Cloned osteoprogenitor cell from bone marrow stroma produces mineralized matrix in vitro and in vivo

Abstract Abstract 2191 Poster Board II-168 Aims: The stroma-forming cells in a bone marrow are derived from hematopoietic stem cells. We reported previously that non-adherent leukemia blast cells converted into myofibroblasts to create a microenvironment for proliferation of leukemia blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system chronic myelogenous leukemia (CML) cells are also differentiated into myofibroblasts to contribute to a bone marrow-stroma in vivo. Materials and Methods: Bone marrow cells were collected from informed CML patients, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, non-adherent mononuclear cells were injected to the priory 2.5 Gray-irradiated non-obese diabetes (NOD)/SCID mice intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Bcr-Abl transcript, and mice were sacrificed after chimeric mRNA was demonstrated. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select CML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and Discussion: When non-adherent CML cells were transplanted to NOD/SCID mice, CML cells were engrafted after 2 months. In the murine bone marrow human stromal cells were identified, in which BCR and ABL gene was fused with FISH analysis. When the parental CML cells were cultured on the CML-derived myofibroblasts, CML cells grew extensively in a vascular endothelial growth factor-A-dependent fashion. These results indicate that CML cells can create their own microenvironment for proliferation in vivo. Disclosures: No relevant conflicts of interest to declare.

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Bone Marrow Stromal Cells: Characterization and Clinical Application

Critical Reviews in Oral Biology & Medicine ◽

10.1177/10454411990100020401 ◽

1999 ◽

Vol 10 (2) ◽

pp. 165-181 ◽

Cited By ~ 193

Author(s):

P.H. Krebsbach ◽

S.A. Kuznetsov ◽

P. Bianco ◽

P. Gehron Robey

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Hematopoietic Cells ◽

Marrow Stromal Cells ◽

Proliferative Potential ◽

Bone Marrow Stroma ◽

Marrow Stroma

The bone marrow stroma consists of a heterogeneous population of cells that provide the structural and physiological support for hematopoietic cells. Additionally, the bone marrow stroma contains cells with a stem-cell-like character that allows them to differentiate into bone, cartilage, adipocytes, and hematopoietic supporting tissues. Several experimental approaches have been used to characterize the development and functional nature of these cells in vivo and their differentiating potential in vitro. In vivo, presumptive osteogenic precursors have been identified by morphologic and immunohistochemical methods. In culture, the stromal cells can be separated from hematopoietic cells by their differential adhesion to tissue culture plastic and their prolonged proliferative potential. In cultures generated from single-cell suspensions of marrow, bone marrow stromal cells grow in colonies, each derived from a single precursor cell termed the colony-forming unit-fibroblast. Culture methods have been developed to expand marrow stromal cells derived from human, mouse, and other species. Under appropriate conditions, these cells are capable of forming new bone after in vivo transplantation. Various methods of cultivation and transplantation conditions have been studied and found to have substantial influence on the transplantation outcome The finding that bone marrow stromal cells can be manipulated in vitro and subsequently form bone in vivo provides a powerful new model system for studying the basic biology of bone and for generating models for therapeutic strategies aimed at regenerating skeletal elements.

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New Insights on Properties and Spatial Distributions of Skeletal Stem Cells

Stem Cells International ◽

10.1155/2019/9026729 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11

Author(s):

Jun-qi Liu ◽

Qi-wen Li ◽

Zhen Tan

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stromal Cells ◽

Skeletal Biology ◽

Spatial Distributions ◽

Bone Marrow Stroma ◽

Marrow Stroma

Skeletal stem cells (SSCs) are postnatal self-renewing, multipotent, and skeletal lineage-committed progenitors that are capable of giving rise to cartilage, bone, and bone marrow stroma including marrow adipocytes and stromal cells in vitro and in an exogenous environment after transplantation in vivo. Identifying and isolating defined SSCs as well as illuminating their spatiotemporal properties contribute to our understating of skeletal biology and pathology. In this review, we revisit skeletal stem cells identified most recently and systematically discuss their origin and distributions.

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A Novel Human Bone Marrow Stroma-Derived Cell Line TF274 Is Highly Osteogenic In Vitro and In Vivo

Calcified Tissue International ◽

10.1007/s002239900517 ◽

1998 ◽

Vol 63 (3) ◽

pp. 214-220 ◽

Cited By ~ 20

Author(s):

U. Prabhakar ◽

I. E. James ◽

R. A. Dodds ◽

E. Lee-Rykaczewski ◽

D. J. Rieman ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Human Bone ◽

Human Bone Marrow ◽

Bone Marrow Stroma ◽

Marrow Stroma

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Hedgehog Pathway: A New Target for B-Cell Lymphomas an Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.389.389 ◽

2006 ◽

Vol 108 (11) ◽

pp. 389-389

Author(s):

Christine Dierks ◽

Jovana Grbic ◽

Katja Zirlik ◽

Roland H. Mertelsman ◽

Markus Warmuth

Keyword(s):

