Labeling Mesenchymal Stromal Cells with PKH26 or VybrantDil Significantly Diminishes their Migration, but does not affect their Viability, Attachment, Proliferation and Differentiation Capacities

Kelp, Alexandra; Abruzzese, Tanja; Wöhrle, Svenja; Frajs, Viktoria; Aicher, Wilhelm K.

doi:10.4172/2157-7552.1000199

Cited by 8 publications

(9 citation statements)

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“…However, due to the small size of EVs, their tracking can be challenging and they need to be fluorescently labelled in their native state. Lipophilic labelling with dye molecules such as the PKH family have been widely used to label a range of cell types such as mesenchymal stem cells 38,39 and tumor cells 40 in proliferation and migration studies 39,41 . Since EVs have a lipid bilayer structure similar to that of the cell's plasma membrane, the PKH family has been adapted for EV labelling due to the hydrophobic interactions between the EV lipids and the long alkane tails of PKH.…”

Section: Discussionmentioning

confidence: 99%

Systematic Evaluation of PKH Labelling on Extracellular Vesicle Size by Nanoparticle Tracking Analysis

Dehghani

Gulvin

Flax

et al. 2020

Sci Rep

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flax 3,4 & Thomas R. Gaborski 2,4 ✉ extracellular vesicles (eVs) are membrane vesicles secreted by cells and can modulate biological activities by transferring their content following uptake into recipient cells. Labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. A reliable fluorescent technique needs to preserve the size of EVs since changes in size may alter their uptake and biodistribution. Lipophilic fluorescent dye molecules such as the PKH family have been widely used for EV labelling. Here, the effect of PKH labelling on the size of EVs was systematically evaluated using nanoparticle tracking analysis (NTA), which is a widely used technique for determining the size and concentration of nanoparticles. NTA analysis showed a size increase in all the PKH labelling conditions tested. As opposed to lipophilic dye molecules, no significant shift in the size of labelled EVs was detected with luminal binding dye molecules such as 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, hereinafter CFSE). This finding suggests that PKH labelling may not be a reliable technique for the tracking of EVs.

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Section: Discussionmentioning

confidence: 99%

Systematic Evaluation of PKH Labelling on Extracellular Vesicle Size by Nanoparticle Tracking Analysis

Dehghani

Gulvin

Flax

et al. 2020

Sci Rep

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“…Lipophilic dyes such as the PKH family have been widely used to label a range of cell types such as mesenchymal stem cells [17,32] and tumor cells [33] in proliferation and migration studies [32,34]. Since exosomes have a lipid bilayer structure similar to that of the cell's plasma membrane, PKH dye family have been adapted for exosome labelling.…”

Section: Resultsmentioning

confidence: 99%

Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size

Dehghani

Flax

et al. 2019

Preprint

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Extracellular vesicles (EVs) are membrane vesicles secreted by cells and distributed widely in all biofluids. EVs can modulate the biological activities of cells in a paracrine or endocrine manner, in part by transferring their content, such as miRNA, following uptake in recipient cells. Fluorescent labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. Lipophilic fluorescent dyes such as those in the PKH family have been widely used for EV labelling. One concern with the use of lipophilic dyes is an increase in the EV size. This size shift alone may undermine the validity of EVs tracing studies as small changes in the size of inorganic nanoparticles are known to affect their cellular uptake and biodistribution. Here, the possibility of minimizing the size shift of PKH labelled EVs was systematically studied by changing the labelling condition. Unfortunately, the size shift towards larger particles was observed in all the PKH labelling conditions, including those where the labelled EVs were below the fluorescent detection limit. As opposed to lipophilic dyes, no significant shift in the size of labelled EVs was detected with protein binding dyes. Since the size shifts identified in all the PKH labelling conditions are likely to affect the cellular uptake and biodistribution, PKH may not be a reliable technique for EVs tracking.

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“…In two piglets in the current study, the authors euthanized at 12 h after PKH huMSC administration and were able to visualize cell immunofluorescence, demonstrating a similar time course of cell migration. Such PKH labeling may have, however, diminished the huMSC migration compared with non-labeled huMSCs [41] .…”

Section: Discussionmentioning

confidence: 98%

Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia

et al. 2021

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Background: With therapeutic hypothermia (HT) for neonatal encephalopathy, disability rates are reduced, but not all babies benefit. Pre-clinical rodent studies suggest mesenchymal stromal cells (MSCs) augment HT protection. Aims: The authors studied the efficacy of intravenous (IV) or intranasal (IN) human umbilical cordderived MSCs (huMSCs) as adjunct therapy to HT in a piglet model. Methods: A total of 17 newborn piglets underwent transient cerebral hypoxia-ischemia (HI) and were then randomized to (i) HT at 33.5°C 1À13 h after HI (n = 7), (ii) HT+IV huMSCs (30 £ 10 6 cells) at 24 h and 48 h after HI (n = 5) or (iii) HT+IN huMSCs (30 £ 10 6 cells) at 24 h and 48 h after HI (n = 5). Phosphorus-31 and hydrogen-1 magnetic resonance spectroscopy (MRS) was performed at 30 h and 72 h and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)positive cells and oligodendrocytes quantified. In two further piglets, 30 £ 10 6 IN PKH-labeled huMSCs were administered. Results: HI severity was similar between groups. Amplitude-integrated electroencephalogram (aEEG) recovery was more rapid for HT+IN huMSCs compared with HT from 25 h to 42 h and 49 h to 54 h (P 0.05). MRS phosphocreatine/inorganic phosphate was higher on day 2 in HT+IN huMSCs than HT (P = 0.035). Comparing HT+IN huMSCs with HT and HT+IV huMSCs, there were increased OLIG2 counts in hippocampus (P = 0.011 and 0.018, respectively), internal capsule (P = 0.013 and 0.037, respectively) and periventricular white matter (P = 0.15 for IN versus IV huMSCs). Reduced TUNEL-positive cells were seen in internal capsule with HT+IN huMSCs versus HT (P = 0.05). PKH-labeled huMSCs were detected in the brain 12 h after IN administration. Conclusions: After global HI, compared with HT alone, the authors saw beneficial effects of HT+IN huMSCs administered at 24 h and 48 h (30 £ 10 6 cells/kg total dose) based on more rapid aEEG recovery, improved 31 P MRS brain energy metabolism and increased oligodendrocyte survival at 72 h.

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Labeling Mesenchymal Stromal Cells with PKH26 or VybrantDil Significantly Diminishes their Migration, but does not affect their Viability, Attachment, Proliferation and Differentiation Capacities

Cited by 8 publications

References 0 publications

Systematic Evaluation of PKH Labelling on Extracellular Vesicle Size by Nanoparticle Tracking Analysis

Systematic Evaluation of PKH Labelling on Extracellular Vesicle Size by Nanoparticle Tracking Analysis

Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size

Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia

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