Bone marrow stromal cells induce BMP2/4 production in oxygen-glucose-deprived astrocytes, which promotes an astrocytic phenotype in adult subventricular progenitor cells

Abstract

Bone morphogenetic proteins (BMPs) affect cell proliferation and differentiation. Astrocytes in ischemic brain are highly responsive to bone marrow stromal cell (BMSC) treatment. We investigated the effects of BMSCs on astrocytes cultured under oxygen- and glucose-deprived conditions, which in part simulate in vivo stroke conditions, to test the hypothesis that BMSCs alter astrocytic expression of BMPs which may contribute to neurological functional recovery of stroke. Quantitative real-time RT-PCR showed that the expression of BMP2/4 mRNAs decreased within ischemic astrocytes, In contrast, BMP2/4 mRNA was significantly increased after cocultured with BMSCs. Western blotting also confirmed this increase at the protein level in the medium of ischemic astrocytes after coculture with BMSCs. As a source of neural stem and progenitor cells, cultured subventricular zone (SVZ) neurospheres exposed to medium obtained from ischemic astrocytes cocultured with BMSCs were significantly enriched in cells expressing the astrocytic marker glial fibrillary acidic protein (GFAP), but not at the expense of beta-III-tubulin-positive SVZ neuroblasts. The expression of BMP2/4 subsequently increased the phosphorylation of downstream effector Smad1 and the expression of notch signal pathway-induced protein Hes1 in cultured SVZ neurospheres. BMP antagonist Noggin blocked the elevation of phosphorylated Smad1 and the expression of Hes1 as well as reducing the percentage of astrocytic SVZ progenitor cells. Our results indicate that BMSCs increase BMP2/4 expression in ischemic astrocytes. These changes enhance subventricular progenitor cell gliogenesis by activating relevant signaling pathways. BMSC-stimulated signaling of endogenous astrocytes may alter the ischemic environment, promoting remodeling of brain and hence, improve functional recovery after stroke.

Fig. 1

A: Quantitative RT-PCR mRNA expression level of BMP2, BMP4, and BMP7 in astrocytes. The BMP2 mRNA expression in ischemic astrocytes is increased when cocultured with BMSCs. The BMP4 mRNA decreased in the ischemic astrocytes and, cocultured with BMSCs, can partially recover the mRNA-expressing level of BMP4 in ischemic astrocytes compared with ischemic astrocytes alone. There are no significant changes in BMP7 mRNA expression in normal astrocytes and ischemic astrocytes cocultured with or without BMSCs. B: Protein level of BMP2/4 in various conditioned media. There is no BMP2/4 detected in the normal differentiation medium and low BMP2/4 in BMSCs alone. The expression of BMP2/4 is decreased in the medium of ischemic astrocytes; BMSCs increase the expression of BMP2/4 in the medium of ischemic astrocytes to a level comparable to that in the medium of normal cultured astrocytes. N, normal differentiation medium; M, BMSCs alone cultured medium; A, normal astrocytes alone cultured medium; AD, ischemic astrocytes cultured medium; ADM, ischemic astrocytes with BMSCs cocultured medium. *P < 0.05, **P < 0.01.

Fig. 2

Immunofluorescence shows representative GFAP-positive cells (A) and TUJ1-positive cells (B) differentiated from the SVZ neurospheres. Among all the conditioned media, the media from BMSCs alone and ischemic astrocytes alone have GFAP-positive cells comparable to those with normal differentiation medium, the media collected from normal astrocytes, and ischemic astrocytes cocultured with BMSCs obviously increase the percentage of GFAP-positive cells compare with the normal differntiation medium. The fraction of TUJ1-positive cells either did not change or increased, and, as with the GFAP positive cells, it significantly increased only when the SVZ neurospheres were cultured with medium obtained from BMSCs alone. Noggin inhibits the effect of BMSCs and decreases the percentage of GFAP-positive cells differentiated from stem and progenitor cell cultured with normal astrocyte medium and ischemic astrocytes-BMSCs coculture medium. N, normal differentiation medium; M, BMSCs alone cultured medium; A, normal astrocytes alone cultured medium; AD, ischemic astrocytes cultured medium; ADM, ischemic astrocytes with BMSCs cocultured medium. *Compared with N, P < 0.05; **compared with N, P < 0.01; ⁺⁺compared with AD, P < 0.01; ^#compared with no Noggin, P < 0.01; ^#compared with no Noggin, P < 0.001.

Fig. 3

A: Representative Western blot results show the protein level in the SVZ neurospheres cultured in the various conditioned media. B: Semiquantitative results of viarious protein levels. The levels of BMPR1a and total Smad1/5/8 are constant in the SVZ neurospheres in all culture conditions. The level of p-Smad1 in the SVZ neurospheres decrease after culture with the medium from ischemic cultured astrocytes compared with that from normal cultured astrocytes, but it significantly increased when cultured in the medium of ischemic astrocytes cocultured with BMSCs, and this increase was greater than the effect of p-Smad induced by BMSC-alone medium. A similar response of Hes1 expression in the SVZ neurospheres cultured with conditioned media is found. The expression of Hes5 exhibited no significant change. Noggin can prevent all changes, as above mentioned. N, normal differentiation medium; M, BMSCs alone cultured medium; A, normal astrocytes alone cultured medium; AD, ischemic astrocytes cultured medium; ADM, ischemic astrocytes with BMSCs cocultured medium. *P < 0.01.

Fig. 4

Coronal section sketch of a rat brain subjected to 2 hr of middle cerebral artery occlusion with BMSC treatment. IBZ, ischemic boundary zone, where the majority of BMSCs survive in the brain and localed (Chen et al., 2001). IC, ischemic core; SVZ, subventricular zone, where cells can proliferate throughout the life of the rats (Gage, 2002) and are amplified after ischemia (Chen et al., 2001).