SSEA-3 Research Articles

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.

Immunohistochemical analysis of selected stem cell markers including stage-specific mouse embryonic antigen 1 (SSEA-1) in porcine and bovine endometrium throughout the oestrous cycle

There has been a lack of research regarding endometrial stem cells in farm animals, and particularly, regarding epithelial stem/progenitor cells. We aimed to identify potential stem cell subpopulations in porcine and bovine endometrium by measuring the expression of selected stem cell markers (OCT3/4, CD44, SSEA-1, SOX10, CD73, and CD117) during the follicular and luteal phases of the oestrous cycle. We studied 28 endometrial tissue samples collected from 14 healthy, cycling pigs and cows. The endometrial mucosa of each sample was divided into basal, middle, and luminal portions, and the immunohistochemical staining intensity and percentages of cells that were marker-positive were recorded for each portion of the endometrium. Expression of OCT-3/4 was identified in the glands and stroma, and that of SOX10 and CD117 was identified in stroma of both porcine and bovine endometrium. In porcine endometrium, CD44 was only expressed in the glands, whereas SSEA-1 was expressed in the glands and stroma. In bovine endometrium, CD73 was only expressed in the glands. Differences in the expression of most of these markers were found between phases of the oestrous cycle and/or portions of the endometrial mucosa. Our data indicate the existence of both epithelial and mesenchymal stem cell subpopulations in the endometrium of pigs and cows during their oestrous cycles. The expression pattern of the stem cell marker SSEA-1 in porcine endometrium suggests a possible alternative location of the epithelial stem/progenitor cell population in the middle portion of the endometrial glands.

Conformational alteration in glycan induces phospholipase Cβ1 activation and angiogenesis

BackgroundIn endothelial cells, phospholipase C (PLC) β1-activated Ca2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCβ1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCβ1 activation.MethodsGlobo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCβ1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice.ResultsThe results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with Kd of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCβ1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCβ1 in cells by GHCer derived from EVs.ConclusionsOverall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCβ1, leading to Ca2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.

Single-cell RNA sequencing reveals different signatures of mesenchymal stromal cell pluripotent-like and multipotent populations

Somatic stem cells are advantageous research targets for understanding the properties required to maintain stemness. Human bone marrow-mesenchymal stromal cells (BM-MSCs) were separated into pluripotent-like SSEA-3(+) Muse cells (Muse-MSCs) and multipotent SSEA-3(-) MSCs (MSCs) and were subjected to single-cell RNA sequencing analysis. Compared with MSCs, Muse-MSCs exhibited higher expression levels of the p53 repressor MDM2; signal acceptance-related genes EGF, VEGF, PDGF, WNT, TGFB, INHB, and CSF; ribosomal protein; and glycolysis and oxidative phosphorylation. Conversely, MSCs had higher expression levels of FGF and ANGPT; Rho family and caveola-related genes; amino acid and cofactor metabolism; MHC class I/II, and lysosomal enzyme genes than Muse-MSCs. Unsupervised clustering further divided Muse-MSCs into two clusters stratified by the expression of cell cycle-related genes, and MSCs into three clusters stratified by the expression of cell cycle-, cytoskeleton-, and extracellular matrix-related genes. This study evaluating the differentiation ability of BM-MSC subpopulations provides intriguing insights for understanding stemness.

Role of glycosphingolipid SSEA-3 and FGF2 in the stemness and lineage commitment of multilineage differentiating stress enduring (MUSE) cells.

Multilineage differentiating Stress Enduring (MUSE) cells are endogenous, stress-resistant stem cells, expressing pluripotency master genes and able to differentiate in cells of the three embryonic sheets. Stage-Specific Embryonic Antigen 3 (SSEA-3), a glycosphingolipid (GSL), is the marker for identifying MUSE cells and is used to isolate this population from mesenchymal stromal cells. GSLs modulate signal transduction by interacting with plasma membrane components. The growth factor FGF2, important for MUSE cells biology, may interact with GSLs. Specific cell surface markers represent an invaluable tool for stem cell isolation. Nonetheless their role, if any, in stem cell biology is poorly investigated. Functions of stem cells, however, depend on niche external cues, which reach cells through surface markers. We addressed the role of SSEA-3 in MUSE cell behaviour, trying to define whether SSEA-3 is just a marker or if it plays a functional role in this cell population by determining if it has any relationship with FGF2 activity. We evidenced how the SSEA-3 and FGF2 cooperation affected the self-renewal and clonogenic capacity of MUSE cells. The block of SSEA-3 significantly reduced the multilineage potential of MUSE cells with production of nullipotent clones. We contributed to dissecting the mechanisms underlying MUSE cell properties for establishing successful stem-cell-based therapies and the promotion of MUSE cells as a tool for the in vitro disease model.

