scholarly journals AXIN2 Reduces the Survival of Porcine Induced Pluripotent Stem Cells (piPSCs)

2021 ◽  
Vol 22 (23) ◽  
pp. 12954
Author(s):  
Rui Zhang ◽  
Shuai Yu ◽  
Qiaoyan Shen ◽  
Wenxu Zhao ◽  
Juqing Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Pluripotent Stem Cells ◽  
Wnt Signaling Pathway ◽  
Culture Conditions ◽  
Rna Seq ◽  
Expression Of Genes ◽  
Proliferation And Apoptosis ◽  
Induced Pluripotent

The establishment of porcine pluripotent stem cells (piPSCs) is critical but remains challenging. All piPSCs are extremely sensitive to minor perturbations of culture conditions and signaling network. Inhibitors, such as CHIR99021 and XAV939 targeting the WNT signaling pathway, have been added in a culture medium to modify the cell regulatory network. However, potential side effects of inhibitors could confine the pluripotency and practicability of piPSCs. This study aimed to investigate the roles of AXIN, one component of the WNT pathway in piPSCs. Here, porcine AXIN1 and AXIN2 genes were knocked-down or overexpressed. Digital RNA-seq was performed to explore the mechanism of cell proliferation and apoptosis. We found that (1) overexpression of the porcine AXIN2 gene significantly reduced survival and negatively impacted the pluripotency of piPSCs, and (2) knockdown of AXIN2, a negative effector of the WNT signaling pathway, enhanced the expression of genes involved in cell cycle but reduced the expression of genes related to cell differentiation, death, and apoptosis.

scholarly journals Valproic acid promotes the in vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaotong Wang ◽  
Mengyuan Qu ◽  
Zili Li ◽  
Yuting Long ◽  
Kai Hong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Pluripotent Stem Cells ◽  
Wnt Signaling Pathway ◽  
Human Pluripotent Stem Cells ◽  
Rna Seq ◽  
In Vitro Differentiation ◽  
Upregulated Genes

Abstract Background Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction. Methods We examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols. Results The combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway. Conclusions VPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.

scholarly journals AXIN2 reduces the survival of porcine induced pluripotent stem cells (piPSCs)

2021 ◽  
Author(s):  
rui zhang ◽  
Shuai Yu ◽  
Qiaoyan Shen ◽  
Wenxu Zhao ◽  
Juqing Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Proliferation ◽  
Pluripotent Stem Cells ◽  
Culture Medium ◽  
Wnt Signaling Pathway ◽  
Rna Seq ◽  
Expression Of Genes ◽  
Induced Pluripotent ◽  
Effect Of Inhibitors

Abstract BackgroundThe establishment of porcine pluripotent stem cells (piPSCs) is still a critical topic and challenging issue. However, all piPSCs are extremely sensitive to changes of the culture conditions.In addition, the side effect of inhibitors in culture medium confine the pluripotency and practicability. This study aimed to investigate the roles of AXIN in piPSCs and further explore the mechanism. Here, porcine AXIN1 gene and AXIN2 were knockdown, cloned, and overexpressed in piPSCs. Digital RNA-seq was performed to explore the mechanism of cell proliferation and anti-apoptosis. ResultsHere, we found (1): overexpression of the porcine AXIN2 gene significantly reduce the survivability of piPSCs, meanwhile wreck the pluripotency of piPSCs; (2): The Digital RNA-seq analysis reveals that AXIN2, as a negative effector of the WNT signaling pathway, whom after knockdown enhances the expression of genes involved in cell cycle such as CCND1, and reduced the expression of genes related to cell differentiation, cell death, and cell apoptosis. ConclusionAXIN2 could reduce the pluripotency and survival of piPSCs and also provided a potential to simplify the cultrue medium.

scholarly journals Involvement of the Wnt signaling pathway in feeder-free culture of human induced pluripotent stem cells

2015 ◽  
Vol 12 (5) ◽  
pp. 6797-6800 ◽  
Author(s):  
MINORU TOMIZAWA ◽  
FUMINOBU SHINOZAKI ◽  
YASUFUMI MOTOYOSHI ◽  
TAKAO SUGIYAMA ◽  
SHIGENORI YAMAMOTO ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Induced Pluripotent Stem Cells ◽  
Signaling Pathway ◽  
Pluripotent Stem Cells ◽  
Wnt Signaling Pathway ◽  
Induced Pluripotent

scholarly journals Valproic acid Promotes the in Vitro Differentiation of Human Pluripotent Stem Cells Into Spermatogonial Stem Cell-like Cells

