The study of adhesive properties of multipotent mesenchymal stromal cells evaluated from fibroblast colony-forming units in the bone marrow of adult mice and rats in populations of cells attached and unattached to plastic substrate after 2 h to 7 days in culture demonstrated both similarities and differences. The increase in the fibroblast colony-forming units in the adhesive population peaked on day 7 of in vitro culture in both cases; however, nearly no fibroblast colony-forming units were observed in the nonadhesive population from the mouse bone marrow in this period. Conversely, the number of colonies from the rat bone marrow nonadhesive population on day 7 of culture considerably increased, and this nonadhesive population in long-term culture became the source for subsequent nonadhesive subpopulations containing fibroblast colony-forming units. After 7 days of in vitro culture, the suspension of cells isolated from the liver of 17-day-old rat fetuses also contained a fraction of unattached fibroblast colony-forming units. In the nonadhesive subpopulations from the bone marrow and fetal liver, fibroblast colony-forming units were observed up to day 48 and 30, respectively. Stromal cell precursors of nonadhesive subpopulations from the rat bone marrow featured a period of colony formation reduced to 7 days (i.e., they were formed 1.5-2 times faster compared to the primary culture). The total number of fibroblast colony-forming units from all nonadhesive subpopulations was roughly 6 and 7.4 times that of the adhesive population of the primary culture from the bone marrow and fetal liver, respectively. Considering that the mammalian bone marrow remains the preferred source of mesenchymal stromal cells, using nonadhesive subpopulations in the presented culture system can considerably increase the yield of stromal precursor cells.

Permanent lines of pluripotent stem cells can be obtained from humans and monkeys using different techniques and from different sources--inner cell mass of the blastocyst, primary germ cells, parthenogenetic oocytes, and mature spermatogonia--as well as by transgenic modification of various adult somatic cells. Despite different origin, all pluripotent lines demonstrate considerable similarity of the major biological properties: active self-renewal and differentiation into various somatic and germ cells in vitro and in vivo, similar gene expression profiles, and similar cell cycle structure. Ten years of intense studies on the stability of different human and monkey embryonic stem cells demonstrated that, irrespective of their origin, long-term in vitro cultures lead to the accumulation of chromosomal and gene mutations as well as epigenetic changes that can cause oncogenic transformation of cells. This review summarizes the research data on the genetic and epigenetic stability of different lines of pluripotent stem cells after long-term in vitro culture. These data were used to analyze possible factors of the genome and epigenome instability in pluripotent lines. The prospects of using pluripotent stem cells of different origin in cell therapy and pharmacological studies were considered.

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

Our study showed that protamine (80% w/w to DNA) effectively protected its molecules from degradation by native nucleases of the mammalian blood serum. Exogenous DNA bound to protamine effectively stimulated restoration of cyclophosphamide-induced leukopoiesis in mice. It is suggested that the phenomenon was due to repair processes taking place in hemopoietic stem cells damaged by a cross-linking cytostatic drug such as cyclophosphamide. DNA dosage may be reduced and the original DNA fragment size maintained by DNA binding to protamine. As a result, it might involve longer DNA fragments into repair processes of homologous recombination and eventually increase the cell's chances of getting rid of extensive damage.

Systemic transplantation of mesenchymal stem cells (MSC) is known to promote reparative process in a number of tissue damage as well as mucous healing one. It provided the basis for our study which was aimed to the effect of systemic transplantation of allogenic MSC (intravenous transfusion) in complex therapy of patients with Ulcerative Colitis.

The influence of cytokine LIF (Leukemia Inhibitory Factor) on the viability, and proliferation of mouse R1 line embryonic stem cells (ESC) and their distribution by cell cycle stages has been investigated. LIF (5-20 ng/ml) increased growth of colonies and maintained high proliferative and pluripotent properties of R1. LIF was also involved into the inhibition of spontaneous cell differentiation and apoptotic cell death; it also decreased the rations of S/G2+M cell cycle and doubling-time of cell population.

The study undertaken 3 years ago examined the effect of systemic transplantation of autologous mesenchymal stem cells (MSC) in the complex therapy of 27 patients with pulmonary tuberculosis, including 15 patients with multidrug-resistant pulmonary tuberculosis and 12 with extensive drug resistance of Mycobacterium tuberculosis. All the patients were bacteria-discharging persons with disseminated destructive processes in lung tissue, most (n=17) of them had chronic fibrocavernous tuberculosis. In all the patients, previous long specific antituberculous treatment was ineffective or inadequately effective. After systemic MSC transplantation, 16 patients were followed up for 1.5-2 years or more and the remaining 11 patients for at least 6 months. After MSC administration, a positive clinical effect was observed in all 27 cases; bacterial discharge stopped in 20 patients after 3-4 months; resolution of sustained lung tissue cavities further occurred in 11 patients. At present, a persistent remission of a tuberculous process may be stated in 9 of the 16 patients in whom MSCs were transplanted 1.5-2 years, significant positive bacteriological and morphological changes are observed in 6 patients. Thus, inclusion of transplantation of the autologous MSCs propagated in the culture into a course of antituberculous therapy may be a promising procedure for enhancing the efficiency of therapy in patients with resistant forms of pulmonary tuberculosis.