Expression of Fucosyltransferase 4 (FUT4) mRNA Is Increased in Endometrium from Women with Endometriosis.

Endometriosis is a common gynecological disorder defined as the presence of endometrial-like tissue (glands and stroma) outside the uterus. The etiopathogenesis of endometriosis is still poorly recognized. It is speculated that stage-specific embryonic antigen 1 (SSEA-1)-positive stem-like glandular epithelial cells may contribute to the development of the disease. The synthesis of SSEA-1 is mediated by fucosyltransferase 4 encoded by the FUT4 gene. Therefore, this study aimed to evaluate the specific expression of FUT4 mRNA in biopsies of the endometrium from women with and without endometriosis. FUT4 mRNA levels were examined in 49 women with laparoscopically confirmed endometriosis and 28 controls by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expression of FUT4 mRNA was significantly increased in the endometrium of patients with endometriosis when compared to the controls (p < 0.0001). Expression of FUT4 mRNA in the endometrium was correlated with the severity of endometriosis (rs = 0.5579, p < 0.0001); however, there were no differences in endometrial FUT4 mRNA expression when comparing endometriotic lesions from various locations. The discriminatory ability of FUT4 mRNA expression was evaluated by receiver-operating characteristics (ROC), which showed high statistical significance (AUC = 0.90, p < 0.0001), thus indicating that an increased level of endometrial FUT4 mRNA may serve as a specific marker for endometriosis.

Injectable Peptide Hydrogel Encapsulation of Mesenchymal Stem Cells Improved Viability, Stemness, Anti-Inflammatory Effects, and Early Stage Wound Healing.

Human-adipose-derived mesenchymal stem cells (hADMSCs) are adult stem cells and are relatively easy to access compared to other sources of mesenchymal stem cells (MSCs). They have shown immunomodulation properties as well as effects in improving tissue regeneration. To better stimulate and preserve the therapeutic properties of hADMSCs, biomaterials for cell delivery have been studied extensively. To date, hyaluronic acid (HA)-based materials have been most widely adopted by researchers around the world. PGmatrix is a new peptide-based hydrogel that has shown superior functional properties in 3D cell cultures. Here, we reported the in vitro and in vivo functional effects of PGmatrix on hADMSCs in comparison with HA and HA-based Hystem hydrogels. Our results showed that PGmatrix was far superior in maintaining hADMSC viability during prolonged incubation and stimulated expression of SSEA4 (stage-specific embryonic antigen-4) in hADMSCs. hADMSCs encapsulated in PGmatrix secreted more immune-responsive proteins than those in HA or Hystem, though similar VEGF-A and TGFβ1 release levels were observed in all three hydrogels. In vivo studies revealed that hADMSCs encapsulated with PGmatrix showed improved skin wound healing in diabetic-induced mice at an early stage, suggesting possible anti-inflammatory effects, though similar re-epithelialization and collagen density were observed among PGmatrix and HA or Hystem hydrogels by day 21.

Inhibition of Gap Junctional Intercellular Communication Upregulates Pluripotency Gene Expression in Endogenous Pluripotent Muse Cells.

