Protoplasmic Astrocytes Enhance the Ability of Neural Stem Cells to Differentiate into Neurons In Vitro

Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific marker β-tubulin III, were dramatically increased at 7 days in the co-culture condition. Blocking the effects of brain-derived neurotrophic factor (BDNF) with an anti-BDNF antibody reduced the number of neurons differentiated from NSCs when co-cultured with protoplasmic astrocytes. In fact, the content of BDNF in the supernatant obtained from protoplasmic astrocytes and NSCs co-culture media was significantly greater than that from control media conditions. These results indicate that protoplasmic astrocytes promote neuronal differentiation of NSCs, which is driven, at least in part, by BDNF

The growth and differentiation of NSCs with different media at 3 days <i>in vitro</i>.

<p>(A) Differentiation of NSCs co-cultured with protoplasmic astrocytes. (B) Differentiation of NSCs in NB+N2 medium. (C) Differentiation of NSCs in co-culture+BDNF antibody medium. (D) Differentiation of NSCs in DMEM/F12+10% FBS medium. The scale bar = 150 µm.</p

BDNF secretion in supernatant solution of different media.

<p>Units = pg/ml for all data in the table.</p><p>N = 3 for all groups;</p>*<p>indicates p<0.05 compared to the protoplasmic astrocyte group.</p

Quantification of differentiated cells cultured in various media at 7 days <i>in vitro</i>.

<p>Protoplasmic astrocytes grown in co-culture medium (A), NB+B27 medium (B), co-culture medium+BDNF antibody (C), and DMEM/F12+10% FBS medium (D). Scale bar = 75 µm. (E) Quantification of differentiated neurons in the various media conditions. β-tubulin III staining (green) indicates neurons; GFAP staining (red) indicates astrocytes. The nuclei were counterstained with DAPI (blue). *p<0.05 indicates statistical significance compared with the D/F+FBS group. ^# p<0.05 indicates statistical significance compared with the co-culture+antibody group.</p

Identification of NSCs and protoplasmic astrocytes <i>in vitro</i>.

<p>(A) Representative photomicrograph of neurospheres in culture. (B) Immunocytochemical staining of purified NSCs with Nestin(bar = 82.91 µm). (C) Immunocytochemical staining of NSCs with anti-Brd-U antibody (bar = 48.93 µm). (D) Immunocytochemical staining of differentiated cells from NSCs (green indicates neuron-specific label β-tubulin; red indicates the astrocyte-specific label GFAP; blue indicates the nucleus-specific label DAPI; bar = 75 µm). (E) Representative photomicrograph of purified protoplasmic astrocytes (×100). (F) Immunocytochemical staining of purified protoplasmic astrocytes with GFAP. (G) Nucleus staining of purified protoplasmic astrocytes with DAPI. (H) Merge of F and G. The scale bar = 37.5 µm for F, G, and H.</p

Western blot analysis of β-tubulin III protein in differentiated cells cultured with various media at 7 days <i>in vitro</i>.

<p>(A) Western blots showing the relative amount of β-tubulin <b>III</b> protein in all groups. β-actin was used to control for loading. (B) Data represent mean ± SD of three independent experiments. *p<0.05 indicates statistical significance compared with the D/F+FBS group. ^# p<0.05 indicates statistical significance compared with the co-culture+antibody group.</p

Integration of metabolome and transcriptome reveals flavonoid accumulation in the intergeneric hybrid between Brassica rapa and Raphanus sativus

Brassica rapa and Raphanus sativus are two important edible vegetables that contain numerous nutritional ingredients. However, the agronomic traits and nutritional components of the intergeneric hybrid of B. rapa and R. sativus remain poorly understood. In this study, we used a stably inherited intergeneric hybrid of B. rapa and R. sativus as a model to study its metabolome and transcriptome profiles. Morphological and cytological analysis showed the intergeneric hybrid had the expected chromosome number and normal meiosis behavior. Moreover, the metabolome analysis showed multiple important secondary metabolites, including flavonoids and glucosinolates, were significantly upregulated in the hybrid. Furthermore, transcriptome data revealed that the expression level of the important genes involved in phenylpropanoid and flavonoid pathways was significantly upregulated in the hybrid. Ultimately, our data indicate the intergeneric hybrid will be a valuable bioengineering resource and promise to become a new-type hybrid vegetable with great medicinal value in future