Methods for obtaining embryonic stem cell dna methylation signatures

A method for determining effects of experiential exposure on stem cell maturation in a subject, comprising:

• obtaining an exposure sample and a control sample from the subject and analyzing extent of methylation of at least one CpG dinucleotide in DNA of each sample within a plurality of oligonucleotides sequences selected from at least one of the group of cg10338787 (SEQ ID No:
• 68), cg22497969 (SEQ ID No:
• 83), cg11968804 (SEQ ID No:
• 71), cg10237252 (SEQ ID No:
• 67), cg17310258 (SEQ ID No:
• 78), cg13485366 (SEQ ID No:
• 72), cg03455765 (SEQ ID No:
• 62), cg04193160 (SEQ ID No:
• 63), cg27367526 (SEQ ID No:
• 85), cg03384000 (SEQ ID No:
• 61), cg15575683 (SEQ ID No:
• 76), cg17471939 (SEQ ID No:
• 79), cg11199014 (SEQ ID No:
• 70), cg13948430 (SEQ ID No:
• 73), cg01567783 (SEQ ID No:
• 60), cg01278041 (SEQ ID No:
• 59), cg19005955 (SEQ ID No:
• 80), cg16154155 (SEQ ID No:
• 77), cg14652587 (SEQ ID No:
• 75), cg19659741 (SEQ ID No:
• 81), cg06705930 (SEQ ID No:
• 65), cg23009780 (SEQ ID No:
• 84), cg22130008 (SEQ ID No:
• 82), cg05840541 (SEQ ID No:
• 64), cg06953130 (SEQ ID No:
• 66), cg11194994 (SEQ ID No:
• 69), and cg14375747 (SEQ ID No:
• 74), thereby determining a methylation status of at least one CpG dinucleotide in the DNA of the exposure sample and a methylation status of at least one CpG dinucleotide in the DNA of the control sample;
• and,
• deconvoluting the methylation array data from the control sample and the exposure sample to obtain methylation status of individual leukocyte subtypes in the samples, and comparing methylation status of the at least one CpG dinucleotide within a leukocyte subtype of the control sample to the methylation status of the at least one CpG dinucleotide within the same leukocyte subtype of the exposure sample, to determine sites of differential methylation, and correlating a difference in methylation status between the control sample and the exposure sample to obtain the effect of the exposure on stem cell methylation signature.

A kit for determining embryonic stem cell methylation signatures, comprising:

• an array with a plurality of DNA probes attached to a surface or a plurality of surfaces at known addressable locations on the array, wherein the probes hybridize to a DNA sequence of each of a methylated form and an unmethylated form of a CpG dinucleotide in a sequence of a gene of the plurality of genes in the sample;
• primers and reagents for detecting the hybridized probes and for detecting the reaction products derived from the hybridized probes to obtain methylation data;
• and
• instructions for analyzing at least one sample on the array, and instructions for preparing a stem cell methylation signature.

A method for identifying progenitor cell lineages, comprising:

• comparing DNA methylation profiles of a leukocyte subtype between a prenatal or neonatal sample and an adult sample;
• identifying CpG sites differentially methylated between the prenatal or neonatal sample and the adult sample for the leukocyte subtype;
• filtering to select a lineage invariant subset of CpG loci, the subset loci having consistent differential methylation between the leukocyte subtype and an absolute change in methylation greater than a pre-determined threshold between the prenatal or neonatal sample and the adult sample, thereby forming a candidate list of CpG loci for a stem cell methylation signature;
• and
• reducing the candidate list of CpG loci for the stem cell methylation signature by selecting CpGs with minimal residual cell-specific effects, thereby forming a block of differentially methylated regions (DMRs) across the progenitor cell axis of multipotency to terminal differentiation, to identify the progenitor cell lineages.

A method of using an array to determine an embryonic stem cell (ESC) methylation signature in a biological sample, comprising:

• analyzing extent of DNA hybridization in an adult sample and a prenatal or neonatal sample to each of a plurality of oligonucleotide probes, the probes being affixed to at least a first surface for methylated CPG sequences and a second surface for unmethylated CpG sequences, the DNA sequences of the oligonucleotides on the first surface and the second surface being otherwise identical, the plurality of the nucleotide sequences selected from at least one of the group of cg10338787 (SEQ ID No:
• 68), cg22497969 (SEQ ID No:
• 83), cg11968804 (SEQ ID No:
• 71), cg10237252 (SEQ ID No:
• 67), cg17310258 (SEQ ID No:
• 78), cg13485366 (SEQ ID No:
• 72), cg03455765 (SEQ ID No:
• 62), cg04193160 (SEQ ID No:
• 63), cg27367526 (SEQ ID No:
• 85), cg03384000 (SEQ ID No:
• 61), cg15575683 (SEQ ID No:
• 76), cg17471939 (SEQ ID No:
• 79), cg11199014 (SEQ ID No:
• 70), cg13948430 (SEQ ID No:
• 73), cg01567783 (SEQ ID No:
• 60), cg01278041 (SEQ ID No:
• 59), cg19005955 (SEQ ID No:
• 80), cg16154155 (SEQ ID No:
• 77), cg14652587 (SEQ ID No:
• 75), cg19659741 (SEQ ID No:
• 81), cg06705930 (SEQ ID No:
• 65), cg23009780 (SEQ ID No:
• 84), cg22130008 (SEQ ID No:
• 82), cg05840541 (SEQ ID No:
• 64), cg06953130 (SEQ ID No:
• 66), cg11194994 (SEQ ID No:
• 69), and cg14375747 (SEQ ID No:
• 74), for determining methylation status of at least one CpG dinucleotide in the DNA of each of the adult and the prenatal or neonatal sample sample;
• deconvoluting the methylation array data from the adult sample and the prenatal or neonatal sample to obtain methylation data of a plurality of leukocyte subtypes in the samples;
• comparing methylation status of the at least one CpG dinucleotide for a leukocyte subtype in the adult sample to the methylation status of the at least one CpG dinucleotide of the leukocyte subtype of the prenatal or neonatal sample, to determine differentially methylated regions (DMRs);
• and
• analyzing the DMRs to determine the fraction of sequences from progenitor cell lineage origin which constitutes the ESC methylation signature.

An array for efficient and economical determination of embryonic stem cell (ESC) content in a biological sample, comprising a surface containing a plurality of nucleotide sequences, each sequence at an addressable location, the sequences selected from at least one of the group of:

• cg10338787 (SEQ ID No:
• 68), cg22497969 (SEQ ID No:
• 83), cg11968804 (SEQ ID No:
• 71), cg10237252 (SEQ ID No:
• 67), cg17310258 (SEQ ID No:
• 78), cg13485366 (SEQ ID No:
• 72), cg03455765 (SEQ ID No:
• 62), cg04193160 (SEQ ID No:
• 63), cg27367526 (SEQ ID No:
• 85), cg03384000 (SEQ ID No:
• 61), cg15575683 (SEQ ID No:
• 76), cg17471939 (SEQ ID No:
• 79), cg11199014 (SEQ ID No:
• 70), cg13948430 (SEQ ID No:
• 73), cg01567783 (SEQ ID No:
• 60), cg01278041 (SEQ ID No:
• 59), cg19005955 (SEQ ID No:
• 80), cg16154155 (SEQ ID No:
• 77), cg14652587 (SEQ ID No:
• 75), cg19659741 (SEQ ID No:
• 81), cg06705930 (SEQ ID No:
• 65), cg23009780 (SEQ ID No:
• 84), cg22130008 (SEQ ID No:
• 82), cg05840541 (SEQ ID No:
• 64), cg06953130 (SEQ ID No:
• 66), cg11194994 (SEQ ID No:
• 69), and cg14375747 (SEQ ID No:
• 74), for analyzing a fraction of sequences of progenitor cell lineage origin having an ESC methylation signature.

