Impact of hypoxia and long-term cultivation on the genomic stability and mitochondrial performance of ex vivo expanded human stem/stromal cells.
Recent studies have described the occurrence of chromosomal abnormalities and mitochondrial dysfunction in human stem/stromal cells (SCs), particularly after extensive passaging in vitro and/or expansion under low oxygen tensions. To deepen this knowledge we investigated the influence of hypoxia (2% O(2)) and prolonged passaging (>P10) of human bone marrow stromal cells (BMSCs) and adipose-derived stromal cells (ASCs) on the expression of genes involved in DNA repair and cell-cycle regulation pathways, as well as on the occurrence of microsatellite instability and changes in telomere length. Our results show that hypoxic conditions induce an immediate and concerted down-regulation of genes involved in DNA repair and damage response pathways (MLH1, RAD51, BRCA1, and Ku80), concomitantly with the occurrence of microsatellite instability while maintaining telomere length. We further searched for mutations occurring in the mitochondrial genome, and monitored changes in intracellular ATP content, membrane potential and mitochondrial DNA content. Hypoxia led to a simultaneous decrease in ATP content and in the number of mitochondrial genomes, whereas the opposite effect was observed after prolonged passaging. Moreover, we show that neither hypoxia nor prolonged passaging significantly affected the integrity of the mitochondrial genome. Ultimately, we present evidence on how hypoxia selectively impacts the cellular response of BMSCs and ASCs, thus pointing for the need to optimize oxygen tension according to the cell source.
Department of Bioengineering and Institute for Biotechnology and Bioengineering, Instituto Superior Técnico (IST), Technical University of Lisbon, Lisboa, Portugal.
Pedro H
PH
Oliveira
Joana S
JS
Boura
Manuel M
MM
Abecasis
Jeffrey M
JM
Gimble
Cláudia Lobato
CL
da Silva
Joaquim M S
JM
Cabral
D000255:Adenosine Triphosphate / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000328:Adult; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D042822:Genomic Instability; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D016615:Telomere / Q000378:metabolism; D013997:Time Factors
D000255:Adenosine Triphosphate; D010100:Oxygen