[ULTRASTRUCTURAL CHANGES OF THE STEM CELLS IN THE CYCLE MONOLAYER--SPHERES--MONOLAYER].

Sphere formation can be used to prepare stem cells (SCs) prior to transplantation. Here SCs isolated from human subepicardial adipose tissue were analyzed at different stages of the monolayer-spheres-monolayer cycle by transmission electron microscopy. The results obtained with both adherent-induced and hanging-drop induced spheres were similar. At first 2-3 passages (stage 1), isolated SCs displayed embryonal cell-like ultrastructure. With increasing passage times (stage 2), SCs became bigger and more electron-dark with a multilobed nucleus, well-developed rough endoplasmic reticulum (RER), prominent Golgi apparatus and numerous vacuoles. After 2 h from the initiation of the formation of spheres (stage 3), SCs gathered into clusters and formed desmosome-like intercellular contacts. Their nucleus possessed a large loose fibrillo-granular nucleoli, the cytoplasm was densely packed with disintegrated cisternae of RER, Golgi apparatus was not detected. After 24 h from the initiation of spheres (stage 4), SCs in well-formed spheres exhibited large dense nucleoli and poorly developed Golgi apparatus and RER. One day after sphere dissociating (stage 5), SCs were embryonal cell-like and morphologically similar to the cells of the first stage except for the presence of a large nucleolus and numerous Golgi complexes. After 48 h from sphere dissociating (stage 6), SCs became electron-dark and resembled the SCs of the second stage by the presence of irregularly shaped nuclei and the cetoplasm filled with RER. We interpreted the results as senescence of the SCs with the number of passages after isolation from tissue and a day after dissociation of the spheres and as rejuvenation of the SCs just after sphere dissociation. Further research is needed to reveal the genetic, biochemical and physiological parameters of the SCs on established morphologically distinct stages in order to provide higher-quality cellular material for disease cell therapy.

[Comparative characteristics of the stem cells isolated from subcutaneous and subepicardial adipose tissue].

BACKGROUND: Stem cells (SCs) considerably vary in morphological, immunophenotypic, proliferative, and differentiation characteristics depending on their tissue source. The comparative analysis of their biological properties is essential for the optimal choice of SCs for regenerative therapies. METHODS: Using immunocytochemistry, flow cytometry, histochemistry and real-time RT-PCR, we have investigated SCs obtained from human subepicardial (SEC-AT) and subcutaneous (SC-AT) adipose tissue and cultured under similar culture conditions without any differentiation-promoting factors. RESULTS: The cultures were similar in the high proportion of proliferating cell nuclear antigen (PCNA)-positive cells. In both cultures, immunophenotyping has revealed high expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105, low expression of CD31, CD34 and CD45, and wide variability in CD117, CD146 and CD309 expression. The only distinction in CD marker profile was significantly lower expression of CD90 in SCs from SEC-AT. Histochemical analysis has shown the lack of Oil Red O-positive cells in both cultures and about ten-fold higher number of alkaline phosphatase-positive cells among SCs from SC-AT. In the both cultures, immunocytochemistry has detected similar low expression of slow myosin heavy chain marker MAB1628 and smooth muscle actin marker α-hSMA. Gap junctional protein Connexin-43 expression was markedly higher in SCs from SC-AT, and epithelial cell marker Cytokeratin-19 expression was detected only in these cells. By RT-PCR, GATA4 mRNA was found to be highly expressed only in SCs from SEC-AT. CONCLUSIONS: Our results suggest that SC-AT, as compared with SEC-AT, is richer in epithelial cell and osteogenic progenitors. In turn, SEC-AT possesses cardiomyogenic SCs, and can be considered as an alternative to SC-AT as a source of SCs for cell cardiotherapy.

[The presence and localization of heat shock protein 70 in rat mast cells].

Heat shock protein 70 (Hsp70) is considered not only as a cytosolic stress protein, but also as an extracellular molecule with immunomodulatory and signaling functions that play a role in adaptation to stress on cellular and systemic levels. The active involvement of mast cells in adaptation to stress may be associated with the presence of Hsp70 in secretory granules. Using immunoelectron microscopy, we showed that Hsp70 localized in secretory granules of rat pericardial and peritoneal mast cells. Localization of Hsp70 in rat perinoneal mast cells isolated by centrifugation on Percoll was confirmed by immunoblotting. The proposed involvement of mast cells in production of extracellular Hsp70 and possible functions of Hsp70 inside the mast cells granules are discussed.

