Laboratory of Stem Cell Biology:2
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| − | + | == Study of Tissue Regeneration Mechanisms Involving Adult Stem Cells == | |
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| − | + | Group under the leadership of Igor Prudnikov | |
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| − | + | Using a model of subtotal radiation injury in experimental rats, we have demonstrated that human mesenchymal stromal cells (hMSCs) restore the functional capabilities of vascular endothelial cells damaged by radiation. No traces of donor cells were detected in recipient vessels, suggesting that this effect is mediated by endocrine or paracrine mechanisms. These experiments are described in detail below. The effect of intravenous administration of hMSCs was evaluated by measuring the activity of large-conductance calcium-activated potassium channels (BKCa) in thoracic aortic smooth muscle cells (SMCs) obtained from non-lethally irradiated rats. Irradiation significantly increased blood pressure and reduced acetylcholine (ACh)-induced relaxation responses in irradiated rats compared to the control group. Simultaneous measurements of contractile force and intracellular calcium concentration ([Ca²⁺]i) revealed increased myofilament sensitivity to Ca²⁺ following irradiation. Intravenous administration of hMSCs effectively restored BKCa current and the amplitude of ACh-induced endothelium-dependent vasodilation in vascular tissues from irradiated rats. Additionally, hMSC administration normalized elevated blood pressure and myofilament Ca²⁺ sensitivity in these animals. In healthy rats, hMSCs did not affect these parameters. | |
| − | + | The effect of intravenous administration of hMSCs was evaluated by measuring the activity of large-conductance calcium-activated potassium channels (BKCa) in thoracic aortic smooth muscle cells (SMCs) obtained from non-lethally irradiated rats. Irradiation significantly increased blood pressure and reduced acetylcholine (ACh)-induced relaxation responses in irradiated rats compared to the control group. Simultaneous measurements of contractile force and intracellular calcium concentration ([Ca²⁺]i) revealed increased myofilament sensitivity to Ca²⁺ following irradiation. Intravenous administration of hMSCs effectively restored BKCa current and the amplitude of ACh-induced endothelium-dependent vasodilation in vascular tissues from irradiated rats. Additionally, hMSC administration normalized elevated blood pressure and myofilament Ca²⁺ sensitivity in these animals. In healthy rats, hMSCs did not affect these parameters. No immunohistochemical evidence of hMSC engraftment was observed in host rats. Polymerase chain reaction (PCR) analysis confirmed that hMSCs were negative for hematopoietic cell markers and positive for hMSC-specific markers. No clinical signs of graft-versus-host disease were observed during the 30-day experimental period. These findings indicate that hMSCs effectively normalize vascular function impaired by irradiation. Similar effects were observed in other pathological models, including genetically determined arterial hypertension and diabetes mellitus. Physiological experiments exploring the effects of stem cells in treating various pathological conditions have provided a broader understanding of their role in tissue viability. We propose that these effects are mediated by mechanisms involving exosomes—vesicles generated by stem cells (and other cell types) during endocytosis. Our research revealed that adult human bone marrow stem cells lack the ability to undergo apoptosis involving cytochrome c and caspases. However, their direct descendants, neutrophils, generate activated caspase-3, which is present in exosomes. These exosomes can transmit signals locally at the site of vessel or tissue damage or distribute activated caspase-3 systemically. Thus, various physiological processes, from regeneration to cell death, rely on exosomes as carriers of functional signals throughout the body. | |
