Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition

Clinical studies have recently demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficacious potential therapy for pulp regeneration. However, some limitations need to be addressed, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more efficient in vitro culturing of human dental pulp stem cells might be useful for providing low cost and high reliability testing for pulp regeneration therapy. In this study, we established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original diploid chromosomes and stemness characteristics and exhibited an enhanced proliferation rate. In addition, we showed the immortalized human dental pulp stem cells still keeps their osteogenic and adipogenic differentiation abilities under appropriate culture conditions even though the cell proliferation was accelerated. Taken together, our established cell lines could serve as a useful in vitro tool for pulp regeneration therapy, and can contribute to reproducibility and ease of cell handling, thereby saving time and costs associated with safety and quality control tests.


Introduction
Human dental pulp stem cells exhibit high proliferation, greater tissue regeneration capabilities, lower immunogenicity, and greater plasticity than those of other mesoderm-derived mesenchymal stem cells [1]. Furthermore, unlike other mesoderm-derived mesenchymal stem cells, human dental pulp stem cells are isolated easily from extracted teeth without causing secondary damage or ethical controversy. Paino  cells are a good option for applications in human bone tissue engineering without the use of scaffolds in vitro and in vivo [2]. Therefore, human dental pulp stem cells have attracted attention as candidate cells for stem cell therapy for various disorders, including the regeneration of lost pulp and dentin in the root canal space [3,4]. Recently, a pilot clinical study and a phase I clinical trial in humans have been reported that demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficient therapeutic approach [5][6][7]. However, there are some limitations to this approach, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more effective tools are needed to provide low cost and high reliability for stem cell-mediated regeneration therapy of lost pulp.
Our research group has previously reported efficient immortalization in multiple species via co-expression of R24C mutant cyclin-dependent kinase 4 (CDK4 R24C ), Cyclin D1, and telomere reverse transcriptase (TERT) [8][9][10][11][12][13][14]. This immortalization method using mutant CDK4, Cyclin D1, and TERT was termed "K4DT" in reference to the introduced genes. The chromosomal pattern of cells established using the K4DT method is retained, along with the nature of primary cells, possibly due to the intact function of p53 [13,[15][16][17]. We also recently demonstrated that our corneal epithelial cell line, established with the K4DT immortalization method, can be a useful tool to detect eye toxicity, and it can be used as a new resource for in vitro ocular toxicity testing [18]. These findings indicated that applying the K4DT immortalization method to human dental pulp stem cells might be useful in generating a more effective tool to evaluate the safety and quality of isolated individual dental pulp cell products before transplantation. We speculated that in vitro culturing of human dental pulp stem cells immortalized by the K4DT method might be useful as a biological resource to reduce the cost of pulp regeneration therapy.
With this aim in mind, we transduced CDK4 R24C , Cyclin D1, and TERT into human dental pulp stem cells via retrovirus. We successfully established immortalized human dental pulp stem cells and evaluated the characteristics of the cells.

Cell Culture
Human dental pulp stem cells (PT-5025) were purchased from Lonza Japan Ltd (Tokyo, Japan) and were cultured according to the manufacturer's instructions.

Preparation and infection of recombinant retroviruses into human dental pulp stem cells
To immortalize primary human dental pulp stem cells, we prepared recombinant retroviruses expressing R24C mutant cyclin-dependent kinase 4 (CDK4 R24C ), Cyclin D1, and TERT. PQCXIP-CDK4R24C (puromycin-resistant), pQCXIN-Cyclin D1 (G418-resistant), and pCLXSH-TERT (hygromycin B-resistant) retroviral plasmids as well as pQCXIN-EGFP (G418-resistant) as a control expressing EGFP to monitor the efficiency of infection were constructed as described previously [16]. These retroviral plasmids were co-transfected into 293T cells together with packaging plasmids, pCL-GagPol and pCMV-VSV-G-RSV-Rev, by using the lipofection method [19]. Viral fluids recovered from the transfected cells were filtered through 0.45 μm disks (Sartorius, Goettingen, Germany; product code, 17598 K). Primary human dental pulp stem cells were inoculated with individual or mixed recombinant viruses in the presence of 8 μg/mL of polybrene (hexadimethrine bromide, Sigma-Aldrich, #H9268). The cell culture medium was replaced with fresh medium one day post-inoculation followed by selection with 1 mg/mL G418, 0.8 μg/mL puromycin and/or 40 μg/mL hygromycin according to drug resistance of each vector and its combination. One week post-selection, the obtained resistant cells were expanded into new plates. We named the retrovirus-infected human dental pulp cells WT (no treatment), EGFP (infected with EGFP-expressing retrovirus), K4D (infected with mutant CDK4 and Cyclin D1), TERT (infected with TERT), and K4DT (infected with mutant CDK4, Cyclin D1, and TERT) cells according to the introduced genes. The infected mass population of cells was subjected to further analysis, such as western blotting and extension of the life span.