Gap junctions (GJ) are suggested to support stem cell differentiation. The Muse cells that are applied in clinical trials are non-tumorigenic pluripotent-like endogenous stem cells, can be collected as stage-specific embryonic antigen 3 (SSEA-3+) positive cells from multiple tissues, and show triploblastic differentiation and self-renewability at a single cell level. They were reported to up-regulate pluripotency gene expression in suspension. We examined how GJ inhibition affected pluripotency gene expression in adherent cultured-Muse cells. Muse cells, mainly expressing gap junction alpha-1 protein (GJA1), reduced GJ intercellular communication from ~85% to 5–8% after 24 h incubation with 120 μM 18α-glycyrrhetinic acid, 400 nM 12-O-tetradecanoylphorbol-13-acetate, and 90 μM dichlorodiphenyltrichloroethane, as confirmed by a dye-transfer assay. Following inhibition, NANOG, OCT3/4, and SOX2 were up-regulated 2–4.5 times more; other pluripotency-related genes, such as KLF4, CBX7, and SPRY2 were elevated; lineage-specific differentiation-related genes were down-regulated in quantitative-PCR and RNA-sequencing. Connexin43-siRNA introduction also confirmed the up-regulation of NANOG, OCT3/4, and SOX2. YAP, a co-transcriptional factor in the Hippo signaling pathway that regulates pluripotency gene expression, co-localized with GJA1 (also known as Cx43) in the cell membrane and was translocated to the nucleus after GJ inhibition. Adherent culture is usually more suitable for the stable expansion of cells than is a suspension culture. GJ inhibition is suggested to be a simple method to up-regulate pluripotency in an adherent culture that involves a Cx43-YAP axis in pluripotent stem cells, such as Muse cells.

Glycosphingolipids in human embryonic stem cells and breast cancer stem cells, and potential cancer therapy strategies based on their structures and functions.

Expression profiles of glycosphingolipids (GSLs) in human embryonic stem cell (hESC) lines and their differentiated embryoid body (EB) outgrowth cells, consisting of three germ layers, were surveyed systematically. Several globo- and lacto-series GSLs were identified in undifferentiated hESCs and during differentiation of hESCs to EB outgrowth cells, and core structure switching of these GSLs to gangliosides was observed. Such switching was attributable to altered expression of key glycosyltransferases (GTs) in GSL biosynthetic pathways, reflecting the unique stage-specific transitions and mechanisms characteristic of the differentiation process. Lineage-specific differentiation of hESCs was associated with further GSL alterations. During differentiation of undifferentiated hESCs to neural progenitor cells, core structure switching from globo- and lacto-series to primarily gangliosides (particularly GD3) was again observed. During differentiation to endodermal cells, alterations of GSL profiles were distinct from those in differentiation to EB outgrowth or neural progenitor cells, with high expression of Gb4Cer and low expression of stage-specific embryonic antigen (SSEA)-3, -4, or GD3 in endodermal cells. Again, such profile changes resulted from alterations of key GTs in GSL biosynthetic pathways. Novel glycan structures identified on hESCs and their differentiated counterparts presumably play functional roles in hESCs and related cancer or cancer stem cells, and will be useful as surface biomarkers. We also examined GSL expression profiles in breast cancer stem cells (CSCs), using a model of epithelial-mesenchymal transition (EMT)-induced human breast CSCs. We found that GD2 and GD3, together with their common upstream GTs, GD3 synthase (GD3S) and GD2/GM2 synthase, maintained stem cell phenotype in breast CSCs. Subsequent studies showed that GD3 was associated with epidermal growth factor receptor (EGFR), and activated EGFR signaling in breast CSCs and breast cancer cell lines. GD3S knockdown enhanced cytotoxicity of gefitinib (an EGFR kinase inhibitor) in resistant MDA-MB468 cells, both in vitro and in vivo. Our findings indicate that GD3S contributes to gefitinib resistance in EGFR-positive breast cancer cells, and is a potentially useful therapeutic target in drug-resistant breast cancers.

Abstract P5-08-01: Globo H: A Globo series glycosphingolipid target antigen in breast cancer

