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. 2022 Feb 22;74(2):293–307. doi: 10.1007/s10616-022-00529-z

Surface coating materials regulates the attachment and differentiation of mouse embryonic stem cell derived embryoid bodies into mesoderm at culture conditions

Derya Sağraç 1, Selinay Şenkal 1, Taha Bartu Hayal 1, Fikrettin Şahin 1, Zehra Çobandede 1, Ayşegül Doğan 1,✉
PMCID: PMC8976036  PMID: 35464166

Abstract

Abstract

Pluripotent stem cells as a promising cell source with unlimited proliferation and differentiation capacity hold great promise for cell-based therapies in regenerative medicine. Establishment of appropriate culture conditions might enable the control of cellular fate decision in cell culture. Transfer of three-dimensional (3D) embryoid bodies to two-dimensional (2D) monolayer culture systems for initiation of cell differentiation and specialization requires an adaptation of cells which can be managed by extracellular matrix (ECM) materials. Here we compare the characteristics of four different cell culture coating materials and their effect on attachment and differentiation of cells spreading from mouse embryonic stem cell (mESC) derived embryoid bodies (EBs) in mesoderm inducing culture conditions. Atomic force microscope (AFM) and scanning electron microscope (SEM) analysis along with Water Contact Angle technique were used to analyze physical properties of ECM materials and to evaluate cellular behavior on surfaces. Cell migration and differentiation were performed initially by using mesoderm inducing culture conditions and then three germ layer specification conditions. We investigated properties of coating materials such as roughness and wettability control cell attachment, migration and differentiation of mESCs. Matrigel-Gelatin combination is suitable for cell attachment and migration of cells spreading from 3D EBs followed by transfer onto coated surfaces. Matrigel-Gelatin coating enhanced differentiation of cells into mesoderm like cells via EMT process. Our data demonstrated that the Matrigel-Gelatin combination as a cell culture coating matrix might serve as a suitable platform to transfer EBs for differentiation and might influence pluripotent stem cell fate decision into mesoderm and further mesoderm derivative cell populations.

Graphical abstract

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Supplementary Information

The online version contains supplementary material available at 10.1007/s10616-022-00529-z.

Keywords: Mouse embryonic stem cells, Extracellular matrix, Matrigel, Laminin, Gelatin, Mesoderm

Introduction

Cellular proliferation, growth, morphological changes, migration and rearrangement occur during development at various stages in sequential order or in combination based on the status of the embryo (Cooper 2000). Determination of germ layers during gastrulation and cellular differentiation are critical for establishment of future body axis and organogenesis. Cellular movements and Epithelial-Mesenchymal Transition (EMT) process occur during gastrulation to form mesoderm from pluripotent epithelial epiblast cells (Acloque et al. 2009) which then specialize into axial, paraxial, intermediate and lateral mesoderm. Formation of mesoderm and subsequent organogenesis including the heart is one of the best examples of cell migration and differentiation by EMT (Kim et al. 2018). Similar processes such as cell migration, invasion and EMT are observed at ex vivo cell culture conditions during ES cell differentiation (Eastham et al. 2007; Kim et al. 2014).

Pluripotent stem cell-based regenerative medicine has been of interest since the discovery of embryonic stem cells. Establishment of appropriate culture conditions, which mimic embryonic development, is required to control embryonic stem cell differentiation in vitro and to obtain adequate cell sources for research or therapy (Valamehr et al. 2011). Extracellular matrix materials are crucial to generate the appropriate environment to simulate the actual tissue support for cellular attachment and differentiation. Although pluripotent stem cell differentiation begins with the formation of embryoid bodies as 3D cellular aggregates, derivation of a desired cell type by differentiation is conducted by using 2D monolayer culture systems. Transfer of EBs to adherent culture systems followed by supplementation of growth factors and small molecules is one of the major strategies to induce cellular differentiation (Valamehr et al. 2008).

Various kinds of matrix materials including Gelatin (Tamm et al. 2013; Li et al. 2009), Fibronectin (Lam et al. 2012), Vitronectin (Chen et al. 2011; Prowse et al. 2010), Laminin (Taelman et al. 2019) and Matrigel have been used for not only pluripotent stem cells but also different types of adult stem cells and cell lines in vitro. Selection of appropriate extracellular matrix material (ECM) for coating might facilitate cell attachment and enable the differentiation of cells into the desired lineage.

