Human Umbilical Cord-Derived Schwann-Like Cell Transplantation Combined with Neurotrophin-3 Administration in Dyskinesia of Rats with Spinal Cord Injury
- First Online:
- Cite this article as:
- Yan-Wu, G., Yi-Quan, K., Ming, L. et al. Neurochem Res (2011) 36: 783. doi:10.1007/s11064-011-0402-9
Mesenchymal stem cells are capable of differentiating into Schwann-like cells. In this study, we induced human umbilical-cord mesenchymal stem cells (HUMSCs) in vitro into neurospheres constituted by neural stem-like cells, and further into cells bearing strong morphological, phenotypic and functional resemblances with Schwann-like cells. These HUMSC-derived Schwann-like cells, after grafting into the injured area of the rats’ spinal cord injury (SCI), showed a partial therapeutic effect in terms of improving the motor function. Neurotrophin-3 (NT-3) was reported to improve the local microenvironment of the grafted cells, and we, therefore, further tested the effect of Schwann-like cell grafting combined with NT-3 administration at the site of cell transplantation. The results showed that NT-3 administration significantly promoted the survival of the grafted cells in the host-injured area. Significant improvement in rats treated by Schwann-like cell grafting combined with NT-3 administration was demonstrated in the behavioral test as compared with that in animal models received the cell grafting only. These results suggest that transplantation of the Schwann-like cells combined with NT-3 administration may represent a new strategy of stem cell therapy for spinal cord injury.
KeywordsHuman umbilical cord mesenchymal stem cellsSchwann-like cellsCell differentiationCell transplantationSpinal cord injury
The mesenchymal stem cells (MSCs) isolated from the cushioning matrix between the umbilical blood vessels, like other MSCs, possess strong proliferation and multipotent differentiation capacities in vitro [1, 2]. With the protection from the placental barrier, these MSCs can lower the risks of viral or bacterial contamination in the cell isolation process; human umbilical cord MSCs (HUMSCs), with few ethical concerns and relatively low immunogenicity, may serve as good candidate seed cells in the cell replacement therapy for spinal cord injury.
HUMSCs are capable of differentiating into mesoblastemas, and in the presence of appropriate chemical factors, these cells may transform into dopaminegic-like cells , oligodendrocyte, osteocytes, chondrocytes, or adipocytes [4–7]. A recent study comparing several stem cells for their differentiation capacities shows that HUMSCs have multipotent differentiation ability in vitro . In this study, we examined the potential of HUMSCs in differentiating into Schwann-like cells in vitro and tested the therapeutic effect of these differentiated cells on rat models with spinal cord injury.
Current evidence suggests that in vitro HUMSCs transplantation alone can not totally replace the damaged or degenerated neurons in the nerve tissue. Most of the transplanted cells failed to survive, and only a very small fraction of the cells may eventually function to serve the therapeutic purpose [8, 9]. As an essential chemical factor in allowing the normal growth of nerves, neurotrophin-3 (NT-3) has been shown to have obvious neuroprotective effects and promote the survival of both the grafted cells and the host neurons . In this study, we tested the effect of HUMSC-derived Schwann-like cell transplantation combined with NT-3 in improving the motor dysfunction of the rats with spinal cord injury.
Materials and Methods
Isolation and Culture of HUMSCs
This study was approved by the ethical committee of Zhujiang Hospital affiliated to Southern Medical University. Umbilical cords (n = 10; gestational ages, 39–40 weeks) were obtained from local maternity hospitals after normal deliveries. With the written consent of parents, the fresh human umbilical cords were obtained after birth and were collected into 0.01 mmol/l Hanks’ balanced salt solution (HBSS) (Gibco, USA) at a temperature of 4°C. Following disinfection in 75% ethanol for 30 S, the umbilical cord vessels were got rid off while still in HBSS. The mesenchymal tissue (in Wharton’s jelly) was then diced into cubes of about 0.5 cm3 and centrifuged at 1,200 rmp for 5 min. Following removal of supernatant, the precipitate (mesenchymal tissue) was washed with serum-free DMEM/F12 (Gibco, USA) and centrifuged at 1,200 rmp for 5 min. The precipitate was then enzymatically dissociated for 18 h at 37°C using 0.075% collagenase type II (Sigma, St. Louis, MO, USA), and further digested with 0.125% trypsin/EDTA (Gibco) at 37°C for 30 min. The suspension was neutralized by DMEM/F12 containing 10% (v/v) fetal bovine serum (FBS; Gibco) and counted under a microscope with the aid of a hemocytometer. Cultures were maintained at subconfluent levels at 37°C in 5% CO2 in a humidified incubator. The culture medium was changed 48 h later and every 3 days subsequently. After reaching 80% confluency, the cells were digested with 0.25% trypsin and passaged at the ratio of 1:3.
Immunofluorescen Detection of the Adherent Cells
The HUMSCs in the second to fifth passages, after washing 3 times with PBS for 5 min, were fixed in 4% paraformaldehyde for immunocytochemical detection of the cytoskeletal protein vimentin using standard protocols . Mouse anti-human vimentin antibody and goat anti-mouse IgG-FITC conjugate (Chemicon International, Inc. Temecula, CA) were used as the primary and the secondary antibody with a dilution of 1:400, respectively. The samples were examined using a fluorescent microscope (Vertrieb, Deutschland, Leica, Germany) and photographed with IM50 imaging system. All the immunocytochemical experiments were repeated for 3 times.
The phenotypic characteristics of HUMSCs was detected as following: cells at passage 4 were trypsinized into single-cell suspensions and stained with fluorescein isothiocyanate (FITC)-labeled antibodies, including anti-human CD29-FITC, CD31-FITC, CD34-FITC, CD44-PE, CD105-PE and CD166-PE (Becton–Dickinson, Franklin Lakes, NJ) at a temperature of 4°C for 30 min; nonspecific anti-human IgG-FITC under the same cultured condition was used as an isotype control; and then, they were performed flow cytometric analysis.
Induction of Cell Differentiation
The second to the fifth passages of HUMSCs in exponential growth (80–90% confluence) were digested with 0.25% trypsin for 10 min at room temperature and centrifuged at 1,500 r/min for 15 min. After washing with PBS for 3 times, the cell deposit was re-suspended in the induction culture medium for neural stem cells (DMEM/F12 supplemented with 20 ng/ml epidermal growth factor [EGF, Peprotech, UK], 20 ng/ml basic fibroblast growth factor [bFGF, Peprotech, UK], and N2 [1:50, Gibco, USA]). The cells were plated in 25 cm2 tissue culture flasks at the density of (1 − 2) × 105/ml and incubated at 37°C in the presence of 5% CO2. The culture medium was changed weekly, and the growth factors were supplemented twice every weeks .
To induce the cell differentiation into Schwann-like cells, HUMSC-derived neurospheres growing to 100 μm in the neural stem cell induction medium were digested with trypsin and cultured for 4 days in Schwann-like cell induction medium, namely DMEM/F12 containing 35 ng/ml retinoic acid (Sigma), 5 microM forskolin (Peprotech),10 ng/ml basic human fibroblast growth factor (Peprotech),10 ng/ml recombinant human platelet-derived growth factor-AA (Sigma), and 200 ng/ml heregulin (R&D Systems, Minneapolis, Minnesota, USA).
Immunofluorescent Identification of the Cells
The neurospheres cultured for 8 h in DMEM/F12 containing 10% FBS and 20 ng/ml N2 (1:50) were used for immunofluorescent identification. Briefly, the neurospheres were washed 3 times with PBS (5 min each time) and fixed in 4% paraformaldehyde for 15 min at room temperature. Immunofluorescent detection, using rabbit anti-human nestin antibody (1:400) as the primary antibody and Cy3-labeled goat anti-rabbit IgG (1:400) as the secondary antibody, was carried out according to the descriptions by Chaturvedi et al. . Both of the antibodies were the products of Chemicon International, Inc. (Temecula, CA). The cells were observed under a fluorescent microscope, and 10 visual fields were randomly selected for cell counting and image analysis.
