Embryonic definitive hematopoiesis generates hematopoietic stem and progenitor cells (HSPCs) that are essential for the establishment and maintenance of the adult blood system. This process requires the specification of a subset of vascular endothelial cells (ECs) to become hemogenic ECs and to have subsequent endothelial-to-hematopoietic transition (EHT), and the underlying mechanisms are largely undefined. We identified microRNA (miR)-223 as a negative regulator of murine hemogenic EC specification and EHT. Loss of miR-223 leads to increased formation of hemogenic ECs and HSPCs, which is associated with increased retinoic acid signaling, which we previously showed as promoting hemogenic EC specification. Additionally, loss of miR-223 leads to the generation of myeloid-biased hemogenic ECs and HSPCs, which results in an increased proportion of myeloid cells throughout embryonic and postnatal life. Our findings identify a negative regulator of hemogenic EC specification and highlight the importance of this process for the establishment of the adult blood system.
Copyright © 2023 Elsevier Inc. All rights reserved.

Understanding the murine fetal liver (FL) hematopoietic microenvironment, which promotes HSC proliferation, warrants identifying innate relationships between stem cells and the niche. An inclusive study of these cell associations remains elusive. Here, we optimized a protocol to immunolabel HSCs alongside the FL vasculature, a promising niche component. We provide a comprehensive plan from tissue processing, immunohistochemistry, and confocal microscopy, to three-dimensional distance analyses between HSCs and vasculature. This technique can be adapted for achieving congruous outcomes for other cell types. For complete details on the use and execution of this protocol, please refer to Biswas et al. (2020).
Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%-90% of the cells die when placed in medium where the major growth factor is granulocyte-macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.