Pulpbow: Novel Approach to Trace The Vasculogenic Potential of DPSC

Objectives: The absence of a vascular network capable of distributing nutrients and oxygen is a major limiting factor in generating bioengineered tissues, particularly the dental pulp due to the anatomy of the root canal. We have shown that dental pulp stem cells (DPSC) can differentiate into endothelial cells and form functional blood vessels through vasculogenesis. However, it is not clear how sub-populations of cells contribute to the formation of these blood vessels in 3D models. The objective of this study was to develop a new strategy to track the distribution of individual clones of pulp cells over time (named Pulpbow), and to use this strategy to evaluate DPSC-mediated vasculogenesis.
Methods: The Pulpbow was created by permanently labeling individual dental pulp stem cells with different ratios of red, green and blue derivatives of fluorescent proteins using lentiviral vectors. The superimposition created by the simultaneous expression of these 3 basic hues create an array of colors (rainbow effect) that is permanent, unique to each cell and passed through generations (i.e. a color tag).These cells were them used in in vitro sprouting assays, ex vivo live-cell tracking assays and in in vivo transplantations. In addition, a cell cycle live reporter was used to analyze proliferation during sprout formation.
Results: Simultaneous tracking of cells during sprout formation revealed that no single population is more prone to vasculogenesis, and that there is a clonal organization mediated by proximity. Cells with different hues can start or participate in the progression of the growing sprout. During this process, there is intense cell motility and minimal cell proliferation. Blood vessels formed by pulpbow-transplanted cells confirmed the random patterns of cells’ colors.
Conclusions: Dental pulp stem cell mediated vasculogenesis is a polyclonal process that shows minimal cell proliferation, high motility and dynamic cell-cell interactions.