Barbara J. Muller-Borer

Quantum dot labeling of mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, fat and muscle cells and are being investigated for their utility in cell-based transplantation therapy. Yet, adequate methods to track transplanted MSCs in vivo are limited, precluding functional studies. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. These nanoparticles are resistant to chemical and metabolic degradation, demonstrating long term photostability. Here, we investigate the cytotoxic effects of in vitro QD labeling on MSC proliferation and differentiation and use as a cell label in a cardiomyocyte co-culture.ResultsA dose-response to QDs in rat bone marrow MSCs was assessed in Control (no-QDs), Low concentration (LC, 5 nmol/L) and High concentration (HC, 20 nmol/L) groups. QD yield and retention, MSC survival, proinflammatory cytokines, proliferation and DNA damage were evaluated in MSCs, 24 -120 hrs post QD labeling. In addition, functional integration of QD labeled MSCs in an in vitro cardiomyocyte co-culture was assessed. A dose-dependent effect was measured with increased yield in HC vs. LC labeled MSCs (93 ± 3% vs. 50% ± 15%, p < 0.05), with a larger number of QD aggregates per cell in HC vs. LC MSCs at each time point (p < 0.05). At 24 hrs >90% of QD labeled cells were viable in all groups, however, at 120 hrs increased apoptosis was measured in HC vs. Control MSCs (7.2% ± 2.7% vs. 0.5% ± 0.4%, p < 0.05). MCP-1 and IL-6 levels doubled in HC MSCs when measured 24 hrs after QD labeling. No change in MSC proliferation or DNA damage was observed in QD labeled MSCs at 24, 72 and 120 hrs post labeling. Finally, in a cardiomyocyte co-culture QD labeled MSCs were easy to locate and formed functional cell-to-cell couplings, assessed by dye diffusion.ConclusionFluorescent QDs label MSC effectively in an in vitro co-culture model. QDs are easy to use, show a high yield and survival rate with minimal cytotoxic effects. Dose-dependent effects suggest limiting MSC QD exposure.

Influence of Dynamic Gap Junction Resistance on Impulse Propagation in Ventricular Myocardium: A Computer Simulation Study

The gap junction connecting cardiac myocytes is voltage and time dependent. This simulation study investigated the effects of dynamic gap junctions on both the shape and conduction velocity of a propagating action potential. The dynamic gap junction model is based on that described by Vogel and Weingart (J. Physiol. (Lond.). 1998, 510:177-189) for the voltage- and time-dependent conductance changes measured in cell pairs. The model assumes that the conductive gap junction channels have four conformational states. The gap junction model was used to couple 300 cells in a linear strand with membrane dynamics of the cells defined by the Luo-Rudy I model. The results show that, when the cells are tightly coupled (6700 channels), little change occurs in the gap junction resistance during propagation. Thus, for tight coupling, there are negligible differences in the waveshape and propagation velocity when comparing the dynamic and static gap junction representations. For poor coupling (85 channels), the gap junction resistance increases 33 MOmega during propagation. This transient change in resistance resulted in increased transjunctional conduction delays, changes in action potential upstroke, and block of conduction at a lower junction resting resistance relative to a static gap junction model. The results suggest that the dynamics of the gap junction enhance cellular decoupling as a possible protective mechanism of isolating injured cells from their neighbors.

Adult-derived liver stem cells acquire a cardiomyocyte structural and functional phenotype ex vivo

We examined the differentiation potential of an adult liver stem cell line (WB F344) in a cardiac microenvironment, ex vivo. WB F344 cells were established from a single cloned nonparenchymal epithelial cell isolated from a normal male adult rat liver. Genetically modified, WB F344 cells that express beta-galactosidase and green fluorescent protein or only beta-galactosidase were co-cultured with dissociated rat or mouse neonatal cardiac cells. After 4 to 14 days, WB F344-derived cardiomyocytes expressed cardiac-specific proteins and exhibited myofibrils, sarcomeres, and a nascent sarcoplasmic reticulum. Further, rhythmically beating WB F344-derived cardiomyocytes displayed calcium transients. Fluorescent recovery after photobleaching demonstrated that WB F344-derived cardiomyocytes were coupled with adjacent neonatal cardiomyocytes and other WB F344-derived cardiomyocytes. Fluorescence in situ hybridization experiments suggested that fusion between WB F344 cells and neonatal mouse cardiomyocytes did not take place. Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment ex vivo acquiring a cardiomyocyte phenotype and function. The identification ex vivo of microenvironmental signals that appear to cross germ layer and species specificities should prove valuable in understanding the molecular basis of adult stem cell differentiation and phenotypic plasticity.

Electrical coupling and impulse propagation in anatomically modeled ventricular tissue

Computer simulations were used to study the role of resistive couplings on flat-wave action potential propagation through a thin sheet of ventricular tissue. Unlike simulations using continuous or periodic structures, this unique electrical model includes random size cells with random spaced longitudinal and lateral connections to simulate the physiologic structure of the tissue. The resolution of the electrical model is ten microns, thus providing a simulated view at the subcellular level. Flat-wave longitudinal propagation was evaluated with an electrical circuit of over 140,000 circuit elements, modeling a 0.25 mm by 5.0 mm sheet of tissue. An electrical circuit of over 84,000 circuit elements, modeling a 0.5 mm by 1.5 mm sheet was used to study flat-wave transverse propagation. Under normal cellular coupling conditions, at the macrostructure level, electrical conduction through the simulated sheets appeared continuous and directional differences in conduction velocity, action potential amplitude and V/spl dot//sub max/ were observed. However, at the subcellular level (10 /spl mu/m) unequal action potential delays were measured at the longitudinal and lateral gap junctions and irregular wave-shapes were observed in the propagating signal. Furthermore, when the modeled tissue was homogeneously uncoupled at the gap junctions conduction velocities decreased as the action potential delay between modeled cells increased. The variability in the measured action potential was most significant in areas with fewer lateral gap junctions, i.e., lateral gap junctions between fibers were separated by a distance of 100 /spl mu/m or more.<<ETX>>

Mechanisms controlling the acquisition of a cardiac phenotype by liver stem cells

The mechanisms underlying stem cell acquisition of a cardiac phenotype are unresolved. We studied early events during the acquisition of a cardiac phenotype by a cloned adult liver stem cell line (WB F344) in a cardiac microenvironment. WB F344 cells express a priori the transcription factors GATA4 and SRF, connexin 43 in the cell membrane, and myoinositol 1,4,5-triphosphate receptor in the perinuclear region. Functional cell–cell communication developed between WB F344 cells and adjacent cocultured cardiomyocytes in 24 h. De novo cytoplasmic [Ca2+]c and nuclear [Ca2+]nu oscillations appeared in WB F344 cells, synchronous with [Ca2+]i transients in adjacent cardiomyocytes. The [Ca2+] oscillations in the WB F344 cells, but not those in the cardiomyocytes, were eliminated by a gap junction uncoupler and reappeared with its removal. By 24 h, WB F344 cells began expressing the cardiac transcription factors Nkx2.5, Tbx5, and cofactor myocardin; cardiac proteins 24 h later; and a sarcomeric pattern 4–6 days later. Myoinositol 1,4,5-triphosphate receptor inhibition suppressed WB F344 cell [Ca2+]nu oscillations but not [Ca2+]c oscillations, and L-type calcium channel inhibition eliminated [Ca2+] oscillations in cardiomyocytes and WB F344 cells. The use of these inhibitors was associated with a decrease in Nkx2.5, Tbx5, and myocardin expression in the WB F344 cells. Our findings suggest that signals from cardiomyocytes diffuse through shared channels, inducing [Ca2+] oscillations in the WB F344 cells. We hypothesize that the WB F344 cell [Ca2+]nu oscillations activate the expression of a cardiac specifying gene program, ushering in a cardiac phenotype.

Calcium signals induce liver stem cells to acquire a cardiac phenotype

Heart failure is a major cause of premature death and disability in the United States. Stem cell therapy has attracted great interest for the treatment of myocardial infarction and heart failure. Some tissue-specific adult-derived stem cells demonstrate plasticity in that they are multipotent, react to inductive signals provided by a new micro-environment, and acquire the phenotype of cells endogenous to the new micro-environment. The mechanism through which this phenotype is acquired is unknown. We have demonstrated that a liver-derived clonal stem cell line, WB F344, differentiate into cardiomyocytes in vivo and in vitro. Using a co-culture model of neonatal heart cells and WB F344 cells, we have found that cytosolic communication between the two cell types results in calcium-induced transcription of cardiac transcription factors and appears to usher in the cardiac phenotype. Functional gap junctions and IP3 receptors appear to be required for this process. We propose that the observed low frequency of stem cell differentiation into cardiomyocytes when transplanted into the injured heart is due, in part, to their inability to establish functioning intercellular communications with healthy cardiomyocytes and receive instructive signals needed to activate a cardiac gene program.

pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy

Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHi) or extracellular pH (pHo) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 micron were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pHo was measured during continuous perfusion of SNARF-1 free acid. pHi measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pHi and pHo were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing pHi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pHi and pHo changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pHi regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHi) or extracellular pH (pHo) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 μm were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pHo was measured during continuous perfusion of SNARF-1 free acid. pHi measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pHi and pHo were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing pHi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pHi and pHo changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pHi regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.

