Elfren Ray Baclagon

Frizzled 7 Maintains the Undifferentiated State of Human Limbal Stem/Progenitor Cells

Wnt signaling pathway plays an important role in the regulation of human limbal stem/progenitor cells (LSCs). To examine the possible function of Frizzled (Fz) receptors in LSCs, the expression of 10 Fz receptors was profiled in the limbus and cornea. Only Fz7 had preferential expression in the basal limbal epithelium which contains the LSCs. The expression of Fz7 was colocalized with the putative LSC markers including p63α, N‐cadherin and keratin (K) 14, and was minimum in cells expressing the corneal maturation marker K12. The expression of Fz7 was higher in the enriched LSCs population and decreased in cultured LSCs when there was a loss of progenitor phenotype. When the Fz7 was knocked down (FzKD) using shRNA in primary LSCs, the expression of putative LSC markers ABCG2, ΔNp63α, and K14 was decreased significantly. The colony forming efficiency of the Fz7KD LSCs was significantly decreased in the subsequent passage 1 and 2 compared to the control. Our finding suggests that Wnt signaling is one of the factors of LSC niche, and Fz7 helps to maintain the undifferentiated state of LSCs. Stem Cells 2014;32:938–945

A 3D culture system enhances the ability of human bone marrow stromal cells to support the growth of limbal stem/progenitor cells.

The standard method of cultivating limbal epithelial progenitor/stem cells (LSCs) on a monolayer of mouse 3T3 feeder cells possesses the risk of cross-contamination in clinical applications. Human feeder cells have been used to eliminate this risk; however, efficiency from xenobiotic-free cultures on a monolayer appears to be lower than in the standard method using 3T3 cells. We investigated whether bone marrow stromal cells (BMSCs), also known as bone marrow-derived mesenchymal stem cells, could serve as feeder cells for the expansion of LSCs in the 3-dimensional (3D) system. Primary single human LSCs on a monolayer of 3T3s served as the control. Very poor growth was observed when single LSCs were cultured on BMSCs. When LSC clusters were cultured on a BMSC monolayer (CC-BM), 3D culture system (3D CC-BM) and fibrin 3D system (fibrin 3D CC-BM), the 3D CC-BM method supported a greater LSC expansion. The 3D CC-BM system produced a 2.5-fold higher cell growth rate than the control (p<0.05). The proportion of K14(+) and p63α(bright) cells was comparable to those in the control (p>0.05), whereas the proportion of K12(+) cells was lower (p<0.05). These results indicate that BMSCs can efficiently support the expansion of the LSC population in the 3D culture.

Human adipose-derived stem cells support the growth of limbal stem/progenitor cells

The most efficient method to expand limbal stem cells (LSCs) in vitro for clinical transplantation is to culture single LSCs directly on growth-arrested mouse fibroblast 3T3 cells. To reduce possible xenobiotic contamination from 3T3s, primary human adipose-derived stem cells (ASCs) were examined as feeder cells to support the expansion of LSCs in vitro. To optimize the ASC-supported culture, freshly isolated limbal epithelial cells in the form of single cells (SC-ASC) or cell clusters (CC-ASC) were cultured using three different methods: LSCs seeded directly on feeder cells, a 3-dimensional (3D) culture system and a 3D culture system with fibrin (fibrin 3D). The expanded LSCs were examined at the end of a 2-week culture. The standard 3T3 culture served as control. Expansion of SC-ASC showed limited proliferation and exhibited differentiated morphology. CC-ASC generated epithelial cells with undifferentiated morphology in all culture methods, among which CC-ASC in 3D culture supported the highest cell doubling (cells doubled 9.0 times compared to cells doubled 4.9 times in control) while maintained the percentage of putative limbal stem/progenitor cells compared to the control. There were few cell-cell contacts between cultured LSCs and ASCs in 3D CC-ASC. In conclusion, ASCs support the growth of LSCs in the form of cell clusters but not in single cells. 3D CC-ASC could serve as a substitute for the standard 3T3 culture to expand LSCs.