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WO2003078611A1 - Cellules nourricieres a fibroblastes embryonnaires humains - Google Patents

Cellules nourricieres a fibroblastes embryonnaires humains Download PDF

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Publication number
WO2003078611A1
WO2003078611A1 PCT/GB2003/001092 GB0301092W WO03078611A1 WO 2003078611 A1 WO2003078611 A1 WO 2003078611A1 GB 0301092 W GB0301092 W GB 0301092W WO 03078611 A1 WO03078611 A1 WO 03078611A1
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WO
WIPO (PCT)
Prior art keywords
cells
cell
stem cells
human
embryo
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Application number
PCT/GB2003/001092
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English (en)
Inventor
Peter Andrews
Harry Moore
Jon Draper
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University Of Sheffield
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Sheffield filed Critical University Of Sheffield
Priority to AU2003229867A priority Critical patent/AU2003229867A1/en
Publication of WO2003078611A1 publication Critical patent/WO2003078611A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • the invention relates to the isolation of a population of cells comprising human embryonic fibroblasts and their use in culture to maintain embryonic stem cells in a pluripotential state.
  • each cell has the developmental potential to form a complete embryo and all the cells required to support the growth and development of said embryo.
  • the cells that comprise the inner cell mass are said to be pluripotential (e.g. each cell has the developmental potential to form a variety of tissues).
  • Embryonic stem cells may be principally derived from two embryonic sources. Cells isolated from the inner cell mass are termed embryonic stem (ES) cells. In the laboratory mouse, similar cells can be derived from the culture of primordial germ cells isolated from the mesenteries or genital ridges of days 8.5-12.5 post coitum embryos. These would ultimately differentiate into germ cells and are referred to as embryonic germ cells (EG cells). Each of these types of pluripotential cell has a similar developmental potential with respect to differentiation into alternate cell types, but possible differences in behaviour (eg with respect to imprinting) have led to these cells to be distinguished from one another.
  • WO96/22362 An indication that conditions may be determined which could allow the establishment of human ES/EG cells in culture is described in WO96/22362, which is incorporated by reference.
  • the application describes cell lines and growth conditions which allow the continuous proliferation of primate ES cells which" exhibit a range of characteristics or markers which are associated with stem cells having pluripotent characteristics.
  • hES human embryonic stem-cells
  • they must be cultured in the presence of mouse fetal fibroblast cells, typically which have been pre-treated to inhibit cell division.
  • Co-incubation of hES cells with those of a different animal species is undesirable as it leads to the potential exposure and incorporation of infectious agents and genetic material from animal cells (e.g. from mouse to human cells).
  • hES cells should be maintained in defined medium without other cell types or the products of other cell types but currently this is not possible. Maintaining hES cells in the presence of a human feeder cell or the products of the human feeder cells is also acceptable as exposure with animal cells is avoided.
  • hES cells maintained in culture have a number of characteristic markers which identify them as pluripotential. These include, but are not limited to; maintenance in culture for at least 20 passages when maintained on fibroblast feeder layers; production of clusters of cells referred to as embryoid bodies; when cultured in suspension, an ability to differentiate into multiple cell types in monolayer culture; the formation of xenograft teratomas with multiple differentiated cell types when injected into immunodeficient mice, and the expression of ES/EG cell specific markers, notably SSEA3, SSEA4, TRA-1-60, TRA-1-81, alkaline phosphatase, and Oct4 (Andrews et ah, 2001; Draper et al, 2002 and Henderson et al. 2002).
