Bone-marrow-derived chondrogenesis in vitro

1992 ◽  
Vol 101 (2) ◽  
pp. 333-342 ◽  
Author(s):  
L. Berry ◽  
M.E. Grant ◽  
J. McClure ◽  
P. Rooney
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Marrow Stromal Cells ◽  
Bone Marrow Stroma ◽  
Collagen Types ◽  
Positive Matrix ◽  
Marrow Stroma ◽  
Neonatal Chicks

Bone marrow stromal cells from embryonic, neo-natal and adult chickens were grown in vitro over a 21-day period. Marrow stromal cells from embryonic and neonatal chicks produced clonally derived chondrocytic colonies. The cells within the colonies were surrounded by a refractile, Alcian-blue-positive matrix and their cartilagenous nature was shown biochemically and immunocytochemically by the synthesis of collagen types II and X. The ability of chick bone marrow cells to form chondrocytic colonies decreased during development and was lost by adulthood. In addition to chondrocytic colonies, fat cells and fibroblasts were also observed in the cultures. Our data demonstrate that chick bone marrow stroma contains cells that are capable of differentiating along different pathways within the same culture, providing further evidence for the presence in bone marrow of a stromal stem cell.

scholarly journals Bone Marrow Stromal Cells: Characterization and Clinical Application

1999 ◽  
Vol 10 (2) ◽  
pp. 165-181 ◽  
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.

scholarly journals Interference with Bone Marrow Stroma Derived Factors CXCL12 and TGFBI Increases Apoptosis in ALL Cells and Enhances Antileukemia Effects of Dexamethasone, Methotrexate, Vincristine and 6-Mercaptopurine

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4774-4774
Author(s):  
Sana Usmani ◽  
Olena Tkachencko ◽  
Leti Nunez ◽  
Craig A. Mullen
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Low Dose ◽  
Marrow Stromal Cells ◽  
Bone Marrow Stroma ◽  
Marrow Stroma ◽  
The Impact

Abstract Background: Bone marrow stroma provides a favorable microenvironmental niche for ALL cell survival. We and others have demonstrated that bone marrow stromal cells contribute to prevention of apoptosis in ALL cells. Objective: Identify potentially "drug-able" molecules derived from marrow stromal cells that contribute to prevention of ALL cell apoptosis. Methods: We have developed an in vitro system to identify stromal gene products that deliver antiapoptotic signals to ALL cells. Primary human ALL cells are co-cultured with human bone marrow stromal cells. We manipulate stromal cells with siRNA directed against candidate stromal cell genes. Two days later the siRNA is washed out of culture and primary ALL cells are added to the stromal cells. Controls include irrelevant siRNA. Five days later we measure viability and apoptosis in ALL cells by flow cytometry. Results: (1) Knockdown of stroma cell CXCL12 or TGFBI reduces ALL survival. We performed global gene expression analysis upon human marrow stromal cells using RNASeq technology. Using bioinformatic approaches we are selecting some of the expressed stromal genes as candidates for the molecular mechanisms by which stromal cells prevent ALL apoptosis. We present preliminary results for two of our early candidates. (A) CXCL12 is a paracrine chemokine known to have activity in the marrow microenvironment upon hematopoietic cells and we hypothesized it may participate in the effect. Knockdown of CXCL12 with siRNA increased ALL cell death in the co-culture system. As measured by quantitative reverse transcriptase PCR stromal cell CXCL12 mRNA was reduced approximately 75% by siRNA treatment. Figure 1 displays representative results of the impact of CXCL12 knockdown in stromal cell on the survival of ALL cells in the coculture. The magnitude of effect was ~40% increase in ALL cell death. (B) TGFBI (transforming growth factor beta induced) is expressed by stromal cells. The gene is involved in cell-collagen interactions and we hypothesized it played a role. siRNA reduced stromal gene expression by about 90%. Figure 2 displays representative results in which ALL cell death increased by about 50%. (2) Validation of results using inhibitors to CXCL12. The gene knockdown experiments suggested a potential role for CXCL12 in prevention of ALL cell apoptosis. To further test this we tested the effect of plerixafor, a specific inhibitor of CXCL12/CXCR4 interactions, on survival of ALL. ALL cells express CXCR4. In a dose dependent manner (25 - 400 micromolar) we observed a 31-39% reduction in ALL survival in stromal co-cultures including plerixafor. Figure 3 depicts representative results with plerixafor 200 micromolar. We are evaluating small molecules to block TGFBI. (3) Potential augmentation of chemotherapy drug effects on ALL. We hypothesize that interference with stromal cell molecules that prevent apoptosis in ALL cells may increase the effectiveness of conventional antileukemia drugs. In our stromal cell/ALL coculture system we have identified the effective in vitro concentrations of the most commonly used ALL drugs. We measured the impact of combination of low dose plerixafor (LD10) and these individual drugs (used at approximately the LD50 concentrations). Figure 4 demonstrates increased antileukemia effects related to plerixafor for dexamethasone, vincristine, and 6-mercaptopurine. Results are plotted as a percentage of ALL cells surviving in the absence of any drugs. The low dose plerixafor alone control did not produce a statistically significant reduction in ALL survival. Conclusions: Marrow stromal cell-produced CXCL12 may contribute to prevention of apoptosis in human ALL cells. Pharmacological interference with its effect may enhance the effectiveness of some conventional chemotherapy drugs. Marrow stromal cell-produced TGFBI may also contribute to prevention of apoptosis in human ALL cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of proteoglycan core proteins in human bone marrow stroma

