Temporal changes guided by mesenchymal stem cells on a 3D microgel platform enhance angiogenesis in vivo at a low-cell dose.
Proceedings of the National Academy of Sciences of the United States of America
Modeling Secondary Iron Overload Cardiomyopathy with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
2020; 32 (2): 107886
Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 104 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).
View details for DOI 10.1073/pnas.2008245117
View details for PubMedID 32709748
Allogeneic Mesenchymal Stromal Cells (MSCs) are of Comparable Efficacy to Syngeneic MSCs for Therapeutic Revascularization in C57BKSdb/db Mice Despite the Induction of Alloantibody.
Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.
View details for DOI 10.1016/j.celrep.2020.107886
View details for PubMedID 32668256
Toward Customized Extracellular Niche Engineering: Progress in Cell-Entrapment Technologies.
Advanced materials (Deerfield Beach, Fla.)
2018; 30 (1)
Intramuscular administration of mesenchymal stromal cells (MSCs) represents a therapeutic option for diabetic critical limb ischemia. Autologous or allogeneic approaches may be used but disease-induced cell dysfunction may limit therapeutic efficacy in the former. Our aim was to compare the efficacy of allogeneic and autologous MSC transplantation in a model of hindlimb ischemia in diabetes mellitus and to determine whether allogeneic transplantation would result in the activation of an immune response. MSCs were isolated from C57BL/6 (B6) and diabetic obese C57BKSdb/db mice. Phosphate-buffered saline (control group), and MSCs (1 × 106) from B6 (allogeneic group) or C57BKSdb/db (syngeneic group) were administered intramuscularly into the ischemic thigh of C57BKSdb/db mice following the induction of hindlimb ischemia. MSCs derived from both mouse strains secrete several angiogenic factors, suggesting that the potential therapeutic effect is due to paracrine signaling. Administration of allogeneic MSCs significantly improved blood perfusion as compared with the control group on week 2 and 3, post-operatively. In comparison with the control group, syngeneic MSCs significantly improved blood perfusion at week 2 only. There was no statistical difference in blood perfusion between allogeneic and syngeneic MSC groups at any stages. There was no statistical difference in ambulatory and necrosis score among the three groups. Amputation of toes was only observed in the control group (one out of seven animals). Alloantibody was detected in three out of the eight mice that received allogeneic MSCs but was not observed in the other groups. In summary, we demonstrated comparable efficacy after transplantation of autologous and allogeneic MSCs in a diabetic animal model despite generation of an immune response.
View details for DOI 10.1177/0963689718784862
View details for PubMedID 30016879
The Functional Response of Mesenchymal Stem Cells to Electron-Beam Patterned Elastomeric Surfaces Presenting Micrometer to Nanoscale Heterogeneous Rigidity.
Advanced materials (Deerfield Beach, Fla.)
The primary aim in tissue engineering is to repair, replace, and regenerate dysfunctional tissues to restore homeostasis. Cell delivery for repair and regeneration is gaining impetus with our understanding of constructing tissue-like environments. However, the perpetual challenge is to identify innovative materials or re-engineer natural materials to model cell-specific tissue-like 3D modules, which can seamlessly integrate and restore functions of the target organ. To devise an optimal functional microenvironment, it is essential to define how simple is complex enough to trigger tissue regeneration or restore cellular function. Here, the purposeful transition of cell immobilization from a cytoprotection point of view to that of a cell-instructive approach is examined, with advances in the understanding of cell-material interactions in a 3D context, and with a view to further application of the knowledge for the development of newer and complex hierarchical tissue assemblies for better examination of cell behavior and offering customized cell-based therapies for tissue engineering.
