Expression Of Stage-specific Embryonic Antigen 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.

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.

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.

Effects of prostaglandin E2 on clonogenicity, proliferation and expression of pluripotent markers in human periodontal ligament cells

Background and objectiveBased 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 methodsHuman PDL cells were cultured and treated with various doses of PGE2, and the aforementioned characteristics of PDL stemness were analyzed. ResultsThe 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). ConclusionOur 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.

SSEA-4+ CD34- Cells in the Adult Human Heart Show the Molecular Characteristics of a Novel Cardiomyocyte Progenitor Population

Stage-specific embryonic antigen (SSEA) expression is used to describe the differentiation state of an embryonic stem cell (ESC). In human ESCs, SSEA-3 and SSEA-4 are highly expressed in undifferentiated cells and downregulated upon differentiation. SSEA-4 has also been described as a marker for adult stem cells in various tissues, including human neonatal cardiac tissue. However, there is currently little data on the expression of SSEAs in human adult cardiac tissue. We obtained right and left atrial biopsies from patients undergoing cardiac surgery. These were dissociated, stained for SSEAs and other cardiac stem cell markers and analyzed by flow cytometry. Directly isolated cells expressed variable levels of SSEA-1, SSEA-3 and SSEA-4. The SSEA-1+ population was established as contaminating hematopoietic cells. The SSEA-4+ population, on the other hand, could be subdivided based on the endothelial progenitor marker CD34. The SSEA-4+ CD34- population in the right atrium had a high gene expression of both early (TBX5, NKX2.5) and late (TNNT2) cardiomyocyte markers. The SSEA-4+ CD34+ population, on the other hand, overlapped with previously described C-kit+ CD45- cardiac stem cells. Primary monolayer-cultured cells retained expression of SSEAs while the cardiomyogenic specification in the SSEA-4+ CD34- population was lost. In tissue sections, SSEA-4+ cells could be identified both within and outside the myocardium. Within the myocardium, some SSEA-4+ cells coexpressed cardiomyogenic markers. In conclusion, the results show that the adult human heart expresses SSEAs and that there is a subpopulation of SSEA-4+ CD34- cells that show features of a cardiomyocyte progenitor population.

Neuronal differentiation of induced pluripotent stem cells in hybrid polyester scaffolds with heparinized surface

This study presents the differentiation capability of induced pluripotent stem (iPS) cells in heparinized binary scaffolds composed of poly(ɛ-caprolactone) (PCL) and poly(β-hydroxybutyrate) (PHB). The neuronal differentiation of iPS cells in scaffolds with surface heparin was characterized with immunochemical staining. The results revealed that an increase in the weight percentage of PCL and in the gel concentration enhanced the porosity of scaffolds. Based on the carbon quantity, an increase in the grafting concentration of heparin raised the content of sulfur, nitrogen, and oxygen on the scaffolding surface. In addition, a higher grafting concentration of heparin yielded a weaker expression of stage-specific embryonic antigen 1 in constructs, indicating that the grafted heparin accelerated differentiation and reduced the number of phenotypic iPS cells after cultivation. When the grafting concentration of heparin increased, the staining against βIII tubulin exhibited a stronger fluorescent intensity, demonstrating that iPS cells in constructs differentiated toward neurons. The heparinized PCL–PHB scaffolds can be efficient biomaterials in guiding the differentiation of iPS cells for nerve tissue engineering.

Expression of Globo H and SSEA3 in breast cancer stem cells and the involvement of fucosyl transferases 1 and 2 in Globo H synthesis

We examined the expression in breast cancer stem cells (BCSCs) of Globo H, a potential tumor-associated antigen for immunotherapy of epithelial cancers including breast cancer. Flow cytometry revealed Globo H expression in 25/41 breast cancer specimens (61.0%). Non-BCSCs from 25/25 and BCSCs from 8/40 (20%) expressed Globo H. We showed the expression of stage-specific embryonic antigen 3 (SSEA3), the pentasaccharide precursor of Globo H, in 31/40 (77.5%) tumors. Non-BCSCs from 29/40 [corrected] and BCSCs from 25/40 (62.5%) expressed SSEA3. Like Globo H, SSEA3 expression in normal tissues was predominately at the secretory borders of epithelium, where access to the immune system is restricted. Immunization of mice with Globo H-KLH and alpha-GalCer induced antibodies reactive with Globo H and SSEA3, suggesting that a Globo H-based vaccine will target tumor cells expressing Globo H or SSEA3. We next sought to reduce Globo H expression by siRNA targeting fucosyltransferase (FUT) 1 and 2, which mediate alpha-1,2 linkage of fucose to SSEA3 to generate Globo H. We showed both genes to be involved in the biosynthesis of Globo H. Moreover, FUT2 expression in BCSCs was significantly lower than in non-BCSCs harvested from a primary human breast cancer in NOD/SCID mouse, whereas FUT1 was slightly lower in BCSCs. Thus, the lower expression of Globo H in BCSCs may be attributed to less FUT2/FUT1, and to reduced SSEA3 in BCSCs compared with non-BCSCs. Our findings provide insight into further development of a Globo H-based vaccine and FUT1/FUT2-targeted therapy for breast cancer.

