A Computational Model of Mechanical Stretching of Cultured Cells on a Flexible Membrane.

Mechanical forces applied to cells are known to regulate a wide variety of biological processes. Recent studies have supported that mechanical forces can cause nuclear deformation, leading to significant alterations in the gene expression and chromatin landscape of the cell. While the stresses and strains applied to cells is it is often known or controlled experimentally on a macroscopic length scale, it is often unclear what the actual forces and displacements are at the microscopic level of the cell. In this work, we created a model of cell deformation during application of mechanical stretch to cultured cells growth on a flexible membrane. This configuration is commonly used is in experimental studies as a means to apply controlled mechanical strains to adherent cultured cells. The parameters used in the study were used for application of strain to a mesenchymal stem cell stretched on a membrane. computational model was created to simulate the stresses and strains within the cell under a variety of stain amplitudes, waveforms and frequencies of mechanical loading with the range of commonly used experimental systems. The results demonstrate the connection between mechanical loading parameters applied through the flexible membrane and the resulting stresses and strains within the cell and nucleus. Using a viscoelastic model of chromatin, we connected the results provide to a rough model of resulting deformation within chromatin from the forces applied to the nucleus. Overall, the model is useful in providing insight between experimentally applied mechanical forces and the actual forces within the cell to better interpret the results of experimental studies. Statement of Significance: In this work, we created a computational model of the mechanical stretching of cell on a flexible membrane under cyclic mechanical loading. This model provides insight into the forces and displacements inside of cell that result from that application of stretch. As many experiments use this set up, our work is relevant to interpreting many studies that use mechanical stretch to stimulate mechanotransduction.

Mechanical Rejuvenation of Mesenchymal Stem Cells from Aged Patients.

Mesenchymal stem cells (MSC) are an appealing therapeutic cell type for many diseases. However, patients with poor health or advanced age often have MSCs with poor regenerative properties. A major limiter of MSC therapies is cellular senescence, which is marked by limited proliferation capability, diminished multipotency, and reduced regenerative properties. In this work, we explored the ability of applied mechanical forces to reduce cellular senescence in MSCs. Our studies revealed that mechanical conditioning caused a lasting enhancement in proliferation, overall cell culture expansion potential, multipotency, and a reduction of senescence in MSCs from aged donors. Mechanistic studies suggested that these functional enhancements were mediated by oxidative stress and DNA damage repair signaling with mechanical load altering the expression of proteins of the sirtuin pathway, the DNA damage repair protein ATM, and antioxidant proteins. In addition, our results suggest a biophysical mechanism in which mechanical stretch leads to improved recognition of damaged DNA in the nucleus. Analysis of the cells through RNA-seq and ATAC-seq, demonstrated that mechanical loading alters the cell's genetic landscape to cause broad shifts in transcriptomic patterns that related to senescence. Overall, our results demonstrate that mechanical conditioning can rejuvenate mesenchymal stem cells derived from aged patients and improve their potential as a therapeutic cell type.

Transmembrane stem factor nanodiscs enhanced revascularization in a hind limb ischemia model in diabetic, hyperlipidemic rabbits.

Therapies to revascularize ischemic tissue have long been a goal for the treatment of vascular disease and other disorders. Therapies using stem cell factor (SCF), also known as a c-Kit ligand, had great promise for treating ischemia for myocardial infarct and stroke, however clinical development for SCF was stopped due to toxic side effects including mast cell activation in patients. We recently developed a novel therapy using a transmembrane form of SCF (tmSCF) delivered in lipid nanodiscs. In previous studies, we demonstrated tmSCF nanodiscs were able to induce revascularization of ischemia limbs in mice and did not activate mast cells. To advance this therapeutic towards clinical application, we tested this therapy in an advanced model of hindlimb ischemia in rabbits with hyperlipidemia and diabetes. This model has therapeutic resistance to angiogenic therapies and maintains long term deficits in recovery from ischemic injury. We treated rabbits with local treatment with tmSCF nanodiscs or control solution delivered locally from an alginate gel delivered into the ischemic limb of the rabbits. After eight weeks, we found significantly higher vascularity in the tmSCF nanodisc-treated group in comparison to alginate treated control as quantified through angiography. Histological analysis also showed a significantly higher number of small and large blood vessels in the ischemic muscles of the tmSCF nanodisc treated group. Importantly, we did not observe inflammation or mast cell activation in the rabbits. Overall, this study supports the therapeutic potential of tmSCF nanodiscs for treating peripheral ischemia.

Transmembrane Stem Factor Nanodiscs Enhanced Revascularization in a Hind Limb Ischemia Model in Diabetic, Hyperlipidemic Rabbits.

Therapies to revascularize ischemic tissue have long been a goal for the treatment of vascular disease and other disorders. Therapies using stem cell factor (SCF), also known as a c-Kit ligand, had great promise for treating ischemia for myocardial infarct and stroke, however clinical development for SCF was stopped due to toxic side effects including mast cell activation in patients. We recently developed a novel therapy using a transmembrane form of SCF (tmSCF) delivered in lipid nanodiscs. In previous studies, we demonstrated tmSCF nanodiscs were able to induce revascularization of ischemia limbs in mice and did not activate mast cells. To advance this therapeutic towards clinical application, we tested this therapy in an advanced model of hindlimb ischemia in rabbits with hyperlipidemia and diabetes. This model has therapeutic resistance to angiogenic therapies and maintains long term deficits in recovery from ischemic injury. We treated rabbits with local treatment with tmSCF nanodiscs or control solution delivered locally from an alginate gel delivered into the ischemic limb of the rabbits. After eight weeks, we found significantly higher vascularity in the tmSCF nanodisc-treated group in comparison to alginate treated control as quantified through angiography. Histological analysis also showed a significantly higher number of small and large blood vessels in the ischemic muscles of the tmSCF nanodisc treated group. Importantly, we did not observe inflammation or mast cell activation in the rabbits. Overall, this study supports the therapeutic potential of tmSCF nanodiscs for treating peripheral ischemia.

The effects of strain history on aortic valve interstitial cell activation in a 3D hydrogel environment.

Aortic valves (AVs) undergo unique stretch histories that include high rates and magnitudes. While major differences in deformation patterns have been observed between normal and congenitally defective bicuspid aortic valves (BAVs), the relation to underlying mechanisms of rapid disease onset in BAV patients remains unknown. To evaluate how the variations in stretch history affect AV interstitial cell (AVIC) activation, high-throughput methods were developed to impart varied cyclical biaxial stretch histories into 3D poly(ethylene) glycol hydrogels seeded with AVICs for 48 h. Specifically, a physiologically mimicking stretch history was compared to two stretch histories with varied peak stretch and stretch rate. Post-conditioned AVICs were imaged for nuclear shape, alpha smooth muscle actin (αSMA) and vimentin (VMN) polymerization, and small mothers against decapentaplegic homologs 2 and 3 (SMAD 2/3) nuclear activity. The results indicated that bulk gel deformations were accurately transduced to the AVICs. Lower peak stretches lead to increased αSMA polymerization. In contrast, VMN polymerization was a function of stretch rate, with SMAD 2/3 nuclear localization and nuclear shape also trending toward stretch rate dependency. Lower than physiological levels of stretch rate led to higher SMAD 2/3 activity, higher VMN polymerization around the nucleus, and lower nuclear elongation. αSMA polymerization did not correlate with VMN polymerization, SMAD 2/3 activity, nor nuclear shape. These results suggest that a negative feedback loop may form between SMAD 2/3, VMN, and nuclear shape to maintain AVIC homeostatic nuclear deformations, which is dependent on stretch rate. These novel results suggest that AVIC mechanobiological responses are sensitive to stretch history and provide insight into the mechanisms of AV disease.

