Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05

    • In this study we dissected the role of RA signaling

    2018-10-24

    In this study, we dissected the role of RA signaling in human hematopoietic development, using a human pluripotent stem cell differentiation system as an in vitro model, to generate hematopoietic progenitors. We have identified multiple stages in development that are influenced by RA signaling, where modulation of RA signaling directs the generation of hematopoietic polo-like kinase 1 from pluripotent stem cells in vitro. We report that RA decreases blood generation from human pluripotent stem cells. RA signaling inhibition before the onset of hematopoiesis establishes an increase in the final output of cells with an HSC-like phenotype that confirms the role of RA signaling in the early stages of blood generation. qRT-PCR analysis for genes marking specific developmental cell types identified the stages during development where decreased RA signaling increased commitment toward the hematopoietic lineage, including the initial commitment of pluripotent stem cells to mesoderm, and by increased specification toward hemangiogenic mesoderm, while reducing cardiac mesoderm differentiation. The suppressing effect of RA on mesodermal commitment has been described in the murine embryo (Bain et al., 1996; Okada et al., 2004), and RA has been demonstrated to direct mesoderm toward cardiac tissue (anterior LPM) at the expense of hematopoietic development in Xenopus (Deimling and Drysdale, 2009), zebrafish (de Jong et al., 2010), and mouse (Szatmari et al., 2010), but this is the first time that these functions of RA have been verified in human development. Taken together, these results suggest that high but physiological levels of RA antagonize blood development and that substantially lower amounts of RA signaling relative to other tissues in the developing embryo are required to facilitate blood development. We show that RA accelerates the differentiation of generated hematopoietic progenitors to more mature blood cells, in agreement with findings in the adult human bone marrow where RA scavenging stroma cells is crucial to prevent rapid differentiation of hematopoietic progenitors (Ghiaur et al., 2013). By restricting RA signaling using DEAB, we demonstrate a significant increase in the generation of HSC-like progenitors capable of lymphoid and myeloid differentiation (∼3-fold). In this study, we performed transplantation experiments to assess the capacity of these cells to repopulate immunocompromised mice but found no significant difference in engraftment capacity in progenitors derived from human pluripotent stem cells (data not shown). Our results using DEAB may appear to contradict findings that RA can increase the progenitor output generated from human ESCs (Yu et al., 2010); however, Yu et al. only assessed the effect of RA on the general CD34+ progenitors and did not address the action of RA to differentiate and ultimately reduce the numbers of more immature hematopoietic progenitors; indeed, we observed that adding low amounts of RA increased total blood output but at the expense of the HSC-like progenitor fraction (the fraction with the highest lymphoid/myeloid differentiation potential). Our findings indicate that reduced RA signaling preserves the generated progenitor fraction from differentiation into more mature cell types, similar to the finding that DEAB delayed the differentiation of subcultured human cord blood and bone marrow HSCs (Chute et al., 2006). Regarding the role of RA signaling on blood development, a recent study on the cephalochordate Amphioxus (Branchiostoma lanceolatum), considered to be one of the most closely related invertebrates to the vertebrate family (Pascual-Anaya et al., 2013), reported that HSC-equivalent Pdvegfr+ Scl+ cells in the aorta-gonad-mesonephros (AGM) during embryonic development were abrogated when the embryos were treated with RA. This indicates that first, RA, at concentrations above what is naturally found at embryonic sites of hematopoiesis, can be a strong negative regulator of blood development, and second, that the relationship between RA signaling and development toward the hematopoietic lineage is a shared feature across all members of the vertebrate subphylum. Therefore, it is possible that, from an evolutionary perspective, blood development depends on an RA-low environment with controlled low levels of RA being critical during separate stages during this process, a process mimicked in our human model in the presence of DEAB. While our data demonstrate that increasing concentrations of RA abrogate the development of blood, it is important to appreciate that RA signaling is needed in this developmental process, albeit at controlled levels, since full abrogation of RA signaling, as demonstrated in the murine setting (Chanda et al., 2013; Goldie et al., 2008), is incompatible with hematopoietic development and the emergence of HSCs at the AGM. In addition, Chanda et al. demonstrate that increases in RA signaling around the time of HSC emergence from hemogenic endothelium increase transplantable HSCs in the murine setting; however, whether any expansion of already-committed HSCs occurred in the culture was not addressed. Our concept that RA is required at low levels during blood development helps to explain the contradicting views on RA in blood development and provides a more unified model for this process. As RA has been shown to have pleiotropic effects on separate hematopoietic cell types (Purton, 2007), and by the evidence that signaling through specific receptor elements confers separate responses in different populations (Chanda et al., 2013; Purton et al., 2006), it would be fitting to describe the nature of RA signaling to be context dependent. Identification and manipulation of specific RA receptors in specific cell populations will allow for further understanding and better recapitulation of development toward blood. Taken together, we have provided evidence identifying multiple developmental stages that depend on restricted RA signaling for the efficient differentiation toward blood, and we have shown that RA signaling inhibition can prevent differentiation and loss of more immature blood progenitors generated in vitro. Our findings show the utility of our pluripotent stem cell differentiation system to study human hematopoietic development, modeling human embryonic development, and to determine the effects and functions of RA during this process.