scholarly journals Hematopoiesis in 3 dimensions: human and murine bone marrow architecture visualized by confocal microscopy

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. e41-e55 ◽  
Author(s):  
Tomoiku Takaku ◽  
Daniela Malide ◽  
Jichun Chen ◽  
Rodrigo T. Calado ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Three Dimensional ◽  
Cell Types ◽  
Bone Marrow Tissue ◽  
Murine Bone Marrow ◽  
3 Dimensional ◽  
Vascular Structures ◽  
Bone Structures ◽  
Surrounding Bone

AbstractIn many animals, blood cell production occurs in the bone marrow. Hematopoiesis is complex, requiring self-renewing and pluripotent stem cells, differentiated progenitor and precursor cells, and supportive stroma, adipose tissue, vascular structures, and extracellular matrix. Although imaging is a vital tool in hematology research, the 3-dimensional architecture of the bone marrow tissue in situ remains largely uncharacterized. The major hindrance to imaging the intact marrow is the surrounding bone structures are almost impossible to cut/image through. We have overcome these obstacles and describe a method whereby whole-mounts of bone marrow tissue were immunostained and imaged in 3 dimensions by confocal fluorescence and reflection microscopy. We have successfully mapped by multicolor immunofluorescence the localization pattern of as many as 4 cell features simultaneously over large tiled views and to depths of approximately 150 μm. Three-dimensional images can be assessed qualitatively and quantitatively to appreciate the distribution of cell types and their interrelationships, with minimal perturbations of the tissue. We demonstrate its application to normal mouse and human marrow, to murine models of marrow failure, and to patients with aplastic anemia, myeloid, and lymphoid cell malignancies. The technique should be generally adaptable for basic laboratory investigation and for clinical diagnosis of hematologic diseases.

Hematopoiesis in Three-Dimensions: Tissue Architecture of Murine and Human Bone Marrow Visualized to a Depth of 100 Micron by Confocal Microscopy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4781-4781
Author(s):  
Tomoiku Takaku ◽  
Daniela Malide ◽  
Ning Zhi ◽  
Rodrigo Calado ◽  
Jichun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Laser Scanning ◽  
Cell Types ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Bone Marrow Tissue ◽  
Murine Bone Marrow ◽  
3 Dimensional ◽  
Whole Mount

Abstract Three-dimensional (3D) reconstruction of organs and tissues is a powerful tool to establish anatomical and functional relationships of microscopic structures. We developed whole-mount tissue processing methods for 3D in situ visualization of murine and human bone marrow; our methods are compatible with fluorescent labeling of different cell types and other structures of interest in the tissue microenvironment. The major technical problems addressed were the conditions for tissue fixation in the absence of permeabilization and sectioning; antibody penetration and binding; and the acquisition of high quality images by adequate laser scanning confocal microscope. For murine bone marrow, the sternum was bisected sagitally; for human tissue, 2–3 mm fragments of core biopsies were utilized. Bone marrow tissue and cells were exposed to fluorescence labeled nucleic acid dyes and antibodies, with or without prior chemical fixation. Single and double labeling of cells was feasible with combinations of various antibodies and direct and indirect immunofluorescent techniques. In some experiments, cells were visualized from transgenic mice with cell populations expressing green fluorescence protein (GFP). Series of two dimensional (xy) images 600 μm × 600 μm were collected along the z-axis at 5 μm z-intervals to depths of 60–100 μm using a Zeiss LSM 510 confocal microscope. Two dimensional images were assembled to reconstruct 3-dimensional volumes by Bitplane’s Imaris 3D computer software. Antigenicity was preserved, allowing simultaneous labeling of cell types and structures by immunohistochemistry or nuclear dyes. Different hematopoietic cell types as well as blood vessels, adipose cells, and extracellular matrix were visualized in complex 3-dimensional organization of intact bone marrow tissue revealing unknown features of multicellular architecture. Normal murine bone marrow, after brief fixation formaldehyde, is shown in Figure A. Rat anti-mouse basement-membrane monoclonal antibody (MAb) and fluorescent isothiocyanate (FITC)-labeled donkey anti-rat monoclonal antibody were used to visualize the extracellular matrix and micro-vessels (appearing green). Allophycocyanin (APC)-labeled rat anti-mouse CD45R cells permitted visualization of B lymphocytes (red). 4’,6-diamidino-2-phenylindole(DAPI) stained all nuclei (blue). Nests of lymphocytes appeared encased by extracellular matrix, fed by microvessels running from the bone edge. An example of the architecture of a human hematologic malignancy is shown in figure B, from a marrow biopsy of a patient with multiple myeloma prior to therapy. Mouse anti-human CD20 MAb and FITC-labeled donkey anti-mouse IgG were used to visualize mature B cells (green). APC-conjugated mouse anti-human CD38 MAb identified plasma cells (red). DAPI stained nuclei (blue). The large tumor cells appeared in unevenly distributed cell clumps. In mouse experiments, (not illustrated), marrow cells were easily observed in animals in which GFP was driven by the ubiquitin-C promoter. In humans (also not illustrated), we observed malignant cell populations stained with appropriate lineage-specific antibodies in patients with leukemia and compared CD34 cell numbers in normal with aplastic bone marrow. Confocal laser scanning microscopy, a powerful technique to generate serial sections of whole-mount tissue and their digital reassembly into virtual 3-dimensional structures, has been readily adapted to examination of murine and human bone marrow. The wide variety of MAbs available for specific antigens in combination with this imaging method should aid in conceptualizing microanatomical relationships among hematopoietic cells, stroma, blood vessels, and extracellular matrix in normal and diseased bone marrow. Figure Figure

