Nov 23, 2022
Version 2
Senescence Cocktail V.2
- Sakthikumar Mathivanan1
- 1University of Wisconsin System
- Daniel's workspace
Document Citation: Sakthikumar Mathivanan 2022. Senescence Cocktail. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l24xmxg1y/v2Version created by Daniel El Kodsi
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: November 23, 2022
Last Modified: May 31, 2024
Document Integer ID: 73188
Keywords: ASAPCRN, senescence in fibroblast, senescence of ipsc, neurodegenerative disorders with human stem cell, aged fibroblast, senescence cocktail, features characteristic of aged fibroblast, senescence, axonal degeneration, related neurodegenerative disorder, phenotypes in neuron, human stem cell, derived neuron, stem cell, als patient ipsc, embryonic fibroblast, fibroblast, motor neuron, neuron, generation of reliable drug discovery platform
Abstract
Modeling age-related neurodegenerative disorders with human stem cells is difficult due to the embryonic nature of stem cell derived neurons. We developed a chemical cocktail to induce senescence of iPSC-derived neurons to address this challenge. We first screened small molecules that induce embryonic fibroblasts to exhibit features characteristic of aged fibroblasts. We then optimized a cocktail of small molecules that induced senescence in fibroblasts and cortical neurons without causing DNA damage. The utility of the “senescence cocktail” was validated in motor neurons derived from ALS patient iPSCs which exhibited protein aggregation and axonal degeneration substantially earlier than those without cocktail treatment. Our “senescence cocktail” will likely enhance the manifestation of disease-related phenotypes in neurons derived from iPSCs, enabling the generation of reliable drug discovery platforms.
Troubleshooting
SENESCENCE COCKTAIL- ZHANG LAB
Our senescence cocktail (SLO) is a combination of SBI-0206965 (10mM) + Lopinavir (1mM) + O151 (1mM).
Cell types tested: In our experiments, we tested the activity of SLO on Fibroblasts, H9 derived cortical neurons, hiPSC-TDP43 (Mutant and Isogenic) derived motor neurons.
Final timepoint tested: Neurons were treated with SLO on Day 7 post plating and performed analysis on Day 12.
Final Concentration: 1:1000 dilution (add the senescence cocktail directly to the fresh neuronal media and then feed to the cells).
Workflow:
Workflow
User Optimization needed:
1) The user must optimize the concentration and timing for their experiments.
2) This depends on the cell type (neurons/ astrocytes) and timepoint (short term or long term).
3) We tested SLO on neuronal culture alone. If you are using neuron astrocyte co-culture, the concentration needs to be optimized.
Reagents:
1) SBI-0206965: Cat.no: S7885, Selleckchem
2) Lopinavir: Cat.no: S1380, Selleckchem
3) O151: Cat.no: 5153248, Hit2Lead (Chembridge)
4) Solvent- DMSO
5) Neuronal media- DMEM/ F12.
For more information please refer to our biorxiv preprint: