COMPOSITIONS AND METHODS OF GENERATING REPROGRAMMED ADIPOCYTE CELLS AND METHODS OF USE THEREFORE
The invention provides therapeutic compositions comprising reprogrammed adipocyte cells for use as disease models, therapeutic compositions comprising reprogrammed adipocyte cells for the treatment of conditions characterized by a reduction in cell number or tissue mass, and methods of generating such cells.
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This application claims the benefit of the following U.S. Provisional Application No. 61/100,946, filed Sep. 29, 2008, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTIONObesity is a disease in which the natural energy reserve, stored in the adipose tissue of humans and other mammals, is increased to a point where it is associated with adverse health effects and mortality. Obesity is a complex, multi-factorial disease involving environmental, genetic, and behavioral components. It is also the second leading cause of preventable death in the U.S. Adipose tissue secretes exocrine mediators that lead to endothelial dysfunction and atherosclerosis. Increased triglycerides, decreased HDL levels and abnormal LDL composition characterize the primary dyslipidemia related to obesity and no doubt play a major role in the development of atherosclerosis and cardiovascular disease in obese individuals. Obesity is also associated with type 2 diabetes, metabolic disorders, and premature mortality.
Despite recognition of the risks associated with obesity, methods for preventing or treating obesity and associated metabolic disorders are inadequate. Obesity continues to pose a significant public health problem. The identification of effective compositions for the prevention or treatment of obesity requires a better understanding of adipocyte biology. While mouse models for obesity exist, they represent rare single gene deletions that do not adequately recapitulate human disease processes, in part due to differences in mouse and human metabolism. Research into adipocyte biology has also been limited by the fact that a renewable source of adipocytes is unavailable. Although human adipose tissue is easily obtained, primary adipocytes are difficult to maintain in culture and are not amenable to expansion. As a consequence, in vitro systems for understanding mature primary adipocyte function do not exist.
SUMMARY OF THE INVENTIONAs described below, the present invention features methods for generating reprogrammed adipocyte cells, compositions comprising the reprogrammed adipocyte cells, and methods of using such cells.
In one aspect, the invention generally provides a method for generating a reprogrammed adipocyte, the method involving exogenously expressing in a pluripotent stem cell one or more adipogenic transcription factor polypeptides (e.g., PPARγ, CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBPβ, C/EBPδ C/EBPα and CDEC); and contacting the cell with one or more of insulin, rosiglitazone, dexamethasone and isobutylmethylxanthine, thereby generating a reprogrammed adipocyte.
In another aspect, the invention features a method for generating a reprogrammed adipocyte, the method involving exogenously expressing in a pluripotent stem cell a PPARγ2 polypeptide; and
contacting the cell with one or more of insulin, rosiglitazone, dexamethasone and isobutylmethylxanthine, thereby generating a reprogrammed adipocyte. In one embodiment, the cell is contacted with insulin and rosiglitazone.
In another aspect, the invention features a method for generating a reprogrammed adipocyte, the method involving exogenously expressing in a pluripotent stem cell a C/EBPα polypeptide; and contacting the cell with one or more of insulin, rosiglitazone, dexamethasone and isobutylmethylxanthine, thereby generating a reprogrammed adipocyte. In one embodiment, the cell is contacted with insulin.
In another aspect, the invention features a reprogrammed adipocyte generated according to any previous aspect or any method delineated herein. In one embodiment, the cell comprises a genetic alteration associated with a disease selected from the group consisting of Type 2 diabetes mellitus, insulin resistance, obesity, lipodystrophy, metabolic disorders, cardiac disease, early-onset myocardial infarction and laminopathies. In one embodiment, the cell is characterized for a single nucleotide polymorphism listed in Table 1, Table 2, or Table 3. In another embodiment, the cell is derived from an FPLD2 patient, a patient with type 2 diabetes, or a patient with early onset myocardial infarction In another embodiment, the cell from the FPLD2 subject has a LMNA mutation at R482W, H506D, R399H, R582H, T655fxX49, or L387V L421P. In yet another embodiment, the cell comprises a homologous recombination event, inhibitory nucleic acid molecule, or other genetic alteration that disrupts the function or activity of a polypeptide associated with the disease.
In another aspect, the invention features a reprogrammed adipocyte that exogenously expresses an adipogenic transcription factor polypeptide selected from the group consisting of PPARγ, CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBPβ, C/EBPδ C/EBPα, and CDEC, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function.
In yet another aspect, the invention features a reprogrammed adipocyte that exogenously expresses a PPARγ2 or C/EBPα polypeptide, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function.
In still another aspect, the invention features a reprogrammed adipocyte that exogenously expresses a polypeptide selected from the group consisting of C/EBPα, C/EBPβ and C/EBPδ, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function. In one embodiment, the adipocyte exogenously expresses C/EBPα and further expresses C/EBPβ and/or C/EBPδ.
In another aspect, the invention features a reprogrammed adipocyte that exogenously expresses a C/EBPα and a C/EBPβ polypeptide.
In yet another aspect, the invention features a method for identifying a therapeutic agent, the method involving contacting the reprogrammed adipocyte of any previous aspect with a candidate agent and identifying an alteration in a disease marker.
In another aspect, the invention features a method of ameliorating cell or tissue loss in a subject (e.g., human) in need thereof, the method involving delivering to the subject a cell generated according to a method of a previous aspect or any other method delineated herein.
In one embodiment, the cell or tissue loss is associated with trauma, cell death, or a congenital defect.
In another aspect, the invention features a collection of at least two (e.g., 2, 3, 4, 5, 6, 7) expression vectors, wherein each vector comprises a distinct nucleic acid sequence encoding a polypeptide selected from the group consisting of PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, CREB1 and KROX20. In one embodiment, one of the expression vectors encodes PPARγ2 and the other encodes C/EBPα. In another embodiment, one of the expression vectors encodes C/EBPα and the other encodes C/EBPβ. In another embodiment, one vector encodes PPARγ2 and the other encodes a polypeptide that is any one or more of C/EBPα, C/EBPβ and/or C/EBPδ. In one embodiment, each vector further comprises a promoter operably linked to the nucleic acid sequence. In another embodiment, the promoter is positioned for expression in a mammalian cell.
In another aspect, the invention features a host cell (e.g., human) comprising one or more of the expression vectors of a previous aspect or otherwise delineated herein. In one embodiment, the cell is a pluripotent or multipotent cell. In another embodiment, the cell is an adipocyte derived mesenchymal stem cell, human embryonic stem cell or induced pluripotent stem cell.
In another aspect, the invention features a pharmaceutical composition comprising a reprogrammed adipocyte according to any previous aspect in a pharmaceutically acceptable excipient.
In another aspect, the invention features a kit comprising one or more polynucleotides encoding one or more adipogenic transcription factor polypeptides and instructions for generating a reprogrammed adipocyte in accordance with any previous aspect.
In one embodiment, the adipogenic transcription factor polypeptides are encoded by an expression vector.
In another aspect, the invention features a kit comprising a reprogrammed adipocyte according to any previous aspect, and instructions for engraft went of the reprogrammed adipocyte in a subject.
In one embodiment of any of the above aspects or any other aspect of the invention delineated herein, the pluripotent stem cell is contacted with one or more of insulin, rosiglitazone and dexamethasone. In another embodiment, a pluripotent stem cell is contacted with insulin, rosiglitazone, dexamethasone and isobutylmethylxanthine. In various embodiments of any of the above aspects, the pluripotent stem cell is any one or more of an induced pluripotent stem cell, human embryonic stem cell, mesenchymal stem cell, adipocyte-derived mesenchymal stem cell, bone marrow derived stem cell and other mesenchymal stem cell. In one embodiment, an induced pluripotent stem cell is derived from a somatic cell (e.g., adipocyte, keratinocyte, epidermal cell, fibroblast, hematopoietic cell, peripheral blood mononuclear cell and their progenitor cells). In various embodiments of any of the above aspects, pluripotency is induced by expression of one or more of OCT4, SOX2 and either cMYC and KLF4 or NANOG and LIN28 in a somatic cell. For example, pluripotency is induced by expression of Oct4 and KLF4. In one embodiment of any of the above aspects, the pluripotent stem cell is contacted in vitro. In various embodiments of any of the above aspects, the method further comprises identifying an adipocyte phenotype by detecting an increase in an adipocyte marker, an adipocyte morphology, or adipocyte function that is not detectably expressed or expressed only nominally in a corresponding control cell. In other embodiments of any of the above aspects, the reprogrammed adipocyte expresses one or more adipocyte markers selected from the group consisting of CIDEC, FABP4, PPARγ2, adiponectin, leptin, and perilipin. In still other embodiments of any of the above aspects, the reprogrammed adipocyte comprises lipid droplets. In still other embodiments of any of the above aspects, the reprogrammed adipocyte responds to insulin, has lipolytic activity, displays de novo synthesis of fatty acids and/or incorporates free fatty acids. In still other embodiments of any of the above aspects, the adipogenic transcription factor polypeptide is any one (two, three, four, five, six) or more of an PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, CREB1 and KROX20. In one embodiment of any of the above aspects, the adipogenic transcription factor protein is PPARγ2, C/EBPα, C/EBPβ and/or C/EBPδ.
The invention provides therapeutic compositions comprising reprogrammed adipocyte cells, methods of generating such cells, and methods of using them to treat conditions characterized by a reduction in cell number or tissue mass. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
DEFINITIONSBy “OCT4 polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to OCT4 UniProtKB/Swiss-Prot Q01860 (PO5F1_HUMAN) and having DNA binding activity. An exemplary Oct 4 sequence is provided below:
By “Oct4 polynucleotide” is meant a polynucleotide encoding an Oct4 polypeptide. Exemplary Oct 4 polynucleotides (e.g., mRNA) are provided at DQ486513 NCBI Accession Nos. NM—002701 and NM—203289.
