scholarly journals Tranexamic Acid Promotes Murine Bone Marrow-Derived Osteoblast Proliferation and Inhibits Osteoclast Formation In Vitro

2021 ◽  
Vol 22 (1) ◽  
pp. 449
Author(s):  
Anke Baranowsky ◽  
Jessika Appelt ◽  
Kristina Tseneva ◽  
Shan Jiang ◽  
Denise Jahn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tranexamic Acid ◽  
Bone Cells ◽  
Osteoclast Formation ◽  
Surgical Trauma ◽  
Short Term ◽  
Matrix Mineralization ◽  
Murine Bone Marrow

Despite modern surgical trauma care, bleeding contributes to one-third of trauma-related death. A significant improvement was obtained through the introduction of tranexamic acid (TXA), which today is widely used in emergency and elective orthopedic surgery to control bleeding. However, concerns remain regarding potential adverse effects on bone turnover and regeneration. Therefore, we employed standardized cell culture systems including primary osteoblasts, osteoclasts, and macrophages to evaluate potential effects of TXA on murine bone cells. While osteoblasts derived from calvarial digestion were not affected, TXA increased cell proliferation and matrix mineralization in bone marrow-derived osteoblasts. Short-term TXA treatment (6 h) failed to alter the expression of osteoblast markers; however, long-term TXA stimulation (10 days) was associated with the increased expression of genes involved in osteoblast differentiation and extracellular matrix synthesis. Similarly, whereas short-term TXA treatment did not affect gene expression in terminally differentiated osteoclasts, long-term TXA stimulation resulted in the potent inhibition of osteoclastogenesis. Finally, in bone marrow-derived macrophages activated with LPS, simultaneous TXA treatment led to a reduced expression of inflammatory cytokines and chemokines. Collectively, our study demonstrates a differential action of TXA on bone cells including osteoanabolic, anti-resorptive, and anti-inflammatory effects in vitro which suggests novel treatment applications.

scholarly journals Hematopoietic regulatory factors produced in long-term murine bone marrow cultures and the effect of in vitro irradiation

Blood ◽  
1984 ◽  
Vol 64 (2) ◽  
pp. 516-525 ◽  
Author(s):  
RJ Gualtieri ◽  
RK Shadduck ◽  
DG Baker ◽  
PJ Quesenberry
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Factor Vii ◽  
Murine Bone Marrow ◽  
L Cell ◽  
Bone Marrow Cultures ◽  
Macrophage Colony ◽  
Marrow Cultures

The nature of hematopoietic regulatory factors elaborated by the adherent (stromal) cells of long-term murine bone marrow cultures and the effect of in vitro stromal irradiation (XRT) on the production of these factors was investigated. Using an in situ stromal assay employing a double layer of semisolid agar, it was possible to demonstrate stromal elaboration of at least two colony-stimulating activities, ie, granulocyte/macrophage colony-stimulating activity (G/M- CSA) and megakaryocyte colony-stimulating activity (Meg-CSA). Exposure of the stroma to XRT resulted in dose-dependent elevations of both activities that correlated inversely with total myeloid cell mass as determined by concurrent reductions in total supernatant cell recoveries from irradiated cultures. Mixture experiments that combined control and irradiated stroma revealed that the hematopoietically active control stroma could block detection of XRT-related G/M-CSA elevations. These data implicate a local negative feedback mechanism in the regulation of hematopoiesis. Antiserum directed against purified L cell colony-stimulating factor (CSF) reduced granulocyte/macrophage colony formation in the target layer but did not effect the increased Meg-CSA. While a radioimmunoassay for L-cell type CSF was unable to detect significant differences in concentrated media from control and irradiated cultures, bioassays of these media revealed XRT-related G/M- CSA elevations. These results indicate that the G/M-CSA elaborated in these cultures is immunologically distinct from the Meg-CSA produced, and although distinct from L cell CSF, the G/M-CSA is crossreactive with the L cell CSF antiserum. Morphologic, histochemical, and factor VII antigen immunofluorescent studies were performed on the stromal cell population responsible for production of these stimulatory activities. In addition to “fat” cells, the stromal cells remaining after XRT were composed of two predominant cell populations. These included a major population of acid phosphatase and nonspecific esterase-positive macrophage-like cells and a minor population of factor VII antigen negative epithelioid cells.

scholarly journals No stimulative effect of adipocytes on hematopoiesis in long-term human bone marrow cultures

