Inducing endoderm differentiation by modulating mechanical properties of soft substrates

Authors

  • Maria Jaramillo,

    1. Bioengineering Department, University of Pittsburgh, Pittsburgh, PA, USA
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  • Satish S. Singh,

    1. Chemical and Petroleum Engineering Department, University of Pittsburgh, Pittsburgh, PA, USA
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  • Sachin Velankar,

    1. Chemical and Petroleum Engineering Department, University of Pittsburgh, Pittsburgh, PA, USA
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  • Prashant N. Kumta,

    1. Bioengineering Department, University of Pittsburgh, Pittsburgh, PA, USA
    2. Chemical and Petroleum Engineering Department, University of Pittsburgh, Pittsburgh, PA, USA
    3. Mechanical Engineering and Material Science Department, University of Pittsburgh, Pittsburgh, PA, USA
    4. Department of Oral Biology, Swanson School of Engineering, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA, USA
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  • Ipsita Banerjee

    Corresponding author
    1. Bioengineering Department, University of Pittsburgh, Pittsburgh, PA, USA
    2. Chemical and Petroleum Engineering Department, University of Pittsburgh, Pittsburgh, PA, USA
    • Correspondence to: Ipsita Banerjee, PhD, 1249 Benedum hall, University of Pittsburgh, Pittsburgh, PA, USA. E-mail: Ipb1@pitt.edu

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Abstract

Early embryonic stem cell (ESC) differentiation is marked by the formation of three germ layers from which all tissues types arise. Conventionally, ESCs are differentiated by altering their chemical microenvironment. Recently however, it was established that a mechanical microenvironment can also contribute towards cellular phenotype commitment. In this study, we report how the cellular mechanical microenvironment of soft substrates affects the differentiation and phenotypic commitment of ESCs. Mouse ESCs were cultured in a fibrin hydrogel matrix in 2D and 3D cultures. The gelation characteristics of the substrates were modulated by systematically altering the fibrinogen concentration and the fibrinogen-thrombin crosslinking ratio. Analysis of the ESCs cultured on different substrate conditions clearly illustrated the strong influence that substrate physical characteristics assert on cellular behaviours. Specifically, it was found that ESCs had a higher proliferation rate in gels of lower stiffness. Early differentiation events were studied by analyzing the gene and protein expression levels of early germ layer markers. Our results revealed that lower substrate stiffness elicited stronger upregulation of endoderm related genes Sox17, Afp and Hnf4 compared to stiffer substrates. While both 2D and 3D cultures showed a similar response, the effects were much stronger in 3D culture. These results suggest that physical cues can be used to modulate ESC differentiation into clinically relevant tissues such as liver and pancreas. Copyright © 2012 John Wiley & Sons, Ltd.

1 Introduction

Embryonic stem cells (ESCs) are pluripotent cells isolated from the inner cell mass of the blastocyst. In addition to their ability to differentiate into any tissue cell type, they can be propagated in vitro in an undifferentiated state indefinitely, which makes them perfect candidates for use in regenerative medicine and the study of differentiation (De Vos et al., 2009). The first stage of ESC differentiation is marked by the formation of three germ layers with distinct molecular markers from which all tissue types arise. The ectoderm mainly forms the skin and nervous system; the mesoderm forms muscle, cartilage, bone as well as hematopoietic cells; while the endoderm mainly forms the GI tract and both liver and pancreas (Murry and Keller, 2008).

Studies in the last decade have established the possibility of differentiating ESCs in vitro into early germ layers as well as into mature organ-specific cells. Most of these in-vitro inductions were achieved through modulations of the chemical microenvironment. More recently, studies have examined the effect of mechanical cues such as matrix elasticity on stem cell differentiation (Chowdhury et al., 2010; Evans et al., 2009). Mesenchymal stem cells cultured on substrates of various stiffness were reported to exhibit significant differences in their lineage commitment, which could be correlated to the physiological stiffness of the differentiated phenotype (Engler et al., 2006). Similar studies have been conducted on ESCs; however, most of these studies targeted towards osteogenic differentiation; hence, using substrates with stiffness ranges that were a few orders of magnitude higher than those used in the current study (Evans et al., 2009).

The current study reports how the physical microenvironment of soft substrates affects the early differentiation and phenotypic commitment of ESCs, with emphasis on endodermal differentiation. To demonstrate this effect, fibrin, a biocompatible hydrogel, was selected as the substrate to provide a desired variation in the physical microenvironment, primarily by modification of its gelation characteristics. Fibrin was chosen primarily because of its known attributes as a biocompatible and biodegradable scaffold, a fact that should translate into future clinical studies. Furthermore, fibrin is a biopolymer that plays a key role in the natural process of wound healing (Mosesson et al., 2001), which makes it a good candidate for stem cell transplantation (Rosso et al., 2005). Moreover, it has also been recently studied as a gel carrier system for beta-islet transplantation (Lim et al., 2009) and can also be easily coupled with chemical cues to aid differentiation (Kidszun et al., 2006), making it ideal for stem cell differentiation studies. Additionally, the substrate chemistry of fibrin hydrogels can be easily manipulated to achieve a broad range of soft substrates, making it suitable for the current study.

