Human Mesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC)

Fasudil Induces Early Hepatic Endoderm Differentiation of hUCM-MSCs Inhibition of Further Hepatoblast Maturation.

Human umbilical cord matrix mesenchymal stem cells (hUCM-MSCs) are a promising source for tissue regeneration, however efficient differentiation techniques are still limited. This study examined the ROCK inhibitor fasudil for hepatic differentiation. Screening indicated that 10 µM fasudil for 72 hours was optimum and significantly increased endoderm markers (GATA4, SOX17, FOXA2) compared to controls (Fig. 1a, b). However, fasudil was more effective at day 3 (Fig. S1a and b) than other ROCK inhibitors (Y-27632, ripasudil), which also elevated these genes.

Tomographic studies showed that fasudil-treated cells were more ovoid in form with changed mitochondrial morphology and a considerable increase in lipid droplet (LD) densities (Fig. 1c, d). However, whereas endoderm gene expression was boosted, subsequent hepatoblast differentiation was inhibited. On day 7, the hepatoblast marker AFP was dramatically decreased in the fasudil group, whereas HNF4A was unaffected (Fig. 1e-g). This inhibition was associated with reduced mitochondrial activity at day 3 (Fig. 1h), possibly caused by an excessive accumulation of LDs interfering with energy production. Fasudil was superior to other inhibitors in preserving ATP levels (Fig. S1c, d), although its creation of LDs ultimately impeded differentiation at later stages. These results suggest that fasudil stimulates transcriptional activation of early endoderm but disturbs organelle homeostasis, preventing their further development into hepatoblasts.