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Isolation of putative corneal epithelial stem cells from cultured limbal tissue.

Kim MK, Lee JL, Shin KS, Jung GA, Wee WR, Lee JH, Park KS, Son YS - Korean J Ophthalmol (2006)

Bottom Line: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus.Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS.This technique may be very useful in tissue engineered stem cell therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea.

ABSTRACT

Purpose: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue.

Methods: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2 U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation.

Results: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3 x 10(4) cell/ml and 8.06 x 10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21 x 10(6) cell/ml with a trypsin/EDTA treatment (p < 0.05). CFE was 9.67 +/- 2.13% and 6.63 +/- 2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61 +/- 0.42% and 5.21 +/- 4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67 +/- 2.24% and 1.17 +/- 6.13%, respectively (p < 0.05).

Conclusions: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.

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(A) The Hoechst 33342 exclusion assay revealed that Hoechst negative cells were 5.21±4.91% in human cells. (B) The Hoechst Sp assay showed Sp cells were 1.15±0.94% in human cells).
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Figure 3: (A) The Hoechst 33342 exclusion assay revealed that Hoechst negative cells were 5.21±4.91% in human cells. (B) The Hoechst Sp assay showed Sp cells were 1.15±0.94% in human cells).

Mentions: The Hoechst 33342 exclusion assay revealed that Hoechst negative cells with low Hoechst blue and red were 3.61±0.42% and 5.21±4.91% in rabbit and human, respectively (Fig. 3A). According to the Hoechst Sp assay, the mean percentage of Sp cells was 1.15±0.94% in human (Fig. 3B). Each fraction of rabbit cells isolated by Hoechst exclusion was cultured until P4 (nearly 28 days). The Hoechst negative fraction maintained the small and round morphology well, and formed colonies at high CFEs. However, while the Hoechst positive fraction formed colonies, the cells became large in size and were differentiated in colonies with low CFEs (Fig. 4A-F). The CFEs of Hoechst negative and Hoechcst positive fraction after FACS in rabbit were 12.67±2.24% and 1.17±6.13%, respectively (p<0.05, Mann Whitney U test). The Hoechst positive cell fraction grew relatively rapidly and was over 85% of confluence between 6 and 7 days of culture, while the Hoechst negative cell fraction grew very slowly and displayed a similar confluence between about 9 and 12 days of culture.


Isolation of putative corneal epithelial stem cells from cultured limbal tissue.

Kim MK, Lee JL, Shin KS, Jung GA, Wee WR, Lee JH, Park KS, Son YS - Korean J Ophthalmol (2006)

(A) The Hoechst 33342 exclusion assay revealed that Hoechst negative cells were 5.21±4.91% in human cells. (B) The Hoechst Sp assay showed Sp cells were 1.15±0.94% in human cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908817&req=5

Figure 3: (A) The Hoechst 33342 exclusion assay revealed that Hoechst negative cells were 5.21±4.91% in human cells. (B) The Hoechst Sp assay showed Sp cells were 1.15±0.94% in human cells).
Mentions: The Hoechst 33342 exclusion assay revealed that Hoechst negative cells with low Hoechst blue and red were 3.61±0.42% and 5.21±4.91% in rabbit and human, respectively (Fig. 3A). According to the Hoechst Sp assay, the mean percentage of Sp cells was 1.15±0.94% in human (Fig. 3B). Each fraction of rabbit cells isolated by Hoechst exclusion was cultured until P4 (nearly 28 days). The Hoechst negative fraction maintained the small and round morphology well, and formed colonies at high CFEs. However, while the Hoechst positive fraction formed colonies, the cells became large in size and were differentiated in colonies with low CFEs (Fig. 4A-F). The CFEs of Hoechst negative and Hoechcst positive fraction after FACS in rabbit were 12.67±2.24% and 1.17±6.13%, respectively (p<0.05, Mann Whitney U test). The Hoechst positive cell fraction grew relatively rapidly and was over 85% of confluence between 6 and 7 days of culture, while the Hoechst negative cell fraction grew very slowly and displayed a similar confluence between about 9 and 12 days of culture.

Bottom Line: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus.Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS.This technique may be very useful in tissue engineered stem cell therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea.

ABSTRACT

Purpose: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue.

Methods: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2 U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation.

Results: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3 x 10(4) cell/ml and 8.06 x 10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21 x 10(6) cell/ml with a trypsin/EDTA treatment (p < 0.05). CFE was 9.67 +/- 2.13% and 6.63 +/- 2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61 +/- 0.42% and 5.21 +/- 4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67 +/- 2.24% and 1.17 +/- 6.13%, respectively (p < 0.05).

Conclusions: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.

Show MeSH