E.G. Olivares

Human umbilical cord stromal stem cell express CD10 and exert contractile properties

BACKGROUNDnIt has been demonstrated that human umbilical cord stromal stem cells (UCSSCs) are bio-equivalent to bone marrow mesenchymal stem cells. However, little is known about their tissue origin or in vivo functions, and data on their expansion properties are limited due to early senescence in the culture methods described to date.nnnMETHODSnUC sections and cultured UCSSCs were analyzed with a panel of 12 antibodies. UCSSCs were grown in low-FCS containing medium at 5% or 21% oxygen and were assayed for their clonogenic properties, karyotype stability, expression of specific cellular markers, and multi-lineage potential. UCSSC contractile properties were evaluated by using collagen gel contraction assays under cytokine stimulus.nnnRESULTSnImmunohistochemistry studies showed that the UCSSCs were derived from the Whartons jelly and not from the vascular smooth muscle sheath of the blood vessels. UCSSC growth properties were increased in a 5% oxygen atmosphere in comparison to normoxic culture conditions. In both culture conditions, UCSSCs were CD14-, CD34-, and CD45-negative while expressing high levels of CD73, CD90 and CD105 and maintaining their differentiation potentialities. UCSSCs expressed alpha smooth muscle actin and behaved as functional myofibroblasts when cellular contraction was challenged with appropriate stimuli.nnnCONCLUSIONSnUCSCs are mesenchymal stem cells that reside in the perivascular area of Whartons jelly and are phenotypically and functionally related to myofibroblasts.

Liaison between natural killer cells and dendritic cells in human gestation

A successful pregnancy relies on immunological adaptations that allow the fetus to grow and develop in the uterus, despite being recognized by maternal immune cells. Among several immunocompetent cell types present within the human maternal/fetal interface, DC-SIGN+ dendritic cells (DCs) and CD56+ natural killer (NK) cells are of major importance for early pregnancy maintenance, not only generating maternal immunological tolerance but also regulating stromal cell differentiation. Previous reports show the presence of NK–DC cell conjugates in first trimester human decidua, suggesting that these cells may play a role in the modulation of the local immune response within the uterus. While effective immunity is necessary to protect the mother from harmful pathogens, some form of tolerance must be activated to avoid an immune response against fetal antigens. This review article discusses current evidence concerning the functions of DC and NK cells in pregnancy and their liaison in human decidua.

Fetal–Placental Hypoxia Does Not Result from Failure of Spiral Arterial Modification in Mice

OBJECTIVESnTo determine if fetal-placental hypoxia is a primary outcome of defective spiral artery remodeling.nnnSTUDY DESIGNnPregnancies in Rag2(-/-)Il2rg(-/-) double knock-out mice, which fail to undergo normal physiological spiral arterial remodeling, were compared to syngeneic BALB/c control pregnancies. Mice at gestation day (gd)6, 8, 10, 12 and 18 were infused with Hypoxyprobe-1 before euthanasia to enable detection of cellular hypoxia by immunohistochemistry.nnnRESULTSnIn implantation sites of both phenotypes, trophoblast cells were reactive to Hypoxyprobe-1. No major differences were observed between the phenotypes in decidua or placenta at any gd or in gd18 fetal brain, lung, heart, liver or intestine or in maternal heart, brain, liver or spleen. Maternal kidneys from BALB/c were significantly hypoxic to Rag2(-/-)Il2rg(-/-) kidneys.nnnCONCLUSIONSnIn mice, lack of pregnancy-associated spiral artery remodeling does not impair oxygen delivery to the conceptus, challenging the concept that deficient spiral arterial remodeling leads to fetal hypoxia in human gestational complications such as preeclampsia and fetal growth restriction. The isolated hypoxic response of normal kidney has revealed that renal lymphocytes may have unique, tissue-specific regulatory actions on vasoconstriction that are pregnancy independent.

Expression of the vasoactive proteins AT1, AT2, and ANP by pregnancy-induced mouse uterine natural killer cells.

Angiotensin II receptor type 1 (AT1) activation leads to vasoconstriction and type 2 receptor (AT2) leads to vasodilation. Atrial natriuretic peptide (ANP) antagonizes the effects of AT1. In human and murine pregnancies, uterine natural killer (uNK) cells closely associate with decidual blood vessels. Protein localization of AT1, AT2, and ANP to mouse uNK cells was examined between gestation days (gds) 6 and 12, the interval of uNK cell expansion. Percentages of uNK cells expressing AT1 or AT2 changed between gd6 and gd10. Atrial natriuretic peptide did not localize to uNK cells at gd6 or 8, but did colocalize to uNK cells at gd10 and 12, times immediately after spiral arterial modification. This is the first report of AT1, AT2, and ANP expression in uterine immune cells. Expression of these molecules suggests that uNK cells have the potential to contribute to the changes in blood pressure that occur between days 5 and 12 of pregnancy in mice.

