Journal: Stem Cells International

Article Title: Survivin Improves Reprogramming Efficiency of Human Neural Progenitors by Single Molecule OCT4

doi: 10.1155/2016/4729535

Figure Lengend Snippet: Morphology of NiPS colonies by 1F-OCT4. Low (a) and high (b) magnification of NiPS colonies. 1F-OCT4 human NiPS colonies were stained for alkaline phosphatase (c); immunofluorescent analysis of pluripotency and surface markers (d). The pluripotent markers (OCT4, SOX2, and NANOG) and surface markers (SSEA4, TRA-1-60, and TRA-1-81) were stained with antibodies in human 1F-OCT4 NiPS cells. Nuclei are stained with DAPI (blue). Scale bars, 100 μ m.

Article Snippet: The episomal vector containing pCXLE-OCT4 (1F-OCT4) was introduced into human NPC by electroporation (Amaxa Nucleofector 2B, LabWrench, Canada) on day 0.

Techniques: Staining

Journal: Stem Cells International

Article Title: Survivin Improves Reprogramming Efficiency of Human Neural Progenitors by Single Molecule OCT4

doi: 10.1155/2016/4729535

Figure Lengend Snippet: Analyses of DNA methylation and gene expression profiling in NiPS, NPC, and hES cells. (a) Analysis of gene expression profiling: the MvA plot is a comparison plot comparing two microarrays. The y -axis is displayed on a log2 scale with green threshold lines for two-fold changes. The lower parts of MvA plot represent signals of NiPS cells, while the upper parts represent signals of NPC or hES cells. The color coding of the plot indicates the density of probes represented by that data point; (b) bisulphite sequencing analysis of OCT4 and NANOG promoter regions in human NSCs, 1F-OCT4 human NiPS clones, and human ES cells; (c) Hematoxylin and Eosin (HE) stain of teratomas generated from 1F-OCT4 derived iPS cells injected subcutaneously into immunocompromised SCID mice. Structures derivative of all three germ layers could be identified. (i) Neural tissue (ectoderm, left), (ii) cartilage (mesoderm, middle), and (iii) gut-like epithelium (endoderm, right).

Article Snippet: The episomal vector containing pCXLE-OCT4 (1F-OCT4) was introduced into human NPC by electroporation (Amaxa Nucleofector 2B, LabWrench, Canada) on day 0.

Techniques: DNA Methylation Assay, Expressing, Bisulfite Sequencing, Clone Assay, H&E Stain, Generated, Derivative Assay, Injection

Journal: Stem Cells International

Article Title: Survivin Improves Reprogramming Efficiency of Human Neural Progenitors by Single Molecule OCT4

doi: 10.1155/2016/4729535

Figure Lengend Snippet: AP positive numbers in Survivin overexpression or Survivin knockdown in the reprogramming of NPCs. (a) AP positive numbers of 1F-OCT4, mock (vector control), and Survivin overexpression on 3.5 cm diameter well from the left to right; (b) AP positive numbers of 1F-OCT4, nonsilence, and Survivin-RNAi on the same size dishes (from the left to right). A representative experiment is shown in the left panels. Counting AP+ colonies in the same experiment, mean values + SD are shown in the right panels. (c) The relative expression of Survivin in mRNA level.

Article Snippet: The episomal vector containing pCXLE-OCT4 (1F-OCT4) was introduced into human NPC by electroporation (Amaxa Nucleofector 2B, LabWrench, Canada) on day 0.

Techniques: Over Expression, Plasmid Preparation, Expressing

Journal: Stem Cells International

Article Title: Survivin Improves Reprogramming Efficiency of Human Neural Progenitors by Single Molecule OCT4

doi: 10.1155/2016/4729535

Figure Lengend Snippet: SOX2 and OCT4 synergistically regulate expression of Survivin in ES cells. ChIP-qPCRs were conducted using SOX2 and OCT4 antibodies and primers specific for promoter of NANOG, Survivin (Birc5), and the β -actin genes. DNA lysates of cells were as input. NANOG was the positive control while β -actin was the negative control.

Article Snippet: The episomal vector containing pCXLE-OCT4 (1F-OCT4) was introduced into human NPC by electroporation (Amaxa Nucleofector 2B, LabWrench, Canada) on day 0.

