WO2010007031A2 - Procédés pour améliorer la différenciation cardiaque des cellules souches embryonnaires humaines - Google Patents

Procédés pour améliorer la différenciation cardiaque des cellules souches embryonnaires humaines Download PDF

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WO2010007031A2
WO2010007031A2 PCT/EP2009/058936 EP2009058936W WO2010007031A2 WO 2010007031 A2 WO2010007031 A2 WO 2010007031A2 EP 2009058936 W EP2009058936 W EP 2009058936W WO 2010007031 A2 WO2010007031 A2 WO 2010007031A2
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cells
wnt3a
stem cells
serum
culture medium
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WO2010007031A3 (fr
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Thanh Tran
Mark Burcin
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Novartis Ag
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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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • 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
    • C12N2501/105—Insulin-like growth factors [IGF]
    • 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
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • cardiomyocytes The post-mitotic nature of isolated cardio myocytes does not allow the generation of cell lines for in vitro studies. For this reason most of the knowledge of cardiomyocyte biology is based on non-primate primary cardiomyocytes. Often times this knowledge does not translate to human cardiomyocytes. Due to their ability to propagate indefinitely (self-renewing) and develop into all cell types (pluripotency) human embryonic stem cells (hESC) could serve as an alternative source for generating human cardiac cells.
  • hESC human embryonic stem cells
  • Human ESCs can spontaneously differentiate into cardiomyocytes that genotypically, phenotypically and functionally resemble those of native cardiomyocytes.
  • the conventional method for differentiating human embryonic stem cells into cardiomyocytes include forming cell aggregates termed embryoid bodies (EBs) in suspension, maintaining cells in medium containing 20% fetal bovine serum, and attaching EBs on gelatin-coated plate. Differentiating ESCs using these conditions, however, results in an extremely low and inconsistent cardiomyocyte yield.
  • EBs embryoid bodies
  • Wnts are lipid-modified glycoproteins that, upon binding to their respective receptor/co-receptor (Frizzled/LRP5-6, respectively), activate either the canonical or non-canonical signaling pathway.
  • FGF fibroblast growth factor
  • BMP bone morphogenic protein
  • Wnt increases intracellular Ca 2+ and/or activity of c-Jun N-terminal kinase (JNK) or protein kinase C (PKC).
  • JNK c-Jun N-terminal kinase
  • PKC protein kinase C
  • Treatment of mouse ESC with recombinant Wnt3a accelerates the regulation of brachyury gene expression (a known marker gene for mesoderm cell lineage determination) and enhances upregulation of cardiac-specific gene expression (cardiomyogenesis).
  • brachyury gene expression a known marker gene for mesoderm cell lineage determination
  • cardiac-specific gene expression cardiac-specific gene expression
  • ESCs e.g., human ESCs (hESCs)
  • the methods described herein provide an efficient way for large-scale generation of differentiated human cardiomyocytes, one of the prerequisites for robust and predictable assay development.
  • methods for improving cardiomyocyte cell population by inducing differentiation of embryonic stem cells into embryonic bodies and cultivating these embryonic bodies in culture medium without serum.
  • methods for generating linage specific cells are also disclosed herein.
  • hESCs with recombinant Wnt3a enhances brachyury gene expression, this is indicative of mesendoderm formation, and mesendoderm can give rise to not only mesoderm lineages specifying cardiac, blood and bone cells, but also to endoderm lineages leading to lung, hepatic and pancreatic cells.
  • Cardiomyocytes generated in culture from ESCs can be used, for example, as a research tool to better understand human cardiovascular disorders and to provide improved predictability compared to existing models used for target validation in drug development. Cardiomyocytes generated under these conditions can be used, for example, for cell replacement therapy in patients with cardiovascular disease. Additionally, factors useful for inducing cardiomyocyte differentiation can also be useful as therapeutic agents or drug targets for treating, for example, patients who suffer from heart disease, e.g., myocardial infarction.
  • the present invention is directed to a method for inducing differentiation of embryonic stem cells, precursor cells, pluripotent stem cells or induced pluripotent stem cells into cardiomyocytes, comprising contacting stem cells with Wnt3a protein or a functional fragment thereof under conditions suitable for the differentiation of stem cells, wherein the Wnt3a protein causes the stem cells to differentiate into cardiomyocytes at a greater proportion than in the absence of Wnt3a or functional fragment thereof.
  • the method further comprises cultivating the differentiated cells in culture medium without serum such that the population of cardiomyocytes increases compared with cardiomyocytes in the presence of serum.
  • the methods of the present invention optionally comprise inhibiting the activity of VEGF or BMP4, e.g., wherein noggin is used to inhibit the activity of BMP4 or neuropilin-1 is used to inhibit the activity of VEGF.
  • the methods of the present invention optionally comprise aggregation of the human embryonic stem cells into embryoid bodies, and optionally contacting the embryoid bodies with ⁇ -catenin.
  • the methods of the present invention optionally comprise monitoring the state of differentiation by monitoring the level of a bio marker, wherein the bio marker is indicative of a particular differentiated or partially differentiated cell type, e.g., wherein the bio marker is brachyury or ⁇ -catenin.
  • Wnt3a protein or functional fragment thereof is provided at a concentration of at least about 30 ng/niL.
  • the effect of Wnt3a is monitored by determining the expression level of one or more genes that are upregulated or downregulated in response to Wnt3a, e.g., wherein the genes that are upregulated are selected from the group consisting of the genes in Table 2, and wherein the genes that are downregulated are selected from the group consisting of the genes in Table 3.
  • the expression levels of the one or more genes are determined using a hybridization assay.
  • the expression levels of the one or more genes are determined using a microarray.
  • the Wnt3a protein or a functional fragment thereof causes the stem cells to differentiate into cardiomyocytes at about 10-100%, about 30-70%, or about 40% greater proportion than in the absence of the Wnt3a, or functional fragment thereof.
  • the present invention is directed to a method of inhibiting Wnt3a comprising contacting Wnt3a with an inhibitor.
  • the inhibitor is selected from the group consisting of a small molecule, an antibody and an siRNA molecule.
  • the present invention is directed to a method of activating the Wnt3a signaling pathway comprising inhibiting one or more inhibitors of Wnt3a.
  • the one or more inhibitors of Wnt3a are inhibited by an inhibitor selected from the group consisting of: a small molecule, and antibody and an siRNA molecule.
