Extracellular matrices modulate differentiation of human embryonic stem cell-derived hepatocyte-like cells with spatial hepatic features

doi:10.21203/rs.3.rs-3157108/v1

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Research Article

Extracellular matrices modulate differentiation of human embryonic stem cell-derived hepatocyte-like cells with spatial hepatic features

https://doi.org/10.21203/rs.3.rs-3157108/v1

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Journal Publication

published 01 Nov, 2023

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Background

Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) can provide a valuable in vitro model for disease modelling and drug development. However, it is challenging to generate these cells with functions comparable to hepatocytes in vivo. Extracellular matrices (ECM) play an important role in supporting liver development and hepatocyte functions, but their functions in hepatocyte differentiation and maturation during hPSC differentiation remain unclear. Here, we investigate the effects of two ECM - Matrigel and type I collagen on hepatic differentiation of human embryonic stem cells (hESCs).

Methods

hESCs-derived HLCs were generated through multistage differentiation in 2D and 3D cultures, incorporating either type I collagen or Matrigel during hepatic specification and maturation. The resulting cells were characterized with various molecular and cellular techniques for their hepatic functionality.

Results

Our results showed that HLCs cultured with collagen exhibited a significant increase in albumin and alpha 1 anti-trypsin accompanied with reduced AFP compared to HLCs cultured with Matrigel and that they also secreted more urea than Matrigel cells. However, these HLCs exhibited lower CYP3A4 activity and glycogen storage than those cultured with Matrigel. The functional differences in HLCs between collagen and Matrigel cultures closely resembled the hepatocytes of periportal and pericentral zones, respectively.

Conclusion

Our study demonstrates that Matrigel and collagen have differential effects on the differentiation and functionality of HLCs, which resemble, to an extent, hepatic zonation in the liver lobules. Our finding has an important impact on the generation of hPSC-HLCs for biomedical and medical applications.

Extracellular matrix

Hepatocyte

hESC differentiation

hepatic zonation

Matrigel

Collagen.

Liver is an important organ in our body as it plays an essential role in metabolism and detoxification and is responsible for the maintenance of body homeostasis [1]. Therefore, it is not surprising that liver disease is one of the major contributors to human morbidity and mortality [2]. Furthermore, the liver is also associated with the pathogenesis of diseases like malaria as parasites require liver cells to amplify themselves before causing the disease [3]. Developing new treatments or prevention strategies for liver and liver-associated diseases will greatly reduce the burden on healthcare services and improve human health. As such, it is important to understand the mechanisms of human liver pathogenesis and establishing in vitro and in vivo liver models can greatly facilitate such studies.

Since hepatocytes constitute over 70% of the liver mass and carry out most of the liver functions, obtaining functional hepatocytes is vital for the establishment of an effective liver model [1]. With the limited availability of primary human hepatocytes from donors and the defective nature of hepatocarcinoma cell lines, human pluripotent stem cell (hPSC)-derived hepatocytes offer a valuable consistent source for human hepatocytes as hPSCs, derived from normal human cells, can self-renew indefinitely and thus are available to produce hepatocytes on demand [4–6]. The differentiation procedure from hPSCs to hepatocytes emulates the embryonic development of the liver, in which the hepatocytes originate from the definitive endodermal epithelium of the embryonic foregut and then become hepatic endoderm influenced by signals from adjacent mesodermal cells [7]. The hepatic endoderm gives rise to non-polarized hepatoblasts that invade adjacent mesenchyme to generate the liver bud. The hepatoblasts can then differentiate into both hepatocytes and biliary epithelial cells (BECs) depending on signalling regulation [8–11]. Although our current differentiation approaches are able to efficiently generate hepatocyte-like cells (HLCs) from hPSCs, expressing most of the hepatocyte markers, the maturation and functionalities of these HLCs are still not comparable to primary hepatocytes [3, 12–16].

