Search Results (60)

We present a detailed, xeno-free protocol for the rapid differentiation of human induced pluripotent stem cells (hiPSCs) into microglia using the well-characterized KOLF2.1J reference line. This system employs doxycycline-inducible expression of six transcription factors (6-TF), stably integrated into the CLYBL safe harbor locus, to drive uniform microglial differentiation within two weeks. Building upon an established transcription factor-driven approach, our protocol includes key optimizations for KOLF2.1J, including culture on Laminin-521 to support xeno-free conditions. The resulting i-Microglia exhibit hallmark features of mature microglia, including expression of P2RY12, loss of the pluripotency marker SSEA4, phagocytic activity, and upregulation of immune markers (e.g., CD80, CD83) upon LPS stimulation. We also demonstrate compatibility with co-culture systems using iPSC-derived neurons. Additionally, we describe a modification of the line to include a constitutive mCherry reporter integrated into the SH4-2 safe harbor locus, enabling fluorescent tracking of microglia in mixed cultures or in vivo. This protocol provides a reproducible and scalable platform for generating functional human microglia from a widely used hiPSC line, supporting applications in brain tumors and disease modeling, neuroinflammation research, and therapeutic screening. Full article

Urethral stricture is a disease of fibrotic narrowing that compromises the urethral mucosa and spongiosum. Oral mucosal graft urethroplasty delivers excellent outcomes in complex cases, yet its procedural demands restrict availability beyond specialized centers. Endoscopic transplantation of oral mucosa has been proposed; while feasibility is shown, clinical efficacy remains suboptimal. We asked whether extracellular vesicles from stem cells of human exfoliated deciduous teeth (SHED-EVs) promote oral mucosa fibroblast (OMF) growth under urethra-mimetic paracrine conditions and whether culture growth phase tunes EV function. SHED-EVs were collected during logarithmic (SHED-EV-L) or stationary (SHED-EV-S) phases under xeno-free conditions, isolated by a standardized workflow, and characterized by nanoparticle tracking analysis. miRNA cargo was profiled with a human miRNA microarray platform and normalized for comparative analyses. OMF proliferation was quantified in a horizontal indirect co-culture with urethral epithelial cells using incubator-based time-lapse imaging. SHED-EV-L produced a sustained pro-proliferative effect across 24–96 h, whereas SHED-EV-S showed a weaker early effect with a late catch-up; both exceeded vehicle at 96 h. Fibrosis-related miRNA heat maps showed culture growth phase-dependent patterns: SHED-EV-L displayed relatively higher signals for miR-31-3p, miR-146b-3p, several let-7 members, and selected miR-181 isoforms, whereas SHED-EV-S showed a marked relative increase of miR-486-3p; miR-21, miR-99/100, and miR-205 were broadly comparable between phases. These findings indicate that culture growth phase is a practical design lever that orients SHED-EV cargo and function, supporting phase-matched formulations for adjunctive transurethral applications and motivating in vivo validation and manufacturing-oriented quality controls. Full article

Cell-based therapeutics are redefining interventions for vision loss by enabling tissue replacement, regeneration, and neuroprotection. This review surveys contemporary cellular strategies in ophthalmology through the lenses of therapeutic effectiveness, translational readiness, and governance. We profile principal sources—embryonic and induced pluripotent stem cells, mesenchymal stromal cells, retinal pigment epithelium, retinal progenitor and limbal stem cells—and enabling platforms including extracellular vesicles, encapsulated cell technology and biomaterial scaffolds. We synthesize clinical evidence across age-related macular degeneration, inherited retinal dystrophies, and corneal injury/limbal stem-cell deficiency, and highlight emerging applications for glaucoma and diabetic retinopathy. Delivery routes (subretinal, intravitreal, anterior segment) and graft formats (single cells, sheets/patches, organoids) are compared using standardized structural and functional endpoints. Persistent barriers include GMP-compliant derivation and release testing; differentiation fidelity, maturation, and potency; genomic stability and tumorigenicity risk; graft survival, synaptic integration, and immune rejection despite ocular immune privilege; the scarcity of validated biomarkers and harmonized outcome measures and ethical, regulatory, and health-economic constraints. Promising trajectories span off-the-shelf allogeneic products, patient-specific iPSC-derived grafts, organoid and 3D-bioprinted tissues, gene-plus-cell combinations, and cell-free extracellular-vesicle therapeutics. Overall, cell-based therapies remain investigational. With adequately powered trials, methodological harmonization, long-term surveillance, scalable xeno-free manufacturing, and equitable access frameworks, they may eventually become standards of care; at present, approvals are limited to specific products/indications and regions, and no cell therapy is the standard of care for retinal disease. Full article

