Transplantation of novel human GDF5-expressing CHO cells is neuroprotective in models of Parkinson's disease

Abstract

Growth/differentiation factor 5 (GDF5) is a neurotrophic factor that promotes the survival of midbrain dopaminergic neurons in vitro and in vivo and as such is potentially useful in the treatment of Parkinson's disease (PD). This study shows that a continuous supply of GDF5, produced by transplanted GDF5-overexpressing CHO cells in vivo, has neuroprotective and neurorestorative effects on midbrain dopaminergic neurons following 6-hydroxydopamine (6-OHDA)-induced lesions of the adult rat nigrostriatal pathway. It also increases the survival and improves the function of transplanted embryonic dopaminergic neurons in the 6-OHDA-lesioned rat model of PD. This study provides the first proof-of-principle that sustained delivery of GDF5 in vivo may be useful in the treatment of PD.

Fig 1

Characterization of GDF5 expression and secretion by the GDF5-CHO cell line in vitro. (A) Representative photomicrograph of a single GDF5-CHO cell grown for 3 DIV, immunocytochemically stained for GDF5. Scale bar = 10 μm. (B) Western blot detection (using MP52 antibody) of high molecular weight GDF5 precursor proteins in a GDF5-CHO cell extract after 15 DIV. (C) Western blot detection (run under reducing conditions, using MP52 antibody) of GDF5 monomeric proteins in conditioned medium from mock-CHO and GDF5-CHO cell cultures after 1, 3, 5, 10, 15, and 21 DIV, as indicated. (D) Western blot detection (run under non-reducing conditions, using aMP5 antibody) of GDF5 dimeric proteins in conditioned medium from mock-CHO and GDF5-CHO cell cultures after 1, 3, 5, 10, 15, and 21 DIV, as indicated. rhGDF5 was run as a positive control in both (C) and (D). Graphical representation of the number of (E) dopaminergic (TH-positive) neurons, (F) total (β-III tubulin-positive) neurons and (G) total cells per field in E14 rat VM cultures treated with conditioned medium from mock-CHO or GDF5-CHO cells for 3 DIV. Data are shown as mean ± SEM (*P < 0.05 compared with mock-CHO cell group; Student's t-test; n = 3 experiments).

Fig 2

Characterization of GDF5 expression by the GDF5-CHO cell line in vivo and of the immune response of the host striatum to GDF5-CHO cell transplantation. (A)–(D) Representative photomicrographs showing (A) CD 8, (B) MHC I, (C) MHC II and (D) IgM immunoreactivity at the graft sites at 10 days after intrastriatal grafting of CHO cells. Graphical representation of the extent of the immunoreactivity to (E) CD 4, (F) CD 8, (G) MHC I, (H) MHC II, (I) IgM and (J) Ed 1 in the adult rat striatum at 5, 10, 21 and 42 days after grafting of E14 VM cells, or CHO cells with or without oral CyA, as indicated. (K)–(N) Representative photomicrographs showing intrastriatal transplants of mock-CHO cells and GDF5-CHO cells at 1 and 7 days post-grafting, as indicated, immunocytochemically stained for GDF5 using aMP5 antibody. Scale bar = 100 μm.

Fig 3

Neuroprotective and neurorestorative effects of GDF5-CHO cells in two adult rat models of PD. (A) Outline of experiment to analyse the neuroprotective effect of GDF5-CHO cells in the 6-OHDA MFB lesion model of PD. (B) Amphetamine-induced rotational rates at 21 days post-surgery for each treatment group (n = 5 per group). (C) Survival of dopaminergic neurons in the SN at 28 days post-surgery expressed as the number of TH-positive neurons in the left SN as a percentage of those in the right. Data are expressed as mean ± SEM. ***P < 0.001 compared with control (lesion only) and mock-CHO groups, anova with post-hoc Tukey's test. (D) Outline of experiment to analyse the neurorestorative effect of GDF5-CHO cells in the 6-OHDA striatal lesion model of PD. (E) Amphetamine-induced rotational rates at 28 days post-surgery for each treatment group (n = 5 per group). (F) Survival of dopaminergic neurons in the SN at 35 days post-surgery, expressed as the number of TH-positive neurons in the left SN as a percentage of those in the right. Data are expressed as mean ± SEM. *P < 0.05 compared with control (lesion only), mock-CHO (SN) and mock-CHO (striatum) groups; anova with post-hoc Tukey's test.

Fig 4

GDF5-CHO cells improve the survival and function of grafts of E14 rat VM in an adult rat model of PD. (A) Outline of experiment to analyse the effects of co-grafting GDF5-CHO cells with E14 rat VM grafts in the 6-OHDA MFB lesion model of PD. (B) Amphetamine-induced rotational rates at 3 weeks after lesion surgery and at 1, 2, 3 and 6 weeks post-grafting surgery for each treatment group (n = 5 per group). (C) Representative photomicrographs of grafted TH-positive neurons in the ‘graft alone’, ‘graft + GDF5’, ‘graft + GDF5-CHO without CyA’ and ‘graft + GDF5-CHO with CyA’ groups at 7 weeks post-grafting. (D) Survival of grafted dopaminergic neurons (expressed as a percentage of the total number grafted) at 7 weeks post-grafting in each treatment group (n = 5 per group). Data are expressed as mean ± SEM. (**P < 0.01, ***P < 0.001 versus control (‘graft alone’), GDF5-CHO without CyA, mock-CHO with or without CyA groups; anova with post-hoc Tukey's test). (E) Representative line drawings showing individual TH-positive neurons in control (‘graft alone’) and treated ‘graft + GDF5-CHO with CyA’ grafts. Scale bar = 100 μm.