scholarly journals Inhibition of ROCK–Myosin II Signaling Pathway Enables Culturing of Human Pluripotent Stem Cells on Microcarriers Without Extracellular Matrix Coating

2014 ◽  
Vol 20 (3) ◽  
pp. 227-238 ◽  
Author(s):  
Allen Kuan-Liang Chen ◽  
Xiaoli Chen ◽  
Yu Ming Lim ◽  
Shaul Reuveny ◽  
Steve Kah Weng Oh
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Signaling Pathway ◽  
Pluripotent Stem Cells ◽  
Myosin Ii ◽  
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.

scholarly journals Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0129855 ◽  
Author(s):  
Chiemi Takenaka ◽  
Hiroshi Miyajima ◽  
Yusuke Yoda ◽  
Hideo Imazato ◽  
Takako Yamamoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Pluripotent Stem Cells ◽  
Defined Medium ◽  
Human Pluripotent Stem Cells ◽  
Controlled Growth

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.

Mechanotransduction of human pluripotent stem cells cultivated on tunable cell-derived extracellular matrix

Biomaterials ◽  
2018 ◽  
Vol 150 ◽  
pp. 100-111 ◽  
Author(s):  
In Gul Kim ◽  
Chang-Hyun Gil ◽  
Joseph Seo ◽  
Soon-Jung Park ◽  
Ramesh Subbiah ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells

scholarly journals Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Aki Stubb ◽  
Camilo Guzmán ◽  
Elisa Närvä ◽  
Jesse Aaron ◽  
Teng-Leong Chew ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Pluripotent Stem Cells ◽  
Three Dimensional ◽  
Focal Adhesions ◽  
Structural Features ◽  
Human Pluripotent Stem Cells ◽  
Actin Ring ◽  
Localisation Microscopy

Abstract While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin β5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

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