Biology subjects

Zeug, A.

Publications and source records attributed to Zeug, A..

3 recordsLinked to original sources

bioRxiv2021

Targeted biallelic integration of an inducible Caspase 9 suicide gene for safer iPSC-based cellular therapies

Drug-inducible suicide systems may help to minimize risks of cellular therapies due to the tumor forming potential of human induced pluripotent stem cells (hiPSCs). Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed that showed elimination or silencing of the transgene. We have introduced a drug-inducible Caspase9 suicide system (iCASP9) into the AAVS1 safe harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. In mice, drug treatment generally led to rapid elimination of teratomas, but individual animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ~ 3x10-8. Transgene elimination, presumably via Loss of Heterozygosity (LoH), or methylation of the CAG promoter but not methylation of the O_SCPLOWPPPC_SCPLOWO_SCPCAP1C_SCPCAPO_SCPLOWRC_SCPLOWO_SCPCAP12C_SCPCAPO_SCPLOWCC_SCPLOW locus were identified as underlying mechanisms. In contrast, we never observed any escapees from biallelic iCASP9 hiPSCs, even after treatment of up to 0.8 billion hiPSCs. In conclusion, biallelic integration of an iCASP9 system in the AAVS1 locus may substantially contribute to the safety level of iPSC-based therapies, which should be calculated by relating clonal escapee frequencies to the cell number in tumors of a size that is readily detectable during routine screening procedures.

molecular biology↗

bioRxiv2020

Deciphering spatio-temporal fluorescence changes using multi-threshold event detection (MTED)

Recent achievements in indicator optimization and imaging techniques promote the exploration of Ca2+ activity patterns as a main second messenger in many organs. Astrocytes are important regulators of brain activity and well known for their Ca2+-dependent modulation of neurons. However, standardized methods to analyze and interpret Ca2+ activity recordings are missing and hindering global comparisons. Here, we present a biophysics-based concept to analyze Ca2+signals, which includes multiple thresholds and provides the experimenter with a comprehensive toolbox for a differentiated and in-depth characterization of Ca2+ signals. We analyzed various ex vivo and in vivo imaging datasets and verify the validity of our multi-threshold event detection (MTED) algorithm across Ca2+ indicators, imaging setups, and model systems from primary cell culture to awake, head-fixed mice. Applying our MTED concept enables standardized analysis and advances research using optical readouts of cellular activity to decrypt brain function. It allowed us to obtain new insights into the complex dependence of Ca2+activity patterns on temperature and neuronal activity. Highlights[->] We present a robust pixel-based algorithm to analyze multidimensional fluorescence data. [->]Automated multiple-threshold analysis accurately quantifies changes in fluorescence across magnitudes. [->]It characterizes the complexity of dynamic and overlapping activity patterns of Ca2+ activity of astrocytes in vitro, in situ, and in vivo. [->]Its application provides quantitative parameters how temperature and neuronal activity determine astrocytic Ca2+ activity.

neuroscience↗

bioRxiv2019

3dSpAn: an interactive software for 3D segmentation and analysis of dendritic spines

Three dimensional segmentation and analysis of dendritic spines involve two major challenges: 1) how to segment individual spines from the dendrites and 2) how to quantitatively assess the morphology of individual spines. We developed a software named 3dSpAn to address these two issues by implementing our previously published 3D multiscale opening algorithm in shared intensity space and using effective morphological features for individual dendritic spine plasticity analysis. 3dSpAn consists of four modules: Preprocessing and ROI selection, Intensity thresholding and seed selection, Multiscale segmentation and Quantitative morphological feature extraction. We show the results of segmentation and morphological analysis for different observation methods, including in vitro and ex vivo imaging with confocal microscopy, and in vivo samples, using high-resolution two-photon microscopy. The software is freely available, the source code, windows installer, the software manual and video tutorial can be obtained from: https://sites.google.com/view/3dSpAn/.

neuroscience↗