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Rotating culture regulates the formation of HepaRG-derived liver organoids via YAP translocation

Abstract

Background

Liver organoid serves as an alternative model for liver pathophysiology in carbohydrate or lipid metabolism and xenobiotic metabolism transformation. Biomechanical cues including spaceflight mission can affect liver organoid construction and their related functions, but their underlying mechanisms are not fully understood yet. Here, a rotating cell culture device, namely Rotating Flat Chamber (RFC), was specifically designed for adhering cells or cell aggregated to elucidate the effects of altered gravity vector on HepaRG-derived liver organoids construction.

Results

The organoids so formed under RFC presented the fast growth rate and large projection area. Meanwhile, the expressions of two pluripotency markers of SOX9 and CD44 were enhanced. This finding was positively correlated with the increased YAP expression and nuclear translocation as well as the elevated α4β6-integrin expression. Inhibition of YAP expression and nuclear translocation decreased the expression of SOX9 and CD44 under RFC, thereby attenuating the pluripotency of HepaRG-derived liver organoids.

Conclusions

In conclusion, we proposed a novel liver organoid construction method using rotating culture, by which the pluripotency of liver organoids so constructed is mediated by α4β6-integrin and YAP translocation. This work furthered the understanding in how the gravity vector orientation affects the construction of liver organoids and the related mechanotransductive pathways.

Background

The liver plays a vital role in various physiological processes within the body, including nutrient anabolism, endocrine control, immune regulation, and lipid homeostasis [1]. Given the importance of hepatic functions, liver diseases, such as hepatitis, fibrosis, and cirrhosis, often result in morbidity and mortality [2], and yet the primary treatment for end-stage liver diseases highly relies on orthotopic liver transplantation. Due to the scarcity of liver donors and other constraints, only less than 2% of liver failure patients undergo liver transplantation, making liver cell transplantation as an effective alternative to the whole liver organ transplantation procedure [2]. Evidently, primary human hepatocytes derived from donor organs cannot meet the demand for liver cell therapy, and, thus, the liver organoid serves as a potential strategy not only for mimicking physiological functions in vivo but also potentiating the pathological therapy of liver diseases.

Liver organoid is usually constructed in vitro through self-assembly of stem cells [3], as first observed in the intestinal organoids [4]. In addition to those biochemical factors that can regulate various behaviors of morphology, spreading, migration, proliferation, and differentiation of composing cells [5,6,7], mechanical factors are also critical in modulating liver organoid formation since those composing liver (progenitor) cells reside within a mechanical microenvironment of the liver organ. Typically, these factors encompass mechanical loads (such as shear stress, mechanical stretch, or blood pressure), extracellular matrix (ECM) stiffness, substrate microtopography, and cell or clone shape control. Thus, the related mechanical regulation is meaningful for liver organoid construction, not only on-ground but also in space when gravity alters. After 13.5 days spaceflight, for example, the mouse accumulates more lipid droplets with reduced retinol content in hepatocytes, increased liver weight and enhanced liver ECM deposit, leading to a sigh or phenotype of non-alcoholic fatty liver disease (NAFLD) and fibrosis [8]. In fact, astronauts also face the challenges of liver enlargement and the increased risk of liver diseases due to the dysregulation of liver metabolism (such as fatty liver, diabetes) after several months of spaceflight [9].

Meanwhile, ground-based clinostats can apply continuous rotation to alter the sample’s orientation against gravity vector, used to refer as “time-averaged zero gravity vector condition” [10]. Based on the hypothesis that cells possess “gravity receptors” and the gravity vector alters much faster than cellular gravity perception time, clinostats could potentially be utilized to mimic typical biological effects under microgravity, as indicated that rotating cell culture can influence the proliferation and differentiation of liver (progenitor) cells or tissue morphology [11], and even lead to abnormal liver function [12, 13]. However, hepatocyte proliferation seems to exhibit varied readouts when using different clinostat tools, as exemplified by promoting the proliferation of HepG2 cells cultured in a rotating wall vessel (RWV) [14, 15] but inhibiting the growth of CCL-13 or HepG2 cells cultured in random positioning machine (RPM) [16, 17]. While these cues offer basic insights into understanding the regulation of hepatocyte functions by gravity vector orientation, it remains unclear whether the construction of liver organoids is affected by the alteration in gravity vector.

Moreover, cell–cell interactions and the relevant molecular mechanisms are crucial for liver organoid formation and function. For instance, the Hippo signaling pathway inside the composing cells is associated with proliferative growth and stemness maintenance of stem cells [18], in which Yes-associated protein (YAP) is a key effector in regulating organogenetic development, homeostasis, and regeneration [19, 20]. Upon activation, YAP tends to translocate into the nucleus, binds to the transcriptional factors, and induces the transcription of target genes, thereby regulating various cell behaviors [21, 22]. Specifically, YAP can also respond to gravitational alteration. Adult cardiovascular progenitor cells cultured on the International Space Station or two-dimensional clinostat can induce gene expression of YAP and its target superoxide dismutase 2 (SOD2), a marker of cell survival [23]. In contrast, YAP nuclear translocation is impaired in glioblastoma cells or mesenchymal stem cells when the cells are cultured in RPM or so-called microgravity-on-a-chip respectively [24, 25].

