Hongling Du

Experimental Murine Endometriosis Induces DNA Methylation and Altered Gene Expression in Eutopic Endometrium

Abstract The eutopic endometrium in women with endometriosis demonstrates diminished endometrial receptivity and altered gene expression. It is unknown if the endometrium being defective gives rise to a predisposition toward endometriosis and infertility or, alternatively, if endometriosis causes the altered endometrial receptivity. Here we created experimental endometriosis in mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. Methylation of Hoxa10 was also evaluated as a potential mechanism responsible for altered gene expression. Expression of each gene was measured using quantitative real-time RT-PCR at 14 wk after induction of endometriosis. Expression of Hoxa10 and Hoxa11, which are necessary for endometrial receptivity, were decreased in the endometriosis group. Insulin-like growth factor binding protein-1 (Igfbp1) mRNA was decreased in the endometriosis group. However, there was no change in Integrin beta3 (Itgb3) mRNA expression. Total progesterone receptor (Pgr-AB) was increased in the endometriosis group and the ratio of Pgr-B to Pgr-AB was increased, indicating a shift from Pgr-A to Pgr-B expression. Basic transcription element-binding protein-1 (Bteb1), official symbol and name Klf9, Kruppel-like factor 9, which functionally interacts with Pgr in endometrium, was also decreased in the endometriosis group. In addition, hypermethylation of Hoxa10 in the endometriosis group was shown by methylation-specific PCR and confirmed by bisulfite sequencing. These findings demonstrate that normal endometrium, when placed in an ectopic location to create experimental endometriosis, led to characteristic changes in gene expression in eutopic endometrium. These data suggest the existence of a signal conduction pathway from endometriosis that alters endometrial gene expression through altered Pgr signaling and epigenetic programming.

Endometrial stem cell transplantation restores dopamine production in a Parkinson’s disease model

Parkinson’s disease (PD) is a neurodegenerative disorder caused by the loss of dopaminergic neurons. Adult human endometrial derived stem cells (HEDSC), a readily obtainable type of mesenchymal stem‐like cell, were used to generate dopaminergic cells and for transplantation. Cells expressing CD90, platelet derived growth factor (PDGF)‐Rβ and CD146 but not CD45 or CD31 were differentiated in vitro into dopaminergic neurons that exhibited axon projections, pyramidal cell bodies and dendritic projections that recapitulate synapse formation; these cells also expressed the neural marker nestin and tyrosine hydroxylase, the rate‐limiting enzyme in dopamine synthesis. Whole cell patch clamp recording identified G‐protein coupled inwardly rectifying potassium current 2 channels characteristic of central neurons. A 1‐methyl 4‐phenyl 1,2,3,6‐tetrahydro pyridine induced animal model of PD was used to demonstrate the ability of labelled HEDSC to engraft, migrate to the site of lesion, differentiate in vivo and significantly increase striatal dopamine and dopamine metabolite concentrations. HEDSC are a highly inducible source of allogenic stem cells that rescue dopamine concentrations in an immunocompetent PD mouse model.

Demonstration of Multipotent Stem Cells in the Adult Human Endometrium by In Vitro Chondrogenesis

Stem cells are defined by their unique capacity for self-renewal and multilineage differentiation. Stem cells have been obtained from multiple extramedullary tissues. Recently, a population of progenitor cells have been identified in the endometrium. However, multilineage differentiation of endometrial stem cells has not been reported.Endometrial tissue was obtained from reproductive-aged women undergoing surgery for benign disease, from which monolayer endometrial stromal cell (ESC), myometrial, fibroid, fallopian tube, and uterosacral ligament tissue cultures were generated. Once confluent, cells were trypsinized and centrifuged in conical tubes to form a cell pellet. Cell pellets were cultured in a defined chondrogenic media (CM) containing dexamethasone and transforming growth factor (TGF)—β2 or TGF-β3 for 3 to 21 days. Samples were analyzed for markers of human articular cartilage, including sulfated glycosaminoglycans and expression of type II collagen. ESC pellets cultured in CM were found to contain cells that resemble chondrocytes. These cells expressed sulfated glycosaminoglycans and type II collagen typical of human articular cartilage. Myometrial, fibroid, fallopian tube, and uterosacral ligament cells were unable to undergo chondrogenic differentiation using the pellet culture method. Cells derived from the endometrium were able to differentiate into a heterologous cell type: chondrocytes, thus demonstrating the presence of multipotent stem cells. Endometrium is a potential source of multipotent stem cells.

