text:
|Publication number
|Publication type
|Application number
|Publication date
|2 Jun 1992
|Filing date
|14 Feb 1991
|Priority date
|6 Jun 1990
|Fee status
|Publication number
|Henry J. Lee
|Original Assignee
|Asten Group, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (22), Referenced by (34), Classifications (15), Legal Events (11)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation-in-part of my copending application Ser. No. 07/534,164 entitled PAPERMAKERS FABRIC WITH STACKED MACHINE DIRECTION YARNS, filed Jun. 6, 1990, which application is incorporated by reference herein as if fully set forth.
The present invention relates to papermakers fabrics and in particular to fabrics comprised of flat monofilament yarns having a high aspect ratio of cross-sectional width to height.
Papermaking machines generally are comprised of three sections: forming, pressing, and drying. Papermakers fabrics are employed to transport a continuous paper sheet through the papermaking equipment as the paper is being manufactured. The requirements and desirable characteristics of papermakers fabrics vary in accordance with the particular section of the machine where the respective fabrics are utilized.
With the development of synthetic yarns, shaped monofilament yarns have been employed in the construction of papermakers fabrics. For example, U.S. Pat. No. 4,290,209 discloses a fabric woven of flat monofilament warp yarns; U.S. Pat. No. 4,755,420 discloses a non-woven construction where the papermakers fabric is comprised of spirals made from flat monofilament yarns.
Numerous weaves are known in the art which are employed to achieve different results. For example, U.S. Pat. No. 4,438,788 discloses a dryer fabric having three layers of cross machine direction yarns interwoven with a system of flat monofilament machine direction yarns such that floats are created on both the top and bottom surfaces of the fabric. The floats tend to provide a smooth surface for the fabric.
Permeability is an important criteria in the design of papermakers fabrics. In particular, with respect to fabrics made for running at high speeds on modern drying equipment, it is desirable to provide dryer fabrics with relatively low permeability.
U.S. Pat. No. 4,290,209 discloses the use of flat monofilament warp yarns woven contiguous with each other to provide a fabric with reduced permeability. However, even where flat warp yarns are woven contiguous with each other, additional means, such as stuffer yarns, are required to reduce the permeability of the fabric. As pointed out in that patent, it is desirable to avoid the use of fluffy, bulky stuffer yarns to reduce permeability which make the fabric susceptible to picking up foreign substances or retaining water.
U.S. Pat. No. 4,290,209 and U.S. Pat. No. 4,755,420 note practical limitations in the aspect ratio (cross-sectional width to height ratio) of machine direction warp yarns defining the structural weave of a fabric. The highest practical aspect ratio disclosed in those patents is 3:1, and the aspect ratio is preferably, less than 2:1.
U.S. Pat. No. 4,621,663, assigned to the assignee of the present invention, discloses one attempt to utilize high aspect ratio yarns (on the order of 5:1 and above) to define the surface of a papermakers dryer fabric. As disclosed in that patent, a woven base fabric is provided to support the high aspect ratio surface yarns. The woven base fabric is comprised of conventional round yarns and provides structural support and stability to the fabric disclosed in that patent.
U.S. Pat. No. 4,815,499 discloses the use of flat yarns in the context of a forming fabric. That patent discloses a composite fabric comprised of an upper fabric and a lower fabric tied together by binder yarns. The aspect ratio employed for the flat machine direction yarns in both the upper and lower fabrics are well under 3:1.
The present invention is directed to a papermakers fabric which provides for usage of high aspect ratio yarns as structural weave components. The fabric comprises a system of flat monofilament machine direction yarns (hereinafter MD yarns) which are woven in a selected weave construction which provides stability to the fabric.
In a preferred embodiment, the system of MD yarns comprises upper and lower yarns which are vertically stacked. Preferably, the upper MD yarns define floats on the upper surface of the fabric and each upper MD yarn is paired in a vertically stacked orientation with a lower MD yarn. The lower MD yarns may weave in an inverted image of the upper MD yarns to provide floats on the bottom fabric surface or may weave with a different repeat to provide a different surface on the bottom of the fabric.
At least the upper MD yarns are flat monofilament yarns woven contiguous with each other to reduce the permeability of the fabric and to lock in the machine direction alignment of the stacking pairs of MD yarns. In the preferred embodiment, the same type and size yarns are used throughout the machine direction yarn system and both the top and the bottom MD yarns weave contiguously with adjacent top and bottom MD yarns, respectively. The stacked, contiguous woven machine direction system provides stability and permits the MD yarns to have a relatively high aspect ratio, cross-sectional width to height, of greater than 3:1; the aspect ratio preferably ranging from about 2:1 to 6:1.
It is an object of the invention to provide a papermakers fabric which utilizes high aspect yarns as structural weave components.
Other objects and advantages will become apparent from the following description of presently preferred embodiments.
FIG. 1 is a schematic diagram of a papermakers fabric made in accordance with the teachings of the present invention;
FIG. 2 is a cross-sectional view of the fabric depicted in FIG. 1 along line 2--2;
FIG. 3 is a cross-sectional view of the fabric depicted in FIG. 1 along line 3--3;
FIG. 4 is a cross-sectional view of a prior art weave construction;
FIG. 5 illustrates the actual yarn structure of the fabric depicted in FIG. 1 in the finished fabric showing only two representative stacked MD yarns;
FIG. 6 is a schematic view of a second embodiment of a fabric made in accordance with the present invention;
FIG. 7 is a cross-sectional view of the fabric depicted in FIG. 6 along line 7--7;
FIG. 8 is a cross-sectional view of the fabric depicted in FIG. 6 along line 8--8; and
FIG. 9 is a perspective view of a portion of the fabric illustrated in FIGS. 6-8.
Referring to FIGS. 1, 2, and 3, there is shown a papermakers dryer fabric 10 comprising upper, middle and lower layers of cross machine direction (hereinafter CMD) yarns 11, 12, 13, respectively, interwoven with a system of MD yarns 14-19 which sequentially weave in a selected repeat pattern. The MD yarn system comprises upper MD yarns 14, 16, 18 which interweave with CMD yarns 11, 12 and lower MD yarns 15, 17, 19 which interweave with CMD yarns 12, 13.
The upper MD yarns 14, 16, 18 define floats on the top surface of the fabric 10 by weaving over two upper layer CMD yarns 11 dropping into the fabric to weave in an interior knuckle under one middle layer CMD yarn 12 and under one CMD yarn 11 and thereafter rising to the surface of the fabric to continue the repeat of the yarn. The floats over upper layer CMD yarns 11 of upper MD yarns 14, 16, 18 are staggered so that all of the upper and middle layer CMD yarns 11, 12 are maintained in the weave.
As will be recognized by those skilled in the art, the disclosed weave pattern with respect to FIGS. 1, 2, and 3, results in the top surface of the fabric having a twill pattern. Although the two-float twill pattern represented in FIGS. 1, 2, and 3 is a preferred embodiment, it will be recognized by those of ordinary skill in the art that the length of the float, the number of MD yarns in the repeat, and the ordering of the MD yarns may be selected as desired so that other patterns, twill or non-twill, are produced.
As best seen in FIGS. 2 and 3, lower MD yarns 15, 17, 19, weave directly beneath upper MD yarns 14, 16, 18, respectively, in a vertically stacked relationship. The lower yarns weave in an inverted image of their respective upper yarns. Each lower MD yarn 15, 17, 19 floats under two lower layer CMD yarns 13, rises into the fabric over one CMD yarn 13 and forms a knuckle around one middle layer CMD yarn 12 whereafter the yarn returns to the lower fabric surface to continue its repeat floating under the next two lower layer CMD yarns 13.
