Retaining mTeSR1 Medium during Hepatic Differentiation Facilitates Hepatocyte-Like Cell Survival by Decreasing Apoptosis

Background/Aims: Hepatocyte-like cells derived from human pluripotent stem cells could be an important cell source for hepatocyte transplantation. The present study investigated the effect of retaining mTeSR1 medium during hepatic differentiation on hepatocyte-like cells in vitro. Methods: Human embryonic stem cell line H1 were treated with activin A and bone morphogenetic protein 4 (BMP4) for definitive endoderm (DE) cell induction and subsequently treated with BMP2 and fibroblast growth factor 4 (FGF4) for early hepatic cell induction. Hepatocyte growth factor (HGF) and fibroblast growth factor (KGF) were added for early hepatic cell expansion and then mixed with oncostatin-M for maturation. During DE induction, 0%, 25%, 50% and 75% concentrations of mTeSR1 medium were separately added for early hepatic induction and expansion. For optimization, the expression levels of SRY-related HMG-box 17 (SOX17) and forkhead box A2 (FOXA2) at day 4, alpha fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α) at day 15, and albumin (ALB) at day 25 were quantified in differentiated cells by qRT-PCR. The ALB-positive cell proportion was measured by flow cytometry. Functional tests including ALB secretion and indocyanine green (ICG) angiography uptake and release by ELISA, urea production by urea assay kit, and glycogen storage ability by periodic acid Schif reaction (PAS) staining were performed in the differentiated cells. The induced pluripotent stem (iPS) cells were used to examine whether the optimized method was suitable for differentiating iPS cells. DE and hepatic markers were detected by immunostaining, and functional testing was performed as described above. Flow cytometry with an Annexin V-FITC apoptosis detection kit and fluorescence microscopy with Hoechst 33258 were used to analyze apoptosis in differentiated cells derived from H1 cells. Results: All differentiated cells with retention of 0%, 25%, 50% and 75% mTeSR1 expressed SOX17, FOXA2, AFP, HNF4α, and ALB, while higher expression levels were observed in differentiated cells in the 0% and 25% groups. The flow cytometry results showed that the proportion of ALB-positive differentiated cells derived from H1 cells was higher in the 25% mTeSR1 group than in other groups. However, no significant difference in ALB secretion, urea production, ICG uptake and release and glycogen storage ability was detected between the 25% and 0% groups. The iPS cells could differentiate into hepatocyte-like cells with 25% mTeSR1 retention. The apoptosis ratio of differentiated cells was lower in the 25% mTeSR1 group than in the 0% mTeSR1 group. Conclusion: Retaining 25% mTeSR1 medium during hepatic differentiation has been proposed to increase the percentage of ALB-positive cells and cell survival by decreasing cell apoptosis

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 TLR2 homozygous knockout human embryonic stem cell line WAe001-A-64 using CRISPR/Cas9 editing

Toll-like receptor 2 (TLR2) is a pattern recognition receptor which plays an important role in innate immune system. In humans it’s encoded by the TLR2 gene and also been designated as CD282. Using CRISPR/Cas9 gene editing technology, we have established a TLR2 mutant WAe001-A-64 cell line from the original embryonic stem cell line H1. It has adopted two biallelic deletions in exon 3 of TLR2 which resulted in a frame shift and early termination in the translation of TLR2. Moreover, WAe001-A-64 has maintained the normal karyotype, pluripotent phenotype, parental cell morphology and the ability to differentiate into three germ layers

Additional file 1 of Kupffer cells abrogate homing and repopulation of allogeneic hepatic progenitors in injured liver site

Additional file 1. Fig. S1. KCs ablation supports survival of allo-HPs at the injury site. Fig. S2. Engrafted allo-HPs migrate and navigate to the injury site through portal vein (PV). Fig. S3. Depletion of neutrophils in peripheral blood. Fig. S4. Corresponding full-length immunoblot. Supplementary Table S1: List of primers for qPCR

A spatiotemporal atlas of mouse liver homeostasis and regeneration.

The mechanism by which mammalian liver cell responses are coordinated during tissue homeostasis and perturbation is poorly understood, representing a major obstacle in our understanding of many diseases. This knowledge gap is caused by the difficulty involved with studying multiple cell types in different states and locations, particularly when these are transient. We have combined Stereo-seq (spatiotemporal enhanced resolution omics-sequencing) with single-cell transcriptomic profiling of 473,290 cells to generate a high-definition spatiotemporal atlas of mouse liver homeostasis and regeneration at the whole-lobe scale. Our integrative study dissects in detail the molecular gradients controlling liver cell function, systematically defining how gene networks are dynamically modulated through intercellular communication to promote regeneration. Among other important regulators, we identified the transcriptional cofactor TBL1XR1 as a rheostat linking inflammation to Wnt/β-catenin signaling for facilitating hepatocyte proliferation. Our data and analytical pipelines lay the foundation for future high-definition tissue-scale atlases of organ physiology and malfunction