The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-␤ pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC-and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:83-95

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An aspiration system, incorporating a regulated vacuum pump, was used to examine, in vitro, some factors that may affect oocyte collection. In an open aspiration system, as the length of the needle was increased, or the internal diameter decreased, the velocity (and flow rate) of aspirated fluid decreased. There was a difference, however, between experimental flows and those predicted by Hagen-Poiseuille's Law. Upon application of vacuum to a closed aspiration system, employing isolated bovine ovaries, there was an initial rapid increase in the collection tube vacuum to 85% of the selected pump vacuum followed by a more gradual rise to 100%. The vacuum within the needle similarly rose rapidly to approximately half the selected vacuum, while the vacuum at the needle tip was approximately 5% of selected vacuum. The vacuums throughout the system briefly equilibrated as maximum flow/velocity was reached. Flow/velocity slowed dramatically as the follicle collapsed, and stopped as the needle tip was blocked. If vacuum was maintained during the withdrawal of the needle from the follicle, there was a dramatic forward flow of fluid toward the collection tube. The morphological appearance of bovine cumulus after in-vitro aspiration was generally unaltered by vacuums commonly utilized in oocyte collection, providing the cumulus was regular, compact and refractile. The cumulus was less resistant to aspiration if it was damaged or had degenerated. These results suggest that an intact cumulus may offer protection during oocyte collection.

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