Molecular mechanisms regulating the establishment of hepatocyte polarity during human hepatic progenitor cell differentiation into a functional hepatocyte-like phenotype

Abstract

SummaryThe correct functioning of hepatocytes requires the establishment and maintenance of hepatocyte polarity. However, the mechanisms regulating the generation of hepatocyte polarity are not completely understood. The differentiation of human fetal hepatic progenitor cells (hFHPCs) into functional hepatocytes provides a powerful in vitro model system for studying the molecular mechanisms governing hepatocyte development. In this study, we used a two-stage differentiation protocol to generate functional polarized hepatocyte-like cells (HLCs) from hFHPCs. Global gene expression profiling was performed on triplicate samples of hFHPCs, immature-HLCs and matureHLCs. When the differential gene expression was compared based on the differentiation stage, a number of genes were identified that might be essential for establishing and maintaining hepatocyte polarity. These genes include those that encode actin filament-binding protein, protein tyrosine kinase activity molecules, and components of signaling pathways, such as PTK7, PARD3, PRKCI and CDC42. Based on known and predicted protein-protein interactions, the candidate genes were assigned to networks and clustered into functional categories. The expression of several of these genes was confirmed using real-time RT-PCR. By inactivating genes using small interfering RNA, we demonstrated that PTK7 and PARD3 promote hepatic polarity formation and affect F-actin organization. These results provide unique insight into the complex process of polarization during hepatocyte differentiation, indicating key genes and signaling molecules governing hepatocyte differentiation.

Direct Differentiation of Homogeneous Human Adipose Stem Cells Into Functional Hepatocytes by Mimicking Liver Embryogenesis

“…Together, these results suggested that, after two initial steps aimed at mimicking gastrulation and hepatic endoderm formation in vivo, homogenous hASCs could differentiate into hepatic progenitor cells. From day 10, the differentiated cells were matured using our previous protocol based on hFHPCs differentiation by the sequential exposure to HGF, OSM, and DEX (Hua et al, ). As commitment progresses, differentiation of hepatic progenitor cells along the hepatic cell lineage is promoted by essential extracellular signals.…”

“…RT-PCR and real-time RT-PCR were performed as previously described (Hua et al, 2012;Zhang et al, 2012). The primers used are listed in supplemental materials Table S1.…”

Activation of Wnt/β-catenin signalling via GSK3 inhibitors direct differentiation of human adipose stem cells into functional hepatocytes

“…siRNA transfection was performed following the manufacturer’s protocol as previously described34. Briefly, ON-TARGET SMARTpool siRNAs directed against β-catenin (L-003482-00-0005, Dharmacon, Lafayette, USA) or Non-targeting siRNAs (D-001810-10-05, Dharmacon) were mixed with Transfection DharmaFECT 1 (Dharmacon), respectively.…”

“…Real-time RT-PCR was performed as previously described1034. Total cellular RNA was extracted from 2.5 × 10 5 cells with the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.…”

Microanatomy of the digestive system of Supachai's caecilian,Ichthyophis supachaiiTaylor, 1960 (Amphibia: Gymnophiona)

“…In urodeles, the hepatic parenchymal arrangement exhibits interspecific variation, in having the one-cell-thick plate type, combined one-and two-cell-thick plate type and combined two-and multilayered-cell-thick plate type (Akiyoshi and Inoue 2012). In addition, the hepatocytes of I. supachaii are the major epithelial cell type of the liver and exhibit cell polarity by forming the apical and basal poles, as observed in other vertebrates (Hua et al 2012;M€ usch 2014). In addition, the hepatocytes of I. supachaii are the major epithelial cell type of the liver and exhibit cell polarity by forming the apical and basal poles, as observed in other vertebrates (Hua et al 2012;M€ usch 2014).…”