Establishment of an Immortalized Endometriotic Stromal Cell Line from Human Ovarian Endometrioma

The primary Ec-ESCs that were successfully isolated from a human endometrioma, exhibited the typical fibroblast-like morphology as shown in Figure 1A . The stromal origin was verified by immunofluorescent staining of vimentin, cytokeratin and the endometriotic stromal cell marker hIFITM1 [ 23 ]. Figure 1B shows that Ec-ESC were positive for vimentin and hIFITM1 and were negative for cytokeratin. The Ec-ESCs were transfected with the hTERT expressing plasmid and selection was done with media containing 25μg/ml hygromycin-B for 8 days. The selected cells were expanded with 10μg/ml hygromycin-B and were maintained at the same concentration. As shown in Figure 2A and Fig 2B , the Ec-ESCs maintained a fibroblast-like appearance with a vimentin-positive and cytokeratin-negative status after hTERT immortalization. iEc-ESC had a bipolar or multipolar shape and grew in a parallel arrangement at confluence. After 1 passage and 20 passages, the iEc-ESC still exhibited the fibroblastic spindle-shape morphology. The hTERT expression was analyzed by RT-qPCR to confirm immortalization. Compared to primary Ec-ESC, the hTERT mRNA level of iEc-ESC was over 50000 fold, which indicated a successful hTERT immortalization ( Figure 2C ). TP53 mRNA level did not show a significant change after hTERT immortalization compared with primary Ec-ESC, indicating no changes in DNA damage. We examined primary and immortalized Ec-ESC for the expression of ERβ, PR and SF-1, which have been shown to be expressed in endometriotic lesions[ 24 ]. As shown in Figure 3 , the primary Ec-ESC expressed strong ERβ and SF-1 and weak PR. Five and 20 passages after hTERT immortalization, expression levels of ERβ, PR and SF-1 in iEc-ESC were not changed compared to the primary Ec-ESC. Ec-ESCs, iEc-ESCs and Eu-ESCs were decidualized following treatment with EPC in vitro . Following EPC treatment for six days, normal Eu-ESCs exhibited a cuboidal appearance with increased cell volume and ambiguous cell boundaries, which are typical features of decidual cells. Significantly higher mRNA expression of decidual cell markers Prolactin ( PRL ) and IGFBP1 were evident in the EPC treated group compared to the vehicle treated group ( Figure 4 ). In contrast, following EPC treatment for six days, both Ec- ESC and iEc-ESC rarely showed a cuboidal appearance. PRL and IGFBP1 were significantly increased in the EPC treated group compared to the vehicle group in both Ec-ESC and iEc-ESC ( Figure 4A and 4B ).There was no significant change in PRL and IGFBP1 mRNA expression when iEc-ESC were compared with Ec-ESC ( Supplemental Figure 1 ). However, when compared with normal Eu-ESC, PRL mRNA level in iEc-ESC was about 70% less; IGFBP1 mRNA levels in iEc-ESC was 99% less ( Figure 4C ), which suggests both primary and immortalized Ec-ESC had an impaired decidualization response. Karyotypic analysis was performed on iEc-ESC ( Fig 5 ). Cytogenetic analysis was performed on nineteen G-banded metaphase cells from human iEc-ESC. Fifteen cells demonstrated an apparently normal female karyotype ( Fig 5A ), four cells demonstrated an unbalanced translocation between the long-arm of chromosome 1 at band q44 and the long-arm of chromosome 11 at band q13, resulting in partial trisomy 11q (11q13 ->qter) ( Fig 5B ). The PCR method was used to detect mycoplasma contamination in the iEc-ESC using appropriate positive and negative controls. Following PCR amplification, the results obtained from mycoplasma analysis revealed that media harvested from iEc-ESCs showed single band on semi-quantitative gel suggesting that these cells did not have mycoplasma contamination ( Supplemental Figure 2 ).