The main objective of this study was to investigate the effect of physical properties of soft substrates on specific characteristics of ESCs. ESC cultures differentiated on fibrin gel were analyzed in detail to ascertain their proliferative potential and differentiation patterning behaviour in response to variations in substrate mechanical properties. Two different culture configurations were analyzed: cells seeded on top of pre-formed fibrin gel (2D) and cells embedded inside the fibrin gel (3D). To the best of our knowledge, this is the first study illustrating the effect of physical properties of soft substrates on endodermal differentiation of ESCs. This could have a significant impact in the derivation of hepatic and pancreatic cells from ESCs for use in the treatment of hepatic and diabetes disorders.

2 Materials and methods

2.1 Gel synthesis

Soft fibrin hydrogels comprised of 1, 2, 4 and 8 mg/ml of fibrinogen were synthesized. Fibrinogen/thrombin ratios (F/T) of 10, 2.5, and 1.25 mg/U were synthesized for each fibrinogen concentration as previously described (Ko, 2008) and shown in Table 1. For convenience, these ratios are referred to throughout the text as 0.25, 1 and 2x, corresponding to increased crosslinking and thrombin concentrations. Instead of simply using fibrinogen or thrombin concentration, the F/T ratio was altered to ensure that a wide range of stiffness values could be studied for the fibrinogen concentrations chosen. This was systematically done to ensure a complete range of low, medium and high concentrations of fibrinogen and thrombin, respectively, as outlined in Table 1; 1 mg/ml represents a low fibrinogen concentration and 8 mg/ml represents a high fibrinogen concentration. Thrombin concentrations represent three F/T ratios: low (1.25), medium (2.5) and high (10), corresponding to low, medium and high units of thrombin.

Table 1. Concentration of thrombin in NIH units of activity/ml for all fibrin hydrogel conditions
 0.25x*1x#2x@
  • *

    Fibrinogen/Thrombin = 10.

  • #

    Fibrinogen/Thrombin = 2.5.

  • @

    Fibrinogen/Thrombin = 1.25.

1 mg/ml0.10.40.8
2 mg/ml0.20.81.6
4 mg/ml0.41.63.2
8 mg/ml0.83.26.4

2.2 Rheology measurements

Gel discs of 35-mm diameter prepared as described for 2D gel synthesis were deposited onto glass slides pre-rinsed with DI water. The samples were then allowed to gel fully at 4 °C. After complete gelation, they were fully immersed in the same media used for differentiation studies. The glass slides were then secured to the Peltier cell of a AR2000 stress-controlled rheometer (TA Instruments) and kept at 37 °C throughout the measurements. A frequency sweep was then performed using 25-mm stainless steel in parallel plate geometry with sandpaper glued to the plate to avoid slippage. Samples were subjected to an oscillatory strain described by Equation (1), where is the amplitude of the oscillatory strain (5%), f is the frequency and t is the time. Frequencies used ranged from 0.1 to 100 rad/s.

display math(1)

The stress required to achieve the specified strain was measured and the components of the complex modulus, storage (G’) and loss (G”) moduli were accordingly determined.

2.3 Scanning electron microscopy (SEM)

The fibrin gel microstructure was analyzed using SEM. Gels and samples were prepared for SEM analysis as previously described (Ko, 2008). SEM images were collected using a Philips XL30 field emission gun SEM (FEI Company). Further image analysis was performed using ImageJ software (http://rsbweb.nih.gov/ij/).

2.4 Maintenance and differentiation of ES cells

Murine ESD3 cells were cultured according to previously published methods (Banerjee et al., 2010).

2.5 2D Cell culture

For differentiation of ESCs on fibrin substrate, cells were trypsinized, washed and re-plated in appropriate configurations. For 2D culture, 30 000 cells in 200μl media were plated on top of the pre-formed fibrin gels prepared on 48-well plates and polymerized overnight at 4 °C.

2.6 3D Cell culture

For 3D cell culture, 100 000 cells were re-suspended in the fibrinogen solution before adding thrombin and plated on 48-well plates. The gel with the entrapped cells was then allowed to polymerize for 1 h at 4 °C, after which the culture media was added and subsequently incubated. For both cases, cells were maintained in DMEM medium (GIBCO) supplemented with 10% FBS, 4 mM L-glutamine (Cambrex) and 100 U/ml penicillin with media changed every day. As control, embryoid bodies were formed in rotary culture by adding 100 000 cells in ultralow attachment 35 mm-dishes with the same media above. Dishes were placed in a rotator during incubation and maintained at a constant speed of 40 rpm.

2.7 qRT-PCR analysis

Mouse embryonic stem cells (ESD3) cultured in 2D and 3D configurations were harvested by trypsin after five days of culture and RNA was extracted using the NucleoSpin kit (Macherey Nagel) according to manufacturer protocols. Sample absorbance at 280 and 260 was measured using a Smart Spec spectrophotometer (BioRad) to obtain RNA concentration and quality. Reverse transcription was performed using a ImProm II reverse transcription kit (Promega) following manufacturer recommendations. qRT-PCR analysis was performed for pluripotency and early germ layer markers using the primers listed in Table 2. Each sample was then run in duplicates (how many times?) and average values were accordingly used for analysis.

