Stem cell homing and repopulation are not well understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 were found to be critical for murine bone marrow engraftment by human severe combined immunodeficient (SCID) repopulating stem cells. Treatment of human cells with antibodies to CXCR4 prevented engraftment. In vitro CXCR4-dependent migration to SDF-1 of CD34+CD38-/low cells correlated with in vivo engraftment and stem cell function. Stem cell factor and interleukin-6 induced CXCR4 expression on CD34+ cells, which potentiated migration to SDF-1 and engraftment in primary and secondary transplanted mice. Thus, up-regulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.

IntroductionTransplanted human hematopoietic stem cells must retain specific adhesive capacity to interact with the vascular endothelium that lines the bone marrow (BM) (1-3). The mechanisms by which these cells home to and engraft the BM are still obscure. Murine hematopoietic progenitors (HPCs) have been shown to interact in vivo with both P-selectin and E-selectin on vascular endothelium of murine BM (4). In addition, VCAM-1 has been shown to support rolling of HPC on BM endothelium in the absence of endothelial selectins (4). Nevertheless, optimal recruitment of HPC to the BM requires the combined action of both selectins and VCAM-1 (5). Thus, the initiation of primary rolling adhesion of blood-borne cells to the lining of the BM microvessels under shear flow is critical for the recruitment of HPC to the BM. HPC rolling on BM endothelium is likely to be accompanied by a coordinated sequence of adhesive and activation events leading to cell arrest, a key step in the successful extravasation of blood-borne cells to extravascular beds (6, 7). In these prior studies, the mechanisms by which HPCs develop firm adhesion to BM endothelium in the presence of physiological shear flow have not been elucidated.We recently established a key role for the chemokine SDF-1 and its receptor CXCR4 in murine BM engraftment by human severe combined immunodeficiency (SCID) repopulating stem cells (8). In light of the growing evidence that chemokines can regulate the arrest of leukocytes on blood vessels through integrin-dependent interactions with IgSF ligands, we asked whether SDF-1 regulates CD34 + progenitors interaction with BM endothelium, and if so, how. We now report that human BM endothelium constitutively expresses high levels of SDF-1. Using in vitro flow chamber assays, we have tested the specific effects of SDF-1 on the ability of human cord blood CD34 + progenitors to initiate rolling interactions and arrest on human vascular endothelium and on isolated endothelial receptors, including E-selectin, P-selectin, ICAM-1, and VCAM-1, which have all been shown to be constitutively expressed on both murine and human BM endothelium (9). Our results suggest that SDF-1 is a key player The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34 + progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34 + cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34 + cells under flow, but, in the presence of surface-bound SDF-1, CD34 + progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction ...

Trafficking of human CD34 ؉ stem/progenitor cells (HSCs/HPCs) is regulated by chemokines, cytokines, proteolytic enzymes, and adhesion molecules. We report that the adhesion receptor CD44 and its major ligand, hyaluronic acid (HA), are essential for homing into the bone marrow (BM) and spleen of nonobese diabetic/ severe combined immunodeficient (NOD/ SCID) mice and engraftment by human HSCs. Homing was blocked by anti-CD44 monoclonal antibodies (mAbs) or by soluble HA, and it was significantly impaired after intravenous injection of hyaluronidase. Furthermore, stromal cellderived factor-1 (SDF-1) was found to be a rapid and potent stimulator of progenitor adhesion to immobilized HA, leading to formation of actin-containing protrusions with CD44 located at their tips. HPCs migrating on HA toward a gradient of SDF-1 acquired spread and polarized morphology with CD44 concentrating at the pseudopodia at the leading edge. These morphologic alterations were not observed when the progenitors were first exposed to anti-CD44 mAbs, demonstrating a crosstalk between CD44 and CXCR4 signaling. Unexpectedly, we found that HA is expressed on human BM sinusoidal endothelium and endosteum, the regions where SDF-1 is also abundant. Taken IntroductionThe outcome of hematopoietic stem cell transplantation is influenced by the ability of the cells to home and repopulate their specialized bone marrow (BM) niches. The crosstalk between the hematopoietic stem/progenitor cells (HSCs/HPCs) and the microenvironment, which regulates homing to the BM, is not fully elucidated. Data indicate that transplanted HSCs/HPCs lodge into their BM niches by a sequence of highly regulated events that mimic the migration of leukocytes to inflammatory sites. This process includes tethering and rolling on E-and P-selectins, firm adhesion to the vessel wall, transendothelial extravasation, and migration through the extracellular matrix (ECM). [1][2][3] This multistep process is mediated by an interplay between chemokines, growth factors, proteolytic enzymes, and adhesion molecules. 4,5 The chemokine stromal cell-derived factor-1 (SDF-1), also named CXCL-12, and its receptor, CXCR4, play key roles in human HSC trafficking and repopulation. 6 This chemokine, expressed by both human and murine BM endothelium and stroma, 7,8 is the most powerful chemoattractant of HSCs/HPCs 9,10 that also regulates their survival. 11,12 It induces the integrin-mediated firm arrest of human HPCs under physiologic shear flow, facilitates their transendothelial migration, 3,8 and regulates homing 13 and BM engraftment. 14 Furthermore, SDF-1 is also required for the retention of murine stem and progenitor cells within the BM. 15,16 HSCs/HPCs express several types of adhesion molecules that are responsible for cell-cell and cell-ECM interactions 17 ; among them CD44 is of particular interest.The importance of CD44 in cell migration is reported for a variety of normal and malignant cells. 18 CD44 is a multifunctional and multistructural receptor that has a large array of isoforms....

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAMneg and EpCAMdim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24+CD133+ cells comprise >50% of HFK cells and predominantly co-express EpCAMbright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM+EpCAM- and to a lesser extent in NCAM+EpCAM+ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM+EpCAM+FZD7+), MM stem cells (NCAM+EpCAM-FZD7+) or both (NCAM+FZD7+). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.

1. Post-natch mucosal development was examined in the chick small intestinal epithelium using immunostaining with proliferating cell nuclear antigen (PCNA) and 5-bromo-2-deoxyuridine (BrdU). 2. On the day of hatching jejunal crypts were small and a single crypt per villus was observed. However, during the 108 h post-hatch crypts developed rapidly branching and increasing in size, cell numbers and cell size. 3. Almost all epithelial cells in the small intestine of the hatching chick were proliferating, as indicated by PCNA and BrdU, while more than 80% of proliferating cells were localised in the crypts after 108 h post hatch. 4. Estimate of villus cell transit time using BrdU was only possible from 48 h post-hatch when villus transit time was 72 h in the jejunum, whereas at 336 h transit time was 96 h. 5. In the 108 h post hatch a rapid transition occurs from total jejunal epithelial cell proliferation and immature crypts to a defined proliferative zone in the crypts, with constant division and migration.