Establishment of Three Types of Immortalized Human Skin Stem Cell Lines Derived from the Single Donor

Abstract

Currently, human-skin derived cell culture is a basic technique essential for dermatological research, cellular engineering research, drug development, and cosmetic development. But the number of donors is limited, and primary cell function reduces through cell passage. In particular, since adult stem cells are present in a small amount in living tissues, it has been difficult to obtain a large amount of stem cells and to stably culture them. In this study, skin derived cells were isolated from the epidermis, dermis, and adipose tissue collected from single donor, and immortalization was induced through gene transfer. Subsequently, cell lines that could be used as stem cell models were selected using the differentiation potential and the expression of stem cell markers as indices, and it was confirmed that these could be stably cultured. The immortalized cell lines established in this study have the potential to be applied not only to basic dermatological research but also to a wide range of fields such as drug screening and cell engineering.

INTRODUCTION

The skin tissue is the largest organ that protects the whole body against the peripheral environment or dryness. It functions as a physical, chemical, and antimicrobial barrier and a sensor to mechanical stimuli, pain, and temperature. Its structure consists of three layers: the epidermis, dermis, and adipose tissue. Various cells exist in each tissue. Furthermore, it has been shown that stem cells are present as the source of supply of these cells.¹⁾

Among the 3 layers of the skin, the epidermis, existing in the outer layer, plays an extremely important role as a biological defense mechanism to promote water retention and prevent external stimuli/foreign body invasion. The epidermis is composed of multiple layers of keratinocytes (KCs) that are at various differentiated stages (basal, spinous, granular, and cornified layers).²⁾ Stem cells for KCs (epidermal stem cells) exist in the basal layer, the lowest layer of the epidermis. They repeatedly proliferate and differentiate if necessary, and supply new cells to the epidermis.³⁾ Therefore, the cells in the epidermis are continuously regenerating. The process from the production of KCs in the basal layer to exfoliation as dirt is termed “epidermal turnover.” The dermis is an extracellular-matrix-rich tissue, consisting of collagen fibers, primarily comprising type I/III collagen produced by fibroblasts (FBs), and elastic fibers such as elastin. Furthermore, a large volume of glycosaminoglycans, such as hyaluronic acid, and proteoglycans, exist, contributing to dermal flexibility through water retention and fiber stabilization. Based on recent studies, there may be mesenchymal-stem-cell-like cells that may differentiate into various cells, such as adipocytes, chondrocytes, and osteoblasts, among FBs existing in the dermis tissue.^4–6) In the adipose tissue below the dermis, adipocytes and adipose stromal cells (ASCs) primarily exist. Adipocytes play a role in accumulating neutral fat, cushioning physical forces, and regulating the body temperature. Among ASCs, there may be mesenchymal stem cells that have a multi-differentiation ability.^7–10) Subcutaneous-fat collection is less invasive than bone-marrow collection, and a large number of cells can be collected by small-volume fat aspiration; therefore, adipose-tissue-derived stem cells may be applied in the field of regenerative medicine.¹¹⁾

Currently, human-skin derived cell culture is a basic technique essential for dermatological research, cellular engineering research, drug development, and cosmetic development. These cells can be isolated from the human skin through plastic surgical procedures, but the number of donors is limited, and primary cell function reduces in the early phase through cell passage; therefore, it has been difficult to obtain cells stably. In particular, only a small number of somatic stem cells exist in the biological tissue; therefore, it has been difficult to obtain a large number of such cells or culture them for a long period while maintaining their proliferation/differentiation abilities. Furthermore, the cellular ability and genetic background markedly differ among donors, and there was a limitation that no reproducible experiment can be conducted in any study with primary cells. On the other hand, recent advances in cell immortalization technology have facilitated the establishment of immortalized cell lines of various cell types that can be infinitely proliferated.^12–15) If such immortalized cell lines are used, stable study results can be permanently obtained. In addition, further genetic modification may facilitate the preparation of purpose-matched cells. Furthermore, immortalized cells are also extremely useful as a tool for promptly screening a large amount of compound by high-throughput screening; they may contribute to searching for seeds for drug discovery.

