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WO2008115087A1 - Procédé de production de cellules souches embryonnaires - Google Patents

Procédé de production de cellules souches embryonnaires Download PDF

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Publication number
WO2008115087A1
WO2008115087A1 PCT/RS2007/000009 RS2007000009W WO2008115087A1 WO 2008115087 A1 WO2008115087 A1 WO 2008115087A1 RS 2007000009 W RS2007000009 W RS 2007000009W WO 2008115087 A1 WO2008115087 A1 WO 2008115087A1
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WIPO (PCT)
Prior art keywords
stem cell
embryonic stem
arrested
embryo
cells
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PCT/RS2007/000009
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English (en)
Inventor
Miodrag Stojkovic
Original Assignee
Miodrag Stojkovic
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Priority to PCT/RS2007/000009 priority Critical patent/WO2008115087A1/fr
Publication of WO2008115087A1 publication Critical patent/WO2008115087A1/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
    • 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)

Definitions

  • the present invention relates to a process for providing embryonic stem cells derived from an arrested embryo and in particular a process for providing human embryonic stem cells. Furthermore, there is provided embryonic stem cells and cell lines provided by the process of the invention, and methods of culturing said cells and cell lines.
  • hESC Human embryonic stem cells
  • This invention makes use of the novel and surprising observation that arrested embryos, which never reach the morula or blastocyst stages under described growth conditions, can be used for derivation of pluripotent embryonic stem cell line.
  • a first aspect of the present invention provides an in vitro method of deriving at least one embryonic stem cell (ESC), capable of expressing markers of pluripotency and / or of differentiation, comprising the step of propagating an embryonic stem cell derived from an arrested embryo.
  • ESC embryonic stem cell
  • an embryo is either a fertilized or reconstructed (after nuclear transfer) and cleaved oocyte. Cleavage of developing embryos occurs approximately every 24 hours. Arrested embryos do not cleave any more.
  • the present invention provides the use of an arrested embryo to provide an embryonic stem cell.
  • the method of the invention includes the step of deriving at least one viable blastomere from an arrested embryo.
  • An embryonic stem cell may then be propagated from said at least one viable blastomere or whole embryo.
  • the present inventor has surprisingly demonstrated that arrested embryos, which never reach the morula or blastocyst stages and are generally regarded as "dead” (Landry DW, Zucker HA. Embryonic death and the creation of human embryonic stem cells. J Clin Invest 2004; 114: 1184-1186), nonetheless still have proliferative potential and may be used for the derivation of ESC, in particular hESC under suitable in vitro conditions.
  • Said arrested embryos can be defined as embryos having at least one of the following:
  • arrested embryos also labelled as dead embryos
  • the present inventors have determined that not all blastomeres within the arrested embryo are abnormal nor are all blastomeres responsible for developmental arrest.
  • Embryonic stem cells more particularly human embryonic stem cells (hESC) derived from arrested embryos have been determined by the inventors to possess a normal karyotype. The inventor considers this finding indicates that arrested embryos, although possessing some blastomeres with chromosomal or other abnormalities might preserve a self-normalization mechanism(s) and protect the subsequently derived ESC, for example hESC, from these abnormalities.
  • hESC human embryonic stem cells
  • the method of the present invention of using an arrested embryo to provide an embryonic stem cell offers an attractive option to further refine strategies for research and derivation of ESC and in particular hESC, for example, but not limited to: using vital staining to identify and isolate viable blastomeres; to set up conditions for derivation of clonal lines from the same embryo (and to find out how clonal they are); to study and compare normal and abnormal early human development; to study genetic/epigenetic profiles of embryos and derived hESC; and to derive new hESC lines of clinical grade for cell replacement therapies or drug discoveries.
