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US 20140004512A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0004512 A1 Santourlidis (43) Pub. Date: Jan. 2, 2014 (54) QUALITY DETERMINATION OF STEM Publication Classi?cation CELLS (51) Int. Cl. (75) Inventor: Simeon Santourlidis, Neuss (DE) C12Q 1/68 (200601) , __ (52) US. Cl. (73) Asslgnees: Peter _W‘?r“e__t’ Dusseldorf (DE); F‘med CPC .................................. .. C12Q 1/6888 (2013.01) Ghanjatl, Dusseldorf (DE); Simeon USPC 435/6 11 Santourlidis, Neuss (DE); Nicole ....................................................... .. Groth, Grefrath (DE) (21) Appl. No.: 13/878,915 (57) ABSTRACT (22) PCT Filed: 06L 11, 2011 The invention relates to a method for determining the quality of a pluripotent stem cell, comprising the following steps: (86) PCT NOJ PCT/EP11/67697 measuring the DNA methylation of at least one CpG in a CpG §371 (OX1), island in at least tWo genes of the pluripotent stem cell, (2)’ (4) Date; sep_ 18, 2013 comparing it to the DNA methylation of the at least one CpG in a CpG island in the at least tWo genes of at least one ( 30 ) Forei g n APP lication Priorit y Data reference cell, Wherein the genes are located on different chromosomes and belong to the gene family of olfactory Oct. 11, 2010 (EP) ................................ .. 101871374 receptor genes. Patent Application Publication Jan. 2, 2014 Sheet 2 0f 6 US 2014/0004512 A1 B8595 mmmH ____A_a 216m321 .1 41.__~ wwwmmHmw 00000O00O RRRRRR Patent Application Publication Jan. 2, 2014 Sheet 5 0f 6 US 2014/0004512 A1 mmm —— 0R1E1 2.065051 . mHmEnoEE‘mm: OR3M OR3A1 ——. ORBAZ 0mm UR1A2 Patent Application Publication Jan. 2, 2014 Sheet 6 0f 6 US 2014/0004512 A1 on!“ 3592mm .mm2 OM‘ OM comm US 2014/0004512 A1 Jan. 2, 2014 QUALITY DETERMINATION OF STEM is assumed that the methylated CpGs in?uence the binding of CELLS transcription factors through their steric position in the major grooves of the DNA. Thus, an incompletely methylated CpG [0001] The present invention relates to a method for deter mining the quality of pluripotent stem cells. island can result in a transcription block or reduced transcrip tion depending on Which CpGs are methylated, but there may [0002] In regenerative medicine, a Wide variety of also be no repressing in?uence on transcription at all. approaches are intensively pursued to be able to administer stem cells to patients suffering from degenerative diseases in [0017] According to the invention, the DNA methylation of applications of cell replacement therapy. Already noW, thera at least one CpG in a CpG island in at least three genes of a pies using stem cells serving for cartilage regeneration are pluripotent stem cell to be examined is analyZed, i.e., genes being performed in various hospitals. In the foreseeable are selected, and methylation is measured on the CpG islands future, the application of stem cells Will increase exponen knoWn from these genes on at least one position of the CpG tially. island. Preferably, a position in a CpG island that is as relevant [0003] The method of reprogramming endogenous termi as possible to transcription control is measured. This is fol nally differentiated cells by inducing pluripotency Will con loWed by comparing such methylation With the methylation tribute to this development. This circumvents both problems of a reference cell, in Which the same CpG islands in the same of immune rejection and ethical aspects. genes and the same positions Within the CpG islands of the [0004] HoWever, in all existing approaches, this requires in reference cell are compared. vitro cell culturing, for Which no uniform standards exist. For [0018] In one embodiment of the invention, the pluripotent application With humans, it is required to determine the per stem cell examined may be an induced pluripotent stem cell, formance or quality of the stem cells. in Which case a knoWn pluripotent stem cell is suitable as the [0005] Chuanying Pan et al., J. Genet. Genomics 37 (2010) reference cell. 241-248, examined the demethylation of the promoters of [0019] If the stem cell examined and the reference cell have NANOG and OCT4 in induced pluripotent stem cells of similar or identical methylation patterns, this is indicative of ?broblasts. successful reprogramming and thus a high quality of the [0006] Prashant Mali et al., Stem Cells 28 (2010) 713-720, induced pluripotent stem cell obtained. Additionally or alter examined the demethylation of induced pluripotent stem cells natively, the starting cell, for example, a ?broblast cell, may by means of a HumanMethylation27 BeadChip. also be employed as the reference cell. The extent of alter [0007] It is the object of the present invention to provide a ation of the DNA methylation may also be used to conclude simple method for determining the performance and quality. the success of induction therefrom, i.e., the more similar the [0008] This object is achieved by a method for determining terminally differentiated cell and the presumably induced the quality of pluripotent stem cells, comprising the steps of: stem cell are, the Worse is the quality of the induced stem cell. [0009] measuring the DNA methylation of at least one [0020] Stem cells are frequently kept in culture for CpG in a CpG island in at least three genes of the pluri extended periods of time. There are stem cell lines, in part potent stem cell; originating from embryonic stem cells, that are kept in culture [0010] comparing With the DNA methylation of said at for years. In such cases, it cannot be excluded that the stem least one CpG in a CpG island in said at least three genes cells got damaged during the culturing, Which may remain of at least one reference cell; unnoticed for a long time. In such a case, the method accord [0011] Wherein said genes are located on different chromo ing to the invention can be employed to observe the degree of somes and belong to the gene family of olfactory receptor methylation of one or more CpG islands from tWo or more genes. genes in the course of a culture, and to conclude an alteration [0012] The manifestation of a speci?c cell type, for of the cultured cell from the occurrence of differences. In example, of a stem cell, requires cell-type speci?c gene other Words, a cell that exhibits a DNA methylation pattern expression. This requires qualitatively and quantitatively deviating from that of its originally cultured cell has a lesser adapted gene regulation. quality. [0013] One of the essential mechanisms for gene regulation is DNA methylation. Approximately 60% of the human genes [0021] According to the invention, it is preferred that not only the DNA methylation of three genes is determined, but are in?uenced by DNA methylation in differentiated cells. In DNA, cytosine that is in the context of a palindromic CpG that more genes are employed in order to make differences more pronounced. According to the invention, the number of dinucleotide can have an additional methyl group. Such cor respondingly methylated genes are not expressed, or only so genes may be three, or four, or ?ve, or seven, or ten, or more. in a greatly reduced Way. [0022] In order to obtain a representative impression of the [0014] HoWever, the methylation is not uniformly distrib methylome, genes that are located on different chromosomes uted in the genes throughout the genome, but is increased in are examined, because this gives an improved overvieW of the so-called CpG islands, Which are in the 5' regions of the situation of the cell. genes. [0023] In many cases, it Will be reasonable to examine [0015] If a CpG island is completely methylated, transcrip genes that at least in part belong to one family of genes. In tion of the corresponding gene is not possible. In the case of other embodiments, it may also be reasonable to examine ten a non-methylation, the gene has transcriptional competence, genes, for example, Wherein some genes belong to one family i.e., it can be transcribed. of genes and the other genes belong to another family of [0016] HoWever, the CpG dinucleotides that can be methy genes. lated are not equivalent for the regulation of the gene to Which [0024] It is important to the method according to the inven they belong. The methylation of particular CpGs, Which act tion that alWays the degree of methylation only of identical through their association With target sequences of transcrip positions in identical CpG islands of identical genes can be tion factors, exert the greatest in?uence on gene expression. It compared. US 2014/0004512 A1 Jan. 2, 2014 [0025] According to the invention, it is preferred that not only a single position is analyzed in each of the measured CpG islands, but that several positions, or if several CpG islands exist, several positions in several CpG islands, are analyZed. [0026] The family of genes that is employed according to the invention is the gene family of olfactory receptor genes (The human olfactory receptor gene family, Malnic B, God frey P A, Buck L B. Proc Natl Acad Sci USA. 2004 Feb 24; 101(8): 2584-9. Erratum in: Proc Natl Acad Sci USA. 2004 May 4; 101(18): 7205, and The mouse olfactory receptor gene family. Godfrey P A, Malnic B, Buck L B. Proc Natl Acad Sci USA. 2004 Feb. 17; 101(7): 2156-61. Epub 2004 Feb 9). The olfactory receptor genes are the largest gene family in the human genome (about 1,000 genes). These genes are associated With a CpG island in their 5' region in embryonic and induced pluripotent stem cells, and this island is densely methylated in both types of stem cells.
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  • Us 2018 / 0305689 A1

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    US 20180305689A1 ( 19 ) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2018 /0305689 A1 Sætrom et al. ( 43 ) Pub . Date: Oct. 25 , 2018 ( 54 ) SARNA COMPOSITIONS AND METHODS OF plication No . 62 /150 , 895 , filed on Apr. 22 , 2015 , USE provisional application No . 62/ 150 ,904 , filed on Apr. 22 , 2015 , provisional application No. 62 / 150 , 908 , (71 ) Applicant: MINA THERAPEUTICS LIMITED , filed on Apr. 22 , 2015 , provisional application No. LONDON (GB ) 62 / 150 , 900 , filed on Apr. 22 , 2015 . (72 ) Inventors : Pål Sætrom , Trondheim (NO ) ; Endre Publication Classification Bakken Stovner , Trondheim (NO ) (51 ) Int . CI. C12N 15 / 113 (2006 .01 ) (21 ) Appl. No. : 15 /568 , 046 (52 ) U . S . CI. (22 ) PCT Filed : Apr. 21 , 2016 CPC .. .. .. C12N 15 / 113 ( 2013 .01 ) ; C12N 2310 / 34 ( 2013. 01 ) ; C12N 2310 /14 (2013 . 01 ) ; C12N ( 86 ) PCT No .: PCT/ GB2016 /051116 2310 / 11 (2013 .01 ) $ 371 ( c ) ( 1 ) , ( 2 ) Date : Oct . 20 , 2017 (57 ) ABSTRACT The invention relates to oligonucleotides , e . g . , saRNAS Related U . S . Application Data useful in upregulating the expression of a target gene and (60 ) Provisional application No . 62 / 150 ,892 , filed on Apr. therapeutic compositions comprising such oligonucleotides . 22 , 2015 , provisional application No . 62 / 150 ,893 , Methods of using the oligonucleotides and the therapeutic filed on Apr. 22 , 2015 , provisional application No . compositions are also provided . 62 / 150 ,897 , filed on Apr. 22 , 2015 , provisional ap Specification includes a Sequence Listing . SARNA sense strand (Fessenger 3 ' SARNA antisense strand (Guide ) Mathew, Si Target antisense RNA transcript, e . g . NAT Target Coding strand Gene Transcription start site ( T55 ) TY{ { ? ? Targeted Target transcript , e .