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| + | We also investigated models to study the responses of the body’s own adult stem cells activation. Neural stem cells in the cerebral cortex of fish serve as a suitable model. In response to eye injury, silent neural stem cells in the fish nervous system become activated, initiating regeneration through the formation of new neuronal and glial cells from the neuroblast pool of adult stem cells. The fish brain exhibits a unique vertebrate characteristic: it grows throughout the organism’s life. This makes fish an ideal model for studying embryonic and postembryonic central nervous system (CNS) development and the influence of various factors on these processes. A system of progenitor cells persists in the brain of adult vertebrates, enabling the replenishment of neuronal and glial cell populations long after birth. However, the mechanisms of pre- and postnatal brain morphogenesis in fish, which maintain a larval state for an extended period, remain largely unstudied. During postembryonic development in teleost fish, certain neurotransmitters and gaseous mediators (e.g., nitric oxide [NO] and hydrogen sulfide [H2S]) act as factors that initiate and regulate cellular and tissue processes of the genetic program during brain development. Neuroblasts in the periventricular region of the brain release gamma-aminobutyric acid (GABA), providing negative feedback control of stem cell proliferation and regulating the size of the neuroblast pool. We propose that classical neurotransmitters (e.g., GABA, catecholamines) and gasotransmitters (NO and H2S) not only modulate neuronal activity and synaptic transmission in mature neural networks but also serve as morphogenetic factors in postembryonic brain development. The presence of enzymes synthesizing gasotransmitters in brain regions expressing proliferative cell nuclear antigen (PCNA) confirms their role in regulating postembryonic neurogenesis. | ||
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| + | A superficially located periventricular proliferative zone with PCNA-positive (PCNA+) cells, corresponding to the pallial periventricular zone (PVZ) of other fish species, was identified in the telencephalon of juvenile masu salmon (Oncorhynchus masou). PCNA+ cells were also detected in the brain parenchyma, with the highest concentration in the medial zone. Following mechanical injury, zones of induced neurogenesis—neurogenic niches and sites of secondary neurogenesis surrounded by radial glial fibers—appeared in the masu salmon telencephalon. The PVZ of the juvenile masu salmon pallium contains clusters of undifferentiated HuCD-positive (HuCD+) neurons. After mechanical injury, changes in HuCD+ cell topography were observed, including the formation of neurogenic niches in the lateral zone and increased cell distribution density and migration in the medial zone. These findings demonstrate that brain plasticity persists, resembling embryonic or early developmental stages. | ||
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| + | Further studies of apoptosis as a key regulator of cellular homeostasis during ontogenesis and embryogenesis prompted us to revise its classical mechanisms in normal stem cells. We investigated 2′-deoxyadenosine 5′-triphosphate (dATP) and cytochrome c-induced apoptosome formation as a reliable and natural process of programmed cell death in the brain of newborn rats. We explored which factors contribute to the nonlinear increase in apoptosome formation during initiation. Stimulation of actin assembly by substances such as Ribonuclease A (RNase A) or cations (Na⁺ or K⁺) was found to induce apoptosome formation. Conversely, the actin polymerization inhibitor cytochalasin D disrupted this process. For the first time, we demonstrated that apoptosome organization is directly linked to the cytoskeleton and propose that beta-actin serves as a key regulator of apoptosome formation, warranting its inclusion among proteins critical for programmed cell death. | ||
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| + | Although existing data suggest the involvement of cytoskeletal proteins, particularly actin, in apoptosis, their specific role remains unclear. We used fluorescent labeling of amino acid residues in purified rat brain beta/gamma-actin in monomeric form (G-actin) with 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) to monitor induced conformational changes in the protein status. We observed the formation of a fluorescent derivative upon reaction of CBQCA with the phenol group of free tyrosine and its residues in peptides and proteins, independent of cyanide presence. Analysis of labeled G-actin revealed changes in fluorescence intensity in the spectrum characteristic of the tyrosine reaction product, but not amino groups. Notably, cytochrome c in micromolar concentrations reduced fluorescence in a dose-dependent manner, indicating direct interaction with G-actin. Using this novel CBQCA labeling of tyrosine residues, we studied conformational changes in the actin molecule and confirmed its interaction with cytochrome c. We propose that actin regulates apoptosome formation, particularly through its interaction with cytochrome c. | ||
Latest revision as of 14:52, 1 October 2025
| About | Study of Tissue Regeneration Mechanisms Involving Adult Stem Cells. Group under the leadership of Igor Prudnikov |
Mitochondrial Pathways in Cell Death via Apoptosis. Group under the leadership of Olga Akopova. |
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Study of Tissue Regeneration Mechanisms Involving Adult Stem Cells
Group under the leadership of Igor Prudnikov
Using a model of subtotal radiation injury in experimental rats, we have demonstrated that human mesenchymal stromal cells (hMSCs) restore the functional capabilities of vascular endothelial cells damaged by radiation. No traces of donor cells were detected in recipient vessels, suggesting that this effect is mediated by endocrine or paracrine mechanisms. These experiments are described in detail below. The effect of intravenous administration of hMSCs was evaluated by measuring the activity of large-conductance calcium-activated potassium channels (BKCa) in thoracic aortic smooth muscle cells (SMCs) obtained from non-lethally irradiated rats. Irradiation significantly increased blood pressure and reduced acetylcholine (ACh)-induced relaxation responses in irradiated rats compared to the control group. Simultaneous measurements of contractile force and intracellular calcium concentration ([Ca²⁺]i) revealed increased myofilament sensitivity to Ca²⁺ following irradiation. Intravenous administration of hMSCs effectively restored BKCa current and the amplitude of ACh-induced endothelium-dependent vasodilation in vascular tissues from irradiated rats. Additionally, hMSC administration normalized elevated blood pressure and myofilament Ca²⁺ sensitivity in these animals. In healthy rats, hMSCs did not affect these parameters. The effect of intravenous administration of hMSCs was evaluated by measuring the activity of large-conductance calcium-activated potassium channels (BKCa) in thoracic aortic smooth muscle cells (SMCs) obtained from non-lethally irradiated rats. Irradiation significantly increased blood pressure and reduced acetylcholine (ACh)-induced relaxation responses in irradiated rats compared to the control group. Simultaneous measurements of contractile force and intracellular calcium concentration ([Ca²⁺]i) revealed increased myofilament sensitivity to Ca²⁺ following irradiation. Intravenous administration of hMSCs effectively restored BKCa current and the amplitude of ACh-induced endothelium-dependent vasodilation in vascular tissues from irradiated rats. Additionally, hMSC administration normalized elevated blood pressure and myofilament Ca²⁺ sensitivity in these animals. In healthy rats, hMSCs did not affect these parameters. No immunohistochemical evidence of hMSC engraftment was observed in host rats. Polymerase chain reaction (PCR) analysis confirmed that hMSCs were negative for hematopoietic cell markers and positive for hMSC-specific markers. No clinical signs of graft-versus-host disease were observed during the 30-day experimental period. These findings indicate that hMSCs effectively normalize vascular function impaired by irradiation. Similar effects were observed in other pathological models, including genetically determined arterial hypertension and diabetes mellitus. Physiological experiments exploring the effects of stem cells in treating various pathological conditions have provided a broader understanding of their role in tissue viability. We propose that these effects are mediated by mechanisms involving exosomes—vesicles generated by stem cells (and other cell types) during endocytosis. Our research revealed that adult human bone marrow stem cells lack the ability to undergo apoptosis involving cytochrome c and caspases. However, their direct descendants, neutrophils, generate activated caspase-3, which is present in exosomes. These exosomes can transmit signals locally at the site of vessel or tissue damage or distribute activated caspase-3 systemically. Thus, various physiological processes, from regeneration to cell death, rely on exosomes as carriers of functional signals throughout the body.
