Perspective on This Article from Metformin Prevents Liver Tumorigenesis by Inhibiting Pathways Driving Hepatic Lipogenesis

Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that NF-kappa B promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF-kappa B activation in the pulmonary vasculature are unknown. To identify mechanisms that activate proangiogenic NF-kappa B signaling in the developing pulmonary vasculature, proteomic analysis of the lung secretomewas performed using two-dimensional difference gel electrophoresis. NF-kappa B activation and angiogenic function was assessed in primary pulmonary endothelial cells (PECs) and TGFBI (transforming growth factor-beta-induced protein)-regulated genes identified using RNA sequencing. Alveolarization and pulmonary angiogenesis was assessed in wild-type and Tgfbi null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Secreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PECs. Proteomic analysis identified TGFBI as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NF-kB via avb3 integrins. RNA sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PECs. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. Our studies identify TGFBI as a developmentally regulated protein that promotes NF-kappa B-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

Synthetic nerve guidance conduits (NGCs) offer an alternative to harvested nerve grafts for treating peripheral nerve injury (PNI). NGCs have been made from both naturally derived and synthesized materials. While naturally derived materials typically have an increased capacity for bioactivity, synthesized materials have better material control, including tunability and reproducibility. Protein engineering is an alternative strategy that can bridge the benefits of these two classes of materials by designing cell-responsive materials that are also systematically tunable and consistent. Here, we tested a recombinantly derived elastin-like protein (ELP) hydrogel as an intraluminal filler in a rat sciatic nerve injury model. We demonstrated that ELPs enhance the probability of forming a tissue bridge between the proximal and distal nerve stumps compared to an empty silicone conduit across the length of a 10 mm nerve gap. These tissue bridges have evidence of myelinated axons, and electrophysiology demonstrated that regenerated axons innervated distal muscle groups. Animals implanted with an ELP-filled conduit had statistically higher functional control at 6 weeks than those that had received an empty silicone conduit, as evaluated by the sciatic functional index. Taken together, our data support the conclusion that ELPs support peripheral nerve regeneration in acute complete transection injuries when used as an intraluminal filler. These results support the further study of protein engineered recombinant ELP hydrogels as a reproducible, off-the-shelf alternative for regeneration of peripheral nerves.

Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material-based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell-mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC-secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter-responsive neurons. Matrix remodeling modulates expression of the transcriptional co-activator Yes-associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin-dependent manner. Thus, cell-remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.

Background Metabolic reprogramming is a key feature of malignant cells. While glucose is one of the primary substrates for malignant cells, cancer cells also display a remarkable metabolic flexibility. Depending on nutrient availability and requirements, cancer cells will utilize alternative fuel sources to maintain the TCA cycle for bioenergetic and biosynthetic requirements. Lactate was typically viewed as a passive byproduct of cancer cells. However, studies now show that lactate is an important substrate for the TCA cycle in breast, lung, and pancreatic cancer. Methods Metabolic analysis of colorectal cancer (CRC) cells was performed using a combination of bioenergetic analysis and 13 C stable isotope tracing. Results We show here that CRC cells use lactate to fuel the TCA cycle and promote growth especially under nutrient-deprived conditions. This was mediated in part by maintaining cellular bioenergetics. Therefore targeting the ability of cancer cells to utilize lactate via the TCA cycle would have a significant therapeutic benefit. Phosphoenolpyruvate carboxykinase (PEPCK) is an important cataplerotic enzyme that promotes TCA cycle activity in CRC cells. Treatment of CRC cells with low micromolar doses of a PEPCK inhibitor (PEPCKi) developed for diabetes decreased cell proliferation and utilization of lactate by the TCA cycle in vitro and in vivo. Mechanistically, we observed that the PEPCKi increased nutrient stress as determined by decreased cellular bioenergetics including decreased respiration, ATP levels, and increased AMPK activation. 13 C stable isotope tracing showed that the PEPCKi decreased the incorporation of lactate into the TCA cycle. Conclusions These studies highlight lactate as an important substrate for CRC and the use of PEPCKi as a therapeutic approach to target lactate utilization in CRC cells.

A key feature of peripheral arterial disease (PAD) is damage to endothelial cells (ECs), resulting in lower limb pain and restricted blood flow. Recent preclinical studies demonstrate that the transplantation of ECs via direct injection into the affected limb can result in significantly improved blood circulation. Unfortunately, the clinical application of this therapy has been limited by low cell viability and poor cell function. To address these limitations we have developed an injectable, recombinant hydrogel, termed SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) for cell transplantation. SHIELD provides mechanical protection from cell membrane damage during syringe flow. Additionally, secondary in situ crosslinking provides a reinforcing network to improve cell retention, thereby augmenting the therapeutic benefit of cell therapy. In this study, we demonstrate the improved acute viability of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following syringe injection delivery in SHIELD, compared to saline. Using a murine hind limb ischemia model of PAD, we demonstrate enhanced iPSC-EC retention in vivo and improved neovascularization of the ischemic limb based on arteriogenesis following transplantation of iPSC-ECs delivered in SHIELD.