1999 ◽  
Vol 343 (3) ◽  
pp. 663-668 ◽  
Author(s):  
Karen P. SCHOFIELD ◽  
John T. GALLAGHER ◽  
Guido DAVID
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Marrow Stromal Cells ◽  
Bone Marrow Stroma ◽  
Core Proteins ◽  
Stem And Progenitor Cells ◽  
Marrow Stroma

Heparan sulphate proteoglycans (HSPGs) present on the surface of bone marrow stromal cells and in the extracellular matrix (ECM) have important roles in the control of adhesion and growth of haemopoietic stem and progenitor cells. The two main groups of proteoglycans which contain heparan sulphate chains are members of the syndecan and glypican families. In this study we have identified the main surface membrane and matrix-associated HSPGs present in normal human bone marrow stroma formed in long-term culture. Proteoglycans were extracted from the adherent stromal layers and treated with heparitinase and chondroitinase ABC. The core proteins were detected by Western blotting using antibodies directed against syndecans-1-4, glypican-1 and the ECM HSPG, perlecan. Stromal cell expression at the RNA level was detected by Northern blotting and by reverse transcription PCR. Glypican-1, syndecan-3 and syndecan-4 were the major cell-membrane HSPG species and perlecan was the major ECM proteoglycan. There was no evidence for expression of syndecan-1 protein. Syndecan-3 was expressed mainly as a variant or processed 50-55 kDa core protein and in lower amounts as the characteristic 125 kDa core protein. These results suggest that syndecan-3, syndecan-4 and glypican-1 present on the surface of marrow stromal cells, together with perlecan in the ECM, may be responsible for creating the correct stromal ‘niche’ for the maintenance and development of haemopoietic stem and progenitor cells. The detection of a variant form of syndecan-3 as a major stromal HSPG suggests a specific role for this syndecan in haemopoiesis.

Abstract P120: Increased [ca 2+ ] e Enhances Adipocyte Development In Bone Marrow Stroma

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Ryota Hashimoto ◽  
Youichi Katoh ◽  
Seigo Itoh ◽  
Takafumi Iesaki ◽  
Hiroyuki Daida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Bone Marrow Stroma ◽  
Age Related ◽  
Marrow Stroma ◽  
High Concentrations ◽  
Adipocyte Development ◽  
Oil Red O

Background: Bone marrow stroma contains adipocytes, osteoblasts, and lymphohematopoietic donor cells. With age, fatty marrow gradually predominates in bone marrow stroma and is a factor underlying age-related fracture and anemia. Thus, it is important to understand the mechanism of adipocyte development in bone marrow stroma. Bone marrow Ca 2+ levels can reach high concentrations of 8 to 40 mM, while circulating plasma Ca 2+ levels normally range from 2.3 to 2.6 mM. However, the effects of a high extracellular calcium concentration ([Ca 2+ ] e ) on adipocyte development in bone marrow stroma remain largely unknown. Methods and Results: We studied the effects of high [Ca 2+ ] e on adipocyte development in bone marrow stroma. First, we used the fura-2 method to examine whether a change in [Ca 2+ ] e alters [Ca 2+ ] i levels in bone marrow stromal cells. Changes of [Ca 2+ ] e from 1.8 mM to 5.4 mM and 10.8 mM significantly increased [Ca 2+ ] i by 1.1 and 1.3 times, respectively. Next, bone marrow stromal cells were cultured for 14 days in high [Ca 2+ ] e (5.4 mM and 10.8 mM) and normal [Ca 2+ ] e (1.8 mM) conditions. Adipocyte development was monitored by Oil Red O staining of cytoplasmic lipids and by the activity of glycerol-3-phosphate dehydrogenase (GPDH). In 5.4 mM and 10.8 mM [Ca 2+ ] e , Oil Red O-stained cells increased significantly by 1.4 and 2.3 times, respectively, and GPDH activity increased significantly by 1.7 and 2.3 times, respectively, compared with the respective values in 1.8 mM [Ca 2+ ] e . Conclusions: These results indicate that high [Ca 2+ ] e induces an increase of [Ca 2+ ] i , which enhances adipocyte development in bone marrow stroma. Further studies are required to determine the influx pathway of Ca 2+ , since prevention of Ca 2+ influx into bone marrow stromal cells might suppress development of fatty marrow and reduce age-related fracture and anemia.