scholarly journals Three-dimensional model of bone marrow

Blood ◽  
1978 ◽  
Vol 51 (4) ◽  
pp. 633-643 ◽  
Author(s):  
N Mohandas ◽  
M Prenant
Keyword(s):  
Bone Marrow ◽  
Close Association ◽  
Three Dimensional ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Scale Models ◽  
Marrow Space ◽  
Normal Rat

Abstract Three-dimensional scale models of bone marrow from a hypertransfused and a normal rat were constructed. The model of marrow from the hypertransfused rat demonstrated the existence of distinct erythroblastic islands in situ in which the erythroblasts underwent sychronous maturation. Macrophages were found in close association with the developing erythroblasts. The immature erythroblasts were tightly grouped, but as they matured they began to move apart. Erythroblasts in individual clusters were found to be at the same stage of morphologic maturation. In contrast, the model of marrow from the normal rat showed a majority of clusters containing erythroblasts at various stages of maturation. Erythropoiesis was not spatially restricted to the area proximal to the sinuses but was found to occur over the entire marrow space. Thrombopoiesis, however, was found to take place exclusively in the immediate vicinity of the marrow sinuses.

In Situ Visualization of Human MSC Engraftment in Bone Marrow: Integration into Murine Microenvironment and Interaction with Human HSC.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1283-1283
Author(s):  
Yukari Muguruma ◽  
Takashi Yahata ◽  
Hiroko Miyatake ◽  
Kiyoshi Ando ◽  
Tomomitsu Hotta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Stromal Cells ◽  
Fluorescent Protein ◽  
Hematopoietic Cells ◽  
Human Mscs ◽  
Murine Bone Marrow ◽  
Visual Evidence

Abstract Bone marrow is a complex organ system composed of two distinct lineages of cells: the hematopoietic cells and the supporting stromal cells, often referred as hematopoietic microenvironment (HME). Mesenchymal stem cells (MSCs) in bone marrow are shown to give rise to some of the components of HME, including osteoblasts, adipocytes and stromal fibroblasts in vitro, and to endothelial cells in vivo. It is a well accepted, but not definitely proven, concept that the HME provides structural niches, where dormant hematopoietic stem cells (HSCs) reside, and controls their renewal and differentiation. Although cotransplantation of human MSCs together with human HSCs resulted in increased chimerism of HSCs in animal models, existence of donor MSCs could only be detected using sensitive PCR-based analysis. Until this date, there is no physical evidence that transplanted MSCs have indeed engrafted in bone marrow and directly participated in that biological effect. In this study, we present the visual evidence for the sustained integration of human MSCs in murine bone marrow. Furthermore, we are able to delineate the physical interaction of injected human MSCs and cord blood derived CD34-positive HSCs (CBCD34). In order to assess the spatial distribution, lineage commitment and interaction of MSCs and HSCs in situ, we transplanted green fluorescent protein (GFP)-transduced MSCs and yellow fluorescent protein (YFP)-transduced CBCD34 into tibia of NOD/SCID mice. Ten weeks after intramedullary injection, longitudinal sections of mouse tibiae were made and stained with various antibodies for multicolor immunofluorescent analysis using a confocal microscope. We detected not only the existence of GFP-expressing MSCs in bone marrow, but also differentiation into several cell lineages. GFP-expressing cells exhibited phenotype and morphplogy of N-cadherin-positive bone lining osteoblasts, osteocalcin-positive osteocytes in bone, cells lining abluminal surface of vasculature, and in rare occasion, CD34 and CD31-positive endothelial cells. We then quantitatively evaluated the proportion of GFP-MSCs interacted with primitive YFP-CD34 and lineage committed YFP-CD15 and -Glycophorin-expressing cells as well as the proportion of above mentioned hematopoietic cells interacted with GFP-MSC. Approximately 50% of MSCs associated with CD34-posititive stem cells compared to only 2% and 3% of those with CD15 and Glycophorin-positive cells, respectively. It was also evident that the frequency of CD34-positive cells interacted with MSCs was significantly higher than those with CD15 and Glycophorin-positive cells. The results were consistent with a long appreciated notion that more primitive cells closely interact with hematopoietic supporting stromal cells. Furthermore, we quantitatively proved that the majority of YFP-CD34-positive HSCs were found close proximity to the bone. By transplanting GFP-MSCs together with YFP-HSCs, this study provided direct visual evidence that transplanted human MSCs engrafted in murine bone marrow and integrated into HME, which physically interacted with human HSC.

Quality-enhanced GaN epitaxial films on Si(111) substrates by in situ deposition of SiN on a three-dimensional GaN template

RSC Advances ◽  
2016 ◽  
Vol 6 (88) ◽  
pp. 84794-84800 ◽  
Author(s):  
Yunhao Lin ◽  
Meijuan Yang ◽  
Wenliang Wang ◽  
Zhiting Lin ◽  
Guoqiang Li
Keyword(s):  
Vapor Deposition ◽  
Three Dimensional ◽  
Chemical Vapor ◽  
Epitaxial Films ◽  
Organic Chemical ◽  
3 Dimensional ◽  
In Situ Deposition ◽  
Metal Organic ◽  
Organic Chemical Vapor Deposition

High-quality crack-free GaN epitaxial films were successfully grown on Si(111) substrates using metal–organic chemical vapor deposition by in situ depositing SiN on a 3-dimensional (3D) GaN template.

Meis1-mediated apoptosis is caspase dependent and can be suppressed by coexpression of HoxA9 in murine and human cell lines

Blood ◽  
2005 ◽  
Vol 105 (3) ◽  
pp. 1222-1230 ◽  
Author(s):  
Peter J. Wermuth ◽  
Arthur M. Buchberg
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cell Types ◽  
Selective Advantage ◽  
Myeloid Differentiation ◽  
Homeodomain Protein ◽  
Murine Bone Marrow ◽  
Differentiation Pathways

AbstractCoexpression of the homeodomain protein Meis1 and either HoxA7 or HoxA9 is characteristic of many acute myelogenous leukemias. Although Meis1 can be overexpressed in bone marrow long-term repopulating cells, it is incapable of mediating their transformation. Although overexpressing HoxA9 alone transforms murine bone marrow cells, concurrent Meis1 overexpression greatly accelerates oncogenesis. Meis1-HoxA9 cooperation suppresses several myeloid differentiation pathways. We now report that Meis1 overexpression strongly induces apoptosis in a variety of cell types in vitro through a caspase-dependent process. Meis1 requires a functional homeodomain and Pbx-interaction motif to induce apoptosis. Coexpressing HoxA9 with Meis1 suppresses this apoptosis and provides protection from several apoptosis inducers. Pbx1, another Meis1 cofactor, also induces apoptosis; however, coexpressing HoxA9 is incapable of rescuing Pbx-mediated apoptosis. This resistance to apoptotic stimuli, coupled with the previously reported ability to suppress multiple myeloid differentiation pathways, would provide a strong selective advantage to Meis1-HoxA9 coexpressing cells in vivo, leading to leukemogenesis.

scholarly journals In situelectro-sequencing in three-dimensional tissues

2021 ◽  
Author(s):  
Qiang Li ◽  
Zuwan Lin ◽  
Ren Liu ◽  
Xin Tang ◽  
Jiahao Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Three Dimensional ◽  
Cell Types ◽  
Developmental Trajectory ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Gene Expression ◽  
Cell Gene

