text:
Nasik: Naga Sadhus today expressed their resentment after Sonu Nigam compared them to Radhe Maa. What looked like a comment from the singer in support of the beleaguered God-woman has now snowballed into a controversy.
Speaking to Faking News, a Naga Sadhu said, "How can Sonu Nigam compare us with Radhe Maa. I mean how. We are way better dancers than Radhe Maa. I have seen the video of Radhe Maa which was flashed across news channels last week and I must say that lady has got horrible moves. She was prancing around like Little Red Riding Hood. Do you call that dancing?"
"We Sadhus are better dancers. DJ's from across the world shoot their trance videos with us. Did you know the head spin move was inspired by a Sadhu doing the headstand? We strongly protest this comparison by Sonu Nigam and ask him to take back his statement or else will protest outside his house," he threatened.
Apparently, the Association of Naga Sadhus has challenged Radhe Maa for a dance competition to settle this issue for good. "Yes we will do all we can to prove our point. We don't indulge in masquerade. We have nothing to hide, literally," said the chief of the association.
Retracting into damage control mode, Sonu Nigam clarified that he has been misinterpreted. "All I said was that people should not criticize Radhe Maa for her attire. I respect Naga Sadhus and very well know about their dancing prowess. I have attended Kumbh Mela previously and seen a moon-walking Naga Sadhu," said the singer.
Meanwhile, Bollywood choreographer and part-time director Remo D'Souza is planning to make ABCD 3 with Radhe Maa and Naga Sadhus in lead roles. "I shared this idea with Prabhu Sir (Prabhu Deva) and he immediately liked it. Initially we thought of making it Varun Dhawan and Shraddha Kapoor, but clearly Naga Sadhu and Radhe Maa would be a bigger draw for the audience," he said.


text:
Univision taps South America nets for shows
Univision struck long-term programming deals with networks in Colombia and Venezuela to supply shows for its U.S. and Puerto Rican viewers.
Both deals focus on novelas produced by Colmbia's RCN Television and Venezuala's Raio Caracas Television (RCTV). Univision's deal with RCTV spans 10 years, including 800 hours of original programing per year, while the RCN deal is a five-year pact, covering 300 hours of programming per year.
Terms of the deals were not disclosed. The deals come on the heels of Univision's proposed acquisition of two Puerto Rican TV stations from Raycom last week in a $59 million cash and stock transaction. - Richard Tedesco

subset: commoncrawl


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Dermalogica Age Smart Skin Resurfacing Cleanser 150ml
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11 Reviews
11 Reviews
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Love this cleanser, gets deep into the skin & cleanses without drying my skin out. Highly recommend!Report this review
I'm disappointed with this product overall. After using for about four days my normally smooth skin started to become very dry and flaking around my chin and cheeks. Will only use for a day or so if I run out of my usual ASAP cleanser which is brilliant.Report this review
I was going to purchase and try this product today but after reading that one ingredient is Sodium Laurel Sulphate will definitely not buy. Find it hard to believe that in this day and age a replacement ingredient cannot be found for SLS. We should all be taking a stand against this ingredient - please Dermalogica try and find something else to add to your product.Report this review
Love this product. I've been using it for years and love how it makes my skin feel smooth and gives it a healthy glow.Report this review
I am very happy with the product. I am only using once a day, and feel that it starts my day with a smooth skin ready for my make up application.Report this review
I'm quite disappointed in Dermalogica this time. The second ingredient, after water, is sodium lauryl sulphate...the same ingredient in my dishwashing detergent. This makes it a wildly overpriced, very average cleanser. It's so drying I'll have to buy a better cleanser straight away. The instructions do note that dryness and sensitivity may arise, in which case you should only use it 2-3 times a week.Report this review
I was given some samples and this product is brilliant. Unfortunately I purchased the massive bottle of special cleansing gel which I need to finish first, but next time I'll be buying this instead.Report this review
I am a convert to this cleanser. It was reccommended to me by my beautician but ofcourse I found it at a much better price here! I am onto my 4th bottle and panic when I am running low! It has cleared and smoothed my skin and is the best it was ever looked!Report this review
I am only part way through my first bottle, but feel the need to have a second ready on hand. That's how good it is. Almost a face-life in a bottle - leaves skin clean and plump.Report this review
this is a great gentle cleanser that does not strip your skin of moisture. i have combination skin and am concerned with premature ageing.Report this review


text:
|United States Patent
|Patent No:
|Rossant et al.
|Date of Patent:
|Oct. 07, 2003
|Trophoblast cell preparations
|Janet Rossant, Toronto (Canada); Satoshi Tanaka, Tokyo (Japan); and Tilo Kunath, Toronto (Canada)
|Mount Sinai Hospital, Toronto (Canada)
|( * )
|Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days.
|Appl. No.:
|Mar. 07, 2001
|PCT Filed:
|Sep. 23, 1999
|PCT No.:
|PCT Pub. No.:
|PCT Pub. Date:
|Mar. 30, 2000
|Related U.S. Application Data
|Provisional Application No. 60/101483 Filed on Sep. 23, 1998
|Int. Cl.7
|U.S. Cl.
|435/325; 435/373; 435/377; 435/455; 800/21
|Field of Search
|435/325; 435/455; 435/373; 435/377; 800/21
|References Cited
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|* cited by examiner
|Primary Examiner —Anne M. Wehbe′
|Assistant Examiner — Q Janice Li
|Attorney, Agent, or Firm — Howson and Howson
|Exemplary claim number — 1
|Art Unit — 1632
[00001] Stable pluripotent trophoblast stem (TS) cell lines and uses of the cell lines are described. The cell lines comprise cells that (i) are capable of indefinite proliferation in vitro in an undifferentiated state, and (ii) are capable of differentiation into cells of the trophoblast lineage in vivo.
|16 Claims, 4 Drawing Sheets, and 19 Figures
|[00002] This application was filed pursuant to 35 USC §371 as a national phase of PCT/CA99/00867, filed Sep. 23, 1999, which claims the benefit of U.S. patent application Ser. No. 60/101,483, filed Sep. 23, 1998.
|[00003] The invention relates to trophoblast cell preparations and uses of the cell preparations.
|[00004] In mammals, the earliest developmental decision specifies the trophoblast cell lineage. In mice, this lineage appears at the blastocyst stage as the trophectoderm, a sphere of epithelial cells surrounding the inner cell mass (ICM) and the blastocoel. After implantation, the ICM gives rise to the embryo proper and some extraembryonic membranes. However, the trophectoderm is exclusively restricted to form the fetal portion of the placenta and the trophoblast giant cells. The polar trophectoderm (the subset of trophectoderm in direct contact with the ICM) maintains a proliferative capacity and gives rise to the extraembryonic ectoderm (ExE), the ectoplacental cone (EPC), and secondary giant cells of the early conceptus (1). The rest of the trophectoderm ceases to proliferate and becomes primary giant cells. Studies in primary culture and chimeric mice have suggested that stem cells exist in the extraembryonic ectoderm which contribute descendants to the EPC and the polyploid giant cells (2). Further evidence indicated that maintenance of these stem cell-like characteristics was dependent on signals from the ICM and later from the epiblast (3), since diploid trophoblast cells transformed into giant cells when removed from the embryonic environment (4). However, the nature of the embryo-derived signal was not known and all attempts at routine long-term culture of mouse trophoblast stem cells have been unsuccessful.
|[00005] Expression and functional analyses indicated that Fgf4 and Fgfr2 may be involved in trophoblast proliferation (5, 6, 7). The reciprocal expression domains of Fgfr2 and Fgf4 suggested that the trophoblast could be a target tissue for an embryonic FGF signal. Fgfr2-null and Fgf4-null mice show similar peri-implantation lethal phenotypes (6, 7). This may result from defects in the ICM and its endoderm derivatives. However, it is also consistent with the possibility that FGF4 acts on the trophoblast through FGFR2 to maintain a proliferating population of trophoblast cells. Support for this latter possibility is provided by recent studies showing that inhibiting FGF signaling blocked cell division in both the ICM and trophectoderm (8).
|[00006] The present inventors have found that FGF4 can promote sustained proliferation of primary cultures of diploid trophoblast cells and it permits isolation of stable FGF4-dependent mouse trophoblast stem (TS) cell lines from both the ExE of 6.5 dpc embryos and the trophectoderm of 3.5 dpc blastocysts. TS cell lines expressed many diploid trophoblast markers and retained the capacity to differentiate into other trophoblast subtypes in vitro upon removal of FGF4. Most importantly, when these stem cells were introduced into chimeras they exclusively contributed to all trophoblast subtypes in vivo. Availability of trophoblast stem cell lines opens up new possibilities for understanding the genetic regulation of placental development and placental insufficiencies and modulating the same. The cell lines also enable the treatment of placental insufficiencies by pharmacological intervention or gene-based therapy.
