Studying global gene expression changes during hematopoietic development to identify novel regulators of hematopoietic stem cell function

Self-renewal and differentiation are the two primary attributes of all kinds of stem cells. Hematopoietic stem cells (HSCs) are multi-potent stem cells that are responsible for producing all blood cells throughout the lifetime of an organism. Hematopoietic stem cell transplantation (HSCT) is an effective method for treatment of hematopoietic disorders including leukemia and other hematopoietic malignancies, as well as immunodeficiencies. The first HSCT was performed more than 50 years ago1. Today, more than 30,000 autologous and 15,000 allogeneic transplantations are performed annually2. Bone marrow (BM), mobilized peripheral blood (MPB) and umbilical cord blood (UCB) are three major sources for HSC. Although HSC transplantation can cure a significant proportion of patients, it still has certain limitations like availability of a matched donor, graft-versus-host diseases (GvHD) and delayed engraftment post-transplantation.

The process of finding donors that have compatible histocompatibility antigens (HLA) is often a challenge. In this regard, UCB derived HSCs are considered an alternative source for HSCT, it requires less stringent HLA matching as compared with BM grafts3. One of the problems related to the use of UCB as cell source is the limited number of HSCs per graft, such that one UCB unit is insufficient to treat adult patients4-6. Some approaches could be useful to overcome the limitation such as ex vivo expansion of HSCs or improving the homing capacity of HSCs. Even though a lot of information is available on various regulators that control their function in vivo, maintenance and expansion of HSCs is still a major challenge and also the biggest hurdle in order to exploit their immense potential.

During development, HSCs appear in various transient sites before they finally migrate to the bone marrow, which is a major site of hematopoiesis. In adult BM, long-term repopulating HSCs are mostly quiescent and are in G0 stage of cell cycle, while an expansion of HSC pools occurs in the fetal liver during development. Therefore, we aim at studying HSCs and their niche during hematopoietic development.

Both intrinsic and extrinsic factors can influence the self-renewal capacity of HSCs. In order to uncover important regulators that govern self-renewal and proliferation of HSCs, we performed genome-wide transcriptome analysis of HSCs and their niche from various stages

of development using RNA-sequencing (RNA-Seq).

The cell-intrinsic regulators of HSCs were shortlisted after comparing gene expression profile of HSCs isolated from fetal liver (FL) and adult bone marrow (BM) reflecting the proliferating and quiescence state, respectively. These intrinsic regulators were further validated for their functional role in hematopoiesis using a flk1:GFP/gata1:dsRed double transgenic Zebrafish line, in which the cells from blood and endothelial lineages are fluorescently labeled. Our study identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish.

Most of the studies on the hematopoietic niche have been done using adult BM as a model system where HSCs are mostly in a quiescent state. However, the niche in the FL has been less studied than the BM niche. Here, we performed transcriptome analysis of niche cells that directly surround HSCs by using laser capture microdissection from FL of E12.5, E14.5, and E16.5. The differentially regulated genes between FL 12.5 and FL16.5 relative E14.5 were shortlisted. From these lists of potential extrinsic factors, we identified a role of VEGF-C and S100A8 in increasing HSC number by an addition of recombinant proteins to the ex vivo culture of HSCs. Although the ex vivo expansion of HSC in the presence of these factors was achieved, These HSCs had reduced in long-term repopulation potential in vivo. The discrepancy between these findings will need further experimentation, including evaluation of homing or of possible exhaustion of HSCs in response to these factors.

Lastly, we investigated the role of differentially regulated biological pathways between FL 14.5 and BM HSCs based on gene expression data obtained from RNA-seq. Interestingly, we observed that FL HSCs showed increased expression levels of genes involved in oxidative phosphorylation (OxPhos) and the tricarboxylic acid cycle  (TCA). On other hand, OxPhos pathways have been demonstrated to cause exhaustion of BM HSC; but this has not been extensively studied in fetal liver HSC. We here demonstrated that FL HSCs use the OxPhos pathway in addition to glycolysis, without apparent HSC exhaustion.

The results presented in this thesis form a foundation for further functional validation of novel transcriptional regulators, extrinsic growth factors and metabolic regulators in mammalian models of hematopoiesis, to ultimately provide novel and clinically relevant

methods that will allow ex vivo expansion of HSCs.