Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin.

Abstract

SummaryA high proportion of the CD34+CD38 -cells in normal human marrow are defined as longterm culture-initiating cells (LTC-IC) because they can proliferate and differentiate when cocultured with cytokine-producing stromal feeder layers. In contrast, very few CD34+CD38 -cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC), although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38 -cells in the latter type of culture showed that Flt3-1igand (FL), Steel factor (SF), and interleukin (IL)-3 were both necessary and sufficient to obtain an N30-fold amplification of the input LTC-IC population within 10 d. As single factors, only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly, a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of >500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38 -cells. However, this required the presence oflL-6 and/or granulocyte/colonystimulating factor and/or nerve growth factor 13 in addition to FL, SF, and IL-3. Also, for this response, the most potent single-acting factor tested was IL-3, not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facihtate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols.

Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation

“…Indeed, data reported so far on LTC-IC amplification are limited to incubation periods of a few weeks. [16][17][18] In our experiments, after a 2 week-incubation, the magnitude of LTC-IC expansion did not appear to be different for several growth factor combinations. However, when the same culture conditions were prolonged, quite the opposite occurred with the LTC-IC pool, in that only a few growth factor combinations could further amplify it, while others would lead to its exhaustion.…”

“…17 The most relevant finding that emerges from our experiments is that a completely different situation is observed when the suspension cultures are extended for prolonged periods of time. The kinetics of LTC-IC expansion demonstrated that the stem cell pool could be further expanded by increasing the length of incubation: afer a 5-week incubation only some growth factor combinations could sustain a prolonged and increasingly greater expansion of LTC-IC.…”

“…FL was already known as a growth factor capable of stimulating LTC-IC maintenance 16 and amplification 17,18 from peripheral blood CD34 + Lin − and bone marrow CD34 + 38 − subpopulations, respectively. In our studies, FL by itself was capable of sustaining a 72-fold increase of CB LTC-IC number after 14 days of culture and this value was not significantly improved by the addition of the other growth factors studied.…”

“…Even then, the CFC output can be observed for some weeks and their amplification is obtained without a concomitant net expansion or even a maintenance of the stem cell pool. 14,15,17,22,23 The decline and disappearance of clonogenic precursors reflect the exhaustion of a more primitive cell population. The data reported here show a CFC production that increased week by week for over 30 weeks.…”

Regulation of FLT3 and its ligand in normal hematopoietic progenitor cells

“…Soluble forms activate the corresponding FLT3 receptor [3] Although its messenger RNA is detected in most hematopoietic and nonhematopoietic tissues, protein expression is restricted to T lymphocytes and BM stromal cells composing the hematopoietic microenvironment [15][16][17]. Generally, FL acts in combination with other cytokines, resulting in strongly synergistic effects; isolated addition of FL to stem/progenitor cell cultures has no significant effect on proliferation of myeloid and lymphoid progenitors [18][19][20][21].…”