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Co-culture with endometrial stromal cells enhances the differentiation of human embryonic stem cells into endometrium-like cells.

ABSTRACT: In vitro differentiation of human embryonic stem cells (hESCs) into endometrium-like cells may provide a useful tool for clinical treatment. The aim of the present study was to investigate the differentiation potential of hESCs into endometrium-like cells using three methods, which included induction by feeder cells, co-culture with endometrial stromal cells and induction with embryoid bodies. Following differentiation, the majority of cells positively expressed cytokeratin and epithelial cell adhesion molecule (EPCAM). Factors associated with endometrium cell function, namely the estrogen and progesterone receptors (ER and PR), were also detected. At day 21 following the induction of differentiation, the expression levels of cytokeratin, EPCAM, ER and PR were significantly increased in the co-culture method group, as compared with the other two methods. Furthermore, these cells became decidualized in response to progesterone and prolactin. In addition, the number of cytokeratin-positive or EPCAM-positive cells significantly increased following the induction of differentiation using the co-culture method, as compared with the other two methods. The mRNA expression levels of Wnt members that are associated with endometrial development were subsequently examined, and Wnt5a was found to be significantly upregulated in the differentiated cells induced by feeder cells and co-culture with endometrial stromal cells; however, Wnt4 and Wnt7a expression levels were unaffected. Additionally, the mRNA expression levels of Wnt5a in the differentiated cells co-cultured with endometrial stromal cells were higher when compared with those induced by feeder cells. In conclusion, the present findings indicated that the co-culture system is the optimal protocol for the induction of hESC differentiation into endometrium-like cells, and Wnt5a signaling may be involved in this process.

SUBMITTER: Yu W

PROVIDER: S-EPMC4486790 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Co-culture with endometrial stromal cells enhances the differentiation of human embryonic stem cells into endometrium-like cells.

Yu Wenzhu W Niu Wenbin W Wang Shuna S Chen Xuemei X Sun B O BO Wang Fang F Sun Yingpu Y

Experimental and therapeutic medicine 20150512 1

<i>In vitro</i> differentiation of human embryonic stem cells (hESCs) into endometrium-like cells may provide a useful tool for clinical treatment. The aim of the present study was to investigate the differentiation potential of hESCs into endometrium-like cells using three methods, which included induction by feeder cells, co-culture with endometrial stromal cells and induction with embryoid bodies. Following differentiation, the majority of cells positively expressed cytokeratin and epithelial ...[more]

PMID: 26170910

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Project description:Embryonic stem cells (ESCs), due to their intrinsic capability to generate somatic cells of all three germ layers, are potential sources of neural cells for cell replacement therapies. However, the empirical differentiation protocols and the lack of mechanistic understanding of the neural differentiation of ESCs have limited the utility of ESCs as a developmental model or as a cell source for neural cell populations for replacement therapies. Co-culturing ESCs with stromal cells is one of the extensively used methods to induce neural differentiation. Despite several studies to identify neural inducing factors in stromal cell induced neural differentiation, the self-regulatory effects of ESCs in the neural differentiation process remain unexplored. For the first time, we elucidate the self-regulatory role of mESCs in their neural cell differentiation by supplementing conditioned media from differentiating mESCs to mESC-PA6 co-cultures and quantitatively evaluating the change in neural differentiation. Moreover, we use statistical tools to analyze the expression of various growth and trophic factors and distinguish the factors produced primarily by PA6 cells versus mESCs in co-culture. We observe that addition of the medium containing mESC-secreted factors to a single mESC colony co-cultured with PA6 cells significantly enhances the neural differentiation of mESCs compares to the medium extracted from the stromal cells only. Hierarchical clustering of gene expression data from PA6 and co-cultured mESCs segregates two groups of factors that are produced by the stromal cells and differentiating mESCs. Identifying the major soluble factors that drive and regulate the neural differentiation process in the mESC-PA6 co-culture niche will help understand molecular mechanisms of neural development. Moreover, it can be a major step toward developing novel protocols to differentiate stem cells with mESC derived factor supplementation without using feeder cells and with greater efficiency compared to existing approaches.

Project description:BackgroundThe monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown.MethodsElectrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test.ResultsHuman eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining.ConclusionsOur findings suggest eMSC improves regeneration of injured endometrium in mice.

Project description:Endometrial-factor induced infertility remains one of the most significant pathology among all fertility disorders. Stem cell-based therapy is considered to be the next-generation approach. However, there are still issues about successfully retrieving human endometrium-derived mesenchymal stem/stromal cells (hEnMSCs). Moreover, we need to establish a better understanding of the effect of hEnMSCs on the endometrial recovery and the clinical outcome. According to these challenges we created a multi-step study. Endometrium samples were collected from females undergoing assisted reproductive technology (ART) procedure due to couple infertility. These samples were obtained using an endometrium scratching. The hEnMSCs were isolated from endometrium samples and characterized with flow cytometry analysis. Groups of endometrium injured female mice were established by the mechanical injury to uterine horns and the intraperitoneal chemotherapy. The hEnMSCs suspension was injected to some of the studied female mice at approved time intervals. Histological changes of mice uterine horns were evaluated after Masson's trichrome original staining, hematoxylin and eosin (H&E) staining. The fertility assessment of mice was performed by counting formed embryo implantation sites (ISs). The expression of fibrosis related genes (Col1a1, Col3a1, Acta2, and CD44) was evaluated by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results showed that endometrium scratching is an effective procedure for mesenchymal stem/stromal cells (MSCs) collection from human endometrium. Isolated hEnMSCs met the criteria for defining MSCs. Moreover, hEnMSCs-based therapy had a demonstrably positive effect on the repair of damaged uterine horns, including a reduction of fibrosis, intensity of inflammatory cells such as lymphocytes and polymorphonuclear cells (PMNs) and the number of apoptotic bodies. The injured mice which recieved hEnMSCs had higher fertility in comparison to the untreated mice. Gene expression was reflected in histology changes and outcomes of conception. In conclusion, hEnMSCs demonstrated a positive impact on endometrium restoration and outcomes of endometrial-factor induced infertility. Further exploration is required in order to continue exploring the multifactorial associations between stem cell therapy, gene expression, endometrial changes and reproductive health, so we can identify individually effective and safe treatment strategies for endometrial-factor induced infertility, which is caused by mechanical effect or chemotherapy, in daily clinical practise.

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Co-culture with endometrial stromal cells enhances the differentiation of human embryonic stem cells into endometrium-like cells.

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Co-culture with endometrial stromal cells enhances the differentiation of human embryonic stem cells into endometrium-like cells.

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