Isolation, Cultivation Of Human Fetal Sebocytes And Sweat Eccrine Gland Cells In Vitro, And The Preliminary Study On Differentiation From Human Fetal Epidermal Stem Cells To Hair Follicle, Sebaceous Gland And Eccrine Sweat Gland

[Background]The local or general lesions after burns are related to the loss of skin barrier, so the main purpose of burns therapy is to heal the burns wound as early as possible, and restore the function and appearance of the burned skin as much as possible. The skin autografting which is widely used at present has cured numerous patients, but it still confront many cruxes, such as lack of donor sites, increasing in wound area caused by donor sites, contracture of grafted thin split skin and shortage of skin appendages, etc. Tissue engineering skin technique provides a new method to repair the burned wound. Up to now, many kinds of the tissue engineering skin have been developed, in which the epidermis is substituted with sheets of keratinocytes cultured in vitro or autogeneous thin-split graft, while the dermis is substituted with acellular dermisor artificial dermis seeded with fibroblasts. Some of the tissue engineering skin have been commercialized and obtained good results clinically. However, all of them also have a common disadvantages, that is, they are all lack of skin appendages, such as hair follicle, sebaceous gland and sweat gland, and the "functional tissue engineering skin" can not be constructed. The skin and its appendages are all developed from ectoderm. As the specific adult stem cells in skin tissues, the epidermal stem cells which posses high proliferative potential and multipotent, are considered as the key source of occurrence, repairing and rebuilding of skin appendages, such as hair follicle, sebaceous gland and sweat gland, and can differentiate intomany kinds of cells in skin and structures such as hair follicle, sebaceous gland and sweat gland. The establishment of the culture model of sebocytes and eccrine sweat gland cells in vitro will help us get the message about their growth characteristics in details, and it is a important pathway to eatablish functional tissue engineering skin including skin appendages such as hair follicle, sebaceous gland and sweat gland by simulating a certain specific microenvironment(niche) to modulate the differentiation direction and induce the occurence of hair follicle, sebaceous gland and sweat gland. Studies have showed that there are abounding hemopoietic stem cells in umbilical cord blood, and their survival needs the support of many kinds of cytokines, in other words, the denomination and proportion of cytokines in umbilical cord serum are all suitable. Dermal papilla cells are necessary in inducing epidermis stem cells to differentiate into hair follicle by providing optimal condition for the niches. Androgen could stimulate the proliferation of sebocytes. Epidermal growth factor(EGF) plays a important role in the occunrence of eccrine sweat gland.Another crux that confronts us is how to relieve the immunological rejection of the tissue engineering skin. For example, the acellular dermis(ACM), which is most widely used clinically, was processed by a series procedures to lower its immunity by removing the cellular components and cross-linking the collagens with glutaraldehyde. While the angiogenesis of the grafted ACM needs more time than that of autograft, this would induce the collagen degenerated and its antigenic determinant exposed, as a result, in some cases ACM caused certain immunological rejection response.As for artificial dermis, the seeded adult homogeneous fibroblast is a strong antigen.The fetal skin is low immunogenic, thus cause weak immunological rejection when fetal dermis being used to repair burns wound.Fetal fibroblasts also were seeded in some tissue engineering skin to lessen the immunological rejection.So the construction of tissue engineering skin with fetal epidermis stem cells, keratinocytes, fibroblasts and dermal papilla cells which are used as epidermis and seed cells, and combined with suitable dermal cage and the above factors will be more similar to the real niche. It will be helpful to the construction of "functional tissue engineering skin" with skin appendages such as hair follicle, sebaceous gland and sweat gland etc. and the lowestimmunogenicity. [Aim]The aims of this study are as follows: To obtain primary culture of human fetal sebocytes and eccrine sweat gland cells with organ culture in vitro, and to summarize their biological characteristics of culture in vitro; To simulate the niche which could induce human fetal epidermis stem cells differentiate into skin appendages such as hair follicle, sebaceous gland and sweat gland etc. with umbilical cord serum, dermal papilla cells, 5a-dihydrotestosterone /3-isobutyl-l-methyl-xanthine and EGF; To construct tissue engineering skin with human fetal skin cells as seed cells to decrease the immunogenicity as low as possible. [Methods]1. Isolation and culture of human fetal keratinocytes in vitro. Dispase was used to separate the epidermis and dermis. Suspension of epidermal cells was prepared by trypsin digestion and reserved in liquid nitrogen to ensure that all the keratinocytes and epidermal stem cells used in following experiments were from one single individual.Primary culture of keratinocytes was done with DMEM containing serum and