Isolation, culture, purification and ultrastructural investigation of cardiac telocytes

Abstract

Telocytes (TCs), a novel type of stromal cells, are crucial to cardiac renovation and regeneration. To dissect the pathophysiological effects of cardiac TCs in heart disease, it is essential to develop an effective method to isolate, culture, purify and characterize these cells. In the present study, cardiac TCs were isolated from the hearts of rats by enzymatic digestion. Histology and CD34/PDGFRα expression by flow cytometric assay were used to characterize the cultured cardiac TCs, which were purified by flow cytometric sorting and confirmed by immunofluorescence and electron microscopy. Typical TCs were observed in primary culture, with these exhibiting typical fusiform cell bodies with long moniliform telopodes. Based on flow cytometric sorting with antibodies to CD34 and PDGFRα, there was a substantial increase in the purity of cardiac TCs. Furthermore, immunofluorescence demonstrated that almost all the sorted TCs expressed vimentin, a marker of TCs. Moreover, electron micrographs showed typical TCs based on their ultrastructural features. Using this method, we developed a reproducible protocol for the isolation and purification of cardiac TCs from rat hearts, which yielded TCs with typical characteristics.

Figure 1

TCs shown under a light microscope in primary culture. (A) A few cells had adhered 72 h after isolation. (B) Arrows show typical TCs with long, thin Tps at 96 h in primary culture. (C) TCs with typical moniliform aspects: Alternation of podoms and podomeres (as shown in black frame). (D) TC connections were formed by Tps and the cell body. TCs, telocytes; Tps, telopodes.

Figure 2

Flow cytometry revealed double positive expression of CD34 and PDGFRα (A) before and (B) after cell sorting. (C) Quantification of CD34/PDGFRα double positive cells. Data are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05. CD34, cluster of differentiation 34; PDGFRα, platelet derived growth factor receptor α.

Figure 3

Immunofluorescent staining for vimentin in the sorted cardiac TCs. (A) Fluorescence inverted microscopy showed that nearly all sorted TCs are positive for vimentin (red). Nuclei were counterstained with DAPI (blue). (B) Higher magnification of the telocytes. Staining was concomitant on cell body and thin telopode. Original magnification, ×400. TCs, telocytes.

Figure 4

Transmission electron micrograph of a TC. (A) TCs had a small oval body, mostly occupied by the nucleus, encircled by a small amount of cytoplasm. (B) The structures in the cytoplasm were: m, mitochondria; li, lipid droplets; g, Golgi apparatus; er, endoplasmic reticulum; and NC, nucleus. (C) Higher magnification of a telopode. TC, telocyte.

Figure 5

Transmission electron micrograph showing connections of Tp with TC. (A) Black arrows show direct contacts (atypical junctions) between Tp and TC cell body. Extracellular vesicles: (B) Exosomes, intraluminal vesicles filled with mvbs; (C) ectosomes (arrow) released from the plasma membrane of the Tps; (D) multivesicular cargo (arrow), clusters of endomembrane vesicles encircled by the plasma membrane from a Tp. Tp, telopode; TC, telocyte; mvb, multivesicular body.