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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.Metodom nanoporovogo sekvenirovaniia proveden analiz transkriptov tsitokhromov R450 v obraztsakh tkani pecheni trekh donorov i kletkakh linii HepG2 (gepatoblastoma cheloveka). Pokazano, chto priamoe sekvenirovanie mRNK s ispol'zovaniem nanoporovogo sekvenatora MinION (Oxford Nanopore Technologies) pozvoliaet poluchat' kolichestvennye profili transkriptov (i ikh splaĭs-variantov) dlia genov supersemeĭstva tsitokhroma R450, kodiruiushchikh izoformy, pri uchastii kotorykh metaboliziruetsia bol'shaia chast' (~80%) lekarstvennykh veshchestv. Profili splaĭs-variantov sushchestvenno razlichaiutsia dlia raznykh donorov. Ékspressiia genov tsitokhromov R450 na urovne transkriptov v kletkakh linii HepG2 znachitel'no slabee v sravnenii s ikh ékspressieĭ v normal'noĭ tkani pecheni, chto zatrudniaet issledovanie al'ternativnogo splaĭsinga transkriptov tsitokhromov R450 v kletkakh HepG2 metodom priamogo nanoporovogo sekvenirovaniia mRNK. Profili ékspressii genov supersemeĭstva tsitokhroma R450 na urovne trankriptov v tkani pecheni cheloveka i kletkakh HepG2 znachitel'no razlichaiutsia kak kolichestvenno, tak i kachestvenno.

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.

The calcium phosphate particles can be used as building blocks for fabrication of 3D scaffolds intended for bone tissue engineering. This work presents for the first time a rapid creation of 3D scaffolds using magnetic levitation of calcium phosphate particles. Namely, tricalcium phosphate particles of equal size and certain porosity are used, which undergo the process of recrystallization after magnetic levitational assembly of the scaffold to ensure stitching of the scaffold. Label-free levitational assembly is achieved by using a custom-designed magnetic system in the presence of gadolinium salts, which allows the levitation of calcium phosphate particles. Chemical transformation of tricalcium- to octacalcium phosphate under the condition of magnetic levitation in non-homogeneous magnetic field is also demonstrated. This approach allows obtaining rapidly the octacalcium phosphate phase in the final 3D product, which is biocompatible.

Simple Summary A new technology has been recently developed by Oxford Nanopore Technologies, enabling researchers to investigate the structure and relative abundance of specific molecules, ribonucleic acids. The ribonucleic acids carry information from the genes to proteins, which are responsible for virtually every process in the human organism, including disease progression and response to therapies. Special computational methods allow identification of various activated biological processes by analyzing the changes in concentrations of ribonucleic acids. This is of particular interest for precision medicine which aims at single-patient analysis. Here we evaluated whether ribonucleic acid abundances measured by new technology are suited for robust predictions of activated biological processes in single samples. We performed simulations varying the number of experimental replicates and analysed activated biological processes’ predictions using two algorithms. In brief, we found that at least two replicates are required to obtain reproducible results. We hope that our findings may be of interest to researchers planning their nanopore experiments and may stimulate further development of clinical applications of this technology. Abstract Long-read direct RNA sequencing developed by Oxford Nanopore Technologies (ONT) is quickly gaining popularity for transcriptome studies, while fast turnaround time and low cost make it an attractive instrument for clinical applications. There is a growing interest to utilize transcriptome data to unravel activated biological processes responsible for disease progression and response to therapies. This trend is of particular interest for precision medicine which aims at single-patient analysis. Here we evaluated whether gene abundances measured by MinION direct RNA sequencing are suited to produce robust estimates of pathway activation for single sample scoring methods. We performed multiple RNA-seq analyses for a single sample that originated from the HepG2 cell line, namely five ONT replicates, and three replicates using Illumina NovaSeq. Two pathway scoring methods were employed—ssGSEA and singscore. We estimated the ONT performance in terms of detected protein-coding genes and average pairwise correlation between pathway activation scores using an exhaustive computational scheme for all combinations of replicates. In brief, we found that at least two ONT replicates are required to obtain reproducible pathway scores for both algorithms. We hope that our findings may be of interest to researchers planning their ONT direct RNA-seq experiments.

The chromosome-centric dataset was created by applying several technologies of transcriptome profiling. The described dataset is available at NCBI repository (BioProject ID PRJNA635536). The dataset referred to the same type of tissue, cell lines, transcriptome sequencing technologies, and was accomplished in a period of 8 years (the first data were obtained in 2013 while the last ones - in 2020). The high-throughput sequencing technologies were employed along with the quantitative PCR (qPCR) approach, for data generation using the gene expression level assessment. qPCR was performed for a limited group of genes, encoded on human chromosome 18, for the Russian part of the Chromosome-Centric Human Proteome Project. The data of high-throughput sequencing are provided as Excel spreadsheets, where the data on FPKM and TMP values were evaluated for the whole transcriptome with both Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing.

Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA) treatment underlies differentiation therapy of acute myeloid leukemia. Knowing the regulation of this process it is possible to identify potential targets for antileukemic drugs and develop novel approaches to differentiation therapy. In this study, we have performed transcriptomic and proteomic profiling to reveal up- and down-regulated transcripts and proteins during time-course experiments. Using data on differentially expressed transcripts and proteins we have applied upstream regulator search and obtained transcriptome- and proteome-based regulatory networks of induced granulocytic differentiation that cover both up-regulated (HIC1, NFKBIA, and CASP9) and down-regulated (PARP1, VDR, and RXRA) elements. To verify the designed network we measured HIC1 and PARP1 protein abundance during granulocytic differentiation by selected reaction monitoring (SRM) using stable isotopically labeled peptide standards. We also revealed that transcription factor CEBPB and LYN kinase were involved in differentiation onset, and evaluated their protein levels by SRM technique. Obtained results indicate that the omics data reflect involvement of the DNA repair system and the MAPK kinase cascade as well as show the balance between the processes of the cell survival and apoptosis in a p53-independent manner. The differentially expressed transcripts and proteins, predicted transcriptional factors, and key molecules such as HIC1, CEBPB, LYN, and PARP1 may be considered as potential targets for differentiation therapy of acute myeloid leukemia.

Introduction. As an alternative to autochondral transplantation, variants of chondrocyte replacement with mesenchymal stromal cells (MSCs) were considered, since these cells are present in all organs and tissues of human body and possess multilinear potential for differentiation. A number of studies demonstrate that the ability for chondrogenic differentiation of MSCs from different tissues varies, however, those studies are few and controversial. In accordance with the ethical principles and the technical ease of obtaining, adipose tissue, Wharton’s jelly (stroma) and dental pulp are the most attractive sources of MSCs for tissue engineering.Aim: to compare the chondrogenic potential of MSCs allocated from adipose tissue, Wharton’s jelly of the umbilical cord, and human deciduous teeth pulp cultured in the composition of microspheres (pellets).Materials and methods. The phenotype of primary cultures of MSCs was studied via flow microscopy. Chondrogenic differentiation was performed with 3D-culture in microspheres in the presence of TGFβ1 for two weeks under standard conditions. Human chondroblasts were used as a positive control. Cell viability was determined by fluorescent staining. Morphological study was performed using histological and immunohistochemical staining methods.Results. Cultures of MSCs from all sources had similar phenotypes CD29+, CD34–, CD44+, CD49b+, CD45–, CD73+, CD90+, HLADR. In the mass of living cells in microspheres only singular dead cells were visualized. A significant production of extracellular matrix (ECM) was observed in the chondroblast and adipose tissue microspheres, whereas in the microspheres of dental pulp and umbilical cord stroma derived MSCs, no significant ECM was detected. Among the MSCs, the highest production of collagen and glycosaminoglycans (GAG) in the ECM was observed in the microspheres of adipose tissue-derived MSCs, and the lowest production – in the microspheres of dental pulp-derived MSCs. Conclusion. A histological analysis of all microspheres after 14 days of culturing in the chondrogenic medium revealed the signs of differentiation in the chondrogenic direction, a progressive increase of the ECM produced by cells, and the presence of total collagen and GAG in it. Of all the MSCs studied, the greatest chondrogenic potential in vitro (the intensity of development of ECM components) is possessed by the adipose tissue-derived MSCs.

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.

