Isolation of cells from the feto-maternal interface

Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
Current protocols in immunology / edited by John E. Coligan ... [et al.] 04/2012; Chapter 7:Unit 7.40.1-11. DOI: 10.1002/0471142735.im0740s97
Source: PubMed

ABSTRACT The mucosal lining of the human uterus is host to a specialized population of leukocytes, which, during pregnancy, interact with invading placental cells (trophoblast) of fetal origin. Of particular interest are uterine natural killer cells, which account for around 70% of the leukocytes at this site during the first trimester of pregnancy, and seem to be specially adapted to recognize invading trophoblast cells. In order to understand the interactions between mucosal immune cells and trophoblast, and those among the immune cells themselves, it is useful to be able to isolate and culture these cells. Here, we describe protocols for the isolation of leukocytes, stromal cells, and trophoblast cells from the feto-maternal interface.

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Available from: Ashley Moffett, Mar 23, 2015
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    • "Placental villi samples were obtained from women undergoing surgical termination of pregnancy in the first trimester. Primary human trophoblasts were isolated as previously described [18]–[19]. Briefly, the collected placental villi were minced and digested in 0.25% trypsin for 15 minutes with constant shaking. "
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    ABSTRACT: Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05) and increased urokinase (uPA) and matrix metalloproteinases (MMPs) transcripts and their activity (p<0.05) in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.
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    Human Reproduction 09/2013; 28(11). DOI:10.1093/humrep/det355 · 4.57 Impact Factor
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