br Experimental Procedures See Supplemental Experimental Pro

Experimental Procedures See Supplemental Experimental Procedures for details on mice, flow cytometry and cell sorting, cell-cycle and apoptosis analysis, myeloid progenitor and Lys-GFP in vitro differentiation assay, and gene expression.
Author Contributions
Acknowledgments
Introduction In utero hematopoietic stem cell transplantation (IUHSCT) is a clinically viable therapeutic option, which could potentially provide successful treatment for many genetic and developmental diseases affecting the immune and hematopoietic systems (MacKenzie et al., 2015). IUHSCT has safely been performed for decades in humans and is the only approach that can promise the birth of a healthy infant (Muench and Barcena, 2004; Nijagal et al., 2012). To date, its success has been limited to recipients with severe combined immunodeficiency disorders in which there is a selective advantage of donor cell engraftment/survival over host integrin inhibitors (Flake et al., 1996; Gotherstrom et al., 2014; Le Blanc et al., 2005; Touraine et al., 1989; Wengler et al., 1996). Because IUHSCT must be performed without myeloablation or immunosuppression, immunologic barriers and absence of stress-induced signaling have been considered as significant contributors to the limited donor HSC engraftment (Merianos et al., 2009; Nijagal et al., 2011; Peranteau et al., 2007). Other challenges observed with IUHSCT result from the unique intricacies of fetal hematopoietic stem/progenitor cell (HSC) biology and the fetal microenvironment. It has been postulated that transplanted adult cells could potentially be outcompeted by endogenous fetal HSC, since the latter are actively cycling and undergo symmetric self-renewal divisions more efficiently than adult HSC (Bowie et al., 2007). Also, the fetal microenvironment might not be appropriate to support engraftment and/or expansion of donor HSC derived from ontogenically disparate sources, as differences in membrane composition and response to cytokines exist between fetal and adult cells (Arora et al., 2014; Bowie et al., 2007; Derderian et al., 2014). MCAM/CD146, within the adult human bone marrow (BM), is a marker of stromal progenitors/pericytes (Sacchetti et al., 2007), which produce stromal cell-derived factor 1 (SDF-1/CXCL12) and stem cell factor (SCF), and mediate HSC maintenance/retention (Corselli et al., 2013; Sugiyama et al., 2006), while VEGFR2/Flk-1 was shown to specifically define a continuous network of arterioles and sinusoidal endothelial cells within the BM, which are essential for HSC engraftment and reconstitution of hematopoiesis (Butler et al., 2010; Hooper et al., 2009; Kiel et al., 2005). Moreover, in an adult setting, CD146-expressing subendothelial cells have been shown, upon transplantation, to be able to transfer the hematopoietic microenvironment to heterotopic sites (Sacchetti et al., 2007). Here, we investigated whether transplantation of allogeneic adult BM-derived CD146-expressing mesenchymal (CD146+CXCL12+VEGFR2−) or endothelial (CD146+CXCL12+VEGFR2+) cells resulted in stable long-term contribution/integration into specific fetal BM niches, and whether administration of these cells, simultaneously with, or prior to, HSC transplantation, improved levels of HSC engraftment in an in utero setting. In addition, since information about the preferential engraftment sites of adult-derived HSC within the fetal microenvironment after IUHSCT is scarce, we also investigated whether and where donor-derived HSC localized in the fetal BM, and whether they underwent cell cycling. We also evaluated, in the co-transplantation approach, whether cell-cell interactions with CD146+CXCL12+VEGFR2− or CD146+CXCL12+VEGFR2+ cells played a role in altering the patterns or levels of engraftment of subsequently transplanted HSC, and sought to identify the responsible factors. Our results show that, in a non-myeloablative fetal setting, allogeneic adult donor HSC engraft within the metaphysis, and proliferate efficiently beside endogenous hematopoietic cells, while CD146+CXCL12+VEGFR2+and CD146+CXCL12+VEGFR2− cells integrate in a different anatomic area, the bone, and/or vasculature of the diaphysis. Mechanistically, we demonstrate that CD146+CXCL12+VEGFR2+ and CD146+CXCL12+VEGFR2− cells contribute to robust CXCL12 production, and that increased expression of VEGFR2 in the microvasculature of CD146+CXCL12+VEGFR2+ transplanted animals paralleled enhanced levels of donor-derived hematopoietic cells in circulation. These studies provide additional insights into IUHSCT biology, and demonstrate the feasibility of enhancing donor HSC engraftment to levels that would likely be therapeutic in many of the diseases that are candidates for IUHSCT.