Reduced proportion of decidual DC-SIGN+ cells in human spontaneous abortion

Recent studies showed that some functions of decidual dendritic cells appear to be essential for pregnancy. In humans, decidual dendritic cells are identifiable by their expression of DC-SIGN. We compared the subpopulations of human decidual DC-SIGN+ cells from first-trimester normal pregnancies and spontaneous abortions by flow cytometry. In normal decidua, DC-SIGN+ cells expressed antigens associated with immature myeloid dendritic cells. In samples from spontaneous abortions, we detected decidual DC-SIGN+ cells with an antigen phenotype equivalent to that of DC-SIGN+ cells from normal pregnancies, but at a significantly lower proportion (P < 0.01). Our results support the hypothesis that dendritic cells play a role in normal or pathological human pregnancy outcomes.

Human decidual stromal cells secrete C-X-C motif chemokine 13, express B cell-activating factor and rescue B lymphocytes from apoptosis: distinctive characteristics of follicular dendritic cells.

BACKGROUNDnDecidual stromal cells (DSCs) have classically been considered fibroblastic cells, although their function, cell lineage and origin are not fully understood. We previously demonstrated that human DSCs showed similarities with follicular dendritic cells (FDCs): DSCs expressed FDC-associated antigens, both types of cells are contractile and both are related to mesenchymal stem cells (MSCs). To further characterize DSCs, we investigated whether DSCs and FDCs share any distinctive phenotypical and functional characteristics.nnnMETHODSnHuman FDC lines were obtained from tonsillectomy samples, human DSC lines from elective termination of pregnancy samples and human MSC lines from bone marrow aspirates. We isolated DSC, FDC and MSC lines and compared their characteristics with flow cytometry and enzyme-linked immunosorbent assay. Cell lines were cultured with tumour necrosis factor (TNF) and lymphotoxin (LT)α(1)β(2), cytokines involved in FDC differentiation. Cell lines were also differentiated in culture after exposure to progesterone and cAMP, factors involved in the differentiation (decidualization) of DSC.nnnRESULTSnLike MSCs, DSCs and FDCs expressed MSC-associated antigens (CD10, CD29, CD54, CD73, CD106, α-smooth muscle actin and STRO-1) and lacked CD45 expression, and all three types of cell line showed increased expression of CD54 (ICAM-1) and CD106 (VCAM-1) when cultured TNF and LTα(1)β(2). DSCs and FDCs, however, exhibited characteristics not observed in MSCs: DSCs expressed FDC-associated antigens CD14, CD21 and CD23, B cell-activating factor and secreted C-X-C motif chemokine 13. Moreover, DSC lines but not MSC lines inhibited the spontaneous apoptosis of B lymphocytes, a typical functional attribute of FDC. During culture with progesterone and cAMP, FDCs, like DSCs but in contrast to MSCs, changed their morphology from a fibroblastic to a rounder shape, and cells secreted prolactin.nnnCONCLUSIONSnOur results suggest that DSCs and FDCs share a common precursor in MSCs but this precursor acquires new capacities when it homes to peripheral tissues. We discuss these shared properties in the context of immune-endocrine regulation during pregnancy.

Orphan Receptor Kinase ROR2 is Expressed in the Mouse Uterus

OBJECTIVEnWingless-type mouse mammary tumor virus integration site family, member 5A (WNT5A), is expressed in mouse decidua and is thought to play an important role in decidualization. We examined expression of the receptor for WNT5A, receptor tyrosine kinase-like orphan receptor 2 (ROR2), in the uteri of cycling and pregnant mice.nnnSTUDY DESIGNnReverse transcription (RT)-PCR and immunohistochemistry were performed.nnnRESULTSnRT-PCR revealed that transcripts for Ror2, Wnt3a, Wnt5a and inhibitor of WNT signaling, Dickkopf homolog 1 (Dkk1), were present in the pregnant uterus. Immunohistochemistry revealed that in the virgin uterus, ROR2 is expressed in stromal cells and on the basal side of uterine gland and endometrial epithelial cells. During pregnancy, both the luminal and basal side of uterine gland epithelial cells expressed ROR2, stromal cell expression of ROR2 became more frequent and ROR2 expressing uterine Natural Killer (NK) cells and cells lining the maternal vascular space emerged. Immunofluorescence imaging and flow cytometry revealed that although uterine NK cells expressed ROR2, NK cells of the spleen were ROR2 negative.nnnCONCLUSIONnThe expression of ROR2 by endometrial epithelial cells may suggest WNT signaling has roles in uterine epithelial cell polarity or implantation. Expression of ROR2 by uterine NK cells may suggest WNT signaling regulates uterine NK cell functions such angiogenesis and regulation of trophoblast migration. In summary, our results show that ROR2 expression by maternal uterine cells is influenced by pregnancy.

Apoptotic DC-SIGN+ cells in normal human decidua

BACKGROUNDnNormal pregnancy and spontaneous abortion in humans and mice are associated with immune responses. The decidua harbors dendritic cells identifiable in humans by their expression of DC-SIGN. Because dendritic cells are essential for immune response regulation, decidual DC-SIGN+ cells may play a role in normal or pathological pregnancy outcomes. Previous reports suggested that DC interact with NK cells in decidua, although the functional significance of this phenomenon remains unknown.nnnOBJECTIVEnWe studied the presence of conjugates of DC-SIGN+ cells with CD56+ NK cells in normal human decidua.nnnMETHODSnConjugates of DC-SIGN+ cells with CD56+ NK cells were studied in leukocyte suspensions of normal human decidua (6-11 weeks) by flow cytometry and confocal microscopy. The presence of apoptotic cells was determined by the TUNEL assay, incubation with annexin V and confocal microscopy in decidual leukocyte suspensions and by the TUNEL assay in decidual sections.nnnRESULTSnWe observed conjugates of decidual DC-SIGN+ cells with CD56+ NK cells (40.2±26.1% of all the DC-SIGN+ cells by flow cytometry and 52.3±10.2% by confocal microscopy). We also found that a proportion of DC-SIGN+ cells were in apoptosis, since they were TUNEL+ (40.2±7.2% of all DC-SIGN+ cells in decidual sections) and annexin V+ (34.4±15.2% in leukocyte suspensions). And sorted DC-SIGN+ cells had multilobulated nuclei.nnnCONCLUSIONSnThe conjugates of decidual DC-SIGN+ cells with CD56+ NK cells strongly suggest that these latter cells induce apoptosis in DC-SIGN+ cells during normal pregnancy. We discuss this possibility in the context of maternal-fetal tolerance.

Human decidual stromal cells secrete soluble pro-apoptotic factors during decidualization in a cAMP-dependent manner

STUDY QUESTIONnIs there a relationship between decidualization and apoptosis of decidual stromal cells (DSC)?nnnSUMMARY ANSWERnDecidualization triggers the secretion of soluble factors that induce apoptosis in DSC.nnnWHAT IS KNOWN ALREADYnThe differentiation and apoptosis of DSC during decidualization of the receptive decidua are crucial processes for the controlled invasion of trophoblasts in normal pregnancy. Most DSC regress in a time-dependent manner, and their removal is important to provide space for the embryo to grow. However, the mechanism that controls DSC death is poorly understood.nnnSTUDY DESIGN, SIZE, DURATIONnThe apoptotic response of DSC was analyzed after exposure to different exogenous agents and during decidualization. The apoptotic potential of decidualized DSC supernatants and prolactin (PRL) was also evaluated.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnDSC lines were established from samples of decidua from first trimester pregnancies. Apoptosis was assayed by flow cytometry. PRL production, as a marker of decidualization, was determined by enzyme-linked immunosorbent assay.nnnMAIN RESULTS AND THE ROLE OF CHANCEnDSCs were resistant to a variety of apoptosis-inducing substances. Nevertheless, DSC underwent apoptosis during decidualization in culture, with cAMP being essential for both apoptosis and differentiation. In addition, culture supernatants from decidualized DSC induced apoptosis in undifferentiated DSC, although paradoxically these supernatants decreased the spontaneous apoptosis of decidual lymphocytes. Exogenously added PRL did not induce apoptosis in DSC and an antibody that neutralized the PRL receptor did not decrease the apoptosis induced by supernatants.nnnLIMITATIONS, REASONS FOR CAUTIONSnFurther studies are needed to examine the involvement of other soluble factors secreted by decidualized DSC in the induction of apoptosis.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe present results indicate that apoptosis of DSC occurs in parallel to differentiation, in response to decidualization signals, with soluble factors secreted by decidualized DSC being responsible for triggering cell death. These studies are relevant in the understanding of how the regression of decidua, a crucial process for successful pregnancy, takes place.nnnSTUDY FUNDING/COMPETING INTERESTSnThis work was supported by the Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (Grant CTS-6183, Proyectos de Investigación de Excelencia 2010 to C.R.-R.) and the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain (Grants PS09/00339 and PI12/01085 to E.G.O.). E.L.-D. was supported by fellowships from the Ministerio de Educación y Ciencia, Spain and the University of Granada. The authors have no conflict of interest.