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.Promielotsitarnye kletki linii HL-60 iavliaiutsia shiroko ispol'zuemoĭ model'iu dlia izucheniia indutsiruemoĭ granulotsitarnoĭ differentsirovki. Issledovanie belkov iadernoĭ fraktsii, v osobennosti transkriptsionnykh faktorov, neobkhodimo dlia luchshego ponimaniia molekuliarnykh mekhanizmov sozrevaniia kletok. Mass-spektrometriia iavliaetsia moshchnym metodom dlia analiza proteoma v silu vysokoĭ chuvstvitel'nosti, proizvoditel'nosti i spetsifichnosti analiza. V rabote, s ispol'zovaniem metoda monitoringa vybrannykh reaktsiĭ (SRM), provedena otsenka urovnia soderzhaniia iadernykh belkov RBPJ, STAT1, CEBPB, CASP3, VAV1, PRKDC, PARP1 i UBC9, vydelennykh s primeneniem gipertonicheskogo bufera, detergentov (dodetsilsul'fata natriia (SDS), dezoksikholata natriia (DOC) i rasshchepliaemogo detergenta ProteaseMAX™) i s pomoshch'iu tsentrifugirovaniia v gradiente plotnosti sakharozy. Minimal'noe i maksimal'noe soderzhanie belkov sostavilo 1,13±0,28 i 14,34±1,63 fmol'/mkg obshchego belka dlia transkriptsionnogo faktora RBPJ i ubikvitin-proteinligazy pervogo tipa UBC9 sootvetstvenno. Po rezul'tatam panoramnogo mass-spektrometricheskogo analiza iadernykh fraktsiĭ udalos' identifitsirovat' 2356 belkov, iz nikh 106 belkov byli annotirovany kak transkriptsionnye faktory. Tret' transkriptsionnykh faktorov (37 belkov) byli identifitsirovany tol'ko vo fraktsii, poluchennoĭ s pomoshch'iu tsentrifugirovaniia v gradiente plotnosti sakharozy, v to vremia kak lish' 9 i 8 transkriptsionnykh faktorov bylo identifitsirovano tol'ko vo fraktsiiakh iader, poluchennykh s ispol'zovaniem gipertonicheskogo bufera i detergentov, sootvetstvenno. Transkriptsionnye faktory, identifitsirovannye v kletkakh linii HL-60, predstavliaiut soboĭ reguliatornye molekuly, napravlennoe profilirovanie kotorykh pod deĭstviem induktorov differentsirovki pozvolit prolit' svet na mekhanizm sozrevaniia granulotsitov.

The aim of this work was to identify the spectrum of possible protein-protein interactions (PPI) for six target proteins (CYB5A, RAB27B, SMAD4, CXXC1, RNMT, ТТR) encoded by genes of human chromosome 18, which have a certain medical significance. For this purpose, a comprehensive approach based on the combined application of three technologies, direct molecular fishing based on affinity chromatography, protein mass spectrometry and SPR (surface plasmon resonance) analysis, was used. As a result, from 1 to 11 potential protein partners were isolated from the lysate of the human hepatocellular carcinoma HepG2 cell line for each target protein. Using the Biacore 3000 SPR biosensor, 10 potential PPIs, for which preparations of recombinant proteins were available, were validated. Positive results, which confirm direct interaction of the identified protein partners with target proteins, were obtained for five protein pairs (СYB5A/CPR, СYB5A/CYP2C9, СYB5A/CYCS, CPR/СYP2C9 and CXXC1/CYCS). The values of equilibrium dissociation constants of protein complexes (Kd values ranged from 10–7 M to 10–5 M) were determined for these PPIs. To evaluate the specificity of identified PPIs, 16 additional different PPIs of the target proteins were tested. The effectiveness of direct molecular fishing was evaluated using cytochrome b5 (CYB5A) as the most studied target protein in the context of structural and functional relationships with its protein partners. The new data extend our knowledge in the field of interactomics of proteins encoded by human chromosome 18 genes and reduces the number of unverified bioinformatic predictions of possible PPIs presented in accessible Internet resources.

