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Telocytes accompanying cardiomyocyte in primary culture: two- and three-dimensional culture environment

Abstract Recently, the presence of telocytes was demonstrated in human and mammalian tissues and organs (digestive and extra-digestive organs, genitourinary organs, heart, placenta, lungs, pleura, striated muscle). Noteworthy, telocytes seem to play a significant role in the normal function and regeneration of myocardium. By cultures of telocytes in two- and three-dimensional environment we aimed to study the typical morphological features as well as functionality of telocytes, which will provide important support to understand their in vivo roles. Neonatal rat cardiomyocytes were isolated and cultured as seeding cells in vitro in two-dimensional environment. Furthermore, engineered myocardium tissue was constructed from isolated cells in three-dimensional collagen/Matrigel scaffolds. The identification of telocytes was performed by using histological and immunohistochemical methods. The results showed that typical telocytes are distributed among cardiomyocytes, connecting them by long telopodes. Telocytes have a typical fusiform cell body with two or three long moniliform telopodes, as main characteristics. The vital methylene blue staining showed the existence of telocytes in primary culture. Immunohistochemistry demonstrated that some c-kit or CD34 immuno-positive cells in engineered heart tissue had the morphology of telocytes, with a typical fusiform cell body and long moniliform telopodes. Also, a significant number of vimentin+ telocytes were present within engineered heart tissue. We suggest that the model of three-dimensional engineered heart tissue could be useful for the ongoing research on the functional relationships of telocytes with cardiomyocytes. Because the heart has the necessary potential of changing the muscle and non-muscle cells during the lifetime, telocytes might play an active role in the heart regeneration process. Moreover, telocytes might be a useful tool for cardiac tissue engineering.

The current study confirmed that telocytes exist in the heart, including myocard, endocardium and epicardium [1,4,5,[22][23][24][25]. Located between cardiomyocytes, blood capillaries and nerve endings, they appear to create a network with roles for the function and regeneration of heart [1,4,23,25]. Nevertheless, the mechanism has not yet been entirely understood due to very few reports on telocytes in vitro. Our study attempts to isolate and culture telocytes from neonatal rat cardiac tissues in vitro to study the appearance and characteristics of telocytes and to get more biological insights of the roles of telocytes in heart. # These authors contributed equally to this work.  [26]. Considering the positive reports of telocytes on the normal heart structure, function and regeneration in vivo, we assumed that telocytes may have important effects to improve the quality of engineered heart tissue and to ensure the normal function of cardiomyocytes. However, there are no reports on telocytes in engineered heart tissue so far.
The purpose of this study was: (1) to culture telocytes in vitro in two-dimensional environment and identify their morphology and characteristics; (2) to investigate them in three-dimensional environment combined with the construction of engineered cardiac tissues and (3) to provide an in vitro model to investigate the roles of telocytes in heart.

Newborn rat cardiac myocytes
We used the method as previously described in our lab [26]. In brief, cardiac myocytes were isolated from 1-day-old Wistar rats. The ventricles were minced and digested with 0.1% trypsin (Sigma, St. Louis, MO, USA) solution to release cells. The isolated cells were pre-plated on the tissue culture dishes for 1 hour to purify the cell suspension with cardiac myocytes by allowing fibroblasts attachment. The unattached cells were collected and cultured in medium.

Engineered cardiac tissue construction
In order to investigate telocytes in a three-dimensional environment, we constructed engineered cardiac tissue in collagen/Matrigel scaffolds using a refined method previously described [27]. In brief, 0.5 ml concentrated 2ϫ H-DMEM (Invitrogen, Carlsbad, CA, USA) culture medium containing 20% FBS (Invitrogen) was mixed with 0.5 ml of liquid collagen type I and a basement membrane protein mixture (Matrigel; Becton Dickinson Biosciences, San Jose, CA, USA) in 4:1 (v/v). The pH of the mixture was neutralized immediately by titration with 0.1 mol/l NaOH. A total of 1.0 ϫ 10 7 freshly isolated cardiomyocytes were mixed with the mixture and pipetted into casting moulds for incubation. After 60 min., 1.0 ml serum-containing culture medium (DMEM, 10% FBS) was added to the dish. The culture medium was changed daily.

