Effect of Substrate on Proliferation and Differentiation of Stem Cells: Discertation
Author
Botha, Jaco
Term
4. term
Publication year
2013
Submitted on
2013-05-31
Pages
56
Abstract
Baggrund: Hornhindens overflade fornyes løbende af limbale epitelstamceller, som findes ved kanten af hornhinden (limbus). Hvis disse stamceller går tabt på grund af sygdomme, skader, medfødte forhold, infektioner eller medicinske indgreb, opstår limbal stamcellemangel (LSCD). Det kan føre til karnydannelse, kronisk betændelse og uklar hornhinde med ubehag, smerter, lysoverfølsomhed og svært nedsat syn. Den mest anerkendte behandling ved LSCD med uklar hornhinde er hornhindetransplantation kombineret med transplantation af dyrket limbalt epitel (CLET). Nye erstatningsmaterialer til hornhindens støttevæv og forbedrede CLET‑procedurer er lovende, men selve cellelaget er skrøbeligt og kræver et bæremateriale. Der er afprøvet flere bæreløsninger, bl.a. amnionmembran (guldstandard), kollagenbaserede membraner og siloxan‑hydrogel kontaktlinser, med varierende resultater. Formål: At sammenligne, hvordan stamceller hæfter, deler sig (prolifererer) og bevarer stamcelleegenskaber på naturlige og syntetiske underlag, der kan støtte udvikling mod en hornhindecelletype, i et serum- og feeder‑frit system. Metoder: Vi undersøgte fedtvævsafledte stamceller (ASCs) som model for limbale stamceller på flere underlag: intakt amnionmembran, epitel‑fri amnionmembran (dAM), acellulær amnionmembran (aAM), plastisk komprimeret kollagenmembran samt to specialiserede dyrkningsskåle (CCC og PIPAA). Derudover isolerede vi limbale epitelceller (LECs) fra corneosklerale ringe med enten Dispase eller kollagenase og dyrkede dem i et xeno‑ og feeder‑frit system. Resultater: ASCs hæftede på alle testede underlag inden for 24 timer. Celler på dAM og aAM viste den højeste vækstrate (p < 0,05). Efter tre dages dyrkning var der ingen signifikante forskelle mellem de øvrige underlag og kontrol. Kollagenase gav flere LECs end Dispase, men donorvævets levedygtighed var ofte utilstrækkelig. Konklusion: Valget af underlag påvirker vigtige celleegenskaber som form og vækst i denne model. Begrænset levedygtighed af donorvæv forhindrede dog vellykket dyrkning af LECs på de testede underlag. Der er behov for yderligere forskning.
Background: The cornea’s surface is continuously renewed by limbal epithelial stem cells located at the edge of the cornea (the limbus). When these cells are lost due to tumors, autoimmune disease, trauma, congenital conditions, infections, or medical treatments, limbal stem cell deficiency (LSCD) develops. This can cause blood vessel ingrowth, chronic inflammation, and corneal clouding, leading to discomfort, pain, light sensitivity, and severely reduced vision. The most accepted treatment for LSCD with corneal opacification is corneal transplantation combined with transplantation of cultured limbal epithelium (CLET). New supporting materials and optimized CLET procedures are promising, but the cell layer is fragile and needs a carrier scaffold. Several carriers have been explored, including amniotic membrane (the current gold standard), collagen-based membranes, and silicone hydrogel contact lenses, with mixed results. Aim: To compare cell adhesion, proliferation, and maintenance of stem cell properties on natural and synthetic substrates that could support differentiation toward a corneal cell type, using a serum- and feeder-free system. Methods: We used adipose-derived stem cells (ASCs) as a model for limbal stem cells on several substrates: intact amniotic membrane; epithelially denuded amniotic membrane (dAM); acellular amniotic membrane (aAM); plastic-compressed collagen membrane; and two specialized culture surfaces (CCC and PIPAA). We also isolated limbal epithelial cells (LECs) from donor corneoscleral rims with either Dispase or collagenase and cultured them in a xeno- and feeder-free system. Results: ASCs attached to all substrates within 24 hours. Cells on dAM and aAM showed the highest growth rates (p < 0.05). After three days, there were no significant differences between the other substrates and controls. Collagenase yielded more LECs than Dispase, but donor tissue viability was often inadequate. Conclusion: In this model, the substrate influences key cell features such as shape and proliferation. However, limited viability of donor tissue prevented successful growth of LECs on the tested substrates. Further research is needed.
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