Search

Konstantin N Yarygin, Alexei Y Lupatov, Irina V Kholodenko Laboratory of Cell Biology, Institute of Biomedical Chemistry, Moscow, Russia Abstract: The scope of this review is to revise recent advances of the cell-based therapies of liver diseases with an emphasis on cell donor’s and patient’s age. Regenerative medicine with cell-based technologies as its integral part is focused on the structural and functional restoration of tissues impaired by sickness or aging. Unlike drug-based medicine directed primarily at alleviation of symptoms, regenerative medicine offers a more holistic approach to disease and senescence management aimed to achieve restoration of homeostasis. Hepatocyte transplantation and organ engineering are very probable forthcoming options of liver disease treatment in people of different ages and vigorous research and technological innovations in this area are in progress. Accordingly, availability of sufficient amounts of functional human hepatocytes is crucial. Direct isolation of autologous hepatocytes from liver biopsy is problematic due to related discomfort and difficulties with further expansion of cells, particularly those derived from aging people. Allogeneic primary human hepatocytes meeting quality standards are also in short supply. Alternatively, autologous hepatocytes can be produced by reprogramming of differentiated cells through the stage of induced pluripotent stem cells. In addition, fibroblasts and mesenchymal stromal cells can be directly induced to undergo advanced stage hepatogenic differentiation. Reprogramming of cells derived from elderly people is accompanied by the reversal of age-associated changes at the cellular level manifesting itself by telomere elongation and the U-turn of DNA methylation. Cell reprogramming can provide high quality rejuvenated hepatocytes for cell therapy and liver tissue engineering. Further technological advancements and establishment of national and global registries of induced pluripotent stem cell lines homozygous for HLA haplotypes can allow industry-style production of livers for immunosuppression-free transplantation. Keywords: cell aging, rejuvenation, liver engineering, liver cell therapy

Screening of cell surface markers of three glioma cell lines (astrocytoma 1321N1, glioblastoma T98g, and glioblastoma astrocytoma U373 MG) was performed. Glioma cells expressed common mesenchymal cell markers, although the expression levels varied between the cell lines. The expression of proneural markers and glioma cancer stem cell markers was very low and also varied. Induction of differentiation towards the mesodermal cell lineages showed effective adipogenic and osteogenic differentiation for only the U373 MG cell line, while the 1321N1 and T98g lines demonstrated weak adipogenic potential and failed to undergo osteogenic differentiation. The obtained results point to the intratumor phenotypical heterogeneity of cells in gliomas and to the differences between the three studied types of gliomas with regard to the content of cells with mesenchymal phenotype., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-β1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-β1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

A culture of cells expressing markers of mesenchymal stem cells (MSC) (CD73, CD90, CD44, CD29, and CD49b), but not hematopoietic cell markers, and capable of multilineage differentiation was isolated from the deciduous tooth pulp. Co-culturing with immature dendritic cells in the presence of LPS did not reveal an ability of the MSC to suppress the maturation of dendritic cells. On the contrary, co-culturing of MSC with monocytes in the presence of granulocyte-macrophage CSF and IL-4 led to complete suppression of monocyte differentiation into dendritic cells. However, long-term culturing of MSC from dental pulp showed that by the passage 11, they almost completely lose their suppressor ability. These results indicate that the immunological properties of MSC can change during culturing without changing their phenotypic markers. This should be taken into account when creating biomedical cell products., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

In translational animal study aimed at evaluation of the effectiveness of innovative methods for treating cerebral stroke, including regenerative cell technologies, of particular importance is evaluation of the dynamics of changes in the volume of the cerebral infarction in response to therapy. Among the methods for assessing the focus of infarction, MRI is the most effective and convenient tool for use in preclinical studies. This review provides a description of MR pulse sequences used to visualize cerebral ischemia at various stages of its development, and a detailed description of the MR semiotics of cerebral infarction. A comparison of various methods for morphometric analysis of the focus of a cerebral infarction, including systems based on artificial intelligence for a more objective measurement of the volume of the lesion, is also presented., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

The use of drug-loaded nanoparticles is an actively developed approach in targeted cancer therapy. Prevascularized spheroids generated from mesenchymal stem cells and endotheliocytes are considered as a model to evaluate the tropism of therapeutic nanoparticles to a specific tissue. Nanoparticles based on co-polymer of lactic and glycolic acids (poly(lactic-co-glycolic acid; PLGA) labeled with cyanine dye (Cy5) were incubated with prevascularized spheroids, and the rate of their penetration and their distribution in the spheroid-forming cells were evaluated. Endotheliocytes more intensively accumulated nanoparticles than mesenchymal stem cells: the number of nanoparticles in mixed-cell spheroids of mesenchymal stem cells and endotheliocytes was greater than in spheroids built solely of mesenchymal stem cells by 5±1.2 times. The developed 3D in vitro cell model provides a low-cost way to assess tissue tropism of therapeutic nanoparticles under conditions closer to natural in comparison with 2D culture., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Non-alcoholic steatohepatitis (NASH) is a serious public health issue associated with the obesity pandemic. Obesity is the main risk factor for the non-alcoholic fatty liver disease (NAFLD), which progresses to NASH and then to end-stage liver disease. Currently, there are no specific pharmacotherapies of NAFLD/NASH approved by the FDA or other national regulatory bodies and the treatment includes lifestyle adjustment and medicines for improving lipid metabolism, enhancing sensitivity to insulin, balancing oxidation, and counteracting fibrosis. Accordingly, further basic research and development of new therapeutic approaches are greatly needed. Mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles prevent induced hepatocyte death in vitro and attenuate NASH symptoms in animal models of the disease. They interact with hepatocytes directly, but also target other liver cells, including Kupffer cells and macrophages recruited from the blood flow. This review provides an update on the pathogenesis of NAFLD/NASH and the key role of macrophages in the development of the disease. We examine in detail the mechanisms of the cross-talk between the MSCs and the macrophages, which are likely to be among the key targets of MSCs and their derivatives in the course of NAFLD/NASH cell therapy.

