Local Cellular Microenvironment Research Articles

Advances in Material-Assisted Electromagnetic Neural Stimulation.

Bioelectricity plays a crucial role in organisms, being closely connected to neural activity and physiological processes. Disruptions in the nervous system can lead to chaotic ionic currents at the injured site, causing disturbances in the local cellular microenvironment, impairing biological pathways, and resulting in a loss of neural functions. Electromagnetic stimulation has the ability to generate internal currents, which can be utilized to counter tissue damage and aid in the restoration of movement in paralyzed limbs. By incorporating implanted materials, electromagnetic stimulation can be targeted more accurately, thereby significantly improving the effectiveness and safety of such interventions. Currently, there have been significant advancements in the development of numerous promising electromagnetic stimulation strategies with diverse materials. This review provides a comprehensive summary of the fundamental theories, neural stimulation modulating materials, material application strategies, and pre-clinical therapeutic effects associated with electromagnetic stimulation for neural repair. It offers a thorough analysis of current techniques that employ materials to enhance electromagnetic stimulation, as well as potential therapeutic strategies for future applications.

In vivo magnetic resonance spectroscopy by transverse relaxation encoding with narrowband decoupling

Cell pathology in neuropsychiatric disorders has mainly been accessible by analyzing postmortem tissue samples. Although molecular transverse relaxation informs local cellular microenvironment via molecule-environment interactions, precise determination of the transverse relaxation times of molecules with scalar couplings (J), such as glutamate and glutamine, has been difficult using in vivo magnetic resonance spectroscopy (MRS) technologies, whose approach to measuring transverse relaxation has not changed for decades. We introduce an in vivo MRS technique that utilizes frequency-selective editing pulses to achieve homonuclear decoupled chemical shift encoding in each column of the acquired two-dimensional dataset, freeing up the entire row dimension for transverse relaxation encoding with J-refocusing. This results in increased spectral resolution, minimized background signals, and markedly broadened dynamic range for transverse relaxation encoding. The in vivo within-subject coefficients of variation for the transverse relaxation times of glutamate and glutamine, measured using the proposed method in the human brain at 7 T, were found to be approximately 4%. Since glutamate predominantly resides in glutamatergic neurons and glutamine in glia in the brain, this noninvasive technique provides a way to probe cellular pathophysiology in neuropsychiatric disorders for characterizing disease progression and monitoring treatment response in a cell type-specific manner in vivo.

Comprehensive visualization of cell-cell interactions in single-cell and spatial transcriptomics with NICHES.

Recent years have seen the release of several toolsets that reveal cell-cell interactions from single-cell data. However, all existing approaches leverage mean celltype gene expression values, and do not preserve the single-cell fidelity of the original data. Here, we present NICHES (Niche Interactions and Communication Heterogeneity in Extracellular Signaling), a tool to explore extracellular signaling at the truly single-cell level. NICHES allows embedding of ligand-receptor signal proxies to visualize heterogeneous signaling archetypes within cell clusters, between cell clusters and across experimental conditions. When applied to spatial transcriptomic data, NICHES can be used to reflect local cellular microenvironment. NICHES can operate with any list of ligand-receptor signaling mechanisms, is compatible with existing single-cell packages, and allows rapid, flexible analysis of cell-cell signaling at single-cell resolution. NICHES is an open-source software implemented in R under academic free license v3.0 and it is available at http://github.com/msraredon/NICHES. Use-case vignettes are available at https://msraredon.github.io/NICHES/. Supplementary data are available at Bioinformatics online.

Molecular tension in syndecan-1 is regulated by extracellular mechanical cues and fluidic shear stress

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 has higher mechanical tension in regions of cell adhesion, on and in response to nanotopographical cues. In addition, we found that substrate stiffness also regulated the mechanical tension of syndecan-1 and altered its binding to actin, myosin iiB and signaling intermediates including Src, PKA and FAK. Application of fluidic shear stress created a gradient in tension in syndecan-1 and led to enhanced association with actin, Src, myosin IIb and other cytoskeleton related molecules. Overall, our studies support that syndecan-1 is responsive to the mechanical environment of the cells and alters its association with actin and signaling intermediates in response to mechanical stimuli.

DNA Homeostasis and Senescence: Lessons from the Naked Mole Rat.