The purpose of this study was to establish a human-skin-derived stem cell line that has essential cell characteristics and can be stably cultured over a long period using cell immortalization technology. Concretely, KCs, FBs, and ASCs were collected from the single donor, and monoclonal lines were established after an immortalization-inducing gene was transferred into each cell. Subsequently, cell lines that could be used as stem cell models from these monoclonal strains were selected using the differentiation potential and the expression of stem cell markers as indices, and it was confirmed that these could be stably cultured. The immortalized cell lines established in this study may contribute to the future development of dermatological/skin stem cell research.

MATERIALS AND METHODS

Separation of Skin-Derived Cells and Cell Culture

This study was conducted with an ethical approval from the Research and Ethics Committee of Fujita Health University (Approval No. HG19-057). After obtaining informed consent, skin tissue was obtained from excess normal skin of a Japanese patient (male, 63 years old) who underwent a dermatological surgery. Keratinocytes (KCs), Fibroblasts (FBs), and adipose stromal cells (ASCs) were isolated from epidermis, dermis, and adipose tissue. Briefly, the epidermal tissue peeled from the dermis by Dispase treatment (4 °C, over night, Godo Shusei, Tokyo, Japan) was reacted with trypsin (37 °C, 1 h, Wako Pure Chemical Corporation, Osaka, Japan), and then KCs were obtained using a 100 µm nylon cell strainer. In addition, the dermis was shredded and treated with 0.5% collagenase (37 °C, 1 h, Nitta Gelatin, Osaka, Japan), and FBs were separated using a 100 µm nylon cell strainer. Erythrocytes were removed by suspending FBs in ACTB (Tris-buffered ammonium chloride). ASCs were separated from adipose tissue using the same method as FBs. KCs were cultured in low-Ca²⁺ medium Keratinocyte-serum free medium (KSFM, Invitrogen, Carlsbad, CA, U.S.A.) for propagation. FBs were grown in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, U.S.A.). ASCs were cultured in complete medium (D/α medium), which consists of DMEM and α minimum essential medium (α MEM, Invitrogen) with a 1 : 1 ratio, supplemented with 1% FBS, 1× ITS-X (Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF, PeproTech Inc, Rocky Hill, NJ, U.S.A.) and 0.4 µg/mL hydrocortisone (Sigma-Aldrich). During the culture period, the cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air, and the medium was replaced every 2–3 d.

Viral Transduction and Establishment of Immortalized Cells

Previous studies demonstrated that immortalization by telomerase reverse-transcriptase (TERT), mutant cyclin-dependent kinase 4 (CDK4^R24C), or cyclin D1 (CCND1) is superior to the use of simian vacuolating virus 40 T antigen (SV40) or human papilloma virus-derived oncoprotein E6/E7 in terms of maintaining the characteristics of primary cells.^16,17) Therefore, in this study, we used TERT, CDK4^R24C, and CCND1 as immortalization-inducing genes. To overexpress genes in KCs, FBs, ASCs, lentiviral gene expression vectors expressing TERT, CDK4^R24C and CCND1 were purchased (VectorBuilder, Chicago, IL, U.S.A.). Lenti-X™ 293T cells (TaKaRa Bio, Shiga, Japan) and Lenti-X™ HTX Packaging System (TaKaRa Bio) were used to prepare lentiviral particles. Briefly, non-confluent Lenti-X™ 293T cells in 10-cm culture dishes were transfected with lentiviral gene expression vectors with Lenti-X™ HTX Packaging System. Lentiviruses were harvested at 72 h post-transfection and filtered through a 0.45 µm filter to remove cellular debris. Early-passage KCs (P1), FBs (P3), ASCs (P3) were transduced with Lentiviruses at multiplicity of infection (MOI) of 10 in culture media containing 4 µg/mL polybrene (Sigma-Aldrich). To obtain monoclonal cell lines, we used limiting dilution method.

Three-Dimensional (3D) Human KCs Culture

The culture was based on the Millicell hanging cell culture insert (Millipore, Billerica, MA, U.S.A.). Primary and immortalized KCs were seeded at a density of 2.0 × 10⁵ cell/insert in 0.4 mL expansion medium (CNT-07; CELLnTEC, Berne, Switzerland). The inserts were then placed in a 24-well plate with 1 mL CNT-07 medium being added into the well. Cells were first grown in CNT-07 medium for 48 h until 100% confluence and were then maintained in 3D medium (CNT-02-3DP5, CELLnTEC). The medium in the insert was aspirated after 16 h, and the cells were exposed to air. The 3D culture was then maintained for 9 d. The medium was replaced every 2–3 d.

