Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells

Abstract

The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O2) and hypoxic (1 % O2) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2. Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.

Application of Bioreactor in Stem Cell Culture

“…The gas with normal oxygen fraction is also known as normoxia; the gas with lower and higher oxygen fractions is also known as hypoxia and hyperoxia, respectively. Oxygen concentration is an important component of the niche in the in vitro SC expansion, and also crucial for the proliferation and differentiation of SCs and the stemness maintenance [42,43].…”

“…Oxygen concentration can regulate the proliferation and differentiation of MSCs and affect the osteogenic, chondrogenic and adipogenic capabilities of MSCs [43,46]. Dos Santos et al cultured hMSCs with 2% oxygen, and they found that 2% oxygen was better to promote the growth and expansion of MSCs as compared to normoxia.…”

Hypoxia promotes adipose-derived stem cell proliferation via VEGF

“…In fact, hypoxia was demonstrated as a stimulator of ADSC proliferation, but its effects did not change the ADSC phenotype (Fotia et al, 2015;Wan Safwani et al, 2016). Hypoxia has also been demonstrated as a protector from senescence , genomic stability inducer (Bigot et al, 2015), maintainer of stemness (Fotia et al, 2015;Petrangeli et al, 2016;Wan Safwani et al, 2016), and enhancer of viability, motility and tropism (Feng et al, 2015). These hypoxia effects have been demonstrated through stimulation of hypoxia-inducible factor 1- (HIF-1) expression (Kakudo et al, 2015), generation of reactive oxygen species (ROS) and downstream phosphorylation of platelet-derived growth factor receptor  (PDGFR), extracellular signal-regulated ki-nases 1/2 (ERK1/2) and Akt (Kang et al, 2014;Kim et al, 2011).…”

“…In fact, hypoxia was demonstrated as a stimulator of ADSC proliferation, but its effects did not change the ADSC phenotype (Fotia et al, 2015;Wan Safwani et al, 2016). Hypoxia has also been demonstrated as a protector from senescence , genomic stability inducer (Bigot et al, 2015), maintainer of stemness (Fotia et al, 2015;Petrangeli et al, 2016;Wan Safwani et al, 2016), and enhancer of viability, motility and tropism (Feng et al, 2015).…”

Stemness of Mesenchymal Stem Cells

“…Mimicking endogeneous niche of MSCs is able to delay the cell aging and maintain MSC stemness, including hypoxia [16][17][18], coating with extra-cellular matrix (ECM) [19,20], and 3D culture [21,22] among others. Hypoxic environment has been suggested as physiologic niche to maintain stemness of stem cells.…”