John O'neill

Efficient generation of lung and airway epithelial cells from human pluripotent stem cells

The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP—agonists to signaling pathways critical for early lung development in the mouse—recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.

The regulation of growth and metabolism of kidney stem cells with regional specificity using extracellular matrix derived from kidney

Native extracellular matrix (ECM) that is secreted and maintained by resident cells is of great interest for cell culture and cell delivery. We hypothesized that specialized bioengineered niches for stem cells can be established using ECM-derived scaffolding materials. Kidney was selected as a model system because of the high regional diversification of renal tissue matrix. By preparing the ECM from three specialized regions of the kidney (cortex, medulla, and papilla; whole kidney, heart, and bladder as controls) in three forms: (i) intact sheets of decellularized ECM, (ii) ECM hydrogels, and (iii) solubilized ECM, we investigated how the structure and composition of ECM affect the function of kidney stem cells (with mesenchymal stem cells, MSCs, as controls). All three forms of the ECM regulated KSC function, with differential structural and compositional effects. KSCs cultured on papilla ECM consistently displayed lower proliferation, higher metabolic activity, and differences in cell morphology, alignment, and structure formation as compared to KSCs on cortex and medulla ECM, effects not observed in corresponding MSC cultures. These data suggest that tissue- and region-specific ECM can provide an effective substrate for in vitro studies of therapeutic stem cells.

Rapid retraction of microvolume aqueous plugs traveling in a wettable capillary.

We report a transport behavior-specifically, rapid retraction movement-of small (∼μL) deionized water plugs traveling in series within a small wettable tubular geometry. In this study, two water plugs separated by a certain distance in a dry cylindrical glass capillary were moved by positive pressure airflow applied at the tube inlet. As the plugs travel, a thin aqueous film is generated between the plugs as a result of the leading plugs aqueous deposition onto the inner surface of the tube. The leading plug continuously loses volume by film deposition onto the surface and eventually ruptures. Then, the lagging plug quickly travels the distance initially separating the two plugs (plug retraction). Our studies show that the rapid retraction of the lagging plug is caused by surface tension in addition to the positive pressure applied. Furthermore, the plug retraction speed is strongly affected by tube radius and the distance between the plugs.