Chronic Myelogenous Leukemia Cells Contribute to a Bone Marrow-Stroma in In Vivo NOD/SCID Mouse System.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2191-2191
Author(s):  
Ryosuke Shirasaki ◽  
Haruko Tashiro ◽  
Yoko Oka ◽  
Toshihiko Sugao ◽  
Nobu Akiyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Scid Mouse ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Myelogenous Leukemia ◽  
Bone Marrow Stroma ◽  
Marrow Stroma ◽  
Mouse System

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.

scholarly journals Stromal colony-stimulating activity production and myeloid colony- forming cells in human hemopoietic and nonhemopoietic bone marrow

Blood ◽  
1981 ◽  
Vol 57 (4) ◽  
pp. 771-780
Author(s):  
RS Schwartz ◽  
PL Greenberg
Keyword(s):  
Bone Marrow ◽  
Cancellous Bone ◽  
Stromal Cells ◽  
Concentration Gradient ◽  
Long Bone ◽  
Bone Marrow Stroma ◽  
Colony Stimulating Activity ◽  
Marrow Stroma

In order to evaluate the role of the stromal bone marrow microenvironment in regulating granulopoiesis, we have examined the capacity of adult human proximal hemopoietic (PH) and distal nonhemopoietic (DNH) long bone to produce colony-stimulating activity (CSA), characterized the cellular sources of CSA, and quantitated the colony-forming cells (CFU-GM) of marrow from these sites. Stromal elements were obtained from slices of cancellous bone. PH bone marrow stroma contained CFU-GM concentrations similar to aspirated PH marrow and significantly more CFU-GM than DNH bone marrow: 20.7 +/- 4.8/10(5) cells and 25.8 +/- 12.0/mg bone versus 0.81 +/- 0.34/10(5) cells and 0.02 +/- 0.01/mg bone (p less than 0.001). Conditioned media prepared from PH and DNH bone were quantitated for CSA by their ability to promote in vitro granulocyte colony formation of nonadherent human marrow cells. Stromal CSA production was destroyed by freeze--thawing and was radioresistant (4400 rad). Of DNH stromal cells, 15%--30% were monocyte-macrophage, but the slow absolute numbers of these cells suggested alternative CSA cellular sources in distal bones. PH stroma produced significantly more CSA than DNH bone stroma: 0.72 +/- 0.10 versus 0.30 +/- 0.06 U/mg bone (p less than 0.01). The CSA concentration gradient between PH and DNH bones may contribute to the regulation of granulopoiesis in marrow and to the absence of hemopoiesis distally.

scholarly journals Bone marrow stromal cells produce thrombopoietin and stimulate megakaryocyte growth and maturation but suppress proplatelet formation

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1309-1316 ◽  
Author(s):  
H Nagahisa ◽  
Y Nagata ◽  
T Ohnuki ◽  
M Osada ◽  
T Nagasawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Bone Marrow Cells ◽  
Marrow Stromal Cells ◽  
Orphan Receptor ◽  
Liver And Kidney ◽  
Proplatelet Formation ◽  
Cytoplasmic Processes

