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Stage-specific embryonic antigens (SSEAs) are carbohydrate markers that have diverse roles in embryonic development. However, the exact roles of SSEAs remain unclear. To obtain mechanistic insights into their roles, we aimed to develop functionalized SSEA glycan analogs via chemical synthesis. Herein, we report a convergent synthetic approach for SSEA-3 and SSEA-4 analogs using readily available versatile building blocks. A key step, namely the stereoselective glycosylation of a common tetrasaccharide acceptor, was successfully achieved using a 4-O-Bn Gal donor for SSEA-3 and a Neu-Gal donor for SSEA-4, which were previously developed by our group. The obtained SSEA-3 and SSEA-4 glycans were further functionalized with biotin and deuterated lipid for applications in biological studies. Thus, the findings of this study will facilitate further research on SSEAs.

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Background: Breast cancer (BC) is the most common malignant tumors and the leading cause of cancer deaths among women. The early diagnosis and treatment of BC are effective measures that can increase survival rates and reduce mortality. Carbohydrate antigens 15-3 (CA15-3) and carcinoma embryonic antigens (CEA) have been regarded as the most two valuable tumor markers of BC. The combined detection of CA15-3 and CEA could improve the sensitivity and accuracy of early diagnosis for BC. Methods: The multi-channel double-gate silicon nanowire field effect transistor (SiNW-FET) biosensors were fabricated by using the top-down semiconductor manufacturing technology. By surface modification of the different SiNW surfaces with monoclonal CA15-3 and CEA antibodies separately, the prepared SiNW-FET was processed into biosensor for dual-channel detection of CA15-3 and CEA. Results: The prepared SiNW-FET biosensors were proved to have high sensitivity and specificity for the dual-channel detection of CA15-3 and CEA, and the detection limit is as low as 0.1U/mL CA15-3 and 0.01 ng/mL CEA. Moreover, the SiNW-FET biosensors were able to detect CA15-3 and CEA in serum by connecting a miniature hemodialyzer. Conclusion: The present study reported a SiNW-FET biosensor for dual-channel detection of breast cancer biomarkers CA15-3 and CEA in serum, which has potential clinical application value for the early diagnosis and curative effect observation of BC.

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INTRODUCTION: Cartilage regeneration is a promising therapy for restoring joint function in patients with cartilage defects. The limited availability of autologous chondrocytes or chondrogenic progenitor cells is an obstacle to its clinical application. We investigated the existence and chondrogenic potential of synovial membrane-derived multilineage-differentiating stress-enduring (Muse)-like cells as an alternative cell source for cartilage regeneration. METHODS: Cells positive for stage-specific embryonic antigen-3 (SSEA-3), a marker of Muse cells, were isolated from the synovial membranes of 6 of 8 patients (median age, 53.5 years; range 36-72 years) by fluorescence-activated cell sorting. SSEA-3-positive cells were cultured in methylcellulose to examine their ability to form Muse clusters that are similar to the embryoid bodies formed by human embryonic stem cells. Muse clusters were expanded and chondrogenic potential of M-cluster-derived MSCs examined using a pellet culture system. Chondrogenic differentiation was evaluated by proteoglycan, safranin O, toluidine blue and type II collagen staining. To evaluate the practicality of the procedure for isolating Muse-like cells, we compared chondrogenic potential of M-cluster derived MSCs with expanded cells derived from the clusters formed by unsorted synovial cells. RESULTS: Synovial membranes contained SSEA-3-positive cells that after isolation exhibited Muse-like characteristics such as forming clusters that expressed NANOG, OCT3/4, and SOX2. In the pellet culture system, cell pellets created from the M-cluster-derived MSCs exhibited an increase in wet weight, which implied an increase in extracellular matrix production, displayed metachromasia with toluidine blue and safranin O staining and were aggrecan-positive and type II collagen-positive by immunostaining. Unsorted synovial cells also formed clusters in methylcellulose culture, and the expanded cell population derived from them exhibited chondrogenic potential. The histological and immunohistochemical appearance of chondrogenic pellet created from unsorted synovial cell-derived cells were comparable with that from M-cluster-derived MSCs. CONCLUSIONS: Muse-like cells can be isolated from the human synovial membrane, even from older patients, and therefore may provide a source of multipotent cells for regenerative medicine. In addition, the cluster-forming cell population within synovial cells also has excellent chondrogenic potential. These cells may provide a more practical option for cartilage regeneration.