The study was approved by the Institutional Review Boards of Michigan State University, Spectrum Health Medical System (Grand Rapids, MI) and Prisma Healthcare (Greenville, SC). Written informed consent was obtained from participating patients. Normal eutopic endometrial stromal cells (Eu-ESCs) were isolated from endometrial biopsy collected from a 32 year old Hispanic woman without endometriosis at Prisma Healthcare (Greenville, SC)[ 21 ]. The ovarian endometrioma was obtained from a 25-year-old Caucasian woman with dysmenorrhea. The pathology report indicated an endometriotic cyst with a fragment of fallopian tube with serosal endometriosis on the left ovary and a benign simple cyst on the right ovary. Primary endometriotic stromal cells were isolated from a portion of the endometriotic cyst on the left ovary as previously described[ 19 ] with some modifications. Fresh endometrioma specimens were collected in HBSS (Gibco) and were rinsed to remove blood cells. The tissues were chopped into small pieces and digested in phenol-red free DMEM/F-12 containing collagenase IV (final concentration 1mg/ml), 0.5% Trypsin (final concentration 0.01%), DNase I (final concentration 40μg/ml) for 60 min on a rotator in a 37°C incubator. The digestion was neutralized by the addition of 0.5ml FBS. After the red blood cells were lysed using red blood cell lysis buffer, the mixture was first filtered through a 70μm cell strainer. The cells were then separated through a 40μm cell strainer, so that the dispersed stromal cells passed through the strainer into filtrate. Endometriotic stromal cells were cultured with complete growth medium (phenol red–free DMEM/F12 medium supplemented with 10% charcoal dextran stripped fetal bovine serum, 100 units/ml of penicillin/streptomycin, and 1 mM sodium pyruvate) in a T25 culture flask at 37°C in an atmosphere of 5% CO2/95% air. To prepare the lentiviral vector, 293FT cells were plated on a 6-well plate and cultured in DMEM with 10% FBS. 1.5 mL of OptiMEM (Gibco) was combined with 42 μL of Lipofectamine-3000 (Invitrogen #11558–019) and kept at rest for 5 min. hTERT plasmid DNA (4.5μg, Addgene plasmid #85140) was mixed with 13.5μg Trans-Lentiviral packaging mix (TLP4606), 36μl P3000 and 1.5 mL of OptiMEM and then combined with Lipofectamine–OptiMEM mixture and kept at rest for 20 min at room temperature. 293FT cells had a media replacement with 1 mL of basal DMEM, at which point 500 μL of the plasmid DNA–OptiMEM–Lipofectamine mixture was added. After 24 hours, each well was replaced with fresh medium. After 48 hours, the viral supernatant was collected, centrifuged to remove debris, and frozen at −80 °C until use. Primary Ec-ESC were seeded onto six-well plates until they reached 80% confluence. The cells were infected with the hTERT lentiviral vector supernatant in the presence of 8μg/ml polybrene. Eight hours later, the medium from the first infection was removed and the cells were re-infected with fresh lentiviral supernatant. Eighteen hours later, the medium was removed, and the complete growth medium was added to the cells. After 72 hours of incubation, cells were cultured in fresh medium with 25μg/ml hygromycin. Following selection for 8 days, the cells were maintained in medium with 10μg/ml hygromycin for expansion. Total RNA was isolated from cultured cells using TRIzol reagent (Life Technologies). RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA). qPCR was then performed to measure gene expression levels with Power Up SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) using the ViiA7 qPCR System (Applied Biosystems, Foster City, CA). The following primers were used: hTERT (forward 5′-CGGAAGAGTGTCTGGAGCAA-3′ and reverse 5′-GGATGAAGCGGAGTCTGGA-3′), TP53 (forward 5′-CAGCACATGACGGAGGTTGT-3’ and reverse 5′-TCATCCAAATACTCCACACGC-3’), IGFBP1 (forward 5′-CCTGCCAAACTGCAACAAGA-3’ and reverse 5′-TCCCATTCCAAGGGTAGACG-3’), PRL ( forward 5′-ACCCTGTCTGGTCGGGACTT-3′ and reverse 5′-TGTTGTGGATGATTCGGCAC-3’), RLP17 (forward 5’-ACGAAAAGCCACGAAGTATCTG −3’ and reverse 5’-GACCTTGTGTCCAGCCCCAT −3’. RPL17 was used for normalization. Cells were grown on glass coverslips to 80–90% confluency. The coverslips were washed with PBS and fixed with 2% paraformaldehyde in PBS for 10 min at 37°C. The fixed cells were treated with blocking buffer (PBS /5% goat normal serum/ 0.3% of Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 30 minutes at room temperature. The cells were then incubated with the primary antibodies overnight at 4°C and secondary antibodies for one hour at room temperature. The primary antibody information is as follows: Vimentin polyclonal antibody (#5741S) was from Cell Signaling Technology, Inc. (Waltham, MA). Cytokeratin, pan (Mixture) monoclonal antibody ( C2562 ) was from Sigma-Aldrich (St. Louis, MO). Estrogen receptor beta polyclonal antibody (PA1–310B) and progesterone receptor monoclonal antibody (MA5–14505) were from Thermo Fisher Scientific (Cambridge, MA). Human Polyclonal IFITM1 antibody (AF4827) was from R&D Systems (Minneapolis, MN). The secondary antibody information is as follows: DyLight 594 goat anti-rabbit IgG antibody (DI-1594) and DyLight 488 goat anti-rabbit IgG antibody (DI-1488) were from Vector Laboratories (Burlingame, CA). Alexa Fluor® 594 AffiniPure donkey anti-goat IgG (H+L) and Alexa Fluor® 488 AffiniPure donkey anti-mouse IgG (H+L) were from Jackson ImmunoResearch Laboratories Inc (West Grove, PA). PBS washed coverslips were then mounted onto microscope slides with a DAPI-impregnated mounting media (Vector Laboratories, Burlingame, CA) to enable nuclear visualization and images captured with a fluorescent microscope (Nikon Instruments Inc., Melville, NY) using software from NIS Elements, Inc. (Nikon, Melville, NY). The immortalized endometriotic stromal cells (iEc-ESC) were cultured in antibiotic-free growth media. When the cells reached confluency, the supernatant was collected and the mycoplasma status was checked following the manufacturer’s instructions (MP0025, Millipore Sigma, Louis, MO). Immortalized endometriotic stromal cells were cultured in T25 flasks. When the cells reached 60–70% confluence with large colonies, the cells were sent to Cell Line Genetics, Inc. (Madison, WI) for karyotype analysis. Decidualization was induced in primary normal eutopic endometrial stromal cells, endometriotic stromal cells and immortalized endometriotic stromal cells (in triplicate) at 80% to 90% confluence Cells were incubated in phenol red–free DMEM/F12 medium supplemented with 2% charcoal dextran stripped fetal bovine serum with EPC (36 nM 17β-estradiol, 1 μM medroxyprogesterone acetate and 0.5 mM dibutyryl-cAMP ) for up to 6 days with a media change every 2 days[ 22 ]. Controls were treated with vehicle (100% ethanol) and the media were changed every 2 days. Statistical analysis was performed using SPSS version 18.0 (SPSS). All data were expressed as mean±SD. The Student’s t-test was used for comparisons between the two groups. P< 0.05 was considered statistically significant (two tailed).

Endometriosis is an inflammatory gynecological disorder dominated by estrogen. As an effective model for endometriosis, it is important that immortalized endometriotic cell lines preserve the inherent characteristics of endometriotic stromal cells and are differentiated from a cancer cell phenotype. Our results show that iEc-ESCs were positive for vimentin but negative for cytokeratin. TP53 gene expression also did not change compared to the primary Ec-ESCs, which indicates that iEc-ESCs maintained their normal stromal phenotype after hTERT immortalization. Different groups have developed eutopic and/or ectopic cell lines using various techniques[ 25 , 26 ]. Chen et al. generated hEM15A (eutopic) and hEM5B2 (ectopic) cell lines using PCD2SV40T plasmids. hEM15A cells have normal chromosome structure and exhibit eutopic endometrial stromal characteristics, however, hEM5B2 cells had a polyploid karyotype and demonstrated epithelial characteristics[ 25 ]. The simian virus 40 (SV40) expression induced immortalization is routinely used to generate cell lines, however, this technique can cause aberrant differentiation, compromised DNA damage response and altered chromosome numbers[ 27 ]. The changes acquired during SV40 immortalization suggest that these immortalized cells resemble tumor cells[ 27 ]. Similarly, human papillomavirus (HPVs) E6/E7 induced immortalization can also lead to chromosomal imbalance and could adapt a malignant phenotype[ 28 ]. End1/E6E7 and VK2/E6E7 are two HPV-16 E6/E7 transformed epithelial cell lines available through American Type Culture Collection (ATCC). It is important to note that End1/E6E7 and VK2/E6E7 were developed from the endocervix and vaginal tissue respectively from women with endometriosis and are not representative of endometriotic lesions. Considering the pros and cons of various immortalization techniques, we immortalized ovarian endometriotic stromal cells using hTERT. Data obtained from the cytogenetic analysis suggest that iEc-ESCs have no major karyotypic change and have 46, XX genotype. We observed that 15/19 cells population have a normal female karyotype and 4/19 cells demonstrated an unbalanced translocation between the long-arm of chromosome 1 at band q44 and the long arm of chromosome 11 at band q13, resulting in partial trisomy 11q (11q13 ->qter). The observed partial trisomy in the fraction of immortalized