  • characteristic markers include, but are not limited to; maintenance in culture for at least 20 passages when maintained on fibroblast feeder layers; production of clusters of cells referred to as embryoid bodies; when cultured in suspension, an ability to differentiate into multiple cell types in monolayer culture; the formation
  • a further group of cells which have relevance to developmental biology are embryonal carcinoma (EC) cells, the stem cells of terato carcinoma cells. These cells form tumours referred to as teratomas and have many features in common with hES cells. The most important of these features is the characteristic of pluripotentiality.
  • EC embryonal carcinoma
  • Teratomas contain a wide range of differentiated tissues, and have been known in humans for many hundreds of years. They typically occur as gonadal tumours of both men and women. The gonadal forms of these tumours are generally believed to originate from germ cells, and the extra gonadal forms, which typically have the same range of tissues, are thought to arise from germ cells that have migrated incorrectly during embryogenesis. Teratomas are therefore generally classed as germ cell tumours which encompasses a number of different types of cancer. These include seminoma, embryonal carcinoma, yolk sac carcinoma and choriocarcinoma.
  • a method for the derivation and culture of a human fetal cell line with the capacity to maintain primate ES cells, in particular, hES cells, or EC cells in a proliferative and pluripotent state as defined by cell morphology and specific marker antibodies is therefore very desirable.
  • a method to prepare a cell sample comprising proliferating human embryonic fibroblasts comprising the steps of: i) preparing a cell suspension from a dis-aggregated human embryo, or part thereof; ii) culturing the cells obtained in (i) under growth conditions to establish a proliferating population of human fibroblast cells; and optionally iii) cloning a population of said human fibroblasts.
  • said embryo is between about 4 weeks to 10 weeks old, preferably about a 4-6 week old embryo. More preferably a 5 week old embryo.
  • said part thereof is the rib-region of said embryo.
  • said fibroblasts are cloned and substantially free of other contaminating human embryonic cells.
  • said dis-aggregation is facilitated by addition of a protease, preferably a collagenase.
  • a protease preferably a collagenase.
  • embryonic fibroblasts are pre-cultured prior to cloning, preferably for at least 1 week before cloning.
  • said dis-aggregated embryos are resuspended in a medium comprising 10-20% fetal bovine serum, 0.05- 0.2 mM non- essential amino acids, 0.05-0.2mM mercaptoethanol, l-3mM glutamine, 0.5-2.0mM pyruvate, 50-100 units/ml penicillin, 50-100 ug/ml streptomycin, 750-1250 units/ml Leukaemia inhibiting factor, 0.5-2 ng/ml basic fibroblast growth factor (bFGF) and 5-15 uM forskolin.
  • a medium comprising 10-20% fetal bovine serum, 0.05- 0.2 mM non- essential amino acids, 0.05-0.2mM mercaptoethanol, l-3mM glutamine, 0.5-2.0mM pyruvate, 50-100 units/ml penicillin, 50-100 ug/ml streptomycin, 750-1250 units/ml Leukaemia inhibiting factor, 0.5-2 ng/
  • said dis-aggregated embryos are resuspended in a medium comprising about 15% fetal bovine serum, about 0.1 mM non-essential amino acids, about 0.1 mM mercaptoethanol, about 2 mM glutamine, about ImM pyruvate, about 100 units/ml penicillin, about 100 ug/ml streptomycin, about 1000 units/ml Leukaemia inhibiting factor, about 1 ng/ml basic fibroblast growth factor (bFGF) and about 10 uM forskolin.
  • a medium comprising about 15% fetal bovine serum, about 0.1 mM non-essential amino acids, about 0.1 mM mercaptoethanol, about 2 mM glutamine, about ImM pyruvate, about 100 units/ml penicillin, about 100 ug/ml streptomycin, about 1000 units/ml Leukaemia inhibiting factor, about 1 ng/ml basic fibroblast growth factor (bFGF) and about 10 u