Telocytes in two-dimensional cultures Phase contrast microscopy
In primary culture of cardiomyocytes isolated from myocardial tissues, the typical telocytes were detected. They have a fusiform cell body and two long prolongations (Fig. 1A). The morphology of telocytes in vitro is different from cardiac cells. After 7 days of culture, typical morphological features of telocytes appeared under light microscope: a small oval-shaped cell body with extremely long and thin moniliform prolongations (telopodes) extended from the cell body (Figs. 1 and 2). Telocytes often accompanied the cardiomyocytes and their telopodes ended up on cardiomyocytes. However, telocytes with their long prolongations interconnected cardiomyocytes and accelerated their synchronously beating (Figs. 3 and 4 and Movie S1).

Methylene blue staining
Methylene blue staining further confirmed the existence of telocytes in primary culture. We used supravital methylene blue staining to identify telocytes in vitro, as recommended in [12][13][14]. Methylene blue staining showed that telocytes cultured in vitro had long and extremely thin prolongations -telopodes (Fig. 5A).  They connected neighbouring cardiac cells making a consistent beating 'syncytium' (Fig. 5B).

Telocytes in three-dimensional engineered heart tissues Histology
Conventional histological procedures revealed two type of cells in engineered heart tissue: (i) cardiac myocytes (with rod-shaped body) and nuclei (centrally located and elongated) oriented longitudinally (Fig. 6A); (ii) telocytes, as a distinct cell type with two or three long telopodes. These cells appeared with spindle or triangle shape, depending on the number of the telopodes. There were dense nodular or vacuolar structures along the prolongation (Fig.  6B-C) -podoms [2].

Immunohistochemistry
We used immunohistochemical staining for antigens expressed in telocytes: c-kit, CD34 and vimentin. c-kit or CD34 ϩ telocytes existed in engineered heart tissue with long cell processes (Figs 7  and 8). In addition, a number of vimentin ϩ cells were observed in engineered heart tissues (Fig. 9A). Among these cells, telocytes appeared with long and extremely thin telopodes. Vimentin is discontinuously expressed along these telopodes (Fig. 9B), presumably due to the tortuous telopodes' three-dimensional path.

Discussion
We isolated and cultured cardiomyocytes in primary culture in vitro and observed typical telocytes among the cardiomyocytes.      The typical morphological characteristics of telocytes cells include a fusiform or a triangular cell body and two or three long moniliform prolongations (telopodes). The telocytes distributed among cardiomyocytes seem to connect them to beat synchronously. Furthermore, we have detected telocytes in three-dimensional engineered heart tissues. Histological staining showed that telocytes with two or three long prolongations were present in engineered heart tissue. Immunohistochemistry confirmed that telocytes were immuno-positive for c-kit, CD34 and vimentin in engineered heart tissue. Our study has confirmed that typical telocytes do exist in primary culture in vitro. We have also established the model of three-dimensional engineered heart tissues for the ongoing research of the mechanism of telocytes to cardiomyocytes.
We used immunohistochemical identification c-kit, CD34 and vimentin antibodies to identify telocytes in engineered heart tissues. However, telocytes shared some markers with other types of cells [1], e.g. cardiac stem cells, which express c-kit [26][27][28][29][30], endothelial cells known for CD34 positivity [1,31] and fibroblasts, which may express vimentin [16]. In addition, although we used pre-plating for 1 hr to remove fibroblast from primary culture, we failed to completely clear the fibroblasts. Therefore, as a component of seeding cells, fibroblast existed in engineered heart tissues with strong vimentin expression. As an identifying method for telocytes, it will also depend on different markers in bi-labelling or tri-labelling immunohistochemistry to this end. A certainty in identification of telocytes is based upon the results of transmission electron microscopy examination.   Any queries (other than missing material) should be directed to the corresponding author for the article.