http://repub.eur.nl/res/aut/9063/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/32052/ 2002-11-27T00:00:00Z The goal of this thesis research is to establish ex vivo expansion conditions for HSCs derived from UCB. To realize the expansion of HSCs, CD34+ or ACJ33+ UCB cells were cultured in the absence or presence of various cocktails of early acting cytokines including Flt3-L, Tpo, SCF or IL6 under stroma-free or stroma-supported conditions. The HSC and progenitor expansion was assessed using in vitro and in vivo long-term repopulating cells. First, the experiments were designed to test whether HSC expansion would alter the in vivo long-term engraftment potential of CD34+ UCB cells in the presence of EMderived stromal cells during two weeks. Also the cytokines required for expansion of HSCs and progenitors in either the presence or absence of stroma have been evaluated. The experiments described in chapter 3 are closely linked to the work described in the previous chapter. They were designed to investigate whether HSC expansion could be improved when cultured for more than two weeks, and whether the presence of EMderived stromal cells, and combinations of specific cytokines could affect the HSC and progenitor maintenance or expansion. In chapter 4 the effect of a new fusion protein ofiL6 and the soluble IL6R, H-IL6, has been evaluated on the long-term ex vivo expansion ofHCSs derived from AC133+ UCB cells. To do this, we used stroma-free and stroma-supported LTCs and compared several cytokine combinations in the presence or absence of this chimeric protein, or IL6, and estimated the HSC and progenitor output by multiparameter FACS analysis and CAFC assays. Following these experiments, nineteen newly established murine embryonic stromal clones have been investigated for their ability to sustain human HSCs and progenitors in extended LTCs of CD34+ UCB cells in the absence or presence of the cytokines Flt3- L and Tpo for periods as long as twelve weeks. A significant proportion of HSC and progenitor subsets was found in the non-adherent compartment of these co-cultures. With an interest to elucidate the factors that determine the proportion of adherent and non-adherent compartments, we evaluated in chapter 6 the chemoattractive activity of different stromal cells and the effect of exogenously added cytokines herein. http://repub.eur.nl/res/pub/8153/ 2002-01-01T00:00:00Z The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSC-supportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs. http://repub.eur.nl/res/pub/8365/ 2002-01-01T00:00:00Z Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers, isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage.