EXP. 1 aimed to investigate and compare the differentiation ability of mouse embryonic stem (ES) and induced pluripotent stem (iPS) cells into neuronal lineage using embryoid body (EB) formation. We determined for the first time the differentiation potential of mouse Sleeping beauty transposon (SB)-mediated iPS cells to form neuronal progenitor cells (NPCs) and neurons. Both mouse ES and SB-iPS cells exhibited the neuronal characteristics detected by Pax6, Nestin and Tuj-1. However, SB-iPS cells produced less nestin-positive cells than ES cells (6.12±1.61 vs 74.36±1.65, respectively). In conclusion, ES and SB-iPS cells show a difference in their capacity to differentiate towards the neuronal lineage. Even though the neuronal differentiation rates of iPS cells needs to be improved, our results are encouraging and show that SB-iPS cells are capable of forming neurons.
EXP. 2 aimed to investigate the ability of D11 Rybp-deficient ES cells to generate the neuronal population in vitro via EB formation comparing with R1 wild type ES cell line. After 8 days of EB suspension culture, the rosette-liked structures contained Pax6 and Musashi-1 positive cells has been found in both R1 and D11-derived EBs. Several neuronal gene expression both NPCs (Nestin and Musashi-1) and neurons specific markers (Tuj-1 and NeuroD1) of D11 derived cells were lower than those in controls. The percentage of nestin-positive cells derived from D11 ES cells were found to be significantly lower than those derived from R1 ES cells (42.62±8.84 vs 80.42±4.27, respectively This results suggested that Rybp-deficient ES cells can be induced to generate NPCs and neurons in vitro. However, the efficiency was poorer when compared to the wild-type.
EXP. 3 aimed to investigate the effect of TGF-β1 inhibitor using A83-01 on the development and neuronal differentiation potential of mouse ES cells during EB suspension period. Our results showing that the diameters of EBs treated with A83-01 (592.6±27 µm) were significantly smaller than the control group (631.7±14.1 µm, P< 0.01). Eight-day-old EBs derived from the two groups similarly contained a complex network of mixed population of early and mature neurons as indicated by Pax-6 and NeuN staining, respectively. Comparing to the control, TGF-β1 inhibitor potentially suppressed transcription factor Oct4 while rapidly up-regulation of neuronal associated genes (Sox1 and MAP2). Furthermore, this inhibitor also down-regulated astrocyte related gene (GFAP) compared favorably to non-treated control. It is concluded that selective TGF-β1/ALK inhibitor efficiently stimulates the cell fate alteration from ES state toward neuronal lineages.
EXP. 4 aimed to examine the efficacy of motor neuron differentiation of ES cells treated with TGF-β1 inhibitor (A83-01) during EB suspension culture. The neuronal characteristic of 8 day-old EBs was examined by immunohistochemical analysis on cross-sectioned EBs. Our result demonstrated that the aggregated ES cells differentiated into NPCs as they expressed Pax-6 and Tuj-1. Quantiative RT-PCR analysis revealed that treatment the EB with selective TGF-β1 inhibitor up-regulated the motor neuron progenitor Olig2 at higher levels than that obtained from the control (4.20±0.20 vs. 0.73±0.09, P<0.01). In contrast, mRNA expression levels of motor neuron Hoxc8 of a control group were significantly higher than the TGF-β1 inhibitor treated group (14.73±2.6 vs. 2.37±0.42, P<0.01). the differentiated cells expressed a neuronal marker (Tuj-1), motor neuron progenitor marker (Olig2), developing motor neuron progenitor (Isl-1) and functional motor neuron marker (ChAT). We concluded that TGF-β1 signaling appears to affect generation and differentiation fate of motor neuron progenitors.