JP2002517982A - Cell differentiation / proliferation and maintenance factors and their use - Google Patents

Abstract

  (57) [Summary] The present invention relates to biologically active factors that can affect the differentiation, proliferation and / or maintenance of pluripotent cells. The present invention also provides a method of producing a pluripotent cell having different properties from a pluripotent cell (more specifically, an EPL cell) using a biologically active factor; It relates to a method for producing partially or terminally differentiated cells. The invention also relates to pluripotent cells and partially or terminally differentiated cells, as well as human cells and gene therapy, and their use in generating transgenic animals.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to biologically active factors, and more particularly to factors that can affect the differentiation, proliferation and / or maintenance of pluripotent cells, including embryonic stem (ES) cells. The present invention also provides a method for producing pluripotent cells having different properties (more particularly, EPL cells) from pluripotent cells using biologically active factors; To a method of producing terminally differentiated cells or terminally differentiated cells. The invention also relates to pluripotent cells and partially or terminally differentiated cells, and uses thereof.

[0002] Early developmental events in the mammalian embryo elaborate the extra-embryonic cell lineage and result in the formation of blastocysts, which are responsible for the trophectoderm, primitive endoderm and pluripotent cells. Pool, containing inner cell mass (ICM / primordial ectoderm). As outbreaks continue, ICM
They undergo rapid proliferation, selective apoptosis, differentiation and remodeling so that cells of the / primordial ectoderm develop and form primitive ectoderm. In mice, the cellular I
CM begins to grow rapidly around the time of blastocyst implantation. The resulting pluripotent cell mass extends to the blastocoel. 5.0 and 5.5d. p. In between c, the endoderm of the primordial ectoderm undergoes apoptosis and forms the primordial amniotic cavity. Outer surviving cells or early primitive ectoderm continue to proliferate and 6.0-6.5 d. p. c. Up to the pseudo-stratified epithelial layer of pluripotent cells called primitive ectoderm or germ ectoderm
forming a reinforced epithelial layer. Primitive ectoderm gives rise to germ cells and acts as a substrate for the production of embryo-specific early and extra-embryonic mesoderm during gastrulation.

[0003] Analysis of the developmentally latent and differentially expressed genes of a temporally distinct pool of pluripotent cells in the pre-gastrulation embryo has led to the analysis of two distinct pluripotencies of the ICM and primitive ectoderm A population of cells was identified. Cells within each population appear to be homologous as evidenced by transplantation studies and analysis of gene expression markers. Establishment of heterogeneity of pluripotent cells and determination of a subpopulation of pluripotent cells in the primitive ectoderm, as defined by differential gene expression, is 6.5 d. p. c. Has not been demonstrated in embryos prior to the formation of primordial streaks. 4.5 and 6.5 d. p. c. The lack of genetic markers in the uterine environment and the small size, complexity and relative difficulty of achieving embryos did not allow a comprehensive analysis of the development of pluripotent cells.

[0004] Pluripotent cells can be isolated from pre-transplant mouse embryos as embryonic stem (ES) cells. ES cells can be maintained in vitro as a pluripotent cell population in vitro, and
When reintroduced into the host blastocyst, it can contribute to all adult tissues of the mouse, including germ cells. Thus, ES cells retain the ability to respond to all signals that regulate normal mouse development, and mechanisms based on the biology and differentiation of pluripotent cells within the early embryo, and embryo manipulation, and Potentially represents a powerful model system for the study of mechanisms that offer opportunities for the resulting commercial, medical and agricultural applications. Other pluripotent cells and cell lines share some or all of these properties and applications.

[0005] Successful isolation, long-term clonal maintenance, genetic manipulation and germline transmission of pluripotent cells from species other than rodents are generally difficult to date and the cause is unknown. It is. International Patent Application WO 97/32033 and US Pat.
No. 453,357 describes pluripotent cells, including cells from species other than rodents, and primitive pluripotent cells are described in International Patent Applications WO 98/43679 and WO 96/43.
No. 23362 and U.S. Pat. No. 5,843,780. However, primordial pluripotent cells are useful if these pluripotent cells can be transformed into pluripotent cells having different properties.

[0006] ES cell differentiation can be regulated in vitro by the cytokine leukemia inhibitory factor (LIF), which promotes self-renewal and prevents stem cell differentiation, and other gp130 agonists. However, the biological molecules that can induce the differentiation of ES cells into specific cell types, excluding retinoic acid, in the presence or absence of LIF, are currently unknown.

It is an object of the present invention to overcome, or at least mitigate, one or more difficulties or deficiencies associated with the prior art.

[0008] Applicants have identified biologically active factors that can affect the differentiation, proliferation and / or maintenance of pluripotent cells, including ES cells. More particularly, this factor is a pluripotent cell with different properties of pluripotent cells (eg, ES cells in adherent and suspension cultures), more particularly early primitive ectoderm-like (EPL) can cause conversion to cells. In addition, the factor may maintain and support the growth of these cells in vitro. It also allows the isolation and maintenance of EPL cells derived from primitive ectoderm in vitro and in vivo.

[0009] While Applicants do not want to be limited by theory, EPL cells have 6.0 levels.
p. d. c. Alternatively, it is thought to represent an in vitro equivalent of pluripotent cells after embryo transfer prior to early primordial ectoderm.

Thus, the term “EPL cell” refers to a cell that retains pluripotency and
4 and / or conversion to cells expressing Fgf5 and / or Oct4 and Fgf
It should be understood that it refers to cells from pluripotent cells that are maintained as cells that express 5: (a) biologically active factors of the invention or their high or low molecular weight components; Or (b) a conditioned medium according to the invention; or (c) an extracellular matrix in the presence or absence of a low molecular weight component according to the invention.

[0011] Pluripotent cells (EPL cells can be derived) include in vitro or in vivo induced ICM / primordial ectoderm, in vitro or in vivo derived primitive ectoderm, teratocarcinoma cells, EC cells, primordial germ The cells can be, but are not limited to, cells, EG cells, and ES cells from pluripotent cells induced by dedifferentiation or by nuclear transfer. EPL cells can also be derived from differentiated cells by dedifferentiation.

In addition, EPL cells can be reverted to ES cells by removal of (a), (b) or (c) in the presence of a gp130 agonist, but is not limited to such.

[0013] The biologically active agent preferably comprises two components: a low molecular weight component and a high molecular weight component.

[0014] The low molecular weight component can be an amino acid or a functionally active analog thereof, or a peptide or a functionally active fragment or analog thereof. Preferably, the low molecular weight component is proline or a functionally active analog thereof, or a proline-containing peptide or functionally active fragment or analog thereof. Even more preferably, the low molecular component is L-proline or L-proline.
-A peptide containing proline. The peptide preferably has a molecular weight of less than about 5 kD, and more preferably has a molecular weight of less than about 3 kD. Alternatively or additionally, the peptide is preferably about 1-11, more preferably about 1-7.
, Most preferably about 1 to 4 amino acids in size. Most preferably, this peptide is selected from the group: Pro-ala Ala-pro Ala-pro-gly Pro-OH-pro Pro-gly Gly-pro Gly-pro-ala Gly-pro-OH-pro. Gly-pro-arg-pro Gly-pro-gly-gly Val-ala-pro-gly substance P-frag1 to 4 (Arg-pro-lys-pro) Substance P free acid (arg-Pro-Lys-Pro-Gln) -Gln-Phe-Phe-gl
y-Leu-metOH) Protease digested (including collagenase digested) collagen fragments; and functionally active fragments and analogs thereof; and molecules that compete for biological activity.

[0015] The term "functionally active" means that the fragment or analog can affect the differentiation, proliferation and / or maintenance of pluripotent cells, including ES cells.

[0016] The high molecular weight component can be a polypeptide or a protein. Preferably, the high molecular weight component has a molecular weight greater than about 10 kD, more preferably about 50 to 1 kD.
It has a molecular weight between 000 kD, most preferably between about 100-500 kD.
The high molecular weight component can be in solution or contained in the extracellular matrix.

In a preferred form of this aspect of the invention, the high molecular weight component can be an extracellular matrix protein, or a functionally active fragment or analog thereof (eg, fibronectin or laminin or a functionally active thereof). Fragments or analogs). In certain preferred forms,
The high molecular weight component is cellular fibronectin. Cellular fibronectin
About 210-250 k, as measured on a 10% reduced / denatured polyacrylamide gel.
It may have a molecular weight of D.

In a further aspect of the present invention there is provided a composition capable of affecting the differentiation, proliferation and / or maintenance of a pluripotent cell, including an ES cell, wherein said composition is as hereinbefore described. Such biologically active factors and / or low and / or high molecular weight components are included with the adjuvant, excipient or carrier.

[0019] Biologically active factors can be isolated from the medium conditioned by the cultured cells. Alternatively, the components of the biologically active agent can be produced from other sources,
It can be produced synthetically or recombinantly.

Thus, in a further aspect of the invention, a conditioned medium capable of affecting the differentiation, proliferation and / or maintenance of pluripotent cells, or a fraction thereof comprising a medium component of less than about 5 kDa, and / or a fraction of about 10 kDa. A fraction thereof comprising the above media components is provided.

[0021] Preferably, the conditioned medium comprises a biologically active agent, as described herein above, or a low or high molecular weight component thereof.

Preferably, the conditioned medium is prepared by a method as described later herein.

Preferably, the conditioned medium is a hepatocyte or hepatoma cell or hepatoma cell line, more preferably a human hepatocellular carcinoma cell line (eg, HepG2 cells (ATCC HB-8
065) or Hepa-1c1c-7 cells (ATCC CRL-2026))
Primary embryonic mouse liver cells, primary adult mouse liver cells, or primary chicken liver cells, or extraembryonic endoderm cells or extraembryonic endoderm cell lines (eg, cell line EN
D-2 and PYS-2). However, biologically active factors can be isolated from media conditioned by liver cells or other cells from any suitable species, preferably mammals or birds. Alternatively, the activity is also 1
It can be induced by the contribution of two or more conditioned media different from cells expressing one or more other components.

Thus, in a further aspect of the present invention, there is provided a method of preparing a conditioned medium capable of affecting the differentiation, proliferation and / or maintenance of a pluripotent cell, said method comprising: a cell, and a cell culture medium Culturing the cells in the cell culture medium for a time sufficient to produce a conditioned medium; and separating the cells from the culture medium to provide the conditioned medium. I do.

[0025] The cells can be of any suitable type. Preferably, the cell is a hepatocyte or hepatoma cell or hepatocyte cell line or hepatoma cell line. More preferably, they are human hepatocellular carcinoma cell lines, primary embryonic mouse liver cells, primary adult mouse liver cells,
And primary chicken liver cells. Most preferably, a human hepatocellular carcinoma cell line (eg, Hep G2 cells (ATCC HB-8065) or Hepa-1c-1c-7 cells (ATCC CRL-2026)) is used. Alternatively, the cells may be endoderm cells, more preferably primary extraembryonic cells, or cultured extraembryonic cells or cell lines (eg, endodermal cell lines END-2 and PYS-2).

The cells can be cultured under conditions suitable for their growth and maintenance in vitro. This may involve the use of serum (including fetal calf serum and bovine serum), or the medium may be serum free. Other growth enhancing components such as insulin, transferrin and sodium selenite can be added to improve the growth of cells that can be derived from conditioned media or extracellular matrix. As will be readily apparent to one skilled in the art, the growth enhancing component will depend on the cell type cultured, the presence of other growth factors, the amount of adhesin and the amount of serum present.

[0027] The cells may be cultured for a time sufficient to establish the cells in culture. This contemplates the time at which cells equilibrate in the culture medium.
Preferably, the cells are cultured for about 3-5 days.

[0028] The cell culture medium may be any cell culture medium suitable for identifying cells used. If the cells are liver cells or a liver cell line, the culture medium is preferably DMEM with high glucose supplemented with 10% FCS, 40 μg / ml gentamicin, 1 mM L-glutamine, 37 ° C., 5% CO _2. is there.

[0029] Separation of the cell culture medium from the cells can be accomplished by any suitable technique, such as decanting the medium from the cells. Preferably, the cell culture medium is clarified by centrifugation or filtration (eg, through a 0.22 μM filter),
Remove excess cells and cell debris. If this separation method does not remove growth components from the medium, other known methods for separating cells from culture can be used.

[0030] For yet a further aspect of the invention, there is provided a substantially or partially purified extracellular matrix. This extracellular matrix can affect the differentiation, proliferation, and / or maintenance of pluripotent cells.

[0031] Preferably, the extracellular matrix comprises a biologically active agent as described herein above, or a high molecular weight component thereof.

[0032] Preferably, the extracellular matrix is prepared by a method as described later herein.

Thus, in a further aspect of the invention, the differentiation, proliferation and / or proliferation of pluripotent cells
Alternatively, a method is provided for preparing an extracellular matrix that can affect maintenance, comprising: providing a cell, a support substrate, and a cell culture medium; the cell culture in the presence of a support substrate. Culturing the cells in the medium for a time sufficient to produce an extracellular matrix; and separating the cells and the culture medium from the support substrate to provide an extracellular matrix. I do.

[0034] The cells can be of any suitable type. Preferably, the cells are hepatocytes or hepatoma cells or hepatocyte or hepatoma cell lines. More preferably, they are selected from the group consisting of human hepatocellular carcinoma cell lines, primary embryonic mouse liver cells, primary adult mouse liver cells, and primary chicken liver cells. Most preferably, a human hepatocellular carcinoma cell line (eg, Hep G2 cells (ATCC HB-8065) or Hepa-1c-1c-7 cells (ATCC CRL-2026)) is used. Alternatively, the cells may be endoderm cells, more preferably primary extraembryonic cells, or cultured extraembryonic cells or cell lines (eg, endodermal cell line END-2)
And PYS-2).

The cells can be cultured according to the conditions described for preparing a conditioned medium. Isolation of matrix-related activity involves the isolation of cell culture media and appropriate cells or cell lines from the ECM and the underlying support. This involves washing the matrix with a suitable buffer. Preferably, cells are cultured in 0.2% azide, 0.1 mM PMSF in phosphate buffered saline (PBS) for 6-18 hours to kill and dissociate cells or wash further with PBS Using the cells and debris removed by culturing, the cells were cultured in 0.5 mM EGTA for 15 minutes to separate the cells.

Alternatively, extracellular matrices that can affect the differentiation, proliferation and / or maintenance of pluripotent cells are derived from animal sources (eg, visceral, wall or primitive endoderm) or animal cells and tissues (eg, placenta) Can be prepared. Extracellular matrices can also be prepared by artificial means, such as drying the purified extracellular matrix or semi-purified extracellular matrix components on tissue culture plates.

Applicants have reported that biologically active factors prepared using human hepatoma cell lines, primary embryonic mouse liver cells, primary adult mouse liver cells and primary chicken liver cells can be used to differentiate mouse ES cells, It has been found that growth and / or maintenance can be affected. Thus, although Applicants do not wish to be limited by theory, it appears that this factor is biologically active across species.

[0038] The components of the biologically active agent can be partially or substantially purified. Purification of this component involves removal of cells followed by cation exchange chromatography, hydrophobic interaction chromatography, anion exchange chromatography, heparin affinity chromatography, size exclusion chromatography, ultrafiltration, normal phase chromatography. And / or by techniques such as reverse phase chromatography. Preferably, purification of this component is performed using FPLC and HPLC chromatography techniques.

In a preferred form of this aspect of the invention, the low molecular weight components can be purified by a combination of fractions as required (eg, ultrafiltration, gel filtration chromatography (preferably on a Sepharose G10 column). ), Normal phase chromatography and gel filtration chromatography (preferably on a Superdex peptide gel filtration column); and preferably, can be purified sequentially in a defined order.

Thus, the present invention provides a method for partially or substantially purifying the low molecular weight component of a biologically active factor that can affect the differentiation, proliferation and / or maintenance of a pluripotent cell. And the method comprises the following steps: providing a source of the low molecular weight component, a first gel filtration chromatography support, a normal phase chromatography support, and a second gel filtration chromatography support; Contacting a source of the components with a first gel filtration chromatography support to produce a first fraction; contacting the first fraction with a normal phase chromatography support to form a second fraction; Contacting the second fraction with a second gel filtration chromatography support to produce a partially or substantially purified low molecular weight component; Is included.

Preferably, the method comprises the step of fractionating the source of the low molecular weight component (eg,
Ultrafiltration) to obtain a fraction containing components having a molecular weight of less than about 3 kD.

In a further preferred form of this aspect of the invention, the high molecular weight component is subjected to affinity chromatography (preferably on heparin Sepharose affinity chromatography (eg on a heparin Sepharose CL-6B column)),
Anion exchange chromatography (eg, using a Resource Q anion exchange column) and gel filtration chromatography (preferably, Super
(on a rose 6 gel filtration column), and preferably by continuing in the stated sequence.

Thus, the present invention provides a method for partially or substantially purifying the high molecular weight component of a biologically active factor that can affect the differentiation, proliferation, and / or maintenance of pluripotent cells. I do. The method comprises the steps of: providing a source of a high molecular weight component as described above, a heparin affinity chromatography support, an anion exchange chromatography support, and a gel filtration chromatography support; Contacting a source of molecular weight components with a heparin affinity chromatography support to produce a first fraction; contacting said first fraction with an anion exchange chromatography support to produce a second fraction;
Contacting the second fraction with a gel filtration chromatography support to produce a partially or substantially purified high molecular weight component.

Alternatively, the high molecular weight component is optionally fractionated (eg, by ultrafiltration (eg, Ami
con DiaFlo YM10 membrane), anion exchange chromatography (eg, using a Sepharose Q anion exchange column), hydrophobic interaction chromatography (eg, using a phenyl sepharose hydrophobic interaction column), Heparin affinity chromatography (e.g., using heparin Sepharose affinity chromatography (e.g., using a heparin Sepharose CL-6B column)) and gel filtration (e.g., Super
(using an O.S. 6 gel filtration column), and preferably by continuing in the stated order.

Thus, the present invention provides a method for partially or substantially purifying the high molecular weight component of a biologically active factor that can affect the differentiation, proliferation and / or maintenance of a pluripotent cell. I do. The method includes the following steps: a source of the high molecular weight components described above, an anion exchange chromatography support, a hydrophobic interaction chromatography support, a heparin affinity chromatography support, and a gel filtration chromatography support. Contacting the source of the high molecular weight component with an anion exchange chromatography support to produce a first fraction; hydrophobic interaction chromatography of the first fraction Contacting a support to produce a second fraction; contacting said second fraction with a heparin affinity chromatography support to produce a third fraction; Contacting the fraction with a gel filtration chromatography support to produce a partially or substantially purified high molecular weight component Process.

Preferably, the method comprises the steps of:
Ultrafiltration) to obtain a fraction containing components of molecular weight greater than about 10 kD.

Alternatively, the high molecular weight component can be purified by performing gelatin affinity chromatography (preferably gelatin sepharose affinity chromatography), followed by dialysis, if necessary.

Thus, the present invention provides a method for partially or substantially purifying the high molecular weight component of a biologically active factor that can affect the differentiation, proliferation, and / or maintenance of a pluripotent cell. I do. The method includes the steps of providing a source of the high molecular weight component, a gelatin affinity chromatography support, and a dialysis system; a source of the high molecular weight component, a gelatin affinity chromatography support. Contacting the first fraction to produce a first fraction; and subjecting the first fraction to dialysis to produce a partially or substantially purified high molecular weight component.

Preferably, in the method for purifying a high molecular weight component, the source of the high molecular weight component is subjected to a fractionation step (for example, ultrafiltration) to obtain a fraction containing a component having a molecular weight of more than about 10 kD. , And further steps. This step may be included at any suitable point in the purification procedure.

Preferably, the source of the high or low molecular weight component is a partially purified biologically active agent as described herein above, a conditioned medium as described herein above. Or an extracellular matrix as described herein above.

A conditioned medium as described hereinabove can be used to induce and maintain pluripotent cells, including EPL cells, or the medium can be fractionated to produce a biologically active factor or its The components may be produced and added either alone or in combination with other media to provide a media for inducing and maintaining pluripotent cells (eg, EPL cells). Alternatively, a partially or substantially purified or synthetic or recombinant form of a biologically active agent or a component thereof is added, alone or in combination, to another medium. This may provide a medium for inducing and maintaining pluripotent cells (eg, EPL cells). The conditioned medium may be used undiluted or may be used diluted (eg, about 10% to 80%).

When the low molecular weight component is proline, its concentration in the cell maintenance medium preferably ranges from about 40 μM or more. When the high molecular weight component is cellular fibronectin, its concentration in the cell maintenance medium preferably ranges from about 2 μg / ml or more. Extracellular matrices derived from related cells and related cell lines or artificially prepared extracellular matrices can also be used to provide biologically active high molecular weight components. Conditioned media, factors, or high molecular weight components thereof, may also be used to coat the culture plate to provide the desired biological activity.

The biologically active factor of the invention or its high or low molecular weight components can be used to generate pluripotent cells, including EPL cells, which are derived from rodents As well as from other more commercially important species, including humans.

Thus, in a further aspect of the present invention, there is provided a method of generating and / or maintaining early primitive ectoderm-like (EPL) cells. The method comprises the steps of: providing a pluripotent cell and a biologically active factor according to the invention, or a high or low molecular weight component thereof; or a conditioned medium according to the invention, or Providing an extracellular matrix according to the invention and optionally a low molecular weight component of a biologically active agent of the invention; and Contacting with a molecular weight component, or a conditioned medium, or an extracellular matrix to produce or maintain EPL cells.

Pluripotent cells include embryonic stem (ES) cells, in vitro or in vitro induced ICM / primordial ectoderm, in vivo or in vitro derived primitive ectoderm, primordial germ cells, EG cells, teratocarcinoma cells , EC cells, and pluripotent cells induced by dedifferentiation or by nuclear transfer. EPL cells also
It can be derived from differentiated cells by dedifferentiation.

The addition of a biologically active factor or its high or low molecular weight components or a conditioned medium or extracellular matrix, if necessary, to low molecular weight components can be used to isolate EPL cells in vitro, It can be used for growth or maintenance.
EPL cells can be produced in adherent culture or as cell aggregates in suspension culture. Biologically active factors or components of biologically active factors, or conditioned media or extracellular matrices with the addition of low molecular weight components as needed, alone or in combination with other factors Can be used to generate differentiated cells, tissues or organs by techniques known to those skilled in the art. Differentiated cells derived from EPL cells can be used in allografts, concordant or xenografts, cell therapy, tissue or organ expansion or replacement, and gene therapy.

In one form of this aspect of the invention, the pluripotent cells are treated with a biologically active agent or a high or low molecular weight component thereof or a conditioned medium, or a low- The molecular weight component is added, preferably about 10
A gp130 agonist at a concentration greater than 0 units / ml, and more preferably greater than about 1000 units / ml (eg, cytokine leukemia (leuk)
aemia) inhibitor (LIF)). Oncostatin M,
CNTF, CT1 or IL6 and soluble IL6 receptor, and IL11 as well as other equal levels of gp130 agonists may also be used.

In the mouse, the pluripotent cells can be ES cells or cells derived from ICM / protoectodermal pluripotent cells of an embryo or cell aggregate (embryoid body) Alternatively, it can be either embryonic-derived primitive ectoderm or primitive ectoderm derived from in vitro differentiation of ES cells as embryoid bodies or cell aggregates. In other species, the pluripotent cells may be from an equivalent source of cells at various relevant stages. Sources of pluripotent cells from all species may include primordial germ cells or cells from teratocarcinoma. In addition, pluripotent cells can be differentiated (eg, by returning differentiated cells to a pluripotent stage) or by the application of nuclear transfer techniques (eg, of differentiated cells or partially differentiated cells). (If the nucleus is transferred to an oocyte or early embryonic cell). Pluripotent cells can be from any vertebrate, including mice, humans, cows, sheep, pigs, goats, horses, and chickens. The pluripotent cells can be isolated by any method known to one of skill in the art.

In a preferred form of this aspect of the invention, the method comprises the following additional step: identifying EPL cells.

The conversion of pluripotent cells into EPL cells is dependent on the expression of marker genes (RNA transcripts and cell surface markers), cell morphology, cytokine responsiveness, and / or
Alternatively, it can be evaluated by differentiation in vitro or in vivo.

[0061] Marker genes that can be used to evaluate the conversion of pluripotent cells to EPL cells include known markers (eg, Rex1, Fgf5, Oct4, alkaline phosphatase, uvomorulin, AFP, H19, Evx1, brachy).
uri) and novel marker genes identified by the present inventors (eg, L
17, Psc1 and K7). Marker genes that are down-regulated during the transition from ES cells to EPL cells include Rex1, L17 and Psc1. Fgf5 and K7 are up-regulated during this transition. The pluripotent cell markers Oct4, alkaline phosphatase and uvomorulin are expressed at similar levels by both ES and EPL cells. Other genes expressed in the partially or differentiated embryonic or extraembryonic lineage (eg, AFP, H19, Evxl and bra
nchyury) is not expressed on any ES or EPL cells.

As cytokines and growth factors that differentially act on EPLs and other pluripotent cells, members of the FGF family that induce EPL cell differentiation but do not induce ES cell differentiation (eg, , AFGF, bFGF and FGF4)
And TGFβ that induces EPL cell differentiation but not ES cell differentiation
Family members (eg, activin A). In addition, EPL
Cells are maintained in culture with LIF levels about 10-fold lower than required for ES cell maintenance.

The differentiation of EPL cells depends on the rate and structure ratio and the differentiated cell types formed in cell aggregates or embryoid bodies (eg, primitive ectoderm, visceral endoderm, developing mesoderm, myocardium, Macrophages, and neurons) can be distinguished from the differentiation of other pluripotent cells. When compared to ES cells, EPL cells are characterized by accelerated formation of late primordial ectoderm, developing mesoderm, beating heart cells (cardiocytes).
And experience accelerated and increased formation of macrophages, and reduced or no formation of visceral endoderm and neurons. EPL cells do not contribute to embryo development after blastocyst injection, whereas ES and EG cells do. EPL cells do not contribute to embryonic development after blastocyst injection, but ES cells and E
G cells contribute.

EPL cells can be grown in the presence or absence of an additional gp130 agonist in the presence of a biologically active factor or component thereof or conditioned media, or extracellular matrix, optionally with low molecular weight components. Culturing below may maintain a pluripotent state until further differentiation induced by factors, conditions or procedures is initiated.

Thus, in a further aspect of the present invention there is provided a method of producing a partially differentiated cell and / or a terminally differentiated cell. The method comprises the steps of: a pluripotent cell and a biologically active factor according to the invention, or a high or low molecular weight component thereof, or a conditioned medium according to the invention, or a cell according to the invention. Providing an outer matrix and optionally a low molecular weight component of a biologically active factor of the invention; transforming the pluripotent cell with a biologically active factor, or a high or low molecular weight component thereof; Or conditioned medium, or extracellular matrix, and additional gp130
Contacting in the presence or absence of an agonist to produce early primordial embryonic-like (EPL) cells; and manipulating the environment of the EPL cells to reduce partially differentiated cells and / or terminally differentiated cells. The process of generating.

Pluripotent cells were expressed in embryonic stem (ES) cells, in vivo or in vitro induced I
The group consisting of CM / primordial ectoderm, primitive ectoderm derived in vivo or in vitro, primordial germ cells, EG cells, teratocarcinoma cells, EC cells, and pluripotent cells induced by dedifferentiation or by nuclear transfer You can choose from. EPL cells also
It can be derived from differentiated cells by dedifferentiation.

In a preferred form of this aspect of the invention, the environment of the EPL cells is altered by the maintenance or removal of biologically active factors or components thereof or conditioned media or extracellular matrix, such as one or more differentiation factors (eg, , Growth factors). For example, contacting EPL cells with conditioned media, biologically active factors, or components thereof, is preferably performed in cell aggregates grown in suspension, preferably growth factors from the FGF family (eg, FGF4) can be used to generate at least partially differentiated cell types that are equivalent to embryonic neuroectoderm, which are further expanded into a range of neuronal cell types And can be differentiated. Neuroectoderm is induced via cell types equivalent to embryonic ectoderm, which can further differentiate into a range of other ectodermal cell types.

