scholarly journals Hydrogels as feeder-free scaffolds for long-term self-renewal of mouse induced pluripotent stem cells

2012 ◽  
Vol 9 (4) ◽  
pp. 375-388 ◽  
Author(s):  
Jing Jing Yang ◽  
Jian Fang Liu ◽  
Takayuki Kurokawa ◽  
Kazuhiro Kitada ◽  
Jian Ping Gong
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Self Renewal ◽  
Induced Pluripotent

scholarly journals Differential Coupling of Self-Renewal Signaling Pathways in Murine Induced Pluripotent Stem Cells

PLoS ONE ◽  
2012 ◽  
Vol 7 (1) ◽  
pp. e30234 ◽  
Author(s):  
Luca Orlando ◽  
Yolanda Sanchez-Ripoll ◽  
James Foster ◽  
Heather Bone ◽  
Claudia Giachino ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Signaling Pathways ◽  
Pluripotent Stem Cells ◽  
Self Renewal ◽  
Induced Pluripotent ◽  
Differential Coupling

scholarly journals Noncanonical NOTCH Signaling Limits Self-Renewal of Human Epithelial and Induced Pluripotent Stem Cells through ROCK Activation

2013 ◽  
Vol 33 (22) ◽  
pp. 4434-4447 ◽  
Author(s):  
Takashi Yugawa ◽  
Koichiro Nishino ◽  
Shin-ichi Ohno ◽  
Tomomi Nakahara ◽  
Masatoshi Fujita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Fate ◽  
Pluripotent Stem Cells ◽  
Human Keratinocytes ◽  
Small Gtpase ◽  
Keratinocyte Differentiation ◽  
Growth Defect ◽  
Self Renewal ◽  
Induced Pluripotent

NOTCH plays essential roles in cell fate specification during embryonic development and in adult tissue maintenance. In keratinocytes, it is a key inducer of differentiation. ROCK, an effector of the small GTPase Rho, is also implicated in keratinocyte differentiation, and its inhibition efficiently potentiates immortalization of human keratinocytes and greatly improves survival of dissociated human pluripotent stem cells. However, the molecular basis for ROCK activation is not fully established in these contexts. Here we provide evidence that intracellular forms of NOTCH1 trigger the immediate activation of ROCK1 independent of its transcriptional activity, promoting differentiation and resulting in decreased clonogenicity of normal human keratinocytes. Knockdown of NOTCH1 abrogated ROCK1 activation and conferred sustained clonogenicity upon differentiation stimuli. Treatment with a ROCK inhibitor, Y-27632, or ROCK1 silencing substantially rescued the growth defect induced by activated NOTCH1. Furthermore, we revealed that impaired self-renewal of human induced pluripotent stem cells upon dissociation is, at least in part, attributable to NOTCH-dependent ROCK activation. Thus, the present study unveils a novel NOTCH-ROCK pathway critical for cellular differentiation and loss of self-renewal capacity in a subset of immature cells.

308 Tet1 PLAYS IMPORTANT ROLES IN MAINTAINING CHARACTERIZATIONS OF PORCINE INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 301
Author(s):  
A. R. Fan ◽  
K. Y. Ma ◽  
T. C. Zhao ◽  
P. P. An ◽  
B. Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Self Renewal ◽  
Qrt Pcr ◽  
Post Transfection ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent

It was recently found that the ten-eleven-translocation (TET) family of Fe(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and thus promotes active demethylation of genomes. Tet1 is highly expressed in mouse embryonic stem cells (mESC) and has been demonstrated to involve in mESC maintenance. Here we used small interference RNA (siRNA) to transiently knockdown expression of Tet1 in porcine induced pluripotent stem cells (iPSC) in order to identify its functions. The fetal fibroblasts were isolated from a 30-day-old porcine fetus and induced into iPSC with defined transcription factors, namely Oct-4, Sox-2, Klf-4, and C-myc. The colonies appeared on Day 12 and were picked up on Day 14. These colonies had normal ES-like morphology and alkaline phosphatase activity. Specifically, they were positively stained for pluripotency-specific markers, including Oct4, Sox2, Nanog, Rex1, and SSEA1. When cultured in vitro, the cells formed embryoid bodies (EB), and all 3 germ layer markers (endoderm: AFP, alphaAT; mesoderm: BMP4, Enolase; ectoderm: GFAP, Neurod) were detected positively in EB. For siRNA transfections, iPSC from the colonies were transfected with 40 pmol of siRNA and 2 µL of Lipofectamine 2000 in 1 well of a 24-well plate. After transfection, iPSC were subjected to fluorescence-activated cell sorting to determine the fraction of FAM-positive cells in order to confirm transfection efficiency; the percentage of positive cells reached 48 ± 4.96. We observed obvious knockdown of Tet1 after short-term transfection of siRNA, and the knockdown efficiency was confirmed using qRT-PCR and immunofluorescence staining. Notably, knockdown of Tet1 resulted in morphological abnormality and loss of undifferentiated state of porcine iPSC. However, no obvious morphological changes were observed in the negative control (transfected with nonsense-siRNA), positive control (transfected with GAPDH-siRNA), or mock control (transfected with DEPC-treated water). To gain insight into the molecular mechanism underlying the self-renewal defect, we analysed the effects of Tet1 knockdown on the expression of key stem cell factors and differentiation markers of different embryonic layers using qRT-PCR. We found that knockdown of Tet1 resulted in downregulated expression of pluripotency-related genes, such as Lefty-2, Klf-2, and Sox-2 (the expression ratios of post-transfection to pre-transfection were 0.31 ± 0.21, 0.48 ± 0.072, and 0.65 ± 0.046, respectively), and upregulated expression of differentiation-related genes, including Pitx-2, Hand-1, Gata-6, and Lef-1 (the expression ratios of post-transfection to pre-transfection were 4.35 ± 1.36, 2.56 ± 0.68, 2.91 ± 1.47, and 2.33 ± 1.11, respectively). However, Oct-4, C-myc, Klf-4, and Nanog were not downregulated (the expression ratios of post-transfection to pre-transfection were 0.91 ± 0.15, 1.12 ± 0.26, 1.15 ± 0.21, and 1.08 ± 0.08, respectively). Taken together, Tet1 plays important roles in porcine iPSC self-renewal and characterization maintenance. This study was financed by National Basic Research Program of China (NO.2009CB941001).

scholarly journals Expansion and long-term maintenance of induced pluripotent stem cells in stirred suspension bioreactors

2011 ◽  
Vol 6 (6) ◽  
pp. 462-472 ◽  
Author(s):  
Mehdi Shafa ◽  
Kirsten Sjonnesen ◽  
Akihiro Yamashita ◽  
Shiying Liu ◽  
Marek Michalak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent ◽  
Term Maintenance
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