Hanging-drop Method Research Articles

Abstract 2303: In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models

Abstract Clinical studies using TCR-transgenic T cells for adoptive T cell therapy revealed the efficacy of this therapeutic approach but also uncovered possible toxic effects against healthy tissues, such as neuronal or cardiac cells, caused by on-target/off-tumor or off-target toxicity (Morgan et al., J Immunother. 2013; Linette et al., Blood 2013). Due to the lack of adequate in vivo models for prediction of TCR efficacy or potential TCR-mediated toxicity against healthy tissues, physiologically relevant in vitro models need to be developed. The usage of 3D spheroids as targets for TCR-transduced T cells represents a powerful strategy to test killing capacity or possible safety issues of TCRs, which could fill the gap between 2D co-cultures and animal models. Compared to 2D cell layers, cells within a 3D spheroid structure more closely resemble the physiological in vivo situation regarding cell-cell-interactions, proliferation rates, hypoxia, and gene expression. Here we describe the use of 3D spheroids generated from tumor cell lines for assessment of TCR efficacy or healthy primary cells/iPSC-derived cells for evaluation of potential TCR-derived toxicity. HLA-A2-positive cells served as target cells in functional co-culture assays with CD8+ T cells transduced with an HLA-A2-restricted TCR. 3D spheroids have been generated using ULA (ultra-low attachment) plates or via the hanging-drop culture method. Spheroids were co-cultured with TCR-transduced T cells and killing of spheroids was analyzed in real-time using the IncuCyte live cell imaging system. For better visualization of spheroid killing, tumor cell lines were transduced using a lentivirus encoding a nuclear-restricted red fluorescent protein or by adding Annexin V red fluorescent reagent for detection of apoptotic cells. Thereby, spheroid killing could be quantified via decrease or increase in red fluorescence over time. Killing of 3D spheroids derived from healthy cells was analyzed via phase contrast and evaluation of spheroid morphology. TCR-transduced T cells showed efficient killing of tumor cell spheroids, confirming the results generated in a 2D system. Furthermore, 3D spheroids generated from healthy cells (e.g. normal human lung fibroblasts) or iPSC-derived cells (e.g. cardiomyocytes or neurons) were not killed via TCR-transduced T cells, whereas exogenous loading of the spheroids with the specific peptide led to efficient killing, showing the general susceptibility to TCR-mediated killing of healthy cell 3D spheroids.In summary, we showed that functional in vitro assays using 3D spheroid structures using tumor cell lines or healthy cells represent an elegant approach to assess both efficacy and potential toxicity of a given TCR in vitro. The analyzed TCR showed potent efficacy as well as a favorable safety pattern in 3D in vitro cultures. Citation Format: Maja Buerdek, Kathrin Mutze, Kai Pinkernell, Dolores J. Schendel. In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2303.

Comparative evaluation of sedative and analgesic effects of sacro-coccygeal epidural administration of detomidine, lidocaine and lidocaine/detomidine in dromedary calves

The present study aims to comparatively evaluate the effects of sacro-coccygeal epidural detomidine, lidocaine and lidocaine/detomidine on sedation and anti-nociception in dromedary calves. This is experimental, prospective, crossover (Latin square), randomized and blinded study. A total of ten clinically healthy male dromedary calves with the age of 15 ± 2 weeks and weight of 125 ± 5.5 kg were assigned to five treatments groups (4 experimental and one control group). Calves in groups D25 and D50 received a low (25 μg kg−1) and high (50 μg kg−1) dose of detomidine, respectively. Groups LID and LD50 received lidocaine (0.22 mg kg−1) and lidocaine/detomidine (0.22 mg kg-1 / 50 μg kg−1), respectively. Calves in the control group received normal saline. Correct spinal needle (18-gauge, 3.7 cm) insertion in the epidural space was confirmed by hanging drop method and loss of resistance to the drug injection. Superficial pain was test by pinprick method. There were no significant differences in time to onset of tail paralysis and perineal anti-nociception among LID, D50 and LD50 groups. Calves receiving epidural lidocaine/detomidine were observed to have long-lasting and reliable sedation and perineal anti-nociception (median = 75, ranges = 60–90 min) in comparison with other studied groups. There were no significant differences in time to onset of ataxia among LID, D50 and LD50 groups. Sacro-coccygeal epidural administration of lidocaine/detomidine could be use for long-duration and manipulative procedures in perineal region of dromedary calves.