Histochemistry

3D reconstructed epidermis were fixed with 4% paraformaldehyde (Wako Pure Chemical Corporation) and embedded in paraffin. Paraffin-embedded 3D reconstructed epidermis were sectioned at 4 µm using a sliding microtome CTM-180 (Sakura Finetek, Tokyo, Japan). Sectioned samples were deparaffinized and processed for Hematoxylin and Eosin (H&E) staining. The experiments were performed in triplicate, and representative images are shown in each Figure.

Immunocytochemistry

Primary and immortalized FBs were washed with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 1 h at 4 °C. The media with the anti-type I collagen (COL1) rabbit polyclonal (LSL Corporation, Tokyo, Japan) at a dilution of 1 : 100 was introduced into culture dish, and cells were incubated for 1 h at 37 °C. After being washed in PBS, the cells were stained with Alexa Fluor 594 donkey anti-rabbit immunoglobulin G (IgG) (Molecular Probes, Eugene, OR, U.S.A.) at a dilution of 1 : 1000 for 30 min at 37 °C. Cells were then washed in PBS, and 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution (Dojindo Laboratories, Kumamoto, Japan) was added to confirm the presence of cell nuclei. The experiments were performed in triplicate, and representative images are shown in each Figure.

Induction of Immortalized FBs and ASCs Differentiation into Adipocytes

Differentiation of immortalized FBs and ASCs into adipocytes was performed as described previously.¹⁸⁾ Differentiation induction was carried out using adipogenic induction medium consisting of DMEM supplemented with 10% FBS, 33 µM Biotin (Sigma-Aldrich), 10 µg/mL Insulin (Sigma-Aldrich), 1 µM dexamethasone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) and 0.2 mM indomethacin (Sigma-Aldrich) for 6 d. Additionally, cells were cultured using adipogenic differentiation medium consisting of DMEM supplemented with 10% FBS, 33 µM Biotin and 10 µg/mL Insulin for 4 d. The medium was replaced every 2–3 d. Adipogenesis was assayed by lipid accumulation in differentiated cells with Oil Red O staining. The experiments were performed in triplicate, and representative images are shown in each Figure.

Induction of Immortalized FBs and ASCs Differentiation into Osteoblasts

Differentiation was carried out using osteogenic differentiation medium consisting of DMEM supplemented with 10% FBS, 50 µM ascorbic acid 2-phosphate (Sigma-Aldrich) and 0.01 µM dexamethasone for 21 d. The medium was replaced every 2–3 d. Osteogenesis was assayed by matrix mineralization in differentiated cells and with Alizarin Red S staining. The experiments were performed in triplicate, and representative images are shown in each Figure.

Induction of Immortalized FBs and ASCs Differentiation into Chondrocytes

Cells were seeded in a 15 mL polypropylene conical tube at a density of 1 × 10⁵. After centrifuged, cells were suspended by chondrogenic differentiation medium consisting of DMEM supplemented with 10% FBS, 50 µM ascorbic acid 2-phosphate, 0.01 µM dexamethasone, 0.5 × ITS-X (Invitrogen), 10 ng/mL BMP-2 (PeproTech Inc.) and 10 ng/mL TGF-β3 (PeproTech Inc.). Cells were further centrifuged for 2 min at 500 g and incubated at the bottom of a conical tube containing 0.5 mL of chondrogenic differentiation medium for 14 d. The medium was replaced every 2–3 d. The experiments were performed in triplicate, and representative images are shown in each Figure.

Quantitative Real-Time PCR Analysis

Total RNA was extracted from cells using TRIZOL Reagent (Invitrogen), and cDNA was synthesized by reverse transcription. Real-time PCR was performed with the SuperScript III Platinum Two-Step qRT-PCR kit (Invitrogen), using the 7300 Real Time PCR System (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s protocol. The primer sequences are shown in Table 1. The contents of the selected genes were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Differences between samples were quantified based on the ∆∆Ct method. All PCR products were checked by melting curve analysis to exclude the possibility of multiple products or an incorrect product size.

