Identifizierung humaner GJB2 35delG modifizierender Gene unter Verwendung von induzierten pluripotenten Stammzellen
Humangenetik
Molekulare Biologie und Physiologie von Nerven- und Gliazellen
Zusammenfassung der Projektergebnisse
About 50% of the cases dealing with recessive hereditary hearing loss are caused by a mutation in the GJB2 gene in the DFNB1 locus encoding a protein that allows cells in the inner ear to exchange small molecules. Although genetic reasons for deafness in those patients are rather obvious, the degree of hearing loss varies surprisingly between different cases. This is of particular interest as success in acquiring spoken language skills highly depends on functional hearing and therefore we aimed to resolve the molecular basis of GJB2-releated hearing loss in order to develop novel treatment strategies. Our research was aimed to identify the molecular reasons why patients that carry exactly the same mutation end up with different levels of hearing loss. We speculate that there might be other genetic factors involved that modulate the impact of the known DFNB1 mutation on patient’s ability to perceive sound. To achieve this goal, we applied stem cell-based technology that was specifically tailored to answer our questions, which are virtually impossible to address in an animal model. We started with a patient’s skin biopsy and by applying different factors in the culture dish, we generated a stem cell population that is capable of differentiating into nearly every tissue present in the human body including the crucial inner ear cell types. Testing of the in vitro differentiated inner ear cell types revealed functional properties like the capacity to transport small molecules just as cells from the human inner ear would. Further investigating those cells we found that the method applied not only produced our target cell type but a variety of other cells present in the sensory part of the inner ear. A prerequisite to study the genetic disease as mentioned earlier is to generate affected cells in large quantities, because off-target cells may mask disease related effects. We tried to overcome this bottleneck by systematically changing the cell culture conditions in order to tease out best growth of the target cell type. As cells did not respond as predicted, we concluded that the culture protocol was not properly mirroring inner ear development and most likely missing a critical developmental signal. In an effort to learn about the molecular composition of all cell types present in the inner ear sensory epithelium we developed a novel method that allowed us to generate a cellular map of the organ of Corti featuring the expression of 192 different genes at single cell resolution. This method combined multiparallel qRT-PCR with bioinformatics-based reconstruction of a spatial tissue map. Because this approach allows researchers to look at cellular processes at unprecedented resolution it was acknowledged beyond our field and highlighted in social media. Currently we are maximizing the benefit of this approach by not only looking at a limited number of 192 genes but assessing all genes actively transcribed at a given time per cell, which add up to thousands of different genes depending on the cell type investigated. The application of this new technology will educate us about how different inner ear cell types develop over time and ultimately allow us to resemble the same processes in the culture dish. Once this critical step is taken we’re confident to identify and quantify all genes expressed in cochlear cells and compare profiles from normal hearing people with DFNB1 patients. This strategy will enable us to gain in depth understanding of complex genetic diseases and to develop rational treatment strategies in the near future.
Projektbezogene Publikationen (Auswahl)
- Inner ear hair cell-like cells from human embryonic stem cells. Stem Cells Dev. 2014 Jun 1;23(11):1275-84
Ronaghi M, Nasr M, Ealy M, Durruthy-Durruthy R, Waldhaus J, Diaz GH, Joubert LM, Oshima K, Heller S
(Siehe online unter https://doi.org/10.1089/scd.2014.0033) - Reconstruction of the mouse otocyst and early neuroblast lineage at single-cell resolution. Cell. 2014 May 8;157(4):964-78
Durruthy-Durruthy R, Gottlieb A, Hartman BH, Waldhaus J, Laske RD, Altman R, Heller S
(Siehe online unter https://doi.org/10.1016/j.cell.2014.03.036) - Changes in the regulation of the Notch signaling pathway are temporally correlated with regenerative failure in the mouse cochlea. Front Cell Neurosci. 2015;9:110
Maass JC, Gu R, Basch ML, Waldhaus J, Lopez EM, Xia A, Oghalai JS, Heller S, Groves AK
(Siehe online unter https://doi.org/10.3389/fncel.2015.00110) - Quantitative High-Resolution Cellular Map of the Organ of Corti. Cell Rep. 2015 Jun 9;11(9):1385-99
Waldhaus J, Durruthy-Durruthy R, Heller S
(Siehe online unter https://doi.org/10.1016/j.celrep.2015.04.062) - Comparative functional characterization of novel non-syndromic GJB2 gene variant p.Gly45Arg and lethal syndromic variant p.Gly45Glu. PeerJ. 2016 Oct 11;4:e2494
Rodriguez-Paris J, Waldhaus J, Gordhandas J, Pique L, Schrijver I
(Siehe online unter https://doi.org/10.7717/peerj.2494)
