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Abstract
Bone marrow derived mesenchymal stem cells (MSCs) are regularly utilized for translational therapeutic strategies including cell therapy, tissue engineering, and regenerative medicine and are frequently used in preclinical mouse models for both mechanistic studies and screening of new cell based therapies. Current methods to culture murine MSCs (mMSCs) select for rapidly dividing colonies and require long-term expansion. These methods thus require months of culture to generate sufficient cell numbers for feasibility studies in a lab setting and the cell populations often have reduced proliferation and differentiation potential, or have become immortalized cells. Here we describe a simple and reproducible method to generate mMSCs by utilizing hypoxia and basic fibroblast growth factor supplementation. Cells produced using these conditions were generated 2.8 times faster than under traditional methods and the mMSCs showed decreased senescence and maintained their multipotency and differentiation potential until passage 11 and beyond. Our method for mMSC isolation and expansion will significantly improve the utility of this critical cell source in pre-clinical studies for the investigation of MSC mechanisms, therapies, and cell manufacturing strategies.
Highlights
For the chondrogenic differentiation assay, 2–3 weeks after chondrogenic induction, chondrogenic pellets were stained with a type II collagen antibody and showed expression throughout the pellet. These results demonstrate that murine MSCs (mMSCs) cultured under hypoxic conditions classify as mesenchymal stem cells by cell surface marker expression and properly undergo tri-lineage differentiation
We demonstrate that the enhanced proliferation rates and Colony Forming Unity (CFU) promoted by culturing MSCs in hypoxia or hypoxia+Basic fibroblast growth factor (bFGF) conditions are sustained out to p9-12 (Supplemental Fig. 2A, B)
The ability to culture mMSCs in a simple, effective, and timely fashion from existing transgenic mouse models would fill a significant unmet need by allowing researchers to investigate the function of specific genes in various translational and differentiation processes in the pre-clinical lab setting
Summary
Basic fibroblast growth factor (bFGF) supplementation was previously linked to the maintenance of stemness and the suppression of senescence with an optimal effect at 10 ng/mL, making bFGF at this dose a viable growth factor candidate[14] It was established recently by Fábián, et al that the combination of hypoxia and bFGF can improve human MSC proliferation and that this is mediated, in part, by ERK pathway activation and increased expression of HIF-1α15. We found that low oxygen tension (5% O2 = hypoxia) in combination with bFGF supplementation (10 ng/mL) has an additive effect on mMSC proliferation, resulting in fast expansion, while simultaneously preserving stemness and suppressing senescence This new approach generates mMSCs faster than existing protocols, is highly reproducible, allows MSCs to maintain their multipotency and differentiation potential beyond passage 11, and produces cells that demonstrate therapeutic potential in vivo
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