Key Points Perivascular cells maintain HSPCs ex vivo.

Summary The ability to generate T cells from self-renewing pluripotent stem cells (PSC) has the potential to transform the current practice of autologous T cell immunotherapy into universal off-the-shelf products. However, differentiation of human PSCs into mature, conventional T cells has been challenging with existing methods. We report that a 3D artificial thymic organoid (PSC-ATO) system induced efficient differentiation of human embryonic stem cell and induced pluripotent stem cell-derived mesoderm progenitors to mature, functional T cells with a diverse T cell receptor (TCR) repertoire. This continuous culture system supported both hematopoietic specification and terminal differentiation to naïve CD3+CD8αβ+ and CD3+CD4+ conventional T cells. Introduction of an MHC class I-restricted TCR in PSCs produced naïve, antigen-specific cytotoxic CD8αβ+ T cells which lacked endogenous TCR Vβ expression. Functional assays and RNA sequencing aligned PSC-derived T cells with primary naïve CD8+ T cells. The PSC-ATO system presented here is an efficient platform for generating functional, mature T cells from human PSCs.

Key Points• Human HSPCs expressing CD34 exhibit E-selectin binding activity, whereas those lacking CD34 do not.• CD34 is a unique E-and P-selectin ligand on human HSPCs that binds with kinetics comparable to other known selectin ligands.

Summary Human pluripotent stem cells (PSCs) are critical in vitro tools for understanding mechanisms that regulate lineage differentiation in the human embryo as well as a potentially unlimited supply of stem cells for regenerative medicine. Pluripotent human and mouse embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts share a similar transcription factor network to maintain pluripotency and self-renewal, yet there are considerable molecular differences reflecting the diverse environments in which mouse and human ESCs are derived. In the current study we evaluated the role of Protein arginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewal and pluripotency given its critical role in safeguarding mouse ESC pluripotency. Unlike the mouse, we discovered that PRMT5 has no role in hESC pluripotency. Using microarray analysis we discovered that a significant depletion in PRMT5 RNA and protein from hESCs changed the expression of only 78 genes, with the majority being repressed. Functionally, we discovered that depletion of PRMT5 had no effect on expression of OCT4, NANOG or SOX2, and did not prevent teratoma formation. Instead, we show that PRMT5 functions in hESCs to regulate proliferation in the self-renewing state by regulating the fraction of cells in Gap 1 (G1) of the cell cycle and increasing expression of the G1 cell cycle inhibitor P57. Taken together our data unveils a distinct role for PRMT5 in hESCs and identifies P57 as new target.

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.