Currently, no medical therapies exist to augment stroke recovery. Stem cells are an intriguing treatment option being evaluated, but cell-based therapies have several challenges including developing a stable cell product with long term reproducibility. Since much of the improvement observed from cellular therapeutics is believed to result from trophic factors the stem cells release over time, biomaterials are well-positioned to deliver these important molecules in a similar fashion. Here we show that essential trophic factors secreted from stem cells can be effectively released from a multi-component hydrogel system into the post-stroke environment. Using our polymeric system to deliver VEGF-A and MMP-9, we improved recovery after stroke to an equivalent degree as observed with traditional stem cell treatment in a rodent model. While VEGF-A and MMP-9 have many unique mechanisms of action, connective tissue growth factor (CTGF) interacts with both VEGF-A and MMP-9. With our hydrogel system as well as with stem cell delivery, the CTGF pathway is shown to be downregulated with improved stroke recovery.

Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials.

The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2 cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2 cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported in vivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by pharmaceutical companies without major changes to their current testing protocols.

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell–cell contact and promote β-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

Introduction: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are a promising cell source for enhancing blood perfusion for treatment of peripheral arterial disease (PAD). However, poor cell survival is a critical bottleneck to the efficacy of stem cell therapy. Hypothesis: We hypothesize that encapsulation of cells within an injectable shear-thinning hydrogel with controllable properties will prolong cell survival under ischemic conditions and maintain cellular phenotype, in comparison to cell injection in saline. Methods: The protein hydrogel is comprised of two complementary engineered proteins that self-assemble upon simple mixing. The hydrogel network incorporates a polyethylene glycol physical crosslinker that modulates hydrogel stiffness and degradation. Bioluminescent iPSC-ECs were encapsulated within the hydrogel with controllable stiffness ( G’ ~10-800 Pa) under conditions of hypoxia (1% O 2 ). The cells within hydrogel were then subjected to an in vitro model of injection and assayed for cell survival, proliferation, and endothelial phenotype for up to 14 days. To verify these results in an experimental model of PAD, 10 6 cells were injected in saline or in 400 Pa hydrogel into the ischemic limb of SCID mice for assessment of cell viability and blood flow recovery. Results: Acutely after injection in vitro , cell survival in saline was 65%, in comparison to 94% in hydrogels with stiffnesses of 10-800 Pa. Bioluminescence imaging demonstrated the highest cell proliferation in the hydrogel with 400 Pa stiffness after 14 days. In the 400 Pa hydrogel, iPSC-ECs maintained elongated morphology with robust expression of endothelial phenotypic marker, CD31. In the ischemic hindlimb, iPSC-EC retention was markedly increased when encapsulated in the 400 Pa hydrogel, compared to saline delivery. Concomitantly, mean blood perfusion recovery in the ischemic limb after 14 days was 0.68 when treated with cells in hydrogel, in contrast to 0.61 when cells were injected in saline. Conclusions: These findings demonstrate that stem cell encapsulation within the 400 Pa protein hydrogel improves cell viability and blood perfusion, which may have therapeutic benefit for treatment of PAD.

