Abstract
BackgroundHuman fibroblasts can be reprogrammed into induced hepatocyte-like cells through the expression of a set of transcription factors. Although the generation of induced hepatocyte-like cells by HNF4A, HNF1A, and FOXA3 expression has proven to be a robust experimental strategy, using multiple lentivirus results in a highly variable heterogeneous population.MethodsWe designed and implemented a novel approach based on the delivery of reprogramming factors and green fluorescent protein in a single doxycycline-inducible lentiviral vector using 2A self-cleaving peptides.ResultsFibroblasts infected with the lentiviral vector can be amplified in basic fibroblast culture media in the absence of doxycycline without induction of hepatic genes. Upon switching to hepatic maturation media containing doxycycline, cells stop proliferating, activate hepatic gene transcription, and perform metabolic functions characteristic of hepatocytes.ConclusionOur strategy can generate an unlimited source of homogeneously induced hepatocyte-like cells from different genetic background donors, capable of performing typical hepatic functions suitable for drug research and other in vitro applications.
Highlights
Human fibroblasts can be reprogrammed into induced hepatocyte-like cells through the expression of a set of transcription factors
DOX-inducible coordinated expression of hepatic transcription factors A tetracycline-inducible vector (TetO-HHFG) was constructed based on the Tet operator (TetO)-FUW-eGFP plasmid where gene expression is controlled from seven copies of the tetracycline operator (7xTetO)
Cells were cultured in hepatic maturation media (HMM) media as described in the “Methods” section and the expression of the exogenous genes and albumin evaluated by immunocytochemistry
Summary
Human fibroblasts can be reprogrammed into induced hepatocyte-like cells through the expression of a set of transcription factors. The generation of induced hepatocyte-like cells by HNF4A, HNF1A, and FOXA3 expression has proven to be a robust experimental strategy, using multiple lentivirus results in a highly variable heterogeneous population. To achieve high expression of multiple transcription factors, most of the reprogramming protocols use a combination of individual lentivirus each expressing a specific factor. This experimental setting unavoidably results, for each experiment, in a heterologous population of cells expressing multiple transcription factor stoichiometry that severely affects cell homogeneity of the reprogramming experiment [7]. The necessity to perform a new round of lentiviral infection in each experiment, limits the use of iHEP in pharmaceutical industry, where adaptation to automated high-throughput screening and Good Manufacturing Practices (GMP) facilities are mandatory [2]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
