Circulating SSEA-1+ stem cell-mediated tissue repair in allergic airway inflammation.

Structural changes known as airway remodeling characterize chronic/severe asthma and contribute to lung dysfunction. We previously reported that neonatal SSEA-1+ pulmonary stem/progenitor cells (PSCs) ameliorated airway inflammation in asthmatic mice. However, the molecular mechanisms by which endogenous SSEA-1+ PSC of adult mice afford beneficial effects in alveolar homeostasis and lung repair after allergen challenge remain incompletely understood. To analyze the expression profile and clarify the biological significance of endogenous adult lung SSEA-1+ cells in asthmatic mice. Lung SSEA-1+ cells and circulating SSEA-1+ cells in peripheral blood were determined by confocal microscopy and cytometric analysis. GFP chimeric mice were used to trace cell lineage in vivo. The roles of circulating SSEA-1+ cells were verified in ovalbumin-induced and house dust mite-induced allergic asthmatic models. In asthmatic mice, endogenous lung SSEA-1+ cells almost disappeared; however, a unique population of circulating SSEA-1+ cells was enriched after the challenge phase. In asthmatic mice, adoptive transfer of circulating SSEA-1+ cells had a specific homing preference for the lung in response to inhaled antigen through upregulating CXCR7-CXCL11 chemokine axis. Circulating SSEA-1+ cells can transdifferentiate in the alveolar space and ameliorate lung inflammation and structural damage through inhibiting the infiltration of inflammatory cells into peribronchovascular and goblet cell hyperplasia areas, reducing the thickened smooth muscle layers and PAS-positive mucus-containing goblet cells. Reinforcing bone marrow-derived circulating SSEA-1+ cells from peripheral blood into lung tissue which create a rescue mechanism in maintaining alveolar homeostasis and tissue repair to mediate lung protection for emergency responses after allergen challenge in asthmatic conditions.

Menstrual fluid endometrial stem/progenitor cell and supernatant protein content: cyclical variation and indicative range.