Table 2. Primers used for qPCR experiments
TISSUENAMEDIRSEQUENCE
ControB-ActinLcagcagttggttggagca
  Rtgggagggtgagggactt
PLURIPOTENCYrex1Laaggtcatccacggcaca
  Rtgggagtcatcgcttggt
 0ct4Lggagaagtgggtggaggaa
  Rgctgattggcgatgtgag
 sox2Lctggactgcgaactggaga
  Rttggatgggattggtggt
MesodermBrachyury TLaagaacggcaggaggatg
  Rgcgagtctgggtggatgta
 FGF8Lacggcaaaggcaaggact
  Rtgaagggcgggtagttga
 GSCLgcaccgcaccatcttca
  Rtcgcttctgtcgtctcca
EndodermSox 17Latccaaccagcccactga
  Racaccacggaggaaatgg
 AFPLctctggcgatgggtgttt
  Raactggaagggtgggaca
 HNF4Lcatcgtcaagcctccctct
  Rccctcagcacacggtttt
EctodermNestinLggaggatgtggtggaggat
  Rttcccgtctgctctggtt
 FGF5Lttcaagcagtccgagcaa
  Rtaggcacagcagagggatg
 BMP4Latctggtctccgtccctga
  Rcgctccgaatggcacta

2.8 Cell proliferation assay

The proliferative potential of ES cells was analyzed using Alamar™ blue assay. For the 2D culture, 15 000 ESD3 cells were plated on fibrin gels of different configurations. Twenty-four hours after plating, the culture media was replaced by fresh media containing 10% Alamar blue and incubated for 4 h. Fluorescence was obtained from a multi-well reader (BioTek Synergy 2, Winooski, VT, USA) at an excitation wavelength of 570 nm and emission wavelength of 585 nm according to manufacturer instructions. Results were normalized to the values obtained for the gels with the highest thrombin concentration or most crosslinked (2x) condition.

2.9 Immunohistochemical analysis

Staining was performed on differentiated cells. The fibrin gels containing cells in 3D format were formed on coverslips in a 24-well plate. Staining was performed following company recommendations. The antibodies used were AFP goat polyclonal antibody and Sox17 rabbit polyclonal antibody (Santa Cruz Biotechnologies). The secondary antibodies used were donkey anti-rabbit IgG Texas Red (Santa Cruz Biotechnology) and Alexa Fluor® 488 donkey anti-goat IgG (Invitrogen). Confocal images were taken using an Fluoview 1000 system (Olympus).

2.10 Statistical analysis

Each experiment was performed twice with duplicates each time. Average and standard error were measured and Student t-test was performed for significance.

3 Results

3.1 SEM characterization of fibrin microstructure

Appropriate characterization of ESC interaction with a substrate microenvironment requires a thorough understanding of the microstructural features of the substrate under various conditions. Figure 1A and 1B illustrate SEM images of the fibrin gels taken at two very high values of fibrinogen concentrations of 1 and 8 mg/ml, respectively selected as representative concentrations. It was observed that for a fixed fibrinogen concentration, there was a decrease in fibre diameter as thrombin concentration increased (Figures. 1A: a, b, c) and the fibres appeared to be less bundled. It can also be seen that there was no significant difference in fibre diameter between 1x and 2x compared to 0.25x. The difference in diameter also appeared to become less significant as fibrinogen concentration increased.

Figure 1.

(A) Effect of various the fibrinogen concentration and F/T ratios on the microstructure of the gels imaged at 20 KX. (a)-(c): 1 mg/ml of fibrinogen at F/T ratios of 0.25, 1 and 2x respectively. (d)-(f): 8 mg/ml fibrinogen at F/T ratios of 0.25, 1 and 2x respectively. (B) Effect of various the fibrinogen concentration and F/T ratios on the microstructure of the gels observed at 5 KX. (a)-(c): 1 mg/ml fibrinogen at F/T ratios of 0.25, 1 and 2x, respectively. (d)-(f): 8 mg/ml fibrinogen at F/T ratios of 0.25, 1 and 2x, respectively

As fibrinogen concentration decreased for a fixed fibrinogen to thrombin (F/T) ratio, the fibres formed more bundle type thicker structures, as can be seen in Figure 1A: e compared to 1A: b. As a result, each fibril appears to have a larger fibre diameter: ~ 0.5-0.75 µm for 1 mg/ml fibrinogen compared to ~ 0.1 µm for a fibrinogen concentration of 8 mg/ml at a fixed F/T ratio of 1x.

Furthermore, at a fixed fibrinogen concentration, the substrate appeared to have a more open pore structure with a larger pore size as thrombin concentration decreased from 2x to 1x and 0.25x, as can be seen for both 1 and 8 mg/ml fibrinogen concentrations in Figures 1B: a-c and d-f, respectively. To better analyze the effect of thrombin concentration on the porosity of the substrate, ImageJ software was used to quantify the average pore size of the gel at fixed fibrinogen concentrations. SEM images were binary and ImageJ software was used to estimate the average size of spaces between fibres. This value was obtained for each fibrinogen concentration and was normalized to the values obtained for gels synthesized with the same fibrinogen concentration at a F/T ratio of 2x. As illustrated in Figure 2, pore size decreased with increasing thrombin concentrations for both 1 and 8 mg/ml values of fibrinogen concentrations. However, the effect of thrombin concentration on pore size was much stronger between 0.25x and the higher ratios, while between 1x and 2x, the changes were minimal as can be seen in Figure 1.