With respect to each pair of stacked yarns, the interior knuckle, formed around the middle layer CMD yarns 12 by one MD yarn, is hidden by the float of the other MD yarn. For example, in FIGS. 1 and 3, lower MD yarn 15 is depicted weaving a knuckle over CMD yarn 12 while MD yarn 14 is weaving its float over CMD yarns 11, thereby hiding the interior knuckle of lower MD yarn 15. Likewise, with respect to FIGS. 1 and 3, upper MD yarn 18 is depicted weaving a knuckle under yarn CMD yarn 12 while it is hidden by lower MD yarn 19 as it floats under CMD yarns 13.
The upper MD yarns 14, 16, 18, are woven contiguous with respect to each other. This maintains their respective parallel machine direction alignment and reduces permeability. Such close weaving of machine direction yarns is known in the art as 100% warp fill as explained in U.S. Pat. No. 4,290,209. As taught therein (and used herein), actual warp count in a woven fabric may vary between about 80%-125% in a single layer and still be considered 100% warp fill.
The crowding of MD yarns 14, 16, and 18 also serves to force MD yarns 15, 17, 19, into their stacked position beneath respective MD yarns 14, 16, 18. Preferably MD yarns 15, 17, and 19 are the same size as MD yarns 14, 16, and 18 so that they are likewise woven 100% warp fill. This results in the overall fabric of the preferred embodiment having 200% warp fill of MD yarns.
Since the lower MD yarns 15, 17, 19 are also preferably woven 100% warp fill, they likewise have the effect of maintaining the upper MD yarns 14, 16, 18 in stacked relationship with the respect to lower MD yarns 15, 17, 19. Accordingly, the respective MD yarn pairs 14 and 15, 16 and 17, 18 and 19 are doubly locked into position thereby enhancing the stability of the fabric.
As set forth in the U.S. Pat. No. 4,290,209, it has been recognized that machine direction flat yarns will weave in closer contact around cross machine direction yarns than round yarns. However, a 3:1 aspect ratio was viewed as a practical limit for such woven yarns in order to preserve overall fabric stability. The present stacked MD yarn system preserves the stability and machine direction strength of the fabric and enables the usage of yarns with increased aspect ratio, in a preferred range of 2:1 to 6:1, to more effectively control permeability.
The high aspect ratio of the MD yarns translates into reduced permeability. High aspect ratio yarns are wider and thinner than conventional flat yarns which have aspect ratios less than 3:1 and the same cross-sectional area. Equal cross-sectional area means that comparable yarns have substantially the same linear strength. The greater width of the high aspect ratio yarns translates into fewer interstices over the width of the fabric than with conventional yarns so that fewer openings exist in the fabric through which fluids may flow. The relative thinness of the high aspect ratio yarns enables the flat MD yarns to more efficiently cradle, i.e. brace, the cross machine direction yarns.
For example, as illustrated in FIG. 4, a fabric woven with a single layer system of a flat machine direction warp having a cross-sectional width of 1.5 units and a cross-sectional height of 1 unit, i.e. an aspect ratio of 1.5:1, is shown. Such fabric could be replaced by a fabric having the present dual stacked MD yarn system with MD yarns which are twice the width, i.e. 3 units, and half the height, i.e. 0.5 units. Such MD yarns thusly having a fourfold greater aspect ratio of 6:1, as illustrated in FIG. 3.
The thinner, wider MD yarns more efficiently control permeability while the machine direction strength of the fabric remains essentially unaltered since the cross-sectional area of the MD yarns over the width of the fabric remains the same. For the above example, illustrated by FIGS. 4 and 3, the conventional single MD yarn system fabric has six conventional contiguous flat yarns over 9 units of the fabric width having a cross-sectional area of 9 square units, i.e. 6*(1u.*1.5u.). The thinner, wider high aspect ratio yarns, woven as contiguous stacked MD yarns, define a fabric which has three stacked pairs of MD yarns over 9 units of fabric width. Thus such fabric also has a cross-sectional area of 9 square units, i.e. (3*(0.5u.*3u.))+(3*(0.5u.*3u.)), over 9 units of fabric width.
In one example, a fabric was woven in accordance with FIGS. 1, 2 and 3, wherein the CMD yarns 11, 12, 13 were polyester monofilament yarns 0.6 mm in diameter interwoven with MD yarns 14-19 which were flat polyester monofilament yarns having a width of 1.12 mm and a height of 0.2 mm. Accordingly, the aspect ratio of the flat MD yarns was 5.6:1. The fabric was woven at 48 warp ends per inch with a loom tension of 40 PLI (pounds per linear inch) and 12.5 CMD pick yarns per inch per layer (three layers).
The fabric was heat set in a conventional heat setting apparatus under conditions of temperature, tension and time within known ranges for polyester monofilament yarns. For example, conventional polyester fabrics are heat set within parameters of 340� F.-380� F. temperature, 6-15 PLI (pounds per linear inch) tension, and 3-4 minutes time. However, due to their stable structure, the fabrics of the present invention are more tolerant to variations in heat setting parameters.
The fabric exhibited a warp modulus of 6,000 PSI (pounds per square inch) measured by the ASTM D-1682-64 standard of the American Society for Testing and Materials. The fabric stretched less that 0.2% in length during heat setting. This result renders the manufacture of fabrics in accordance with the teachings of the present invention very reliable in achieving desired dimensional characteristic as compared to conventional fabrics.
The resultant heat set fabric had 12.5 CMD yarns per inch per layer with 106% MD warp fill with respect to both upper and lower MD yarns resulting in 212% actual warp fill for the fabric. The finished fabric has a permeability of 83CFM (cubic feet per minute) as measured by the ASTM D-737-75 standard.
As illustrated in FIG. 5, when the fabric 10 is woven the three layers of CMD yarns 11, 12, 13 become compressed. This compression along with the relatively thin dimension of the MD yarns reduces the caliper of the fabric. Accordingly, the overall caliper of the fabric can be maintained relatively low and not significantly greater than conventional fabrics woven without stacked MD yarn pairs. In the above example, the caliper of the finished fabric was 0.050 inches.
It will be recognized by those of ordinary skill in the art that if either top MD yarns 14, 16, 18 or bottom MD yarns 15, 17, 19 are woven at 100% warp fill, the overall warp fill for the stacked fabric will be significantly greater than 100% which will contribute to the reduction of permeability of the fabric. The instant fabric having stacked MD yarns will be recognized as having a significantly greater percentage of a warp fill than fabrics which have an actual warp fill of 125% of non-stacked MD yarns brought about by crowding and lateral undulation of the warp strands. Although the 200% warp fill is preferred, a fabric may be woven having 100% fill for either the upper or lower MD yarns with a lesser degree of fill for the other MD yarns by utilizing yarns which are not as wide as those MD yarns woven at 100% warp fill. For example, upper yarns 14, 16, 18 could be 1 unit wide with lower layer yarns 15, 17, 19 being 0.75 units wide which would result in a fabric having approximately 175% warp fill.
Such variations can be used to achieve a selected degree of permeability. Alternatively, such variations could be employed to make a forming fabric. In such a case, the lower MD yarns would be woven 100% warp fill to define the machine side of the fabric and the upper MD yarns would be woven at a substantially lower percentage of fill to provide a more open paper forming surface.
Referring to FIGS. 6, 7 and 8, there is shown a second preferred embodiment of a fabric 20 made in accordance with the teachings of the present invention. Papermakers fabric 20 is comprised of a single layer of CMD yarns 21a, 21b interwoven with a system of stacked MD yarns 22-25 which weave in a selected repeat pattern. The MD yarn system comprises upper MD yarns 22, 24 which define floats on the top surface of the fabric 20 by weaving over three CMD yarns, under the next one CMD yarn 21a to form a knuckle, and thereafter returning to float over the next three CMD yarns in a continuation of the repeat.