Hepatocyte maturation is a complex process, requiring interaction between cells, extracellular matrices (ECM) and various substances secreted by other cell types, such as Kupffer cells, endothelial cells, or stellate cells [17–19]. Furthermore, the liver is a structurally complex and highly organized organ composed of repeating hexagonally shaped functional lobules with a portal triad in each corner and a central vein in the centre (Fig. S1A). Blood enters the lobules from the portal triad and flows radially toward a draining central vein, which divides the intra-lobular hepatocytes into 3 distinct functional zones, with zone 1 close to the portal triad and zone 3 around the central vein [20]. Increasing evidence shows that the functionality of a hepatocyte is also regulated by spatial signals relevant to its position in the liver lobules [21–23]. As yet, it is a huge challenge to generate functional hepatocytes and liver tissues from hPSCs.

ECM play an important role in liver development and hepatocyte differentiation through cell-ECM interactions, so evidently, aberrant ECM expression can lead to liver disorders [24, 25]. The liver contains multiple ECM components with collagens and fibronectin as the main constituents, while laminin is predominantly found in the bile ductules [26]. However, the effects of these ECM in the modulation of hepatocyte maturation and functions are not completely understood. In this study, we investigated the effects of growth factor-reduced Matrigel (abbreviate as MG hereafter) and type I collagen (as COL hereafter) on the differentiation, maturation, and function of human embryonic stem cell-derived HLCs in both monolayer and 3D spheroid culture systems. We show that MG and COL have differential effects on the functions of the HLCs and generate HLCs with functional features of different hepatic zones.

hESCs culture and hepatic differentiation

H1 and H7 hESC lines, purchased from WiCell Research Institute (Madison, WI, http://www.wicell.org), were routinely cultured on MG-coated plates in mouse embryonic fibroblast-conditioned knockout serum replacement (KSR) medium supplemented with 10 ng/ml of bFGF (brief as MEF-CM) [27]. All the cells were cultured in incubator at 37°C, 5% CO₂.

Hepatic differentiation (Fig. 1A) was modified from the previously published method [28] and initiated by plating accutase-dissociated 5 × 10⁵ hESCs onto 6-well plates coated with MG (Corning), allowing them to adhere in MEF-CM medium. The next day, the medium was changed to RPMI-B27 supplemented with 100 ng/ml Activin A (Qkine) and 15 nM Torin2 (Bio-Techne) for three days, followed by a 5-day treatment with 20 ng/ml BMP4 and 20 ng/ml bFGF (Both from PeproTech) in DMEM containing 20% KSR. Finally, the maturation medium of Hepatocyte culture medium (Lonza) supplemented with 20 ng/ml OSM (Biotech) and 20ng/ml HGF (PeproTech) was applied for 5 days. For 3D culture, the hepatic differentiating cells at day 4 of the differentiation shown above were collected and plated into 24-well AggreWell plates (STEMCELL Technologies) following the manufacturer’s instruction, then continued to be culture in the same medium for 3–4 days with indicated ECM. The resulting spheroids were transferred into suspension culture in hepatic maturation medium for 5 days (Fig. 2A).

Quantitative reverse transcription–PCR (qRT–PCR).

Total RNA was isolated from cells using the Tri Reagent (Sigma) and reverse transcription was performed with Protoscript II Reverse Transcriptase (NEB). PCR was carried out using SYBR Green JumpStar Taq ReadyMix (Merck) in the StepOnePlus real-time PCR system (Applied Biosystems). Primer sequences are provided in Table S1 and two housekeeping genes, β-ACTIN and RPL22 were used, which showed similar patterns. Thus, the relative quantification of target gene expression was calculated using the 2^−ΔCt method with RPL22 as the normalizer.

