Study On The Distribution, Isolation And Culture Of The Epidermal Stem Cells Of Rats In Vitro

Although there are different stem cells in many tissues, it is very difficult to distinguish them clearly because of their small amount and unconspicuous discrimination from others cells around them. How to isolate these infrequent stem cells from different tissues is a very important thing among researchers to solve the low purity and insufficient quantitity of stem cells in vitro.Skin tissue has high ability of regeneration. Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life.Balancing the rate of cell division with the rate of cell loss is essential for epidermal homeostasis and must be maintained for life. With the development of molecular biology, cell biology, tissue engineering and biotechnology, epidermal stem cells(ESCs) are thinken highly of more and more on the gene therapy and cell therapy because of their typical biological characters .How to isolate and culture the ESCs is of great importance not only to the repair in trauma but also to the study on the skin cancer. Although the existence of the ESCs has been known for some decades, their specific makers, identification standards, distribution and the culture conditions in vitro remain unelucidated ,which hinder the study of the ESCs from lab bench to clinical applications.At present, there are two main methods in isolating the ESCs. One method bases on rapid attachment to type IV collagen; the other bases on the markers present on the ESCs isolated by FCM. The former method is easy to apply, but has a lower purity; The latter method has a higher purity, but the isolated cells activity is affected and costs much, which hinders this method to spread in our country.MACS technology is an extremely efficient cell sorting technology which contains immunology, cell biology and magnetomechanics technologies. The MACS MicroBeads are very small, 50 nanometers in diameter. They are about one million times smaller in volume than an eukaryotic cell and comparable to the size of a virus. They are too small to be detected by optical methods. Therefore, they neither change the scatter properties of the cell in the FCM nor do they influence the lightmicroscopic appearance of the cell. The MACS MicroBeads are biodegradable due to their small size and their biochemical composition of iron oxide and polysaccharide. They decompose when cells are cultured, and it is therefore not necessary to remove them from cells after the separation process. Nowadays, MACS technology is generally applied on cell sorting of the hematological system.The skin is the largest organ in the human body and it is the outermost barrier that protects us against a variety of environmental assaults. The ESCs play an important role in the skin renewing, repair in skin trauma, generation of skin canner and so on. Among the studies of the ESCs, research objects are usually human skin or mouse skin, while rat skin is seldom applied. In this experimental study, rat skin was applied to investigate the distribution, isolation and culture of the ESCs of rats in vitro; The main methods and results are as follows:1. The tissue location and characterization of the ESCsImmnohistomchemical methods were used to confirm the location of the epidermal stem cells of rats;α6-integrin,β1-integrin and K15 were expressed in the basal layer cells and hair follicle bulge cells,CD71and K10 were negatively or poorly expressed in the basal layer cells while they were strongly expressed in the superbasal layer cells;CD34 was expressed in the hair follicle bulge cells while not expressed in the basal layer cells; These result suggested that stem cells located at the basal layer of epidermis and the hair follicle bulge, The ESCs locating at basal layer maybe have different characters from the hair follicle stem cells.The ESCs were isolated by rapid adhesion to type IV collagen. The ESCs growthed well on type IV collagen. Cultured cells had homogeneous slabstone-shape. Cells were small and round and had a large nuclear: cytoplasm ratio. The characteristics suggested a primitive morphologic feature. Compared to no rapidly adherent cells,cultured ESCs had higher colony-forming efficiency. Immunocytochemical stain data showed thatα6-integrin,β1-integrin and K15 were strongly expressed in the cultured epidermal stem cells,while CD71 and K10 were weak or negative. These results suggested the ESCs could be cultured in vitro by this ways. 2. Isolation of the ESCs of rat in vitro by VarioMACSIt was the first time we isolated the ESCs of rat in vitro by VarioMACS. The ESCs were isolated by first depleting the non-target cells by CD71 and then magnetically labeling and positively selecting the subset cells byα6-integrin. The isolating efficiency was 8.62/1. Scanning electron microscope(SEM) indicated that the isolated ESCs were round or oval-shape, regular appearance and some granulose cell processes, The small MACS MicroBeads(about 50 nanometers in diameter) were found onα6briCD71dim after isolation by SEM.3. The characterization of the ESCs isolated by VarioMACSThe ESCs isolated by VarioMACS growthed slowly in vitro.Cell viability was low. On the condition of KSFM medium, cultured cells had homogeneous slabstone-shape. Cells were small and round and had a large nuclear: cytoplasmi ratio. Immunocytochemical stain data showed thatα6-integrin,β1-integrin and K15 were strongly expressed in the cultured ESCs,while CD71 and K10 were weak or negative. With the software IPP analysis, the AOD of the ESCs isolated by Vario MACS was significantly different from the ESCs isolated by type IV collagen(P<0.05).These characteristics suggested a primitive feature.On the condition of DMEM+10%FBS medium, cultured cells were larger and polygon-shaped,gradually appeared delamination. Immunocytochemical stain data showed that K10 was expressed whileα6-integrin,β1-integrin and K15 were negative. The results suggested that the ESCs isolated by Vario MACS have the potential of differentiation to nomal epithelial cells.In this study, the ESCs of rat were successfully isolated in vitro.It was the first time we isolated the ESCs of rat by VarioMACS. This study opened a new path for the isolation of the ESCs in vitro. Also, this study provided a technology platform for the ESCSs isolation by MACS in vitro.