Phenomenon of Tissue-Specific Stem Cell Plasticity

Summary: It has been suggested that bone marrow (BM)-derived hematopoietic stem cells transdifferentiate into tissue-specific stem cells (the so-called phenomenon of  stem cell plasticity), but the possibility of committed tissue-specific stem cells pre-existing in BM has not been given sufficient consideration. We hypothesized that tissue-committed stem cells i) circulate at a low level in the peripheral blood (PB) under normal steady-state conditions, maintaining a pool of stem cells in peripheral tissues, and their levels increase in PB during stress/tissue injury; ii) they could be chemoattracted to the BM where they find a supportive environment and that the SDF-1–CXCR4, HGF/SF-c-MET and LIF-LIF R axes play an prominent role in the homing/retention of these cells to BM niches. SDF-1 chemotaxis combined with real-time RT-PCR analysis revealed that i) these early tissue-specific cells reside in normal murine BM, ii) express CXCR4 on their surface and iii) can be enriched (up to 60x) after chemotaxis to an SDF-1, HGF/SF or LIF gradient. These cells were also highly enriched within purified populations of murine Sca-1+ BM MNC as well as of human CD34+, AC133+ and CXCR4-positive cells. We also found that the expression of mRNA for SDF-1, HGF/SF and LIF is upregulated in damaged heart, kidney and liver. Hence our data provide a new perspective on bone  marrow not only as a home for hematopoietic stem cells but also a „hideout” for already differentiated tissue-committed stem/progenitor cells that fpllow an SDF-1, HGF/SF and LIF gradient, could be mobilized into PB, and subsequently take part in organ/tissue regeneration.

Key words: LIF, HGF, SDF-1, plasticity, cell fusion, chemotactic isolation, regeneration.
 

Isolation of Human Skeletal Muscle Stem Cells

Summary: We have shown that early muscles precursors cells (satellite cells) express on their surface CXCR4 receptor. These cells isolated by preplating method combine with cell sorting of CXCR4 positive cells, in in vitro cultures form characteristic for skeletal muscle miotubes. We believe that these cells could be use in the future in regeneration of muscle tissues. Right now we carry on experiments to develop the isolation strategy for clinical use.

Key words: satellitecells, isolation, cell plasticity, CXCR4, SDF-1, regeneration.
 

Endothelial Progenitor Cells: Origin, Characteristics and Their Future Therapeutic Use

Summary: Differentiation of endothelial cells and hematopoiesis are closely linked processes due to common ancestors, hemangioblasts. Endothelial progenitor cells (EPCs) are defined as CD34+ CD133+ Flk-1+ stem cells, which differentiate into endothelial cells in vitro and could be incorporated into sites of pathological neovascularization in vivo. Endothelial progenitors have been isolated from human bone marrow, umbilical cord blood and GM-CSF-mobilized peripheral blood. To date, most studies on EPCs differentiation concerned their phenotypic and biological characteristics. Presently, the possibilities of therapeutic use of the isolated endothelial progenitor cells are considered. Methods are developed to isolate and propagate these cells for their future application, for repairing of damaged or degenerating endothelia in ischemia and, particularly, myocardial infarction. On the other hand, inhibiting the recruitment of endothelial progenitors might provide a novel approach to limit tumor angiogenesis.

Key words: endothelial progenitor cells, CD34, VEGF-R2, neovascularization, angiogenesis, ischemia, cancer.
 

Advances in Methods of Isolation And Expansion of Human Epidermal Stem Cells

Summary: Human skin is an organ that is highly enriched in stem cells forming holoclones in in vitro cultures. They highly express??1- and ?6- integrins, p63 protein, Delta1 and ?-catenin. Recently these cells acquire a lot of attention both in research and clinical trials because of their potential use in healing of burns and trophic ulcers. However keratinocytes expansions ex vivo from small biopsies are successful employed in the clinic, our knowledge of mechanisms that regulate a renewal and differentiation of skin stem cells is very limited.  Thus aim of our studies was to isolate pure population of epidermal stem cells and expand it subsequently for clinical application.  First we found that the c-kit antigen is a very useful tool to separate keratinocytes from melanocytes in primary cultures. Next, to identify surface markers that could be employed for isolation of skin stem cells we phenotype early keratinocytes with sets of different antibodies (against CXCR1, CD44H, c-met,??3-,5-,6-integrin, FAS). Subsequently to isolate the fraction of early keratinocytes forming in vitro holoclones we stained isolated epidermal cells with antibodies against ?1-integrin in the presence of Rh123. We observed that Rh123 dull ?1-integrin bright cells are highly enriched in cells growing in vitro holoclones. Using this subpopulation of keratinocytes we evaluated a panel of growth factors and cytokines that could influence survival and expansion of early keratinocytes and limit their differentiation. Finally we tried to correlate  our in vitro data with activation of several signaling pathways that are crutial for cell proliferation/survival.

