| Hair follicle stem cells (HSCs) play crucial roles in hair follicle morphogenesis and hair cycling. These stem cells are self-renewal and have the multi-lineage potential to generate epidermis, sebaceous glands, and hair follicle. It is evident that hair follicle stem cells are located in the bulge region of outer root sheath of hair follicle. Investigation of hair follicle stem cells is important and promising, in terms of its great application value in hair regenerative medicine and its prospective function as a model system where stem cell reprogramming, plasticity and characterization of niches can be studied. However, it is extremely difficult to distinguish stem cells from their immediate progenies since stem cells are rare in tissue and there is no conspicuous difference between these cells. This becomes a great obstacle to conduct further research on stem cells. How to collect enough hair follicle stem cells with high purity and viability with immature status is a problem need to be solved urgently.Currently, adherence separation and immunity separation are two main techniques for isolating hair follicle stem cells. The first approach is not an ideal choice because it suffers from low precision and experimental reproducibility. In contrast, in immunity separation, special antigens on the stem cells' surface, called stem cell markers, are identified and utilized to separate target cells. Currently, keratin 15,β1-integrin,α6-integrin and CD34 are the most common positive markers used in follicle stem cell isolation and CD71 is the main negative marker. Immunity separation is carried out with flow cytometry separation technique and immunomagnetic microbeads separation technique.Magnetic activated cell sorting (MACS) is a novel and efficient cell sorting technology developed in recent years. MACS technology has many advantages over other cell sorting methods, such as high efficiency, high cell viability and easiness to operate. Nowadays, MACS has been widely applied on hematological system cell sorting. However, its application on epithelial stem cells has not been carried out successfully.In our study, Vario MACS was used to separate hair follicle stem cells in nature tissue sections and satisfactory results were obtained. The general protocol and results are listed below:①Tissue localization and characterization of hair follicle stem cells In order to define the tissue localization of hair follicle stem cells in rat vibrissa skin, we used immunohistochemical staining and immunofluorescence staining to detect the expression of hair follicle stem cells' positive markers CD34,α6-integrin,β1-integrin and the negative marker CD71 in rat vibrissa hair follicle tissue. CD34,α6-integrin andβ1-integrin were strongly expressed in cytoplasm and cell membrane of cells in hair follicle outer root sheath but absent in hair bulb. In contrast, CD71 was expressed weakly in hair follicle outer root sheath and strongly expressed in epidermis and hair bulb.Primary hair follicle bulge cells were successfully cultured using tissue culture method and improved digested method with KSFM medium. Cultured cells grew well and had high colony-forming ability. These cells displayed homogeneous cobblestone-like shape and expressed follicle stem cells' markers CD34,α6-integrin andβ1-integrin, which indicated that follicle bulge cells could be successfully cultured by the methods we used.②Enrichment of rat hair follicle stem cells by Vario MACSIn this part of study, we chose three surface markers of rat hair follicle to design four isolation strategies: positive selection with single marker of CD34, positive selection with single marker ofα6-integrin, CD71 depletion followed by CD34 positive selection and CD71 depletion followed byα6-integrin positive selection. In order to screen the best strategy, hair follicle stem cells were enriched from cell suspension digested from rat vibrissa follicle bulge region with MACS technology and immunofluorescence staining was conducted. Then, fluorescence intension was detected by flow cytometry to identify our target cells and estimate the isolation efficiencies.The result of flow cytometry analysis showed that all the four strategies had ideal effect. The positive rates of CD34bri cells,α6bri cells, CD34briCD71dim cells andα6briCD71dim cells were 81.24%, 100.00%, 82.54% and 89.24%, respectively.CD34 and CD71 compound selection was considered to be the most ideal strategy. On one hand, purer stem cells could be obtained with combined selection than single positive selection since double selection could distinguish stem cells from their progenies. On the other hand, although there was no obvious difference between the isolation efficiency of the double strategies, it was more convenient to conduct the protocol of CD34 selection than that ofα6 selection.③The biological characteristics of CD34bri hair follicle stem cells A series of researches were conducted on CD34bri cells and CD34dim cells to investigate the biological characteristics of hair follicle stem cells and to further evaluate the Vario MACS technology. We detected the viability of isolated cells, analyzed the expression of the markers in the separated cells by immunofluorescence staining. Furthermore, the isolated CD34bri cells and CD34dim cells were cultured, the cell growth curve was plotted, and expression of CD34,α6-integrin,β1-integrin in the two types of cells was detected. In addition, scanning electron microscope (SEM) and transmission electron microscope (TEM) were employed to observe the morphology and ultrastructures of these cells.Rat hair follicle stem cells selected by Vario MACS grew slowly, because of the impairment of their viability. The cell growth curve showed that CD34bri cells had greater proliferating potential and higher colony-forming ability than CD34dim cells. CD34,α6-integrin andβ1-integrin were expressed in cytoplasm and cell membrane, and expression in CD34bri cells were much stronger than in CD34dim cells. CD34 only had weak expression in CD34dim cells. SEM indicated that the CD34bri follicle stem cells were round in shape and with some microbeads attached to the cell membrane. In contrast, no microbeads adhered to other three kinds of cells: follicle bulge cells primary cultured for 7 days, cells digested directly from follicle bulge region and CD34dim cells. TEM suggested that CD34bri cells had some typical characteristics as progenitor cells, such as big nucleus, obvious nucleolus, large nuclear-cytoplasm ratio and few organelles in cytoplasm.The significance of our study was to isolate and enrich rat hair follicle stem cells successfully and effectively by Vario MACS technology, and to obtain these stem cells' electron microscope morphological data. Most importantly, the isolated hair follicle stem cells had high viability and could be cultured successfully for the first time. It serves as the foundation for the further research on hair follicle stem cell line. |
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