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

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.

Download Full-text

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

10.21203/rs.3.rs-769984/v1 ◽

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.

Download Full-text

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

International Journal of Molecular Sciences ◽

10.3390/ijms222312954 ◽

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.

Download Full-text

Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo

Molecular Medicine Reports ◽

10.3892/mmr.2015.4586 ◽

2015 ◽

Vol 13 (1) ◽

pp. 720-730 ◽

Cited By ~ 8

Author(s):

LIPING OU ◽

LIAOQIONG FANG ◽

HEJING TANG ◽

HAI QIAO ◽

XIAOMEI ZHANG ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Wnt Signaling ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells

Download Full-text

Single cell transcriptome analysis reveals markers of naïve and lineage-primed hematopoietic progenitors derived from human pluripotent stem cells

10.1101/602565 ◽

2019 ◽

Author(s):

Antonella Fidanza ◽

Nicola Romanò ◽

Prakash Ramachandran ◽

Sara Tamagno ◽

Martha Lopez-Yrigoyen ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Single Cells ◽

Human Pluripotent Stem Cells ◽

In Vitro Differentiation ◽

Hematopoietic Progenitors ◽

Lineage Priming ◽

Cell Transcriptome ◽

Single Cell Transcriptome

AbstractDuring embryogenesis the hematopoietic system develops through distinct waves that generate progenitors with increasing lineage potential, ultimately producing haematopoietic stem cells (HSCs). In vitro differentiation of human pluripotent stem cells (hPSCs) follows the early steps of haematopoietic development but the production of HSCs has proven more challenging. To study the dynamics and heterogeneity of hematopoietic progenitor cells generated in vitro from hPSCs, we performed RNA sequencing of over 10000 CD235a-CD43+single cells. We identified the transcriptome of naïve progenitors and those primed toward erythroid, megakaryocyte and leukocyte lineages, and revealed their markers by clustering, trajectory analyses and functional assays. CD44 marks naïve clonogenic progenitors that express the transcription factor, LMO4 and can be expanded upon BMP4 stimulation. Naïve progenitors give rise to primed CD326+erythroid, ICAM2+CD9+megakaryocyte, and monocyte, neutrophil and eosinophil progenitors. We have generated an online dataset of human hematopoietic progenitors and their transcriptional remodelling upon lineage priming.

Download Full-text

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

10.1101/2021.05.12.443828 ◽

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.

Download Full-text

Lithium chloride promotes proliferation of neural stem cells in vitro, possibly by triggering the Wnt signaling pathway

Animal Cells and Systems ◽

10.1080/19768354.2018.1487334 ◽

2018 ◽

Vol 23 (1) ◽

pp. 32-41 ◽

Cited By ~ 7

Author(s):

Jian Zhang ◽

Lu He ◽

Zhong Yang ◽

Lihong Li ◽

Wenqin Cai

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Wnt Signaling ◽

Signaling Pathway ◽

Lithium Chloride ◽

Wnt Signaling Pathway

Download Full-text

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

eLife ◽

10.7554/elife.70992 ◽

2021 ◽

Vol 10 ◽

Author(s):

Benjamin D Gastfriend ◽

Hideaki Nishihara ◽

Scott G Canfield ◽

Koji L Foreman ◽

Britta Englehardt ◽

...

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, decreased PLVAP and decreased permeability. 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.

Download Full-text