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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.

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Co-culture of QD-labelled KSC and KSC-GFP cells showed minimal QD transfer.A–C) Following 24 h co-culture, very few GFP+ cells were labelled with QDs (arrowheads in C); A) QD+ cells (red); GFP+ cells (green); B) phase contrast and fluorescence overlay; C) Zoom of boxed area in B. D) Flow cytometric analysis shows that following 24 h of co-culture, only 3% (+/−0.09%) of cells were GFP+QD+; n = 3.
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pone-0032650-g006: Co-culture of QD-labelled KSC and KSC-GFP cells showed minimal QD transfer.A–C) Following 24 h co-culture, very few GFP+ cells were labelled with QDs (arrowheads in C); A) QD+ cells (red); GFP+ cells (green); B) phase contrast and fluorescence overlay; C) Zoom of boxed area in B. D) Flow cytometric analysis shows that following 24 h of co-culture, only 3% (+/−0.09%) of cells were GFP+QD+; n = 3.

Mentions: Our findings are markedly different from those described in a recent report by Pi et al., where it was shown that QDs excreted by ESCs could re-label >20% of a fresh ESC population [12]. However, in the Pi et al. study, only one wash step was used to remove the QDs from the cell suspension following re-labelling, whereas we routinely perform four washes. It is thus possible that the excess of QD aggregates in the Pi et al. study might have remained stuck to the outside of the cells, leading to false positive results when the cells were analysed using flow cytometry [12]. To investigate if QDs could be transferred to neighbouring cells under direct co-culture conditions, QD-labelled cells were mixed with a population of KSCs that constitutively expressed GFP (KSC-GFP cells). Following 24 h of co-culture, very few GFP+ cells contained QDs (Figure 6A, B and C) and flow cytometric analysis showed that the percentage of QD-labelled GFP+ cells was <4% (Figure 6D). Our findings contrast with those of Ranjbarvarizi et al, who showed 100% transfer efficiency of QDs from labelled cells derived from umbilical cord blood and bone marrow to neighbouring cells in co-culture experiments [11]. It has been reported that the behaviour of QDs inside cells can vary depending on the size of the QDs and their surface chemistry [26]. However, the QDs used in the current study were coated with the same positively charged peptides as those used by Ranjbarvarizi et al. [11]. Furthermore, although the QDs used here (QD 655 nm) were of a different size, this is unlikely to account for the differences in results, as Ranjbarvarizi et al. observed QD transfer to unlabelled cells when both smaller (585 nm) and larger (800 nm) QDs were used. Given that human umbilical cord blood-derived CD34+ cells, such as those used by Ranjbarvarizi et al. [11], display a high incidence of cell-cell fusion in 2D culture [27], then cell-specific fusion may account for the differing observations. Consistent with this notion, it has recently been shown that QDs did not transfer to neighbouring human bone marrow-derived MSCs [10].


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)

Co-culture of QD-labelled KSC and KSC-GFP cells showed minimal QD transfer.A–C) Following 24 h co-culture, very few GFP+ cells were labelled with QDs (arrowheads in C); A) QD+ cells (red); GFP+ cells (green); B) phase contrast and fluorescence overlay; C) Zoom of boxed area in B. D) Flow cytometric analysis shows that following 24 h of co-culture, only 3% (+/−0.09%) of cells were GFP+QD+; n = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032650-g006: Co-culture of QD-labelled KSC and KSC-GFP cells showed minimal QD transfer.A–C) Following 24 h co-culture, very few GFP+ cells were labelled with QDs (arrowheads in C); A) QD+ cells (red); GFP+ cells (green); B) phase contrast and fluorescence overlay; C) Zoom of boxed area in B. D) Flow cytometric analysis shows that following 24 h of co-culture, only 3% (+/−0.09%) of cells were GFP+QD+; n = 3.
Mentions: Our findings are markedly different from those described in a recent report by Pi et al., where it was shown that QDs excreted by ESCs could re-label >20% of a fresh ESC population [12]. However, in the Pi et al. study, only one wash step was used to remove the QDs from the cell suspension following re-labelling, whereas we routinely perform four washes. It is thus possible that the excess of QD aggregates in the Pi et al. study might have remained stuck to the outside of the cells, leading to false positive results when the cells were analysed using flow cytometry [12]. To investigate if QDs could be transferred to neighbouring cells under direct co-culture conditions, QD-labelled cells were mixed with a population of KSCs that constitutively expressed GFP (KSC-GFP cells). Following 24 h of co-culture, very few GFP+ cells contained QDs (Figure 6A, B and C) and flow cytometric analysis showed that the percentage of QD-labelled GFP+ cells was <4% (Figure 6D). Our findings contrast with those of Ranjbarvarizi et al, who showed 100% transfer efficiency of QDs from labelled cells derived from umbilical cord blood and bone marrow to neighbouring cells in co-culture experiments [11]. It has been reported that the behaviour of QDs inside cells can vary depending on the size of the QDs and their surface chemistry [26]. However, the QDs used in the current study were coated with the same positively charged peptides as those used by Ranjbarvarizi et al. [11]. Furthermore, although the QDs used here (QD 655 nm) were of a different size, this is unlikely to account for the differences in results, as Ranjbarvarizi et al. observed QD transfer to unlabelled cells when both smaller (585 nm) and larger (800 nm) QDs were used. Given that human umbilical cord blood-derived CD34+ cells, such as those used by Ranjbarvarizi et al. [11], display a high incidence of cell-cell fusion in 2D culture [27], then cell-specific fusion may account for the differing observations. Consistent with this notion, it has recently been shown that QDs did not transfer to neighbouring human bone marrow-derived MSCs [10].

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