDM and X-FDMs, Gx0.5 is considered an optimal substratum to cultivate hPSCs; cells passaged up to 5 on Gx0.5 would maintain the typical morphologic feature of undifferentiated hPSCs (Fig. S12). When the expression of key pluripotent markers (OCT4, SSEA4, TRA-1-60, and SOX2) was examined by immunostaining, OCT4 was strongly expressed throughout the colony of passage 5 (Fig. 7A). Quantitative analysis of OCT4 expression showed a gradual decrease up to passage 3, which then reached a plateau at 92%; this was not the case with the Gx0, which exhibited a sharp and continuous decline of OCT4 expression in each serial passage (Fig. 7A). Other markers of pluripotency (SSEA4, TRA-1-60, and SOX2) were also strongly expressed from the hPSCs
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Fig. 5. Comparison of Epithelial-Mesenchymal Transition related genes for hPSCs cultured on FDM. (A) qRT-PCR scatter plot analysis of EMT-related genes of cells cultured on matrigel (mTeSR™1), Gx0, and Gx0.5. (B) Differential gene expression under functional gene groupings and (C) all genes that showed a more than 2-fold difference in expression (Gx0.5 vs Gx0).
colony on Gx0.5 for up to passage 10 (Fig. S13). Immunostaining embryoid body (EB) aggregates for AFP, TuJ1, and PECAM showed that EBs maintained three-germ layer potential (Fig. 7c). Chromosomal analysis also showed that the cells retained a normal karyotype (Fig. 7d). In addition, histochemical analysis of the teratoma
formed after subcutaneous dorsal transplantation evidenced the presence of three-germ layers; endoderm, ectoderm, and mesoderm lineages (Fig. 7e). These results demonstrate that hPSCs cultured on Gx0.5 are capable of preserving their pluripotent capacity. Hence, Gx0.5 may provide some favorable signaling cues
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Fig. 6. ZEB1 induced EMT and loss of E-cadherin expression in migrating cells. (A) In silico construction of EMT-gene regulatory network based on the information collated from Gene Network Pro. (B) Immunostaining of hPSCs for ZEB1 and (C) Vimentin, along with E-cadherin on Gx0 and Gx0.5. (Scale bars, 100 mm).
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Fig. 7. Pluripotency confirmation of hPSCs cultivated on Gx0.5. (A) Immunostaining of nuclear (DAPI) and OCT4 expression on day 35. Scale bars, 500 mm (B) Percentage of OCT4expressing cells in the colony from day 7e35. (C) Embryoid body formation (scale bars, 200 mm) and immunostaining for three-germ layer potential: AFP (endoderm), TuJ1 (ectoderm), and PECAM (mesoderm). Scale bars, 100 mm (D) Karyotype analysis of hPSCs (P10) displaying normal chromosomes. (E) Teratoma formation in vivo 12 weeks posttransplantation and analysis of hPSC pluripotency: gut epithelial (endoderm), neural rossettes (ectoderm), and cartilage (mesoderm). Scale bars, 100 mm Statistically significant difference is indicated as ***(p < 0.001).
(not clear yet) that enables PSCs to retain its adhesive potential but discourages cell migration and subsequent EMT, which is ideal for hPSC self-renewal and expansion. 4. Discussion In this work, we demonstrate that FDM elasticity can affect hPSC plasticity by influencing adhesion, motility, and EMT-related behavior. We further show that Gx0.5 can support robust expansion and long-term (up to 14 days) hPSC culture in a naturally derived microenvironment. The proliferative and pluripotent capacity of hPSCs seem to be significantly influenced by substrate rigidity, favoring a specific elastic modulus as opposed to a soft or rigid matrix. This study indicates that current FDM-based platforms are very advantageous because hPSCs can grow in a homogenous monolayer with high efficiency of cell survival and self-renewal, and are independent of highly specialized, costly culture medium
such as mTeSR™1. Furthermore X-FDMs barely dissociated in the presence of proteolytic enzyme, i.e., Trypsin-EDTA (Fig. S14) and more importantly, FDM is reproducible using human cell sources (Fig. S15). Among the many variables that direct stem cell fate [21], our results indicate that X-FDMs influenced the spatial information and focal adhesion formation of hPSCs which would otherwise undergo migration and differentiation. Rigid environments are able to inhibit the EMT process by restricting motility but it also undermines the adhesive and proliferative capacity of hPSCs. Therefore, modulation should be optimally adjusted to balance this trade-off in achieving robust expansion while retaining focal adhesion for proper colony integration (Fig. 8). Based on the experimental results, it seems that an elastic moduli of 800 ± 200 Pa) is optimal for long-term hPSC cultivation on FDM which is interestingly, close to the range of values reported on Matrigel® via the Hertz contact model [22,23]. Naturally derived biomaterials have shown great promise as it is
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Fig. 8. Schematic illustration of hPSC plasticity when cultivated on tunable matrix microenvironment. The biophysical features of FDM and X-FDMs play a critical role in regulating hPSCs adhesion, proliferation, or maintenance. While the pristine FDM (Gx0) possesses adhesive, proliferative, and EMT-inducing features, the crosslinked FDMs conserve the epithelial and pluripotent characteristics of hPSCs at the expense of growth and expansion.