Population doublings
To measure the cell proliferation rates of WT, K4D, and K4DT cells, we sequentially passaged the cells. All of the cell lines were cultured in DMEM with 10% FBS containing penicillin/ streptomycin. Each cell line was initially seeded into 6-well plates at a density of 5.0 × 10 4 cells in triplicate. When the cell line reached confluency, the cells were dispersed using 0.05% trypsin-EDTA (Life Technologies). We recorded the total number of cells in each dish using an automatic cell counter (Thermo Fisher Scientific, Waltham, MA, USA). Cells (5.0 × 10 4 ) were then seeded into a new dish to evaluate their growth rate by determining the population doubling level (PDL). The PDL was calculated using the following equation: PDL = log2 (A/B), where A is the number of cells harvested at each passage and B is the number of seeded cells.

Genomic polymerase chain reaction
Genomic DNA was extracted using a NucleoSpin Tissue kit (TaKaRa Bio, Shiga, Japan) according to the manufacturer's protocol. PCR amplification was performed using Go Taq Green Master Mix (Promega, Madison, USA) based on the protocol provided by the manufacturer (2 min of pre-denaturation at 95˚C, 30 cycles of 15 sec at 95˚C, 15 sec at 55˚C, and 1 min at 72˚C). For the detection of the Cyclin D1 cassette, the R24C mutant CDK4 cassette, and the TERT cassette, the PCR primer sequences used were described in our previous publication [14]. Tuberous sclerosis type II (TSC2) was used as an internal control since the TSC2 gene is a unique gene in the human genome, and furthermore there are no TSC2 pseudogenes. For internal control of genomic amplification, we designed forward and reverse primers for TSC2, (5'-ACT AGG CAG CAA CCA GCG TCAC -3') and (5'-TGG ACC CCA TCT CGG CAC CA-3'), respectively. PCR products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide.

Cell cycle assay
The cell cycle phase was analyzed using a Muse Cell Cycle Assay Kit (#MCH100106; Merck) and Muse Cell Analyzer (#0500-3115; Merck). The detailed method for the analysis was based on the protocol provided by the manufacturer. Statistical significance was evaluated using a non-parametric Steel-Dwass test with six samples for each experimental group.

Senescence-associated β-galactosidase staining
At passage 7, WT, K4D, and K4DT cells were seeded at a density of 5.0 x 10 4 cells/well in 6-well plates. After 4 days of culture, they were fixed and stained using a Senescence Detection Kit (#K320-250; Biovision, Inc., Milpitas, CA, USA) according to the manufacturer's protocol.

Karyotype analysis
Karyotype analyses were carried out in the K4DT cells. Standard G-banding chromosome analysis was performed in the Nihon Gene Research Laboratories, Inc. (Sendai, Japan). The chromosome number was determined from 50 mitotic cells, and the detailed chromosomal condition was evaluated using G-banding in 20 mitotic cells.

Flow cytometric analysis
Cells (1.0 × 10 6 cells) were suspended in PBS containing 2% FBS and 2 mM EDTA, and stained with FITC conjugated antibodies against human CD90, CD45, and CD34 (BioLegend, San Diego, CA, USA) to characterize the K4DT cells compared with the WT cells. FITC mouse IgG1, κ Isotype control antibody and FITC mouse IgG2a, κ Isotype control antibody (BioLegend, San Diego, CA, USA) were used for the negative control experiments. Data were analyzed with a CytoFlex flow cytometry system (Beckman Coulter, Brea, CA, USA).

Establishment of cell lines of human dental pulp stem cells transduced with CDK4 R24C , Cyclin D1, and TERT
We transduced EGFP or human CDK4, Cyclin D1, and TERT into primary human dental pulp stem cells using retroviruses. We monitored the gene delivery efficiency of the retroviruses in the dental pulp stem cells using the recombinant retrovirus QCXIN-EGFP as a control. We estimated that the transduction of these genes into the dental pulp stem cells by retroviruses had an efficiency of approximately 50% (Fig 1, middle panels). Due to the presence of a neomycin-resistance gene downstream of QCXIN-EGFP, we performed selection using G418 antibiotics to purify the EGFP-expressing cells. The surviving cells selected by treatment with 1 mg/mL G418 were almost all positive for EGFP expression (Fig 1, lower panels), indicating that antibiotic selection worked properly. To evaluate the potential toxicity of these genes in human dental pulp cells, we compared the cell morphologies of the recombinant cells with those of primary cells. Although the recombinant cells' sizes were relatively smaller in K4D, K4DT, and TERT cells, the recombinant cells had a similar morphology to WT cells (Fig 2), indicating that transduction of human CDK4 R24C , Cyclin D1, and TERT via retroviruses did not cause any toxicity in these cells.

Detection of the genomic expression cassettes with PCR and detection of protein expression
To monitor the genomic insertion of exogenous genes, we performed PCR using genomic DNA extracted from EGFP, WT, K4D, K4DT, and TERT cells. The amplification products specific to the Cyclin D1, CDK4, and TERT expression cassettes were detected with expected ratios (Fig 3A), indicating that the expression cassettes were integrated into the cells. The results of TSC2 PCR amplification as a control showed that all genomic DNA produced sufficient amplification products, indicating that the recovery of genomic DNA and the amplification reaction worked properly. Furthermore, we carried out western blotting to detect the expression of Cyclin D1 and CDK4 proteins with specific antibodies. K4D and K4DT cells showed specific bands at the expected molecular weight, whereas WT showed a weak signal, which could be attributed to endogenous dental pulp stem cell-derived Cyclin D1 and CDK4 proteins (Fig 3B). Taken together, these data showed that the exogenous genes were inserted into the genomic DNA of human dental pulp stem cells and that functional proteins were produced by retroviral transduction.

K4DT cells continued to proliferate without cellular senescence
We compared proliferation of the established cells with or without transgenes. At the beginning, a specific number (5 x 10 4 cells/35 mm diameter dish) of WT, K4D, and K4DT cells were seeded. The K4D and K4DT cells showed an increase in cell growth speed compared to WT cells (Fig 4A). The K4DT cells did not exhibit a decrease in cell proliferation, whereas K4D cells showed slower rates of proliferation at around passage 4. Furthermore, the WT and K4D cells showed enlarged cytoplasm, and the cells stained positively for senescence-associated beta-galactosidase (SA-β-Gal) at passage 7 (Fig 4B, upper and middle panels), indicating the presence of cellular senescence in WT and K4D cells. However, the K4DT cells showed no morphological changes and did not stain positively for SA-β-Gal (Fig 4B, lower panel). These results indicate that, although the expression of CDK4 R24C and Cyclin D1 induces dramatically enhanced cell proliferation speed until senescence at an early passage, co-expression of these genes is not enough to immortalize human dental pulp stem cells. We therefore concluded that the combined expression of CDK4 R24C , Cyclin D1, and TERT allowed us to efficiently establish immortalized cells from human dental pulp cells.