|[00007] Broadly stated, the present invention relates to a stable pluripotent trophoblast stem (TS) cell line. In particular, the invention relates to a purified preparation of trophoblast stem cells which (i) arc capable of indefinite proliferation in vitro in an undifferentiated state; and (ii) are capable of differentiation into cells of the trophoblast lineage in vivo. The preparation of trophoblast stem cells is also characterized by expression of genetic markers of diploid trophoblast stem cells.
|[00008] A trophoblast stem cell preparation of the invention may be induced to differentiate into cells of the trophoblast lineage in vitro or in vivo. The invention therefore also relates to a purified trophoblast stem cell preparation of the invention (preferably cultured in vitro) induced to differentiate into cells of the trophoblast lineage. This differentiated cell preparation is characterized by expression of genetic markers of trophoblast cell lineages (e.g. diploid trophoblast cells of the ectoplacental cone (EPC), and the secondary giant cells of the early conceptus). In an embodiment of the invention a purified trophoblast cell preparation comprises cells of the trophoblast lineage including diploid trophoblast cells.
|[00009] A cell preparation of the invention may be derived from or comprised of cells that have been genetically modified either in nature or by genetic engineering techniques in vivo or in vitro.
|[00010] Cell preparations or cell lines of the invention can be modified by introducing mutations into genes in the cells or by introducing transgenes into the cells. Insertion or deletion mutations may be introduced in a cell using standard techniques. A transgene may be introduced into cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. By way of example, a transgene may be introduced into cells using an appropriate expression vector including but not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses). Transfection is easily and efficiently obtained using standard methods including culturing the cells on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
|[00011] A gene encoding a selectable marker may be integrated into cells of a cell preparation of the invention. For example, a gene which encodes a protein such as β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or a fluorescent protein marker may be integrated into the cells. Examples of fluorescent protein markers are the Green Fluorescent Protein (GFP) from the jellyfish A. victoria, or a variant thereof that retains its fluorescent properties when expressed in vertebrate cells. (Examples of GFP variants include a variant of GFP having a Ser65Thr mutation of GFP (S65T) that has longer wavelengths of excitation and emission, 490 nm and 510 nm, respectively, compared to wild-type GFP (400 nm and 475 nm): a blue fluorescent variant of GFP (e.g. N66H-GFP) (Heim et al. Proc. Natl. Acad. Sci. 91:12501, 1994). MmGFP (M. Zernicka-Goetz et al, Development 124:1133-1137, 1997), enhanced GFP ("EGFP") (Okabe, M. et al. FEBS Letters 407:313-319, 1997; Clontech Palo Alto, Calif.), EGFP which has a Phe to Leu mutation at position 64 resulting in the increased stability of the protein at 37° C. and a Ser to Thr mutation at position 65 resulting in an increased fluorescence, and, EGFP commercially available from Clontech incorporating a humanised codon usage rendering it "less foreign" to mammalian transcriptional machinery and ensuring maximal gene expression.)
|[00012] The invention also relates to a method for producing a purified trophoblast stem (TS) cell preparation i.e. a cell line, comprising the steps of culturing early postimplantation trophoblast cells or cells of a blastocyst, preferably from the trophectoderm on a feeder layer (e.g. a fibroblast layer or a medium conditioned by fibroblasts) in the presence of FGF4 and a co-factor. The method may additionally comprise inducing differentiation of the trophoblast stem cells by removing the FGF4, the co-factor, or the feeder layer. In an embodiment of the invention, the method comprises isolating a blastocyst, culturing the blastocyst on a fibroblast layer in the presence of FGF4 and a co-factor, removing a blastocyst outgrowth and dissociating the outgrowth, selecting flat colonies i,e. epithelial-like cells, and culturing the colonies. The invention also contemplates trophoblast cell preparations or lines derived at all stages of development under the same culture conditions.
|[00013] The term "blastocyst" used herein refers to the structure during early embryonic development comprising an inner cluster of cells, the inner cell mass (ICM), which gives rise to the embryo, and an outer layer, the trophectoderm, which gives rise to extra-embryonic tissues. Preferably, cells from the trophectoderm of a 3.5 dpc blasotocyst are used in the method of the invention. The term "postimplantation trophoblasts" used herein refers to cells derived from extraembryonic extoderm (ExE) cells preferably isolated from 6.5 days post coitum conceptuses. The term "epithelial-like cells" refers to the flat colonies obtained after dissociation of a blastocyst outgrowth and which are like the cells which sometimes appear during the isolation of embryonic stem cells from blastocysts as described in B. Hogan et al (10).
|[00014] The blastocysts or early postimplantation trophoblasts may be derived or isolated from any mammalian or marsupial species including but not limited to rodents (e.g. mouse, rat, hamster, etc.), rabbits, sheep, goats, pigs, cattle, primates, and humans are preferred. Mutant or transgenic blastocysts and postimplantation trophoblasts may be used to prepare a cell preparation or cell line of the invention. For example, a cell preparation or cell line of the invention may be derived from a Fgf4 or Errβ mutant blastocyst. Cells used to prepare a cell preparation or cell line of the invention can be engineered to contain a selectable marker or they may be genetically altered using techniques well known in the art.
|[00015] The cells derived from a blastocyst or postimplantation trophoblast cells are cultured on a feeder layer. The feeder layer may be a confluent fibroblast layer, preferably primary mouse embryonic fibroblast (EMFI) cells. Embryonic fibroblasts may be obtained from 12 day old fetuses from outbred mice, but other strains may be used as an alternative. STO cells (i.e. a permanent line of irradiated mouse fibroblasts) can also be used as a feeder layer. The feeder layer may also comprise medium conditioned by primary embryonic fibroblast cells.
|[00016] Cells from a blastocyst or early postimplantation trophoblast cells are preferably cultured in medium comprising RPMI 1640 with 20% fetal bovine serum, sodium pyruvate, β-mercaptoethanol, L-glutamine, and penicillin/streptomycin. The FGF4 used in the method of the invention may be recombinant FGF4 (preferably recombinant human FGF4) which may be produced using standard recombinant techniques or it may be obtained from commercial sources (e.g. Sigma). The co-factor used in the method of the invention is preferably heparin. Once established the cell lines may be grown on a feeder layer such as a fibroblast layer (e.g. EMFI cells) or in a conditioned medium prepared from a fibroblast layer (See for example the medium described in note 13, page 15).
|[00017] Cells from the cell preparations may be introduced into a blastocyst or aggregated with an early stage embryo to produce chimeric conceptuses. A chimeric conceptus may be allowed to grow to term, or sacrificed during gestation to observe the contribution of the stem cell line. In an embodiment, the invention provides a chimeric placenta wherein the trophoblast lineage is repopulated by cells from a cell preparation of the invention. The conceptuses and placenta can be engineered to carry selectable markers or genetic alterations. Cell lines can be derived from the chimeric conceptuses and placenta. Therefore, the invention further provides a chimeric conceptus, differentiated trophoblast cells, mutant trophoblast stem cells, or a chimeric placenta derived from a purified preparation of the invention.
|[00018] The cell preparations, chimeric conceptuses, and chimeric placentas may be used to screen for potential therapeutics that modulate trophoblast development or activity e.g. Invasion or proliferation. In particular, the cell preparations, chimeric embryos, or chimeric placenta may be subjected to a test substance, and the effect of the test substance may be compared to a control (e.g. in the absence of the substance) to determine if the test substance modulates trophoblast development or activity. Cell preparations of the invention derived from mouse mutants can be used to identify genes and substances that are important for the trophoblast cell lineage, and in vitro differentiation of mutant cell preparations can identify genes and substances important for selected trophoblast subtypes. Selected substances may be useful in regulating trophoblasts in vivo and they may be used to treat various conditions requiring regulation of trophoblast development or activity such as the conditions described below.
|[00019] The cell preparations of the invention may be transplanted into animals to treat specific conditions requiring modulation of trophoblast development or activity. For example, the cell preparations may be used to prolong fetal survival in conditions of placental insufficiency, or to reduce uncontrolled trophoblast invasion and abnormal trophoblast growth associated with conditions such as hydatiform mole and choriocarcinoma. The cell preparations may be used for therapeutic treatment of placental defects in humans by transplantation of the cell preparations at any stage of pregnancy to generate chimeric placenta.
|[00020] The cell preparations may be used to prepare model systems of disease for conditions such as precclampsia, hydatiform mole, or choriocarcinoma.
|[00021] The cell preparations or cell lines of the invention can be used to produce growth factors, hormones, etc. relevant to human placenta. The cell preparations or cell lines of the invention can also be used to produce therapeutics such as human Chorionic Gonadotropin (hCG).
|[00022] The cell preparations or cell lines of the invention can be used to screen for genes expressed in or essential for trophoblast differentiation. Screening methods that can be used include Representational Difference Analysis (RDA) or gene trapping with for example SA-lacZ (D. P. Hill and W. Wurst, Methods in Enzymology, 225: 664, 1993). Gene trapping can be used to induce dominant mutations (e.g. by deleting particular domains of the gene product) that affect differentiation or activity of trophoblast cells and allow the identification of genes expressed in or essential for trophoblast differentiation.
|[00023] The invention will now be described in relation to the drawings in which:
|[00024] FIGS. 1a-c. Trophoblast stem (TS) cell lines cultured in the presence and absence of FGF4 and EMFI-conditioned medium (EMFI-CM). (A) Differential interference contrast (DIC) micrograph (100×) of TS3.5 cell colonies cultured on gelatinized glass in the presence of FGF4 and EMFI-CM (13). The cells grew as tight epithelial sheets with distinctly defined borders. Differentiated giant cells are indicated (arrows). (B) DIC micrograph (100×) of TS3.5 cells cultured for 4 days on gelatinized glass in the absence of FGF4 and EMFI-CM. Large nuclei and dark, perinuclear deposits are characteristic of giant cells. Bar, 5 mm. (C) DNA content was analyzed by flow cytometric studies of cells stained with propidium iodide (PI) (14). TS cells were analyzed 0, 2, 4, and 6 days after the removal of FGF4 and EMFI-CM. Diploid (2N), tetraploid (4N), and octaploid (8N) DNA contents are indicated.
|[00025] FIGS. 2a-b. RNA analysis of TS cell lines. (A) Northern blot analysis of gene expression in TS cell lines. TS cells were grown in 70% EMFI-CM and 30% TS medium supplemented with FGF4 and heparin for 2 days (13). The undifferentiated samples (undiff) were allowed to proliferate further in the same conditions for 0, 2, and 4 days (day 0, day 2, and day 4, respectively). The differentiated samples (diff) had FGF4, heparin, and EMFI-CM removed for 2 and 4 days (day 2 and day 4, respectively) and total RNA was prepared at each time point indicated. Total RNA (10 μg) from TS cells, undifferentiated ES cells, and 7.5 dpc embryos was fractionated on a 1% denaturing agarose gel and blotted onto a nylon membrane. Three blots were made for each cell line and sequentially probed/reprobed with antisense RNA probes as indicated (15). All three blots were finally reprobed with the GAPDH antisense RNA and confirmed to contain essentially equal amounts of RNA (only one blot is shown for each cell line). mEomes, mouse eomesodermin; T, brachyury (B) RT-PCR analysis of Hnf4 expression in the TS cells. From 0.5 μg of total RNA, first-strand cDNA was synthesized with (+) or without (−) reverse transcriptase. Primers specific for β-actin and Hnf4 were added in a single reaction tube to amplify both β-actin and Hnf4-specific fragments simultaneously (15). Amplification of Hnf4-specific fragments was never observed in TS cell samples. The predicted sizes of the β-actin and Hnf4 bands are 321 bp and 270 bp, respectively. Similar results were obtained from a TS3.5 cell line.
|[00026] FIGS. 3a-e. TS cell chimeras generated by EGFP-TS3.5 cell blastocyst injections. (A to D) A 6.5 dpc chimera. The intact conceptus revealed TS cell contributions to the extraembryonic ectoderm (ExE), a patch in the ectoplacental cone (EPC), and a few giant cells on Reichert's membrane (RM) (arrow) (A and B). Removal of RM and separation of the EPC from the ExE further illustrated the TS cell contributions to extraembryonic regions and not the epiblast (Epi) (C and D). (E to H) An 8.5 dpc chimera. A large contribution of TS cells to the placenta (Pl) was observed in the intact conceptus (E and F). A patch of EGFP-positive giant cells was also observed at the distal tip of the conceptus (enlarged in the inset). Removal of RM exposed the embryo proper (Emb) and the yolk sac (YS) which did not exhibit any TS cell contributions (G and H). (I and J) A 9.5 dpc chimera. The giant cell layer, yolk sac, and amnion have been removed. A substantial TS cell contribution was observed at the center of the placenta with a speckling of EGFP-positive cells emanating from it. This contribution is largely confined to the labyrinthine trophoblast. (K and L) A chimeric term placenta. Embryos were observed under partial bright-field (A, C, E, G, I, K) and dark-field optics (B, D, F, H, J, L). Green fluorescence was observed as described (26) and all photographs were taken with Kodak P1600 film at 1600 ASA.
|[00027] FIGS. 4a-b. A model for embryonic-trophoblast interactions and the maintenance of TS cells if) vivo. (A) A schematic drawing of a 3.5 dpc blastocyst (inset) emphasizing a region where the polar and mural trophectoderm meet with the ICM. FGF4 and at least one other unidentified factor produced in the ICM may signal to the overlying polar trophectoderm, maintaining it in a proliferative state. As the trophectoderm cells move away from the ICM to become mural trophectoderm, they cease to receive the ICM-derived signals and consequently differentiate. (B) A schematic drawing of a 6.5 dpc conceptus (inset) emphasizing the embryonic-extraembryonic boundary. Similar to the blastocyst scenario, this suggested that FGF4 and an unknown factor(s) from the epiblast signal to the extraembryonic ectoderm (ExE) and directly or indirectly mediate the expression of genes such as Errβ, Cdx2, and mEomes (eomesodermin). These signals maintain a trophoblast stem cell population in the ExE nearest to the epiblast. As trophoblast cells move away from the embryonic-extraembryonic border, they no longer receive the epiblast signals and initiate a differentiation pathway.
|[00028] TS cell lines were first derived from early postimplantation embryos. ExE cells were isolated from 6.5 dpc conceptuses as previously described (4), disaggregated by trypsin, and cultured on a feeder layer of primary mouse embryonic fibroblast (EMFI) cells in the presence of various combinations of growth factors (data not shown). The combination of FGF4 (25 ng/ml) and heparin (1 μg/ml) in TS cell medium (9) proved successful in allowing the passage of colonies with a tight epithelial morphology (FIG. 1A). Removal of either FGF4, heparin, or the EMFFI cells resulted in a rapid decline in proliferation with subsequent differentiation into a giant cell-like phenotype (FIG. 1B). Some giant cells also consistently appeared at the edges of colonies after each passage even under optimal conditions suggesting that a small percentage of the cells underwent spontaneous differentiation (FIG. 1A). Since the giant cells were relatively trypsin-resistant, they were left behind after each passage and so remained at a relatively constant level in the cultures.
|[00029] Under the identical culture conditions used for isolating TS cell lines from ExE, cell lines were derived from 3.5 dpc blastocysts which exhibited a morphology and behavior indistinguishable from that of ExE-derived TS cell lines (12).The blastocyst-derived and ExE-derived lines are referred to as TS3.5 and TS6.5 cell lines, respectively, to distinguish their tissues of origin. Generation of TS3.5 and TS6.5 cell lines was efficient and reproducible; 58 clonal TS3.5 cell lines were obtained from 91 blastocysts (64%) and 17 TS6.5 cell lines from 39 ExEs of 6.5 dpc embryos (44%); they were derived from different strain backgrounds (129/sv and ICR) and of both sexes Some of these TS cell lines were stably maintained for more than 50 passages over a period of more than six months with no apparent change in their morphology or viability.
|[00030] To address the possibility that FGF4 stimulated the proliferation of TS cells indirectly by inducing the secretion of mitotic factors from the feeder cells, conditioned medium from EMFI cells (EMFI-CM) was prepared in the absence of FGF4. TS cells were maintained in an undifferentiated state on gelatin-coated plates in medium supplemented with 70% EMFI-CM and FGF4/heparin; lower concentrations of EMFI-CM were not effective (13). Leukemia inhibitory factor (LIF), the critical factor produced by EMFI cells that maintains ES cells undifferentiated, could not substitute for EMFI-CM even at five-times the concentration used in ES cell medium. These results suggest that a) EMFI cells secrete an unidentified factor(s) (EMFI-factor) that acts along with FGF4 to maintain the TS cells in a proliferative and undifferentiated state, b) secretion of this factor(s) is not a result of the addition of FGF4 to the media, and c) FGF4 acts directly on the TS cells.