becoming increasingly evident that topography, adhesion proteins, and extracellular cues from niche environments regulate stem cell behavior in maintaining a balance between quiescence, selfrenewal and differentiation [24,25]. Recent studies continue to implicate that CDMs are capable of modeling a stem cell niche by retaining signaling properties dependent on cell type and maturational stage. For example, ECM deposited by hMSCs accelerated proliferation and enhanced the maintenance of stemness of naïve hMSCs but ECM deposited by osteogenic hMSCs induced differentiation into osteoblasts even in the absence of dexamethasone [26]. The rigidity of the landscape also impacts stem cell plasticity as shown in this study as well as bone marrow derived MSCs, where soft matrices favor neurogenic commitment, while stiffer or rigid matrices favor myogenic or osteogenic commitment, respectively [27]. Taken together, it seems possible to manipulate the extrinsic signals of CDMs through physiochemical means to regulate its affinity and synergy with different types of cells. Since the ability of a stem cell to seed in its niche represents one of the most important features of the niche itself, it is important to elucidate how cell-ECM interactions dictate cellular behavior and how topographic alterations or other physiochemical modifications shape the relationship dynamic at play.
epithelial and pluripotent character of hPSCs at the cost growth and expansion in a concentration-dependent manner. Overall, these findings highlight the independent regulation of hPSC plasticity by the elasticity of a niche-like microenvironment, which can be properly adjusted to prolong cultivation. This could be very useful for the culture of hPSCs in a feeder-free and naturally derived microenvironment as well as basic translational research that explore the complexities of cell-ECM dynamics. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant (No. 2015R1A2A2A04004469) and (No.2015M3A9C7030091) from the Ministry of Science, ICT and Future Planning, and by the grant (715003071HD120) from the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.biomaterials.2017.10.016. Conflict of interest
5. Conclusions We present that the physical features of FDM plays a critical role in regulating pluripotent stem cell adhesion, motility, growth, and differentiation, The pristine FDM possessed adhesive, proliferative, and EMT-inducing features while X-FDMs adamantly conserved the
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Mechanotransduction of human pluripotent stem cells cultivated on tunable cell-derived extracellular matrix In Gul Kim a, 1, Chang-Hyun Gil b, 1, Joseph Seo b, 1, Soon-Jung Park b, Ramesh Subbiah a, Taek-Hee Jung b, Jong Soo Kim b, Young-Hoon Jeong b, Hyung-Min Chung b, Jong Ho Lee a, Man Ryul Lee d, Sung-Hwan Moon b, *, 1, Kwideok Park a, c, **, 1 a
Center for Biomaterials, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea Department of Stem Cell Biology, Konkuk University, School of Medicine, Seoul, 05029, Republic of Korea Biomedical Engineering Major, Korea University of Science and Technology (UST), Daejeon, 34113, Republic of Korea d Soonchunhyang Institute of Medi-bio Science (SIMS), Institute of Tissue Regeneration, College of Medicine, Soon Chun Hyang University, Cheonan, 31151, South Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2017 Accepted 7 October 2017 Available online 9 October 2017
Cell-derived matrices (CDM) are becoming an attractive alternative to conventional biological scaffolding platforms due to its unique ability to closely recapitulate a native extracellular matrix (ECM) de novo. Although cell-substrate interactions are recognized to be principal in regulating stem cell behavior, very few studies have documented the acclimation of human pluripotent stem cells (hPSCs) on pristine and altered cell-derived matrices. Here, we investigate crosslink-induced mechanotransduction of hPSCs cultivated on decellularized fibroblast-derived matrices (FDM) to explore cell adhesion, growth, migration, and pluripotency in various biological landscapes. The results showed either substrate-mediated induction or inhibition of the Epithelial-Mesenchymal-Transition (EMT) program, strongly suggesting that FDM stiffness can be a dominant factor in mediating hPSC plasticity. We further propose an optimal FDM substratum intended for long-term hPSC cultivation in a feeder-free niche-like microenvironment. This study carries significant implications for hPSC cultivation and encourages more in-depth studies towards the fundamentals of hPSC-CDM interactions. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Human pluripotent stem cells (hPSCs) are traditionally cultivated on feeder cells such as mouse embryonic fibroblasts (MEFs) but there has been a progressive move away from the use of feeders because they can introduce xenogeneic contaminants or other undefined factors into the culture systems [1]. For this reason, Matrigel® (Corning, Corning, NY) is one of the most widely used extracellular components for feeder-free culture of hPSCs but it suffers from limited customization and inherent heterogeneity as it is derived from Engelbreth-Holm-Swarm mouse sarcomas which
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (S.-H. Moon), [email protected] (K. Park). 1 These authors contributed equally to this work as first and corresponding authors, respectively. https://doi.org/10.1016/j.biomaterials.2017.10.016 0142-9612/© 2017 Elsevier Ltd. All rights reserved.
may also contain unwanted xenogeneic materials [2]. While recent advancements continue to demonstrate synthetic and biological alternatives ranging from nanopatterns [3], hydrogels [4], and single ECM components such as vitronectin [5], another caveat is that unlike cellular secretions, these platforms are unable to recapitulate a complex, organized, and heterogeneous mixture of macromolecules reminiscent of native microenvironments. The extracellular matrix (ECM) is the non-cellular component of tissues and organs composed of various macromolecules secreted by cells to serve as a natural scaffold with structural integrity and biological cues. While its specific composition and function may vary tremendously depending on the type of particular tissue, it is mainly composed of two interlocking macromolecules known as proteoglycans, such as glycosaminoglycans (GAGs), and fibrous proteins, such as collagen, vitronectin, elastin, fibronectin, and laminin [6]. In essence, it is a highly dynamic structure that is constantly being remodeled and subjected to a myriad of posttranslational modifications as it plays a pivotal role in the
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biophysical and biochemical dialogue between various cells [7]. It possesses a wide range of distinct properties that depend on the inhabitant cells but it is widely known that homeostasis, cell adhesion, cell-to-cell communication, and differentiation are fundamental functions of the ECM [8]. There has been a growing interest in engineering cell-derived matrices (CDM) to create a biomimetic microenvironment that resembles a native niche. Early studies have shown that the biophysical and biochemical cues can be reserved after the decellularization process, which makes the generated matrix a naturally derived culture substratum for stem cell adhesion, proliferation, and differentiation [9,10]. Substrate features are of particular interest because it has a profound impact on stem cell lineage specification. For example, substrate stiffness is an essential characteristic by which cells sense the external forces and subsequently respond to the environment in an appropriate manner. Our previous study demonstrated the effects of mechanotransduction on the fate of human mesenchymal stem cells (hMSCs) and recent reports continue to highlight the importance of cell-ECM interactions in regulating stem cell fate [11,12]. However, even though hPSCs possess enormous potential in regenerative therapy, little attention has been paid to some fundamental aspects of hPSC-CDM interactions partly due to the lack of proper tools. Hence, we prepared a natural ECM analog that is engineered de novo from in vitro cultured fibroblasts to explore the acclimation of hPSCs cultivated on pristine and altered decellularized fibroblastderived matrices (FDM). Mechanotransduction was conducted via non-cytotoxic crosslinking to modulate FDM rigidity to various degrees. Both substrate types were utilized to investigate cell adhesion, growth, migration, and pluripotency. This study shows that simple yet effective physiological controls can dictate hPSCECM dynamics in a closely recapitulated natural environment. Based on our findings, we further propose an optimal FDM platform intended for feeder-free and long-term hPSC cultivation (up to 14 days).