The cells cultured for 14 days in the induction medium for Schwann-like differentiation were also identified by immunofluorescent assay following the same procedures, using mouse anti-GFAP antibody, rabbit anti-S100 antibody and rabbit anti-P75 antibody as the primary antibodies, and FITC-labeled goat anti-mouse IgG (1:400) and Cy3-labeled goat anti-rabbit IgG (1:400) as the secondary antibodies. All the antibodies were the products of Chemicon International, Inc. (Temecula, CA). The cells were observed under a fluorescent microscope, and 10 visual fields were randomly selected for cell counting and image analysis.
The procedures for Western blotting were carried out following the protocols described previously , using rabbit anti-GFAP antibody (1:400), mouse anti-S100 and anti-P75 antibodies (1:400) as the primary antibodies and biotin-labeled goat anti-mouse antibody as the secondary antibody (all the antibodies were the products of Chemicon International, Inc. Temecula, CA). The film was developed by electrochemiluminescence, and β-actin was used as the internal control.
Stem Cell and NG108-15 Neuron Coculture
Undifferentiated HUMSCs and differentiated HUMSCs were plated at a density of 10,000 cells per slide flask and allowed to settle for 24 h. NG108-15 cells were then added to the HUMSCs monolayer at a density of 1,000 cells and the co-cultures maintained for a further 24 h followed by fixation with 4% (w/v) paraformaldehyde for 20 min at room temperature. Fluorescent immunocytochemistry (as above) using mouse anti-neurofilament protein antibody (1:500, Chemicon). The percentage of neurite-bearing neurons and the lengths of longest neurites were measured to assess neurites outgrowth .
The cells cultured for 14 days in the induction medium for Schwann-like differentiation was infected by a recombinant lentivirus carrying green fluorescent protein gene (GFP) for fluorescent cell labeling, and further cultured in the medium containing 5% FBS for 48 h. The construction of the recombinant lentiviral vector and the subsequent cell infection procedures were carried out following the protocols described previously . The detail is the plasmids pMD.2G (Addgene plasmid 12259), pCMVΔR8.91,and pWPXL-EGFP (Addgene plasmid 12257) kindly provided by Dr. Xu (Beijing, China). High-titer GFP lentiviral supernatants were generated by transient cotransfection of three plasmids in 293T cells using Fugene 6 (Roche, CH, Switzerland). About 293T cells (2.5 × 106cells) were transfected with 12 μg of pWPXL-EGFP, 12 μg of pCMVΔR8.91, and 5 μg of pMD.2G. Supernatants of transfected 293T cells were collected 2 days posttransfection, filtered through 0.45 μm pore size filters, and stored at −70°C. The viral titer was determined on HeLa cells infected with serial dilutions of each viral supernatant in presence of protamine sulfate (5 μg/ml; Sigma, St. Louis, MO) by flow cytometry analysis. The titer was calculated according to the following formula: Transduction Units (TU)/ml = (Percentage of GFP-positive cells/100) × (Number of cells infected) × (Vector dilution factor), as described previously. Viral titer typically ranged around 3.7 × 106TU/ml after 2 days and 2.0 × 106TU/ml after 3 days. The cell lines 293T cells and HeLa cells were maintained in DMEM containing 10% FBS, 100U/ml penicillin, and 100 μg/ml streptomycin.
Establishment of Rat Models with Spinal Cord Injury and Cell Transplantation
In this experiment, 48 adult female SD rats, weighing 220–250 g, were chosen. The rats were anesthetized with 3.6% chloral hydrate by intraperitoneal injection. Under aseptic conditions, the animal’s back was shaved, and a 2-cm-long midline incision was made. The laminectomy was performed using a surgical microscope (Zeiss, Germany) at the level of T9-T10 to expose the spinal cord, which was then transected with a micro scissor. Subsequently, the muscles and skin were sutured. The body temperature of the animals was kept at 37°C with a thermal blanket. After operation, the rats were housed in pairs to reduce the stress of depression, on a 12-h light/dark with standard rat diet and water ad libitum. Meanwhile, ampicillin was administered at a daily dose of 0.1 g/kg for 3 days to prevent infection. Manual bladder compression was performed two to three times daily for 1 week, then once daily until the bladder reflex in the model animals was re-established.
