Bone marrow-derived cells (BMDCs) can differentiate into nonhematopoietic cells, suggesting that BMDCs may contribute to the maintenance of multiple tissues. Donor-derived bone marrow cells have been identified in human uterine endometrium. Here, two murine models were used to investigate the contribution of nonendometrial stem cells to endometrium. We investigate whether BMDCs can localize to uterine endometrium and to endometriosis. After bone marrow transplantation, male donor-derived bone marrow cells were found in the uterine endometrium of female mice. Although uncommon (<0.01%), these cells can differentiate into epithelial cells. After generation of experimental endometriosis by ectopic endometrial implantation in the peritoneal cavity, bone marrow from LacZ transgenic mice was used for transplantation. LacZ expressing cells were found in the wild-type ectopic endometrium implanted in the peritoneal cavity of hysterectomized LacZ transgenic mice. The repopulation of endometrium with bone marrow-derived stem cells may be important to normal endometrial physiology and also may help to explain the cellular basis for the high long-term failure of conservative alternatives to hysterectomy. The examination of a sexually dimorphic organ such as the uterus demonstrates the ability of male bone marrow, which cannot harbor circulating endometrial cells, to generate endometrium de novo and proves their mesenchymal stem cell origin. Finding Y chromosome bearing endometrial cells demonstrates the potential to recapitulate embryonic developmental pathways that were never activated in males; BMDCs may have vast regenerative capacity. Additionally, the ability of stem cells to engraft endometriosis has implications for the origin and progression of this disease. Ectopic differentiation of stem cells may be a novel mechanism of disease.
Disclosure of potential conflicts of interest is found at the end of this article.
Regeneration of the endometrium in each reproductive cycle is essential for the continued survival of most mammalian species, including humans. De novo development of endometrial stroma, glands, and vasculature occurs in a predictable fashion, presumably from endogenous stem cells located in the endometrium [1, 2]. Recent evidence has implicated bone marrow-derived cells as possible endometrial progenitors. Bone marrow-derived cells have been identified in the endometrium of women who were bone marrow transplant recipients; these cells appear histologically indistinguishable from endogenous endometrial cells and express markers of glandular and stromal differentiation . It is unknown whether these cells originate from bone marrow mesenchymal stem cells or, alternatively, are circulating endometrial cells originally derived from the endometrium and harbored in bone marrow. These cells, regardless of their origin, may serve as a source of reparative cells for the reproductive tract.
Endometriosis is the development of endometrial tissue outside of the uterus. It is a common medical problem, occurring in 10%–15% of reproductive-age women. Manifestations include pelvic pain and infertility . The traditional explanation for the existence of endometrium in ectopic locations is based on the common occurrence of retrograde menstruation, where endometrial tissue flows out of the fallopian tubes and into the peritoneal cavity [5, 6]. Implants of endometrium likely persist in some individuals. This theory does not account for the existence of endometriosis in areas far removed from the pelvis; endometriosis is reported to occur in locations that do not communicate with the peritoneal cavity, such as the lung and brain, where retrograde menstruation cannot account for the presence of this tissue. The existence of a circulating source of endometrial progenitor cells suggests an alternative theory for the etiology of endometriosis.
The sexually dimorphic reproductive tract provides a unique opportunity to determine whether cells that are derived from bone marrow are truly bone marrow mesenchymal cells or merely transient circulating cells derived from differentiated tissue. We used male-to-female bone marrow transplants to demonstrate that the stem cells colonizing the uterus are not of uterine origin, as a male would not have a circulating source of these uterine cells. Additionally, we demonstrate that bone marrow-derived cells contribute to endometriosis, providing an alternate explanation for the origin of these ectopic cells. Both of these findings have implications for the prevention and treatment of several common reproductive disorders.
Materials and Methods
Ten one-week-old LacZ transgenic mice (strain B6.129S7-Gt(ROSA)26Sor) and an equal number of wild-type C57BL mice were obtained from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) and maintained in the Animal Facility of Yale University School of Medicine. Wild-type mice were used as uterine tissue donors for implantation of ectopic uterine endometrium (experimental endometriosis). C57BL/6 male and female mice were obtained from Charles River Laboratories (Wilmington, MA, http://www.criver.com) and maintained in the Animal Facility of Yale University School of Medicine. They were used for bone marrow transplantation.
