Metadichol, a natural ligand for the expression of Yamanaka reprogramming factors in somatic and primary cancer cell lines

Abstract

The conversion of somatic cells back into induced pluripotent stem cells (iPSCs) or embryonic-like stem cells involves the introduction of four genes commonly called Yamanaka factors, i.e., Sox2, Oct4, Klf4, and c-Myc, into the cells. Because these genes and the viral vectors used to introduce them into cells have the potential to cause cancer, iPSC lines are not clinically useful. Most instances of direct reprogramming have been achieved by the forced expression of defined factors using viral vectors. Here, we show that Metadichol® has the potential to generate iPSCs nonvirally and may be helpful in clinical applications. Metadichol is a nanoformulation of long-chain alcohols derived from food. Quantitative real-time PCR (qRT-PCR) and western blotting showed that OCT4, SOX2, and Nanog are expressed when fibroblasts were treated with Metadichol at one picogram to 100 nanograms. Reverse-transcription PCR (RT-PCR) also revealed that OCT4, KLF4, Nanog, and Sox2 levels increased compared to controls by 4.01-, 3.51-and 1.26-, and 2.5-fold, respectively, in A549 cancer cells. In Colo-205 cells, OCT4, KLF4, and Sox2 were increased by 1.79-, 13.17-, and 2.25-fold, respectively. Metadichol treatment with triple-negative primary breast cancer (HCAF-TNPBC) primary cancer cells led to multifold increases in OKSM factors by 19-, 6-, 8.07-, 2.45-, and 6.91-fold in concentration ranges of 1 picogram to 100 nanograms. Metadichol is a natural product that induces the expression of Yamanaka factors needed for reprogramming and Klotho, an antiaging gene, and curbs the expression of the TP53 gene, which is critical for reprogramming somatic cells into IPSCs. Metadichol increases endogenous vitamin C levels, leading to the efficient reprogramming of somatic cells into iPSCs. Metadichol is nontoxic and commercially available as a nutritional supplement. Thus, it can be directly tested in vivo in human subjects to confirm that cells can indeed be programmed into a state of induced pluripotency and cause the mitigation of disease conditions.

Full text

Metadichol, a natural ligand for the expression of Yamanaka reprogramming factors in somatic and primary
cancer cell lines
P.R. Raghavan
NANORX INC
Correspondence: raghavan@nanorxinc.com
PO Box 131
Chappaqua, NY 10514
Abstract.
The conversion of somatic cells back into induced pluripotent stem cells (iPSCs) or embryonic-like stem cells
involves the introduction of four genes commonly called Yamanaka factors, i.e., Sox2, Oct4, Klf4, and c-Myc,
into the cells. Because these genes and the viral vectors used to introduce them into cells have the potential to
cause cancer, iPSC lines are not clinically useful. Most instances of direct reprogramming have been achieved
by the forced expression of defined factors using viral vectors. Here, we show that Metadichol® has the
potential to generate iPSCs nonvirally and may be helpful in clinical applications. Metadichol is a
nanoformulation of long-chain alcohols derived from food. Quantitative real-time PCR (qRT-PCR) and western
blotting showed that OCT4, SOX2, and Nanog are expressed when fibroblasts were treated with Metadichol at
one picogram to 100 nanograms. Reverse-transcription PCR (RT-PCR) also revealed that OCT4, KLF4, Nanog,
and Sox2 levels increased compared to controls by 4.01-, 3.51- and 1.26-, and 2.5-fold, respectively, in A549
cancer cells. In Colo-205 cells, OCT4, KLF4, and Sox2 were increased by 1.79-, 13.17-, and 2.25-fold,
respectively. Metadichol treatment with triple-negative primary breast cancer (HCAF-TNPBC) primary cancer
cells led to multifold increases in OKSM factors by 19-, 6-, 8.07-, 2.45-, and 6.91-fold in concentration ranges
of 1 picogram to 100 nanograms. Metadichol is a natural product that induces the expression of Yamanaka
factors needed for reprogramming and Klotho, an antiaging gene, and curbs the expression of the TP53 gene,
which is critical for reprogramming somatic cells into IPSCs. Metadichol increases endogenous vitamin C
levels, leading to the efficient reprogramming of somatic cells into iPSCs. Metadichol is nontoxic and
commercially available as a nutritional supplement. Thus, it can be directly tested in vivo in human subjects to
confirm that cells can indeed be programmed into a state of induced pluripotency and cause the mitigation of
disease conditions.
Keywords Yamanaka factors, OCT4, SOX2, MYC, OKSM factors, VDR, P53, Reprogramming cells, Somatic,
IPSC, Klotho, Primary cancer cell lines