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 770-774
Author(s):  
I Touw ◽  
B Lowenberg
Keyword(s):  
Bone Marrow ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Adherent Cell ◽  
Murine Bone Marrow ◽  
Bone Marrow Cultures ◽  
Human Bone Marrow Cultures ◽  
Marrow Cultures

Long-term cultures of human bone marrow were established for 5–13 wk to study the role of adipocytes in sustaining hematopoiesis. At weekly intervals, the numbers of nucleated cells and granulocyte-macrophage progenitor cells (GM-CFU) in culture were estimated in relation to the numbers of fat-containing cells present in the adherent stroma layer. In these quantifications, the numbers of GM-CFU trapped in the adherent cell layer were considered separately. It was found that the presence of adipocytes did not correlate with more active hematopoiesis. Fat cells appeared at late stages when successful cultures were being exhausted or early in cultures with poor activity. These observations suggest that human marrow continuous hematopoiesis in vitro, unlike hematopoiesis in the analogous murine bone marrow cultures, does not depend on the presence of adipocytes.

scholarly journals Regulation of megakaryopoiesis in long-term murine bone marrow cultures

Blood ◽  
1978 ◽  
Vol 51 (2) ◽  
pp. 245-255 ◽  
Author(s):  
N Williams ◽  
H Jackson ◽  
AP Sheridan ◽  
MJ Jr Murphy ◽  
A Elste ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Leukemic Cell ◽  
Precursor Cells ◽  
Mouse Bone Marrow ◽  
Murine Bone Marrow ◽  
Marrow Cultures

Abstract Megakaryocytes and their precursor cells were sustained in mouse bone marrow suspension cultures for over 4–6 wk. Megakaryocyte precursor cells were detected by their capacity to form colonies of megakaryocytes in semisolid agar cultures. Colony formation was dependent on the presence of medium conditioned by a myelomonocytic leukemic cell line (WEHI-3CM). Megakaryocytes from the liquid and semisolid cultures were identified by cytoplasmic acetylcholine esterase and by ultrastructural analysis. The suspension medium from the bone marrow liquid cultures which sustained megakaryopoiesis was not directly acitive in stimulating megakaryocyte colony formation in the semisolid agar cultures, but potentiated the number of colonies detected when WEHI-3CM was present. Bone marrow-conditioned medium increased the sensitivity of megakaryocyte progenitor cells to the stimulus in WEHI-3CM. Addition of the activities present in the two sources produced a quantitative assay for the detection of mouse megakaryocyte progenitor cells. These studies showed: (1) that no inductive regulator of in vitro clones of megakaryocytes was present in the supernatants from the long-term marrow cultures and, (2) that at least two factors were necessary for the induction of megakaryocyte progenitors to proliferate and differentiate in semisolid cultures in vitro.

scholarly journals Interleukin-11 stimulates multiple phases of erythropoiesis in vitro

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1218-1223
Author(s):  
VF Quesniaux ◽  
SC Clark ◽  
K Turner ◽  
B Fagg
Keyword(s):  
Bone Marrow ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Murine Bone Marrow ◽  
Kit Ligand ◽  
B Cell Maturation ◽  
Stromal Cell Line ◽  
Interleukin 11

Interleukin-11 (IL-11), a pleiotropic cytokine originally isolated from a primate bone marrow stromal cell line, has been shown to stimulate T- cell-dependent B-cell maturation, megakaryopoiesis, and various stages of myeloid differentiation, but to inhibit adipogenesis. Because stromal cells are essential for the maintenance of early hematopoietic progenitor cells in long-term culture, we investigated the effects of IL-11 on multipotent and erythroid precursors from murine bone marrow in vitro in suspension and semisolid cultures. Our results show that in the presence of IL-3 or c-kit ligand (KL), IL-11 has profound stimulatory effects on primitive multilineage hematopoietic progenitors, pre-CFC(multi), as well as on precursors representing various stages of erythroid differentiation observable in vitro, including CFC(multi), BFU-E, and CFU-E. In addition, the combination of KL with IL-11 also stimulated highly proliferative erythroid progenitors that yield remarkable macroscopic erythroblast colonies in culture. These results indicate that IL-11 is likely to play a pivotal role in early hematopoiesis and at multiple stages of erythropoiesis.

