A Novel Stem Cell Model to Study Preeclampsia Endothelial Dysfunction

Abstract

Preeclampsia is a common pregnancy complication affecting 5% to 7% of all pregnancies worldwide annually. While the pathogenesis is not fully understood, maternal endothelium dysfunction is thought to be a central component to preeclampsia development. Studies to dissect maternal endothelial dysfunction, particularly on a patient-specific basis, are hampered by limited access to systemic primary endothelial cells (ECs). The objective of this study was to establish a replenishable, patient-specific in vitro EC model to allow robust mechanistic studies to dissect endothelial dysfunction in preeclampsia. Induced pluripotent stem cells (iPSCs) from three women with a history of normotensive pregnancies were differentiated into ECs. The established ECs were exposed to pooled sera from normotensive pregnancies, preeclamptic pregnancies, normotensive postpartum for non-pregnant comparison and controls. Endothelial functions including nitric oxide (NO) release, cell migration, tube formation and viability were evaluated. Levels of NO release were significantly lower after incubation with preeclamptic sera compared to the fetal bovine serum (FBS) control, and normotensive and non-pregnant (postpartum) sera treatments were also lower than FBS but higher than preeclamptic sera treatments. Tube formation and cell migration were also impaired with preeclamptic sera compared to FBS controls. Cell viabilities remained unaffected by any sera treatment. Consistent outcomes were obtained across all three patient-specific lines treated with the same pooled sera. Establishment of patient-derived iPSC-ECs treated with pregnancy sera serves as a novel model to explore the interplay between individual maternal endothelial health and circulating factors that lead to endothelial dysfunction in preeclampsia.

Keywords: Endothelial dysfunction; Hypertension; Induced pluripotent stem cells (iPSCs); Preeclampsia; Pregnancy.

Fig. 1

Workflow of generation of patient-derived iPSC-endothelial cells (ECs) and assessment for endothelial functions. Peripheral blood mononuclear cells (PBMCs) were isolated from normotensive maternal blood and were reprogrammed into induced pluripotent stem cells (iPSCs). Then, iPSCs were induced into the mesoderm cells by activating WNT signaling and were further differentiated into ECs with addition of growth factors. Cell morphologies of iPSCs and differentiated ECs are shown. The differentiated ECs were pretreated with basal media (B) or 5% of fetal bovine sera (FBS), pooled patients’ sera from normotensive pregnancy (N), preeclampsia (PE) and normotensive 6 + weeks postpartum (PP-N), respectively, with functional assays analyzed subsequently

Fig. 2

Characterization of patient-derived ECs. Successful ECs differentiation was indicated by elevation of mRNA levels and positive immunofluorescence staining of specific EC markers. A Gene expression of vWF, CD31, Notch 1 and eNOS in iPSCs compared to the three established ECs. B Immunofluorescence staining of vWF, CD31, Notch 1 and eNOS in one representative EC line. The cells were counterstained with DAPI as a nuclear marker. vWF, von Willebrand factor; eNOS, endothelial NOS; ECs, endothelial cells; DAPI, 4’,6-diamidino-2-phenylindole. Quantitative data are expressed as means ± SEM (n = 3). *P < 0.05, **P < 0.001

Fig. 3

Viability and nitric oxide (NO) release after patients’ sera treatment in three iPSC-EC lines. ECs were differentiated from three individuals with normotensive pregnancy. ECs were pretreated with basal media or 5% of sera (FBS, PE, N, and PP-N) before preforming the functional assays. A Cellular viabilities were not affected among all the treatments. B Levels of NO release upon preeclamptic sera treatment were significantly reduced when compared with FBS treatment. Quantitative data are expressed as means ± SEM (n = 3–4). *P < 0.05, **P < 0.001

Fig. 4

Cell migration in response to pooled patients’ sera. iPSC-ECs were pretreated with basal media or 5% of sera before performing wound healing assay. A Representative pictures demonstrated cell migration under each treatment condition at 24 h. The shaded areas in yellow represent the initial wound scratch. B Quantitative data are expressed as mean ± SEM (n = 4). *P < 0.05

Fig. 5

Tube formation in response to pooled patients’ sera. iPSC-ECs were pretreated with basal media or 5% of sera before splitting and plating on Matrigel with continuous treatment. A Representative pictures demonstrated EC tube formation under each treatment condition at 8 h. B Quantitative data are expressed as mean ± SEM (n = 3). *P < 0.05