Development of Dermal cell line has great scope in the field of skin related diseases and regenerative medicine. Alopecia leads to a skin disorder causing balding and its mechanism is not yet understood. In the present study, we have developed and characterized a heterogeneous population of human dermal mesenchymal-like stem cell line from scalp biopsy of androgenetic alopecia patient with a view to isolate cells from the bulge region of the hair follicle. Our study showed that the dermal cells isolated from dermis of skin showed epithelial-like cells expressing CD34 and Keratin 18, which are characteristic of bulge hair follicle cells. These cells also expressed mesenchymal phenotypes and pluripotency markers such as Oct4, Nanog and SOX2. These cells were designated as “Human Dermal Mesenchymal-like Stem Cells (hDMSCs)”. To confirm their epithelial phenotypes, we have grown these cells at low serum concentration and it was observed that 3% serum concentration provided optimum conditions for their growth and maintenance of characteristics. The hDMSCs cells are presently at passage 10. This study reports the establishment of human dermal mesenchymal-like cell line from the dermis of Alopecia patient, which may be used as an in vitro model system to study the mechanism of Alopecia and other related skin disorders.
The stem cell discovery and research has brought about a revolution to the field of science and technology. It has built a horizon for new ventures in tissue engineering and regenerative medicine [1,2]. Enormous methods have been put forth to isolate stem cells from various tissues which opened doors to the idea of selfcell repair and has emerged as a new technology for therapeutic and clinical applications [3-5].
Androgenetic Alopecia (AGA) is a common hair disorder, which is hereditary and androgen-dependent process, seen in genetically predisposed male and female [
Recent studies have suggested that skin-derived stem cells would have potential contributions in studies related to dermatology and dermatopathology such as the skin aging, wound healing disorders and skin neoplasms; and also in the field of tissue repair, regenerative medicine, gene and cell-based therapies [9-11]. Development of different types of skin cell lines under in vitro conditions can be achieved using different components supplemented in the growth media which would give rise to different types of skin cells [
CD34+ MSC-like cells may be suggested to be present in the human adult dermis and it is observed that the perivascular region in the dermis was rich in CD34+ cells which were capable of differentiating into mesenchymal lineages [
The serum concentration in the growth media plays a critical role in development and differentiation of various cell types under in vitro conditions. Studies have shown that epithelial cells grow better in serum free media as compared to media containing serum as it was found that the activity of transforming growth factor-beta (TGFbeta) could differentiate epithelial cells and reduces its growth rate [
The present study, aimed at developing and characterizing a human dermal cell line from an androgenetic alopecia patient to understand the mechanism of alopecia and related disorder. We characterized these cells by using morphological and molecular stem cell markers to confirm the cell phenotypes. Development of Dermal skin cell line from human could be important as an in vitro model system for in-depth studies on the mechanism of skin diseases at molecular level and in the current situation dermal skin cells can be useful for regenerative therapies and for bone and cardiac related disorders.
Fresh scalp biopsy from a patient (33/F) suffering from Androgenetic Alopecia was obtained in sterile 1× Phosphate Buffer Saline (PBS) (Himedia, India) with prior consent of the patient as per the guidelines of the ethical committee at Jaslok Hospital and Research Centre, Mumbai, India. The tissue explant was sent to the stem cell laboratory for further processing. This explant was washed several times with sterile 1× PBS containing 1% Penstrep (Himedia, India). The explant was dissected so as to separate the epidermal layer from the dermal layer. The dermal layer was further processed. The tissue was minced well and digested with 0.25% Trypsin-EDTA (Himedia, India) at 37˚C for half an hour in the CO2 incubator. The explant was then placed onto 65 mm Nunc culture dish which were scratched so as to support the adherence of the tissue on to the petri dish. The dishes were fed with freshly prepared complete growth medium (Dulbecco’s Modified Eagle’s Medium, Himedia, India) that was supplemented with 10% FBS (Fetal Bovine Serum, GIBCO), 1% Penicillin—Streptomycin, 1 μl/ml Insulin (Sigma, USA), 2 μl/ml·L—Glutamine (Himedia, India). The dishes were incubated at 37˚C in the CO2 incubator with 5% CO2. Regular monitoring of these dishes was done to check for outgrowth of cells from the tissue explant using the phase contrast microscope. Within 3 to 4 days, outgrowth of cells from the explant was observed and the cells adhered to the culture dish without any feeder layer or substratum. Every alternate day, the cultures were fed with growth medium and these cells achieved confluence within two weeks. Each time before the transfer of cells for an increase in passage, these cells were given an initial partial trypsinization using 0.25% Trypsin-EDTA for 1minute to remove the fibroblasts cells from the pool of other cells. Once these cells were removed, the rest of the cells were kept in trypsin for another 2 minutes, neutralized with fresh growth media and then transferred to 50 mm Nunc culture flasks for expansion of these cultured cells. This process was repeated when the cells were confluent. A heterogeneous population of skin cells was obtained after several weeks of culturing. Presently, the cell line is at passage 10. Excessive cells were cryopreserved at −86˚C for further characterization by cellular and molecular markers [3,20].
The Epithelial Dermal cells such as the keratinocytes which grow better in serum free or low serum containing medium and therefore we have studied the effect of different concentrations of serum containing growth medium on this cell line. The sub-confluent dermal cell cultures growing in T50 flask were taken for this study. 2 × 104 cells were transferred to each five 65 mm Nunc petri dishes and were fed with normal growth media and incubated in the CO2 incubator for 24 hr. Next day, these cells were washed with 1× PBS and fed with growth media containing 10%, 5%, 3%, 1% Fetal Bovine Serum (FBS) and serum free media. The cells were monitored after an interval of 2 hr, 4 hr and 8 hr, then after every 24 hr for 8 days to check for any morphological changes under phase contrast microscope. The cells before and after treatment were photographed before they were sacrificed for RNA extraction after 8 days in culture to study the expression of molecular markers.
Total RNA was extracted using Trizol method (Invitrogen, Carlsbad, USA) from the dermal layer of the skin biopsy obtained from the Alopecia patient (in vivo study), dermal cell line (in vitro study) and dermal cells treated with different concentration of serum. The RNA was reverse transcribed to complementary DNA (cDNA) using Applied Biosystems high capacity cDNA kit (Applied Biosystems, USA) and used for RT/PCR of various molecular markers such as CD34, Keratin 18, IL-6, CD 105, CD73, CD13, Oct4, Nanog, SOX2, and β-Actin (internal control) as described in previous studies [3-5,20]. The amplified PCR products were run on 2% Agarose gel electrophoresis and observed under the gel documentation system (Cell Biosciences) and were photographed.
Isolation of human dermal cells from scalp biopsy of Androgenetic Alopecia patient was previously described in the materials and methods. Regular monitoring of the culture dishes was done every day and within 3 - 4 days of incubation, outgrowth of cells was observed. Phase contrast microscopy of the development of dermal cell line was shown in Figures 1(a)-(d).
In our present study, we have studied 9 molecular stem cell markers for the characterization of Human Dermal skin biopsy (in vivo) and isolated dermal cells (in vitro) by RT/PCR technology.