The Role of Primary Cilia in Mesenchymal Stem Cell Differentiation: A Pivotal Switch in Guiding Lineage Commitment

Abstract

Primary cilia are sensory organelles that have been shown to play a critical role in lineage commitment. It was our hypothesis that the primary cilium is necessary for chemically induced differentiation of human mesenchymal stem cells (MSC). To investigate this, polaris siRNA was used to inhibit the primary cilia and the mRNA levels of transcription factors Runx2, PPARgamma were measured by RT PCR as markers of osteogenic, adipogenic and chondrogenic differentiation, respectively. MSCs with inhibited primary cilia had significantly decreased basal mRNA expression levels of all three lineages specific transcription factors indicating that primary cilia are critical in multiple differentiation pathways. Furthermore, to determine if primary cilia play a role in the differentiation potential of MSCs, progenitor cells transfected with either scrambled or polaris siRNA were cultured in osteo-inductive, chondro-inductive, or adipo-inductive media and lineage commitment was ascertained. Interestingly, within 24 h of culture, cells transfected with polaris siRNA in both osteogenic and adipogenic media lost adhesion and released from the slides; however MSCs in chondrogenic media as well as cells transfected with scrambled siRNA did not. These results suggest that the primary cilium is necessary for the normal progression of chemically induced osteogenic and adipogenic differentiation. As a control, the experiment was repeated with NIH3T3 fibroblasts and none of the effects of inhibited primary cilia were observed indicating that the loss of adhesion may be specific to MSCs. Furthermore after biochemically inducing the cells to differentiate, polaris knockdown resulted in abrogation of both Runx2 and PPARgamma mRNA while SOX9 mRNA expression was significantly lower. These results suggest that primary cilia play an essential role not only in the initiation of both osteogenic and adipogenic differentiation, but also in maintaining the phenotype of differentiated cells. Interestingly, chondrogenic differentiation appeared less dependent on a functional primary cilium.

FIGURE 1

Primary cilia stained with anti acetylated tubulin (red) and DAPI (blue) in human mesenchymal stem cells. Cells were imaged on a Nikon C-1 confocal microscope (20×).

FIGURE 2

Western blot showing reduced levels of polaris protein in MSC cells transfected with polaris siRNA relative to cells transfected with control siRNA (right lane). Actin served as a loading control.

FIGURE 3

Baseline transcription factor mRNA levels in MSC cells with or without primary cilia. The relative gene expression levels were quantified by real time RT-PCR and normalized to 18s rRNA levels. Bars represent mean ± SEM (N ≥ 8 for all groups).

FIGURE 4

Loss of adhesion when MSCs were placed in adipogenic differentiation media. (a) Cells transfected with scrambled siRNA sequence. (b) Cells transfected with siRNA against polaris (20×).

FIGURE 5

mRNA levels in MSC cells with or without primary cilia after differentiation. The relative gene expression levels were quantified by real time RT-PCR and normalized to 18s rRNA levels. Bars represent mean ± SEM (N ≥ 8 for all groups).