Missing (MP) and functionally uncharacterized proteins (uPE1) comprise less than 5% of the total number of proteins encoded by human Chr18 genes. Within half a year, since the January 2020 version of NextProt, the number of entries in the MP+uPE1 datasets changed, mainly due to the achievements of antibody-based proteomics. Assuming that the proteome is closely related to the transcriptome scaffold, quantitative PCR, Illumina HiSeq, and Oxford Nanopore Technology were applied to characterize the liver samples of three male donors in comparison with the HepG2 cell line. The data mining of the Expression Atlas (EMBL-EBI) and the profiling of biopsy samples by using orthogonal methods of transcriptome analysis have shown that in HepG2 cells and the liver, the genes encoding functionally uncharacterized proteins (uPE1) are expressed as low as for the missing proteins (less than 1 copy per cell), except the selected cases of HSBP1L1, TMEM241, C18orf21, and KLHL14. The initial expectation that uPE1 genes might be expressed at higher levels than MP genes, was compromised by severe discrepancies in our semi-quantitative gene expression data and in public databanks. Such discrepancy forced us to revisit the transcriptome of Chr18, the target of the Russian C-HPP Consortium. Tanglegram of highly expressed genes and further correlation analysis have shown the severe dependencies on the mRNA extraction method and the analytical platform. Targeted gene expression analysis by quantitative PCR (qPCR) and high-throughput transcriptome profiling (Illumina HiSeq and ONT MinION) for the same set of samples from normal liver tissue and HepG2 cells revealed the detectable expression of 250+ (92%) protein-coding genes of Chr18 (at least one method). The expression of slightly more than 50% protein-coding genes was detected simultaneously by all three methods. Correlation analysis of the gene expression profiles showed that the grouping of the datasets depended almost equally on both the type of biological material and the experimental method, particularly cDNA/mRNA isolation and library preparation., Отсутствующие белки и функционально не охарактеризованные белки (в англоязычной литературе обозначенные как missing (MP) и functionally uncharacterized proteins (uPE1), соответственно) составляют менее 5% от общего числа белков, кодируемых генами 18 хромосомы человека. В течение полугода, начиная с января 2020 года, в версии NextProt выросло количество записей в наборах данных MP+uPE1. Подобные изменения обусловлены преимущественно достижениями протеомики на основе антител. В данной работе количественная ПЦР, технологии секвенирования Illumina HiSeq и Oxford Nanopore Technologies были применены для сравнительного анализа транскриптомного профиля образцов печени трех доноров мужского пола и клеточной линии HepG2. Анализ данных атласа экспрессии (Expression Atlas, EMBL-EBI) и полученных результатов по биологическим образцам с использованием ортогональных методов анализа транскриптома показал, что в клетках печени и HepG2 уровень экспрессии генов, кодирующих функционально не охарактеризованные белки (uPE1), находится на таком же низком уровне, как и в случае генов MP (в количестве менее 1 копии на клетку). Исключение составили несколько генов: HSBP1L1, TMEM241, C18orf21 и KLHL14. Согласно существенным расхождениям в ранее полученных полуколичественных данных по экспрессии генов и данным в открытых базах данных, изначально предполагалось, что экспрессия генов uPE1 может быть выше, чем генов MP. Подобное расхождение побудило обратиться к транскриптому 18 хромосомы человека, являющейся целевой для России в проекте «Протеом человека». Полученные результаты о наиболее экспрессируемых генах и дальнейший корреляционный анализ показал существование зависимости от метода экстракции мРНК и аналитической платформы. Анализ экспрессии целевых генов 18 хромосомы с применением количественной ПЦР (qPCR) и методов высокопроизводительного профилирования транскриптома (Illumina HiSeq и ONT MinION) для одинаковых наборов образцов нормальной ткани печени и клеточной линии HepG2 выявил более 250 (92%) белок-кодирующих генов, детектируемых хотя бы одним методом. Экспрессия более чем 50% белок-кодирующих генов была детектирована всеми тремя методами. Корреляционный анализ профилей экспрессии генов показал, что результаты «группируются» в зависимости от типа биологического материала и экспериментальных методов, в частности от способа подготовки библиотеки (выделения кДНК, мРНК). Зависимость от выбора способа биоинформатической обработки была отмечена в значительно меньшей степени.

Missing (MP) and functionally uncharacterized proteins (uPE1) comprise less than 5% of the total number of human Chr18 genes. Within half a year, since the January 2020 version of NextProt, the number of entries in the MP+uPE1 datasets has changed, mainly due to the achievements of antibody-based proteomics. Assuming that the proteome is closely related to the transcriptome scaffold, quantitative PCR, Illumina HiSeq, and Oxford Nanopore Technology were applied to characterize the liver samples of three male donors compared with the HepG2 cell line. The data mining of Expression Atlas (EMBL-EBI) and the profiling of our biospecimens using orthogonal methods of transcriptome analysis have shown that in HepG2 cells and the liver, the genes encoding functionally uncharacterized proteins (uPE1) are expressed as low as for the missing proteins (less than 1 copy per cell), except for selected cases of HSBP1L1, TMEM241, C18orf21, and KLHL14. The initial expectation that uPE1 genes might be expressed at higher levels than MP genes, was compromised by severe discrepancies in our semi-quantitative gene expression data and in public databanks. Such discrepancy forced us to revisit the transcriptome of Chr18, the target of Russian C-HPP Consortia. Tanglegram of highly expressed genes and further correlation analysis have shown the severe dependencies on the mRNA extraction method and analytical platform.Targeted gene expression analysis by quantitative PCR (qPCR) and high-throughput transcriptome profiling (Illumina HiSeq and ONT MinION) for the same set of samples from normal liver tissue and HepG2 cells revealed the detectable expression of 250+ (92%) protein-coding genes of Chr18 (at least one method). The expression of slightly more than 50% protein-coding genes was detected simultaneously by all three methods. Correlation analysis of the gene expression profiles showed that the grouping of the datasets depended almost equally on both the type of biological material and the experimental method, particularly cDNA/mRNA isolation and library preparation. The dependence on the choice of bioinformatics analysis pipeline was also noticeable but significantly less. Furthermore, the combination of Illumina HiSeq and ONT MinION sequencing to validate proteotypic peptides of missing and uPE1 proteins was performed for the heat-shock factor binding protein HSBP1L1 (missing protein, recently transferred to PE1 category) and uncharacterized protein C18orf21 (uPE1). We observed that a nonsynonymous SNP led to the loss of the site of trypsinolysis in HSBP1L1. The modified version of HSBP1L1 was included in the sequence database and searched against the MS/MS dataset from Kulak, Geyer & Mann (2017), but delivered no significant identification. Thus, HSBP1L1 is still missing for the MS-pillar of C-HPP, although its existence at the protein level has been confirmed.