The Bone Marrow "Mystery Population" of Stem Cells 20 Years Later - a Puzzle Solved?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2392-2392
Author(s):  
Malwina Suszynska ◽  
Daniel Pedziwiatr ◽  
Magdalena J Kucia ◽  
Mariusz Z Ratajczak ◽  
Janina Ratajczak
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Receptor Expression ◽  
Stem Cell Population ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Low Levels

Abstract Background . Almost 20 years ago, a "mystery" population of small stem cells with many of the phenotypic characteristics attributed to resting hematopoietic stem cells was identified in murine bone marrow (BM) (Stem Cells 1998, 16, 38-48). These cells expressed high levels of Sca-1, H-2K, and CD38 and low levels of Thy-1.1; they expressed CD45 antigen but were lineage-negative (lin-) for other hematopoietic markers. These cells incorporated only low levels of Rh123 and were resistant to the cytotoxic effects of 5-fluorouracil. The only phenotypic characteristic that distinguishes these cells from Sca-1+, Lin-, CD45+ Thy-1.1low long-term-reconstituting hematopoietic stem cell population is the lack of c-kit expression. In sum, this "mystery" population of small Sca-1+, lin-, c-kit- but CD45+ stem cells do not respond to hematopoietic growth factors in vitro, form in vivo spleen colonies, or reconstitute lethally irradiated mice. With our discovery of Sca-1+ Lin- CD45- very small embryonic-like stem cells (VSELs) in murine bone marrow (BM) (Leukemia 2006, 20, 857-869), we became interested in this "mystery" population of stem cells. VSELs, like the "mystery" population, are c-kit - and, if freshly isolated from BM, do not show any hematopoietic activity in standard in vitro and in vivo assays. In order to become specified to hematopoiesis, they need to be expanded over an OP-9 stromal support (Exp Hematol 2011;39:225-237). Hypothesis. Since (1) very small CD45- VSELs can be specified in OP-9 co-cultures into long-term reconstituting CD45+ HSCs, (2) the size of the "mystery" population is intermediate between VSELs and HSCs, and (3) VSELs and HSCs differ in cell surface receptor expression, we hypothesized that the "mystery" population is a missing developmental intermediate between VSELs and HSCs. Materials and Methods . Multicolor FACS analysis was employed to compare size and expression of surface markers between murine BM HSCs, the unknown population of stem cells, and VSELs. Next, the populations of small Sca-1+ H2-K+ lin- c-kit+ CD38+/- CD45+ cells (HSCs), smaller Sca-1+ H-2K+ lin- c-kit- CD38+ CD45+ cells (the "mystery" population), and very small in size Sca-1+ H-2K+ lin- c-kit- CD38+/- CD45- cells (VSELs) were purified by FACS from BM (Figure 1) and tested for in vitro colony formation. All these cell populations were primed/expanded over OP-9 support and subsequently evaluated for their hematopoietic potential after passaging in consecutive methylocellulose cultures (passages 1-4). RQ-PCR analysis was employed for detection of pluripotency marker expression as well as hematopoietic gene expression. Results . We found that, in contrast to HSCs, neither freshly sorted stem cells from the "mystery" BM population nor, as expected, VSELs grew hematopoietic colonies in standard methylcellulose cultures. This was also an important step in excluding contamination of our sorted populations with clonogenic cells. We also found that, while VSELs highly expressed Oct-4, this transcription factor was expressed at very low levels in the "mystery" population and was not detectable in HSCs. The most important observation was that the "mystery" population of stem cells became specified in OP-9-supported cultures into clonogenic HSPCs, and this specification occurred faster than the delayed specification of VSELs. VSELs first became enriched for HSPCs after acquiring CD45 antigen expression. Conclusions . Based on the results presented, we propose that the "mystery" population in murine BM is a population of stem cells intermediate between the most primitive population of BM-residing stem cells (VSELs) and the population of stem cells already specified to lympho-hematopoietic development (HSCs). Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic regulatory factors produced in long-term murine bone marrow cultures and the effect of in vitro irradiation

Blood ◽  
1984 ◽  
Vol 64 (2) ◽  
pp. 516-525 ◽  
Author(s):  
RJ Gualtieri ◽  
RK Shadduck ◽  
DG Baker ◽  
PJ Quesenberry
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Factor Vii ◽  
Murine Bone Marrow ◽  
L Cell ◽  
Bone Marrow Cultures ◽  
Macrophage Colony ◽  
Marrow Cultures

