University of Pittsburgh

Kazuki Takeishi

Yang Wang

Alejandro Soto-Gutierrez

Kan Handa

Alexandra Sylvie Collin De l'Hortet

Jorge Guzman Lepe

Ira Jacob Fox

Tomoji Mashimo

Branimir Popovic

Eduardo Cervantes Alvarez

Alexandra Sylvie Collin De I'HORTET

Methods are disclosed herein for efficiently generating human induced pluripotent stem cells (iPSC) containing a nucleic acid including a doxycycline promoter operably linked to a nucleic acid encoding Cas9. These methods include transfecting a human somatic cell with a nucleic acid molecule comprising a doxycycline promoter operably linked to a nucleic acid encoding a Cas9, and constitutive promoter operably linked to a tetracycline responsive element and inducing the somatic cell to form an iPSC, thereby producing an iPSC that can undergo CRISPR/Cas9-mediated recombination at a high efficiency. The human iPSC, or a cell differentiated therefrom, is cultured in the presence of doxycycline to induce expression of the Cas9. These cells can then be used to target in any gene of interest by introducing nucleic acids encoding sgRNAs. Induced pluripotent stem cells produced by these methods are also disclosed.

Human induced pluripotent stem cells for high efficiency genetic engineering

Methods are disclosed herein for producing human hepatocytes from human induced pluripotent stem cells. Also provided are transgenic rats for the expansion of human hepatocytes, such as those produced using the methods disclosed herein.

Methods of engineering human induced pluripotent stem cells to produce liver tissue

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Pittsburgh, PA, United States of America

Kazuki Takeishi