Characterization and efficacy of PKH26 as a probe to study the replication history of the human hematopoietic KG1a progenitor cell line

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).     

Phototoxicity of the fluorescent membrane dyes PKH2 and PKH26 on the human hematopoietic KG1a progenitor cell line.

BACKGROUND: The phototoxic effects of the well-known fluorescent membrane dyes PKH2 and PKH26 have been unknown, although their use in cell tracking experiments has increased dramatically. To eliminate the phototoxicity-induced alteration in cell function and morphology, it is essential to examine the suspicious phototoxicity of these dyes. METHODS: Chemical and phototoxic effects of PKH dyes on the human hematopoietic KG1a cell line were examined. To minimize phototoxicity in long-term cell tracking experiments lasting up to 18 h with a fluorescence microscope system, time-lapse monitoring with different time intervals and exposure times was introduced. RESULTS: There were no significant effects of the two PKH dyes on cell viability and growth when using dye concentrations up to 5 microM. However, when stained cells were exposed to excitation light, cell viability decreased dramatically, showing the phototoxicity of the PKH dyes. More than 60% of cells stained with 5 microM PKH26 died after 5 min of continuous light exposure. The phototoxic effect was more extensive in cells stained with higher concentrations of the dyes. CONCLUSIONS: We present guidelines for the optimal use of these dyes by using a defined hardware configuration.     

Biologic properties of gadolinium diethylenetriaminepentaacetic acid-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells

Background aimsThis study was conducted to characterize gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA)-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells (HuMSCs) and to track them with magnetic resonance imaging (MRI) in vitro and in vivo.MethodsHuMSCs were isolated from umbilical cords and expanded in vitro. Cells were sequentially labeled with Gd-DTPA and PKH26. The labeling efficiency was determined by spectrophotometry measurements, and the longevity of Gd-DTPA maintenance was measured with MRI. The influence of double labeling on cellular biologic properties was assessed by cell proliferation, viability, differentiation, cycle and apoptosis. Transplantation of double-labeled HuMSCs or placebo was performed in 39 female Sprague-Dawley rats. Leak point pressure and maximal bladder capacity were measured in animals 6 weeks after injection.ResultsThe T1 values and signal intensity on T1-weighted imaging of labeled cells were significantly higher than the control group (P < 0.05). The signal intensity on T1-weighted imaging of labeled cells was retained >14 days in vitro and in vivo. There was no significant difference in the cell cycle, cell apoptosis, cell proliferation and cell viability between labeled and unlabeled HuMSCs (P > 0.05). After double labeling, HuMSCs were still capable of differentiating into osteoblasts and adipocytes. Periurethrally injected HuMSCs in the rats significantly improved leak point pressure and maximal bladder capacity.ConclusionsHuMSCs were successfully labeled with Gd-DTPA and PKH26. This labeling method is reliable and efficient and can be applied for tracking cells in vitro and in vivo without altering cellular biologic properties.     

PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.     

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.     

PKH26标记的人羊膜间充质干细胞在宫腔粘连大鼠中的迁移示踪

为了观察PKH26标记的人羊膜间充质干细胞(hAMSCs)在宫腔粘连大鼠子宫内膜中的迁移情况,文中提取鉴定及PKH26标记hAMSCs,检测PKH26染色剂对hAMSCs生物学特性的影响;利用机械感染双重法建立大鼠宫腔粘连模型并经尾静脉移植PKH26标记的hAMSCs,荧光共聚焦显微镜下观察PKH26标记的hAMSCs移植后在大鼠子宫内膜中的分布情况。结果显示,PKH26染色剂对细胞的活性、周期、凋亡等无明显影响,PKH26标记的阳性细胞主要分布在大鼠受损的子宫内膜中。表明PKH26标记技术是一种安全有效的示踪方法,可用于hAMSCs移植在治疗宫腔粘连时的示踪研究。     

