Limits...
Quantum dots do not affect the behaviour of mouse embryonic stem cells and kidney stem cells and are suitable for short-term tracking.

Rak-Raszewska A, Marcello M, Kenny S, Edgar D, Sée V, Murray P - PLoS ONE (2012)

Bottom Line: We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types.Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion.Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking.

View Article: PubMed Central - PubMed

Affiliation: Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom. a.rakraszewska@googlemail.com

ABSTRACT
Quantum dots (QDs) are small nanocrystals widely used for labelling cells in order to enable cell tracking in complex environments in vitro, ex vivo and in vivo. They present many advantages over traditional fluorescent markers as they are resistant to photobleaching and have narrow emission spectra. Although QDs have been used effectively in cell tracking applications, their suitability has been questioned by reports showing they can affect stem cell behaviour and can be transferred to neighbouring cells. Using a variety of cellular and molecular biology techniques, we have investigated the effect of QDs on the proliferation and differentiation potential of two stem cell types: mouse embryonic stem cells and tissue-specific stem cells derived from mouse kidney. We have also tested if QDs released from living or dead cells can be taken up by neighbouring cells, and we have determined if QDs affect the degree of cell-cell fusion; this information is critical in order to assess the suitability of QDs for stem cell tracking. We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types. Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion. However, although the QDs have a high labelling efficiency (>85%), they are rapidly depleted from both stem cell populations. Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking.

Show MeSH
Flow cytometric analysis shows that QDs excluded from cells display a poor re-labelling efficiency.A–B) Fluorometer analysis of the medium collected following each of the 4 washes immediately after QD labelling, and of medium collected from KSC (A) and ESC (B) following 48 h of culture. Note that the medium was not filtered, only centrifuged in order to eliminate cells from the suspension; n = 3 for each timepoint. C) QDs (0.134 nM) collected from ESC culture medium following 48 h of initial labelling, labelled 4.45+/−0.12% of a fresh ESC population. D) QDs (0.134 nM) collected from KSC culture medium following 48 h of initial labelling, labelled 2.4+/−0.3% of a fresh KSC population; n = 3 for each experiment.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3293847&req=5

pone-0032650-g005: Flow cytometric analysis shows that QDs excluded from cells display a poor re-labelling efficiency.A–B) Fluorometer analysis of the medium collected following each of the 4 washes immediately after QD labelling, and of medium collected from KSC (A) and ESC (B) following 48 h of culture. Note that the medium was not filtered, only centrifuged in order to eliminate cells from the suspension; n = 3 for each timepoint. C) QDs (0.134 nM) collected from ESC culture medium following 48 h of initial labelling, labelled 4.45+/−0.12% of a fresh ESC population. D) QDs (0.134 nM) collected from KSC culture medium following 48 h of initial labelling, labelled 2.4+/−0.3% of a fresh KSC population; n = 3 for each experiment.

Mentions: To determine if QD depletion was due to cell division, mitomycin C was used to block cell proliferation and the number of QD-labelled cells was determined using flow cytometry. If the decrease in the number of QD-labelled cells was entirely due to signal dilution following cell division, it would be expected that mitomycin C treatment should prevent signal loss. In the presence of mitomycin C, the percentage of QD-labelled KSCs did not significantly decrease between days 1 and 3, leading to the conclusion that cell division was the main reason for the reduction in the number of labelled KSCs (Figure 4C). In contrast, the mitomycin C had a negative impact on ESC resulting in excessive cell death (Figure 5F, Movie S4); therefore, FACS analysis beyond day 1 was not possible (Figure 4D). To confirm that QD loss over time is due to cell division rather than excretion, as suggested by Pi et al. [12], we have investigated the amount of QD in the medium after 2 days in culture using fluorometry. We found that the amount of QD present in the medium after 2 days in culture was negligible (<0.15 nM), suggesting that the QD are not released from cells (Figure 5A and 5B). In order to confirm the above results, time lapse studies were performed of QD-labelled ESC and KSC, both with and without treatment with mitomycin C (MMC). They revealed that over the time-course of the experiment, neither KSC, nor ESC, appeared to excrete QDs (Movie S1, S2 and S3). Moreover, upon MMC-induced cell death of ESC, we did not observe any QD release (Movie S4). Taken together, these results show that QDs are diluted in ESCs and KSCs by cell division and not by QD excretion from cells. However, due to the fact that this depletion is quite rapid, QDs are only suitable for relatively short-term tracking.


