Endothelial-to-hematopoietic transition (EHT) is a unique morphogenic event in which flat, adherent hemogenic endothelial (HE) cells acquire round, non-adherent blood cell morphology. Investigating the mechanisms of EHT is critical for understanding the development of hematopoietic stem cells (HSCs) and the entirety of the adult immune system, and advancing technologies for manufacturing blood cells from human pluripotent stem cells (hPSCs). Here we describe a protocol to (a) generate and isolate subsets of HE from hPSCs, (b) assess EHT and hematopoietic potential of HE subsets in bulk cultures and at the single-cell level, and (c) evaluate the role of NOTCH signaling during HE specification and EHT. The generation of HE from hPSCs and EHT bulk cultures are performed in xenogen- and feeder-free system, providing the unique advantage of being able to investigate the role of individual signaling factors during EHT and the definitive lympho-myeloid cell specification from hPSCs.
© 2022. Springer Science+Business Media, LLC, part of Springer Nature.

The inability of Nef to downmodulate the CD3-T cell receptor (TCR) complex distinguishes HIV-1 from other primate lentiviruses and may contribute to its high virulence. However, the role of this Nef function in virus-mediated immune activation and pathogenicity remains speculative. Here, we selectively disrupted this Nef activity in SIVmac239 and analyzed the consequences for the virological, immunological, and clinical outcome of infection in rhesus macaques. The inability to downmodulate CD3-TCR does not impair viral replication during acute infection but is associated with increased immune activation and antiviral gene expression. Subsequent early reversion in three of six animals suggests strong selective pressure for this Nef function and is associated with high viral loads and progression to simian AIDS. In the absence of reversions, however, viral replication and the clinical course of infection are attenuated. Thus, Nef-mediated downmodulation of CD3 dampens the inflammatory response to simian immunodeficiency virus (SIV) infection and seems critical for efficient viral immune evasion.
Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

Human brain evolution is associated with expansion and folding of the neocortex. Increased diversity in neural progenitor (NP) populations (such as basally located radial glia [RG], which reside in an enlarged outer subventricular zone [OSVZ]) likely contributes to this evolutionary expansion, although their characteristics and relative contributions are only partially understood. Through single-cell transcriptional profiling of sorted human NP subpopulations, we identified the primate-specific TMEM14B gene as a marker of basal RG. Expression of TMEM14B in embryonic NPs induces cortical thickening and gyrification in postnatal mice. This is accompanied by SVZ expansion, the appearance of outer RG-like cells, and the proliferation of multiple NP subsets, with proportional increases in all cortical layers and normal lamination. TMEM14B drives NP proliferation by increasing the phosphorylation and nuclear translocation of IQGAP1, which in turn promotes G1/S cell cycle transitions. These data show that a single primate-specific gene can drive neurodevelopmental changes that contribute to brain evolution.
Copyright © 2017 Elsevier Inc. All rights reserved.

Elderly HIV-1 infected individuals progress to AIDS more frequently and rapidly than people becoming infected at a young age. To identify possible reasons for these differences in clinical progression, we performed comprehensive phenotypic analyses of CD4+ T cells from uninfected young and elderly individuals, and examined their susceptibility to HIV-1 infection and programmed death.
Peripheral blood mononuclear cells (PBMCs) from older people contain an increased percentage of central memory and Th17 CD4+ T cells that are main target cells of HIV-1 and strongly reduced proportions of naïve T cells that are poorly susceptible to HIV-1. Unstimulated T cells from elderly individuals expressed higher levels of activation markers, death receptors, and the viral CXCR4 co-receptor than those from young individuals but responded poorly to stimulation. CD4+ T cells from older individuals were highly susceptible to CXCR4- and CCR5-tropic HIV-1 infection but produced significantly lower quantities of infectious virus than cells from young individuals because they were highly prone to apoptosis and thus presumably had a very short life span. The increased susceptibility of T cells from the elderly to HIV-1 infection correlated directly with CXCR4 and inversely with CD4 expression. The levels of apoptosis correlated with the cell surface expression of FAS but not with the expression of programmed death receptor 1 (PD1) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
Increased levels of activated and highly susceptible HIV-1 target cells, reduced CD4 and enhanced CXCR4 cell surface expression, together with the high susceptibility to FAS-induced programmed cell death may contribute to the rapid CD4+ T cell depletion and accelerated clinical course of infection in elderly HIV-1-infected individuals.