[0068] In the absence of a biologically active agent or a component thereof, and preferably FG
In the presence of F family members (eg, FGF4), growth factors from the TGF-β family are added to EPL cells to generate at least partially differentiated cell types equivalent to embryonic nascent mesoderm. This can further differentiate into a range of mesodermal cell types.

[0069] In a further preferred aspect of this aspect of the invention, the conditioned medium, or embryoid bodies or aggregates formed from pluripotent cells in the presence of biologically active factors or components thereof, are disaggregated. It can be a single cell suspension. When reaggregated in conditioned media, or in the absence of biologically active factors or components thereof, preferably in the presence of a member of the FGF family (eg, FGF4), these cells predominately differentiate, An at least partially differentiated cell type equivalent to embryonic nascent mesoderm can be generated, which can further differentiate into a range of mesoderm cell types, including blood or muscle lineages.

Pluripotent cells were expressed in embryonic stem (ES) cells, in vivo or in vitro induced I
The group consisting of CM / primordial ectoderm, primitive ectoderm derived in vivo or in vitro, primordial germ cells, EG cells, teratocarcinoma cells, EC cells, and pluripotent cells induced by dedifferentiation or by nuclear transfer You can choose from. EPL cells also
It can be derived from differentiated cells by dedifferentiation.

In a further preferred form of this aspect of the invention, the differentiation of the EPL cells first forms a monolayer culture of the EPL cells, and then the FGF family or TGFβ
This can be accomplished by the process of further culturing the cell in the presence of a member of the family. Cells induced by this process are specialized morphologies of differentiated cells.

Further, suspension culture by aggregation of EPL cells formed in adherent or suspension cultures in the absence of conditioned media or biologically active factors or components thereof The formation of EPL cell aggregates in an organism can differentiate these cells in the absence of cell types such as visceral endoderm, which is known to affect the differentiation of pluripotent cells in the embryo. Occurs. Aggregated EPL cells differentiated in this manner form high levels of the developing mesoderm, which is further differentiated to beating muscle and blood as well as of mesoderm origin Other tissues can be formed.

In a further preferred form of this aspect of the invention, the method comprises the following further steps: Partially differentiated by a procedure involving cell surface markers and gene expression markers, morphology, and differentiation potential Identifying differentiated or terminally differentiated cells.

[0074] Marker genes that can be used to evaluate the conversion of pluripotent cells to neuroectodermal cells and neural lineages include known markers, such as Gbx2, Sox
1, Sox2, Nestin, N-Cam, Oct4, Fgf5 and brachy
ury. Markers that are down-regulated during the transition from pluripotent cells to neuroectodermal cells include Oct4 and Fgf5. Markers up-regulated during this transition include Gbx2, Sox1,
Sox2, Nestin and N-Cam. Markers that are not expressed during this transition include brachyury.

Marker genes that can be used to assess the conversion of pluripotent cells to mesoderm and derivatives include known markers such as Oct4, Fgf5 and brachyury, and N controlled down during the transition to
kx 2.5 markers include Oct4 and Fgf5. Markers that are up-regulated during this transition include brachyury and, if the mesoderm differentiates into muscle precursors, include Nkx 2.5.

[0076] Mesodermal and ectodermal germ layers can be distinguished based on their ability to differentiate in vitro. Mesoderm germ layers can differentiate into muscle and blood lineages, but not neuroectodermal or nervous lineages. Ectodermal germ layers can differentiate into neuroectodermal and neural lineages, but not muscle and blood lineages.

In a further aspect of the invention, for producing ectoderm cells preferentially,
Then, preferentially, to produce partially differentiated ectodermal and neuroectodermal cells, and also to produce terminally differentiated ectodermal cells, including but not limited to skin cells And, also, a method for preferentially producing terminally differentiated neurons.

Including, but not limited to, preferentially producing mesodermal germ cells, producing preferentially partially differentiated mesoderm cells therefrom, and also blood cells and beating heart cells Also provided are methods for preferentially producing non-limiting terminally differentiated mesoderm cells.

In a further aspect of the invention, ectodermal, neuroectodermal, partially differentiated or partially differentiated neuroectodermal cells, and terminally differentiated ectodermal cells (eg, skin) Cells), and terminally differentiated neural cells produced by the method of the present invention.

In a further aspect of the invention, mesoderm cells, partially differentiated mesoderm cells, and terminally differentiated mesoderm cells produced by the methods of the invention (eg,
Blood cells and muscle cells).

[0081] In a further aspect of the invention, cultured EPL cells are provided.

Alternatively, EPL cells can be grown in conditioned media, or biologically, in the presence of a gp130 agonist, such as LIF, preferably at a level of about 1000 units / ml or more, or other gp130 agonists at equivalent levels. It can be returned to ES cells by removing active factors or their components. This is accompanied by re-establishment of ES cell gene expression, cytokine response, morphology and differentiation potential both in vivo and in vitro. This is a means for generating ES cells by reversion or dedifferentiation of primitive ectoderm or EPL cells from any desired species, and in particular from species where ES cells were not previously available. I will provide a.

Thus, in a further aspect of the present invention there is provided a method of producing an ES cell,
This method comprises: a pluripotent cell, a biologically active factor according to the invention or a high or low molecular weight component thereof, or a conditioned medium according to the invention, or an extracellular matrix according to the invention, and optionally Accordingly, providing a low molecular weight component of a biologically active factor according to the invention; and a gp130 agonist; a pluripotent cell and a biologically active factor or a high or low molecular weight component thereof. Or contacting the conditioned medium or extracellular matrix; and producing EPL cells in the presence or absence of an additional gp130 agonist; and converting the EPL cells and gp130 agonist to a biologically active agent or Its high or low molecular weight components, or conditioned media, or extracellular matrix Contacting in the presence; involves returning the EPL cells into ES cells.

The pluripotent cells can be of any suitable type, as described herein above.

The method may include the additional step of differentiating the ES cells to produce partially or terminally differentiated cells, as described herein above.

[0086] Preferably, the gp130 agonist is a leukemia inhibitory factor (LIF). However, other gp130 agonists (eg, Oncostatin M, CNTF, CT
1, or IL6 with soluble IL6 receptor at the levels required to maintain pluripotent ES cells in vitro may also be used.

In a further aspect of the present invention there is provided a method of producing a genetically modified ES cell, comprising: a pluripotent cell, a biologically active factor according to the invention or a high thereof. A molecular weight or low molecular weight component, or a conditioned medium according to the present invention, or an extracellular matrix according to the present invention, and optionally, a low molecular weight component of a biologically active factor according to the present invention; and a gp130 agonist. Providing, in the presence or absence of an additional gp130 agonist to produce EPL cells, a pluripotent cell and a biologically active factor or a high or low molecular weight component thereof, or a conditioned medium, or Contacting with an extracellular matrix; modifying one or more genes in the EPL cells; Contacting the modified EPL cells with a gp130 agonist in the absence of a biologically active factor or a high or low molecular weight component thereof, or a conditioned medium, or an extracellular matrix; Converting the EPL cells into genetically modified ES cells.

The pluripotent cells can be of any suitable type, as described herein above.

It should be noted that genetically modified ES cells can also be produced according to the present methods when reverted ES cells (but not EPL cells) are genetically modified.

The method comprises generating a genetically modified partially differentiated or terminally differentiated cell by the methods described herein above, including the formation of EPL cells. Additional step of differentiating the obtained ES cells.

Alternatively, the genetically modified partially differentiated or terminally differentiated cells may be produced by a method comprising: a pluripotent cell, a biologically active agent or a biologically active agent according to the invention. A high or low molecular weight component, or a conditioned medium according to the present invention, or an extracellular matrix according to the present invention, and optionally, a low molecular weight component of a biologically active factor according to the present invention; and a gp130 agonist Contacting the pluripotent cells with a biologically active agent or a high or low molecular weight component thereof, or a conditioned medium, or an extracellular matrix; the presence or absence of an additional gp130 agonist. Producing EPL cells in the presence; modifying one or more genes in the EPL cells; To produce genetically modified partially differentiated cells or terminally differentiated cells to beauty, it is differentiated genetically modified EPL cells.

[0092] Alternatively, partially or terminally differentiated cells can be produced as described herein above and then genetically modified.

[0093] Pluripotent cells can be of any suitable type, as described herein above.

The present invention also provides a method for producing a genetically modified EPL cell, comprising: a pluripotent cell, and a biologically active agent according to the invention or a high molecular weight thereof. Or providing a low molecular weight component, or a conditioned medium according to the present invention, or an extracellular matrix according to the present invention, and optionally, a low molecular weight component of a biologically active factor according to the present invention; Modifying one or more genes in sexual cells; and genetically modified pluripotent cells and biologically active factors or their high or low molecular weight components, or conditioned media, or extracellular matrix. Contacting with an EPL cell; producing a genetically modified EPL cell in the presence or absence of an additional gp130 agonist.

[0095] Pluripotent cells can be of any suitable type, as described herein above.

[0096] Alternatively, EPL cells can be produced as described herein above and then genetically modified.

The method can include the further step of differentiating the genetically modified EPL cells to produce a genetically modified partially differentiated or terminally differentiated cell.

Alternatively, the genetically modified EPL cells and / or ES cells can be produced by dedifferentiating a genetically modified partially or terminally differentiated cell, the method comprising: Includes: partially or terminally differentiated cells; and a biologically active factor according to the invention or a high or low molecular weight component thereof, or a conditioned medium according to the invention, or according to the invention Providing an extracellular matrix and, optionally, a low molecular weight component of a biologically active agent according to the invention; modifying one or more genes in the partially or terminally differentiated cells And a genetically modified dedifferentiated cell and a biologically active factor or a high or low molecular weight component thereof. Dedifferentiating the genetically modified partially or terminally differentiated cells by a method that involves contacting the cells with a conditioned medium or extracellular matrix; or the presence of an additional gp130 agonist or Producing genetically modified EPL cells in the absence.

[0099] The genetically modified EPL cells so formed can be reverted to genetically modified ES cells and / or as described herein above. Can be differentiated to form partially differentiated or terminally differentiated cells.

In one aspect of the method, the genetically modified partially differentiated or terminally differentiated cells are nuclear transplanted to form a genetically modified dedifferentiated pluripotent cell. Can be used as a nucleoplasm. The dedifferentiated pluripotent cells so formed are, in the presence or absence of an additional gp130 agonist,
It can be used to produce genetically modified ES and EPL cells by a method that includes contacting with: A biologically active agent according to the invention or a high or low molecular weight component thereof. Or a conditioned medium according to the invention, or an extracellular matrix according to the invention, and optionally, a low molecular weight component of a biologically active factor according to the invention.

The genetically modified ES and EPL cells so formed can form a genetically modified partially or terminally differentiated cell, as described herein above. Can be differentiated.

Modifications of the genes of these cells can be performed by any means known to those of skill in the art, and these modifications can be accomplished by introducing foreign DNA, removing DNA, or removing DNA from these cells. Generating a mutation. Modification of this gene
Includes any change in the genetic makeup of the cell that results in a cell that is genetically different from the original cell.

Thus, the method may comprise a pluripotent cell maintained in its pluripotent state or a genetically modified pluripotent cell, or a genetically modified pluripotent cell maintained in its partially differentiated state. The modified partially differentiated cells can be provided and used to form chimeric and transgenic animals, including animals created by nuclear transfer.

In a further aspect of the invention, an ES cell, a genetically modified ES cell, an EPL cell, a genetically modified EPL cell, produced by a method of the invention.
Partially or terminally differentiated cells and genetically modified partially or terminally differentiated cells are provided.

In a further aspect of the present invention, there is provided a method of producing a chimeric animal, comprising: a pluripotent cell or a genetically modified pluripotent cell according to the invention, and a protogastrutum Providing a developing embryo; introducing a pluripotent cell or a genetically modified pluripotent cell into the protogastrulating embryo; and monitoring the ability to form a chimera.

Thus, in a still further aspect of the invention, a chimeric or transgenic animal (including an animal derived from nuclear transfer) produced using a cell according to the invention or a genetically modified cell. Is provided.

The biologically active agents, components thereof, conditioned media, extracellular matrices, cells and methods of the invention have numerous applications, including: (1) Cytoplasmic or nucleoplasmic in nuclear transfer supply source. For example: EPL cells of any origin and cells derived from EPLs (ES cells, partially differentiated cells, terminally differentiated cells) can be used as cytoplasts or nucleoplasm in nuclear transfer Genetically modified EPL cells, genetically modified EPL cells, and genetically modified partially differentiated cells and genetically modified terminally differentiated cells are obtained by cytoplasmic or nucleoplasmic nuclear transfer EPL derived from dedifferentiation of partially differentiated or terminally differentiated cells
Cells can be used as a source of nuclear material for nuclear transfer.Genetically modified from dedifferentiation of genetically modified partially differentiated cells or genetically modified terminally differentiated cells Modified EPL cells can be used as a source of nuclear material for nuclear transfer. Unmodified or genetically modified pluripotent cells, partially differentiated cells, terminally differentiated Infected cells, tissues, organs and animals can be derived from nuclear transfer when EPL cells are used as cytoplasts or nucleoplasm. (2) Use in human medicine. For example: EPL cells obtained from any source and, preferably, differentiated progeny of EPL cells obtained by programmed or directed differentiation, are not modified for human cell therapy It can be used in form.

A preferred mode of use is to use autologous EPL cells and their progeny: cells programmed to form an ectodermal lineage include neuronal and skin cell treatment procedures It can be used for cell therapy procedures that are not limited to this. For example, cell therapy using neuronal cells can be used to treat Parkinson's disease-cells programmed for mesodermal lineage include bone marrow therapy and muscle cell therapy (
For example, for the treatment of cancer and for bone marrow rescue), including but not limited to genetically modified E obtained by any means described in this application.
PL cells, and preferably, their differentiated progeny obtained by directed or programmed differentiation, can be used for human gene therapy.

[0109] Such gene therapy is preferably performed using autologous EPL cells or differentiated progeny thereof.

Examples of genetic diseases that can be treated include hemophilia, type I diabetes, Duchenne dystrophy and other muscular dystrophy, Gauche's disease and other mucopolysaccharide diseases, cystic fibrosis EPL cells obtained from any source and, preferably, their differentiated progeny obtained by programmed or directed differentiation, for example, confer resistance to infection by a virus EPL cells obtained from any source, and preferably, programmed differentiation or indication, by genetic engineering of the viral receptor that provides the cell, to confer on the cell its resistance to viral infection Those differentiated progeny obtained by the differentiated differentiation are genetically modified. Can enable cells to act as drug delivery systems for biologically active protein drugs, such as cytokines or lymphokines, such as interleukin-2 for treating cancer and other diseases. -Unmodified or genetically modified EPL cells obtained from any source, and preferably their differentiated progeny obtained by programmed or directed differentiation, are transplanted. Can be used to create components of cells and tissues and organs.

The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, that the following description is illustrative only and should not be understood in any way limiting the generality of the invention described above.

Table 1. Summary of gene expression patterns in ES and EPL cells compared to gene expression in ICM and primitive ectoderm of the embryo. RNA was isolated from ES and EPL cells grown for 2 days in the presence of MEDII, and Oct4, Uvomorulin, Fgf5, Rex1, AFP, H19, Evxl and Brachy.
Ury expression was analyzed by Northern blot. Gbx2 expression
Detection was performed using an RNA protection assay. Alkaline phosphatase activity was detected using enzyme staining in ES and EPL cell layers. Embryonic expression patterns were determined by Rosner et al., 1990, Schueller et al., 1990, Yeom et al.
991 (Oct4); Hahnel et al., 1990 (alkaline phosphatase);
Sefton et al., 1992 (Uvomorulin); Haub and Goldfarb.
1991, He'bert et al., (Fgf5); Rogers et al., 1991 (R
ex1); Bulfone et al., 1993; Chapman et al., 1997 (Gbx
2); Dziadek and Adamson, 1978 (AFP); Poiri
er et al., 1991 (H19); Bastian and Gruss, 1990, D.
ush and Martin, 1992 (Evx1); and Herrmann
, 1991 (Brachyury).

Table 2. ES cells and reverted EPL cells contribute to the development of chimeric mice when injected into CBA / C57 black host blastocysts, whereas EPL cells do not.
E14TG2a ES cells, their EPL cell derivatives (EPL; 2 and EPL; 4 respectively) and reverted EPL cells grown in 50% MEDII for 2 and 4 days (medium containing mLIF but no MEDII) E in
Formed by culturing 6 days PL cells) (EPL; 2 ^R and EPL; 4 ^R) Summary of results from injection of.

Table 3. Effect of purified ECM components on EPL cell stability in culture. ES
Cells were seeded in DMEM + 50% MEDII on tissue culture plastic pretreated with the ECM components gelatin, laminin, plasma fibronectin, type IV collagen, and a mixture of laminin, plasma fibronectin and type IV collagen. After 5 days, cultures were stained with alkaline phosphatase and the percentage of EPL cell colonies without associated differentiation was determined.

Table 4. Individual 5.5 d. p. c. Embryos are cultured in embryo culture medium or embryo culture medium + 50%
In any of the MEDIIs, seeded into tissue culture wells treated with 2 ml of type IV collagen. After 5 days, explants were fixed in 4% PFA and stained for Oct4 expression by in situ hybridization.

Table 5. Summary of physiochemical properties of low molecular weight components of EPL cell inducing activity.

Table 6. Amino acid analysis of samples induced by purification (Figure 15) of sfMEDII (active sample) and unconditioned medium (control sample). sfMED
Samples purified from II were analyzed with and without hydrolysis.

Table 7. Summary of EPL cell-inducing activity of amino acids, proline analogs and peptides tested in the presence of R. + = EPL cell inducing activity (minimum active concentration
-= Lack of EPL cell formation; ^* = high concentration (500 μM AZ)
ES cell death with ET, 100 μM 3,4-dehydro-L-proline, 50 μM sarcosine and substance P).

Table 8. Summary of EPL cell formation from ES cells against ECM components. ECM components were added in solution (± 40 μM L-proline) or in matrix (± 40 μM
(L-proline). Purified biological activity = Protocol 2
MedII = Hep G2 conditioned medium or ECM; + = EPL cell formation; − = no EPL cell formation; ± = scattered flat pluripotent cell colonies. ECM components were used in solutions at 10 μg / ml (type IV collagen; vitronectin and plasma fibronectin), 3 μg / ml (laminin) or 2 μg / ml (cellular fibronectin and purified bioactivity). Gelatin was used at 0.01% and MEDII at 50%.

Example 1 Formation of a Primitive Ectoderm-like Cell Population. EPL Cells Derived from ES Cells in Response to Biologically Induced Factors Materials and Methods Cell Culture Conditions Cultures were performed in the absence of feeders on tissue culture grade plasticware (Falcon) pretreated with 0.2% gelatin / PBS for a minimum of 30 minutes. Cells were grown in Dulbecco's Modified Eagle's Medium (Gibco BRL) supplemented with 1000 units of LIF, pH 7.4 (containing high glucose and
% Fetal calf serum (FCS; Commonwealth Serum Labor)
atories), 40 μg / ml gentamicin, 1 mM L-glutamine,
Cultures (supplemented with 0.1 mM β-mercaptoethanol (β-ME)) (referred to herein as DMEM) in a humidified incubator under 10% CO ₂ . Routine tissue culture was performed as described by Smith (1991). E14 ES cells (Hooper et al., 1987) were transferred to Anna
Michelska (Murdoch Institute Melbour
ne). CCE ES cells (Robertson et al., 1986)
Richard Harvey (Walter and Eliza Hall)
Institute, Melbourne). The MBL5 (Pease et al., 1990) and D3 (Doetschman et al., 1985) ES cell lines were
Lindsay Williams (Ludwig Institute, Me
lbourne). CGR8 and E14TG2a (Hooper et al.,
1987) ES cells were obtained from Austin Smith (Centre for G).
(Enome Research, Edinburgh).

COS transfected with the mouse LIF expression plasmid pDR10 as described by Smith (1991) with the following modifications:
-1 (ATCC CRL-1650) cells. COS-1 cells,
Bio-rad Gene Pulser at 270 volts and 250 μFD
Was transfected by electroporation using a capacitance of Transfected cells containing high glucose and 10% FCS,
DME supplemented with 40 μg / ml gentamicin and 1 mM L-glutamine
Plated at 7 × 10 ⁴ cells / cm ² in M, pH 7.4. Media was harvested and assayed for LIF expression as described by Smith (1991). Alternatively, 1000 units of recombinant LIF (ESGR
O, AMRAD).

Hep G2 cells (Knowles et al., 1980; ATCC HB-8065)
) Was maintained in culture in DEME and passaged at confluence. HepG2 cells were seeded in conditioned medium (MEDII) at a density of 5 × 10 ⁴ cells / cm ^{2 in} DMEM. Media was harvested 4-5 days later, sterilized by filtration through a 0.22 μm membrane, and supplemented with 0.1 mM β-ME before use. ME
DII was stored at 4 ° C for 1-2 weeks or at -20 ° C for up to 6 months without apparent loss of activity.

EPL cells were isolated from 50% MEDI in DMEM with or without the addition of LIF.
Formed and maintained in media containing I-conditioned media. EPL formation was evident at between 10% and 80% addition of MEDII, which was the optimal culture condition at 50% MEDII (data not shown).

EPL cells were formed from ES cells and maintained as follows.

Adhesive culture: ES cells were grown at a density of 1.3 × 10 ⁴ cells / cm ² as described above.
Inoculated on tissue culture grade plasticware (Falcon) pretreated with 0.2% gelatin / PBS in DMEM with 50% MEDII for a minimum of 30 minutes. EPL cells were maintained in 50% MEDII using conventional tissue culture techniques (as described in Smith, 1991).

In suspension aggregates: ES cells were transferred to D containing 50% MEDII as described above.
Seed at a density of 1 × 10 ⁵ cells / cm ² in suspension culture in bacterial Petri dishes in MEM. The resulting EPL cell aggregates were split 1: 2 after 2 days and
Seed into fresh DMEM containing 0% MEDII. Control aggregates (EB
) Was formed by aggregating ES cells in the same manner in DMEM without MEDII.

DNA manipulations All DNA manipulations were performed using standard protocols (Sambrook
k et al., 1989). The DNA was transferred to a T ₇ Sequenase kit (Pharma
cia) was used as ordered by the manufacturer and sequenced. Sequencing reaction
Separated on a 6% polyacrylamide / urea gel and exposed to X-ray film as described in Sambrook et al., 1989.

(Northern blot and ribonuclease protection analysis) Cytoplasmic RNA was obtained from cultured ES cell layers and EPL cell layers by Edward.
isolated using the method of S. et al. (1985). Total RNA was isolated from EPL cell aggregates washed once with 1 × PBS, pelleted by centrifugation, and
Stored at 20 ° C. The pellet is mixed with 1 ml of extraction buffer (50 mM NaCl;
0 mM Tris · Cl, pH 7.5; 5 mM EDTA, pH 8.0; 0.5
% SDS) (supplemented with 200 μg proteinase K (Merck)). Sterile, acid-washed glass beads (0.5 mm) were added just below the meniscus and vortexed vigorously. An additional 3 ml of extraction buffer / proteinase K was added and incubated at 37 ° C. for 1 hour before phenol / chloroform extraction. Nucleic acid was added to 400 μl of 3 M Na acetate and 4 ml
Of isopropanol was added for 30 minutes at −80 ° C. and collected by centrifugation at 3500 rpm in a bench top centrifuge. Nucleic acid was added to 400 μl DNase I buffer (50 mM Tris · Cl, pH 8.
0; 1 mM EDTA, pH 8.0; 10 mM MgCl ₂ ; 0.1 mM DT
T), DNase I-free RNase (Boehringer Mannh)
eim) resuspended in 20 units and incubated at 37 ° C. for 1 hour. RNA was precipitated by the addition of 40 μl of 3M Na acetate and 1 ml of ethanol and collected by centrifugation.

[0129] Northern blot analysis was performed as described in Thomas et al., 1995. Gigaprime labeling kit (BresaGen)
Alternatively, it was prepared from DNA fragments using the Megaprime kit (Amersham). DNA fragments were isolated from the following plasmids. H
The 19 cDNA fragment was converted to LC10-8 (Poirier et al., 1991).
Was cut out as a 778 bp fragment using PvuII. Blues
The 462 bp Oct4 StuI cDNA fragment (residues 491 to 593) in the script KS + was purified from H. var. Dr. Schoeler
Et al., 1990). The 484 bp fragment was converted to XhoI / HindI
Released by II digestion and used for probe generation. A Brachyury-specific probe was constructed from pSK75 (Herrman, 1991) by 1600 bp.
It was excised as an EcoRI fragment. The Uvomorulin probe is used for pUC
8 from F20A (Ringwald et al., 1987), a 620 bp EcoR
It was cut out as an I fragment. 700bp PstI / BamHI Evx
1 The cDNA fragment was converted to pAB11 (Dush and Martin, 19
92). Fgf5 was obtained as a full length coding region clone in Bluescript (He'bert et al., 1990). From now on, 800b
The pEcoRI / BamHI cDNA fragment was isolated and used as a probe. The AFP probe was used to encode the first 350 bp of mouse AFP cDNA cloned in pBluescript KSII +.
Generated from the 00 bp EcoRI fragment (Dr. R. Krumlauf,
NIMR, donated by London). 848 bp Rex1 fragment (this is pCR ^™ II (pRex1, Dr. N. Clarke, Departmen)
to of Genetics, Cambridge University, U
. K. ) Was cut out by EcoRI digestion. The mGAP probe was synthesized by labeling the entire plasmid (Rathjen et al., 1990) containing the 300 bp mGAP cDNA sequence. L17, K7 and Psc1 of 737 bp, 232 bp and 458 bp respectively
Of the ddPCR fragment by EcoRI digestion into Bluescript
II Released from KS + and used as probe.

The RNase protection assay methodology and antisense probes for detection of Gbx2 and mGAP were as described in Chapman et al., (1997). Antisense riboprobes for Oct4 detection were transcribed from the above Oct4-containing plasmid linearized with NciI using T3 RNA polymerase.

[0131] Northern blots and RNase protectants were exposed to phosphorescent major screens (Molecular Dynamics) and
Developed on a Dynamics Phosphor Imager instrument. The level of gene expression was measured using ImageQuant (Molecular Dynam).
ics) using software.

In Situ Hybridization In situ hybridization was performed on cell layers, cell aggregates and whole mouse embryos using the method of Rosen and Bedington (1993) with the following modifications. Cell layers and cell aggregates are prehybridized, hybridized and washed at 60 ° C., and embryos are prehybridized, hybridized, and 65 ° C. (L17; K7) and 63
. Washing was performed at 5 ° C. (Psc1). Cell layers, cell aggregates, and embryos were blocked with 10% heat inactivated FCS, and antibodies were added in TBST / 1% FCS. This antibody was not pre-adsorbed in the case of in situ hybridization of cell layers or cell aggregates. Antibodies were pre-adsorbed using embryo powder (Harlow and Lane, 1988) prior to incubation with mouse embryos.

Out-crossed Swiss embryos were time-matched to Swi
Collected from ss female mice on specific days. 0.5 days after mating was designated as noon on the day of filling. Mated female mice were sacrificed by cervical dislocation or CO ₂ asphyxiation. The uterus was then maintained at 37 ° C. in DMEM supplemented with 10 mM HEPES, pH 7.4 until the embryos were cut. Embryos are cut using standard cutting techniques
Removed in PBS. The Reichart membrane is removed and the embryos are
Direct transfer to 4% paraformaldehyde (PFA) in S fixative.

The antisense Oct4 probe was replaced with a 462 bp StuI Oct4 cDN
Synthesized by T3 RNA polymerase as a run-off transcript from Bluescript containing the A fragment (Schoeler et al., 1990) and HindII
I linearized. The sense transcript used as a control was obtained from the same plasmid linearized with XhoI and transcribed using T7 RNA polymerase. Sense and antisense Fgf5 transcripts were cloned into EcoRI or B
generated from plasmid clones containing the full-length Fgf5 coding sequence linearized with either of the amHI (He'bert et al., 1990) and T7 RNA, respectively.
Transcription was performed using polymerase or T3 RNA polymerase. L17, Ps
The sense and antisense probes for cl and K7 were
It was generated from a plasmid clone containing fragments of 7 bp, 458 bp and 232 bp. Antisense transcripts of L17, Psc1 and K7 were
Produced by digestion and HindIII digestion with transcription with T3 RNA polymerase (L17, Psc1) and transcription with T7 RNA polymerase (K7). The sense probe was digested with BamHI of the clone, and T7 RNA polymerase for L17 and Psc1 and T7 for K7.
3 Produced by transcription using RNA polymerase. An antisense Brachyury probe was synthesized from pSK75 (Herrman,
1991), linearized with BamHI and transcribed using T7 polymerase. Sense probes were generated from the same plasmid linearized with HindIII and transcribed using T3 polymerase.

(Alkaline Phosphatase Staining) Alkaline phosphatase was visualized using a diagnostic kit 86-R (Sigma). This kit was used as described by the manufacturer with the following modifications.
The cell layer was fixed in 4.5 mM citric acid, 2.25 mM sodium citrate, 3 mM sodium chloride, 65% methanol and 4% paraformaldehyde before washing and staining.

Histological Analysis Embryoid bodies were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4 ° C., then embedded in paraffin wax and Hogan Were sectioned as described in 1994. Sections were stained with hematoxylin: eosin.

(DDPCR) The identification of transcripts differentially expressed by differential presentation PCR between ES and EPL cells was determined by Schulz (1997) (Psc1) and Liang and Pardee (1992) (L17, K7). Performed as described in Primers were designed to incorporate an EcoRI restriction site and
The A product was cloned into EcoRI digested pBluescript II KS +.

Results (MEDII results in the transfer of ES cells to EPL cells in adherent cultures) In media and feeder cell layer supplemented with recombinant LIF (LIF) (FIG. 1A)
ES cells grow as a homogeneous population with greater than 95% of the colonies exhibiting a pronounced hemispherical colony morphology when grown in the absence of C. To assay for factors that can induce ES cell differentiation, ES cells were seeded and cultured in the presence of mLIF and conditioned media from mammalian cell lines. Five days later, the cells were evaluated for divergence from ES cell colony morphology.

Hep G2 cells (MEDII) conditioned on tissue culture grade plasticware (Falcon) pretreated with 0.2% gelatin / PBS
When cultured in the presence of 80% medium, the ES cells gave rise to a cell population that was morphologically different from the cells we named early primitive ectoderm-like or EPL cells (FIG. 1B). . EPL cell formation was specific for MEDII and no response was seen to any of the other 13 conditioned media assayed. In contrast to the characteristic ES cell colony morphology, EPL cells grow as monolayer colonies, where individual cells containing nuclei with one or more prominent nucleoli can be easily recognized. Was. Morphologically, EPL cells are similar to P19 EC cells (McBu
nery and Rogers, 1982; Rudnicki and McBurn
ey, 1987). The cells are thought to be similar to cells of the primitive ectoderm. 5
Culture of ES cells in the presence of 0% MEDII resulted in a relatively homogeneous cell population. In this population, more than 95% of the colonies were of EPL cell colony morphology, and no remaining ES cell colonies could be detected (FIG. 1D).
). This occurred in the presence or absence of added LIF (FIG. 1D). Within the EPL cell population, sporadic colonies of differentiated cells similar to those found in ES cell cultures were detected (FIG. 1D). The level of differentiated colonies was 4 times higher than EPL cell cultures formed in the absence of added LIF (FIG. 1D). This suggests a possible role for LIF in EPL stability. The formation of a relatively uniform cell population from ES cells indicates that ES cells
This was in contrast to the differentiated cell type variants that were generated when spontaneously differentiated in response to the elimination of E. coli (FIGS. 1C, D). However, a small population of EPL-like colonies was found in naturally differentiated ES cell cultures (FIG. 1D). This suggests that EPL cells are normal derivatives of ES cells. EPL cell morphology can be maintained in long-term culture for more than 40 passages, ie, more than 100 days (data not shown), and was dependent on the continued presence of MEDII in its culture medium. ME
Removal of DII and LIF resulted in the generation of an array of differentiated cell types, similar to that generated from spontaneous ES cell differentiation (FIG. 1C) (data not shown).

Metastasis from ES cells to EPL cells in response to MEDII was induced by a number of independently induced ES cell lines, including MBL5 D3, CCE, E14 and CGR8.
(Data not shown). EPL generated from each ES cell line
The appearance of the cells was similar.

(EPL Cells Are Pluripotent) The spontaneous generation of differentiated cells in culture (FIG. 1D) and the spontaneous differentiation of EPL cells following removal of MEDII indicates that these cells are It suggested that the ability was retained.

Early mouse embryo and germ-line pluripotent cells, as well as ES cells in culture, are characterized by the expression of the homeobox gene Oct4 and alkaline phosphatase activity. Oct4 expression is restricted to pluripotent cells of early mouse embryos and is down-regulated for pluripotent cell differentiation both in vivo and in vitro. EPL cells were converted to ES cells by MEDII or MEDII +
It was formed by culturing in the presence of LIF for two, four, six and sixteen days, with passage every two days. Northern analysis of RNA from these populations showed Oct4 expression at levels equivalent to those found in ES cells (
FIG. 2Ai), and suggests that EPL cell formation is not equivalent to the process of spontaneous differentiation (FIG. 2Aii). In situ hybridization of ES4 and EPL cell monolayers with an Oct4-specific antisense RNA probe distributes evenly across the EPL cells, but a subpopulation of cells in the culture Unrestricted Oct4 expression was shown (FIGS. 2B, C). Cells that differentiated spontaneously within the population did not express Oct4 (data not shown). Analysis of alkaline phosphatase activity also showed uniform expression by EPL cells (FIG. 2D, E
). Here, there was down regulation of activity in differentiated cells (data not shown). The uniform distribution of these pluripotent cell markers in EPL cell cultures has demonstrated the homogeneity of the EPL cell population.

(EPL cell formation is defined as primitive ectoderm.
Expression of pluripotent cell marker genes by EPL cells indicates that these cells are down-regulated with pluripotent cell populations in early embryos, or perhaps Oct4 expression Suggested that it could be equivalent to a non-differentiated cell line. Embryonic pluripotent cell populations can be distinguished from differentiated cell lineages by morphological and developmental criteria and by temporal and spatial expression of marker genes. Embryonic equivalents of EPL cells were studied by analyzing the expression of marker genes that identify pluripotent cell populations of the embryo and differentiated cells of the extraembryonic lineage and gastrulation embryo.

The three genes Fgf5, Rexl and Gbx2 have been reported to be differentially transcribed between ICM and primitive ectoderm cells (summarized in Table 1). Although expression of Fgf5 is upregulated in the formation of ICM-derived primitive ectoderm, expression of both Rexl and Gbx2 can be detected in ICM, although 6.5 d. p.
c. Up to no detectable in the primitive ectoderm. Expression of Fgf5 and Rex1 in ES cells and EPL cells was evaluated by Northern blot (FIG. 2).
A; Table 1). Gbx2 expression was analyzed by RNase protection assay (FIG. 2F; Table 1). The slightly detectable expression of Fgf5 in ES cells was increased 50-fold in EPL cell proliferation in MEDII for 2 days. F
gf5 expression increases in EPL cells with time in culture, resulting in Fg
Maximal expression, representing a 340-fold induction of f5, was reached by day 6 and maintained in culture for at least 16 days. Rex1 was expressed at high levels by ES cells, but this expression was down-regulated by 50% within 2 days of EPL cell formation (FIG. 2A; Table 1). Rex1 expression was observed in EPL cultured for longer periods of time.
Further reduced in cells, as a result, EPL cells grown in MEDII for 6 days showed a 5-fold reduction in Rex1 expression compared to ES cells. Gbx
2 expression was high in ES cells and maintained in culture in MEDII for 2 days. However, this Gbx2 expression was reduced to undetectable levels by day 6 with further culture in MEDII. Changes in the expression of Fgf5, Rexl and Gbx2 are compared to MEDII + LIF compared to EPL cells formed in MEDII.
Was delayed when migration of ES cells to EPL cells was performed in the cell (FIG. 2A; 2F)
. This suggests that LIF delays the transition from ES cells to EPL cells. Maintenance of EPL cell morphology and high levels of Fgf5 and Oct4
(FIG. 2A) demonstrates the ability of MEDII to maintain EPL cells as a stable cell population, and is distinctly different from the altered gene expression observed during spontaneous differentiation of ES cells (FIG. 2Aii).

EPL cell monolayers cultured in MEDII for 4 days were probed for Fgf5 expression using DIG-labeled antisense Fgf5 transcripts. Equivalent levels of Fgf5-specific staining were observed in all EPL cells in culture (
(FIG. 2G). This indicates that the uniformity of the transition from ES cells to EPL cells and the EP
Confirm both homogeneity of the L cell population.

Uvomorulin (expressed by pluripotent cells of the ICM and primitive ectoderm,
Expression of cadherin, which is down-regulated in primordial germline cells, was evaluated in ES and EPL cell cultures by Northern blot analysis. Uvomorulin expression was maintained in EPL cells at levels equivalent to or greater than those observed in ES cells (FIG. 2H). This suggests that these cells do not represent an in vitro equivalent of primordial germ cells.

[0147] Consistent with the expression pattern of the pluripotent cell marker, EPL cells were transformed into early embryonic (H1
9, AFP), primitive streak (Evx1), or developing mesoderm (Brachyur
y) did not express detectable levels of the marker gene specific for the extraembryonic lineage (Table 1; data not shown).

The morphological transfer of ES cells to EPL cells has been found to be accompanied by differential regulation of marker genes that identify pluripotent cell populations in vivo. Although gene expression in ES cells was equivalent to their origin from pluripotent cells of the pre-implantation embryo, EPL cells were shared by the primitive ectoderm, only one cell population in the early embryo (Table 1).

(Expression of a novel pluripotent cell marker gene supports an association between EPL cells and early primordial ectoderm) ES cell to EPL cell migration is a transition of ICM to primordial ectoderm It appears to mimic gene expression changes associated with. Screening was performed to identify genes that are differentially expressed between these cell populations. 2 in MEDII + LIF
Isolated from ES and EPL cells cultured for 4, 6, and 8 days
NA is compared with Differential Display Polymerase Chain Reaction (ddPCR)
Was analyzed using. CDNA differentially expressed between ES cells and EPL cells
The fragment was purified, cloned, and sequenced.

As three novel partial cDNAs, L17, Psc1 and K7 were identified (FIG. 3). RNA and EPL of ES cells prepared as outlined above
Northern blot expression analysis of cellular RNA showed that L17 was highly expressed in ES cells, but was rapidly down-regulated with EPL formation and maintenance in culture (FIG. 4A). Psc1 is expressed in ES cells and L1
It was down-regulated in EPL cells more gradually than 7 (FIG. 4B). K7 was expressed at low levels in ES cells, but was upregulated in extended EPL cell cultures (4, 6, and 8 days; FIG. 4C).

In vivo expression of these genes in pre-implantation and peri-implantation mouse embryos was determined at 3.5 d. p. c. ~ 5.5d
. p. c. Mouse embryos, including the transfer of ICM to primitive ectoderm
Were analyzed by whole mount in situ hybridization. Expression of all three novel genes was localized to pluripotent cells at these stages in vivo.

L17 is 3.5 d. p. c. ICM, and 4.5 d. p. c. ICM
/ Highly expressed in pluripotent cells of primordial ectoderm (FIG. 5A). L17 expression is down-regulated as the ICM / protoectoderm begins to proliferate (day 4.75);
And it was undetectable after pro-amniotic cavity formation.

Psc1 is 3.5 d. p. c. Of the inner cell mass and 4.5 d. p
. c. Was expressed in pluripotent cells of the ICM / primordial ectoderm (FIG. 5B). L17
Unlike Psc1, expression is maintained in proliferating primordial ectodermal buds (4.7).
5 to 5.0 d. p. c. ) And 5.0 d. p. c. Later it was down-regulated after initiating the formation of the retroamniotic cavity.

The expression of K7 was 4.5 d. p. c. It was not detected in pluripotent cells of previous ICM / protoectoderm (FIG. 5C). K7 expression was 5.25 d. p. c. Was detected in pluripotent cells after formation of the amniotic cavity in
Form a columnar epithelial sheet characteristic of primitive ectoderm 5.5d. p. c. Was controlled down by.

The stringent restriction of L17, Psc1 and K7 expression on embryonic pluripotent cells in vivo supports the identification of EPL cells as pluripotent. The differential expression of these genes between the ICM / primordial ectoderm and the primordial ectoderm population of the embryo identifies the EPL cells as distinct from EC cells and more closely related to cells of the early primordial ectoderm And matched.

Role of gp130 Signaling in EPL Cell Maintenance Maintain E without the addition of mLIF compared to EPL cultures with the addition of mLIF.
The increased levels of differentiated cell colonies seen in PL cell cultures (FIG. 1)
C, D; FIG. 6), suggesting a possible role for LIF, or other cytokines signaling through gp130, in maintaining EPL cells. Mouse gp neutralizes the activity of mLIF, hOSM, hIL-6 and hIL-11 but not hLIF in myeloid leukemic M1 cells and ES cells (Lake, 1996)
The role of gp130 signaling in the maintenance of EPL cells was evaluated using an antibody to RPL130 (RX-19; Koshimizu et al., 1996) and an antibody to hLIF that neutralizes hLIF activity (R & D Systems).

EPL cell formation was determined by anti-gp130 antibody (10 μg / ml) or anti-hLIF
Observed in the presence of antibody (10 μg / ml). On the other hand, the addition of anti-hLIF antibody resulted in significant destabilization of EPL cells compared to cells cultured in MEDII alone (FIG. 6). This suggested that the presence of hLIF in MEDII, but not mLIF expressed by the cells, was important in maintaining EPL cells. Anti-hLIF antibody and 1,000 units of mLIF (ESGRO
Amrad) reduced the level of differentiation to the level observed in EPL cell cultures formed in MEDII + LIF. This means that gp
Demonstrates that 130 signaling could restore EPL cell stability in culture.

ES cells were isolated from MEDII, anti-hLIF antibody (10 μg / ml), and mLI
F was seeded at a concentration of 10 to 1000 units / ml in a medium containing (ESGRO; Amrad). The maintenance of EPL cells is approximately 50-100 units / ml m
Achieved by the addition of LIF (data not shown), which is 10-20 times lower than the 1000 units / ml required for ES cell maintenance.

EPL Cell Formation in Suspension Culture Aggregates (EBM) formed from ES cells in 50% MEDII for 4 days were formed from ES cells in the absence of MEDII for 4 days. Control aggregates (EB) were compared at the morphological and gene expression levels. EB is E
Internal tissue collapse was shown in comparison with BM. This is indicated by a random distribution of cell death in some internal regions that are randomly distributed through the EB compared to a uniform central region of cell death in the EBM (FIG. 7A). This central area of cell death in the EBM was surrounded by a distinctly homogeneous cell layer of uniform thickness. Furthermore, the outer layer of extraembryonic endoderm formed in ES cells EB cannot be distinguished by microscopy. This suggests that EBM consists of a single cell type.

[0160] Gene expression analysis was performed on EBs and EBMs generated in culture for 4 days by in situ hybridization and Northern blot analysis (Figure 7B). Aggregates were analyzed for the expression of primitive ectoderm markers Oct4 and Fgf5, and Brachyury, a gene that is up-regulated in the differentiation of pluripotent cells into developing mesoderm. The expression of Oct4 in the EB is patchy (cells in the area are not stained), suggesting that some of the cells in this aggregate have undergone differentiation. In contrast, Oct4 expression was detected uniformly throughout the cell layer containing EBM. This suggests that these cells maintained pluripotency and did not undergo differentiation. This conclusion is supported by the expression of the differentiation-specific marker Brachyury in about 10% of EBs, and suggests that some cells in these aggregates have undergone differentiation to the developing mesoderm. In contrast, no detection of Brachyury expression in the EBM indicates that cells in these aggregates did not undergo any differentiation to the developing mesoderm.

The expression of Fgf5 in aggregates was analyzed to assess EPL cell / primordial ectoderm formation (FIG. 7B). In EBM, Fgf5 was uniformly expressed throughout the cell layer. This indicates that the pluripotent cells in these aggregates undergo migration and EPL cells /
Figure 4 shows that it represented a homogeneous population of pluripotent cells that formed primitive ectoderm. In contrast,
The expression of Fgf5 in EB was heterogeneous.

[0162] Northern blot analysis of RNA extracted from EB and EBM supported the findings of in situ analysis (Figure 8). At day 4, EBM gene expression was characterized by high levels of Oct4 and Fgf5 that are diagnostic for primitive ectoderm. Brachyury expression, which is diagnostic for developing mesoderm, could not be detected. ES cell EBs expressed a range of genes consistent with the formation of heterogeneous cell types.

[0163] Summarizing the expression data, EBM is a distinction between EPL cells and embryonic primordial embryos, in contrast to EBs, where different cell types ranging from early primordial ectoderm to extraembryonic endoderm can be detected on differentiated cells. It was suggested that it consisted of a homogeneous cell population equivalent to the germ layer. Control aggregates generated in 100 units of LIF without MEDII did not develop as EBM. This demonstrates that the alterations observed in the differentiation of pluripotent cells in the presence of MEDII cannot be responsible for the low levels of LIF present in MEDII (data not shown).

Summary The data in this example demonstrate that biological activity directing the uniform differentiation of ES cells into an alternative stable pluripotent cell population called early primordial ectoderm-like cells or EPL cells Is included in the conditioned medium, MEDII. EPL cells were identified as most closely related to cells of the early primitive ectoderm based on gene expression and morphology. Transfer from ES cells to EPL cells can be performed in adherent cultures or in suspension aggregates.

Example 2 ES cells and EPL cells represent different but interchangeable pluripotent cell states Materials and Methods Unless otherwise stated, all cell and tissue culture techniques were performed As described in Example 1. EPL cells were transformed with 1000 units of LIF in the absence of MEDII.
Were reverted by seeding a single cell suspension of EPL cells at a density of 1.3 × 10 ⁴ cells / cm ² in a medium containing.

Blastocyst injection ES and EPL cells were introduced into CBA / C57F2 blastocysts using standard blastocyst injection techniques as described in Stewart, 1993.

GPI Analysis Glucose phosphate isomerase (GPI) analysis of blood and tissues was performed using Brad
ley, 1987. Blood samples were collected from the tail. The analyzed tissue samples were brain, eye, thigh, heart, intestine, kidney, liver, lung, muscle, stomach, skin,
Obtained from spleen, tongue, thymus, and ovaries or testes. Tissue samples were prepared by freeze / thaw and by homogenization.

(Results) EPL cells seeded and cultured in media containing LIF but not MEDII were observed to be compatible with ES cell-like colony morphology (we found that reversion was Call process). Furthermore, removal of MEDII from established EPL cell colonies in the presence of LIF resulted in the formation of a three-dimensional colony structure suggestive of ES cell phenotype (data not shown). Northern blot analysis was used to test the expression of Oct4, Fgf5 and Rex1 in ES, EPL, and reverted EPL cells (FIG. 9A). E cultured and passaged in MEDII and MEDII + LIF for 2, 4, and 6 days
PL cells were reverted by passage of the cells into medium containing LIF alone for 6 days. The reverted EPL cells showed low expression of Fgf5 and high expression of Rex1. This is comparable to the gene expression profile observed in ES cells and differs from high levels of Fgf5 expression and low levels of Rex1 expression in parental EPL cells. These data indicated that phenotypic reversion of EPL cells was associated with the establishment of ES cell gene expression profiles. These data also demonstrated the need for MEDII for both establishing and maintaining EPL cell properties.

[0169] Cloned EPL cell lines were generated and reverted to confirm that reversion was not a result of residual ES cells within the EPL cell population. Contains MEDII but added L
EPL cells grown in media without IF for 4 days were seeded at limiting dilution in MEDII + LIF to generate clonal EPL cell colonies. Two clones were grown in MEDII + LIF for 3 weeks before seeding in media containing mLIF alone or MEDII + LIF. The resulting culture is
Evaluated for the presence of S cells, EPL cells and differentiated colonies (FIG. 9B)
. A high percentage of cells from both lines form alkaline phosphatase positive colonies with ES cell morphology in cultures seeded and maintained in LIF alone,
This was not seen in cultures maintained in MEDII + LIF. This indicated efficient reversion of this clonal strain.

(Reverted EPL cells, but not EPL cells, contribute to chimeric mice after blastocyst injection) Pluripotent cells derived from pre-implantation and post-implantation embryos were not blastocyst-injected but blastocyst-injected. Depends on their ability to contribute to later embryonic development. ES cells retain the ability to contribute to all embryonic and adult tissues. E14TG2a ES cells and their EPL cell derivatives were tested for their ability to contribute to chimeric mice when injected into CBA / C57F2 blastocysts. The contribution of ES and EPL cell derivatives to mouse progeny was assessed by coat color contribution and blood and tissue GPI analysis. E14TG2a ES cells contributed to the development of 44% of injected blastocysts (Table 2). In contrast, E14T grown in MEDII without added LIF for 2 and 4 days to establish EPL cell gene expression
G2a-induced EPL cells did not contribute to chimerism when the coat color of 33 and 50 surviving offspring was evaluated, respectively (Table 2). GPI analysis of blood samples taken from 20 of these mice also failed to detect any contribution from EPL cells. Analysis of 15 tissue samples taken from two mice also failed to detect any EPL cell contribution. This is because the proportion of surviving offspring is comparable from blastocysts injected with ES and EPL cells,
Side effects of EPL cells on the viability of injected blastocysts cannot be explained (Table 2).

The EPL cells used in the blastocyst injection experiments described above were reverted by culturing for 6 days in medium containing LIF but not MEDII (2 ^R and 4 ^R ). These cells represent 36% (2 ^R ) and 63% (4 ^R ) of injected blastocysts.
) Contributed to embryo development (Table 2). G of blood and tissue samples taken from 10 and 2, respectively, of chimeric mice produced using reverted EPL cells
PI analysis confirmed that these cells could contribute to mesodermal, endodermal, and ectodermal-derived adult mouse cell lines (data not shown). These data indicate that the transition from ES cells to EPL cells reflects cells of various developmental potentials that contribute to development when introduced into host blastocysts, as reflected by the ability of ES cells, but not EPL cells. It is shown to occur. The ability of reverted EPL cells to contribute to chimera development suggests that this loss in the ability of EPL cells to form chimeras cannot be responsible for the loss of pluripotency in EPL cell formation.

SUMMARY The ability of EPL cells to revert to ES cells depends on the behavior of other putative and demonstrated pluripotent cell types, such as the formation of EG cells from primordial germ cells (PGC) in culture. ) And support the inference that EPL cells are pluripotent. further,
Reversion to ES cell type is a predicted characteristic of primitive ectoderm. Combined with the demonstrated ability of EPL cells to not contribute to chimeric mice after blastocyst injection to progeny, this suggests that EPL cells differ from ES cells and are most closely associated with primitive ectoderm. Support identification. Ultimately, reversion demonstrates that EPL cells can be used as substrates for the production of ES cells in vitro by dedifferentiation or reversion.

Example 3 Isolation of EPL and ES Cells from Embryonic Primordial Ectoderm and In Vitro-Derived Primordial Ectoderm Introduction Existing procedures and culture conditions may be extraprimitive for any mammalian species. It could not support the maintenance and / or proliferation of germ layer-derived pluripotent cells. Successful isolation and maintenance of pluripotent cells and cell lines derived from primordial ectoderm has led to the development of genetically engineered pluripotent cells with potential for commercial, medical, and agricultural applications. An alternative methodology is provided for isolation. In mice, such strains are designated as pre-implantation embryos (
It has only been isolated from pluripotent cells of the ES cells) or primordial germline (EG cells). Efficient isolation of pluripotent cell lines is currently limited to a limited number of inbred mouse strains (eg, 129) and has proven successful in other mammals. Absent.

MEDII is the E cell most closely associated with pluripotent cells of the primitive ectoderm of mammalian embryos.
Supports the formation and maintenance of PL cells. In this example, we discuss the development of a methodology that utilizes the biological activity of MEDII to isolate and maintain pluripotent cells from primitive ectoderm.

Materials and Methods Cells and Cell Cultures Cell culture conditions for ES and EPL cells were, unless otherwise specified,
As described in Example 1. Cells isolated from primitive ectoderm were treated with high glucose and 15% fetal calf serum (FCS; Commonwealth).
h Serum Laboratories), 40 μg / ml gentamicin, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (β-M
E), and Dulbecco's modified Eagle's medium (G
ibco BRL), pH 7.4 (embryonic culture medium) in a humidified incubator under 10% CO ₂ . The inactivated feeder layer was replaced with STO cells (AT
CC CRL-1503) or primary mouse embryonic fibroblasts (Abbondan)
zo et al., 1993). Inactivate feeder cells by 30
This was achieved by exposing the cells to gray ionizing radiation. Feeder cells were 1x1 in DMEM at least 6 hours before use according to manufacturer's instructions.
Plated at a density of 0 ⁵ cells / cm ² on tissue culture plastic treated with human placenta type IV collagen (Sigma). The feeder cell layer was washed once with DMEM before the addition of culture medium and embryonic explants. Pluripotent cells were identified using the pluripotency specific markers alkaline phosphatase and Oct4 and in situ analysis techniques as described in Example 1.

[0176] Embryoid bodies (EB) were formed as described in Example 1.

Source of Embryonic Cells CBA / C57 F2 embryos were mated on specific days in estrus (t
Im-mated) CBA / C57 F1 female mice.
0.5 days after coitum was designated as noon on the day of vaginal plugging. Mated female mice were sacrificed by cervical dislocation or CO ₂ asphyxiation, and uteri were kept at 37 ° C. in DMEM supplemented with 10 mM HEPES, pH 7.4 until embryo dissection. Remove the embryo using standard incision techniques
Transferred in DMEM supplemented with 0 mM HEPES, pH 7.4. Removal of extraembryonic ectoderm was achieved mechanically by pipetting the embryo through a Pasteur pipette stretched in flame to a slightly smaller caliber than the embryo.

(Results) (Isolation of pluripotent cells derived from primitive ectoderm formed in vitro) Embryoid bodies (EBs) formed by the aggregation of ES cells in suspension culture have extracellular cell types It follows a differentiation pathway equivalent to early mouse embryogenesis, with an ordered appearance of differentiated cell types. The appearance of these cells can be monitored by changes in gene expression. By day 4 of EB development, the pluripotent cell population in the embryoid body has
It consists mostly or completely of the primitive ectoderm, as determined by the expression of the primitive ectoderm markers Fgf5 and Oct4, and down-regulation of the ES / ICM cell marker Rex1 (FIG. 10A). Pluripotent cells in EBs differentiate largely by day 8/9 of culture.

Each EB developed in culture for 4-8 days was diluted with 0.5 mM in 1 × PBS.
The cells were treated with EGTA for 5 minutes and then trypsinized to a single cell suspension. Each single cell suspension was added to DMEM + LIF or DMEM + 50% MEDI
Equally divided between two 2 ml tissue culture wells containing either I + LIF.
After 5 days, cultures were stained for alkaline phosphatase activity to identify pluripotent cell colonies. The presence of MEDII in the medium resulted in the isolation of a large number of pluripotent cell colonies (FIG. 10B). This indicates that this factor allowed efficient isolation and maintenance of pluripotent EPL cells from pluripotent cells in EB. These EPL cells are at these stages by expression of Fgf5 and Rex1 (FIG. 10A) and by failure to support pluripotent cell growth and maintenance in medium supplemented with LIF alone (FIG. 10B). Produced from primitive ectoderm indicated as being the only pluripotent cell population in the EB. LIF has been shown to be sufficient for maintaining pluripotent ICM cells or pluripotent ES cells. Pluripotent cells
It can be isolated from EB-derived primitive ectoderm in either the presence or absence of LIF, but was dependent on the presence of MEDII.

These data indicate that MEDII can assist in the maintenance and growth of pluripotent cells from EB primitive ectoderm. Pluripotent cells isolated from primitive ectoderm are equivalent in morphology and gene expression to EPL cells and can revert to ES cells when cultured in the presence of LIF and in the absence of MEDII (Data not shown; Example 2). Thus, pluripotent cells isolated from EB primitive ectoderm and converted back to ES cells have been shown to contribute to chimeric mice when introduced into host blastocysts.

Isolation and maintenance of pluripotent cells from primitive ectoderm of mouse embryo The purpose of this study was to isolate and maintain pluripotent cells from primitive ectoderm of mouse non-129 strain. Was. Successful isolation and maintenance of pluripotent cells from this lineage that do not normally produce ES cells at a high frequency provides a general technique that can be extended to other mammals.

(Primal ectoderm from 5.5 dpc embryos is a preferred material for pluripotent cell isolation) 5.5, 6.5, and 7.5 (dpc The whole embryos dissected from female mice mated in estrus in) were dissected free of Reichart's membrane and individually in 1 ml embryo culture medium + 50% MEDII.
Placed in 2 ml tissue culture wells (pre-treated with 0.1% gelatin for 30 minutes). After three days of culture at 37 ° C., 10% CO ₂ , embryonic explants were stained for alkaline phosphatase to assess the presence and amount of alkaline phosphatase positive cells. The number of embryonic explants containing alkaline phosphatase positive cells was 5.5 d. p. c. Maximum in day explants (varied between 25% and 40%) and 7.5 d. p. c. It decreased significantly with increasing embryonic age to less than 5% in day embryo explants. 5.5 d. p. c. Although the amount of alkaline phosphatase positive cells in embryo explants fluctuated, most explants
It contained more than 20% of alkaline phosphatase positive cells. 5.5d. p.
c. Embryos were used as a source of primitive ectoderm in subsequent experiments.

Successful isolation and maintenance of pluripotent cells from primitive ectoderm was confirmed at 5.25 d. p.
c. And 5.5d. p. c. This was achieved from embryos collected between. Embryo collection after this opportunity can compromise this procedure, even by 1-2 hours. Explants from these embryos differentiate rapidly and the primitive ectoderm cell layer cannot be morphologically identified. 5.25d. p. c. No attempt was made to isolate cells from earlier embryos.

(Purified Extracellular Matrix (ECM) Components Stabilize EPL Cells in Culture) The effects of alternative extracellular matrix activity on the stability of EPL / primordial ectoderm cells in culture were determined by (2.5 × 10 ² cells / cm ² ) in DMEM + LIF or DMEM + 50% MEDII, gelatin (Example 1), plasma fibronectin, laminin, collagen type IV (all obtained from Sigma and manufacturer's instructions And plasma fibronectin, laminin,
E on tissue culture plastic pretreated with a combination of and type IV collagen
Tested by seeding S cells. Selected and purified ECM components have been shown to be present in the extracellular matrix of early embryos. Cells were cultured for 5 days and stained for alkaline phosphatase. Colonies were classified for the presence or absence of differentiated (alkaline phosphatase negative) cell types in the colonies, and the presence of differentiated cells was used as a measure of pluripotent cell stability (Table 3). In the conditions tested, gelatin was least preferred for EPL cell stability, with 78% of EPL cell colonies containing at least some differentiated cells. All of the purified matrix components, compared to gelatin,
The more stable EPL cell culture resulted, and the type IV collagen and matrix mixture containing type IV collagen resulted in 59% and 62.5% undifferentiated EPL cell colonies, respectively. ES cells cultured in the absence of MEDII were stable with all tested matrices.

Tissue culture plastic pretreated with type IV collagen was used for the culture of pluripotent cells from primitive ectoderm in the next experiment.

(Biological activity in MEDII is required for the growth and maintenance of embryonic primitive ectoderm.
Yes) The role of MEDII in maintaining primitive ectoderm in culture was determined by dissecting 5.5 d. p
. c. Embryos are individually cultured in embryo culture medium or embryo culture medium + 50% MEDII
Place in 2 ml tissue culture wells pretreated with type V collagen, 37 ° C, 10% CO _Two Was evaluated by maintaining After 5 days of culture, explants were fixed with 4% PFA
And the presence of pluripotent Oct4 positive cells by in situ hybridization
(FIG. 11; Table 4). Cultured in the presence of LIF
The embryo quickly becomes disordered and becomes necrotic, apparently a primitive ectodermal cell layer
And after 5 days did not contain any Oct4 positive cells. Contrast
In addition, many of the embryos maintained in the presence of 50% MEDII + LIF
Strong, homogeneously stained, distinct, marker for Oct4 (FIG. 11B)
The embryonic tissue, including the identifiable primitive ectoderm cell layer, was retained. 50% MED
54% of surviving explants cultured in the presence of II + LIF were Oct4 positive
Active cells. This indicates that MEDII is derived from primitive ectoderm in culture.
It promotes the maintenance of pluripotent cells.

A comparative analysis of a number of experiments indicates that fresh MEDII, in contrast to frozen MEDII, is more effective for maintaining in vitro pluripotent cells from embryonic primitive ectoderm. Indicated.

Other Parameters Related to Successful Isolation and Maintenance of Primitive Ectoderm from Embryos In Vitro Visceral endoderm is an inducible signal involved in the differentiation of pluripotent cells during gastrulation Is recognized as a source of Removal of visceral endoderm from embryonic explants was performed to remove the source of this differentiation-inducing signal and to promote pluripotent cell stability in culture. Although the effect of this step was not quantified, successful isolation and maintenance of pluripotent cells from embryos was not achieved from explants containing primitive and extraembryonic endoderm.

In addition, primordial ectoderm explants that failed to attach to the tissue culture substratum during the first stages of in vitro culture should contain growing primordial ectoderm with fewer mixed differentiated cells It has been shown. These explants were a preferred source of pluripotent cells for further culture. Suspension culture of primitive ectoderm explants was performed using DMEM medium (
0.5 ml of 0.5% agarose in low melting point agarose, Sigma), 2 m
1 achieved by addition to tissue culture wells. This is 3 × 30 in embryo culture medium.
Min, then pre-equilibrated against embryo culture medium containing 50% MEDII.

(Isolation and maintenance of pluripotent cells from the primitive ectoderm of the 5.5 dpc embryo (FIG. 12)) On the morning of the sixth day from pregnancy (approximately 5.4 dpc) On), embryos were dissected from the uterus from female mice mated in estrus between 10 and 11 am. Each embryo is dissected to remove the Reichart membrane, extraplacental cone and visceral endoderm layer, resulting in a cup-like structure containing primitive ectoderm without the presence of mixed cell types. (FIG. 12B). All tissues were mechanically removed from the embryo. Primitive ectoderm explants were individually placed in 2 ml wells packed with agarose and 1
Cultured in ml of embryo culture medium + 50% MEDII. Embryos are grown at 37 ° C. in 10%
The cells were cultured in a CO ₂ humidified incubator.

On day 2 or 3 of culture, surviving explants are removed from suspension, re-dissected to remove any differentiation that occurs in culture, and prepared as described. Done 2
The ml well was relocated.

On day 5 of culture, surviving explants (seeded on day 0) that usually contain an identifiable epithelial sheet of Oct4 positive cells that are in many respects equivalent to EPL cells (FIG. 12B) (Approximately 25% of the explants) were removed from the suspension, re-dissected, and plated in tissue culture plastic pretreated with collagen type IV in embryo culture medium + 50% MEDII. Explants were further cultured for 2 days in embryo culture medium + 50% MED
Maintained in II. Explants at this stage consisted of a normally intricate epithelial sheet (FIG. 12B). Explants were then cultured in embryo culture medium in the presence of LIF and in the absence of MEDII. This causes the sheet to flatten and
It had an S cell-like appearance (FIG. 12B). Analysis of these cells by in situ hybridization showed that these cells were pluripotent as assessed by Oct4 (FIG. 12B) and alkaline phosphatase expression.

On day 9 or 10 of culture, explants were broken up into single cells and small clumps of cells by trypsinization and pretreated with type IV collagen or inactivated feeder cells. Seeded in 2 ml wells previously seeded. Cells obtained by this method are apparently equivalent to ES cells (FIG. 12B), express the pluripotent cell markers alkaline phosphatase and Oct4 (FIG. 13), and retain the ability to differentiate spontaneously; It grew during culture for at least 4 weeks. Pluripotent cells were isolated from approximately 10% of the embryos. Its efficiency can be compared to isolation of ES cells from ICM of 129 mouse blastocyst stage embryos by standard techniques.

Summary The studies described in this example demonstrate that pluripotent cells from embryonic primordial ectoderm can be isolated in culture as pluripotent cells similar to EPL and ES cells for long periods of time. Provides first demonstration that it can be separated and maintained. Culture and passage of these cells was dependent on the biological activity contained in MEDII. Pluripotent cells obtained in this manner appear to be amenable to genetic manipulation and represent a source with potential commercial, medical, and agricultural applications. Demonstration that pluripotent EPL cells and ES cells can be isolated from the primitive ectoderm of mouse species in which ES and EG cells are difficult to isolate by other methodologies has been demonstrated from other mammalian and avian species. Potentially offers an opportunity for the isolation of pluripotent cell lines.

Demonstration that cells equivalent to EPL cells can be isolated and maintained from embryonic primitive ectoderm cultured in the presence of the biological activity contained in MEDII,
Provides further support for the proposed similarity between EPL cells and embryonic primitive ectoderm.

Example 4 Purification of Components of Biologically Active Factor from Serum-Free Conditioned Medium Materials and Methods All cell and tissue culture techniques were used in the Examples unless otherwise noted. 1 as described. Purification of EPL cell-inducing factors was performed using standard FPLC and H
PLC chromatography techniques were utilized. It assesses activity by a morphology-based assay for the conversion of ES cells to EPL cells. Amino acids, proline analogs, and peptides were purchased from Sigma.

[0197] Serum-free MEDII (sfMEDII) was used as a source of biologically active factors in all purification protocols. SfMEDII was shown to cause cell migration from ES to EPL in a manner similar to MEDII (Example 1, data not shown). To produce sfMEDII, Hep G2 cells were seeded at a density of 5 × 10 ⁴ cells / cm ² and cultured for 3 days. Cells are 1x
Twice with PBS and serum-free medium (1 mM L-glutamine, 0.1 mM β-
ME, 1 × ITSS supplement (Boehringer Mannheim), 10
DMEM with high glucose but no phenol red supplemented with mM HEPES, pH 7.4 and 110 mg / L sodium pyruvate
) Once for 2 hours. Fresh serum-free medium was added at a ratio of 0.23 ml / cm ² and cells were cultured for an additional 3-4 days. SfMEDII was collected, sterilized, and stored as for MEDII (Example 1). sfMEDII
Large-scale production of corrugated roller bottles
er bottle) (Falcon).

Cellular Assay for EPL Cell Inducing Activity A morphology-based assay for testing EPL cell inducing activity of purified fractions was performed using D3 ES cells. The assay was performed in 2 ml wells (Falcon)
Medium, the total volume was 1 ml / well. ES cells (2.5 × 10 ² cells / cm ² )
The cells were cultured in the presence of the semi-purified fraction and scored microscopically for EPL cell morphology 4 days after culture and macroscopically scored for alkaline phosphatase activity 5 days after staining.

The extracellular matrix preparation was formed as follows. Hep G2 cells were grown to confluency under the conditions described in Example 1. Cells were grown at 0 in PBS.
. 6% at 37 ° C. or room temperature in 5% sodium azide / 0.1 mM PMSF.
Incubate for 18 hours to kill and detach cells, or 0.5 m
The cells were cultured in EGTA at room temperature for 15 minutes to detach the cells. Cells and debris were removed by washing with PBS. Formation of a matrix from semi-purified or purified protein was achieved by drying 1-2 μg of protein on 2 ml tissue culture wells. 1000 cells / ml of ES cells
2.5% in DMEM supplemented with LIF and 10 μg / ml L-proline
The cells were seeded on a matrix at a concentration of × 10 ² cells / cm ² . On day 4, cell morphology was assayed microscopically, followed on day 5 by staining for alkaline phosphatase activity.

[0200] The presence of proline in the semi-purified sample was determined by 0.2 m in 80% propanol.
The samples were detected directly by thin layer chromatography on mSilica gel 60 F ₂₅₄ plates (Merck), followed by staining with ninhydrin.

(Protease digestion) 250 mg of type IV collagen (Sigma) was added to a final volume of 10 ml of 50 m
M Tris. Digestion was performed using 3 FALGPA units of type IV collagenase (Sigma) in 100 mM CaCl ₂ buffer with Cl pH 7.4, overnight incubation at 37 ° C. with continuous mixing. Amicon Diaflow using a 400 ml ultrafiltration cell (Amicon) at 4 ° C. under nitrogen pressure.
Digested collagen fragments were separated from enzymes and undigested proteins by ultrafiltration through a YM3 membrane. The eluate obtained from the ultrafiltration was used for 0.1 mL.
Passed through a 22 μm filter and a 1.5 ml aliquot was lyophilized and resuspended in 200 μl water. 4 × 50 μl of the resuspended filtrate was equilibrated in water and applied to a Superdex peptide gel filtration column connected to a SMART micropurification system (Pharmacia). 25 μl fractions were collected and assayed directly for EPL cell inducing activity in the presence of R.

For trypsin digestion of the high molecular weight component of the EPL cell inducing activity, 80 μl of R
(3.6 mg / ml) with 20 mM Tris. Cl pH 8.5, 20 mM
Using 100 units of trypsin (Difco) in CaCl ₂ at 25 ° C.
Digested for hours. The reaction was stopped by adding 100 μM PMSF. The sample was concentrated on a Centricon-10 column (Amicon) and assayed for high molecular weight components of EPL cell inducing activity.

Protein Analysis by Reduced PAGE and Western Blot Protein samples were obtained directly from conditioned media or chromatographic fractions were desalted on Centricon-10 columns (Amicon) to give
mM Tris. The original volume was reached using Cl pH 8.5. Samples were electrophoresed on a 10% reducing SDS polyacrylamide gel as described by Laemmli, 1970, and proteins were analyzed by Heukesho.
Visualization was by silver staining using the method of ven and Dernick (1985).

For Western blotting, gel was transferred to Western Transfer Buffer (39 mM).
 Glycine, 48 mM Tris. Cl, 20% methanol, 0.037%
Wash for 30 minutes in SDS) and Mini-Protean II instrument (B
ioRad) on this buffer in nitrocellulose at 400 mAmps,
Electroblot was performed at a constant voltage for 3 hours. The filter was washed overnight with PBT (0.1
% Triton X-100, 1 × PBS), 5% skim milk powder
Locked. Monoclonal antibody 3E2 (Sigma) (cellular human fibrone
(Specific for the EDA region of Kuching) in 1% skim milk powder, PBT
Dilute to 1/1000 and incubate for 2 hours at room temperature, then filter
For 2 hours at room temperature. Filter in PBT 3
1x skim milk, 1/10 in PBT for 2 hours at room temperature
In goat anti-mouse alkaline phosphatase antibody (Dako) diluted to
Incubated. Western buffer 1 (100 mM Tris.Cl pH
7.4, 100 mM NaCl) for 20 minutes, followed by Western buffer
2 (Tris.Cl pH 9.5, 100 mM NaCl, 5 mM MgCl _Two ) Was performed for 2 × 20 minutes. The reaction is performed with a Western substrate mixture (10 m
l Western buffer 2, 40 μl NBT (75 mg / ml in 70% DMF
) In 40 μl BCIP (50 mg / ml in 100% DMF) in the dark
Proceed and add 10 ml Western Buffer 1, 100 mM EDTA
Was stopped by

[0205] (Results) (soluble biological agent in MEDII is responsible for conversion to EPL cells from ES) SfMEDII is, 10 × 10 ³ M _r cutoff membrane; limited through a (Centricon-3 units Amicon) Separation into two fractions by external filtration. Both fractions were assayed for EPL-forming activity, defined as the ability to cause complete conversion of D3 ES cells to alkaline phosphatase-positive EPL cells in the presence of mLIF (FIG. 14). Fraction retained at a concentration equivalent to 50% MEDII (
ES cells seeded at> 3 × 10 ³ M _r ) did not form EPL cells, but
The size of the S cell colony increased. ES cells (<3 × 10 ³ M _r ) seeded into the eluted fraction at a concentration equivalent to 50% MEDII resulted in an array of colony morphologies containing ES, EPL, and differentiated cells. The presence of ES cells and the higher ratio of differentiated cells was not characteristic of the conversion of ES to EPL cells, demonstrating that eluted material alone could not induce EPL cell formation. ES cells,
50% MEDII in medium containing both retained and eluted fractions
Seeding at a concentration equivalent to that produced uniform EPL cells equivalent to those observed for sfMEDII. These data indicated that two separable biological agents were required for the conversion of ES to EPL cells.

Large Scale Preparation of R and E Fractions from sfMEDII Starting material for purification and analysis of bioactive factors from MEDII was 400
Amicon using 4 ml of ultrafiltered cells (Amicon) under nitrogen pressure
Obtained by ultrafiltration of sfMEDII through Diaflo YM3 membrane. Retained fraction (R) (> 3 × 10 ³ M _r ) was used immediately or aliquoted and stored at −20 ° C. The eluted fraction (E) (<3 × 10 ³ M _r ) was used immediately or stored at 4 ° C.

(Cell assay for detection of low molecular weight component of EPL cell inducing activity) PD10 column (Pharmacia) was used to remove low molecular weight components of EPL cell inducing activity in 1 ml of culture medium to remove low molecular weight contaminants. 20μ of R passed through
1 (50-100 μg of protein), but assayed as described above.

Physicochemical Properties of the Low Molecular Weight Component The physicochemical properties of the low molecular weight component of EPL cell inducing activity were estimated from the treatments and assays of E (Table 5). Activity was maintained following repeated acid treatments at pH 2.0, freeze-thaw cycles, boiling for 1 hour, and reduction with 50 mM DTT at room temperature. The active ingredient was soluble in water, acetonitrile, methanol, and propanol, and was amenable to HPLC purification, but did not bind to ion exchange or reverse phase HPLC columns using standard techniques.

(Purification of Low-Molecular-Weight Component of EPL Cell Inducing Activity) 220 ml of E was added to a Sephadex G10 column (110
0 ml bed volume, 110 x 113 mm). Elution was performed at room temperature at a flow rate of 35.
Performed with water at ml / min. 45 ml fractions were collected and a 1 ml aliquot of each fraction was lyophilized. Lyophilized fractions were resuspended in 100 μl of water and 25 μl were assayed for EPL cell inducing activity. Activity in fractions 6-10
In, the detection was performed 19 to 25.2 minutes after the injection (FIG. 15A).

Fractions 7-9 were pooled, lyophilized and resuspended in 1 ml of 30:70 methanol: acetonitrile. The sample was centrifuged at 14,000 rpm for 10 minutes to remove the precipitate, and a 10 ml Waters radial packed normal phase silica column (8 mm ID) coupled to a Waters 510 HPLC instrument.
. ). The column was washed with 30:70 methanol: acetonitrile at a flow rate of 0.2 ml / min for 15 min, after which the material was eluted with a linear gradient of 20 min against water using a flow rate of 0.5 ml / min. The eluted material was detected with a Waters 490E programmable multi-wavelength detector set at 215 nm. 1 ml
Fractions were collected, lyophilized, resuspended in 50 μl of DMEM and assayed for EPL cell-inducing activity eluted from the column with 70% water / 30% (30:70 methanol: acetonitrile) (FIG. 15B). ).

The fraction with the highest activity from normal phase chromatography between 32 and 35 minutes was lyophilized, resuspended in 50 μl of water and 10 μl of SMART
Superdex peptide gel filtration columns (Pharmacia) coupled to a micropurification system (Pharmacia) and equilibrated in water at room temperature. The column was eluted with water at a flow rate of 25 μl / min (FIG. 15C) and 25 μl
Of samples were collected. This was repeated five times to obtain a sample suitable for analysis. Individual samples were run in a single peak or several closely eluting peaks (ie, fractions 8, 9, and 10) from about 71.04 to 74.0 after injection.
Fractions eluting at 4 minutes were assayed directly for biological activity detected. The estimated molecular weight of the active fraction was <700D according to elution volume.

Characterization of Purified Low Molecular Weight Components Fraction 9 from the Superdex peptide gel filtration column was lyophilized and subjected to FM
Derivatized with OC and OPA and amino acid analysis was performed using Hewlett-Pa
Performed with and without hydrolysis using a ckard Amicon-Quant II analyzer. The results were compared to a control sample of unconditioned medium subjected to the same purification (Table 6). The amino acids alanine and proline imino acid were abundant in both hydrolyzed and unhydrolyzed samples compared to controls. This indicates that these amino acids were present in the purified sample as free amino acids (and not as peptides).

L-proline (3 × 10 ⁻⁴ M) and L-alanine (3.9 × 10 ⁻⁴ M)
Assay for EPL cell inducing activity. L-proline, in the presence of R,
The conversion of ES cells to EPL cells was effected in a manner that distinguished the low molecular weight components of fMEDII. This was due to morphological changes, as well as to Fgf5 reported in Example 1.
And Rexl expression (data not shown). L-alanine had no effect on ES cells in the presence or absence of R.

The addition of 10 ⁻⁴ M L-proline to ES cells in the absence of R resulted in a heterogeneous population containing several cells / colony morphologically similar to EPL cells. R
Was required for complete and uniform EPL cell formation.

The minimum active molar concentration of proline was found to be about 40 μM. The optimal concentration in the biological assay was found to be 100 μM.

Effect of Proline Analogs and Proline-Containing Peptides on ES and EPL Cells Proline analogs and proline-containing peptides reported to have proline-like biological activity in other cell lines were tested over a range of concentrations. , R
Was evaluated for its ability to form EPL cells from ES cells in the presence or absence. Table 7 shows the results.

The proline analogs D-proline, trans-4-hydroxy-L-proline, pyrrolidine, N-acetyl-L-proline, Nt-BOC-proline,
And L-pipecolic acid (PCA) had no observable effect on ES cell growth or differentiation in the presence or absence of R. Sarcosine, L
-Azetidine-2-carboxylic acid (AZET) and 3,4 dehydro-L-proline inhibit ES cell growth at low concentrations and at higher concentrations in the presence or absence of R, EPL cells Caused ES cell death without inducing formation. These data point to structural requirements for proline activity in EPL cell formation that differ from those described for other proline biological activities.

The proline-containing peptides ala-pro, gly-pro, pro-OH
-Pro, ala-pro-gly, gly-pro-ala, gly-pro
-Arg-pro, val-ala-pro-gly, gly-pro-gly
-Gly, substance P free acid (arg-pro-lys-pro-gln-gln-
phe-phe-gly-leu-met-OH), substance P fragments 1-4
(Arg-pro-lys-pro) and Bradykinin (arg-p
ro-pro-gly-phe-ser-pro-phe-arg; data not shown), in combination with R, was able to induce the conversion of ES to EPL cells at similar molar concentrations as L-proline. (Table 7). Peptide pro in the presence of R
-Ala, pro-gly, and gly-pro-OH-pro are also ES
Could be converted to EPL cells, but at a molar concentration approximately 6 times higher than that of proline (Table 7). The lack of correlation between the specific activity and the molar amount of proline in these peptides provides further evidence for structural requirements for the action of this factor and points out the possibility of interacting with the receptor.

EPL cells were also formed when ES cells were cultured in the presence of R and partially purified collagen IV hydrolyzate resulting from collagenase digestion of collagen IV. Free L-proline could not be detected by TLC in the partially purified collagen IV hydrolysate. The biological activity of collagen IV hydrolyzate is probably due to the repeated G in collagen IV protein.
Reflects the presence of the ly-Pro-X motif and provides a possible source of this biological activity in vivo by degradation of ECM components.

Carboxy terminus of substance P (fragments 5-11; gln-gln-phe-
phe-gly-leu-met-OH; data not shown), integrin-binding peptide RGD, neurokinin A and B receptor antagonists (neurokinin A and senktide), and the amino acids L-alanine and L-lysine are represented by R Has no observable effect on ES cells in the presence or absence of and has been used at a concentration equal to or greater than the bioactive peptide described above.

At concentrations greater than 50 μM, tachykinin substance P induced apoptosis of ES cells. Compared with substance P free acid and substance P fragments 1-4, substance P
Has a significantly increased affinity for the NK-1 receptor.

These results indicate that L-proline and the proline-containing peptide were sfMEDI
It has been shown to possess the biological activity associated with low molecular weight components of I. The identification of multiple factors with similar but different specific activities has been directed to structural requirements for biological activity, suggesting the involvement of receptor interactions and having a low molecular weight component of EPL cell-inducing activity Allows further peptide predictions.

(Purification of high molecular weight component of EPL cell-inducing activity) (Cell assay for detection of high molecular weight component of EPL-cell-inducing activity) (
Assays described previously, except supplemented with 10% FCS) or supplemented with 40 μM L-proline.

Unless otherwise indicated, fractions were prepared for inclusion in the assay as follows. The protein concentration of the semi-purified fraction of R from the chromatographic purification step was estimated using the BioRad protein assay kit (BioRad # 500-0001). Aliquots of each fraction containing 100 μg of protein were
concentrated with centricon-10 units (Amicon), 2 ml of PBS
And concentrated again. 0.1 μg to 10 μg of protein
Were assayed for the high molecular weight component of EPL cell inducing activity at concentrations ranging from.

(Physicochemical Characteristics of High Molecular Weight Component) The physicochemical characteristics of the low molecular weight component having the activity of inducing EPL cells were measured using Amicon Di.
Extrapolated from treatment and assay of R prepared as above, except that aflo YM10 membrane was used for ultrafiltration. R is converted to a PD10 column (Ph
armacia) to remove low molecular weight contaminants and to remove
g / ml was used in the assay. Treatment of R by incubation at> 56 ° C. for 1 hour or digestion with the protease trypsin resulted in loss of biological activity. The high molecular weight component of the EPL cell inducing activity was also unstable below pH 5.5 or after treatment with a reducing agent such as 100 mM DTT for 4 hours at room temperature. These data indicated that the high molecular weight component of the EPL cell inducing activity was proteinaceous.

(Biological activity of high molecular weight components can be detected in the extracellular matrix (ECM) of cultured Hep G2 cells.) ES cells were placed in 2 ml tissue culture wells at a low cell density of 2.5 x 10 ² Cell / c
seeded on Hep G2 ECM m ^2, and the and in DMEM + LIF L-
Cultured for 5 days with or without proline. Cultures were stained for alkaline phosphatase activity after 5 days and the cultures were assayed by morphology for EPL cell formation. Matrix from Hep G2 cells could act as a source of high molecular weight components of EPL cell inducing activity and, when used in combination with L-proline, effectively induced the formation of EPL cells (Table 8). . When used alone, the matrix failed to induce complete conversion of ES to EPL cells, but many colonies adapted to flat or spread morphology.

(Purification of high molecular weight components from sfMEDII: Protocol 1) R prepared as described above from 4 l of
. Made in Cl pH 8.5 and passed through a 300 ml Sepharose Q anion exchange column (Pharmacia). The bound protein is
00 ml of 20 mM Tris. Cl pH 8.5, 200 mM NaCl, 2
00 ml of 20 mM Tris. Cl pH 8.5, 400 mM NaCl, and 200 ml of 20 mM Tris. Elution was via a step gradient of Cl pH 8.5, 1 M NaCl (FIG. 16A). 200 ml samples were collected and assayed for their ability to form EPL cells in the presence of 40 μM L-proline. Activity eluted from the column at 400 mM NaCl.

The active fraction from anion exchange chromatography was analyzed using 20 mM Tris. C
1 It was made to have a final concentration of 0.5 M ammonium sulfate in pH 8.5. The sample was filtered through a 0.22 μm filter to remove precipitated material before passing through a 40 ml phenyl Sepharose rapid hydrophobic interaction column (Pharmacia). 50 ml of bound protein in one step
0 mM Tris. The column eluted at Cl pH 8.5 (FIG. 16B) and was assayed to contain a high molecular weight component of EPL cell inducing activity.