In the current study, we aimed to identify an appropriate coating material for in vitro differentiation of mouse ES cells. We selected a medium composition to direct cells into mesodermal lineages to trigger EMT. We characterized and compared the four different extracellular matrix coating (Matrigel, Gelatin, Matrigel-Gelatin Mixture, Laminin) for surface properties, cellular attachment, migration and differentiation. Matrigel-Gelatin mixture enhanced cell attachment, migration and induced EMT which directs cells into mesoderm differentiation. Matrigel-Gelatin mixture might be used as an efficient, safe and inexpensive technique for mesoderm-derived cell differentiation for future therapies.

Materials and methods

Cell culture

Mouse embryonic stem cells (R1/E ATCC, SCRC-1036) were cultured on Mitomycin C treated mouse embryonic fibroblast (MEF) by using DMEM medium (4.5 g/L glucose) supplemented with 15% ES cell qualified fetal bovine serum (FBS, Gibco), 1% l-Glutamine (100X, Gibco), 1% penicillin–streptomycin- amphotericin (PSA, Gibco), 1% non-essential amino acid (NEAA 100X, Gibco) and 10 ng/ml leukemia inhibitor factor (LIF, Abm, Richmond, USA). Growth medium was replaced every day to grow mESC cell colonies. mESC culture and MEF isolation was conducted as described previously by our group (Şişli et al. 2021). The animal experiments and procedure were approved by the Yeditepe University Animal Experiments Ethics Committee (Approval number/date: 714/, 21.12.2018). Animals were housed at a constant temperature (23 ± 1 °C) and humidity (60 ± 10%), maintained at a 12 h light/dark cycle and fed with food and water ad libitum.

Preparation of coating materials

Four different coating materials were used to coat tissue culture plastics prior to culture. Laminin-521 (STEMCELL Technologies, Vancouver, Canada) was diluted in serum-free medium as final concentration of 10 µg/ml and incubated 1 h at 37 °C for coating. Matrigel (ESC qualified, Corning, USA) was prepared in serum-free medium as final concentration of 0.5 mg/6well and incubated 1 h at room temperature for coating (Hayal et al. 2021). Gelatin powder (Bioshop, USA) was dissolved in distilled water as 1% (w/v) and autoclaved for further use. Gelatin solution was diluted in PBS as 0.1% (w/v) and incubated 1 h at room temperature for coating. Matrigel-Gelatin mixture (1:200) was prepared with the dilution of Matrigel in 0.1% (w/v) Gelatin solution as a ratio of 1:200 (Matrigel:Gelatin) and incubated 1 h at room temperature for coating. Coated tissue culture well plates were immediately used for cell culture experiments.

Determination of optimum cell number for embryoid body (EB) formation

mESCs were detached and seperated from MEFs as described previously by our group (Şişli et al. 2021). Plastic polyprene V bottom wells of PCR plates (BioRad, California, USA) were used to generate EBs. Four different number of cells (1000–2000–3000–4000 cells/well of PCR plate) were used to optimize EB formation. Cells were mixed with N2B27 medium supplemented with 12 ng/ml bFGF (Peprotech, UK) and 10 µM ROCK Inhibitor (Y-27632, STEMCELL Technologies, Vancouver, Canada). Cells were placed inside the wells of PCR plates with a multichannel pipetor. The plate was sealed and centrifuged at 200×g for 3 min to spin down the cells. Cells were incubated in a humidified incubator at 37 °C for 2 days (Fig. 1A). EBs were visualized under a light microscope equipped with AxioCam camera system (Axio Vert.A1; Zeiss, Heidelberg, Germany). The area and diameter of EBs were measured by Image J (NIH, Bethesda, USA) (Schneider et al. 2012).

Fig. 1.

Fig. 1

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Determination of appropriate cell number for mESC-derived EB generation. A The experimental protocol of epiblast-like cell differentiation for EB generation. B Light microscope images of EBs. C Area and diameter of EBs and spreading cells. D Gene expression analysis of SOX-2, T/Bra and SOX-17. E Gene expression analysis of OCT4, NANOG, c-Myc. *P < 0.05, Scale bar = 100 µm

Transfer of EBs from to 2D culture conditions and cell migration analysis

Optimum number of cells was determined as 4000 cells/well. EBs, generated in N2B27 medium after 2 days incubation, were transferred to surfaces coated with Laminin, Matrigel, Gelatin, and Matrigel-Gelatin mixture. EBs were cultured in N2B27 medium supplemented with 3 µM CHIR99021 to direct cells into mesoderm like cell differentiation for another 4 days. Migratory phenotype was analyzed as described in Fig. 3. Adherence and migration patterns were analyzed on images taken by a light microscope equipped with AxioCam camera system (Axio Vert.A1; Zeiss, Heidelberg, Germany). The area of EBs were measured by Image J (NIH, Bethesda, USA) (Schneider et al. 2012). Samples at different times points were collected for further analysis.