Immunostaining of cells

Cells were fixed with 4% paraformaldehyde for 15 min, followed by incubation for 1 hour with PBS containing 10% donkey serum, 3% BSA and 0.3% triton (Sigma-Aldrich) for permeabilization and blocking non-specific binding. The cells were then incubated overnight with primary antibodies at 4°C and 1 hour with fluorescence-conjugated secondary antibodies with PBS washes between. Finally, the cells were stained with DAPI (Sigma-Aldrich) for nuclei and mounted using Mowiol 4–88 solution. All procedures were performed at room temperature unless stated. Antibodies used in this study: mouse anti-albumin (Merck, A6684, 1:100), rabbit anti-AFP (Abcam, ab169552, 1:500), rabbit anti-E-cadherin (CST, #3195, 1:200), rabbit anti-Glucokinase (abcam, ab88056, 1:100), rabbit anti-LEF1 (CST, #2230, 1:200), donkey anti-rabbit IgG (H + L), Alexa Fluor™ 488 (Fisher, A21206, 1:500), donkey anti-Rabbit IgG (H + L), Alexa Fluor® 568 conjugate (Fisher, A10042, 1:500).

Periodic acid-Schiff (PAS) staining

Following 4% paraformaldehyde fixation and PBS washes, cells were stained using the PAS kit following the manufacturer’s instruction (Merck). Briefly, cells were incubated for 5 min in 1% periodic acid solution followed by washing with distilled water for several minutes and then immersing into Schiff’s reagent for 20 min. After washing with tap water, cells were counterstained with haematoxylin solution for 20 sec before visualizing them under Nikon TE2000 microscope.

Albumin ELISA, Urea Assay and CYP3A4 activity assay

Supernatants of the HLC cultures were collected at day 13 of the differentiation after 24 hours incubation with cells and the secreted ALB protein levels were measured by the human Albumin ELISA kit (Merck) according to the manufacturer’s instructions. Medium incubated for 24 hours in the absence of cells was used as the blank controls. After collection of supernatants, cells were collected and counted. Urea assay was performed similarly using the urea assay kit (Abcam) following the instructions of the manufacturer. CYP3A4 activities were measured at the end of differentiation (day 13) in either untreated or after rifampicin (25 µM) stimulation using the P450-Glo CYP3A4 assay (luciferin-IPA) kit (Promega) following the manufacturer’s instructions.

BODIPY staining

4% paraformaldehyde-fixed HLCs were incubated in the dark with BODIPY 493/503 (Fisher, 1:1000 in PBS) for 30 min and then counterstained with DAPI after washing. The LD signals were finally captured using a Leica SP5 II confocal fluorescent microscope. Quantification and image analysis were performed using CellProfiler software (https://cellprofiler.org/).

Statistical analysis

Unpaired two-tail t-test, welch t-test, one-way or two-way ANOVA were used for statistical analysis with GraphPad Prism 8 or 9 (GraphPad, USA) on at least three independent experimental samples. P < 0.05 was considered a statistically significant difference.

COL improved the albumin secretion of hESCs-derived HLCs

COL is one of the most abundant ECM in the liver, while MG is the most used ECM for hESC culture and hepatocyte differentiation [12, 13, 29]. MG is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma and is primarily composed of laminin, collagen IV, and heparan sulphate proteoglycan [30]. Given the distinct composition between MG and COL, we asked whether different components of ECM may contribute differently to the maturation and function of hESCs-derived HLCs. To address this, early hepatic differentiating cells that were generated following the definitive endoderm formation from hESCs (both H1 and H7 lines) were split and plated onto either MG- or COL-coated plates and continued being cultured for hepatic specification and maturation (Fig. 1A). During this process, cells from both MG and COL cultures showed no clear difference in their overall morphology although COL cells looked slightly bigger than the MG cells (Fig. 1B; Fig. S1B). However, the expression of key hepatocyte markers, albumin (ALB), alpha 1 anti-trypsin (A1AT), and alpha-fetoprotein (AFP) showed significant differences between HLCs cultured on MG and COL (Fig. 1C-E; Fig. S1C). The transcripts of ALB and A1AT were significantly more abundant in HLCs on COL-coated dishes than in MG-coated ones, whereas AFP mRNA was higher in MG HLCs (Fig. 1C; Fig. S1C). The protein expression of ALB and AFP corresponded to the transcript expression, showing higher ALB and lower AFP in HLCs cultured on COL than MG (Fig. 1D, E; Fig. S1D). Moreover, albumin secretion was higher in COL HLCs than in MG (Fig. 1F; Fig. S1E). Since adult hepatocytes produce more ALB while foetal hepatocytes express higher AFP [31], the data indicate that COL may enhance the maturation of hESCs-derived HLCs.

