Transition of Human Embryonic Stem Cell-Derived Endothelial Cells to Smooth Muscle Cells in Culture as a Model for Vascular Development.

There is now growing evidence that embryonic stem cells provide an important resource to define the cellular and molecular mechanism of vascular development, and may serve as a potential source of cells for vascular repair. In our study, human ES cells (hESCs), H9 cell line, were differentiated by stromal cell co-culture with mouse bone marrow-derived stromal cell line S17 for 10–15 days. After this time, flow cytometry analysis confirmed the presence of a cell population with expression of surface antigens typical of endothelial cells (ECs). The hESC-derived cells were sorted for specific subpopulations of CD34+, CD31+, Flk1+ and Tie2+ cells using immunomagnetic selection to enrich for endothelial precursors. These sorted cells were cultured on fibronectin-coated plates in EGM2 media. Under these conditions, the cells assume EC morphology. The putative hESC-derived ECs expressed several EC markers including Flk1, Tie2, CD143, CD146, and bound to the lectin UEA-1. Gene expression analysis by RT-PCR further confirmed expression of transcripts for endothelial genes: Flk1, CD31, CD34, Tie2, eNOs, vWF and VE-Cadherin. Furthermore, the ECs were functional as shown by their ability to take up acLDL and form capillary-like structures when replated on Matrigel. To evaluate the smooth muscle cell (SMC) potential of this hESC-derived EC population, culture conditions were changed to media containing FBS, TGF β and PDGF-BB. Under these SMC-conditions, the cell populations converted to a flatter morphology and acquired intracellular fibrils. These cells expressed smooth muscle specific markers, as determined by immunohistochemistry: α-SMC actin, calponin and SM22. Q-RT-PCR confirmed a remarkable increase in expression of transcripts specific for SMC: α-SMC actin, calponin, SM22, smoothelin, myocardin. Importantly, we also found concomitant increased expression of 2 genes APEG-1and CRP2/SmLIM, preferentially expressed in arterial SMCs. At the time when SMC-gene expression increased, there was a corresponding dramatic decrease in expression of transcripts for endothelial genes. Notably, HUVEC cells treated with the same SMC-conditions did not develop into SMCs, suggesting this transition potential is unique to hESC-derived cells. Next, we used two functional tests to further evaluate the hESC-derived SMCs. First, we examined increase in intracellular calcium concentration evoked by 9 different agonists. The majority of the SMC population responded to bradykinin, oxytocin, endothelin-1, histamine and ATP, with fewer cells demonstrating a response to serotonin, vasopressin, norephinephrine and carbachol, consistent with a smooth muscle phenotype. In contrast, the hES-derived ECs responded to endothelin-1, histamine, bradykinin, as well as carbachol, with little response to oxytocin or the other agonists. Finally, we demonstrate that co-culture of hESC-derived SMCs together with hESC-derived ECs form ordered vascular structures composed of both cell types when cultured in a 3-dimensional Matrigel. These studies demonstrate that populations of hESC-derived ECs can convert to SMCs based on defined culture conditions. Further studies are now needed to identify whether this transition is the result of bipotential progenitor cells; or, if specific differentiated cells switch between these lineages. Alternatively, differentiated hESCs may produce progenitor cells specific for each lineage that are retained within the EC population.