Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

Cardiomyocyte regeneration is limited in adults. The adipose tissue-derived stromal vascular fraction (Ad-SVF) contains pluripotent stem cells that rarely transdifferentiate into spontaneously beating cardiomyocyte-like cells (beating CMs). However, the characteristics of beating CMs and the factors that regulate the differentiation of Ad-SVF toward the cardiac lineage are unknown. We developed a simple culture protocol under which the adult murine inguinal Ad-SVF reproducibly transdifferentiates into beating CMs without induction. The beating CMs showed the striated ventricular phenotype of cardiomyocytes and synchronised oscillation of the intracellular calcium concentration among cells on day 28 of Ad-SVF primary culture. We also identified beating CM-fated progenitors (CFPs) and performed single-cell transcriptome analysis of these CFPs. Among 491 transcription factors that were differentially expressed (≥ 1.75-fold) in CFPs and the beating CMs, myocyte-specific enhancer 2c (Mef2c) was key. Transduction of Ad-SVF cells with Mef2c using a lentiviral vector yielded CFPs and beating CMs with ~ tenfold higher cardiac troponin T expression, which was abolished by silencing of Mef2c. Thus, we identified the master gene required for transdifferentiation of Ad-SVF into beating CMs. These findings will facilitate the development of novel cardiac regeneration therapies based on gene-modified, cardiac lineage-directed Ad-SVF cells

Establishment and Clinical Applications of a Portable System for Capturing Influenza Viruses Released through Coughing

Coughing plays an important role in influenza transmission; however, there is insufficient information regarding the viral load in cough because of the lack of convenient and reliable collection methods. We developed a portable airborne particlecollection system to measure the viral load; it is equipped with an air sampler to draw air and pass it through a gelatin membrane filter connected to a cone-shaped, megaphone-like device to guide the cough airflow to the membrane. The membrane was dissolved in a medium, and the viral load was measured using quantitative real-time reverse transcriptasepolymerase chain reaction and a plaque assay. The approximate viral recovery rate of this system was 10% in simulation experiments to collect and quantify the viral particles aerosolized by a nebulizer. Using this system, cough samples were collected from 56 influenza A patients. The total viral detection rate was 41% (23/56), and the viral loads varied significantly (from <10, less than the detection limit, to 2240 viral gene copies/cough). Viable viruses were detected from 3 samples with ?18 plaque forming units per cough sample. The virus detection rates were similar among different groups of patients infected with different viral subtypes and during different influenza seasons. Among patients who did not receive antiviral treatment, viruses were detected in one of six cases in the vaccinated group and four of six cases in the unvaccinated group. We found cases with high viral titers in throat swabs or oral secretions but very low or undetectable in coughs and vice versa suggesting other possible anatomical sites where the viruses might be mixed into the cough. Our system is easy to operate, appropriate for bedside use, and is useful for comparing the viral load in cough samples from influenza patients under various conditions and settings. However, further large-scale studies are warranted to validate our results

Serial photographs of a sneeze extracted from the video image.

<p>Original (upper rows) and enhanced (lower rows) images of the sneeze of a healthy adult male volunteer. The photographs were extracted from the video image recorded by a digital high-vision, high-speed video system at 0.01 s and every 0.05 sec. The mist of the sneeze advanced forward as a mass, associated with gradual diffusion and fading followed by disappearance. A part of the mist cloud looked swirled in the peripheral area (arrows).</p

Position and velocity of particles at the distal margin of the sneeze and cough.

<p>Measured maximum horizontal velocity Vh (♦,▴) and its position: horizontal distance h, from the mouth (<b>◊</b>,△) of the sneeze (A) and cough (B). Values for Vh (solid line) calculated from the approximate equations for the sneeze (I) and for the cough (II), and their relative positions acquired by integration of Vh (dotted line) with respect to the elapsed time t after release.</p

Velocity distribution of particles moving forward as a mass in the sneeze.

<p>Information on the position and horizontal velocity of each particle/particle cluster was extracted from the results of the vector analysis of the sneeze and is plotted along with the elapsing time on a two-dimensional graph. The velocity levels are expressed by color graduation.</p

Vector analysis of particles movement in the sneeze.

<p>The video image was converted to digital images collected every 1/300 s. Each brightness pattern of the aerosol particles or particle clusters, recognized as a particular granular signal, was automatically traced during 1/300 s using image processing software. The vectors are color graduated according to their velocity levels.</p

Image of cough movement represented by smoke cough.

<p>Image analysis of the cough of a smoker healthy adult male volunteer after one breath of smoke. The image of the natural cough was substituted with that of cigarette smoke used as the tracing marker. The photographs were extracted from the video image using a digital high-vision, high-speed video system (upper rows). Vector analysis was performed on the microcloud identified with various densities of white tones of the smoke (lower rows), as it was done for the particles/particle clusters of sneeze.</p