the corresponding supernatant was collected and used in the following experiments. Subcultures were done with SFM-KC. Cultured keratinocytes were reserved in liquid nitrogen. Adult keratinocytes were cultured with the same methods mentioned above as control.2. Isolation of human fetal sebaceous gland was obtained by Dispase digestion and microdissection. The primary culture of human fetal sebocytes was established with organ culture in vitro, and observation was made to summarize the biological characteristics of human fetal sebocytes cultured in vitro. The colony-forming efficiency was calculated under microsope, and the cycle analysis of human fetal sebocytes was tested with flow cytometer. The cells were identified by oil red staining, immunohistochemical staining of CK4.62 and EMA, and double-labelling technique. Cultured human fetal sebocytes were reserved in liquid nitrogen.3. Isolation of human fetal eccrine sweat gland was obtained by collagenase II digestion and microdissection. The primary culture of human fetal eccrine sweat gland cells was established with organ culture in vitro, and observation was made to summarize the biological characteristics of human fetal eccrinesweat gland cells cultured in vitro. The colony-forming efficiency was calculated under microsope, and the cycle analysis of human fetal eccrine sweat gland cells was tested with flow cytometer. The cells were identified by immunohistochemical staining of K7 and K14. Cultured cells of human fetal eccrine sweat gland were reserved in liquid nitrogen.4. Isolation and culture of human fetal fibroblasts in vitro. Dispase was used to separate the epidermis and dermis. Fibroblasts were cultured after isolation by trypsin digestion and the corresponding supernatant was collected and used in the following experiments. Cultured fibroblasts were reserved in liquid nitrogen as seed cells. Adult fibroblasts were cultured with the same methods mentioned above as control.5. Isolation and identification of human fetal epidermal stem cells. The suspension of fetal epidermal cells was prepared from epidermal sheet separated with Dispase or the epidermal cells of the same individual reserved in liquid nitrogen. The epidermal stem cells were isolated and gathered by rapid adhering to type IV collagen, thus obtained cells were cultured in the stem cell growth medium prepared with the fibroblasts conditioned medium obtained in above experiment. The cells were identified as epidermal stem cells according to cycle analysis with flow cytometer, testing of colony-forming efficiency, as well as immunohistochemical staining of keratin 19 and integrin pi. The keratinocytes same individual were used as control.6. Isolation and culture of human fetal dermal papilla cells in vitro. The dermal papillae were isolated by microdissection followed by collagenase digestion. The dermal papilla cells were cultured in kerationcyte's conditioned medium. Observation was made to summarize the biological characteristics of human fetal dermal papilla cells cultured in vitro, and the colony-forming efficiency, and the cycle analysis of cells with flow cytometer were tested.The cultured dermal papilla cells were identified from fibroblasts with immunohistochemical staining of a-SMA and Vimentin. The dermal papilla cells were reserved in liquid nitrogen as seed cells.7. The induction experiment of human fetal epidermal stem cells. 5 ct-dihydrotestosterone or 3-isobutyl-l-methyl-xanthine plus dexamethasone and insulin was used as induction stimulating factors respectively, and umbilical cord serum and high dose of EGF were supplemented in induction medium to culture and induce human fetal epidermal stem cells. Observation was made tosummarize the biological characteristics of cells cultured in vitro. The cells were identified by oil red staining, immunohistochemical staining of CK4.62, EMA and K19.8. Regeneration experiment of hair follicle, sebaceous gland and sweat gland. Tissue engineering skin was fabricated by using the epidermal stem cells sheet after induction as epidermis substitute, the PGA+fibronogen seeded with dermal papilla cells and fibroblasts as dermis substitute.The artificial skin was cultured in vitro, then was transplanted to repair the full-thickness wound on the back of athymic rice.The re-formed skin in transplantation area was biopsied to observe the structure and the regeneration of hair follicle, sebaceous gland and sweat gland. Immunohistochemical staining with anti-human keratin 14 monoclonal antibody was done to identify the origin of re-formed skin, hi the control group, the epidermis sheet was replaced by epidermis stem cells without induction and keratinocytes sheet as the epidermal substitute, the dermis was replaced by the PGA+fibronogen seeded with fibroblasts alone as dermis substitute. [Results]1. The human fetal keratinocytes grew well in vitro and could be subcultured over 10 passages, while the adult keratinocytes could be only subcultured to 5 passages. The culture and growth characteristics and of the epidermal cells after reserved by cells suspension in liquid nitrogen was same as that of the fresh sample.2. The cultured human fetal sebaceous gland cells proliferated slowly in vitro, and the shape of them was similar to keratinocytes. The colone growth tendency was observed after subculture. Oil red staining showed that the nuclei is blue, and