Limits...
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.

No MeSH data available.


Related in: MedlinePlus

Automated purification of differentiated cells without cell sorting.(a) Schematic and time course of the puromycin-purification experiment. In the representative dot plot both untransfected (UT) and 302-pos populations can be removed by the addition of puromycin as they do not express the puroR gene. (b) LEFT Bar chart shows the total cell number from the entire cell suspension volume counted in P1/whole-cell- (white), 7-ADD neg- (grey), EGFP pos- (green) and TRA-1-60 pos- (purple) gates after the stated treatments. UPPER RIGHT Representative histograms of EGFP fluorescence (7-ADD neg-gated cells) under conditions 2 μg of puromycin with either 50 ng Ctrl-puroR switch (black shade) or 302a-puroR switch (green shade) (n = 3 for all groups). LOWER RIGHT Representative histograms of TRA-1-60 staining (7-ADD-neg gated cells) under conditions 2 μg of puromycin with either Ctrl-puroR switch (black shade) or 302a-puroR switch (purple shade). Error bars represent the SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016789&req=5

f6: Automated purification of differentiated cells without cell sorting.(a) Schematic and time course of the puromycin-purification experiment. In the representative dot plot both untransfected (UT) and 302-pos populations can be removed by the addition of puromycin as they do not express the puroR gene. (b) LEFT Bar chart shows the total cell number from the entire cell suspension volume counted in P1/whole-cell- (white), 7-ADD neg- (grey), EGFP pos- (green) and TRA-1-60 pos- (purple) gates after the stated treatments. UPPER RIGHT Representative histograms of EGFP fluorescence (7-ADD neg-gated cells) under conditions 2 μg of puromycin with either 50 ng Ctrl-puroR switch (black shade) or 302a-puroR switch (green shade) (n = 3 for all groups). LOWER RIGHT Representative histograms of TRA-1-60 staining (7-ADD-neg gated cells) under conditions 2 μg of puromycin with either Ctrl-puroR switch (black shade) or 302a-puroR switch (purple shade). Error bars represent the SEM of three independent experiments.

Mentions: A major advantage of our strategy is that we can place any gene as the reporter on the miR-302a switch. Therefore, we hypothesised that placing a simple puromycin selection circuit using puromycin-resistant mRNA (puroR) under the translational regulation of miR-302a switch would allow for automated elimination of residual hiPSCs without the need for cell sorting (Fig. 6a). We chose puromycin selection because of the ease of the design and puromycin is already used globally to form stable clones and has very little bystander effects on neighbouring cells. Firstly this design allows us to remove untransfected cells by adding puromycin. In cells that express active miR-302a, the miR-switch will repress the translation of the downstream puroR gene. Thus, upon supplementing the medium with puromycin, these cells will die. In contrast cells unresponsive to miR-302a switch will survive (Fig. 6a). Initially, we optimised the experimental conditions to assess toxicity of the puromycin. We found 2 μg/ml was sufficient to give complete toxicity for both hiPSCs and differentiated mDA cells (Supplementary Fig. 5). Approximately 10 ng of puroR mRNA (in 96-well plate scale) was enough to restore cell viability under 2 μg/mL puromycin. When regulating the puroR translation with miR-302a switch, we observed significant cell toxicity in hiPSCs but not in differentiated mDA neuronal cultures (Supplementary Fig. 5b).


MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells
Automated purification of differentiated cells without cell sorting.(a) Schematic and time course of the puromycin-purification experiment. In the representative dot plot both untransfected (UT) and 302-pos populations can be removed by the addition of puromycin as they do not express the puroR gene. (b) LEFT Bar chart shows the total cell number from the entire cell suspension volume counted in P1/whole-cell- (white), 7-ADD neg- (grey), EGFP pos- (green) and TRA-1-60 pos- (purple) gates after the stated treatments. UPPER RIGHT Representative histograms of EGFP fluorescence (7-ADD neg-gated cells) under conditions 2 μg of puromycin with either 50 ng Ctrl-puroR switch (black shade) or 302a-puroR switch (green shade) (n = 3 for all groups). LOWER RIGHT Representative histograms of TRA-1-60 staining (7-ADD-neg gated cells) under conditions 2 μg of puromycin with either Ctrl-puroR switch (black shade) or 302a-puroR switch (purple shade). Error bars represent the SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016789&req=5

f6: Automated purification of differentiated cells without cell sorting.(a) Schematic and time course of the puromycin-purification experiment. In the representative dot plot both untransfected (UT) and 302-pos populations can be removed by the addition of puromycin as they do not express the puroR gene. (b) LEFT Bar chart shows the total cell number from the entire cell suspension volume counted in P1/whole-cell- (white), 7-ADD neg- (grey), EGFP pos- (green) and TRA-1-60 pos- (purple) gates after the stated treatments. UPPER RIGHT Representative histograms of EGFP fluorescence (7-ADD neg-gated cells) under conditions 2 μg of puromycin with either 50 ng Ctrl-puroR switch (black shade) or 302a-puroR switch (green shade) (n = 3 for all groups). LOWER RIGHT Representative histograms of TRA-1-60 staining (7-ADD-neg gated cells) under conditions 2 μg of puromycin with either Ctrl-puroR switch (black shade) or 302a-puroR switch (purple shade). Error bars represent the SEM of three independent experiments.
Mentions: A major advantage of our strategy is that we can place any gene as the reporter on the miR-302a switch. Therefore, we hypothesised that placing a simple puromycin selection circuit using puromycin-resistant mRNA (puroR) under the translational regulation of miR-302a switch would allow for automated elimination of residual hiPSCs without the need for cell sorting (Fig. 6a). We chose puromycin selection because of the ease of the design and puromycin is already used globally to form stable clones and has very little bystander effects on neighbouring cells. Firstly this design allows us to remove untransfected cells by adding puromycin. In cells that express active miR-302a, the miR-switch will repress the translation of the downstream puroR gene. Thus, upon supplementing the medium with puromycin, these cells will die. In contrast cells unresponsive to miR-302a switch will survive (Fig. 6a). Initially, we optimised the experimental conditions to assess toxicity of the puromycin. We found 2 μg/ml was sufficient to give complete toxicity for both hiPSCs and differentiated mDA cells (Supplementary Fig. 5). Approximately 10 ng of puroR mRNA (in 96-well plate scale) was enough to restore cell viability under 2 μg/mL puromycin. When regulating the puroR translation with miR-302a switch, we observed significant cell toxicity in hiPSCs but not in differentiated mDA neuronal cultures (Supplementary Fig. 5b).

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