High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.     

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%± 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%± 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.     

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.     

Volumetric imaging of erythrocytes using label‐free multiphoton photoacoustic microscopy

Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high‐resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Performance of individual vs. group sampling for inferring dispersal under isolation‐by‐distance

Models of isolation‐by‐distance formalize the effects of genetic drift and gene flow in a spatial context where gene dispersal is spatially limited. These models have been used to show that, at an appropriate spatial scale, dispersal parameters can be inferred from the regression of genetic differentiation against geographic distance between sampling locations. This approach is compelling because it is relatively simple and robust and has rather low sampling requirements. In continuous populations, dispersal can be inferred from isolation‐by‐distance patterns using either individuals or groups as sampling units. Intrigued by empirical findings where individual samples seemed to provide more power, we used simulations to compare the performances of the two methods in a range of situations with different dispersal distributions. We found that sampling individuals provide more power in a range of dispersal conditions that is narrow but fits many realistic situations. These situations were characterized not only by the general steepness of isolation‐by‐distance but also by the intrinsic shape of the dispersal kernel. The performances of the two approaches are otherwise similar, suggesting that the choice of a sampling unit is globally less important than other settings such as a study's spatial scale.     

Acute responses of regional vascular conductance to oral ingestion of fructose in healthy young humans

Background

Recently, it was reported in healthy young subjects that fructose containing drinks increased blood pressure acutely, without any apparent change in total vascular conductance (TVC). However, because it is well known that the splanchnic vasculature is dilated by oral fructose ingestion, it is assumed to be the concomitant vasoconstriction in other peripheral region(s) that is responsible for this finding. Thus, the purpose of this study was to determine the acute response of regional VC to oral fructose ingestion in young healthy humans.

Results

In 12 healthy young subjects, mean arterial blood pressure (MAP), heart rate, cardiac output, and blood flow (BF) in the superior mesenteric (SMA), brachial (BA), and popliteal (PA) arteries, in addition to forearm skin BF, were measured continuously for 2 h after ingestion of 400 ml fructose solution (containing 50 g fructose). Regional VC was calculated as BF/MAP. MAP increased for 120 min after fructose ingestion without any change in TVC. While VC in the SMA was elevated after ingestion, VC in BA and PA and forearm skin decreased.

Conclusions

While TVC was apparently unchanged during the 2 h after fructose ingestion, there were coincident changes in regional VCs in the peripheral circulation, but no net change in TVC.     

Understanding and modeling alternating tangential flow filtration for perfusion cell culture

Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post‐run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1291–1300, 2014     

Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014     

Evaluating two process scale chromatography column header designs using CFD

Chromatography is an indispensable unit operation in the downstream processing of biomolecules. Scaling of chromatographic operations typically involves a significant increase in the column diameter. At this scale, the flow distribution within a packed bed could be severely affected by the distributor design in process scale columns. Different vendors offer process scale columns with varying design features. The effect of these design features on the flow distribution in packed beds and the resultant effect on column efficiency and cleanability needs to be properly understood in order to prevent unpleasant surprises on scale‐up. Computational Fluid Dynamics (CFD) provides a cost‐effective means to explore the effect of various distributor designs on process scale performance. In this work, we present a CFD tool that was developed and validated against experimental dye traces and tracer injections. Subsequently, the tool was employed to compare and contrast two commercially available header designs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:837–844, 2014     

Effect of different training mode on Interleukin-6 (IL-6) and C-reactive protein (CRP) in type 2 diabetes mellitus (T2DM) patients

[Purpose]

This study was carried out to investigate the effects of different training modes on IL-6 and CRP in patients with type 2 diabetes mellitus (T2DM).

[Methods]

The subjects consisted of 16 middle-aged women with type 2 diabetes mellitus (T2DM), all of whom had no other complications. The 16 subjects were randomly assigned to two experimental groups: the circuit training group (CTG, n = 8) and aerobic training group (ATG, n = 8). Based on measured THR (target heart rate) for maximum oxygen consumption rate, the circuit training group (CTG) exercised at 60% intensity, 60 min/day, 5 sets, 3 days/week for 12 weeks. Based on measured THR (target heart rate) for maximum oxygen consumption rate, the aerobic training group (ATG) exercised at 60% intensity (which was increased gradually in weeks 4, 7, and 10) 60 min/day, 3 days/week for 12 weeks.

[Results]

The results are as follows. Significant decreases in the post training values of weight, % Fat, BMI, IL-6 and CRP (p < .05) were observed in the CTG compared to pre-training. However, there were no differences in the physical characteristic and blood inflammatory factors between the groups (ATG & CTG).

[Conclusion]

In conclusion, the results of this study suggest that circuit training (CT) may be considered as an effective training mode for helping to decrease the blood inflammatory factors (IL-6 and CRP) in patients with type 2 diabetes mellitus (T2DM).