Inhibition of Hedgehog Delays Liver Regeneration through Disrupting the Cell Cycle.

Liver regeneration is a complicated biological process orchestrated by various liver resident cells. Hepatic cell proliferation and reconstruction of the hepatic architecture involve multiple signaling pathways. It has been reported that the Hh signal is involved in liver regeneration. However, the signal transduction pathways and cell types involved are ill studied. This study aimed to investigate hedgehog signal response cell types and the specific molecular mechanism involved in the process of liver regeneration. Partial hepatectomy (PH) of 70% was performed on ICR (Institute of Cancer Research) mice to study the process of liver regeneration. We found that the hedgehog signal was activated significantly after PH, including hedgehog ligands, receptors and intracellular signaling molecules. Ligand signals were mainly expressed in bile duct cells and non-parenchymal hepatic cells, while receptors were expressed in hepatocytes and some non-parenchymal cells. Inhibition of the hedgehog signal treated with vismodegib reduced the liver regeneration rate after partial hepatectomy, including inhibition of hepatic cell proliferation by decreasing Cyclin D expression and disturbing the cell cycle through the accumulation of Cyclin B. The current study reveals the important role of the hedgehog signal and its participation in the regulation of hepatic cell proliferation and the cell cycle during liver regeneration. It provides new insight into the recovery of the liver after liver resection.

Generation of a DKK1 homozygous knockout human embryonic stem cell line WAe001-A-21 using CRISPR/Cas9.

Dickkopf1 (DKK1) is a secreted inhibitor for the Wnt signalling, which is involved in cell proliferation, tissue regeneration and embryonic development. Using CRISPR/Cas9 editing, we established a homozygous mutant DKK1 human embryonic stem cell line (WAe001-A-21). It has a 41 bp deletion in exon 2 of DKK1, leading to its coding frame shift. The WAe001-A-21 cell line maintains a normal karyotype, pluripotency markers, typical stem cell morphology and the ability to differentiate into three germ layers.

Construction of a GLI3 compound heterozygous knockout human embryonic stem cell line WAe001-A-20 by CRISPR/Cas9 editing.

The human GLI3 protein has a dual function as a transcriptional activator or repressor of hedgehog signaling, depending on the proteolytic processing forms of GLI3. In this study, we established a compound heterozygous GLI3 mutant human embryonic stem cell line (WAe001-A-20) through CRISPR/Cas9 editing. The WAe001-A-20 cells carried two deletions on two different alleles of exon 2 of GLI3, respectively, which resulted in a frame shift and early termination in the translation of GLI3. Moreover, WAe001-A-20 maintains a normal karyotype, parental cell morphology, pluripotent phenotype and the ability to differentiate into three germ layers. Resource table.

Generation of WAe001-A-15, a human embryonic stem cell line with miR-122 doxycycline-inducible expression.

MiR-122 is the most abundant miRNA in the human liver accounting for 52% of the entire hepatic miRNome. Previous studies have demonstrated that miR-122 is a valuable therapeutic target for liver diseases, including viral hepatitis, fibrosis, steatosis, and hepatocarcinoma. Here, we constructed a miR-122 doxycycline-inducible expression human embryonic stem cell line WAe001-A-15 using the piggyBac transposon system. The cell line retained its pluripotency, in vitro differentiation potential, normal morphology, and karyotype.