Long-Term Efficacy And Safety Of VEGF-Expressing Human Umbilical Cord Mesenchymal Stem Cells In Hemiparkinsonian Models

Part Ⅰ Stereotaxical infusion of rotenone: a reliable rodent model forParkinson’s DiseaseObjective A clinically-related animal model of PD may enable the elucidation of theetiology of the disease and assist the development of medications. However, none ofthe current neurotoxin-based models recapitulates the main clinical features of thedisease or the pathological hallmarks, such as DA neuron specificity of degenerationand Lewy body formation, which limits the use of these models in PD research. Theaim of this study is to develop a parkinsonian animal model which is reliable andeasily accessible, clinically-related and better mimics the clinical, pathological andpathophysiological features of idiopathic PD.Methods We developed rat models by stereotaxically (ST) infusing small doses (3,6,12μg in1μl DMSO) of the mitochondrial complex-I inhibitor, rotenone, into twobrain sites: the right VTA and the SNc. For the SYS infusion, and by subcutaneouslyinjecting rotenone (SYS, dissolved in sunflower oil,2mg/kg/d) into the back of therats daily. Four weeks after the infusion, the behavioral profiles (apomorphine,APO-induction rotations), biogenic amine levels in the striatum, oxidative stresslevels 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/SNCAexpression and the ultrastructural changes in SNc.Results All the ST model rats showed typical behavioral features of PD, such as backhunching and stiffness, face-washing behaviors, bradykinesia, or hypokinesia. Afterrecovery from anesthesia, spontaneous rotations to the opposite side of the infusionsite were observed in all ST rats. Moreover, APO-induced rotations progressedgradually until the24thweek. Immunostaining data showed that SNCA expressionwas increased while the number of TH-positive neuron was decreased, both in adose-dependent fashion. SYS model reproduced the typical behaviors of PD but wasassociated with peripheral toxicity. HPLC analysis indicated rotenone depleted DAbut not5-HT at the beginning in the low-dose groups. Spectrophotometric studydemonstrated that the glutathione (GSH) and superoxide dismutase (SOD) activitieswere lowered, and the generation of malondialdehyde (MDA) was increased in the midbrain treated with rotenone. In addition, the infused SNc developed characteristicultrastructural changes including mitochondrial swelling, mitochondrial crest fracture,mitochondrial vacuolar degeneration, dilated and broken rough endoplasmic reticulaand perinuclear space augmentation.Conclusions We successfully developed rat PD models by ST and SYSadministration of rotenone. The SYS model can reproduce the typical behaviors of PDbut is associated with limitations such as peripheral toxicity. Unlike the mostcommonly used6-OHDA and MPTP models, our ST model can reproduce slow andspecific nigrostriatal DA degeneration in association with typical behavioral featuresof PD and the progression in APO-induced rotational behavior until the24thweek.Moreover, rotenone depleted DA but not5-HT indicating selectively DA degeneration.This rotenone-based ST model thus recapitulates the slow and specific loss of DAneurons and better mimics the clinical features of idiopathic PD, representing areliable and more clinically-related model for PD. Part Ⅱ VEGF-Expressing Human Umbilical Cord MesenchymalStem Cells, an improved therapy strategy for Parkinson’s DiseaseSection Ⅰ Isolation, Identification and Adenoviral Infection of HumanUmbilical Cord Mesenchymal Stem CellsObjective Several previous studies have focused on the treatment effectiveness ofembryonic stem cells and neural stem cells in parkinsonian animal models. However,embryonic stem cells have associated caveats including formation of teratomas andelicitation of immune rejection in transplantation and the supply of human neural stemcells is limited. In this study, we aimed to find a stem cell used for transplantation inneurodegenerative diseases such as PD which is reliable and easily accessible, legallyand ethically noncontroversial, and have low immunogenicity.Methods With the permission of the parturient (Gynecology and Obstetricsdepartment, Union Hospital, HUST), umbilical cords were aseptically collectedduring Cesarean sections. The cords were further cut into1-2mm3fragments and the explants were transferred into6-well plates containing the DMEM/F12along with20%fetal bovine serum