State of organelles in differentiated cells influence the early stage of hepatic differentiation
In our initial work, we tested how the ROCK inhibitor, fasudil, influences the differentiation of hUCM–MSCs into hepatic endoderm, a crucial process in the initial stages of cellular differentiation. Previous studies were reported that ROCK inhibitors not only improve stem cell viability but also promote the induction of human pluripotent stem cells (hPSC) into the endoderm [19]. Moreover, differentiation efficiency increased when small molecules were used rather than only protein-used [20]. Thus, in order to confirm the effect of fasudil on the hepatic differentiation of hUCM-MSCs, we first verified a proper concentration and treatment time of fasudil. Unlike the previously reported concentration of fasudil used in hPSCs [21], endoderm markers (GATA4, SOX17 and FOXA2) were significantly increased at a high concentration of 10 µM, and it was effective when treated for 72 hrs (P < 0.05, Fig. 1a, b). We also conducted tomographic analysis to confirm the state of organelles, such as LDs and mitochondria, within differentiated cells, which were known to be important for metabolism and regulation of stem cell fate [16, 22]. As a result, the stem cells changed to a more ovoid shape and the mitochondrial morphology was different (Fig. 1c). Moreover, LDs were induced inside the cells, and the number of LDs increased (P < 0.001, Fig. 1c, d).
Next, we examined whether the use of fasudil increased the differentiation efficiency of hepatoblasts, as an evaluation of the next step in the hepatic endoderm (Fig. 1e). The phenotype of differentiated cells was similar in both the control and fasudil-treated groups (Fig. 1f). However, the expression of hepatoblast-related genes (AFP and HNF4A) was suppressed in the fasudil-treated group (P < 0.001, Fig. 1g). To explain the hepatoblast differentiation efficiency decreased despite endodermal gene upregulation, we performed mitochondrial function test. Mitochondrial functions, such as adenosine triphosphate (ATP) production, are related to the efficiency of differentiation and are reduced when excessive LDs are induced [23]. Therefore, we hypothesized that LDs induced by fasudil would affect mitochondrial function. As we expected, mitochondrial function was diminished in the cells treated with fasudil on the day 3 of differentiation (P < 0.001, Fig. 1h). Collectively, these results indicated that while fasudil transcriptionally impacts the early stages of hepatic differentiation in hUCM–MSCs, it does not influence next stage of endoderm due to organelle conditions.
The effect of gelatin viscosity on hepatoblast induction of hUCM-MSCs
Based on fasudil-treated results, we hypothesized that mitochondrial function correlates with differentiation efficiency. Therefore, next, we aimed to enhance mitochondrial function through an extracellular matrix (ECM) component. Gelatin is known to a common biomaterial for cell culture that provides a suitable biological signal for host cells [24]. Moreover, as confirmed in our result, when hUCM-MSCs were cultured on 0.1% or 1% gelatin-coated dishes, mitochondrial activation levels, such as basal, maximal oxidative phosphorylation, ATP production, and proton leak, were increased (P < 0.001, Fig. 2a). Previous studies have shown that enhancement of oxidative phosphorylation levels and ATP production in differentiated cells is necessary for specific lineage differentiation [25, 26]. Therefore, we expected that gelatin used as ECM would synergistically improve the efficiency of differentiation with fasudil.
To assess gelatin’s impact on hepatic differentiation, we cultured hUCM-MSCs on 0.1% or 1% gelatin-coated dishes, and differentiation proceeded under these conditions (Fig. 2b). The phenotype of differentiated cells was similar on day 7 between all groups (Fig. 2c). Next, we analyzed the dynamics of gene expression associated with hepatic endoderm and hepatoblasts on the differentiation day 0, 3, and 7. As a result, endoderm-related genes (SOX17 and FOXA2) except for GATA4 and hepatoblast-related genes (AFP and HNF4A) exhibited significant upregulation in the 1% gelatin and fasudil group compared to other groups (Fig. 2d, e). However, interestingly, when hepatoblast differentiation was progressed, the hepatoblast markers (AFP and HNF4A) were decreased in the 0.1% gelatin and fasudil group, in contrast to the 1% gelatin and fasudil group (#a and #b, P < 0.01; Fig. 2e).
To explain the variation in differentiation efficiency observed between the low-viscosity and high-viscosity gelatin groups, we examined the early differentiation fate of stem cells. Notably, fasudil has been documented as a promoter for the differentiation of cells originating from the ectoderm and mesoderm, including neurons and cardiomyocytes [27, 28]. Thus, we confirmed the gene expression of ectoderm (PAX6, SOX1 and OTX2) and mesoderm (T, MIXL2 and CDX2) markers in differentiated cells on day 3. The results showed that the expression of ectoderm genes (PAX6 and OTX2) were suppressed in the 1% gelatin and fasudil group (P < 0.05; Fig. 2f). Moreover, the endoderm gene, MIXL2, was downregulated, but the mesendoderm gene, T, was upregulated in the 1% gelatin and fasudil group (P < 0.01; Fig. 2g). These results indicated that high-viscosity gelatin repressed the gene expression of ectoderm- and endoderm-related genes, leading to endodermal fate.