  • the method of the present invention comprises the steps of: removing the zona pellucida of an arrested embryo, plating out the arrested embryo on inactivated feeder cells and / or on extracellular matrix components (ECM), growing the plated embryo in embryonic stem cell (ESC) medium to form primary outgrowth, dispersion of the primary outgrowth into dispersed cells, and replating of dispersed cells.
  • ECM extracellular matrix components
  • the zona pellucida may be removed using any suitable means as known in the art, for example, but not limited to, Tyrode's solution or pronase.
  • the method includes the step of performing a biopsy (micromanipulation) of the arrested embryo.
  • the outgrowth may be replated on fresh feeder cells and / or ECM using non-conditioned embryonic stem cell medium.
  • dispersion of the primary outgrowth can be into several clumps, wherein a clump may include at least 50 ESCs.
  • the step of dispersing the primary outgrowth may be performed by mechanical means (for example using a needle or flushing) or by chemical means (for example using collagenase) or by a combination of mechanical and / or chemical means.
  • the arrested embryo can be an arrested mammalian embryo.
  • the arrested embryo is an arrested human embryo.
  • the arrested embryo may be an early, 2 to 10 cells, embryo or a late, 16 to 24 cells, embryo.
  • a late arrested embryo having 16 to 24 cells is used in the method of the invention.
  • a late arrested embryo is considered to be advantageous as it is likely to contain more viable blastomeres.
  • the inventor considers that the likelihood of deriving an embryonic stem cell is increased, the greater the number of viable blastomeres which are present in the arrested embryo.
  • the arrested embryo for use in a method of the present invention may be obtained by nuclear transfer (NT).
  • therapeutic cloning may allow for treatment of neurological diseases, for example Alzheimer's, or Parkinson's or metabolic diseases, for example diabetes.
  • neurological diseases for example Alzheimer's, or Parkinson's or metabolic diseases, for example diabetes.
  • such cells may be useful as in vitro human disease models or, for example, to study oocyte-derived mitochondrial proteins or mitochondrial DNA.
  • an arrested embryo used in the method can express the intracellular markers OCT4, NANOG and REX 1.
  • an embryonic stem cell produced by the method can express at least one pluripotency marker selected from; cell surface markers TRA1-60, TRA1- 81, or SSEA4 and intracellular markers OCT4, NANOG, REX 1, or hTERT.
  • pluripotency marker selected from; cell surface markers TRA1-60, TRA1- 81, or SSEA4 and intracellular markers OCT4, NANOG, REX 1, or hTERT.
  • the feeder cells of the method can include inactivated mouse embryonic (MEF) feeder cells, human fetal lung (HFL) feeder cells, foreskin feeder cells, fetal muscle feeder cells or the like.
  • MEF mouse embryonic
  • HFL human fetal lung
  • foreskin feeder cells fetal muscle feeder cells or the like.
  • feeder-free conditions using ECM components may be provided.
  • the feeder cells can be inactivated mouse feeder cells or human feeder cells.
  • cells can be inactivated by gamma irradiation, X ray, or using chemical inactivation (mitomycin, 10 micrograms/ml).
  • the ESC medium of the method may be any suitable medium as known in the art.
  • Said medium should preferably be conditioned by fibroblast cells , embryonic stem cells or the like or a combination of fibroblast cells and embryonic stem cells to allow an arrested embryo to attach and proliferate.
  • an extracellular matrix (ECM) derived from feeder cells or ECM components for example, collagen, vitronectin, laminin, or fibronectin, provides one or more substances necessary to promote the growth of ES cells and / or prevents or inhibits the rate of differentiation of such cells.
  • the ESC medium is precultured.
  • Preconditioning of the growth medium is performed for 48 hours on feeder cells, by ESC grown on feeder cells, more particularly on hESC grown on feeder cells, or on ECM.
  • feeder cells and ECM may be used.
  • the cell culture media includes a growth medium effective to support the growth of ES cells, a nutrient serum effective to support growth of ES cells, non-essential and essential amino acids and a reducing agent. Additionally a growth factor may be provided to assist in the maintenance and / or derivation of cultures of ES cells. Moreover, the media may contain a ligand(s) capable of binding receptors present on the ES cells or other suitable agent(s) which may modulate, for example activate, signal transduction pathways in the ES cell. The identities of effective concentrations of such components may be determined by those of skill in the art in culturing cells.
  • the ESC Medium comprises knockout- Dulbecco's Modified Eagle's Medium (DMEM), 100 ⁇ M 2-mercaptoethanol, 1 mM L- glutamine, 100 mM non-essential amino acids, 10% serum replacement, 1% penicillin-streptomycin and 4 ng/mL to 80 ng/mL basic fibroblast growth factor (bFGF).
  • DMEM knockout- Dulbecco's Modified Eagle's Medium
  • 2-mercaptoethanol 100 ⁇ M 2-mercaptoethanol
  • 1 mM L- glutamine 100 mM non-essential amino acids
  • 10% serum replacement 1% penicillin-streptomycin
  • 4 ng/mL to 80 ng/mL basic fibroblast growth factor (bFGF) basic fibroblast growth factor
  • said growth medium is ESC medium containing knockout Dulbecco's Modified Eagle's Medium (DMEM), 100 ⁇ M beta-mercaptoethanol, 1 imM L-glutamine, 100 mM non-essential amino acids, 10% serum replacement, 1 % penicillin-streptomycin and 4 ng/ml bFGF which was previously conditioned for 48 hours on hESC grown on feeder cells.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the conditioned medium before use of the conditioned medium, the conditioned medium is spun for 5 minutes at 1 ,000 rpm and subsequently filter sterilized (0.22 ⁇ m), to remove any cellular material.
  • conditioned medium is kept at 4°C until needed.
  • the growth medium may be changed every second day or as required.
  • the embryonic stem cells may be cultured on suitable feeder cells or on ECM in the presence of non-conditioned or conditioned ESC medium.
  • the method further comprises the step of culturing the replated dispersed cells on suitable feeder cells, for example fibroblast cells and / or embryonic stem cells, or ECM or ECM components to prevent their differentiation.
  • suitable feeder cells for example fibroblast cells and / or embryonic stem cells, or ECM or ECM components to prevent their differentiation.
  • the method further comprises the step of at least one of removal of feeder cells or ECM and or ECM components, replating the dispersed cells on plates without ECM components or human serum, and removal of bFGF. These further optional steps allow for the spontaneous differentiation of ESC.
  • the method of the invention may optionally comprise analysing at least one of the arrested embryos, or the stem cells derived therefrom, for gene-expression of at least one pluripotency gene to demonstrate the presence of specific pluripotency markers.
  • analysis can be performed using RT- PCR.
  • the step of analysing gene-expression can detect gene expression of at least one of OCT4, NANOG, REX ⁇ , TERT or a combination thereof.
  • the method of the invention may comprise immunocytochemical staining of the at least one stem cell provided by the method to determine a differentiation state thereof.
  • said immunocytochemical staining can detect surface markers on the stem cell: TRA-1-60, TRA-1-81, SSEA-4.
  • the method of the present invention may comprise the step of staining the embryo for identification of at least one viable blastomere in the arrested embryo.
  • the method of the present invention may comprise karyotype analysis of the stem cell(s) provided by the method.
  • an embryonic stem cell provided by the method of the present invention is a substantially undifferentiated embryonic stem cell.
  • an embryonic stem cell provided by the method of the present invention is a pluripotent mammalian embryonic stem cell.
  • the stem cell is a pluripotent human stem cell.
  • the method of the present invention further includes the step of differentiating the embryonic stem cell produced to derivates of the three germ layers in-vitro and in vivo.
  • an embryonic stem cell derived from an arrested embryo.
  • an embryonic stem cell line derived from an arrested embryo.
