Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres

Fernández-Shaw, S.; Shorter, S.C.; Naish, Caroline; Barlow, David H.; Starkey, Phyllis M.

doi:10.1093/oxfordjournals.humrep.a137609

Cited by 54 publications

(33 citation statements)

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“…Samples were maintained in DMEM-F12, 10% FCS and 1% P/S at 4˚C overnight or processed immediately. Extraction of stromal cells was performed as described in Fernandez-Shaw et al (40) and Zhang et al (41). Briefly, tissue was cut in 2-3 mm pieces with a scalpel and incubated with 1 mg/ml collagenase (Invitrogen; Thermo Fisher Scientific, Inc.) in complete DMEM medium for 2 h at 37˚C.…”

Section: Isolation Of Endometrial Stromal Cells From Tissue Samplesmentioning

confidence: 99%

Quantitative PCR marker genes for endometrial adenocarcinoma

Kölbl

Victor

Birk

et al. 2016

Molecular Medicine Reports

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Abstract. Endometrial adenocarcinoma is a common malignancy in women worldwide, with formation of remote metastasis occurring following oncological treatment. Circulating tumor cells (CTCs) are regarded to be the origin of haematogenous metastasis formation. The present study aimed to identify suitable marker genes using a quantitative polymerase chain reaction (qPCR) approach to detect CTCs from blood samples of patients with endometrial carcinoma. Therefore, RNA was isolated from endometrial adenocarcinoma cell lines and from healthy endometrial tissue and reverse transcribed to cDNA, which was then used in qPCR on a number of marker genes. Cytokeratin 19 and claudin 4 were identified as suitable marker genes for CTCs in endometrial adenocarcinoma, due to their high expression in the majority of the cell lines investigated. The expression values of the genes examined varied widely between the different cell lines, which is similar to the variation in the patient samples. Therefore, the necessity for a set of genes for CTC detection and not one single marker gene is demonstrated. qPCR is a fast, cost-efficient and easy to perform technique, which may be used in the detection of CTCs. Investigation of the occurrence of CTCs in cancer patients would aid in the prevention of metastasis and thereby refine treatment.

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Section: Isolation Of Endometrial Stromal Cells From Tissue Samplesmentioning

confidence: 99%

Quantitative PCR marker genes for endometrial adenocarcinoma

Kölbl

Victor

Birk

et al. 2016

Molecular Medicine Reports

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“…The tissue was chopped finely and incubated at 37 C in 5 ml Dulbecco's modified Eagle's medium (DMEM) containing 0·2% collagenase (type 1A). After 60 min of digestion, filtration through a 250 µm sieve (Nylon Bolting Cloth; Lockortex, Warrington, Cheshire, UK) was carried out to separate the undigested pieces of tissue and the mucous material from the cells, followed by a second filtration through a 40 µm sieve to separate the epithelial cells (predominantly present as glands) from the stromal and red blood cells (single cells) (Fernandez-Shaw et al 1992). The single cells were collected by centrifugation at 300 g and the pellet was resuspended in culture medium (DMEM containing 10% fetal calf serum, 4 mM glutamine and antibiotics).…”

Section: Cell Culturementioning

confidence: 99%

Expression and signalling characteristics of the corticotrophin-releasing hormone receptors during the implantation phase in the human endometrium

Karteris

Papadopoulou²,

Grammatopoulos³

et al. 2004

Journal of Molecular Endocrinology

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Corticotrophin-releasing hormone (CRH) has been identified in several peripheral tissues, including the female reproductive organs. CRH is expressed in the placenta, myometrium, epithelial endometrium and the endometrial stromal cells at all phases of the menstrual cycle. Similarly, CRH receptors are present in pregnant and non-pregnant myometrium, placenta and endometrium. Putative roles of CRH in the endometrium include involvement in implantation, decidualisation and maintenance of pregnancy. In this study we sought to investigate in detail the CRH receptor repertoire expressed in the human endometrium and their signalling characteristics. Using RT-PCR we were able to demonstrate the expression of CRH receptor 1 (CRH-R1 ) and CRH-R2 in the human endometrium. CRH-R1 was present in 40% of endometrial cDNAs examined. No apparent expression of CRH-R2 , CRH-R2 or any other CRH-R1 splice variants was detected. Chemical cross-linking studies with 125 I-ovine CRH revealed that the endometrial CRH receptor has a molecular weight of 45 kDa. Using the non-hydrolysable photoreactive analogue [ -32 P]GTP-azidoanilide and peptide antisera raised against G-protein -subunits, we then studied coupling of endometrial CRH receptors to G proteins. Treatment of endometrial membranes with human CRH (100 nM) increased the labelling of Gq and Gs, but not Gi or Go. These results were supported by experiments in epithelial cells of the non-pregnant human endometrium in the secretory phase which showed that CRH induced increases in both cAMP and inositol trisphosphate levels. These results suggested that CRH may exert multiple effects in the human endometrium via distinct signalling cascades. These events are possibly mediated via different receptor subtypes.

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“…Separation of epithelial and stromal compartments of the endometriii was performed, using a modification of a protocol described previous (Satyaswaroop et al, 1979;Fernandez-Shaw et al, 1992). Brief endometrial tissue was digested in 0.25% collagenase type (Worthington Biochemical Corporation, New Jersey, USA) Dulbecco's modified Eagle's medium (DMEM) for 1 h at 37°S tromal cells were separated from glands by filtration of digested tissue through 40 |im gauze (Lockoertex, Warrington, UK), and enriched by passage of the filtrate through a step wise gradient of 20% over 60 % percoll.…”

Section: Isolation Of Endometrial Stromal and Epithelial Cellsmentioning

confidence: 99%

Endocrinology and paracrinology

et al. 1996

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o whom correspondence should be addressed Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. In rodents HB-EGF has been found to mediate the mrtogenic effects of oestrogen on the endometrial glandular cells and those of progesterone on the stromal cells. HB-EGF has also been found to be expressed at the site of implantation before any other discernible sign of blastocyst attachment in rodents. Our aim was to determine whether HB-EGF mRNA was expressed in the human pregnant and non-pregnant endometrial tissues. Normal endometrial tissues at different stages of the menstrual cycle, first trimester chorionic villi and decidua, second trimester chorionic villi and term placental tissues were snap frozen and stored at -80°C. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyse the expression of HB-EGF mRNA in these tissues. HB-EGF mRNA was found to be expressed in human endometrium throughout the menstrual cycle, ft was also present in first trimester chorionic villi and decidua, second trimester villi and term placenta. These findings suggest that HB-EGF may have a role in the growth or function of the human uterus, and that it may be important at the feto-maternal interface throughout pregnancy.

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Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres

Cited by 54 publications

References 0 publications

Quantitative PCR marker genes for endometrial adenocarcinoma

Quantitative PCR marker genes for endometrial adenocarcinoma

Expression and signalling characteristics of the corticotrophin-releasing hormone receptors during the implantation phase in the human endometrium

Endocrinology and paracrinology

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