Measuring stem cell frequency in epidermis: a quantitative in vivo functional assay for long-term repopulating cells.

Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulation in vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cells in vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104 basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.

In vitro observations of mechanical heart valve cavitation.

The in vitro cavitation thresholds and locations were studied on ten different heart valve designs. The valves were mounted in a mock circulation flow loop which simulated a cardiovascular system. All the tests were run at 70 beats per minute with a cardiac output varying between 2 l/min and 6 l/min in increments of 1 l/min. In vitro cavitation phenomena generated at the closing instant of mechanical heart valves were captured using a video photographic technique. Cavitation locations and intensity on different valve designs were analyzed from the cavitation images recorded on a video tape. When cavitation occurs on a bileaflet valve, it can occur in the same localized area of the leaflet from cycle to cycle thus producing a cumulative effect. In a single disc valve, the free rotation of the valve disc during operation provides a means of distributing a localized cavitation activity over an ever changing disc surface. Thus any cavitation-induced damage on the disc surface can be reduced or eliminated even though a single disc valve may have a lower cavitation threshold. Cavitation locations and thresholds are primarily a function of valve design. Smaller size valves have higher cavitation thresholds than larger ones. The cavitation thresholds of all the valves tested were above the physiological left ventricular maximum dp/dt at rest. If in vivo cavitation occurs under some extreme conditions, this study suggests possible locations on mechanical heart valves which could be examined for traces of cavitation activity.