Signature to trace human cell lineages.

1. CREDIT SEMINAR
DNA methylation signature
for tracing Human stem cell
lineages during development
Speaker :
NIKUNJ TYAGI
Ph.D Scholar

2. Content
 Introduction
 Epigenetics & stem cell differentiation
 Dynamism of CpG Methylation
 Effect of epigenetic drug treatment
 FCO signature
 Analytical pipeline
 Validation of FCO signature
 Dynamics of FCO signature
 Conclusion
 References
1

3. Introduction
2
• Lineage tracing –A Powerful Tool.
• Criteria of a lineage tracer.
• Direct Observation
Advantages- Noninvasive.
Disadvantage - Requires a transparent embryo
When cell-fate decisions are not autonomous.
Accessibility of the tissues
Naturally occurring epigenetic marks such as DNA methylation
provide a promising alternative

4. Epigenetics & Stem cell Differentiation
• Key Players are histone modifications and DNA methylation
• Cellular identity is indeed close reflection of cellular epigenetic status
• The requirements for cellular differentiation of an stem cell are a loss of
proliferation potential and a gain of cell-type identity.
• Works in harmony ,to affect expression profiles of stem cells
3

5. Lineage restriction of human developmental potency
4

6. Dynamism of CpG Methylation
• DNA methylation in the sequence of 5’-CpG-3’ is critical.
• 1-2% of the genome comprises CpG islands ,70% of Cytosine residues are
methylated
• Involved in transcriptional silencing and developmental processes
• High-throughput strategies demonstrate that human ES cells have a unique CpG
methylation signature
• Silencing of pluripotency-related genes occurs by means of CpG promoter
hyper methylation
5

7. DNA methylation and stemness
In ES cells and multipotent progenitor cells, HCP promoters are characterized by low
DNA methylation levels, whereas LCP promoters are enriched in DNA methylation
6

8. Effects on differentiation potential of multipotent/pluripotent stem
cells after treatment with epigenetic drugs
Stem cell classification Epigenetic drug Differentiation after
treatment
Multipotent stem cells
Adipose-derived stem cells 5-aza-2'-deoxycytidine Cardiomyocytes
Adipose-derived stem cells 5-aza-2'-deoxycytidine;
trichostatin A
Cardiomyocytes
Bone marrow mesenchymal
stem cells
5-aza-2'-deoxycytidine Osteocytes
Pluripotent stem cells
Embryonic stem cells 5-aza-2'-deoxycytidine Cardiomyocytes
Embryonic stem cells 5-aza-2'-deoxycytidine Endothelial cells
Embryonic stem cells Trichostatin A Cardiomyocytes
7

9. Embryonic and adult hematopoietic stem cells contain methylated CpG
loci that are unique to each of these types of stem cells but that are
invariant with respect to the lineage specification of their progeny.
Hypothesis
9

10. Blood cells arise from hematopoietic stem cells
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11. FCO Signature
• Common invariant biomarker of a cell that originated during the
prenatal period.
• A subset of CpGs represent differentially methylated
regions(DMRs), common among cell lineage subtypes.
• But different among fetal and adult progenitors.
Can be applied to trace proportion of cells in a mixture of cell types
that are of fetal cell origin
10

12. A novel analytical pipeline
11

13. Selection of invariant loci for the fetal cell origin-FCO signature
Panel B, the reduced library (27 CpGs), showed strong separation of
UCB and APB samples, however the residual variability from cell
type was attenuated.
Lucas A. Salas et al. Genome Res. 2018;28:1285-1295 12

14. Gene Name(s) CHR NO.
WWC2-AS2 4
14
NAGS;PYY 17
CADM2;CADM2 3
OBSCN;OBSCN 1
TTLL8 22
PAX6;PAX6;PAX6 11
PROSER2 10
TOM1L1 17
OTOP3 17
PEA15 1
LRFN1 19
1
9
2
FBXL14 12
17
1
8
11
TP73 1
FSTL4 5
ADAM5 8
4
PRDM16;PRDM16 1
2
STEAP2;STEAP2;STEAP2 7
List of candidate loci identified and selected