  • Explorations in Olfactory Receptor Structure and Function by Jianghai

    Explorations in Olfactory Receptor Structure and Function by Jianghai Ho Department of Neurobiology Duke University Date:_______________________ Approved: ___________________________ Hiroaki Matsunami, Supervisor ___________________________ Jorg Grandl, Chair ___________________________ Marc Caron ___________________________ Sid Simon ___________________________ [Committee Member Name] Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Neurobiology in the Graduate School of Duke University 2014 ABSTRACT Explorations in Olfactory Receptor Structure and Function by Jianghai Ho Department of Neurobiology Duke University Date:_______________________ Approved: ___________________________ Hiroaki Matsunami, Supervisor ___________________________ Jorg Grandl, Chair ___________________________ Marc Caron ___________________________ Sid Simon ___________________________ [Committee Member Name] An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Neurobiology in the Graduate School of Duke University 2014 Copyright by Jianghai Ho 2014 Abstract Olfaction is one of the most primitive of our senses, and the olfactory receptors that mediate this very important chemical sense comprise the largest family of genes in the mammalian genome. It is therefore surprising that we understand so little of how olfactory receptors work. In particular we have a poor idea of what chemicals are detected by most of the olfactory receptors in the genome, and for those receptors which we have paired with ligands, we know relatively little about how the structure of these ligands can either activate or inhibit the activation of these receptors. Furthermore the large repertoire of olfactory receptors, which belong to the G protein coupled receptor (GPCR) superfamily, can serve as a model to contribute to our broader understanding of GPCR-ligand binding, especially since GPCRs are important pharmaceutical targets.
  • Download from the Sequence Read Archive Repository ( 421 Under the Accession Number SRP070526

    Download from the Sequence Read Archive Repository ( 421 Under the Accession Number SRP070526

    bioRxiv preprint doi: https://doi.org/10.1101/058446; this version posted June 12, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Article 2 Discoveries 3 4 Title 5 Deciphering the wisent demographic and adaptive histories from individual 6 whole-genome sequences 7 8 Authors and Affiliations 9 Mathieu GAUTIER1,2§, Katayoun MOAZAMI-GOUDARZI3, Hubert LEVEZIEL4, Hugues 10 PARINELLO5, Cécile GROHS3, Stéphanie RIALLE5, Rafał KOWALCZYK6, Laurence 11 FLORI3,7§ 12 1 CBGP, INRA, CIRAD, IRD, Supagro, 34988 Montferrier-sur-Lez, France 13 2 IBC, Institut de Biologie Computationnelle, 34095 Montpellier, France 14 3 GABI, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France 15 4 UGMA, INRA, Université de Limoges, 87060 Limoges, France 16 5 MGX-Montpellier GenomiX, c/o Institut de Génomique Fonctionnelle, Montpellier, France 17 6 Mammal Research Institute, Polish Academy of Sciences, 17-230 Białowieża, Poland 18 7 INTERTRYP, CIRAD, IRD, 34398 Montpellier, France 19 20 §Corresponding authors 21 Emails: 22 [email protected] 23 [email protected] 24 1 bioRxiv preprint doi: https://doi.org/10.1101/058446; this version posted June 12, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 Abstract 26 As the largest European herbivore, the wisent (Bison bonasus) is emblematic of the continent 27 wildlife but has unclear origins. Here, we infer its demographic and adaptive histories from 28 two individual whole genome sequences via a detailed comparative analysis with bovine 29 genomes.