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

View Article: PubMed Central - PubMed

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.

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miR-302a and 367 switches specifically detect hiPSC cells.(a) miR-302a switch can remove undifferentiated or partially differentiated cells before transplantation. miRNA binding to the 5′UTR of the hmAG reporter causes translation repression. The dotted outline on the dot-plot corresponds to the miR-pos fraction. (b) hsa-miR-302a-5p and -367-3p are specifically expressed in 201B7 hiPSCs relative to NHDF and downregulated in spontaneously differentiated 201B7 cells and 201B7-derived mDA cells (n = 3 for all groups). (c) Representative dot plots of 201B7 and HeLa transfected with either 45 ng of Ctrl- (black dots), miR-302a (green) or miR-367 (purple) switches mRNA and 90 ng of tagBFP internal control. Right panel shows the percentage of 302-pos and 302-neg cells (n = 3 for all groups). (d) Percentage of translation efficiency (T.E., geometric mean of hmAG/geometric mean of tagBFP) of three hiPSC lines and NHDF cells transfected with Ctrl- (black), miR-302a (green) or miR-367 (purple) switches (n = 3 for all groups). (e) Representative histograms of the translation efficiency of the raw fluorescence signal (hmAG/tagBFP) for 201B7, HeLa and NHDF transfected with either miR-302a (top) or miR-367 (bottom). Error bars represent the SEM of three independent experiments.
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f1: miR-302a and 367 switches specifically detect hiPSC cells.(a) miR-302a switch can remove undifferentiated or partially differentiated cells before transplantation. miRNA binding to the 5′UTR of the hmAG reporter causes translation repression. The dotted outline on the dot-plot corresponds to the miR-pos fraction. (b) hsa-miR-302a-5p and -367-3p are specifically expressed in 201B7 hiPSCs relative to NHDF and downregulated in spontaneously differentiated 201B7 cells and 201B7-derived mDA cells (n = 3 for all groups). (c) Representative dot plots of 201B7 and HeLa transfected with either 45 ng of Ctrl- (black dots), miR-302a (green) or miR-367 (purple) switches mRNA and 90 ng of tagBFP internal control. Right panel shows the percentage of 302-pos and 302-neg cells (n = 3 for all groups). (d) Percentage of translation efficiency (T.E., geometric mean of hmAG/geometric mean of tagBFP) of three hiPSC lines and NHDF cells transfected with Ctrl- (black), miR-302a (green) or miR-367 (purple) switches (n = 3 for all groups). (e) Representative histograms of the translation efficiency of the raw fluorescence signal (hmAG/tagBFP) for 201B7, HeLa and NHDF transfected with either miR-302a (top) or miR-367 (bottom). Error bars represent the SEM of three independent experiments.

Mentions: For certain cell types, there are no effective cell-surface or intracellular markers for their positive selection by cell sorting. Furthermore, in some cases, a mix-culture of cells, that excludes harmful cells to cause teratoma formation or graft overgrowth, is required. In the above cases, ideally we would use a general tool that can remove the undifferentiated or partially differentiated cells, while also being applicable to any differentiation protocol (Fig. 1a, top). Here we have established such a method, which can selectively identify undifferentiated and partially differentiated cells with high-resolution. The method is simple and cost-effectively, and can also be easily scaled up to handle millions of cells. It is noteworthy that our method is the only one capable of interrogating the intracellular information of living cells. Comparatively, most existing technologies are restricted to information displayed on the cell surface.


MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells
miR-302a and 367 switches specifically detect hiPSC cells.(a) miR-302a switch can remove undifferentiated or partially differentiated cells before transplantation. miRNA binding to the 5′UTR of the hmAG reporter causes translation repression. The dotted outline on the dot-plot corresponds to the miR-pos fraction. (b) hsa-miR-302a-5p and -367-3p are specifically expressed in 201B7 hiPSCs relative to NHDF and downregulated in spontaneously differentiated 201B7 cells and 201B7-derived mDA cells (n = 3 for all groups). (c) Representative dot plots of 201B7 and HeLa transfected with either 45 ng of Ctrl- (black dots), miR-302a (green) or miR-367 (purple) switches mRNA and 90 ng of tagBFP internal control. Right panel shows the percentage of 302-pos and 302-neg cells (n = 3 for all groups). (d) Percentage of translation efficiency (T.E., geometric mean of hmAG/geometric mean of tagBFP) of three hiPSC lines and NHDF cells transfected with Ctrl- (black), miR-302a (green) or miR-367 (purple) switches (n = 3 for all groups). (e) Representative histograms of the translation efficiency of the raw fluorescence signal (hmAG/tagBFP) for 201B7, HeLa and NHDF transfected with either miR-302a (top) or miR-367 (bottom). Error bars represent the SEM of three independent experiments.
© Copyright Policy - open-access
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f1: miR-302a and 367 switches specifically detect hiPSC cells.(a) miR-302a switch can remove undifferentiated or partially differentiated cells before transplantation. miRNA binding to the 5′UTR of the hmAG reporter causes translation repression. The dotted outline on the dot-plot corresponds to the miR-pos fraction. (b) hsa-miR-302a-5p and -367-3p are specifically expressed in 201B7 hiPSCs relative to NHDF and downregulated in spontaneously differentiated 201B7 cells and 201B7-derived mDA cells (n = 3 for all groups). (c) Representative dot plots of 201B7 and HeLa transfected with either 45 ng of Ctrl- (black dots), miR-302a (green) or miR-367 (purple) switches mRNA and 90 ng of tagBFP internal control. Right panel shows the percentage of 302-pos and 302-neg cells (n = 3 for all groups). (d) Percentage of translation efficiency (T.E., geometric mean of hmAG/geometric mean of tagBFP) of three hiPSC lines and NHDF cells transfected with Ctrl- (black), miR-302a (green) or miR-367 (purple) switches (n = 3 for all groups). (e) Representative histograms of the translation efficiency of the raw fluorescence signal (hmAG/tagBFP) for 201B7, HeLa and NHDF transfected with either miR-302a (top) or miR-367 (bottom). Error bars represent the SEM of three independent experiments.
Mentions: For certain cell types, there are no effective cell-surface or intracellular markers for their positive selection by cell sorting. Furthermore, in some cases, a mix-culture of cells, that excludes harmful cells to cause teratoma formation or graft overgrowth, is required. In the above cases, ideally we would use a general tool that can remove the undifferentiated or partially differentiated cells, while also being applicable to any differentiation protocol (Fig. 1a, top). Here we have established such a method, which can selectively identify undifferentiated and partially differentiated cells with high-resolution. The method is simple and cost-effectively, and can also be easily scaled up to handle millions of cells. It is noteworthy that our method is the only one capable of interrogating the intracellular information of living cells. Comparatively, most existing technologies are restricted to information displayed on the cell surface.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus