Journal: Cell Transplantation

Article Title: Establishment of Immortalized Human Amniotic Mesenchymal Stem Cells

doi: 10.3727/096368912x655055

Figure Lengend Snippet: Figure 3. Immunohistochemical analysis of fHAM cells and iHAM cells. The expressions of stem cell markers on the fHAM cells and iHAM cells were detected by immunofluorescence. Most of the upper layer was fHAM cells, including some octamer binding transcription protein 3/4 (Oct3/4)-positive cells. From the third layer, they were iHAM cells. The nuclei of most cells were stained by Oct3/4, sex-determining region Y box 2 (Sox2), Lin28, and Krüppel-like factor 4 (Klf4). Some nuclei were stained strongly by Nanog, nestin, and stage-specific embryonic antigen-4 (SSEA-1). Vimentin and anti-a-smooth muscle (a-SM) actin were clearly expressed in the cytoplasm. No cytokeratin (CK)-positive cells were observed. Scale bar: 50.0 mm.

Article Snippet: After blocking for nonspecific reaction with BLOCK ACE (Dainippon Pharmaceutical, Osaka, Japan) for 45 min, cells were incubated in the following primary antibodies diluted 1:200 in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 24 h at 4°C: octamer binding transcription factor 3/4 (Oct3/4), Nanog, Krüppel-like factor 4 (KLF4), stage-specific embryonic antigen 4 (SSEA4), a-smooth muscle (a-SM) actin, nestin, c-myc (all from Santa Cruz Biotechnology, Inc., CA, USA), sex-determining region Y box 2 (Sox2), anti-human Lin28 (R&D System, Inc., Minneapolis, MN, USA), vimentin (Dako, Glostrup, Denmark), and cytokeratin 18 (CK18; Progen, Heidelberg, Germany).

Techniques: Immunohistochemical staining, Immunofluorescence, Binding Assay, Staining

Journal: Cell Transplantation

Article Title: Establishment of Immortalized Human Amniotic Mesenchymal Stem Cells

doi: 10.3727/096368912x655055

Figure Lengend Snippet: Figure 4. (A) Expression of representative stem cell marker genes by RT-PCR. Samples are as follows: lane -RT, reagent control; lane 1, freshly isolated HAM cells (fHAM cells); lane 2, HAM cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at passage 2 (HAM cells P2); lanes 3–6, iHAM cells lines (iHAM3 542, iHAM3 542-344, iHAM4 136-344, and iHAM4 344-96). All iHAM cells lines showed stronger reactions than fHAM cells and HAM cells P2 for Oct-4, Klf4, c-myc, breast cancer resistance gene (BCRP), and nestin (neural progenitor cell marker). However, expression was weaker than fHAM cells and HAM cells P2 for Sox2 and Nanog. b-Actin mRNA was used as a positive control. (B) Formation of spheres. iHAM cells made spheres easily after they were cultured on the ultralow attachment dish. There were various sizes of spheres. Notice that they indicated alkaline phosphatase (ALP) activity (scale bar: 500 mm) and Oct3/4 reaction (scale bar: 200 mm).

Article Snippet: After blocking for nonspecific reaction with BLOCK ACE (Dainippon Pharmaceutical, Osaka, Japan) for 45 min, cells were incubated in the following primary antibodies diluted 1:200 in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 24 h at 4°C: octamer binding transcription factor 3/4 (Oct3/4), Nanog, Krüppel-like factor 4 (KLF4), stage-specific embryonic antigen 4 (SSEA4), a-smooth muscle (a-SM) actin, nestin, c-myc (all from Santa Cruz Biotechnology, Inc., CA, USA), sex-determining region Y box 2 (Sox2), anti-human Lin28 (R&D System, Inc., Minneapolis, MN, USA), vimentin (Dako, Glostrup, Denmark), and cytokeratin 18 (CK18; Progen, Heidelberg, Germany).

Techniques: Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Control, Isolation, Cell Culture, Modification, Positive Control, Activity Assay

Journal: PLoS ONE

Article Title: A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells

doi: 10.1371/journal.pone.0161229

Figure Lengend Snippet: Human hESC line WA07 was serially subcultured for five passages. The number of viable cells per passage were compared by initially seeding 2×10 4 viable cells per cm 2 into three wells of a six-well plate for each medium, using respective passaging solution and matrix. Panel A shows cell attachment and growth of WA07 hESCs at passage 1 in L7 ™ hPSC, mTeSR1 TM , and E8 TM media. Panel B shows cell attachment and growth of WA07 hESCs at the end of passage 5 in L7 ™ hPSC, mTeSR1 ™ , and E8 ™ media. Panel C Panel A shows the viability and total viable cells of WA07 hESCs grown in L7 ™ hPSC, mTeSR1 ™ , and E8 ™ media, demonstrating comparative growth for the cells grown in each media. Panel D shows immunocytochemistry analysis of WA07 hESCs grown in L7 ™ hPSC, mTeSR1 ™ , and E8 ™ media, demonstrating comparative expression of OCT4 (red), Nanog (red), SSEA4 (green), TRA1-60 (green), and TRA1-81 (green) in each media. Following differentiation of hPSCs into embryoid bodies (Panel E), differentiated WA07 hESCs readily expressed the markers for early ectoderm (detected TUJ1, green), endoderm (detected Alpha-Feto Protein (AFP), green), and mesoderm (detected Smooth Muscle Actin (SMA), green) in L7 ™ hPSC and E8 TM media. Cell nuclei are shown by DAPI (blue).

Article Snippet: Primary antibodies raised against pluripotency-associated antigens OCT4 (Abcam, ab19857; 1:350) and Nanog (R&D Systems, AF1997; 6.7μg/ml) were used in combination with the secondary antibodies Cy3-conjugated Donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-165-152; 1:200) and Cy3-conjugated donkey anti-Goat IgG (H+L) (Jackson ImmunoResearch, 805-165-180; 1:200), respectively.

Techniques: Passaging, Cell Attachment Assay, Immunocytochemistry, Expressing

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 2 Vps26a knockdown prolongs the maintenance of stemness under ND conditions in P19 ECCs. a, b The effect of Vps26a knockdown on the expression of ESC stemness genes were determined by semi-qPCR (a) and western blot (b) analyses of Oct3/4 and Nanog using shCTL- and shVps26a-ECCs. c Morphological changes of shCTL (shC)- and shVps26a (shV)-ECCs during retinoic acid-induced neurogenesis (RA-ND) for the indicated time periods. The yellow arrowheads indicate cells with differentiated morphologies. d–f Expression kinetics of ESC stemness and neuronal markers examined by semi-qPCR (d), qPCR (e), and western blotting (f)

Article Snippet: Subsequently, antibodies against Oct3/4 (SC-5279, Santa Cruz Biotechnology), Nanog (NBP2-19469, Novus Biological), MAP2 (AB5622, Millipore), Tubb3 (MAB1637, Millipore), pERK1/2 (4370, Cell Signaling), Hif2α (NB100-122, Novus Biological), Nox4 (PA1-46014, Thermo Fisher Scientific), and Vps26a (AB23892, Abcam) were incubated with the prepared cells at 4 °C overnight.

Techniques: Knockdown, Expressing, Western Blot