Maintenance of Osteogenic Differentiation Capacity of MSPC Despite Amplified Proliferation Under Elevated Oxgen Conditions

Abstract 1916

Clinical trials are underway to test the safety and efficacy of mesenchymal stem/progenitor cells (MSPCs) in various diseases. Due to their low frequency in situ, MSPC expansion is the prerequisite for dose finding studies as well as for most applications in adult patients. Notably, cultured MSPCs are a mixture of heterogeneous cells in various stages of cell cycle, proliferation and differentiation activity. A major safety concern for MSPC propagation is the risk of malignant transformation or premature senescence hampering MSPC function. The in vitro and consequently in vivo cellular characteristics may be influenced by factors as tissue source, age of the donor, materials and media, growth factors and oxygen pressure, arguing for standardized culture procedures at least in clinical trials. Defining the optimal conditions for efficient expansion of clinical grade cell therapeutics is still a challenge.

We have previously shown that long-term expanded human bone marrow-derived MSPCs acquired senescence-related gene expression changes independent of culture conditions (Haematologica 2010). It has been speculated that elevated oxygen (20% air O₂) contributes to genomic instability and malignant transformation in vitro. We therefore analyzed the influence of different oxygen conditions during long-term expansion on MSPC behavior focussing osteogenic differentiation. A gene panel previously defined as senescence markers was tested for differential expression after varying culture conditions.

Bone marrow-derived MSPCs were expanded in α-MEM supplemented with 10% human platelet lysate replacing fetal bovine serum under physiologic conditions (5% O₂) or air oxygen (20% O₂) until spontaneous cessation of proliferation. Osteogenic induction was analyzed by Alizarin red. RNA was isolated from corresponding early and late passages and analyzed by qRT-PCR for p16^ink4a, PARG1, CDKN2B, PTN and MCM6.

In total, MSPCs could be cultured for 5 passages at 30 cells/cm² and for 10 passages at 3,000 cells/cm² for up to 85 days resulting in more cumulative population doublings (PDs) of MSPCs at air O₂ compared to 5% O₂ and in cultures with low compared to standard seeding density. Long-term cultured MSPCs after 40 PDs (air O₂) and 35 PDs (5% O₂) retained their osteogenic differentiation capacity. Compared to early passages, RT-PCR in late passages revealed an up-regulation of p16^ink4a, PARG1 and CDKN2B without specific influence of culture conditions. PTN and MCM6 were significantly down-regulated, mainly in air O₂ cultures with high seeding density correlating with diminished cell proliferation compared to low density cultures. There was no evidence of immortalization or malignant transformation. The capacity for in vivo bone formation of long term cultured MSPCs is currently tested in a novel humanized mouse model for bone and marrow niche formation (Blood 2012).

Long term expansion of MSPCs under animal serum-free air oxygen conditions was safe and most efficient at low seeding density. Even in late passages (>30 PDs) MSPCs preserved their potential for osteogenic differentiation in vitro. At air oxygen delayed replicative senescence was observed, mainly at low seeding density. There was no evidence for immortalization or transformation indicating applicability of standardized ambient air culture conditions for pre-clinical cell expansion.