Journal: Regenerative therapy

Article Title: Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

doi: 10.1016/j.reth.2014.12.004

Figure Lengend Snippet: Fig. 4. Pluripotent marker expression of xenogeneic-free SEES cell lines In the undifferentiated state, xenogeneic-free SEES cell lines expressed markers characteristic of pluripotent hESCs, including OCT4, NANOG, SOX2, SSEA4, and TRA1-60. SEES-4: scale bars are 200 mm; SEES-5: scale bars are 50 mm in OCT3/4 and TRA1-60 and 200 mm in SOX2 and SSEA4; SEES-6: scale bars are 50 mm in OCT3/4 and SOX2 and 200 mm in SSEA4; SEES-7: scale bars are 200 mm.

Article Snippet: Antibodies against human SSEA-1 (R&D Systems; #FAB2155C), SSEA4 (R&D Systems; #FAB1435F), TRA1-60 (Merck Millipore; #MAB4360), and TRA1-81 (Merck Millipore; #MAB4381) were used as primary antibodies in hESCs.

Techniques: Marker, Expressing

Journal: Regenerative therapy

Article Title: Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

doi: 10.1016/j.reth.2014.12.004

Figure Lengend Snippet: Fig. 6. Characterization of the pluripotency of SEES-2 maintained using a modified conventional hESC culture medium SEES-2 cells were stably maintained over 20 passages on the qualified MEF feeder layer in the modified medium, which contained pharmaceutical-grade recombinant human bFGF (trafermin) and high-dose (35-K) gamma-irradiated KO-SR without antibiotics. A) Typical hESC colony morphology was readily visible. ALP activity was detected. B) SEES-2 cells expressed undifferentiated hESC markers, including OCT4, NANOG, SOX2, SSEA4, and TRA1-60. SEES-2 cells could differentiate into three embryonic germ layers in vitro and in vivo. Scale bars are 200 mm. C) SEES cells that had been differentiated in vitro via EBs expressed markers of the primary germ layers, ectoderm (TUJ1), mesoderm (aSMA), and endoderm (AFP). Scale bars are 100 mm. D) Histological analysis of teratomas containing multidifferentiated tissues derived from SEES-2 cells. Pigmented epithelium (ectoderm), cartilage (mesoderm), and gut epithelial tissues (endoderm). E) Chromosomal analysis of SEES-2 cells cultivated through 16 passages using a modified conventional hESC culture medium showed a normal 46,XX karyotype.

Article Snippet: Antibodies against human SSEA-1 (R&D Systems; #FAB2155C), SSEA4 (R&D Systems; #FAB1435F), TRA1-60 (Merck Millipore; #MAB4360), and TRA1-81 (Merck Millipore; #MAB4381) were used as primary antibodies in hESCs.

Techniques: Stable Transfection, Recombinant, Irradiation, Activity Assay, In Vitro, In Vivo, Derivative Assay

Journal: Regenerative therapy

Article Title: Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

doi: 10.1016/j.reth.2014.12.004

Figure Lengend Snippet: Fig. 7. Expression of pluripotency markers in SEES-2 cells was maintained using a modified conventional hESC culture medium Flow cytometric analysis of hESC-specific marker expression in SEES-2 cells. The isotype control is indicated by the blue line, and the unlabeled sample, which was used as a control, is indicated by the red line. Surface staining is shown by the yellow line for SSEA4, SSEA1, TRA-1-60, and TRA-1-81.

Article Snippet: Antibodies against human SSEA-1 (R&D Systems; #FAB2155C), SSEA4 (R&D Systems; #FAB1435F), TRA1-60 (Merck Millipore; #MAB4360), and TRA1-81 (Merck Millipore; #MAB4381) were used as primary antibodies in hESCs.