Cellular populations with phenotype similar to multipotent mesenchymal stromal cells have been isolated from two different sources: a human bone marrow and adipose tissue. The comparative analysis of differentiation efficiency of these cells in the direction of osteogenesis revealed morphological changes confirmed by Alizarin red and von Kossa staining in bone marrow cells on the 14th day, and in adipose tissue cells on the 28th day of culturing in the medium with inductors. The analysis of osteopontin, osteocalcin and bone sialoprotein gene expression in RT-PCR reactions detected essential distinctions in the potency of these cells to differentiate into bone tissue cells.

At present there exists a limited understanding of mechanisms of differentiation and specialization of cardiac conduction system in mammals. For characterization of its development and differentiation on early stages we used as experimental model murine embryonic stem cells expressing enhanced green fluorescent protein (eGFP) under transcriptional control of human atrial natriuretic peptide (ANP) as promoter. Cardiac nature of ANP-eGFP was confirmed by immuno staining with antibodies to troponin I and a-actinin. In ANP- eGFP expressing embryonic stem cells it is possible to distinguish subpopulation of spindle-shaped pacemaker cells capable to higher frequency of spontaneous contractions, velocity of activation and amplitude compared with those of triangular and polygonal atrial-like cardiomyocytes. These results show that expression of ANP-eGFP cells allows to identify pacemaker cells among embryonic stem cells by their morphological and electrophysiological properties.

Failure of injured axons to regenerate in the central nervous system (CNS) is the main obstacle for repair of stroke and traumatic injuries to the spinal cord and sensory roots. This regeneration failure is highlighted at the dorsal root transitional zone (DRTZ), the boundary between the peripheral (PNS) and central nervous system where sensory axons enter the spinal cord. Injured sensory axons regenerate in the PNS compartment of the dorsal root but are halted as soon as they reach the DRTZ. The failure of regenerating dorsal root axons to re-enter the mature spinal cord is a reflection of the generally non-permissive nature of the CNS environment, in contrast to the regeneration supportive properties of the PNS. The dorsal root injury paradigm is therefore an attractive model for studying mechanisms underlying CNS regeneration failure in general and how to overcome the hostile CNS environment Here we review the main lines which have been pursued to achieve growth of injured dorsal root axons into the spinal cord: 1) modifying the inhibitory nature of the DRTZ by breaking down or blocking the effect of growth repelling molecules, 2), stimulate elongation of injured dorsal root axons by a prior conditioning lesion or administration of specific growth factors, 3) implantation of olfactory ensheathing cells to provide a growth supportive cellular terrain at the DRTZ and 4) replacing the regeneration deficient adult dorsal root ganglion neurons with embryonic neurons or neural stem cells.

Proteomic studies of some human tissues and organs (skeletal muscles, myometrium, motor zone of the brain, prostate), and also cultivated myoblasts revealed 41 of 300 identified proteins, in which the present of certain variants of amino acids ("conflicts") was recognized at several positions. Among the 93 registered amino acids "conflicts", seven cases represented the results of the protein polymorphisms caused by corresponding substitution of individual amino acid. Moreover, among prostate proteins the proteomic analysis revealed two isoforms of prostate-specific antigen, formed due to alternative splicing. Thus, our results have shown, that proteomic technologies allow to specify effectively the features of primary structures and to characterize various kinds of polymorphism in many human proteins.

The insufficiency of liver functions remains one of the major causes of death. The liver transplantation is the most effective method for treating severe liver diseases. The shortage of donor organs and high risk of graft rejection are the main problems for liver transplantation. Stem cells and isolated hepatocytes are alternative means for repopulating the liver after various injuries instead of liver transplantation. This review analyses the experimental and clinical advantage and perspectives on the stem cells use in clinical trials for treating hepatic failure.

The application of etoposide for chemical enucleation of rat oocytes was tested. The reconstruction efficiency after chemical and mechanical enucleation was comparatively analyzed. The obtained data indicate similar viability of reconstructed rat embryos irrespective of the enucleation technique.

The paper analyzes wide range of studies which used the ability of stem cells to exclude vital dyes Rhodamin 123 and Hoechst 33342 as a marker. Data suggest that this marker reflects phenotypical differences between stem cells fractions but is not a characteristic of sternness.

The mesenchymal stromal cell is a multipotent precursor of osteoblasts, adipocytes, and some other cell types. In this study, a comparative analysis of cultured mesenchymal stromal cells from the rat bone marrow at the early and late stages of subculturing has been performed using molecular genetic and cytological methods. The culture has undergone 11 passages during 140 days. Upon long-term culturing, the mesenchymal stromal cells have proved to lose their potential for adipogenic differentiation but preserve the potential for osteogenesis. Morphological characters typical of osteogenic differentiation can be observed at the earlier stages of culturing (passages 1-4) but disappear at later stages (passages 9-11), despite mineralization of the extracellular matrix and the expression of osteogenic differentiation markers. A comparative analysis of the proliferation potential of stromal cells has shown that differences in the period of cell population doubling at the early and later stages of culturing are insignificant. An almost complete arrest of cell growth has been observed in the middle of the culture period (passages 5 and 6).