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.

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.

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.

The aim of this work was to test modifications of the standard protocol for the sample preparation of cell/tissue lysate before performing the affinity isolation of lysate protein partners for the target protein (bait protein) which is covalently immobilized on an inert sorbent (e.g. BrCN-, SH-Sepharose 4B) or a carrier (e.g. paramagnetic nanoparticles). The series of our previous works on applying the approach to direct molecular fishing procedure with combination of affinity chromatography and LC-MS/MS analysis using a number of proteins, encoded by the genes of human chromosome 18, have shown that there are at least two problems affecting the specificity and the effectiveness of this procedure. These include: (i) redundancy of the background proteins in the eluates from an affinity sorbent (carrier) due to isolation of multiprotein complexes “labeled” with a direct protein partner which binds with a bait protein immobilized on the sorbent; (ii) low enrichment of the eluates with appropriate protein partners due to the fact that some direct protein partners in the lysate exist in stable “wild type” complexes with the bait protein itself. This means that latter group of protein partners will not be sufficiently isolated from lysate. Therefore, in order to increase the specificity and efficiency of affinity isolation of protein partners for the bait protein, we modified the standard protocol of lysate preparation and the preliminary step on dissociation of lysate protein complexes was added. Several model experiments for the choice of regeneration solution, assessment of their efficiency in the dissociation of lysate protein complexes as well as the stability and binding capacity of proteins were performed under the control of surface plasmon resonance (SPR) biosensor Biacore 3000 using HepG2 cell lysate. It was shown that acid treatment and incubation of the cell lysate for one min on ice (final lysate dilution 20 times) and subsequent neutralization (pH shift from 2.0 to 7.4) resulted in maximal dissociation of the lysate protein complexes without significant negative effects on the protein-protein interactions tested., Целью работы было экспериментальное тестирование модификации стандартного протокола пробоподготовки клеточного/ тканевого лизата перед выполнением процедуры аффинного выделения из него белков-партнеров для целевого белка (белка-наживки), иммобилизованного на инертном сорбенте или парамагнитных наночастицах. Цикл наших предыдущих работ, посвященных прямому молекулярному фишингу с сопряжением хроматографических и масс-спектрометрических методов и технологии парамагнитных наночастиц c использованием ряда белков 18-ой хромосомы человека и также других белков показал, что существуют, по крайне мере, две проблемы, влияющие на специфичность и эффективность данной процедуры: (i) избыточность фоновых белков в элюатах с аффинного сорбента, обусловленная выделением мультибелковых комплексов, меченых прямым партнером, который связывается с целевым белком на сорбенте; (ii) низкая обогащенность элюатов белками-партнерами целевой группы обусловленная тем, что та или иная часть прямых белков-партнеров в лизате находится в составе стабильных комплексов «дикого типа» с самим белком-наживкой и не будет в достаточной степени выделена из лизата. Поэтому для повышения специфичности и эффективности аффинного выделения белков-партнеров целевого белка нами предложена модификация стандартной пробоподготовки, заключающаяся в предварительной диссоциации белковых комплексов лизата. Модельные эксперименты по выбору регенерационного раствора, оценке стабильности и связывающей способности белков при его воздействии, а также оценка эффективности диссоциации комплексов в лизате были выполнены под контролем оптического биосенсора Biacore 3000 («GE Healthcare», США) с использованием лизата клеточной культуры гепатокарциномы человека (HepG2) и рекомбинантных препаратов белков, кодируемых генами 18-ой хромосомы человека, Показано, что кислотная обработка разбавленного в 20 раз лизата с кратковременной экспозицией в течение 1 мин на льду и с последующей нейтрализацией (с рН 2.0 до рН 7.4) приводила к максимальной диссоциации белковых комплексов лизата, не оказывая существенного негативного влияния на тестируемые белок- белковые взаимодействия.

A gene-centric approach was applied for a large-scale study of expression products of a single chromosome. Transcriptome profiling of liver tissue and HepG2 cell line was independently performed using two RNA-Seq platforms (SOLiD and Illumina) and also by Droplet Digital PCR (ddPCR) and quantitative RT-PCR. Proteome profiling was performed using shotgun LC-MS/MS as well as selected reaction monitoring with stable isotope-labeled standards (SRM/SIS) for liver tissue and HepG2 cells. On the basis of SRM/SIS measurements, protein copy numbers were estimated for the Chromosome 18 (Chr 18) encoded proteins in the selected types of biological material. These values were compared with expression levels of corresponding mRNA. As a result, we obtained information about 158 and 142 transcripts for HepG2 cell line and liver tissue, respectively. SRM/SIS measurements and shotgun LC-MS/MS allowed us to detect 91 Chr 18-encoded proteins in total, while an intersection between the HepG2 cell line and liver tissue proteomes was ∼66%. In total, there were 16 proteins specifically observed in HepG2 cell line, while 15 proteins were found solely in the liver tissue. Comparison between proteome and transcriptome revealed a poor correlation (R

To obtain more information about human proteome, especially about proteoforms (protein species) coded by 18th chromosome, we separated proteins from human cancer cell line (HepG2) by two-dimensional gel electrophoresis (2DE). Initially, proteins in major spots were identified by MALDI-MS peptide mass fingerprinting. According to parameters (pI/Mw) of identified proteins the gel was calibrated. Using this calibrated gel, a virtual 2D map of proteoforms coded by Chromosome 18 was constructed. Next, the produced gel was divided into 96 sections with determined coordinates. Each section was cut, shredded, and treated by trypsin according to mass-spectrometry protocol. After protein identification by shotgun mass spectrometry using ESI LC-MS/MS, a list of 20 462 proteoforms (product of 3774 genes) was generated. Among them, 165 proteoforms are representing 39 genes of 18th chromosome. The 3D graphs showing the distribution of different proteoforms from the same gene in 2D map were generated. This is a first step in creation of 2DE-based knowledge database of proteins coded by 18th chromosome.

Virtual and experimental 2DE coupled with ESI LC-MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI-TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC-MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw (http://web.expasy.org/compute_pi/pi_tool-doc.html). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.

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.Issledovanie proteoma iadernoĭ fraktsii iavliaetsia vazhnoĭ zadacheĭ, poskol'ku bol'shaia chast' protsessov, proiskhodiashchikh v kletke, initsiiruetsia v iadre. Razrabotka i vybor usloviĭ fraktsionirovaniia kletochnykh lizatov i parametrov mass-spektrometricheskogo detektirovaniia i obrabotki dannykh v nastoiashchee vremia prodolzhaiut ostavat'sia aktual'nymi. Predstavlennyĭ v rabote mass-spektrometricheskiĭ analiz aprobirovan na liniiakh kletok, proiskhodiashchikh iz raznykh tkaneĭ: HL-60 (perifericheskaia krov'), HepG2 (pechen'), EA.hy926 (éndoteliĭ sosudov). Prodemonstrirovana vysokaia vosproizvodimost' rezul'tatov i ikh soglasovannost' s biologicheskimi svoĭstvami issledovannykh ob"ektov. Blagodaria ispol'zovaniiu raznogo tipa kletochnykh liniĭ vyiavlen nabor iz 16 belkov, ch'i kolichestvennye pokazateli (LFQ – Label-free Quantification Intensity) pozvoliaiut diskriminirovat' iadernuiu i tsitozol'nuiu fraktsii vne zavisimosti ot proiskhozhdeniia kletok. Takzhe predlozhen nabor iz 16 belkov, ch'i LFQ sviazany s individual'nymi priznakami kletochnykh liniĭ vne zavisimosti ot fraktsii. Éti paneli belkov mogut ispol'zovat'sia kak indikatory uspeshnogo fraktsionirovaniia kletochnogo ili tkanevogo lizata.

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.

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.

Targeted mass spectrometry represents a powerful tool for investigation of biological processes. The convenient approach of selected reaction monitoring using stable isotope-labeled peptide standard (SIS) is widely applied for protein quantification. Along with this method, high-resolution parallel reaction monitoring has been increasingly used for protein targeted analysis. Here we applied two targeted approaches (selected reaction monitoring with SIS and label-free parallel reaction monitoring) to investigate expression of 11 proteins during all-trans retinoic acid-induced differentiation of HL-60 cells. In our experiments, we have determined the proteins expression ratio at 3, 24, 48, and 96 h after all-trans retinoic acid treatment in comparison with 0 h, respectively. Expression profiles of four proteins (VAV1, PRAM1, LYN, and CEBPB) were highly correlated ( r > 0.75) and FGR expression was detected on proteome level starting from 24 h by both techniques. For prominent differences (fold change ≥ 2) label-free parallel reaction monitoring is not inferior to selected reaction monitoring with isotopically labeled peptide standards. Differentially expressed proteins, that have been determined in our study, can be considered as potential drug targets for acute myeloid leukemia (AML) treatment.

The study presents results of development and investigation of processes of three-dimensional inkjet printing of scaffold structures based on microgranules of tricalcium phosphate and their further chemical modification in buffer solutions with the purpose of manufacturing bioactive ceramic scaffolds for bone tissue engineering.

