J Vet Sci.  2007 Dec;8(4):377-382. 10.4142/jvs.2007.8.4.377.

Enhanced tyrosine hydroxylase expression in PC12 cells co-cultured with feline mesenchymal stem cells

• Jin GZ
• Yin XJ
• Yu XF
• Cho SJ
• Lee HS
• Lee HJ
• Kong IK

• KMID: 1106218
• DOI: http://doi.org/10.4142/jvs.2007.8.4.377

Abstract

Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson's disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p < 0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.

Keyword

co-culture; feline bone marrow; mesenchymal stem cell; rat PC12 cell; tyrosine hydroxylase

MeSH Terms

Animals
Antigens, Surface/metabolism
Blotting, Western
Cats/*physiology
Cell Culture Techniques
Cells, Cultured
*Gene Expression Regulation, Enzymologic
Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism
Immunohistochemistry
Mesenchymal Stem Cells/*cytology/metabolism
Microscopy, Phase-Contrast
PC12 Cells/cytology/*enzymology
Rats
Tyrosine 3-Monooxygenase/*metabolism

Figure

• 

  Fig. 1 Identification of isolated bone marrow-derived MSCs. (A) Phase contrast image of heterogeneous MSCs after passage 1. (B) Phase contrast image of fibroblast-like, homogeneous MSCs after passage 4. (C) Immunostaining of vimentin antigen, a marker expressed by BMCs. Scale bar = 200 µm, A and B; 50 µm, C.

• 

  Fig. 2 Flow cytometric determination of cell-surface antigen profiles for feline MSC. Feline bone marrow MSCs were stained with feline specific antibodies to a variety of cell-surface antigens. The percentage of cells positive for each antibody is listed in the corresponding histogram. An isotype control is used to identify background fluorescence. MSC from 3 separate cats were tested with similar results. Representative results from 1 cat are shown.

• 

  Fig. 3 Phase contrast image of PC12 cells and MSCs. (A) MSCs cultured alone for 3 days. The cells exhibited an elongated spindle shape resembling fibroblasts. (B) PC12 cells cultured alone for 3 days. The cells tended to grow in small clusters. (C) PC12 cells were co-cultured with MSCs for 3 days. The PC12 cells were not significantly changed, but the MSCs possessed a longer, more complex processes (white arrowheads). Scale bar = 200 µm.

• 

  Fig. 4 Representative Western blot analysis of TH proteins. (A) Immunoblots of TH and GAPDH protein expression. M, molecular marker, Lane 1, MSC group; Lane 2, PC12 group; Lane 3, MSC + PC12 group. (B) Ratio of TH to GAPDH density values in A. TH expression was significantly increased in the MSC + PC12 group. **p<0.001 vs. PC12 group.

• 

  Fig. 5 Fluorescence micrographs of co-cultures of PC12 cells (green) and MSCs (red) for three days. (A) MSCs prelabeled with Dil-Ac-LDL (red). (B) PC12 cells stained with a rabbit anti-TH antibody (green). (C) Merged image of A and B. MSCs were not TH positive. Scale bar = 100 µm.

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