Production of blood cells is regulated by the interplay of various cytokines and bone marrow stromal cells. Recently, a ligand for the orphan receptor Mpl was identified as thrombopoietin (TPO), which specifically regulates megakaryocyte differentiation, and it was reported to be expressed mainly in liver and kidney. As it was found that thrombopoietin is also produced in bone marrow stromal cells, we studied further the roles of bone marrow stromal cells on megakaryocytopoiesis and platelet formation. The stromal cells stimulated growth and maturation of bone-marrow-derived megakaryocytes in the presence of thrombopoietin, and also supported growth of BaF3 cells expressing exogenous Mpl without thrombopoietin. Thrombopoietin induces drastic morphological change of megakaryocytes in bone marrow cells in vitro, ie, the formation of lengthy beaded cytoplasmic processes (proplatelet formation). However, when the purified megakaryocytes were cocultured with the stromal cells with or without thrombopoietin, most of the megakaryocytes adhered to the stromal cells and remained unchanged, while free megakaryocytes induced proplatelet formation. These observations indicated that the stromal cells in a hematopoietic microenvironment in bone marrow secrete thrombopoietin and stimulate proliferation and maturation of megakaryocytes, but the interaction of megakaryocytes with the stromal cells may suppress proplatelet formation.

scholarly journals Stromal colony-stimulating activity production and myeloid colony- forming cells in human hemopoietic and nonhemopoietic bone marrow

Blood ◽  
1981 ◽  
Vol 57 (4) ◽  
pp. 771-780 ◽  
Author(s):  
RS Schwartz ◽  
PL Greenberg
Keyword(s):  
Bone Marrow ◽  
Cancellous Bone ◽  
Stromal Cells ◽  
Concentration Gradient ◽  
Long Bone ◽  
Bone Marrow Stroma ◽  
Colony Stimulating Activity ◽  
Marrow Stroma

Abstract In order to evaluate the role of the stromal bone marrow microenvironment in regulating granulopoiesis, we have examined the capacity of adult human proximal hemopoietic (PH) and distal nonhemopoietic (DNH) long bone to produce colony-stimulating activity (CSA), characterized the cellular sources of CSA, and quantitated the colony-forming cells (CFU-GM) of marrow from these sites. Stromal elements were obtained from slices of cancellous bone. PH bone marrow stroma contained CFU-GM concentrations similar to aspirated PH marrow and significantly more CFU-GM than DNH bone marrow: 20.7 +/- 4.8/10(5) cells and 25.8 +/- 12.0/mg bone versus 0.81 +/- 0.34/10(5) cells and 0.02 +/- 0.01/mg bone (p less than 0.001). Conditioned media prepared from PH and DNH bone were quantitated for CSA by their ability to promote in vitro granulocyte colony formation of nonadherent human marrow cells. Stromal CSA production was destroyed by freeze--thawing and was radioresistant (4400 rad). Of DNH stromal cells, 15%--30% were monocyte-macrophage, but the slow absolute numbers of these cells suggested alternative CSA cellular sources in distal bones. PH stroma produced significantly more CSA than DNH bone stroma: 0.72 +/- 0.10 versus 0.30 +/- 0.06 U/mg bone (p less than 0.01). The CSA concentration gradient between PH and DNH bones may contribute to the regulation of granulopoiesis in marrow and to the absence of hemopoiesis distally.

TC-PTP–deficient bone marrow stromal cells fail to support normal B lymphopoiesis due to abnormal secretion of interferon-γ

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4220-4228 ◽  
Author(s):  
Annie Bourdeau ◽  
Nadia Dubé ◽  
Krista M. Heinonen ◽  
Jean-François Théberge ◽  
Karen M. Doody ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Bone Marrow Stroma ◽  
B Lymphopoiesis ◽  
Marrow Stroma ◽  
Ifn Γ

Abstract The T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of the Jak/Stat cytokine signaling pathway. Our study shows that the absence of TC-PTP leads to an early bone marrow B-cell deficiency characterized by hindered transition from the pre-B cell to immature B-cell stage. This phenotype is intrinsic to the B cells but most importantly due to bone marrow stroma abnormalities. We found that bone marrow stromal cells from TC-PTP−/− mice have the unique property of secreting 232-890 pg/mL IFN-γ. These high levels of IFN-γ result in 2-fold reduction in mitotic index on IL-7 stimulation of TC-PTP−/− pre-B cells and lower responsiveness of IL-7 receptor downstream Jak/Stat signaling molecules. Moreover, we noted constitutive phosphorylation of Stat1 in those pre-B cells and demonstrated that this was due to soluble IFN-γ secreted by TC-PTP−/− bone marrow stromal cells. Interestingly, culturing murine early pre-B leukemic cells within a TC-PTP–deficient bone marrow stroma environment leads to a 40% increase in apoptosis in these malignant cells. Our results unraveled a new role for TC-PTP in normal B lymphopoiesis and suggest that modulation of bone marrow microenvironment is a potential therapeutic approach for selected B-cell leukemia.

scholarly journals New Insights on Properties and Spatial Distributions of Skeletal Stem Cells

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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