  • the present invention is directed to a method for inducing differentiation of mesoderm specific pluripotent cells comprising contacting mesoderm specific pluripotent cells with Wnt3a protein, or a functional fragment thereof, under conditions suitable for the differentiation of mesoderm specific pluripotent cells, wherein the Wnt3a protein causes the mesoderm specific pluripotent cells to differentiate at a greater proportion than in the absence of Wnt3a, or functional fragment thereof.
  • the present invention is directed to a method for improving a cardiomyocyte cell population comprising inducing differentiation of embryonic stem cells, precursor cells, pluripotent stem cells or induced pluripotent stem cells into embryonic bodies in a culture medium comprising a Wnt3a protein or a functional fragment thereof; cultivating the embryonic bodies in a culture medium with a reduced serum level; and further cultivating the embryonic bodies in a culture medium without serum, such that the culture medium with no serum increases the population of cardiomyocytes compared with proliferating none-cardiac cells.
  • Wnt3a protein or functional fragment thereof is provided at a concentration of at least about 30 ng/mL.
  • the Wnt3a protein or fragment thereof increases the population of embryonic bodies by about 10-100%, by about 30-70%, and by about 40%, compared with the absence of the Wnt3a or functional fragment thereof.
  • the stem cells are initially grown in culture medium comprising about 2- 40% serum, and preferably about 15% serum.
  • the serum in the culture medium is then reduced to about 2-7%, and preferably about 5%.
  • the step of cultivating the embryonic bodies in culture medium with reduced serum increases the population of cardiomyocytes by about 10-100%, and about 50%, compared to culture medium in the presence of serum.
  • the embryonic bodies are cultivated in the culture medium with the reduced serum level for at least 24 hours, and preferably about 48 hours.
  • the embryonic bodies are then cultivated in culture medium without serum.
  • the step of cultivating the embryonic bodies in culture medium without serum increases the population of cardiomyocytes by about 10-100%, and about 50% compared to the presence of reduced serum.
  • the embryonic bodies are cultivated in the culture medium without serum for at least 30 days, and preferably for at least 60 days.
  • the present invention is directed to a method for generating linage specific cells, comprising inducing differentiation of embryonic stem cells, precursor cells, pluripotent stem cells or induced pluripotent stem cells into mesoendodermal cells in a culture medium comprising a Wnt3a protein or a functional fragment thereof, and adding cell differentiating factors to the culture medium, wherein the differentiating factors generate linage specific cells.
  • the differentiating factors are selected from the group consisting of nodal pathway (e.g., activin A), Hedgehog and Notch inhibitors (e.g., cyclopamine, DAPT), FGF, retinoic acid, GLP-I, IGF-I, HGF, PDX-I, PAX6, PAX4, Nkx ⁇ .l, BMP pathway members (e.g., Noggin, BMP4), VEGF and antagonists, wnt pathway members (e.g., Wnt8) and Wnt antagonists (e.g., DKK).
  • the linage specific cells can be cardiac cells, or none- cardiac cells such as liver cells, pancreatic cells, lung cells, blood cells, neural cells, skeletal muscle cells, thyroid cells and gut cells.
  • FIGS IA-D are graphs and images showing effects of ⁇ -catenin/canonical signaling pathway on mesendoderm formation.
  • Human embryonic stem cells hESC were treated with vehicle, rWntl (300 ng/ml), rWnt2 (300 ng/ml), rWnt3a (100 ng/ml), rWnt7a (300 ng/ml), or BIO (300 nM) for 48 hours.
  • Cells were harvested and subjected to qRT-PCR analysis detecting Brachyury gene expression (IA).
  • IB immunoblot analysis probing for total or active ⁇ -catenin
  • FIGS 2A-F are graphs showing effects of ⁇ -catenin/canonical signaling pathway on mesendoderm formation.
  • Human ESC were treated as described in IA. Cells were harvested for RNA and subjected to SYBR Green RT-PCR. Fold differences against control-treated cells are shown for Eomes (2A), Mespl (2B), Mesp2 (2C), Soxl7 (2D), FoxA2 (2E) and Soxl (2F). Data are shown as means of two independent experiments.
  • FIGS 3A-D are graphs showing Wnt3 a- specific induction of mesendoderm formation.
  • RNA was isolated and subjected to qRT-PCR. Data represent mean ⁇ SD; n triplicate; *p ⁇ 0.05 (3D).
  • FIGS 4A-C are micrographs showing effects of ⁇ -catenin/canonical signaling pathway on cell survival.
  • Human embryonic stem cells hESC
  • hESC Human embryonic stem cells
  • cells were visualized under the microscope (4A).
  • Dkkl 100 ng/ml
  • Wnt3a CM 100 ng/ml
  • cells were visualized under the microscope (4B).
  • PCNA proliferating cell nuclear antigen
  • FIGS 5A-D are graphs showing effects of ⁇ -catenin/canonical signaling pathway on hESC cardiomyogenesis.
  • Human ESC were treated with rWntl (300 ng/ml), rWnt2 (300 ng/ml), rWnt3a (100 ng/ml), rWnt7a (300 ng/ml), or BIO for 48 hours and cultivated as described in 4 A.
  • Beating clusters were tabulated on day 12 of differentiation (5A).
  • Cells were treated with increasing concentration of rWnt3a for 48 hours and cultivated as described in 4A. Beating clusters were quantified on day 12 of differentiation (5B).
  • Human ESC were preincubated with increasing concentration of Dkkl for an hour, treated with control or Wnt3a CM for 48 hours, and cultivated as described in 4 A. Beating clusters were tabulated on day 12 of differentiation (5C). Data are shown as means of two independent experiments. Cells were treated with Wnt3a for 48 hours and harvested at indicated time. Total RNA were obtained and subjected to qRT-PCR. Data represent means of two independent experiments (5D).
  • FIGS 6A-F are graphs and images showing cell line independent effects of Wnt3a on mesendoderm formation, cell viability, and cardiomyogenesis.
  • Hl (6A-C) or H7 (6D-F) ESC were treated with control or Wnt3a CM for 48 hours and analyzed for mesendoderm formation (6A & 6D), cell viability (6B & 6E), and cardiomyogenesis (6C & 6F).
  • FIGS 7 A is a micrograph showing the immunohistochemical analysis of human embryonic stem cell derived cardiomyocytes.
  • Human embryonic stem cells hESC
  • Wnt3a CM Human embryonic stem cells
  • Beating clusters day 16-30
  • Cells were fixed and immunostained with antibodies against cTnl, Nkx2.5, ⁇ -actinin, or Mef2 as indicated above. Images were taken using Zeiss confocal microscope with 63X objective lens (7A).