To further validate this finding, we also examined the effects of MG and COL on HLCs in 3D cultures. hESCs were initially differentiated into early hepatic cells as in 2D culture, which were then collected on day 4 of the differentiation (Fig. 2A) and plated into 24-well AggreWell plates in hepatic specification medium supplemented with MG or COL for further differentiation and maturation until day 13 (Fig. 2A, B). The resulting spheroids were analysed for the expression of AFP, ALB, and A1AT. Like the 2D cultures, higher expression of ALB and A1AT as well as higher albumin secretion were detected in spheroids of COL cultures than that of MG, although supplementing MG exhibited improved ALB and A1AT expression compared to no ECM (Fig. 2C-E; Fig.S2A, B). These data further support that COL enhances the maturity of the HLCs, and moreover, they also revealed that 3D cultures showed lower AFP and higher ALB in comparison to the monolayer cultures with the same ECM (Fig. 1 vs Fig. 2), indicative of a more efficient hepatocyte maturation in 3D cultures.

COL and MG differentially affect ureagenesis and CYP3A4 activity in hESC-HLCs

The liver is an important organ for detoxification; thus, to further explore the impact of ECM on these functions in hESCs-derived HLCs, we compared ureagenesis in HLCs cultured with COL and MG using both monolayer and 3D culture systems. In both 2D and 3D cultures, HLCs cultured with COL produced more urea than those cultured with MG (Fig. 3A, B; Fig. S3A, B). Interestingly, MG did not improve ureagenesis in the 3D culture (Fig. 3A, B; Fig. S3A, B). Moreover, ureagenesis was over 10-fold higher in 3D culture than in 2D regardless of the ECM supplementation. These results support our observations in Figs. 1 & 2, indicating that both COL and 3D culturing improve hESCs-derived HLCs maturation and functionality.

Next, the expression of cytochrome P450 enzymes, particularly CYP3A4 and CYP3A7 were examined in these cells. CYP3A4 is the most abundant drug-metabolizing enzyme in the human liver, responsible for the phase I metabolism of dietary compounds and over 50% of prescribed drugs, while CYP3A7 is the predominant P450 enzyme in human foetal and infant liver tissues [32, 33]. Surprisingly, both CYP3A4 and CYP3A7 transcripts were higher in the HLCs cultured with MG than those with COL, regardless of whether they were grown in 2D or 3D cultures (Fig. 2C, D; Fig. S3C, D). Although there was no difference in the basal activity of CYP3A4 in HLCs between MG and COL cultures, a significantly higher CYP3A4 activity was detected in the HLCs of MG cultures than those of COL upon rifampicin stimulation, despite all of them showing an overall increase in response to rifampicin (Fig. 3E, F; Fig. S3E, F).

Together, these data indicate that HLCs maturation and function might be differentially regulated by COL and MG and cannot be simply described as positive effects of either COL or MG on HLCs culture.

MG showed stronger glycogen accumulation in HLCs than in COL

Since MG and COL revealed diverse effects on different functions of hESCs-derived HLCs, we asked whether they may also have differential effects on cell metabolism. Glucose and lipid metabolism are important functions of hepatocytes. The conversion of glucose into glycogen by the liver is a major pathway to maintain blood glucose homeostasis. Therefore, we studied glycogen synthesis and storage by the HLCs cultured with MG or COL. Interestingly, the HLCs cultured on MG showed stronger PAS (periodic acid-Schiff) staining than cells grown on COL (Fig. 4A; Fig. S4A). Moreover, liver-specific glycogen synthase (GYS2) also exhibited higher expression in MG cultured HLCs than COL HLCs (Fig. 4B, C; Fig S4B, C). Together, these data indicate that HLCs cultured on MG have stronger glucose-glycogen conversion than those cultured on COL. Additionally, we also looked at glucokinase (GK) expression in our cultures because it plays a key role in glycogen synthesis and glycolysis by catalysing the reaction to convert glucose to glucose-6-phosphate and is tightly regulated, only being active when it is in the cytoplasm [34]. In our 2D cultures, we detected GK in the cytoplasm of HLCs on both COL and MG (Fig. 4D) whereas, in 3D cultures, GK showed distinct localisation depending on whether the cells were cultured with COL or MG (Fig. 4E). It revealed a strong nuclear expression in COL 3D cultures whereas in MG cultures, it had more diffuse, predominantly cytoplasmic localization (Fig. 4E). These data suggest that MG may promote more glycogen synthesis and glycolysis in HLCs than COL, particularly in the 3D culture system.