View details for PubMedID 29194781
Stimulation of 3D Osteogenesis by Mesenchymal Stem Cells Using a Nanovibrational Bioreactor
Nature Biomedical Engineering
Towards Customised Extracellular Niche Engineering: Progress in Cell Entrapment Technologies
Scaffold and scaffold-free self-assembled systems in regenerative medicine
BIOTECHNOLOGY AND BIOENGINEERING
2016; 113 (6): 1155-1163
Cells directly probe and respond to the physicomechanical properties of their extracellular environment, a dynamic process which has been shown to play a key role in regulating both cellular adhesive processes and differential cellular function. Recent studies indicate that stem cells show lineage-specific differentiation when cultured on substrates approximating the stiffness profiles of specific tissues. Although tissues are associated with a range of Young's modulus values for bulk rigidity, at the subcellular level, tissues are comprised of heterogeneous distributions of rigidity. Lithographic processes have been widely explored in cell biology for the generation of analytical substrates to probe cellular physicomechanical responses. In this work, it is shown for the first time that that direct-write e-beam exposure can significantly alter the rigidity of elastomeric poly(dimethylsiloxane) substrates and a new class of 2D elastomeric substrates with controlled patterned rigidity ranging from the micrometer to the nanoscale is described. The mechanoresponse of human mesenchymal stem cells to e-beam patterned substrates was subsequently probed in vitro and significant modulation of focal adhesion formation and osteochondral lineage commitment was observed as a function of both feature diameter and rigidity, establishing the groundwork for a new generation of biomimetic material interfaces.
View details for PubMedID 28861921
Variability in Endogenous Perfusion Recovery of Immunocompromised Mouse Models of Limb Ischemia
TISSUE ENGINEERING PART C-METHODS
2016; 22 (4): 370-381
Self-assembly in tissue engineering refers to the spontaneous chemical or biological association of components to form a distinct functional construct, reminiscent of native tissue. Such self-assembled systems have been widely used to develop platforms for the delivery of therapeutic and/or bioactive molecules and various cell populations. Tissue morphology and functional characteristics have been recapitulated in several self-assembled constructs, designed to incorporate stimuli responsiveness and controlled architecture through spatial confinement or field manipulation. In parallel, owing to substantial functional properties, scaffold-free cell-assembled devices have aided in the development of functional neotissues for various clinical targets. Herein, we discuss recent advancements and future aspirations in scaffold and scaffold-free self-assembled devices for regenerative medicine purposes. Biotechnol. Bioeng. 2016;113: 1155-1163. © 2015 Wiley Periodicals, Inc.
View details for DOI 10.1002/bit.25869
View details for Web of Science ID 000375119900001
View details for PubMedID 26498484
Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture
Immunocompromised hind limb ischemia (HLI) murine models are essential for preclinical evaluation of human cell-based therapy or biomaterial-based interventions. These models are used to generate proof of principle that the approach is effective and also regulatory preclinical data required for translation to the clinic. However, surgical variations in creation of HLI models reported in the literature introduce variability in the pathological manifestation of the model, in consequence affecting therapeutic endpoints. This study aims to compare the extent of vascular regeneration in HLI-induced immunocompromised murine models to obtain a stable and more reproducible injury model for testing. Athymic and Balb/C nude mice underwent HLI surgery with single and double ligation of femoral artery (FA). The recovery from surgery was observed over a period of 2 weeks with respect to ischemia reperfusion using laser Doppler and clinical signs of necrosis and ambulatory impairment. Double ligation of the FA results in a more severe response to ischemia in Balb/C with endogenous perfusion recovery up to 50% ± 10% compared with 75% ± 20% in athymic nude mice. Single iliac artery (IA) and FA lead to creation of mild ischemia compared with femoral artery-vein (FAV) pair ligation in Balb/C. Microcirculatory parameters indicate significantly lower capillary numbers (26 ± 3/mm(2)) and functional capillary density (203 ± 5 cm/cm(2)) in the FAV group. In this study, we demonstrate a reproducible, arterial double ligation in an immunocompromised Balb/C nude mouse model that exhibits characteristic pathological signs of ischemia with impaired endogenous recovery.
View details for DOI 10.1089/ten.tec.2015.0441
View details for Web of Science ID 000373237600008
View details for PubMedID 26830861
An injectable elastin-based gene delivery platform for dose-dependent modulation of angiogenesis and inflammation for critical limb ischemia
2015; 65: 126-139
Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.
View details for DOI 10.1038/srep20922
View details for Web of Science ID 000369828800001
View details for PubMedID 26860065
View details for PubMedCentralID PMC4748261
Three-Dimensional Microgel Platform for the Production of Cell Factories Tailored for the Nucleus Pulposus
2015; 26 (7): 1297-1306
Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ-forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 μg) and IL-10 (10 μg)/eNOS (20 μg) and reduced inflammation with IL-10 (10 μg) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELP injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia.
