Comparison of Organoids from Menstrual Fluid and Hormone-Treated Endometrium: Novel Tools for Gynecological Research

Endometrial organoids (EMO) are an important tool for gynecological research but have been limited by generation from (1) invasively acquired tissues and thus advanced disease states and (2) from women who are not taking hormones, thus excluding 50% of the female reproductive-aged population. We sought to overcome these limitations by generating organoids from (1) menstrual fluid (MF; MFO) using a method that enables the concurrent isolation of menstrual fluid supernatant, stromal cells, and leukocytes and (2) from biopsies and hysterectomy samples from women taking hormonal medication (EMO-H). MF was collected in a menstrual cup for 4–6 h on day 2 of menstruation. Biopsies and hysterectomies were obtained during laparoscopic surgery. Organoids were generated from all sample types, with MFO and EMO-H showing similar cell proliferation rates, proportion and localization of the endometrial basalis epithelial marker, Stage Specific Embryonic Antigen-1 (SSEA-1), and gene expression profiles. Organoids from different disease states showed the moderate clustering of epithelial secretory and androgen receptor signaling genes. Thus, MFO and EMO-H are novel organoids that share similar features to EMO but with the advantage of (1) MFO being obtained non-invasively and (2) EMO-H being obtained from 50% of the women who are not currently being studied through standard methods. Thus, MFO and EMO-H are likely to prove to be invaluable tools for gynecological research, enabling the population-wide assessment of endometrial health and personalized medicine.

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody

Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5–2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.

Development of Fluorescently Labeled SSEA-3, SSEA-4, and Globo-H Glycosphingolipids for Elucidating Molecular Interactions in the Cell Membrane

Glycosphingolipids (GSLs), such as the globo-series GSLs stage-specific embryonic antigen 3 (SSEA-3), SSEA-4, and Globo-H, are specifically expressed on pluripotent stem cells and cancer cells, and are known to be associated with various biological processes such as cell recognition, cell adhesion, and signal transduction. However, the behavior and biological roles of these GSLs are still unclear. In our previous study, we observed the interactions between the lipid raft and GSLs in real-time using single-molecule imaging, where we successfully synthesized various fluorescent analogs of GSLs (e.g., GM1 and GM3). Here, we have developed fluorescent analogs of SSEA-3, SSEA-4, and Globo-H using chemical synthesis. The biophysical properties of these analogs as raft markers were examined by partitioning giant plasma membrane vesicles from RBL-2H3 cells into detergent-resistant membrane fractions and liquid-ordered/liquid-disordered phases. The results indicated that the analogs were equivalent to native-type GSLs. The analogs could be used to observe the behavior of globo-series GSLs for detailing the structure and biological roles of lipid rafts and GSL-enriched nanodomains during cell differentiation and cell malignancy.

ISOLATION OF AMNIOTIC FLUID MESENCHYMAL STEM CELLS (AF-MSCs) OBTAINED FROM CAESAREAN SECTIONS

Amniotic fluid is a liquid that fills the amniotic cavity which has defense and nutritional functions in fetal development. Human aterm amniotic fluid can be an ideal alternative as a source of mesenchymal stem cells, originating from the neonate. Preclinical studies of second and third trimester amnion fluid cells confirmed the number of potential donors from this wasted material. In several studies, AF-MSCs express mesenchymal markers such as CD90, CD73 (SH3, SH), CD105 (SH2), CD29, CD166, CD49e, CD58 and CD44 (MHC class I). These cells also express HLA-ABC antigens, CD 34, CD 45 which are hematopoietic markers, and endothelial CD31 markers. There is no expression of CD10, CD11b, CD14, CD34, CD117, EMA and HLA-DR, DP, DQ antigens. Most of AF-MSCs have pluripotent properties which are characterized by the discovery of octamer binding protein 3/4 (Oct-3/4), transcription factors Nanog (Nanog), and stage-specific embryonic antigen 4 (SSEA-4) on RT-PCR examination. From this study, 8 million cells was isolated. These cells will be used for research on pelvic organ prolapse therapy by using AF-MSCs. AF-MSCs isolation totally takes 6 weeks. From 1 flask, 2 million of stem cells was obtained. Keywords: amniotic fluid, AF-MSCs

ISOLATION OF AMNIOTIC FLUID MESENCHYMAL STEM CELLS (AF-MSCs) OBTAINED FROM CAESAREAN SECTIONS

Amniotic fluid is a liquid that fills the amniotic cavity which has defense and nutritional functions in fetal development. Human aterm amniotic fluid can be an ideal alternative as a source of mesenchymal stem cells, originating from the neonate. Preclinical studies of second and third trimester amnion fluid cells confirmed the number of potential donors from this wasted material. In several studies, AF-MSCs express mesenchymal markers such as CD90, CD73 (SH3, SH), CD105 (SH2), CD29, CD166, CD49e, CD58 and CD44 (MHC class I). These cells also express HLA-ABC antigens, CD 34, CD 45 which are hematopoietic markers, and endothelial CD31 markers. There is no expression of CD10, CD11b, CD14, CD34, CD117, EMA and HLA-DR, DP, DQ antigens. Most of AF-MSCs have pluripotent properties which are characterized by the discovery of octamer binding protein 3/4 (Oct-3/4), transcription factors Nanog (Nanog), and stage-specific embryonic antigen 4 (SSEA-4) on RT-PCR examination. From this study, 8 million cells was isolated. These cells will be used for research on pelvic organ prolapse therapy by using AF-MSCs. AF-MSCs isolation totally takes 6 weeks. From 1 flask, 2 million of stem cells was obtained.