Search

Maintenance of hematopoietic stem cell (HSC) quiescence is critical for self-renewal and differentiation into mature lineages. Therefore, the ability to reliably detect abnormal HSC cycling is essential for experiments that seek to investigate abnormalities of HSC function. The ability to reproducibly evaluate cell cycle status in a rare cell subset requires careful optimization of multiple parameters during cell preparation and sample processing. Here, we describe a method where data acquisition parameters and fluorochrome combination for long-term HSC staining have been specifically designed for concurrent use with DAPI and Ki-67 antibodies. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Regulation of stem and progenitor cell populations is critical in the development, maintenance, and regeneration of tissues. Here, we define a novel mechanism by which a niche-secreted RNase, angiogenin (ANG), distinctively alters the functional characteristics of primitive hematopoietic stem/progenitor cells (HSPCs) compared with lineage-committed myeloid-restricted progenitor (MyePro) cells. Specifically, ANG reduces the proliferative capacity of HSPC while simultaneously increasing proliferation of MyePro cells. Mechanistically, ANG induces cell-type-specific RNA-processing events: tRNA-derived stress-induced small RNA (tiRNA) generation in HSPCs and rRNA induction in MyePro cells, leading to respective reduction and increase in protein synthesis. Recombinant ANG protein improves survival of irradiated animals and enhances hematopoietic regeneration of mouse and human HSPCs in transplantation. Thus, ANG plays a non-cell-autonomous role in regulation of hematopoiesis by simultaneously preserving HSPC stemness and promoting MyePro proliferation. These cell-type-specific functions of ANG suggest considerable therapeutic potential., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation rates than CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside in several, perhaps overlapping, niches that produce regulatory molecules and signals necessary for homeostasis and for increased output after stress or injury. Despite considerable advances in the specific cellular or molecular mechanisms governing HSC-niche interactions, little is known about the regulatory function in the intact mammalian haematopoietic niche. Recently, we and others described a positive regulatory role for prostaglandin E2 (PGE2) on HSC function ex vivo. Here we show that inhibition of endogenous PGE2 by non-steroidal anti-inflammatory drug (NSAID) treatment in mice results in modest HSC egress from the bone marrow. Surprisingly, this was independent of the SDF-1-CXCR4 axis implicated in stem-cell migration. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin. Haematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in other species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced E-prostanoid 4 (EP4) receptor signalling. These results not only uncover unique regulatory roles for EP4 signalling in HSC retention in the niche, but also define a rapidly translatable strategy to enhance transplantation therapeutically.

Stem cells reside in a specialized regulatory microenvironment or niche, where they receive appropriate support for maintaining self-renewal and multi-lineage differentiation capacity. The niche may also protect stem cells from environmental insults including cytotoxic chemotherapy and perhaps pathogenic immunity. The testis, hair follicle and placenta are all sites of residence for stem cells and are immune-suppressive environments, called immune-privileged sites, where multiple mechanisms cooperate to prevent immune attack, even enabling prolonged survival of foreign allografts without immunosuppression. We sought to determine if somatic stem-cell niches more broadly are immune-privileged sites by examining the haematopoietic stem/progenitor cell (HSPC) niche in the bone marrow, a site where immune reactivity exists. We observed persistence of HSPCs from allogeneic donor mice (allo-HSPCs) in non-irradiated recipient mice for 30 days without immunosuppression with the same survival frequency compared to syngeneic HSPCs. These HSPCs were lost after the depletion of FoxP3 regulatory T (T(reg)) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with T(reg) cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. T(reg) cells seem to participate in creating a localized zone where HSPCs reside and where T(reg) cells are necessary for allo-HSPC persistence. In addition to processes supporting stem-cell function, the niche will provide a relative sanctuary from immune attack.

Appropriate transcriptional regulation is critical for the biological functions of many key regulatory genes, including the stem cell leukemia (SCL) gene. As part of a systematic dissection of SCL transcriptional regulation, we have previously identified a 5,245-bp SCL +18/19 enhancer that targeted embryonic endothelium together with embryonic and adult hematopoietic progenitors and stem cells (HSCs). This enhancer is proving to be a powerful tool for manipulating hematopoietic progenitors and stem cells, but the design and interpretation of such transgenic studies require a detailed understanding of enhancer activity in vivo. In this study, we demonstrate that the +18/19 enhancer is active in mast cells, megakaryocytes, and adult endothelium. A 644-bp +19 core enhancer exhibited similar temporal and spatial activity to the 5,245-bp +18/19 fragment both during development and in adult mice. Unlike the +18/19 enhancer, the +19 core enhancer was only active in adult mice when linked to the eukaryotic reporter gene human placental alkaline phosphatase. Activity of a single core enhancer in HSCs, endothelium, mast cells, and megakaryocytes suggests possible overlaps in their respective transcriptional programs. Moreover, activity in a proportion of thymocytes and other SCL-negative cell types suggests the existence of a silencer elsewhere in the SCL locus.

To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside in several, perhaps overlapping, niches that produce regulatory molecules and signals necessary for homeostasis and for increased output after stress or injury. Despite considerable advances in the specific cellular or molecular mechanisms governing HSC-niche interactions, little is known about the regulatory function in the intact mammalian haematopoietic niche. Recently, we and others described a positive regulatory role for prostaglandin E2 (PGE2) on HSC function ex vivo. Here we show that inhibition of endogenous PGE2 by non-steroidal anti-inflammatory drug (NSAID) treatment in mice results in modest HSC egress from the bone marrow. Surprisingly, this was independent of the SDF-1-CXCR4 axis implicated in stem-cell migration. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin. Haematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in other species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced E-prostanoid 4 (EP4) receptor signalling. These results not only uncover unique regulatory roles for EP4 signalling in HSC retention in the niche, but also define a rapidly translatable strategy to enhance transplantation therapeutically. [ABSTRACT FROM AUTHOR]

Fetal liver (FL) hematopoietic progenitors have superior blood engraftment competence compared with adult bone marrow (BM), however less is known about FL in vivo vascular capacity. Here we show in transplantation assays that FL cells possess enhanced vascular endothelial potential compared with adult bone marrow. We generated high-level hematopoietic chimeras using donor cells from mice transgenic for the stem cell leukaemia 3′ enhancer human placental alkaline phosphatase (SCL3′Enh-PLAP) reporter construct, active in vascular endothelium, and blood progenitor and stem cells. Long-term lineage tracing analysis revealed PLAP+ vascular-like patches in FL-derived chimeras, whereas adult BM-derived chimeras presented only rare and scattered PLAP1 cells. PLAP+ vascular-like patches were formed following transplantation into both newborn and adult recipient mice, although their frequency was reduced in adult recipients. Confocal analysis of multiple labeled tissues revealed that whereas most liver and heart PLAP+ vascular patch-associated cells were endothelial, PLAP+ vascular patches in the kidney contained endothelial, hematopoietic, and putative hemangioblastic cells. Moreover, fluorescenceactivated cell sorting assays showed that only FL PLAPbright+ donor cells can generate PLAP+ vascular patches upon transplantation. Taken together, these data demonstrate superior vascular contribution potential of FL cells, and not only provide new insights into the developmental pathways controlling endothelial development but also may prove informative when addressing the mechanisms involved in vascular regeneration and hemangiogenic recovery in a clinical context. [ABSTRACT FROM AUTHOR]