Influence of nitrosamines and ionizing radiation on the chondrogenic differentiation potential of neonatal and adult stromal cells

Background: Comprehensive analyses comparing individual DNA damage response (DDR) of distinct stromal cell types with respect to their developmental age are limited. The imperative necessity of providing developmental age-matched cell sources for meaningful toxicological drug safety assessments in replacement of animal-based testing strategies is evident. Here, DDR after radiation or treatment with N-methyl-N-nitrosurea (MNU) is to be determined in neonatal and adult stromal cells compared with iPSCs. The project focuses on a differentiation-dependent regulation of repair capacity during chondrogenic differentiation of neonatal and adult stromal cells and iPSC as control.

Aim: The project aims to characterize responses to exogenous genotoxic insults essential for maintenance of genomic integrity with particular physiological relevance in the context of development and differentiation, since the sensitivity between distinct cell types at distinct developmental stages is likely variable.

Experimental procedure / working program: Neonatal stromal cells from cord blood and adult mesenchymal stromal cells from bone marrow are applied as a osteo-chondrogenic differentiation model upon genotoxic treatment with N-Methyl-Nitrosurea (MNU) and ionizing radiation in comparison to an embryonal iPSC control. Published data of our group analyzing osteogenesis (closely connected to chondrogenesis) suggest developmental age-dependent differences in DNA damage repair capacity with the highest expression levels of DDR and repair genes in iPSCs (closest to embryonic stem cells), followed by neonatal stromal cells, with adult stromal cells having with the lowest overall expression. In addition activation of apoptosis will be analyzed in close cooperation with AG Wesselborg applying the Nicoletti method (quantification of DNA content) as well as activation of caspases by Western Blot and fluorimetric caspase 3 assays. Moreover, the impact of the time the genotoxic insult takes place during chondrogenesis will be analyzed. The project will be accomplished in close cooperation with other members of the GRK 2578.

Recent publications:

1. Cruet-Hennequart S, Drougard C, Shaw G, et al.: Radiation-induced alterations of osteogenic and chondrogenic differentiation of human mesenchymal stem cells. PLoS One 2015;10:e0119334.
2. Liedtke S, Biebernick S, Radke TF, et al.: DNA damage response in neonatal and adult stromal cells compared with induced pluripotent stem cells. Stem Cells Transl Med 2015;4:576-89.
3. Pham CD, Hartmann R, Bohler P, et al.: Callyspongiolide, a cytotoxic macrolide from the marine sponge Callyspongia sp. Org Lett 2014;16:266-9.
4. Sacchetti B, Funari A, Remoli C, et al.: No Identical "Mesenchymal Stem Cells" at Different Times and Sites: Human Committed Progenitors of Distinct Origin and Differentiation Potential Are Incorporated as Adventitial Cells in Microvessels. Stem Cell Reports 2016;6:897-913.
5. Stuhldreier F, Kassel S, Schumacher L, et al.: Pleiotropic effects of spongean alkaloids on mechanisms of cell death, cell cycle progression and DNA damage response (DDR) of acute myeloid leukemia (AML) cells. Cancer Lett 2015;361:39-48.