We also investigated models to study the responses of the body’s own adult stem cells activation. Neural stem cells in the cerebral cortex of fish serve as a suitable model. In response to eye injury, silent neural stem cells in the fish nervous system become activated, initiating regeneration through the formation of new neuronal and glial cells from the neuroblast pool of adult stem cells. The fish brain exhibits a unique vertebrate characteristic: it grows throughout the organism’s life. This makes fish an ideal model for studying embryonic and postembryonic central nervous system (CNS) development and the influence of various factors on these processes. A system of progenitor cells persists in the brain of adult vertebrates, enabling the replenishment of neuronal and glial cell populations long after birth. However, the mechanisms of pre- and postnatal brain morphogenesis in fish, which maintain a larval state for an extended period, remain largely unstudied. During postembryonic development in teleost fish, certain neurotransmitters and gaseous mediators (e.g., nitric oxide [NO] and hydrogen sulfide [H2S]) act as factors that initiate and regulate cellular and tissue processes of the genetic program during brain development. Neuroblasts in the periventricular region of the brain release gamma-aminobutyric acid (GABA), providing negative feedback control of stem cell proliferation and regulating the size of the neuroblast pool. We propose that classical neurotransmitters (e.g., GABA, catecholamines) and gasotransmitters (NO and H2S) not only modulate neuronal activity and synaptic transmission in mature neural networks but also serve as morphogenetic factors in postembryonic brain development. The presence of enzymes synthesizing gasotransmitters in brain regions expressing proliferative cell nuclear antigen (PCNA) confirms their role in regulating postembryonic neurogenesis.
A superficially located periventricular proliferative zone with PCNA-positive (PCNA+) cells, corresponding to the pallial periventricular zone (PVZ) of other fish species, was identified in the telencephalon of juvenile masu salmon (Oncorhynchus masou). PCNA+ cells were also detected in the brain parenchyma, with the highest concentration in the medial zone. Following mechanical injury, zones of induced neurogenesis—neurogenic niches and sites of secondary neurogenesis surrounded by radial glial fibers—appeared in the masu salmon telencephalon. The PVZ of the juvenile masu salmon pallium contains clusters of undifferentiated HuCD-positive (HuCD+) neurons. After mechanical injury, changes in HuCD+ cell topography were observed, including the formation of neurogenic niches in the lateral zone and increased cell distribution density and migration in the medial zone. These findings demonstrate that brain plasticity persists, resembling embryonic or early developmental stages.
Further studies of apoptosis as a key regulator of cellular homeostasis during ontogenesis and embryogenesis prompted us to revise its classical mechanisms in normal stem cells. We investigated 2′-deoxyadenosine 5′-triphosphate (dATP) and cytochrome c-induced apoptosome formation as a reliable and natural process of programmed cell death in the brain of newborn rats. We explored which factors contribute to the nonlinear increase in apoptosome formation during initiation. Stimulation of actin assembly by substances such as Ribonuclease A (RNase A) or cations (Na⁺ or K⁺) was found to induce apoptosome formation. Conversely, the actin polymerization inhibitor cytochalasin D disrupted this process. For the first time, we demonstrated that apoptosome organization is directly linked to the cytoskeleton and propose that beta-actin serves as a key regulator of apoptosome formation, warranting its inclusion among proteins critical for programmed cell death.
Although existing data suggest the involvement of cytoskeletal proteins, particularly actin, in apoptosis, their specific role remains unclear. We used fluorescent labeling of amino acid residues in purified rat brain beta/gamma-actin in monomeric form (G-actin) with 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) to monitor induced conformational changes in the protein status. We observed the formation of a fluorescent derivative upon reaction of CBQCA with the phenol group of free tyrosine and its residues in peptides and proteins, independent of cyanide presence. Analysis of labeled G-actin revealed changes in fluorescence intensity in the spectrum characteristic of the tyrosine reaction product, but not amino groups. Notably, cytochrome c in micromolar concentrations reduced fluorescence in a dose-dependent manner, indicating direct interaction with G-actin. Using this novel CBQCA labeling of tyrosine residues, we studied conformational changes in the actin molecule and confirmed its interaction with cytochrome c. We propose that actin regulates apoptosome formation, particularly through its interaction with cytochrome c.
