Abstract
The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.
Highlights
Three passages after cell sorting by Magnetic Activated Cell Sorting (MACS) both p75NTR+ve and p75NTR-ve populations were analyzed by Fluorescence-Activated Cell Sorting (FACS)
When the co-expression of other epithelial stem cells markers (Integrin β1 and α6) was studied on the same passages of the p75NTR-ve and p75NTR-ve populations, it was possible to observe that 88% of the p75NTR+ve cells were co-expressing both integrins in contrast with just 28% of p75NTR-ve cells
Keratinocytes from oral mucosa have been successfully used for many proposes, including reconstruction of the oral epithelium after tumor resection[17] and oral implants,[18] for the prevention of stenosis after endoscopic mucosal resection,[19] for re-epithelialization of the corneal surfaces in patients with total limbal stem-cell deficiency,[20] reconstruction of urethra,[21] regeneration of skin,[22] dental tissue engineering[23] and creation of induced pluripotent stem cells.[24]
Summary
Isolation of progenitor epithelial cells has been proposed by using many different techniques such as the capacity of rapidly adherence to collagen IV,[1] the morphology of the colonies[2] or the ability to efflux Hoechst 33342 (side population).[3,4] Likewise, a variety of cell markers for isolating progenitor epithelial cells either by using Magnetic Activated Cell Sorting (MACS)[5] or by Fluorescence-Activated Cell Sorting (FACS) have been described.[6]The p75NTR is member of the TNF receptor superfamily,[7] and its has been suggested as a potential epithelial stem cell marker in different tissues and organs including esophagus,[8] uterine cervix,[9] epithelial odontogenicBraz. The p75NTR is member of the TNF receptor superfamily,[7] and its has been suggested as a potential epithelial stem cell marker in different tissues and organs including esophagus,[8] uterine cervix,[9] epithelial odontogenic. We isolated different populations of keratinocytes by MACS and FACS based on the expression of the epithelial stem cell markers P75NTR and CD71, respectively. We analyzed the expression of these and other epithelial stem cells markers in the positive and negative populations. To assess the stem cell proprieties, we performed functional assays such as long-term growth potential, determination of the colony-forming efficiency and in vitro epithelial reconstruction
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