STUDY QUESTION: Does natural variation exist in the endometrial stem/progenitor cell and protein composition of menstrual fluid across menstrual cycles in women? SUMMARY ANSWER: Limited variation exists in the percentage of some endometrial stem/progenitor cell types and abundance of selected proteins in menstrual fluid within and between a cohort of women. WHAT IS KNOWN ALREADY: Menstrual fluid is a readily available biofluid that can represent the endometrial environment, containing endometrial stem/progenitor cells and protein factors. It is unknown whether there is natural variation in the cellular and protein content across menstrual cycles of individual women, which has significant implications for the use of menstrual fluid in research and clinical applications. STUDY DESIGN, SIZE, DURATION: Menstrual fluid was collected from 11 non-pregnant females with regular menstrual cycles. Participants had not used hormonal medications in the previous 3 months. Participants collected menstrual fluid samples from up to five cycles using a silicone menstrual cup worn on Day 2 of menstrual bleeding. PARTICIPANTS/MATERIALS, SETTING, METHODS: Menstrual fluid samples were centrifuged to separate soluble proteins and cells. Cells were depleted of red blood cells and CD45+ leucocytes. Menstrual fluid-derived endometrial stem/progenitor cells were characterized using multicolour flow cytometry including markers for endometrial stem/progenitor cells N-cadherin (NCAD) and stage-specific embryonic antigen-1 (SSEA-1) (for endometrial epithelial progenitor cells; eEPC), and sushi domain containing-2 (SUSD2) (for endometrial mesenchymal stem cells; eMSC). The clonogenicity of menstrual fluid-derived endometrial cells was assessed using colony forming unit assays. Menstrual fluid supernatant was analyzed using a custom magnetic Luminex assay. MAIN RESULTS AND THE ROLE OF CHANCE: Endometrial stem/progenitor cells are shed in menstrual fluid and demonstrate clonogenic properties. The intraparticipant agreement for SUSD2+ menstrual fluid-derived eMSC (MF-eMSC), SSEA-1+ and NCAD+SSEA-1+ MF-eEPC, and stromal clonogenicity were moderate-good (intraclass correlation; ICC: 0.75, 0.56, 0.54 and 0.52, respectively), indicating limited variability across menstrual cycles. Endometrial inflammatory and repair proteins were detectable in menstrual fluid supernatant, with five of eight (63%) factors demonstrating moderate intraparticipant agreement (secretory leukocyte protein inhibitor (SLPI), lipocalin-2 (NGAL), lactoferrin, follistatin-like 1 (FSTL1), human epididymis protein-4 (HE4); ICC ranges: 0.57-0.69). Interparticipant variation was limited for healthy participants, with the exception of key outliers of which some had self-reported menstrual pathologies. LARGE SCALE DATA: N/A. There are no OMICS or other data sets relevant to this study. LIMITATIONS, REASONS FOR CAUTION: The main limitations to this research relate to the difficulty of obtaining menstrual fluid samples across multiple menstrual cycles in a consistent manner. Several participants could only donate across <3 cycles and the duration of wearing the menstrual cup varied between 4 and 6 h within and between women. Due to the limited sample size used in this study, wider studies involving multiple consecutive menstrual cycles and a larger cohort of women will be required to fully determine the normal range of endometrial stem/progenitor cell and supernatant protein content of menstrual fluid. Possibility for selection bias and true representation of the population of women should also be considered. WIDER IMPLICATIONS OF THE FINDINGS: Menstrual fluid is a reliable source of endometrial stem/progenitor cells and related endometrial proteins with diagnostic potential. The present study indicates that a single menstrual sample may be sufficient in characterizing a variety of cellular and protein parameters across women's menstrual cycles. The results also demonstrate the potential of menstrual fluid for identifying endometrial and menstrual abnormalities in both research and clinical settings as a non-invasive method for assessing endometrial health. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Australian National Health and Medical Research Council to C.E.G. (Senior Research Fellowship 1024298 and Investigator Fellowship 1173882) and to J.E. (project grant 1047756), the Monash IVF Research Foundation to C.E.G. and the Victorian Government's Operational Infrastructure Support Program. K.A.W., M.L.D.-T., S.G.S. and J.E. declare no conflicts of interest. C.E.G. reports grants from NHMRC, during the conduct of the study; grants from EndoFound USA, grants from Ferring Research Innovation, grants from United States Department of Defence, grants from Clue-Utopia Research Foundation, outside the submitted work. CEF reports grants from EndoFound USA, grants from Clue-Utopia Research Foundation, outside the submitted work.

SSEA-1-positive fibronectin is secreted by cells deviated from the undifferentiated state of human induced pluripotent stem cells.

Quality control for human induced pluripotent stem cells (hiPSCs) is important for efficient and stable production of hiPSC-derived cell therapy products to be used for transplantation. During cell culture, hiPSCs spontaneously undergo morphological changes and lose pluripotent properties. Such cells are termed deviated cells, which are altered from the undifferentiated state of hiPSCs, and express the early differentiation marker stage-specific embryonic antigen 1 (SSEA-1). In this study, we searched for soluble SSEA-1+ glycoproteins secreted from deviated cells generated by culturing hiPSCs in cell culture medium containing heat-inactivated supplements. Glycoproteins obtained from cell culture supernatants of SSEA-1+ deviated cells were enriched by an O-glycan binding lectin and blotted with anti-SSEA-1 antibody. A single protein band at >250 kDa specifically detected by anti-SSEA-1 antibody was identified as fibronectin (FN) by LC-MS/MS analysis and immunoprecipitation combined with western blotting, indicating that FN is a carrier protein of SSEA-1. We then constructed a sandwich enzyme-linked immunosorbent assay to detect SSEA-1+ FN secreted from deviated cells. This FN-SSEA-1 test proved to be both sensitive and specific, allowing for non-destructive detection of SSEA-1+ deviated cells within mixed cell population, with a lower limit of detection of 100 cells/mL. The developed assay may provide a standard technology for quality control of hiPSCs used for regenerative medicine.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells.

