All-trans Retinoic Acid Promoted Human Umbilical Cord Blood Derived Multipotent Stem Cells To Differentiate Into Dopaminergic Neurons

Pakinson’s disease (PD) is a common neurodegenerative disease in the aged population. The pathophysiology of PD is a chronic process that dopamine neurons in the substantia nigra area degenerate and lose progressively. The current treatment measures mostly in order to control symptoms, not to improve the degeneration and lost of dopamine neurons, and therefore can not delay the progress of PD. Stem cells with a potential of differentiating into neurons could be transplanted for degenerated and lost neurons replacement, providing a novel and available way for PD treatment. However, choosing a safe, effective and without ethnic issues stem cell source must be further explored for PD transplantation.Objective:To explore whether human cord blood-derived stem cells (CB-SCs) with multipotential of differentiation and low immunogenicity could be induced into dopamine neurons in the presence of all-trans retinoic acid (ATRA) in vitro.Methods:1. Cord blood samples collection:10human umbilical cord blood samples were collected from healthy donors after informed consents signed in the obstetrical ward of Jinan central hospital.2. Divided CB-SCs into different groups:when isolated CB-SCs proliferated to a certain number, CB-SCs were divided into three groups:Serum-Free control group, Neuro-Medium control group and Inductive group. CB-SCs in Serum-Free control group kept culturing in the serum free medium for other12-14days; in the Neuro-Medium control group and Inductive group, CB-SCs were cultured in the neuronal medium for other12-14days and10μmol/L ATRA was contained in the medium of Inductive group.3. Real-time PCR test:the expression of Nurrl, Wntl and Enl which manipulate dopamine neuronal differentiation was estimated through real-time PCR test.4. Immunocytochemistrical test:cells in the Neuro-Medium control group and Inductive group were fixed and the expression of NeuN (neuronal nuclear, the specail marker of neuron), TH (tyrosine hydroxylase, the special enzyme of dopamine neuron) and DAT (dopamine transporter, the special marker of dopamine neuron) was analyzed through immunocytochemistrical tests.5. ELISA test:after neurons derived from CB-SCs were depolarized by challenging with Hank’s balanced salt solution containing56mmol/L potassium, supernatants of Neuro-Medium control group and Inductive group were collected for ELISA test. Then, the content of dopamine in supernatants was analyzed through ELISA test.Results:1. Untreated CB-SCs were established to express Nurrl, Wntl, Enl mRNA through real-time PCR analysis.2. Differentiation of CB-SCs into neuron-like cells after ATRA treatment With the treatment of ATRA, most of CB-SCs were induced into cells with typical neuronal morphology. But this change was not obvious in the Neuro-Medium control group. Cells in the Serum-Free control group kept proliferating and retained morphological features of CB-SCs.3. Differentiated CB-SCs showed special markers of dopamine neuron Immunostaining results revealed that about70±7%of ATRA-treated cells were positive for NeuN,48±11%of ATRA-treated cells expressed TH, and36±9%positive for DAT. In contrast, only a minority of CB-SCs in the Neuro-Medium control group expressed TH and DAT maker, and the proportion of NeuN positive cells was<15%in total cells.4. Differentiated CB-SCs displayed a potential of releasing dopamine neurotransmitter The ELISA results established that after potassium stimulated cell depolarization, dopamine in the supernatants of Inductive group was up to940±42pg/mL higher than in the Neuro-Medium control group (P<0.001). The dopamine in the supernatant of Neuro-Medium control group was just370±15pg/mL as shown by the ELISA data analysis.Conclusions:1. The expression of Nurrl, Wntl, Enl gene has indicated that CB-SCs possess the potential for dopaminergic differentiation.2. ATRA could promoted CB-SCs to differentiate into dopaminergic neurons with a function of releasing dopamine neurotransmitter.Significance:In this study, we demonstrated CB-SCs could be induced into dopaminergic neurons in vitro. This result provided an experimental and theoretical basis for further studies of CB-SCs transplantation in animal models. Furthermore, this study also developed a new inductive method and source available in stem cell transplantation for Parkinson’s disease treatment.