Transmembrane Stem Factor Nanodiscs Enhanced Revascularization in a Hind Limb Ischemia Model in Diabetic, Hyperlipidemic Rabbits.

Therapies to revascularize ischemic tissue have long been a goal for the treatment of vascular disease and other disorders. Therapies using stem cell factor (SCF), also known as a c-Kit ligand, had great promise for treating ischemia for myocardial infarct and stroke, however clinical development for SCF was stopped due to toxic side effects including mast cell activation in patients. We recently developed a novel therapy using a transmembrane form of SCF (tmSCF) delivered in lipid nanodiscs. In previous studies, we demonstrated tmSCF nanodiscs were able to induce revascularization of ischemia limbs in mice and did not activate mast cells. To advance this therapeutic towards clinical application, we tested this therapy in an advanced model of hindlimb ischemia in rabbits with hyperlipidemia and diabetes. This model has therapeutic resistance to angiogenic therapies and maintains long term deficits in recovery from ischemic injury. We treated rabbits with local treatment with tmSCF nanodiscs or control solution delivered locally from an alginate gel delivered into the ischemic limb of the rabbits. After eight weeks, we found significantly higher vascularity in the tmSCF nanodisc-treated group in comparison to alginate treated control as quantified through angiography. Histological analysis also showed a significantly higher number of small and large blood vessels in the ischemic muscles of the tmSCF nanodisc treated group. Importantly, we did not observe inflammation or mast cell activation in the rabbits. Overall, this study supports the therapeutic potential of tmSCF nanodiscs for treating peripheral ischemia.

Mechanical conditioning of human mesenchymal stem cells for enhancing vascular regeneration.

Human mesenchymal stem cells (hMSCs) are an appealing cell type for therapeutic applications but remain limited by poor efficacy in clinical trials. Here, we describe a conditioning technique that enhances the vascular regenerative properties of hMSCs and increases their expression of endothelial cell and pericyte markers. We also describe an alginate gel encapsulation protocol for delivering the conditioned cells. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).1.

Transmembrane stem cell factor protein therapeutics enhance revascularization in ischemia without mast cell activation.

Stem cell factor (SCF) is a cytokine that regulates hematopoiesis and other biological processes. While clinical treatments using SCF would be highly beneficial, these have been limited by toxicity related to mast cell activation. Transmembrane SCF (tmSCF) has differential activity from soluble SCF and has not been explored as a therapeutic agent. We created novel therapeutics using tmSCF embedded in proteoliposomes or lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and improved the revascularization in the ischemic hind limb. Proteoliposomal tmSCF preferentially acted on endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization and recruitment to the site of injury. The type of lipid nanocarrier used altered the relative cellular uptake pathways and signaling in a cell type dependent manner. Overall, we found that tmSCF-based therapies can provide therapeutic benefits without off target effects.

Mechanobiological conditioning of mesenchymal stem cells for enhanced vascular regeneration.

Using endogenous mesenchymal stem cells for treating myocardial infarction and other cardiovascular conditions typically results in poor efficacy, in part owing to the heterogeneity of the harvested cells and of the patient responses. Here, by means of high-throughput screening of the combinatorial space of mechanical-strain level and of the presence of particular kinase inhibitors, we show that human mesenchymal stem cells can be mechanically and pharmacologically conditioned to enhance vascular regeneration in vivo. Mesenchymal stem cells conditioned to increase the activation of signalling pathways mediated by Smad2/3 (mothers against decapentaplegic homolog 2/3) and YAP (Yes-associated protein) expressed markers that are associated with pericytes and endothelial cells, displayed increased angiogenic activity in vitro, and enhanced the formation of vasculature in mice after subcutaneous implantation and after implantation in ischaemic hindlimbs. These effects were mediated by the crosstalk of endothelial-growth-factor receptors, transforming-growth-factor-beta receptor type 1 and vascular-endothelial-growth-factor receptor 2. Mechanical and pharmacological conditioning can significantly enhance the regenerative properties of mesenchymal stem cells.