As we age, our bodies accrue damage in the form of DNA mutations. These mutations lead to the generation of sub-optimal proteins, resulting in inadequate cellular homeostasis and senescence. The build-up of senescent cells negatively affects the local cellular micro-environment and drives ageing associated disease, including neurodegeneration. Therefore, limiting the accumulation of DNA damage is essential for healthy neuronal populations. The naked mole rats (NMR) are from eastern Africa and can live for over three decades in chronically hypoxic environments. Despite their long lifespan, NMRs show little to no biological decline, neurodegeneration, or senescence. Here, we discuss molecular pathways and adaptations that NMRs employ to maintain genome integrity and combat the physiological and pathological decline in organismal function.

Dengue Virus Induces Increased Activity of the Complement Alternative Pathway in Infected Cells.

Severe dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infection in vitro were investigated. mRNA for factor H (FH), a major negative regulator of the AP, was significantly increased in DENV-infected endothelial cells (EC) and macrophages, but, in contrast, production of extracellular FH protein was not. This discord was not seen for the AP activator factor B (FB), with DENV induction of both FB mRNA and protein, nor was it seen with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface-bound and intracellular FH protein was, however, induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalized cell lines (ARPE-19 and human retinal endothelial cells), FH protein was induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there was an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells, with lower FH relative to FB protein, an increased ability to promote AP-mediated lytic activity, and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease.IMPORTANCE Dengue virus (DENV) is a significant human viral pathogen with a global medical and economic impact. DENV may cause serious and life-threatening disease, with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however, overactivity of the complement alternative pathway has been suggested to play a role. In this study, we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells) in vivo Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease.

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

Syndecan-1 in mechanosensing of nanotopological cues in engineered materials

The cells of the vascular system are highly sensitive to biophysical cues from their local cellular microenvironment. To engineer improved materials for vascular devices and delivery of cell therapies, a key challenge is to understand the mechanisms that cells use to sense biophysical cues from their environment. Syndecans are heparan sulfate proteoglycans (HSPGs) that consist of a protein core modified with heparan sulfate glycosaminoglycan chains. Due to their presence on the cell surface and their interaction with cytoskeletal and focal adhesion associated molecules, cell surface proteoglycans are well poised to serve as mechanosensors of the cellular microenvironment. Nanotopological cues have become recognized as major regulators of cell growth, migration and phenotype. We hypothesized that syndecan-1 could serve as a mechanosensor for nanotopological cues and can mediate the responsiveness of vascular smooth muscle cells to nanoengineered materials. We created engineered substrates made of polyurethane acrylate with nanogrooves using ultraviolet-assisted capillary force lithography. We cultured vascular smooth muscle cells with knockout of syndecan-1 on engineered substrates with varying compliance and nanotopology. We found that knockout of syndecan-1 reduced alignment of vascular smooth muscle cells to the nanogrooves under inflammatory treatments. In addition, we found that loss of syndecan-1 increased nuclear localization of Yap/Taz and phospho-Smad2/3 in response to nanogrooves. Syndecan-1 knockout vascular smooth muscle cells also had elevated levels of Rho-associated protein kinase-1 (Rock1), leading to increased cell stiffness and an enhanced contractile state in the cells. Together, our findings support that syndecan-1 knockout leads to alterations in mechanosensing of nanotopographical cues through alterations of in rho-associated signaling pathways, cell mechanics and mediators of the Hippo and TGF-β signaling pathways.

Lessons From 30 Years of Teaching About the Microenvironment of Tumors.

