Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth

Abstract

Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell-cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In this study, we found that MSC treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR-133b) level in the ipsilateral hemisphere. In vitro, miR-133b levels in MSCs and in their exosomes increased after MSCs were exposed to ipsilateral ischemic tissue extracts from rats subjected to MCAo. miR-133b levels were also increased in primary cultured neurons and astrocytes treated with the exosome-enriched fractions released from these MSCs. Knockdown of miR-133b in MSCs confirmed that the increased miR-133b level in astrocytes is attributed to their transfer from MSCs. Further verification of this exosome-mediated intercellular communication was performed using a cel-miR-67 luciferase reporter system and an MSC-astrocyte coculture model. Cel-miR-67 in MSCs was transferred to astrocytes via exosomes between 50 and 100 nm in diameter. Our data suggest that the cel-miR-67 released from MSCs was primarily contained in exosomes. A gap junction intercellular communication inhibitor arrested the exosomal microRNA communication by inhibiting exosome release. Cultured neurons treated with exosome-enriched fractions from MSCs exposed to 72 hours post-MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR-133b to neural cells via exosomes.

Figure 1

MSC administration increases the miR-133b after stroke. Real-time reverse-transcribed PCR assay showed microRNA 133b (miR-133b) significantly decreased in the ipsilateral brain tissues of rats subjected to MCAo compared to the normal rats, and MSC administration significantly increased the miR-133b level in the MCAo rat brain. NOR: normal rats, MCAo: rats subjected to MCAo, MSCs: rats subjected to MCAo with MSC administration. *, p < .05 compared with NOR. ^#, p < .05 compared with MCAo (n = 6 per group). Abbreviations: MCAo, middle cerebral artery occlusion; MSC, mesenchymal stromal cell.

Figure 2

Culture of mesenchymal stromal cells (MSCs) with ischemic tissue brain extracts increased the miR-133b expression in MSCs and their generated exosomes. Real-time reverse-transcribed PCR data showed that compared to the normal rat brain tissue extract treated group, miR-133b levels in MSCs exposed to middle cerebral artery occlusion (MCAo) brain tissue extracts and their exosomes were significantly increased, and the exosome miR-133b level reached a peak after MSCs cultured with the 72 hours post-MCAo brain tissue extract (A). The TEM image showed that the morphology of MSC released exosomes within a size range of 40–100 nm (B). Western blot detected that the exosome marker protein, Alix, was primary located in the range of density from 1.19 to 1.221 g/ml (C), and miR-133b also primary presented a high level in these two density fractions (D). MC, EC: MSCs and their exosomes exposed to the normal rat brain tissue extract as controls; M24, M72, M168, E24, E72, and E168: MSCs and their exosomes treated with the brain tissue extracts after MCAo at 24, 72, and 168 hours, respectively. *, p < .05, compared with MC in (A) and compared with 1.032 g/ml density fraction in (D). ^#, p < .05 compared with EC (n = 3 per group). Abbreviation: miR-133b, microRNA 133b.

Figure 3

MSC exosomes containing microRNA 133b (miR-133b) are transferred to neural cells. Real-time reverse-transcribed PCR revealed that compared to exosome-enriched fraction deprived media, exosome-enriched fractions collected from MSCs exposed to middle cerebral artery occlusion (MCAo) brain extracts significantly increased the miR-133b level in astrocytes (A) and neurons (B), respectively. Compared with astrocytes and neurons treated with exosome-enriched fractions from MSCs exposed to normal rat brain tissue extracts, miR-133b levels were significantly increased in astrocytes and neurons treated with exosome-enriched fractions from MSCs exposed to 24, 72, or 168 hours post-MCAo brain tissue extracts, and the miR-133b level was maximum at the 72 hours post-MCAo brain tissue extract exposure (A, B). To further confirm that the increased parenchymal cell miR-133b is attributed to the transfer of exosomes generated by MSCs, MSCs were transfected with a miR-133b inhibitor and the miR-133b level was significantly decreased in the MSCs and their exosome-enriched fractions (C). Compared with normal cultured astrocytes, treatment with exosome-enriched fractions from MSCs transfected with the miR-133b inhibitor negative control significantly increased the miR-133b level in the astrocytes, but this increase in miR-133b was significantly diminished when astrocytes were treated with exosome-enriched fractions from MSCs transfected with miR-133b inhibitor. The exosome-enriched fraction deprived media either from MSCs transfected with miR-133b inhibitor negative control or from MSCs transfected with miR-133b inhibitor had no obvious effects on the miR-133b level in astrocytes (D). MeC, EC: media and exosome-enriched fractions from MSCs treated with the normal rat brain tissue extracts as controls, respectively; Me24, Me72, Me168, E24, E72, and E168: media- and exosome-enriched fractions from MSCs treated with the MCAo rat brain tissue extracts for 24, 72, and 168 hours, respectively; NOR: normal cultured astrocytes; MeN, MeI: astrocytes treated with exosome-enriched fraction deprived media from MSCs transfected with miR-133b inhibitor negative control and miR-133b inhibitor, respectively; EN, EI: astrocytes treated with exosome-enriched fractions from MSCs transfected with miR-133b inhibitor negative control and miR-133b inhibitor, respectively. *, p < .05 compared with EC in (A) and (B); compared with negative control, respectively, in (C); compared with NOR in (D). ^#, p < .05 compared with MeC in (A) and (B); compared with EN in (D) (n = 3 per group). Abbreviation: MSC, mesenchymal stromal cell.

Figure 4

Exosomes mediate the microRNA (miRNA) communication between noncontacting MSCs and astrocytes mainly through the gap junction intercellular communication. Data show that cel-miR-67 is expressed in cel-miR-67 expression plasmid-transfected MSCs (A). Luciferase expression in the astrocytes was significantly increased after coculture with MSCs. The luciferase expression significantly decreased in astrocytes cocultured with cel-miR-67 expression plasmid-transfected MSCs compared with negative control cel-miR-239 expression plasmid-transfected MSCs. Fifty nanometer filter but not the 100 nm filter significantly inhibited the decrease of luciferase expression in astrocytes cocultured with cel-miR-67 expression plasmid-transfected MSCs. miRIDIAN miRNA inhibitor of cel-miR-67 counteracted the effect of cel-miR-67 on the decrease of luciferase expression in astrocytes. Furthermore, 150 μM carbenoxolone also substantially inhibited the downregulation of luciferase expression in astrocytes cocultured with cel-miR-67 expression plasmid-transfected MSCs (B). The cel-miR-67 was primarily located in exosome-enriched fractions with very low levels in the media (C).⁺, p < .01 compared with cel-miR-67 transfected medium group. *, p < .05 compared with the astrocytes cocultured with cel-miR-67 expression plasmid-transfected MSCs; ^#, p < .05, compared with the 100 nm filter applied.⁺, p < .01 compared with cel-miR-67 transfected medium group (n = 3 per group). Abbreviation: MSC, mesenchymal stromal cell.

Figure 5

GJIC mediates the exosomal microRNA transfer via affecting the exosome release from MSCs but not their intake into astrocytes. Data showed that the cel-miR-67 level was significantly decreased in the exosome-enriched fractions released from MSCs treated with 150 μM carbenoxolone (A), but the cel-miR-67 level in MSCs was not changed (B). Concomitantly, after 150 μM carbenoxolone, the total protein that indicates exosome quantity significantly decreased (C). The exosome-enriched fractions from cel-miR-67 expression plasmid-transfected MSCs downregulated the luciferase expression in cel-miR-67 pMIR-reporter luciferase plasmid-transfected astrocytes, but treatment of astrocytes with 150 μM carbenoxolone had no obvious effect on the luciferase expression downregulation by the exosome released from cel-miR-67 expression plasmid-transfected MSCs (D). EC: exosome-enriched fractions from non-carbenoxolone-treated MSCs, EI: exosome-enriched fractions from carbenoxolone-treated MSCs, MC: non-carbenoxolone-treated MSCs, MI: carbenoxolone-treated MSCs, AN: non-carbenoxolone-treated astrocytes without exosome-enriched fractions treatment, AC: non-carbenoxolone-treated astrocytes with exosome-enriched fractions treatment, AI: carbenoxolone-treated astrocytes with exosome-enriched fractions treatment. *, p < .05 compared with EC. ^#, p < .05 compared with AN (n = 3 per group).

Figure 6

Exosomal miR-133b from middle cerebral artery occlusion (MCAo)-modified mesenchymal stromal cells (MSCs) increases neurite outgrowth. Cortical neuron neurite outgrowth is shown using fluorescence microscopy (A). Compared with the normal cultured (nonexosome treatment) group, administering exosome-enriched fractions from MSCs exposed to the 72 hours post-MCAo brain extract significantly increased the neurite branch number (B) and total neurite length (C). The miR-133b inhibitor substantially decreased the neurite branch number and total neurite length compared with normal cultured or exosome-enriched fraction treated neurons. Western blot (D) data showed that the RhoA expression significantly decreased after MSC exosome treatment in control neurons, and the RhoA level significantly increased in miR-133b inhibitor transfected neurons (E). Data also showed that increased miR-133b expression by miR-133b direct transfection (F) in neurons significantly increased the neurite branch number (G) and the total neurite length (H), respectively. NG: the miR-133b inhibitor negative control-transfected neurons, EX+NG: the miR-133b inhibitor negative control-transfected neurons treated with exosome-enriched fractions from MSCs exposed to MCAo brain extracts, IN: the miR-133b inhibitor-transfected neurons, EX+IN: the miR-133b inhibitor-transfected neurons treated with exosome-enriched fractions from MSCs exposed to MCAo brain extracts. Scale bars = 50 μm. *, p < .01, compared with NG; ^#, p < .01, compared with EX+NG. Data are presented as mean ± SE (neurons n = 50 per group). Abbreviation: miR-133b, microRNA 133b.