Table 1. Primers Used in Real-Time RT-PCR Analysis

Gene	Sequences
	Forward	Reverse
GAPDH	5′-TGCACCACCAACTGCTTAGC-3′	5′-TCTTCTGGGTGGCAGTGATG-3′
TERT	5′-TGTACTTTGTCAAGGTGGATGTG-3′	5′-CTGGAGGTCTGTCAAGGTAGAG-3′
CDK4	5′-ATCTTTGACCTGATTGGGCT-3′	5′-AAAGTCAGCATTTCCAGCAG-3′
CCND1	5′-CCTCTAAGATGAAGGAGACCA-3′	5′-AAATGAACTTCACATCTGTGGC-3′
ITGA6	5′-ACCAAGGTTCTGAGCCCAAA-3′	5′-AGCCACAGGGTTTCCTCCAT-3′
ITGB1	5′-AATGAGGAGGATTACTTCGGACTTC-3′	5′-GTTCCTGAGCTTAGCTGGTGTTG-3′
CD44	5′-CCAGCAACCCTACTGCTGATGA-3′	5′-CTCCTGAAGTGCTGCTCCTTTC-3′
CD90	5′-CCGCTCCCGAACCAACT-3′	5′-GGCGGATAAGTAGAGGACCTTCA-3′

Cell Proliferation Assay

Cell proliferation rates were calculated using IncuCyte ZOOM™ (Essen BioScience, Ann Arbor, MI, U.S.A.). Briefly, cells were seeded in 96-well plates at a density of 3000 cells, cultured in a growth medium suitable for each cell, and cell density was measured at each time point.

DNA Content Analysis

DNA content analysis was performed using Flow cytometry (FACSCanto; BD Biosciences, San Jose, CA, U.S.A.) to determine the ploidy levels of immortalized cells. Specifically, cells were fixed in 70% ethanol for 2 h at 4 °C, and were subsequently treated with 0.25 mg/mL ribonuclease (RNase) solution (Sigma-Aldrich) for 30 min at 37 °C. The cells were then stained with 50 µg/mL Propidium Iodide (PI) (Sigma-Aldrich) at 4 °C and analyzed using Flow cytometry.

RESULTS

Isolation of Skin Cells from the Single Donor

In this study, KCs, FBs, and ASCs were isolated from the epidermis, dermis, and adipose tissue of the single donor, respectively, and cultured using media appropriate for the respective cells (Supplementary Fig. 1).

Immortalization of KCs

When KCs are exposed to air on a cell-culture insert for differentiation, a layer structure resembling the biological epidermis can be formed. In this study, primary KCs collected from the donor’s skin tissue also formed a three-dimensional layer structure. On the other hand, the epidermis-forming ability markedly reduced through repeated cell passage (Figs. 1A, B). In addition, the proliferation ability markedly reduced with cell passage, and there was no proliferation of P5 cells, making the continuation of culture impossible (Fig. 1C). We transferred three genes: TERT, CDK4^R24C, and CCND1, into P1 KCs before the loss of epidermis-forming or proliferation abilities to induce immortalization (polyclone). Furthermore, three monoclonal lines were established through single-cell cloning from this polyclone, and termed Asian derived immortalized keratinocyte 1 (AIK1), AIK2, and AIK3, respectively. It was confirmed that the expressions of TERT, CDK4, and CCND1 in these monoclonal lines were markedly enhanced (Figs. 2A, B). It was possible to culture P27 or later cells. Furthermore, the three-dimensionally cultured epidermis was prepared using these monoclonal lines, and the epidermis-forming ability was confirmed in the AIK1 line, as demonstrated in early passage primary KCs (P1). On the other hand, in the AIK2 line, nuclei were present in the stratum corneum, and incomplete cornification was observed. In the AIK3 line, the epidermis was slightly thinner than in the other 2 clones (Fig. 2C). Furthermore, we analyzed the expressions of epidermal stem cell markers (ITGA6, ITGB1)¹⁹⁾ in an undifferentiated state. Their expressions were the highest in the AIK1 line (Supplementary Fig. 2). We focused on the AIK1 line with a normal differentiation ability (epidermis-forming ability) and high stem cell marker levels, and examined the stability of cell proliferation and differentiation abilities through cell passage. As a result, the proliferation ability of the AIK1 line was stable even on the 37th cell passage, and the three-dimensional epidermis-synthesizing ability was maintained (Figs. 3A–C). Thus, the AIK1 line could be established as immortalized KCs that may proliferate stably over a long period, with their potent differentiation ability.