Abstract Cancer cells may be differentiated from normal cells by the surface expression of unusual glycosylation patterns: tumor-associated carbohydrate antigens (TACAs) may be expressed as membrane-bound glycoproteins or glycolipids. Differential expression of TACAs in multiple cancer types represents a potential therapeutic target for anticancer therapies. At least 16 different types of epithelial cancers have been found to be Globo H–positive, with 60% to 65% of patients with breast cancer having Globo H–positive tumors. It has been shown that tumor-infiltrating lymphocytes in breast cancer tissues could take up Globo H ceramide (GHCer) released by tumor cells masking cancer cells from immune surveillance through the suppression of Notch 1 signaling. Globo series antigens, including Globo H and stage-specific embryonic antigen 4 could associate with the FAK/CAV1/AKT/RIP complex to trigger the downstream β-catenin signaling pathway to promote tumor cell survival. Moreover, exogenous addition of GHCer promoted tumor cell migration. Thus, treatment-mediated depletion of GHCer may overcome the immunosuppressive mechanism of cancer cells, with resulting decreases in tumor cell survival and migration. OBI Pharma, Inc., in collaboration with NeoGenomics, has developed a validated immunohistochemistry assay to measure the expression of Globo H in clinical specimens. The intensity and extent of Globo H expression are determined microscopically using the histochemical scoring system (H-score), defined as the sum of the products of the staining intensity (score of 0-3) multiplied by the percentage of cells (0-100) stained at a given intensity. To date, 420 patients with triple-negative breast cancer (TNBC) have been screened across 4 separate clinical trials. Of those patients, 173 (41%) had an H-score of ≥15, with a median H-score of 70. Of the 173 patients with an H-score of ≥15, 69 (40%) had an H-score of ≥100. Adagloxad simolenin (AS/OBI-822) is a Globo H conjugate vaccine that when administered in combination with the adjuvant OBI-821 results in anti–Globo H IgM and IgG humoral immune responses. These responses have been assessed in all clinical trials of AS/OBI-821 to date. In the early trials that administered up to 9 doses, early and robust IgM responses were noted but with delayed and lower-titer IgG responses. When the number of doses was increased to 9-12 doses over an extended time (up to either 37 or 68 weeks), it became apparent that the peak IgG titers occurred at around the time of the last AS/OBI-821 dose. The time course for the development of anti–Globo H IgM and IgG humoral responses showed that following injection, anti–Globo H IgM and IgG antibody titers demonstrated a geometric mean ratio (GMR) increase of 10.26 and 12.47, respectively, from almost nondetectable levels at baseline. The GMR in the placebo group remained low in comparison. Mean time to peak titer was approximately 4 weeks for IgM and 40 weeks for IgG. These data were employed in the design of the phase 3, randomized, open-label study of the anti–Globo H vaccine AS/OBI-821 in the adjuvant treatment of patients with high-risk, early-stage Globo H–positive TNBC (The GLORIA Study). To enter the GLORIA study, patients must have a centrally confirmed Globo H IHC H-score of ≥15, and it is anticipated that approximately 40% of patients screened will have this level of expression. The GLORIA study is presently enrolling patients in 7 countries, with another 9 countries anticipated to begin recruiting in 2021. Citation Format: Darren S. Sigal, Apostolia Maria Tsimberidou, I-Ju Chen, Pei Hsu, Tillman Pearce. Globo H: A Globo series glycosphingolipid target antigen in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-01.

Application prospects of multilineage differentiating stress-enduring (Muse) cells in the treatment of skin diseases

Multilineage differentiating stress-enduring (Muse) cells are a subgroup of mesenchymal stem cells (MSCs) that express stage-specific embryonic antigen-3 (SSEA-3) and CD105. It is capable of generating cells representing all three germ layers from a single cell and is non-tumorigenic. Muse cells can efficiently migrate and integrate into damaged tissues that produce sphingosine-1-phosphate (S1P), while Muse cells specifically express S1P receptor 2. The factors secreted by Muse cells play anti-inflammatory, anti-fibrosis, and anti-apoptotic effects, and cooperate to complete the function and structure repair of the damaged group. This review summarizes the basic characteristics of Muse cells and describes their application in skin repair.

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.

PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD39)1 Sep 2021PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS Meghan Robinson, Ryan Flannigan, and Luke Witherspoon Meghan RobinsonMeghan Robinson More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Luke WitherspoonLuke Witherspoon More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002049.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS), however, animal components introduce the risk of contaminating with pathogens, making them unsuitable for medical use. This study set out to develop xeno-free culture conditions for the expansion of human SSCs. METHODS: SSCs were derived from human induced pluripotent stem cells (hiPSCs) and tested in various xeno-free media conditions in combination with growth factors Prostaglandin D2 (PDG2) and Insulin-Like Growth Factor 1 (IGF1). Primary SSCs then underwent 3 passages in the best condition, were cryopreserved and thawed, and then analysed for changes in gene and protein expression by real time polymerase chain reaction (RT-qPCR) and immunocytochemistry. RESULTS: 10 ng/mL PDG2 with 10 ng/mL IGF1 was found to replace FBS and BSA without loss of viability or growth. ROCK inhibitor Y-27632 was determined to be necessary for viability upon thawing. Compared to established conditions, the SSCs shared identical protein expression profiles for the SSC markers Glial Cell-Derived Neurotrophic Factor Family Receptor Alpha 1 (GFRA1), G-Protein Coupled Receptor 125 (GPR125), Thy-1 Cell Surface Antigen (CD90), and Stage-Specific Embryonic Antigen 4 (SSEA4) (Figure 1A-B). RT-qPCR analyses revealed no significant variation in gene expression of undifferentiated germ cell markers and Inhibitor Of Differentiation 4 (ID4), and Fibroblast Growth Factor Receptor 3 (FGFR3). The greatest change seen was an increase in Deleted In Azoospermia Like (DAZL), a regulator of both SSC proliferation and differentiation (23-fold) (Figure 1C). CONCLUSIONS: This study identified a combination of growth factors capable of replacing FBS and BSA components in established SSC expansion cell culture. This xeno-free defined formulation allows standardized SSC culture and removes the risk of animal pathogens. Source of Funding: VCHRI, CUASF, CIHR, UBC © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e671-e672 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Meghan Robinson More articles by this author Ryan Flannigan More articles by this author Luke Witherspoon More articles by this author Expand All Advertisement Loading ...

Abstract 3176: Inhibitory activity of Globo-H and SSEA-4 on activated T-cells

Abstract Background/Aim: Globo-H (GH) and stage-specific embryonic antigen-4 (SSEA-4) are globo-series glycosphingolipid (GSL) antigens highly expressed on a variety of epithelial cancers. GH and SSEA-4 are reported to play an important role in cancer cell development and survival and thus are promising targets for developing immunotherapeutic agents for cancer treatment. Shedding of GSLs by tumor cells and uptake by neighboring cells may generate a preferable environment for tumor progression and survival within the tumor microenvironment. GH shed from tumor cells was shown to promote angiogenesis and suppress immune responses in vitro. In addition, exogenous Globo-H ceramide (GHCer) inhibits proliferation of human PBMCs and murine CD4+ T-cells. Given the important role of sialic acid in regulating the immune system, SSEA-4, a polysaccharide with sialic acid at the terminal, may play an important role in the regulation of T-cell responses. This study aimed to investigate the GHCer and SSEA-4 ceramide (SSEA-4Cer) effects on T-cell activation. Methods: C57BL/6 mice bearing murine B16F10 melanoma were treated with GHCer by peritumoral injection and monitored for 26 days. T-cell tumor subpopulations were evaluated by FACS and immunohistochemical analysis. Presence of GHCer and SSEA-4Cer expression on human T cells after the treatment with GHCer and SSEA-4Cer was monitored by FACS analysis. Cell proliferation and apoptosis of T cells after treatment with GHCer, SSEA-4Cer, and glucoceramide (GlcCer) were assessed using ATP assay and annexin V staining, respectively. After treatment with GHCer/SSEA-4Cer/GlcCer, expression levels of CD25, cytokine IFN-γ, and Ki-67 in human PBMCs stimulated with anti-CD3/CD28 were measured by flow cytometry. Results: B16F10 tumor growth rate increased 2.54-fold (P&amp;lt;0.05, n=6) in GHCer-treated mice with a lower number of tumor-infiltrating CD3+ cells (0.45-fold; P&amp;lt;0.01, n=6) compared with control mice. Not surprisingly, addition of GHCer and SSEA-4Cer to human T cells was shown to increase GH and SSEA-4 levels on the surface of T cells. Furthermore, addition of GHCer and SSEA-4Cer to the activated T cells showed a 26.4% and 36.9% decrease in cell proliferation, respectively, while no induction of apoptosis was observed. In contrast, GHCer had no impact on cell viability and proliferation of resting T cells. Exogenous GHCer and SSEA-4Cer significantly reduced CD3+CD25+ and CD3+IFNγ+ T cells in anti-CD3/CD28-stimulated PBMCs. SSEA-4Cer exhibited higher activity in the suppression of CD8+CD25+ T cells in PBMCs than GHCer did. In addition, suppression of the proliferation of activated T-cells by SSEA-4Cer was abolished in the presence of NF-κB inhibitor. Conclusion: GHCer inhibits T-cell infiltration into the tumor in vivo and SSEA-4 exhibits immunosuppressive activity on activated T cells, suggesting that both GHCer and SSEA-4Cer may play a role in regulating immunologic responses in the tumor microenvironment. Citation Format: Tzer-Min Kuo, Chin-Chan Lee, Jiann-Shiun Lai, Chung-Chen Su, Ming-Tain Lai. Inhibitory activity of Globo-H and SSEA-4 on activated T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3176.