  • said cell or cell line can be provided as a culture which is capable of proliferating in vitro whilst maintaining the potential to differentiate to derivates of endoderm, mesoderm and ectoderm tissue.
  • a stem cell library comprising embryonic stem cell lines derived from at least one arrested embryo.
  • said library consists of embryonic stem cell lines derived from at least one arrested embryo.
  • said stem cell library can comprise pluripotent stem cells derived from at least one arrested embryo and / or differentiated stem cells derived from at least one arrested embryo.
  • an embryonic stem cell or embryonic stem cell line of the invention can be provided using the methods of the present invention.
  • the embryonic stem cell or stem cell line can be provided in purified or isolated form.
  • an embryonic stem cell or embryonic stem cell line of the invention can be a mammalian stem cell or mammalian stem cell line.
  • an embryonic stem cell or embryonic stem cell of the invention is selected from a mouse embryonic stem cell or stem cell line, a primate embryonic stem cell or stem cell line, or a human embryonic stem cell or stem cell line.
  • a stem cell or stem cell line of the invention may be a human embryonic stem cell or human embryonic stem cell line.
  • a stem cell or stem cell line of the invention can be modified by in wf/utreatment or genetically modified to provide a particular characteristic for a specific industrial application.
  • an embryonic stem cell or embryonic stem cell line of the invention can be pluripotent.
  • an embryonic stem cell or embryonic stem cell line of the invention may comprise a cell surface marker selected from TRA1-60, TRA1-81, SSEA4 and intracellular marker selected from OCT4, NANOG, REX 1, hTERT.
  • an embryonic stem cell or embryonic stem cell line of the invention comprises the cell surface markers TRA1-60, TRA1-81, SSEA4 and intracellular markers OOT4, NANOG, REX 1, hTERT.
  • the embryonic stem cell or embryonic stem cell line can be capable of differentiating into cells derived from mesoderm, endoderm and ectoderm germ layers.
  • an embryonic stem cell or embryonic stem cell line of the invention is differentiated to provide cells of ectoderm, mesoderm or endoderm origin (for instance blood cells, neuron cells, or muscle cells.
  • a differentiated cell line derived from an arrested embryo derived from an arrested embryo.
  • the differentiated cell line is provided using the methods of the present invention.
  • the differentiated cell line is selected from cells of ectoderm, mesoderm or endoderm origin (for instance blood cells, neuron cells, or muscle cells).
  • hESC derived from arrested and hESC derived from developing embryos are equivalent i.e. are pluripotent and able to differentiate under both in vitro and in vivo conditions and there are no differences in their characteristics (expression of different markers).
  • embryonic stem cell or stem cell line of the invention there is provided the use of an embryonic stem cell or stem cell line of the invention.
  • said use can be; to analyse mammalian development or misdevelopment, to analyse pluripotency, analyse differentiation, to analyse cell proliferation, to analyse signalling pathways, to analyse genetic and epigenetics of embryos, the production of transgenic non-human animals, ESC and hESC replacement therapy, human or non-human tissue replacement therapy, or drug development.
  • the use includes a method of screening to identify an agent that affects ES cell function.
  • the method includes incubating components comprising the agent and at least one ES cell derived from an arrested embryo under conditions sufficient to allow the agent and ES cell to interact, and determining the effect of the agent on ES cell function before and after incubating in the presence of the agent.
  • a cell function that may be modulated, for example inhibited or stimulated, by the agent includes, differentiation, gene expression, production of growth factors, response to growth factors, modulation of the cell membrane and the like.
  • an embryonic stem cell or embryonic stem cell line of the invention in medicine, in particular to treat a specific disease.
  • Said embryonic stem cell or embryonic stem cell line may be provided in a composition or medicament to treat disease.
  • “Treatment” or “therapy” includes any regime that can benefit a human or non-human animal.
  • the treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviation or prophylactic effects.
