Biology subjects

Raab, C.

Publications and source records attributed to Raab, C..

3 recordsLinked to original sources

bioRxiv2026

Minimizing time in culture: A prototypic autologous manufacturing workflow for monoclonal iPSC lines within seven weeks

Induced pluripotent stem cells (iPSCs) hold great promise for both allogeneic and autologous cellular therapies. However, broad application and clinical translation is hindered by fragmented, complex and time-intensive workflows, resulting in high manufacturing costs, poor standardization and increased risk of genomic aberrations in derived iPSCs. In this study we developed a standardizable, automatable and time-efficient process for the derivation of monoclonal iPSC lines straight from skin including a comprehensive and cascaded QC strategy. We generated monoclonal iPSC lines derived from human skin punch biopsies of ten donors (age 49-81) via mRNA-based reprogramming that subsequently underwent comprehensive and thorough characterization of phenotypic and genetic properties. The use of a combined mechanical and enzymatic fibroblast isolation protocol and a transient non-integrative reprogramming technology allowed us to obtain 78 monoclonal iPSC lines, ready for banking, molecular characterization and further differentiation within seven weeks from initial sample processing to passage four iPSC lines. The phenotypical characterization via flow cytometry-based pluripotency marker expression and 2D-directed differentiation into the three germ layers showed low intra- and inter-donor variability over all generated lines. A combination of SNP array based CNV analysis followed by whole exome sequencing proved to be the most efficient approach for assessment of genomic integrity. Proof-of-concept experiments for closed system processing revealed that a substantial part of the most error-prone and technically demanding steps can be transferred to semi-automated, closed systems. In conclusion, the described protocol allows for time-efficient, standardizable and automatable generation of high-quality monoclonal iPSC lines from human skin punch biopsies within seven weeks, thus moving the field of autologous iPSC manufacturing one step further towards cost-efficient clinical implementation.

cell biology↗

bioRxiv2020

Innervation modulates the functional connectivity between pancreatic endocrine cells

Direct modulation of pancreatic endocrine cell activity by autonomic innervation has been debated. To resolve this question, we established an in vivo imaging model which also allows chronic and acute neuromodulation. Starting at a stage when zebrafish islet architecture is reminiscent of that in adult rodents, we imaged calcium dynamics simultaneously in multiple islet cell types. We first find that activity coupling between beta cells increases upon glucose exposure. Surprisingly, glucose exposure also increases alpha-alpha, alpha-beta and beta-delta coordination. We further show that both chronic and acute loss of nerve activity diminish activity coupling, as observed upon gap junction depletion. Notably, chronic loss of innervation severely disrupts delta cell activity, suggesting that delta cells receive innervation which coordinates its output. Overall, these data show that innervation plays a vital role in the establishment and maintenance of homotypic and heterotypic cellular connectivity in pancreatic islets, a process critical for islet function.

cell biology↗

bioRxiv2018

Doxorubicin induces caspase-mediated proteolysis of KV7.1

The voltage-gated potassium channel Kv7.1 (KCNQ1) co-assembles with KCNE1 to generate the cardiac potassium current IKs. Gain- and loss-of-function mutations in KCNQ1 are associated with atrial fibrillation and long-QT (LQT) syndrome, respectively, highlighting the importance of modulating IKS activity for proper cardiac function. On a post-translational level, IKS can be regulated by phosphorylation, ubiquitination and sumoylation. Here, we report proteolysis of Kv7.1 as a novel, irreversible posttranslational modification. The identification of two C-terminal fragments (CTF1 and CTF2) of Kv7.1 led us to identify an aspartate critical for the generation of CTF2 and caspases as responsible for mediating Kv7.1 proteolysis. Activating caspases by apoptotic stimuli significantly reduced Kv7.1/KCNE1 currents, which was abrogated in cells expressing caspase-resistant Kv7.1 D459A/KCNE1 channels. An increase in cleavage of Kv7.1 could be detected in the case of LQT mutation G460S, which is located adjacent to the cleavage site. Application of apoptotic stimuli or doxorubicin-induced cardiotoxicity provoked caspase-mediated cleavage of endogenous Kv7.1 in human cardiomyocytes. In summary, our findings establish caspases as novel regulatory components for modulating Kv7.1 activity which may have important implications for the molecular mechanism of doxorubicin-induced cardiotoxicity.\n\nNon-standard Abbreviations and Acronyms

biochemistry↗