Mesenchymal stromal cells (MSCs) have demonstrated therapeutic efficacy across numerous clinical applications, with evidence suggesting their paracrine effects, particularly through extracellular vesicles (EVs), possibly driving functional outcomes. In this study we perform the comprehensive characterization of microRNA expression profiles in human MSC-derived EVs (MSC-EV) compared to their parental cells, cultured under clinically relevant xeno-free conditions. MSCs were isolated from the bone marrows of healthy donors and characterised according to the International Society for Cellular Therapy criteria, while MSC-EVs were isolated using differential ultracentrifugation and validated according to the International Society for Extracellular Vesicle guidelines. NanoString profiling identified 590 mature microRNAs expressed across both populations, with 42 being significantly differentially expressed between MSC-EVs and parental MSCs. Five microRNAs were distinctly highly expressed in MSCs and five in MSC-EVs, while fifteen of the top twenty most abundant microRNAs showed high expression in both populations. MicroRNA expression patterns were validated in an independent cohort. Functional pathway analysis of differentially expressed microRNAs showed enrichment of key biological processes including cell proliferation, differentiation, and immune regulation. This standardised profiling approach develops our understanding of MSC/MSC-EV microRNA cargo, using a transparent methodological approach that allows for the improved comparability of datasets for the development and advancement of MSC-EV therapeutics. Full article

Background: Mesenchymal stem cell-based therapy may be indicated in ischaemic heart disease. The use of autologous adipose-derived mesenchymal stromal cells (AdMSCs) offers regenerative potential due to their paracrine effects. The aim of this study was to expand and characterise adult human thymus-derived MSCs harvested during open heart surgery with respect to their stem cell and paracrine properties. Methods: Enzymatically and non-enzymatically isolated human thymic AdMSCs (ThyAdMSCs) were cultured in xeno-free media containing pooled human platelet lysate (pPL). MSC characterisation was performed. Ex vivo expanded ThyAdMSCs were differentiated into three lineages. Proliferative capacity and immunomodulatory properties were assessed by proliferation assays and mixed lymphocyte reaction, respectively. Gene expression analysis was performed by qPCR. Results: Both isolation methods yielded fibroblast-like cells with plastic adherence and high proliferation. Flow cytometry revealed distinct expression of MSC markers in the absence of haematopoietic cell surface markers. Ex vivo expanded ThyAdMSCs could be differentiated into adipocytes, osteocytes, and chondrocytes. Activated peripheral blood mononuclear cells were significantly reduced when co-cultured with ThyAdMSCs, indicating their ability to inhibit immune cells in vitro. Gene expression analysis showed significantly less IFNγ and TNFα, indicating an alteration of the activated and pro-inflammatory state in the presence of ThyAdMSCs. Conclusions: These results demonstrate an efficient method to generate AdMSCs from human thymus. These MSCs have a strong immunomodulatory capacity and are, therefore, a promising cell source for regenerative medicine. The culture conditions are crucial for cells to proliferate in culture. Further research could explore the use of ThyAdMSCs or their secretome in surgical procedures. Full article

Osteoarthritis (OA) remains a leading cause of disability worldwide, with no disease-modifying therapies currently available for treatment. The infrapatellar fat pad (IFP) harbors mesenchymal stem/stromal cells (MSC) with potent immunomodulatory and regenerative properties, making them a promising candidate for OA treatment. A growing body of evidence suggests that the therapeutic effects of MSC are largely mediated by their extracellular vesicles (EVs), which carry bioactive cargo that modulates inflammation and tissue repair. However, optimizing MSC-derived EVs as a cell-free therapeutic approach requires an in-depth understanding of how culture conditions and inflammatory/hormonal priming influence their functional properties. In this study, IFP-MSC were expanded in regulatory-compliant human platelet lysate (HPL) and xeno-/serum-free (XFSF) media and primed with an inflammatory/fibrotic cocktail (TIC) with oxytocin (OXT) to assess the impact on their immunophenotypic profile and EV cargo. The immunophenotype confirmed that TIC+OXT-primed MSC retained key immunomodulatory surface markers, while EV characterization verified the successful isolation of CD63+/CD9+ vesicles. Pathway enrichment analysis of both HPL- and XFSF- TIC+OXT EVs cargo identified key miRNAs associated with immune regulation, tissue repair, and anabolic signaling. Functional assays revealed that TIC+OXT EVs promoted M2-like anti-inflammatory macrophage polarization and exhibited chondroprotective properties in chondrocytes/synoviocytes inflammatory osteoarthritic assay. These findings highlight the therapeutic potential of TIC+OXT-primed IFP-MSC-derived EVs as immunomodulatory and chondroprotective agents, offering a promising strategy for OA treatment through a clinically viable, cell-free approach. Full article

