Telocytes in the Spleen

Abstract

Telocytes, a novel type of interstitial cells with very long and thin prolongations, have been identified in many organs in mammals. At present, the ultrastructural, immunocytochemical and electrophysiological properties of telocytes in multiple organs have been understood. However, telocytes in spleen, especially their roles in spleen have not been reported. The aim of this study was to investigate the ultrastructure, distribution and immunophenotypes of splenic telocytes. Rat spleen was harvested for the ultrastructure analysis by transmission electron microscopy (TEM). The primary culture of telocytes was performed after combined enzymatic digestion. The characteristic morphology was analyzed by a scanning electron microscopy (SEM). It was shown that telocytes displayed a piriform/spindle/triangular shape with long and slender telopods and extremely long prolongation contracting with surrounding cells in the spleen. Their dynamic profiles of cytoplasmic separation were recorded by the Live Cell Imaging System. The length of telopods was mostly distributing in 20-30 μm, in accordance with normal distribution. Most telocytes had three or two telopods (28.71% and 22.58% respectively). Immunostaining indicated that these cells were positive for vimentin, CD34, nanog and sca-1, but negative for c-kit. These data prove the existence of telocytes in the spleen, which may serve as the experimental base for exploring their roles in the spleen.

Fig 1. Ultrastructure of splenic TCs under transmission electron microscopy.

A. A TC with telopodes (Tps) in close contact with lymphocytes. B. A TC with Tps embraces with leukocyte. C. Tps with two branches distribution. D. Clusters of TCs between leukocytes and red blood cells in spleen.

Fig 2. Isloated splenic TCs under phase-contrast microscope.

A. Splenic TCs with typical morphology of piriform or triangular and small cell bodies and slender TPs; B. TCs forming a 3 D structure anchored by hetero- and homocellular junctions with their TPs; C. circle-like structure in TCs cultured in vitro, in which the long prolongations extended to support the wall of the circle-like structure. Scale bar = 20μm.

Fig 3. Dynamic morphology and movement of TCs captured by Live Cell Imaging System.

Dynamic cytokinesis process in TCs under Live Cell Imaging System at 0 min in A, at 2 min in B, at 4 min in C, at 6 min in D, at 8 min in E, at 12 min in F, at 18 min in G, at 20 min in H, respectively; DAPI labeling nuclei of TCs excited with ultraviolet light(blue). Scale bar = 20μm.

Fig 4. Isloated splenic TCs by scanning electron microscopy.

A. An example of TC with 6 Tps. B. An example of two TCs keeping in touch with each other and growing their TPs with dichotomous branch out forming. C. An example of TC with TPs in different length (tens to hundreds of micrometres). D. An example of TC with two TPs and one of TPs reaching as 297.5μm. Black arrows indicate the piriform and small cell body and black arrowheads indicate the podoms arranged alternately on Tps.

Fig 5. Positive skewed distribution of TP’s length.

Fig 6. Detection of splenic TCs by immunofluorescence for vimentin, CD34, nanog and sca-1.

A. Vimentin immunostaining (FITC fluorescence labeling, green); D,G,J. CD34, nanog and sca-1 immunostaining respectively (Cy3 fluorescence labeling, red); B,E,H,K. nuclei of TCs (DAPI staining, blue), C. merged image of A and B; F. merged image of D and E; I. merged image of G and H; L. merged image of J and K. Scale bar = 100μm.

Fig 7. Double immunofluorescence staining for splenic TCs cultured in vitro A,E,I.

Vimentin immunostaining (A. Cy3 fluorescence labeling, red; E,I. FITC fluorescence labeling, green); B,F,J. CD34, nanog and sca-1 immunostaining respectively (B. FITC fluorescence labeling, green; F,J. Cy3 fluorescence labeling, red); C,G,K. nuclei of TCs (DAPI staining, blue), D. merged image of A, B and C; H. merged image of E, F and G; L. merged image of I, J and K. Scale bar = 20μm.