Cell cycle assay
To compare the cell cycle distribution of cells, we performed cell cycle analysis of WT, K4D, and K4DT cells at passage 2. We compared the percentages of cells in each phase of the cell cycle. K4D and K4DT cells showed similar cell cycle profiles as WT cells, with a small fraction of S phase cells (Fig 5A). We also used six samples from each group to evaluate the potential effect of cell cycle stages. Both K4D and K4DT cells showed a significant decrease in the ratio of G0/G1 phase and an increase in the ratio of G2/M phase compared to WT cells (Fig 5B). These results indicated that cell proliferation was accelerated by the expression of mutant CDK4 and Cyclin D1.

Karyotype analysis
We evaluated the chromosomal karyotype of K4DT cells. To obtain the chromosome number, we evaluated 50 mitotic cells. The results showed that K4DT cells displayed a chromosome number of 2n = 46, indicating that K4DT cells retained the original number of chromosomes (Fig 6A, 6B and 6C). In G-banding analysis, two samples out of 20 mitotic cells exhibited a chromosomal abnormality, indicating that more than 90% of K4DT cell samples had normal G banding patterns of human chromosomes (Fig 6D).

Characterization of immortalized human dental pulp stem cells
To determine whether our immortalized cells keep the original characteristic of dental pulp stem cells, the stemness and their differentiation abilities were assessed by using the K4DT cells at passage 8, 15, or 16. Flow cytometry analysis of the K4DT cells showed more than 70% expressed CD90, which is cell surface markers in mesenchymal stem cells, whereas less than 1.0% of the K4DT expressed CD45 or CD34, which is hematopoietic cell surface markers. The WT cells showed similar results that CD90 expression was positive, whereas CD45 and CD34 expression were negative (Fig 7). These data indicated that the K4DT cells keep the original cell surface markers. In addition, K4DT cells at passages 15 showed positive staining of calcified materials by Alizarin red S after 17 days of culture in the osteoinduction medium ( Fig  8A). Furthermore, we performed the immunofluorescence staining with anti-osteocalcin antibody. Osteocalcin was used for the mature osteoblast differentiation marker due to encode a bone specific protein synthesized by osteoblast [20]. The K4DT cells after 25 days of culture in the osteoinduction medium showed the osteocalcin-positive expression cells, while the cells of culture in the normal medium showed negative (Fig 8B). The data indicated that K4DT cells have the potency to differentiate into osteoblasts. We also observed that the K4DT cells at passages 16 developed numerous lipid droplets stained with Oil Red O at 23 days after the cells were induced to differentiate in the adipoinduction medium (Fig 9). The data indicate that the K4DT cells have the differentiation ability to adipocytes. From these results, we concluded that our established immortalized human dental pulp stem cells preserved the expression of stemness associated markers and their multi-differentiability even after passages.

