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Hypoxic Mesenchymal Stem Cell

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raditio ghifiardi
raditio ghifiardi

This document discusses how growing human mesenchymal stem cells at lower, physiological oxygen concentrations of 3% instead of the standard 20% can increase their lifespan and genetic stability by reducing oxidative stress and DNA damage. Specifically, lower oxygen tension limits telomere shortening and chromosomal aberrations. It is concluded that stem cells for human therapy should be cultured under low oxygen conditions to increase their biosafety.

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Yuyun Rindiastuti
SUMMARY Expansion of human stem cells before cell therapy is typically performed at 20% O2. Growth in these pro-oxidative conditions can lead to oxidative stress and genetic instability. Culture of human mesenchymal stem cells at lower, physiological O2 concentrations significantly increases lifespan, limiting oxidative stress, DNA damage, telomere shortening and chromosomal aberrations. Growth at reduced oxygen tensions favors a natural metabolic state of increased glycolysis and reduced oxidative phosphorylation. These observations indicate that bioenergetic pathways are intertwined with the control of lifespan and decisively influence the genetic stability of human primary stem cells. We conclude that stem cells for human therapy should be grown under low oxygen conditions to increase biosafety.
INTRODUCTION hMSC Regenerative medicine Standard Physiological Expansion culture nonphysiological oxygen tension oxygen tension -Increase oxidative -Improves cell growth stress -Extend life span -Telomerase -Alters differentiation shortening process -Increase DNA -Reduce damage, chromosomal chromosomal abnormalities aberration
Oxygen levels & Oxygen Genetic exogenous oxidative sensing, HIF stability stress 1 response Cytogenetic analysis, gene expression, bioenergetics studies Culture grown in physiologycal 20% -Increase genetic instability oxygen -Decrease glycolysis -transcriptional activation in Culture grown in HIF 1A target genes standard 3% oxygen - Inhibition oxygen consumption
RESULT Growth of hMSC at 20% O2 reduces lifespan, increases oxidative stress and the rate of telomere shortening Cell growth Telomerase shortening & cell senescence
Carbonyl & MDA levels Superoxide accumulation
Growth of hMSC at 20% O2 increases DSB generation and chromosomal instability. DSB level (53BP1 marker) DNA damage & chromosomal aberration
Oxidative stress increases aneuploidy in hMSC Aneuploidy levels
Metaphase chromosomal analysis for aneuploidy confirmation
Exogenous source of ROS cause growth defects & genetic istability
Culture of hMSC at 20% O2 significantly increases oxygen consumption and decreases glycolytic metabolism Transcriptional upregulation of glycolysis genes impact mitochondrial oxygen consumption rate & extra cellular acidification rate
Higher oxygen tension promotes oxygen consumption, medium acidification drop, decrease glycolysis,
DISCUSSION Culture of -Promotes DNA damage & senescence mammalian cells at -Chromosomal aberration during long term 20% oxygen culture Importance of biosafety in cell Culture ADMSC at 3% therapy 0xygen -Increase growth rate -No evidence of spontaneous immortalization -- reduce telomere shortening rate - decrease structural chromosomal aberration
Significant upregulation of gene expression in Balance cells grown at 3% oxygen tension related to HIF bioenergetics 1A stabilization pattern HIF 1A decreases OXPHOS system by downregulating mitochondrial ATP production & oxygen consumption Autocrine & paracrine effect in cell growth & senescence Implication of gene ontology analysis (HIF 1 dependent gene expression)
MATERIAL & METHODS Cell & cell cuture conditions -Four independent human ADMSC -Cultured under 3% & 20% oxygen tension -PQ & H2O2 treatment for confirmation of exogenous oxidative stress exposure Analysis -Cell characterization by surface marker staining -Aneuploidy analysis -Telomere length quantification by Q-FISH -Chromosomal aberration -TRAP assay --DNA damage immunofluorescence staining 53BP1 -ROS detection by flowcytomwtry -Microarray gene profilling - detection of protein carbonyls and MDA -OCR &ECAR measurement
CONCLUSSION Stem cell for human therapy should be grown under low oxygen conditions to increase biosafety
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Hypoxic Mesenchymal Stem Cell

