Figure 1.
Isolation of A2B5+ cells and formation of neurospheres in the proliferation condition.
(A): FACS confirmed a high purity of A2B5+ MACS separation. A2B5+cell group after MACS separation contained 90.21% of A2B5+ cells (right). The 1.47% population is the same cells without secondary antibody. (B): A2B5+ cells cultured in ITSFn formed numerous neurospheres at one week after. (C): Secondary neurospheres were derived from cultured A2B5+ cells, after monthly passages. (D): A2B5+ cells were plated onto fibronectin to record SOCE in proliferation medium, (E): One or no sphere was found in cultured A2B5- cells until 4 weeks (F): Neurospheres were A2B5+/Nestn+. (G and H): Cultured A2B5- population consists of already-differentiated neuron, astrocyte, and oligodendrocyte. Scale bars, 50 µm.
Table 1.
Differential expression of TRPC subtype between differentiated cells and A2B5 + NPCs.
Figure 2.
Identification of neural stem cell characteristics of A2B5+ NPCs and differentiation of A2B5+ NPCs into mature neuronal and glial cells.
Many cells in the neurospheres stem cell-like characteristics. (A,C,and D): Neurosphere was A2B5+/Nestn+, GFAP+/Nestin+, and A2B5+/Tuj1+ confirming their stem cell-like characteristics. Although there is a difference in intensity, each single channel describes co-expression (B): Two percent of A2B5+ NPCs adhered to plates and were A2B5+/Nestn+. (E): Many cells in these cultures manifested progressive neuronal morphological maturation during 3 weeks in differentiation condition. (F-I): A2B5+ NPCs cultured in differentiation media developed cellular processes and differentiated into neuronal cells (MAP2+/Tuj1+), astrocytes (GFAP+) and oligodendrocytes (O4+). (J and K): The differentiated neuronal cells were also positive for calbindin D28k, intracellular calcium-binding proteins of the EF-hand related to calmodulin and troponin-C. Scale bars, 50 µm.
Figure 3.
High KCl-induced Ca2+ transient in A2B5++ NPCs and differentiated neuronal cells.
(A, B): Cells were pre-loaded with Fura 2-AM, washed and pre-incubated for at least 10 min prior to the addition of KCl (133 mM) for 60 sec. Upper panel show false-colour images of the cells illuminated at 340 nm. Lower trace shows high KCl-induced Ca2+ transient, in A2B5+NPCs (A) and differentiated neuronal cells (B) respectively. The time of KCl addition is indicated by the red. Error bars represent SEM. The data are representative of at least 3 separate experiments.
Figure 4.
TG-stimulated Ca2+-influx measured in A2B5+ NPCs versus differentiated neuronal cells.
(A): In the absence of calcium, Ca2+ stores were released with 1 µM thapsigargin and then 2 mM Ba2+ added. TG-stimulated Ba2+ influx was monitored in A2B5+ NPCs and differentiated neuronal cells. (B): TG-stimulated Ba2+ influx was measured for the control (0 µM) or the experimental groups for which 2-APB (50 or 100 µM) was added into the camber. After store depletion, TG-stimulated Ba2+ influx was inhibited by the addition of 2-APB. Each trace is the average of A2B5+ NPCs or differentiated neuronal cells. The time of Ba2+ addition is indicated by the dark grey and that of 2-APB is done by the grey. Error bars represent SEM. The data are representative of at least 3 separate experiments.
Figure 5.
Effect of 2-APB on TG-stimulated Ca2+-influx and cell viability in differentiated neuronal cells.
The A2B5+ NPCs were cultured in NM media supplemented with 10, 50 and 100 µM 2-APB for five days, and then the cells were switched to media without 2-APB. (A): After 3 weeks, TG-stimulated Ba2+ entry was monitored in A2B5+ NPCs and differentiated neuronal cells. After store depletion, TG-stimulated Ba2+ influx was inhibited by the addition of 2-APB in differentiated media. Each trace is the average of A2B5+ NPCs or differentiated neuronal cells. The time of Ba2+ addition is indicated by the dark grey and that of 2-APB is done by the grey. Error bars represent SE. The data are representative of at least 3 separate experiments. (B): After 3 weeks, the A2B5+ NPCs treated with 10 µM 2-APB normally differentiated into mature neuronal cells. However, the rate of differentiation of the cells significantly reduced in the media supplemented with 50 µM 2-APB, and the cells hardly differentiated into mature neuronal cells in the media supplemented with 100 µM 2-APB. (C): The proliferation assay was done to show effects of SOCE inhibitor on the fate determination and survival of A2B5+ NPCs in the differentiation condition. For proliferation assay, the A2B5+ NPCs were treated with 2-APB at the indicated concentrations (10, 50 and 100 µM) 24 h after plating. Data was expressed as the mean optical density of 3 readings. Error bars indicate the standard deviation. Scale bars, 50 µm.
Figure 6.
Effect of siRNA targeting TRPC5 and TRPC6 on neuronal differentiation of NPCs in the differentiation conditions.
(A, B): A2B5+ NPCs were transfected with siRNA against TRPC5, TRPC6, and both of them, respectively, and cultured in both proliferation and differentiation condition for 3 weeks. The proliferation rate in the treated cells was lower than control, and there was no significant difference in morphology or immunophenotype. (C): Confocal microscopic image shows that most cells treated with siRNA of TRPC5 and TRPC6 were Nestin+/A2B5+ in the differentiation condition. (D): To assess transfection efficiency, the BLOCK-iT™ Alexa Fluor® Red Fluorescent Oligo was used. Although complete knockdown is difficult, prevalent red fluorescence proved high transfection efficiency. Scale bars, 50 µm.
Figure 7.
Effect of expressing siRNA targeting TRPC5 and TRPC6 on TG-stimulated Ca2+-influx in the neuronal differentiation.
A2B5+ NPCs were transfected with siRNA of TRPC5, TRPC6, and both of them, respectively and cultured in the differentiation condition for 3 weeks. (A-C): TG-stimulated Ba2+ entry was monitored in control cells and cells transfected with siRNA of TRPC5 (A) or TRPC6 (B) or both of them (C). TG-stimulated Ba2+ influx was inhibited by siRNA of TRPC5 as well as siRNA of both TRPC5 and TRPC6. After recording, we performed single cell PCR in neuronal differentiation (black; siRNA-untransfected cell, red; siRNA-transfected cell). Cell transfected with siRNA of TRPC5 was Nestin+/MAP2-, although that transfected siRNA of TRPC6 was Nestin-/MAP2+. (D): For average data, A2B5+ NPCs were transfected with siRNA and cultured in the differentiation condition. TG-stimulated Ba2+ influx was significantly inhibited by siRNA TRPC5 and TRPC5+TRPC6. The time of Ba2+ addition is indicated by the dark grey. Error bars represent SEM. The data are representative of at least 3 separate experiments. *, P<0.01 compared with corresponding control without siRNA TRPC.