Browsing by Subject "Cell therapy"
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Item Open Access Biologically Improved Electrotransfection for Gene Delivery and Genome Editing(2019) Mao, MaoSuccessful transfection of genetically active materials is essential to gene delivery and genome editing. Electrotransfection, also known as electroporation, is a fast, safe, and convenient non-viral method for introducing materials such as proteins and nucleic acids into cells and tissues. It has been widely used in academic research, industrial manufacturing, and clinical therapeutics. Particularly, electrotransfection is one of the most commonly used method in gene delivery into mammalian cells. However, despite its many advantages comparing to other gene delivery methods, the application of electrotransfection is limited by inconsistent transfection efficiency, which is caused by the poor understanding of the mechanism of electrotransfection.
The goal of my research is to understand the fundamental biological mechanisms of electrotransfection and to develop novel strategies that can improve the transfection efficiency of gene delivery and genome editing. To this end, this study is divided into two phases. Phase 1 aims at understanding the key cellular components involved in the transport process. Phase 2 focuses on the development of strategies to enhance electrotransfection by controlling the biological pathways that are involved in electrotransfection.
In the first phase of my study, we investigated the dependence of electrotransfection efficiency on endocytosis. Data from this study demonstrated that macropinocytosis is involved in electrotransfection. The results revealed that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles formed from macropinocytosis. Furthermore, electrotransfection efficiency was reduced significantly by lowering temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis.
Second phase of this study focuses on the intracellular transport of plasmid DNA, especially the transport of DNA molecules towards degradative compartments. Our data elucidated that components in both endocytic and autophagic pathways are responsible for intracellular trafficking and processing of transfected materials such as pDNA. In addition, we also characterized a new type of vesicle named amphisome-like vesicle (ALB) and revealed its involvement in electrotransfection. Based on these findings, we propose a novel strategy to enhance electrotransfection by blocking degradative routes within the endocytic pathways, which led to the development of a new technique called transfection by redirection of endocytic and autophagic traffic (TREAT). Transfection of plasmid DNA (pDNA), messenger RNA (mRNA), sleeping beauty transposon system (SB), and different forms of CRISPR/Cas9 system by TREAT achieved superior efficiency in various cell lines including difficult-to-transfect human primary cells. In addition, we successfully applied TREAT method to improve clinically relevant applications including SB-based gene integration and CRISPR/Cas9-based editing of T cell receptor alpha constant (TRAC). In summary, we studied the biological mechanism of electrotransfection and developed a general, flexible, and reliable technique to enable highly efficient non-viral gene delivery and genome editing. Furthermore, the insights gained on the mechanism of electrotransfection provide better understanding of cellular response to exogenous materials. In the future, our study could potentially pave new paths for a wide range of research and therapeutic applications such as CRISPR/Cas9 mediated high-throughput loss-of-function gene screening analysis, correction of disease-related mutations, as well as genetic engineering of immune cells and stem cells for transplantation.
Item Open Access Cellular and Biomaterial Engineering for Orthopaedic Regenerative Medicine(2015) Brunger, Jonathan M.The ends of long bones that articulate with respect to one another are lined with a crucial connective tissue called articular cartilage. This tissue plays an essential biomechanical function in synovial joints, as it serves to both dissipate load and lubricate articulating surfaces. Osteoarthritis is a painful and debilitating disease that drives the deterioration of articular cartilage. Like many chronic diseases, pro-inflammatory cytokines feature prominently in the onset and progression of osteoarthritis. Because cartilage lacks physiologic features critical for regeneration and self-repair, the development of effective strategies to create functional cartilage tissue substitutes remains a priority for the fields of tissue engineering and regenerative medicine. The overall objectives of this dissertation are to (1) develop a bioactive scaffold capable of mediating cell differentiation and formation of extracellular matrix that recapitulates native cartilage tissue and (2) to produce stem cells specifically tailored at the scale of the genome with the ability to resist inflammatory cues that normally lead to degeneration and pain.
Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. By immobilizing lentivirus driving the expression of transforming growth factor-β3 to a highly structured, three dimensionally woven tissue engineering scaffold, we developed a technique for producing cell-instructive scaffolds that control human mesenchymal stem cell differentiation and possess biomechanical properties approximating those of native tissues. This work represents an important advance, as it establishes a method for generating constructs capable of restoring biological and mechanical function that may circumvent the need for ex vivo conditioning of engineered tissue substitutes.
Any functional cartilage tissue substitute must tolerate the inflammation intrinsic to an arthritic joint. Recently emerging tools from synthetic biology and genome engineering facilitate an unprecedented ability to modify how cells respond to their microenvironments. We exploited these developments to engineer cells that can evade signaling of the pro-inflammatory cytokine interleukin-1 (IL-1). Our study provides proof-of-principle evidence that cartilage derived from such engineered stem cells are resistant to IL-1-mediated degradation.
Extending on this work, we developed a synthetic biology strategy to further customize stem cells to combat inflammatory cues. We commandeered the highly responsive endogenous locus of the chemokine (C-C motif) ligand 2 gene in pluripotent stem cells to impart self-regulated, feedback-controlled production of biologic therapy. We demonstrated that repurposing of degradative signaling pathways induced by IL-1 and tumor necrosis factor toward transient production of cytokine antagonists enabled engineered cartilage tissue to withstand the action of inflammatory cytokines and to serve as a cell-based, auto-regulated drug delivery system.
In this work, we combine principles from synthetic biology, gene therapy, and functional tissue engineering to develop methods for generating constructs with biomimetic molecular and mechanical features of articular cartilage while precisely defining how cells respond to dysfunction in the body’s finely-tuned inflammatory systems. Moreover, our strategy for customizing intrinsic cellular signaling pathways in therapeutic stem cell populations opens innovative possibilities for controlled drug delivery to native tissues, which may provide safer and more effective treatments applicable to a wide variety of chronic diseases and may transform the landscape of regenerative medicine.
Item Open Access Electrical Coupling Between Cardiomyocytes and Unexcitable Cells: The Effect of Cardiac Fibroblasts and Genetically Engineered HEK-293 Cells on Cardiac Action Potential Shape and Propagation(2011) McSpadden, Luke ChristopherExcess cardiac myofibroblasts in fibrotic heart diseases as well as cell-based therapies involving implantation of stem cells or genetically engineered somatic cells in the heart may all lead to a situation where a cardiomyocyte becomes electrically coupled to an unexcitable cell. In these settings, electrotonic loading of cardiomyocytes by unexcitable cells can affect cardiac action potential generation, propagation, and repolarization depending on the properties of both cardiomyocytes and unexcitable cells. The objective of this dissertation was to advance our understanding of the electrical interactions between cardiomyocytes and unexcitable cells using a variety of electrophysiological, molecular, and cell culture techniques.
First, we utilized aligned cardiomyocyte monolayers covered with unexcitable cardiac fibroblasts or human embryonic kidney-293 (HEK) cells that expressed similar levels of the gap junction protein connexin-45. These cells weakly coupled to cardiomyocytes and marginally slowed cardiac conduction only at high coverage density, while producing no other measurable electrophysiological changes in cardiomyocytes. In contrast, unexcitable HEK cells genetically engineered to stably express the more conductive connexin-43 channels (Cx43 HEK) strongly coupled to cardiomyocytes, depolarized cardiac resting membrane potential, significantly slowed impulse propagation, decreased maximum capture rate, and increased action potential duration (APD) at high coverage density. None of the studied unexcitable cells significantly altered conduction velocity anisotropy ratio or the relatively low incidence of pacemaking activity of cardiac monolayers at any coverage density.
Next, we utilized individual micropatterned cell pairs consisting of a cardiomyocyte and an unexcitable Cx43 HEK cell with or without stably overexpressed inward rectifier potassium channels (Kir2.1+Cx43 HEK). By systematically varying the relative sizes of micropatterned cells, we showed that Cx43 HEK cells significantly depolarized cardiomyocytes, reduced maximum upstroke velocity and action potential amplitude, prolonged APD, and modulated beating rate as a function of HEK:CM area ratio. In contrast, in cell pairs formed between cardiomyocytes and Kir2.1+Cx43 HEK cells we observed significant reduction in cardiomyocyte action potential amplitude, duration, and maximum upstroke velocity, but no change in other measured parameters.
Finally, we utilized a hybrid dynamic clamp setting consisting of a live micropatterned cardiomyocyte coupled in real time to a virtual model of capacitive and/or ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells. We found that coupling of cardiomyocytes to the ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells was sufficient to reproduce the dependence of cardiomyocyte maximal diastolic potential and pacemaking behavior on HEK:CM area ratio observed in micropatterned cell pairs, but did not replicate the observed changes in action potential upstroke or duration. The pure capacitance model with no ionic current, on the other hand, significantly decreased cardiomyocyte maximum upstroke velocity and prolonged cardiomyocyte APD as function of HEK:CM area ratio without affecting maximal diastolic potential or pacemaking behavior. When the unexcitable cell model containing both capacitive and ionic currents was connected to cardiomyocytes, all changes in action potential shape observed in micropatterned cell pairs were accurately reproduced.
These studies describe how coupling of unexcitable cells to cardiomyocytes can alter cardiomyocyte electrophysiological properties dependent on the unexcitable cell connexin isoform expression, ion channel expression, and cell size. This knowledge is expected to aid in the design of safe and efficient cell and gene therapies for myocardial infarction, fibrotic heart disease, and cardiac arrhythmias.
Item Open Access Functional Maturation of Engineered Myocardium for Studies of Development and Regeneration(2017) Jackman, ChristopherIschemic heart disease is the leading cause of death worldwide, in part due to the heart’s limited capacity to regenerate. Transplantation of exogenous cells into the heart is a promising approach to restore cardiac function in ischemic disease. Pre-engineering of cells into a functional cardiac tissue patch prior to implantation is expected to maximize therapeutic benefits, however, the electrical and mechanical properties of engineered cardiac tissues are currently far inferior to those of native myocardium. Furthermore, the levels of functionality of engineered tissues following implantation on the heart have not been studied. To further the state-of-the-art in the field, the primary goals of this dissertation have been to engineer cardiac tissue with functional properties comparable to those of adult myocardium and to quantify electrical function of such engineered tissues following epicardial implantation.
To achieve these goals, we first developed dynamic, free-floating culture conditions for engineering "cardiobundles", 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes embedded in fibrin-based hydrogel. Compared to static conditions, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (∼2.1-fold), contractile force (∼3.5-fold), and conduction velocity of action potentials (∼1.4-fold). The average contractile force per cross-sectional area (59.7 mN/mm2) and conduction velocity (CV=52.5 cm/s) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions. This functional rescue, which could be blocked by rapamycin, was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy.
We then combined 0.2 Hz electrical stimulation with application of thyroid hormone (5 nM triiodothyronine) to further mature dynamically cultured cardiobundles during 5-week culture. These conditions further increased myocardial volume and contractile force by ~40%, shortened action potential and twitch durations and increased maximum capture rate. Additional evidence of maturation included polarization of N-cadherin junctions, a switch to troponin isoforms expressed in the adult heart, and development of sarcolemmal T-tubular structures. Since cardiomyocytes in this system exited cell cycle by two weeks of culture (<1% of cycling cells per day), we utilized cardiobundles to screen factors that reactivate cardiomyocyte proliferation following injury by hydrogen peroxide (H2O2). Specifically, we expressed a pro-proliferative transcription factor, constitutively active Yes-associated protein 1 (caYAP), under the control of an enhancer element selectively activated during injury in zebrafish hearts. Application of H2O2 resulted in a transient activation of the injury-responsive enhancer in a subset of cardiomyocytes 1-2 days post-injury, but the resulting caYAP expression was insufficient to induce a significant mitogenic effects. Nonetheless, in vitro matured cardiobundles hold promise for use as a relatively high-throughput system for discovery of novel pro-regenerative factors in various cardiac injury settings.