By “Sox2 polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to Sox2 UniProtKB/Swiss-Prot UniProtKB/Swiss-Prot P48431 and having DNA binding activity. An exemplary Sox2 amino acid sequence follows:
By “Sox2 polynucleotide” is meant a polynucleotide encoding a Sox2 polypeptide. An exemplary Sox2 polynucleotide sequence is provided at NCBI Accession No. NM—003106.
By “cMYC polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to cMYC UniProtKB/Swiss-Prot P01106 (MYC_HUMAN) and having DNA binding activity. An exemplary cMyc amino acid sequence follows:
By “cMYC polynucleotide” is meant a polynucleotide encoding a cMYC polypeptide. An exemplary cMYC polynucleotide sequence is provided at NCBI Accession No. NM—002467
By “KLF4 polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot 043474 (KLF4_HUMAN) and having DNA binding activity. An exemplary KLF4 amino acid sequence follows:
By “KLF4 polynucleotide” is meant a polynucleotide encoding a KLF4polypeptide. An exemplary KLF4 polynucleotide sequence is provided at NCBI Accession No.—NM—004235.
By “Nanog polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot Q9H9S0 (NANOG_HUMAN) and having DNA binding activity. An exemplary Nanog amino acid sequence follows:
By “Nanog polynucleotide” is meant a polynucleotide encoding a KLF4polypeptide. An exemplary Nanog polynucleotide sequence is provided at NCBI Accession No. NM—024865.
By “Lin28 polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot Q9H9Z2 (LN28A_HUMAN) and having DNA binding activity. An exemplary Lin28 amino acid sequence follows:
By “Lin28 polynucleotide” is meant a polynucleotide encoding a Lin28 polypeptide. An exemplary Lin28 polynucleotide sequence is provided at NCBI Accession No. NM—024674.
By “PPARγ2 polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P37231 (PPARG_HUMAN) and having DNA binding activity. An exemplary PPARγ2 amino acid sequence follows:
By “PPARγ2 polynucleotide” is meant a polynucleotide encoding a PPARγ2 polypeptide. An exemplary PPARγ2 polynucleotide sequence is provided at NCBI Accession No. NM—015869
By “CEBPα_polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P49715 (CEBPα_HUMAN) and having DNA binding activity. An exemplary CEBPα amino acid sequence follows:
By “CEBPα polynucleotide” is meant a polynucleotide encoding a CEBPα polypeptide. An exemplary CEBPα polynucleotide sequence is provided at NCBI Accession No. NM—004364
By “CEBPβ polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P17676 (CEBPβ_HUMAN) and having DNA binding activity. An exemplary CEBPβ amino acid sequence follows:
By “CEBPβ polynucleotide” is meant a polynucleotide encoding a CEBPβ polypeptide. An exemplary CEBPβ polynucleotide sequence is provided at NCBI Accession No. NM—005194.
By “CEBPδ polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P49716 (CEBPD_HUMAN) and having DNA binding activity. An exemplary CEBPδ amino acid sequence follows:
By “CEBPδ polynucleotide” is meant a polynucleotide encoding a CEBPδ polypeptide. An exemplary CEBPδ polynucleotide sequence is provided at NCBI Accession No. NM—005195.
By “SREBP1c polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P36956 (SRBP1_HUMAN) and having DNA binding activity. An exemplary SREBP1c amino acid sequence follows:
By “SREBP1c polynucleotide” is meant a polynucleotide encoding a SREBP1c polypeptide. An exemplary SREBP1c polynucleotide sequence is provided at NCBI Accession No. NM—001005291.
By “CREB1 polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P16220 (CREB1_HUMAN) and having DNA binding activity. An exemplary CREB1 amino acid sequence follows:
By “CREB1polynucleotide” is meant a polynucleotide encoding a CREB1polypeptide. An exemplary CREB1polynucleotide sequence is provided at NCBI Accession No. BC010636.
By “KROX20(EGR2) polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P11161 (EGR2_HUMAN) and having DNA binding activity. An exemplary KROX20 amino acid sequence follows:
By “KROX20 polynucleotide” is meant a polynucleotide encoding a KROX20 polypeptide. An exemplary KROX20 polynucleotide sequence is provided at NCBI Accession No.—BC35625.
By “KLF5 polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot Q13887 (KLF5_HUMAN) and having DNA binding activity. An exemplary KLF5 amino acid sequence follows:
By “KLF5 polynucleotide” is meant a polynucleotide encoding a KLF5 polypeptide. An exemplary KLF5 polynucleotide sequence is provided at NCBI Accession No.—NM—001730.
By “KLF15 polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot Q9UIH9 (KLF15_HUMAN) and having DNA binding activity. An exemplary KLF15 amino acid sequence follows:
By “KLF15 polynucleotide” is meant a polynucleotide encoding a KLF15 polypeptide. An exemplary KLF15 polynucleotide sequence is provided at NCBI Accession No. NM—014079
By “CIDEC polypeptide” is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot Q96AQ7 (CIDEC_HUMAN) and having DNA binding activity. An exemplary CIDEC amino acid sequence follows:
By “CIDEC polynucleotide” is meant a polynucleotide encoding a CIDEC polypeptide. An exemplary CIDEC polynucleotide sequence is provided at NCBI Accession No. NM—022094.
By “adipogenic” is meant inducing one or more of an adipocyte marker, adipocyte function, or adipocyte morphology in a multipotent or pluripotent stem cell.
By “induced pluripotent stem cell” is meant a differentiated somatic cell that acquires pluripotency by the exogenous expression of one or more transcription factors in the cell.
By “reprogrammed adipocyte” is meant a pluripotent cell that is induced to express one or more of an adipocyte marker, adipocyte function, or adipocyte morphology by the exogenous expression of one or more transcription factors in the cell.
By “adipocyte marker” is meant one or more of Glut4, PPARy, C/EBPα, C/EBPβ, C/EBPδ, leptin, adiponectin, SREBP1c, FABP4, CIDEC and/or perilipin.
By “adipocyte morphology” is meant the presence of lipid droplets.
By “adipocyte function” is meant the ability to respond to insulin, have lipolytic activity, display de novo synthesis of fatty acids, and or incorporate free fatty acids. In one embodiment, the adipocyte is engrafted in a host.
By “alteration” is meant a change (increase or decrease) in the expression levels of a gene or polypeptide as detected by standard art known methods such as those described above. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
By “analog” is meant a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.
By “autologous” is meant cells from the same subject.
By “compound” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
The term “engraft” as used herein refers to the process of stem cell incorporation into a tissue of interest in vivo through contact with existing cells of the tissue.
By “exogenously expressed” is meant expressing a polypeptide or polynucleotide that is not naturally expressed at a functionally significant level in the cell. For example, a recombinant polypeptide that is introduced into the cell using an expression vector is an example of an exogenously expressed polypeptide. In other example, the cell expresses a heterologous polypeptide or polynucleotide.
A “labeled nucleic acid or polypeptide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic bonds, van der Waals forces, electrostatic attractions, hydrophobic interactions, or hydrogen bonds, to a label such that the presence of the nucleic acid or probe may be detected by detecting the presence of the label bound to the nucleic acid or probe.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
By “fusion protein” is meant a protein that combines at least two amino acid sequence that are not naturally contiguous.
By “identity” is meant the amino acid or nucleic acid sequence identity between a sequence of interest and a reference sequence. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes listed in Tables 1 and 2), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and most preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “increases or decreases” is meant a positive or negative alteration. Such alterations are by 5%, 10%, 25%, 50%, 75%, 85%, 90% or even by 100% of a reference value.
By “isolated” is meant a material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings.
By “isolated nucleic acid molecule” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In one embodiment, the preparation is at least 75%, 85%, 90%, 95%, or at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
By “matrix” is meant a medium that provides for the survival, proliferation, or growth of one or more cells. In one embodiment, a matrix is a cell scaffold comprising a biodegradable medium.
By “naturally occurs” is meant is endogenously expressed in a cell of an organism.
By “obtaining” as in “obtaining the polypeptide” is meant synthesizing, purchasing, or otherwise acquiring the polypeptide.
By “operably linked” is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.
By “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification.
By “positioned for expression” is meant that the polynucleotide of the invention (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant polypeptide of the invention, or an RNA molecule).
By “promoter” is meant a polynucleotide sufficient to direct transcription. Exemplary promoters include nucleic acid sequences of lengths 100, 250, 300, 400, 500, 750, 900, 1000; 1250, and 1500 nucleotides that are upstream (e.g., immediately upstream) of the translation start site.
The term “self renewal” as used herein refers to the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter cells with development potentials that are indistinguishable from those of the mother cell. Self renewal involves both proliferation and the maintenance of an undifferentiated state.
The term “stem cell” is meant a pluripotent cell or multipotent stem cell having the capacity to self-renew and to differentiate into multiple cell lineages.
By “stem cell generation” is meant any biological process that gives rise to stem cells. Such processes include the differentiation or proliferation of a stem cell progenitor or stem cell self-renewal.
By “stem cell progenitor” is meant a cell that gives rise to stem cells.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
By “syngeneic,” as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
By “reference” is meant a standard or control condition.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
The invention provides therapeutic compositions comprising reprogrammed adipocyte cells for use as disease models, therapeutic compositions comprising reprogrammed adipocyte cells for the treatment of conditions characterized by a reduction in cell number or tissue mass, and methods of generating such cells.
The invention is based, at least in part, on the discovery of a method for the directed differentiation of human pluripotent cells into adipocytes. Known regulators of adipogenesis were cloned into a doxycycline-inducible lentiviral backbone (e.g. PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP-1c, CREB1, and KROX20). As reported herein, the adipogenic activity of PPARγ2, C/EBPα, C/EBPβ or C/EBPδ was demonstrated by ectopically expressing them in human pluripotent cells. The viral transduction and inducible expression of PPARγ2 and C/EBP factors in human pluripotent cells combined with the addition of insulin, rosiglitazone, dexamethasone, and isobutylmethylxanthine to the cells' growth medium resulted in the appearance of lipid filled cells with large monolocular lipid droplets. These characteristics are hallmarks of human adipose tissue. Importantly, these cells were positive for the mature adipocyte markers CIDEC, FABP4 and perilipin.
Accordingly, the invention provides a renewable source of reprogrammed adipocyte cells (also termed reprogrammed adipocytes). Using this protocol, pluripotent cell derived adipocytes were routinely generated at an efficiency of ˜20%. Such cells are useful not only for in therapeutic applications, but also for the study of diseases associated with adipose dysfunction. Somatic cells may be obtained from subjects having a genetic disorder of interest or genetic mutations may be introduced, for example, by homologous recombination. Adipocytes or reprogrammed adipocyte cells are then generated from these genetically altered cells according to the methods of the invention. Cells having one or more genetic alterations are useful for the study of diseases. In particular, the invention provides cellular disease models for all diseases related to metabolic syndrome, including metabolic syndrome, Type 2 diabetes mellitus, insulin resistance, obesity, lipodystrophy, metabolic disorders, cardiac disease, early-onset myocardial infarction, and laminopathies.
ObesityObesity is a disease in which natural energy reserves that are stored in the fatty (adipose) tissue of humans and other mammals is substantially increased to a point where it is associated with adverse health effects and mortality. Obesity is a complex, multi-factorial disease'involving environmental, genetic, and behavioral components. It is also the second leading cause of preventable death in the U.S. Obesity substantially increases the risk of developing a number of related diseases, such as cardiovascular disease, type 2 diabetes, and cancer. Current estimates suggest that as many as 60 million Americans are obese (1 in every 3), and 9 million are severely obese. Alarmingly, the prevalence of obesity has almost tripled in adults and children over the past 50 years. Each year, obesity causes at least 300,000 excess deaths in the U.S., and healthcare costs associated with obesity are approximately $100 billion. Statistics such as these have caused many to view obesity as a national pandemic.
Adipose Tissue and Adipogensis.Adipose tissue is present in all mammals, as well as in a variety of non-mammalian species. Adipocytes play a central role in energy homeostasis, and they act as an integrator of various physiological pathways. Adipocytes store and release energy and regulate the balance of nutrients in the blood. Through the release of adipokines, such as leptin and adiponectin, adipose tissue communicates with other regulators of energy homeostasis like the central nervous system, pancreas, and liver. Adipose tissues differ from many other tissues in that they occur in multiple, dispersed sites around the body. In general, there are two types of adipose tissue, brown adipose tissue and white adipose tissue. Brown adipose tissue is only present in significant amounts peri-naturally in humans, where its primary function is to dissipate energy in the form of heat. White adipose tissue (WAT) consists of deposits of fat cells (adipocytes) and supporting tissue types that are located principally in three anatomical areas—subcutaneous, dermal, and intraperitoneal. It is the last of these depots, also known as visceral adipose tissue that poses the greatest health risk when enlarged. The combination of various adipose tissue depots is often referred to as the ‘adipose organ’.
In general, adipose depots are comprised of five cell types: adipocytes, endothelia, fibroblast/stromal vascular cells, immune cells and nerves. Adipocytes are the primary cellular component of adipose tissue. Adipocytes comprise as much as 80% of the adipose depot. Each adipocyte is in close proximity to a blood vessel (capillary) and the adipose tissue is surrounded by and often interlaced with fibroblast/stromal vascular cells and a number of immune cells such as macrophages. Adipose tissue is innervated by sympathetic and sensory nerves. White adipose tissue is the only tissue in the body that can markedly change its mass after adult size is reached. In fact, fat mass can range from 2-3% of body weight to as much as 60-70% of body weight in humans. In obesity, fat mass typically exceeds 22% of body weight in males and 32% in females. The development of obesity is thought to be dependent on both an increase in fat cell size (hypertrophy) and fat cell number (hyperplasia).
LipodystrophyLipodystrophy is a disorder of adipose tissue characterized by the selective loss of body fat. Patients with lipodystrophy have a tendency to develop insulin resistance, diabetes, high triglyceride levels (hypertriglyceridemia), and fatty liver. There are numerous forms of lipodystrophy. These forms may be characterized as genetic or acquired forms of the disease. The genetic forms of lipodystrophy include congenital generalized lipodystrophy (the Berardinelli-Seip syndrome) and several types of familial partial lipodystrophy (the Dunnigan type, the Kobberling type, and the mandibuloacral dysplasia type). The acquired forms of lipodystrophy include acquired generalized lipodystrophy (the Lawrence syndrome), acquired partial lipodystrophy (the Barraquer-Simons syndrome), and lipodystrophy induced by protease inhibitors used to treat HIV.
By far the most common form of lipodystrophy is HIV-associated lipodystrophy. This syndrome occurs in individuals with HIV infection who are being treated with antiretroviral medications. Although the term HIV-associated lipodystrophy refers to abnormal central fat accumulation (lipohypertrophy) and localized loss of fat tissue (lipoatrophy), some patients have only lipohypertrophy, some have only lipoatrophy, and, less commonly, a subset of patients exhibits a mixed clinical presentation. Lipohypertrophy in this syndrome is characterized by the presence of an enlarged dorsocervical fat pad, circumferential expansion of the neck, breast enlargement, and abdominal visceral fat accumulation. Lipoatrophy is exemplified by peripheral fat wasting with loss of subcutaneous tissue in the face, arms, legs, and buttocks. Involvement of the face is most common and carries a social stigma that may negatively affect the quality of life of patients with HIV disease and may pose a barrier to treatment and reduce medical adherence. Other features of HIV lipodystrophy syndrome include hyperlipidemia, insulin resistance, hyperinsulinemia, and hyperglycemia. Patients with HIV lipodystrophy syndrome are at increased risk for the development of atherosclerosis and diabetes mellitus.
Laminopathy and Dunnigan-Type Familial Partial Lipodystrophy Syndrome Type 2A group of human genetic disorders, referred to as laminopathies, are associated with defects in A-type lamins (lamin A/C, or LMNA) and their binding partners. The lamin protein is found at the inner surface of the nuclear envelope where it supports the structural stability of the nucleus and binds to numerous nuclear proteins. Mutations in LMNA are responsible for nine distinct clinical syndromes (e.g., Hutchinson-Guilford progeria, Emery-Dreifuss muscular dystrophy, and dilated cardiomyopathy). It is still unclear how mutations in LMNA promote particular disease phenotypes, and why certain mutations can give rise to tissue-specific effects. It has been proposed that the different phenotypes are due to the different influences on each mutation in LMNA: 1) structural integrity of nuclear envelope, 2) interaction with nuclear proteins, and 3) regulation of the expression of transcription factors.
Dunnigan-type familial partial lipodystrophy syndrome type 2 (FPLD2) is an autosomal dominant genetic disorder caused by a number of mutations in LMNA gene. Interestingly, mutations associated with FPLD2 are clustered in the C-terminal domain of the protein, which has been implicated in DNA binding as well as binding to nuclear scaffold proteins and transcription factors such as SREBP1, which is an adipogenic transcription factor. The major clinical feature of FPLD2 is fat loss in the limbs and trunk, with elevated fat storage in the neck and face. Patients with FPLD2 have normal peripheral adipose tissue but experience fat loss at the onset of puberty. Due to the selective fat loss, they exhibit metabolic dysfunction such as insulin resistance, glucose intolerance, lowered plasma high-density-lipoprotein cholesterol, and accumulation of plasma triglycerides. They often develop diabetes mellitus, hypertriglyceridemia, and early-onset atherosclerosis. While the physiological consequences of LMNA mutations have been described, very little about the molecular events underlying the disease is known because of the absence of an accurate model system. Although animal models have informed our understanding of the molecular mechanisms underlying human diseases, they often fail to produce relevant and informative phenotypes. Indeed, Lamin A null mice do not phenocopy human FPLD2. Reprogrammed adipocyte cells may be generated according to the methods of the invention from subjects identified as having an LMNA mutations.
Stem CellsAs reported herein stem cells are generated from any of a variety of sources. As reported herein below, induced pluripotent stem cells are generated from somatic cells by introducing a combination of two, three or four of the following transcription factors (OCT4, SOX2, and either cMYC and KLF4 or NANOG and L1N28). In one embodiment, OCT4 and KLF4 are used to induce pluripotency. Virtually any somatic cell known in the art can be induced to become a pluripotent cell. Somatic cells particularly useful in the methods of the invention include but are not limited to cells (fibroblasts, keratinocytes) obtained in skin punch biopsies, hair follicles, and peripheral blood mononuclear cells (PBMC) fractions from blood draws. Such cells can be induced to become stem cells using the methods described herein. Other cells useful in the methods of the invention include embryonic stem cells, adipose stem cells, mesenchymal stem cells, and hematopoietic stem cells, and all those known in the art that have been identified in mammalian organs or tissues.
The hematopoietic stem cell, isolated from bone marrow, blood, cord blood, fetal liver and yolk sac, is the progenitor cell that generates blood cells or following transplantation reinitiates multiple hematopoietic lineages and can reinitiate hematopoiesis for the life of a recipient. (See Fei, R., et al., U.S. Pat. No. 5,635,387; McGlave, et al., U.S. Pat. No. 5,460,964; Simmons, P., et al., U.S. Pat. No. 5,677,136; Tsukamoto, et al., U.S. Pat. No. 5,750,397; Schwartz, et al., U.S. Pat. No. 5,759,793; DiGuisto, et al., U.S. Pat. No. 5,681,599; Tsukamoto, et al., U.S. Pat. No. 5,716,827; Hill, B., et al. 1996.)
It is well known in the art that hematopoietic cells include pluripotent stem cells and multipotent progenitor cells. Hematopoietic stem cells can be obtained from blood products. A “blood product” as used in the present invention defines a product obtained from the body or an organ of the body containing cells of hematopoietic origin. Such sources include unfractionated bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph and spleen. It will be apparent to those of ordinary skill in the art that all of the aforementioned crude or unfractionated blood products can be enriched for cells having “hematopoietic stem cell” characteristics in a number of ways. For example, the blood product can be depleted from the more differentiated progeny.