Bone Marrow ◽

B Cell ◽

Hedgehog Pathway ◽

Bone Marrow Stroma ◽

Apoptosis Induction ◽

B Cell Lymphomas ◽

Lymphoma Cells ◽

Marrow Stroma ◽

Hh Signaling

Abstract The hedgehog pathway (Hh) is one of several key developmental pathways whose deregulation is known to induce tumor formations in mice and humans including medulloblastoma or basal cell carcinoma. Certain human solid tumors (prostate cancer, pancreatic cancer) have been identified which require sustained Hh-Gli signaling for proliferation. Here, we show for the first time a crucial role of Hedgehog signalling in hematopoietic malignancies. In our experiments, we demonstrate that the Hh pathway is essential for survival of more than 70% of primary lymphomas extracted from transgenic Eμ-Myc mice or Cdkn2a−/− mice (INK4Arf−/−). 34 Myc-lymphomas (10 B-cell lymphomas, 12 plasmablastomas, 10 plasmocytomas, 2 mixed lymphomas) and 10 Cdkn2a−/− lymphomas were isolated from bone marrow, spleen or lymph nodes from diseased mice and expanded on bone marrow stroma cells from Cdkn2a−/− mice. Inhibition of Hh signaling by Cyclopamine (2–5 μM), an alkaloid which inhibits smoothened activation, induced apoptosis in 80% of B-cell lymphomas, 82% of plasmablastomas and 60% of multiple myelomas without affecting stroma proliferation. Protein or transcript levels of Hh pathway downstream targets, such as Gli1, Ptch or Bcl2 but not BMI1 were downregulated after 24h treatment with Cyclopamine. Overexpression of Bcl2 or Gli3 could inhibit apoptosis induction by Cyclopamine. High levels of Ihh protein could be detected in our stroma cell line as well as in bone marrow stroma from various mouse strains. Stimulation of lymphoma cells with recombinant Shh or Ihh protein could overcome apoptosis induction induced by removal of the stromal layer, indicating Ihh as a mediator between stroma and lymphoma cells. Injection of luciferazed lymphoma cells into C57Bl6 mice induced lymphoma formation within 2–6 weeks, as shown by Xenogen luciferase imaging. Subcutaneous treatment of lymphoma injected mice with Cyclopamine could inhibit lymphoma formation and even reduce lymphoma mass in mice with already fully developed lymphomas, indicating stroma-lymphoma interaction through hedgehog signaling as an important survival mechanism for lymphoma cells also in vivo. Our data demonstrates that proliferation and survival of a majority of B-cell lymphomas and plasmacytomas is dependent on intact Hh signaling in vitro and in vivo, suggesting disruption of this pathway as a novel approach in lymphoma therapy.

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Stem-cell factor in aplastic anemia: in vitro expression in bone marrow stroma and fibroblast cultures

European Journal Of Haematology ◽

10.1111/j.1600-0609.1995.tb00681.x ◽

2009 ◽

Vol 54 (4) ◽

pp. 262-269 ◽

Cited By ~ 13

Author(s):

M. Slanicka Krieger ◽

C. Nissen ◽

A. Wodnar-Filipowicz

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Aplastic Anemia ◽

Stem Cell Factor ◽

Bone Marrow Stroma ◽

In Vitro Expression ◽

Fibroblast Cultures ◽

Marrow Stroma

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Hematopoietic-Extrinsic Cues Dictate Circadian Redistribution of Mature and Immature Hematopoietic Cells in Blood and Spleen

Cells ◽

10.3390/cells8091033 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1033 ◽

Cited By ~ 4

Author(s):

Miriam Stenzinger ◽

Darja Karpova ◽

Christian Unterrainer ◽

Sabine Harenkamp ◽

Eliza Wiercinska ◽

...

Keyword(s):

Bone Marrow ◽

Clock Genes ◽

Hematopoietic Cells ◽

Underlying Mechanism ◽

Constant Darkness ◽

Intrinsic Cues ◽

Extrinsic Cues ◽

Marrow Stroma

Circadian oscillations in circulating leukocyte subsets including immature hematopoietic cells have been appreciated; the origin and nature of these alterations remain elusive. Our analysis of wild-type C57BL/6 mice under constant darkness confirmed circadian fluctuations of circulating leukocytes and clonogenic cells in blood and spleen but not bone marrow. Clock gene deficient Bmal1−/− mice lacked this regulation. Cell cycle analyses in the different hematopoietic compartments excluded circadian changes in total cell numbers, rather favoring shifting hematopoietic cell redistribution as the underlying mechanism. Transplant chimeras demonstrate that circadian rhythms within the stroma mediate the oscillations independently of hematopoietic-intrinsic cues. We provide evidence of circadian CXCL12 regulation via clock genes in vitro and were able to confirm CXCL12 oscillation in bone marrow and blood in vivo. Our studies further implicate cortisol as the conveyor of circadian input to bone marrow stroma and mediator of the circadian leukocyte oscillation. In summary, we establish hematopoietic-extrinsic cues as causal for circadian redistribution of circulating mature/immature blood cells.

