Biology of bone marrow-derived endothelial cell precursors

Over the past decade, the old idea that the bone marrow contains endothelial cell precursors has become an area of renewed interest. While some still believe that there are no endothelial precursors in the blood, even among those who do, there is no consensus as to what they are or what they do. In this review, we describe the problems in identifying endothelial cells and conclude that expression of endothelial nitric oxide synthase may be the most reliable antigenic indicator of the phenotype. The evidence for two different classes of endothelial precursors is also presented. We suggest that, though there is no single endothelial cell precursor, we may be able to use these phenotypic variations to our advantage in better understanding their biology. We also discuss how a variety of genetic, epigenetic, and methodological differences can account for the seemingly contradictory findings on the physiological relevance of bone marrow-derived precursors in normal vascular maintenance and in response to injury. Data on the impact of tumor type and location on the contribution of bone marrow-derived cells to the tumor vasculature are also presented. These data provide hope that we may ultimately be able to predict those tumors in which bone marrow-derived cells will have a significant contribution and design therapies accordingly. Finally, factors that regulate bone marrow cell recruitment to and function in the endothelium are beginning to be identified, and several of these, including stromal derived factor 1, monocyte chemoattractant factor-1, and vascular endothelial growth factor are discussed.     

Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.     

Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8alphaalpha+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.     

Bone marrow-derived cells as progenitors of lung alveolar epithelium.

We assessed the capacity of plastic-adherent cultured bone marrow cells to serve as precursors of differentiated parenchymal cells of the lung. By intravenously delivering lacZ-labeled cells into wild-type recipient mice after bleomycin-induced lung injury, we detected marrow-derived cells engrafted in recipient lung parenchyma as cells with the morphological and molecular phenotype of type I pneumocytes of the alveolar epithelium. At no time after marrow cell injection, did we detect any engraftment as type II pneumocytes. In addition, we found that cultured and fresh aspirates of bone marrow cells can express the type I pneumocyte markers, T1alpha and aquaporin-5. These observations challenge the current belief that adult alveolar type I epithelial cells invariably arise from local precursor cells and raise the possibility of using injected marrow-derived cells for therapy of lung diseases characterized by extensive alveolar damage.     

Inhibition of human bone marrow lymphoid progenitor colonies by antibodies to VLA integrins.

Studies in animal models suggest that the integrin adhesion protein VLA-4 may play an important role in lymphopoiesis. The relationship between cell adhesion and lymphopoiesis in humans has been difficult to study because of the relative rarity and stringent in vitro growth requirements of lymphoid progenitors from normal adult human bone marrow. To determine the functional significance of VLA-4-mediated adhesion in human lymphopoiesis, we developed a culture system in which a bone marrow-derived adherent layer supports the formation of colonies of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursor cells from normal adult human bone marrow. Limiting dilution studies were consistent with clonal origin of these colonies. CFU-TdT were enriched in the CD34+ bone marrow fraction, consistent with CD34 expression by other hematopoietic progenitors. CD34 expression and lack of lineage-specific markers in a significant proportion of the TdT+ colony cells suggest that the TdT+ CFU may represent an uncommitted lymphoid progenitor cell. Development of TdT+ colonies required direct contact with the adherent layer and was significantly inhibited by specific anti-VLA-4 alpha chain antibody, suggesting a functional role for the previously reported VLA-4-dependent adhesion of human B cell precursors to bone marrow-derived fibroblasts.     

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.     

