Serial Imaging of Luciferase Positive Bone Marrow Stromal Cell Migration to Form Radiation Pulmonary Fibrosis

Abstract Abstract 4734 Thoracic irradiation of C57BL/6 mice leads to an acute reaction phase in the lungs characterized by increased cytokine production and inflammation days 1–14 post irradiation. This is followed by a latent period where inflammation, histologic appearance and cytokine response returns to control levels. The late reaction phase occurs 100+ days post irradiation and is characterized by organizing alveolitis/fibrosis and involves migration of marrow origin macrophages and proliferating mesenchymal stem cells (fibroblasts), a subpopulation which migrates from marrow to the lungs. To quantitate migration in real time, thoracic irradiated mice were either made chimeric for luciferase positive (luc+) whole marrow or were injected with cells from a positive luc+ bone marrow stromal cell line and serially imaged at day 7, 60 or 120 using an IVIS.. 200 Optical Imaging System. As a control for migration to the lung, another group of mice received 20Gy to the right hind leg and 1.5 ×106 luc+ bone marrow stromal cells i.p. Imaging of chimeric mice revealed luc+ cell lung migration only after day 120. C57BL/6NTac female mice that received 20Gy thoracic irradiation followed by an i.p. injection 1.5 × 106 luc+ positive bone marrow stromal cells revealed no migration of luc+ cells to the lungs at day 7 or day 60. Furthermore there was no migration to 20Gy irradiated leg at any timepoint. In marked contrast, at the time of the late reaction phase, at day 100, fibrosis was revealed as an increase in luc+ cell migration in lungs. The lung fibrosis model in C57BL/6 mice combined with live imaging allows sequential measurement of the effect of agents which may alter migration of bone marrow cells that contribute to radiation pulmonary fibrosis. Disclosures: No relevant conflicts of interest to declare.

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In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system

Journal of Clinical Neuroscience ◽

10.1016/j.jocn.2009.10.013 ◽

2010 ◽

Vol 17 (7) ◽

pp. 908-913 ◽

Cited By ~ 10

Author(s):

Xu Qi ◽

Ming Shao ◽

Haisheng Peng ◽

Zhenggang Bi ◽

Zhiqiang Su ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Culture System ◽

Stromal Cell ◽

Bone Marrow Stromal Cell ◽

Marrow Stromal Cells ◽

In Vitro Differentiation ◽

Differential Protein Expression

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A Method for Measurement of Drug Sensitivity of Myeloma Cells Co-Cultured with Bone Marrow Stromal Cells

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113478168 ◽

2013 ◽

Vol 18 (6) ◽

pp. 637-646 ◽

Cited By ~ 18

Author(s):

Kristine Misund ◽

Katarzyna A. Baranowska ◽

Toril Holien ◽

Christoph Rampa ◽

Dionne C. G. Klein ◽

...

Keyword(s):

Bone Marrow ◽

Cell Survival ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Large Scale ◽

Stromal Cell ◽

Marrow Stromal Cells ◽

Cell Nuclei ◽

Myeloma Cells

The tumor microenvironment can profoundly affect tumor cell survival as well as alter antitumor drug activity. However, conventional anticancer drug screening typically is performed in the absence of stromal cells. Here, we analyzed survival of myeloma cells co-cultured with bone marrow stromal cells (BMSC) using an automated fluorescence microscope platform, ScanR. By staining the cell nuclei with DRAQ5, we could distinguish between BMSC and myeloma cells, based on their staining intensity and nuclear shape. Using the apoptotic marker YO-PRO-1, the effects of drug treatment on the viability of the myeloma cells in the presence of stromal cells could be measured. The method does not require cell staining before incubation with drugs, and less than 5000 cells are required per condition. The method can be used for large-scale screening of anticancer drugs on primary myeloma cells. This study shows the importance of stromal cell support for primary myeloma cell survival in vitro, as half of the cell samples had a marked increase in their viability when cultured in the presence of BMSC. Stromal cell–induced protection against common myeloma drugs is also observed with this method.

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A novel 37-Kd adhesive membrane protein from cloned murine bone marrow stromal cells and cloned murine hematopoietic progenitor cells

Blood ◽

10.1182/blood.v82.5.1436.1436 ◽

1993 ◽

Vol 82 (5) ◽

pp. 1436-1444 ◽

Cited By ~ 3

Author(s):

