Navigating challenges: optimising methods for primary cell culture isolation.

Primary cell lines are invaluable for exploring cancer biology and investigating novel treatments. Despite their numerous advantages, primary cultures are laborious to obtain and maintain in culture. Hence, established cell lines are still more common. This study aimed to evaluate a range of techniques for isolating primary breast cancer cultures, employing distinct enzymatic compositions, incubation durations, and mechanical approaches, including filtration. Out of several protocols, we opted for a highly effective method (Method 5) that gave rise to a primary cell culture (BC160). This method combines mechanical disaggregation and enzymatic digestion with hyaluronidase and collagenase. Moreover, the paper addresses common issues in isolating primary cultures, shedding light on the struggle against fibroblasts overgrowing cancer cell populations. To make primary cell lines a preferred model, it is essential to elaborate and categorise isolation methods, develop approaches to separate heterogeneous cultures and investigate factors influencing the establishment of primary cell lines.

Understanding Hypoxia-Driven Tumorigenesis: The Interplay of HIF1A, DNA Methylation, and Prolyl Hydroxylases in Head and Neck Squamous Cell Carcinoma.

Hypoxia-inducible factor 1-alpha (HIF1A) is a key transcription factor aiding tumor cells' adaptation to hypoxia, regulated by the prolyl hydroxylase family (EGLN1-3) by directing toward degradation pathways. DNA methylation potentially influences EGLN and HIF1A levels, impacting cellular responses to hypoxia. We examined 96 HNSCC patients and three cell lines, analyzing gene expression of EGLN1-3, HIF1A, CA9, VEGF, and GLUT1 at the mRNA level and EGLN1 protein levels. Methylation levels of EGLNs and HIF1A were assessed through high-resolution melting analysis. Bioinformatics tools were employed to characterize associations between EGLN1-3 and HIF1A expression and methylation. We found significantly higher mRNA levels of EGLN3, HIF1A, GLUT1, VEGF, and CA9 (p = 0.021; p < 0.0001; p < 0.0001; p = 0.004, and p < 0.0001, respectively) genes in tumor tissues compared to normal ones and downregulation of the EGLN1 mRNA level in tumor tissues (p = 0.0013). In HNSCC patients with hypermethylation of HIF1A in normal tissue, we noted a reduction in HIF1A mRNA levels compared to tumor tissue (p = 0.04). In conclusion, the differential expression of EGLN and HIF1A genes in HNSCC tumors compared to normal tissues influences patients' overall survival, highlighting their role in tumor development. Moreover, DNA methylation could be responsible for HIF1A suppression in the normal tissues of HNSCC patients.

Primary cancer-associated fibroblasts exhibit high heterogeneity among breast cancer subtypes.

Background: Cancer-associated fibroblasts (CAFs) are a diverse subset of cells, that is recently gaining in popularity and have the potential to become a new target for breast cancer (BC) therapy; however, broader research is required to understand their mechanisms and interactions with breast cancer cells. The goal of the study was to isolate CAFs from breast cancer tumour and characterise isolated cell lines. We concentrated on numerous CAF biomarkers that would enable their differentiation. Materials and methods: Flow cytometry, immunofluorescence, and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were used to phenotype the primary CAFs. Results/Conclusions: According to our findings, there was no significant pattern in the classification of cancer-associated fibroblasts. The results of biomarkers expression were heterogeneous, thus no specific subtypes were identified. Furthermore, a comparison of cancer-associated fibroblasts derived from different BC subtypes (luminal A and B, triple-negative, HER2 positive) did not reveal any clear trend of expression.

Development of a quasi-humanoid phantom to perform dosimetric and radiobiological measurements for out-of-field doses from external beam radiation therapy.

