Abstract 15048: Klf4 Acts as a Molecular Switch During Stem Cell Differentiation Into Endothelial and Smooth Muscle Cells.
Background: Use of embryonic-derived vascular progenitor cells is hampered by an incomplete understanding of molecular switches that control their differentiation either to endothelial (EC) or smooth muscle (SM) cells. To further clarify this issue, we firstly investigated the vascular potential of cKit+ cells isolated from mouse embryonic stem cells and secondly identified a novel mechanism involved in specifying vascular lineage during differentiation.
Methods and Results: cKit+ cells were isolated from mouse stem cells after 4 days culture on collagen IV and induced to differentiate towards EC or SM cells by treatment with VEGF or PDGF, respectively. After 4 days on collagen IV-coated plates, stem cells differentiated into a mixed population containing about 30–50% cKit+ cells. When sorted and stimulated with PDGF cKit+ cells gave rise to α-SM actin, calponin and SM myosin heavy chain positive SMCs. In the presence of VEGF, cKit+ cells differentiate towards EC lineage expressing Flk1, CD31, VE-cadherin and vWF. In particular, exposure to shear stress between day 3 to 5 significantly increased the expression of EC markers and improved tube formation capacity as shown in a 3-dimensional matrix assay. Quantitative PCR showed increasing levels of Klf4 expression during initial stage of EC differentiation and strong reduction during SMC differentiation (10 fold change in both cases). Indeed, infection of cKit+ cells with Klf4 adenovirus strongly induced expression of EC markers even in absence of VEGF stimulation. Interestingly, while transcriptional levels of early EC markers such as CD31 and Flk1 were only mildly increased after Klf4 over-expression, more mature markers such as vWF and VE-cadherin were increased respectively of 20 and 200 fold, suggesting a possible direct bind of Klf4 to their promoters. As expected Klf4 expression strongly decreased SM markers.
Conclusions: Our data demonstrated for the first time that cKit+ cells, derived from stem cells are a suitable source of vascular progenitor cells for potential use in the future clinical application. We provided an insight of the molecular mechanism governing the switch towards either EC or SMC differentiation, i.e. Klf4 as a key transcription factor.
- © 2010 by American Heart Association, Inc.