Usefulness of PKHs for studying cell proliferation

Classical methods for proliferative assessment (such as tritiated thymidine or bromodeoxyuridine (BrdUrd) incorporation) need sample fixation. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine the rate and extent of proliferation in cell lines. Flow cytometric analysis associated with modelling software makes it possible to estimate the number of cells having undergone different numbers of cell divisions by sampling the cell population at varying times post-labelling. Two major questions were addressed in these studies, (i) Does PKH26 give a stable and reproducible labelling? (ii) Does labelling with PKH26 alter cellular proliferation characteristics? We conclude that the methods developed here provide a simpler, more complete means for assessment of cell proliferation in patients with hematological malignancies.     

Fluorescent dyes for lymphocyte migration and proliferation studies

Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long-term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, both in vitro and in vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short-term (up to 3 day) lymphocyte migration experiments, namely the DNA-binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.     

In Vitro and In Vivo Magnetic Resonance Tracking of Sinerem-Labeled Human Umbilical Mesenchymal Stromal Cell-Derived Schwann Cells

Tracking of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles-labeled embryonic stem cells, neural stem cells, or adult mesenchymal stem cells in vitro and in vivo by using magnetic resonance (MR) imaging have been reported. However, whether the transdifferentiated cells can be effectively labeled by USPIO has not yet been investigated. The requirement for nerve donor material evokes additional morbidity and inability to generate a sufficiently large number of cells in a short time to hamper the clinic application of Schwann cells (SCs) transplantation. These limitations may be avoided if SCs can be generated from clinically accessible sources, such as bone marrow and umbilical cord. However, a reliable means of inducing the selective differentiation of human mesenchymal stromal cells isolated from the umbilical cord (HUMSCs) into SCs in vitro has not yet been established. In this study, we induce HUMSCs into Schwann-like cells in terms of morphology, phenotype, and function by an improved protocol basing on our previous studies. Furthermore, HUMSCs-derived SCs are labeled efficiently in vitro with ultrasmall superparamagnetic iron oxide contrast agent (USPIO) Sinerem and poly-l-lysine (PLL) without affecting morphology, cell cycle, proliferation, and differentiation ability of the labeled cells between the concentration of 200 to 800 μg/ml. Importantly, when grafted into the intact cerebral cortex and striatum, the survival and migration of these Sinerem-labeled cells were observed using MRI. Our study suggest the effective concentration field for Sinerem use in tracking transdifferentiated HUMSCs, and Sinerem labeling transdifferentiated HUMSCs is feasible, efficient, and safe for MRI tracing following grafting into nervous system.     

Concurrent measurement of the survival of two populations of rabbit platelets labeled with either two PKH lipophilic dyes or two concentrations of biotin

BACKGROUND: To avoid radioisotopic labeling and permit comparison of the survival of two platelet populations concurrently in one animal, we compared simultaneous recoveries and survival times of homologous rabbit platelets labeled in vitro with the lipophilic dyes PKH26 (red fluorescing) and PKH67 (green fluorescing) and with two levels of biotin (low, 1 microg/ml; high, 10 microg/ml). METHODS: Blood samples were drawn up to 96 h postinfusion and analyzed by flow cytometry. Biotin-labeled samples were incubated with phycoerythrin-streptavidin before analysis. RESULTS: Recovery of PKH26-labeled platelets at 1 h was lower (37.5%) than that of PKH67-labeled platelets (47.3%; P < 0.001). Platelet survival times were 62.4 and 61.9 h. Recoveries at 1 h of platelets labeled with two levels of biotin were similar (86.6% and 84.6%) and greater than those of PKH-labeled platelets (P < 0.001). Survival of platelets labeled with biotin did not differ (low, 83.3 h; high, 85.2 h) and was longer than for PKH-labeled platelets (P < 0.01). Labeling methods did not activate platelets (measured by P-selectin expression), nor did they affect platelet responses to adenosine diphosphate (ADP), collagen, or thrombin. CONCLUSIONS: Labeling with two levels of biotin is superior to labeling with PKH dyes, and is useful for measuring concurrently the survival of two differing platelet populations.     

