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Stable transgene insertion into a host genome irrevocably and unambiguously marks individual cells and all their descendants, i.e., the respective cell clone. Based thereon, retroviral gene marking has become an important tool for investigating the in vivo fate of different cell types, both in animal models and in clinical gene transfer. Moreover, identification of (vector) insertion sites in malignant clones transformed because of insertional activation of proto-oncogenes after experimental as well as therapeutic retroviral gene transfer has resulted in new insights into oncogenic transformation of hematopoietic stem cells (HSCs). However, because of the high sensitivity of the PCR-based methods for insertion site detection, researchers are often confronted with large numbers of different insertion sites/cell clones whose contribution to the given state is hard to judge. A relatively simple ligation-mediated polymerase chain reaction (LM-PCR) method allows the preferential analysis of insertion sites in those cell clones that significantly contributed to the cell pool analyzed. In murine bone marrow transplantation models, we have shown that this method is very useful to analyze the impact of retroviral insertion sites on both malignant and benign clonal dominance of individual repopulating HSC. [ABSTRACT FROM AUTHOR]

Maintenance and differentiation of hematopoietic stem and progenitor cells are controlled by complex interactions with the stroma microenvironment. Stroma-cell interactions can be supported by locally expressed membranespanning cell-surface growth factors. Ligands of the tyrosine receptor kinases subclass III like SCF or CSF-1 are expressed by stroma as soluble glycoproteins, proteoglycans or membrane-bound glycoproteins. SCF synergizes with other growth factors in enhancing growth of early progenitor cells whereas CSF-1 is known to regulate the survival, proliferation and differentiation of mononuclear phagocytes. Whereas the biological role of the soluble isoforms of SCF and CSF- 1 are well characterized, the function of the membrane-bound ligands remain unclear. To analyze the biological significance of membrane-bound SCF and -CSF-1 in vitro we used an epithelial cell line to ectopically express the different isoforms. In cocultures of SCF- or CSF-1 transduced epithelial cells with primary early hematopoietic progenitor cells we examined whether interaction between the membrane-bound isoforms of SCF and CSF-1 and their receptors mediate cell proliferation, self-renewal or differentiation. We show here that the membranebound isoforms of SCF and CSF-1 both have functions in inducing self-renewal of early hematopoietic cells. In contrast, soluble SCF and CSF-1 exert specific functions: SCF by itself causes clonal extinction whereas CSF-1 is involved in the macrophage differentiation. In context with the stroma-hematopoietic cell interaction we also show that CSF-1 can sustain the self-renewal of a murine stem cell line Myl-D7. Keywords: Stroma-hematopietic cell interaction; Stroma-encoded membrane-bound and soluble ligands; Stroma-independent mutants [ABSTRACT FROM AUTHOR]

The hematopoietic system in mammals is comprised of a heterogeneous population of cells which range in function from mature cells of different lineages with limited proliferative potential to multipotent stem cells with extensive proliferative, differentiative and self-renewal capacities. Over a lifetime, the human body produces a trillion blood cells per day. To sustain that enormous cell output, functionally mature cells are generated from highly proliferative, but short-lived progenitors, which in turn arise from a rare population of quiescent hematopoietic stem cells (HSC) that sustain the entire hematopoietic hierarchy. Understanding the composition of HSC compartment is critical for clinical applications such as gene therapy or ex vivo expansion of small hematopoietic samples, for example umbilical cord blood (CB). [ABSTRACT FROM AUTHOR]

Allogeneic hemopoietic stem cell transplantation of matched unrelated donors carries an increased risk of graft versus host disease (GvHD) and transplant related mortality (TRM). We introduced ATG Fresenius at median dose of 90 mg/kg body weight as part of the conditioning regimen for prevention of serious GvHD. We compared 48 recipients of mismatched transplants with 170 recipients of an HLA-matched transplant. The mismatches involved one or two loci. The groups differed in age [HLA-matched: 33 years (0,9-61) HLA-mismatched: 21 years (0,9-51)] and graft source, bone marrow versus peripheral blood stem cell (matched 67% bone marrow, mismatched 83% bone marrow). They were comparable in diagnosis, stage of disease and conditioning. Key words: Mismatched unrelated donor, anti-thymocyte-globuline, stem cell transplantation [ABSTRACT FROM AUTHOR]

The given article gives a short overview about the development and the current structure of the bone marrow transplantation center in Kyiv. A summary on HSCT between 2001 and 2005 in relation to underlying diseases is provided. Keywords: allogeneic and autologous HSCT, Kyiv BMT center [ABSTRACT FROM AUTHOR]

Selectively shaping the T cell repertoire in hemopoietic stem cell transplantation and leukemia immunotherapy towards specific recognition and elimination of malignant and virus-infected cells represents a new therapeutic concept. This concept takes advantage of selectively tolerizing or depleting graft versus host disease (GvHD)-mediating T lymphocytes, while preserving or selecting cytotoxic T cell responses specific for malignant and viral disease. Two specific strategies have been extensively explored in preclinical models for this particular purpose. One strategy is to equip recipient-derived T lymphocytes with "off the shelve" available T cell antigen receptors (TCRs) specific for leukemia-associated as well as human cytomegalovirus (hCMV)- specific antigenic epitopes in order to tackle leukemic relapse and hCMV infection. This strategy is obviously as attractive in immunotherapy of malignant disease by transferring specificity and affinity of TCRs for broadspectrum tumor- and leukemia-associated antigens into T lymphocytes of patients and thus breaking their state of cancer- and leukemia-specific T cell tolerance. Another strategy is to selectively deplete the donor stem cell graft of GvHD-mediating alloreactive T lymphocytes while preserving the integrity of a leukemia- and virus-reactive T cell repertoire within the stem cell inoculum. As these strategies have been successfully developed at the preclinical level, the opportunity of transferring them into clinical application represents a possible current challenge and endeavor. Keywords: Cytotoxic T lymphocytes; T cell antigen receptor; allogeneic hemopoietic stem cell transplantation; acute myeloid leukemia [ABSTRACT FROM AUTHOR]

Retroviral vectors are powerful tools for genetic analysis of stem cells and their progenitors. They have been used both as gene vectors, to both up or down regulate gene expression - as well as mutagens, to identify genes modulating a specific phenotype. Furthermore, their importance in the clinic is currently being tested in several on-going gene therapy trials. Understanding the basic biology of retrovirus is tantamount to developing efficacious tools for the laboratory and the clinic. Here we summarize the characterization of a novel γ-retrovirus isolate from feral mice. The M813 isolate was shown to have a unique host range and belong to a novel interference group. Our analysis also revealed the highly fusogenic potential of this virus. Finally, we were able to identify the sodium myo-inositol transporter as its receptor. The unique characteristics of this viral isolate open several venues for the development of novel research tools. Keywords: retroviruses, virus-cell interaction, cellular receptor, myo-inositol transporter, Mus cervicolor [ABSTRACT FROM AUTHOR]

Institutional achievements in research of low temperature preservation of stem cells derived from fetal and adult sources are presented in the report. Special attention is attended to cryopreservation of pretenders on hemopoietic stem cells from human cord blood and fetal liver. Examining of viability of cryopreserved with DMSO fetal liver cells of specific phenotype by parallel determining with vital dye has demonstrated that CD133+ and CD34+cell candidates occurred to be more sensitive to programmed cryopreservation in comparison with more differentiated erythroid precursors (glycophorin-A - positive cells). No differences in viabilities between CD 45-, CD 133- and CD 34- positive cells after cryopreservation of primary suspension of fetal liver cells was revealed. Cryopreservation of cord blood nucleated cells with PEO-1500 allowed to obtain higher viability of hemopoietic stem CD 34+-cells in respect of CD45+-cells. Presented data demonstrated that hemopoietic cells of human fetal liver and cord blood of various phenotypes were characterized with different sensitivity to cryopreservation. Candidates to stem hemopoietic cells, obtained from two different sources: human fetal liver and cord blood, demonstrate quite a high viability after cryopreservation with various methods using cryoprotectants with different mechanisms of action. Keywords: HSC; cryopreservation; viability [ABSTRACT FROM AUTHOR]

The hybrid cell lines, which we have obtained, can be widely used in veterinary virology and biotechnology for preparing vaccines, test-systems for viruses. Any strains of hybrid cells are producing the biological active proteins (enzymes and others). We have obtained hybrid cell lines (P?-?К?C?, P?- ?К?H?), which are sensitive to prion protein, and also hybrid culture with β-cells of the pancreas of rabbit. Keywords: Stem cells; hybridoma; biotechnology [ABSTRACT FROM AUTHOR]

Development of the long-term models of hematopoietic stem cells (HSCs) cultures and investigation of cell interactions are the important tasks in modern biotechnology. Strategies of hematopoietic progenitor cell expansion in the past decades have made considerable progress. Successful ex vivo expansion of hematopoietic and progenitor cells could be implemented for a variety of clinical applications. Increasing cells numbers of HSC is of a great benefit for transplantation purposes or genetic modification. However, ex vivo HSC expansion for clinical applications encounters many difficulties. It is suggested, that stromal presence is important for hematopoiesis alongside with cytokines in vitro and in vivo, but the question remains: whether diffusible factors produced by stromal cells are sufficient for the regeneration of primitive hematopoietic cells, or whether direct cell-to-cell contacts would be required. Keywords: stem cell expansion; stem cell culture in vitro; diffusion chambers [ABSTRACT FROM AUTHOR]

The RUNX1 gene, which encodes a transcription factor, is a common target of genetic mutations in acute leukemia. We propose that RUNX1 is a gatekeeper gene, the disruption of which leads to the exodus of a subset of selfrenewing "stem" cells from the normal environmental controls of homeostasis. This pool of "escaped" cells is the target of secondary mutations, accumulating over time to induce the aggressive manifestation of acute leukemia. Evidence from patient and animal studies support the concept that RUNX1 mutations are the initiating event in different leukemia subtypes, but also suggests that diverse mechanisms are used to subvert RUNX1 function. One common result is the inhibition of differentiation - but its effect impinges on different lineages and stages of differentiation, depending on the mutation. A number of different approaches have led to the identification of a few secondary events that lead to the overt acute phase, however, the majority are unknown. Finally, the concept of the "leukemic stem cell" and its therapeutic importance is discussed in light of the RUNX1 gatekeeper function. Key words: leukemic stem cell, core-binding factor, differentiation, self-renewal [ABSTRACT FROM AUTHOR]

It is generally accepted that tumor development is a multifactorial process which is caused by a sequential accumulation of different genetic alterations leading to aberrant cell cycle control, instability of genomic integrity as well as decrased recognition by the immune system. During the last decade, the tumor-host interaction has been well defined. It has been demonstrated that professional antigen presenting cells (APC), macrophages, natural killer (NK) cells, NKT cells and in particular T-lymphocytes play a key role in anti-tumor immunity. In general, immune cells monitor MHC class I-presented antigenic peptides. Presentation of self peptides by MHC class I molecules results in the generation of tolerance. In contrast, presentation of viral or tumor-derived foreign antigens leads to the induction of lysis by CD8+ cytotoxic T lymphocytes (CTL). Keywords: tumor immunology, escape mechanisms, MHC class I, CTL, NK [ABSTRACT FROM AUTHOR]

The measurement of telomere length in peripheral blood cells can give an insight into the replicative history of their respective precursor cells, the hematopoietic stem cells (HSC). Much of the observed telomere loss occurs at the stem and progenitor cell level even though these populations express the enzyme telomerase. Investigations in normal steady state hematopoiesis provided the basis for follow up studies in model disorders with increased turnover rates of HSC either due malignant transformation or due to depletion of the stem cell compartment like in defined bone marrow failure syndromes or as a consequence of reduced bone marrow reserve e.g. due to chemotherapy-induced hematological toxicity. In model scenarios like in Chronic myelogeneous leukemia (CML), the degree of telomere shortening can be correlated both with disease duration, disease stage and severity as well as with response to disease-modifying treatment strategies. Whether alterations in telomere biology are secondary phenomena or play a causal role for replicative senescence and/or the induction of genetic instability linked to disease progression in these acquired HSC disorders is under investigation. Keywords: Telomere; Telomerase; stem cell; chronic myeloid leukemia [ABSTRACT FROM AUTHOR]

