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