Korean J Physiol Pharmacol.  2016 Jan;20(1):53-62. 10.4196/kjpp.2016.20.1.53.

Effects of CoCl2 on multi-lineage differentiation of C3H/10T1/2 mesenchymal stem cells

• Yoo HI
• Moon YH
• Kim MS

• KMID: 2150473
• DOI: http://doi.org/10.4196/kjpp.2016.20.1.53

Abstract

Mesenchymal stem cells (MSCs) in the bone marrow and other somatic tissues reside in an environment with relative low oxygen tension. Cobalt chloride (CoCl2) can mimic hypoxic conditions through transcriptional changes of some genes including hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). This study evaluated the potential role of CoCl2 preconditioning on multi-lineage differentiation of C3H/10T1/2, a murine MSC line to understand its possible molecular mechanisms in vitro. CoCl2 treatment of MSCs markedly increased HIF-1alpha and VEGF mRNA, and protein expression of HIF-1alpha. Temporary preconditioning of MSCs with CoCl2 induced up-regulation of osteogenic markers including alkaline phosphatase, osteocalcin, and type I collagen during osteogenic differentiation, followed by enhanced mineralization. CoCl2 also increased chondrogenic markers including aggrecan, sox9, and type II collagen, and promoted chondrocyte differentiation. CoCl2 suppressed the expression of adipogenic markers including PPARgamma, aP2, and C/EBPalpha, and inhibited adipogenesis. Temporary preconditioning with CoCl2 could affect the multi-lineage differentiation of MSCs.

Keyword

Differentiation; Hypoxia; Mesenchymal stem cells

MeSH Terms

Adipogenesis
Aggrecans
Alkaline Phosphatase
Anoxia
Bone Marrow
Chondrocytes
Cobalt
Collagen Type I
Collagen Type II
Mesenchymal Stromal Cells*
Osteocalcin
Oxygen
PPAR gamma
RNA, Messenger
Up-Regulation
Vascular Endothelial Growth Factor A
Aggrecans
Alkaline Phosphatase
Cobalt
Collagen Type I
Collagen Type II
Osteocalcin
Oxygen
PPAR gamma
RNA, Messenger
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Effects of CoCl2 on cell viability of C3H/10T1/2 cells.C3H/10T1/2 cells were cultured in the 2% serum medium in the presence of different CoCl2 concentrations for 48 h (A) and in the presence of 0.1 or 0.25 mM CoCl2 for a designated time (B). Cell viability was determined by the MTT assay. Each value is the mean±SD of triplicate independent experiments. *p<0.05, as compared to control.

• 

  Fig. 2 Effect of CoCl2 in the expression of HIF-1α and VEGF mRNA.C3H/10T1/2 cells were seeded in 60-mm culture dishes at a density of 1×105 cells and incubated in the growth medium containing 0.1 mM CoCl2 for 48 h. At the indicated times, total RNA and cell lysates were isolated and RT-PCR analysis (A), quantitative real time RT-PCR analysis (B), and Western blotting analysis (C) was done.

• 

  Fig. 3 Effects of treatment of CoCl2 on osteogenic differentiation.(A) C3H/10T1/2 cells were pre-incubated with 0.1 mM CoCl2 for 0 h, 24 h or 48 h. After incubation, the cells were replaced with osteogenic medium and cultured for 18 days, prior to staining with Alizarin red S. (B) Results from (A) were quantified by spectrophotometry. (C) C3H/10T1/2 cells were preincubated with 0.1 mM CoCl2 for 0 h, 24 h, or 48 h. After incubation, cells were cultured with osteogenic medium for 3 days. Total cellular RNA was extracted, and gene expression of osteogenic markers was detected by semi-quantitative RT-PCR. Expression of actin was examined in the same sample as a control for the amount of present reverse-transcribed cDNA. (D) Effects of treatment of CoCl2 during osteoblast differentiation. C3H/10T1/2 cells were pre-incubated with 0.1 mM CoCl2 for 0 h, 24 h, or 48 h. After incubation, the cells were cells were replaced with an osteogenic medium and cultured for 10 days. At indicated times, total cellular RNA was extracted and gene expression of osteogenic markers was assessed by qRT-PCR. Values shown are normalized to β-actin levels. The data represent the mean±S.D. from triplicate independent experiments (*p<0.05).

• 

  Fig. 4 Effects of treatment of CoCl2 on the chondrogenic differentiation.(A) C3H/10T1/2 cells were pre-incubated with 0.1 mM CoCl2 for 0 h, 24 h, or 48 h. After incubation, the cells shifted to a chondrogenic medium and cultured for 14 days. The cells were then stained with Alcian blue. (B) C3H/10T1/2 cells were pre-incubated with 0.1 mM CoCl2 for 0 h, 24 h, or 48 h. After incubation, the cells were shifted to a chondrogenic medium and cultured for 3 days. Total cellular RNA was extracted and gene expression of the chondrogenic markers aggrecan, sox9, and Col 2A1 was assessed by semi- quantitative RT-PCR. Expression of actin was examined in the same sample as a control for the amount of present reverse-transcribed cDNA. (C) Effects of treatment of CoCl2 during chondrocyte differentiation. C3H/ 10T1/2 cells were pre-incubated with 0.1 mM CoCl2 for 0 h, 24 h, or 48 h. After incubation, the cells were replaced with chondrogenic medium and cultured for 10 days. At indicated times, total cellular RNA was extracted and gene expression of the chondrogenic makers aggrecan, sox9, and Col 2A1 was assessed by qRT-PCR. Values shown are normalized to β-actin levels.

• 

  Fig. 5 Effects of treatment of CoCl2 on the adipogenic differentiation.(A) C3H/10T1/2 cells were pre-incubated with 0.1 mM CoCl2 for 0 h, 24 h, or 48 h. After incubation, the cells were cultured with adipogenic medium for 5 days then stained with Oil red s. (B) The degree of lipid accumulation as quantified by spectrophotometry. (C) Total cellular RNA was extracted and gene expression of the adipogenic markers PPARγ, aP2, and C/EBPα was assessed by semi-quantitative RT-PCR. Expression of actin was examined in the same sample as a control for the amount of present reverse-transcribed cDNA. (D) Effects of treatment of CoCl2 during adipocyte differentiation. C3H/10T1/2 cells were preincubated with 0.1 mM CoCl2 for 0 h, 24 h, or 48 h. After incubation, the cells were cells were replaced with adipogenic medium and cultured for 10 days. At indicated times, total cellular RNA was extracted and gene expression of the adipogenic makers PPARγ, aP2, and C/EBPα was analyzed by qRT-PCR. Values shown are normalized to β-actin levels. The data represent the mean±S.D. from triplicate independent experiments (*p<0.05).

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