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

Increased Expression of Cell Surface SSEA-1 is Closely Associated with Naïve-Like Conversion from Human Deciduous Teeth Dental Pulp Cells-Derived iPS Cells.

Stage-specific embryonic antigen 1 (SSEA-1) is an antigenic epitope (also called CD15 antigen) defined as a Lewis X carbohydrate structure and known to be expressed in murine embryonal carcinoma cells, mouse embryonic stem cells (ESCs), and murine and human germ cells, but not human ESCs/induced pluripotent stem cells (iPSCs). It is produced by α1,3-fucosyltransferase IX gene (FUT9), and F9 ECCs having a disrupted FUT9 locus by gene targeting are reported to exhibit loss of SSEA-1 expression on their cell surface. Mouse ESCs are pluripotent cells and therefore known as "naïve stem cells (NSCs)." In contrast, human ESCs/iPSCs are thought to be epiblast stem cells (EpiSCs) that are slightly more differentiated than NSCs. Recently, it has been demonstrated that treatment of EpiSCs with several reprograming-related drugs can convert EpiSCs to cells similar to NSCs, which led us to speculate that SSEA-1 may have been expressed in these NSC-like EpiSCs. Immunocytochemical staining of these cells with anti-SSEA-1 revealed increased expression of this epitope. RT-PCR analysis also confirmed increased expression of FUT9 transcripts as well as other stemness-related transcripts such as REX-1 (ZFP42). These results suggest that SSEA-1 can be an excellent marker for human NSCs.

Phenotypic and Molecular Characterization of Domestic Cat (Felis catus) Spermatogonial Stem Cells.

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations. We concluded that SSC surface markers used in other mammalian species were not specific for identifying cat SSCs. However, the pluripotent markers we evaluated were more specific to cat spermatogonia, and the presence of SSEA-1 and SSEA-4 in fewer and primarily individual cells suggests that these two markers may be used for enrichment of cat SSCs. The expression of pluripotent transcription factors at mRNA level by single-stained cells positive for SSEA-4 and by dual-stained cells positive for both GFRalpha1 and SSEA-4 reflects the undifferentiated stage of cat SSCs. The absence of transcription factors in double-stained cells positive for only one marker implies the loss of the stem cell-like identity with the loss of either GFRalpha1 or SSEA-4. Further investigation is warranted to elucidate the biological characteristics of these spermatogonial subpopulations.

Isolation and Purification of Viable PGCs from Mouse Embryos.

In all organisms with sexual reproduction, sperm and oocytes derive from embryonic precursors termed primordial germ cells (PGCs) which pass on genetic information to subsequent generations. Studies aimed to unravel PGC development at molecular level in mammals can be traced at the early 1980s and were hampered by the difficulty in obtaining both sufficient quantities and purity of PGCs. For many laboratories, the isolation and purification methods of PGCs at different stages from embryos are the most shortcut and affordable tool to study many aspects of their development at cellular and molecular levels. In the present chapter, I focus on immunomagnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS) methods used in my laboratory for the purification of mouse PGCs from 10.5 to 12.5 dpc embryos before their differentiation in oogonia/oocytes in female and prospermatogonia in male.

SSEA-1 isolates human endometrial basal glandular epithelial cells: phenotypic and functional characterization and implications in the pathogenesis of endometriosis.