Fig. 3.

Fig. 3

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Cell migration analysis of EBs on coated wells. A Timeline and medium composition of differentiation protocol to obtain mesoderm-like cells. B Light microscopy analysis of cell migration (Scale bar = 100 µm). C Illustration showing the calculated regions for cell migration. D Cell outgrowth area of the migration zone. *P < 0.05, Scale bar = 100 µm

Scanning electron microscope (SEM) analysis

EBs attached to the coated surfaced were analysed by SEM at day 6. Samples were fixed with 2% glutheraldehyde prepared in 0.1 M sodium cacodylate buffer at 4 °C for 1 h. Fixed samples were washed with PBS and dehydrated in alcohol series (60% to 100%). Dried samples were coated with gold-platinum (Au/Pt) with a 7.5 nm thickness via BALTEC SCD005 sputter and then SEM images were taken using Zeiss Evo40 (Zeiss, Heidelberg, Germany). Images were analyzed by “Interactive 3D Surface Plot “ plugin of Image J and plots were prepared to show cell-surface interactions (NIH, Bethesda, USA) (Schneider et al. 2012).

Atomic force microscope (AFM) analysis

Tissue Culture Plastic (TCP) glass slides were coated with aforementioned coating materials (Matrigel, Gelatin, Matrigel-Gelatin Mixture, Laminin). After coating, the slides were washed twice with deionized water and dried at room temperature. Uncoated TCP glass slides were used as control. Sample surface topographies were analyzed using XE-100 AFM system (Park Systems, Korea) equipped with NSC36B silicon cantilever on contact mode. Scan fields of 40 × 40 µm2 on all surfaces were processed using the XEI software (1.18).

Water contact angle (WCA) assay

The wettability of coated surfaces was investigated using the water contact angle assay as mentioned before (Aliuos et al. 2014). Briefly, the TCP glass slides were incubated for 1 h at 37 °C in a humidified atmosphere. Surface tension was analyzed by using a Hamilton syringe and a KSV CAM 101 surface tension meter (KSV Instruments Ltd., Finland). Contact angle (theta angle, Θ) of the water droplet with the surface was measured at 3rd second. If Θ is less than 90°, surfaces are considered as hydrophilic.

Determination of actin rearrangement

filamentous Actin (F-Actin) immunocytochemistry analysis were performed to observe actin rearrangement of cells on coated surfaces. Cells at day 6 were fixed with 4% PFA for 30 min at 4 °C and permeabilized with Triton-X-100 for 5 min at room temperature. Cells were washed with PBST and incubated with Phalloidin-FITC (Abcam, UK) for 40 min. The staining of Actin cytoskeleton structure was analyzed by Confocal microscope (LSM900; Zeiss, Germany). Intensity of protein expression was measured by ZEN 3.2 Blue software (Zeiss, Germany).

Trilineage differentiation

Embryoid bodies were transferred onto Matrigel-Gelatin coated surfaces at day 2 to initiate three germ layer differentiation. Endoderm, mesoderm and ectoderm differentiation media were directly applied to the cells starting from day 2. Medium components are listed in Supplementary Table 1. Differentiation protocol is illustrated in Fig. 7. At the end of the differentiation, samples were collected for quantitative PCR and immunocytochemistry experiments for characterization of differentiated cells.

Fig. 7.

Fig. 7

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Three germ layer differentiation of cells on Matrigel-Gelatin mixture coated wells. A Timeline and medium composition of three germ layer differentiation protocol. B Morphological analysis of differentiated cells (Scale bar = 100 µm). C FOXA2, α-SMA, NURR1 gene expression analysis Definitive endoderm marker, Foxa2 was upregulated in differentiated group. D SOX-17, α-SMA, MAP2 protein expression analysis (Scale bar = 50 µm). *P < 0.05

Quantitative real-time PCR

Primers for epithelial to mesenchymal transition and pluripotency related pathways were selected from PrimerBank (Harvard, USA) and β-Actin, Cdh1, Cdh2, ZEB1/2, Fibronectin, Col1, Snail, Slug, Twist, TGF-β, β-Catenin, MMP2, MMP9, VEGF, Nurr1, Mixl-1, Flk-1 and FOXA2, Akt, WNT3, PI3K, JNK were designed using Primer-BLAST software (NCBI, Bethesda, Maryland, USA) and synthesized by Sentegen (Ankara, Turkey) (Supplementary Table 2). The β-Actin gene was used as the housekeeping gene. Total RNA from migrated cells at Day 3 and Day 6 were isolated by TRI reagent (Sigma-Aldrich, USA) according to the manufacturer’s instructions. cDNA was synthesized by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, California, USA). SYBR Green (Applied Biosystems, Thermo Scientific, USA) qPCR method was used as described previously (Şişli et al. 2020). CFX96 RT-PCR system (Bio-Rad, USA) was used in all RT-PCR experiments.

