Int J Stem Cells.  2021 May;14(2):191-202. 10.15283/ijsc20213.

Valproic Acid Enhance Reprogramming of Bactrian Camel Cells through Promoting the Expression of Endogenous Gene c-Myc and the Process of Angiogenesis

• Li Z
• Ge W
• Li Y
• Zhang Y
• Zhao X
• Hu J

• KMID: 2515995
• DOI: http://doi.org/10.15283/ijsc20213

Abstract

Background and Objectives
Induced pluripotent stem cells (iPSCs) are usually generated by reprogramming differentiated cells through the introduction of specific transcription factors, but this is a difficult and inefficient process. Valproic acid (VPA) is a histone deacetylase inhibitor that significantly improves the efficiency of iPSC generation. But its role and mechanism are still unclear.
Methods and Results
We transduced Bactrian camel fetal fibroblasts (BCFFs) with retroviruses carrying defined factors (OCT4, SOX2, KLF4, c-MYC and EGFP; OSKMG) in the presence of VPA. Cells were collected (Day 7) and analyzed using RNA-seq technology. Afterwards, different groups of cells and transcriptomics results were detected by PCR and qRT-PCR technology. The results showed that VPA promoted the expression of the endogenous gene c-Myc and inhibited cell proliferation; at the same time, it promoted the expression of VEGF and other genes related to angiogenesis.
Conclusions
When VPA is added to the culture medium, only the cells that have begun to reprogram can break the G2/M repression through the expression of the endogenous gene c-Myc, and use the nutrients and space in the culture dish to proliferate normally, which can achieve the purpose of directly improving the efficiency of reprogramming. Another new discovery for Bactrian camels, VPA significantly increased the expression of VEGFC and other genes, promoting the transformation of fibroblasts to endothelial cells (different from the mesenchymal-to-epithelial transition process of other species) to accelerate the early induction of Bactrian camels iPSc process. Overall, this study proved the new mechanism of VPA in enhancing the induction of pluripotency from the transcriptome level.

Keyword

Induced pluripotent stem cells; Bactrian camel; Valproic acid; RNA-seq

Figure

• 

  Fig. 1 VPA cell treatments. (A) Sche-matic diagram of iPSC culture medium containing VPA. (B) The EGFP retroviralpackaging 48-h after trans-duction. (C) The infection efficiency in CEFs was observed 48-h after the second viral infection. (D) Cells in the treatment (iPSC medium with VPA) and control (iPSC medium without VPA) groups were cultured in the iPSC medium on day 7.

• 

  Fig. 2 Dynamic changes of processed cells. (A) Detection of the expression of exogenous genes in the three groups of BCFFs, OSKM and OSKM＋VPA. (B) Detection of the expression of endogenous genes in the three groups of BCFFs, OSKM and OSKM＋VPA. (C) Heat map of the four endogenous genes. (D, E) The expressions of the three germ layer genes NCSTN (endoderm), DES (mesoderm), PAX6 and CRABP2 (ectoderm) were detected by qRT-PCR.

• 

  Fig. 3 Transcriptomic sequencing, basic analysis of correlations, and differences between groups. (A) Co-rrelation heat map of the samples. In the figure, the abscissa and ordinate are the respective samples and the color depth (intensity) indicates the correlation coefficient between the two samples. (B) Genes from different samples are expressed in different colors; the redder the color, the higher the expression; whereas, the bluer the color, the lower the ex-pression. (C) KEGG pathway annota-tion. The map is plotted with different pathways and the number of target genes contained within. The pathways belonging to the same KEGG A-class classification share the same color. Each pillar represents a pathway and the height of the pillar represents the number of genes involved in each pathway. (D) Top 20 genes obtained after KEGG enrichment analysis. Each bubble represents a pathway; the size of the bubble represents the number of target genes contained in the pathway and the color of the bubble represents the significant degree of enrichment of the pathway.

• 

  Fig. 4 Participate in the screening of key pathways for reprogramming of BCFFs. (A) Represents a protein-protein interaction network made according to different requirements. Different nodes represented different enzymes. The interactions among these enzymes were represented by different colorful lines. (B) Twelve genes were verified by qRT-PCR. (C) Heat map of key genes of the PI3K-Akt and MAPK signaling pathways. (D) Heat map of key genes of p53 signaling pathway. (E) TP53 gene expression was detected by qRT-PCR.

• 

  Fig. 5 Effects on proliferation and apoptosis genes. (A) The heat map made using enriched genes in cell cyclesignaling pathways. (B) The changes of proliferation and apoptosis genes in the three groups of BCFFs, OSKM and OSKM＋VPA.

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  Fig. 6 The influence of VPA on the mesenchymal-to-epithelial transition process. (A) The heat map made using enriched genes in cancer signaling pathways. (B) The results of qRT-PCR detection of the expression of fibroblast marker genes S1004A, VIM, HSP47 and ACTA2 in the three groups of BCFFs, OSKM and OSKM＋VPA. (C, D) The results of qRT-PCR detection of the expression of endothelial cell marker genes ICAM1, VEGFC and LYVE1 in the three groups of BCFFs, OSKM and OSKM＋VPA. (E) The qRT-PCR results of the expression of the epithelial cell marker gene CLDN7 in the three groups of BCFFs, OSKM and OSKM＋VPA. (F) PCR detection of the expression of epithelial cell marker genes EPCAM and CDH1 in the three groups of BCFFs, OSKM and OSKM＋VPA.

Reference

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