VEGF-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for Parkinson’s disease


The umbilical cord provides a rich source of primitive mesenchymal stem cells (human umbilical cord mesenchymal stem cells (HUMSCs)), which have the potential for transplantation-based treatments of Parkinson's Disease (PD). Our pervious study indicated that adenovirus-associated virus-mediated intrastriatal delivery of human vascular endothelial growth factor 165 (VEGF 165) conferred molecular protection to the dopaminergic system. As both VEGF and HUMSCs displayed limited neuroprotection, in this study we investigated whether HUMSCs combined with VEGF expression could offer enhanced neuroprotection. HUMSCs were modified by adenovirus-mediated VEGF gene transfer, and subsequently transplanted into rotenone-lesioned striatum of hemiparkinsonian rats. As a result, HUMSCs differentiated into dopaminergic neuron-like cells on the basis of neuron-specific enolase (NSE) (neuronal marker), glial fibrillary acidic protein (GFAP) (astrocyte marker), nestin (neural stem cell marker) and tyrosine hydroxylase (TH) (dopaminergic marker) expression. Further, VEGF expression significantly enhanced the dopaminergic differentiation of HUMSCs in vivo. HUMSC transplantation ameliorated apomorphine-evoked rotations and reduced the loss of dopaminergic neurons in the lesioned substantia nigra (SNc), which was enhanced significantly by VEGF expression in HUMSCs. These findings present the suitability of HUMSC as a vector for gene therapy and suggest that stem cell engineering with VEGF may improve the transplantation strategy for the treatment of PD.


Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting 107–187 people per 100 000 population.1 In China, PD affects 1.7% of the population aged 65 years or older.2 PD patients suffer from rest, rigidity, bradykinesia and postural abnormalities. Current pharmacotherapies only provide temporary relief from early symptoms without stopping disease progression, and the beneficial effects wear off with long-term use. Moreover, non-motor symptoms, including cognitive, autonomic and psychiatric symptoms, are not sufficiently ameliorated by current therapies. Although there are many promising candidate neuroprotective agents based on pathological and laboratory studies, it is not yet possible to determine whether any drug has a disease-modifying effect on PD.3, 4 Thus, any treatment that can reverse or stop disease progression is critically needed.

At present, replacement of the progressively degenerated nigrostriatal dopaminergic (DA) neurons through transplantation is considered to have the most potential as a therapy.3, 5 It is acknowledged that stem cells used for transplantation in neurodegenerative diseases such as PD should be reliable and easily accessible, legally and ethically non-controversial, and have low immunogenicity.6 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 teratomas7 and elicitation of immune rejection in transplantation,3 and the supply of human neural stem cells is limited. Human umbilical cord mesenchymal stem cells (HUMSCs) seem to meet all these requirements and have already manifested therapeutic potential in a PD model.8 However, information on the differentiation fate of HUMSCs after transplantation is sparse, and converting these cells into a stable neuron phenotype remains unexplored. Furthermore, safety is still the first factor to be considered during therapeutic use of stem cells, especially in terms of the stem-cell tumorigenesis problem.9, 10 Thus, we still need to assess the safety of HUMSC treatment for which there is a lack of published evidence.

On the other hand, modulation of neurotrophic factors may reduce dopaminergic vulnerability. The loss of neurotrophic factors, such as vascular endothelial growth factor (VEGF) generated by the vascular system, has been suggested as one of the causes of neurodegenerative disease.11 VEGF was proven to promote the growth and survival of DA neurons in ventral mesencephalon explants12, 13 and animal models for PD.14, 15 Our previous work indicated that relatively low-level expression of VEGF mediated by adeno-associated virus in the striatum protected the DA neurons of parkinsonian rats.16 In this study, we attempt to develop a more effective neurorestorative and neuroregenerative therapy on the basis of a combination of VEGF expression and HUMSC therapy.

In this study, a new method to isolate and culture HUMSCs, which is easy to master and of high reproducibility, provided an efficient alternative to traditional enzymatic digestion-based methods. In addition, safety, reliability and differentiation of HUMSCs after transplantation were assessed.


