Screening Methods for

Study Anti-Adipogenesis
Using Cellular Models
Ms. Ploychat Chamnanthongpiwan
PST Ph.D. student
6473002033

1
 Outlines

2
 Important of Adipocyte may be pathogenic through the
adverse consequences of excessive fat
Study mass through deleterious endocrinologic
and immunologic activity
Adipose hypertrophy and visceral adipose
tissue accumulation are associated with
the most common metabolic disease
found in clinical practice including type-2
Diabetes mellitus, Hypertension,
Dyslipidemia, Atherosclerosis and
Cellulite

3
 What is adipogenesis?

4
 What is adipogenesis?
Adipogenesis is a muti-step process
involving a cascade of transcription factors and
cell cycle proteins regulating gene expression
and leading to adipocyte development.
Several positive and negative regulators
of this network have been elucidated.

Preadipocytes Adipocytes

5 (Al-Sayegh, 2023)
(Ghaben, 2019)
Overview of molecular mechanisms of adipogenesis

activated

Adipocyte marker

α-smooth muscle actin (αSMA)
platelet-derived growth factor receptors
(PDGFRα and/or PDGFRβ)
Bone morphogenetic protein (BMP)
6 peroxisome proliferator-activated receptor-γ (PPARγ)
enhancer-binding protein α and β (C/EBPα and C/EBPβ)
lipases adipose triglyceride lipase (ATGL)
Lipoprotein lipase (LPL)
zinc finger protein 423 (ZFR423)
7 (Klusoczki, 2019; Nagy, 2019; Toth, 2020; Ziqubu, 2023)
 Screening Methods ?

8
 Modeling of Anti-Adipogenesis
2D and 3D In Vitro Models In Vivo Animal Models
Cell Line Models Wagyu cattle
3T3-L1 Mouse
3T3-F442A
Ob17
Mice
OP9 Rat
C3H10T1/2 Rabbit
Porcine preadipocytes
Adipose-derived stem cells
characterized by an extensive lipid
deposition in skeletal muscles that is
Adipose-derived Stem Cells Models
often seen in several human pathologies
embryonic stem cells
like myopathies may be considered as
mesenchymal stem cells
acceptable models for studying the
Induced pluripotent stem cells
mechanisms behind adipogenesis.

9 (Ruiz-Ojeda, 2016)
Cellular models
Murine cell lines

used for studies

useful in understanding
related to cellular
the molecular events
determination of the
responsible for
separate cell fates,
preadipocyte conversion
including adipocytes
Unipotent cell lines Pluripotent cell lines

Swiss 3T3 Swiss 3T3 C3H/HeM C3H 10T1/2 C57BL/6J C3H C57BL/6J/C3H Pregnant Inner cell mass
17-19 days 17-19 days Adipose Embryonic Genetically 14-17 days Newborn 13.5-15.5 During
old embryos old embryos tissue fibroblast with obese fat embryo calavarial embryo day blastocyst
female adult 5-azacytidine pads bone marrow
3T3-L1 cells 3T3-F442A AP-18 cells TA1 cells Ob1771 C3H10T1/2 OP9 cells MEF cells mESC cells
cells cells cells
(Kim ,2020; Zhao, 2019; Hemmeryckx, 2019; Khalilpourfarshbafi, 2019; Shinohara,1992; Ninomiya-Tsuji, 1993; Chen, 2010; Abderrahim-Ferkoune, 2004;
10
Schwind, 2017; Hussain, 2020; Wolins, 2006; Rosen, 2006; Ota, 2017; Yusuf, 2013; Hou, 2020))
Summary of adipose cell lines discussed
Cell Lines Characteristics References
- Non-embryonic.
Ob1771 Abderrahim-Ferkoune, 2004.
- Low fatty acid biosynthesis
- Suitable for high-throughput studies.
- 72-hour rapid adipogenic differentiation (fast)
OP9 - Confluent following many passages Boyer, 2015; Wolins, 2006.
- Long culture times appropriate for high-throughput
screening
Porcine
- Resembles more human preadipocytes.
preadipocyt
- Suitable for the study of metabolic hormones.
es
Adipose-
derived
stem - Suitable for adipogenic commitment studies.
cells
(ADSCs)
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Summary of adipose cell lines discussed
Cell Lines Characteristics References
- Most frequently used preadipocyte model.
- Relatively homogenous.
- simpler and less expensive to use than freshly Green, 1974; Green, 1976; Kim,
3T3-L1 isolated cells 2020; Matsuo , 2015; Vishwanath,
- Many passages are standing. 2013; Zebisch, 2012; Zhao, 2019.
- Homogenous response to treatments and
experiments

