The iPSC secretome is beneficial for in vitro propagation of primary osteoarthritic chondrocytes cell lines.

BACKGROUND: One of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells. METHODS: We added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles. RESULTS: iPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time. CONCLUSION: Our preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion.

Imidazole-Based Lithium Salt LiHDI as a Solid Electrolyte Interphase-Stabilising Additive for Lithium-Conducting Electrolytes.

Lithium salt LiHDI (lithium 4,5-dicyano-2-(n-heptafluoropropyl)imidazolide) is proposed as a solid electrolyte interphase-stabilising additive for lithium-ion batteries, which can be added in a smaller amount than fluoroethylene carbonate (FEC) and vinylene carbonate (VC) additives. Electrolytes containing either lithium 4,5-dicyano-2-(trifluoromethyl)imidazolide (LiTDI) or battery-standard LiPF6 were tested with various amounts of LiHDI additive. Chemical stability in the presence of water and the thermal stability of LiHDI are on par with LiTDI. LiHDI additive does not negatively affect the properties of electrolytes. Conductivity measurements of solutions, galvanostatic cycling of graphite-LiFePO4 cells at room temperature, cells' cycling at 60 °C, internal cell resistance monitoring during cycling, and XPS analysis of electrodes' surfaces after cycling have been performed. LiHDI, unlike the FEC-VC mixture, does not negatively affect the properties of the electrolyte. Cycling showed improved capacity retention with LiHDI additive with both graphite and LiFePO4 as capacity-limiting electrodes over samples without additives. At elevated temperatures, samples with LiHDI exhibited better capacity retention during cycling than those with FEC-VC. Internal cell resistance can be correlated with capacity retention. XPS results show changes in the composition of SEI depending on the composition of the electrolyte and the duration of cycling.

Sex-specific differences in telomere length of patients with primary knee osteoarthritis.

Accelerated telomere shortening is associated with age-related diseases, including osteoarthritis (OA). We aimed to determine the relative telomere length (TL) in leukocytes and cartilage of patients with primary knee OA and to investigate factors that may affect TL in OA. Relative TL measurements were performed using qPCR in leukocytes of 612 individuals (310 patients with primary knee OA undergoing total knee arthroplasty (TKA) and 302 unaffected controls). We also analysed cartilage in 57 of the 310 OA patients, measuring relative TL in severely affected and less affected (control) cartilage collected from the same knee. Cartilage TLs were compared to leukocyte TLs in all 57 patients. A significant sex-by-disease-status interaction was found in regard to relative TL. Controlling for age, the average difference of leukocyte TL between female OA patients versus female controls was 0.217 units greater than that between male OA patients versus male controls (95% CI; [0.014, 0.421]). Relative TL comparison of severely and less affected cartilage samples from the same joint showed attrition of telomeres corresponding to disease severity (0.345 mean TL difference with 95% CI of [0.151, 0.539]) in the joint. We also noted that both severely and less affected cartilage had shorter telomeres than leukocytes collected from the same patient. Severe and moderate pain in OA patients was associated with shorter TL in leukocytes, but there was no association with depression or smoking in leukocytes and cartilage. Our study indicates that sex is an important factor in OA contributing to leukocyte and cartilage TL and that pain in OA shows an inverse association only with leukocyte TL.

Oxidative Stress in Long-Term Exposure to Multi-Walled Carbon Nanotubes in Male Rats.

