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Abstract: Ustilago maydis, a Basidiomycete fungus that infects maize, exhibits two basic morphologies, a yeast-like and a filamentous form. The yeast-like cell is elongated, divides by budding, and the bud grows by tip extension. The filamentous form divides at the apical cell and grows by tip extension. The repertoire of morphologies is increased during interaction with its host, suggesting that plant signals play an important role in generation of additional morphologies. We have used Saccharomyces cerevisiae and Schizosaccharomyces pombe genes known to play a role in cell polarity and morphogenesis, and in the cytoskeleton as probes to survey the U. maydis genome. We have found that most of the yeast machinery is conserved in U. maydis, albeit the degree of similarity varies from strong to weak. The U. maydis genome contains the machinery for recognition and interpretation of the budding yeast axial and bipolar landmarks; however, genes coding for some of the landmark proteins are absent. Genes coding for cell polarity establishment, exocytosis, actin and microtubule organization, microtubule plus-end associated proteins, kinesins, and myosins are also present. Genes not present in S. cerevisiae and S. pombe include a homolog of mammalian Rac, a hybrid myosin–chitin synthase, and several kinesins that exhibit more similarity to their mammalian counterparts. We also used the U. maydis genes identified in this analysis to search other fungal and other eukaryotic genomes to identify the closest homologs. In most cases, not surprisingly, the closest homolog is among filamentous fungi, not the yeasts, and in some cases it is among mammals. [Copyright &y& Elsevier]

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

Human pluripotent stem cells (PSCs), in particular induced PSCs, are very difficult to derive, grow, and bank. They require extensive amounts of resources and time to render them useful for basic and applied research. As the derivation methods, culture systems and tissues of origin differ, so does the quality of the PSCs themselves. Consequently, there are generally accepted molecular and cellular markers that serve as benchmarks of pluripotency. PSCs undergo rigorous qualification before they can be truly considered a stem cell or completely reprogrammed into a stem-like cell as in the case of iPSCs. Morphology is a good indicator of PSCs but the further qualification of cellular markers of pluripotency and differential potential is necessary. The standard methods enclosed in this chapter delineate the techniques necessary to qualify PSCs at the cellular level.