Differentiation of Human Induced Pluripotent Stem Cells into Definitive Endoderm Using Simple Dialysis Culture Device

2021 ◽  
Author(s):  
Hyun Jin Choi ◽  
Fuad Gandhi Torizal ◽  
Marie Shinohara ◽  
Yasuyuki Sakai
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Definitive Endoderm ◽  
Induced Pluripotent ◽  
Dialysis Culture

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.

Small Molecules Differentiate Definitive Endoderm from Human Induced Pluripotent Stem Cells on PCL Scaffold

2014 ◽  
Vol 173 (7) ◽  
pp. 1727-1736 ◽  
Author(s):  
Elham Hoveizi ◽  
Sirus Khodadadi ◽  
Shima Tavakol ◽  
Oveis Karima ◽  
Mohammad Ali Nasiri-Khalili
Keyword(s):  
Stem Cells ◽  
Small Molecules ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Definitive Endoderm ◽  
Induced Pluripotent

scholarly journals Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity

2020 ◽  
Vol 8 ◽  
pp. 205031212096645
Author(s):  
Estela Cruvinel ◽  
Isabella Ogusuku ◽  
Rosanna Cerioni ◽  
Sirlene Rodrigues ◽  
Jéssica Gonçalves ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Definitive Endoderm ◽  
Body Formation ◽  
Differentiation Capacity ◽  
High Efficient ◽  
Induced Pluripotent

Objectives: To establish a straightforward single-cell passaging cultivation method that enables high-quality maintenance of human induced pluripotent stem cells without the appearance of karyotypic abnormalities or loss of pluripotency. Methods: Cells were kept in culture for over 50 passages, following a structured chronogram of passage and monitoring cell growth by population doubling time calculation and cell confluence. Standard procedures for human induced pluripotent stem cells monitoring as embryonic body formation, karyotyping and pluripotency markers expression were evaluated in order to assess the cellular state in long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes, cardiomyocytes and definitive endoderm to evaluate its differentiation capacity. Results: Human induced pluripotent stem cells clones maintained its pluripotent capability as well as chromosomal integrity and were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes, cardiomyocytes and definitive endoderm. Conclusions: Our findings support the routine of human induced pluripotent stem cells single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy human induced pluripotent stem cells to be used in drug discovery, toxicity, and disease modeling as well as for therapeutic approaches.

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.

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