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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

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

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

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).

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

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

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

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

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

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