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 <5% adherent cells for DPPC surfaces and a plateau of ~70% adherent cells at 1.0 mol% TPOm-1L. There was much less adhesion to TPOm-2L (a maximum of ~25% adhesion). Selective adhesion to the TPOm lipopeptides persisted after 6 days of culture, both in the presence and absence of serum. Culture surfaces with TPOm lipopeptides elicit similar M07e cell signaling response kinetics via the ERK1,2 and STAT5 pathways as compared to soluble TPOm and recombinant human TPO (rhTPO). It is interesting that surface presentation of TPOm synergizes more extensively with stem cell factor (SCF) for the activation of STAT5 than does soluble TPOm. Experiments with bone marrow (BM) CD34+ cells show that surfaces incorporating TPOm-2L supplemented with SCF and flt-3 ligand (FL) support similar overall expansion and protection from apoptosis as controls of soluble TPOm or rhTPO with SCF and FL. Further, there was no difference in the ability of TPOm to retain CD34+ cells or CD34+Thy1+ cells. Also, BM CD34+ cell cultures supplemented with TPOm-1L alone supported similar megakaryocyte maturation, evidenced by the appearance of polyploid CD41+ cells after 9 and 12 days of culture, as those supplemented with soluble TPOm. An advantage of this presentation strategy is the potential to save on cytokines during long-term culture. Feeding cultures stimulated by TPOm lipopeptides requires only exchange of basal media. In summary, we have developed a method to present immobilized TPOm in an active conformation that supports cell adhesion and signaling as well as the expansion and differentiation of CD34+ cells.