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Abstract
Purpose
To investigate the characteristics of cultured rabbit corneal keratocytes in vitro and evaluate the possibility of differentiation of mesenchymal stem cells to keratocytes using the keratocyte conditioned medium (KCM).
Methods
Isolated keratocytes were seeded on the stromal side of amniotic membranes (AM) or plastic dishes, and morphologic changes were evaluated. Rabbit mesenchymal stem cells were cultured on AM with α-MEM (minimum essential medium alpha) and KCM. The gene expression patterns of specific keratocyte markers (keratocan, lumican, and aldehyde dehydrogenase family, member A1 (ALDH1A1)) of cultured cells were evaluated by RT-PCR.
Results
Keratocytes on AM showed dendritic morphology with slow proliferation in contrast, cells on dishes were stellate in shape with fast proliferation. Cultured keratocytes on AM maintained the expression of keratocan, lumican and ALDH1A1 while keratocytes on plastic dishes steadily lost their keratocyte marker gene expression. Additionally, mesenchymal stem cells cultured with KCM on AM induced expression of keratocan and ALDH1A1.
References
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Figure 1.
Morphologic difference between primary cells cultured on amniotic membrane (AM) or plastic dish. (A) Cells were dendritic when cultured on AM stroma. (B) In contrast, cells cultured on plastic were stellate shaped. Cells grew more rapidly on plastic than on AM. Times to confluence were compared. After reaching 40% confluence, the cells were observed. Cells on AM reached 40% confluence at 7 days after culture, but cells on dish at 2 days after culture. D7, 7 days of culture; D2, 2 days of culture. Magnification: ×200.
Figure 2.
RT-PCR analysis of expression of specific marker transcripts in rabbit keratocytes. Compared with β-actin as a loading control, keratocyte marker transcripts was expressed in all AM cultures but keratocan was largely lost when sub-cultured on dish, lumican and ALDH transcripts was lost steadily in dish cultures. K3 and K12, cornea epithelium markers, were regarded as negative markers for keratocytes. Corneal epithelium cells were used to confirm the specificity of keratocytes markers. AM=amniotic membrane; T=cornea tissue; K=keratocyte; E=epithelium cell; P=passage.
Figure 3.
Mesenchynal stem cells (MSCs) grown on dish were continuously subcultured on amniotic membrane (AM). MSCs were cultured in α-MEM, and the medium was switched to containing keratocyte conditioned medium (KCM). Cells reached confluence faster on plastic than on AM. Cells on AM reached 50% confluence after 10 days but cells on dish grown to 90% confluence at 6 days. D10=10 days of culture; D6=6 days of culture. Magnification: ×200.
Figure 4.
Mesenchymal stem cells induced expression of keratocyte marker when cultured in α-MEM medium containing KCM. The expression of keratocan and ALDH transcripts were readily detected by RT-PCR after incubation with KCM. But lumican was represented no expressional change. K3 and K12, cornea epithelium markers, were regarded as negative markers for keratocytes. Corneal epithelium cells were used to confirm the specificity of keratocytes markers. AM, amniotic membrane; T=cornea tissue; K=keratocyte; M=mesenchymal stem cell; 1st=first passage after exposure to KCM; 2nd=sec-ond passage after exposure to KCM; E=epithelium cell.
Table 1.
Sequence of RT-PCR primer
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