Stage-specific embryonic antigen-3 (SSEA-3) and ß3GalT5 are cancer specific and significant markers for breast cancer stem cells.

The discovery of cancer stem cells (CSCs), which are responsible for self-renewal and tumor growth in heterogeneous cancer tissues, has stimulated interests in developing new cancer therapies and early diagnosis. However, the markers currently used for isolation of CSCs are often not selective enough to enrich CSCs for the study of this special cell population. Here we show that the breast CSCs isolated with CD44(+)CD24(-/lo)SSEA-3(+) or ESA(hi)PROCR(hi)SSEA-3(+) markers had higher tumorigenicity than those with conventional markers in vitro and in vivo. As few as 10 cells with CD44(+)CD24(-/lo)SSEA-3(+) formed tumor in mice, compared with more than 100 cells with CD44(+)CD24(-/lo). Suppression of SSEA-3 expression by knockdown of the gene encoding ß-1,3-galactosyltransferase 5 (ß3GalT5) in the globo-series pathway, led to apoptosis in cancer cells specifically but had no effect on normal cells. This finding is further supported by the analysis of SSEA-3 and the two related globo-series epitopes SSEA4 and globo-H in stem cells (embryonic stem cells and induced pluripotent stem cells) and various normal and cancer cells, and by the antibody approach to target the globo-series glycans and the late-stage clinical trials of a breast cancer vaccine.

Isolation and characterization of human mesenchymal stem cells from gingival connective tissue.

BACKGROUND AND OBJECTIVE: The main purpose of this study was to isolate and characterize gingival connective tissue-derived mesenchymal stem cells (GMSCs). The secondary purpose was to present a modified isolation method for the GMSCs. MATERIAL AND METHODS: Collected healthy gingival tissue samples were de-epithelialized and minced into small fragments. The tissues were digested by dispase and collagenase IV for 30 min. The first digested cell suspension was discarded, and then additional digestion was performed to the remaining cells in the same solution for 90 min. The isolated cells from gingiva was incubated in 37°C humidified condition and observed by inverted microscope. Cytoskeletal morphology was evaluated by phalloidin immunofluorescence. Potency of the cells was tested by colony-forming unit fibroblast assay. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometric, immunofluorescence analysis. RESULTS: GMSCs showed spindle-shaped, fibroblast-like morphology, colony-forming abilities, adherence to plastic and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency. GMSCs expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 in flow cytometry. Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and a mesenchymal marker (vimentin) were observed by immunofluorescence. CONCLUSIONS: In conclusion, we isolated and characterized stem cells from human gingival connective tissue with modified protocol. GMSCs showed multipotency with high proliferation and characteristics of mesenchymal stem cells. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs' proliferation and cryopreservation.

Activin A is essential for Feeder-free culture of human induced pluripotent stem cells.

Feeder-free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder-free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA-1-60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA-1-60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder-free culture of hiPS cells requires A or AC to maintain pluripotency.

Isolation, characterization and differentiation of cells expressing pluripotent/multipotent markers from adult human ovaries.

Pluripotent stem cells are still generally accepted not to exist in adult human ovaries, although increasing studies confirm the presence of pluripotent/multipotent stem cells in adult mammalian ovaries, including those of humans. The aim of this study is to isolate, characterize and differentiate in vitro stem cells that originate from the adult human ovarian cortex and that express markers of pluripotency/multipotency. After enzymatic degradation of small ovarian cortex biopsies retrieved from 18 women, ovarian cell cultures were successfully established from 17 and the formation of cell colonies was observed. The presence of cells/colonies expressing some markers of pluripotency (alkaline phosphatase, surface antigen SSEA-4, OCT4, SOX-2, NANOG, LIN28, STELLA), germinal lineage (DDX4/VASA) and multipotency (M-CAM/CD146, Thy-1/CD90, STRO-1) was confirmed by various methods. Stem cells from the cultures, including small round SSEA-4-positive cells with diameters of up to 4 µm, showed a relatively high degree of plasticity. We were able to differentiate them in vitro into various types of somatic cells of all three germ layers. However, these cells did not form teratoma when injected into immunodeficient mice. Our results thus show that ovarian tissue is a potential source of stem cells with a pluripotent/multipotent character for safe application in regenerative medicine.

Multiparameter flow cytometry for the characterization of human embryonic stem cells.

Using multiparameter staining methods and flow cytometry to investigate the pluripotency of HUES7 human embryonic stem cell cultures, it was found that the multidimensional approach of marker co-expression allowed the different cell populations to be easily identified and demonstrated cross reactivity between the SSEA 4 and SSEA 1 antibodies, resulting in a substantial false positive SSEA 1 population. It is the accepted norm to apply control gates at a 95 % confidence level of the isotype control; however, this study found that adjusting the control gate to a 99 % confidence level significantly reduced the effect of this cross reactivity. Though conversely, this gating shift also decreased the positive marker expression of SSEA 4 and Tra-1-60, indicating that there is a need for strongly expressing markers coupled with increased optimization of fluorophore/antibody combinations before a gating strategy of 99 % can be implemented on a more routine basis.

Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media.

OBJECTIVES: Despite constant advances in the field of biology and medical application of human embryonic stem cells, the molecular mechanism of pluripotency remains largely unknown. So far, definitions of pluripotent stem cells (SC) have been based on a limited number of antigenic markers and have not allowed for unambiguous determination of the homogeneity of each subpopulation. Moreover, the use of some crucial pluripotency markers such as SSEA-3 and SSEA-4 has recently been questioned due to the possibility that the pattern of surface glycans may be changed depending on the content of the cell culture medium. AIM: Quantitative analysis of amniotic SC subpopulations cultured in different media, based on the following pluripotency surface markers: SSEA-3, SSEA-4, TRA- 1-60 and TRA- 1-81 expression and co-expression. MATERIAL AND METHODS: Immunofluorescence and fluorescence microscopy were used to identify and localize SC within a normal human placenta at term. The number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells with co-expression of the above mentioned markers, cultured in media containing different protein supplements of animal origin, was counted by flow cytometry RESULTS AND CONCLUSIONS: Cells with characteristics of embryonic SC were identified in the amniotic epithelium and the chorion, but not in the decidua basalis. Amniotic epithelium contained various types of SC, with SSEA-4+ as the most numerous. Disproportion in the number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells characterized by co-expression of these antigens, as well as lack of quantitative differences between SC subpopulations cultured in different media, was observed. In conclusion, the amniotic epithelium is composed of SC at different stages of the development but human amnion might become an alternative source of SSEA-4+ embryonic-like SC. The composition of the evaluated media, characterized by different content of animal-derived proteins, does not influence the number of cells identified within the SC subpopulations.

No evidence for the presence of oogonia in the human ovary after their final clearance during the first two years of life.

BACKGROUND: Conflicting results of studies on mouse and human have either verified or refuted the presence of oogonia/primordial germ cells in the post-natal ovary. The aim of this study was to trace whether oogonia recognized by immunohistochemical methods in the first trimester human ovary were present also in peri- and post-natal ovaries. METHODS: For this study, 82 human ovaries were collected: 25 from embryos from 5 to 10 weeks post conception (wpc), 2 at 18 wpc, 32 from 32 wpc to 2 years and 23 from 2 to 32 years. Of these, 80 ovaries were fixed and paraffin-embedded and 2 (8 year-old) ovaries were processed for plastic sections. Serial sections were prepared for immunohistochemical detection of markers for oogonia: tyrosine kinase receptor for stem cell factor (SCF)(C-KIT), stage-specific embryonic antigen-4 (SSEA4), homeobox gene transcription factor (NANOG), octamer binding transcription factor 4 (OCT4) and melanoma antigen-4 (Mage-A4), while noting that C-KIT also stains diplotene oocytes. RESULTS: Almost all oogonia exclusively stained for SSEA4, NANOG, OCT4 and C-KIT, whereas MAGE-A4 only stained a small fraction. At birth only a few oogonia were stained. These disappeared before 2 years, leaving only diplotene oocytes stained for C-KIT. From 18 wpc to 2 years, the medulla contained conglomerates of healthy and degenerating oogonia and small follicles, waste baskets (WBs) and oogonia enclosed in growing follicles (FWB). Medulla of older ovaries contained groups of primordial, healthy follicles. CONCLUSIONS: We found no evidence for the presence of oogonia in the human ovary after their final clearing during the first 2 years. We suggest that perinatal medullary WB and FWB give rise to the groups of small, healthy follicles in the medulla.

