Transcriptome analysis of hiHepPCs in various culture conditions, human liver-derived hepatocytes, human liver-derived cholangiocytes, HUVECs, and HPBECs
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ABSTRACT: Transcriptome analysis of human induced hepatic progenitor cells (hiHepPCs) in various culture conditions, human liver-derived hepatocytes, human liver-derived cholangiocytes, human umbilical vein endothelial cells (HUVECs), and human peripheral blood-derived endothelial cells (HPBECs) We found that a specific combination of three transcription factors, FOXA3, HNF1A, and HNF6, could convert HUVECs and HPBECs into cells that closely resembled hepatic progenitor cells in vitro. These hiHepPCs were expandable in long-term culture and able to differentiate into hepatocytes and cholangiocytes in accordance with their culture conditions. We conducted RNA-seq analyses to investigate the characteristincs of hiHepPCs and their progenies, in addition to those of parental HUVECs, HPBECs, and human liver-derived cells.
Project description:Transcriptome analysis of hiHepPCs in various culture conditions, human liver-derived hepatocytes, human liver-derived cholangiocytes, HUVECs, and HPBECs
Project description:Generation of expandable induce hepatic stem cells (iHSCs) has a potential for cell-based therapy and drug screening in liver disease. Recently many groups reported reprogramming of somatic cells into hepatocyte-like cells by direct conversion. However, the induced hepatocytes have limitations in proliferation, function and viability for cell therapy and drug development. We generated iHSCs which have properties including self-renewal and bipotency into hepatocytes and cholangiocytes. iHSCs-derived hepatocytes showed typical morphology and functions which include glycogen storage, low-density lipoprotein (LDL) uptake, Indocyanine green (ICG) metabolism, and Albumin secretion, and iHSCs-derived cholangiocytes also have functional characteristics of cholangiocytes, including cyst and tubules formation, cyst polarity, and the secretion of small substances by transmembrane channel protein. In addition, iHSCs attenuated liver injury, such as alcohol induced steatosis and carbon tetrachloride (CCl4) induced fibrosis models. Taken together, generation of functional iHSCs would be used in therapeutic applications.
Project description:To characterise liver cells from wt, untreated adult male mice. In particular we are interested on hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinuisoidal endothelial cells (LSECs), Kupffer cells (KCs) and cholangiocytes (CHs).
Project description:We report the generation of human pluripotent-stem-cell-derived (hPSC), expandable hepatic organoids (hEHOs) using a newly established method that consists of subjecting hPSCs to a sequence of distinct wholly defined (serum-free, feeder free) media lineage restricting the cells to become determined hepatic stem cells followed by a process of shifting the cells from monolayer (2D) to organoid (3D ) cultures. The hEHOs stably keep phenotypic features of a bi-potent hepatic lineage that can differentiate into functional hepatocytes or cholangiocytes. The hEHOs can expand for over 20 passages enabling industrial scaling to amounts requisite for industry or clinical programs. The cells from culture are able to engraft rapidly into injured liver parenchyma of FRG mice following transplantation and to differentiate in vivo into mature hepatocytes. If implanted into the epididymis fat pads of immune-deficiency mice, they do not generate non-hepatic lineages nor teratomas. We further developed a derivative model by incorporating human fetal liver mesenchymal cells (hFLMCs) into the hEHOs, referred as hFLMC/hEHO, and used the organoids to model alcohol liver disease-associated pathophysiologic changes, such as oxidative stress generation, steatosis, inflammatory mediators release and fibrosis, following treatment with alcohol. Our work demonstrates that the hFLMC/hEHO provide a novel ex vivo pathophysiological model for studying alcohol liver disease as well as many other non-genetic liver diseases.
Project description:The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.
Project description:We generated a high-resolution cellular atlas of the healthy human liver by profiling the transcriptome of more than 25,000 individual liver cells using droplet-based RNA-sequencing. Recently published datasets and in situ hybridization were integrated to confirm, validate and locate newly identified cell populations. We identified, annotated and characterized a total of 23 cell subpopulations that represent the degree of heterogeneity of parenchymal (i.e. hepatocytes and cholangiocytes) and non-parenchymal liver cells (i.e. endothelial cells, stellate cells, macrophages and lymphoid cells). We successfully classified human hepatocytes and liver sinusoidal endothelial cells along the porto-central axis and for the first time reveal the existence of functionally specialized pericentral GPC3+ and periportal HHIP+ DBH+ hepatic stellate cells in the healthy human liver. Our study provides a description of the different cell compartments that enter into the composition of a healthy human liver and currently constitutes the biggest single-cell RNA sequencing dataset available on human healthy hepatocytes and hepatic stellate cells. We identified subsets of hepatic stellate cells characterized by distinct localization and physiological functions.
Project description:Recently, we have shown that after partial hepatectomy (PHx), an increased hepatic blood flow initiates liver growth in mice by vasodilation and mechanically-triggered release of angiocrine signals. Here, we use mass spectrometry to identify a mechanically-induced angiocrine signal in human hepatic endothelial cells, that is, myeloid-derived growth factor (MYDGF). We show that it induces proliferation and promotes survival of primary human hepatocytes derived from different donors in two-dimensional cell culture, via activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3). MYDGF also enhances proliferation of human hepatocytes in three-dimensional organoids. In vivo, genetic deletion of MYDGF decreases hepatocyte proliferation in the regenerating mouse liver after PHx; conversely, adeno-associated viral delivery of MYDGF increases hepatocyte proliferation and MAPK signaling after PHx. We conclude that MYDGF represents a mechanically-induced angiocrine signal and that it triggers growth of, and provides protection to, primary mouse and human hepatocytes
Project description:The mammalian liver possesses a remarkable regenerative ability. 1) The 'oval cell' response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. 2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). We establish a long-term 3D organoid culture system from mouse and human fetal/pediatric/adult hepatocytes that retain key morphological and functional features of hepatocytes fate. We report the mRNA and single cell sequencing of Hep-Orgs from different donors in different passages. We compared Hep-Orgs with primary hepatocytes, proliferative hepatocytes or Chol-Orgs derived from Epcam+ biliary cells. By analyzing, we determine similar gene expression profile of Hep-Orgs with primary hepatocytes and make genes lists distinguished with undamaged hepatocytes as well. We find the Hep-Orgs resemble proliferative damage-response of hepatocytes after partial hepatectomy while Chol-Orgs express high cholangiocytes markers. The sequencing data constitutes a valuable resource to understand liver organoids especially Hep-Orgs.
Project description:The molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. In addition, the gene expression changes associated with activation of primary human hepatic stellate cells, a key event during fibrogenesis, remain poorly characterized. Here, we provide the transriptomic profile underpinning the healthy phenotype of human hepatocytes, liver sinusoidal endothelial cells (LSECs) and quiescent hepatic stellate cells (qHSCs) as well as activated HSCs (aHSCs) We assess the transcriptome for purified, non-cultured human hepatocytes, liver sinusoidal cells (LSECs) and quiescent hepatic stellate cells (qHSCs) as well as culture-activated HSCs (aHSCs).