Innocuousness and intracellular distribution of PKH67: A fluorescent probe for cell proliferation assessment

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.     

大鼠脂肪间充质干细胞的鉴定及肝内移植的研究

目的:从脂肪组织中获取间充质干细胞(ADMSCs)并验证其多向分化潜能,探讨ADMSCs在肝再生中的作用。方法:获取大鼠脂肪组织,用胶原酶消化法获取干细胞,并进行体外扩增、传代,取第3代细胞分别用不同诱导培养液进行成骨、成脂诱导,诱导后通过细胞形态学和特殊染色观察诱导效果。用PKH26标记细胞,制作部分肝切除模型,将标记的自体ADMSCs经门静脉植入体内,2周后切下取肝脏制成冰冻切片,荧光显微镜观察植入细胞在肝脏的定位,免疫荧光染色观察其白蛋白的表达。结果:从脂肪组织中分离出的细胞能在体外大量扩增,能被诱导分化为成骨细胞、脂肪细胞,ADMSCs移植2周后,可见PKH26标记细胞散在分布于肝内,免疫荧光染色显示标记细胞白蛋白染色阳性。结论:大鼠脂肪组织中可以获取具有多向分化潜能的间充质干细胞,该细胞在肝再生环境中能向肝细胞分化,参与肝再生。     

Visualizing Endocytotic Pathways at Transmission Electron Microscopy via Diaminobenzidine Photo-Oxidation by a Fluorescent Cell-Membrane Dye

The endocytotic pathway involves a complex, dynamic and interacting system of intracellular compartments. PKH26 is a fluorescent dye specific for long-lasting cell membrane labelling which has been successfully used for investigating cell internalization processes, at either flow cytometry or fluorescence microscopy. In the present work, diaminobenzidine photo-oxidation was tested as a procedure to detect PKH26 dye at transmission electron microscopy. Our results demonstrated that DAB photo-oxidation is a suitable technique to specifically visualise this fluorescent dye at the ultrastructural level: the distribution of the granular dark reaction product perfectly matches the pattern of the fluorescence staining, and the electron density of the fine precipitates makes the signal evident and precisely detectable on the different subcellular compartments involved in the plasma membrane internalization routes.Key words: Endocytosis, PKH26 dye, diaminobenzidine photo-oxidation, transmission electron microscopy     

Comparison of transdifferentiated and untransdifferentiated human umbilical mesenchymal stem cells in rats after traumatic brain injury

Transdifferentiated and untransdifferentiated mesenchymal stem cells (MSCs) have shown therapeutic benefits in central nervous system (CNS) injury. However, it is unclear which would be more appropriate for transplantation. To address this question, we transplanted untransdifferentiated human umbilical mesenchymal stem cells (HUMSCs) and transdifferentiated HUMSCs (HUMSC-derived neurospheres, HUMSC-NSs) into a rat model of traumatic brain injury. Cognitive function, cell survival and differentiation, brain tissue morphology and neurotrophin expression were compared between groups. Significant improvements in cognitive function and brain tissue morphology were seen in the HUMSCs group compared with HUMSC-NSs group, which was accompanied by increased neurotrophin expression. Moreover, only few grafted cells survived in both the HUMSCs and HUMSC-NSs groups, with very few of the cells differentiating into neural-like cells. These findings indicate that HUMSCs are more appropriate for transplantation and their therapeutic benefits may be associated with neuroprotection rather than cell replacement.     

