Emanuel Nečas | Charles University in Prague (original) (raw)
Papers by Emanuel Nečas
Annals of Hematology, 1992
Normal as well as hypertransfused BDF1 mice were exposed to 300 ppm of benzene, 6 h/day, 5 days p... more Normal as well as hypertransfused BDF1 mice were exposed to 300 ppm of benzene, 6 h/day, 5 days per week for 2, 4, and 6 weeks respectively. Erythroid-committed hematopoietic progenitor cells CFU-E were determined in the bone marrow, and 59Fe incorporation was measured in the peripheral blood 48 h after its injection, as an indicator of active erythroid cell production. CFU-E numbers were reduced in benzene-exposed mice at all intervals, as was SgFe incorporation in the peripheral blood after 2 weeks of exposure. In hypertransfused mice the CFU-E suppression, caused primarily by the hypertransfusion, was aggravated by benzene. After injection of 1 IU Ep 4 days before killing the CFU-E numbers and the 59Fe incorporation increased in controls as well as in benzene-exposed animals, but the difference persisted. After nine consecutive Ep injections the difference concerning the femur disappeared after 2 and 6 weeks of benzene exposure but was still present in the peripheral blood. These results suggest that chronic benzene exposure has a negative effect on early erythroidcommitted, Ep-responsive hematopoietic cells.
STEM CELLS, 1998
Murine hematopoiesis has been analyzed by many authors, and available data allow for quantitative... more Murine hematopoiesis has been analyzed by many authors, and available data allow for quantitative evaluation of this dynamic process. In this study, the capacity of several populations of the bone marrow clonogenic cells (progenitors) to produce blood cells was compared with their actual production. The cell cycle progression rate was directly measured in the following types of hematopoietic progenitors: day 8 colony-forming units-spleen, GMcolony-forming cells, BFU-E, and CFU-E in normal mice. The cell cycle progression rates of the individual progenitors, together with their numbers in the whole hematopoietic tissue, were used to calculate the absolute numbers produced daily in each population. The data reviewed from literature were analyzed in parallel. The capacity of the progenitors to produce mature blood cells was derived from the daily production of progenitors multiplied by their clonogenic potential. This theoretical capacity to produce blood cells was compared to the actual blood cell production determined from the turnover of circulating blood elements. The comparison strongly suggested an intensive cell death rate occurring at the early stages of differentiation and its decline as the hematopoietic cells become more differentiated and mature.
Cell Proliferation, 1990
The haemopoietic stem cells forming spleen colonies (CFU-S) had on average 30 to 40% of cells eng... more The haemopoietic stem cells forming spleen colonies (CFU-S) had on average 30 to 40% of cells engaged in the DNA synthesis in normal mice continuously over 4 years. A majority of experiments aimed at the suppression of the CFU-S proliferation, which included suppression of the T-lymphocytes by means of cyclosporin A or by adult thymectomy, administration of antibacterial and antifungal agents and maintainance of mice in a sterile environment, suppression of antibody-producing cells by a successive administration of the bacterial lipopolysaccharide and cyclophosphamide and attempts to increase the total number of CFU-S in the body through massive transfusions of bone marrow cells or by grafting plugs of the bone marrow under the kidney capsulae, have not been sufficiently effective. A transient suppression of CFU-S proliferation occurred during recovery of the haemopoietic tissue from damage caused by cyclophosphamide. The results support the view that changes in CFU-S numbers and in the proportion of them in DNA synthesis may be positively correlated when CFU-S numbers fluctuate physiologically about their normal values. The failure to manipulate the CFU-S proliferation rate easily suggests that proliferation of these cells may not be under a strong 'switch on - switch off' control.
Haematology and Blood Transfusion / Hämatologie und Bluttransfusion, 1979
The method available at present for the measurement of the pluripotential stem cells (CFUs) is ba... more The method available at present for the measurement of the pluripotential stem cells (CFUs) is based on the formation of the macroscopically visible colonies on the spleen surface of the lethally irradiated mice recipients of the bone marrow cell suspension [9]. This method demonstrates a fraction of the pluripotential stem cells injected [7].
Advances in Experimental Medicine and Biology, 1999
The oxygen sensors monitor oxygen tension in the organism and trigger appropriate feedback respon... more The oxygen sensors monitor oxygen tension in the organism and trigger appropriate feedback responses when the tension declines. Peripheral chemoreceptors in the carotid and aortic bodies, and erythropoietin (Epo) producing cells in the kidney are the most notoriously known oxygen sensors.
Ceskoslovenská fysiologie / Ústrední ústav biologický, 1980
Acta Haematologica, 2003
The goal of the study was to investigate changes in expression of selected growth factors tentati... more The goal of the study was to investigate changes in expression of selected growth factors tentatively involved in regeneration of haematopoietic tissues (bone marrow and spleen) following cyclophosphamide (CY) damage in the mouse. The bone marrow (BM) and spleen were examined separately, since the regenerating pattern for haematopoietic progenitor cells (HPC) markedly differs in these two haematopoietically active organs after CY. Cytokines assumed to have a stimulatory effect on HPC – stem cell factor (SCF), fetal liver tyrosine kinase 3-ligand (flt3-ligand), thrombopoietin (TPO), stromal cell-derived factor 1 (SDF-1), oncostatin M (OSM) –, a suppressive effect on HPC proliferation – macrophage inflammatory protein-1α (MIP-1α), transforming growth factor-β1 (TGFβ1), tumour necrosis factor-α (TNFα) – and to be involved in migration of HPC (SCF, flt3-ligand, MIP1α, SDF-1) were examined at the level of mRNA expression by means of real-time RT-PCR. The expression of a particular cytoki...
Biology of Blood and Marrow Transplantation, 2013
Significant controversy exists regarding the impact of hematopoietic stroma damage by irradiation... more Significant controversy exists regarding the impact of hematopoietic stroma damage by irradiation on the efficiency of engraftment of intravenously transplanted stem cells. It was previously demonstrated that in normal syngenic mice, all intravenously transplanted donor stem cells, present in the bone marrow, compete equally with those of the host. In this study, we comprehensively compared the blood cell production derived from transplanted donor stem cells with that from the host stem cells surviving various doses of submyeloablative irradiation. We compared the partial chimerism resulting from transplantation with theoretical estimates that assumed transplantation efficiencies ranging from 100% to 20%. The highest level of consensus between the experimental and the theoretical results was 100% for homing and engraftment (ie, the utilization of all transplanted stem cells). These results point to a very potent mechanism through which intravenously administered hematopoietic stem cells are captured from circulation, engraft in the hematopoietic tissue, and contribute to blood cell production in irradiated recipients. The damage done to hematopoietic stroma and to the trabecular bone by submyeloablative doses of ionizing radiation does not negatively affect the homing and engraftment mechanisms of intravenously transplanted hematopoietic progenitor and stem cells.
Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. W... more Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNA-synthesizing cells by two thymidine analogues, optimized for <i>in-vivo</i> use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1<sup>+</sup> and Sca-1<sup>−</sup> HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1<sup>+</sup> and Sca-1<sup>−</sup> myeloid progenitors self-renew and differentiate. Division of the Sca-1<sup>+</sup> progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1<sup>−</sup> c...
International Journal of Molecular Sciences
Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the ... more Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2); however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection; liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increase...
Frontiers in Cell and Developmental Biology
The immense regenerative power of hematopoietic tissue stems from the activation of the immature ... more The immense regenerative power of hematopoietic tissue stems from the activation of the immature stem cells and the progenitor cells. After partial damage, hematopoiesis is reconstituted through a period of intense regeneration when blood cell production originates from erythro-myeloid progenitors in the virtual absence of stem cells. Since the damaged hematopoiesis can also be reconstituted from transplanted hematopoietic cells, we asked whether this also leads to the transient state when activated progenitors initially execute blood cell production. We first showed that the early reconstitution of hematopoiesis from transplanted cells gives rise to extended populations of developmentally advanced but altered progenitor cells, similar to those previously identified in the bone marrow regenerating from endogenous cells. We then identified the cells that give rise to these progenitors after transplantation as LSK CD48– cells. In the submyeloablative irradiated host mice, the transpla...
Frontiers in Cell and Developmental Biology
Regeneration of severely damaged adult tissues is currently only partially understood. Hematopoie... more Regeneration of severely damaged adult tissues is currently only partially understood. Hematopoietic tissue provides a unique opportunity to study tissue regeneration due to its well established steady-state structure and function, easy accessibility, well established research methods, and the well-defined embryonic, fetal, and adult stages of development. Embryonic/fetal liver hematopoiesis and adult hematopoiesis recovering from damage share the need to expand populations of progenitors and stem cells in parallel with increasing production of mature blood cells. In the present study, we analyzed adult hematopoiesis in mice subjected to a submyeloablative dose (6 Gy) of gamma radiation and targeted the period of regeneration characterized by massive production of mature blood cells along with ongoing expansion of immature hematopoietic cells. We uncovered significantly expanded populations of developmentally advanced erythroid and myeloid progenitors with significantly altered immunophenotype. Their population expansion does not require erythropoietin stimulation but requires the SCF/c-Kit receptor signaling. Regenerating hematopoiesis significantly differs from the expanding hematopoiesis in the fetal liver but we find some similarities between the regenerating hematopoiesis and the early embryonic definitive hematopoiesis. These are in (1) the concomitant population expansion of myeloid progenitors and increasing production of myeloid blood cells (2) performing these tasks despite the severely reduced transplantation capacity of the hematopoietic tissues, and (3) the expression of CD16/32 in most progenitors. Our data thus provide a novel insight into tissue regeneration by suggesting that cells other than stem cells and multipotent progenitors can be of fundamental importance for the rapid recovery of tissue function.
Methods in Molecular Biology
The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are ro... more The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters. In the current step-by-step protocol, we present' two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.
Cell Cycle
Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. W... more Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNAsynthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1 + and Sca-1 − HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1 + and Sca-1 − myeloid progenitors self-renew and differentiate. Division of the Sca-1 + progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1 − cells arising from cell division entered a new round of the cell cycle, corresponding to symmetric self-renewing cell division. The novel data also enabled the estimation of the cell production rates in Sca-1 + and in three subtypes of Sca-1 − HSPCs and revealed Sca-1 negative cells as the major amplification stage in the blood cell development.
PLOS ONE
Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload an... more Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload and decreased by accelerated erythropoiesis or iron deficiency. The purpose of the study was to examine the effect of these stimuli, either alone or in combination, on the main signaling pathway controlling hepcidin biosynthesis in the liver, and on the expression of splenic modulators of hepcidin biosynthesis. Liver phosphorylated SMAD 1 and 5 proteins
STEM CELLS
Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experime... more Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experiments. When we used ubiquitin-GFP (UBC-GFP) transgenic mice to study the availability of niches for transplanted hematopoietic stem and progenitor cells, the results were strikingly different from the corresponding experiments that used congenic mice polymorphic in the CD45 antigen. Analysis of these unexpected results revealed that the hematopoiesis of UBC-GFP mice was outcompeted by the hematopoiesis of wild-type (WT) mice. Importantly, UBC-GFP mice engrafted the transplanted bone marrow of WT mice without conditioning. There was a significant bias toward lymphopoiesis in the WT branch of chimeric UBC-GFP/WT hematopoiesis. A fraction of immature Sca-1 1 cells in the spleen of UBC-GFP mice expressed GFP at a very high level. The chimeric hematopoiesis was stable in the long term and also after transplantation to secondary recipient mice. The article thus identifies a specific defect in the hematopoiesis of UBC-GFP transgenic mice that compromises the lymphoid-primed hematopoietic stem cells in the bone marrow and spleen. STEM CELLS 2018; 00:000-000 SIGNIFICANCE STATEMENT Tagging cells with genetic markers may unintentionally alter tissue function. Ubiquitin-green fluorescent protein transgenic mice have a diminished population of lymphoid-primed hematopoietic stem cells and engraft transplanted wild-type bone marrow without conditioning. The stem cell niches for lymphoid-primed stem cells are also in the extramedullary spleen tissue.
Folia biologica
The objective of this study was to determine whether human auricular chondrocytes can also expres... more The objective of this study was to determine whether human auricular chondrocytes can also express α α− −-smooth muscle actin. Immunohistochemistry using monoclonal antibodies for α α-smooth actin, muscle-specific actin, β β-actin, S-100 protein, CD34, and desmin was performed on samples of human ear cartilage obtained from 20 individuals during a partial resection of the ear for different reasons. Moreover, the RT-PCR analysis of actin isoforms in auricular chondrocytes was performed. Approximately 60 % of the chondrocytes of the ear cartilage expressed α α-smooth muscle actin as demonstrated by immunohistochemistry in all the examined samples. Actin-positive chondrocytes occurred in both external subperichondrial layers of the auricular cartilage. This finding was confirmed by the RT-PCR technique. The knowledge of this fact could help us to better understand the chondrocyte changes occurring during the healing and transplantation of auricular cartilage. The question of whether it is necessary to refer to these predominating cells in ear cartilage as myochondrocytes is considered. This is the first report of an unusual immunophenotype and contractile potential for human auricular chondrocytes.
Annals of Hematology, 1992
Normal as well as hypertransfused BDF1 mice were exposed to 300 ppm of benzene, 6 h/day, 5 days p... more Normal as well as hypertransfused BDF1 mice were exposed to 300 ppm of benzene, 6 h/day, 5 days per week for 2, 4, and 6 weeks respectively. Erythroid-committed hematopoietic progenitor cells CFU-E were determined in the bone marrow, and 59Fe incorporation was measured in the peripheral blood 48 h after its injection, as an indicator of active erythroid cell production. CFU-E numbers were reduced in benzene-exposed mice at all intervals, as was SgFe incorporation in the peripheral blood after 2 weeks of exposure. In hypertransfused mice the CFU-E suppression, caused primarily by the hypertransfusion, was aggravated by benzene. After injection of 1 IU Ep 4 days before killing the CFU-E numbers and the 59Fe incorporation increased in controls as well as in benzene-exposed animals, but the difference persisted. After nine consecutive Ep injections the difference concerning the femur disappeared after 2 and 6 weeks of benzene exposure but was still present in the peripheral blood. These results suggest that chronic benzene exposure has a negative effect on early erythroidcommitted, Ep-responsive hematopoietic cells.
STEM CELLS, 1998
Murine hematopoiesis has been analyzed by many authors, and available data allow for quantitative... more Murine hematopoiesis has been analyzed by many authors, and available data allow for quantitative evaluation of this dynamic process. In this study, the capacity of several populations of the bone marrow clonogenic cells (progenitors) to produce blood cells was compared with their actual production. The cell cycle progression rate was directly measured in the following types of hematopoietic progenitors: day 8 colony-forming units-spleen, GMcolony-forming cells, BFU-E, and CFU-E in normal mice. The cell cycle progression rates of the individual progenitors, together with their numbers in the whole hematopoietic tissue, were used to calculate the absolute numbers produced daily in each population. The data reviewed from literature were analyzed in parallel. The capacity of the progenitors to produce mature blood cells was derived from the daily production of progenitors multiplied by their clonogenic potential. This theoretical capacity to produce blood cells was compared to the actual blood cell production determined from the turnover of circulating blood elements. The comparison strongly suggested an intensive cell death rate occurring at the early stages of differentiation and its decline as the hematopoietic cells become more differentiated and mature.
Cell Proliferation, 1990
The haemopoietic stem cells forming spleen colonies (CFU-S) had on average 30 to 40% of cells eng... more The haemopoietic stem cells forming spleen colonies (CFU-S) had on average 30 to 40% of cells engaged in the DNA synthesis in normal mice continuously over 4 years. A majority of experiments aimed at the suppression of the CFU-S proliferation, which included suppression of the T-lymphocytes by means of cyclosporin A or by adult thymectomy, administration of antibacterial and antifungal agents and maintainance of mice in a sterile environment, suppression of antibody-producing cells by a successive administration of the bacterial lipopolysaccharide and cyclophosphamide and attempts to increase the total number of CFU-S in the body through massive transfusions of bone marrow cells or by grafting plugs of the bone marrow under the kidney capsulae, have not been sufficiently effective. A transient suppression of CFU-S proliferation occurred during recovery of the haemopoietic tissue from damage caused by cyclophosphamide. The results support the view that changes in CFU-S numbers and in the proportion of them in DNA synthesis may be positively correlated when CFU-S numbers fluctuate physiologically about their normal values. The failure to manipulate the CFU-S proliferation rate easily suggests that proliferation of these cells may not be under a strong 'switch on - switch off' control.
Haematology and Blood Transfusion / Hämatologie und Bluttransfusion, 1979
The method available at present for the measurement of the pluripotential stem cells (CFUs) is ba... more The method available at present for the measurement of the pluripotential stem cells (CFUs) is based on the formation of the macroscopically visible colonies on the spleen surface of the lethally irradiated mice recipients of the bone marrow cell suspension [9]. This method demonstrates a fraction of the pluripotential stem cells injected [7].
Advances in Experimental Medicine and Biology, 1999
The oxygen sensors monitor oxygen tension in the organism and trigger appropriate feedback respon... more The oxygen sensors monitor oxygen tension in the organism and trigger appropriate feedback responses when the tension declines. Peripheral chemoreceptors in the carotid and aortic bodies, and erythropoietin (Epo) producing cells in the kidney are the most notoriously known oxygen sensors.
Ceskoslovenská fysiologie / Ústrední ústav biologický, 1980
Acta Haematologica, 2003
The goal of the study was to investigate changes in expression of selected growth factors tentati... more The goal of the study was to investigate changes in expression of selected growth factors tentatively involved in regeneration of haematopoietic tissues (bone marrow and spleen) following cyclophosphamide (CY) damage in the mouse. The bone marrow (BM) and spleen were examined separately, since the regenerating pattern for haematopoietic progenitor cells (HPC) markedly differs in these two haematopoietically active organs after CY. Cytokines assumed to have a stimulatory effect on HPC – stem cell factor (SCF), fetal liver tyrosine kinase 3-ligand (flt3-ligand), thrombopoietin (TPO), stromal cell-derived factor 1 (SDF-1), oncostatin M (OSM) –, a suppressive effect on HPC proliferation – macrophage inflammatory protein-1α (MIP-1α), transforming growth factor-β1 (TGFβ1), tumour necrosis factor-α (TNFα) – and to be involved in migration of HPC (SCF, flt3-ligand, MIP1α, SDF-1) were examined at the level of mRNA expression by means of real-time RT-PCR. The expression of a particular cytoki...
Biology of Blood and Marrow Transplantation, 2013
Significant controversy exists regarding the impact of hematopoietic stroma damage by irradiation... more Significant controversy exists regarding the impact of hematopoietic stroma damage by irradiation on the efficiency of engraftment of intravenously transplanted stem cells. It was previously demonstrated that in normal syngenic mice, all intravenously transplanted donor stem cells, present in the bone marrow, compete equally with those of the host. In this study, we comprehensively compared the blood cell production derived from transplanted donor stem cells with that from the host stem cells surviving various doses of submyeloablative irradiation. We compared the partial chimerism resulting from transplantation with theoretical estimates that assumed transplantation efficiencies ranging from 100% to 20%. The highest level of consensus between the experimental and the theoretical results was 100% for homing and engraftment (ie, the utilization of all transplanted stem cells). These results point to a very potent mechanism through which intravenously administered hematopoietic stem cells are captured from circulation, engraft in the hematopoietic tissue, and contribute to blood cell production in irradiated recipients. The damage done to hematopoietic stroma and to the trabecular bone by submyeloablative doses of ionizing radiation does not negatively affect the homing and engraftment mechanisms of intravenously transplanted hematopoietic progenitor and stem cells.
Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. W... more Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNA-synthesizing cells by two thymidine analogues, optimized for <i>in-vivo</i> use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1<sup>+</sup> and Sca-1<sup>−</sup> HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1<sup>+</sup> and Sca-1<sup>−</sup> myeloid progenitors self-renew and differentiate. Division of the Sca-1<sup>+</sup> progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1<sup>−</sup> c...
International Journal of Molecular Sciences
Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the ... more Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2); however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection; liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increase...
Frontiers in Cell and Developmental Biology
The immense regenerative power of hematopoietic tissue stems from the activation of the immature ... more The immense regenerative power of hematopoietic tissue stems from the activation of the immature stem cells and the progenitor cells. After partial damage, hematopoiesis is reconstituted through a period of intense regeneration when blood cell production originates from erythro-myeloid progenitors in the virtual absence of stem cells. Since the damaged hematopoiesis can also be reconstituted from transplanted hematopoietic cells, we asked whether this also leads to the transient state when activated progenitors initially execute blood cell production. We first showed that the early reconstitution of hematopoiesis from transplanted cells gives rise to extended populations of developmentally advanced but altered progenitor cells, similar to those previously identified in the bone marrow regenerating from endogenous cells. We then identified the cells that give rise to these progenitors after transplantation as LSK CD48– cells. In the submyeloablative irradiated host mice, the transpla...
Frontiers in Cell and Developmental Biology
Regeneration of severely damaged adult tissues is currently only partially understood. Hematopoie... more Regeneration of severely damaged adult tissues is currently only partially understood. Hematopoietic tissue provides a unique opportunity to study tissue regeneration due to its well established steady-state structure and function, easy accessibility, well established research methods, and the well-defined embryonic, fetal, and adult stages of development. Embryonic/fetal liver hematopoiesis and adult hematopoiesis recovering from damage share the need to expand populations of progenitors and stem cells in parallel with increasing production of mature blood cells. In the present study, we analyzed adult hematopoiesis in mice subjected to a submyeloablative dose (6 Gy) of gamma radiation and targeted the period of regeneration characterized by massive production of mature blood cells along with ongoing expansion of immature hematopoietic cells. We uncovered significantly expanded populations of developmentally advanced erythroid and myeloid progenitors with significantly altered immunophenotype. Their population expansion does not require erythropoietin stimulation but requires the SCF/c-Kit receptor signaling. Regenerating hematopoiesis significantly differs from the expanding hematopoiesis in the fetal liver but we find some similarities between the regenerating hematopoiesis and the early embryonic definitive hematopoiesis. These are in (1) the concomitant population expansion of myeloid progenitors and increasing production of myeloid blood cells (2) performing these tasks despite the severely reduced transplantation capacity of the hematopoietic tissues, and (3) the expression of CD16/32 in most progenitors. Our data thus provide a novel insight into tissue regeneration by suggesting that cells other than stem cells and multipotent progenitors can be of fundamental importance for the rapid recovery of tissue function.
Methods in Molecular Biology
The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are ro... more The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters. In the current step-by-step protocol, we present' two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.
Cell Cycle
Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. W... more Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNAsynthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1 + and Sca-1 − HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1 + and Sca-1 − myeloid progenitors self-renew and differentiate. Division of the Sca-1 + progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1 − cells arising from cell division entered a new round of the cell cycle, corresponding to symmetric self-renewing cell division. The novel data also enabled the estimation of the cell production rates in Sca-1 + and in three subtypes of Sca-1 − HSPCs and revealed Sca-1 negative cells as the major amplification stage in the blood cell development.
PLOS ONE
Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload an... more Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload and decreased by accelerated erythropoiesis or iron deficiency. The purpose of the study was to examine the effect of these stimuli, either alone or in combination, on the main signaling pathway controlling hepcidin biosynthesis in the liver, and on the expression of splenic modulators of hepcidin biosynthesis. Liver phosphorylated SMAD 1 and 5 proteins
STEM CELLS
Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experime... more Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experiments. When we used ubiquitin-GFP (UBC-GFP) transgenic mice to study the availability of niches for transplanted hematopoietic stem and progenitor cells, the results were strikingly different from the corresponding experiments that used congenic mice polymorphic in the CD45 antigen. Analysis of these unexpected results revealed that the hematopoiesis of UBC-GFP mice was outcompeted by the hematopoiesis of wild-type (WT) mice. Importantly, UBC-GFP mice engrafted the transplanted bone marrow of WT mice without conditioning. There was a significant bias toward lymphopoiesis in the WT branch of chimeric UBC-GFP/WT hematopoiesis. A fraction of immature Sca-1 1 cells in the spleen of UBC-GFP mice expressed GFP at a very high level. The chimeric hematopoiesis was stable in the long term and also after transplantation to secondary recipient mice. The article thus identifies a specific defect in the hematopoiesis of UBC-GFP transgenic mice that compromises the lymphoid-primed hematopoietic stem cells in the bone marrow and spleen. STEM CELLS 2018; 00:000-000 SIGNIFICANCE STATEMENT Tagging cells with genetic markers may unintentionally alter tissue function. Ubiquitin-green fluorescent protein transgenic mice have a diminished population of lymphoid-primed hematopoietic stem cells and engraft transplanted wild-type bone marrow without conditioning. The stem cell niches for lymphoid-primed stem cells are also in the extramedullary spleen tissue.
Folia biologica
The objective of this study was to determine whether human auricular chondrocytes can also expres... more The objective of this study was to determine whether human auricular chondrocytes can also express α α− −-smooth muscle actin. Immunohistochemistry using monoclonal antibodies for α α-smooth actin, muscle-specific actin, β β-actin, S-100 protein, CD34, and desmin was performed on samples of human ear cartilage obtained from 20 individuals during a partial resection of the ear for different reasons. Moreover, the RT-PCR analysis of actin isoforms in auricular chondrocytes was performed. Approximately 60 % of the chondrocytes of the ear cartilage expressed α α-smooth muscle actin as demonstrated by immunohistochemistry in all the examined samples. Actin-positive chondrocytes occurred in both external subperichondrial layers of the auricular cartilage. This finding was confirmed by the RT-PCR technique. The knowledge of this fact could help us to better understand the chondrocyte changes occurring during the healing and transplantation of auricular cartilage. The question of whether it is necessary to refer to these predominating cells in ear cartilage as myochondrocytes is considered. This is the first report of an unusual immunophenotype and contractile potential for human auricular chondrocytes.