Y Shiota ◽

JG Wilson ◽

K Harjes ◽

ED Zanjani ◽

M Tavassoli

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Stromal Cell ◽

Hematopoietic Cells ◽

Marrow Stromal Cells ◽

Hematopoietic Progenitor Cells ◽

Single Chain ◽

Hematopoietic Progenitor

Abstract The adhesion of hematopoietic progenitor cells to bone marrow stromal cells is critical to hematopoiesis and involves multiple effector molecules. Stromal cell molecules that participate in this interaction were sought by analyzing the detergent-soluble membrane proteins of GBI/6 stromal cells that could be adsorbed by intact FDCP-1 progenitor cells. A single-chain protein from GBI/6 cells having an apparent molecular weight of 37 Kd was selectively adsorbed by FDCP-1 cells. This protein, designated p37, could be surface-radiolabeled and thus appeared to be exposed on the cell membrane. An apparently identical 37- Kd protein was expressed by three stromal cell lines, by Swiss 3T3 fibroblastic cells, and by FDCP-1 and FDCP-2 progenitor cells. p37 was selectively adsorbed from membrane lysates by a variety of murine hematopoietic cells, including erythrocytes, but not by human erythrocytes. Binding of p37 to cells was calcium-dependent, and was not affected by inhibitors of the hematopoietic homing receptor or the cell-binding or heparin-binding functions of fibronectin. It is proposed that p37 may be a novel adhesive molecule expressed on the surface of a variety of hematopoietic cells that could participate in both homotypic and heterotypic interactions of stromal and progenitor cells.

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Distinct Stress Responses of Bone Marrow Stromal Cell Subgroups Defined by Unbiased Single-Cell Mass-Cytometry Analysis

Blood ◽

10.1182/blood.v128.22.430.430 ◽

2016 ◽

Vol 128 (22) ◽

pp. 430-430 ◽

Cited By ~ 1

Author(s):

Nicolas Severe ◽

Murat Karabacak ◽

Ninib Baryawno ◽

Karin Gustafsson ◽

Youmna Sami Kfoury ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Stromal Cell ◽

Marrow Stromal Cells ◽

Osteoblastic Cells ◽

Mouse Bone Marrow ◽

Bone Marrow Niche ◽

Mass Cytometry

Abstract The bone marrow niche is a heterogeneous tissue comprised of multiple cell types that collectively regulate hematopoiesis. It is thought to be a critical stress sensor, integrating information at the level of the organism down to signals at the level of the single cell. In so doing, the niche orchestrates hematopoietic stem and progenitor cell (HSPC) responses to organismal stress. However, most studies of the niche have depended on genetic marker or deletion studies that inherently limit analysis to the selected indicator genes or cells. While this has greatly enhanced our understanding of bone marrow function, it does not permit systems level evaluation of subgroups of cells and their relative response to a particular challenge. We therefore sought a less biased strategy to study bone marrow stromal cells and the cytokines they elaborate under homeostatic and stress conditions. We used Mass-Cytometry (CyTOF) to resolve protein levels at single cell resolution in mouse bone marrow. We established a panel of 36 antibodies: 20 surface and intracellular phenotypic markers, 12 cytokines regulating hematopoiesis, 1 marker of proliferation, 1 marker for DNA damage, 1 viability marker and 1 nucleated cell marker. We intentionally selected antibodies that recognize antigens already defined by others as bone marrow stromal markers. Freshly isolated non-hematopoietic cells from long bones and pelvis were analyzed and clustered into subgroups based on their protein expression signature. We applied k-means clustering using common markers to group bone marrow stromal cells into phenotypical subtypes. At steady state, analysis of over 20.000 mouse bone marrow stromal single-cells negative for the hematopoietic markers CD45 and Ter119 revealed 4 large clusters: an endothelial population expressing CD31, Sca1 and CD105, a mesenchymal stromal cell population expressing Sca1, CD140a, Nestin and LeptinR, a bone marrow stromal progenitor population expressing CD105, CD271 and Runx2 and a mature bone cell population expressing Osteocalcin and CD140a. Within these clusters, sub-populations were evident by adding CD106, CD90, CD73, Embigin, CD29, CD200, c-Kit and CD51. In total, 28 distinct populations of bone marrow stromal cells were identified based on their phenotypic signature. Only one cluster of cells was negative for all the markers we selected. Therefore, the complex heterogeneity of the bone marrow niche cells can be resolved to 28 subpopulations by single-cell protein analysis. Assessing the response of these groups to systemic challenges of medical relevance, we evaluated cells prior to whole body lethal irradiation (9.5Gy), one hour and one day later (the time of transplantation) and 3 days after irradiation (2d post transplantation) with and without transplanted cells. Notably, LeptinR+CD106+Sca1+ cells putatively essential for hematopoiesis and stem cell support were highly sensitive to and largely killed by irradiation. In contrast, endothelial cells and osteoblastic cells were resistant to irradiation. In particular, osteoblastic cells expressing osteocalcin (GFP+), embigin, NGFR and CD73 increased their expression of multiple hematopoietic cytokines including SDF-1, kit ligand, IL-6, G-CSF and TGF-b one day after irradiation. These data indicate that LeptinR+CD106+Sca1 stromal cells are unlikely to participate in HSPC engraftment post-irradiation while a subset of osteoblastic cells are. Unbiased single cell analysis can resolve subsets of bone marrow cells that respond differently to organismal stress. This method enables comprehensively quantifying subpopulation changes with specific challenges to begin defining the systems biology of the bone marrow niche. Disclosures No relevant conflicts of interest to declare.

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