and10ng/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, CD44andCD45were further accessed by immuocytochemistry staining. Moreover, theultrastructure of the HUMSCs was examined with a transmission electron microscope.Differentiation of HUMSCs was assessed in cultures of the fifth passage. Two weekslater, osteogenic differentiation was assessed by alizarin red staining, and intracellularlipid accumulation was visualized using Oil-Red-O staining. Differentiatedneurocytes from the HUMSCs were confirmed by immunostaining with NSE, glialfibrillary acidic protein (GFAP) or Nestin antibody. HUMSCs were infected byAd-VEGF-EGFP or Ad-EGFP at a MOI of0,10,20,50,100,200and400plaquesforming units per cell (pfu/cell). After48hours of infection, the efficiency ofinfection of adenovirus was investigated by flow cytometric detection of the EGFPmarker. The MTT colorimetric assay was employed to evaluate the effect of theVEGF gene transfection on the proliferation of HUMSCs. The coexpression of VEGFand EGFP was observed under confocal microscopy by immunofluorescent staining.VEGF secretion from HUMSCs into culture medium and striatal VEGF level weremeasured by an enzyme linked immunosorbent assay (ELISA) kit.Results Approximately1×107HUMSCs were harvested from every5cm of umbilicalcord2weeks later. The HUMSCs looked like long spindle-shaped fibroblasts, formedcolonies and became confluent. The HUMSCs were positive for CD10(24.33%),CD13(98.68%), and CD49e (99.17%), but negative for hematopoietic markers CD14,CD45and HLA-DR (MHC II), myeloid marker CD33and endothelial/hematopoieticstem cell marker CD34. By immunofluorescent staining, HUMSCs were furthervalidated to be positive for CD44and negative for CD34and CD45. In HUMSCs,high nucleo-cytoplasmic ratio, large nucleolus, the extensiveness of roughendoplasmic reticulum and generous numbers of ribosomes on the rough endoplasmicreticulum were observed using transmission electron microscopy. Adipogenicdifferentiation was identified by the accumulation of lipid-rich vacuoles within thecells, which were further visualized by the oil red O staining. And calciumdepositions were observed by the alizarin red staining. Two weeks after neurogenic,immunofluorescence results showed that some of the induced cells expressedNeuron-Specific Enolase (NSE,21.2%), GFAP (36.4%), or Nestin (48.4%). The ’efficiency of infection of adenovirus in HUMSC was91.9±2.6%for48hour-transfection at Mol400pfu/cell. MTT colorimetric assay showed that theAd-VEGF-EGFP infection promoted the proliferation and survival of HUMSCs. Theexpression of VEGF protein was visualized in Ad-VEGF-EGFP infected cells,co-expressing with EGFP. ELISA data indicated that the release of VEGF intomedium 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 mostof the problems remaining in the clinical stem cell treatment. In addition, a newmethod to isolate and culture HUMSCs, which is easy to master and with highreproducibility, provided an efficient alternative to traditional enzymaticdigestion-based methods. Moreover, HUMSCs can be differentiated into osteoblasts,adipocytes and neurocytes. Transfection of VEGF gene by adenovirus can excreteVEGF which stimulates the proliferation of HUMSCs. In summary, the capacity ofHUMSCs differentiated into neurocytes holds much promise for the treatment of anumber of neurological diseases, especially for PD. Section Ⅱ VEGF-Expressing Human Umbilical Cord Mesenchymal Stem Cellstransplatation for rotenone parkinsonian animal modelObjective The umbilical cord provides a rich source of primitive mesenchymal stemcells (HUMSCs), which have the potential for transplantation-based treatments for PD.Our pervious study indicated that adenovirus-associated virus (AAV)-mediatedintrastriatal delivery of human vascular endothelial growth factor165(VEGF)conferred molecular protection to the DA system. Since VEGF and HUMSCs eachdisplayed limited neuroprotection, here we investigated whether HUMSCs combinedwith VEGF expression could offer a more effective neurorestorative andneuroregenerative therapy for PD.Methods First of all, we had constructed the replication-deficient adenovirus vectorcontaining the cDNA for VEGF and enhanced green fluorescent protein (Ad-VEGF-EGFP). HUMSCs were modified by adenovirus-mediated VEGF genetransferring for48hours, and subsequently transplanted into rotenone-lesioned striataof hemiparkinsonian rats. Four weeks after surgery, parkinsonian rodents weredivided randomly into3groups: the saline group (10μl