Introduction
In 2006, Dr. Yamanaka and his group (1) showed that somatic cells could be programmed using just four genes,
i.e., Oct4, Sox2, Klf4, and c-Myc (OSKM; also called Yamanaka factors). These genes are used to reprogram
adult mouse fibroblasts (connective tissue cells) into an embryonic state known as pluripotency. Pluripotent
cells are similar to embryonic stem cells and can become any other cell type in the body.
In 2011, a French researcher, Jean-Marc Lemaitre, added two additional factors (Nanog and LIN28) to the
Yamanaka factors and reported comparing aged fibroblasts from healthy older adults and healthy people over
100 years old. These factors lead to cellular rejuvenation. The six factors mentioned above can reprogram cells
into induced pluripotent stem cells (iPSCs), which can be converted to any other cell type in the body (2).
Kromer et al. (3) showed that aged cells such as fibroblasts have short telomeres and dysfunctional
mitochondria.
It is relatively easy to isolate cells in a dish, revert them to a developmental state, and then reprogram them into
the desired cells using Yamanaka factors. However, this is not practical in living animals because cell memory
cannot be erased and reverted to a pluripotent state. It is also possible that the expression of Yamanaka factors
causes cancer in animals (4).
Belmonte and his team reported that the cells and organs of living animals could be rejuvenated (5). The
research group used a specially engineered mouse strain designed to age more rapidly than usual and an
engineered normally aging mouse strain. These mice were engineered to express Yamanaka factors upon
exposure to the antibiotic doxycycline in drinking water. Moreover, allowing transient expression of Yamanaka
factors for two days> was followed by silencing of these factors upon the removal of doxycycline from the
animals’ drinking water.
In late 2020, David Sinclair restored vision in aged mice and mice with damaged retinal nerves using partial
cellular reprogramming (6). Only three factors were used, and MYC was removed to reduce cancer risk. This
strategy also alleviated age-related vision impairment in treated mice and mice that experienced increased eye
pressure, an indicator of glaucoma.
In 2021, researchers exposed cells to OSKM via a doxycycline-inducible lentiviral system, as in previous
animal studies (7). This strategy reversed fibroblast cell aging by 30 years, allowing aged cells to function
similarly to those in a person approximately 25 years of age.
For the translation of partial cellular reprogramming strategies in humans, it is necessary to identify a method
for inducing Yamanaka factor expression in cells that do not require drugs such as doxycycline.
Other critical challenges related to the clinical application of these cells are their safety and tumorigenic
potential when transplanted back into patients (8).
The generation of induced pluripotent stem cells (iPSCs) with viral integration and the use of viruses to deliver
transcription factors are associated with tumorigenesis (9). There is a need for reprogramming protocols that do
not require permanent genomic integration or viral vectors. Such protocols represent the future of regenerative
medicine.
Deng et al. (10) reported that a combination of specific small molecules could reprogram mouse fibroblasts into
iPSCs via a single transcription factor, Oct4, eliminating the need for Sox2, Klf4, and c-Myc expression. This
finding brought us closer to achieving the generation of iPSCs using small molecules without any genetic
modification. Other small molecules that can eliminate the need for exogenous Oct4 expression are needed.
Herein, we show that metadichol (11), a small nontoxic molecule, naturally induces the expression of Yamanaka
factors in vitro in fibroblasts and A549 and Colo-205 cancer cells and primary cancer cell lines.

Experimental procedures – quantitative PCR (qPCR), western blotting, and cell culture
All work was carried out by a service provider, Skanda Life sciences, Bangalore, India.
Chemicals and reagents
A549 cells, Colo-205 cells, and human cardiac fibroblast cells were obtained from ATCC (USA). Primary breast
cancer cells were obtained from BIOIVT, Detroit, Michigan, USA. Primary antibodies were purchased from
ABclonal, Woburn, Massachusetts, USA, and E-lab Science, Maryland, USA. Primers were sourced from
SahaGene, Hyderabad, India (Table 1). Other molecular biology reagents were from Sigma Aldrich.
Table 1
1
OCT4
F
GTTGATCCTCGGACCTGGCTA
134
R
GGTTGGCTCACTCGGTTCT
2
P53
F
GGCCCACTTCACCGTACTAA
278
R
GTGGTTTCAAGGCCAGATGT
3
KLF4
F
CCCACACAGGTGAGAAACCT
199
R
ATGTGTAAGGCGAGGTGGTC
4
SOX2
F
AACCCCAAGATGCACAACTC
234
R
CGGGGCCGGTATTTATAATC
5
c-MYC
F
CCATCCAGGTGAACCACCTA
241
R
ATCTCCGAACACATCACTTC
6
NANOG
F
ATCTGCTGGAGGCTGAGGTA
180
R
GTGGTTTCAAGGCCAGATGT
7
KLF 2
F
CTTCTCTCGACGCCATCTCC
160
R
AGCCATCCAAAAGCCCCATT
8
KLOTHO
F
AGGGTCCTAGGCTGGAATGT
158
R
CCTCAGGGACACAGGGTTTA
Maintenance and seeding
The cells were maintained in the appropriate medium, with or without the required supplements and 1%
antibiotics, in a humidified atmosphere of 5% CO2 at 37 °C. The medium was changed every other day until the
cells reached confluency. The viability of the cells was assessed using a hemocytometer.
When the cells reached 70–80% confluence, single-cell suspensions containing 106 cells/mL were prepared and
seeded in 6-well plates at a density of 1 million cells per well. The cells were incubated for 24 h at 37 °C in 5%
CO2. After 24 h, the cell monolayer was rinsed with serum-free medium and treated with Metadichol at
predetermined concentrations.
Cell treatments
Different concentrations of Metadichol (1 pg/mL, 100 pg/mL, 1 ng/ml, and 100 ng/mL) were prepared in
serum-free medium. Subsequently, a Metadichol-containing medium was added to predesignated wells. Control
cells received medium without the drug. The cells were incubated for 24 h. After treatment, the cells were
gently rinsed with sterile PBS. Whole-cell RNA was isolated using TRIzol reagent (Invitrogen) per the
manufacturer’s instructions, and cDNA was prepared. The samples were analyzed by qPCR and western
blotting for various biomarkers.
Quantitative real-time PCR (qRT-PCR)
RNA isolation
Total RNA was isolated from each treatment group using TRIzol. Approximately ~1×106 cells were collected in
1.5 mL microcentrifuge tubes. The cells were centrifuged at 5000 rpm for 5 min at four °C, and the cell
supernatant was discarded. Then, 650 µL of TRIzol was added to the pellet, and the contents were mixed well
and incubated on ice for 20 min. Subsequently, 300 µL of chloroform was added to the mixture, and the samples

