Abstract
Purpose
Bone marrow multi-potent stromal cells represent a heterogenous source of cells with great promise in joint cartilage regenerative medicine. However, due to their low numbers upon harvesting, MSCs need to be expanded without compromising their capacity to form chondrocytes (cartilage cells). To date there is no consensus on how to expand MSCs in order to maximize their potential for cartilage repair and nor are there any specific cell signatures of MSCs with chondrogenic propensity. Emerging evidence suggest that marrow stem cells exist in a hypoxic microenvironment. On this basis and in addition to cartilages natural existence in hypoxic environment (1–7% O2), we hypothesized that MSC expansion under hypoxia will result in the enrichment of MSCs with predilection to chondrocytes compared to expansion under the conventional culture conditions of 21% O2.
Method
Bone marrow was harvested from the iliac crest of 4 donors (mean age 43.5 years) post informed consent and local ethical approval. Fifteen million mono-nucleated (MNCs) cells were seeded into T150cm2 culture flasks in the presence of alpha MEM plus 10% FBS and 5 ng/ml FGF2. Similarly, 0.25 million MNCs were seeded in 10cm petri dishes for colony forming unit-fibroblastic (CFU-f) assay. The seeded flasks and petri dishes were cultured under normoxia (21% O2) and hypoxia (3% O2). Petri dished cells were cultured for 14 days and those in flasks were cultured until passage 2 (P2). Developed cell colonies per dish were revealed after crystal violet staining. Colony counts and diameters were recorded. P2 cells were treated with a panel of antibodies for cell surface marker analysis by fluorescent activated cell sorting (FACS) flow cytometry. P2 cell pellets were formed and induced towards cartilage in a defined serum free medium containing TGFβ1. Pellets were cultured for 3 weeks under normoxia and were then processed for histological, biochemical and gene expression analyses.
Results
The mean number of cell colonies was 1.25-fold higher after hypoxia culture relative to normoxia. There were no differences in colony diameters. A panel of common protein signatures (CD29, CD90, CD105 and CD151) for stem cells declined in expression after expansion in hypoxia. However, other signatures (CD13, CD34 and CD44) expression level increased under hypoxia, whilst CD73 expression was unchanged. Pellets from hypoxia-expanded MSCs showed on average a 1.4-fold increase in chondrogenic capacity as judged by glycosaminoglycan (GAG) matrix per DNA content relative to normoxia pellets. The gene expression of collagen II, SOX9, aggrecan and matrillin-3 increased by 1.2-, 2-, 1.3- and 1.5-fold, respectively, in pellets formed from hypoxia-expanded stem cells relative to their normoxia counterparts.
Conclusion
Expansion of stem cells under hypoxia potentiates their capacity to form cartilage with improved cartilage properties. However, there is a need for signatures to identify stem cells with propensity to form cartilage.