Characterization of HUMSCs

Distinctive morphology and surface antigens of HUMSCs. After the initial 9–12 days of primary culture, a small population of single HUMSCs adhered to the plastic surface (Figure 1a), were similar in shape to long spindle-shaped fibroblasts, subsequently formed colonies and became confluent (Figures 1a–d). After being cultured for another 2 weeks, 1 × 107 HUMSCs were harvested from every 5 cms of umbilical cord, and the number of HUMSCs doubled every 2 or 3 days (data not shown). 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 (MHC II) (Figure 1i), myeloid marker CD33 and endothelial/hematopoietic stem cell marker CD34. By immunofluorescent staining, HUMSCs were further validated to be positive for CD44 (Figure 1g) and negative for CD34 and CD45 (Figure 1h), indicating that HUMSCs are mesenchymal stem cells and not hematopoietic or endothelial cells. In HUMSCs, the extensiveness of rough endoplasmic reticulum and generous numbers of ribosomes on the rough endoplasmic reticulum were observed using transmission electron microscopy. In addition, HUMSCs had a high nucleo-cytoplasmic ratio and a large nucleolus (Figures 1e and f), indicating that HUMSC is a kind of cell with vigorous metabolism and still in an early stage.

Figure 1

Typical morphological appearance and immunophenotyping of HUMSCs. (ac) Each panel shows the growth of HUMSCs after 9, 15 and 18 days in culture. (d) Indicates the morphological appearance of HUMSCs by HE staining, which is a good way to show the long spindle-shaped and unique vortex shape of HUMSCs. (e and f) Demonstrate the ultrastructure of HUMSCs, which indicates the extension of rough endoplasmic reticulum surrounded by a large number of ribosomes, and the large nucleolus with a high nucleo-cytoplasmic ratio. (i) Analysis on FACScalibur shows HUMSCs positive for CD10 (24.33%), CD13 (98.68%) and CD49e (99.17%), but negative for CD14, CD33, CD34, CD45 and HLA-DR. Immunofluorescent staining further validated that HUMSCs were positive for CD44 (g) and negative for CD34 and CD45 (h).

Neuronal differentiation of HUMSCs. When HUMSCs were cultured under conditions favorable for adipogenic, neurogenic or osteogenic differentiation, the expanded cells were highly differentiated and the appearance of lineage-specific cell types was easily distinguished under an inverted phase contrast microscope (Supplementary Figure S1a, adipogenic differentiation for 2 weeks; Supplementary Figure S1d and Supplementary Figure S1e, neurogenic differentiations for 2 weeks). Adipogenic differentiation was identified by the accumulation of lipid-rich vacuoles within the cells (Supplementary Figure S1a), which were further visualized by the Oil-Red-O staining at 3 weeks (Supplementary Figure S1b). After 3 weeks of osteogenic supplementation, calcium depositions were observed by Alizarin red staining (Supplementary Figure S1c). Two weeks after induction of the neurogenic medium, immunofluorescence results showed that some of the induced cells expressed neuron-specific enolase (NSE, 21.2%, Supplementary Figure S1i and Figure 1), glial fibrillary acidic protein (GFAP, 36.4%, Supplementary Figure S1h and k) or nestin (48.4%, Supplementary Figure S1g and j), suggesting that HUMSCs could be differentiated into neuron-, glial- and neural stem cell-like cells.

Effective infection of HUMSCs by adenovirus

The efficiency of infection of adenovirus in HUMSC was 91.9±2.6% for 48 h transfection at multiplicity of infection (MOI) 400 plaque-forming units per cell (Figure 2i), and the efficiency increased with increasing MOI as indicated by fluorescence microscopy (Figures 2a–f). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay showed that the Ad-VEGF-EGFP infection did not have any negative effects on HUMSCs, but rather promoted their proliferation and survival (Figure 2g). The expression of VEGF protein was visualized in Ad-VEGF-EGFP-infected cells (Figure 2j), coexpressing with EGFP (Figures 2j–m).

Figure 2

Transient expression of VEGF and EGFP in HUMSCs. The efficiency of infection of Ad-VEGF-EGFP in HUMSCs increased with increasing MOI, as indicated by fluorescence microscopy (af), and the efficiency of infection was 91.9±2.6% at MOI 400 pfu per cell at 48 h of infection (i). MTT assay showed that MOI 400 pfu of cell promoted HUMSC proliferation without any side effects (g), which could be attributed to the VEGF secretion into the medium from Ad-VEGF-EGFP-infected HUMSCs (h). Immunofluorescence with confocal microscopy was used to observe the coexpression of EGFP and VEGF (VEGF 165 antibody and CY3-conjugated donkey antimouse IgG), and nuclear matter was stained by Hoechst33258 (jm). Statistics consisted of one-way ANOVA; *P<0.05, compared with the control group.