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BACKGROUND AND OBJECTIVE: Based on our earlier work on the response of periodontal ligament (PDL) cells to mechanical stress by induction of cyclooxygenase expression and production of prostaglandin PGE2 that could regulate mineralization of PDL cells, it was hypothesized that PGE2 had potential effects on PDL stemness. In this study, we aimed to investigate clonogenicity, proliferation and expression of certain pluripotent markers, considered to be characteristics of PDL stemness, in response to treatment with exogenously-added PGE2. MATERIAL AND METHODS: Human PDL cells were cultured and treated with various doses of PGE2, and the aforementioned characteristics of PDL stemness were analyzed. RESULTS: The clonogenicity and proliferation were significantly enhanced by PGE2 at low concentrations (0.01, 0.1 and 1ng/ml; P<0.05), but only the proliferation was significantly diminished by PGE2 at a high concentration (100ng/ml; P<0.05). Expression of NANOG and OCT4 mRNA and protein was increased by PGE2 treatment at 0.1 and 1ng/ml. Consistently, expression of stage-specific embryonic antigen 4, a putative stem cell marker, was significantly augmented by PGE2 treatment at 1ng/ml (P<0.05). CONCLUSION: Our findings suggest that although a high dose of PGE2 (100ng/ml) inhibits proliferation of PDL cells, PGE2 at low doses appears to play a role in the maintenance of PDL stemness.

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Acute myeloid leukemia is driven by leukemic stem cells which can be identified by cross lineage expression or arrest of differentiation compared to normal hematopoietic stem cells. Self-renewal and lack of differentiation are also features of stem cells and have been associated with the expression of embryonic genes. The aim of our study was to evaluate the expression of embryonic antigens (OCT4, NANOG, SOX2, SSEA1, SSEA3) in hematopoietic stem cell subsets (CD34+CD38- and CD34+CD38+) from normal bone marrows and in samples from acute myeloid leukemia patients. We observed an upregulation of the transcription factors OCT4 and SOX2 in leukemic cells as compared to normal cells. Conversely, SSEA1 protein was downregulated in leukemic cells. The expression of OCT4, SOX2, and SSEA3 was higher in CD34+CD38- than in CD34+CD38+ subsets in leukemic cells. There was no correlation with biological characteristics of the leukemia. We evaluated the prognostic value of marker expression in 69 patients who received an intensive treatment. The rate of complete remission was not influenced by the level of expression of markers. Overall survival was significantly better for patients with high SOX2 levels, which was unexpected because of the inverse correlation with favorable genetic subtypes. These results prompt us to evaluate the potential role of these markers in leukemogenesis and to test their relevance for better leukemic stem cell identification.

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The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1- and SSEA-1+ cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

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Glycosphingolipids (GSLs) are composed of complex glycans linked to sphingosines and various fatty acid chains. Antibodies against several GSLs designated as stage-specific embryonic antigens (SSEAs), have been widely used to characterize differentiation of embryonic stem (ES) cells. In view of the cross-reactivities of these antibodies with multiple glycans, a few laboratories have employed advanced mass spectrometry (MS) technologies to define the dynamic changes of surface GSLs upon ES differentiation. However, the amphiphilic nature and heterogeneity of GSLs make them difficult to decipher. In our studies, systematic survey of GSL expression profiles in human ES cells and differentiated derivatives was conducted, primarily with matrix-assisted laser desorption/ionization MS (MALDI-MS) and MS/MS analyses. In addition to the well-known ES-specific markers, SSEA-3 and SSEA-4, several previously undisclosed globo- and lacto-series GSLs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer were identified in the undifferentiated human ES and induced pluripotent stem cells. Furthermore, during differentiation to embryoid body outgrowth, the core structures of GSLs switched from globo- and lacto- to ganglio-series. Lineage-specific differentiation was also marked by alterations of specific GSLs. During differentiation into neural progenitors, core structures shifted to primarily ganglio-series dominated by GD3. GSL patterns shifted to prominent expression of Gb4Cer with little SSEA-3 and- 4 or GD3 during endodermal differentiation. Several issues relevant to MS analysis and novel GSLs in ES cells were discussed. Finally, unique GSL signatures in ES and cancer cells are exploited in glycan-targeted anti-cancer immunotherapy and their mechanistic investigations were discussed using anti-GD2 mAb and Globo H as examples.

