The Therapeutic Potential Of Human Cord Blood-Derived Multipotent Stem Cells In Parkinson's Disease

BackgroundParkinson's disease (PD) results from the dysfunction and chronic degeneration of dopamine neurons in the substantia nigra of the mesencephalon. Dopamine neurons play key roles in controlling voluntary movements and regulating body gestures. To date, pharmacological agents have shown only limited therapeutic potential for slowing the progression of PD. Therefore, finding alternative therapies may be necessary to provide a cure or a long-lasting, effective treatment of symptoms. Stem cells, which possess the ability to self-renew and give rise to different cell lineages to replenish the damaged and aged tissue cells, may provide a solution. Stem cells derived from human cord blood have unique advantages over other stem cell sources including (1) a large source,(2)no ethical concerns,(3) no risk to the donors, and (4) low risk of graft-versus-host disease (GVHD). A novel type of stem cells from human cord blood, designated cord blood multipotent stem cells (CB-SCs) display ES-like cell characteristics, including multi-potential differentiation. Of particular relevance to neurodegenerative disease research, CBSCs have been shown to differentiate into neurons when exposed to neuronal growth factor (NGF). To explore the therapeutic potential of CB-SCs in PD, we examined whether CB-SCs could be induced to differentiate toward dopamine neurons in the presence of all-trans retinoic acid (ATRA), a well-established inducer.Method1. CB-SC preparationsHuman umbilical cord blood samples were collected from healthy donors at Jinan Central Hospital. Mononuclear cells were isolated with Ficoll-Hypaque, and red blood cells were removed using red blood cell lysis buffer. The remaining mononuclear cells were washed three times with PBS and seeded. Cells were cultured in serum-free culture medium.2. Cell differentiation CB-SCs grown to70%confluence were treated with5u mol/L or10μmol/L ATRA in the presence of Neurobasal-Amedium. CB-SCs cultured in Neurobasal-A medium as control. CB-SCs cultured in serum-free culture medium served as an additional control. After treatment for12days, cells were subjected to examination for specific markers of dopamine neurons.3. Quantitative real-time PCRQuantitative real-time PCR was used to quantify mRNA expression for specific markers of dopamine neurons. Total RNA was extracted using a Qiagen kit. First-strand cDNAs were synthesized from total RNA using a QuantiTect Reverse Transcription kit according to the manufacturer's instructions. Real-time PCR was performed on each sample in triplicate with the ABI Prism7900HT Fast Real-Time PCR System. The validated gene-specific RT2PCR Primer sets for each gene were designed and purchased from SABiosciences. Expression levels were determined relative to β-actin as an internal control.4. Western blot analysisTo determine the expression of dopaminergic transcription factors, we performed Western blotting as previously described. Briefly, cells were washed with cold PBS and solubilized with a lysis buffer and a cocktail of protease inhibitors. Samples were mixed with a loading buffer, then boiled, loaded, and separated by electrophoresis. The separated proteins were transferred to a nitrocellulose membrane and blocked with5%non-fat dry milk in TBST for1h followed by incubation with rabbit anti-human Nurrl, Enl, or Wntl Abs.β-Actin served as an internal loading control.5. ImmunocytochemistryThe ATRA-treated CB-SCs and cells from control groups were fixed with4%paraformaldehyde and subsequently permeabilized with0.5%Triton X-100, incubated with, and blocked with2.5%horse serum for20min. Fixed cells were immunostained with rabbit anti-human TH Ab, rat anti-DAT Ab, mouse anti-NeuN (Neuronal nuclear) Ab, mouse anti-GFAP (glial fibrillary acidic protein) Ab, mouse anti-β Ⅲ tubulin, or mouse anti-MAP-2(microtubule-associated protein-2) Ab. Appropriate secondary antibodies anti-rabbit/rat IgG-TRITC, anti-mouse IgG-FITC, and mounting medium with DAPI were used for detection and visualization. Images were acquired using an Olympus IX710Camera with the manufacturer's software and edited using Adobe Photoshop CS3.6. Dopamine enzyme-linked immunosorbent assayFollowing12days of treatment with ATRA or control medium, cells were examined for dopamine release. Dopamine levels were quantitated using an enzyme-linked immunosorbent assay it. Briefly, cells were washed twice with PBS to remove all culture medium and then placed in Hank's balanced salt solution containing5.33mmol/L K+or56mmol/L K+concentration. High concentrations of potassium induce secretion of dopamine through cell depolarization. After incubation for5min, supernatants were collected for ELISA following the manufacturer's instructions.Result1. CB-SCs possess the potential for dopaminergic differentiationCB-SC cultures were established from multiple human cord blood preparations. To evaluate the CB-SCs'potential for differentiation into dopamine neurons, we examined basal