Quiescence regulation by normal haematopoietic stem cells and leukaemia stem cells.

The haematopoietic system is maintained by rare haematopoietic stem cells (HSCs), which are quiescent most of the time and only divide occasionally to self-renew and/or to undergo commitment to clonal expansion via the generation of highly proliferative progenitor cells. The latter is responsible for the generation of all mature cells of the system through subsequent lineage commitment and terminal differentiation. Cells with similar properties also exist in leukaemias and are known as leukaemia stem cells (LSCs). Quiescence provides essential protection for both HSC and LSC from cytotoxic stress and DNA damage and, in the case of LSCs, likely causes therapy resistance and disease relapse in leukaemia patients. Specific inhibition of LSC quiescence has been considered a promising strategy for eliminating LSCs and curing leukaemias. Although the understanding of mechanisms responsible for quiescence maintenance in these cells remains limited, particularly for LSCs, recent studies have suggested potential differences in their dependency on certain pathways and their levels of stress and DNA damage caused by increased cycling. Such differences likely stem from oncogenic mutations in LSCs and could be specifically exploited for the elimination of LSCs while sparing normal HSCs in the future.

Gene Inactivation in Adult Long-Term Hematopoietic Stem Cells Using Inducible Mouse Models.

The availability of mouse models that allow inducible long-term hematopoietic stem cell (LT-HSC)-specific gene deletion in adult mice has been limited. Therefore, analysis of gene function in adult LT-HSCs has mostly relied on models such as the interferon inducible Mx1-Cre model. Unfortunately, the Mx1-Cre strain has significant drawbacks due to lack of specificity towards the hematopoietic system, adverse effects of interferon induction on the interpretation of data, and Cre expression leakage. In this chapter, we will describe the use of other inducible models, the tamoxifen-inducible Cre-ERT and Cre-ERT2 strains, and how these mice can be used to study gene function in LT-HSC.

Analyzing gene function in adult long-term hematopoietic stem cells using the interferon inducible Mx1-Cre mouse system.

Long-term hematopoietic stem cells (LT-HSCs) have the ability to self-renew and differentiate into all blood cell lineages. Understanding the genetic networks that regulate LT-HSC function in the adult bone marrow requires inducible gene targeting and bone marrow transplantations. In this chapter we describe the use of the inducible Mx1-Cre mouse model to delete genes in LT-HSCs and methodologies for examining the function of LT-HSCs following deletion.

Transplantation of mouse fetal liver cells for analyzing the function of hematopoietic stem and progenitor cells.

Hematopoietic stem cells are defined by their ability to self-renew and differentiate through progenitor cell stages into all types of mature blood cells. Gene-targeting studies in mice have demonstrated that many genes are essential for the generation and function of hematopoietic stem and progenitor cells. For definitively analyzing the function of these cells, transplantation studies have to be performed. In this chapter, we describe methods to isolate and transplant fetal liver cells as well as how to analyze donor cell reconstitution. This protocol is tailored toward mouse models where embryonic lethality precludes analysis of adult hematopoiesis or where it is suspected that the function of fetal liver hematopoietic stem and progenitor cells is compromised.

RapGEF2 is essential for embryonic hematopoiesis but dispensable for adult hematopoiesis.

RapGEF2 is one of many guanine nucleotide exchange factors (GEFs) that specifically activate Rap1. Here, we generated RapGEF2 conditional knockout mice and studied its role in embryogenesis and fetal as well as adult hematopoietic stem cell (HSC) regulation. RapGEF2 deficiency led to embryonic lethality at ~ E11.5 due to severe yolk sac vascular defects. However, a similar number of Flk1(+) cells were present in RapGEF2(+/+) and RapGEF2(-/-) yolk sacs indicating that the bipotential early progenitors were in fact generated in the absence of RapGEF2. Further analysis of yolk sacs and embryos revealed a significant reduction of CD41 expressing cells in RapGEF2(-/-) genotype, suggesting a defect in the maintenance of definitive hematopoiesis. RapGEF2(-/-) cells displayed defects in proliferation and migration, and the in vitro colony formation ability of hematopoietic progenitors was also impaired. At the molecular level, Rap1 activation was impaired in RapGEF2(-/-) cells that in turn lead to defective B-raf/ERK signaling. Scl/Gata transcription factor expression was significantly reduced, indicating that the defects observed in RapGEF2(-/-) cells could be mediated through Scl/Gata deregulation. Inducible deletion of RapGEF2 during late embryogenesis in RapGEF2(cko/cko)ER(cre) mice leads to defective fetal liver erythropoiesis. Conversely, inducible deletion in the adult bone marrow, or specific deletion in B cells, T cells, HSCs, and endothelial cells has no impact on hematopoiesis.

