16 research outputs found
Intracellular Delivery of Exogenous Macromolecules into Human Mesenchymal Stem Cells by Double Deformation of the Plasma Membrane
No full textPhysical techniques for intracellular delivery of exogeneous materials offer an attractive strategy to enhance the therapeutic efficiency of stem cells. However, these methods are currently limited by poor delivery efficiency as well as cytotoxic effects. Here, a high throughput microfluidic device is designed for efficient (approximate to 85%) cytosolic delivery of exogenous macromolecules with minimal cell death (less than 10%). The designed microfluidic device enables the generation of transient pores as the cells pass through the micron-sized constrictions (6-10 mu m) leading to the passive diffusion of extracellular cargos into the cell cytosol. Specifically, the microfluidic system is designed to induce a double deformation on the cell membrane at the squeezing zones to maximize intracellular delivery. Additionally, the flow rate, ionic concentration, and the molecular weight of the cargo are optimized for maximum efficiency. The optimized device enables cytosolic diffusion of small (3 kDa) and large molecules (70 kDa) without inducing any apoptotic effect. Overall, this double cell deformation platform offers new opportunities to rapidly and efficiently deliver extracellular cargo into stem cells without affecting their viability and functionality
Development of MicroRNA-146a-Enriched Stem Cell Secretome for Wound-Healing Applications
No full textSecretome-based therapies have the potential to become the next generation of viable therapeutic wound repair treatments. However, precise strategies aimed to refine and control the secretome composition are necessary to enhance its therapeutic efficacy and facilitate clinical translation. In this study, we aim to accomplish this by transfecting human adipose-derived stem cells (hASCs) with microRNA-146a, which is a potent regulator of angiogenesis and inflammation. The secretome composition obtained from the transfected hASCs (secretome(146a)) was characterized and compared to nontransfected hASCs secretome to evaluate changes in angiogenic and anti-inflammatory growth factor, cytokine, and miRNA content. In vitro proliferation, migration, and tubular morphogenesis assays using human umbilical vein endothelial cells (HUVECs) were completed to monitor the proangiogenic efficacy of the secretome(146a). Finally, the anti-inflammatory efficacy of the secretome(146a) was assessed using HUVECs that were activated to an inflammatory state by IL-1 beta. The resulting HUVEC gene expression and protein activity of key inflammatory mediators were evaluated before and after secretome treatment. Overall, the secretome(146a) contained a greater array and concentration of therapeutic paracrine molecules, which translated into a superior angiogenic and anti-inflammatory efficacy. Therefore, this represents a promising strategy to produce therapeutic secretome for the promotion of wound repair processes
Strategies to develop endogenous stem cell-recruiting bioactive materials for tissue repair and regeneration
No full textA leading strategy in tissue engineering is the design of biomimetic scaffolds that stimulate the body's repair mechanisms through the recruitment of endogenous stem cells to sites of injury. Approaches that employ the use of chemoattractant gradients to guide tissue regeneration without external cell sources are favored over traditional cell-based therapies that have limited potential for clinical translation. Following this concept, bioactive scaffolds can be engineered to provide a temporally and spatially controlled release of biological cues, with the possibility to mimic the complex signaling patterns of endogenous tissue regeneration. Another effective way to regulate stem cell activity is to leverage the inherent chemotactic properties of extracellular matrix (ECM)-based materials to build versatile cell-instructive platforms. This review introduces the concept of endogenous stem cell recruitment, and provides a comprehensive overview of the strategies available to achieve effective cardiovascular and bone tissue regeneration. © 2017 Elsevier B.V
Controlling Adult Stem Cell Behavior Using Nanodiamond-Reinforced Hydrogel: Implication in Bone Regeneration Therapy