Lower MD yarns 23, 25, weave directly beneath respective upper MD yarns 22, 24 in a vertically stacked relationship. The lower MD yarns weave in an inverted image of their respective upper MD yarns. Each lower MD yarn 23, 25 floats under three CMD yarns, weaves upwardly around the next one CMD yarn 21a forming a knuckle and thereafter continues in the repeat to float under the next three CMD yarns.
As can be seen with respect to FIGS. 6 and 8, the knuckles formed by the lower MD yarns 23, 25 are hidden by the floats defined by the upper MD yarns 22, 24 respectively. Likewise the knuckles formed by the upper MD yarns 22, 24 are hidden by the floats of the lower MD yarns 23, 25 respectively.
The caliper of the fabric proximate the knuckle area shown in FIG. 8, has a tendency to be somewhat greater than the caliper of the fabric at non-knuckle CMD yarns 21b, shown in FIG. 7. However, the CMD yarns 21a around which the knuckles are formed become crimped which reduces the caliper of the fabric in that area as illustrated in FIG. 8. Additionally, slightly larger diameter CMD yarns are preferably used for CMD yarns 21b, shown in FIG. 7, which are not woven around as knuckles by the MD yarns to eliminate any difference in fabric caliber. Preferably the diameter of the larger CMD yarn 21b equals the diameter d of the smaller CMD yarns 21a plus the thickness t of the MD yarns.
In one example, a fabric was woven in accordance with FIGS. 6-9, wherein the CMD yarns 21a, 21b were polyester monofilament yarns 0.6 mm and 0.8 mm, respectively, in diameter interwoven with MD yarns 22-25 which were flat polyester monofilament yarns having a width of 1.12 mm and a height of 0.2 mm. Accordingly, the aspect ratio of the flat MD yarns was 5.6:1. The fabric was woven at 48 total warp ends per inch with a loom tension of 40 PLI (pounds per linear inch) and 20 CMD total pick yarns per inch. The permeability averaged 90 CFM in the resultant fabric.
As best shown in FIG. 9, the high aspect ratio yarns 22-24 effectively brace the CMD yarns 21a in the weave construction. This bracing effect can be quantified in terms of the degree of contact arc Θ and contact bracing area, CBA, as follows: ##EQU1## where d=diameter of the CMD yarn Θ=the degree of arc over which there is contact between the MD and CMD yarns
w=width of the MD yarn
π=the constant pi.
The degrees of arc over which MD yarns 22-25 are in contact with CMD yarns 21a is dependent upon the spacing of the CMD yarns within the weave. For the above example, employing alternating 0.6 mm and 0.8 mm diameter CMD yarns with 0.2 mm thick MD yarns, the degree of contact arc can be maintained in a preferred range of between 60� to 180� by varying the pick count of the CMD yarns from 14 picks per inch to a maximum of 28.22 picks per inch.
In the preferred embodiment where the pick count is 20 picks per inch, the degree of contact arc Θ is approximately 101�. This results in a bracing contact area of approximately 0.79 mm2 at each knuckle in the fabric.
Applicant's use of high ratio aspect yarns, i.e. yarns having a width:thickness ratio of at least 3:1, provides for increased bracing contact of the flat MD yarns with the CMD yarns 21a. This is comparatively exemplified by modifying the equation for contact bracing area, CBA, to be defined in terms of the thickness of the MD yarns.
Since the MD yarn width w equals the thickness t of the MD yarn multiplied by the aspect ratio, w>3t for yarns having an aspect ratio greater than 3:1. Accordingly, fabrics made in accordance with the teachings of the present invention utilizing high aspect ratio MD yarns exhibit enhanced bracing of the CMD yarns by the MD yarns such that: ##EQU2##
The preferred inverted image weave of the lower MD yarns facilitates the creation of seaming loops at the end of the fabric which enable the fabric ends to be joined together. In forming a seaming loop, the upper MD yarns extend beyond the end of the fabric and the respective lower yarns are trimmed back a selected distance from the fabric end. The upper MD yarns are then bent back upon themselves and rewoven into the space vacated by the trimmed lower MD yarns. When the upper MD yarns are backwoven into the space previously occupied by the lower MD yarns, their crimp matches the pattern of the lower MD yarns, thereby locking the resultant end loops in position. Similarly, alternate top MD yarns can be backwoven tightly against the end of the fabric such that loops formed on the opposite end of the fabric can be intermeshed in the spaces provided by the non-loop forming MD yarns to seam the fabric via insertion of a pintle through the intermeshed end loops.
Since the top and bottom machine direction yarns are stacked, the resultant end loops are orthogonal to the plane of the fabric surface and do not have any twist. In conventional backweaving techniques, the loop defining yarns are normally backwoven into the fabric in a space adjacent to the yarn itself. Such conventional loop formation inherently imparts a twist to the seaming loop, see U.S. Pat. No. 4,438,788, FIG. 6.
A variety of other weave patterns employing the paired stacked weave construction of the instant invention may be constructed within the scope of the present invention. For example, in some applications it may be desirable to have MD yarn surface floats over four or more CMD yarns. Such fabrics are readily constructed in accordance with the teachings of the present invention.
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|U.S. Classification
|International Classification
|Cooperative Classification
|European Classification
|14 Feb 1991
Owner name: ASTEN GROUP, INC., A CORP. OF DE, SOUTH CAROLIN
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:LEE, HENRY J.;REEL/FRAME:005607/0392
Effective date: 19910208
|16 Nov 1993
|Certificate of correction
|22 Jun 1995
Owner name: ASTEN, INC., A CORP. OF DE, SOUTH CAROLINA
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text:
|Publication number
|Publication type
|Application number
|PCT number
|Publication date
|Nov 14, 2000
|Filing date
|Apr 21, 1994
|Priority date
|Apr 21, 1993
|Fee status
|Also published as
|Publication number
|Austin Gerard Smith, Peter Scott Mountford
|Original Assignee
|The University Of Edinburgh
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (4), Non-Patent Citations (18), Referenced by (116), Classifications (62), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a 371 of PCT/GB94/00848 filed Apr. 21, 1994.
This invention relates to methods of isolating and/or enriching and/or selectively propagating animal stem cells, genetically modified animal cells and animals for use in said method, transgenic animals providing a source of such cells and selectable marker constructs for producing genetically modified cells and transgenic animals.
Stem cells are progenitor cells which have the capacity both to self-renew and to differentiate into mature somatic tissues.
Embryonic stem cells are the archetypal stem cell, being capable of differentiating to form the whole gamut of cell types found in the adult animal. Such stem cells are described as pluripotential as they are capable of differentiating into many cell types. Other types of stem cells, for example bone marrow stem cells and epidermal stem cells, persist in the adult animal. These stem cells have a more restricted capacity for differentiation.
In general, when required for research purposes or for medical use, stem cells have to be isolated from tissue samples by various fractionation procedures, but even after careful segregation of cell types, these stem cell preparations consist of mixed cell types and while enriched for stem cells, include high proportions of differentiated cells which are not categorised as stem cells.
Furthermore, most stem cells cannot be grown readily in culture and when attempts are made to culture stem cells, the cells being cultured (which ordinarily contain a mixed population of cell types) grow at different rates and stem cells rapidly become overgrown by non-stem cell types. An exception is that embryonic stem cells from two specific strains of mice (129 and Black 6) can be cultured in vitro. Thus established lines of embryonic stem cells can be obtained by culturing early (31/2 day) embryonic cells from murine strain 129 and Black 6, or hybrids thereof.
There has developed a pressing need to isolate and maintain in vitro embryonic stem cells from other murine strains and more especially from other species including other laboratory animals (e.g. rats, rabbits and guinea pigs), domesticated animals (e.g. sheep, goats, horses, cattle, pigs, birds, fish, etc.) and primates. Similarly, numerous medical applications for other pluripotential cells such as haematopoictic stem cells also demand their isolation and culture in vitro.