Studies performed at RCRM have shown that hematopoietic and immune systems' reconstitution after irradiation depends greatly on the functional abilities of the stem cells. Subset analysis and expression of CD34+ antigens on bone marrow and peripheral blood cells were studied in Chernobyl accident clean-up workers including patients with leukemia and myelodysplasia and patients exposed to the natural levels of irradiation. In myelodysplasia the elevation of early CD34+ cells was detected in bone marrow and peripheral blood. In leukemia the CD34+117+38- primitive progenitor cell counts were elevated mainly in patients with proliferation of poorly differentiated cells while in ALL's the CD34+ counts were smaller. Circulating HSC and progenitors after radiation exposure in a wide range of doses have are preserved in a number and with proliferation potencies sufficient for the onset of clonal proliferation. In AML FLT3 mutations are the most abundant single-gene mutations. There is no difference in prevalence of FLT3 mutations in groups of radiation-associated and spontaneous AML cases. LOH/deletions at 5q and/or 7q and 7? tend to be more frequent in radiation-associated AML cases. Bone marrow and bone tissue microenvironment plays a key role in normal and neoplastic HSC changes. Differentiation to B-lineage isn't changed and is associated with B-cell compartment growth. Keywords: stem cells; radiation; leukemia; Chernobyl [ABSTRACT FROM AUTHOR]

Recent experimental results in culture and in vivo are summarized that show the existence of developmental relationships between cells that build up blood vessel walls and some previously unrelated tissues and organs. It was formerly demonstrated, in lower vertebrates as well as mammals, including humans, that discrete subsets of blood-forming endothelial cells play a key role in the emergence of the definitive hematopoietic system. We have also documented the existence in human skeletal muscle of endothelium-borne, extremely potent myogenic progenitor cells. Finally, we have characterized and purified perivascular cells - or pericytes - from human tissues and demonstrated their ability to give rise to mesodermal differentiated derivatives, principally skeletal muscle. Keywords: Endothelial cell; pericyte; stem cell; blood vessel; skeletal muscle [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) correlates with poor survival of patients due to delayed diagnosis and frequent recurrence after adjuvant treatment. The majority of malignant hepatic lesions shows a tremendous heterogeneity in differentiation patterns and molecular signatures which challenges efficient therapeutic intervention. Here we discuss aspects on which route hepatocytes progress towards epithelial lineage commitment during liver repopulation and further resume configurations of hepatocyte differentiation during liver tumorigenesis. We particularly focus on the epithelial to mesenchymal transition (EMT) of hepatocytes and its consequences upon the progression of HCC which depends on the synergy of transforming growth factor (TGF)-β signaling and the hyperactivation of Ras-subeffectors. Recent insights into the epithelial plasticity of malignant hepatocytes revealed that activation of platelet-derived growth factor (PDGF) links TGF-β signaling to nuclear β-catenin accumulation upon EMT. PDGF-dependent activation of β- catenin reduces cellular turnover and provides protection of malignant hepatocytes against anoikis, the latter known as a prerequisite for dissemination of carcinoma and a feature of metastatic cancer stem cells. Finally, we discuss the cancer cell fate determination by integrating the repertoire of hepatocellular differentiation in a novel concept of liver carcinoma progression. Keywords: Hepatocyte; liver progenitor cell; TGF-β; β-catenin; cancer stem cell [ABSTRACT FROM AUTHOR]

Novel therapies for the treatment of heart and kidney diseases are urgently needed. Because bone marrow derived cells have shown tremendous promise we utilized common animal models for myocardial infarction (MI) and acute kidney injury (AKI) to test the potential of mesenchymal stem cells (MSC) as a tool for organ regeneration. Cell tracking was accomplished in vivo with MRI after iron loading of MSC and postmortem by EGFP or Y-PCR. MSC did ameliorate tissue injury and enhance recovery in both models. MSC treated rats with MI showed a decreased infarct area and thickened myocardium compared to controls, however, no significant amounts of cells could be found in hearts of treated animals after 10 weeks of observation. Rats with AKI had improved recovery of renal function as determined by serum creatinine. Initially, MSC could be located in the renal cortex, however numbers declined progressively and MSC could not be shown to differentiate into tubular cells, thereby excluding replacement of injured kidney cells as mechanism of action. Notably, MSC treated kidneys had higher proliferative and lower apoptotic indices. Expression of proinflammatory cytokines IL-1β, TNF-α, IFN-γ, and Keywords: myocardial infarction; acute kidney injury; cell therapy; adult stem cells [ABSTRACT FROM AUTHOR]

