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Project

Understanding the role of Base Excision Repair mechanisms in fetal liver and bone marrow hematopoietic stem cells.

Hematopoietic stem cells (HSCs) ensure the lifelong process of hematopoiesis, which continuously generates all mature blood cells. Self-renewal potential and long-term multilineage differentiation are critical functional properties of HSCs, which underlie the clinical therapeutic utility of hematopoietic stem and progenitor cell (HSPC) transplantation in immunocompromised and/or cancer patients. Consequently, HSPC functionality is tightly controlled by a complex interplay of intrinsic and extrinsic factors. Despite HSC biology being the most studied stem cell field for decades, full understanding of all molecular mechanisms regulating HSPC self-renewal and differentiation remains elusive. Novel insights will not only enhance understanding of hematopoiesis and HSPC physiology, but will also help to disentangle the molecular mechanisms underlying hematologic diseases and malignancies, as well as improve the still challenging ex vivo expansion of HSCs, without functional exhaustion.

The first aim of this thesis was to evaluate different culture strategies allowing functional ex vivo mouse bone marrow (BM) HSPC expansion. Mimicking the in vivo embryonic fetal liver (FL) HSC niche in a dish, using 3D fibrin-based hydrogels or co-culture with pluripotent stem cell-derived FL-like cells, did not support ex vivo HSC expansion, in contrast to a recently published albumin-free HSC expansion medium. In line with that publication, we observed that the albumin-free polyvinyl alcohol (PVA)-supplemented medium supported extensive ex vivo expansion of HSCs with in vivo repopulating capacity, even when the culture was initiated from less purified Lin-cKit+ progenitors. Additionally, our results showed that the PVA supplementation alone is insufficient to allow functional in vitro culture HSC expansion and that the Ham’s F12 medium composition itself is also critical, which is not the case for the more commonly used IMDM medium. Although further research is needed to elucidate the exact underlying mechanisms, the F12-PVA-based functional HSPC expansion greatly enhanced our abilities to study intrinsic HSPC regulator(s) also during in vitro expansion.

The second aim of this thesis was to assess the intrinsic role of 4 different Base Excision Repair (BER) genes in adult HSPCs using a CRISPR/Cas9 knockout (KO) approach. Even though BER resolves oxidative and other frequent endogenous DNA lesions, the role of key BER genes, such as Apex1, Xrcc1, Lig1 and Polβ, has previously not been studied in HSPCs in contrast to all other DNA repair pathways. The most severe phenotype was observed when we knocked out Apex1, as Apex1-KO HSPCs failed to repopulate irradiated recipient mice shortly after transplantation. This in vivo phenotype was recapitulated in vitro, where reduced expansion of Apex1 KO BM cells was observed in the PVA-supplemented F12 culture medium maintaining repopulating HSCs. Although less severe compared to Apex1 loss, Lig1 KO also caused an HSPC expansion defect and in vivo HSPC dysfunction, as Lig1 KO HSPCs showed impaired contribution to different committed progenitors and lineages. By contrast, knockout of Xrcc1 had only minor effects on HSPC expansion and reconstitution potential. However, Xrcc1 elimination might give some HSC clones a competitive advantage and favor myeloid-biased differentiation, which might correspond to clonal hematopoiesis. Finally, knockout of Polβ did not affect proliferating HSPCs. Overall, this study identifies for the first time BER genes – in particular Apex1 and Lig1 – as essential BM HSPC regulators supporting hematopoietic regeneration and functional ex vivo expansion.

As the Apex1 KO resulted in a severe phenotype, not yet reported in adult hematopoietic cells, and because little is known regarding the importance of its two main catalytic domains - APEX1 nuclease (involved in BER) and the redox effector function (REF-1) (involved in transcriptional regulation) - in non-transformed primary cells, subsequent studies aimed at further delineating the role of APEX1 in HSPC biology. Using specific APEX1 domain inhibitors, we demonstrated that both the APEX1 nuclease and REF-1 activities are crucial for the support of HSPC and lineage-committed progenitor survival and proliferation. Single-cell transcriptomics of HSPCs and their progeny identified distinct transcriptional changes underlying hematopoietic defects induced by APEX1 nuclease and REF-1 inhibition. While inhibition of the APEX1 nuclease function induced an early activation of differentiation programs, inhibition of the APEX1 REF-1 function significantly downregulated interferon regulated genes (IRGs) and regulons in HSPCs. Thus, our study also indicates that a basal interferon signaling, regulated by REF-1, is required to maintain and expand in vivo repopulating HSPCs in F12-PVA-based culture.

Date:19 Apr 2017 →  27 Sep 2023
Keywords:BER
Disciplines:Genetics, Systems biology, Molecular and cell biology
Project type:PhD project