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.

24-h Langendorff-perfused neonatal rat heart used to study the impact of adenoviral gene transfer.

The human genome project has increased the demand for simple experimental systems that allow the impact of gene manipulations to be studied under controlled ex vivo conditions. We hypothesized that, in contrast to adult hearts, neonatal hearts allow long-term perfusion and efficient gene transfer ex vivo. A Langendorff perfusion system was modified to allow perfusion for >24 h with particular emphasis on uncompromised contractile activity, sterility, online measurement of force of contraction, inotropic response to beta-adrenergic stimulation, and efficient gene transfer. The hearts were perfused with serum-free medium (DMEM + medium 199, 4 + 1) supplemented with hydrocortisone, triiodothyronine, ascorbic acid, insulin, pyruvate, l-carnitine, creatine, taurine, l-glutamine, mannitol, and antibiotics recirculating (500 ml/2 hearts) at 1 ml/min. Hearts from 2 day-old rats beat constantly at 135-155 beats/min and developed active force of 1-2 mN. During 24 h of perfusion, twitch tension increased to approximately 165% of initial values (P < 0.05), whereas the inotropic response to isoprenaline remained constant. A decrease in total protein content of 10% and histological examination indicated moderate edema, but actin and calsequestrin concentration remained unchanged and perfusion pressure remained constant at 7-11 mmHg. Perfusion with a LacZ-encoding adenovirus at 3 x 108 active virus particles yielded homogeneous transfection of approximately 80% throughout the heart and did not affect heart rate, force of contraction, or response to isoprenaline compared with uninfected controls (n = 7 each). Taken together, the 24-h Langendorff-perfused neonatal rat heart is a relatively simple, inexpensive, and robust new heart model that appears feasible as a test bed for functional genomics.

Fluid migration through the tail of the Dalkon shield intrauterine device.

X-ray radiographs were used to monitor the migration of Renouve -dip radiopaque tracer through the multifilament tail of Dalkon Shield intrauterine devices (IUDs). The movement of the tracer was more rapid and migrated a greater distance through the long end of the tail than through the short end of the tail. The double knot serves as at least a temporary barrier to the migration of tracer from the long end of the tail, but this barrier was circumvented when drops of Renouve -dip radiotracer were placed at both ends of the tail. Migration of Renouve -dip radiotracer was prevented when the end of the Dalkon Shield tail was fused by heating. No migration of this tracer occurred on the tails of commonly used IUDs which possessed monofilament tails, thus demonstrating that fluid migration occurs within and not on the surface of the multifilament tail.

PIP: X-ray radiographs were used to monitor the migration of Renouve-dip radiopaque tracer through the multifilament tail of the Daldon Shield IUD. The movement of the tracer was more rapid and migrated a greater distance through the long end of the tail than through the short end. The double knot serves as at least a temporary barrier to the migration of the tracer from the long end of the tail, but this barrier was circumvented when drops of Renouve-dip radiotracer were placed at both ends of the tail. Migration of Renouve-dip radiotracer was prevented when the end of the Dalkon Shield tail was fused by heating. No migration of this tracer occurred on the tails of commonly used IUDs which possessed monofilament tails, thus demonstrating that fluid migration occurs within and not on the surface of the multifilament tail.