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 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 A PP lication Priorit y Data reference cell, Wherein the genes are located on different and belong to the family of olfactory Oct. 11, 2010 (EP) ...... 101871374 receptor genes.

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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 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 (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. In contrast, the same CpG islands of the same genes in ?broblasts are essentially non-methylated. Since this difference can be found several times in each , it represents a pro nounced re?ection of the DNA methylation of the genome (“methylome”). The particular advantage of the gene family of olfactory receptor genes resides in their large number, being distributed over all chromosomes, and the dense methy lation in embryonic and induced pluripotent stem cells. Thus, it is possible to determine methylation on a large number of genomic sites that can be differentially methylated. Prefer ably, at least 10 sites, more preferably at least 20, at least 50, at least 100 or at least 1000 sites are analyZed and employed for the evaluation. [0027] The nomenclature of the genes is “ORnXm”, Where [0028] “OR” represents the olfactory receptor superfamily; [0029] “n” is an integer representing the family, Wherein the members have a sequence identity of more than 40%; [0030] “X” is a single letter representing a subfamily, Wherein the members have a sequence identity of more than 60%; and [0031] “m” is an integer designating an individual family member. [0032] Thus, for example, OR1A1 is the ?rst isoform of the subfamily A of olfactory receptor family 1. [0033] It is considered that receptors of the same subfamily recogniZe similar molecules. [0034] There are tWo large groups, class I (?sh-like recep tor) With the OR families 51 to 56, and class II (tetrapod) With the OR families 1 to 13. US 2014/0004512 A1 Jan. 2, 2014

[0036] A Wide variety of methods are commonly used as methods for determining DNA methylation. One common method is methylation-speci?c PCR. In this method, methy lated cytosine is converted to uracil using bisul?te. By using speci?c primers, it can be examined Whether or not the sites to be examined are methylated. The measuring method is a real time PCR in Which the labeled marker or labeled probes are employed. [0037] An alternative determining method is the so-called Nimble-Gene by the Roche company. In this method, DNA fragments are precipitated by means of 5'-methylcytidine speci?c antibodies, isolated and detected after ampli?cation on an array. [0038] Other methods, for example, by radioactive label ing, Southern blotting or the like, are also possible. [0039] When several CpG islands or several positions in one CpG island or several genes are analyZed, the methyla tion-speci?c PCR reactions employed can also be performed in one PCR With a large number of primers in principle. In order to increase the speci?city of PCR, it is preferred that the respective examinations are performed separately. [0040] In many embodiments, it Will be helpful to addition ally introduce controls in order to check the quality of the measurement. [0041] Since differences may occur betWeen the measure ments, it Will also be often reasonable to check the reference cell and the stem cell simultaneously in tWo reactions. HoW ever, in many cases, it Will suf?ce to recur to earlier measure ments and correspondingly stored data for the methylation of the stem cells. [0042] The invention also relates to a method for determin ing the quality of a pluripotent stem cell, comprising the steps of: [0043] measuring the DNA methylation of at least one CpG in a CpG island in at least tWo genes of the pluri potent stem cell; [0044] comparing With the DNA methylation of said at least one CpG in a CpG island in said at least tWo genes of at least one reference cell. [0045] FIG. 1 shoWs the DNA methylation status in the 5' region of three olfactory receptor genes each on three differ ent chromosomes in ?broblasts, IPS ?broblasts and ES cells (I3). [0046] FIG. 2 shoWs the DNA methylation status in the 5' region of olfactory receptor genes on in ?bro blasts, IPS ?broblasts and ES cells (I3). US 2014/0004512 A1 Jan. 2, 2014