Fig. 1. Influence of Cell Passage on the Function of Primary KCs

A. Microscopic image of primrary KCs (P3–5). B. H&E-stained sections of 3D reconstructed epidermis using keratinocyte (P3–5). C. Changes in the cell proliferation ability. The cell density was normalized to that on day 0 (control). Data are expressed as the mean ± standard deviation (S.D.) of five experiments. Bars: (A) 250 µm, (B) 50 µm. (Color figure can be accessed in the online version.)

Fig. 2. Establishment of Immortalized Monoclonal Lines of KCs

A. Gene expression analysis using real-time PCR. The expression level of each gene was normalized to that on polyclone (control). Data are expressed as the mean ± S.D. of three experiments. B. Microscopic image of AIK1, AIK2, and AIK3. C. H&E-stained sections of 3D reconstructed epidermis using AIK1, AIK2, and AIK3. Bars: (B) 250 µm, (C) 50 µm. The arrowheads indicate nuclei that are retained in cells in the cornified layers. (Color figure can be accessed in the online version.)

Fig. 3. Influence of Cell Passage on the Function of AIK1

A. Changes in the cell proliferation ability. The cell density was normalized to that on day 0 (control). B. Microscopic image of AIK1 (P37). C. H&E-stained sections of 3D reconstructed epidermis using AIK1 (P37). Bars: (B) 250 µm, (C) 50 µm. (Color figure can be accessed in the online version.)

Immortalization of FBs

FBs produce collagen, which plays an important role in the elasticity of the skin. A previous study indicated that, when FBs were stimulated with ascorbic acid, type I collagen synthesis increased, promoting collagen matrix assembly formation.²⁰⁾ In this study, early passage primary FBs (P5) collected from the donor’s skin tissue, collagen matrix assembly formation was promoted in the presence of stimulation with ascorbic acid. On the other hand, in P25 cells, the responsiveness to ascorbic acid reduced (Figs. 4A, B). Furthermore, there was a marked reduction in the proliferation ability of P25 cells (Fig. 4C). We transferred three genes: TERT, CDK4^R24C, and CCND1, into early passage primary FBs (P3) to induce immortalization (polyclone). Furthermore, three monoclonal lines were established through single-cell cloning from this polyclone, and termed Asian derived immortalized fibroblast 1 (AIF1), AIF2, and AIF3, respectively. It was confirmed that the expressions of TERT, CDK4, and CCND1 in these monoclonal lines were markedly enhanced. It was possible to culture P25 or later cells (Figs. 5A, B). When investigating the responsiveness to ascorbic acid using these monoclonal lines, a collagen matrix assembly was confirmed (Fig. 5C). In the AIF3 line, collagen matrix assembly formation was slightly less marked than in the other two lines. Based on recent studies, there may be mesenchymal-stem-cell-like cells that can differentiate into various cells, such as adipocytes, chondrocytes, and osteoblasts, among FBs existing in the dermis tissue.^4–6) We examined differentiation into adipocytes, osteoblasts, and chondrocytes using immortalized FBs. As a result, the AIF1 and AIF3 lines were confirmed to differentiate into adipocytes (Fig. 5D). The AIF1 and AIF2 lines were confirmed to differentiate into osteoblasts. In the AIF3 line, there was no differentiation (Fig. 5E). Concerning chondrocytes, the formation of a cartilage-like structure was confirmed in all monoclonal lines (Fig. 5F). When confirming the expressions of mesenchymal stem cell marker genes (CD44, CD90), their expressions in the AIF1 line were slightly higher than in the other two lines (Supplementary Fig. 3). We focused on the AIF1 line with a potent collagen-producing ability, multi-differentiation ability, and stem cell marker expressions, and examined the stability of the cell proliferation ability. As a result, the proliferation ability of the AIF1 line was stable even on the 40th cell passage. At this point, the collagen-producing ability was also maintained (Figs. 6A–C). Thus, the AIF1 line could be established as immortalized FBs that may proliferate stably over a long period, with their potent differentiation ability.

Fig. 4. Influence of Cell Passage on the Function of Primary FBs

A. Microscopic image of primary FBs (P5–25). B. Immunostaining of COL1. blue: DAPI, green: COL1. C. Changes in the cell proliferation ability. The cell density was normalized to that on day 0 (control). Data are expressed as the mean ± S.D. of five experiments. Bars: (A) 250 µm, (B) 100 µm. (Color figure can be accessed in the online version.)

