Figure 1.
Isolation of ESP and EMP cells.
(A) Summary of procedures for the preparation of epithelial-enriched and stromal-enriched fractions and the mixture of both fractions from human cycling endometria. (B) Flow cytometric distribution of ESP cells, EMP cells, and the endometrial replicative population (ERP) in each of the three fractions stained with Hoechst 33342 indicated in Figure S1. Addition of 50 µM reserpine resulted in the disappearance of the ESP fraction (inset in each panel). (C) Proportion of ESP in the whole fraction dissociated from human endometria at different phases of the menstrual cycle. * P<0.01 and ** P<0.00005, versus early proliferative phase. Each bar indicates the mean ± SEM. n = 48.
Figure 2.
Recreation of the human endometrium and its components from ESP cells in immunodeficient mice.
(A) The macroscopic appearance of an ESP-initiated lesion (surrounded by white arrowheads) in the kidney of a NOD/SCID/γcnull (NOG) mouse treated with E2 pellets for 10 weeks. Bar, 1 mm. (B) H&E-stained section of the same lesion indicated in (A). A small box marks a region shown at a higher magnification in the adjacent panel as indicated. Bars, 500 µm (left) and 100 µm (right). (C) H&E-stained and immunofluorescence images of ESP- or EMP-initiated lesions co-stained with DAPI and antibodies against CK and Vm where various endometrial cell components were formed. The right table shows the number and frequency of the ESP- or EMP- initiated mice which predominantly possessed human endometrium with glandular structures, vessel-like structures consisting of endothelial cells, migrating endothelial cells, or stromal cell components. Bars, 500 µm. (D) Immunofluorescence images of serial sections of the ESP-initiated lesion co-stained with DAPI and antibodies against αSMA and hCD31. Note that ESP-initiated vessel-like structures positive for hCD31 and Vm were surrounded by muscle-like layers positive for αSMA and Vm. Bars, 100 µm. (E) Immunofluorescence images of the same lesion as (A) co-stained with DAPI and antibodies against CK and vimentin (Vm). Bars, 500 µm. The borders between the reconstituted tissue and the mouse kidney (MK) are indicated by the dotted lines. (F) Immunofluorescence images of the ESP-initiated lesion co-stained with DAPI and antibodies against human CD31 (hCD31) and Vm in NOG mice. Yellow arrowheads indicate hCD31-positive cells. Bars, 200 µm. (G) Immunofluorescence images of the ESP-initiated lesion co-stained with DAPI and antibodies against PR and Vm. Bars, 50 µm.
Figure 3.
Growth and differentiation potentials of ESP and EMP cells.
(A) Fluorescence images of DiI-labeled ESP cells co-cultured with DiO-labeled EMP cells following Hoechst DNA dye staining. Bars, 200 µm. (B) Cloning efficiencies of ESP cells and EMP cells in EGM-2MV medium. * P<0.001. Each bar indicates the mean ± SEM. N ≥4. (C) Phase contrast micrographs and fluorescence images (insets) of colonies generated from ESP and EMP cultures. The ESP cells and EMP cells were separately seeded at a clonal density, cultured in EGM-2MV medium for two weeks, and subjected to immunofluorescence studies using antibodies against the indicated markers. CK, cytokeratin. Bars, 500 µm.
Figure 4.
Subpopulations of ESP cells: surface markers, and localization in human endometrium.
(A) RT-PCR analyses of various transcripts in ESP and EMP cells before and after two weeks of culture. MDR1, multidrug resistance 1; KDR, kinase insert domain-containing receptor. (B) Flow cytometric analysis of ESP and EMP cells immunostained with isotypic control antibodies or antibodies against endothelial cell surface markers (CD31, CD34, and CD144). (C) An immunofluorescence merged image of human endometrium co-stained with Hoechst and antibodies against ABCG2 and αSMA. White and yellow dotted lines indicate the endometrium-myometrium junction and the luminal surface of the uterine epithelium, respectively. Bars, 200 µm. (D) Immunofluorescence images of human endometrium co-stained with Hoechst and antibodies against hCD31 and ABCG2. A small box marks a region shown at a higher magnification in the adjacent panel as indicated. Yellow arrowheads indicate endothelial cells doubly positive for hCD31 and ABCG2. Bars, 200 µm (upper), 100 µm (lower left) and 50 µm (lower right).
Figure 5.
Proposed model for ESP-driven endometrial regeneration and the establishment and progression of endometriosis.