Assessment of Fallopian Tube Epithelium Features Derived from Induced Pluripotent Stem Cells of Both Fallopian Tube and Skin Origins

Fallopian tube epithelial cells (FTECs) play a significant role in the development of high-grade serous ovarian cancer (HGSOC), but their utilization in in vitro experiments presents challenges. To address these limitations, induced pluripotent stem cells (iPSCs) have been employed as a potential solution, driven by the hypothesis that orthologous iPSCs may offer superior differentiation capabilities compared with their non-orthologous counterparts. Our objective was to generate iPSCs from FTECs, referred to as FTEC-iPSCs, and compare their differentiation potential with iPSCs derived from skin keratinocytes (NHEK). By introducing a four-factor Sendai virus transduction system, we successfully derived iPSCs from FTECs. To assess the differentiation capacity of iPSCs, we utilized embryoid body formation, revealing positive immunohistochemical staining for markers representing the three germ layers. In vivo tumorigenesis evaluation further validated the pluripotency of iPSCs, as evidenced by the formation of tumors in immunodeficient mice, with histological analysis confirming the presence of tissues from all three germ layers. Quantitative polymerase chain reaction (qPCR) analysis illuminated a sequential shift in gene expression, encompassing pluripotent, mesodermal, and intermediate mesoderm-related genes, during the iPSC differentiation process into FTECs. Notably, the introduction of WNT3A following intermediate mesoderm differentiation steered the cells toward a FTEC phenotype, supported by the expression of FTEC-related markers and the formation of tubule-like structures. In specific culture conditions, the expression of FTEC-related genes was comparable in FTECs derived from FTEC-iPSCs compared with those derived from NHEK-iPSCs. To conclude, our study successfully generated iPSCs from FTECs, demonstrating their capacity for FTEC differentiation. Furthermore, iPSCs originating from orthologous cell sources exhibited comparable differentiation capabilities. These findings hold promise for using iPSCs in modeling and investigating diseases associated with these specific cell types.


Introduction
Epithelial ovarian cancer, specifically high-grade serous ovarian cancer (HGSOC), is the fifth leading cause of cancer-related deaths in women worldwide, with a global annual diagnosis of over 220,000 cases [1,2].Most patients with ovarian cancer have late-stage disease, with a five-year survival rate of only 30-40% [1].Unfortunately, most Cells 2023, 12, 2635 2 of 17 ovarian cancer patients are diagnosed at an advanced stage, bearing a five-year survival rate of merely 30-40%.Notably, HGSOC has been found to originate from the fallopian tube epithelium (FTE) [3], but the scarcity of an appropriate research model due to the challenges in retrieving and sustaining FTE in vitro has posed a significant hurdle.
FTE encompasses two crucial types of epithelial cells: secretory and ciliated.The current research models for fallopian tubes, including ex vivo and organoid formation models, strive to maintain cell polarity by reestablishing ciliated and secretory cells [4][5][6].However, these models face limitations due to the absence of luminal architecture and a conducive microenvironment, leading to restricted fallopian tube epithelial cell (FTEC) proliferation and increased senescence [4][5][6].An alternative involves employing FTE research in animal models, such as genetically engineered mice and patient-derived xenografts, yet these models may not fully replicate the complex evolution of human tumors [7][8][9][10][11].
Furthermore, obtaining FTE requires surgical intervention, making it challenging to retrieve repeatedly [12].Thus, there is a compelling need for a consistent FTEC line, potentially offering an enduring resource for cancer research.While current FTEC lines primarily focus on cancer initiation, they may not precisely mirror actual genetic changes, emphasizing the necessity for fresh FTECs as a renewable source for research.
Interestingly, FTECs demonstrate stemness, self-renewal capabilities, and Wnt-related organoid formation [13].They display a cuboidal cell morphology and consistent proliferation up to nine passages [13].Nevertheless, they tend to become senescent over time.Therefore, genetically modified cell lines have been developed as an alternative [14].However, the application of fresh FTECs necessitates further exploration.
Recently, researchers have made strides in differentiating FTECs from induced pluripotent stem cells (iPSCs) derived from fibroblasts, offering potential advantages due to the pluripotency, self-renewal, and limitless proliferation of iPSCs [15].This flexibility has led to successfully modeling various inherited human diseases, including familial cancers [16,17].It is essential to note that iPSCs derived from different cellular origins may exhibit varying differentiation capacities, underlining the significance of considering the cellular source of iPSCs during clinical translation [18,19].
With the profound potential of iPSCs, this study aimed to develop a new iPSC line originating from FTECs and to compare its differentiation capabilities with those of NHEK-iPSCs.This research seeks to provide a robust foundation for future investigations in the field.