Fig. 5. Establishment of Immortalized Monoclonal Lines of FBs

A. Gene expression analysis using real-time PCR. The expression level of each gene was normalized to that on polyclone (control). Data are expressed as the mean ± S.D. of three experiments. B. Microscopic image of AIF1, AIF2, and AIF3. C. Immunostaining of COL1. blue: DAPI, green: COL1. D. Induction of differentiation into adipocytes. The bottom panels in D are magnified views of the top panels. E. Induction of differentiation into osteoblasts. F. Induction of differentiation into chondrocytes. Bars: (B) 250 µm, (C) 100 µm, (D) upper: 100 µm, lower: 50 µm, (E) 100 µm, (F) 1 mm. (Color figure can be accessed in the online version.)

Fig. 6. Influence of Cell Passage on the Function of AIF1

A. Changes in the cell proliferation ability. The cell density was normalized to that on day 0 (control). B. Microscopic image of AIF1 (P40). C. Immunostaining of COL1. blue: DAPI, green: COL1. Bars: (B) 250 µm, (C) 100 µm. (Color figure can be accessed in the online version.)

Immortalization of ASCs

Previous studies indicated that mesenchymal stem cells were present among ASCs existing in the adipose tissue, differentiating into adipocytes, osteoblasts, and chondrocytes.^7–10) In this study, the morphology of primary ASCs collected from the donor’s skin tissue was flattened through cell passage (Fig. 7A). To confirm the influence of cell passage on the ability of ASCs, differentiation into adipocytes, osteoblasts, and chondrocytes was induced using P5 and P10 cells. As a result, differentiation into adipocytes, osteoblasts, and the chondrocytes was confirmed (Figs. 7B–D). But, concerning the cell proliferation ability, P10 cells showed a marked reduction (Fig. 7E). We transferred three genes: TERT, CDK4^R24C, and CCND1, into early passage primary ASCs (P3) to induce immortalization (polyclone). Furthermore, three monoclonal lines were established through single-cell cloning from this polyclone, and termed Asian derived immortalized ASC 1 (AIA1), AIA2, and AIA3, respectively. It was confirmed that the expressions of TERT, CDK4, and CCND1 in these monoclonal lines were markedly enhanced. It was possible to culture P20 or later cells (Figs. 8A, B). When investigating the multi-differentiation ability using these monoclonal lines, it was confirmed in all monoclonal lines (Figs. 8C–E). When confirming the expressions of mesenchymal stem cell markers (CD44, CD90), their expressions in the AIA3 line were slightly higher than in the other two lines (Supplementary Fig. 4). We focused on the AIA3 line, and examined the stability of cell proliferation and differentiation abilities. As a result, the proliferation ability of the AIA3 line was stable even on the 40th cell passage. Furthermore, the multi-differentiation ability was also maintained (Figs. 9A–E). Thus, the AIA3 line could be established as immortalized ASCs that may proliferate stably over a long period, with their potent differentiation ability.

Fig. 7. Influence of Cell Passage on the Function of Primary ASCs

A. Microscopic image of primary ASCs (P5–10). B. Induction of differentiation into adipocytes. The right panels in B are magnified views of the left panels. C. Induction of differentiation into osteoblasts. D. Induction of differentiation into chondrocytes. E. Changes in the cell proliferation ability. The cell density was normalized to that on day 0 (control). Data are expressed as the mean ± S.D. of five experiments. Bars: (A–C) 250 µm, (B) (enlarged image) 50 µm, (D) 1 mm. (Color figure can be accessed in the online version.)

Fig. 8. Establishment of Immortalized Monoclonal Lines of ASCs

A. Gene expression analysis using real-time PCR. The expression level of each gene was normalized to that on polyclone (control). Data are expressed as the mean ± S.D. of three experiments. B. Microscopic image of AIA1, AIA2, and AIA3. C. Induction of differentiation into adipocytes. D. Induction of differentiation into osteoblasts. E. Induction of differentiation into chondrocytes. Bars: (B–D) 250 µm, (E) 1 mm. (Color figure can be accessed in the online version.)

Fig. 9. Influence of Cell Passage on the Function of AIA3

A. Changes in the cell proliferation ability. The cell density was normalized to that on day 0 (control). B. Microscopic image of AIA3 (P40). C. Induction of differentiation into adipocytes. D. Induction of differentiation into osteoblasts. E. Induction of differentiation into chondrocytes. Bars: (B–D) 250 µm, (C) (enlarged image) 50 µm, (E) 1 mm. (Color figure can be accessed in the online version.)

