A novel iPSC-derived 3D organoid in vitro model of human Fallopian Tube Epithelium

An outstanding result achieved by Regenerative Medicine Institute team in Cedars-Sinai Medical Center (Los Angeles, CA, USA) and published in Nature Scientific Report in September 2017, is going to open new perspective in ovarian cancer research. Indeed, the establishment of a novel 3D human fallopian tube epitheliul organoid in vitro model from induced pluripotent stem cells (iPSC) represents a significant breakthrough to study the earliest stages of high-grade serous ovarian cancer (HGSC).

High-grade serous carcinoma (HGSC), the most common subtype of epithelial ovarian cancer (~70%), has the highest mortality rate among all gynecological cancers (Kurman RJ, 2013) due to late stage at diagnosis and development of drug resistance. It is now accepted that the majority of HGSCs arise from the secretory cells of the fallopian tube epithelium (FTE) (Folkins AK et al., 2008). The discovery of an extra-ovarian origin of ovarian cancer is a fundamental advance toward improving early detection, prevention and treatment of this lethal disease. However, the lack of relevant in vitro human models that can recapitulate tissue-specific architecture and study early alterations has hindered the further understanding of FTE transformation as well as the initiation and progression of HGSC (Yucer et al., 2017).

Current fallopian tube models, including ex vivo and three-dimensional (3D) spheroid models, demonstrate the importance of cell polarity in recreating secretory and ciliated cells (Eddie S. et al., 2015; Lawrenson K et al., 2013; Levanon K et al., 2010). However, FTE cells in these models have a reduced proliferation rate and induced senescence because of the lack of convoluted luminal architecture and the ectopic microenvironment (Eddie S. et al., 2015; Lawrenson K et al., 2013; Levanon K et al., 2010). Alternatively, induced pluripotent stem cell (iPSC) technology and 3D-tissue engineering provide powerful tools to recapitulate physiologically relevant aspects of disease progression in vitro. However, no successful protocol exists, to date, for iPSC development into the fallopian tube epithelial cells (Yucer et al., 2017). Yucer N. and colleagues (2017) from Cedars-Sinai Medical Center (CA, USA) describe a rapid and efficient method to create an iPSC- derived 3D model of human FTE with the desired cell types and luminal architecture. The female reproductive tract, including FTE, arises from the Müllerian duct in parallel to the urinary system from intermediate mesoderm (IM) of the urogenital ridge in the posterior primitive streak. Scientists recapitulated Müllerian development in vitro by inducing iPSC differentiation in Intermediate Mesoderm-Like Cells and consequently driving differentiation of Intermediate Mesoderm into Fallopian Tube Epithelium, by adding pro-Müllerian growth factors. Correct differentiation process was monitored through gene expression quantification and immunocytochemistry of intermediate mesoderm markers (PAX2, PAX8, GATA3, OSR1, WT1, and OVGP1) and subsequently FTE markers (TUBB4A, FOXJ1, PAX8, OVGP1).

Generation of Human Fallopian tube-like organoid was obtained on a solid growth platform i.e. 3D Matrigel along with pro- Müllerian growth factors which enabled the FTE organoid to self-organize into a convoluted luminal structure. Fallopian tube organoids were also exposed to estrogen and

progesterone to increase the cellular architectural complexity. Importantly, staining of secretory and ciliated cellular components demonstrated that these structures accurately model fallopian tube architecture. Indeed, the cellular phenotype and organization of the iPSC-derived FTE organoids were comparable to fresh human fallopian tube tissue (Fig. 3f). Importantly, mature organoids contained TUBB4A-positive and PAX8-negative ciliated cells as well as PAX8-positive secretory cells (Fig. 3g). The differentiation of iPSC-derived organoids into fallopian tube mature cells over time was further demonstrated using heat map analysis, which showed increased expression of the ciliated cell marker FOXJ1 and secretory cell markers OVGP1and PAX8, which were similar to human fallopian tube tissue (Fig. 3h). Critically, iPSC-derived FTE organoids not only expressed both ciliated and secretory cell markers but also formed visible cilia, further demonstrating that this novel model closely mimics the proper physiology and anatomy of the human FTE.

In summary, this iPSC-derived fallopian tube epithelium organoid provides a powerful in vitro model of ovarian cancer that can recapitulate early de novo genomic alterations and germline mutations, faithfully model disease progression and ultimately uncover novel treatments.

Fig.3 Development and Characterization of an iPSC-Derived Fallopian Tube Organoid. (a) Schematic of factors involved in the differentiation of fallopian tube organoids. (b) Bright field image and H&E staining of FTE organoid at day 14. (c–e) Immunocytochemistry for FTE markers TUBB4A, FOXJ1 and PAX8 and epithelial marker CDH1 (E-Cadherin) at organoid culture day 14. (f) Immunocytochemistry for FTE markers PAX8, TUBB4A, OVGP1 and epithelial marker CDH1 at FTE organoid culture day 45, along with human fallopian tube tissue. (g) Immunocytochemistry for FTE markers TUBB4A and PAX8 at FTE organoid culture day 45. (h) Gene expression of fallopian tube markers OVGP1 (for 2 different primers), FOXJ1, TNFaIP2 and PAX8, as well as kidney markers SALL1 and FOXD1 at organoids culture day 45, human fallopian tube and kidney. The color matrix of the heat map represents the log2(Ratio) of each individual gene relative to its expression at the iPSC stage. Relative gene expression to iPSC stage (day 0) was calculated using ΔΔCt method and normalized to endogenous GAPDH level for 87iCTR-n3 iPSC line (i) H&E staining of FTE organoid at culture day 45 and day 180, and human fallopian tube tissue (from Yucer N. et al., 2017. Directed Differentiation of Human Induced Pluripotent Stem Cells into Fallopian Tube Epithelium. Sci Rep. 2017 Sep 6;7(1):10741).

References

  • Kurman, R. J. Origin and molecular pathogenesis of ovarian high-grade serous carcinoma. Ann Oncol 24(Suppl 10), x16–21, (2013).
  • Yucer N et al. Directed Differentiation of Human Induced Pluripotent Stem Cells into Fallopian Tube Epithelium. Sci Rep. 2017 Sep 6;7(1):10741.
  • Folkins, A. K. et al. A candidate precursor to pelvic serous cancer (p53 signature) and its prevalence in ovaries and fallopian tubes from women with BRCA mutations. Gynecol Oncol 109, 168–173, (2008).
  • Eddie, S. L. et al. Three-dimensional modeling of the human fallopian tube fimbriae. Gynecol Oncol 136, 348–354, (2015).
  • Lawrenson, K. et al. In vitro three-dimensional modeling of fallopian tube secretory epithelial cells. BMC Cell Biol 14, 43, (2013).
  • Levanon, K. et al. Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis. Oncogene 29, 1103–1113, (2010).

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