Ectopic expression of factor VIII in MSCs and hepatocytes derived from rDNA targeted hESCs.

Hemophilia A is an X-linked recessive bleeding disorder caused by FVIII gene deficiency, which may result in spontaneous joint hemorrhages or life-threatening bleeding. Currently, cell-based gene therapy via ex vivo transduction of transplantable cells with integrating gene-expressing vectors offers an attractive treatment for HA. In present study, we targeted an expression cassette of B-domain-deleted FVIII into the ribosomal DNA (rDNA) locus of human embryonic stem cells (hESCs) by transfection with a nonviral targeting plasmid pHrn. The targeted hESCs clone could be expanded and retained the main pluripotent properties of differentiation into three germ layers both in vitro and in vivo. Importantly, under defined induction conditions, the targeted hESCs could differentiated into functional mesenchymal stem cells (MSCs) and hepatocytes, as validated by relevant specific cell markers and functional examination. Tumorgenesis assay demonstrated that these cells are relatively safe for future applications. Analysis on gene expression revealed that exogenous FVIII mRNA and FVIII proteins were both present in differentiated MSCs and hepatocytes. These results indicated that through gene targeting at hESCs rDNA locus a persistent cell source of transplantable genetic-modified cells can be accomplished for HA therapy.