2. Materials and methods 2.1. Preparation of fibroblast-derived extracellular matrix (FDM) To prepare FDM, NIH3T3 mouse fibroblasts (ATCC, Manassas, VA) or human foreskin BJ1 fibroblasts (ATCC) were seeded at the density of 2 104 cells/cm2 on either plastic dishes or gelatincoated glass in 12-well plates, and cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin streptomycin (P/S; Thermo Fisher Scientific). After 1 week, confluent cells were washed twice with phosphate buffered saline (PBS; Thermo Fisher Scientific), then decellularized by incubation with a detergent solution containing 0.25% Triton X-100 and 10 mM NH4OH (SigmaAldrich Co., St. Louis, MO) in PBS at 37 C. The samples were then treated with 50 U/ml DNase I and 2.5 mL/ml RNase A (Thermo Fisher Scientific) for 2 h at 37 C. The decellularized samples were gently washed with PBS three times and the resulting FDM was used immediately or stored at 4 C prior to use. Major FDM components are identified by immunofluorescence of mouse anti-fibronectin (FN; Santa Cruz Biotechnology, Inc., Dallas, Texas) and mouse anti-laminin (LN; Santa Cruz Biotechnology, Inc.). Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) and Alexa Fluor 594-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Inc., West Grove, PA) were used as a secondary antibody.
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2.2. Preparation of crosslinked FDMs The prepared FDM was then treated with different amounts of genipin (GN; Wako Chemicals USA, Richmond, VA) to induce a crosslinking of the FDM. Briefly, a stock solution of 5% (w/v) GN was prepared in dimethyl sulfoxide (DMSO) and diluted to different concentrations (0.5, 1, and 2%) with PBS. GN solution was added to FDM, and the samples were incubated at room temperature for 4 h to allow the crosslinking of FDM itself. The crosslinked FDM (XFDM) was then washed five times with PBS and stored at 4 C before use. The X-FDM was named as follows: Gx0.5, Gx1.0 and Gx2.0, respectively. Gx0 represents the non-crosslinked or pristine FDM. 2.3. Quantitative analysis of ECM proteins To quantify the ECM constituents of the FDM and X-FDMs, fibronectin (FN), laminin (LN), collagen type I (Col I), and vitronectin (VN) were selected. Each sample in 6-well plates was incubated overnight with the following primary antibodies: mouse anti-FN (Santa Cruz Biotechnology, Inc.), mouse anti-LN (Santa Cruz Biotechnology, Inc.), rabbit anti-Col I (Abcam, Cambridge, UK), and rabbit anti-VN (Santa Cruz Biotechnology, Inc.), respectively and followed by the treatment of goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (HRP; Santa Cruz Biotechnology, Inc.). After several washing, the samples were added with 1 ml of 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) and incubated for 20 min. The reaction was stopped using 0.16 M sulfuric acid, after which 100 mL aliquots were taken for the absorbance measurements at 405 nm (Multiskan Microplate Reader, Thermo Fisher Scientific). For the comparison, the fluorometric readouts are converted to the protein levels that are normalized to the surface area (9.6 cm2) of one well per each sample. 2.4. Characterization of FDM and X-FDMs: SEM and AFM The surface morphology of FDM and X-FDM was observed using a scanning electron microscope (SEM; Model S-3000 N, Hitachi). Bio-atomic force microscope (Bio-AFM; NanoWizard II, JPK Instruments, Germany) equipped with an inverted optical microscope (Nikon) was also used in a liquid contact mode to investigate the surface topography and morphology of FDM and X-FDM, respectively. In addition, the AFM-nano indentation technique was employed to measure the Young's modulus (E) of each sample, as described previously [13]. A 10 mm diameter SiO2 particle attached to PT.SiO2.AU.SN10 cantilevers (Novascan Technologies, Ames, USA) with a spring constant of 0.01 N/m was utilized. Based on the force spectra curves, E was calculated using Hertz's contact model in JPK data processing software (v3.3.25). The Poisson ratio of cells was set to 0.5. Approximately 5 indentations on each sample (n ¼ 3, each group) at different regions were carried out for E measurement. 2.5. Cell culture Various cell types are used: human pluripotent stem cell line; H9 (WiCell Research Institute, Madison, WI) [14], mouse embryonic fibroblasts (MEFs), and primary MEFs (Orient Bio Inc., Seongnam, Korea). H9-hPSCs were cultured in 80% DMEM: Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, Waltham, MA) supplemented with 20% KnockOut™ Serum Replacement, 1% P/S (Thermo Fisher Scientific), 1% Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA; Thermo Fisher Scientific), 0.1% beta-mercaptoethanol (b-ME;
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Thermo Fisher Scientific and 4 ng/ml recombinant human basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ). hPSCs culture media was changed daily and cultures were passaged every 5e7 days. The primary CF1 MEFs were treated with Mitomycin C (MMC; M-MEF; Sigma-Aldrich Co.) and served as a feeder layer for hPSCs. M-MEFs were cultured in 90% high-glucose DMEM (Thermo Fisher Scientific supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 1% P/S, 1% MEM-NEAA, and 0.1% b-ME. The cell culture media was changed every 2 days. For subculture of hPSCs onto FDM and X-FDM, hPSCs were treated with Dispase® (Thermo Fisher Scientific) or mechanically dissected with a glass Pasteur pipette (Corning). All the cells were cultivated in a thermostat incubator at 37 C under 5% CO2. 2.6. Alkaline phosphatase staining and immunocytochemistry To confirm alkaline phosphatase (AP) expression in the hPSC colonies, AP staining was performed with an ES Cell Characterization Kit (EMD Millipore, Billerica, MA). For immunostaining, the cells were fixed for 20 min at room temperature in 4% paraformaldehyde (PFA) and permeabilized with 0.03% Triton X-100 in PBS for 5 min. After the treatment with 5% normal goat serum for 30 min, they were incubated overnight at 4 C with primary antibodies; OCT4, SOX2, SSEA-4, and TRA-1-60 (EMD Millipore). Alexa Fluor® 488, 555 or 594 secondary antibodies (Molecular Probes Inc., Sunnyvale, CA) were used to visualize the hPSCs-specific markers. Cell nuclei were stained with DAPI (DAKO, Carpentaria, CA). All images were acquired using a fluorescence microscope (Nikon, Chiyoda-ku, Japan). 2.7. Quantitative polymerase chain reaction (qPCR) For analysis of messenger ribonucleic acid (mRNA) expression, total RNA was extracted using TRIzol® Reagent (Thermo Fisher Scientific). The quantity and purity of extracted RNA were examined via a NanoDrop® ND-1000 spectrophotometer (NanoDrop Products, Wilmington, DE). Total RNA (1 mg) was reversetranscribed into complementary deoxyribonucleic acid (cDNA) using a Maxim RT premix kit (iNtRon Biotechnology, Sungnam, Korea). The target genes and associated primers are as follows: OCT4 (forward) 50 -AAC TCG AGC AAT TTG CCA AGC TCC-30 and (reverse) 50 -TTC GGG CAC TGC AGG AAC AAA TTC-30 , E-cadherin (forward) 50 -CGA GAG CTA CAC GTT CAC GG-30 and (reverse) 50 GGG TGT CGA GGG AAA AAT AGG-30 , N-cadherin (forward) 50 -TCA CAG ATT CGG GTA ATC CTC-30 and (reverse) 50 -TGC AGC TGG CTC AAG TCA TA-30 , Vimentin (forward) 50 -AAG TTT GCT GAC CTC TCT GAG GCT-30 and (reverse) 50 -TTC CAT TTT CAC GCA TCT GGC GTT TC30 , and GAPDH (forward) 50 -GTG GGG CGC CCC AGG CAC CAG GGC30 and (reverse) 50 -CTC CTT AAT GTC ACG CAC GAT TTC-3’. 2.8. Transmission electron microscope For the sub-cellular level examination of hPSCs, cells cultured on the non-crosslinked FDM (Gx0) and crosslinked FDMs (Gx0.5, Gx1.0, Gx2.0), respectively were fixed overnight at 4 C in 2.5% glutaraldehyde/0.1 M PBS and then treated with 1% osmium tetroxide for 2 h. Samples were dehydrated using increased concentrations of ethanol (70%e100%) and infiltrated with a 1:1 mixture of epoxy resin (Polysciences, Inc., Warrington, PA) and propylene oxide for 2 h. Samples were desiccated under a vacuum for 3 h, put in fresh Epoxy resin, and then kept at 60 C overnight for polymerization of the embedding medium. Vertical sections were prepared by thin-sectioning of each sample using an ultramicrotome (Ultracut S;Reichert Technologies, Depew, NY) and observed via transmission electron microscope (TEM; Tecnai F20;
FEI, Hillsboro, OR). 2.9. Quantitative PCR and RT2 profiler PCR array Upon the cultivation of hPSCs on FDM (Gx 0) and X-FDM (Gx 0.5), the expression of 84 genes associated with the epithelialmesenchymal transition (EMT) and focal adhesion (FA) were analyzed via EMT and Focal Adhesion RT2 Profiler PCR Array kit (Qiagen, Hilden, Germany). Target genes were screened using a LightCycler® 96 Real-Time PCR System (F. Hoffmann-La Roche Ltd., Basel, Switzerland) following the manufacturer's instructions. Total RNA was isolated using Invitrogen™ TRIzol™ reagent (Thermo Fisher Scientific) and the RNA concentrations were determined using a spectrophotometer (NanoDrop One; Thermo Fisher Scientific). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Data are representative of the means of two separate experiments. The genes with a fold change more than two were identified amongst the differentially expressed genes. All the data were normalized to the average values of five housekeeping genes, ACTB, B2M, GAPDH, HPRT1, and RPLP0. Data analysis was performed using SA Biosciences and Cluster software (RT2 profiler PCR array data analysis version 3.5, Qiagen). The interactive network during EMT and FA was constructed using Gene Network Central Pro™ (Qiagen). 2.10. Karyotype analysis Chromosome analysis of hPSCs was performed according to the previously described method with slight modifications [15]. Briefly, hPSCs were re-plated and after 3 days, the cells were incubated with 100 ml of colcemid (Thermo Fisher Scientific) for 3 h at 37 C, and then detached using 0.25% trypsin-EDTA. Cells were treated with a hypotonic solution (1% citrate buffer) and the cell lysates were fixed in a fixation solution (ethanol: glacial acetic acid ¼ 3:1). The chromosomes were identified through G-banding and karyotype analysis was performed by a genome diagnosis company (GenDix Inc., Seoul, Korea). 2.11. Teratoma formation and histochemical analysis To confirm the pluripotency of hPSCs cultivated on the crosslinked FDM (Gx0.5), passage 10 hPSCs (3 106) were injected into the dorsal region of 6-week-old, non-obese, diabetic/severe combined immunodeficiency (NOD/SCID) mice (The Jackson Laboratory, Bar Harbor, ME). When the resulting teratomas were removed 12 weeks later, the samples were paraffin-embedded, then serially sectioned (5 mm) using a microtome (Leica Microsystems, Wetzlar, Germany). The three-germ layers were confirmed by histochemical staining via hematoxylin and eosin for the gut epithelium and by some special stains as follows: Periodic acid shiff (PAS) stain for secretory epithelium, Alcian blue stain for cartilage, and Masson's trichrome stain for muscle fibers. Images were analyzed using an inverted microscope (Nikon, Chiyoda-ku, Japan). 2.12. Statistical analysis Data are represented as mean ± standard deviation. Statistical analysis was performed using student t-tests or one-way analysis of variance (ANOVA), with Tukey's post-hoc multiple comparisons (GraphPad Prism 5). Statistically significant difference is marked as *(p < 0.05), **(p < 0.01), or ***(p < 0.001). Histograms of the obtained data were generated using Microsoft Excel 2010 software (Microsoft Corporation, Redmond, WA).