No full textAbstractNanodiamonds (NDs) have attracted considerable attention as drug delivery nanocarriers due to their low cytotoxicity and facile surface functionalization. Given these features, NDs have been recently investigated for the fabrication of nanocomposite hydrogels for tissue engineering. Here we report the synthesis of a hydrogel using photocrosslinkable gelatin methacrylamide (GelMA) and NDs as a three-dimensional scaffold for drug delivery and stem cell-guided bone regeneration. We investigated the effect of different concentration of NDs on the physical and mechanical properties of the GelMA hydrogel network. The inclusion of NDs increased the network stiffness, which in turn augmented the traction forces generated by human adipose stem cells (hASCs). We also tested the ability of NDs to adsorb and modulate the release of a model drug dexamethasone (Dex) to promote the osteogenic differentiation of hASCs. The ND-Dex complexes modulated gene expression, cell area, and focal adhesion number in hASCs. Moreover, the integration of the ND-Dex complex within GelMA hydrogels allowed a higher retention of Dex over time, resulting in significantly increased alkaline phosphatase activity and calcium deposition of encapsulated hASCs. These results suggest that conventional GelMA hydrogels can be coupled with conjugated NDs to develop a novel platform for bone tissue engineering.</jats:p
Deciphering the role of substrate stiffness to enhance internalization efficiency of plasmid DNA in stem cells using lipid-based nanocarriers
No full textThis study investigates the role of substrate stiffness on non-viral transfection of human adipose-derived stem cells (hASCs) with the aim to maximize hASCs expression of vascular endothelial growth factor (VEGF). The results confirm the direct effect of substrate stiffness in regulating cytoskeletal remodeling and corresponding plasmid internalization
Engineering a biomimetic environment to pre-condition stem cells for efficacious cardiovascular repair
No full textStatement of Purpose: Myocardial infarction (MI) is amongst the main causes of mortality worldwide, affecting people across a wide range of ages. Stem cell-based therapies are a viable approach to repair cardiovascular tissues due to their advantageous effect in promoting angiogenesis and decreasing scar tissue development. But, their translation to the clinic is hindered by their lack of ability to secrete sufficient therapeutic factors and poor differentiation in vivo. To combat this problem, various strategies like genetic modification and biophysical pre-conditioning have aimed at enhancing the efficacy of stem cells for cardiovascular tissue regeneration
Nanodiamond-based injectable hydrogel for sustained growth factor release: Preparation, characterization and in vitro analysis
No full textInvestigating carrier-based methods and permeabilization strategies to efficiently deliver extracellular cargo into stem cells
No full textIntracellular delivery of extracellular cargo into stem cells is an active area of research with enormous potential in field of regenerative medicine. Different types of cargo can be delivered, including genetic materials (DNA, RNA, siRNA), proteins, and small molecules that are not permeable to the cell membrane. Based on the macromolecule delivered, it is possible to directly control stem cell gene expression, activate specific intracellular pathways, or induce the secretion of therapeutic growth factors. Over the years, several strategies have been investigated to promote efficient internalization of external cargos, and they can be subdivided into two main groups according to the method of delivery. In the first category, we commonly include carrier-based approaches where the physical and chemical properties of the carrier dictate the route of internalization and the delivery efficiency. Aside from the carrier, the cell microenvironment can also impact the process of internalization when using these strategies. Additionally, several parameters such as chemistry, stiffness, and topography of the cell-substrate need to be properly selected for efficient delivery of the external cargo. The second group consists of all the other possible carrier-free based strategies that aim to promote intracellular delivery by increasing the permeability of the cell membrane. Transient pores can be induced in the cell membrane by using different physical and mechanical methods that enable the passive diffusion of extracellular cargo. In this case, the method of permeabilization should not be harmful to the cell and should not elicit any permanent damage to the plasma membrane. Based on these scientific premises, which will be discussed in detail in Chapter 1, the aim of this thesis was to investigate both types of delivery methods to identify novel approaches for effective intracellular delivery of genes as well as small polar molecules into stem cells using human adipose-derived stem cells (hASCs) as a model cell