Optimization of the proliferation of individual cells from defined populations in vitro requires simultaneous in situ monitoring and control of the cell environment (e.g., the composition and concentration of growth factors bathing the cells). In this proposal, a novel single-cell bioreactor will be used which continuously monitors individual wells in a 96-well plate by optical microscope imaging and pattern recognition sofware to optimize the self-renewal of human CD34+Thy1+Lin- stem cells from umbilical cord blood. A robotocized liquid-dispensing system will replace one combination of growth factors with a second combination of growth factors in individual wells; the exchange of these media will be correlated to specific events in the cell cycle as recognized by the pattern recognition software. Stem cells will be directed through two cell divisions--with conservation of the pluripotent phenotype--with medium change replacing stimulatory growth factors for cell division with quiescence growth factors to hold cells between divisions. The goal is to identify the optimal conditions for self-renewal of these cells at the single-cell level. This information will be critical to generate large numbers of pluripotent stem cells ex vivo.
