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Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells,followed by the migration,differentiation,and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens,such as FGF8. Disruption of this developmental balance can lead to brain malformations,which underlie a range of complex neurodevelopmental disorders,including epilepsy,autism,and intellectual disabilities. Studying the early stages of human brain development,whether under normal or pathological conditions,remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently,human brain organoids have emerged as a powerful in vitro alternative,allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures,where neural progenitors and neurons are grown on flat surfaces,3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However,3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore,few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning. • To address these limitations,we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs),our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation,NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors,where they self-organize into neural rosettes and neuroepithelial structures,surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids,leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity,enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas. Key features • This protocol builds on the method established by Chambers et al. [1] for generating 2D neural progenitors,followed by dissociation and reaggregation into 3D brain organoids. • For optimal growth and maturation,telencephalic organoids are cultured in spinning mini-bioreactors [2] or on orbital shakers. • The protocol enables the generation of telencephalic neural progenitors in 10 days and produces 3D telencephalic organoids containing neocortical neurons within one month of culture. • Addition of morphogens in the culture medium (example: FGF8) enhances cellular heterogeneity,promoting the emergence of distinct brain domains within a single organoid. View Publication

ABSTRACTCell therapy based on chimeric antigen receptor (CAR) T cells has represented a revolutionary new approach for treating tumors,especially hematological diseases. Complete remission rates (CRR) > 80%–97% and 50%–90% overall response rates (ORR) have been achieved with a treatment based on CAR‐T cells in patients with malignant B‐cell tumors that have relapsed or are refractory to previous treatments. Toxicity remains the major problem. Most patients treated with CAR‐T cells develop high‐grade cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity syndrome (ICANS). However,the unprecedentedly high CRR and ORR have led to the approval of six CAR‐T cell therapeutics by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA),prompting researchers to improve existing products and develop new ones. By now,around 1000 clinical trials based on CAR‐T cells are registered at ClinicalTrials.gov: 82% are for hematological diseases,while the remaining 16% are for solid tumors. As a result of this increased research,an enormous amount of conflicting information has been accumulated in the literature,and each group follows its manufacturing protocols and performs specific in vitro testing. This review aimed to combine and compare clinical and preclinical information,highlighting the most used protocols to provide a comprehensive overview of the in vitro world of CAR‐T cells,from manufacturing to their characterization. The focus is on all steps of the CAR‐T cell manufacturing process,from the collection of patient or donor blood to the enrichment of T cells,their activation with anti‐CD3/CD28 beads,interleukin‐2 (IL‐2) or IL‐7 and IL‐15 (induction of a more functional memory phenotype),and their transfection (viral or non‐viral methods). Automation is crucial for ensuring a standardized final product. View Publication

Seven female individuals with multiple congenital anomalies,developmental delay and/or intellectual disability have been found to have a genetic variant of uncertain significance in the mediator complex subunit 12 gene ( MED12 c.3412C>T,p.Arg1138Trp). The functional consequence of this genetic variant in disease is undetermined,and insight into disease mechanism is required. We identified a de novo MED12 p.Arg1138Trp variant in a female patient and compared disease phenotypes with six female individuals identified in the literature. To investigate affected biological pathways,we derived two induced pluripotent stem cell (iPSC) lines from the patient: one expressing wildtype MED12 and the other expressing the MED12 p.Arg1138Trp variant. We performed neural disease modelling,transcriptomics and protein analysis,comparing healthy and variant cells. When comparing the two cell lines,we identified altered gene expression in neural cells expressing the variant,including genes regulating RNA polymerase II activity,transcription,pre-mRNA processing,and neural development. We also noted a decrease in MED12L expression. Pathway analysis indicated temporal delays in axon development,forebrain differentiation,and neural cell specification with significant upregulation of pre-ribosome complex gene pathways. In a human neural model,expression of MED12 p.Arg1138Trp altered neural cell development and dysregulated the pre-ribosome complex providing functional evidence of disease aetiology and mechanism in MED12-related disorders. The online version contains supplementary material available at 10.1186/s10020-025-01365-5. View Publication

The breakthrough development of induced pluripotent stem cells (iPSCs) raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells. However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear. In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with autogenous peripheral blood mononuclear cells (PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation. However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs. Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells (CD3+CD8− T cells,CD3+CD8+ T cells or CD3−CD56+ NK cells) by NPCs in both PBMC and T cell co-culture systems. These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs. View Publication

Patients with Fanconi anemia (FA) have a high risk of developing acute myeloid leukemia (AML). In this study,we attempted to identify cell-surface markers for leukemia-initiating cells in FA-AML patients. We found that the IL-3 receptor-α (IL-3Rα) is a promising candidate as an leukemia-initiating cell-specific antigen for FA-AML. Whereas IL-3Rα expression is undetectable on normal CD34(+)CD38(-) HSCs,it is overexpressed on CD34(+)CD38(-) cells from FA patients with AML. We examined the leukemia-initiating cell activity of IL-3Rα-positive FA-AML cells in a humanized" FA xenotransplant model in which we separated AML cells into IL-3Rα-positive and IL-3Rα-negative CD34 fractions and transplanted them into irradiated recipient mice. In all 3 FA-AML samples View Publication

The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However,the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs,no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process,we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study,we used a sized-based,label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (ie,>3 million cells/minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining,flow cytometry analysis,and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the “downstream processing” of cell manufacturing workflow,ensuring better quality and safety of transplanted cells. View Publication

Adipose-derived stromal cells (ASCs) possess significant regenerative potential,playing a key role in tissue repair and angiogenesis. During wound healing,ASC interacts with the extracellular matrix by recognizing arginylglycylaspartic acid (RGD) motifs,which are crucial for mediating these functions. This study investigates how RGD exposure influences ASC behavior,with a focus on angiogenesis. To mimic the wound-healing environment,ASC were cultured in a porcine gelatin sponge,an RGD-exposing matrix. Transcriptomics revealed that ASC cultured in gelatin exhibited an upregulated expression of genes associated with inflammation,angiogenesis,and tissue repair compared to ASC in suspension. Pro-inflammatory and pro-angiogenic factors,including IL-1,IL-6,IL-8,and VEGF,were significantly elevated. Functional assays further demonstrated that ASC-conditioned media enhanced endothelial cell migration,tubulogenesis,and reduced endothelial permeability,all critical processes in angiogenesis. Notably,ASC-conditioned media also promoted vasculogenesis in human vascular organoids. The inhibition of ASC-RGD interactions using the cyclic peptide cilengitide reversed these effects,underscoring the essential role of RGD-integrin interactions in ASC-mediated angiogenesis. These findings suggest that gelatin sponges enhance ASC’s regenerative and angiogenic properties via RGD-dependent mechanisms,offering promising therapeutic potential for tissue repair and vascular regeneration. Understanding how RGD modulates ASC behavior provides valuable insights into advancing cell-based regenerative therapies. View Publication

The recent advances,offered by cell therapy in the regenerative medicine field,offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors,such as myoblasts or satellite cells,possess a marked regenerative capacity,but the exploitation of this potential still encounters significant challenges in clinical application,due to low rate of proliferation in vitro,as well as a reduced self-renewal capacity. In this scenario,induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches,but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs,generated from pericytes and fibroblasts,exploiting skeletal muscle-derived extracellular vesicles (EVs),in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches." View Publication