Transition of Human Embryonic Stem Cell-Derived Endothelial Cells to Smooth Muscle Cells in Culture as a Model for Vascular Development.
Authors: Obrtlikova Petra , Ross Jeffrey J. , Luttun Aernout , Beckman Joan D. , Keirstead Susan A. , Kaufman Dan S. 
Published: April 2020

Abstract
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.