Immunofluorescence Microscopy and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Including Primary Cilia Associated Signaling Pathways

2014 ◽  
pp. 123-140 ◽  
Author(s):  
Maj Linea Vestergaard ◽  
Aashir Awan ◽  
Caroline Becker Warzecha ◽  
Søren Tvorup Christensen ◽  
Claus Yding Andersen
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Signaling Pathways ◽  
Human Embryonic Stem Cells ◽  
Primary Cilia ◽  
Immunofluorescence Microscopy ◽  
Embryonic Stem ◽  
Mrna Analysis

CDK11 safeguards the identity of human embryonic stem cells via fine‐tuning signaling pathways

2019 ◽  
Vol 235 (5) ◽  
pp. 4279-4290
Author(s):  
Jianyi Ding ◽  
Zhuoqing Fang ◽  
Xinyuan Liu ◽  
Zhexin Zhu ◽  
Chunsheng Wen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Signaling Pathways ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Fine Tuning

scholarly journals Expression profiles of protein tyrosine kinase genes in human embryonic stem cells

Reproduction ◽  
2008 ◽  
Vol 136 (4) ◽  
pp. 423-432 ◽  
Author(s):  
Mi-Young Son ◽  
Janghwan Kim ◽  
Hyo-Won Han ◽  
Sun-Mi Woo ◽  
Yee Sook Cho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Signaling Pathways ◽  
Human Embryonic Stem Cells ◽  
Tyrosine Kinases ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Degenerate Primers ◽  
Protein Tyrosine ◽  
Hesc Lines

Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid-treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases’ activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs.

scholarly journals Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

2008 ◽  
Vol 180 (5) ◽  
pp. 897-904 ◽  
Author(s):  
Enko N. Kiprilov ◽  
Aashir Awan ◽  
Romain Desprat ◽  
Michelle Velho ◽  
Christian A. Clement ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Embryonic Stem ◽  
Serum Starvation ◽  
Mechanistic Basis ◽  
Patched 1 ◽  
Hesc Lines

Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.

scholarly journals Analysis of alternative signaling pathways of endoderm induction of human embryonic stem cells identifies context specific differences

2012 ◽  
Vol 6 (1) ◽  
pp. 154 ◽  
Author(s):  
Shibin Mathew ◽  
Maria Jaramillo ◽  
Xinan Zhang ◽  
Li Zhang ◽  
Alejandro Soto-Gutiérrez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Signaling Pathways ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Context Specific
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