STUDY QUESTION: Can the basal epithelial compartment of the human endometrium be defined by specific markers? SUMMARY ANSWER: Human endometrial epithelial cells from the basalis express nuclear SOX9 and the cell-surface marker SSEA-1, with some cells expressing nuclear ß-catenin. In vitro, primary endometrial epithelial cells enriched for SSEA-1+ show some features expected of the basalis epithelium. WHAT IS KNOWN ALREADY: The endometrial glands of the functionalis regenerate from the basalis gland stumps following menstruation. Endometriosis is thought to originate from abnormal dislocation of the basalis endometrium. In the highly regenerative intestinal epithelium, SOX9 and nuclear ß-catenin are more highly expressed in the intestinal crypt, the stem/progenitor cell region. STUDY DESIGN, SIZE, DURATION: A large prospective observational study analysing full-thickness human endometrial hysterectomy samples from 115 premenopausal women, 15 post-menopausal women and ectopic endometriotic lesions from 20 women with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Full-thickness endometrium from hysterectomy tissues was analysed by immunohistochemistry for SSEA-1, SOX9 and ß-catenin. Primary human endometrial epithelial cells from short-term cultures were sorted into SSEA1+/- fractions with a cell sorter or magnetic beads and analysed for markers of differentiation and pluripotency and telomere lengths (TLs) using qPCR, telomerase activity [telomere repeat amplification protocol (TRAP)] and growth in 3D culture. MAIN RESULTS AND THE ROLE OF CHANCE: Similar to the intestinal crypt epithelium, human endometrial basal glandular epithelial cells expressed nuclear SOX9 and contained a rare subpopulation of cells with nuclear ß-catenin suggestive of an activated Wnt pathway. The embryonic stem cell-surface marker, SSEA-1, also marked the human endometrial basal glandular epithelial cells, and isolated SSEA-1(+) epithelial cells grown in monolayer showed significantly higher expression of telomerase activity, longer mean TLs, lower expression of genes for steroid receptors and produced a significantly higher number of endometrial gland-like spheroids in 3D culture compared with SSEA-1(-) epithelial cells (P = 0.009). Cells in ectopic endometriosis lesions also expressed SSEA-1 and nuclear SOX9, suggesting that the basalis contributes to ectopic lesion formation in endometriosis following retrograde menstruation. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study with only short-term culture of the primary human epithelial cells in vitro. WIDER IMPLICATIONS OF THE FINDINGS: The surface marker SSEA1 enriches for an endometrial epithelial cell subpopulation from the basalis. Since the functional endometrium originates from these cells, it is now possible to study basalis epithelium for stem/progenitor cell activity to extend our current understanding of endometrial biology in health and diseases. STUDY FUNDING/COMPETING INTEREST(S): The work included in this manuscript was funded by Wellbeing of Women project grant RG1073 (D.K.H. and C.G.). We also acknowledge the support by National Health and Medical Research Council, RD Wright Career Development Award 465121 and Senior Research Fellowship 1042298, and the Victorian Government's Operation Infrastructure Support Program to C.G. and MRC G0601333 to T.V.Z. All authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Characterization of Oogonial Stem Cells in Adult Mouse Ovaries with Age and Comparison to In Silico Data on Human Ovarian Aging.

Many adult somatic stem cell lineages are comprised of subpopulations that differ in gene expression, mitotic activity, and differentiation status. In this study, we explored if cellular heterogeneity also exists within oogonial stem cells (OSCs), and how chronological aging impacts OSCs. In OSCs isolated from mouse ovaries by flow cytometry and established in culture, we identified subpopulations of OSCs that could be separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Levels of aldehyde dehydrogenase (ALDH) activity were inversely related to OSC differentiation, whereas commitment of OSCs to differentiation through transcriptional activation of stimulated by retinoic acid gene 8 was marked by a decline in ALDH activity and in SSEA1 expression. Analysis of OSCs freshly isolated from ovaries of mice between 3 and 20 months of age revealed that these subpopulations were present and persisted throughout adult life. However, expression of developmental pluripotency associated 3 (Dppa3), an epigenetic modifier that promotes OSC differentiation into oocytes, was lost as the mice transitioned from a time of reproductive compromise (10 months) to reproductive failure (15 months). Further analysis showed that OSCs from aged females could be established in culture, and that once established the cultured cells reactivated Dppa3 expression and the capacity for oogenesis. Analysis of single-nucleus RNA sequence data sets generated from ovaries of women in their 20s versus those in their late 40s to early 50s showed that the frequency of DPPA3-expressing cells decreased with advancing age, and this was paralleled by reduced expression of several key meiotic differentiation genes. These data support the existence of OSC subpopulations that differ in gene expression profiles and differentiation status. In addition, an age-related decrease in Dppa3/DPPA3 expression, which is conserved between mice and humans, may play a role in loss of the ability of OSCs to maintain oogenesis with age.

Stemness potency and structural characteristics of thyroid cancer cell lines.

BACKGROUND: Thyroid cancer is the most frequent type of endocrine malignancy. Thyroid carcinomas are derived from the follicular epithelium and classified as papillary (PTC) (85%), follicular (FTC) (12%), and anaplastic (ATC) (<3%). Thyroid cancer could arise from thyroid cancer stem-like cells (CSCs). CSCs are cancer cells that feature stem-like properties. Kruppel-like factor (KLF4) and Stage-spesific embryonic antigen 1 (SSEA-1) are types of stem cell markers. Filamentous actin (F-actin) is an essential part of the cellular cytoskeleton. The purpose of this study was to evaluate the stem cell potency and the spatial distribution of the cytoskeletal element F-actin in PTC, FTC, and ATC cell lines. MATERIALS AND METHODS: Normal thyroid cell line (NTC) Nthy-ori-3-1, PTC cell line BCPAP, FTC cell line FTC-133 and ATC cell line 8505c were stained with SSEA-1 and KLF4 for stem cell potency and F-actin for cytoskeleton. The morphological properties of cells were assessed by a scanning electron microscope (SEM) and elemental ratios were compared with EDS. RESULTS: PTCs had greater percentages of SSEA-1 and KLF4 protein intensity (0.32% and 0.49%, respectively) than NTCs. ATCs had a greater proportion of KLF4 expression (0.8%) than NTCs. NTCs and FTCs had increased F-actin intensity across the cell, but PTCs had the lowest among these four cell lines. NTCs and PTCs, as well as NTCs and FTCs, have statistically identical aspect ratios and round values. These values, however, were statistically different in ATCs. CONCLUSION: The study of stem cell markers and the cytoskeletal element F-actin in cancer and normal thyroid cell lines may assist in the identification of new therapeutic targets and contribute in the understanding of treatment resistance mechanisms.

High-Dimensional Mass Cytometry Analysis of Embryonic Antigens and Their Signaling Pathways in Myeloid Cells from Bone Marrow Aspirates in AML Patients at Diagnosis.

BACKGROUND: Embryonic antigens (EA) regulate pluripotency, self-renewal, and differentiation in embryonic stem (ES) cells during their development. In adult somatic cells, EA expression is normally inhibited; however, EAs can be re-expressed by cancer cells and are involved in the deregulation of different signaling pathways (SPs). In the context of AML, data concerning the expression of EAs are scarce and contradictory. METHODS: We used mass cytometry to explore the expression of EAs and three SPs in myeloid cells from AML patients and normal bone marrow (NBM). Imaging flow cytometry was used for morphological assessment of cells in association with their OCT3/4 expression status (positive vs. negative). RESULTS: An overall reduction in or absence of EA expression was observed in immature myeloid cells from AML patients compared to their normal counterparts. Stage-specific embryonic antigen-3 (SSEA-3) was consistently expressed at low levels in immature myeloid cells, whereas SSEA-1 was overexpressed in hematopoietic stem cells (HSCs) and myeloblasts from AML with monocytic differentiation (AML M4/M5). Therefore, these markers are valuable for distinguishing between normal and abnormal myeloid cells. These preliminary results show that the exploration of myeloid cell intracellular SPs in the setting of AML is very informative. Deregulation of three important leukemogenic SPs was also observed in myeloid cells from AML. CONCLUSIONS: Exploring EAs and SPs in myeloid cells from AML patients by mass cytometry may help identify characteristic phenotypes and facilitate AML follow-up.

Endometrial stem/progenitor cells: Properties, origins, and functions.

The endometrium is the inner mucosal lining of the uterus that undergoes extensive cyclic growth, regeneration, differentiation, and shedding throughout the menstrual cycle in response to steroid hormones. It repeatedly undergoes approximately 450 cycles of degeneration and regeneration in a woman's lifetime. Endometrial abnormalities can be associated with repeated embryo implantation failure, recurrent spontaneous abortion, and other physiological features responsible for female infertility. This significant regenerative capacity may occur as a result of tissue-resident stem cell populations within the endometrium. Indeed, the existence of endometrial stem cells was only observed in humans and rodents through several isolation and characterization methods in the last few years. Although endometrial stem cells share various biological characteristics with other types of mesenchymal stem cells, they also show some differences in phenotype, self-renewal, and multilineage differentiation potential. Extensive studies over many years on endometrial stem cells will provide new insights into the physiology and mechanisms underlying various gynaecological diseases related to endometrial abnormalities such as female infertility, endometriosis, and endometrial cancer. Here we summarized recent studies about cellular origins and biological characteristics of endometrial stem cells. We also reviewed various recent studies to improve our understanding of their physiological roles. Many preclinical studies on their potential therapeutic applications to various endometrial diseases that could lead to reproductive dysfunction were also reviewed.

Long-term maintenance of human endometrial epithelial stem cells and their therapeutic effects on intrauterine adhesion.

BACKGROUND: The human endometrium is a highly regenerative tissue that is believed to have two main types of stem cells: endometrial mesenchymal/stromal stem cells (eMSCs) and endometrial epithelial stem cells (eESCs). So far, eMSCs have been extensively studied, whereas the studies of eESCs are constrained by the inability to culture and expand them in vitro. The aim of this study is to establish an efficient method for the production of eESCs from human endometrium for potential clinical application in intrauterine adhesion (IUA). RESULTS: Here we developed a culture condition with a combination of some small molecules for in vitro culturing and expansion of human SSEA-1+ cells. The SSEA-1+ cells exhibited stem/progenitor cell activity in vitro, including clonogenicity and differentiation capacity into endometrial epithelial cell-like cells. In addition, the SSEA-1+ cells, embedded in extracellular matrix, swiftly self-organized into organoid structures with long-term expansion capacity and histological phenotype of the human endometrial epithelium. Specifically, we found that the SSEA-1+ cells showed stronger therapeutic potential than eMSCs for IUA in vitro. In a rat model of IUA, in situ injection of the SSEA-1+ cells-laden chitosan could efficiently reduce fibrosis and facilitate endometrial regeneration. CONCLUSIONS: Our work demonstrates an approach for isolation and expansion of human eESCs in vitro, and an appropriate marker, SSEA-1, to identify eESCs. Furthermore, the SSEA-1+ cells-laden chitosan might provide a novel cell-based approach for IUA treatment. These findings will advance the understanding of pathophysiology during endometrial restoration which may ultimately lead to more rational clinical practice.

Elevated transcription and glycosylation of B3GNT5 promotes breast cancer aggressiveness.

BACKGROUND: Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer because of its aggressive biological characteristics and no effective targeted agents. However, the mechanism underlying its aggressive behavior remain poorly understood. ß1,3-N-acetylglucosaminyltransferase V (B3GNT5) overexpression occurs specifically in BLBC. Here, we studied the possible molecular mechanisms of B3GBT5 promoting the aggressiveness of BLBC. METHODS: The potential effects of B3GNT5 on breast cancer cells were tested by colony formation, mammosphere formation, cell proliferation assay, flow cytometry and Western blotting. The glycosylation patterns of B3GNT5 and associated functions were determined by Western blotting, quantitative real-time PCR and flow cytometry. The effect of B3GNT5 expression on BLBC was assessed by in vitro and in vivo tumorigenesis model. RESULTS: In this study, we showed that B3GNT5 copy number amplification and hypomethylation of B3GNT5 promoter contributed to the overexpression of B3GNT5 in BLBC. Knockout of B3GNT5 strongly reduced surface expression of SSEA-1 and impeded cancer stem cell (CSC)-like properties of BLBC cells. Our results also showed that B3GNT5 protein was heavily N-glycosylated, which is critical for its protein stabilization. Clinically, elevated expression of B3GNT5 was correlated with high grade, large tumor size and poor survival, indicating poor prognosis of breast cancer patients. CONCLUSIONS: Our work uncovers the critical association of B3GNT5 overexpression and glycosylation with enhanced CSCs properties in BLBC. These findings suggest that B3GNT5 has the potential to become a prognostic marker and therapeutic target for BLBC.