Our understanding of low dose, out-of-field radiation and their radiobiological effects are limited, in part due to the rapid technological advances in external beam radiotherapy, especially for non-coplanar and dynamic techniques. Reliable comparisons of out-of-field doses produced by advanced radiotherapy techniques are difficult due to the limitations of commercially available phantoms. There is a clear need for a functional phantom to accurately measure the dosimetric and radiobiological characteristics of out-of-field doses, which would in turn allow clinicians and medical physicists to optimize treatment parameters. We designed, manufactured, and tested the performance of a quasi-humanoid (Q-H) adult phantom. To test the physics parameters, we used computed tomography (CT) scans of assembled Q-H phantom. Static open field and dynamic techniques were measured both in- and out-of-field with ionization chambers and radiochromic films for two configurations (full solid and with water-filled containers). In the areas simulating soft tissues, lung, and bones, median Hounsfield units and densities were, respectively: 129.8, -738.7, 920.8 HU and 1.110, 0.215, 1.669 g/cm3 . Comparison of the measured to treatment planning systems (TPS) in-field dose values for the sample volumetric arc therapy (VMAT) (6 MV flattening filter-free (FFF)) plan, 96.4% of analyzed points passed the gamma evaluation criteria (L2%/2 mm, threshold (TH) 10%) and less than 1.50% for point dose verification. In the two phantom configurations: full poly(methyl) methacrylate (PMMA) and with water container, the off-axis median doses for open field, relative to the central axis of the beam (CAX) were similar, respectively: 0.900% versus 0.907% (15 cm distance to CAX); 0.096% versus 0.120% (35 cm); 0.018% versus 0.018% (52 cm); 0.009% versus 0.008% (74 cm). For VMAT 6 MV FFF, doses relative the CAX were, respectively: 0.667% (15 cm), 0.062% (35 cm), 0.019% (52 cm), 0.016% (74 cm). The Q-H phantom meets the International Commission on Radiation Units and Measurements (ICRU) and American Association of Physicists in Medicine (AAPM) recommended phantom criteria, providing medical physicists with a reliable, comprehensive system to perform dose calculation and measurements and to assess the impact on radiobiological response and on the risk of secondary tumor induction.

FLASH radiotherapy: an emerging approach in radiation therapy.

FLASH radiotherapy (RT) is a technique involving the delivery of ultra-high dose rate radiation to the target. FLASH-RT has been shown to reduce radiation-induced toxicity in healthy tissues without compromising the anti-cancer effects of treatment compared to conventional radiation therapy. In the present article, we review the published data on FLASH-RT and discuss the current state of knowledge of this novel approach. We also highlight the technological constraints and complexity of FLASH-RT and describe the physics underlying this modality, particularly how technology supports energy transfer by ionising radiation (e.g., beam on/off sequence, pulse-energy load, intervals). We emphasise that current preclinical experience is mostly based on FLASH electrons and that clinical application of FLASH-RT is very limited. The incorporation of FLASH-RT into routine clinical radiotherapy will require the development of devices capable of producing FLASH photon beams.

Currently used in clinical practice beam rate changes have no significant effect on the reduction of clonogenic capacity of PNT1A cells in vitro.

BACKGROUND: Due to the lack of selectivity of ionizing radiation between normal and cancer cells, it is important to improve the existing radiation patterns. Lowering the risk of cancer recurrence and comfort during treatment are priorities in radiotherapy. MATERIALS AND METHODS: In the experiment we used dose verification to determine the irradiation time calculated by a treatment planning system for 6XFFF and 10XFFF beams. Cells cultured under standard conditions were irradiated with a dose of 2 Gy at different beam rates 400 MU/min, 600 MU/min, 800 MU/min, 1000 MU/min, 1400 MU/min, 1600 MU/min and 2400 MU/min using 6XFFF, 10XFFF and 6XFF beams. RESULTS: The experiment was aimed at comparing the biological response of normal prostate cells after clinically applied radiation patterns. No statistically significant differences in the cellular response were observed. The wide range of beam rates as well as the beam profiles did not significantly affect cell proliferation. CONCLUSIONS: High beam rates, without significantly affecting the clonogenic capacity of cells, have an impact on the quality of patient's treatment. With the increasing beam rate the irradiation time is shortened, which has an important impact on patients' health. This experiment can have a practical significance.

The Role of Gold Nanorods in the Response of Prostate Cancer and Normal Prostate Cells to Ionizing Radiation-In Vitro Model.