15. Discovery (A) and replication (B) of the deconvolution algorithm using lineage-invariant, CpG loci
in new born and adult peripheral blood leukocytes.
Lucas A. Salas et al. Genome Res. 2018;28:1285-1295
Proportion of cells exhibiting FCO signature is higher
in cord blood samples, mean(SD) 85.4%(6.0)
13

16. Synthetic Mixture experiment
Lucas A. Salas et al. Genome Res. 2018;28:1285-1295
(B)
Validation of FCO Signature ,CCC=0.97 (P<0.05)
14

17. Methylation signature deconvolution in pluripotent, fetal progenitors, and
adult CD34+ stem/progenitor cells
Lucas A. Salas et al. Genome Res. 2018;28:1285-1295
Concordance of the leukocyte-derived FCO signature, mean (SD) 75.9% (8.5)
15

18. Estimation of FCO in ESC and iPSC through different number of cell
culture passages
Reduction of 0.14% on average for every additional passage
Lucas A. Salas et al. Genome Res. 2018;28:1285-1295 16

19. FCO Signature as an indicator of ESC Lineage
Lucas A. Salas et al. Genome Res. 2018;28:1285-1295 17
High FCO fraction in diverse fetal tissues (3-26 weak of gestational age)

20. Dynamics of FCO Signature with gestational age
Lucas A. Salas et al. Genome Res. 2018;28:1285-1295 18

21. FCO methylation signature deconvolution in blood leukocytes
sampled at birth through childhood and adult ages
Lucas A. Salas et al. Genome Res. 2018;28:1285-1295
Dramatic and rapid decrease in the FCO cell fraction occurred over the
first 5 yrs. of life
19

22. Conclusion
• This work defined and applied a DNA methylation signature common among
human fetal hematopoietic progenitor cells.
• It has also shown that this signature traces the lineage of cells and informs the
study of stem cell heterogeneity in humans under homeostatic conditions.
20

23. References
• Salas et al.: Tracing human stem cell lineage during development using DNA
methylation. Genome research 2018, 28:1285-1295
• Sørensen AL, Jacobsen BM, Reiner AH, Andersen IS, Collas P: Promoter
DNA methylation patterns of differentiated cells are largely programmed at the
progenitor stage. Mol Biol Cell 2010, 21: 2066-2077
• Berdasco María and Esteller Manel : DNA methylation in stem cell renewal
and multipotency. Stem Cell Research & Therapy 2011, 2:42
• Fouse et al. Promoter CpG Methylation Contributes to ES Cell Gene
Regulation in Parallel with Oct4/Nanog,PcG Complex, and Histone H3 K4/K27
Trimethylation. Cell stem cell 2007, 2:160-169
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25. We hypothesized that invariant
methylation marks with high potential to be indicative
of a FCO would be differentially methylated in newborns compared
with adults and shared across six major blood cell lineages
(granulocytes [Gran], monocytes [Mono], B lymphocytes [Bcell],
CD4+ T lymphocytes [CD4T], CD8+ T lymphocytes [CD8T], and
natural killer lymphocytes [NK]).

26. DIRECT TRACING
• lineage-tracing experiment always needs to fulfill the following three
requirements: (1) A careful assessment of the cells that are marked at the initial
timepoint, so that the starting populations are clearly defined. (2) The markers
used to mark the cells remain exclusively in the original cells and their progeny
and will not diffuse to the neighboring cells. (3) These markers are sufficiently
stable and are not toxic to the cells during the entire tracing period.
• The advantages of lineage tracing by direct observation include the speed and
ease of establishing the technique and the fact that it is noninvasive. A major
limitation is that, for an intact organism, continuous observation requires a
transparent embryo with a small number of cells. It is also harder to interpret
results when cell-fate decisions are not autonomous. The tissue to be studied
must also be accessible, which precludes studies of postimplantation
mammalian embryos, unless the cells or embryos are placed in culture, which
potentially alters their behavior. Nevertheless, the transparency of zebrafish
(Danio rerio) embryos has enabled lineage analysis by direct observation to be
performed during early vertebrate development

