Silencing of microRNA-21 in vivo ameliorates autoimmune splenomegaly in lupus mice

MicroRNAs (miRNAs) have been implicated in B cell lineage commitment, regulation of T cell differentiation, TCR signalling, regulation of IFN signalling, and numerous other immunological processes. However, their function in autoimmunity, and specifically in systemic lupus erythematosus (SLE), remains poorly understood. B6.Sle123 is a spontaneous genetic mouse model of SLE characterized by autoantibody production, lymphosplenomegaly, and glomerulonephritis. We identified several differentially regulated miRNAs in B and T lymphocytes of B6.Sle123 mice. We found that miR-21 expression in lupus B and T cells is up-regulated and that in vivo silencing of miR-21 using a tiny seed-targeting LNA reversed splenomegaly, one of the cardinal manifestations of autoimmunity in B6.Sle123 mice, and de-repressed PDCD4 expression in vivo and in vitro. In addition, treatment with anti-miR-21 altered CD4/CD8 T cell ratios and reduced Fas receptor-expressing lymphocyte populations. Our study shows that tiny LNAs can be used to efficiently antagonize endogenous miRNAs in peripheral lymphocytes in vivo and in primary lymphocytes cultured ex vivo and can alter the course of a spontaneous genetic disease in mice

Contraction pressure analysis using optical imaging in normal and MYBPC3-mutated hiPSC-derived cardiomyocytes grown on matrices with tunable stiffness

Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems. Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with β-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines. Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. β-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure. Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.</p

Tissue-Engineered Vascular Rings from Human iPSC-Derived Smooth Muscle Cells

There is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust, patient-specific tissue model systems for studying the pathogenesis of vascular disease, and for developing novel therapeutic interventions. Here, we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore, we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system, extendable to study other vascular proliferative diseases for drug screening. Thus, this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications

Bmp4 signaling enhances transcription factor <i>Tbx5</i> and <i>Tbx20</i> mRNA expression.

<p>FACS-sorted GFP+ Isl1-CPCs were cultured in the presence of Bmp4 or vehicle control. mRNA was collected following 2, 4 and 6 days of Bmp4 treatment. qRT-PCR analysis of duplicates from three independent experiments for <i>tbx5</i> (A) and <i>tbx20</i> (B) expression is normalized by housekeeping gene, Gapdh. *p<0.05; **p<0.01; n = 3 independent experiments.</p

Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

<div><p>As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs) could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs) could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs) may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP) expression that is directed by the <i>mef2c</i> gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of Isl1 expressing cardiac progenitors, which could lead to enhancement of cardiac differentiation and engraftment, holding a significant therapeutic value for cardiac repair in the future.</p></div

Calcium transients of cardiomyocytes derived from Isl1-CPC after cell loading with Ca²⁺ indicator fura-2.

<p>(A) Representative recordings for increase in [Ca²⁺]i transient amplitude and frequency was measured following loading of control and Bmp4-treated cardiomyocyte cultures with Ca²⁺ indicator fura-2. Cytosolic Ca²⁺ of spontaneous beating cardiomyocyte was measured by ratio of fluorescence intensity at 340 nm and 380 nm (F340/F380). The measurements were recorded 6 days of incubation after Isl1-CPC isolation. (B) The quantitative representation of beating frequency (BF) of calcium transients per minute is shown by bar graphs (n = 3). Experiments were repeated 3 times and 10–15 cells were measured in each experiment. (C) Change of [Ca²⁺] was calculated from Ca²⁺ transient recordings for cardiomyocyte cultures differentiated in the absence or presence of Bmp4. *p<0.05; n = 3 independent experiments.</p

Isl1-CPC derived cultures have cardiomyocyte-like electrophysiological properties.

<p>Action potentials of differentiated Isl1-CPCs from control (A) or Bmp4-treated cultures (B) were recorded after 6 days in culture under whole-cell current clamp mode. (A, B) Representative action potentials demonstrate nodal-like, atrial-like and ventricular-like cardiomyocytes in the absence and presence of Bmp4. (C) Action potential amplitude (APA), action potential duration at the 90% of repolarization (APD₉₀) and maximum diastolic potential (MDP) for ventricular-like cardiomyocytes were quantified and depicted as bar graph for both control and Bmp4-treated cultures. A total of 29 cardiomyocytes derived from Isl1-CPCs was analyzed from 4 independent experiments. There were no significant differences for APA, MDP and APD₉₀ between control and Bmp4 treated samples. The details for AP measurement setting are under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110752#s2" target="_blank">Materials and Methods</a>.</p

BMP signaling enhances Isl1-CPC cardiac differentiation.

<p>Freshly sorted EB day 5.5 GFP⁺ Isl1-CPC were treated with control (A, D) or 25 ng/ml Bmp4 (B, E) for 6 days, followed by immunostaining with antibodies against cardiomyocyte-specific protein cTnT (red in A and B) or labelled with cTnT and Alexa fluor 647 for FACS analysis (D, E). Nuclei: blue (Hoechst). (C) Quantification of A and B (over 2000 cells counted from 3 independent experiments). (F) Quantification of D and E (20000 cells FACS analyzed for each of 3 independent experiments). Scale bar: 100 µm; *statistical significance <i>p<0.05</i>, Bmp4 –treated cultures vs. control.</p

Comparison of cardiomyocyte (CM) types from mESC-derived Isl1-CPCs in the absence or presence of Bmp4 treatment. The number of cardiomyocytes (# of CM) and the proportion of cardiomyocytes (% of total) exhibiting nodal-, atrial- and ventricular-like properties are shown (n = 4).

<p>Comparison of cardiomyocyte (CM) types from mESC-derived Isl1-CPCs in the absence or presence of Bmp4 treatment. The number of cardiomyocytes (# of CM) and the proportion of cardiomyocytes (% of total) exhibiting nodal-, atrial- and ventricular-like properties are shown (n = 4).</p

Comparison of action potential parameters for Isl1-CPC derived cardiomyocyte cultures in the absence or presence of Bmp4 treatment (n = 4).

<p>Comparison of action potential parameters for Isl1-CPC derived cardiomyocyte cultures in the absence or presence of Bmp4 treatment (n = 4).</p