A kit for determining embryonic cell content, the kit comprising a plurality of primers for custom bisulfate sequencing library preparation, each primer directing amplification of a hyper methylatable CpG dinucleotide located in a DNA sequence selected from cg10338787 (SEQ ID No:

• 68), cg22497969 (SEQ ID No:
• 83), cg11968804 (SEQ ID No:
• 71), cg10237252 (SEQ ID No:
• 67), cg17310258 (SEQ ID No:
• 78), cg13485366 (SEQ ID No:
• 72), cg03455765 (SEQ ID No:
• 62), cg04193160 (SEQ ID No:
• 63), cg27367526 (SEQ ID No:
• 85), cg03384000 (SEQ ID No:
• 61), cg15575683 (SEQ ID No:
• 76), cg17471939 (SEQ ID No:
• 79), cg11199014 (SEQ ID No:
• 70), cg13948430 (SEQ ID No:
• 73), cg01567783 (SEQ ID No:
• 60), cg01278041 (SEQ ID No:
• 59), cg19005955 (SEQ ID No:
• 80), cg16154155 (SEQ ID No:
• 77), cg14652587 (SEQ ID No:
• 75), cg19659741 (SEQ ID No:
• 81), cg06705930 (SEQ ID No:
• 65), cg23009780 (SEQ ID No:
• 84), cg22130008 (SEQ ID No:
• 82), cg05840541 (SEQ ID No:
• 64), cg06953130 (SEQ ID No:
• 66), cg11194994 (SEQ ID No:
• 69), and cg14375747 (SEQ ID No:
• 74).

A kit for determining embryonic stem cell methylation signatures, comprising:

• an array with a plurality of DNA probes attached to a surface or a plurality of surfaces at known addressable locations on the array, wherein the probes hybridize to a DNA sequence of each of a methylated form and an unmethylated form of a CpG dinucleotide in a sequence of a gene of the plurality of genes in the sample or;
• a set of oligonucleotide primers comprising a plurality of sequences each having a CpG dinucleotide within each primer sequence;
• primers and reagents for detecting the hybridized probes and for detecting the reaction products derived from the hybridized probes to obtain methylation data;
• and
• instructions for analyzing at least one sample on the array, and instructions for preparing a stem cell methylation signature.

A kit for quantifying embryonic stem cells in a biological sample, the kit comprising:

• at least one of
• (i) an array with a plurality of DNA probes attached to a surface or a plurality of surfaces at known addressable locations on the array, wherein the probes hybridize to a DNA sequence of each of a methylated form and an unmethylated form of a CpG dinucleotide in a stem cell signature sequence in the sample;
• and/or
• (ii) a plurality of oligonucleotide primers comprising a plurality of gene sequences in the stem cell signature for amplification of genomic DNA at a plurality of loci corresponding to hypermethylated CpG sites;
• and
• reagents comprising at least one of:
• primers for amplifying DNA in the sample, for detecting sample DNA hybridized with probes, and for detecting reaction products derived from the hybridized probes to obtain methylation data;
• and
• instructions for analyzing at least one sample on the array, and instructions for quantifying embryonic stem cells based on the stem cell methylation signature.

A method for quantifying effects of experiential exposure on stem cell maturation in a subject, comprising:

• obtaining an exposure sample and a control sample from the subject and analyzing extent of methylation of at least one CpG dinucleotide in DNA of each sample within a plurality of CpG dinucleotide locations selected from at least one of the group of cg10338787, cg22497969, cg11968804, cg10237252, cg17310258, cg13485366, cg03455765, cg04193160, cg27367526, cg03384000, cg15575683, cg17471939, cg11199014, cg13948430, cg01567783, cg01278041, cg19005955, cg16154155, cg14652587, cg19659741, cg06705930, cg23009780, cg22130008, cg05840541, cg06953130, cg11194994, and cg14375747, thereby determining a methylation status of at least one CpG dinucleotide in the DNA of the exposure sample and a methylation status of at least one CpG dinucleotide in the DNA of the control sample;
• and,
• deconvoluting the methylation array data from the control sample and the exposure sample to obtain methylation status of individual leukocyte subtypes in the samples, and comparing methylation status of the at least one CpG dinucleotide within a leukocyte subtype of the control sample to the methylation status of the at least one CpG dinucleotide within the same leukocyte subtype of the exposure sample, to determine sites of differential methylation, and correlating a difference in methylation status between the control sample and the exposure sample to obtain the effect of the exposure on stem cell methylation signature.