View details for DOI 10.1016/j.biomaterials.2015.06.037
View details for Web of Science ID 000358806600014
View details for PubMedID 26151745
Microgel Microenvironment Primes Adipose-Derived Stem Cells Towards an NP Cells-Like Phenotype
ADVANCED HEALTHCARE MATERIALS
2014; 3 (12): 2012-2022
Intradiscal injection of growth factors or cells has been shown to attenuate symptoms of intervertebral disc degeneration. However, different approaches are needed to overcome limitations such as short-term efficacy and leakage of the injected solutions. The current study aims at creating a platform for the realization of functional cell factories by using in parallel cell delivery and gene therapy approaches. Superfect, a transfecting agent, was used as nonviral gene vector because of its ability to form complexes with plasmid DNA (polyplexes). Polyplexes were loaded into collagen hollow microsphere reservoirs, and their ability to transfect cells was ascertained in vitro. Adipose-derived stem cells were then embedded in three-dimensional (3D) microgels composed of type II collagen/hyaluronan, which mimics the environmental cues typical of the healthy nucleus pulposus. These were functionalized with polyplex-loaded collagen hollow spheres and the secretion of the target protein was assessed quantitatively. Delivery of polyplexes from a reservoir system lowered their toxicity significantly while maintaining high levels of transfection in a monolayer culture. In 3D microgels, lower levels of transfection were observed, however; increasing levels of luciferase were secreted from the microgels over 7 days of culture. These results indicate that 3D microgels, functionalized with polyplex-loaded reservoirs offer a reliable platform for the production of cell factories that are able to manufacture targeted therapeutic proteins for regenerative therapies that have applications in nucleus pulposus repair.
View details for DOI 10.1021/bc5004247
View details for Web of Science ID 000358186500014
View details for PubMedID 25290910
A shape-controlled tuneable microgel platform to modulate angiogenic paracrine responses in stem cells
2014; 35 (31): 8757-8766
Cell therapy of the degenerated intervertebral disc is limited by the lack of appropriate cell sources, thus new strategies for the differentiation of stem cells towards a nucleus pulposus (NP)-like phenotype need investigation. In the current study, it is hypothesized that spherical niche-like structures composed of type II collagen and hyaluronan (HA) mimic the NP microenvironment and promote the differentiation of adipose-derived stem cells (ADSCs) towards an NP-like phenotype. ADSCs are embedded in microgels of different concentrations of collagen II/HA. Cells' response to the different environments is studied by characterizing differences in cells' viability, morphology, and gene expression. After 21 days of culture, ADSCs maintain ± 80% viability in all the conditions tested. Moreover, microgels with higher concentration of collagen are stable and maintain cells in a rounder shape. In presence of differentiation media, cells are able to differentiate in all the conditions tested, but in a more pronounced manner in the microgel with a higher concentration of collagen. By tuning microgels' properties, it is possible to influence ADSCs' phenotype and ability to differentiate. Indeed, when cultured in high concentrations of collagen, ADSCs expresses high levels of collagen II, aggrecan, SOX9, and low levels of collagen I.
View details for DOI 10.1002/adhm.201400175
View details for Web of Science ID 000346171100007
View details for PubMedID 25100329
Development of cell delivery platforms have been driven based on an empirical cytoprotective design. While cell-matrix and cell-cell interactions that influence biochemical effects beyond survival has been limited and overshadowed in an effort to incrementally improve biomimicking properties of the tissue-engineered constructs. Here we demonstrate fabrication of a shape controlled 3D type-I collagen-based microgel platform that can be tuned to modulate angiogenic paracrine- 'angiocrine' responses of human mesenchymal stem cells (hMSCs). Furthermore, these microgels were characterized as a 3D cell culture tool to assess optimal biological response as a function of cell-matrix and cell-cell interactions. Finally, optimised hMSC embedded microgels were shown to induce vascular repair and functional improvement in vivo in a mouse model of hind-limb ischemia. The approach described here in designing a tuneable cell delivery platform using naturally occurring extracellular matrix molecules highlights the need for highly customised matrices with an array of self-assembling proteins that dictate specific cell function resembling the native tissue of interest for repair.
View details for DOI 10.1016/j.biomaterials.2014.06.053
View details for Web of Science ID 000340984600001
View details for PubMedID 25047627