  • FIGS 8A-B shows the global gene expression analysis of Wnt3a effects.
  • Cluster a stem cell marker genes
  • cluster b mesendoderm marker genes
  • cluster c TGF ⁇ -family related genes
  • cluster d cardiac specific genes (8B).
  • FIGS HA-D are graphs showing Wnt3a enhanced expression of mesendoderm, mesoderm and endoderm, but not ectoderm, marker genes. Genes shown are derived from global gene expression analysis data.
  • FIGS 14A-B are graphs showing effects of ⁇ -catenin regulating factors on mesendoderm formation.
  • Human ESC were treated with vehicle, IGFl (100 ng/ml), PMA (200 nM), ISO (5 mM), or rWnt3a (30 ng/ml) for 48 hours, harvested and subjected to RT-PCR analysis detecting Brachyury gene expression (IA). Cells were treated as in IA and subjected to immunoblot analysis probing for ⁇ -catenin (IB).
  • stem cells refer to cells that can differentiate into a diverse range of specialized cell types.
  • Pluripotent refers to the ability of a single stem cell to give rise to all of the various cell types that make up the body. Pluripotent cells cannot make so-called “extra-embryonic” tissues such as the amnion, chorion and other components of the placenta.
  • totipotent refers to a stem cell that can give rise to all the cell types that make up the body plus all of the cell types that make up the extraembryonic tissues such as the placenta.
  • multipotent refers to the ability of a single stem cell to develop into more than one cell type of the body.
  • embryonic stem cells typically, upon aggregation, differentiation is initiated and the cells begin to a limited extent to recapitulate embryonic development.
  • myocardial infarction refers to acute myocardial infarction a medical condition that occurs when the blood supply to a part of the heart is interrupted, most commonly due to rupture of a vulnerable plaque. The resulting ischemia or oxygen shortage causes damage and potential death of heart tissue. Acute myocardial infarction (AMI or MI) is sometimes commonly referred to as heart attack.
  • AMI acute myocardial infarction
  • myocardial damage refers to death (e.g., apoptosis) of myocardial cells and/or tissue in a subject, e.g., after myocardial infarction.
  • death e.g., apoptosis
  • biological sample refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • body fluids including but not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • a biological sample further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. Most often, the sample has been removed from an animal, but the term “biological sample” can also refer to cells or tissue analyzed in vivo, e.g., without removal from animal.
  • a biological sample will contain cells from the subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to measure the cancer-associated polynucleotide or polypeptides levels.
  • a biological sample further refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses a nucleic acid containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally-occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acids, and which are metabolized in a manner similar to the reference nucleotides.
  • nucleic acid sequence also encompasses naturally-occurring allelic variants of said nucleic acid.
  • oligonucleotide refers to a nucleic acid molecule consisting of two or more deoxyribonucleotides or ribonucleotides joined by phosphodiester bonds, and preferably containing between about 6 and about 300 nucleotides in length.
  • the size of the oligonucleotide will depend on many factors, including the ultimate function or use of the oligonucleotide.
  • an oligonucleotide that functions as an extension primer or probe will be sufficiently long to prime the synthesis of extension products in the presence of a catalyst, e.g., DNA polymerase, and deoxynucleotide triphosphates.
  • oligonucleotide further refers to an oligonucleotide that has been modified structurally ("modified oligonucleotide”) but functions similarly to the unmodified oligonucleotide.
  • modified oligonucleotide can contain non-naturally occurring portions, such as, for example, altered sugar moieties or inter- sugar linkages, such as a phosphorothioate.
  • the term "gene” refers to a nucleic acid sequence that encodes and regulates expression of a polypeptide.
  • a gene includes, therefore, regulatory elements, e.g., promoters, splice sites, enhancers, repressor binding sites, etc.
  • a gene can have many different "alleles,” which are sequence variations that can affect the polypeptide sequence or expression level, or have no effect on the polypeptide.
  • a gene can include one or more "open reading frames,” which are nucleic acid sequences that encode a contiguous polypeptide.
  • a gene can be present either endogenously or exogenously.
  • polypeptide refers to a polymer in which monomer amino acids ("amino acid residues") are joined together through peptide or disulfide bonds. It also refers to either a full-length naturally-occurring amino acid sequence or a fragment thereof between, for example, about 8 and about 500 amino acids in length. Unnatural amino acids, for example, ⁇ -alanine, phenyl glycine and homoarginine, can be additionally included. Commonly-encountered amino acids that are not gene-encoded can also be used in the present invention. All of the amino acids used in the present invention can be either the D- or L-optical isomer. The L- isomers are preferred. A polypeptide sequence also encompasses naturally-occurring allelic variants of said polypeptide.
  • the term "subject" as used herein refers to any living organism in which an immune response is elicited.
  • the term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the term “efficacy” refers to the degree to which a desired effect is obtained. Specifically, the term refers to the degree to which hESCs are induced to differentiate into cardiomyocytes or the degree to which a patient is treated for myocardial damage.
  • the term “efficacy” as used in the context of the present invention also refers to relief or reduction of one or more symptoms or clinical events associated with myocardial damage or disease.
  • a significant change in the expression level refers to either an increase or a decrease of the expression level from the control level by an amount greater than the standard error of the assay employed to assess expression.
  • the term also refers to a change by preferably at least about 2%, about 5%, about 10%, about 20%, about 25%, about 30%, preferably at least about 40%, about 50%, more preferably at least about 60%, about 70%, or about 90%, about 100%, about 150%, or about 200%, or greater.
  • control level refers to a standard level of a biomarker by which a change is measure against.
  • the "control level” can be a normal level of a biomarker nucleic acid expression, or a biomarker polypeptide, or a biomarker biological activity from normal or healthy cells, tissues, or subjects, or from a population of normal or healthy cells, tissues, or subjects.
  • the biomarker can also be indicative of the differentiation state of, for example, a stem cell that has been induced to differentiate.
  • hESCs human embryonic stem cells
  • Conventional methods for differentiating human embryonic stem cells (hESCs) into cardiomyocytes include incubating cells in KnockOut DMEM medium containing 20% serum (v/v), 1% glutamine (wt/v), 1% non-essential amino acids (wt/v), and 0.1 mM ⁇ -mercaptoethanol (v/v).