line. The overall work of this thesis is iv subdivided into two separate sections based on the strategy adopted for the delivery of the external cargo. The first part of the thesis, discussed in Chapter 2, aimed to investigate the modulatory role of substrate stiffness in the transfection of stem cells by using conventional lipid-based carriers complexed with plasmid DNA (lipoplexes). Precisely, we investigated whether the changes in cellular morphology and cytoskeletal rearrangement, induced by a variation in the stiffness of the cell-substrate, had any significant impact on the process of transfection. The findings of this work are important to advance the understanding of how the physical properties of the cell microenvironment can be controlled to enhance the efficacy of conventional carrier-based strategies for the transfection of stem cells. The second part of the thesis, discussed in Chapter 3 and 4, was focused on the design of a novel carrier-free approach for the intracellular delivery of small polar molecules, such as trehalose. Trehalose is a cryoprotecting agent (CPA) that could be used as a replacement of more toxic molecules such as dimethylsulfoxide (DMSO). However, the poor permeability of trehalose through cell membranes limits its applicability as a CPA for stem cell banking applications. Therefore, to promote efficient intracellular delivery of trehalose, we fabricated and optimized a microfluidic device that could create temporary pores in the cell membranes to enable the uptake of extracellular cargo without the use of any carrier. The safety and efficiency of this new platform were tested by studying several parameters, including cell viability, apoptosis, cellular morphology, and differentiation potential of hASCs loaded with trehalose after cryopreservation. Overall, the results and findings of this final section of the thesis provide a novel valuable strategy for the internalization of trehalose to promote efficient cryopreservation of stem cells without the use of DMSO
Author Correction: AHR is a Zika virus host factor and a candidate target for antiviral therapy
No full textFil: Giovannoni, Federico. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Estrategias Antivirales; Argentina. CONICET-Instituto de Química Biológica; Argentina.Fil: Bosch, Irene. Massachusetts Institute of Technology. Institute for Medical Engineering and Science; Estados Unidos. Mount Sinai School of Medicine. Department of Medicine; Estados Unidos.Fil: Manganeli Polonio, Carolina. University of São Paulo. Immunology Department-ICB IV. Neuroimmune Interactions Laboratory; Brasil. University of São Paulo. Scientific Platform Pasteur-USP; Brasil.Fil: Torti, María F. University of São Paulo. Immunology Department-ICB IV. Neuroimmune Interactions Laboratory; Brasil. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Estrategias Antivirales; Argentina. CONICET-Instituto de Química Biológica; Argentina.Fil: Wheeler, Michael A. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Li, Zhaorong. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Romorini, Leonardo. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Rodríguez Varela, María Soledad. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Rothhammer, Veit. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Barroso, Andreia. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Tjon, Emily C. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Sanmarco, Liliana M. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Takenaka, Maisa C. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Sadegh Modaresi, Seyed Mohamad. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Gutiérrez-Vázquez, Cristina. Harvard Medical School. Brigham and Women's Hospital. Ann Romney Center for Neurologic Diseases; Estados Unidos.Fil: Ghabdan Zanluqui, Nágela. University of São Paulo. Scientific Platform Pasteur-USP; Brasil. University of São Paulo. School of Medicine. Immunopathology and Allergy Post Graduate Program; Brasil.Fil: Barreto Dos Santos, Nilton. University of São Paulo. Institute of Biomedical Science. Department of Pharmacology; Brasil.Fil: Demarchi Munhoz, Carolina. University of São Paulo. Institute of Biomedical Science. Department of Pharmacology; Brasil.Fil: Wang, Zhongyan. Boston University School of Public Health. Dept. of Environmental Health; Estados Unidos.Fil: Damonte, Elsa B. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Estrategias Antivirales; Argentina. CONICET-Instituto de Química Biológica; Argentina
Evaluation of midcarpal capitate contact mechanics in normal, injured and post-operative wrists
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