were mixed well by gentle inversion for 1–2 min and incubated on ice for 10 min. The samples were
centrifuged at 12000 rpm for 15 min at four °C. The upper aqueous layer was transferred to a new, sterile 1.5
mL centrifuge tube, and an equal amount of prechilled isopropanol was added to the tube. The samples were
incubated at -20 °C for 60 min. After incubation, the mixture was centrifuged at 12000 rpm for 15 min at four
°C. The supernatant was discarded carefully, and the RNA pellet was retained. The pellet was washed with 1.0
mL of 100% ethanol, followed by 700 µL of 70% ethanol via centrifugation, as described above, after each step.
The RNA pellet was air-dried at RT for approximately 15–20 min and then resuspended in 30 µL of DEPC-
treated water. The RNA concentration was quantified using a Spectradrop (SpectraMax i3x, USA)
spectrophotometer (Molecular Devices), and cDNA was synthesized using reverse-transcription PCR (RT-
PCR).
cDNA synthesis
cDNA was synthesized from 2 µg of RNA using the PrimeScript cDNA synthesis kit (Takara) and oligo dT
primers per the manufacturer’s instructions. The reaction volume was 20 μL, and cDNA synthesis was
performed on an Applied Biosystems instrument (Veritii). The cDNA was used for qPCR (50 °C for 30 min
followed by 85 °C for 5 min).
Primers and qPCR
The PCR mixture (final volume of 20 µL) contained 1 µL of cDNA, 10 µL of SYBR green Master Mix, and one
µM complementary forward and reverse primers specific for the respective target genes. The samples were run
under the following conditions: initial denaturation at 95 °C for 5 min, followed by 30 cycles of secondary
denaturation at 95 °C for 30 s, annealing at the optimized temperature for 30 s, and extension at 72 °C for 1
min. The number of cycles that allowed amplification in the exponential range and without reaching a plateau
was selected as the optimal number of cycles. The obtained results were analyzed using CFX Maestro software.
Fold change was calculated using the following equation.
(ΔΔCT Method)
The comparative CT method determined the relative expression of target genes to the housekeeping gene (β-
actin) and untreated control cells.
The delta CT for each treatment was calculated using the formula.
Delta Ct = Ct (target gene) – Ct (reference gene)
To compare the delta Ct of individually treated samples with the untreated control sample, the Ct was subtracted
from the control to obtain the delta delta CT.
Delta delta Ct = delta Ct (treatment group) – delta Ct (control group)
The fold change in target gene expression for each treatment was calculated using the formula. Fold change =
2^ (−delta delta Ct)
Protein isolation
Total protein was isolated from 106 cells using RIPA buffer supplemented with the protease inhibitor PMSF.
The cells were lysed for 30 min at four °C with gentle inversion. The cells were centrifuged at 10,000 rpm for
15 min, and the supernatant was transferred to a fresh tube. The Bradford method was used to determine the
protein concentration, and 25 µg of protein was mixed with 1× sample loading dye containing SDS and loaded
onto a gel. The proteins were separated under denaturing conditions in Tris-glycine buffer.
The proteins were transferred to methanol-activated PVDF membranes (Invitrogen) using a Turbo transblot
system (Bio-Rad, USA). Nonspecific binding to the membranes was blocked by incubation in 5% BSA for 1 h.
The membranes were incubated overnight with the respective primary antibody at 4 °C, followed by a species-
specific secondary antibody for 1 h at RT. The blots were washed and incubated with ECL substrate (Merck) for
1 min in the dark. Images were captured at appropriate exposure settings using a ChemiDoc XRS system (Bio-
Rad, USA).

