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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. We describe a serum-free, chemically defined 3D in vitro model using iHEP cells, which is compatible with automation and conventional assay plates. The iHEP cells cultured in this model form polarized aggregates with functional bile canaliculi and strongly increased expression of albumin, urea and genes encoding phase I and II drug metabolism enzymes and bile transporters. Cytochrome P450-mediated metabolism is significantly higher in 3D iHEP aggregates compared to 2D iHEP culture. Furthermore, addition of human liver sinusoidal endothelial cells (sECs) and iPS-derived endothelial cells (iECs) improved mature hepatocyte function and CYP450 enzyme activity. Also, ECs formed endothelial networks within the hepatic 3D cultures, mimicking aspects of an in vivo architecture. Collectively, these results suggest that the iHEP/EC aggregates described here may have the potential to be used for many applications, including as an in vitro model to study liver diseases associated with sinusoidal endothelial cells. STATEMENT OF SIGNIFICANCE: iPS-derived hepatocytes provide an inexhaustible source of cells for drug screening, toxicology studies and cell-based therapies, but lack mature phenotype of adult primary human hepatocytes (PHH). Herein, we show that 3D culture of iPS-derived hepatocytes and their co-culture with human sinusoidal endothelial cells (sECs) to improve their maturity.

Significance Generating fully functional arterial endothelial cells is a critical problem for vascular development and disease research. Currently, the arterial endothelial cells derived from human pluripotent stem cells lack the range of arterial-specific functions in vitro and the protective function for ischemic tissues in vivo. Here, we combine single-cell RNA sequencing and CRISPR-Cas9 technology to identify pathways for regulating arterial endothelial cell differentiation. We then manipulate these pathways and generate arterial endothelial cells that demonstrate unprecedented arterial-specific functions as well as improve survival of myocardial infarction. These findings facilitate the understanding of vascular development and disease and provide a source of cells that have broad applications for vascular disease modeling and regenerative medicine.

Summary 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. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines., Highlights • Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1 induce and maintain a hemangioblast state • FGF2 promotes the expansion of these progenitors and impacts their potency, Thomson, Vereide, et al. employ six transcription factors to induce and maintain a hemangioblast state over numerous cell generations in culture. The “expandable hemangioblasts” (eHBs) are controlled by fibroblast growth factor, which stimulates their proliferation and poises the cells to produce endothelial cells and leukocytes.

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. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats., National Institutes of Health (U.S.) (NIH 1UH2 TR000506-01), National Institutes of Health (U.S.) (3UH2 TR000506-02S1), National Institutes of Health (U.S.) (T32 HL007936-12), National Institutes of Health (U.S.) (RO1 HL093282), National Institutes of Health (U.S.) (R21 EB016381-01)

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.

Cell culture has played a central role in developing our understanding of angiogenesis, and a wide variety of culture systems have been adapted for this purpose. Despite the value of this approach, many of the systems employed have suffered from a lack of precise control over culture conditions, an inability to visualize the process of angiogenesis in real time, and limitations in the ability to replicate the in vivo situation in which multiple cell types interact over distances of 100s of microns. With the advent of microfluidics, many of these obstacles can be overcome, and in vitro experiments can be produced with closer relevance to the in vivo situation. In this chapter, we describe the evolution of these microfluidic devices in the context of angiogenesis and describe current capabilities.

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). Liver and vascular cells were cultured on each sidewall of a collagen gel scaffold between two microfluidic channels under static or flow conditions. Morphogenesis of 3D hepatocyte cultures was found to depend on diffusion and convection across the nascent tissue. Furthermore, rMVECs formed 3D capillary-like structures that extended across an intervening gel to the hepatocyte tissues in hepatocyte-rMVEC coculture while they formed 2D sheet-like structures in rMVEC monoculture. In addition, diffusion of fluorescent dextran across the gel scaffold was analyzed, demonstrating that secreted proteins from the hepatocytes and MVECs can be exchanged across the gel scaffold by diffusional transport. The experimental approach described here is useful more generally for investigating microvascular networks within 3D engineered tissues with multiple cell types in vitro.—Sudo, R., Chung, S., Zervantonakis, I. K., Vickerman, V., Toshimitsu, Y., Griffith, L. G., Kamm, R. D. Transport-mediated angiogenesis in 3D epithelial coculture.

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. Here, the developed platform is used to evaluate and quantify capillary growth and endothelial cell migration from an intact cell monolayer. We also evaluate the endothelial cell response when placed in co-culture with physiologically relevant cell types, including cancer cells and smooth muscle cells. This resulted in the following observations: cancer cells can either attract (MTLn3 cancer cell line) endothelial cells and induce capillary formation or have minimal effect (U87MG cancer cell line) while smooth muscle cells (10T 1/2) suppress endothelial activity. Results presented demonstrate the capabilities of this platform to study cellular morphogenesis both qualitatively and quantitatively while having the advantage of enhanced imaging and internal biological controls. Finally, the platform has numerous applications in the study of angiogenesis, or migration of other cell types including tumor cells, into a three-dimensional scaffold or across an endothelial layer under precisely controlled conditions of mechanical, biochemical and co-culture environments.

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. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling.

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. Here we introduce a microfluidic platform for 3D cell culture in biologically derived or synthetic hydrogels with the capability to monitor cellular dynamics in response to changes in their microenvironment. Direct scaffold microinjection, was employed to incorporate 3D matrices into microfluidic devices. Our system geometry permits a unique window for studying directional migration, e.g. sprouting angiogenesis, since sprouts grow predominantly in the microscopic viewing plane. In this study, we demonstrate the ability to generate gradients (non-reactive solute), surface shear, interstitial flow, and image cells in situ. Three different capillary morphogenesis assays are demonstrated. Human adult dermal microvascular endothelial cells (HMVEC-ad) were maintained in culture for up to 7 days during which they formed open lumen-like structures which was confirmed with confocal microscopy and by perfusion with fluorescent microspheres. In the sprouting assay, time-lapse movies revealed cellular mechanisms and dynamics (filopodial projection/retraction, directional migration, cell division and lumen formation) during tip-cell invasion of underlying 3D matrix and subsequent lumen formation.

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. Macro- and microvascular endothelial monolayers were cultured on type I collagen gels in a microfluidic cell culture device and subjected to apical-to-basal (A–B) and B–A transendothelial flows. Samples were perfusion fixed and analyzed for morphological responses, localization and degree of phosphorylation of certain signaling proteins. Application of B–A, but not A–B flow, to cultured endothelial monolayers was found to promote capillary morphogenesis and resulted in distinct localization patterns of VE-cadherin and increased FAK phosphorylation. These results suggest that B–A flow triggers the transition of vascular endothelial cells from a quiescent to invasive phenotype and that the flow-mediated response involves signaling at cell–cell and cell–matrix interfaces. These results support the hypothesis that transendothelial pressure gradients resulting in B–A flow promotes sprouting angiogenesis and are consistent with early observations that tumor angiogenesis occurs from the venous side of the circulation., National Institute for Biomedical Imaging and Bioengineering (U.S.) (EB003805), National Science Foundation (U.S.) (STC CBET-0939511), National Science Foundation (U.S.). Office of Emerging Frontiers in Research and Innovation (0735997)