Bone Marrow Stromal Precursor Research Articles

Damage of the Bone Marrow Stromal Precursors in Patients with Acute Leukemia at the Onset of the Disease and During Treatment.

In patients with acute leukemia (AL), malignant cells and therapy modify the properties of multipotent mesenchymal stromal cells (MSCs) and their descendants, reducing their ability to maintain normal hematopoiesis. The aim of this work was to elucidate the alterations in MSCs at the onset and after therapy in patients with AL. The study included MSCs obtained from the bone marrow of 78 AL patients (42 AML and 36 ALL) and healthy donors. MSC growth characteristics, gene expression pattern, proteome and secretome were studied using appropriate methods. The concentration of MSCs in the bone marrow, proliferative potential, the expression of several genes, proteomes and secretomes were altered in AL-MSCs. Stromal progenitors had been affected differently in ALL and AML patients. In remission, MSC functions remain impaired despite the absence of tumor cells and the maintenance of benign hematopoietic cells. AL causes crucial and, to a large extent, irreversible changes in bone marrow MSCs.

Linoleic acid blunts early osteoblast differentiation and impairs oxidative phosphorylation in vitro

BackgroundLinoleic acid (LNA), an essential polyunsaturated fatty acid (PUFA), plays a crucial role in cellular functions. However, excessive intake of LNA, characteristic of Western diets, can have detrimental effects on cells and organs. Human observational studies have shown an inverse relationship between plasma LNA concentrations and bone mineral density. The mechanism by which LNA impairs the skeleton is unclear, and there is a paucity of research on the effects of LNA on bone-forming osteoblasts. MethodsThe effect of LNA on osteoblast differentiation, cellular bioenergetics, and production of oxidized PUFA metabolites in vitro, was studied using primary mouse bone marrow stromal cells (BMSC) and MC3T3-E1 osteoblast precursors. ResultsLNA treatment decreased alkaline phosphatase activity, an early marker of osteoblast differentiation, but had no effect on committed osteoblasts or on mineralization by differentiated osteoblasts. LNA suppressed osteoblast commitment by blunting the expression of Runx2 and Osterix, key transcription factors involved in osteoblast differentiation, and other key osteoblast-related factors involved in bone formation. LNA treatment was associated with increased production of oxidized LNA- and arachidonic acid-derived metabolites and blunted oxidative phosphorylation, resulting in decreased ATP production. ConclusionOur results show that LNA inhibited early differentiation of osteoblasts and this inhibitory effect was associated with increased production of oxidized PUFA metabolites that likely impaired energy production via oxidative phosphorylation.

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced μCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt−/−, KitW/W−v) and development of specific BMA deletion models would be highly desirable for this purpose.

Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight.

Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45+MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.

Hydrogel encapsulation to improve cell viability during syringe needle flow.

This work examines pluronic F-127 poloxamer for cell protection during injection through a syringe needle. Direct cell injection is a minimally invasive method for cell transplantation; however, it often results in poor cell viability. We proposed that encapsulating cells in this hydrogel would protect cells from detrimental mechanical forces during injection and increase cell viability. The hydrogel was tested at multiple weights and carbon nanobrush concentrations to determine how gel weight affects cell viability as well as to allow the gels to remain as electrically conductive scaffolds. This work assessed the ability of the hydrogel to prevent cell membrane bursting. We used D1 multipotent mouse bone marrow stromal precursor cells for this study. We found that the pressure drop increases with increasing weight of the gels. However, cell viability also increases as the weight of the gels increases. These results support the proposition that hydrogels can be used to protect cells during syringe needle injection. Since these hydrogels undergo a reverse phase transition, the gels can be used to transplant cells into the body in solution form through injection. The gels will then harden in situ to allow for cell proliferation and tissue regeneration at the desired site.