  • a cell culture comprising human embryonic fibroblasts prepared by the method according to the invention.
  • a cell culture composition comprising conditioned cell culture medium prepared by the method according to the invention.
  • a method to maintain embryonic stem cells comprising the steps of:
  • said cell sample is treated to substantially inhibit cell division of cells comprised in said sample, preferably fibroblasts.
  • said cells are treated with mitomycin, typically at a concentration of 5 ⁇ g - 15 ⁇ g per ml. More preferably said mitomycin is 1 O ⁇ g/ml.
  • said cell sample is treated by ⁇ - irradiation, preferably at least 3000 rads, to inhibit cell division, preferably fibroblast cell division.
  • said embryonic stem cells are primate cells, ideally human cells.
  • said embryonic stem cells are rodent, ideally murine cells.
  • stem cells are embryonal teratocarcinoma cells.
  • a cell culture vessel comprising an embryonic fibroblast cell culture according to the invention.
  • said vessel also comprises embryonic stem cells.
  • Table 1 represents a selection of antibodies used to monitor stem cell differentiation
  • Table 2 represents protein markers of stem cell differentiation
  • Figure 1 represents a micrograph of human embryonic fibroblasts as herein disclosed.
  • Figure 2 represents a graph of comparative surface antigen expression on hES cells propagated on HEF1 vs hES propagated embryonic fibroblasts.
  • Dulbecco's modified eagles medium (DMEM) at 5°C and transported to the laboratory. Tissue from the rib area was removed and incubated in 1 mg/ml collagenase in DMEM for 1-2 hours with agitation. A cell suspension was made by aspiration. The cells were washed x3 with medium and finally resuspended in
  • DMEM medium containing 15% fetal bovine serum, 0.1 mM non-essential amino acids, 0.1 mM mercaptoethanol, 2 mM glutamine, lmM pyruvate, 100 units/ml penicillin, 100 ug/ml streptomycin, 1000 units/ml Leukaemia inhibiting factor, 1 ng/ml basic fibroblast growth factor (bFGF) and 10 uM forskolin.
  • bFGF basic fibroblast growth factor
  • Fibroblast-like cells were allowed to proliferate for 1 week before being passaged at low density 2 times. These cells were capable of being cryopreserved in liquid nitrogen using standard protocols (10% DMSO in 90%) Fetal calf serum, slow cool to -80°C overnight, plunge in liquid nitrogen). A cell line produced in this way was generated and given the designation HEF1 (human embryonic feeder 1). Maintenance of hES cells
  • HEFl cells To assess the capacity of HEFl cells to maintain hES cells in a proliferative and pluripotent state, the former were cultured to confluency, treated with mitomyocin C (standard protocol) to inhibit cell proliferation, and replated into culture wells and flasks at a density of 3.6 x 10 4 per cm 2 .
  • hES cells were recovered from mouse feeder cells and replated either on new mouse feeder cells or HEFl cells and cultured for 7 days. The status of hES cells was assessed after this time by fluorescent activated cell sorting (FACS) by standard protocol (Fenderson et al, 1987 and Andrews et al, 1987) and by morphological observation.
  • FACS fluorescent activated cell sorting
  • Samples were prepared for SDS-PAGE by adding 4 times Laemmli electrophoresis sample buffer and boiling for 5 min. After electrophoresis with 16 ⁇ g of protein on a 10% polyacrylamide gel (Laemmli, 1970) the proteins were transferred to nitro-cellulose membrane with a pore size of 0.45 ⁇ m. The blots were washed with PBS and 0.05%) Tween (PBS-T). Blocking of the blots occurred in 5% milk powder in PBS-T (60 min, at RT). Blots were incubated with the appropriate primary antibody. Horseradish peroxidase labelled secondary antibody was used to visualise antibody binding by ECL (Amersham, Bucks., UK). Materials used for SDS-PAGE and western blotting were obtained from Biorad (California, USA) unless stated otherwise.
  • Table 1 Antibodies used to detect stem cell differentiation
  • Table 2 Protein markers of differentiation, detected by Western Blot and/or immunofluorescence