However hitherto the problems associated with producing cultures of stem cells including the problem of producing cell populations of a satisfactorily low degree of heterogeneity and the problem of overgrowth in culture of non-pluripotent cells have not been solved. A particular problem associated with the continuing presence of certain differentiated cell types is that these can cause elimination of stem cells from the culture by inducing their differentiation or programmed cell death.
We have now developed a technique by which the aforementioned problems can be overcome.
According to one aspect of the invention there is provided a method of isolating and/or enriching and/or selectively propagating animal stem cells, which comprises maintaining a source of said cells under culture conditions conducive to cell survival, characterised in that the source of cells includes cells containing a selectable marker which is capable of differential expression in (a) stem cells and (b) cells other than the desired stem cells, whereby differential expression of said selectable marker results in preferential isolation and/or survival and/or division of the desired stem cells. In the context of this invention, the term "animal cell" is intended to embrace all animal cells, especially of mammalian species, including human cells.
Examples of stem cells include both unipotential and pluripotential stem cells, embryonic stem cells, gonadal stem cells, somatic stem/progenitor cells, haematopoietic stem cells, epidermal stem cells and neuronal stem cells.
In carrying out the method of the invention, the source of cells may include pluripotential cells having a positive selectable marker and expression of the said marker is used to permit isolation and maintenance of the plunrpotential cells. Alternatively, the source of cells may include a negative selectable marker which is expressed in cells other than the desired pluripotential cells and is used selectively to deplete the source of cells of cells other than the desired pluripotential cells.
The selectable marker may, for example, be a foreign gene, a cellular gene or an antibiotic resistance gene such as for example the bacterial neomycin resistance gene.
Alternatively the selectable marker may be a growth stimulating gene, for example an immortalising gene, an oncogene or a gene coding for the polyoma or SV40 T antigens or derivatives thereof, or the selectable marker may be a gene coding for a growth factor or a growth factor receptor or a signal transducing molecule or a molecule that blocks cell death.
In one particular embodiment the isolation and/or enrichment and/or selective propagation of the desired pluripotential cells is dependent on the presence of cells other than the desired pluripotential cells and the simultaneous maintenance of both cell types is dependent on expression of a selectable marker, in one or the other cell population, which is capable of rescuing cells that do not express the marker but which neighbour cells which do themselves express the marker. In this instance, the selectable marker may, for example, be the hypoxanthinc phosphoribosyl tranferase (HPRT) gene.
In another embodiment the selectable marker may be a gene encoding a product which is toxic per se, or a toxic gene product which is conditionally active in combination with a suicide substrate. An example of such a gene product is a herpes simplex virus thymidine kinase (HSV-TK) in combination with ganciclovir.
Expression of the selectable marker may be achieved by operatively inserting the selectable marker into an expression construct prior to introduction to the cell source, in which case expression of the selectable marker can result from the introduction of either a stable or transiently integrated construct. Alternatively, expression of the selectable marker results from operatively inserting the selectable marker into an endogenous gene of the cell source.
Various means of introducing the selectable marker may be employed, including introduction into the cells by transfection, lipofection, injection, ballistic missile, viral vector or by electroporation.
The source of the cells may be a single cell such as a fertilized oocyte, or it may comprise a mixture of cells, such as cells derived from an embryo, blood or somatic tissue of a normally bred or transgenic animal or cell line. In the latter case the selectable marker may be incorporated into the transgenic animal's genome.
Most preferably, in carrying out the method of the invention a gene or gene fragment operatively linked to and regulating expression of the selectable marker is/are associated with a pluripotential stage of cellular development. Such a gene or gene fragment may be active in pluripotential cells of the developing embryo, especially in the inner cell mass and/or primitive ectoderm, or may be active in adult stem cells.
In preparing a source of cells for use in accordance with the invention one of the following protocols may advantageously be adopted:
introducing into a source of cells containing stem cells, a selectable marker construct, wherein said selectable marker construct is adapted to operatively link to an endogenous gene which provides said differential expression, or
introducing into a source of cells containing stem cells, a selectable marker construct, wherein said selectable marker construct has been previously linked to one or more gene fragments which provide said differential expression.
The genetic marker preferably comprises a selectable marker operatively linked to a promoter which is differentially active in the desired pluripotent cells (e.g. primitive ectoderm). By "selectable marker" is meant a selectable gene which may be a foreign gene or a cellular gene which is not naturally expressed, or such a gene which is naturally expressed, but at an inappropriate level, in the target cell populations. This gene in use acts as a selection marker by adapting the phenotype of the target cell population in such a way that cells with the so-adapted phenotype may be enriched or depleted under particular culture conditions.
In the case where stem cells are embryonic cells it is preferred that the selectable marker is operatively linked to a promoter which is differentially active in stem (e.g. primitive ectoderm, primordial germ cells) and non-stem cells. Promoter and other cis-regulatory elements may be included in the expression construct prior to introduction into the cells or by targeting promoter-less constructs into specific genes by site specific recombination.
A wide variety of gene products may be relied upon for selective isolation and propagation of the desired stem cells, including markers which are designed to protect the desired cells from the effects of an inhibiting factor present in the culture medium. In this instance, the inhibiting factor can, for example, be an antibiotic substance which inhibits growth or reproduction of cultured cells, not expressing the gene (i.e. cells other than the desired cells). The selectable marker (e.g. HPRT) may also provide protection both for the desired cells in which it is expressed as well as other closely associated cells by means of metabolic rescue.
Alternatively the selectable marker may selectively permit the growth of stem cells. In this instance the marker may encode a growth factor, a growth factor receptor, a transcription factor, an immortalising or an oncogenic product (e.g. temperature sensitive simian virus 40 T antigen).
Alternatively, the selectable marker may be a cell surface antigen or other gene product which allows purification or depletion of expressing cells for example by panning or fluorescence-activated cell sorting (FACS). The invention thus enables stem cell populations to be obtained/maintained having a satisfactory degree of homology.
Alternatively the selectable marker may be a conditionally toxic gene for instance herpes simplex virus thymidine kinase [HSV-TK]. In this instance expression of the selectable marker is directed to cells other than the desired cells and not to stem cells. Cells other than the desired phenotype may be selectively depleted by addition of a lethal substrate (e.g. ganciclovir).
The genetic marker may be introduced into the source of cells by a variety of means, including injection, transfection, lipofection, electroporation or by infection with a viral vector.
Further, the source of cells may be produced by transfection extemporaneously, or the source of cells may be derived from a transgenic animal, e.g., the founder transgenic animal or an animal at least one ancestor of which has had the aforementioned genetic marker introduced into its genetic complement. In such transgenic animals it is possible for the marker to pass down the germ line and eventually results in the production of progeny, from the tissues of which (especially from the embryonic tissue) the required source of cells can be derived.
Thus according to further aspects of the invention, there is provided an animal cell capable of being cultured under appropriate selective culture conditions so as to enable isolation and/or enrichment and/or selective propagation of stem cells, characterised in that said cell contains a selectable marker wherein differential expression of the selectable marker in (a) the desired stem cells and (b) cells other than the desired stem cells enables selective growth of the desired stem cell to occur.
The invention further provides an animal cell capable of being cultured under selective culture conditions so as to grow as stem cells, characterised in that said cells contain stem cells containing a genetic marker, whereby a gene product associated with the genetic marker is produced and which under said culture conditions causes selective survival and/or division of the desired stem cells to occur.
The animal cells according to this aspect of invention are preferably characterised by possessing the preferred characteristics described above.
The invention further provides according to another aspect thereof, a transgenic animal having genetic characteristics such that it or its progeny, during embryonic development or later life, constitute a source of animal pluripotential cells as defined above.
