Louie Ye

Endometrial regeneration and endometrial stem/progenitor cells

The functional layer of the human endometrium is a highly regenerative tissue undergoing monthly cycles of growth, differentiation and shedding during a woman’s reproductive years. Fluctuating levels of circulating estrogen and progesterone orchestrate this dramatic remodeling of human endometrium. The thin inactive endometrium of postmenopausal women which resembles the permanent basal layer of cycling endometrium retains the capacity to respond to exogenous sex steroid hormones to regenerate into a thick functional endometrium capable of supporting pregnancy. Endometrial regeneration also follows parturition and endometrial resection. In non menstruating rodents, endometrial epithelium undergoes rounds of proliferation and apoptosis during estrus cycles. The recent identification of adult stem cells in both human and mouse endometrium suggests that epithelial progenitor cells and the mesenchymal stem/stromal cells have key roles in the cyclical regeneration of endometrial epithelium and stroma. This review will summarize the evidence for endometrial stem/progenitor cells, examine their role in mouse models of endometrial epithelial repair and estrogen-induced endometrial regeneration, and also describe the generation of endometrial-like epithelium from human embryonic stem cells. With markers now available for identifying endometrial mesenchymal stem/stromal cells, their possible role in gynecological diseases associated with abnormal endometrial proliferation and their potential application in cell-based therapies to regenerate reproductive and other tissues will be discussed.

Endometrial reconstruction from stem cells

Adult stem cells have been identified in the highly regenerative human endometrium on the basis of their functional attributes. They can reconstruct endometrial tissue in vivo suggesting their possible use in treating disorders associated with inadequate endometrium. The identification of specific markers for endometrial mesenchymal stem cells and candidate markers for epithelial progenitor cells enables the potential use of endometrial stem/progenitor cells in reconstructing endometrial tissue in Asherman syndrome and intrauterine adhesions.

Reepithelialization of the Uterine Surface Arises from Endometrial Glands: Evidence from a Functional Mouse Model of Breakdown and Repair

The human endometrium is highly regenerative undergoing monthly cycles of growth and regression. Endometrial repair after menses is a critical component of the cycle; however, little is understood about the mechanisms behind this rapid process. Adult stem/progenitor cells identified in human and mouse endometrium may be responsible for its remarkable regenerative capacity; however, a functional role for stem/progenitor cells in menstruation is yet to be established. This study aimed to identify label retaining cells as candidate epithelial stem or progenitor cells involved in the rapid reepithelization of the uterine surface in our functional mouse model of endometrial breakdown and repair. Adult mice were pulse labeled with bromodeoxyuridine before endometrial breakdown and repair was induced. Throughout endometrial breakdown and repair, very rapid dilution of bromodeoxyuridine label was observed in the luminal epithelium, whereas label within the glandular epithelium remained constant. Importantly, glandular epithelial cells were shown to proliferate selectively in response to endometrial repair, and the majority strongly expressed estrogen receptor-alpha at this time. This is the first study to demonstrate a functionally diverse response during endometrial repair from the anatomically connected luminal and glandular epithelium and highlights the likelihood that the endometrial glands are the residence of epithelial progenitor cells contributing to reepithelialization of the uterine surface after menses.

Placental-Specific sFLT-1 e15a Protein Is Increased in Preeclampsia, Antagonizes Vascular Endothelial Growth Factor Signaling, and Has Antiangiogenic Activity

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a–specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a.

Placental specific mRNA in the maternal circulation are globally dysregulated in pregnancies complicated by fetal growth restriction.

CONTEXT Fetal growth restriction (FGR) is a leading cause of perinatal mortality, yet no reliable screening test exists. Placental specific mRNA in the maternal circulation may reflect changes in the placental transcriptome in FGR and could be a novel biomarker for FGR. OBJECTIVE The aim of the study was to identify placental specific RNA detectable in the maternal circulation and examine whether they are differentially expressed in severe preterm FGR. DESIGN In silico screening was used to identify placental specific RNAs. Their expression in cases of severe FGR vs controls was examined in both maternal blood and placenta by microarray, RT-PCR, and in situ hybridization. RESULTS Via in silico analysis, we identified 137 genes very highly expressed in the placenta relative to other tissues. Using microarray, we found that they were detectable in the maternal blood and were globally dysregulated with preterm FGR; 75 genes (55%) had a ≥1.5-fold differential expression compared to controls. Eight genes (ERVWE-1, PSG1, PLAC4, TAC3, PLAC3, CRH, CSH1, and KISS1) were validated by RT-PCR to be significantly increased in both maternal blood and placenta in a larger cohort of severe FGR compared to controls. In situ hybridization confirmed PAPPA2 and ERVWE-1 localized to the syncytiotrophoblast. CONCLUSION There is global differential expression of placental specific mRNA in the maternal blood in pregnancies complicated by severe preterm FGR. Placental specific mRNA in maternal blood may represent a new class of biomarkers for preterm FGR.

Generation of Human Female Reproductive Tract Epithelium from Human Embryonic Stem Cells

Background Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT). Methodology/Principal Findings We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1+ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA). Conclusions/Significance These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.