Quantum dots do not affect the behaviour of mouse embryonic stem cells and kidney stem cells and are suitable for short-term tracking.

Rak-Raszewska A, Marcello M, Kenny S, Edgar D, Sée V, Murray P - PLoS ONE (2012)

Flow cytometric analysis shows that QDs excluded from cells display a poor re-labelling efficiency.A–B) Fluorometer analysis of the medium collected following each of the 4 washes immediately after QD labelling, and of medium collected from KSC (A) and ESC (B) following 48 h of culture. Note that the medium was not filtered, only centrifuged in order to eliminate cells from the suspension; n = 3 for each timepoint. C) QDs (0.134 nM) collected from ESC culture medium following 48 h of initial labelling, labelled 4.45+/−0.12% of a fresh ESC population. D) QDs (0.134 nM) collected from KSC culture medium following 48 h of initial labelling, labelled 2.4+/−0.3% of a fresh KSC population; n = 3 for each experiment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293847&req=5

pone-0032650-g005: Flow cytometric analysis shows that QDs excluded from cells display a poor re-labelling efficiency.A–B) Fluorometer analysis of the medium collected following each of the 4 washes immediately after QD labelling, and of medium collected from KSC (A) and ESC (B) following 48 h of culture. Note that the medium was not filtered, only centrifuged in order to eliminate cells from the suspension; n = 3 for each timepoint. C) QDs (0.134 nM) collected from ESC culture medium following 48 h of initial labelling, labelled 4.45+/−0.12% of a fresh ESC population. D) QDs (0.134 nM) collected from KSC culture medium following 48 h of initial labelling, labelled 2.4+/−0.3% of a fresh KSC population; n = 3 for each experiment.
Mentions: To determine if QD depletion was due to cell division, mitomycin C was used to block cell proliferation and the number of QD-labelled cells was determined using flow cytometry. If the decrease in the number of QD-labelled cells was entirely due to signal dilution following cell division, it would be expected that mitomycin C treatment should prevent signal loss. In the presence of mitomycin C, the percentage of QD-labelled KSCs did not significantly decrease between days 1 and 3, leading to the conclusion that cell division was the main reason for the reduction in the number of labelled KSCs (Figure 4C). In contrast, the mitomycin C had a negative impact on ESC resulting in excessive cell death (Figure 5F, Movie S4); therefore, FACS analysis beyond day 1 was not possible (Figure 4D). To confirm that QD loss over time is due to cell division rather than excretion, as suggested by Pi et al. [12], we have investigated the amount of QD in the medium after 2 days in culture using fluorometry. We found that the amount of QD present in the medium after 2 days in culture was negligible (<0.15 nM), suggesting that the QD are not released from cells (Figure 5A and 5B). In order to confirm the above results, time lapse studies were performed of QD-labelled ESC and KSC, both with and without treatment with mitomycin C (MMC). They revealed that over the time-course of the experiment, neither KSC, nor ESC, appeared to excrete QDs (Movie S1, S2 and S3). Moreover, upon MMC-induced cell death of ESC, we did not observe any QD release (Movie S4). Taken together, these results show that QDs are diluted in ESCs and KSCs by cell division and not by QD excretion from cells. However, due to the fact that this depletion is quite rapid, QDs are only suitable for relatively short-term tracking.

Bottom Line: We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types.Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion.Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking.

View Article: PubMed Central - PubMed

Affiliation: Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom. a.rakraszewska@googlemail.com

ABSTRACT
Quantum dots (QDs) are small nanocrystals widely used for labelling cells in order to enable cell tracking in complex environments in vitro, ex vivo and in vivo. They present many advantages over traditional fluorescent markers as they are resistant to photobleaching and have narrow emission spectra. Although QDs have been used effectively in cell tracking applications, their suitability has been questioned by reports showing they can affect stem cell behaviour and can be transferred to neighbouring cells. Using a variety of cellular and molecular biology techniques, we have investigated the effect of QDs on the proliferation and differentiation potential of two stem cell types: mouse embryonic stem cells and tissue-specific stem cells derived from mouse kidney. We have also tested if QDs released from living or dead cells can be taken up by neighbouring cells, and we have determined if QDs affect the degree of cell-cell fusion; this information is critical in order to assess the suitability of QDs for stem cell tracking. We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types. Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion. However, although the QDs have a high labelling efficiency (>85%), they are rapidly depleted from both stem cell populations. Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking.

Show MeSH