Cardiomyocyte precursors and telocytes in epicardial stem cell niche: electron microscope images

Abstract

A highly heterogeneous population of stem and progenitor cells has been described by light immunohistochemistry in the mammalian adult heart, but the ultrastructural identity of cardiac stem cells remains unknown. Using electron microscopy, we demonstrate the presence of cells with stem features in the adult mouse heart. These putative cardiac stem cells are small (6-10 microm), round cells, with an irregular shaped nucleus, large nucleolus, few endoplasmic reticulum cisternae and mitochondria, but numerous ribosomes. Stem cells located in the epicardial stem cell niche undergo mitosis and apoptosis. Cells with intermediate features between stem cells and cardiomyocyte progenitors have also been seen. Moreover, electron microscopy showed that cardiomyocyte progenitors were added to the peripheral working cardiomyocytes. Telocytes make a supportive interstitial network for stem cells and progenitors in the stem cell niche. This study enhances the hypothesis of a unique type of cardiac stem cell and progenitors in different stages of differentiation. In our opinion, stem cells, cardiomyocyte progenitors and telocytes sustain a continuous cardiac renewal process in the adult mammalian heart.

Fig 1

High resolution light microscopy on toluidine blue stained semithin section (∼1 μm thick ultramicrotome section) of Epon-embedded mouse heart (6 month old) shows the limited space where cardiomyocyte progenitors have been found by electron microscopy. Cardiac stem cell niche is located in the subepicardial area surrounding coronary artery next to the emergence from aorta (rectangle red mark).

Fig 2

(A) Electron microscopy of the niche depicted in Fig. 1 shows the presence of putative cardiac stem cells (CSC), isolated or in small groups, cardiomyocyte progenitors (CMP) and cells with intermediate features (CSC-CMP). All these cells are placed in a loose extracellular matrix. (B) Details at higher magnification from area marked in (A) with a dotted square. The CMP have characteristic leptofibrils (arrow) and is closely assisted by telocytes processes (TCp) which contain few dense granules (asterisk) suggesting paracrine signalling. CM – cardiomyocyte; macro – macrophage; coll – collagen fibres.

Fig 3

Electron microscopy images show putative cardiac stem cells (CSC) in mouse cardiac stem cell niche. (A, B) CSC are small, round cells, with discrete irregular shaped nucleus, large nucleolus (nu), few endoplasmic reticulum cisternae (ER) and mitochondria (m), dense granules (arrows) and numerous ribosomes. Telocyte processes (TCp) are in close proximity or applied on the plasma membrane of CSC (small arrows in Inset). n – nerve fibres. (C) Committed cell with more abundant ER and numerous long mitochondria (m), well-developed Golgi apparatus (G), clusters of vesicles (v) but few filaments (f) and no basal lamina.

Fig 4

Electron microscopy images from epicardial stem cell niche show more differentiated CMP with characteristic leptofibrils (lf). (A) Telocyte (TC) chaperone a low differentiated CMP with distinctive leptofibrils (lf), unorganized myofibrils (f), Golgi apparatus and clusters of mitochondria (m). (B) Higher magnification of CMP from A on a successive ultrathin section demonstrates the presence of well-developed Golgi apparatus (G), cytoplasmic vesicles (v), mitochondria (m), myofibrils (f) and incomplete basal lamina (arrows). (C) Low differentiated CMP with leptofibrils (lf), mitochondria (m), numerous free ribosomes, myofilaments (f) and a continuous basal lamina (arrows). TCp – telocyte processes; A – adipocytes.

Fig 5

Cardiac stem cells in epicardial stem cell niche maintain their dynamic equilibrium through mitosis (A, B) and apoptosis (C, D). (A, B) CSC in prophase of mitosis: condensed chromatin (chr), nuclear envelope breaks (small arrows). Detached nuclear envelope fragments make parallel pairs with ER cisternae in the cytoplasm (arrowhead). Nucleolus (nu) is still visible in (B). (C, D) Apoptosis of stem cells: hypercondensation of chromatin and nuclear fragmentation (nc1–3). Plasma membrane scission (arrows) generates apoptotic bodies containing nuclear fragments. Note the chromatin clumping in a nuclear fragment (nc2 in D) and preserved nuclear envelope. CM – cardiomyocyte; TCp – telocyte processes.

Fig 6

Cardiomyocyte progenitors (CMP) merge with adult mouse cardiomyocytes (CM). Rectangular marked areas in A, C, E are enlarged in B, D and F, respectively. (A, B) Direct cellular contact (arrows) between a committed cell and a cardiomyocyte shows none distinctive junctional structure. Basal lamina of cardiomyocytes encloses the CMP cellular projection (asterisks in B). (C, D) Finger-like cellular projections (dashed arrow) of CMP interacts with neighbouring cardiomyocytes to form adhesive contacts. Asymmetric reinforced contacts (immature desmosomes – arrows) and electron-dense lamellar material (asterisks) in between adjoining cells. (E, F) Completely differentiated desmosomes (arrows) and reduced electron-dense material (asterisk) in gap between contacting membranes of CMP and cardiomyocytes.