Role of telomerase in reactivation of macrophage nuclei in heterokaryons

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.     

Enhanced Natural Killers with CISH and B2M Gene Knockouts Reveal Increased Cytotoxicity in Glioblastoma Primary Cultures

Molecular Biology - In an experimental study using the CRISPR/Cas9 system, “enhanced” NK cell lines with knockout of CISH, the gene for the CIS protein (a negative regulator of NK...     

Enhanced control of proliferation in telomerized cells

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.     

Enhanced control of proliferation in telomerized cells

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but “escape” to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Nondividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytize debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.     

Role of Telomerase in Reactivation of Macrophage Nuclei in Heterokaryons

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.     

Induction of telomerase activity increase reprogramming efficiency of human dermal fibroblasts

Reprogramming of human cells is a perspective direction of development of Cell Biology with the distant prospect of using these methods for cellular-replacing therapy in clinical practice. One of the problems rising in this regard is low reprogramming efficiency and application of undesirable oncogenes in initial techniques. In this research, we had offered the alternative modified method of reprogramming human skin fibroblasts. It has been shown that the telomerase induction increases an exit of reprogrammed cells and allows excluding oncogene c-Myc from a used set of genes.     

Influence of oxygen on three different types of telomerized cells derived from a single donor

Three primary cell cultures were derived from a single donor: skin fibroblasts (SF), dermal papilla cells (DP), and mesenchimal stromal cells from lipoaspirate (LA). After a high efficiency introduction of the gene of human telomerase catalytic component (hTERT) in lentiviral construct, we obtained three different strains of immortalized cells. All cells were cultured in a low oxygen (3%) environment. When telomerized cells were grown in atmospheric conditions (21% oxygen), growth retardation was observed after a period of 18–40 days. SF-hTERT and DP-hTERT overcame this retardation. In 30–45 days the rate of their growth became as high as it was in 3% oxygen. LA-hTERT cells were incapable to restore the previous growth rate and after several passages ceased proliferation. We found that telomerized cells were heterogeneous in their ability to form colonies at low density (3 cells per 1 cm²): they made 0–9 population doublings in 7 days. At the same time, mean growth rate of SF-hTERT did not change, while the growth of DP-hTERT and LA-hTERT cells was considerably accelerated in comparison with their growth in mass culture. Transfer of telomerized cells to 21% oxygen reduced their ability to form colonies to a different extent. The total number of cells grown in 21% oxygen was 3 times less than the number grown in 3% oxygen for SF-hTERT and DP-hTERT, and 6 times less for LA-hTERT. Original cells (with the exception of LA) were more sensitive to oxygen level than the corresponding telomerized cells. The total number of cells of all three strains (SF, DP, LA) grown in 21% oxygen was 6–7 times less. We discuss the reasons of incapability of LA-hTERT cells to adapt to atmospheric oxygen.     

IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals

Neurochemical Research - The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the...