All the rats with successfully established spinal cord transaction were equally randomized into 4 groups (n = 12): control group was performed 10 μl saline injection into the lesion site of SCI; sham-operated group were similar to that of transection injury, but left the spinal cord untouched; Schwann-like cells group performed 1 × 106 Schwann-like neuron-like cells in 10 μl PBS into the lesion site of the SCI; and Schwann-like cells plus neurotrophin-3 (NT-3) group received both Schwann-like neuron-like cells injection (1 × 106 in 10 μl PBS) into the SCI site and 100 ng NT3 administration(Invitrogen, California, USA). After spinal cord injury or cell transplantation, all the lesion sites were covered with a piece of gelfoam. Before experimental treatment, all of the model animals must have shown the symptoms of full paraplegia in order to ensure the standardization during the effect analysis.
Immunofluorescence Assay of the Transplanted Cells
Twelve weeks after the cell transplantation, the SCI tissue was obtained from 3 rats of each group., fixed in paraformaldehyde at 4°C for 24 h, and placed in 0.1 mol/L PBS containing 20% sucrose. When the tissue sank to the bottom of the container, a tissue block 2.0 cm to −2.0 cm relative to transplanted site was prepared for serial frozen Sects. 25 μm in thickness. One out of every 5 serial sections was taken for immunohistochemical staining. The sections were observed under a fluorescent microscope for green fluorescence signals emitted by the transplanted cells to assess the cell survival.
Retrograde Neuronal Labeling
About 7 weeks after lesion, biotin dextran amine was surgically introduced caudal to the lesion site to trace dorsal column fibers: 5 animals from each group were anesthetized and injected with 2 μl 10% BDA in 0.01 mol l−1 PBS into the spinal cord 1 cm caudal to the lesion site. About 7 days after BDA injection, the animals were killed by transcardiac perfusion. Longitudinal sections (30 μm) of the spinal cord were subject to BDA histochemistry. Sections were viewed and photographed using a fluorescent microscope (Vertrieb, Deutschland, Leica, Germany) and photographed with IM50 imaging system.
Assessments of neurological outcome and hind-limb motor function recovery of all the model animals were evaluated by 2 independent researchers weekly after spinal cord injury until the 8th week. BBB open-field locomotor test was performed to assess the hind-limb motor function, as established by Basso et al. . The BBB scores ranged from 0 to 21, in which 0 reflects no movement of the hind limbs and 21 implies normal locomotion. If the score was below 8, the rat could only move its hind-limb joint without supporting its body weight. Scores of 9–13 represented that the rats were able to support its body weight without coordination. A score of 14–21 signified that the rat could stabilize its trunk and coordinate the movement. In this study, the assessment of hind-limb motor function was strictly based on the objective criteria.
The measurement data were presented as Mean ± SD. The difference between the mean values was analyzed using t test (for one or two factors) or ANOVA (for multiple factors) using SPSS13.0 software. A P value less than 0.05 was considered to indicate a significant difference.
Induced Cell Differentiation and Immunofluorescent Identification
Functional Properties of Differentiated Cells
GFP Labeling and Retrograde Neuronal Labeling
Immunofluorescence Assay of the Transplanted Cells
As shown in Fig. 6a, the pathological extent of injured spinal cord in the controls was much larger than that in the other 2 treatment groups at the 12th week after SCI. This suggested that all treatments, including Schwann-like cells and Schwann-like cells + NT3, could play a role in lesion repair. In particular, the extent of the damage was least in Schwann-like cells + NT3 treatment group among all the groups (Fig. 6).
