Neuronal differentiation of human UCB-MSCs in four-step neuronal induction medium. (A) Schematic of four-step neuronal induction condition for human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) differentiation. (B) to (E) Differentiation of the control RCB 2080 hUCB-MSC cell line with the four-step induction protocol showed transdifferentiation of MSCs to neurons as ascertained by β-III tubulin immunostaining. (F) to (I) β-III tubulin immunostaining of neurons transdifferentiated from hUCB-MSC SCB4 cells with the four-step neuronal induction protocol. (J) to (M) Immunostaining of β-III tubulin, a neuronal marker on neurons from hUCB-MSC SCB10 upon four-step medium treatment. (N) Comparison of percentage of β-III tubulin-positive cells in control RCB 2080 with hUCB-MSC SCB4 and hUCB-MSC SCB10: ~35% β-III tubulin-positive cells were in RCB 2080, whereas ~15% cells in hUCB-MSC SCB4 and 7.4% cells in hUCB-MSC SCB10 were positive for β-III tubulin - showing a significant difference in pattern of differentiation between the hUCB-MSC cell line RCB 2080 and primary hUCB-MSC SCB4 and SCB10, indicating that neuronal differentiation potential of primary hUCB-MSCs vary from batch to batch. Data represented as mean ± standard deviation of triplicates (n = 3) from three different experiments. Scale bar = 100 μm. aFGF, acidic fibroblast growth factor; BF, Bright Feild; bFGF, basic fibroblast growth factor; βME, beta-mercaptoethanol; DAPI, 4',6-diamidino-2-phenylindole; IBMX, 3-isobutyl-1-methylxanthine; IMDM, Iscove's modified Dulbecco's medium; NGF, nerve growth factor; PMA, phorbol myristate acetate.