Abstract 306: Large-scale Contractile Smooth Muscle Cell Differentiation from Tissue-specific Red Fluorescence Tagged Mouse Embryonic Stem Cells
Background: Molecular pathways that promote or obstruct specification of precursors to smooth muscle cells (SMC), and maturation to the contractile form remain poorly understood. Moreover, lack of reliable methods to derive SMC from stem cells in a predictable manner and in sufficient quantities precludes their use in in vivo therapeutic and tissue engineering applications.
Results: We have generated transgenic mouse embryonic stem (ES) cells to facilitate studies of lineage commitment and differentiation of SMC and the regulatory networks responsible. Given that upregulation of SMC-specific markers was found to precede and parallel contractile SMC appearance (spontaneous beat frequency <10/min) in an in vitro embryoid body (EB) model, we endeavored to track SMC precursors. To date, no definitive SMC extracellular markers exist. Consequently, we introduced a smooth muscle myosin heavy chain (SM-MHC) promoter-driven bi-cistronic Cre recombinase and enhanced green fluorescent protein (EGFP) genes into mouse ES cells harboring a Cre-activatable red fluorescent protein (DsRed). Select clones were differentiated, 7 of 9 displayed floxed-stop element excision and consequent robust DsRed expression in areas of SMC-like contraction beginning at day 18 of differentiation. Immunofluorescent studies confirmed DsRed and EGFP co-localization. Flow cytometric analysis of enzymatically dissociated mature EB at days 32–37 revealed low abundance of DsRed+ (1–2%), DsRed+/EGFP+ (<0.2%) and EGFP+ (<0.05%) cells. Viable DsRed+ cells were sorted, re-plated and their proliferative, migratory and contractile functions are under investigation. Suspension bioreactor-based large-scale differentiation of reporter-tagged ES cell-derived EB recapitulated the process of SMC differentiation yielding comparable percentage of DsRed+ cells (1–2%) as early as day 15. Initial input of 8e6 ES cells typically generated 25,000 viable EB (~200 cells), which at day 15 reached 70–95e3 cells each.
Conclusions: Our approach is the first to allow live tracking of differentiating SMC and combined with large-scale bioreactor synthesis complemented with SM-specific antibiotic selection strategy has the potential to generate therapeutic quantities of cells.