With the advent of state-of-the-art genomic and proteomic techniques, much has been learned about the origins and drivers of human cancers. But a solid tumor is more than a conglomerate of malignant cells that have gone awry. A solid tumor is an abnormal organ composed of cancer cells and host stromal cells, which are nourished by the vasculature and embedded in an extracellular matrix. The interaction among these cells, the surrounding matrix, and the local cellular microenvironment influences the expression of certain genes, whose products control the pathophysiological characteristics of the tumor, govern tumor progression, and affect the tumor’s response to various therapies. In recent years, some of the attempts to overcome the barriers to treatment in tumors have come to fruition and led to multiple novel therapeutic strategies. Along these lines, we are delighted to announce a special issue of The Cancer Journal, for which we serve as guest editors. It will be dedicated to the 30th anniversary Annual Tumor Course “Critical Issues in Tumor Microenvironment: Angiogenesis, Metastasis and Immunology” scheduled for October 5 to 8, 2015, at Harvard Medical School in Boston. For the last 3 decades, this course has continuously offered the best in critical analysis of what is currently known about the tumor microenvironment, metabolism, angiogenesis, metastasis, immunology, and targeted therapies. The goal has been to educate clinicians and scientists alike by highlighting key gaps in our present knowledge of cancer and outline future directions for research at the bench, in the clinic, and in drug development. Over the years, the faculty included many internationally recognized experts in these areas, including Drs Lewis C. Cantley, Peter Carmeliet, Lisa M. Coussens, Glenn Dranoff, Harold F. Dvorak, Napoleone Ferrara, Isaiah J. Fidler, Judah Folkman, Michael A. Gimbrone, Pietro M. Gullino, Richard O. Hynes, Robert S. Kerbel, Raju Kucherlapati, Robert S. Langer, Thomas Maciag, Joan Massague, Marsha A. Moses, Drew M. Pardoll, Jeffrey W. Pollard, Erkki Ruoslahti, George W. Sledge, Thomas P. Stossel, A. Gregory Sorensen, Ian F. Tannock, Thomas F. Tedder, Daniel Von Hoff, Robert A. Weinberg, Zena Werb, and Bruce R. Zetter. In the upcoming July/August 2015 issue of The Cancer Journal, some of these experts have kindly agreed to provide their unique perspectives and will summarize their latest findings from their clinics and laboratories. We think that the readership of The Cancer Journal will greatly benefit from the unique insights of these true leaders in their fields.

Cell replacement therapy: Lessons from teleost fish

Many disorders of the CNS are characterized by a massive loss of neurons. A promising therapeutic strategy to cure such conditions is based on the activation of endogenous stem cells. Implementation of this strategy will benefit from a better understanding of stem cell dynamics and the local CNS microenvironment in regeneration-competent vertebrate model systems. Using a spinal cord injury paradigm in zebrafish larvae, Briona and Dorsky (2014) have provided evidence for the existence of two distinct neural stem cell populations. One population has the characteristics of radial glia and expresses the homeobox transcription factor Dbx. The other lacks Dbx but expresses Olig2. These results are placed in the context of other studies that also support the notion of heterogeneity of adult stem cells in the CNS. The implication that differences among stem cell populations, in combination with specific factors from the local cellular microenvironment, might have a decisive impact on the fate choices of the new cells, is discussed. Reviewed evidence suggests that rather few modifications in the signaling pathways involved in the control of stem cell behavior have led, in the course of evolution, to the pronounced differences between mammals and regeneration-competent organisms. As a consequence, rather minor pharmacological manipulations may be sufficient to reactivate the hidden neurogenic potential of the mammalian CNS, and thus make it available for therapeutic applications.

Rotary suspension culture enhances mesendoderm differentiation of embryonic stem cells through modulation of Wnt/β-catenin pathway.

Recently, physical factors in the local cellular microenvironment have been confirmed with strong influences on regulating stem cell fate. Despite the recent identification of the rotary cell culture system (RCCS) as a bioreactor for culturing stem cells, the underlying biological role provided by RCCS in the lineage differentiation of embryonic stem cells (ESCs) remains largely undefined. Here, we explored the embryoid body (EB) formation and subsequent differentiation of mouse ESCs in RCCS. We demonstrated that EBs formed in RCCS were more homogeneous and bigger in size compared with those in the static condition. Further, we determined that mesendoderm differentiation was prominently enhanced, while neuroectodermal differentiation was significantly suppressed in RCCS. Surprisingly, we found that Wnt/β-catenin signaling was greatly enhanced mainly due to the increased expression of Wnt3 during ESC differentiation in RCCS. Inhibition of Wnt/β-catenin signaling by DKK1 decreased the expression of Brachyury and attenuated mesendoderm differentiation in RCCS. Intriguingly, Wnt3a markedly increased Brachyury expression under static condition rather than in RCCS. Taken together, our findings uncover a new role of rotary suspension culture in initializing the early differentiation of ESCs.

Co-regulation of primary mouse hepatocyte viability and function by oxygen and matrix.

