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In JoVE (1)
Other Publications (6)
Articles by Bonnie Barrilleaux in JoVE
JoVE General
Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.
Other articles by Bonnie Barrilleaux on PubMed
Review: Ex Vivo Engineering of Living Tissues with Adult Stem Cells
Adult stem cells have the potential to revolutionize regenerative medicine with their unique abilities to self-renew and differentiate into various phenotypes. This review examines progress and challenges in ex vivo tissue engineering with adult stem cells. These rare cells are harvested from a variety of tissues, including bone marrow, adipose, skeletal muscle, and placenta, and differentiate into cells of their own lineage and in some cases atypical lineages. Insight into the stem cell niche leads to the identification of matrix components, soluble factors, and physiological conditions that enhance the ex vivo amplification and differentiation of stem cells. Scaffolds composed of metals, naturally occurring materials, and synthetic polymers organize stem cells into complex spatial groupings that mimic native tissue. Cell signals from covalently bound ligands and slowly released regulatory factors in scaffolds direct stem cell fate. Future advances in stem cell biology and scaffold design will ultimately improve the efficacy of tissue substitutes as implants, in research, and as extracorporeal devices.
Small-molecule Antagonist of Macrophage Migration Inhibitory Factor Enhances Migratory Response of Mesenchymal Stem Cells to Bronchial Epithelial Cells
Human mesenchymal stem cells (MSCs) from bone marrow stroma can home to and repair injured tissue, but the rate of engraftment is generally low. Regulating migration-related signaling of MSCs may be a powerful strategy to enhance this process. To gain insight into the molecular mechanisms governing homing, we identified negative factors affecting MSC migration using an in vitro model of injured lung. Heat-labile factors in bovine pituitary extract, a component of serum-free epithelial medium, inhibited more than 97% of MSC migration. This was partly due to a dose-dependent response to macrophage migration inhibitory factor (MIF). Eighty-five ng/mL recombinant MIF, the concentration found in the epithelial medium, inhibited about 50% of MSC migration. Media conditioning by uninjured or bleomycin-injured bronchial epithelial cells partially attenuated this suppressive effect. Additionally, the anti-inflammatory agent ISO-1, a small-molecule MIF antagonist, further increased MSC migration by nearly fourfold in conditioned epithelial media. This is the first report of the effect of MIF and ISO-1 on MSC migration, and the data suggest that MIF and its antagonists may have therapeutic applications in controlling MSC homing during repair of injured lung and in other clinically relevant systems.
Migratory Response of Mesenchymal Stem Cells to Macrophage Migration Inhibitory Factor and Its Antagonist As a Function of Colony-forming Efficiency
Human mesenchymal stem cells (MSCs) are capable of repairing pulmonary disorders, but their efficacy is limited by poor engraftment. A strategy is proposed to augment MSC migration to lung tissue by antagonizing macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine. Recombinant MIF (85 ng/ml) inhibited in vitro chemokinesis of multipotent MSCs by nearly 50 and 20% for donor preparations with colony-forming efficiencies of 22 +/- 4% and 66 +/- 3%, respectively (P < 0.05). The small-molecule MIF antagonist, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1, 85 microg/ml), restored MSC migration for all donors to levels found in the absence of MIF. At this concentration, ISO-1 increased migration to conditioned medium from bronchial epithelial cell cultures by >or=3-fold for all donor MSC preparations (P < 0.05). Transcript levels for the MIF receptor, CD74, in MSCs were independent of colony-forming efficiency. These data suggest that MIF and its antagonists may be relevant to the control of MSC homing and efficacy of stem cell therapies in a variety of clinical scenarios.
In Vitro High-capacity Assay to Quantify the Clonal Heterogeneity in Trilineage Potential of Mesenchymal Stem Cells Reveals a Complex Hierarchy of Lineage Commitment
In regenerative medicine, bone marrow is a promising source of mesenchymal stem cells (MSCs) for a broad range of cellular therapies. This research addresses a basic prerequisite to realize the therapeutic potential of MSCs by developing a novel high-capacity assay to quantify the clonal heterogeneity in potency that is inherent to MSC preparations. The assay utilizes a 96-well format to (1) classify MSCs according to colony-forming efficiency as a measure of proliferation capacity and trilineage potential to exhibit adipo-, chondro-, and osteogenesis as a measure of multipotency and (2) preserve a frozen template of MSC clones of known potency for future use. The heterogeneity in trilineage potential of normal bone marrow MSCs is more complex than previously reported: all eight possible categories of trilineage potential were detected. In this study, the average colony-forming efficiency of MSC preparations was 55-62%, and tripotent MSCs accounted for nearly 50% of the colony-forming cells. The multiple phenotypes detected in this study infer a more convoluted hierarchy of lineage commitment than described in the literature. Greater cell amplification, colony-forming efficiency, and colony diameter for tri- versus unipotent clones suggest that MSC proliferation may be a function of potency. CD146 may be a marker of multipotency, with approximately 2-fold difference in mean fluorescence intensity between tri- and unipotent clones. The significance of these findings is discussed in the context of the efficacy of MSC therapies. The in vitro assay described herein will likely have numerous applications given the importance of heterogeneity to the therapeutic potential of MSCs.
Activation of CD74 Inhibits Migration of Human Mesenchymal Stem Cells
Therapeutic administration of mesenchymal stem cells (MSCs) by systemic delivery utilizes the innate ability of the cells to home to damaged tissues, but it can be an inefficient process due to a limited knowledge of cellular cues that regulate migration and homing. Our lab recently discovered that a potent pro-inflammatory cytokine, macrophage migration inhibitory factor (MIF), inhibits MSC migration. Because MIF may act on multiple cellular targets, an activating antibody (CD74Ab) was employed in this study to examine the effect of one MIF receptor, CD74 (major histocompatibility complex class II-associated invariant chain), on MSC motility. CD74 activation inhibits in a dose-dependent manner up to 90% of in vitro migration of MSCs at 40 mug/ml CD74Ab (p < 0.001), with consistent effects observed among three MSC donor preparations. A blocking peptide from the C-terminus of CD74 eliminates the effect of CD74Ab on MSCs. This suggests that MIF may act on MSCs, at least in part, through CD74. Late-passage MSCs exhibit less chemokinesis than those at passage 2. However, MSCs remain responsive to CD74 activation during ex vivo expansion: MSC migration is inhibited approximately 2-fold in the presence of 5 microg/ml CD74Ab at passage 9 vs. approximately 3-fold at passage 2 (p < 0.001). Consistent with this result, there were no significant differences in CD74 expression at all tested passages or after CD74Ab exposure. Targeting CD74 to regulate migration and homing potentially may be a useful strategy to improve the efficacy of a variety of MSC therapies, including those that require ex vivo expansion.
Inducing IPSCs to Escape the Dish
Induced pluripotent stem cells (iPSCs) hold great promise for autologous cell therapies, but significant roadblocks remain to translating iPSCs to the bedside. For example, concerns about the presumed autologous transplantation potential of iPSCs have been raised by a recent paper demonstrating that iPSC-derived teratomas were rejected by syngeneic hosts. Additionally, the reprogramming process can alter genomic and epigenomic states, so a key goal at this point is to determine the clinical relevance of these changes and minimize those that prove to be deleterious. Finally, thus far few studies have examined the efficacy and tumorigenicity of iPSCs in clinically relevant transplantation scenarios, an essential requirement for the FDA. We discuss potential solutions to these hurdles to provide a roadmap for iPSCs to "jump the dish" and become useful therapies.
