Anti-KRT18 monoclonal antibody, clone FQS2722

Objective: To develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eECs) retaining molecular and functional characteristics of endometrial epithelium in vivo. Design: In vitro study using human endometrial cells. Setting: University research laboratory. Patient(s): Endometrial biopsies were obtained from premenopausal women undergoing benign gynecologic procedures. Intervention(s): Primary eECs were cryopreserved in 1% fetal bovine serum/10% dimethylsulfoxide in Defined Keratinocyte SerumFree Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium. Main Outcome Measure(s): Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity. Result(s): Endometrial epithelial cells recovered after cryopreservation (n = 5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared with eSF, recovered eECs displayed increased (P<.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eECs secreted levels of cytokines and growth factors similarly to freshly cultured eECs. Recovered eECs could form a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN. Conclusion(s): We have developed a protocol for cryopreservation of eECs in which recovered cells after thawing demonstrate morphologic, transcriptomic, and functional characteristics of human endometrial epithelium in vivo. (C) 2016 by American Society for Reproductive Medicine.

Lipotransfer has been applied in breast augmentation surgery for several years and the resident adipose-derived stem cells (ASCs) play an important role in enhancing fat graft survival. However, the interaction between ASCs and mammary epithelium is not fully understood. Many studies have shown that ASCs have a tumor-supportive effect in breast cancer. To the best of our knowledge, this is the first study on the effect of mammary epithelial cells on the human ASCs in 3D culture. ASCs were cultivated on matrigel in the conditioned medium (CM) prepared from a human breast epithelial cell line (HBL-100). The ASCs formed KRT18-positive acini-like structures after stimulation with breast epithelial cells. The expression of epithelial genes (CDH1 and KRT18) was up-regulated while the expression of mesenchymal specific genes (CDH2 and VIM) was down-regulated as determined by qRT-PCR. The stemness marker (CD29) and angiogenic factors (CD31 and VEGF) were also down-regulated as examined by immunofluorescence. In addition, the CM obtained from HBL-100 enhanced the migration and inhibited the adipogenic differentiation of ASCs. These results demonstrate that ASCs have the ability to transform into epithelial-like cells when cultured with mammary epithelial cells. Given these observations, we infer that ASCs have a positive effect on lipotransfer, not only due to their ability to secrete growth factors, but also due to their direct participation in the formation of new breast tissue.