Identification and characterization of repopulating spermatogonial stem cells from the adult human testis.

BACKGROUND: This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes. METHODS: Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers. RESULTS: Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics. CONCLUSIONS: This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.

Lifespan of human amniotic fluid-derived multipotent mesenchymal stromal cells.

BACKGROUND AIMS: Human multipotent mesenchymal stromal cells (hMSC) have become one of the main interests in regenerative medicine because of their ability to differentiate into different lineages. Human amniotic fluid is reported to contain MSC (hAMSC) and therefore may be a useful source of cells for clinical applications. However, our understanding of the behavior of these cells in indefinite in vitro culture conditions is very limited. METHODS: We systematically evaluated and characterized, throughout their whole lifespan, the expansion potential, chromosomal stability, surface and intracellular phenotype and differentiation potential of fibroblastoid hAMSC (F-type hAMSC). RESULTS: Nine F-type hAMSC cultures could be expanded in in vitro culture conditions for 223.25 ± 24.44 days (mean ± SD), during which time 28.96 ± 1.5 passages were made giving rise to 54.95 ± 3.17 population doublings (PD) and an estimated number of accumulated cells of between 1.0 × 10(22) and 9.7 × 10(23), with no visible alterations in the chromosome during their lifespan. All the cultures showed unchanged percentages of strongly positive expressions of the surface markers CD29, CD44, CD73, CD90, CD95, CD105 and HLA-ABC, as well as the embryonic intracellular markers Nanog and Sox2, during their lifespan, whereas the expression of the embryonic surface markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 fell until it disappeared with progression of the culture. These cells retained their differentiation capacities to adipogenic, chondrogenic and osteogenic lineages throughout their lifespan. CONCLUSIONS: F-type hAMSC exhibit reproducible biologic characteristics, confirming that these cells are ideal candidates for use in regenerative medicine.

Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts.

BACKGROUND AND OBJECTIVE: Human induced pluripotent stem (iPS) cells, which have similar properties to human embryonic stem (hES) cells, have been generated from neonatal and adult human dermal fibroblasts by reprogramming. iPS cells have high pluripotency and differentiation potential, and may be a potential autologous stem cell source for future regenerative therapy. MATERIAL AND METHODS: iPS cell lines from human gingival fibroblasts and, for the first time, from periodontal ligament fibroblasts, were generated by reprogramming using a retroviral transduction cocktail of OCT3/4, SOX2, KLF4 and c-MYC. iPS induction was investigated through expression of the embryonic stem cell markers SSEA4, OCT4, NANOG, GCTM-2, TG30 and TRA-1-60. Following in vitro differentiation, the expression of genes for differentiation markers for ectoderm (SOX1, PAX6), mesoderm [RUNX1, T(Brachyury)] and endoderm (GATA4, AFP) was assessed by real-time RT-PCR. The ability to form teratomas following implantation into mouse testes was assessed by histology. RESULTS: Human gingival fibroblast- and periodontal ligament fibroblast-derived iPS cells showed similar characteristics to hES cells. Both sets of iPS cells displayed colony morphology comparable to that of hES cells and expressed the hES cell-associated cell-surface antigens, SSEA3, SSEA4, GCTM-2, TG30 (CD9) and Tra-1-60, and the hES cell marker genes, OCT4, NANOG and GDF3. These iPS cells showed differentiation potential to form embryoid bodies in vitro and expressed genes for endoderm, ectoderm and mesoderm. Teratoma formation following implantation into mouse testes was observed. CONCLUSION: These results demonstrate that iPS cells can be successfully generated from adult human gingival and periodontal ligament fibroblasts.