Optimized fluorescent probe combinations for evaluation of proliferation and necrosis in anthracycline-treated leukaemic cell lines

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukaemia (AML). Anthracyclines such as daunorubicin (DNR) are typically used to treat AML and can induce drug resistance. The goal of the studies described here was to select a combination of fluorescent probes that could be used in combination with flow cytometry to monitor cell proliferation vs. cell death/necrosis as a function of anthracycline uptake. Propidium iodide (PI), the most commonly used marker of membrane integrity, cannot be used to evaluate necrosis in DNR-containing cells because of spectral overlap. A membrane integrity probe compatible with the use of a dye dilution method using PKH67 to study cell proliferation was also selected. The results show that DAPI and Cascade Blue (CB), like PI, were able to detect necrotic cells when no DNR was present, although CB gave less resolution between viable and necrotic cells than PI or DAPI. In the presence of DNR, DAPI cannot be used owing to the fluorescence quenching by DNR. However, it was found that a combination of DNR, CB, and PKH67 allows simultaneous identification of chemoresistant cells, based on reduced DNR accumulation, necrotic cells based on CB incorporation, and proliferating cells based on partitioning of PKH67 fluorescence between daughter cells. It was also found that unless a marker of necrosis is used in combination with the dye dilution assay, a moderate decrease of fluorescence as a result of necrosis may be incorrectly interpreted as proliferation.     

人体皮肤成纤维细胞的快速分离及多向分化能力鉴定

目的建立快速分离人皮肤成纤维细胞的方法,并探讨成纤维细胞在成脂、成骨、成软骨和成神经的多向分化潜能。 方法利用组织块培养法分离人体皮肤成纤维细胞,通过形态学观察、流式分析、Vimentin蛋白染色鉴定成纤维细胞;再利用生长曲线、核型分析、线粒体染色分析不同传代细胞的增殖速度,线粒体及染色体形态的改变;最后进行成纤维细胞成脂、成骨、成软骨和成神经的诱导分化实验,鉴定其多向分化潜能。两代细胞增殖速度及线料体相对量的比较采用t检验统计分析。 结果分离的皮肤成纤维细胞呈典型梭状及多角形;高表达细胞表面标记物CD90 （NCF1,NCF2占比分别为99.9%,98.7%）和CD73 （NCF1,NCF2占比分别为98.2%,85.6%）,但极少表达造血干细胞标记物CD34 （NCF1,NCF2占比分别为1.8%,2.6%）;细胞Vimentin蛋白表达呈阳性,阳性率为100%;对细胞生长曲线进行分析,表明分离后不同代次细胞增殖差异无统计学意义（t?= 1.586,P?= 0.1567）;线粒体相对含量统计分析,同一株细胞系第5代（相对荧光强度值：6876±577.8）与第10代（相对荧光强度值：7371±471.9）之间的差异无统计学意义（t?= 0.664,P?= 0.543）;核型分析分别显示传代后保持染色体数目为正常46条且形态无明显异常;经诱导后成纤维细胞可向成脂、成骨、成软骨和类神经分化。 结论利用组织块培养法分离出的人体皮肤成纤维细胞状态稳定,增殖能力强,具有成脂、成骨、成软骨和成神经多向分化诱导潜能,为细胞移植修复骨损伤、软骨损伤和神经损伤性疾病的临床研究提供细胞来源及实验依据。     

PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.     

Wheat germ agglutinin uptake by cytotoxic lymphocytes,a quantitative model of receptor-mediated endocytosis

Summary The binding and uptake of fluorescence labeled wheat germ agglutinin into cytotoxic T-cells was measured by single cell cytophotometric analysis. The intensity of fluorescence in these cells increased continuously over 24 hrs, indicating a permanent turnover of the ligands for WGA. Although the labeling of the cells was intense, no change in the proliferation rate of this interleukin-2 dependent cell line was observed. Therefore no interaction between the interleukin-2 receptor and other receptors regulating the cellular proliferation with the lectin is likely.Abbreviations au arbitary units - CTLL-1 murine cytotoxic interleukin-2 dependent cell line - FCS fetal calf serum - FITC fluoresceinisothiocyanate - HEPES hydroxyethylpiperazine-ethanesulfonic acid - MHC major histocompatibility complex - WGA wheat germ agglutinin