Therapeutic Potential of DsRed Porcine Amniotic Fluid Stem Cells in Parkinsonian Model Rats
Date Issued
2016
Date
2016
Author(s)
Lin, Tzu-Hsuan
Abstract
Parkinson''s disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra that leads to dopamine level decreases in the brain. Its clinical symptoms include resting tremor, rigidity, bradykinesia and postural instability. However, present pharmacological and surgical therapies can only postpone the emergence of motor symptoms, none have been able to reverse the degeneration of dopaminergic neurons. Recent studies have shown that amniotic fluid stem cells (AFSCs) are capable of differentiating into cell types from all three embryonic germ layers and expressing pluripotent stem cell marker, Oct-4. Most importantly, AFSCs won''t induce teratoma formation in vivo when applied on clinical transplantation. Therefore, to investigate the therapeutic potential and possible mechanisms of AFSCs in PD is beneficial to developing a new and more effective way for treating PD. In this study, we used the AFSCs isolated from transgenic DsRed pigs for xenotransplantation in parkinsonian rats. First, we found that DsRed porcine AFSCs (pAFSCs) could differentiate into dopaminergic neuron-like cells in vitro. After induction with dopaminergic neuronal differentiation medium which was composed of sonic hedgehog, fibroblast growth factor 8, basic fibroblast growth factor, and brain-derived neurotrophic factor, the cells developed a neuronal morphology expressing the neuronal marker β-III tubulin. Additionally, the differentiated DsRed pAFSCs could express dopaminergic neuronal specific marker tyrosine hydroxylase (TH) 12 days after induction. These results implied the feasibility of using pAFSCs as a cell source for developing therapies of PD. Next, Sprague Dawley rats unilaterally lesioned by 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle were used as the parkinsonian model animal to evaluate the therapeutic effect of AFSCs in PD. After 2 weeks of 6-OHDA lesioning, the immunoreactivity of dopaminergic neuronal marker-TH reduced in the nigrostriatal pathway compared with the sham lesioned control group. Following DsRed pAFSCs transplantation into striatum, apomorphine-induced rotations were significantly less than the PD model control and sham grafted control group that only received PBS injection 2 weeks after transplantation (p < 0.01). Moreover, the rotations number of the DsRed pAFSCs transplanted group were reduced by about 40% and showed very significantly lower than the other two groups 4 weeks after transplantation (p < 0.001). TH positive dopaminergic neurons and fibers could be observed in the graft side by immunohistochemistry of the brain sections and the density of ipsilateral TH positive fibers were significantly increased after 4 weeks following transplantation of DsRed pAFSCs (p < 0.001). These results suggested that transplantation of DsRed pAFSCs could significantly alleviate the asymmetric rotational behavior of the PD rats and prevent further deterioration. Furthermore, we tracked the grafted cells by immunofluorescence staining of the brain sections at 24, 48, 72 hours and 4 weeks after cell transplantation in order to figure out the possible therapeutic mechanism. We found that the transplanted DsRed pAFSCs stayed around the graft site in striatum but had not yet started to differentiate into neural lineage at 24, 48 and 72 hours after transplantation. Until 4 weeks after transplantation, the xenografted cells could survive and differentiate into dopaminergic neurons though most of the grafts become cellular debris that left in the graft site. In conclusion, the results show that DsRed pAFSCs could differentiate into dopaminergic neuron-like cells in vitro under the specific culture condition, and when transplanted in vivo, DsRed pAFSCs could not only survive and differentiate into dopaminergic neurons but also significantly promote the functional behavioral recovery of the PD rats 4 weeks after transplantation. Hence, AFSCs might be a promising cell source for clinical cell therapy development of Parkinson''s disease.
Subjects
Amniotic fluid stem cells
Stem cell-based therapy
Regenerative medicine
DsRed transgenic pigs
Type
thesis
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