SSEA4 Monoclonal Antibody (MC-813-70)
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Immunofluoroscent analysis of Shef 4 Human ES cell line using mouse anti-SSEA4 antibody (Product # 41-4000).
Histogram from flow cytometric analysis of NTERA-2 cells stained with either SSEA4 Monoclonal Antibody, Mouse (clone MC813-70) (Product # 41-4000) (black line) or mouse IgG isotype control (gray line) followed by detection with FITC-conjugated goat anti-mouse IgG (Product # M32601).
Figure S1 Characterization of F49B7 iPSCs and Retinal Organoids from Several iPSC Lines, Related to Figures 1 , 2 , and 3 and Table S1 (A) Bright-field image of F49B7 iPSC colony. (B) Bright-field image of iPSC colony. Blue stain, alkaline phosphatase (pluripotency marker). (C - F) Confocal images of iPSCs. Green, antibody for pluripotency markers; white, Hoechst (nucleus marker). (C) SOX2, (D) NANOG, (E) OCT4 and (F) SSEA4. (G - I) Confocal images of iPSCs directly differentiated into the three germ layers. White, Hoechst. (G) Ectoderm; magenta, Nestin; green, PAX6 (ectoderm markers). (H) Endoderm; magenta, SOX17; green, FOXA2 (endoderm markers). (I) Mesoderm; magenta, NCAM; green, Brachyury (mesoderm markers). (J) G-banded karyotyping of F49B7 iPSCs. (K) Different iPSC lines generating 5-layered retinal organoids. Confocal images. Green, Bassoon antibody (synaptic marker); white, Hoechst (nucleus marker). Boxed area, outline of F49B7 image shown cropped in Figure 1 . (L) Retinal neuroepithelium (arrows) on a five-week old organoid. (M) Embryoid body diameter (day 7) versus the number of cells seeded per microwell. Points, mean diameter of embryoid bodies (n = 12) within an independent experiment. Line, quadratic fit. (N) Percentage of all organoids that were retinal organoids (week 6) versus embryoid body diameter (day 7). Points, experiments. Line, quadratic fit. (O) Confocal image. Green, RHO antibody (rod outer segment marker); white, Hoechst (nucleus marker). OS, outer
4 Characterization of isolated human spermatogonial stem cells (SSCs) from cryopreserved tissues. A, Morphology of cultured spermatogonia cells on days 6, 8, and 10. Scale bars: 200 um. B, Immunofluorescence staining of PLZF, SSEA4, and GFRalpha1 as markers for human SSCs. Scale bars: 50 um. C,D, Hematoxylin and eosin (H&E) staining of mouse testis sections before and after injection of busulfan. E,F, transplantation of hSSCs to seminiferous tubules and detection of PKH26-labeled cells in mouse testis tubules 8 weeks after transplantation. Scale bars: 50 um
Figure 6. At P7, PF-S cells still maintain cobblestone-like shape (b), but PF-C cells stay highly elongated (a). The cell shape factor defined by the cell aspect ratio is significantly high in PF-C cells (g). PF-C cells lose stemness ((c), (e)) quickly than PF-S cells ((d), (f)), as evidenced by the low-level expression of stem cell markers, NS and SSEA-4 in PF-C (h). The white box insets show enlarged images of NS staining ((c), (d)). *p < 0.01.
Figure 5 Osteogenesis differentiation of rabbit BMSCs in vitro (A) A typical colony was formed in the primary culture of rabbit BMCs. (B) More than 90% of BMCs expressed Oct-4; (C) More than 95% of BMCs presented nucleostemin; ( D ) more than 80% of BMCs were positively stained by SSEA-4. Alizarin Red S staining showed that less than 20% of BMCs either cultured in tissue culture plate with normal medium (Group-1, E ) or LDI-glycerol scaffold with normal medium (Group-3, G ) were positively stained by Alizarin Red S. However, more than 80% of BMCs either cultured in tissue culture plate with osteogenesis medium (Group-2, F ) or in bioactive molecules containing (LDI-glycerol-AA-GP-DEX) polymer scaffold with normal culture medium (Group-4, H ) were positively stained by Alizarin red S (Bars: 100 mum). Both regular RT-PCR (I) and qRT-PCR (J) showed that three osteogenesis gene markers, collagen I, Runx-2, and osteocalcin were increased in rabbit BMSCs cultured either in osteogenesis medium (Group 2) or bioactive molecules containing polymer scaffold (Group 4) compared to the BMSCs cultured in tissue culture plate with normal medium (Group 1) and LDI-glycerol polymer scaffold (Group 3). *p<0.05 compared to Group 1; #p<0.05 compared to Group 3.