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3. Results
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matrix roughness and stiffness is directly influenced by genipin concentration.
3.1. Preparation and characterization of pristine and altered FDMs FDM was generated by cultivating fibroblasts which were subsequently decellularized through a chemical approach. FDM was further treated with a natural crosslinking agent (genipin) in different concentrations and the resultant FDMs (X-FDMs) with distinct rigidity were prepared accordingly (Fig. 1). Optical images taken before or after the decellularization process showed distinct surface morphologies that suggest the near removal of the cellular components out of confluent cells. Major ECM proteins (fibronectin and laminin) were found in the FDM via immunofluorescence (Fig. 2A). When the amount of ECM proteins in the FDM before or after decellularization was quantified using ELISA, Col I remained unchanged but other ECM components showed reduced levels in comparison to that of native cells (Fig. 2B). In addition, the same experiment using X-FDMs exhibited that FN, LN, Col I, and VN were well conserved even after genipin treatment. The surface texture of both substrate types were highly magnified by SEM which showed self-assembled fine fibrils randomly dispersed across the entire surface irrespective of genipin concentrations (Fig. S1A). The fibrous structure of FDM was also visualized via AFM which revealed topographic differences between each group (Fig. S1B). The pristine FDM (Gx0) showed an average root mean square (RMS) of 135 ± 45 nm but X-FDMs with 0.5, 1, or 2% genipin treatment had increased roughness values of 189 ± 35, 228 ± 27, and 344 ± 35 nm, respectively (Fig. 2C). Furthermore, the Young's modulus (E) measurements found a trend of increasing matrix stiffness in genipin concentrationdependent manner; 118 ± 51 Pa for Gx0 and 800 ± 180, 5600 ± 1100, and 8900 ± 1500 Pa for Gx0.5, Gx1.0, and Gx2.0 respectively (Fig. 2D). There were statistically significant differences among the four groups. The force-displacement curves also present significantly different patterns; the indentation force of Gx0 s gradually increased with displacement of AFM probe, whereas the stiffer matrix (e.g., Gx2.0) exhibited very sharp increase of indentation force at specific points which is indicative of a more rigid surface (Fig. 2E). These results show that genipininduced crosslinking of FDM is effective because the degree of
3.2. Cell adhesion, colony formation, and preservation of hPSCs on FDMs Cell adhesion was examined with FDM and crosslinked FDMs by seeding hPSCs at a cell density of 1 105/cm2 (Fig. S2). Gx0 appeared most favorable for cell adhesion, whereas the number of cells attached to the matrix significantly declined with increasing matrix stiffness (Fig. 3A). When each group was live-stained with alkaline phosphatase (AP) to quantify cell adhesion and to confirm early pluripotency, an inverse relationship was clear between the number of AP-positive colonies and the degree of matrix stiffness. The number of positively stained colonies determined the cell attachment rate, which was 85%, 80%, 55%, and 43% for the Gx0, 0.5, 1.0, and 2.0, respectively (Fig. 3B). As hPSCs grow and expand in defined medium for 5 days, Gx0 favored cell proliferation the most in terms of colony formation (Fig. S3, Supplementary live image 1). This is sharply contrasted with the images of colony size on the XFDMs (Fig. 3C). A fold difference in colony size from day 1e5 showed that Gx0 had the highest growth rate, with a 5.7-fold increase, but that of Gx2.0 had the lowest, with a 2.3-fold increase on day 5 (Fig. 3D). Supplementary video related to this article can be found at https://doi.org/10.1016/j.biomaterials.2017.10.016. To investigate the maintenance potential of hPSCs cultivated on FDM or X-FDMs, AP staining was performed on day 7. Interestingly, the Gx0 group began to show a loss of AP expression as indicated by black arrows, which was less apparent for X-FDMs (Fig. 3E). Although the colony size on Gx0.5 was smaller than that of Gx0, AP expression was maintained for most hPSCs and stagnant growth was not observed as seen in Gx1.0 and Gx2.0 (Fig. 3E, white arrows). Quantification of AP-positive hPSCs revealed that the number of APþ colonies correlated with genipin concentration; AP expression increased to 65%, 85%, 90%, and 95% for the Gx0, 0.5, 1.0, and 2.0 group, respectively (Fig. 3F). In addition, hPSCs on Gx0 began to exhibit not only migratory activity of peripheral cells on day 8 (Fig. S4A, red arrows) but also morphological changes within the colony by day 10 (Fig. S4A, blue arrows). In contrast, cells on Gx0.5
Fig. 1. Schematic of FDM-based platform for hPSCs cultivation in a feeder-free system. Fibroblast-derived extracellular matrix (FDM) is prepared via decellularization of in vitrocultured fibroblasts. The decellularized matrix is subsequently treated using a natural cross-linker, genipin at different concentrations (0.5, 1.0, and 2.0%, w/v), respectively. hPSCs are then seeded and cultivated on the non-crosslinked, natural FDM (Gx0) and crosslinked FDMs, respectively.
