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Isolation, culture, purification and ultrastructural investigation of cardiac telocytes.

Li YY, Zhang S, Li YG, Wang Y - Mol Med Rep (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Diseases, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai 200092, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f2-mmr-14-02-1194: 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.

Mentions: Following 72 h of primary culture, single adherent cells were observed in a distinct area of the culture dish. These cells were rhombic or irregular in shape, highly refractive, inconsistent in size and shape, clear at boundaries, but with obvious characteristic projections (Fig. 1A). After 96 h of culture, a cell monolayer was observed, with TCs exhibiting the characteristic outline of a small cell body and long moniliform Tps. Additionally, connections between the cells by Tps were observed, with these interconnected in the form of a network (Fig. 1B). TCs are characterized by a small cell body and the presence of Tps. As a distinct feature, Tps have extremely long and thin prolongations, with alternation of podoms and podomeres (Fig. 1C and D). After 7 days, the cells grew to 80% confluence in culture. Following the initial culture of cardiac primary cells, adherent cells were digested and then stained for the markers CD34 and PDGFRα, with positive expression identified by flow cytometry (Fig. 2). The proportions of cells were as follows: CD34−/PDGFRα−, 15.4%; CD34+/PDGFRα−, 0.91%; CD34−/PDGFRα+, 52.2%; and CD34+/PDGFRα+, 31.5% (Fig. 2A). Following cell sorting, the proportion of CD34+/PDGFRα+ cells reached 99.3% (Fig. 2B). The sorted cells were plated and cultured under normoxic conditions.


Isolation, culture, purification and ultrastructural investigation of cardiac telocytes.

Li YY, Zhang S, Li YG, Wang Y - Mol Med Rep (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940097&req=5

f2-mmr-14-02-1194: 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.
Mentions: Following 72 h of primary culture, single adherent cells were observed in a distinct area of the culture dish. These cells were rhombic or irregular in shape, highly refractive, inconsistent in size and shape, clear at boundaries, but with obvious characteristic projections (Fig. 1A). After 96 h of culture, a cell monolayer was observed, with TCs exhibiting the characteristic outline of a small cell body and long moniliform Tps. Additionally, connections between the cells by Tps were observed, with these interconnected in the form of a network (Fig. 1B). TCs are characterized by a small cell body and the presence of Tps. As a distinct feature, Tps have extremely long and thin prolongations, with alternation of podoms and podomeres (Fig. 1C and D). After 7 days, the cells grew to 80% confluence in culture. Following the initial culture of cardiac primary cells, adherent cells were digested and then stained for the markers CD34 and PDGFRα, with positive expression identified by flow cytometry (Fig. 2). The proportions of cells were as follows: CD34−/PDGFRα−, 15.4%; CD34+/PDGFRα−, 0.91%; CD34−/PDGFRα+, 52.2%; and CD34+/PDGFRα+, 31.5% (Fig. 2A). Following cell sorting, the proportion of CD34+/PDGFRα+ cells reached 99.3% (Fig. 2B). The sorted cells were plated and cultured under normoxic conditions.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Diseases, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai 200092, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus