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Nanoparticle labeling identifies slow cycling human endometrial stromal cells.

Xiang L, Chan RW, Ng EH, Yeung WS - Stem Cell Res Ther (2014)

Bottom Line: It remains unclear whether slow-cycling cells exist in the human endometrium.They also differentiate into multiple mesenchymal lineages and the expression of lineage specific markers was lower than that of non-FPC.In summary, nanoparticle labeling is a promising tool for the identification of putative somatic stem or progenitor cells when their surface markers are undefined.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Evidence suggests that the human endometrium contains stem or progenitor cells that are responsible for its remarkable regenerative capability. A common property of somatic stem cells is their quiescent state. It remains unclear whether slow-cycling cells exist in the human endometrium. We hypothesized that the human endometrium contains a subset of slow-cycling cells with somatic stem cell properties. Here, we established an in vitro stem cell assay to isolate human endometrial-derived mesenchymal stem-like cells (eMSC).

Methods: Single-cell stromal cultures were initially labeled with fluorescent nanoparticles and a small population of fluorescent persistent cells (FPC) remained after culture of 21 days. Two populations of stromal cells, namely FPC and non-FPC were sorted.

Results: Quantitative analysis of functional assays demonstrated that the FPC had higher colony forming ability, underwent more rounds of self-renewal and had greater enrichment of phenotypically defined prospective eMSC markers: CD146+/CD140b+ and W5C5+ than the non-FPC. They also differentiate into multiple mesenchymal lineages and the expression of lineage specific markers was lower than that of non-FPC. The FPC exhibit low proliferation activities. A proliferation dynamics study revealed that more FPC had a prolonged G1 phase.

Conclusions: With this study we present an efficient method to label and isolate slow-proliferating cells obtained from human endometrial stromal cultures without genetic modifications. The FPC population could be easily maintained in vitro and are of interest for tissue-repair and engineering perspectives. In summary, nanoparticle labeling is a promising tool for the identification of putative somatic stem or progenitor cells when their surface markers are undefined.

No MeSH data available.


Related in: MedlinePlus

Nanoparticle-labeled endometrial stromal cells. Qtracker® label of endometrial stromal cells. Quantitation of nanoparticle-labeled endometrial stromal cells (A) with different duration of chase (n = 4 per time point) and (B) at different menstrual phases (n = 3, proliferative, white; n = 3, secretory, black). Fluorescence expressing cells are reported as means ± SEM of the percentage of total stromal cells seeded. Day 1 post-labeled stromal cells stained with DAPI nuclei stain (blue) indicating the nanoparticles (red) are located in the cytoplasm (C). Representative phase contrast images of nanoparticle-labeled stromal cells at different days in culture (D – H). Nanoparticle-labeled cells (arrows) detected on day 1 (D) and then among the unlabeled stromal cells on day 3 (E), day 6 (F) and day 15 (G). Fluorescent signal retained at day 21 (H) (E - H). Negative controls of unlabeled stromal cells are shown in the insets. Representative photographs of post-labeled day 21 endometrial stromal cells after FACS analysis as FPC (I) and non-FPC (J) population in culture for five days. Culture dish displaying distribution of stromal colony forming units (CFU) (K) after 15 days of culture. Morphology of large (L) and small (M) CFU. Scale bars = 50 μm (C, I, J) and 100 μm (D - H, L - M). DAPI, 4',6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; SEM, standard error of the mean.
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Figure 1: Nanoparticle-labeled endometrial stromal cells. Qtracker® label of endometrial stromal cells. Quantitation of nanoparticle-labeled endometrial stromal cells (A) with different duration of chase (n = 4 per time point) and (B) at different menstrual phases (n = 3, proliferative, white; n = 3, secretory, black). Fluorescence expressing cells are reported as means ± SEM of the percentage of total stromal cells seeded. Day 1 post-labeled stromal cells stained with DAPI nuclei stain (blue) indicating the nanoparticles (red) are located in the cytoplasm (C). Representative phase contrast images of nanoparticle-labeled stromal cells at different days in culture (D – H). Nanoparticle-labeled cells (arrows) detected on day 1 (D) and then among the unlabeled stromal cells on day 3 (E), day 6 (F) and day 15 (G). Fluorescent signal retained at day 21 (H) (E - H). Negative controls of unlabeled stromal cells are shown in the insets. Representative photographs of post-labeled day 21 endometrial stromal cells after FACS analysis as FPC (I) and non-FPC (J) population in culture for five days. Culture dish displaying distribution of stromal colony forming units (CFU) (K) after 15 days of culture. Morphology of large (L) and small (M) CFU. Scale bars = 50 μm (C, I, J) and 100 μm (D - H, L - M). DAPI, 4',6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; SEM, standard error of the mean.

Mentions: Freshly isolated endometrial stromal cells at different menstrual phases were loaded with the Qtracker® dye. The distribution and pattern of the fluorescence signal in the cells were assessed for 21 days. All the cells showed 100% intracellular punctuate red fluorescent signal at 24-hour post-labeling and the nanoparticles accumulated in the perinuclear cytoplasm (Figure 1A, C, D). The nanoparticles did not contribute to adverse effects on proliferation or viability of post-labeled cells (data not included). The percentage of cells declined rapidly to 3.6 ± 0.8% over 15 days of culture (Figure 1A, E, F, G). Only 0.8 ± 0.4% of the endometrial stromal cells retained the fluorescence on D21 post-labeling (Figure 1A, H). These stromal cells retaining the fluorescence are termed FPC. The temporal change in the fluorescence labeling index was similar in the endometrial stromal cells from the proliferative and the secretory phases (Figure 1B). Based on these results, chase periods of 15 and 21 days were selected for comparison of the efficiency in obtaining an enriched population of slow-proliferating cells from the culture.