  • a differentiated or non-differentiated embryonic stem cell or embryonic stem cell line of the invention may be transplanted into a human or non-human animal, in particular embodiments an adult human or non-human animal, to treat a disease selected from haematopoietic disorders, endocrine deficiencies, degenerative neurological disorders or other disorders of tissue loss or organ damage, for example tissue loss or organ damage due to disease or hair loss.
  • the present invention provides a useful pharmaceutical composition
  • a useful pharmaceutical composition comprising an ES cell derived from an arrested embryo or a product derived therefrom.
  • a product can be protein expressed from an ES cell or a cell line derived from an arrested embryo.
  • a method of culturing a cell derived from an arrested embryo comprising at least one step selected from: i) culturing a pluripotent cell derived from an arrested embryo in the presence of at least one of: feeder cells and extracellular matrix components such that said cell is able to proliferate in an undifferentiated state, ii) culturing a pluripotent cell derived from an arrested embryo in the absence of at least one or both of feeder cells and extracellular matrix components to cause differentiation of the cell, and iii) culturing a cell derived from an arrested embryo in the presence of an agent to promote or retain said cell in a differentiated state
  • Figure 1 illustrates developmental stages and chronological time of normal early human development up to blastocyst stage.
  • figure 1 shows oocyte retrieval assessed as Day 0, early developing embryos of good quality possessing equal blastomeres, absence of fragmentation, and which do not show delayed cleavage or do not arrest under in vitro conditions; typically these embryos cleave to morulae on Day 4 and to blastocysts on Day 5;
  • Figure 2 illustrates different stages and ages of arrested human embryos, wherein these embryos do not form blastocyst even after prolonged culture and arrest at early or late embryonic stages - (A).
  • Early and late arrested embryos could stop their cleavage with all equal (normal) blastomeres, but very frequently they have unequal or fragmented blastomeres; however, both groups of arrested and developing embryos show similar expression of pluripotency genes (B) when compared with early (a) or late (b) developing embryos, (a): ea1, two cell early arrested embryo; ea2, five cell early arrested embryo; ea3, eight cell early arrested embryos, recovered on Days 3-5. Two cell, 4 cell, 8 cell, 12 cell, and 16 cell are normal developing early embryos.
  • e Representative micrograph of late arrested embryo (16 cell stage) stained with DAPI (f), note the presence of unequal or fragmented but not stained (uf) blastomeres in both, early, and late arrested embryos (Scale bar: 100 ⁇ m. Abbreviations: ea, early arrested; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ia, late arrested);
  • Figure 3 Developing and arrested embryos used for derivation of new human embryonic stem cell (hESC) lines - (A): Phase contrast micrograph of developing and good quality compacting Day 4 morula, showing the presence of equal blastomeres (A and schematically a1). After chemical removal of the zona pellucida, the embryo was plated on mouse feeder cells and attached after 1 day (B). After mechanical dissection, primary outgrowth and hESC-like colony was observed 15 (C) and 21 (D) days after initial plating, respectively.
  • A Phase contrast micrograph of developing and good quality compacting Day 4 morula, showing the presence of equal blastomeres (A and schematically a1). After chemical removal of the zona pellucida, the embryo was plated on mouse feeder cells and attached after 1 day (B). After mechanical dissection, primary outgrowth and hESC-like colony was observed 15 (C) and 21 (D) days after initial plating, respectively.
  • FIG. 4 Characterization of hES-NCL9 line derived from late arrested embryo -
  • A Human embryonic stem cell (hESC) grown on mouse embryonic fibroblasts (MEF) stained with antibody recognizing the GTCM2 (a), TRA-1-60 (b), TRA-1-81 (c), SSEA-4 (d), and alkaline phosphates (e) epitopes (passages 17-21).
  • B Reverse transcription-polymerase chain reaction (RT-PCR) analysis of undifferentiated hES-NCL9. PCR products were obtained using primers specific for OCT4, NANOG, REX1, TERT, and GAPDH (passages 17- 21).
