Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.
OBJECTIVE
To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons.


METHODS
Bone marrow from 6 adult dogs.


METHODS
BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx.


RESULTS
Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons.


CONCLUSIONS
Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.
Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan.
Rei
R
Nakano
Kazuya
K
Edamura
Hiroshi
H
Sugiya
Takanori
T
Narita
Ken
K
Okabayashi
Tadaaki
T
Moritomo
Kenji
K
Teshima
Kazushi
K
Asano
Tomohiro
T
Nakayama
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D015220:Calcium Channels / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D004285:Dogs; D007150:Immunohistochemistry / Q000662:veterinary; D059630:Mesenchymal Stem Cells / Q000378:metabolism* / Q000502:physiology; D064231:Nestin / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
D015220:Calcium Channels; D064231:Nestin; D016900:Neurofilament Proteins; D012333:RNA, Messenger