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Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve injury and disease. Stem cell differentiation has emerged as a key human cell model and disease motivating development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains controversial,we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived Schwann cells generated with our previously reported protocol. We identified only modest transcriptome differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally,both cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol are molecularly comparable and functionally equivalent. Subject areas: Neuroscience,Cell biology,Stem cells research,Transcriptomics View Publication

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes,but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR,which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends,capturing the target region for sequencing on PacBio’s Sequel,Sequel II,or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized,even for repeats in the thousands. Key features • This protocol is adapted from PacBio’s previous “no-amp targeted sequencing utilizing the CRISPR-Cas9 system.” • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type,blood,or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio. View Publication

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by the accumulation of pathological proteins and synaptic dysfunction. This study aims to investigate the molecular and functional differences between human induced pluripotent stem cells (hiPSCs) derived from patients with sporadic AD (sAD) and age-matched controls (healthy subjects,HS),focusing on their neuronal differentiation and synaptic properties in order to better understand the cellular and molecular mechanisms underlying AD pathology. Skin fibroblasts from sAD patients ( n = 5) and HS subjects ( n = 5) were reprogrammed into hiPSCs using non-integrating Sendai virus vectors. Through karyotyping,we assessed pluripotency markers (OCT4,SOX2,TRA-1–60) and genomic integrity. Neuronal differentiation was evaluated by immunostaining for MAP2 and NEUN. Electrophysiological properties were measured using whole-cell patch-clamp,while protein expression of Aβ,phosphorylated tau,Synapsin-1,Synaptophysin,PSD95,and GluA1 was quantified by western blot. We then focused on PAK1-LIMK1-Cofilin signaling,which plays a key role in regulating synaptic structure and function,both of which are disrupted in neurodegenerative diseases such as AD. sAD and HS hiPSCs displayed similar stemness features and genomic stability. However,they differed in neuronal differentiation and function. sAD-derived neurons (sAD-hNs) displayed increased levels of AD-related proteins,including Aβ and phosphorylated tau. Electrophysiological analyses revealed that while both sAD- and HS-hNs generated action potentials,sAD-hNs exhibited decreased spontaneous synaptic activity. Significant reductions in the expression of synaptic proteins such as Synapsin-1,Synaptophysin,PSD95,and GluA1 were found in sAD-hNs,which are also characterized by reduced neurite length,indicating impaired differentiation. Notably,sAD-hNs demonstrated a marked reduction in LIMK1 phosphorylation,which could be the underlying cause for the changes in cytoskeletal dynamics that we found,leading to the morphological and functional modifications observed in sAD-hNs. To further investigate the involvement of the LIMK1 pathway in the morphological and functional changes observed in sAD neurons,we conducted perturbation experiments using the specific LIMK1 inhibitor,BMS-5. Neurons obtained from healthy subjects treated with the inhibitor showed similar morphological changes to those observed in sAD neurons,confirming that LIMK1 activity is crucial for maintaining normal neuronal structure. Furthermore,administration of the inhibitor to sAD neurons did not exacerbate the morphological alterations,suggesting that LIMK1 activity is already compromised in these cells. Our findings demonstrate that although sAD- and HS-hiPSCs are similar in their stemness and genomic stability,sAD-hNs exhibit distinct functional and structural anomalies mirroring AD pathology. These anomalies include synaptic dysfunction,altered cytoskeletal organization,and accumulation of AD-related proteins. Our study underscores the usefulness of hiPSCs in modeling AD and provides insights into the disease's molecular underpinnings,thus highlighting potential therapeutic targets. The online version contains supplementary material available at 10.1186/s13195-024-01632-3. View Publication

The development of immunocompetent skin models marks a significant advancement in in vitro methods for detecting skin sensitizers while adhering to the 3R principles,which aim to reduce,refine,and replace animal testing. This study introduces for the first time an advanced immunocompetent skin model constructed entirely from induced pluripotent stem cell (iPSC)-derived cell types,including fibroblasts (iPSC-FB),keratinocytes (iPSC-KC),and fully integrated dendritic cells (iPSC-DC). To evaluate the skin model’s capacity,the model was treated topically with a range of well-characterized skin sensitizers varying in potency. The results indicate that the iPSC-derived immunocompetent skin model successfully replicates the physiological responses of human skin,offering a robust and reliable alternative to animal models for skin sensitization testing,allowing detection of extreme and even weak sensitizers. By addressing critical aspects of immune activation and cytokine signaling,this model provides an ethical,comprehensive tool for regulatory toxicology and dermatological research. View Publication

