citations

                    Genome-wide CRISPR Screening to Identify Drivers of TGF-ß-Induced Liver Fibrosis in Human Hepatic Stellate Cells
                

Authors:

Shan Yu,*† Matthew Ericson,† Andrea Fanjul,† Derek M. Erion,‡ Maria Paraskevopoulou,‡ Erin N. Smith,† Banumathi Cole,§ Ryan Feaver,§ Corine Holub,† Narender Gavva,† Shane R. Horman,† and Jie Huang†

In:

Source: Chem Biol
Publication Date: (2022)
Issue: 17: 4

Research Area:

Cancer Research/Cell Biology

Gastroenterology

Basic Research

Drug Discovery

Cells used in publication:

Stellate cell, hepatic, human: Species: human; Tissue Origin: liver

Platform:

384-well HT Nucleofector® System

Experiment

Stellate cells: Human hepatic stellate cells (HSCs) were purchased from either Lonza (Cat #: HUCLS1) or Sciencell (Cat #: 5300) and were cultured according to manufacturers’ instructions. Briefly, for cryopreserved HSCs from Lonza, vials of HSCs were taken out of liquid nitrogen storage and were thawed in a water bath. The cells were gently transferred to a conical tube with 5 mL MCST250 medium (Lonza Cat #: MSCT250) under sterile conditions.

Nucleofection wiht the High Troughput Nucleofector (HTN): concentrated single-cell HSC suspension (12,500–50,000 cells/20 µL) was prepared using either the P3 primary cell 4D-Nucleofector X Kit (Cat #: V4XP-3032, Lonza) or P2 primary cell 4D-Nucleofector X Kit S (Cat #: V4XP-2032, Lonza) according to manufacturer’s instructions. Cells were then added to the RNP complex and electroporated using a Lonza 4D-Nucleofector X Unit (Cat #: AAF-1003X). The electroporation programs used were described in each figure description. After 10 min postelectroporation, complete culture medium without antibiotic was then added into each well and gently pipetted up and down.

Abstract

Liver fibrosis progression in chronic liver disease leads to cirrhosis, liver failure, or hepatocellular carcinoma and often ends in liver transplantation. Even with an increased understanding of liver fibrogenesis and many attempts to generate therapeutics specifically targeting fibrosis, there is no approved treatment for liver fibrosis. To further understand and characterize the driving mechanisms of liver fibrosis, we developed a high-throughput genome-wide CRISPR/Cas9 screening platform to identify hepatic stellate cell (HSC)-derived mediators of transforming growth factor (TGF)-ß-induced liver fibrosis. The functional genomics phenotypic screening platform described here revealed the novel biology of TGF-ß-induced fibrogenesis and potential drug targets for liver fibrosis.

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