Given that stem cell fate determinations according to gelatin viscosity can be influenced by the organelle states, next, we investigated the impact of high-viscosity gelatin on organelles in the differentiated cells.
High-viscosity gelatin inhibited the induction of LDs and enhanced mitochondrial function
To analyze the effects of high-viscosity gelatin on stem cell fate through regulation of organelles, tomographic and mitochondrial function analyses were performed. It is known that tomographic analysis can be used to observe cell organelles more accurately than confocal laser scanning microscopy [29]. Therefore, we tomographically analyzed LDs and morphology of the mitochondria in differentiated cells on day 3. As a result, the group treated with 1% gelatin and fasudil showed lower LDs induction than the group treated with 0.1% gelatin and fasudil (P < 0.001; Fig. 3a, b), suggesting that high-viscosity gelatin suppressed the production of LDs induced by fasudil. Moreover, in the 0.1% gelatin and fasudil group, mitochondrial morphology appeared to be hyperfusion induced (Fig. 3a).
Next, we performed a seahorse assay to determine how excessive LDs induction and hyperfusion form of mitochondrial affect cellular mitochondrial function. The result showed that overall OCR levels were highest in the 1% gelatin and fasudil group (P < 0.05, Fig. 3c, d). Moreover, we measured the mtDNA copy number across all groups. A previous study reported that mtDNA levels gradually increased to support differentiation [30]. Thus, we expected that the more efficient the differentiation, the higher the mtDNA copy number would be. In our study, the mtDNA copy number was higher in the 1% gelatin and fasudil group (P < 0.001, Fig. 3e).
In summary, the enhanced differentiation efficiency in the 1% gelatin and fasudil group can be attributed not only to the downregulation of ectodermal and mesodermal gene expression but also to the modulation of organelle states, including reduced LD production and activated mitochondrial function.
Low-viscosity gelatin synergistically enhanced the efficiency and function of HLCs
Finally, we differentiated the hepatoblasts, which were induced using 1% gelatin coating and fasudil, into HLCs. At this stage, we used gelatin by adding step-2 differentiation medium rather than coating the dishes. Gelatin is known for its use in the long-term maintenance of human hepatocytes [31]. Thus, we assumed that it would help in the maturation of HLCs from hUCM-MSCs. To confirm the effect of gelatin on hepatocyte maturation, we induced HLCs from the hepatoblasts using OSM for 8 days, and we conducted the maturation by adding gelatin at different viscosities: 0% (no gelatin added) or 0.1% or 1% for 5 days (Fig. 4a). The HLC phenotypes were similar in all groups on differentiation day 20 (Fig. 4b). However, transcription analysis revealed that the expression of mature hepatocyte-related genes (ALB, CYP3A4, CYP1A2, HNF1A, and HNF4A) was significantly elevated in the low-viscosity (0.1%) gelatin group compared to the others (P < 0.01, Fig. 4c). We also confirmed the expression of these proteins (Albumin; ALB and CYP3A4) by Western blotting, wherein ALB levels in the 0.1% gelatin group were comparable to those yielded in the 0% gelatin group, but CYP3A4 expression was notably higher (P < 0.01, Fig. 4d, e). Moreover, the maturation efficiency was assessed through the expression of hepatocyte-specific miRNAs, such as miR-122 and miR-192 [32]. These results also showed that hepatocyte-specific miRNAs expressed significantly upregulated in the presence of 0.1% gelatin (Fig. 4f).
Next, considering the primary roles of hepatocytes in protein synthesis and detoxification, we measured albumin secretion and CYP3A4 activity. Consistent with previous findings, hepatic function was significantly enhanced in HLCs with the addition of 0.1% gelatin (P < 0.001, Fig. 5a, b). Overall, the specification and maturation of hepatocytes were more efficient with low-viscosity gelatin, in contrast to hepatoblast differentiation. Therefore, we attempted to understand this phenomenon by analyzing the state of differentiated cell organelles, focusing on LDs and mtDNA copy number in the HLCs on day 20. Confocal imaging analysis confirmed that fewer LDs were induced when 0.1% gelatin was used for hepatocyte maturation (P < 0.05, Fig. 5c, d), and a significant increase in mtDNA copy number was observed in the same group (P < 0.001, Fig. 5e). These findings imply that low-viscosity gelatin is favorable for the maturation of HLCs, diverging from its role in early differentiation stages, and highlight that organelle states are pivotal in determining the efficiency and functionality of HLCs.