Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications.

As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Blood outgrowth endothelial cells overexpressing eNOS mitigate pulmonary hypertension in rats: a unique carrier cell enabling autologous cell-based gene therapy.

We have investigated a unique cell type, blood outgrowth endothelial cells (BOEC), as a cell-based gene therapy approach to pulmonary hypertension. BOEC are bona fide endothelial cells, obtained from peripheral blood, that can be expanded to vast numbers, and are amenable to both cryopreservation and genetic modification. We established primary cultures of rat BOEC and genetically altered them to over-express human eNOS plus green fluorescent protein (rBOEC/eNOS) or to express GFP only (rBOEC/GFP). We gave monocrotaline to rats on day 0, and they developed severe pulmonary hypertension. As a Prevention model, we infused saline or rBOEC/GFP or rBOEC/eNOS on day 3, and then examined endpoints on day 24. The rBOEC/eNOS recipients developed elevated NOx (serum and lung) and less severe: elevation of right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary arteriolar muscularization and loss of alveolar density. As an Intervention model, we waited until day 21 to give the test infusions, and we examined endpoints on day 35. The rBOEC/eNOS recipients again developed elevated NOx and manifested the same improvements. Indeed, rBOEC/eNOS infusion not only prevented worsening of RVSP but also partially reversed established arteriolar muscularization. These data suggest that BOEC may be useful as a carrier cell for genetic strategies targeting pulmonary hypertension. Their properties render BOEC amenable to preclinical and scale-up studies, available for autologous therapies, and tolerant of modification and storage for potential future use in patients at risk for PAH, eg, as defined by genetics or medical condition.

Detection of viruses in used ventilation filters from two large public buildings.

BACKGROUND: Viral and bacterial pathogens may be present in the air after being released from infected individuals and animals. Filters are installed in the heating, ventilation, and air-conditioning (HVAC) systems of buildings to protect ventilation equipment and maintain healthy indoor air quality. These filters process enormous volumes of air. This study was undertaken to determine the utility of sampling used ventilation filters to assess the types and concentrations of virus aerosols present in buildings. METHODS: The HVAC filters from 2 large public buildings in Minneapolis and Seattle were sampled to determine the presence of human respiratory viruses and viruses with bioterrorism potential. Four air-handling units were selected from each building, and a total of 64 prefilters and final filters were tested for the presence of influenza A, influenza B, respiratory syncytial, corona, parainfluenza 1-3, adeno, orthopox, entero, Ebola, Marburg, Lassa fever, Machupo, eastern equine encephalitis, western equine encephalitis, and Venezuelan equine encephalitis viruses. Representative pieces of each filter were cut and eluted with a buffer solution. RESULTS: Attempts were made to detect viruses by inoculation of these eluates in cell cultures (Vero, MDCK, and RK-13) and specific pathogen-free embryonated chicken eggs. Two passages of eluates in cell cultures or these eggs did not reveal the presence of any live virus. The eluates were also examined by polymerase chain reaction or reverse-transcription polymerase chain reaction to detect the presence of viral DNA or RNA, respectively. Nine of the 64 filters tested were positive for influenza A virus, 2 filters were positive for influenza B virus, and 1 filter was positive for parainfluenza virus 1. CONCLUSION: These findings indicate that existing building HVAC filters may be used as a method of detection for airborne viruses. As integrated long-term bioaerosol sampling devices, they may yield valuable information on the epidemiology and aerobiology of viruses in air that can inform the development of methods to prevent airborne transmission of viruses and possible deterrents against the spread of bioterrorism agents.

The establishment of murine blood outgrowth endothelial cells and observations relevant to gene therapy.

Endothelial cells are an attractive vehicle for gene therapy because they may be used in an autologous fashion and may allow for direct exposure of the gene product into the intravascular space. To explore this future potential, a reproducible system was developed for the culture of murine blood outgrowth endothelial cells. These cells demonstrated acetylated low-density lipoprotein (LDL) incorporation, matrigel tube formation, and specific endothelial staining characteristics, namely P1H12, VeCAD, vascular cell adhesion molecule (VCAM), vWF, platelet endothelial cell adhesion molecule (PECAM-1), and vascular endothelial growth factor receptor-2 (VEGFR2). They were also negative for smooth muscle actin and monocytic markers CD11b, CD14, and CD16. Moreover, these cells were amendable to gene transfer with red fluorescent and green fluorescent expression vectors as well as human Factor VIII (hFVIII) while maintaining endothelial characteristics. Both source- and gene-introduced cells also manifested excellent proliferative potential. Furthermore, murine blood outgrowth endothelial cells (BOECs) demonstrated persistent in vivo seeding in the liver, lung, spleen, and bone morrow of recipient mice.