Bone Marrow-Derived Cell Isolation and Transplantation
Donor bone marrow was flushed from the femurs, tibias, and humeri of 8–10-week-old male mice with cold sterile phosphate-buffered saline (PBS). The marrow suspension was filtered through sterile 70-μM Nitex mesh (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Ten recipient female mice (8–10-weeks old) were lethally irradiated with two doses of 4.8 Gy 3 hours apart and transplanted with 1 × 107 unfractionated bone marrow cells by tail veil injection within 1 hour of the second irradiation dose . Female mice (10) of the same age were also injected with 1 × 107 unfractionated bone marrow cells into the uterus directly. An equal number of mice were transplanted with female-derived marrow as a control. Vaginal cytology was assessed after transplant to determine ovarian function and estrogen production. Female transplanted mice were sacrificed after 6 months and evaluated by Y chromosome fluorescent in situ hybridization (FISH).
Polymerase Chain Reaction
DNA was extracted from tissue using QIAamp DNA Mini Kit in accordance with the manufacturer's guidelines (Qiagen, Hilden, Germany, http://www1.qiagen.com). Primers used for sex-determining region of the Y chromosome (SRY) polymerase chain reaction (PCR) were 5′-CTGACATCACTGGTGAGCATACAC-3′ (forward) and 5′-AAGCTGTTTGCTGTCTTTGTGC-3′ (reverse). Primers were a generous gift from Dr. Jeffray Sklar (Yale University). To amplify the 397-base-pair region of SRY, high-fidelity Taq polymerase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) was used. Polymerase chain reaction was performed as follows: 5 minutes at 95°C, 40 cycles of 95°C for 45 seconds; 60°C for 30 seconds; 72°C for 45 seconds.
Y FISH and Immunofluorescence
Formalin-fixed paraffin-embedded biopsy specimens were cut into serial sections 3-μm thick, placed on coated slides, and deparaffinized through a series of xylene and ethanol washes. For Y FISH and CD45 and cytokeratin analysis, sections were incubated in BD Biosciences (San Diego, http://www.bdbiosciences.com) Retrievagen A solution for 30 minutes at 100°C and then 20 minutes at room temperature. Y FISH was modified from Donnelly et al.  using a digoxigenin-labeled Y chromosome probe and anti-digoxigenin-rhodamine antibody (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). The digoxigenin-labeled murine Y probe was kindly supplied by Dr. Diane Krause (Yale University). After Y FISH, slides were incubated simultaneously with both 1:20 rat anti-mouse CD45 (BD Biosciences) and 1:100 rat anti-mouse F4/80 (eBioscience Inc., San Diego, http://www.ebioscience.com) at 4°C overnight followed by 1:500 anti-rat-Alexa 488 (Molecular Probes, Eugene, OR, http://www.probes.invitrogen.com) for 1 hour at 37°C [9, –11]. Alternatively, slides were incubated with 1:100 anti-cytokeratin (Z0622; Dako, Glostrup, Denmark, http://www.dako.com) for 1 hour at room temperature followed by 1:500 anti-rabbit-Alexa 647 (Molecular Probes) for 1 hour at 37°C. All slides were coverslipped by using Vectashield/4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Negative and positive controls for Y chromosome consisted of sections of uterus from female-to-female bone marrow (BM) transplants and testis, respectively. Negative and positive control tissues were processed simultaneously in each staining run. For each cell type, at least 150,000 cells were counted.
Endometrial transplants were created as previously described [12, –14]. Briefly, after administration of ketamine xylene by intraperitoneal injection, a vertical incision was made in the abdominal wall and the uterus excised. Segments of ten wild-type mouse uteri were transferred into the peritoneal cavity of previously hysterectomized wild-type mice as a control. Segments of ten LacZ expressing uteri were transplanted ectopically under the peritoneum of non-LacZ producing mice and, similarly, ten non-LacZ uteri were transferred to the peritoneal cavity of ten LacZ transgenic mice. The ovaries were left in situ. After 10 weeks, the ectopic implants were identified and removed. Frozen sections were stained with 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) to distinguish cells originating in the transplanted donor uteri from cells derived from the recipient.
