These data are in agreement with prior studies

These data are in agreement with prior studies demonstrating that hyaluronan levels play a critical role in determining the site of HSC lodgment/engraftment following transplantation (Avigdor et al., 2004; Ellis et al., 2007, 2009, 2011; Nilsson et al., 2003), and that HSC exhibit preferential engraftment within sites of active bone remodeling (Chan et al., 2009). In the adult, SDF-1/CXCL12 plays a critical role in BM repopulation by circulating CD34+ progenitors (Peled et al., 1999a, 1999b), and during development, SDF-1 induces the migration of primitive HSC from the fetal liver to the BM (Ma et al., 1998) and promotes HSC colonization (Ara et al., 2003). Likewise, membrane-bound SCF is important for HSC maintenance (Barker, 1997) and myeloid development after transplantation (Takagi et al., 2012). Both CD146++− and CD146+++ gsk-3 expressed, in vitro and after in vivo engraftment, SDF-1 and SCF. However, our analyses also showed that SCF is ubiquitously present within the fetal microenvironment, suggesting that SCF provided by the transplanted cells is not likely to be responsible for the observed enhancement in engraftment. By contrast, BM immunohistochemistry of transplanted animals showed robust SDF-1/CXCL12 production by transplanted CD146+++ cells. The production of SDF-1/CXCL12 at the site of HSC entrance/exit provides a potential explanation for the increased HSC engraftment seen in the co-transplanted animals over those receiving HSC alone. CD146+++ and CD146++− cells seem to affect HSC engraftment through some additional cell-specific mechanism, since CD146+++ cells only produced a statistically significant increase in BM donor hematopoietic engraftment if they were administered 3 days prior to the HSC. In contrast, CD146++− cells enhanced BM hematopoietic engraftment whether they were administered 3 days prior to the HSC graft or concomitantly with it. The ability of CD146++−, but not CD146+++, cells to enhance hematopoietic engraftment when transplanted simultaneously with the HSC, suggests that the effect of CD146++− cells could be mediated, at least in part, by their ability to alter the innate and adaptive immune responses (Domev et al., 2014; English et al., 2010; Le Blanc and Davies, 2015; Le Blanc et al., 2007), perhaps protecting the transplanted HSC from the rudimentary immune elements present within the fetus at this stage of gestation (Skopal-Chase et al., 2009). Our data also show that HSC3+CD146+++ animals exhibit significantly higher levels of overall hematopoietic cell engraftment than any other group. In vitro, and after engraftment, CD146+++ cells strongly expressed VEGFR2 while CD146++− cells did not, suggesting that, even in the absence of injury to the vascular niche (Hooper et al., 2009), VEGFR2 seems to play a role in increasing hematopoietic engraftment in the fetal recipient. This could be due to VEGFR2\'s ability to promote survival of hematopoietic progenitors through the activation of anti-apoptotic pathways (Larrivee et al., 2003) and/or stimulating the formation of hematopoietic-supporting hemospheres (Wang et al., 2013). It is also of note that the data presented here corroborate reports demonstrating that mesenchymal stromal cells transplanted in utero contribute to the forming bone, and could therefore be of clinical benefit for patients with osteogenesis imperfecta (Gotherstrom et al., 2014). More importantly, we demonstrate that a substantial fraction of transplanted stromal cells were found in the liver and never reached the BM, underscoring the need for defining strategies to increase bone engraftment of mesenchymal cells after in utero transplantation (Jones et al., 2012; Millard et al., 2015). Overall, these studies demonstrate that in a fetal setting, transplanted HSC locate primarily in the metaphysis, and that both CD146++− and CD146+++ stromal cells contribute, long-term, to the different BM niches and influence hematopoiesis through CXCL12, crucial for successful HSC engraftment in a fetal setting. Moreover, we show the feasibility of using a co-transplantation strategy that significantly (>10-fold) enhances donor HSC engraftment following IUHSCT. Because stromal cells have been transplanted safely in fetal recipients, confirmation of these results in another preclinical model would allow this straightforward and clinically viable approach to be used in a clinical setting to achieve levels of hematopoietic cell engraftment that would likely be therapeutic in many of the diseases that are candidates for treatment by IUHSCT.