In mammals, a lifetime supply of mature blood cells by a process known as hematopoiesis is maintained by differentiation and proliferation of hematopoietic stem/progenitor cells (HSPCs) in response to physiological or pathological stimuli. Removal of aging hematopoietic cells by phagocytes is a physiological stimulus for blood cell generation, while massive loss of mature blood cells due to infection, inflammation or bleeding functions as a pathological stimulus for hematopoiesis. Both types of stimuli alter gene expression and/or post-transcriptional events that prompt cell cycle activation or changes in cell fate decisions by hematopoietic stem cells (HSCs) to produce more fate-restricted progenitors. Those cells then produce mature blood cells to supply lost populations. Based on analysis of the hematopoietic system, which emerges from HSCs, one trillion blood cells are reportedly produced daily in an average human weighting 70 kg under physiological conditions (Ogawa, 1993). The detailed analysis of spatiotemporal regulation of hematopoiesis could foster development of novel therapies and diagnostics for infection, immunological disease, and hematological malignancies.

Use of imaging techniques has revealed that hematopoietic activities in both steady state and pathological conditions are dynamic and that their sequence is regulated spatiotemporally by interaction with the niche. Further development and application of imaging techniques, including in vivo HSC labeling, has revealed critical details relevant to the biology of the hematopoietic system (Kataoka et al., 2011; Chen et al., 2012; Koechlein et al., 2016; Sawai et al., 2016). Here, we review recent advances relevant to in vivo and in vitro imaging analysis of HSCs and their niches and discuss future directions.

Imaging technology can reveal spatiotemporal cellular interactions and dynamics in the HSC niche. In particular, transgenic animal lines expressing fluorescent proteins in niche cells enable visualization of HSPC interaction with the hematopoietic microenvironment (Table 3). Zebrafish are now a powerful model in which to define mechanisms relevant to hematopoiesis and characterize HSC interactions with the microenvironment that govern development (Lieschke and Currie, 2007). In zebrafish, hematopoietic cell lineages are derived from posterior lateral mesoderm (PLM) cells, and Notch signaling between PLM cells and their scaffold, somitic cells, is required for hematopoietic development (Kobayashi et al., 2014). Using two-dimensional (2D) time-lapse live imaging of zebrafish embryo has revealed that HSCs are derived directly from aortic endothelium during development (Bertrand et al., 2010; Kissa and Herbomel, 2010). HSPCs then enter the bloodstream and arrive at the endothelial network in the ventral region of the embryo called the caudal hematopoietic tissue (CHT). HSPCs subsequently attach to the endothelium and remodel the CHT vascular plexus to form a surrounding pocket serving as a site for HSPC division (Tamplin et al., 2015).

Zebrafish models have also been useful to define embryonic HSC niche function. For example, nitric oxide production in the aorta-gonad-mesonephros (AGM) region is critical for a larval hematopoietic response to bacterial infection, as shown by studies using three-dimensional (3D) confocal live imaging (Hall et al., 2012). Since zebrafish embryos are relatively easy to manipulate, some have employed clonal mapping using multi-color genetic labeling and reported evidence suggesting that a limited number of HSC clones contributes to life-long hematopoiesis (Henninger et al., 2017). While wild-type zebrafish embryos are of high clarity, a transgenic line is now available with a body transparent enough for imaging analysis in adult fish (White et al., 2008).

In mammals, bone marrow HSCs are derived from embryonic hemogenic ECs in AGM. Fetal HSCs from AGM migrate to fetal liver (FL) or spleen and then expand their number. Analysis of Ly6a (Sca-1)-GFP transgenic mice, in which HSPCs are GFP-positive (Ma et al., 2002), combined with 3D confocal microscopy, has revealed the precise timing of HSC emerging can be visualized at the embryonic aortic endothelium (Boisset et al., 2010). Confocal microscopy-based 3D imaging of Ly6a-GFP embryos has revealed that HSPCs from FL interact with ECs (Tamplin et al., 2015). By improving sample preparation and imaging technology, longer time-lapse imaging of developmental stages will provide a more complete picture of HSC migration between organs.

Other imaging analysis has suggested that portal vessel-associated pericytes serve as critical HSC niche components in mouse FL (Khan et al., 2016). Specifically, in mice at birth, portal vessels change from a Neuropilin-1⁺Ephrin-B2⁺ artery to EphB4⁺ vein phenotype, resulting in pericyte loss and HSC release from FL. Perivascular lodgment of HSPCs induces active remodeling of the perivascular niche to promote HSPC expansion and maintenance in FL during development.

Post-natal hematopoiesis in mammals occurs mainly in bone marrow. Essential processes of bone development and ossification precede bone marrow development and begin embryonically. In the case of long bones, mineralization of cartilage is followed by blood vessel invasion of the central region of that tissue. Blood then perfuses bones, and actively dividing HSPCs arrive as early as E16.5 in mice, as revealed by 2D immunohistochemical analysis (Coskun et al., 2014). A recent study using in vitro imaging system reported that these HSPCs in fetal bone marrow switch from actively-dividing to quiescent, a transition mediated by osteoblast activity, as loss of osteolineage cells in Osx^−/− mice perturbs induction of HSPC quiescence (Coskun et al., 2014). Another study reports active division of murine HSCs in bone marrow until 3 weeks of age, but after 4 weeks HSCs stop dividing and become quiescent (Bowie et al., 2006). However, molecular and environmental cues that induce these phenotypic changes remain unclear.

In vivo imaging of bone marrow using a custom-built fluorescence confocal/multiphoton microscope revealed that pre-B-cell acute lymphoblastic leukemia (ALL) cells preferentially home to bone marrow vessels that express the adhesion molecule E-selectin and Cxcl12 (Sipkins et al., 2005). ALL cells also locally metastasize to Cxcl12-expressing vascular niche cells (Colmone et al., 2008). ALL cells also alter niche cell properties, decrease Cxcl12 production and induce SCF overexpression in bone marrow. In vivo time-lapse imaging of the T-ALL niche also reveals that T-ALL cells directly induce osteoblast shrinking and blebbing (Hawkins et al., 2016). Acute myelogenous leukemia and myeloproliferative neoplasms remodel the bone marrow microenvironment by disrupting niche cells, such as MSCs, neurons and Schwann cells (Arranz et al., 2014; Hanoun et al., 2014). As part of the host defense system, immune cells are consumed during infection, activating hematopoietic stem cells to supply blood cells (King and Goodell, 2011). Toll-like receptors and interferon receptors on HSPCs sense infection stress and activate a myeloid differentiation pathway called “emergency myelopoiesis” (Nagai et al., 2006). HSPCs also directly recognize the bacterial product bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) through the innate immune sensor STING. Activation of the c-di-GMP/STING pathway mobilizes HSPCs to peripheral blood (Kobayashi et al., 2015). c-di-GMP also suppresses expression of niche factors (namely, Cxcl12, SCF and Angiopoietin-1) in various non-hematopoietic niche cells. c-di-GMP induces expansion of the sinusoidal area of bone marrow, as revealed by 2D immunohistochemical analysis. Furthermore, in vivo time-lapse imaging during acute infection shows that HSC motility is more significantly activated after infection than in steady state (Rashidi et al., 2014). Infectious stress induces HSPC niche remodeling and facilitates HSPC mobilization.

Physiological aging also changes properties of the HSPC niche. Confocal imaging techniques have revealed that bone marrow arteries covered with the α-smooth muscle actin⁺ cells decrease in number and become more permeable with aging in mice (Kusumbe et al., 2016). Imaging analysis of animal models of hematological disease or aging will provide pathophysiological insights with potential therapeutic application.

The dynamics of hematopoiesis are tightly regulated by HSPCs and their niches within the bone marrow. Imaging techniques provide novel methods to define spatiotemporal regulation of complex multicellular microenvironments like bone marrow that every year we know more and more (Joseph et al., 2013). Although various methodological and technological hurdles remain, use of diverse techniques brings increasing insight into HSC interaction with niche cells and reveals how hematopoietic homeostasis is achieved in a dynamic manner.

Here, we have provided examples of imaging-based investigation of various hematopoietic activities, including developmental, physiological and pathological conditions and aging. Studies discussed here focus not only on stem cell location but on properties of the niche environment, such as local oxygen conditions. We anticipate that visualization of HSC cellular status in the niche will define additional mechanisms underlying hematopoiesis and leukemogenesis and potentially suggest novel therapies for blood cell diseases. Achieving this aim will require development of novel chemical and genetic probes of the cell cycle, metabolism, and signaling status and application of those methods to HSPC biology.

TM and KT wrote the manuscript; and KT conceived and supervised the project.