Summary of results: qRT-PCR
Table 2: RT-PCR results of Metadichol treatment with human cardiac fibroblasts (HCFs)
Table 3: RT-PCR results following Metadichol treatment in A-549 and COLO205 cancer cells
Cancer cell lines (Fold Increase)
A549 lung carcinoma
cell line
P53
Oct4
(Pou5F1)
Klf4
Nanog
SOX2
Klotho
Concentration of
Metadichol
Fold increase
Fold increase
Fold increase
Fold increase
Fold increase
Fold increase
Control
1
1
1
1
1
1
1 picogram
No expression
0.27
1.05
0.82
0.47
0.37
100 picograms
No expression
0.92
0.44
0.82
0.80
0.60
1 nanogram
No expression
4.01
3.51
1.26
2.51
1.99
100 nanograms
No expression
1.25
1.58
0.81
0.73
0.83
Colo-205 pancreatic
cancer cell line
Concentration of
Metadichol
Fold increase
Fold increase
Fold increase
Fold increase
Fold increase
Fold increase
Control
1
1
1
1
1
1
1 picogram
No expression
0.41
2.55
1.74
2.16
1.89
100 picograms
No expression
0.88
2.98
0.68
2.21
1.22
1 nanogram
No expression
1.79
13.17
0.41
2.25
2.58
100 nanograms
No
expression
0.81
6.77
1.19
1.71
0.86
Human cardiac
fibroblasts
(HCFs)
Metadichol
concentration
NANOG
OCT 04
(POU5F1)
SOX2
KLF2
Klotho
Fold Increase
Control
1.00
1.00
1.00
1
1
Fold Increase
1 pg
2.39
0.28
0.85
1.265
2.82
Fold Increase
100 pg
5.86
10.36
10.21
2.468
10.13
Fold Increase
1 ng
1.40
1.67
1.04
6.052
0.26
Fold Increase
100 ng
0.22
0.96
2.55
14.81
0.19
!

Table 4. RT-PCR results following metadichol treatment in primary cancer cells (
triple-negative primary breast
cancer (HCAF-TNPBC) cells)
Human cancer associated
fibroblasts (HCAF) triple
negative primary breast
cancer (TNPBC
c-MYC
Oct4
(Pou5F1)
Klf4
SOX2
Klotho
Concentration of
Metadichol
Fold
increase
Fold
increase
Fold
increase
Fold
increase
Fold
increase
Control
1
1
1
1
1
1 picogram
3.36
4.56
3.36
0.57
2.13
100 picograms
4.39
4.94
4.39
0.62
1.10
1 nanogram
4.04
19.63
8.07
2.45
0.64
100 nanograms
6.91
9.32
6.91
1.16
1.02
Discussion
Metadichol is a nanoemulsion of a mixture of the long-chain chain alcohols C26, C28, and C30; C28 is the
primary alcohol (greater than 85% of the mixture). We observed that 100 picograms of Metadichol increased the
expression of Yamanaka factors in fibroblasts, whereas one nanogram of Metadichol increased Yamanaka factor
expression in cancer cell lines. We did not observe p53 expression in the cancer cell lines. Published reports (12,
13) suggest that the inactivation of p53 markedly increases the efficiency of iPSC production. This shows that
p53 deficiency simplifies iPSC generation by allowing the production of iPSCs expressing Oct4 and Sox2.
However, reprogramming human primary cancer cells remains a challenge. Hu et al. (14) successfully
reprogrammed primary human lymphoblasts from a BCR–ABL+ CML patient using trans-free gene IPSC
technology to ectopically express OKSM factors.
The effect of Metadichol on human cardiac fibroblasts (see Table 2) and on cancer cell lines (A-549) and
pancreatic cancer cell lines COLO 205 are shown in Table 3 and on triple-negative primary breast cancer cells
(HCAF-TNPBC primary cancer cells). There were multifold increases in OKSM factors in all these, but
interestingly, there was no increase in p53 expression. Interestingly, Metadichol also induced the expression of
the Klotho gene. A mutation in the Klotho gene (KL (−/−)) significantly decreases the proliferation of adipose-
derived stem cells, which leads to lower expression of pluripotent transcription factors (Nanog, Sox-2, and Oct-
4) in mice (15, 16). The expression of these factors is vital for maintaining the pluripotency of stem cells. As a
longevity hormone, Klotho protects cells from oxidative stress; simultaneously, as a tumor suppressor, it inhibits
the growth of cancer cells (17).
Another critical factor involved in the reprogramming of cells is vitamin C. Numerous studies have shown that
adding vitamin C to the culture medium of somatic cells during reprogramming improves the efficiency and
quality of iPSC formation (18, 19). Metadichol, as shown in previous studies, increases vitamin C levels in vivo
in patients (20-22). iPSC generation via the forced expression of OSKM leads to increased production of
reactive oxygen species (ROS), which can cause DNA damage and senescence (23). Owing to its antioxidant
properties, vitamin C was initially added to the culture medium of mouse and human cells during
reprogramming to mitigate the effects of ROS, which can potentially hamper the efficiency and quality of
reprogramming (24).

Compared with other antioxidants, such as glutathione, N-acetylcysteine, vitamin E, and lipoic acid, vitamin C
is substantially more efficient at enhancing the generation of mouse ESCs and iPSCs from mouse or human
fibroblasts (25). Vitamin C can facilitate reprogramming by increasing histone demethylation, and histone
demethylases are essential for expressing the ESC master transcription factor Nanog (26). Other studies have
shown that vitamin C increases the embryonic stem cell population in humans and leads to DNA demethylation
at genomic loci known to undergo widespread loss of methylation during the reprogramming of somatic cells
into iPSCs (27).
Analysis of gene networks using Pathway Studio (28, 29) and protein-protein interaction maps shows the
formation of a loop feedback network, as shown in Figure 1. The top 25 most highly significant (p<E-9) cell
processes regulated by the gene set are shown in Table 5. A complete list of processes regulated by the subset of
genes is available in the supplementary material. The gene network shows the role of TP53, which inhibits the
key Yamanaka factors Oct4, KLF2, Nanog, and Sox2. Metadichol is an inverse agonist or, more likely, a protean
agonist (30) of the vitamin D receptor (11). Binding to VDR leads to increased Klotho gene expression (31),
which blocks the actions of TP53, potentially clearing the way for IPSC conversion
The cell processes regulated by the genes in Figure 1 are shown in Table 5. There were over 700 significantly
changed genes, and almost all were involved in cell differentiation pluripotency maintenance, cellular
senescence, regulation of DNA methylation, fibroblast differentiation, and other vital cellular processes. And
stem cell development. These genes have more interactions among themselves than expected for a random set
of genes of the same size and degree distribution drawn from the genome. Such enrichment indicates that the
group of genes shares a significant biological connection through their involvement in cell pluripotency
processes.
Summary and conclusion
Our study shows that the forced programming of external Yamanaka factors and grafting with viruses are not
needed for reprogramming. Metadichol is a safe compound (with an LD50 of over 5000 mg/kg in rats (32-34))
and is commercially available as a supplement. The knockdown of tumor suppressor genes, such as p53,
enhances reprogramming efficiency (35, 36). Cancer cell reprogramming can safely, precisely, and effectively
reactivate silenced tumor suppressor genes, e.g., Klotho, as we have demonstrated. Additionally, vitamin C
plays a critical role in cell reprogramming by mitigating ROS effects, which can increase the expression of
Yamanaka factors. Given the effect of Metadichol on the expression of critical genes involved in the
reprogramming of somatic cells, the effects of Metadichol should be assessed in patients to determine if the in
vitro results translate to in vivo effects. Previous studies (37, 38) treating various skin diseases with Metadichol
showed skin renewal, including wound healing and rejuvenation. Such a strategy to harness the body’s cells,
thus avoiding the incompatibility issues associated with external donor cell sources, would likely be effective in
mitigating many human diseases, including cardiovascular and neurological disorders.
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Gene network of reprogramming factors