The embryonic stem (ES) cell has unlimited self-renewal and pluripotent differentiation potential (Thomson, J. et al. 1995; Thomson, J. A. et al. 1998; Shamblott, M. et al. 1998; Williams, R. L. et al. 1988; Orkin, S. 1998; Reubinoff, B. E., et al. 2000). These cells are derived from the inner cell mass (ICM) of the pre-implantation blastocyst (Thomson, J. et al. 1995; Thomson, J. A. et al. 1998; Martin, G. R. 1981), or can be derived from the primordial germ cells from a post-implantation embryo (embryonal germ cells or EG cells). ES and/or EG cells have been derived from multiple species, including mouse, rat, rabbit, sheep, goat, pig and more recently from human and human and non-human primates (U.S. Pat. Nos. 5,843,780 and 6,200,806).
Embryonic stem cells are well known in the art. For example, U.S. Pat. Nos. 6,200,806 and 5,843,780 refer to primate, including human, embryonic stem cells. U.S. Patent Applications Nos. 20010024825 and 20030008392 describe human embryonic stem cells. U.S. Patent Application No. 20030073234 describes a clonal human embryonic stem cell line. U.S. Pat. No. 6,090,625 and U.S. Patent Application No. 20030166272 describe an undifferentiated cell that is stated to be pluripotent. U.S. Patent Application No. 20020081724 describes what are stated to be embryonic stem cell derived cell cultures.
Stem cells of the present invention also include mesenchymal stem cells. Mesenchymal stem cells, or “MSCs” are well known in the art. MSCs, originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. During embryogenesis, the mesoderm develops into limb-bud mesoderm, tissue that generates bone, cartilage, fat, skeletal muscle and endothelium. Mesoderm also differentiates to visceral mesoderm, which can give rise to cardiac muscle, smooth muscle, or blood islands consisting of endothelium and hematopoietic progenitor cells. Primitive mesodermal or MSCs, therefore, could provide a source for a number of cell and tissue types. A number of MSCs have been isolated. (See, for example, Caplan, A., et al., U.S. Pat. No. 5,486,359; Young, H., et al., U.S. Pat. No. 5,827,735; Caplan, A., et al., U.S. Pat. No. 5,811,094; Bruder, S., et al., U.S. Pat. No. 5,736,396; Caplan, A., et al., U.S. Pat. No. 5,837,539; Masinovsky, B., U.S. Pat. No. 5,837,670; Pittenger, M., U.S. Pat. No. 5,827,740; Jaiswal, N., et al., (1997). J. Cell Biochem. 64(2):295-312; Cassiede P., et 4 (1996). J Bone Miner Res. 9:1264-73; Johnstone, B., et al., (1998) Exp Cell Res. 1:265-72; Yoo, et 4 (1998) J Bon Joint Surg Am. 12:1745-57; Gronthos, S., et al., (1994). Blood 84:4164-73); Pittenger, et al., (1999). Science 284:143-147.
Mesenchymal stem cells are believed to migrate out of the bone marrow, to associate with specific tissues, where they will eventually differentiate into multiple lineages. Enhancing the growth and maintenance of mesenchymal stem cells, in vitro or ex vivo will provide expanded populations that can be used to generate new tissue, including breast, skin, muscle, endothelium, bone, respiratory, urogenital, gastrointestinal connective or fibroblastic tissues.
Adipocyte stem cells are isolated from adipose tissue. Adipose tissue has been shown to contain a population of cells that retain a high proliferation capacity in vitro and the ability to undergo differentiation into multiple cell lineages in vitro. These cells are referred to as adipose stem cells and are biologically similar, although not identical, to mesenchymal stem cells derived from the bone marrow. Differentiation causes stem cells to adopt the phenotypic, biochemical, and functional properties of more terminally differentiated cells. Such differentiation is achieved using the methods described herein.
Biological samples may comprise mixed populations of cells, which can be purified to a degree sufficient to produce a desired effect. Those skilled in the art can readily determine the percentage of stem cells or their progenitors in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Purity of the stem cells can be determined according to the genetic marker profile within a population.
In several embodiments, it will be desirable to purify the cells before, during, or after the differentiation protocol. Cells of the invention (e.g., induced pluripotent stem cells, embryonic, mesenchymal, hematopoietic, adipose stem cells, differentiated adipocytes or reprogrammed adipocyte cells) preferably comprise a population of cells that have about 50-55%, 55-60%, 60-65% and 65-70% purity (e.g., non-stem and/or non-progenitor cells have been removed or are otherwise absent from the population). More preferably the purity is about 70-75%, 75-80%, 80-85%; and most preferably the purity is about 85-90%, 90-95%, and 95-100%.
Methods of Characterizing Induced Pluripotent Stem CellsA number of standard analyses are used to determine whether human iPS cells are completely reprogrammed to a stable pluripotent state. At a molecular level, human iPS cells are tested for the expression of pluripotency marker genes (e.g., SSEA-3/4, Tra-1-60/−81) and the down-regulation of lineage-specific genes associated with fibroblasts. Such alterations in gene expression may be confirmed by qRT-PCR and/or immunostaining. Epigenetic profiling is performed to determine whether the human iPS cells from FPLD2 patients are similar to hES cells. Pluripotency is also assessed by gene expression analysis and compared to similar analysis of human embryonic stem cell lines. At a functional level, human iPS cells are tested for the ability to differentiate into all three germ layers in vitro via embryoid body formation and in vivo by the formation of teratomas when injected into nude mice. To ensure that the cells have acquired genetic aberrations during in vitro culture, karyotyping and DNA fingerprinting is carried out. Fully reprogrammed human iPS are expanded and stocks are frozen for subsequent use.
Reprogrammed Adipocyte CellsOnce obtained from a desired source, a stem cell or stem progenitor cell is maintained in culture. Employing the culture conditions described in greater detail in the Examples, it is possible to preserve stem cells of the invention and to stimulate the expansion of stem cell number in their undifferentiated state. Differentiation is accomplished by reprogramming pluripotent (e.g. human embryonic stem cells (hESC) and human induced pluripotent stem cells (hIPS)) or multipotent cells (e.g. adipose tissue-derived mesenchymal stem cells (ADMSCs)) into adipocytes. In general, the method involves the ectopic expression of an adipogenic transcription factors that is any one or more of KROX20, C/EBPalpha, C/EBPbeta C/EBPdelta, CREB1, PPARg2 and SREBP1. These transcription factors are associated with adipocyte differentiation and adipocyte identity. Overexpression of one or more of these factors reprograms the pluripotent or multipotent cells into a fat storing, reprogrammed adipocyte cell fate. In addition to the expression of one or more of the genes listed above, donor cells are cultured in adipogenic differentiation media. In specific embodiments, the adipogenic differentiation media includes KO (knockout)-DMEM, KO-DMEM, Plasmanate, antibiotics (e.g., Penicillin/Streptomycin), Gluta-Max™, 2-Mercaptoethanol, non-essential amino acids, insulin (e.g., 10 μg/ml), dexamethasone (e.g., 1.2 μM), rosiglitazone (e.g., 0.5 μM) and IBMX (e.g., 0.5 mM).
Cells have been transduced with a virus that allows the inducible over-expression of PPARγ2. This results in the generation of large lipid filled structures (as determined by Oil Red O staining) and the production of adipocyte specific proteins such as Perilipin and FABP4/aP2 (as demonstrated by immunocytochemistry).
In certain embodiments, except as otherwise provided, the media used is that which is conventional for culturing cells. Appropriate culture media can be a chemically defined serum-free media such as the chemically defined media RPMI, DMEM, Iscove's, etc or so-called “complete media”. Typically, serum-free media are supplemented with human or animal plasma or serum. Such plasma or serum can contain small amounts of growth factors. The media used according to the present invention, however, can depart from that used conventionally in the prior art.
The cells are treated with agents to induce differentiation. Treatment of the stem cells or support cells of the invention may involve variable parameters depending on the particular type of agent used. In one embodiment, known regulators of adipogenesis are expressed in the stem cells. Such regulators include any one or more of PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, CREB1, and KROX20. In one embodiment, PPARγ2, C/EBPα, C/EBPβ and/or C/EBPδ are expressed in human stem cells where they induce adipogenesis. In yet another embodiment, inducible forms of PPARγ2 and C/EBP factors are expressed in the stem cells. The stem cells are contacted with any one or more of insulin, rosiglitazone, dexamethasone, and isobutylmethylxanthine. It is well within the level of ordinary skill in the art for practitioners to vary the parameters accordingly.
Cells of the invention are cultured on a commercially available extracellular matrix, such as Mantel.
Methods of Characterizing Reprogrammed Adipocyte CellsAdipocytes display a number of morphological and molecular hallmarks.
Characteristic adipocyte features are assayed in reprogrammed adipocyte cells by characterizing general cell morphology by microscopy using immunostainings, BODIPY neutral lipid dye, Oil-Red-O staining and/or electron microscopy. Reprogrammed adipocyte cells may also be characterized using qRT-PCR assays, global transcriptional profiling, and Western blot analysis of key adipose proteins.
In one embodiment, reprogrammed adipocyte cells are assayed for the expression of one or more of the following adipocyte markers: PPARy, C/EBPa, C/EBPb, C/EBPd, leptin, adiponectin, SREBP1c, FABP4, CIDEC. Each of these adipocyte markers is normalized to the housekeeping gene HPRT. Levels of these markers can be assayed at the polypeptide or polynucleotide level.
In another embodiment, immunocytochemistry is used to assay for any one or more of the following adipocyte markers: FABP4, CIDEC, PPARy, and/or Glut4.