Activation of vascular endothelial growth factor receptor-2 in bone marrow leads to accumulation of myeloid cells: role of granulocyte-macrophage colony-stimulating factor

Vascular endothelial growth factor (VEGF) is a secreted cytokine that plays a major role in the formation and maintenance of the hemopoietic and vascular compartments. VEGF and its receptors, VEGFR-1 and VEGFR-2, have been found to be expressed on subsets of normal and malignant hemopoietic cells, but the role of the individual receptors in hemopoiesis requires further study. Using a VEGFR-2 fusion protein that can be dimerized with a synthetic drug, we were able to specifically examine the effects of VEGFR-2 signaling in hemopoietic cells in vivo. Mice transplanted with bone marrow transduced with this inducible VEGFR-2 fusion protein demonstrated expansion of myeloid cells (Gr-1+, CD11b+). Levels of myeloid progenitors were also increased following VEGFR-2 activation, through autocrine and paracrine mechanisms, as measured by clonogenic progenitor assays. VEGFR-2 activation induced expression of GM-CSF and increased serum levels in vivo. Abrogation of GM-CSF activity, either with neutralizing Abs or by using GM-CSF-null hemopoietic cells, inhibited VEGFR-2-mediated myeloid progenitor activity. Our findings indicate that VEGF signaling through VEGFR-2 promotes myelopoiesis through GM-CSF-dependent and -independent mechanisms.     

Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.     

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.     

Endothelial progenitor cells in health and disease

There is currently great excitement and expectation in the stem cell community following the discovery that multipotent stem cells can be cultured from human fetal tissue and retain their ability to give rise to a variety of differentiated cell types found in all three embryonic germ layers. Although the earliest sites of hematopoietic cell and endothelial cell differentiation in the yolk sac blood islands were identified about 100 years ago, cells with hemangioblast properties have not yet been identified in vivo. Endothelial cells differentiate from angioblasts in the embryo and from endothelial progenitor cells, mesoangioblasts and multipotent adult progenitor cells in the adult bone marrow. Circulating endothelial progenitor cells (EPC) have been detected in the circulation after vascular injury and during tumor growth. The molecular and cellular mechanisms underlying EPC recruitment and differentiation are not yet understood, and remain as one of the central issues in stem cell biology. For many years, the prevailing dogma stated that the vessels in the embryo develop from endothelial progenitors, whereas sprouting of vessels in the adult results only from division of differentiated endothelial cells. Recent evidence, however, indicates that EPC contribute to vessel growth in the embryo and in ischemic, malignant or inflammed tissues in the adult, and can even be therapeutically used to stimulate vessel growth in ischemic tissues.     

Bone marrow-derived hemopoietic precursors commit to the T cell lineage only after arrival in the thymic microenvironment

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study, we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed, soluble Notch ligands that allows the titration of T cell lineage commitment, we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast, their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically, this is not due to differences in receptor expression, because early T lineage precursors, bone marrow lineage marker-negative, Sca-1-positive, c-Kit-positive and common lymphoid progenitor cells, express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment.     

Myelomonocytic cells are sufficient for therapeutic cell fusion in liver

Liver repopulation with bone marrow-derived hepatocytes (BMHs) can cure the genetic liver disease fumarylacetoacetate hydrolase (Fah) deficiency. BMHs emerge from fusion between donor bone marrow-derived cells and host hepatocytes. To use such in vivo cell fusion efficiently for therapy requires knowing the nature of the hematopoietic cells that fuse with hepatocytes. Here we show that the transplantation into Fah(-/-) mice of hematopoietic stem cells (HSCs) from lymphocyte-deficient Rag1(-/-) mice, lineage-committed granulocyte-macrophage progenitors (GMPs) or bone marrow-derived macrophages (BMMs) results in the robust production of BMHs. These results provide direct evidence that committed myelomonocytic cells such as macrophages can produce functional epithelial cells by in vivo fusion. Because stable bone marrow engraftment or HSCs are not required for this process, macrophages or their highly proliferative progenitors provide potential for targeted and well-tolerated cell therapy aimed at organ regeneration.     

