High-Throughput Flow Cytometry Purification of Transduced Progenitors Expressing Defined Levels of Vascular Endothelial Growth Factor Induces Controlled Angiogenesis In Vivo

Abstract

Delivery of therapeutic genes by genetically modified progenitors is a powerful tool for regenerative medicine. However, many proteins remain localized within or around the expressing cell, and heterogeneous expression levels can lead to reduced efficacy or increased toxicity. For example, the matrix-binding vascular endothelial growth factor (VEGF) can induce normal, stable, and functional angiogenesis or aberrant angioma growth depending on its level of expression in the microenvironment around each producing cell, and not on its total dose. To overcome this limitation, we developed a flow cytometry-based method to rapidly purify transduced cells expressing desired levels of a therapeutic transgene. Primary mouse myoblasts were transduced with a bicistronic retrovirus expressing VEGF linked to a nonfunctional, truncated form of the syngenic molecule CD8a. By using a clonal population uniformly expressing a known VEGF level as a reference, cells producing similar VEGF amounts were rapidly sorted from the primary population on the basis of their CD8a fluorescence intensity. A single round of sorting with a suitably designed gate yielded a purified population that induced robust, normal, and stable angiogenesis, and completely avoided angioma growth, which was instead always caused by the heterogeneous parent population. This clinically applicable high-throughput technique allowed the delivery of highly controlled VEGF levels in vivo, leading to significantly improved safety without compromising efficacy. Furthermore, when applied to other suitable progenitor populations, this technique could help overcome a significant obstacle in the development of safe and efficacious vascularization strategies in the fields of regenerative medicine and tissue engineering.