Nanoparticle labeling identifies slow cycling human endometrial stromal cells.

Xiang L, Chan RW, Ng EH, Yeung WS - Stem Cell Res Ther (2014)

Nanoparticle-labeled endometrial stromal cells. Qtracker® label of endometrial stromal cells. Quantitation of nanoparticle-labeled endometrial stromal cells (A) with different duration of chase (n = 4 per time point) and (B) at different menstrual phases (n = 3, proliferative, white; n = 3, secretory, black). Fluorescence expressing cells are reported as means ± SEM of the percentage of total stromal cells seeded. Day 1 post-labeled stromal cells stained with DAPI nuclei stain (blue) indicating the nanoparticles (red) are located in the cytoplasm (C). Representative phase contrast images of nanoparticle-labeled stromal cells at different days in culture (D – H). Nanoparticle-labeled cells (arrows) detected on day 1 (D) and then among the unlabeled stromal cells on day 3 (E), day 6 (F) and day 15 (G). Fluorescent signal retained at day 21 (H) (E - H). Negative controls of unlabeled stromal cells are shown in the insets. Representative photographs of post-labeled day 21 endometrial stromal cells after FACS analysis as FPC (I) and non-FPC (J) population in culture for five days. Culture dish displaying distribution of stromal colony forming units (CFU) (K) after 15 days of culture. Morphology of large (L) and small (M) CFU. Scale bars = 50 μm (C, I, J) and 100 μm (D - H, L - M). DAPI, 4',6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; SEM, standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230801&req=5

Figure 1: Nanoparticle-labeled endometrial stromal cells. Qtracker® label of endometrial stromal cells. Quantitation of nanoparticle-labeled endometrial stromal cells (A) with different duration of chase (n = 4 per time point) and (B) at different menstrual phases (n = 3, proliferative, white; n = 3, secretory, black). Fluorescence expressing cells are reported as means ± SEM of the percentage of total stromal cells seeded. Day 1 post-labeled stromal cells stained with DAPI nuclei stain (blue) indicating the nanoparticles (red) are located in the cytoplasm (C). Representative phase contrast images of nanoparticle-labeled stromal cells at different days in culture (D – H). Nanoparticle-labeled cells (arrows) detected on day 1 (D) and then among the unlabeled stromal cells on day 3 (E), day 6 (F) and day 15 (G). Fluorescent signal retained at day 21 (H) (E - H). Negative controls of unlabeled stromal cells are shown in the insets. Representative photographs of post-labeled day 21 endometrial stromal cells after FACS analysis as FPC (I) and non-FPC (J) population in culture for five days. Culture dish displaying distribution of stromal colony forming units (CFU) (K) after 15 days of culture. Morphology of large (L) and small (M) CFU. Scale bars = 50 μm (C, I, J) and 100 μm (D - H, L - M). DAPI, 4',6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; SEM, standard error of the mean.
Mentions: Freshly isolated endometrial stromal cells at different menstrual phases were loaded with the Qtracker® dye. The distribution and pattern of the fluorescence signal in the cells were assessed for 21 days. All the cells showed 100% intracellular punctuate red fluorescent signal at 24-hour post-labeling and the nanoparticles accumulated in the perinuclear cytoplasm (Figure 1A, C, D). The nanoparticles did not contribute to adverse effects on proliferation or viability of post-labeled cells (data not included). The percentage of cells declined rapidly to 3.6 ± 0.8% over 15 days of culture (Figure 1A, E, F, G). Only 0.8 ± 0.4% of the endometrial stromal cells retained the fluorescence on D21 post-labeling (Figure 1A, H). These stromal cells retaining the fluorescence are termed FPC. The temporal change in the fluorescence labeling index was similar in the endometrial stromal cells from the proliferative and the secretory phases (Figure 1B). Based on these results, chase periods of 15 and 21 days were selected for comparison of the efficiency in obtaining an enriched population of slow-proliferating cells from the culture.

Bottom Line: It remains unclear whether slow-cycling cells exist in the human endometrium.They also differentiate into multiple mesenchymal lineages and the expression of lineage specific markers was lower than that of non-FPC.In summary, nanoparticle labeling is a promising tool for the identification of putative somatic stem or progenitor cells when their surface markers are undefined.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Evidence suggests that the human endometrium contains stem or progenitor cells that are responsible for its remarkable regenerative capability. A common property of somatic stem cells is their quiescent state. It remains unclear whether slow-cycling cells exist in the human endometrium. We hypothesized that the human endometrium contains a subset of slow-cycling cells with somatic stem cell properties. Here, we established an in vitro stem cell assay to isolate human endometrial-derived mesenchymal stem-like cells (eMSC).

Methods: Single-cell stromal cultures were initially labeled with fluorescent nanoparticles and a small population of fluorescent persistent cells (FPC) remained after culture of 21 days. Two populations of stromal cells, namely FPC and non-FPC were sorted.

Results: Quantitative analysis of functional assays demonstrated that the FPC had higher colony forming ability, underwent more rounds of self-renewal and had greater enrichment of phenotypically defined prospective eMSC markers: CD146+/CD140b+ and W5C5+ than the non-FPC. They also differentiate into multiple mesenchymal lineages and the expression of lineage specific markers was lower than that of non-FPC. The FPC exhibit low proliferation activities. A proliferation dynamics study revealed that more FPC had a prolonged G1 phase.

Conclusions: With this study we present an efficient method to label and isolate slow-proliferating cells obtained from human endometrial stromal cultures without genetic modifications. The FPC population could be easily maintained in vitro and are of interest for tissue-repair and engineering perspectives. In summary, nanoparticle labeling is a promising tool for the identification of putative somatic stem or progenitor cells when their surface markers are undefined.

No MeSH data available.


Related in: MedlinePlus