Initial Assessment of Variability of Responses to Toxicants in Donor-Specific Endothelial Colony Forming Cells

Abstract

There is increased interest in using high throughput in vitro assays to characterize human population variability in response to toxicants and drugs. Utilizing primary human endothelial colony-forming cells (ECFCs) isolated from blood would be highly useful for this purpose because these cells are involved in neonatal and adult vasculogenesis. We characterized the cytotoxicity of four known toxic chemicals (NaAsO2, CdCl2, tributyltin [TBT], and menadione) and their four relatively nontoxic counterparts (Na2HAsO4, ZnCl2, SnCl2, and phytonadione, respectively) in eight ECFC clones representing four neonatal donors (2 male and 2 female donors, 2 clones per donor). ECFCs were exposed to 9 concentrations of each chemical in duplicate; cell viability was evaluated 48 h later using the fluorescent vital dye fluorescent dye 5-Carboxyfluorescein Diacetate (CFDA), yielding concentration-effect curves from each experiment. Technical (day-to-day) variability of the assay, assessed from three independent experiments, was low: p-values for the differences of results were 0.74 and 0.64 for the comparison of day 2 vs. day 1 and day 3 vs. day 1, respectively. The statistical analysis used to compare the entire concentration-effect curves has revealed significant differences in levels of cytotoxicity induced by the toxic and relatively nontoxic chemical counterparts, demonstrating that donor-specific ECFCs can clearly differentiate between these two groups of chemicals. Partitioning of the total variance in the nested design assessed the contributions of between-clone and between-donor variability for different levels of cytotoxicity. Individual ECFC clones demonstrated highly reproducible responses to the chemicals. The most toxic chemical was TBT, followed by NaAsO2, CdCl2, and Menadione. Nontoxic counterparts exhibited low cytotoxicity at the higher end of concentration ranges tested. Low variability was observed between ECFC clones obtained from the same donor or different donors for CdCl2, NaAsO2, and TBT, but for menadione, the between-donor variability was much greater than the between-clone variability. The low between-clone variability indicates that an ECFC clone may represent an individual donor in cell-based assays, although this finding must be confirmed using a larger number of donors. Such confirmation would demonstrate that an in vitro ECFC-based testing platform can be used to characterize the inter-individual variability of neonatal ECFCs exposed to drugs and/or environmental toxicants.

Multifunctional Heterometallic IrIII−AuI Probes as Promising Anticancer and Antiangiogenic Agents

“…Therefore, they are primary cells that can reflect characteristics of the donor they come from, which is an interesting property to analyse variance of biological responses to drugs and to develop personalised medicine approaches. [ 48 , 49 , 50 ] It has been hypothesised that their functionality can be co‐opted by tumours in order to become vascularised, making them a target for therapies aimed to inhibit neovascularization as a strategy to control tumour growth. [51] In fact, several reports have shown their possible involvement in the development of tumours by supporting tumour angiogenesis and metastasis.…”

“…Main differences between ucb‐ECFCs and apb‐ECFCs are their proliferative potential and the maturation degree, which can give rise to different physiological features. Therefore, they are primary cells that can reflect characteristics of the donor they come from, which is an interesting property to analyse variance of biological responses to drugs and to develop personalised medicine approaches [48–50] . It has been hypothesised that their functionality can be co‐opted by tumours in order to become vascularised, making them a target for therapies aimed to inhibit neovascularization as a strategy to control tumour growth [51] .…”

Multi-omics delineation of cytokine-induced endothelial inflammatory states

“…However, BOECs have been shown to express mature EC markers over multiple passages, are preferred for metabolic labeling strategies due to their extensive proliferative ability and can be directly derived from adult donors and patients 24 , 25 , 67 – 69 . Variability in donor-to-donor responses is a point of concern as well 70 , 71 , but throughout this study, we employed BOECs derived from 19 different donors (Supplementary Table 2 ) and observed consistent inflammatory signatures throughout. Growth conditions of cultured ECs such as used matrix, 3D culturing, or flow could also affect inflammatory outcome.…”