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.
Highlights
We found the expression of miR-302/367 in the Induced pluripotent stem cell (iPSC) lines to be 102–6fold greater than spontaneously differentiated 201B7 cells (cells cultured without basic growth factor for 2 weeks), 201B7-derived midbrain dopaminergic (mDA) cells, standard culture lines of Normal human dermal fibroblast cells (NHDF) and HeLa, and lastly primary hepatocytes and renal cells (Fig. 1b and Supplementary Fig. 1a)
With the presence of the miRNA, translation from the reporter is repressed, causing a downward shift in hmAG fluorescence on the dot-plot as illustrated in Fig. 1a, with cells within dotted-gate being the positive fraction responding to the miR-switch. 201B7 cells transfected with either miR-302a or miR-367 switches are spatially resolved from cells transfected with miR switch containing no miRNA antisense sequence (Ctrl-miR switch, black dots), whereas most HeLa cells transfected with the three switches overlapped each other (Fig. 1c)
We decided to focus on miR-302a switch for the following reasons: (1) From three independent experiments, the percentage of hiPS cells responsive to the miR-302a switch than those that responded to the miR-367 switch (Fig. 1c); (2) When measuring the translational efficiency from the dot plots the miR-302a switched-transfected hiPS cells gave a higher fold-change than miR-367 switch-transfected cells (Fig. 1d); (3) And the miR-302a switch could completely separate 201B7 from NHDF and HeLa cells, whereas the miR-367 switch did not (Fig. 1e)
Summary
MicroRNA switches (miR-switches), were encoded on modified mRNA (modRNA)[12,13] that post-transcriptionally regulates fluorescent reporters in response to the activity of the human miRNA-302/367 cluster expressed in living cells (Fig. 1a, bottom). This cluster is important for maintaining the self-renewal of stem cells and in the primed state of pluripotency[14,15,16,17]. By sorting hiPSC spiked midbrain dopaminergic (mDA)-like neuronal cell cultures with miR-302a switch, we could prevent teratoma formation in a standard in vivo tumorigenicity assay on mice. We placed the miR-302a switch in the control of puromycin selection to automatically and selectively remove contaminating hiPSCs, removing the necessity for cell sorting, which is time consuming and can damage the cells
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