Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches

Abstract

Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p-, and miR-375-switches purified endothelial cells, hepatocytes, and insulin-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable.

Sall1 transiently marks undifferentiated heart precursors and regulates their fate

“…To generate time dependent Sall1 overexpressing cells, A DOX inducible SALL1 expressing piggybac vector and a PBEF1a-mSALL1-IRES-mcherry vector were co-electroporated with the piggybac transposase vector PBASE2 into 201B7 cells [10] with NEPA21 (NEPA GENE) [11]. DOX-SALL1 hiPSCs were maintained and differentiated as described [12]. …”

Mammalian synthetic biology in the age of genome editing and personalized medicine

“…Furthermore, coupling a β-catenin–binding RNA aptamer to microRNA (miRNA) targeted against a green fluorescent protein (GFP) mRNA construct has been shown to enable quantification of nuclear β-catenin concentrations [33]. Interestingly, a similar strategy can be employed in reverse to detect endogenous expression of miRNAs involved in cell development [34,35•]. By expressing an mRNA targeted by miRNA-302a-5p, Parr et al demonstrated the ability to sort or selectively eliminate undifferentiated human iPSCs from heterogeneous cell populations, on the basis of retained miR-302a-5p expression in undifferentiated cells [35].…”

microRNAs Distinctively Regulate Vascular Smooth Muscle and Endothelial Cells: Functional Implications in Angiogenesis, Atherosclerosis, and In-Stent Restenosis

“…Most recently, miR-126 has been identified as an efficient marker in the detection and purification of EC [81]. This miR plays a critical role in modulating vascular development and homeostasis, targeting specific mRNAs including the Sprouty-related protein 1 (SPRED-1), CXCL12, SDF-1, and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) [82–87].…”