Long-term Efficacy And Safety Of VEGF-expressing Human Umbilical Cord Mesenchymal Stem Cells In Rotenone-induced Hemiparkinsonian Rats

PartⅠStereotaxical infusion of rotenone:a reliable rodent model for Parkinson’s DiseaseObjective A clinically-related animal model of PD may enable the elucidation of the etiology of the disease and assist the development of medications. However, none of the current neurotoxin-based models recapitulates the main clinical features of the disease or the pathological hallmarks, such as DA neuron specificity of degeneration and Lewy body formation, which limits the use of these models in PD research. The aim of this study is to develop a parkinsonian animal model which is reliable and easily accessible, clinically-related and better mimics the clinical, pathological and pathophysiological features of idiopathic PD.Methods We developed rat models by stereotaxically (ST) infusing small doses (3,6, 12μg in 1μl DMSO) of the mitochondrial complex-Ⅰinhibitor, rotenone, into two brain sites:the right VTA and the SNc. For the SYS infusion, and by subcutaneously injecting rotenone (SYS, dissolved in sunflower oil,2 mg/kg/d) into the back of the rats daily. Four weeks after the infusion, the behavioral profiles (apomorphine, APO-induction rotations), biogenic amine levels in the striatum, oxidative stress levels in the midbrain, and TH immunoreactivities in the SNc and VTA were assessed. Importantly, the ST model was further validated by the detection of TH/SNCA expression and the ultrastructural changes in SNc.Results All the ST model rats showed typical behavioral features of PD, such as back hunching and stiffness, face-washing behaviors, bradykinesia, or hypokinesia. After recovery from anesthesia, spontaneous rotations to the opposite side of the infusion site were observed in all ST rats. Moreover, APO-induced rotations progressed gradually until the 24th week. Immunostaining data showed that SNCA expression was increased while the number of TH-positive neuron was decreased, both in a dose-dependent fashion. SYS model reproduced the typical behaviors of PD but was associated with peripheral toxicity. HPLC analysis indicated rotenone depleted DA but not 5-HT at the beginning in the low-dose groups. Spectrophotometric study demonstrated that the glutathione (GSH) and superoxide dismutase (SOD) activities were lowered, and the generation of malondialdehyde (MDA) was increased in the midbrain treated with rotenone. In addition, the infused SNc developed characteristic ultrastructural changes including mitochondrial swelling, mitochondrial crest fracture, mitochondrial vacuolar degeneration, dilated and broken rough endoplasmic reticula and perinuclear space augmentation.Conclusions We successfully developed rat PD models by ST and SYS administration of rotenone. The SYS model can reproduce the typical behaviors of PD but is associated with limitations such as peripheral toxicity. Unlike the most commonly used 6-OHDA and MPTP models, our ST model can reproduce slow and specific nigrostriatal DA degeneration in association with typical behavioral features of PD and the progression in APO-induced rotational behavior until the 24th week. Moreover, rotenone depleted DA but not 5-HT indicating selectively DA degeneration. This rotenone-based ST model thus recapitulates the slow and specific loss of DA neurons and better mimics the clinical features of idiopathic PD, representing a reliable and more clinically-related model for PD.PartⅡVEGF- Expressing Human Umbilical Cord Mesenchymal Stem Cells, an improved therapy strategy for Parkinson’s Disease SectionⅠIsolation, Identification and Adenoviral Infection of Human Umbilical Cord Mesenchymal Stem CellsObjective Several previous studies have focused on the treatment effectiveness of embryonic stem cells and neural stem cells in parkinsonian animal models. However, embryonic stem cells have associated caveats including formation of teratomas and elicitation of immune rejection in transplantation and the supply of human neural stem cells is limited. In this study, we aimed to find a stem cell used for transplantation in neurodegenerative diseases such as PD which is reliable and easily accessible, legally and ethically noncontroversial, and have low immunogenicity.Methods With the permission of the parturient (Gynecology and Obstetrics department, Union Hospital, HUST), umbilical cords were aseptically collected during Cesarean sections. The cords were further cut into 1-2 mm3 fragments and the explants were transferred into 6-well plates containing the DMEM/F12 along with 20% fetal bovine serum and 10 ng/ml basic fibroblast growth factor. Immunophenotyping of the HUMSCs CD10, CD13, CD14, CD33, CD34, CD44, CD45, CD49E and HLA-DR were accessed by flow cytometry. CD34, CD44 and CD45 were further accessed by immuocytochemistry staining. Moreover, the ultrastructure of the HUMSCs was