Publications

To exclude bacteria- or animal-derived factors from cultured fabrication of transplantable epithelial cell sheets, primary human oral mucosal epithelial cells were seeded on temperature-responsive culture inserts having submicron-scale pores. Supplying culture medium containing human autologous serum to both apical and basal sides of human epithelial cells allows these cells to grow to confluence. These proliferating cells created stratified epithelial layers even when 3T3 feeder layers and fetal bovine serum were eliminated from culture. Normal keratin expression profiles were obtained with these cells, and basal and midlayer cells expressed p63, a putative stem/progenitor marker. These results suggest that temperature-responsive culture inserts can be useful in clinical settings that require the exclusion of xenogeneic factors.

Cell-based therapies have now generated significant interest as novel drug delivery systems, with various adult cell types used in treating a wide range of diseases. To overcome the limits that restrict treatments for corneal surface dysfunction, corneal epithelial stem cells expanded ex vivo have been applied as an alternative approach. While previous studies used various carrier substrates, we present a novel method using cell sheet engineering with temperature-responsive culture dishes to create carrier-free corneal epithelial stem cell sheets that can be transplanted without sutures. Results from clinical trials reveal successful transplantation with the recovery of lost visual acuity in all cases. Cell sheet engineering, therefore, presents a novel method for the delivery of corneal epithelial stem cells, and can also be applied for other approaches of cellular therapeutics.

BACKGROUND

With recent advances in breast cancer risk reduction practices, it is increasingly important to assess both the breadth of and disparities in use across different racial/ethnic groups.

METHODS

We conducted telephone interviews with 1,700 women ages 40 to 74, from four racial/ethnic groups, without prior history of breast cancer, who received mammograms at one of five mammography facilities in San Francisco. Main outcomes measured included recognition of tamoxifen, raloxifene, genetic testing, and prophylactic surgery. Global indicators (recognition of any therapy, discussion of breast cancer risk) were developed from original outcome measures and analyzed using logistic regression.

RESULTS

Multivariate analyses indicate that race/ethnicity and interview language affected recognition of therapies and discussion of risk. White women were more likely than all other women to recognize any therapy and more likely than Asian-Americans to discuss risk. Women at high risk, who had a prior abnormal mammogram, who perceived themselves to be at high risk, or who were exposed to breast health information were more likely to discuss risk.

CONCLUSIONS

Women are aware of preventive therapies, although discussion and use is limited. Interventions to increase use of therapies should focus on those at high risk.

Current guidelines recommend that postmenopausal hormone therapy (HT) be used primarily for treatment of vasomotor and urogenital symptoms associated with the menopausal transition and that women use the lowest effective dose for the shortest time necessary. Vasomotor symptoms improve or resolve spontaneously within a few months to a few years of onset in the majority of women, suggesting that most women should be able to discontinue HT within a few years of starting treatment. Approximately 75% of women who try to stop are able to stop HT without major difficulty. However, some women who would like to stop HT are unable to do so, mainly owing to the development of vasomotor symptoms. Troublesome symptoms associated with stopping HT appear to be more common among women who start HT for treatment of symptoms, but they also are reported by women who started HT for other reasons, such as prevention of osteoporosis. Unfortunately, little information is available to guide physicians in helping women who have difficulty stopping HT. Many clinicians recommend slowly tapering HT or adding another drug for treatment of hot flashes, but the effectiveness of these approaches has not been evaluated. For women who cannot tolerate even a slow taper, the value of symptom relief likely outweighs any increased risks due to HT use.

This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.