|[00031] Chromosome spreads from two TS cell lines passaged over 20 times revealed an apparently normal euploid karyotype. The ploidy of the stem cells and differentiated giant cells were determined by FACS analysis of cells stained with propidium iodide (14). The profile for cells maintained in EMFI-CM supplemented with FGF4heparin (13) revealed prominent peaks at 2N and 4N indicative of the G1 and G2/M DNA content of a diploid cell line (FIG. 1C). A small shoulder of higher ploidy cells (>4N) was also observed and was likely due to the presence of spontaneously differentiating giant cells in the culture. Upon removal of FGF4 and EMFI-CM a distinct 8N peak appeared within 4 days. The 2N peak was reduced and the 4N peak, which would include diploid G2/M cells and tetraploid G1 cells, increased in size. By day 6, cells of higher than 8N ploidy were seen in the analysis. These observations are consistent with the morphological differentiation of TS cells to giant cells.
|[00032] Several genetic markers were analyzed during stem cell and differentiative culture conditions to confirm the trophoblast identify of the TS3.5 and TS6.5 cell lines and characterize their differentiation in the absence of FGF4 (15). Markers of the diploid ExE were highly expressed in TS cells. Errβ, an orphan nuclear receptor, is specifically expressed in the ExE nearest to the extraembryonic-embryonic boundary at early postimplantation stages and later in the chorionic ectoderm (16). This gene was highly expressed in TS cells grown in the presence of FGF4 and 70% EMFI-CM, but was down-regulated when differentiation was induced by removing FGF4 and EMFI-CM (FIG. 2A). This was also the case for other genes known to be highly expressed in the ExE, such as Cdx2 (17), Fgfr2 (6), and the mouse homologue of eomesodermin (18) (FIG. 2A). In contrast to the ExE-specific genes, 4311, an EPC-specific gene (19), was not detected in the undifferentiated cells, but was induced 4 days after the removal of FGF4 and EMFI-CM. Mash2, encoding a basic helix-loop-helix (bHLH) transcription factor, has been shown to be required in diploid trophoblast cells of the EPC to allow development of the spongiotrophoblast layer (20). Consistent with this, Mash2 was upregulated in differentiating TS cells prior to the expression of 4311 (FIG. 2A). Mash2 transcripts were also progressively induced in TS cells cultured in stem cell conditions. Placental lactogen1 (Pl-1), a specific marker for giant cells (21), is progressively induced in cultures after removal of FGF4, consistent with the predicted increase in giant cell content. As observed for the Mash2 gene, the increasing expression of Pl-1 during stem cell culture conditions may reflect the presence of spontaneously differentiating cells that accumulate after each passage (FIG. 2A). Hand1, another bHLH transcription factor that is known to play an important role in the development of giant cells but is not expressed in the ExE (22), was detected throughout the culture periods analyzed regardless of the presence of FGF4 and EMFI-CM (FIG. 2A). Oct3/4, Brachyury, and Hnf4, genes specific for ICM/epiblast (23), mesoderm (24), and primitive endoderm (25), respectively, were not detected in TS cells (FIG. 2). Thus, these established cell lines conserve a gene expression profile largely characteristic of trophoblast cells in the ExE and they express distinctive markers of other trophoblast cell lineages upon differentiation.
|[00033] The most definitive test for the trophoblast identity and stem cell capacity of TS cells is to investigate their potential to incorporate into trophoblast lineages in vivo. Rossant et al. (2) have shown that the cells isolated from the ExE of 6.5 dpc embryos can contribute to the EPC and giant cells when directly injected into blastocysts, despite temporal asynchrony between donor and host cells. To investigate the potency of TS cells to contribute to trophoblast lineages in vivo, chimeric embryos were made by the aggregation method (26) and blastocyst injection. A TS3.5 and a TS6.5 cell line were derived from B5/EGFP transgenic mice (27) that ubiquitously express enhanced-green fluorescent protein (EGFP, Clontech) in all embryonic and extraembryonic tissues. These lines were passaged more than 20 times (two months) before they were used for the chimera experiments. Chimeras were obtained from each cell line using both methods (Table 1). EGFP-positive cells were only observed in tissues of the trophoblast lineage in the 61 chimeric embryos analyzed (FIG. 3). TS cells contributed to the ExE, EPC, and giant cells, but were never observed in the epiblast, primitive endoderm, or other ICM-derived extraembryonic tissues, such as the allantois, yolk sac, and amnion (Table 2, FIG. 3). High contributions to chimeric placentae at term were also observed, indicating that these cells could functionally support fetal development (FIG. 3K, L). There was no significant difference between the EGFP-TS3.5 and EGFP-TS6.5 cell lines in their ability to contribute to trophoblast subtypes. However, blastocyst injections gave a higher frequency of chimeras than the aggregation method (Table 1). These results clearly show that TS cells retain the potency to differentiate into all trophoblast cell types in vivo despite being cultured in vitro for extended periods of time. Taken together with the results of the Northern analyses it was concluded that a stable pluripotent mouse trophoblast stem (TS) cell line was established.
|[00034] It has been proposed that the ExE is the first tissue to be formed from the polar trophectoderm and that it may act as a stem cell population that subsequently gives rise to the EPC which generates new secondary giant cells (2, 3). Successful derivation of TS cell lines expressing trophoblast markers from the ExE of 6.5 dpc embryos and 35 dpc blastocysts is consistent with this model. FGF4 produced by the ICM and later by the epiblast is one of the critical signals required for the maintenance of the proliferative undifferentiated state of ExE (FIG. 4). From the expression pattern and null phenotype of the Fgfr2 gene, this receptor tyrosine kinase is the best candidate to functionally receive the FGF4 signal in the trophoblast. This model predicts that the lethality observed in homozygous null mutants for both Fgf4 and Fgfr2 (6, 7) may in part be caused by the loss of the proliferating population of the ExE soon after implantation. During normal implantation the blastocyst first adheres to the uterine wall through its mural trophectoderm at the abembryonic pole; however, Fgfr2 −/− blastocysts implanted randomly implying that the trophectoderm surrounding the embryo is not polarized. The components downstrean of the trophoblast FGF response are not known, but the T-box gene, mouse eomesodermin, and the caudal-related gene, Cdx2, are good candidates since they are expressed in the appropriate cells and members of these gene families have been shown to be regulated by FGF-signaling (28, 29). As trophoblast cells continue to proliferate and move distally from the ICM/epiblast, they cease to receive the mitotic and differentiation-inhibitory signals from the embryo proper. This would result in differentiation into EPC and finally to giant cells.
|[00035] The above model makes a number of testable predictions about the involvement of FGF-signaling in trophoblast development. For example, the model predicts that TS cell lines could be derived from Fgf4, but not Fgfr2 mutant blastocysts. Establishing TS cell lines from other mouse mutants will reveal the genes essential for this stem cell lineage, while in vitro differentiation of mutant lines will identify genes important for other trophoblast subtypes. In summary, the establishment of FGF4-dependent TS cell lines from blastocysts and the ExE of 6.5 dpc embryos has revealed that a stem cell population exists within the trophoblast lineage for at least a 3-day window during early development and that the essential embryo-derived signals for trophoblast proliferation include FGF4. These cell lines are an invaluable tool to further dissect the function of genes and signaling pathways important to the development of the mammalian trophoblast lineage and its interactions with the embryo. The ability of wild type TS cells to make high contributions in chimeras indicates that these cells have the potential to rescue mutant embryos with placental defects. Such "TS cell rescue" analysis could be an alternative to the "tetraploid rescue" technique (27) currently used. Finally, obtaining similar trophoblast stem cell lines from human embryos opens up new avenues to future cell-based therapies for placental insufficiencies.
|[00036] While the present invention has been described with reference to what is presently coincided to be a preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiment. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
|[00037] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
References and Notes
|[00038] 1. J. Rossaint, in Experimental Approaches to Mammalain Embryonic Development, J. Rossant and R. A. Pederson. Eds (Cambridge Univ. Press, London, 1986), pp. 97-120; J. Rossant, Sem. Dev. Biol. 6, 237 (1995).
|[00039] 2. J. Rossant, R. L. Gardner, H. L. Alexandre, J. Embryol. Exp. Morpihol. 48, 239 (1978); M. H. Johnson and J. Rossant, ibid. 61, 103 (1981).
|[00040] 3. R. L. Gardner and M. H. Johnson, ibid. 28,279 (1972); J. Rossant and W. Iamura-Lis. ibid. 62, 217 (1981); E. B. ligren, Placenta 4, 307 (1983).
|[00041] 4. J. Rossant and L. Ofer, J. Embryol. Exp. Morphol. 39, 183 (1977); E. B. llgren, ibid. 62,183 (1981).
|[00042] 5. L. Niswander and G. R. Martin, Development 114, 755 (1992); D. A. Rappolee, C. Basilico, Y. Patel, Z. Werb, ibid. 120, 2259 (1994).
