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.

, , , , , ,
This thesis was financially sponsored by Amgen B.V .. Sanquin bloedvoorziening (CLB) and SanverTECH B.V.
B. Löwenberg (Bob)
Erasmus University Rotterdam
Erasmus MC: University Medical Center Rotterdam

Kusadasi, N. (2002, November 27). Ex vivo expansion of human umbilical cord blood hematopoietic stem and progenitor cells. Retrieved from