Compositions And Methods Of Generating Reprogrammed Adipocyte Cells And Methods Of Use Therefore

Abstract

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.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] 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 INVENTION

[0002] Obesity 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.

[0003] 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 INVENTION

[0004] As described below, the present invention features methods for generating reprogrammed adipocyte cells, compositions comprising the reprogrammed adipocyte cells, and methods of using such cells.

[0005] 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., PPAR.gamma., CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBP.beta., C/EBP.delta. C/EBP.alpha. and CDEC); and contacting the cell with one or more of insulin, rosiglitazone, dexamethasone and isobutylmethylxanthine, thereby generating a reprogrammed adipocyte.

[0006] In another aspect, the invention features a method for generating a reprogrammed adipocyte, the method involving exogenously expressing in a pluripotent stem cell a PPAR.gamma.2 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.

[0007] 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/EBP.alpha. 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.

[0008] 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.

[0009] In another aspect, the invention features a reprogrammed adipocyte that exogenously expresses an adipogenic transcription factor polypeptide selected from the group consisting of PPAR.gamma., CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBP.beta., C/EBP.delta. C/EBP.alpha., and CDEC, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function.

[0010] In yet another aspect, the invention features a reprogrammed adipocyte that exogenously expresses a PPAR.gamma.2 or C/EBP.alpha. polypeptide, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function.

[0011] In still another aspect, the invention features a reprogrammed adipocyte that exogenously expresses a polypeptide selected from the group consisting of C/EBP.alpha., C/EBP.beta. and C/EBP.delta., wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function. In one embodiment, the adipocyte exogenously expresses C/EBP.alpha. and further expresses C/EBP.beta. and/or C/EBP.delta..

[0012] In another aspect, the invention features a reprogrammed adipocyte that exogenously expresses a C/EBP.alpha. and a C/EBP.beta. polypeptide.

[0013] 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.

[0014] 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.

[0015] In one embodiment, the cell or tissue loss is associated with trauma, cell death, or a congenital defect.

[0016] 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 PPAR.gamma.2, C/EBP.alpha., C/EBP.beta., C/EBP.delta., SREBP1c, CREB1 and KROX20. In one embodiment, one of the expression vectors encodes PPAR.gamma.2 and the other encodes C/EBP.alpha.. In another embodiment, one of the expression vectors encodes C/EBP.alpha. and the other encodes C/EBP.beta.. In another embodiment, one vector encodes PPAR.gamma.2 and the other encodes a polypeptide that is any one or more of C/EBP.alpha., C/EBP.beta. and/or C/EBP.delta.. 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.

[0017] 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.

[0018] In another aspect, the invention features a pharmaceutical composition comprising a reprogrammed adipocyte according to any previous aspect in a pharmaceutically acceptable excipient.

[0019] 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.

[0020] In one embodiment, the adipogenic transcription factor polypeptides are encoded by an expression vector.

[0021] 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.

[0022] 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, PPAR.gamma.2, 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 PPAR.gamma.2, C/EBP.alpha., C/EBP.beta., C/EBP.delta., SREBP1c, CREB1 and KROX20. In one embodiment of any of the above aspects, the adipogenic transcription factor protein is PPAR.gamma.2, C/EBP.alpha., C/EBP.beta. and/or C/EBP.delta..

[0023] 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.

DEFINITIONS

[0024] By "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:

TABLE-US-00001 1 maghlasdfa fspppggggd gpggpepgwv dprtwlsfqg ppggpgigpg vgpgsevwgi 61 ppcpppyefc ggmaycgpqv gvglvpqggl etsqpegeag vgvesnsdga spepctvtpg 121 avklekekle qnpeesqdik alqkeleqfa kllkqkritl gytqadvglt lgvlfgkvfs 181 qtticrfeal qlsfknmckl rpllqkwvee adnnenlqei ckaetlvgar krkrtsienr 241 vrgnlenlfl qcpkptlqqi shiaqqlgle kdvvrvwfcn rrqkgkrsss dyaqredfea 301 agspfsggpv sfplapgphf gtpgygsphf talyssvpfp egeafppvsv ttlgspmhsn

[0025] 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.sub.--002701 and NM.sub.--203289.

[0026] 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:

TABLE-US-00002 1 mynmmetelk ppgpqqtsgg gggnstaaaa ggnqknspdr vkrpmnafmv wsrgqirkma 61 qenpkmhnse iskrlgaewk llsetekrpf ideakrlral hmkehpdyky rprrktktlm 121 kkdkytlpgg llapggnsma sgvgvgaglg agvnqrmdsy ahmngwsngs ysmmqdqlgy 181 pqhpglnahg aaqmqpmhry dvsalqymsn tssqtymngs ptysmsysqq gtpgmalgsm 241 gsvvkseass sppvvtsssh srapcgagdl rdmismylpg aevpepaaps rlhmsqhyqs 301 gpvpgtaing tiplshm

[0027] By "Sox2 polynucleotide" is meant a polynucleotide encoding a Sox2 polypeptide. An exemplary Sox2 polynucleotide sequence is provided at NCBI Accession No. NM.sub.--003106.

[0028] 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:

TABLE-US-00003 1 mpinvsftnr nydldydsvq pyfycdeeen fyqqqqqsel qppapsediw kkfellptpp 61 lspsrrsglc spsyvavtpf slrgdndggg gsfstadqle mvtellggdm vngsficdpd 121 detfikniii qdcmwsgfsa aaklvsekla syqaarkdsg spnparghsv cstsslylqd 181 lsaaasecid psvvfpypin dssspkscas qdssafspss dsllsstess pqgspeplvl 241 heetppttss dseeeqedee eidvvsvekr qapgkrsesg spsagghskp phsplvlkrc 301 hvsthqhnya appstrkdyp aakrvkldsv rvlrqisnnr kctsprssdt eenvkrrthn 361 vlerqrrnel krsffalrdq ipelenneka pkvvilkkat ayi1svgaee qkliseedll 421 rkrreqlkhk leqlrnsca

[0029] By "cMYC polynucleotide" is meant a polynucleotide encoding a cMYC polypeptide. An exemplary cMYC polynucleotide sequence is provided at NCBI Accession No. NM.sub.--002467

[0030] 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:

TABLE-US-00004 1 mrqppgesdm aysdallpsf stfasgpagr ektlrqagap nnrwreelsh mkrlppvlpg 61 rpydlaaatv atdlesggag aacggsnlap lprreteefn dlldldfils nslthppesv 121 aatvsssasa ssssspsssg pasapstcsf typiragndp gvapggtggg llygresapp 181 ptapfnladi ndvspsggfv aellrpeldp vyippqqpqp pggglmgkfv lkaslsapgs 241 eygspsvisv skgspdgshp vvvapynggp prtcpkikqe aysscthlga gpplsnghrp 301 aandfplgrq lpsrttptlg leevlssrdc hpalplppgf hphpgpnyps flpdqmqpqv 361 pplhyqgqsr gfvaragepc vcwphfgthg mmltppsspl elmppgscmp eepkpkrgrr 421 swprkrtath tcdyagcgkt ytksshlkah lrthtgekpy hcdwdgcgwk farsdeltrh 481 yrkhtghrpf qcqkcdrafs rsdhlalhmk rhf

[0031] By "KLF4 polynucleotide" is meant a polynucleotide encoding a KLF4polypeptide. An exemplary KLF4 polynucleotide sequence is provided at NCBI Accession No.--NM.sub.--004235.

[0032] 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:

TABLE-US-00005 1 msvdpacpqs lpcfeasdck esspmpvicg peenypslqm ssaemphtet vsplpssmdl 61 liqdspdsst spkgkqptsa eksvakkedk vpvkkqktrt vfsstqlcvl ndrfqrqkyl 121 slqqmqelsn ilnlsykqvk twfqnqrmks krwqknnwpk nsngvtqkas aptypslyss 181 yhqgclvnpt gnlpmwsnqt wnnstwsnqt qniqswsnhs wntqtwctqs wnnqawnspf 241 yncgeeslqs cmqfqpnspa sdleaaleaa geglnviqqt tryfstpqtm dlflnysmnm 301 qpedv

[0033] By "Nanog polynucleotide" is meant a polynucleotide encoding a KLF4polypeptide. An exemplary Nanog polynucleotide sequence is provided at NCBI Accession No. NM.sub.--024865.

[0034] 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:

TABLE-US-00006 1 mgsysnqqfa ggcakaaeea peeapedaar aadepqllhg agickwfnvr mgfgflsmta 61 ragvaldppv dvfvhqsklh megfrslkeg eaveftfkks akglesirvt gpggvfcigs 121 errpkgksmq krrskgdrcy ncggldhhak ecklppqpkk chfcqsishm vascplkaqq 181 gpsaqgkpty freeeeeihs ptllpeagn

[0035] By "Lin28 polynucleotide" is meant a polynucleotide encoding a Lin28 polypeptide. An exemplary Lin28 polynucleotide sequence is provided at NCBI Accession No. NM.sub.--024674.

[0036] By "PPAR.gamma.2 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 PPAR.gamma.2 amino acid sequence follows:

TABLE-US-00007 1 mgetlgdspi dpesdsftdt lsanisqemt mvdtempfwp tnfgissvdl svmedhshsf 61 dikpfttvdf ssistphyed ipftrtdpvv adykydlklq eyqsaikvep asppyysekt 121 qlynkpheep snslmaiecr vcgdkasgfh ygvhacegck gffrrtirlk liydrcdlnc 181 rihkksrnkc qycrfqkcla vgmshnairf grmpqaekek llaeissdid qlnpesadlr 241 alakhlydsy iksfpltkak arailtgktt dkspfviydm nslmmgedki kfkhitplqe 301 qskevairif qgcqfrsvea vqeiteyaks ipgfvnldln dqvtllkygv heiiytmlas 361 lmnkdgvlis egqgfmtref lkslrkpfgd fmepkfefav kfnaleldds dlaifiavii 421 lsgdrpglln vkpiediqdn llqalelqlk lnhpessqlf akllqkmtdl rqivtehvql 481 lqvikktetd mslhpllgei ykdly

[0037] By "PPAR.gamma.2 polynucleotide" is meant a polynucleotide encoding a PPAR.gamma.2 polypeptide. An exemplary PPAR.gamma.2 polynucleotide sequence is provided at NCBI Accession No. NM.sub.--015869

[0038] By "CEBP.alpha._polypeptide" is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P49715 (CEBP.alpha._HUMAN) and having DNA binding activity. An exemplary CEBP.alpha. amino acid sequence follows:

TABLE-US-00008 1 mesadfyeae prppmsshlq spphapssaa fgfprgagpa qppappaape piggicehet 61 sidisayidp aafndeflad lfqhsrqqek akaavgptgg ggggdfdypg apagpggavm 121 pggahgpppg ygcaaagyld grleplyery gapairpivi kqepreedea kqlalaglfp 181 yqppppppps hphphpppah laaphlqfqi ahcgqttmhl qpghptpppt pvpsphpapa 241 lgaaglpgpg salkglgaah pdlrasggsg agkakksvdk nsneyrvrre rnniavrksr 301 dkakqrnvet qqkvleltsd ndrlrkrveq lsreldtlrg ifrqlpessl vkamgnca

[0039] By "CEBP.alpha. polynucleotide" is meant a polynucleotide encoding a CEBP.alpha. polypeptide. An exemplary CEBP.alpha. polynucleotide sequence is provided at NCBI Accession No. NM.sub.--004364

[0040] By "CEBP.beta. polypeptide" is meant a polypeptide having at least 85% amino acid sequence identity to UniProtKB/Swiss-Prot P17676 (CEBP.beta._HUMAN) and having DNA binding activity. An exemplary CEBP.beta. amino acid sequence follows:

TABLE-US-00009 1 mqrlvawdpa clplpppppa fksmevanfy yeadclaaay ggkaapaapp aarpgprppa 61 gelgsigdhe raidfspyle plgapqapap atatdtfeaa ppapapapas sgqhhdflsd 121 lfsddyggkn ckkpaeygyv slgrlgaakg alhpgcfapl hppppppppp aelkaepgfe 181 padckrkeea gapgggagma agfpyalray lgygavpsgs sgslstssss sppgtpspad 241 akapptacya gaapapsqvk skakktvdkh sdeykirrer nniavrksrd kakmrnletq 301 hkvleltaen erlqkkveql srelstlrnl fkglpeplla ssghc

[0041] By "CEBP.beta. polynucleotide" is meant a polynucleotide encoding a CEBP.beta. polypeptide. An exemplary CEBP.beta. polynucleotide sequence is provided at NCBI Accession No. NM.sub.--005194.