Enzyme-linked immunosorbent assay data indicated that the release of VEGF into medium from Ad-VEGF-EGFP-infected HUMSCs was in a MOI-dependent manner (Figure 2h). The VEGF in the medium of Ad-EGFP-infected HUMSCs was 0.20±0.04 ng ml−1 48 h after adenoviruses infection, whereas it was 9.78±0.82 ng ml−1 in the medium of Ad-VEGF-EGFP (400 MOI)-treated HUMSCs. Striatal VEGF levels on an average were 17.07±2.50, 16.53±1.20, 24.72±1.68 and 58.74±10.38 ng per mg of wet tissue in the contralateral striatum of each group, in the lesioned side of the saline group, Ad-EGFP group and Ad-VEGF-EGFP group, respectively, 12 weeks after transplantation. Striatal VEGF levels were 49.58 and 255.46% higher in the Ad-EGFP and Ad-VEGF-EGFP groups compared with that in the saline group (Figure 4am). Semi-quantitative immunohistochemical analysis of striatal VEGF expression (Figure 4am, Ad-VEGF-EGFP group: Figure 4y–aa; Ad-EGFP group: Figures 4ab–ad; saline group: Figure 4ak) consistently indicated that the striatal VEGF expression levels were 48.45 and 312.18% higher than that in saline group.

Effect of transplantation on parkinsonian rats

HUMSCs and VEGF-expressing HUMSCs improved apomorphine-induced rotations in parkinsonian rats. A unilateral rotenone lesion of the VTA and the SNc induced turning behavior as expected in all animals after apomorphine treatment.17 In the saline group, a progressive degradation in performance was observed following rotenone infusion. However, the lesioned rats receiving a HUMSC transplant showed a significant decrease in the number of rotations (Figure 3a). Our data showed that at the twelfth posttransplant week, the Ad-EGFP group decreased rotations by 53.21%, whereas the Ad-VEGF-EGFP group decreased rotations by 72.44%, compared with the saline group. In some Ad-VEGF-EGFP group animals, the rotations were decreased by more than 90%.

Figure 3

Effectiveness and safety of HUMSC transplantation. (a) Shows apomorphine-induced rotations of three groups every 2 weeks after transplantation, and both HUMSC groups showed a significant difference compared with the saline group (P<0.05). At 6, 8, 10 and 12 weeks after transplantation, there was a significant difference between the Ad-EGFP group and the Ad-VEGF-EGFP group (statistics consisted of one-way ANOVA; *P<0.05 compared with the Ad-EGFP group). (dk) Show TH immunoreactivity (rabbit polyclonal antibody and FITC-conjugated goat antirabbit IgG) in the striatum and the SNc; more TH-stained cells (c) in the SNc and higher TH immunoreactivity in the striatum (b) were observed in HUMSC-treated animals compared with the saline group animals, and significant difference was observed between the Ad-EGFP group and Ad-VEGF-EGFP group (*P<0.05 compared with the saline group, #P<0.05 compared with the Ad-EGFP group). (lo) Indicate coexpression of TH (rabbit polyclonal antibody and CY3-conjugated goat antirabbit IgG) and EGFP in the grafted SNc. No tumor-like formations were detected in the transplanted striatum. (pr) Show the transplanted striatum and (s) shows the contralateral striatum.

HUMSCs and VEGF-expressing HUMSCs prevent tyrosine hydroxylase (TH) immunoreactivity decrease in the striatum and SNc. Asymmetric staining of TH in the striatum and the SNc showed evidence of the toxic effect of rotenone on DA neurons and the nigrostriatal tract on the side ipsilateral to the injection site (Figures 3d, e, h and i). The quantification study indicated that rotenone induced the degeneration of 73.13% of TH-positive cells in the lesioned SNc and destroyed 89.61% of the nerve terminals in the lesioned striatum at 16 weeks after surgery. However, the animals with HUMSC transplantation showed significantly more TH-stained cells in the lesioned SNc than those in the saline group (Figure 3j: Ad-EGFP group, 106.42%; Figure 3k: Ad-VEGF-EGFP group, 233.16%). Consequently, a correlation was observed between behavioral recovery and TH immunoreactivity in the striatum (Figure 3f: Ad-EGFP group, 301.24%; Figure 3g: Ad-VEGF-EGFP group, 648.75%). A significant difference in striatal (Figure 3b) or nigral TH immunoreactivity (Figure 3c) was observed between the Ad-EGFP group and the Ad-VEGF-EGFP group. Furthermore, EGFP coexpression with TH in the SNc was observed using confocal microscopy (Figures 3l–o), indicating that HUMSCs migrated from the striatum to the SNc and differentiated into TH-positive cells in the lesioned SNc. Quantitative analysis indicated that Ad-VEGF-EGFP group animals displaying behavioral improvement had 150–2000 TH- and EGFP-positive cells in SNc, suggesting that HUMSCs migrated to the SNc and differentiated into TH-positive cells.