Study the Anti-MUC1 antibody-based iron oxide nanoparticles on three-dimension spheroid and breast cancer (MCF-7) cell imaging

Abstract Non-invasive methods for breast cancer detection in early stages may help to increase the survival rate of patients. This study aimed to evaluate the application of Anti-MUC1 antibody-based iron oxide nanoparticle (SPIONs-C595) which was assessed in vivo as a molecular imaging probe for breast cancer (MCF-7) detection using MRI. Nine groups of female NRC NU/Nu mice (each group of 3), 6 to 8 weeks old were used and MCF-7 cells were injected subcutaneously into both flanks of nude mice. After two weeks the mice received an intravenous injection of different concentrations of SPIONs-C595. The uptake ability of SPIONs-C595 on three-dimension (3D) macrostructure is exploited a modified hanging drop method using Prussian blue for MCF-7 cells. The iron content was measured in liver, kidney, spleen, and tumor. The MR imaging features and biodistribution of nanoprobe was also investigated. The MR images obtained from digested tumor after nanoprobe administration in different time-period revealed that enhancement of T1 and T2 relaxation time. Moreover, the storage stability test was shown great application and no sedimentation of nanoparticles within two months storage at 4°C. Additionally, great validation of SPIONs-C595 on the 3D spheroid of MCF-7 was observed. The biodistribution analysis showed that iron content of the spleen was more than the other studied organs. These results highlighted the feasibility of an in-vivo model for detection of breast cancer MUC1 expression. Current researches are ongoing to further enhancement of relaxation times for classification of MUC1 status using clinical specimens.

A systematic comparison of sitting and hanging-drop crystallization using traditional and cross-diffusion microbatch crystallization plates

Obtaining good quality protein crystals is still desirable for high resolution structural determination of the proteins using crystallography. The crystal quality is affected by the growth environment and growth methods. As frequently used methods in practical protein crystallization, sitting and hanging-drop methods are seldom compared in terms of crystal quality in the literatures. We performed a systematic comparison on the quality of the protein crystals grown in sitting and hanging-drop methods using different crystallization plates: cross-diffusion microbatch plate (CDM SD: CDM plate, sitting drop method; CDM HD: CDM plate, hanging drop method), traditional sitting and hanging-drop vapor diffusion plates (T SD: traditional plate, sitting drop method; and T HD: traditional plate, hanging drop method). Five different proteins, proteinase K (prk), lysozyme (lys), concanavalin A VI (con), catalase (cat) and α-Chymotrypsinogen A II (chy), were used. The crystal quality was compared in terms of the resolution limit, mosaicity and Wilson plot B factor. It was found that the crystals grown in CDM plate using the hanging drop method (CDM HD) exhibited the best morphology and the best crystal quality. X-ray diffraction tests showed that the CDM plate using hanging drop method is indeed a practical and useful method for obtaining high-quality protein crystals.

A Net Mold-Based Method of Biomaterial-Free Three-Dimensional Cardiac Tissue Creation.

Ischemic cardiomyopathy poses a significant public health burden due to the irreversible loss of functional cardiac tissue. Alternative treatment strategies include creation of three-dimensional (3D) cardiac tissues to both replace and augment injured native tissue. In this study, we utilize a net mold-based method to create a biomaterial-free 3D cardiac tissue and compare it to current methods using biomaterials. Cardiomyocytes, fibroblasts, and endothelial cells were combined using a hanging drop method to create spheroids. For the net mold patch method, spheroids were seeded into a net mold-based system to create biomaterial-free 3D cardiac patches. For the gel patch, spheroids were embedded in a collagen gel. Immunohistochemistry revealed increased alignment, vascularization, collagen I expression, cell viability, and higher density of cells in the net mold patch compared with the gel patch. Furthermore, in vivo testing in a left anterior descending artery ligation rat model found increased ejection fraction and smaller scar area following implantation of the net mold patch. We present a novel and simple reproducible method to create biomaterial-free 3D net mold patches that may potentially improve the treatment of heart failure in the future.

Fabrication of three-dimensional islet models by the geometry-controlled hanging-drop method

The hanging-drop method has been widely used to fabricate three-dimensional (3D) in vitro tissue models due to its advantages such as being easy to perform, inexpensive, and permitting precise control of cell spheroid formation. The geometry of hanging drop may play a critical role on the formation of cell spheroids, which, however, has not been explored. In this study, we developed a modified hanging-drop platform that enables the production of cell spheroids in a high-throughput manner by controlling hanging drop geometry with a defined spreading ring. The surface tension force is proportional to the spreading ring and the gravitational force is determined by droplet volume, and the geometry can be determined by the balance between surface tension and gravity. β-TC-6 cell spheroids with optimized diameters were fabricated as 3D in vitro islet models. The models showed morphology similar to primary islets and had functionality that more closely resembled primary islets than two-dimensional cell culture. The developed platform holds great potential for engineering well-controlled in vitro tissue models for various applications such as physiological and pathological studies, drug screening, as well as transplantation for treatment purpose.

Determination of Pollen Germination Rates and Pollen Quantities of Some Hybrid Walnut Genotypes

The aim of this research is to determine the rate of pollen germination and pollen production of 29 hybrid walnut genotypes. For this aim, the germination percentage of the pollens was examined by using the agar in the petri and hanging drop methods. Pollen production amount of the genotypes was determined with the hemacytometric method. The highest germination ratio of genotype 30 was obtained by the agar in the petri method in 1% agar+10% sucrose (54.65%). In 15% sucrose concentration in the hanging drop method, which is the best medium for all 29 hybrid walnut genotypes, pollen germination rate was 51.84% (genotype 9). The amount of pollen in a flower and an anther is very high in all genotypes. The consequence indicates that different treatments had a significant effect on the germination percentage. As a result of the findings obtained from this study, genotypes can be used as a pollinator.

Design, 3D printing and validation of a novel low-cost high-capacity sitting-drop bridge for protein crystallization

Sitting-drop protein crystallization is not used as commonly as the hanging-drop method for crystal optimization owing to the limitations of commercially available sitting-drop bridges, particularly when they are used in conjunction with 24-well crystallization plates. The commercially available sitting-drop bridge, containing space for only a single drop, restricts their wider use. Proteins that preferentially crystallize under sitting-drop conditions therefore require more work, time and resources for their optimization. In response to these limitations, and using 3D printing, a new sitting-drop bridge has been designed and developed, where five crystallization drops can be placed simultaneously in each well of a 24-well crystallization plate. This significantly simplifies the process and increases the potential of sitting drops in crystal optimization, reducing costs and hence overcoming the limitations of current approaches.

Crystal structure of the aromatic-amino-acid aminotransferase from Streptococcus mutans.

Streptococcus mutans, a facultatively aerobic and Gram-positive bacterium, is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. In this study, the aromatic-amino-acid aminotransferase from Streptococcus mutans (SmAroAT) was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The recombinant protein was crystallized using the hanging-drop vapor-diffusion method with PEG 3350 as the primary precipitant. The crystal structure of SmAroAT was solved at 2.2 Å resolution by the molecular-replacement method. Structural analysis indicated that the proteins of the aromatic-amino-acid aminotransferase family have conserved structural elements that might play a role in substrate binding. These results may help in obtaining a better understanding of the catabolism and biosynthesis of aromatic amino acids.

Combinatorial Utilization of Murine Embryonic Stem Cells and In Vivo Models to Study Human Congenital Heart Disease.

