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Identification of quiescent, stem-like cells in the distal female reproductive tract.

Wang Y, Sacchetti A, van Dijk MR, van der Zee M, van der Horst PH, Joosten R, Burger CW, Grootegoed JA, Blok LJ, Fodde R - PLoS ONE (2012)

Bottom Line: LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation.Upon serum stimulation spheroid cells are induced to differentiate and form glandular structures which express markers of mature műllerian epithelial cells.Overall, the results indicate that quiescent cells located in the distal oviduct have stem-like properties and can differentiate into distinct cell lineages specific of endometrium, proximal and distal oviduct.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics & Gynecology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
In fertile women, the endometrium undergoes regular cycles of tissue build-up and regression. It is likely that uterine stem cells are involved in this remarkable turn over. The main goal of our current investigations was to identify slow-cycling (quiescent) endometrial stem cells by means of a pulse-chase approach to selectively earmark, prospectively isolate, and characterize label-retaining cells (LRCs). To this aim, transgenic mice expressing histone2B-GFP (H2B-GFP) in a Tet-inducible fashion were administered doxycycline (pulse) which was thereafter withdrawn from the drinking water (chase). Over time, dividing cells progressively loose GFP signal whereas infrequently dividing cells retain H2B-GFP expression. We evaluated H2B-GFP retaining cells at different chase time points and identified long-term (LT; >12 weeks) LRCs. The LT-LRCs are negative for estrogen receptor-α and express low levels of progesterone receptors. LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation. Upon serum stimulation spheroid cells are induced to differentiate and form glandular structures which express markers of mature műllerian epithelial cells. Overall, the results indicate that quiescent cells located in the distal oviduct have stem-like properties and can differentiate into distinct cell lineages specific of endometrium, proximal and distal oviduct. Future lineage-tracing studies will elucidate the role played by these cells in homeostasis, tissue injury and cancer of the female reproductive tract in the mouse and eventually in man.

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Pulse-chase experiment using the doxycycline-inducible H2B-GFP system.A schematic representation of the experiment is given in A, where a 7-day treatment with doxycycline (pulse) results in expression of H2B-GFP throughout the entire organism. At different chase time points (12 and 47 weeks after pulse), the GFP signal is progressively diluted from dividing cells and retained in quiescent cells in the distal oviduct (B). In the proximal oviduct (C) and the endometrium (D), label-retaining cells (LRCs) are lost at both 12 and 47 weeks chase time points. FACSorting was performed on single cell digestions of oviducts from different mice. In (E), the GFP signal is plotted against the number of cells. Animals used were: untreated mice as negative controls (black line, Negative); mice pulsed for 7 days (no chase) as positive controls (red line, Pulse); mice pulsed for 7 days and chased for 12 weeks (green line, 12 weeks); and mice pulsed for 7 days and chased for 47 weeks (light-green line, 47 weeks). The scale bar represents 50 μM.
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pone-0040691-g001: Pulse-chase experiment using the doxycycline-inducible H2B-GFP system.A schematic representation of the experiment is given in A, where a 7-day treatment with doxycycline (pulse) results in expression of H2B-GFP throughout the entire organism. At different chase time points (12 and 47 weeks after pulse), the GFP signal is progressively diluted from dividing cells and retained in quiescent cells in the distal oviduct (B). In the proximal oviduct (C) and the endometrium (D), label-retaining cells (LRCs) are lost at both 12 and 47 weeks chase time points. FACSorting was performed on single cell digestions of oviducts from different mice. In (E), the GFP signal is plotted against the number of cells. Animals used were: untreated mice as negative controls (black line, Negative); mice pulsed for 7 days (no chase) as positive controls (red line, Pulse); mice pulsed for 7 days and chased for 12 weeks (green line, 12 weeks); and mice pulsed for 7 days and chased for 47 weeks (light-green line, 47 weeks). The scale bar represents 50 μM.

Mentions: H2B-GFP labeling of the vast majority of uterine cells was observed after 7 days of doxycycline pulse both by immunohistochemistry (IHC; Figure 1) and immunofluorescence (Figure S1A). Notably, the H2B signal appeared much higher in epithelial than in stromal or myometrial cells. In Figure 1, IHC analysis of H2B-GFP after 7 days of doxycycline treatment showed clear and complete epithelial staining in the distal and proximal oviduct, and in the endometrium (Figure 1B–D; left panels). Upon doxycycline withdrawal (chase), it is expected that dividing cells progressively lose their H2B-GFP signal, while quiescent or infrequently dividing cells will retain the label for longer chase periods (Figure 1A). In the endometrium, epithelial cells appeared to completely lose H2B-GFP expression within 2 to 4 weeks, whereas stromal LRCs lost H2B-GFP expression between 8 and 12 weeks of chase (Figure S1). These results are largely in agreement with those by Chan et al. [16] although in our pulse-chase analysis the glandular epithelium appeared to loose its label at a slower rate than the luminal epithelium [16]. In the proximal oviduct no label retaining cells were observed after 12 weeks of chase (Figure 1C). Remarkably however, many LRCs were found after 12 weeks of chase in the distal oviduct (Figure 1B, Figure S2). Furthermore, after an extensive 47 week chase multiple LRCs are still present in the distal oviduct (Figure 1B). Here, we will refer to LRCs persisting for at least 12 weeks of chase and onwards, as long-term label-retaining cells (LT-LRCs).