The active fraction from hydrophobic interaction chromatography was analyzed using 20 mM Tris.
Diluted in Cl pH 8.5 so that the conductivity was less than 100 mM NaCl. The sample was applied to a 10 ml heparin Sepharose CL-6B column (P
harmacia). The bound protein was reduced to 10 ml of 0.
5M NaCl, 20mM Tris. Elute from the column with Cl pH 8.5 (
FIG. 16C) and the assay showed that it contained a high molecular weight component of EPL cell inducing activity.

The active fraction from heparin Sepharose affinity chromatography was applied to a Superose 6 gel filtration column (Pharmacia) using SMAR.
T system (Pharmacia) with 20 mM Tris. Cl pH
8.5 Passed in 150 mM NaCl (FIG. 16D). 50 μl fractions were collected and assayed directly. A fraction containing biological activity equivalent to the high molecular weight component of sfMEDII eluted between 500-1000 kDa as a single peak.
EPL cell formation was observed only in the presence of E or L-proline.

Reduced SDS PAGE revealed the presence of a highly purified protein of approximately 210-260 kDa in samples containing the biologically active high molecular weight component (FIG. 16E).

(Purification of high molecular weight components from sfMEDII: Protocol 2) One liter of sfMEDII was added to 20 mM Tris. Cl, pH 8.5, equilibrated in a 50 ml heparin Sepharose CL-6B column (P
harmacia). The column was loaded with 200 ml of 20 mM Tris.
Cl, pH 8.5, 200 ml of 20 mM Tris. Cl, pH 8.5 + 15
0 mM NaCl, 200 ml of 20 mM Tris. Cl, pH 8.5 + 50
0 mM NaCl, and 200 ml of 20 mM Tris. Cl, pH 8.5
Washed sequentially with +1 M NaCl (FIG. 17A). An aliquot of each wash was assayed for biological activity on ES cells. The high molecular weight component of the EPL cell inducing activity eluted from the column in the 500 mM NaCl fraction.

The active fraction from heparin Sepharose affinity chromatography was converted to 150 mM NaCl with 20 mM Tris. Cl, pH 8.5, and diluted with 20 mM Tris. 1 ml Resource Q anion exchange column (Pharmac) pre-equilibrated with Cl, pH 8.5 + 150 mM NaCl.
ia). The protein was reduced from 150 mM to 500 mM over 60 minutes.
The column was eluted with a gradient of NaCl followed by a wash at 1 ml / min in 1M NaCl for 5 minutes (FIG. 17B). Alternate 1.5 ml fractions, 200-300 m
The high molecular weight component of EPL cell inducing activity eluted from the column with M NaCl was assayed.

Fractions 10-17 from anion exchange chromatography were pooled and
0 mM Tris. Cl, pH 8.5 + 150 mM NaCl in Super
It was passed through anose6 gel filtration column (Pharmacia) (FIG. 17C). 50μ
One fraction was collected and assayed directly for the presence of the high molecular weight component of EPL cell-inducing activity, which eluted as a single peak between 500-1000 kDa.

The active fraction was analyzed by reduced SDS PAGE and was shown to contain a highly purified protein of approximately 210-260 kDa equivalent to the purified protein in Protocol 1 (FIG. 17D).

(Purification of polymer component from sfMEDII: Protocol 3) 4 liters of sfMEDII was purified by equilibrating 25 liters of gelatin Sepharose affinity chromatography column (Pharma) pre-equilibrated in PBS.
cia) at a flow rate of about 5 ml / min. The column was re-equilibrated in PBS and the protein was eluted with a 50 ml volume of 6M urea in PBS (FIG. 18).
A). The eluate was dialyzed four times at 4 ° C. against 4 liters of PBS to remove urea. The protein concentration of the eluate was determined using a BioRad protein assay kit, and 0.1-10 μg was assayed for biological activity.

The eluate was analyzed by reducing SDS PAGE and approximately highly purified about 210-210 corresponding to the protein purified in protocols 1 and 2.
It was shown to contain a 260 kDa protein (FIG. 18B). Purified protein yield was determined by Bradford analysis to be approximately 1 mg / liter sfME
It was determined to be DII.

Characterization and Identification of High Molecular Weight Activity The highly purified active fraction obtained from purification protocols 1-3 was 2 μl in solution.
EPL cell formation in the presence of E-proline or L-proline, either when used at g / ml (FIG. 19C) or when pre-coated on tissue culture plastic (data not shown). Could be induced. This was consistent with previous identification of this protein as a component of ECM. The activity of inducing EPL cells is E
-Could not be detected in the absence of proline or L-proline.

Extracellular matrix proteins human laminin (Sigma), bovine vitronectin (Dr. Z. Upton, CRC for Tissue Growth)
and Repair, Adelaide) Collagen IV (Sigma),
Human plasma fibronectin (Sigma) and human cellular fibronectin (
JP Levesque, IMVS, Adelaide) were tested for the ability to promote the formation of EPL cells from ES cells in tissue culture medium in the presence of 40 μM proline and compared to the active fraction (Table 8). Of these,
Only cellular fibronectin at a concentration of 1 μg / ml or higher could result in the formation of EPL cells from ES cells when added in solution (FIGS. 19A, B). Cellular fibronectin is a homodimer of a 250 kDa disulfide-linked protein, the activity of which is sfMEDII identified by reduced SDS PAGE.
Consistent with the size and characteristics of the highly purified bioactive factor.

Using antibodies specific for cellular fibronectin, Western blot analysis of active fractions from Superose 6 gel filtration (protocol 2) yielded 240k
The purified protein of Da was identified and confirmed as cellular fibronectin (
(FIG. 20).

(Multiple ECM proteins can be used to provide high molecular weight components that are active in inducing EPL cells when presented to cells as a matrix.) E when pre-coated on top
The ability to promote PL cell formation was tested. Morphological induction of EPL cell morphology was restricted to cellular and plasma fibronectin and laminin, a basement membrane component, in the presence of L-proline (Table 8).

[0242] Fibronectin and laminin bind to cells via cell surface integrin receptors, and the specificity of this ligand binding is determined by the combination of various heterodimers between alpha and beta integrins. These data are
It suggests that a range of ECM components that activate a reasonable integrin receptor can induce EPL cell formation when presented to ES cells as extracellular matrix. However, it has been shown that only cellular fibronectin induces the formation of EPL cells from ES cells when presented in a soluble form.

Summary The size fraction of sfMEDII represented the requirements for at least two biologically active factors (high and low molecular weight components) in EPL cell formation. The low molecular weight component of sfMEDII was identified as active L-proline at concentrations above 40 μM. Analysis of proline analogs and small peptides demonstrated that many small peptides containing proline could be substituted for L-proline in the formation of EPL cells.

Chromatographic purification by various protocols identified the high molecular weight component of sfMEDII as cellular fibronectin. In the presence of L-proline, the high molecular weight component, cellular fibronectin, could result in the formation of EPL cells, both in solution and as a matrix. The ability of some extracellular matrix components to induce the formation of EPL cells when associated with the matrix but not in a soluble form and presented to ES cells in combination with L-proline, depends on the cellular fibronectin biology. Activity was mediated by integrins at the cell surface, most probably suggesting a heterodimer of α1: β5.

In summary, the data presented in this example identifies factors that, when presented in a soluble or matrix-related form, can induce the formation of EPL cells from ES cells, and provide similar receptors / molecules. Provides sufficient information for predicting factors with similar biological activities that act via interactions.

Example 5 Biologically active components of EPL cell-inducing activity can be isolated from different sources and species Materials and Methods (primary cell line) Preparation Primary liver cells were prepared using a procedure based on a slightly modified procedure described by Giger and Myer (1981). Slaughter the mouse,
And immediately dissected to expose the liver. Each liver was passed through the portal vein with 10 ml of PBS followed by Hank's buffered saline (HBSS; 0.8 g in 2 liters).
KCl, 0.12 g KH ₂ PO ₄ , 16 g NaCl, 0.1 g Na ₂ HPO ₄ , 2 g glucose, 4 ml 1% phenol red, 0.035 g NaHC
Perfused with 10 ml 0.05% collagenase in O ₃ ). The perfused liver was removed, macerated with 10 ml / liver 0.05% collagenase / HBSS, and
Incubated for 30 minutes at 7 ° C. The liver suspension was regularly agitated by pipetting to assist in liver separation. Cells are washed with HBSS (10 ml
) To remove collagenase and remove contaminating erythrocytes from Sassa
Solution (6.95 g NH ₄ Cl, 2.058 g Tris-
Base, 1 g KHCO ₃ ). Williams E medium (1 ml
The cells were seeded in gelatin-treated tissue culture flasks in DMEM after 2 washes in 1 g / l, Gibco BRL) and incubated at 37 ° C., 5% CO ₂ .

Primary avian hepatocytes were prepared from 17-18 day old chicken embryos. Briefly, chicken embryos were removed from eggs, decapitated, and dissected to expose the heart and liver. The liver was perfused via heart cannulation with 10 ml of 0.9% NaCl containing 2 mM EDTA to remove blood cells, followed by H2
Perfused with 4 ml of 0.05% collagenase in BSS. When all livers were perfused and harvested, livers were removed, pooled in HBSS and transferred into fresh 0.05% collagenase / HBSS. Pooled livers are macerated with fine tipped scissors and incubated for 30 minutes with gentle agitation every 5 minutes, and 10 ml every 10 minutes
Gently pipetted with a pipette to aid in cell separation. Collagenase was removed by washing with HBSS (2 ml / liver). Contaminating red blood cells were lysed in Sassa solution. Williams E (2ml /
After two washes with (liver), cell debris and hemoglobin from lysed erythrocytes were removed, the hepatocytes were resuspended in Williams E medium (1 ml / liver) and the yield was measured. Typically 2 × 10 ⁷ cells / liver were obtained and plated out into 175 cm ² tissue culture flasks and cultured in 20 ml DMEM at 37 ° C./5% CO ₂ . .

(Preparation of Conditioned Medium) Primary mouse hepatocytes and primary chicken hepatocytes were subjected to DM prior to collection of conditioned medium.
Cultured in EM0 for 4 days.

The mouse hepatocellular carcinoma cell line Hepa-1c1c 7 (ATCC CRL 202
6) and Hep 3B (ATCC HB-8064), and P19 embryonic stage carcinoma-derived cell line END2 (visceral endoderm-like, Mummery et al., 1985) and P
YS2 (mural endoderm-like, Lehman et al., 1974) was maintained in DMEM.
Conditioned media was collected after culturing the cell lines in non-gelatinized tissue culture flasks at 37 ° C., 5% CO ₂ for 4 days.

The conditioned medium was processed as described for MEDII in Example 1. Conditioned media was assayed for biological activity as described in Examples 1 and 4.

(Visualization of Bioactive Ingredients) The presence of proline in the active fraction of the END-2 medium was detected by thin layer chromatography as described in Example 4. Cellular fibronectin was detected by reduced SDS PAGE as described in Example 4.

(Results) Conditioned medium from primary mammalian hepatocytes and primary avian hepatocytes, a liver-derived cell line (H
epa-1c1c 7 and Hep 3B) and embryonic endoderm-like cell lines (EN
D-2 and PYS-2) were assayed for their ability to form EPL cells from ES cells.

Conditioned media from both mouse and chicken primordial hepatocytes was cultured during culture with ES
This could result in the migration of cells into EPL cells (FIG. 21A). This was only true media taken from seeded primary hepatocytes. Passaging resulted in loss of hepatocytes overgrown by cells of fibroblast appearance and loss of biological activity inducing EPL cells. No differences in biological activity were detected in primary embryonic (chicken) and adult (mouse) hepatocytes and in conditioned media from MEDII.

Media conditioned by the cultured cell lines Hepa-1c1c7, Hep 3B, END-2 and PYS-2 did not induce the formation of EPL cells from ES cells during culture. However, some of these cell lines could be shown to express one component of biological activity. Conditioned media from Hepa-1c1c 7 cells, when used in combination with L-proline, resulted in the migration of ES to EPL cells (FIG. 21B). This suggests that these cells express a high molecular weight component of EPL cell inducing activity. This means that Hepa-1c1c
7 was confirmed by reduced SDS PAGE analysis of the conditioned media, indicating the presence of a protein with equivalent mobility to cellular fibronectin (FIG. 21).
C). Conditioned media from Hep 3B, alone or in combination with the biologically active component purified from MEDII, could not result in the transfer of ES to EPL cells. These cells, and some other liver-derived cell lines, did not express detectable levels of cellular fibronectin (FIG. 2).
1C). This indicated that the expression of the high molecular weight component of the EPL cell-inducing activity was not unevenly distributed among the liver-derived cell lines.

Conditioned medium from END-2 cells induced the formation of EPL cells from ES cells when used in combination with cellular fibronectin (FIG. 21B). This suggested that these cells secreted L-proline or functional analogs but did not secrete cellular fibronectin (FIG. 21C). Semipurification of this conditioned medium by normal phase chromatography (Example 4) and analysis by thin layer chromatography demonstrated the presence of L-proline in the END-2 conditioned medium (data not shown).

Overview The biological activities that are equivalent to the activity found in MEDII and that can induce the transformation of ES cells into EPL cells are from cells of different species (including mammals and birds). It has been identified in conditioned media and in tissues from both embryos and adults. This demonstrates that this activity is conserved across these species during evolution, and demonstrates the fundamental importance of this activity in embryonic development. Furthermore, the individual components of EPL cell-inducing activity are expressed by cells from different lineages.

Example 6 (Alternative Differentiation of ES and EPL Cells In Vitro) Materials and Methods Cell Culture and In Vitro Differentiation Assays All cell and tissue culture techniques are used unless otherwise stated. As described in Example 1. All EPL cells used for the differentiation assay were
It was formed from ES cells cultured for 2 days in the presence of EDII. The presence or absence of additional LIF was identified for each example.

KSF4 ES cell line, which constitutively expresses nuclear-localized LacZ (Berger et al., 1995; Patrick Tam, CMIR, New S
out Wales, obtained from Australia)), as described above, at 40 μg / ml gentamicin, 20% fetal calf serum (FCS), 0.1 mM
β-mercaptoethanol, 1 mM L-glutamine and 1000 units of L
Maintained on inactivated primordial mouse fetal fibroblast feeder layer (Abbondanzo et al., 1993) in DMEM (high glucose) supplemented with IF. EP
Prior to L cell formation, KSF4 cells were cultured on gelatinized tissue culture plastic for two passages in the absence of a feeder layer.

For most of the experiments detailed herein, embryoid bodies (EBs) were prepared using a partial trypsinization method (Robertson,
1987) without addition of MEDII or LIF (DMEM) in the medium. Alternatively, EBs were plated at 1 × 10 ⁵ cells / ml in bacteriological dishes in ES DMEM or at 1.6 × 10 ⁴ on the inner surface of a Petri dish lid in hanging drops of medium (50 ml). Formed from a single cell suspension. Cell aggregates formed in the suspended drop were transferred to a bacteriological dish for further culture after 48 hours. EBs were maintained with regular replenishment of the medium. The mixed cells EB are
As previously described, the two cell populations were formed from a single suspension mixed at a specific ratio.

Results consistent with those presented herein were obtained for various ES cell lines (D3 (D
oetschman et al., 1985), MBL5 (Pease et al., 1990), K
SF-4 (Berger et al., 1995) and E14 (Hooper et al., 198).
7)) was obtained using EPL cells from, as well as using alternative methods of EB formation, including single cell suspension, hanging drops and partial trypsinization. In addition, the potential differentiation reported for EPL cells would be associated with these cells if they could not be regenerated by ES cells that had been spontaneously differentiated by culturing in the absence of LIF for an equal amount of time. Specifically associated (data not shown).

Retinoic acid (RA) differentiation of pluripotent cell aggregates was performed by plating ES or EPL cells in bacteriological dishes at a density of 1 × 10 ⁵ cells / ml in DMEM supplemented with 1 μM RA. Was carried out. After 48 hours, aggregates were transferred to DMEM and maintained for 2 days, then individually seeded into 2 ml tissue culture wells. The presence of neurons was assessed by microscopy two days after seeding.
Identification of neurons was determined using the neurofilament 200 antibody N-4142 (Sigma).
Was confirmed by positive staining.

Cytokine / growth factor assay Assays for the effects of cytokines / growth factors were performed on ES cells and EP seeded at low density (75 cells / cm ² ) in 4-well multidishes (Nunc).
For each L cell, 0.8 ml of DMEM + LIF or 0.8 ml of D
Performed in MEM + 50% MEDII + LIF. Cytokines / growth factors were added at the indicated concentrations and the assay was stained for alkaline phosphatase activity 5 days after culture as described in Example 1.

Gene Expression Analysis RNA was purified from ES cells and EP using the method of Edwards et al. (1985).
Isolated from L cells. The RNA was purified from Chomczynski and Sacchi (
1987) from EB and RA treated aggregates.

[0264] Northern blot analysis and whole mount in situ hybridization were performed as described in Example 1. Riboprobes were synthesized as described by Kreig and Melton (1987).

The probe fragments used for Northern blot analysis and / or in situ hybridization were performed as detailed in Example 1, with the addition of: AFP (pBluescript K with HindIII)
Linearization of a plasmid containing the 400 bp EcoRI fragment encoding the first 350 bp of mouse AFP cDNA in SII + (obtained from Dr. R. Krumlauf, NIMR, London), followed by transcription using T3 polymerase; 57 from 13G43 (Mason et al., 1986)
Goosecoid (linearization of a plasmid containing 909 bp goosecoid genomic DNA with HindIII (Blum et al., 1992) and transcription using T3 polymerase); Nkx2.5 (
1.6 kb Nkx2.5 cDNA with HindIII (Lints et al.
993) and transcription using T3 polymerase).

Histological Analysis EBs were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4 ° C. and processed and sectioned as described in Example 1. Sections are 10
For 2 seconds, stained with toluidine blue, Histoclear (National
Diagnostics) and covered with a coverslip using xylene-based mounting media. E for in situ hybridization
B was immobilized overnight with 4% PFA, washed several times with PBS, 0.1% Tween-20, treated with 100% methanol for 5 minutes and then with isopropanol for 10 minutes.
Minutes. The mass was then embedded and sectioned as described in Example 1.

To detect β-galactosidase activity, EBs were fixed in 0.2% glutaraldehyde in PBS for 15 minutes on ice and detergent rinse (0.1 M
3 × 15 using a phosphate buffer (pH 7.3), 2 mM MgCl ₂ , 0.01% sodium deoxycholate, 0.02% Nonidet P-450)
Wash for 30 minutes and wash with a surfactant rinse containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg / ml X-gal.
Stained at 2 ° C. for 2 hours. The EB is then washed three times (3 ×) with a surfactant rinse.
Washed and transferred to 70% ethanol. For sectioning, EBs were dehydrated against 100% ethanol and processed as described in Example 1.

(Immunological Detection) AFP expression in sectioned EBs was determined by Dziadek with the following modifications:
And essentially as described by Adamson (1978). Prior to antibody incubation, endogenous peroxidase activity was blocked by incubating sections in 3% H ₂ O ₂ in methanol for 30 minutes. Sections were incubated with a 1/200 dilution of rabbit anti-AFP (ICN) for 2 hours, washed several times with PBS, and then HRP-conjugated anti-rabbit IgG (S
Ilenus).

Pectoral Muscle Formation from ES and EPL Cells Pectoral muscle formation was determined by pre-equilibrated agarose plugs in DMEM (37 ° C., 10 ° C. in 2 ml tissue culture wells against DMEM). Evaluated in individual EBs, formed from either ES or EPL cells, plated on DMEM medium-based 1% agarose, equilibrated for 3 hours at% CO ₂ . The presence or absence of the beating muscle was assessed by microscopic examination on days 4, 6, 8, 10 and 12. Alternatively, beating muscle formation was assessed in individual EBs seeded into 2 ml wells on day 6 of development and scored microscopically on days 7, 8, 10, 12, 14, and 16. Seeded aggregates were also scored on days 8, 10 and 12 for the presence of terminally differentiated neurons.

Results Embryoid bodies (formed by the aggregation of pluripotent cells in suspension) follow a differentiation pathway similar to early embryonic development (Example 1), and It offers an opportunity for extensive evaluation of the differentiation of pluripotent cells into both extraembryonic cell types. The term EB is used herein to describe pluripotent cell aggregates cultured in DMEM.

Preliminary Characterization of EPL and ES Embryoid Body Formation When EBs were formed from either ES or EPL cells, clear morphological differences were revealed. The EBs of ES cells are round, relatively smooth,
And contained a homogeneous population of regular aggregates (FIG. 22A). EBs formed from EPL cells also formed a homogeneous population, but their aggregates appeared irregular and unorganized (FIG. 22B). By sectioning and staining, the EBs of ES cells contain a relatively homogeneous population of cells, characterized by a compact round morphology (FIG. 22C), whereas the EPL is reminiscent of undifferentiated pluripotent cells. The internal cells in the EBs are shown to be loosely confluent and heterogeneous, probably reflecting differentiation (FIG. 22D). This observation prompted a detailed comparative study of the differentiation of ES and EPL cells.

Accelerated Differentiation of Pluripotent Cells in EPL Cell Embryoid Bodies The progression from ICM to primitive ectoderm, germ layer formation, gene expression both during in vivo and in vitro embryoid body differentiation Change can be monitored. Rex1 is expressed by ICM and ES cells, and EPL cells, 6.0 d. p. c
. Is down-regulated in the primitive ectoderm at the time point and during primitive ES cell differentiation in vitro. Fgf5 is not expressed by either ICM or ES cells, but is expressed in EPL cells and in primitive ectoderm before and during gastrulation, and is transient during ES cell differentiation in vitro. Is adjusted upward. Oct4 is expressed by all pluripotent cell populations of embryos, EPL cells and ES cells, and is downregulated in the differentiation of these cells in vivo and in vitro.

Although Rex1 expression was found to be down-regulated in both ES cell embryoid bodies and EPL cell embryoid bodies, the kinetics of Rex1 regulation differed between the two cell populations. . Down-regulation to a slightly detectable basal level resulted in day 1 EP
It was observed in L cell embryoid bodies (FIG. 23). In contrast, similar levels of Rex
1 expression was not observed in ES cell embryoid bodies until day 2 and on day 1 an intermediate level of expression was observed. Similarly, the kinetics of Fgf5 regulation depends on ES cells or EPL.
Different between embryoid bodies derived from cells. Fgf5, already expressed in EPL cells, is rapidly upregulated from day 1 in EPL cell embryoid bodies and
Peak reached on day / 3 and expression was significantly reduced on day 4 thereafter. Up-regulation of Fgf5 expression in ES cell embryoid bodies was not observed by day 3. Expression levels of these embryoid bodies increased further on day 4, but their levels were
Fgf5 levels on L-cell embryoid bodies on days 2 and 3 remained lower. Oct4 expression decreased over time in both ES and EPL embryoid bodies, but was more rapidly down-regulated in EPL cell embryoid bodies,
A four-fold reduction was found between day 4 and day 4 (FIG. 23). Following this decrease in Oct4 expression, Fgf5 expression was at the highest level. This probably means
It reflects the differentiation of pluripotent cells within their embryoid bodies.

Rex1, Fgf observed in the development of both ES and EPL embryoid bodies
Changes in 5 and Oct4 expression suggested that differentiation reflected normal embryonic development events and proceeded through the formation of late primitive ectoderm. Furthermore, a rapid increase in Fgf5 expression and earlier initiation of differentiation, as detected by a decrease in Oct4 and Fgf5 expression, indicates that differentiation of pluripotent cells in EPL cell embryoid
It was shown to be promoted compared to cell embryoid bodies. These properties are consistent with our earlier 6.0 d. p. c. Consistent with alignment with a pluripotent cell population that occurs earlier.

(Environment of EPL cell embryoid body is not permissive for visceral endoderm formation) 4.5 d. p. c. By then, pluripotent cells exposed to the blastocoel cavity had differentiated to form primitive endoderm. Primitive endoderm is two distinct endoderm cell populations, visceral endoderm (which is in contact with primitive ectoderm) and body wall endoderm (
Migrate from pluripotent cells to form a layer of endoderm adjacent to the vegetative ectoderm). Others analyzed extraembryonic endoderm formation at a relatively late stage of embryoid body development (days 9-12) but found that endoderm cells contained both visceral endoderm cells and body wall endoderm cells. The formation of the outer layer can be observed in embryoid bodies by day 4 or day 5 of development. This timing is consistent with the formation of primitive ectoderm and the generation of lumen (ie, events that result in endodermal specification in the embryo). Thus, analysis of endoderm formation at this stage appears to reflect normal embryonic events.

The formation of body wall and visceral endoderm during development of EPL and ES embryoid bodies,
Analyzed by SPARC and AFP expression, respectively. SPARC expression is expressed in ES
And showed similar kinetics in EPL cell embryoid bodies with an approximately 2-fold increase over the first 4 days (FIG. 24A). Whole mount of ES and EPL cell embryoid bodies (w
holemount) in situ hybridization and sectioning is performed by S
It shows that PARC expression is restricted to the outer layer of endoderm cells of both embryoid bodies of both ES and EPL cells at day 4 (FIGS. 24B, C), indicating the formation of body wall endoderm.
The levels of AFP are so low that Northern or RN during these stages.
AFP expression was not detected by ase protection, so whole mount in situ hybridization of embryoid bodies was used to detect AFP expression. By the third day, 1
% Of ES cell embryoid bodies showed dispersed patches of AFP expression on their surface. This level increased to 52.9 +/- 9.5% of ES cell embryoid bodies by day 4 (FIG. 24D). Sectioning confirmed that AFP expression was restricted to external cells, thus indicating visceral endoderm (FIG. 24F). AFP expression could not be detected on the surface or internal cells of EPL cell embryoid bodies on day 3 or 4 of embryoid body development (FIG. 24E).

A cell mixing experiment was performed to determine whether the failure of EPL cells to form visceral endoderm reflected an innate limitation of the developmental potential of these cells or changes in their embryoid body environment. did. The KSF-4 ES cell line constitutively expresses the LacZ gene in its nucleus, modified to target the β-galactosidase protein.
Analysis of embryoid bodies formed from KSF-4 ES cells demonstrated a level of visceral endoderm formation comparable to D3 ES cells (data not shown). After passage in the absence of mouse embryonic fibroblast feeder cells, KSF-4 ES cells were
After culturing in MEDII for 2 days, KSF-4 EPL cells (EPL (LZ +)) were formed. Embryoid bodies generated by coaggregation of D3 ES cells and EPL (LZ +) cells at a 1: 1 ratio were evaluated on day 4 by AFP expression for visceral endoderm formation. ES and EPL (LZ +) cell embryoid bodies produced visceral endoderm at levels consistent with their previously described levels (65% and 0% of embryoid bodies, respectively). Embryoid bodies formed from the mixed cell population gave rise to visceral endoderm at a level (51%) comparable to ES embryoid bodies. Double staining for β-galactosidase activity and AFP expression revealed the presence of both LacZ + and LacZ- cells within its visceral endoderm (FIG. 25). This suggests that the inability of the EPL embryoid to produce visceral endoderm results from a defect in its embryoid body environment, rather than an innate developmental limitation of EPL cells, and 2 shows the presence of an inducible signal for visceral endoderm formation.

Differentiation of Pluripotent Cells in the Absence of Visceral Endoderm Formation of EBs from EPL cells provides a methodology for differentiation of pluripotent cells in the absence of visceral endoderm I do. Visceral endoderm is known to express signals that affect the differentiation of pluripotent cells in vivo. Differentiation in the absence of this cell type offers the opportunity to specifically control the differentiation of pluripotent cells by adding factors and environmental changes. This cannot be achieved in EBs of ES cells, where the differentiation of pluripotent cells is at least partially controlled by signals from visceral endoderm spontaneously differentiating from external ES cells.

Mesoderm Formation in EBs of EPL Cells Promoted and Enhanced The formation of early mesoderm in EBs of ES and EPL cells was analyzed by analyzing the expression of early mesoderm markers brachyury and goosecoid. Monitored by Consistent with previous reports, brachyury expression was expressed in ES
In cell EBs, little was detected on days 0-3 of development, but was up-regulated on day 4 (FIG. 26). Goosecoid expression could not be detected in the EBs of ES cells during the course of this experiment. In contrast, brachyury
And goosecoid expression were upregulated 30- and 6-fold on days 2 and 3, respectively, of EPL cell EB development, and then on day 4 expression increased 9-fold (
brachyury) and 6.5-fold (goosecoid) (FIG. 26)
). In both ES cell embryoid bodies and EPL cell embryoid bodies, expression of mesoderm markers occurs just prior to a decrease in expression levels of the primitive ectodermal markers Fgf5 and Oct4 (FIG. 23). Brachyury expression in ES cell embryoid bodies did not reach the levels found in 3/4 day EPL cell bodies, even after further extended culture.

[0280] Whole mount in situ hybridization was used to determine the degree of brachyury expression within the embryoid body population. Brachyury expression,
It was detected in 1% of ES cells EB on day 3 and 16% of its cell bodies on day 4 (FIGS. 26B, D). In contrast, 98% and 92% of EPL cell EBs showed brachyury expression on days 3 and 4, respectively (FIG. 26C, E
). A similar expression pattern was observed for goosecoid (data not shown). Oct4 expression in ES and EPL cell embryoid bodies showed an expected inverse correlation with the onset and extent of brachyury expression. Oct4 expression was relatively uniform throughout control EBs on days 3 and 4 (FIGS. 26F, H
), Which were dispersed in the EBs of EPL cells in which mesoderm differentiation started (FIG. 26G,
I). Comparison of markers specific to the developing mesoderm indicates that the EPL cell embryoid body has an enhanced differentiation program resulting in earlier appearance and more extensive mesoderm-like formation compared to the ES cell embryoid body Has suggested that

Terminal differentiation of the developing mesoderm was monitored by the appearance of beating cardiomyocytes (FIG. 27).
A). This terminal differentiation was first detected in the EBs of ES cells at day 8 of development (8%) and increased over time to reach 36% of the cell body on day 12. E
In the EBs of PL cells, the beating muscle is observed in 14% of the embryoid bodies by day 6 (2 days before its appearance in the EBs of the ES cells) and on days 10 and 12 of embryoid body development By the eye, it stably increased to 60%. The percentage of EBs in EPL cells, including the beating muscle, was higher than EBs in ES cells at all time points. Consistent with this profile, expression of Nkx2.5 was higher in EPL compared to EB in ES cells (day 8).
It was induced earlier (by day 6) in the EBs of the cells (FIG. 27B). In addition, the expression level of Nkx2.5 increased in both ES and EPL cell EBs up to day 12, and throughout this period the levels in ES cell EBs were higher than those observed in EPL cell EBs. About three times lower. E
The enhanced ability of PL cells to form myocardium, when differentiated as embryoid bodies, likely affects promoted and increased development of developing mesoderm.

Thus, the formation and differentiation of EPL cells as EBs provides a methodology for efficient programming of differentiation of pluripotent cells into mesodermal lineages. The mesodermal precursor is E
It is formed earlier and at higher levels in the EBs of S cells.

EPL cell embryoid bodies show reduced ability to form neurons Differentiation of EPL cells into ectoderm-derived lineages was assessed by the presence of neurons in individual embryoid bodies (FIG. 28A) And it compared with EB of ES cell. Neurons were not detected in any embryoid body population prior to day 10. On day 10, 26% of the EBs of the ES cells retained a clear neural network. This neural network is 1
By day 2, it had risen to 41%. Embryoid bodies derived from EPL cells did not form neurons.

ES cells and P19 embryonal carcinoma (EC) cells form neurons when differentiated as aggregates in the presence of retinoic acid (RA). To test whether the failure of EPL cell EBs to give rise to neurons results from an innate limitation of the ability of the neurons to differentiate, the ability of the EPL cells to differentiate into neurons after aggregation in the presence of RA was evaluated. . Aggregates of individual RA-treated ES and EPL cells were scored for the presence of neurons (FIG. 28B), and 63% and 6%, respectively.
It was found to show a similar frequency of neuron formation of 8%. This is the EP
The absence of neurons in the EBs of L cells does not affect the innate limitations in the ability of EPL cells to form neurons, but has resulted in a decrease in neuron specification,
It has been shown to affect the altered embryoid body environment.

In short, EPL cell EBs formed nascent mesoderm and mesoderm derivatives with high efficiency, but did not form neurons, despite retaining their ability to form neurons. Differentiation of EPL cells in EBs can affect the directed formation of mesoderm germ layers from pluripotent cells cultured under these conditions. This may be due to the fact that the EBs of EPL cells do not form the outer layer of visceral endoderm, which has been demonstrated to play a role in the specification of ectodermal lineages.

Differentiation of EPL cells in response to cytokines / growth factors Differentiation of ES and EPL cells in response to members of the FGF growth factor family ES and EPL cells were isolated from DMEM + LIF or DMEM + 50%
MEDII + LIF was seeded at a density of 75 cells / cm ² . Cells were incubated with increasing concentrations (0.1-100 ng / ml) of recombinant bovine basic fibroblast growth factor (bFG).
F: Boehringer Mannheim) for 5 days.
Addition of bFGF to ES cells in the presence of LIF had no effect on ES cell morphology, differentiation or proliferation. In contrast, bFG at concentrations above 10 ng / ml
Addition of F induced the differentiation of EPL cells into a characteristic cell type (cell type A; FIG. 17A), which did not stain for alkaline phosphatase.
Similar results were obtained with human recombinant acidic FGF (R & D Systems; 10 ng / m
1), obtained with other members of the FGF family, including human recombinant kFGF / FGF4 (Sigma). Human recombinant FGF5 (R & D Systems)
Concentrations up to 500 ng / ml did not induce ES or EPL cell differentiation.

Individual colonies in a representative differentiation culture induced with 10 ng / ml bFGF were classified as ES cells, EPL cells, differentiated cells, or mixed EPL / differentiated cells based on morphology and alkaline phosphatase staining. (FIG. 29B). The percentage of undifferentiated ES cell colonies was similar regardless of the presence of bFGF. EP
Addition of bFGF to L cell cultures resulted in a marked reduction in the level of EPL cell colonies (58% to 21%) and a corresponding increase in colonies containing differentiation (
42% to 79%). This indicates that bFGF induces EPL cell differentiation but not ES cell differentiation in culture.

(Activin A induces EPL cell differentiation but does not induce ES cell differentiation) ES cells and EPL cells were seeded at a density of 75 cells / cm ² . Cells
Increasing concentrations of recombinant human activin A (1-200 ng / ml; Flinders
Medical Center, S.M. A. R. (Provided by Dr. Rogers) in DMEM + LIF or DMEM + 50% MEDII + LIF
Each was cultured for 5 days. Activin A did not affect ES cell morphology, differentiation or proliferation at any of the concentrations tested. Addition of activin A to EPL cells in culture induced their differentiation. After 5 days, most of the colonies in the activin A-treated wells have a different colony morphology characterized by the presence of a fibroblast cell type (FIG. 29C), which is stained for alkaline phosphatase activity. Did not. This cell type is morphologically different from cell type A, formed by the differentiation of EPL cells with bFGF, and is named cell type B.

Individual colonies in a representative differentiation culture induced with 150 ng / ml activin A were identified as ES cells, EPL cells, differentiated cells, or mixed EPL / differentiated cells based on morphology and alkaline phosphatase staining. Classified (FIG. 29D)
. The percentage of undifferentiated ES cell colonies was similar regardless of the presence of activin A. Addition of activin A to EPL cell cultures significantly reduced the percentage of EPL cell colonies (52% to 3%) and increased the percentage of colonies containing differentiated cells (
(48% to 97%), resulting in efficient cell differentiation.

Reverted EPL cells restore the differentiation potential of ES cells. EPL cells, when cultured in the absence of MEDII but in the presence of LIF, have morphology, gene expression, cytokine response and scutellum Revert to ES cells as assessed by their ability to contribute to the chimera after vesicle injection (Example 2). ES cells,
The cells are differentiated into EPL cells by culturing in MEDII for 2 days, and then passaged to a medium containing LIF to form reverted EPL cells (EPL ^R ). Embryoid body
The S, EPL and EPL ^R cell populations were formed and their differentiation was compared.

Embryoid bodies derived from EPL ^R cells were morphologically identical to EBs of ES cells and could be easily distinguished from EBs of EPL cells on day 4 (FIG. 22; data shown). Zu). Expression of Brachyury in ES and EPL cell EBs (FIG. 30A)
, Consistent with the profile previously described (FIG. 26). The expression of Brachyury in EBs of EPL ^R cells is similar to the expression observed for EBs in ES cells,
Its expression was detected at low levels on day 4 of development and not high on days 2 and 3 as described for EPL cells. Compared to EPL EBs that did not produce visceral endoderm, EPL ^R EBs produced visceral endoderm at levels comparable to ES EBs as shown by whole mount in situ hybridization (respectively, 31% and 46%). E seeded individually
Microscopic analysis of the EBs of S, EPL and EPL ^R (FIGS. 30B, C) revealed EP
The reversion of L to EPL ^R cells was shown to be accompanied by a high level of restoration of neuronal formation and a decrease in the level of beating cardiomyocytes and the appearance of late stage. Thus, reverted EPL cells have a similar ability to differentiate into ES cells both in vitro and in vivo (Example 2).

Summary A comparative analysis of ES and EPL differentiation, confirming the pluripotent nature of EPL cells,
Supports the identification of EPL cells as primitive ectoderm-like, and ES cells and EPL
Cells were defined as developmentally different populations based on alternative differentiation capacity and growth factor response. In combination with Example 2, this means that ES and EPL cells
4 shows different differentiation potential both in vitro and in vivo.

The products of ES and EPL cell differentiation within the embryoid body differ in both the proportion of ectodermal and mesodermal germ layers and derivatives, and the formation of extraembryonic lineage visceral endoderm. Thus, the formation of EPL cell embryoid bodies provides: a methodology for the differentiation of pluripotent cells in the absence of visceral endoderm and inducible signals generated by visceral endoderm. This offers an unprecedented opportunity for the control of pluripotent cell differentiation, including directed lineage-specific differentiation-differentiation into EPL cells and subsequent formation of embryoid bodies, or mesodermal-derived growth factors Methodology for efficient programming of pluripotent cell differentiation on mesodermal fate / treatment with cytokines.

Example 7 Alternative formation of ectoderm and mesoderm germ layers and derivatives by directed differentiation of pluripotent cells in vitro The use of the factor in MEDII is based on As any of the aggregates in suspension typical of primitive ectoderm, it has enabled the development of a strategy for the in vitro production of a homogeneous population of pluripotent EPL cells. Furthermore, EP
The L embryoid body is an instructional cell type visceral endoderm (instructive cell type)
inability to form an E. pediatric endoderm) and EPL
The response of cells to inducible signals that do not differentiate ES cells provides an opportunity to directly control the differentiation of pluripotent cells.

In this example, we control the differentiation of pluripotent EPL cells in vitro to generate cell types typical for certain embryonic germ layers, especially ectoderm and mesoderm Demonstrate what you can do. These germ cells are used for the production of differentiated derivatives and may have potential for commercial, medical and agricultural uses. In particular, manipulation of environmental stimuli (eg, altering the extracellular matrix and / or adding exogenous soluble factors) can be used for efficient and programmed alternative germ layer formation. Further manipulation of this environment allows for the directional formation of individual cell lines derived from the germ layer, which allows for the production of specific cell types and differentiation intermediates. An example of this strategy is outlined below.

Materials and Methods Cell Culture Conditions All cell and tissue culture techniques were described in Examples 1 and 6, unless otherwise specified.

Lineage-Specific Differentiation: Formation of Neuroectoderm For neuroectoderm production, EBM (Example 1) was harvested on day 4 and in a bacterial Petri dish at 20 ng / ml. Resuspended in DMEM with 50% MEDII with or without FGF4 (Sigma) and maintained for another 3 days.

Subsequently, cell aggregates used for analysis of gene expression by in situ hybridization were seeded on tissue culture plates previously coated with gelatin for 16 hours before fixing with 4% PFA (8 Day). Control aggregates were ES cell EBs formed from ES cells in DMEM without MEDII. Cell aggregates at day 8 used for analysis of gene expression by in situ hybridization were gelatinized in 50:50 DEME: F12 with 10% FCS (F12, Gibco BRL). Seeded on tissue culture plastic. After 16 hours, the medium was replaced with ITSS (Boehri
nger Mannheim), 1 mM L-glutamine and 10-20 ng
Changed to 50:50 DEME: F12 supplemented with / ml recombinant human FGF4. Aggregates were maintained in this medium for 1-4 days before fixation with 4% PFA.

Day 7 aggregates were individually seeded into 2 ml tissue culture wells for a functional differentiation assay to assess the ability of ES cells formed to differentiate into neuroectodermal neurons, and Maintained as above. Neuron formation on day 8, 1
Evaluations were made on day 0 and day 12.

Lineage-Specific Differentiation: Mesoderm Formation The formation of cells of the mesoderm lineage was achieved using several similar approaches.

From EPL cells produced in adherent cultures: Mesoderm was formed from EPL cells formed by adherent culture in the presence of MEDII as described in Example 1. As described in Example 6, these cells were trypsinized to a single cell suspension and seeded in bacterial plates at a density of 1 × 10 ⁵ cells / ml to obtain EPL cell EBs. Formed.

From EPL cells formed in suspension (EMB): EPL cells formed as cell aggregates (EBM) in suspension culture are trypsinized to a single cell suspension, and Differentiation to mesoderm was achieved by reaggregation in DMEM in bacterial dishes or DMEM containing 10 ng / ml FGF4. Cells were seeded at a density of 1 × 10 ⁵ cells / ml.

[0303] (formation of macrophages from ES cells and EPL cells) day 0 ES cells and EPL cells were trypsinized and seeded at a density of 1 × 10 ⁵ cells / ml in DMEM in a Petri dish of bacterial grade And allowed for the formation of aggregates. On day 2, about 100 aggregates from each plate were collected and 4
00 U / ml IL-3 (provided by Dr. T. Gonda, IMVS, Adelaide) and 10 ng / ml recombinant human M-CSF (R & D System).
s) supplemented with 1.25 ml of MC medium (Iscoves modified Dulbecco medium (
IMDM), 15% FCS, 50 mg / ml ascorbic acid, and 4.5 ×
^10-4 M monothioglycerol (0.9% methylcellulose in MTG)). On days 14 and 18, 50 colonies from each cell type were
The macrophages were scored as containing the above macrophages (positive) or less than 5 macrophages (negative).

[0304] Cells formed in MC cultures were collected by centrifugation and cytospin (
cytospin) and air dried. Macrophages were identified by morphology when stained with May-Grunwald-Giemsa dye and by positive staining with the macrophage-specific antibody F4 / 80 (Austyn).
And Gordon, 1981).

Gene Expression Analysis Gene expression analysis by in situ hybridization, Northern blot and RNase protection was performed as described in Examples 1 and 6. Additional probes were: Sox1 riboprobes for whole mount in situ hybridization were linearized with BamHI and transcribed with T3 RNA polymerase (antisense), or Hind.
Plasmid # 1022 (Sense, Robin Lovell-Badge, NIMR, Lo) linearized with III and transcribed with T7 RNA polymerase.
(obtained from Ndon). An antisense Sox1 probe for RNase protection was prepared using the approximately 450 bp from Sox1 cDNA in plasmid # 1022 subcloned into pBluescript II KS +.
Xho1 / EcoR1 fragment. The resulting plasmid was linearized with Kpn1 and transcribed with T7 RNA polymerase. The Sox2 probe for use in in situ hybridization was plasmid # 1015 linearized with AccI and transcribed with T3 RNA polymerase (antisense), or linearized with NotI and transcribed with T7 RNA polymerase (sense). (Obtained from Dr. Robin Lovell-Badge, NIMR, London). An antisense Gbx2 riboprobe for use in in situ hybridization,
PGbx2c7 linearized with AluI and transcribed with T3 RNA polymerase
. 1 (Chapman et al., 1997).

Immunohistological analysis Seeded cell aggregates are fixed with 4% paraformaldehyde in PBS and blocked in an appropriate blocking buffer for 30 minutes, followed by primary antibody in blocking buffer. At 4 ° C. The aggregates were then rinsed for 1 hour with a species-specific secondary antibody in blocking buffer. For FITC-conjugated secondary antibody, 5 mg / ml propyl gallate was added. Slide Ni
Tested using fluorescence on a kon TE300 microscope.

Nestin: Blocking buffer: 1% goat serum in PBS, 1 mg / ml BS
A, Triton X100. Primary antibody: Developmental St
udies Hybridoma Bank, reference rat 401, used at a dilution of 1: 100. Secondary antibody: FITC-conjugated goat anti-mouse IgM (μ specificity (
μ-specific): Sigma), used at a concentration of 1: 100.

N-Cam: blocking buffer: 1% FCS in PBS, 1 mg / ml B
SA, 1% Triton X100. Primary antibody: Santa Cruz B
iotech, SC-1507, used at a concentration of 1:20. Secondary antibody: FIT
C-conjugated rabbit anti-goat IgG (Sigma) was used at a dilution of 1: 700.

NF200: NF200 was detected by indirect immunofluorescence as described by Robertson (1987). Primary antibody: anti-neural filament 2
00 (Sigma Immunochemicals N-4142), 1: 2
Used at a dilution of 00. Secondary antibody: FITC-conjugated anti-rabbit IgG (Sile
nu.), used at a dilution of 1:60.

Results (Programmed formation of ectoderm, neuroectoderm and neural lineage by differentiation of pluripotent cells in vitro) ES cells in suspension culture for 4 days in DMEM containing 50% MEDII EBM (Example 1) formed by agglomerating 50% MEDII
Or in DMEM with 50% MEDII and 20 ng / ml FGF4 for an additional 3 days in suspension. Under these conditions, about 10
0% of the EBM developed into a cell aggregate with a unique laminar structure with the appearance of pseudo-stratification (FIG. 31A). These layers were observed in aggregates cultured with 50% MEDII only, but were enhanced in the presence of FGF4. However, these layers were not found at all in EPL cell EBs, but were only sporadically observed in ES cell EBs containing a subset of cells within the body.

The identity of cells in these layers was tested by transcript expression and analysis of diagnostic cell surface markers for embryonic cell types. For analysis of gene expression by in situ hybridization, aggregates were incubated 50 days after suspension culture.
% MEDII and seeded on gelatin-treated tissue culture plastic in DMEM with or without FGF4 for 16 hours (FIG. 31B). No expression of Oct4, a marker for pluripotent cells, could be detected in the cell layer, indicating that these cells were not pluripotent. Brachyury expression was not detected by in situ hybridization. This demonstrates that these structures did not include developing mesoderm. Furthermore, cell types in the form of mesoderm cannot be recognized in aggregates.

Sox1 is initially expressed in vivo by neuroectoderm / neural plate and undifferentiated neurons. It is not expressed in cell types of mesodermal or endodermal origin. Gbx2 is expressed in the primordial striatum and then in neural tube closure (neura)
It is expressed in the neuroectoderm prior to (l tube closure) and subsequently at the midbrain / hindbrain boundary. Sox2 is expressed in the neuroectoderm following neural tube closure and persists until the terminal differentiation of neural cells. In situ hybridization demonstrated that Sox1 was expressed extensively by cells in the layer and Gbx2 was expressed by a subset of these cells. This means that the cell types produced by these culture conditions are neuroectodermal,
In particular, it suggested that it corresponds to the initial neural plate around the time of neural tube closure.

Analysis of the expression of neuronal genes in these aggregates revealed in situ hybridization (Sox1, Sox2 and Gbx2;
32A) and immunohistochemistry (Nestin and N-CAM; FIG. 32B) refined by analysis of nerve-specific markers. At day 8 of development, Sox1 expression was detected in more than 90% of the aggregates, but expression was observed only in some cells of each aggregate. Sox1 expression increased at days 9 and 10 of development, with approximately 100% of cells in each aggregate expressing Sox1. Similarly,
At day 10, most cells also expressed Sox2. Gbx2 expression was observed in some cells at day 8 of development and was down-regulated at days 9 and 10. This expression pattern was consistent with the identification of the cell layer as having neuroectodermal origin.

The identification of these cells as neuroectoderm was supported by the expression of neuronal proteins (FIG. 32B, data not shown). Nestin, an intermediate filament protein expressed by undifferentiated neural stem cells, was expressed in the cell layer on days 8, 9, and 10, similar to Sox1. N-Cam is a marker expressed by all neural lineages, including both undifferentiated and differentiated cells. N-Cam staining was detected throughout the aggregates on days 8, 9, and 10, and the number of cells expressing N-Cam increased to almost 100%. N-Cam is expressed in both the cell layer and the differentiated cells surrounding the layer, indicating that most of the differentiated cells were of neural origin.

[0315] Northern blot analysis and RNase protection analysis of gene expression
Supporting the conclusion that EBM was programmed to form neuroectoderm in the presence of MEDII. Oct4 and Fgf5 expression (FIG. 33A) was significantly down-regulated in EBM aggregates between days 4 and 5, indicating loss of pluripotent cells. However, low but consistent levels of Oct4 transcription were detected in EBM aggregates after 5 days. In situ hybridization (FIG. 31B) showed that low Oct4 transcripts were not expressed from residual pluripotent cells in aggregates, and this may be a characteristic of early neuroectoderm in these aggregates. Proven. This residual expression could not be detected in ES cells EB forming low levels of neuroectoderm. The expression of brachyury is O
Consistent with the deletion of ct4 expression, it was observed from day 4 in ES cell EBs,
It could not be detected in EBM (FIG. 8). Finally, Gbx2 expression and S
ox1 expression (FIG. 33B) was detected by RNase protection in aggregates formed from EBM on days 6, 7, and 8. Gbx2 expression declined on day 8, while Sox1 expression continued to increase by day 10.

These results demonstrate that pluripotent cells can be specifically programmed for ectodermal and neuroectodermal developmental fate by factors within MEDII. The ectoderm cells are formed in the absence of mesoderm cell types and show a temporal pattern of gene expression that corresponds to neuroectoderm in vivo. This pattern involves progression from an early neuroectodermal cell type, which corresponds to the neural plate, to a late neuroectodermal cell type, which appears after neural tube closure. In the embryo, these cells are precursors for all neural lineages.

(Neuroectoderm induced by in vitro directed pluripotent cell differentiation can be further differentiated into neuronal cell types) MEDII, or individual aggregates generated from EBM in the presence of ES cell EBs And neurons, which were morphologically identified by the presence of axonal processes (and confirmed by expression of NF200; data not shown) and the presence of beating cardiocytes About 8
, Were evaluated on days 10 and 12. The beating heart cells (FIG. 34A) had 50
% Of ES cell EBs, and these
Survived for two days. In contrast, the majority of E retained in MEDII
BM-derived aggregates (99.6%) did not contain beating heart cells. The loss of beating heart cells (mesoderm-derived tissue) in these aggregates is consistent with the loss of brachyury expression at early stages of development (Example 1, FIGS. 7, 8), and Shows that no mesoderm line is formed in these aggregates. Neurons were not detected in any aggregate population on day 8 of development (FIG. 34B).
) Was evident in more than 60% of EBM-derived aggregates on day 10. By day 12, over 90% of these aggregates contained neurons. By comparison, neuron formation was observed in 10% (day 10) and 25% (day 12) of ES cell EBs. These data show that neuroectoderm,
Supports the previous conclusion that EBM cultured in the presence of MEDII forms at high levels and in the absence of mesoderm cells, and this neuroectoderm gives rise to terminally differentiated neuronal cell types Prove that you get. Therefore, E in MEDII
Aggregates generated from BM are enriched in undifferentiated neurons.

The formation of neuroectoderm in vivo proceeds through the formation of terminal ectoderm.
Although there are no markers for the final ectoderm in vivo, gene expression analysis indicates that EBMs that express low levels of Oct4 and are programmed to form neuroectoderms that cannot express neuroectoderm markers Cell populations were identified. These cells contain primitive ectoderm (high Oct4 expression) and neuroectoderm (high Sox1,
Gbx2), and suggests that neuroectoderm formation proceeds through a population of intermediates that may represent the final ectoderm.

The formation of the neuroectoderm described herein promotes neurogenesis without relying on the addition of chemical inducers (eg, retinoic acid) or genetic engineering. Instead, it relies on biological inducers found in the conditioned medium MEDII. The neural precursors formed in this manner are thought to be differentiated from pluripotent cells in a manner similar to the formation of neural cells during embryogenesis, and thus are considered commercially, medically and agriculturally. Ideal for producing differentiated neurons useful in the above applications. Furthermore, in contrast to the formation of a restricted nervous system by chemical inducers such as retinoic acid, the identity of neuronal cell types produced using these methodologies does not appear to be limited.

Neural ectoderm programming of pluripotent cell differentiation requires the high molecular weight component of MEDII but not the low molecular weight component of MEDII. During embryogenesis, the neuroectodermal / Anterior primitive ectoderm cells that maintain association with the extracellular matrix
m cell). Bioactive EC in high molecular weight components of MEDII
The identification of the M protein has prompted studies of the role of this activity in the formation of directed neuroectoderm from pluripotent cells. This was tested by the ability of this component to induce neuron formation in EPL EBs, where neuronal cell types are not normally formed (Example 6).

EPL cells were formed by culturing ES cells for 2 days in the presence of MEDII and in the absence of LIF. EPL cells EB were formed in DMEM or DMEM + R (Example 4). On the fourth day of development, the tissue embryoid body
2 ml tissue culture wells treated with gelatin were individually inoculated. Beating heart cells and neurons were evaluated at days 7, 8, 9, and 10 of development (FIG. 35).
.

EPL cell EBs formed beating muscle with high efficiency in the absence of high molecular weight components, and these EBs did not contain neurons. Encapsulation of high molecular weight components (50-100 μg / ml) during tissue embryoid body formation was observed in about 5% of EBs on day 9 and 10 days and 12 days.
12% resulted in neuronal development. Only 10-12% of EPL cell EBs formed in the presence of high molecular weight components contain pulsatile muscle, and pulsatile muscle formation was delayed by 9 days. These experiments were performed in the presence of 1 μg / ml anti-human LIF neutralizing antibody (R & D Systems). Thus, an altered developmental program cannot account for the low levels of LIF expressed by Hep G2 cells.

This experiment demonstrates the role of the high molecular weight component of MEDII, and possibly the ECM component, in the program of differentiation in pluripotent cell aggregates, such as slowing and reducing mesoderm formation and promoting neuron formation. Demonstrate.

Efficient programmed formation of mesoderm lineages derived from pluripotent cells. Pluripotent cells are identified by differentiation through homologous EPL cell intermediates, as shown in Example 6. Can be programmed to adopt the mesoderm developmental fate. This may reflect the absence of visceral ectoderm in EPL cells EB. Developmental mesoderm formation can be detected in about 100% of EPL cells EB, and by scoring beating heart cells containing only one of the possible nascent mesoderm cell developmental fate, Probably underestimated. The previous experiments were performed with EPL cells formed from ES cells in adherent culture. Here we describe an alternative methodology for efficient mesoderm formation from pluripotent cells in vitro.

(Mesodermal Formation by Reaggregation of EPL Cells Formed in Suspension) EPL cells formed in suspension in the presence of MEDII (EBM, Example 1) were extraembryonic Embryonic primitive ect
oderm), comprising a stable and homogenous population of cells having properties corresponding to the above. Programming the differentiation of these cells to a mesodermal developmental fate would require both removal of the neuroectoderm-inducing activity of MEDII and dissociation from the ECM component. This mimics the behavior of pluripotent cells destined to form mesoderm during primitive streak formation in vivo.