- beyond 3T3-L1 cells commitment to advanced Green, 1976; Hemmeryckx, 2019;

3T3-F442A
adipocyte differentiation. Khalilpourfarshbafi, 2019; Wu, 2012.

- Fibroblast-like stem cells.

C3H10T1/2 Hussain, 2020; Schwind, 2017
- Suitable for adipogenic commitment studies.

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Adipogenic applications of murine in vitro cell line model
Differentiation
Cell Model Adipogenic agents Reported applications References
time
AP-18 Combination of DEX and 7-21 days Potential in identification of mechanism for Chen, 2010
IBMX or Insulin subcutaneous adipocytes biology
Ob1771 Insulin and T3 6–8 days Examination of different molecules in Abderrahim-
differentiation process of adipogenesis Ferkoune,200
4
C3H10T1/2 IBMX, DEX, Insulin, and 12 days • Screening natural compounds and crude extract for Schwind,
Troglitazone/ anti-adipogenic effects 2017;
Rosiglitazone • Examination of the role and function of adipocyte Hussain,
related proteins in adipogenesis 2020
• Estimation of the regulatory effects of non-coding RNA
in adipogenic differentiation
OP9 Serum replacement 2–3 days • Identification of key regulators in adipocyte related Wolins, 2006
method (SRM), Insulin disease conditions
oleate method (IOM) and • Screening compounds on early and late
Adipogenic cocktail differentiation of adipogenesis
method (ACM) • Examination of natural crude extracts for
antiadipogenic effects
• Used in high-throughput RNA screening and
techniques

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Adipogenic applications of murine in vitro cell line model
Differentiation
Cell Model Adipogenic agents Reported applications References
time
3T3-L1 Dexamethasone (DEX), 7–10 days • Understanding role of adipocyte-related proteins and Kim, 2020;
3-isobutyl-1- genes Zhao,2019
methylxanthine (IBMX) • Used in co-culture and three-dimensional culture
and Insulin system for adipose tissue
• Screening anti-adipogenic compounds, antiadipogenic
peptides, adipogenic agents in food products and anti-
adipogenic crude extracts
3T3-F442A Insulin, Fetal bovine 10-12 days • Examination of adipogenic agents in differentiation Hemmeryckx,
serum (FBS), processes 2019;
Triiodothyronine (T3), • Screening anti-adipogenic compounds, effective Khalilpourfars
IBMX and DEX adipogenic peptides, adipogenesis transcriptional hbafi, 2019
factors and anti-adipogenic crude extracts
TA1 DEX, Insulin and 6–10 days • Understanding role and function of adipocyte related (Shinohara,
Indomethacin proteins 1992;
• Screening effective agents in adipose differentiation Ninomiya-
process Tsuji, 1993
• Estimating pre-existing and new genes involved in
adipogenesis
• Used in identification of early adipogenic markers

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Adipogenic applications of murine in vitro cell line model
Differentiation
Cell Model Adipogenic agents Reported applications References
time
Mouse Retinoic acid (RA), 21 days • Characterization of pre-existing genes and new Rosen, 2006;
Embryonic Insulin, T3 and adipogenic regulatory genes Ota, 2017
Stem cells Rosiglitazone • Used in advanced and high-throughput techniques
(mESCs) • Identification of genetic and epigenetic mechanisms
involved in adipogenesis
• Potential in exploring developmental fate of
adipocytes origin and screening compounds on
differentiation of adipogenesis
Mouse Insulin, DEX, IBMX, 14 days • MEFs from genetically modified or knockout mice used Yusuf , 2013;
Embryonic Troglitazone and FBS for study the effects of genes in adipogenesis Hou, 2020
Fibroblasts • Evaluation of the effects of proteins or genes in
(MEFs) adipogenesis
• Screening anti-adipogenic compounds and
antiadipogenic crude extracts

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 Why 3T3-L1 ?

01 They are abundant supply of homogeneous cells through culture.

good model to screen compounds for their potential antilipolytic effects.
(Pereira-Fernandes et al., 2014)

02 They have adherent properties.

suitable models to study molecular mechanisms and transcription factors.

(Poulos et al., 2010)

03 They provide a monolayer culture of newly differentiated fat cells.

ideal for the study of long-term regulation of adipocyte functions.
(Adler-Wailes et al., 2010)

04 They are significantly lower-costs than other mature adipocytes cell line models.

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 3T3-L1 Cell line

transformed

Swiss 3T3 mouse embryo 3T3-L1 cells Adipocyte-like cells

17-19 days old (Pre-adipocyte) spontaneous lipid
accumulation

17 (Green, 1979; Kuri-Harcuch, 2019; Novikoff, 1980; Xiu, 2017)

 Differentiation process of 3T3-L1

DMEM+FBS+Antibiotic
humidified
atmosphere 37 ͦ C
5% CO₂

3T3-L1 cell line Basal media Incubate

Day 0 Day 2 Day 6 Day 7-10

IBMX + DEX + Insulin DMEM + FBS + Insulin DMEM 3T3-L1 cells

Induction media Standard Growth Un-supplemental Adipocyte-like

media growth media cells
Dexamethasone : DEX
Dulbecco's modified Eagle's medium : DMEM
fetal bovine serum: FBS
18 (Kim, 2020; Zhao., 2019; M.S. Vohra, 2020)
3-isobutyl-1-methylxanthine :IBMX
 Evaluating Adipogenesis
The morphological appearance
01 of lipid-laden fat cells
determined by light microscopy and lipid
staining

02 expression of molecular
markers of adipocytes.
Assaying lipid accumulation alone may
not reveal the extent of adipocyte
differentiation

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 In vitro cell viability of the active compound
Day 0 Day 5 Day 7 Day 9 Day 11
IBMX + DEX + Insulin DMEM + FBS + Supplement

Induction media Supplemental Supplemental Supplemental Supplemental

growth media growth media growth media growth media

Oil Red O staining

Day 7-10
3T3-L1 cells

Dexamethasone : DEX
Dulbecco's modified Eagle's medium : DMEM
fetal bovine serum: FBS
20 (Kim, 2020; Zhao., 2019; M.S. Vohra, 2020)
3-isobutyl-1-methylxanthine :IBMX
 Oil Red O staining Method

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Oil red O staining and morphological changes in 3T3-L1

3T3-L1 cells at 0, 2, 4, and 8 days after adipogenic stimulation.

Bars 50 μm
22 (Nobusue et al., 2008)
 Previous
study

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 Previous
study

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 Research Topic Chemical Propose mechanism Effects References

Butylated Hydroxyanisole Isomers 2-tertbutyl-4- Regulated transcriptional and Induced the differentiation of Sunl, 2019
Induce Distinct Adipogenesis in 3T3-L1 hydroxyanisole (3-BHA) protein level of adipogenic bio adipocytes and increase cellular
Cells. markers upstream lipid accumulation
Evaluation of a Screening System for Bisphenol A PPARɣ activator Induced the differentiation of Pereira-Fernandes et al, 2013
Obesogenic Compounds: Screening of adipocytes
Endocrine Disrupting Compounds and
Paraben PPARɣ activator Induced the differentiation of
Evaluation of the PPAR Dependency of
adipocytes
the Effect.
Phthalates PPARɣ activator Induced the differentiation of
adipocytes
Tonalide Act via non-PPARɣ mediated Induced the differentiation of
adipocytes
Bisphenol A Diglycidyl Ether Induces Bisphenol A Propose to act through a Induce adipogenesis Chamorro-Garcia, 2012
Adipogenic Differentiation of Multipotent mechanism that is downstream
Stromal Stem Cells Through a of/parallel to PPARɣ
Peroxisome Proliferator-Activated
Receptor Gamma-Independent
Mechanism.
Effects of Crude Oil/Dispersant Mixture Bisphenol A Propose to act through a Induce adipogenesis Chamorro-Garcia, 2012
and Dispersant Components on mechanism that is downstream
PPARgamma Activity in Vitro and in of/parallel to PPARɣ
Vivo: Identification of Dioctyl Sodium
Sulfosuccinate (DOSS; CAS #577-11-7)
as a Probable Obesogen.
Substitute for Bisphenol A in Terms of Bisphenol S Targets the PGC1α and the ERRɣ Increase cellular lipid Helies-Toussaint, 2014
Metabolic Function? Vitro Study. genes accumulation, increase glucose
uptake and increase leptin
production