iEc-ESCs might be due to hTERT induced immortalization. Furthermore, there is also a probability that the partial trisomy was retained from the original primary endometriotic stromal cells harvested from the endometrioma tissue. It has been reported that there is a higher frequency of chromosomal aberrations in ovarian endometriosis compared to extragonadal endometriosis and the normal endometrium[ 29 ]. Cells were further analyzed for the presence of endometriotic stromal cells markers ER-β, PR and SF-1 before, and at 5 and 20 passages following hTERT immortalization. We observed that both the original primary Ec-ESC and iEc-ESC had strong ERβ and SF-1 expression and weak PR expression. Moreover, we also observed that there was cytoplasmic ERβ staining in the iEc-ESCs. ERβ is well known for its role as a transcription factor involved in biological functions. However, ERβ in the cytoplasm plays an important role in maintaining cell survival by interacting with cytoplasmic apoptotic machinery to prevent TNF-a-induced apoptosis[ 30 ]. Additionally, in vitro decidualization induction with EPC induced a significant increase of IGFBP1 and PRL mRNA levels compared to the vehicle treated group in both primary and immortalized Ec-ESC, suggesting that these cells are hormone-responsive. In contrast, IGFBP1 and PRL levels were significantly lower in iEc-ESC compared to disease-free normal eutopic endometrial stromal cells exposed to the decidualization treatment. Those observations are in agreement with previously published studies [ 10 ] and revealed that iEc-ESCs maintain the inherent characteristic of endometriotic stromal cells by exhibiting a diminished decidualization response. Endometriosis is categorized into three subtypes, OE, PE, and DIE. A recent bioinformatic study found that, although there are some common features amongst the different subtypes of endometriosis, PE tended to be more associated with dysregulated peritoneal immune and inflammatory microenvironment while OE and DIE seemed to be at more risk of malignant development [ 31 ]. In addition, that report also suggested differences in the lesion microenvironment of three endometriosis subtypes[ 32 ]. Compared to DIE, OE lesions showed increased ER-β but reduced PR-B levels, as well as significantly lower fibrotic content and greater vascularity[ 32 ]. These findings indicate the need for subtype specific in vitro model systems to study this enigmatic disease. Zeitvogel et al [ 18 ] have previously generated series of endometriotic cell lines including 22B (immortalized endometriotic stromal cells). The primary cells in this study were obtained from a light red color peritoneal lesion and subsequently immortalized using SV40. The 22B cells have been used to investigate endometriosis associated invasion, migration and adhesion[ 33 , 34 ]. In another study[ 19 ], the ectopic stromal cells were harvested from deep endometriotic tissues and immortalized using a hTERT expressing retroviral vector. This immortalized stromal cell line retained steroid hormone receptor expression and function. The endometriotic stromal cell lines from these two groups make in vitro tools available for both peritoneal endometriosis and deep infiltrative endometriosis[ 18 , 19 ]. However, to the best our knowledge, there are no immortalized endometriotic stromal cell line available to study ovarian endometriosis which is observed in about 20% of the patients with the disease[ 20 ]. The iEc-ESC generated from ovarian endometrioma in the present study would be a valuable in vitro tool to investigate the pathology of ovarian endometriosis. This together with the stromal cell lines previously generated from peritoneal and deep infiltrating disease[ 18 , 19 ] will serve as useful tools to study cellular differences between the three subtypes of this disease. Collectively, these data support the utility of iEc-ESC as a new, potentially ideal in vitro endometriotic stromal cell model to study endometriotic lesion pathophysiology. These well-characterized iEc-ESC will likely serve as a vital resource to study cellular processes and molecular mechanisms pertaining to ectopic stromal cell biology. Due to the feasibility of in vitro gene manipulation, iEc-ESC may also become a valuable tool to study loss-of-function and gain-of-function regulation in the context of endometriotic lesion pathology. The immortalized ectopic stromal cell line we have developed is physiologically similar to primary endometriotic stromal cells from endometriomas. These cells will serve as a valuable resource to investigators for in vitro studies designed to understand underlying mechanisms associated with the pathophysiology of endometriosis.

