Effect of Reprogramming on Primary Cilia Mechanics

Abstract

The advent of human induced pluripotent stem cell (hiPSC) reprogramming has revolutionized the field of developmental biology. To realize the full potential of this promising technology, it is imperative to understand mechanical and chemical signaling pathways that coordinate the process of reprogramming. Primary cilia have been shown to play a critical role in mechano-chemical signaling across a wide spectrum of cell types, with major implications in maintaining homeostasis. Their functions in hiPSCs and their characteristic changes during the reprogramming process remain largely vague. This work focused on understanding how reprogramming affects the mechanical characteristics of primary cilia. Using immunofluorescence imaging assays and electron microscopy studies, we established for the first time the presence of primary cilia on fibroblasts derived hiPSCs. Using quantitative PCR assays, we identified changes of expression levels of pluripotency markers Nanog and Cripto as well as primary cilia signaling partners Hh, Ptch1, Gli1, Gli2, and Gli3. Furthermore, morphometric analysis revealed that reprogramming resulted in an increase in curvature of primary cilia from ∼0.16 μ−1 to ∼0.78 μ−1 (p<0.02), indicating a significant change in Young's modulus ratio of at least 5-fold, and a decrease in the length of primary cilia from ∼2.64 μ to ∼0.7 μ (p<1.76x10−13). Taken together, this was the first systematic study showing the presence of primary cilia on human reprogrammed cells and demonstrating significant effects of the reprogramming process on primary cilia mechanics.