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 Research Topic Chemical Propose mechanism Effects References

Imidacloprid, a Neonicotinoid Insecticide, Imidacloprid Purpose to be mediated via the Increase adipocyte Park et al., 2013; Mesnage et
Potentiates Adipogenesis in 3T3-L1 pregnane X receptor differentiation and lipogenesis al.,2018
Adipocytes.; Evaluation of Neonicotinoid
Insecticides for Oestrogenic,
Thyroidogenic and Adipogenic Activity
Reveals Imida cloprid Causes Lipid
Accumulation.
Activation of PPARalpha and Monoethylhexyl PPARɣ and PPARα activator Increase adipocyte Hurst and Waxman, 2003
PPARgamma by Environmental Phththalate differentiation and insulin
Phthalate Monoesters. sensitization
Quizalofop-P-Ethyl Induces Quizalofop-p-ethyl PPARɣ activator Induce lipid accumulation Biserni et al., 2019
Adipogenesis in 3T3-L1 Adipocytes.
The Commonly Used Nonionic Span 80 Transactivates PXRα Promotes adipogenesis Bowers et al., 2016
Surfactant Span 80 has RXRalpha
Transactivation Activity, Which Likely
Increases the Obesogenic Potential of
Oil Dispersants and Food Emulsifiers.
Endocrine-Disrupting Organotin Tributyltin PPARɣ and PXRα agonist Induces adipogenesis, increase Grun et al., 2006; Inadera and
Compounds are Potent Inducers of triglyceride storage and increase Shimomura, 2005
Adipogenesis in Vertebrates; the expression of adipogenic
Environmental Chemical Tributyltin marker genes
Augments Adipocyte Differentiation.
Organotin Compounds Promote Adipocyte Dibutyltin (DBT) PPARɣ and PXRα activator Stimulate adipocyte differentiation Kanayama et al., 2005
Differentiation as Agonists of the Peroxisome and increase the expression of
Proliferator-Activated Receptor adipocyte marker gene
Gamma/Retinoid X Receptor Pathway.
Effects of Crude Oil/Dispersant Mixture and Dioctyl Sodium PPARɣ activator Induce adipogenesis and increases Temkin et al., 2016
Dispersant Components on PPARgamma Sulfosuccinate cellular lipid accumulation
Activity in Vitro and in Vivo: Identification of
Dioctyl Sodium Sulfosuccinate (DOSS; CAS
27
#577-11-7) as a Probable Obesogen.
Future Plan
Anti-Adipogenesis for Anti-cellulite Activity of Caesalpinia sappan L. extract in
vitro study and Anti-Cellulite Emulgel form

In Vitro Evaluation of Adipogenesis

- Cytotoxic effect of C. sappan ethanolic extract
Cell Culture
• HaCaT cell line
• 3T3-L1 cell line
- Cytotoxicity Determination—MTT Assay
- 3T3-L1 Differentiation Assay—Oil Red O
- Quantification of the Triglyceride Content

Formulation as anti-cellulite

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Acknowledgement

Asst.Prof. Dusadee Charnvanich, Ph.D.

Dr. Sakan Warinhomhoun, Ph.D.
Faculty of Pharmaceutical Sciences,
School of Medicine, Walilak University
Chulalongkorn University
THANK
THANK YOU
YOU
For Your Attention
Any Questions?
Addition Information
 Work In Progress
Anti-Adipogenesis for Anti-cellulite Activity of Caesalpinia sappan L. extract in
vitro study and Anti-Cellulite Emulgel form
Aims Formulations
To determine the anti-cellulite effects of C. sappan
ethanolic extract a phytocosmetic formulation In Vitro Evaluation of Adipogenesis
containing - Cytotoxic effect of C. sappan ethanolic extract
Cell Culture
Plant materials • HaCaT cell line Doing !!!
C. sappan wood were obtained from Thai herbal drug • 3T3-L1 cell line
store of Thailand. The voucher specimens were - Cytotoxicity Determination—MTT Assay
prepared and kept at faculty of pharmaceutical - 3T3-L1 Differentiation Assay—Oil Red O
science, Chulalongkorn University - Quantification of the Triglyceride Content
- RNA Extraction and cDNA Synthesis
Plant extraction
- EtOH crude extract
- Semi-purification of crude extract
• Partition of crude extract
MAJOR MECHANISM INVOLVE IN CELLULITE

(Dupont et al., 2014)

 1 2 3

ADIPOSE OEDEMATOUS FIBROTIC

CELLULITE CELLULITE CELLULITE
Firm cellulite, orange Fluid retention, soft cellulite Hard, compact cellulite
peel effect on loose skin. often on loose skin. with orange peel effect.
3 Cellulite when standing and lying down.

Visible when standing, but not disappear

2 when lying down.

- No cellulite when lying down or standing.

1 - An orange peel texture can be seen
when the skin is pinched.

0 Dimples are visible upon pinch test

 Genetics Lack of physical activity
Overweight Hormone changes
Poor diets Dehydration

Slow metabolism Total body fat

Bad dieting Thickness and color of your skin

 Modeling Adipogenesis

2D cell culture 3D cell culture

Hydrogel with tunable

Tissue culture plastic Elastic properties Spheroid models Biomimetic tissue models

High complexity and Physical relevance

Reproducibility
2D cell culture models are simple and reproducible, Physiological relevance exhibited in 3D cell culture
they lack the complexity models.
(Ruiz-Ojeda, 2016)
3T3-L1 will be plated
in well plate Western Blotting

treated with 50 g/mL of Radioimmunoprecipitation Sodium dodecyl sulfate–polyacrylamide gel

active compound assay buffer electrophoresis will be used to separate proteinsanalyzed using the
ChemiDocTM XRS+
instrument (Bio-Rad).

4°C overnight 2 h at room temp.

blocked with 5% skim milk
washed
3 times
primary antibody secondary antibody
transferred to β-catenin, GSK-3β (1:1000),
Locked in Tris-buffered saline goat anti-rabbit IgG
polyvinylidene phospho-GSK-3β (Ser9), p44/42
containing 0.08 % Tween 20 labeled with horseradish
difluoride membranes MAPK (Erk1/2) (1:1000), phospho-
peroxidase (HRP)
p44/42 MAPK (ERK1/2)
(1:2500)
(Thr202/Tyr204) (1:1000), and
glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) (1:5000)
Determination of lipid accumulation by Oil Red O staining
Oil Red O Staining
 Cellulite
3
 Adipocyte Differentiation via Signaling Pathways

01 AKT/PKB Signaling STIMULATORY PPARɣ: Transcriptional factors : regulates

adipocyte differentiation

02 WNT Signaling INHIBITORY

Master regulator of adipocyte
differentiation with C/ERPα
03 TGF - ꞵ Signaling INHIBITORY

ADIPOCYTE : Fat Storing Cells (Lipocyte)

Adipose Tissue Differentiation form Mesenchymal stem cells

Pre-adipocytes
 Differentiation and
trans-differentiation

α-smooth muscle actin

(αSMA)
platelet-derived growth factor receptors
(PDGFRα and/or PDGFRβ)
Bone morphogenetic protein
(BMP)
peroxisome proliferator-activated receptor-γ
(PPARγ)
enhancer-binding protein α and β
(C/EBPα and C/EBPβ)
lipases adipose triglyceride lipase
(ATGL)
Lipoprotein lipase
(LPL)
zinc finger protein 423
(ZFR423)
Adipogenesis
signaling pathway
in 3T3-L1

serine/threonine protein kinase B (Akt)