Table 5 Cell processes regulated by the input genes
Cell processes
regulated by
entities enriched
in input genes
Tot al # o f
Neighbors
Overlap
Percent
Overlap
Overlapping Entities
p-value
Protein regulators
of messenger RNA
synthesis
615
9
1
POU5F1,KLF4,NANOG,MYC,KL,KLF2,VDR,SOX2,TP53
4.2643E-13
Protein regulators
of cancer stem
cell population
203
7
3
POU5F1,KLF4,NANOG,MYC,VDR,SOX2,TP53
3.383E-12
Protein regulators
of cancer stem
cell differentiation
100
6
5
POU5F1,KLF4,NANOG,MYC,SOX2,TP53
7.7156E-12
Protein regulators
of cell renewal
938
9
0
POU5F1,KLF4,NANOG,MYC,KL,KLF2,VDR,SOX2,TP53
1.9437E-11
Protein regulators
of
dedifferentiation
969
9
0
POU5F1,KLF4,NANOG,MYC,KL,KLF2,VDR,SOX2,TP53
2.6076E-11
Protein regulators
of stem cell
development
529
8
1
POU5F1,KLF4,NANOG,MYC,KL,KLF2,SOX2,TP53
2.6336E-11
Protein regulators
of cell
transdifferentiation
976
9
0
POU5F1,KLF4,NANOG,MYC,KL,KLF2,VDR,SOX2,TP53
2.7829E-11
Protein regulators
of cancer stem
cell development
48
5
10
POU5F1,NANOG,MYC,SOX2,TP53
4.0368E-11
Protein regulators
of cancer stem
cell renewal
144
6
4
POU5F1,KLF4,NANOG,MYC,SOX2,TP53
7.156E-11
Protein regulators
of cancer stem
cell proliferation
147
6
4
POU5F1,KLF4,NANOG,MYC,SOX2,TP53
8.1118E-11
Protein regulators
of cell maturation
1156
9
0
POU5F1,KLF4,NANOG,MYC,KL,KLF2,VDR,SOX2,TP53
1.284E-10
Protein regulators
of endothelial cell
homeostasis
64
5
7
KLF4,NANOG,MYC,KL,KLF2
1.7908E-10
Protein regulators
of trophoblast
differentiation
375
7
1
POU5F1,KLF4,NANOG,MYC,VDR,SOX2,TP53
2.5516E-10
Protein regulators
of cancer stem
cell phenotype
383
7
1
POU5F1,KLF4,NANOG,MYC,KLF2,SOX2,TP53
2.9585E-10
Protein regulators
of cell plasticity
385
7
1
POU5F1,KLF4,NANOG,MYC,KL,SOX2,TP53
3.0685E-10
Protein regulators
of cell
dedifferentiation
389
7
1
POU5F1,KLF4,NANOG,MYC,KL,SOX2,TP53
3.2989E-10
Protein regulators
of gene repression
737
8
1
POU5F1,KLF4,NANOG,MYC,KLF2,VDR,SOX2,TP53
3.7447E-10

Protein regulators
of stem cell
function
764
8
1
POU5F1,KLF4,NANOG,MYC,KL,VDR,SOX2,TP53
4.9918E-10
Protein regulators
of stem cell
maintenance
779
8
1
POU5F1,KLF4,NANOG,MYC,KL,KLF2,SOX2,TP53
5.8304E-10
Protein regulators
of stem cell fate
determination
81
5
6
POU5F1,KLF4,NANOG,MYC,SOX2
5.9944E-10
Protein regulators
of blastocyst
formation
208
6
2
POU5F1,KLF4,NANOG,KL,SOX2,TP53
6.6392E-10
Protein regulators
of stem cell fate
473
7
1
POU5F1,KLF4,NANOG,MYC,VDR,SOX2,TP53
1.2955E-09
Protein regulators
of nuclear
reprogramming
497
7
1
POU5F1,KLF4,NANOG,MYC,KLF2,SOX2,TP53
1.8304E-09
Protein regulators
of cellular
senescence
1563
9
0
POU5F1,KLF4,NANOG,MYC,KL,KLF2,VDR,SOX2,TP53
1.9549E-09
Protein regulators
of stem cell fate
commitment
262
6
2
POU5F1,KLF4,NANOG,MYC,SOX2,TP53
2.6661E-09
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Symbol
Full Name
KL
klotho
KLF2
Kruppel like factor 2
KLF4
Kruppel like factor 4
MYC
MYC proto-oncogene, bHLH transcription factor
NANOG
Nanog homeobox
POU5F1 ( OCT 4)
POU class 5 homeobox 1
SOX2
SRY-box transcription factor 2
TP53
tumor protein p53
VDR
vitamin D receptor
IPSC
Induced Pluripotent Stem Cell

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RAW Q-RT-PCR- data and Western Blots

Evaluation of gene regulation in Human Cardiac Fibroblasts
cells treated with Metadichol cells treated with Metadichol®

NANOG Gene Expression
Sample
Actin
NaNoG
Delta ct
Delta Delta ct
Fold change 2^DDct
Control
20.85
22.50
1.66
0.00
1.00
1pg
20.53
20.93
0.40
-1.26
2.39
100pg
21.34
20.44
-0.89
-2.55
5.86
1ng
20.62
21.79
1.17
-0.48
1.40
100ng
20.75
24.58
3.84
2.18
0.22

OCT 4 Gene expression
Sample
Actin
Oct-04
Delta ct
Delta Delta ct
Fold change
2^DDct
Control
20.85
26.94
6.09
0.00
1.00
1pg
20.53
28.49
7.95
1.86
0.28
100pg
21.34
24.06
2.72
-3.37
10.36
1ng
20.62
25.97
5.35
-0.74
1.67
100ng
20.75
26.90
6.15
0.06
0.96

Sox2 Gene Expression
Sample
Actin
SOX2
Delta ct
Delta Delta
ct
Fold
change
Control
20.85
20.49
-0.36
0.00
1.00
1pg
20.53
20.41
-0.13
0.23
0.85
100pg
21.34
17.63
-3.71
-3.35
10.21
1ng
20.62
20.20
-0.42
-0.05
1.04
100ng
20.75
19.03
-1.71
-1.35
2.55