AbstractPairwise mapping of single-cell gene expression and electrophysiology in intact three-dimensional (3D) tissues is crucial for studying electrogenic organs (e.g., brain and heart)1–5. Here, we introducein situelectro-sequencing (electro-seq), combining soft bioelectronics within situRNA sequencing to stably map millisecond-timescale cellular electrophysiology and simultaneously profile a large number of genes at single-cell level across 3D tissues. We appliedin situelectro-seq to 3D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches, precisely registering the CM gene expression with electrophysiology at single-cell level, enabling multimodalin situanalysis. Such multimodal data integration substantially improved the dissection of cell types and the reconstruction of developmental trajectory from spatially heterogeneous tissues. Using machine learning (ML)-based cross-modal analysis,in situelectro-seq identified the gene-to-electrophysiology relationship over the time course of cardiac maturation. Further leveraging such a relationship to train a coupled autoencoder, we demonstrated the prediction of single-cell gene expression profile evolution using long-term electrical measurement from the same cardiac patch or 3D millimeter-scale cardiac organoids. As exemplified by cardiac tissue maturation,in situelectro-seq will be broadly applicable to create spatiotemporal multimodal maps and predictive models in electrogenic organs, allowing discovery of cell types and gene programs responsible for electrophysiological function and dysfunction.

scholarly journals Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 753-763
Author(s):  
P Van Vlasselaer ◽  
N Falla ◽  
H Snoeck ◽  
E Mathieu
Keyword(s):  
Bone Marrow ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Three Dimensional ◽  
The Other ◽  
Light Scatter ◽  
Murine Bone Marrow ◽  
Osteogenic Cells ◽  
Mineralized Matrix ◽  
Binding Intensity

Osteogenic cells were sorted from bone marrow of 5-fluorouracil (5-FU)- treated mice based on light scatter characteristics, Sca-1 expression, and their binding to wheat germ agglutinin (WGA). Four sort gates were established using forward (FSC) and perpendicular (SSC) light scatter and were denominated as FSChigh SSClow, FSClow SSChigh, FSClow SSClow, and FSChigh SSChigh cell. Cells from the FSChigh SSChigh gate, but not from the other gates, synthesized alkaline phosphatase, collagen, and osteocalcin and formed a mineralized matrix in culture. The number of osteoprogenitor cells was significantly enriched after depleting the 5- FU bone marrow from cells of the lymphoid and myeloid lineage, eg, T cells, B cells, natural killer cells, granulocytes, macrophages, and erythrocytes. Approximately 95% of the FSChigh SSChigh cell population of this “lineage-negative” (Lin-) marrow expressed the Sca-1 antigen (Sca-1+) and bound WGA. Three additional sort windows were established based on WGA binding intensity and were denominated as Sca-1+ WGAdull, Sca-1+ WGAmedium, and Sca-1+ WGAbright. Cells from the Sca-1+ WGAbright gate, but not from the other gates, synthesized bone proteins and formed a mineralized matrix. However, they lost this capacity upon subcultivation. Further immunophenotypic characterization showed that FSChigh SSChigh Lin- Sca-1+ WGAbright cells expressed stromal (KM16) and endothelial (Sab-1 and Sab-2) markers, but not hematopoietic surface markers such as c-kit and Thy1.2. Sorted FSChigh SSChigh Lin- Sca-1+ WGAbright cells form three-dimensional nodules that stain with the von Kossa technique and contain osteoblast and osteocyte-like cells.

scholarly journals In Vitro Comparison of 2D-Cell Culture and 3D-Cell Sheets of Scleraxis-Programmed Bone Marrow Derived Mesenchymal Stem Cells to Primary Tendon Stem/Progenitor Cells for Tendon Repair

2018 ◽  
Vol 19 (8) ◽  
pp. 2272 ◽  
Author(s):  
Chi-Fen Hsieh ◽  
Zexing Yan ◽  
Ricarda Schumann ◽  
Stefan Milz ◽  
Christian Pfeifer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Sheet ◽  
Tendon Repair ◽  
Three Dimensional ◽  
Cell Types ◽  
Tendon Tissue ◽  
Cell Sheets ◽  
Tendon Tissue Engineering ◽  
Dimensional Cell

