Stromal cell identity modulates vascular morphogenesis in a microvasculature-on-a-chip platform.

Supportive stromal cells of mesenchymal origins regulate vascular morphogenesis in developmental, pathological, and regenerative contexts, contributing to vessel formation, maturation, and long-term stability, in part via the secretion of bioactive molecules. In this work, we adapted a microfluidic lab-on-a-chip system that enables the formation and perfusion of microvascular capillary beds with connections to arteriole-scale endothelialized channels to explore how stromal cell (SC) identity influences endothelial cell (EC) morphogenesis. We compared and contrasted lung fibroblasts (LFs), dermal fibroblasts (DFs), and bone marrow-derived mesenchymal stem cells (MSCs) for their abilities to support endothelial morphogenesis and subsequent perfusion of microvascular networks formed in fibrin hydrogels within the microfluidic device. We demonstrated that while all 3 SC types supported EC morphogenesis, LFs in particular resulted in microvascular morphologies with the highest total network length, vessel diameter, and vessel interconnectivity across a range of SC-EC ratio and density conditions. Not only did LFs support robust vascular morphology, but also, they were the only SC type to support functional perfusion of the resultant capillary beds. Lastly, we identified heightened traction stress produced by LFs as a possible mechanism by which LFs enhance endothelial morphogenesis in 3D compared to other SC types examined. This study provides a unique comparison of three different SC types and their role in supporting the formation of microvasculature that could provide insights for the choice of cells for vascular cell-based therapies and the regulation of tissue-specific vasculature.

Injectable pre-cultured tissue modules catalyze the formation of extensive functional microvasculature in vivo.

Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.

A systems mechanobiology model to predict cardiac reprogramming outcomes on different biomaterials.

During normal development, the extracellular matrix (ECM) regulates cell fate mechanically and biochemically. However, the ECM's influence on lineage reprogramming, a process by which a cell's developmental cycle is reversed to attain a progenitor-like cell state followed by subsequent differentiation into a desired cell phenotype, is unknown. Using a material mimetic of the ECM, here we show that ligand identity, ligand density, and substrate modulus modulate indirect cardiac reprogramming efficiency, but were not individually correlated with phenotypic outcomes in a predictive manner. Alternatively, we developed a data-driven model using partial least squares regression to relate short-term cell states, defined by quantitative mechanosensitive responses to different material environments, with long-term changes in phenotype. This model was validated by accurately predicting the reprogramming outcomes on a different material platform. Collectively, these findings suggest a means to rapidly screen candidate biomaterials that support reprogramming with high efficiency, without subjecting cells to the entire reprogramming process.

Evaluating the potential of endothelial cells derived from human induced pluripotent stem cells to form microvascular networks in 3D cultures.

A major translational challenge in the fields of therapeutic angiogenesis and regenerative medicine is the need to create functional microvasculature. The purpose of this study was to assess whether a potentially autologous endothelial cell (EC) source derived from human induced pluripotent stem cells (iPSC-ECs) can form the same robust, stable microvasculature as previously documented for other sources of ECs. We utilized a well-established in vitro assay, in which endothelial cell-coated (iPSC-EC or HUVEC) beads were co-embedded with fibroblasts in a 3D fibrin matrix to assess their ability to form stable microvessels. iPSC-ECs exhibited a five-fold reduction in capillary network formation compared to HUVECs. Increasing matrix density reduced sprouting, although this effect was attenuated by distributing the NHLFs throughout the matrix. Inhibition of both MMP- and plasmin-mediated fibrinolysis was required to completely block sprouting of both HUVECs and iPSC-ECs. Further analysis revealed MMP-9 expression and activity were significantly lower in iPSC-EC/NHLF co-cultures than in HUVEC/NHLF co-cultures at later time points, which may account for the observed deficiencies in angiogenic sprouting of the iPSC-ECs. Collectively, these findings suggest fundamental differences in EC phenotypes must be better understood to enable the promise and potential of iPSC-ECs for clinical translation to be realized.

Sprouting angiogenesis induces significant mechanical heterogeneities and ECM stiffening across length scales in fibrin hydrogels.

Matrix stiffness is a well-established instructive cue in two-dimensional cell cultures. Its roles in morphogenesis in 3-dimensional (3D) cultures, and the converse effects of cells on the mechanics of their surrounding microenvironment, have been more elusive given the absence of suitable methods to quantify stiffness on a length-scale relevant for individual cell-extracellular matrix (ECM) interactions. In this study, we applied traditional bulk rheology and laser tweezers-based active microrheology to probe mechanics across length scales during the complex multicellular process of capillary morphogenesis in 3D, and further characterized the relative contributions of neovessels and supportive stromal cells to dynamic changes in stiffness over time. Our data show local ECM stiffness was highly heterogeneous around sprouting capillaries, and the variation progressively increased with time. Both endothelial cells and stromal support cells progressively stiffened the ECM, with the changes in bulk properties dominated by the latter. Interestingly, regions with high micro-stiffness did not necessarily correlate with remodeled regions of high ECM density as shown by confocal reflectance microscopy. Collectively, these findings, especially the large spatiotemporal variations in local stiffness around cells during morphogenesis in soft 3D fibrin gels, underscore that characterizing ECM mechanics across length scales. provides an opportunity to attain a deeper mechanobiological understanding of the microenvironment's roles in cell fate and tissue patterning.

