Age and dose dependent changes to the bone and bone marrow microenvironment after cytotoxic conditioning with busulfan.

Preparative regimens before Hematopoietic Cell Transplantation (HCT) damage the bone marrow (BM) microenvironment, potentially leading to secondary morbidity and even mortality. The precise effects of cytotoxic preconditioning on bone and BM remodeling, regeneration, and subsequent hematopoietic recovery over time remain unclear. Moreover, the influence of recipient age and cytotoxic dose have not been fully described. In this study, we longitudinally investigated bone and BM remodeling after busulfan treatment with low intensity (LI) and high intensity (HI) regimens as a function of animal age. As expected, higher donor chimerism was observed in young mice in both LI and HI regimens compared to adult mice. Noticeably in adult mice, significant engraftment was only observed in the HI group. The integrity of the blood-bone marrow barrier in calvarial BM blood vessels was lost after busulfan treatment in the young mice and remained altered even 6 weeks after HCT. In adult mice, the severity of vascular leakage appeared to be dose-dependent, being more pronounced in HI compared to LI recipients. Interestingly, no noticeable change in blood flow velocity was observed following busulfan treatment. Ex vivo imaging of the long bones revealed a reduction in the frequency and an increase in the diameter and density of the blood vessels shortly after treatment, a phenomenon that largely recovered in young mice but persisted in older mice after 6 weeks. Furthermore, analysis of bone remodeling indicated a significant alteration in bone turnover at 6 weeks compared to earlier timepoints in both young and adult mice. Overall, our results reveal new aspects of bone and BM remodeling, as well as hematopoietic recovery, which is dependent on the cytotoxic dose and recipient age.

Intravital two-photon microscopy of the native mouse thymus.

The thymus, a key organ in the adaptive immune system, is sensitive to a variety of insults including cytotoxic preconditioning, which leads to atrophy, compression of the blood vascular system, and alterations in hemodynamics. Although the thymus has innate regenerative capabilities, the production of T cells relies on the trafficking of lymphoid progenitors from the bone marrow through the altered thymic blood vascular system. Our understanding of thymic blood vascular hemodynamics is limited due to technical challenges associated with accessing the native thymus in live mice. To overcome this challenge, we developed an intravital two-photon imaging method to visualize the native thymus in vivo and investigated functional changes to the vascular system following sublethal irradiation. We quantified blood flow velocity and shear rate in cortical blood vessels and identified a subtle but significant increase in vessel leakage and diameter ~24 hrs post-sublethal irradiation. Ex vivo whole organ imaging of optically cleared thymus lobes confirmed a disruption of the thymus vascular structure, resulting in an increase in blood vessel diameter and vessel area, and concurrent thymic atrophy. This novel two-photon intravital imaging method enables a new paradigm for directly investigating the thymic microenvironment in vivo.

Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing.

Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine.

Deletion of Vhl in Dmp1-Expressing Cells Causes Microenvironmental Impairment of B Cell Lymphopoiesis.

The contributions of skeletal cells to the processes of B cell development in the bone marrow (BM) have not been completely described. The von-Hippel Lindau protein (VHL) plays a key role in cellular responses to hypoxia. Previous work showed that Dmp1-Cre;Vhl conditional knockout mice (VhlcKO), which deletes Vhl in subsets of mesenchymal stem cells, late osteoblasts and osteocytes, display dysregulated bone growth and reduction in B cells. Here, we investigated the mechanisms underlying the B cell defects using flow cytometry and high-resolution imaging. In the VhlcKO BM, B cell progenitors were increased in frequency and number, whereas Hardy Fractions B-F were decreased. VhlcKO Fractions B-C cells showed increased apoptosis and quiescence. Reciprocal BM chimeras confirmed a B cell-extrinsic source of the VhlcKO B cell defects. In support of this, VhlcKO BM supernatant contained reduced CXCL12 and elevated EPO levels. Intravital and ex vivo imaging revealed VhlcKO BM blood vessels with increased diameter, volume, and a diminished blood-BM barrier. Staining of VhlcKO B cells with an intracellular hypoxic marker indicated the natural existence of distinct B cell microenvironments that differ in local oxygen tensions and that the B cell developmental defects in VhlcKO BM are not initiated by hypoxia. Our studies identify novel mechanisms linking altered bone homeostasis with drastic BM microenvironmental changes that dysregulate B cell development.