  • C Karyotyping of hES-NCL9 cells grown on MEF show a normal female karyotype (passage 19).
  • D Spontaneous differentiation of hES-NCL9 into neuronal (a), fat (b), and endoderm-like (c) cells demonstrating their differentiation ability under in vitro growth conditions. Cells stained with nestin (a) or ⁇ -fetoprotein antibodies (c). Fat cells stained with oil red O staining (b).
  • E Histological analysis of differentiated tissues found in teratomas formed in the testis of severe combined immunodeficient (CB17/ICR-Prkdc sc ⁇ d /Crl) mice following transplantation of NCL9 hESC.
  • Histological staining Weigerts (I 1 IV-VI) and hematoxylin and eosin (II, III). (Scale bars: (I 1 II) 500 ⁇ m; (III) 100 ⁇ m; (IV-VI) 200 ⁇ m. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase);
  • Figure 5 illustrates whole embryos treated with acidified Tyrode's solution to remove zona pellucida and then plating of same on feeder cells in the presence of growth medium;
  • Figure 6 shows the deviation of LES-NCL6 line, wherein whole day 6 expand blastocysts (a) of good quality was freed from zona pellucida and plated on human fetal lung feeder cells - primary outgrowth was noted (white arrow) and replated on fresh feeder cells. Typical hESC colony was noted three days after replating (white arrow). (Scale bars: 100 ⁇ m (a, b); 200 ⁇ m (c));
  • Figure 7 shows quantitative expression ⁇ SD (by real-time RT-PCR) of pluripotency genes in old (HI and hES-NCLI) and new (hES-NCL7-9) hESC lines -new hESC lines derived from morulae or arrested embryo expressed similar or higher level of different pluripotency genes (OCT4, NANOG, REX1, TERT) when compared with HI or hES-NCLI lines derived from blastocysts; and
  • arrested embryos were freed from ZP and plated on inactivated mouse or human feeder cells as discussed in Strelchenko N, Verlinsky O, Kucharenko V et al. Morula-derived human embryonic stem cells. RBM Online 2004;9:623-629.
  • hESC clumps were cultured either on inactivated mouse embryonic (MEF) or human fetal lung (HFL) feeder cells in the presence of ESC medium containing Knockout-DMEM (Invitrogen, Carlsbad, CA), 100 ⁇ M ⁇ -mercaptoethanol (Sigma-Aldrich, St.
  • hES-NCL1 line grown on MEF 1 mM L- glutamine (Invitrogen), 100 mM nonessential amino acids, 10% serum replacement (SR, Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 4 ng/ml basic fibroblast growth factor (Invitrogen) which was previously conditioned for 48 hours on hES-NCL1 line grown on MEF. Before use, conditioned medium was spun for 5 minutes at 1,000 rpm and subsequently filter-sterilized (0.22 ⁇ m) to remove any cellular material. This conditioned medium was kept at 4°C until needed. Growth medium was changed every second day. Each hESC line was passaged mechanically and then transferred to freshly prepared feeders.
  • RT-PCR reverse transcription- polymerase chain reaction
  • SEQ ID NO 2 OCT 4 Reverse ⁇ '-CTTAATCCAAAAACCCTGG-S' SEQ ID NO 3: NANOG Forward ⁇ '-CTGAGATGCCTCACACGGAGACTG-S'
  • SEQ ID NO 5 REX1 Forward ⁇ '-GCGTACGCAAATTAAAGTCCAGA-S'
  • SEQ ID NO 6 REX1 Reverse ⁇ '-CAGCATCCTAAACAGCTCGCAGAAT-S'
  • SEQ ID NO 8 Tert Reverse ⁇ '-GAACAGTGCCTTCACCCTCGA-S'
  • SEQ ID NO 10 GAPDH 1 Reverse ⁇ '-CACCACCCTGTTGCTGTAGC-S'
  • Real-time PCR analysis was carried out using QuantiTect SYBR Green PCR Master mix (Qiagen, Valencia, CA). The reaction was performed with 1 ⁇ l cDNA per 20 ⁇ l reaction, and each reaction was performed in triplicate.
