Luminal and Glandular Epithelial Cells from the Porcine Endometrium maintain Cell Type-Specific Marker Gene Expression in Air-Liquid Interface Culture

Abstract

Two different types of epithelial cells constitute the inner surface of the endometrium. While luminal epithelial cells line the uterine cavity and build the embryo-maternal contact zone, glandular epithelial cells form tubular glands reaching deeply into the endometrial stroma. To facilitate investigations considering the functional and molecular differences between the two populations of epithelial cells and their contribution to reproductive processes, we aimed at establishing differentiated in vitro models of both the luminal and the glandular epithelium of the porcine endometrium using an air-liquid interface (ALI) approach. We first tested if porcine luminal endometrium epithelial cells (PEEC-L) reproducibly form differentiated epithelial monolayers under ALI conditions by monitoring the morphology and the trans-epithelial electrical resistance (TEER). Subsequently, luminal (PEEC-L) and glandular epithelial cells (PEEC-G) were consecutively isolated from the endometrium of the uterine horn. Both cell types were characterized by marker gene expression analysis immediately after isolation. Cells were separately grown at the ALI and assessed by means of histomorphometry, TEER, and marker gene expression after 3 weeks of culture. PEEC-L and PEEC-G formed polarized monolayers of differentiated epithelial cells with a moderate TEER and in vivo-like morphology at the ALI. They exhibited distinct patterns of functional and cell type-specific marker gene expression after isolation and largely maintained these patterns during the culture period. The here presented cell culture procedure for PEEC-L and -G offers new opportunities to study the impact of embryonic signals, endocrine effectors, and reproductive toxins on both porcine endometrial epithelial cell types under standardized in vitro conditions. Created with BioRender.com .

Keywords: Air–liquid interface; Cell culture; Endometrium; Glandular epithelium; Luminal epithelium.

Fig. 1

ALI-PEEC-L morphology and barrier function in long-term culture. (A) Cell height of PEEC-L after 1, 2 and 3 weeks of culture. (B) TEER of PEEC-L cells after 1, 2 and 3 weeks of culture. (A, B) The data are presented as a boxplot. Significant differences (p ≤ 0.05) between the time points are indicated by different superscript letters. (C) Correlation of cell height and TEER. The correlation was considered significant at p < 0.05. (D) Representative cross-sections of ALI-PEEC-L at 1, 2 and 3 weeks of culture, HE staining, magnification × 40, scale bar = 10 µm. n = 5. TEER, transepithelial electrical resistance; ALI, air–liquid interface; PEEC-L, porcine endometrial epithelial cells luminal; HE, hematoxylin–eosin

Fig. 2

Morphology and barrier function of ALI-PEEC-L depend on medium composition. (A) Representative cross-sections of PEEC-L at the ALI after 3 weeks of culture in UM, CM, SF and NU, HE staining, magnification × 40, scale bar = 10 µm. (B) Transepithelial electrical resistance of ALI-PEEC-L after 3 weeks of culture. The results are presented as mean ± SEM. Significant differences (p ≤ 0.05) between the media are indicated by different superscript letters, n = 3–4. TEER, transepithelial electrical resistance; ALI, air–liquid interface; PEEC-L, porcine endometrial epithelial cells luminal; UM, unconditioned medium; CM, conditioned medium; SF, serum free medium; NU Nu-Serum medium; HE, hematoxylin–eosin

Fig. 3

Luminal and glandular cell clusters during cell isolation. (A) Representative picture of PEEC-L displaying planar structure during cell isolation before the last enzymatic digestion step, scale bar = 100 µm. (B) Representative picture of PEEC-G displaying tubular structure during cell isolation before the last enzymatic digestion step, scale bar = 100 µm

Fig. 4

Morphology and barrier function of ALI-PEEC-L and -G after long term culture. (A) Cell height of PEEC-L and -G after 3 weeks at the ALI. (B) TEER of PEEC-L and -G after 3 weeks at the ALI. (A, B) The data are presented as a boxplot. Significant differences (p ≤ 0.05) between ALI-PEEC-L and -G are indicated by different superscript letters. (C) Representative pictures of PEEC-L and -G in vivo and in vitro. HE staining, magnification × 40, scale bar = 10 µm, n = 4. TEER, transepithelial electrical resistance; ALI, air–liquid interface; PEEC-L, porcine endometrial epithelial cells luminal; PEEC-G, porcine endometrial epithelial cells glandular; HE, hematoxylin–eosin

Fig. 5

mRNA expression of PEEC-L and PEEC-G at isolation and at the ALI. (A) mRNA expression of the functional endometrial epithelial markers Estrogen receptor 1 (ESR1), Progesterone receptor (PGR), Mucin 16 (MUC16), Mucin 1 (MUC1) at isolation and at the ALI. (B) mRNA expression of the luminal epithelial markers Stanniocalcin 1 (STC1), Angiopoietin-related protein 1 (ANGPTL1) and Insulin-like growth factor-binding protein 2 (IGFBP2) at isolation and at the ALI. (C) mRNA expression of the glandular epithelial markers Wnt inhibitory factor 1 (WIF1), Follistatin (FST) and Forkhead box A2 (FOXA2) at isolation and at the ALI. (A, B, C) The results are presented as a boxplot. A high ΔCq represents a high transcript abundance. Significant differences (p ≤ 0.05) between PEEC-L and -G at isolation and at the ALI are indicated by different superscript letters. (D) mRNA expression pattern of ESR1, PGR, MUC16, MUC1, STC1, ANGPTL1, IGFBP2, WIF1, FST and FOXA2 at isolation (PEEC) and after 3 weeks in culture (ALI-PEEC). The results are presented as mean delta delta quantitative cycle (ΔΔCq) ± SEM. ΔΔCq > 0 indicates higher transcript abundance in PEEC-L than in PEEC-G. Significant differences (p ≤ 0.05) between PEEC and ALI-PEEC are indicated by an asterisk, n = 4. ALI, air–liquid interface; PEEC-L, porcine endometrial epithelial cells luminal; PEEC-G, porcine endometrial epithelial cells glandular