A Robust Longitudinal Co-culture of Obligate Anaerobic Gut Microbiome With Human Intestinal Epithelium in an Anoxic-Oxic Interface-on-a-Chip.

The majority of human gut microbiome is comprised of obligate anaerobic bacteria that exert essential metabolic functions in the human colon. These anaerobic gut bacteria constantly crosstalk with the colonic epithelium in a mucosal anoxic-oxic interface (AOI). However, in vitro recreation of the metabolically mismatched colonic AOI has been technically challenging. Furthermore, stable co-culture of the obligate anaerobic commensal microbiome and epithelial cells in a mechanically dynamic condition is essential for demonstrating the host-gut microbiome crosstalk. Here, we developed an anoxic-oxic interface-on-a-chip (AOI Chip) by leveraging a modified human gut-on-a-chip to demonstrate a controlled oxygen gradient in the lumen-capillary transepithelial interface by flowing anoxic and oxic culture medium at various physiological milieus. Computational simulation and experimental results revealed that the presence of the epithelial cell layer and the flow-dependent conditioning in the lumen microchannel is necessary and sufficient to create the steady-state vertical oxygen gradient in the AOI Chip. We confirmed that the created AOI does not compromise the viability, barrier function, mucin production, and the expression and localization of tight junction proteins in the 3D intestinal epithelial layer. Two obligate anaerobic commensal gut microbiome, Bifidobacterium adolescentis and Eubacterium hallii, that exert metabolic cross-feeding in vivo, were independently co-cultured with epithelial cells in the AOI Chip for up to a week without compromising any cell viability. Our new protocol for creating an AOI in a microfluidic gut-on-a-chip may enable to demonstrate the key physiological interactions of obligate anaerobic gut microbiome with the host cells associated with intestinal metabolism, homeostasis, and immune regulation.

Engineering of microscale vascularized fat that responds to perfusion with lipoactive hormones.

Current methods to treat large soft-tissue defects mainly rely on autologous transfer of adipocutaneous flaps, a method that is often limited by donor site availability. Engineered vascularized adipose tissues can potentially be a viable and readily accessible substitute to autologous flaps. In this study, we engineered a small-scale adipose tissue with pre-patterned vasculature that enables immediate perfusion. Vessels formed after one day of perfusion and displayed barrier function after three days of perfusion. Under constant perfusion, adipose tissues remained viable and responded to lipoactive hormones insulin and epinephrine with lipid accumulation and loss, respectively. Adipocyte growth correlated inversely with distance away from the feeding vessel, as predicted by a Krogh-type model.

Neutrophil Elastase Regulates Emergency Myelopoiesis Preceding Systemic Inflammation in Diet-induced Obesity.

Inflammation plays a significant role in the development of obesity-related complications, but the molecular events that initiate and propagate such inflammation remain unclear. Here, we report that mice fed a high-fat diet (HFD) for as little as 1-3 days show increased differentiation of myeloid progenitors into neutrophils and monocytes but reduced B lymphocyte production in the bone marrow. Levels of neutrophil elastase (NE) and the nuclear factors CCAAT/enhancer-binding protein α (C/EBPα) and growth factor-independent 1 (GFI-1) are elevated in hematopoietic stem and progenitor cells from HFD-fed mice, but mice lacking either NE or C/EBPα are resistant to HFD-induced myelopoiesis. NE deletion increases expression of the inhibitory isoform of p30 C/EBPα, impairs the transcriptional activity of p42 C/EBPα, and reduces expression of the C/EBPα target gene GFI-1 in hematopoietic stem and progenitor cells, suggesting a mechanism by which NE regulates myelopoiesis. Furthermore, NE deletion prevents HFD-induced vascular leakage. Thus, HFD feeding rapidly activates bone marrow myelopoiesis through the NE-dependent C/EBPα-GFI-1 pathway preceding vascular damage and systemic inflammation.