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. 2008 Jan;212(1):19-30.
doi: 10.1111/j.1469-7580.2007.00834.x. Epub 2007 Nov 23.

Orthotopic transplantation of immortalized mesencephalic progenitors (CSM14.1 cells) into the substantia nigra of hemiparkinsonian rats induces neuronal differentiation and motoric improvement

Stefan Jean-Pierre Haas  1 , Stanislav Petrov, Golo Kronenberg, Oliver Schmitt, Andreas Wree
Affiliations

Orthotopic transplantation of immortalized mesencephalic progenitors (CSM14.1 cells) into the substantia nigra of hemiparkinsonian rats induces neuronal differentiation and motoric improvement

Stefan Jean-Pierre Haas et al. J Anat. 2008 Jan.

Abstract

Neural progenitor cell grafting is a promising therapeutic option in the treatment of Parkinson's disease. In previous experiments we grafted temperature-sensitive immortalized CSM14.1 cells, derived from the ventral mesencephalon of E14-rats, bilaterally in the caudate putamen of adult hemiparkinsonian rats. In these studies we were not able to demonstrate either a therapeutic improvement or neuronal differentiation of transplanted cells. Here we examined whether CSM14.1 cells grafted bilaterally orthotopically in the substantia nigra of hemiparkinsonian rats have the potential to differentiate into dopaminergic neurons. Adult male rats received 6-hydroxydopamine into the right medial forebrain bundle, and successful lesions were evaluated with apomorphine-induced rotations 12 days after surgery. Two weeks after a successful lesion the animals received bilateral intranigral grafts consisting of either about 50 000 PKH26-labelled undifferentiated CSM14.1 cells (n = 16) or a sham-graft (n = 9). Rotations were evaluated 3, 6, 9 and 12 weeks post-grafting. Animals were finally perfused with 4% paraformaldehyde. Cryoprotected brain slices were prepared for immunohistochemistry using the freeze-thaw technique to preserve PKH26-labelling. Slices were immunostained against neuronal epitopes (NeuN, tyrosine hydroxylase) or glial fibrillary acidic protein. The CSM14.1-cell grafts significantly reduced the apomorphine-induced rotations 12 weeks post-grafting compared to the sham-grafts (P < 0.05). There was an extensive mediolateral migration (400-700 microm) of the PKH26-labelled cells within the host substantia nigra. Colocalization with NeuN or glial fibrillary acidic protein in transplanted cells was confirmed with confocal microscopy. No tyrosine hydroxylase-immunoreactive grafted cells were detectable. The therapeutic effect of the CSM14.1 cells could be explained either by their glial cell-derived neurotrophic factor-expression or their neural differentiation with positive effects on the basal ganglia neuronal networks.