The poor and slow healing capacity of tendons requires novel strategies to speed up the tendon repair process. Hence, new and promising developments in tendon tissue engineering have become increasingly relevant. Previously, we have established a tendon progenitor cell line via ectopic expression of the tendon-related basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) in human bone marrow mesenchymal stem cells (hMSC-Scx). The aim of this study was to directly compare the characteristics of hMSC-Scx cells to that of primary human tendon stem/progenitors cells (hTSPCs) via assessment of self-renewal and multipotency, gene marker expression profiling, in vitro wound healing assay and three-dimensional cell sheet formation. As expected, hTSPCs were more naive than hMSC-Scx cells because of higher clonogenicity, trilineage differentiation potential, and expression of stem cell markers, as well as higher mRNA levels of several gene factors associated with early tendon development. Interestingly, with regards to wound healing, both cell types demonstrate a comparable speed of scratch closure, as well as migratory velocity and distance in various migration experiments. In the three-dimensional cell sheet model, hMSC-Scx cells and hTSPCs form compact tendinous sheets as histological staining, and transmission electron microscopy shows spindle-shaped cells and collagen type I fibrils with similar average diameter size and distribution. Taken together, hTSPCs exceed hMSC-Scx cells in several characteristics, namely clonogenicity, multipotentiality, gene expression profile and rates of tendon-like sheet formation, whilst in three-dimensional cell sheets, both cell types have comparable in vitro healing potential and collagenous composition of their three-dimensional cell sheets, making both cell types a suitable cell source for tendon tissue engineering and healing.

High Levels of Acetylated Low Density Lipoprotein Uptake and Low Tie2 Promoter Activity Distinguishes Sinusoids from Other Vessel Types in the Murine Bone Marrow

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5465-5465
Author(s):  
Xiaomiao Li ◽  
Zhongbo Hu ◽  
Marda Jorgenson ◽  
William Slayton
Keyword(s):  
Bone Marrow ◽  
Blood Vessels ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Cell Types ◽  
Blood Stream ◽  
Low Density ◽  
Murine Bone Marrow ◽  
Different Types

Abstract Background: The bone marrow contains a variety of blood vessels that have different functions in maintaining the bone marrow as the major blood producing organ in adulthood. For instance, arterioles function to control the flow of blood into bone marrow compartments, and the sinusoids serve as a conduit to the blood stream and niches for megakaryocyte development. Most current studies of the bone marrow vasculature, including studies quantifying changes in the marrow vascular by microvascular density, do not differentiate between different types of marrow vessels. Recognizing the changes in different types of blood vessels has important physiologic implications. Here we report a new method to distinguish sinusoids from arterioles in the murine bone marrow. Methods and Results: We used transgenic mice with GFP expressed downstream of the Tie-2 promoter, combined with in vivo acetylated low-density lipoprotein (Ac-LDL) uptake method to differentiate sinusoids from arterioles. We found that Ac-LDL was specifically endocytosed by sinusoids, and Tie-2 expression was more pronounced in the arteries, arterioles, and transitional capillaries. Combining these two functional endothelial markers and using confocal microscopy to obtain three dimensional images, we identified transitional zones where arterioles emptied into the sinusoids. Conclusions: These results demonstrate that the marrow vasculature and specific endothelial cell types are functionally heterogeneous. Methods to study changes in the marrow vasculature and particularly the vascular niche, a function of sinusoids, need to take into account this heterogeneity.

Computer-Aided Three-Dimensional Reconstruction and Measurement of the Vestibular End-Organs

1988 ◽  
Vol 98 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Akira Takagi ◽  
Isamu Sando ◽  
Akira Takagi ◽  
Isamu Sando
Keyword(s):  
Temporal Bone ◽  
Three Dimensional ◽  
Three Dimensions ◽  
Small Computer ◽  
Lateral Semicircular Canal ◽  
3 Dimensional ◽  
Dimensional Reconstruction ◽  
Vestibular Endorgans ◽  
Length And Area ◽  
Bone Structures

It is very valuable for temporal bone morphologists to be able to recognize temporal bone serial sections in three dimensions and to be able to measure temporal bone structures three-dimensionally. We can now do 3-dimensional reconstruction to visualize the structures of vestibular endorgans (utricular and saccular maculae) and measure these endorgans in space by means of a small computer system and software that we developed. As well as obtaining the dimensions—such as length and area—of the utricular and saccular maculae, we also found that (1) most of the utricular macula lies in one plane, which is the same as the plane of the lateral semicircular canal, (2) the saccular macula is shaped like part of a sphere, and (3) the angle between the two maculae is less than a right angle. Such knowledge is indispensable to the evaluation of the function of the utricular and saccular maculae.)

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