Such transgenic animal may be produced according to the invention by introducing a genetic marker into a fertilised oocyte or an embryonic cell, or an embryonic stem cell in vitro, the genetic marker having the characteristics defined above, and utilising the resulting transformed oocyte or embryonic cell as a progenitor cell for the desired transgenic animal.
Vectors for use in producing an animal cell defined above form a further aspect of the invention.
Thus the invention further provides vectors for use in genetically modifying animal cells so as to produce transformed cells suitable for use as the source of cells for the method referred to above, said vector comprising a first genetic component corresponding to said selectable marker and a second genetic component which in the genetically modified animal cells (1) results in the said differential expression of the selectable marker from either a transiently or stably integrated construct or (2) enables site-directed integration of the selectable marker into a specific gene so as to provide operative coupling of the selectable marker with targeted endogenous gene regulatory elements.
Such vectors may be in the form of expression vectors in which said second genetic component includes control sequences which are differentially activated (a) in stem cells and (b) in cells other than the desired stem cells.
The invention covers vectors which when used to transform animal cells become integrated into the animal genome as well as vectors which do not become so integrated.
The expression vectors referred to above may comprise a DNA sequence coding for the afore-mentioned selectable marker operatively linked to a genetic control element, or sequence enabling targeting of a promoterless marker to an endogenous gene which is expressed differentially in the said stem cells and in cells other than the desired stem cells.
For the generation of pluripotential embryonic stem cells the expression constructs preferably comprise a DNA sequence coding for said selectable marker operatively linked or targeting to a genetic control element(s) which is associated with a stage of embryonic development prior to differentiation of pluripotential embryonic cells. Most preferably the genetic control elements derive from a gene specifically active in the inner cell mass of the mouse blastocyst, in primitive ectoderm, and in primordial germ cells of the early embryo.
In more detail, the present invention has resulted in the development of expression constructs which direct specific expression of selectable markers in stem cells and not in differentiated cell types. Having introduced an expression construct by transfection or via the generation of transgenic animals, stem cells present within mixed cell populations can be isolated by culturing in the presence of the selection agent in vitro, or by otherwise manipulating the culture conditions.
One example of a gene which displays a suitably restricted stem cell expression pattern and therefore may provide suitable "stem cell specific" regulatory elements for the expression of a selectable marker in accordance with the invention is the Oct4 gene.
Octamer binding transcription factor 4 is a member of the POU family of transcription factors (reviewed by Scholer, 1991). Oct4 transcription is activated between the 4- and 8-cell stage in the developing mouse embryo and it is highly expressed in the expanding blastocyst and then in the pluripotent cells of the egg cylinder. Transcription is down-regulated as the primitive ectoderm differentiates to form mesoderm (Scholer et al., 1990) and by 8.5 d.p.c. (days post coitum) is restricted to migrating primordial germ cells. High level Oct4 gene expression is also observed in pluripotent embryo carcinoma and embryonic stem cell lines, and is down-regulated when these cells are induced to differentiate (Schmoler et al., 1989; Okamoto et al., 1990).
Selectable marker genes under the control of the Oct4 promoter may, according to the in vention, be applied to the isolation of embryonic stem cell lineages. Furthermore, reports describing low level Oct4 expression in some adult tissues (Takeda et al., 1992) may extend the utility of these expression constructs beyond embryonic stem cells to include other stem cells essential to tissue homeostasis and repair in other systems including the haematopoietic system. In the event that Oct4 is not expressed in somatic stem cells, other transcriptional regulatory elements, such as those associated with the haematopoietic stem cell specific antigen CD34, may be utilised in a similar manner.
Two specific approaches are provided according to the invention for generating the desired spatial and temporal restrictions in transgenic expression. The first approach is through the generation of transgenic animals in which a partially characterised Oct4 gene promoter fragment (Okazawa et al., 1991) is employed to drive stem cell specific transcription of the selectable marker. An appropriate selectable marker is the neomycin phosphotransferase gene which confers resistance to the antibiotic G418. An alternative is to utilise a selectable marker which is associated with the production of a gene product which can counteract a deficiency in a metabolite, e.g. the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in HPRT-deficient cells (Hooper et al., 1987). This approach may be advantageous in situations where stem cells require continuous support from closely associated differentiated cells. In this instance direct cell contact will permit metabolic rescue of the neighboring support cells by the stem cells despite the lack of selectable marker gene expression in the support cells.
The second approach utilise the endogenous Oct4 gene locus, and therefore the associated Oct4 gene regulatory elements, to link resistance marker gene expression as closely as possible with the endogenous Oct4 gene expression profile. This may be accomplished by high efficiency gene trap targeted mutagenesis of the Oct4 gene in embryonic stem cells. This approach provides more tightly regulated control of selectable marker gene expression by avoiding random integration site effects which often result in unpredictable expression patterns of randomly integrated constructs.
FIG. 1 depicts the Oct4-Neo-BS plasmid.
FIG. 2 depicts the Oct4-Neo-B-cfos plasmid.
FIG. 3 depicts the Oct4-tgtvec plasmid.
The invention will now be described in more detail in the following Example, with particular reference to the accompanying drawings of which FIG. 1 illustrates the structure of plasmid Oct-4-Neo-βS, FIG. 2 illustrates the structure of plasmid Oct-4-Neo-βfos and FIG. 3 illustrates the structure of the plasmid Oct4-tgtvec.
1. Isolation of OCT4 Promoter Sequences:
We screened a strain 129 mouse genomic lambda library with a 330 bp 5'Oct4 cDNA fragment. Several clones were isolated and screened by restriction analysis and Southern blot hybridization. A 1.4 kb Bam HI-Hind III fragment containing the Oct4 promoter element (Okazawa et al., 1991) was isolated from clone 1 and ligated into pBluescript II KS(-) (Stratagene) to generate pOct4 (5' genomic).
2. Construction of Plasmids:
To generate the Oct4-Neo promoter constructs an engineered Neomycin resistance gene (neo), designed to provide an Nco I restriction site at the translation initiation codon, was isolated from pLZIN (Ghattas et al., 1991) as a 1.1 kb Xba I-Sph I fragment encompassing encephalomyocarditus virus internal ribosome entry site sequence (EMCV-IRES, Ghattas et al., 1991) and 5'-Neo coding sequences and cloned into pSP72 (Promega Biotech). The Kpn I-Nco I EMCV-IRES sequence was replaced with a 1.3 kb Oct4 promoter fragment isolated from pOct4 (5' genomic) by Kpn I and subsequent partial Nco I restriction digest. Neo3'-coding, rabbit β-globin gene (intron) and SV40 polyadenylation sequences were isolated as a 1.7 kb Sph I fragment from 6P-IRESNeo-βS and ligated into the Sph I site to generate Oct4-Neo-βS (FIG. 1). To generate the Oct4-Neo-βfos construct (FIG. 2), an Oct4-Neo-βS Bam HI fragment incorporating the Oct4 promoter, neo gene and the rabbit β-globin intron was inserted 5' to a human c-fos genomic sequence. This 1.7 kb genomic sequence (Bal I-Sph I) encodes human c-fos mRNA 3' coding and non-coding sequences previously associated with mRNA destabilization (Wilson and Triesman, 1988), and, the c-fos polyadenylation sequence.
The Oct4-neo construct (Oct4-tgtvec) is designed for targetted integration into the Oct4 gene (FIG. 3). The Oct4 targetting construct contains 1.7 kb of 5'Oct4 gene sequence and 4.2 kb of 3'Oct4 gene sequence. Following homologous recombination this construct incorporates a lacZ-neomycin fusion gene (βgeo, encoding a bifunctional protein, Freidrich and Soriano, 1991) into the first intron of the Oct4 gene. Splicing from the splice donor sequence of the first exon-intron boundary to the integrated IRES-βgeo sequence is facilitated by the inclusion of a murine engrailed-2 splice acceptor sequence (Skarnes et al., 1992) immediately 5' to the IRES-βgeo sequence. Translation of the βgeo cistron of the Oct4-βgeo fusion transcript is facilitated by the inclusion of the EMCV-IRES immediately 5' to the βgeo coding sequence.
