Carlijn Voermans

Identification of Adiponectin as a Novel Hemopoietic Stem Cell Growth Factor

The hemopoietic microenvironment consists of a diverse repertoire of cells capable of providing signals that influence hemopoietic stem cell function. Although the role of osteoblasts and vascular endothelial cells has recently been characterized, the function of the most abundant cell type in the bone marrow, the adipocyte, is less defined. Given the emergence of a growing number of adipokines, it is possible that these factors may also play a role in regulating hematopoiesis. Here, we investigated the role of adiponectin, a secreted molecule derived from adipocytes, in hemopoietic stem cell (HSC) function. We show that adiponectin is expressed by components of the HSC niche and its’ receptors AdipoR1 and AdipoR2 are expressed by HSCs. At a functional level, adiponectin influences HSCs by increasing their proliferation, while retaining the cells in a functionally immature state as determined by in vitro and in vivo assays. We also demonstrate that adiponectin signaling is required for optimal HSC proliferation both in vitro and in long term hemopoietic reconstitution in vivo. Finally we show that adiponectin stimulation activates p38 MAPK, and that inhibition of this pathway abrogates adiponectin’s proliferative effect on HSCs. These studies collectively identify adiponectin as a novel regulator of HSC function and suggest that it acts through a p38 dependent pathway.

Wnt signaling in the stem cell niche

Purpose of reviewAll the cells present in the blood are derived from the hematopoietic stem cell (HSC). Because mature blood cells have a limited life span, HSCs must perpetuate themselves through self-renewal to maintain a functional hematopoietic compartment for the lifetime of an organism. This review focuses on studies that identify the Wnt signaling pathway as a mediator of HSC self-renewal and maintenance and analyzes its potential influence in context of the HSC niche. Recent findingsThe Wnt signaling pathway has emerged as a potential regulator of self-renewal for HSCs. Recent reports have demonstrated that Wnt signaling can directly promote HSC self-renewal and ability to reconstitute the hematopoietic system of lethally irradiated mice. The recent findings that osteoblasts are an important regulatory component of the HSC microenvironment, and that elements of the Wnt signaling pathway can influence osteoblast frequency, raise the possibility that Wnt signaling may influence HSC function indirectly through the niche as well. SummaryIn this review, the authors evaluate the experimental evidence for a direct role of Wnt signaling HSCs as well as an indirect role through its influence on the HSC niche. Defining the mechanism of action of Wnt signaling in HSC maintenance in context of the surrounding microenvironment and determining how this signal may integrate with other niche derived signals represents the next challenge in HSC biology.

The C-terminal Domain of Rac1 Contains Two Motifs That Control Targeting and Signaling Specificity

Rho-like GTPases control a wide range of cellular functions such as integrin- and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the C-terminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.

Increased migration of cord blood-derived CD34+ cells, as compared to bone marrow and mobilized peripheral blood CD34+ cells across uncoated or fibronectin-coated filters

Hematopoietic progenitor cells (CD34+ cells) migrate to the bone marrow after reinfusion into peripheral veins. Stromal cell-derived factor-1 (SDF-1) is a chemokine produced by bone marrow stromal cells that induces migration of CD34+ cells. In this study we compared spontaneous and SDF-1-induced migration of CD34+ cells from bone marrow (BM), peripheral blood (PB), and cord blood (CB) across Transwell filters. Under all circumstances, CB CD34+ cells showed significantly more migration than did BM or PB CD34+ cells. SDF-1 induced migration of BM CD34+ cells was higher than that of PB CD34+ cells, possibly due to differences in sensitivity towards SDF-1. Indeed, PB CD34+ cells showed a significantly lower expression of the receptor for SDF-1 (CXCR-4) than did BM and CB CD34+ cells. The sensitivity to SDF-1, as measured by migration towards different concentrations of SDF-1, was identical for BM and CB-derived CD34+ cells and correlated with their equal CXCR-4 receptor expression. Coating of the filters with the extracellular matrix protein fibronectin (FN) strongly enhanced the SDF-1-induced migration of PB CD34+ cells (2.5 times) and of BM CD34+ cells (1.5 times). SDF-1 induced migration of PB CD34+ cells over FN-coated filters was blocked by antibodies against beta1 integrins. Subsequently, analysis was performed to determine whether SDF-1 preferentially promoted migration of subsets of CD34+ cells. Actively cycling CD34+ cells, which were present in BM (14%) but hardly in PB (2.2%) or CB (1.2%), were found to migrate preferentially towards SDF-1. In the input, 14%+/-2.5% of the BM CD34+ cells were in G2/M and S phase, whereas in the migrated fraction 20%+/-5.7% of the cells were actively cycling (p < 0.05). We did not observe preferential migration of phenotypically recognizable primitive CD34+ subsets, despite the fact that CB CD34+ cells are thought to contain a higher percentage of immature subsets. In conclusion, the relatively lower migration of PB CD34+ cells seems to be due to a lower sensitivity towards SDF-1, and the higher migrational capacity of CB CD34+ cells, in comparison to BM and PB CD34+ cells, seems to have an as yet unknown intrinsic cause. The increased migration of CB CD34+ cells may favor homing of these cells to the bone marrow, which might reduce the number of cells required for hematological reconstitution after transplantation.

Adhesion Molecules Involved in Transendothelial Migration of Human Hematopoietic Progenitor Cells

In the process of homing, CD34+ hematopoietic progenitor cells migrate across the bone marrow endothelium in response to stromal cell‐derived factor (SDF)‐1. To develop more efficient stem cell transplantation procedures, it is important to define the adhesion molecules involved in the homing process. Here, we identified the adhesion molecules that control the migration of primary human CD34+ cells across human bone marrow endothelial cells.

Migration of Human Hematopoietic Progenitor Cells Across Bone Marrow Endothelium Is Regulated by Vascular Endothelial Cadherin

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34+ cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34+ cells in response to stromal cell-derived factor-1α. Stromal cell-derived factor-1α-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34+ cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.

Migratory behavior of leukemic cells from acute myeloid leukemia patients.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 contribute to stem cell homing and may play a role in the trafficking of leukemic cells. Therefore, we analyzed migration across Transwell filters of cells derived from bone marrow (BM) or peripheral blood (PB) from 26 acute myeloid leukemia (AML) patients. The presence of the extracellular matrix protein fibronectin (FN) strongly enhanced the spontaneous and SDF-1-induced migration of leukemic PB and BM cells. No differences in spontaneous, SDF-1-induced migration or CXCR-4 expression were observed between the different AML subtypes. Subsequently, it was determined whether SDF-1 preferentially promoted migration of subsets of leukemic cells. Leukemic cells expressing CD34, CD38 and HLA-DR were preferentially migrating, whereas cells expressing CD14 and CD36 showed diminished migration. Analysis of paired PB and BM samples indicated that significantly higher SDF-1-induced migration was observed in AML for CD34+ BM-derived cells compared to CD34+ PB-derived cells, suggesting a role for SDF-1 in the anchoring of leukemic cells in the BM or other organs. The lower percentage of circulating leukemic blasts in patients with a relatively high level of SDF-1-induced migration also supports this hypothesis. In conclusion, we have shown that primary AML cells are migrating towards SDF-1 independent of the AML subtypes.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271brightCD146− and CD271brightCD146+ subsets correlates with donor age. The main subset in adults was CD271brightCD146−, whereas the CD271brightCD146+ population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271−CD146+ cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions.

SDF-1–induced actin polymerization and migration in human hematopoietic progenitor cells

OBJECTIVE The capacity of hematopoietic progenitor cells (HPCs; CD34(+) cells) to respond to chemotactic stimulation is essential for their homing efficiency, e.g., during stem cell transplantation. Previous studies established that stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 play an important role in the homing of HPCs. The aim of the present study was to analyze SDF-1-induced actin polymerization and migration of HL-60 cells and primary human CD34(+) cells. MATERIALS AND METHODS SDF-1-induced migration of CD34(+) cells from cord blood (CB) and peripheral blood (PB) across fibronectin-coated filters was measured in a Transwell assay. Actin polymerization was detected using fluorescent phalloidin and analyzed by confocal microscopy and FACS analysis. RESULTS SDF-1 induced a rapid and transient increase in actin polymerization and in polarization of the actin cytoskeleton in primary CD34(+) cells and HL-60 cells. SDF-1 was found to induce significantly more actin polymerization in CB CD34(+) cells that show fast migration in vitro compared to slow migrating PB CD34(+) cells. Moreover, CB CD34(+) cells that had migrated toward SDF-1 showed an elevated and prolonged rise in F-actin upon second exposure to SDF-1 compared to nonmigrated cells, although both cell types expressed equal levels of the SDF-1 receptor CXCR-4. CONCLUSIONS The relatively high migratory capacity of CB-derived human HPCs is not related to cellular polarization or high expression of the SDF-1 receptor but is largely determined by their capacity to efficiently polymerize F-actin in response to SDF-1.

Impact of interferon-γ on hematopoiesis

The proinflammatory cytokine interferon-γ (IFN-γ) is well known for its important role in innate and adaptive immunity against intracellular infections and for tumor control. Yet, it has become clear that IFN-γ also has a strong impact on bone marrow (BM) output during inflammation, as it affects the differentiation of most hematopoietic progenitor cells. Here, we review the impact of IFN-γ on hematopoiesis, including the function of hematopoietic stem cells (HSCs) and more downstream progenitors. We discuss which hematopoietic lineages are functionally modulated by IFN-γ and through which underlying molecular mechanism(s). We propose the novel concept that IFN-γ acts through upregulation of suppressor of cytokine signaling molecules, which impairs signaling of several cytokine receptors. IFN-γ has also gained clinical interest from different angles, and we discuss how chronic IFN-γ production can lead to the development of anemia and BM failure and how it is involved in malignant hematopoiesis. Overall, this review illustrates the wide-ranging effect of IFN-γ on the (patho-)physiological processes in the BM.