248 Engineering Novel Human Sweat Gland Cell Lines Using CRISPR/Cas9 and Primary Cell Technologies

Sweating plays a fundamental role in human thermoregulation, and various disorders such as hyperhidrosis are associated with dysregulated sweat formation. To study the molecular mechanisms in health and disease, primary cell cultivation techniques and cell-based models for the different cell types that are forming the eccrine sweat gland are required. Currently, the immortalized NCL-SG3 cell line is widely used as a cellular sweat gland model, which allowed us recently to identify and characterize TMEM16A as major Ca2+-activated Cl- channel (CaCC) and key player in sweat glands. However, not only the exact cell type which gave rise to NCL-SG3 cells is unclear, but also the major cholinergic signaling pathway via the muscarinic acetylcholine receptor M3 is inactive. Here, we present two approaches to generate advanced cellular sweat gland models. We applied CRISPR-Cas9 genome editing to re-activate silent endogenous genes in NCL-SG3 cells via a modified Cas9-SAM transactivator complex. Additionally, we generated NCL-SG3 cells that re-express M3 acetylcholine receptor, which allowed us for the first time to characterize Ca2+ signaling and downstream activation of CaCCs in response to physiologic stimulation with cholinergic neurotransmitters. In a second approach we are currently generating novel cell lines from human primary sweat gland isolates. We are establishing sweat gland isolation and primary cell cultivation techniques in order to obtain native primary cells for all sweat gland cell types. Further analyses will reveal, if these cells remain in a stable differentiated state with conserved functional properties.