Integrin α4-positive human trophoblast progenitors: functional characterization and transcriptional regulation.

STUDY QUESTION: What are the functional characteristics and transcriptional regulators of human trophoblast progenitor cells (TBPCs)? SUMMARY ANSWER: TBPC lines established from the human smooth chorion by cell sorting for integrin α4 expressed markers of stemness and trophoblast (TB) stage-specific antigens, invaded Matrigel substrates and contributed to the cytotrophoblasts (CTBs) layer of smooth chorion explants with high-mobility group protein HMGI-C (HMGA2) and transcription factor GATA-4 (GATA4) controlling their progenitor state and TB identity. WHAT IS KNOWN ALREADY: Previously, we reported the derivation of TBPC lines by trypsinization of colonies that formed in cultures of chorionic mesenchyme cells that were treated with an activin nodal inhibitor. Microarray analyses showed that, among integrins, α4 was most highly expressed, and identified HMGA2 and GATA4 as potential transcriptional regulators. STUDY DESIGN, SIZE, DURATION: The aim of this study was to streamline TBPC derivation across gestation. High-cell surface expression of integrin α4 enabled the use of a fluorescence-activated cell sorter (FACS) approach for TBPC isolation from the human smooth chorion (n = 6 lines). To confirm their TBPC identity, we profiled their expression of stemness and TB markers, and growth factor receptors. At a functional level, we assayed their invasive capacity (n = 3) and tropism for the CTB layer of the smooth chorion (n = 3). At a molecular level, we studied the roles of HMGA2 and GATA4. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Cells were enzymatically disassociated from the human smooth chorion across gestation. FACS was used to isolate the integrin α4-positive population. In total, we established six TBPC lines, two per trimester. Their identity was determined by immunolocalization of a suite of antigens. Function was assessed via Matrigel invasion and co-culture with explants of the human smooth chorion. An siRNA approach was used to down-regulate HMGA2 and GATA4 expression and the results were confirmed by immunoblotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The endpoints analyzed included proliferation, as determined by 5-bromo-2'-deoxyuridine (BrDU) incorporation, and the expression of stage-specific antigens and hormones, as determined by qRT-PCR and immunostaining approaches. MAIN RESULTS AND THE ROLE OF CHANCE: As with the original cell lines, the progenitors expressed a combination of human embryonic stem cell and TB markers. Upon differentiation, they primarily formed CTBs, which were capable of Matrigel invasion. Co-culture of the cells with smooth chorion explants enabled their migration through the mesenchyme after which they intercalated within the chorionic CTB layer. Down-regulation of HMGA2 showed that this DNA-binding protein governed their self-renewal. Both HMGA2 and GATA4 had pleitropic effects on the cells' progenitor state and TB identity. LIMITATIONS, REASONS FOR CAUTION: This study supported our hypothesis that TBPCs from the chorionic mesenchyme can contribute to the subpopulation of CTBs that reside in the smooth chorion. In the absence of in vivo data, which is difficult to obtain in humans, the results have the limitations common to all in vitro studies. WIDER IMPLICATIONS OF THE FINDINGS: The accepted view is that progenitors reside among the villous CTB subpopulation. Here, we show that TBPCs also reside in the mesenchymal layer of the smooth chorion throughout gestation. We theorize that they can contribute to the CTB layer in this region. This phenomenon may be particularly important in pathological situations when CTBs of the smooth chorion might provide a functional reserve for CTBs of the placenta proper. STUDY FUNDING/COMPETING INTERESTS: Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award P50HD055764. O.G., N.L., K.O., A.P., T.G.-G., M.K., A.B., M.G. have nothing to disclose. S.J.F. received licensing fees and royalties from SeraCare Life Sciences for trisomic TBPC lines that were derived according to the methods described in this manuscript. TRIAL REGISTRATION NUMBER: N/A.

The role of chorionic cytotrophoblasts in the smooth chorion fusion with parietal decidua.