A method for surfaceselective laser sintering which produces mineralpolymeric materials based on calcium phosphates and aliphatic polyesters was developed. Threedimensional matrices of the given architectonics for replacement of bone defects and bone tissue engineering were obtained. The microstruc� ture of the experimental samples obtained and their surface morphology and internal structure were investi� gated by scanning electron microscopy (SEM). The characteristic values of the compressive strength and rel� ative deformation of the mineral-polymer composite samples obtained by surfaceselective laser sintering of fine powders consisting of 80 wt % ceramic granules based on tricalcium phosphate and 20 wt % D,Lpoly� lactide PDL04 corresponded to the characteristic indices of the similar parameters for the trabecular bone tissue. As a result of the initial study of the biological properties of mineral-polymer composite scaffolds made by surfaceselective laser sintering, it was shown that they had low cytotoxicity and no adverse effects on the proliferative potential of mesenchymal stem cells. The technology of surfaceselective laser sintering suggested could be effectively used to create scaffolds for bone tissue engineering.

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 fi broblast-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 identifi cation of the multipotent mesenchymal stromal cells. Fetal liver fi broblast-like cells were positive for CD54; this marker was absent in skin fi broblast 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.

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.Ispol'zuia obratnuiu transkriptsiiu sovmestno s kolichestvennoĭ PTsR v real'nom vremeni ili tsifrovoĭ kapel'noĭ PTsR, provedeno profilirovanie transkriptoma khromosomy 18 cheloveka v gepatotsitakh pecheni i kletkakh gepatoblastomy (kletochnaia liniia HepG2) v terminakh absoliutnogo chisla kopiĭ kazhdogo transkripta na kletku. Kolichestvennaia predstavlennost' transkriptov var'iruet v predelakh ot 0,006 do 9635 i ot 0,011 do 4819 kopiĭ na kletku dlia kletochnoĭ linii HepG2 i gepatotsitov pecheni, sootvetstvenno. Obnaruzhena znachitel'naia korreliatsiia mezhdu ékspressieĭ genov khromosomy 18 v gepatotsitakh i kletkakh HepG2: koéffitsient korreliatsii Spirmana sostavil 0,81. Raspredelenie chastoty transkriptov po ikh kolichestvennoĭ predstavlennosti iavliaetsia bimodal'nym dlia kletok linii HepG2 i unimodal'nym dlia gepatotsitov pecheni. Bioinformaticheskiĭ analiz differentsial'noĭ ékspressii genov vyiavil, chto geny khromosomy 18, pokazyvaiushchie povyshennuiu ékspressiiu v kletkakh HepG2 po sravneniiu s gepatotsitami, assotsiirovany s protsessami kletochnogo deleniia i kletochnoĭ adgezii. Predpolagaetsia, chto usilennaia ékspressiia étikh genov sviazana s proliferativnoĭ aktivnost'iu kul'tiviruemykh kletok HepG2. Razlichiia transkriptomnykh profileĭ dolzhny uchityvat'sia pri ispol'zovanii kletok linii HepG2 v kachestve modeli gepatotsitov pecheni.

In vivo tracking of transplanted mesenchymal stem cells (MSCs) migration and homing is vital for understanding the mechanisms of beneficial effects of MSCs transplantation in animal models of diseases and in clinical trials. Transplanted cells can be labeled with superparamagnetic iron oxide (SPIO) particles and visualized in vivo using a number of iron sensitive MRI techniques. However, the applicability of those techniques for SPIO-labeled MSCs tracking in live brain has not been sufficiently investigated. The goal of this study was to estimate the efficiency of various MRI techniques of SPIO-labeled cell tracing in the brain. To achieve that goal, the precision and specificity of T2WI, T2*WI and SWI (Susceptibility-Weighted Imaging) techniques of SPIO-labeled MSCs tracing in vitro and in live rat brain were for the first time compared in the same experiment. We have shown that SWI presents the most sensitive pulse sequence for SPIO-labeled MSCs MR visualization. After intracerebral administration due to limitations caused by local micro-hemorrhages the visualization threshold was 102 cells, while after intra-arterial transplantation SWI permitted detection of several cells or even single cells. There is just one publication claiming detection of individual SPIO-labeled MSCs in live brain, while the other state much lower sensitivity, describe detection of different cell types or high resolution tracing of MSCs in other tissues. This study confirms the possibility of single cell tracing in live brain and outlines the necessary conditions. SWI is a method convenient for the detection of single SPIO labeled MSCs and small groups of SPIO labeled MSCs in brain tissue and can be appropriate for monitoring migration and homing of transplanted cells in basic and translational neuroscience.

The developments of three-dimensional printing of osteoconductive ceramic matrices based on tricalcium phosphate for tissue engineering constructions intended for acceleration of the reparative processes and effective osteointegration with tissues of the living body are studied in the present work.

We report the results obtained in 2012-2013 by the Russian Consortium for the Chromosome-centric Human Proteome Project (C-HPP). The main scope of this work was the transcriptome profiling of genes on human chromosome 18 (Chr 18), as well as their encoded proteome, from three types of biomaterials: liver tissue, the hepatocellular carcinoma-derived cell line HepG2, and blood plasma. The transcriptome profiling for liver tissue was independently performed using two RNaseq platforms (SOLiD and Illumina) and also by droplet digital PCR (ddPCR) and quantitative RT-PCR. The proteome profiling of Chr 18 was accomplished by quantitatively measuring protein copy numbers in the three types of biomaterial (the lowest protein concentration measured was 10(-13) M) using selected reaction monitoring (SRM). In total, protein copy numbers were estimated for 228 master proteins, including quantitative data on 164 proteins in plasma, 171 in the HepG2 cell line, and 186 in liver tissue. Most proteins were present in plasma at 10(8) copies/μL, while the median abundance was 10(4) and 10(5) protein copies per cell in HepG2 cells and liver tissue, respectively. In summary, for liver tissue and HepG2 cells a "transcriptoproteome" was produced that reflects the relationship between transcript and protein copy numbers of the genes on Chr 18. The quantitative data acquired by RNaseq, PCR, and SRM were uploaded into the "Update_2013" data set of our knowledgebase (www.kb18.ru) and investigated for linear correlations.

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.

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.

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.

The results of the development and introduction of universal production standards for cell products of mesenchymal origin are presented: technology for obtaining and culturing of primary cell cultures from human postnatal organs and tissues, cell product quality and safety control procedures, methods for cell product storage and transportation, and the necessary files.

We compared the capacity of cultured human skin fibroblasts, human umbilical cord cells obtained after normal delivery on gestation week 38-40, and mesenchymal bone marrow stem cells to differentiation into adipocytes, osteoblasts, and chondrocytes. Our findings suggest that mesenchymal stem cells are multipotent cells and can differentiate into adipose, cartilaginous, and bone tissue. Umbilical cord fibroblast-like cells can differentiate into adipocytes and chondrocytes, and only few cells in this culture can differentiate into osteoblasts. Skin fibroblasts differentiate only into adipocytes.

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.

Cultures of mesenchymal cells from human deciduous 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. Deciduous tooth pulp mesenchymal cells populate biodegradable 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.

Cell-mediated cytotoxicity was studied in vitro during the interaction of bone marrow mesenchymal stem cells, fibroblast-like cells from newborn umbilical cord, and skin fibroblasts of an adult donor with peripheral blood mononuclear cells. Independently on the origin, mesenchymal cells were not lysed with allogeneic natural killer cells and cytotoxic lymphocytes. Mixed cultures of mesenchymal cells had no cytotoxic effect on peripheral blood mononuclear cells and did not activate proliferation of T and B lymphocytes, natural killer cells, and CD14+ lymphocytes. In vitro experiments showed that mesenchymal cells of different origin and allogeneic immunocompetent cells are tolerant to each other.

The expression of cytoplasmic and surface proteins in cultured human skin fibroblasts, human umbilical cord cells obtained after normal delivery on gestation week 38-40, and mesenchymal bone marrow stem cells was compared by the methods of immunocytochemistry and flow cytofluorometry. Bone marrow mesenchymal stem cells expressed a great variety of marker proteins typical of stem and progenitor cells and did not express proteins typical of differentiated cells. Fibroblast-like umbilical cord cells expressed markers of both stem cells and differentiated cells. Fibroblasts of dermal origin were characterized by intensive expression of proteins typical of differentiated cells.

Background Human hepatoma HepG2 cells are used as an in vitro model of the human liver. High-throughput transcriptomic sequencing is an advanced approach for assessing the functional state of a tissue or cell type. However, the influence of experimental factors, such as the sample preparation method and inter-laboratory variation, on the transcriptomic profile has not been evaluated. Results The whole-transcriptome sequencing of HepG2 cells was performed using the SOLiD platform and validated using droplet digital PCR. The gene expression profile was compared to the results obtained with the same sequencing method in another laboratory and using another sample preparation method. We also compared the transcriptomic profile HepG2 cells with that of liver tissue. Comparison of the gene expression profiles between the HepG2 cell line and liver tissue revealed the highest variation, followed by HepG2 cells submitted to two different sample preparation protocols. The lowest variation was observed between HepG2 cells prepared by two different laboratories using the same protocol. The enrichment analysis of the genes that were differentially expressed between HepG2 cells and liver tissue mainly revealed the cancer-associated gene signature of HepG2 cells and the activation of the response to chemical stimuli in the liver tissue. The HepG2 transcriptome obtained with the SOLiD platform was highly correlated with the published transcriptome obtained with the Illumina and Helicos platforms, with moderate correspondence to microarrays. Conclusions In the present study, we assessed the influence of experimental factors on the HepG2 transcriptome and identified differences in gene expression between the HepG2 cell line and liver cells. These findings will facilitate robust experimental design in the fields of pharmacology and toxicology. Our results were supported by a comparative analysis with previous HepG2 gene expression studies. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1108) contains supplementary material, which is available to authorized users.