US009732128B2 (12 ) United States Patent ( 10 ) Patent No. : US 9 ,732 , 128 B2 West et al . (45 ) Date of Patent : Aug. 15 , 2017 ( 54 ) METHODS OF MODIFYING FOREIGN PATENT DOCUMENTS TRANSCRIPTIONAL REGULATORY WO WO 01/ 00650 1 / 2001 NETWORKS IN STEM CELLS wo WO 2007 /019398 2 / 2007 WO WO 2009101407107 A2 * 8 /2009 ( 75 ) Inventors : Michael D . West , Mill Valley, CA WO 2010033906 3 /2010 ( US ) ; Karen B . Chapman , Mill Valley , WO 2011103343 8 / 2011 CA (US ) ( 73 ) Assignee : BioTime, Inc. , Alameda, CA (US ) OTHER PUBLICATIONS Takahashi et al. Induction of Pluripotent Stem Cells from Adult ( * ) Notice : Subject to any disclaimer, the term of this Human Fibroblasts by Defined Factors . Cell, 2007 . 131: 861 -872 . * patent is extended or adjusted under 35 Darlington et al. Expression of RESP18 in Peptidergic and Catecholaminerigic Neurons . The Journal of Histochemistry & U . S . C . 154 ( b ) by 270 days . Cytochemistry , 1997 . 45 ( 9 ) :1265 - 1277 . * Gonzales et al, Distribution patterns of estrogen receptor alpha and (21 ) Appl. No. : 13/ 279 , 123 beta in the human cortex and hippocampus during development and adulthood . J Comp Neurol. 2007 . 503 ( 6 ) : 790 - 802 . * ( 22 ) Filed : Oct. 21, 2011 “ Comparable ” downloaded from http :/ /www .merriam -webster . com / dictionary /comparable on Mar . 6 , 2014 . * (65 ) Prior Publication Data Brugmann et al . Induction and Specification of the Vertebrate Ectodermal Placodes : precursors of the cranial sensory organs . US 2012 /0129262 A1 May 24 , 2012 Biology of the Cell , 2005 . 97 ( 5 ) : 303 - 315 . * Graham et al . SOX2 Functions to Maintain Neural Progenitor Identity . Neuron 2003 . 39 :749 - 765 . * Sansom et al . The Level of the Transcription Factor PAX6 is Related U . S . Application Data Essential for Controlling the Balance between Neural Stem Cell (60 ) Provisional application No . 61/ 415 , 244, filed on Nov. Self -Renewal and Neurogenesis . PLOS Genetics , 2009 . * 18 , 2010 , provisional application No. 61 /406 , 064 , Negorev et al. Sp100 as a Potent Tumor Suppressor: Accelerated Senescence and Rapid Malignant Transformation of Human Fibro filed on Oct . 22 , 2010 . blasts through Modulation of an Embryonic Stem Cell Program , ( 2010 ) Cancer Research 70 : 9991 . ( 51 ) Int. Cl. Niwa et al. , “ Quantative expression of Oct - 3 / 4 defines differentia C12N 5 / 00 ( 2006 . 01 ) tion , dedifferentiation or self - renewal of ES cells " Nature Genetics C12N 5 / 02 ( 2006 . 01 ) (2000 ) vol. 24 , pp . 372 - 378 . AOIN 63 /00 ( 2006 . 01 ) COOK 14 /47 ( 2006 .01 ) * cited by examiner C12N 5 / 074 ( 2010 . 01 ) (52 ) U . S . CI. Primary Examiner — Christopher M Babic CPC .. CO7K 14 /4702 (2013 . 01 ) ; C12N 570696 Assistant Examiner — Kimberly A Aron ( 2013. 01 ) ; CI2N 2501/ 602 ( 2013 .01 ) ; C12N ( 74 ) Attorney , Agent , or Firm — Jennifer Fleischer 2501 /603 (2013 . 01 ) (58 ) Field of Classification Search ( 57 ) ABSTRACT None The vast differentiation potential of human embryonic and See application file for complete search history . induced pluripotent stem cells , including their potential to cascade through all of the somatic cell lineages and to ( 56 ) References Cited display the complete transcriptional regulatory network of U . S . PATENT DOCUMENTS human biology, has generated interest in deriving scalable , purified , and identified cell types and methods of discover 7 ,033 ,831 B2 4 / 2006 Fisk et al . ing the precise structure of the human regulatory network . 7 ,510 , 870 B2 * 3 / 2009 Oh .. 435 / 325 2002 /0001842 A11 / 2002 Chapman et al . However, the innate capacity of pluripotent cells to display 2002 /0142397 A1 10 / 2002 Collas et al . all these lineages is not necessarily reflected during their 2003 / 0046722 Al 3 / 2003 Collas et al. culture in vitro . The clonal isolation and propagation of 2004 /0014210 A1 * 1 / 2004 Jessell et al. 435 / 368 progenitors greatly facilitates the generation of highly puri 2004 /0199935 Al 10 / 2004 Chapman et al . fied and identified formulations for research and therapeutic 2005 /0014258 AL 1 / 2005 Collas et al. purposes . Nevertheless , other cell types have yet to be 2005 /0074880 Al * 4 / 2005 Sang et al . 435 /455 2006 / 0212952 AL 9 / 2006 Collas et al. isolated and propagated from normal cells and methods of 2006 / 0246446 A1 * 11/ 2006 Evans et al . .. .. .. 435 / 6 isolating said novel cell types as well as methods for 2006 /0251642 A1 * 11/ 2006 Wolffe et al. .. .. .. .. .. 424 / 94 .65 introducing perturbations into the transcriptional regulatory 2007/ 0259423 AL 11/ 2007 Odorico et al. network in order to construct a computer model of the entire 2008/ 0070303 AL 3 / 2008 West et al . 2009 / 0047263 A1 * 2 / 2009 Yamanaka et al . .. .. 424 /93 . 21 human transcriptional regulatory network would greatly 2009 /0263896 A1 * 10 / 2009 Kelly et al. .. 435 / 366 benefit basic research as well as manufacturing technology 2009 / 0280096 A1 * 11/ 2009 Kubo et al. 424 /93 . 7 for cell- based therapies. 2010 /0167404 AL 7 / 2010 West et al. 2010 / 0184033 A17 / 2010 West et al . 5 Claims, 6 Drawing Sheets atent Aug . 15 , 2017 Sheet 1 of 6 US 9 ,732 , 128 B2 POUSF1 (OCT4 ) * * * * * * * * * Constitutive* * * * * * * * * * * * . .og OCT4. .* . * .* . * * . *. * . *( * * . +* * *) * * * * * *PS * * * * * * * * * * -* * * derived* * * * * * * * * * * .* . *. * . *. * .* .* . *. * . *. * .* .* . *. * .* EPS . * .* . *. * .* . * .* . *. * . * .* . *. * . *. *. * . *. *. * . *. *. * . *. * . *. * . 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SelectLinesof DES8nd . hoprosent ovention PSCels . wwwwwwwwww w . 01bis253577 . 8008887998000 9 . FIG.4 . Wwwwwwwwwwwwwwwwwww GR . DiverseNomalhes-derviedClonalProgeniors CellLines . 9(92020080129283293322230382070- . 1000010 . 6000 5000 3000 2000 1000 0 wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww SO atent Aug . 15 , 2017 Sheet 6 of 6 US 9 ,732 , 128 B2 Wwwwwwwwwwwwwwwwwww wwwwwwwwwww * * * SelectLneofthemivojjueul11888) * ESand PSCols * * * * * .* * . .4 . *- . YU . FIG.5 . 9815139827379323202123292820193022X5127404841467565249879832son67 . pogammaxonamgommoname%2522575000mm CellLines DiverseNormalES-derivedClonalProgenitors . *. - * U UG L L KOU . 1 SO US 9 ,732 , 128 B2 METHODS OF MODIFYING describe a technology to generate such novel and scalable TRANSCRIPTIONAL REGULATORY cell lines through the exogenous or endogenous up or NETWORKS IN STEM CELLS down - regulation of the activity of transcriptional regulators . Such exogenously or endogenously - introduced modifica CROSS REFERENCE TO RELATED 5 tions to the activity of transcriptional regulators may include APPLICATIONS transcription factors constitutively expressed in pluripotent stem cells such as hES cells or somatic cells reprogrammed This application claims the benefit under 35 U . S . C . $ 119 to pluripotency such as hiPS cells . Such transcriptional ( e ) to U . S . Provisional Patent Application Ser. No . 61 /406 , regulators can be introduced in a manner that allows the 064 , filed Oct. 22 , 2010 and U . S . Provisional Patent Appli - 10 precise regulation of the level and timing of their expression cation Ser. No . 61 /415 , 244 , filed Nov . 18 , 2010 . The entirety including but not limited to the use of an inducible promoter of each of these applications is incorporated herein by driving the expression or inhibition of expression of a reference . number of transcription factors.