Kim D-S, Cho H-J, Choi H-R, Kwon S-B, Park K-C
Research Division for Human Life Sciences, Seoul National University, 28 Yongon-Dong, 110-744, Chongno-Gu, Seoul, Korea.
Cell Mol Life Sci. 2004 Nov;61(21):2774-81. doi: 10.1007/s00018-004-4288-4.
The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-alpha(6) integrin and anti-CD71 antibodies. RA cells express high levels of alpha(6) integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed alpha(6) integrin and beta(1) integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.
人表皮干细胞的分离对于其临床应用至关重要。在本研究中，我们根据表皮角质形成细胞对IV型胶原的黏附能力分离出了三类细胞群体，即快速黏附（RA）细胞、缓慢黏附（SA）细胞和非黏附（NA）细胞。本研究的目的是鉴定RA细胞，并研究使用这些细胞进行表皮重建的可能性。为了鉴定RA细胞，我们使用抗α(6)整合素和抗CD71抗体进行了流式细胞术分析。RA细胞高表达α(6)整合素且低表达CD71，这些被认为是表皮干细胞性质的标志物。此外，电子显微镜显示RA细胞体积小，核质比高，而SA和NA细胞具有发育良好的细胞器和丰富的张力丝。蛋白质免疫印迹分析显示RA细胞增殖缓慢且表达p63（一种假定的表皮干细胞标志物），而SA和NA细胞表达c-Myc，已知其可调节干细胞命运。为了比较表皮再生能力，我们使用RA、SA和NA细胞构建了皮肤替代物（SEs）。与由SA或NA细胞形成的表皮相比，由RA细胞构建的表皮形成良好。此外，只有含有RA细胞的SEs在基底层表达α(6)整合素和β(1)整合素。这些结果表明RA细胞代表表皮干细胞，且主要由干细胞组成。因此，使用一种简单技术分离RA细胞为其临床应用提供了一条潜在途径，因为它们易于分离且能提供高产率的表皮干细胞。