A common pathway for dendritic cell and early B cell development

B cells and dendritic cells (DCs) each develop from poorly described progenitor cells in the bone marrow (BM). Although a subset of DCs has been proposed to arise from lymphoid progenitors, a common developmental pathway for B cells and BM-derived DCs has not been clearly identified. To address this possibility, we performed a comprehensive analysis of DC differentiative potential among lymphoid and B lymphoid progenitor populations in adult mouse BM. We found that both the common lymphoid progenitors (CLPs), shown here and elsewhere to give rise exclusively to lymphocytes, and a down-stream early B-lineage precursor population devoid of T and NK cell precursor potential each give rise to DCs when exposed to the appropriate cytokines. This result contrasts with more mature B-lineage precursors, all of which failed to give rise to detectable numbers of DCs. Significantly, both CLP and early B-lineage-derived DCs acquired several surface markers associated with functional DCs, and CLP-derived DCs readily induced proliferation of allogeneic CD4(+) T cells. Surprisingly, however, DC differentiation from both lymphoid-restricted progenitors was accompanied by up-regulation of CD11b expression, a cell surface molecule normally restricted to myeloid lineage cells including putative myeloid DCs. Together, these data demonstrate that loss of DC developmental potential is the final step in B-lineage commitment and thus reveals a previously unrecognized link between early B cell and DC ontogeny.     

Vascular Integration of Endothelial Progenitors During Multistep Tumor Progression

Bone marrow-derived endothelial precursor cells contribute to tumor neovascularization. However, it is unclear when during progressive tumor growth circulating precursors are recruited into the preexisting vascular network, and how they home specifically into the tumor microenvironment. Here, we summarize recent findings from mouse models of multistage carcinogenesis, which reveal distinct phases of angiogenic activity. Only advanced tumors with a highly heterogeneous, sprouting vasculature recruite endothelial progenitors into neovessels. Surprisingly, during progressive tumor growth endothelial cells acquire new characteristics and secrete CC chemokines, a group of chemoattractants with adjacent cysteins, which play a dual role by enhancing neovascularization in an autocrine and endocrine fashion. Locally, chemokines stimulate endothelial proliferation; systemically, they guide chemokine receptor-positive circulating progenitors into the tumor bed. Subsequently, endothelial progenitors are truly integrated into the network of pre-existing vessel. This mechanism represents a novel concept where not the tumor itself, but endothelial cells as components of the tumor-induced stroma foster neovascularization in a self-amplifying loop.     

Selective thymus settling regulated by cytokine and chemokine receptors

To generate T cells throughout adult life, the thymus must import hemopoietic progenitors from the bone marrow via the blood. In this study, we establish that thymus settling is selective. Using nonirradiated recipient mice, we found that hemopoietic stem cells were excluded from the thymus, whereas downstream multipotent progenitors (MPP) and common lymphoid progenitors rapidly generated T cells following i.v. transfer. This cellular specificity correlated with the expression of the chemokine receptor CCR9 by a subset of MPP and common lymphoid progenitors but not hemopoietic stem cells. Furthermore, CCR9 expression was required for efficient thymus settling. Finally, we demonstrate that a prethymic signal through the cytokine receptor fms-like tyrosine kinase receptor-3 was required for the generation of CCR9-expressing early lymphoid progenitors, which were the most efficient progenitors of T cells within the MPP population. We conclude that fms-like tyrosine kinase receptor-3 signaling is required for the generation of T lineage-competent progenitors, which selectively express molecules, including CCR9, that allow them to settle within the thymus.     

Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression

Accumulating studies support the idea that a common progenitor, termed the hemangioblast, generates both hematopoietic and endothelial cell lineages. To better define the relationship between these cell lineages, we have generated knock-in embryonic stem (ES) cells carrying a non-functional human CD4 at the Scl locus. By using in vitro differentiated Scl(+/CD4) ES cells, we demonstrate that FLK1 and SCL are molecular determinants of the hemangioblast. Furthermore, our studies demonstrate that hematopoietic and endothelial cells develop via distinct, sequential generation of FLK1 and SCL-expressing cells. FLK1(+)CD4(-) cells first arise in developing embryoid bodies. The Scl gene is turned on within FLK1(+)CD4(-) cells to give rise to FLK1(+)CD4(+) cells. Alternatively, a subpopulation of the initial FLK1(+)CD4(-) cells remains as SCL negative. Within the FLK1(+)CD4(+) cells, FLK1 is down regulated to generate FILK1(-)CD4(+) cells. Replating studies demonstrate that hematopoietic progenitors are enriched within FLK1(+)CD4(+) and FLK1(-)CD4(+) cells, while endothelial cells develop from FLK1(+)CD4(+) and FLK1(+)CD4(-) cell populations.     