Like neural stem cells, HUMSCs are capable of multilineage differentiation into dopaminergic-like cells, oligodendrocyte, osteocytes, chondrocytes, or adipocytes in vitro. We have for the first time induced HUMSCs to differentiate into neurospheres and further into functional Schwann-like cells with typical Schwann-like cell phenotype. So far, numerous reports have been available to describe the success in inducing fetal stem cells to differentiate into such functional Schwann-like cells [14, 17, 18] as bone morrow mesenchymal stem cells and HUMSCs. As currently the major source of MSCs, bone marrow-derived MSCs are not restricted by ethical concerns or immune rejection, but they easily undergo the aging process as the age of the donor increases, without mentioning the painful process of cell harvesting and the anesthetic risks for the donors . For human umbilical cord blood mesenchymal stem cells, the low successful rate of isolation and culture is limited to clinical application. HUMSCs combine the merits of both fetal stem cells and adult stem cells . We transplanted HUVMSC-derived Schwann-like cells into the site of the SCI and found no malignant changes of the cells giving rise to brain tumors, suggesting that HUMSCs can be a good candidate seed cells for cell therapy of SCI.
In this experiment, HUMSCs in passage 4 showed positive staining of CD29, CD44, CD105, CD166, negative staining of CD31,CD34. which suggest the HUMSCs cultured by our protocol expressing the markers of MSCs,negative for vascular endothelial markers.The induction of HUMSCs into Schwann-like cells involves the use of multiple neurotrophic factors. Kingham et al.  reported successful induction of HUMSCs into Schwann-like neurons by pre-inducing the mesenchymal stem cells using β-mercaptoethanol, and then, supplementing with GGF-2, bFGF, PDGF and forskolin. This induction system resulted in a rate of 42.9 ± 3.3% S100-positive cells. Lin et al.  reported pre-induction using β-mercaptoethanol, and then supplementing with some related factors could result in a rate of 25% S100-positive cells. We adopted a two-step induction protocol of the HUMSCs: at first, the long spindle-shaped HUMSCs were induced into neural stem cell like structures (neurospheres), and then some factors were added into the culture medium to further induce the neurospheres to differentiate into neuron-like cells bearing morphological, immunophenotypic and functional resemblances with Schwann-like cells, which avoid the harmfulness of β-mercaptoethanol to the researchers. The nearly 45% S100 positive rate provides guarantee for therapeutic effects; however, much work remains to be done to explore differentiation mechanism.
Studies indicate that only 5–10% of the transplanted cells may survive the transplantation procedure , a quantity far insufficient to produce significant therapeutic effects against animal models, therefore, new strategies of cell transplantation are needed. So far, measures being available to enhance the survival of the grafted cells include neurotrophic support, antioxidant and antiopoptotic protection of the cells. Experimental evidences had shown that administration of such factors as GDNF, BDNF and bFGF provided neurotrophic support of the grafted tissues or cells, significantly enhanced the survival of the grafted neural stem cells or neurons in the host brain, and improved the motor function of the animal models with PD [21–23].
As a neurotrophic factor found widely in mature and developing neural tissues, NT3 plays important roles in the neurogenesis and neural cell survival and repair . Our results demonstrate that Schwann-like cell grafting combined with NT3 administration significantly promoted the survival and migration of HUMSC-derived Schwann-like cells grafted into the site of the SCI rats, also resulting in increased density of the grafted cells at the cell transplantation site as compared with Schwann-like cell grafting alone. HE staining of the rats lent further support to the finding. More importantly, behavioral tests of the SCI rats showed significant behavioral improvements after the cell grafting combined with NT3 administration. All these data suggest that NT3 can significantly improve the therapeutic effects of HUMSC-derived Schwann-like cell grafting against SCI, which are accorded with the neurotrophic and protective effects . In summary, all the currently available data suggest its bright future of stem cell therapy in combination with the use of neurotrophic factors for treatment of SCI.
The neurospheres derived from HUMSCs are capable of further differentiation into functional Schwann-like cells in vitro. Transplantation of the Schwann-like cells combined with NT3 administration produces obvious behavioral improvement of the SCI rats, which sheds light on a new strategy for stem cell therapy of SCI. But before the clinical application of HUMSC-derived Schwann-like cells, rigorous laboratory tests are needed for thorough evaluation of neurotransmitter secretion and potential tumorigenicity.
This study was supported by the National Natural Science Foundation of China (No. 30901546/H0912) and Natural Science Foundation of Guangdong (No. 9451051501002508).