Abstract 13136: Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Into Lymphatic Endothelial Cells
Backgrounds: Lymphatic vessels play a crucial role in fluid homeostasis and cancer metastasis. Therefore, understanding the process of lymphatic vessel formation is essential for investigating the related biology and treatment. Embryonic stem (ES) and induced pluripotent stem (iPS) cells have been increasingly used as model systems for studying differentiation processes. However, no prior studies have reported lymphatic vascular differentiation from human ES (hES) or hiPS cells. In this study, we explored whether human pluripotent stem cells can be efficiently differentiated into lymphatic endothelial cells (LECs), and whether these cells can contribute to lymphatic vessels in vivo.
Methods and Results: To induce lymphatic differentiation, we evaluated three different culture conditions, spontaneous differentiation through EB formation, co-culture with OP9 cells and a feeder free system using hES cell lines (H1 and H9) and hiPS cells (BJ1). Quantitative RT-PCR, immunocytochemistry and FACS analyses were employed to measure the generation of LECs from these pluripotent stem cells. We identified that co-culture with OP9 cells together with VEGF-A, VEGF-C and EGF can efficiently differentiate hES and hiPS cells into the lymphatic endothelial lineage. FACS analyses showed that after 14 days of the co-culture, about 40–70% of these cells expressed PROX-1, LYVE-1 and PODOPLANIN (POD), and about 10–14% expressed VEGFR−3. Cells double-positive for LYVE-1 and POD, VEGFR-3 and POD, or LYVE-1 and VEGFR-3 were observed at 3–30% at day 14. About 10% of total cells at day 14 were positive for all four LEC markers. Injection of LYVE-1+POD+ cells isolated from differentiating hES and hiPS cells into a wound model demonstrated their contribution to newly developed lymphatic vessels.
Conclusions: This is the first report to demonstrate that hES/hiPS cells can be uniformly and efficiently differentiated into the lymphatic endothelial lineage in vitro by a newly developed protocol and that these cells can contribute to lymphatic vessel formation in vivo. This system will be a useful platform for studying human LEC biology and therapy such as lymphedema.
- © 2010 by American Heart Association, Inc.