Characterization of thymic progenitors in adult mouse bone marrow

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1(-)) (Thy1.1(-) progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1(-) progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1(-) population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.     

Dietary isoflavones act on bone marrow osteoprogenitor cells and stimulate ovary development before influencing bone mass in pre-pubertal piglets

Food containing soybeans provide isoflavone phytoestrogens that can preserve bone mass in postmenopausal women, and prevent bone loss in ovariectomized rats. But their effects on bone remain unclear, particularly on bone formation during growth. Two groups of eight pre-pubertal piglets were fed a basal or an isoflavone-enriched (S800) diet for 6 weeks. The S800 diet contained 800 mg SoyLifetrade mark/kg, providing 2.8 mg isoflavones/kg body weight/day. Several bones were collected and tested for bone strength and density. Bone marrow was collected from humeri together with blood samples and genital tracts. The plasma concentrations of isoflavones were increased in the pigs fed S800, but growth rate, body weight, plasma bone markers, bone mineral density, and strength were all unaffected. In contrast, cultured stromal cells from S800 pigs had more alkaline phosphatase-rich cells and mineralized nodules, secreted more osteocalcin, osteoprotegerin and RANK-L, synthesized more osteoprotegerin, and RANK-L. Cultured mononucleated nonadherent bone marrow cells from S800 pigs developed fewer tartrate-resistant acid phosphatase mononucleated cells (osteoclast progenitors) when cultured with 1,25(OH)(2)D(3), and resorbed a smaller area of dentine slices. Freshly isolated bone marrow osteoclast progenitors from S800 pigs had more caspase-3 cleavage activity, and synthesized less RANK. Both osteoclast and osteoblast progenitors had ERalpha and ERbeta, whose syntheses were stimulated by the S800 diet. The S800 piglets had heavier ovaries with more follicles, but their uterus weight was unaffected. We conclude that dietary isoflavones have no detectable effect on the bone mass of growing female piglets, but act on bone marrow osteoprogenitors via ERs--mainly ERbeta, and stimulate ovary development.     

The mast cell-committed progenitor. In vitro generation of committed progenitors from bone marrow

Mast cell-committed progenitors are detected in the unique microenvironment of the mesenteric lymph node (MLN) of Nippostrongylus brasiliensis-infected mice but not in naive bone marrow. We have determined that MLN cells, after infection, produce high levels of IL-3, IL-4, and IgE, presumably in the form of immune complexes with antigens produced by the infecting helminth. After N. brasiliensis infection, peak production of these factors occurs several days before the peak appearance of mast cell-committed progenitors in the MLN. To determine if these factors play a role in mast cell commitment, we recreated these conditions, in vitro. Naive bone marrow cells were cultured with combinations of IL-3, IL-4, and IgE immune complexes, or on IgE-coated plates, and then assayed for acquisition of the ability to form mast cell colonies when supplemented with fibroblast-conditioned medium alone. IL-3 and IgE immune complexes, and, unexpectedly, IgE immune complexes alone were found to be capable of producing mast cell-committed progenitors, i.e., cells responsive to fibroblast-conditioned medium alone, from bone marrow, whereas IL-4 did not enhance production of mast cell-committed progenitors from bone marrow. Production of IFN-gamma peaked at the same time point as committed progenitor activity and may be responsible for down regulating the response.     

Population balance model of in vivo neutrophil formation following bone marrow rescue therapy

In this paper, we develop a simple four parameter population balance model of in vivo neutrophil formation following bone marrow rescue therapy. The model is used to predict the number and type of neutrophil progenitors required to abrogate the period of severe neutropenia that normally follows a bone marrow transplant. The estimated total number of 5 billion neutrophil progenitors is consistent with the value extrapolated from a human trial. The model provides a basis for designing ex vivo expansion protocols. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro structural and functional relationships between preosteoclastic and bone endothelial cells: A juxtacrine model for migration and adhesion of osteoclast precursors

The role of vascularization in the process of bone resorption has not been clarified. The interactions between vascular endothelium and osteoclast progenitors were analyzed using clonal cell lines of bone-derived endothelial and preosteoclastic cells. Insulin-like growth factor I is a major chemotactic stimulator of preosteoclastic cell migration mediated by bone endothelial cells. Osteoclast precursors rapidly adhered to bone endothelial monolayers. This phenomenon appeared to be cell-specific and mediated through the binding of vitronectin and fibronection receptors to fibronectin. In addition, direct contact with bone endothelial cells induced osteoclast progenitors to differentiate into more mature elements, with the tendency to cluster together to form large multinucleated cells. These findings demonstrated specific in vitro interactions between bone endothelial cells and osteoclast progenitors, offering a new model for understanding the molecular mechanisms which direct the processes of osteoclast recruitment and ontogeny. © 1995 Wiley-Liss, Inc.     