Transplantation of Bone Marrow Stromal Cell-Derived Neural Precursor Cells Ameliorates Deficits in a Rat Model of Complete Spinal Cord Transection

After severe spinal cord injury, spontaneous functional recovery is limited. Numerous studies have demonstrated cell transplantation as a reliable therapeutic approach. However, it remains unknown whether grafted neuronal cells could replace lost neurons and reconstruct neuronal networks in the injured spinal cord. To address this issue, we transplanted bone marrow stromal cell-derived neural progenitor cells (BM-NPCs) in a rat model of complete spinal cord transection 9 days after the injury. BM-NPCs were induced from bone marrow stromal cells (BMSCs) by gene transfer of the Notch-1 intracellular domain followed by culturing in the neurosphere method. As reported previously, BM-NPCs differentiated into neuronal cells in a highly selective manner in vitro. We assessed hind limb movements of the animals weekly for 7 weeks to monitor functional recovery after local injection of BM-NPCs to the transected site. To test the sensory recovery, we performed functional magnetic resonance imaging (fMRI) using electrical stimulation of the hind limbs. In the injured spinal cord, transplanted BM-NPCs were confirmed to express neuronal markers 7 weeks following the transplantation. Grafted cells successfully extended neurites beyond the transected portion of the spinal cord. Adjacent localization of synaptophysin and PSD-95 in the transplanted cells suggested synaptic formations. These results indicated survival and successful differentiation of BM-NPCs in the severely injured spinal cord. Importantly, rats that received BM-NPCs demonstrated significant motor recovery when compared to the vehicle injection group. Volumes of the fMRI signals in somatosensory cortex were larger in the BM-NPC-grafted animals. However, neuronal activity was diverse and not confined to the original hind limb territory in the somatosensory cortex. Therefore, reconstruction of neuronal networks was not clearly confirmed. Our results indicated BM-NPCs as an effective method to deliver neuronal lineage cells in a severely injured spinal cord. However, reestablishment of neuronal networks in completed transected spinal cord was still a challenging task.

Understanding the role of the microenvironment during definitive hemopoietic development

Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow have identified osteoblasts and endothelial cells as two important supportive cell types within the niche and demonstrated that interactions between HSCs and cellular and extracellular components within the endosteal and perivascular regions are critical for HSC regulation. However, the understanding of the role of the microenvironment in definitive HSC establishment, expansion, and maintenance during embryonic development is extremely limited. This review focuses on what is known about the components of each HSC microenvironment at various developmental stages and their known functional roles.

Mineralization and Osteoblast Cells Response of Nanograde Pearl Powders

The main objective of this study is to characterize the thermal, mineralization, and osteoblast cells response of pearl nanocrystallites. The results obtained from X‐ray diffraction, FTIR spectra demonstrate that the pearl nano‐crystallites can induce the formation of an HA layer on their surface in SBF, even after only short soaking periods. The in vitro cell response to nano‐grade pearl powders is assessed by evaluating the cytotoxicity against a mouse embryonic fibroblast cell line and by characterizing the attachment ability and alkaline phosphatase activity of mouse bone cells (MC3T3‐E1, abbreviated to E1) and bone marrow stromal precursor (D1) cells. The cytotoxicities of pearls were tested by the filtration and culture of NIH‐3T3 mouse embryonic fibroblast cells. The viability of the cultured cells in media containing pearl crystallites for 24 and 72 h is greater than 90%. The bone cells seen in these micrographs are elongated and align predominately along the pearl specimen. The cells observed in these images also appeared well attached and cover the surface almost completely after 1 h. The pearl nanocrystallites had a positive effect on the osteogenic ability of ALP activity, and this promoted the osteogenic differentiation of MSCs significantly at explanations.

Catecholamine regulation of stromal precursors and hemopoietic stem cells in cytostatic myelosuppression

Effects of a sympatholytic drug on bone marrow stromal and hemopoietic precursors were studied on the model of cyclophosphamide-induced myelosuppression. Sympatholytic treatment increased the content of hemopoietic stem cells of different classes in the bone marrow. Selective stimulation of differentiation of polypotent precursors into granulocyte-macrophage precursors was noted. Acceleration of proliferation and maturation of granulocytic precursors was observed at later terms during regeneration of the hemopoietic tissue. The sympatholytic inhibited proliferation of stromal precursors and reduced feeder activity of fibroblasts for granulocyte precursors.

