Enhancers are cis-acting elements capable of regulating transcription in a distance and orientation-independent manner. A subset of enhancers are occupied by RNA polymerase II (RNAP II) and transcribed to produce long non-coding RNAs termed eRNAs. We thoroughly investigated the association between eRNA productivity and various chromatin marks and transcriptional regulators in mouse embryonic stem cells (ESCs) through an integrative approach. We found that eRNA-producing enhancers exhibited elevated levels of the active mark H3K27Ac, decreased DNA methylation, and enrichment for the DNA hydroxylase Tet1. Many eRNA-producing enhancers have recently been characterized as "super-enhancers," suggesting an important role in the maintenance of pluripotency. Using experimental methods, we focally investigated a well-characterized enhancer linked to the Nanog locus and confirmed its exclusive eRNA productivity in ESCs. We further demonstrate that the binding of Sall4 and Tet family proteins were required for eRNA productivity at this locus. Collectively, we demonstrate that Tet1 binding and DNA hypomethylation are hallmarks of eRNA production.

Cell-based immunotherapy has been gaining interest as an improved means to treat HIV/AIDS. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) could become a potential resource. Our previous studies have shown hESC and iPSC-derived natural killer (NK) cells can inhibit HIV-infected targets in vitro. Here, we advance those studies by expressing a HIV chimeric receptor combining the extracellular portion of CD4 to the CD3ζ intracellular signaling chain. We hypothesized that expression of this CD4ζ receptor would more efficiently direct hESC- and iPSC-derived NK cells to target HIV-infected cells. In vitro studies showed the CD4ζ expressing hESC- and iPSC-NK cells inhibited HIV replication in CD4+ T cells more efficiently than their unmodified counterparts. We then evaluated CD4ζ-hESC- and iPSC-NK cells in vivo anti-HIV activity using a humanized mouse model. We demonstrated significant suppression of HIV replication in mice treated with both CD4ζ-modified and unmodified hESC-/iPSC-NK cells compared to control mice. However, we did not observe significantly increased efficacy of CD4ζ expression in suppression of HIV infection. These studies indicate that hESC/iPSC-based immunotherapy can be utilized as a unique resource to target HIV/AIDS.

OBJECTIVE Previous studies have demonstrated development of endothelial cells (ECs) and smooth muscle cells (SMCs) as separate cell lineages derived from human embryonic stem cells (hESCs). We demonstrate CD34+ cells isolated from differentiated hESCs function as vascular progenitor cells capable of producing both ECs and SMCs. These studies better define the developmental origin and reveal the relationship between these two cell types, as well as provide a more complete biological characterization. MATERIALS AND METHODS hESCs are co-cultured on M2-10B4 stromal cells or Wnt1 expressing M2-10B4 for 13–15 days to generate a CD34+ cell population. These cells are isolated using a magnetic antibody separation kit and cultured on fibronectin coated dishes in EC medium. To induce SMC differentiation, culture medium is changed and a morphological and phenotypic change occurs within 24–48 hours. RESULTS CD34+ vascular progenitor cells give rise to ECs and SMCs. The two populations express respective cell specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when co-cultured in Matrigel. Human umbilical vein endothelial cells (HUVEC) cultured under SMC conditions do not exhibit a change in phenotype or genotype. Wnt1 overexpressing stromal cells produced an increased number of progenitor cells. CONCLUSIONS The ability to generate large numbers of ECs and SMCs from a single vascular progenitor cell population is promising for therapeutic use to treat a variety of diseased and ischemic conditions. The step-wise differentiation outlined here is an efficient, reproducible method with potential for large scale cultures suitable for clinical applications.

Sall4 is a transcription factor that exists in two splice isoforms – Sall4a and Sall4b – and regulates transcription in embryonic stem cells, hematopoiesis and acute myeloid leukemia. Constitutive overexpression of Sall4 in mice induces acute myeloid leukemia. Interestingly, a potential benefit of using Sall4 to facilitate ex vivo hematopoietic stem cell expansion has been proposed. However, distinct roles for how Sall4 contributes to normal versus malignant processes remain undefined. Here we show that Sall4b is the predominant isoform in murine hematopoietic stem cells and progenitors. Overexpression of either Sall4 isoform in HSCs or progenitors impairs hematopoietic colony formation and expansion in vitro. Lineage negative bone marrow overexpressing Sall4b fails to engraft and reconstitute hematopoiesis when transplanted. We found that both Sall4a and Sall4b overexpression impair hematopoiesis in part through dose-dependent repression of Bmi1. Additionally we have identified the following potential novel Sall4 target genes in hematopoiesis: Arid5b (Sall4a and Sall4b), Ezh2 and Klf2 (Sall4a). Lastly, we found that Sall4 expression is variable in acute myeloid leukemia, ranging from no expression to levels comparable to embryonic stem cells. These results show that Sall4 isoforms contribute to only a subset of acute myeloid leukemia and that overexpression of Sall4 isoforms impairs hematopoiesis through repression of Bmi1. Together these data demonstrate the sensitivity of hematopoiesis to appropriately balanced Sall4 expression, highlighting the importance of regulating this dynamic in potential therapeutic applications such as ex vivo stem cell expansion.

No abstract