Stage-specific Embryogenic Antigen-4 Expression in Castration-resistant Prostate Cancer and its Correlation With the Androgen Receptor.

Stage-specific embryonic antigen (SSEA)-4 plays important roles in the malignant aggressiveness of various cancers. The aim of this study was to investigate the pathological characteristics of SSEA-4 in castration-resistant prostate cancer (CRPC). SSEA-4 expression and its pathological roles were evaluated in five prostate cancer (PC) cell lines and 27 CRPC tissues. The relationship between SSEA-4 and the androgen receptor (AR) was also examined. SSEA-4 expression was detected in AR-negative cells (PC3, DU145, and AICaP1) but was not detected in AR-positive cells (LNCaP and AICaP2). SSEA-4 expression in human CRPC tissues was significantly higher than that in locally advanced or metastatic hormone sensitive PC (HSPC) tissues. A negative correlation was also detected between SSEA-4 and AR in CRPC tissues but not in HSPC tissues. SSEA-4 was over-expressed in CRPC and the changes were mediated by complex mechanisms that related to the AR and hormonal therapy.

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

Does natural variation exist in the endometrial stem/progenitor cell and protein composition of menstrual fluid across menstrual cycles in women? 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. 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. 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. 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. 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. N/A. There are no OMICS or other data sets relevant to this study. 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. 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. 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.

Elimination of cells deviated from human induced pluripotent stem cells with a photoactivatable IR700-labelled antibody

Human induced pluripotent stem cells (hiPSCs) are important starting materials for cell therapy products (CTPs) used for transplantation. During cell culture, hiPSCs often spontaneously undergo morphological changes and lose pluripotency. Such cells are called ‘deviated cells’, which are deviated from the undifferentiated state of hiPSCs, lack the expression of hiPSC markers and become positive for the early differentiation marker SSEA1 (stage-specific embryonic antigen 1, Lewis X glycan). Previously, we identified fibronectin (FN) as a predominant carrier protein of SSEA1 secreted from deviated cells, but not hiPSCs. A sandwich assay using antibodies (Abs) against FN and SSEA1 was developed for non-destructive quantitative evaluation of deviated cells present in hiPSC cultures. In this study, a novel technology was developed to specifically eliminate deviated cells using an anti-FN Ab along with a near-infrared (NIR) photoabsorber, IRDye700DX N-hydroxysuccinimide ester (IR700), which has been used for cancer photoimmunotherapy. The anti-FN Ab conjugated with the IR700 dye (IR700-αFN) bound to and induced the death of deviated cells upon NIR irradiation. In contrast, IR700-αFN failed to stain the hiPSCs, and IR700-αFN/NIR had little or no effect on survival. Finally, IR700-αFN/NIR irradiation induced selective removal of deviated cells from a mixed culture with hiPSCs, demonstrating that the proposed method is suitable for the removal of unwanted deviated cells present in hiPSC culture for the production of CTPs.

