α1,3-fucosylation treatment improves cord blood CD34 negative hematopoietic stem cell navigation.

For almost two decades, clinicians have overlooked the diagnostic potential of CD34neg hematopoietic stem cells because of their limited homing capacity relative to CD34posHSCs when injected intravenously. This has contributed to the lack of appeal of using umbilical cord blood in HSC transplantation because its stem cell count is lower than bone marrow. The present study reveals that the homing and engraftment of CD34negHSCs can be improved by adding the Sialyl Lewis X molecule via α1,3-fucosylation. This unlocks the potential for using this more primitive stem cell to treat blood disorders because our findings show CD34negHSCs have the capacity to regenerate cells in the bone marrow of mice for several months. Furthermore, our RNA sequencing analysis revealed that CD34negHSCs have unique adhesion pathways, downregulated in CD34posHSCs, that facilitate interaction with the bone marrow niche. Our findings suggest that CD34neg cells will best thrive when the HSC resides in its microenvironment.

CD34+ HSPCs-derived exosomes contain dynamic cargo and promote their migration through functional binding with the homing receptor E-selectin.

Exosomes are tiny vesicles released by cells that carry communications to local and distant locations. Emerging research has revealed the role played by integrins found on the surface of exosomes in delivering information once they reach their destination. But until now, little has been known on the initial upstream steps of the migration process. Using biochemical and imaging approaches, we show here that exosomes isolated from both leukemic and healthy hematopoietic stem/progenitor cells can navigate their way from the cell of origin due to the presence of sialyl Lewis X modifications surface glycoproteins. This, in turn, allows binding to E-selectin at distant sites so the exosomes can deliver their messages. We show that when leukemic exosomes were injected into NSG mice, they traveled to the spleen and spine, sites typical of leukemic cell engraftment. This process, however, was inhibited in mice pre-treated with blocking E-selectin antibodies. Significantly, our proteomic analysis found that among the proteins contained within exosomes are signaling proteins, suggesting that exosomes are trying to deliver active cues to recipient cells that potentially alter their physiology. Intriguingly, the work outlined here also suggests that protein cargo can dynamically change upon exosome binding to receptors such as E-selectin, which thereby could alter the impact it has to regulate the physiology of the recipient cells. Furthermore, as an example of how miRNAs contained in exosomes can influence RNA expression in recipient cells, our analysis showed that miRNAs found in KG1a-derived exosomes target tumor suppressing proteins such as PTEN.

Nanoscopic Characterization of Cell Migration under Flow Using Optical and Electron Microscopy.

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that takes place through essential interactions with adhesion molecules on an endothelial cell layer. The homing process of HSPCs begins with the tethering and rolling of the cells on the endothelial layer, which is achieved by the interaction between selectins on the endothelium to the ligands on HSPC/leukemic cells under shear stress of the blood flow. Although many studies have been based on in vitro conditions of the cells rolling over recombinant proteins, significant challenges remain when imaging HSPC/leukemic cells on the endothelium, a necessity when considering characterizing cell-to-cell interaction and rolling dynamics during cell migration. Here, we report a new methodology that enables imaging of stem-cell-intrinsic spatiotemporal details during its migration on an endothelium-like cell monolayer. We developed optimized protocols that preserve transiently appearing structures on HSPCs/leukemic cells during its rolling under shear stress for fluorescence and scanning electron microscopy characterization. Our new experimental platform is closer to in vivo conditions and will contribute to indepth understanding of stem-cell behavior during its migration and cell-to-cell interaction during the process of homing.

Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms.

In contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34- stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by ST and LT-HSCs to migrate to the bone marrow(BM). Specifically focusing on murine ST-HSCs (Flk2-CD34+) and LT-HSCs (Flk2-CD34-), we observed a distinctive expression pattern of BM homing effectors necessary for the first step, namely sialyl Lewis-X (sLex) (ligand for E-selectin), and the second step, namely CXCR4 chemokine receptor (receptor for SDF-1). sLex expression was higher on Flk2-CD34+ ST-HSCs (>60%) compared with Flk2-CD34- LT-HSCs (<10%), which correlated to binding to E-selectin. Higher concentrations of CXCR4 were observed on Flk2-CD34+ ST-HSCs compared with Flk2-CD34- LT-HSCs. Interestingly, the expression of CD26, a peptidase known to deactivate chemokines (ie, SDF-1), was higher on Flk2-CD34- LT-HSCs. Given that both E-selectin-binding and CXCR4-mediated migration are compromised in Flk2-CD34- LT-HSCs, we aimed to enhance their ability to migrate using recombinant human fucosyltransferase 6 (rhFTVI) and the CD26 inhibitor, Dip A (diprotin A). To this end, we observed that although LT-HSCs expressed low concentrations of sLex, they were able to engraft when transplanted into recipient mice. Moreover, although both CD26 inhibition and fucosylation enhanced migration of both HSC populations in vitro, only pretreatment of LT-HSCs with Dip A enhanced engraftment in vivo after transplantation into recipient mice. Remarkably, fucosylation of Flk2-CD34+ ST-HSCs consistently led to their ability to transplant secondary recipients. These data suggest that using fucosylation and Dip A to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs differentially influences their abilities to migrate and engraft in vivo and promotes the ability of ST-HSCs to engraft secondary recipient mice, the gold standard for testing functionality of LT-HSCs.

Single-molecule imaging and microfluidic platform reveal molecular mechanisms of leukemic cell rolling.

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that occurs through key interactions between adhesion molecules. Tethering and rolling of the cells on endothelium, the crucial initial step of the adhesion cascade, is mediated by interactions between selectins expressed on endothelium to their ligands expressed on HSPCs/leukemic cells in flow. Although multiple factors that affect the rolling behavior of the cells have been identified, molecular mechanisms that enable the essential slow and stable cell rolling remain elusive. Here, using a microfluidics-based single-molecule live cell fluorescence imaging, we reveal that unique spatiotemporal dynamics of selectin ligands on the membrane tethers and slings, which are distinct from that on the cell body, play an essential role in the rolling of the cell. Our results suggest that the spatial confinement of the selectin ligands to the tethers and slings together with the rapid scanning of a large area by the selectin ligands, increases the efficiency of selectin-ligand interactions during cell rolling, resulting in slow and stable rolling of the cell on the selectins. Our findings provide novel insights and contribute significantly to the molecular-level understanding of the initial and essential step of the homing process.

Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells.

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.

Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy.

The parallel plate flow chamber assay is widely utilized to study physiological cell-cell adhesive interactions under dynamic flow that mimics the bloodstream. In this technique, the cells are perfused under defined shear stresses over a monolayer of endothelial cells (expressing homing molecules, e.g., selectins) or a surface (expressing recombinant homing molecules). However, with the need to study multiple samples and multiple parameters per sample, using a traditional bright-field microscope-based flow assay allows only one sample at a time to be analyzed, resulting in high interexperiment variability, the need for normalization, waste of materials, and significant consumption of time. We developed a multiplexing approach using a three-color fluorescence staining method, which allowed for up to seven different combination signatures to be run at one time. Using this fluorescent multiplex cell rolling (FMCR) assay, each sample is labeled with a different signature of emission wavelengths and mixed with other samples just minutes before the flow run. Subsequently, real-time images are acquired in a single pass using a line-scanning spectral confocal microscope. To illustrate the glycan-dependent binding of E-selectin, a central molecule in cell migration, to its glycosylated ligands expressed on myeloid-leukemic cells in flow, the FMCR assay was used to analyze E-selectin-ligand interactions following the addition (fucosyltransferase-treatment) or removal (deglycosylation) of key glycans on the flowing cells. The FMCR assay allowed us to analyze the cell-adhesion events from these different treatment conditions simultaneously in a competitive manner and to calculate differences in rolling frequency, velocity, and tethering capability of cells under study.

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44.

CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.