Human white adipose tissue vasculature contains endothelial colony-forming cells with robust in vivo vasculogenic potential

Epub ahead of print.-- The final publication is available at link.springer.comBlood-derived endothelial colony-forming cells (ECFCs) have robust vasculogenic potential that can be exploited to bioengineer long-lasting human vascular networks in vivo. However, circulating ECFCs are exceedingly rare in adult peripheral blood. Because the mechanism by which ECFCs are mobilized into circulation is currently unknown, the reliability of peripheral blood as a clinical source of ECFCs remains a concern. Thus, there is a need to find alternative sources of autologous ECFCs. Here we aimed to determine whether ECFCs reside in the vasculature of human white adipose tissue (WAT) and to evaluate if WAT-derived ECFCs (watECFCs) have equal clinical potential to blood-derived ECFCs. We isolated the complete endothelial cell (EC) population from intact biopsies of normal human subcutaneous WAT by enzymatic digestion and selection of CD31+ cells. Subsequently, we extensively compared WAT-derived EC phenotype and functionality to bonafide ECFCs derived from both umbilical cord blood and adult peripheral blood. We demonstrated that human WAT is indeed a dependable source of ECFCs with indistinguishable properties to adult peripheral blood ECFCs, including hierarchical clonogenic ability, large expansion potential, stable endothelial phenotype, and robust in vivo blood vessel-forming capacity. Considering the unreliability and low rate of occurrence of ECFCs in adult blood and that biopsies of WAT can be obtained with minimal intervention in an ambulatory setting, our results indicate WAT as a more practical alternative to obtain large amounts of readily available autologous ECFCs for future vascular cell therapies.This work was supported by a National Institutes of Health Grant (R00EB009096, J. M.-M).Peer reviewe

Cyclin and DNA distributed cell cycle model for GS-NS0 cells.

Mammalian cell cultures are intrinsically heterogeneous at different scales (molecular to bioreactor). The cell cycle is at the centre of capturing heterogeneity since it plays a critical role in the growth, death, and productivity of mammalian cell cultures. Current cell cycle models use biological variables (mass/volume/age) that are non-mechanistic, and difficult to experimentally determine, to describe cell cycle transition and capture culture heterogeneity. To address this problem, cyclins-key molecules that regulate cell cycle transition-have been utilized. Herein, a novel integrated experimental-modelling platform is presented whereby experimental quantification of key cell cycle metrics (cell cycle timings, cell cycle fractions, and cyclin expression determined by flow cytometry) is used to develop a cyclin and DNA distributed model for the industrially relevant cell line, GS-NS0. Cyclins/DNA synthesis rates were linked to stimulatory/inhibitory factors in the culture medium, which ultimately affect cell growth. Cell antibody productivity was characterized using cell cycle-specific production rates. The solution method delivered fast computational time that renders the model's use suitable for model-based applications. Model structure was studied by global sensitivity analysis (GSA), which identified parameters with a significant effect on the model output, followed by re-estimation of its significant parameters from a control set of batch experiments. A good model fit to the experimental data, both at the cell cycle and viable cell density levels, was observed. The cell population heterogeneity of disturbed (after cell arrest) and undisturbed cell growth was captured proving the versatility of the modelling approach. Cell cycle models able to capture population heterogeneity facilitate in depth understanding of these complex systems and enable systematic formulation of culture strategies to improve growth and productivity. It is envisaged that this modelling approach will pave the model-based development of industrial cell lines and clinical studies

A framework for the design, modeling and optimization of biomedical systems

We present an overview of the key building blocks of a design framework for modeling and optimization of biomedical systems with main focus on leukemia, that we have been developing in the Biological Systems Engineering Laboratory and the Centre for Process Systems Engineering at Imperial College. The framework features the following areas: (i) a three-dimensional, biomimetic, in vitro platform for culturing both healthy and diseased blood; (ii) a novel, hollow fiber bioreactor that upgrades this in vitro platform to enable expansion and continuous harvesting of healthy and diseased blood; (iii) a global optimization-based approach for the design and operation of the aforementioned bioreactor; (iv) a pharmacokinetic / pharmacodynamic model representing patient response to Acute Myeloid Leukemia treatment; (v) an experimental framework for cell cycle modeling and quantitative analysis of environmental stress. This manuscript recapitulates the progress made in the different areas as well as the way in which these areas are connected, finally leading to a hybrid in vitro/in silico platform which allows the optimization of the ex vivo expansion of healthy and diseased blood. © 2014 Elsevier B.V

Modelling of control experiments.

<p>A) Cell growth and viability, B) Glucose and lactate concentration profiles, C) Glutamate and mAb concentration profiles, D) Cell cycle distribution.</p

Proliferation assay—Cyclin expression profiles.

<p>A) Cyclin E1 expression—EdU positive cells, B) Cyclin E1 expression—EdU negative cells, C) Cyclin B1 expression—EdU positive cells, D) Cyclin B1 expression—EdU negative cells, E) Average cyclin E1 (for G₁/G₀ phase) and cyclin B1 (for G₂/M) expression before and after glutamate exhaustion.</p