Mesenchymal stem cells (MSCs) hold great promise for regenerative medicines such as cell therapy and tissue engineering (1,2). These approaches require cells to be grown in culture and differentiated into specific cell types such as osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), adipocytes (fat cells) or neurons (nerve cells). Here, we use Nanolive’s automated microscope, the CX-A, to make novel observations about the various stages involved in neuronal differentiation.
Human MSCs were grown in low-serum cell growth media in 35 mm dishes pre-coated with fibronectin. The first video in the compilation shows a healthy population of MSCs undergoing mitosis (00:00 to 00:17). At this pre-differentiation stage cells are motile, and their (large, flat) morphology is homogenous.
When neurogenic cell differentiation media was added the cells stop dividing (00:17 to 00:42). No acute changes in morphology were observed over the first couple of hours, but after approximately 6 h differentiation starts to occur. Most cells have the long, thin, spindle-shaped morphology of neural progenitor cells, while a small minority assume the radial morphology of glial cells (00:42 to 00:59).
Differentiation success appears to be related to confluency: at low confluency, cells are highly mobile and seem to differentiate less. Conversely, at high confluency almost all cells show morphological signs of differentiation (00:59 to 01:28).
Several days after the differentiation media is added, more complex, advanced changes in morphology become visible (01:28 to 02:18). These changes in morphology are a consequence of the retraction of the actin cytoskeleton. Branched protoplasmic extensions resembling dendrites are formed, some of which (specifically those connected to neighboring cells) eventually form axons.
(1) Xu, Y., Shi, Y. and Ding, S. 2008. A chemical approach to stem-cell biology and regenerative medicine. Nature, 453(7193), 338-344.
(2) Wu, S.M. and Hochedlinger, K. 2011. Harnessing the potential of induced pluripotent stem cells for regenerative medicine. Nature Cell Biology, 13(5), 497-505.
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