Liver diseases, characterized by high morbidity and mortality, represent a substantial medical problem globally. The current therapeutic approaches are mainly aimed at reducing symptoms and slowing down the progression of the diseases. Organ transplantation remains the only effective treatment method in cases of severe liver pathology. In this regard, the development of new effective approaches aimed at stimulating liver regeneration, both by activation of the organ's own resources or by different therapeutic agents that trigger regeneration, does not cease to be relevant. To date, many systematic reviews and meta-analyses have been published confirming the effectiveness of mesenchymal stromal cell (MSC) transplantation in the treatment of liver diseases of various severities and etiologies. However, despite the successful use of MSCs in clinical practice and the promising therapeutic results in animal models of liver diseases, the mechanisms of their protective and regenerative action remain poorly understood. Specifically, data about the molecular agents produced by these cells and mediating their therapeutic action are fragmentary and often contradictory. Since MSCs or MSC-like cells are found in all tissues and organs, it is likely that many key intercellular interactions within the tissue niches are dependent on MSCs. In this context, it is essential to understand the mechanisms underlying communication between MSCs and differentiated parenchymal cells of each particular tissue. This is important both from the perspective of basic science and for the development of therapeutic approaches involving the modulation of the activity of resident MSCs. With regard to the liver, the research is concentrated on the intercommunication between MSCs and hepatocytes under normal conditions and during the development of the pathological process. The goals of this review were to identify the key factors mediating the crosstalk between MSCs and hepatocytes and determine the possible mechanisms of interaction of the two cell types under normal and stressful conditions. The analysis of the hepatocyte-MSC interaction showed that MSCs carry out chaperone-like functions, including the synthesis of the supportive extracellular matrix proteins; prevention of apoptosis, pyroptosis, and ferroptosis; support of regeneration; elimination of lipotoxicity and ER stress; promotion of antioxidant effects; and donation of mitochondria. The underlying mechanisms suggest very close interdependence, including even direct cytoplasm and organelle exchange.

Glioblastoma is a tumor characterized by pronounced hypoxia. Hypoxia produces diverse effects on tumor cells, and the results of experimental studies available so far are contradictory. In vitro hypoxia can be modeled in two ways: by reducing the level of atmospheric oxygen (physically induced hypoxia) or by using hypoxia-inducing chemicals such as cobalt chloride (II) (CoCl 2 ) (chemically induced hypoxia). In the present work, we analyzed the effect of CoCl 2 on the viability, proliferation, and apoptosis of cells of three glioblastoma cell lines: 1321N1, T98g, and U373 MG. It was shown that CoCl 2 induced a dose-dependent decrease in cell viability and proliferation, and at high concentrations (200 and 400 μM) stimulated cell death. CoCl 2 had no effect on the cytotoxic activity of doxorubicin in two cell lines T98g and U373 MG, and enhanced the effect of the chemotherapeutic agent on the 1321N1 cell line, though no synergistic cytotoxic effect of the two agents was observed., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

We studied therapeutic efficacy and migration characteristics of mesenchymal stem cells isolated from the human placenta after their intracerebral (stereotactic) administration to rats with the experimental ischemic stroke. It was shown that cell therapy significantly improved animal survival rate and reduced the severity of neurological deficit. New data on the migration pathways of transplanted cells in the brain were obtained., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

We compared the ability of SW837, SW480, HT-29, Caco-2, and HCT116 colorectal cancer lines and cancer-associated fibroblasts obtained from a colorectal adenocarcinoma biopsy specimen to modulate differentiation and maturation of dendritic cells in co-culture. The expression of surface markers of dendritic cell differentiation (CD1a) and maturation (CD83), as well as the expression of CD14 monocyte marker was evaluated by flow cytometry. Cancer-associated fibroblasts completely suppressed dendritic cell differentiation from peripheral blood monocytes induced by granulocyte-macrophage CSF and IL-4, but had no significant effect on their maturation under the influence of bacterial LPS. On the contrary, tumor cell lines did not interfere with monocyte differentiation, although some of them significantly reduced the level of CD1a expression. In contrast to cancer-associated fibroblasts, tumor cell lines and conditioned medium from primary tumor cell culture suppressed LPS-induced maturation of dendritic cells. These results suggest that tumor cells and cancer-associated fibroblasts can modulate different stages of the antitumor immune response., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

More than 50% cells isolated from the endometrial cavity scraping and the myometrium of the rudimentary horn of an underdeveloped uterus removed from a patient with uterine aplasia and maintained under culturing conditions normal for mesenchymal stem cells (MSC) expressed embryonic transcription factors Oct4 and Nanog, embryonic cell membrane sialyl glycolipid SSEA4, and MSC markers. After 2-3 passages, the cells lost the expression of the early embryogenesis markers, but retained MSC markers. The presence of dormant stem cells in the underdeveloped endometrium and in the uterus indicates that this tissue has a regenerative potential that can be activated and used for completion of organ morphogenesis. This task requires the development of methods of early diagnosis of morphogenesis impairment and tools for safe reactivation of the ontogenesis., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Systemic transplantation of mesenchymal stem cells (MSCs) is a promising approach for the treatment of ischemia-associated disorders, including stroke. However, exact mechanisms underlying its beneficial effects are still debated. In this respect, studies of the transplanted cells distribution and homing are indispensable. We proposed an MRI protocol which allowed us to estimate the dynamic distribution of single superparamagnetic iron oxide labeled MSCs in live ischemic rat brain during intravenous transplantation after the transient middle cerebral artery occlusion. Additionally, we evaluated therapeutic efficacy of cell therapy in this rat stroke model. According to the dynamic MRI data, limited numbers of MSCs accumulated diffusely in the brain vessels starting at the 7th minute from the onset of infusion, reached its maximum by 29 min, and gradually eliminated from cerebral circulation during 24 h. Despite low numbers of cells entering brain blood flow and their short-term engraftment, MSCs transplantation induced long lasting improvement of the neurological deficit, but without acceleration of the stroke volume reduction compared to the control animals during 14 post-transplantation days. Taken together, these findings indicate that MSCs convey their positive action by triggering certain paracrine mechanisms or cell-cell interactions or invoking direct long-lasting effects on brain vessels.

Over the past two decades, mesenchymal stem cells (MSCs) have shown promising therapeutic effects both in preclinical studies (in animal models of a wide range of diseases) and in clinical trials. However, the efficacy of MSC-based therapy is not always predictable. Moreover, despite the large number of studies, the mechanisms underlying the regenerative potential of MSCs are not fully elucidated. Recently, it has been reliably established that transplanted MSCs can undergo rapid apoptosis and clearance from the recipient's body, still exhibiting therapeutic effects, especially those associated with their immunosuppressive/immunomodulating properties. The mechanisms underlying these effects can be mediated by the efferocytosis of apoptotic MSCs by host phagocytic cells. In this concise review, we briefly describe three types of MSC-generated extracellular vesicles, through which their therapeutic functions can potentially be carried out; we focused on reviewing recent data on apoptotic MSCs and MSC-derived apoptotic bodies (MSC-ApoBDs), their functions, and the mechanisms of their therapeutic effects., Competing Interests: The authors declare no conflict of interest.

Stem cells serve as a source of cellular material in embryogenesis and postnatal growth and regeneration. This requires significant proliferative potential ensured by sufficient telomere length. Telomere attrition in the stem cells and their niche cells can result in the exhaustion of the regenerative potential of high-turnover organs, causing or contributing to the onset of age-related diseases. In this review, stem cells are examined in the context of the current telomere-centric theory of cell aging, which assumes that telomere shortening depends not just on the number of cell doublings (mitotic clock) but also on the influence of various internal and external factors. The influence of the telomerase and telomere length on the functional activity of different stem cell types, as well as on their aging and prospects of use in cell therapy applications, is discussed.

CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 in CD133 expression modulation was confirmed experimentally in the Caco2 cell line clones: the knockout, though not the knockdown, of the TRIM28 gene downregulated CD133. These results for the first time highlight an important role of the TRIM28 transcription factor in the regulation of CD133-associated cancer cell heterogeneity., Competing Interests: The authors declare no conflict of interest.

Intravenous transplantation of mesenchymal stem (stromal) cells (MSC) is a promising approach to the treatment of ischemic stroke. In the published reports of the already completed preclinical and clinical studies the dosages of transplanted MSC greatly vary. However, the optimal dosage has not been determined. The dose-dependent effect of intravenous MSC transplantation was studied, in rats with experimental cerebral infarction. To this end, 5×10 5 and 2×10 6 MSC were intravenously administered 24 h after modeling of acute focal ischemia followed by complex assessment of the therapeutic efficacy over 60 days. The rate and degree of the recovery of neurological functions in rats increased with increasing the dose of injected cells, which confirms the dose-dependent effect of intravenous MSC transplantation., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl 2 and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.

Intra-arterial (IA) mesenchymal stem cells (MSCs) transplantation providing targeted cell delivery to brain tissue is a promising approach to the treatment of neurological disorders, including stroke. Factors determining cell distribution after IA administration have not been fully elucidated. Their decoding may contribute to the improvement of a transplantation technique and facilitate translation of stroke cell therapy into clinical practice. The goal of this work was to quantitatively assess the impact of brain tissue perfusion on the distribution of IA transplanted MSCs in rat brains. We performed a selective MR-perfusion study with bolus IA injection of gadolinium-based contrast agent and subsequent IA transplantation of MSCs in intact rats and rats with experimental stroke and evaluated the correlation between different perfusion parameters and cell distribution estimated by susceptibility weighted imaging (SWI) immediately after cell transplantation. The obtained results revealed a certain correlation between the distribution of IA transplanted MSCs and brain perfusion in both intact rats and rats with experimental stroke with the coefficient of determination up to 30%. It can be concluded that the distribution of MSCs after IA injection can be partially predicted based on cerebral perfusion data, but other factors requiring further investigation also have a significant impact on the fate of transplanted cells.

