Neuronal differentiation of Mesenchymal Stem Cells (MSCs)

Bone-marrow derived MSCs were obtained from Lonza Group Ltd. (Basel, Switzerland), and maintained in culture, as per manufacturer’s instructions. For dopaminergic neuronal differentiation of MSCs, Trzaska K. et al.’s protocol was adopted⁴³, and is depicted in Fig. 1A. Briefly, MSCs were plated in MSC-culture media, and allowed to adhere to Poly-D-lysine coated cell culture vessel’s surface at 37 °C for 24 hours, in a 5% CO₂ incubator. Neuronal differentiation was initiated by replacing the MSC-culture media with neurobasal media supplemented with 0.5% B27 (Invitrogen^TM, Life Technologies, Carlsbad, CA, USA), 250 ng/ml SHH, 100 ng/ml FGF-8, and 50 ng/ml bFGF (R&D Systems, Inc., Minneapolis, MN). The cells were maintained in the same media throughout the 12-day differentiation protocol. On day 9 of differentiation, 50 ng/ml BDNF was spiked into the differentiation media. Undifferentiated MSCs were maintained as negative controls, and SH-SY5Y cells (American Type Culture Collection, Manassas, VA, USA) differentiated into neurons with the help of retinoic acid (10 µM for 7 days), were used as positive controls in the functional characterization experiments.

MSCs induced with growth factors show characteristics of dopaminergic neurons. MSCs were induced with growth factors; b-FGF, FGF-8 and SHH ligand for 9 days, followed by addition of BDNF for next 3 days (A). The induced MSCs were analyzed for dopaminergic neuron characteristics on day 12 of differentiation. TH and DAT protein expression was analyzed in the protein lysate obtained from SH-SY5Y (induced into neuronal like cells with retinoic acid), un-induced MSCs and induced MSCs on day 9 and 12 of differentiation, via western blot (B). Ptx3 and Nurr1 expression was analyzed in differentiated MSCs via immunofluorescence (C). Dopamine release was quantified in the supernatant of MSCs and differentiated MSCs on day 12 of differentiation via ELISA (D). Bars represent mean ± SE. *p value < 0.05, as compared with MSCs (D). The electrical signature of MSCs and differentiated MSCs (E) and in mouse dopaminergic slices indicated that a hyperpolarizing pulse of 60 mV induced a large inward current in differentiated MSCs. Western blots showing expression of proteins involved in calcium entry are shown in (F). Antibodies used for detection are labeled in the figure. Traces of Tg (1 µM) induced currents were measured in MSCs and differentiated MSCs (G). The holding potential for current recordings was −80 mV and the relative current-voltage (I–V) curves along with the current density (average from 7–10 cells) is shown as bar graph.