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.


Sorting of partially differentiated mDA cells using the miR-302a switch.(a) Schematic of the experimental procedure. hiPSCs were partially differentiated through the mDA differentiation protocol for 4 days and passaged to a new 6-well plate for reverse transfection of miR-302a switch and sorted into 302-pos and 302-neg fractions for RT-qPCR analysis or recultured for later alkaline phosphatase staining. (b) Fold change in miR-302a-5p and 367-3p expression for the 302-neg fraction relative to the 302-pos fraction and normalized with RNU6B expression from snap-frozen cells sorted on day 5 (n = 3 for all groups). (c) Relative RT-qPCR gene expressions from 302-neg to 302-pos fractions and normalized by GADPH from snap-frozen cells sorted on day 5 (n = 3 for all groups). (d) Representative bright-field images of sorted cells recultured in hiPSC medium for a further 7 days and (below) the counted number of ALP-pos colonies. The TRA-1-60-pos and -neg fractions were sorted from non-transfected cells stained with an anti-TRA-1-60 antibody. Error bars represent the SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Sorting of partially differentiated mDA cells using the miR-302a switch.(a) Schematic of the experimental procedure. hiPSCs were partially differentiated through the mDA differentiation protocol for 4 days and passaged to a new 6-well plate for reverse transfection of miR-302a switch and sorted into 302-pos and 302-neg fractions for RT-qPCR analysis or recultured for later alkaline phosphatase staining. (b) Fold change in miR-302a-5p and 367-3p expression for the 302-neg fraction relative to the 302-pos fraction and normalized with RNU6B expression from snap-frozen cells sorted on day 5 (n = 3 for all groups). (c) Relative RT-qPCR gene expressions from 302-neg to 302-pos fractions and normalized by GADPH from snap-frozen cells sorted on day 5 (n = 3 for all groups). (d) Representative bright-field images of sorted cells recultured in hiPSC medium for a further 7 days and (below) the counted number of ALP-pos colonies. The TRA-1-60-pos and -neg fractions were sorted from non-transfected cells stained with an anti-TRA-1-60 antibody. Error bars represent the SEM of three independent experiments.

Mentions: As a proof of principle that our miR-302a switch can be used to make cultures safer for transplantation, we deliberately suspended the mDA differentiation of hiPSC cells at day 4 (cells after 4 days of differentiation contain undifferentiated and/or partially differentiated cells in our mDA induction protocol). These cells were then transfected with miR-302a switch and underwent FACS the following day to separate 302-pos and 302-negative (302-neg) cells (see scheme in Fig. 4a). We then re-cultured an equal number of 302-pos and 302-neg sorted cells to new plates and grew them for a further 7 days in hiPSC-culture medium to promote the growth of any residual harmful cells. We first confirmed the 302-pos and 302-neg sorted fractions from cells on day 5 for RNA analysis. We measured the expression of miR-302a and miR-367 and then expression of key pluripotent-, neuroectoderm- and mDA-associated mRNAs. As expected, the 302-neg sorted fraction contained a reduced level of both miR-302a and miR-367 relative to 302-pos sorted fraction (Fig. 4b). Furthermore, the 302-neg sorted fraction had a lower expression level of the pluripotency marker OCT4, higher expression levels of the neuroectoderm marker PAX6 and of the midbrain marker EN1 (Fig. 4c). After 7 days of continuous culture in hiPSC culture medium, the cells were fixed and stained with an alkaline phosphatase (ALP) kit to detect hiPSC colony formation. This staining is often used to screen for colonies during iPSC reprogramming21. Over three independent sorting experiments, a significant number of ALP+ iPS-like colonies were observed in wells seeded with either non-sorted (5 ± 3 colonies) or 302-pos sorted cells (22 ± 6) (Fig. 4d). In contrast, we saw no colonies in the wells re-cultured with 302-neg sorted cells. For comparison, we also sorted day 5 mDA cells based on TRA-1-60 staining and as above re-cultured for 7 days an equal number of the TRA-1-60-pos and TRA-1-60-neg cells. As expected, wells seeded with TRA-1-60-pos cells had an even higher number of colonies (45 ± 5 colonies). Interestingly, TRA-1-60-neg cells also went on to form ALP+ colonies (21 ± 3 colonies), illustrating the high sensitivity of miR-302a switch, which can be used to remove harmful overgrowing cells.


MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells
Sorting of partially differentiated mDA cells using the miR-302a switch.(a) Schematic of the experimental procedure. hiPSCs were partially differentiated through the mDA differentiation protocol for 4 days and passaged to a new 6-well plate for reverse transfection of miR-302a switch and sorted into 302-pos and 302-neg fractions for RT-qPCR analysis or recultured for later alkaline phosphatase staining. (b) Fold change in miR-302a-5p and 367-3p expression for the 302-neg fraction relative to the 302-pos fraction and normalized with RNU6B expression from snap-frozen cells sorted on day 5 (n = 3 for all groups). (c) Relative RT-qPCR gene expressions from 302-neg to 302-pos fractions and normalized by GADPH from snap-frozen cells sorted on day 5 (n = 3 for all groups). (d) Representative bright-field images of sorted cells recultured in hiPSC medium for a further 7 days and (below) the counted number of ALP-pos colonies. The TRA-1-60-pos and -neg fractions were sorted from non-transfected cells stained with an anti-TRA-1-60 antibody. 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

f4: Sorting of partially differentiated mDA cells using the miR-302a switch.(a) Schematic of the experimental procedure. hiPSCs were partially differentiated through the mDA differentiation protocol for 4 days and passaged to a new 6-well plate for reverse transfection of miR-302a switch and sorted into 302-pos and 302-neg fractions for RT-qPCR analysis or recultured for later alkaline phosphatase staining. (b) Fold change in miR-302a-5p and 367-3p expression for the 302-neg fraction relative to the 302-pos fraction and normalized with RNU6B expression from snap-frozen cells sorted on day 5 (n = 3 for all groups). (c) Relative RT-qPCR gene expressions from 302-neg to 302-pos fractions and normalized by GADPH from snap-frozen cells sorted on day 5 (n = 3 for all groups). (d) Representative bright-field images of sorted cells recultured in hiPSC medium for a further 7 days and (below) the counted number of ALP-pos colonies. The TRA-1-60-pos and -neg fractions were sorted from non-transfected cells stained with an anti-TRA-1-60 antibody. Error bars represent the SEM of three independent experiments.
Mentions: As a proof of principle that our miR-302a switch can be used to make cultures safer for transplantation, we deliberately suspended the mDA differentiation of hiPSC cells at day 4 (cells after 4 days of differentiation contain undifferentiated and/or partially differentiated cells in our mDA induction protocol). These cells were then transfected with miR-302a switch and underwent FACS the following day to separate 302-pos and 302-negative (302-neg) cells (see scheme in Fig. 4a). We then re-cultured an equal number of 302-pos and 302-neg sorted cells to new plates and grew them for a further 7 days in hiPSC-culture medium to promote the growth of any residual harmful cells. We first confirmed the 302-pos and 302-neg sorted fractions from cells on day 5 for RNA analysis. We measured the expression of miR-302a and miR-367 and then expression of key pluripotent-, neuroectoderm- and mDA-associated mRNAs. As expected, the 302-neg sorted fraction contained a reduced level of both miR-302a and miR-367 relative to 302-pos sorted fraction (Fig. 4b). Furthermore, the 302-neg sorted fraction had a lower expression level of the pluripotency marker OCT4, higher expression levels of the neuroectoderm marker PAX6 and of the midbrain marker EN1 (Fig. 4c). After 7 days of continuous culture in hiPSC culture medium, the cells were fixed and stained with an alkaline phosphatase (ALP) kit to detect hiPSC colony formation. This staining is often used to screen for colonies during iPSC reprogramming21. Over three independent sorting experiments, a significant number of ALP+ iPS-like colonies were observed in wells seeded with either non-sorted (5 ± 3 colonies) or 302-pos sorted cells (22 ± 6) (Fig. 4d). In contrast, we saw no colonies in the wells re-cultured with 302-neg sorted cells. For comparison, we also sorted day 5 mDA cells based on TRA-1-60 staining and as above re-cultured for 7 days an equal number of the TRA-1-60-pos and TRA-1-60-neg cells. As expected, wells seeded with TRA-1-60-pos cells had an even higher number of colonies (45 ± 5 colonies). Interestingly, TRA-1-60-neg cells also went on to form ALP+ colonies (21 ± 3 colonies), illustrating the high sensitivity of miR-302a switch, which can be used to remove harmful overgrowing 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.