Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with rising global prevalence. Current management strategies predominantly address behavioral symptoms rather than core neurobiological dysfunctions, highlighting an unmet need for mechanism-targeted interventions. This review comprehensively evaluates the therapeutic potential of mesenchymal stem/stromal cell (MSC)-based therapies for ASD. We discuss the multi-faceted mechanisms through which MSCs and their secretomes (particularly MSC-EVs or MSC-exos) may ameliorate ASD pathophysiology, including modulation of neuroinflammation via anti-inflammatory cytokine and adenosine signaling, promotion of neurogenesis and synaptic plasticity through neurotrophic factors (e.g., BDNF, GDNF), restoration of gut-brain axis communication, and enhancement of cerebral angiogenesis via VEGF. We synthesize a decade of preclinical evidence (2015–2025) from various ASD animal models (e.g., BTBR, VPA-induced, MIA, Shank3B KO), detailing improvements in social behavior, repetitive actions, and cognitive function following MSC or MSC-derived product administration via routes such as intravenous, intranasal, or intracerebroventricular delivery. Furthermore, we analyze all registered early-phase clinical trials (Phase I and II, n = 9 as of March 2026), comparing different MSC sources, their safety profiles, and preliminary efficacy outcomes. Key challenges are examined, including heterogeneity in trial design, small sample sizes, the predominance of open-label studies, and the gap between promising preclinical results and established clinical translation. MSC-based therapy represents a transformative, pathophysiology-informed strategy for ASD. While preclinical and early clinical data are encouraging, significant hurdles remain in standardization, dose optimization, and demonstration of long-term efficacy and safety. We emphasize the necessity for rigorous, large-scale randomized controlled trials (RCTs) to advance this promising therapeutic approach toward clinical application.

De novo pathogenic variants in KCNQ2 are a well-established cause of neurodevelopmental disorders, particularly developmental and epileptic encephalopathies. The KCNQ2 gene encodes the Kv7.2 subunit of a voltage-gated potassium channel that mediates the neuronal M-current and plays a key role in stabilizing the resting membrane potential. In this study, we established three induced pluripotent stem cell (iPSC) lines from dermal fibroblasts from a 4-year-old female patient carrying the KCNQ2 c.783A > C; p.Phe261Leu variant and a sibling control. These lines were characterized using standard validation methods, including confirmation of the pathogenic variant, SNP-based karyotyping, STR profiling, assessment of pluripotency marker expression, and evaluation of trilineage differentiation potential.

Mitochondrial DNA (mtDNA) damage and accumulation activate the cGAS-STING DNA-sensing pathway, which promotes immune clearance of tumor cells. Maintenance of the cytosolic level of mtDNA is key to sustain immune activation. T cell malignancies (T-CMs) are a general name of diseases with abnormal clonal proliferation of T lymphocytes at various stages. Immunotherapy of T-CMs is challenged by the lack of specific antigens to discriminate T-CMs from normal T cells. As intrinsic STING activation can promote the clearance of T-CMs by immune cells, we herein explored whether isoliensinine (IsoL), a natural compound from Nelumbinis Plumula could enhance NK clearance by mtDNA-mediated immune responses in tumor cells. To investigate whether IsoL modulated immune recognition and clearance of T-CMs, we pre-treated three T-CM cell lines (Jurkat, Molt4 and Hut102) with IsoL then co-cultured with NK-92MI cells. We showed that IsoL pre-treatment promoted cytosolic mtDNA accumulation by inducing ROS-dependent mitochondrial damage and inhibiting mitophagy via peroxiredoxin 1 (PRDX1), an antioxidant enzyme. Loss of PRDX1 in T-CMs also induced ROS-dependent mitochondrial DNA damage, and blocked mitophagy by preventing accumulation of mature PINK1, which was required to initiate mitophagy via recruiting Parkin to the damaged mitochondria. Remarkably, IsoL could induce expression of activating ligands in vitro, enhance NK cell infiltrations, and increase apoptosis of T-CMs. Moreover, we demonstrated that IsoL could sensitize T-CMs for NK clearance in vitro and in vivo. These results suggest that IsoL could be a potential therapeutic agent to enhance immune therapy of T-CMs.