Sprouting angiogenesis is the common response of live tissues to physiological and pathological angiogenic stimuli. Its accurate evaluation is of utmost importance for basic research and practical medicine and pharmacology and requires adequate experimental models. A variety of assays for angiogenesis were developed, none of them perfect. In vitro approaches are generally less physiologically relevant due to the omission of essential components regulating the process. However, only in vitro models can be entirely non-xenogeneic. The limitations of the in vitro angiogenesis assays can be partially overcome using 3D models mimicking tissue O 2 and nutrient gradients, the influence of the extracellular matrix (ECM), and enabling cell-cell interactions. Here we present a review of the existing models of sprouting angiogenesis that are based on the use of endothelial cells (ECs) co-cultured with perivascular or other stromal cells. This approach provides an excellent in vitro platform for further decoding of the cellular and molecular mechanisms of sprouting angiogenesis under conditions close to the in vivo conditions, as well as for preclinical drug testing and preclinical research in tissue engineering and regenerative medicine., Competing Interests: The authors declare no conflict of interest.

Animal model studies and first clinical trials have demonstrated the safety and efficacy of the mesenchymal stem cells' (MSCs) transplantation in stroke. Intra-arterial (IA) administration looks especially promising, since it provides targeted cell delivery to the ischemic brain, is highly effective, and can be safe as long as the infusion is conducted appropriately. However, wider clinical application of the IA MSCs transplantation will only be possible after a better understanding of the mechanism of their therapeutic action is achieved. On the way to achieve this goal, the study of transplanted cells' fate and their interactions with the blood-brain barrier (BBB) structures could be one of the key factors. In this review, we analyze the available data concerning one of the most important aspects of the transplanted MSCs' action-the ability of cells to cross the blood-brain barrier (BBB) in vitro and in vivo after IA administration into animals with experimental stroke. The collected data show that some of the transplanted MSCs temporarily attach to the walls of the cerebral vessels and then return to the bloodstream or penetrate the BBB and either undergo homing in the perivascular space or penetrate deeper into the parenchyma. Transmigration across the BBB is not necessary for the induction of therapeutic effects, which can be incited through a paracrine mechanism even by cells located inside the blood vessels.

A comparative analysis of the cell surface markers and immunological properties of cell cultures originating from normal endometrium and endometrioid heterotopias of women with extragenital endometriosis was carried out. Both types of cell cultures expressed surface molecules typical of mesenchymal stromal cells and did not express hematopoietic and epithelial markers. Despite similar phenotype, the mesenchymal stromal cells derived from the two sources had different immunomodulation capacities: the cells of endometrioid heterotopias but not eutopic endometrium could suppress dendritic cell differentiation from monocytes as well as lymphocyte proliferation in allogeneic co-cultures. A comparative multiplex analysis of the secretomes revealed a significant increase in the secretion of pro-inflammatory mediators, including IL6, IFN-γ, and several chemokines associated with inflammation by the stromal cells of ectopic lesions. The results demonstrate that the stromal cells of endometrioid heterotopias display enhanced pro-inflammatory and immunosuppressive activities, which most likely impact the pathogenesis and progression of the disease.

Visualization of transplanted stem cells in the brain is an important issue in the study of the mechanisms of their therapeutic action. MRI allowing visualization of single transplanted cells previously labeled with superparamagnetic iron oxide particles is among the most informative methods of non-invasive intravital imaging. Verification of MRI data using pathomorphological examination at the microscopic level helps to avoid errors in data interpretation. However, making serial sections of the whole brain and searching for transplanted cells under the microscope is laborious and time-consuming. We have developed a method for 3D modeling of the distribution of transplanted cells in the brain allowing navigating through various brain structures and identifying the areas of accumulation of transplanted cells, which significantly increases the efficiency and reduces the time of histological examination., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Extensive studies of the rudimental tissues taken from patients with aplasia of the uterus and vagina are aimed at elucidation of the mechanisms of the genesis of these malformations and at the search of the ways of their correction. We performed a histological examination of human uterine rudiments and immunohistochemical analysis of the expression of estrogen and progesterone receptors, VEGF, and stem/progenitor cell markers in these tissues. We found that the rudimental tissues show signs of disorganized histogenesis, but retain activity and contain cells expressing estrogen and progesterone receptors and VEGF as well as poorly differentiated precursor cells or stem cells. The presented data contribute to the in-depth studies of the mechanisms of formation of uterine rudiments and development of methods of their correction.

A correlation was found between chemoresistance of HT-29CD133 + and HT-29CD133 - sublines obtained after cell sorting and high expression of CD133. On the other hand, knockout of the PROM1 gene and, as a consequence, the absence of CD133 expression did not increase the sensitivity of tumor cells to chemotherapy, which indicates the absence of a direct effect of CD133 on the formation of chemoresistance in colorectal cancer cells. Variants of the HT-29 line with complete or partial knockout of the PROM1 gene were equally sensitive to protein kinase inhibitors sorafenib and sunitinib. Notably, the highest resistance to mTOR inhibitors, temsirolimus and everolimus, was shown by cells with complete knockout of the PROM1 gene (KO-HT-29 (P1)). These findings suggest that CD133 is associated with the chemoresistance of colorectal cancer cells, but is not involved in its formation.

