ABSTRACT
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.
Subject(s)
Cell Differentiation , Coculture Techniques , Dendritic Cells , Dental Pulp , Mesenchymal Stem Cells , Tooth, Deciduous , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Dental Pulp/cytology , Dendritic Cells/cytology , Humans , Tooth, Deciduous/cytology , Cells, Cultured , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Monocytes/cytology , Monocytes/immunology , Interleukin-4/metabolism , Interleukin-4/pharmacology , Lipopolysaccharides/pharmacologyABSTRACT
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.
Subject(s)
Magnetic Resonance Imaging , Magnetic Resonance Imaging/methods , Animals , Stroke/diagnostic imaging , Stroke/pathology , Brain Ischemia/diagnostic imaging , Brain Ischemia/pathology , Disease Models, Animal , Cerebral Infarction/diagnostic imaging , Cerebral Infarction/pathology , Ischemic Stroke/diagnostic imaging , Ischemic Stroke/pathology , Artificial IntelligenceABSTRACT
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.
ABSTRACT
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.
Subject(s)
Brain Ischemia , Ischemic Stroke , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Stroke , Pregnancy , Female , Rats , Humans , Animals , Ischemic Stroke/metabolism , Brain Ischemia/therapy , Brain Ischemia/metabolism , Brain , Mesenchymal Stem Cells/metabolism , Stroke/therapy , Stroke/metabolism , Disease Models, Animal , Infarction, Middle Cerebral Artery/metabolismABSTRACT
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.
Subject(s)
Cancer-Associated Fibroblasts , Cell Differentiation , Colorectal Neoplasms , Dendritic Cells , Humans , Caco-2 Cells , Colorectal Neoplasms/metabolism , Dendritic Cells/metabolism , Lipopolysaccharides/pharmacology , Lipopolysaccharides/metabolism , Monocytes , Stromal Cells , Cancer-Associated Fibroblasts/metabolismABSTRACT
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.
Subject(s)
Embryonic Stem Cells , Mesenchymal Stem Cells , Uterus , Female , Humans , Cell Differentiation/physiology , Embryonic Stem Cells/metabolism , Endometrium/metabolism , Mesenchymal Stem Cells/metabolism , Myometrium , Uterus/metabolism , Uterus/pathologyABSTRACT
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.
Subject(s)
Mesenchymal Stem Cells , Nanoparticles , Polylactic Acid-Polyglycolic Acid Copolymer , Polyglycolic Acid , Lactic Acid/pharmacology , Glycols , Cells, CulturedABSTRACT
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.
Subject(s)
Brain Ischemia , Mesenchymal Stem Cell Transplantation , Stroke , Animals , Brain Ischemia/therapy , Disease Models, Animal , Infarction, Middle Cerebral Artery , RatsABSTRACT
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.
Subject(s)
Brain/pathology , Cell Tracking/methods , Ischemic Stroke/therapy , Mesenchymal Stem Cell Transplantation/methods , Animals , Brain/blood supply , Brain/metabolism , Cells, Cultured , Disease Models, Animal , Female , Ferric Compounds/chemistry , Ferric Compounds/pharmacokinetics , Humans , Imaging, Three-Dimensional , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/pathology , Infarction, Middle Cerebral Artery/therapy , Infusions, Intra-Arterial , Ischemic Stroke/metabolism , Ischemic Stroke/pathology , Magnetic Resonance Imaging , Magnetite Nanoparticles/chemistry , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Pregnancy , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Progesterone , Receptors, Estrogen , Female , Humans , Progesterone/metabolism , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Uterus/metabolism , Vagina/pathologyABSTRACT
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.
Subject(s)
Adenocarcinoma , Colorectal Neoplasms , AC133 Antigen/genetics , AC133 Antigen/metabolism , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Drug Resistance, Neoplasm/genetics , HT29 Cells , Humans , Neoplastic Stem Cells/metabolismABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters , Fluorescent Dyes , ATP-Binding Cassette Transporters/genetics , Benzimidazoles , Caco-2 Cells , Cell Line, Tumor , Flow Cytometry , HumansABSTRACT
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.
Subject(s)
Chondrocytes/cytology , Chondrogenesis/physiology , Mesenchymal Stem Cells/cytology , Cell Culture Techniques , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Chondrocytes/physiology , Chondrogenesis/genetics , Humans , Immunohistochemistry , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Wharton JellyABSTRACT
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.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation/physiology , Cell Lineage , Humans , Induced Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/metabolismABSTRACT
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.
Subject(s)
Antigens, CD/genetics , Antigens, Neoplasm/genetics , Epithelial Cell Adhesion Molecule/genetics , Gene Expression Regulation, Neoplastic , Spheroids, Cellular/metabolism , Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Caco-2 Cells , Cell Line, Tumor , Epithelial Cell Adhesion Molecule/metabolism , HCT116 Cells , Humans , Spheroids, Cellular/pathologyABSTRACT
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.
Subject(s)
Induced Pluripotent Stem Cells/transplantation , Neural Stem Cells/transplantation , Neurodegenerative Diseases/therapy , Precision Medicine/methods , Stem Cell Transplantation/methods , Animals , Cell Differentiation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Lentivirus/genetics , Lentivirus/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Retroviridae/genetics , Retroviridae/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Transduction, Genetic/methodsABSTRACT
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.
Subject(s)
Corpus Striatum/cytology , Lateral Ventricles/cytology , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/cytology , Neural Stem Cells/cytology , Placenta/cytology , Animals , Carotid Artery, Internal/cytology , Cell Movement , Cell Proliferation , Corpus Striatum/surgery , Female , Humans , Injections, Intra-Arterial , Injections, Intraventricular , Lateral Ventricles/surgery , Male , Mesenchymal Stem Cells/physiology , Middle Cerebral Artery/cytology , Neural Stem Cells/physiology , Placenta/physiology , Pregnancy , Primary Cell Culture , Rats , Rats, Wistar , Stereotaxic Techniques , Transplantation, HeterologousABSTRACT
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.
Subject(s)
Adenocarcinoma/metabolism , Colorectal Neoplasms/metabolism , Gangliosides/metabolism , Caco-2 Cells , HT29 Cells , HumansABSTRACT
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.
Subject(s)
AC133 Antigen/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Caco-2 Cells , Cell Proliferation/physiology , Colorimetry , HT29 Cells , Humans , Ki-67 Antigen/metabolismABSTRACT
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.