[0047] FIG. 3 shows the DNA methylation status in the 5' (c) a knoWn embryonic cell line 1-3. CpG methylation on region of olfactory receptor genes on chromosome 11 in olfactory receptor genes from different chromosomes Was ?broblasts, IPS ?broblasts and ES cells (I3). analyZed. The results of the analyses are shoWn in FIGS. 1 to [0048] FIG. 4 shoWs the DNA methylation status in the 5' 6. region of olfactory receptor genes on in 1. A method for determining the quality of a pluripotent ?broblasts, IPS ?broblasts and ES cells (I3). stem cell, comprising the steps of: [0049] FIG. 5 shoWs the DNA methylation status in the 5' measuring the DNA methylation of at least one CpG in a region of olfactory receptor genes on chromosome 17 in CpG island in at least three genes of the pluripotent stem ?broblasts, IPS ?broblasts and ES cells (I3). cell; [0050] FIG. 6 shoWs the DNA methylation status in the 5' comparing With the DNA methylation of said at least one region of olfactory receptor genes on chromosome 3 in ?bro CpG in a CpG island in said at least three genes of at least blasts, IPS ?broblasts and ES cells (I3). one reference cell; [0051] The invention is further illustrated by the folloWing Wherein said genes are located on different chromosomes and Examples: belong to the gene family of olfactory receptor genes. 2. The method according to claim 1, Wherein said pluripo 1. Isolation of Genomic DNA tent stem cell is an induced pluripotent stem cell. 3. The method according to claim 1, Wherein said reference [0052] Genomic DNA Was isolated as folloWs: The cell is a pluripotent stem cell. genomic DNA Was isolated from the cells by means of the 4. The method according to claim 1, Wherein said reference Qiagen DNeasy blood&tissue DNA isolations kit. cell is a differentiated cell. 5. The method according to claim 1, Wherein at least tWo 2. Isolation of Methylated DNA reference cells are employed. [0053] 1 pg of genomic DNA Was converted by ultrasoni 6. The method according to claim 1, Wherein the methyla cation to a fragment siZe of about 300 to 1,000 base pairs. The tion of at least one CpG island each in at least four or at least methylated DNA fragments Were precipitated by means of a ?ve genes of said pluripotent stem cell is measured and methylcytosine-speci?c antibody; a Methylamp Methylated respectively compared With the methylation in genes of the DNA Capture Kit (MeDIP from Diagenode) Was used. reference cells. 7. The method according to claim 1, Wherein said olfactory 3. Analysis of Methylation receptor genes are human members of class I or II. 8. The method according to claim 1, Wherein several CpG [0054] The precipitates Were hybridized on a Nimble-Gene islands are measured in each gene. 3 85K Ref. Seq. PromoterArray HG1 8 (Roche). The promoter 9. The method according to claim 1, Wherein the methyla regions of all knoWn genes rich in CpG dinucleotides are tion of at least one CpG island each in at least seven or at least present on this array, covalently bonded in the form of 50 mer ten genes of saidpluripotent stem cell is measured and respec oligonucleotide samples. tively compared With the methylation in genes of the refer [0055] The hybridiZed arrays Were scanned With a microar ence cells. ray scanner (Molecular Devices), and images Were generated 10. The method according to claim 1, Wherein said olfac With the Axon Genepix softWare. Nimble-Scan Version 2.5 tory receptor genes are selected from OR6K6, OR6N1, and Signal-Map Version 1.9 Were employed for analysis. OR6N2, OR51A7, 0R5 1 G2, OR51G1, OR1M1, OR7G2, OR7G1. 4. Determination of Quality 11. The method according to claim 1, Wherein methyla [0056] The cells employed Were cells obtained from (a) tion-speci?c PCR is employed for measuring the DNA ?broblasts, (b) pluripotent stem cells induced from ?bro methylation. blasts by retroviral transfer of the four transcription factors 12. The method according to claim 1 1, Wherein methylated Oct3/ 4, Sox2, c-Myc, and Klf4 (by analogy With: Induction of cytosines are converted to uracil using bisul?te. pluripotent stem cells from mouse embryonic and adult ?bro 13. The method according to claim 11, Wherein real-time blast cultures by de?ned factors. Takahashi K, Yamanaka S. PCR With labeled markers or labeled probes is employed. Cell. 2006 Aug. 25; 126(4): 663-76. Epub 2006 Aug. 10), and * * * * *