To characterize bone marrow contribution to the uterus, we studied the endometrium from human and primate peripheral blood stem cell transplant (PBSCT) models. In a Rhesus macaque long-term stable mixed bone marrow chimerism model after transplantation with autologous CD34+ cells labeled with green fluorescent protein (GFP), a hysterotomy was performed to obtain endometrial tissue. Rare GFP+ cells (indicating transduced hematopoeitic stem cell, or HSC,-derivation) were observed in adherent stromal culture;however, none were double positive for the mesenchymal stem cell marker CD146. Similarly, when bone marrow was examined from this autologous rhesus model, rare GFP+ cells (indicating transduced HSC-derivation) were observed in culture. However, GFP+ cells were not CD146 double positive indicating that donor cells only engrafted in the hematopoietic compartment, but not the stromal compartment of the bone marrow. One transplanted animal was used for immunohistochemistry (IHC). PDGFrbeta (CD140b), an endometrial stem cell marker, did not co-localize with donor-derived (GFP) cells in the endometrium. Donor derived cells residing within the endometrium were not CD45+, which is consistent with previous findings. Two animals were used for fresh digestion and FACS analysis;primary cell cultures were attempted, but none of the GFP+ sorted cells were able to establish a primary cell culture, while GFP- sorted fraction readily established primary cultures. In humans, 14 PBSCT recipients were assessed. Nine of ten endometrial samples were able to generate adherent stromal cells cultures. Endometrial and stromal cell exhibited high levels of mesenchymal stem cell surface markers CD146 and PDGFrbeta. As fresh endometrial and bone marrow samples have blood contamination (which is known to be donor derived), microchimerism testing on these samples would result in a false positive result for donor cells. To verify that culturing stromal cells from endometrium and bone marrow cultures had low levels of donor blood cell contamination, we determined the percentage of cells that were positive for the white blood cell (WBC) marker CD45 by FACS analysis. After confirming that culturing the samples purified the stromal compartment of interest, with low levels of WBC contamination, samples were analyzed for donor microchimerism using PCR to detect percentage of donor and recipient STR sequences. There was 0% donor chimerism of the stromal cells from the endometrial samples as compared to 91.2% chimerism from the peripheral blood cells (p<0.001). Similarly, in the bone marrow cohort, there was 0% donor microchimerism of the stromal cells from the bone marrow samples as compared to 97.3% microchimerism from the peripheral blood cell (positive control) samples (p<0.001). In summary, PBSCT did not result in the engraftment of donor derived endometrial stromal cells. Our results here suggest that the bone marrow does not contribute to the endometrial stem cell niche.

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