NRXN1 deletion (NRXN1 del) is a rare copy number variant associated with several neurodevelopmental, neuropsychiatric, and cognitive outcomes. The NRXN1 gene encodes for a pre-synaptic cell adhesion molecule that is important for synapse formation, regulation and neurotransmission. We used a gene-first approach to investigate neurocognitive and brain phenotypes in NRXN1 del carriers. Forty-two participants (21 NRXN1 del carriers and 21 neurotypical age and sex-matched comparisons) completed IQ assessments, and a neurocognitive battery, including, executive function, attention, and social cognition tasks. Magnetic resonance imaging (MRI) data, including T1-weighted anatomical scans, resting state functional MRI and diffusion tensor imaging, were acquired in 36 participants (17 NRXN1 del carriers and 19 comparisons). NRXN1 del carriers had lower mean IQ and poorer spatial working memory performance compared to comparisons (p ≤ 0.05). Neuroimaging results revealed group differences in visual and ventral attention resting state networks (p < 0.05). Network-based statistical analysis showed a significant effect of group status for 28/115 connections, with poorer segregation between visual and default networks in NRXN1 del carriers relative to comparisons. No differences in brain structural volume or cortical thickness, or diffusion measures of white matter structural architecture were observed between groups. This exploratory study provides evidence for neurocognitive impacts and brain functional differences related to underlying synaptic mechanisms. Brain functional differences in NRXN1 del carriers may support altered excitation/inhibition dynamics within the brain. Gene-first approaches may establish brain-based translational markers to identify neurobiologically informed subgroups within neurodevelopmental and neuropsychiatric conditions, and ultimately transdiagnostic therapeutic strategies.

Human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) provide a critical source for the study of motor neuron diseases (MNDs), which has been hindered by the lack of appropriate disease models for many years. Although many spinal MN differentiation protocols have been established by mimicking in vivo neurogenesis using extrinsic signaling molecules, substantial variations in the duration and efficiency persist due to inconsistencies in concentrations, timing, and delivery methods of these molecules. Here, we present an efficient monolayer culture differentiation strategy that enables the generation of enriched CHAT+ spinal MNs (sMNs) in 18 days and functional sMNs exhibiting extensive network activities, as confirmed by multielectrode array (MEA), within 28 days. Therefore, this optimized MN differentiation protocol facilitates the production of mature sMNs for MND research, high-throughput drug screening, and potential cell replacement therapies.

Since its inception, induced pluripotent stem cell (iPSC) technology has been hailed as a powerful tool for comprehending disease etiology and advancing drug screening across various domains. While earlier iPSC-based disease modeling and drug assessment primarily operated at the cellular level, recent years have witnessed a significant shift towards organoid-based investigations. Organoids derived from iPSCs offer distinct advantages, particularly in enabling the observation of disease progression and drug metabolism in an in vivo-like environment, surpassing the capabilities of iPSC-derived cells. Furthermore, iPSC-based cell therapy has emerged as a focal point of clinical interest. In this review, we provide an extensive overview of non-integrative reprogramming methods that have evolved since the inception of iPSC technology. We also deliver a comprehensive examination of iPSC-derived organoids, spanning the realms of the nervous system, cardiovascular system, and oncology, as well as systematically elucidate recent advancements in iPSC-related cell therapies.