Cells are able to sense the changes in extracellular mechanical cues via various mechanoreceptor. For instance, integrin, composed of a heterodimer protein with α and β subunits linked by non-covalent bonds, serves as the first sensing protein along the mechanosignaling axis. Its headpiece can interact with various ECM ligands, such as collagen I, laminin, or fibronectin, leading to the conformational changes in the intracellular tail of β subunit and recruiting the ankle protein talin that binds to vinculin and, in turn, F-actin [26]. While integrin is known to provide a mechanical connection between the ECM and the cytoskeleton [27], it is not clear whether YAP and integrin play a role in hepatic cell-derived organoid formation under gravitational alteration.

Here we aimed to elucidate how rotating culture regulates liver organoid formation and what underlying mechanotransductive mechanisms are. The roles of YAP and related mechanosensing protein integrin were discussed specifically. We found that rotating culture favored the stemness maintenance in liver organoids, mainly attributed to the enhanced YAP expression and translocation along α4β6-integrin mediated mechanotransductive pathway.

Results

Rotating culture promotes the formation of HepaRG-derived liver growth organoids

A rotating device, namely Rotating Flat Chamber (RFC), was used for adhering cells or cell aggregates, as reported previously [28]. This RFC was in-house developed by the National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, consisting of the cell culture chamber(s), a rotating platform, a motor, and other accessory components and allowing rotation at varied speeds along the long axis (Additional file: Fig. S1A, B). Human liver progenitor HepaRG cells were mixed with matrigel domes in the culture chamber for 7 days for self-assembly to form liver organoids (HepaRG-derived liver growth organoids, HGOs) (Fig. 1A). To elucidate the effect of rotating culture on the promotion of HGO formation, time courses were tested at days 1, 3, 5, and 7 (1, 3, 5, and 7 day). Optical imaging showed that HepaRG cells under both RFC and static control formed irregular clusters of grape strings right after 1 day and the oval-like organoids were then presented at 3 days (Fig. 1B). Morphological parameters of HGOs also indicated that the maximum projected area and circularity increased but the aspect ratio decreased with time (Fig. 1C–E). Compared with static control, the HGOs grown under RFC presented a larger projected area on the first 5 days and tended to be stable on 7 days without significant difference (Fig. 1C), implying that the rotating culture promoted the formation of HGOs especially at the early stage. Similar time-dependence was observed for HGOs circularity and aspect ratio, in which the circularity was higher but the aspect ratio was lower on the first 3 days for HGOs under RFC and comparable beyond the 5 days. Specifically, the HGOs under RFC were rounder than those in the control, yielding the values of circularity and aspect ratio closer to unity (round shape) at the early stage of 3 days followed by stable shape at 7 days (Fig. 1D, E).

Fig. 1
figure 1

Rotating culture promotes the growth and proliferation of HepaRG-derived liver growth organoids (HGOs). A Schematic of HGO construction using a rotating flat chamber (RFC) device. B Images of HGOs at different growing stages under RFC. Cells cultured in static culture chamber served as control. Arrows indicated the typical HGOs. Scale bar = 50 μm. C–E Quantification of maximum projected area (C), circularity (D) and aspect ratio (E) of HGOs at different stages. n ≥ 30 (n represents the number of all samples tested). F Representative stained images of EdU+ cells at 3 or 7 days under RFC. Cells cultured in static culture chamber served as control. Scale bar = 20 μm. G Quantification of EdU+ cells proportion normalized to Hoechst+ cells. n ≥ 45. Data were presented as the mean ± SEM. P < 0.05*, 0.01**, 0.0001****, compared to the control at the same time point in C–E with two-way ANOVA analysis

In addition, the proliferation capability of HGOs at 3 and 7 days was tested. Data indicated a higher rate of EdU+ cells under RFC compared to the control at 3 days, while the rate even decreased at 7 days under RFC compared to that at 3 days, mainly due to long-term cell proliferation, but remained significantly higher than the control (Fig. 1F, G). No dead cells were observed in HGOs under both culture conditions at 3 or 7 days through live and dead staining (Additional file: Fig. S1C, D), indicating that the device was suitable for long-term cultivation of HGOs. These results indicated that the HGOs were more sensitive to the mechanical rotation at the early stage (3 days), with higher proliferation rates and larger and rounder shapes, and reach to the steady status at the late stage (7 days). From these observations, two typical time points of 3 days and 7 days were selected for the following functional tests.

YAP and α6β4-integrin are the key proteins in rotation-induced HGO formation

To map the potential signaling molecules during the formation of HGOs, genome-wide RNA-seq was performed for the gene expression profiles of HGOs after rotating culture for 1, 3, or 7 days when static culture served as control. Results from the cluster heat map of differentially expressed genes (DEGs) showed that no significant differences of gene expression profiles were found between RFC and control cultures at 1 or 3 days. Only 7-day-rotating culture induced obvious clustering changes in the gene expression of HGOs, presumably due to the time-accumulated effects of such the rotating culture (Additional file: Fig. S2A). Compared with the data at 1 or 3 days where only a few DEGs were identified (Additional file: Fig. S2B), total 948 DEGs (620 up- and 328 down-regulated) were identified at 7 days (Fig. 2A), with well-defined clustering heat map (Fig. 2B). Among those upregulated DEGs of interest were typically stemness markers (e.g., SOX9 and CD44), mechanosensitive molecules (e.g., integrins and YAP1), skeleton proteins (e.g., PXN, TUBB4A, and FLNB), and a marker of cell survival (SOD2) (Fig. 2F).

