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With each breath, oxygen diffuses across remarkably thin alveolar type I (AT1) cells into underlying capillaries. Interspersed cuboidal AT2 cells produce surfactant and act as stem cells. Even transient disruption of this delicate barrier can promote capillary leak. Here, we selectively ablated AT1 cells, which uncovered rapid AT2 cell flattening with near-continuous barrier preservation, culminating in AT1 differentiation. Proliferation subsequently restored depleted AT2 cells in two phases, mitosis of binucleated AT2 cells followed by replication of mononucleated AT2 cells. M phase entry of binucleated and S phase entry of mononucleated cells were both triggered by AT1-produced hbEGF signaling via EGFR to Wnt-active AT2 cells. Repeated AT1 cell killing elicited exuberant AT2 proliferation, generating aberrant daughter cells that ceased surfactant function yet failed to achieve AT1 differentiation. This hyperplasia eventually resolved, yielding normal-appearing alveoli. Overall, this specialized regenerative program confers a delicate simple epithelium with functional resiliency on par with the physical durability of thicker, pseudostratified, or stratified epithelia.

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The spindle is a dynamic structure that changes its architecture and size in response to biochemical and physical cues. For example, a simple physical change, cell confinement, can trigger centrosome separation and increase spindle steady-state length at metaphase. How this occurs is not understood, and is the question we pose here. We find that metaphase and anaphase spindles elongate at the same rate when confined, suggesting that similar elongation forces can be generated independent of biochemical and spindle structural differences. Furthermore, this elongation does not require bipolar spindle architecture or dynamic microtubules. Rather, confinement increases numbers of astral microtubules laterally contacting the cortex, shifting contact geometry from "end-on" to "side-on." Astral microtubules engage cortically anchored motors along their length, as demonstrated by outward sliding and buckling after ablation-mediated release from the centrosome. We show that dynein is required for confinement-induced spindle elongation, and both chemical and physical centrosome removal demonstrate that astral microtubules are required for such spindle elongation and its maintenance. Together the data suggest that promoting lateral cortex-microtubule contacts increases dynein-mediated force generation and is sufficient to drive spindle elongation. More broadly, changes in microtubule-to-cortex contact geometry could offer a mechanism for translating changes in cell shape into dramatic intracellular remodeling.

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It is important to understand the role played by endogenous signals in shaping stem cell fate decisions to develop better culture systems and to improve understanding of development processes. In this study, we describe the behavior of mouse embryonic stem cells (mESCs) inside microfluidic chambers (microchambers) operated under conditions of minimal perfusion. mESCs inside microchambers formed colonies and expressed markers of pluripotency in the absence of feeders or pluripotency-inducing signals such as leukemia inhibitory factor (LIF), while mESCs in standard cultureware differentiated rapidly. In a series of experiments, we demonstrate that remarkable differences in stem cell phenotype are due to endogenous production of LIF and other growth factors brought upon by cultivation in confines of a microchamber in the absence of perfusion (dilution). At the protein level, mESCs produced ∼140 times more LIF inside microchambers than under standard culture conditions. In addition, we demonstrate that pluripotent phenotype of stem cells could be degraded by increasing the height (volume) of the microchamber. Furthermore, we show that inhibition of LIF in microchambers, via the JAK/STAT3 pathway, leads to preferential differentiation into mesoderm that is driven by bone morphogenetic protein (BMP)-4. Collectively, we demonstrate for the first time that it is possible to design a cell culture system where stem cell fate is controlled solely by the endogenous signals. Our study may help shift the paradigm of stem cell cultivation away from relying on expensive exogenous molecules such as growth factors and toward designing culture chambers for harnessing endogenous signals. Stem Cells 2016;34:1501-1512.

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