  • v/v human embryonic stem cells
  • the present invention is based on the discovery and development of two cultivation methods using Wnt3a to derive improved yields of cardiomyocytes from hESC.
  • the timeframe for adding the Wnt3 is important to initiate differentiation. Addition too early may not activate the correct pathway, while addition too late may activate a different pathway.
  • the present invention identifies the appropriate timeframe to add Wtn3 resulting in a high yield of cardiomyocytes.
  • Using the methods described herein results in a highly efficient and reproducible way of generating high yields of cardiomyocytes from hESC.
  • the use of serum free conditions provides an enriched population of cardiomyocytes. Cultivating the differentiated EB in serum-free medium for several days, e.g., about 60 days results in the elimination of highly proliferating none-cardiac cells such as lung, pancreatic, hepatic, and blood cells, which are more sensitive to the lack of serum.
  • This approach yields many advantages over the conventional differentiation protocol including an improvement in cardiac differentiation by excluding anti-cardiomyogenic factor(s) such as insulin, and the elimination of highly proliferating none-cardiac cells.
  • anti-cardiomyogenic factor(s) such as insulin
  • the serum free cultivation conditions therefore provide a selection method for deriving a pure cell population of cardiomyocytes.
  • These cardiomyocytes can be used for pharmaceutical screens and therapy.
  • These methods described herein substantially increase cell survival and proliferation of differentiating hESCs and provide an efficient way to differentiate hESCs towards the mesoderm and cardiac lineage. These methods allow for the development of improved protocols for ESC cultivation and differentiation, and for generating cardiomyocytes from ESC.
  • Wnt3a conditioned medium or recombinant Wnt3a enhances cell survival (EB size and number), mesendoderm formation (Brachyury gene expression), and cardiomyogenesis (beating clusters).
  • the Wnt3a protein or a functional fragment thereof causes the cells to differentiate into cardiomyocytes at about 10-100%, about 30-70%, or about 40% greater proportion than in the absence of the Wnt3a, or functional fragment thereof.
  • hESCs Human embryonic stem cells
  • Wnt3a a member of the Wnt-pathway during differentiation
  • Wnt3a is added at a specific time point during the differentiation process of embryonic stem cells (48 hours after cultivation in culture medium with serum) and under these conditions Wnt3a induces a) stem cell/progenitor cell survival; b) stem cell/progenitor cell proliferation; c) induction of stem cells towards mesendoderm progenitor cells that in part develop towards the cardiac lineage causing an overall increase in cardiac myocyte yield.
  • Wnt3a could also be inducing cardiac regeneration after, for example, a condition leading to cardiomyocyte damage, e.g., myocardial infarction or heart failure patients, by a) decreasing myocardial cell death, b) improving endogenous regeneration of cardiomyocytes or c) improving regeneration of endogenous stem cells.
  • Methods for inducing differentiation of stem cells include, but are not limited to, embryonic stem cells, precursor or progenitor cells, pluripotent stem cells, induced pluripotent stem cells, etc., include, for example, serum free and insulin free methods in addition to conventional cell culture methods.
  • the invention is directed to methods for inducing differentiation of hESCs to generate cardiomyocytes.
  • the invention is directed to contacting hESCs, under conditions suitable for differentiation, e.g., as an embryoid body (EB) in an appropriate medium, with Wnt3a protein, or a fragment thereof.
  • the contact can occur either by adding recombinant proteins (rWnt3a) to the medium, or by using Wnt3a-conditioned media (Wnt3a-CM).
  • the invention is not limited to hESC and can include, for example, pluripotent stem cells, totipotent stem cells, adult stem cells, haematopoietic stem cells, mesenchymal stem cells, neural stem cells, stem cell isolated from amniotic fluid or induced pluripotent stem cells.
  • pluripotent stem cells totipotent stem cells, adult stem cells, haematopoietic stem cells, mesenchymal stem cells, neural stem cells, stem cell isolated from amniotic fluid or induced pluripotent stem cells.
  • Methods for producing and substantially purifying are known in the art.
  • Methods for producing Wnt3a-CM are likewise known in the art.
  • conditioned media refers to cell culture media containing biologically active components from previously cultured cells or tissues that have released the components, e.g., Wnt3a, into the media.
  • the invention is directed to a method for expanding and protecting EBs.
  • hESCs when aggregated to form EBs, are protected and allowed to form larger EBs in the presence of Wnt3a, either as rWnt3a or Wnt3a-CM.
  • Wnt3a either as rWnt3a or Wnt3a-CM.
  • Wnt3a protein or a functional fragment thereof causes the EBs to differentiate into cardiomyocytes at about 10-100%, about 30-70%, or about 40% greater proportion than in the absence of the Wnt3a, or functional fragment thereof.
  • Serum such as animal serum e.g., fetal bovine serum
  • the invention is directed to methods for improving cardio myocyte cell population by inducing differentiation of embryonic stem cells into embryonic bodies and cultivating these embryonic bodies in culture medium without serum.
  • methods for generating linage specific cells are also disclosed herein.
  • the invention is directed to methods for treating a patient after cardiac damage, for example, damage caused by myocardial infarction.
  • the method of treatment can be, for example, a molecularly-mediated method designed to protect and/or regenerate cardiomyocytes.
  • the molecularly-mediated method includes an expression construct comprising a Wnt3a coding sequence, or active fragment thereof.
  • the coding sequence or fragment(s) thereof is operably linked to expression control sequences.
  • the expression construct comprising the molecularly- mediated treatment can confer either transient or stable expression of the translated product.
  • the coding sequence can encode either the endogenous protein or a heterologous protein that is sufficiently homologous to the endogenous protein to possess biological activity in the recipient host cell.
  • the construct can comprise at least one coding sequence operably linked to expression control sequences suitable for transient expression under the control of a cardiac tissue specific promoter, which can be either constitutive or inducible.
  • a cardiac tissue specific promoter which can be either constitutive or inducible.
  • the cardiac tissue promoter can be specific for ventricular or atrial tissue.
  • the present invention is directed to a method of treating a subject afflicted with heart disease, comprising administering a construct comprising a Wnt3a coding sequence or fragment thereof, e.g., by direct cardiac injection, wherein the construct is capable of expressing a Wnt3a protein or fragment thereof capable of protecting and/or regenerating cardiomyocytes.
  • the present invention is directed to a method of treating a patient after myocardial infarction or heart failure, comprising contacting myocardial tissue damaged by myocardial infarction with one or more cells capable of expressing and secreting Wnt3a or a functional fragment thereof.
