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The generation of functional gametes, both eggs and sperm, from murine pluripotent stem cell (PSC) sources, has set the stage for the eventual use of this emerging technology in other species. With the field enthusiastically embracing this eventuality, in particular for animal conservation efforts, there are a number of key factors to consider regarding the applicability of these methods across species, particularly with regard to the generation of eggs. To date, published studies point to the need for fetal somatic tissue and primitive granulosa cells to serve as a niche for the growth and maturation of oocytes generated from PSCs. In practice, the need for such tissue represents a major limitation when attempting to apply this to species in which access to fetal ovaries is limited or unethical. To circumvent this, we and others have derived methods to generate ovarian granulosa cells from PSCs, albeit with low yield. Herein we present an update on the status of generating early stage granulosa cells from PSCs, and provide evidence for improvements based on a stepwise, 2-dimensional protocol for the directed differentiation of human PSCs. 

Abstract Mitochondria are well-characterized regarding their function in both energy production and regulation of cell death; however, the heterogeneity that exists within mitochondrial populations is poorly understood. Typically analyzed as pooled samples comprised of millions of individual mitochondria, there is little information regarding potentially different functionality across subpopulations of mitochondria. Herein we present a new methodology to analyze mitochondria as individual components of a complex and heterogeneous network, using a nanoscale and multi–parametric flow cytometry-based platform. We validate the platform using multiple downstream assays, including electron microscopy, ATP generation, quantitative mass-spectrometry proteomic profiling, and mtDNA analysis at the level of single organelles. These strategies allow robust analysis and isolation of mitochondrial subpopulations to more broadly elucidate the underlying complexities of mitochondria as these organelles function collectively within a cell.