Hypoxic Mesenchymal Stem Cell
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Transcript of Hypoxic Mesenchymal Stem Cell
Yuyun Rindiastuti
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.
SUMMARY
hMSC Regenerative medicine
Expansion cultureStandard
“nonphysiological oxygen tension”
Physiological oxygen tension
-Improves cell growth-Extend life span-Alters differentiation process-Reduce chromosomal abnormalities
-Increase oxidative stress-Telomerase shortening-Increase DNA damage, chromosomal aberration
INTRODUCTION
Oxygen levels & exogenous oxidative
stress
Genetic stability
Cytogenetic analysis, gene expression,
bioenergetics studies
Culture grown in physiologycal 20%
oxygen
Culture grown in standard 3% oxygen
-transcriptional activation in HIF 1A target genes- Inhibition oxygen
consumption
- Increase genetic instability- Decrease glycolysis
Oxygen sensing, HIF 1 response
RESULTGrowth of hMSC at 20% O2 reduces lifespan, increases oxidative stress and the rate of telomere shortening
Telomerase shortening & cell
senescence
Cell growth
Superoxide accumulation
Carbonyl & MDA levels
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 mammalian cells at
20% oxygen
- Promotes DNA damage & senescence- Chromosomal aberration during long term
culture
Importance of biosafety in cell
therapyCulture ADMSC at 3%
0xygen
- Increase growth rate- No evidence of spontaneous immortalization
- - reduce telomere shortening rate- decrease structural chromosomal aberration
Significant upregulation of gene expression in cells grown at 3% oxygen tension related to HIF
1A stabilization
Balance bioenergetics
pattern
Implication of gene ontology analysis (HIF 1 dependent gene
expression)
Autocrine & paracrine effect in cell growth &
senescence
HIF 1A decreases OXPHOS system by downregulating
mitochondrial ATP production & oxygen
consumption
MATERIAL & METHODSCell & 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
Thank You