saline alone without donorcells; n=12), the Ad-EGFP group (implanted1.0×106Ad-EGFP infected HUMSCs;n=10) and the Ad-VEGF-EGFP group (implanted1.0×106Ad-VEGF-EGFP infectedHUMSCs; n=10). The treatment efficacy study was carried out by the APO-inducedrotational behaviors and TH immunoreactivity in the striata and SNc; thedifferentiated fate of HUMSCs was assessed by the immunostaining of Nestin (neuralstem cell marker), NSE (neuronal marker), GFAP (astrocyte marker) and TyrosineHydroxylase (TH, DA marker); the safety study was measured by HE staining of thewhole striatum region.Results The efficiency of infection of adenovirus in HUMSCs was91.9±2.6%for48hour-transfection at Mol400pfu/cell. MTT colorimetric assay showed that theAd-VEGF-EGFP infection did not cause any side effects, but rather promotedHUMSCs proliferation and survival. APO-induced rotations showed the Ad-EGFPgroup decreased rotations by53.21%, whereas the Ad-VEGF-EGFP group decreasedrotations by72.44%, compared to the saline group. Animals with HUMSCstransplantation showed significantly more TH-stained cells in the lesioned SNc(Ad-EGFP group,106.42%; Ad-VEGF-EGFP group,233.16%) and stronger THimmunoreactivity in the lesioned striata (Ad-EGFP group,301.24%; Ad-VEGF-EGFPgroup,648.75%) than those in the saline group. Compared to the Ad-EGFP group,more Nestin (by21.64%more), NSE (by18.80%more), TH (by30.27%more) andVEGF (by92.03%more) positive cells, but fewer GFAP (by36.92%less) positivecells were observed in the Ad-VEGF-EGFP group. The striatal VEGF level was49.58%and255.46%higher in Ad-EGFP group and Ad-VEGF-EGFP group than thatin Saline group.Conclusion In summary, this study presents that VEGF-expressing HUMSCs make asignificant behavioral improvement in a chronic PD model, which is more significantthan HUMSCs transplantation alone and corresponds to TH immunoreactivity in thestriata and SNc. Importantly, VEGF expression enhances the neuroprotective effectsby 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. Part Ⅲ Long-term efficacy and safety of human umbilical cordmesenchymal 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 unresolvedquestions and problems regarding the safety, feasibility and the long-term efficacy ofthis approach. In this study, we investigated migration, therapeutic, tumorigenesis andepileptogenic effects of HUMSCs12months after transplantation to rotenone-inducedhemiparkinsonian rats.Methods HUMSCs were labeled by DiI (10μM) for30minutes at37oC and thenused for transplantation. Rotenone-induced parkinsonian rodent models were dividedrandomly into two groups4weeks after surgery: Saline group,10μl saline alone(without donor cells, n=12); HUMSCs group,10μl saline containing1.0×106DiI-labeled HUMSCs (n=18). After a period of12months, the migration study wasdetected by monitoring the red flourescence of DiI from the continuous frozen sectionof brain tissue via a confocal microscopy; the efficacy study was carried out by theAPO-induced rotational behavior and TH immunoreactivity in the striatum and SNc;the differentiated fate of HUMSCs was assessed by the immunohistochemical stainingof 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 CD50andCD97value of pentetrazole.Results After co-culture with DiI for30minutes,99.8%±0.1%of HUMSCs werelabeled. DiI-labeled HUMSCs majorly distributed in the lesioned striatum, and partsof the HUMSCs migrated to the lesioned SNc and VTA. HUMSCs even migrated tothe contralateral cerebral hemisphere through the corpus callosum.12months after transplantation, HUMSCs group decreased rotations by67.48%, reduced SNc THimmunoreactivity by56.95%and striatum immunoreactivity by48.40%. Humanoriginal Nestin, NSE, GFAP and TH were observed in HUMSCs group. Theimmunofluorescence staining showed DiI-labeled HUMSCs were migrated to SNcand differentiated into TH-positive cells in SN. The expression levels of Bcl-2andP53in the grafted striata were up-regulated by281%and200%, respectively, ascompared to ungrafted striata. PCNA, β-catenin, C-myc and NF-κB expressions wereno difference between HUMSCs implanted and un-implanted striatum. HE staining ofthe striata, especially besides the needle track and the cortex, no tumor-like structureswas detected. The CD50and CD97of PTZ showed no significant difference betweenHUMSCs 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. Furtherconfirmation is required for the efficacy, safety and underlying mechanism oflong-term HUMSCs transplanted to non-human primates.