BACKGROUND/PURPOSE: Human placenta and chorion are rapidly growing transient embryonic organs built from diverse cell populations that are of either, ectodermal [placenta and chorion specific trophoblast (TB) cells], or mesodermal origin [villous core and chorionic mesenchyme]. The development of placenta and chorion is synchronized from the earliest phase of implantation. Little is known about the formative stages of the human chorion, in particular the steps between the formation of a smooth chorion and its fusion with the parietal decidua. METHODS: We examined the available histological material using immunohistochemistry, and further analyzed in vitro the characteristics of the recently established and reported human self-renewing trophoblast progenitor cells (TBPC) derived from chorionic mesoderm. RESULTS: Here, we provided evidence that the mechanism by which smooth chorion fuses with parietal decidua is the invasion of smooth chorionic cytotrophoblasts (schCTBs) into the uterine wall opposite to the implantation side. This process, which partially replicates some of the mechanisms of the blastocyst implantation, leads to the formation of a new zone of contacts between fetal and maternal cells. CONCLUSION: We propose the schCTBs invasion of the parietal decidua as a mechanism of 'fusion' of the membranes, and that schCTBs in vivo contribute to the pool of the invasive schCTB.

Hair cycle and wound healing in mice with a keratinocyte-restricted deletion of FAK.

Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAKK5 KO mice). FAK(K5 KO) mice displayed three major phenotypes--irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis--pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and three-dimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAKloxP/+ and FAKK5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.

Baseline toxicity of several pesticides to Hyaliodes vitripennis (Say) (Hemiptera: Miridae).

Hyaliodes vitripennis (Say) is a univoltine indigenous predacious mirid. It has been reported in several orchards where IPM programmes are used. It is a generalist, and feeds on phytophagous mites in addition to other arthropods. In Quebec, a foliar application of imidacloprid, deltamethrin or lambda-cyhalothrin is used at least once per season to manage arthropod pests such as leafhoppers and leaf-eating caterpillars. Meanwhile, several applications of metiram, flusilazole, myclobutanil and mancozeb are made to control apple scab [Venturia inaequalis (Cooke) Winter]. In laboratory trials, comparison of lethal concentrations of the three insecticides against H vitripennis nymphs and adults showed no significant difference. However, when lethal concentrations were compared between two growth stages for each insecticide, a significant difference was noted between adults and nymphs treated with lambda-cyhalothrin, adults being more susceptible than nymphs. No such difference could be detected for imidacloprid or deltamethrin. When LC50 values were compared with the manufacturer's label rates, deltamethrin and imidacloprid were toxic to the nymphs and adults, and lambda-cyhalothrin was slightly toxic to the nymphs and moderately toxic to the adults. Among the fungicides evaluated in the laboratory, myclobutanil showed moderate toxicity to adults at the manufacturer's label rate. The remaining fungicides had no toxic effects to adults or nymphs, even at four times the manufacturer's label rate.

Effects of five insecticides used in apple orchards on Hyaliodes vitripennis (Say) (Hemiptera: Miridae).

Azinphos-methyl, carbaryl, dimethoate, phosmet and phosalone were used in apple orchards to manage apple aphid, apple maggot, woolly apple aphid and leaf eating caterpillars. Among the five insecticides evaluated, dimethoate, carbaryl and azinphosmethyl were the most toxic to the nymphs and adults of Hyaliodes vitripennis (Say) from two regions. Phosalone was the least toxic. Nymphs were more resistant than the adults. While the LC50 for dimethoate was 130 ppm for nymphs, it was 3 ppm for adults from St. Jean-Baptiste-de-Rouville. There were also significant differences in the level of resistance between the two regions where the H. vitripennis were collected. At St. Alexandre the LC50 for phosalone on nymphs was 19,250 ppm whereas, at St. Jean-Baptiste-de-Rouville it was 160,000 ppm.