CACNA1A encodes the pore-forming α 1A subunit of the Ca V 2.1 calcium channel,whose altered function is associated with various neurological disorders,including forms of ataxia,epilepsy,and migraine. In this study,we generated isogenic iPSC-derived neural cultures carrying CACNA1A loss-of-function mutations differently affecting Ca V 2.1 splice isoforms. Morphological,molecular,and functional analyses revealed an essential role of CACNA1A in neurodevelopmental processes. We found that different CACNA1A loss-of-function mutations produce distinct neurodevelopmental deficits. The F1491S mutation,which is located in a constitutive domain of the channel and therefore causes a complete loss-of-function,impaired neural induction at very early stages,as demonstrated by changes in single-cell transcriptomic signatures of neural progenitors,and by defective polarization of neurons. By contrast,cells carrying the Y1854X mutation,which selectively impacts the synaptically-expressed Ca V 2.1[EFa] isoform,behaved normally in terms of neural induction but showed altered neuronal network composition and lack of synchronized activity. Our findings reveal previously unrecognized roles of CACNA1A in the mechanisms underlying neural induction and neural network dynamics and highlight the differential contribution of the divergent variants Ca V 2.1[EFa] and Ca V 2.1[EFb] in the development of human neuronal cells. The online version contains supplementary material available at 10.1007/s00018-025-05740-7. View Publication

Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here,we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently,CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically,CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis,glycolytic capacity,maximal mitochondrial respiration,ATP-linked respiration,and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together,these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity. View Publication

Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity,their large size often leads to necrotic core formation,limiting reproducibility and challenging the integration of microglia. Here,we present a detailed,reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons,astrocytes,and optionally microglia,all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells,generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling),microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications,including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories,demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes. View Publication

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in COL7A1. Patient-derived induced pluripotent stem cells (iPSCs) enable the personalized study of RDEB pathogenesis and potential therapies. However,current skin cell differentiation protocols via 2D culture perform suboptimally when applied to engineered 3D skin constructs (ESC). Here,we present an approach to source fibroblasts (iFBs) and keratinocytes (iKCs) from iPSC-derived skin organoids using an optimized differentiation protocol,and utilize them to engineer ESCs modeling wild-type and RDEB phenotypes. The resulting iPSC-derived skin cells display marker expression consistent with primary counterparts and produce ESCs exhibiting significant extracellular matrix remodeling,protein deposition,and epidermal differentiation. RDEB constructs recapitulated hallmark disease features,including absence of collagen VII and reduced iFB proliferation. This work establishes a robust and scalable strategy for generating physiologically-relevant,iPSC-derived skin constructs,offering a powerful model for studying RDEB mechanisms and advancing personalized regenerative medicine. View Publication

The potential of the immune system to decrease cancer progression is widely recognized and has led to the development of innovative anti-cancer immunotherapies. Here,we studied human macrophages derived from genetically engineered iPSCs (iMac) with angiotensin-converting enzyme (ACE) expression regulatable by a doxycycline (dox)-inducible promoter as a novel anti-cancer immunotherapy. Increased ACE expression in iMac (cells now termed ACE-iMac) augments polarization towards an M1 macrophage phenotype characterized by increased production of proinflammatory cytokines,reactive oxygen species,nitric oxide,and an RNA profile indicating an aggressive immune response. ACE-iMac kills tumor cells in vitro significantly better than iMac. In vivo,studies using tumor xenografts for melanoma,breast cancer,and head and neck squamous cell carcinoma (HNSCC) showed a highly significant 3.4- to 7.2-fold reduction in solid tumor size following ACE-expressing ACE-iMac immunotherapy as compared to results with iMac. To further investigate the impact of ACE on human anti-tumor responses,we developed a humanized BLT-NSG mouse model with a fully functional adaptive immune system. Here,ACE-iMac treatment significantly reduced the growth of human melanoma xenografts by enhancing the activation of human T cells and NK cells. In conclusion,enhancing ACE expression in human-derived macrophages (ACE-iMac) greatly amplifies their anti-cancer phenotype,offering a compelling new therapeutic strategy with the potential to improve clinical outcomes for cancer patients. View Publication