Morphological and Phenotypic Analyses of the Human Placenta Using Whole Mount Immunofluorescence1

Abstract

The human placenta performs multiple essential functions required for successful pregnancy. Alterations in the placental vasculature have been implicated in severe complications of pregnancy. Despite the importance of placental vascular function during pregnancy, there are gaps in our knowledge regarding the molecular pathways that control vessel development. Furthermore, there are limited tools available to simultaneously examine the morphology, phenotype, and spatial arrangement of cells within intact placental structures. To overcome these limitations, we developed whole mount immunofluorescence (WMIF) of the human placenta. Morphological analyses using WMIF revealed that blood vessel structures were consistent with an immature, angiogenic morphology in first-trimester placentas and mature, remodeled endothelium at term. To investigate placental expression of factors that control blood vessel development, we utilized WMIF to examine gestation age-specific expression of 1) the receptors for vascular endothelial growth factor (VEGFR-1, VEGFR-2, and VEGFR-3), which are required for placental vascular development in mice, and 2) activated, tyrosine phosphorylated STAT3 (pSTAT3), a transcription factor that mediates VEGFR2 signaling. We detected high levels of VEGFR2, VEGFR3, and pSTAT3 expression in early placental blood vessels that were significantly diminished by term. VEGFR1 was expressed primarily in trophoblast and Hofbauer cells throughout gestation. Based on our collective results, we propose that VEGFR2, VEGFR3, and STAT3 play essential roles in the development of the human placental vasculature. In addition, we anticipate that WMIF will provide a powerful approach for comparing placental morphology and protein expression in normal versus pathological pregnancies and for investigating the effects of environmental factors on placental function.

Biomimetic Micropatterned Adhesive Surfaces To Mechanobiologically Regulate Placental Trophoblast Fusion

“…To approximate stresses in both the villous tree tips and in micropatterned adhesive islands, we simulated a uniform volumetric contraction of the cells against a rigid underlying hemispherical substrate. The geometry of the bud diameters was estimated to vary between 50 and 200 μm, based on reported characteristics of the villous tree developmental stages . We hence developed models of physiological (<200 μm diameter) and supraphysiological (500 μm diameter).…”

“…The geometry of the bud diameters was estimated to vary between 50 and 200 μm, based on reported characteristics of the villous tree developmental stages. 32 We hence developed models of physiological (<200 μm diameter) and supraphysiological (500 μm diameter). Although the absolute values for stresses reported are dependent on multiple assumptions that are specific to certain cell type and culture conditions, the characteristic stress profiles observed should be reasonably accurate.…”

Analysis of homeobox gene action may reveal novel angiogenic pathways in normal placental vasculature and in clinical pregnancy disorders associated with abnormal placental angiogenesis.

“…Recent studies have provided evidence for transcriptional control of VEGF signaling by Notch ligand as well as hypoxia-inducible factor ( HIF1a ) in placental angiogenesis ( Fang et al, 2013 ). Morphological and phenotypic analyses of the human placenta using whole mount immunofluorescence technique were employed to demonstrate that early human placental blood vessels express high levels of the pro-angiogenic receptors VEGFR1, VEGFR3 and the activated signal transduction and activator of transcription 3 (pSTAT3) suggesting that these molecules play a role in regulation of placental vascular development ( Bushway et al, 2014 ). Thus, an understanding of transcriptional mechanisms would afford a valuable insight into the downstream angiogenic signaling cascades in the placenta, which as yet, remain largely unexplored in the human placenta.…”

Single-cell RNA-seq reveals the diversity of trophoblast subtypes and patterns of differentiation in the human placenta

“…The villus is the functional unit of the placenta and consists of an outer epithelial trophoblast layer and a stromal cell core, derived from the trophectoderm and the extraembryonic mesoderm, respectively. 6 The stromal cell core contains fetal endothelial cells, mesenchymal stromal cells (MSCs), Hofbauer cells 7 amongst others. MSCs in the human placenta have been reported to be fibroblast-like cells with differentiation capabilities and immunomodulatory properties.…”