Cell therapy is an emerging approach to stroke treatment with a potential to limit brain damage and enhance its restoration after the acute phase of the disease. In this study we tested directly reprogrammed neural precursor cells (drNPC) derived from adult human bone marrow cells in the rat middle cerebral artery occlusion (MCAO) model of acute ischemic stroke using human placenta mesenchymal stem cells (pMSC) as a positive control with previously confirmed efficacy. Cells were infused into the ipsilateral (right) internal carotid artery of male Wistar rats 24 h after MCAO. The main goal of this work was to evaluate real-time distribution and subsequent homing of transplanted cells in the brain. This was achieved by performing intra-arterial infusion directly inside the MRI scanner and allowed transplanted cells tracing starting from their first pass through the brain vessels. Immediately after transplantation, cells were observed in the periphery of the infarct zone and in the brain stem, 15 min later small numbers of cells could be discovered deep in the infarct core and in the contralateral hemisphere, where drNPC were seen earlier and in greater numbers than pMSC. Transplanted cells in both groups could no longer be detected in the rat brain 48-72 h after infusion. Histological and histochemical analysis demonstrated that both the drNPC and pMSC were localized inside blood vessels in close contact with the vascular wall. No passage of labeled cells through the blood brain barrier was observed. Additionally, the therapeutic effects of drNPC and pMSC were compared. Both drNPC and pMSC induced substantial attenuation of neurological deficits evaluated at the 7th and 14th day after transplantation using the modified neurological severity score (mNSS). Some of the effects of drNPC and pMSC, such as the influence on the infarct volume and the survival rate of animals, differed. The results suggest a paracrine mechanism of the positive therapeutic effects of IA drNPC and pMSC infusion, potentially enhanced by the cell-cell interactions. Our data also indicate that the long-term homing of transplanted cells in the brain is not necessary for the brain's functional recovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Namestnikova, Gubskiy, Revkova, Sukhinich, Melnikov, Gabashvili, Cherkashova, Vishnevskiy, Kurilo, Burunova, Semkina, Abakumov, Gubsky, Chekhonin, Ahlfors, Baklaushev and Yarygin.)

DyeCycle Violet efflux, caused by ATP-binding cassette transporters activity, was analyzed in human colorectal adenocarcinoma cell lines SW480, HT-29, Caco-2 by neans of FACSAria III flow cytometer and ImageStreamX Mk II imaging flow cytometer. Along with similarity of cytometry data obtained on the two instruments, the use of imaging flow cytometry made it possible to characterize the morphology of side population cells, as well as morphology of other cell populations differing in the degree of dye accumulation. The population of cells, which are smaller than the side population cells and practically do not take the dye, is of the special interest. Probably, this population may contribute to the tumor resistance to chemotherapy.

We performed a comparative study of the proliferative potential of human mesenchymal stromal cells (MSC) from three sources (tooth pulp, adipose tissue, and Wharton's jelly) in spheroid culture; human chondroblasts served as the positive control. Histological examination revealed signs of chondrogenic differentiation in all studied cell cultures and the differences in the volume and composition of the extracellular matrix. Spheroids formed by MSC from the tooth pulp and Wharton's jelly were characterized by low content of extracellular matrix and glycosaminoglycans. Spheroids from adipose tissue MSC contained maximum amount of the extracellular matrix and high content of glycosaminoglycans. Chondrocytes produced glycosaminoglycan-enriched matrix. Type II collagen was produced by chondrocytes (to a greater extent) and adipose tissue MSC (to a lesser extent). The results of our study demonstrate that MSC from the adipose tissue under conditions of spheroid culturing exhibited maximum chondrogenic potential.

Almost quarter of a century long studies aimed at identification, isolation, culturing, and use of postnatal pluripotent cells for the development of cell-based technologies have not met with success and failed to provide reliable and reproducible protocols of cell isolation, identification, and culturing. At the same time, experimental data in this field suggest that postnatal pluripotent cells are not the copies of embryonic cells and, therefore, the tests routinely used for identification of embryonic pluripotent cells are not fully adequate for characterization of their postnatal analogues. Therefore, cell lineage tracing methods showing the differentiation routes of the studied cells in human or animal body after birth should be developed and used.

Cell therapy of neurological diseases is gaining momentum. Various types of stem/progenitor cells and their derivatives have shown positive therapeutic results in animal models of neurological disorders and in clinical trials. Each tested cell type proved to have its advantages and flaws and unique cellular and molecular mechanism of action, prompting the idea to test combined transplantation of two or more types of cells (combined cell therapy). This review summarizes the results of combined cell therapy of neurological pathologies reported up to this point. The number of papers describing experimental studies or clinical trials addressing this subject is still limited. However, its successful application to the treatment of neurological pathologies including stroke, spinal cord injury, neurodegenerative diseases, Duchenne muscular dystrophy, and retinal degeneration has been reported in both experimental and clinical studies. The advantages of combined cell therapy can be realized by simple summation of beneficial effects of different cells. Alternatively, one kind of cells can support the survival and functioning of the other by enhancing the formation of optimum environment or immunomodulation. No significant adverse events were reported. Combined cell therapy is a promising approach for the treatment of neurological disorders, but further research needs to be conducted.

One of the main goals of the Chromosome-Centric Human Proteome Project (C-HPP) is detection of "missing proteins" (PE2-PE4). Using the UPS2 (Universal proteomics standard 2) set as a model to simulate the range of protein concentrations in the cell, we have previously shown that 2D fractionation enables the detection of more than 95% of UPS2 proteins in a complex biological mixture. In this study, we propose a novel experimental workflow for protein detection during the analysis of biological samples. This approach is extremely important in the context of the C-HPP and the neXt-MP50 Challenge, which can be solved by increasing the sensitivity and the coverage of the proteome encoded by a particular human chromosome. In this study, we used 2D fractionation for in-depth analysis of the proteins encoded by human chromosome 18 (Chr 18) in the HepG2 cell line. Use of 2D fractionation increased the sensitivity of the SRM SIS method by 1.3-fold (68 and 88 proteins were identified by 1D fractionation and 2D fractionation, respectively) and the shotgun MS/MS method by 2.5-fold (21 and 53 proteins encoded by Chr 18 were detected by 1D fractionation and 2D fractionation, respectively). The results of all experiments indicate that 111 proteins encoded by human Chr 18 have been identified; this list includes 42% of the Chr 18 protein-coding genes and 67% of the Chr 18 transcriptome species (Illumina RNaseq) in the HepG2 cell line obtained using a single sample. Corresponding mRNAs were not registered for 13 of the detected proteins. The combination of 2D fractionation technology with SRM SIS and shotgun mass spectrometric analysis did not achieve full coverage, i.e., identification of at least one protein product for each of the 265 protein-coding genes of the selected chromosome. To further increase the sensitivity of the method, we plan to use 5-10 crude synthetic peptides for each protein to identify the proteins and select one of the peptides based on the obtained mass spectra for the synthesis of an isotopically labeled standard for subsequent quantitative analysis. Data are available via ProteomeXchange with the identifier PXD019263.

We studied the formation of spheroids by Caco-2, SW480, and HCT116 human colorectal adenocarcinoma cell lines under low-adhesion culturing conditions. Of these three cell lines, only HCT116 formed stable tumor spheroids. Flow cytometry analysis of 19 surface markers in monolayer HCT116 culture and spheroids formed by these cells revealed considerable similarity of the expression profiles in these two culturing modes. The only exception was EpCAM molecule: its expression in spheroids was 3-fold higher than in the monolayer culture. Scanning confocal laser microscopy showed equal EpCAM distribution in the inner mass of the spheroids.

Human placenta mesenchymal stromal cells were injected to healthy rats either stereotaxically into the striatum or intra-arterially through the internal carotid artery. Some cells injected into the brain migrated along the corpus callosum both medially and laterally or concentrated around small blood vessels. A small fraction of MSC injected intra-arterially adhered to the endothelium and stayed inside blood vessels for up to 48 hours mostly in the basin of the middle cerebral artery. Neither stereotaxic, nor intra-arterial transplantation of mesenchymal stromal cells modulated the proliferation of neural stem cells in the subventricular zone of the brain, but stereotaxic transplantation suppressed activation of their proliferation in response to traumatization with the needle.

The use of induced pluripotent stem cells (IPSC) is a promising approach to the therapy of CNS diseases. The undeniable advantage of IPSC technology is the possibility of obtaining practically all types of somatic cells for autologous transplantation bypassing bioethical problems. The review presents integrative and non-integrative methods for obtaining IPSC and the ways of their in vitro and in vivo application for the study and treatment of neurological diseases.

Using flow cytometry GD2 ganglioside expression was evaluated both on colorectal adenocarcinoma cell lines and on tumor tissue samples from colorectal cancer patients. The marker was found on EpCAM-positive tumor cells in 6 of 12 patients' samples but not on the HT29 and CaCo-2 cell lines. GD2 expression was not an exceptional feature of cancer stem cells, since its expression level was similar on CD133-positive and CD133-negative tumor cells. Thus, the presence of GD2 ganglioside was revealed on colorectal adenocarcinoma cells for the first time. This finding makes it possible to use targeted therapy to treat this disease.

Chronic liver diseases constitute a significant economic, social, and biomedical burden. Among commonly adopted approaches, only organ transplantation can radically help patients with end-stage liver pathologies. Cell therapy with hepatocytes as a treatment for chronic liver disease has demonstrated promising results. However, quality human hepatocytes are in short supply. Stem/progenitor cells capable of differentiating into functionally active hepatocytes provide an attractive alternative approach to cell therapy for liver diseases, as well as to liver-tissue engineering, drug screening, and basic research. The application of methods generally used to isolate mesenchymal stem cells (MSCs) and maintain them in culture to human liver tissue provides cells, designated here as liver MSCs. They have much in common with MSCs from other tissues, but differ in two aspects-expression of a range of hepatocyte-specific genes and, possibly, inherent commitment to hepatogenic differentiation. The aim of this review is to analyze data regarding liver MSCs, probably another type of liver stem/progenitor cells different from hepatic stellate cells or so-called hepatic progenitor cells. The review presents an analysis of the phenotypic characteristics of liver MSCs, their differentiation and therapeutic potential, methods for isolating these cells from human liver, and discusses issues of their origin and heterogeneity. Human liver MSCs are a fascinating object of fundamental research with a potential for important practical applications.

We studied proliferative activity of colorectal cancer cells with different expression level of CD133 molecule associated with cancer stem cells phenotype. Analysis of BrdU incorporation into Caco-2 and HT-29 cell lines showed that the percentage of cells in the DNA synthesis phase in the CD133 +/high population is higher than in CD133 -/low population. The expression of proliferation marker Ki-67 and the percentage of Ki-67 + cells were also higher in the CD133 +/high population. Colorimetric analysis with crystal violet dye showed that the number of cells after 10-days culturing was higher in the CD133 +/high population in both cell lines. These findings suggest that cells with high level of CD133 expression are characterized by higher proliferative activity, which can contribute to the tumor progression.

HL-60 promyelocytic cells are a widely used as a model for studying induced granulocytic differentiation. Investigation of proteins of the nuclear fraction, particularly transcription factors, is necessary for a better understanding of molecular mechanisms of cell maturation. Mass spectrometry is a powerful tool for analyzing a proteome due to its high sensitivity, specificity and performance. In this paper, using the selected reaction monitoring (SRM) method, we have assessed the levels of RBPJ, STAT1, CEBPB, CASP3, VAV1, PRKDC, PARP1 and UBC9 nuclear proteins isolated using hypertonic buffer, detergents (sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and fissionable detergent ProteaseMAX™) and using centrifugation in a sucrose density gradient. The minimum and maximum protein content was 1.13±0.28 and 14.34±1.63 fmol/mkg of total protein for the transcription factor RBPJ and ubiquitin-protein ligase type I UBC9, respectively. According to the results of shotgun mass spectrometric analysis of nuclear fractions, 2356 proteins were identified, of which 106 proteins were annotated as transcription factors. 37 transcription factors were uniquely identified in the fraction obtained by centrifugation in a sucrose density gradient, while only 9 and 8 transcription factors were uniquely identified in the nuclear fractions obtained using hypertonic buffer and detergents, respectively. The transcription factors identified in the HL-60 cell line represent regulatory molecules; their directed profiling under the influence of differentiation inducers, will shed light on the mechanism of granulocyte maturation.

Induction of direct cell death is one of the mechanisms of the antitumor effect of GD2-specific antibodies used for the therapy of high-risk neuroblastoma. The mechanisms of the cytotoxic signal triggered by antibody binding to GD2 ganglioside on the surface of the tumor cell remain insufficiently studied. Using inhibitor analysis we demonstrated that actin microfilaments are involved in the cell death induced by GD2-specific antibodies. Specifically, a strong antagonistic influence of cytochalasin D on the cytotoxic effect induced by GD2-specific antibodies was demonstrated in GD2+ tumor cell lines, which was expressed in at least 20% increase in cell survival and a significant decrease of the fraction of cells with fragmented DNA.

We compared the effects of placental mesenchymal stromal cells and neural progenitor cells derived from induced human pluripotent cells after their intravenous administration to rats in 24 h after transitory occlusion of the middle cerebral artery. The therapeutic effects were evaluated by the dynamics of animal survival, body weight, neurological deficit, and the volume of infarction focus in 7, 14, 30, and 60 days after surgery. Intravenous injection of neural progenitor cells produced a therapeutic effect on the course of experimental ischemic stroke by increasing animal survival in the most acute period and accelerating compensation of neurological deficit and body weight recovery. Neural progenitor cells were more effective than mesenchymal stromal cells from human placenta. The effectiveness of intravenous transplantation of neural progenitor cells in the model of occlusion of the middle cerebral artery is shown by us for the first time, although the therapeutic effect of their direct transplantation into the brain has already been described.

The data on cancer stem cell surface molecular markers of 27 most common cancer diseases were analyzed using natural language processing and data mining techniques. As a source, 8933 full-text open-access English-language scientific articles available on the Internet were used. Text mining was based on searching for three entities within one sentence, namely a tumor name, a phrase "cancer stem cells" or its synonym, and a name of differentiation cluster molecule. As a result, a list of surface molecular markers was formed that included markers most frequently mentioned in the context of certain tumor diseases and used in studies of human and animal tumor cells. Based on similarity of the associated markers, the tumors were divided into five groups.

Transplantation of solid organs, including liver, induces a number of serious complications related to immune incompatibility and requiring long-term use of immunosuppressive drugs. Finding the ways to inducing recipient immunological tolerance to the grafts is a top priority in organ transplantation and immunology. Along with the search for immunosupressive therapy, the development of alternative approaches to induction of immunological tolerance based on cell technologies is now in progress. In this regard, studies of the so-called spontaneous operational tolerance observed in ~20% patients after orthotopic liver transplantation is a promising trend. Understanding of this phenomenon can shed light on the mechanisms of immunological tolerance to allografts and will help to identify specific tolerance biomarkers and cell types with the aptitude for the induction of tolerance to liver allografts.

Acute promyelocytic leukemia, a form of acute myeloid leukemia, is characterized by cell differentiation arrest at the promyelocyte stage. Current therapeutic options include administration of all trans-retinoic acid (ATRA), but this treatment produces many side effects. ATRA is known to induce differentiation of leukemic cells into granulocytes, but the mechanism of this process is poorly studied. We performed comparative proteomic profiling of HL-60 promyelocytic cells at different stages of ATRA-induced differentiation to identify differentially expressed proteins by high-resolution mass spectrometry and relative quantitative analysis without isotope labels. A total of 1162 proteins identified by at least two unique peptides were analyzed, among them 46 and 172 differentially expressed proteins were identified in the nuclear and cytosol fractions, respectively. These differentially expressed proteins can represent candidate targets for combination therapy of acute promyelocytic leukemia.