[0042] By "CEBP.delta. 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 CEBP.delta. amino acid sequence follows:

TABLE-US-00010 1 msaalfsldg pargapwpae papfyepgra gkpgrgaepg algepgaaap amyddesaid 61 fsayidsmaa vptlelchde lfadlfnsnh kaggagplel lpggparplg pgpaaprllk 121 repdwgdgda pgsllpaqva acaqtvvsla aaggptppts pepprssprq tpapgparek 181 sagkrgpdrg speyrqrrer nniavrksrd kakrrnqemq qklvelsaen eklhqrveql 241 trdlaglrqf fkqlpsppfl paagtadcr

[0043] By "CEBP.delta. polynucleotide" is meant a polynucleotide encoding a CEBP.delta. polypeptide. An exemplary CEBP.delta. polynucleotide sequence is provided at NCBI Accession No. NM.sub.--005195.

[0044] 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:

TABLE-US-00011 1 mdeppfseaa legalgepcd ldaalltdie dmlqlinnqd sdfpglfdpp yagsgaggtd 61 paspdtsspg slspppatls ssleaflsgp qaapsplspp qpaptplkmy psmpafspgp 121 gikeesvpls ilqtptpqpl pgallpcisf apappqfsst pvlgypsppg gfstgsppgn 181 tqqplpglpl asppgvppvs lhtqvqsvvp qqlltvtaap taapvtttvt sqiqqvpvll 241 gphfikadsl lltamktdga tvkaaglspl vsgttvqtgp lptivsggti latvplvvda 301 eklpinrlaa gskapasaqs rgekrtahna iekryrssin dkiielkdlv vgteaklnks 361 avlrkaidyi rflqhsnqkl kgenlslrta vhkskslkdl vsacgsggnt dvlmegvkte 421 vedtltppps dagspfqssp lslgsrgsgs ggsgsdsepd spvfedskak peqrpslhsr 481 gmldrsrlal ctivflclsc nplasllgar glpspsdtts vyhspgrnvl gtesrdgpgw 541 aqwllppvvw llngllvlvs lvllfvygep vtrphsgpav yfwrhrkqad ldlargdfaq 601 aaqqlwlalr algrplptsh ldlacsllwn lirhllqrlw vgrwlagrag glqqdcalry 661 dasasardaa lvyhklhqlh tmgkhtgghl tatnlalsal nlaecagdav svatlaeiyv 721 aaalrvktsl pralhfltrf flssarqacl aqsgsvppam qwlchpvghr ffvdgdwsvl 781 stpweslysl agnpvdplaq vtqlfrehll eralncvtqp npspgsadgd kefsdalgyl 841 qllnscsdaa gapaysfsis ssmatttgvd pvakwwaslt avvihwlrrd eeaaerlcpl 901 vehlprvlqe serplpraal hsfkaarall gcakaesgpa slticekasg ylqdslattp 961 asssidkavq lflcdlllvv rtslwrqqqp papapaaqgt ssrpqasale lrgfqrdlss 1021 lrrlaqsfrp amrrvflhea tarlmagasp trthqlldrs lrrragpggk ggavaelepr 1081 ptrrehaeal llascylppg flsapgqrvg mlaeaartle klgdrrllhd cqqmlmrlgg 1141 gttvtss

[0045] By "SREBP1c polynucleotide" is meant a polynucleotide encoding a SREBP1c polypeptide. An exemplary SREBP1c polynucleotide sequence is provided at NCBI Accession No. NM.sub.--001005291.

[0046] 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:

TABLE-US-00012 1 mtmesgaenq qsgdaavtea enqqmtvqaq pqiatlaqvs mpaahatssa ptvtivqlpn 61 gqtvqvhgvi qaaqpsviqs pqvqtvqssc kdlkrlfsgt qistiaesed sqesvdsvtd 121 sqkrreilsr rpsyrkilnd lssdapgvpr ieeekseeet sapaittvtv ptpiyqtssg 181 qyiaitqgga iqlanngtdg vqglqtltmt naaatqpgtt ilqyaqttdg qqilvpsnqv 241 vvqaasgdvq tyqirtapts tiapgvvmas spalptqpae eaarkrevrl mknreaarec 301 rrkkkeyvkc lenrvavlen qnktlieelk alkdlychks d

[0047] By "CREB1polynucleotide" is meant a polynucleotide encoding a CREB1polypeptide. An exemplary CREB1polynucleotide sequence is provided at NCBI Accession No. BC010636.

[0048] 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:

TABLE-US-00013 1 mmtakavdki pvtlsgfvhq lsdniypved laatsvtifp naelggpfdq mngvagdgmi 61 nidmtgekrs ldlpypssfa pvsaprnqtf tymgkfsidp qypgascype giinivsagi 121 lqgvtspast tasssvtsas pnplatgplg vctmsqtqpd ldhlyspppp pppysgcagd 181 lyqdpsafls aattstsssl ayppppsyps pkpatdpglf pmipdypgff psqcqrdlhg 241 tagpdrkpfp cpldtlrvpp pltplstirn ftlggpsagv tgpgasggse gprlpgsssa 301 aaaaaaaaay nphhlplrpi lrprkypnrp sktpvherpy pcpaegcdrr fsrsdeltrh 361 irihtghkpf qcricmrnfs rsdhltthir thtgekpfac dycgrkfars derkrhtkih 421 lrqkerkssa psasvpapst ascsggvqpg gticssnsss lgggplapcs srtrtp

[0049] By "KROX20 polynucleotide" is meant a polynucleotide encoding a KROX20 polypeptide. An exemplary KROX20 polynucleotide sequence is provided at NCBI Accession No.--BC35625.

[0050] 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:

TABLE-US-00014 1 matrvasmsa rlgpvpqppa pqdepvfaql kpvlgaanpa rdaalfpgee lkhahhrpqa 61 qpapaqapqp aqppatgprl ppedlvqtrc emekyltpql ppvpiipehk kyrrdsasvv 121 dqfftdtegl pysinmnvfl pdithlrtgl yksqrpcvth iktepvaifs hqsettappp 181 aptqalpeft sifsshqtaa pevnnifikq elptpdlhls vptqqghlyq llntpdldmp 241 sstnqtaamd tlnvsmsaam aglnthtsav pqtavkqfqg mppctytmps qflpqqatyf 301 ppsppssepg spdrqaemlq nitpppsyaa tiasklaihn pnlpttlpvn sqniqpvryn 361 rrsnpdlekr rihycdypgc tkvytksshl kahlrthtge kpykctwegc dwrfarsdel 421 trhyrkhtga kpfqcgvcnr sfsrsdhlal hmkrhqn

[0051] By "KLF5 polynucleotide" is meant a polynucleotide encoding a KLF5 polypeptide. An exemplary KLF5 polynucleotide sequence is provided at NCBI Accession No.--NM.sub.--001730.

[0052] 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:

TABLE-US-00015 1 mvdhllpvde nfsspkcpvg ylgdrlvgrr ayhmlpspvs eddsdasspc scsspdsqal 61 cscyggglgt esqdsildfl lsgatlgsgg gsgssigass gpVawgpwrr aaapvkgehf 121 clpefplgdp ddvprpfqpt leeieeflee nmepgvkevp egnskdldac sqlsagphks 181 hlhpgssgre rcspppggas aggaqgpggg ptpdgpipvl lqiqpvpvkg esgtgpaspg 241 qapenvkvaq llvniqgqtf alvpqvvpss nlnlpskfvr iapvpiaakp vgsgplgpgp 301 agllmgqkfp knpaaelikm hkctfpgcsk mytksshlka hlrrhtgekp factwpgcgw 361 rfsrsdelsr hrrshsgvkp yqcpvcekkf arsdhlskhi kvhrfprssr svrsvn

[0053] By "KLF15 polynucleotide" is meant a polynucleotide encoding a KLF15 polypeptide. An exemplary KLF15 polynucleotide sequence is provided at NCBI Accession No. NM.sub.--014079

[0054] 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:

TABLE-US-00016 1 meyamkslsl lypkslsrhv svrtsvvtqq llsepspkap rarpervsta drsvrkgima 61 ysledlllkv rdtlmladkp fflvleedgt tveteeyfqa lagdtvfmvl qkgqkwqpps 121 eqgtrhplsl shkpakkidv arvtfdlykl npqdfigcln vkatfydtys lsydlhccga 181 krimkeafrw alfsmqatgh vllgtscylq qlldateegq ppkgkassli ptclkilq

[0055] By "CIDEC polynucleotide" is meant a polynucleotide encoding a CIDEC polypeptide. An exemplary CIDEC polynucleotide sequence is provided at NCBI Accession No. NM.sub.--022094.

[0056] By "adipogenic" is meant inducing one or more of an adipocyte marker, adipocyte function, or adipocyte morphology in a multipotent or pluripotent stem cell.

[0057] 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.

[0058] 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.

[0059] By "adipocyte marker" is meant one or more of Glut4, PPARy, C/EBP.alpha., C/EBP.beta., C/EBP.delta., leptin, adiponectin, SREBP1c, FABP4, CIDEC and/or perilipin.

[0060] By "adipocyte morphology" is meant the presence of lipid droplets.

[0061] 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.

[0062] 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.

[0063] By "analog" is meant a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0064] By "autologous" is meant cells from the same subject.

[0065] By "compound" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] By "fusion protein" is meant a protein that combines at least two amino acid sequence that are not naturally contiguous.

[0073] 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.sup.-3 and e.sup.-100 indicating a closely related sequence.

[0074] 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).

[0075] 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.degree. C., more preferably of at least about 37.degree. C., and most preferably of at least about 42.degree. 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.degree. C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37.degree. C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42.degree. C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 .mu.g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0076] 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.degree. C., more preferably of at least about 42.degree. C., and most preferably of at least about 68.degree. C. In a preferred embodiment, wash steps will occur at 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42.degree. 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.degree. 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

[0082] 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.

[0083] By "naturally occurs" is meant is endogenously expressed in a cell of an organism.

[0084] By "obtaining" as in "obtaining the polypeptide" is meant synthesizing, purchasing, or otherwise acquiring the polypeptide.

[0085] 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.

[0086] By "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification.

[0087] 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).

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] By "stem cell progenitor" is meant a cell that gives rise to stem cells.

[0093] 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.

[0094] By "syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison.

[0095] 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.

[0096] 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.

[0097] By "reference" is meant a standard or control condition.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIGS. 1A and 1B provide vector maps of the following lentiviral constructs: TRE-PPAR.gamma.2 based on FUGW vector (FIG. 1A) and ubiquitin rtTA M2 (FIG. 1B).

[0103] FIGS. 2A-2C are micrographs showing results from viral efficiency assays. ADMSC were infected with supernatant eGFP/rtTA M2 in a 1:2 ratio. Micrographs were generated 24 hours after doxycycline induction. FIG. 2A shows an overlay of FIGS. 2B and 2C. FIG. 2B shows cells in brightfield. FIG. 2C shows GFP expression.

[0104] FIG. 3A-3F are micrographs showing the morphology of adipocytes derived from human ES cells: Pictures A-B taken in 200.times., C-D in 40.times., E-F 100.times.--magnification. FIGS. 3A-D show cells from the HUES 8 line that were infected with C/EBP.alpha. and differentiated for 21 days in adipogenic differentiation medium. FIGS. 3E-3F show cells from the HUES 8 line that were infected with control rtTA virus and differentiated for 21 days in adipogenic differentiation medium. FIGS. 3A, 3C, and 3E are bright field images. FIGS. 3B, 3D, and 3F are corresponding images stained with Bodipy, a fluorescent marker of neutral lipids.

[0105] FIGS. 4A-4H are micrographs showing the morphology of adipocytes derived from human ES cells. Pictures were taken in 200.times. magnification. All lines were infected with PPAR.gamma.2 and rtTA M2 and differentiated for 14 days in adipogenic differentiation medium. FIGS. 4A-4D show the morphology of human embryonic stem cell HUES-6 line infected with PPAR.gamma.-rtTA). FIGS. 4E-4H the morphology of human embryonic stem cell ADMSC infected with PPAR.gamma.-rtTA. FIGS. 4A and 4B show cells in bright field and corresponding immunostaining for Fatty acid binding protein 4 (FABP4). FIGS. 4C and 4D show bright field and corresponding immunostaining for perilipin. FIGS. 4E and 4F show brightfield and corresponding immunostaining for FABP4; FIGS. 4G and 4H show brightfield and corresponding immunostaining for perilipin.

[0106] FIG. 5 provides graphs showing the relative expression of key adipogenic genes depicted as fold change normalized to HPRT. Expression was assayed by qRT-PCR of human induced pluripotent stem cells (BJ#8, blue bars) transduced HUES 8 with either C/EBP.alpha. left panel), C/EBP.beta. and .delta., or PPAR.gamma. and C/EBP.beta. and .delta. (right panel), differentiated for 21 days in adipogenic differentiation medium.

[0107] FIGS. 6A-6G illustrate the generation and characterization of human induced pluripotent stem (hiPS) cells. FIG. 6A is a schematic diagram illustrating the experimental scheme for the generation of hiPS cells. FIG. 6B includes 3 micrographs showing the morphology of primary human somatic cells from skin biopsy, hair follicle pull, and peripheral blood mononuclear cell (PBMC) fraction. FIG. 6C shows the morphology and marker expression in hiPS colonies. FIG. 6D illustrates results obtained using bisulfite sequencing of the NANOG and the OCT4 promoter regions containing differentially methylated CpGs in BJ fibroblasts, BJ fibroblast-derived hiPS, and WA09 hES cells. Open circles represent unmethylated CpGs; closed circles denote methylated CpGs. FIG. 6E shows a microarray analysis of gene expression in hiPS cells. Genes with greater than two-fold expression level between HUES8 hES cells and BJ fibroblasts were analyzed. Shown are BJ fibroblasts, HUES8 hES cells, and BJ fibroblast-derived hiPS clones. FIG. 6F provides micrographs showing the in vitro differentiation of fibroblast and keratinocyte-derived hiPS cells into lineages from all three germ layers. Immunostaining for (i) Tuj1 (neuronal), (ii) cardiac troponin T (cTnT; cardiac muscle) or myosin heavy chain (MF20; skeletal muscle), and (iii) alpha-fetoprotein (AFP; epithelial, early endodermal). FIG. 6G provides micrographs showing hematoxylin and eosin staining of teratomas generated from fibroblast-derived hiPS cells. Differentiated structures from all three germ layers were present. (i) Pigmented epithelium (ectoderm), (ii) cartilage (mesoderm), (iii) gut-like epithelium (endoderm), and (iv) muscle (mesoderm).

[0108] FIG. 7 is a schematic diagram illustrating the differentiation of human ES cells into adipocytes. The images show ES-derived adipocytes with 20.times. brightfield (top) and DAPI (blue), perilipin (red) antibody staining.

[0109] FIG. 8 is a schematic diagram illustrating the directed differentiation of human pluripotent cells to adipocytes using adipogenic transcription factors. Depicted above is the strategy to inducibly express transcription factors in combination with an adipogenic media cocktail. Along the timeline of differentiation are gene expression and morphological changes that are expected to occur as human pluripotent cells adopt an adipocyte cell fate.

[0110] FIG. 9 is a schematic diagram illustrating the transcriptional events leading to adipocyte formation. This figure shows how transcriptional events and protein interactions could lead a human embryonic stein cell to differentiate to an adipocyte. It shows some important pathways that are known to play a role in adipocyte differentiation in mouse and human systems. It also portrays the mechanisms by which growth factors and chemical compounds interact with these pathways. The underlying network of effectors is highly complex; for example, there are more than 100 specific transcription factors active in adipocytes and many of them form complexes with different co-activators and histone-remodeling complexes.

[0111] FIG. 10 describes the 9p21.3 DM and MI loci, with superimposed HapMap CEU (European) linkage disequilibrium map and local genes/transcripts.

[0112] FIGS. 11A-11B show the strategy for performing homologous recombination in HUES or iPS cells. FIG. 11B is a photograph of an agarose gel with PCR products, which confirm that successful recombination occurred in a HUES-8 clone (#3) with dual PCR screening at 5' and 3' ends [primer positions indicated in (A)].

[0113] FIGS. 12A-E document the generation and characterization of human induced pluripotent stem (human iPS) cells. FIG. 12A is a schematic diagram illustrating an experimental scheme for the generation of human iPS cells. FIG. 12B provides micrographs of primary human somatic cells from skin biopsy, hair follicle pull, and PBMC fraction. FIG. 12C shows morphology and marker expression in human iPS colonies.

[0114] FIG. 12D shows the in vitro differentiation of fibroblast and keratinocyte-derived human iPS cells into lineages from all three germ layers. Immunostaining for (i) Tuj1 (neuronal), (ii) cardiac troponin T (cTnT; cardiac muscle) or myosin heavy chain (MF20; skeletal muscle), and (iii) alpha-fetoprotein (AFP; epithelial, early endodermal). (E) Hematoxylin and eosin stain of teratomas generated from fibroblast-derived human iPS cells. Differentiated structures from all three germ layers were present. (i) Pigmented epithelium (ectoderm), (ii) cartilage (mesoderm), (iii) gut-like epithelium (endoderm), and (iv) muscle (mesoderm).

DETAILED DESCRIPTION OF THE INVENTION

[0115] 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.

[0116] 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. PPAR.gamma.2, C/EBP.alpha., C/EBP.beta., C/EBP.delta., SREBP-1c, CREB1, and KROX20). As reported herein, the adipogenic activity of PPAR.gamma.2, C/EBP.alpha., C/EBP.beta. or C/EBP.delta. was demonstrated by ectopically expressing them in human pluripotent cells. The viral transduction and inducible expression of PPAR.gamma.2 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.

[0117] 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 .about.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.

Obesity

[0118] Obesity 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.

[0119] 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`.

[0120] 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).

Lipodystrophy

[0121] Lipodystrophy 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.

[0122] 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 2

[0123] A 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.

[0124] 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 Cells

[0125] As 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.

[0126] 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.)

[0127] 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.

[0128] 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).

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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 Cells

[0135] A 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 Cells

[0136] Once 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.TM., 2-Mercaptoethanol, non-essential amino acids, insulin (e.g., 10 .mu.g/ml), dexamethasone (e.g., 1.2 .mu.M), rosiglitazone (e.g., 0.5 .mu.M) and IBMX (e.g., 0.5 mM).

[0137] Cells have been transduced with a virus that allows the inducible over-expression of PPAR.gamma.2. 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).

[0138] 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.

[0139] 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 PPAR.gamma.2, C/EBP.alpha., C/EBP.beta., C/EBP.delta., SREBP1c, CREB1, and KROX20. In one embodiment, PPAR.gamma.2, C/EBP.alpha., C/EBP.beta. and/or C/EBP.delta. are expressed in human stem cells where they induce adipogenesis. In yet another embodiment, inducible forms of PPAR.gamma.2 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.

[0140] Cells of the invention are cultured on a commercially available extracellular matrix, such as Mantel.

Methods of Characterizing Reprogrammed Adipocyte Cells

[0141] Adipocytes display a number of morphological and molecular hallmarks.

[0142] 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.

[0143] 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.

[0144] In another embodiment, immunocytochemistry is used to assay for any one or more of the following adipocyte markers: FABP4, CIDEC, PPARy, and/or Glut4.

[0145] 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.

[0146] In other embodiments, reprogrammed adipocyte cells of the invention are analyzed for the response to insulin by measuring (U-.sup.14C)-D-glucose uptake (Kashiwagi et al., J. Clin Invest 72 (4), 1246-1254 (1983)1983), lipolytic activity by inducing lipolysis using .beta.-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-.sup.14C) acetate and incorporation of free fatty acids using (.sup.3H) oleate or (.sup.14C) palmitate.

[0147] 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 Repair

[0148] The 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 Malnutrition

[0149] Patients 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 Cells

[0150] An 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.

[0151] 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 Compositions

[0152] An 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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 LD.sub.50 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 Cells

[0159] Genetic 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.

[0160] 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 Expression

[0161] As 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., PPAR.gamma.2, C/EBP.alpha., C/EBP.beta., C/EBP.delta., 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-CaPO.sub.4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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).

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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 Analogs

[0176] The Examples herein describe the expression of adipogenic polypeptides (e.g., PPAR.gamma.2, C/EBP.alpha., C/EBP.beta., C/EBP.delta., 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.sup.-3 and e.sup.-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., .beta. or .gamma. amino acids.

[0177] 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).

[0178] 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 Extracts

[0179] Reprogrammed 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.).

[0185] 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.

Kits

[0186] The 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.

[0187] 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.

[0188] In another aspect, the invention provides kits that feature expression vectors useful for the differentiation of an reprogrammed adipocyte cell.

[0189] 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.

[0190] 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 Cells

[0191] Known regulators of adipogenesis were cloned into a doxycycline-inducible lentiviral backbone (e.g. PPAR.gamma.2, C/EBP.alpha., C/EBP.beta., C/EBP.delta., SREBP1c, CREB1, and KROX20) (FIG. 1). To test viral titers human adipocyte-derived mesenchymal stem cells (ADMSCs) and human embryonic stem cells were transduced with enhanced green fluorescent protein (eGFP) and reverse tetracycline-controlled transcriptional activator M2 (rtTA-M2) expressing lentiviruses. Fluoresence microscopy of ADMSCs followed by fluorescence-activated cell sorting (FACS) revealed a transfection efficiency of 93% (FIGS. 2A-2C) with a similar efficiency in human embryonic stem cells.

[0192] The adipogenic potential of lentiviral PPAR.gamma.2 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.

[0193] Ectopic expression of PPAR.gamma.2, C/EBP.alpha., C/EBP.beta. or C/EBP.delta. showed that each of these has adipogenic activity in human pluripotent cells. In particular, the viral transduction and inducible expression of PPAR.gamma.2 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 (FIGS. 3A-3F).

[0194] Importantly, these cells expressed the mature adipocyte markers CIDEC, FABP4 and perilipin (FIGS. 4A-4H). These methods provide for the generation of pluripotent cell derived adipocytes at an efficiency of .about.20%.