  • 2. SUMMARY Expansion of human stem cells before cell therapy is typically performed at 20% O2. Growth in these pro-oxidative conditions can lead to oxidative stress and genetic instability. Culture of human mesenchymal stem cells at lower, physiological O2 concentrations significantly increases lifespan, limiting oxidative stress, DNA damage, telomere shortening and chromosomal aberrations. Growth at reduced oxygen tensions favors a natural metabolic state of increased glycolysis and reduced oxidative phosphorylation. These observations indicate that bioenergetic pathways are intertwined with the control of lifespan and decisively influence the genetic stability of human primary stem cells. We conclude that stem cells for human therapy should be grown under low oxygen conditions to increase biosafety.
  • 3. INTRODUCTION hMSC Regenerative medicine Standard Physiological Expansion culture nonphysiological oxygen tension oxygen tension -Increase oxidative -Improves cell growth stress -Extend life span -Telomerase -Alters differentiation shortening process -Increase DNA -Reduce damage, chromosomal chromosomal abnormalities aberration
  • 4. Oxygen levels & Oxygen Genetic exogenous oxidative sensing, HIF stability stress 1 response Cytogenetic analysis, gene expression, bioenergetics studies Culture grown in physiologycal 20% -Increase genetic instability oxygen -Decrease glycolysis -transcriptional activation in Culture grown in HIF 1A target genes standard 3% oxygen - Inhibition oxygen consumption
  • 5. RESULT Growth of hMSC at 20% O2 reduces lifespan, increases oxidative stress and the rate of telomere shortening Cell growth Telomerase shortening & cell senescence
  • 6. Carbonyl & MDA levels Superoxide accumulation
  • 7. Growth of hMSC at 20% O2 increases DSB generation and chromosomal instability. DSB level (53BP1 marker) DNA damage & chromosomal aberration
  • 8. Oxidative stress increases aneuploidy in hMSC Aneuploidy levels
  • 9. Metaphase chromosomal analysis for aneuploidy confirmation
  • 10. Exogenous source of ROS cause growth defects & genetic istability
  • 11. Culture of hMSC at 20% O2 significantly increases oxygen consumption and decreases glycolytic metabolism Transcriptional upregulation of glycolysis genes impact mitochondrial oxygen consumption rate & extra cellular acidification rate
  • 12. Higher oxygen tension promotes oxygen consumption, medium acidification drop, decrease glycolysis,
  • 13. DISCUSSION Culture of -Promotes DNA damage & senescence mammalian cells at -Chromosomal aberration during long term 20% oxygen culture Importance of biosafety in cell Culture ADMSC at 3% therapy 0xygen -Increase growth rate -No evidence of spontaneous immortalization -- reduce telomere shortening rate - decrease structural chromosomal aberration
  • 14. Significant upregulation of gene expression in Balance cells grown at 3% oxygen tension related to HIF bioenergetics 1A stabilization pattern HIF 1A decreases OXPHOS system by downregulating mitochondrial ATP production & oxygen consumption Autocrine & paracrine effect in cell growth & senescence Implication of gene ontology analysis (HIF 1 dependent gene expression)
  • 15. MATERIAL & METHODS Cell & cell cuture conditions -Four independent human ADMSC -Cultured under 3% & 20% oxygen tension -PQ & H2O2 treatment for confirmation of exogenous oxidative stress exposure Analysis -Cell characterization by surface marker staining -Aneuploidy analysis -Telomere length quantification by Q-FISH -Chromosomal aberration -TRAP assay --DNA damage immunofluorescence staining 53BP1 -ROS detection by flowcytomwtry -Microarray gene profilling - detection of protein carbonyls and MDA -OCR &ECAR measurement
  • 16. CONCLUSSION Stem cell for human therapy should be grown under low oxygen conditions to increase biosafety