Hyaluronic acid and chondrogenesis of murine bone marrow mesenchymal stem cells in chitosan sponges

To establish the dose-dependent effects of high-molecular-weight hyaluronic acid (HA) supplementation on chondrogenesis by mesenchymal stem cells (MSCs) cultured on chitosan sponges and to determine the extent to which MSC matrix production (chondrogenesis) can be influenced by incorporation of high-molecular-weight HA into chitosan scaffolds. Murine MSCs derived from a multipotent bone marrow stromal precursor. MSCs were seeded on chitosan and chitosan-HA scaffolds in chondrogenic medium with various HA concentrations. Scanning electron microscopy, fluorescence microscopy (viability assay), and DNA quantification were used to assess cell attachment, distribution, and viability 48 hours after seeding. Constructs were cultured for 3 weeks prior to evaluation of cell distribution and chondrogenic differentiation via histologic evaluation and quantification of DNA, glycosaminoglycan, and collagen II. 48 hours after MSC seeding, cell viability and DNA content were similar among groups. Three weeks after seeding, HA supplementation of the culture medium improved matrix production in a dose-dependent manner, as indicated by matrix glycosaminoglycan and collagen II concentrations. The scaffold composition, however, had no significant effect on matrix production. High-molecular-weight HA supplementation in culture medium had a dose-dependent effect on matrix production and thus chondrogenic differentiation of MSCs cultured on chitosan sponges. The addition of HA in the surrounding fluid during chondrogenesis should improve cartilage production and may be useful for producing engineered cartilage tissues.

Effect of Adrenomimetics and Serotonin on Polypotent Stromal and Hemopoietic Precursors in Cytostatic Myelosuppression

Effects of serotonin and adrenomimetics (phenylephrine and isoprenaline) on bone marrow stromal and polypotent hemopoietic precursors were studied in vitro on the model of cyclophosphamide-induced myelosuppression. It was found that under conditions of myelosuppression, adrenomimetics potentiate differentiation of polypotent hemopoietic precursors into mature precursors (granulocyte-macrophage and granulocyte CFU) initiated by granulocytic CSF, while serotonin suppresses these processes. Adrenomimetics (especially, isoprenaline) abolish high rate of division of stromal precursors and suppress the growth of granulocytic CSF induced by fibroblast-like cells. Serotonin does not affect proliferation of stromal precursors, but potentiates the granulocytopoiesis-stimulating effects of fibroblasts.

Characterization of the Differentiation and Leptin Secretion Profile of Adult Stem Cells on Patterned Polylactide Films

Several issues need to be better understood before breast tissue engineering becomes a clinically viable option. One of the most important aspects is the interaction between cells and the microtopography of the implant surface. The aim of this study was to evaluate the efficacy of D1 cells, multipotent mouse bone marrow stromal precursors, in differentiating to adipocytes and to characterize their metabolic activity (lactic acid released and glucose consumed), leptin secretion and lipid production when cultured on patterned poly(L-lactide) (PLLA) films. It was determined that, by appropriate stimulation, the D1 cells displayed morphological characteristics of adipocytes and produced lipid. The results showed that a patterned surface did affect the rate of lipid production. Polynomial models were proposed to predict the amount of leptin secreted by the cells over a period of time.

Effect of Macrophage Migration Inhibition Factor on the Content of Stromal Precursor Cells in Mouse Bone Marrow and Efficiency of Bone Marrow Precursor Cell Cloning In Vitro

The content of stromal precursor cells in the bone marrow of mice decreased 2-5.7 times 24 h after injection of macrophage migration inhibition factor in doses of 0.1-50 ng/kg, this reduction depending on the dose of inhibition factor. The content of precursor cells in the bone marrow of mice increased 2-fold 24 h after injection of S. typhimurium bacterial mass. One day after injection of S. typhimurium bacterial mass, the count of precursor cells in mouse spleen was 7-fold higher than 24 h after injection of macrophage migration inhibition factor. The efficiency of cloning of mouse bone marrow stromal precursor cells in vitro was suppressed 1.7-2.8 times in the presence of macrophage migration inhibition factor in doses of 0.1 to 50 ng/ml culture medium. The effect of cloning inhibition was preserved, if macrophage migration inhibition factor was added to the culture medium after 2 days of bone marrow cell culturing. In general, macrophage migration inhibition factor inhibits stromal precursor cells in vivo and in vitro. The data also indicate that macrophage migration inhibition factor is not responsible for rapid and sharp increase in the count of stromal precursor cells after immunization of animals.