Targeted Nanoparticle Delivery of Doxorubicin Into Placental Tissues to Treat Ectopic Pregnancies

Abnormal trophoblast growth can cause life-threatening disorders such as ectopic pregnancy, choriocarcinoma, and placenta accreta. EnGeneIC Delivery Vehicles (EDVs) are nanocells that can promote tissue-specific delivery of drugs and may be useful to medically treat such disorders. The objective of this study was to determine whether EDVs loaded with the chemotherapeutic doxorubicin and targeting the epidermal growth factor receptor (EGFR, very highly expressed on the placental surface) can regress placental cells in vitro, ex vivo, and in vivo. In female SCID mice, EGFR-targeted EDVs induced greater inhibition of JEG-3 (choriocarcinoma cells) tumor xenografts, compared with EDVs targeting an irrelevant antigen (nontargeted EDVs) or naked doxorubicin. EGFR-targeted EDVs were more readily taken up by human placental explants ex vivo and induced increased apoptosis (M30 antibody) compared with nontargeted EDVs. In vitro, EGFR-targeted EDVs administered to JEG-3 cells resulted in a dose-dependent inhibition of cell viability, proliferation, and increased apoptosis, a finding confirmed by continuous monitoring by xCELLigence. In conclusion, EGFR-targeted EDVs loaded with doxorubicin significantly inhibited trophoblastic tumor cell growth in vivo and in vitro and induced significant cell death ex vivo, potentially mediated by increasing apoptosis and decreasing proliferation. EDVs may be a novel nanoparticle treatment for ectopic pregnancy and other disorders of trophoblast growth.

MMP-15 Is Upregulated in Preeclampsia, but Does Not Cleave Endoglin to Produce Soluble Endoglin

Preeclampsia is a major pregnancy complication, characterized by severe endothelial dysfunction, hypertension and maternal end-organ damage. Soluble endoglin is an anti-angiogenic protein released from placenta and thought to play a central role in causing the endothelial dysfunction and maternal organ injury seen in severe preeclampsia. We recently reported MMP-14 was the protease producing placentally-derived soluble endoglin by cleaving full-length endoglin present on the syncytiotrophoblast surface. This find identifies a specific drug target for severe preeclampsia; interfering with MMP-14 mediated cleavage of endoglin could decrease soluble endoglin production, ameliorating clinical disease. However, experimental MMP-14 inhibition alone only partially repressed soluble endoglin production, implying other proteases might have a role in producing soluble endoglin. Here we investigated whether MMP-15–phylogenetically the closest MMP relative to MMP-14 with 66% sequence similarity–also cleaves endoglin to produce soluble endoglin. MMP-15 was localized to the syncytiotrophoblast layer of the placenta, the same site where endoglin was localized. Interestingly, it was significantly (p = 0.03) up-regulated in placentas from severe early-onset preeclamptic pregnancies (n = 8) compared to gestationally matched preterm controls (n = 8). However, siRNA knockdown of MMP-15 yielded no significant decrease of soluble endoglin production from either HUVECs or syncytialised BeWo cells in vitro. Importantly, concurrent siRNA knockdown of both MMP-14 and MMP-15 in HUVECS did not yield further decrease in soluble endoglin production compared to MMP-14 siRNA alone. We conclude MMP-15 is up-regulated in preeclampsia, but does not cleave endoglin to produce soluble endoglin.

Identification of Label-Retaining Perivascular Cells in a Mouse Model of Endometrial Decidualization, Breakdown, and Repair

ABSTRACT The human endometrium is incredibly dynamic, undergoing monthly cycles of growth and regression during a womans reproductive life. Endometrial repair at the cessation of menstruation is critical for reestablishment of a functional endometrium receptive for embryo implantation; however, little is understood about the mechanisms behind this rapid and highly efficient process. This study utilized a functional mouse model of endometrial breakdown and repair to assess changes in endometrial vasculature that accompany these dynamic processes. Given that adult endometrial stem/progenitor cells identified in human and mouse endometrium are likely contributors to the remarkable regenerative capacity of endometrium, we also assessed label-retaining cells (LRC) as candidate stromal stem/progenitor cells and examined their relationship with endometrial vasculature. Newborn mouse pups were pulse-labeled with bromodeoxyuridine (BrdU) and chased for 5 wk before decidualization, endometrial breakdown, and repair were induced by hormonal manipulation. Mean vessel density did not change significantly throughout breakdown and repair; however, significantly elevated endothelial cell proliferation was observed in decidual tissue. Stromal LRC were identified throughout breakdown and repair, with significantly fewer observed during endometrial repair than before decidualization. A significantly higher percentage of LRC were associated with vasculature during repair than before decidualization, and a proportion were undergoing proliferation, indicative of their functional capacity. This study is the first to examine the endometrial vasculature and candidate stromal stem/progenitor cells in a functional mouse model of endometrial breakdown and repair and provides functional evidence suggesting that perivascular LRC may contribute to endometrial stromal expansion during the extensive remodeling associated with this process.

PAPPA2 is increased in severe early onset pre-eclampsia and upregulated with hypoxia

Severe early onset pre-eclampsia is a serious pregnancy complication, believed to arise as a result of persistent placental hypoxia due to impaired placentation. Pregnancy-associated plasma protein A2 (PAPPA2) is very highly expressed in the placenta relative to all other tissues. There is some evidence that PAPPA2 mRNA and protein are increased in association with pre-eclampsia. The aim of the present study was to characterise the mRNA and protein expression, as well as localisation, of PAPPA2 in an independent cohort of severe early onset pre-eclamptic placentas. We also examined whether exposing placental explants to hypoxia (1% oxygen) changed the expression of PAPPA2. Expression of PAPPA2 mRNA and protein was upregulated in severe early onset pre-eclamptic placentas compared with preterm controls and localised to the syncytiotrophoblast. Interestingly, protein localisation was markedly reduced in term placenta. Syncytialisation of BeWo cells did not change PAPPA2 expression. However, hypoxia upregulated PAPPA2 mRNA and protein expression in primary placental explants. Together, our data suggest that PAPPA2 may be upregulated in severe pre-eclampsia and, functionally, this may be mediated via increased placental hypoxia known to occur with this pregnancy disorder.