Isolation and distribution of rabbit keratocyte precursors

Abstract

Purpose

To isolate multipotent precursors from the rabbit corneal stroma and to compare the distribution and proliferative capacity of keratocyte precursors obtained from the central and peripheral regions of the corneal stroma.

Methods

The rabbit corneal stroma was divided into a peripheral region (6.0–10.0 mm in diameter) and a central region (6.0 mm in diameter). A sphere-forming assay was then performed to isolate precursors from the stroma of each region. To promote differentiation, isolated sphere colonies were plated in wells with a medium containing fetal bovine serum. Expression of various markers by the sphere colonies and their progeny was examined using immunocytochemistry and/or reverse-transcription polymerase chain reaction (RT–PCR).

Results

The rate of primary sphere formation by cells from the peripheral stroma (51.4±10.1/10,000 cells) was significantly higher than by cells from the central stroma (35.9±3.0/10,000 cells; p=0.00021). Secondary sphere formation rate was significantly higher in the peripheral stroma (45.6±6.4/10,000 cells) than in the central stroma (33.4±2.1/10,000 cells; p=0.00002). Cells from the spheres were positive for CD34 and nestin. Their progeny showed a keratocyte-like spindle shape and expressed vimentin, α-smooth muscle actin, and two neural differentiation markers (microtubule-associated protein-2 and neuron-specific enolase). Expression of nestin and vimentin was confirmed by RT–PCR.

Conclusions

Our findings demonstrate that both the peripheral and central regions of the corneal stroma contain a significant number of precursors, but the peripheral stroma has more precursors with a stronger proliferative capacity than that of cells from the central stroma.