HLCs cultured on MG and COL exhibited different patterns of lipid droplets

Next, we examined the effects of MG and COL on lipid metabolism by studying the presence and size of lipid droplets (LDs) because hepatocytes store both dietary fatty acids and de novo-generated lipids in the form of LDs (Gluchowski et al., 2019). BODIPY staining, a method that stains LDs, was applied to HLCs on day 13 of their differentiation on MG or COL. Both MG and COL HLCs exhibited positive staining of LDs (Fig. 5A; Fig. S5A) and their quantitation showed that HLCs cultured on COL had significantly more and larger LDs than HLCs cultured on MG (Fig. 5B,C). However, the genes involved in lipogenesis, such as sterol regulatory element-binding protein 1 (SREBP1), its downstream lipogenic enzyme fatty acid synthase (FASN) and the fatty acid esterification enzyme DGAT2 (diacyl glycerol transferase-2), were expressed significantly higher in MG-cultured HLCs than in COL HLCs (Fig. 5D, E; Fig. S5B, C), indicating a lower lipogenesis in the COL HLCs. These data suggest that higher LD contents in COL-cultured HLCs may not result from increased lipogenesis but possibly from high fatty acid (FA) uptake from the medium. It has been reported before that COL increases triglyceride content and free FA uptake in human podocytes by activating discoidin domain receptor 1 (DDR1) and consequently increasing FA uptake [35, 36]. Excessive FA is esterified to LD with the help of the endoplasmic reticulum (ER) enzyme, DGAT1, to prevent lipotoxicity [37, 38]. Indeed, when we checked DGAT1 expression in our cultures, HLCs on COL expressed significantly higher DGAT1 than on MG (Fig. 5F, G; Fig. S5D, E), supporting that higher LDs in COL-cultured HLCs might be associated with an increased FA uptake.

MG and COL differentially modulate HLCs maturation and functionality representing different hepatic zones

Our data above clearly demonstrate that MG and COL differentially regulate the differentiation, maturation and functionality of the hESCs-derived HLCs. COL revealed enhancement in ALB production and ureagenesis in HLCs, while MG showed improvement in the expression and function of CYP3A and promoted glycolysis and glucose-glycogen conversion (Fig. 6A). Altogether, it appeared that the differential effects of MG and COL on HLCs correspond well with hepatic zonation in the liver lobules [21, 39, 40]. MG facilitated the differentiation of early-differentiating hepatic cells into HLCs of perivenous areas, whilst COL aided their differentiation into hepatocytes of periportal areas (Fig. 6A). Moreover, glutamine synthetase (GS), an established hepatocyte zonation marker abundantly expressed in the perivenous area [40], had significantly higher expression in the HLCs cultured with MG than with COL (Fig. 6B; Fig. S6A), supporting that MG and COL in HLC cultures affect their differentiation and function to resemble hepatocytes of different hepatic zones.

Furthermore, it has been reported previously that the Wnt/β-catenin signalling pathway plays a vital role in the establishment of hepatocyte zonation and is more active in the perivenous area [41–43]. To explore whether Wnt signalling could be different in MG and COL cultured HLCs, we verified the expression of Wnt direct target genes - LEF1 and AXIN2 and showed that both were expressed significantly higher in HLCs of MG cultures than COL (Fig. 6C, D; Fig. S6B, C). Correspondingly, immunostaining also showed that LEF1 protein was more abundant in HLCs from MG cultures than COL (Fig. 6E; Fig. S6D), supporting that MG and COL may affect hepatocyte zonation through regulating Wnt signalling.