Mitogen activated protein kinases (MAPKs)
cluster of differentiation 36 (CD36)
Peroxisome proliferation activated receptor γ (PPARγ)
fatty acid synthase (FAS)
acetyl-coenzyme A carboxylase (ACC)
adenosine monophosphate-activated kinase (AMPK)
insulin receptor substrate (IRS1)
Sterol regulatory element binding proteins (SREBP)
Phosphatidylinositol 3-kinase (PI3K)
Adipocyte specific genes such as FAS,
FABP4, and acetyl-coenzyme A carboxylase (ACC) (Guru et al., 2020)
 What is different between Triglycerol
synthesis and Adipogenesis?
Triglycerol synthesis and adipogenesis are two different biological processes

Triglycerol synthesis is the process by Adipogenesis, on the other hand, is the

which triglycerides are formed from
glycerol and three fatty acid
molecules. This process can occur in the
liver and adipose tissue, where fatty
acids are synthesized and then combined
with glycerol to form triglycerides.
process of forming adipocytes (fat cells)
from stem cells. It involves two phases:
determination and terminal
differentiation.

So while triglycerol synthesis is the process of forming triglycerides,

adipogenesis is the process of forming fat cells.
https://en.wikipedia.org/wiki/Triglyceride [online];
https://themedicalbiochemistrypage.org/synthesis-of-fatty-acids-triglycerides-and-phospholipids/ [online]
(Kapałczyńska, 2018)

Comparison between 2D VS 3D Adipocyte Cell Models?

Type of
2D 3D References
culture
Time of culture With in minutes to a few hours From a few hours to a few days Baker, 2012; Chen,
formation 2012;

Culture quality High performance, Worse performance and

reproducibility, reproducibility,
long-term culture, easy to difficult to interpret, cultures more Hickman, 2014
interpret, difficult to carry out
simplicity of culture
In vivo Do not mimic the natural In vivo tissues and organs are in
imitation structure of the tissue or tumour 3D form Griffith, 2006
mass
Cells Deprived of cell-cell and cell Proper interactions of cell-cell and
interactions extracellular environment cell-extracellular environment, Bissell, 2003;
Cawkill, 2007;
interactions, no in vivo-like environmental “niches” are Engler, 2006; Gilbert,
microenvironment and no created 2010; Lee, 2008
“niches”
(Kapałczyńska, 2018)

Comparison between 2D VS 3D Adipocyte Cell Models?

Type of
2D 3D References
culture
Changed morphology and way Preserved morphology and way of Kilian, 2010;
Characteristics Mahmud, 2009;
of divisions; loss of diverse divisions, diverse phenotype and Mseka, 2007;
of cells
phenotype and polarity polarity Yamada, 2007
Unlimited access to oxygen, Variable access to oxygen,
Access to
nutrients, metabolites and nutrients, metabolites and
essential Breslin, 2013
signaling molecules signaling molecules
compounds
(in contrast to in vivo) (same as in vivo)
Changes in gene expression, Expression of genes, splicing, Birgersdotter, 2005;
Molecular
mRNA splicing, topology and topology and biochemistry of cells Fuchs, 2004; Li,
mechanisms 2006
biochemistry of cells as in vivo
Cost of More expensive, more time- Aggarwal, 2009;
Cheap, commercially available
maintaining consuming, fewer commercially Krishnamurthy, 2013;
tests and the media Weiswald, 2015.
a culture available tests
 Modeling Adipogenesis

Two-Dimensional (2D) Models

Cells Lines Models
3T3-L1
3T3-F442A
Ob17
Hormonal cocktails
OP9
trans-gene induction
C3H10T1/2
extracellular matrix
Porcine preadipocytes
properties
Adipose-derived stem cells

Adipose-derived Stem Cells Models

embryonic stem cells Adipocyte
mesenchymal stem cells
Induced pluripotent stem cells

(Armani, 2010)
 References
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