Long-Term Trypsin Treatment Promotes Stem Cell Potency of Canine Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) isolated from adipose tissue (adipose-derived stem cells [ADSCs]) are considered one of the most promising cell types for applications in regenerative medicine. However, the regenerative potency of ADSCs may vary because of heterogeneity. Long-term trypsin treatment (LTT) is known to significantly concentrate multilineage-differentiating stress-enduring (Muse) cells from human MSCs. In this study, we aimed to generate cells with high stem cell potency from canine ADSCs using LTT. After 16 h of treatment with trypsin, surviving ADSCs (LTT-tolerant cells) had significantly enhanced expression of stage-specific embryonic antigen (SSEA)-1, a mouse embryonic stem cell marker, and fucosyltransferase 9, one of several fucosyltransferases for SSEA-1 biosynthesis. However, LTT-tolerant cells did not enhance the expression of SSEA-3, a known human Muse cell marker. LTT-tolerant cells, however, showed significantly higher self-renewal capacity in the colony-forming unit fibroblast assay than ADSCs. In addition, the LTT-tolerant cells formed cell clusters similar to embryoid bodies and expressed undifferentiated markers. Moreover, these cells differentiated into cells of all three germ layers and showed significantly higher levels of α 2-6 sialic acid (Sia)-specific lectins, known as differentiation potential markers of human MSCs, than ADSCs. LTT-tolerant cells had a normal karyotype and had low telomerase activity, showing little carcinogenetic potency. LTT-tolerant cells also showed significantly increased activity of transmigration in the presence of chemoattractants and had increased expression of migration-related genes compared with ADSCs. In addition, LTT-tolerant cells had stronger suppressive activity against mitogen-stimulated lymphocyte proliferation than ADSCs. Overall, these results indicated that the LTT-tolerant cells in canine ADSCs have similar properties as human Muse cells (although one of the undifferentiated markers is different) and are expected to be a promising tool for regenerative therapy in dogs.

Minimal influence of chronic inflammation on the potency and differentiation characteristics of gingiva-derived mesenchymal stem cells-An in vitro study.

Objective:Gingiva-derived mesenchymal stem cells (GMSCs) have been identified and characterized from healthy tissues. However, reports on the influence of chronic inflammation on their stemness characteristics are sparse. The present study evaluated the potency and differentiation ability of GMSCs from periodontally healthy GMSC (H-GMSC) and inflamed GMSC (I-GMSC) tissues.Materials and Methods:Established H-GMSCs and I-GMSCs were evaluated on their potency characteristics, such as morphology, viability, proliferation rate, population doubling time, colony-forming ability, expression of stemness markers, and mesenchymal differentiation potential.Results:H-GMSCs and I-GMSCs exhibited fibroblast-like morphology and showed >95% viability with high proliferation potential and shorter doubling time. H-GMSCs showed fewer and smaller colonies, whereas I-GMSCs exhibited multiple and larger colonies. The evaluation of stemness markers revealed that both H-GMSCs and I-GMSCs were weakly positive for stage-specific embryonic antigen-4, Stro1, and CD105 (Endoglin), strongly positive for CD73 and CD90, and negative for the hematopoietic cell markers, CD34 and CD45. H-GMSCs showed a slightly higher osteogenic potential when compared to I-GMSCs, while I-GMSCs had a higher adipogenic potential than H-GMSCs.Conclusion:The findings showed that the inflammatory environment might have a stimulatory effect on the growth kinetics and ability of colony formation in GMSCs. However, varied osteogenic and adipogenic differentiation was observed between H-GMSCs and I-GMSCs.

Intravenously delivered multilineage-differentiating stress enduring cells dampen excessive glutamate metabolism and microglial activation in experimental perinatal hypoxic ischemic encephalopathy

Perinatal hypoxic ischemic encephalopathy (HIE) results in serious neurological dysfunction and mortality. Clinical trials of multilineage-differentiating stress enduring cells (Muse cells) have commenced in stroke using intravenous delivery of donor-derived Muse cells. Here, we investigated the therapeutic effects of human Muse cells in an HIE model. Seven-day-old rats underwent ligation of the left carotid artery then were exposed to 8% oxygen for 60 min, and 72 hours later intravenously transplanted with 1 × 104 of human-Muse and -non-Muse cells, collected from bone marrow-mesenchymal stem cells as stage-specific embryonic antigen-3 (SSEA-3)+ and −, respectively, or saline (vehicle) without immunosuppression. Human-specific probe revealed Muse cells distributed mainly to the injured brain at 2 and 4 weeks, and expressed neuronal and glial markers until 6 months. In contrast, non-Muse cells lodged in the lung at 2 weeks, but undetectable by 4 weeks. Magnetic resonance spectroscopy and positron emission tomography demonstrated that Muse cells dampened excitotoxic brain glutamatergic metabolites and suppressed microglial activation. Muse cell-treated group exhibited significant improvements in motor and cognitive functions at 4 weeks and 5 months. Intravenously transplanted Muse cells afforded functional benefits in experimental HIE possibly via regulation of glutamate metabolism and reduction of microglial activation.