Ethics
The experimental protocol was approved by the Research Ethics Committee of Hualien Tzu Chi Hospital (IRB110-224-C).NHEK-iPSC was already derived in the previous study [20].

FTEC Culture
The culture medium for FTECs consisted of Dulbecco's Modified Eagle Medium (DMEM, cat.No. D5030, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, cat.No. F7524, Sigma) and 5 µg/mL insulin (Sigma) [13].The FTECs were then cultured in uncoated culture dishes.The medium was changed every three days.The cells were passaged at a 1:3 ratio when they reached 90% confluence after seven days of culture.Passages 4-6 of the FTECs were used for iPSC induction.

qPCR
qPCR was performed to confirm the expression of the pluripotency genes.Table 1 lists the primer sequence of the tested genes.Real-time qPCR (RT-qPCR) was performed and monitored using a FastStart Universal SYBR Green Master Mix (cat.No. 03003230001, Roche, Indianapolis, IN, USA) and a qPCR detection system (ABI StepOnePlus system, Applied Biosystems, Foster City, CA, USA).Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.Each target gene's expression level was calculated using the 2 −∆∆Ct method [21].Three readings were obtained for each experimental sample and each gene of interest.All of the experiments were performed in triplicate.
For iPSC differentiation, EB formation was performed for five days.The resulting EBs were plated onto gelatin-treated chamber slides and fixed.Antibodies specific for the three germ layers, namely the ectoderm (microtubule associated protein 2 (MAP2) and beta-III tubulin), mesoderm (brachyury), and endoderm (ATBF1), were identified (1:200).

Chromosomal Analysis
Karyotyping of iPS-FTEC cells was carried out at the Cytogenetics Laboratory of Ko's Obstetrics and Gynecology Clinic (Taipei, Taiwan).In brief, the cells were cultured until they reached exponential growth and were then treated with colchicine (cat.No. C9754, Sigma) to arrest them at metaphase.Subsequently, the cells were subjected to a hypotonic solution to induce bursting.Following bursting, the cells were affixed to a glass slide and stained with Giemsa stain.A cytogeneticist reviewed the chromosomes, which were organized into karyograms.The distribution of chromosome numbers was determined by examining 50 metaphases.The karyotypes of these 50 metaphases were analyzed, and the results were reported following the guidelines of the 2016 International System for Human Cytogenetic Nomenclature.

Animal Teratoma Formation Experiments
All of the animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Hualien Tzu Chi Hospital.Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and regulations were followed.
Three 6-8-week-old female NOD-SCID mice were purchased from Tzu Chi University.The mice were fed in a temperature-controlled environment (Animal Center at Tzu Chi University) under a 12 h light/12 h dark cycle.Professional personnel at the Animal Center were responsible for animal care.
For the teratoma generation assay, iPSCs were removed from the dish by mechanical slicing using glass capillaries, pelletized, and resuspended in PBS.
iPSCs (5 × 10 5 ) mixed with Matrigel (1:1) were injected into the subcutaneous tissue of the mice.Tumor formation was followed by palpation.After six months, or after reaching a tumor size of 0.5 cm 3 in volume, the mice were euthanized with CO 2 inhalation.After euthanization, the tumors were dissected, fixed, embedded in paraffin, and processed for histological examination.

Histological Examination
The resultant teratomas were fixed in 10% formalin and embedded in 4% paraformaldehyde.The specimens were sectioned into 4 µm thick slices.The tissue sections were stained with hematoxylin and eosin (H&E) (Sigma, St. Louis, MO, USA) to evaluate the cellular architecture.The structures of the germ layers were examined and photographed using a light microscope (Nikon, Tokyo, Japan).

iPSC Differentiation to FTE
The differentiation protocol was followed according to a previous study, with slight modification [15].The newly developed FTE-iPSCs and previously established NHEK-iPSCs were used for the FTE differentiation experiments (Figure 1).

Growing FTE Organoids from Spheroids in the Matrigel
On day 4, spheroid growth in the Matrigel was collected using a 200 µL pipette tip and pooled into a 1.5 mL tube.The collected spheroids were mixed with 50 µL Matrigel (BD Biosciences, Franklin Lakers, NJ, USA), estrogen (1 ng/mL), and progesterone (33 ng/mL), and slowly placed into the well of a 24-well dish.The droplets were allowed to solidify for 10-15 min in an incubator at 37 • C. FTM supplemented with growth factors was then added.The medium was changed every 3-4 days.The cells were replated every two weeks.