HUMSC treatment as a safe approach. After transplantation, no immunosuppressant was administrated to the animals and none of the 20 transplanted animals showed immunological rejection in the total period of 12 weeks. On the basis of an hematoxylin and eosin (HE) stain of the striatum (Figures 3p–s), no tumor-like formations (duct-like structures, irregular nests, malignant cell infiltration or specific ultrastructural organization) were observed, not even in proximity to the pin track.

In vivo neuronal differentiation of HUMSCs. Twelve weeks after implantation, human original Nestin-, NSE-, GFAP- and TH-positive cells were detected in the grafted striatum of Ad-EGFP and Ad-VEGF-EGFP group rats (Figures 4a–ad and Figure 4al). Compared with the Ad-EGFP group, more Nestin (Figures 4a–f, by 21.64% more)-, NSE (Figures 4g–l, by 18.80% more)-, TH (Figures 4m–r, by 30.27% more)- and VEGF (Figure 4y–ad, by 92.03% more)-positive cells, but fewer GFAP (Figure 4s–x, by 36.92% less)-positive cells were observed in the Ad-VEGF-EGFP group. In addition, hyperplasia of GFAP (rabbit polyclonal antibody, Santa Cruz, Santa Cruz, CA, USA)-positive cells proximal to the needle track was observed in the grafted striatum (Figures 4ae–ag), compared with the contralateral striatum (Figures 4ah–aj).

Figure 4

Differentiated fate of HUMSCs after transplantation. Human original Nestin (af), NSE (gl), GFAP (sx), TH (mouse monoclonal antibody, mr) and VEGF (rabbit polyclonal antibody, y–ad and ak) were visualized by double staining of EGFP and CY3-conjugated donkey antimouse IgG. (al) Shows the percentage of neuronal cells, identified by CY3-positive (CY3+) cells within the total number of EGFP-positive (EGFP+) cells in each transplanted rat at 12 weeks after transplantation. All the detected neurocyte markers showed significant differences between the Ad-EGFP and the Ad-VEGF-EGFP group. (*P<0.05, compared with the Ad-EGFP group). (am and an) show the semiquantitative analysis of striatal VEGF (rabbit polyclonal antibody, the Ad-VEGF-EGFP group: y–aa; the Ad-EGFP group: ab–ad; the saline group: ak) immunohistochemical staining and enzyme-linked immunosorbent assay of striatal VEGF (rabbit polyclonal antibody) levels in the contralateral side of each group and in the lesioned side of the saline, Ad-EGFP and Ad-VEGF-EGFP groups. (*P<0.05, compared with the contralateral side or the saline group; #P<0.05, compared with Ad-EGFP group). (ae) (Nuclear, Hoechst33258), (ag) (CY3-conjugated goat antirabbit IgG) and (af) (merged) show GFAP (rabbit polyclonal antibody)-positive cells proximal to the pin track, whereas (ah–aj) (CY3-conjugated goat antirabbit IgG) show GFAP-positive cells in the contralateral striatum.


In this study, we showed that intrastriatal infusion of either HUMSCs or VEGF-expressing HUMSCs to rotenone-induced parkinsonian rats resulted in a reduction of apomorphine-induced rotations, and a revival of TH immunoreactivity in the lesioned striatum and SNc, which indicated therapeutic effects of these cells in parkinsonian rats. Our primary finding is that VEGF protein expression in HUMSCs enhanced their treatment effectiveness, as further behavioral recovery and TH immunoreactivity protection were observed in the Ad-VEGF-EGFP group compared with the Ad-EGFP group. These data suggest that VEGF gene-modified HUMSCs confer more powerful protection for PD, and VEGF-expressing HUMSCs are an improved transplantation and gene therapy strategy for PD.