|[00043] 6. A. Orr-Urtreger et al., Dev. Biol. 158, 475 (1993); E. Arnan , R. Haffner-Krausz, Y. Chen, J. K. Heath, P. Lonai, Proc. Natl Acad. Sci. U.S.A. 95, 5082 (1998); J. Partanen and J. Rossant, unpublished data.
|[00044] 7. B. Feldman, W. Poueymirou, V. E. Papaioanriou, T. M. DeChiara, M. Goldfarb, Science 267, 246 (1995).
|[00045] 8. N. Chai et al., Dev. Biol. 198, 105 (1998).
|[00046] 9. TS cell medium is RPMI 1640 supplemented with 20% fetal bovine serum (HyClone), sodium pyruvate (1 mM, GibcoBRL), β-mercaptoethanol (100 μM, Sigma), L-glutamine (2 mM, GibcoBRL), and penicillin/streptomycin (50 μg/ml each). Human recombinant FGF4 (25 ng/ml, Sigma) and heparin (1 μg/ml) were added to aliquots of TS cell medium and used immediately.
|[00047] 10. B. Hogan, R. Beddington, F. Costantini, E. Lacy, Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., ed. 2, 1994), pp. 265-272; E. J. Robertson, in Teratocarcinomas and Embryonic Stem Cells, E. J. Robertson, Ed. (IRL Press, Oxford, 1987), pp. 71-112.
|[00048] 11. J.-E. Fléchon, S. Laurie, E. Notarianni, Placenta 16, 643 (1995).
|[00049] 12. TS3.5 cell lines were obtained using similar techniques for ES cell line derivation (10). Briefly, 3.5 dpc blastocysts were individually plated into 4-well plates on EMFI cells and cultured in TS media with FGF4 and heparin (9). The medium was changed after two days and the blastocyst outgrowth was trypsinized on the third day. On day 5 or 6, flat colonies, referred to as "epithelial-like cells" in (10). were picked and passaged. Once established, the cell lines were grown without EMFI cells, but in the presence of EMFI conditioned medium (13). Under the current culture conditions ES cell colonies were not observed.
|[00050] 13. Conditioned medium from EMFI cells (EMFI-CM) was prepared by incubating TS medium (9) without FGF4 or heparin on confluent plates of mitomycin-treated EMFI cells for 72 hours. The conditioned medium was filtered (0.45 μm) and stored at −20° C. Established TS cell lines were routinely cultured in 70% EMFI-CM, 30% TS medium, 25 ng/ml hrFGF4 and 1 μg/ml heparin on gelatin-coated plates. The medium was changed every two days and the cells were passaged (1 in 25) every four days or at 80%-90% confluency.
|[00051] 14. TS cells were grown in the absence of EMFI cells (13) and collected by cell scraping at 0, 2, 4, and 6 days after the removal of FGF4, heparin, and EMFI-CM. The cells were fixed and stained with propidium iodide (Molecular Probes) as described [Z. Darzynkiewicz and G. Juan, in Current Protocols in Cytometry (John Wiley & Sons, Inc., New York, 1997), pp. 7.5.2-7.5.3]. Cell fluorescence was measured by a flow cytometry with an argon ion laser (488 nm). The data was analyzed with Coulter EXPO Cytometer Software version 2.0 by Applied Cytometry Systems, 1998.
|[00052] 15. Total RNA was prepared from cells and embryos with TRIzol (GibcoBRL) according to the manufacturer's instructions. Northern blotting was performed by a standard protocol. Antisense RNA probes for Errβ(16), eomesodermin (18), Cdx2 [E. Suh, L. Chen, J. Taylor, P. G. Traber, Mol Cell. Biol. 14, 7340 (1994)], Fgfr2, Mash2 (20), 4311 (19), Handl (22), Pl-1 [P. Colosi, F. Talamantes, D. I. H. Linzer, Mol. Endocrinol. 1, 767 (1987)], Oct-3/4 (23), Brachyury (24), and GAPDH [P. Fort et al., Nucleic Acids Res. 13, 1431 (1985)] were labeled with either [α-32P]UTP or DIG-11-UTP (Boehringer Mannheim) by using Strip-EZ RNA kit (Ambion). Blots were hybridized overnight at 65° C. in NorthernMax Prehybridization/hybridization Buffer (Ambion) and were finally washed in 0.1×SSC/0.1% SDS at 65° C. DIG-labeled probes were detected with the DIG Luminescent Detection Kit (Boehringer Mannheim). Removal of hybridized RNA probes was performed with the Strip-EZ RNA kit (Ambion) according to manufacturer's recommendations. To assess the expression of Hnf4 in the TS cell lines, first strand cDNA synthesized from 0.5 μg total RNA of TS cells and 7.5 dpc embryos with random hexamers was subjected to 35 cycles of PCR (62° C. annealing temperature) by using 0.2 μM each of Hnf4-specific primers (5′-CACGTCCCCATCTGAAGGTG-3′ and 5′-CTTCCTTCTTCATGCCAGCCC-3′) and 0.1 μM each of β-actin-specific primers (5′-GACAACGGCTCCGGCATGTGCAAAG-3′ and 5′-TTCACGGTTGGCCTTAGGGTTCAG-3′). The primer sequences were adapted from D. loannis et al., Development 125, 1529 (1998).
|[00053] 16. K. Pettersson et al., Mech. Dev. 54, 211 (1996); J. Luo et al., Nature 388, 778 (1997).
|[00054] 17. F. Beck, T. Erler, A. Russell, R. James, Dev. Dyn. 204, 219 (1995).
|[00055] 18. B. G. Ciruna and J. Rossant, Mech. Dev. 81, 199 (1999).
|[00056] 19. K. R. Lescisin, S. Varmuza, J. Rossant, Genes Dev. 2, 1639 (1988).
|[00057] 20. F. Guillemot, A. Nagy, A. Auerbach, J. Rossant. A. L. Joyner, Nature 371, 333 (1994); M. Tanaka, M. Gertsenstein. J. Rossant, A. Nagy, Dev. Biol. 190, 55 (1997).
|[00058] 21. T. N. Faria. L. Ogreni. F. TalamanIes, D. I. Linzer. M. J. Soares, Biol. Reprod. 44, 327 (1991).
|[00059] 22. J. C. Cross et al., Development 121, 2513 (1995); P. Riley. L. Anson-Cartwright, J. C. Cross. Nat. Genet. 18, 271 (1998).
|[00060] 23. S. L. Palmieri, W. Peter, H. Hess, H. R. Schöler. Dev. Biol. 166, 259 (1994); H. R. Schöler. G. R. Dressier, R. Balling. H. Rohdewohld, P. Gruss. EMBO J. 9, 2185 (1990).
|[00061] 24. D. G. Wilkinson, S. Bhatt, B. G. Herrmann, Nature 343, 657 (1990).
|[00062] 25. S. A. Duncan et al., Proc. Natl. Acad. Sci. U.S.A. 91, 7598 (1994).
|[00063] 26. A. Nagy and J. Rossant, in Gene Targeting, A. L. Joyner Ed. (IRL Press. Oxford, 1993), pp. 147-180; S. A. Wood, N. D. Allen, J. Rossant, A. Auerbach, A. Nagy, Nature 365, 87 (1993).
|[00064] 27. A.-K. Hadjantonakis, M. Gertsenstein, I. Ikawa, M. Okabe, A. Nagy, Mech Dev. 1998 August;76(1-2):79-90.
|[00065] 28. K. J. P. Griffin, S. L. Amacher, C. B. Kimmel, D. Kimelman, Development 125, 3379 (1998); K. Griffin, R. Patient, N. Holder, ibid. 121, 2983 (1995).
|[00066] 29. M. E. Pownall, H. V. Isaacs, J. M. Slack, Curr. Biol. 8, 673 (1998); H. V. Isaacs, M. E. Pownall, J. M. Slack, EMBO J. 17, 3413 (1998).
We claim:
|1. An isolated preparation of mouse trophoblast stem cells which (i) are capable of indefinite proliferation in vitro in an undifferentiated state; (ii) are capable of differentiation into cells of the trophoblast lineage in vivo, and (iii) are diploid trophoblast cells.
|2. An isolated preparation as claimed in claim 1 wherein the cells are characterized by expression of the genetic markers Errβ, Cdx2 and Fgfr2.
|3. The isolated preparation according to claim 1 which is derived from or comprised of cells that have been genetically modified by genetic engineering techniques.
|4. An isolated cell preparation as claimed in claim 3 modified by introducing mutations into genes in the cells or by introducing transgenes into the cells.
|5. A method for producing a preparation of mouse trophoblast stem cells as claimed in claim 1 comprising culturing early postimplantation trophoblast cells or cells of a blastocyst from a mouse on a feeder laser in the presence of exogenous FGF4 and heparin, said feeder layer selected from the group consisting of fibroblasts or medium conditioned by fibroblasts.
|6. A method for producing differentiated cells of trophoblast lineages comprising culturing early postimplantation trophoblast cells or cells of a blastocyst from a mouse as claimed in claim 5 and inducing differentiation of the stern cells to cells of trophoblast lineages by removing the FGF4, the heparin, or the feeder layer.
|7. A method as claimed in claim 5 wherein the early postimplantation trophoblast cells or cells of a blastocyst are transgenic.
|8. A method as claimed in claim 5 wherein the feeder layer is a confluent fibroblast layer or a medium conditioned by primary embryonic fibroblast cells.
|9. A method as claimed in claim 5 wherein the feeder layer comprises primary mouse embryonic fibroblast (EMFI) cells or STO cells.
|10. A method as claimed in claim 5 wherein the FGF4 is recombinant FGF4.
|11. A method for producing chimeric conceptuses or placenta comprising the steps of producing a preparation of mouse trophoblast stem cells as claimed in claim 5 and further comprising the step of introducing cells frown the preparation into a non-human blastocyst or aggregating the cells with an early stage non-human embryo to produce chimeric conceptuses or placenta.
|12. A method as claimed in claim 11 wherein the chimeric conceptuses or placenta are engineered to carry selectable markers or genetic alterations.
|13. A method as claimed in claim 11 wherein cell lines are derived from the chimeric conceptuses or chimeric placenta.
|14. A method for producing a preparation of mouse trophoblast stem cells as claimed in claim 1 comprising:
(a) isolating extraembryonic cells from embryos or blastocysts from a mouse;
(b) culturing the extraembryonic cells on a feeder layer in the presence of exogenous FGF4 and heparin to thereby obtain an isolated preparation of mouse trophoblast stem cells, said feeder layer selected from the group consisting of fibroblasts or medium conditioned by fibroblasts.
|15. A method for producing differentiated cells of trophoblast lineages comprising culturing extraembryonic cells according to claim 14, and inducing differentiation of the stem cells to cells of trophoblast lineages by removing the FGF4, the heparin, or the feeder layer.
|16. A method for screening for potential therapeutics that modulate trophoblast development or activity comprising subjecting an isolated preparation of mouse trophoblast stem cells which (i) are capable of indefinite proliferation in vitro in an undifferentiated state; (ii) are capable of differentiation into cells of the trophoblast lineage in vivo, and (iii) are diploid trophoblast cells, to a test substance, and comparing the effect of the test substance to a control to determine if the test substance modulates trophoblast development or activity.


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Watch this weekend's weather forecast: Frosty, cold, but mostly dry
PUBLISHED: 10:00 16 December 2017
Wondering what the weather has in store for us this weekend? Watch our three-minute Met Office video forecast.
A repeat of last weekend's snowy scenes is unlikely but not impossible, but Saturday is expected to be the colder of the two days.


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Original post by: Yasir ,
I also have the same problem with my 5d mark iii error 30 keeps popping out even if I replace the battery. So I removed the lens and raised the camera mirror up, I found that the sensor cover was squeezed. I straightened it. Buh the upper one is not as tight as it was. So every 3-4 clicks then error pops out again..


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- You have a project or an idea that we can help you implement.
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The best and most comfortable way to talk to us is to send us an email to firstname.lastname@example.com.
Please, point out what are your goals, what are the deadlines, and what budget you have. We'll get back to you within 24-48 hours.


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A striking neo-modernist design, Cube Lofts speaks to its name by displaying a cube-like structure made of dark steel and glass. A great work of architecture among Toronto's loft buildings, Cube Lofts is also a boutique residence. Rising a sizeable 6 storeys and home to just 21 units, Cube Lofts could be the exclusive residence in Little Italy you've been looking for. Presenting a great diversity of layouts, at Cube Lofts you'll find one and two bedroom suites as well as both single and multi-level loft spaces. The top floor of the building has two penthouse suites with over 2,000sqft of space. All lofts paces are home to open concept living spaces and kitchens, with floor-to-ceiling windows, hardwood floors and cabinetry. With space for a home office, as well as coming equipped with a rooftop deck, this condo is really at the forefront of the Toronto Real Estate scene. Known for its quaint streets, Little Italy can offer you a never-ending list of possibilities. Mod Club Theatre is a great spot for musical concerts and The Royal has all the latest films. Situated on College Street, Cube Lofts is also a short walk to spending your night bar hopping. Visit Trinity Bellwoods Park is a great place for a stroll or picnic, as well as Dufferin Grove Park, which is also home to a weekly Farmer's Market.
Building Details 799 College St
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About 799 College St, Toronto
There are a lot of great restaurants nearby 799 College St, like Bistro in a Box, Ghazale and The Fish Store & Sandwiches, just to name a few. Grab your morning coffee at Tim Hortons located at 668 College Street. Groceries can be found at Metro which is nearby and you'll find Stephanian Pharmacy a 11-minute walk as well. The Royal Cinema is only at a short distance from Cube Lofts. If you're an outdoor lover, condo residents of 799 College St are nearby from Fred Hamilton Park, Bickford Park and Dufferin Grove Park.
Living in this Little Italy condo is made easier by access to the TTC. OSSINGTON STATION - EASTBOUND PLATFORM Subway stop is only a 9 minute walk. There is also COLLEGE ST AT OSSINGTON AVE Light RailStop, a short walk, with (Bus) route 306 Carlton, and (Light Rail) route 506 Carlton nearby. If you need to get on the highway often from Cube Lofts, Gardiner Expressway and Dowling Ave has both on and off ramps and is within a few minutes drive.


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ALLETE Clean Energy to Build Montana Wind Energy Facility to Supply Electricity to NorthWestern Energy under Power Sale Agreement
Project will expand wind portfolio to more than 700 megawatts across six states
ALLETE Clean Energy acquired the South Peak project from Peak Clean Energy and will manage construction of the wind facility, scheduled to be online by late 2019. Energy from South Peak will be delivered to
"We're excited to be working with our newest customer in
The South Peak project will utilize GE wind turbines and will be built adjacent to NorthWestern Energy's existing Spion Kop wind facility. It will deliver energy via a direct connection to NorthWestern's transmission grid, and has landowner agreements and design elements in place.
"We are delighted that ALLETE Clean Energy has selected GE's advanced technology 2.X MW platform for this project, along with a 15-year servicing agreement," said
To qualify for federal renewable energy production tax credits, the project will utilize a share of wind turbines ALLETE Clean Energy purchased in 2016 that meet the standards for the production tax credit "safe harbor" provision. The safe harbor turbine investment allows ALLETE Clean Energy to pursue its three-pronged production tax credit strategy that includes building and operating new wind farms based on long-term power sale agreements, building wind farms for other companies through a build-transfer structure, and refurbishing existing wind farms while extending power sale agreements.
"ALLETE recently raised its average annual earnings per share growth outlook, and a big reason for that is the solid performance of
In addition to developing this new project with
ALLETE Clean Energy was established in 2011 to acquire or develop capital projects to create energy solutions by way of wind, solar, biomass, hydro, natural gas, shale resources, clean coal technology and other emerging innovations.
The statements contained in this release and statements that
View source version on businesswire.com: http://www.businesswire.com/news/home/20180315005094/en/
ALLETE Inc.
Media Contact:
Amy Rutledge, 218-723-7400
Manager, Corporate Communications
Investor Contact:
Vince Meyer, 218-723-3952
Director, ALLETE Investor Relations & Treasury


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We're so confident we'll match you with the right mattress, you can take it home and sleep on it for 365-nights. If it's not a perfect fit, we'll exchange it for one that is.
- Months 1-4: 100% credit toward exchange to a different mattress.
- Months 5-8: 75% credit toward exchange to a different mattress.
- Months 9-12: 50% credit toward exchange to a different mattress.
1-Year Comfort Guarantee – It will take time for your body to adjust to your new mattress, so you should sleep on it for a minimum of 30-nights to allow for this adjustment period. If you are not comfortable within 365-nights of the original delivery date, simply come into one of our locations with your original receipt and we'll exchange it for one that is. Subject to the above restrictions, our 365-night comfort guarantee is valid for one exchange per purchase, which must done within 365-nights of the delivery date of your original mattress. You may exchange your mattress once, with a $149 sanitation fee and re-delivery fee of $79. Exchanges done within 30-days of delivery will be subject to additional $99 fee. Exchanges will only be accepted on mattresses in good condition – no tears, stains, bed bugs, holes, odors (such as pet and smoke), or soiling of any kind. We highly recommend our water proof mattress protector to keep your mattress clean and hypoallergenic.
