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.

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.

[Splice variants of mRNA of cytochrome P450 genes: analysis by the nanopore sequencing method in human liver tissue and HepG2 cell line]. / Splais-varianty mRNK genov tsitokhromov R450: analiz metodom nanoporovogo sekvenirovaniia v tkani pecheni cheloveka i kletkakh linii HepG2.

The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.

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.

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.

Effect of Bioactive Peptide Complex Isolated from Bovine Serum on Proliferation and Migration of Mesenchymal Stromal Cells In Vitro and Reparation of Bone Defects In Vivo.

We studied the effect of a bioactive peptide complex isolated from bovine serum on the proliferative potential and migration rate of mesenchymal stromal cells in vitro, as well as on the healing of modeled bone defects in rats. This bioregulatory preparation stimulated proliferation of mesenchymal stromal cells from deciduous tooth pulp in vitro, but did not affect the rate of their migration in two-dimensional cultures. In vivo experiments showed that application of this preparation in combination with hydroxyapatite and chitosan gel accelerated bone tissue regeneration, thus ensuring restoration of morphologically normal bone matrix. Thus, cattle blood serum is an available source for the production of bioregulatory preparations for medical purposes.

[Proteomics of transcription factors: identification of pool of HL-60 cell line-specific regulatory proteins]. / Proteomika transkriptsionnykh faktorov: identifikatsiia pula reguliatornykh belkov, spetsifichnykh dlia kletok linii HL-60.

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.

Effect of Low-Level Laser Irradiation on Proliferative Activity of Wharton's Jelly Mesenchymal Stromal Cells.

We studied the effect of low-level laser irradiation on proliferative activity of cultured human Wharton's jelly mesenchymal stromal sells. Cells were irradiated with a solid-state laser emitting at 650 nm; irradiation doses were 0.04, 0.4, or 4 J/cm2. Laser irradiation was performed once at the start of the cell proliferation experiment or daily throughout the experiment. Cells were cultured for 7 days. The number of viable cells was assessed using the MTT test. An increase in cell proliferative activity was detected after daily laser irradiations; the maximum stimulating effect was achieved at a dose of 0.04 J/cm2. These results substantiate medical use of lasers for expansion of cells intended for transplantation.

A Cell Model of Human Small Intestinal Wall Based on Genetically Modified Caco-2 Cells.

We propose a cell model of the human small intestinal wall based on genetically modified Caco-2 cells that allows visualization and quantitative assessment of activation of NF-κB factor and related intracellular pathway by using fluorescence microscopy. A dose-dependent increase in fluorescence intensity of the obtained cells in response to TNFα exposure in concentrations of 1-100 ng/ml was demonstrated. It was found that this parameter correlates with a decrease in the transepithelial resistance of the cell monolayer in response to TNFα and can be used to assess the toxic effects of substances on epithelial cells of the human small intestine.

Proteomic Profiling of HL-60 Cells during ATRA-Induced Differentiation.

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.

[Comparative proteomic profiling of nuclear and cytosolic fractions from cell lines of different origin]. / Sravnitel'noe proteomnoe profilirovanie iadernoi i tsitozol'noi fraktsii kletochnykh linii razlichnogo proiskhozhdeniia.

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.

[PCR analysis of the absolute number of copies of human chromosome 18 transcripts in liver and HepG2 cells]. / PTsR-analiz absoliutnogo chisla kopii transkriptov khromosomy 18 cheloveka v kletkakh pecheni i linii HepG2.

Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

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.

Variability of the Phenotype and Proliferation and Migration Characteristics of Human Mesenchymal Stromal Cells Derived from the Deciduous Teeth Pulp of Different Donors.

We performed a comparative study of cell phenotype and proliferation and migration activities in vitro of mesenchymal stromal cells from human exfoliated deciduous teeth (SHED cells) from three donors. In the primary cultures, the cells of different donors had the same morphology and cytophenotype, but differed by proliferative and migration capacities. The results indicate that individual mesenchymal stromal cells cultures can differ considerably by important cell properties, and this should be considered when evaluating their potential therapeutic efficacy and in experimental studies.

Comparative analysis of the expression of surface markers on fibroblasts and fibroblast-like cells isolated from different human tissues.

Expression of 20 surface markers was analyzed in cultures of mesenchymal stromal cells of the umbilical cord, fibroblasts from adult and fetal human skin, and fibroblast-like cells of fetal liver was analyzed by fl ow cytometry. The studied cultures did not express hemopoietic cells markers, but were positive for CD73, CD90, and CD105 markers recommended by the International Society of Cell Therapy for the identification of the multipotent mesenchymal stromal cells. Fetal liver fibroblast-like cells were positive for CD54; this marker was absent in skin fibroblast cultures, but was expressed by umbilical cord mesenchymal stromal cells. Further study of these cells revealed a minor subpopulation of cells co-expressing CD24 and CD90 or CD24 and CD54. We hypothesized that these cells probably participate in epithelial mesenchymal transition.

Effect of calcium phosphate materials on multipotent mesenchymal cells from exfoliated deciduous teeth (SHED cells) in vitro.

Various calcium phosphate ceramic materials were created and their effect on cultured mesenchymal cells from exfoliated deciduous tooth pulp was evaluated. Tricalcium phosphate ceramics provides best cell survival and is an optimal material for bone tissue engineering. Analysis of the effects of tricalcium phosphate ceramics on osteogenic differentiation of SHED cells suggests that this material potentiated dexamethasone-induced osteogenic differentiation, which manifested in the increased number of ossification foci and enhanced extracellular matrix production by cells. Thus, the tricalcium phosphate ceramics created by us is a promising biomedical material that can be used for tissue-engineered bone analogs.

Design of tissue engineering implants for bone tissue regeneration of the basis of new generation polylactoglycolide scaffolds and multipotent mesenchymal stem cells from human exfoliated deciduous teeth (SHED cells).

Cultures of multipotent mesenchymal stromal cells from the pulp of human deciduous teeth (SHED cells) were characterized. The cells were used for population of 3D biodegradable polylactoglycolide scaffolds; their osteogenic potential was preserved under these conditions. Implantation of the scaffolds to mice induced no negative reactions in the recipients. These results suggest that the use of polylactoglycolide scaffolds populated with SHED cells is a promising approach for creation of implants for bone defect replacement.

Mesenchymal cells of the decidual tooth pulp: cytophenotype and initial evaluation of possibility of their use in bone tissue engineering.

Cultures of mesenchymal cells from human decidual tooth pulp were derived. The phenotype and capacity to osteogenic and adipogenic differentiation of these cells are close to those of bone marrow mesenchymal stem cells. Decidual tooth pulp mesenchymal cells populate biodegraded polylactide scaffolds and hence, can be used for the creation of tissue engineering transplants for bone defect repair. Storage of decidual tooth pulp mesenchymal cells in the stem cell cryobanks together with umbilical blood will appreciably extent the periods of age for collection of juvenile autologous stem cells for use throughout the life span.