Reconstruction of endometrium from human endometrial side population cell lines

Irene Cervelló, Aymara Mas, Claudia Gil-Sanchis, Laura Peris, Amparo Faus, Philippa T K Saunders, Hilary O D Critchley, Carlos Simón, Irene Cervelló, Aymara Mas, Claudia Gil-Sanchis, Laura Peris, Amparo Faus, Philippa T K Saunders, Hilary O D Critchley, Carlos Simón

Abstract

Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1-7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45⁻) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Morphology and phenotype of endometrial…
Figure 1. Morphology and phenotype of endometrial somatic stem cell lines (ICE lines).
A. Epithelial cell line (ICEp). Upper panel shows aspect of cell growth in hypoxic conditions with typical epithelial features as polygonal/cuboidal shape. Lower panel, expression patterns related flow cytometric analysis confirms positive expression (highlighted in red) of ICEp for epithelial CD9 (76.6%) and for mesenchymal stem cell markers like CD73 (93.3%), CD90 (88.4%) and CD105 (67.8%). Negative expression in ICEp was distinguished for stromal origin (Vimentin), hematopoietic stem cell markers (CD45 and CD34), endothelial cells (CD31), bone marrow stromal (stro-1) and endothelial progenitors (CD133). B. Stromal cell line (ICEs). Upper panel shows the confluence cell culture with fibroblast-like appearance in hypoxic conditions. Lower panel, expression patterns related flow cytometric analysis confirms positive expression (highlighted in red) of ICEs for stromal Vimentin (91.4%) and for mesenchymal stem cell markers like CD73 (48.2%) and CD90 (53.4%). Negative expression in ICEs was distinguished for epithelial origin (CD9), mesenchymal stem cell marker (CD105), hematopoietic stem cell markers (CD45 and CD34), endothelial cells (CD31), bone marrow stromal (stro-1) and endothelial progenitors (CD133). In all the cases mouse FITC-labeled IgG1 (Millipore), FITC-labeled IgG2b (Chemicon), APC-labeled IgG1 (Milteny Biotec), Alexa647-labeled IgM (Biolegend), and PE-labeled IgG1(Abcam) were used as isotypic controls (black) for staining of endometrial somatic stem cell lines (ICEp and ICEs).
Figure 2. Molecular and cytogenetic characterization of…
Figure 2. Molecular and cytogenetic characterization of ICE lines.
A. Results from PCR assay demonstrated the expression of undifferentiated genes in both cell lines (highlighted in figure by asterisk). In addition the absence of typical endoderm and ectoderm markers suggest their commitment to mesenchymal lineage throughout the presence of mesoderm genes. To assess the integrity of the samples analysed GAPDH gene expression was performed. ICEp: Epithelial somatic stem cell line; ICEs: Stromal somatic stem cell line; C+: hESC, human embryonic stem cell line (VAL-9) and C−: water. B. Normal karyotypes 46XX were obtained in both cases. C. Telomerase activity associated with length of telomeres was performed in order to known the undifferentiated status of the cell lines. Telomerase activity of positive control, VAL, showed a ladder of amplification products with six base increments starting at 50 nucleotides. An intermediate telomerase pattern was observed in both cases in comparison with hESC (VAL-9) and differentiated cell line (Fsk).
Figure 3. Immunostaining for ERalpha and progesterone…
Figure 3. Immunostaining for ERalpha and progesterone receptor on cytospins of ICE and control cell lines.
Immunopositive staining for ERalpha was detected in the nuclei of HTB222 cells (lower left panel, insets high magnification and negative control). Immunopositive staining for progesterone receptor was detected in nuclei of Ishikawa cells grown in the presence of E2 (lower left panel, insets high magnification and negative control). In both the positive contol (+ve) cell lines a proportion of the cell nuclei were immunonegative (*). No immunopositive cell nuclei were detected in any of the ICEp or ICEs cell lines tested. All ICE cell cytospins were stained in parallel with the appropriate positive control cell line using identical conditions.
Figure 4. In vitro differentiation of ICE…
Figure 4. In vitro differentiation of ICE lines into mesenchymal lineages.
A. Upper panel, adipogenic differentiation assay was visualised by the presence of Oil Red O staining in lipids vacuoles. Cells non-treated with differentiation media were included as control, being negative for Oil Red staining. Adipocytes from culture explant were included as positive control. Lower box indicated over-expression of lipoprotein lipase at mRNA levels in induced differentiated cells. B. Upper panel, osteogenic differentiation process was detected by the reactivity against bone sialoprotein. Cells non-treated with differentiation media were included as control being negative for bone sialoprotein expression. Osteocytes from culture explant were included as a positive control. Lower box shows over-expression of osterix mRNA in induced differentiated cells.
Figure 5. Reconstruction of human endometrial-like tissue…
Figure 5. Reconstruction of human endometrial-like tissue from ICE lines.
A. Macroscopic and microscopic visualisation of the transplanted site (arrow). H&E staining to assess the presence of endometrial reconstruction in the murine kidney after 60 days of xenotransplantation. B. Pictures showing some results for human vimentin protein expression (green fluorescence signal) in mice treated with P4 and injected with stromal (ICEs) and epithelial+stromal (ICEp+ICEs) cell lines. Nuclei stained with DAPI (blue fluorescence signal) and autofluorescence of kidney cells (red fluorescence signal) are shown. C. Table summarizing the results of the samples analysed. In the first column H&E results concerning visual analysis are assigned as + (reconstructed endometrial tissue), ++ (high appearance of endometrial-like tissue) and +/− (poor observation of reconstructed tissue) in all the mice injected with human cells. Serial markers for immunohistochemistry assays (hVimentin, hCD9, hCD31 and PR) were performed in a subset of mice injected with total endometrium, ICEp, ICEs, ICEp+ICEs, ICEp+P4, ICEs+P4 and ICEp+ICEs+P4. Note positive expression is indicated as + and negative expression as −.
Figure 6. Immunohistochemical analysis of endometrial xenografts…
Figure 6. Immunohistochemical analysis of endometrial xenografts from mice.
Panel summarizing the immunohistochemical analysis for hvimentin, hCD9, hCD31 and PR in endometrial-like tissues obtained after the injection of total endometrial cell suspensions (End = positive control of the cellular injection procedure) treated with E2, putative epithelial somatic stem cell lines from human endometrium (ICEp) treated with E2 and E2+P, putative stromal somatic stem cell lines from human endometrium (ICEs) treated with E2 and E2+P and ICEp+ICEs treated with E2 and E2+P. Human endometrial tissue sections were used as positive controls for all the antibodies described above (upper section of the panel).

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