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Fig. 2. Characterization of FDM and crosslinked FDMs. (A) ECM components (e.g., fibronectin and laminin) are examined before or after decellularization via immunofluorescence, along with DAPI staining. Scale bars, 50 mm. (B) Various ECM proteins (FN, LN, VN, Col I) from FDM (Gx0) and X-FDMs (Gx0.5, Gx2.0) are quantitatively determined and compared to those of native cells (before decellularization). (C) Average root mean square for roughness measurement by AFM and (D) matrix elasticity (stiffness) as indicated by Young's modulus respectively (at least 20 independent sites probed from n ¼ 5 FDM and X-FDM). (E) Representative of force-displacement curves of each sample. Statistically significant difference is indicated as *(p < 0.05), **(p < 0.01), or ***(p < 0.001).
remained stationary and maintained morphologic consistency. Continuous hPSC cultivation on Gx0.5 lasted until day 14 which suggested that matrix stiffness could significantly extend the maintenance period (Fig. S4B). Even though hPSCs exhibited prolonged maintenance on Gx2.0 (up to day 18), most hPSCs were unable to adhere or slackly attached to Gx2.0 post plating (Fig. S5). Taken together, when accounting for both cell growth and maintenance, Gx0.5 was the most favorable platform for hPSC preservation, expansion and long-term culture (Fig. S6).
3.3. Epithelial-mesenchymal transition analysis by microscopy In order to determine whether FDM stiffness can alter the characteristics of hPSCs, we examined the relative expression levels of four specific genes related to epithelial and mesenchymal properties (OCT4, E-cadherin, N-cadherin, and vimentin) via quantitative PCR on day 7. Both OCT4 and E-cadherin were highly expressed on both Gx0.5 and Gx2.0 but Gx0 showed a downregulation of the pluripotent and epithelial markers. Interestingly mesenchymal markers, N-cadherin and vimentin were significantly upregulated on Gx0 while their expression levels were relatively much weaker on X-FDMs that were similar to those of the control (Fig. 4A). This shift in gene expression was also examined at the cellular level by immunostaining of each group for E-cadherin and DAPI. For Gx0, downregulated E-cadherin but upregulated migratory activities were apparently observed at the marginal areas of the colony (Fig. 4B and C). While the Gx0.5 group presented a slight loss of E-cadherin expression in comparison to Gx2.0, both Gx0.5 and Gx2.0 would maintain localized expression of E-cadherin within the hPSC colony and exhibit little to no migratory activity (Fig. 4B and C). For a more in-depth analysis of cell migration, a
closer inspection of the hPSCs colony was taken by SEM. At the micro level, it was clear that ECM cleavage occurred at the marginal domain of Gx0 (Fig. 4C, white arrows) while it remained intact for both Gx0.5 and 2.0 (Fig. 4C, black arrows). This observation was consistent with the TEM images at the sub-cellular level. The Gx0 group experienced a loss of epithelial barrier function, as evidenced by cell-cell detachment mostly at the margins of the colony (Fig. 4D, white arrows). However the cells on X-FDMs were closely bound to each other via tight junctions in both the marginal and central domain (Fig. 4D, black arrows). These results demonstrate that the pristine FDM induced cell migration and a cadherin switch, whereas X-FDMs preserved E-cadherin expression and colony integration.
3.4. Differential gene expression analysis of the epithelialmesenchymal transition program To further investigate the mechanisms behind the unique hPSC behavior on FDM and X-FDM, qRT-PCR array was conducted to profile 84 genes that either change or regulate the EMT. Differential gene expression of hPSCs was examined on mTeSR™1- Matrigel®, Gx0, and Gx0.5 at 5 day (Fig. S7). A scatter plot analysis of foldchange differences showed that the expression levels were genedependent but the majority of EMT-related genes were upregulated in Gx0 when compared to those of mTeSR™1. This pattern was similar between Gx0 and Gx0.5 but lesser variance and lower degrees of fold-changes were reflected. Interestingly, expression levels remained analogous between mTeSR™1 and Gx0.5 (Fig. 5a). When we analyzed differential gene expression categorized under functional groupings, many regulatory factors that are often involved in EMT induction and cell migration, such as VIM, TGFB,
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Fig. 3. Colony formation and maintenance of hPSCs on various FDMs. (A) AP staining of adherent colonies and (B) Quantitative comparison of cell attachment rate. (C) Morphology of hPSCs colony on day 5. Scale bars, 500 mm. (D) Comparison of colony size with time for up to 5 days as indicated by fold difference. (E) AP staining of hPSCs colony on day 7 shows the sign of differentiation (black arrows) and poor cell growth (white arrows). Scale bars, 1000 mm. (F) Maintenance potential as determined by the ratio between APþ and AP colony. Statistically significant difference is indicated as **(p < 0.01) or ***(p < 0.001).
SLUG (SNAI2), and EGFR were upregulated in Gx0 while potent tumor suppression genes, such as CDH1 and CAV2 were downregulated (Fig. 5B) [16e18]. Furthermore, a list of all genes that exhibited greater than 2-fold difference found that the EMTinducing transcription factor ZEB1 and its paralog ZEB2 were also upregulated in Gx0 as compared to Gx0.5 (Fig. 5C) [19]. In order to better understand gene interactions based on a predetermined list of known associations, an EMT-gene regulatory network was constructed in silico and it showed that ZEB1 was coexpressed either directly or indirectly with other EMT-inducing genes (Fig. 6A). ZEB1 is involved in regulatory roles with respect to TGF-b signaling, which is involved in numerous cellular processes including EMT. In addition, cell migration and the early stage of EMT were also confirmed by immunostaining of ZEB1, E-cadherin, and vimentin for the Gx0 and Gx0.5 group. Gx0 revealed stronger expression of vimentin in the periphery and ZEB1in migrating cells (Fig. 6B, white arrows) as well as weaker expression of E-cadherin. In contrast, Gx0.5 showed sparse ZEB1 (Fig. 6C, white arrows) localization and condensed E-cadherin expression within the colony (Fig. 6B and C). This was consistent with the PCR data, as
the master gene (ZEB1) that drives EMT and SLUG gene that represses E-cadherin transcription were both downregulated in the Gx0.5 group. These results are indicative of substrate-mediated cell migration and EMT induction inGx0, which can be suppressed by increasing FDM stiffness as evidenced by Gx0.5. 3.5. Differential gene expression analysis of focal adhesion Since cell adhesion and signal transduction molecules, such as integrins and protein kinases are major regulators of hPSCs focal adhesion and behavior, we further profiled 84 additional genes involved in focal adhesion. Similar to that of EMT array, the scatter plot highlighted upregulation of the majority of focal adhesionrelated genes in Gx0 with amplified fold-change differences relative to mTeSR™1 (Fig. S8). Differential gene expression analysis revealed that many integrin-related genes including ITGAV as well as its subunits ITGB1 (fibronectin) and ITGB3 (vitronectin) were strongly expressed in Gx0, and genes associated with focal adhesion kinase (FAK) signaling and its downstream target AKT were also upregulated (Fig. S9) [20].
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Fig. 4. Analysis of pluripotent or differentiation genes and colony stability. (A) Differentiation related genes in hPSCs are analyzed via q-PCR as they are cultured on MEF, control, and FDMs, respectively. (B) Immunostaining of E-cadherin shows decreased expression at the marginal region of colony. Scale bars, 100 mm (C) SEM images exhibit significantly different colony pattern, either separated (white arrows) or integrated (black arrows) on specific matrix microenvironment. The area marked in white box is enlarged. Scale bars, 200 mm (D) TEM images at the subcellular level display either loose or stable cell-cell tight junctions at the margin and center of hPSCs colony. Scale bars, 500 nm. Statistically significant difference is indicated as *(p < 0.05), **(p < 0.01), or ***(p < 0.001).
In addition, among the genes that exhibited greater than 2-fold difference, CRK, which is known for its diverse roles in cell motility and differentiation, was identified as strongly expressed in Gx0 (Fig. S10). Although the exact mechanism that governs signal transduction is not fully understood at this time, the focal adhesion-gene regulatory network suggests the dynamics of associated genes for which the cumulative responses contributed to the adhesive and migratory ability of cells on Gx0, which is consistent with our functional assays and EMT analysis. This was further evidenced upon staining prominent focal adhesion markers phalloidin and vinculin, which showed proper anchorage of actin stress fibers to focal adhesions, but only Gx0 exhibited branching focal adhesion behavior (Fig. S11). Current data suggest that Gx0 possesses some signaling cues that are oriented towards cell adhesion, migration, and differentiation, whereas Gx0.5 holds significantly altered signaling outputs with respect to cell adhesion
and motility. 3.6. Characterization of hPSCs long-term cultured on Gx0.5 Among the tested FDM and X-FDMs, Gx0.5 is considered an optimal substratum to cultivate hPSCs; cells passaged up to 5 on Gx0.5 would maintain the typical morphologic feature of undifferentiated hPSCs (Fig. S12). When the expression of key pluripotent markers (OCT4, SSEA4, TRA-1-60, and SOX2) was examined by immunostaining, OCT4 was strongly expressed throughout the colony of passage 5 (Fig. 7A). Quantitative analysis of OCT4 expression showed a gradual decrease up to passage 3, which then reached a plateau at 92%; this was not the case with the Gx0, which exhibited a sharp and continuous decline of OCT4 expression in each serial passage (Fig. 7A). Other markers of pluripotency (SSEA4, TRA-1-60, and SOX2) were also strongly expressed from the hPSCs
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Fig. 5. Comparison of Epithelial-Mesenchymal Transition related genes for hPSCs cultured on FDM. (A) qRT-PCR scatter plot analysis of EMT-related genes of cells cultured on matrigel (mTeSR™1), Gx0, and Gx0.5. (B) Differential gene expression under functional gene groupings and (C) all genes that showed a more than 2-fold difference in expression (Gx0.5 vs Gx0).
colony on Gx0.5 for up to passage 10 (Fig. S13). Immunostaining embryoid body (EB) aggregates for AFP, TuJ1, and PECAM showed that EBs maintained three-germ layer potential (Fig. 7c). Chromosomal analysis also showed that the cells retained a normal karyotype (Fig. 7d). In addition, histochemical analysis of the teratoma
formed after subcutaneous dorsal transplantation evidenced the presence of three-germ layers; endoderm, ectoderm, and mesoderm lineages (Fig. 7e). These results demonstrate that hPSCs cultured on Gx0.5 are capable of preserving their pluripotent capacity. Hence, Gx0.5 may provide some favorable signaling cues
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Fig. 6. ZEB1 induced EMT and loss of E-cadherin expression in migrating cells. (A) In silico construction of EMT-gene regulatory network based on the information collated from Gene Network Pro. (B) Immunostaining of hPSCs for ZEB1 and (C) Vimentin, along with E-cadherin on Gx0 and Gx0.5. (Scale bars, 100 mm).
