| Summary: | 碩士 === 國立臺灣大學 === 生化科技學系 === 101 === Hemophilia A is the most common type of hemophilia and is also known as factor VIII (FVIII) deficiency. FVIII is an essential blood clotting factor, and its defects result in the formation of fibrin deficient clots, causing bleeding. liver is a major source of FVIII and the liver sinusoidal endothelial cells (LSEC) are endothelial cells (EC) that line the hepatic microvasculature, sinusoids, which are the major cell type of FVIII production. Previous reports showed that transplantation of LSEC improved the hemophilia phenotype of mice deficient for FVIII. Thus, LSEC is expected to be one of the source for cell therapy of Hemophilia A. My research can be divided into two parts, the generation of human induced pluripotent stem cell (hiPSC) and the differentiation toward LSEC, which can express FVIII, from hiPSC.
In the first part, I infect c-Myc, Klf4, Oct4, and Sox2 to human somatic cell line HS68 by lentivirus for human iPSC formation. However, the results from analysis of cell surface antigen and mRNA expression showed that there is no complete pluripotent-specific expressed in cells.
LSEC is a specified EC which emerged into liver bud at early development stage. In my second part of thesis, on the basis of differentiation from human embryonic stem cell (hESC) toward EC, associated the previous study: the inhibition of TGFβ/activin signaling could generate LESC-like from mouse ESC. I designed the protocol for inducing hiPSC toward LSEC through conditional medium with specific cytokines such as BMP4, ActivinA, bFGF, VEGF and TGFβ receptor inhibitor (Tbr1ki2) at different stages. By adopting 253G1 into embryonic body (EB) form, I used the protocol for LSEC induction.16 days later, about 55%-differentiated cells expressed a LESC marker, stabilin-2, a major hyaluronan clearance receptor, and incorporated FITC-labeled hyaluronan. Most importantly, those cells produced FVIII as evidenced by mRNA expression and protein secretion.
In conclusion, FVIII-producing LSEC can be derived from human iPS cells and those cells may be an excellent source for cell therapy of hemophilia and the application of regeneration medicine.
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