  • the present invention is directed to a method of treating a subject afflicted with heart disease, comprising administering cardiomyocytes generated from human embryonic stem cells into a damaged myocardial tissue, wherein the cardiomyocytes are generated by contacting human embryonic stem cells with Wnt3a protein; or a functional fragment thereof, under conditions suitable for the differentiation of stem cells, and wherein the Wnt3a protein causes the stem cell to differentiate into cardiomyocytes at a greater proportion than in the absence of Wnt3a, or functional fragment thereof.
  • the subject is treated after myocardial infarction or heart failure.
  • the present invention is directed to a method of personalized therapy comprising: a) isolating one or more cells from a subject that are altered for their state of differentiation; b) contacting the isolated cells with Wnt3a, thereby inducing the cells to differentiate into cardiomyocytes; and c) reintroducing the isolated and differentiated cells into the subject.
  • the isolated cells are fibroblasts.
  • the invention is also directed to a cellular-based biological treatment for a patient who has suffered myocardial damage, e.g., after myocardial infarction.
  • a cellular-based cardiac treatment includes providing cardiomyocytes generated by the methods described herein, directly to the damaged tissue.
  • the cardiomyocytes can be, for example, derived from stem cells obtained directly from the patient, thereby eliminating the risk of graft versus host disease (GVHD).
  • the patient-derived cells can be genetically manipulated or chemically stimulated to control their state of differentiation.
  • Such a patient-derived cell for example, can be an induced pluripotent stem cell (iPS cell).
  • the cardiomyocytes can be generated from stem cells that have been genetically modified, e.g., transformed stem cells, transgenic stem cells, knockout stem cells, or stem cells otherwise engineered to produce and/or secrete, for example, Wnt3a.
  • the invention also encompasses methods of protecting and regenerating cardiac tissue in a patient suffering from myocardial damage, e.g., after myocardial infarction, by introducing one of the biologic constructs described herein, e.g., a construct comprising a Wnt3a coding sequence or functional fragment thereof, into a node region of the patient's heart.
  • the construct can be, for example, directly injected into the heart of the patient, for example, into the right atrium at a site that is localized to a region surrounding the sinoatrial node.
  • Hl, H7, and H9 human embryonic stem cells were obtained from WiCeIl Research Institute (Madison, WI). Cells (passages 35-60) were maintained on irradiated CFl feeder cells with complete growth medium (CGM) containing 20% KO-Serum Replacement, 0.5% glutamine, 1% non-essential amino acids, 0.1 mM ⁇ - mercaptoethanol, and 10 ng/ml basic fibroblast growth factor in DMEM/F12 medium.
  • CGM complete growth medium
  • hESC For differentiation of hESC, cells were treated as described above with some modifications. Briefly, cells were triturated into larger aggregates and reconstituted with differentiation medium (DM) containing 15% fetal bovine serum, 1% glutamine, 1% non-essential amino acids, and 0.1 mM ⁇ -mercatoethanol in KO-DMEM medium. Cells were distributed onto Ultralow binding 6-well plate and treated with varying factors for 48 hours. Cells were maintained in DM medium for 6 days in suspension as they formed cell aggregates termed embryoid bodies (EB). On day 6, EB were attached onto 0.1% gelatin-coated 6-well plate with DM medium containing 5% serum.
  • DM differentiation medium
  • SF serum-free medium
  • BSA bovine serum albumin
  • taurine 5 mM taurine
  • carnitine 5 mM carnitine
  • 5 mM creatine in high-glucose DMEM medium (36).
  • Microdissected beating clusters were dissociated into single cells with 0.05%
  • the reactions were run in a 384-well optical plate with the following settings: stage 1, 2 minutes at 5O 0 C; stage 2, 10 minutes at 95 0 C; stage 3, 15 seconds at 95 0 C and 1 minute at 6O 0 C for 40 cycles; and stage 4, 1 minute at 95 0 C and then 6O 0 C.
  • PCR reactions were set in 4 stages: stage 1, 2 minutes at 5O 0 C; stage 2, 10 minutes at 95 0 C; stage 3, 15 seconds at 95 0 C and 1 minute at 6O 0 C for 40 cycles; and stage 4, 1 minute at 95 0 C and then 6O 0 C.
  • ⁇ -Actin was used as loading control.
  • Table 4 is a list of primers for Taqman real time PCR analysis.
  • Table 5 is a list of primers for SYBR Green real time PCR analysis.
  • Membrane was incubated with 5% BSA in 0.01% Tween-20 containing Tris Bufferred Saline (Fisher Scientific, Pittsburgh, PA) for an hour at room temperature; with primary antibody against ⁇ -catenin (BD Biosciences, San Jose, CA), PCNA (Cell Signaling Technology Inc., Danvers, MA), or GAPDH (Novus Biologicals Inc., Littleton, CO) overnight at 4 0 C; and with HRP conjugated anti-mouse or -rabbit IgG (Cell Signaling) for 2 hours. Membrane was exposed to ECL Plus (GE Healthcare) for two minutes and developed using autoradiographic film.
  • Tris Bufferred Saline Tris Bufferred Saline
  • Undifferentiated hESC, cells treated with or without Wnt3a (day 2), and beating clusters (day 16 or 23) were collected.
  • RNA was isolated using RNeasy Mini Kit.
  • Global gene expression was profiled using Affymetrix Human Genome 133 plus set v2.0 Array.
  • the raw intensities from Affymetrix eel files downloaded from GDL were normalized and summarized into gene expression indices at probe-set level using Plier method implemented in the "plier" package with quantile normalization available at Bioconductor in R statistical computing environment (37). A constant number (16) were added on summarized data before the final Iog2 transform were performed.
  • the MAS5 present/absent calls were also generated for gene filtering purposes.
  • NCBI Entrez gene (genome build 36.46, Sept 11, 2006) based custom CDF (version 10.2, Feb 2008) published by the Microarray Lab of Neuroscience Institute at the University of Michigan was used to summarize the probe-set level signals (38). In total, 17589 Entrez gene probe-sets expression values were generated. Probe-sets that were absent across all conditions were eliminated from further statistical analyses. To identify differentially expressed genes, moderated F-test implemented in the "limma" package were used to compare expression values across different groups with the contrast matrix (39). The two group comparison analyses in specified in the contrast matrix were performed using moderated T-test. P-values generated by these tests were adjusted for multiple hypothesis testing using the method of Benjamini & Hochberg (40). Hierachical clustering was perform in R using 1 -Pearson correlation distance and Ward linkage method to generate the dendrogram.