Figure 3 Stem cell marker expression of nucleostemin (NS; (a), (b)), Oct-4 (c, d), SSEA-4 (e, f), and stro-1 (g, h) for AFSCs (a, c, e, and g) and NPSCs (b, d, f, and h) determined by immunocytochemistry (a)-(h) and analyzed by semiquantification (i). Less than 40% of AFSCs expressed nucleostemin (a, i), whereas more than 90% of NPSCs were positively stained indicating nucleostemin (b, i). About 28% of AFSCs presented Oct-4 (c, i) compared to 86% for NPSCs (d, i). There was no significant difference in the expression of SSEA-4 and stro-1 for AFSCs (e, g, i) and NPSCs (f, h, i). Inset images were the enlarged images in the respective square. Bar: 100 mu m.
Figure 1 Generation and characterization of STEMCCA transduced hiPSC . Primary human induced pluripotent stem cells (hiPSC) colonies derived from non-ALS (A) and ALS patients of sporadic form (B-H) after transduction with STEMCCA Cre-Excisable Constitutive Polycistronic Lentivirus expressing the embrionary genes OCT4, SOX2, KLF4 , and CMYC after several passages in E8 medium. hiPSCs were derived from fibroblasts of extensor hallucis brevis nerve obtained by biopsy from sporadic ALS patients and from distal fragments of the accessory nerve from a non-ALS subject. Immunostaining of cultured hiPSC for SSEA-4 (B) , TRA1-60 (E) , TRA1-81 (C) , and OCT-4 (F) . The presence of OCT-4 immunoreactivity in the nucleus of hiPSC samples that were not counterstained with nuclear DAPI is shown in the box inside of the (F) . Of note, OCT-4 has been described in cytoplasmic vesicles (F) , an event associated to regulation of pluripotency-associated protein homeostasis of pluripotent cells (Cho et al., 2014 ; Muratore et al., 2014 ). Cell nuclei were stained with DAPI (blue). hiPSC colonies were analyzed for alkaline phosphatase activity using the Alkaline Phosphatase Live Stain (G) . Karyotyping sampling of hiPSC clone from an ALS patient, in which metaphase plates showed the normal male chromosomal content ( H ; 42,XY). The same pattern was observed for other subjects of the study, including the non-ALS patients (data not shown). Scale bars, 50 mum.
Figure 2 Expression of stem cell markers for BMSCs and MMSCs. Both types of MSCs exhibited high expression of stem cell markers, SSEA-4 (A, B) , Nanog (C, D) , and nucleostemin (E, F) , respectively. Insets show enlarged view of positive staining with three stem cell markers. There was no great difference in the expression of these three recognized stem cell markers. (Magnification of microscopy: 20x) (Bar: 50 mum).
Figure 3 Immunofluorescence assays for stem cell marker protein expression in AF-derived colony forming cells. The AF-derived cells were positive for Oct-4 ( A-C ), nucleostemin ( D-F ) and SSEA-4 ( G-I ). Scale bars, 200 um.
Figure 3 Expression of the stem cell marker SSEA-4 by hTSCs cultured in vitro in various concentrations of PGE 2 . A : without PGE 2 treatment; B : 0.01 ng/ml PGE 2 ; C : 0.1 ng/ml PGE 2 ; D : 1 ng/ml PGE 2 ; E : 10 ng/ml PGE 2 ; and F : 100 ng/ml PGE 2 . hTSCs were seeded in 12-well plates, cultured with six different concentrations of PGE 2, incubated with mouse anti-human SSEA-4 primary antibody, and detected with Cy3-conjugated goat anti-mouse IgG. Nuclei were stained with Hoechst (Blue). Expression of SSEA-4 (red) is dose-dependent, with more robust expression seen in hTSCs treated with low levels of PGE 2 ( A-D ) than expression levels seen in those treated with high levels ( E, F ). Positively stained cells were also counted to calculate percentage staining ( G ) (*p<0.05 with respect to hTSCs not treated with PGE 2 ). Bar: 100 um.
Figure 6 Lin - CD45 - cells show a high nuclear/cytoplasm ratio. ( A ) Immunocytochemistry shows small cells (<=10 um) with high nuclear (blue)/cytoplasm ratio positive for CD34 (red). ( B ) Note one CD34-positive and one CD34-negative cell and an example of cell debris present in the sample (arrow). ( C ) Rare SSEA-4-positive cell. Scale bars = 10 um ( A - B ) and 5 um ( C ).
Figure 6 Self-renewal of hACL-SCs and hMCL-SCs . At passage 5, hACL-SCs had already become highly elongated in confluent culture, a typical fibroblast phenotype ( A ). In contrast, even at passage 13, confluent hMCL-SCs remained cobblestone-like ( B ). Moreover, hACL-SCs no longer expressed nucleostemin ( C ) or SSEA-4 ( E ) at passages > 5, whereas hMCL-SCs expressed both stem cell markers at passage 13 ( D, F ). Note, however, that hMCL-SCs at this high passage exhibited a lesser degree of nucleostemin expression compared to the cells at passage 1 (see Figure 3). The results shown here were obtained from a male donor of 27 years old (see Table 1). (Bar: 100 mum).
