Experimental Study On Immortalization Of Human Dermal Papilla Cells

Background:Dermal papilla cells (DPC) play an essential role in embryonic formation of hair follicle as well as post-natal hair cycling and growth. It has been proved that the Cultured DPC had the ability to induce hair follicle regeneration in vivo and in vitro. The dysfunction of DPC usually abolishes the initiation of subsequent hair cycle to result in follicle-associated illness. Therefore, the study on the control of hair follicle growth and cycle should take functional status of DPC into consideration. With the rapid advancement in the technology of tissue engineering over the years, many kinds of tissue engineering skin have been put into clinic in many countries. At present, the main origin of seed cells of tissue engineering skin is usually dermal fibroblasts and keratinocytes. In consequence, there are great differences between normal skin and tissue engineering skin, with the later have no skin adnexa such as hair follicle and sweat gland. The skin defects will be well healed by using DPC as seed cell to construct the tissue engineering skin with hair follicles. However, DPC cultured in vitro lost their inductive properties at high passage and showed growth retardation known as replicative senescence after a certain number of cell divisions, which greatly restricted the further use of DPC in basic research and clinical.The general way to solve the problem of replicative senescence of cultured cells in vitro is to establish an immortalized cell line. However, the most methods of establishing cell lines were transfection with oncogene and viral genes in last the century. These cell lines have some disadvantages: the exogenous genes interfere with cells'normal physiology, they are transformant, their phenotypes and karyotypes have changed. Moreover, not every cell line was really immortalized as some of them had only overcome senescence to prolong the life span. Ectopic expression of human telomerase reverse transcriptase (hTERT) gene, which encodes the catalytic subunit of the telomerase holoenzyme, can activate the telomerase of cells, enables extension of life spans of normal human cells and even lead to cell immortalization. Because the way of activating the human telomerase is similar to the physiological way in the embryo stem cells, the immortalized cells have normal biological behaviors and characters of embryo stem cell. This study will establish the immortalized normal human DPC line by transfection of hTERT gene mediated with cationic liposome and then investigate the biological and functional characteristics of the cell line, with a view to provide sufficient, stable and normal cells for basic research and clinical usages of DPC.Objective:1. To establish an immortalized human DPC line.2. To investigate the biological and functional characteristics of the immortalized human DPC in vitro.3. To examine the hair follicle-inducing ability of the immortalized human DPC in vivo.Methods:1. The human dermal papilla cells were firstly isolated and cultured from scalp hair follicle via enzyme digestive method established by our previous work. Then the morphology of cultured DPC was observed, and the character of cultured DPC was identified with the immunohistochemical staining ofα-smooth muscle actin (α-SMA).2. The plasmid pIRES2-EGFP and plasmid pIRES2-EGFP-hTERT encoding hTERT were transfected into cultured normal human DPC at passage 3 with cationic liposome, then the positive cell clones were selected with G418. After the transfected DPC were expanded in culture, integration and expression of hTERT gene were detected with polymerase chain reaction (PCR) and reverse transcription (RT)-PCR, and the telomerase activity was detected with telomeric repeat amplification protocol-enzyme linked immunosorbent assay (TRAP-ELISA).3. We chose the pIRES2-EGFP-hTERT transfected DPC at passage 30 for the following studies. The morphology of the cultured cells was observed with inverted microscope and the expression ofα-SMA in the cells was detected with immunohistochemical staining. The growth and proliferation of the cells were investigated with cell growth curve and cell cycle analysises. The karyotype, growth in soft agar and tumorigenicity in nude mice of the cells were analyzed. The vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in the cultured medium of the cells was detected with ELISA.4. The pIRES2-EGFP-hTERT transfected DPC at passage 30 and the fresh isolated hair follicle epithelial cells were co-injected subcutaneously into nude mice to induce hair follicle regeneration.Results:1. The human dermal papilla cells were isolated and cultured from scalp hair follicle via enzyme digestive method established by our previous work. Cultured DPC were similar to fibroblasts in appearance with larger cell size, exhibited an aggregative growth pattern and expressedα-SMA.2. The plasmid pIRES2-EGFP and plasmid pIRES2-EGFP-hTERT encoding hTERT were transfected into cultured normal human DPC with cationic liposome and gained 1 clone respectively after selection with G418 for 3 weeks, which were named as DPC-EGFP and DPC-hTERT. After continuous culture, the DPC-EGFP cells showed expected slowing of growth that is associated with aging and ceased proliferation at passage 12, while the DPC-hTERT cells showed strong proliferation and have been maintained in culture for passage 65 to date without significant growth retardation. The hTERT wasn't integrated and expressed and the telomerase activity was negative in untransfected DPC and DPC-EGFP cells. However, the hTERT was integrated and expressed and meanwhile the telomerase activity was positive in DPC-hTERT cells.3. The DPC-hTERT cells exhibited little difference in morphology as compared with the earlier passages DPC and expressedα-SMA, growing and proliferating more actively than DPC. They were normal karyotype, had nontumorigenic in nude mice, and had no anchorage-independent growth. They also retained the secretory function of normal DPC.4. Co-injected the DPC-hTERT cells and fresh isolated hair follicle epithelial cells into nude mice formed epidermal cyst structure with many cells and pigment granule.Conclusion:1. An immortalized human DPC line (DPC-hTERT) has been established.2. DPC-hTERT is a normal cell line without obvious transformed phenotype. It retains the biological and functional characteristics of normal human DPC which may be afforded to be an ideal cell model for further studying of DPC, and provided sufficient, stable and normal seed cells for constructing of tissue engineering skin with hair follicles.