Clinical features of particular diseases or conditions are characterized in reprogrammed adipocyte cells having a desired genetic alteration. In one embodiment, such cells are generated from patient's having the disease or condition. Adipocyte pathology may be assayed by detecting any one or more of the following: alterations in lipid droplet size within a cell, alterations in the accumulation of triglyceride as an indication of lipogenesis, alterations in the release of glycerol as an indication of lipolysis. An increase or decrease in any of the aforementioned parameters is detected by comparing normal adipocytes to adipocytes obtained from a subject having a particular disease or condition, or to adipocytes containing a desired genetic mutation. Patients affected with FPLD2 often present with metabolic complications such as glucose intolerance, insulin resistant, and fatty liver. Reprogrammed adipocyte cells derived from such patients may be characterized for glucose uptake. Of particular interest are cells comprising a L387V and L421P mutations.
In other embodiments, reprogrammed adipocyte cells of the invention are analyzed for the response to insulin by measuring (U-14C)-D-glucose uptake (Kashiwagi et al., J. Clin Invest 72 (4), 1246-1254 (1983)1983), lipolytic activity by inducing lipolysis using β-adrenergic receptor agonists and measuring glycerine concentration using a glycerol assay kit that employs an ELISA-based coupled enzymatic reaction, de novo synthesis of fatty acids using (1-14C) acetate and incorporation of free fatty acids using (3H) oleate or (14C) palmitate.
In other embodiments, reprogrammed adipocyte cellular function is evaluated by transplanting the reprogrammed adipocyte cells into an immunocompromised mouse, sectioning the implanted fat pads, and looking at vascularization by the host, and/or assaying the blood for human leptin using an immunoassay (e.g., ELISA).
Tissue RepairThe invention features methods of repairing damaged tissues using reprogrammed adipocytes. Reprogrammed adipocytes are cultured and expanded in vitro. Therapeutic compositions comprising the cells are administered to a damaged or diseased tissue. For example, adipocytes of the invention are useful for the treatment of congenital deformities, posttraumatic repair, cancer rehabilitation, and other soft tissue defects. Cosmetic surgery often requires the application of adipose tissue. Traditional methods of soft tissue reconstruction, as described in U.S. Pat. No. 5,716,404, can be improved by administering reprogrammed adipocytes. For example, engineered soft tissue is useful for cosmetic surgery or for reconstruction of the breast, face, or other body part after cancer surgery or trauma.
Clinical Correction of MalnutritionPatients with profound malnutrition often require a source of metabolic energy, which has been traditionally provided by enteral or venous administration of lipids. These are damaging procedures with known complications including, fat emboli syndrome, TPN-induced hepatitis, and cholestatis. The present invention provides transplantable cells, which can contribute metabolic energy.
Administration of Reprogrammed Adipocyte CellsAn reprogrammed adipocyte cell of the invention is administered according to methods known in the art. Such compositions may be administered by any conventional route, including injection or by gradual infusion over time. The administration may, depending on the composition being administered, for example, be, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. The stem cells are administered in “effective amounts”, or the amounts that either alone or together with further doses produces the desired therapeutic response.
Administered cells of the invention can be autologous (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). Generally, administration of the cells can occur within a short period of time following differentiation in culture (e.g. 1, 2, 5, 10, 24 or 48 hours after treatment) and according to the requirements of each desired treatment regimen.
Stem Cell Related Pharmaceutical CompositionsAn reprogrammed adipocyte cell of the invention may be combined with pharmaceutical excipients known in the art to enhance preservation and maintenance of the cells prior to administration. In some embodiments, cell compositions of the invention can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the cells utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
A method to potentially increase cell survival when introducing the cells into a subject in need thereof is to incorporate stem cells of interest into a biopolymer or synthetic polymer. Depending on the subject's condition, the site of injection might prove inhospitable for cell seeding and growth because of scarring or other impediments. Examples of biopolymer include, but are not limited to, cells mixed with fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. This could be constructed with or without included expansion or differentiation factors. Additionally, these could be in suspension, but residence time at sites subjected to flow would be nominal. Another alternative is a three-dimensional gel with cells entrapped within the interstices of the cell biopolymer admixture. Again, expansion or differentiation factors could be included with the cells. These could be deployed by injection via various routes described herein.
Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the stem cells or their progenitors as described in the present invention. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.
One consideration concerning the therapeutic use of stem cells is the quantity of cells necessary to achieve an optimal effect. Different scenarios may require optimization of the amount of cells injected into a tissue of interest. Thus, the quantity of cells to be administered will vary for the subject being treated. The precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, sex, weight, and condition of the particular patient. As few as 100-1000 cells can be administered for certain desired applications among selected patients. Therefore, dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.
The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or carrier in compositions and to be administered in methods of the invention. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.
Methods for Creating Genetically Altered Stem CellsGenetic alteration of a stem cell includes all transient and stable changes of the cellular genetic material relative to a wild-type reference sequence. In one embodiment, stem cells having genetic alterations of interest are isolated from subjects that have the genetic alteration. In another embodiment, stem cells having a desired mutation are generated by homologous recombination. In still other embodiments, genetic alterations are created by the addition of exogenous genetic material. In one embodiment, a population of cells that includes stem cells are transfected with an inhibitory nucleic acid molecule (e.g., siRNA, shRNA, antisense oligonucleotides). Such nucleic acid molecules inhibit the expression of a gene of interest (e.g., a gene associated with metabolic syndrome, Type 2 diabetes mellitus, insulin resistance, obesity, lipodystrophy, metabolic disorders, cardiac disease, early-onset myocardial infarction, and laminopathies). In one approach, an inhibitory nucleic acid molecule is introduced directly into a target cell, such as an induced pluripotent stem cell, human embryonic stem cell or other stem cell, such that the inhibitory nucleic acid molecule reduces expression of a gene of interest in the cell. In another approach, the target cell is transduced with an expression vector that encodes an inhibitory nucleic acid molecule. Expression of the inhibitory nucleic acid molecule in the target cell reduces target gene expression. Other exemplary genetic alterations include any gene therapy procedure, such as introduction of a mutated gene to replace an wild-type gene or introduction of a vector that encodes a dominant negative gene product. Exogenous genetic material includes nucleic acids or oligonucleotides, either natural or synthetic, that are introduced into the stem cells.
In particular, the invention provides adipocyte cellular disease models having genetic alterations in genes associated with metabolic syndrom, Type 2 diabetes mellitus, insulin resistance, obesity, lipodystrophy, metabolic disorders, cardiac disease, early-onset myocardial infarction, and laminopathies.
Exogenous Polypeptide ExpressionAs described herein, pluripotency is induced by expressing in a somatic cell a lentiviral vector encoding OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. In one embodiment, Oct4 and KLF4 are introduced. The stem cells and induced pluripotent cells described herein are then modified to express transcription factor polypeptides (e.g., PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, CREB1, and KROX20). If desired, cells of the invention can be further modified to express any other polypeptide of interest. Expression of these polypeptides is not limited to the vectors and methods described herein. Various techniques may be employed for introducing nucleic acids into cells. Such techniques include transfection of nucleic acid-CaPO4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like.
One method of introducing exogenous genetic material into cells involves transducing the cells in situ using replication-deficient retroviruses. Replication-deficient retroviruses are capable of directing synthesis of all virion proteins, but are incapable of making infectious particles. Accordingly, these genetically altered retroviral vectors have general utility for high-efficiency transduction of genes in cultured cells, and specific utility for use in the method of the present invention. Retroviruses have been used extensively for transferring genetic material into cells. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with the viral particles) are provided in the art.
Because viruses insert efficiently a single copy of the gene encoding the agent into the host cell genome, retroviruses permit the exogenous genetic material to be passed on to the progeny of the cell when it divides. In addition, gene promoter sequences in the LTR region have been reported to enhance expression of an inserted coding sequence in a variety of cell types. Delivery of an effective amount of an agent via a retrovirus can be efficacious if the efficiency of transduction is high and/or the number of target cells available for transduction is high.
Yet another viral candidate useful as an expression vector for transformation of cells is the adenovirus, a double-stranded DNA virus. Like the retrovirus, the adenovirus genome is adaptable for use as an expression vector for gene transduction, i.e., by removing the genetic information that controls production of the virus itself. Because the adenovirus functions usually in an extrachromosomal fashion, the recombinant adenovirus does not have the theoretical problem of insertional mutagenesis. On the other hand, adenoviral transformation of a target cell may not result in stable transduction. However, more recently it has been reported that certain adenoviral sequences confer intrachromosomal integration specificity to carrier sequences, and thus result in a stable transduction of the exogenous genetic material.
Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77 S-83S, 1995).
Thus, as will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring exogenous genetic material into cells. The selection of an appropriate vector to deliver an agent and the optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation. The promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) required to obtain the desired gene transcription activity. For the purpose of this discussion an “enhancer” is simply any nontranslated DNA sequence which works contiguous with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. Preferably, the exogenous genetic material is introduced into the cell genome immediately downstream from the promoter so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence. A preferred retroviral expression vector includes an exogenous promoter element to control transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and inducible promoters.
Naturally-occurring constitutive promoters control the expression of essential cell functions. As a result, a gene under the control of a constitutive promoter is expressed under all conditions of cell growth. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or “housekeeping” functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al., 1991, Proc. Natl. Acad. Sci. USA, 88:4626-4630), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the actin promoter (Lai et al., 1989, Proc. Natl. Acad. Sci. USA, 86:10006-10010), and other constitutive promoters known to those of skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: the early and late promoters of SV40; the long terminal repeats (LTRS) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of Herpes Simplex Virus, among many others. Accordingly, any of the above-referenced constitutive promoters can be used to control transcription of a heterologous gene insert.
Genes that are under the control of inducible promoters are expressed only or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions). Inducible promoters include responsive elements (REs) which stimulate transcription when their inducing factors are bound. For example, there are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP. Promoters containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive versus inducible; strong versus weak), it is possible to control both the existence and level of expression of an agent in the genetically modified cell. Selection and optimization of these factors for delivery of an is deemed to be within the scope of one of ordinary skill in the art without undue experimentation, taking into account the above-disclosed factors.
