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Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation.

Li Z, Liu C, Xie Z, Song P, Zhao RC, Guo L, Liu Z, Wu Y - PLoS ONE (2011)

Bottom Line: We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP.Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly.Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Life Science Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen, China.

ABSTRACT

Background: Mesenchymal stem cells (MSCs) hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood.

Methodology/principal findings: Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes.

Conclusions/significance: Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

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Gene expression, histone acetylation and DNA methylation in early and late passage MSCs.(A) Real-Time PCR analysis of the expression of Nanog, REX1 and CD133 in culture passage (P) 1, passage 6 MSCs cultured in growth medium or passage 6 MSCs growth medium supplemented with bFGF (P6-bFGF). bFGF treatment started from passage 1 cells. (B) TERT histone H3 acetylation (** P<0.01 versus P1 and P6 in bFGF-supplemented culture). (C) TERT gene expression (Real-Time PCR analysis) and, (D) DNA methylation in CpG islands in the promoter region of TERT in passage 1 and 6 MSCs cultured in growth medium versus passage 6 MSCs cultured in growth medium supplemented with bFGF (P6-bFGF).
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pone-0020526-g004: Gene expression, histone acetylation and DNA methylation in early and late passage MSCs.(A) Real-Time PCR analysis of the expression of Nanog, REX1 and CD133 in culture passage (P) 1, passage 6 MSCs cultured in growth medium or passage 6 MSCs growth medium supplemented with bFGF (P6-bFGF). bFGF treatment started from passage 1 cells. (B) TERT histone H3 acetylation (** P<0.01 versus P1 and P6 in bFGF-supplemented culture). (C) TERT gene expression (Real-Time PCR analysis) and, (D) DNA methylation in CpG islands in the promoter region of TERT in passage 1 and 6 MSCs cultured in growth medium versus passage 6 MSCs cultured in growth medium supplemented with bFGF (P6-bFGF).

Mentions: Cultured in growth medium, MSCs underwent modest but progressive morphological changes with successive passages. The cells became larger and fatter in general (Fig. 3A and B). Meanwhile, a small portion of cells became extremely larger and flatter, which were similar to osteoblasts in morphology and positive for ALP stain (Fig. 3C), indicating the presence of spontaneous differentiation of MSCs into osteoblasts. In accordance with changes in morphology, the expression of genes associated with osteogenesis such as collagen type I, ALP, bone sialoprotein (BSP), osteocalcin (OCN) and osteopontin (OPN) increased progressively with successive cell passages (Fig. 3D), while the expression of genes associated with stem cell pluripotency and proliferation decreased markedly, which included Oct4, Sox2, Nanog, REX1, CD133 and TERT (Fig. 4A and C; Fig. 5B and E).


Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation.

Li Z, Liu C, Xie Z, Song P, Zhao RC, Guo L, Liu Z, Wu Y - PLoS ONE (2011)

Gene expression, histone acetylation and DNA methylation in early and late passage MSCs.(A) Real-Time PCR analysis of the expression of Nanog, REX1 and CD133 in culture passage (P) 1, passage 6 MSCs cultured in growth medium or passage 6 MSCs growth medium supplemented with bFGF (P6-bFGF). bFGF treatment started from passage 1 cells. (B) TERT histone H3 acetylation (** P<0.01 versus P1 and P6 in bFGF-supplemented culture). (C) TERT gene expression (Real-Time PCR analysis) and, (D) DNA methylation in CpG islands in the promoter region of TERT in passage 1 and 6 MSCs cultured in growth medium versus passage 6 MSCs cultured in growth medium supplemented with bFGF (P6-bFGF).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111432&req=5

pone-0020526-g004: Gene expression, histone acetylation and DNA methylation in early and late passage MSCs.(A) Real-Time PCR analysis of the expression of Nanog, REX1 and CD133 in culture passage (P) 1, passage 6 MSCs cultured in growth medium or passage 6 MSCs growth medium supplemented with bFGF (P6-bFGF). bFGF treatment started from passage 1 cells. (B) TERT histone H3 acetylation (** P<0.01 versus P1 and P6 in bFGF-supplemented culture). (C) TERT gene expression (Real-Time PCR analysis) and, (D) DNA methylation in CpG islands in the promoter region of TERT in passage 1 and 6 MSCs cultured in growth medium versus passage 6 MSCs cultured in growth medium supplemented with bFGF (P6-bFGF).
Mentions: Cultured in growth medium, MSCs underwent modest but progressive morphological changes with successive passages. The cells became larger and fatter in general (Fig. 3A and B). Meanwhile, a small portion of cells became extremely larger and flatter, which were similar to osteoblasts in morphology and positive for ALP stain (Fig. 3C), indicating the presence of spontaneous differentiation of MSCs into osteoblasts. In accordance with changes in morphology, the expression of genes associated with osteogenesis such as collagen type I, ALP, bone sialoprotein (BSP), osteocalcin (OCN) and osteopontin (OPN) increased progressively with successive cell passages (Fig. 3D), while the expression of genes associated with stem cell pluripotency and proliferation decreased markedly, which included Oct4, Sox2, Nanog, REX1, CD133 and TERT (Fig. 4A and C; Fig. 5B and E).

Bottom Line: We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP.Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly.Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Life Science Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen, China.

ABSTRACT

Background: Mesenchymal stem cells (MSCs) hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood.

Methodology/principal findings: Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes.

Conclusions/significance: Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

Show MeSH