Dermama Stem Cell Research Progress

1. Research Progress
Yuyun

2. MSC synchronization
(24h) 300.000cell/ml
in 60mm dish
Treatment under Treatment under
hypoxyc condition normoxyc condition
(2.2%oxygen, 5%CO2 (20% oxygen)
)
24h incubation
24h incubation
MSC proliferation &
viability, MSC
characterization, pluri
48h incubation 48h incubation
potency marker
72h incubation 72h incubation

3. Material & Method
Cell culture
24h 48h 72h
incub incub incub
Four independent
MSC 1 ation ation ation
MSC
MSC 2
24h Normoxyc, 20% O2
Media: DMEM- synchro
high glucose, FBS nizati0n Hypoxic 2.2%O2
MSC 3
10%, NEAA
0.1mM, b-
mercaptoethanol MSC 4 24h 48h 72h
0.01mM, bFGF incub incub incub
5ng/ml, EGF ation ation ation
5ng/ml

4. Analysis Method
Trypan blue stained cell counted by
Cell number & automatic cell counting machine
viability (invitrogen)
MSC marker CD 44, CD 106 PCR
Energy metabolism
HIF 1 A, HIF 1B, GLUT-1
gene related glycolisis
PCR
mechanism
HIF 2A, Oct-4, Sox-
Pluripotency marker
2, Rex-1 PCR

5. RESULT
Cell proliferation & viability
5,000,000
4,500,000
4,000,000
3,500,000
3,000,000
Cell number
2,500,000
2,000,000
1,500,000
1,000,000
500,000
0
Graph 1. Cell number profile

6. 98%
96%
94%
92%
Cell viability
90%
88%
86%
Graph 2. Cell viability

7. ANOVA test comparing differences of cell proliferation rate &
viability between group
Compared group Mean between Significancy (p)
group
Normoxyc & hypoxic viability 27.875 0.000*
Normoxyc & hypoxic 1.093 0.000*
proliferation rate
Anova statistical analysis of cell proliferation rate & viability
expression, significant p<0.05, *statistically significant

8. MSC surface marker expression

9. HIF 1A & GLUT 1 expression
Normoxic group Hypoxic group

10. Densitometric analysis of HIF 1A
Densitometric analysis of
expression (ImageJ) N=normoxyc,
HIF 1A expression (ImageJ)
H=hypoxic

11. ANOVA test comparing differences HIF 1 A expression group (based on
densitometric analysis of HIF 1A expression by ImageJ)
Compared group Mean between group Significancy (p)
MSC, normoxyc &hypoxic 1.044 0.02*
treatment, 24hours
MSC, normoxyc &hypoxic 0.811 0.02*
treatment, 48hours
MSC, normoxyc &hypoxic 0.000 0.991
treatment, 72hours
Anova statistical analysis of HIF 1A expression, significant p<0.05, *statistically significant
ANOVA test comparing differences GLUT-1 expression group (based on
densitometric analysis of GLUT 1 expression by ImageJ)
Compared group Mean between group Significancy (p)
MSC, normoxyc &hypoxic 2.617 0.031*
treatment, 24hours
MSC, normoxyc &hypoxic 3.234 0.046*
treatment, 48hours
MSC, normoxyc &hypoxic 2.403 0.040*
treatment, 72hours
Anova statistical analysis of GLUT-1 expression, significant p<0.05, *statistically significant

12. HIF 2A Expression
Normoxic group Hypoxic group
Densitometric analysis of HIF 1A expression
(ImageJ) N=normoxyc, H=hypoxic

13. ANOVA test comparing differences of HIF 2A
expression between group (based on densitometric
analysis of HIF 2A expression by ImageJ)
Compared group Mean between group Significancy (p)
MSC, normoxyc &hypoxic 0.765 0.040*
treatment, 24hours
MSC, normoxyc &hypoxic 1.887 0.047*
treatment, 48hours
MSC, normoxyc &hypoxic 2.698 0.047*
treatment, 72hours
Anova statistical analysis of HIF 2A expression, significant p<0.05, *statistically significant

14. Pluripotency gene expression
Normoxic group Hypoxic group

15. Densitometric analysis of HIF 1A expression
(ImageJ) N=normoxyc, H=hypoxic

16. ANOVA test comparing differences of Oct 4
expression between group (based on densitometric
analysis of Oct4 expression by ImageJ)
Compared group Mean between group Significancy (p)
MSC, normoxyc 0.347 0.029*
&hypoxic treatment,
24hours
MSC, normoxyc 0.492 0.015*
&hypoxic treatment,
48hours
MSC, normoxyc 0.603 0.005*
&hypoxic treatment,
72hours
Anova statistical analysis of Oct4 expression, significant p<0.05, *statistically significant

17. ANOVA test comparing differences of Rex 1
expression between group (based on
densitometric analysis of Rex 1 expression by
ImageJ)
Compared group Mean between group Significancy (p)
MSC, normoxyc &hypoxic 1.779 0.049*
treatment, 24hours
MSC, normoxyc &hypoxic 2.881 0.029*
treatment, 48hours
MSC, normoxyc &hypoxic 2.117 0.006*
treatment, 72hours
Anova statistical analysis of Rex1 expression, significant p<0.05, *statistically significant

18. RESEARCH PLAN
MTOR/ HYPOXIA
MAPK
NORMOXIA acute chronic
HIF 3A
Proteosomal
degradation HIF 1A HIF 1A HIF 2A
degradation HIF 1B
HIF 1A
FIH
HIF 1B P3oo/CBP
HIF target gene
HIF 1A Cell cycle
progression,
HRE pluripotency,
Unstable HIF 1A lack of protooncogene
binding to p300/CBP
HIF-target gene
(glycolysis, cell
survival)

19. Plan
1. Focusing the study in cell proliferation and survival under
hypoxic condition
2. Screen out glucose transporter gene expression: GLUT 1,
GLUT 2, GLUT3, GLUT4
3. Check the expression of HIF 1B and HIF 3A
4. Confirming the cell culture in low glucose and high glucose
medium
5. Screen out the expression of MAPK/MTOR related to
energy metabolism sensing under hypoxic condition
6. Confirmation study using Real time PCR

20. Thank
you