Nov/CCN3 Enhances Cord Blood Engraftment by Rapidly Recruiting Latent Human Stem Cell Activity.

Umbilical cord blood (UCB) has had considerable impact in pediatric stem cell transplantation, but its wider use is limited in part by unit size. Long-term ex vivo culture offers one approach to increase engraftment capacity by seeking to expand stem and progenitor cells. Here, we show brief incubation (8 h) of UCB CD34+ cells with the matricellular regulator Nov (CCN3) increases the frequency of serially transplantable hematopoietic stem cells (HSCs) 6-fold. This rapid response suggests recruitment rather than expansion of stem cells; accordingly, in single-cell assays, Nov increases the clonogenicity of phenotypic HSCs without increasing their number through cell division. Recruitment is associated with both metabolic and transcriptional changes, and tracing of cell divisions demonstrates that the increased clonogenic activity resides within the undivided fraction of cells. Harnessing latent stem cell potential through recruitment-based approaches will inform understanding of stem cell state transitions with implications for translation to the clinic.

Activation of platelets in whole blood by recombinant factor VIIa by a thrombin-dependent mechanism.

Using a diluted whole blood method of flow cytometric analysis, we have shown that platelets could be activated in vitro in the presence of high concentrations (100 nmol/l) of recombinant factor (F) VIIa (rFVIIa; NovoSeven(R)) and 2.5 mmol/l calcium chloride. This was demonstrated by a significant increase in the mean percentage of platelets expressing CD62P and their mean fluorescent intensity (MFI) after 30 min versus platelets incubated with calcium or rFVIIa alone or diluted blood alone. The presence of rFVIIa and calcium increased the exposure of the PAC-1 activation epitope of glycoprotein (Gp) IIb/IIIa. This effect was equally influenced by the presence of calcium alone but not by rFVIIa. The effect of rFVIIa was time and concentration dependent. Thrombin generation was also necessary, as the effect of rFVIIa was completely abrogated by the additional presence of hirudin. Furthermore, soy bean trypsin inhibitor (SBTI) but not corn trypsin inhibitor (CTI) abrogated CD62P exposure, suggesting that thrombin was derived via FX but not FXII activation. Exposure of CD62P demonstrated a significant lag phase, sometimes of the order of > 30 min, as well as large intersubject variation. Significant platelet activation was observed at a concentration as low as 25 nmol/l rFVIIa. Platelet-leucocyte aggregation was also increased in the presence of 25 nmol/l rFVIIa and calcium. No significant difference was observed between levels of CD62P in diluted whole blood and platelet-rich plasma adjusted to an identical platelet count after their exposure to rFVIIa and calcium for 30 min.

Lymphocyte trafficking through the blood-brain barrier is dependent on endothelial cell heterotrimeric G-protein signaling.

We have previously shown that the engagement of ICAM-1 on brain endothelial cells (EC) results in the propagation of EC signaling pathways that are necessary for efficient lymphocyte migration across the tight vascular barriers of the brain. Signaling via this receptor alone, however, is unlikely to explain the differential recruitment of leukocytes at different vascular beds. In this study, we investigated the role of EC heterotrimeric G-protein-mediated signaling in supporting transendothelial migration of T lymphocytes. Treatment of brain EC monolayers with pertussis toxin (PTX) resulted in ADP-ribosylation of G-protein alpha subunits and inhibition (>80%) of lymphocyte migration without affecting lymphocyte adhesion. Aortic and high endothelial venule EC treated identically resulted in only partial inhibition of lymphocyte migration (<40%). Expression of ribosylation-resistant (PTX-insensitive) G-protein alpha subunits in brain EC restored their ability to support lymphocyte migration after pretreatment with PTX. Treatment of brain EC with PTX did not inhibit ICAM-1-stimulated tyrosine phosphorylation of focal adhesion kinase, suggesting the effects of PTX in inhibiting EC facilitation of lymphocyte migration are distinct from activation of EC through ICAM-1. We conclude that a heterotrimeric G-protein-mediated signaling pathway in brain EC is essential for efficient transendothelial migration of T lymphocytes into the brain.