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Hypoxia enhances chondrogenesis and prevents terminal differentiation through PI3K/Akt/FoxO dependent anti-apoptotic effect.

Lee HH, Chang CC, Shieh MJ, Wang JP, Chen YT, Young TH, Hung SC - Sci Rep (2013)

Bottom Line: Here, we compared the effects of hypoxia (1% oxygen) and normoxia (air) on chondrogenic differentiation of human mesenchymal stem cells (MSCs).MSCs induced for differentiation under hypoxia increased in chondrogenesis, but decreased in endochondral ossification compared to those under normoxia.The hypoxia-dependent protection of MSCs from chondrogenesis-induced apoptosis correlated with an increase in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan [2] Department of Orthopedics, Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan [3] Department of Orthopedics, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.

ABSTRACT
Hypoxia, a common environmental condition, influences cell signals and functions. Here, we compared the effects of hypoxia (1% oxygen) and normoxia (air) on chondrogenic differentiation of human mesenchymal stem cells (MSCs). For in vitro chondrogenic differentiation, MSCs were concentrated to form pellets and subjected to conditions appropriate for chondrogenic differentiation under normoxia and hypoxia, followed by the analysis for the expression of genes and proteins of chondrogenesis and endochondral ossification. MSCs induced for differentiation under hypoxia increased in chondrogenesis, but decreased in endochondral ossification compared to those under normoxia. MSCs induced for differentiation were more resistant to apoptosis under hypoxia compared to those under normoxia. The hypoxia-dependent protection of MSCs from chondrogenesis-induced apoptosis correlated with an increase in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO pathway. These results suggest that the PI3K/Akt/FoxO survival pathway activated by hypoxia in MSCs enhances chondrogenesis and plays an important role in preventing endochondral ossification.

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Hypoxic culture enhanced chondrogenesis and suppressed expression of markers of endochondral ossification in MSCs.MSCs (aliquots of 2.5 × 105) were pelleted and induced in chondrogenic differentiation medium under normoxic (Nor, 21% O2) and hypoxic (Hyp, 1% O2) conditions. (a) Quantitative RT-PCR for mRNA levels on day 7 of induction. (b) (Left panel) Alcian blue staining on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. (c) (Left panel) ICC staining for type II collagen on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. (d) (Left panel) ICC staining for type X collagen on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. Scale bars = 100 microns. [Values are mean ± SE; *, p < 0.05 and **, p < 0.01 indicate significant variance (Mann Whitney U test) compared to MSCs incubated under Nor conditions] (Three pellets were conducted at different times for each MSC line. Experiments were performed using MSCs from three individuals and representative data from individual #2 are shown).
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f1: Hypoxic culture enhanced chondrogenesis and suppressed expression of markers of endochondral ossification in MSCs.MSCs (aliquots of 2.5 × 105) were pelleted and induced in chondrogenic differentiation medium under normoxic (Nor, 21% O2) and hypoxic (Hyp, 1% O2) conditions. (a) Quantitative RT-PCR for mRNA levels on day 7 of induction. (b) (Left panel) Alcian blue staining on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. (c) (Left panel) ICC staining for type II collagen on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. (d) (Left panel) ICC staining for type X collagen on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. Scale bars = 100 microns. [Values are mean ± SE; *, p < 0.05 and **, p < 0.01 indicate significant variance (Mann Whitney U test) compared to MSCs incubated under Nor conditions] (Three pellets were conducted at different times for each MSC line. Experiments were performed using MSCs from three individuals and representative data from individual #2 are shown).

Mentions: To determine the effects of hypoxic culture on MSC chondrogenesis, we first examined the mRNA levels of Sox9, Col2a1, aggrecan, Runx2, and Col10a1 in MSCs induced for chondrogenesis under normoxic (21% O2, the air) and hypoxic (1% O2) conditions by quantitative RT-PCR. MSCs induced under hypoxic conditions exhibited significantly higher mRNA levels of Sox9, Col2a1 and aggrecan compared to those of MSCs under normoxic conditions (p < 0.05) (Figure 1a). By contrast, hypoxic conditions decreased the mRNA levels of Runx2 and Col10a1 compared to normoxic culture (p < 0.01). Moreover, histological sections stained with Alcian blue also demonstrated that hypoxic conditions had increased proteoglycan synthesis compared to normoxic culture on day 7 and 14 of chondrogenesis (Figure 1b). ICC further demonstrated that hypoxic culture of MSCs increased type II collagen expression (Figure 1c), but decreased type X collagen expression compared to normoxic culture (Figure 1d). Together, these data suggested that hypoxic conditions increased chondrogenesis and suppressed expression of markers associated with endochondral ossification.