Key words: epidermis, stem cell, in vitro expansion.
 

Participation of Human Stem Cells in Regeneration of Skeletal Muscles –the Mouse Model

Summary: SCID mice have ability to accept ksenogenic grafts. It let us to investigate participation of human stem cells in tissue regeneneration. Regeneration of skeletal muscle is a good model for examining the participation of cells which take part in this process. Regeneration of skeletal muscle depends on presence of the satellite cells (muscle progenitor cells). Except them, stem cells from different tissues can be involved in the regeneration. The target of the project is investigate of the participation of stem cells in regeneration in ksenogenic system. Regeneration of skeletal muscle of SCID mice was described. Also preleminary results of experiment consisting in injection human stem cells from cord blood into SCID mice were described.

Key words: regeneration, stem cells, skeletal muscle..

Characteristic of Maturation of Megakaryocyte Progenitor Cells in in Vitro Culture of Hematopoietic Stem Cells

Summary:  The possibility of isolation of stem cells and in vitro expansion of hematopoietic progenitors creates the possibility of a new generation cellular therapy in treating hematopoietic defects including thrombocytopenia. No in vitro culture has been developed yet, which might yield a sufficient number of megakaryopoietic precursors for clinical purposes. Hence, the aim of the study is to elaborate a technique of ex vivo   expansion of megakaryocyte progenitor cells. The results made the initial optimalisation of culture conditions possible. The analysis of phenotype and morphology confirmed thr differentiation and maturation of cultured cells into megakaryocyte progenitors. The correlation of activity of ?1.6-fucosyltransferase (6FucT) with the number of CD41a+  cells indicates that the activity of this enzyme might be an additional, new sign of differentiation of CD34+ cells into megakaryocytes.

Key words: megakaryocyte progenitor cells, ex  vivo  expansion, ?1.6-fucosyltransferase.
 

Sten Cells in Therapy of Neurologic Disorders

Summary: Although neurodegenerative diseases have different and highly specific causes, the dysfunction or loss of a vulnerable group of neurons is common to all these disorders. It may allow the development of similar therapeutic strategies based on “neural cell replacement concept”. Cell trans plantation has over the last two decades emerged as a promising approach for restoration of function in neurodegenerative diseases, in particular Parkinson’s and Huntington’s disease. Clinical trials have so far focused on the use of implants of embryonic tissue containing already fate-committed dopaminergic neuroblasts with the capacity to develop into fully mature neurons in their new location in the host brain. The recent demonstration that immature neural progenitor cells with multipotent properties can be isolated from both the developing and adult CNS and that these cells can be maintained and propagated in culture, has provided a new interesting tool for restorative cell replacement and gene transfer therapies. The existence of stem/progenitor cells in postnatal bone marrow and umbilical cord blood and their possibility to proliferate and differentiate into nervous system cells, opens up the possibility of using stem cells found in these tissues to treat degenerative and post-traumatic diseases of the central nervous system. The purpose of this review is to discuss the prospects of the emerging progenitor cell technology for cell replacement and restorative therapies in neurodegenerative diseases, and consider some of the critical issues that must be solved in order to make stem cells useful in brain repair.

Key words: neurodegeneration, ischemia, stem cells, neuronal progenitors, endogenous stem cells.
 

An Optimization of Isolation of Early Hematopoietic Cells From Heparinized Cadaveric Organ Donors (HCOD)

