GRDS Conferences

1. Comparative analysis of cardiovascular development
related genes in stem cells isolated from
deciduous pulp and adipose tissue
Loo Zhang Xin
(Phd candidate)
ICAET, 26-27 December 2014

2. Introduction
AMI = acute myocardial infarction

3. Cell therapy as an alternative?
• Left ventricular of the human heart contains ~5.8 x 109
cardiomyocytes.
• Upon the onset of AMI, 25% of the cardiomyocytes will be lost.
• Regeneration of the infarcted heart potentially requires:
– a billion cardiomyocytes,
– along with other supporting cells such as endothelial and smooth vascular cells
for functional integration.
• Possible few mechanisms for which these cells may influence the cardiovascular
regeneration:
– Differentiation of stem cells into cardiomyoctes,
– Fusion of stem cells with endogenous cells,
– Stem cells secretome signaling

4. List of suitable candidates
Embryonic stem cells
(ESC)
Induced pluripotetent
stem cells (IPS)
Adult stem cells
Pros • Totipotency
• Laflamme et al.
showsed that ESC were
able to generate
cardiomyocytes
and had limited death
rate in animal model.
• Can generate a
patient’s specific stem
cells.
• Ieda et al. used a
combination of cardiac
transcription factors to
direct reprogram
cardiac fibroblast into
cardiomyocytes.
• Cardiac stem cells
(CSCs) offers better
prospects.
• Bolli et al. reported
successful clinical trial
using CSCs in human
subjects with ischemic
cardiomyopathy had
been reported.
Cons • Ethical issues
 destruction of embryo
• Complicated isolation
methods
• Tendency to form
tumours
• Risk of insertional
mutagenesis is of
particular concern.
•Invasive procedures in
isolating and culturing
the cells
• Escalating production
cost due to autologous
settings.

5. Upcoming candidates

6. Methodology
• Characterized MSCs sources: human dental pulp (SHED) and human
adipose stem cells (ASC)
 Morphology, Tri-lineage differentiation and immunophenotyping
 Human Stem Cell Transcription Factors Array for initial screening
 Ingenuity Pathway Analysis
• Cardiac differentiation using defined media
 SHED and ASC were seeded onto 6-well plates at a density of 100,000 cells/cm2
.
 Cardiac media: KO-DMEM containing 0.5% penicillin/streptomycin and
1% 1X Glutamax were supplemented with 100 ng/ml human recombinant Activin A
for 24 hours followed by 10 ng/ml human recombinant BMP2 for 4 days.
 The medium was then exchanged for KO-DMEM without supplementary cytokines,
and cultures were refed every 2–3 days in a humidified incubator at 37°C and 5 %
CO2 for up to 14 days.
 Cell morphology was captured using an inverted microscope, RNA was extracted at
day 7 and day 14, immunocytochemisty at day 14.

7. ADIPOSE DENTAL
MSCs CARDIOMYOCYTES

8. Results & Discussion

9. MSCs derived from SHED
and ASC:
Fibroblastic morphology
Expressed MSCs markers
 Capable of differentiating
into osteoblasts, adipocytes
and chondroblasts

10. WHY Human stem cell
transcription factor array ?
REASON: Plenty of
experimental works
emphasized on
understanding stem cell
characteristics such as
proliferation pathways,
quality of the cells under in
vitro culture conditions &
lack of information
pertaining to stem cells at the
basal level.
Observation:
Higher expression level for
the HOX group of genes in
the ASC [Development of
obesity and body fat
distribution]
SHED expressed many
markers related to
pluripotency [More primitive

12. • Both types of cells are prone to angiogenesis as there are more
interconnected molecules involved in formation of blood vessels and
vasculogenesis.
• This provides an explanation of how stem cells are able to improve or
preserve cardiac function in an animal model through angiogenesis and not
due to cardiomyocyte differentiation.
• In hypoxic conditions, expression of HIF-α increases rapidly which in turn
activates genes that encode for pro-angiogenic growth factors, such as
VEGF, BGFG, Ang-1 and 2, PFG and PDGF-B.
• These released cytokines encourage angiogenesis and the angiogenic effects
of human multipotent stromal cell which explains the underlying
improvement of cardiac function through any individual or a combination of
mechanisms such as apoptosis inhibition, increase in survival, and
angiogenesis stimulation by activation of the PI3K-Akt pathway.

13. ASC: Assumed more of a polygonal
shape.
SHED: Elongated and irregular
There has been no reports yet of a
successful protocol using dental stem
cells to generate beating cardiomyocytes.
Prior examples utilized dental pulp
stem cells with a combination of VEGF,
BFGF and IGF-1.
In this case, spontaneous cellular
beating was also not detected and the
morphology similar to what we observed
in our experiments.
Only GATA4 and NKX 2.5 were
detected in differentiated ASC

14. ASC expressed significantly more
cardiomyocyte biomarkers compared
to SHED at both day 7 and day 14
[Could be both adipose and heart
development are mesoderm origin.
This indicates that not all sources of
MSCs are suitable for cardiac
differentiation & selection of the MSC
source for treating a disease highly
depended on its origin.
Spontaneous cellular beating, the
hallmark indicator of successful
cardiomyocyte differentiation was not
detected in our experiment
[Could be cTnI was suppressed in this
condition whereby it can only be
activated when the cells were cultured
with neonatal rat cardiomyocytes, or
when these cells were delivered in
vivo to murine models of myocardial
infarction]

15. Acknowledgement
This work is part of a research collaboration between Hygieia
Innovation and the Faculty of Dentistry, University of Malaya and is
supported by the University of Malaya, High Impact Research Grant,
Ministry of Higher Education, Malaysia.
(Grant No UM.C/HIR/MOHE/DENT/02)