title: High-Throughput Platform for Identifying Molecular Factors Involved in Phenotypic Stabilization of Primary Human Hepatocytes In Vitro.
abstract: Liver disease is a leading cause of morbidity worldwide and treatment options are limited, with organ transplantation being the only form of definitive management. Cell-based therapies have long held promise as alternatives to whole-organ transplantation but have been hindered by the rapid loss of liver-specific functions over a period of days in cultured hepatocytes. Hypothesis-driven studies have identified a handful of factors that modulate hepatocyte functions in vitro, but our understanding of the mechanisms involved remains incomplete. We thus report here the development of a high-throughput platform to enable systematic interrogation of liver biology in vitro. The platform is currently configured to enable genetic knockdown screens and includes an enzyme-linked immunosorbent assay-based functional assay to quantify albumin output as a surrogate marker for hepatocyte synthetic functions as well as an image-based viability assay that counts hepatocyte nuclei. Using this platform, we identified 12 gene products that may be important for hepatocyte viability and/or liver identity in vitro. These results represent important first steps in the elucidation of mechanisms instrumental to the phenotypic maintenance of hepatocytes in vitro, and we hope that the tools reported here will empower additional studies in various fields of liver research.
authors.affiliation: Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, MA, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Shan
authors.affiliation: The Broad Institute of MIT and Harvard, Cambridge, MA, USA Pfizer Neuroscience and Pain Research Unit, Cambridge, MA, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Logan
authors.affiliation: The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Root
authors.affiliation: The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Carpenter
authors.affiliation: Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, MA, USA The Broad Institute of MIT and Harvard, Cambridge, MA, USA Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA Institute for Medical Engineering and Science, MIT, Cambridge, MA, USA Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA sbhatia@mit.edu.
authors.forename: Sangeeta N
authors.initials: SN
authors.lastname: Bhatia
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D022781:Hepatocytes / Q000166:cytology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008099:Liver / Q000166:cytology; D061251:Primary Cell Culture / Q000379:methods*
keywords: RNA interference; cell-based assays; hepatocytes; high-content screening; image analysis