Figure 2.

Average pore size calculated using ImageJ for 1 and 8 mg/ml fibrinogen concentrations and three F/T ratios normalized to 2x for both fibrinogen concentrations. * p < 0.05 compared to highest stiffness group

3.2 Rheological characterization of fibrin hydrogels

The effect of microstructural variations of the gel on the macroscopic property of the gel substrate was investigated by conducting a rheological characterization of the gel. Table 3 illustrates the storage modulus (G’), a measure of energy stored by the sample under oscillatory deformation conditions, while Table 4 illustrates the loss modulus (G”), a measure of energy dissipated under vibratory conditions. Both storage and loss moduli showed little dependence on frequency of oscillation, indicative of a solid-like behaviour: therefore, their values at a frequency of 0.5 Hz was arbitrarily chosen as a representative value.

Table 3. G’ values in Pa for various fibrinogen concentrations and all three crosslinking ratios at a frequency of 0.5 Hz
 0.25x1x2x
  • *

    p < 0.05 compared to highest stiffness group.

1 mg/ml4.0 ± 0.9*14.1 ± 4.0*24.8 ± 4.5
2 mg/ml13.0 ± 0.9*35.8 ± 8.742.0 ± 7.1
4 mg/ml72.1 ± 6.0*89.2 ± 9.197.9 ± 11.9
8 mg/ml171.1 ± 20.3*193.9 ± 17.7*247.3 ± 15.5
Table 4. G” values in Pa for various fibrinogen concentrations and all three crosslinking ratios at a frequency of 0.5 Hz
 0.25x1x2x
  • *

    p < 0.05 compared to highest stiffness group.

1 mg/ml0.7 ± 0.1*1.5 ± 0.32.7 ± 0.6
2 mg/ml3.3 ± 0.44.6 ± 1.64.8 ± 1.7
4 mg/ml9.4 ± 1.410.8 ± 3.011.5 ± 2.5
8 mg/ml24.7 ± 4.6*28.2 ± 2.833.7 ± 3.9

After being cleaved by plasmin, fibrinogen is converted into the monomeric form of fibrin and self-assembles to form an insoluble fibrin clot that is stabilized by the crosslinking of factor XIII. In the current study, the scaffold storage modulus was varied by two orders of magnitude from 4 Pa, corresponding to a fibrinogen concentration of 1 mg/ml, and an F/T ratio of 0.25x to 247 Pa, corresponding to a fibrinogen concentration of 8 mg/ml and an F/T ratio of 2x. Furthermore, upon increasing thrombin concentration for a fixed fibrinogen concentration, gel stiffness increased by as much as 76 Pa (8 mg/ml of fibrinogen for F/T ratios of 0.25 and 2x). Mechanical stiffness was varied by either keeping the fibrinogen concentration fixed or by selecting four different fibrinogen concentrations: 1 mg/ml representing the low range and 8 mg/ml representing the high range. Similarly, for a fixed fibrinogen concentration, the thrombin concentration was varied correspondingly to F/T ratios of 0.25, 1 and 2x, representing low, medium and high units of thrombin, respectively. Based on this, rationale, it was thus possible to prepare fibrin gels that exhibited a wide range of mechanical stiffness.

Moreover, despite altering the F/T ratio, conditions with the same thrombin concentration and different fibrinogen concentrations also existed. Similarly, after increasing fibrinogen concentration, gel stiffness also increased by as much as 95 Pa (4 mg/ml fibrinogen with F/T 2x and 8 mg/ml fibrinogen with F/T 1x). The three cases in which fibrinogen concentration was doubled were as follows: 1) 1 mg/ml fibrinogen with an F/T ratio of 2x and 2 mg/ml of fibrinogen with an F/T ratio of 1x; 2) 2 mg/ml of fibrinogen with an F/T ratio of 2x and 4 mg/ml of fibrinogen with an F/T ratio of 1x; and 3), 4 mg/ml of fibrinogen with an F/T ratio of 2x and 8 mg/ml of fibrinogen with an F/T ratio of 1x). Gel stiffness nearly doubled in all three cases, while thrombin concentration remained fixed except at low fibrinogen concentrations as illustrated in Table 3.

In comparing this trend where thrombin concentration was doubled for a fixed fibrinogen concentration (1-2x for all fibrinogen concentrations), a much lower relative increase in storage modulus was observed, except at lower fibrinogen concentrations. This suggests that fibrinogen concentration was more influential on the manipulation of gel moduli, except under dilute conditions. Interestingly, the storage modulus for all conditions was not exclusive and an overlap in modulus was observed between the two conditions. More specifically, the moduli of gels prepared with 1 mg/ml fibrinogen and an F/T ratio of 1x and 2 mg/ml fibrinogen with an F/T ratio of 0.25x were both ~ 14 Pa, indicative of the influence of other microstructural forces affecting mechanical stiffness such as fibre diameter, porosity and orientation.

3.3 Effect of substrate properties on ESC proliferation

ESCs plated on soft fibrin substrates were found to attach well under all substrate conditions and were alive and proliferating. However, cells did not spread out significantly and retained a spherical clumped-up morphology. Observation of the cells after three days of culture consistently showed the formation of cell clusters (Figure 3). When cell morphology was compared across substrates of different thrombin concentrations for the same fibrinogen concentration, it was typically observed that substrates with lower thrombin concentrations resulted in larger cell clusters compared to their more crosslinked counterparts.

