No abstract

Identification of prevalent specific markers is crucial to stem/ progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However, the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP-binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDH Bright (ALDH Br ) cell subsets for their phenotype and proliferative capability. In this study, we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin 2 ) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin 2 CD34 1 CD38 Low/2 cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDH Br cells when associated with SP functionality (SP/ALDH Br fraction). Furthermore, the SP marker identified G 0 cells in all ALDH fractions, allowing us to sort quiescent cells regardless of ALDH activity. Moreover, we show that, within the Lin 2 CD34 1 CD38 2 ALDH Br population, the SP marker identifies cells with higher primitive characteristics, in terms of stemness-related gene expression and in vitro and in vivo proliferative potential, than the Lin 2 CD34 1 CD38 2 ALDH Br main population cells. In conclusion, our study shows that the coexpression of SP and ALDH markers refines the Lin 2 CD34 1 CD38 2 hematopoietic compartment and identifies an SP/ALDH Br cell subset enriched in quiescent primitive HSCs/HPCs.

Bisphosphonates are widely used as therapeutic agents in bone disorders including cancer metastasis due to their osteoclast inhibitory effect. Recent data shows that bisphosphonates may also induce bone-building by stimulating osteoblast activity. Clinical observations, however, have revealed that bisphosphonates may cause necrosis in the oral cavity which questions their usefulness in bone regeneration during the consolidation of inorganic implants. Here we report the investigation of bone neogenesis following chronic amine bisphosphonate (Zometa) treatment in a novel experimental model, using the rat tail vertebra as a support. This method involves (1) implantation of titan screw into the tail vertebrae, (2) systemic bisphosphonate treatment and (3) quantitative biophysical measurements which mirrors consolidation of implant, i.e. strength of fixation and changes in newly formed bone architecture using micro Computer Tomograph (micro-CT). The degree of fixation of titan implants (osseointegration) increased by 36% on the effect of Zometa and the structure of newly formed bone became robust. The mass of new bone increased 3.1-fold at 6 weeks of regeneration, as compared to controls. Thus, Zometa, a potent aminobisphosphonate used in therapy of cancer metastases, osteoporosis and bone marrow transplantation, significantly increased bone neogenesis and enforced osseointegration of titan implants as measured quantitatively in the rat tail vertebra. Our data support the usefulness of aminobisphosphonates in the rehabilitation of bone loss as well as in improvement osseointegration of implants. We emphasise that this novel method may open up new possibilities for screening the effects of local and systemic treatments.