Control of Vascular Smooth Muscle Cell Growth by Connexin 43

Connexin 43 (Cx43), the principal gap junction protein in vascular smooth muscle cells (VSMCs), regulates movement of ions and other signaling molecules through gap junction intercellular communication (GJIC) and plays important roles in maintaining normal vessel function; however, many of the signaling mechanisms controlling Cx43 in VSMCs are not clearly described. The goal of this study was to investigate mechanisms of Cx43 regulation with respect to VSMC proliferation. Treatment of rat primary VSMCs with the cAMP analog 8Br-cAMP, the soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY), or the Cx inducer diallyl disulfide (DADS) significantly reduced proliferation after 72 h compared with vehicle controls. Bromodeoxyuridine uptake revealed reduction (p < 0.05) in DNA synthesis after 6 h and flow cytometry showed reduced (40%) S-phase cell numbers after 16 h in DADS-treated cells compared with vehicle controls. Cx43 expression significantly increased after 270 min treatment with 8Br-cAMP, 8Br-cGMP, BAY or DADS. Inhibition of PKA, PKG or PKC reversed 8Br-cAMP-stimulated increases in Cx43 expression, whereas only PKG or PKC inhibition reversed 8Br-cGMP- and BAY-stimulated increases in total Cx43. Interestingly, stimulation of Cx43 expression by DADS was not dependent on PKA, PKG or PKC. Using fluorescence recovery after photobleaching, only 8Br-cAMP or DADS increased GJIC with 8Br-cAMP mediated by PKC and DADS mediated by PKG. Further, DADS significantly increased phosphorylation at MAPK-sensitive Serine (Ser)255 and Ser279, the cell cycle regulatory kinase-sensitive Ser262 and PKC-sensitive Ser368 after 30 min while 8Br-cAMP significantly increased phosphorylation only at Ser279 compared with controls. This study demonstrates that 8Br-cAMP- and DADS-enhanced GJIC rather than Cx43 expression and/or phosphorylation plays important roles in the regulation of VSMC proliferation and provides new insights into the growth-regulatory capacities of Cx43 in VSM.

A superfusion system to study border zones in confluent cultures of neonatal rat heart cells

We present a new experimental method to study intracellular ion regulation in cultured cardiomyocytes at a border zone separating two different and distinct environments. Our system uses a dual-flow superfusion chamber to produce two different but adjacent environments over a monolayer of cardiomyocytes. Fluorescent microscopy of fluorescein showed that the transition between the two environments was nearly linear and was 220-320 micron wide depending on fluid viscosity and velocity. We superfused cultured monolayers on one side with a solution at pH 6.5 and on the other side with a solution at pH 7.4. We observed a sharply demarcated difference in intracellular pH (pHi) between the two halves of the cell monolayer as measured with the fluorescent pHi indicator carboxy-seminaphthorhodafluor-1. The demarcation of pHi corresponded well with the demarcation of the border measured with fluorescein. We conclude that our superfusion system will facilitate the study of intercellular communication and interactions across boundaries of cardiac tissue where different ionic or metabolic conditions are present, for example, between ischemic and nonischemic myocardium.We present a new experimental method to study intracellular ion regulation in cultured cardiomyocytes at a border zone separating two different and distinct environments. Our system uses a dual-flow superfusion chamber to produce two different but adjacent environments over a monolayer of cardiomyocytes. Fluorescent microscopy of fluorescein showed that the transition between the two environments was nearly linear and was 220-320 μm wide depending on fluid viscosity and velocity. We superfused cultured monolayers on one side with a solution at pH 6.5 and on the other side with a solution at pH 7.4. We observed a sharply demarcated difference in intracellular pH (pHi) between the two halves of the cell monolayer as measured with the fluorescent pHi indicator carboxy-seminaphthorhodafluor-1. The demarcation of pHi corresponded well with the demarcation of the border measured with fluorescein. We conclude that our superfusion system will facilitate the study of intercellular communication and interactions across boundaries of cardiac tissue where different ionic or metabolic conditions are present, for example, between ischemic and nonischemic myocardium.

Labeling and imaging mesenchymal stem cells with quantum dots.

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, -cartilage, adipose, and muscle cells. Adult derived MSCs are being actively investigated because of their potential to be utilized for therapeutic cell-based transplantation. Methods to track MSCs in vivo are -limited, preventing long-term functional studies of transplanted cells. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. Nanoparticles are resistant to chemical and metabolic degradation, demonstrating long-term photostability. Here, we describe the technique to label MSCs with QDs and demonstrate intracellular QD distribution in the labeled MSCs with laser scanning confocal fluorescent microscopy.