MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells

Abstract

The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information.

Viral Vector-Based Innovative Approaches to Directly Abolishing Tumorigenic Pluripotent Stem Cells for Safer Regenerative Medicine

“…This fusion protein has the potential to completely eliminate hPSCs. Yet another group synthesized a molecular switch for microRNA-302a-5p, a molecule that is highly and specifically expressed in undifferentiated hPSCs but gradually decreases to basal levels during differentiation 34 . This switch sensitively detected undifferentiated and partially differentiated hiPSCs.…”

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].…”

“…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]. …”

Synthetic gene circuits and cellular decision-making in human pluripotent stem cells

“…Another exciting study reverse engineered the temporal and dose specific interplay of Pdx1, Ngn3 and MafA (based on knowledge from mouse studies) to increase the hPSC-derived production of human beta-like cells [74]. Finally, another key interest in the stem cell field is to produce clinical-grade hPSC using genetically engineered fail-safe switches that ablate hPSC but not differentiated cells upon transplantation [75,76]. For example, one recently established switch puts a resistance gene under the control of an endogenous miRNA (miR-302) that is expressed only in hPSC but not in differentiated cells [76].…”

“…Therefore this opens new opportunities for generating cellular therapeutics that have synthetically integrated mechanisms for studying or regenerating disordered cellular functions [10] and at the same time could contain safety mechanisms to remove cells that become tumorigenic or do not function properly in vivo [75,76].…”