Stroma as a target to prevent stress-induced cellular senescence of hematopoietic stem cells in vitro and in vivo

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Adult stem cells are characterized by their ability to self-renew and regenerate tissues. Under different in vitro and in vivo conditions of stress, hematopoietic stem cells (HSCs) their ability to self-renew is poorly maintained, which ultimately leads to HSC exhaustion. Our previous work shows that selected stromal cells, or their conditioned medium preserve functional HSCs in culture. Gene expression studies using our co-cultures of stromal cells with early hematopoietic cells: Lineage- Sca-1+ Kit+ cells (LSKs) showed a highly dynamic reciprocal interaction of stromal cells and LSK cells. For instance, LSK cells up-regulate the stromal production of connective tissue growth factor (Ctgf), which, in turn, is required to maintain HSCs with long-term repopulating ability in cultures. The underlying mechanism was that HSC-enriched CD34- CD48- CD150+ LSK (CD34- SLAM) cells cultured on Ctgf-knockdown stroma increases the fraction of these cells in G0, which show delayed time to first cell division and a DNA damage response-associated senescence. This study unveils important feedback mechanisms by which hematopoietic cells promote the ability of niche cells to maintain HSC activity by antagonizing HSC senescence. Here, we are interested in translating these studies in vivo, and, more importantly, use the previous results to define strategies, which prevent DNA damage-response-associated HSC senescence under stress conditions in vivo. For this purpose, we will focus on stress-inducible Ctgf to dissect the relevance of different niche populations in preventing HSC senescence using genetic reporter and conditional knockout models. Also, since CTGF is a known regulator of stromal cell proliferation and differentiation in vitro, we will assess the role of stress-induced genes, such as Ctgf, in stromal cell function and regeneration in vivo. In the second part of the project, we will put our previous studies in a broader perspective and translate newly proposed and already existing gene expression analyses to aid in the formulation of stress-induced signaling pathways in niche cells and critical nodes therein. Screening will be performed using single CD34- SLAM cultures in conditioned media of genetically altered stromal cells, and, in a later step, in the presence of small molecule inhibitors. These analyses will be used to define specific niche targets to antagonize HSC senescence. The results of this project may have broader implications for prevention of exhaustion of normal HSC under different conditions of chronic stress, such as observed in in vitro culture. Interestingly, the defined targets may also be of relevance for combating HSC exhaustion in vivo as a result of stress due to wounding, infections, leukemia, or cytostatic therapy, as well as during diabetes or ageing.