Gene expression analysis of hematopoietic progenitor cells identifies Dlg7 as a potential stem cell gene.

Inducible hematopoietic stem/progenitor cell lines represent a model for studying genes involved in self-renewal and differentiation. Here, gene expression was studied in the inducible human CD34+ acute myelogenous leukemia cell line KG1 using oligonucleotide arrays and suppression subtractive cloning. Using this approach, we identified Dlg7, the homolog of the Drosophila Dlg1 tumor suppressor gene, as downregulated at the early stages of KG1 differentiation. Similarly, Dlg7 was expressed in normal purified umbilical cord blood CD34+CD38- progenitors but not in the more committed CD34+CD38+ population. Dlg7 expression was not detected in differentiated cells obtained from hematopoietic colonies, nor was expression detected in purified T-cells, B-cells, and monocytes. When analyzed in different types of stem cells, Dlg7 expression was detected in purified human bone marrow-derived CD133+ progenitor cells, human mesenchymal stem cells, and mouse embryonic stem (ES) cells. Overexpression of Dlg7 in mouse ES cells increased their growth rate and reduced the number of EBs emerging upon differentiation. In addition, the EBs were significantly smaller, indicating an inhibition in differentiation. This inhibition was further supported by higher expression of Bmp4, Oct4, Rex1, and Nanog in EBs overexpressing Dlg7 and lower expression of Brachyury. Finally, the Dlg7 protein was detected in liver and colon carcinoma tumors but not in normal adjacent tissues, suggesting a role for the gene in carcinogenesis. In conclusion, our results suggest that Dlg7 has a role in stem cell survival, in maintaining stem cell properties, and in carcinogenesis. Disclosure of potential conflicts of interest is found at the end of this article.

Flt3/Flk-2 ligand in combination with thrombopoietin decreases apoptosis in megakaryocyte development.

The growth factors thrombopoietin (TPO) and Flt3/Flk-2-ligand (FL), either independently or in combination, modulate megakaryocyte development. Our results show that bone marrow CD34+ cells cultured with TPO and FL differentiate at a slower rate into CD41+ cells and are delayed in apoptosis at the later stages of the cultures compared to cells cultured with TPO alone. Our data also show that FL in synergy with TPO may inhibit apoptosis in megakaryocyte development by up-regulating bcl-2 and inducing conformational changes of p53, in MK progenitors. FL in combination with TPO slows down maturation and consequently delays apoptosis of MK progenitor cells.

Flt3/Flk-2-ligand in synergy with thrombopoietin delays megakaryocyte development and increases the numbers of megakaryocyte progenitor cells in serum-free cultures initiated with CD34+ cells.

Megakaryocytopoiesis involves proliferation and maturation of committed precursors that increase their size by polyploidy, a process that is believed to be critical for the efficient production and release of platelets. Thrombopoietin has been shown to act on proliferation, maturation, and survival pathways in megakaryocytopoiesis. Less is known about the role of Flt3/Flk-2-ligand in this development. Apoptosis has an important role in hematopoiesis in general. It has been shown to have an effect on senescent megakaryocytes but not megakaryocyte progenitor cells. In this study, a serum-free culture model was developed, differentiating bone marrow CD34(+) hematopoietic stem cells into megakaryocytes, using thrombopoietin and Flt3/Flk-2-ligand. The model was used to study the effect of these growth factors on expansion of megakaryocyte progenitor cells, differentiation of megakaryocytes, and ploidy. Our results demonstrate that bone marrow CD34(+) cells cultured with thrombopoietin and Flt3/Flk-2-ligand show a lower developmental rate into MK cells compared to cells cultured with thrombopoietin alone. Cells cultured with thrombopoietin and Flt3/Flk-2-ligand expressed less CD41, the ploidy level was lower, and they appeared less mature. On the other hand, the cells showed up to 10-fold increase in cell numbers compared to five-fold increase when cultured with thrombopoietin alone. These results suggest that Flt3/Flk-2-ligand in synergy with thrombopoietin may slow down megakaryocyte development by causing increased proliferation of megakaryocyte progenitor cells.