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The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1⁻ and SSEA-1⁺ cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

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BACKGROUND: The prion protein (PrP) might be useful as a tool to collect cardiac progenitor cells derived from embryonic stem (ES) cells. It is also possible that PrP(+) cells include undifferentiated cells with a capacity to develop into tumors. METHODS: PrP(+) cells isolated from embryoid bodies (EB) formed by mouse AB1 ES cells were examined using RT-PCR analysis and clonogeneic cell assay. To assess their potential to differentiate into cardiomyocytes, Nkx2.5(GFP/+) (hcgp7) cells, another ES cell line that carries the GFP reporter gene in the Nkx2.5 loci, were used. RESULTS: PrP(+) cells isolated from EB of day 7 and 14 did not express pluripotency markers, but expressed cardiac cell markers, while PrP(+) cells isolated from EB of day 21 expressed pluripotency markers. Cultured PrP(+) cells isolated from EB of day 21 expressed pluripotency markers to form colonies, whereas those isolated from EB of day 7 and 14 did not. To exclude proliferating cells from PrP(+) cells, stage specific embryo antigen 1 (SSEA1) was employed as a second marker. PrP(+)/SSEA1(-) cells did not proliferate and expressed cardiac cell markers, while PrP(+)/SSEA1(+) did proliferate. CONCLUSION: PrP(+) cells isolated from EB included undifferentiated cells in day 21. PrP(+)/SSEA1(-) cells included cardiomyoctes, suggesting PrP and SSEA1 may be useful as markers to enrich the fraction of cardiomyocytes.

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STUDY QUESTION: Can human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia? SUMMARY ANSWER: Germ cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1-2 weeks) but not late (> 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures. WHAT IS KNOWN ALREADY: The development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures. STUDY DESIGN, SIZE, DURATION: Testicular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured in vitro for 1-2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (UTF1, FGFR3, ZBTB16, GPR125, DAZL, GATA4 and VIM) and flow cytometry (CD9 and SSEA4). MAIN RESULTS AND THE ROLE OF CHANCE: UTF1, FGFR3, DAZL and ZBTB16 qRT-PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, GPR125 mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in early primary testicular cell cultures (1-2 weeks), their expression steadily declined over time in vitro. After 6 weeks in culture only somatic cells were detected. LIMITATIONS, REASONS FOR CAUTION: Different groups attempting SSC culture have utilized different sources of human testes and minor differences in the preparation and maintenance of the testicular cell cultures. Differences in outcome may be explained by genetic background of the source tissue or technical differences. WIDER IMPLICATIONS OF THE FINDINGS: The ability to propagate human SSCs in vitro is a prerequisite for proposed autologous transplantation therapy aimed at restoring fertility to men who have been treated for childhood cancer. By applying the assays validated here it will be possible to quantitatively compare human SSC culture conditions. The eventual development of conditions for long-term propagation of human SSCs in vitro will greatly facilitate learning about the basic biology of these cells and in turn the ability to use human SSCs in therapy. STUDY FUNDING/COMPETING INTERESTS: The experiments presented in this manuscript were funded by a Project Development Team within the ICTSI NIH/NCRR Grant Number TR000006. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

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A review is given of studies on human teratocarcinoma cell lines in culture. A human ovarian teratocarcinoma line (PA-1) has been characterized in detail (Zeuthen et al. 1980) and has been shown to exhibit some properties of human teratocarcinoma stem-cells. This line forms embryoid bodies with a distinct outer cell layer analogous to endodermal cells formed by some mouse teratocarcinoma cell lines, and also is capable of a certain limited differentiation in culture as well as upon transplantation to nude mice. A panel of human testicular teratocarcinoma cell lines has been studied by Andrews et al. (1980): While these cells also share some embryonic characteristics, their differentiation in culture is more limited. Studies of various markers suggest that these lines are equivalent to a pre-blastocyst stage derivation. The PA-1 cell line and possibly two other testicular teratocarcinoma lines could possibly be derived at a stage equivalent to a later stage of development.