Discussion
In this study, by co-expressing mutated cyclin-dependent kinase 4 (CDK4 R24C ), Cyclin D1, and telomerase reverse transcriptase (TERT), we established a novel immortalized human dental pulp stem cell line, termed K4DT cells, which do not have major chromosomal abnormalities or a transformed phenotype. Analysis of cell proliferation over sequential passages showed that K4DT cell lines had a more accelerated proliferation speed than K4D cell lines and primary human dental pulp stem cells. During these experiments, K4DT cells showed continuous proliferation without affecting differentiation potential and signs of senescence. This result indicated that using a combination of CDK4 R24C , Cyclin D1, and TERT efficiently immortalized human dental pulp stem cells.
Cellular stress activates the p16-Rb (retinoblastoma protein) pathway [21]. The p16 protein binds the Cyclin D1-CDK4 complex and inactivates its kinase activity, which inhibits endogenous CDK4, resulting in a slowdown of cell cycle turnover [17]. Although the overexpression of wild-type CDK4 sequesters p16, CDK4 alone does not completely suppress Rb, because its kinase activity is inhibited by p16. Overexpression of mutant CDK4 (CDK4 R24C ), which is not bound by p16, in cooperation with Cyclin D1 can phosphorylate and suppress Rb in the presence of high levels of p16 [17]. However, to immortalize some cell types in culture, in addition to inactivation of the p16-Rb pathway, the expression of TERT is required to prevent progressive telomere shortening [22]. Sasaki et al. showed that neither CDK4 R24C nor Cyclin D1 alone or in combination with TERT could efficiently immortalize primary human ovarian surface epithelium cells [14]. In fact, our present study showed that combining the expression of CDK4 R24C and Cyclin D1 is insufficient to immortalize human dental pulp stem cells, termed K4D. We therefore concluded that the combined expression of the three genes successfully immortalized human dental pulp stem cells. We believe that our established K4DT cell line has the advantage of being easy to handle, which should increase the reproducibility and save time and costs for the safety and quality control tests needed for universal dental pulp regeneration therapy.
A previous study showed that human pulp-derived cells can be immortalized with Simian Virus 40 T-antigen (SV40) [23]. Although SV40 is well known to be able to effectively immortalize most cell types by inactivating the p53 and Rb pathways and interacting with a wide range of cellular proteins [24], the expression of SV40 frequently leads to chromosomal abnormalities and polyploidy in transformed human cells [25][26][27]. Our established immortalized human dental pulp stem cells showed no change in chromosome number, 46+XY. These results indicated that the K4DT immortalization method, which uses co-expression of CDK4 R24C , Cyclin D1, and TERT, is better able to preserve the original nature of dental pulp stem cells without chromosomal instability compared to other immortalization methods, such as the use of SV40. However, in the G-banding analysis, we noted chromosomal aberrations in two out of 20 mitotic cells. We previously showed that 6.5% of cells exhibited abnormal chromosome patterns, even in wild type pig embryonic fibroblasts [26]. Therefore, there is a possibility that the~10% observed chromosome abnormalities could be an artifact caused by sample preparation for chromosome analysis. On the other hand, immortalization driven by the expression of TERT alone was recently reported in cell types related to the dental field, including dental pulp cells [28,29], human deciduous tooth dental pulp cells [30], and gingival fibroblasts [31]. Although we also tried to obtain immortalized dental pulp stem cells through the expression of TERT alone, we were unable to, even after confirming the successful integration of TERT into cells by PCR. Dental pulp stem cells expressing TERT alone stopped proliferating soon after expanding into new plates for further analysis assays, whereas K4D and K4DT cells showed constant proliferation. We used the pCLXSH-TERT plasmid that utilizes the viral LTR promoter for TERT expression, whereas others reported that immortalized dental pulp cells using TERT could be generated using the pQCXIP plasmid [32]. There is a possibility that dental pulp stem cells expressing TERT alone using the CMV promoter, which is stronger than the LTR promoter, or changing culture conditions that stimulate the stress signaling pathway [33] might promote continuous proliferation in cells transformed with TERT alone.
We performed flow cytometric analysis and the differentiation experiments used as methods for the identification and characterization of dental pulp stem cells [34], and observed our established immortalized human dental pulp stem cells, K4DT cells maintained its original stemness characteristics without affecting differentiation potential. The results suggest that coexpression of CDK4 R24C , Cyclin D1, and TERT retain the original differentiation abilities of human dental pulp cells even though the cell proliferation was accelerated. Shiomi et al. reported that the co-expression of these three genes keeps the original cellular differentiation ability of human myogenic cells [17]. Our present results in differentiation experiments exhibited higher differentiation potential in the K4DT cells compared to that in primary human dental pulp stem cells, termed WT cells for both osteogenesis and adipogenesis. In these experiments, we used the WT cells at passage 8, which is passage number the cells have exhibited slower rates of cell proliferation as shown in Fig 4A. Therefore, the exact mechanism of the higher differentiation potential in K4DT cells has been unknown, the differentiation potential in K4DT cells might be due to keep the differentiation-inducing condition as primary human dental pulp stem cells in the early passages.

Conclusion
We established novel human dental pulp stem cell lines without major chromosomal abnormalities or a transformed phenotype and losing their original characteristics by co-expressing mutant CDK4, Cyclin D1, and TERT. Our experimental results will contribute to the reduction of the therapeutic cost of pulp regeneration.