examined with a transmission electron microscope. Differentiation of HUMSCs was assessed in cultures of the fifth passage. Two weeks later, osteogenic differentiation was assessed by alizarin red staining, and intracellular lipid accumulation was visualized using Oil-Red-O staining. Differentiated neurocytes from the HUMSCs were confirmed by immunostaining with NSE, glial fibrillary acidic protein (GFAP) or Nestin antibody. HUMSCs were infected by Ad-VEGF-EGFP or Ad-EGFP at a MOI of 0,10,20,50,100,200 and 400 plaques forming units per cell (pfu/cell). After 48 hours of infection, the efficiency of infection of adenovirus was investigated by flow cytometric detection of the EGFP marker. The MTT colorimetric assay was employed to evaluate the effect of the VEGF gene transfection on the proliferation of HUMSCs. The coexpression of VEGF and EGFP was observed under confocal microscopy by immunofluorescent staining. VEGF secretion from HUMSCs into culture medium and striatal VEGF level were measured by an enzyme linked immunosorbent assay (ELISA) kit.Results Approximately 1×107 HUMSCs were harvested from every 5 cm of umbilical cord 2 weeks later. The HUMSCs looked like long spindle-shaped fibroblasts, formed colonies and became confluent. The HUMSCs were positive for CD10 (24.33%), CD13 (98.68%), and CD49e (99.17%), but negative for hematopoietic markers CD14, CD45 and HLA-DR (MHCⅡ), myeloid marker CD33 and endothelial/hematopoietic stem cell marker CD34. By immunofluorescent staining, HUMSCs were further validated to be positive for CD44 and negative for CD34 and CD45. In HUMSCs, high nucleo-cytoplasmic ratio, large nucleolus, the extensiveness of rough endoplasmic reticulum and generous numbers of ribosomes on the rough endoplasmic reticulum were observed using transmission electron microscopy. Adipogenic differentiation was identified by the accumulation of lipid-rich vacuoles within the cells, which were further visualized by the oil red O staining. And calcium depositions were observed by the alizarin red staining. Two weeks after neurogenic, immunofluorescence results showed that some of the induced cells expressed Neuron-Specific Enolase (NSE,21.2%), GFAP (36.4%), or Nestin (48.4%). The efficiency of infection of adenovirus in HUMSC was 91.9±2.6% for 48 hour-transfection at Mol 400 pfu/cell. MTT colorimetric assay showed that the Ad-VEGF-EGFP infection promoted the proliferation and survival of HUMSCs. The expression of VEGF protein was visualized in Ad-VEGF-EGFP infected cells, co-expressing with EGFP. ELISA data indicated that the release of VEGF into medium from Ad-VEGF-EGFP infected HUMSCs was in a MOI-dependent manner.Conclusions HUMSCs are reliable, easily accessible, readily expanded in culture, legally and ethically noncontroversial, and have low immunogenicity, resolving most of the problems remaining in the clinical stem cell treatment. In addition, a new method to isolate and culture HUMSCs, which is easy to master and with high reproducibility, provided an efficient alternative to traditional enzymatic digestion-based methods. Moreover, HUMSCs can be differentiated into osteoblasts, adipocytes and neurocytes. Transfection of VEGF gene by adenovirus can excrete VEGF which stimulates the proliferation of HUMSCs. In summary, the capacity of HUMSCs differentiated into neurocytes holds much promise for the treatment of a number of neurological diseases, especially for PD.SectionⅡVEGF-Expressing Human Umbilical Cord Mesenchymal Stem Cells transplatation for rotenone parkinsonian animal modelObjective The umbilical cord provides a rich source of primitive mesenchymal stem cells (HUMSCs), which have the potential for transplantation-based treatments for PD. Our pervious study indicated that adenovirus-associated virus (AAV)-mediated intrastriatal delivery of human vascular endothelial growth factor 165 (VEGF) conferred molecular protection to the DA system. Since VEGF and HUMSCs each displayed limited neuroprotection, here we investigated whether HUMSCs combined with VEGF expression could offer a more effective neurorestorative and neuroregenerative therapy for PD.Methods First of all, we had constructed the replication-deficient adenovirus vector containing the cDNA for VEGF and enhanced green fluorescent protein (Ad-VEGF-EGFP). HUMSCs were modified by adenovirus-mediated VEGF gene transferring for 48 hours, and subsequently transplanted into rotenone-lesioned striata of hemiparkinsonian rats. Four weeks after surgery, parkinsonian rodents were divided randomly into 3 groups:the saline group (10μl saline alone without donor cells; n= 12), the Ad-EGFP group (implanted 1.0×106 Ad-EGFP infected HUMSCs; n=10) and the Ad-VEGF-EGFP group (implanted 1.0×106 Ad-VEGF-EGFP infected HUMSCs; n=10). The treatment efficacy study was carried out by the APO-induced rotational behaviors and TH immunoreactivity in the striata and SNc; the differentiated