Michael C. Winter

Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

In this study, we describe a simple system in which human keratinocytes can be redirected to an alternative differentiation pathway. We transiently transfected freshly isolated human skin keratinocytes with the single transcription factor OCT4. Within 2 days these cells displayed expression of endogenous embryonic genes and showed reduced genomic methylation. More importantly, these cells could be specifically converted into neuronal and contractile mesenchymal cell types. Redirected differentiation was confirmed by expression of neuronal and mesenchymal cell mRNA and protein, and through a functional assay in which the newly differentiated mesenchymal cells contracted collagen gels as efficiently as authentic myofibroblasts. Thus, to generate patient-specific cells for therapeutic purposes, it may not be necessary to completely reprogram somatic cells into induced pluripotent stem cells before altering their differentiation and grafting them into new tissues.

Paracellular Permeability Restricts Airway Epithelial Responses to Selectively Allow Activation by Mediators at the Basolateral Surface

Respiratory pathogens and toxins often assault the lung from the airway lumen. Airway epithelia may initiate and amplify inflammation in response to these attacks, but under certain conditions confinement of inflammation to the airway lumen may be beneficial to the host. Accordingly, we hypothesized that airway epithelial polarity allows different responses to basolateral vs apical stimuli that may modulate inflammation. Using primary human airway epithelial cells differentiated at an air-liquid interface in culture, we found that responses to several cytokines required basolateral mediator application. In contrast, responses to Haemophilus influenzae occurred after either basolateral or apical interaction with airway epithelia. Experiments focused on IFN-γ receptor polarity confirmed its predominant basolateral location in cultured airway epithelia as well as in normal human airway tissue. Furthermore, physical and pharmacologic disruption of barrier function in airway epithelia allowed responses to apical application of IFN-γ and other cytokines. These in vitro studies directly correlated with experiments in mice in which an airway epithelial response to IFN-γ injected into the airway lumen was seen only after disruption of barrier function. The results indicate that airway epithelia with intact barrier function restrict inflammatory responses by limitation of cell activation through requiring interaction of selected mediators with the basolateral surface. However, loss of barrier integrity allows epithelial responses to these mediators if located in the airway lumen to amplify airway defenses.

Histamine alters cadherin-mediated sites of endothelial adhesion

We tested the hypothesis that histamine alters the focal apposition of endothelial cells by acting on sites of cadherin-mediated cell-cell adhesion. Focal apposition was measured as the impedance of a cell-covered electrode, which was partitioned into a cell-matrix resistance, a cell-cell resistance, and membrane capacitance. Histamine causes an immediate, short-lived decrease in the impedance of an electrode covered with human umbilical vein endothelial (HUVE) cells. ECV304 cells are a line of spontaneously transformed HUVE cells that do not express the endothelial cadherin, cadherin-5. Histamine increased ECV304 cell calcium to 600 nM. Histamine did not increase myosin light chain phosphorylation of control or transfected ECV304 cells. ECV304 cells transfected with either E-cadherin or cadherin-5 on a dexamethasone-responsive plasmid (pLKneo) increased their cell-cell resistance when stimulated with dexamethasone, whereas ECV304 cells transfected with pLKneo-lacZ did not. Histamine did not affect the impedance of ECV304 cells transfected with pLKneo-lacZ. In contrast, histamine decreased the cell-cell resistance of ECV304 cells transfected with either pLKneo-E-cadherin or pLKneo-cadherin-5. From these data, we conclude that histamine acts on sites of cadherin-mediated cell-cell apposition.We tested the hypothesis that histamine alters the focal apposition of endothelial cells by acting on sites of cadherin-mediated cell-cell adhesion. Focal apposition was measured as the impedance of a cell-covered electrode, which was partitioned into a cell-matrix resistance, a cell-cell resistance, and membrane capacitance. Histamine causes an immediate, short-lived decrease in the impedance of an electrode covered with human umbilical vein endothelial (HUVE) cells. ECV304 cells are a line of spontaneously transformed HUVE cells that do not express the endothelial cadherin, cadherin-5. Histamine increased ECV304 cell calcium to 600 nM. Histamine did not increase myosin light chain phosphorylation of control or transfected ECV304 cells. ECV304 cells transfected with either E-cadherin or cadherin-5 on a dexamethasone-responsive plasmid (pLKneo) increased their cell-cell resistance when stimulated with dexamethasone, whereas ECV304 cells transfected with pLKneo-lacZ did not. Histamine did not affect the impedance of ECV304 cells transfected with pLKneo-lacZ. In contrast, histamine decreased the cell-cell resistance of ECV304 cells transfected with either pLKneo-E-cadherin or pLKneo-cadherin-5. From these data, we conclude that histamine acts on sites of cadherin-mediated cell-cell apposition.