On day four, EMB was trypsinized to a single cell suspension and DM
EM +/− 10 ng / ml FGF4 or DMEM + 50% MEDII + /
Reaggregated in -10 ng / ml FGF4. Four days after the outbreak, DME
Clear morphological differences were evident between reaggregates cultured in M and aggregates cultured in DMEM + 50% MEDII (FIG. 36). On days 2 and 4, RNA from these aggregates was analyzed by Northern blot for expression of brachyury and Oct4 (FIG. 36). Oc
t4 expression was down-regulated in day 4 reaggregates. This indicated a loss of pluripotency when differentiation occurred. Consistent with the fact that reaggregates are destined to form neuroectoderm, brachyury expression is
No detectable in reaggregates generated in MEM + 50% MEDII.
Brachyury expression, indicative of mesoderm formation, was 2% in the absence of FGF4.
It was strongly up-regulated in re-aggregates generated in DMEM on days 4 and 4. In the presence of FGF4, brachyury expression peaked on day 2 and was down-regulated by day 4. This means that FGF
4 suggests that the addition of 4 results in accelerated mesodermal differentiation of pluripotent living cells. Marker gene expression was confirmed by in situ hybridization of Oct4 and brachyury specific probes with reaggregates (data not shown). These data indicate that EPL cells formed in suspension can be programmed to differentiate into mesoderm.

(Mesodermal progenitors induced by the differentiation of committed pluripotent cells can be programmed for alternative developmental fate.) The programmed formation of high levels of mesoderm in EPL cells EB This was demonstrated by the formation of mesodermal and beating heart cells (Example 6). EPL cell E
Response to exogenous cytokines to establish whether elevated levels of nascent mesoderm in B were developmentally restricted to myogenic lineages or could be programmed to an alternative developmental fate The formation of growing macrophages was evaluated.

EPL cells and control ES cell EBs were isolated from mIL-3 and hM-CS.
Differentiation in MC medium supplemented with F, and on days 12, 15, and 1
On day 8, the presence of macrophages was recorded (FIG. 37). On day 12, 32.4% of EPL cell EBs were found to contain macrophages as compared to 4.8% of ES cell EBs. The proportion of tissue embryoid bodies containing macrophages on days 15 and 18 increased to 43% of EPL cell EBs and 9-10% of ES cell EBs. Consistent with the early formation of mesoderm in EPL cell EBs, macrophage formation was initiated approximately 2 days earlier in these tissue embryoid bodies compared to ES cell EBs (data not shown). The increased formation of multiple mesoderm lineages in the EPL cell EBs indicates that increased nascent mesoderm in these (tissue germ layers) bodies may be programmed for alternative developmental fate in response to specific environmental cues. It suggests that it contains a potential mesodermal precursor.

(Pluripotent cell differentiation can be specifically programmed for the formation of selective mesodermal and ectodermal lineages) Mesodermal differentiation of pluripotent cells (EB formation of EPL cells) and extracellular A comparative analysis of the methodology developed to direct germ layer differentiation (EBM formation) is shown in FIG. The differentiated population was scored for the presence of neurons and beating muscle as representative cell types for the mesoderm and ectoderm lineages, respectively.

The EBs of EPL cells formed the beating muscle with high efficiency, but failed to form neurons. EBM showed opposing patterns and formed neurons with high efficiency, but failed to form pulsatile muscle. In particular, mesoderm formation is probably underestimated in this analysis. This is because only a single mesodermal cell type (pulsatile muscle) was scored. Thus, the proper use of factors within MEDII to form and differentiate pluripotent cell populations directs the specific formation of selective mesoderm and ectoderm germ layers from pluripotent cells in vitro I can do it.

Overview We describe a method for directing the differentiation of pluripotent cells into selective germ layer (ie, ectoderm and mesoderm) cell lines. (In MEDII
The formation of EPL cells in response to biological activity (in adherent or suspension culture) is an essential step in these lineage-specific differentiation protocols. The pluripotent cells used in the above examples were obtained by culturing ES cells in MEDII, but we have determined that primitive pluripotent cells isolated from embryonic ICM, primitive ectoderm or PGCs, or It will be appreciated that pluripotent cells obtained by dedifferentiation or nuclear transfer could be used as a source of material for controlled lineage-specific differentiation in vitro.

Neuroectoderm formation in the absence of mesoderm formation occurred when EPL cells differentiated in suspension culture in the presence of MEDII. This suggested that the conditioned media contained the components necessary for neural determination. Fractionation of the conditioned medium indicates that the component is present in the fraction containing media components greater than 10 kDa and the ECM component is
It was suggested that it could be identified as an active component for L cell formation. The formation of neuroectoderm from primitive ectoderm by contacting ECM components is consistent with the determination of this lineage during embryogenesis.

[0333] Efficient formation of mesoderm from EPL cells in the absence of neuroectoderm can be achieved in adherent or suspension cultures and removes MEDII and potentially removes E.
It may be necessary to eliminate contact with the CM protein. This is MEDII
Can be achieved by aggregation or reaggregation of EPL cells in medium without the addition of
Alternatively, it can be achieved by seeding EPL cells on gelatin-treated plastic-ware in the presence of a TGFβ or FGF family member. EPL cell differentiation proceeds in the absence of visceral endoderm as cell aggregation and results in the formation of a mesoderm cell lineage, which is assessed by gene expression and termination of differentiation.

Importantly, the inclusion or elimination of EPL cells and MEDII or MEDII activity provides an approach for the production of cell populations that are highly enriched in progenitors and differentiated cells from a single germ layer. provide. This approach does not involve the addition of a chemical inducer (eg, retinoic acid for neuronal cell formation) or prior genetic modification of pluripotent cells. Instead, this approach relies on factors thought to be involved in the formation of neuroectoderm and mesoderm during normal embryonic development in vivo. Precursors derived in this manner are ideal for human medicine, veterinary applications and planned applications in agriculture.

Example 8 Application of Gene Therapy The utility of the present invention in gene therapy is as follows. It relies on the use of nuclear transfer techniques that have already been developed and used in several animal species, including sheep, cattle and mice. The donor human ovum is fertilized in vitro, but then the nucleus is removed and replaced with nuclei from cells from the intended gene therapy recipient. The nuclear transferred eggs are developed into early embryos in vitro, and cells from the early embryos are then used to generate EPL or ES cell lines using the procedures and products of the present invention. This cell line is then genetically modified using DNA transfer techniques known to those of skill in the art to correct for genetic deficiencies, or to coagulate for the desired gene (eg; hemophilia). A functional copy of the factor gene, the CF gene for cystic fibrosis, the insulin gene for type I diabetes) is replaced or added. The modified ES or EPL cell line is then differentiated in vitro into the desired cell, tissue, or organ. This is reintroduced into the recipient in a therapeutic transplant procedure. All genes (except for the modified or introduced gene) of the modified cell line are identical to the recipient individual's own gene, which is, after all, a perfectly matched transplantation, There seems to be no rejection of the organization.

Alternatively, modified cells from incompatible human ES or EPL cell lines are genetically modified and then encapsulated by cell encapsulation techniques known to those skilled in the art. They are then introduced into the recipient as a graft so as to produce in the individual a gene product that is deleted or missing in the treated individual (eg, clotting factors, insulin, etc.). It is also possible to genetically modify a donor's established ES cell line or EPL cell line to be non-immunological to any recipient, thereby providing a "universal ES cell line or EPL cell line.
Cell line ". This cell line is further modified and used in the therapeutic procedures described above without using encapsulation techniques.

Using such “universal” donor cells, xenograft procedures (eg, xenograft organ vessels (eg, pig kidney or heart) with human endothelium to reduce or eliminate delayed transplant rejection Generate human endothelial cells that are used for coating.

Example 9 Transplantation of Pluripotent Cell-Derived Nerve Cells Despite the promise given by long-term therapeutic transplantation of neural cells and the cure of neurological disorders, a critical The hurdle still exists. Allografts and xenografts are rejected even if immunological protection is provided by the blood-brain barrier. In addition, the availability of fetal cells, which are often used in nerve transplant studies, is rare. Nerve cells, including neural stem cells generated by in vitro controlled differentiation of pluripotent cells along defined neural pathways, provide an ideal source of cells for neural cell therapy and nerve transplantation.

Transplantation of neurodopamine cells in humans has been attempted to alleviate some of the symptoms of Parkinson's disease, and transplantation of neuronal cells may also provide benefits for neurodegenerative disorders. As the first step in providing these cells for human therapy,
Transplantation using neural cells from pluripotent cells can be studied in a mouse model. Differentiation of ES cell lines expressing β-galactosidase directed to nuclear location (KSF4 cell line) or cytoplasm (ROSA 26 cell line) is a neural pathway using MEDII as described herein. Along. Neuroectoderm (
The neuroderm or differentiated nerve cells can be injected into the neonatal cerebral vesicles. Survival of the transplanted cells is indicated by maintenance of cells expressing β-galactosidase in the brain. The ability of the transplanted cells to assimilate can be determined by studying the relationships established by marker cells that express the cytoplasmic form of β-galactosidase (ie, cells from ROSA 26 ES cells).

[0340]

[Table 9] Finally, it should be understood that various modifications, changes and / or additions can be made without departing from the spirit of the invention as outlined herein.

[0341]

[Table 1]

[0342]

[Table 2]

[0343]

[Table 3]

[0344]

[Table 4]

[0345]

[Table 5]

[0346]

[Table 6]

[0347]

[Table 7]

[0348]

[Table 8]

[Brief description of the drawings]

FIG. 1. MEDII affects migration from ES cells to EPL cells. ES cells are grown after 4 days in culture in media containing LIF (A), 50% MEDII + LIF (B) or without additives (C). ES cells cultured in the presence of MEDII show characteristic morphologies associated with the formation of EPL cells. This bar shows 50 μm. (D) ES cells were LIF, 50% MEDII + LIF, 50% MEDI
Seeded at a density of 250 cells / cm ² in DMEM containing I and no additives. After 5 days, cultures were stained for alkaline phosphatase and hematoxylin, and the ES, EPL and percentage of differentiated colonies were determined. Alkaline phosphatase positive colonies were subdivided into ES cell colonies and EPL cell colonies based on morphology, and differentiated colonies (D) were determined to be alkaline phosphatase negative. The plating efficiency (%) for each state was 42.8 +/- 8.7 (LIF); 47.26+
/-2.6 (50% MEDII + LIF); 40.5 +/- 5.9 (50% ME
DII); 41.4 +/- 6.6 (no addition).

(A) Northern blot analysis of RNA of ES cells and EPL cells. ME
E, cultured for 2, 4, 6, and 16 days in DII or MEDII + LIF
14 ES cells, EPL cell derivative (i) and 20 μg of total RNA isolated from spontaneously differentiated ES cells (ii) cultured for 6 days in the absence of exogenous factors
Was analyzed for expression of Fgf5, Rex1, Oct4 and mGAP. Fg
The f5 transcript is 2.7 and 1.8 kb (Hebert et al., 1990), Rex
1, 1.9 kb (Hosler et al., 1989), and Oct4, 1.55 kb (
(Rosner et al., 1990) and 1.5 kb for mGAP. (B, C
) Shows ES cell layer (B) and EPL cell layer (C) for Oct4 expression.
In-situ analysis of. (D, E): ES cells (D) and EPL cells (E)
Stained for the presence of alkaline phosphatase. (F): MEDII + LI
10 μg of RNA from ES cells and EPL cells cultured for 2, 4 and 6 days in F and MEDII showed Gbx2, Oct4 and m4 by RNase protection.
GAP expression was analyzed. (G) EPL for Fgf5 expression.
In situ analysis of cells. (H): 20 μg of total RNA from ES and EPL cells maintained in MEDII + LIF for 2 and 6 days was analyzed by Northern blot for expression of Uvomorulin (Uvo). mGAP expression was used to normalize RNA levels. The rod is 32 μm for BE,
And, F indicates 25 μm.

FIG. 3A: Sequence of L17 ddPCR product.

FIG. 3B. Sequence of the ddPCR product of K7.

FIG. 3C: Sequence of the ddPCR product of Psc1.

FIG. 4A. 5 μg of poly A RNA (L17 and Psc1) or 20 isolated from ES and EPL cells maintained in a culture of MEDII + LIF for up to 8 days.
L17 (A), Psc1 (B) and K7 (4) in μg of total RNA (K7)
) Northern analysis of expression. The transfer size is shown in the figure. Northern blots
Quantified and normalized to Oct4 expression.

FIG. 4B. 5 μg of poly A RNA (L17 and Psc1) or 20 isolated from ES and EPL cells maintained in a culture of MEDII + LIF for up to 8 days.
L17 (A), Psc1 (B) and K7 (4) in μg of total RNA (K7)
) Northern analysis of expression. The transfer size is shown in the figure. Northern blots
Quantified and normalized to Oct4 expression.

FIG. 4C. 5 μg of poly A RNA (L17 and Psc1) or 20 isolated from ES and EPL cells maintained in a culture of MEDII + LIF for up to 8 days.
L17 (A), Psc1 (B) and K7 (4) in μg of total RNA (K7)
) Northern analysis of expression. The transfer size is shown in the figure. Northern blots
Quantified and normalized to Oct4 expression.

Fig. 5A 3.5d. p. c. And 5.5d. p. c. In vivo expression of novel marker genes L17 (A), Psc1 (B) and K7 (C) by whole mount in situ hybridization analysis of mouse embryos cleaved between and. PE is
Primitive ectoderm; PC is primitive amniotic cavity; VE is visceral ectoderm.

Fig. 5B 3.5d. p. c. And 5.5d. p. c. In vivo expression of novel marker genes L17 (A), Psc1 (B) and K7 (C) by whole mount in situ hybridization analysis of mouse embryos cleaved between and. PE is
Primitive ectoderm; PC is primitive amniotic cavity; VE is visceral ectoderm.

Fig. 5C 3.5d. p. c. And 5.5d. p. c. In vivo expression of novel marker genes L17 (A), Psc1 (B) and K7 (C) by whole mount in situ hybridization analysis of mouse embryos cleaved between and. PE is
Primitive ectoderm; PC is primitive amniotic cavity; VE is visceral ectoderm.

FIG. 6. hLIF is required for maintenance of EPL cells in culture. ES cells were seeded at a density of 250 cells / cm ² in DMEM containing: 50% ME
DII or 50% MEDII + mLIF (1000 units / ml), hLIF (
1000 units / ml), anti-hLIF antibody (10 μg / ml), hLIF (100
0 units / ml) and anti-hLIF antibody (10 μg / ml), mLIF (1000
Unit / ml) and anti-hLIF antibody (10 μg / ml), anti-gp130 antibody (1
0 μg / ml), or hLIF (1000 units / ml) and anti-gp130 antibody (10 μg / ml). After 5 days in culture, colonies were stained for alkaline phosphatase and the percentage of EPL cell-containing colonies (EPL) and differentiated colonies (D) was determined. The plating efficiency (%) for each state was 32.5 +/- 1.5 (50% MEDII); 39.8 +/-
1.3 (50% MEDII + mLIF); 35.0 +/- 0.2 (MEDII +
hLIF); 25.5 +/- 1.0 (MEDII + anti-hLIF antibody); 28.8
+/- 3.8 (MEDII + hLIF + anti-hLIF antibody); 40.0 +/- 0.
5 (MEDII + mLIF + anti-hLIF antibody); 36.8 +/- 3.3 (MED
II + anti-gp130 antibody); 36.3 +/- 2.3 (MEDII + hLIF + anti-gp130 antibody).

FIG. 7 (A) 5 μm sections stained with hematoxylin: eosin of aggregates formed from ES cells and grown in DMEM (EB, i) or DMEM; MEDII (EBM, ii) for 4 days . (B): formed from ES cells and DMEM (EB, i, iii, v) or DMEM; MEDII (4 days)
Aggregates grown in EBM, ii, iv, vi) were analyzed by Oct4 (i, ii), brachy by whole mount in situ hybridization.
uri (iii, iv) and Fgf5 (v, vi) expression were analyzed.

FIG. 8. 20 μg of total R isolated from EB and EBM at 2, 3 and 4 days of development
NA was determined by Northern blot analysis from Fgf5, brachyury, Oct4.
And analyzed for mGAP expression. Fgf5 transcripts were 2.7 and 1
. 8 kb (Hebert et al., 1990), and in brachyury 2.1 kb
(Lake, 1996), 1.54 kb in Oct4 (Rosner et al., 199).
0) and 1.5 kb for mGAP.

(A) Northern blot analysis of RNA from EPL cells and reverted EPL cells. Of EPL cells cultured in the presence of MEDII or MEDII + LIF for 2, 4 and 6 days, and of their reverted derivatives (2 ^R , 4 ^R , and 6 ^R ) reverted in a medium containing only LIF 20 μg of total RNA was transferred to Fgf5
, Rex1, Oct4 and mGAP expression were analyzed by Northern blot. (B): Clonal EPL cell lines # 1 and # 2 were seeded at a density of 250 cells / cm ² and 500 cells / cm ² , respectively, in media containing mLIF and 50% MEDII + LIF. After 5 days, the cultures were stained for alkaline phosphatase and the percentage of ES, EPL and differentiated colonies was determined. Alkaline phosphatase positive colonies were subdivided into ES cell colonies and EPL cell colonies based on morphology, and differentiated colonies (D) were determined to be alkaline phosphatase negative. 34. Plating efficiency (%): 18.4 +/- 2.62 (clone # 1, LIF);
8 +/- 4.56 (clone # 1, 50% MEDII + LIF); 20.4 + /
-1.71 (clone # 2, LIF); 31.16 +/- 2.93 (clone #
2,50% MEDII + LIF).

(A): 20 μg of total RNA isolated from ES cells (day 0) and ES cell EBs at 1, 2, 3 and 4 days of development, Rex1 by Northern blot analysis.
, Fgf5, Oct4 and mGAP expression were analyzed. (B): LI
Alkaline phosphatase positive from EB, 5, 6, 7 and 8 day single cell suspension cultures in DMEM supplemented with F (grey) or DMEM supplemented with LIF + 50% MEDII (black) Number of pluripotent cell colonies.

FIG. 11. Maintenance of embryo-derived primitive ectoderm in culture requires MEDII. (A) Embryo culture medium (LIF / CIV) or embryo culture medium + 50% MEDII (MEDII /
CIV) at 5.5 d. p. Morphology of embryonic implants in c. (B
) 5.5 d. Cultured in embryo culture medium + 50% MEDII for 5 days. p. In situ hybridization analysis of Oct4 expression in embryonic explants of c. The same implant is found in both panels. The right panel is four times larger.

(A) Schematic of the strategy used for isolation and maintenance of pluripotent cells from primitive ectoderm of mouse embryo. (B) Representative embryonic explants at different stages of the isolation procedure. Explants from several different isolation experiments are shown.

(A) Schematic of the strategy used for the isolation and maintenance of pluripotent cells from primitive ectoderm of mouse embryos. (B) Representative embryonic explants at different stages of the isolation procedure. Explants from several different isolation experiments are shown.

FIG. p. c. Isolated from embryonic primordial ectoderm (EEPL cells)
Pluripotent cell colonies established in long-term culture on the TO feeder layer were analyzed for alkaline phosphatase and Oct4 expression by cell staining and in situ hybridization.

FIG. 14. Two soluble factors in MEDII are responsible for the formation of EPL cells from ES cells. sfMEDII was separated into a retention fraction (R) and an elution fraction (E) via ultrafiltration on a centricon-3 unit. ES cells, LIF
At a density of 250 cells / cm ² in media containing E, R, or E + R. After 5 days, cultures were stained for alkaline phosphatase and the percentage of ES, EPL (alkaline phosphatase positive) and differentiated colonies (alkaline phosphatase negative; D) was determined. Plating efficiency (%): 48.4% (elution fraction); 41.6% (retention fraction); 49
. 2% (elution fraction and retention fraction); 32.8% (mLIF).

FIG. 15. Purification of low molecular weight components of EPL cell-inducing activity. (A): Sephadex G
10 Fractionation of E by gel filtration. (B): fractionation of the active fraction from A by normal phase chromatography. (C): fractionation of the active fraction from B by Superdex peptide gel filtration. The eluted material was determined by absorbance at 215 nm. Chromatographic fractions were assayed for the presence of low molecular weight components of EPL cell-inducing activity. The solid line under each chromatograph indicates the fraction containing this activity.

FIG. 16: Purification of high molecular weight components of sfMEDII (Protocol 1). Sequential purification of R via anion exchange chromatography (A), hydrophobic interaction chromatography (B), heparin affinity chromatography (C) and Superose 6 gel filtration chromatography (D). Protein is 280 nm
Detected in. Solid bars indicate fractions containing biological activity. (E) Silver-stained reduced SDS 10% acrylamide gel of the active fraction from each stage of purification. Samples contained either 5 μg protein (100 KD; anion exchange; hydrophobic interactions) or 2 μg protein (heparin affinity; gel filtration).

FIG. 17: Purification of high molecular weight components of sfMEDII (Protocol 2). Heparin Seph
arose CL-6B chromatography (A), anion exchange chromatography (B), and Superose 6 gel filtration chromatography (C)
Purification of R by HPLC. Protein was detected at 280 nm. The solid bar is
Shows fractions containing biological activity. (D) Silver-stained reduced SDS 10% acrylamide gel of the active fraction from each stage of purification. Samples contained either 5 μg protein (sfMEDII), 2 μg protein (heparin affinity; anion exchange) or 1 μg protein (gel filtration).

FIG. 18. Purification of high molecular weight components of sfMEDII (Protocol 3). (A) shows sfMEDII by gelatin Sepharose affinity chromatography.
Purification. Protein was detected at 280 nm. Elution of biological activity is indicated by the solid bar. (B) shows 5 μg of protein from sfMEDII, and the effluent fraction of gelatin Sepharose, and 1 μg of cellular fibronectin from stromal cells (cFN stromal cells), and gelatin S from sfMEDII.
Silver-stained reduced SDS 10% acrylamide gel of epharose purified high molecular weight component (cFSNSFM2).

FIG. 19: Human plasma fibronectin (2 μg / m 2) in the presence of 87 μM L-proline
l) (A), human cellular fibronectin (2 μg / ml) (B), and the high molecular weight component of sfMEDII purified by protocol 2 (2 μg / ml) (C
2) Morphology of the ES cells cultured in the above.

FIG. 20 shows a monoclonal antibody against human cell fibronectin (3E2, Sigma).
), Purified human plasma fibronectin (1 μg, 0.5 μg
), Purified human cell fibronectin (1.0, 0.5, 0.25 μg) and high molecular weight components of sfMEDII purified by Protocol 2 (1.0, 0.
5, 0.25 μg) Western blot. The position of the protein size marker is shown.

FIG. 21. Components of EPL cell-inducing activity can be identified in dispersed cell types and species. (A) Morphology of ES cells derived from primary adult mouse hepatocytes or primary embryonic chicken hepatocytes after 3 days in culture and seeded in DMEM + 50% conditioned medium.
(B) shows D from Hepa-1c1c 7 cells +/− 40 μM L proline.
ES cells seeded in MEM + 50% conditioned medium, or END-2 cells +/- 1
E seeded in DMEM + 50% conditioned medium from 0 μg cell fibronectin
Morphology of S cells. (C) 5 μg protein from conditioned DMEM, 1, no cells; 2, Hep G2; 3, Hep 3B; 4, Hepa-1.
Electrophoresed on a 10% reduced SDS polyacrylamide gel like c1c 7; 5, END-2; 6, PYS-2. Proteins were visualized by silver staining. Arrows indicate the location of cellular fibronectin monomers.

FIG. 22. Morphology of ES and EPL cell EBs at day 4 of differentiation. (A) is the EB of the ES, and (B) is the EB of the EPL. (C, D): EBs were sectioned and stained with toluidine-blue; (C) is ES EB, (D) is EPL EB.

FIG. 23. Pluripotent cell markers in the EBs of differentiating ES and EPL cells,
Expression of Fgf5, Rex1, and Oct4. Northern blot analysis was performed on 20 μg of total RNA from EBs of ES and EPL cells on the indicated days of differentiation. mGAP expression was used as a loading control.

FIG. 24. When EPL cells are differentiated as EBs, they form paretal endoderm but not visceral endoderm. (A) SPARC expression in ES and EPL cell-derived EBs at day 1-4 of differentiation as determined by Northern blot analysis of 20 μg of total RNA. (B, C) Antisense DIG labeled SPAR
Sections of EBs from day 4 ES cells (B) and EPL cells (C) subjected to whole mount in situ hybridization using C-specific riboprobes.
(D, E) Whole mount in situ hybridization of ES cells (D, F) and EPL cells (E) EB on day 4 using antisense DIG-labeled AFP-specific riboprobes. The region that expresses AFP is indicated by an arrow. (F)
Section of ES cell expression.

FIG. 25. EPL cells restore their ability to form visceral endoderm when mixed with ES cells during EB formation. Day 4 chimeric EPL formed by 1: 1 cell mixing
(LZ +): ES EBs were stained for β-galactosidase activity (cells in circled areas), sectioned and stained for AFP protein by immunohistochemistry (black).

FIG. 26. Enhancement and promotion of mesoderm induction in EBs of EPL cells compared to EBs of ES cells. (A) Brachyury in ES and EPL cell EBs at day 0-4 of differentiation, determined by Northern blot analysis of 20 μg of total RNA,
Expression of goosecoid and mGAP. (BI): Day 3 (B, C, F,
G) and on day 4 (D, E, H, I), ES cells (B, D, F, H) and EB whole mount in situ hybridization of EPL cells (C, E, G, I).

FIG. 27. Enhancement and promotion of beating heart cell formation in EBs of EPL cells compared to EBs of ES cells. (A) Percentage of EBs of ES and EPL cells showing beating muscle during days 4-12 of differentiation. Mean percentages and standard deviations were derived from two separate experiments in each experiment (n = 48). (B) 2
Nkx2.5 expression in EBs of ES and EPL cells from day 4-12 as determined by Northern blot analysis of 0 μg of total RNA.

FIG. 28. The ability of EPL cells to form neurons depends on the in vitro differentiation regime used. (A) ES forming neurons at 8, 10 and 12 days of differentiation
EB percentage of cells and EPL cells. (B) Percentage of RA-treated aggregates of ES and EPL cells, including neurons. Mean percentages and standard deviations were derived from four separate experiments. N = 32 in each experiment
.

FIG. 29A. ES cells and EPL in response to FGF and TGFβ family growth factors
Cell differentiation. (A) EPL cell colonies differentiated in the presence of bFGF, showing characteristic differentiation morphology of cell type A. (B) After 5 days, ES cells after culture of ES cells and EPL cells in the presence or absence of bFGF (10 ng / ml) (
ES), EPL cells (EPL), differentiated cells (D) and ratio of mixed EPL cells and differentiated cells (SD) colonies. (C) EPL cell colonies differentiated in the presence of activin A. This is characteristic of the differentiated morphology of cell type B. (D) 5 days later, after culturing of ES cells and EPL cells in the presence or absence of activin A (150 ng / ml), ES cells (ES), EP
Percentage of colonies of L cells (EPL), differentiated cells (D) and mixed EPL cells and differentiated cells (SD).

FIG. 29B. ES cells and EPL in response to FGF and TGFβ family growth factors.
Cell differentiation. (A) EPL cell colonies differentiated in the presence of bFGF, showing characteristic differentiation morphology of cell type A. (B) After 5 days, ES cells after culture of ES cells and EPL cells in the presence or absence of bFGF (10 ng / ml) (
ES), EPL cells (EPL), differentiated cells (D) and ratio of mixed EPL cells and differentiated cells (SD) colonies. (C) EPL cell colonies differentiated in the presence of activin A. This is characteristic of the differentiated morphology of cell type B. (D) 5 days later, after culturing of ES cells and EPL cells in the presence or absence of activin A (150 ng / ml), ES cells (ES), EP
Percentage of colonies of L cells (EPL), differentiated cells (D) and mixed EPL cells and differentiated cells (SD).