Although oxygen and extracellular matrix cues both influence differentiation state and metabolic function of primary rat and human hepatocytes, relatively little is known about how these factors together regulate behaviors of primary mouse hepatocytes in culture. To determine the effects of pericellular oxygen tension on hepatocellular function, we employed two methods of altering oxygen concentration in the local cellular microenvironment of cells cultured in the presence or absence of an extracellular matrix (Matrigel) supplement. By systematically altering medium depth and gas phase oxygen tension, we created multiple oxygen regimes (hypoxic, normoxic, and hyperoxic) and measured the local oxygen concentrations in the pericellular environment using custom-designed oxygen microprobes. From these measurements of oxygen concentrations, we derived values of oxygen consumption rates under a spectrum of environmental contexts, thus providing the first reported estimates of these values for primary mouse hepatocytes. Oxygen tension and matrix microenvironment were found to synergistically regulate hepatocellular survival and function as assessed using quantitative image analysis for cells stained with vital dyes, and assessment of secretion of albumin. Hepatocellular viability was affected only at strongly hypoxic conditions. Surprisingly, albumin secretion rates were greatest at a moderately supra-physiological oxygen concentration, and this effect was mitigated at still greater supra-physiological concentrations. Matrigel enhanced the effects of oxygen on retention of function. This study underscores the importance of carefully controlling cell density, medium depth, and gas phase oxygen, as the effects of these parameters on local pericellular oxygen tension and subsequent hepatocellular function are profound.

Mechanobiology: a new frontier for human pluripotent stem cells.

Research on human pluripotent stem cells (hPSCs) has expanded rapidly over the last two decades, owing to the promises of hPSCs for applications in regenerative medicine, disease modeling, and developmental biology studies. While most studies of hPSCs have so far focused on identifying extrinsic soluble factors, intracellular signaling pathways, and transcriptional networks that are involved in regulating hPSC self-renewal and differentiation, a few promising studies have emerged in recent years to reveal some unique mechano-sensitive and -responsive properties of hPSCs and the effect of the physical aspects of the local cellular microenvironment on regulating hPSC behaviors. This Frontier Review is to highlight these recent studies of mechanobiology in hPSCs and to discuss the impact of advancing our understanding of mechanoregulation of hPSC behaviors on improving survival, self-renewal and differentiation of hPSCs using well-controlled synthetic micro/nanoscale cell culture tools.

A simple route to functionalize polyacrylamide hydrogels for the independent tuning of mechanotransduction cues

Physico-chemical and biochemical factors in the local cellular microenvironment are known to impact on multiple aspects of cell behaviour through specific signal pathways. These mechanotransduction cues can couple each other to regulate cell fate, and it remains unclear whether mechanotransduction in different contexts shares common mechanisms. Undoubtedly, a challenge will involve the further characterization of such cooperative mechanisms, as well as clearly defining the individual role of each mechanical and biochemical parameter. To control these mechanotransduction cues in an independent manner, we developed a simple and efficient strategy to immobilize any desired nature of proteins on polyacrylamide hydrogels and independently control various parameters of the cell microenvironment, such as matrix stiffness, cell-binding ligand density and confined adhesiveness. This novel platform is validated by conducting single-cell experiments and opens a broad avenue for studying complex interplays involved in mechanotransduction with a facile and versatile approach.

Mechanics Regulates Fate Decisions of Human Embryonic Stem Cells

Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.

Forcing Stem Cells to Behave: A Biophysical Perspective of the Cellular Microenvironment

Physical factors in the local cellular microenvironment, including cell shape and geometry, matrix mechanics, external mechanical forces, and nanotopographical features of the extracellular matrix, can all have strong influences on regulating stem cell fate. Stem cells sense and respond to these insoluble biophysical signals through integrin-mediated adhesions and the force balance between intracellular cytoskeletal contractility and the resistant forces originated from the extracellular matrix. Importantly, these mechanotransduction processes can couple with many other potent growth-factor-mediated signaling pathways to regulate stem cell fate. Different bioengineering tools and microscale/nanoscale devices have been successfully developed to engineer the physical aspects of the cellular microenvironment for stem cells, and these tools and devices have proven extremely powerful for identifying the extrinsic physical factors and their downstream intracellular signaling pathways that control stem cell functions.

High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment

Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinson's disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis.