Buffalo (Bubalus bubalis) embryonic stem cell-like cells and preimplantation embryos exhibit comparable expression of pluripotency-related antigens.

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin-C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2- to 4-cell, 8- to 16-cell, morula and blastocyst stages strongly expressed stage-specific embryonic antigen (SSEA)-4 but lacked expressions of SSEA-1 and SSEA-3. Putative ES cells also expressed tumour rejection antigen (TRA)-1-60, TRA-1-81 and Oct4. Whereas in all early embryonic stages, TRA-1-60 was observed only in the periplasmic space, and TRA-1-81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency-related surface antigen phenotype, which resembles that of the ICM.

Prognostic and predictive value of ALDH1, SOX2 and SSEA-4 in bladder cancer.

Transurethral resection of bladder tumor (TUR-BT) and radical cystectomy (RC) are standard treatment options for bladder cancer (BC). Neoadjuvant chemotherapy (NAC) prior to RC improves outcome of some patients but currently there are no valid biomarkers to identify patients who benefit from NAC. Presence of cancer stem cells (CSC) has been associated with poor outcome and resistance to chemotherapy in various cancers. Here we studied the expression of stem cell markers ALDH1, SOX2 and SSEA-4 with immunohistochemistry in tissue microarray material consisting of 195 BC patients treated with RC and 74 patients treated with TUR-BT followed by NAC and RC. Post-operative follow-up data of up to 22 years was used. Negative to weak cytoplasmic SOX2 staining was associated with lymphovascular invasion and non-organ confined disease. It was also associated with shortened cancer-specific survival, but the finding was not statistically significant. Contrary to previous reports, none of the other tested biomarkers were associated with cancer-specific mortality or clinicopathological characteristics. Neither were they associated with response to NAC. Despite the promising results of previously published studies, our results suggest that CSC markers ALDH1, SOX2 and SSEA-4 have little if any prognostic or predictive value in BC treated with RC.

Optimization of isolation and further characterization of umbilical-cord-blood-derived very small embryonic/ epiblast-like stem cells (VSELs).

Because of their small size and density, umbilical cord blood (UCB)-derived very small embryonic/epiblast-like stem cells (VSELs) are usually lost at various steps of UCB preparation. Accordingly, we noticed that a significant number of these cells, which are smaller than erythrocytes, are lost during gradient centrifugation over Ficoll-Paque as well as during routine volume depletion of UCB units before freezing. To preserve these cells in final UCB preparations, we propose a relatively short and economical three-step isolation protocol that allows recovery of approximately 60% of the initial number of Lin(-)/CD45(-)/CD133(+) UCB-VSELs present in freshly harvested UCB units. In this novel approach (i) UCB is lysed in a hypotonic ammonium chloride solution to deplete erythrocytes; (ii) CD133(+) including VSELs cells are enriched by employing immunomagnetic beads; and subsequently (iii) Lin(-)/CD45(-)/CD133(+) cells are sorted by fluorescence-activated cell sorting. The whole isolation procedure takes approximately 2-3 h per UCB unit and isolated cells are highly enriched for an Oct-4(+) and SSEA-4(+) population of small Lin(-)/CD45(-)/CD133(+) cells.

Stage-specific embryonic antigen 4 expression in epithelial ovarian carcinoma.