For the first time, we showed that HUMSCs were differentiated into TH-, Nestin-, NSE- and GFAP-positive cells after transplantation into a parkinsonian corpus striatum. Interestingly, VEGF promoted HUMSC differentiation into TH-, NSE- and Nestin-positive cells, but inhibited HUMSC conversion into GFAP-positive cells, which demonstrated VEGF expression enhancing the dopaminergic differentiation of HUMSCs in vivo. TH and EGFP coexpression was observed in cells of the lesioned SNc, indicating that HUMSCs could migrate from corpus striatum to the SNc and differentiate into TH-positive cells in the lesioned SNc. Importantly, none of the 20 animals under HUMSCs treatment showed immunological rejection throughout the entire period of 12 weeks, and no tumor-like formations were detected among those animals at the end of 12 weeks.

Another interesting finding was that Ad-EGFP-HUMSC transplantation significantly increased VEGF level in the striatum, as shown by enzyme-linked immunosorbent assay and VEGF immunohistochemistry staining, which was similar to a previous study that showed human bone marrow-derived mesenchymal stem cells could stimulate the expression of brain-derived neurotrophic factor.18 Moreover, the striatal VEGF level in the Ad-VEGF-EGFP group was significantly higher than that in the saline group and Ad-EGFP group. Previous studies showed the direct and indirect neuroprotective effects of VEGF.14, 15, 16, 19, 20 Directly, VEGF is neuroprotective and even induces neurogenesis of DA neurons,14, 15, 16, 21 and VEGF displayed the neuroprotective effects by activating VEGF receptor 1, VEGF receptor 2 and the neuropilin receptor.21 Other well-known functions of VEGF include angiogenesis, accentuation of vessel permeability and glial proliferation, which promote the expression of neurotrophic factors like glial cell line-derived neurotrophic factor.14, 15, 16, 21 In this study, immunofluorescence staining of GFAP proximal to the pin track showed the proliferation of GFAP-positive cells compared with contralateral striatum, the same findings as our previous study.16 These data are evidence that the activity of gliocytes and the increased glial cell line-derived neurotrophic factor levels contribute to the protective effect.16 Further, we found that VEGF not only protected DA neurons but also promoted directional differentiation of HUMSCs into TH-positive cells.

In this study, we introduced a simple, reliable and effective method of preparing HUMSCs. HUMSCs have been isolated by enzymatic digestion methods22 and by the culturing of minced tissue.23 However, the majority of subsequent studies used enzymatic digestion methods,8, 24, 25, 26 and the concentration and species of enzyme used for digestion varied dramatically among different studies. Using the method we described, HUMSCs could be isolated successfully from every umbilical cord we collected, suggesting that the culturing of minced tissue method is reliable for the isolation of HUMSCs. Moreover, this method is easy to perform. HUMSCs share many surface markers with adult MSCs. Consistent with previous reports,8, 27, 28 we found that HUMSCs are positive for CD13, CD29, CD44, CD90 and CD10 and negative for CD14, CD28, CD31, CD34, CD45, CD56 and CD133. Data from transmission electron microscopy, the ‘gold standard’ for the morphology of cells, showed the extensiveness of rough endoplasmic reticulum and generous numbers of ribosomes on the rough endoplasmic reticulum, demonstrating that HUMSC is a type of early-stage cell and is capable of rapid expansion and long-term survival. Successful adipogenic, osteogenic and neurogenic differentiation further validated the pluripotency of HUMSCs, indicating the functional characteristic of HUMSC as a kind of stem cell, as also observed for MSC in previous studies.27, 28

For the parkinsonian animal model, we used the rotenone-based stereotaxic model for the symptoms to progress gradually until 24 weeks, recapitulating the slow and specific loss of DA neurons and better mimicking the clinical features of idiopathic PD.17 Previous studies failed to provide evidence of neuronal differentiation of HUMSCs after transplantation.8, 24 In the present study, EGFP was used as a primary stem cell label. Furthermore, mouse monoclonal antibodies were used to detect the expression of specific human original Nestin, NSE, GFAP, TH and VEGF. The expression of all these markers in the striatum was significantly different between the Ad-EGFP group and the Ad-VEGF-EGFP group. For example, the percentage of human original NSE-positive cells was 80±10% in the Ad-VEGF-EGFP group, but only 61±10% in the Ad-EGFP group. These data by no means indicate that 80±10% of the HUMSCs converted to NSE-positive cells, as the adenovirus-mediated non-genetically modified cells were unable to maintain long-term expression of exogenous genes. Portions of HUMSCs did not express EGFP after transplantation for 12 weeks; namely, the total number of EGFP-labeled cells in the implanted brain was less than 1 × 106 in each rat. Thus, the data are only a good indicator to show the difference between the Ad-VEGF and Ad-VEGF-EGFP groups.17

In agreement with previous studies and on the basis of the results of this study, the mechanisms of the neuroprotective effect of the VEGF-expressing HUMSCs may include the following: (1) release of VEGF, which is neuroprotective to DA neurons, directly acting on target cells;16, 29, 30 (2) replacement of the degenerated DA neurons by HUMSCs in the SNc; (3) activity of gliocytes and the increased glial cell line-derived neurotrophic factor level;16 (4) promotion of the levels of possible neuroprotective trophic factors;31 and (5) anti-inflammatory functionality.32, 33

In summary, this study shows that VEGF-expressing HUMSCs make a significant behavioral improvement in a chronic PD model, which is more significant than HUMSC transplantation alone and corresponds to TH immunoreactivity in the striatum and SNc. Importantly, VEGF expression enhanced the neuroprotective effects by promoting dopaminergic 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.

Materials and methods

Isolation and identification of HUMSCs

Preparation of HUMSCs. The experiments described in this study were approved by the Ethical Committee of Tongji Medical College, Huazhong University of Science and Technology, China (HUST). With the permission of the parturient (Gynecology and Obstetrics department, Union Hospital, HUST), umbilical cords were aseptically collected during cesarean sections, stored at 4 °C in 0.01 M sterilized phosphate-buffered solution (PBS) with heparin (20–30 u ml−1) until further processing. To avoid endothelial cell contamination, umbilical arteries and vein were removed and the cords were cut into small pieces (0.5–1 cm). Subsequently, the cords were further cut into 1–2 mm3 fragments and the explants were transferred into six-well plates containing DMEM/F12 (Invitrogen, Carlsbad, CA, USA), along with 20% fetal bovine serum (Invitrogen) and 10 ng per ml of basic fibroblast growth factor (Peprotech, Rocky Hill, NJ, USA). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The fragments were left undisturbed for 10–12 days to allow cells to migrate from the explants. They were re-fed every 48–72 h and passaged (1:3) by trypsin (0.25%, Invitrogen) digestion until cells reached 80–90% confluency.

Immunophenotyping of HUMSCs by flow cytometry. HUMSCs were harvested by treatment with 0.25% trypsin, washed with PBS and incubated for 30 min at 4 °C in darkness with the following antibodies: CD10-FITC, CD13-PE, CD14-FITC, CD33-PE, CD34-FITC, CD45-PE, CD49e-PE and HLA-DR-FITC (Becton Dickinson, Temse, Belgium). Subsequently, the tissues were washed in PBS and fixed with an FIX solution (Becton Dickinson). The specific fluorescence of 10 000 cells was analyzed on FACScalibur (Becton Dickinson).

Immunofluorescence. HUMSCs grown on coverslips were fixed in 4% paraformaldehyde for 30 min at 4 °C, washed with PBS and permeabilized with 0.1% Triton-X100 (Amresco, Solon, OH, USA) and 5% bovine serum albumin (Invitrogen) in PBS, followed by an overnight incubation (at 4 °C) with monoclonal antibodies against CD34, CD44 and CD45 (1:100 dilution, Santa Cruz).34 The corresponding secondary FITC-conjugated donkey-antimouse IgG (Proteintech, Chicago, IL, USA) diluted 1:100 in PBS was applied for 1 h in darkness at room temperature. Cells were visualized under an epifluorescent microscope (Olympus, Japan).

Ultrastructure of the HUMSCs. After being harvested by 0.25% trypsin digestion, HUMSCs were washed twice (1000 r.p.m., 5 min) in PBS and fixed in 0.01 M PBS containing 2.5% glutaraldehyde.17 The fragments were postfixed in 1% osmium tetroxide in the same buffer, dehydrated in graded alcohols, embedded in Epon 812, sectioned with ultramicrotome and stained with uranyl acetate and lead citrate. The sections were examined with a transmission electron microscope (TEM; Technai 10, Philips, The Netherlands).