Similar biological characteristics of human embryonic stem cell lines with normal and abnormal karyotypes

BACKGROUNDHuman embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar.METHODSSeven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines.RESULTSAll seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines.CONCLUSIONSThe present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research.

Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment

AbstractIt is believed that a primitive cell type that maintains the mesenchymal compartment exists in the bone marrow. However, this putative mesenchymal stem/progenitor cell is yet to be identified and isolated. We are reporting the identification, isolation, and detailed characterization of the most primitive mesenchymal progenitor cells in the adult murine bone marrow, based on the expression of stage-specific embryonic antigen–1 (SSEA-1). This primitive subset can be identified in mesenchymal cell cultures and also directly in the bone marrow, thus ascertaining for the first time their existence in an adult organism. Characterization of SSEA-1+ mesenchymal cells revealed that upon purification these cells gave rise to SSEA-1− mesenchymal cells, whereas the reverse could not be observed. Also, these SSEA-1+ cells have a much higher capacity to differentiate than their negative counterparts, not only to several mesenchymal cell types but also to unconventional cell types such as astrocyte-, endothelial-, and hepatocyte-like cells in vitro. Most importantly, a single-cell–derived population was capable of differentiating abundantly into different mesenchymal cell types in vivo. Altogether we are proposing a hierarchical organization of the mesenchymal compartment, placing SSEA-1+ cells at the apex of this hierarchy.

Gene Expression Profiling of Mouse Embryonic Stem Cell Subpopulations1

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.

Expression of SSEA-1 (Lewis(x)) on transitional cell carcinoma of the bladder.

The expression of stage-specific embryonic antigen 1 (SSEA-1) in transitional cell carcinomas of the bladder (TCCB) has been reported to correlate with tumor grade and the likelihood of lymphatic metastases. We examined the expression of this antigen in TCCBs to evaluate if staining correlated with grade, stage, recurrence, progression and response to intravesical chemotherapy. We studied the expression of SSEA-1 in TCCBs from 74 patients by staining with two different monoclonal antibodies (Mabs), P-12 and anti-SSEA-1, to evaluate if staining correlated with grade, stage and tumor recurrence. Staining was considered as positive or negative irrespective of the intensity of staining. Extent of tumor staining was measured in quartiles of 100% (25, 50, 75 and 100). Follow-up was available in 47/74 (63%) patients and ranged from 6 months to 13 years (median 2 years). Staining with one or both Mabs was observed in 57/75 (76%) tumors. None of the grade I tumors showed > 50% staining while 26% of grade II and only 33% of grade III tumors showed staining of > 50% of cells. 21% of patients whose tumors showed staining with both Mabs were free of recurrence after resection of the primary tumor; 41% of patients with tumors staining negative with both Mabs showed no recurrence. Expression of SSEA-1 does correlate with tumor recurrence especially in grade II tumors, but the correlation is not very strong (0.05 < p < 0.1). Expression of this antigen is a weak indicator of recurrence in superficial TCCBs.

Developmental fate in chimeras derived from highly asynchronous murine blastomeres.

The developmental fate of single blastomeres from eight-cell murine embryos reaggregated with intact two-cell embryos was evaluated after culture. Fluorescein isothiocyanate was used to follow developmental fate in preblastocyst chimeric embryos. Expression of stage-specific embryonic antigen 3 was used to assay developmental fate at the blastocyst stage, and glucosephosphate isomerase variants were used to assay at the blastocyst stage and after implantation. The results suggest that the descendents of the 1/8 component stay in a patch area and do not selectively migrate to the inner cell mass (ICM). This is in contrast to many studies that indicate that smaller blastomeres, which are more advanced in development, migrate to the ICM. The differences in experimental designs are discussed. Possible mechanisms for this phenomena are that the eight-cell blastomere is physically excluded from the ICM by position or polarization, or that it is differentiating ahead of the two-cell component and becomes trophectoderm.