fate of HUMSCs was assessed by the immunostaining of Nestin (neural stem cell marker), NSE (neuronal marker), GFAP (astrocyte marker) and Tyrosine Hydroxylase (TH, DA marker); the safety study was measured by HE staining of the whole striatum region.Results The efficiency of infection of adenovirus in HUMSCs was 91.9±2.6% for 48 hour-transfection at Mol 400 pfu/cell. MTT colorimetric assay showed that the Ad-VEGF-EGFP infection did not cause any side effects, but rather promoted HUMSCs proliferation and survival. APO-induced rotations showed the Ad-EGFP group decreased rotations by 53.21%, whereas the Ad-VEGF-EGFP group decreased rotations by 72.44%, compared to the saline group. Animals with HUMSCs transplantation showed significantly more TH-stained cells in the lesioned SNc (Ad-EGFP group,106.42%; Ad-VEGF-EGFP group,233.16%) and stronger TH immunoreactivity in the lesioned striata (Ad-EGFP group,301.24%; Ad-VEGF-EGFP group,648.75%) than those in the saline group. Compared to the Ad-EGFP group, more Nestin (by 21.64% more), NSE (by 18.80% more), TH (by 30.27% more) and VEGF (by 92.03% more) positive cells, but fewer GFAP (by 36.92% less) positive cells were observed in the Ad-VEGF-EGFP group. The striatal VEGF level was 49.58% and 255.46% higher in Ad-EGFP group and Ad-VEGF-EGFP group than that in Saline group.Conclusion In summary, this study presents that VEGF-expressing HUMSCs make a significant behavioral improvement in a chronic PD model, which is more significant than HUMSCs transplantation alone and corresponds to TH immunoreactivity in the striata and SNc. Importantly, VEGF expression enhances the neuroprotective effects by promoting DA neuron-orientated differentiation of the HUMSCs. Therefore, transplantation of VEGF-expressing HUMSCs represents a novel stem cell engineering-based therapeutic approach for the treatment of PD. PartⅢLong-term efficacy and safety of human umbilical cord mesenchymal stem cells in rotenone-induced hemiparkinsonian ratsObjective Several studies have demonstrated functional improvements, neuroprotective and neuroregenerative effects after mesenchymal stem cells (MSCs) transplantation to parkinsonian animal models. However, there are many unresolved questions and problems regarding the safety, feasibility and the long-term efficacy of this approach. In this study, we investigated migration, therapeutic, tumorigenesis and epileptogenic effects of HUMSCs 12 months after transplantation to rotenone-induced hemiparkinsonian rats.Methods HUMSCs were labeled by DiI (10μM) for 30 minutes at 37℃and then used for transplantation. Rotenone-induced parkinsonian rodent models were divided randomly into two groups 4 weeks after surgery:Saline group,10μl saline alone (without donor cells, n=12); HUMSCs group,10μl saline containing 1.0×106 Dil-labeled HUMSCs (n=18). After a period of 12 months, the migration study was detected by monitoring the red flourescence of Dil from the continuous frozen section of brain tissue via a confocal microscopy; the efficacy study was carried out by the APO-induced rotational behavior and TH immunoreactivity in the striatum and SNc; the differentiated fate of HUMSCs was assessed by the immunohistochemical staining of Nestin (neural stem cell marker), NSE (neuronal marker), GFAP (astrocyte marker) and TH (DA marker); the safety study was measured by the p53, C-myc, H-RAS,β-catenin, Bcl-2, PCNA and NF-κB immunofluorescent staining, and the CD50 and CD97 value of pentetrazole.Results After co-culture with DiI for 30 minutes,99.8%±0.1% of HUMSCs were labeled. DiI-labeled HUMSCs majorly distributed in the lesioned striatum, and parts of the HUMSCs migrated to the lesioned SNc and VTA. HUMSCs even migrated to the contralateral cerebral hemisphere through the corpus callosum.12 months after transplantation, HUMSCs group decreased rotations by 67.48%, reduced SNc TH immunoreactivity by 56.95% and striatum immunoreactivity by 48.40%. Human original Nestin, NSE, GFAP and TH were observed in HUMSCs group. The immunofluorescence staining showed DiI-labeled HUMSCs were migrated to SNc and differentiated into TH-positive cells in SN. The expression levels of Bcl-2 and P53 in the grafted striata were up-regulated by 281% and 200%, respectively, as compared to ungrafted striata. PCNA,β-catenin, C-myc and NF-κB expressions were no difference between HUMSCs implanted and un-implanted striatum. HE staining of the striata, especially besides the needle track and the cortex, no tumor-like structures was detected. The CD50 and CD97 of PTZ showed no significant difference between HUMSCs treated and untreated groups.Conclusion The results of this long-term study presents that HUMSCs transplantation, one of the most potential treatments for PD, is an effective and safe approach. Further confirmation is required for the efficacy, safety and underlying mechanism of long-term HUMSCs transplanted to non-human primates.