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Improving Culture Conditions to Boost hMSC Therapeutic Potential



Review of “Density-Dependent Metabolic Heterogeneity in Human Mesenchymal Stem Cells” from Stem Cells by Stuart P. Atkinson

Packed like sardines in a train on the way to work, walking out in the middle of the countryside with a few of your nearest and dearest, or alone, shipwrecked on a desert island. How we act is completely different in each situation, and a new study from Teng Ma (Florida State University, USA) has recently suggested that human mesenchymal stem cells (hMSCs) share this feature. Their new study suggests that the metabolic characteristics of hMSCs in in vitro culture are dependent on how many other hMSCs are present in close proximity. This finding points to new strategies to boost hMSCs expansion and enhance their clinical potential [1].

hMSCs are an intrinsically heterogeneous cell type [2, 3] who are prone to undesirable culture-induced changes in phenotype [4]. Their characteristics are also known to be reliant on culture density [5, 6], but quite how this occurs is relatively unstudied. The current study found that clonal density (CD) and low density (LD) growth of hMSCs boosted the number of the clonogenic, and most therapeutically useful, hMSC sub-population of cells, as measured by cell surface expression of CD146 and colony forming (CFU-F) ability.

While this in itself was not a novel finding, the researchers then discovered that when they compared CD and LD to medium density (MD) and high density (HD) hMSC growth they saw differences in metabolic profiles. Following extraction of polar metabolites and analysis using gas chromatography-mass spectrometry the group found that lower density hMSCs relied on glycolysis for ATP production while higher density hMSCs utilized oxidative phosphorylation instead. Previous studies have linked stem cell proliferation and multipotency to glycolytic ATP synthesis, and so, the observed metabolic profiles point to the existence of hMSCs with a greater therapeutic potential at low growth densities.

Additionally, the study found that higher density hMSC cultures contained higher levels of reactive oxygen species (ROS), a byproduct of oxidative phosphorylation. They correlated this to a higher level of hMSC apoptosis, as measured by higher caspase 3/7 expression, and a higher level of senescence, as measure by SA-βGal activity, which both combine to reduce the therapeutic value of hMSC cultures.

Taken together, this study suggests that tweaking initial cell densities, controlling oxygen tensions, and the supply of appropriate cellular “foodstuffs” can enhance the therapeutic potential of hMSCs expanded in vitro. So much like us humans, this study suggests that what surrounds MSCs has an important influence on their characteristics.


  1. Liu Y, Munoz N, Bunnell BA, et al. Density-Dependent Metabolic Heterogeneity in Human Mesenchymal Stem Cells. Stem Cells 2015;33:3368-3381.
  2. Russell KC, Phinney DG, Lacey MR, et al. In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem Cells 2010;28:788-798.
  3. Muraglia A, Cancedda R, and Quarto R Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000;113 ( Pt 7):1161-1166.
  4. Bara JJ, Richards RG, Alini M, et al. Concise review: Bone marrow-derived mesenchymal stem cells change phenotype following in vitro culture: implications for basic research and the clinic. Stem Cells 2014;32:1713-1723.
  5. Lee RH, Hsu SC, Munoz J, et al. A subset of human rapidly self-renewing marrow stromal cells preferentially engraft in mice. Blood 2006;107:2153-2161.
  6. Lee RH, Seo MJ, Pulin AA, et al. The CD34-like protein PODXL and alpha6-integrin (CD49f) identify early progenitor MSCs with increased clonogenicity and migration to infarcted heart in mice. Blood 2009;113:816-826.