Adult mesenchymal stem cells (aMSCs) are a stem cell population present in bone marrow, which can be isolated and expanded in culture and characterized. Due to the lack of specific surface markers, it is difficult to separate the MSCs from bone marrow directly. Here, we present a novel method using high-specific nucleic acids called aptamers. Porcine MSCs were used as a target to generate aptamers by combinatorial chemistry out of a huge random library with in vitro technology called systematic evolution of ligands by exponential enrichment (SELEX). After cloning and sequencing, the binding affinity was detected using a cellsorting assay with streptavidin-coated magnetic microbeads. We also used 12-well plates immobilized with aptamers to fish out MSCs from the cell solution and a fluorescein isothiocyanate-labeled aptamer to sort MSCs from bone marrow using high-speed fluorescence-activated cell sorting. The cells showed high potency to differentiate into osteogenic, as well as into adipogenic, lineages with typical morphological characteristics. Surface marker staining showed that the attached cells were CD29؉ , SLA class I ؉ , SLA DQ ؊ , and SLA DR ؊ . Compared with existing methods, this study established a novel, rapid, and efficient method for direct isolation of aMSCs from porcine bone marrow by using an aptamer as a probe to fish out the aMSCs. This new application of aptamers can facilitate aMSC isolation and enrichment greatly, thereby enhancing the rate of aMSC-derived cells after in vitro differentiation for various applications in the emerging field of tissue engineering and regenerative medicine. STEM CELLS 2006;24:2220 -2231

SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.

Highlights d RhoA/Cdc42 DKO mice display severe macrothrombocytopenia and defective hemostasis d DKO megakaryocytes show defective cytoplasmic maturation but normal endomitosis d Loss of RhoA/Cdc42 signaling abolishes proplatelet formation d RhoA/Cdc42 signaling regulates gene expression during megakaryocyte maturation

Megakaryocytes are large cells in the bone marrow, which give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics were not affected in the absence of RhoB. However, in vitro generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, non-redundant functions in the megakaryocyte lineage.