Marrow Mesenchymal Stem Cells Have Potential To Differentiate By Supernatant Of Cultured Hair Follicle

ObjectiveThe skin is the biggest organ of human body. It is very important to resist microorganism and ultraviolet radiation, to prevent water loss, and to regulate body temperature. At the same time, the skin is one part of immune system. The regeneration and repair was achieved mainly by proliferation and division of healthy cells from the surrounding tissue. However, this self-reconstruction was not sufficient for serious damage such as severe burns, widespread cutting skin ulcer, traumatic defect and skin ulcer. Therefore, enough graftskin is needed to repair skin trauma. Tissue engineering skin is the first product which is approved by FDA. This product made good therapeutic effect in curing telephium and severe burns. But now we could not manufacture tissue engineering skin which contains hair follicle, sweat gland, and sebaceous gland. For this reason, at present there is no artificial skin which satisfies the repair of traumatic defect in function, which depresses the adaptive capacity of the skin to the environment. It is a hot research spot how to realize the functional reconstruction of the skin.The development of stem cell theory and technology could bring a hope for recovering the physiological function together with rebuilding anatomic structure of skin injuries. Stem cells have the infinite reproductive activity all their life, and retain their ability to differentiate into mature functional cells under certain condition. There are two categories: adult and embryonic stem cells. The research in embryonic stem cells is restricted by its limited source and ethical controversy, so research on adult stem cells attracts more attentions. Though hair follicle stem cells, as a new kind of seed cells for tissue engineering skin, are gradually paid more attention to, the technology of segregation and purification is not very perfect and there is no perfect cultivated amplification system. MSCs (mesenchymal stem cells), which have self-renewal ability and multi-directional differentiation potentiality, have multiple potential to differentiation into bone, cartilage, fat, cardiac muscle cells and hepatic cells and so on in the same germinal layer, and bestriding the germinal layer into pulmonary epithelial cells, enteric epithelial cells, nerve cells and epidermic cells and so on. MSCs are easy to obtain and separate. MSCs have high proliferating and retain the stable bionomics. they are convenient to expand and transfect. We can get MSCs by bone marrow aspiration. Cells induced from MSCs have no tissue matching and immune rejection. At present, MSCs is preferable seed cells during cell transplantation. It was reported that MSCs could be differentiated into vascular endothelial cells, epidermal cells and sebaceous gland duct cells, when they have been transplanted on the trauma. Thus, MSCs have the potential to repair the trauma in the practical application.It is not very clear that the differentiated mechanism and induced condition of MSCs. The differentiation of MSCs is closely related to the microenvironment which is called "niche" they grow in. It is probably because certain factors in the microenvironment are necessary for MSCs to differentiate into target cell lineages. There is a research that MSCs have potential to differentiate into neurons, glials and retinal cells induced by supernatant of cultured retinal cells. In this study, to investigate the potentiality of induced MSCs into hair follicle stem cells by supernatant of cultured hair follicle, we have identified MSCs induced by supernatant of cultured hair follicle by morphology, immunohistochemical staining technique, immunofluorescence staining technique and RT-PCR. The potentiality of MSCs in repairing the skin trauma urged us to investigate how to rebuild the anatomic structure and physiologic function by using induced differentiation of MSCs.Materials and methodsOne male Wistar rat weighing 100g was to put to death by cutting the cervical vertebrae. MSCs were isolated and cultured by complete adherence. We removed the hematopoietic cell after changing the culture fluid and transferring the culture. Then the stem cells creeped the glass slides. They were fixed and characterized by immunostained with CD44 and CD29. Under the sterile condition, We got the hair follicles from the whisker skin of the rat with microsurgical instruments in the 24 well tissue culture plate. Supernatant of cultured hair follicles were collected, filtrated and mixed with DMEM at a ratio of 2: 3 respectively. MSCs were induced by two different mixed culture medium, observed under phase contrast microscope, the characterized by immnostained with K15 and K19. RT-PCR was used to further confirm the immnocytochemical results.Results一,Isolated culture and identification of MSCsMSCs were isolated culture by complete adherence. A few cells began to adhere to the culture flask. The morphology of the stem cells was like fibroblasts. They quickly formed the colony. We changed the culture fluid of MSCs at the first time. MSCs fast propagated after three days and mixed together at 80%. After passage 2 to 3, MSCs were fusiform shape. Cell nuclei of MSCs were very big and nucleoli were manifest. Above ninety-five percent of MSCs was uniformity in the morphology. MSCs of Passage three were characterized with CD44 and CD29 by immunohistochemical staining technique. Cell membranes of MSCs were buffy. We could see the positive cells of CD29 and CD44 in the cell membranes.