BACKGROUND:Diabetic cardiomyopathy (DCM) is a serious health-threatening complication of diabetes mellitus characterized by myocardial fibrosis and abnormal cardiac function. Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are a potential therapeutic tool for DCM and myocardial fibrosis via mechanisms such as the regulation of microRNA (miRNA) expression and inflammation. It remains unclear, however, whether hUC-MSC therapy has beneficial effects on cardiac function following different durations of diabetes and which mechanistic aspects of DCM are modulated by hUC-MSC administration at different stages of its development. This study aimed to investigate the therapeutic effects of intravenous administration of hUC-MSCs on DCM following different durations of hyperglycemia in an experimental male model of diabetes and to determine the effects on expression of candidate miRNAs, target mRNA and inflammatory mediators. METHODS:A male mouse model of diabetes was induced by multiple low-dose streptozotocin injections. The effects on severity of DCM of intravenous injections of hUC-MSCs and saline two weeks previously were compared at 10 and 18 weeks after diabetes induction. At both time-points, biochemical assays, echocardiography, histopathology, polymerase chain reaction (PCR), immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to analyze blood glucose, body weight, cardiac structure and function, degree of myocardial fibrosis and expression of fibrosis-related mRNA, miRNA and inflammatory mediators. RESULTS:Saline-treated diabetic male mice had impaired cardiac function and increased cardiac fibrosis after 10 and 18 weeks of diabetes. At both time-points, cardiac dysfunction and fibrosis were improved in hUC-MSC-treated mice. Pro-fibrotic indicators (α-SMA, collagen I, collagen III, Smad3, Smad4) were reduced and anti-fibrotic mediators (FGF-1, miRNA-133a) were increased in hearts of diabetic animals receiving hUC-MSCs compared to saline. Increased blood levels of pro-inflammatory cytokines (IL-6, TNF, IL-1β) and increased cardiac expression of IL-6 were also observed in saline-treated mice and were reduced by hUC-MSCs at both time-points, but to a lesser degree at 18 weeks. CONCLUSION:Intravenous injection of hUC-MSCs ameliorated key functional and structural features of DCM in male mice with diabetes of shorter and longer duration. Mechanistically, these effects were associated with restoration of intra-myocardial expression of miRNA-133a and its target mRNA COL1AI as well as suppression of systemic and localized inflammatory mediators.

Mesenchymal stromal cells (MSCs) a potentially effective disease-modulating therapy for diabetic nephropathy (DN) but their clinical translation has been hampered by incomplete understanding of the optimal timing of administration and in vivo mechanisms of action. This study aimed to elucidate the reno-protective potency and associated mechanisms of single intravenous injections of human umbilical cord-derived MSCs (hUC-MSCs) following shorter and longer durations of diabetes. A streptozotocin (STZ)-induced model of diabetes and DN was established in C57BL/6 mice. In groups of diabetic animals, human (h)UC-MSCs or vehicle were injected intravenously at 8 or 16 weeks after STZ along with vehicle-injected non-diabetic animals. Diabetes-related kidney abnormalities was analyzed 2 weeks later by urine and serum biochemical assays, histology, transmission electron microscopy and immunohistochemistry. Serum concentrations of pro-inflammatory and pro-fibrotic cytokines were quantified by ELISA. The expression of autophagy-related proteins within the renal cortices was investigated by immunoblotting. Bio-distribution of hUC-MSCs in kidney and other organs was evaluated in diabetic mice by injection of fluorescent-labelled cells. Compared to non-diabetic controls, diabetic mice had increases in urine albumin creatinine ratio (uACR), mesangial matrix deposition, podocyte foot process effacement, glomerular basement membrane thickening and interstitial fibrosis as well as reduced podocyte numbers at both 10 and 18 weeks after STZ. Early (8 weeks) hUC-MSC injection was associated with reduced uACR and improvements in multiple glomerular and renal interstitial abnormalities as well as reduced serum IL-6, TNF-α, and TGF-β1 compared to vehicle-injected animals. Later (16 weeks) hUC-MSC injection also resulted in reduction of diabetes-associated renal abnormalities and serum TGF-β1 but not of serum IL-6 and TNF-α. At both time-points, the kidneys of vehicle-injected diabetic mice had higher ratio of p-mTOR to mTOR, increased abundance of p62, lower abundance of ULK1 and Atg12, and reduced ratio of LC3B to LC3A compared to non-diabetic animals, consistent with diabetes-associated suppression of autophagy. These changes were largely reversed in the kidneys of hUC-MSC-injected mice. In contrast, neither early nor later hUC-MSC injection had effects on blood glucose and body weight of diabetic animals. Small numbers of CM-Dil-labeled hUC-MSCs remained detectable in kidneys, lungs and liver of diabetic mice at 14 days after intravenous injection. Single intravenous injections of hUC-MSCs ameliorated glomerular abnormalities and interstitial fibrosis in a mouse model of STZ-induced diabetes without affecting hyperglycemia, whether administered at relatively short or longer duration of diabetes. At both time-points, the reno-protective effects of hUC-MSCs were associated with reduced circulating TGF-β1 and restoration of intra-renal autophagy.