Localization of β-Galactosidase
Ectopic endometrium were fixed in 2% paraformaldehyde, 7.5% sucrose, and 100 mM sodium phosphate (Na2PO4), pH 7.2, overnight at 4°C. After 24 hours, the tissues were washed in 3% sucrose, 100 mM Na2PO4, pH 7.2, for 2 hours, after which they were cryoprotected in 30% sucrose, 100 mM Na2PO4, pH 7.2, for 3 hours. Subsequently, they were frozen in ornithine carbamyl transferase compound on dry ice and sectioned at 10–15 mm on a cryostat. β-Galactosidase expression was identified by immunohistochemistry as described above using a mouse monoclonal anti-β-galactosidase antibody. Alternatively, sections were attached to poly(l-lysine)-coated slides and incubated directly in X-gal. The slides were then rinsed in PBS, mounted with Hydromount (National Diagnostics, Atlanta, https://www.nationaldiagnostics.com), and viewed using a microscope equipped with Nomarski optics [15, 16].
Bone Marrow-Derived Cells Localized to Uterine Endometrium
To confirm that bone marrow-derived stem cells can engraft the uterus, we transplanted 1 × 107 unfractionated bone marrow cells from male donors into female recipients through either the tail vein or uterus. All mice survived the transplant. Daily vaginal cytology was obtained to determine estrogen production in transplanted mice; all showed absent cytologic maturation, indicating lack of cyclic ovarian sex steroid production. Six months after bone marrow transplantation, we attempted to identify the Y chromosome by both FISH and PCR in the recipients' uteri. As shown in Figure 1A, PCR amplification of SRY in uterine samples was seen in female mice transplanted with male bone marrow but not in those transplanted with female bone marrow. In uterine tissue sections, the Y chromosome was visualized with the digoxigenin-labeled murine Y probes. Testis was used as a positive control and demonstrated approximately 85% of cells staining positive for Y chromosome; this is consistent with prior reports and attributed to sectioning artifact as well as inefficiency of hybridization. Cells were counted by two observers blinded to the experimental group. CD45 is a pan-white cell marker used here to distinguish endometrial cells from transient leukocytes present in the endometrium. F4/80 is a macrophage-specific marker simultaneously used to assure the ability to distinguish endometrial cells. We detected 1/5,000 Y chromosome positive (red) and CD45 and F4/80 negative signals in epithelial cells and 1/3,000 Y chromosome positive (red) and CD45 and F4/80 negative signals in stromal cells in female mice with transplanted BM through the tail vein. None of the controls transplanted with female marrow demonstrated a CD45 and F4/80 negative Y chromosome-containing cell. We also did not find any Y chromosome signal or SRY amplification in female uteri that had been infused with BM directly into the uterus.
Bone Marrow-Derived Cells Engraft as Cytokeratin Expressing Cells in Endometrium
To investigate whether bone marrow-derived cells (BMDCs) that engraft endometrium are capable of epithelial cell differentiation, we assayed the uterus for coexpression of cytokeratin and Y chromosome; cytokeratin is a marker of differentiated epithelial cells. As shown in Figure 2, donor-derived cells in the epithelial layer that contain the Y chromosome are also cytokeratin positive, indicating epithelial differentiation.
BMDCs Engraft Ectopic Endometrium
To determine whether bone marrow-derived stem cells can engraft ectopic endometrium, a murine model of endometriosis was created by transplanting segments of the uterus into the peritoneum of previously hysterectomized mice. Here, wild-type uterine segments were transplanted into the peritoneal cavity of LacZ transgenic mice. Uteri derived from wild-type mice, when transplanted into LacZ positive mice, showed cells that expressed LacZ (Fig. 3). β-Galactosidase expression was confirmed in both differentiated epithelial cells and stromal cells. Note the X-gal staining in cells at two stages of differentiation in Figure 3E and 3F, suggesting that the incorporated cell is capable of differentiation. These data were confirmed with isopropyl β-d-thiogalactoside staining as demonstrated in Figure 4. After ten weeks, approximately 0.1% of stromal cells and 0.04% of epithelial cells were of donor origin. A source of uterine cells from outside of the uterus (as all animals were hysterectomized) suggests that cells of extrauterine origin can contribute to ectopic endometrium as well.
