Mesenchymal Stem Cells from the Deciduous Tooth Pulp Lose their Ability to Suppress the Differentiation of Dendritic Cells during Long-Term Culturing.

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.

Suppressive Effect of Chemically Induced Hypoxia on Glioblastoma Cell Proliferation.

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) (CoCl2) (chemically induced hypoxia). In the present work, we analyzed the effect of CoCl2 on the viability, proliferation, and apoptosis of cells of three glioblastoma cell lines: 1321N1, T98g, and U373 MG. It was shown that CoCl2 induced a dose-dependent decrease in cell viability and proliferation, and at high concentrations (200 and 400 µM) stimulated cell death. CoCl2 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.

Therapeutic Efficacy and Migration of Mesenchymal Stem Cells after Intracerebral Transplantation in Rats with Experimental Ischemic Stroke.

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.

Isolation of a Population of Cells Co-Expressing Markers of Embryonic Stem Cells and Mesenchymal Stem Cells from the Rudimentary Uterine Horn of a Patient with Uterine Aplasia.

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.

Colorectal Cancer and Tumor Stromal Cells Have Different Effects on the Differentiation and Maturation of Dendritic Cells In Vitro.

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.

Selective Accumulation of Poly(Lactic-Co-Glycolic Acid) Nanoparticles in Endotheliocytes and Mesenchymal Stromal Cells Cultured as Mixed-Cell Spheroids.

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.

Dose-Dependent Effects of Intravenous Mesenchymal Stem Cell Transplantation in Rats with Acute Focal Cerebral Ischemia.

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, 5×105 and 2×106 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.

MRI-Based and Histologically Verified 3D Modeling of Spatial Distribution of Intra-Arterially Transplanted Cells in Rat Brain.

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.

Human Uterine Rudiments: Histological and Immunohistochemical Study.

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.

Analysis of the Correlation between CD133 Expression on Human Colorectal Adenocarcinoma Cells HT-29 and Their Resistance to Chemotherapeutic Drugs.

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.

[Identification of the side population associated with ATP-binding cassette transporters activity using imaging flow cytometry]. / Vyiavlenie "bokovoi populiatsii", assotsiirovannoi s aktivnost'iu ATP-zavisimykh transporterov, pri pomoshchi protochnoi tsitometrii s vizualizatsiei.

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.

Chondrogeneic Potential of MSC from Different Sources in Spheroid Culture.

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.

Expression of Epithelial Cell Adhesion Molecule (EpCAM) in Tumor Spheroids of Human Colorectal Adenocarcinoma Cells.

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.

Methods of Generation of Induced Pluripotent Stem Cells and Their Application for the Therapy of Central Nervous System Diseases.

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.

Distribution and Migration of Human Placental Mesenchymal Stromal Cells in the Brain of Healthy Rats after Stereotaxic or Intra-Arterial Transplantation.

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.

[Expression of ganglioside GD2 on colorectal adenocarcinoma cells]. / Ékspressiia gangliozida GD2 na kletkakh kolorektal'noi adenokartsinomy.

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.

Proliferative Activity of Colorectal Cancer Cells with Different Levels of CD133 Expression.

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.

Involvement of Actin Filaments in the Cytotoxic Effect of GD2-Specific Antibodies.

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.

Comparative Analysis of the Effects of Intravenous Administration of Placental Mesenchymal Stromal Cells and Neural Progenitor Cells Derived from Induced Pluripotent Cells on the Course of Acute Ischemic Stroke in Rats.

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.

[Cell therapy for ischemic stroke. Stem cell types and results of pre-clinical trials]. / Kletochnaia terapiia ishemicheskogo insul'ta. Tipy stvolovykh kletok i rezul'taty doklinicheskikh issledovanii.

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.