Background Long‐QT syndrome is a primary cardiac ion channelopathy predisposing a patient to ventricular arrhythmia through delayed repolarization on the resting ECG. We aimed to establish a patient‐specific, human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes model of long‐QT syndrome type 3 (LQT3) using clustered regularly interspaced palindromic repeats (CRISPR/Cas9), for disease modeling and drug challenge. Methods and Results HiPSCs were generated from a patient with LQT3 harboring an SCN5A pathogenic variant (c.1231G>A; p.Val411Met), and an unrelated healthy control. The same SCN5A pathogenic variant was engineered into the background healthy control hiPSCs via CRISPR/Cas9 gene editing to generate a second disease model of LQT3 for comparison with an isogenic control. All 3 hiPSC lines were differentiated into cardiomyocytes. Both the patient‐derived LQT3 ( SCN5A +/− ) and genetically engineered LQT3 ( SCN5A +/− ) hiPSC‐derived cardiomyocytes showed significantly prolonged cardiomyocyte repolarization compared with the healthy control. Mexiletine, a cardiac voltage‐gated sodium channel (Na V 1.5) blocker, shortened repolarization in both patient‐derived LQT3 and genetically engineered LQT3 hiPSC‐derived cardiomyocytes, but had no effect in the control. Notably, calcium channel blockers nifedipine and verapamil showed a dose‐dependent shortening of repolarization, rescuing the phenotype. Additionally, therapeutic drugs known to prolong the corrected QT in humans (ondansetron, clarithromycin, and sotalol) demonstrated this effect in vitro, but the LQT3 clones were not more disproportionately affected compared with the control. Conclusions We demonstrated that patient‐derived and genetically engineered LQT3 hiPSC‐derived cardiomyocytes faithfully recapitulate pathologic characteristics of LQT3. The clinical significance of such an in vitro model is in the exploration of novel therapeutic strategies, stratifying drug adverse reaction risk and potentially facilitating a more targeted, patient‐specific approach in high‐risk patients with LQT3.

While previous research has demonstrated the therapeutic efficacy of telomerase reverse transcriptase (TERT) overexpression using adeno-associated virus and cytomegalovirus vectors to combat aging, the broader implications of TERT germline gene editing on the mammalian genome, proteomic composition, phenotypes, lifespan extension, and damage repair remain largely unexplored. In this study, we elucidate the functional properties of transgenic mice carrying the Tert transgene, guided by precise gene targeting into the Rosa26 locus via embryonic stem (ES) cells under the control of the elongation factor 1α ( EF1α ) promoter. The Tert knock-in ( TertKI ) mice harboring the EF1α-Tert gene displayed elevated telomerase activity, elongated telomeres, and extended lifespan, with no spontaneous genotoxicity or carcinogenicity. The TertKI mice showed also enhanced wound healing, characterized by significantly increased expression of Fgf7, Vegf, and collagen. Additionally, TertKI mice exhibited robust resistance to the progression of colitis induced by dextran sodium sulfate (DSS), accompanied by reduced expression of disease-deteriorating genes. These findings foreshadow the potential of TertKI as an extraordinary rejuvenation force, promising not only longevity but also rejuvenation in skin and intestinal aging.

Developmental and epileptic encephalopathies (DEEs) are a group of severe, early-onset epilepsies which are often caused by genetic mutations in ion channels. Mutations in KCNQ2, which encodes the voltage-gated potassium channel Kv7.2, is known to cause DEE. Here, we generated three iPSC lines from dermal fibroblasts of a 5 year-old male patient with the KCNQ2 c.881C > T (p.Ala294Val) pathogenic heterozygous variant and three iPSC lines from a healthy sibling control. These iPSC lines have been validated by SNP karyotyping, STR analysis, expression of pluripotent genes, the capacity to differentiate into three germ layers and confir-mation of the mutation in the patient.

