Research progress by dr. Yuyun Rindiastuti
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This document summarizes a research study on the effects of hypoxic and normoxic conditions on mesenchymal stem cells (MSCs). Key findings include: 1. Cell proliferation and viability were significantly higher in hypoxic conditions compared to normoxic conditions. 2. Expression of genes related to glycolysis and energy metabolism (HIF-1A, GLUT-1) were significantly higher in hypoxic MSCs. 3. Pluripotency marker expression (Oct-4, Rex-1) was also significantly higher in hypoxic MSCs. 4. The results suggest that hypoxic conditions promote MSC proliferation, maintain stem cell properties, and induce metabolic pathways that
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Research progress by dr. Yuyun Rindiastuti
- 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