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CD56(+)CD146(+) Cells in Normal Human Bone Marrow-Stroma Proliferated to Form Tumors in severe Combined Immunodeficiency Mice

Blood ◽

10.1182/blood.v118.21.1328.1328 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1328-1328

Author(s):

Ryosuke Shirasaki ◽

Haruko Tashiro ◽

Yoko Oka ◽

Tadashi Yamamoto ◽

Nobu Akiyama ◽

...

Keyword(s):

Bone Marrow ◽

Severe Combined Immunodeficiency ◽

Tumor Formation ◽

Scid Mice ◽

Myelogenous Leukemia ◽

Cell Fraction ◽

Bone Marrow Stroma ◽

Normal Human ◽

Marrow Stroma

Abstract Abstract 1328 Background and Aims: We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and in vivo. Recently, we also reported that myelogenous leukemia-derived myofibroblasts formed blastomas in non-obese diabetes severe combined immunodeficiency (NOD/SCID) mice, in which the CD56-positive cell-fraction was selectively proliferated (16th EHA). To ascertain whether normal human bone marrow-stroma-derived cells also behave like leukemia-derived cells, stromal cells were injected to NOD/SCID mice, and tumor-formation was observed. Materials and Methods: Bone marrow cells were collected from informed normal individuals, whose adherent cells were separated and were cultured for one month to eliminate monocyte/macrophage, vascular cell, and pericyte to prepare stromal myofibroblast-rich fraction. Cells were injected to NOD/SCID mice intra-venously, peritoneally, and subcutaneously (3 × 106/mice). When the tumor formation was observed or mice were dead, they were sacrificed and the engrafted cells were analyzed. Results and Discussion: Between at day 70 and 90 after injection mice were dead. Autopsy findings revealed tumor formation at subcutaneous injection sites, and cells were also infiltrated to the liver and ascitic fluid. These cells expressed CD56, CD146, and Nestin strongly, but not other lineage-specific markers including CD133; however, the ratio of CD56(+)CD146(+) fraction in the injected stromal cells was below 0.1%. When the tumor cells were cultured in vitro, they exhibited spindle-shaped appearance, and doubling time was 12 to 16 hours. They expressed c-Myc, Klf4, Nanog, Sox2, and other molecules that are expressed in an immature somatic stem cell. These observations indicate that CD56(+)CD146(+) cell-fraction in bone marrow-stroma is very immature and highly proliferative. We are now analyzing biological characteristics of this specific cell-fraction, and determining the contribution to normal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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1204. Gene-Modified Autologous Bone Marrow Stroma Embedded in Matrigel™ Leads to a Neovascularized Implant for Prolonged In Vivo Systemic Delivery of Functional Erythropoietin

Molecular Therapy ◽

10.1016/s1525-0016(16)44034-7 ◽

2002 ◽

Vol 5 (5) ◽

pp. S391

Keyword(s):

Bone Marrow ◽

Autologous Bone Marrow ◽

Bone Marrow Stroma ◽

Systemic Delivery ◽

Marrow Stroma ◽

Autologous Bone

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Some observations of the hematopoietic status in vivo and in vitro on mice of genotype S1/S1d

Blood ◽

10.1182/blood.v48.4.601.601 ◽

1976 ◽

Vol 48 (4) ◽

pp. 601-608 ◽

Cited By ~ 6

Author(s):

FD Wilson ◽

L O'Grady

Keyword(s):

Bone Marrow ◽

Colony Growth ◽

Hematopoietic Stem ◽

Quantitative Studies ◽

Bone Marrow Cultures ◽

Marrow Stroma ◽

Constant Cell ◽

Hematopoietic Stroma

Abstract Studies on the mechanism of anemia in mice of genotype S1/S1d have implicated the hematopoietic stroma (the hematopoietic inductive microenvironment, HIM) rather than hematopoietic stem cells as the site of the defect. Using methylcellulose-supported bone marrow culture systems, we have observed, in addition to classical hematopoietic colonies, the formation of surface associated fibroblastic plaques that could stimulate hematopoietic colony growth. These plaques were hypothesized to be derived from bone marrow stroma precursors. In view of the reported stromal-based defect in S1/S1d mice, studies were initiated, using our culture system, to determine if abnormalities exist in the plaque-forming potentials of these mice. Relative to controls, bone marrow derived from S1/S1d mice exhibited a significant decrease in hematopoietic colonly-forming units in culture, but no differences were apparent in the absolute numbers of fibroblastic plaque-forming units or in the ability of such plaques once derived to stimulate hematopoietic colony growth when overlain with fresh normal bone marrow preparations. Quantitative studies on the bone marrow of the S1/S1d mice revealed a marked reduction in total nucleated cells per femur. The importance of evaluating the results of bone marrow cultures in an absolute (i.e., number of units per femur) rather than a relative (i.e., number of units forming in a constant cell inoculum) term was underlined by these studies.

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