Immobilized thrombopoietin (TPO) lipopeptide mimic supports similar signaling and CD34+cell differentiation as soluble TPO

Ex vivo expansion of hematopoietic stem cells (HSCs) would greatly facilitate cell and gene therapies. However, HSC division in culture is associated with differentiation. This contrasts with sustained HSC expansion in vivo, and has led to the hypothesis that a stem cell niche supports self-renewal. It is likely that multiple aspects of the niche will have to be mimicked to substantially enhance HSC self-renewal. We are developing a defined culture surface for the presentation of cytokines and cell adhesion molecule (CAM) ligands that are thought to be in the HSC niche. Peptide mimics of CAM ligands and cytokines conjugated to dipalmitoyl glycerol via a polyethylene glycol tether are incorporated into dipalmitoylphosphatidylcholine (DPPC) vesicles and deposited onto a hydrophobic surface to create a lipid monolayer. We have previously shown that this system effectively presents adhesive peptide ligands (Jensen et al., JACS 126:15223, 2004). The strategy for immobilizing lipopeptides has been extended to the presentation of a peptide mimetic for the hematopoietic growth factor thrombopoietin (TPO). The lipopeptide mimetic of TPO is based on the branched dimer mimic (TPOm) developed by Cwirla et al. (Science 276:1696, 1997). We have synthesized two versions of TPOm lipopeptide, the first linked to a lipid at both of the amine termini (TPOm-2L) and the second is linked by a single lipid at the carboxy terminus (TPOm-1L). This immobilization strategy does not interfere with the bioactivity of the TPOm as evidenced by cell adhesion and signaling assays. Adhesion was measured with a normal force assay at 30g using the TPO-responsive M07e cell line. We observed a dose-dependent increase in adhesion, with