Stem Cell Engineering Center Seminar Series
Controlling the expansion and differentiation of stem and progenitor cells in vivo and in vitro is an important goal in regenerative medicine and for the development of patient-based, disease-relevant cellular models. This usually achieved by exposing stem and progenitor cells to specific types and doses of cytokines, growth factors and extracellular matrix proteins. This can also be achieved by directly modulating the activity of signaling enzymes or transcription factors using genetic engineering strategies or by delivering bioactive molecules in the cell cytosol or nucleus. In this seminar, I will first describe our investigation of the mechanisms responsible for the combinatorial cytokine dependence of stem/progenitor cell fate decisions. During the development of embryonic and adult tissues, there is stage-specific, sequential expression of cytokine receptors during differentiation. We hypothesized that combinatorial cytokine dependence arises in cell subsets in unique transitional stages where cell fate decision can be regulated by combinations of early- and late-acting factors. We used adult bone marrow erythropoiesis (red blood cell development) as a model system since it depends on the sequential action of stem cell factor (SCF), an early-acting cytokine and erythropoietin (EPO), a late-acting cytokine. We have identified a subset of hematopoietic progenitor cells which fate decisions depend on both the SCF and EPO signals. The integration of the EPO and SCF signals was found to take place in the MAPK pathway and was found depend on a molecular mechanism that controls the dynamics of the ERK signal. Next, I will describe some work on the differentiation of neural precursor cells isolated from human olfactory epithelium (OE). These neural precursor cells can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. We examined the impact of changes in glycogen synthase kinase-3 (GSK-3) activity on the fate of adult human OE neural precursors in vitro. In these experiments, ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) and substrate-competitive (TAT-eIF2B) inhibitors were used in order to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. The results indicated that GSK-3 inhibition promotes the early stages of neuronal differentiation and decrease proliferation in cultures of adult human neural precursors. This finding provides insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders. The different investigations described above both required the use of tools to examine how signal transduction operates in single live cells. We mostly used multicolor flow cytometry in these studies but we have also explored several applications for single-cell capillary electrophoresis (CE). Therefore, in the last part of the seminar, I will explain how single-cell CE and mass spectrometry (MS) can be combined to examine the fate of enzyme activity reporters and inhibitors in single mammalian cells with a resolution at the single amino acid level.