Multi-walled carbon nanotubes (MWCNTs) serve as nanoparticles due to their size, and for that reason, when in contact with the biological system, they can have toxic effects. One of the main mechanisms responsible for nanotoxicity is oxidative stress resulting from the production of intracellular reactive oxygen species (ROS). Therefore, oxidative stress biomarkers are important tools for assessing MWCNTs toxicity. The aim of this study was to evaluate the oxidative stress of multi-walled carbon nanotubes in male rats. Our animal model studies of MWCNTs (diameter ~15-30 nm, length ~15-20 µm) include measurement of oxidative stress parameters in the body fluid and tissues of animals after long-term exposure. Rattus Norvegicus/Wistar male rats were administrated a single injection to the knee joint at three concentrations: 0.03 mg/mL, 0.25 mg/mL, and 0.5 mg/mL. The rats were euthanized 12 and 18 months post-exposure by drawing blood from the heart, and their liver and kidney tissues were removed. To evaluate toxicity, the enzymatic activity of total protein (TP), reduced glutathione (GSH), glutathione S-transferase (GST), thiobarbituric acid reactive substances (TBARS), Trolox equivalent antioxidant capacity (TEAC), nitric oxide (NO), and catalase (CAT) was measured and histopathological examination was conducted. Results in rat livers showed that TEAC level was decreased in rats receiving nanotubes at higher concentrations. Results in kidneys report that the level of NO showed higher concentration after long exposure, and results in animal serums showed lower levels of GSH in rats exposed to nanotubes at higher concentrations. The 18-month exposure also resulted in a statistically significant increase in GST activity in the group of rats exposed to nanotubes at higher concentrations compared to animals receiving MWCNTs at lower concentrations and compared to the control group. Therefore, an analysis of oxidative stress parameters can be a key indicator of the toxic potential of multi-walled carbon nanotubes.

Conditioned Medium - Is it an Undervalued Lab Waste with the Potential for Osteoarthritis Management?

BACKGROUND: The approaches currently used in osteoarthritis (OA) are mainly short-term solutions with unsatisfactory outcomes. Cell-based therapies are still controversial (in terms of the sources of cells and the results) and require strict culture protocol, quality control, and may have side-effects. A distinct population of stromal cells has an interesting secretome composition that is underrated and commonly ends up as biological waste. Their unique properties could be used to improve the existing techniques due to protective and anti-ageing properties. SCOPE OF REVIEW: In this review, we seek to outline the advantages of the use of conditioned media (CM) and exosomes, which render them superior to other cell-based methods, and to summarise current information on the composition of CM and their effect on chondrocytes. MAJOR CONCLUSIONS: CM are obtainable from a variety of mesenchymal stromal cell (MSC) sources, such as adipose tissue, bone marrow and umbilical cord, which is significant to their composition. The components present in CMs include proteins, cytokines, growth factors, chemokines, lipids and ncRNA with a variety of functions. In most in vitro and in vivo studies CM from MSCs had a beneficial effect in enhance processes associated with chondrocyte OA pathomechanism. GENERAL SIGNIFICANCE: This review summarises the information available in the literature on the function of components most commonly detected in MSC-conditioned media, as well as the effect of CM on OA chondrocytes in in vitro culture. It also highlights the need to standardise protocols for obtaining CM, and to conduct clinical trials to transfer the effects obtained in vitro to human subjects.

The Induced Pluripotent Stem Cells in Articular Cartilage Regeneration and Disease Modelling: Are We Ready for Their Clinical Use?

The development of induced pluripotent stem cells has brought unlimited possibilities to the field of regenerative medicine. This could be ideal for treating osteoarthritis and other skeletal diseases, because the current procedures tend to be short-term solutions. The usage of induced pluripotent stem cells in the cell-based regeneration of cartilage damages could replace or improve on the current techniques. The patient's specific non-invasive collection of tissue for reprogramming purposes could also create a platform for drug screening and disease modelling for an overview of distinct skeletal abnormalities. In this review, we seek to summarise the latest achievements in the chondrogenic differentiation of pluripotent stem cells for regenerative purposes and disease modelling.

Effect of continuous passive motion on the early recovery outcomes after total knee arthroplasty.

INTRODUCTION: Continuous passive motion (CPM) is a frequently used method in the early post-operative rehabilitation of patients after knee surgery. In this study, the effectiveness of the CPM method was evaluated after primary total knee arthroplasty during an early recovery period. METHODS: Eighty patients undergoing total knee arthroplasty were assigned into two groups. The experimental group received CPM and active exercises, while the control group active exercises only. All subjects were evaluated once before the surgery and at a discharge, in terms of mean active range of motion (AROM), mean Knee Society Score (KSS), and Western Ontario and MacMaster Universities Osteoarthritis Index (WOMAC). RESULTS: The mean AROM for the experimental group was 82.3° ± 14.3° and 76.1° ± 22.2° for the control. The mean KSS score was 136.4 ± 19.3 points for the experimental group, and 135.7 ± 15.1 for the control. There were no statistical differences between the two groups. The KSS functional score was 66.4 ± 8.1 points for the experimental group compared to 62.2 ± 7.3 points for the control, but there was a statistically significant difference between the groups at discharge from the hospital (p = 0.009). A subjective estimation of the pain level, joint stiffness and function also showed a statistically significant difference between the two groups (38.6 ± 14.3 points for the CPM group and 21.2 ± 15.7 for the control). CONCLUSION: These findings show that there is no significant effect of CPM in terms of improving clinical measurements. However, there was a significant beneficial effect on the subjective assessment of pain level, joint stiffness, and functional ability.