Molecular regulation of müllerian development by Hox genes

Abstract: HOX genes are a family of regulatory molecules that encode conserved transcription factors controlling aspects of morphogenesis and cell differentiation during normal embryonic development. All metazoans possess a common genetic system for embryonic patterning, and this system is also used in the reproductive tract. Hox genes are also expressed in the adult uterus. Hox genes are essential both for the development of müllerian tract in the embryonic period and adult function. Sex steroids regulate Hox gene expression during embryonic and endometrial development in the menstrual cycle. EMX2 and β3‐integrin acting downstream of Hoxa10 gene are likely involved in both these developmental processes. This article reviews the role and molecular regulation of Hox genes in reproductive tract development.

Ischemia/Reperfusion Injury Promotes and Granulocyte-Colony Stimulating Factor Inhibits Migration of Bone Marrow-Derived Stem Cells to Endometrium

The endometrium is a dynamic tissue that undergoes repeated rounds of regeneration in each reproductive (estrous or menstrual) cycle. We have previously shown that bone marrow (BM)-derived stem cells engraft the endometrium in rodents and humans; however, it is not known if these cells contribute physiologically to uterine cyclic regeneration or alternatively are primarily involved in uterine repair in response to injury. Here we performed male-to-female BM transplant and tested the ability of uterine injury to recruit BM-derived cells to endometrium in the presence and absence of sex steroids. Uterine ischemia/reperfusion injury resulted in an ~2-fold increase in BM-derived stem cell recruitment to the endometrium. The effect was independent of sex steroids or the existence of an estrous cycle. BM-derived mesenchymal stem cells (MSCs) are involved in uterine repair after injury, but not the cyclic regeneration of the endometrium in the estrous/menstrual cycle. Granulocyte-colony stimulating factor (G-CSF) is used to increase BM mobilization for transplant and has been proposed as a means of mobilizing stem cells to the uterus. Here G-CSF treatment led to decreased BM engraftment of the uterus after injury, likely by favoring mobilization of hematopoietic stem cells over the MSCs. G-CSF is unlikely to be of benefit in repair of uterine injury in humans. Taken together, we demonstrate that ischemic injury drives BM MSC engraftment of the uterus, independent of estrous cycle, sex steroids, or G-CSF.

Stem cells and female reproduction.

Several recent findings in stem cell biology have resulted in new opportunities for the treatment of reproductive disease. Endometrial regeneration can be driven by bone marrow derived stem cells. This finding has potential implications for the treatment of uterine disorders. It also supports a new theory for the etiology of endometriosis. The ovaries have been shown to contain stem cells that form oocytes in adults and can be cultured in vitro to develop mature oocytes. Stem cells from the fetus have been demonstrated to lead to microchimerism in the mother and implicated in several maternal diseases. Additionally the placenta may be another source of hematopoietic stem cell. Finally endometrial derived stem cells have been demonstrated to differentiate into non-reproductive tissues. While we are just beginning to understand stem cells and many key questions remain, the potential advantages of stem cells in reproductive biology and medicine are apparent.

Bone Marrow-Derived Stem Cell (BMDSC) Transplantation Improves Fertility in a Murine Model of Asherman's Syndrome

Ashermans Syndrome is characterized by intrauterine adhesions or fibrosis resulting as a consequence of damage to the basal layer of endometrium and is associated with infertility due to loss of normal endometrium. We have previously shown that bone marrow derived stem cells (BMDSCs) engraft the endometrium in mice and humans and Ischemia/reperfusion injury of uterus promoted BMDSCs migration to the endometrium; however, the role of BMDSCs in Ashermans syndrome has not been characterized. Here a murine model of Ashermans syndrome was created by traumatizing the uterus. We evaluate stem cell recruitment and pregnancy after BMDSCs transplantation in a model of Ashermans syndrome. In the Ashemans syndrome model, after BMDSC transplant, the Y chromosome bearing CD45-cells represented less than 0.1% of total endometrial cells. Twice the number of Y+CD45- cells was identified in the damaged uterus compared to the uninjured controls. There was no significant difference between the damaged and undamaged uterine horns in mice that received injury to a single horn. In the BMDSC transplant group, 9 of the 10 mice conceived, while only 3 of 10 in the non-transplanted group conceived (Chi-Square p = 0.0225); all mice in an uninjured control group conceived. The time to conception and mean litter size were not different between groups. Taken together, BMDSCs are recruited to endometrium in response to injury. Fertility improves after BMDSC transplant in Ashermans Syndrome mice, demonstrating a functional role for these cells in uterine repair. BMDSC transplantation is a potential novel treatment for Ashermans Syndrome and may also be useful to prevent Ashermans syndrome after uterine injury.

Stem cells and reproduction.

Purpose of review To review the latest developments in reproductive tract stem cell biology. Recent findings In 2004, two studies indicated that ovaries contain stem cells which form oocytes in adults and that can be cultured in vitro into mature oocytes. A live birth after orthotopic transplantation of cyropreserved ovarian tissue in a woman whose ovaries were damaged by chemotherapy demonstrates the clinical potential of these cells. In the same year, another study provided novel evidence of endometrial regeneration by stem cells in women who received bone marrow transplants. This finding has potential for the use in treatment of uterine disorders. It also supports a new theory for the cause of endometriosis, which may have its origin in ectopic transdifferentiation of stem cells. Several recent studies have demonstrated that fetal cells enter the maternal circulation and generate microchimerism in the mother. The uterus is a dynamic organ permeable to fetal stem cells, capable of transdifferentiation and an end organ in which bone marrow stem cells may differentiate. Finally stem cell transformation can be an underlying cause of ovarian cancer. Summary Whereas we are just beginning to understand stem cells, the potential implications of stem cells to reproductive biology and medicine are apparent.

Chemoattraction of bone marrow-derived stem cells towards human endometrial stromal cells is mediated by estradiol regulated CXCL12 and CXCR4 expression.

Bone marrow derived cells engraft to the uterine endometrium and contribute to endometriosis. The mechanism by which these cells are mobilized and directed to the endometrium has not been previously characterized. We demonstrate that human endometrial stromal cells (hESCs) produce the chemokine CXCL12 and that bone marrow cells (BMCs) express the CXCL12 receptor, CXCR4. Treatment with physiological levels of estradiol (E2) induced both CXCL12 and CXCR4 expression in hESCs and BMCs, respectively. BMCs migrated towards hESCs conditioned media; a CXCR4 antagonist blocked migration indicating that CXCL12 acting through its receptor, CXCR4, is necessary for chemoattraction of BM cells to human endometrial cells. E2 increased both CXCL12 expression in endometrial cells and CXCR4 expression in BM cells, further enhancing chemoattraction. E2 induced CXCL12/CXCR4 expression in endometrium and BM, respectively, drives migration of stem cells to the endometrium. The E2-CXCL12/CXCR4 signaling pathway may be useful in determining treatments for endometrial disorders, and may be antagonized to block stem cell migration to endometriosis.

The Role of Hox Genes in Female Reproductive Tract Development, Adult Function, and Fertility

HOX genes convey positional identity that leads to the proper partitioning and adult identity of the female reproductive track. Abnormalities in reproductive tract development can be caused by HOX gene mutations or altered HOX gene expression. Diethylstilbestrol (DES) and other endocrine disruptors cause Müllerian defects by changing HOX gene expression. HOX genes are also essential regulators of adult endometrial development. Regulated HOXA10 and HOXA11 expression is necessary for endometrial receptivity; decreased HOXA10 or HOXA11 expression leads to decreased implantation rates. Alternation of HOXA10 and HOXA11 expression has been identified as a mechanism of the decreased implantation associated with endometriosis, polycystic ovarian syndrome, leiomyoma, polyps, adenomyosis, and hydrosalpinx. Alteration of HOX gene expression causes both uterine developmental abnormalities and impaired adult endometrial development that prevent implantation and lead to female infertility.