3-D culture and endothelial cells improve maturity of human pluripotent stem cell-derived hepatocytes.

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes (iHEP) offer an attractive alternative to primary human hepatocytes (PHH) for drug toxicity studies, as PHHs are limited in supply, vary in their metabolic activity between donors, and rapidly lose their functionality in vitro. However, one of the major drawbacks with iHEP cells in drug safety studies is their decreased phenotypic maturity, with lower liver specific enzyme activity compared with that of PHH. Here we evaluated the effects of 3D culture and non-parenchymal cells on the maturation of iHEPs.

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells.

An expandable, inducible hemangioblast state regulated by fibroblast growth factor.

During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that "trap" murine cells in a proliferative state and endow them with a hemangioblast potential. These "expandable" hemangioblasts (eHBs) are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells.

Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells.

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices.

Mechanism of a flow-gated angiogenesis switch: early signaling events at cell-matrix and cell-cell junctions.

A bias towards angiogenesis from the venous circulation has long been known, but its cause remains unclear. Here we explore the possibility that high interstitial pressure in tumors and the resultant net filtration pressure gradient that would induce flow from the interstitium into the venous circulation or lymphatics could also be an important mechanical regulator of angiogenesis. The objective of this study was to test the hypothesis that basal-to-apical (B-A) transendothelial flow promotes angiogenesis and to investigate potential mechanisms.

Microfluidic platforms for studies of angiogenesis, cell migration, and cell-cell interactions. Sixth International Bio-Fluid Mechanics Symposium and Workshop March 28-30, 2008 Pasadena, California.

Recent advances in microfluidic technologies have opened the door for creating more realistic in vitro cell culture methods that replicate many aspects of the true in vivo microenvironment. These new designs (i) provide enormous flexibility in controlling the critical biochemical and biomechanical factors that influence cell behavior, (ii) allow for the introduction of multiple cell types in a single system, (iii) provide for the establishment of biochemical gradients in two- or three-dimensional geometries, and (iv) allow for high quality, time-lapse imaging. Here, some of the recent developments are reviewed, with a focus on studies from our own laboratory in three separate areas: angiogenesis, cell migration in the context of tumor cell-endothelial interactions, and liver tissue engineering.

Transport-mediated angiogenesis in 3D epithelial coculture.

Increasing interest has focused on capturing the complexity of tissues and organs in vitro as models of human pathophysiological processes. In particular, a need exists for a model that can investigate the interactions in three dimensions (3D) between epithelial tissues and a microvascular network since vascularization is vital for reconstructing functional tissues in vitro. Here, we implement a microfluidic platform to analyze angiogenesis in 3D cultures of rat primary hepatocytes and rat/human microvascular endothelial cells (rMVECs/hMVECs).

Cell migration into scaffolds under co-culture conditions in a microfluidic platform.

Capillary morphogenesis is a complex cellular process that occurs in response to external stimuli. A number of assays have been used to study critical regulators of the process, but those assays are typically limited by the inability to control biochemical gradients and to obtain images on the single cell level. We have recently developed a new microfluidic platform that has the capability to control the biochemical and biomechanical forces within a three dimensional scaffold coupled with accessible image acquisition.

Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling.

Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells.

Design, fabrication and implementation of a novel multi-parameter control microfluidic platform for three-dimensional cell culture and real-time imaging.

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigation. While the importance of the extracellular microenvironment is clear, the ability to investigate the effects of physiologically relevant biophysical and biochemical factors is restricted in traditional cell culture platforms. Moreover, the versatility for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small population is lacking.