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Figures

Fig. 1
Fig. 1
Tyrosine hydroxylase distribution in the Hemiparkinsonian rat model. (a) Frontal section through the region of the CPu. The right hemisphere, i.e. the intact side, displays a strong TH-ir. The contralateral lesioned hemisphere contains no more obvious TH-ir after dopaminergic deafferentiation. (b) Frontal section through the mesencephalon containing the SNc. Immunoreactivity was found on the intact side at the right. On the lesioned side only a limited TH-ir was observed in the SNc and the VTA. (c) Sagittal section of an intact control hemisphere demonstrating TH-ir of the CPu, SNc, VTA and medial forebrain bundle (MFB). The arrows points to the intact MFB and SNc. (d) Sagittal section of a 6-OHDA lesioned hemisphere demonstrating abundance of TH-ir. The asterisk is placed over the location of 6-OHDA injection. Scale bar: 160 µm (a), 80 µm (b), 250 µm (c,d).
Fig. 2
Fig. 2
Neurotrophine contents of CSM14.1 cells in vitro. (a) 50 µg protein lysate of CSM14.1 cells and the control probe of 5 ng recombinant rat GDNF were analyzed by Western blotting for GDNF. Specific bands in both probes around 15 kDa can be detected. A second specific band in protein lysates of CSM14.1 cells around 42 kDa belongs to the protein product of the housekeeping-gene β-actin, demonstrating the correct loading. Moreover, a distinct positive immunocytochemical reaction for GDNF in undifferentiated CSM14.1 cells was found (b), whereas the negative control (c) shows clear immunonegativity. (d) In a Western blot of another probe of CSM14.1 cells no immunoreactivity for CNTF can be detected, whereas 0.5 ng of recombinant rat CNTF shows a specific band around 22.8 kDa. Again in the CSM14.1-cell lysates the band around 42 kDa belongs to β-actin, demonstrating clearly the correct loading of the gel. The immunocytochemical staining for CNTF also does not show any immunopositive reaction (e), similar to the immunonegative control in (c). The Nissl-stain (f) demonstrates the morphology of adherent undifferentiated CSM14.1 cells. Scale bar: 100 µm (b,c,e,f).
Fig. 3
Fig. 3
Time scale of experiments and development of apomorphine-induced rotation behavior. The diagram shows the mean net-rotations/min of the experimental groups. Gray bars represent CSM14.1-transplanted animals (n = 16) and black bars the results of the sham-transplanted group (n = 9). The arrow below illustrates the time scale of experiments and behavioral measurements. It starts with the event of lesioning (0 days), the 6-OHDA injection effect was evaluated 12 days later by an initial rotation test and 2 days after these initial rotation measurements, (sham-)transplantations were performed. Three weeks, 6 weeks, 9 weeks and 12 weeks after (sham-)transplantation the rotations were further registered. Subtracting the number of contralateral rotations after the (sham-)transplantation from the number of initial rotations showed a trend (P = 0.053) towards a significant decrease of rotations after 9 weeks in the CSM14. 1-transplanted group. This significant decrease declined further at 12 weeks (P
Fig. 4
Fig. 4
Graft localisation and morphology in the SNc. Immunofluorescence micrographs of the lesioned hemisphere (left) and the contralateral intact control side (right). Immunofluorescence of GFAP (green) and PKH26 (red) marked CSM14.1 cells in the substantia nigra are shown for the lesioned side (a) and the contralateral intact side (b) as an overview at low magnification. PKH26-labelled transplanted cells migrated over a distance of about 400 mm and no obvious differences between the lesioned (c) or the control side (d) were observed. (e,f) Images represent a magnification of the specimen of the top. Parallel sections stained for TH (green fluorescence), demonstrate the correct placement of the PKH26-labelled grafts (red fluorescence) in the region of the lesioned SNc (g) or the intact contralateral control hemisphere (h). The distinct vertical transplantation needle tract can be detected in the upper part (g). Single dopaminergic TH-immunoreactive neurons, lacking PKH26-labelling survived the 6-OHDA lesion as demonstrated by green immunofluorescence (i), whereas the intact control side shows many TH-ir neurons (green) and also the survival of transplanted PKH26-labelled CSM14.1 cells (j). As well as in the lesioned SNc (i) and the intact control SNc (j) no colocalizations of PKH26 and TH of transplanted CSM14.1 cells within the VTA were observed (g,h). The yellow fluorescence signal (j) mimicking a PKH26/TH colocalization stems from different cells lying on top of another. Scale bars: 1000 µm (a,b), 200 µm (c,d, g,h), 70 µm (e,f,i,j).
Fig. 5
Fig. 5
Neuronal differentiation of CSM14.1 cells. NeuN-stained sections (green fluorescence) from the region of the SNc in the lesioned (left) and intact control side (right) are illustrated. Immunofluorescent micrographs of PKH26-labelled cells show a distinct NeuN-immunoreactivity, indicating an obvious neural phenotype of transplanted CSM14.1 cells in the lesioned (a) or intact control side (b). The core of the graft in the lesioned hemisphere appears with an intense red fluorescence in the upper right corner (a). Further evidence at higher magnification is displayed in (c) and (d) as c′, d′ and d″. CSM14.1 cells in the lesioned side are labelled by PKH26 and contain NeuN (c and c′). PKH26 (d′) and NeuN-colocalization (d″) of a transplanted CSM14.1 cell in the intact control side is also shown. CLSM-imaging (2-µm-thick optical sections) supplies further evidence for CSM14.1-cell survival and differentiation into NeuN-immunoreactive neurons in the lesioned side (e). The arrow points to a cell which is also documented at higher magnification (e′). Due to the lack of PKH26-labelled cells sham-transplanted animals never showed a PKH26/NeuN colocalization (f). In these animals only red autofluorescent host-derived macrophages, mainly lying around blood vessels (arrows), were detectable. These cells never contained NeuN. Moreover, due to their location and morphology they were clearly distinguishable from PKH26-labelled and transplanted NeuN-immunoreactive cells from transplanted animals. Scale bars: 70 µm (a,b), 50 µm (c,d), 20 µm (c′,d′,d″), 30 µm (e,f).
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