Isolation of Functional Human Endothelial Cells from Small Volumes of Umbilical Cord Blood

Abstract

Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential as a therapeutic tool to promote vasculogenesis and angiogenesis and treat cardiovascular diseases. However, current methods to isolate ECs are limited by a low yield with few colonies appearing during isolation. In order to utilize blood-derived ECs for therapeutic applications, a simple method is needed that can produce a high yield of ECs from small volumes of blood without the addition of animal-derived products. For the first time, we show that human endothelial cells can be isolated without the prior separation of blood components through the technique of diluted whole blood incubation (DWBI) utilizing commercially available human serum. We isolated ECs from small volumes of blood (~ 10 ml) via DWBI and characterized them with flow cytometry, immunohistochemistry, and uptake of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). These ECs are functional as demonstrated by their ability to form tubular networks in Matrigel, adhere and align with flow under physiological fluid shear stress, and produce increased nitric oxide under fluid flow. An average of 7.0 ± 2.5 EC colonies that passed all functional tests described above were obtained per 10 ml of blood as compared to only 0.3 ± 0.1 colonies with the traditional method based on density centrifugation. The time until first colony appearance was 8.3 ± 1.2 days for ECs isolated with the DWBI method and 12 ± 1.4 days for ECs isolated with the traditional isolation method. A simplified method, such as DWBI, in combination with advances in isolation yield could enable the use of blood-derived ECs in clinical practice.

Increased Yield of Endothelial Cells from Peripheral Blood for Cell Therapies and Tissue Engineering

“…MNCs were then resuspended in full EC medium and incubated in collagen precoated 12-well tissue culture polystyrene plates (Corning Costar, MA, USA, with Type-1 calf skin collagen, Sigma-Aldrich, as previously described [6]) at 37°C, 5% CO 2 . Medium was changed every 24 h for the first 4 days, and subsequently every other day.…”

“…pBD-ECs were further tested for uptake of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL, Biomedical Technologies, MA, USA) according to manufacturer’s instructions [6]. …”

“…The latter are the most promising cells for ex vivo expansion and endothelial cell replacement. Late-outgrowth EPCs are highly proliferative, lack the leukocyte marker CD45, and have also been referred to as endothelial outgrowth cells (EOC) [2–4], endothelial colony-forming cells (ECFC) [5], and blood-derived endothelial cells (EC) [6]. We will use the term peripheral-blood derived endothelial cells (pBD-EC) in this study.…”

“…We recently developed the diluted whole blood incubation (DWBI) method in which diluted whole blood with the addition of human serum and growth factors is directly incubated on surfaces. This method avoids positive or negative selection steps that invariably lead to the loss of putative progenitor cells and resulted in yields of 7.0 ±2.5 EC colonies derived from 10 ml of human umbilical cord blood [6]. …”

“…We chose pigs for our in vivo study because of the following reasons: they are the preferred large animal model for cardiovascular diseases [15], we have previously characterized pBD-ECs and found them to be phenotypically and functionally analogous to human blood-derived ECs [6,16,17], and pigs enabled us to transplant autologous cells into the pigs’ aortae and inferior vena cavae (IVCs) to test cell functionality under flow in vivo. In vivo studies presented here provide proof-of-concept for utilizing DWBI and AMD3100-mobilized pBD-ECs for autologous cell therapies by demonstrating that pBD-ECs remain functional under flow on the surface of implanted intravascular titanium (Ti) tubes, prevent thrombosis in the IVC locally and also reduce the coagulation response systemically.…”