In this study, we investigated the effect of COL and MG on hESCs-derived HLCs maturation and function using various assays and showed that COL and MG differentially affect the specification and functions of hESCs-derived HLCs. Interestingly, the differential effects of MG and COL appear to be associated with liver zonation [20, 22, 39]. The finding that COL promotes differentiating HLCs more toward periportal zone 1 cells corresponds with higher expression of COL in the portal stroma (Sánchez-Romero et al., 2019). Therefore, our data demonstrate that MG and COL differentially modulate HLCs with zonation characteristics in the liver lobules and that ECM play an important role in regulating the differentiation, maturation, and function of hepatocytes from early hepatic progenitors.

Many factors are thought to contribute to the formation of liver lobule zonation, such as nutrients, oxygen, hormones, and morphogens. Among them, nutrients and oxygen are high in periportal areas, and form a gradient along the portal-central axis [21, 44, 45]. In our experiments, both MG and COL are applied to parallel HLC cultures with the same source of differentiating cells and culture medium, therefore, cell-autonomous and other environmental factors, except MG and COL, are unlikely to account for the differential zonal functions in these HLCs. Additionally, our results show that HLCs cultured with MG exhibit higher Wnt activity than cells cultured with COL, indicating that MG and COL may differentially regulate HLCs zonal functions through divergent effects on Wnt signalling since it is crucial for hepatic zonation and is highly active in the perivenous region of the liver lobules [41–43]. However, a previous work report that purified individual ECM, including laminin, type I or IV collagen, and fibronectin, have no differential effects on mouse hepatic stem cells regarding the generation of ALB-producing cells [46]. Given that MG is not only an assortment of ECM proteins but also contains multiple growth factors [30], we cannot completely exclude the possibility that residue growth factors might still affect Wnt signalling although a growth factor-reduced MG was used in our study. Therefore, further studies are required to dissect the exact mechanisms by which COL and MG may regulate Wnt signalling and hepatic functional zonation during hepatocyte differentiation, possibly using a defined individual or combination of ECM.

Our studies provide strong evidence that MG and COL differentially affect HLCs functions, demonstrating the importance of ECM in modulating differentiation and functionality of human hepatocytes derived from hPSCs. These findings will benefit researchers working in the field of hPSCs to derive human hepatocytes for regenerative medicine, disease modelling and drug discovery. It becomes increasingly clear that a single hepatocyte does not perform all the hepatic functions as the spatial localization in the liver lobules influences their functionality. Application of different ECM may facilitate the generation of specific groups of hepatocytes for the functions of our interests. In addition, our findings, together with tissue engineering technology, could facilitate the generation of liver tissues that resemble the liver lobular structure by applying different ECM along the periportal-pericentral axis.

A1AT, alpha-1 antitrypsin; AFP, α-fetoprotein; ALB, albumin; COL, type I collagen; CYP450, cytochrome P450; ECM, extracellular matrix; FA, fatty acid; GS, glutamine synthetase; GK, glucokinase; GYS2, glycogen synthase 2; hESCs, human embryonic stem cells; hPSCs, human pluripotent stem cells; HLCs, hepatocyte-like cells; LD, lipid droplet; MG, Matrigel.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

All data and materials except hESCs will be available. The hESCs are under original MTA restrictions.

Competing interests

The authors declare no potential conflict of interest.

Funding

Faiza Farhan is sponsored by the Punjab Educational Endowment Fund (PEEF) PhD scholarship; The study is supported by the Genesis Research Trust (Genesis fund 119) and Imperial Confidence in Concept (ICiC, P80898). The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Author contributions

FF and WC conceived the study and experimental design. FF, MT, and PW executed the experiments. WC, FF, and MT contributed to the writing or editing of the manuscript.

Acknowledgement

The authors thank all the members of the Stem Cell Differentiation group for their support throughout the project.

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Extracellular matrices modulate differentiation of human embryonic stem cell-derived hepatocyte-like cells with spatial hepatic features

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