KLotho gene Expression.
Sample
Actin
Klotho
Delta ct
Delta
Delta ct
Fold
change
Control
20.85
26.61
5.76
0.00
1.00
1pg
20.53
24.80
4.26
-1.50
2.82
100pg
21.34
23.76
2.42
-3.34
10.13
1ng
20.62
28.30
7.68
1.92
0.26
100ng
20.75
28.90
8.15
2.39
0.19

Relative expression of KLF 2 gene in HCF cells
Sample
Actin
KLF 2
Delta ct
Delta Delta ct
Fold change
2^DDct
Control
20.08
28.654
8.58
0.000
1.000
1pg
19.62
27.860
8.24
-0.339
1.265
100pg
19.42
26.694
7.27
-1.304
2.468
1ng
19.19
25.165
5.98
-2.597
6.052
100ng
19.23
23.921
4.69
-3.888
14.810

Evaluation of gene regulation in A-549 Cancer cell line treated with Metadichol® !

Relative expression of SOX 2 gene A-549 Cell line
Relative expression of KLF 4 gene:
Concentration
Beta
Actin
SOX 2
Delta cq
Delta (Delta)
cq
2^-
DD(Cq)
Control
21.68
20.29
-1.39
0.00
1.00
1 pg
21.19
20.88
-0.31
1.09
0.47
100 pg
21.39
20.31
-1.08
0.31
0.80
1 ng
21.82
19.10
-2.72
-1.33
2.51
100 ng
21.63
20.70
-0.93
0.46
0.73
Concentration
Beta
Actin
KLF 4
Delta cq
Delta (Delta) cq
2^-DD(Cq)
Control
21.68
17.12
-4.56
0.00
1.00
1 pg
21.19
16.56
-4.63
-0.07
1.05
100 pg
21.39
18.03
-3.36
1.20
0.44
1 ng
21.82
15.46
-6.36
-1.81
3.51
100 ng
21.63
15.42
-5.21
-0.66
1.58
1.00
0.47
0.80
2.51
0.73
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control 1 pg 100 pg 1 ng 100 ng
Fold change
Expression of SOX 2 gene in A549
cells
Treatment

Relative expression of OCT 4 gene in A-549 cell lines
Concentration
Beta
Actin
OCT 4
Delta cq
Delta (Delta) cq
2^-DD(Cq)
Control
21.68
20.20
-1.48
0.00
1.00
1 pg
21.19
21.60
0.41
1.89
0.27
100 pg
21.39
20.03
-1.36
0.12
0.92
1 ng
21.82
18.34
-3.48
-2.00
4.01
100 ng
21.63
19.83
-1.80
-0.32
1.25
1.00 1.05
0.44
3.51
1.58
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
Control 1 pg 100 pg 1 ng 100 ng
Fold change
Expression of KLF 4 gene in A549 cells
Treatment
1.00
0.27
0.92
4.01
1.25
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Control 1 pg 100 pg 1 ng 100 ng
Expression of OCT gene in A549 cells
Treatment

Relative expression of NANOG gene:
Concentration
Beta
Actin
NANOG
Delta cq
Delta (Delta)
cq
2^-
DD(Cq)
Control
21.68
15.58
-6.11
0.00
1.000
1 pg
21.19
15.37
-5.83
0.28
0.82
100 pg
21.39
15.58
-5.81
0.30
0.82
1 ng
21.82
15.39
-6.44
-0.33
1.26
100 ng
21.63
15.83
-5.81
0.30
0.81
Concentration
Beta Actin
KLOTHO
Delta cq
Delta
(Delta) cq
2^-DD(Cq)
Control
22.46
18.31
-4.15
0.00
1.00
1 pg
22.98
20.25
-2.73
1.43
0.37
100 pg
23.19
19.77
-3.42
0.73
0.60
1 ng
22.45
17.30
-5.15
-0.99
1.99
100 ng
22.87
18.47
-4.40
-0.24
1.18
1.000
0.82 0.82
1.26
0.81
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
Control 1 pg 100 pg 1 ng 100 ng
Fold change
Expression of NANOG gene in A549 cells
Treatment

1.00
0.37
0.60
1.99
1.18
0.00
0.50
1.00
1.50
2.00
2.50
Control 1 pg 100 pg 1 ng 100 ng
Fold change
Expression of Klotho gene in A549 cells
Treatment

Evaluation of gene regulation in COLO 205 Cancer cell lines treated with Metadichol®
COLO-205 cell lines
Relative expression of SOX 2 gene:
Concentration
Beta Actin
SOX 2
Delta cq
Delta (Delta) cq
2^-DD(Cq)
Control
22.43
16.71
-5.72
0.00
1.00
1 pg
23.173
16.34
-6.83
-1.11
2.16
100 pg
23.26
16.40
-6.86
-1.14
2.21
1 ng
23.51
16.62
-6.89
-1.17
2.25
100 ng
23.705
17.22
-6.49
-0.77
1.71
1.00
2.16 2.21 2.25
1.71
0.00
0.50
1.00
1.50
2.00
2.50
Control 1 pg 100 pg 1 ng 100 ng
Fold change
Expression of SOX 2 gene in COLO205
cells
Treatment

