Why Optimize MSC Cell Culture Conditions? Feed a Fibroblast, Starve a Stem Cell

Alicia D. Henn, CSO BioSpherix

A new publication in Stem Cell Research & Therapy (open access) from Alan Wells’ group at University of Pittsburgh has some highly relevant findings for Mesenchymal stem/stromal cell (MSC) culture optimization. MSC are an incredibly valuable cell type for both industrial and research scale cell-based therapeutics. The Wells group reported that in their investigations of the relationships between MSC starvation, autophagy, and differentiation, they stumbled upon a strikingly high glucose consumption rate in comparison with other cell types.1

 

The Findings
The Wells group used an immortalized MSC cell line as well as primary human bone marrow MSC and started with looking at serum deprivation and autophagy. They were surprised to find no effect on immortalized MSC when they starved them of serum, pyruvate, or individual amino acids. They did, however, find a large effect when MSC were starved of glucose.

Using the Agilent Seahorse analyzer, the authors found that in addition to a high rate of glucose consumption, MSC exhibited high extracellular acidification rates, also suggesting active glycolysis. The MSC responded to increased glucose levels by reduced autophagy. Conversely, autophagy was restored in response to reduction of glucose back to lower levels. However, these experiments were done without controlling room air oxygen levels to physiologically relevant oxygen levels.

As we all learned in biochemistry, cells have two major mechanisms for generating ATP for cellular activities, aerobic oxidative phosphorylation (cellular respiration) and anaerobic glycolysis. Oxygen is the final electron acceptor in the electron transport chain used for ATP generation by mitochondria during cellular respiration.

The Wells group went on to confirm the anaerobic nature of the immortalized MSC. Using BioSpherix equipment to control the oxygen levels for cell culture, they found that the changes in MSC autophagy they observed under different glucose availability conditions were not at all affected by culture at different oxygen levels (21%, 4%, or 1% O2). This helps strengthen the association between autophagy and the dependence of MSC upon the glycolytic metabolic pathways that do not require oxygen as the final electron acceptor.

 

The Significance

The fate of implanted MSC is of intense research interest right now because of the high clinical value of MSC in a wide variety of disease interventions. The authors point out that there are many publications on the effects of low oxygen on MSC function, and that their work indicates that MSC don’t need high oxygen levels for survival. MSC are derived from low-oxygen in vivo environments such as bone marrow, and in many applications, are returned to extremely low-oxygen environments, like knee cartilage, after expansion in vitro under supraphysiologic room air oxygen conditions. High oxygen levels can be extremely stressful to human MSC, inducing exosome release which may help drive the anti-inflammatory activities of MSC-based cell therapies.

 

The work of the Wells’ group and others to define the best metabolic conditions for MSC expansion, differentiation, and ultimate in vivo function, is critical to the success of these new therapies.

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1. Nuschke, A., Rodrigues, M., Wells, A. W., Sylakowski, K. & Wells, A. Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation. Stem Cell Research & Therapy 7, 179, doi:10.1186/s13287-016-0436-7 (2016).