Immunocytochemistry analysis

Fixed cells were incubated with pluripotency and EMT marker antibodies (Supplementary Table 3). Cells were incubated for 16 h at 4 °C with the primary antibodies diluted in 1% BSA-PBS at a 1:200 ratio. The next day, cells were incubated with Alexa Fluor-488 goat anti-mouse or Alexa Fluor-594 goat anti-rabbit secondary antibodies (Invitrogen, USA). Cells were observed using a Confocal microscope (LSM900; Zeiss, Germany). Intensities were analyzed by ZEN 3.2 Blue software (Zeiss, Germany).

Gelatin zymography

Gelatin zymography was conducted according to the literature (Toth et al. 2012). Briefly, supernatants were collected from experimental groups and concentrated with Amicon 10 kDa centrifugal filter tubes (Merck, USA) by centrifugation in 4000×g for 20 min at room temperature. Protein concentration of samples was then quantified by BCA assay according to manufacturer instructions (Thermo, UK). 30 µg/ml of total protein was loaded in each well of hand-casted 7.5% gelatin-SDS PAGE gels. Protein bands were separated at 150 V for 1 h. The gel was soaked in incubation buffer overnight. The next day, gel was stained with 0.5% Coomassie Blue R-250 for 30 min at room temperature. Imaging was performed with ChemiDoc Imaging System (BioRad, USA).

Statistical analysis

Experiments were conducted as triplicates (3 replicates in each experiment) and statistical analyses were performed using Kruskal–Wallis test (as a non-parametric method) post hoc Dunn's multiple comparisons test. P values less than 0.05 were considered statistically significant. All statistical analysis and Correlation matrix analysis were performed using the GraphPad Prism version 9.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com”. Scatter plots were generated by an online tool ClustVis (https://biit.cs.ut.ee/clustvis/) (Metsalu et al. 2015).

Results

Cell number is important for embryoid body (EB) formation and gene expression

Embryoid bodies were generated by two days of bFGF treatment which induces the epiblast like stage in mESC differentiation protocol (Fig. 1A). mESCs derived EBs were generated by using four different cell number per EB (1 × 103–2 × 103–3 × 103–4 × 103 cells/well). Embryoid bodies were formed as single EBs/well of the 96 well PCR plates for the all experimental groups (Fig. 1B). Total area and diameter of EBs were increased in a cell concentration dependent manner (Fig. 1C). There was a positive correlation between EB size and cell number as expected. SOX-2, T/Bra and SOX-17 gene expressions were analyzed to determine ectoderm, mesoderm and endoderm germ layer specification in EBs. SOX-2 expression was enhanced in a cell number dependent manner compared to 1 × 103 cells/well group and significant increase was observed in 4 × 103cells/well group. On the contrary SOX-17 expression was decreased while cell number to generate EBs increased. T/Bra gene expression was similar for 1 × 103 and 4 × 103cells/well groups and showed an insignificant decrease in 2 × 103 and 3 × 103 cells/well groups (Fig. 1D). OCT4, NANOG and c-MYC gene expression levels were detected to confirm decrement of pluripotency. OCT4 and NANOG gene expression were reduced when size of the EB increased and c-MYC expression was enhanced insignificantly (Fig. 1E). EBs were formed with 4 × 103cells/well for further experiments.

Physical properties of ECM materials are important for cell attachment and differentiation

Roughness as an important factor for cell adhesion and movement was determined by AFM. Laminin, Matrigel-Gelatin, and Gelatin had high roughness values, 14.68 µm, 9.6 µm, and 11.61 µm respectively indicating the favorable environment for cell migration (Fig. 2A). Laminin, Matrigel-Gelatin, and Gelatin demonstrated insignificant higher roughness values compared to TCP. Hydrophilic and hydrophobic characteristics of the ECM materials were evaluated by WCA analysis. All coating materials can be considered as hydrophilic with a contact angle (Θ) less than 90°. Matrigel, Laminin, and Matrigel-Gelatin coated surfaces were more hydrophobic than TCP and Gelatin coated surfaces (Fig. 2B). Contact angle of Laminin and Matrigel were 63.7° ± 4.3° and 72.5° ± 0.9° respectively which were highly hydrophobic compared to other groups (Fig. 2B). Contact angle of Gelatin and Matrigel-Gelatin was similar to TCP.

Fig. 2.

Fig. 2

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Surface Characteristics of ECM Materials. A AFM analysis to measure roughness of the coated surfaces. B WCA analysis of the coated surfaces *P < 0.05, Scale bar = 40 µm

Cell spreading and migration of EBs were depended on coating of cell culture surfaces with ECM material

EBs were transferred onto well plates coated with Laminin, Matrigel, Gelatin and Matrigel-Gelatin mixture at day 2. Cell spreading and migration were monitored until day 6 in medium supplemented with 3 µM CHIR99021 to induce a mesoderm like cellular differentiation (Fig. 3A). EBs were completely attached to the surface one day after transfer (at day 3) and started to spread and migrate outwards. Cell migration was analyzed at day 3 and day 6. Spreading cell morphology was different between groups. Migrated cells were more cubic in the Laminin coated group, proliferated rapidly and spread to the surrounding area. Cells were more spindle-like with sharp edges in Matrigel, Gelatin and Matrigel-Gelatin mixture coated groups. EB attachment was not strong and cell migration was reduced in Gelatin coated group compared to other experimental groups (Fig. 3B). Cell migration was measured by analysis of the migration zone on light microscope images (Fig. 3C) and the migration zone was represented as outgrowth area (Fig. 3D). The outgrowth area as an indicator of cell migration rate showed that Laminin, Matrigel and Matrigel-Gelatin mixture coated wells facilitated cell migration compared to Gelatin group. There was not a significant change between Laminin, Matrigel and Matrigel-Gelatin groups (Fig. 3D).

The SEM analyses were used to understand the cellular shape and attachment to confirm light microscopy analysis (Fig. 4A). Results were similar to light microscope analysis. Gelatin coating did not favor cell spreading and movement. EB attachment was weak which made the cell migration difficult. Laminin, Matrigel, Matrigel-Gelatin facilitated cell migration. Cells spreading from the mESC derived EBs were more cubic and flatter in the Laminin coated group and had a spindle morphology in the Matrigel and Matrigel-Gelatin groups. It was noticed that the morphology of cells on Laminin coated surfaces was very superficial and closely adhere to the surface. In contrast, cells on the Matrigel, Gelatin and Matrigel-Gelatin coated surfaces showed fibroblast-like morphology. In addition, cells on Gelatin coated surfaces tended to detach from the surface (Fig. 4A). Cell density and attachment on coated well plates were analyzed by “Interactive 3D Surface Plot” to show the cell arrangement, dispersion and movement based on ECM coating (Supplementary Fig. 1). Three-dimensional (3D) interactive plots demonstrated the difference for cellular attachment. The color scale as an indicator of cell existence and density at zone and migration regions showed that Matrigel and Matrigel-Gelatin mixture coating favors cell proliferation and movement (Supplementary Fig. 1).

Fig. 4.

Fig. 4

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Cell attachment and movement on coated wells. A SEM analysis of cell attachment and migration on coated surfaces. B F-actin polymerization of cells cultured on coated wells. Arrowhead indicates branched F-actin proteins. Scale bar = 50 µm

Differences related to cell attachment and migration which depend on coating materials were further identified through F-Actin staining which is the marker of cell migration (Fig. 4B). Pericentric nucleus location, lamella, and lamellipodia were clearly seen in all groups except for Gelatin coated surfaces. The cells were spread out from the center of EBs and showed flat shape morphology on Laminin, Matrigel and Matrigel-Gelatin coated surfaces. Matrigel-Gelatin coating enhanced the cell attachment and induced a more branched-like lamellipodia which indicated the strong attachment to the surface (Fig. 4B).

ECM materials regulate the cellular differentiation of mECS derived EBs via EMT process

Pluripotency and differentiation related markers were detected by gene and protein expression analyses to observe cellular behavior on Laminin, Matrigel, Gelatin and Matrigel-Gelatin mixture coated surfaces. OCT4 and KLF4 gene expression were reduced significantly in all coated groups compared to regular tissue culture well plates (tissue culture plastic-TCP). Gelatin and Laminin coating induced NANOG expression in EBs at day 6. c-Myc expression enhanced in coated groups excluding EBs placed on Matrigel-Gelatin mixture coated surfaces (Fig. 5A). SOX-2, T/Bra and SOX-17 as early differentiation markers of three germ layers were detected by qPCR and immunocytochemistry analysis (Fig. 5B–D). Although SOX2 and T/Bra gene expression were reduced in EBs placed onto Laminin, Matrigel, Gelatin and Matrigel-Gelatin mixture coated well plates compared to TCP, this change was not significant. SOX-17 expression were reduced in Laminin and Gelatin groups but enhanced in Matrigel and Matrigel-Gelatin groups (Fig. 5B). Similar results were obtained in the protein expression analysis (Fig. 5C, D). Although SOX-2 and T/Bra expressions were not very prominent, SOX-17 expression was distinct with a markedly expression at the spreading cell population (Fig. 5C) and enhanced in EBs placed onto Laminin, Matrigel and Matrigel-Gelatin coated wells (Fig. 5D). SOX-2 and T/Bra expression were observed at the spreading cells and edge regions of the EB in Laminin, Matrigel and Matrigel-Gelatin groups while their expression was only observed in the core of EB in Gelatin coated group (Fig. 5C). Because we induce the mesoderm-like cell differentiation in our experimental system to observe the effects of four different ECM materials, we checked the EMT markers by gene and protein expression levels. Overall, EBs at day 6 showed an increase in gene expression of the selected panel during 4 days of mesoderm inducing differentiation protocol which was initiated by transfer of EBs at day 2 followed by their attachment to the ECM materials at day 3 (Fig. 5E). Gene expression profile of cells at day 3 and day 6 clustered together as expected which was related to the gene regulatory networks of pluripotent stem cells during differentiation protocol. Matrigel-Gelatin mixture coating enhanced the EMT genes such as MMP2, MMP2 TGFβ1 and ZEB-1/2 indicating the mesoderm fate decision (Fig. 5E). Matrigel and Matrigel-Gelatin were clustered together which was confirmed by correlation matrix analysis (Fig. 5F). Correlation coefficient values clearly showed that the gene expression profile of day 3 and day 6 EBs were completely different. Although cell attachment and migration results were completely different for Matrigel-Gelatin and Gelatin, the gene expression profile had a similar pattern for EMT markers. However, the same situation was not observed in protein expression analysis. Interestingly, α-SMA, TWIST, SLUG, CATENIN, SNAIL were significantly upregulated in Gelatin coating group which was not observed in protein expression analysis (Fig. 6).

Fig. 5.

Fig. 5

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Molecular characteristics of cells cultured on coated wells. A Gene expression analysis of OCT4, KLF4, NANOG, c-Myc. B Gene expression and C Immunocytochemistry analysis of SOX-2, T/Bra and SOX-17. D Quantification of protein expression intensities from staining analysis. E Heat map representation and Cluster analysis of EMT marker gene panel. F Correlation coefficient analysis of EMT marker gene panel of day 3 and day 6. *P < 0.05, Scale bar = 50 µm

Fig. 6.

Fig. 6

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EMT marker protein expression analysis. A Immunocytochemistry analysis of EMT marker proteins panel. B Heat map representation and Cluster analysis of EMT marker proteins C Correlation coefficient analysis of EMT marker proteins at day 6. D Gelatin zymography analysis of pro-MMP2 and pro-MMP9 activity E MMP2 and MMP9 gene expression levels of cells cultured on coated surfaces. *P < 0.05, Scale bar = 50 µm

According to the protein expression and localization analysis of selected EMT proteins, Matrigel-Gelatin and Laminin coating groups clustered together (Fig. 6A, B). Mesenchymal morphology markers such as α-SMA, Slug, Snail, and CDH-2 (N-Cadherin) were elevated and epithelial markers, CDH-1 (E-Cadherin), Mucin-1 were reduced in EBs located onto Matrigel-Gelatin coated wells compared to other ECM materials indicating the EMT (Fig. 6A, B). Correlation matrix analysis showed that Matrigel, Matrigel-Gelatin and Laminin had a similar EMT protein marker expression profile (Fig. 6C).

Gelatin Zymography was conducted to demonstrate MMP enzyme activity as an indicator of EMT process. Results demonstrated that Matrigel coating enhanced pro-MMP-2 activity compared to TCP and other ECM materials. pro-MMP-9 increased in all experimental groups compared to TCP indicating the EMT on coated surfaces (Fig. 6D). In addition, Matrigel-Gelatin mixture coating enhanced MMP-2 and MMP-9 gene expression of EBs at day 6 compared to other groups suggesting the potential of this ECM coating for mesoderm cell differentiation (Fig. 6E).