Our large selection of mattresses makes the exchange process easy, therefore there are no refunds.
Do not remove the law tag from the mattress or foundation, as it will void the 365-night comfort guarantee. Please note that our 365-night comfort guarantee is not valid on special order sizes (split king, cal king, full xl, and twin xl), warranty exchanges, clearance items, floor models, motion bases, bed frames, pillows, mattress protectors, sheets, toppers, furniture, and other accessories. See store associate details.


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"This body is made for being bold, powerful, and sexy," model Ashley Graham says in the latest commercial for Lane Bryant's #ThisBody campaign. "This body is made for proving them wrong," says Precious Lee. But, apparently, certain major television networks aren't of the belief that #ThisBody was even made to be seen.
According to TMZ, the clothing retailer's new ad has been rejected by both NBC and ABC. The 30-second spot features several plus-size models, including Sports Illustrated swimsuit issue cover girls Graham and Lee, posing, working out, laughing, and, in one instance, nursing a baby — all in various states of undress.
TMZ says its sources "firmly believe" that the commercial was turned down due to the size of the women it depicts, not the nudity — citing similar ads for retailers like Victoria's Secret that feature nearly nude "straight-size" models. But I'm not so sure.
One thing I've definitely never seen in a Victoria's Secret ad is a model breastfeeding. Not that there's anything wrong with it — there isn't! But public breastfeeding has been such a controversial, hot-button issue for so long that including it in the commercial almost feels like Lane Bryant is making some kind of political or social statement. In that respect, I can kind of see where a TV exec who's trying to avoid controversy is coming from in not wanting to air it.
I also can't think of a Victoria's Secret ad where the models are hanging out together completely naked, like they are in Lane Bryant's. Again, I'm not personally offended — but, you know, we're talking national television, and this is a really big country.
Yes, I do think it's important to our society that a wider array of body types is shown onscreen — we can't all be Angels. But I also wouldn't say that Lane Bryant was avoiding controversy when it created this commercial. Just look at all the attention it's getting now.
In a statement to Cosmopolitan, the brand neither confirmed nor denied that NBC and ABC had banned the ad, saying, "The THIS BODY campaign was meant to be a fun way for us to celebrate and honor women of all shapes and sizes. What is too much for some does not hold true for others. All women should be celebrated and feel empowered to express themselves as they see fit. We want her to know she can attract as much media attention, look just as striking as any woman, and decide what beautiful means to her. The THIS BODY commercial holds nothing back. It is a true celebration of women of all sizes doing what makes THEM feel beautiful whether it's breastfeeding their newborn, flaunting their bodies the way they see fit, breaking down barriers all around and simply being who they are or want to be!"
We've reached out to Lane Bryant ourselves and will update this post when we hear back.
UPDATE, 4:30pm:
When asked specifically about whether Lane Bryant's commercial had, in fact, been rejected by NBC and ABC, a spokesperson said:
"The first edit was turned down out of hand and while we clearly created the commercial to air we will not edit it as we believe it's a beautiful and appropriate expression of women's bodies. We do plan to air this through our own media and through digital channels where they find it as acceptable."
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Imagine being the head football coach of the Penn State Nittany Lions, having a generational type of talent like Saquon Barkley, and knowing you have the former number-one running back recruit in the country, Miles Sanders. That's exactly the situation James Franklin was in two seasons ago. Following the most recent NFL Draft, where the Eagles took two Nittany Lions with their five picks (they'd sign two more after the draft's conclusion), PSU's head coach, James Franklin spoke with Fran Duffy and Amy Franklin of Eagles Draft Central.
Two Pennsylvania guys, who were former teammates, now play on opposite sides, and hopefully, we'll see some great duels over the next few seasons with Sanders donning the midnight green.
'You're going to love them (both Sanders and defensive end Shareef Miller). They're great players, but they're better people and you're going to really enjoy getting to know these guys over the next couple of years' Franklin said on Saturday.
Sanders didn't earn a starting spot in the Nittany Lions offense until this past season when he was a junior. Once he was given his opportunity, he responded with 220 rushing attempts, 1,274 yards, and nine touchdowns.
These days, the NFL doesn't seem as excited as they once were to go with running backs early unless they're certain they're once in a generation types of tailbacks. Sanders may not be seen as that, which is why he slipped to Round 2, but by joining a nice stable with Philadelphia, and by having Duce Staley to guide him, we may be looking at one of those 'steals' a la Alvin Kamara.
Philly's style of play seems to be suited for Sanders' skill set. Might he flourish in Year 4 of the Doug Pederson and Carson Wentz era? We say he's got an awesome shot to do so.


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The Winter Not-So Blues (Kat)
If you're a first year like me, when you think of fresh starts, you probably think of flipping the calendar to February; where a 'first day back' filled with whispers of summer adventure, long queues at the Co-Op and the remnants of patchy sunburn, awaits. A completely clean slate, with new subjects, new classes and a certain apprehensiveness when it comes to locating those new tutorial rooms…
But, as most of you reading probably realise, at uni you only have to flip the calendar to July for the chance at a clean slate. Being the jaffy (try urban dictionary if you're confused) that I am, it feels bizarre to start again mid-year. 12 weeks go by and you feel like you're finally starting to make friends in your classes, then *poof*, the break happens and you don't know anyone all over again. Strangely enough though, I feel excited about semester two. I've got the kind of renewed motivation that only comes after a decent break – and even if it disappears within the first week like always, it's enough to remind me why I'm studying in the first place.
The thing about this 'fresh start' is that it's not quite like all the others. Yes, I'll probably still get lost trying to find my new classes, and that first day back will probably still involve a long queue at the Co-Op (!!), but this time, a little experience is on our side. This time, I already know I can drop by House of Cards when I'm keen for caffeine. This time, I know that the Systems Gardens is the best place to go when you need a little peace and quiet. This time, I even know where to locate my favourite study nook (I think I could write an ode to Arts West, honestly!). Because this time, the things that were so dazzlingly new a few months ago, are now refreshingly familiar. And because of that, I wonder what I will see and do this semester, now that I'm not quite so overwhelmed?
What do you think?


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In the event that you are encountering sensitivities or experiencing skin illnesses because of the garments you wear, the time has come to reevaluate and change to an alternate texture or apparel material to buy. Move to natural cotton to stay away from different skin concerns.
In the event that the coconut tree is viewed as the "tree of life," at that point cotton is the "texture of life." This is a direct result of a wide utilization of cotton, from the space to the kitchen. In any case, because of business developing systems, regular cotton may contain contaminations that could trigger ominously helpless reactions in a couple people. This is the place characteristic cotton ends up being valuable.
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Therefore, a kid needs to be comfortable with his parents – something parents leave no stone unturned to ensure. Kids love TV, no chance cartoons could ever make them unhappy. They make friends based on their tastes and although friends do argue, they fight and forget in a matter of minutes. What remains to take care then is their wardrobe. The chemicals in kids' clothes can cause irritation, itching and other harmful effects on the sensitive skin of kids. Not only does this lead to skin infections and rude behavior, it may also result in an unpleasant personality due to constant irritation. We (organicandmore.com) are organic cotton fabric supplier across worldwide.
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Source: 3 Benefits of Organic Cotton Baby Clothing | Why it is better than others?


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If you told me Joel Stave would pass for almost 300 yards and two touchdowns against the Ohio State secondary, I probably would've told you the Badgers' shot at pulling off the upset were pretty high.
Unfortunately for Stave, his one interception of the day allowed the Buckeyes to extend the lead to 31-14 late in the third quarter. Even taking into account the wildly productive connection between Stave and Jared Abbrederis all game long, Wisconsin is not built to operate at such a deficit.
With vague questions lingering about Braxton Miller's ability to jump back into the action and operate like the fully operational Death Star—and not the partially built one in Episode 6—the opening minutes would prove crucial. Miller quickly dispelled any notions of rust, zinging a laser of a touchdown pass on a skinny post to Evan Spencer, who zoomed by freshman corner Sojourn Shelton as if he wasn't there.
Later in the quarter, Stave found Abbrederis as the pocket began to collapse around him (who finished with a whopping 207 yards on 10 receptions), resulting in a 36-yard score and Bradley Roby on the ground in the 6'2'' receiver's wake.
Wisconsin defensive coordinator Dave Aranda's side of the ball had a rough go of it in terms of getting pressure on Miller. A few minutes after the Abbdrederis score, Miller rolled to his left and rocketed yet another laser, this time to Devin Smith in the corner of the end zone for six.
