Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells

Abstract

IntroductionThe capacity of bone marrow mesenchymal stromal cells (BMSCs) to be induced into chondrocytes has drawn much attention for cell-based cartilage repair. BMSCs represent a small proportion of cells of the bone marrow stromal compartment and, thus, culture expansion is a necessity for therapeutic use. However, there is no consensus on how BMSCs should be isolated nor expanded to maximize their chondrogenic potential. During embryonic development pluripotent stem cells differentiate into chondrocytes and form cartilage in a hypoxic microenvironment.MethodsFreshly harvested human BMSCs were isolated and expanded from the aspirates of six donors, under either hypoxic conditions (3% O2) or normoxic conditions (21% O2). A colony-forming unit fibroblastic (Cfu-f) assay was used to determine the number of cell colonies developed from each donor. BMSCs at passage 2 (P2) were characterized by flow cytometry for the phenotypic expression of cell surface markers on mesenchymal stem cells. BMSCs at P2 were subsequently cultured in vitro as three-dimensional cell pellets in a defined serum-free chondrogenic medium under normoxic and hypoxic conditions. Chondrogenic differentiation of the BMSCs was characterized by biochemical and histological methods and by quantitative gene-expression analysis.ResultsAfter 14 days of culture, the number of BMSC colonies developed under hypoxia was generally higher (8% to 38% depending on donor) than under normoxia. BMSCs were positive for the cell surface markers CD13, CD29, CD44, CD73, CD90, CD105 and CD151, and negative for CD34. Regardless of the oxygen tension during pellet culture, hypoxia-expanded BMSC pellets underwent a more robust chondrogenesis than normoxia-expanded BMSC pellets after three weeks of culture, as judged by increased glycosaminoglycan synthesis and Safranin O staining, along with increased mRNA expression of aggrecan, collagen II and Sox9. Hypoxic conditions enhanced the mRNA expression of hypoxia inducible factor-2 alpha (HIF-2α) but suppressed the mRNA expression of collagen X in BMSC pellet cultures regardless of the oxygen tension during BMSC isolation and propagation.ConclusionsTaken together, our data demonstrate that isolation and expansion of BMSCs under hypoxic conditions augments the chondrogenic potential of BMSCs. This suggests that hypoxia-mediated isolation and expansion of BMSCs may improve clinical applications of BMSCs for cartilage repair.

Mesenchymal stem cells produce functional cartilage matrix in three-dimensional culture in regions of optimal nutrient supply

“…Chondrocytes are well suited to operate in this context, with robust pathways (including hypoxia inducible factor -1alpha (Schipani et al, 2001) and glucose transporters (Mobasheri et al, 2005)) that are tuned for operation in this native state of duress. While the prochondrogenic effects of low oxygen tension have been noted (Adesida et al, 2012), MSCs within the center of the constructs would likely experience both low oxygen and low nutrient conditions, the combinatorial effect of which has been shown to cause marked cell death in this cell type (Potier et al, 2007).…”

Long-term correction of diabetic hyperglycemia through glucose-responsive hepatic insulin production using lentivirus

“…Interestingly, transient hypoxia has been found to be a critical environmental cue for the acquisition and maintenance of stem cell-like qualities [25]. For instance, mesenchymal stem cells (MSCs) tend to be found throughout the body in areas of low oxygen tension [26,27], and when cultured ex vivo, hypoxic conditions have been shown to extend their replicative lifespan [28,29], maintain their multipotent differentiation potential into later passages [30,31], and upregulate the expression of stemness markers [32]. More remarkably, hypoxia has been shown to enhance the reprogramming efficiency of somatic cells into induced pluripotent stem cells (iPSCs) [33,34].…”

Genetic Networks in Osseointegration

“…If so, the oxygen level of the microenvironment near the Ti surface may be depleted, thus potentially creating a hypoxic zone. It is well established that a hypoxic environment can induce chondrogenic differentiation as indicated by the expression of the chondrogenic transcription factor Sox9 and cartilage-related ECM molecules (Kanichai et al, 2008;Adesida et al, 2012). Although this mechanism is speculative, BMSCs may thus undergo chondrogenic differentiation in the postulated hypoxic microenvironment at the Ti implant surface (Figure 5).…”