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    2018-10-22

    These findings are consistent with a low SRC activity which is increased after ex vivo culture (Fig. 4A). In a parallel project, we found that CD34+CD38−/low and not CD34+CD38+ fraction of cultured steady state PB protease inhibitors hiv contains the cells exhibiting the capacity to engraft NSG mice (giving human CD45+, CD33+ (myeloid) and CD19+ (lymphoid) cells in murine bone marrow) (work in progress). Thus, the SP cells were analyzed with respect to CD38 expression (Figs. 3B, C). Taking into account only CD34+CD38−/low SP cells, the phenomenon of increase in absolute cell number after culture persists (10 fold) (Fig. 3F), which is similar to the increase in SRC activity in the same condition (human chimerism increased about 8 fold), suggesting that the HSCs are really highly enriched in this SP CD34+CD38−/low cell fraction. In line with this observation are also our results showing a numerical increase in CD90+ (5.9 fold) and CD133+ (28 fold) subpopulations of SP CD34+CD38−/low cells. We can thus conclude that the CD34+ cells purified from LRF steady state PB contain a restricted population of short- and long-term repopulating HSC, a finding completely consistent with the one of a low-frequency SP and SP CD34+CD38−/low populations (as well as its CD90+ and CD133+ subpopulations). These populations increase after culture as does the SRC activity. The following is the supplementary data related to this article.
    Acknowledgments The authors are thankful to Mrs Elisabeth Doutreloux-Volkmann and Dr Ivana Gadjanski for the language corrections. This project was supported by the grant “No”.
    Introduction The ability to generate forebrain neurons from embryonic stem cells (ESCs) has provided a new tool for studying the complex relationships between molecular systems and neuronal fate determination (Petros et al., 2011). Several studies have successfully generated cortical interneurons (cINs) from mouse (Maroof et al., 2010; Danjo et al., 2011) and human ESCs (Goulburn et al., 2011), but the derivation efficiencies and ability to produce cIN subgroups is limited. Forced expression of key fate-determining genes can promote the differentiation of ESCs into specific cell fates. There are two primary origins of cINs; those that originate in the medial ganglionic eminence (MGE) or preoptic area and require the transcription factor Nkx2.1 for their fate determination, and those that originate within the caudal ganglionic eminence and are Nkx2.1-independent. Downstream of Nkx2.1, the transcription factor Lhx6 is expressed from cell cycle exit through postnatal development (Liodis et al., 2007), and ectopic Lhx6 expression is sufficient to rescue the loss cIN subgroup markers in Nkx2.1−/− mice (Du et al., 2008). Upstream of Nkx2.1, the morphogen Sonic hedgehog (Shh) is required for the induction and maintenance of Nkx2.1 expression (Fuccillo et al., 2004; Xu et al., 2005). However, it is unknown if the only role for Shh in cIN fate determination is to induce Nkx2.1 expression, or whether Shh functions via additional factors that are required to specify cIN fate. In this paper we engineered a mouse ESC (mESC) line to both inducibly express Nkx2.1 via doxycycline (Dox) (Kyba et al., 2002), and to express GFP under the control of the Lhx6 promoter. We used this mESC line to (1) determine whether inducible Nkx2.1 expression enhances generation of GFP+ cINs, and (2) study the role of Shh signaling in cIN fate determination. Our results indicate that induced Nkx2.1 expression increases the generation of mESC-derived cINs. Additionally, forced Nkx2.1 expression is sufficient to induce cIN fates in the absence of Shh signaling, suggesting that the primary role of Shh is to maintain Nkx2.1 expression. These findings demonstrate the potential for utilizing temporally controlled gene expression in combination with fate markers to enhance the ESC-derivation of neuronal subgroups and to study the fate determination of forebrain neurons.