This is consistent with our previous studies where we found multipotency within both CD11a- and CD11a+ fractions at e12.5 (Inlay et al., 2014), and found 4-Guanidinobutanoic acid both CD11a- and CD11a+ fetal HSCs at e17.5 (Fathman et al., 2014). use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed 11a- eKLS (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs. maturation and neonatal transplantation. In the former, candidate populations or tissues are harvested from the embryo and cultured with the addition of exogenous factors to induce maturation of these cells into HSCs, which is then confirmed by adult transplantation (Taoudi et al., 2008; Rybtsov et al., 2011). However, these maturation assays rely on the presence of cultured stromal lines as well as 4-Guanidinobutanoic acid potent exogenous factors such as SCF, TPO, IL-3, and Flt3L. Accordingly, these assays can potentially drive HSC formation from cell-types that are more primitive than pre-HSCs, such as hemogenic endothelium (Hadland et al., 2017). An alternative approach to reveal pre-HSC activity is intravenous injection of embryonic cells directly into irradiated recipients (Yoder and Hiatt, 1997; Yoder et al., 1997b,a). While less sensitive than cultures, neonatal transplantation presents minimal risk of introducing artifacts by bypassing the non-physiological concentrations of cytokines and growth factors used (Yoder et al., 1997a; Boisset et al., 2010; Arora et al., 2014). Adult HSCs can be precisely identified by a combination of different markers expressed (or unexpressed) on their surface. While many different combinations can work, a commonly used definition for murine HSCs is Lineage- Kit+ Sca1+ CD150+ and CD34-. However, many adult HSC markers are not similarly expressed in the early embryo and can change depending on the tissue and timepoint examined (Cumano and Godin, 2007). Alternative assays have identified potential pre-HSC markers including hematopoietic markers CD41 (Rybtsov et al., 2011), CD43 (Inlay et al., 2014), and CD45 (Taoudi et al., 2008; Boisset et al., 2010), progenitor markers Kit (Boisset et al., 2010) and Sca1 (Inlay et al., 2014), and endothelial markers CD31 (Inlay et al., 2014), VE-Cadherin (CD144) (Taoudi et al., 2008), and EPCR (CD201) (Zhou et al., 2016). This has resulted in the identification of populations such as Type I (CD144+ CD41+ CD45-) and Type II (CD144+ CD45+) pre-HSCs (Rybtsov et al., 2011), or rarer CD201hi subsets within these populations (Zhou et al., 2016) or a CD27+ subset within Type II pre-HSCs (Li et al., 2017). However, a strictly-defined pre-HSC cell type has not 4-Guanidinobutanoic acid been described to the same resolution as that in adult HSCs. CD11a (integrin alpha L, or multipotency assay, we determined that only a rare CD11a- population we termed CD11a- KLS cells (defined as Ter119- CD43+ Kit+ Sca1+ CD144+ CD11a-) contained all multipotent progenitor activity, regardless of what timepoint or tissue it was isolated from Inlay et al. (2014). Neonatal 4-Guanidinobutanoic acid transplantation demonstrated these cells produce a variety of lineages multipotency. In the present study, we use an neonatal NSG Cd200 transplantation system to prospectively identify pre-HSCs in e10.5 and e11.5 tissues. In line with our previous work, the absence of CD11a expression on pre-HSCs (defined as Ter119- CD43+ Kit+ Sca1+ CD144+ CD11a-) was critical for distinguishing them from downstream progenitors which were all CD11a+. Moreover, our data suggest the neonatal liver serves as an essential temporary niche for the maturation of embryonic progenitors which lack the expression of the BM homing receptor CXCR4 prior to seeding the BM. These findings establish CD11a as a key marker to identify and isolate a highly purified pre-HSC population, beyond what has been achieved, therefore paving the way for more detailed characterization of these immature progenitors. Materials and Methods Antibodies A detailed list of all antibodies used in this study is shown in Supplementary Table 1. Mice In our experiments, we used embryos from a male crossed to a (C57Bl/6; Jackson Laboratory; stock no. 00664) female. males were generated from a cross.