二,Culture and observation of hair follicleMost of hair follicles began to grow at 1d after cultured. The hair follicles grew day and day. The growth of hair follicle mostly concentrated at one to four day. Most of hair follicles began to adhere to the tissue culture plate. After 7 days, fibroblasts and epithelioid cells grew from hair follicles. They propagated day by day, like paving a road. At 10d, most of hair follicles had been adherence. Hair follicles grew slowly.三,Observation and identification of MSCs after induced by conditioned medium.1. When MSCs of passage 3 were induced by the conditioned medium at 7d, the morphology of a few cells began to change. The morphology of the cells transformed from fusiform shape to keratinocytes day by day.2. After 3 weeks induction, MSCs could be partially identified by the positive staining for keratin 15 and CD34, specific antibodies of hair follicle stem cells.3. After 3 weeks induction, K15 and CD34 were detected in most of MSCs induced by supernatant of cultured hair follicles by RT-PCR.DiscussionThis self-reconstruction was not sufficient for serious damage such as severe burns, widespread cutting skin ulcer, traumatic defect and skin ulcer. Therefore, enough graftskin is needed to repair skin trauma. But now we could not manufacture tissue engineering skin which contains hair follicle, sweat gland, and sebaceous gland. For this reason, at present there is no artificial skin which satisfies the repair of traumatic defect in function, which depresses the adaptive capacity of the skin to the environment. It is a hot research spot how to realize the functional reconstruction of the skin.MSCs (mesenchymal stem cells), which have self-renewal ability and multi-directional differentiation potentiality, have multiple potential to differentiation into bone, cartilage, fat, cardiac muscle cells and hepatic cells and so on in the same germinal layer, and bestriding the germinal layer into pulmonary epithelial cells, enteric epithelial cells, nerve cells and epidermic cells and so on. MSCs are easy to obtain and separate. MSCs have high proliferating and retain the stable bionomics. they are convenient to expand and transfect. We can get MSCs by bone marrow aspiration. Cells induced from MSCs have no tissue matching and immune rejection. At present, MSCs have been the preference during cell transplantation. MSCs have been induced into cardiomyocytes by 5-aza by Wang Haiping, and so on. And MSCs have been induced into osteoblast by Pan Feng et all in my laboratory. It was confirmed that MSCs were differentiated into vascular endothelial cells,epidermal cells and sebaceous duct by autotransplanting into the wound surface. Nakagawa, and so on repaired the cutaneous deficiency of nude mouse by transplanting MSCs into the artificial skin. He confirmed that MSCs had the potentiality to promote the wound healing. McFarlin, and so on, injected MSCs into the wound. It confirmed that MSCs could promote the wound healing. Though the study of hair follicle stem cells as seed cells has been advanced, the separation and purification about hair follicle stem cells would be studied deeply. We simulated the internal microenvironment by collecting the supernatant of cultured hair follicle. MSCs were induced by the cell factors (KCF,FCF,VEGF and so on). After 3 weeks, we could observe that MSCs show the typical keratinocytes, polygon and nucleus is large. The cells expressed K15 and CD34 especially.K15 and CD34 were the specific markers of hair follicle stem cells .My research indicated that MSCs have the potential differentiate into hair follicle stem cells by supernatant of cultured hair follicle which offered the foundation that MSCs were induced into epidermis,dermis and skin appendages.MSCs were induced into hair follicle stem cells by supernatant of cultured hair follicle . It is possibility that we can synthesis the skin which has complete anatomic structure and good function. The research proved that MSCs have the great potential differentiate into hair follicle stem cells and offered the foundation that MSCs were induced into epidermis,dermis and skin appendages. But we have to take the strict animal experiment. Besides we have to study deeply the molecule mechanism and cell function after differentiation into hair follicle stem cells.ConclusionMSCs have potential to differentiate into hair follicle stem cells induced by supernatant of cultured hair follicle in vitro.