In addition to at least one promoter and at least one heterologous nucleic acid, the expression vector preferably includes a selection gene, for example, a neomycin resistance gene, for facilitating selection of cells that have been transfected or transduced with the expression vector. Alternatively, the cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the therapeutic agent(s), the other vector containing a selection gene. The selection of a suitable promoter, enhancer, selection gene and/or signal sequence is deemed to be within the scope of one of ordinary skill in the art without undue experimentation.
A variety of expression systems exist for the production of the polypeptides of the invention. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.
One particular bacterial expression system for polypeptide production is the E. coli pET expression system (e.g., pET-28) (Novagen, Inc., Madison, Wis.). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.
Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system that is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.
Alternatively, recombinant polypeptides of the invention are expressed in Pichia pastoris, a methylotrophic yeast. Pichia is capable of metabolizing methanol as the sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde by the enzyme, alcohol oxidase. Expression of this enzyme, which is coded for by the AOX1 gene is induced by methanol. The AOX1 promoter can be used for inducible polypeptide expression or the GAP promoter for constitutive expression of a gene of interest.
Once the recombinant polypeptide of the invention is expressed, it is isolated, for example, using affinity chromatography. In one example, an antibody (e.g., produced as described herein) raised against a polypeptide of the invention may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). Alternatively, the polypeptide is isolated using a sequence tag, such as a hexahistidine tag, that binds to nickel column.
Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).
Adipogenic Polypeptides and AnalogsThe Examples herein describe the expression of adipogenic polypeptides (e.g., PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, CREB1, and KROX20) in stem cells (iPS, HUES). Also included in the invention are polypeptides or fragments thereof that are modified in ways that enhance their ability to reprogram a cell. In other embodiments, variations in the sequence increase protein solubility or yield. For example, the invention provides a modified adipogenic transcription factor polypeptide having an enhanced ability to reprogram a stem cell to an reprogrammed adipocyte cell. The invention provides methods for optimizing an adipogenic amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogs of any naturally-occurring polypeptide of the invention. Analogs can differ from a naturally-occurring polypeptide of the invention by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino, acid sequence of the invention. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, preferably at least 25, 50, or 75 amino acid residues, and more preferably more than 100 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.
In addition to full-length polypeptides, the invention also provides fragments of any one of the polypeptides or peptide domains of the invention. As used herein, the term “a fragment” means at least 5, 10, 13, or 15 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80, 100, 200, 300 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
Adipogenic polypeptide analogs have a chemical structure designed to mimic the naturally-occurring adipogenic transcription factor polypeptide's functional activity. Such analogs are administered according to methods of the invention. Adipogenic transcription factor polypeptide analogs may exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the reprogramming activity of a reference adipogenic polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference adipogenic polypeptide. Assays for measuring adipocyte morphology, phenotype, or functional activity include, but are not limited to, those described in the Examples below.
Test Compounds and ExtractsReprogrammed adipocyte cells having a genetic alteration are particularly useful in methods of drug screening. In the field of pharmaceutical research, the use of high-throughput screening has been an essential tool used to identify therapeutics. Prior to the present invention, a source of human adipocytes that can generate the large number of cells necessary for screening has been lacking. Furthermore, metabolic syndrome and adipose-related diseases, such as obesity and obesity induced diabetes, are sporadic and multifactorial. The present invention provides a cellular model for such diseases, which exhibits the morphological and functional disease phenotypes useful for disease modeling. Pluripotent or multipotent (e.g., hESCs/hIPS) that carry a disease genotype are differentiated into reprogrammed adipocyte cells thus permitting the in vitro modeling of adipose-related diseases.
In one embodiment, hESC and/or hIPS that carry a disease genotype can be differentiated into adipocytes thus permitting the in vitro modeling of adipose related diseases. Adipocytes derived from hESC/hIPS provide a system for the discovery of therapeutics to treat metabolic syndrome, Type 2 diabetes mellitus, insulin resistance, obesity, lipodystrophy, metabolic disorders, cardiac disease, early-onset myocardial infarction, and laminopathies and other disorders involving adipose tissue. Functional adipocytes can be differentiated in quantities that allow high-throughput screening of small compounds, siRNAs or proteins with desirable pharmacotherapeutic effects. The availability of hESC/hIPS derived adipocytes will also allow for the genome wide analysis of gene transcription, epigenetic status and protein synthesis as well as metabolic activity for varying genetic backgrounds and disease states.
Compounds that ameliorate a symptom of a disease delineated herein (e.g., metabolic syndrome, Type 2 diabetes mellitus, insulin resistance, obesity, lipodystrophy, metabolic disorders, cardiac disease, early-onset myocardial infarction, and laminopathies) are identified from large libraries of natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. The compounds are then screened for the desired activity. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Agents used in screens may include known compounds (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds.
Libraries of natural agents in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Such agents can be modified using methods known in the art and described herein. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. 37:1233, 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.
When a crude extract is found to have a desired activity further fractionation of the positive lead extract is necessary to isolate molecular constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that reprograms a cell (e.g., an adult cell or embryonic stem cell) or that enhances regeneration. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful as therapeutics are chemically modified according to methods known in the art.
KitsThe invention provides kits for promoting reprogrammed adipocyte cell differentiation, as well as kits for the engraft went of a reprogrammed adipocyte cell into a tissue of a subject. In one embodiment, the kit includes a therapeutic composition containing an effective amount of an reprogrammed adipocyte cell in unit dosage form. In one embodiment, the kit comprises a sterile container which contains a number of multipotent or induced pluripotent stem cells or reprogrammed adipocyte cells; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired an multipotent or induced pluripotent stem cell is provided together with compositions and instructions for differentiating it in vitro. In another embodiment, the kit includes instructions for administering an reprogrammed adipocyte cell to a tissue of a subject. The instructions will generally include information about the use of the composition or the engraftment of the reprogrammed adipocyte cell in a tissue. In other embodiments, the instructions include at least one of the following: description of the adipogenic vectors; dosage schedule and administration; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
In another aspect, the invention provides kits that feature expression vectors useful for the differentiation of an reprogrammed adipocyte cell.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989); “Oligonucleotide Synthesis” (Gait, IRL Press, Oxford 1984); “Animal Cell Culture” (Freshney, Alan R. Liss, Inc., N.Y. 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, Blackwell Scientific Publication, Oxford, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, Cold Spring Harbor Laboratory, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, Birkhauser Boston, Cambridge, Mass. 1994); “Current Protocols in Immunology” (Coligan, Current Protocols in Immunology Wiley/Greene, NY1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES Example 1 Reprogrammed Adipocyte Cells were Obtained by the Directed Differentiation of Human Embryonic Stem CellsKnown regulators of adipogenesis were cloned into a doxycycline-inducible lentiviral backbone (e.g. PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, CREB1, and KROX20) (
The adipogenic potential of lentiviral PPARγ2 overexpression in ADMSCs and human embyronic stem cells was tested. In both cases there was differentiation into adipocytes at high efficiency. Of note, discernible differences in cellular morphology was observed between adipocytes derived from ADMSCs and those derived from human embryonic stem cells. The ADMSC-derived adipocytes show numerous small-sized lipid droplets. In contrast, the adipocytes derived from stem cells showed a mainly monolocular morphology, characteristic of mature adipocytes.
Ectopic expression of PPARγ2, C/EBPα, C/EBPβ or C/EBPδ showed that each of these has adipogenic activity in human pluripotent cells. In particular, the viral transduction and inducible expression of PPARγ2 and C/EBP factors in human pluripotent cells combined with the addition of insulin, rosiglitazone, dexamethasone, and isobutylmethylxanthine to the cells' growth medium resulted in the appearance of lipid filled cells with large monolocular lipid droplets reminiscent of cells found in human adipose tissue (
Importantly, these cells expressed the mature adipocyte markers CIDEC, FABP4 and perilipin (
The analysis of 3 hES lines infected with C/EBPα (no rtTA), PPARγ—C/EBPβ-C/EBPδ and C/EBPβ-C/EBPδ is shown in comparison to a control line BJ-iPS without a transgene. As shown in
Differentiated reprogrammed adipocyte cells are compared to primary human adipocytes using microarrays for gene expression analysis. Comparison will be made between cells from primary fat tissue, adipocytes derived from iPS cells and ADMSCs, and the starting populations of undifferentiated iPS and ADMSCs. These cells will also be functionally characterized by assaying (1) the response to insulin by measuring (U-14C)-D-glucose uptake (Kashiwagi, supra), (2) lipolytic activity by inducing lipolysis using β-adrenergic receptor agonists and measuring glycerine concentration using a glycerol assay kit that employs an ELISA-based coupled enzymatic reaction, (3) de novo synthesis of fatty acids using (1-14C) acetate and (4) incorporation of free fatty acids using (3H) oleate or (14C) plamitate. Finally, it will be of interest to assess whether the in vitro differentiated adipocytes can be maintained in culture without further induction with doxycyline and standard adipogenic differentiation medium, or whether these cells will be similarly difficult to propagate as primary adipocytes.
Example 2 Reprogrammed Adipocyte Cells are Useful for Identifying Genetic Alterations Associated with Cardiac PathologyThe methods of the invention provide for the generation of subject-specific tissues for genetic profiling and other studies through the use of reprogrammed induced pluripotent stem (iPS) cells. iPS cell lines can be generated from patient-derived fibrobasts using methods described by (Park et al., Cell. 2008; 134:877-886; Maherali et al., Cell Stem Cell 3, 340-345). Fibroblasts suitable for reprogramming have been obtained from three different sources: skin punch biopsies, hair follicles, and peripheral blood mononuclear cells (PBMC) fractions from blood draws (
The lentivirus harbors loxP sites that allow for the use of Cre recombinase to remove the four genes from cells after reprogramming is complete. These cells can be used to generate adipocytes using a doxycycline-inducible, lentivirus-based differentiation protocol that involves the addition of insulin, rosiglitazone, dexamethasone, and isobutylmethylxanthine to the cells' growth medium, along with viral transduction of the reverse tetracycline transactivator (rtTA) and inducible forms of PPARγ2 and C/EBP factors (
This provides for the comparison of molecular profiles obtained from iPS-derived adipocytes to eQTL, analyses of surgically derived adipose tissue samples. The medical history of the subjects from which the cells are isolated is defined according to the following characteristics: body mass index, history of smoking, age of myocardial infarction, age, gender, blood pressure, cholesterol level, history of diabetes, or any other medical condition that may affect adipocyte cellular function.
Adipocytes having alterations at genetic loci associated with early-onset myocardial infarction are of particular interest. In one approach, SNPs in MI-associated genetic loci, for example, rs4977574 in the 9p21.3 locus are associated with alterations in the expression of nearby transcripts.
While studies of genomic variation can identify novel genetic contributors to cardiac disease and myocardial infarction, such studies fail to address how particular genetic loci affect gene expression in tissue types relevant to myocardial infarction (MI). Expression quantitative trait locus (eQTL) analyses of genotype vs. gene expression can be carried out using adipose and liver tissue samples surgically obtained from patients; however, these studies are limited by the number of samples available and, more importantly, by the samples being obtained from patients undergoing surgical procedures unrelated to cardiovascular disease. The present invention provides a renewable source of adipose tissue that can be readily obtained from subjects who have suffered premature MI.
Example 3 PPARγ2 Expression is Sufficient for Adipocyte Differentiation In Vitro from both hEs Cells and ADMSCsAs described above, human somatic cells can be directly reprogrammed in vitro into a pluripotent embryonic stem cell-like state by introducing a combination of four transcription factors (OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28) (Takahashi et al., Nat Protoc, 2007. 2(12):3081-9, Yu et al., Science, 2007. 318(5858): 1917-20, Lowry et al., Proc Natl Acad Sci USA, 2008. 105(8): p. 2883-8). These induced pluripotent stem (iPS) cells are nearly identical to embryonic stem (ES) cells in gene expression, DNA methylation and chromatin modifications (Takahashi et al., Nat Protoc, 2007. 2(12):3081-9, Yu et al., Science, 2007. 318(5858): 1917-20, Maherali supra). Using a retroviral system, human iPS cells have successfully been generated from patients with a variety of genetic diseases, including Parkinson disease, Huntington disease, and type 1 diabetes mellitus. A tetracycline regulatable lentivirus containing each of the four transcription factors, OCT4, SOX2, cMYC, and KLF4, has been successfully used to generate human iPS cells from human fibroblasts and keratinocytes. These methods are also useful to generate human iPS cells from FPLD2 patient fibroblasts in order to build a cell-based model of lipodystrophy to address the molecular events involved in adipogenesis and obesity.
Fibroblasts cell lines with LMNA mutations from FPLD2 patients were obtained from Dr. Corinne Vigouroux at the University Pierre et Marie Curie-Paris in Paris, France. The initial phenotype and genotype characterization was carried out as described (Lowry et al., Proc Natl Acad Sci USA, 2008. 105(8): p. 2883-8). The following cell lines were obtained: LMNA mutation at 1) R482W, typical FPLD2 mutation found most of patients, 2) H506D, 3) R399H, 4) R582H, and 5) T655fxX49, those mutations exhibit partial lipodystrophy, 6) L387V with insulin-resistant diabetes, 7) L421P, patient with obesity (BMI 40.5) and diabetes. These cell lines are reprogrammed by introducing four transcription factors (OCT4, SOX2, KLF4, and cMYC) using both a floxed lentiviral system and piggyBac transposon/transposase system (Soldner, et al., Cell, 2009. 136(5): 964-77, Kaji et al., Nature, 2009. 458(7239):771-5, Woltjen et al., Nature, 2009. 458(7239): p. 766-70, Yusa et al., Nat Methods, 2009. 6(5):363-9). After transgene expression, cells are cultured in human embryonic stem cell conditions as previously described (Cowan, et al., N Engl J Med, 2004. 350(13): p. 1353-6). The generation of human iPS cells using these systems takes advantage of current retrovirus or lentivirus systems. It has been demonstrated that the delivery of four factors by these system are relatively efficient in order to generate human iPS cells. More importantly, transgene expression can be removed from the genome once cells have achieved a pluripotent state.
The foxed lentiviral system is based on a polycistronic lentiviral vector that allows expression of OC4, SOX2, KLF4, and cMYC which are flanked by two loxP sites. This cassette can be efficiently excised by the transient expression of Cre recombinase after successful human iPS cell generation. The piggyBac transposon is a mobile genetic unit originally found in insects that has been modified for use in human cells. This transposon can efficiently integrate into the genome to express multiple transgenes. Our piggyBac system is designed to express OCT4, SOX2, KLF4, and cMYC and can be removed by the expression of transposase leaving an unmarked genome. Using these vector technologies, iPS colonies are isolated and expanded from FPLD2 patient fibroblasts.
To differentiate human pluripotent (iPS and hES) cells into adipocytes, transcription factors involved in adipogenesis are overexpressed, in combination with an adipose differentiation protocol originally developed for mouse preadipocytes (3T3-L1 cells) (Jessen, Gene, 2002. 299(1-2):95-100).
After the cells have achieved a morphology consistent with mature adipocytes (large unioccular fat droplets) doxycycline will be removed from the media to ascertain whether this cell state can be stably maintained without the overexpression of adipogenic transcription factors. While the initial molecular events of adipogenesis remain unclear, it is widely accepted that the transcription factor PPARG is the “master regulator” of terminal adipocyte differentiation. Indeed, viral overexpression of PPARG2 is sufficient for adipocyte differentiation in vitro from both hES cells and ADMSCs. However, it is likely that concomitant expression of other factors will increase the efficiency of adipocyte differentiation and generate a more robust adipocyte phenotype. A combinatorial approach will be taken to overexpress the set of adipogenic transcription factors listed above (
Type 2 diabetes mellitus (DM) is rapidly becoming a leading cause of morbidity and mortality worldwide, with hundreds of millions of people projected to develop the disease in the coming decades. Although much of this increase in incidence is attributed to environmental factors, e.g., the “Western lifestyle” comprising increase caloric intake and decreased physical activity, there is a strong genetic component to the disease as well. Genome-wide association studies (GWAS) of common single nucleotide polymorphisms (SNPs) have been reported for both type 1 DM (Barrett et al., Nat. Genet. 2009; 41:703-707) and type 2 DM (Frayling, Nat Rev Genet. 2007; 8:657-662; Zeggini et al., Nat. Genet. 2008; 40:638-645; Yasuda et al., Nat. Genet. 2008; 40:1092-1097; Unoki et al., Nat. Genet. 2008; 40:1098-1102; Lyssenko et al., Nat. Genet. 2009; 41:82-88; Bouatia-Naji et al., Nat. Genet. 2009; 41:89-94), with more than 20 genetic loci identified for the latter. Achieving an understanding of how these genetic loci contribute to the pathogenesis of type 2 DM may identify molecular pathways for which novel antidiabetic therapeutics can be developed to curb the incidence of the disease and better treat those already with the disease.
Among the most highly associated genetic loci for type 2 DM is a region on chromosome 9p21.3 harboring the SNP rs10811661 (P=8×10−15, odds ratio=1.4 for homozygotes for the risk allele compared to homozygotes for the protective allele). Of the common genetic variants identified in GWAS to date, this SNP is the second largest genetic influence on the incidence of type 2 DM (Table 3) by odds ratio. Linkage disequilibrium defines a minimal region of =10 kb containing rs10811661, flanked by recombination hotspots and presumably containing the causal DNA variant(s) (
Neither the DM locus nor the MI locus harbors any known genes, although the MI locus does harbor predicted exons of a non-coding RNA, termed ANRIL, of unknown significance. The closest genes are two cell cycle regulators, the cyclin-dependent kinase inhibitors CDKN2A and CDKN2B, which lie more than 120 kb upstream of rs10811661 in the DM locus, and the 5′-methylthioadenosine phosphorylase (MTAP) gene, which lies even further upstream. It is unclear whether the causal DNA variants in the diabetes and MI loci affect the activity of one or more of these genes, or whether they work through a different mechanism, and how the loci are able to independently contribute to two very different diseases. Of relevance may be a study in mice demonstrating that overexpression of CDKN2A results in decreased pancreatic beta cell proliferation, whereas knockout of the gene increases beta cell proliferation (Krishnamurthy et al., 2006).
Human embryonic stem (HUES) cells offer a powerful model in which the functional consequences of human genetic variation at the 9p21.3 locus confers risk for DM can be analysed. Human embryonic stem (HUES) cells offer a more faithful model in which the functional consequences of human genetic variation can be measured at the cellular level. By virtue of being pluripotent, HUES cells can in principal serve as a renewable source of differentiated cells of any tissue type for analysis. HUES cells and iPS approach are useful to create an allelic series of cell lines (such as adipocytes and pancreatic beta cells) at the 9p21.3 DM locus, offering a novel and unique tool with which to study the pathogenesis of DM.
Recognizing that rs10811661 may be in linkage disequilibrium with the causal variant related to DM—somewhere in the ˜10 kb locus—rather than being the causal variant itself, two complementary strategies are used to establish that DNA variants in the DM locus affect local genes in cis—CDKN2A, CDKN2B, MTAP, ANRIL—or other genes in trans in such a way as to contribute to disease. Such cells are developed using human embryonic stem (HUES) cell lines and/or induced pluripotent stem (iPS) cell lines with deletion of either the entire ˜10 kb DM locus or with naturally-occurring differing genotypes at rs10811661 and (2) perform gene expression profiling in these pluripotent cell lines as well as HUES/iPS-derived adipose and pancreatic beta cells.
Homologous recombination in a human embryonic stem (HUES) cell line (HUES-8) was successfully performed to knock out the entire ˜10 kb DM locus on one of the chromosomes 9 in the cell clone. The homologous recombination strategy and confirmation of the recombinant clone is shown in
The results reported herein were obtained using the following materials and methods.