CFTR Influences Beta Cell Function and Insulin Secretion Through Non-Cell Autonomous Exocrine-Derived Factors

&NA; Although &bgr;‐cell dysfunction in cystic fibrosis (CF) leads to diabetes, the mechanism by which the cystic fibrosis transmembrane conductance regulator (CFTR) channel influences islet insulin secretion remains debated. We investigated the CFTR‐dependent islet‐autonomous mechanisms affecting insulin secretion by using islets isolated from CFTR knockout ferrets. Total insulin content was lower in CF as compared with wild‐type (WT) islets. Furthermore, glucose‐stimulated insulin secretion (GSIS) was impaired in perifused neonatal CF islets, with reduced first, second, and amplifying phase secretion. Interestingly, CF islets compensated for reduced insulin content under static low‐glucose conditions by secreting a larger fraction of islet insulin than WT islets, probably because of elevated SLC2A1 transcripts, increased basal inhibition of adenosine triphosphate‐sensitive potassium channels (K‐ATP), and elevated basal intracellular Ca2+. Interleukin (IL)‐6 secretion by CF islets was higher relative to WT, and IL‐6 treatment of WT ferret islets produced a CF‐like phenotype with reduced islet insulin content and elevated percentage insulin secretion in low glucose. CF islets exhibited altered expression of INS, CELA3B, and several &bgr;‐cell maturation and proliferation genes. Pharmacologic inhibition of CFTR reduced GSIS by WT ferret and human islets but similarly reduced insulin secretion and intracellular Ca2+ in CFTR knockout ferret islets, indicating that the mechanism of action is not through CFTR. Single‐molecule fluorescent in situ hybridization, on isolated ferret and human islets and ferret pancreas, demonstrated that CFTR RNA colocalized within KRT7+ ductal cells but not endocrine cells. These results suggest that CFTR affects &bgr;‐cell function via a paracrine mechanism involving proinflammatory factors secreted from islet‐associated exocrine‐derived cell types.

Treatment with the cancer drugs decitabine and doxorubicin induces human skin keratinocytes to express Oct4 and the OCT4 regulator mir-145

Previously, we showed that transient transfection with OCT4 not only produced high expression of Oct4 in skin keratinocytes, but also caused a generalized demethylation of keratinocyte DNA. We hypothesized that DNA demethylation alone might allow expression of endogenous OCT4. Here, we report that treatment with the cancer drug decitabine results in generalized DNA demethylation in skin keratinocytes, and by 48 h after treatment, 96% of keratinocytes show expression of the endogenous Oct4 protein and the OCT4 repressor mir‐145. This is true for keratinocytes only, as skin fibroblasts treated similarly show no OCT4 or mir‐145 expression. Decitabine‐treated keratinocytes also show increased mir‐302c and proliferation similar to other Oct4+ cells. Treatment with doxorubicin, another cancer drug, induces expression of mir‐145 only in cells that already express OCT4, suggesting that Oct4 regulates its own repressor. Co‐treatment with decitabine and doxorubicin results first in increased OCT4 and mir‐145, then a decrease in both, suggesting that OCT4 and mir‐145 regulate each other. The novel strategy presented here provides a regulatable system to produce Oct4+ cells for transformation studies and provides a unique method to study the effects of endogenous Oct4 in cancer cells and the surrounding somatic cells.

The indirect effect of Tityus discrepans on rabbit pulmonary vasculature.

Serum (IS) was obtained 0.5, 2, 4 or 6 h after inoculating s.c. six rabbits (approximately 2 kg) in each time period with 1 mg/kg of Tityus discrepans (Td) venom; the control was serum obtained from four rabbits 4 h after injecting them 1 ml s.c. of 0.9% NaCl. IS produced a transient (<25 min) rise in pulmonary artery pressure of isolated and perfused rabbit lungs, other lung parameters were not altered. We found that both scorpion venom and IS produced a approximately 50% transient increase of transendothelial electric resistance in cultured tissue human umbilical cord vein. Neither venom nor IS changed the transepithelial electrical resistance of tissue cultured human airway epithelia. The experiments suggest that humoral factors contained in the inoculated serum modify vascular endothelium in a much more effective manner than the venom by itself. These experiments also make it unlikely that vascular endothelium is the source of the humoral factors contained in inflammatory serum.

Aging Epidermis Is Maintained by Changes in Transit-Amplifying Cell Kinetics, Not Stem Cell Kinetics

It has been assumed that the slow rate of healing in aging epidermis is due to slowing of the epidermal stem cell proliferative rate. In this issue, Charruyer et al. report that this may not be the case. Using a long-term repopulating model, they demonstrate that epidermal stem cell kinetics are maintained. Instead, it is the compensatory action of the transit-amplifying (TA) cells that changes in aging skin and thus bears responsibility for slowed wound healing.

Human Skin Keratinocytes Can Be Reprogrammed to Express Neuronal Genes and Proteins After a Single Treatment with Decitabine

Abstract Patient-specific cell replacement therapy is fast becoming the future of medicine, requiring safe, effective methods for reprogramming a patients own cells. Previously, we showed that a single transient transfection with a plasmid encoding Oct4 was sufficient to reprogram human skin keratinocytes (HSKs), and that this transfection resulted in a decrease in global DNA methylation. In more recent work we showed that decreasing global DNA methylation using the U.S. Food and Drug Administration–approved cancer treatment drug decitabine was sufficient to induce expression of endogenous Oct4. Here we report that a single treatment with decitabine, followed by 5 days in a defined neuronal transformation medium, then 7 days in a neuronal maintenance medium is sufficient to convert HSKs into cells that change their morphology substantially, gain expression of neuronal markers, and lose expression of keratinocyte markers. Within 1 week of treatment the cells express mRNA for β3-tubulin and doublecortin, and at the end of 2 weeks express mRNA for NeuN, FOXP2, and NCAM1. Additionally, at the end of this protocol, neurofilament-1, nestin, synapsin, FOXP2, and GluR1 proteins are detectable by immunostaining. Thus, we demonstrate a simple method that begins the process for producing cells for cell replacement therapies without using exogenously introduced DNA.