expression of the dopamine neuron-specific transcription factors Nurrl, Wntl, and En1. Real time PCR analysis showed that untreated CB-SCs expressed Nurrl, Wntl, and En1mRNA. Western blot analysis further confirmed protein expression of all three. Real-time PCR analysis also revealed that CB-SCs displayed a low level of TH mRNA, a key enzyme responsible for catalyzing the conversion of the tyrosine to dihydroxyphenylalanine (DOPA, a precursor for dopamine). The presence of these markers indicated that CB-SCs possess the potential for dopaminergic differentiation.2. Differentiation of CB-SCs into neuron-like cells after treatment with ATRAWe evaluated the response of CB-SCs to exposure to two ATRA concentrations. Both concentrations induced morphological changes consistent with differentiation to neurons, and the higher concentration induced changes in a larger proportion of cells. Most cells (90%) exposed to101M ATRA displayed typical neuronal morphologies within5-8days, followed by the formation of more elongated and branched cell processes after10-12days. In contrast,35%of cells in51M ATRA group also turn into neuron-like morphologies after10-12days, but with relatively shorter cell processes. Most of the Neuro-medium control cells (>95%) failed to display neuronal differentiation. CB-SCs cultured in serum-free culture medium continued to grow with round morphologies at high confluence. Cells from each group were immunostained for specific neuronal markers including bⅢ tubulin, microtubule-associated protein2(MAP2), NeuN (mature neuronal marker), and glial fibrillary acidic protein (GFAP, an astrocyte marker). Immunostaining showed that90±4%of ATRA-treated cells strongly expressed βⅢ tubulin and MAP-2panels), Most about70±7%of ATRA-treated cells were positive for NeuN, and only a few cells (5±2%) expressed GFAP. In contrast, a few cells (5%) in the Neuro-medium-treated group displayed only background levels of bⅢ tubulin, MAP-2, NeuN, and GFAP. These data indicate that treatment with10μ mol/L ATRA induces differentiation of CB-SCs into neuronal-like cells.3. CB-SCs gave rise to functional dopamine neurons after treatment with ATRA, and these cells release dopamine in response to stimulationWe performed immunocytochemical analysis to examine the expression of dopamine neuron-specific proteins including TH (a marker of dopamine neurons) and DAT (a dopamine transporter). Immunostaining results revealed that48±11%of ATRA-treated cells expressed TH, while36±9%of ATRA-treated cells were positive for DAT. In contrast, CB-SCs in the medium control groups only showed background levels of TH and DAT expression (5±1%). These data indicate that CB-SCs exposed to ATRA produce proteins indicative of dopamine neurons. To examine whether ATRA-induced CB-SCs produce and are capable of secreting dopamine, we stimulated the cultured cells with potassium to initiate depolarization. Results from ELISA demonstrated that dopamine levels were markedly increased in the10μ mol/L ATRA-treated groups compared to the control groups (P<0.001). These data confirm that CB-SCs can give rise to functional dopamine neuron-like cells after treatment with101M ATRA in Neuro-medium.ConclusionThis study demonstrated that CB-SCs can be induced to differentiate into functional DA neurons in the presence of10μ mol/L ATRA combined with Neurobasal medium. These ATRA-induced CB-SCs displayed neuronal morphology, expressed tyrosine hydroxylase (TH) and other DA neuron-specific molecular markers, and secreted DA in response to potassium. These findings indicate ATRA-induced CB-SCs may provide an alternative to other stem cell-based approaches to generating DA neurons for the treatment of PD. Parkinson's disease (PD) results from the chronic degeneration of dopaminergic neurons. A replacement for these neurons has the potential to provide a clinical cure and/or lasting treatment for symptoms of the disease. Human cord blood-derived multipotent stem cells (CB-SCs) display embryonic stem cell characteristics, including multi-potential differentiation. To explore their therapeutic potential in PD, we examined whether CB-SCs could be induced to differentiate into dopamine neurons in the presence of all-trans retinoic acid (ATRA). Prior to treatment, CB-SCs expressed mRNA and protein for the key dopaminergic transcription factors Nurrl, Wntl, and En1. Following treatment with10μmol/L ATRA for12days, CB-SCs displayed elongated neuronal-like morphologies. Immunocytochemistry revealed that48±11%of ATRA-treated cells were positive for tyrosine hydroxylase (TH), and36±9%of cells were positive for dopamine transporter (DAT). In contrast, control CB-SCs (culture medium only) expressed only background levels of TH and DAT. Finally, ATRA-treated CB-SCs challenged with potassium released increased levels of dopamine compared to control. These data demonstrate that ATRA induces differentiation of CBSCs into dopaminergic neurons. This finding may lead to the development of an alternative approach to stem cell therapy for Parkinson's disease.