Metabolic shift in density-dependent stem cell differentiation.

BACKGROUND: Vascular progenitor cells (VPCs) derived from embryonic stem cells (ESCs) are a valuable source for cell- and tissue-based therapeutic strategies. During the optimization of endothelial cell (EC) inductions from mouse ESCs using our staged and chemically-defined induction methods, we found that cell seeding density but not VEGF treatment between 10 ng/mL and 40 ng/mL was a significant variable directing ESCs into FLK1+ VPCs during stage 1 induction. Here, we examine potential contributions from cell-to-cell signaling or cellular metabolism in the production of VPCs from ESCs seeded at different cell densities. METHODS: Using 1D 1H-NMR spectroscopy, transcriptomic arrays, and flow cytometry, we observed that the density-dependent differentiation of ESCs into FLK1+ VPCs positively correlated with a shift in metabolism and cellular growth. RESULTS: Specifically, cell differentiation correlated with an earlier plateauing of exhaustive glycolysis, decreased lactate production, lower metabolite consumption, decreased cellular proliferation and an increase in cell size. In contrast, cells seeded at a lower density of 1,000 cells/cm2 exhibited increased rates of glycolysis, lactate secretion, metabolite utilization, and proliferation over the same induction period. Gene expression analysis indicated that high cell seeding density correlated with up-regulation of several genes including cell adhesion molecules of the notch family (NOTCH1 and NOTCH4) and cadherin family (CDH5) related to vascular development. CONCLUSIONS: These results confirm that a distinct metabolic phenotype correlates with cell differentiation of VPCs.

Organ-on-a-chip model of vascularized human bone marrow niches.

Bone marrow niches (endosteal and perivascular) play important roles in both normal bone marrow function and pathological processes such as cancer cell dormancy. Unraveling the mechanisms underlying these events in humans has been severely limited by models that cannot dissect dynamic events at the niche level. Utilizing microfluidic and stem cell technologies, we present a 3D in vitro model of human bone marrow that contains both the perivascular and endosteal niches, complete with dynamic, perfusable vascular networks. We demonstrate that our model can replicate in vivo bone marrow function, including maintenance and differentiation of CD34+ hematopoietic stem/progenitor cells, egress of neutrophils (CD66b+), and niche-specific responses to doxorubicin and granulocyte-colony stimulating factor. Our platform provides opportunities to accelerate current understanding of human bone marrow function and drug response with high spatial and temporal resolution.

Functional characterization of embryonic stem cell-derived endothelial cells.

Endothelial cells (EC) derived from embryonic stem cells (ESC) require additional functional characterization before they are used as a cell therapy in order to enhance their potential for engraftment and proliferation. We explore several physiologically relevant functions of ESC-derived EC (ESC-EC), such as its capacity to produce nitric oxide (NO), regulate permeability, activate and express surface molecules for the recruitment of leukocytes in response to inflammatory stimuli, migrate and grow new blood vessels, lay down extracellular matrix, and take up low-density lipoproteins. We also examined the ESC-EC ability to upregulate NO in response to shear stress and downregulate NO in response to pro-inflammatory TNF-α activation. Functional responses of ESC-EC were compared with those of cultured mouse aortic ECs. The ESC-EC exhibit most aspects of functional endothelium, but interesting differences remain. The ESC-EC produced less NO on a per cell basis, but the same amount of NO if quantified based on the area of endothelial tissue. They also exhibit increased angiogenic sprouting and are more resistant to inflammatory signals. We further characterized the subphenotype of our ESC-EC and observed both venous and arterial markers on individual cells with a larger percentage of the cells exhibiting a venous phenotype. These data support the hypothesis that the developmental default pathway is toward a venous EC, and that refinement of methods for differentiation towards arterial EC is required to maintain a homogeneous population.

Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells.

Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors-utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification.

Specialized mouse embryonic stem cells for studying vascular development.

Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

Specialized tip/stalk-like and phalanx-like endothelial cells from embryonic stem cells.

Endothelial cells (EC) generated in vitro from stem cells are desirable for their potential in a variety of in vitro models and cell-based therapeutic approaches; however, EC can take on a number of functionally and phenotypically distinct specializations. Here, we show the generation of functionally distinct EC subpopulations, including (1) the pro-angiogenic migrating tip-like and proliferative stalk-like EC, and (2) the less migratory cobblestone-shaped phalanx-like EC. Both embryonic stem cell (ESC)-derived EC subpopulations are generated from outgrowths of Flk-1+ vascular progenitor cells with high levels of vascular endothelial growth factor treatment, while the phalanx-like ESC-derived EC (ESC-EC) are subsequently isolated by selecting for cobblestone shape. Compared with the ESC-derived angiogenic endothelial cells (named ESC-AEC) that contain only 14% Flt-1+ and 25% Tie-1+ cells, the selected phalanx-like ESC-EC express higher numbers of cells expressing the phalanx markers Flt-1+ and Tie-1+, 89% and 90%, respectively. The ESC-AEC also contain 35% CXCR4+ tip cells, higher expression levels of stalk marker Notch-1, and lower expression levels of Tie-2 compared with the phalanx-type ESC-EC that do not contain discernible numbers of CXCR4+ tip cells. Perhaps most notably, the ESC-AEC display increased cell migration, proliferation, and 3 times more vessel-like structures after 48 h on Matrigel compared with the phalanx-like ESC-EC. This work analyzes, for the first time, the presence of distinct EC subtypes (tip/stalk, and phalanx) generated in vitro from ESC, and shows that phalanx-like EC can be purified and maintained in culture separate from the tip/stalk-like containing EC.

Endothelial cells derived from embryonic stem cells respond to cues from topographical surface patterns.

The generation of micro- and nano-topography similar to those found in the extra cellular matrix of three-dimensional tissues is one technique used to recapitulate the cell-tissue physiology found in the native tissues. Despite the fact that ample studies have been conducted on the physiological significance of endothelial cells alignment parallel to shear stress, as this is the normal physiologic arrangement for healthy arterial EC, very few studies have examined the use of topographical signals to initiate endothelial cell alignment. Here, we have examined the ability for our mouse embryonic stem cell-derived endothelial cells (ESC-EC) to align on various microchip topographical systems. Briefly, we generated metal molds with 'wrinkled' topography using 1) 15 nm and 2) 30 nm of gold coating on the pre-strained polystryene (PS) sheets. After thermal-induced shrinkage of the PS sheets, polydimethylsiloxane (PDMS) microchips were then generated from the wrinkled molds. Using similar Shrink™-based technology, 3) larger selectively crazed acetone-etched lines in the PS sheets, and 4) fully crazed acetone-treated PS sheets of stochastic topographical morphology were also generated. The 15 nm and 30 nm gold coating generated 'wrinkles' of uniaxial anisotropic channels at nano-scaled widths while the crazing generated micron-sized channels. The ESC-EC were able to respond and align on the 320 nm, 510 nm, and the acetone-etched 10.5 µm channels, but not on the fully 'crazed' topographies. Moreover, the ESC-EC aligned most robustly on the wrinkles, and preferentially to ridge edges on the 10.5 µm-sized channels. The ability to robustly align EC on topographical surfaces enables a variety of controlled physiological studies of EC-EC and EC-ECM contact guidance, as well as having potential applications for the rapid endothelialization of stents and vascular grafts.