A cell culture consisting mainly of satellite cells and mononuclear myoblasts was derived from femoral muscles of infant (aged 3-7 days) and adult rats. Satellite cells identified by expression of the specific marker Pax7 accounted for approximately 80% of the isolated cell fraction. Mononuclear myoblasts represented by proliferating and postmitotic cell pools were identified immunocytochemically by the expression of markers Ki67 and desmin. Differentiation of satellite cells and myoblasts in the culture depended on the concentration of Ca2+ in the culture medium (F12 with different Ca2+ concentrations or DMEM). Differentiation of myogenic cells manifested in myoblasts fusion, formation of myotubes, and expression of myosin in myofibrils was observed only in the medium with a high Ca2+ concentration (2 mM). Satellite cells and myoblasts from the muscles of newborn and adult rats did not differ noticeably in their capacity for differentiation.

The goal of this study was the search for and structure-function analysis of the regulatory genes specific for pluripotent embryonic stem cells (ESCs). This was the first study in which PCR was used to obtain DNA fragments with primers constructed on the basis of OCT4 and NANOG mRNA. cDNA synthesized on mRNA isolated from human embryonic eye in the 9.5th week of development was used as a template in PCR analysis. PCR fragment DNA was sequenced. A comparative analysis of the nucleotide sequences demonstrated their 100% homology with the OCT4-pg1 retrogene and NANOG gene. Expression of the genes of interest was reliably detected in the cornea, crystalline lens, retina, and eye tunics in the 10.5th week of development. The nuclear localization of the products of the NANOG gene and OCT4-pg1 retrogene indicates that these proteins are classified with transcription factors. The role of the OCT4-pg1 retrogene and NANOG gene in self-renewal and differentiation of pluripotent cells in a developing eye is discussed.

The studies of CNS neural stem and progenitor cell differentiation both in vitro and in vivo, require the application of highly specific markers of neural and glial cells. The aim of the present investigation was to study the distribution of differentiating neuron marker doublecortin (DCX) expression in different structures of embryonic rat brain and spinal cord before cortical plate formation, using immunocytochemical methods, light and confocal microscopy. The presence of DCX was demonstrated in three types of cells of the developing central nervous system at days 13-14 of embryonic development: neurons which demonstrate positive reaction for nuclear marker of differentiated neural cells NeuN; migrating and maturing neuroblasts; some cells belonging to radial glial cell population. Sufficiently high selectivity of DCX expression allows recommending of its usage in the mammalian CNS early development investigations.

When hybrid cells are created, not only the nuclear genomes of parental cells unite but their cytoplasm as well. Mitochondrial DNA (mtDNA) is a convenient marker of cytoplasm allowing one to gain insight into the organization of hybrid cell cytoplasm. We analyzed the parental mtDNAs in hybrid cells resulting from fusion of Mus musculus embryonic stem (ES) cells with splenocytes and fetal fibroblasts of DD/c mice or with splenocytes of M. caroli. Identification of the parental mtDNAs in hybrid cells was based on polymorphism among the parental mtDNAs for certain restrictases. We found that intra- and inter-specific ES cell-splenocyte hybrid cells lost entirely or partially mtDNA derived from the somatic partner, whereas ES cell-fibroblast hybrids retained mtDNAs from both parents in similar ratios with a slight bias. The lost of the “somatic” mitochondria by ES-splenocyte hybrids implies non-random segregation of the parental mitochondria as supported by a computer simulation of genetic drift. In contrast, ES cell-fibroblast hybrids show bilateral random segregation of the parental mitochondria judging from analysis of mtDNA in single cells. Preferential segregation of “somatic” mitochondria does not depend on the differences in sequences of the parental mtDNAs but depends on replicative state of the parental cells.

Proteoglycans were isolated from extracellular matrix of L6J1 rat myoblasts and their influence on myoblast adhesion was studied. Proteoglycan digestion with chondroitinase AC and heparinase III degrading the polysaccharide moieties revealed that chondroitin sulfate proteoglycans are the main class of myoblast extracellular matrix proteoglycans. Electrophoresis of enzymatically processed proteoglycans was used to examine their core proteins. Myoblast adhesion was suppressed by proteoglycans or the mixture of proteoglycans and fibronectin/extracellular matrix. When being processed with chondroitinase AC the combined substrate of fibronectin and proteoglycans lost the capability of myoblast adhesion suppression. Thus, as a result of presented work the proteoglycans of L6J1 rat myoblast extracellular matrix were isolated and purified. The main class of proteoglycans was chondroitin sulphate proteoglycans. Isolated proteoglycans suppressed myoblast adhesion and this effect was mediated by polysaccharide moieties of proteoglycans.