Long-Term Cryopreservation May Cause Genomic Instability and the Premature Senescence of Cells.

Cryopreservation is an essential step for utilizing various cell types for biological research and medical purposes. At the same time, there is a lack of data on the effect of cryopreservation, especially when prolonged, on the karyotype of cells. In the present work, we analyzed the genetic stability of cells subjected to a cryopreservation procedure. The objects were immortalized Chinese hamster lung fibroblasts (CHL V-79 RJK line) and human endometrial mesenchymal stem/stromal cells (eMSCs). We showed that short-term cryopreservation in liquid nitrogen for up to 6 months did not affect the karyotype stability of CHL V-79 RJK and eMSCs. On the contrary, karyotyping of G-banded metaphase chromosomes in cells underwent 10-year cryopreservation, which revealed genomic instability in both cell lines associated with the variability of chromosome number in cells, random chromosomal rearrangements, and condensation disorder in homologs. In addition, we found out that long-term cryopreservation of eMSCs does not affect the expression of their typical surface markers and morphology, but results in a significant reduction in proliferative potential and early manifestation of cellular senescence features upon eMSCs culturing. Thus, we concluded that the long-term cryopreservation of cells of different types and biological origin can lead to irreversible changes of their karyotype and acceleration of cellular senescence.

Induction of Premature Cell Senescence Stimulated by High Doses of Antioxidants Is Mediated by Endoplasmic Reticulum Stress.

In our previous study, we found that high doses of several substances with antioxidant capacities (Tempol, resveratrol, diphenyleneiodonium) can cause genotoxic stress and induce premature senescence in the human mesenchymal stem cells (MSCs). Here, using whole-transcriptome analysis, we revealed the signs of endoplasmic reticulum stress and unfolded protein response (UPR) in MSCs stressed with Tempol and resveratrol. In addition, we found the upregulation of genes, coding the UPR downstream target APC/C, and E3 ubiquitin ligase that regulate the stability of cell cycle proteins. We performed the molecular analysis, which further confirmed the untimely degradation of APC/C targets (cyclin A, geminin, and Emi1) in MSCs treated with antioxidants. Human fibroblasts responded to antioxidant applications similarly. We conclude that endoplasmic reticulum stress and impaired DNA synthesis regulation can be considered as potential triggers of cell damage and premature senescence stimulated by high-dose antioxidant treatments.

Chromothripsis-Explosion in Genetic Science.

Chromothripsis has been defined as complex patterns of alternating genes copy number changes (normal, gain or loss) along the length of a chromosome or chromosome segment (International System for Human Cytogenomic Nomenclature 2020). The phenomenon of chromothripsis was discovered in 2011 and changed the concept of genome variability, mechanisms of oncogenic transformation, and hereditary diseases. This review describes the phenomenon of chromothripsis, its prevalence in genomes, the mechanisms underlying this phenomenon, and methods of its detection. Due to the fact that most often the phenomenon of chromothripsis occurs in cancer cells, in this review, we will separately discuss the issue of the contribution of chromothripsis to the process of oncogenesis.

Impact of Polyallylamine Hydrochloride on Gene Expression and Karyotypic Stability of Multidrug Resistant Transformed Cells.

The synthetic polymer, polyallylamine hydrochloride (PAA), is found in a variety of applications in biotechnology and medicine. It is used in gene and siRNA transfer, to form microcapsules for targeted drug delivery to damaged and tumor cells. Conventional chemotherapy often does not kill all cancer cells and leads to multidrug resistance (MDR). Until recently, studies of the effects of PAA on cells have mainly focused on their morphological and genetic characteristics immediately or several hours after exposure to the polymer. The properties of the cell progeny which survived the sublethal effects of PAA and resumed their proliferation, were not monitored. The present study demonstrated that treatment of immortalized Chinese hamster cells CHLV-79 RJK sensitive (RJK) and resistant (RJKEB) to ethidium bromide (EB) with cytotoxic doses of PAA, selected cells with increased karyotypic instability, were accompanied by changes in the expression of p53 genes c-fos, topo2-α, hsp90, hsc70. These changes did not contribute to the progression of MDR, accompanied by the increased sensitivity of these cells to the toxic effects of doxorubicin (DOX). Our results showed that PAA does not increase the oncogenic potential of immortalized cells and confirmed that it can be used for intracellular drug delivery for anticancer therapy.

Cell Cycle-Dependent Expression of Bk Channels in Human Mesenchymal Endometrial Stem Cells.

The study of ion channels in stem cells provides important information about their role in stem cell fate. Previously we have identified the activity of calcium-activated potassium channels of big conductance (BK channels) in human endometrium-derived mesenchymal stem cells (eMSCs). BK channels could have significant impact into signaling processes by modulating membrane potential. The membrane potential and ionic permeability dynamically changes during cycle transitions. Here, we aimed at verification of the role of BK channels as potassium transporting pathway regulating cell cycle passageway of eMSCs. The functional expression of native BK channels was confirmed by patch-clamp and immunocytochemistry. In non-synchronized cells immunofluorescent analysis revealed BK-positive and BK-negative stained eMSCs. Using cell synchronization, we found that the presence of BK channels in plasma membrane was cell cycle-dependent and significantly decreased in G2M phase. However, the study of cell cycle progression in presence of selective BK channel inhibitors showed no effect of pore blockers on cycle transitions. Thus, BK channel-mediated K+ transport is not critical for the fundamental mechanism of passageway through cell cycle of eMSCs. At the same time, the dynamics of the presence of BK channels on plasma membrane of eMSCs can be a novel indicator of cellular proliferation.

Cytogenetic and Transcriptomic Analysis of Human Endometrial MSC Retaining Proliferative Activity after Sublethal Heat Shock.

Temperature is an important exogenous factor capable of leading to irreversible processes in the vital activity of cells. However, the long-term effects of heat shock (HS) on mesenchymal stromal cells (MSC) remain unstudied. We investigated the karyotype and DNA repair drivers and pathways in the human endometrium MSC (eMSC) survived progeny at passage 6 after sublethal heat stress (sublethal heat stress survived progeny (SHS-SP)). G-banding revealed an outbreak of random karyotype instability caused by chromosome breakages and aneuploidy. Molecular karyotyping confirmed the random nature of this instability. Transcriptome analysis found homologous recombination (HR) deficiency that most likely originated from the low thermostability of the AT-rich HR driving genes. SHS-SP protection from transformation is provided presumably by low oncogene expression maintained by tight co-regulation between thermosensitive HR drivers BRCA, ATM, ATR, and RAD51 (decreasing expression after SHS), and oncogenes mTOR, MDM2, KRAS, and EGFR. The cancer-related transcriptomic features previously identified in hTERT transformed MSC in culture were not found in SHS-SP, suggesting no traits of malignancy in them. The entrance of SHS-SP into replicative senescence after 25 passages confirms their mortality and absence of transformation features. Overall, our data indicate that SHS may trigger non-tumorigenic karyotypic instability due to HR deficiency and decrease of oncogene expression in progeny of SHS-survived MSC. These data can be helpful for the development of new therapeutic approaches in personalized medicine.

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells.

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated ß-galactosidase (SA-ß-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny.

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.