Figure 3.

Representative images of cells plated on gels of identical fibrinogen concentration while various crosslinking F/T ratios: 0.25x (left) and 2x (right). Cells attach but do not spread out, forming cell clusters instead. Cell cluster size greater in gels with lower crosslinking ratios

Since differences in cell cluster size can be attributed to differences in cell proliferation rate, proliferation analysis was performed by Alamar blue assay 24 h after plating the cells on fibrin gels. A duration of 24 h was chosen primarily to minimize the effect of fibrin degradation by the cells and to avoid the effect of differentiation on proliferation. The effect of thrombin concentration of the substrates on cellular proliferation is illustrated in Figure 4. For each fibrinogen concentration, the proliferation rate was normalized to the condition with the lowest F/T ratio. It was consistently observed that for a specific fibrinogen concentration, ESCs cultured on lower crosslinked substrates exhibited a higher proliferation rate compared to parallel cultures on a more crosslinked structure.

Figure 4.

Comparison of proliferation of mouse ES cells plated on gels of different fibrinogen concentrations and various crosslinking ratios after 24 h of plating. Y-Axis represents Alamar blue fluorescence. Results normalized to the lowest crosslinking ratio for each group. At all fibrinogen concentrations, proliferation was found to decrease as crosslinking increased. * p < 0.05 compared to highest stiffness group

3.4 Effect of substrate physical properties on embryonic stem cell differentiation

3.4.1 Substrate stiffness

ESCs were plated on the gels and allowed to spontaneously differentiate for five days, after which samples were analyzed by qRT-PCR for three different marker for each of the germ layers and for pluripotency assessment. The markers used were: undifferentiating markers Rex1, Oct4 and Sox2 (Ellis et al., 2004; Jin et al., 2002; Palmqvist et al., 2005); early endoderm markers Afp, Sox17 and Hnf4 (Dziadek and Adamson, 1978 Hudson et al., 1997, Weber et al., 1996; Brachyury et al.,(not in reference section); early mesoderm markers Gsc and Fgf8 (Blum et al., 1992 MacArthur et al., 1995, Wilkinson et al., 1990); and early ectoderm markers Nestin, Fgf5 and Bmp4 (Lobo et al., 2004, Monsoro-Burq et al., 1996, Rathjen et al., 1999). During differentiation, cells were cultured in DMEM/FBS without further lineage specific induction.

The effect of substrate properties on ESC differentiation was analyzed by comparing the relative variation of all germ layer markers across the entire range of substrate stiffness. Figure 5A illustrates the sensitivity of each germ layer along with pluripotency to changes in substrate stiffness. It was observed that pluripotency markers maintained similar expression levels in all substrates. As to individual germ layers, Figure 5A shows that mesoderm and ectoderm markers were relatively insensitive to changes in substrate stiffness in the specific stiffness ranges examined. Endoderm markers, however, were found to respond strongly to changes in substrate properties. It was observed that substrates of lower stiffness of ~ 13 Pa resulted in stronger endoderm upregulation. However, a higher stiffness range of 171 Pa was observed to upregulate both Afp and Sox17. Interestingly, the other germ layers were also slightly elevated under higher stiffness conditions, indicating an overall increase in differentiation under those conditions. Moreover, preferential upregulation of endoderm markers was observed only at lower stiffness conditions. Of the endoderm markers, the magnitude of upregulation of Afp was thousands of folds stronger than that of Sox17, while both Sox17 and Afp elicited a similar trend in expression.

Figure 5.

Effect of changing substrate stiffness on ESC pluripotency and germ layer commitment in 2D (a) and 3D (b). Results are normalized to undifferentiated cells. Most significant effect was observed in the upregulation of endoderm markers SOX17 and AFP in lower range stiffness gels. Effect was more prominent in 3D

3.4.2 Substrate composition

As earlier mentioned, fibrin substrate properties were modified by changing both fibrinogen concentration and F/T ratio. As a result, it was important to analyze the effect of gel composition on stem cell differentiation. Differentiation patterns were compared across different thrombin concentrations for a fixed value of fibrinogen concentration (Figures 6a, b). Very high fibrinogen concentrations of 1 and 8 mg/ml were chosen to assess differentiation patterns across a wide spectrum of fibrinogen concentrations. For ease of comparison, the fold changes in gene expression levels were represented by normalizing to 2x crosslinking for each fibrinogen concentration.

Figure 6.

Effect of changing substrate crosslinking ratios on ESC cell pluripotency and germ layer commitment analyzed at very high fibrinogen concentrations of 1 mg/ml (a) and 8 mg/ml 1 mg/ml (b) in 2D Results are normalized to highest crosslinking ratio (2x). Most significant effect was observed in upregulation of endoderm markers compared to cells differentiated on gels of lower crosslinking (0.25x, 1x) and higher crosslinking (2x) ratios. * p < 0.05 compared to highest stiffness group

Analogous to Figure 5, the mesoderm and ectoderm markers were found to be relatively insensitive to changes in crosslinking ratio in the 1 mg/ml fibrinogen concentration (Figure 6a). In contrast, the endodermal markers, particularly Sox17 and Afp, were found to be extremely sensitive to changes in crosslinking ratio. For 1 mg/ml fibrinogen concentration, we observed approximately 5- and 9-fold increases in Afp expression levels for softer gel conditions of ratios 0.25 and 1x, respectively, compared to the stiffer gels synthesized at 2x. Sox17 expression levels exhibited a moderate 3- and 5-fold increase when thrombin concentration was lowered from 2 to 1x in both fibrinogen concentration groups, respectively. Similar patterns were found across all fibrinogen concentrations, where lowering the crosslinking ratio resulted in stronger expression of endoderm markers (Supplementary figs. 1, 2).

While the above analysis focuses on the effect of changing crosslinking ratios for fixed fibrinogen concentrations, it was worthwhile to compare the effect of changing fibrinogen concentrations for a fixed cross-linking ratio, since both cases resulted in differences in substrate stiffness (Table 3). Figure 9 represents the changes in gene expression levels when the F/T ratio remained constant (1x) while the concentration of fibrinogen varied between 1 and 8 mg/ml (Figure 9A). It was observed that lower values of fibrinogen concentration preferentially favoured endoderm differentiation.

3.4.3 Substrate microstructure

In all analyses, we consistently observed that endoderm upregulation correlated with lower substrate stiffness conditions and was achieved either by lowering the crosslinking ratio for fixed fibrinogen concentrations or by lowering fibrinogen concentrations for a fixed crosslinking ratio. To further analyze whether substrate stiffness was uniquely controlling the differentiation patterning, we examined two fibrin substrates of similar stiffness but of various fibrinogen and thrombin concentrations and analyzed ESC differentiation patterns on them. As illustrated in Tables 1 and 3, gels synthesized with 1 mg/ml fibrinogen with an F/T ratio of 1x and 2 mg/ml fibrinogen with an F/T ratio of 0.25x resulted in a similar gel stiffness value of ~ 14 Pa. ESCs differentiated on these two conditions were analyzed for their differentiation patterns, as illustrated in Figure 10. Of interest, it was observed that the mesoderm and ectoderm markers elicited quite similar behaviour under these conditions, although endoderm markers were strongly altered. These results indicate that while substrate stiffness strongly influenced differentiation patterns, it was clearly not the sole player in this complex process. Careful observation of the fibrin gel under these two conditions by SEM (Supplementary figure 3) revealed significant differences in their microstructural characteristics despite comparable stiffness values. Analysis revealed that some of the major structural differences included differences in fibre density, fibrin and pore size. Following this analysis, it is reasonable to suggest that along with macroscopic stiffness, microstructural features are also likely to influence differentiation patterns. A more detailed characterization of the gel microstructural features and its possible correlation with ESC differentiation is currently being investigated by our group.

3.4.5 Culture configuration

Experiments were performed by plating cells on pre-formed fibrin gels in 2D configurations. However, to better elucidate the effects of stiffness, composition and microstructure in ESC differentiation, ESCs were also cultured in 3D configurations in which ESCs were suspended within the fibrin substrate during synthesis of the gels.

Similarly to 2D experiments, the effect of substrate properties on ESC differentiation was analyzed by comparing the relative variation of all germ layer markers across the entire range of substrate stiffness. Figure 5B illustrates the sensitivity of each germ layer along with pluripotency to changes in substrate stiffness. Pluripotency, mesoderm and ectoderm markers were relatively insensitive to changes in substrate stiffness in the specific stiffness ranges considered in this study. Endoderm markers were found to respond strongly to changes in substrate property. It was observed that substrates of lower stiffness (~ 13 Pa) resulted in stronger endoderm upregulation. In contrast to 2D configurations, the positive effect of higher stiffness range was less obvious in 3D cultures, where the strongest effect was in the lower range of 13 Pa. Both Sox17 and Afp elicited a similar trend in expression; the magnitude of upregulation of Afp was thousands of folds stronger than that of Sox17, with the upregulation under 3D culture being more dominant than 2D configuration. An additional control of ESC differentiation through EB formation was included in the 3D cultures to account for the possible effect of biochemical induction arising from differential swelling of the gels. As illustrated in Figure 5, the magnitude of upregulation in softer substrates was much stronger than that of EB, which suggests that the substrate played a more significant role than biochemical induction through the media.

We also studied the effect of substrate composition in 3D configurations (Figure 7). When the fibrinogen concentration was maintained constant and thrombin ratios were varied, the 3D culture resulted in a much stronger effect on Sox17 expression levels, which was upregulated by 3- and 7-fold for gel conditions of 0.25 and 1x respectively, compared to the stiffer gels synthesized at 2x for the 1 mg/ml fibrinogen gels. In the case of the 8 mg/ml gels, an even more pronounced effect was found with 5- and 15-fold upregulation in the 1 and 0.25x conditions, respectively. Furthermore, in the 8 mg/ml fibrinogen conditions, the endoderm marker Hnf4 also exhibited a significant upregulation comparable to Sox17. Additionally, while the 2D cultures led to somewhat elevated expression levels of many of the germ layer markers, 3D cultures were consistently more specific for endoderm markers along with Fgf and Nestin. Hence, while the overall effect of softer substrate (~ 13 Pa) was more pronounced than the stiffer substrates, lower crosslinking always resulted in stronger endoderm expression compared to higher crosslinking for invariant fibrinogen concentrations in the entire range of substrate stiffness considered in this study,

Figure 7.