  • the Light-Cycler experimental run protocol used was as follows: PCR activation step (95 0 C for 15 minutes), amplification with data acquisition repeated 50 times (94 0 C for 15 seconds, annealing temperature for primers for 30 seconds, 72 0 C for 20 seconds with a single fluorescence data collection at 76 0 C for 10 seconds), melting curve (6O 0 C to 95 0 C with a temperature transition rate of 0.1 0 C per second and continuous fluorescence data collection), and finally cooling to 4O 0 C.
  • the crossing point (CP) for each transcript was determined using second derivative maximum method in the LightCycler software v3.5.3 (Roche Diagnostics). GAPDH CP for each sample was used as the internal control of these real-time analyses. The data were analysed
  • Real-Time RT-PCR analysis for TERT quantification was carried out using the LightCycler TeIoTAGGG hTERT quantification kit according to the manufacturer's instructions (Roche Diagnostics, Indianapolis). For each reaction, 100 ng total RNA was used and all reactions were performed in duplicate and normalized to porphobilinogen deaminase in the same run.
  • the karyotype of 10 metaphase cells was determined by standard G-banding procedure. Differentiation of hESC under in vitro conditions and after tumour formation in severe combined immunodeficient (SCID) mice was done as previously described in Stojkovic M, Lako M, Stojkovic P et al. Derivation of human embryonic stem cells from Day 8 blastocysts recovered after three-step in vitro culture. Stem Cells 2004;22:790-797 and Stojkovic P, Lako M, Stewart R et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 2005;23:306-314
  • Example 1 Surplus and donated human embryos were used to determine whether early (2-10 cells) and late (16-24 cells) arrested embryos (schematically Figure 2A) could be used for derivation of new hESC lines.
  • Oocyte retrieval Day 0. Percentage based on number of used embryos.

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Abstract

L'invention concerne des cellules souches embryonnaires, en particulier des cellules souches embryonnaires humaines, très demandées comme objets de recherche du fait de leur capacité à se reproduire et à se différencier en plusieurs types sinon en tous les types de cellules du corps. La présente invention concerne un procédé par lequel des embryons non développés peuvent être utilisés pour produire des cellules souches embryonnaires.
PCT/RS2007/000009 2007-03-20 2007-03-20 Procédé de production de cellules souches embryonnaires WO2008115087A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHANG KAREN H ET AL: "Quantitative screening of embryonic stem cell differentiation: Endoderm formation as a model", BIOTECHNOLOGY AND BIOENGINEERING, vol. 88, no. 3, 5 November 2004 (2004-11-05), pages 287 - 298, XP002471259, ISSN: 0006-3592 *
EVANS M ET AL: "Source and nature of embryonic stem cells", COMPTES RENDUS - BIOLOGIES, ELSEVIER, PARIS, FR, vol. 325, no. 10, October 2002 (2002-10-01), pages 1003 - 1007, XP004395811, ISSN: 1631-0691 *
SHUFARO Y ET AL: "Therapeutic applications of embryonic stem cells", BAILLIERE'S BEST PRACTICE AND RESEARCH. CLINICAL OBSTETRICS AND GYNAECOLOGY, BAILLIERE TINDALL, LONDON, GB, vol. 18, no. 6, December 2004 (2004-12-01), pages 909 - 927, XP004664907, ISSN: 1521-6934 *
ZHANG XIN ET AL: "Derivation of human embryonic stem cells from developing and arrested embryos", STEM CELLS (MIAMISBURG), vol. 24, no. 12, December 2006 (2006-12-01), pages 2669 - 2676, XP002471258, ISSN: 1066-5099 *

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