Here, we demonstrate that bone marrow-derived stem cells engraft the murine endometrium. Both stromal and epithelial cells were derived from bone marrow origin. These data show the potential for stem cells to have a role in the regeneration or repair of this tissue after injury. However, the small number of engrafted cells limits their potential to significantly contribute to cyclic endometrial function during each estrus cycle. In other organs, the homing and engraftment of stem cells are influenced by injury and inflammation, presumably through the generation of a signal emanating from the damaged tissue [17, –19]. A more significant engraftment of endometrium by bone marrow is likely to occur after endometrial injury or inflammatory insult. Additionally, the proliferation and development of endometrium are entirely regulated by hormonal stimuli. Ovarian estrogen and progesterone drive endometrial growth and apoptosis [20, 21]. As the radiation used prior to bone marrow transplantation typically renders these mice sterile and compromises sex steroid production as demonstrated by vaginal cytology, the endometrium of transplanted mice may be subject to decreased turnover compared with that typical of untreated mice. The recruitment of stem cells to the uterus in hormonally intact animals may be more robust than indicated here.
The examination of a sexually dimorphic organ such as endometrium demonstrates that the engrafted cells are true stem cells; an alternative source of cells that can serve to regenerate any tissue are cells originating in the tissue under study that have entered the circulation. These cells may take up residence in bone marrow or other sites and then serve as a source of progenitor cells. Here, we demonstrate that the cells are not of uterine origin; in males, the primordial mullerian duct degenerates under the influence of mullerian inhibiting factor (MIF) prior to differentiation of endometrial cells. Males therefore cannot harbor circulating endometrial cells. The identification of Y chromosome containing endometrial cells assures that these cells are true stem cells. Additionally, this finding has significant implications for reproductive tract development. Although the Y chromosome results in MIF production, it does not lead to inhibition of the differentiation of stem cells in the adult. Y-driven MIF blocks uterine development during embryonic development but does not prevent the engraftment and differentiation of bone marrow-derived uterine progenitor cells in the adult. The uncoupling of embryonic signaling from adult stem cell differentiation suggests a potential for wide use of these cells. Perhaps multiple cell types can be produced outside the normal embryonic environment in which they normally arise.
Finally, the ectopic stem cell engraftment of endometrium has significant implications for disease. Although it is clear that trafficking of BM-derived circulating progenitor cells may contribute to diseases such as cancer and adiposity [22, 23], ectopic stem cell trafficking may also result in pathology. In a murine model we have demonstrated that cells of nonuterine origin can give rise to endometrial cells in ectopic uterine transplants. Although we have not demonstrated that ectopic differentiation of stem cells causes disease, the data suggest that they can contribute to its progression. The endometrium itself must be capable of signaling to and directing the homing of stem cells despite its ectopic location. Ectopic stem cell engraftment may be a novel mechanism of disease.
The ectopic location of the endometrium is characteristic of endometriosis. Endometriosis is a common disease, occurring in approximately 15% of women, causing infertility and pelvic pain . The predominant theory for the origin of endometriosis is retrograde menstruation through the fallopian tubes with ectopic implantation; however, this cannot explain foci of endometriosis outside the peritoneal cavity [5, 6]. A nonendometrial source of stem cells, which can result in endometrial cells, suggests an alternative origin of some endometriosis. In some instances, endometriosis may arise by differentiation of bone marrow-derived cells into endometrium in ectopic locations. There is an association between endometriosis and immune disorders, perhaps indicating that a single bone marrow disorder may be common to both bone marrow-derived ectopic endometrium and other immune phenomenon [24, –26]. Disorders of bone marrow-derived stem cell homing, engraftment, and differentiation may contribute to disease.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.