A functional genomics and candidate gene validation approach to identify genes regulating pancreatic development and/or stem cell fitness
Final Report Abstract
The successful development of an alternative pancreatic differentiation screening platform of the current project had to overcome a series of significant obstacles. First, we labour-intensively had to realise that mouse embryonic stem cells do not fulfil the requirements for an shRNA-based development screen. This was time-consuming and strongly perturbed the current proposal. Second, we have successfully established a human differentiation platform and here provide proof of concept by showing original data across a large set of human pluripotent stem cell lines. Showing results for that many cell lines and extensive experimental protocols aims to erase any doubts regarding technique and feasibility of the human screening platform with respect to our application for further funding. Thirdly, we have already conducted a human DE screen in replicates. Finally, we have been able to demonstrate the successful application of such functional genomics screens to identify candidate genes, and simultaneously validate candidate genes as outlined with Dkk3. Therefore we hope to convince the reviewers of our data, and of the fact that our current platform is much more robust compared to the start of our previous funding, even though we gained this knowledge in hindsight. At this stage we would like to underline that granting us follow-up funding would allow us to proceed and progress with the evaluation of the definitive endoderm screen we have already performed in human embryonic stem cells, which would otherwise be lost. Regarding this screen, we would like to stress the fact that (i) all quality controls have been fulfilled, (ii) the screen has already been conducted in replicates and (iii) the Solexa PCR DNA quality is of sufficiently high quality for deep sequencing. The only relevant missing information before we can proceed are the results of the sequencing procedure, which usually takes several months until completion (long queue time). However, according to our collaborator, we will receive results soon. In turn, hit validation of this screen and our robust differentiation platform is the foundation for Aim1 in the follow-up proposal. However, we envision not only going on with the human pluripotent stem cell screen, but also with our efforts to validate Dkk3 as a candidate gene for the regulation of stem cell fitness. The dual beauty of the so-called "Tumor Suppressor Gene" Library, has been previously illustrated in several studies identifying on the one hand novel tumour suppressors, but on the other hand novel regulators of stem cell fitness and organ regeneration 52"55. Thus, previous literature and our own work on Dkk3 support the milestones described in Aim2 of the follow-up proposal, where we plan to complete our knowledge on the regulatory functions of Dkk3 in the stem cell compartment.
Publications
- Increased Reprogramming Capacity of Mouse Liver Progenitor Cells, Compared With Differentiated Liver Cells, Requires the BAF Complex. Gastroenterology. 2012 Apr;142(4):907-17
Kleger A, Mahaddalkar P, Katz SF, Lechel A, Ju JY, Loya K, Lin Q, Hartmann D, Liebau S, Kraus J, Cantz T, Kestler HA, Zaehres H, Schöler H, Rudolph KL
(See online at https://doi.org/10.1053/j.gastro.2012.01.004) - A Hierarchy in Reprogramming Capacity in Different Tissue Microenvironments: What We Know and What We Need to Know. Stem Cells Dev. 2013 Mar 1;22(5):695-706
Liebau S, Mahaddalkar PU, Kestler HA, Illing A, Seufferlein T, Kleger A
(See online at https://doi.org/10.1089/scd.2012.0461) - Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation. Stem Cells Int, 2013;2013:360573
lling A, Stockmann M, Telugu NS, Linta L, Russell R, Müller M, Seufferlein T, Liebau S, Kleger A
(See online at https://doi.org/10.1016/j.stemcr.2013.08.002) - Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny. Stem Cells Int, 2013, 2013:784629
Linta L, Stockmann M, Lin Q, Lechel A, Proepper Ch, Boeckers TM, Kleger A, Liebau L
(See online at https://doi.org/10.1155/2013/784629) - TBX3 Directs Cell-Fate Decision toward Mesendoderm. Stem Cell Reports, 2013 Aug 29;1(3):248-65
Weidgang CE, Russell R, Tata PR, Kühl SJ, Illing A, Müller M, Lin Q, Brunner C, Boeckers TM, Bauer B, Kartikasari AER, Guo Y, Radenz M, Bernemann C, Weiß M, Seufferlein S, Zenke M, lacovino M, Kyba M, Schöler HR, Kühl M, Liebau S, Kleger A
(See online at https://doi.org/10.1016/j.stemcr.2013.08.002) - A fresh look on T-box factor action in the early embryogenesis. Stem Cells Dev. 2015 May 8
Lourenço MB, Linta L, Seufferlein T, Kleger A, Liebau S
(See online at https://doi.org/10.1089/scd.2015.0102) - Loss of ATM accelerates pancreatic cancer formation and Epithelial-Mesenchymal-Transition. Nature Communications, May 2015
Russell R, Perkhofer L, Liebau S, Lin Q, Lechel A, Feld FM, Zenke M, Illing A, Hartmann D, von Figura G, Weissinger SE, Rudolph KL, Möller P, Lennerz JK, Seufferlein T, Wagner M, Kleger A
(See online at https://doi.org/10.1038/ncomms8677)