In addition, Akt, PI3K, WNT and JNK gene expression was upregulated in Matrigel-Gelatin mixture coating group indicating the activation of Planar cell polarity pathway via JNK and some of the important mediators such as Akt and PI3K (Supplementary Fig. 2). Akt and JNK were significantly upregulated in Matrigel-Gelatin coating group.

Matrigel-Gelatin coated surface can be used for endoderm, mesoderm and ectoderm differentiation

We observed cell attachment and movement by directing cells into mesoderm-like phenotype via CHIR99021. After selection of Matrigel-Gelatin as a coating material which enhanced cell attachment, migration and triggered an EMT process, we induced three germ layer differentiations on EBs transferred onto coated well plates. We used a more specific mesoderm differentiation medium, ectoderm and endoderm differentiation media to start germ layer specification (Fig. 7A). Cell morphology changed depending on endoderm, mesoderm and ectoderm inducing conditions (Fig. 7B). FOXA2, α-SMA, NURR1 as a markers endoderm, mesoderm and ectoderm respectively were upregulated at the end of the differentiation experiments (Fig. 7C). SOX17, α-SMA, MAP2 immunocytochemistry analysis showed a successful endoderm, mesoderm and ectoderm differentiation (Fig. 7D).

Discussion

Pluripotent stem cell culture has been improved over the years since the discovery of embryonic stem cells and induced pluripotent stem cells. Determination of appropriate culture conditions are not only important for keeping pluripotency but also required for cell differentiation, modelling of embryonic development and diseases in culture. ECM materials are crucial to model differentiation of ES cells. As differentiation of ES cells generally starts with the formation of 3D EBs and then their transfer to monolayer culture systems, we analyzed the four different ECM materials for their physical properties, cell attachment and differentiation potential.

The size of the EBs and gene expression profile changed based on the cell number which was used to generate EBs. It has been reported in the literature that the size of the EBs affect the differentiation potential of both human (Mohr et al. 2010) and mouse (Messana et al. 2008) ES cells. Increasing the EB size definitely reduces pluripotency related genes and induced neurogenic SOX-2 significantly. Although c-MYC expression is related to pluripotency of ES cells (Varlakhanova et al. 2010), increasing the cell number to generate EB resulted in an insignificant increase in the c-MYC gene level. This might be related to the activity of c-MYC in cell proliferation which was shown in a study by Melnik et al. using mesenchymal stromal cells (Melnik et al. 2019). In addition, suppression of SOX-17 which is an endoderm marker might be related to high expression of c-Myc. It has been shown that c-MYC suppressed primitive endoderm by repressing GATA6 (Smith et al. 2010).

Roughness as an adhesion force measurement (Marrese et al. 2018) was analyzed via AFM and the roughness of the surface was positively correlated with cell migration. Matrigel-Gelatin demonstrated the highest roughness value and positively regulated cell adhesion and migration of cells spreading from mESC derived EBs. It was previously shown that increasing the surface roughness enhanced cellular attachment of osteosarcoma cells (Li et al. 2012). Although the relation between cell attachment and surface roughness or hydrophobicity might be a cell dependent manner, mESC derived EBs attached to the rough and hydrophilic Matrigel-Gelatin coated surface easily in the current study. Cell attachment and movement morphology were different on coated surfaces. Laminin coating induced a flatter and more cubic-like cell morphology in the presence of CHIR99021, and Matrigel-based coatings induced a more spindle like morphology which is a characteristic of mesoderm and mesenchymal cells.

Cellular attachment and migration might be explained with the actin filament reorganization pattern which is essential for cytoskeletal reconstruction and cell movement (Svitkina 2018). During EMT, cytoskeletal structure alters in favor of the leading edge by forming filopodia and lamelliopodia which facilitate movement of cells on the surface (Ji et al. 2020). F-actin expression and placement showed that Matrigel-Gelatin coating might be used as a favorable ECM material for cellular attachment and differentiation.