Finally, we analyzed electrical function and integration of engineered cardiac tissues following epicardial implantation. Cardiac patches were generated from neonatal rat cardiomyocytes expressing a genetically-encoded calcium indicator (GCaMP6) and implanted in adult rats with normal heart function for up to 6 weeks. After 2 weeks of in vitro culture, engineered cardiac patches contained robustly coupled cardiomyocytes, generated maximum active forces of 18.0 ± 1.4 mN, and propagated action potentials with a conduction velocity of 32.3 ± 1.8 cm/s. From dual optical mapping of GCaMP6-labelled patch and RH237-stained heart, 85% patches survived implantation and conducted action potential with velocities not different from those pre-implantation. Asynchronous activation of the patch and the heart indicated a lack of graft-host electrical coupling consistent with the formation of non-cardiomyocyte scar tissue between the patch and heart. In a subcutaneous implantation model, scar tissue formation between the patch and native muscle could not be reduced by enhancement of patch-muscle contact area with a surgical mesh or co-implantation of bone marrow-derived macrophages within the patch.
In summary, using neonatal rat cardiomyocytes, we developed a novel methodology for engineering cylindrical cardiac tissues (cardiobundles) with a near-adult functional output. mTOR signaling was identified as an important mechanism for advancing cardiobundle maturation and function in vitro, along with the application of electrical stimulation and thyroid hormone supplementation. Cardiobundle injury model was established to allow screening of pro-regenerative factors and approaches in vitro. Epicardial implantation of engineered cardiac tissue patches served to develop an enhanced analysis method for graft-host integration in animal models of cell-based cardiac repair. Collectively, these methods and results are expected to aid advances in the field of cell-based cardiac therapy towards eventual clinical applications.
Item Open Access Genome Engineering in Stem Cells for Skeletal Muscle Regeneration(2020) Kwon, JenniferSkeletal muscle has the innate ability to robustly regenerate in a highly orchestrated fashion that is initiated by satellite cells, the resident stem cell population. These cells are defined by their uniform expression of the transcription factor, PAX7, which plays a key role in myogenesis through specification and maintenance of satellite cells, as well as regulation of myogenic differentiation. In conditions of skeletal muscle wasting such as cachexia, sarcopenia, and muscular dystrophies, the deterioration of muscle overwhelms the regenerative capabilities of satellite cells, which are believed to undergo early senescence due to exhaustive proliferation. There is significant potential for harnessing satellite cells for gene and cell therapies for such diseases; however, satellite cell specification and regulation is still poorly understood.
The CRISPR/Cas9 system has been established as a multifaceted tool that can be used as a platform for a variety of applications, including sequence-specific genome and epigenome editing for cell differentiation and treatment of genetic diseases. The objective of my research proposal was to use CRISPR/Cas9-based genome engineering technologies toward applications for skeletal muscle regeneration. First, I used a CRISPR/Cas9-based transcriptional activator to direct differentiation of human pluripotent stem cells into functional skeletal muscle progenitor cells. Next, I conducted a high-throughput CRISPR activation screen to identify novel upstream regulators of myogenic progenitor cell differentiation. Lastly, I demonstrated that satellite cells can be targeted in vivo with AAV and subsequently gene-edited to correct the dystrophin reading frame in a mouse model for Duchenne muscular dystrophy. Together, this work provides novel contributions to the field of satellite cell biology and highlights the utility of CRISPR/Cas9 genome engineering in stem cells for skeletal muscle regeneration.
Item Open Access Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations.(Stem cell research & therapy, 2021-11-04) Medrano-Trochez, Camila; Chatterjee, Paramita; Pradhan, Pallab; Stevens, Hazel Y; Ogle, Molly E; Botchwey, Edward A; Kurtzberg, Joanne; Yeago, Carolyn; Gibson, Greg; Roy, KrishnenduBackground
Human Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs.Methods
13 hMSC samples derived from 10 "healthy" donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis.Results
scRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion.Conclusions
This study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.