INTRODUCTION: Stage-specific embryonic antigen 4 (SSEA-4) is a widely used marker to monitor the differentiation of pluripotent embryonic stem cells. Little is known about the expression pattern of SSEA-4 in solid tumors up to now. METHODS: In this study, we investigated the expression of SSEA-4 in 479 cases of various degrees of ovarian epithelial lesions by immunohistochemistry, consisting of 45 normal ovarian epithelia, 110 benign serous ovarian cystadenomas, 68 borderline serous ovarian cystadenomas, 104 invasive serous ovarian carcinomas, 64 benign serous mucinous cystadenomas, 48 borderline mucinous ovarian cystadenomas, and 40 invasive mucinous carcinomas. Moreover, the association between SSEA-4 expression and clinicopathological parameters was also evaluated. RESULTS: The expression of SSEA-4 was found to be increased from normal epithelium to benign cystadenoma and to borderline cystadenoma and adenocarcinoma in both serous and mucinous group. The loss or reduction of the expression of SSEA-4 was significantly correlated with more advanced tumor stage and poorer tumor cell differentiation. CONCLUSIONS: We therefore proposed that SSEA-4 may play a role during the oncogenesis of epithelial ovarian carcinoma and posses a tumor suppressor effect during malignancy promotion. It could be a potential therapy target in patients with epithelial ovarian carcinoma.

Generation and characterization of embryonic stem-like cell lines derived from in vitro fertilization Buffalo (Bubalus bubalis) embryos.

In the present study, buffalo embryonic stem-like (ES-like) cell lines were successfully isolated, cultured and characterized. From a total of 92 normal buffalo embryos obtained by in vitro fertilization, 18 were morulae, 33 were blastocyst and 41 were hatched blastocyst, the inside of morulae or inner cell masses of blastocysts were isolated mechanically and cultured onto mitomocin-C-inactivated buffalo embryonic fibroblasts as feeder layers. Alkaline phosphatase (AP) of ES-like cells, as well as the specific stage embryonic antigen SSEA-1, SSEA-3, SSEA-4 and transcription factor OCT-4, was used to evaluate the characterization of the cells. The spontaneous differentiation of ES-like cells was induced by culturing on leukaemia inhibitory factor-free medium for more than 2 weeks without passage. To evaluate mark gene expression, total RNA was extracted from cells, and specific primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR). After 8-10 days of culture, primary ES-like cell colonies were formed in 0% (0/18) of morulae, 24.24% (8/33) of blastocysts and 60.98% (25/41) of hatched blastocysts, respectively. The forming rate of primary ES-like cells colonies in hatched blastocyst group was significantly (p < 0.05) higher than the obtained for other groups. Two ES-like cell lines could survive to eight passages at least by using the method of mechanical dissociation, but just three passages by using the method of enzymatic dissociation. The cells formed large, multicellular colonies with distinct boundaries, exhibited many important features of ES/ES-like cells, including positive AP, SSEA-1, SSEA-3 and SSEA-4 activity. Undifferentiated buffalo ES-like cells expressed Oct-4, Nanog, Sox2 gene mRNA. In vitro differentiation experiments had demonstrated that those cells were pluripotent.

Establishment of the long-term in vitro culture system for chicken primordial germ cells.

The objective of this study was to establish the long-term in vitro culture system for chicken gonadal primordial germ cells (gPGCs). Primitive gonads collected from 5.5-day-old chicken embryos were dissociated and explanted onto plates pre-coated with 0.1% gelatin. Each of the four different conditioned media from proliferating and mitotically inactivated chicken embryonic fibroblast (CEF) cells and murine embryonic fibroblasts (STO cells, CRL-1053, ATCC, USA), respectively, was supplemented with growth factors and used to support the growth of gPGCs. The result showed that all the conditioned media could promote the growth and colony formation of gPGCs in vitro, in particular the medium conditioned by inactivated CEF cells. The gPGC-derived colonies maintained in inactivated CEF cells-conditioned medium up to 281 days were positively stained by periodic acid Schiff reaction and antibodies specific to anti-SSEA-1, SSEA-3, SSEA-4, integrin alpha6 and integrin beta1. Their capacities of migration via vascular system and taking up residence in the primary gonadal ridge were further demonstrated by transferring to the dorsal aorta of stage 17 recipient embryos. These results suggested that our culture system is able to maintain chicken gPGCs for long-term in vitro culture without losing their capacity to express pluripotent markers and to integrate into the gonads.

