| Summary: | The increasing appreciation of the enormous developmental potential of stem cells has
raised hopes that one day they can be manipulated in vitro to generate desired cell
populations for clinical applications. To provide a quantitative understanding of cytokine
effects that affect the maintenance of hematopoietic stem cell (HSC) function in vitro,
changes in HSC numbers in serum-free cultures were evaluated with a modified
competitive repopulation unit (CRU) assay based on the level of donor-derived
engraftment. A factorial design was used to screen the effects of thrombopoietin, flt-3
ligand (FL), Steel factor (SF), interleukin (IL)-11, incubation temperature and
hydrocortisone in cultures of Sca-1⁺lin⁻ mouse bone marrow. The results indicated that
IL-11 and SF were the most important stimulators of CRU expansion. When response
surface methodology was used to analyze cytokine effects, IL-11 was found to have a
bell-shaped dose-response with a maximum effect at 20 ng/mL. High concentration
saturation or decrease in the effect of SF or FL was not observed and their stimulatory
effect increased continuously, beyond 300 ng/mL. A negative interaction between SF and
FL was also discovered that resulted in a significant inhibitory effect when both were
added to cultures at high concentrations (>100 ng/mL). When the response of CRUs was
compared with colony-forming cells and total cells, a surprisingly similar pattern of
cytokine effects was found. However, the populations differed dramatically in their
responsiveness to cytokine doses such that the maintenance of CRU numbers in vitro
required > 50-fold greater concentrations. The observed pattern of cytokine effects guided
the development of a mathematical model of cell expansion based on Monod kinetics.
The HSC proliferation and self-renewal response requirements for gpl30 activation were
also examined in single-cell cultures. HSC survival and mitogenesis were maximized by
exposure to SF and FL, but optimal retention of their functional capacity required the
additional presence of a cytokine that will activate gpl30. Taken together, these results
have demonstrated that HSCs mitogenesis and self-renewal can be independently
manipulated by the activation of different cell surface cytokine receptors, with additional
differential effects dictated by the intensity of signaling.
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