KCNQ2 encodes the potassium-gated voltage channel Kv7.2, responsible for the M−current, which contributes to neuronal resting membrane potential. Pathogenic variants in KCNQ2 cause early onset epilepsies, developmental and epileptic encephalopathies. In this study, we generated three iPSC lines from dermal fibroblasts of a 5 year-old female patient with the KCNQ2 c.638C > T (p.Arg213Gln) pathogenic heterozygous variant and three iPSC lines from a healthy sibling control. These iPSC lines were validated by confirming the targeted mutation, SNP karyotyping, STR analysis, pluripotent gene expression, differentiation capacity into three germ layers, and were free of transgene integration and Mycoplasma.

Juvenile osteochondritis dissecans (JOCD) is a pediatric disease, which begins with an osteonecrotic lesion in the secondary ossification center which, over time, results in the separation of the necrotic fragment from the parent bone. JOCD predisposes to early-onset osteoarthritis. However, the knowledge gap in JOCD pathomechanisms severely limits current therapeutic strategies. To elucidate its etiology, we conducted a study with induced pluripotent stem cells (iPSCs) from JOCD and control patients. iPSCs from skin biopsies were differentiated to iMSCs (iPSC-derived mesenchymal stromal cells) and subjected to chondrogenic and endochondral ossification, and endoplasmic reticulum (ER)-stress induction assays. Our study, using 3 JOCD donors, showed that JOCD cells have lower chondrogenic capability and their endochondral ossification process differs from control cells; yet, JOCD- and control-cells accomplish osteogenesis of similar quality. Our findings show that endoplasmic reticulum stress sensing and response mechanisms in JOCD cells, which partially regulate chondrocyte and osteoblast differentiation, are related to these differences. We suggest that JOCD cells are more sensitive to ER stress than control cells, and in pathological microenvironments, such as microtrauma and micro-ischemia, JOCD pathogenesis pathways may be initiated. This study is the first, to the best of our knowledge, to realize the important role that resident cells and their differentiating counterparts play in JOCD and to put forth a novel etiological hypothesis that seeks to consolidate and explain previously postulated hypotheses. Furthermore, our results establish well-characterized JOCD-specific iPSC-derived in vitro models and identified potential targets which could be used to improve diagnostic tools and therapeutic strategies in JOCD.

Multiple molecular pathways and cellular processes have been implicated in the neurobiology of autism and other neurodevelopmental conditions. There is a current focus on synaptic gene conditions, or synaptopathies, which refer to clinical conditions associated with rare genetic variants disrupting genes involved in synaptic biology. Synaptopathies are commonly associated with autism and developmental delay and may be associated with a range of other neuropsychiatric outcomes. Altered synaptic biology is suggested by both preclinical and clinical studies in autism based on evidence of differences in early brain structural development and altered glutamatergic and GABAergic neurotransmission potentially perturbing excitatory and inhibitory balance. This review focusses on the NRXN-NLGN-SHANK pathway, which is implicated in the synaptic assembly, trans-synaptic signalling, and synaptic functioning. We provide an overview of the insights from preclinical molecular studies of the pathway. Concentrating on NRXN1 deletion and SHANK3 mutations, we discuss emerging understanding of cellular processes and electrophysiology from induced pluripotent stem cells (iPSC) models derived from individuals with synaptopathies, neuroimaging and behavioural findings in animal models of Nrxn1 and Shank3 synaptic gene conditions, and key findings regarding autism features, brain and behavioural phenotypes from human clinical studies of synaptopathies. The identification of molecular-based biomarkers from preclinical models aims to advance the development of targeted therapeutic treatments. However, it remains challenging to translate preclinical animal models and iPSC studies to interpret human brain development and autism features. We discuss the existing challenges in preclinical and clinical synaptopathy research, and potential solutions to align methodologies across preclinical and clinical research. Bridging the translational gap between preclinical and clinical studies will be necessary to understand biological mechanisms, to identify targeted therapies, and ultimately to progress towards personalised approaches for complex neurodevelopmental conditions such as autism.

