The exogenous addition of osteogenically derived EVs outperf

The exogenous addition of osteogenically derived EVs outperformed the effect of current osteoinductive strategies in terms of the type and/or intensity of the signaling, with the early activation of the key osteogenic commitment genes SP7 (Figure 3B) and BMP2 (Figure 3C), considered necessary and sufficient to induce bone formation (Noel et al., 2004). In addition, EVs induced a transient upregulation of downstream matrix-associated genes and protein (Figures 3D, 3E, and 2C) that may be required to promote cell-cell and cell-matrix interactions and support long-term differentiation and mineralization (Langenbach and Handschel, 2013). Notably, no major differences were noted between the exogenous addition of OM- or RUNX2-EVs, suggesting that the inherent cargo of hBMSC-EVs may also contribute to the observed effect. Indeed, the selective cargo of EVs derived from uncommitted hBMSCs suggests their involvement in the osteogenic differentiation. MSC-EVs were described to carry tRNA species putatively targeting the transcription factor RUNX2 and SOX11 (Baglio et al., 2015), miRNA, miR-22, that indirectly targets RUNX2 (Baglio et al., 2015) and several proteins involved in the BMP, mitogen-activated protein kinase, transforming growth factor β, and Wnt pathways (Kim et al., 2012). Furthermore, our experimental design, comprising the addition of a pool of OM-/RUNX2-EVs to hBMSCs, does not exclude the contribution of the EVs derived from recipient JWH 073 and a possible synergistic signaling.
Experimental Procedures
Authors Contributions
Acknowledgments The authors thank the financial support of QREN (RL1-ABMR-NORTE-01-0124-FEDER-000016 and RL3–TECT–NORTE-01-0124-FEDER-000020) co-financed by North Portugal Regional Operational Program (ON.2–O Novo Norte), under the NSRF, through the ERDF; the European Union\'s Seventh Framework Program (FP7/2007–2013), Grant No. REGPOT-CT2012-316331- POLARIS; and the laboratory assistance of Elsa Ribeiro, Magda Graça, and Sónia Zacarias.
Introduction Embryonic stem cells (ESCs), derived from pre-implantation embryos, share two unique properties: the ability to grow indefinitely in culture and to differentiate into all cell types (Evans and Kaufman, 1981). ESC self-renewal is regulated by a complex network of transcription factors and signaling pathways (Ng and Surani, 2011). The transforming growth factor β (TGF-β) pathway plays a pivotal role in cell fate determination during mouse embryonic development, such as primitive streak formation (Oshimori and Fuchs, 2012). Both Smad1/5/8 and Smad2/3 branches are involved in pluripotency and differentiation of ESCs. Activin/Nodal/Smad2/3 signaling is important for proper differentiation toward the mesendoderm lineage (Fei et al., 2010), whereas bone morphogenetic protein (BMP)/Smad1/5/8 signaling promotes self-renewal in mouse ESCs (mESCs) (Ying et al., 2003). Accumulating evidence reveals that microRNAs (miRNAs) are crucial in controlling the pluripotent stem cell state. Their important regulatory role in mouse and human ESCs has been identified using Dicer and DGCR8 knockout mice. Dicer and DGR8 deletion resulted in embryonic lethality (Bernstein et al., 2003), while DGCR8-deficient mESCs were viable but defective in proliferation and differentiation (Wang et al., 2007). Several studies reported on miRNAs maintaining the ESC state, whereas others reported miRNAs as promoting differentiation. miR-290–295 and miR-302–367 clusters include the most abundant miRNAs in mouse and human ESCs and are characterized as ES cell-specific cell cycle miRNAs (Gangaraju and Lin, 2009; Melton et al., 2010). In contrast, miR-134, miR-296, and miR-470 are related to ESC differentiation and self-renewal silencing (Tay et al., 2008). Although there is no doubt that miRNAs regulate ESC self-renewal and lineage commitment, their role in relevant signaling pathways that determine ESC function remains unclear. In this study, we report the identification of four miRNAs as critical regulators of ESC fate. miR-16-1 (miR-16-1/15a cluster) and miR-191 (miR-191/425 cluster), which are highly expressed in mESCs, directly target Smad2, an Activin/Nodal signaling important mediator, leading to the inhibition of mesendoderm lineage. Another miRNA expressed in the undifferentiated state, miR-23a (miR-27/24a/23a cluster), inhibits the endodermal and ectodermal differentiation. On the contrary, miR-421 (miR-421/374b/c cluster) was identified as a differentiation regulator, by suppressing BMP signaling and the critical pluripotency factor, Oct4. Altogether, the mechanisms incorporating the two branches of TGF-β signaling pathway and miRNAs are highlighted, unraveling their importance to ESC lineage commitment.