2021 ◽  
Author(s):  
Xiaotong Wang ◽  
Mengyuan Qu ◽  
Zili Li ◽  
Yuting Long ◽  
Kai Hong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Pluripotent Stem Cells ◽  
Wnt Signaling Pathway ◽  
Human Pluripotent Stem Cells ◽  
Sequencing Analysis ◽  
Upregulated Genes

Abstract Background: Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on the SSCLC induction. Methods: We examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. The percentage of haploid cells and cells expressed meiotic and spermatid genes were also detected. RNA-sequencing analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols.Results: The combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. High concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-sequencing analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and the expression of genes in Wnt signaling pathway. Conclusions: VPA robustly promotes the differentiation of human pluripotent stem cell lines into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that inactivation of Wnt signaling pathway might be a cause of SSC depletion in azoospermia testes. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.

scholarly journals A Small Molecule Modulating Monounsaturated Fatty Acids and Wnt Signaling Confers Maintenance to Induced Pluripotent Stem Cells Against Endodermal Differentiation

2021 ◽  
Author(s):  
Vahid Hosseini ◽  
Ashkan Kalantary-Charvadeh ◽  
Maryam Hajikarami ◽  
Parisa Fayyazpour ◽  
Reza Rahbarghazi ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Stem Cells ◽  
Wnt Signaling ◽  
Induced Pluripotent Stem Cells ◽  
Click Chemistry ◽  
Signaling Pathway ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Endodermal Differentiation ◽  
Induced Pluripotent

Abstract Background: Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. This enzyme can orchestrate posttranslational modification of proteins involved in the development and differentiation of cells through the fatty acid acylation process. In this study, we evaluated whether a small molecule modulating unsaturated fatty acids influences early endodermal differentiation of induced pluripotent stem cells, using biochemical methods and immunostaining.Methods: The hiPSCs were cultured in an endoderm-inducing medium containing activin A and low defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The yield of three germ layers endoderm, mesoderm, and ectoderm, and the cell cycle analysis were assessed using flow cytometry. The expression of endoderm and pluripotency markers, as well as the expression of Wnt signaling pathway proteins, were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction.Results: The population of cells showing endoderm features was decreased at the end of differentiation when SCD1 was inhibited on the first day. Moreover, early SCD1 inhibition preserved hiPSCs properties without a shift toward mesoderm or ectoderm. Treatment of cells with SCD1 inhibitor only on the first day decreased the β-catenin gene expression and intensity of fluorescent emission in the click chemistry. These effects were effectively rescued by cotreatment with oleate. Late treatment at two subsequent days of endoderm induction induced no significant effect on endoderm-specific markers and fluorescent intensity. Reproducible results were also obtained with a human embryonic stem cell line. Conclusion: The small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring maintenance to hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in endoderm commitment of pluripotent stem cells may be eminent in disorders of endoderm-derived organs and dysregulated metabolism.

scholarly journals A small molecule modulating monounsaturated fatty acids and Wnt signaling confers maintenance to induced pluripotent stem cells against endodermal differentiation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vahid Hosseini ◽  
Ashkan Kalantary-Charvadeh ◽  
Maryam Hajikarami ◽  
Parisa Fayyazpour ◽  
Reza Rahbarghazi ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Stem Cells ◽  
Wnt Signaling ◽  
Induced Pluripotent Stem Cells ◽  
Click Chemistry ◽  
Signaling Pathway ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Endodermal Differentiation ◽  
Induced Pluripotent

Abstract Background Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs). Methods The hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction. Results When SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased β-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells. Conclusion The small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism. The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. β-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, β-catenin increases the expression of the endoderm markers Sox17 and CXCR4.

scholarly journals Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

2021 ◽  
Vol 22 (9) ◽  
pp. 5011
Author(s):  
Daehwan Kim ◽  
Sangho Roh
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Stem Cell Research ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Human Diseases ◽  
Induced Pluripotent

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.

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