Cellular extract facilitates nuclear reprogramming by altering DNA methylation and pluripotency gene expression

Abstract

The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in disease mechanisms and regenerative medicine. Epigenetic modifications enable differentiated cells to perpetuate molecular memory to retain their identity. Therefore, the aim of this study was to investigate the reprogramming modification of yak fibroblast cells that were permeabilized and incubated in the extracts of mesenchymal stem cells derived from mice adipose tissue [adipose-derived stem cells (ADSCs)]. According to the results, the treatment of ADSC extracts promoted colony formation. Moreover, pluripotent gene expression was associated with the loss of repressive histone modifications and increased global demethylation. The genes Col1a1 and Col1a2, which are typically found in differentiated cells only, demonstrated decreased expression and increased methylation in the 5'-flanking regulatory regions. Moreover, yak fibroblast cells that were exposed to ADSC extracts resulted in significantly different eight-cell and blastocyst formation rates of cloned embryos compared with their untreated counterparts. This investigation provides the first evidence that nuclear reprogramming of yak fibroblast cells is modified after the ADSC extract treatment. This research also presents a methodology for studying the dedifferentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells toward a pluripotent state without genetic alteration.

FIG. 1.

Morphological observation of yak fibroblasts after with or without ADSC extract incubated. (A, A′) Yak fibroblasts without ADSC extract incubated (control). (B, B′) Yak fibroblasts 2 days after exposure to ADSC extract. (C, C′) Yak fibroblasts 3 days after exposure to ADSC extract. (D, D′) Yak fibroblasts 5 days after exposure to ADSC extract.

FIG. 2.

Quantitative analysis of the histone acetylation of H3K9 (A, B) and methylation status of 5MeC (C, D) in control and extract-treated cells using immunostaining. (A, a) H3K9ac in yak fibroblasts without ADSC extract incubated (control). (B, b) H3K9ac in yak fibroblasts with ADSC extract incubated. (C, c) 5MeC in yak fibroblasts without ADSC extract incubated (control). (D, d) 5MeC in yak fibroblasts with ADSC extract incubated. The histogram represents average optical intensity and an asterisk (*) indicates significant differences (p<0.05).

FIG. 3.

Relative expression of pluipotent associated genes (Oct-4, Sox-2, c-Myc, and Klf-4) and fibroblast marker genes (Col1a1 and Col1a2) in ADSCs and yak fibroblast cells treated with extracts or untreated groups. Transcript levels in yak fibroblast cells untreated were used as the calibrator (relative expression=1.0). Values with different superscripts (a, b, and c) are significantly different (p<0.05).

FIG. 4.

Exposure of yak fibroblast cells to ADSC extracts elicits DNA demethylation of Col1a1 and Col1a2. (A) Bisulfite sequencing analysis of Col1a1 and Col1a2 methylation in control and treated groups. Global percentages of methylated cytosines (%Me) are shown. Each row of circles for a given amplicon represents the methylation status of each CpG in one bacterial clone for that region. (B) Percentages of methylated cytosines in each position in Col1a1 and Col1a2 determined from data shown in A. On the x axes, CpG No. 1 is the 5′ cytosine examined in each region. Positions of genomic regions examined are shown.