Abstract The nature of hematopoietic regulatory factors elaborated by the adherent (stromal) cells of long-term murine bone marrow cultures and the effect of in vitro stromal irradiation (XRT) on the production of these factors was investigated. Using an in situ stromal assay employing a double layer of semisolid agar, it was possible to demonstrate stromal elaboration of at least two colony-stimulating activities, ie, granulocyte/macrophage colony-stimulating activity (G/M- CSA) and megakaryocyte colony-stimulating activity (Meg-CSA). Exposure of the stroma to XRT resulted in dose-dependent elevations of both activities that correlated inversely with total myeloid cell mass as determined by concurrent reductions in total supernatant cell recoveries from irradiated cultures. Mixture experiments that combined control and irradiated stroma revealed that the hematopoietically active control stroma could block detection of XRT-related G/M-CSA elevations. These data implicate a local negative feedback mechanism in the regulation of hematopoiesis. Antiserum directed against purified L cell colony-stimulating factor (CSF) reduced granulocyte/macrophage colony formation in the target layer but did not effect the increased Meg-CSA. While a radioimmunoassay for L-cell type CSF was unable to detect significant differences in concentrated media from control and irradiated cultures, bioassays of these media revealed XRT-related G/M- CSA elevations. These results indicate that the G/M-CSA elaborated in these cultures is immunologically distinct from the Meg-CSA produced, and although distinct from L cell CSF, the G/M-CSA is crossreactive with the L cell CSF antiserum. Morphologic, histochemical, and factor VII antigen immunofluorescent studies were performed on the stromal cell population responsible for production of these stimulatory activities. In addition to “fat” cells, the stromal cells remaining after XRT were composed of two predominant cell populations. These included a major population of acid phosphatase and nonspecific esterase-positive macrophage-like cells and a minor population of factor VII antigen negative epithelioid cells.

scholarly journals Stromal cell progeny of murine bone marrow fibroblast colony-forming units are clonal endothelial-like cells that express collagen IV and laminin

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 620-625 ◽  
Author(s):  
S Perkins ◽  
RA Fleischman
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Lineage ◽  
Fibroblast Cell ◽  
Collagen Iv ◽  
Short Term ◽  
Murine Bone Marrow ◽  
Colony Forming Units

Abstract Studies of human and murine bone marrow explants have demonstrated the existence of stromal cell precursors that give rise to colonies of adherent cells in short-term cultures. Because previous data suggested that these colonies were composed of fibroblasts, the precursor cells were termed fibroblast colony-forming units (CFU-F). However, we have recently shown that the stromal cells which support hematopoiesis in murine long-term bone marrow cultures (LTBC) express collagen IV and laminin, markers associated with an endothelial cell lineage, but are negative for collagen I and III, markers associated with a fibroblast cell lineage. Because these conflicting results suggest major functional differences between the stromal cells observed in long-term cultures and the short-term assay, we re-examined the lineage of CFU-F- derived stromal cells. Using two-color immunofluorescence, we characterized virtually all of the cells comprising individual “CFU-F” colonies derived from mouse radiation chimeras. Identification of donor (hematopoietic) or host (stromal) origin was based on surface staining for strain-specific H-2 surface antigens, and, for endothelial or fibroblast properties, on cytoplasmic staining for laminin and collagen IV, or collagens I and III, respectively. The results demonstrate that a large proportion of the cells in CFU-F colonies are donor-derived and fail to stain with any of the antisera specific for nonhematopoietic cells. In addition, these donor-derived cells exhibit marked phagocytic capacity and stain positively with monoclonal antibodies characteristic of the monocyte-macrophage hematopoietic cell lineage (anti-T200, anti- Mac-1, F4/80). However, the remainder of the cells are host-derived cells that stain positively with antisera to collagen IV and laminin. In contrast, stains for collagen types I and III were negative under conditions that allowed for strong staining of control skin fibroblasts. In separate studies, using mixtures of two genetically distinct bone marrows, the cells expressing collagen IV were further shown to be clonal in origin within individual colonies, directly demonstrating that the CFU-F assay provides a quantitative measure of the numbers of marrow stromal cell precursors. Thus, the current studies establish a remarkable similarity between the hematopoietic microenvironment in the short-term CFU-F assay and the long-term culture system: the majority of adherent cells are hematopoietic cells of the monocyte-macrophage lineage, while the remainder are stromal cells whose precise lineage remains uncertain, but whose pattern of collagen expression is more consistent with an endothelial rather than a fibroblast cell origin.

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