  • Example 2 Method for inducing hESC differentiation into cardiomyocytes.
  • the hESCs were cultivated in suspension culture in the presence of Wtn3 and the absence of serum, which is essential for differentiation of the cells to cardiomyocytes.
  • Wtn3 is time dependent because if it is added to late, it will activate different proteins.
  • the stem cells are cultivated in suspension to form cell aggregates termed embryoid bodies using growth medium containing 15% serum and Wnt3a protein.
  • the next 4 days the embryoid bodies are kept in suspension with medium containing 15% serum but without Wnt3a protein.
  • the embryoid bodies are attached to plates using culture medium with a reduced serum level of 5%.
  • the embryoid bodies are cultivated in the culture medium with 5% serum level for at least 24 - 48 hours.
  • the embryoid bodies are then cultivated in the culture medium with no serum level for about 60 days. Accordingly, on the day of initiating differentiation (day 0), human embryonic stem cells were incubated with collagenase for 10 minutes at 37 0 C and dissociated into small cell aggregates by trituration.
  • the aggregated stem cells were transferred onto a low binding tissue culture plate and cultivated in Basic Myocyte Medium (BMM) containing DMEM (high glucose) supplemented with 1 x MEM non-essential amino acids, 2 mM L-gluthamine, 5.5 ⁇ g/ml transferrin, 5 ng/ml sodium selenite, 0.1 mM ⁇ -mercaptoethanol, 5 ⁇ M p38 MAP kinase inhibitor SB203580, [serum].
  • BMM Basic Myocyte Medium
  • Wnt3a conditioned medium was added at this time point for 24 hours with varying concentrations of Wtn3, preferably at least about 30ng/mL.
  • Example 3 Effects of fi-catenin/canonical signaling pathway on mesendoderm formation.
  • Wnt2 increased the protein level of total ⁇ - catenin ⁇ 4-fold difference, but exerted negligible effect on the Brachyury gene expression and ⁇ -catenin activation ( Figure 1).
  • Wnt3a, BIO and IGFl equivalent Iy enhanced ⁇ -catenin stabilization; however, only Wnt3a highly increased Brachyury gene upregulation and ⁇ -catenin activation ( ⁇ 2.5 fold induction), whereas, BIO and IGFl had little or no effect (Figure 1 & Figure 14B).
  • Example 4 Wnt3a-specific induction of mesendoderm formation.
  • Example 5 Effects of fi-catenin/canonical signaling pathway on cell survival and EB formation.
  • hESC were treated with various Wnt iso forms, BIO, IGFl, PMA or ISO for 48 hours and cells were maintained in suspension as they formed embryoid bodies.
  • Wnt3a substantially increased EB formation and size, whereas the other factors had little or no effect. This effect by Wnt3a was dose-dependent, with the optimal efficacy between 30-100 ng/ml.
  • Example 6 Effects offi-catenin/canonical signaling pathway on cardiomyogenesis.
  • the differentiated cells were cultured in serum free medium.
  • Human ESC were treated as described above with various factors for the first 48 hours, the EB are attached to the gelatin-coated plate on day 6 and cultivated in medium containing 5% serum for two days. The EBs are further cultivated in serum- free medium for up to 60 days. Beating clusters usually emerged on day 8 and progressively increased with time until day 12 of differentiation. Without any treatment with Wnt3 for the first 48 hours, there were little or no beating cluster observed on day 12 of differentiation; however, after Wnt3a treatment, there were substantially more beating clusters observed (Figure 5A).
  • Example 7 Invariant effects of Wnt3a among different hESC lines. To determine whether Wnt3a could exert similar effects on other hESC lines including Hl and H7, these cell lines were treated with or without Wnt3a and analyzed for mesendoderm formation, cell viability and cardiomyogenesis. The results showed that Wnt3a highly induced gene upregulation of mesendoderm (Eomes), mesoderm (Mespl and Mesp2), and endoderm (Sox 17 and FoxA2), but not ectoderm (Otx2 and Soxl), lineages in both Hl and H7 (Figure 6A and 6D). Wnt3a also enhanced EB formation and cardiac differentiation in both of these cell lines ( Figure 6B-C and 6E-F, respectively).
  • Example 8 Characterization of beating clusters.
  • beating clusters contained cells expressing cardiac-related proteins such as cTnl (cardiac troponin I), ⁇ -actinin, Nkx2.5, and Mef2a. Quantitative scoring showed that there were approximately 50% cardio myocytes ( ⁇ -actinin positive cells) within the beating clusters ( Figure 7B&C). These beating clusters also highly expressed cardiac- related genes such as ⁇ -MHC and Nkx2.5 ( Figure 7D-E).
  • Example 9 Genome wide analysis of Wnt3a-inducing cardiomyogenesis.
  • Wnt3a Wnt3a. Many of these genes have been implicated as direct or indirect targets of the canonical/ ⁇ -catenin pathway (Table 1).
  • Table 1 is a list of significantly changed genes that are known as direct or indirect targets of Wnt3a.
  • CLDN11 claudin 11 oligodendrocyte transmembrane protein
  • DKK3 dickkopf homolog 3 (Xenopus laevis) 0.9 ⁇ 0.11 2.1 ⁇ 0.41 2.4 ⁇ 0.70**
  • WNT3 wingless-type MMTV integration site family member 3 1.0 ⁇ 0.18 3.2 ⁇ 0.99 3.2 ⁇ 0.58**
  • WNT8A wingless-type MMTV integration site family member 8A 1.0 ⁇ 0.18 4.7 ⁇ 1.29 4.9 ⁇ 1.42**
  • CDX1 caudal type homeobox 1 1.1 ⁇ 0.24 3.3 ⁇ 0.77 3.2 ⁇ 0.67**
  • DKK4 dickkopf homolog 4 (Xenopus laevis) 1.1 ⁇ 0.25 56.4 ⁇ 23.49 54.1 ⁇ 30.87*
  • WNT5B wingless-type MMTV integration site family member 5B 1.2 ⁇ 0.24 3.8 ⁇ 0.29 3.2 ⁇ 1.03*
  • MMP2 matrix metallopeptidase 2 1.5 ⁇ 0.12 3.9 ⁇ 0.69 2.6 ⁇ 0.3**
  • LEF1 lymphoid enhancer-binding factor 1 2.0 ⁇ 0.35 9.1 ⁇ 2.24 4.8 ⁇ 1.71**
  • ISL1 ISL LIM homeobox 1 2.1 ⁇ 1.20 7.2 ⁇ 2.97 3.9 ⁇ 1.79*
  • DKK1 dickkopf homolog 1 (Xenopus laevis) 2.3 ⁇ 0.89 65.4 ⁇ 20.24 30.2 ⁇ 7.93*
  • WNT5A wingless-type MMTV integration site family member 5A 3.1 ⁇ 0.46 15.7 ⁇ 2.79 5.1 ⁇ 1.44**
  • Table 2 is a list of significantly changed genes that are up-regulated by
  • CDH11 cadherin 11 type 2, OB-cadherin (osteoblast) NM_001797 4 0 ⁇ 1 7*
  • PDGFR platelet-derived growth factor receptor
  • alpha polypeptide NM_006206 9 4 ⁇ 2 1**
  • GAT A4 and GATA6 are zinc finger transcription factors that are essential for embryonic development. Homozygous GATA4 or GATA6 knockout mice died at embryonic day 9.5 or 6.5, respectively.