Differentiation studies. Differentiation of HUMSCs was assessed in cultures of the fifth passage. The cells were cultured in a medium that contained osteogenic (10−7 mol l−1 dexamethasone, 10 mM β-glycerophosphate and 50 mg l−1 ascorbate-phosphate), adipogenic (10−6 mol l−1 dexamethasone and 10 mg l−1 Insulin) or neurogenic (DMEM/F12 with 0.5% B27, 1% fetal bovine serum, 5% horse serum, 0.5 mM retinoic acid, 20 ng per ml of epidermal growth factor, 50 ng per ml of basic fibroblast growth factor, 50 ng per ml of nerve growth factor) materials.35, 36, 37 Regents were mostly ordered from Sigma Aldrich (St Louis, MO, USA), except B27 (Invitrogen), epidermal growth factor and nerve growth factor (Peprotech). Two weeks later, osteogenic differentiation was assessed by Alizarin red staining, and intracellular lipid accumulation was visualized using Oil-Red-O staining and inverted phase contrast microscopy. Differentiated neurocytes from the HUMSCs were confirmed by positive staining with NSE (mouse monoclonal antibody, Abcam, Cambridge, MA, USA), GFAP (mouse monoclonal antibody, Santa Cruz) and Nestin (mouse monoclonal antibody, Santa Cruz), which were detected by immunofluorescence staining and assessed by either fluorescence microscopy or flow cytometry.

Adenovirus infection

Adenoviral vectors and adenovirus infection. The replication-deficient adenovirus vector containing the complementary DNA for VEGF and enhanced green fluorescent protein (Ad-VEGF-EGFP) has been described previously.38 The replication-deficient adenovirus vector that carried the EGFP gene was used as a control. HUMSCs were seeded at a density of 5 × 105 cells per well of a six-well cell culture plate. HUMSCs were exposed to infectious viral particles in 1 ml serum-free DMEM/F12 at 37 °C medium for 2 h. HUMSCs were infected by Ad-VEGF-EGFP or Ad-EGFP at a MOI of 0, 10, 20, 50, 100, 200 and 400 plaque-forming units per cell. Subsequently, the normal medium was added to the plates for 48 h incubation. Thereafter, HUMSCs were used for transplantation or immunostaining and the medium was used for the VEGF enzyme-linked immunosorbent assay test.

In vitro and in vivo expression of VEGF and EGFP. After 48 h of infection, the efficiency of infection of adenovirus was investigated by flow cytometric detection of the EGFP marker. The MTT colorimetric assay was used to evaluate the effect of VEGF gene transfection on the proliferation of HUMSCs.39, 40 The coexpression of VEGF (mouse monoclonal antibody, Millipore, Billerica, MA, USA) and EGFP was observed under a confocal microscope by immunofluorescent staining. VEGF secretion from HUMSCs into culture medium and striatal VEGF levels were spectrophotometrically measured at 450 and 570 nm by an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).41 Basal media were also analyzed as a negative control.

Rodent hemiparkinsonian model and transplantation

Rotenone-induced hemiparkinsonian rat model. A rotenone-induced slowly progressive hemiparkinsonian rat model was chosen for this study.17 Briefly, inbred adult female Sprague-Dawley rats (220–260 g; from the Center of Experimental Animals, Tongji Medical College, HUST; 12-h light/dark cycles, 22±2 °C, 60±5% humidity; standard laboratory chow and water ad libitum) were anesthetized with chloral hydrate (400 mg kg−1 in 0.9% NaCl, I.P.) and fastened to a cotton bed over a stereotaxic frame (RWD Life Science, Shenzhen, China). Rotenone dissolved in DMSO was infused into the right VTA (AP: 5.0 mm; L: 1.0 mm; DV: 7.8 mm) at a flow rate of 0.2 μl min−1. The needle was left in place for an additional 5 min to complete the diffusion of the drug. Rotenone was subsequently infused into the right SNc (AP: 5.0 mm; L: 2.0 mm; DV: 8.0 mm) at a flow rate of 0.2 μl min−1, with a 5-min needle retention. After needle withdrawal, proper postoperative care was given until the animals recovered completely. The animals were given ibuprofen and penicillin in their drinking water for 24 h to alleviate potential postsurgical discomfort and prevent infection. Four weeks after surgery, the animals were used for HUMSC transplantation.