Fig. 2
figure 2

Genome-wide RNA-seq assays and GO enrichment and KEGG pathways analyses. A Volcanic maps of differentially expressed genes (DEGs) between rotating and static cultures at 7 day. Blue, red, and gray dots represent downregulated, upregulated, and unchanged genes, respectively. B The heat map of expression patterns of the whole genome at 7 days. C, D The Gene Ontology (GO) analysis of biological processes (C) or cell components (D) at 7 days. D The bar chart of those enriched KEGG pathways at 7 day. The length depends on the number of genes enriched in the pathway, and the color indicates the significance of the enrichment of the pathway. F The heat map of interest of gene expression patterns of the whole genome at 7 days

Next, gene ontology (GO) enrichment analyses were conducted on the identified DEGs to reveal biological processes (BP) and cell components (CC). For the former, DEGs at 7 days were enriched in the cellular processes, biological processes, cell communication, multicellular biological development, and signal transduction regulation (Fig. 2C). For the latter, the DEGs were enriched in different localized organelles, ECM, cytoskeleton, cell junction, and cell–cell junction (Fig. 2D). Here the cell junction is essentially meaningful for organoid formation, enabling tight connections among cells or between cells and ECM to maintain the structure and function of HGOs. Additionally, the Kyoto Encyclopaedia of Genes and Genomes enrichment (KEGG) analysis defined the top 20 important pathways from those identified DEGs, including PI3K-Akt, HIF-1, MAPK, Hippo, PPAR, JAK-STAT, TNF, and FOXO-related signaling pathways (Fig. 2E). These pathways were involved in biological processes such as cell proliferation, growth, and differentiation. In this work, we mainly focused on the mechanosensitive molecule (α6β4-integrin) and the key effector YAP in the Hippo pathway, and explored whether the rapid growth of HGOs under rotating culture was caused by increased expressions of stemness markers SOX9 and CD44 and the regulation of mechanosensitive α6β4.

Rotating culture enhances the stemness of HGOs

Based on the above RNA-seq data, we performed the first functional test to determine if the HGOs formed under rotating culture is able to maintain their stemness when considering that HepaRG is a type of hepatic bipotent progenitor cell. Immunostaining of two typical stemness markers, SOX9 and CD44, presented distinct distributions under RFC or in static control. At the early stage (3 day), SOX9 expression pattern was seemingly identical from cell to cell throughout the organoids for both cultures, and uniformly distributed within individual cells in the control but prominently polarized at the edges of individual cells under RFC (Fig. 3A). Again, uniform distribution of CD44 expression was visualized throughout the organoids, with the polarized expression at the edges of individual cells under RFC and in the control (Fig. 3B). At the late stage (7 day), the number of cells inside the organoid was increased and a large three-dimensional (3D) sphere was formed. SOX9 expression yielded uniform distributions throughout the organoids and within individual cells in the control but was accumulated at the periphery of the organoids and uniformly distributed within individual cells (Fig. 3C). CD44 expression was likely concentrated at the periphery of the organoids in the control but uniformly distributed throughout the organoids under RFC, all polarized at the edges of individual cells (Fig. 3D). The protein expressions were detected by simple western (Wes) test [29]. In addition, the relevant quantitative analysis indicated that SOX9 and CD44, at 3 days, were significantly increased under RFC compared to those in the control, consistent with the enhancement in mRNA expression of SOX9 and CD44 (Fig. 3E, F). Similarly, both the protein and mRNA expressions of the two markers at 7 days were also significantly increased (Fig. 3G, H), suggesting that rotating culture is beneficial to the stemness maintenance during HGO formation. Finally, time course comparisons of SOX9 and CD44 expressions at 3 and 7 days showed that SOX9 expression was enhanced with time under RFC but not in the control (Additional file: Fig. S3A, B). Similar enhancement of CD44 expression with time was also observed under RFC, except that CD44 gene expression tended to decrease with time slightly in the control (Additional file: Fig. S3C, D). These data implied that rotating culture could maintain or even enhance the multi-potentiality of liver organoids for a relatively long period in the current settings.

Fig. 3
figure 3

Rotating culture maintains the stemness of HGOs at 3 or 7 day. A–D Representative stained images of SOX-9 (red, A, C), CD44 (yellow, B, D), F-actin (green), and Hoechst (blue) in HGOs at 3 (A, B) or 7 days (C, D) under RFC. Cells cultured in static culture chamber served as control. Scale bar = 20 μm. E, G Simple western analysis and quantification of SOX9 (left) and CD44 (right) protein expression in HGOs at 3 (E) or 7 days (G). F, H qPCR analysis of SOX9 (left) and CD44 (right) mRNA expression in HGOs at 3 (F) or 7 days (H). Data were presented as the mean ± SEM. N ≥ 3 (N represents the biological repeats). P < 0.05*, 0.01**, 0.0001****, compared to the control in E–H with two-tailed Student’s t test

Rotating culture promotes YAP translocation translocation via α6β4-integrin signaling