  • Eomesodermin and brachyury are transcription factors containing DNA- binding domain (T-domain) that are expressed early during gastrulation. They are shown to be important for mesoderm specification.
  • Goosecoid is a transcription factor containing a homeodomain. It also plays important roles in mesoderm specification.
  • Mixll is a transcription factor transiently expressed during embryogenesis that is implicated in mesoderm and endoderm specification.
  • Cadherin- 11 may be involved in the metastatic process of osteosarcoma. The correlation of the expression levels of CDHI l in osteosarcoma samples with the risk of disease progression and metastasis was examined. CDHI l may be useful as a prognostic marker of disease progression and survival in osteosarcoma. In addition, cadherin proteins in the developing olfactory system are positioned to underlie the formation of the odorant map and local circuits within the olfactory bulb.
  • PDGFRa The PDGF system comprises four isoforms (PDGF-A, -B, -C, and -
  • PDGFR-alpha and -beta two receptor chains
  • This growth factor plays important roles in wound healing, atherosclerosis, fibrosis, and malignancy.
  • PDGFRa might transmit a mitogen effect from the epicardium as source of secreted factors that influence cardiomyocyte proliferation.
  • Mespl is expressed in almost all precursors of the cardiovascular system and plays an essential role in cardiac morphogenesis. Mespl may play a key role in the early specification for cardiac precursor cells.
  • Mesp 2 The Notch signaling pathway and its regulators are major components of most of the events required for temporally- and spatially-coordinated somite formation. The link between the clock and segmental border formation is of fundamental importance during somitogenesis. During this process, Mesp2, a basic HLH protein, plays a critical role in the anterior presomitic mesoderm.
  • CXCR4 is uniquely associated with mesendoderm formation.
  • CXCR4(+) progenitors are mesoderm-restricted progeny according to co-expression with the early mesoderm marker FIk-I.
  • the CXCR4(+)/Flk-l(+) subpopulation overexpresses cardiac lineage transcription factors (Mef2C, Myocardin, Nkx2.5), while pluripotent genes (Oct4, Fgf4, Sox2) as well as neuroectoderm (Soxl) and endoderm (AFP) markers are depleted.
  • the CXCR4(+)/Flk-l(+) biomarker combination identified embryonic stem cell progeny significantly enriched with Mesp-1, GATA-4 and Tbx5, indicative of pre-cardiac mesoderm and the primary heart field.
  • Hhex expression starts soon after that of VEGFR2 in the blood islands. It is later expressed in the angioblasts and the developing vascular network and can be used as an early marker for endothelial precursor cells. Hhex is required for hemangioblast differentiation into both endothelial and hematopoietic cells.
  • CERl orthologs are evolutionarily conserved target of WNT and NODAL signaling pathways in non-rodent mammals.
  • Human CERl mRNA is expressed in embryonic stem (ES) cells in the undifferentiated state and in the early endodermal lineage.
  • CERl upregulation in human ES cells leads to Nodal signaling inhibition associated with differentiation of human ES cells.
  • the molecular evolution of CERl orthologs contributes to the significantly divergent scenarios of early embryogenesis in primates and rodents. Foxa2 is required for the formation of the node and notochord, and in its absence severe defects in gastrulation, neural tube patterning, and gut morphogenesis result in embryonic lethality.
  • Foxal and Foxa2 cooperate to establish competence in foregut endoderm and are required for normal development of endoderm-derived organs such as the liver, pancreas, lungs, and prostate.
  • endoderm-derived organs such as the liver, pancreas, lungs, and prostate.
  • members of the Foxa family control glucose metabolism through the regulation of multiple target genes in the liver, pancreas, and adipose tissue.
  • LHXl LIM homeobox 1.
  • Studies of mouse models and human patients have established that the LIM-HD factors are critical for the development of specialized cells in multiple tissue types, including the nervous system, skeletal muscle, the heart, the kidneys and endocrine organs such as the pituitary gland and the pancreas.
  • HMG-box (Sox 17) transcription factors are involved with the formation of definitive endoderm: both Mixll and the Soxl7 mutant embryos show a paucity of this tissue. In the Mixll mutant, very few Sox 17 and Cerl -expressing cells are present in the poorly formed embryonic foregut and none in the prospective hindgut. In Sox 17 mutants, the population of definitive endoderm is reduced severely and the domain normally occupied by the definitive endoderm is replaced by cells displaying molecular and functional characteristics of the visceral endoderm. Embryonic stem (ES) cells lacking either Mixll or Soxl7 activity are impaired in their ability to colonize the gut endoderm of the chimera.
  • ES Embryonic stem
  • Table 3 is a list of significantly changed genes that are down-regulated by Wnt3a.
  • FAM71 F1 family with sequence similarity 71 member F1 NM_032599 2 5 2 ⁇ 2 44*
  • DPP A5 developmental pluripotency associated 5
  • stem cells are down-regulated by Wnt3a and are described in more detail below.
  • DPP A5 developmental pluripotency associated 5
  • stem cells could serve as a potential stem cell marker.
  • ATP6V0A4 encodes H + -ATPase subunit whose destructive mutations lead to kidney disease known as autosomal recessive distal renal tubular acidosis (rdRTA).
  • LECTl also known as chondromodulin-I, is a secreted protein that possesses antiangiogenic activity. It has been shown to inhibit endothelial cell proliferation and angiogenesis.
