Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

Efforts to advance fish health diagnostics have been highlighted in many studies to improve the detection of pathogens in aquaculture facilities and wild fish populations. Typically, the detection of a pathogen has required sacrificing fish; however, many hatcheries have valuable and sometimes irreplaceable broodstocks, and lethal sampling is undesirable. Therefore, the development of non-lethal detection methods is a high priority. The goal of our study was to compare non-lethal sampling methods with standardized lethal kidney tissue sampling that is used to detect Renibacterium salmoninarum infections in salmonids. We collected anal, buccal, and mucus swabs (non-lethal qPCR) and kidney tissue samples (lethal DFAT) from 72 adult brook trout (Salvelinus fontinalis) reared at the Colorado Parks and Wildlife Pitkin Brood Unit and tested each sample to assess R. salmoninarum infections. Standard kidney tissue detected R. salmoninarum 1.59 times more often than mucus swabs, compared to 10.43 and 13.16 times more often than buccal or anal swabs, respectively, indicating mucus swabs were the most effective and may be a useful non-lethal method. Our study highlights the potential of non-lethal mucus swabs to sample for R. salmoninarum and suggests future studies are needed to refine this technique for use in aquaculture facilities and wild populations of inland salmonids.

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Feeder-Dependent/Independent Mouse Embryonic Stem Cell Culture Protocol

10.1007/7651_2021_402 ◽

2021 ◽

Author(s):

Hatice Burcu Şişli ◽

Selinay Şenkal ◽

Derya Sağraç ◽

Taha Bartu Hayal ◽

Ayşegül Doğan

Keyword(s):

Cell Culture ◽

Stem Cell ◽

Embryonic Stem Cell ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Stem Cell Culture ◽

Cell Culture Protocol ◽

Culture Protocol

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

Scientific Reports ◽

10.1038/s41598-020-80848-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shinichiro Takashima ◽

Soichiro Usui ◽

Oto Inoue ◽

Chiaki Goten ◽

Kosei Yamaguchi ◽

...

Keyword(s):

Adipose Tissue ◽

Lentiviral Vector ◽

Troponin T ◽

Stromal Vascular Fraction ◽

Cardiac Regeneration ◽

Cardiac Lineage ◽

Cell Transcriptome ◽

Single Cell Transcriptome ◽

Culture Protocol ◽

Enhancer Factor

AbstractCardiomyocyte 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.

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