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Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.
Citation:
Majunder, A., S. K. Dhara, J. HARRILL, W. R. MUNDY, AND S. L. Stice. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells. Presented at Society for Neuroscience Annual meeting, San Diego, CA, November 13 - 17, 2010.
Impact/Purpose:
In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to result in adverse health effects. Cell cultures derived from nervous system tissue can be used in automated, high content screening assays to examine the chemical effects on neuronal outgrowth. However, it is not clear which neuronal cell culture is the most appropriate model for screening. The NAS report on "Toxicity Testing in the 21st Century" emphasizes the use of in vitro models derived-from or based-upon human biology. This study characterizes the use of LiF to influence the growth and phenotype of the hN2TM human embryonic stem cell (hESC)-derived neural cells.
Description:
Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer culture and readily differentiate into neural cells. Previous studies have shown that hNP cells have a high potential for both basic and applied neural research. Cytokines that enhance or otherwise modify the differentiation potential of these cells are thus of great interest. Human Neural Progenitor cells (hNP1, ArunA Biomedical) were differentiated for 2 weeks either in presence or absence of L1F, and assayed for neural progenitor and neuronal markers by flow cytometry and immunocytochemistry. Further, differentiated cells were assayed for viability and neurite outgrowth, using a Cellomics Array Scan VT1 platform. Flow-cytometric analysis demonstrated that before differentiation, > 80% of hNP cells expressed neural progenitor markers Nestin (82%±2.6) and SOX2 (88.5%±0.8). After 2 weeks of differentiation decreases in Nestin expression was similar both in the presence (42.9%±4.3, P<0.001) and absence (42.7%±2.7, P<0.0002) of L1F. However, the decrease in SOX2 expression was significantly greater (P<0.02) in presence of L1F; control: 88.5%±0.8, L1F+: 41.1%±3.2 (P<0.001), L1F-: 59.6±1.5 (P<0.0002) . Differentiation was also associated with changes in the expression of neuronal markers MAP2, Tuj1 and HuC/D. Cells differentiated with and without L1F expressed the neuronal marker TuJ!. In the absence of L1F a subset of Tuj1+ cells expressed MAP2 (11.1%±1.3) and/or HuC/D (8.9%±O.9). However, in the presence of L1F, the proportion 'OfMAP2+ cells (37.9%±1.3, P<0.003) and HUC/D+ cells (38.9%±1.0, P<0.002) were significantly higher than in cells differentiated in the absence of L1F. High Content Image analysis demonstrated quantitative differences in cell viability and neurite outgrowth between cells differentiated either in presence or absence of L1F. The average number of neurites per neuron significantly increased in L1F+, but not L1F-, cultures between 2 h (0.23 ± 0.09) and 28 h (0.58 ± 0.09, P< 0.0001). Likewise, the percentage of cells with at least one neurite increased significantly in L1F+, but not L1F-, cultures between 2 h (3.4 % ± 1.9) and 48 h (12.3 % ± 2.5, P < 0.0001). Moreover, total neurite length per viable cell increased significantly over time in both L1F+ (2 h: 5.2 ± 2.3 11m, 48 h: 23 ± 3.7 11m) and L1F-(2 h: 1.6 ± 0.9 11m, 48 h: 3.9 ± 2.3 11m) cultures. Similarly, average neurite length increased significantly over time in both L1F+ (2 h: 21.2 ± 211m,48 h: 39.3 ± 2.4 11m) and L1F-(2 h: 20.5 ± 3.2 11m, 48 h: 32.6 ± 6.7 11m) cultures. In all cases, increases in neurite outgrowth parameters were greater in magnitude in L1F+ as compared to L1F-cultures. We conclude that L1F plays an important role during differentiation of hNP cells, significantly enhancing the proportion of MAP2+ and HuC/D+ neurons, cell viability, and neurite outgrowth. (This abstract does not necessarily reflect USEPA policy.)