FIG. 29C. ES cells and EPL in response to FGF and TGFβ family growth factors.
Cell differentiation. (A) EPL cell colonies differentiated in the presence of bFGF, showing characteristic differentiation morphology of cell type A. (B) After 5 days, ES cells after culture of ES cells and EPL cells in the presence or absence of bFGF (10 ng / ml) (
ES), EPL cells (EPL), differentiated cells (D) and ratio of mixed EPL cells and differentiated cells (SD) colonies. (C) EPL cell colonies differentiated in the presence of activin A. This is characteristic of the differentiated morphology of cell type B. (D) 5 days later, after culturing of ES cells and EPL cells in the presence or absence of activin A (150 ng / ml), ES cells (ES), EP
Percentage of colonies of L cells (EPL), differentiated cells (D) and mixed EPL cells and differentiated cells (SD).

FIG. 29D. ES cells and EPL in response to FGF and TGFβ family growth factors.
Cell differentiation. (A) EPL cell colonies differentiated in the presence of bFGF, showing characteristic differentiation morphology of cell type A. (B) After 5 days, ES cells after culture of ES cells and EPL cells in the presence or absence of bFGF (10 ng / ml) (
ES), EPL cells (EPL), differentiated cells (D) and ratio of mixed EPL cells and differentiated cells (SD) colonies. (C) EPL cell colonies differentiated in the presence of activin A. This is characteristic of the differentiated morphology of cell type B. (D) 5 days later, after culturing of ES cells and EPL cells in the presence or absence of activin A (150 ng / ml), ES cells (ES), EP
Percentage of colonies of L cells (EPL), differentiated cells (D) and mixed EPL cells and differentiated cells (SD).

FIG. 30. Reverted EPL cells have the same differentiation ability as ES cells when differentiated as EB. (A): ES cells, EPL cells and EPL ^R at 1-4 days of differentiation
Northern blot analysis of 20 μg of total RNA for expression of Fgf5 and brachyury in EBs from cells. (B): Percentage of EB of ES, EPL and EPL ^R cells showing beating muscles from day 7-12 of differentiation (n
= 36). (C): ES cells forming neurons from 7-12 days of differentiation, E
Percentage of EBs in PL and EPL ^R cells (n = 36).

FIG. 31. Formation of directed pluripotent cells into the neuroectoderm through the formation of EBM. (A) formed from ES cells and DMEM + 50% M
4 days in EDII, and additionally DMEM + 50% MEDII + 20 ng / m
l EBM grown in FGF4 for 3 days. A unique convoluted cell layer with a pseudo-stratified epithelial appearance formed in the aggregate. (B) Aggregates grown as A were placed on gelatin-treated plastic on DMEM + 50% MEDII + 20 ng / ml
Seeded in FGF4 for 16 hours and Oct4 (i), brachyury (ii), Gbx2 (iii) by in situ hybridization
And Sox1 (iv) expression.

FIG. 32. Expression of neural ectoderm and neural stem cell markers in EBM-derived aggregates.
EBM (Day 4) was DMEM + 50% MEDII + 20 ng / ml FGF4
And seeded on gelatin for analysis of gene expression on days 8, 9, and 10. (A) In situ hybridization of aggregates using Gbx2, Sox1 and Sox2 antisense probes. (
B) Immunohistochemistry of aggregates on day 9 using antibodies against nestin and N-Cam.

FIG. 33A. Expression of pluripotent cells and neuroectodermal markers during programmed formation of EBM-derived neuroectoderm. (A) ES cell EB (IC + B, 4 days), DMEM
EB (I) of ES cells cultured from day 4 in +20 ng / ml FGF4
C + B / FGF, days 5-8), EBM (MEDII, day 4) and DMEM
10 μg of total RNA from EBM (MEDII / FGF, days 5-8) cultured from day 4 in + 50% MEDII + 20 ng / ml FGF4 was analyzed by Northern blot for expression of Oct4, Fgf5 and mGAP.

FIG. 33B. Expression of pluripotent cells and neuroectodermal markers during programmed formation of EBM-derived neuroectoderm. (B) EBM (Day 4) and DMEM + 50% MED
II + EBM cultured in 20 ng / ml FGF4 (days 6, 7, 8)
RNase protection for Gbx2 and Sox1 on 20 μg of total RNA from.

FIG. 34. Formation of differentiated cell types of mesodermal and ectodermal origin by directed differentiation of pluripotent cells in vitro. ES cell EB (control EB) and EBM
Individually seeded aggregates arising from (EBM) can be identified by (A) beating heart cells (
8, 10, the presence of neurons (derived from mesoderm) and (B) neurons (derived from ectoderm)
And 12 days.

FIG. 35. High molecular weight component of MEDII promotes neuron formation from EPL cells. 1 and 4, DMEM; 2 and 5, DMEM + anti-human LIF antibody; 3 and 6, DMEM + anti-human LIF antibody + 100 μg / ml E cultured in R
Individually seeded aggregates arising from PL cell EBs can be found in beating heart cells (1,
2,3) and the presence of neurons were evaluated on days 7, 8, 9 and 10.

FIG. 36. Formation of mesoderm by reaggregation of EPL cells formed in suspension. Trypsinize to single cells and DMEM (IC + B) +/− 10 ng / ml F
GF4 or DMEM + 50% MEDII (MEDII) +/− 10 ng / m
l 20 μg of total RNA from EBM reaggregated in FGF4 (day 4)
Was analyzed by Northern blot after 2 and 4 days for expression of brachyury and Oct4. Day 4 DMEM and DMEM + 50% M
A representative aggregate in EDII is shown.

FIG. 37. Embryonic mesoderm in the EB of EPL cells can be programmed for the formation of hematopoietic cells. EBs of ES and EPL cells were cultured in methylcellulose supplemented with IL-3 and M-CSF to induce macrophage formation. The proportion of aggregates containing macrophages was determined on days 12, 15 and 18.

FIG. 38. Comparative analysis of methodologies for directed formation of mesoderm (EPL EB) and ectoderm (EBM) germ layers from pluripotent cells. Seeded aggregates were scored at day 10 for beating heart cell formation and at day 12 for neuron formation.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/06 C12N 5/00 B 15/09 E 15/02 15/00 A C12Q 1/68 B (81 ) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Lasgen, Joy Australia 5051 South Australia, Blackwood, Mimosa Avenue 1 (72) Inventor Lake, Julie-Ann Australia 5083 South Australia, Broadview, Beven Avenue 1 (72) Inventor Washington, Jennifer Australia 5031 South Australia A, Sebalton, Valentine Street 54 Sea F-term (Reference) 4B024 AA01 AA20 BA21 DA02 GA03 GA18 HA03 HA17 4B063 QA05 QQ08 QQ15 QQ33 QQ79 QQ80 QS36 4B065 AA91X AA93X AB01 AB05 AB06 BA08 BB01 BB19 CA23 BC14ABC BA11 BA12 BA13 BA17 CA40 DA01 EA20 FA72 GA22 GA23 GA26 HA02 HA05

Claims (86)

[Claims] 1. A partially or substantially purified biologically active factor that can affect the differentiation, proliferation and / or maintenance of pluripotent cells in the presence or absence of an additional gp130 agonist. The factor is a low molecular weight component selected from the group consisting of proline and functionally equivalent analogs thereof, proline-containing peptides and functionally active fragments and analogs thereof; Molecules that compete with the low molecular weight component; and high molecular weight components selected from the group consisting of extracellular matrix proteins and functionally active fragments or analogs thereof; and molecules that compete with the high molecular weight component for biological activity. A partially or substantially purified biologically active agent, including. 2. The biologically active agent of claim 1, wherein said low molecular weight component has a molecular weight of less than about 5 kD, and wherein said high molecular weight component has a molecular weight of greater than about 10 kD. 3. The biologically active agent according to claim 1, wherein said low molecular weight component is selected from the group consisting of: Protease-digested (including collagenase-digested) collagen fragments; and their functionally active fragments and analogs; and molecules that compete with them for biological activity. 4. The biologically active component according to claim 1, wherein the high molecular weight component is fibronectin or a functionally active fragment or analog thereof, or laminin or a functionally active fragment or analog thereof. factor. 5. The biologically active agent according to claim 4, wherein said high molecular weight component is cellular fibronectin or a functionally active fragment or analog thereof. 6. A method for influencing the differentiation, proliferation and / or maintenance of a pluripotent cell, comprising a biologically active agent according to claim 1 and an adjuvant, diluent or carrier for said agent. Composition to obtain. 7. A method for preparing a conditioned medium comprising a biologically active agent according to claim 1, comprising the steps of: providing a hepatocyte or hepatoma cell, a hepatocyte cell line or A cell selected from the group consisting of a liver cancer cell line, an extraembryonic endoderm cell and an extraembryonic endoderm cell line, and a cell culture medium; culturing the cell in the cell culture medium for a time sufficient to produce a conditioned medium And separating the cells from the culture medium to provide the conditioned medium. 8. The cell or cell line, wherein the cell line is a human or mouse hepatocellular carcinoma cell line, a primary embryonic mouse hepatocyte, a primary adult mouse hepatocyte, a primary chicken hepatocyte,
8. The method of claim 7, wherein the method is selected from the group consisting of and extraembryonic endoderm cells or extraembryonic endoderm cell lines. 9. The cell or cell line is a human hepatocellular carcinoma cell line Hep G.
2 (ATCC HB-8065), mouse hepatocellular carcinoma cell line Hepa 1c1c
The method according to claim 8, wherein the method is selected from the group consisting of -7 (ATCC CRL-2026), visceral endoderm cell line END-2, and mural endoderm cell line PYS-2. 10. The cell or cell line is a human hepatocellular carcinoma cell line Hep.
The method according to claim 9, which is G2 (ATCC HB-8065). 11. A conditioned medium comprising the biologically active agent according to claim 1 and capable of affecting the differentiation, proliferation and / or maintenance of pluripotent cells. 12. A method for preparing an extracellular matrix comprising a biologically active agent according to claim 1, wherein the method comprises the steps of: providing a hepatocyte or hepatoma cell, a liver A cell line or a cell line selected from the group consisting of a liver cancer cell line, an extraembryonic endoderm cell and an extraembryonic endoderm cell line, a support substrate, and a cell culture medium; Culturing the cells in the presence of a support substrate for a sufficient time; and separating the cells and the cell culture medium from the support substrate to provide the extracellular matrix. ,Method. 13. The cell or cell line is a human or mouse hepatocellular carcinoma cell line, a primary embryonic mouse hepatocyte, a primary adult mouse hepatocyte, a primary chicken hepatocyte, and a human or mouse extraembryonic endoderm cell 13. The method of claim 12, wherein the method is selected from the group consisting of: and an extraembryonic endoderm cell line. 14. The cell or cell line is a human hepatocellular carcinoma cell line Hep G2 (ATCC HB-8065) or a mouse hepatocellular carcinoma cell line Hepa 1c1.
14. A selected from the group consisting of c-7 (ATCC CRL-2026).
The method described in. 15. The method of claim 14, wherein said cell or cell line is the human hepatocellular carcinoma cell line Hep G2 (ATCC HB-8065). 16. An extracellular matrix comprising the biologically active agent according to claim 1 and capable of affecting differentiation, proliferation and / or maintenance of pluripotent cells. 17. A method for partially or substantially purifying the high molecular weight component of a biologically active factor that can affect the differentiation, proliferation and / or maintenance of a pluripotent cell, said method comprising: The method comprises: providing a source of the high molecular weight component, a heparin affinity chromatography support, an anion exchange chromatography support, and a gel filtration chromatography support; a source of the high molecular weight component, and the heparin Contacting with an affinity chromatography support to produce a first fraction; contacting the first fraction with the anion exchange chromatography support;
Producing a second fraction; and contacting the second fraction with the gel filtration chromatography support to produce the partially or substantially purified high molecular weight component; A method comprising: 18. The partially purified biologically active agent of claim 1, the conditioned medium of claim 11, or the extracellular of claim 16, wherein the high molecular weight source is 18. The method according to claim 17, which is a matrix. 19. The method of claim 18, wherein the heparin affinity chromatography support is a heparin sepharose CL-6B column and the gel filtration support is a superose 6 gel filtration column. 20. A method for partially or substantially purifying a high molecular weight component of a biologically active factor that can affect the differentiation, proliferation and / or maintenance of a pluripotent cell, said method comprising: The method comprises providing a source of the high molecular weight component, an ultrafiltration membrane, an anion exchange chromatography support, a hydrophobic interaction chromatography support, a heparin affinity chromatography support, and gel filtration chromatography. Contacting the source of the high molecular weight component with the ultrafiltration membrane to obtain a fraction having a component greater than about 10 kD; a fraction having a component greater than about 10 kD and the anion Contacting with an exchange chromatography support to obtain a first fraction; said first fraction and said hydrophobic interaction chromatography Contacting the carrier with a support to generate a second fraction; contacting the second fraction with the heparin affinity chromatography support to generate a third fraction; Contacting the third fraction with the gel filtration chromatography support to produce the partially or substantially purified high molecular weight component. 21. The partially purified biologically active agent of claim 1, the conditioned medium of claim 11, or the extracellular of claim 16, wherein the high molecular weight source is 21. The method according to claim 20, which is a matrix. 22. The ultrafiltration membrane as defined in Amicon DiaFlo YM1.
0 membrane, wherein the anion exchange chromatography support is Seph.
arose Q anion exchange column, wherein the hydrophobic interaction chromatography support is a phenyl sepharose hydrophobic interaction column, and the heparin affinity chromatography support is heparin sepharose CL.
-6B column and the gel filtration chromatography support is
22. The method of claim 21 which is a rose 6 gel filtration column. 23. A method for partially or substantially purifying the high molecular weight component of a biologically active factor that can affect the differentiation, proliferation and / or maintenance of a pluripotent cell, said method comprising: The method comprises providing a source of the high molecular weight component, a gelatin affinity chromatography support, and a dialysis system; contacting the source of the high molecular weight component with the gelatin affinity chromatography support, Obtaining a first fraction; and subjecting the first fraction to dialysis to produce the partially or substantially purified high molecular weight component. 24. The partially purified biologically active agent of claim 1, the conditioned medium of claim 11, or the extracellular of claim 16, wherein the high molecular weight source is 24. The method of claim 23, wherein the method is a matrix. 25. The method of claim 24, wherein said gelatin affinity chromatography support is a gelatin sepharose affinity chromatography support. 26. A method for partially or substantially purifying low molecular weight components of a biologically active factor that can affect the differentiation, proliferation and / or maintenance of a pluripotent cell, said method comprising: The method comprises providing a source of the low molecular weight component, an ultrafiltration membrane, a first gel filtration chromatography support, a normal phase chromatography support, and a second gel filtration chromatography support; Contacting a source of low molecular weight components with the ultrafiltration membrane to obtain a fraction having a component of less than about 3 kD; a fraction having a component of less than about 3 kD; and the first gel filtration Contacting a chromatography support to obtain a first fraction; contacting the first fraction with the normal phase chromatography support to generate a second fraction; And the second fraction; Encompasses contacting the second gel filtration chromatographic supports, to produce the partially or substantially purified low molecular weight components, the method. 27. The method of claim 26, wherein the low molecular weight source is the partially purified biologically active factor of claim 1 or the conditioned medium of claim 11. Method. 28. The ultrafiltration membrane comprising Amicon DiaFlo YM3
A membrane, wherein the first gel filtration chromatography support is a Sepharose gel filtration chromatography support, and the second gel filtration chromatography support is a Superdex peptide gel filtration chromatography support, The method of claim 26. 29. A method for generating and / or maintaining early primitive ectoderm-like (EPL) cells, said method comprising: providing a pluripotent cell; and the biology of claim 1 Or a conditioned medium according to claim 11; or an extracellular matrix according to claim 16, and said pluripotent cells in the presence or absence of an additional gp130 agonist;
Contacting said biologically active agent or said conditioned medium or said extracellular matrix to generate or maintain said EPL cells. 30. The pluripotent cell may be an embryonic stem (ES) cell, an ICM / primordial ectoderm derived in vivo or in vitro, a primitive ectoderm derived in vivo or in vitro, a primordial germ cell, an EG cell, 30. The method of claim 29, wherein the method is selected from the group consisting of teratocarcinoma cells, EC cells, and pluripotent cells obtained by differentiation and / or nuclear transfer. 31. The method of claim 29, wherein said low molecular weight component is selected from the group consisting of: Protease-digested (including collagenase-digested) collagen fragments; and their functionally active fragments and analogs; and molecules that compete with them for biological activity. 32. The method of claim 29, wherein said high molecular weight component is fibronectin or a functionally active fragment or analog thereof, or laminin or a functionally equivalent fragment or analog thereof. 33. The method of claim 32, wherein said high molecular weight component is cellular fibronectin or a functionally equivalent fragment or analog thereof. 34. The method of claim 29, wherein said pluripotent cells are contacted with said biologically active agent, or said conditioned medium, or said extracellular matrix in the presence of an additional gp130 agonist. Method. 35. The biologically active agent, or the conditioned medium, or the extracellular matrix, wherein the human or mouse hepatocellular carcinoma cell line, primary embryonic mouse hepatocytes, primary adult mouse hepatocytes, primary 30. The method of claim 29, wherein the method is produced from a cell or cell line selected from the group consisting of chicken hepatocytes and extraembryonic endoderm cells or extraembryonic endoderm cell lines. 36. The cell or cell line is a human hepatocellular carcinoma cell line Hep G2 (ATCC HB-8065) or a mouse hepatocellular carcinoma cell line Hepa 1c1.
36. The method of claim 35, wherein the method is selected from the group consisting of c-7 (ATCC CRL-2026), visceral endoderm cell line END-2, and mural endoderm cell line PYS-2. 37. The method of claim 36, wherein said cell or cell line is the human hepatocellular carcinoma cell line Hep G2 (ATCC HB-8065). 38. The method of claim 29, comprising identifying EPL cells, preferably by expression of Oct4 and Fgf5. 39. The EPL cell is a mammal or a bird.
9. The method according to 9. 40. A cultured EPL cell. 41. The cell of claim 40, which is a mammal or a bird. 42. A method for producing a partially differentiated cell and / or a terminally differentiated cell, said method comprising the steps of: providing a pluripotent cell, and the biology of claim 1 Or a conditioned medium according to claim 11, or an extracellular matrix according to claim 16, and said pluripotent cells in the presence or absence of an additional gp130 agonist;
Contacting said biologically active factor or said conditioned medium or said extracellular matrix to produce said EPL cells; and said biologically active factor or a high or low molecular weight component thereof, Or in the presence or absence of the conditioned medium, or the extracellular matrix, and 1
Culturing said EPL cells in the presence or absence of one or more differentiation factors to produce partially differentiated cells and / or terminally differentiated cells. 43. The pluripotent cells are embryonic stem (ES) cells, in vivo or in vitro induced ICM / primordial ectoderm, in vivo or in vitro derived primitive ectoderm, primordial germ cells, EG cells, 43. The method of claim 42, wherein the method is selected from the group consisting of teratocarcinoma cells, EC cells, and pluripotent cells obtained by differentiation and / or nuclear transfer. 44. The method of claim 42, wherein said low molecular weight component is selected from the group consisting of: Protease digested (including collagenase digested) collagen fragments; and their functionally active fragments and analogs; and molecules that compete with them for biological activity. 45. The method of claim 42, wherein said high molecular weight component is fibronectin or a functionally active fragment or analog thereof, or laminin or a functionally active fragment or analog thereof. 46. The method of claim 45, wherein said high molecular weight component is cellular fibronectin or a functionally active fragment or analog thereof. 47. The method of claim 42, further comprising identifying partially differentiated and / or terminally differentiated cells by expression, morphology, and / or differentiation potential of cell surface markers. 48. The method of claim 42, wherein said partially and / or terminally differentiated cells are mammals or birds. 49. The EPL cell is preferably derived from the FGF family in the presence of the biologically active factor, or a high or low molecular weight component thereof, or the conditioned medium, or the extracellular matrix. And wherein said partially differentiated cells and / or terminally differentiated cells are predominantly ectoderm cells, predominantly partially differentiated ectoderm, dominant 43. The method of claim 42, wherein the method comprises neuroectoderm or predominantly neural stem cells and / or predominantly terminally differentiated skin or neuronal cells. 50. The EPL cells are cultured in suspension in the absence of the biologically active factor, or a high or low molecular weight component thereof, or the conditioned medium, or the extracellular matrix. And the partially differentiated and / or terminally differentiated cells are predominantly mesoderm cells, predominantly partially differentiated cells and / or predominantly terminally differentiated developing mesoderm cells (eg, blood cells 43) and a muscle cell). 51. The method according to claim 51, wherein the EPL cells formed in suspension are free of the biologically active agent or the high or low molecular weight component thereof, or the conditioned medium, or the extracellular matrix. In the presence, preferably in the presence of a growth factor from the FGF family, the cells dissociate and reaggregate, and the cells and partially and / or terminally differentiated cells are predominantly mesoderm germ layers 43. The method of claim 42, comprising cells, predominantly partially differentiated cells and / or predominantly terminally differentiated nascent mesoderm cells (e.g., blood cells and muscle cells). 52. The EPL cell is derived from the FGF family in the absence of the biologically active factor or the high or low molecular weight component thereof, or the conditioned medium, or the extracellular matrix. Growth factors and TGF
43. The method of claim 42, wherein the cells are cultured in adherent culture in the presence of a growth factor from the beta family and the cells are differentiated. 53. The method of claim 50 or 51, wherein the pluripotent cells are differentiated in the absence of visceral endoderm. 54. The differentiation of a pluripotent cell according to claim 49, wherein predominantly ectodermal cells, predominantly neuroectodermal cells, and predominantly partially differentiated cells and / or terminally differentiated neuroectodermal cells How to generate cells. 55. The differentiation of a pluripotent cell according to claim 50 or 51, wherein predominantly early or developing mesoderm cells and predominantly partially and / or terminally differentiated mesoderm cells are obtained. How to generate. 56. Generated by the method of claim 42 or 54,
Neural ectoderm cells, partially differentiated neuroectoderm cells or neural stem cells or terminally differentiated neural cells. 57. A mesoderm cell and a partially differentiated or terminally differentiated mesoderm cell (eg, a muscle cell or a mesoderm cell) produced by the method of claim 42, 50, 51 or 55. blood cell). 58. Produced by the method of claim 42 or 54,
Ectodermal germ cells, partially differentiated ectodermal cells, or terminally differentiated ectodermal cells (eg, skin cells). 59. A method for producing embryonic stem (ES) cells, the method comprising: providing a pluripotent cell; a biologically active agent according to claim 1; or The conditioned medium according to claim 11, or the extracellular matrix according to claim 16, and a gp130 agonist; the pluripotent cell and the biologically active agent, or the conditioned medium, or the extracellular matrix. Contacting in the presence or absence of an additional gp130 agonist to generate EPL cells; and combining the EPL cell with the gp130 agonist with the biologically active agent, or the high molecular weight thereof. Contact in the absence of a component or low molecular weight component, or the conditioned medium, or a component of the extracellular matrix, to allow the EPL cells to revert to ES cells Degree, including,, way. 60. The pluripotent cells are embryonic stem (ES) cells, in vivo or in vitro induced ICM / primordial ectoderm, in vivo or in vitro induced primitive ectoderm, primordial germ cells, EG cells, 60. The method of claim 59, wherein the method is selected from the group consisting of teratocarcinoma cells, EC cells, and pluripotent cells obtained by differentiation and / or nuclear transfer. 61. The method of claim 59, wherein said low molecular weight component is selected from the group consisting of: Protease-digested (including collagenase-digested) collagen fragments; and their functionally active fragments and analogs; and molecules that compete with them for biological activity. 62. The method of claim 59, wherein said high molecular weight component is fibronectin or a functionally active fragment or analog thereof, or laminin or a functionally active fragment or analog thereof. 63. The method of claim 62, wherein said high molecular weight component is cellular fibronectin or a functionally active fragment or analog thereof. 64. The method of claim 59, wherein said ES cells are mammals or birds. 65. The method of claim 59, further comprising differentiating said ES cells to produce partially or terminally differentiated cells. 66. An ES cell produced by the method of claim 59. 67. The cell of claim 66, which is a mammal or a bird. 68. A method for generating a genetically modified ES cell, comprising: providing a pluripotent cell; a biologically active agent according to claim 1; Or the conditioned medium of claim 11, or the extracellular matrix of claim 16, and a gp130 agonist; the pluripotent cells and the biologically active agent, or the conditioned medium, or the extracellular. Contacting a matrix with or without an additional gp130 agonist to produce EPL cells; modifying one or more genes in said EPL cells; and said genetically modified EPL cells And the gp130 agonist, the biologically active factor, or the high or low molecular weight component thereof, or the conditioned medium, or the extracellular matrix. Contacting the genetically modified EPL cells back into ES cells in the absence of any of the components of the cells. 69. A method for producing a genetically modified EPL cell, comprising:
The method provides: a pluripotent cell, a biologically active agent according to claim 1, or a conditioned medium according to claim 11, or an extracellular matrix according to claim 16. Modifying one or more genes in the pluripotent cell; and the genetically modified pluripotent cell and the biologically active agent, or the conditioned medium, or the gp130 agonist. Contacting an extracellular matrix in the presence or absence of an additional gp130 agonist to produce said genetically modified EPL cells. 70. The pluripotent cell is an embryonic stem (ES) cell, the ES cell of claim 59, an in vivo or in vitro induced ICM / protoderm, an in vivo or in vitro induced extraprimordial cell. 70. The method of claim 69, wherein the method is selected from the group consisting of germ layers, primordial germ cells, EG cells, teratocarcinoma cells, EC cells, and pluripotent cells obtained by differentiation or nuclear transfer. 71. A method for producing a genetically modified EPL cell, comprising the steps of producing the EPL cell according to claim 29, and genetically modifying the EPL cell. 72. A method for producing a genetically modified, partially differentiated or terminally differentiated cell, said method comprising the steps of: providing a pluripotent cell. A conditioned medium according to claim 11, or an extracellular matrix according to claim 16, and a gp130 agonist; the pluripotent cell and the biologically active agent. Or contacting the conditioned medium, or the extracellular matrix, in the presence or absence of an additional gp130 agonist, to produce EPL cells; modifying one or more genes in the EPL cells; And differentiating the EPL cells to produce a genetically modified, partially differentiated or terminally differentiated cell. 73. Generating a genetically modified, partially differentiated or terminally differentiated cell comprising preparing the partially or terminally differentiated cell of claim 42. how to. 74. A genetically modified ES cell produced by the method of claim 68. 75. Produced by the method of claim 69 or 70.
Genetically modified EPL cells. 76. Produced by the method of claim 71 or 72,
Genetically modified, partially or terminally differentiated cells. 77. A method for producing a chimeric animal, the method comprising: providing an ES cell according to claim 62 or a genetically modified ES cell according to claim 73; A protogastrogenic embryo; introducing the ES cells or genetically modified ES cells into the protogastrulating embryo; and monitoring the ability to form chimeras. 78. A chimeric or transgenic animal produced by the method of claim 77. 79. The unmodified EPL of claim 40 or 41.
77. The method of using the cells, or their differentiated progeny of claim 76, for use in human cell therapy or transgenic animal production. 80. The use of the genetically modified EPL cells of claim 75, or their differentiated progeny of claim 76, for use in human gene therapy or transgenic animal production. Method. 81. A method for preparing a nuclear transfer cell, the method comprising: providing EPL cells, or partially or fully differentiated cells derived from EPL, and enucleated. Transplanting EPL cells or partially or fully differentiated cells derived from EPL cells, or nuclei derived from these cells, into enucleated recipients; Fusing and activating to form a nuclear transfer cell. 82. The method of claim 81, wherein said enucleated recipient cell is an oocyte, single cell embryo or other pluripotent cell. 83. A method for inducing a nuclear transfer cell, the method comprising: providing a cell, an enucleated recipient EPL cell or an enucleated cell derived from an EPL cell; Transplanting the enucleated EPL cells or enucleated cells derived from the EPL cells; fusing and activating the combined cells to form nuclear transfer cells. 84. A nuclear transfer cell derived according to the method of claims 81 and 83. 85. Mammals and birds derived from the nuclear transfer cells of claim 84. 86. A pluripotent cell derived from the nuclear transfer cell according to claim 84.