We have established an in vitro model of the human congenital heart defect (CHD)-associated mutation NKX2.5 R141C. We describe the use of the hanging drop method to differentiate Nkx2.5R141C/+ murine embryonic stem cells (mESCs) along with Nkx2.5+/+ control cells. This method allows us to recapitulate the early stages of embryonic heart development in tissue culture. We also use qRT-PCR and immunofluorescence to examine samples at different time points during differentiation to validate our data. The in vivo model is a mouse line with a knock-in of the same mutation. We describe the isolation of RNA from embryonic day 8.5 (E8.5) embryos and E9.5 hearts of wild-type and mutant mice. We found that the in vitro model shows reduced cardiomyogenesis, similar to Nkx2.5R141C/+ embryos at E8.5, indicating a transient loss of cardiomyogenesis at this time point. These results suggest that our in vitro model can be used to study very early changes in heart development that cause CHD. © 2018 by John Wiley & Sons, Inc.

190 Effects of endocrine-disrupting chemical on calcium signalling in cardiomyocyte differentiation from mouse embryonic stem cells

Endocrine-disrupting chemicals (EDC) are substances similar to steroid hormones that can disturb normal physiological functions of male and female reproductive organs. Endocrine-disrupting chemicals tend to bind to steroid hormone receptors. Sex steroid hormones modulate calcium signalling in the cardiac muscle in early embryo development. Among the steroid hormones, progesterone (P4) has been reported to affect blood pressure and other aspects of the cardiovascular system. The EDC that have similar structure to P4, such as octyl-phenol (OP) and bisphenol A (BPA), are potentially harmful to development of the heart. To confirm the effect of OP and BPA on early differentiation of mouse embryonic stem cells (mES) into cardiomyocytes, the hanging-drop method with mESC cell line (ES-E14TG2a) was used for forming embryoid bodies. Pluripotent mESC were cultured in basal medium with leukemia inhibitory factor and grown on mitomycin C-treated mouse embryonic fibroblasts in a 60-mm plate at 37°C in a 5% CO2 humidified tissue culture incubator with basal medium consisting of DMEM/F-12 supplemented with nonessential amino acids, 10% heat-inactivated and certified fetal bovine serum (FBS), 2-mercaptoethanol, penicillin, and streptomycin. The mouse embryoid bodies (mEB) were suspended onto 6-well plates and cultured with differentiation medium containing steroid-free FBS without leukemia inhibitory factor. Progesterone, OP, and BPA were added on Day 2 from mEB attachment. In addition, mifepristone (RU486), an antagonist for progesterone receptor (PR), was used to confirm the effect of P4 through PR. To determine whether RU486 is capable of attenuating the inhibition effect of P4, RU486 was added for 1 day starting on Day 11. Assessment of cardiomyocyte differentiation was determined by checking the number of beating cell populations divided by the total number of attached mEB. Total RNA was extracted using Trizol reagent and synthesised to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase. Messenger RNA level was assessed using quantitative real-time PCR. To investigate calcium signalling, the mRNA levels of calcium channel gene Trpv2 and contraction-related genes Ryr2, Cam2, and Mylk3 were analysed. Beating ratio was decreased in P4, OP, and BPA treatments. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison tests, and P<0.05 was considered statistically significant in least 3 different replicates. The mRNA level of Pgr was significantly increased in P4, OP, and BPA-treated group. However, the mRNA level of calcium channel gene Trpv2 was significantly decreased in the P4, OP, and BPA-treated group. Expression of contraction-related genes such as Ryr2, Cam2, and Mlck3 were significantly decreased in the P4, OP, and BPA-treated group. In addition, treatment with RU486 rescues altered calcium channel gene and contraction-related genes. Taken together, these results suggest that OP and BPA may affect the differentiation of mESC into cardiomyocyte and disrupts differentiation of cardiomyocytes.

Determination of male flower characteristics of Turkish hazelnut (Corylus avellanaL.) cultivars

Hazelnut (Corylus avellana L.) is a monoecious, self-incompatible and wind-pollinated species. This study was carried out to determine the catkin number and pollen germination rate of 19 Turkish cultivars. In vitro germination was assessed with two methods under two temperature regimes. Pollen germination was between 8.8 and 65.0% in the medium containing 15% sucrose, 20 ppm boric acid and 0.75% agar among the cultivars while large variation (11.4-72.2%) was observed in hanging drop method. The cultivar 'Karafindik' had the largest number of catkins (3.7 per node) and gave the highest germination rate while 'Giresun Melezi' gave the lowest rate. It is suggested that 'Karafindik' has a high pollenizer potential for other cultivars with round shaped nuts. On the other hand recently registered cultivar 'Giresun Melezi' would be considered not good pollenizer due to its low pollen viability.

Acoustic assembly of cell spheroids in disposable capillaries

Multicellular spheroids represent a promising approach to mimic 3D tissues in vivo for emerging applications in regenerative medicine, therapeutic screening, and drug discovery. Conventional spheroid fabrication methods, such as the hanging drop method, suffer from low-throughput, long time, complicated procedure, and high heterogeneity in spheroid size. In this work, we report a simple yet reliable acoustic method to rapidly assemble cell spheroids in capillaries in a replicable and scalable manner. Briefly, by introducing a coupled standing surface acoustic wave, we are able to generate a linear pressure node array with 300 trapping nodes simultaneously. This enables us to continuously fabricate spheroids in a high-throughput manner with minimal variability in spheroid size. In a proof of concept application, we fabricated cell spheroids of mouse embryonic carcinoma (P19) cells, which grew well and retained differentiation potential in vitro. Based on the advantages of the non-invasive, contactless and label-free acoustic cell manipulation, our method employs the coupling strategy to assemble cells in capillaries, and further advances 3D spheroid assembly technology in an easy, cost-efficient, consistent, and high-throughput manner. This method could further be adapted into a novel 3D biofabrication approach to replicate compilated tissues and organs for a wide set of biomedical applications.

Characterization of Human Dermal Papilla Cells in Alginate Spheres

Maintenance of trichogenecity of dermal papilla cells (DPCs) have been a problem during cell therapy for androgenic alopecia, as they lose their regenerative potential in in vitro culture. Various spheroid culture techniques are used to increase and maintain trichogenecity of these cells. However, there are some critical drawbacks in these methods. Applying a hydrocell plate for sphere formation or hanging drop methods by hand would be difficult to control the size and cell density inside it. It would be difficult to commercialize or mass production for clinical therapy. In aim to address and overcome these drawbacks, we have introduced alginate sphere. The alginate sphere of DPCs were prepared by electrospinning at different voltages to control the size of sphere. Then the obtained alginate spheres were evaluated for cellular dynamics and density of DPCs under different conditions. In this study, we found that DPCs do not proliferate in alginate sphere. However, the number of DPCs were maintained and found to be in dormant state. Further, the dormant DPCs in the alginate sphere have upregulated DPC signature genes (SOX2, ALPL, WIF1, Noggin, BMP4 and VCAN) and proliferative capacity. Thus, we speculate that alginate sphere environment maintains the dormancy of DPCs with increased trichogenecity.

Resveratrol restores sensitivity of glioma cells to temozolamide through inhibiting the activation of Wnt signaling pathway.

Malignant gliomas are aggressive primary neoplasms that originate in the glial cells of the brain or the spine with notable resistance to standard treatment options. We carried out the study with the aim to shed light on the sensitization of resveratrol to temozolomide (TMZ) against glioma through the Wnt signaling pathway. Initially, glioma cell lines with strong resistance to TMZ were selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the glioma cells were subjected to resveratrol, TMZ, Wnt signaling pathway inhibitors, and activators. Cell survival rate and inhibitory concentration at half maximum value were detected by MTT, apoptosis by flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, in vitro proliferation by hanging drop method and β-catenin translocation into nuclei by TOP/FOP-FLASH assay. The expressions of the Wnt signaling pathway-related and apoptosis-related factors were determined by western blot analysis. Nude mice with glioma xenograft were established to detect tumorigenic ability. Glioma cell lines T98G and U138 which were highly resistant to TMZ were selected for subsequent experiments. Resveratrol increased the efficacy of TMZ by restraining cell proliferation, tumor growth, and promoting cell apoptosis in glioma cells. Resveratrol inhibited Wnt2 and β-catenin expressions yet elevated GSK-3β expression. Moreover, the Wnt signaling pathway participates in the sensitivity enhancing of resveratrol to TMZ via regulating O 6 -methylguanine-DNA methyltransferase (MGMT) expression. Resveratrol sensitized TMZ-induced glioma cell apoptosis by repressing the activation of the Wnt signaling pathway and downregulating MGMT expression, which may confer new thoughts to the chemotherapy of glioma.

Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model

Porphyromonas gingivalis-induced inflammatory effects are mostly investigated in monolayer cultured cells. The aim of this study was to develop a 3D spheroid model of gingiva to take into account epithelio-fibroblastic interactions. Human gingival epithelial cells (ECs) and human oral fibroblasts (FBs) were cultured by hanging drop method to generate 3D microtissue (MT) whose structure was analyzed on histological sections and the cell-to-cell interactions were observed by scanning and transmission electron microscopy (SEM and TEM). MTs were infected by P. gingivalis and the impact on cell death (Apaf-1, caspase-3), inflammatory markers (TNF-α, IL-6, IL-8) and extracellular matrix components (Col-IV, E-cadherin, integrin β1) was evaluated by immunohistochemistry and RT-qPCR. Results were compared to those observed in situ in experimental periodontitis and in human gingival biopsies. MTs exhibited a well-defined spatial organization where ECs were organized in an external cellular multilayer, while, FBs constituted the core. The infection of MT demonstrated the ability of P. gingivalis to bypass the epithelial barrier in order to reach the fibroblastic core and induce disorganization of the spheroid structure. An increased cell death was observed in fibroblastic core. The development of such 3D model may be useful to define the role of EC–FB interactions on periodontal host-immune response and to assess the efficacy of new therapeutics.

In Vitro Expansion and Characterization of Mesenchymal Stromal Cells from Peritoneal Dialysis Effluent in a Human Protein Medium.

The therapeutic potential of mesenchymal stromal cells (MSCs) from various tissue origins have extensively been explored in both experimental and clinical studies, and peritoneal dialysis effluent-derived MSC (pMSC) may be an easily obtainable MSC source for clinical applications. In this study, we expanded and characterized the pMSCs after expansion in a human protein culture medium. The pMSCs were expanded in plastic dishes with the human protein medium. MSC marker expression was examined by flow cytometry. Spherical formation was tested by hanging drop method, and osteogenic, adipogenic, and chondrogenic differentiation capacities were confirmed by positive staining with Alizarin red, Oil red O, and Alcian blue, respectively. Here, we showed that after four passages of culturing in plastic dishes, pMSCs in the human protein medium displayed a homogeneous pattern of classical MSC markers (positive: CD29, CD44, CD73, CD90, and CD166; negative: CD14, CD34, CD45, CD79a, CD105, CD146, CD271, HLA-DR, SSEA-4, and Stro-1), while in the standard medium, pMSCs from some donors were CD45 or HLA-DR positive. For nonclassical MSC markers, pMSCs were CD200 positive from all the donors, negative for CD163, CD271, CD36, and CD248, and either positive or negative for CD274 and CD140b. Further, pMSCs from the human protein medium had the spherical formation capacity and multipotent differentiation capacity in vitro. In conclusion, upon expansion in a human protein medium, pMSCs showed a differential MSC marker expression profile from those of bone marrow or adipose tissue-derived MSCs and could maintain the multipotency. The therapeutic potential of the pMSCs requires further investigation.

Pentraxin 3 promotes cardiac differentiation of mouse embryonic stem cells through JNK signaling pathway.

Cardiovascular disease is a leading cause of death worldwide, requiring the development of new therapeutic strategies including stem cell therapy. Pentraxins (PTXs) are a superfamily of proteins highly involved in different myocardial disorders, and thus this study aimed to identify the modulation of long pentraxin 3 (PTX3) in the differentiation of mouse embryonic stem cells (mESCs) toward cardiomyocytes. Cell toxicity of PTX3 was detected by MTT and LDH assays in mESCs. Embryoid bodies (EBs) were differentiated using hanging drop method, and the beating was observed under microscope. Expressional levels of early cardiac progenitor marker genes were assessed by qRT-PCR. Expression of marker proteins in early myocardial development and the activation of JNK signaling pathway was evaluated by Western blot. PTX3 treatment at 50 ng/mL significantly promoted the expression of cardiac-specific marker genes including Nkx2.5, Mef2c, Tbx5, dHand, and αMHC, and increased the expression of cardiac maturity indicative markers including connexin 43 and troponin C1. PTX3 enhanced the phosphorylation of JNK across the incubation duration, whereas the activation of p38 remained the same as control group. Co-treatment of JNK signaling pathway inhibitor SP600125 impaired the PTX3-promoted transcription of Nkx2.5, Mef2c, Tbx5, dHand, and αMHC. This study revealed the promotion of PTX3 in the differentiation of mESCs into cardiomyocytes and the underlying mechanism.

MSMEG_6292, a Mycobacterium smegmatis RNA polymerase secondary channel-binding protein: purification, crystallization and X-ray diffraction analysis.

The transcriptional activity of RNA polymerase (RNAP) is controlled by a diverse set of regulatory factors. A subset of these regulators modulate the activity of RNAP through its secondary channel. Gre factors reactivate stalled elongation complexes by enhancing the intrinsic cleavage activity of RNAP. In the present study, the protein MSMEG_6292, a Gre-factor homologue from Mycobacterium smegmatis, was expressed heterologously in Escherichia coli and purified using standard chromatographic techniques. The hanging-drop vapour-diffusion crystallization method yielded diffraction-quality crystals. The crystals belonged to the trigonal space group P3121 (or its enantiomorph P3221), with unit-cell parameters a = b = 83.15, c = 107.07 Å, α = β = 90, γ = 120°. The crystals diffracted to better than 3.0 Å resolution. Molecular-replacement attempts did not yield any phasing models; hence, platinum derivatization was carried out with K2PtCl4 and derivative data were collected to 3.4 Å resolution.

Abstract 342: The Absence of S-nitrosoglutathione Reductase (GSNOR -/- ) Reduces Maturation of iPSC-derived Cardiomyocytes

Introduction: Induced pluripotent stem cells (iPSCs) are a potential source of cardiac regenerative therapy. However, incomplete structural and functional maturation of iPSC-derived cardiomyocytes (iPSC-CMs), including lack of T-tubules, immature sarcoplasmic reticulum (SR), and inefficient Ca 2+ -induced Ca 2+ release remain major limitations. We assessed the influence of nitric oxide (NO) on differentiation and maturation of iPSC-CMs. Objective: We hypothesized that enhanced S-nitrosylation impairs SR calcium (Ca 2+ ) handling (storage and uptake) in iPSC-CMs. Methods: iPSCs were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR -/- ), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into CMs from embryoid bodies via the hanging-drop method. Analyses of cell morphology and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) were performed at an early stage of cardiac development (day 21 of cell culture). The involvement of the SR Ca 2+ ATPase (SERCA2) in iPSC-CM contraction was tested by blocking Ca 2+ re-uptake with 1 μM Thapsigargin and SR Ca 2+ stores were estimated by addition of 20 mM caffeine. The role of the cardiac ryanodine receptor (RyR2) was assessed by blocking the channel with 10 μM ryanodine. The dependence of Ca 2+ transients on L-type Ca 2+ channels (LTCC) was demonstrated using 10 μM nifedipine, which eliminated [Ca 2+ ] i transients and cell contraction. Length of sarcomeres within the contractile apparatus was determined by staining cells with α-actinin. Results: Sarcomeres were more organized in WT iPSC-CMs than in GSNOR -/- iPSC-CMs after 21 days in culture. The loss of GSNOR activity reduced SR Ca 2+ content and sarcomere organization in iPSC-CMs. While iPSC-CMs are relatively immature in terms of ultrastructure and Ca 2+ handling; GSNOR –/– iPSC-CMs were less able to recycle intracellular Ca 2+ . Conclusions: Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that the absence of GSNOR affects CM differentiation. These findings have important implications for regenerative medicine and provide new targets for iPSC-based therapy.