VEGF(165), but not VEGF(189), stimulates vasculogenesis and bone marrow cell migration into Ewing's sarcoma tumors in vivo

We previously showed that bone marrow stem cells participate in the tumor vessel expansion that supports the growth of Ewing's sarcoma tumors in vivo. The purpose of this study was to determine the relative importance of two isoforms of vascular endothelial growth factor (VEGF) in tumor vessel expansion and recruitment of bone marrow-derived cells during tumor growth. We injected VEGF(165)-siRNA-transfected cells (TCsi/7-1), control siRNA-transfected cells (TC/si-control), or TC71 parental Ewing's sarcoma cells into nude mice. The TCsi/7-1 tumors were then treated with adenoviral vectors expressing VEGF(165) (Ad-VEGF(165)), VEGF(189) (Ad-VEGF(189)), or beta-galactosidase (Ad-beta-gal). Bone marrow cells labeled with fluorescent tracker dye were injected into the mice 3 weeks later. The TCsi/7-1 tumors were significantly smaller (P < 0.05), had decreased vessel density, and showed significantly lower bone marrow cell migration than did TC71 parental and TC/si-control tumors. Treatment with Ad-VEGF(165), but not Ad-VEGF(189) or Ad-beta-gal, resulted in a significant increase in bone marrow cell infiltration, tumor vessel density, and tumor growth. Immunohistochemical staining revealed that the injected bone marrow cells migrated to and incorporated into the expanding CD31(+) tumor vessel network. Taken together, these data show that VEGF(165) is a chemoattractant that recruits bone marrow cells into the tumor area. These data provide a mechanism by which Ewing's sarcoma cells induce vasculogenesis.     

The origin of mesoderm in phoronids

Integrin alphaIIb is a cell adhesion molecule expressed in association with beta3 by cells of the megakaryocytic lineage, from committed progenitors to platelets. While it is clear that lymphohemopoietic cells differentiating along other lineages do not express this molecule, it has been questioned whether mammalian hemopoietic stem cells (HSC) and various progenitor cells express it. In this study, we detected alphaIIb expression in midgestation embryo in sites of HSC generation, such as the yolk sac blood islands and the hemopoietic clusters lining the walls of the major arteries, and in sites of HSC migration, such as the fetal liver. Since c-Kit, which plays an essential role in the early stages of hemopoiesis, is expressed by HSC, we studied the expression of the alphaIIb antigen in the c-Kit-positive population from fetal liver and adult bone marrow differentiating in vitro and in vivo into erythromyeloid and lymphocyte lineages. Erythroid and myeloid progenitor activities were found in vitro in the c-Kit(+)alphaIIb(+) cell populations from both origins. On the other hand, a T cell developmental potential has never been considered for c-Kit(+)alphaIIb(+) progenitors, except in the avian model. Using organ cultures of embryonic thymus followed by grafting into athymic nude recipients, we demonstrate herein that populations from murine fetal liver and adult bone marrow contain T lymphocyte progenitors. Migration and maturation of T cells occurred, as shown by the development of both CD4(+)CD8- and CD4-CD8(+) peripheral T cells. Multilineage differentiation, including the B lymphoid lineage, of c-Kit(+)alphaIIb(+) progenitor cells was also shown in vivo in an assay using lethally irradiated congenic recipients. Taken together, these data demonstrate that murine c-Kit(+)alphaIIb(+) progenitor cells have several lineage potentialities since erythroid, myeloid, and lymphoid lineages can be generated.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.