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Fig. 7. Pluripotency confirmation of hPSCs cultivated on Gx0.5. (A) Immunostaining of nuclear (DAPI) and OCT4 expression on day 35. Scale bars, 500 mm (B) Percentage of OCT4expressing cells in the colony from day 7e35. (C) Embryoid body formation (scale bars, 200 mm) and immunostaining for three-germ layer potential: AFP (endoderm), TuJ1 (ectoderm), and PECAM (mesoderm). Scale bars, 100 mm (D) Karyotype analysis of hPSCs (P10) displaying normal chromosomes. (E) Teratoma formation in vivo 12 weeks posttransplantation and analysis of hPSC pluripotency: gut epithelial (endoderm), neural rossettes (ectoderm), and cartilage (mesoderm). Scale bars, 100 mm Statistically significant difference is indicated as ***(p < 0.001).
(not clear yet) that enables PSCs to retain its adhesive potential but discourages cell migration and subsequent EMT, which is ideal for hPSC self-renewal and expansion. 4. Discussion In this work, we demonstrate that FDM elasticity can affect hPSC plasticity by influencing adhesion, motility, and EMT-related behavior. We further show that Gx0.5 can support robust expansion and long-term (up to 14 days) hPSC culture in a naturally derived microenvironment. The proliferative and pluripotent capacity of hPSCs seem to be significantly influenced by substrate rigidity, favoring a specific elastic modulus as opposed to a soft or rigid matrix. This study indicates that current FDM-based platforms are very advantageous because hPSCs can grow in a homogenous monolayer with high efficiency of cell survival and self-renewal, and are independent of highly specialized, costly culture medium
such as mTeSR™1. Furthermore X-FDMs barely dissociated in the presence of proteolytic enzyme, i.e., Trypsin-EDTA (Fig. S14) and more importantly, FDM is reproducible using human cell sources (Fig. S15). Among the many variables that direct stem cell fate [21], our results indicate that X-FDMs influenced the spatial information and focal adhesion formation of hPSCs which would otherwise undergo migration and differentiation. Rigid environments are able to inhibit the EMT process by restricting motility but it also undermines the adhesive and proliferative capacity of hPSCs. Therefore, modulation should be optimally adjusted to balance this trade-off in achieving robust expansion while retaining focal adhesion for proper colony integration (Fig. 8). Based on the experimental results, it seems that an elastic moduli of 800 ± 200 Pa) is optimal for long-term hPSC cultivation on FDM which is interestingly, close to the range of values reported on Matrigel® via the Hertz contact model [22,23]. Naturally derived biomaterials have shown great promise as it is
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Fig. 8. Schematic illustration of hPSC plasticity when cultivated on tunable matrix microenvironment. The biophysical features of FDM and X-FDMs play a critical role in regulating hPSCs adhesion, proliferation, or maintenance. While the pristine FDM (Gx0) possesses adhesive, proliferative, and EMT-inducing features, the crosslinked FDMs conserve the epithelial and pluripotent characteristics of hPSCs at the expense of growth and expansion.
becoming increasingly evident that topography, adhesion proteins, and extracellular cues from niche environments regulate stem cell behavior in maintaining a balance between quiescence, selfrenewal and differentiation [24,25]. Recent studies continue to implicate that CDMs are capable of modeling a stem cell niche by retaining signaling properties dependent on cell type and maturational stage. For example, ECM deposited by hMSCs accelerated proliferation and enhanced the maintenance of stemness of naïve hMSCs but ECM deposited by osteogenic hMSCs induced differentiation into osteoblasts even in the absence of dexamethasone [26]. The rigidity of the landscape also impacts stem cell plasticity as shown in this study as well as bone marrow derived MSCs, where soft matrices favor neurogenic commitment, while stiffer or rigid matrices favor myogenic or osteogenic commitment, respectively [27]. Taken together, it seems possible to manipulate the extrinsic signals of CDMs through physiochemical means to regulate its affinity and synergy with different types of cells. Since the ability of a stem cell to seed in its niche represents one of the most important features of the niche itself, it is important to elucidate how cell-ECM interactions dictate cellular behavior and how topographic alterations or other physiochemical modifications shape the relationship dynamic at play.
epithelial and pluripotent character of hPSCs at the cost growth and expansion in a concentration-dependent manner. Overall, these findings highlight the independent regulation of hPSC plasticity by the elasticity of a niche-like microenvironment, which can be properly adjusted to prolong cultivation. This could be very useful for the culture of hPSCs in a feeder-free and naturally derived microenvironment as well as basic translational research that explore the complexities of cell-ECM dynamics. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant (No. 2015R1A2A2A04004469) and (No.2015M3A9C7030091) from the Ministry of Science, ICT and Future Planning, and by the grant (715003071HD120) from the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.biomaterials.2017.10.016. Conflict of interest
5. Conclusions We present that the physical features of FDM plays a critical role in regulating pluripotent stem cell adhesion, motility, growth, and differentiation, The pristine FDM possessed adhesive, proliferative, and EMT-inducing features while X-FDMs adamantly conserved the
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