Rotating culture provides centrifuge forces on the HGOs seeded on the substrate of the RFC chamber, and, thus, those well-known mechanosensing proteins were first tested, based on the above RNA-seq data for the enhanced mRNA expressions of α6β4-integrin and YAP (cf. Fig. 2), to decipher the molecular mechanisms in the increased expression of stemness markers of HGOs under RFC. On the one hand, α6β4-integrin is specifically involved in signal transduction during cell movement, growth, and even cell survival [30] and also found here to play a significant role in the rotating culture-induced organoid formation (cf. Fig. 2). Here both Wes and qPCR data showed the significantly enhanced expression of α6 and β4 subunits (Fig. 4A, B; Additional file: Fig. S4 A, B). Immunostaining of these two subunits presented that α6β4-integrin was highly expressed at the periphery of the organoids and diffusely distributed inside the organoids, with diffuse distributions within individual cells. This expression pattern was consistent between 3 and 7 days or between rotating and static cultures (Fig. 4C–F; Additional file: Fig. S4C, D). Semi-quantitative analysis also showed that, at 3 days, both α6 and β4 subunit expressions were significantly higher under RFC (Additional file: Fig. S4E). Moreover, α6β4-integrin expression at both protein and gene levels was relatively higher under RFC at 7 days (Fig. 4A, B; Additional file: Fig. S4F), suggesting that α6β4-integrin might sense the external forces via rotating culture and then mediate the stemness maintenance of HGOs under RFC, especially at the early stage.

Fig. 4
figure 4

Rotating culture promotes α6- (ITGA6) and β4- (ITGB4) integrin subunit expressions in HGOs at 7 days. A Simple western analysis and quantification of ITGA6 (left) and ITGB4 (right) protein expression in HGOs. B qPCR analysis of ITGA6 (left) and ITGB4 (right) mRNA expression in HGOs. N ≥ 3. C, D Representative stained images of ITGA6 (orange, C), ITGB4 (red, D), F-actin (green) and Hoechst (blue) in HGOs under RFC. Cells cultured in static culture chamber served as control. Scale bar = 20 μm. E, F Mean fluorescence intensity (MFI) along the dotted arrow lines (left) in C or D and MFI ratio inform the periphery to the interior along dot lines across the organoids (right). n ≥ 15. Data were presented as the mean ± SEM. P < 0.05*, 0.01**, 0.001***, 0.0001****, compared to the control in A, B and E, F with two-tailed Student’s t test

On the other hand, YAP is an important transcriptional regulator of the Hippo signaling pathway and can serve as a mechanosensitive checkpoint for nuclear translocation and transcriptional regulation [19]. Immunostaining test presented that, at 3 days, YAP was uniformly expressed within the organoids under RFC or in the control (Fig. 5A), whereas, at 7 days, it was evenly expressed throughout the organoids in the control but polarized at the periphery of the organoids under RFC (Fig. 5B). Semi-quantitative analysis indicated that YAP expression was significantly increased under RFC at 3 days, as seen in the total YAP, nuclear YAP, and YAP nuclear/cytoplasmic ratio (Fig. 5C; Additional file: Fig. S5A). In addition, Wes and qPCR data also supported the significant enhancement in total YAP and YAP mRNA expressions, corresponding to a significant decrease in the p-YAP/YAP ratio, indicating that YAP was activated and translocated into nuclei under RFC (Fig. 5D, E; Additional file: Fig. S5B). Consistently, all data at 7 days confirmed the remarkable enhancement in total YAP and YAP nuclear/cytoplasmic ratio as well as YAP mRNA expression in HGOs under RFC, together with the significantly decreased p-YAP/YAP ratio (Fig. 5F–H; Additional file: Fig. S5C, D). Taken together, rotating culture could increase the YAP expression and promote the nuclear translocation of YAP, thereby promoting the stemness maintenance of HGOs.

Fig. 5
figure 5

Rotating culture fosters YAP expressions and nuclear translocation in HGOs. A, B Representative stained images of YAP (magenta), F-actin (green), and Hoechst (blue) in HGOs under RFC at 3 (A) or 7 days (B). Cells cultured in static culture chamber served as control. Scale bar = 20 μm. C, F Quantification of nucleus/cytoplasm intensity ratio of HGOs in A or B. n ≥ 80. D, G Simple western analysis and quantification of p-YAP/YAP expression in HGOs at 3 (D) or 7 days (G). N ≥ 3. E, H qPCR analysis of YAP mRNA expression in HGOs at 3 (E) or 7 days (H). N ≥ 3. Data were presented as the mean ± SEM. P < 0.05*, 0.01**, 0.001***, 0.0001****, compared to the control in C–E and F–H with two-tailed Student’s t test

Transcriptional YAP is key to the formation and stemness of HGOs

To further decipher the correlation between YAP and rotating culture in enhancing the expression of stemness markers SOX9 and CD44, a small molecule inhibitor VP was used to abolish the binding of YAP to its transcriptional factor TEAD. Immunostaining and immunoblotting tests indicated that VP treatment induced a decrease in nuclear/cytoplasmic ratio of YAP, total YAP protein expression, and mRNA expression of YAP, compared to those without VP treatment, together with the correspondingly increased ratio of p-YAP/YAP (Fig. 6A, B; Additional file: Fig. S6A-C). The differences in the above YAP expression pattern between rotating and static cultures disappeared, even though the p-YAP/YAP ratio was still higher under RFC (Fig. 6A, B). Consistently, the nuclear/cytoplasmic ratio of YAP, total YAP protein expression, and mRNA expression of YAP were decreased significantly at 7 days, compared to those control without VP treatment, together with the significantly increased ratio of p-YAP/YAP (Fig. 6C, D; Additional file: Fig. S6D-F). The differences in above YAP expression pattern between rotating and static cultures also disappeared (Fig. 6C, D), with sharply reduced YAP mRNA expression (Additional file: Fig. S6C). Additionally, VP treatment led to the decreased expression of downstream YAP target genes CCND (mainly at 3 days) and CYR61, eliminating the high expression of CCND and CYR61 under RFC (Fig. 6E, F). These results implied that abolishing YAP expression of HGOs under RFC could reduce the expression of YAP downstream target genes.