[0195] The analysis of 3 hES lines infected with C/EBP.alpha. (no rtTA), PPAR.gamma.--C/EBP.beta.-C/EBP.delta. and C/EBP.beta.-C/EBP.delta. is shown in comparison to a control line BJ-iPS without a transgene. As shown in FIG. 3 C, almost no lipid accumulation was found in the Bj-IPS. However endogenous PPAR.gamma. and C/EBP.beta., is found in cells cultured in the differentiation medium. This did not lead to the formation of adipocytes and subsequently very low levels of the maturation marker FABP4, or the mature marker CIDEC were found in quantitative RT-PCR. The best results were achieved when C/EBP.alpha., was transduced, but expressed at a relatively low level, due to the transgene expression via the viral LTRs, independent of doxycycline induction. PPAR.gamma.transgene expression on the other hand resulted in robust differentiation when PPAR gamma was strongly expressed using the rtTA doxycycline induction, as shown in FIG. 2 and corresponding qRT-PCR (FIG. 5).

[0196] 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-.sup.14C)-D-glucose uptake (Kashiwagi, supra), (2) lipolytic activity by inducing lipolysis using .beta.-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-.sup.14C) acetate and (4) incorporation of free fatty acids using (.sup.3H) oleate or (.sup.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 Pathology

[0197] The 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 (FIG. 6B). The cells are transduced with a single doxycycline-inducible lentivirus that transiently expresses all of the four genes needed for fibroblast programming into iPS cells (FIGS. 6A-6D). Vectors and methods for generating induced pluripotent stem cells are known in the art and described, for example, in Maherali et al., Cell Stem Cell 3, 340-345, which is incorporated herein by reference in its entirety. FIG. 6F provides micrographs showing the in vitro differentiation of fibroblast and keratinocyte-derived hiPS cells into lineages from all three germ layers. Pluripotency was also analysed by a microarray analysis of gene expression in hiPS cells (FIGS. 6E and 6F). Genes with greater than two-fold expression level between HUES8 hES cells and BJ fibroblasts were analyzed. Shown are BJ fibroblasts, HUES8 hES cells, and BJ fibroblast-derived hiPS clones. Immunostaining for (i) Tuj1 (neuronal), (ii) cardiac troponin T (cTnT; cardiac muscle) or myosin heavy chain (MF20; skeletal muscle), and (iii) alpha-fetoprotein (AFP; epithelial, early endodermal) (FIG. 6F). FIG. 6G provides micrographs showing hematoxylin and eosin staining of teratomas generated from fibroblast-derived hiPS cells. Differentiated structures from all three germ layers were present. (i) Pigmented epithelium (ectoderm), (ii) cartilage (mesoderm), (iii) gut-like epithelium (endoderm), and (iv) muscle (mesoderm).

[0198] 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 PPAR.gamma.2 and C/EBP factors (FIG. 7).

[0199] 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.

[0200] 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.

TABLE-US-00017 TABLE 1 Replication evidence for four previously reported common variants associated with premature myocardial infarction. Studies (maximum available effective Previously reported SNPs with sample size) convincing replication evidence SNP rs4977574 rs646776 rs17465637 rs1746048 Chr 9p21 1p13 1q41 10q11 Position 22,088,574 109,530,572 220,890,152 44,095,830 NCBI35 (bp) Non-risk allele A C A T Risk allele G T C C Risk allele 0.56 0.81 0.72 0.84 frequency Gene(s) of CDKN2A CELSR2 MIA3 CXCL12 interest in CDKN2B PSRC1 associated SORT1 Studies (maximum interval

TABLE-US-00018 TABLE 2 Five new common variants associated with early-onset myocardial infarction. Studies (maximum available Newly-identified effective sample common variants at size) Newly-identified loci previously reported loci SNP rs9982601 rs12526453 rs6725887.sup.c rs1122608 rs11206510 Chr 21q22 6p24 2q33 19p13 1p32 Position NCBI35 34,520,998 13,035,530 203,454,130 11,024,601 55,268,627 (bp) Non-risk C G T T C allele Risk allele T C C G T Risk allele 0.13 0.65 0.14 0.75 0.81 frequency Gene(s) of SLC5A3 PHACTR1 WDR12 LDLR PCSK9 interest in MRPS6 associated KCNE2 interval

[0201] 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

PPAR.gamma.2 Expression is Sufficient for Adipocyte Differentiation In Vitro from both hEs Cells and ADMSCs

[0202] As 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.

[0203] 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.

[0204] 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.

[0205] 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). FIG. 8 provides a schematic diagram illustrating this strategy. Using a doxycycline-inducible lentivirus system, the genes in the transcriptional network demonstrated to control terminal adipocyte differentiation (e.g. PPAR.gamma., CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBP.beta., C/EBP.delta. and C/EBP.alpha.) (FIG. 9) will be transduced into human pluripotent cells. The infected cells were cultured for three days under standard human embryonic stem cell conditions (Cowan supra). Three days following infection, the cells were cultured in standard adipocyte differentiation cocktail containing insulin, dexamethasone, isobutylmethylxanthine (IBMX), and rosiglitazone, a synthetic agonist for PPAR.gamma., and lentiviral gene expression were induced by the addition of doxycycline.

[0206] 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 (FIG. 9).

Example 4

HUES Cells and iPS are Used to Generate an Allelic Series of Cell Lines on 9p21.3

[0207] 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.

TABLE-US-00019 TABLE 3 SNPs associated with type 2 DM identified in GWAS. Odds ratio per allele SNP Closest gene P value (95% CI) rs7901695 TCF7L2 1 .times. 10.sup.-48 1.37 (1.31-1.43) rs10811661 CDKN2A-CDKN2B 8 .times. 10.sup.-15 1.20 (1.14-1.25) rs8050136 FTO 1 .times. 10.sup.-12 1.17 (1.12-1.22) rs13266634 SLC30A8 1 .times. 10.sup.-19 1.15 (1.12-1.19) rs1111875 HHEX-IDE 7 .times. 10.sup.-17 1.15 (1.10-1.19) rs10946398 CDKAL1 2 .times. 10.sup.-18 1.14 (1.11-1.17) rs4402960 IGF2BP2 9 .times. 10.sup.-16 1.14 (1.11-1.18)

[0208] Among the most highly associated genetic loci for type 2 DM is a region on chromosome 9p21.3 harboring the SNP rs10811661 (P=8.times.10.sup.-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) (FIG. 10). Remarkably, in an independent set of GWAS studies with myocardial infarction (MI), SNPs on chromosome 9p21.3 were also identified as being strongly associated with DM. However, these SNPs do not coincide with the DM-associated SNPs (Helgadottir et al., 2008), and they define a .about.60 kb region just proximal to the DM locus, separated by a recombination hotspot (FIG. 10)

[0209] 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).

[0210] 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.

[0211] Recognizing that rs10811661 may be in linkage disequilibrium with the causal variant related to DM--somewhere in the .about.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 .about.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.

[0212] Homologous recombination in a human embryonic stem (HUES) cell line (HUES-8) was successfully performed to knock out the entire .about.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 FIG. 11. Genome-wide genotyping of a Wide array of HUES cells and identified cell lines of various genotypes was performed at rs10811661. Induced pluripotent stem (iPS) cells are derived from patient samples by a virus-based reprogramming of patient-derived fibroblasts. Fibroblasts suitable for reprogramming were obtained from three different sources: skin punch biopsies, hair follicles, and peripheral blood mononuclear cells (PBMC) fractions from blood draws (FIG. 12). A single doxycycline-inducible lentivirus was developed that transiently expresses all of the four genes needed for fibroblast programming into iPS cells. The lentivirus harbors loxP sites that allow for the use of Cre recombinase to subsequently remove the four genes.

[0213] The results reported herein were obtained using the following materials and methods.

[0214] Cell culture media can be made as follows:

Adipogenic Differentiation Media

TABLE-US-00020 [0215] Volume: 651 ml 325.5 ml KO-DMEM 500 ml 250 ml KO-Serum replacer 65 ml 32.5 ml Plasmanate (human) 65 ml 32.5 ml Penicillin-Streptomycin 100x Solution 6.5 ml 3.3 ml Gluta-Max .TM. 6.5 ml 3.3 ml 2-Mercaptoethanol Sol. (13.8M) 3.5 .mu.l 1.8 .mu.l Non-essential Amino Acids Solution 6.5 ml 3.3 ml Insulin 50 nM-25 .mu.g (e.g., 1, 5, 10, 15, 20, 25 .mu.g/ml) Dexamethasone 500 nM-5 .mu.M (e.g., 500 nM, 1.2 .mu.M, 2 .mu.M, 5 .mu.M) Rosiglitazone 100 nM-20 .mu.M (e.g., 100 nM, 5 .mu.M, 10 .mu.M, 20 .mu.M) If desired, IBMX 500 nM-500 .mu.M (e.g., 500 nm, 100 .mu.M, 250 .mu.M, 500 .mu.M)

hESC Cell Culture Media

TABLE-US-00021 KO-DMEM 500 mL KO serum replacement 65 mL Human Plasmanate 65 mL Penicillin-Streptomycin 100x Solution 6.5 mL Gluta-Max .TM. 6.5 mL 2-Mercaptoethanol 1000x (or 3.5uL of 13.8M solution) 0.65 mL Non-essential Amino Acids Solution 6.5 mL bFGF ~10 ng/mL Alternatively, mTESR culture media (Stem Cell Technologies) may be used.

ADMSC Cell Culture Media

TABLE-US-00022 [0216] DMEM media 500 mL Fetal Bovine Serum (FBS) 55 mL Penicillin-Streptomycin 100x Solution 5.5 mL

Maintenance and Expansion of ADMSCs

[0217] 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 Conditions

[0218] hESCs 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)

[0219] 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

[0220] 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 Conditions

[0221] hESCs 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)

[0222] 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

[0223] 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.

[0224] 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

[0225] 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

[0226] The cell medium was removed and replaced with PBS containing BODIPY dye (used as a 10 000.times. 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.

[0227] Nuclear Staining with DAPI

[0228] 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 Embodiments

[0229] From 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.

[0230] 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.

[0231] 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.

