Sarah Hale

Transcriptional Profiling of Human Cord Blood CD133+ and Cultured Bone Marrow Mesenchymal Stem Cells in Response to Hypoxia

Umbilical cord blood (UCB) and bone marrow (BM)‐derived stem and progenitor cells possess two characteristics required for successful tissue regeneration: extensive proliferative capacity and the ability to differentiate into multiple cell lineages. Within the normal BM and in pathological conditions, areas of hypoxia may have a role in maintaining stem cell fate or determining the fine equilibrium between their proliferation and differentiation. In this study, the transcriptional profiles and proliferation and differentiation potential of UCB CD133+ cells and BM mesenchymal cells (BMMC) exposed to normoxia and hypoxia were analyzed and compared. Both progenitor cell populations responded to hypoxic stimuli by stabilizing the hypoxia inducible factor (HIF)‐1α protein. Short exposures to hypoxia increased the clonogenic myeloid capacity of UCB CD133+ cells and promoted a significant increase in BMMC number. The differentiation potential of UCB CD133+ clonogenic myeloid cells was unaltered by short exposures to hypoxia. In contrast, the chondrogenic differentiation potential of BMMCs was enhanced by hypoxia, whereas adipogenesis and osteogenesis were unaltered. When their transcriptional profiles were compared, 183 genes in UCB CD133+ cells and 45 genes in BMMC were differentially regulated by hypoxia. These genes included known hypoxia‐responsive targets such as BNIP3, PGK1, ENO2, and VEGFA, and other genes not previously described to be regulated by hypoxia. Several of these genes, namely CDTSPL, CCL20, LSP1, NEDD9, TMEM45A, EDG‐1, and EPHA3 were confirmed to be regulated by hypoxia using quantitative reverse transcriptase polymerase chain reaction. These results, therefore, provide a global view of the signaling and regulatory network that controls oxygen sensing in human adult stem/progenitor cells derived from hematopoietic tissues.

Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.

Stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. We have used iron labeling of bone marrow stromal cells (BMSCs) to noninvasively track cell location in the infarcted rat heart over 16 weeks using cine‐magnetic resonance imaging (cine‐MRI) and to isolate the BMSCs from the grafted hearts using the magnetic properties of the donor cells. BMSCs were isolated from rat bone marrow, characterized by flow cytometry, transduced with lentiviral vectors expressing green fluorescent protein (GFP), and labeled with iron particles. BMSCs were injected into the infarct periphery immediately following coronary artery ligation, and rat hearts were imaged at 1, 4, 10, and 16 weeks postinfarction. Signal voids caused by the iron particles in the BMSCs were detected in all rats at all time points. In mildly infarcted hearts, the volume of the signal void decreased over the 16 weeks, whereas the signal void volume did not decrease significantly in severely infarcted hearts. High‐resolution three‐dimensional magnetic resonance (MR) microscopy identified hypointense regions at the same position as in vivo. Donor cells containing iron particles and expressing GFP were identified in MR‐targeted heart sections after magnetic cell separation from digested hearts. In conclusion, MRI can be used to track cells labeled with iron particles in damaged tissue for at least 16 weeks after injection and to guide tissue sectioning by accurately identifying regions of cell engraftment. The magnetic properties of the iron‐labeled donor cells can be used for their isolation from host tissue to enable further characterization.

Bone marrow-derived stromal cells home to and remain in the infarcted rat heart but fail to improve function: an in vivo cine-MRI study

Basic and clinical studies have shown that bone marrow cell therapy can improve cardiac function following infarction. In experimental animals, reported stem cell-mediated changes range from no measurable improvement to the complete restoration of function. In the clinic, however, the average improvement in left ventricular ejection fraction is around 2% to 3%. A possible explanation for the discrepancy between basic and clinical results is that few basic studies have used the magnetic resonance (MR) imaging (MRI) methods that were used in clinical trials for measuring cardiac function. Consequently, we employed cine-MR to determine the effect of bone marrow stromal cells (BMSCs) on cardiac function in rats. Cultured rat BMSCs were characterized using flow cytometry and labeled with iron oxide particles and a fluorescent marker to allow in vivo cell tracking and ex vivo cell identification, respectively. Neither label affected in vitro cell proliferation or differentiation. Rat hearts were infarcted, and BMSCs or control media were injected into the infarct periphery (n = 34) or infused systemically (n = 30). MRI was used to measure cardiac morphology and function and to determine cell distribution for 10 wk after infarction and cell therapy. In vivo MRI, histology, and cell reisolation confirmed successful BMSC delivery and retention within the myocardium throughout the experiment. However, no significant improvement in any measure of cardiac function was observed at any time. We conclude that cultured BMSCs are not the optimal cell population to treat the infarcted heart.

The impact of proliferative potential of umbilical cord-derived endothelial progenitor cells and hypoxia on vascular tubule formation in vitro.

Revascularization of the damaged tissue is pivotal to tissue repair. Here, by bringing together two in vitro model systems, we have been able to examine (1) the ability of human umbilical vein endothelial cells (HUVEC) containing a complete hierarchy of endothelial progenitors derived from the human umbilical cord to generate vascular tubules within a human stromal niche in vitro and (2) the effects of exposure to low oxygen tensions on endothelial progenitor cell proliferation and tubule formation in vitro. Our results demonstrate that high proliferative potential endothelial colony forming cells (HPP-ECFC) from cultured HUVEC preferentially contribute to vascular tubule formation in vitro and that these progenitor cells are concentrated in the CD34(lo/-) fraction. HUVEC were initially resistant when exposed to hypoxia (1.5% O(2)) for short periods (1-2 days), but sustained chronic hypoxia (4-14 days) inhibited their ability to proliferate. This was reflected by a loss in their ability to form tubules in cocultures of human dermal fibroblasts (hDFs). In contrast, an acute exposure to low oxygen tensions (1.5% O(2) for 24 h) followed by reoxygenation did not adversely affect the capacity of these cells to both proliferate and form vascular tubules in vitro.These studies therefore provide a model system to study the influences of the microenvironmental niche and modification of this niche on vascular tubule formation in vitro from HPP-ECFC.

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

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

CXCR2 modulates bone marrow vascular repair and haematopoietic recovery post‐transplant

Murine models of bone marrow transplantation show that pre‐conditioning regimens affect the integrity of the bone marrow endothelium and that the repair of this vascular niche is an essential pre‐requisite for successful haematopoietic stem and progenitor cell engraftment. Little is known about the angiogenic pathways that play a role in the repair of the human bone marrow vascular niche. We therefore established an in vitro humanized model, composed of bone marrow stromal and endothelial cells and have identified several pro‐angiogenic factors, VEGFA, ANGPT1, CXCL8 and CXCL16, produced by the stromal component of this niche. We demonstrate for the first time that addition of CXCL8 or inhibition of its receptor, CXCR2, modulates blood vessel formation in our bone marrow endothelial niche model. Compared to wild type, Cxcr2−/− mice displayed a reduction in bone marrow cellularity and delayed platelet and leucocyte recovery following myeloablation and bone marrow transplantation. The delay in bone marrow recovery correlated with impaired bone marrow vascular repair. Taken together, our data demonstrate that CXCR2 regulates bone marrow blood vessel repair/regeneration and haematopoietic recovery, and clinically may be a therapeutic target for improving bone marrow transplantation.

Jam‐a is highly expressed on human hematopoietic repopulating cells and associates with the key hematopoietic chemokine receptor cxcr4

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

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

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