  • PRDM 14 is a nuclear protein with potential histone methytransferase activity. It is shown to inhibit differentiation and maintain stem cell self-renewal capability.
  • INDO encodes the indoleamine-pyrrole 2,3 dioxygenase that plays important roles in the immune escape mechanism of tumors, probably by regulating the metabolism of tryptophan (an essential amino acid).
  • ADRA2A encodes the adrenergic receptor alpha 2A whose promoter polymorphisms are linked to the pathophysiology of attention deficit/hyperactivity disorder (ADHD).
  • TMEM 15 IB and FAM71F1 are putative genes with unknown function. Genes such as Nanog, CYP26A1, Lin28 and PRMD14, putative stem cell marker genes, were markedly decreased in later stage of differentiation ( Figure 9).
  • Wnt3a highly induced genes characteristic of mesendoderm (Wnt3, Gata4, Gsc, Mixll, T, Eomes, and Gata6), mesoderm (Tbx6, Kdr, Cdx2, Cdhl l, Mespl, Pdgfra, and Mesp2), and endoderm (Sox7, Pthrl, Cxcr4, Hhex, Lhxl, Foxa2, Cerl, and Soxl7) lineages (Figure 11). Wnt3a, on the other hand, had little or no effects on the expression of ectodermal marker genes (Pax6, Zicl, Soxl, Sox3, En2, Otx2, and Ncaml).
  • beating clusters exhibited enrichment of cardiac-specific genes ( Figure 12) such as transcription factors (Mef2a, Mef2c, Nkx2.5, Handl and Hand2), structural proteins (Tnni3, Tnnt2, Myh6, Myh7, Myl2, and MyB), calcium homeostatic proteins (PIn, ATP2A2, RYR2), and cardiac- related channels/receptors (Cavl.2, NCXl, and Adrb2).
  • transcription factors Mef2a, Mef2c, Nkx2.5, Handl and Hand2
  • structural proteins Tni3, Tnnt2, Myh6, Myh7, Myl2, and MyB
  • calcium homeostatic proteins PIn, ATP2A2, RYR2
  • cardiac-related channels/receptors Cavl.2, NCXl, and Adrb2
  • these beating clusters also exhibited little or no increase in the expression of marker genes for liver, pancreas, lung (endoderm lineage), blood (mesoderm lineage),
  • the findings reported here show the functional disparity among ⁇ -catenin regulating factors in specifying the differentiation of human embryonic stem cells toward the cardiac lineage.
  • the results show that (1) Wnt3a specifically enhanced hESC mesendoderm formation by inducing the expression of genes characteristic of mesendoderm, mesoderm, and endoderm, but not ectoderm, cell lineages; (2) Wnt3a substantially increased hESC EB formation and size without increasing cell proliferation; (3) Wnt3a significantly improved the differentiation of human ESC towards the cardiac lineage; (4) Wnt3a exerted these effects in a cell line independent manner; and (5) other Wnt isoforms (1, 2, or 7a), BIO, IGFl, PMA, or ISO, on the other hand, exerted little or no effect.
  • Treating hESC during the first 48 hours of EB formation with Wnt3a generated a large quantity of mesendodermal precursor cells.
  • the present findings using human embryonic stem cells showed that Wnt3a, but not a GSK3 ⁇ inhibitor, substantially increased the expression of genes characteristic of mesendoderm, mesoderm and endoderm lineages. This functional inconsistency of GSK3 ⁇ inhibition might simply be due to treatment duration (transient vs. chronic) and analysis time (day 2 vs. day 7).
  • Wnt3a's similar effect in promoting mouse and human ESC mesendoderm formation suggest an evolutionarily-conserved canonical Wnt/ ⁇ -catenin signaling pathway in the mammalian system.
  • Wnt3a enhanced EB formation and size, a result which was consistent with the cell survival studies reported by Dravid et al. and Davidson et al..
  • Dravid et al. show that treatment of hESC with Wnt3a increases the percentage of BrdU positively- stained cells from 23.3% to 34.0% with no apparent effect on the level of apoptosis.
  • Davidson et al. show that Wnt3a treatment increases hESC- derived neurosphere formation and size, whereas, Dkkl treatment has the opposite effect. They additionally demonstrated that Wnt3a promotes cell proliferation and prevents apoptosis.
  • neither study showed differentiation of the cells to cardiomyocytes using hESC.
  • BIO has been shown to increase cell proliferation in neonatal and adult rat cardiomyocytes; however, no such effect of BIO was observed on human EB formation and size in the present study. This discrepancy could probably be due to differences in cell type and cell maturity.
  • differentiated EB were cultivated in serum-free medium for up to at least 60 days.
  • This approach yields many advantages over the conventional differentiation protocol including an improvement in cardiac differentiation by excluding anti-cardiomyogenic factor(s) such as insulin, and the elimination of highly proliferating none-cardiac cells such as lung, pancreatic, hepatic, and blood cells, which are more sensitive to the lack of serum.
  • anti-cardiomyogenic factor(s) such as insulin
  • highly proliferating none-cardiac cells such as lung, pancreatic, hepatic, and blood cells, which are more sensitive to the lack of serum.
  • the results show that after 60 days in cultivation, beating clusters usually appeared as isolated clusters surrounded by small areas of cellular monolayers. Therefore, such serum free cultivation conditions can be used as a selection method for deriving a pure cell population of cardiomyocytes.
  • the Wnt3a directed differentiation method disclosed herein may facilitate technological advancements in the generation of cell line-specific homogenous induced pluripotent stem cell (iPS)-derived cardiac, pancreatic, skeletal muscle, lung, thyroid, blood, gut or hepatic cells by generating significant quantities of mesendodermal precursor cells, enhancing their viability, and increasing their potential to become cardiomyocytes and other mesendoderm derived cell lineages.
  • iPS induced pluripotent stem cell

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Abstract

La présente invention concerne des procédés pour induire une différenciation des cellules souches dans des cardiomyocytes ou autres lignées cellulaires différenciées définies par mise en contact des cellules souches avec Wnt3a ou un fragment fonctionnel de celui-ci. L’invention décrit également des méthodes de traitement de patients atteints de lésions myocardiques, par ex., après un infarctus du myocarde ou une insuffisance cardiaque.
PCT/EP2009/058936 2008-07-14 2009-07-13 Procédés pour améliorer la différenciation cardiaque des cellules souches embryonnaires humaines WO2010007031A2 (fr)

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