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Endometrial organoids: in vitro models for endometrial research and personalized medicine †

The endometrium is one of the most dynamic tissues in the human body, undergoing hormonally regulated cycles of growth and shedding each month. There is evidence that endometrial stem/progenitor cells are responsible for the regenerative properties of the endometrium, and there has been a great deal of interest in characterizing these cells so that they can be harnessed to treat infertility, disease, and injury, or modulated to treat proliferative disorders of the endometrium such as cancer and endometriosis [1]. Many of the tools and techniques used to study stem/progenitor cells in other tissues have proved invaluable in endometrial research. Colony-forming assays have been used to detect epithelial and stromal cells with the stem cell-related ability to expand clonally in vitro [2]. In vivo transplantation assays have demonstrated that certain endometrial populations are capable of reconstituting endometrial epithelium and stroma in vivo [3]. Label retention studies in the mouse have identified quiescent populations that are activated during regeneration [4]. Stem/progenitor markers have been used to identify cell types that may be involved in endometrial regeneration [5]. Several studies have also touched on the utility of three-dimensional culture systems in endometrial research [6]. However, recent studies by Turco et al [7] and Boretto et al [8] represent the first systematic examination of the conditions and protocols required to initiate, maintain, and cryopreserve endometrial epithelial organoids. Organoids are defined as three-dimensional structures generated in vitro from stem cells that self-organize through cell sorting into multicellular structures that have functionality representative of the organ or tissue from which they were derived [9]. They are a miniature and simplified version of their parent organ/tissue and may contain multiple differentiated cells types. Organoids are often derived from tissue fragments containing tissue specific progenitor cells and can be clonally propagated. Organoids represent a powerful test of the regenerative and differentiation potential of the initiating cell type and the developmental processes they recapitulate, cell arrangement in the niche and lineage commitment, as the various cell types self-organize into mini-organs or tissues [9]. Organoid systems have proved to be an invaluable tool for investigating stem/progenitor cells in organs including intestine, liver, pancreas, prostate, fallopian tube, and kidney [7, 10]. Studies by Turco et al [7] and Boretto et al [8] are detailed examinations of methods for generating epithelial organoids from endometrium. Turco et al generated organoids from normal and malignant human endometrium, and Boretto et al from normal human and mouse endometrium. Both studies used matrigel and a defined culture medium supplemented with factors that promote organoid formation and maintenance by cells from other tissues (Figure 1A). Key factors promoting organoid formation and maintenance were shown to include activators/amplifiers of WNT signaling (WNT ligands and/or RSPO (R-spondin-1)), growth factors (EGF, FGF10) acting as epithelial mitogens, inhibitors of TGFβ (A83–01) and BMP (Noggin) signaling, and nicotinamide (commonly used in iPS and embryonic stem cell differentiation). Organoids generated could be maintained for many passages and several months (>6 months), and remained viable after cryopreservation. Both studies provide evidence that organoids are of clonal origin, and Turco et al also show that human endometrial organoids are genetically stable after passaging for more than 5 months. Endometrial organoids show physiologically relevant responses to ovarian hormones, with estrogen increasing proliferation and estrogen-regulated gene expression, and progesterone slowing proliferation, increasing progesteroneregulated gene expression and promoting differentiation of secretory and ciliated cells (Figure 1B). Mouse endometrial organoids survive when transplanted under the kidney capsule and expand in response to ovarian hormones [8]. Endometrial epithelium has historically been difficult to maintain in vitro, and the ability to reliably produce, maintain, and cryopreserve physiologically relevant epithelial organoids from