Organoid IHC
The staining procedure was the same as described in the previous section.The fixed organoids were then embedded in an optimal cutting temperature (OCT) compound (25608-930, Tissue-Tek, Torrance, CA, USA).Cryostat was used on frozen sections (thickness = 12 µm), placed on a glass slide, and stored at −80 • C. The sections were rehydrated with 1X PBS for 5 min and blocked with PBS containing 10% FBS and 0.05% Triton X-100 (PBST) for 1 h at 25 • C, followed by 2 h of incubation with primary antibodies in a blocking solution at 25 • C. The slides were washed thrice with PBS-T for 15 min at a room temperature of 25 • C and incubated with specific AF488-or AF594-conjugated secondary antibodies, followed by 4 ,6-diamidino-2-phenylindole (DAPI) counterstaining.After washing thrice with PBST, the slides were covered with a cover slide and imaged using a light microscope.The primary antibody concentration was 1:200, and that of the secondary antibodies was 1:400.The primary antibodies used were PAX8 (CL48860145, Proteintech, Rosemont, IL, USA), FOXJ1 (14-9964-82, Invitrogen), LGR5 (373804, BioLegend, San Diego, CA, USA), and DAPI (D1306, Invitrogen).The complete differentiation protocol is illustrated in Figure 1.

Statistical Analysis
All of the analyses were performed using SPSS Statistics, version 25 (IBM, Armonk, NY, USA).Gene expression at different stages of differentiation was compared in both cell lines.Data are presented as mean ± standard deviation.Means were compared using a one-way analysis of variance followed by Tukey's correction.The level of statistical significance was set at p < 0.05.
The pluripotency marker SOX2 was downregulated on day 2. In contrast, the other two markers, OCT4 and NANOG, did not show decreased expression following differentiation in FTEC-iPSCs (Figure 3C, left panel).In NHEK-iPSCs, the expression levels of all three pluripotency markers decreased after differentiation (Figure 3C, right panel).
In Figure 3D, the left panel represents FTEC-iPSC and the right panel represents NHEK-iPSC.There were five culture conditions on days 0, 2, 4, and 6 (WNT3 or WNT4).The mesoderm-related genes (Brachyury and MIXL1) should be expressed on day 2 of differentiation.The results demonstrated an increased expression of Brachyury and MIXL1 on day 2, which means both iPSCs could differentiate into the mesoderm on day 2. The mesoderm gene expressions (Brachyury and MIXL) seemed higher in NHEK-iPSC than in FTEC-iPSC with mesoderm differentiation.chromosome analysis of iPSC showed normal female karyotypes (Figure 2G).Tumorigenesis was evaluated to prove the pluripotency of iPSCs in vivo.Tumors were formed after three months in nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice (Figure 2H).The teratoma histology showed three germ layer tissues: endoderm (cartilage and fat tissue), endoderm, and ectoderm (Figure 2I,J).The pluripotency marker SOX2 was downregulated on day 2. In contrast, the other two markers, OCT4 and NANOG, did not show decreased expression following differentiation in FTEC-iPSCs (Figure 3C, left panel).In NHEK-iPSCs, the expression levels of all three pluripotency markers decreased after differentiation (Figure 3C, right panel).The intermediate mesoderm-related genes, OSR1, PAX2, and GATA3, increased in expression on days 4 and 6 of differentiation.In Figure 3E, there was increased expression of OSR1, PAX2, and GATA3 on days 4 and 6, which means both iPSCs were differentiated into the mesoderm on days 4 and 6.The intermediate mesoderm gene expressions, OSR1 and PAX2, were higher in FTEC-iPSC than in the NHEK-iPSC differentiation.The GATA3 expression was comparable between FTEC-iPSC and NHEK-iPSC.
Taken together, FTEC-iPSC could differentiate toward the mesoderm on day 2 and intermediate mesoderm on days 4 and 6.However, pluripotency genes OCT4 and NANOG were still expressed after differentiation.Compared with FTEC-iPSC, NHEK-iPSC could differentiate into the mesoderm and intermediate mesoderm with decreasing pluripotency gene expressions.

