Abstract
BackgroundIn the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions.ResultsMorphologic studies, differentiation and transcriptional profiling experiments were performed on MSC cultured in normoxia (21% O2) versus hypoxia (5% O2) for up to passage 2. Cells at passage 0 and at passage 2 were compared, and those at passage 0 in hypoxia generated fewer and smaller colonies than in normoxia. In parallel, MSC displayed (>4 fold) inhibition of genes involved in DNA metabolism, cell cycle progression and chromosome cohesion whereas transcripts involved in adhesion and metabolism (CD93, ESAM, VWF, PLVAP, ANGPT2, LEP, TCF1) were stimulated. Compared to normoxic cells, hypoxic cells were morphologically undifferentiated and contained less mitochondrias. After this lag phase, cells at passage 2 in hypoxia outgrew the cells cultured in normoxia and displayed an enhanced expression of genes (4-60 fold) involved in extracellular matrix assembly (SMOC2), neural and muscle development (NOG, GPR56, SNTG2, LAMA) and epithelial development (DMKN). This group described herein for the first time was assigned by the Gene Ontology program to "plasticity".ConclusionThe duration of hypoxemia is a critical parameter in the differentiation capacity of MSC. Even in growth promoting conditions, hypoxia enhanced a genetic program that maintained the cells undifferentiated and multipotent. This condition may better reflect the in vivo gene signature of MSC, with potential implications in regenerative medicine.
Highlights
In the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low
mesenchymal stem cells (MSC) may interact with hemopoietic stem cells (HSC), which reside in a specific microenvironment formed by various stromal precursor cells and osteoblasts, called the niche [4,5,6]
STRO-1 was transiently expressed on 50% of the cells at P0 under hypoxia and diminished thereafter as expected from previous observations [18]
Summary
The effect of hypoxia on MSC expansion and phenotype Bone marrow mononuclear cells (MNC) were cultured and passaged until P3. Culture of MSC in hypoxia inhibited cell differentiation and mitochondrial biogenesis The number of mitochondria was evaluated by flow cytometry, by Mitotracker staining and by transmission electron microscopy TEM at P2, under hypoxia and normoxia in 3 independent experiments. Comparative analysis of transcriptome from MSC cultured at P0 in hypoxia versus normoxia revealed 386 dysregulated genes (1% out of 41,000 genes), of which 174 were up regulated (45%) and 212 were down regulated (55%). Comparative analysis of transcriptome from MSC cultured at P2 in hypoxia versus normoxia revealed 519 dysregulated genes, of which 264 were up regulated (50.9%) and 255 were down regulated (49.1%). The results matched that of the gene arrays with enhanced expression of HOXA11, KIT, WNT4, OXCT2 and inhibited expression of CCL2, CX3CL1 under hypoxia
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