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Introduction: Africa, home to 1.4 billion people and the highest genetic diversity globally, harbors unique genetic variants crucial for understanding complex diseases like neurodegenerative disorders. However, African populations remain underrepresented in induced pluripotent stem cell (iPSC) collections, limiting the exploration of population-specific disease mechanisms and therapeutic discoveries. Methods: To address this gap, we established an open-access African Somatic and Stem Cell Bank. Results: In this initial phase, we generated 10 rigorously characterized iPSC lines from fibroblasts representing five Nigerian ethnic groups and both sexes. These lines underwent extensive profiling for pluripotency, genetic stability, differentiation potential, and Alzheimer's disease and Parkinson's disease risk variants. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology was used to introduce frontotemporal dementia-associated MAPT mutations (P301L and R406W). Discussion: This collection offers a renewable, genetically diverse resource to investigate disease pathogenicity in African populations, facilitating breakthroughs in neurodegenerative research, drug discovery, and regenerative medicine. Highlights: We established an open-access African Somatic and Stem Cell Bank. 10 induced pluripotent stem cell lines from five Nigerian ethnic groups were rigorously characterized. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology was used to introduce frontotemporal dementia-causing MAPT mutations. The African Somatic and Stem Cell Bank is a renewable, genetically diverse resource for neurodegenerative research.

Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids, T lymphocytes, and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac, neurological, or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling, and variant-preferred healthy control lines were identified for specific disease settings.

Accurate modeling of human neuronal cell biology has been a long-standing challenge. However, methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately, these methods entail numerous challenges, including poor efficiency, variable cell type heterogeneity, and lengthy, expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol, these lines can be inducibly differentiated into either cortical (i3 Neurons) or lower motor neurons (i3 LMN) in a rapid, efficient, and scalable manner (Wang et al., 2017). In this manuscript, we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i3 Neurons or i3 LMNs, and we present neuronal culture conditions for various experimental applications. {\textcopyright} 2018 by John Wiley & Sons, Inc.

Advances in differentiation of cardiomyocytes from human induced pluripotent stem cell (hiPSC) were emerged as a tool for modeling of cardiovascular disease that recapitulates the phenotype for the purpose of drug screening, biomarker discovery, and testing of single-nucleotide polymorphism (SNP) as a modifier for disease stratification. Here, we describe the (1) retroviral reprogramming strategies in the generation of human iPSC, (2) methodology in characterization of iPSC in order to identify the stem cell clones with the best quality, and (3) protocol of cardiac differentiation by modulation of Wnt signaling and $\$-catenin pathway.

Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes, in vitro testing of novel therapies, and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward, scientists can expend considerable resources and time to establish this technology. A major hurdle is the accurate determination of valid iPSC-like colonies that can be selected for further cloning and characterization. In this study, we describe the use of a gammaretroviral vector encoding a fluorescent marker, mRFP1, to not only monitor the efficiency of initial transduction but also to identify putative iPSC colonies through silencing of mRFP1 gene as a consequence of successful reprogramming.

Although distinct human induced pluripotent stem cell (hiPSC) lines can display considerable epigenetic variation, it has been unclear whether such variability impacts their utility for disease modeling. Here, we show that although low-passage female hiPSCs retain the inactive X chromosome of the somatic cell they are derived from, over time in culture they undergo an erosion" of X chromosome inactivation (XCI). This erosion of XCI is characterized by loss of XIST expression and foci of H3-K27-trimethylation�

Microglia are the brain's resident immune cells, essential for homeostasis and implicated in common neurodegenerative diseases like Alzheimer's and Parkinson's disease (PD), where their early activation and sustained inflammatory mediator release contribute to neuronal loss. However, their role in rare disorders is unclear. β-propeller protein-associated neurodegeneration (BPAN), caused by WDR45 mutations, shares key features with PD, including iron accumulation and dopaminergic neuron loss, but the impact of microglia and mutant WDR45 in BPAN pathophysiology remains unexplored. To address this, we established the first induced pluripotent stem stell (iPSC)-derived microglia model from BPAN patients. Parallel targeted transcriptomic and secretomic profiling revealed a shift from a homeostatic microglial toward a stress-adapted and transcriptionally reprogrammed state characterized by selective remodeling of immune signaling pathways and dysregulation of autophagy and cellular stress responses. Complementary secretomic analysis identified reduced secretion of lysosomal enzymes alongside increased shedding of immune-associated surface proteins, indicating altered lysosomal trafficking and remodeling of microglial immune signaling. These findings identify a distinct microglial phenotype in BPAN and implicate microglial dysfunction as a potential contributor to disease mechanisms, highlighting new avenues for therapeutic strategies targeting neuroimmune pathways.

The SIRPα-CD47 "don't eat me" checkpoint axis plays a critical role in shaping antitumor activities of macrophages within the tumor microenvironment (TME). However, targeting this axis with anti-CD47 antibodies to enhance antitumor responses in clinical trials has been challenging. Here, we demonstrated that SIRPA-knockout (KO) iPSC-derived macrophages (iMacs) exhibit superior antitumor activity against various CD47-expressing tumors in vitro when combined with cancer-targeted monoclonal antibodies (mAbs) or chimeric antigen receptors (CARs). Moreover, SIRPA-KO protected macrophages from mAb- and CAR-driven hypophagia, enabling efficient tumoricidal effects even after serial tumor exposures. Retention of phagocytic activities in SIRPA-KO iMacs was associated with heightened surface expression of Fc receptors and GD2-CAR compared to their SIRPA-expressing counterparts. Despite the powerful impact of SIRPA-KO on iMac antitumor activities in vitro, only modest efficacy was observed in human xenograft mouse models of SK-OV3 ovarian carcinoma and CHLA-163 neuroblastoma treated with mAb or CAR-iMac therapy, indicating further engineering or combinatorial therapeutic strategies are needed for potent in vivo antitumor efficacy. Together, these findings identify SIRPα as a regulator in macrophage hypophagia and underscore the advantages of using SIRPA-KO macrophage therapeutic strategies to modulate the SIRPα-CD47 checkpoint to unleash macrophage antitumor activity within the TME.

Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) offer a promising model system for understanding motor neuron diseases (MNDs) and advancing drug discovery. However, variability in differentiation outcomes presents a major barrier to reproducibility and model reliability. This study evaluates a widely adopted small molecule protocol for MN differentiation to quantify variability and identify its sources within an industrial setting. Analysing data from 15 differentiation sets across 8 cell lines, we found that non-genetic factors – particularly induction set and operator – were the predominant sources of variability, outweighing the contribution from cell line genetics. We further demonstrated that iPSC genomic instability, as assessed by a targeted RT-qPCR assay for common karyotypic abnormalities, significantly affected differentiation efficiency and purity. Cultures derived from genomically stable iPSCs exhibited reduced variance and improved MN marker expression profiles. These findings support routine genomic assessment of iPSCs as a practical and effective strategy to enhance the reliability of iPSC-derived MN models, thereby improving their utility in preclinical MND research and therapeutic development.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-23378-0.

The use of blood vessel organoids (BVOs), or vascular organoids derived from human induced pluripotent stem cells (hiPSC), provides a valuable tool for research on vascular regeneration. Vascular organoid culture relies on the use of Matrigel, a murine sarcoma-derived matrix, which limits its translational potential and reproducibility. This study evaluated the potential alternatives to Matrigel for both 2D hiPSC and 3D vascular organoid culture to enhance the clinical applicability of hiPSC-derived vascular organoids. Vitronectin is a suitable replacement for Matrigel in hiPSC culture and expansion, maintaining pluripotency and facilitating subsequent differentiation into vascular organoids. Additionally, we demonstrated that fibrin-based hydrogels effectively support vascular organoid differentiation, promoting vascular network formation and endothelial cell sprouting comparable to Matrigel-based cultures. Through gene expression analysis, surface area quantification, and immunohistochemistry, we validated the efficacy of Vitronectin and fibrin in supporting hiPSC-derived vascular organoid differentiation. This Matrigel-free protocol not only enhances reproducibility but is a step forward in xeno-free culture conditions, offering a versatile platform for disease modelling, vascular tissue engineering, and patient-specific therapeutic screening.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20091-w.

ObjectiveThere is currently no long-term treatment for the repair of damaged cartilage and osteoarthritis (OA). Induced pluripotent stem cells (iPSCs) are an ideal cell source for screening platforms due to their ability to self-renew and differentiate to cell types that would otherwise require invasive surgeries to obtain, such as chondrocytes and mesenchymal stromal cells (MSCs). Here, we developed an iPSC-based screening platform and tested previously described pro-chondrogenic small molecule compounds, to determine their potential to identify hits.DesigniPSC derived chondroprogenitors (iCPs) and neural crest cell (NCC) derived MSCs (iNCC-MSCs) were generated, and their chondrogenic potential was confirmed. The iPSC derived cells and a primary bone marrow derived MSC (BM-MSC) line were cultured as pellets and treated with different concentrations of small molecule compounds, in the presence of chondrogenic inducing growth factors, over 14 days at 2 ​% O2. Glycosaminoglycan (GAG) synthesis was quantified by a 1,9- dimethylmethylene blue (DMMB) assay.ResultsAfter 14 days of chondrogenesis, forskolin, baicalin and sesamin enhanced GAG synthesis in the iCPs, and forskolin enhanced GAG synthesis in the iNCC-MSCs, while no small molecule compounds enhanced GAG synthesis in the BM-MSCs.ConclusionOur findings further demonstrate how the small molecules pro-chondrogenic effects are dependent on the screening platform conditions, including the cell type, molecule concentration, 3D culture, hypoxia, and the inclusion of additional growth factors. The iPSC-based screening platform developed has the potential to identify disease modifying OA drugs (DMOADs) in novel compound screening libraries.