Fig. 6
figure 6

Inhibitor VP diminishes rotating culture-induced YAP activation and abolishes stemness maintenance in HGOs. A, C Quantification of stained nucleus/cytoplasm intensity ratio of HGOs in Additional file: Fig. S5 A or D at 3 (A) or 7 days (C). n ≥ 40. B, D Simple western analysis and quantification of p-YAP/YAP protein expression in HGOs at 3 (B) or 7 days (D). E, F qPCR analysis of CCND1 and CYR61 mRNA expressions in HGOs under RFC at 3 (E) or 7 days (F) with VP treatment. G, I Simple western analysis and quantification of SOX9 (left) and CD44 (right) protein expression in HGOs at 3 (G) or 7 days (I). H, J qPCR analysis of SOX9 (left) and CD44 (right) mRNA expression in HGOs at 3 (H) or 7 days (J). Cells cultured in static culture chamber served as control. Each bar represented the value for VP-treated group normalized by control. Data were presented as the mean ± SEM. N ≥ 3. P < 0.05*, 0.01**, 0.001***, 0.0001****, compared to the control with two-tailed Student’s t test

Finally, we examined whether the expression of stemness markers was also altered after VP treatment. Results showed that, at 3 days, the protein expressions of SOX9 and CD44 were significantly decreased after VP treatment in HGOs (Fig. 6G; Additional file: Fig. S6G-I), consistent with those remarkably reduced mRNA expressions of SOX9 and CD44 (Fig. 6H). Similarly, at 7 days, SOX9 and CD44 expressions were significantly lowered at both protein and gene levels (Fig. 6I, J; Additional file: Fig. S6J-L). These findings suggested that abolishing YAP via VP treatment and its downstream target genes could reverse the increased expression of stemness markers SOX9 and CD44 induced by rotating culture under RFC.

Discussion

In this work, an in-house built clinostat, RFC, was used to investigate effects of altered gravity vector on HGOs in vitro. Our results suggested that this specialized rotating culture approach for adhesive cells is beneficial to the formation of HGOs. In addition, the enhanced expression of stemness markers SOX9 and CD44 in the HGOs under RFC was mainly induced by YAP nuclear translocation together with the upstreaming mechanosensing of α4β6-integrin. By contrast, the SOX9 and CD44 expressions were reduced by YAP inhibitor VP to inhibit YAP activation and its downstream target genes. A working model was also proposed in Fig. 7.

Fig. 7
figure 7

A working model proposed for illustrating the mechanism of YAP-mediated HGOs formation under RFC. Under rotating culture of HGOs that serves as a typical model for simulated microgravity effects, integrin expression is significantly increased, YAP nuclear translocation is remarkably enhanced via mechanotransductive pathways, and the expressions of stemness markers SOX9 and CD44 are evaluated, finally enhancing the multipotency of HGOs. Addition of YAP inhibitor VP decreases SOX9 and CD44 expressions in HGOs, which abolishes HGO formation

Biologically, the rotating cell culture protocol is known to favor cell proliferation and functions. For example, the requirement for feeding layers, serum, and leukemia suppressor to prevent spontaneous differentiation of mouse ESCs in those conventional methods is eliminated in a clinostat culture [31] and the cell proliferation is thus enhanced for human bone marrow mesenchymal stem cells in the RPM [32], with the increased expressions of HGF and TGF-β factors that are associated with cell migration and proliferation [33]. In a RWV culture, the proliferation of human umbilical cord blood stem cells is increased as 3D tissue-like aggregates and the vascular tubular assemblies are also developed [34]. In a RCCS culture, the proliferation and viability of human epidermal stem cells and spermatogonial stem cells are enhanced, in which human epidermal stem cells are able to form multi-layer 3D epidermal structures and spermatogonial stem cells tend to maintain clonal-forming capacity and differentiation ability before differentiating into round spermatids with flagella [35, 36]. Consistently, our results indicated that the rotating cell culture using RFC promoted the formation of HGOs with high growth rate at the early stage (Fig. 1). In addition, the expressions of stemness markers SOX9 and CD44 were also increased under RFC, indicating that the rotating cell culture was conducive to the stemness maintenance of HGOs (Figs. 2 and 3). It is known that promoting the expression of hypoxia induced factor 1A (HIF1A) is beneficial for maintaining the stemness of neural stem cells [37] and positively correlated with SOX9 in liver stem cells [38, 39]. In this work, our RNAseq data showed an upregulation of HIF1A at 7 days (Fig. 2F) but not at 3 days (Additional file: Fig. S2C). Considering the higher projection area and proliferation rate of HGOs at the early stage caused by rotating culture, more oxygen was consumed under RFC than the control, leading to the high HIF1A expression and the converse promotion of the stemness. This observation was also meaningful for a therapeutic purpose, if the potency of these HGOs could be well maintained until use.