Techniques: Expressing, Marker, Control, Staining

Journal: Bioengineering (Basel, Switzerland)

Article Title: Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions.

doi: 10.3390/bioengineering10090999

Figure Lengend Snippet: Figure 3. Characterization of hPSC after long-term culture on W10% HSt-PMEDSAH-g dishes. hPSC cultured on W10% HSt-PMEDSAH-g dishes for 20 consecutive passages, maintaining self-renewal and pluripotency. (A) Representative micrographs of immunocytochemistry every 5 weeks showing strong positive staining for human pluripotency markers NANOG, OCT4, SOX2, TRA1-60, and TRA-181. (B) Dot plots of hPSC after 20 passages of culture on W10% HSt-PMEDSAH-g dishes, showing more than 99% co-expression between SSEA3/OCT4 and SSEA4/OCT4. Isotype controls for the respective antibodies were used. The threshold/gate was set to a maximum of 0.8% positive cells in the unstained control and every signal above was counted as a positive signal. FMO controls were used to assess the spread of the fluorochrome into the missing channel and the gates were set accordingly. (C) Pluripotency was confirmed by qPCR hPSC ScoreCard assay quantifying the self-renewal and trilineage differentiation potential of passage 20 hPSC cultured on W10% HSt- PMEDSAH-g and 10 days old EB developed from the same pool of cells. (D) G-banding karyogram of cells cultured on W10% HSt-PMEDSAH-g dishes at passage 20 confirming genetic integrity. Scale bars, 200 µm.

Article Snippet: After this, cells were incubated for 30 min in dark at 4 ◦C, first with human IgG to block un-specific binding and then with human/mouse NANOG APC-conjugated antibody (Biolegend, San Diego, CA, USA), OCT4 PE-conjugated antibody (Biolegend), SSEA3 APC-conjugated antibody (R&D systems), SSEA4 PE-conjugated antibody (R&D systems, Minneapolis, MN, USA), TRA-1-60 FITC-conjugated antibody (Biolegend), and TRA-1-81 PE-conjugated antibody (Biolegend).

Techniques: Cell Culture, Immunocytochemistry, Staining, Expressing, Control

Journal: PLoS ONE

Article Title: Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

doi: 10.1371/journal.pone.0042838

Figure Lengend Snippet: ( a ) Schematic summarizes the experimental strategy for determining 4F reprogramming efficiencies of FACS-purified hematopoietic progenitors of ( b ) BMSC-primed lineage-committed or ( c ) +/− BMSC-primed transgene-enriched myeloid populations. Experimental details are provided in . The %CD34 + gradient symbol above the schematic reflects the concept that multipotent primitive CD34+CD38lo stem-progenitors are enriched during Days -3 to -2, but differentiate rapidly in culture thereafter (see also Fig. 1c and Fig. S2a) . CD34 + CD38 lo stem-progenitors give rise to lineage-committed CD34 + CD38 + progenitors which subsequently differentiate further to CD34-negative CD33 + CD45 + myeloid cells ( e.g ., promyelocytes). Post-sort analysis of FACS-purified Day 0 CD34 + CD38 + CB fractions verified that >95% of this populations consisted of CD33 + CD13 + CD45 + myeloid cells (see also Fig. 1c ). Both reprogramming efficiency (AP and live TRA-1-81 staining of hESC-like colonies) and reprogramming completion (bulk SSEA4 + TRA-1-81 + and NANOG + FACS staining) assays were conducted 4 weeks following 4F nucleofections on P 0 MEF cultures. ( b ) Shown is a representative AP staining (plates done in triplicate, with indicated average number of hESC-like colonies emerging per the number of single sorted day 3 BMSC-primed CB cells plated on MEF (i.e., unsorted CB vs. CD34 + CD38 lo vs. CD34 + CD38 + fractions. The averaged results of two independent experiments are indicated. In lower panels are shown representative FACS staining of surface TRA-1-81 and intracellular NANOG pluripotency markers of the same cultures demonstrating that 50–80% of AP + hESC-like colonies possessed a Type III TRA-1-81 + NANOG + phenotype . ( c ) To determine the more accurate reprogramming efficiency of purified +/− BMSC-primed episome-expressing myeloid populations, CB progenitors were co-nucleofected on day 0 with both the 4F pEP4 EO2S EM2K episome, as well as a pCEP4-GFP episomal GFP reporter construct. Episomal transgene expressing-only populations were subsequently FACS-purified by GFP expression prior to plating on day 3 MEF following with (+BMSC) or without (-BMSC) stromal co-culture. This was done by staining +/− BMSC-primed CB cells on day 3 with CD34-PE and FACS-purifying them into episome-expressing (GFP + CD34 + , GFP + CD34 − ), and non episome-expressing (GFP − ) populations prior to MEF plating. The results of AP stained plates shown are representative of independent sorting experiments using pooled donor CB samples for each 4F nucleofection, with the averaged results of two independent experiments indicated below. FACS analysis of these same experiments (data not shown) revealed 60% TRA-1-81 and NANOG expression for +BMSC AP + colonies (GFP + CD34 − ), and 55% TRA-1-81 expression for –BMSC AP + colonies (GFP + CD34 − ).