Effect of changing substrate crosslinking ratios on ESC pluripotency and germ layer commitment analyzed at very high fibrinogen concentrations of 1 mg/ml (a) and 8 mg/ml 1 mg/ml (b) in 3D. Results are normalized to highest crosslinking ratio of 2x. Most significant effect was observed in the upregulation of endoderm markers compared to cells differentiated on gels of lower crosslinking (0.25x, 1x) and higher crosslinking (2x) ratios. * p < 0.05 compared to highest stiffness group

To further analyze the differentiated cell population for its protein expression levels, immunohistochemical analysis was performed for the case eliciting the strongest upregulation in gene expression: the 3D culture for Sox17 and Afp expression. As illustrated in Figure 8, the differentiated cells cultured in the softer substrates that resulted in highest upregulation of both Afp and Sox17 (1 mg/ml, 1x) stained strongly for both Sox17 and Afp. High co-expression of these markers was also observed in the cell clusters formed. Parallel analysis of differentiated cells on gels of higher thrombin concentrations of 2x showed negligible stain (data not included).

Figure 8.

Immunocytochemistry images of cells plated at 1 mg/ml and 1x. High expression of endoderm markers AFP (Red) and Sox17 (Green) was observed in the cell clusters that were found to be highly co-expressed

When crosslinking ratios were maintained constant and fibrinogen concentrations were modified, we once again found that lower values of fibrinogen concentration (Figure 9b) preferentially favoured endoderm differentiation and this effect was more accentuated in the 3D culture compared to the 2D culture, with upregulation of endoderm Afp and Sox17 being 4 and 17x higher in 1 mg/ml fibrinogen gels, respectively.

Figure 9.

Pluripotency and germ layer markers of ESC cells differentiated in substrates of various and very high fibrinogen concentrations with same crosslinking ratios of 1x in (A) 2D and (B) 3D cultures. Significant upregulation of endoderm markers was found when cells were differentiated on gels obtained at lower fibrinogen concentrations. * p < 0.05 compared to highest stiffness group

4 Discussion

This study presented an analysis of the effects of a mechanical microenvironment on ESC culture and differentiation. ESCs were maintained on soft fibrin substrates of various physical characteristics modulated by altering both of its components, fibrinogen and thrombin, involved in the formation of fibrin gels.

Analysis of the microscopic architecture of the fibrin gels using SEM revealed a strong effect of both fibrinogen concentration and crosslinking on fibre diameter, bundling and relative pore size, as demonstrated in Figures 1 and 2. Such findings are in agreement with previously published data (Blombäck and Bark, 2004; Weisel, 2004). Mechanical characterization of the fibrin gels was performed using a plate rheometer to measure both storage and loss modulus, as summarized in Table 3. While comparing Tables 3 and 4, it should be noted that G’> > G” for all samples considered, which suggests as expected a more solid-like than liquid-like behaviour for all conditions consistent with low loss characteristics of the solids. These results illustrate that increases in fibrinogen or thrombin concentrations resulted in an increase in substrate stiffness. While a greater response was observed by increasing fibrinogen concentrations, chemical variation was also greater, which led us to focus on the effects of various thrombin concentrations for most of our observations.

ESCs were cultured for five days on these substrates, at the end of which the cells were analyzed for their proliferative and differentiation potential. Since we were interested in early germ layer commitment, differentiation was performed over a relatively short duration. Our first observation after cell plating was that instead of spreading out, the cells formed clusters that varied in size depending on thrombin concentration (Figure 3). A possible explanation could be that fibre thickness decreased as the substrate became more crosslinked, thus decreasing pore size and exhibiting a higher modulus as shown in Figures 1 and 2 and Tables 3 and 4. A similar trend was consistently observed across all four fibrinogen concentrations analyzed.

Cell proliferation assay also confirmed that gels synthesized with lower thrombin concentrations facilitated increased proliferation compared to stiffer substrates (Figure 4). A similar trend has also been reported with mesenchymal stem cells and fibroblasts grown in fibrin gels (Duong et al., 2009; Ho et al., 2006). Studies using neural stem cells have shown that when plated in 3D hydrogel cultures, there was a significant increase in proliferation as stiffness of the scaffolds decreased (Banerjee et al., 2009). Similar results were obtained for neural cells in 2D matrices with various elasticity (Engler et al., 2006).

Analysis of differentiation patterns of the cells cultured on the substrates revealed that while mesoderm and ectoderm remained relatively insensitive to changes in substrate physical properties, the endoderm markers elicited quite a strong response. More specifically, substrates in the range of 13 Pa showed the strongest preferential upregulation of the endoderm markers under both 2D and 3D culture configurations. The overall effect of the substrate, however, was more pronounced in 2D than in 3D cultures. We would like to note that a similar study has been recently reported. However, the study used substrates with stiffness ranges that were four orders of magnitude higher than ours and their results were targeted towards osteogenic differentiation (Evans et al., 2009). It was observed while evaluating the effect of substrate composition that for a fixed fibrinogen concentration, softer gels were preferentially upregulating endoderm related markers compared to more crosslinked counterparts. Figures 6 and 7 are representative of fibrinogen concentrations of 1 and 8 mg/ml, while similar analyses on substrates of different fibrinogen concentrations consistently showed a similar trend of softer substrates, leading to a stronger upregulation of endoderm related markers. Notwithstanding the variability in the extent of the particular effect, there was an overall remarkable consistency in preferential upregulation of endodermal expression in less crosslinked substrates for both 2D and 3D culture configurations. For 2D, ESCs cultured on 0.25x showed stronger upregulation compared to 1x, while the difference between 1x and 2x was more subtle. The 3D culture showed a clearer trend of higher upregulation with softer substrates.