DNA Content Analysis

In order to determine the ploidy levels of the AIK1, AIF1, AIA3 lines, we stained the cells with PI and performed DNA content analysis using flow cytometry to compare the fluorescence peak of PI with the respective primary cells. The AIF1 and AIA3 lines showed the same fluorescence intensity peaks compared with the primary cells, indicating that these cells are a homogeneous population of diploid cells. On the other hand, the fluorescence peak for PI shifted in the AIK1 line compared with its primary cells. This indicated that the cells had polyploid abnormality (Supplementary Fig. 5).

DISCUSSION

In this study, primary KCs, FBs, and ASCs were isolated from the epidermis, dermis, and adipose tissue collected from the skin of the single donor, and immortalization was induced through gene transfer (TERT, CDK4^R24C, and CCND1) into respective cells. In a previous study, human immortalized KCs (HDK1) were also established through gene transfer (TERT, CDK4^R24C, and CCND1). They are used in various studies.¹⁶⁾ HDK1 can proliferate over a long period while maintaining its normal differentiation ability. In addition, it is also used as a stem cell model due to the expressions of KC stem cell markers (ITGB1, ITGA6).^19,21,22) The AIK1 line, immortalized KCs established in this study, proliferate over a long period while maintaining its normal differentiation ability, as demonstrated for HDK1. In addition, the expressions of stem cell markers were higher than in the other monoclonal lines (AIK2, AIK3) (Figs. 2, 3, Supplementary Fig. 2). Therefore, the AIK1 line established in this study may be available as an epidermis-derived stem cell model. Our findings also indicated that there was a polyploid abnormality in the AIK1 line. Further studies are needed to understand the mechanism underlying the polyploid abnormality in the AIK1 line and its effect on cellular function.

In previous studies, human immortalized FBs were established using various methods,^23,24) but no stem cell feature- or multi-differentiation ability-confirmed cell line has been established. Interestingly, monoclonal lines of immortalized FBs established in this study had a multi-differentiation ability. In particular, the AIF1 line differentiated into adipocytes, osteoblasts, and chondrocytes, and the expressions of mesenchymal stem cell marker genes were detected (Figs. 5, 6, Supplementary Fig. 3). Previous studies suggested that the population of various primary FBs contains a specific proportion of mesenchymal stem cell-like cells capable of differentiating into adipocytes, osteoblasts, and chondrocytes.^4–6) The AIF1 established in this study may be derived from mesenchymal stem cell-like cells present in dermal tissue. The AIF1 line may be available as a dermis-derived stem cell model. In previous studies, human immortalized ASCs were established by transferring various immortalization-inducing genes (TERT, E6/E7, Bmi-1, and SV40).^25–29) On the other hand, no study has reported a cell line immortalized by transferring a combination of TERT, CDK4^R24C, and CCND1, which was established in this study. In the immortalized ASCs established in this study (AIA3), long-term proliferation/multi-differentiation abilities and stem cell marker expressions were confirmed (Figs. 8, 9, Supplementary Fig. 4). Therefore, the AIA3 line may be available as an adipose-tissue-derived stem cell model.

Thus, in this study, stem cell models (AIK1, AIF1, and AIA3) that can be stably cultured for a long period were established from the epidermis, dermis, and adipose tissue derived from the single donor (Supplementary Fig. 6). AIK1, AIF1, and AIA3 have been confirmed to be negative for pathogenic viruses such as hepatitis C (HCV) and hepatitis B virus (HBV). And these cells also have been confirmed to be negative for mycoplasma (data not shown). These cells may be useful for research on the mechanism of stem cell differentiation, which remains to be clarified, and drug screening. Furthermore, in this study, immortalized cells were established from the skin derived from the same donor; the genetic background (nuclear genome, mitochondrial genome) is common. Therefore, these cells may also be useful for investigating cell-type-related differences in the gene expression pattern, DNA methylation, and responsiveness to various drugs without considering individual differences. Each immortalized cell line established in this study may contribute to the development of dermatology and drug discovery research.

Acknowledgments

We thank Natsuko Goshima (Cellisis Co., Ltd., Aichi, Japan) for support in preparing the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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