Accelerating stem cell proliferation by down-regulation of cell cycle regulator p21

Tissue engineering is often limited by the time required for culture expansion of cells necessary for scaffold seeding. Cell cycle regulators control entry and exit into the cell cycle and as such regulate cellular proliferation rates. The authors hypothesized that transient alteration in cell cycle regulators can be utilized as a means to accelerate stem cell proliferation.

Mesenchymal stem cells were harvested from wild-type mice and mice deficient in the cell cycle regulator p21. Wild-type cells were treated with small interfering RNA against p21 in two- or three-dimensional cultures in vitro. Cellular proliferation and the potential for cellular differentiation into the bone or fat lineage were assessed.

Mesenchymal stem cells treated with small interfering RNA targeting p21 demonstrated a significant decrease in p21 protein and mRNA expression 96 hours after treatment. They also proliferated significantly faster than control cells (2.5 to three times) in both two- and three-dimensional culture. Similarly, cells harvested from p21-deficient mice demonstrated a significant acceleration in cellular proliferation. Inhibition of p21 expression was not associated with significant changes in spontaneous cellular differentiation. However, transient p21 inhibition promoted both osteoblastic and adipogenic differentiation when cells were exposed to differentiation medium.

Transient inhibition of the cell cycle regulator p21 results in significant acceleration of mesenchymal stem cell proliferation without promoting spontaneous cellular differentiation. Exposure to differentiation medium results in increased cellular differentiation toward the osteoblast and fat lineage. Manipulation of cell cycle regulators may represent a novel means by which stem cell proliferation can be accelerated, thereby decreasing the time required for scaffold synthesis in tissue engineering.