[Localization of substance P- and FMRFamide-like immunoreactivity in the atrium of the snail Achatina fulica].

By immunohistochemical and immunocytochemical methods localization of Substanse P (SP) and FMRFamide in the atrium of the snail Achatina fulica was investigated. Nerve fibers innervating the snail atrium contact tightly with the granular cells (GC) situated between muscle and endocardial cells, forming neuroendocrine units. Both neuromediators were found in the cells of the neuroendocrine units. By immunohistochemistry SP- and FMRFamide-immunoreactive material was revealed in the granules of the atrial GC. Elecrtonmicroscopical immunocytochemistry has confirmed the presence of SP- and FMRFamide-immunoreactive material in the granules of the GC and shown their presence in the neurosecretory granules of the nerve endings contacting both the atrial GC and cardiomyocytes.

[Immunolocalization of ANP in mast cells of rat and human pericardium].

It is known that many heart diseases are accompanied by a significant increase in the level of atrial natriuretic peptide (ANP), a regulator of cardiovascular homeostasis, in the pericardial fluid. Cellular sources of ANP in pericardial cavity remain uncertain. By EM immunocytochemistry, we have examined the presence and localization of ANP in rat and human pericardium. ANP-immunoreactive material was revealed in granules of mast cells (MCs) situated in connective tissue of the pericardium. MCs have an oval form and measure about 6.5 x 12.5 and 9.1 x 13.6 microm in the rat and human pericardium, respectively. Density of MC population makes up about 50 and 10 cells/mm2 in the rat and human pericardium, respectively. Our data suggest possible participation of the pericardial MCs in endocrine function of pericardium and in control of the ANP level in pericardial cavity.

[Synthesis of nucleic acids and localization of atrial natriuretic peptide in the crayfish haemocytes].

Three types of cells circulate in haemolymph of the crayfish Astacus astacus: agranular haemocytes (HCs I), small-granule haemocytes (HCs II) and large-granule haemocytes (HCs III). Their proliferation, differentiation and function remain poorly understood. By means of light and electron microscopic autoradiography using [3H]-thymidine, we have revealed that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To determine whether the HCs I are proliferating progenitor cells for the granular HCs, we have analyzed autographs of HC population in 1, 2, 7 and 21 days after a single [3H]-thymidine administration. Contrary to the expectation, we have failed to find labeled HCs II and HCs III. These findings raise doubts on the capacity of the HCs I to differentiate into two other types of HCs. By autoradiography using 3H-uridine, it has been detected that intensity of the RNA synthesis was the greatest in HCs I and less by a factor of two and four in HCs II and HCs III, respectively. Additionally, by EM immunocytochemistry, ANP-like immunoreactivity was revealed in the large granules of the HCs III. We assume that availability of ANP in secretory granules extends the possible functions of the crayfish HCs and suggests their participation in regulation of water-salt balance and immune responses.

[The density of myocardial and pericardial rat mast cells in isoproterenol-induced heart failure].

Myocardial mast cells (MC) respond to cardiovascular pathology. The behavior of MC population in myocardium and pericardium of rats has been studied 24 h, 14, 28 and 60 days after two isoproterenol injections (at 24 h intervals). The extent of heart failure has been estimated by supersonic inspection 28 and 60 days after isoproterenol injections. The density of MCs of different degrees of maturity was estimated on paraffin sections stained with Alcian blue--Safranin. The MC density in myocardium of intact and experimental rats was relatively low: from 4 to 6 cells/mm2. The MC density in pericardium of intact rats was several times higher than in myocardium: 48.6 +/- 13.0 cells/mm2. In 24 h and 14 days after isoproterenol injections the pericardial MC density was 1.5 times higher than in control rats (P < 0.05) at the expense of increase in the number of mature MCs with Safranine-positive granules without the increase in the number of immature cells with Alcian blue-positive granules. In 28 days the pericardial MC density was 2 times higher than in intact rats (P < 0.05) at the expense of increase in number of immature and mature cells. In 60 days after isoproterenol injections the pericardial MC density and the ratio of immature and mature cells compared with control did not reach statistical significance. The changes in pericardial MC population corresponded to severity of heart failure according to functional indices. The findings show active reaction of pericardial MCs on myocardium dysfunction that stimulates the maturation of resident immature MCs in pericardium and migration of immature cells to pericardium of damage heart.

Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica.

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.

[Activation of mast cells after experimental myocardial infarction in 3 week-old rats].

It is known that mast cells (MC) take an active part in regeneration processes in postinfarction heart in adult rats and humans. Behaviour of population of cardial MCs has been studied 20, 60, 75 and 90 days after experimental myocardial infraction induced in 3 week-old and adult rats by ligation of left coronary artery. The density of MC of different degrees of maturity was estimated in atrium and ventricle on paraffin sections stained with Alcian blue - Safranin. Findings were compared with MC density obtained in hearts of intact rats. The MC density in intact 1.5-2.5 month-old rats in atrium and ventricle was about 0.6 cells/mm2, in intact 3.5-4.0 month-old rats in atrium--1.2 cells/mm2, in ventricle--0.6 cells/mm2. The MC density in 3 week-old rats with infarction was significantly higher than in intact rats: 5-fold increase in 20 and 60 days in atrium, and 2-fold increase in 60 and 75 days in ventricle. In 60 days after infarction the MC density in adult rats was 3 times lower in atrium and 2 times lower in ventricle than in the same heart compartments of 3 week-old rats with infarction. After infarction in 3 week-old rats, a relative share of young cells with alcian-positive granules sharply increased in 20 days and then decreased by 60-75 days. This indicates a migration of immature MCs to infracted myocardium and their subsequent differentiation. The MC activation after infraction in young rats may result from a more active immune reaction in younger rats and/or functional peculiarities of their MC.

[Immunocytochemical localization of atrial natriuretic peptide in endothelial and granular cells of the heart of lophotrochozoa]. / Immunotsitokhimicheskaia lokalizatsiia predserdnogo natriiureticheskogo peptida v éndotelial'nykh i granuliarnykh kletkakh serdtsa lofotrokhoforovykh.

In parallel with contraction, vertebrate cardiomyocytes perform endocrine function and produce natriuretic peptides (NP)--ANP and BNP--involved in cardiovascular homeostasis maintenance. ANP-like peptides have been reported also in hearts of some invertebrates, however, their cellular localization was not determined. By electron microscopical immunocytochemistry with polyclonal monospecific antibodies raised against ANP and protein A-gold technique, we have localized ANP-like immunoreactivity in granules within endothelial cells in the heart of the brachiopod Rhynchonella psittacea, the polychaete Arenicola marina, and the gastropod mollusc Achatina fulica--all being representatives of the major phylogenetic group Lophotrochozoa. ANP-like immunoreactivity was also revealed in one of 3 morphologically distinguishable types of granules in the snail heart granular cells. By electron microscopical autoradiography with the use of [3H]-thymidine, the ability for DNA synthesis was demonstrated in heart endothelial cells of the investigated animals. Forms of NP-system organization in hearts of Lophotrochozoa and Vertebrates, and close histogenetic relationships of endothelial and granular cells in the snail heart are discussed.

Ploidy levels and the number of nuclei in cardiomyocytes of the lamprey and fish.

It is well known that polyploidization of cardiomyocytes (CMC) is an essential component of heart growth in the warm-blooded vertebrates. Using the Feulgen cytophotometry of alkali-dissociated cells, we determined the ploidy in CMC of the lower vertebrates: lamprey Lampetra fluviatilis (Cyclostomata), skate Bathyraja maculata (Chondrostei), sterlet Acipenser ruthenus, and Russian sturgeon Acipenser güldenstädti (Ganoids), as well as paradise fish Macropodus opercularis, Amur sleeper Perccottus glehni, and Atlantic salmon Salmo solar (Teleostei). The data obtained have demonstrated a wide variety in CMC ploidy of both cyclostomata and fishes. About 85% of the lamprey CMC contain 2 or more (up to 17) nuclei per cell; with 90 and 10% of the nuclei being, respectively, diploid and tetraploid. Hearts of the skate and sturgeons contain mononucleated diploid CMC. In the perch-like fishes, mononucleated diploid and mononucleated tetraploid CMC make, respectively, 95 and 5%. The salmon heart contains near 50% of mononucleated diploid CMC, 13% of mononucleated tetra- and octaploid CMC, the rest CMC being multinucleated (up to 6 nuclei per cell). In all the examined species, the increased nuclear ploidy is accompanied with a significant increase in the nuclear volume. The number of nucleoli per nucleus does not correlate with the nuclear ploidy level. Evolutionary aspects of CMC polyploidy in chordates are discussed.

Atrial natriuretic peptide in the granular cells of the snail heart.

