The Caenorhabditis elegans germ line provides a model for understanding how signaling from a stem cell niche promotes continued mitotic divisions at the expense of differentiation. Here we report cellular analyses designed to identify germline stem cells within the germline mitotic region of adult hermaphrodites. Our results support several conclusions. First, all germ cells within the mitotic region are actively cycling, as visualized by bromodeoxyuridine (BrdU) labeling. No quiescent cells were found. Second, germ cells in the mitotic region lose BrdU label uniformly, either by movement of labeled cells into the meiotic region or by dilution, probably due to replication. No label-retaining cells were found in the mitotic region. Third, the distal tip cell niche extends processes that nearly encircle adjacent germ cells, a phenomenon that is likely to anchor the distal-most germ cells within the niche. Fourth, germline mitoses are not oriented reproducibly, even within the immediate confines of the niche. We propose that germ cells in the distal-most rows of the mitotic region serve as stem cells and more proximal germ cells embark on the path to differentiation. We also propose that C. elegans adult germline stem cells are maintained by proximity to the niche rather than by programmed asymmetric divisions. INTRODUCTIONStem cells are responsible for generating tissues during development and maintaining them during adulthood. To accomplish these tasks, stem cells must produce additional stem cells (self-renewal) as well as differentiated cells. Over the past few years, considerable progress has been made in the analysis of individual stem cells in several organisms, including hematopoietic stem cells (HSC) in mammals (Kiel et al., 2005;Shizuru et al., 2005) and germline stem cells (GSC) in Drosophila (Wong et al., 2005). In this article, we investigate the germline mitotic region in the Caenorhabditis elegans adult hermaphrodite, which contains GSC. Parallels between C. elegans GSC and other stem cell systems include use of Notch signaling to control both HSC and C. elegans GSC (Calvi et al., 2003;Kimble and Crittenden, 2005) and use of Puf proteins to control both Drosophila and C. elegans GSC .C. elegans adult GSC are found at the distal end of the gonadal arm within the "mitotic region," which is defined by the presence of mitotically dividing germ cells (see Figure 1, A and B). In adults, the single somatic distal tip cell (DTC) is located at the tip of the mitotic region and forms a stem cell niche (Kimble and White, 1981). The distal sheath cells are important for larval germline proliferation (Killian and Hubbard, 2005), but they have little or no contact with the mitotic region in adults (Hall et al., 1999;Killian and Hubbard, 2005). The DTC and the mitotic germline cells are encapsulated by a thin extracellular matrix, which separates them from neighboring organs (e.g., intestine; Hall et al., 1999;Lints and Hall, 2004). Proximal to the mitotic region, the "transition zone" contains germ cells in early...

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Remarkable interest in the epigenetic status of human induced pluripotent stem (iPS) cells inspired numerous studies of their X-inactivation patterns. However, both the presence and the absence of X-inactivation have been described to date in undifferentiated iPS cells. The reasons for the discordant results between different studies are unclear, and further X-inactivation testing is warranted for all female human iPS cell lines. Some of the inconsistency in the current data most likely results from the use of different X-inactivation assays by different authors. We provide a detailed protocol for a simple, reliable and affordable X-inactivation assay based on promoter methylation and CAG-repeat polymorphism in the human androgen receptor (AR) gene at Xq11.2. This assay is commonly used in clinical genetic laboratories and we propose that it could be ideal for routine assessment and monitoring of the X-inactivation status in female human iPS cell lines.

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Pluripotent stem cell (PSC) cultures are subjected to selective pressures that can result in acquisition and expansion of recurrent genetic abnormalities at any time. These recurrent abnormalities enhance the variant cells harboring them with a competitive advantage over wild‐type cells. Variant cells can eventually supplant wild‐type cells entirely and become fixed in culture. Such variants can impact the efficacy of PSCs in research and clinical applications. Therefore, routine genomic characterization is required for reliable and effective use of PSCs. In this article we describe the capabilities and limitations of several assays commonly used for assessing PSC genomic stability. Based on this analysis, we provide a recommendation for integrating assays into a comprehensive testing regimen that maximizes coverage while minimizing cost. © 2020 by John Wiley & Sons, Inc.