Characterizing how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for therapeutic manipulation of stem cells1. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types2–4. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis5, expression of HIF-1 and related genes6, and staining with surrogate hypoxic markers (e.g. pimonidazole)6–8. Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow (BM) of live mice. Using two-photon phosphorescence lifetime microscopy (2PLM), we determined the absolute pO2 of the BM to be quite low (<32 mmHg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mmHg, 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 dramatically after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Stem cells reside in a specialized, regulatory environment termed the niche that dictates how they generate, maintain and repair tissues1,2. We have previously documented that transplanted haematopoietic stem and progenitor cell populations localize to subdomains of bone-marrow microvessels where the chemokine CXCL12 is particularly abundant3. Using a combination of high-resolution confocal microscopy and two-photon video imaging of individual haematopoietic cells in the calvarium bone marrow of living mice over time, we examine the relationship of haematopoietic stem and progenitor cells to blood vessels, osteoblasts and endosteal surface as they home and engraft in irradiated and c-Kit-receptor-deficient recipient mice. Osteoblasts were enmeshed in microvessels and relative positioning of stem/progenitor cells within this complex tissue was nonrandom and dynamic. Both cell autonomous and non-autonomous factors influenced primitive cell localization. Different haematopoietic cell subsets localized to distinct locations according to the stage of differentiation. When physiological challenges drove either engraftment or expansion, bone-marrow stem/progenitor cells assumed positions in close proximity to bone and osteoblasts. Our analysis permits observing in real time, at a single cell level, processes that previously have been studied only by their long-term outcome at the organismal level.

Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for realtime monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH 2 stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.nonlinear microscopy ͉ vibrational imaging ͉ back scattering

The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation and regeneration, yet relatively little is known about the architecture of microenvironments that support malignant metastasis. 1,2 Using dynamic in vivo confocal imaging, we show that the murine bone marrow (BM) contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant SDF-1 in discrete, discontinuous areas that localize the homing of a variety of tumor cell lines. Disruption of SDF-1/CXCR4 interactions inhibits Nalm-6 cell (acute lymphoblastic leukaemia) homing to these vessels. Further studies revealed that circulating leukemic cells engraft surrounding these vessels, suggesting that this molecularly distinct vasculature denotes a microenvironment for early metastatic tumor spread in BM. Finally, purified hematopoietic stem/progenitor cells and lymphocytes also localize to the same microdomains, indicating that this vasculature may function in benign states to demarcate specific portals for entry of cells into the marrow space. Specialized vascular structures therefore appear to delineate a microenvironment with unique physiology that is exploited by circulating malignant cells.It has been thought that tumor cells derive their ability to transit to specific organs by co-opting the same tissue-homing mechanisms used by benign leukocytes. 3 Substantial in vitro and more limited in vivo data provide evidence that tumors depend on selectin-, integrin-, and chemokinemediated vascular cell adhesion events in order to identify and bind to vascular beds at sites of tissue entry. 4,5 These molecular mechanisms are thought to enable the efficient spread of malignancies to target organs. Differential expression of these endothelial signals among tissues is known to control the destination of cellular traffic, but the contributions of the vascular molecular framework to the regulation of complex cellular microenvironments remain to be fully elucidated. Competing interests statementThe authors declare that they have no competing financial interests. The bone marrow (BM) is a frequent site for solid tumor spread. It can also be considered the most ubiquitous site for leukemic cell metastasis, as disease is seen to migrate from the initial birthplace of the leukemic clone to marrow spaces in distant sites throughout the body. These observations suggest that BM provides an avid environment for circulating tumor lodgement and growth. Moreover, the BM is commonly the source of latent or "minimal residual disease" following treatment, raising the possibility that specific anti-apoptotic "niches" for metastatic growth may exist. Understanding the biologic architecture of this host microenvironment therefore has significant implications for our approach to tumor treatment. NIH Public AccessWhile a variety of in vitro and in vivo techniques exist to study cell transit through BM, these are limited in their ability...

Stem cells reside in a specialized regulatory microenvironment or niche1,2, where they receive appropriate support for maintaining self-renewal and multi-lineage differentiation capacity1-3. The niche may also protect stem cells from environmental insults3 including cytotoxic chemotherapy and perhaps pathogenic immunity4. The testis, hair follicle, and placenta are all sites of residence for stem cells and are immune suppressive environments, called immune privileged (IP) sites, where multiple mechanisms conspire to prevent immune attack, even enabling prolonged survival of foreign allografts without immunosuppression (IS)4. We sought to determine if somatic stem cell niches more broadly are IP sites by examining the hematopoietic stem/progenitor cell (HSPC) niche1,2,5-7 in the bone marrow (BM), a site where immune reactivity exists8,9. We observed persistence of allo-HSPCs in non-irradiated recipients for 30 days without IS with the same survival frequency compared to syngeneic HSPCs. These HSPCs were lost after the depletion of FoxP3 regulatory T cells (Tregs). High resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with Tregs that accumulated on the endosteal surface in the calvarial and trabecular BM. Tregs appear to participate in creating a localized zone where HSPCs reside and where Tregs are necessary for allo-HSPC persistence. In addition to processes supporting stem cell function, the niche will provide a relative sanctuary from immune attack.

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The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1-dependent migration was regulated by the PI3K and ERK/ MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be IntroductionMultiple myeloma (MM) is the second most prevalent hematologic malignancy; it remains incurable, and the median survival time is 3 to 5 years. 1,2 It is characterized by the presence of multiple lytic lesions and widespread involvement of the bone marrow at diagnosis, implying a continuous (re)circulation of MM cells in the peripheral blood and (re)entrance into the bone marrow. 1 Studies have demonstrated the presence of circulating malignant plasma cells in more than 70% of patients diagnosed with MM. 3,4 Migration of cells through the blood to the bone marrow niches requires active navigation, a process termed homing.Chemokines are small chemoattractant cytokines that bind to specific G-protein-coupled 7-span transmembrane receptors present on the plasma membranes of target cells. [5][6][7] Chemokines play a central role in lymphocyte trafficking and homing. [8][9][10][11] One of the most extensively studied chemokines in migration is SDF-1 and its receptor, CXCR4. 12,13 SDF-1 is primarily produced by stromal cells. CXCR4 is expressed on the surfaces of normal cells such as hematopoietic stem cells and T and B lymphocytes and on malignant cells such as breast cancer cells and lymphoid malignancies. 6,11,[14][15][16] To date, the role of CXCR4 in homing of MM cells to the bone marrow has not been fully elucidated. Inhibitors of CXCR4, such as AMD3100 (AnorMED, Toronto, ON, Canada), have been shown to induce the mobilization of stem cells. 17,18 AMD3100 (AnorMED) is a bicyclam molecule that reversibly blocks the binding of CXCR4 with SDF-1. 19 Because SDF-1/CXCR4-dependent signaling differs between cell types and between malignant and normal counterparts, 20 it is critical to investigate the unique role of CXCR4/SDF-1 in MM. In this study, we sought to determine the effect of CXCR4 and its specific inhibitor, AMD3100, on the migration and in vivo ...

The results suggest that child abuse, in part through epigenetic reprogramming of oligodendrocytes, may lastingly disrupt cortical myelination, a fundamental feature of cerebral connectivity.