Technically, this rotating cell culture device RFC is advantageous for those adherent cells. It is noticed that the current in vitro cell culture is mostly conducted using those clinostats with different designs, such as a horizontally rotating coverslip inserted into the fixture of chambers [40] or a horizontally rotating sealed culture chamber with a medium chamber and a gas-permeable membrane [28]. In this work, our RFC device is easy to operate for liver organoid construction, allowing a 20 μL mixture of cell and matrigel to form domes in the cell culture chamber and supporting HepaRG cells to form a grape-like cell aggregate as early as 1 day after inoculation. Such the well-operated device and the simplified procedure are biologically significant, as compared to those complicated assays with the large gradient high magnetic field (i.e., ~ 12 Tesla) for human mesenchymal stem cells [41] or long-term (240-min) clinostat culture for bipotential murine oval liver stem cells [42]. It is also indicated here that no necrosis was observed in the central region of the organoids from 3D HepaRG culture in the matrix under RFC up to 7 days (Additional file: Fig. S1D), suggesting that this RFC device may be favorable for 3D culture of pluripotent stem cell-derived organoids. Considering that these HGOs are 3D formed and pre-seeded onto the substrate, this rotating culture-based procedure is advantageous to elucidate the effects of 3D cell culture in organoid formation and maintenance.

Mechanistically, liver organoids are assumed to be the miniature livers cultured in the laboratory that possess biological characteristics similar to those of real livers, which are valuable for liver development, diseases, and drug screening. Thus, understanding the molecular mechanisms are key to optimize the organoid construction in vitro. In this work, we found that PI3K-Akt, HIF-1, MAPK, and Hippo signaling pathways are enriched under RFC. Typically, Hippo signal transduction is known to govern the organ size by regulating cell proliferation, apoptosis, and stem cell self-renewal, in which the core effector YAP/TAZ is able to transport into the nucleus after dephosphorylation and interacts with TEAD 1–4 or other transcriptional factors, thereby inducing the gene expression that promotes cell proliferation and inhibits apoptosis. Our RNA-seq data presented the elevated gene expressions of Akt signaling cascade amplification factors (e.g., α6β4-integrin) as well as signaling molecules of Akt, VEGFA, SRC, ABL, YAP1, and YES1, from HGOs under RFC (Fig. 2), and, accordingly, we focused on this mechanosensitive molecule of YAP due to its crucial role in hepatocyte proliferation, differentiation, and polarity establishment [19]. Recently, a series methods of liver organoid construction are developed based on the role of YAP in liver development and regeneration [43,44,45], mainly through promoting hepatocyte proliferation and differentiation by overexpressing YAP or its activated form (e.g., phosphorylated YAP) and thus achieving the formation of liver organoids. Nevertheless, challenges still exist such as the limited scale and functional maturity of organoids and the difficulty and complexity in mimicking the physiological structure of the liver. By elucidating the potential role of α6β4-integrin and Hippo signaling in HGOs, here we proposed that such the rotating cell culture serves as one of the effective ways, which can promote the formation of HGOs and increase the expression of hepatic progenitor cell markers by fostering YAP nuclear translocation and enhancing α4β6-integrin expression (Figs. 3, 4, 5, and 6), potentiating a new strategy for elaborating liver development and constructing liver organoids.

Finally, selecting appropriate composing cells is crucial in liver organoid construction. Stem cells usually serve as the high-priority candidate with the capacity for self-renewal and multipotent differentiation. For example, HepaRG cells are adult stem cells with multipotent differentiation ability and can fully differentiate into functional hepatocytes and biliary-like cells [46], which is beneficial to simulate in vitro the complicated microenvironment of the liver in vivo [47]. They are often used to build liver-on-a-chip platforms and the flux- or 3D-culture improves their viability and functions [48,49,50]. 3D cell aggregates formed from the self-organization of HepaRG cells can be obtained by embedding in ultra-low adhesion plates or after embedding in substrates such as alginate, cellulose hydrogels, or gelatin [51, 52]. The liver organoids formed in 3D culture significantly improve the expression profile of liver-specific genes through strong cell–cell and cell–matrix interactions and the maintenance of cell polarity [53, 54], which is conducive to this work. In fact, our results in organoid morphology, stemness biomarker expression, and typical mechanosensitive molecules supported that HepaRG cells could serve as the seed cells in constructing liver organoid especially using rotating cell culture approach. Meanwhile, our RNA-seq data also indicated an increase in the expression of a newly reported stemness marker CD63 [55] under RFC, implying that these progenitor cells present the strong proliferation capability and also the high differentiation potentials. In the future, mechanical regulation and parameter optimization are to improve the generation efficiency and quality of liver organoids, and a more physiologically like 3D culture system could be developed to improve the functional maturity of liver organoids.

Conclusions

The formation of liver progenitor cell-derived liver organoids appears to be regulated by YAP nuclear translocation under RFC with rotating cell culture methods for adherent HepaRG cells. Both YAP signaling pathway and α4β6-integrin sensation to mechanical cues could be of potential interests in the improvement of liver organoid formation and efficiency, where the rotating culture method may favor liver organoid construction. Our results also provide clues for understanding cell gravity perception.

