scholarly journals The Expression of Wnt5a Gene Throughout Definitive Endoderm Induction Process in Induced Pluripotent Stem Cells

2020 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Shahrokh Lorzadeh ◽  
Negar Azarpira ◽  
Saeid Ghavami ◽  
Leila Kohan
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Induced Pluripotent Stem Cells ◽  
Mrna Expression ◽  
Pluripotent Stem Cells ◽  
Definitive Endoderm ◽  
Expression Level ◽  
Induction Step ◽  
Induced Pluripotent ◽  
Canonical Wnt

Background: Induced pluripotent stem cells (iPSCs) have the ability to proliferate indefinitely and differentiate into three germ layers of ectoderm, mesoderm, and endoderm. Definitive induction is the first and the most delicate stage of differentiation of various iPSC-derived organs. It has been found that the Wnt signaling pathway implicates in embryogenesis, organogenesis, and cell communication. Objectives: In the present study, we aimed to investigate the expression pattern of the Wnt5a gene as an indicator of non-canonical Wnt signaling activity during definitive endoderm induction of iPSCs. Methods: Human iPSCs (RSCB0042) were acquired from Royan stem cell bank of Royan Institute (Tehran, Iran). The iPSCs were cultured on a feeder layer of mitomycin-inactivated mouse embryonic fibroblasts (MEF), and iPSC colonies were collected for embryoid body (EB) generation by suspension culture method. Then endoderm induction step was performed using a series of small molecules. The quantitative real-time PCR was used to assess the mRNA expression of wnt5a, Nanog, OCT4, SOX17, and FOXA2 genes. Results: The production of efficient EBs confirmed by a decrease in Nanog and Oct4 gene expression and the success of DE (definite endoderm) induction step was confirmed by a high expression level of DE specific genes, Sox17, and FoxA2. A significant upregulation of Wnt5a in EB samples and a minor decrease at day 4 was observed. However, the differentiation process followed by an incremental fashion in Wnt5a mRNA expression starting from day 4 of differentiation among the samples of days 6 and 8 (DE stage). Conclusions: Our results suggest that Wnt5a is more activated at the later steps of endoderm induction rather than the early steps, which may be due to the stimulation of canonical Wnt signaling. Finding the expression level of Wnt5a could rise insights for developing more efficient differentiation induction protocols.

scholarly journals Evaluation of ATG5 Gene Expression in Human Induced Pluripotent Stem Cells During Endoderm Induction

2020 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Alice Sabet ◽  
Negar Azarpira ◽  
Saeid Ghavami ◽  
Leila Kohan
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Mrna Expression ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Metabolic Stress ◽  
High Rate ◽  
Human Ipscs ◽  
Induced Pluripotent

Background: Autophagy is a vital cell survival mechanism that authorizes cells to assort to metabolic stress and is essential for the development and maintenance of cellular and tissue homeostasis, as well as the prevention of human disease. It has also been shown that autophagy plays a significant role in the development and differentiation of stem cells, as well as induced pluripotent stem cells (iPSCs). Objectives: The present study aimed to examine the mRNA expression of the ATG5 gene, one of the key markers of autophagy in human iPSCs (hiPSCs) during endoderm induction. Methods: In this study, we cultured the human iPSC line (R1-hiPSC1) on mitomycin-C, inactivated mouse embryonic fibroblasts (MEF) layer, and used hanging drop protocol to generate embryoid body (EB) and expose differentiation. The Real-time PCR method was used to examine the mRNA expression level of ATG5 in hiPSC during endoderm induction. Results: Our results demonstrated the high mRNA expression of ATG5 in the MEI stage, which shows the high rate of autophagy in MEI days rather than the other stages of differentiation. Conclusions: The modification of ATG5 gene expression within hiPSC during endoderm induction shows the importance of autophagy assessments in hiPSC differentiation. Therefore, subsequent studies are needed to clarify the details of autophagy effects on hiPSC differentiation.

scholarly journals Stage-Specific Regulation of Reprogramming to Induced Pluripotent Stem Cells by Wnt Signaling and T Cell Factor Proteins

Cell Reports ◽  
2013 ◽  
Vol 3 (6) ◽  
pp. 2113-2126 ◽  
Author(s):  
Ritchie Ho ◽  
Bernadett Papp ◽  
Jackson A. Hoffman ◽  
Bradley J. Merrill ◽  
Kathrin Plath
Keyword(s):  
Stem Cells ◽  
T Cell ◽  
Wnt Signaling ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
T Cell Factor ◽  
Specific Regulation ◽  
Induced Pluripotent

scholarly journals Regulation of WNT Signaling by VSX2 During Optic Vesicle Patterning in Human Induced Pluripotent Stem Cells

Stem Cells ◽  
2016 ◽  
Vol 34 (11) ◽  
pp. 2625-2634 ◽  
Author(s):  
Elizabeth E. Capowski ◽  
Lynda S. Wright ◽  
Kun Liang ◽  
M. Joseph Phillips ◽  
Kyle Wallace ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Optic Vesicle ◽  
Induced Pluripotent

scholarly journals Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Anett Illing ◽  
Marianne Stockmann ◽  
Narasimha Swamy Telugu ◽  
Leonhard Linta ◽  
Ronan Russell ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Cell Fate ◽  
Pluripotent Stem Cells ◽  
Human Hair ◽  
Definitive Endoderm ◽  
Sk Channel ◽  
Induced Pluripotent

Pluripotent stem cells present an extraordinary powerful tool to investigate embryonic development in humans. Essentially, they provide a unique platform for dissecting the distinct mechanisms underlying pluripotency and subsequent lineage commitment. Modest information currently exists about the expression and the role of ion channels during human embryogenesis, organ development, and cell fate determination. Of note, small and intermediate conductance, calcium-activated potassium channels have been reported to modify stem cell behaviour and differentiation. These channels are broadly expressed throughout human tissues and are involved in various cellular processes, such as the after-hyperpolarization in excitable cells, and also in differentiation processes. To this end, human induced pluripotent stem cells (hiPSCs) generated from plucked human hair keratinocytes have been exploitedin vitroto recapitulate endoderm formation and, concomitantly, used to map the expression of the SK channel (SKCa) subtypes over time. Thus, we report the successful generation of definitive endoderm from hiPSCs of ectodermal origin using a highly reproducible and robust differentiation system. Furthermore, we provide the first evidence that SKCas subtypes are dynamically regulated in the transition from a pluripotent stem cell to a more lineage restricted, endodermal progeny.

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