Fluorescent nano-switch probes: Monitoring of microRNA level in induced pluripotent stem cells without affecting pluripotency

The isolation of highly pluripotent induced pluripotent stem cells (iPSCs) and increasing reprogramming efficiency are goals of reprogramming into iPSCs. It has been studied to isolate highly pluripotent iPSCs without... [ view full abstract ]

The isolation of highly pluripotent induced pluripotent stem cells (iPSCs) and increasing reprogramming efficiency are goals of reprogramming into iPSCs. It has been studied to isolate highly pluripotent iPSCs without affecting pluripotency. In general, highly pluripotent iPSCs has been isolated by surface markers and morphology of cells with confirming generation embryonic body and teratoma. However, surface markers and morphology do not show endogenous condition. microRNA (miRNA) has been drawn lots of interest as a biomarker, because it has been proved that miRNA plays a role as a regulator in various biological functions; metastasis, proliferation, and especially differentiation state or pluripotency level in iPSCs. Therefore, miRNA expression level can be combined with conservative method (surface markers and morphology) for efficiently distinguish highly pluripotent iPSCs during reprogramming somatic cells into iPSCs. A non-invasive imaging system that can monitor miRNA expression level without affecting pluripotency of iPSCs provides a useful tool to identify and analyze specific cell populations during reprogramming into iPSCs.
Herein, we designed a miRNA imaging probe (miP) containing sequences of positively and negatively expressed miRNAs that determine the differentiation status of somatic cells during reprogramming. miP consists of a complementary sequence to target miRNA, a fluorophore and a quencher at the end of each oligonucleotide. When miP meets with target miRNA, quencher is detached from fluorophore and miP has fluorescence signal. Also, polyethylene glycol (PEG)-grafted polyethylene imine (PEI-PEG) was utilized as a non-invasive delivery carrier of a fluorescence imaging probe, which enabled repeated evaluation of miRNA expression.
miP loaded PEI-PEG (miP-P) showed ~96 % of miP untake efficiency, and had fluorescence signal within 30 minutes in vitro when it met with endogenous target miRNA. We call it as a fluorescent nanoswicth, because it turns “ON” with target miRNA. The fluorescence signal results were corresponded with qRT-PCR analysis. In addition, we confirmed that miP-P system does not affect pluripotency of iPSCs.
Consequently, the fluorescent nanoswitch has a potent to isolate highly pluripotent iPSCs without affecting pluripotency of iPSCs. We envisage that this miRNA imaging system could be valuable methodology for identifying and sorting pluripotent stem cells during somatic cell reprogramming.