Event Abstract Back to Event Supramolecular hydrogels with double network formation for cell transplantation therapies Lei Cai1, Ruby Dewi1 and Sarah Heilshorn1 1 Stanford University, Materials Science & Engineering, United States Introduction: Stem cell transplantation via direct injection is a minimally invasive strategy for the potential treatment of a variety of injuries and diseases. Transplanted cells are hypothesized to exert their regenerative potential through secretion of paracrine factors that stimulate endogenous cells. However, preclinical studies indicate that transplanted cell retention is typically only ~1% one week post-injection, regardless of cell type and dose[1]. Thus, our goal was to design a supramolecular hydrogel that provides both (1) mechanical shielding of cells during injection and (2) an optimal matrix post-transplantation to promote paracrine factor secretion. This required fine-tuning of two distinct, noncovalent crosslinking steps to optimize the hydrogel mechanical properties during two separate phases of biomaterial use: (1) initial cell encapsulation and delivery and (2) in situ cell scaffolding. Materials and Methods: The supramolecular hydrogel is composed of a recombinant, engineered protein with multiple repeats of a molecular-recognition domain and a PEG-PNIPAM copolymer decorated with complementary molecular-recognition peptides[2]. The first crosslinking step occurs ex vivo through peptide-based molecular recognition to encapsulate cells within a weak hydrogel, while the second crosslinking step occurs in situ through a thermally-induced, hydrophobic phase transition to form a reinforcing network. This programmed stiffening effect was characterized using rheology and FRET analysis of diffusivity and mesh size. Encapsulation of human adipose-derived stem cells (hASCs, obtained with informed consent as approved by IRB oversight) was characterized by Live/Dead labeling, immunocytochemistry and confocal microscopy. Cell proliferation was quantified by DNA analysis. Paracrine factor production was monitored by mRNA expression, Western blotting, ELISA, and functional endothelial cell tubulogenesis assays. In vivo retention of transplanted material and cells was followed by noninvasive imaging of a near-infrared Cy5.5 reporter and bioluminescent firefly luciferase reporter, respectively. Results and Discussion: A family of seven supramolecular materials was prepared with nearly identical weak hydrogel properties ex vivo (G' ~10 Pa) and a range of mechanical properties spanning three orders of magnitude upon secondary crosslinking in vivo (G' ~10-1000 Pa). All seven hydrogels were able to provide significant mechanical shielding to encapsulated hASCs when injected through a 28-gauge syringe needle, resulting in >90% viability. In contrast, hASC delivery in saline resulted in 69% viable cells. Following injection, 3D cell proliferation over two weeks was significantly enhanced within hydrogels of intermediate stiffness (G' ~200-400 Pa). Interestingly, levels of mRNA expression and protein secretion of several pro-angiogenic factors including vascular endothelial growth factor, angiopoietin, fibroblast growth factor-2, hepatocyte growth factor, and platelet-derived growth factor were significantly upregulated in hydrogels of intermediate stiffness. Conditioned medium from encapsulated hASCs was functionally bioactive, as it actively promoted endothelial cell network formation in an in vitro model system. As expected, stiffer hydrogels resulted in longer material and cell retention in in vivo mouse models. Conclusion: Together, these data suggest that hydrogel mechanical properties may need to be separately optimized for each phase of cell transplantation, with weaker gels preferred during implantation and intermediate to stiffer gels favored post-transplantation to promote the secretion of regenerative paracrine factors. Supramolecular hydrogels are well suited to achieving this type of multi-stage control over material properties. California Institute for Regenerative Medicine RT3-07948; National Institutes of Health NRSA F32HL128094References:[1] Mathiasen, A. B.; Kastrup, J. Theranostics 2013, 3, 561-572.[2] Cai, L.; Dewi, R. E.; Heilshorn, S. C. Advanced Functional Materials, 2015, 25, 1344-1351. Keywords: stem cell, 3D scaffold, Biodegradable material, biomacromolecule Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Extracellular matrices for therapeutic delivery Citation: Cai L, Dewi R and Heilshorn S (2016). Supramolecular hydrogels with double network formation for cell transplantation therapies. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00809 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lei Cai Ruby Dewi Sarah Heilshorn Google Lei Cai Ruby Dewi Sarah Heilshorn Google Scholar Lei Cai Ruby Dewi Sarah Heilshorn PubMed Lei Cai Ruby Dewi Sarah Heilshorn Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A family of shear-thinning hydrogels for injectable encapsulation and long-term delivery (SHIELD) has been designed and synthesized with controlled in situ stiffening properties to regulate the stem cell secretome. The authors demonstrate that SHIELD with an intermediate stiffness (200-400 Pa) could significantly promote the angiogenic potential of human adipose-derived stem cells.

Exposing myoblasts to basic fibroblast growth factor (bFGF), which is released after muscle injury, results in receptor phosphorylation, faster migration, and increased proliferation. These effects occur on time scales that extend across three orders of magnitude (10(0)-10(3) minutes). Finite element modeling of Transwell assays, which are traditionally used to assess chemotaxis, revealed that the bFGF gradient formed across the membrane pore is short-lived and diminishes 45% within the first minute. Thus, to evaluate bFGF-induced migration over 10(2) minutes, we employed a microfluidic assay capable of producing a stable, linear concentration gradient to perform single-cell analyses of chemokinesis and chemotaxis. We hypothesized that the composition of the underlying extracellular matrix (ECM) may affect the behavioral response of myoblasts to soluble bFGF, as previous work with other cell types has suggested crosstalk between integrin and fibroblast growth factor (FGF) receptors. Consistent with this notion, we found that bFGF significantly reduced the doubling time of myoblasts cultured on laminin but not fibronectin or collagen. Laminin also promoted significantly faster migration speeds (13.4 μm h(-1)) than either fibronectin (10.6 μm h(-1)) or collagen (7.6 μm h(-1)) without bFGF stimulation. Chemokinesis driven by bFGF further increased migration speed in a strictly additive manner, resulting in an average increase of 2.3 μm h(-1) across all ECMs tested. We observed relatively mild chemoattraction (∼67% of myoblast population) in response to bFGF gradients of 3.2 ng mL(-1) mm(-1) regardless of ECM identity. Thus, while ECM-bFGF crosstalk did impact chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. These data suggest that the main physiological effect of bFGF on myoblast migration is chemokinesis and that changes in the surrounding ECM, resulting from aging and/or disease may impact muscle regeneration by altering myoblast migration and proliferation.

Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.