Bioimaging of microRNA124a-independent neuronal differentiation of human G2 neural stem cells

Abstract

Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell-specific miRNAs in living organisms. Here, we examined the microarray-based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron-specific miR-9 and miR-124a during neuronal differentiation of G2 cells in vitro and in vivo. Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline-dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real-time PCR showed that expression of endogenous mature miR-9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron-specific mature miR-124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons. However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.

Keywords: Fluc, Firefly Luciferase; G2 human neural stem cells; Gluc, Gaussia luciferase; In vivo imaging; Neuronal differentiation; Reporter gene; miR, microRNA; microRNAs.

Fig. 1

Characterization of neuronal differentiation in G2 human neural stem cells. (A) Schematic illustration of the tetracycline-dependent neuronal differentiation of the G2 cell line. The HB2.G2 cell line was infected with two different viral vector systems, CMV-regulated tetracycline regulatory gene and tetracycline responsive element-dependent v-myc expression vector. In the presence of doxycycline, tTA and doxycycline complex binds to 7× tetO sequences within the tetracycline responsive element, causing the v-myc oncogene to be expressed and thereby maintaining the self-renewal of the immortalized human neural stem cells, HB2.G2. In contrast, the neuronal differentiation of the G2 cell line can be induced by the removal of doxycycline. (B) G2 cells maintained in DMEM containing 10% FBS with doxycycline (2 μg/ml) were transferred into DMEM containing 5 ng/ml of epidermal growth factor (EGF) in serum-free conditions to induce neuronal differentiation. Neurite-like processes were observed at 4 days after induction of neuronal differentiation in G2 cells. (C) G2 cells at 6 days after induction of the neuronal lineage showed an immunoreactive pattern to Tuj1, MAP2, GFAP, and O4. While a high fluorescence signal using the Oct4 antibody was detected in undifferentiated G2 cells, only very weak non-specific signals of Oct4 were found in differentiated G2 cells due to reduced expression of this protein. Scale bar, 20 μm.

Fig. 2

Microarray-based microRNA expression analysis during neurogenesis in G2 cells. Clustering analysis represented relative expression levels of miRNAs at the neuronal differentiation stage in G2 cells. 68 miRNAs showing both a greater than twofold increase in expression between differentiation time points and a gradual increase expression pattern during progression of neuronal differentiation were selected and classified into four differentiation time point groups. Of the 68 miRNAs, several miRNAs including the miR-335, miR-26 and let-7 families showed distinct expression patterns at each stage of neuronal differentiation in G2 cells.

Fig. 3

In vitro validation of miR-9 and miR-124a expression patterns in neuronal differentiation of G2 cells. (A) Small RNA was extracted from G2 cells at 0, 2, 4, and 6 days after neuronal induction by the withdrawal of doxycycline. Quantitative RT-PCR analysis showed that the endogenous mature miR-9 expression level was gradually increased following the induction of neuronal differentiation of G2 cells. In contrast, there was no significant change in the expression level of mature miR-124a during neurogenesis. U6 snRNA was used as an internal control. Results are expressed as the mean (SD) of triplicate experiments. (B) qRT-PCR analysis was performed to assess the expression level of an miR-124a target gene using specific primers for VAMP3, a putative target gene for miR-124a. Similar expression patterns of VAMP3 were detected at each neuronal differentiation time point in G2 cells. (C) The constructed CMV/Gluc/3×PT_miR-9 (or CMV/Gluc/3×PT_miR-124a) plasmid vector was transiently transfected into undifferentiated G2 cells, and Gaussia luciferase activity was measured at each neuronal differentiation time point in G2 cells. A gradual decrease in the level of Gaussia luciferase was found in the treatment group of CMV/Gluc/3×PT_miR-9, due to the increase in expression of miRNA-9 during neuronal differentiation of G2 cells. CMV/Gluc/3×PT_miR-124a-treated G2 cells showed similar activity levels of Gaussia luciferase until 2 days after neuronal differentiation. (^*P < 0.05, ^**P < 0.01).

Fig. 4

In vivo monitoring of miR-9 (or miR-124a) expression levels during the progression of neuronal differentiation of G2 cells in nude mice. (A and B) CMV/Gluc/3×PT_miR-9 and CMV-Fluc (firefly luciferase vector regulated by CMV promoter) were co-transfected into undifferentiated G2 cells, and harvested G2 cells were incorporated into a pre-wetted PLLA scaffold. To induce neuronal differentiation, G2 cells were resuspended with PBS buffer without doxycycline. As soon as the G2 cell/PLLA complex was surgically implanted into the thighs of mice, the in vivo Gaussia luciferase signal was visualized and showed that a more strongly decreased signal was present in the group with induced neuronal differentiation in the right thigh of the mice, compared to that in the undifferentiated group (left thigh). In contrast, CMV/Gluc/3×PT_miR-124a-transfected G2 cells inside the PLLA scaffold in nude mice showed similar Gaussia luciferase signals between undifferentiated and differentiated G2 cells until 2 days after neuronal differentiation. Firefly luciferase signal was used to detect the survival of G2 cells for an in vivo normalization study. (C) Quantitative ROI data acquired from bioluminescence image results showed a continuously decreasing pattern for the ratio value between undifferentiated (Undiff.) and differentiated (Diff.) groups in CMV/Gluc/3×PT_miR-9-transfected G2 cells at 3 days, compared to that of CMV/Gluc/3×PT_miR-124a-transfected G2 cells (^*P < 0.05, ^**P < 0.01).