The middle cerebral artery occlusion (MCAO) model in rats closely imitates ischemic stroke and is widely used. Existing instrumental methods provide a certain level of MCAO guidance, but monitoring of the MCA-occluding intraluminal filament position and possible complications can be improved. The goal of this study was to develop a MRI-based method of simultaneous control of the filament position, blood flow in the intracranial vessels, and hemorrhagic complications. Rats were subjected to either MRI-guided MCAO (group 1, n = 51) or MCAO without MRI control (group 2, n = 38). After operation, group 1 rats were transferred into a MRI scanner for the control of the filament position and possible complications. Ninety minutes after the onset of MCAO, the filament was removed in rats of both groups and MRI control of the infarct volume and hemorrhagic complications performed. High-resolution T1- and T2-weighted imaging performed immediately after filament insertion provided visualization of the filament position, blood flow in brain arteries, and complications related to inappropriate filament insertion. It permitted replacement of wrongly positioned filaments and exclusion of animals with complications from the experiment. MRI-based MCAO guiding provided real-time intra-operational monitoring of crucial parameters determining MCAO suitability for stroke modeling, including better assessment of the operation outcomes in individual animals and significant enhancement of the model success rate. The possibility of simultaneous visualization of the filament, blood flow in the arteries, brain tissue, and hemorrhagic complications is the principal advantage of the proposed method over other instrumental methods of MCAO quality control. Graphical Abstract MRI-guided middle cerebral artery occlusion technique permits intra-operational monitoring via direct non-invasive simultaneous visualization of the filament, blood flow in the arteries, brain tissue, and hemorrhagic complications. It provides better assessment of MCAO outcomes in individual animals and significant enhancement of MCAO success rate.

Proteomic analysis of the nuclear fraction is of great importance, since many cellular processes are initiated in the nucleus. Refinement and choice of experimental procedures for cell lysate fractionation and parameters for mass spectrometric detection and data processing continue to be of current interest. The mass spectrometry analysis presented here was tested on human cell lines derived from different tissues: HL-60 (peripheral blood); HepG2 (liver); EA.hy926 (vascular endothelium). High reproducibility of results and their consistency with biological properties of the objects under study were demonstrated. The use of cells of different types made it possible to reveal a set of 16 proteins whose LFQ-values allow for the discrimination between proteome fractions regardless of cell origin. Also, a set of 16 proteins is suggested which are associated with individual characteristics of cell lines regardless of cell fraction. These protein panels can serve as parameters to verify the proteomic analysis done was of sufficient quality, in particular as indicators of successful fractionation of cell or tissue lysate.

The literature review addresses the use of stem cells (SC) in ischemic stroke (IS). Part 1 of the paper overviews the results of experimental animal studies. Characteristics of different SC types and results of their studies in experimental models of IS are presented in the first section, the second section considers pros and cons of the methods of SC injection.

The first part of the review summarized the results of preclinical animal studies using stroke models that demonstrated the efficacy of cell therapy. The second part presents the proposed mechanisms of action of stem cells, optimal therapeutic window for cell transplantation, the results of completed clinical trials on humans in the period from 2010 to 2017, as well as the legal aspects of the use of cell technologies in the Russian Federation.

Cell therapy of the nervous system disorders using neural stem/progenitor cells (NSPCs) proved its efficacy in preclinical and pilot clinical studies. The mechanisms of the beneficial effects of NSPCs transplantation include replacement of damaged cells, paracrine activation of the regeneration, and immunomodulation. Detailed assessment of NSPCs-induced immunomodulation can contribute to better control of autoimmune reactions and inflammation in patients with neurodegenerative diseases. Interactions of NSPCs with dendritic cells (DCs), the key players in the induction of the immune system response to antigens are of particular interest. Here, we demonstrate that co-culturing of monocytes with NSPCs obtained and grown utilizing serum-containing medium instead of growth factor-containing serum-free medium, results in total suppression of monocyte differentiation into DCs. The effect is similar to the action of mesenchymal stem cells (MSCs). No significant effect on DCs maturation was observed. Cultures of NSPCs set up and maintained in serum-free medium have no influence on monocyte differentiation and DCs maturation. Therefore, the effects of NSPCs upon DC differentiation from monocytes strongly depend on culture conditions, whereas the molecular marker expression patterns are similar in both types of NSPCs cultures. In broader prospective, it means that cells with almost identical phenotypes can display opposite immunological properties depending upon culture conditions. It should be taken into account when developing NSPCs-based cell products for regenerative medicine.

Stromal liver cells obtained from liver biopsy specimens of a patient with alcoholic cirrhosis can proliferate for a long time in culture passing more than 30 passages. In the course of culturing from early to late passages, acceleration of cell proliferation, decrease of the expression of some markers, and loss of hepatogenic differentiation potential were observed. On passage 30, induced pluripotent stem cells were obtained from these cells and comparative analysis of adipogenic and hepatic differentiation potencies of these cells and original liver stromal cells was performed. Induced pluripotent stem cells differentiated into both directions more efficiently and more rapidly than initial cells. Under conditions of hepatic differentiation, liver stromal cells started to express markers of definitive endoderm, but not markers of immature/mature hepatocytes, whereas induced pluripotent stem cells consistently expressed markers of definitive endoderm, immature/mature hepatocytes.

We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.