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Isolation and distribution of rabbit keratocyte precursors.

Mimura T, Amano S, Yokoo S, Uchida S, Usui T, Yamagami S - Mol. Vis. (2008)

Bottom Line: 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).Their progeny showed a keratocyte-like spindle shape and expressed vimentin, alpha-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan. mimurattky@umin.ac.jp

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, alpha-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.

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Formation of secondary sphere. (A, B) Secondary spheres were generated after the dissociation of primary spheres derived from peripheral or central keratocytes. Scale bar=100 μm. (C) The re-plating efficiency from primary to secondary colonies was higher for spheres derived from the peripheral stroma than for those from the central stroma (p=0.000025, unpaired t-test).
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f3: Formation of secondary sphere. (A, B) Secondary spheres were generated after the dissociation of primary spheres derived from peripheral or central keratocytes. Scale bar=100 μm. (C) The re-plating efficiency from primary to secondary colonies was higher for spheres derived from the peripheral stroma than for those from the central stroma (p=0.000025, unpaired t-test).

Mentions: To further evaluate the proliferative capacity of the keratocytes, cells from the primary spheres were passaged under the same culture conditions as those used for the initial growth of the spheres. Secondary spheres were generated after the dissociation of the primary spheres that were derived from the peripheral or central stroma. Photographs of representative secondary spheres are shown in Figure 3A and B. The number of secondary spheres per 10,000 cells was significantly higher when primary spheres that were derived from the peripheral stroma were passaged than spheres from the central stroma (45.6±6.4 versus 33.4±2.1, respectively; p=0.000025; unpaired t-test; Figure 3C). Re-plating to generate secondary sphere colonies was less efficient than the generation of primary spheres, indicating that the precursor cells had a limited proliferative capacity.


Isolation and distribution of rabbit keratocyte precursors.

Mimura T, Amano S, Yokoo S, Uchida S, Usui T, Yamagami S - Mol. Vis. (2008)

Formation of secondary sphere. (A, B) Secondary spheres were generated after the dissociation of primary spheres derived from peripheral or central keratocytes. Scale bar=100 μm. (C) The re-plating efficiency from primary to secondary colonies was higher for spheres derived from the peripheral stroma than for those from the central stroma (p=0.000025, unpaired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254968&req=5

f3: Formation of secondary sphere. (A, B) Secondary spheres were generated after the dissociation of primary spheres derived from peripheral or central keratocytes. Scale bar=100 μm. (C) The re-plating efficiency from primary to secondary colonies was higher for spheres derived from the peripheral stroma than for those from the central stroma (p=0.000025, unpaired t-test).
Mentions: To further evaluate the proliferative capacity of the keratocytes, cells from the primary spheres were passaged under the same culture conditions as those used for the initial growth of the spheres. Secondary spheres were generated after the dissociation of the primary spheres that were derived from the peripheral or central stroma. Photographs of representative secondary spheres are shown in Figure 3A and B. The number of secondary spheres per 10,000 cells was significantly higher when primary spheres that were derived from the peripheral stroma were passaged than spheres from the central stroma (45.6±6.4 versus 33.4±2.1, respectively; p=0.000025; unpaired t-test; Figure 3C). Re-plating to generate secondary sphere colonies was less efficient than the generation of primary spheres, indicating that the precursor cells had a limited proliferative capacity.

Bottom Line: 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).Their progeny showed a keratocyte-like spindle shape and expressed vimentin, alpha-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan. mimurattky@umin.ac.jp

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, alpha-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.

Show MeSH
Related in: MedlinePlus