Improved Models of Human Endometrial Organoids Based on Hydrogels from Decellularized Endometrium

Emilio Francés-Herrero, Elena Juárez-Barber, Hannes Campo, Sara López-Martínez, Lucía de Miguel-Gómez, Amparo Faus, Antonio Pellicer, Hortensia Ferrero, Irene Cervelló, Emilio Francés-Herrero, Elena Juárez-Barber, Hannes Campo, Sara López-Martínez, Lucía de Miguel-Gómez, Amparo Faus, Antonio Pellicer, Hortensia Ferrero, Irene Cervelló

Abstract

Organoids are three-dimensional (3D) multicellular tissue models that mimic their corresponding in vivo tissue. Successful efforts have derived organoids from primary tissues such as intestine, liver, and pancreas. For human uterine endometrium, the recent generation of 3D structures from primary endometrial cells is inspiring new studies of this important tissue using precise preclinical models. To improve on these 3D models, we decellularized pig endometrium containing tissue-specific extracellular matrix and generated a hydrogel (EndoECM). Next, we derived three lines of human endometrial organoids and cultured them in optimal and suboptimal culture expansion media with or without EndoECM (0.01 mg/mL) as a soluble additive. We characterized the resultant organoids to verify their epithelial origin, long-term chromosomal stability, and stemness properties. Lastly, we determined their proliferation potential under different culture conditions using proliferation rates and immunohistochemical methods. Our results demonstrate the importance of a bioactive environment for the maintenance and proliferation of human endometrial organoids.

Keywords: ECM hydrogel; decellularization; endometrium; organoids; proliferation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Experimental design. Human endometrial organoids were cultured under four different experimental conditions (ExM, ExM+EndoECM, ExM-NA, ExM-NA+EndoECM) and evaluated microscopically, histologically, immunohistochemically, and for chromosomal stability. Abbreviations: EndoECM, endometrial extracellular matrix; IHC: immunohistochemistry; IHF: immunohistofluorescence; N-CAD: N-cadherin; SSEA-1: stage-specific embryonic antigen-1; ExM: expansion medium; NA; nicotinamide. Created with BioRender.com.
Figure 2
Figure 2
Characterization of glandular origin in human endometrial organoids by histological and immunohistochemical staining. H&E, PAS staining, and MUC-1 expression in positive controls (endometrium and kidney) and in human endometrial organoids cultured in the four different experimental conditions: ExM, ExM+EndoECM, ExM-NA, ExM-NA+EndoECM. Negative controls for MUC-1 are shown at the bottom of the figure in small size. Scale bars are 100 µm.
Figure 3
Figure 3
Characterization of epithelial phenotype in human endometrial organoid by immunofluorescence staining. Epithelial (pan-cytokeratin) and stromal (vimentin) marker expressions in human endometrium and organoids cultured in the four different experimental conditions: ExM, ExM+EndoECM, ExM-NA, and ExM-NA+EndoECM. Cell’s nuclei stained with DAPI. Scale bars are 100 µm.
Figure 4
Figure 4
Chromosomal analysis of human endometrial organoids by cytogenetic microarray. (a) Genomic DNAs from early (p3), mid (p5), and late (p8) passage organoids exposed to ExM and EndoECM culture conditions are compared with a human reference genome. Intensity of each probe (log ratio) is expressed on the Y-axis and its location on chromosomes (1–22, X and Y) on the X-axis. A signal ratio of 0 indicates copy number equivalence. Reduced signal on the Y chromosome is not significant due to the absence of this chromosome in the samples. (b) Summary of genetic analyses.
Figure 5
Figure 5
Comparative proliferation of human endometrial organoids exposed to different conditions. (a) Representative images of endometrial organoids in all culture conditions: ExM, ExM+EndoECM, ExM-NA, ExM-NA+EndoECM. (b) Days 2–7, 2–5, and 5–7 proliferation rates. * p < 0.05, ** p < 0.001, *** p < 0.0001. Scale bars are 1 mm.
Figure 6
Figure 6
Human endometrial organoids proliferation assay. (a) Immunohistochemistry for Ki67 expression in breast cancer and organoids cultured in the four different experimental conditions ExM, ExM+EndoECM, ExM-NA, ExM-NA+EndoECM. (b) Percentage of Ki67 positive cells in organoids. * p < 0.05, ** p < 0.01, *** p < 0.0001. Scale bars are 100 µm.
Figure 7
Figure 7
Epithelial progenitor cell markers in human endometrial organoids. N-cadherin (green) and SSEA-1 (red) expressions in positive controls (human endometrium) and human endometrial organoids cultured in the four different experimental conditions: ExM, ExM+EndoECM, ExM-NA, ExM-NA+EndoECM. A higher magnification of the boxed areas is shown in all panels. Cell’s nuclei stained with DAPI. Scale bars are 10 µm.

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