toll like receptors However there are still some

However, there are still some challenges that need to be addressed in order for our findings to be developed as an effective treatment for autoimmunity prevention. First, although islet function was preserved in the EV-treated mice, decreased β cell mass in association with insulitis was still observed in the EV-treated mice as shown in Figures 2A and 2B, suggesting that further study is needed to optimize the injection frequency and dose to maintain the long-lasting immunomodulation effects of EVs. Furthermore, the main problem with MSC-derived EV-based therapy is that EVs are highly heterogeneous, depending on the cellular source, state, and environmental conditions. Our previous study showed that MSCs isolated from different donors exhibit huge variation in their therapeutic efficacy in suppressing inflammation in vivo, and some MSCs even fail to show any therapeutic effects in sterile inflammation-mediated disease models (Lee et al., 2014). But, we found that the therapeutic efficacy of MSCs in suppressing sterile inflammation correlates with the TSG-6 mRNA level in MSCs (Lee et al., 2014). Therefore, we selected MSCs expressing a high level of TSG-6 to prepare EVs for the current study, considering the marked donor variation of their therapeutic efficacy in vivo. However, further study is needed to investigate whether therapeutic efficacy of MSC-derived EVs correlates with their parent cells, and the TSG-6 level in MSCs can also be used as a biomarker to select the cell source for EV production. Hence, pre-selecting the most effective cellular source for EV production will help to avoid variation in the therapeutic efficacy of MSC-derived EVs and is essential for successful clinical translation. Lastly, defining the therapeutic factors responsible for the immunomodulation effect in EVs will also help to develop a biomarker to select the effective cellular source for EV preparation and provide a strategy to maximize their therapeutic efficacy, for example, by manipulating the cellular source of EVs by overexpressing the defined therapeutic factors.
Experimental Procedures
Author Contributions
Acknowledgments This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: lHI15C3134); the NIH (grant number: P40RR17447).
Introduction Hematopoietic stem cells (HSCs) are defined by their lifelong ability to self-renew and generate each blood cell type. In vertebrates, HSCs first emerge from the ventral wall of the dorsal toll like receptors (VDA) in the aorta-gonad-mesonephros (AGM) region of the developing embryo (Dzierzak and Speck, 2008). The process of de novo HSC production, termed endothelial-to-hematopoietic transition (EHT), involves the specification, budding, and egress of select hemogenic endothelial cells into circulation. As this process proceeds, hemogenic endothelial cells acquire markers of HSC identity, and once EHT is complete, begin migration to secondary niches to expand in number and differentiate (Bertrand et al., 2010; Kissa and Herbomel, 2010). Endothelial cells slated to undergo EHT are specified by sequential actions of the Hedgehog and vascular endothelial growth factor (VEGF) pathways, which in turn activate Notch (Carroll and North, 2014). Notch signaling in the VDA is responsible for runx1 induction via scl and gata2; Runx1 function is required for EHT and HSC production (Chen et al., 2009; Kissa and Herbomel, 2010; North et al., 1999). While factors necessary for VDA specification and EHT are increasingly defined (Carroll and North, 2014), the mechanisms by which hemogenic endothelium is structurally remodeled to allow hematopoietic stem and progenitor cell (HSPC) budding, egress, and subsequent migration to intermediate and adult niches are incompletely understood. In the mouse, HSCs are present in the AGM, umbilical and vitelline arteries by embryonic day 10.5 (E10.5), followed shortly by the placenta. Newly formed HSCs migrate via the circulation to the fetal liver (FL), a transient niche, to mature and proliferate. After leaving the FL, HSCs migrate to sites of adult hematopoiesis, seeding the thymus at E11.5, the spleen at E12.5, and finally the bone marrow (BM) by E15 (Dzierzak and Speck, 2008). In zebrafish embryos, de novo specified HSCs begin to bud from the VDA by 28 hours post fertilization (hpf), then migrate to the caudal hematopoietic tissue (CHT; mammalian fetal liver equivalent) from 36 to 72 hpf to proliferate and differentiate into committed progenitors. Shortly thereafter, HSPCs leave the CHT and populate the adult sites of hematopoiesis, the thymus and kidney marrow (Carroll and North, 2014). This general pattern of sequential migrations between hematopoietic niches is evolutionarily conserved (Dzierzak and Speck, 2008) and presumed to be essential for proper HSPC development and function. We and others have determined that sterile inflammatory signaling regulates HSPC production in vertebrate embryos (Espín-Palazón et al., 2014; He et al., 2015; Li et al., 2014; Sawamiphak et al., 2014). Similarly, we have previously shown that the well-known inflammatory factor prostaglandin E2 (PGE2) increases HSC numbers (North et al., 2007) and affects CXCL12/CXCR4-mediated HSPC homing to the adult niche across species (Goessling et al., 2011). However, the detailed mechanism(s) by which inflammatory signals influence HSPC development, including EHT and migratory behavior, are largely unknown.