Background . Non-small cell lung cancer (NSCLC) is still a slightly less orphan disease after immunotherapy, and routine treatment has low efficiency and adverse events. Ginseng is commonly used in the treatment of NSCLC. The purpose of this study is to assess the efficacy and hemorheological indexes of ginseng and its active components in patients with non-small cell lung cancer. Methods . A comprehensive literature search was performed in PubMed, the Cochrane Library, Medline (Ovid), the Web of Science, Embase, CKNI, Wan Fang, VIP, and SinoMed up to July 2021. Only randomized controlled trials evaluating ginseng in combination with chemotherapy versus chemotherapy alone in NSCLC patients were included. Primary outcomes included patients’ condition after using ginseng or its active components. Secondary outcomes included changes in immune cells, cytokines, and secretions in serum. Data were extracted by two independent individuals, and the Cochrane Risk of Bias tool version 2.0 was applied for the included studies. Systematic review and meta-analysis were performed by RevMan 5.3 software. Results . The results included 1480 cases in 17 studies. The results of the integration of clinical outcomes showed that the treatment of ginseng (or combination of ginseng with chemotherapy) can improve the quality of life for patients with NSCLC. Analysis of immune cell subtypes revealed that ginseng and its active ingredients can upregulate the percentages of antitumor immunocyte subtypes and downregulate the accounts of immunosuppressive cells. In addition, a reduction of the inflammatory level and an increase of antitumor indicators in serum were reported. Meta-analysis showed that Karnofsky score: WMD = 16, 95% CI (9.52, 22.47); quality-of-life score: WMD = 8.55, 95%CI (6.08, 11.03); lesion diameter: WMD = −0.45, 95% CI (−0.75, −0.15); weight: WMD = 4.49, 95% CI (1.18, 7.80); CD3 + : WMD = 8.46, 95% CI (5.71, 11.20); CD4 + : WMD = 8.45, 95% CI (6.32, 10.57)+; CD8 + : WMD = −3.76, 95% CI (−6.34, −1.18); CD4 + /CD8 + : WMD = 0.32, 95% CI (0.10, 0.53); MDSC: WMD = −2.88, 95% CI (−4.59, −1.17); NK: WMD = 3.67, 95% CI (2.63, 4.71); Treg: WMD = −1.42, 95% CI (−2.33, −0.51); CEA: WMD = −4.01, 95% CI (−4.12, −3.90); NSE: WMD = −4.00, 95% CI (−4.14, −3.86); IL-2: WMD = 9.45, 95% CI (8.08, 10.82); IL-4: WMD = −9.61, 95% CI (−11.16, −8.06); IL-5: WMD = −11.95, 95% CI (−13.51, −10.39); IL-6: WMD = −7.65, 95% CI (−8.70, −6.60); IL-2/IL-5: WMD = 0.51, 95% CI (0.47, 0.55); IFN- γ : WMD = 15.19, 95% CI (3.16, 27.23); IFN- γ /IL-4: WMD = 0.91, 95% CI (0.85, 0.97); VEGF: WMD = −59.29, 95% CI (−72.99, −45.58); TGF- α : WMD = −10.09, 95% CI (−12.24, −7.94); TGF- β : WMD = −135.62, 95% CI (−147.00, −124.24); TGF- β 1: WMD = −4.22, 95% CI (−5.04, −3.41); arginase: WMD = −1.81, 95% CI (−3.57, −0.05); IgG: WMD = 1.62, 95% CI (0.18, 3.06); IgM: WMD = −0.45, 95% CI (−0.59, −0.31). All results are statistically significant. No adverse events were reported in the included articles. Conclusion . It is a reasonable choice to use ginseng and its active components as adjuvant therapy for NSCLC. Ginseng is helpful for NSCLC patients’ conditions, immune cells, cytokines, and secretions in the serum.

Sporadic amyotrophic lateral sclerosis (sALS) is the majority of ALS, and the lack of appropriate disease models has hindered its research. Induced pluripotent stem cell (iPSC) technology now permits derivation of iPSCs from somatic cells of sALS patients to investigate disease phenotypes and mechanisms. Most existing differentiation protocols are time-consuming or low efficient in generating motor neurons (MNs). Here we report a rapid and simple protocol to differentiate MNs in monolayer culture using small molecules, which led to nearly pure neural stem cells in 6 days, robust OLIG2+ pMNs (73%-91%) in 12 days, enriched CHAT+ cervical spinal MNs (sMNs) (88%-97%) in 18 days, and functionally mature sMNs in 28 days. This simple and reproducible protocol permitted the identification of hyperexcitability phenotypes in our sALS iPSC-derived sMNs, and its application in neurodegenerative diseases should facilitate in vitro disease modeling, drug screening, and the development of cell therapy.

Motile cilia lining on the ependymal cells are crucial for cerebrospinal fluid (CSF) flow and its dysfunction is often associated with hydrocephalus. Unc51-like-kinase 4 (Ulk4) was previously linked to CSF flow and motile ciliogenesis in mice, as the hypomorph mutant of Ulk4 (Ulk4(tm1a/tm1a)) developed hydrocephalic phenotype resulted from defective ciliogenesis and disturbed ciliary motility, while the underling mechanism is largely obscure. Here, we report that serine/threonine kinase 36 (STK36), a paralog of ULK4, directly interacts with ULK4 and this was demonstrated by yeast two-hybrid (Y2H) in yeast and coimmunoprecipitation (co-IP) assays in HEK293T cells, respectively. The interaction region was confined to their respective N-terminal kinase domain. The hypomorph mutant of Stk36 (Stk36(tmE4-/-)) also developed progressive hydrocephalus postnatally and dysfunctional CSF flow, with multiple defects of motile cilia, including reduced ciliary number, disorganized ciliary orientation, defected axonemal structure and inconsistent base body (BB) orientation. Stk36(tmE4-/-) also disturbed the expression of Foxj1 transcription factor and a range of other ciliogenesis-related genes. All these morphological changes, motile cilia defects and transcriptional dysregulation in the Stk36(tmE4-/-) are practically copied from that in Ulk4(tm1a/tm1a) mice. Taken together, we conclude that both Stk36 and Ulk4 are crucial for CSF flow, they cooperate by direct binding with their kinase domain to regulate the Foxj1 transcription factor pathways for ciliogenesis and cilia function, not limited to CSF flow. The underlying molecular mechanism probably conserved in evolution and could be extended to other metazoans.

Induced pluripotent stem cell (iPSC) technology, in combination with electrophysiological characterization via multielectrode array (MEA), has facilitated the utilization of iPSC-derived motor neurons (iPSC-MNs) as highly valuable models for underpinning pathogenic mechanisms and developing novel therapeutic interventions for motor neuron diseases (MNDs). However, the challenge of MN adherence to the MEA plate and the heterogeneity presented in iPSC-derived cultures raise concerns about the reproducibility of the findings obtained from these cellular models. We discovered that one novel factor modulating the electrophysiological activity of iPSC-MNs is the extracellular matrix (ECM) used in the coating to support in vitro growth, differentiation and maturation of iPSC-MNs. The current study showed that two coating conditions, namely, Poly-L-ornithine/Matrigel (POM) and Polyethyleneimine (PEI) strongly promoted attachment of iPSC-MNs on MEA culture dishes compared to three other coating conditions, and both facilitated the maturation of iPSC-MNs as characterized by the detection of extensive electrophysiological activities from the MEA plates. POM coating accelerated the maturation of the iPSC-MNs for up to 5 weeks, which suits modeling of neurodevelopmental disorders. However, the application of PEI resulted in more even distribution of the MNs on the culture dish and reduced variability of electrophysiological signals from the iPSC-MNs in 7-week cultures, which permitted the detection of enhanced excitability in iPSC-MNs from patients with amyotrophic lateral sclerosis (ALS). This study provides a comprehensive comparison of five coating conditions and offers POM and PEI as favorable coatings for in vitro modeling of neurodevelopmental and neurodegenerative disorders, respectively.

Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant ( SCN5A ± ) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development. Graphical Abstract