ABSTRACT
Objective • To generate a doxycycline (Dox)-inducible multiplexed CRISPR interference (CRISPRi) system for multiple gene inhibition in human embryonic stem cells (hESCs) to explore the function of gene families and model multigene diseases. Methods • A Dox-inducible multiplexed CRISPRi system was developed by Golden Gate assembly in hESCs. This system consisted of two plasmids, one expressing modified repressive nuclease-deactivated CRISPR-associated protein 9 (dCas9) and Krüppel-associated box (KRAB) transcriptional repressor domain under the control of Dox, the other carrying eight independent guide RNA (gRNA) expression cassettes. PCR was conducted using total genomic DNA as a template to confirm whether these two plasmids were integrated into genome. Western blotting was performed to confirm whether the expression of dCas9-KRAB could be induced by Dox treatment. Results • Using this tunable CRISPRi system, multiple genes were successfully silenced simultaneously in hESCs. The silence of genes and related to hESC self-renewal caused obvious cell differentiation in terms of changed cell morphology, decreased activity of alkaline phosphatase, and reduced expression of stage-specific embryonic antigen 4 (SSEA4), a marker of undifferentiated hESCs. Conclusion • This Dox-inducible multiplexed CRISPRi system can be used for quick and efficient silence of multiple genes in hESCs in a highly controlled manner.
ABSTRACT
PURPOSE: Whole liver transplantation has limitation including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients. However, hepatocytes have limitation in proliferation and lose their property during culture period. To over come these problems, here we performed differentiation of human embryonic stem cells (hESCs) into definitive endoderm in order to differentiate into hepatocytes efficiently. METHODS: Undifferentiated hESCs were maintained on mouse embryo fibroblast feeder (MEF) layer for 5~7 days. For endoderm differentiation, we used modified Kevin A D'Amour's method that added 100 ng/mL Activin A for 5 days. After differentiation, differentiated endodermal cells were collected and RT-PCR and immunostain analysis were performed. RESULTS: After 5 days of differentiation period, hES cells showed endoderm committed-cells and increased expression of endoderm-specific marker genes (Sox17 and Foxa2). Also differentiated endoderm cells were stained with Sox17 and Foxa2 whereas undifferentiated hES cells were not stained with Sox17, Foxa2. CONCLUSION: In vitro differentiotion from hES cells to definitive endoderm was done repetitively by our methods. Further well defined protocol for differentiation of definitive endoderm to hepatocytes should be made.