Abstract
BackgroundAlthough extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation – allowing for an increase in percentage yield of neuronal cells.ResultsUncommitted GFAP and SOX2 positive neural progenitors – so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100β-, SOX2-], or mixture of neural and non-neural cells.In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells – i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively.ConclusionWe suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells – such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.
Highlights
Extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict
GFAP positive neural progenitors (NHA) derivatives differentiating to the neuronal lineage demonstrated silencing of glial and progenitor markers [GFAP, CD44, NESTIN] expression, while those differentiating to the glial lineage showed silencing of neuronal and progenitor markers [β-III-TUBULIN, MAP2, NESTIN] expression
We have shown a co-expression of CD44, GFAP and MAP2 by means of triple staining and SOX2 expression by a single staining (Figure 1)
Summary
Extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. Expression of neuronal and glial markers during differentiation of these cells is regulated in accordance with the "model of discordant phenotypes suppression" [1,2,3] This model states that before differentiation, markers belonging to diverse lineages are expressed by stem cells or progenitors; whereas during differentiation, genes superfluous in derived lines are silenced. In accordance with this concept, uncommitted neural progenitors (NHA) co-express glial [GFAP, CD44], neuronal [β-III-TUBULIN, MAP2] and progenitor [NESTIN] markers. Expression of MAP2 and βIII-TUBULIN is definitely increased in neuronal derivatives of NHA [1,2]
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