Effect of microgravity on the mesenchymal stem cell characteristics of limbal fibroblasts.

BACKGROUND: Mesenchymal stem cells (MSCs) are important for regenerative medicine. Limbal fibroblasts (LFs), present in the corneal limbus, have been shown to possess MSC characteristics, and can differentiate into other cell types. The current study sought to investigate the effect of microgravity on the proliferation and differentiation of LFs, and identify culture conditions to obtain a high proportion of LFs possessing MSC characteristics. METHODS: A rotary cell culture system was used to generate microgravity. Cellular proliferation and MSC marker (CD14, CD45, CD90, CD105, and SSEA4) expression were evaluated by WST-1 test and flow cytometry, respectively. Differentiation of LFs into adipocytes, osteocytes, and chondrocytes was examined. The effects of LF-conditioned medium on limbal stem cell differentiation were assessed. RESULTS: The cellular proliferation rates under microgravity were significantly lower than those under normal gravity (0.44 vs. 0.18 at 24 h, and 0.70 vs. 0.44 at 48 h, both P ≤ 0.004). Higher proportions of cells expressed CD90 (95.33% vs 81.69%), CD105 (95.32% vs 87.96%), and SSEA4 (68.26% vs 26.13%) under microgravity than under normal gravity. The differentiation potential of LFs was more prominent under microgravity. The LF-conditioned medium attenuated the differentiation of limbal corneal epithelial stem cells. CONCLUSION: Under microgravity, LFs showed a higher proportion of MSC characteristics and were easily induced into different linage cells. Culture in a microgravity environment may allow harvesting a greater number of MSC-like LFs for stem cell therapy in ocular surface reconstruction.

Novel surface-enhanced Raman scattering-based assays for ultra-sensitive detection of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 10(6) cells, a sensitivity (0.0001%) which was ∼2000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5(+) and TRA-1-60(+) cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications.

CD44 and SSEA-4 positive cells in an oral cancer cell line HSC-4 possess cancer stem-like cell characteristics.

BACKGROUND: Cancer may be derived from cancer stem-like cells (CSCs), which are tumor-initiating cells that have properties similar to those of stem cells. Identification and isolation of CSCs needs to be improved further. MATERIALS AND METHODS: CSCs markers were examined in human oral cancer cell lines by flow cytometry. The stem cell properties of subpopulations expressing different markers were assessed further by in vitro sphere formation assays, expression of stemness genes, drug resistance assays, and the ability to form tumors in nude mice. RESULTS: We demonstrated that CSCs could be isolated by the cell surface markers CD44 and stage-specific embryonic antigen-4 (SSEA-4). CD44+SSEA-4+ cells exhibited cancer stem-like properties, including extensive self-renewal into the bulk of cancer cells. In vivo xenograft experiments indicated that CD44+SSEA-4+ cells exhibit the highest tumorigenic capacity compared with the remaining subpopulations and parental cells. Double-positive cells for CD44 and SSEA-4 exhibited preferential expression of some stemness genes and were more resistant to the anticancer drugs, cisplatin and 5-fluorouracil (5-FU). In addition, cells expressing CD44 and SSEA-4 were detected in all tumor specimens analyzed, while coexpression of CD44 and SSEA-4 was not detectable in normal oral mucosa. CONCLUSION: Our findings suggest that CD44+SSEA-4+ cells exhibit the characteristics of CSCs in oral squamous cell carcinoma and provide a target for the development of more effective therapies.