Sequence CWU 1

1

171360PRTHomo sapiens 1Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly1 5 10 15Gly Gly Gly Asp Gly Pro Gly Gly Pro Glu Pro Gly Trp Val Asp Pro 20 25 30Arg Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly 35 40 45Pro Gly Val Gly Pro Gly Ser Glu Val Trp Gly Ile Pro Pro Cys Pro 50 55 60Pro Pro Tyr Glu Phe Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val65 70 75 80Gly Val Gly Leu Val Pro Gln Gly Gly Leu Glu Thr Ser Gln Pro Glu 85 90 95Gly Glu Ala Gly Val Gly Val Glu Ser Asn Ser Asp Gly Ala Ser Pro 100 105 110Glu Pro Cys Thr Val Thr Pro Gly Ala Val Lys Leu Glu Lys Glu Lys 115 120 125Leu Glu Gln Asn Pro Glu Glu Ser Gln Asp Ile Lys Ala Leu Gln Lys 130 135 140Glu Leu Glu Gln Phe Ala Lys Leu Leu Lys Gln Lys Arg Ile Thr Leu145 150 155 160Gly Tyr Thr Gln Ala Asp Val Gly Leu Thr Leu Gly Val Leu Phe Gly 165 170 175Lys Val Phe Ser Gln Thr Thr Ile Cys Arg Phe Glu Ala Leu Gln Leu 180 185 190Ser Phe Lys Asn Met Cys Lys Leu Arg Pro Leu Leu Gln Lys Trp Val 195 200 205Glu Glu Ala Asp Asn Asn Glu Asn Leu Gln Glu Ile Cys Lys Ala Glu 210 215 220Thr Leu Val Gln Ala Arg Lys Arg Lys Arg Thr Ser Ile Glu Asn Arg225 230 235 240Val Arg Gly Asn Leu Glu Asn Leu Phe Leu Gln Cys Pro Lys Pro Thr 245 250 255Leu Gln Gln Ile Ser His Ile Ala Gln Gln Leu Gly Leu Glu Lys Asp 260 265 270Val Val Arg Val Trp Phe Cys Asn Arg Arg Gln Lys Gly Lys Arg Ser 275 280 285Ser Ser Asp Tyr Ala Gln Arg Glu Asp Phe Glu Ala Ala Gly Ser Pro 290 295 300Phe Ser Gly Gly Pro Val Ser Phe Pro Leu Ala Pro Gly Pro His Phe305 310 315 320Gly Thr Pro Gly Tyr Gly Ser Pro His Phe Thr Ala Leu Tyr Ser Ser 325 330 335Val Pro Phe Pro Glu Gly Glu Ala Phe Pro Pro Val Ser Val Thr Thr 340 345 350Leu Gly Ser Pro Met His Ser Asn 355 3602317PRTHomo sapiens 2Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln1 5 10 15Thr Ser Gly Gly Gly Gly Gly Asn Ser Thr Ala Ala Ala Ala Gly Gly 20 25 30Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn Ala Phe 35 40 45Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu Asn Pro 50 55 60Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu Trp Lys65 70 75 80Leu Leu Ser Glu Thr Glu Lys Arg Pro Phe Ile Asp Glu Ala Lys Arg 85 90 95Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr Arg Pro 100 105 110Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr Leu Pro 115 120 125Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly Val Gly 130 135 140Val Gly Ala Gly Leu Gly Ala Gly Val Asn Gln Arg Met Asp Ser Tyr145 150 155 160Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met Gln Asp 165 170 175Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly Ala Ala 180 185 190Gln Met Gln Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln Tyr Asn 195 200 205Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr Tyr Ser 210 215 220Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly Ser Met225 230 235 240Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val Val Thr 245 250 255Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu Arg Asp 260 265 270Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro Ala Ala 275 280 285Pro Ser Arg Leu His Met Ser Gln His Tyr Gln Ser Gly Pro Val Pro 290 295 300Gly Thr Ala Ile Asn Gly Thr Leu Pro Leu Ser His Met305 310 3153439PRTHomo sapiens 3Met Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr1 5 10 15Asp Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr 20 25 30Gln Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp 35 40 45Ile Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser 50 55 60Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe65 70 75 80Ser Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala 85 90 95Asp Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn 100 105 110Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile 115 120 125Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu 130 135 140Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly145 150 155 160Ser Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu 165 170 175Tyr Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser 180 185 190Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys 195 200 205Ala Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu 210 215 220Ser Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu225 230 235 240His Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln 245 250 255Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala 260 265 270Pro Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser 275 280 285Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr 290 295 300His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro305 310 315 320Ala Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile 325 330 335Ser Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu 340 345 350Asn Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn 355 360 365Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu 370 375 380Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr385 390 395 400Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu 405 410 415Glu Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu 420 425 430Gln Leu Arg Asn Ser Cys Ala 4354513PRTHomo sapiens 4Met Arg Gln Pro Pro Gly Glu Ser Asp Met Ala Val Ser Asp Ala Leu1 5 10 15Leu Pro Ser Phe Ser Thr Phe Ala Ser Gly Pro Ala Gly Arg Glu Lys 20 25 30Thr Leu Arg Gln Ala Gly Ala Pro Asn Asn Arg Trp Arg Glu Glu Leu 35 40 45Ser His Met Lys Arg Leu Pro Pro Val Leu Pro Gly Arg Pro Tyr Asp 50 55 60Leu Ala Ala Ala Thr Val Ala Thr Asp Leu Glu Ser Gly Gly Ala Gly65 70 75 80Ala Ala Cys Gly Gly Ser Asn Leu Ala Pro Leu Pro Arg Arg Glu Thr 85 90 95Glu Glu Phe Asn Asp Leu Leu Asp Leu Asp Phe Ile Leu Ser Asn Ser 100 105 110Leu Thr His Pro Pro Glu Ser Val Ala Ala Thr Val Ser Ser Ser Ala 115 120 125Ser Ala Ser Ser Ser Ser Ser Pro Ser Ser Ser Gly Pro Ala Ser Ala 130 135 140Pro Ser Thr Cys Ser Phe Thr Tyr Pro Ile Arg Ala Gly Asn Asp Pro145 150 155 160Gly Val Ala Pro Gly Gly Thr Gly Gly Gly Leu Leu Tyr Gly Arg Glu 165 170 175Ser Ala Pro Pro Pro Thr Ala Pro Phe Asn Leu Ala Asp Ile Asn Asp 180 185 190Val Ser Pro Ser Gly Gly Phe Val Ala Glu Leu Leu Arg Pro Glu Leu 195 200 205Asp Pro Val Tyr Ile Pro Pro Gln Gln Pro Gln Pro Pro Gly Gly Gly 210 215 220Leu Met Gly Lys Phe Val Leu Lys Ala Ser Leu Ser Ala Pro Gly Ser225 230 235 240Glu Tyr Gly Ser Pro Ser Val Ile Ser Val Ser Lys Gly Ser Pro Asp 245 250 255Gly Ser His Pro Val Val Val Ala Pro Tyr Asn Gly Gly Pro Pro Arg 260 265 270Thr Cys Pro Lys Ile Lys Gln Glu Ala Val Ser Ser Cys Thr His Leu 275 280 285Gly Ala Gly Pro Pro Leu Ser Asn Gly His Arg Pro Ala Ala His Asp 290 295 300Phe Pro Leu Gly Arg Gln Leu Pro Ser Arg Thr Thr Pro Thr Leu Gly305 310 315 320Leu Glu Glu Val Leu Ser Ser Arg Asp Cys His Pro Ala Leu Pro Leu 325 330 335Pro Pro Gly Phe His Pro His Pro Gly Pro Asn Tyr Pro Ser Phe Leu 340 345 350Pro Asp Gln Met Gln Pro Gln Val Pro Pro Leu His Tyr Gln Gly Gln 355 360 365Ser Arg Gly Phe Val Ala Arg Ala Gly Glu Pro Cys Val Cys Trp Pro 370 375 380His Phe Gly Thr His Gly Met Met Leu Thr Pro Pro Ser Ser Pro Leu385 390 395 400Glu Leu Met Pro Pro Gly Ser Cys Met Pro Glu Glu Pro Lys Pro Lys 405 410 415Arg Gly Arg Arg Ser Trp Pro Arg Lys Arg Thr Ala Thr His Thr Cys 420 425 430Asp Tyr Ala Gly Cys Gly Lys Thr Tyr Thr Lys Ser Ser His Leu Lys 435 440 445Ala His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr His Cys Asp Trp 450 455 460Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp Glu Leu Thr Arg His465 470 475 480Tyr Arg Lys His Thr Gly His Arg Pro Phe Gln Cys Gln Lys Cys Asp 485 490 495Arg Ala Phe Ser Arg Ser Asp His Leu Ala Leu His Met Lys Arg His 500 505 510Phe5305PRTHomo sapiens 5Met Ser Val Asp Pro Ala Cys Pro Gln Ser Leu Pro Cys Phe Glu Ala1 5 10 15Ser Asp Cys Lys Glu Ser Ser Pro Met Pro Val Ile Cys Gly Pro Glu 20 25 30Glu Asn Tyr Pro Ser Leu Gln Met Ser Ser Ala Glu Met Pro His Thr 35 40 45Glu Thr Val Ser Pro Leu Pro Ser Ser Met Asp Leu Leu Ile Gln Asp 50 55 60Ser Pro Asp Ser Ser Thr Ser Pro Lys Gly Lys Gln Pro Thr Ser Ala65 70 75 80Glu Lys Ser Val Ala Lys Lys Glu Asp Lys Val Pro Val Lys Lys Gln 85 90 95Lys Thr Arg Thr Val Phe Ser Ser Thr Gln Leu Cys Val Leu Asn Asp 100 105 110Arg Phe Gln Arg Gln Lys Tyr Leu Ser Leu Gln Gln Met Gln Glu Leu 115 120 125Ser Asn Ile Leu Asn Leu Ser Tyr Lys Gln Val Lys Thr Trp Phe Gln 130 135 140Asn Gln Arg Met Lys Ser Lys Arg Trp Gln Lys Asn Asn Trp Pro Lys145 150 155 160Asn Ser Asn Gly Val Thr Gln Lys Ala Ser Ala Pro Thr Tyr Pro Ser 165 170 175Leu Tyr Ser Ser Tyr His Gln Gly Cys Leu Val Asn Pro Thr Gly Asn 180 185 190Leu Pro Met Trp Ser Asn Gln Thr Trp Asn Asn Ser Thr Trp Ser Asn 195 200 205Gln Thr Gln Asn Ile Gln Ser Trp Ser Asn His Ser Trp Asn Thr Gln 210 215 220Thr Trp Cys Thr Gln Ser Trp Asn Asn Gln Ala Trp Asn Ser Pro Phe225 230 235 240Tyr Asn Cys Gly Glu Glu Ser Leu Gln Ser Cys Met Gln Phe Gln Pro 245 250 255Asn Ser Pro Ala Ser Asp Leu Glu Ala Ala Leu Glu Ala Ala Gly Glu 260 265 270Gly Leu Asn Val Ile Gln Gln Thr Thr Arg Tyr Phe Ser Thr Pro Gln 275 280 285Thr Met Asp Leu Phe Leu Asn Tyr Ser Met Asn Met Gln Pro Glu Asp 290 295 300Val3056209PRTHomo sapiens 6Met Gly Ser Val Ser Asn Gln Gln Phe Ala Gly Gly Cys Ala Lys Ala1 5 10 15Ala Glu Glu Ala Pro Glu Glu Ala Pro Glu Asp Ala Ala Arg Ala Ala 20 25 30Asp Glu Pro Gln Leu Leu His Gly Ala Gly Ile Cys Lys Trp Phe Asn 35 40 45Val Arg Met Gly Phe Gly Phe Leu Ser Met Thr Ala Arg Ala Gly Val 50 55 60Ala Leu Asp Pro Pro Val Asp Val Phe Val His Gln Ser Lys Leu His65 70 75 80Met Glu Gly Phe Arg Ser Leu Lys Glu Gly Glu Ala Val Glu Phe Thr 85 90 95Phe Lys Lys Ser Ala Lys Gly Leu Glu Ser Ile Arg Val Thr Gly Pro 100 105 110Gly Gly Val Phe Cys Ile Gly Ser Glu Arg Arg Pro Lys Gly Lys Ser 115 120 125Met Gln Lys Arg Arg Ser Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly 130 135 140Leu Asp His His Ala Lys Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys145 150 155 160Cys His Phe Cys Gln Ser Ile Ser His Met Val Ala Ser Cys Pro Leu 165 170 175Lys Ala Gln Gln Gly Pro Ser Ala Gln Gly Lys Pro Thr Tyr Phe Arg 180 185 190Glu Glu Glu Glu Glu Ile His Ser Pro Thr Leu Leu Pro Glu Ala Gln 195 200 205Asn 7505PRTHomo sapiens 7Met Gly Glu Thr Leu Gly Asp Ser Pro Ile Asp Pro Glu Ser Asp Ser1 5 10 15Phe Thr Asp Thr Leu Ser Ala Asn Ile Ser Gln Glu Met Thr Met Val 20 25 30Asp Thr Glu Met Pro Phe Trp Pro Thr Asn Phe Gly Ile Ser Ser Val 35 40 45Asp Leu Ser Val Met Glu Asp His Ser His Ser Phe Asp Ile Lys Pro 50 55 60Phe Thr Thr Val Asp Phe Ser Ser Ile Ser Thr Pro His Tyr Glu Asp65 70 75 80Ile Pro Phe Thr Arg Thr Asp Pro Val Val Ala Asp Tyr Lys Tyr Asp 85 90 95Leu Lys Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala Ser 100 105 110Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Lys Pro His Glu 115 120 125Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys Gly Asp 130 135 140Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys145 150 155 160Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Ile Tyr Asp Arg Cys 165 170 175Asp Leu Asn Cys Arg Ile His Lys Lys Ser Arg Asn Lys Cys Gln Tyr 180 185 190Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn Ala Ile 195 200 205Arg Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu 210 215 220Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg225 230 235 240Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu 245 250 255Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr Thr Asp Lys 260 265 270Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met Gly Glu Asp 275 280 285Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu 290 295 300Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala305 310 315