From Donor to the Lab: A Fascinating Journey of Primary Cell Lines.

Primary cancer cell lines are ex vivo cell cultures originating from resected tissues during biopsies and surgeries. Primary cell cultures are objects of intense research due to their high impact on molecular biology and oncology advancement. Initially, the patient-derived specimen must be subjected to dissociation and isolation. Techniques for tumour dissociation are usually reliant on the organisation of connecting tissue. The most common methods include enzymatic digestion (with collagenase, dispase, and DNase), chemical treatment (with ethylene diamine tetraacetic acid and ethylene glycol tetraacetic acid), or mechanical disaggregation to obtain a uniform cell population. Cells isolated from the tissue specimen are cultured as a monolayer or three-dimensional culture, in the form of multicellular spheroids, scaffold-based cultures (i.e., organoids), or matrix-embedded cultures. Every primary cell line must be characterised to identify its origin, purity, and significant features. The process of characterisation should include different assays utilising specific (extra- and intracellular) markers. The most frequently used approaches comprise immunohistochemistry, immunocytochemistry, western blot, flow cytometry, real-time polymerase chain reaction, karyotyping, confocal microscopy, and next-generation sequencing. The growing body of evidence indicates the validity of the usage of primary cancer cell lines in the formulation of novel anti-cancer treatments and their contribution to drug development.

Long noncoding RNA CCAT1 rs67085638 SNP contribution to the progression of gastric cancer in a Polish population.

The role of the long noncoding RNA CCAT1 NC_000008.10:g.128220661C > T (rs67085638) in the development of colon cancer has been reported. Therefore, we assessed the prevalence of rs67085638 in patients with gastric cancer (GC). We also evaluated the effect of rs67085638 on B-cell-specific Moloney leukaemia virus insertion site 1 (BMI1) transcripts in primary GC and counterpart histopathologically confirmed disease-free margin tissue. Using high-resolution melting analysis, we evaluated rs67085638 frequency in patients with the GC genotype (n = 214) and controls (n = 502) in a Polish Caucasian population. qRT-PCR was used to determine BMI1 transcripts. We observed the trend of rs67085638 association in all patients with GC (ptrend = 0.028), a strong risk of the GC genotype in male (ptrend = 0.035) but not female (ptrend = 0.747) patients, and the association with non-cardia GC (ptrend = 0.041), tumour stages T3 (ptrend = 0.014) and T4 (ptrend = 0.032), differentiation grading G3 (ptrend = 0.009), lymph node metastasis stage N3 (ptrend = 0.0005) and metastasis stage M0 (ptrend = 0.027). We found that significantly increased BMI1 transcripts were associated with the primary GC genotype classified as grade G3 (p = 0.011) and as lymph node metastasis N3 (p = 0.010) and counterpart marginal tissues (p = 0.026, p = 0.040, respectively) from carriers of the T/T versus C/C genotypes. rs67085638 may contribute to increased BMI1 transcripts and the progression and rapid growth of GC.

Aesculus hippocastanum L. as a Stabilizer in Hemp Seed Oil Nanoemulsions for Potential Biomedical and Food Applications.