To increase the efficiency of therapy via enhancing its selectivity, the usage of gold nanorods (GNR) as a factor sensitizing cancer cells to radiation was proposed. Due to gold nanoparticles' characteristics, the smaller doses of radiation would be sufficient in the treatment, protecting the healthy tissue around the tumor. The aim of this study was to investigate the effect of gold nanorods on cancer and normal prostate cells and the role of nanorods in the cell response to ionizing radiation. The effect was evaluated by measuring the toxicity, cell cycle, cell granularity, reactive oxygen species (ROS) level, and survival fractions. Nanorods showed a strong toxicity dependent on the concentration and incubation time toward all used cell lines. A slight effect of nanorods on the cycle distribution was observed. The results demonstrated that the administration of nanorods at higher concentrations resulted in an increased level of generated radicals. The results of cellular proliferation after irradiation are ambiguous; however, there are noticeable differences after the application of nanorods before irradiation. The obtained results lead to the conclusion that nanorods affect the physiology of both normal and cancer cells. Nanorods might become a potential tool used to increase the effectiveness of radiation treatment.

Surgical Wound Fluids from Patients with Breast Cancer Reveal Similarities in the Biological Response Induced by Intraoperative Radiation Therapy and the Radiation-Induced Bystander Effect-Transcriptomic Approach.

In patients with breast cancer who undergo breast-conserving surgery (BCS), more than 90% of local recurrences occur in the same quadrant as the primary cancer. Surgical wound fluids (SWF) are believed to play a role in this process by inducing an inflammatory process in the scar tissue area. Despite strong clinical data demonstrating the benefits of intraoperative radiotherapy (IORT), the biological basis underlying this process remains poorly understood. Ionizing radiation (IR) directly affects cells by damaging DNA, thereby altering the cell phenotype. IR directly affects cancer cells and also influences unirradiated cells located nearby, a phenomenon known as the radiation-induced bystander effect (RIBE), significantly modifying the tumor microenvironment. We hypothesized that SWF obtained from patients after BCS and IORT would induce a radiobiological response (due to RIBE) in unirradiated cells, thereby modifying their phenotype. To confirm this hypothesis, breast cancer cells were incubated with SWF collected from patients after BCS: (1) without IORT (wound fluid (WF) group), (2) with IORT (radiotherapy wound fluid (RT-WF) group), and (3) WF with conditioned medium from irradiated cells (WF+RIBE group) and then subjected to microarray analysis. We performed gene set enrichment analysis to determine the biological processes present in these cells. This analysis showed that the RT-WF and WF+RIBE groups shared common biological processes, including the enhancement of processes involved in cell-cycle regulation, DNA repair, and oxidative phosphorylation. The WF group was characterized by overrepresentation of pathways involved in the INF-α and INF-γ response, inflammatory response, and the IL6 JAK/STAT3 signaling pathway. These findings show that MDA-MB-468 cells stimulated with surgical wound fluids obtained from patients who underwent BCS plus IORT and from cells stimulated with SWF plus RIBE share common biological processes. This confirms the role of the radiation-induced bystander effect in altering the biological properties of wound fluids.

Overcoming Resistance to Platinum-Based Drugs in Ovarian Cancer by Salinomycin and Its Derivatives-An In Vitro Study.

Polyether ionophore salinomycin (SAL) and its semi-synthetic derivatives are recognized as very promising anticancer drug candidates due to their activity against various types of cancer cells, including multidrug-resistant populations. Ovarian cancer is the deadliest among gynecologic malignancies, which is connected with the development of chemoresistant forms of the disease in over 70% of patients after initial treatment regimen. Thus, we decided to examine the anticancer properties of SAL and selected SAL derivatives against a series of drug-sensitive (A2780, SK-OV-3) and derived drug-resistant (A2780 CDDP, SK-OV-3 CDDP) ovarian cancer cell lines. Although SAL analogs showed less promising IC50 values than SAL, they were identified as the antitumor agents that significantly overcome the resistance to platinum-based drugs in ovarian cancer, more potent than unmodified SAL and commonly used anticancer drugs-5-fluorouracil, gemcitabine, and cisplatin. Moreover, when compared with SAL used alone, our experiments proved for the first time increased selectivity of SAL-based dual therapy with 5-fluorouracil or gemcitabine, especially towards A2780 cell line. Looking closer at the results, SAL acted synergistically with 5-fluorouracil towards the drug-resistant A2780 cell line. Our results suggest that combinations of SAL with other antineoplastics may become a new therapeutic option for patients with ovarian cancer.

Radiobiological models in prediction of radiation cardiotoxicity.