Origination of stromal precursor cells replenishing the population of these cells in heterotopic mouse bone marrow transplants after curettage in recipients

Curettage of bone marrow cavities of two bones (femoral and crural) in recipient mice causes a drastic (more than 7-fold) increase in the count of stromal precursor cells in heterotopic bone marrow transplants. Stromal colonies in cell cultures from these transplants consist of fibroblasts with an appreciable admixture of macrophages. All y chromosome-typed colonies from cultures of female donor bone marrow transplants in recipient males (intact and subjected to curettage) contained cells carrying and not carrying y chromosome. Quantitative results of Y chromosome typing of cells from colonies corresponded to the fibroblast/macrophage ratio in colonies and the predominant localization of the label corresponded to predominant localization of macrophages (at the periphery of colonies). The results indicate that the pool of bone marrow stromal precursor cells under conditions of increased demands originates from local sources, which confirms ample data on inability of these cells to migration.

Surface patterning: Tool to modulate stem cell differentiation in an adipose system

There are several issues that need to be better understood before breast tissue-engineering becomes viable clinically. One of the key issues is the interaction between cells and the microtopography of the implant surface. The aim of this study was to evaluate the efficacy of D1 cells, multipotent mouse bone marrow stromal precursors, in differentiating to fat and to characterize their metabolic activity (lactic acid released and glucose consumed) and lipid production when cultured on patterned poly-L-lactide (PLLA) films. It was determined that, with appropriate stimulation, the D1 cells displayed morphological characteristics of adipocytes and produced lipid. The results show that the patterned surfaces did affect the rate of lipid production. Polynomial models were proposed to predict the metabolic activity of the cells over a period of time.

Effect of α-fetoprotein on the count of bone marrow and splenic stromal precursor cells and proliferation of their cultural descendants

The efficiency of cloning of stromal precursor cell increased more than 2-fold in splenic cultures and more than 3-fold in bone marrow cultures 24 h after injection of Profetal preparation to mice in vivo. The number of nucleated cells did not change in the bone marrow and slightly increased in the spleen. Addition of Profetal in vitro 2-fold decreased the efficiency of stromal precursor cells colony formation in mouse splenic cultures and dose-dependently decreased this process in bone marrow cultures derived from these animals, the maximum (5-fold) inhibitory effect was observed in a dose of 50 microg/ml. Addition of Profetal to cultured human bone marrow fibroblasts did not change the content of stromal fibroblasts in cultures. These data indicate the possibility of indirect effect of alpha-fetoprotein on the number of stromal precursor cell in hemopoietic and lymphoid organs.

Posttransplant hematopoiesis in patients undergoing sibling allogeneic stem cell transplantation reflects that of their respective donors although with a lower functional capability

We tested the principle of whether patient long-term hematopoiesis following allogeneic stem cell transplantation (allo-SCT) reflects the characteristics of the hematopoiesis of their respective donor. For this purpose, we analyzed bone marrow (BM) hematopoiesis using long-term cultures (LTC), delta assays, and clonogeneic assays as well as CD34+ cells and their subsets by flow cytometry in a series of 37 patients undergoing allo-SCT, and we compared it to that of their respective human leukocyte antigen-matched sibling donors in a paired study performed more than 1 year after the transplant procedure. Interestingly, the main factor that influenced post-allo-SCT BM hematopoiesis in the long term was donor hematopoiesis. Nevertheless, compared to their respective donors, patients exhibited a significantly lower number of colony-forming units granulomonocytic, burst-forming units erythroid, and immature progenitors (CD34++/CD38dim/CD90+/CD133+ cells, LTC-initiating cells, and colonies generated in the delta assay). Moreover, BM stromal function was diminished in patients undergoing allo-SCT compared to their donors. In addition, the presence of chronic graft-versus-host disease under immunosuppressive treatment also conditioned an impaired hematopoietic function. In summary, our study shows that BM hematopoiesis evaluated more than 1 year after an allo-SCT mainly reproduces that of their respective donors, although with a significantly decreased in vitro activity.

