Pluripotent stem cells (PSC) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had CD34+CD38−/loCD90+CD45+GPI-80+ foetal liver (FL) HSC immunophenotype, but displayed poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, retinoic acid signalling-induced medial HOXA gene expression marks the establishment of the definitive HSC fate and controls HSC identity and function.

Reversible ubiquitination is essential for the endocytic sorting and down-regulation of G protein-coupled receptors, such as the chemokine receptor CXCR4. The deubiquitinating enzyme AMSH has been implicated in the endocytic sorting of both G protein-coupled receptors and receptor-tyrosine kinases. Herein, we examine the role of AMSH in the regulation of CXCR4 stability and trafficking and characterize protein-protein interactions critical for this function. Loss of AMSH catalytic activity or depletion by RNAi results in increased steady-state levels of CXCR4 under basal conditions. Analysis of truncation and point mutation of AMSH reveal the importance of an RXXK motif for CXCR4 degradation. The RXXK motif of AMSH interacts with the SH3 domains of the STAM and Grb2 families of adaptor proteins with high affinity. Cells expressing a catalytically inactive mutant of AMSH show basal hyperubiquitination, but not increased degradation, of the ESCRT-0 components STAM1 and Hrs. This is dependent on the RXXK motif of AMSH. Ubiquitination of endocytic machinery modulates their activity, suggesting that AMSH may directly regulate endocytic adaptor protein function. This is reflected in CXCR4 trafficking and provides a mechanism by which AMSH specifies the fate of endocytosed receptors. Taken together, these studies implicate AMSH as a key modulator of receptor fate determination through its action on components of the endocytic machinery.The sorting and trafficking of cell surface receptors through endosomal compartments is a highly regulated process that is essential for maintaining cellular homeostasis and generating adaptive and coordinated responses to external stimuli. To avoid prolonged receptor activation and signaling, receptor-ligand complexes are endocytosed and either recycled back to the plasma membrane or sorted to lysosomes for degradation (1-4). This process is mediated by reversible ubiquitination. The modification of a target protein with ubiquitin moieties is extensively utilized to regulate the assembly of endosomal machinery as well as being a sorting signal for transmembrane proteins within the endosomal system (5-7). The crucial role for ubiquitination in the trafficking of endocytosed receptors has been documented for receptor-tyrosine kinases and a number of G protein-coupled receptors including the chemokine receptor CXCR4 (8 -10). CXCR4 has been extensively studied as a co-receptor for the entry of T-trophic human immunodeficiency virus into CD4ϩ T-cells (11,12). Along with its cognate ligand, Stromal cell-derived factor-1␣ (SDF-1/ CXCL12), CXCR4 plays important roles in hematopoiesis, development, and organization of the immune system and stem cell homing (13-15). CXCR4 deregulation is associated with various pathologies, including human immunodeficiency virus infection, cardiovascular disease, and neurodegenerative diseases and is associated with metastatic disease in a number of cancers (16 -20). Mutations that result in truncation of the C-terminal intracellular region of CXCR4 cause WHIM sy...

Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the earlyto-late endosome transition.Endocytosis and trafficking of cell surface receptors is essential for organized signal transduction and maintenance of homeostasis. Malfunctions along the molecular pathways governing endocytosis can lead to a wide range of human pathologies including metabolic disorders, autoimmune diseases, and cancer (1, 2). Ubiquitination, a reversible post-translational modification of proteins (3), mediates spatial and temporal aspects of endocytosis by dictating macromolecular complex assembly and cargo fate (4). Although polyubiquitination linked through Lys 48 of ubiquitin targets substrates for proteasomal degradation (5), mono-and Lys 63 -linked ubiquitination signal cargo trafficking (6), and modulate function of endocytic sorting machinery (7). The reversible nature of ubiquitination is imparted by deubiquitinating enzymes (DUBs) 4 and enables dynamic regulation of these ubiquitin-dependent processes in the cell (8).Trafficking of endocytosed cargo for degradation by the lysosome occurs in a series of discrete selection stages that utilize the endosomal sorting complexes required for transport (ESCRTs) -0, -I, -II, and -III. The ESCRT-0 complex, consisting of the adaptor proteins hepatocyte growth factor-regulated substrate (Hrs) and signal transducing adaptor molecule (STAM), resides at the sorting endosome and functions in the first step of cargo selection. ESCRT-0 partitions the endocytosed material arriving on the early endosomes between recycling and the multivesicular body (9 -11) and interacts with downstream ESCRT machinery (12, 13) respon...

Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38−CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38−CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.