Coronary disease induced by previous radiotherapy is the most common cause of death among patients treated with radiotherapy for cancer. Risk factors that may affect the frequency and intensity of radiotherapy's cardiac toxicity are primarily the radiation dose and the volume of the heart exposed to radiation. The prolonged survival time of patients after radiotherapy, but also the intensive development of modern radiotherapy techniques results in the necessity of precise estimation of both tumor control probability, and the risk of normal tissue damage, thus the models describing the probability of complications in normal tissues have also been developed. The response from the cardiovascular system to high-dose radiation is known and associated with a pro-inflammatory response. However, the effect of low doses may be completely different because it induces an anti-inflammatory response. Also, there is no unambiguous answer to the question of whether RICD is a deterministic effect. Moreover, there is a lack of literature data on the use of known radiobiological models to assess the risk of cardiovascular complications. The models described are general and concerns any healthy tissue. Therefore, when planning treatment for patients, particular attention should be paid to the dose and area of ​​the heart to be irradiated.

Interplay between inflammation and cancer.

Tumor-promoting inflammation is one of the hallmarks of cancer. It has been shown that cancer development is strongly influenced by both chronic and acute inflammation process. Progress in research on inflammation revealed a connection between inflammatory processes and neoplastic transformation, the progression of tumour, and the development of metastases and recurrences. Moreover, the tumour invasive procedures (both surgery and biopsy) affect the remaining tumour cells by increasing their survival, proliferation and migration. One of the concepts explaining this phenomena is an induction of a wound healing response. While in normal tissue it is necessary for tissue repair, in tumour tissue, induction of adaptive and innate immune response related to wound healing, stimulates tumour cell survival, angiogenesis and extravasation of circulating tumour cells. It has become evident that certain types of immune response and immune cells can promote tumour progression more than others. In this review, we focus on current knowledge on carcinogenesis and promotion of cancer growth induced by inflammatory processes.

MicroRNA Profiling During Neural Differentiation of Induced Pluripotent Stem Cells.

MicroRNAs (miRNA) play an essential role in the regulation of gene expression and influence signaling networks responsible for several cellular processes like differentiation of pluripotent stem cells. Despite several studies on the neurogenesis process, no global analysis of microRNA expression during differentiation of induced pluripotent stem cells (iPSC) to neuronal stem cells (NSC) has been done. Therefore, we compared the profile of microRNA expression in iPSC lines and in NSC lines derived from them, using microarray-based analysis. Two different protocols for NSC formation were used: Direct and two-step via neural rosette formation. We confirmed the new associations of previously described miRNAs in regulation of NSC differentiation from iPSC. We discovered upregulation of miR-10 family, miR-30 family and miR-9 family and downregulation of miR-302 and miR-515 family expression. Moreover, we showed that miR-10 family play a crucial role in the negative regulation of genes expression belonging to signaling pathways involved in neural differentiation: WNT signaling pathway, focal adhesion, and signaling pathways regulating pluripotency of stem cells.

The Role of MicroRNAs in Early Chondrogenesis of Human Induced Pluripotent Stem Cells (hiPSCs).

Human induced pluripotent stem cells (hiPSCs) play an important role in research regarding regenerative medicine. Particularly, chondrocytes differentiated from hiPSCs seems to be a promising solution for patients suffering from osteoarthritis. We decided to perform chondrogenesis in a three-week monolayer culture. Based on transcriptome analysis, hiPSC-derived chondrocytes (ChiPS) demonstrate the gene expression profile of cells from early chondrogenesis. Chondrogenic progenitors obtained by our group are characterized by significantly high expression of Hox genes, strongly upregulated during limb formation and morphogenesis. There are scanty literature data concerning the role of microRNAs in early chondrogenesis, especially in chondrogenic differentiation of hiPSCs. The main aim of this study was to investigate the microRNA expression profile and to select microRNAs (miRNAs) taking part in early chondrogenesis. Our findings allowed for selection crucial miRNAs engaged in both diminishing pluripotency state and chondrogenic process (inter alia hsa-miR-525-5p, hsa-miR-520c-3p, hsa-miR-628-3p, hsa-miR-196b-star, hsa-miR-629-star, hsa-miR-517b, has-miR-187). These miRNAs regulate early chondrogenic genes such as: HOXD10, HOXA11, RARB, SEMA3C. These results were confirmed by RT-qPCR analysis. This work contributes to a better understanding of the role of miRNAs directly involved in chondrogenic differentiation of hiPSCs. These data may result in the establishment of a more efficient protocol of obtaining chondrocyte-like cells from hiPSCs.

