A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle.

The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ-organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies.

Paracrine Pathways in Uterine Leiomyoma Stem Cells Involve Insulinlike Growth Factor 2 and Insulin Receptor A.

Context: Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. Objective: To define differential gene expression and signaling pathways in leiomyoma cell populations. Design: Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Setting: Research laboratory. Patients: Eight African American women. Interventions: None. Main Outcomes Measures: Gene expression patterns, cell proliferation, and differentiation. Results: A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. Conclusions: IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.

Fenretinide: A Potential Treatment for Endometriosis.

OBJECTIVE: Fenretinide is a synthetic retinoid analogue that promotes apoptosis but has decreased toxicity when compared to other retinoids. We have previously shown that retinoic acid (RA) production in endometriotic tissue is decreased, resulting in reduced estrogen metabolism and apoptotic resistance. We hypothesize fenretinide may induce apoptosis in endometriotic cells and tissues, thereby reducing disease burden. MATERIALS AND METHODS: Primary endometriotic stromal cells were collected, isolated, cultured, and treated with fenretinide in doses from 0 to 20 µmol/L. Cell count, viability, and immunoblots were performed to examine apoptosis. Quantitative reverse transcription-polymerase chain reaction from endometriotic cells treated with fenretinide was used to examine expression of genes involved in RA signaling including stimulated by RA 6 (STRA6), cellular RA binding protein 2 (CRABP2), and fatty acid binding protein 5 (FABP5). Endometriotic tissue was xenografted subcutaneously into the flanks of mice which were treated with fenretinide for 2 weeks, after which the mice were killed and lesion volumes calculated. Statistical analysis was performed using t test and analysis of variance. RESULTS: Treatment with fenretinide significantly decreased total cell count (doses 5-20 µL) and viability (doses 10-20 µmol/L). Fenretinide increased protein levels of the apoptotic marker poly (ADP ribose) polymerase (starting at 10 µmol/L) and decreased proliferation marker proliferating cell nuclear antigen (10 µmol/L, starting at 8-day treatment). Examination of genes involved in retinoid uptake and action showed that treatment induced STRA6 expression while expression of CRABP2 and FABP5 remained unchanged. Fenretinide also significantly decreased the endometriotic lesion xenograft volume. CONCLUSIONS: Fenretinide increases STRA6 expression thereby potentially reversing the pathological loss of retinoid availability. Treatment with this compound induces apoptosis. In vivo treatments decrease lesion volume. Targeting the RA signaling pathway may be a promising novel treatment for women with endometriosis.

Aberrant expression and localization of deoxyribonucleic acid methyltransferase 3B in endometriotic stromal cells.

OBJECTIVE: To define the expression and function of DNA methyltransferases (DNMTs) in response to decidualizing stimuli in endometriotic cells compared with healthy endometrial stroma. DESIGN: Basic science. SETTING: University research center. PATIENT(S): Premenopausal women with or without endometriosis. INTERVENTION(S): Primary cultures of stromal cells from healthy endometrium (E-IUM) or endometriomas (E-OSIS) were subjected to in vitro decidualization (IVD) using 1 µM medroxyprogesterone acetate, 35 nM 17ß-estradiol, and 0.05 mM 8-Br-cAMP. MAIN OUTCOME MEASURE(S): Expression of DNMT1, DNMT3A, and DNMT3B in E-IUM and E-OSIS were assessed by quantitative real-time polymerase chain reaction and immunoblotting. Recruitment of DNMT3B to the promoters of steroidogenic factor 1 (SF-1) and estrogen receptor α (ESR1) was examined by chromatin immunoprecipitation. RESULT(S): IVD treatment reduced DNMT3B messenger RNA (74%) and protein levels (81%) only in E-IUM; DNMT1 and DNMT3A were unchanged in both cell types. Significantly more DNMT3B bound to the SF-1 promoter in E-IUM compared with E-OSIS, and IVD treatment reduced binding in E-IUM to levels similar to those in E-OSIS. Enrichment of DNMT3B across 3 ESR1 promoters was reduced in E-IUM after IVD, although the more-distal promoter showed increased DNMT3B enrichment in E-OSIS after IVD. CONCLUSION(S): The inability to downregulate DNMT3B expression in E-OSIS may contribute to an aberrant epigenetic fingerprint that misdirects gene expression in endometriosis and contributes to its altered response to steroid hormones.

Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo.

CONTEXT: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. OBJECTIVE: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. DESIGN: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. RESULTS: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CONCLUSIONS: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

Paracrine activation of WNT/ß-catenin pathway in uterine leiomyoma stem cells promotes tumor growth.

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/ß-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of ß-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of ß-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/ß-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.