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3A). The reunion between PCK+ cells and PCK?/Vim+ cells noted on Day 1 led to a gradual increase of cell number by Day 3 and Day 10 (Fig. 3B). To further confirm the proliferation of PCK+ and PCK? cells, we added EdU to the culture medium on Day 5 for 24 hours. The results showed that 10.3 �� 2.3% PCK+ cells and 2.7 �� 0.8% PCK? cells were EdU+ (n = 5, p http://www.selleckchem.com/products/pf-562271.html single cells from Day 10 spheres showed that small p63��+ epithelial cells were also Pax6+ in the nucleus, while a few larger cells were cytoplasmic CK12+ (Fig. 3C), indicating that such sphere growth http://www.selleck.cn/products/azd6738.html represented expansion of progenitors and maintained a corneal fate [29�C31]. Expression of CK10, an epidermal differentiation marker, was not detected (data not shown). Collectively, we have developed a novel in vitro model, in which limbal epithelial progenitors reunited with their presumed NCs to yield sphere growth that exhibited a corneal fate. To determine the mechanism whereby limbal epithelial progenitors reunited with their putative NCs in the above sphere culture, we chose to study the SDF-1/CXCR4 signaling, which is known to control homing of hematopoietic SCs to the bone marrow niche [11, 12]. To know whether this signaling was involved, we characterized their in vivo cytolocalization. Double immunostaining of human limbal http://www.selleckchem.com/products/VX-765.html sections showed that positive SDF-1 staining was found in limbal but not corneal epithelial cells and in corneal but not limbal stromal cells (Fig. 4A). In contrast, weak CXCR4 staining was found in limbal and corneal epithelial cells as well as corneal stromal cells, while strong CXCR4 staining was found in limbal stromal cells (Fig. 4A). To quantify the difference in expressing SDF-1 and CXCR4 between the limbus and the cornea, we used collagenase to isolate both limbal and corneal clusters. Western blot analysis showed that SDF-1 was predominantly expressed in the limbal but not corneal cluster, and CXCR4 was also strongly expressed in the limbal but not corneal cluster (Fig. 4B). To verify whether the above difference indeed existed in limbal SCs and NCs, we removed limbal epithelial sheets by dispase and released cells from the remaining limbal stroma by collagenase. Double immunostaining of cytospin preparation confirmed that SDF-1 was expressed only by p63��+ or Vim? epithelial cells but not by the surrounding p63��? or Vim+ stromal mesenchymal cells (Fig. 4C). In contrast, CXCR4 was expressed by both cell types (Fig. 4C).