Cell culture media can be made as follows:
Adipogenic Differentiation Media
hESC Cell Culture Media
Before transduction cells were expanded until passage four and were kept at less than 85% confluence. ADMSC Medium was changed every day 2 days. Passaging was carried out at ratios of 1:3 to 1:4.
Maintenance and Expansion of Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells on Feeder ConditionshESCs and KIPS were cultured as previously described (Cowan, et al., N Engl J Med, 2004. 350(13): p. 1353-6). Specifically, cultures were maintained on mitotically inactive MEFs plated on 0.1% gelatin-coated cell culture plates. Cells were passaged upon reaching confluency while still maintaining phase-bright colonies and sharp borders. To passage, cells were rinsed twice in DPBS, and then incubated in 0.05% trypsin at room temperature. Using culture media, cells were gently rinsed off the plate and briefly triturated to break up large colonies. Cells were then split directly onto a new feeder layer. WA09 cells were routinely passaged every 3-4 days at ratios of 1:6 to 1:8.
Propagation of hES Cells on Matrigel or Geltrex in Chemical Defined Medium (mTESR, Nutristem, Chem-D)
To transfer cells hESC from feeder conditions to chemical defined medium, cells were trypsinized and replated on gelatin coated dishes in MEF medium. Cells were allowed to settle for 30 minutes to subtract MEF cells. The supernatant and the floating cells were collected and transferred on matrigel coated TC plates (6 mg Matrigel for a 15 cm plate) and maintained in the chemical defined medium. Passaging was carried out before cells reached confluency. To passage, cells were washed with DMEM, Dispase 1 mg/ml was added and cells were incubated at room temperature until white ring appears at the outside of colonies. Cells were washed twice with DMEM and detached from the plates using a cell scraper. Cells were passaged 1:3 or 1:4 onto new Matrigel coated dishes.
Transduction of ADMSCs and hESCs
hESC/hIPS or ADMSC were transduced in 6 well format at sub-confluency by applying equal amounts of rtTA and Tet-(Transcription factor) virus supernatant. Plates were centrifuged at 1000 RPM for 50 min at RT. Plates were incubated for additional 6 hours. Viral supernatant was removed and cells were cultured until confluency to continue propagation of transduced cells or to begin differentiation.
Maintenance and Expansion of Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells on Feeder ConditionshESCs and KIPS were cultured as previously described (Cowan, supra). Specifically, cultures were maintained on mitotically inactive MEFs plated on 0.1% gelatin-coated cell culture plates. Cells were passaged upon reaching confluency while still maintaining phase-bright colonies and sharp borders. To passage, cells were rinsed twice in DPBS, and then incubated in 0.05% trypsin at room temperature. Using culture media, cells were gently rinsed off the plate and briefly triturated to break up large colonies. Cells were then split directly onto a new feeder layer. WA09 cells were routinely passaged every 3-4 days at ratios of 1:6 to 1:8.
Propagation of hES Cells on Matrigel or Geltrex in chemical defined medium (mTESR, Nutristem, Chem-D)
To transfer cells hESC from feeder conditions to chemical defined medium, cells were trypsinized and replated on gelatin coated dishes in MEP medium. Cells were allowed to settle for 30 minutes to subtract MEF cells. The supernatant and the floating cells were collected and transferred on matrigel coated tissue culture plates (6 mg Matrigel for a 15 cm plate) and maintained in the chemical defined medium. Passaging was carried out before cells reached confluency. To passage, cells were washed with DMEM, Dispase 1 mg/ml was added and cells were incubated at room temperature until white ring appears at the outside of colonies. Cells were washed twice with DMEM and detached from the plates using a cell scraper. Cells were passaged 1:3 or 1:4 onto new Matrigel coated dishes.
Transduction of ADMSCs and hESCs
hESC/hIPS or ADMSC were transduced in 6 well format at sub-confluency by applying equal amounts of rtTA and Tet-(Transcription factor) virus supernatant. Plates were centrifuged at 1000 RPM for 50 min at RT. Plates were incubated for additional 6 hours. Viral supernatant was removed and cells were cultured until confluency to continue propagation of transduced cells or begin of differentiation.
Following differentiation, cells are assayed for any one or more of the following adipogenic marker polypeptides or polynucleotides: PPARy, C/EBPa, C/EBPb, C/EBPd, leptin, adiponectin, SREBP1c, FABP4, CIDEC, and Glut4.
Staining of Adipocytes with Oil-Red-O
Media was removed from the culture dishes, and cells were fixed with 10% formalin. Wells were then washed with 60% isopropanol followed by air-drying. Oil-Red-O working solution was applied for 10 minutes. The wells were washed repeatedly using dH2O and were then imaged.
Adipocyte Staining with BODIPY
The cell medium was removed and replaced with PBS containing BODIPY dye (used as a 10 000× stock). The staining solution was removed after 30 minutes and replaced by cell culture medium. Imaging was done using a fluorescence scope in the GFP channel.
Nuclear Staining with DAPI
A DAPI stock solution (1 mg/ml) was used in a 1:1000 dilution. DAPI solution was re-moved after 10 minutes and cells washed with dH2O.
Other EmbodimentsFrom the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
Claims
1-46. (canceled)
47. A method for generating a reprogrammed adipocyte, the method comprising
- exogenously expressing in a pluripotent stem cell one or more adipogenic transcription factor polypeptides; and
- contacting the cell with one or more of insulin, rosiglitazone, dexamethasone and isobutylmethylxanthine, thereby generating a reprogrammed adipocyte.
48. The method of claim 47, wherein the adipogenic transcription factor polypeptide is selected from the group consisting of PPARγ2, CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBPβ, C/EBPδ, C/EBPα and CDEC.
49. The method of claim 48, wherein the pluripotent stem cell is selected from the group consisting of induced pluripotent stem cell, human embryonic stem cell, mesenchymal stem cell, adipocyte-derived mesenchymal stem cell, bone marrow derived stem cell and other mesenchymal stem cell.
50. The method of claim 47, wherein the induced pluripotent stem cell is derived from a somatic cell.
51. The method of claim 50, wherein the somatic cell is selected from the group consisting of adipocyte, keratinocyte, epidermal cell, fibroblast, hematopoietic cell, peripheral blood mononuclear cell and their progenitor cells.
52. The method of claim 47, wherein pluripotency is induced by expression of one or more of OCT4, SOX2 and either cMYC and KLF4 or NANOG and LIN28 in a somatic cell.
53. The method of claim 47, wherein the pluripotent stem cell is contacted in vitro.
54. The method of claim 47, wherein the method further comprises identifying an adipocyte phenotype by detecting an increase in an adipocyte marker, an adipocyte morphology, or adipocyte function that is not detectably expressed or expressed only nominally in a corresponding control cell.
55. The method of claim 47, wherein the reprogrammed adipocyte expresses one or more adipocyte markers selected from the group consisting of CIDEC, FABP4, PPARγ2, adiponectin, leptin, and perilipin.
56. The method of claim 47, wherein the reprogrammed adipocyte comprises lipid droplets.
57. The method of claim 47, wherein the reprogrammed adipocyte responds to insulin, has lipolytic activity, displays de novo synthesis of fatty acids and/or incorporates free fatty acids.
58. A reprogrammed adipocyte generated according to the method of claim 47.
59. The reprogrammed adipocyte of claim 58, wherein the cell comprises a genetic alteration associated with a disease selected from the group consisting of Type 2 diabetes mellitus, insulin resistance, obesity, lipodystrophy, metabolic disorders, cardiac disease, early-onset myocardial infarction and laminopathies.
60. The reprogrammed adipocyte of claim 58, wherein the cell comprises a single nucleotide polymorphism listed in Table 1, Table 2, or Table 3.
61. The reprogrammed adipocyte of claim 58, wherein the cell is derived from an FPLD2 patient, a patient with type 2 diabetes, or a patient with early onset myocardial infarction.
62. The reprogrammed adipocyte of claim 58, wherein the cell from the FPLD2 subject has a LMNA mutation at R482W, H506D, R399H, R582H, T655fxX49, or L387V L421P.
63. A reprogrammed adipocyte that exogenously expresses an adipogenic transcription factor polypeptide selected from the group consisting of PPARγ, CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBPβ, C/EBPδ C/EBPα, and CDEC, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function.
64. A reprogrammed adipocyte that exogenously expresses a PPARγ2 or C/EBPα polypeptide, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function.
65. A reprogrammed adipocyte that exogenously expresses a polypeptide selected from the group consisting of C/EBPα, C/EBPβ and C/EBPδ, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function.
66. A method for identifying a therapeutic agent, the method comprising contacting the reprogrammed adipocyte of claim 65 with a candidate agent and identifying an alteration in a disease marker.
67. A method of ameliorating cell or tissue loss in a subject in need thereof, the method comprising delivering to the subject a cell generated according to the method of claim 47.
68. The method of claim 67, wherein the cell or tissue loss is associated with trauma, cell death, or a congenital defect.
69. A collection of at least two expression vectors, wherein each vector comprises a distinct nucleic acid sequence encoding a polypeptide selected from the group consisting of PPARγ2, C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, CREB1 and KROX20.
70. A host cell comprising one or more of the expression vectors of claim 69.
71. A pharmaceutical composition comprising a reprogrammed adipocyte according to claim 58 in a pharmaceutically acceptable excipient.
Type: Application
Filed: Sep 26, 2011
Publication Date: Aug 30, 2012
Applicant: The General Hospital Corporation (Boston, MA)
Inventors: David H. Lum (Arlington, MA), Tim D. Ahfeldt (Cambridge, MA), Chad Cowan (Boston, MA)
Application Number: 13/245,716
International Classification: A61K 35/12 (20060101); C40B 30/04 (20060101); C12N 1/19 (20060101); C12Q 1/54 (20060101); C12N 15/63 (20060101); C12N 1/21 (20060101); C12N 5/077 (20100101); G01N 33/566 (20060101);