Methods

HepaRG cell culture

Human liver progenitor HepaRG cells (Biochemistry and Cell Biology, Shanghai, China) were cultured in T-flask following the manufacturer’s instructions. Briefly, the cells were maintained in growth medium which contained William’s E medium (Gibco, Thermo-Fisher, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo-Fisher, USA) and 1% penicillin/streptomycin at 37 °C in a 5% CO2 incubator, and renewed the growth medium every 2 days. When the cell density was 80–90% confluent, the cells were trypsinized and inoculated into the cell culture chamber of a rotating flat chamber device, specifically designed for stimulating microgravity effect (as below), or the static culture chamber as control. To inhibit YAP activity, 0.1 μM Verteporfin (VP, MCE, Shanghai, China) was added into the medium during the last 24 h of rotating culture. The cells were then harvested with 0.25% trypsin solution (Gibco, Thermo-Fisher, USA) for 3 min, and then passaged or operated on demand.

Liver organoids formation in rotating flat chamber device

The RFC was setting at the speed of 10 rpm around the axis to counteract the effect of gravity by continuous rotation. In this regard, the orientation of adhered HepaRG cells varied constantly inside the rotating chamber, and the maximum centrifugal force distributed on the culture substrate was estimated to less than 1.5 × 10−3 g (a value that is applied to basically avoid the interference of artificial gravity) based on the rotational speed and the geometric dimensions of the substrate. 8 × 104 to 10 × 104 HepaRG cells were mixed with 400 μL matrigel (Corning, NY, UAS) and 20 domes were inoculated on a commercial cell culture slide (25 mm × 75 mm, Nunc, Thermo-Fisher, USA) that fits well to the external dimensions of the cell culture chamber, that is, 4 × 103 to 5 × 103 cells per dome. After the matrigel was solidified, the culture chambers were filled with growth medium without bubbles to minimize potential shear forces exerted on the cells (Fig. 1A). Total volume of culture medium was determined by the internal dimensions of the chamber (without considering those sealing structures, the internal net space reads 66 mm in length, 19 mm in width and 3 mm in height), yielding a value of ~ 3.8 mL. Inoculated cells were cultured in the culture chamber for 7 days for self-assembly to form liver organoids under RFC, when the cells cultured onto a static culture chamber served as control. Medium was renewed every 2 days. During the cultivation process, optical images were acquired at specific time points (1, 3, 5, and 7 days) and the maximum projected area, aspect ratio, and roundness of liver organoids were calculated automatically by Image J.

Live and dead cell staining assay

Living and dead cells in HGOs were detected by Calcein/PI Cell Viability/Cytotoxicity Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. In short, HGOs were collected into a confocal dish, incubated with an appropriate amount of Calcein AM/PI detection working solution at 37 °C in the dark for 50 min. The nucleus was stained with Hoechst 33342 live cell staining solution. Fluorescence images of HGOs were acquired by a confocal laser microscopy (LSM 880, Zeiss), with Calcein AM stained live cells (green) or propidium iodide (PI) stained dead cells (red).

EdU incorporation and staining

Following the manufacturer’s instructions, the proliferation efficiency of HGOs was detected by Elabscience E-Click EdU Cell Proliferation Imaging assay kit (Elabscience, Wuhan, China). EdU working solution was diluted by culture medium to 50 μM, followed by adding into the cell culture chamber. After incubation at 37 °C in the dark for 2 h, HGOs were collected into a confocal dish and fixed by 4% paraformaldehyde and permeabilize by PBS containing 0.3% Triton X-100. Click reaction solution was added and incubated at room temperature in the dark for 30 min after washing by PBS for 3 times. Hoechst 33342 was used for nuclear staining. Fluorescence images of HGOs were acquired by a confocal laser microscopy (LSM 880, Zeiss), with the stained proliferating cells (red). The EdU+ rate was calculated by the following: EdU+ rate (%) = (number of EdU+ cells) / (number of Hoechst+ cells) × 100.

Quantitative real-time polymerase chain reaction (RT-qPCR) test

HepaRG cells in domes experienced the rotation at 10 rpm in RFC for 3 or 7 days, then washed 3 times with PBS, and the resulted domes with HGOs were collected into a centrifuge tube. Total RNA was extracted from cells using a commercial RNA extraction kit RNAprep Pure Micro Kit (Tiangen, Beijing, China), following the manufacturer’s instruction, and immediately converted to cDNA using commercial ReverTra Ace-a kit (Toyobo, Osaka, Japan). qPCR was performed using GoTaq SYBR Green Master reagents (Promega, Madison, USA) and primers (Invitrogen, USA) in a quantitative real-time amplification system (QuantStudio 7, Thermo-Fisher, USA). Expressions of SOX9, CD44, YAP, α6-integrin (ITGA6), and β4-integrin (ITGB4) were first normalized to housekeeping gene gyceraldehyde-3-phosphate dehydrogenase (GAPDH), and then presented as the fold change to the control (static culture) with three or four biological repeats. The detailed information of primers used are listed in Table 1.

Table 1 Primers sequence used for real-time RT-PCR

Immunofluorescence staining and confocal microscopy

The HGOs collected at given time points under rotating culture were fixed with 4% paraformaldehyde at room temperature for 15 min and permeated by 0.4% Triton X-100 for 10 min. To minimize the non-specific binding of relevant antibodies, the sample was incubated with 1% BSA in a wet box for 1 h, and then covered with a sealing film with a diluting solution of 1% BSA, followed by the incubation with the primary antibodies at 4 °C overnight and the washing with PBS thrice. Appropriate amount of diluted second antibodies was added in the dark, covered with a sealing film, and incubated at 37 °C for 1 h. Finally, Hoechst 33342 was re-stained with 1% BSA solution at 1:1000 dilution and the images were collected using confocal laser microscopy (LSM880, Zeiss). The fluorescence intensity was quantified using the software ImageJ. Antibodies used for immunostaining are summarized in Table 2. In some cases, the outermost single-layer cells of HGOs were defined as the periphery and the rest was termed as the interior. Relative MFI ratio was then obtained by dividing the value for peripheral cells by the one for interior cells.

Table 2 Summaries of antibodies used for immunofluorescence staining

Simple western assay

Simple western (Wes) test was performed via a previously described soft-operation approach [29]. The HGOs were collected at given time points, washed with PBS, digested by Cell Recovery Solution (Corning, NY, USA) to remove the residue matrigel, and lysed with appropriate volume RIPA buffer containing phenylmethanesulfonyl fluoride (PMSF), protease inhibitor (Cell Signaling Technol., Massachusetts, USA), and phosphatase inhibitors (PhosSTOP™, Roche, Switzerland). Cell lysates were kept on ice for 30 min before centrifugation at 12,000 g for 15 min at 4 °C. The supernatants so collected were used to quantify the protein concentration by BCA assay kit (Pierce®, Thermo Scientific, USA), with the Wes™ automatic protein expression analysis system (ProteinSimple, CA, USA) following the manufacturer’s instructions. In brief, the lysates were mixed with SDS-containing Sample Buffer in a 1 × final concentration and heated to 95 °C for 5 min. The total protein amount per 3 μL was loaded on the plate, and proteins of interest were identified using specific primary antibodies and probed with HRP conjugated secondary antibodies against mouse or rabbit (ProteinSimple). Data was analyzed using the Compass software (Version 4.0.0, ProteinSimple). Each protein peak was calculated automatically and the median area under the peak was normalized first to GAPDH and then to the value from static culture at different stages. The antibodies used are summarized in Table 3.

Table 3 Summaries of antibodies used for Wes test

RNA sequencing and analysis

HepaRG cells in domes were collected from each cell culture chamber, and total RNA was extracted using TRIzol reagent (Sigma-Aldrich) for constructing cDNA libraries. RNA concentration of library was measured using Qubit® RNA Assay Kit (Thermo-Fisher) in Qubit® 3.0 for preliminary quantification and then diluted to the final concentration of 1 ng/μL. High-throughput full transcriptome sequencing and bioinformatics analysis were performed by Shandong Xiuyue Biotechnology Co., Ltd (China). Briefly, the insert size was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The Bio-RAD CFX 96 fluorescence quantitative PCR instrument was used to accurately quantify the library effective concentration (> 10 nM) when the reagent Bio-RAD KIT iQ SYBR GRN was used. Finally, the cluster generation and sequencing were performed on Novaseq 6000 S4 platform, using NovaSeq 6000 S4 Reagent kit V1.5. Subsequently, genes with p ≤ 0.05 and foldchange ≥ 1.5 were identified as differentially expressed genes (DEGs). These sequencing data were submitted to NCBI SRA database with BioProject number PRJNA1080078.

Statistical analysis

All the values were presented as mean ± SEM, with a minimum of three independent repeats performed for each assay. Student’s t test was used to analyze the differences between the two groups, and one-way ANOVA was used for multiple comparison. The number of independent repeats and p-values (n.s., not significant; p < 0.05*, 0.01**, 0.001***, 0.0001****) was provided in each figure, and the difference between different groups was considered statistically significant at p < 0.05.

Data availability

Data is provided within the manuscript or supplementary information files.

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Acknowledgements

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Funding

This work was supported by National Key Research and Development Program of China grant 2021YFA0719302, and National Natural Science Foundation of China grants 32130061 and 32371376, Scientific Instrument Developing Project of the Chinese Academy of Sciences GJJSTU20220002, and China Manned Space Flight Technology Project Chinese Space Station Experiment Project YYWT0901EXP0701.

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M.L. and D.L. developed the concept, designed the study; M.L., D.L., S.Z., and L.Z. wrote the paper; S.Z., L.Z., and Y.W. performed experiments; S.Z., L.Z., D.L., S.S., Q.L., and G.S. analyzed the data. All authors reviewed the manuscript.

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Correspondence to Dongyuan Lü or Mian Long.

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12915_2024_2062_MOESM1_ESM.docx

Additional file 1: Fig. S1. Illustration of an in-house built rotating flat chamber (RFC) device. Fig. S2. Genome-wide RNA-seq assays for differentially expressed genes (DEGs). Fig. S3. Rotating culture maintains the stemness of HGOs. Fig. S4. Rotating culture promotes α 6 - (ITGA6) and β 4 - (ITGB4) integrin subunit expressions in HGOs. Fig. S5. Rotating culture fosters total and nuclear YAP expression in HGOs. Fig. S6. Inhibitor VP diminishes rotating culture-induced YAP activation and abolishes stemness maintenance in HGOs.

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Zhong, S., Zheng, L., Wu, Y. et al. Rotating culture regulates the formation of HepaRG-derived liver organoids via YAP translocation. BMC Biol 22, 262 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12915-024-02062-1

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