3. ES Cell Transfection and Colony Selection:
Mouse 129 ES cells (line CGR-8) were prepared and maintained in the presence of Differentiation Inhibiting Activity (DIA) or Leukemia Inhibitory Factor (LIF) as described by Smith (1991). Plasmid DNA for transfection was linearised by Sal I digest, ethanol precipitated and resuspended at 10-14 mg/ml in PBS. Following 10 hours culture in fresh medium, near confluent ES cells were dispersed by trypsinisation, washed sequentially in culture medium and PBS, and resuspended at 1.4�108 /ml in PBS for immediate transfection. Routinely, 0.7 ml of cell suspension was mixed with 0.1 ml DNA containing solution and electroporated at 0.8 kV and 3.0 μFD using a Biorad Gene Pulser and 0.4 cm cuvettes. Transfections were plated on gelatinised tissue culture dishes at 5-8�104 /cm2 in growth medium for 16 hours prior to the addition of selection medium containing 200 μg/ml (active) G418 (Sigma). Single colonies were picked 8-10 days post-transfection and transferred in duplicate into 24 well tissue culture plates for further expansion in growth medium containing 200 μg/ml G418.
Clonal cell lines bearing the Oct4-Neo-βS and Oct4-Neo-βfos constructs (referred to as Oct4-Neo cell lines) were grown for two days, washed twice with PBS and the medium replaced with fresh G418 medium with or without DIA. Cell lines which grew normally in the presence of DIA but did not survive in the absence of DIA were selected for expansion and further analysis.
Clonal cell lines bearing the Oct4-tgtvec targetting construct (referred to as Oct4-targetted cell lines) were expanded in duplicated 24 well plates. Once confluent, one series of cells were frozen for storage while the reminder were analysed by Southern analysis.
4. Further Characterisation of Oct4-Neo and Oct4-targetted Cell Lines:
Selected Oct4-Neo cell lines were assayed for ES cell growth and differentiation in DIA supplemented or non-supplemented medium at various G418 concentrations. Cells were plated at 1�104 /cm2 in 12 well tissue culture plates in the various media preparations and cultured for 6 days. Fresh medium was applied every 2 days until day 6 when cells were fixed and stained as previously described (Smith, 1991.) Oct4-targetted cell lines positive by genomic Southern analysis were analysed by lacZ staining and growth and differentiation in DIA supplemented or non-supplemented medium in 200 μg/ml G418.
5. Production of Embryoid Bodies from Oct4-Neo Cell Lines:
Embryoid bodies were generated by the hanging drop method (Hole and Smith, in press) and maintained in suspension culture for 2, 4, 6 or 8 days in the presence or absence of G418. Control embryoid bodies were generated from the parental cell line CGR-8 in the absence of G418. Embryoid bodies were then collected and transferred to gelatinised tissue culture dishes to enable adherence and expansion of the aggregates for analysis of contributing cell types. All embryoid bodies were maintained for 4 days in the absence of DIA and G418 prior to inspection.
6. Production of Chimeras from Oct4-Neo and Oct-4 Targetted Cell Lines:
Selected Oct4-Neo cell lines were cultured in the absence of G418 for 7 days prior to embryo injection as previously described (Nichols et al., 1990). Briefly, blastocysts for injection were collected 4 d.p.c. from C57BL/6 donors, injected with 10-20 cells and allowed to re-expand in culture prior to transfer to the uteri of pseudopregnant recipients. Chimeras were identified by the presence of patches of sandy coat colour on the C57BL/6 background. Male chimeras were test bred for transmission of the Oct4-Neo transgene. Transgenic mice were then crossed onto different genetic backgrounds.
7. Results
The Oct4-Neo-βS construct generated approximately 50 colonies/106 cells transfected while the Oct4-Neo-βfos construct generated approximately 10 fold fewer colonies. Three clones were selected on the basis of their differential survival in medium containing G418 (200 μg/ml) in the presence or absence of DIA. All three cell lines displayed apparently normal growth rates in DIA-supplemented G418 containing media and died when cultured in the absence of DLA in G418 medium. Cultures maintained in DIA supplemented G418 medium grew as essentially pure ES cells while cultures maintained in DIA supplemented medium in the absence of G418 grew as mixed cultures of ES cells and differentiated progeny closely resembling those of the parental CGR-8 line. Thus G418 selection eliminates differentiated cell types and allows propagation of pure stem cell populations. The three cell lines selected were designated Oct4-Neo-βS18, Oct4-Neo-βS21 and Oct4-Neo-βfos11. These cell lines have been introduced into host blastocysts and resulting chimaeras may be test bred. Similar results were obtained with ES clones targetted with the Oct4-tgtvec construct. In addition, histochemical staining of these cultures for β-galactosidase activity confirmed that expression of βgeo was restricted to undifferentiated stem cells (Mountford et al, 1994).
Embryoid bodies were generated from the Oct4-Neo cell line Oct4-Neo-βfos11 to examine the effect of G418 selection on mixed cell aggregates and to test the utility of the selection system in isolating ES cells from mixed cell populations. Embryoid bodies generated with both the experimental cell line (Oct4-Neo-βfos11) and the parental cell line (CGR-8) and cultured in the absence of G418 were composed almost entirely of differentiated cells with few if any ES like cells. In contrast, visual analysis of the expanded colonies revealed that the Oct4-Neo-βfos11 embryoid bodies cultured in the presence of G418 contained high proportions of ES cells. The feasibility of isolating stem cells from differentiating systems is thus confirmed.
8. Summary
ES cells capable of germ line transmission have previously been established from only 2 inbred strains of mice, 129 and C57BL/6. Combining the Oct4-neomycin selection scheme with established of ES cell isolation and propagation procedures (Evans and Kaufman, 1981; Martin, 1981; Nichols et al., 1990; Yoshida et al, 1994) should enable ES cell line derivation from previously non-productive mouse strains and other mammalian species in which Oct4 is differentially expressed.
Selection against non-stem cell phenotypes in mixed cell populations may be advantageous for several reasons. Firstly, selection against differentiated cells in mixed populations provides a method for extensive stem cell enrichment. Secondly, selective removal of differentiated cells prevents their overgrowth in the cultures. Thirdly, elimination of differentiated cells may enhance stem cell self-renewal due to the loss of differentiation inducing activity associated with differentiated cells.
1. Cell Culture
ES cells were routinely maintained in medium supplemented with Differentiation Inhibiting Activity (DIA) as described by Smith (1991). Embryoid bodies were formed by aggregation of ES cells in the absence of DIA. The aggregates were produced by plating dissociated ES cells in 10 μl or 30 μl drops of medium at a density of 100 cells/drop. Arrays of drops were plated on the lid of a 10 cm tissue culture dishes using a multipipettor. This was then inverted over the base of the dish, which contained 10 ml of water in order to maintain humidity, and the hanging drops were cultured at 37� C. in a 7% CO2 atmosphere.
2. Histology and β-Galactosidase Staining
Embryoid bodies were fixed in Bouin's solution and embedded in agar. Paraffin sections were then prepared by standard procedures and stained with haematoxylin and cosin. Alkaline phophatase staining of embryoid body outgrowths was carried out using Sigma Kit 86-R. Histochemical staining for β-galactosidase was performed with Xgal as described (Beddington et al, 1986).