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention se rapporte à l'isolation d'une population de cellules comprenant des fibroblastes embryonnaires humains et à leur utilisation dans une culture pour maintenir des cellules souches embryonnaires dans un état pluripotentiel.
PCT/GB2003/001092 2002-03-16 2003-03-14 Cellules nourricieres a fibroblastes embryonnaires humains WO2003078611A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003229867A AU2003229867A1 (en) 2002-03-16 2003-03-14 Human embryonic fibroblast feeder cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0206309.7 2002-03-16
GB0206309A GB0206309D0 (en) 2002-03-16 2002-03-16 Isolated cells

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WO2003078611A1 true WO2003078611A1 (fr) 2003-09-25

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2441530B (en) * 2004-02-12 2009-09-23 Univ Newcastle Stem Cells
US8597947B2 (en) 2004-12-29 2013-12-03 Hadasit Medical Research Services & Development Limited Undifferentiated stem cell culture systems
US9005965B2 (en) * 2004-12-29 2015-04-14 Hadasit Medical Research Services & Development Limited Stem cells culture systems

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990008771A1 (fr) * 1989-01-31 1990-08-09 Rubin Jeffrey S Adn codant un facteur de croissance specifique contre des cellules epitheliales
WO1997030147A1 (fr) * 1996-02-16 1997-08-21 Lg Chemical Ltd. Souche de cellules diploides de fibroblastes pulmonaires embryonnaires humaine destinee a la production de virus et procede de production de virus zona-varicelle utilisant cette souche
WO2001051616A2 (fr) * 2000-01-11 2001-07-19 Geron Corporation Techniques pour la croissance et la differenciation de cellules souches pluripotentielles humaines
WO2003029443A1 (fr) * 2001-09-28 2003-04-10 Es Cell International Pte Ltd Procedes de derivation et de propagation de cellules souches embryonnaires humaines (hes) non differenciees sur des matrices sans cellules nourricieres et sur des couches nourricieres humaines

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990008771A1 (fr) * 1989-01-31 1990-08-09 Rubin Jeffrey S Adn codant un facteur de croissance specifique contre des cellules epitheliales
WO1997030147A1 (fr) * 1996-02-16 1997-08-21 Lg Chemical Ltd. Souche de cellules diploides de fibroblastes pulmonaires embryonnaires humaine destinee a la production de virus et procede de production de virus zona-varicelle utilisant cette souche
WO2001051616A2 (fr) * 2000-01-11 2001-07-19 Geron Corporation Techniques pour la croissance et la differenciation de cellules souches pluripotentielles humaines
US20020019046A1 (en) * 2000-01-11 2002-02-14 Carpenter Melissa K. Direct differentiation of human pluripotent stem cells and characterization of differentiated cells
US20020022268A1 (en) * 2000-01-11 2002-02-21 Chunhui Xu Conditioned media for propagating human pluripotent stem cells
WO2003029443A1 (fr) * 2001-09-28 2003-04-10 Es Cell International Pte Ltd Procedes de derivation et de propagation de cellules souches embryonnaires humaines (hes) non differenciees sur des matrices sans cellules nourricieres et sur des couches nourricieres humaines

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FRANSSON L-A ET AL: "EFFECTS OF CYCLOHEXIMIDE, BREFELDIN A, SURAMIN, HEPARIN AND PRIMAQUINE ON PROTEOGLYCAN AND GLYCOSAMINOGLYCAN BIOSYNTHESIS IN HUMAN EMBRYONIC SKIN FIBROBLASTS", BIOCHIMICA ET BIOPHYSICA ACTA, AMSTERDAM, NL, vol. 1137, no. 3, 17 November 1992 (1992-11-17), pages 287 - 297, XP000573708, ISSN: 0006-3002 *
RUBIN JS ET AL: "Purification and characterization of a newly identified growth factor specific for epithelial cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 86, February 1989 (1989-02-01), pages 802 - 806, XP002138002, ISSN: 0027-8424 *
YONEDA T ET AL: "MESENCHYMAL CELLS FROM THE HUMAN EMBRYONIC PALATE ARE HIGHLY RESPONSIVE TO EPIDERMAL GROWTH FACTOR", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 213, no. 4503, 31 July 1981 (1981-07-31), pages 563 - 566, XP000198522, ISSN: 0036-8075 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2441530B (en) * 2004-02-12 2009-09-23 Univ Newcastle Stem Cells
US8597947B2 (en) 2004-12-29 2013-12-03 Hadasit Medical Research Services & Development Limited Undifferentiated stem cell culture systems
US9005965B2 (en) * 2004-12-29 2015-04-14 Hadasit Medical Research Services & Development Limited Stem cells culture systems
US8940537B2 (en) 2007-04-02 2015-01-27 Hadasit Medical Research Services & Development Limited Undifferentiated stem cell culture systems

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AU2003229867A1 (en) 2003-09-29
GB0206309D0 (en) 2002-05-01

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