Figure 3.

Air–Liquid Interface culture methods improve EnAo features at the Pro‐phase. a) Scheme of the air–liquid interface (ALI) and submerged culture (SC) of EnAos. b) Protocol of ALI‐EnAo and SC‐EnAo to mimic Pro‐phase. The time point at which samples were collected for scRNA‐seq and staining analysis is highlighted with arrows. c) Representative staining of indicated endometrial markers. CK7 for EnECs; Vimentin for EnSCs; ZO1 for cell polarity; KI67 for cell proliferation; and E‐cadherin for epithelium. Scale bars: 25 µm. d) Quantification of columnar and squamous cells in the superficial layer of EnAos cultured in the ALI or SC condition on Day 4 and 15, respectively. At least >150 cells were quantified each experiment. e) Quantification of KI67⁺ EnECs in endometrium in vivo and EnAos cultured in the ALI and SC condition. At least >200 cells were quantified each experiment. f,g) Representative staining f) and quantification g) of ciliated cell marker Ac.α‐tubulin in LE and GE of endometrium, ALI‐EnAos, and SC‐EnAos on D15. Scale bars: 25 µm. h) Dynamic expression changes of estrogen receptor (ESR) and progesterone receptor (PGR) along with E₂ and P4 change during menstrual cycle. i) Representative staining of indicated markers in endometrium and ALI‐EnAo on D15 at Pro‐phase. Scale bars: 25 µm. d,e,g) All data were obtained based on three different donor cells. Data are presented as means ± SEMs; Chi‐square test was used to analyze percentage of cell subtypes and two‐sided unpaired Student's t‐test was used to perform the statistical analyses of staining; ^* p ≤ 0.05; ^** p ≤ 0.01; ^*** p ≤ 0.001; ^**** p ≤ 0.0001; ns, no significance. LELS, luminal epithelium‐like structure; GLS, gland‐like structure; SSE, simple squamous epithelium. Endometrium in vivo was obtained from donors during mid‐late Pro‐phase in Figure 3.