Rescue of myeloid lineage-committed preprogenitor cells from cytomegalovirus-infected bone marrow stroma.

The effect of murine cytomegalovirus on myelopoiesis was studied in long-term bone marrow culture to find an in vitro correlate for the lethal virus interference with bone marrow reconstitution (W. Mutter, M. J. Reddehase, F. W. Busch, H.-J. Bühring, and U. H. Koszinowski, J. Exp. Med. 167:1645-1658, 1988). The in vitro generation of granulocyte-monocyte progenitors (CFU-GM) discontinued after infection of the stromal cell layer, whereas the proliferation and differentiation of CFU-GM to granulocyte-monocyte colonies remained unaffected. A protocol was established to probe the functional integrity of earlier hematopoietic cells. Pre-CFU-GM (the progenitors of the CFU-GM) could be recovered from an infected bone marrow donor culture by transfer onto an inductive recipient stromal cell layer. Thus, at least in vitro, infection of bone marrow stroma appears to be the only cause of the defect in myelopoiesis.     

Circadian variations of bone marrow engraftability

Circadian rhythms exist for hematopoiesis, but little is known about circadian variation of bone marrow engraftability and host "acceptability". Using a B6.SJL to C57BL/6J congenic transplant model, we chose 3-times with light on: Hours After Light Onset (HALO) 4, 8, and 12 and 3-times with light off: HALO 16, 20, and 24. The mice were conditioned on a 12-h light/dark cycles. Recipient mice (100 cGy) received 40 million cells. We demonstrated a significant variation of bone marrow engraftability into bone marrow, spleen, and thymus when donor animals were subjected to changes in their light/dark cycles. Two statistically significant nadirs in all three organs were observed at HALO 8 and 24 in experiments carried out in July, while an identical set of experiments in February analyzing engraftment in marrow and spleen showed nadirs at HALO 8, but not at HALO 24. Marrow progenitors from the July experiments showed nadirs at HALO 12 and 24. The percentage of progenitors in S phase peaked at HALO 8 and 24. Interestingly, there were no changes in the ability of host to accept grafts with changes in the light/dark cycles of host animals. Circadian variations of bone marrow engraftability are important and should be considered in bone marrow transplant strategies.     

Role of vitamin D in bone resorption.

The idea that vitamin D must function at the bone site to promote bone mineralization has long existed since its discovery as an anti-rachitic agent. However, the definite evidence for this is still lacking. In contrast, much evidence has accumulated that 1 alpha,25(OH)2D3 in involved in bone resorption. 1 alpha,25(OH)2D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors have been thought to belong to the monocyte-macrophage lineage. 1 alpha,25(OH)2D3 greatly stimulates differentiation and activation of mononuclear phagocytes. Recent reports have indicated that differentiation of mononuclear phagocytes into osteoclasts is strictly regulated by osteoblastic cells, the process of which is also stimulated by 1 alpha,25(OH)2D3. In the differentiation of mononuclear phagocytes into osteoclasts, the target cells for 1 alpha,25(OH)2D3 appear to be osteoblastic stromal cells. Osteoblastic cells produce several proteins such as BGP, MGP, osteopontin and the third component of complement (C3) in response to the vitamin. They appear to be somehow involved in osteoclast differentiation and functions. Thus, 1 alpha,25(OH)2D3 seems to be involved in the differentiation of osteoclast progenitors into osteoclasts directly and also by an indirect mechanism involving osteoblastic cells. The precise role of osteoblastic cells in osteoclast development has to be elucidated in the future.     

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.     

Normal differentiation and functions of mouse dendritic cells derived from RAG-deficient bone marrow progenitors

Dendritic cells (DC) mature upon infectious agent detection to elicit immune responses. It has been suggested that T cells influence peripheral DC function. However, it is not known if lymphocytes influence DC progenitors. Therefore, we determined the ability of bone marrow progenitors from T and B cell-deficient mice to generate functional DC. We report that bone marrow-derived DC from RAG-2(-/-) mice differentiate and proliferate normally. Moreover, such generated DC efficiently internalize particles, mature in response to various Toll-like receptor engagement, and activate allogenic T cells. This work strongly supports that early signals delivered during DC ontogeny by mature lymphocytes do not influence the functional differentiation of DC progenitors.     