320Val Gln Glu Ile Thr Glu Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn 325 330 335Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu 340 345 350Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu 355 360 365Ile Ser Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu 370 375 380Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala Val385 390 395 400Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile 405 410 415Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys 420 425 430Pro Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln 435 440 445Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Leu Leu 450 455 460Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln Leu465 470 475 480Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu 485 490 495Leu Gln Glu Ile Tyr Lys Asp Leu Tyr 500 5058358PRTHomo sapiens 8Met Glu Ser Ala Asp Phe Tyr Glu Ala Glu Pro Arg Pro Pro Met Ser1 5 10 15Ser His Leu Gln Ser Pro Pro His Ala Pro Ser Ser Ala Ala Phe Gly 20 25 30Phe Pro Arg Gly Ala Gly Pro Ala Gln Pro Pro Ala Pro Pro Ala Ala 35 40 45Pro Glu Pro Leu Gly Gly Ile Cys Glu His Glu Thr Ser Ile Asp Ile 50 55 60Ser Ala Tyr Ile Asp Pro Ala Ala Phe Asn Asp Glu Phe Leu Ala Asp65 70 75 80Leu Phe Gln His Ser Arg Gln Gln Glu Lys Ala Lys Ala Ala Val Gly 85 90 95Pro Thr Gly Gly Gly Gly Gly Gly Asp Phe Asp Tyr Pro Gly Ala Pro 100 105 110Ala Gly Pro Gly Gly Ala Val Met Pro Gly Gly Ala His Gly Pro Pro 115 120 125Pro Gly Tyr Gly Cys Ala Ala Ala Gly Tyr Leu Asp Gly Arg Leu Glu 130 135 140Pro Leu Tyr Glu Arg Val Gly Ala Pro Ala Leu Arg Pro Leu Val Ile145 150 155 160Lys Gln Glu Pro Arg Glu Glu Asp Glu Ala Lys Gln Leu Ala Leu Ala 165 170 175Gly Leu Phe Pro Tyr Gln Pro Pro Pro Pro Pro Pro Pro Ser His Pro 180 185 190His Pro His Pro Pro Pro Ala His Leu Ala Ala Pro His Leu Gln Phe 195 200 205Gln Ile Ala His Cys Gly Gln Thr Thr Met His Leu Gln Pro Gly His 210 215 220Pro Thr Pro Pro Pro Thr Pro Val Pro Ser Pro His Pro Ala Pro Ala225 230 235 240Leu Gly Ala Ala Gly Leu Pro Gly Pro Gly Ser Ala Leu Lys Gly Leu 245 250 255Gly Ala Ala His Pro Asp Leu Arg Ala Ser Gly Gly Ser Gly Ala Gly 260 265 270Lys Ala Lys Lys Ser Val Asp Lys Asn Ser Asn Glu Tyr Arg Val Arg 275 280 285Arg Glu Arg Asn Asn Ile Ala Val Arg Lys Ser Arg Asp Lys Ala Lys 290 295 300Gln Arg Asn Val Glu Thr Gln Gln Lys Val Leu Glu Leu Thr Ser Asp305 310 315 320Asn Asp Arg Leu Arg Lys Arg Val Glu Gln Leu Ser Arg Glu Leu Asp 325 330 335Thr Leu Arg Gly Ile Phe Arg Gln Leu Pro Glu Ser Ser Leu Val Lys 340 345 350Ala Met Gly Asn Cys Ala 3559345PRTHomo sapiens 9Met Gln Arg Leu Val Ala Trp Asp Pro Ala Cys Leu Pro Leu Pro Pro1 5 10 15Pro Pro Pro Ala Phe Lys Ser Met Glu Val Ala Asn Phe Tyr Tyr Glu 20 25 30Ala Asp Cys Leu Ala Ala Ala Tyr Gly Gly Lys Ala Ala Pro Ala Ala 35 40 45Pro Pro Ala Ala Arg Pro Gly Pro Arg Pro Pro Ala Gly Glu Leu Gly 50 55 60Ser Ile Gly Asp His Glu Arg Ala Ile Asp Phe Ser Pro Tyr Leu Glu65 70 75 80Pro Leu Gly Ala Pro Gln Ala Pro Ala Pro Ala Thr Ala Thr Asp Thr 85 90 95Phe Glu Ala Ala Pro Pro Ala Pro Ala Pro Ala Pro Ala Ser Ser Gly 100 105 110Gln His His Asp Phe Leu Ser Asp Leu Phe Ser Asp Asp Tyr Gly Gly 115 120 125Lys Asn Cys Lys Lys Pro Ala Glu Tyr Gly Tyr Val Ser Leu Gly Arg 130 135 140Leu Gly Ala Ala Lys Gly Ala Leu His Pro Gly Cys Phe Ala Pro Leu145 150 155 160His Pro Pro Pro Pro Pro Pro Pro Pro Pro Ala Glu Leu Lys Ala Glu 165 170 175Pro Gly Phe Glu Pro Ala Asp Cys Lys Arg Lys Glu Glu Ala Gly Ala 180 185 190Pro Gly Gly Gly Ala Gly Met Ala Ala Gly Phe Pro Tyr Ala Leu Arg 195 200 205Ala Tyr Leu Gly Tyr Gln Ala Val Pro Ser Gly Ser Ser Gly Ser Leu 210 215 220Ser Thr Ser Ser Ser Ser Ser Pro Pro Gly Thr Pro Ser Pro Ala Asp225 230 235 240Ala Lys Ala Pro Pro Thr Ala Cys Tyr Ala Gly Ala Ala Pro Ala Pro 245 250 255Ser Gln Val Lys Ser Lys Ala Lys Lys Thr Val Asp Lys His Ser Asp 260 265 270Glu Tyr Lys Ile Arg Arg Glu Arg Asn Asn Ile Ala Val Arg Lys Ser 275 280 285Arg Asp Lys Ala Lys Met Arg Asn Leu Glu Thr Gln His Lys Val Leu 290 295 300Glu Leu Thr Ala Glu Asn Glu Arg Leu Gln Lys Lys Val Glu Gln Leu305 310 315 320Ser Arg Glu Leu Ser Thr Leu Arg Asn Leu Phe Lys Gln Leu Pro Glu 325 330 335Pro Leu Leu Ala Ser Ser Gly His Cys 340 34510269PRTHomo sapiens 10Met Ser Ala Ala Leu Phe Ser Leu Asp Gly Pro Ala Arg Gly Ala Pro1 5 10 15Trp Pro Ala Glu Pro Ala Pro Phe Tyr Glu Pro Gly Arg Ala Gly Lys 20 25 30Pro Gly Arg Gly Ala Glu Pro Gly Ala Leu Gly Glu Pro Gly Ala Ala 35 40 45Ala Pro Ala Met Tyr Asp Asp Glu Ser Ala Ile Asp Phe Ser Ala Tyr 50 55 60Ile Asp Ser Met Ala Ala Val Pro Thr Leu Glu Leu Cys His Asp Glu65 70 75 80Leu Phe Ala Asp Leu Phe Asn Ser Asn His Lys Ala Gly Gly Ala Gly 85 90 95Pro Leu Glu Leu Leu Pro Gly Gly Pro Ala Arg Pro Leu Gly Pro Gly 100 105 110Pro Ala Ala Pro Arg Leu Leu Lys Arg Glu Pro Asp Trp Gly Asp Gly 115 120 125Asp Ala Pro Gly Ser Leu Leu Pro Ala Gln Val Ala Ala Cys Ala Gln 130 135 140Thr Val Val Ser Leu Ala Ala Ala Gly Gln Pro Thr Pro Pro Thr Ser145 150 155 160Pro Glu Pro Pro Arg Ser Ser Pro Arg Gln Thr Pro Ala Pro Gly Pro 165 170 175Ala Arg Glu Lys Ser Ala Gly Lys Arg Gly Pro Asp Arg Gly Ser Pro 180 185 190Glu Tyr Arg Gln Arg Arg Glu Arg Asn Asn Ile Ala Val Arg Lys Ser 195 200 205Arg Asp Lys Ala Lys Arg Arg Asn Gln Glu Met Gln Gln Lys Leu Val 210 215 220Glu Leu Ser Ala Glu Asn Glu Lys Leu His Gln Arg Val Glu Gln Leu225 230 235 240Thr Arg Asp Leu Ala Gly Leu Arg Gln Phe Phe Lys Gln Leu Pro Ser 245 250 255Pro Pro Phe Leu Pro Ala Ala Gly Thr Ala Asp Cys Arg 260 265111147PRTHomo sapiens 11Met Asp Glu Pro Pro Phe Ser Glu Ala Ala Leu Glu Gln Ala Leu Gly1 5 10 15Glu Pro Cys Asp Leu Asp Ala Ala Leu Leu Thr Asp Ile Glu Asp Met 20 25 30Leu Gln Leu Ile Asn Asn Gln Asp Ser Asp Phe Pro Gly Leu Phe Asp 35 40 45Pro Pro Tyr Ala Gly Ser Gly Ala Gly Gly Thr Asp Pro Ala Ser Pro 50 55 60Asp Thr Ser Ser Pro Gly Ser Leu Ser Pro Pro Pro Ala Thr Leu Ser65 70 75 80Ser