Human neural stem cells (hNSCs) are vital for advancing therapies for neurocognitive disorders. However, standard hNSC culture conditions often lack chemically defined and xeno-free substrates, limiting their clinical applicability. Chitosan, known for its biocompatibility, presents a promising alternative for hNSC culture. Hyaluronic acid (HA) and alginate, with their negative charges, enable effective interaction with positively charged chitosan to form films with enhanced mechanical properties. Incorporating chitosan into substrates creates chitosan–alginate (CA) and chitosan–hyaluronic acid (CHA) composites that meet chemically defined, mechanically tunable, and xeno-free standards. Despite their potential, the effects of these composites’ composition and mechanical properties on hNSC behavior, particularly in film form, remain unexplored. To bridge this gap, we fabricated films with varying chitosan-to-alginate and chitosan-to-hyaluronic acid ratios to assess their influence on hNSC pluripotency under xeno-free conditions. Our results reveal that films with higher chitosan content promote hNSC attachment and proliferation. Conversely, increasing alginate generally decreased cell attachment, proliferation, and multipotency, while increasing HA had no impact on attachment or proliferation but decreased multipotency. This investigation provides insights into the impact of substrate composition and mechanical properties on hNSC behavior, guiding the design of analogous materials for three-dimensional cultures and optimizing stem cell-based therapies for clinical applications. Full article

Cancers constitute a leading cause of mortality. Chimeric antigen receptor (CAR) cell therapies provide breakthrough solutions for various cancers while posing considerable risks of immunological side reactions. Of various cytotoxic lymphocyte subsets, natural killer (NK) cells are considered the least immunogenic. Obtaining viable NK cells with stable phenotypes in quantities sufficient for modification is technologically challenging. The candidate sources include primary mononuclear cell cultures and immortalized NK cell lines; alternatively, the clinical-grade NK cells can be differentiated from induced pluripotent stem cells (iPSCs) by a good manufacturing practice (GMP)-compatible xeno-free protocol. In this review, we analyze existing protocols for targeted differentiation of human iPSCs into NK cells with a focus on xeno-free requirements. Full article

Background and Objectives: Autologous bone grafting is the first choice for reconstructive surgery in bone defects due to trauma or malignant tumors. However, there is an increasing demand for minimally invasive alternatives involving bone regeneration using artificial materials. Biomimetic materials that replicate the body’s microscopic structure, such as Cellnest^®, are gaining attention. Cellnest is a xeno-free recombinant peptide based on human type I collagen, containing a rich Arg-Gly-Asp (RGD) motif related to cell adhesion. The aim of this study was to compare the effects of Cellnest with existing collagen materials (Pelnac^®, Integra^®, Terudermis^®) on bone regeneration and elucidate the underlying mechanisms. Materials and Methods: In vivo experiments involved a rat model of calvarial bone defects, in which Cellnest and other collagen materials were implanted into the defect area. Bone formation was assessed after 4 weeks using micro-computed tomography (micro-CT) and histological analysis. In vitro experiments included the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), adhesion, and migration assays, and a real-time polymerase chain reaction using rapidly expanding cells (RECs) to explore the mechanisms of Cellnest’s bone regenerative capacity. Results: The micro-CT analysis showed that the regenerated bone area was significantly greater in the Cellnest group (72.3%) than in the Pelnac^® (25.5%), Integra^® (31.6%), and Terudermis^® (38.3%) groups. The histological analysis confirmed similar trends, with Cellnest showing 42.2% bone regeneration, outperforming the other materials. The in vitro assays revealed that Cellnest promoted cell proliferation, adhesion, and migration. Gene expression analysis demonstrated that Cellnest significantly increased the levels of the bone formation markers ALP and COL1. Conclusions: Cellnest, a human type I collagen-like peptide rich in RGD motifs, enhances bone regeneration by promoting MSC adhesion and migration, and bone formation-related gene expression. The findings suggest its potential as an effective material for bone defect reconstruction. Full article