UNLABELLED: Cardiomyocytes of vertebrates combine contractile and endocrine functions. They synthesize and secrete atrial natriuretic peptide (ANP), which is localized in their specific granules. The presence of ANP has been shown in some tissues of invertebrates, including the heart of molluscs. We have studied localization of ANP in cells of the snail heart. METHOD: The atrial and ventricular tissues of the snail Helix pomatia were studied by electron microscope immunocytochemistry, using anti-ANP antibodies. ANP-immunoreactivity has been detected in granules of granular cells located on the luminal surface of the snail myocardium. These cells are abundant in the atrium being very rare in the ventricle. Granular cells at different stages of maturation were revealed. Immature granular cells have light granules of moderate size with homogeneous tight content, while mature granular cells are huge in size and all their granules are fused together. The material of these granules loosens up and almost completely fills up the cytoplasm. No ANP-immunoreactivity was observed in muscle cells or nerve fibers. A possible origin of granular cells from the cardiac endothelial cells is discussed. The molluscan heart, similar to that of vertebrates, is a bifunctional organ. However, contrary to the heart of vertebrates, in the molluscan heart contractile and endocrine functions are separated between different types of cells.

DNA-Synthesizing Cells in the Heart of Ascidia obliqua (Tunicata).

In vertebrate cardiomyocytes, DNA synthesis and mitosis occur in the presence of myofibrils. The myocardium of the adult vertebrates has no cambial elements. We have studied the ultrastructural features and replicative abilities of the heart cells of Ascidia obliqua, a representative of the tunicates, which are considered to be closely related to the immediate precursors of vertebrates. The ascidian heart is composed of a pericardium and a myocardium. The pericardium consists of mononucleated cells with well-developed Golgi complexes and expanded channels of rough endoplasmic reticulum. The myocardium consists of mono- and binucleated myoepithelial cells, whose polarity is recognizable in basement lamina underlying the basal surface; centrioles, cilia, and a welldeveloped Golgi complex located at the apical side; and cross-striated myofibers, one per cell, running along the basal membrane. Four hours after injection of tritiated thymidine ([3H]Tdr), labeled nuclei were found both in pericardial and myocardial cells. Electron microscope autoradiographs show that the [3H]Tdr-labeled nuclei in the myocardium belong to myofiber-containing cells. We failed to observe mitosis; however, the occurrence of centrioles and the high number of binucleated myocytes testify to the ability of these cells to undergo karyokinesis. Large numbers of [3H]Tdr-labeled and prophasic nuclei have been observed in the raphe region, the site of transition of the pericardium into myocardium. The morphological features, such as loss of labyrinth junctions and the acquisition of gap junctions, that distinguish the cells of the raphe region from ordinary pericardial cells give evidence of their premyocytic nature. The resemblance of ascidian myocardium to the vertebrate embryonic one and the presence of cambial zone in the ascidian heart are discussed.

DNA and sex chromatin content in nuclei of conductive system and working myocytes of normal and hypertrophied human heart.

DNA content was measured by means of Feulgen cytophotometry in myocyte nuclei of sinoatrial und atrioventricular nodes, Purkinje cells, left and right atria and ventricles from normal and hypertrophied human hearts both on 12 microns sections and on squash preparations of myocardial tissue. In contrast to ordinary atrial and ventricular myocytes of normal and hypertrophied adult hearts, which contain mostly polyploid nuclei, the DNA content in 74 to 88.5% of myocyte nuclei from sinoatrial and atrioventricular nodes does not exceed 2 c. Unlike nodal myocytes in Purkinje cells of hypertrophied adult heart there are 97 to 99.5% of polyploid nuclei, 49 to 88% of them displaying octaploid and higher DNA values. Myocytes of highly hypertrophied atria contain nuclei of considerably higher ploidy level than those of normal atria. Percentage of binucleated cells show a 4-fold increase in hypertrophied atria compared with normal ones. The total area of nucleoli per nucleus is proportional to the degree of ploidy. Number of sex chromatin bodies counted simultaneously with DNA measurements on sections of atrioventricular node and on squash preparations of atria and ventricles is correlated with ploidy level. Judging by correlations between the number of sex chromatin bodies and DNA content in nuclei, on one hand, and between the total area of nucleoli per nucleus and the degree of ploidy, on the other, the mitotic endoreduplication (true polyploidization) is responsible for the increased DNA content in nuclei of conductive system, atrium and ventricle.

Ultrastructural differentiation characteristics of rat atrial and ventricular cardiac muscle cells in culture.

