Non-invasive sources of cells with primary cilia from pediatric and adult patients

Abstract

Background: Ciliopathies give rise to a multitude of organ-specific pathologies; obtaining relevant primary patient material is useful for both diagnostics and research. However, acquisition of primary ciliated cells from patients, particularly pediatric patients, presents multiple difficulties. Biopsies and blood samples are invasive, and patients (and their parents) may be reluctant to travel to medical centers, especially for research purposes. We sought to develop non-invasive methods of obtaining viable and ciliated primary cells from ciliopathy patients which could be obtained in the home environment.

Findings: We introduce two methods for the non-invasive acquisition of primary ciliated cells. In one approach, we collected spontaneously shed deciduous (milk) teeth from children. Fibroblast-like cells were observed after approximately 2 weeks of culture of fragmented teeth. Secondly, urine samples were collected from children or adults. Cellular content was isolated and after approximately 1 week, renal epithelial cells were observed. Both urine and tooth-derived cells ciliate and express ciliary proteins visible with immunofluorescence. Urine-derived renal epithelial cells (URECs) are amenable to 3D culturing, siRNA knockdown, and ex vivo drug testing.

Conclusions: As evidence continues to accumulate showing that the primary cilium has a central role in development and disease, the need for readily available and ciliated patient cells will increase. Here, we introduce two methods for the non-invasive acquisition of cells with primary cilia. We believe that these cells can be used for further ex vivo study of ciliopathies and in the future, for personalized medicine.

Keywords: Cell culture; Cilia; Ciliopathy; Deciduous tooth; Pediatrics; Protocol; Urine.

Fig. 1

Cultures and images of URECs in 2D and 3D conditions. (A) Urine sample 24 h after collection at ×4 magnification. Asterisks indicate squamous cells, and black arrows indicate transitional cells. Scale bar 200 μm. Note that renal epithelial cells are not apparent. (B) Renal epithelial cells in culture 12 days after collection at ×4 magnification. Note that there are two morphologically distinct types of renal epithelial cells, marked with a white or black arrow. Scale bar 200 μm. (C) Wild-type UREC 3D spheroids. Megalin indicates that cells composing spheroids can be derived from the proximal tubule, while AQP2 was used as a marker for collecting duct cells. Nucleus (DAPI, blue); megalin and AQP2 (red); and ZO-1 (green). Scale bar 10 μm. (D) Wild-type UREC 3D spheroids. Cilia are indicated with white arrows. Nucleus (DAPI, blue); Ac. Tubulin (white); and ZO-1 (green). Scale bar 10 μm. (E) Wild-type URECs in 2D monolayer. Nucleus (DAPI, blue); Ac. Tubulin (white); pericentrin (PCNT, red). Scale bar 10 μm

Fig. 2

Cultures of cells derived from deciduous teeth. (A) Tooth fragments are taken into culture in a 12-well plate under standard fibroblast cell culture conditions. (B) Fibroblast-like cells were observed in the culture plate after approximately 2 weeks (left) and expanded ~1 week (right) at ×4 magnification. Scale bar 200 μm. (C) Immunofluorescence imaging of fibroblast-like cells from healthy donors (n = 3) shows 25–51 % ciliation after 24-h serum starvation. Cells from one donor are shown here. Nucleus (DAPI, blue); ARL13B and INPP5E (red); Ac. Tubulin (green). Scale bar 5 or 10 μm. (D). Imaging of fibroblast-like cells from a healthy donor showing cilia and centrosome. Nucleus (DAPI, blue); Ac. Tubulin (green); pericentrin (PCNT; red). Scale bar 10 μm