3. Cell Lines and Selection Systems
Fos11 is a derivative of the ES cell line CGR8 which has been transfected with the Oct4neofos construct. Fos11 cells express low levels of G418 resistance under control of the Oct4 proximal promoter element, but differentiated progeny show no expression of the transgene and are therefore sensitive to G418. OKO160 and OKO8 are derivatives of the ES cell lines CGR8 and E14TG2a respectively in which an IRES-βgeopA cassette has been introduced into one allele of the Oct4 gene by homologous recombination as described. OKO cell lines express high levels of βgeo in the undifferentiated state and therefore stain strongly with Xgal and are G418-resistant. Differentiated progeny lose expression of βgeo and become negative for Xgal staining and sensitive to G418. In monolayer cultures, Fos11 and OKO cells are maintained as pure ES cell populations by culture in the presence of DIA and selection in G418. Under conditions which favour differentiation, however, such as low density and absence of DIA (Smith, 1991), G418 selection results in the complete elimination of these cultures over 3-5 days. Rb40 cells are a derivative of CGR8 which are constitutively resistant to G418 due to expression of neoR directed by the human β-actin promoter.
4. Formation of Embryoid Bodies in the Presence and Absence of Selection against Differentiated Cells
Production of embryoid bodies by the conventional procedure (Doetschman et al, 1986) of detachment of clumps of cells followed by aggregation in bulk suspension culture results in a mixed population of aggregates, heterogeneous in both size and differentiation status. In order to obtain more uniform and consistent development, embryoid bodies in the present study were formed by aggregation of defined numbers of cells in individual drops of culture medium (see Methods). After 48 hours in hanging drop culture, the aggregates were transferred en masse into suspension culture in the presence or absence of G418.
Under G418 selection against differentiated progeny aggregates still formed from both Fos11 cells and the OKO clones. Although some dead cells appeared around the periphery of the aggregates, the bodies themselves increased in size during the culture period. Samples were harvested periodically from t he bulk cultures and processed for histological examination. After several days enbryoid bodies formed in the absence of selection were mostly cystic and contained a variety of morphologically differentiated cell types. Undifferentiated cells were rarely apparent. By contrast, aggregates maintained under selection showed no indications of cellular specialisation and the bodies appeared to consist of solid balls of undifferentiated cells. The great majority of cells in these undifferentiated aggregates appeared healthy and viable and there was no evidence of necrosis, although occasional pyknotic nuclei, suggestive of apoptosis, were seen. Embryoid bodies formed in G418 were noticeably smaller than their counterparts formed in the absence of selection, however. This can be attributed to a combination of the lack of cyst development and the removal of differentiated cells.
5. Persistence of Pluripotential Stem Cells in Embryoid Bodies Formed Under Selection Against Differentiated Cells.
The absence of any undifferentiated aggregates in control cultures implied that it was unlikely that the effect of G418 was due to selection of a subpopulation of non-differentiating aggregates. In order to exclude definitively this possibility, however, and also to facilitate quantitative determination of the effects of G418 selection, a modified protocol was used which allows assessment of the behaviour of individual aggregates. Cultures were initiated in 30 μl hanging drops in the presence or absence of G418 and maintained in drop culture for 7-8 days. Embryoid bodies were then transferred individually to gelatin-coated 96-well tissue culture plates and the media diluted 6-fold with media lacking G418. Th e stem cell maintenance factor DIA was added at this stage to allow expansion of any undifferentiated ES cells which were present. The cultures were allowed to attach and outgrow -for 48 hours then fixed and stained for alkaline phosphatase or for β-galactosidase as appropriate.
The data summarized in Table 1 show that in the absence of any selection undifferentiated stem cells are almost completely eliminated from embryoid bodies within 7 days of suspension culture. Outgrowths contained a variety of morphologically differentiated cell types, but areas of cells with ES cell morphology were not observed. In the OKO cells expression of β-galactosidase is coupled to the stem cell-specific transcription factor Oct4 (Mountford et al, 1994) and therefore serves as a marker of undifferentiated cells. Isolated Xgal-staining cells were occasionally seen in OKO outgrowths, but clusters of staining cells were not detected under these conditions (but see Discussion).
The efficiency of embryoid body formation in G418 was identical to that in non-selected cultures, essentially 100%. In marked contrast to the untreated embryoid bodies, however, embryoid bodies established under continuous G418 selection gave rise to outgrowths consisting largely of ES cells. The undifferentiated nature of these cells was indicated by the characteristic morphology of ES cell colonies a nd by staining with alkaline phosphatase and was confirmed by Xgal staining of the OKO outgrowths.
Several outgrowths from embryoid bodies formed under selection were picked and transferred to 2 cm wells. All of the colonies picked were readily expanded into mass cultures of undifferentiated cells. These cultures remained dependent on DIA and differentiated in similar fashion to parental ES cells when plated in non-supplemented media. Furthermore, these derivatives differentiated efficiently into multiple cell types on aggregation, confirming their pluripotency.
These findings demonstrate that the selective elimination of differentiated progeny results in the persistence of pluripotential stem cells in ES cell aggregates.
6. Stem Cell Extinction in Mixed Aggregates
The implication that differentiated progeny may be directly responsible for stem cell extinction in embryoid bodies was addressed further. The behaviour of OKO cells was assessed following formation of mixed aggregates with Rb40 ES cells which can differentiate in the presence of G418. Rb40 cells express neomycin phosphotransferase constitutively and G418 selection has no discernible effect on their differentiation, either in monolayer culture or in aggregates. Hanging drop cultures were established, using a 3:1 ratio of OKO cells to Rb40 cells. Paraffin sections of mixed embryoid bodies revealed that they underwent extensive differentiation in both the absence and the presence of G418. The effective elimination of undifferentiated stem cells under both conditions was confirmed by Xgal-staining of outgrowths (Table 1).
This result provides direct evidence that the presence of differentiated progeny induces the elimination of pluripotential stem cells. This implies that certain differentiated stem cell progeny are a source of inductive signals which either instruct further differentiation of remaining stem cells or possibly induce them to enter apoptosis.
Aggregation induces ES cells to develop into differentiated structures known as embryoid bodies. Pluripotential stem cells rapidly become extinct in these embryoid bodies due to the efficient induction of differentiation and possibly also to selective cell death. However, if differentiated progeny are specifically eliminated from the aggregates using methods according to the invention, the stem cells persist and can be propagated.
The findings detailed above constitute a clear demonstration that through the use of a stem cell-specific selection system according to the invention it is possible to recover stem cells from conditions which would normally force their elimination by either differentiation or death.
TABLE 1______________________________________Disappearance or Persistence of Oct-4 Expressing ES Cells in Embryoid Bodies. No. No. Xgal % +ve Culture G418* No. Drops Outgrowths +ve Drops______________________________________OKO8 - 25 25 0 0 OKO8 + 25 24 24 96 OKO160 - 30 30 0 0 OKO160 + 30 30 30 100 OKO160:Rb40 - 30 29 0 0 OKO160:Rb40 + 30 30 0 0______________________________________ *500 μg/ml
Lines of transgenic mice were established in which the neomycin phosphotransferase gene conferring resistance to G418 is expressed with the specificity of the Oct4 gene. The βS21 line harbour the Oct4neoβS transgene whilst in the OKO line the neo gene has been incorporated into the endogenous Oct gene via gene targeting with the Oct4-tgtvec construct. These mice were outcrossed for two generations with MF1 outbred albino mice and with inbred CBA mice. Neither of these mouse strains produce ES cells using standard procedures.
Four preferred procedures for isolating stem cells are described. In all cases the embryos are cultured in standard ES cell culture medium supplemented with either Differentiation Inhibiting Activity (Smith, 1991) or interleukin-6 plus soluble interleukin-6 receptor (Yoshida et al, 1994). G418 is added at concentrations of 200 μg/ml-1 mg/ml.