Hypoxia enhances chondrogenesis and prevents terminal differentiation through PI3K/Akt/FoxO dependent anti-apoptotic effect.

Lee HH, Chang CC, Shieh MJ, Wang JP, Chen YT, Young TH, Hung SC - Sci Rep (2013)

Hypoxic culture enhanced chondrogenesis and suppressed expression of markers of endochondral ossification in MSCs.MSCs (aliquots of 2.5 × 105) were pelleted and induced in chondrogenic differentiation medium under normoxic (Nor, 21% O2) and hypoxic (Hyp, 1% O2) conditions. (a) Quantitative RT-PCR for mRNA levels on day 7 of induction. (b) (Left panel) Alcian blue staining on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. (c) (Left panel) ICC staining for type II collagen on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. (d) (Left panel) ICC staining for type X collagen on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. Scale bars = 100 microns. [Values are mean ± SE; *, p < 0.05 and **, p < 0.01 indicate significant variance (Mann Whitney U test) compared to MSCs incubated under Nor conditions] (Three pellets were conducted at different times for each MSC line. Experiments were performed using MSCs from three individuals and representative data from individual #2 are shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3775095&req=5

f1: Hypoxic culture enhanced chondrogenesis and suppressed expression of markers of endochondral ossification in MSCs.MSCs (aliquots of 2.5 × 105) were pelleted and induced in chondrogenic differentiation medium under normoxic (Nor, 21% O2) and hypoxic (Hyp, 1% O2) conditions. (a) Quantitative RT-PCR for mRNA levels on day 7 of induction. (b) (Left panel) Alcian blue staining on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. (c) (Left panel) ICC staining for type II collagen on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. (d) (Left panel) ICC staining for type X collagen on days 7 and 14 of induction. (Right panel) Quantification by Image-Pro Plus. Scale bars = 100 microns. [Values are mean ± SE; *, p < 0.05 and **, p < 0.01 indicate significant variance (Mann Whitney U test) compared to MSCs incubated under Nor conditions] (Three pellets were conducted at different times for each MSC line. Experiments were performed using MSCs from three individuals and representative data from individual #2 are shown).
Mentions: To determine the effects of hypoxic culture on MSC chondrogenesis, we first examined the mRNA levels of Sox9, Col2a1, aggrecan, Runx2, and Col10a1 in MSCs induced for chondrogenesis under normoxic (21% O2, the air) and hypoxic (1% O2) conditions by quantitative RT-PCR. MSCs induced under hypoxic conditions exhibited significantly higher mRNA levels of Sox9, Col2a1 and aggrecan compared to those of MSCs under normoxic conditions (p < 0.05) (Figure 1a). By contrast, hypoxic conditions decreased the mRNA levels of Runx2 and Col10a1 compared to normoxic culture (p < 0.01). Moreover, histological sections stained with Alcian blue also demonstrated that hypoxic conditions had increased proteoglycan synthesis compared to normoxic culture on day 7 and 14 of chondrogenesis (Figure 1b). ICC further demonstrated that hypoxic culture of MSCs increased type II collagen expression (Figure 1c), but decreased type X collagen expression compared to normoxic culture (Figure 1d). Together, these data suggested that hypoxic conditions increased chondrogenesis and suppressed expression of markers associated with endochondral ossification.

Bottom Line: Here, we compared the effects of hypoxia (1% oxygen) and normoxia (air) on chondrogenic differentiation of human mesenchymal stem cells (MSCs).MSCs induced for differentiation under hypoxia increased in chondrogenesis, but decreased in endochondral ossification compared to those under normoxia.The hypoxia-dependent protection of MSCs from chondrogenesis-induced apoptosis correlated with an increase in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan [2] Department of Orthopedics, Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan [3] Department of Orthopedics, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.

ABSTRACT
Hypoxia, a common environmental condition, influences cell signals and functions. Here, we compared the effects of hypoxia (1% oxygen) and normoxia (air) on chondrogenic differentiation of human mesenchymal stem cells (MSCs). For in vitro chondrogenic differentiation, MSCs were concentrated to form pellets and subjected to conditions appropriate for chondrogenic differentiation under normoxia and hypoxia, followed by the analysis for the expression of genes and proteins of chondrogenesis and endochondral ossification. MSCs induced for differentiation under hypoxia increased in chondrogenesis, but decreased in endochondral ossification compared to those under normoxia. MSCs induced for differentiation were more resistant to apoptosis under hypoxia compared to those under normoxia. The hypoxia-dependent protection of MSCs from chondrogenesis-induced apoptosis correlated with an increase in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO pathway. These results suggest that the PI3K/Akt/FoxO survival pathway activated by hypoxia in MSCs enhances chondrogenesis and plays an important role in preventing endochondral ossification.

Show MeSH
Related in: MedlinePlus