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Abstract
BackgroundIn the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells.Methodology/Principal FindingsIn this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact.Conclusions/SignificanceThe sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics.
Highlights
Mouse embryonic stem cells (ES) have the potential to differentiate into many cell types and are considered potential cell therapy candidates to treat neurodegenerative diseases [1,2,3].To avoid teratoma formation in ES cells and prevent damage to fully differentiated mature neurons during transplantation, ES derived neuronal progenitor cells (NPC) are the preferred cell types in neural degenerative disease research [4,5,6,7]
We further studied whether the effect of monolayer cells or mouse embryonic fibroblasts (MEFs) on sphere cell proliferation is a result of cell-cell contact or secreted cytokines
We found that on day 14, stage 4 cells derived from mouse ES can be divided into sphere cells that are neural progenitors, and monolayer cells that are supporting cells playing an important role in accelerating proliferation, reducing apoptosis and prompting differentiation [29]
Summary
Mouse embryonic stem cells (ES) have the potential to differentiate into many cell types and are considered potential cell therapy candidates to treat neurodegenerative diseases [1,2,3].To avoid teratoma formation in ES cells and prevent damage to fully differentiated mature neurons during transplantation, ES derived neuronal progenitor cells (NPC) are the preferred cell types in neural degenerative disease research [4,5,6,7]. Given SDIA method, ES cells cultured on PA6 or MS5 feeder cells for a specific period are used as NPCs [14,15,16,17,18]. The critical time when the neural-progenitors are fully competent to undergo neurogenesis and the time of their isolation from other surrounding cells that are not undergoing neurogenesis are yet to be determined Can these more committed neural progenitors be passaged without losing their potential to differentiate into neurons? The fate and function of cells that do not undergo neurogenesis is yet another interesting question to be answered Are these cells helpful in the differentiation of NPCs into neurons or are they byproducts of the differentiation?. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells
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