Intravital fluorescence microscopy with negative contrast.

Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a "dark side" in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for "filling in the void" by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching.

Two-Photon Phosphorescence Lifetime Microscopy.

Two-photon Phosphorescence Lifetime Microscopy (2PLM) is an emerging nonlinear optical technique that has great potential to improve our understanding of the basic biology underlying human health and disease. Although analogous to 2-photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), the contrast in 2PLM is fundamentally different from various intensity-based forms of imaging since it is based on the lifetime of an excited state and can be regarded as a "functional imaging" technique. 2PLM signal originates from the deactivation of the excited triplet state (phosphorescence) [1, 2]. Typically, this triplet state is a much longer-lived excited state than the singlet excited state resulting in phosphorescence emission times of microseconds to milliseconds at room temperature as opposed to nanoseconds for fluorescence emission [3]. The long-lived nature of the triplet state makes it highly sensitive to quenching molecules in the surrounding environment such as biomolecular oxygen (O2). Therefore, 2PLM can provide not only information on the distribution pattern of the probe in the sample (via intensity) but also determine the local oxygen tension (via phosphorescence lifetime quenching) [1]. The ability to create three-dimensional optical sections in the plane of focus within a thick biological specimen while maintaining relatively low phototoxicity due to the use of near-infrared wavelengths for two-photon excitation gives 2PLM powerful advantages over other techniques for longitudinal imaging and monitoring of oxygen within living organisms [4]. In this chapter, we will provide background on the development of 2PLM, discuss the most common oxygen sensing measurement methods and concepts, and explain the general principles and optical configuration of a 2PLM system. We also discuss the key characteristics and strategies for improvement of the technique. Finally, we will present an overview of the current primary scientific literature of how 2PLM has been used for oxygen sensing in biological applications and how this technique is improving our understanding of the basic biology underlying several areas of human health.

Imaging dynamic mTORC1 pathway activity in vivo reveals marked shifts that support time-specific inhibitor therapy in AML.

Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors.

Live-animal imaging of native haematopoietic stem and progenitor cells.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.

Intravital Imaging of Mouse Bone Marrow: Hemodynamics and Vascular Permeability.

The bone marrow is a unique microenvironment where blood cells are produced and released into the circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden, Nature 505:327-334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originating from arteries and sinusoidal vessels connected to veins. We found the two vessel types to exhibit different vascular permeabilites, hemodynamics, cell trafficking behaviors, and oxygen content (Itkin et al., Nature 532:323-328, 2016; Spencer et al., Nature 508:269-273, 2014). Here, we describe a method to quantitatively measure the permeability and hemodynamics of arterioles and sinusoids in murine calvarial bone marrow using intravital microscopy.

mRNA-mediated glycoengineering ameliorates deficient homing of human stem cell-derived hematopoietic progenitors.

Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell-derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin-bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs.

Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators.

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.

Distinct bone marrow blood vessels differentially regulate haematopoiesis.

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing.

Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (D,L-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad applicability for evaluating vascularization in other engineered tissues as well.

Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.

Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.

Sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Two-photon antenna-core oxygen probe with enhanced performance.

Recent development of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen enabled first noninvasive high-resolution measurements of tissue oxygenation in vivo in 3D, providing valuable physiological information. The so far developed two-photon-enhanced phosphorescent probes comprise antenna-core constructs, in which two-photon absorbing chromophores (antenna) capture and channel excitation energy to a phosphorescent core (metalloporphyrin) via intramolecular excitation energy transfer (EET). These probes allowed demonstration of the methods' potential; however, they suffer from a number of limitations, such as partial loss of emissivity to competing triplet state deactivation pathways (e.g., electron transfer) and suboptimal sensitivity to oxygen, thereby limiting spatial and temporal resolution of the method. Here we present a new probe, PtTCHP-C307, designed to overcome these limitations. The key improvements include significant increase in the phosphorescence quantum yield, higher efficiency of the antenna-core energy transfer, minimized quenching of the phosphorescence by electron transfer and increased signal dynamic range. For the same excitation flux, the new probe is able to produce up to 6-fold higher signal output than previously reported molecules. Performance of PtTCHP-C307 was demonstrated in vivo in pO2 measurements through the intact mouse skull into the bone marrow, where all blood cells are made from hematopoietic stem cells.

Direct measurement of local oxygen concentration in the bone marrow of live animals.

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label.

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4(+) T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.

Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration.

Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.