Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes

Abstract

In many cell types, several cellular processes, such as differentiation of stem/precursor cells, maintenance of differentiated phenotype, motility, adhesion, growth, and survival, strictly depend on the stiffness of extracellular matrix that, in vivo, characterizes their correspondent organ and tissue. In the liver, the stromal rigidity is essential to obtain the correct organ physiology whereas any alteration causes liver cell dysfunctions. The rigidity of the substrate is an element no longer negligible for the cultivation of several cell types, so that many data so far obtained, where cells have been cultured on plastic, could be revised. Regarding liver cells, standard culture conditions lead to the dedifferentiation of primary hepatocytes, transdifferentiation of stellate cells into myofibroblasts, and loss of fenestration of sinusoidal endothelium. Furthermore, standard cultivation of liver stem/precursor cells impedes an efficient execution of the epithelial/hepatocyte differentiation program, leading to the expansion of a cell population expressing only partially liver functions and products. Overcoming these limitations is mandatory for any approach of liver tissue engineering. Here we propose cell lines as in vitro models of liver stem cells and hepatocytes and an innovative culture method that takes into account the substrate stiffness to obtain, respectively, a rapid and efficient differentiation process and the maintenance of the fully differentiated phenotype.

Figure 1

Soft substrate induces a rapid and homogeneous epithelial differentiation of RLSCs. (a) Phase-contrast micrographs of RLSCs grown on Petri plastic dish (CTRL; E > 1 GPa) and on hydrogels with E = 0.4 kPa and 80 kPa, for 24 and 48 hours. Images are representative of three independent experiments. Scale bar: 100 μm. (b) Phase-contrast micrographs and immunofluorescence of cells cultured on plastic (CTRL), 0.4 kPa and 80 kPa for 24 and 48 hours, stained for HNF4α, Vimentin, and E-cadherin. The nuclei were stained with DAPI. Images are representative of three independent experiments. Scale bar: 50 μm. (c) Western blot analysis of Vimentin at 24 and 48 hours after seeding on substrates with the indicated E values. CDK4 was used as a loading control.

Figure 2

RLSCs grown on soft substrate display epithelial/hepatic gene expression and hepatic function. (a) RT-qPCR analysis for the indicated genes of RLSCs grown on 0.4 kPa and 80 kPa at 24 hours (left panel) and 48 hours (right panel). Data are expressed as fold change in gene expression in cells grown on hydrogels versus CTRL (arbitrary value = 1). The graphics are representative of three independent experiments. Note the logarithmic scale. (b) Urea production in RLSCs. Urea levels in supernatant of cells grown on plastic (CTRL) and on 0.4 kPa hydrogel were analysed at 24 and 48 hours. The mean ± SD of two independent experiments is shown.

Figure 3

Substrate rigidity controls pathways of mechanotransduction involved in hepatocyte differentiation. (a) Western blot analysis of phospho-ERK1/2 and total ERK1/2 (used as a loading standard) at 3 and 24 hours after seeding on substrates with the indicated E values. Images are representative of three independent experiments. (b) Flow cytofluorimetric analysis of cell cycle in RLSCs cultured at the indicated conditions for 24 hours. The values, obtained from three independent experiments, are reported as mean ± SD; ^∗∗∗ p ≤ 0.001, ^∗ p ≤ 0.05. (c) Immunofluorescence analysis of RLSCs cultured on Petri dish (CTRL), 0.4 kPa and 80 kPa for 12 hours, stained for YAP protein. The nuclei were stained with DAPI. Images are representative of three independent experiments. Scale bar: 20 μm. (d) RT-qPCR analysis of the YAP target gene, Ctgf. Data are expressed as fold change in gene expression in cells grown on 0.4 kPa and 80 kPa for 24 hours versus CTRL (arbitrary value = 1). The mean ± SD of three independent experiments is shown; ^∗∗∗ p ≤ 0.001.

Figure 4

Differentiation of RLSCs towards hepatocytes correlates with early chromatin modifications on HNF4α promoter. qPCR analysis of ChIP assay performed to quantify H3K9Ac, H3K4me3, and H3K27me3, on the HNF1/HNF6 binding site of HNF4α promoter in RLSCs grown on plastic (CTRL) and on 0.4 kPa hydrogel for 24 and 48 hours. Amplification signals of specific immunoprecipitated samples (IP) and IgG are normalized versus total chromatin (Input) and expressed as % of Input. The mean ± SD of two independent experiments is shown. The upper part of the figure shows a schematic representation of murine HNF4α promoter indicating the binding site for HNF1/HNF6 and the relative positions of the qPCR primers.

Figure 5

Soft substrate improves the differentiation state of hepatocyte cell lines. (a) Phase-contrast micrographs of MMH/E14 hepatocyte cell lines grown on plastic (CTRL) and on 0.4 kPa hydrogel for 24 and 48 hours. Images are representative of three independent experiments. Scale bar: 100 μm. (b) RT-qPCR analysis for the indicated genes. Data are expressed as fold change in gene expression in cells grown on 0.4 kPa versus CTRL (arbitrary value = 1). The graphic represents the mean of three independent experiments ± SD. Asterisks indicate p values in Student's t-test (^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001). Note the logarithmic scale. (c) Urea production in MMH/E14 hepatocytes. Urea levels in supernatant of cells grown on plastic (CTRL) and on 0.4 kPa hydrogel at 24 and 48 hours were analysed. The mean ± SD of two independent experiments is shown. (d) Phase-contrast micrographs of WT/3A hepatocyte cell lines grown on plastic (CTRL) and on 0.4 kPa hydrogel for 48 hours. Images are representative of three independent experiments. Scale bar: 100 μm. (e) RT-qPCR analysis for the indicated genes. Data are expressed as fold change in gene expression in cells grown on 0.4 kPa versus CTRL (arbitrary value = 1). The graphic represents the mean of three independent experiments. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001. Note the logarithmic scale. (f) Urea production in WT/3A hepatocytes. Urea levels in supernatant of cells grown on plastic (CTRL) and on 0.4 kPa hydrogel at 48 hours were analysed. The mean ± SD of two independent experiments is shown.