Nanoemulsion systems receive a significant amount of interest nowadays due to their promising potential in biomedicine and food technology. Using a two-step process, we produced a series of nanoemulsion systems with different concentrations of hemp seed oil (HSO) stabilized with Aesculus hippocastanum L. extract (AHE). Water and commercially-available low-concentrated hyaluronic acid (HA) were used as the liquid phase. Stability tests, including an emulsifying index (EI), and droplet size distribution tests performed by dynamic light scattering (DLS) proved the beneficial impact of AHE on the emulsion's stability. After 7 days of storage, the EI for the water-based system was found to be around 100%, unlike the HA systems. The highest stability was achieved by an emulsion containing 5% HSO and 2 g/L AHE in water, as well as the HA solution. In order to obtain the detailed characteristics of the emulsions, UV-Vis and FTIR spectra were recorded, and the viscosity of the samples was determined. Finally, a visible microscopic analysis was used for the homogeneity evaluation of the samples, and was compared with the DLS results of the water system emulsion, which showed a desirable stability. The presented results demonstrate the possible use of oil emulsions based on a plant extract rich in saponins, such as AHE. Furthermore, it was found that the anti-inflammatory properties of AHE provide opportunities for the development of new emulsion formulations with health benefits.

On the Sensitivity of the Ni-rich Layered Cathode Materials for Li-ion Batteries to the Different Calcination Conditions.

Ni-rich layered oxides, i.e., LiNi0.6Mn0.2Co0.2O2 (NMC622) and LiNiO2 (LNO), were prepared using the two-step calcination procedure. The samples obtained at different calcination temperatures (750-950 °C for the NMC622 and 650-850 °C for the LNO cathode materials) were characterized using nitrogen physisorption, PXRD, SEM and DLS methods. The correlation of the calcination temperature, structural properties and electrochemical performance of the studied Ni-rich layered cathode materials was thoroughly investigated and discussed. It was determined that the optimal calcination temperature is dependent on the chemical composition of the cathode materials. With increasing nickel content, the optimal calcination temperature shifts towards lower temperatures. The NMC-900 calcined at 900 °C and the LNO-700 calcined at 700 °C showed the most favorable electrochemical performances. Despite their well-ordered structure, the materials calcined at higher temperatures were characterized by a stronger sintering effect, adverse particle growth, and higher Ni2+/Li+ cation mixing, thus deteriorating their electrochemical properties. The importance of a careful selection of the heat treatment (calcination) temperature for each individual cathode material was emphasized.

Utilization of Carbon Nanotubes in Manufacturing of 3D Cartilage and Bone Scaffolds.

Cartilage and bone injuries are prevalent ailments, affecting the quality of life of injured patients. Current methods of treatment are often imperfect and pose the risk of complications in the long term. Therefore, tissue engineering is a rapidly developing branch of science, which aims at discovering effective ways of replacing or repairing damaged tissues with the use of scaffolds. However, both cartilage and bone owe their exceptional mechanical properties to their complex ultrastructure, which is very difficult to reproduce artificially. To address this issue, nanotechnology was employed. One of the most promising nanomaterials in this respect is carbon nanotubes, due to their exceptional physico-chemical properties, which are similar to collagens-the main component of the extracellular matrix of these tissues. This review covers the important aspects of 3D scaffold development and sums up the existing research tackling the challenges of scaffold design. Moreover, carbon nanotubes-reinforced bone and cartilage scaffolds manufactured using the 3D bioprinting technique will be discussed as a novel tool that could facilitate the achievement of more biomimetic structures.

Plant Extracts Containing Saponins Affects the Stability and Biological Activity of Hempseed Oil Emulsion System.

In this study, two saponins-rich plant extracts, viz. Saponaria officinalis and Quillaja saponaria, were used as surfactants in an oil-in-water (O/W) emulsion based on hempseed oil (HSO). This study focused on a low oil phase content of 2% v/v HSO to investigate stable emulsion systems under minimum oil phase conditions. Emulsion stability was characterized by the emulsification index (EI), centrifugation tests, droplet size distribution as well as microscopic imaging. The smallest droplets recorded by dynamic light scattering (droplets size v. number), one day after the preparation of the emulsion, were around 50-120 nm depending the on use of Saponaria and Quillaja as a surfactant and corresponding to critical micelle concentration (CMC) in the range 0-2 g/L. The surface and interfacial tension of the emulsion components were studied as well. The effect of emulsions on environmental bacteria strains was also investigated. It was observed that emulsions with Saponaria officinalis extract exhibited slight toxic activity (the cell metabolic activity reduced to 80%), in contrast to Quillaja emulsion, which induced Pseudomonas fluorescens ATCC 17400 growth. The highest-stability samples were those with doubled CMC concentration. The presented results demonstrate a possible use of oil emulsions based on plant extract rich in saponins for the food industry, biomedical and cosmetics applications, and nanoemulsion preparations.