Hematopoietic system in vertebrates hinges on the development of novel organ not present in other organisms and called bone marrow that is located within bones. In order for this organ to develop, first bones have to develop that occurs in bony fish and then cellular mechanism of creating the space inside them. This cellular mechanism is dependent on a single new type of cell called the osteoclast that is resorbing bone. Identification of genes whose products are critical for that process should be helped by the analysis of a disorder that is due to its disturbance. Such disorder called marble bone disease or osteopetrosis was first discovered in man about one hundred years ago and since then identified in many vertebrate species. It is rare and sometimes fatal disease that is due to smaller or absent bone marrow cavities and disturbed development of bone marrow, In extreme cases, there is no bone marrow. Keywords: osteopetrosis; osteoclast; bone resorption [ABSTRACT FROM AUTHOR]

When getting old, a lot of unpleasant events are developing in our organism. Here, we should mention the decline or gradual loss of function and potential and, ultimately, death of cells of different types. Many data exist about age-dependent deterioration of nervous and muscle cells as well as about many proliferating cell populations, such as epithelial, osteogenic, hemopoietic and others. These changes manifest themselves in a number of old age diseases like the Alzheimer's disease, Parkinsonism, heart and brain infarction, osteoporosis, diabetes, immune deficiency, malignancies and others. So, it takes to elaborate some technologies and methods for improving the situation by stimulation or replacement of the impaired organs and tissues. The dream is to launch a medical revolution, in which failing organs and tissues might be repaired not with crude mechanical devices like insulin pumps or titanium joints, but with the living homegrown replacement. And great promises here make now the use of stem cell technologies. Keywords: stem cells; aging, gerontology [ABSTRACT FROM AUTHOR]

The history of the role of hematopoietic growth factors (HGFs) in the regulation of hematopoiesis is briefly reviewed, commencing with the studies that defined the hematopoietic stem cell and led to the development of in vitro assays for HGFs. The nature of HGF synergism and the underlying mechanisms, as well as the "permissive" versus "instructive" models of HGF action, are also discussed. In addition, work on one of the HGFs, colony stimulating factor-1 (CSF-1), is reviewed, including its function in development as revealed by studies of mouse mutants and its role in innate immunity, inflammatory disease and cancer. Keywords: blood cells, macrophages, osteoclasts, review, CSF-1 [ABSTRACT FROM AUTHOR]

G-CSF is a major extracellular regulator of hematopoiesis and the most used cytokine in clinical practice. Coherently with and for a long time after the repeated injections of low doses of G-CSF the study of alterations in hematopoietic precursor cells concentration in the bone marrow of mice was undertaken. G-CSF treatment did not affect the number of granulocytes and oligopotent precursor cells (CFU-C). However, frequency of early multipotent stem cells (LTC-IC) decreased one month after the last (7th) course of G-CSF injections, moreover it halved during the following year. The exhaustion of LTC-IC after G-CSF treatment is discussed. Keywords: granulocyte colony-stimulating factor (G-CSF); LTC-IC; CFU-C; granulocytes [ABSTRACT FROM AUTHOR]

Using a long-term serial bone marrow transplantation assay we have recently observed that retroviral gene marking may result in both benign clonal dominance as well as malignant transformation of single cell clones. The growth advantage of dominant clones has been attributed to insertional mutagenesis, i.e. transcriptional dys-regulation of key growth-regulatory genes due to near-by vector insertions. In order to investigate the physiological role of the CD34 antigen we have of recent performed an analogous serial bone marrow transplantation assay with retroviral vectors encoding murine fulllength or truncated CD34 or, as control, eGFP followed by BM transplantation with long-term follow-up. Therefore, 6 animals were serially transplanted for each of the three transgene groups according to our previously published protocol. Similarly to our earlier results, in long-term repopulating bone marrow stem cells we found insertions into genes shown to be involved in cell cycle regulation and stem cell self-renewal such as a Core-binding factor α group member (Cbfα2t3h), runt-related Runx3, Ras p21 protein activator 4 (RasA4), Hematopoietically expressed homeobox (Hhex) or FBJ Osteosarcoma Oncogene B (Fosb). We detected common insertion sites (CIS) within the three groups, but also within the much larger insertional dominance database (IDDb). However, despite the small group size some differences in insertion site patterns were noted for the different transgenes requiring further investigation. It is therefore tempting to speculate that common features as well as differences in insertion pattern of retroviral vectors expressing different transgenes may allow investigating the mutual influence of retroviral vector insertion sites (RVIS), transgenes and host factors during insertional mutagenesis. Keywords: CD34; bone marrow transplantation; retroviral vector; insertional mutagenesis [ABSTRACT FROM AUTHOR]