Junctional Adhesion Molecule-A Is Highly Expressed on Human Hematopoietic Repopulating Cells and Associates with the Key Hematopoietic Chemokine Receptor CXCR4

Abstract

Hematopoietic stem/progenitor cells (HSPCs) reside in specialized bone marrow microenvironmental niches, with vascular elements (endothelial/mesenchymal stromal cells) and CXCR4‐CXCL12 interactions playing particularly important roles for HSPC entry, retention, and maintenance. The functional effects of CXCL12 are dependent on its local concentration and rely on complex HSPC‐niche interactions. Two Junctional Adhesion Molecule family proteins, Junctional Adhesion Molecule‐B (JAM)‐B and JAM‐C, are reported to mediate HSPC‐stromal cell interactions, which in turn regulate CXCL12 production by mesenchymal stromal cells (MSCs). Here, we demonstrate that another JAM family member, JAM‐A, is most highly expressed on human hematopoietic stem cells with in vivo repopulating activity (p < .01 for JAM‐Ahigh compared to JAM‐AInt or Low cord blood CD34+ cells). JAM‐A blockade, silencing, and overexpression show that JAM‐A contributes significantly (p < .05) to the adhesion of human HSPCs to IL‐1β activated human bone marrow sinusoidal endothelium. Further studies highlight a novel association of JAM‐A with CXCR4, with these molecules moving to the leading edge of the cell upon presentation with CXCL12 (p < .05 compared to no CXCL12). Therefore, we hypothesize that JAM family members differentially regulate CXCR4 function and CXCL12 secretion in the bone marrow niche. Stem Cells 2016;34:1664–1678

Dual TGFβ/BMP Pathway Inhibition Enables Expansion and Characterization of Multiple Epithelial Cell Types of the Normal and Cancerous Breast

“…Similar to results obtained with cells propagated using the previous epithelial reprogramming assay (7), this new culturing method enabled us to document enormous phenotypic heterogeneity in the normal breast. The expanded cell populations are described as CD49f þ /EpCAM À stem/basal-enriched cells, CD49f þ /EpCAM þ luminal progenitor cells, CD49f À / EpCAM þ mature luminal cells, CD44 þ /CD24 À stem/basal cells, CD44 þ /CD24 þ stem/luminal progenitor cells, CD44 À /CD24 þ differentiated cells, CD201 þ /EpCAM À multipotent stem cells, CD271 þ rare basal cells in luminal breast cancer (relevant in antiestrogen resistance models), CD73 þ /EpCAM þ /CD90 À rare endogenous pluripotent somatic stem cells, CD73 þ /CD90 þ / EpCAM À potential mesenchymal stem cells, CD10 þ /EpCAM À basal/myoepithelial cells, ALCAM (CD166) þ cells that are enriched for ERa, and JAM-A þ cells, which are enriched for cancer stem cell phenotype (15)(16)(17). Flow cytometry characterization of breast epithelial cells from six healthy women (KTB200-205) showed remarkable variability in stem/basal, progenitor, and mature/differentiated cell subpopulations ( Fig.…”

Hematopoietic progenitors polarize in contact with bone marrow stromal cells by engaging CXCR4 receptors

“…Interestingly, although ICAM, VCAM and CXCR4 were all segregated in the protrusion at the contact site with the osteoblast, ICAM-and VCAM-mediated adhesions appeared only to have the capacity to induce a morphological polarization of HSPC, whereas CXCR4 engagement with its ligand SDF-1 appeared also to have the capacity to induce the recruitment of the centrosome at the contact site. However, considering that CXCR4, ICAM, VCAM and other adhesion receptors mutually activate each other (Peled et al, 2000) (Glodek et al, 2007) (Petty et al, 2009) (Chang et al, 2016) it is likely that the complete molecular mechanism inducing and establishing the entire internal polarization of HSPC involves the synergy of several signaling pathways associated with the adhesion of the HSPC to the bone-marrow niche cells.…”

A Novel High-Throughput Screening Platform Reveals an Optimized Cytokine Formulation for Human Hematopoietic Progenitor Cell Expansion

“…Details of cytokine changes for each protocol are listed in Supplementary Tables S1, S2 and S3. On day 8, cells were analyzed by flow cytometry for viable nucleated cell count using CountBright beads and DAPI staining and phenotypically characterized for HSPC subsets using nine-color flow cytometry on the BD LSRII as described below and in Chang et al [32].…”

“…+ HSPCs were stained with CD133-APC or -PE and DAPI as described previously [30][31][32]. Multicolor flow cytometry characterization of CD133 + human CB-derived HSPCs was based on the method described by Notta et al [23].…”

“…On days 0 and 8, viable nucleated cells were quantified using CountBright beads to evaluate total cell expansion. The proportion of HSPCs in the expanded cells was determined by multicolor flow cytometric analysis [23,[30][31][32]42]. Figure 4A shows the flow cytometric gating strategy used for these experiments.…”