Positive and negative hematopoietic cytokines produced by bone marrow endothelial cells

Recently, cytokines and interleukins such as SCF, GM-CSF, G-CSF, TGF-beta, IL-6, IL-7, IL-8, IL-11 have been reported to be elaborated by endothelial cells. For further study, serum free bone marrow endothelial cell conditioned medium (BMEC-CM) was collected and ultrafiltrated by using a centriprep 10. The concentrated retentate (R-BMEC-CM) contained some substances whose molecular weight was more than 10 000 daltons. The filtrate (F-BMEC-CM) contained some substances whose molecular weight was less than 10 000 daltons. The effects of R-BMEC-CM and F-BMEC-CM on the growth of haematopoietic progenitors and the expression of cytokine and interleukin mRNAs of BMEC were investigated. The results showed that R-BMEC-CM stimulated the growth of CFU-GM, HPP-CFC, BFU-E, CFU-E, and CFU-Meg; while F-BMEC-CM inhibited the growth of these progenitors. Using the method of hybridizing to the Atlas cDNA Array, we were able to detect the presence of mRNAs of cytokines and interleukins in bone marrow endothelial cells. Our finding of the existence of mRNAs of SCF, GM-CSF, IL-6, TGF-beta, IL-1, and IL-11 in these cells was in agreement with the data reported previously. Furthermore, we detected mRNAs of MIP-2, Thymosion-beta4, PDGF, MSP-1, IFN-gamma, IL-13 and inhibin, which are related to haematopoiesis. Among these cytokines and interleukins, SCF, GM-CSF, IL-6, IL-1, and IL-11 are haematopoietic stimulators which may be responsible for the stimulative effects on the growth of haematopoietic progenitors. One of our new findings, the thymosin-beta4, is a small molecular haematopoietic inhibitor. It may be responsible for the inhibitory effect of F-BMEC-CM on haematopoietic progenitors. The presence of mRNAs of BMP, MSP-1, MIP-2, PDGF and IL-13 suggests that bone marrow endothelial cells might elaborate these substances. Their influence on haematopoietic progenitors needs further study.     

Growth factors in bone marrow transplant recipients

Normal human plasma samples usually support the growth of hemopoietic progenitors in the presence of exogenous growth factors. In contrast, plasma samples collected serially from bone marrow transplant recipients after administration of their preparative regimen demonstrate activities that routinely stimulate the growth of granulocyte-macrophage progenitors (CFU-GM) and precursors for megakaryocytes (CFU-Meg). The peak activities are usually observed between day 7 and day 21. Occasionally growth promoting activities for erythroid bursts are also observed. Activities for the growth of multilineage colonies are only rarely seen. These growth promoting activities for CFU-GM and CFU-Meg usually return to pretransplant values by day 30 in patients with prompt engraftment. Patients with delayed engraftment sustain high levels of growth promoting activities for long periods of time.     

Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow.

The target cell specificity of the B19 parvovirus infection was examined by isolating highly enriched hematopoietic progenitor and stem cells from normal human bone marrow. The efficiency of the B19 parvovirus replication in enriched erythroid progenitor cells was approximately 100-fold greater than that in unseparated bone marrow cells. The more-primitive progenitor cells identical to or closely related to the human pluripotent hematopoietic stem cells, on the other hand, did not support viral replication. The B19 progeny virus produced by the enriched erythroid progenitor cells was infectious and strongly suppressed erythropoiesis in vitro. The susceptibility of both the more-primitive erythroid progenitors (burst-forming units-erythroid) and the more-mature erythroid progenitors (CFU-erythroid) to the cytolytic response of the virus and the lack of effect on the myeloid progenitors (CFU-granulocyte-macrophage) further give evidence to the remarkable tropism of the B19 parvovirus for human hematopoietic cells of erythroid lineage.     

Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow.

Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthy carriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect with HCMV, it has been suggested that they may acquire HCMV during development in the bone marrow. Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers contain endogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infected by clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells become differentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strain AD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry of AD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMV and transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave the circulation and undergo tissue-specific differentiation to macrophages.     

Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.     

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.     

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.