Procedure 1
Blastocysts are flushed on the fourth day of pregnancy. Groups of 4-10 blastocysts are cultured in 1 cm tissue culture wells under G418 selection. Outgrowths are individually detached and dissociated with trypsin as described nichols et al, 1990) after 4-6 days in culture and replated in single wells. G418 selection is maintained. Colonies with the characteristic morphology of ES cells which appear in the cultures over the next 14 days are picked and expanded under continuous selection.
Procedure 2
As Procedure 1, except that blastocysts are put into implantation delay before harvesting by ovariectomy of the dams on the third day of pregnancy. Blastocysts are flushed 4 days after the ovariectomy.
Procedure 3
Post-implantation embryos between 5.5 and 7.5 days post-coitum are isolated and the primitive ectoderm separated by microdissection and/or protease digestion. The primitive ectoderm is gently dissociated into clumps of 20-50 cells which are then cultured as in Procedure 1.
Procedure 4
Embryos prepared as for Procedures 1, 2 or 3 are cultured in hanging drops under G418 selection for a period of 5-7 days before transfer to tissue culture wells and subsequent manipulation as in Procedure 1.
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|U.S. Classification
|435/325, 435/363, 435/353, 435/350, 435/366, 435/372, 435/355, 435/352, 435/351, 435/368, 435/354
|International Classification
|Cooperative Classification
|European Classification
|May 14, 2004
|Fee payment
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|May 2, 2008
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Oil paint in Venice
A review of fresco and tempera and the development of the use of oil paint by artists in Venice. Created by Beth Harris and Steven Zucker.
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in the admin panel? If so, where?
in the admin panel? If so, where?
... Settings -> jQuery Lightbox?
didn't come up ...
jQuery Lightbox For Native Galleries this has the settings so I am fine now.
Downloaded manually, uploaded manually, activated and this time settings visible.
Please don't asked why it didn't work the first time around.
I just figured it out. It's in the WP Settings menu! I was clicking on the Settings link in the plugin page - and got the following error message:
"You do not have sufficient permissions to access this page."
Thanks anyway (I had already seen the post you referred me to and had done that on the install.
Would still be nice if in the next upgrade you gave included some options for captions for non-post images (gallery pages built on nextgen).
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Mark Hurd
Hurd's appointment comes less than two months after HP ousted its former chief executive, Carly Fiorina, and as the industry icon struggles to execute on all cylinders after its megamerger with Compaq.
As earlier reported, Hurd will serve as president and chief executive, with Patricia Dunn retaining the chairwoman title. Hurd will begin in his new post Friday and will join the HP board.
"Mark came to our attention because of his strong execution skills, his proven ability to lead top performing teams and his track record in driving shareholder value," Dunn said. "He demonstrated these skills by turning around NCR, which, while smaller than HP, is a complex organization with multiple business segments."
Dunn also noted: "As we got to know Mark, we were impressed by his emphasis on developing internal talent while reaching outside for new skills, his understanding of the role of culture in a company's success and his personal integrity."
The issue of a cultural fit was one that plagued the Fiorina era. The charismatic executive was accused by a number of employees of killing "the HP way," and was publicly chided during a shareholder meeting by current and former HP employees.
During the search, Hurd emerged as a clear frontrunner within the first month of the effort even though he was not well-known to HP prior to its CEO search. He was not among the candidates reviewed when HP selected Fiorina six years ago.
Three HP board members--Dunn, Jay Keyworth and Tom Perkins--screened a broad field of candidates before submitting the strongest candidates to the full board for their review and selection.
Hurd said he's ready for the challenge of running the larger company, building on a foundation of HP's traditions and current status.
"HP is one of the world's great companies, with a proud history of innovation, outstanding talent and enviable positions in many of its product lines and services," Hurd said.James Ringler, an NCR board member, will serve as interim chief executive at NCR while the company searches for a replacement. At HP, Robert Wayman steps down as acting CEO but remains chief financial officer and a member of the board.
Hurd, 48, initially joined NCR in 1980 and rose up the ranks. He has worked as a senior sales and marketing executive and later as the chief operating officer. In March 2003, Hurd was named CEO.
The company, which last year generated $5.98 billion in revenue, provides software, hardware and services for automated teller machines, data warehousing and IT services.
In the fourth quarter, NCR posted revenue of $1.79 billion, a 9 percent increase over the previous year. And the company


text:
Heather Graham's Apartment Caught on Fire
She's okay!By Caroline Bankoff
She's okay!By Caroline Bankoff
And a nice, flat income tax.By Caroline Bankoff
He allegedly owes $350,000.By Joe Coscarelli
She ate 45 matzo balls! No, just kidding, it wasn't that.By Dan Amira
The breakout star speaks.By Joe Coscarelli
In Florida, too.By Andre Tartar
Make your favorite noise denoting excitement.By Zach Dionne
He should stop squabbling with Obama, for Lindsay's sake.By Margaret Hartmann
The old wrestler would also like the rights to the video, please.By Joe Coscarelli
"It's a long story."By Joe Coscarelli
We doubt that he does this very often.By Caroline Bankoff
Someone made off with ten Cartier pieces from the actress's West Village brownstone.By Joe Coscarelli
New figures provide a preview of a major issue in the upcoming mayoral race.By Chris Smith
Or maybe she's joking.By Margaret Hartmann
White Castle fix after the convention?By Margaret Hartmann
Off of the Long Island Sound.By Caroline Bankoff
His fellow Hamptons churchgoers are fed up.By Joe Coscarelli
She'll be representing the Peace and Freedom Party.By Caroline Bankoff
Vincent D'Onofrio dominated at Coney Island on Sunday.By Joe Coscarelli
Believe it or not, the actor is ready to fight for it.By Joe Coscarelli


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Kathya BriggsShared publicly -
Making this for dinner tomorrow night :) I hope it is a success the chili i made yesterday, was not.
Greek Lemon Chicken. You would think. that I would post a lovely picture with my first recipe post. And it would be a good thought. Alas,. you would be incorrect. But this is yummy and a favorite in m...


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1982 Oilers vs Cowboys
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8 Reviews
.omg......I want to eat fresh all the time NOW but in our northern ontario area tomatillos are not well known. I have been growing them for several years now and this year was the best for size - its a hit and miss with climate and soil effecting growth. I usually do the cooked salsa and freeze or can but this recipe ROCKS!!!!!! Just wanted to add that all ingredients were picked fresh from our garden so it can't get any fresh tasting than that!
Great recipe! I doubled this recipe except for the onion, and added a serrano for extra heat. It was fantastic- my husband and coworkers thought it was really good! Thanks!!
Very tasty as is. Placed over chicken with mango and baked. Is this heaven or what?
Made it without the jalapeno and it tasted great! :) I am using it with an enchilada recipe this evening...can't wait!
Another Recipezaar winner! Really Really good. One of my guests ate this and said next time I'm invited over for a BBQ I should bring this and a bag of chips. Husband also gave this a 10. I too used my immersion blender to mix up part of the 'salsa'. Probably 'mashed' about 2/3. Maybe next time I'll separate and mash only 1/3. I can't believe more folks haven't seen this and rated it high. Normally I'm a bit tentative about making recipes that only have a couple of reviews! I doubled this and husband is glad we have left overs!
Thanks for this recipe! We don't eat bell peppers and this was the first recipe I found for salsa that didn't use them! Only change was that I squeezed the seeds out of the tomato and blended it some. The next time I made it, I took the seeds out of the tomatillos, too, and left the rest as you suggested. Excellent! Everyone loves it.
I adore this recipe, it was so easy to make. I loved the idea of red onion versus regular. I made two slight changes, one I used 3 jalapenos (with seeds) as I like it spicy and two, I used my stick blender and made it more a salsa then a pico consistency. It was great and a wonderful change to regular red salsa. Thank you!