A six-inhibitor culture medium for improving naïve-type pluripotency of porcine pluripotent stem cells.

Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of naïve-state stem cells. The FL6i medium also assisted generation of naïve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state.

Vulnerability of primitive human placental trophoblast to Zika virus.

Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to low titers (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKVU) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKVC). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction.

Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas.

Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall.

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure.

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.

Abnormal oxidative stress responses in fibroblasts from preeclampsia infants.

BACKGROUND: Signs of severe oxidative stress are evident in term placentae of infants born to mothers with preeclampsia (PE), but it is unclear whether this is a cause or consequence of the disease. Here fibroblast lines were established from umbilical cords (UC) delivered by mothers who had experienced early onset PE and from controls with the goal of converting these primary cells to induced pluripotent stem cells and ultimately trophoblast. Contrary to expectations, the oxidative stress responses of these non-placental cells from PE infants were more severe than those from controls. METHODS AND FINDINGS: Three features suggested that UC-derived fibroblasts from PE infants responded less well to oxidative stressors than controls: 1) While all UC provided outgrowths in 4% O2, success was significantly lower for PE cords in 20% O2; 2) PE lines established in 4% O2 proliferated more slowly than controls when switched to 20% O2; 3) PE lines were more susceptible to the pro-oxidants diethylmaleate and tert-butylhydroquinone than control lines, but, unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few genes differentially regulated between PE lines and controls in 4% O2 conditions. However, a more severely stressed phenotype than controls, particularly in the unfolded protein response, was evident when PE lines were switched suddenly to 20% O2, thus confirming the greater sensitivity of the PE fibroblasts to acute changes in oxidative stress. CONCLUSIONS: UC fibroblasts derived from PE infants are intrinsically less able to respond to acute oxidative stress than controls, and this phenotype is retained over many cell doublings. Whether the basis of this vulnerability is genetic or epigenetic and how it pertains to trophoblast development remains unclear, but this finding may provide a clue to the basis of the early onset, usually severe, form of PE.

Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4.

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development, but the soundness of this model has been challenged by others, who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7(+) within 48 h, with minimal expression of mesoderm markers, including T (Brachyury). Instead, they begin to express a series of trophoblast markers, including HLA-G, demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium, and, over time, produce extensive amounts of human chorionic gonadotropin, progesterone, placental growth factor, and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling, which at day 2 provide colonies that are entirely KRT7(+) and in which the majority of cells are transiently CDX2(+). Colonies grown on two chemically defined media, including the one in which BMP4 was reported to drive mesoderm formation, also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts.

Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos.

The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent "primed" induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.

Derivation of induced pluripotent stem cells from pig somatic cells.

For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after approximately 22 days, providing an overall reprogramming efficiency of approximately 0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of approximately 17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.

The contrasting effects of ad libitum and restricted feeding of a diet very high in saturated fats on sex ratio and metabolic hormones in mice.

Skewing of the sex ratio towards males occurs among pups born to mice fed a very high saturated fat (VHF) diet. In the present study, we tested whether the fat content of the VHF diet rather than the number of calories consumed is responsible for this effect. Eight-week-old NIH Swiss mice were placed on the VHF diet either ad libitum (VHF) or in a restricted manner (VHF-R). The VHF-R mice gained weight at a similar rate to controls fed a standard chow diet. Mice were bred at 15 wk and subsequently at 26 wk and 35 wk of age. Overall, the VHF, VHF-R, and control groups delivered 244, 242, and 274 pups, respectively, with male proportions of 0.60, 0.43, and 0.48, respectively. The pup sex ratios of the VHF group (favoring males) and VHF-R group (favoring females) each differed from 0.5 (P < 0.01). The sex ratios also differed (P < 0.0001) between the VHF and control groups, and between the VHF and VHF-R groups. Within the diet groups, maternal body weight had no effect on sex ratio. Serum leptin concentrations among the dams were similar in the VHF and VHF-R groups but higher than in the control group, while the IGF1 and corticosterone levels were comparable in all three groups. Therefore, the atypical sex ratios of offspring born to dams on the VHF diet seem to be influenced by the amount of fat consumed. Since males fed the VHF diet had neither more Y-sperm nor sired more sons than daughters, the dietary effects are manifested exclusively through the female.

Influence of different isoforms of recombinant trophoblastic interferons on prostaglandin production in cultured bovine endometrial cells.

In ruminants, interferon produced by the trophectoderm (IFN-tau) is recognized as the embryonic signal responsible for maternal recognition of pregnancy. IFN-tau is believed to act by down-regulating estrogen receptors, thus preventing appearance of oxytocin receptors responsible for the release of prostaglandin F(2alpha) (PGF(2alpha)) by the endometrium. The present study was undertaken to determine in vitro the biological activities of different IFN-tau isoforms and document putative alternate luteotrophic mechanisms. Endometrial cells in primary cultures were treated with five different rIFN-tau isoforms: two ovine isoforms (ro-4 and ro-11) and three bovine isoforms (rb-1a, rb-2b and rb-3b). Their effect was quantified by measurement of PGE(2) and PGF(2alpha) production by ELISA and induction of cyclooxygenase (COX-2) by Western and Northern analysis and correlated with antiviral activity previously reported. The overall pattern of response to the IFNs tested suggests that low concentrations (<1 microg/ml) reduced the production of both PGs and higher concentrations (>1 microg/ml) stimulated preferentially PGE(2); however, exceptions were noted. Isoform rb-2b with high antiviral activity inhibited PG production in both cell types at all concentrations tested. IFNs rb-1a and ro-11 had similar antiviral activities, inhibiting PG at low concentrations and stimulating them at high concentrations. Isoform rb-3b stands out relative to the other IFNs tested because it induced a variable non-dose-dependent effect on PG production and low antiviral activity. An increase in COX-2 protein expression and messenger was correlated with increased PG production. The results showing two distinct responses to IFN-tau depending on its concentration and/or isoform and the absence of correlation with antiviral activity suggest that complex transduction mechanisms are involved.

Expression of interferon receptor subunits, IFNAR1 and IFNAR2, in the ovine uterus.

Interferon-tau (IFN-tau) is the antiluteolytic factor released by concepti of ruminant ungulate species prior to implantation. All type I interferons, including IFN-tau, exert their action through a common receptor, which consists of two subunits, IFNAR1 and IFNAR2c, but the distribution of the two polypeptides in uterine endometrium has not been examined. In situ hybridization and immunohistochemistry on sections from pregnant and nonpregnant ovine uteri at Days 14 and 15 after estrus and mating showed that both IFNAR1 and IFNAR2 mRNA and protein were strongly expressed in endometrial luminal epithelium (LE), superficial glandular epithelium (GE), and stromal cells, within but not outside caruncles. Similar staining patterns were noted in pregnant and nonpregnant uteri for both subunits. Western blot analysis of membrane fractions from cell lines derived from endometrial LE, GE, and stromal cells, and affinity cross-linking experiments with radioactively labeled IFN-tau performed on crude endometrial membranes indicated the presence of both high ( approximately 110 kDa) and low (75-80 kDa) molecular mass forms of the two receptor subunits. To localize where IFN-tau binds when it is introduced into the uterine lumen, immunohistochemistry with an antiserum against IFN-tau was performed on sections of uteri from Day 14 nonpregnant ewes whose uteri had previously been infused with IFN-tau. Staining was concentrated on the LE and superficial GE cells, and was absent from the deeper regions of the glands and from the stromal tissues. These studies demonstrate the heavy concentration of IFNAR1 and IFNAR2 in cells of the LE and superficial GE, which appear to be the main targets for IFN-tau.