Bone marrow-derived cells from male donors do not contribute to the endometrial side population of the recipient

Abstract

Accumulated evidence demonstrates the existence of bone marrow-derived cells origin in the endometria of women undergoing bone marrow transplantation (BMT). In these reports, cells of a bone marrow (BM) origin are able to differentiate into endometrial cells, although their contribution to endometrial regeneration is not yet clear. We have previously demonstrated the functional relevance of side population (SP) cells as the endogenous source of somatic stem cells (SSC) in the human endometrium. The present work aims to understand the presence and contribution of bone marrow-derived cells to the endometrium and the endometrial SP population of women who received BMT from male donors. Five female recipients with spontaneous or induced menstruations were selected and their endometrium was examined for the contribution of XY donor-derived cells using fluorescent in situ hybridization (FISH), telomapping and SP method investigation. We confirm the presence of XY donor-derived cells in the recipient endometrium ranging from 1.7% to 2.62%. We also identify 0.45-0.85% of the donor-derived cells in the epithelial compartment displaying CD9 marker, and 1.0-1.83% of the Vimentin-positive XY donor-derived cells in the stromal compartment. Although the percentage of endometrial SP cells decreased, possibly being due to chemotherapy applied to these patients, they were not formed by XY donor-derived cells, donor BM cells were not associated with the stem cell (SC) niches assessed by telomapping technique, and engraftment percentages were very low with no correlation between time from transplant and engraftment efficiency, suggesting random terminal differentiation. In conclusion, XY donor-derived cells of a BM origin may be considered a limited exogenous source of transdifferentiated endometrial cells rather than a cyclic source of BM donor-derived stem cells.

Figure 1. Detection and quantification of XY cells from donor origin in the endometria of BMT recipients.

(A&B) Human endometrial sections from 2 different patients, who underwent BMT, showing a positive FISH signal corresponding to chromosome Y (green signal) and chromosome X (orange signal) along both the epithelial and stromal compartments. Presence of donor-derived cells (chromosome Y-positive cells) in the endometrium is indicated by arrows. Lower panels containing control slides: (A) endometrium as a positive control for the chromosome X signal and (B) fetal testes as a positive control for the chromosome XY signal. (C) Table summarizing the characteristics of the recipients, the percentages of XY-positive cells obtained in the whole endometrium, plus the epithelial and stromal fractions. All the images were acquired using a 63×/1.6× oil immersion objective.

Figure 2. Immunophenotyping of the XY donor-derived cells in the endometria of transplanted patients.

(A) XY donor-derived cells (indicated by arrows, orange and green signals) in the absence of the hematopoietic marker CD45+ (blue signal) in the recipients' endometria. Lower panel: spleen as a positive control for CD45 (blue). (B) Co-localization of XY donor-derived cells (indicated by arrows) with epithelial marker CD9 (blue) in recipients' endometria indicate the coexistence of XY+CD9+ cells in epithelial glands. Lower panel: epithelial endometrium as a positive control for CD9 (blue). (C) Co-localization of XY donor-derived cells (indicated by arrows) with stromal marker Vm (blue) in recipients' endometria, indicated by the overlapping of the XY+Vm+ expression in the stromal fraction. Lower panel: stromal endometrium as a positive control for Vm (blue). All the images were acquired using a 63×/1.7× oil immersion objective with a confocal microscope. Note that DAPI signals are shown in gray in all the images.

Figure 3. Analysis of the telomere length regions in the endometria of BMT recipients.

Assessment of telomere length in the endometrial sections from BMT recipients: note that patients 1 and 2 are represented. A color code on the left-hand side of the image symbolizes the degree of colors corresponding to telomeric length, where long and short telomeres are represented in red and blue, respectively. Also note how the regions associated with long telomeres are located mainly in the stromal compartment.

Figure 4. Isolation of endometrial SP cells and FISH analyses from BMT recipients.

(A) Table indicating the percentages of freshly endometrial SP cells obtained in all 5 patients by a cell sorter analysis. Percentages are provided for both the epithelial and stromal fractions. (B) Diagram showing a typical SP graph and the results of the FISH analyses of epithelial and stromal SP cells, revealing the absence of XY donor-derived cells.