the lipid in cell plasm is red. The colony-forming efficiency of them was significantly lower than those of keratinocytes, and the cycle analysis of them didn't show significant difference compared with that of keratinocytes. Positive expression of CK4.62 and EMA was demonstrated by immunohistochemical staining and double-labelling technique.3. The cultured human fetal eccrine sweat gland cells proliferated slowly in vitro, and the shape of them, was similar to keratinocytes. The colone growth tendency was showed after subculture. The colony-forming efficiency and the cycle analysis of of them were no significant difference from those of keratinocytes. The results of immunohistochemical staining of K7 and K14were positive.4. The cultured human fetal fibroblasts grew well and had no difference from adult fibroblasts. The reservation in liquid nitrogen and fusion had no influence on the growth of cultured human fetal fibroblasts.5. Being incubated for 10 min the epidermal stem cells could adhere to type IV collagen.The cells grew slowly and formed large colonies. The colony forming efficiency is 16.9% while the keratinocytes' is 8.0%, which are different significantly.The percent of cells in cell cycle phase Gl, G2, and S are 91.87%, 2.39% and 5.74%respectively, while the keratinocytes' are 73.62%, 7.85% and 18.53%respectively, which are different significantly. The epidermal stem cells were positive in immunohistochemical staining of integrin pi and K19, while the keratinocytes were negative. The reservation in liquid nitrogen and fusion had no influence on the culture of epidermal stem cells.6. The fetal dermal papilla cells could grew well and be subcultured to 20 passages in the keratinocytes conditioned medium. The shape of cultured cells showed no difference from fibroblasts except congregating growth. The results were positive in immunohistochemical staining of a-SMA and Vimentin.7. 5a-dihydrotestosterone and 3-isobutyl-l-methyl-xanthine + insulin + dexamethasone were used as induction stimulating factors respectively, and umbilical cord serum and high dose of EGF were supplemented in induction medium to culture and induce human fetal epidermal stem cells. The cells gradually showed part of characteristics of human fetal sebocytes: some small droplets of lipid appeared, and the results of oil red staining, immunohistochemical staining of CK4.62, EMAand K19 were positive.8. The dermal papilla cells extended after being seeded in PGA +fibronogen and showed multipolar or shuttle shape. The epidermal stem cells could grow to sheet in medium containing high serum, and could be taken off from plate by Dispase digestion. The epidermal substitute could adhere to the dermal substitute after being cultured in vitro for 24-48 hours. The tissue engineering skin repaired the wound about 15 days later after being transplanted to full-thickness wound on the back of athymic mice. Biopsy was done 4-5 weeks after transplantation. The re-formed skin of both the experiment group and the control group showed similar structures to those of normal. The newhair follicles could be seen in the re-formed skin, and gland-mass-like, gland-duct-like structures appeared around. Some results of immunohistochemical staining of EMA, K7 in those gland-mass-like, gland-duct-like structures are positive, while only new hair follicles could be seen in the control without induction, and no new hair follicles in the control of keratinocytes. The cells in basal laver showed positive in immunohistochemical staining by using anti-human K14 monoclonal antibody, indicating that the new epidermis is from human seed cells. [Conclusion]1. The primary culture of human fetal sebocytes can be established by Dispase digestion and microdissection of human fetal sebaceous gland, and organ culture in vitro.2. The primary culture of human fetal eccrine sweat gland cells can be established by collagenase digestion and microdissection of human fetal eccrine sweat gland, and organ culture in vitro.3. Human fetal epidermal stem cells can be isolated and gathered by rapid adhering to type IV collagen, and grow well and keep undifferentiation for long time in the stem cell growth medium prepared with human fetal fibroblast conditioned medium.4. The human fetal epidermal stem cells can be cultured and induced with 5a-dihydrotestosterone or 3-isobutyl-l-methyl-xanthine + dexamethasone + insulin as induction stimulating factors respectively besides umbilical cord serum and high dose of EGF were supplemented to induction medium. The cells showed part of of human fetal sebocyte characteristics gradually and the potency to differentiate into sebocytes.5. The tissue engineering skin fabricated with epidermal substitute, which is made of human fetal epidermal stem cells, and dermal substitute, which is made of PGA+fibronogen seeded with human fetal dermal papilla cells and fibroblasts, can repair the full-thiekness wound of athymic rice efficiently.6. Cultured in medium supplemented with umbilical cord serum, high dose of EGF plus 5a-dihydrotestosterone or 3-isobutyl-l-methyl-xanthine + dexamethasone + insulin as induction stimulating factors respectively, human fetal epidermal stem cells can differentiate into skin appendages including hair follicle, sebaceous gland and sweat gland in the re-formed skin on athymic rice with the induction of human fetal dermal papilla cells.