Chondrogenic Differentiation of Pluripotent Stem Cells under Controllable Serum-Free Conditions.

The repair of damaged articular cartilage using currently available implantation techniques is not sufficient for the full recovery of patients. Pluripotent stem cells (iPSC)-based therapies could bring new perspectives in the treatment of joint diseases. A number of protocols of in vitro differentiation of iPSC in chondrocytes for regenerative purposes have been recently described. However, in order to use these cells in clinics, the elimination of animal serum and feeder cells is essential. In our study, a strictly defined and controllable protocol was designed for the differentiation of pluripotent stem cells (BG01V, ND 41658*H, GPCCi001-A) in chondrocyte-like cells in serum- and a feeder cell-free system, using the embryoid bodies step. The extension of the protocol and culture conditions (monolayer versus 3D culture) was also tested after the initial 21 days of chondrogenic differentiation. Promotion of the chondrogenic differentiation in 3D culture via the elevated expression of genes related to chondrogenesis was achieved. Using immunofluorescence and immunohistochemistry staining techniques, the increased deposition of the specific extracellular matrix was indicated. As a result, chondrocyte-like cells in the early stages of their differentiation using pellet culture under fully controlled and defined conditions were obtained.

Superparamagnetic iron oxide nanoparticles (SPIONs) as a multifunctional tool in various cancer therapies.

Over the past two decades nanotechnology has become an important part of novel medical research. Researchers have made great progress in developing nanotechnology applications used for detecting and treating oncological diseases. Recently, many research groups have focused on the use of superparamagnetic iron oxide nanoparticles (SPIONs) in cancer treatment. Due to the range of therapeutic properties and possibilities of various modifications, SPIONs are a promising and multifunctional tool in various cancer therapies and may help to overcome the limitations of conventional therapies. Moreover, it is still necessary to develop new methods of treatment with expected properties, such as lower toxicity, long-lasting effectiveness and higher selectivity. Analyzing the literature data, we found that currently SPIONs are used in the transport of drugs, immunotherapy and hyperthermia. The main aim of this review is to present various cancer treatment therapies utilizing SPIONs, the importance of the experiments carried out by research groups and further perspectives in the nanotechnological use of SPIONs.

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies.

Fucoidan Exerts Anticancer Effects Against Head and Neck Squamous Cell Carcinoma In Vitro.

Fucoidans have been reported to exert anticancer effects with simultaneous low toxicity against healthy tissue. That correlation was observed in several cancer models, however, it has never been investigated in head and neck cancer before. To magnify the efficacy of conventional therapy, the administration of agents like fucoidan could be beneficial. The aim of this study was to evaluate the anticancer effect of Fucus vesiculosus (FV) extract alone and with co-administration of cisplatin in head and neck squamous cell carcinoma (HNSCC) in vitro. MTT assay results revealed an FV-induced inhibition of proliferation in all tested cell lines (H103, FaDu, KB). Flow cytometric cell cycle analysis showed an FV-induced, dose-dependent arrest in either S/G2 phase (H103, FaDu) or G1 arrest (KB). Furthermore, a dose-dependent gain in apoptotic fraction was observed. Western blot analysis confirmed the induction of apoptosis. A significant dose-dependent increase in reactive oxygen species (ROS) production was revealed in the H103 cell line, while FaDu cells remained unresponsive. On the contrary, an HPV-positive cell line, KB, demonstrated a dose-dependent decrease in ROS synthesis. Moreover, fucoidan enhanced the response to cisplatin (synergistic effect) in all cell lines with the HPV-positive one (KB) being the most sensitive. These results have been confirmed by flow-cytometric apoptosis analysis. In conclusion, we confirmed that fucoidan exhibits anticancer properties against HNSCC, which are manifested by the induction of apoptosis, regulation of ROS production, cell cycle arrest, and inhibition of proliferation.

Wound fluids collected from patients after IORT treatment activates extrinsic apoptotic pathway in MCF7 breast cancer cell line.

OBJECTIVES: Intraoperative radiotherapy (IORT) relates to irradiation of diseased tissue during the surgery within the tumor bed. The reason for this process is based on the fact that the increase in the radiation dose increases local tumor control. It was shown that postoperative fluids obtained from patients after breast cancer conserving surgery, stimulated motility and invasiveness of tumor cells in vitro. The results obtained from TARGIT clinical trial demonstrated that IORT significantly inhibits the stimulatory effect of wound fluids on tumor cells in vitro. We therefore speculated that wound fluids collected from patients after IORT treatment may induce the apoptosis in breast cancer cell lines and it may be a reason for their lower proliferation rate and potential to metastasis. MATERIAL AND METHODS: Breast cancer MCF7 cell line was incubated with wound fluids collected from patients after conserving breast cancer surgery or surgery followed by IORT for 4 days. Then the expression of markers associated with extrinsic or intrinsic apoptosis pathway was established. RESULTS: Our results clearly indicate activation of extrinsic apoptosis pathway by wound fluids collected from patients after IORT treatment. No changes in apoptotic markers were seen in cells treated with wound fluids collected from patients after the surgery alone. CONCLUSIONS: Thus we confirmed that wound fluids collected from patients after IORT treatment may induce the apoptosis in breast cancer cell lines and it may be a reason for their lower proliferation rate and invasiveness of tumor cells in vitro.

Correlations between serum adipocytokine concentrations, disease stage, radiological status and total body fat content in the patients with primary knee osteoarthritis.

PURPOSE: The study was designed to investigate whether serum concentrations of leptin, resistin and adiponectin in obese and normal-weight patients with primary knee osteoarthritis (OA) correlate with clinical and radiological stages of the disease and percentage of total body fat. METHODS: Seventy-three patients with knee OA, divided into obese and normal-weight groups, were clinically evaluated according to the Knee Society Score (KSS), and radiologically assessed using Kellgren and Lawrence scale. The percentage of total body fat and some anthropometric data were also given. Serum leptin, resistin and adiponectin concentrations were measured by Elisa and were correlated with the clinical, radiological and anthropometric parameters. RESULTS: Leptin concentrations were significantly higher (p = 0.001) in the obese patients and positively correlated (R = 0.63) with radiologically assessed OA grade, but only in the normal-weight group. Resistin and adiponectin concentrations were identical in obese and normal-weight patients and negatively correlated (R = -0.41) with the clinical status of obese patients. In both groups, percentage of total body fat positively correlated (R = 0.29 and R = 0.53 for obese and normal-weight respectively) with radiologically assessed OA grade. However, no correlations were found with clinical status of the patients. CONCLUSIONS: It was found that in the obese patients with knee OA, increased percentage of total body fat and elevated serum leptin concentration might favour the advancement of clinical but not radiologically assessed changes in the joint structures, while in normal-weight patients it correlates only with radiologically assessed changes but does not affect to an appreciable extent the clinical status of the patients.

Modified methods for efficiently differentiating human embryonic stem cells into chondrocyte-like cells.

INTRODUCTION: Human articular cartilage has a poor regenerative capacity. This often results in the serious joint disease- osteoarthritis (OA) that is characterized by cartilage degradation. An inability to self-repair provided extensive studies on AC regeneration. The cell-based cartilage tissue engineering is a promising approach for cartilage regeneration. So far, numerous cell types have been reported to show chondrogenic potential, among others human embryonic stem cells (hESCs). MATERIALS AND METHODS: However, the currently used methods for directed differentiation of human ESCs into chondrocyte-like cells via embryoid body (EB) formation, micromass culture (MC) and pellet culture (PC) are not highly efficient and require further improvement. In the present study, these three methods for hESCs differentiation into chondrocyte-like cells in the presence of chondrogenic medium supplemented with diverse combination of growth factors (GFs) were evaluated and modified. RESULTS: The protocols established here allow highly efficient, simple and inexpensive production of a large number of chondrocyte-like cells suitable for transplantation into the sites of cartilage injury. The most crucial issue is the selection of appropriate GFs in defined concentration. The obtained stem-derived cells reveal the presence of chondrogenic markers such as type II collagen, Sox6 and Sox9 as well as the lack or significantly lower level of pluripotency markers including Nanog and Oct3/4. DISCUSSION: The most efficient method is the differentiation throughout embryoid bodies. In turn, chondrogenic differentiation via pellet culture is the most promising method for implementation on clinical scale. The most useful GFs are TGF-ß1, -3 and BMP-2 that possess the most chondrogenic potential. These methods can also be used to obtain chondrocyte-like cells from differentiating induced pluripotent stem cells (iPSCs).