Endometriosis is a chronic gynecological disorder in women of reproductive age. It is characterized by the presence of endometrial-like tissue outside of the uterine cavity[ 1 ]. Endometriosis usually develops in the pelvic and abdominal organs and/cavities, such as the peritoneal wall, ovaries and fallopian tubes[ 1 ]. It is generally categorized into three subtypes, ovarian endometriosis (OE), peritoneal endometriosis (PE), and deep infiltrating endometriosis (DIE). [ 2 ]. Symptoms of endometriosis often include dysmenorrhea, chronic pelvic pain, infertility and an elevated risk of ovarian cancer[ 3 – 5 ]. Current treatments include surgical removal of ectopic tissue and/or hormonal treatment to suppress ovarian function, which provide a temporary but not permanent cure[ 6 ]. The etiology and pathogenesis of endometriosis remain an enigma. Until now, the most widely accepted hypothesis is Sampson’s theory, in which the exfoliated menstrual endometrial cells reflux and attach to the peritoneal membrane and subsequently form endometriotic lesions [ 7 ]. Like the endometrium, endometriotic tissues can respond to the hormonal changes during the menstrual cycle. However, endometriotic cells have certain atypical properties compared to the normal eutopic endometrium. Those include high local estrogen biosynthesis, progesterone resistance and impaired decidualization in endometriotic stromal cells (Ec-ESC)[ 8 – 10 ]. Steroid receptor expression is also altered in endometriotic lesions and in general estrogen receptor α(ERα) and progesterone receptor (PR) are decreased and estrogen receptor β (ERβ) is increased[ 10 – 12 ]. But why these nuclear receptors are abnormally expressed is still not well understood. Thus, dissecting the subtle cellular and molecular mechanisms within endometriotic lesions might help to understand the pathogenesis of endometriosis and explore more effective and thorough therapies. One of the major limitations in understanding the pathological events associated with endometriosis is the long delay from the initiation of the disease until the onset of symptoms and the diagnosis. Therefore, to study the cellular and molecular mechanisms related to endometriotic lesion development, appropriate in vivo and in vitro models are indispensable[ 13 , 14 ]. For in vivo studies, the non-human primate and rodents have been used most exclusively. Non-human primates offer a physiologically relevant model and also provide an opportunity to study the progression of the disease [ 15 , 16 ]. But the high cost, technical skillset and required infrastructure are the major limitations associated with the use of non-human primate models. Due to their size and lower cost, the rodent models permit tissue-specific gene manipulation and are advantageous to initially test therapeutic drugs [ 17 ], but rodents lack natural menstruation and do not develop spontaneous endometriosis. The in vitro model systems allow us to study endometriosis-associated cellular and molecular mechanisms in the cell specific manner. Cell lines can be easily genetically manipulated and tested for various therapeutic targets. Therefore, the in vitro endometrial cell model is still necessary for studying this enigmatic disease. Primary cell cultures obtained from endometriotic lesion biopsies have potential, however, the source and consistent availability of endometriotic tissues from patients are major concerns. Moreover, the short life span of primary cultures of endometriotic cells limit their use. To overcome this obstacle, primary cells can be immortalized by transducing lentiviral vectors expressing the large T antigen of simian virus 40 (SV40 LT) or(and) human telomerase reverse transcriptase (hTERT). At present endometriotic stromal cell lines that are available were derived from peritoneal endometriosis and deep endometriosis[ 18 , 19 ]. However, there is no endometriotic stromal cell line available for other types of endometriosis, especially from ovarian endometriosis which are found in 20% of patients with the disease[ 20 ]. In this study, we successfully generated a human endometriotic stromal cell line from ovarian endometrioma biopsies by transducing cells with lentiviral vectors expressing hTERT. This immortalized cell line has a prolonged life span and retains specific immunological markers and functional properties that primary endometriotic stromal cells have. It will provide researchers one more in vitro tool for studying the dynamic changes of cellular processes associated with the disease.