Project description:Here, we chemically induced liver progenitor cells (CLiPs) from mouse hepatocytes using a previously established protocol. To identify central protein kinases (PKs) that potentially promote the maturation of lpc-Hep cells, we profiled the transcriptomes and phosphoproteomes of freshly isolated primary mouse hepatocytes (MHs) and ALBUMIN+ hepatocytes (CLiP-Heps) cells. Based on the algorithm developed by us and experiment verification, we validated three PKs that promote mouse hepatocyte maturation. Then, we use RNA sequencing technology to identify potential regulators during hepatic reprogramming with samples from HDFs 2.25 days (FHH-2.25d) and 5 days (FHH-5d) after FOXA3, HNF1A, and HNF4A (FHH) transduction, as well as HDFs transduced with GFP for 2.25 days (GFP) as the control group. After combined analysis with phosphoproteomic data with the algorithm and experiment verification, we identified 2 PKs that may be involved in hepatic reprogramming regulation. One of the five potential regulator candidates is PIM1, which can promote the progress remarkably with overexpression. Therefore, we next perform another RNA-seq to drive the molecular mechanisms under undergoing the regulation by PIM1. Finally, we revealed that cell cycle and ferroptosis pathways are involved in the regulation via PIM1.
Project description:Hepatocytes are the predominant cell type in the liver and execute numerous essential biological functions. However, the crucial events and putative regulators during hepatocyte maturation require in-depth investigation. In this study, we performed single-cell and single-nucleus RNA-seq (scRNA-seq and snRNA-seq) to explore the developmental process of hepatocytes. We defined three maturation stages of postnatal hepatocytes, each of which establishes specific metabolic functions and exhibits distinct strategies for regulating proliferation. Hepatic zonation is gradually formed during hepatocyte maturation. The cells or nuclei of different ploidy exhibit zonation preferences in distribution, and asynchrony in the hepatocyte maturation pathway. In addition, combining gene regulatory network analysis and in vivo genetic manipulation, we identified critical maturation- and zonation-related transcription factors. This study not only delineates comprehensive transcriptomic profiles of hepatocyte maturation, but also presents a paradigm to identify functional genes in the development of hepatocyte maturation and zonation by combining genetic manipulation and measuring coordinates in a single-cell developmental trajectory.
Project description:To identify central protein kinases that potentially promote the maturation, we chemically induced liver progenitor cells (CLiPs) from mouse hepatocytes using a previously established protocol, and profiled the phosphoproteome and proteome of freshly isolated primary mouse hepatocytes (MHs) and ALBUMIN+ hepatocytes (CLiP-Hep) cells. To systematically interrogate the early regulatory events of hepatic reprogramming, we profiled the Phosphoproteome and proteome in human dermal fibroblasts (HDFs) at 2.25 days (FHH-2.25d) and 5 days (FHH-5d) after infection of lenti-virus encoding three liver-specific transcription factors, FOXA3, HNF1A and HNF4A (FHH). In this analysis, HDF infected with lenti-virus expressing GFP for 2.25 days (GFP) were used as control.