Elastomeric microposts integrated into microfluidics for flow-mediated endothelial mechanotransduction analysis

Mechanotransduction is known as the cellular mechanism converting insoluble biophysical signals in the local cellular microenvironment (e.g. matrix rigidity, external mechanical forces, and fluid shear) into intracellular signalling to regulate cellular behaviours. While microfluidic technologies support a precise and independent control of soluble factors in the cellular microenvironment (e.g. growth factors, nutrients, and dissolved gases), the regulation of insoluble biophysical signals in microfluidics, especially matrix rigidity and adhesive pattern, has not yet been achieved. Here we reported an integrated soft lithography-compatible microfluidic methodology that could enable independent controls and modulations of fluid shear, substrate rigidity, and adhesive pattern in a microfluidic environment, by integrating micromolded elastomeric micropost arrays and microcontact printing with microfluidics. The geometry of the elastomeric micropost array could be regulated to mediate substrate rigidity and adhesive pattern, and further the elastomeric microposts could be utilized as force sensors to map live-cell subcellular contractile forces. To illustrate the general application of our methodology, we investigated the flow-mediated endothelial mechanotransduction process and examined specifically the involvement of subcellular contractile forces in the morphological realignment process of endothelial cells under a sustained directional fluid shear. Our results showed that the cytoskeletal contractile forces of endothelial cells were spatiotemporally regulated and coordinated to facilitate their morphology elongation process along the direction of flow. Together, our study provided an integrated microfluidic strategy to modulate the in vitro cellular microenvironment with both defined soluble and insoluble signals, and we demonstrated its application to investigate quantitatively the involvement of cytoskeletal contractile forces in the flow-mediated mechanotransduction process of endothelial cells.

Measurement of layer-specific mechanical properties in multilayered biomaterials by micropipette aspiration

Many biomaterials and tissues are complex multilayered structures in which the individual layers have distinct mechanical properties that influence the mechanical behavior and define the local cellular microenvironment. Characterization of the mechanical properties of individual layers in intact tissues is technically challenging. Micropipette aspiration (MA) is a proven method for the analysis of local mechanical properties of soft single-layer biomaterials, but its applicability for multilayer structures has not been demonstrated. We sought to determine and validate MA experimental parameters that would permit measurement of the mechanical properties of only the top layer of an intact multilayer biomaterial or tissue. To do so, we performed parametric nonlinear finite-element (FE) analyses and validation experiments using a multilayer gelatin system. The parametric FE analyses demonstrated that measurement of the properties of only the top layer of a multilayer structure is sensitive to the ratio of the pipette inner diameter (D) to top layer thickness (ttop), and that accurate measurement of the top layer modulus requires D/ttop<1. These predictions were confirmed experimentally by MA of the gelatin system. Using this approach and an inverse FE method, the mean effective modulus of the fibrosa layer of intact porcine aortic valve leaflets was determined to be greater than that of the ventricularis layer (P<0.01), consistent with data obtained by tensile testing of dissected layers. This study provides practical guidelines for the use of MA to measure the mechanical properties of single layers in intact multilayer biomaterials and tissues.

The use of vascular endothelial growth factor functionalized agarose to guide pluripotent stem cell aggregates toward blood progenitor cells

The developmental potential of pluripotent stem cells is influenced by their local cellular microenvironment. To better understand the role of vascular endothelial growth factor (VEGFA) in the embryonic cellular microenvironment, we synthesized an artificial stem cell niche wherein VEGFA was immobilized in an agarose hydrogel. Agarose was first modified with coumarin-protected thiols. Upon exposure to ultra-violet excitation, the coumarin groups were cleaved leaving reactive thiols to couple with maleimide-activated VEGFA. Mouse embryonic stem cells (ESC) aggregates were encapsulated in VEGFA immobilized agarose and cultured for 7 days as free-floating aggregates under serum-free conditions. Encapsulated aggregates were assessed for their capacity to give rise to blood progenitor cells. In the presence of bone morphogenetic protein-4 (BMP-4), cells exposed to immobilized VEGFA upregulated mesodermal markers, brachyury and VEGF receptor 2 (T+VEGFR2+) by day 4, and expressed CD34 and CD41 (CD34+CD41+) on day 7. It was found that immobilized VEGFA treatment was more efficient at inducing blood progenitors (including colony forming cells) on a per molecule basis than soluble VEGFA. This work demonstrates the use of functionalized hydrogels to guide encapsulated ESCs toward blood progenitor cells and introduces a tool capable of recapitulating aspects of the embryonic microenvironment.