Research progress by dr. Yuyun Rindiastuti

Research Progress

Yuyun

MSC synchronization (24h) 300.000cell/ml

in 60mm dish

Treatment under hypoxyc condition

(2.2%oxygen, 5%CO2)

Treatment under normoxyc condition

(20% oxygen)

24h incubation

48h incubation

72h incubation 72h incubation

48h incubation

24h incubationMSC proliferation &

viability, MSC characterization,

pluripotency marker

Material & Method

Cell culture

Four independent MSC MSC 1

MSC 2

MSC 3

MSC 4

24h synchronizati0n

24h incubation

48h incubation

72h incubation

24h incubation

48h incubation

72h incubation

Normoxyc, 20% O2

Hypoxic 2.2%O2Media: DMEM-

high glucose, FBS 10%, NEAA 0.1mM, b-

mercaptoethanol 0.01mM, bFGF

5ng/ml, EGF 5ng/ml

Analysis Method

Cell number & viability

Trypan blue stained cell counted by automatic cell counting machine

(invitrogen)

MSC marker CD 44, CD 106 PCR

Energy metabolism gene related glycolisis

mechanism

HIF 1 A, HIF 1B, GLUT-1 PCR

Pluripotency marker HIF 2A, Oct-4, Sox-2, Rex-1 PCR

RESULT

Cell proliferation & viability

synchroniza-tion

Normoxic 24h

Hypoxic 24h Normoxic 48h


Hypoxic 72h0

500,000

1,000,000

1,500,000

2,000,000

2,500,000

3,000,000

3,500,000

4,000,000

4,500,000

5,000,000

Graph 1. Cell number profile

Cell

num

ber

synchroniza-tion

Normoxic 24h



Hypoxic 72h86%

88%

90%

92%

94%

96%

98%

Graph 2. Cell viability

Cell

viab

ility

Compared group Mean between group

Significancy (p)

Normoxyc & hypoxic viability 27.875 0.000*

Normoxyc & hypoxic proliferation rate

1.093 0.000*

Anova statistical analysis of cell proliferation rate & viability expression, significant p<0.05, *statistically significant

ANOVA test comparing differences of cell proliferation rate & viability between group

MSC surface marker expression

HIF 1A & GLUT 1 expression

Normoxic group Hypoxic group

Densitometric analysis of HIF 1A expression (ImageJ)

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

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 treatment, 24hours

1.044 0.02*

MSC, normoxyc &hypoxic treatment, 48hours

0.811 0.02*


0.000 0.991

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 treatment, 24hours

2.617 0.031*


3.234 0.046*


2.403 0.040*

Anova statistical analysis of GLUT-1 expression, significant p<0.05, *statistically significant

HIF 2A Expression



ANOVA test comparing differences of HIF 2A expression between group (based on densitometric

analysis of HIF 2A expression by ImageJ)


0.765 0.040*


1.887 0.047*


2.698 0.047*

Anova statistical analysis of HIF 2A expression, significant p<0.05, *statistically significant

Pluripotency gene expression



0.347 0.029*


0.492 0.015*


0.603 0.005*

Anova statistical analysis of Oct4 expression, significant p<0.05, *statistically significant

ANOVA test comparing differences of Oct 4 expression between group (based on densitometric

analysis of Oct4 expression by ImageJ)

ANOVA test comparing differences of Rex 1 expression between group (based on

densitometric analysis of Rex 1 expression by ImageJ)


1.779 0.049*


2.881 0.029*


2.117 0.006*

Anova statistical analysis of Rex1 expression, significant p<0.05, *statistically significant

RESEARCH PLAN

NORMOXIA

HYPOXIA

acute chronic

HIF 1AProteosomal degradation

FIH

HIF 1A

Unstable HIF 1A lack of binding to p300/CBP

HIF 1A

P3oo/CBP

HRE

HIF-target gene (glycolysis, cell

survival)

HIF 1B

HIF 2AHIF 1B

HIF target geneCell cycle

progression, pluripotency,

protooncogene

HIF 3A

MTOR/MAPK

HIF 1A degradation

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

Thank you

Research progress by dr. Yuyun Rindiastuti

Health & Medicine

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