Article Snippet: Antibodies included APC conjugated SSEA4 (R&D Systems), PE Mouse anti-Human TRA-1-60 antigen (BD Biosciences) and PE Mouse anti-Human TRA-1-81 antigen (BD Biosciences).

Techniques: Purification, Staining, Expressing, Construct, Co-Culture Assay

Journal: PLoS ONE

Article Title: Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

doi: 10.1371/journal.pone.0042838

Figure Lengend Snippet: ( a ) Emergence of surface pluripotency markers (SSEA4, TRA-1-81) were assayed by FACS at 3 weeks in bulk cultures of 4F episomally-reprogrammed somatic cells briefly co-cultured with (+) or without ( − ) irradiated BMSC ( Fig. S1 ). Fetal fibroblasts (FFB), adult fibroblasts (AdFib), adult keratinocytes (Ker), and GF-activated Day 0 CB (CB) were nucleofected with 4F or 7F on Day 0 ( Fig. S1 ), and reprogrammed bulk cultures were analyzed by FACS 3 weeks later. AP stains of hESC-like colonies were done in parallel of these same experiments, and are presented in Fig. 1d . Shown are the averaged results of 2–5 experiments with averages, and significances ( * ) designated at peak of bar graphs. ( b,c ) The kinetics of pluripotency marker emergence of 4F reprogrammed CB progenitors with (+) and without (−) BMSC priming. ( b ) SSEA4 + , and ( c ) SSEA4 + TRA-1-60 + expressions. ( d ) Enhancement of 4F CB reprogramming with (+BMSC) and without (- BMSC) stromal priming was due to signals that were partially cell contact-dependent, and partially soluble factor-mediated. GF-activated CB cells were cultured as described in Fig. S1 from Day 0 until Day 3 without BMSC co-culture (-BMSC), with BMSC co-culture (+BMSC), or with BMSC co-culture but physically separated from CB cells with a Transwell insert that prevented cell-cell contact between BMSC and CB cells, but allowed diffusion of soluble stromal factors (+BMSC (T)). Shown is the relative fold-increase of reprogramming efficiency (enumerated AP+ hESC-like colonies) from two averaged 4F-reprogramming experiments from baseline efficiencies (-BMSC conditions). Reprogramming efficiency was determined at 3 weeks post-nucleofection with 4F, determined by AP staining of hESC-like colonies (as described in ). ( e ) Gene specific enrichment analysis (GSEA) computation of pathways activated in 4F-nucleofected CB cells by stromal signals. The GSEA algorithm was used to identify curated pathways over-represented among genes with significant (p<0.05, FDR<0.05) differential expression between Day 3 (D3) +BMSC-primed CB samples, vs . D3 unprimed (-BMSC) CB samples that were nucleofected at Day 0 with 4F episome. summarizes the MSigDB v. 3.0 gene set categories that were enriched with FDR <0.05 and nom p <0.05 for these two paired gene set computations. Shown in ( e ) are the major categories of enriched pathways that were significantly and differentially activated in Day 3 BMSC-primed and 4F-nucleofected CB samples.

Article Snippet: Antibodies included APC conjugated SSEA4 (R&D Systems), PE Mouse anti-Human TRA-1-60 antigen (BD Biosciences) and PE Mouse anti-Human TRA-1-81 antigen (BD Biosciences).

Techniques: Cell Culture, Irradiation, Marker, Co-Culture Assay, Diffusion-based Assay, Staining, Quantitative Proteomics

Journal: Stem cell research

Article Title: Establishment of a human embryonic stem cell line with homozygous TP53 R248W mutant by TALEN mediated gene editing

doi: 10.1016/j.scr.2018.04.013

Figure Lengend Snippet: Reagents details.

Article Snippet: Pluripotency Markers , Mouse anti-SSEA4 PE-conjugated , 1:600 , R and D Systems FA1435P-025.

Techniques: Immunocytochemistry, Western Blot, Mutagenesis