27. Suzuki, M. M., & Bird, A. (2008). DNA methylation landscapes: provocative insights from
epigenomics. Nature Reviews Genetics, 9(6), 465–476. doi:10.1038/nrg2341

39. • the multilineage potential of adult stem cells could be imposed early in the
original precursor stem cells through CpG methylation
• DNA methylation is necessary for controlling stem cell proliferation and
differentiation
• role of DNA methylation in controlling the transcriptional activity of genes
important for self-renewal, the dynamism of CpG methylation of tissue-specific
genes during several differentiation programs
• DNA methylation, the addition of a methyl group to the carbon 5 of the
cytosine into CpG contexts, is known to be an essential process in development
and cellular differentiation
• involved in gene regulation of housekeeping and tissue-type genes, silencing
of one allele of imprinted genes, and compensation of the extra copy of the X
chromosome in females

40. effects of CpG methylation on stemness and differentiation
• first piece of evidence came from the observation that important genes for the
maintenance of ES cells, such as Oct4and Nanog genes, are usually
hypomethylated when activated but became hypermethylated during
differentiation
• High-throughput strategies demonstrate that human ES cells have a unique
CpG methylation signature that, in combination with histone modifications,
drives stem cell differentiation through the restriction of the developmental
potential of progenitor cells
• DNA methylation-dependent control of tissue-specific genes (abolishing the
risks of lineage-unrelated gene expression).
• It is suggested that silencing of pluripotency-related genes occurs by means of
CpG promoter hypermethylation
• Expression of the aforementioned(oct 4,nanong,sox 2) transcription regulators
is usually controlled by CpG promoter methylation, and differentiation of ES
cells is accomplished by partial or full methylation of pluripotency-associated
genes, resulting in their downregulation

41. • Cellular differentiation is determined by a loss of proliferation potential and a
gain of cell-type identity
• Characterization of DNA methylation profiles of all human RefSeq promoters
in mesenchymal adult stem cells from various origins, including adipose,
hematopoietic, and neural progenitors and muscle tissue, shows that the
majority of the lineage-specific genes are hypomethylated even if the
progenitor is not able to differentiate into this specific lineage
• the restriction for differentiation in specific programs imposed by means of
DNA methylation is established early in development, in the progenitor state,
and persists after differentiation, as most of the hypermethylated promoters in
undifferentiated cells remain hypermethylated in somatic cells (Sørensen et al
2010)
• treatment with demethylating agents , the use of 5-aza-2'-deoxycytidine (5-
ADC) promotes differentiation of ASCs into cardiac myogenic cells

42. • Genes whose protein products play an essential role during embryonic
development were preferentially methylated, suggesting that gene expression
during development could be regulated by CGI methylation.
• Imprinted gene A gene that is expressed or silenced depending on which
parent contributed it to the zygote. In a mouse cell, for example, the paternal
insulinlike growth factor allele is expressed, but the maternal allele is not. In
some cases, imprinting depends on differential DNA methylation of gene
regulatory regions.

44. • Lineage restriction of human developmental potency. Totipotent cells at the
morula stage have the ability to self-renew and differentiate into all of the cell
types of an organism, including extraembryonic tissues. Pluripotent cells - for
example, in vitro embryonic stem (ES) cells established at the blastocyst stage
and primordial germ cells (PGCs) from the embryo - lose the capacity to form
extraembryonic tissues like placenta. Restriction of differentiation is imposed
during normal development, going from multipotent stem cells (SCs), which
can give rise to cells from multiple but not all lineages, to the well-defined
characteristics of a somatic differentiated cell (unipotent). Specific chromatin
patterns and epigenetic marks can be observed during human development
since they are responsible for controlling transcriptional activation and
repression of tissue-specific and pluripotency-related genes, respectively.
Global increases of heterochromatin marks and DNA methylation occur during
differentiation.