Methods to Manipulate and Monitor Wnt Signaling in Human Pluripotent Stem Cells

2016 ◽  
pp. 161-181 ◽  
Author(s):  
Ian J. Huggins ◽  
David Brafman ◽  
Karl Willert
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells

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.

Biphasic modulation of Wnt signaling supports efficient foregut endoderm formation from human pluripotent stem cells

2016 ◽  
Vol 40 (5) ◽  
pp. 534-548 ◽  
Author(s):  
Jeannine Hoepfner ◽  
Mandy Kleinsorge ◽  
Oliver Papp ◽  
Mania Ackermann ◽  
Susanne Alfken ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Foregut Endoderm

scholarly journals BMP and TGF-β pathway mediators are critical upstream regulators of Wnt signaling during midbrain dopamine differentiation in human pluripotent stem cells

2013 ◽  
Vol 376 (1) ◽  
pp. 62-73 ◽  
Author(s):  
Jingli Cai ◽  
Stephanie Schleidt ◽  
Joshua Pelta-Heller ◽  
Danielle Hutchings ◽  
Gregory Cannarsa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Midbrain Dopamine ◽  
Upstream Regulators

scholarly journals Wnt signaling mediates acquisition of blood-brain barrier properties in naïve endothelium derived from human pluripotent stem cells

2021 ◽  
Author(s):  
Benjamin D Gastfriend ◽  
Hideaki Nishihara ◽  
Scott G Canfield ◽  
Koji L Foreman ◽  
Britta Engelhardt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Blood Brain Barrier ◽  
Pluripotent Stem Cells ◽  
Barrier Properties ◽  
Human Pluripotent Stem Cells ◽  
Brain Barrier ◽  
Blood Brain ◽  
The Central Nervous System

Endothelial cells (ECs) in the central nervous system (CNS) acquire their specialized blood-brain barrier (BBB) properties in response to extrinsic signals, with Wnt/β-catenin signaling coordinating multiple aspects of this process. Our knowledge of CNS EC development has been advanced largely by animal models, and human pluripotent stem cells (hPSCs) offer the opportunity to examine BBB development in an in vitro human system. Here we show that activation of Wnt signaling in hPSC-derived naïve endothelial progenitors, but not in matured ECs, leads to robust acquisition of canonical BBB phenotypes including expression of GLUT-1, increased claudin-5, and decreased PLVAP. RNA-seq revealed a transcriptome profile resembling ECs with CNS-like characteristics, including Wnt-upregulated expression of LEF1, APCDD1, and ZIC3. Together, our work defines effects of Wnt activation in naïve ECs and establishes an improved hPSC-based model for interrogation of CNS barriergenesis.

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.

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