Osteoarthritis Severely Decreases the Elasticity and Hardness of Knee Joint Cartilage: A Nanoindentation Study.

The nanoindentation method was applied to determine the elastic modulus and hardness of knee articular cartilage. Cartilage samples from both high weight bearing (HWB) and low weight bearing (LWB) femoral condyles were collected from patients diagnosed with osteoarthritis (OA). The mean elastic modulus of HWB cartilage was 4.46 ± 4.44 MPa in comparison to that of the LWB region (9.81 ± 8.88 MPa, p < 0.001). Similarly, the hardness was significantly lower in HWB tissue (0.317 ± 0.397 MPa) than in LWB cartilage (0.455 ± 0.434 MPa, p < 0.001). When adjusted to patients' ages, the mean elastic modulus and hardness were both significantly lower in the age group over 70 years (p < 0.001). A statistically significant difference in mechanical parameters was also found in grade 3 and 4 OA. This study provides an insight into the nanomechanical properties of the knee articular cartilage and provides a starting point for personalized cartilage grafts that are compatible with the mechanical properties of the native tissue.

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.

Effect of cellular mass on chondrogenic differentiation during embryoid body formation.

One approach to cell differentiation is to use the natural capacity of pluripotent stem cells to form three germ layers via embryoid bodies (EB). However, unification of this process during in vitro culture remains challenging and many microenvironmental factors including the number of cells in the culture can influence differentiation patterns. The number of cells serves a crucial role as it determines access to nutrients, the distribution of oxygen concentration and cellular interactions, all of which influence the fate of the differentiated cells. The influence of EBs derived from human pluripotent cells on the chondrogenic potential of such cells is not well understood. For this reason, the present study sought to determine the effect of varying amounts of cells on the properties of EBs derived from human embryonic stem cells (BG01V cell line). In the present study, 500­2,000 cells per well were cultivated from 5 to 15 days in suspension cell culture. Expression of pluripotency genes and germ layer markers were evaluated in order to determine the EBs with the greatest and least mesodermal properties. Genes associated with pluripotency and chondrogenesis were also evaluated to assess the influence of suspension culture duration and EB size on chondrogenic differentiation. Immunofluorescence staining for pluripotent and chondrocyte­associated proteins confirmed successful differentiation into chondrocyte­like cells. Alcian blue staining confirmed deposition of proteoglycans. These results suggested that EBs formed in 500­cell wells possess the highest mesodermal and prochondrogenic properties. Differentiation of EBs into chondrocytes on day 5 in 500­cell wells was more efficient than in that observed in larger and older EBs.

Forced differentiation in vitro leads to stress-induced activation of DNA damage response in hiPSC-derived chondrocyte-like cells.

A human induced pluripotent stem cell line (GPCCi001-A) created by our group was differentiated towards chondrocyte-like cells (ChiPS) via monolayer culturing with growth factors. ChiPS are promising because they have the potential to be used in tissue engineering to regenerate articular cartilage. However, their safety must be confirmed before they can be routinely used in regenerative medicine. Using microarray analysis, we compared the ChiPS to both GPCCi001-A cells and chondrocytes. The analysis showed that, compared to both GPCCi001-A cells and chondrocytes, the expression of genes engaged in DNA damage and in the tumor protein p53 signalling pathways was significantly higher in the ChiPS. The significant amount of DNA double strand breaks and increased DNA damage response may lead to incomplete DNA repair and the accumulation of mutations and, ultimately, to genetic instability. These findings provide evidence indicating that the differentiation process in vitro places stress on human induced pluripotent stem cells (hiPSCs). The results of this study raise doubts about the use of stem cell-derived components given the negative effects of the differentiation process in vitro on hiPSCs.

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.