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Isolation of adult progenitor cells with neuronal potential from rabbit corneal epithelial cells in serum- and feeder layer-free culture conditions.

Mimura T, Yamagami S, Uchida S, Yokoo S, Ono K, Usui T, Amano S - Mol. Vis. (2010)

Bottom Line: The primary sphere colonies expressed bromodeoxyuridine (BrdU), a 63-kDa protein (p63), p75 neurotrophin receptor (p75(NTR)), and nestin, whereas their progeny expressed cytokeratin 3, cytokeratin 12, vimentin, alpha-smooth muscle actin, microtubule-associated protein 2, and neuron-specific enolase on immunocytochemical analysis.These markers were confirmed by RT-PCR.These progenitor cells differentiate into the epithelial lineage, and can also produce neuronal protein.

View Article: PubMed Central - PubMed

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

ABSTRACT

Purpose: To isolate progenitor cells from rabbit corneal epithelial cells (CEC) in serum- and feeder layer-free culture conditions and to compare the self-renewal capacity of corneal epithelial progenitor cells obtained from the central and limbal regions of the cornea.

Methods: Tissue samples of New Zealand white rabbit corneas were dissected from the limbal and central regions to obtain CEC for sphere-forming culture, in which the cells formed spheres in serum-free medium containing growth factors. The number of primary and secondary sphere colonies and the size of the primary spheres were compared between the limbal and central regions. To promote differentiation, isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/laminin. The expression of epithelial, neural, and mesenchymal mRNAs was examined in the sphere colonies and their progeny by immunocytochemistry and/or the reverse transcription-polymerase chain reaction (RT-PCR). Adherent differentiated cells from the sphere colonies were also examined morphologically.

Results: Primary spheres were isolated from both the limbal and central regions of the cornea. The rate of primary sphere formation by CEC from the limbal region (55.6+/-10.6/10,000 cells) was significantly higher than that by cells from the central cornea (43.1+/-7.2/10,000 cells, p=0.0028), but there was no significant difference in the size of primary spheres derived from both regions. The self-renewal capacity of cells from the limbal region was higher than that of cells from the central region, as evidenced by the significantly higher secondary sphere formation rate of limbal cells (38.7+/-8.5/10,000 cells) in comparison with that for central cells (31.3+/-5.7/10,000 cells, p=0.013). The primary sphere colonies expressed bromodeoxyuridine (BrdU), a 63-kDa protein (p63), p75 neurotrophin receptor (p75(NTR)), and nestin, whereas their progeny expressed cytokeratin 3, cytokeratin 12, vimentin, alpha-smooth muscle actin, microtubule-associated protein 2, and neuron-specific enolase on immunocytochemical analysis. These markers were confirmed by RT-PCR.

Conclusions: Our findings indicate that limbal CEC contain more progenitor cells with a stronger self-renewal capacity than cells from the central region. These progenitor cells differentiate into the epithelial lineage, and can also produce neuronal protein.

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Comparison of primary sphere formation by rabbit CEC from the periphery and center of the cornea. A: The number of spheres obtained from limbal CEC (n=10) was significantly higher than that from central CEC (n=10) after 7 days of culture. The experiment was repeated twice and representative data are shown. The asterisk indicates a p=0.0028 (unpaired t-test). B: The mean sphere size exceeded 250 μm on day 7.
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f2: Comparison of primary sphere formation by rabbit CEC from the periphery and center of the cornea. A: The number of spheres obtained from limbal CEC (n=10) was significantly higher than that from central CEC (n=10) after 7 days of culture. The experiment was repeated twice and representative data are shown. The asterisk indicates a p=0.0028 (unpaired t-test). B: The mean sphere size exceeded 250 μm on day 7.

Mentions: We adapted the neurosphere-forming assay that was originally devised to enrich neural stem cells and other progenitors [20,21,24,25,27-32] for isolation of adult stem cells from rabbit corneal limbal and central epithelium (Figure 1A). CEC were disaggregated into single cells and plated in uncoated wells with basal medium containing methylcellulose gel matrix to prevent reaggregation at a density of 10 viable cells/μl, as described elsewhere [24,25]. Under these conditions, sphere colonies are derived from proliferation and are not formed by reaggregation of dissociated cells [24,25]. Almost complete disaggregation into single cells was confirmed by counting the percentage of single cells, double cells, and triple cells, which demonstrated that more than 99% of the cells were single. Primary spheres were isolated from CEC derived from both the limbal and central regions. Photographs of representative spheres obtained from the limbal and central regions are shown in Figure 1B,C. When the number of sphere colonies obtained from the CEC was counted, there was a significantly greater number of spheres (55.6±10.6, mean±standard deviation) obtained from the limbal region than from the central region (43.1±7.2) per 10,000 plated cells (Figure 2A). There were no statistically significant differences in the size of primary spheres from the two regions after 3, 5, and 7 days (Figure 2B).


Isolation of adult progenitor cells with neuronal potential from rabbit corneal epithelial cells in serum- and feeder layer-free culture conditions.

Mimura T, Yamagami S, Uchida S, Yokoo S, Ono K, Usui T, Amano S - Mol. Vis. (2010)

Comparison of primary sphere formation by rabbit CEC from the periphery and center of the cornea. A: The number of spheres obtained from limbal CEC (n=10) was significantly higher than that from central CEC (n=10) after 7 days of culture. The experiment was repeated twice and representative data are shown. The asterisk indicates a p=0.0028 (unpaired t-test). B: The mean sphere size exceeded 250 μm on day 7.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927443&req=5

f2: Comparison of primary sphere formation by rabbit CEC from the periphery and center of the cornea. A: The number of spheres obtained from limbal CEC (n=10) was significantly higher than that from central CEC (n=10) after 7 days of culture. The experiment was repeated twice and representative data are shown. The asterisk indicates a p=0.0028 (unpaired t-test). B: The mean sphere size exceeded 250 μm on day 7.
Mentions: We adapted the neurosphere-forming assay that was originally devised to enrich neural stem cells and other progenitors [20,21,24,25,27-32] for isolation of adult stem cells from rabbit corneal limbal and central epithelium (Figure 1A). CEC were disaggregated into single cells and plated in uncoated wells with basal medium containing methylcellulose gel matrix to prevent reaggregation at a density of 10 viable cells/μl, as described elsewhere [24,25]. Under these conditions, sphere colonies are derived from proliferation and are not formed by reaggregation of dissociated cells [24,25]. Almost complete disaggregation into single cells was confirmed by counting the percentage of single cells, double cells, and triple cells, which demonstrated that more than 99% of the cells were single. Primary spheres were isolated from CEC derived from both the limbal and central regions. Photographs of representative spheres obtained from the limbal and central regions are shown in Figure 1B,C. When the number of sphere colonies obtained from the CEC was counted, there was a significantly greater number of spheres (55.6±10.6, mean±standard deviation) obtained from the limbal region than from the central region (43.1±7.2) per 10,000 plated cells (Figure 2A). There were no statistically significant differences in the size of primary spheres from the two regions after 3, 5, and 7 days (Figure 2B).

Bottom Line: The primary sphere colonies expressed bromodeoxyuridine (BrdU), a 63-kDa protein (p63), p75 neurotrophin receptor (p75(NTR)), and nestin, whereas their progeny expressed cytokeratin 3, cytokeratin 12, vimentin, alpha-smooth muscle actin, microtubule-associated protein 2, and neuron-specific enolase on immunocytochemical analysis.These markers were confirmed by RT-PCR.These progenitor cells differentiate into the epithelial lineage, and can also produce neuronal protein.

View Article: PubMed Central - PubMed

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

ABSTRACT

Purpose: To isolate progenitor cells from rabbit corneal epithelial cells (CEC) in serum- and feeder layer-free culture conditions and to compare the self-renewal capacity of corneal epithelial progenitor cells obtained from the central and limbal regions of the cornea.

Methods: Tissue samples of New Zealand white rabbit corneas were dissected from the limbal and central regions to obtain CEC for sphere-forming culture, in which the cells formed spheres in serum-free medium containing growth factors. The number of primary and secondary sphere colonies and the size of the primary spheres were compared between the limbal and central regions. To promote differentiation, isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/laminin. The expression of epithelial, neural, and mesenchymal mRNAs was examined in the sphere colonies and their progeny by immunocytochemistry and/or the reverse transcription-polymerase chain reaction (RT-PCR). Adherent differentiated cells from the sphere colonies were also examined morphologically.

Results: Primary spheres were isolated from both the limbal and central regions of the cornea. The rate of primary sphere formation by CEC from the limbal region (55.6+/-10.6/10,000 cells) was significantly higher than that by cells from the central cornea (43.1+/-7.2/10,000 cells, p=0.0028), but there was no significant difference in the size of primary spheres derived from both regions. The self-renewal capacity of cells from the limbal region was higher than that of cells from the central region, as evidenced by the significantly higher secondary sphere formation rate of limbal cells (38.7+/-8.5/10,000 cells) in comparison with that for central cells (31.3+/-5.7/10,000 cells, p=0.013). The primary sphere colonies expressed bromodeoxyuridine (BrdU), a 63-kDa protein (p63), p75 neurotrophin receptor (p75(NTR)), and nestin, whereas their progeny expressed cytokeratin 3, cytokeratin 12, vimentin, alpha-smooth muscle actin, microtubule-associated protein 2, and neuron-specific enolase on immunocytochemical analysis. These markers were confirmed by RT-PCR.

Conclusions: Our findings indicate that limbal CEC contain more progenitor cells with a stronger self-renewal capacity than cells from the central region. These progenitor cells differentiate into the epithelial lineage, and can also produce neuronal protein.

Show MeSH
Related in: MedlinePlus