Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity

Abstract

Mesenchymal stem cells have the ability to transdifferentiate into neurons and therefore one of the potential adult stem cell source for neuronal tissue regeneration applications and understanding neurodevelopmental processes. In many studies on human mesenchymal stem cell (hMSC) derived neurons, success in neuronal differentiation was limited to neuronal protein expressions which is not statisfactory in terms of neuronal activity. Established neuronal networks seen in culture have to be investigated in terms of synaptic signal transmission ability to develop a culture model for human neurons and further studying the mechanism of neuronal differentiation and neurological pathologies. Accordingly, in this study, we analysed the functionality of bone marrow hMSCs differentiated into neurons by a single step cytokine-based induction protocol. Neurons from both primary hMSCs and hMSC cell line displayed spontaneous activity (�75%) as demonstrated by Ca ++ imaging. Furthermore, when electrically stimulated, hMSC derived neurons (hMd-Neurons) matched the response of a typical neuron in the process of maturation. Our results reveal that a combination of neuronal inducers enhance differentiation capacity of bone marrow hMSCs into high yielding functional neurons with spontaneous activity and mature into electrophysiologically active state. Conceptually, we suggest these functional hMd-Neurons to be used as a tool for disease modelling of neuropathologies and neuronal differentiation studies.

Integrin α5β1 Mediated Cellular Reorganization in Human Mesenchymal Stem Cells During Neuronal Differentiation

“…Both neuronal proteins were expressed in differentiated hMSCs (Figure 1C). The data were correlated with our previous findings in hMd-Neurons (43). To investigate apoptosis, caspase-3 activity was measured in neuronal induced cells, as caspase-3 is involved in the downstream signaling of the apoptotic cascade.…”

“…Considering previously suggested cellular dynamics regulated by integrin α5β1, in this study, involvement of integrin α5β1 in neuronal differentiation of hMSCs was examined based on a differentiation protocol we previously reported (43). The results showed that cytokine based induction of hMSCs into hMd-Neurons requires integrin α5β1 surface localization at specific time points (around day 5), while translocation into the cytosol was observed during further maturation.…”

“…Cell frequency was set at 3,000 cells/cm 2 . The culture was maintained in DMEM containing 10% MSC-FBS and 1% Penicillin/Streptomycin; the cells were incubated at 37˚C in 5% CO 2 for 24 h. Previously defined neuronal induction media, composed of 20 ng/ml hEGF (human epidermal growth factor), 40 ng/ml bFGF (basic fibroblast growth factor), 10 ng/ml FGF-8 (fibroblast growth factor-8), 10 ng/ml human BDNF (human brainderived neurotrophic factor), 40 ng/ml NGF, 0.125 mM dbcAMP (dibutyryl cyclic AMP), 0.5 mM IBMX (3-isobutyl-1methylxanthine), 2 mM L-Glutamine in Neurobasal medium, and B27 supplement in the absence of serum was used for neural differentiation (43). Then, cells were refreshed with neural induction media every 48 h for 12 days.…”

“…As for neuronal induction, a bone marrow derived hMSC cell line (UE7T-13 cells, no RBRC-RCB2161; RIKEN) was used in experimental settings as previously described (43). Firstly, hMSC cells were immunophenotyped and more than 99% of the cells were positive for the mesenchymal stem cell markers CD44, CD73, CD90 and CD105, while negative for hematopoietic lineage markers CD34 and CD45 (Figure 1A).…”

Rapid Induction of Mesenchymal Stem Cells to Neural Cells

“…However the cells were negative for hematopoietic lineage marker CD34. These results indicated that the isolated cells in this study represented HUCB-MSCs and were not mixed with cells of hematopoietic origin (Fig., 5,6,7).…”