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. Full article

Skin aging is a complex, multifaceted process influenced by both intrinsic and extrinsic factors. Understanding the molecular mechanisms underlying skin aging is crucial for developing effective anti-aging strategies. Dermal stem cells play a pivotal role in maintaining skin homeostasis, but their functionality is compromised with aging. This study investigated the impact of aging on dermal stem cells and explored the potential of natural extracts in modulating their biological characteristics. Using bulk RNA barcoding and sequencing (BRB-seq), we identified differentially expressed genes (DEGs) between young and aged dermal stem cells, revealing alterations in cellular processes, including cell proliferation, ECM synthesis, and RNA splicing. We also demonstrated that a natural extract, comprising callus cells and Alpine rose leaf extracts, influenced RNA splicing in aged dermal stem cells, leading to improved dermal structure and integrity in vitro. Our findings suggest that natural extracts may exert their effects through senolytic activity and the modulation of RNA splicing, a process crucial to gene expression and cellular function. This study underscores the potential of integrating high-throughput transcriptomics in understanding skin aging, presenting new avenues for the development of innovative, sustainable, and effective anti-aging strategies. Full article

Differentiation approaches to obtain mesenchymal stem cells (MSCs) have gradually developed over the last few decades. The problem is that different protocols give different MSC types, making further research difficult. Here, we tried three different approaches to differentiate embryonic stem cells (ESCs) from early mesoderm to MSCs using serum-containing or xeno-free differentiation medium and observed differences in the cells’ morphology, doubling rate, ability to form colonies, surface marker analysis, and multilineage differentiation potential of the obtained cell lines. We concluded that the xeno-free medium best fits the criteria of MSCs’ morphology, growth kinetics, and surface marker characterization. In contrast, the serum-containing medium gives better potential for further MSC differentiation into osteogenic, chondrogenic, and adipogenic lineages. Full article

Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5–7%, 1–2%, and 1–2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability. Full article

Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85–92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86–92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97–101%), akin to a Matrigel-based liver model (83–102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes. Full article

Among the available therapeutics for the conservative treatment of osteoarthritis (OA), mesenchymal stromal cells (MSCs)-based products appear to be the most promising. Alongside minimally manipulated cell-based orthobiologics, where MSCs are the engine of the bioactive properties, cell expansion under good manufacturing practice (GMP) settings is actively studied to obtain clinical-grade pure populations able to concentrate the biological activity. One of the main characteristics of GMP protocols is the use of clinical-grade reagents, including the recently released serum-free/xeno-free (SFM/XFM) synthetic media, which differ significantly from the traditional reagents like those based on fetal bovine serum (FBS). As SFM/XFM are still poorly characterized, a main lack is the notion of reliable housekeeping genes (HKGs) for molecular studies, either standalone or in combination with standard conditions. Indeed, the aim of this work was to test the stability of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) in adipose-derived MSCs (ASCs) cultivated in two commercially available SFM/XFM and to compare outcomes with those obtained in FBS. Four different applets widely recognized by the scientific community (NormFinder, geNorm, comparative ΔCt method, and BestKeeper) were used and data were merged to obtain a final stability order. The analysis showed that cells cultured in both synthetic media had a similar ranking for HKGs stability (GAPDH being best), albeit divergent from FBS expanded products (EF1A at top). Moreover, it was possible to identify specific HKGs for side by side studies, with EF1A/TBP being the most reliable normalizers for single SFM/XFM vs. FBS cultured cells and TBP the best one for a comprehensive analysis of all samples. In addition, stability of HKGs was donor-dependent. The normalization effect on selected genes coding for factors known to be involved in OA pathology, and whose amount should be carefully considered for the selection of the most appropriate MSC-based treatment, showed how HKGs choice might affect the perceived amount for the different media or donor. Overall, this work confirms the impact of SFM/XFM conditions on HKGs stability performance, which resulted similarly for both synthetic media analyzed in the study. Full article