Techniques: Expressing, Positive Control, Negative Control

Journal: Stem Cells International

Article Title: Survivin Improves Reprogramming Efficiency of Human Neural Progenitors by Single Molecule OCT4

doi: 10.1155/2016/4729535

Figure Lengend Snippet: The expression of Survivin was decreased with ES cell differentiation. (a) Expression of Survivin was downregulated with ES cell differentiation with Retinoic Acid (RA). The cells of 0 h, 24 h, 36 h, 48 h, and 72 h were collected for Western blot analysis. The proteins of SOX2, OCT4, and Survivin were analyzed in RA-treated differentiation and β -actin as housekeeping control. (b) The change of Survivin mRNA levels in the process of RA-treated differentiation for ES cells. The expressions of mRNA decreased gradually and sharply with prolonged RA-treatment time.

Article Snippet: The episomal vector containing pCXLE-OCT4 (1F-OCT4) was introduced into human NPC by electroporation (Amaxa Nucleofector 2B, LabWrench, Canada) on day 0.

Techniques: Expressing, Cell Differentiation, Western Blot

Journal: Stem Cells Translational Medicine

Article Title: A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

doi: 10.5966/sctm.2014-0214

Figure Lengend Snippet: Introduction of the GFP reporter to the BCL-XL vector helped to monitor nucleofection efficiency and identify fully reprogrammed clones. (A): Diagram of newly constructed GBX vector. (B): GFP expression was used to monitor nucleofection efficiency at day 2 after nucleofection. The microphotograph was taken at 10× magnification. (C): Reprogrammed cells were analyzed at day 13 using flow cytometric analysis. Many cells that gained TRA-1-60 tended to lose GFP expression. (D): Imaging analysis showed that fully reprogrammed iPS-like colonies that gained TRA-1-60 expression (stained in red) lacked the GFP signal (arrow). Scale bar = 200 μm. (E): The GFP-expressing GBX plasmid has potency similar to that of the MBX plasmid expressing BCL-XL alone in reprogramming adult PB MNCs in combination with two other episomal vectors (MOS and MMK). Abbreviations: EBNA1, Epstein-Barr nuclear antigen 1; eGFP, enhanced green fluorescent protein; GBX, modified vector expressing GFP and BCL-XL; GFP, green fluorescent protein; iPS, induced pluripotent stem cells; M, million; MBX, modified vector expressing BCL-XL; MNCs, mononuclear cells; NS, no significant differences on statistical analysis; PB, peripheral blood; SFFV, spleen focus-forming virus; SSC, side scatter.

Article Snippet: To test new EBNA1/OriP episomal vectors compared with the pEB-C5 and pEB-Tg combination or Okita-Yamanaka combination, 2 × 10 6 cultured erythroblasts were mixed with a total of 10 μg of plasmid DNA of episomal vectors for nucleofection (Lonza, Walkersville, MD, http://www.lonza.com ).

Techniques: Plasmid Preparation, Clone Assay, Construct, Expressing, Imaging, Staining, Modification, Virus

Journal: Stem Cells Translational Medicine

Article Title: A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

doi: 10.5966/sctm.2014-0214

Figure Lengend Snippet: Replacement of MEFs by vitronectin as a cell culture substrate maintained high reprogramming efficiency with the improved vector combination. (A): Schematic diagram of using VTN to substitute for MEFs as the coating material for tissue plates used at day 2 after nucleofection. A chemically defined, xeno-free E8 medium was used to replace the human ES cell medium that has animal products in the serum replacement. (B): VTN was shown to sustain high reprogramming efficiency under the MOS/MMK/MBX (pEV) vector combination with six different samples of adult PB MNCs of randomly selected donors. Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; ES, embryonic stem (cell); FBS, fetal bovine serum; M, million; MEF, mouse embryonic fibroblast; MNCs, mononuclear cells; NaB, sodium butyrate; PB, peripheral blood; SFM, serum-free medium; VTN, vitronectin.

Article Snippet: To test new EBNA1/OriP episomal vectors compared with the pEB-C5 and pEB-Tg combination or Okita-Yamanaka combination, 2 × 10 6 cultured erythroblasts were mixed with a total of 10 μg of plasmid DNA of episomal vectors for nucleofection (Lonza, Walkersville, MD, http://www.lonza.com ).

Techniques: Cell Culture, Plasmid Preparation, Modification