The Buckeyes added a field goal with 4:33 to go in the second, extending the lead to 17-7 and necessitating a response from the Badgers. Once again, Stave was up to the challenge, as he found Sam Arneson for an 11-yard touchdown pass with 1:30 to go in the first half. Entering the half down 17-14, on the road, with Miller looking like he was picking up exactly where he left off, would have been a perfect outcome for the underdogs.
Of course, the half did not end without more fireworks. Miller hit the speedy Philly Brown on a 40-yarder with one second left, sending the Badgers into the half down 10 and perhaps reminding UW fans of Miller's late game touchdown heave in the same stadium two years ago.
Other than Miller's ability to exploit UW's vulnerable secondary with ease, the biggest issue confronting the Badgers was the inability to do much on the ground, which is of course where they do their business. Melvin Gordon finished the day with 74 yards on 15 carries, making for a respectable mark of 4.9 yards per carry. However, his longest gain of the day was just 16; for a player who has made a living busting off long gains, the absence of such in this contest was one of several death knells for the Badgers in Columbus.
In even worse news for the Badgers, senior tailback James White managed just 31 yards of his own, albeit on only eight carries. White is no slouch when it comes to making big plays either, as his dynamic touchdown run against Purdue last week showed. Nonetheless, with Gordon unable to shake free, the Badgers needed more tough yardage from their senior back.
With that said, as Michigan fans know, getting production on the ground is contingent upon good blocking. For most of Saturday night, a Buckeye front seven that has been universally deemed talented but young and "raw" stuffed the UW ground game, forcing Stave to beat the Buckeyes through the air.
To Stave's credit, he often did just that, albeit mostly to just one receiver. On that front, I have to say that although I haven't seen nearly as much of Penn State's Allen Robinson—who many deem the best receiver in the conference—it's difficult for me to say that Abbrederis doesn't deserve that distinction.
With the Badgers down 10 entering the second half, an early third quarter score would be a healing salve on a 10-point wound that threatened to become fatal.
The Badgers forced a Buckeye punt, but proceeded to go three-and-out after failing to convert on 3rd & 6. Wisconsin's front seven showed its resolve on the ensuing Buckeye drive, giving up three yards on first, second and third down before stuffing Carlos Hyde on 4th & 1.
Wisconsin thus started their next drive at their own 39, but that field position was immediately squandered. Ohio State linebacker Curtis Grant sacked Stave on first down for a loss of nine. Then, the dam took on its biggest fissure in the game to that point.
Bradley Roby reeled in Stave's only interception of the game, returning it to the Wisconsin 32. The Buckeyes rushed just four on the play, but Ohio State DE Adolphus Washington badly beat left tackle Tyler Marz to the outside. Washington hit Stave right as he threw, leading to a wobbly duck of a pass to an area of the field with nary a Badger to be found. In truth, you can't even fault Stave for this one; Washington is a great player, but you simply can't get beat like that, especially with the Buckeyes only bringing four. Easier said than done, of course.
Nonetheless, with an opportunity to hold the Buckeyes to just a field goal try, Wisconsin gave up a first down on 3rd & 8 at their own 15. This might not have mattered in the end, but being down two scores instead of three would have been a small victory there heading into the final quarter.
Nonetheless, with an opportunity to hold the Buckeyes to just a field goal try, Wisconsin gave up a first down on 3rd & 8 at their own 15. This might not have mattered in the end, but being down two scores instead of three would have been a small victory there heading into the final quarter.
The UW defense had yet another chance to hold the Buckeyes, on third & goal from the one-yard line. Miller pulled the ball from Hyde, and with what appears to be LB Joe Schobert in his face, surgically darted a perfect low ball between Dezmen Southward and Darius Hillary, right into the hands of Philly Brown.
From there, the game of catch-up took an extra order of magnitude of desperation.
I don't remember what OSU was doing defensively (and don't have the game in my DVR), but the Badgers finally found some success on the ground, with Gordon and White picking up gains of eight, four, 10 and five on UW's ensuing drive. Following a holding penalty on Bradley Roby, White popped off a 17-yard score to give the Badgers life with 13:47 to go. The 9-play, 75-yard touchdown drive was Wisconsin's best of the night.
Miller started the next Buckeye drive with a 15-yard scamper, an "oh no" moment if there ever was one. The Buckeyes drove down to the Wisconsin 28, where they were met with a 3rd & 4. Backup nose tackle Warren Herring brought Miller down on the play, forcing a punt. As the announcer in Little Giants said: Mr. Momentum may be changing his address.
The two squads then traded three-and-outs, leaving Gary Andersen's team just 4:38 to score 10 points. Stave completed a big 3rd & 9 pass to Abbrederis (who else) to keep the drive alive. Stave hit Abbrederis again for 23 and Alex Erickson for 11, but the drive fizzled out at the Buckeye 24. This time, placekicker Kyle French put a 42-yarder through the uprights to cut the lead to seven with 2:05 to go.
Fortunately for Andersen, the Badgers had all three of their timeouts, and he used every single one of them. The Badgers forced a punt, and were left with 1:29 to go 90 yards. Given Abbrederis's big play ability, this wasn't an inconceivable outcome. However, the Buckeyes certainly knew this; as such, Stave would need to connect with his secondary targets if the Badgers were truly going to make the Ohio Stadium crowd sweat in the Columbus night.
After an incompletion on first down, a fumble on second, a completion for 13 yards on 3rd down, the Badgers were met with a 4th & 4 for the ballgame. Stave was not able to target Abbrederis on his first three attempts, and the fourth would be no different; OSU's Doran Grant broke up Stave's attempt to Jeff Duckworth, ending the comeback effort in four efficient downs.
The Badgers fought hard, but the Buckeyes were simply too much at the skill positions for the leaky UW secondary to handle, and the Badgers ground game never really took off. For the first time all season, the UW offense was without a single 100+ yard rusher.
If the Badgers are to have a shot at a fourth straight Big Ten title, they will need to win out and hope for two Buckeye conference losses. The way things are going, that seems unlikely; Saturday was likely the coronation of the Buckeyes upon the Leaders division throne.
Miscellaneous Notes:
- Like Michigan, the Badgers' pass rush has been less than fearsome to date. Although Miller was able to avoid trouble a majority of the time, backup NT Warren Herring picked up two sacks on the day.
- Chris Borland, the James Laurinaitis of Badger linebackerdom, picked up a whopping 16 tackles in this one (10 solo), including half of a TFL.
- The Badgers have to be kicking themselves about OSU touchdown at the end of the first half. Keep them off the board there and perhaps the second half plays out another way.
- Kyle French's missed field goal from 32 yards out did nothing to assuage Badgers fans concerns about the placekicking. The miss seemed like a minor footnote of an event at the time, but given that the drive started with a 64-yard reception by Abbrederis (about 47 yards coming via YAC), French's miscue was an ominous moment for a Badger team that was simply looking to hang on as long as possible.
- I have to say it again: Jared Abbrederis is as impressive a receiver as any I've seen this season.
- From the Buckeye perspective: Miller's ability to move around and maintain velocity on his downfield throws is positively Vick-esque. Anyone who was calling for Kenny Guiton to get the start (I didn't pay enough attention to know if they exist, but, they probably did) feels pretty foolish right now.
- Miller's Badger counterpart squashed much of the doubters' sentiments after his performance Saturday night. The lone interception proved costly, but when you figure in the road environment and the absence of the usually robust running game, it's a wonder he only erred once.
- At 3-2 (1-1), the Badgers have a much needed bye week coming up before getting Pat Fitzgerald's Wildcats at Camp Randall on Oct. 12. As important as Saturday night was for the Badgers, the next one will go a long way toward determining the tenor of the rest of the season for UW. I wouldn't have said this at the start of the season, but the upcoming three-game stretch against Northwestern, at Iowa and at Illinois will prove to be a legitimate challenge. I'm sitting here trying to think what would be an acceptable record there for the Badgers, but I'm not really sure anymore. Is Illinois really as good as they've looked against Cincinnati and Miami (OH)? I don't know, but the Illini's trip to Lincoln this week will do a little bit to answer that question. Many, many points will be scored, that much is certain.
- On the other hand, the Buckeyes travel to Evanston this coming Saturday, where College Gameday will be setting up shop. The Wildcats did not exactly impress in their last two victories against Western Michigan and Maine, but Fitzgerald's squad is likely OSU's biggest test until their trip to Ann Arbor, depending on how bullish you feel about Iowa, Illinois (where the Buckeyes play on the road this season) and Penn State, and how down you are vis-a-vis Michigan's recent struggles. Either way, this should be another good one and a major test for a surging Northwestern football program.