Sequential Differentiation of Both iPSC Lines from Intermediate Mesoderm to FTEC
After intermediate mesoderm differentiation, WNT3A or WNT4 were added to induce differentiation into the FTECs.The differentiated FTEC morphology on days 6 and 8 in both cell lines is shown in Figure 4A,B.
In Figure 4C, the upper panel represents FTEC-iPSC and the lower panel represents NHEK-iPSC.There were five culture conditions on days 0, 2, 4, and 6 (WNT3 or WNT4).The Mullerian duct progenitor-related genes (WT1 and OVGP1) were expressed on day 8 of differentiation.The results demonstrated an increased expression of WT1 and OVGP1 on day 8, which means both iPSCs could differentiated into Mullerian duct progenitors by adding WNT3 on day 8.The Mullerian duct progenitor gene expressions (WT1) seemed higher in FTEC-iPSC than in NHEK-iPSC with Mullerian duct progenitor differentiation.The OVGP1 expression was comparable between the two iPSCs.
The nephron progenitor-related genes, SIX2 and FOXD1, showed an increased expression on day 8 of differentiation.In Figure 4D, there was increased expression of SIX2 or FOXD1 on days 8 with adding WNT4, which means both iPSCs differentiated into nephron progenitors on days 8.The nephron progenitor gene expression, SIX2, was higher in FTEC-iPSC than in NHEK-iPSC differentiation.Conversely, the FOXD1 expression was higher in NHEK-iPSC than in FTEC-iPSC.
Taken together, FTEC-iPSC could differentiate toward the Mullerian duct progenitor on day 8 after adding WNT3 and the nephron progenitor on day 8 after adding WNT4.

The FTE Organoid Generation from FTE-iPSC-FTE and NHEK-iPSC-FTE
Organoids were formed by plating the FTECs on Matrigel.We used three methods to generate organoids, namely: cells on the Matrigel, spheroids on the Matrigel, and organoids formed in low-attachment dishes.
The organoids formed under all three conditions, and their morphologies are shown in Figure 5A,B.The WNT3 group exhibited a larger and more organized organoid morphology than the WNT4 group.
The organoid in Figure 5A left panel (+WNT3) was a spherical organoid under a microscope.The organoid comprised densely packed cells with a lumen (cavity) in the center.The cells were arranged in various shapes and sizes, but they all had a similar appearance, with a round nucleus and a granular cytoplasm.A layer of columnar epithelial cells surrounded the lumen.These cells had a long, thin shape and were responsible for lining the lumen and absorbing nutrients (Figure 5A).The organoid morphology in Figure 5A right panel (+WNT4) seemed like the organoid in the left panel.
The organoid in Figure 5B left panel (+WNT3) was a tubular organoid under a microscope.The organoid comprised a single layer of cells arranged in a tubular shape.The cells had a cuboidal or columnar shape, a round nucleus, and a granular cytoplasm (Figure 5B).The lumen of the tubule was filled with a fluid called the extracellular matrix (ECM).ECM provided support for the cells and helped to create a microenvironment that was similar to the environment in the body.After adding WNT4, the organoid morphology became densely packed cells without a lumen in the center.
The two organoids differ in their shape and structure.The first organoid was spherical, while the second organoid was tubular.The first organoid had a lumen in the center, while the second organoid had a lumen that ran the tube length.The cells in the two organoids were also different.The cells in the first organoid were arranged in various shapes and sizes, while the cells in the second organoid were arranged in a single layer.Overall, the two organoid images showed two different types of organoids with different shapes and structures.Both organoids could be regarded as FTEC organoids.IHC also showed that both cell lines formed organoids that expressed the typical FTE markers, namely PAX8, FOXJ1, and LGR5 (Figure 5C).The FTE-related gene expression of PAX8, FOXJ1, and LGR5 increased following organoid differentiation (Figure 5D).

Discussion
We successfully established FTEC-iPSCs, and their morphology reflected the characteristics of stem cell colonies.IHC and qPCR were performed to confirm the expression of pluripotency-related genes.In vitro and in vivo differentiation capabilities were also demonstrated by positive staining for the three germ layer markers and related gene expressions.A sequential differentiation protocol was used to differentiate iPSCs into the mesoderm, intermediate mesoderm, and FTEC organoids.During differentiation, qPCR analysis revealed the presence of pluripotent, mesodermal, and intermediate mesodermalrelated genes.Following intermediate mesodermal differentiation, WNT3A addition drove the cells toward an FTEC morphology, as confirmed by the FTEC-related marker expression and tubule-like structure formation.The Mullerian duct progenitor-related genes were increased earlier, since day 4, in the differentiated iPSC from FTEC than from NHEK.The FTEC-related gene expression was increased in FTECs differentiated from FTEC-iPSCs and those from NHEK-iPSCs in a dish-culture condition, plus WNT3.
The cell type origin may influence the functional and molecular characteristics of iPSCs [19].A previous study reported that the cell type origin could affect the differentiation abilities of iPSCs [19].However, the epigenetic memory of these original cell types is attenuated after an extended passage period [19].Another study showed that the disease status of the cell origin may influence the differentiation potential [22].They further found that iPSCs derived from healthy chondrocytes had better chondrocyte differentiation abilities than those derived from osteoarthritis chondrocytes [22].Furthermore, another study showed that the differentiation ability of iPSCs derived from cardiomyocytes is better than that of iPSCs derived from skin fibroblasts [23].Our analysis agrees with these studies, showing that iPSCs derived from FTECs have better FTEC differentiation capabilities.
WNT signaling is essential for FTEC differentiation [24].A previous study reported adding WNT4 to induce MD progenitors and FTEC differentiation [15].However, in our study, we found that WNT3A addition resulted in improved MD progenitor and FTEC differentiation compared with the addition of WNT4.In our previous studies, WNT3A was established as an essential factor for FTECs or FTEC organoid culturing [13,25].Therefore, WNT3A may play an essential role in FTEC differentiation induction.
In our study, the gene expression levels of FTEC markers (PAX8, FOXJ1, and LGR5) in the organoids were comparable to the original FTEC cells.This is the best reference available and organoids with similar expression levels should be considered for further research [15].
The long-term stability of our model is critical for the pharmacological studies of potential therapy.Proper culture techniques and maintenance protocols are essential to ensure the long-term stability of organoids [26].Cryopreservation techniques can be used to store organoids long term, providing a valuable resource for future experiments [27].Stable organoids are reliable platforms for high-throughput drug screening.They can help identify promising drug candidates and assess their efficacy and safety [28].Improving the long-term stability of organoids by refining culture methods ensures that organoid models remain relevant and reliable for a wide range of applications, including drug development and therapy testing [29].
The main strength of this study lies in the fact that we compared FTEC-iPSCs to primary FTEC in terms of gene expression.Two cell types (FTEC and NHEK) were used to generate iPSCs, and their differentiation capabilities were compared.However, this study has several limitations: (1) We used only two cell types (FTEC and NHEK) for iPSC generation.Therefore, these findings may not apply to iPSCs derived from other cell types.
(2) The study did not investigate the long-term stability of the FTEC-iPSC differentiation capability.This study evaluated only the ability of FTEC-iPSCs to differentiate into FTEC.
(3) The potential of the iPSCs established in this study for other applications, such as disease modeling and drug screening, has not been explored.(4) Finally, this study did not investigate the molecular mechanisms underlying the improved differentiation ability of FTEC-iPSCs compared with NHEK-iPSCs.This study only evaluated the effect of WNT3A addition on FTEC differentiation and did not investigate other factors that may have also influenced this process.We found two different types of organoids with different shapes and structures.A mixture of different types of organoids makes it more difficult to obtain robust and reproducible results.Overall, although this study demonstrates the potential of FTEC-iPSCs for generating FTEC organoids, further studies are required to understand the limitations and potential applications of this approach fully.

Conclusions
Our study successfully generated iPSCs from FTECs, demonstrating their capacity for FTEC differentiation.Furthermore, iPSCs originating from orthologous cell sources exhibited comparable differentiation capabilities.These findings hold promise for using iPSCs in modeling and investigating diseases associated with these specific cell types.

Data Availability Statement:
The data supporting the conclusions of this article is included within the article.

Conflicts of Interest:
The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Cells 2023 , 20 Figure 6 .
Figure 6.Comparison of FTEC-related gene expressions between the organoids derived from iPSCS from fallopian tube epithelial cells (FTECs) and normal human keratinocytes (NHEK).(A) PAX8, (B) FOXJ1, and (C)LGR5 (n = 3).The organoids derived from iPSC originating from FTEC in the dish culture method with the addition of WNT3 exhibit the highest expression for the three genes.The second-highest expression of these three genes is observed in the organoids derived from iPSC originating from NHEK with the supplementation of both WNT3 and WNT4.* p < 0.05, ** p < 0.01, *** p < 0.001.M: Matrigel, D: low attach dish.S: spheroid.

Figure 6 .
Figure 6.Comparison of FTEC-related gene expressions between the organoids derived from iPSCS from fallopian tube epithelial cells (FTECs) and normal human keratinocytes (NHEK).(A) PAX8, (B) FOXJ1, and (C)LGR5 (n = 3).The organoids derived from iPSC originating from FTEC in the dish culture method with the addition of WNT3 exhibit the highest expression for the three genes.The second-highest expression of these three genes is observed in the organoids derived from iPSC originating from NHEK with the supplementation of both WNT3 and WNT4.* p < 0.05, ** p < 0.01, *** p < 0.001.M: Matrigel, D: low attach dish.S: spheroid.

Table 1 .
The primer sequence of the tested genes.