Relative expression of KLF 4 gene
Relative expression of OCT 4 gene
Concentration
Beta
Actin
KLF 4
Delta cq
Delta (Delta) cq
2^-DD(Cq)
Control
22.43
25.17
2.74
0.00
1.00
1 pg
23.17
24.56
1.39
-1.35
2.55
100 pg
23.26
24.42
1.16
-1.58
2.98
1 ng
23.51
22.53
-0.98
-3.72
13.17
100 ng
23.71
23.68
-0.02
-2.76
6.77
Expression of OCT 4 gene in COLO 205 cell lines

Relative expression of NANOG gene:
Concentration
Beta Actin
OCT 4
Delta cq
Delta (Delta) cq
2^-DD(Cq)
Control
22.43
18.61
-3.82
0.00
1.00
1 pg
23.173
20.64
-2.54
1.28
0.41
100 pg
23.26
19.62
-3.64
0.18
0.88
1 ng
23.51
18.85
-4.66
-0.84
1.79
100 ng
23.705
20.20
-3.51
0.31
0.81
Concentratio
n
Beta
Actin
NANOG
Delta cq
Delta (Delta) cq
2^-DD(Cq)
Control
22.46
16.50
-5.97
0.00
1.000
1 pg
22.98
16.22
-6.76
-0.80
1.74
100 pg
23.19
17.78
-5.41
0.56
0.68
1 ng
22.45
17.78
-4.67
1.30
0.41
100 ng
22.87
16.65
-6.22
-0.25
1.19
1.000
1.74
0.68
0.41
1.19
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
1.800
2.000
Control 1 pg 100 pg 1 ng 100 ng
Fold change
Expression of NANOG gene in COLO 205 cells
Treatment

Relative expression of KLOTHO gene Colo-205 cell line
Concentration
Beta Actin
KLOTHO
Delta cq
Delta (Delta) cq
2^-DD(Cq)
Control
22.68
11.03
-11.65
0.00
1.00
1 pg
23.58
11.01
-12.57
-0.92
1.89
100 pg
23.27
11.34
-11.93
-0.28
1.22
1 ng
23.46
10.44
-13.02
-1.37
2.58
100 ng
22.7
11.27
-11.43
0.22
0.86
1.00
1.89
1.22
2.58
0.86
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control 1 pg 100 pg 1 ng 100 ng
Fold change
Expression of KLOTHO gene in Colo205 cells
Treatment

Evaluation of gene regulation in Triple negative Primary Breast
Cancer (HCAF-TNPBC) cells treated with Metadichol®

Expression c-MYC in HCAF-TNPBC Cells
Concentration
Beta Actin
c-MYC
Delta Cq
Delta (Delta) Cq
2^-DD(Cq)
Control
21.68
24.56
2.88
2.88
1.00
1pg/mL
22.04
23.17
1.13
1.13
3.36
100pg/mL
21.845
22.59
0.74
0.74
4.39
1ng/mL
22.475
23.34
0.86
0.86
4.04
100ng/mL
22.31
22.40
0.09
0.09
6.91

Concentration
B-Actin
SOX2
Delta Cq
Delta (Delta) Cq
2^-DD(Cq)
Control
21.32
24.32
3.00
4.44
1.00
1pg/mL
21.68
25.49
3.81
5.25
0.57
100pg/mL
21.49
25.18
3.70
5.14
0.62
1ng/mL
22.12
23.82
1.71
3.15
2.45
100ng/mL
21.95
24.73
2.78
4.22
1.16

Concentration
B-Actin
OCT-4
Delta Cq
Delta (Delta)
Cq
2^-DD(Cq)
Control
21.68
25.48
3.80
2.83
1.00
1pg/mL
22.04
23.65
1.61
0.64
4.56
100pg/mL
21.85
23.34
1.49
0.53
4.94
1ng/mL
22.48
21.98
-0.50
-1.46
19.63
100ng/mL
22.31
22.89
0.58
-0.39
9.32

Concentration
B-Actin
KLF-4
Delta Cq
Delta (Delta)
Cq
2^-DD(Cq)
Control
21.68
26.07
4.39
4.39
1.00
1pg/mL
22.04
24.68
2.64
2.64
3.36
100pg/mL
21.85
24.10
2.26
2.26
4.39
1ng/mL
22.48
23.85
1.38
1.38
8.07
100ng/mL
22.31
23.91
1.60
1.60
6.91

Table 5 Cell processes regulated by the input genes