While pluripotency genes downregulate during differentiation, expression of EMT genes and proteins enhanced in CHIR99021 containing medium. Matrigel-Gelatin exerted a close correlation with Matrigel and Laminin in gene and protein expression analysis respectively. EMT is an important step during early stages of pluripotent stem cells differentiation which is characterized by a series of morphological and molecular events (Eastham et al. 2007) such as formation of a migratory phenotype by cytoskeleton rearrangement. Activation of some of the transcription factors such as ZEB1/2 (Lamouille et al. 2014) and downregulation of CDH1 (Acloque et al. 2009) are initial molecular events observed during EMT (Aban et al. 2021). We also observed the EMT-like gene and protein expression profile in our experiments specifically in Matrigel-Gelatin mixture coated wells. High expression of WNT and JNK indicated the activation of cell polarity pathway in Matrigel-Gelatin group which is in consistent with the PI3K and Akt expression. Both PI3K and Akt genes are related to the attachment dependent survival pathway (Shiojima et al. 2002).

Although Gelatin alone did not favor cell attachment and movement, combination with quite a small amount of Matrigel became an ideal environment for pluripotent stem cell differentiation. This might be related to the combination of roughness provided by Gelatin and attachment provided by Matrigel in culture. Matrigel-Gelatin mixture was not only important for initiation of EMT in culture conditions which triggered mesoderm-like cell differentiation but also acted as a suitable environment for ectoderm and endoderm cell transformation in our study.

In conclusion, Matrigel-Gelatin mixture might be used for pluripotent stem cell differentiation protocols as an alternative option. Protocols involving the transfer of 3D EBs to 2D monolayer culture for mesoderm and further mesenchymal stem cell differentiation can use Matrigel-Gelatin as a coating material. Our results are promising for further studies and generation of mesenchymal stem cells for cell therapy applications.

Supplementary Information

Below is the link to the electronic supplementary material.

10616_2022_529_MOESM1_ESM.docx (15.5KB, docx)

Supplementary file1 (DOCX 15 kb) Supplementary Table 1: Differentiation Medium Composition.

10616_2022_529_MOESM2_ESM.docx (14.4KB, docx)

Supplementary file2 (DOCX 17 kb) Supplementary Table 2: Primers used in qPCR experiment.

10616_2022_529_MOESM3_ESM.docx (14.7KB, docx)

Supplementary file3 (DOCX 14 kb) Supplementary Table 3: Antibodies used in immunocytochemistry experiments.

10616_2022_529_MOESM4_ESM.tif (37.6MB, tif)

Supplementary file4 (TIF 38508 kb) Supplementary Figure 1: Interactive 3D Surface Plot analysis of SEM images. Core zone and Migration zone were marked with yellow lines. Surface plot analyses are shown as x-y-z direction and x-y direction 3D color maps.

10616_2022_529_MOESM5_ESM.tif (963.6KB, tif)

Supplementary file5 (TIF 963 kb) Supplementary Figure 2: Gene expression levels of Akt, PI3K, WNT and JNK. *P<0.05

Acknowledgements

This study was funded by TÜBİTAK 2232 International Fellowship for Outstanding Researchers Program (Project No: 118C186). This study was supported by Yeditepe University. We would like to thank Hatice Burcu Şişli for her help during MEF isolation and culture. We would like to thank Cem Levent Altan for the help during WCA measurements. Authors declare no conflict of interest. We would like to thank Yeditepe University Writing Center and Hakan Şentürk for their proofreading support.

Author contributions

AD conducted the study conception and design. Material preparation, data collection and analysis were performed by DS, SS, TBH and FS. AFM and SEM analysis were conducted by ZC. The first draft of the manuscript was written by DS and AD and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by TÜBİTAK 2232 International Fellowship for Outstanding Researchers Program (Project No: 118C186).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

10616_2022_529_MOESM1_ESM.docx (15.5KB, docx)

Supplementary file1 (DOCX 15 kb) Supplementary Table 1: Differentiation Medium Composition.

10616_2022_529_MOESM2_ESM.docx (14.4KB, docx)

Supplementary file2 (DOCX 17 kb) Supplementary Table 2: Primers used in qPCR experiment.

10616_2022_529_MOESM3_ESM.docx (14.7KB, docx)

Supplementary file3 (DOCX 14 kb) Supplementary Table 3: Antibodies used in immunocytochemistry experiments.

10616_2022_529_MOESM4_ESM.tif (37.6MB, tif)

Supplementary file4 (TIF 38508 kb) Supplementary Figure 1: Interactive 3D Surface Plot analysis of SEM images. Core zone and Migration zone were marked with yellow lines. Surface plot analyses are shown as x-y-z direction and x-y direction 3D color maps.

10616_2022_529_MOESM5_ESM.tif (963.6KB, tif)

Supplementary file5 (TIF 963 kb) Supplementary Figure 2: Gene expression levels of Akt, PI3K, WNT and JNK. *P<0.05

Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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