3D-model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress.

The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell-replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three-dimensional (3D) cell aggregates ("cardiospheres") by putative stem cells isolated from adult dog myocardium using poly-L-ornithine. De novo formation of cardiospheres in growth factor-containing medium occurred over a period of 2-3 weeks, but accelerated to 2-3 days when seeded on poly-L-ornithine. Older cardiospheres developed foci of "beating" cells upon co-culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming "vessel-like" networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post-transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart.

A toxicity index of skin and wound cleansers used on in vitro fibroblasts and keratinocytes.

OBJECTIVE: To determine toxicity indexes of commercially available skin, wound, and skin/wound cleansers on in vitro fibroblasts and keratinocytes. DESIGN: Seventeen cleansers and 3 liquid bath soaps were evaluated for cytotoxic effect on human infant dermal fibroblasts and epidermal keratinocytes. Both skin cell types were exposed to serial 10-fold dilutions of each cleanser until treated cell viability was comparable to untreated controls. RESULTS: The experimental design allowed calculation of relative toxicity indexes ranging from 0 to 100,000. Shur-Clens, SAF-Clens, and saline were found to be the least toxic to fibroblasts (toxicity index 0); Dial Antibacterial Soap and Ivory Liqui-Gel were the most toxic (toxicity index 100,000). Biolex, Shur-Clens, and Techni-Care were the least toxic to keratinocytes (toxicity index 0); hydrogen peroxide, modified Dakin's solution, and povidone (10%) were found to be the most toxic (toxicity index 100,000). CONCLUSIONS: Successful cutaneous tissue repair depends on the viability of the principal cell types involved (fibroblasts and keratinocytes). Toxicity indexes provide helpful guidelines for subsequent in vivo evaluations and clinical applications. The study findings also suggest that judicious use of these supposedly innocuous agents should be considered in a clinical setting.

A new strategy for analysis of phenotype marker antigens in hollow neurospheres.

Changes in expression of phenotypic markers characterizing the cells comprising intact neurospheres are difficult to determine easily and accurately. The problem is compounded by the diversity of cell populations and phenotypes involved, and consequent non-uniform protein expression across the neurosphere wall, or around the circumference. Therefore, interpreting the effects of phenotype modifying conditions has been a complex and demanding task. Here, we report a novel direct method for measuring protein expression in immunofluoerescently labeled intact neurospheres by densitometric image analysis of optical cross-sections, obtained by scanning laser confocal microscopy. To demonstrate our methodology, hollow human neurospheres were exposed to basic fibroblast growth factor (FGF2), which reportedly induces neuronal commitment in monolayer cultures of neuroprogenitor cells derived from neurospheres. We determined that this treatment downregulated nestin and vimentin, protein markers accepted to indicate an immature, uncommitted phenotype. Neuron specific enolase was only marginally affected. Our strategy allows quantitation of changes in expression of marker proteins that is comparable to Western blot analysis. In addition to discriminating heterogeneity in protein expression, suitable optics may allow the resolution down to single cell level. We propose that this novel strategy, with or without confocal microscopy, may be applied to other biological systems. Analysis of protein expression by the cells comprising tubular or cylindrical cellular structures, or approximately spherical cell aggregates, can be performed efficiently using a small sample size.