Summary: The heparinized cadaveric organ donors (HCOD) are an important source of vascularized organs for transplantation purposes. The demand for a new potential sources of hematopoietic stem cells (HSC) for transplantations and gene therapy is growing. Hence the HCODs could be treated as the donors of HSCs, which unfortunately are wasted at this moment. However, few attempts have been reported to use cadavers, but not heparinized, as donors of hemopoietic stem cells. The described methods were time-consuming, technically complicated and relatively expensive. Since HCOD are heparinized before an organ donation, blood in their bone marrow cavities remains liquid and can be easily aspirated.  In our studies we evaluated some selected aspects of optimization of isolation of early hematopoietic cells from HCOD. We determined that heparin was the best anticoagulant, useful for obtaining and short-term storage of marrow cells. We also found that the least toxicity to progenitor cells derived from HCODs, from different culture media evaluated, had RPMI. Our other findings indicated that the presence of air in the container used for blood collection led to better protections of the hematopoietic cells. On the contrary, the presence of higher concentration of oxygen in the container was toxic and should be avoided. We found the positive influence of erythrocytes and granulocytes on the clonogenicity of early hematopoietic cells short-term stored at 4oC. We believe that our findings will support the idea of calling for an international initiative to create bank of hematopoietic stem cells obtained from heparinized cadaveric organ donors, which could be employed in transplantology and gene therapy.

Keywords: hematopoietic stem and progenitor cells, heparinized cadaveric organ donors.
 

Origin of Vascular Smooth Muscle Cells in Aortic Allograft in the Rat

Summary: Neointima formation is a major lesion of vascular remodeling. Using rat aortic allograft we have shown that circulating progenitors of bone marrow origin give rise to cells with smooth muscle-like properties during formation of neointimal thickenings. We try to elucidate the mechanisms that control accumulation of progenitors of smooth muscle cell in the arterial wall.

Key words:  stem cell, vascular smooth muscle cells, vascular remodeling.
 

Optimalization of the Methods of Cd 34+ Cord Blood Cells Isolation And Expansion

Summary: The aim of our study was an optimalization of ex vivo expansion of human monocytes and macrophages for potential clinical application. To establish the most efficient expansion protocol we have compared various cell fractions of human cord blood mononuclear cells (more or less enriched in CD34+ cells), different combinations of growth factors and cytokines as well as medium components.

Key words: ex vivo expansion, CD34+ cells, cord blood, monocytes

Maxillary Bone Augmentation Using Autologic Bone Marrow, Hemopoietic Stem Cells And Platelet Rich Plasma: Fractal Analysis of Radiograms

Summary:

Key words: stem cells, bone marrow transplantation, platelet rich plasma PRP, jaw bone augmentation, Bio-Oss.

Research on the Potential Clinical Usefulness of Stem Cells Collected From the Cord Blood

Summary: In the cord blood there exist stem cells capable of repopulation of hematopoietic system and of production of cells belonging to other tissues. Whereas hematopoietic stem cells are relatively well characterized, a presence of the other (nonhematopoietic) cells still causes many controversies. There are papers describing the cord blood cells being equivalent to adult mesenchymal stem cells, however some authors deny their existence, and according to the other hypothesis assume that the other tissues(nerve, bone, muscle etc.) may be produced as the effect of the so-called stem cell plasticity effect. We have identified plastic-adherent stem cell subpopulation capable to produce at least several types of nerve cells. It was impossible to stimulate in vitro transdifferentiation of hematopoietic cells (CD34+, CD45+, plastic-nonadherent) into neurogenesis, nor neural precursors (CD34–, CD45–, plastic-adherent) into hematopoietic differentiation. We suggest that the presence non-hematopoietic precursors in cord blood results from the existence of separate stem cell populations, not from the hematopoietic stem cell plasticity phenomenon.

Key words: Cord blood, Stem cell, Stem cell plasticity.
 

Possibility of Use of Hematopoietic Cells Collected from Different Donors of Cord Blood for Simultaneous Transplantation Into Adult Recipients. Preliminary Evaluation

Summary: IEssential limitation to the use of cord blood as the source of cell for transplantation is limited cell number, usually below 1 billion, what allows for routine transplantation of only children weighting less than 30 kg, while majority of potential recipients possess higher body mass. This has led to an idea of simultaneous use of several units of cord blood, that combined would fullfill requirements for the necessary cell number for adult recipient. One of the potentially main obstacles is a possibility of mutual and negative interaction between cells derived from different units. Therefore, as the first issue we have tested whether simultaneous culture of mixed hematopoietic cells derived from different units would affect the number of hematopoietic colonies formed. We have not found any differences between mean values, but in particular pairs there were both inhibition and stimulation of colony formation when compared to theoretical, calculated values. Additionally, we have also performed a clinical attempt to simultaneously transplant adult man with acute myeloblastic leukemia utilizing two different cord blood units. Patient has become reconstituted with only one. Combined, these preliminary results may suggest that a method based on this principle may reach the routine application.

Key words:  placental blood, bone marrow transplantation, hematopoiesis.