Figure 3 The expression of stem cell markers in hACL-SCs and hMCL-SCs . Both types of ligament stem cells expressed nucleostemin ( A, B, C ), SSEA-4 ( D, E, F ), CD44 ( G, H, I ), and CD90 ( J, K, L ), but not CD31, CD34, CD45, and CD146 (not shown). Note that negative controls (omission of primary antibodies) were also used in the immunostaining, and no staining signals were seen (data not shown). Also, the results shown here were obtained from a of 26-year-old male donor (see Table 1). The passage 1 cells were used in immunostaining. (Bar: 100 mum).
Figure 6 The testing of stem cell marker expression . A, B . PTSCs and ATSCs at passages 10 expressed Oct-4, respectively. C . No Oct-4 staining was detected on tenocytes. D, E . PTSCs and ATSCs expressed SSEA-4. F . Tenocytes were negative for SSEA-4 staining. G, H . PTSCs and ATSCs expressed nucleostemin. Insets show enlarged view of expressed nucleostemin in pink (arrows). I . Nucleostemin expression was not detected on tenocytes. (bar: 50 mum).
Published figure using SSEA4 monoclonal antibody (Product # 41-4000) in Immunohistochemistry
Fig. 1 H1-CAG-GTRgp cells differentiated into neural progenitor cells with stable expression of GFP in vitro . A. Schematic diagram of the gene knock-in construct targeting the AAVS1 Locus. Donor plasmid containing homology arms to AAVS1 genomic regions (5' and 3' arms), CAG promoter, GFP, TVA, Rgp and PGK-neomycin cassette. B. Genotyping of the H1-CAG-GTRgp cell clones using the Primer-F and Primer-R primers, as depicted in (A). C. Representative GFP expression of the positive clones. Scale bar = 50 mum. D. OCT4 and SSEA4 expression levels in H1 and H1-CAG-GTRgp hES cells determined by FACS. E. SOX2, OCT4 and NANOG expression levels in H1 and H1-CAG-GTRgp hES cells determined by qRT-PCR. F. Hematoxylin and eosin staining of the teratomas formed by H1-CAG-GTRgp hES cells. Scale bar = 50 mum. G. Karyotype of the H1-CAG-GTRgp hES cells. H. qRT-PCR analysis of different NPC markers in H1 hES cells and induced NPCs derived from H1-CAG-GTRgp hES cells. I. Representative immunofluorescent images of induced neuroepithelial cells, immature neuronal cells and neural progenitor cells derived from H1-CAG-GTRgp hES cells. Scale bar = 20 mum. Fig. 1
Fig. 1 Generation of GATA2 -/- human ESCs. a Schematic overview of gene targeting strategy. For GATA2 knockout, a PGK promoter-neomycin cassette replaces exon 3 of the GATA2 locus. Bgl II sites, TALEN sites, primers ( P1 , P2 ), and probes ( probe 1 , probe 2 ) used for genomic PCR and Southern blot are indicated. b Genomic PCR for GATA2 targeting. PCR product by indicated primers is 0.76 kb from WT H1 cells and 2.44 kb from H1- GATA2 -/- . c , d Southern blot analysis of GATA2 targeted H1 cells. The genomic DNAs were digested by Bgl II and detected by indicated probes. One single band detected by probe 1 indicates that there was no random integration of drug cassette. e FACS analysis of the expression of OCT4, SSEA-4, TRA-1-60, and TRA-1-81 in H1 or H1- GATA2 -/- cells. In these and other flow cytometry diagrams, the black line stands for the control and the red line for the experimental plot unless otherwise indicated. f Teratoma formation of H1 or H1- GATA2 -/- cells. g Paired Pearson correlation analysis of global gene expression between WT and GATA2 -/- H1 cells. R Pearson correlation coefficient, TPM transcripts per million. h Karyotype analysis of the H1- GATA2 -/- cell line
Human SSEA4 antigen
See Additional Formats
PBS, pH 7.4
0.1% sodium azide
SSEA-4 (Stage-specific embryonic antigen 4) is a cell surface antigen, expressed along with SSEA-3, TRA-1-60 and TRA-1-81 in embryonic stem cells, embryonal carcinoma cells and induced pluripotent stem cells (iPS). In human cells, these surface markers are down-regulated during the differentiation process, while SSEA-1 is absent in undifferentiated human stem cells and upregulated on the cell surface after retinoic acid mediated differentiation. Conversely, in mouse stem cells, SSEA-1 expression decreases with differentiation while SSEA-3 and SSEA-4 expression increase.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
SSEA-4; stage-specific embryonic antigen 4