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

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.

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.