Platelet-Derived Growth Factor Requirement for Cultured Descendants of Rabbit Stromal Bone Marrow Precursors

Addition of irradiated bone marrow feeder from guinea pigs and rabbits to cultures of passed rabbit bone marrow fibroblasts considerably decreases the efficiency of colony-formation and the total amount of fibroblasts in cultures. The presence of irradiated rabbit blood platelets sharply increases these parameters. The addition of irradiated bone marrow feeder to rabbit bone marrow fibroblasts cultured under conditions promoting cell differentiation also decreases the total content of fibroblasts in cultures. Our results suggest that cultured descendants of rabbit bone marrow stromal precursors preserve both the sensitivity to inhibitory factors produced by bone marrow cells coexisting with stromal precursors in cultures (typical of stromal precursors of these animals) and high sensitivity to growth-stimulating platelet-derived factors (typical of stromal precursors from rabbits and other animal species).

Interleukin-6 deficiency affects bone marrow stromal precursors, resulting in defective hematopoietic support

AbstractInterleukin-6 (IL-6) is a critical factor in the regulation of stromal function and hematopoiesis. In vivo bromodeoxyuridine incorporation analysis indicates that the percentage of Lin-Sca-1+ hematopoietic progenitors undergoing DNA synthesis is diminished in IL-6-deficient (IL-6-/-) bone marrow (BM) compared with wild-type BM. Reduced proliferation of IL-6-/- BM progenitors is also observed in IL-6-/- long-term BM cultures, which show defective hematopoietic support as measured by production of total cells, granulocyte macrophage-colony-forming units (CFU-GMs), and erythroid burst-forming units (BFU-Es). Seeding experiments of wild-type and IL-6-/- BM cells on irradiated wild-type or IL-6-deficient stroma indicate that the hematopoietic defect can be attributed to the stromal and not to the hematopoietic component. In IL-6-/- BM, stromal mesenchymal precursors, fibroblast CFUs (CFU-Fs), and stroma-initiating cells (SICs) are reduced to almost 50% of the wild-type BM value. Moreover, IL-6-/- stromata show increased CD34 and CD49e expression and reduced expression of the membrane antigens vascular cell adhesion molecule-1 (VCAM-1), Sca-1, CD49f, and Thy1. These data strongly suggest that IL-6 is an in vivo growth factor for mesenchymal precursors, which are in part implicated in the reduced longevity of the long-term repopulating stem cell compartment of IL-6-/- mice. (Blood. 2004;103:3349-3354)

Parathyroid hormone activates adhesion in bone marrow stromal precursor cells.

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. However, the underlying mechanisms are unknown. Osteoblasts, the bone-forming cells, derive from multipotential bone marrow stromal precursors called colony-forming units-fibroblastic (CFU-F) upon culture ex vivo. Adhesion of such stromal precursors to bone is likely to be an early event in the anabolic response of bone to PTH. To test this, we measured the number of CFU-F that could be extracted from murine bone marrow after administration of an anabolic dose of PTH. We found that a very early response is a dramatic reduction, starting within 2 h, in the number of CFU-F that could be extracted from their bone marrow. We then tested whether PTH has the ability to activate adhesion of CFU-F in vitro. For this, bone marrow cells were incubated in PTH for varying times. Non-adherent cells were then removed, and the adherent cells were incubated in PTH-free medium for 14 days to assess, as colony formation, the number of CFU-F that had adhered in the preceding period. We found that incubation in PTH caused a substantial increase in the number of CFU-F that adhered within 24 h. This increase was abrogated by peptidic inhibitors of integrins. The increase did not seem to be mediated through a PTH-induced increase in interleukin-6, since interleukin-6 had no effect on CFU-F numbers when substituted for PTH. Similarly, adhesion was unaffected by incubation of bone marrow cells in dibutyryl cyclic AMP, nor by inhibitors or donors of nitric oxide. However, activation of CFU-F in vitro by PTH was strongly inhibited by indomethacin and mimicked by prostaglandin E(2), and indomethacin reversed the PTH-mediated reduction of CFU-F that could be extracted from mouse bone marrow. These results suggested that PTH rapidly activates adhesion of CFU-F to plastic or bone surfaces. This activation may represent an early event in the anabolic response of bone cells to PTH.