Characterization of the crosslinking kinetics of multi-arm poly(ethylene glycol) hydrogels formed via Michael-type addition.

Tunable properties of multi-arm poly(ethylene glycol) (PEG) hydrogel, crosslinked by Michael-type addition, support diverse applications in tissue engineering. Bioactive modification of PEG is achieved by incorporating integrin binding sequences, like RGD, and crosslinking with tri-functional protease sensitive crosslinking peptide (GCYKNRGCYKNRCG), which compete for the same reactive groups in PEG. This competition leads to a narrow range of conditions that support sufficient crosslinking density to provide structural control. Kinetics of hydrogel formation plays an important role in defining the conditions to form hydrogels with desired mechanical and biological properties, which have not been fully characterized. In this study, we explored how increasing PEG functionality from 4 to 8-arms and the concentration of biological moieties, ranging from 0.5 mM to 3.75 mM, affected the kinetics of hydrogel formation, storage modulus, and swelling after the hydrogels were allowed to form for 15 or 60 minutes. Next, human bone marrow stromal cells were encapsulated and cultured in these modified hydrogels to investigate the combined effect of mechano-biological properties on phenotypes of encapsulated cells. While the molar concentration of the reactive functional groups (-vinyl sulfone) was identical in the conditions comparing 4 and 8-arm PEG, the 8-arm PEG formed faster, allowed a greater degree of modification, and was superior in three-dimensional culture. The degrees of swelling and storage modulus of 8-arm PEG were less affected by the modification compared to 4-arm PEG. These findings suggest that 8-arm PEG allows a more precise control of mechanical properties that could lead to a larger spectrum of tissue engineering applications.

Nanotopographic substrates of poly (methyl methacrylate) do not strongly influence the osteogenic phenotype of mesenchymal stem cells in vitro.

The chemical, mechanical, and topographical features of the extracellular matrix (ECM) have all been documented to influence cell adhesion, gene expression, migration, proliferation, and differentiation. Topography plays a key role in the architecture and functionality of various tissues in vivo, thus raising the possibility that topographic cues can be instructive when incorporated into biomaterials for regenerative applications. In the literature, there are discrepancies regarding the potential roles of nanotopography to enhance the osteogenic phenotype of mesenchymal stem cells (MSC). In this study, we used thin film substrates of poly(methyl methacrylate) (PMMA) with nanoscale gratings to investigate the influence of nanotopography on the osteogenic phenotype of MSCs, focusing in particular on their ability to produce mineral similar to native bone. Topography influenced focal adhesion size and MSC alignment, and enhanced MSC proliferation after 14 days of culture. However, the osteogenic phenotype was minimally influenced by surface topography. Specifically, alkaline phosphatase (ALP) expression was not increased on nanotopographic films, nor was calcium deposition improved after 21 days in culture. Ca: P ratios were similar to native mouse bone on films with gratings of 415 nm width and 200 nm depth (G415) and 303 nm width and 190 nm depth (G303). Notably, all surfaces had Ca∶P ratios significantly lower than G415 films. Collectively, these data suggest that, PMMA films with nanogratings are poor drivers of an osteogenic phenotype.

A safe and efficient method to retrieve mesenchymal stem cells from three-dimensional fibrin gels.

Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.

Matrix identity and tractional forces influence indirect cardiac reprogramming.

Heart regeneration through in vivo cardiac reprogramming has been demonstrated as a possible regenerative strategy. While it has been reported that cardiac reprogramming in vivo is more efficient than in vitro, the influence of the extracellular microenvironment on cardiac reprogramming remains incompletely understood. This understanding is necessary to improve the efficiency of cardiac reprogramming in order to implement this strategy successfully. Here we have identified matrix identity and cell-generated tractional forces as key determinants of the dedifferentiation and differentiation stages during reprogramming. Cell proliferation, matrix mechanics, and matrix microstructure are also important, but play lesser roles. Our results suggest that the extracellular microenvironment can be optimized to enhance cardiac reprogramming.

Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6ß1 integrin receptor.

Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6ß1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis.

Expression of Oct4 in human embryonic stem cells is dependent on nanotopographical configuration.

The fate of adult stem cells can be influenced by physical cues, including nanotopography. However, the response of human embryonic stem cells (hESCs) to dimensionally well-defined nanotopography is unknown. Using imprint lithography, we prepared well-defined nanotopography of hexagonal (HEX) and honeycomb (HNY) configurations with various spacings between the nanostructures. In serum-free hESC culture medium, basic fibroblast growth factor (bFGF) is required to maintain expression of Oct4, a pluripotent gene. Unexpectedly, hESCs cultured on nanotopography could maintain Oct4 expression without bFGF supplementation. With bFGF supplementation, the HEX nanotopography maintained Oct4 expression whereas the HNY configuration caused down-regulation of Oct4 expression. Thus, we observed that the lattice configurations of the nanotopography cause hESCs to respond to bFGF in different ways. This differential response to a biochemical cue by nanotopography was unforeseen, but its discovery could lead to novel differentiation pathways. Consistent with studies of other cells, we observed that nanotopography affects focal adhesion formation in hESCs. We posit that this can in turn affect cell-matrix tension, focal adhesion kinase signaling and integrin-growth factor receptor crosstalk, which eventually modulates Oct4 expression in hESCs.