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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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Transplantation of miR-302a switch-sorted cells.(a) Schematic of the experiment and representative dot plots of the sorted cells including gating. (b) Number of teratomas formed in mice transplanted with hiPSCs alone (positive control), mDA day 20 cells alone (negative control), Ctrl-miR switch-sorted cells, and miR-302a switch-sorted cells (n = 4 for all groups). (c) Photos and macroscopic inspection of the testes taken from SCID mice transplanted with sorted or unsorted cells. (d) Representative H&E stained cryo-sections illustrate normal testes morphology of SCID mice transplanted with mDA D20 cells alone or spiked mDA D20 cells sorted with miR-302a switch. (e) Representative H&E stained cryo-sections showing the generation of all three germ layers within the biopsied teratomas.
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f5: Transplantation of miR-302a switch-sorted cells.(a) Schematic of the experiment and representative dot plots of the sorted cells including gating. (b) Number of teratomas formed in mice transplanted with hiPSCs alone (positive control), mDA day 20 cells alone (negative control), Ctrl-miR switch-sorted cells, and miR-302a switch-sorted cells (n = 4 for all groups). (c) Photos and macroscopic inspection of the testes taken from SCID mice transplanted with sorted or unsorted cells. (d) Representative H&E stained cryo-sections illustrate normal testes morphology of SCID mice transplanted with mDA D20 cells alone or spiked mDA D20 cells sorted with miR-302a switch. (e) Representative H&E stained cryo-sections showing the generation of all three germ layers within the biopsied teratomas.

Mentions: To investigate whether miR-302a switch could prevent teratoma formation in iPSC-mixed cell populations, we spiked mDA day 20 cultures with hiPSCs and then transfected the cells with either Ctrl-miRNA switch or miR-302a switch. To show that we could effectively remove the spiked hiPSC cells, we transplanted the sorted miR-302-neg cells into the testes of SCID mice for allogeneic engraftment. If any residual hiPSCs remained after sorting, these cells would form teratomas. Teratoma formation containing all cells of the three germ layers is the main standard to prove an iPSC line is truly pluripotent222324. We chose an initial seeding density in 12-well plates of 60,000 spiked hiPSC cells with 240,000 201B7-dervied mDA cells (20% hiPSC) per well to show the strategy can handle a relatively high number of spiked hiPSCs (Fig. 5a). The sorted cells were transplanted into the testes of the animals and left to engraft for 3 months. miR-302a switch sorted cells (302-neg) effectively removed spiked hiPSCs as no teratomas were observed in all of the tested animals (Fig. 5b,c). Also, the shape and histology of the testes were normal (Fig. 5d). In contrast, all animals transplanted with either Ctrl-miR switch sorted cells or with hiPS cells only developed enlarged teratoma formation. Further microscopic observation by Haematoxylin and Eosin (H&E) staining showed these teratomas contained tissues reminiscent of the three main developmental lineages: ectoderm, mesoderm and endoderm (Fig. 5e).


MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells
Transplantation of miR-302a switch-sorted cells.(a) Schematic of the experiment and representative dot plots of the sorted cells including gating. (b) Number of teratomas formed in mice transplanted with hiPSCs alone (positive control), mDA day 20 cells alone (negative control), Ctrl-miR switch-sorted cells, and miR-302a switch-sorted cells (n = 4 for all groups). (c) Photos and macroscopic inspection of the testes taken from SCID mice transplanted with sorted or unsorted cells. (d) Representative H&E stained cryo-sections illustrate normal testes morphology of SCID mice transplanted with mDA D20 cells alone or spiked mDA D20 cells sorted with miR-302a switch. (e) Representative H&E stained cryo-sections showing the generation of all three germ layers within the biopsied teratomas.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Transplantation of miR-302a switch-sorted cells.(a) Schematic of the experiment and representative dot plots of the sorted cells including gating. (b) Number of teratomas formed in mice transplanted with hiPSCs alone (positive control), mDA day 20 cells alone (negative control), Ctrl-miR switch-sorted cells, and miR-302a switch-sorted cells (n = 4 for all groups). (c) Photos and macroscopic inspection of the testes taken from SCID mice transplanted with sorted or unsorted cells. (d) Representative H&E stained cryo-sections illustrate normal testes morphology of SCID mice transplanted with mDA D20 cells alone or spiked mDA D20 cells sorted with miR-302a switch. (e) Representative H&E stained cryo-sections showing the generation of all three germ layers within the biopsied teratomas.
Mentions: To investigate whether miR-302a switch could prevent teratoma formation in iPSC-mixed cell populations, we spiked mDA day 20 cultures with hiPSCs and then transfected the cells with either Ctrl-miRNA switch or miR-302a switch. To show that we could effectively remove the spiked hiPSC cells, we transplanted the sorted miR-302-neg cells into the testes of SCID mice for allogeneic engraftment. If any residual hiPSCs remained after sorting, these cells would form teratomas. Teratoma formation containing all cells of the three germ layers is the main standard to prove an iPSC line is truly pluripotent222324. We chose an initial seeding density in 12-well plates of 60,000 spiked hiPSC cells with 240,000 201B7-dervied mDA cells (20% hiPSC) per well to show the strategy can handle a relatively high number of spiked hiPSCs (Fig. 5a). The sorted cells were transplanted into the testes of the animals and left to engraft for 3 months. miR-302a switch sorted cells (302-neg) effectively removed spiked hiPSCs as no teratomas were observed in all of the tested animals (Fig. 5b,c). Also, the shape and histology of the testes were normal (Fig. 5d). In contrast, all animals transplanted with either Ctrl-miR switch sorted cells or with hiPS cells only developed enlarged teratoma formation. Further microscopic observation by Haematoxylin and Eosin (H&E) staining showed these teratomas contained tissues reminiscent of the three main developmental lineages: ectoderm, mesoderm and endoderm (Fig. 5e).

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