Expression of the cancer stem cell marker SSEA1 is associated with poor survival in metastatic high-grade serous carcinoma.

The objective of the present study was to perform a quantitative analysis of cancer stem cell (CSC) marker expression in ovarian carcinoma effusions. The clinical role of SSEA1 in metastatic high-grade serous carcinoma (HGSC) was additionally analyzed. CD133, Nanog, SOX2, Oct3/4, SSEA1, and SSEA4 protein expressions were quantitatively analyzed using flow cytometry (FCM) in 24 effusions. SSEA1 expression by immunohistochemistry was analyzed in 384 HGSC effusions. Highly variable expression of CSC markers by FCM was observed, ranging from 0 to 78% of Ber-EP4-positive cells in the case of CD133, with the largest number of negative specimens seen for SSEA4. SSEA1 expression by immunohistochemistry was found in HGSC cells in 336/384 (89%) effusions, most commonly focally (< 5% of cells). SSEA1 was overexpressed in post-chemotherapy disease recurrence specimens compared with chemo-naïve HGSC effusions tapped at diagnosis (p = 0.029). In univariate survival analysis, higher SSEA1 expression was significantly associated with poor overall survival (p = 0.047) and progression-free survival (p = 0.018), though it failed to retain its prognostic role in Cox multivariate survival analysis in which it was analyzed with clinical parameters (p = 0.059 and p = 0.111 for overall and progression-free survival, respectively). In conclusion, CSC markers are variably expressed in ovarian carcinoma effusions. SSEA1 expression is associated with disease progression and poor survival in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

OVOL1 Influences the Determination and Expansion of iPSC Reprogramming Intermediates.

During somatic cell reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts undergo dynamic molecular changes, including a mesenchymal-to-epithelial transition (MET) and gain of pluripotency; processes that are influenced by Yamanaka factor stoichiometry. For example, in early reprogramming, high KLF4 levels are correlated with the induction of functionally undefined, transiently expressed MET genes. Here, we identified the cell-surface protein TROP2 as a marker for cells with transient MET induction in the high-KLF4 condition. We observed the emergence of cells expressing the pluripotency marker SSEA-1+ mainly from within the TROP2+ fraction. Using TROP2 as a marker in CRISPR/Cas9-mediated candidate screening of MET genes, we identified the transcription factor OVOL1 as a potential regulator of an alternative epithelial cell fate characterized by the expression of non-iPSC MET genes and low cell proliferation. Our study sheds light on how reprogramming factor stoichiometry alters the spectrum of intermediate cell fates, ultimately influencing reprogramming outcomes.

Stem Cell Markers Describe a Transition From Somatic to Pluripotent Cell States in a Rat Model of Endometriosis.

OBJECTIVE: To study Thy1 as a fibroblast marker, SSEA1 as a marker of intermediate pluripotency, and Oct4 as a marker of established pluripotency in rat model of endometriosis. DESIGN: In vivo animal study. MATERIALS AND METHODS: Endometriosis was induced in 20 albino female rats through autologous transplantation of one uterine horn to mesentery of intestine. Other 20 rats had their horn removed without transplantation (controls). Rats were sacrificed 4 weeks after induction surgery. Ectopic, eutopic, and control endometria were harvested from endometriosis and control animals respectively. Quantitative syber green based RT-PCR was used to detect expression of Thy-1 (CD90), FUT4 (SSEA1), and POU5F1 (Oct4) genes in tissues. Relative expression was normalized to that of ß actin. Thy1, SSEA1, and Oct4 protein expression were detected by immunohistochemistry. RESULTS: Ectopic endometrium expressed significantly higher mRNA of Oct4 and SSEA1 as compared to control endometrium. Expression levels of Oct4 and SSEA1 were comparable between ectopic and eutopic endometria and between eutopic and control endometria. Thy1 (CD90) gene expression level was comparable among ectopic, eutopic, and control endometria. Oct4 immunoscore were significantly higher in ectopic (6.6±0.91) than eutopic (2.5±0.78) or control endometrium (3.7±0.1) (P value 0.02). Thy1 and SSEA1 immunoscores were comparable among all three types of endometria. CONCLUSIONS: Using rat model of endometriosis, ectopic endometrium showed significantly higher Oct4, and SSEA1, but similar Thy1 gene expression to that of control endometrium. This indicates increased transition from somatic to pluripotent cell states in ectopic endometrium which may play a role in endometriosis pathogenesis.

Chondrocyte proliferation, viability and differentiation is declined following administration of methylphenidate utilized for the treatment of attention-deficit/hyperactivity disorder.

PURPOSE: Methylphenidate (MPH) derivative drugs are used because of psychostimulants effects on attention-deficit hyperactivity disorder in children and adults. As far as we know, toxic or anti-proliferative effects of MPH against cartilage tissue were not studied in the literature. The present study was carried out to investigate the possible effects of MPH on the proliferation, viability and differentiation of primary human chondrocytes, in vitro. METHODS: Monolayer primary chondrocyte cultures were prepared using osteochondral tissue obtained from patients who underwent a total knee prosthesis operation. Stock solution of MPH was prepared and aliquots having 1-1000 µM concentrations of the drug was composed. These solutions were applied to the wells containing cultured chondrocyte samples within the well plates. Control groups were composed of pure chondrocyte culture and no solution was added into them. All groups were evaluated at 24, 48 and 72 h in order to determine the possible negative effects of the drug on the chondrocytes. The data were evaluated by Tukey's honestly significantly different test following analysis of variance. RESULTS: In the group where MPH was applied, it was found that viability, proliferation and stage-specific embryonic antigen-1 protein expression were decreased in comparison to the control group. CONCLUSIONS: It was emphasized that clinicians should not disregard the fact that this drug might suppress chondrocyte cell proliferation and chondrogenic differentiation.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts.

Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore, reprogramming of SSEA-1+ sorted pEFs led to higher reprogramming efficiency. Subsequent transcriptome profiling of the SSEA-1+ vs. the SSEA-1neg cell fraction revealed highly comparable gene signatures. However several genes that were found to be upregulated in the SSEA-1+ cells were similarly expressed in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig.

Stage-Specific Embryonic Antigen-1 (SSEA-1) Expression in Thyroid Tissues.

Stage-specific embryonic antigen-1 (SSEA-1), also known as CD15, is a member of a cluster of differentiation antigens that have been identified in various normal tissues and in different types of cancers including papillary and medullary thyroid carcinoma. SSEA-1 may be expressed in normal stem cells and cancer stem-like cells. To evaluate the potential diagnostic and prognostic utility of SSEA-1 in thyroid tumors, we analyzed the expression of SSEA-1 in normal and neoplastic thyroid tissues by immunohistochemistry (IHC) using a tissue microarray with 158 different tissue cores. To evaluate the potential utility of SSEA-1 as a surface marker, we also assessed the expression of SSEA-1 in thyroid cell lines by flow cytometric analysis. SSEA-1 immunoreactivity was identified in malignant thyroid follicular epithelial cancers but not in the benign thyroid tissues. Anaplastic thyroid (ATC) (80 %) and conventional papillary thyroid carcinoma (PTC) (60.7 %) showed significantly higher percentage of cases that were SSEA-1 immunoreactive than follicular variant of papillary thyroid carcinoma (FVPTC) (20.6 %) and follicular carcinoma (FCA) (32.1 %). Flow cytometric analysis of cultured thyroid cell lines showed that a small subpopulation of ATC and PTC thyroid tumor cells had SSEA-1 immunoreactivity which may represent thyroid cancer stem-like cells. The ATC cells expressed more SSEA-1 immunoreactive cells than the PTC cell lines. Our findings suggest that expression of SSEA-1 immunoreactivity in thyroid neoplasms was associated with more aggressive thyroid carcinomas. SSEA-1 is a marker that detects malignant thyroid neoplasms in formalin-fixed paraffin-embedded thyroid tissue sections and may be a useful marker for thyroid cancer stem-like cells.