Changes in differentiation level of cultured atrial (A) and ventricular (V) cardiac muscle cells from 2-week-old rats have been studied to compare the stability of their morpho-functional characteristics. Cultured A and V cells clearly differ in the form, size, spreading pattern and contractile activity. Comparative electron microscopic analysis of A and V myocytes after 7 days in vitro revealed longitudinally arranged and more numerous myofibrils in V myocytes, whereas A cells show less mature contractile apparatus and greater amounts of free components of sarcomeres scattered about the cytoplasm. Both A and V populations were heterogeneous by differentiation level of individual cells: apart from partial dedifferentiation the degree of the contractile apparatus development depends on de novo formation of myofibrils that underlies the redifferentiation process. Leptofibrils commonly connected to Z-lines of myofibrils were observed in V myocytes. A muscle cells actively produce specific atrial granules in the well-developed Golgi complex. Some of V myocytes also possess rare granules similar to the atrial ones. The data obtained indicate preservation of the differences in biology of A and V myocytes under in vitro conditions and are discussed with special reference to their differentiation state in vitro.

[A morphometric study of myocytes of normal and hypertrophic human atria]. / Morfometricheskoe issledovanie miotsitov normal'nykh i gipertrofirovannykh predserdii cheloveka.

Morphometric study of myocytes of normal and hypertrophic human atria was performed in semithin and paraffin sections. The hypertrophy is followed by the increase of size of both the myocytes and their nuclei: the average nuclei diameter increases from 5.9 to 7.5 microns, the average length from 13.7 to 20.2 microns. The same parameters of the cell change from 13.5 to 18.5 microns and from 121.6 to 143.9 microns. The increase of the nuclei length correlates with the increase of its ploidy. The ratio nucleus cytoplasm remains unchanged as well as the ratio between muscles and connective tissue. The number of binucleated cells in the hypertrophied atrium increases by factor of 3 or 4. The number of myocytes in the atrium was calculated and was equal to 1790.10(6) +/- 241.10(6) under normal conditions and 1890.10(6) +/- 336.10(6) in case of hypertrophy. The mechanisms of the heart weight increase is discussed.

[3H-thymidine and 3H-uridine incorporation into the heart cells of the freshwater crayfish Astacus astacus and the swimming crab Portunus holsatus]. / Vkliuchenie 3H-timidina i 3H-uridina v kletki serdtsa rechnogo raka Astacus astacus i kraba-plavuntsa Portunus holsatus.

DNA and RNA syntheses in the heart cells of two decapod species were investigated with the aid of electron microscopic autoradiography. Isotopes were injected in the cavity of adult animals 4 hours before fixation. 3H-thymidine labeling was found in several satellite cell nuclei and in some particular epicardial cell nuclei. None of myonuclei was labeled. 3H-uridine incorporated in all the nuclei of muscle fibers. Satellite cells were labeled with 3H-uridine very slightly, if at all. Such a peculiarity of biosynthetic processes in the decapod heart satellite cell suggests their myoblastic nature similar to that of satellite cells of somatic muscles. The active 3H-thymidine uptake by the heart satellite cells of adult animals may be accounted for by the permanent growth of the decapods through their whole life span.

[Leptofibrils in rat ventricular cardiomyocytes in vitro]. / Leptofibrilly v zheludochkovykh kardiomiotsitakh krys in vitro.

The presence of leptofibrils in ventricular myocytes of the heart of the 2- and 14-day old rats in 8-day old cultures was shown and their structure was investigated. Leptofibrils are seen only in myocytes on a definite stage of differentiation. Leptofibrils are attached to Z-bands running parallel or perpendicular to myofibrils. The period of their cross striation varies from 135 to 215 nm. Possible functions of the leptofibrils are discussed.

[Quantity of DNA, sex chromatin bodies and nucleoli in the nuclei of the muscle cells of normal and hypertrophied human atria]. / Issledovanie kolichestva DNK, telets polovogo khromatina i iadryshek v iadrakh myshechnykh kletok normal'nykh i gipertrofirovannykh predserdii cheloveka.

Nuclei of myocytes of normal and hypertrophied human heart atrium were studied on squash preparations. Judging by the sex chromatin body pattern, it is the polyploidization that is responsible for the increased DNA contents in these nuclei. The cytophotometric DNA measurements demonstrated the up to 93% occurrence of polyploid nuclei even in myocytes of normal atrium. Myocytes of hypertrophied atrium contained nuclei of higher ploidy degrees. The total area of nucleoli per nucleus was shown to be proportional to a degree of ploidy.