Identification of quiescent, stem-like cells in the distal female reproductive tract.

Wang Y, Sacchetti A, van Dijk MR, van der Zee M, van der Horst PH, Joosten R, Burger CW, Grootegoed JA, Blok LJ, Fodde R - PLoS ONE (2012)

Pulse-chase experiment using the doxycycline-inducible H2B-GFP system.A schematic representation of the experiment is given in A, where a 7-day treatment with doxycycline (pulse) results in expression of H2B-GFP throughout the entire organism. At different chase time points (12 and 47 weeks after pulse), the GFP signal is progressively diluted from dividing cells and retained in quiescent cells in the distal oviduct (B). In the proximal oviduct (C) and the endometrium (D), label-retaining cells (LRCs) are lost at both 12 and 47 weeks chase time points. FACSorting was performed on single cell digestions of oviducts from different mice. In (E), the GFP signal is plotted against the number of cells. Animals used were: untreated mice as negative controls (black line, Negative); mice pulsed for 7 days (no chase) as positive controls (red line, Pulse); mice pulsed for 7 days and chased for 12 weeks (green line, 12 weeks); and mice pulsed for 7 days and chased for 47 weeks (light-green line, 47 weeks). The scale bar represents 50 μM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404087&req=5

pone-0040691-g001: Pulse-chase experiment using the doxycycline-inducible H2B-GFP system.A schematic representation of the experiment is given in A, where a 7-day treatment with doxycycline (pulse) results in expression of H2B-GFP throughout the entire organism. At different chase time points (12 and 47 weeks after pulse), the GFP signal is progressively diluted from dividing cells and retained in quiescent cells in the distal oviduct (B). In the proximal oviduct (C) and the endometrium (D), label-retaining cells (LRCs) are lost at both 12 and 47 weeks chase time points. FACSorting was performed on single cell digestions of oviducts from different mice. In (E), the GFP signal is plotted against the number of cells. Animals used were: untreated mice as negative controls (black line, Negative); mice pulsed for 7 days (no chase) as positive controls (red line, Pulse); mice pulsed for 7 days and chased for 12 weeks (green line, 12 weeks); and mice pulsed for 7 days and chased for 47 weeks (light-green line, 47 weeks). The scale bar represents 50 μM.
Mentions: H2B-GFP labeling of the vast majority of uterine cells was observed after 7 days of doxycycline pulse both by immunohistochemistry (IHC; Figure 1) and immunofluorescence (Figure S1A). Notably, the H2B signal appeared much higher in epithelial than in stromal or myometrial cells. In Figure 1, IHC analysis of H2B-GFP after 7 days of doxycycline treatment showed clear and complete epithelial staining in the distal and proximal oviduct, and in the endometrium (Figure 1B–D; left panels). Upon doxycycline withdrawal (chase), it is expected that dividing cells progressively lose their H2B-GFP signal, while quiescent or infrequently dividing cells will retain the label for longer chase periods (Figure 1A). In the endometrium, epithelial cells appeared to completely lose H2B-GFP expression within 2 to 4 weeks, whereas stromal LRCs lost H2B-GFP expression between 8 and 12 weeks of chase (Figure S1). These results are largely in agreement with those by Chan et al. [16] although in our pulse-chase analysis the glandular epithelium appeared to loose its label at a slower rate than the luminal epithelium [16]. In the proximal oviduct no label retaining cells were observed after 12 weeks of chase (Figure 1C). Remarkably however, many LRCs were found after 12 weeks of chase in the distal oviduct (Figure 1B, Figure S2). Furthermore, after an extensive 47 week chase multiple LRCs are still present in the distal oviduct (Figure 1B). Here, we will refer to LRCs persisting for at least 12 weeks of chase and onwards, as long-term label-retaining cells (LT-LRCs).

Bottom Line: LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation.Upon serum stimulation spheroid cells are induced to differentiate and form glandular structures which express markers of mature műllerian epithelial cells.Overall, the results indicate that quiescent cells located in the distal oviduct have stem-like properties and can differentiate into distinct cell lineages specific of endometrium, proximal and distal oviduct.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics & Gynecology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
In fertile women, the endometrium undergoes regular cycles of tissue build-up and regression. It is likely that uterine stem cells are involved in this remarkable turn over. The main goal of our current investigations was to identify slow-cycling (quiescent) endometrial stem cells by means of a pulse-chase approach to selectively earmark, prospectively isolate, and characterize label-retaining cells (LRCs). To this aim, transgenic mice expressing histone2B-GFP (H2B-GFP) in a Tet-inducible fashion were administered doxycycline (pulse) which was thereafter withdrawn from the drinking water (chase). Over time, dividing cells progressively loose GFP signal whereas infrequently dividing cells retain H2B-GFP expression. We evaluated H2B-GFP retaining cells at different chase time points and identified long-term (LT; >12 weeks) LRCs. The LT-LRCs are negative for estrogen receptor-α and express low levels of progesterone receptors. LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation. Upon serum stimulation spheroid cells are induced to differentiate and form glandular structures which express markers of mature műllerian epithelial cells. Overall, the results indicate that quiescent cells located in the distal oviduct have stem-like properties and can differentiate into distinct cell lineages specific of endometrium, proximal and distal oviduct. Future lineage-tracing studies will elucidate the role played by these cells in homeostasis, tissue injury and cancer of the female reproductive tract in the mouse and eventually in man.

Show MeSH
Related in: MedlinePlus