Ser Leu Glu Ala Phe Leu Ser Gly Pro Gln Ala Ala Pro Ser Pro 85 90 95Leu Ser Pro Pro Gln Pro Ala Pro Thr Pro Leu Lys Met Tyr Pro Ser 100 105 110Met Pro Ala Phe Ser Pro Gly Pro Gly Ile Lys Glu Glu Ser Val Pro 115 120 125Leu Ser Ile Leu Gln Thr Pro Thr Pro Gln Pro Leu Pro Gly Ala Leu 130 135 140Leu Pro Gln Ser Phe Pro Ala Pro Ala Pro Pro Gln Phe Ser Ser Thr145 150 155 160Pro Val Leu Gly Tyr Pro Ser Pro Pro Gly Gly Phe Ser Thr Gly Ser 165 170 175Pro Pro Gly Asn Thr Gln Gln Pro Leu Pro Gly Leu Pro Leu Ala Ser 180 185 190Pro Pro Gly Val Pro Pro Val Ser Leu His Thr Gln Val Gln Ser Val 195 200 205Val Pro Gln Gln Leu Leu Thr Val Thr Ala Ala Pro Thr Ala Ala Pro 210 215 220Val Thr Thr Thr Val Thr Ser Gln Ile Gln Gln Val Pro Val Leu Leu225 230 235 240Gln Pro His Phe Ile Lys Ala Asp Ser Leu Leu Leu Thr Ala Met Lys 245 250 255Thr Asp Gly Ala Thr Val Lys Ala Ala Gly Leu Ser Pro Leu Val Ser 260 265 270Gly Thr Thr Val Gln Thr Gly Pro Leu Pro Thr Leu Val Ser Gly Gly 275 280 285Thr Ile Leu Ala Thr Val Pro Leu Val Val Asp Ala Glu Lys Leu Pro 290 295 300Ile Asn Arg Leu Ala Ala Gly Ser Lys Ala Pro Ala Ser Ala Gln Ser305 310 315 320Arg Gly Glu Lys Arg Thr Ala His Asn Ala Ile Glu Lys Arg Tyr Arg 325 330 335Ser Ser Ile Asn Asp Lys Ile Ile Glu Leu Lys Asp Leu Val Val Gly 340 345 350Thr Glu Ala Lys Leu Asn Lys Ser Ala Val Leu Arg Lys Ala Ile Asp 355 360 365Tyr Ile Arg Phe Leu Gln His Ser Asn Gln Lys Leu Lys Gln Glu Asn 370 375 380Leu Ser Leu Arg Thr Ala Val His Lys Ser Lys Ser Leu Lys Asp Leu385 390 395 400Val Ser Ala Cys Gly Ser Gly Gly Asn Thr Asp Val Leu Met Glu Gly 405 410 415Val Lys Thr Glu Val Glu Asp Thr Leu Thr Pro Pro Pro Ser Asp Ala 420 425 430Gly Ser Pro Phe Gln Ser Ser Pro Leu Ser Leu Gly Ser Arg Gly Ser 435 440 445Gly Ser Gly Gly Ser Gly Ser Asp Ser Glu Pro Asp Ser Pro Val Phe 450 455 460Glu Asp Ser Lys Ala Lys Pro Glu Gln Arg Pro Ser Leu His Ser Arg465 470 475 480Gly Met Leu Asp Arg Ser Arg Leu Ala Leu Cys Thr Leu Val Phe Leu 485 490 495Cys Leu Ser Cys Asn Pro Leu Ala Ser Leu Leu Gly Ala Arg Gly Leu 500 505 510Pro Ser Pro Ser Asp Thr Thr Ser Val Tyr His Ser Pro Gly Arg Asn 515 520 525Val Leu Gly Thr Glu Ser Arg Asp Gly Pro Gly Trp Ala Gln Trp Leu 530 535 540Leu Pro Pro Val Val Trp Leu Leu Asn Gly Leu Leu Val Leu Val Ser545 550 555 560Leu Val Leu Leu Phe Val Tyr Gly Glu Pro Val Thr Arg Pro His Ser 565 570 575Gly Pro Ala Val Tyr Phe Trp Arg His Arg Lys Gln Ala Asp Leu Asp 580 585 590Leu Ala Arg Gly Asp Phe Ala Gln Ala Ala Gln Gln Leu Trp Leu Ala 595 600 605Leu Arg Ala Leu Gly Arg Pro Leu Pro Thr Ser His Leu Asp Leu Ala 610 615 620Cys Ser Leu Leu Trp Asn Leu Ile Arg His Leu Leu Gln Arg Leu Trp625 630 635 640Val Gly Arg Trp Leu Ala Gly Arg Ala Gly Gly Leu Gln Gln Asp Cys 645 650 655Ala Leu Arg Val Asp Ala Ser Ala Ser Ala Arg Asp Ala Ala Leu Val 660 665 670Tyr His Lys Leu His Gln Leu His Thr Met Gly Lys His Thr Gly Gly 675 680 685His Leu Thr Ala Thr Asn Leu Ala Leu Ser Ala Leu Asn Leu Ala Glu 690 695 700Cys Ala Gly Asp Ala Val Ser Val Ala Thr Leu Ala Glu Ile Tyr Val705 710 715 720Ala Ala Ala Leu Arg Val Lys Thr Ser Leu Pro Arg Ala Leu His Phe 725 730 735Leu Thr Arg Phe Phe Leu Ser Ser Ala Arg Gln Ala Cys Leu Ala Gln 740 745 750Ser Gly Ser Val Pro Pro Ala Met Gln Trp Leu Cys His Pro Val Gly 755 760 765His Arg Phe Phe Val Asp Gly Asp Trp Ser Val Leu Ser Thr Pro Trp 770 775 780Glu Ser Leu Tyr Ser Leu Ala Gly Asn Pro Val Asp Pro Leu Ala Gln785 790 795 800Val Thr Gln Leu Phe Arg Glu His Leu Leu Glu Arg Ala Leu Asn Cys 805 810 815Val Thr Gln Pro Asn Pro Ser Pro Gly Ser Ala Asp Gly Asp Lys Glu 820 825 830Phe Ser Asp Ala Leu Gly Tyr Leu Gln Leu Leu Asn Ser Cys Ser Asp 835 840 845Ala Ala Gly Ala Pro Ala Tyr Ser Phe Ser Ile Ser Ser Ser Met Ala 850 855 860Thr Thr Thr Gly Val Asp Pro Val Ala Lys Trp Trp Ala Ser Leu Thr865 870 875 880Ala Val Val Ile His Trp Leu Arg Arg Asp Glu Glu Ala Ala Glu Arg 885 890 895Leu Cys Pro Leu Val Glu His Leu Pro Arg Val Leu Gln Glu Ser Glu 900 905 910Arg Pro Leu Pro Arg Ala Ala Leu His Ser Phe Lys Ala Ala Arg Ala 915 920 925Leu Leu Gly Cys Ala Lys Ala Glu Ser Gly Pro Ala Ser Leu Thr Ile 930 935 940Cys Glu Lys Ala Ser Gly Tyr Leu Gln Asp Ser Leu Ala Thr Thr Pro945 950 955 960Ala Ser Ser Ser Ile Asp Lys Ala Val Gln Leu Phe Leu Cys Asp Leu 965 970 975Leu Leu Val Val Arg Thr Ser Leu Trp Arg Gln Gln Gln Pro Pro Ala 980 985 990Pro Ala Pro Ala Ala Gln Gly Thr Ser Ser Arg Pro Gln Ala Ser Ala 995 1000 1005Leu Glu Leu Arg Gly Phe Gln Arg Asp Leu Ser Ser Leu Arg Arg 1010 1015 1020Leu Ala Gln Ser Phe Arg Pro Ala Met Arg Arg Val Phe Leu His 1025 1030 1035Glu Ala Thr Ala Arg Leu Met Ala Gly Ala Ser Pro Thr Arg Thr 1040 1045 1050His Gln Leu Leu Asp Arg Ser Leu Arg Arg Arg Ala Gly Pro Gly 1055 1060 1065Gly Lys Gly Gly Ala Val Ala Glu Leu Glu Pro Arg Pro Thr Arg 1070 1075 1080Arg Glu His Ala Glu Ala Leu Leu Leu Ala Ser Cys Tyr Leu Pro 1085 1090 1095Pro Gly Phe Leu Ser Ala Pro Gly Gln Arg Val Gly Met Leu Ala 1100 1105 1110Glu Ala Ala Arg Thr Leu Glu Lys Leu Gly Asp Arg Arg Leu Leu 1115 1120 1125His Asp Cys Gln Gln Met Leu Met Arg Leu Gly Gly Gly Thr Thr 1130 1135 1140Val Thr Ser Ser 114512341PRTHomo sapiens 12Met Thr Met Glu Ser Gly Ala Glu Asn Gln Gln Ser Gly Asp Ala Ala1 5 10 15Val Thr Glu Ala Glu Asn Gln Gln Met Thr Val Gln Ala Gln Pro Gln 20 25 30Ile Ala Thr Leu Ala Gln Val Ser Met Pro Ala Ala His Ala Thr Ser 35 40 45Ser Ala Pro Thr Val Thr Leu Val Gln Leu Pro Asn Gly Gln Thr Val 50 55 60Gln Val His Gly Val Ile Gln Ala Ala Gln Pro Ser Val Ile Gln Ser65 70 75 80Pro Gln Val Gln Thr Val Gln Ser Ser Cys Lys Asp Leu Lys Arg Leu 85 90 95Phe Ser Gly Thr Gln Ile Ser Thr Ile Ala Glu Ser Glu Asp Ser Gln 100 105 110Glu Ser Val Asp Ser Val Thr Asp Ser Gln Lys Arg Arg Glu Ile Leu 115 120 125Ser Arg Arg Pro Ser Tyr Arg Lys Ile Leu Asn Asp Leu Ser Ser Asp 130 135 140Ala Pro Gly Val Pro Arg Ile Glu Glu Glu Lys Ser Glu Glu Glu Thr145

150 155 160Ser Ala Pro Ala Ile Thr Thr Val Thr Val Pro Thr Pro Ile Tyr Gln 165 170 175Thr Ser Ser Gly Gln Tyr Ile Ala Ile Thr Gln Gly Gly Ala Ile Gln 180 185 190Leu Ala Asn Asn Gly Thr Asp Gly Val Gln Gly Leu Gln Thr Leu Thr 195 200 205Met Thr Asn Ala Ala Ala Thr Gln Pro Gly Thr Thr Ile Leu Gln Tyr 210 215 220Ala Gln Thr Thr Asp Gly Gln Gln Ile Leu Val Pro Ser Asn Gln Val225 230 235 240Val Val Gln Ala Ala Ser Gly Asp Val Gln Thr Tyr Gln Ile Arg Thr 245 250 255Ala Pro Thr Ser Thr Ile Ala Pro Gly Val Val Met Ala Ser Ser Pro 260 265 270Ala Leu Pro Thr Gln Pro Ala Glu Glu Ala Ala Arg Lys Arg Glu Val 275 280 285Arg Leu Met Lys Asn Arg Glu Ala Ala Arg Glu Cys Arg Arg Lys Lys 290 295 300Lys Glu Tyr Val Lys Cys Leu Glu Asn Arg Val Ala Val Leu Glu Asn305 310 315 320Gln Asn Lys Thr Leu Ile Glu Glu Leu Lys Ala Leu Lys Asp Leu Tyr 325 330 335Cys His Lys Ser Asp 34013476PRTHomo sapiens 13Met Met Thr Ala Lys Ala Val Asp Lys Ile Pro Val Thr Leu Ser Gly1 5 10 15Phe Val His Gln Leu Ser Asp Asn Ile Tyr Pro Val Glu Asp Leu Ala 20 25 30Ala Thr Ser Val Thr Ile Phe Pro Asn Ala Glu Leu Gly Gly Pro Phe 35 40 45Asp Gln Met Asn Gly Val Ala Gly Asp Gly Met Ile Asn Ile Asp Met 50 55 60Thr Gly Glu Lys Arg Ser Leu Asp Leu Pro Tyr Pro Ser Ser Phe Ala65 70 75 80Pro Val Ser Ala Pro Arg Asn Gln Thr Phe Thr Tyr Met Gly Lys Phe 85 90 95Ser Ile Asp Pro Gln Tyr Pro Gly Ala Ser Cys Tyr Pro Glu Gly Ile 100 105 110Ile Asn Ile Val Ser Ala Gly Ile Leu Gln Gly Val Thr Ser Pro Ala 115 120 125Ser Thr Thr Ala Ser Ser Ser Val Thr Ser Ala Ser Pro Asn Pro Leu 130 135 140Ala Thr Gly Pro Leu Gly Val Cys Thr Met Ser Gln Thr Gln Pro Asp145 150 155 160Leu Asp His Leu Tyr Ser Pro Pro Pro Pro Pro Pro Pro Tyr Ser Gly 165 170 175Cys Ala Gly Asp Leu Tyr Gln Asp Pro Ser Ala Phe Leu Ser Ala Ala 180 185 190Thr Thr Ser Thr Ser Ser Ser Leu Ala Tyr Pro Pro Pro Pro Ser Tyr 195 200 205Pro Ser Pro Lys Pro Ala Thr Asp Pro Gly Leu Phe Pro Met Ile Pro 210 215 220Asp Tyr Pro Gly Phe Phe Pro Ser Gln Cys Gln Arg Asp Leu His Gly225 230 235 240Thr Ala Gly Pro Asp Arg Lys Pro Phe Pro Cys Pro Leu Asp Thr Leu 245 250 255Arg Val Pro Pro Pro Leu Thr Pro Leu Ser Thr Ile Arg Asn Phe Thr 260 265 270Leu Gly Gly Pro Ser Ala Gly Val Thr Gly Pro Gly Ala Ser Gly Gly 275 280 285Ser Glu Gly Pro Arg Leu Pro Gly Ser Ser Ser Ala Ala Ala Ala Ala 290 295 300Ala Ala Ala Ala Ala Tyr Asn Pro His His Leu Pro Leu Arg Pro Ile305 310 315 320Leu Arg Pro Arg Lys Tyr Pro Asn Arg Pro Ser Lys Thr Pro Val His 325 330 335Glu Arg Pro Tyr Pro Cys Pro Ala Glu Gly Cys Asp Arg Arg Phe Ser 340 345 350Arg Ser Asp Glu Leu Thr Arg His Ile Arg Ile His Thr Gly His Lys 355 360 365Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His 370 375 380Leu Thr Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys385 390 395 400Asp Tyr Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg His 405 410 415Thr Lys Ile His Leu Arg Gln Lys Glu Arg Lys Ser Ser Ala Pro Ser 420 425 430Ala Ser Val Pro Ala Pro Ser Thr Ala Ser Cys Ser Gly Gly Val Gln 435 440 445Pro Gly Gly Thr Leu Cys Ser Ser Asn Ser Ser Ser Leu Gly Gly Gly 450 455 460Pro Leu Ala Pro Cys Ser Ser Arg Thr Arg Thr Pro465 470 47514457PRTHomo sapiens 14Met Ala Thr Arg Val Leu Ser Met Ser Ala Arg Leu Gly Pro Val Pro1 5 10 15Gln Pro Pro Ala Pro Gln Asp Glu Pro Val Phe Ala Gln Leu Lys Pro 20 25 30Val Leu Gly Ala Ala Asn Pro Ala Arg Asp Ala Ala Leu Phe Pro Gly 35 40 45Glu Glu Leu Lys His Ala His His Arg Pro Gln Ala Gln Pro Ala Pro 50 55 60Ala Gln Ala Pro Gln Pro Ala Gln Pro Pro Ala Thr Gly Pro Arg Leu65 70 75 80Pro Pro Glu Asp Leu Val Gln Thr Arg Cys Glu Met Glu Lys Tyr Leu 85 90 95Thr Pro Gln Leu Pro Pro Val Pro Ile Ile Pro Glu His Lys Lys Tyr 100 105 110Arg Arg Asp Ser Ala Ser Val Val Asp Gln Phe Phe Thr Asp Thr Glu 115 120 125Gly Leu Pro Tyr Ser Ile Asn Met Asn Val Phe Leu Pro Asp Ile Thr 130 135 140His Leu Arg Thr Gly Leu Tyr Lys Ser Gln Arg Pro Cys Val Thr His145 150 155 160Ile Lys Thr Glu Pro Val Ala Ile Phe Ser His Gln Ser Glu Thr Thr 165 170 175Ala Pro Pro Pro Ala Pro Thr Gln Ala Leu Pro Glu Phe Thr Ser Ile 180 185 190Phe Ser Ser His Gln Thr Ala Ala Pro Glu Val Asn Asn Ile Phe Ile 195 200 205Lys Gln Glu Leu Pro Thr Pro Asp Leu His Leu Ser Val Pro Thr Gln 210 215 220Gln Gly His Leu Tyr Gln Leu Leu Asn Thr Pro Asp Leu Asp Met Pro225 230 235 240Ser Ser Thr Asn Gln Thr Ala Ala Met Asp Thr Leu Asn Val Ser Met 245 250 255Ser Ala Ala Met Ala Gly Leu Asn Thr His Thr Ser Ala Val Pro Gln 260 265 270Thr Ala Val Lys Gln Phe Gln Gly Met Pro Pro Cys Thr Tyr Thr Met 275 280 285Pro Ser Gln Phe Leu Pro Gln Gln Ala Thr Tyr Phe Pro Pro Ser Pro 290 295 300Pro Ser Ser Glu Pro Gly Ser Pro Asp Arg Gln Ala Glu Met Leu Gln305 310 315 320Asn Leu Thr Pro Pro Pro Ser Tyr Ala Ala Thr Ile Ala Ser Lys Leu 325 330 335Ala Ile His Asn Pro Asn Leu Pro Thr Thr Leu Pro Val Asn Ser Gln 340 345 350Asn Ile Gln Pro Val Arg Tyr Asn Arg Arg Ser Asn Pro Asp Leu Glu 355 360 365Lys Arg Arg Ile His Tyr Cys Asp Tyr Pro Gly Cys Thr Lys Val Tyr 370 375 380Thr Lys Ser Ser His Leu Lys Ala His Leu Arg Thr His Thr Gly Glu385 390 395 400Lys Pro Tyr Lys Cys Thr Trp Glu Gly Cys Asp Trp Arg Phe Ala Arg 405 410 415Ser Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr Gly Ala Lys Pro 420 425 430Phe Gln Cys Gly Val Cys Asn Arg Ser Phe Ser Arg Ser Asp His Leu 435 440 445Ala Leu His Met Lys Arg His Gln Asn 450 45515416PRTHomo sapiens 15Met Val Asp His Leu Leu Pro Val Asp Glu Asn Phe Ser Ser Pro Lys1 5 10 15Cys Pro Val Gly Tyr Leu Gly Asp Arg Leu Val Gly Arg Arg Ala Tyr 20 25 30His Met Leu Pro Ser Pro Val Ser Glu Asp Asp Ser Asp Ala Ser Ser 35 40 45Pro Cys Ser Cys Ser Ser Pro Asp Ser Gln Ala Leu Cys Ser Cys Tyr 50 55 60Gly Gly Gly Leu Gly Thr Glu Ser Gln Asp Ser Ile Leu Asp Phe Leu65 70 75 80Leu Ser Gln Ala Thr Leu Gly Ser Gly Gly Gly Ser Gly Ser Ser Ile 85 90 95Gly Ala Ser Ser Gly Pro Val Ala Trp Gly Pro Trp Arg Arg Ala Ala 100 105 110Ala Pro Val Lys Gly Glu His Phe Cys Leu Pro Glu Phe Pro Leu Gly 115 120 125Asp Pro Asp Asp Val Pro Arg Pro Phe Gln Pro Thr Leu Glu Glu Ile 130 135 140Glu Glu Phe Leu Glu Glu Asn Met Glu Pro Gly Val Lys Glu Val Pro145 150 155 160Glu Gly Asn Ser Lys Asp Leu Asp Ala Cys Ser Gln Leu Ser Ala Gly 165 170 175Pro His Lys Ser His Leu His Pro Gly Ser Ser Gly Arg Glu Arg Cys 180 185 190Ser Pro Pro Pro Gly Gly Ala Ser Ala Gly Gly Ala Gln Gly Pro Gly 195 200 205Gly Gly Pro Thr Pro Asp Gly Pro Ile Pro Val Leu Leu Gln Ile Gln 210 215 220Pro Val Pro Val Lys Gln Glu Ser Gly Thr Gly Pro Ala Ser Pro Gly225 230 235 240Gln Ala Pro Glu Asn Val Lys Val Ala Gln Leu Leu Val Asn Ile Gln 245 250 255Gly Gln Thr Phe Ala Leu Val Pro Gln Val Val Pro Ser Ser Asn Leu 260 265 270Asn Leu Pro Ser Lys Phe Val Arg Ile Ala Pro Val Pro Ile Ala Ala 275 280 285Lys Pro Val Gly Ser Gly Pro Leu Gly Pro Gly Pro Ala Gly Leu Leu 290 295 300Met Gly Gln Lys Phe Pro Lys Asn Pro Ala Ala Glu Leu Ile Lys Met305 310 315 320His Lys Cys Thr Phe Pro Gly Cys Ser Lys Met Tyr Thr Lys Ser Ser 325 330 335His Leu Lys Ala His Leu Arg Arg His Thr Gly Glu Lys Pro Phe Ala 340 345 350Cys Thr Trp Pro Gly Cys Gly Trp Arg Phe Ser Arg Ser Asp Glu Leu 355 360 365Ser Arg His Arg Arg Ser His Ser Gly Val Lys Pro Tyr Gln Cys Pro 370 375 380Val Cys Glu Lys Lys Phe Ala Arg Ser Asp His Leu Ser Lys His Ile385 390 395 400Lys Val His Arg Phe Pro Arg Ser Ser Arg Ser Val Arg Ser Val Asn 405 410 41516238PRTHomo sapiens 16Met Glu Tyr Ala Met Lys Ser Leu Ser Leu Leu Tyr Pro Lys Ser Leu1 5 10 15Ser Arg His Val Ser Val Arg Thr Ser Val Val Thr Gln Gln Leu Leu 20 25 30Ser Glu Pro Ser Pro Lys Ala Pro Arg Ala Arg Pro Cys Arg Val Ser 35 40 45Thr Ala Asp Arg Ser Val Arg Lys Gly Ile Met Ala Tyr Ser Leu Glu 50 55 60Asp Leu Leu Leu Lys Val Arg Asp Thr Leu Met Leu Ala Asp Lys Pro65 70 75 80Phe Phe Leu Val Leu Glu Glu Asp Gly Thr Thr Val Glu Thr Glu Glu 85 90 95Tyr Phe Gln Ala Leu Ala Gly Asp Thr Val Phe Met Val Leu Gln Lys 100 105 110Gly Gln Lys Trp Gln Pro Pro Ser Glu Gln Gly Thr Arg His Pro Leu 115 120 125Ser Leu Ser His Lys Pro Ala Lys Lys Ile Asp Val Ala Arg Val Thr 130 135 140Phe Asp Leu Tyr Lys Leu Asn Pro Gln Asp Phe Ile Gly Cys Leu Asn145 150 155 160Val Lys Ala Thr Phe Tyr Asp Thr Tyr Ser Leu Ser Tyr Asp Leu His 165 170 175Cys Cys Gly Ala Lys Arg Ile Met Lys Glu Ala Phe Arg Trp Ala Leu 180 185 190Phe Ser Met Gln Ala Thr Gly His Val Leu Leu Gly Thr Ser Cys Tyr 195 200 205Leu Gln Gln Leu Leu Asp Ala Thr Glu Glu Gly Gln Pro Pro Lys Gly 210 215 220Lys Ala Ser Ser Leu Ile Pro Thr Cys Leu Lys Ile Leu Gln225 230 235176PRTArtificial SequenceDescription of Artificial Sequence Synthetic 6xHis tag 17His His His His His His1 5

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 PPAR.gamma.2, CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBP.beta., C/EBP.delta., C/EBP.alpha. 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, PPAR.gamma.2, 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 PPAR.gamma., CREB1, SREBF1, KLF5, KLF15, KROX20, C/EBP.beta., C/EBP.delta. C/EBP.alpha., and CDEC, wherein the expression confers adipocyte-marker expression, adipocyte morphology and/or adipocyte function.

64. A reprogrammed adipocyte that exogenously expresses a PPAR.gamma.2 or C/EBP.alpha. 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/EBP.alpha., C/EBP.beta. and C/EBP.delta., 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 PPAR.gamma.2, C/EBP.alpha., C/EBP.beta., C/EBP.delta., 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.