Functional Differences Between Placental Micro- and Macrovascular Endothelial Colony-Forming Cells

Abstract

Alterations in the development of the placental vasculature can lead to pregnancy complications, such as preeclampsia. Currently, the cause of preeclampsia is unknown, and there are no specific prevention or treatment strategies. Further insight into the placental vasculature may aid in identifying causal factors. Endothelial colony-forming cells (ECFCs) are a subset of endothelial progenitor cells capable of self-renewal and de novo vessel formation in vitro. We hypothesized that ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Human placentas were collected from term pregnancies delivered by cesarean section (n = 16). Placental micro- and macrovasculature was collected from the maternal and fetal side of the placenta, respectively, and ECFCs were isolated and characterized. ECFCs were CD31(+), CD105(+), CD144(+), CD146(+), CD14(-), and CD45(-), took up 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein, and bound Ulex europaeus agglutinin 1. In vitro, macrovascular ECFCs had a greater potential to generate high-proliferative colonies and formed more complex capillary-like networks on Matrigel compared with microvascular ECFCs. In contrast, in vivo assessment demonstrated that microvascular ECFCs had a greater potential to form vessels. Macrovascular ECFCs were of fetal origin, whereas microvascular ECFCs were of maternal origin. ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Although macrovascular ECFCs demonstrated greater vessel and colony-forming potency in vitro, this did not translate in vivo, where microvascular ECFCs exhibited a greater vessel-forming ability. These important findings contribute to the current understanding of normal placental vascular development and may aid in identifying factors involved in preeclampsia and other pregnancy complications.

Keywords: Angiogenesis; Endothelial progenitor cell; Placental vasculature; Preeclampsia; Stem cell.

Figure 1.

Isolation process of micro- and macrovascular endothelial colony-forming cells (ECFCs). (A–C): Microvascular ECFC isolation. (A): Placental tissue samples from maternal side, washed in phosphate-buffered saline. (B): Chopped placental tissue samples in collagenase/dispase digestion solution. (C): Microvascular ECFC colonies with cobblestone appearance at day 14 in culture. (D–F): Macrovascular ECFC isolation. (D): Placental proximal vessels from fetal side, previously flushed with saline and cleaned of adherent tissue. (E): Chopped proximal vessels in collagenase/dispase digestion solution. (F): Macrovascular ECFC colonies with cobblestone appearance at day 14 in culture. Magnification, ×100 (C, F).

Figure 2.

Phenotypic characterization by fluorescence-activated cell sorting and micro- and macrovascular endothelial colony-forming cell (ECFC) staining. Microvascular (A) and macrovascular (B) ECFCs were positive for endothelial-specific cell surface markers CD31, CD105, CD144, and CD146 and negative for monocyte/macrophage-specific marker CD14 and hematopoietic cell specific marker CD45. Microvascular (C) and macrovascular (D) ECFCs incorporated the endothelium-specific stains Dil-acLDL (red) and UEA-1 (green). Counterstaining with Hoechst 33258 (blue). Magnification, ×100; inset, ×400. Abbreviations: Dil-acLDL, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein; UEA-1, Ulex europaeus agglutinin 1.

Figure 3.

Single-cell clonogenic potential. Percentage of low proliferative potential and high proliferative potential (HPP) colonies in micro- and macrovascular ECFCs and control HUVECs generated in the single-cell clonogenic assay 14 days after single-cell plating. Macrovascular ECFCs demonstrate more potency by generating significantly more HPP colonies than microvascular ECFCs and HUVECs in both the first-generation (A) and second-generation (B) plating. Results represent the mean ± SEM of eight individual experiments. ∗, #, Significant differences. Abbreviations: ECFC, endothelial colony-forming cell; HUVEC, human umbilical vein endothelial cell.

Figure 4.

Capillary-like cord formation in vitro. The number of cord intersections (A) and cord length (B) in micro- and macrovascular ECFCs and HUVECs in a Matrigel assay. Results represent the mean ± SEM of eight individual experiments. ∗, #, Significant differences. Abbreviations: ECFC, endothelial colony-forming cell; HUVEC, human umbilical vein endothelial cell.

Figure 5.

In vivo vessel formation in Matrigel implants. Human placental microvascular endothelial colony-forming cell (ECFCs) formed blood vessels in vivo (A). This image depicts microvascular ECFCs forming CD31-expressing endothelial lined vessels that have connected with the mouse vasculature to perfuse the human vessels (note the mouse red blood cells within the vessels). Scale bar = 10 μm. Only vessels containing mouse red blood cells were quantified. The microvascular ECFCs formed significantly more vessels (B) with a significantly greater total vascular area (C) compared with macrovascular ECFCs. Results represent the mean ± SEM. ∗, Significant difference.

Figure 6.

In vivo vessel formation in lung injury model. (A–D): Representative photomicrographs of the vessel density in normoxia controls (A), hyperoxia controls (B), hyperoxia plus macrovascular ECFCs (C), and hyperoxia plus microvascular ECFCs (D). Data for the number of vessels per high power field. (E). Arrows indicate von Willebrand factor-positive vessels. Results represent the mean ± SEM. ∗, Significant difference. Scale bars = 50 μm. Abbreviations: ECFC, endothelial colony-forming cell; HPF, high-power field.