A careful observation of the gels synthesized with lower thrombin concentrations showed that even though no mesoderm markers exhibited significant upregulation, in some cases, Fgf8 was quite significantly upregulated. A possible explanation could be that Fgf, apart from being a mesodermal marker, also plays a role in endothelial development and is an important angiogenic factor (Antoine et al., 2005; West et al., 2001). Upregulation of these markers could probably be attributed to differentiation into endothelial cells due to softer matrices resulting in larger cell clusters. Larger cell clusters resulted in insufficient transport of nutrients and oxygen to the centre of the structures, which resulted in hypoxia and could have triggered the creation of blood vessels, resulting in upregulation of FGF markers.

We consistently observed that for a particular fibrinogen concentration, gels obtained at lower thrombin concentrations favoured endodermal differentiation of ESCs. As revealed by microstructural characterization of the gels, increasing the concentration of thrombin resulted in a dense network of thinner fibres that were less bundled (Figure 2). These microstructural features resulted in increased substrate stiffness because of increased crosslinking for the same fibrinogen concentration (Table 3). Based on the mechanical characteristics of the gel and biological characterization of the cells on the fibrin substrates, it is reasonable to suggest that lower substrate stiffness values preferentially favoured ESC differentiation towards the endodermal lineage.

Even though substrate stiffness clearly played an important role in cellular lineage commitment, it was certainly not the only factor, as shown in Figure 10. Comparison of two substrates of various fibrinogen concentrations and F/T ratios and comparable substrate stiffness revealed that mesoderm and ectoderm markers were almost invariant under these two conditions, indicating that these germ layers were insensitive to the substrate properties at the initial stage of differentiation and within the substrate ranges examined. The endoderm layer however, varied significantly between these conditions, which establishes that substrate stiffness, although important, was not the sole player in the process of germ layer commitment. Careful analysis of the substrate microstructural features revealed that even though macroscopic stiffness properties were similar, the gels differed substantially in their microstructural features. These results warrant a more detailed analysis of the effect of substrate microstructural features on differentiation. This is currently being investigated by the authors. It is important to note that there was variability in the results from the different experiments, as observed by the error bars attributed to population heterogeneity and variable response to global inductive cues (Canham et al., 2010; Gibson et al., 2009). However, despite the variability, all experiments consistently showed a similar trend of softer substrates preferentially favouring differentiation towards endodermal lineage.

Figure 10.

Marker expression comparison between 1 mg/ml fibrinogen obtained at F/T crosslinking ratio of 1x and fibrinogen concentration of 2 mg/ml obtained at F/T crosslinking ratio of 0.25x. Figure shows expression of most markers to be equivalent in both groups except for that of endoderm markers. Results normalized to 1 mg/ml fibrinogen 1x crosslinking ratio.

5 Conclusions

This study presented a thorough analysis of the effects of various substrate mechanical properties on ESC proliferation and differentiation potential. The mechanical properties of fibrin substrate were varied by systematically altering fibrinogen and thrombin concentrations. SEM analysis of the synthesized gels illustrated variations in the microscopic gel microstructure such as fibre thickness and porosity and the overall effective area of fibrin strand available for cell anchorage as a result of altered fibrinogen and thrombin concentrations. Detailed mechanical characterization of the different substrates revealed that variations in substrate concentration or crosslinking resulted in substantial modulations of the substrate stiffness.

Analysis of ESCs cultured on different substrate conditions clearly illustrated the strong influence that substrate mechanical properties assert on cellular proliferation and differentiation potential. ESCs cultured on softer substrates as a result of lower thrombin concentrations were found to be more conducive to proliferation and differentiation. For both 2D and 3D culture configurations, substrates with lower thrombin concentrations elicited a somewhat elevated expression level of most of the gene expression markers. However, the level of upregulation strongly depended on the specific germ layer. It was consistently observed that differential expression of endodermal markers were strongest in gels obtained with lower crosslinking compared to highly crosslinked gels, an effect that was further accentuated in 3D compared to 2D cultures. These results are indicative that lower substrate stiffness values favoured preferential differentiation of ESCs towards endodermal lineage. However, substrate stiffness was not the sole player controlling differentiation, which is probably dependant on both gel microstructure and chemical composition. Furthermore, among the germ layers tested, early mesoderm and ectoderm layers were found to be less responsive to substrate properties.

Author disclosure statement

No competing financial interests exist.

Acknowledgements

IB acknowledges support from NIH New Innovator Award DP2 116520 and ORAU Ralph Powe Junior Faculty Enhancement Award. PNK acknowledges support of the Edward R. Weidlein Chair Professor funds as well as the Center for Complex Engineered Multifunctional Materials (CCEMM) for partial support of this work.

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