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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

The cell proliferation activity (A - E) and G1-phase length (F - H) of human endometrial stromal FPC and non-FPC. Representative photographs of (A) unselected stromal cells, (B) FPC and (C) non-FPC morphology in the proliferation assay at day 5. Cell proliferation assessment on unselected endometrial stromal cells (grey), clonally derived FPC (white) and non-FPC (black) after 15 days in culture using (D) trypan blue assay and (E) CyQUANT NF cell proliferation assay. The absorbance unit (AU) was measured at a wavelength of 480 nm with a reference wavelength of 530 nm. Scale bar: 100 μM. Cells were obtained from the same patient. Results are reported as means ± SEM, n = 3, *P <0.05. Quantification of the G1 length in Cdt-1-expressing stromal cells of FPC and non-FPC populations followed by time-lapse microscopy within a period of 42 hours. The distribution of G1-phase for (F) unselected stromal cells (n = 20 cells), (G) FPC (n = 20 cells) and (H) non-FPC (n = 18 cells) from three patient samples. FPC, fluorescent persistent cells; SEM, standard error of the mean.
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Figure 4: The cell proliferation activity (A - E) and G1-phase length (F - H) of human endometrial stromal FPC and non-FPC. Representative photographs of (A) unselected stromal cells, (B) FPC and (C) non-FPC morphology in the proliferation assay at day 5. Cell proliferation assessment on unselected endometrial stromal cells (grey), clonally derived FPC (white) and non-FPC (black) after 15 days in culture using (D) trypan blue assay and (E) CyQUANT NF cell proliferation assay. The absorbance unit (AU) was measured at a wavelength of 480 nm with a reference wavelength of 530 nm. Scale bar: 100 μM. Cells were obtained from the same patient. Results are reported as means ± SEM, n = 3, *P <0.05. Quantification of the G1 length in Cdt-1-expressing stromal cells of FPC and non-FPC populations followed by time-lapse microscopy within a period of 42 hours. The distribution of G1-phase for (F) unselected stromal cells (n = 20 cells), (G) FPC (n = 20 cells) and (H) non-FPC (n = 18 cells) from three patient samples. FPC, fluorescent persistent cells; SEM, standard error of the mean.

Mentions: The proliferative activity was assessed using two assays. By day 5, more unselected endometrial stromal cells were depicted under the microscope than FPC (Figure 4B) and non-FPC (Figure 4C) populations. This was supported by the quantitative trypan blue assay (Figure 4D), which showed remarkably higher mean cell count for the unselected endometrial stromal cells (29.26 × 104 ± 4.64 × 104) than the clonally derived FPC on day 15 (2.96 × 104 ± 7.41 × 104, P <0.05, n = 3). The mean cell count for the endometrial stromal non-FPC was 20.27 × 104 ± 0.74 × 104 and was not different from that of the unselected endometrial stromal cells but higher than that of FPC. A similar finding was obtained with the fluorescence-based assay by measuring the DNA content of the cultured cells (Figure 4E). The absorbance was significantly higher for the unselected endometrial stromal cells (3.58 × 104 ± 0.18 × 104 AU) than the FPC (1.94 × 104 ± 0.43 × 104 AU, P < 0.05, n = 3). No difference was found between the absorbance for the endometrial stromal non-FPC (3.43 × 104 ± 0.61 × 104 AU) and the unselected endometrial stromal cells. Collectively, these findings together with the passaging efficiency demonstrate the enrichment of slow-proliferating cells in the endometrial stromal FPC subset.


Nanoparticle labeling identifies slow cycling human endometrial stromal cells.

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

The cell proliferation activity (A - E) and G1-phase length (F - H) of human endometrial stromal FPC and non-FPC. Representative photographs of (A) unselected stromal cells, (B) FPC and (C) non-FPC morphology in the proliferation assay at day 5. Cell proliferation assessment on unselected endometrial stromal cells (grey), clonally derived FPC (white) and non-FPC (black) after 15 days in culture using (D) trypan blue assay and (E) CyQUANT NF cell proliferation assay. The absorbance unit (AU) was measured at a wavelength of 480 nm with a reference wavelength of 530 nm. Scale bar: 100 μM. Cells were obtained from the same patient. Results are reported as means ± SEM, n = 3, *P <0.05. Quantification of the G1 length in Cdt-1-expressing stromal cells of FPC and non-FPC populations followed by time-lapse microscopy within a period of 42 hours. The distribution of G1-phase for (F) unselected stromal cells (n = 20 cells), (G) FPC (n = 20 cells) and (H) non-FPC (n = 18 cells) from three patient samples. FPC, fluorescent persistent cells; 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 4: The cell proliferation activity (A - E) and G1-phase length (F - H) of human endometrial stromal FPC and non-FPC. Representative photographs of (A) unselected stromal cells, (B) FPC and (C) non-FPC morphology in the proliferation assay at day 5. Cell proliferation assessment on unselected endometrial stromal cells (grey), clonally derived FPC (white) and non-FPC (black) after 15 days in culture using (D) trypan blue assay and (E) CyQUANT NF cell proliferation assay. The absorbance unit (AU) was measured at a wavelength of 480 nm with a reference wavelength of 530 nm. Scale bar: 100 μM. Cells were obtained from the same patient. Results are reported as means ± SEM, n = 3, *P <0.05. Quantification of the G1 length in Cdt-1-expressing stromal cells of FPC and non-FPC populations followed by time-lapse microscopy within a period of 42 hours. The distribution of G1-phase for (F) unselected stromal cells (n = 20 cells), (G) FPC (n = 20 cells) and (H) non-FPC (n = 18 cells) from three patient samples. FPC, fluorescent persistent cells; SEM, standard error of the mean.
Mentions: The proliferative activity was assessed using two assays. By day 5, more unselected endometrial stromal cells were depicted under the microscope than FPC (Figure 4B) and non-FPC (Figure 4C) populations. This was supported by the quantitative trypan blue assay (Figure 4D), which showed remarkably higher mean cell count for the unselected endometrial stromal cells (29.26 × 104 ± 4.64 × 104) than the clonally derived FPC on day 15 (2.96 × 104 ± 7.41 × 104, P <0.05, n = 3). The mean cell count for the endometrial stromal non-FPC was 20.27 × 104 ± 0.74 × 104 and was not different from that of the unselected endometrial stromal cells but higher than that of FPC. A similar finding was obtained with the fluorescence-based assay by measuring the DNA content of the cultured cells (Figure 4E). The absorbance was significantly higher for the unselected endometrial stromal cells (3.58 × 104 ± 0.18 × 104 AU) than the FPC (1.94 × 104 ± 0.43 × 104 AU, P < 0.05, n = 3). No difference was found between the absorbance for the endometrial stromal non-FPC (3.43 × 104 ± 0.61 × 104 AU) and the unselected endometrial stromal cells. Collectively, these findings together with the passaging efficiency demonstrate the enrichment of slow-proliferating cells in the endometrial stromal FPC subset.

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