HUMSC transplantation. Rodents were divided randomly into three groups 4 weeks after surgery as follows: the saline group (10 μl saline alone without donor cells; n=12); the Ad-EGFP group (implanted with 1.0 × 106 Ad-EGFP-infected HUMSCs; n=10) and the Ad-VEGF-EGFP group (implanted with 1.0 × 106 Ad-VEGF-EGFP-infected HUMSCs; n=10). After 48 h of infection, HUMSCs were trypsinized at room temperature for 5 min with 0.25% trypsin. A total of 1 × 106 cells in a 10 μl suspension were transplanted into the striatum of each rat (anterior 1.0 mm, lateral 3.0 mm, ventral 5.0 mm, relative to the position of the bregma and the skull surface). A 10-min waiting period was implemented before the needle was removed to allow the cells to settle. No immunosuppressive medication was given to the animals.

Apomorphine-induced rotations. Apomorphine-induced rotational behavior was tested every 2 weeks for a total of 12 weeks after HUMSC transplantation.17 Rats were placed on a table (1 m × 1 m) with railings and allowed to acclimatize to the environment for 10 min, after which the animals were intraperitoneally injected with apomorphine (1.5 mg kg−1). Subsequent rotational behavior was recorded for 30 min. Following rotational testing, the animals were placed back in their cages.

Histological examination of grafted HUMSCs. At 12 weeks after transplantation, rats were anesthetized with chloral hydrate and the brains were removed and postfixed for 12 h in 4% paraformaldehyde in PBS at 4 °C. The specimens were then equilibrated in 15% sucrose in PBS for 24 h at 4 °C, then in 20% sucrose in PBS for 24 h at 4 °C and finally in 25% sucrose in PBS for another 24 h at 4 °C. Embedded with OCT compound, brain tissues were cut into serial 10-μm-thick slices with a cryostat. HE staining was performed to detect tumor-like formations.

Immunofluorescent staining was used to label the VEGF, Nestin, NSE, GFAP (mouse monoclonal antibody and rabbit polyclonal antibody, Santa Cruz) and TH (mouse monoclonal antibody, Millipore and rabbit polyclonal antibody, Santa Cruz) expression in the striatum (at 1.2 mm caudal to bregma) and TH expression in the SNc (from −4.5 mm to −6.2 mm caudal to bregma). Expression was visualized by CY3-conjugated donkey-antimouse IgG, FITC-conjugated goat-antirabbit IgG or CY3-conjugated goat-antirabbit IgG (Proteintech). For counting TH-positive cells, a design-based unbiased stereological method and a morphometry/image analysis system (Image-Pro plus 6.0) were used as previously described.25 In each section, the region of interest was outlined (SNc and VTA) and the number of TH-positive cells per mm2 of that region was selected and semiautomatically counted. The qualification study of HUMSCs differentiation involved the identification of CY3-positive cells within the total number of EGFP-positive cells in each transplant.42

Statistical analysis. In tissue immunofluorescence staining, all tissue manipulations were conducted in large batches to avoid batch-to-batch variability. Following experimenter-blinded data collection, analysis of variance (ANOVA) was used to evaluate group differences. In all statistical analyses, the P-value considered statistically significant was P<0.05. All data are presented as mean±s.d., except the immunophenotyping of HUMSCs, which was denoted as the mean. In all graphs, scale bar=50 μm.


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This work was supported by grants from the National Natural Science Foundation of China (30870866), the Wuhan Science and Technology Bureau, China (20066002100) and the Hubei Provincial Science and Technology Department, China (2006ABA130). We are grateful to Drs Li Zou and Weixiang Ouyang (Gynecology and Obstetrics department, Union Hospital, HUST) for assisting with umbilical cord collection, Dr William J Long (MIT Lab for Computer Science, Cambridge, MA, USA) for polishing this manuscript and Dr Edgar (Ned) A Butter (Department of Psychiatry, Harvard Medical School, Boston, MA, USA) for proofreading this manuscript.

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Correspondence to T Wang.

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This manuscript presents original research and has been neither previously published nor submitted for publication elsewhere.

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Xiong, N., Zhang, Z., Huang, J. et al. VEGF-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for Parkinson’s disease. Gene Ther 18, 394–402 (2011).

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  • Parkinson's disease
  • vascular endothelial growth factor
  • umbilical cord mesenchymal stem cells
  • neural differentiation
  • dopaminergic neurons
  • substantia nigra pars compacta

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